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ELSEVIERS

Medical Laboratory Science


Examination Review
This page intentionally left blank
ELSEVIERS

Medical Laboratory Science


Examination Review
Linda J. Graeter
Associate Professor
Medical Laboratory Science Program
University of Cincinnati
Cincinnati, Ohio

Elizabeth G. Hertenstein
Assistant Professor
Medical Laboratory Science Program
University of Cincinnati
Cincinnati, Ohio

Charity E. Accurso
Assistant Professor
Medical Laboratory Science Program
University of Cincinnati
Cincinnati, Ohio

Gideon H. Labiner
Associate Professor
Medical Laboratory Science Program
University of Cincinnati
Cincinnati, Ohio
3251 Riverport Lane
St. Louis, Missouri 63043

Elseviers Medical Laboratory Science Examination ISBN: 978-1-4557-0889-5

Copyright 2015 by Saunders, an imprint of Elsevier Inc. All rights reserved.

No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording,
or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further
information about the Publishers permissions policies, and our arrangements with organizations such as the Copyright Clearance Center and the
Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions.

This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein).

Notices
Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research
methods, professional practices, or medical treatment may become necessary.
Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods,
compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of
others, including parties for whom they have a professional responsibility.
With respect to any drug or pharmaceutical products identified, readers are advised to check the most current information provided (i) on
procedures featured or (ii) by the manufacturer of each product to be administered, to verify the recommended dose or formula, the method and
duration of administration, and contraindications. It is the responsibility of practitioners, relying on their own experience and knowledge of their
patients, to make diagnoses, to determine dosages and the best treatment for each individual patient, and to take all appropriate safety precautions.
To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to
persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or
ideas contained in the material herein.

Library of Congress Cataloging-in-Publication Data


Elseviers medical laboratory science examination review / [edited by] Linda J. Graeter, Elizabeth G. Hertenstein, Charity E. Accurso, Gideon H.
Labiner. First edition.
p.; cm.
Medical laboratory science examination review
Includes bibliographical references and index.
ISBN 978-1-4557-0889-5 (pbk.: alk. paper)
I. Graeter, Linda J., editor. II. Hertenstein, Elizabeth G., editor. III. Accurso, Charity E., editor. IV. Labiner, Gideon H., editor. V. Title: Medical
laboratory science examination review.
[DNLM: 1. Clinical Laboratory TechniquesExamination Questions. QY 18.2]
RB37
616.070 56dc23
2014016504

Executive Content Strategist: Kellie White


Content Development Manager: Billie Sharp
Content Development Specialist: Betsy McCormac
Publishing Services Manager: Catherine Jackson
Senior Project Manager: Rachel E. McMullen
Design Direction: Maggie Reid

Printed in the United States of America


CONTRIBUTORS

Brenda C. Barnes, PhD, MT(ASCP)SBBCM Mark W. Ireton, MA, BS, MLS(ASCP)CM


Director, Medical Laboratory Science Program, Blood Bank Technologist II
Associate Professor Hoxworth Blood Center
Allen College University of Cincinnati
Waterloo, Iowa Cincinnati, Ohio

Janelle M. Chiasera, PhD, MT(ASCP) Paul R. Labbe, MS, MCLT


Chair, The Department of Clinical and Diagnostic Sciences, Vice President Information Resources
Professor CompuNet Clinical Laboratories
The University of Alabama, Birmingham Dayton, Ohio
Birmingham, Alabama
Joel E. Mortensen, PhD, HCLD, FAAM
Sandy Cook, MS, MT(ASCP) Department of Pathology and Laboratory Medicine
Assistant Professor Cincinnati Childrens Hospital Medical Center
Clinical Laboratory Services Cincinnati, Ohio
Ferris State University
Big Rapids, Michigan Susan King Strasinger, DA, MLS(ASCP)
Faculty Associate
Melanie J. Giusti, BS, MLS(ASCP)CM The University of West Florida
Program Manager Pensacola, Florida
Medical Laboratory Science Program
College of Allied Health Sciences
University of Cincinnati
Cincinnati, Ohio

v
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ACKNOWLEDGMENTS

We are grateful to all contributing authors and reviewers who dedicated time and
effort during the development of this book. A special word of recognition and appre-
ciation for their dedication to Medical Laboratory education is offered to Melanie
Giusti, MLS(ASCP)CM; Lara Kolar, MT(ASCP); John Landis, MS, MT(ASCP);
Jennifer Macht, BS MT(ASCP), CHT (ABHI); Ryan McGough, MS, MT(ASCP); Erin
Rumpke, MS, MT(ASCP); and Beth Warning, MS, MLS (ASCP)CM.
Last but not least, our sincere thanks are extended to our Elsevier colleagues: Ellen
Wurm-Cutter, Content Manager; Amy Whittier, Content Development Specialist;
and all others at Elsevier who were involved in this project. Their assistance, thought-
ful advice, and continued support were invaluable as we navigated through the
various steps in completing the book.

vii
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PREFACE

The Medical Laboratory Science Review is intended to certification or licensure examinations. The Evolve web-
serve as a review tool for candidates who are preparing site was created with that in mind and includes printable
to sit for certification or licensure examinations in Med- study worksheets and additional review materials. Stu-
ical Laboratory Science. However, the integral nature of dents are able to generate individualized study files
this review book provides a review for individuals seeking from these materials. The website also includes 1000
to strengthen knowledge in the topics related to the clin- additional multiple-choice questions that are different
ical laboratory. Therefore this text can provide a multi- from those in the book. From these materials, students
purpose review. The text is ideal for those preparing can create additional practice examinations focusing
for Medical Laboratory Science (MLS) or categorical on the content area of their choice.
Technologist/Scientist (Blood Banking, Chemistry,
Hematology, Microbiology, and Molecular Biology) cer-
tification examinations sponsored by the American Soci-
EXAMINATION PREPARATION
ety of Clinical Pathology (ASCP) Board of Certification
or for the American Medical Technologists (AMT)- Preparation for certification and licensure examinations
supported certification examination. Additionally, this is sometimes a daunting and intimidating process.
text will be helpful for those preparing for the Medical We encourage students to recognize the time and effort
Laboratory Technology (MLT) certification examina- placed in successfully completing their respective educa-
tion, although the scope of some of the advanced topics tional programs and the knowledge gained while doing
are outside the required competencies for the MLT level. so. Preparing for the examination then becomes a struc-
The outline format enhances learning and comprehension tured plan that provides for a review of the knowledge
for each professional career entry track. Others who gained.
would benefit from using this review text to support their Shortly before program completion, thoroughly review
studies are those seeking advanced degrees, ASCP Spe- the ASCP Board of Certification website. Be sure to review
cialist certification, Physician Assistant students, and the requirements that must be met to sit for the examina-
Pathology Residents. Additional uses include serving as tion, along with the recommended dates to submit the
a reference book for students and educators, providing application. We encourage students to sit for the examina-
continuing education review and to refresh knowledge. tion within 6 months of program completion. Students
The books preface is followed by review materials who wait longer tend to have a more difficult time review-
encompassing all major areas of the laboratory, divided ing and preparing for the examination. On the website,
into 11 chapters. Each chapter includes a comprehensive you will also find content outlines and a distribution of
bulleted summary of didactic information. The chapter the content areas that will aid in your planning. Details
summary outlines provide a thorough but efficient review are included about the cost, length, and structure of the
of key content information. Each chapter is followed by examination. Review the examination preparation guide.
30 to 100 multiple-choice questions. The questions Complete a practice examination or set of review
include representation of the three question types (I, II, questions. Record your answers on a separate piece
III) to enhance recall, interpretation, and problem-solving of paper so that you can continue to practice with
skills. Each question includes an explanation of the cor- the same questions.
rect answer. The books final section is a comprehensive After reviewing missed questions, make a list of the
practice examination designed using the ASCP Board of specific content areas that were missed (e.g., ane-
Certification guidelines. mias, streptococci, liver enzymes) and then design
The chapters and examination questions were written a study schedule using a calendar that allows more
by Medical Laboratory educators and clinical experts, all time to review the more challenging areas.
of whom are recognized for expertise in their respective Plan 1 to 2 weeks for a thorough review of each
area of practice. All chapters and multiple-choice ques- content area.
tions underwent a peer-review process as the content Create mini study guides for your more challenging
was developed. or weaker areas. The study guides should include
The companion websiteEvolve to accompany Med- the following:
ical Laboratory Science Reviewwas developed to Graphical representations of a disease or process
enhance each candidates preparation process by provid- Concise charts or tables
ing additional review materials. Students often benefit A brief paragraph explaining the topic or ques-
from a variety of study approaches when preparing for tion or a short outline

ix
x Preface

Preparing the guides is an active learning exercise that Practice, practice, practice! It is always helpful to ad-
will help in reviewing the specific content and in main- dress questions you previously reviewed. Use the questions
taining focus on weaker topics. It is human nature to in this text and those from the online companion site.
gravitate toward favorite topics, but it is also necessary The day of the examination . . . breathe deeply! If you
to focus on the areas that are more challenging. Compile are not sure of the location of the testing center, take a
the study guides in a binder organized by content area. test drive to the center a week before the examination.
The guides will be great tools to review the week before Have a scheduled plan for the day. Be well rested and
the actual examination be sure to eat a good meal before arriving at the testing
When studying the review questions or old examina- center. If you have prepared, your efforts will be evident
tions: in your success!
Provide a rationale as to why you can rule out incor-
rect answers and rule in correct ones. NOTE: Although it is this books intent to properly
Pay attention to small details that will help rule out prepare readers for their certification examination, use
a wrong answer. of this book alone does not guarantee passage of certifi-
Writing your rationales out in sentence form is cation or licensure examinations.
another great review tool.
Create your own question rationales.
CONTENTS

Color insert follows p. 20

1 Microbiology, 1
Joel E. Mortensen and Linda J. Graeter

2 Mycology, Virology, and Parasitology, 50


Linda J. Graeter and Joel E. Mortensen

3 Hematology, 90
Sandy Cook

4 Hemostasis, 139
Charity E. Accurso

5 Clinical Fluid Analysis, 159


Sue King Strasinger

6 Immunology and Serology, 180


Elizabeth G. Hertenstein

7 Immunohematology and Blood Transfusion Medicine, 200


Brenda C. Barnes and Elizabeth G. Hertenstein

8 Clinical Chemistry, 228


Janelle M. Chiasera

9 Molecular Diagnostics, 262


Gideon H. Labiner

10 Laboratory Operations, 283


Paul R. Labbe and Linda J. Graeter

11 Laboratory Calculations, 298


Melanie J. Giusti and Mark W. Ireton

APPENDIX
A Answers and Rationales to Certification Preparation Questions, 312
B Mock Examination, 357
C Examination Preparation Worksheet, 367
D Color Insert Figure Credit Lines, 370

xi
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CHAPTER

1
Microbiology
Joel E. Mortensen and Linda J. Graeter

STAINED SMEARS Proper staining technique


Gram stain Underdecolorization
Gram stain history Overdecolorization
Special considerations: Sputum specimens
Developed by Hans Christian Gram in 1884
>25 epithelial cells/lpf saliva
Became the major bacterial staining method
Most bacteria are stained by this method Few epithelial cells, many PMN cells:Specimen
more likely to yield a pathogen
Exceptions include Legionella, Mycoplasma,
Examine properly stained area
Chlamydia, and others
Gram stain procedure Recognize normal oral flora
Report or reject
Crystal violet
Sputum only, may not apply to aspirates or
Grams iodine
children
Decolorizer
Special considerations: Urine
Acetone and alcohol either alone or together
Urine specimens
Safranin
Gram stain mechanism 1 cell per oil immersion field approximately
1  105 CFU/mL
Differences in the microbial cell wall are visualized
Not commonly performed
Cell walls of gram-negative cells have higher
Quantitation
lipid content than gram-positive cells
No organisms seen
Crystal violet penetrates both types
Few per slide Rare
Iodine is added, forming the crystal violetiodine
0 to 2 per field Few
(CV-I) complex (mordant)
2 to 10 per field Moderate
Decolorizer dissolves the lipid layer from the
More than 10 per field Many
gram-negative cells allowing the CV-I complex
to wash out
Counterstain is applied to dye the decolorized GROWING BACTERIA IN THE
gram-negative cells LABORATORYMEDIA
Clinical utility
A true STAT test in microbiology Types of media
Judge adequacy of a specimen Bacteriology
Recognition specific morphologies Routine
Indicate need for additional tests Fastidious
Expand clinical diagnostic picture Anaerobes
Limitations Mycoplasma, Ureaplasma
Only partial bacterial identification Mycobacteriology
Some organisms do not stain Mycology
No organisms seen does not rule out infection Virology
Normal flora can mask pathogens Viruses
Human error Chlamydia
Organisms do not stain as expected Constituents of media
Diagnostic considerations Agar
Cell identification Gelatinous seaweed extract
Epithelial cells 1% to 2% agar in plates
Polymorphonuclear (PMN) cells Nutrients
Bacteria Hydrolyzed proteins

1
2 CHAPTER 1 Microbiology

Animal Chocolate agar


Plant Casein peptones
Carbohydrates, sugars Meat peptones
Enrichments Corn starch
Yeast extracts, blood Hemoglobin (V factor)
Buffers IsoVitaleX Enrichment (X factor)
Stable pH for growth Used for specimens from which fastidious
pH indicators organisms may be isolated
Neutral red: Red to colorless Haemophilus spp., Neisseria spp., Brucella or
Phenol red: Yellow to red Capnocytophaga spp.
MacConkey agar
Thymol blue: Yellow to green/blue
Others Peptone base with lactose, crystal violet, and bile
Inhibitors salts
Lactose to provide fermentable sugar (Lactose
Dyes
positive vs negative)
Crystal violet, eosin, and methylene blue
Crystal violetinhibit gram-positive bacteria
Bile salts
Bile saltsinhibit gram-positive bacteria
Sodium deoxycholate
Neutral red pH indicator
Sodium chloride
Selective for gram-negative organisms
Sodium citrate
Differential for lactose fermentation
Antibiotics
Selective Lactose positive
Escherichia coli: Dry, flat, dark pigment
Contains inhibitory agents to all organisms except
Klebsiella/Enterobacter: Mucoid
one being sought
Selects for certain organisms to the disadvantage of Citrobacter: Late fermenter
Serratia: Late, red pigment (some)
others
Example Lactose negative
Proteus: Swarming
Colistin nalidixic acid agar (CNA)
Morganella, Providencia, Edwardsiella,
MacConkey agar
Hafnia
Differential
Pseudomonas
Allows organism to be morphologically distinguished
Salmonella
from other organisms with different characteristics
Shigella
Examples
Used for various patient and environmental
Sheep blood agar (SBA)
samples
MacConkey agar XLD
Key media for routine aerobic cultures
Yeast extract
Supportive
Sodium deoxycholate
Blood agar Inhibits gram-positive organisms
Chocolate agar Phenol red: pH indicator
Selective/differential for gram-negative bacilli Lactose and sucrose in excess, xylose in lower
MacConkey agar amounts
Xylose lysine desoxycholate (XLD) or Hektoen Lactose and sucrose to provide fermentable
(HE) for stool sugar
Selective/differential for gram-positive organisms Lysine: Lysine decarboxylase
CNA Salmonella decarboxylate the lysine shift the
Phenylethyl alcohol agar (PEA) pH indicator to red
Examples Sodium thiosulfate, ferric ammonium citrate
Blood agar H2S production
Casein peptones: Group of proteins from milk Yellow: Ferments the excess carbohydrates (or
Soybean peptones xylose only), causes large pH drop, yellow
5% sheep blood (E. coli)
Approximately 1% agar-agar Colorless or red: No fermentation, no H2S
Used for general growth of gram-positive and (Shigella and Providencia)
gram-negative aerobes Red with black center
The most common supportive media because Ferments xylose, produces low pH, then
most organisms grow on it, but it is also differ- decarboxylates lysine, produces high pH
ential because of hemolytic pattern H2S production (Salmonella)
CHAPTER 1 Microbiology 3

HE Chopped meatglucose and thioglycolate broth are


Meat peptones and yeast extract most common
Bile salts Use should be limited to fluids and tissue
Inhibit gram-positive organisms Not swabs
Lactose, sucrose, salacin
Lactose and sucrose to provide fermentable
sugar Growth RequirementsOther Important
Indicators: Bromophenol blue and acid fuchsin Components
pH indicators Atmosphere requirements
Ferric ammonium citrate for H2S production 3% to 5% CO2
Differential for Salmonella and Shigella Room air
Yellow-orange colonies: Lactose fermenter Increased CO2 and N, decreased O2
(E. coli) Microaerophilic: 5% O2, 10% CO2, 85% N
Colorless/green colonies with unchanged medium: Anaerobic: 85% N2, 10% H2, 5% CO2
Nonlactose fermenter (Shigella, Providencia) Temperature requirements
Black colonies: H2S production (Salmonella) 35 C Room temperature (25 C to 30 C)
CNA 42 C
Casein peptones 4 C
Digest of animal tissue pH and moisture requirements
Yeast and beef extract pH
Corn starch Most are 6.5 to 7.5
5% sheep blood Buffers maintain pH of media
CNA: inhibits most gram-negative organisms Moisture
Used for samples with mixed flora 70% humidity is optimal for good growth
PEA Prevents drying of media
Casein peptones
Soybean peptones
5% sheep blood STRUCTURE AND METABOLISM
Phenylethyl alcohol to inhibit gram-negative
organisms Bacteria are prokaryotes: Single-cell organisms lacking
Some laboratories use CNA rather than PEA membrane-bound nuclei
Eukaryotes are organisms with a defined nucleus
because of gram-negative breakthrough
Key media for routine anaerobic Mammalian and plant cells are eukaryotic
Basic: Blood agar Reproduction of prokaryotic cells is by binary fission
Simple division of one cell into two cells
Agar base supplemented with 5% sheep blood,
DNA replication and formation of a separating
hemin, and vitamin
Agar base usually trypticase soy or brain heart membrane and cell wall
infusion Can be approximately 20 minutes
Good nonselective medium for initial isolation of
anaerobes
Selective for anaerobes: Supplemented PEA Bacteria
Vitamin K and hemin are added to PEA agar Cells must acquire nutrients, produce energy, and syn-
Suppresses growth of facultative gram-negative thesize macromolecules
bacilli It is important to study these areas so that bacteria can
All anaerobes grow well on this medium be isolated and identified in the laboratory
Selective for gram-negative bacilli: Kanamycin- Bacterial growth requirements
vancomycinlaked blood agar (K-V agar) Nutrients acquired by active transport across the
Aminoglycoside helps separate aerobes and cell membrane from the environment
anaerobes in mixed cultures Requirements for all bacteria
Limit the swarming of Proteus spp. A carbon source (for cellular constituents)
Kanamycin and vancomycin permit growth of A nitrogen source (for proteins)
only gram-negative anaerobes Energy source: Adenosine triphosphate (ATP) (to
Laked blood stimulates the growth of some perform cellular tasks)
anaerobes Trace elements
Back-up broth Iron
Broth medium serves as a check for agar plates Calcium
Useful when the primary shows no growth Zinc
4 CHAPTER 1 Microbiology

Copper Most bacteria are placed into two groups (deter-


Manganese mined by differences in the cell wall)
Cobalt Gram-positive
Phosphorus Gram-negative
Sulfur Chromosome
Potassium Single, long, supercoiled, circular DNA molecule
Magnesium Prokaryotic cell contains no nucleus
Oxygen growth requirements Attached to the cell membrane
Obligate aerobe: Live and grow in air, cannot grow Bacterial chromosomes contain genetic information
anaerobically to code for between 850 to 6500 products
Facultative anaerobe: Can grow aerobically and Enzymes, proteins, and RNA molecules
anaerobically Human chromosome contains approximately
Aerotolerant anaerobe: Grows better anaerobically, 30,000 genes
but can tolerate low levels of air Plasmids
Obligate anaerobe: Grows only anaerobically, poi- Small, circular double-stranded DNA
soned by air Not part of the chromosome
Microaerophilic: Increased CO2 or other enriched May contain several to several hundred genes
environment One plasmid, multiple copies of same plasmid, or
Bacterial metabolism more than one type
Production of ATP Antibiotic resistance genes common
Drives other metabolic processes Can be exchanged between a donor and a recipient
Substrates ! glucose ! metabolic pathway ! energy in conjugation
Fermentation versus respiration Ribosomes
Fermentation Sites of protein synthesis
Glucose is converted into pyruvate A 70S prokaryotic ribosome comprises a 30S subu-
Embdem-Meyerhof pathway or glycolysis nit and a 50S subunit
Fermentation is metabolism in the absence Estimated approximately 15,000 ribosomes in the
of O2 cytoplasm of an E. coli cell
Anaerobic Cell membrane
Net gain: 2ATP, NADH2 Bacterial membrane similar in structure and func-
Pyruvate can then enter several other cycles tion to eukaryotic cell membrane
End-products vary depending on cycle entered Membrane consists of proteins and phospholipids
Aerobic respiration Bilayer, with charged or polar groups facing out-
Glucose usage under aerobic conditions ward and the noncharged portions in between
Pyruvate enters Krebs cycle (tricarboxylic acid Selectively permeable
[TCA] cycle) Many enzymes are attached to membrane
Nicotinamide adenine dinucleotide (NADH) Metabolic reactions take place at membrane
and flavin adenine dinucleotide (FADH) enter Bacterial cell wall
the electron transport chain Cell wall defines the shape of bacterial cells
Net gain: 38 ATP (including ATP from Main constituent is complex polymer
fermentation) peptidoglycan
End-products: CO2 and H2O Many sugar molecules (polysaccharide) linked
Application to the clinical laboratory by small peptide (short protein) chains
Systems to detect fermentation or respiration Peptidoglycan is found only in bacteria
Acid detection (pH indicators) Thickness of the cell wall and its exact composition
Gas detection vary with the species of bacteria
Alcohol detection Gram-positive cell walls have thick layer of
Different carbon sources peptidoglycan
Not all bacteria undergo respiration Gram-negative walls have much thinner layer of
Some lack the enzymes needed peptidoglycan and an outer membrane
Some cannot survive in O2 Within the cell wall of gram-negative bacteria is
Potential biochemical tests for organism lipopolysaccharide (LPS)
identification Part of LPS protrudes from the cell surfaceO
Tests based on presence or absence of these spe- antigen
cific enzymes Cellular morphology and arrangement
Cellular structure Glycocalyx
Bacteria are prokaryotic Some bacteria have layer of material located outside
Bacteria are small (0.2-2 mm diameter, 1-6 mm length) cell wall
CHAPTER 1 Microbiology 5

Glycocalyx is a slimy, gelatinous material produced Capsules


near the cell membrane and secreted outside of the Pili
cell wall Other extracellular proteins
Two types of glycocalyx Bacterial taxonomy: Shared morphologic, physiologic,
Slime layer: Not highly organized, not firmly and genetic traits
attached Species
Pseudomonas and Staphylococcus Basic taxonomic group
Capsule: Highly organized and firmly attached Composed of related individuals with shared
to the cell wall characteristics that resemble one another
Usually polysaccharides, may be combined Complete definition difficult
with lipids and proteins Genus (genera)
Protects from engulfment by white blood Family
cells (WBCs) Order
Both may protect the bacterium from antibiotics Class division
Flagella Kingdom
Enable bacteria to move in liquid environment Bacterial names are binomial
Water, intestinal tract, blood, or urine Genus and species
Consist of three or more protein appendages Treated as Latin and written in italic
twisted together Genus can be abbreviated in written material
Number and arrangement of flagella are character- after first used
istic of species Staphylococcus aureus
Single flagellum at one end to multiple flagella cov- S. aureus
ering the entire cell surface Name changes are designated with ( )
Pili Stenotrophomas (Xanthomonas) maltophilia
Pili or fimbriae are short, hairlike structures
Usually on external surface of gram-negative
FAMILY STAPHYLOCOCCACEAE
organisms
Much thinner than flagella, rigid structure, not General characteristics
associated with motility Gram-positive cocci in clusters or tetrads
Originate in cytoplasm and extend through the Catalase positive
plasma membrane, cell wall, and capsule Aerobic to facultative anaerobic
Two typessex pilus and attachment pilus Nonmotile
Adherence pili anchor to surfaces Staphylococcus: Greek staphyle means bunch of
Tissues in animals body grapes and coccos means granule
Usually quite numerous Genus Staphylococcus: General characteristics
Sex pilus transfers genetic material All ferment glucose
Cell possessing a sex pilus is donor cell Differentiated by coagulase test
Attach to another cell (usually of the same Coagulase-positive are considered S. aureus
species) Coagulase-negative species
Genetic material, usually a plasmid, trans- Staphylococcus epidermidis
ferred through the hollow sex pilus Staphylococcus saprophyticus
Spores Approximately 30 other species
Some bacteria form thick-walled structures
Bacillus and Clostridium
Staphylococcus aureus
Means of survival when moisture or
nutrients low Most clinically important
Formed during sporulation Causes numerous infections
Copy of the chromosome Important hospital pathogen
Some cytoplasm enclosed in thick protein coats Antibiotic resistance has become a major issue (again)
Resistant to heat, cold, drying, most chemicals, Epidemiology
boiling Humans are natural reservoir for S. aureus
Survive for many years in soil or dust Asymptomatic colonization is far more common
Virulence factors than infection
Bacteria that cause disease are termed pathogenic Colonization of nasopharynx and perineum skin
because of various factors occurs shortly after birth and recurs
Examples Transmission occurs by direct contact with a colo-
Exotoxins nized carrier
Endotoxin Carriage rates from 25% to 50%
6 CHAPTER 1 Microbiology

Higher in injection drug users; patients with dia- Abscess


betes, dermatologic conditions, or long-term Fibrin wall around a core of organisms and
indwelling intravascular catheters; and health leukocytes
care workers Pathogenesis
Young children have higher rates Ability to elaborate proteolytic enzymes may facil-
Colonization may be transient or persistent itate the process
Clinical disease Nondisseminated: Local disease (e.g., boils)
Causes suppurative (pus-forming) infections and Dissemination results in pneumonia, bone and joint
toxin diseases infection
Infections can be superficial or invasive Toxin disease: Either toxin alone or in combination
Superficial skin lesions: Boils, sties, furuncles, with invasion
impetigo Toxins
Invasive: Pneumonia, mastitis, arthritis, menin- Enterotoxins
gitis, osteomyelitis, and endocarditis Heat-stable exotoxins that cause diarrhea and
Toxin diseases: Food poisoning, scalded skin syn- vomiting
drome, toxic shock disease Enterotoxins A and D are resistant to gastric and
Scalded skin syndrome digestive acids
Extensive exfoliative dermatitis (skin Toxins are preformed in foods
sloughing) Symptoms (i.e., nausea, vomiting, abdominal
In adults, occurs in chronic renal failure and pain, and cramping) appear 2 to 8 hours after
immunocompromised individuals ingestion and resolve within 8 hours
Mortality in adults can be as high as 50% Enterotoxin F (TSST-1)
Localized Bullous impetigo (large pustule) Epidermolytic toxin
Generalized Profuse peeling of the epider- Sloughing of the skin
mal layer of skin Widespread systemic immune responses
Toxic shock syndrome Exfoliative toxin
Toxic shock syndrome toxin-1 (TSST-1) Similar to TSST-1 but a different site in skin
associated Cytolytic toxins: Extracellular factors that affect
Superantigen red blood cells (RBCs) and WBCs
Characteristic rash Hemolysins
Multisystem disease: High fever, hypotension, Alpha a-Hemolysin: Destroys RBCs, platelets,
and shock tissue
Identified in both sexes Beta b-Hemolysin: Destroys RBCs
Higher prevalence with tampon use Gamma d-Hemolysin: Causes injury, less lethal
Most patients recover: 2% to 5% mortality Leukocidin
Oxacillin-resistant S. aureus Panton-Valentine leukocidin: Exotoxin lethal
Oxacillin-resistant S. aureus (ORSA) and to PMNs
methicillin-resistant S. aureus (MRSA) are resistant May suppress phagocytosis
to antibiotics Enzymes
Methicillin, oxacillin, nafcillin, penicillin, and Coagulase: Causes coagulation of surroundings
amoxicillin Hyaluronidase: Hydrolyzes hyaluronic acid in con-
Frequently other agents nective tissue
Hospital acquired versus community acquired Lipase: Aids colonization by acting on sebaceous
Historically, among persons in hospitals and health glands
care facilities who have weakened immune systems Fatty acidmodifying enzyme: Breaks down antista-
Infections acquired by persons who have not been phylococcal lipids made by the host
recently hospitalized are known as community- Protein A
acquired (CA-ORSA) infections In S. aureus cell wall
Infections in the community are usually skin Binds Fc portion of immunoglobulin (avoid
infections and occur in otherwise healthy people phagocytosis)
May be more pathogenic than hospital-acquired
(HA-ORSA) infection
Pathogenesis and virulence factors Coagulase-Negative Staphylococci
Carriage of the organism
Disseminated via hand to body sites and breaks in Clinically important
the skin Staphylococcus epidermidis
Eczema or minor dermatitis Staphylococcus saprophyticus
CHAPTER 1 Microbiology 7

Normal flora of skin and mucous membranes Beta (b): Lysis of the sheep RBCs in the blood
Approximately 30 other species agar plates
Staphylococcus epidermidis Gamma (g): Nonhemolytic
Infections Classification
Predominantly hospital acquired 85 species at present
Predisposing factors Hemolytic pattern on 5% sheep blood agar
Catheterization, prosthetic heart valves, immu- Serologic group (Lancefield) of the b-hemolytic
nosuppressive therapy group: A, B, C, D, F, G . . . T
Bacteremia Taxonomic/genetic related now used
Endocarditis b-Hemolytic streptococci
Most common cause of hospital-acquired urinary Streptococcus pyogenes: Group A
tract infection (UTI) Streptococcus agalactiae: Group B
Staphylococcus saprophyticus Streptococcus group C, F, G
Normal flora of the mucous membranes of the Others
urogenital tract Many found predominately in animals
Causes UTI
Young, sexually active women
Considered significant in urine cultures even if it is Streptococcus Species
found in small numbers Streptococcus pyogenes
Group A Streptococcus
Not the exact same as S. pyogenes, approximately 5%
Laboratory Diagnosis
Streptococcus anginosus
Media Spread from person to person
Mannitol salt Carriers do exist
CNA Clinical disease
PEA Causes relatively common, significant diseases
CHROMagarmedia containing patented chro- Pharyngitis: Strep throat
mogenic substrates that can be formulated to pro- Skin infections
vide specific colors to develop in colonies of a Otitis
particular genus or species Sinusitis
CHROMagar MRSA Sepsis
S. aureus Scarlet fever
Colonies are medium to large, ivory to yellow, and Some less common diseases
beta-hemolyic Pneumonia
Catalase positive and coagulase positive Meningitis
Mannitol salt positive Fasciitis: Flesh-eating bacteria
S. epidermidis Complications
Colonies are small to medium, nonhemolytic, white Rheumatic heart disease
Coagulase negative Glomerulonephritis
Biochemicals required to identify Rheumatic fever
Often not speciated Complication of pharyngitis
S. saprophyticus Can cause chronic, progressive damage to heart
Colonies are large, with approximately 50% pro- Pathogenesis poorly understood
ducing a yellow pigment Acute glomerulonephritis
Coagulase negative Complication of pharyngitis or cutaneous
Novobiocin resistant Circulating immune complexes deposit in
glomeruli
Inflammatory response causes damage
FAMILY STREPTOCOCCACEAE Pathogenesis and virulence factors
Genus Streptococcus Enters through the respiratory tract or skin contact
Gram-positive cocci in chains or pairs Either local disease or spread
Catalase negative Local disease alone or with S. aureus
Small, grayish colonies on sheep blood agar Systemic spread leads to disease and
Categorized by Lancefield groups complications
Alpha (a): conversion of hemoglobin to methe- M protein and lipoteichoic acid for attachment
moglobin resulting in a green zone in the blood Hyaluronic acid capsule: Inhibits phagocytosis
agar around a colony Extracellular products
8 CHAPTER 1 Microbiology

Pyrogenic (erythrogenic) toxin, which causes the Group D laboratory diagnosis


rash of scarlet fever Small, white colonies with hemolysis on blood
Streptokinase agar
Streptodornase (DNase B) Enterococcus and group D bile esculin positive
Streptolysins Enterococcus grows in higher NaCl concentration
Laboratory diagnosis and is PYR positive
GPC in chains a-Hemolytic streptococci
Small, transparent, smooth, b-hemolytic colonies Streptococcus pneumoniae
Bacitracin susceptible Viridans group streptococci
L-pyrrolidonyl arylamidase (PYR) positive Sometimes enterococcal species
Latex or other grouping tests are usually used in Nutritionally variant streptococci
clinical laboratories Streptococcus pneumoniae
Susceptibility testing and treatment No Lancefield grouping
All isolates are penicillin susceptible Infects humans exclusively, no reservoir is found in
Susceptibility testing not appropriate in almost all nature
settings Carrier rate of S. pneumoniae in the normal human
Exceptions are penicillin-allergic patients nasopharynx is 20% to 40%
Manual method usually used Clinical disease
Streptococcus agalactiae Pneumococcal pneumonia is most common in
Group B Streptococcus elderly, debilitated, or immunosuppressed
Normal flora in female genital tract and gastrointesti- Often after viral infection damages the respira-
nal (GI) tract tory ciliated epithelium
Clinical significance Incidence peaks in the winter
Neonatal sepsis and meningitis Community-acquired pneumonia
UTI Otitis media
Pneumonia in elderly Meningitis (most common cause in adults)
Pathogenesis and virulence factors Septicemia
In early-onset neonatal disease, organism is trans- Laboratory diagnosis
mitted vertically from the mother GPC in pairs, lancet
In late-onset (from 7 days to 3 months age) menin- Colonies are round and usually wet, glistening,
gitis is acquired horizontally, in some instances as a mucoid
nosocomial infection a-Hemolysis
Virulence Optochin susceptible
Capsule Bile soluble
Hemolysin Susceptibility and treatment
Hyaluronidase Susceptibility testing appropriate for isolates from
Proteases normally sterile body sites
Laboratory diagnosis Most automated methods do not work well
Grayish-white, slightly mucoid colonies Penicillin resistance an issue
Small zone of b-hemolysis: Larger colony than Other a-Hemolytic Streptococci
group A Viridans streptococci
Hippurate and CAMP test positive Normal flora
Latex or other grouping tests are usually used in Upper respiratory tract
clinical laboratories Urogenital tract
Susceptibility testing and treatment Clinical significance: Subacute bacterial endo-
All isolates are penicillin susceptible carditis
Other agents are not always active Identification
Susceptibility testing not appropriate in almost a-Hemolytic
all settings Optochin resistance
Exceptions are penicillin-allergic patients Examples: Streptococcus mutans, Streptococcus
Group D Streptococcus salivarius, Streptococcus anginosus group, Strepto-
Normal flora coccus gallolyticus (bovis)
GI tract Susceptibility testing and treatment
Urogenital tract Some penicillin resistance has been reported
Group D antigen, although not always b-hemolytic Susceptibility testing not usually appropriate
Streptococcus gallolyticus because the isolates are rarely clinically significant
Streptococcus equinus If the cause of true disease, susceptibility testing
Clinical significance: Bacteremia and endocarditis may be appropriate
CHAPTER 1 Microbiology 9

Leuconostoc
FAMILY ENTEROCOCCACEAE Micrococcus
Pediococcus
Enterococcus Species General characteristics
38 species Uncommonly isolated
Most common isolates from humans Vitamin B6/pyridoxyl is required for lab growth of
Enterococcus faecalis Abiotrophia and Granulicatella
Enterococcus faecium May be mistaken for Staphylococcus or
Enterococcus avium Streptococcus spp.
Enterococcus casseliflavus Limited pathogenic potential but possible in the
Enterococcus gallinarum young, the old or the immunocompromised
General characteristics May be difficult to identify, rule out Staphylococcus
Gram-positive cocci typically in pairs and short or Streptococcus may be all that is possible
chains
Facultative anaerobe
Catalase negative AEROBIC GRAM-POSITIVE BACILLI
Epidemiology Aerobic gram-positive bacilli represent a tax-
Normal flora of the GI and urogenital tract of onomically and genetically diverse group of organ-
humans and animals isms
One of the top three nosocomial pathogens As human pathogens, notable diseases include liste-
Clinical significance riosis, anthrax, erysipelas, and diphtheria, although
UTI incidence in North America is very low
Wounds General characteristics
Abdominal and pelvic infections Similarities
Nosocomial infections Capable of growth in the presence of O2
Endocarditis and bacteremia Retention of crystal violet after alcohol decolor-
Virulence factors ization step of Gram stain thus appearing purple
Fimbriae: Attachment to epithelial cells in color (gram-positive)
Adhesins: Attachment to intestinal tract Rod shaped
Bacteriocins: Inhibits growth of other intestinal Generally grow easily after 24 hours on non-
bacteria selective agars such as SBA
Gelatinase: Hydrolizes collagen and hemoglobin General characteristics
Laboratory diagnosis Phenotypic diversity includes
Small, white colonies Cell size
a-Hemolytic or nonhemolytic, may be b-hemolytic Approximately 0.2 to 1.5 mm wide, 0.5 to 10 mm
Both Enterococcus and group D are bile esculin long
positive Production of spores
Enterococcus grows in higher NaCl concentration Microscopic appearance may vary
and PYR positive Regular rod shape
Susceptibility testing and treatment Rounded, square, or slightly pointed ends
Inherently resistant to cephalosporins Coryneform with club-shaped cells arranged
Less susceptible than streptococci to penicillin and such that they resemble the letters V and L
ampicillin Filamentous: Cells form long chains
Vancomycin (vancomycin-resistant enterococcus Filamentous with rudimentary or true branching
[VRE]) Initial grouping of aerobic gram-positive bacilli
Resistant to trimethoprim and sulfamethoxazole Spore forming
Most isolates should be tested but limited Bacillus spp.
reporting Nonspore forming
Treatment often limited to vancomycin, aminogly- Listeria, Erysipelothrix, Corynebacterium, and
cosides, and maybe fluoroquinolones others
Some newer agents may be useful: Linezolid,
daptomycin Bacillus Species
General characteristics
MISCELLANEOUS GRAM-POSITIVE COCCI More than 50 species, all are found in soil
Abiotrophia All Bacillus sp. form endospores
Aerococcus All are catalase positive
Granulicatella Spore formation
10 CHAPTER 1 Microbiology

Endospores are unique to Bacillus spp. among aerobes Rhinorrhea (runny nose) rare
Transition from vegetative cells to spores under Incubation period: 1 to 7 days (possibly ranging up
harsh and desiccated environments preserves cell to 42 days)
viability for long periods Case fatality
Not always evident in Gram smears, but visualiza- Without antibiotic treatment: 97%
tion of spores confirms Bacillus genus With antibiotic treatment: 75%
Special stains are used to better visualize spores Anthrax: GI
Spores can be weaponized for use as infectious Abdominal distress, usually accompanied by
aerosols in biological attack bloody vomiting or diarrhea, followed by fever
Highly infectious and signs of septicemia
Bacillus cereus GI illness sometimes seen as oropharyngeal ulcera-
Most common disease is food poisoning tions with cervical adenopathy and fever
Less common opportunistic infection Develops after ingestion of contaminated, poorly
Epidemiology cooked meat
Common agent in food poisoning Incubation period: 1 to 7 days
Two forms of food poisoning Case-fatality: 25% to 60% (role of early antibiotic
Diarrheal: Meat, 24 hours, self-limiting treatment is undefined)
Emetic: Fried rice, 10 hours, self-limiting Anthrax: Complications
Above symptoms caused by two distinct toxins 5% develop meningitis
Opportunistic disease Coma and death occur 1 to 6 days after
exposure
Serious ocular infection
Recovery confers immunity
Wound infection
Vaccines available to high-risk groups
Pathogenesis and virulence
Antibiotics used after exposure
Food poisoning is toxin mediated
Ciprofloxacin, tetracycline (60-day treatment)
Laboratory diagnosis Pathogenesis and virulence factors
Colonies are large, spreading, beta-hemolytic Cutaneous infection remains localized
Catalase positive Inhalation and GI cases often proceed to bacterial
Bacillus anthracis sepsis with high morbidity and mortality
Most virulent and significant human disease Laboratory diagnosis
Epidemiology Colonies are medium to large irregular, gray.
Zoonotic disease in herbivores (e.g., sheep, goats, Medusa head projections, non-hemolytic
cattle) follows ingestion of spores in soil Catalase positive
Human infection typically acquired through con- If suspected, stop working with the isolate and con-
tact with anthrax-infected animals or animal prod- tact State Health Laboratory
ucts (no person-to-person spread)
Less typical is infection through intentional expo-
sure (bioterrorism)
Listeria Species
Clinical presentation: Anthrax General characteristics
Cutaneous: Direct contact with infected material Appear on Gram stain as gram-positive short rods
Inhalation: Aspiration of spore aerosol (wool or coccobacilli
sorters disease) Grow aerobically
Gastrointestinal: Eating of contaminated meat No spores
Anthrax: Cutaneous Not acid fast
Form most commonly encountered in naturally Able to grow at 4 C, unlike many other bacteria
occurring cases Taxonomy and history
Incubation period: 1 to 12 days Six species; type species is Listeria monocytogenes,
Begins as a papule, progresses to a vesicular stage, the most important to human disease
then to a depressed black necrotic ulcer (eschar) Found widespread in nature; habitat is soil and
Edema, redness, or necrosis without ulceration decaying vegetation, but carried by numerous
may occur humans and animals
Case-fatality Epidemiology
Without antibiotic treatment: 20% Easy access to food processing and represents major
With antibiotic treatment: 1% threat to food chain
Anthrax: Inhalation Most virulent of the Listeria and common human
Begins as a viral-like illness, characterized by pathogen is L. monocytogenes
myalgia, fatigue, fever, with or without respiratory Disease: Listeriosis
symptoms, followed by hypoxia Predominantly a food-borne illness (ingestion)
CHAPTER 1 Microbiology 11

Cutaneous: Occupational exposure Lesions painful, with edema, inflammation, and


Meat processors and veterinarians possible local arthritis
High mortality rate Systemic disease possible in immunocompromised
Clinical disease individuals
Affects high-risk populations Laboratory diagnosis
Elderly, immunocompromised hosts Specimen of choice
Pregnant women and their babies Skin biopsy
Complications of pregnancy Gram stain of suspect lesion
Placentitis and/or amnionitis Pleomorphic gram-positive Bacillus
Infection passed to fetus (congenital) Cultivation
Premature birth, abortion, or stillborn birth SBA with 5% to 10% CO2
Neonatal meningitis H2SpositiveonTripleSugarIronAgar(TSI)slant
Meningitis
Sepsis
Corynebacterium Species
Listeria monocytogenes
Laboratory diagnosis A diverse group of gram-positive bacilli commonly
Gram smear of normally sterile body fluids with referred to as coryneforms
characteristic morphology Differentiated by chemotaxonomic means such as
Culture recovery from appropriate specimens analysis of cell wall components
Blood, cerebrospinal fluid (CSF), amniotic Exhibits a clublike morphology that reflects its name
fluid, placenta, genital tract, stool, respiratory (coryne means club in Greek)
secretion General characteristics
Recognition of aerobic gram-positive bacilli Small gram-positive bacilli
b-Hemolytic (clear zone) May resemble Chinese letters
Biochemical profile Characteristic colonial morphology: dry
Motility characteristics: umbrella pattern
Corynebacterium diphtheriae
Growth at 4 C Epidemiology
Erysipelothrix Significant human pathogen is C. diphtheriae,
Nonspore forming gram-positive rod which manifests as a respiratory disease or less com-
Related to Listeria spp. monly as a cutaneous infection
E. rhusiopathiae is the species of clinical interest Acquired by person-to-person contact and spread
Agent of swine erysipelas, widespread disease through close contact with carriers who harbor
in pigs organisms
Cause of erysipeloid, a cutaneous infection in Although a global pathogen, is rare in the United
humans occasionally acquired by contact with States because of childhood vaccination programs
infected animals Clinical disease
General characteristics Normally encountered as a respiratory ailment
Small gram-positive bacilli (0.2-0.5 mm wide  0.8- Common symptoms include sore throat with low-
2.5 mm long) grade fever
Occur singly, in short chains or filaments Adherent membrane of the tonsils, pharynx, or nose
Aerobic or facultative anaerobic O2 requirements is a hallmark of disease
Nonmotile Swelling of neck is usually present in severe disease
Catalase negative Cutaneous diphtheria can occur and manifests as
Wide temperature range growth on complex nutri- infected skin lesions but without a characteristic
ent media such as SBA appearance
Epidemiology and clinical disease Complications
Widespread in nature Myocarditis
Colonizes variety of animals, fish, and birds, but Kidney and liver inflammation
particularly pigs Peripheral neuropathy
Resists cold and alkaline environment Airway obstruction
In animals causes erysipelas, ranging from cutane- Death (occurs in 5% to 10% of respiratory
ous to systemic disease cases)
In humans, erysipeloid is a cutaneous infection con- Pathogenesis and virulence factors
sisting of localized cellulitis after acquisition Invasion
through skin abrasion, injury, or bite Bacteria colonize and proliferate in local tissues
Occupational hazard among animal handlers, of the throat, creating pseudomembrane
farming personnel, veterinarians Toxigenesis
12 CHAPTER 1 Microbiology

Bacteria produce an exotoxin that causes the


FAMILY MYCOBACTERIACEAE
death of eukaryotic cells and tissues by inhibition
of cell protein synthesis General characteristics
Causes heart and nerve damage and is responsi- Slim, rod-shaped organisms that are 1 to
ble for the lethal symptoms of the disease 10 mm long
Vaccination consists of a toxoid, which maintains the Nonmotile and nonspore forming, obligate
antigenicity of the toxin without the toxicity and aerobes
prompts production of toxin-neutralizing antibody Slow growing (3 to 40 days in culture)
Vaccination programs solely are responsible for the Contain mycolic acids, complex, long-chain
control of diphtheria in a population fatty acids
Laboratory diagnosis Possess a cell envelope with a high lipid content
Specimens Although mycobacteria are discussed in the section
Throat and nasopharyngeal on gram-positive bacilli, it is important to note that
Material under pseudomembrane useful with the exception of species classified as rapid
Media growers, mycobacteria do not stain with Gram
General media: 5% SBA reagents
Selective medium: Cystine-tellurite medium All mycobacteria will stain acid fast with Ziehl-
(CTBA), which selects against normal throat flora Neelsen or auromine O stains and are commonly
C. diphtheria colonies are black to gray referred to as an acid-fast bacillus (AFB)
Differential medium: Tinsdale, which allows Acid-fast staining (Ziehl-Neelsen)
recognition of suspected C. diphtheriae Step 1 exposes the smear to carbol fuchsin, a
C. diphtheria colonies are black with red dye
brown halos Step 2 involves decolorization with hydrochloric
Gram stain of sample acid
Gram-positive Chinese letters appearance Step 3 counterstains with methylene blue
Biochemicals An acid-fast organism resists decolorization and
Urease negative remains red, whereas nonacid fast organisms
Nitrate positive stain blue
Catalase positive Cell wall structure
Toxin testing (tests for toxin phage) Mycolic acids are components of a variety of
In vivo: Culture and antitoxin in rabbit lipids found only in mycobacteria, Nocardia, and
In vitro: Immunodiffusion corynebacteria
Corynebacteria Other Than Corynebacterium The chain length of these mycolic acids is longest in
diphtheriae Mycobacterium, intermediate in Nocardia, and
Corynebacteria are widespread as normal flora on the shortest in Corynebacterium spp.
skin and mucosal membranes of humans and animals This explains why mycobacteria are generally acid
Such infections range from local cutaneous to deep- fast, nocardia less acid fast, and corynebacteria are
seated systemic infections nonacid fast
Corynebacterium jeikeium Conversely, Mycobacteria will not stain with the
Important in nosocomial or immunocompromised Gram procedure and Nocardia and Corynebacte-
Line-related infections rium will stain as gram-positive
Antimicrobial resistance Epidemiology and clinical disease
Other Corynebacteria The pulmonary disease tuberculosis, caused by
Because of the common presence of Coryneba- Mycobacterium tuberculosis complex, is an enor-
cterium on human surfaces, significance should mous global problem
be attached to their recovery only in the following Estimated 1.7 billion people, one third of the
cases worlds population, are infected
Isolated from otherwise sterile body sites 8.4 million new cases per year and 2 to 3 million
If clearly predominant in mixed flora culture results deaths
grown from well-collected sample Infection risk proportional to intensity of exposure
If recovered in high count (>104 cfu/mL) from urine Infection does not usually lead to disease
as single organism M. tuberculosis is exclusively a human pathogen
Significance is enhanced in the following cases Mycobacterium bovis is a closely related animal
Multiple specimens positive for same pathogen that causes a disease in people that is
organism indistinguishable from tuberculosis (TB). It is
Coryneform bacteria seen on direct Gram stain acquired from the ingestion of infected meat
with strong leukocytic reaction or milk
CHAPTER 1 Microbiology 13

Mycobacterium Species (Box 1-1) Cell-mediated immunity develops 2 to 6 weeks after


active infection (result of T cell, not B cell,
Mycobacterium tuberculosis proliferation)
Natural history of M. tuberculosis infection At that point, delayed-type hypersensitivity
M. tuberculosis is acquired through the inhalation response is demonstrable through a skin test; the
of droplet nuclei antigen reagent is termed purified protein deriva-
The primary spread of the organism is via aerosol tive, so the test is known as the PPD test
droplets from coughing Patients who cannot raise a sufficient delayed-type
Bacteria not initially killed multiply in the phago- hypersensitivity response will develop miliary
some of a macrophage, destroying the macrophage (disseminated) TB
Released organisms are ingested by other Epidemiology and clinical disease
macrophages Increased risk for clinical disease after primary
Cytokines and chemokines produced attract other infection is found in young children, Native Amer-
phagocytic cells, including monocytes, other alveo- icans, Native Africans, patients infected with the
lar macrophages, and neutrophils human immune deficiency virus (HIV), intravenous
These cells eventually form a nodular structure drug abusers
termed the tubercle or granuloma Infants and the very young have a high mortality
The immunologic response to this infection is cellu- rate from primary infections
lar (rather than humoral), and a delayed type hyper- Highest risk for incidence and reactivation
sensitivity reaction develops against tuberculin Former for current prison inmates
protein that manifests a positive skin test reaction The homeless, the elderly
Dissemination of the organism to the lymph nodes Foreign-born persons from TB-endemic areas
and bloodstream occurs with deposition in liver, Prophylaxis
spleen, kidney, bone, brain, meninges, and other Bacillus Calmette Guerin (BCG) vaccine is 70%
parts of the lung with further granuloma formation. effective in preventing infection in a specific
This is termed disseminated or miliary TB and on population
radiography shows multiple millet seedlike BCG is not used in countries with a low incidence of
lesions TB because the ability to detect infection with M.
Natural history of TB tuberculosis with the PPD is lost
Reactivation of TB results when persisting bacteria After a positive tuberculin test, isoniazid (INH) and
in a host suddenly proliferate, because the granu- other drugs are taken for 6 to 9 months or rifampin
loma formation does not eradicate the organisms for 4 to 9 months to prevent disease
15% to 20% of primary disease reactivates at a Drug-resistant strains
later point, leading to caseous granuloma and cavity Increased resistance to INH and other anti-
formation, during which the disease is highly tuberculosis drugs noted in recent years; in
contagious M. tuberculosis resistance is due to mutation,
Greatest risk for reactivation not plasmid transfer
Within 5 years of primary infection Multiple drugresistant strains pose severe prob-
Those at the extremes of age lems and are seen in populations with HIV
Pregnant women, immunosuppressed patients infection
Malnourished population, alcoholics, and Most nontuberculous Mycobacterium spp. are
patients with diabetes generally more resistant to drugs than M.
Most common presentation of TB is as a chronic pul- tuberculosis
monary disease Laboratory diagnosis
Chronic cough Dual motivation in laboratory detection of M.
Chest pain tuberculosis
Shortness of breath Diagnose and treat patient
Low-grade fever Infection control
Fatigue Stop contagion
Night sweats Protect others
Loss of appetite Nontuberculous Mycobacteria
Weight loss Atypical Mycobacteria and Mycobacteria Other
Sputum production Than Tuberculosis (MOTT)
Immunity to TB Environmentally acquired, not transmitted person to
Infection is contained in 80% to 85% of people person by respiratory means
who recover without ever having symptoms of Generally infects those with weakened immune sys-
disease tems because of age, disease, or other factors
14 CHAPTER 1 Microbiology

B O X 1- 1 Isolation and Identification of Mycobacterium

Mycobacterium characteristics AFB liquid detection systems


Slim rod-shaped organisms that are 1-10 mm long. Specimen Processing
Nonmotile and nonspore forming, obligate aerobes AFB Staining Techniques
Slow growing (3 to 40 days in culture) Carbol fuchsin based
All Mycobacteria will stain Acid-fast with Ziehl-Neelson or Heat fix suspension to slides
auromine/rhodamine stains and are commonly referred to as Acid 15 min @ 80 C or 2 hr @ 65 C
Fast Bacilli or AFB Ziehl-Nielson requires HEAT step during staining (tedious)
With the exception of the rapidly growing AFB, mycobacteria will Kinyoun COLD modification of ZN preferred method
not stain with the Grams procedure Read on oil immersion (1000)
Lab safety is a major concern due to the potential of infectious Auramine Rhodamine fluorescent stain
aerosols Organisms appear yellowish green against a black back-
Containment is crucial ground
Biosafety level 2 hoods Can scan slide at low power (250) and confirm at 400
Gowns, gloves, and masks More sensitive for detection and faster scan of slide than ZN
Sealed centrifuge cups Requires microscope with fluorescent optics
Negative pressure room and BSL 3 containment desirable Incubation
Mycobacterium Sample Preparation Most species require 35 C-37 C, 5%-10% CO2
Common samples: Dark, high humidity, loose caps
NF likely to be present in sputum, bronchial wash or lavage, Optimally at least 2 media
skin lesions, urine, stool Examine weekly for growth
Normally sterile body sites do not contain normal florapleural Hold cultures for 6-8 weeks
fluid, blood, spinal fluid, deep tissue biopsies Lower incubation temperature if infection cutaneous due to
The complex lipid AFB cell wall allows procedures that decontam- possible mycobacteria other than TB (MOTT)
inate normal bacterial flora but allow AFB to remain viable Identification of Mycobacteria
Decontamination Growth Ratetime for visible colonies to appear:
Bacterial flora eliminated or reduced Rapid  7 days
Mycobacteria release from mucus Slow > 7 days
Concentration aids in detection of low numbers AFB Colony morphology
AFB are largely protected from decontamination M. tuberculosis
Use of mildest procedure that controls decontamination rate The color is typically buff, regardless of light
advocated Texture of the colony is rough
Expect contamination rate of 3%-5% M. avium complex
No contamination means procedure is too harsh Color is tan to buff regardless of light
AFB smear and culture follows decontamination Texture of the colony is smooth
Specimen Processing for AFB M. kansasii
Culture MediaSlanted media in tubes preferred for safety reasons. Color is yellow or reddish yellow
Egg based (whole or yolks)Lowenstein-Jensen (LJ) Texture of the colony is smooth
Preferred basic nonselective media AFB Colony morphology
Gruft modificationPenicillin/Nalidixic acidinhibitory to Pigment Production among MOTT
contaminants Scotochromogens: grow with a deep yellow pigment regard-
Malachite greeninhibitory to routine bacteria less of light
Agar based, conventional petri dish M. scofulaceum, M. gordonae
Middlebrook 7H10, 7H11 most popular Photochromogens: develop yellow pigment only after expo-
Salts, vitamins, cofactors, tween/glycerol sure to light
Thin plates for early detection of colonies10-12 days, M. kanasii, M. marinum
microscopically Nonchromogens: no pigment beyond tan to buff colonies
Less commonly used than LJ slants for reasons of containment regardless of light
Liquid media M. avium-intracellulare
7H9 media most common Note: M. tb complex produces buff colonies
Reduces turnaround time to average of 10 days Pigment production among MOTT
Used in BACTEC system (14C labeled palmitic acid-detection of Key Biochemical Tests
free 14CO2), and other continuous monitoring automated Niacin test
systems M. tuberculosis accumulates considerable niacin in media. +
0.5 mL PANTA (polymixin B, amphotericin B, nalidixic acid, Test early sign that isolate may be tuberculosis
trimeth/sulfa, azlocillin)added to processed specimens to Niacin extracted and tested with strip for color
prevent specimens development
CHAPTER 1 Microbiology 15

BOX 1 -1 Isolation and Identification of Mycobacteriumcontd

Semi Quantitative Catalase Additional identification methods for mycobacteria


M. kansasii positive GLC or HPLC
M. scrofulaceum positive Analysis of long-chain fatty acids
M. tb and avium negative Most health departments and CDC prefer method
Hydrogen peroxide added Genetic probes
Positive test bubbles rising above 45 mm from baseline DNA probes specific for hybridization for rRNA sequences
Arylsulfatase use chemiluminometer
Rapid growers are only mycobacterium positive in 3 day test Only single colony of organism needed
Helps to differentiate from similar looking Nocardia species Rapid and specific detection of organism
Nitrate reduction Available for M. tuberculosis, avium, intacellulare, kansasii
M. tb and M. kansasii are positive, M. avium is negative and gordonae
Red color on strip or reagent test is positive

Species Niacin Semi Quant Catalase Nitrate reduct. Aryl sulfatase Tween hydrol
M. tuberculosis + - + - -
M. kansasii - + + - +
M.scrofulaceum - + - - -
M. avium complex - - - - -
M. gordonae - + - V +
M. fortuitum - + + + V

Can cause TB-like, cutaneous, or leprosy-like diseases General Collection Guidelines


Are not susceptible to certain common antituberculo- and Diagnosis
sis antibiotics
M. avium complex (MAC) is the most commonly Sterile body site areas can be set up directly onto cul-
associated HIV-related systemic bacterial infection ture media
CSF, pleural fluid, deep tissue biopsies
and manifests as a pulmonary pathogen, much
Nonsterile sites require special processing: Deconta-
like TB
M. avium infection not associated with acquired mination and digestion
Sputum, bronchial washings, bronchoalveolar
immunodeficiency syndrome is quite rare
Treatment of M. avium involves a long-term regimen lavage, skin biopsy
Likely to be overgrown with routine bacteria
of multiple drug combinations, because this organism
AFB resists decontamination and digestion because
does not always respond to the drug regimens used to
treat M. tuberculosis of cell wall lipid content, whereas routine bacteria
Mycobacterium kansasii: Pulmonary disease in com- are destroyed
AFB: Safety precautions
promised hosts (individuals infected with HIV)
Mycobacterium marinum: Cutaneous disease from Avoid direct contact with organism
Aerosols present greatest hazard
contact with contaminated water
Mycobacterium scrofulaceum: Cervical adenitis in Class 2 biosafety hood
children (contaminated raw milk, soil, daily Maintenance: Airflow checks
products) High-efficiency particulate absorption (HEPA)
Mycobacterium fortuitum, Mycobacterium chelonae, filters: Sterile air current
Sealed centrifuge buckets
Mycobacterium abscessus: Primarily skin and soft tis-
Gown, gloves, masks, foot covers
sue disease in various hosts
Mycobacterium leprae: Agent of leprosy Keep doors closed when specimens are openslight
Other Nontuberculous Mycobacteria negative air pressure created by hood
Mycobacterium gordonae Laboratory diagnosis of TB
Mycobacterium spp. commonly found in tap water AFB visible by Ziehl-Neelsen or fluorochrome
Generally nonpathogenic smears of sputum or appropriate sample
Can confuse the reading of AFB smears if care not Recovery by culture (the gold standard) either on
taken in sample preparation conventional AFB media or that which uses radio-
metric detection (BACTEC)
16 CHAPTER 1 Microbiology

Prolonged incubation time required (up to 8 weeks) Spread person to person through inhalation or con-
Biochemical characterization tact with infected skin
Nucleic acid assays (NAA) Silent phase: Multiplication of bacilli
Represent rapid diagnosis of TB Intermediate phase: Peripheral nerves, sensory
Sensitivity of the NAA is approximately 95% in impairment
patients with a positive AFB smear, but only 50% Organism can be grown in the footpads of mice and
in smear negative cases (U.S. Food and Drug nine-banded armadillos, not on artificial media
Administration data) Diagnosis usually made by clinical findings
Nontuberculous mycobacteria (atypicals, MOTT) and observation of AFB on direct smear of
Classified into Runyon groups based on lesions
Presence or absence of pigmentation
Pigment production: Light dependent or not
FAMILY ENTEROBACTERIACEAE
Growth rate: Slow or fast
Chromatography techniques allow species-level (BOXES 1-2 AND 1-3)
identification of mycobacteria based on their cellu- Largest, most heterogeneous group of clinically impor-
lar fatty acid and/or mycolic acid profiles (reference tant bacteria
laboratories) General characteristics
Specific DNA molecular probes are available for Most are normal flora of the GI tract
species identification Gram-negative bacilli
BCG used outside the United States as an attenuated Facultative anaerobes
vaccine Colony morphology is similar for most
Nontuberculous mycobacteria (MOTT) Large, gray, spreading colonies
All other species not in the M. tuberculosis complex Only Klebsiella and Enterobacter are mucoid
Present everywhere, generally environmental All Enterobacteriaceae
Usually not transmitted person to person Ferment glucose
Pathogenicity varies and often depends on host Reduce nitrates to nitrites (rare exceptions)
immune status (opportunistic infection) Oxidase negative
Runyon: Four groups based on growth rate, Most are motile by peritrichous flagella
pigment production, and reaction of pigment Serologic classification
to light Cell-associated antigens
M. avium complex (MAC) O: Somatic antigens (heat stable)
Complex includes M. avium and Mycoplasma Polysaccharide of the LPS
intracellulare Associated with endotoxin release
Most commonly isolated AFB among the MOTT K: Capsular antigens (heat labile)
Ubiquitous in nature Capsular polysaccharide
Acquired by inhalation or ingestion Strains with K are more pathogenic
Patients with HIV are particularly at risk H: Flagellar antigens (heat labile)
Nonphotochromogens: No pigment Flagellar protein antigens
Smooth, cream-colored colonies Responsible for motility
Greater drug resistance than M. tuberculosis Clinical disease
Rapid growers Based on the clinical infections produced
M. fortuitum-chelonae complex Opportunistic
Grow in 7 days or less on solid media Normal flora
Can grow in routine media and stain as gram- Cause infections outside of natural habitat
positive cells with diphtheroid-like morphology Primary intestinal
Acquired from environmental sources or nosocomi- Salmonella
ally during surgery from contaminated objects or Shigella
fluids Plesiomonas
Enter by inoculation into the skin Yersinia enterocolitica
Can also cause chronic pulmonary infections Opportunistic genera
M. leprae Citrobacter
Cause of leprosy, which is also termed Hansens Edwardsiella
disease Enterobacter
Chronic disease of the skin, mucous membranes, Escherichia
tissue Hafnia
Rare in the United States, but cases exist in Texas Klebsiella
and Louisiana Morganella
CHAPTER 1 Microbiology 17

BOX 1 -2 Tests Used in the Identification of Enteric Gram-Negative Bacilli

Routine Enterobacteriaceae Light orange to red positive


Memebers of the family Enterobacteriaceae are usually divided into Yellow/no changenegative
lactose fermenters (+) and non-lactose fermenters (-) Positive: Proteus spp.
Lactose + Negative: E. coli
E. colidark Indole
Klebsiellamucoid Ability to metabolize tryptophan by testing for indole
Enterobactermucoid Indole + aldehyde yields a red color
Citrobacterlate Indolespot test
Serratialate, red pigment Saturate filter paper with reagent
Lactose Rub portion of colony onto paper
Proteusswarming Rapid development of color is positive test
Morganella Positive: E. coli
Providencia Negative: E. cloacae
Edwardsiella Methyl Red and Voges-Proskauer (MR/VP)
Hafnia Glucose metabolism and metabolic products
Lactose is degraded into glucose and galactose
Important Biochemical Tests Glucose used through EmbdenMeyerhofParnas (EMP pathway)
o-Nitrophenyl-p-D-galactopyranoside (ONPG) to produce pyruvic acid
Some lactose fermenters lack permease and so are slow or Pyruvic acid use produces many mixed acids
late Enterics take two separate pathways
Non-lactose fermenters lack both Mixed acid pathway and butylene glycol pathway
ONPG tests for b-galactosidase Two tests for end-products of these pathways
The substrate is complexed to galactose Methyl Red test and Voges-Proskauer
Cleavage causes a color change Escherichia coli is MR + and VP
Positve: E. coli Enterobacter aerogenes and Klebsiella pneumoniae are MR and
Negative: Salmonella enteritica VP+
Decarboxylase tests Pseudomonas aeruginosa is MR and VP
Detects decarboxylation of specific amino acids Gelatinase
Alkaline end products result Used to determine the production of proteolytic enzyme that
Ornithine decarboxylase (ODC) digests gelatin
Ornithine ! Putrescine Positive: Proteus vulgaris
Lysine decarboxylase (LDC) Negative: Enterobacter aerogenes
Lysine ! Cadaverine Carbohydrate fermentation
Arginine dihydrolase (ADH) Various media used to determine the ability of bacteria to ferment
Arginine ! Citrulline specific carbohydrates
Decarboxylase tests The fermentation pattern can then be used as part of an identifi-
Medium starts purple cation scheme
Fermentation shifts medium acidindictor to yellow Motility Test
Decarboxylation shifts the pH alkalineindicator to purple Single stab of the organism into the gelatin tube
Mineral oil traps alkaline end products Incubate at 37 C for up to 7 days
Control tube lacks amino acidyellow Movement away from initial stab line is positive motility
Simmons Citrate Positive: E. coli
Ability of organism to use sodium citrate for metabolism and Negative: K. pneumoniae
growth Identification of Enterobacteriaceae
Indicatorbromophenol blue Identification is a process or flow
Only streak slant 1. Growth on media
Light inoculum 2. Gram stain
Bluepositive (rise in pH) 3. Lactose fermentation
Greennegative (no change) 4. Oxidase
Positive: Klebsiella pneumoniae and Proteus mirabilis 5. Basic biochemical tests
Negative: Escherichia coli and Shigella dysenteriae Identification of Enterobacteriaceae
Urease Production API 20E
Ability of bacteria to hydrolyze urea to ammonia and CO2 The API-20E test kit is designed for the identification of enteric
Ammonia release causes pH change bacteria
Positivebright pink Plastic strip holding twenty mini-test tubes is inoculated with saline
Ureasemethod suspension of a pure culture
Streak slant Rehydrates the dessicated medium in each tube
Incubate 37 C Incubated 18-24 hours at 37 C
Continued
18 CHAPTER 1 Microbiology

B O X 1- 2 Tests Used in the Identification of Enteric Gram-Negative Bacillicontd

The reactions are converted to a seven-digit code No fermentation


The code is fed into the manufacturers database Alkaline slant/Alkaline butt (K/K) or (K/NC)
Identification usually as genus and species Non-entericsable to degrade peptones
Identification of gastrointestinal pathogens Only glucose fermentation
Screen stool cultures Alkaline slant/Acid butt (K/A)red/yellow
Normal flora in lower bowel 107/mL Too much acid in butt to revert to alkaline
Anaerobes, diptheroids, enterococcus, streptococcus, enterics, yeasts Lactose (sucrose) fermentation
Anaerobes/aerobes at 1000/1 Acid slant/Acid butt (A/A)all yellow
Determine which colonies deserve further attention H2S productionBlack
Initial Screening Steps Gas productionbubbles or split media
Detect suspicious colonies amongst numerous normal flora organisms Lysine Iron Agar (LIA)
Lactose fermenter vs. non-lactose fermenter Lysine, glucose, ferric ammonium citrate, sodium thiosulfate
Growth on selective media Primarily used to detect lysine use
H2S producing colonies Good tool when used with TSI to screen stools for pathogens
Non-sorbitol fermentersE. coli O157 Suspected non-Enterobacteriaceae
Beta hemolytic enteric organisms Vibrio
Oxidase positivesuspected non-Enterobacteriaceae Aeromonas
Suspected Enterobacteriaceae Campylobacter
Conventional identification Tests for other enteric pathogens
Automated identification Growth on specialized medium
Screening tubes Oxidase and catalase
TSI and LIA Gram stain
Triple Sugar Iron (TSI) or KIA Suspected Campylobacter
Screening ID of enteric pathogens Growth on specialized media at 42 C
Identical except TSI sucrose, KIA does not Atmosphere 5% O2, 10% CO2, 85% N2
Detects ability to produce gas and acid from fermentation and H2S Oxidase and catalase positive
Both are used as slants: Curved gram-negative bacilli
Slantaerobic Sea gull shape
Buttanaerobic Stains very lightly
0.1% glucose, 1% sucrose, 1% lactose, phenol red, ferrous sulfate Hippurate hydrolysis positive
Detects fermentation: Suspected Vibrio and Aeromonas
If glucose is the only one fermented, the small amount of acid that Beta hemolytic on Blood agar
is produced on the slant will be oxidized to a neutral product Comma-shaped gram-negative bacilli
Slant remains alkaline (red) Non-lactose fermenter on MacConkey agar
Buttacid is not oxidized and turns yellow Thiosulfate citrate bile sucrose agar (TCBS)
If lactose and sucrose are also fermented, there is so much acid that Yellow green colonies
both the slant and butt become yellow Oxidase and catalase positive
TSI5 Reactions API or commercial systems usually identify
From Tille PM: Bailey & Scotts diagnostic microbiology, ed 13, St Louis, 2014, Mosby.

Proteus Biochemical tests: Many


Providencia Lactose fermentation and utilization of carbohy-
Serratia drates are key biochemical tests
Clinical disease All ferment glucose, so lactose is used for initial
Most cause opportunistic and nosocomial infections differentiation
UTI Rarely, laboratories use traditional tube tests
Pneumonia More often use spot tests or limited workup on
Wound infections some specimen types
Catheter colonization Commercially available in kits
Isolated from almost all body sites API test strips
Laboratory diagnosis Most large laboratories use automated identifi-
Oxidase negative cation systems
Colony morphology and Gram stain Vitek (bioMerieux)
Culture: Use supportive and selective media to MicroScan (Dade)
recover pathogens Phoenix (BD Bioscience)
MacConkey agar Enteric pathogens
XLD Food-borne
Hektoen Salmonella
CHAPTER 1 Microbiology 19

BOX 1 -3 Biochemical Differentiation of Representative Enterobacteriacae

From Tille PM: Bailey & Scotts diagnostic microbiology, ed 13, St Louis, 2014, Mosby.

E. coli Epidemiology
Campylobacter Naturally occur in poultry products and reptiles
Yersinia Food-borne infections account for 1.3 billion cases
Human to human of acute diarrhea with 3 million deaths worldwide
Shigella Ingestion of contaminated food, water, or milk
Salmonella typhi 40,000 cases annually in the United States
Helicobacter pylori Centers for Disease Control and Prevention (CDC)
Water-borne and reports: In recent years a notable increase in
Vibrio cases related to a multidrug-resistant Salmonella
Aeromonas typhimurium
Plesiomonas Case-fatality and hospitalization rates for this
Enterobacteriaceae pathogens strain are twice that of other Salmonella spp.
Salmonella Clinical disease: Gastroenteritis
Shigella Most common type of illness
E. coli Diarrhea, low fever, nausea
Yersinia Symptoms last 1 to 3 days
Plesiomonas Positive stool cultures
Serotyping No systemic involvement
Antigens: Heat stable Clinical disease: Bacteremia/septicemia
A, B, C, etc. Nontyphoidal bacteremia
98% of human isolates are A through G Salmonella choleraesuis
H antigens: Heat sensitive High spiking fever
1, 2, 3, etc. Positive blood cultures
Vi (K) antigens: Virulence antigens Few GI symptoms
Heat sensitive Particularly invasive
May mask O antigens Clinical disease: Enteric fever
Boil to remove Vi, retype S. typhi: Typhoid fever
20 CHAPTER 1 Microbiology

Most serious Dysentery characterized by a small volume of


Fever and GI involvement bloody, mucoid stools, and abdominal pain
Positive blood cultures during first week Pathogenesis
Positive stool cultures during second week Resist gastric acidity
Carrier state Requires very few organisms to infect
Carry bacteria asymptomatically after infection 10 to 200 organisms
Can shed organism unknowingly Organism resistant to acid
Typhoid Mary Cytotoxin (Shiga toxin) causes inflammation and
Pathogenesis ulcerative lesions
Ingested in food Destroys epithelial cell
Survive passage through the gastric acid Bloody, mucus-laden stools
Invade the mucosa of the small and large intestine Laboratory diagnosis
Produce toxins Lactose negative
Invasion of epithelial cells stimulates release of TSI: Alkaline/acid, no gas, no H2S
cytokines, which induce an inflammatory reaction Urease negative
Inflammatory response causes diarrhea Confirm with serotyping
May lead to ulceration and destruction of the Report to health department
mucosa
May disseminate from intestines to systemic disease
Laboratory diagnosis Escherichia coli
Lactose negative May cause several types of diarrheal illnesses
H2S positive: Black colonies Enterohemorrhagic E. coli is most important
Triple Sugar Iron Agar (TSI): Alkaline/acid, H2S, gas E. coli O157:H7
Butt is acid as a result of glucose fermentation Hemorrhagic colitis: Pediatric
Methyl red, citrate, lysine decarboxylase, ornithine Can lead to hemolytic uremic syndrome: Build-up
decarboxylase, arginine dihydrolase positive of toxin in kidneys
Must confirm with serotyping Bloody diarrhea with no PMNs
Reportable organism Fever absent
Water-borne or food-borne: Often transmitted via
Shigella Species ground beef
Serotyping (O antigen) Laboratory diagnosis
A: Shigella dysenteriae (12 serotypes) All E. coli
B: Shigella flexneri (6 serotypes) Ferment glucose, lactose, and xylose
C: Shigella boydii (23 serotypes) Indole and Methyl Red positive
D: Shigella sonnei (1 serotype) No H2S or urease
Groups A to C are physiologically similar; S. sonnei Citrate negative
can be differentiated biochemically Motile or nonmotile strains
Epidemiology E. coli O157:H7
Human to human (fecal-oral route) MacConkey with sorbitol (SMAC)
Very communicable Does not ferment sorbitol (i.e., clear colonies)
Foodinoculated by humans Typical E. coli reactions
S. sonnei: Most common in the United States Indole
S. dysenteriae: Least recovered in the United Confirm identification with routine system
States Confirm with serotyping: O or H
Most severe Reportable organism
Third World countries
Clinical disease
Acute infection with onset of symptoms within 24
Yersinia Species
to 48 hours of ingestion Clinically significant species
Average duration of symptoms in untreated adults Yersinia pestis: Plague
is 7 days Yersinia enterocolitica
Organism may be cultivated from stools for Epidemiology: Y. enterocolitica
30 days or longer Food-borne and water-borne illness
Two basic clinical presentations Blood transfusions
Watery diarrhea associated with vomiting and Y. enterocolitica
mild-to-moderate dehydration Clinical disease
CHAPTER 1 Microbiology 21

Gastroenteritis: May resemble appendicitis Variable from 35 to 42 C


Seasonal and ethnic, chitterlings (small intestines Generally aerobic
of pig or other animals) Campylobacter jejuni
Laboratory diagnosis Epidemiology
Grows better at 25 to 30 C C. jejuni most common
Small, lactose-negative colonies on MacConkey Food-borne gastroenteritis
agar Poultry and raw milk
Cefsulodin-irgasan-novobiocin (CIN) agar may be Water
used: produce pink colonies with red center One of most common causes of human bacterial
TSI: Acid/acid, no gas gastroenteritis in numerous parts of the United
Routine identification system States
Clinical disease
Diarrhea, cramps, abdominal pain, fever within 2
Plesiomonas shigelloides to 5 days of exposure
Epidemiology Bloody stool with high WBC count
Water-borne gastroenteritis: Freshwater Lasts for 7 to 10 days
Mostly in tropics Can be fatal
Clinical disease Pathogenesis and virulence
Usually mild watery diarrhea Susceptibility of host and strain virulence key
Human trial unsuccessful in setting up disease Ingestion of contaminated food or water
Laboratory diagnosis Penetrate the GI tract mucous lining
Routine culture conditions Motility and shape
Gram-negative bacilli Adhere to the gut enterocytes and release toxins
Growth on blood agar: No hemolysis Enterotoxin and cytotoxins
Nonlactose fermenter on MacConkey agar Laboratory diagnosis
Positive oxidase and indole most important initial Special atmosphere and temperature
screening tests 42 C
Routine identification system 5% O2, 10% CO2, 85% N2
Campy blood agar
Brucella agar base with antibiotics
MISCELLANEOUS ENTERIC PATHOGENS Other selective media: Cefoperazone Vancomy-
cin Amphotericin (CVA), Skirrow Medium
Campylobacter Curved gram-negative bacilli
Vibrio
Sea gull shape
Aeromonas
Stains very lightly
Helicobacter pylori Nonhemolytic, flat, gray, mucoid
Oxidase and catalase positive
Family Campylobacteraceae Darting motility on wet preparation
Hippurate hydrolysis positive
Campylobacter
Usually identified as Campylobacter spp.
At least 18 species and subspecies in Campylobacter
Arcobacter
Four species in Arcobacter
Campylobacter Species
FAMILY VIBRIONACEAE
Curved or S-shaped 10 genera, including Vibrio
0.5 to 5.0 mm long  0.5 to 1.0 mm wide
Gram-negative, nonspore forming rods
Motile Vibrio
Single, polar flagellum 76 species currently recognized
Generally microaerophilic Photobacterium damselae previously classified as
Arcobacter Species Vibrio and shares ecology
Curved or S shaped Oxidase-positive, facultative anaerobic, nonspore
0.2 to 0.9 mm wide, 1 to 3 mm long forming, gram-negative bacilli
Gram-negative, nonspore forming bacilli Comma-shaped cells
Motile Typically found in saltwater
Single, polar flagellum All members of genus are motile by single, polar
Grow at 15 to 30 C flagellum
22 CHAPTER 1 Microbiology

Vibrio cholerae Pathogenesis and virulence factors


Epidemiology Produces aerolysin
Water-borne illness Cytotoxic enterotoxin
Found in plankton of fresh, brackish, and salt Causes tissue damage in fish and amphibians
water Unclear pathogenesis in humans
Attached primarily to copepods in the zooplankton Laboratory diagnosis
Coastal outbreaks usually follow zooplankton Routine culture conditions
blooms Grows on MacConkey and blood agar
Pathogenesis and virulence Usually b-hemolytic
Colonizes the GI tract Gram-negative bacilli (straight)
Attaches to villi by pili Lactose fermenter
Secretes a two-part toxin, cholera toxin Positive oxidase and indole good screening tests
Toxin causes increased cyclic adenosine monophos- API or routine identification system
phate (cAMP) synthesis
Massive fluid efflux: Diarrhea
Clinical disease FAMILY HELICOBACTERACEAE
Cholera
Helicobacter, Sulfuricurvum, Sulfurimonas, Sulfuro-
Massive fluid loss: Death within 24 hours
vum, Thiovulum, Wolinella
Rice water stools: WBCs and blood absent
Treatment
Replace fluids, antibiotics Helicobacter
Laboratory diagnosis
29 species of Helicobacter recognized
Comma-shaped gram-negative bacilli
Exact mode of transmission is unknown
Nonlactose fermenter on MacConkey agar
Higher rate in undeveloped countries
Thiosulfate citrate bile sucrose agar (TCBS)
Clinical disease
Yellow-green colonies
One of the most transmitted human infections
Oxidase and catalase positive
String test positive Gastroenteritis
Peptic ulcers
Halophilic Vibrio Organisms Associated with gastric cancer
Require 1% to 2% NaCl for growth Pathogenesis and virulence factors
Cause gastroenteritis, wound infections, septicemia Burrows into gastric mucosa
Vibrio parahaemolyticus: Gastroenteritis from seafood Urease
Vibrio vulnificus: Septicemia from raw shellfish (lac- Converts urea to ammonia and bicarbonate
tose fermenter) Ammonia neutralizes stomach acid
Vibrio alginolyticus: Wound and ear infections Ammonia is toxic to the epithelial cells
Protease, catalase, and phospholipases
Damage to epithelial cells
FAMILY AEROMONADACEAE Elicits powerful immune response (ulcer)
Tissue biopsy is an important specimen in
Diseases mainly in fish and amphibians
Frogs some settings
Laboratory diagnosis
Red leg Curved gram-negative bacilli
Fatal internal hemorrhaging Growth on blood agar in 3 to 5 days
Fish
35 C with high humidity
Develop ulcers, tail rot, fin rot, and hemorrhagic Microaerophilic: 5% O2, 10% CO2, 85% N2
septicemia
Positive: Oxidase, catalase, urease
Not usually grown
Aeromonas hydrophila Urease test on biopsy
Direct stain
Epidemiology and clinical disease
Stool antigen test
Organism ubiquitous in fresh and brackish water
Water-borne illnesses
Gastroenteritis
Wound infections
FAMILY NEISSERIACEAE
Exposure to water, fish hook injuries More than 20 genera
Bacteremia General characteristics
Most often in young or old Aerobic gram-negative diplococci
CHAPTER 1 Microbiology 23

Oxidase and catalase positive Respiratory transmission


Neisseria elongate: Catalase negative, rod shaped Mainly affects adolescents in overcrowded
Human reservoir environments
Respiratory and urogenital tract College dormitories and the military
Sexual transmission (Neisseria gonorrhoeae) Vaccine an important control measure
N. gonorrhoeae and Neisseria meningitidis are the Clinical disease
primary pathogens First or second leading cause of community-
acquired meningitis in the United States
Also causes sepsis, conjunctivitis, disseminated
Pathogenic Neisseria Species
organ infections, pneumonia without meningitis
Neisseria gonorrhoeae Many strains of N. meningitidis; clinically the most
Epidemiology important are A, B, C, Y, and W135
Sexually transmitted by carrier Laboratory diagnosis: Specimen collection and
Causes gonorrhea processing
Clinical disease CSF
Males: Acute urethritis with dysuria and urethral An amount greater than 1 mL of CSF is hand car-
discharge ried to the laboratory
Not commonly asymptomatic Specimen should not be refrigerated
Females: Colonizes endocervix causing CSF specimen may be centrifuged
Vaginal discharge Gram stain is prepared from the sediment
Dysuria Sediment is inoculated to chocolate and blood agar
Abdominal pain Blood
Untreated may lead to pelvic inflammatory Conventional blood culture systems
disease (PID) Skin scraping
Other sites of infection include Petechiae may yield viable or stainable organisms
Eyes Cut open lesion and collect fluid on swab
Throat Laboratory diagnosis: Identification
Rectum Gram-negative diplococci found intracellularly and
Can disseminate if untreated extracellularly
Less than 1% of infections Grow well on sheep blood and chocolate agar
Purulent arthritis and septicemia Grow on Thayer Martin selective agar
Laboratory diagnosis: Culture Catalase and oxidase positive
Rapid transport critical for recovery Cysteine trypticase agar (CTA) sugar oxidation:
Direct plating to selective media at the bedside glucose and maltose positive
Use of transport systems for Neisseria
gonorrhoeae
Selective media to inhibit other bacteria and yeast Other (Nonpathogenic) Neisseria Species
Modified Thayer-Martin (MTM) or other selec-
Neisseria lactamica
tive medium
Neisseria flavescens
Chocolate agar: Allows growth of other Neisseria sicca
saprophytes Clinical significance
Incubation:
Normal flora of human upper respiratory tract
3% to 7% CO2 incubator at 35 to 37 C Cause occasional infections
Humidity is important Occasionally isolated from blood, genital tract,
Laboratory diagnosis: Identification
and CSF
Gram-negative diplococci (some may appear as
Identification not appropriate unless isolated from
tetrads)
systemic site or pure culture
Small gray, translucent, raised colonies
Grows on chocolate, but usually not on blood agar
Catalase and oxidase positive
FAMILY ALCALIGENACEAE
Cysteine trypticase agar (CTA) sugar oxida-
tion: glucose positive General characteristics
Neisseria meningitidis Small (0.2-0.7 mm) coccobacilli
Epidemiology Fastidious, obligate aerobes that require
Human reservoir nicotinic acid
Upper respiratory tract of 3% to 30% of asymp- Bordetella pertussis and Bordetella parapertussis
tomatic individuals are nonmotile, Bordetella bronchiseptica is motile
24 CHAPTER 1 Microbiology

Human respiratory tract is the only source of B. per- Clinical disease


tussis and B. parapertussis Most human infections are wound infections/cellu-
Others infect birds and other mammals litis after cat bites
Pain, swelling, and serosanguinous drainage at
the wound site
Bordetella pertussis Septic arthritis and osteomyelitis may occur after
Epidemiology deep puncture wounds
Worldwide 60 million cases with 500,000 deaths Serious infections may occur in compromised
Endemic in most populations hosts
Respiratory tract infections
Cycle often3 to 4 years
Majority of cases in the United States occur in Bacteremia, endocarditis
Central nervous system (CNS) infection
August to November
No evidence of long-term carriage Eye infections after cat and dog scratches
Laboratory diagnosis
Immunity from vaccine or infection is not
Clinical history important
lifelong
Specimens
Protection wanes after 3 to 5 years
Immunity is undetectable by 12 years Pus, wound swab, tissue, sputum, blood
Subclinical infections in adults may be common Growth requirements
Adults usually the index cases in infants Growth on blood and chocolate agar
Vaccine for adolescents recently approved No growth on MacConkey agar
Clinical disease Incubate for 24 hours in CO2, at 35 C
Typical upper respiratory tract infection for 1 week Small, short, gram-negative bacilli
Paroxysmal cough (out, out, out, whoop) Colonies
Not always present Gray-green, convex, nonhemolytic, odor
Long recovery Biochemicals
Laboratory diagnosis Oxidase and catalase positive
Direct fluorescent antibody (DFA) on nasal Indole positive
specimen Urease negative
Culture of nasal specimen Commercial systems usually appropriate for
Polymerase chain reaction P. multocida
Serologic tests
Not generally available
Not helpful during acute phase Other Pasteurella Species
Difficult to interpret Pasteurella canis
Pasteurella dagmatis
Found in mouths of canines
FAMILY PASTEURELLACEAE Pasteurella caballi
Infections associated with dog bites
Pasteurella multocida Upper respiratory tract of horses
Horse bite wounds
Reported in 1878 in fowl cholerainfected birds
1880: Louis Pasteur
Most common species of Pasteurella isolated from
humans
Haemophilus Species
Grows on blood and chocolate Genus includes
No growth on MacConkey agar Haemophilus influenzae
Oxidase and catalase positive Haemophilus aegyptius
Indole positive Haemophilus haemolyticus
Epidemiology Haemophilus parainfluenzae
Oral cavity of cats and dogs Haemophilus ducreyi
Causative agents of several economically significant New genus Aggregatibacter
veterinary diseases Haemophilus segnis, Haemophilus aphrophilus,
Cattle, buffaloes, sheep, goats, poultry, turkeys, and Actinobacillus actinomycetemcomitans
rabbits, horses, and camels Most members are nonpathogenic or opportunistic
Serious infectious diseases such as fowl cholera, pathogens
bovine hemorrhagic septicemia, and porcine Three major pathogenic species
atrophic rhinitis H. influenzae
CHAPTER 1 Microbiology 25

H. aegyptius Haemophilus ducreyi


H. ducreyi Not normal flora: Sexually transmitted disease
Haemophilus: Derived from Greek for blood (STD)
lover Genital tract pathogen
Require growth factors present in blood Genital chancres, ulcers
X Factor: Hemin, hematin Epidemiology
V Factor: Nicotinamide adenine dinucleotide Chancroid is rare in the United States
(NADH) NADH Localized endemic outbreaks occur in isolated
XV Factor strip test STD and prostitution populations
Both are found in chocolate agar Annual global incidence approximately 6 million
Gram-negative pleomorphic coccobacilli or bacilli per year
Nonmotile Chancroid more common in areas of low soc-
Aerobic or facultative anaerobic ioeconomic status such as Africa, Asia, and the
Oxidase and catalase positive Caribbean
Obligate parasite of mucous membranes of humans More common in areas where the prevalence of
and mammals HIV is high
Haemophilus influenzae Laboratory diagnosis
Often found as part of normal upper respiratory tract 3% to 5% CO2
flora in humans High humidity
Spread by droplets and close contact Must be plated immediately
Clinical disease Blood agar with X factor
Especially in children Does not need V factor
Meningitis Chocolate agar
Septicemia Fastidious, will not satellite on blood agar
Epiglottitis 2 to 10 days needed for growth
Pneumonia Gram-negative coccobacillus: School of fish
Otitis pattern
Vaccine: Single biggest impact on pediatrics in last Gray, yellow, or tan colonies
20 years Nonmucoid
Pathogenesis and virulence Catalase negative, oxidase positive
Polysaccharide capsule Nucleic amplification is definitive test
Seven serogroups: a to f and e0
Capsule type b (Hib) is the most clinically signif- Miscellaneous Haemophilus Species
icant and virulent Examples: H. parainfluenzae, H. haemolyticus
Immunoglobulin A (IgA) proteases Normal flora in humans that occasionally cause upper
Outer membrane proteins and lower respiratory tract infections
Adherence factors May lead to systemic infections resulting from inva-
Laboratory diagnosis
sion of blood and tissue
Grows on chocolate agar
Requires factors X and V
Requires 3% to 5% CO2
Satellites around S. aureus on SBA
Related Organisms
Small translucent colonies Aggregatibacter aphrophilus
Gram-negative coccobacilli Includes both species formally known as factor V
Haemophilus aegyptius independent (H. aphrophilus) and factor Vdependent
Conjunctivitis: Pink eye (H. paraphrophilus) strains
Brazilian purpuric fever Aggregatibacter segnis
Recurrent conjunctivitis Formerly H. segnis
High fever Eikenella corrodens
Vomiting General characteristics
Septicemia All oxidase-positive, fastidious gram-negative
Shock bacilli
Mortality as high as 70% Part of mouth flora in 40% to 70% of humans
Laboratory diagnosis Clinical disease
Requires factors X and V Frequently in infections from human bites
Grows on chocolate in 3% to 5% CO2 May be mixed infections
Biochemical differentiation required Laboratory diagnosis
26 CHAPTER 1 Microbiology

Gram-negative coccobacilli Can cause epidemics and isolated cases


Requires increased CO2 for growth (3%-10%) Occurs sporadically (community-acquired) or
Oxidase positive as an epidemic
Catalase, urease, indole negative Predisposing factorsboth forms
Usually pit the agar during growth Immunocompromised
Increased age
Kingella Species
Heavy smoking
General characteristics
Exposure to high concentration of organisms
Four species in genus
Epidemiology Showers
Air-conditioner cooling towers
Flora of the pharynx in young children
Pathogenesis and virulence factors
Transmitted from child to child
Intracellular pathogen
Clinical disease
Survive and multiply within macrophages
Osteomyelitis
Laboratory diagnosis
Bacteremia
Specimens
Endocarditis
Joint infections Respiratory specimens are preferred
Laboratory diagnosis Urine for antigen testing
Growth requirements
Fastidious short gram-negative coccobacillus
Joint fluid into blood culture bottles Cysteine and iron
Identification with Remel Rapid NH Buffered charcoal yeast extract (BCYE) media
35 C, CO2
Slow growerhold for 2 weeks
FAMILY LEGIONELLACEAE Identification confirmation
Serology testing
Legionella pneumophila DNA probes
L. pneumophila is the primary pathogen Reference laboratory
14 serotypes
01 most common
General characteristics FAMILY BRUCELLACEAE
Nonacid-fast, nonsporulating, and noncapsulated
bacilli
Brucella
Aerobic fastidious gram-negative bacilli; difficult to Zoonotic disease with worldwide distribution
stain Acquired from animals or animal sources; most
Nonfermentative likely to contract are farmers, butchers, veterinar-
Oxidase and catalase positive ians, laboratory workers
Produces b-lactamase Epidemiology
Epidemiology Ingestion of contaminated dairy
Ubiquitous, natural sources Clinical disease
Lakes, ponds, rivers Undulant fever, Mediterranean fever, Malta fever
Human-made sources Lymph nodes, blood, and reticuloendothelial sys-
Cooling towers, air-conditioning units, hot tubs/ tem affected
spas, plumbing fixtures Fever, chills, headache, hepatosplenomegaly
Illness acquired from breathing in organism; no Laboratory diagnosis
human-to-human transfer Safety: BSL III cabinet
Clinical disease Specimens
Described in 1976 Blood, bone marrow, tissue
Epidemic of American Legion members Transport blood and bone marrow in
Two diseases Isolator tube
Legionnaires disease Inoculate solid media and blood culture bottles
Pneumonia: Fever, chills, cough, myalgia, Tape plates
headache, chest pain, sputum Will grow on blood and chocolate agar
Can have a high mortality rate (30%) if not MacConkey negative
treated Requires extended incubation for 7 to 10 days
Symptoms depend on personasymptomatic Automated blood culture systems will detect in
to life-threatening 2 weeks
Pontiac fever Small gram-negative coccobacilli
Milder form of disease, more like influenza, Colonies
no pneumonia, 0% mortality Small, translucent, moist, nonhemolytic
CHAPTER 1 Microbiology 27

All are oxidase and catalase positive Pseudomonas fluorescens


Urea and H2S results vary by species Pseudomonas stutzeri
Do not put suspected Brucella in an automated sys- Pseudomonas alcaligenes
tem for identification General characteristics
Additional tests Oxidase positive
Serology Aerobic gram-negative bacilli
Polymerase chain reaction (PCR) Water and soilubiquitous in environment
Uses numerous substrates as energy
Identification and differentiating beyond P. aeru-
FAMILY FRANCISELLACEAE
ginosa and common species can be time con-
suming
Francisella tularensis
Epidemiology Pseudomonas aeruginosa
Transmission: Handling animals or carcasses
Clinical disease
Contaminated food or water
Wide range of diseases
Highly contagious and invasive
Septicemia
Clinical disease
UTI
Tularemia
Pneumonia
Multiple forms of the disease
Chronic lung infections
Laboratory diagnosis
Specimens Cystic fibrosis
Skin and soft tissue infections
Safety: BSL III cabinet
Chronic lung infections: Cystic fibrosis
Ulcer swabs
Pulmonary infections a major cause of death
Lymph node biopsy
Repeat episodes of airway disease
Sputum
Chronic colonization/infections
Bone marrow
Exacerbations
Rarely isolated from blood
Chronic therapy
Growth requirements
Skin and soft tissue infections
Cysteine and iron
Intact skin is not a good medium for Pseudomonas
Grows on chocolate agar
Water is usually associated with infections
Increased CO2, 35 C
Immunocompromised patients are at risk for
Slow growth: 2 to 5 days
Faint staining, gram-negative coccobacilli infections
Pseudomonas folliculitis
Small, greenish, droplike colonies
Laboratory diagnosis
Biochemically inert
Presumptive identification
Catalase positive
Serologic tests Large colonies
Molecular tests Grapelike odor
Oxidase positive
Pyocyanin
FAMILY PSEUDOMONADACEAE For further confirmation
More than 150 species listed Growth at 42 C
Based on genetic analysis (16S rRNA sequence) Chry- Glucose oxidation
Pyoverdin (fluorescein)
seomonas and Flavimonas are Pseudomonas spp.
Flavimonas oryzihabitans
Chryseomonas luteola, Chryseomonas polytricha Other Pseudomonas Species
Genus previously included most glucose nonferment- P. fluorescens
ing gram-negative bacilli P. putida
Pseudomonas P. stutzeri
Pseudo: Greek for false Pseudomonas oryzihabitans
Monas: Greek for a single unit Water and soil: Ubiquitous in environment
Used early in the history of microbiology to refer to all Uses numerous substrates as energy
germs Identification and differentiating beyond P. aerugi-
Aeruginosa: Latin for oxidized copper nosa difficult
Compares to P. aeruginosa pigments in the laboratory Molecular methods replacing traditional bio-
Most common isolates in the clinical laboratory chemical testing
P. aeruginosa Inherently more resistant to antimicrobial agents
Pseudomonas putida than common gram-negative bacilli
28 CHAPTER 1 Microbiology

General characteristics
FAMILY MORAXELLACEAE Aerobic nonfermentative gram-negative bacilli
Most strains grow well on MacConkey agar
Moraxella Species Oxidase negative
19 species in the genus Nonmotile
Only a few important in human medicine Epidemiology
M. catarrhalis Widely distributed in nature
M. bovis Survive on various surfaces (both moist and dry) in
M. osloensis the hospital environment
Nonmotile, gram-negative coccobacilli Strains have been isolated from food
Moraxella catarrhalis Isolated from healthy human skin
Commensals of the upper respiratory tract Clinical disease
Isolated only from humans Considered nonpathogenic to healthy individuals
Not associated with disease in healthy people Most infections in immunocompromised
Clinical disease individuals
Causes mostly opportunistic infections Frequently isolated in nosocomial infections
Can be isolated from patients with Especially intensive care units
Ear infections A. baumannii is a cause of nosocomial pneumonia
Bronchitis Late-onset ventilator-associated pneumonia
Sinusitis Other infections include
Pneumonia Skin and wound infections
Predisposing pulmonary conditions (e.g., Bacteremia
chronic obstructive pulmonary disease) Meningitis
Laboratory diagnosis Laboratory diagnosis
Grow on simple nutrient agar Coccobacilli
Blood agar at 35 C Oxidase negative
Chocolate agar Unable to reduce nitrate
No growth on Thayer-Martin Difficult to identify some species
Differentiates it from N. gonorrhoeae because Commercial systems appropriate for A. baumannii,
of colistin but questionable for other species
Opaque, gray, smooth and dry colonies Most strains grow well on MacConkey agar
Gram-negative coccobacilli Except some A. lwoffii
Oxidase positive Nonlactose fermenting
CTA sugar oxidation May appear partially lactose fermenting on
Glucose: Negative MacConkey agar
Maltose: Negative
Lactose: Negative
Sucrose: Negative
Other Moraxella Species
FAMILY BURKHOLDERIACEAE
Normal flora and uncommon pathogens of human and
other mammals upper respiratory tract
Burkholderia Species
Speciation is beyond the scope of a routine Burkholderia cepacia
laboratory Epidemiology and clinical disease
Most susceptible to penicillins, cephalosporins, tetra- Opportunistic and nosocomial pathogen
cyclines, and aminoglycosides Major pathogen of patients with cystic fibrosis
Psychrobacter phenylpyruvicus formerly Moraxella Resistant to decontaminating agents
phenylpyruvicus Has been isolated from alcohol and iodine
bottles
Laboratory diagnosis
Aerobic gram-negative bacilli
Acinetobacter Species Growth on blood, chocolate, and MacConkey agar
More than 10 species and multiple genomavars Selective media usually used in patients with cys-
May be as many as 30 species tic fibrosis
Some are important in human medicine Oxidase weakly positive
Acinetobacter baumannii Routine laboratory test may be negative
Acinetobacter lwoffii Commercial systems may identify complex
Acinetobacter haemolyticus Highly resistant antibiotic susceptibility pattern
CHAPTER 1 Microbiology 29

Burkholderia pseudomallei Environmental organisms


Melioidosis Water
Aggressive granulomatous pulmonary disease Soil
Seen in Vietnam veterans Opportunistic and nosocomial infections
Rarely seen in the United States Cystic fibrosis
Automated systems may or may not identify correctly Burns
Clinical history and communication with physician Immunocompromised
critical Pathogenesis and virulence factors
Burkholderia mallei Opportunistic
Glanders Multiple extracellular enzymes used for survival in
Primarily in horses, mules, and donkeys the environment
Usually by ingestion of contaminated food or water Often resistant to multiple antimicrobial agents
Nodular lesions in the lungs and ulceration of the Laboratory diagnosis
mucous membranes Aerobic gram-negative bacilli
Endemic in Africa, Asia, the Middle East, Central and Grows on blood, chocolate, and MacConkey agar
South America Nonfermenters on MacConkey (lactose)
Not to be identified in a routine clinical laboratory Oxidase positive
Motile

Stenotrophomonas maltophilia
Aerobic
Alcaligenes Species
Nonfermentative Alcaligenes faecalis
Gram-negative bacillus Environmental organisms
They are motile by polar flagella Water
Clinical disease Soil
Opportunistic pathogen Opportunistic and nosocomial infections
Serious infections in immunocompromised host Cystic fibrosis
Nosocomial infections Burns
Respiratory isolates are common Immunocompromised
Other sites Pathogenesis and virulence factors
Wounds Opportunistic
Urine Multiple extracellular enzymes used for survival in
Blood the environment
The major risk factor for infection in hospitalized Often resistant to multiple antimicrobial agents
patients is the implantation of medical devices Laboratory diagnosis
Central venous catheters Grow on blood, chocolate, and MacConkey agar
Urinary tract catheters Gram-negative bacilli
Prosthetic heart valves Nonfermenters on MacConkey (lactose)
Intraocular and contact lenses Oxidase positive
Laboratory diagnosis Motile
Growth on blood agar and chocolate agar: Green-
lavender or yellow pigment
Nonlactose fermenter on MacConkey agar FAMILY CHLAMYDIACEAE
Oxidase negative
Catalase and esculin positive Chlamydia and Chlamydophila
Strongly oxidizes maltose
Three species in Chlamydia
Weak oxidation of glucose
Six species in Chlamydophila
Chlamydophila was recognized in 1999
Chlamydia
FAMILY ALCALIGENACEAE
Chlamydia muridarum
Chlamydia suis
Achromobacter Species
Chlamydia trachomatis
Six species Chlamydophila
Achromobacter xylosoxidans most common in clinical Chlamydophila pneumoniae
microbiology Chlamydophila pecorum
Achromobacter denitrificans Chlamydophila psittaci
30 CHAPTER 1 Microbiology

Chlamydophila abortus Humans are the only natural host


Chlamydophila felis Incidence is 300 to 500 cases per year in the
Chlamydophila caviae United States
Obligate intracellular pathogens Male homosexuals are major reservoir of the
Three disease-causing species in humans disease
Chlamydia trachomatis Clinical disease
STDs, eye infections Urethritis, cervicitis, epididymitis
Chlamydophila pneumoniae Pelvic inflammatory disease
Respiratory disease in humans, horses, koalas, STD: Lymphogranuloma venereum
and other animals Trachoma
Chlamydophila psittaci Not an STD, chronic conjunctivitis, blindness
Respiratory disease: Humans, birds Inclusion conjunctivitis
Life cycle Spread to infant from mother
Elementary bodies are the small (0.3-0.4 mm) infec- No blindness
tious form of the chlamydia Pathogenesis
They possess a rigid outer membrane that is Infects nonciliated columnar epithelial cells
extensively cross-linked by disulfide bonds Infiltration of PMNs
Because of their rigid outer membrane the ele- Lymphoid follicle formation and fibrosis
mentary bodies are resistant to harsh environ- Clinical diseases from destruction of the cells and
mental conditions encountered when the the host inflammatory response
Chlamydia organisms are outside of their Does not stimulate long-lasting immunity
eukaryotic host cells
The elementary bodies bind to receptors on host Chlamydophila pneumoniae
cells and initiate infection Formerly classified as Chlamydia
Most Chlamydia organisms infect columnar Humans only source of disease
epithelial cells, but some can also infect Atypical pneumonia
macrophages Epidemiology
Reticulate bodies are the noninfectious intracellular Affects all age groups
form of the Chlamydia organism Most common among older age groups
They are the metabolically active replicating Reinfection is common after a short period of
form of the Chlamydia organism immunity
They possess a fragile membrane lacking the Causes 10% of community-acquired pneumonias
extensive disulfide bonds characteristic of the treated without hospitalization
elementary bodies Clinical disease
Chlamydia trachomatis Commonly causes mild-to-moderate respiratory
General characteristics illness
Only found in humans Pneumonia, bronchitis, sinusitis, flulike illness
Most common bacterial STD pathogen Pneumonia symptoms indistinguishable from other
Infections may be symptomatic or asymptomatic causes of pneumonia
Ocular infections: Biovar trachoma Cough, fever, and difficulties breathing
500 million people are infected worldwide Incidence of asymptomatic appears high
7 to 9 million people are blinded May be severe in immunocompromised
Endemic in Africa, the Middle East, India, and Laboratory diagnosis
Southeast Asia Tissue culture: historical gold standard
Infections occur mostly in children Cultured on McCoy cells
Transmitted by droplets, hands, contaminated Other cell lines will support the growth of
clothing, flies, and by passage through an C. trachomatis
infected birth canal Stain 2 to 6 days after inoculation (iodine or fluo-
Genital tract infections: Biovar trachoma rescent antibody)
May be the most common bacterial STD in the DFA
United States Detects outer membrane of elemental bodies
50 million new cases occur yearly worldwide Can be as sensitive as culture
In the United States, the highest infection rates Molecular techniques
occur in Native and African Americans Nucleic acid amplification
Genital tract infections PCR, ligase chain reaction, strand
STD that occurs sporadically in the United States displacement
Prevalent in Africa, Asia, and South America DNA probes
CHAPTER 1 Microbiology 31

Chlamydophila psittaci Clinical disease: M. hominis


Epidemiology Colonize male and female genital tract
Causative agent of psittacosis: Parrot fever Role in genital tract diseases unclear
Natural reservoir can be any species of bird Associated with adverse pregnancy outcomes
Present in tissues, feces, and feathers of symptom- Clinical diseaseU. urealyticum
atic or asymptomatic birds STD
Veterinarians, zoo keepers, pet shop employees at Urethritis in males
increased risk Infertility, low birth weight, premature delivery
Clinical disease Others
Incubation time of 7 to 15 days Pneumonia, meningitis, bacteremia in newborns
Symptoms include fever, chills, headache, nonpro- Bacteremia, abscesses, arthritis in immunocom-
ductive cough, mild lung inflammation promised patients
Disease usually subsides in 5 to 6 weeks Laboratory diagnosis
Asymptomatic infections are common Mollicutes grow slowly by binary fission and
Seizure, coma, and death (5% mortality rate) produce fried egg colonies on agar plates
can occur Colonies of M. pneumoniae have a granular
Laboratory diagnosis and treatment and prevention appearance
Diagnosis is through serology Because of slow growth, colonies may take up to
Fourfold rise in titer in paired sera 3 weeks to develop
Ureaplasma split urea
FAMILY MYCOPLASMATACEAE
Class Mollicutes: Meaning soft skin FAMILY BARTONELLACEAE
Mycoplasmataceae Bartonella
Grahmella
Mollicutes of Humans Rochalimaea
Grahamella and Rochalimaea may not have valid tax-
Approximately 120 species of the genus Mycoplasma onomic status
Seven species of the genus Ureaplasma
Lack cell walls

Bartonella Species
Very small size
0.2 to 0.8 mm in diameter Approximately 19 described species
Very small genome Three subspecies of Bartonella vinsonii
Require sterols for growth Small (0.6  1.0 mm) gram-negative bacilli
Linked to respiratory infections by Roux and Fastidious
Nocard in 1898 Bartonella henselae
Isolated organism from bovine pleuropneumonia Cat scratch disease
Probably evolved from gram-positive bacteria Acquired after exposure to cats
such as Lactobacillus, Bacillus, Streptococcus, Scratches, bites
Clostridium spp. Disease usually benign
Not found growing freely as living organisms Chronic regional lymphadenopathy
Depend on host for fatty acids, amino acids, nucleic Laboratory diagnosis
acid precursor, and cholesterol Warthin-Starry silver staining
Epidemiology DNA amplification
At least 16 species have been isolated from humans Cultures not recommended
More than half are considered nonpathogenic Blood or tissue transported in Isolator
Mostly Mycoplasma and Ureaplasma spp. Chocolate agar
Associated with mucous membranes Prolonged incubation with increased CO2
Respiratory Serology main testing
Urogenital Cross-reactions with Coxiella burnetii, Chlamydia
Clinical disease spp., and other Bartonella spp.
Clinical disease: M. pneumoniae IgG/IgM EIA
Common cause of mild pneumonia in people Sensitivity 85% and specificity >98%
younger than 40 years of age Laboratory diagnosis, other than cat scratch disease
15% to 50% of all pneumonia in adults and Serology
an even higher percentage of pneumonia in Reference or research laboratory
school-aged children CDC
32 CHAPTER 1 Microbiology

Borrelia
FAMILY RICKETTSIACEAE
Borrelia burgdorferi: Lyme disease
Borrelia hermsii: Relapsing fever
Orientia Treponema pallidum: Syphilis
Orientia tsutsugamushi Other treponemas: Yaws, pinta, bejel
Orientia were formerly classified as Rickettsia General characteristics
Long, thin, spiral
Rickettsia
Approximately 26 species Leptospira interrogans and Leptospira
General characteristics biflexa
Small intracellular bacteria
14 species
0.3 to 0.5  1 to 2 mm
6- to 20-mm coiled rods with hook
Gram-negativelike cell wall
Obligate aerobes
Contains LPS
Fastidious
Independent ATP and host ATP can be used
Epidemiology
No apparent genes for sugar metabolism, lipid syn-
Endemic worldwide
thesis, and amino acid synthesis
Most common zoonotic infection worldwide
Divided into groups based on antigens
Spotted fever group Most prevalent in the tropics and subtropics
Typhus group Contact: Animal urine
Scrub typhus group Directly
Epidemiology: Rocky Mountain Spotted Fever Contaminated fresh water
Clinical disease
(RMSF)
Most common rickettsial disease in the United Leptospirosis
Severity of disease varies with serovar
States
90% of patients mild disease
Approximately 500 to 700 cases per year
Originally described in Rocky Mountain area, but Low fever
more common in South Central states Meningitis
Transmitted by the bite of an infected tick Rash
Highest incidence from April through September 10%: Severe with hepatitis, general organ in-
Principal reservoir for R. rickettsii is hard tick volvement
Laboratory diagnosis
Transovarian passage occurs
Clinical disease: RMSF Clinical presentation
Abrupt onset of fever, chills, headache, and myalgia Culture
5 to 10 days after the tick bite Blood early
Rash common (90%) Urine late (>2 weeks)
Molecular: PCR
2 to 3 days after fever
Serology: Enzyme immunoassay (EIA)
Begins on the hands and feet and spreads toward
the trunk
Palms and soles is common
Complications from vasculitis Borrelia
Respiratory failure, seizures, acute renal failure Approximately 30 species of Borrelia
Mortality rate in untreated patients is 20% Microaerophilic
Laboratory diagnosis Temperature optima 28 to 30 C
Clinical Generation time approximately 18 hr
Fluorescent or PCR for antigen in skin biopsies Flagella determines helical shape
Reference laboratories Flagella-negative mutants are straight bacilli
Serology is the major laboratory test Lyme borreliosis
Weil-Felix test Most common vector-borne disease in the United
Immunoflurorescent antibody (IFA) reagent is States
available Caused by B. burgdorferi
Transmitted by the hard ticks Ixodes spp.
Vector
FAMILY LEPTOSPIRACEAE
Ixodes scapularis
Human disease Deer tick or black-legged tick
Leptospira interrogans: Leptospirosis Ixodes pacificus
CHAPTER 1 Microbiology 33

Clinical disease Noninfectious stage


Stage 1 (3-30 days) May last a lifetime or result in tertiary syphilis
75% develop erythema chronicum migrans Tertiary or late syphilis; noninfectious stage
May be other symptoms Gummas
Stage 2 (1-7 months) Granulomas
Cardiac and neurologic problems No treponemes
Stage 3 (months to years) Causes host response
Arthritis Skin, subcutaneous tissue, deep tissue, bone
Laboratory diagnosis Neurosyphilis
Diagnosis of Lyme borreliosis is clinical diagnosis Congenital syphilis
Laboratory tests should not be used alone, can help Caused by infection in utero with T. pallidum
support a clinical diagnosis A wide spectrum of severity exists
Laboratory tests Laboratory diagnosis
Serology (enzyme immunoassay) Non-treponemal tests include RPR and VDRL
Western blot Treponemal tests include TP-PA and EIA
POS IgM 2 of 3 protein bands
POS IgG 5 of 10 protein bands
ANAEROBIC BACTERIA MOST
Treponema pallidum COMMONLY ISOLATED FROM
Syphilis INFECTION (BOX 1-4)
STD (90%) Bacteria: Relation to O2
Caused by direct contact with lesions that con- Spectrum of sensitivity to O2
tain T. pallidum Strict anaerobes (<5% O2)
T. pallidum enters cracks in mucous membranes of Aerotolerant anaerobes (>5% O2)
genitals, anus, lips, and rectum during vaginal, oral, Facultative anaerobes (aerobes)
and anal sex Strict aerobes
Four untreated stages: 1 > 2 > latent > 3 Strict anaerobic bacteria
Congenital infection possible Superoxide (SO) anion lethal to bacteria
Primary syphilis Facultative and aerobic bacteria handle SO by
Single primary lesion on any cutaneous or mucous producing the enzyme superoxide dismutase
membrane surface (SOD)
Base hard but painless: Hard chancre SO converted to H2O2 and O2
Appears in 3 weeks and disappears in 4 to 12 weeks Catalase and peroxidase production assist in break-
Only diagnostic test is dark-field microscopy ing down H2O2
Secondary syphilis Strict anaerobes lack significant levels of SOD
6 weeks to several months Largely intolerant of O2 in environment
Cutaneous and mucous membrane lesions Lack appropriate cytochrome system to use O2 as
Rough red skin rash terminal electron acceptor
Infectious, live treponemes Energy solely by fermentation
Latent syphilis Gram morphology no different from that in aerobic
Early latent period; 2 yrs or less organisms
Infectious lesions may reappear Many anaerobic bacterial species found among the
Late latent period; over 2 yrs normal microbial flora of humans and animals

BOX 1 -4 Identification of Anaerobic Bacteria

Specimen selection: what not to culture Appropriate specimens


Any specimen likely to be contaminated with normal or coloniz- Blood
ing flora (nasopharyngeal, gingival, bronchial washings, expec- Other normally sterile sites (like pleural fluid)
torated sputum, vaginal or cervical samples, voided urine, Dental/sinusaspiration of abscess or biopsy
surface swabs) Lungaspirate or biopsy, thoracentesis
Minor wounds that are likely to respond to simple drainage Abdomenaspirate of abscess, peritoneal fluid, otherwise
Specimens from acute infections where anaerobes are unlikely sterile tissue
to play a role (bacterial meningitis, routine urinary tract Female genital tractlaparoscopy, surgical biopsy, aspirate of
infection) abscess
Continued
34 CHAPTER 1 Microbiology

B O X 1- 4 Identification of Anaerobic Bacteriacontd

Boneaspirate, biopsy Incubate at 35 C-37 C


Soft tissuesurgical biopsy, aspiration of abscess Use GasPak jars, bags, or anaerobic chambers
Transport of Specimens Use methylene blue or resazurin indicator to validate anaerobic
Tissue specimensanaerobic pouch or anaerobic transport conditions
tube with medium Do not expose plates to air for more than hr
Fluid or aspirated pus Routine media for the interpretation of aerobic growth should
Express into anaerobic transport vials OR be incubated in room air or low CO2 tension if available
Leave in syringe, discard needle & cover with sterile cap Incubation time
Transport of Specimens Inspect at 24 h for rapid growers, 48-72 h for others, 4-5 days
Swabs are strongly discouraged as aspirates or biopsy tissues total
are far better specimens Organisms grow more slowly than aerobes
If swab is unavoidable, it must be collected in a sterile surgical Exceptionsincubate >7 days if Actinomyces is suspected
field and transported in a special anaerobic swab device to lab as it grows very slowly
Primary Media Dissection microscope is helpful to look at tiny colonies
Primary specimens should be plated on non-selective, selective, Approach to Identification
and differential media Complete ID of anaerobes may be complicated, time consum-
All media should be supplemented with vitamin K and hemin ing, and expensive
Media should be fresh General current approach includes:
Routinely used: Focus on Bacteriodes fragilis recognition
Anaerobic SBA, LKV, BBE, CNA or PEA Determination of mixed bacterial flora, both aerobes and
Routine aerobic SBA, MAC, CNA anaerobes
Media has reducing agents to keep O2 levels low Look for toxins of C. difficile
Laked kanamycin-vancomycin (LKV) blood agar Tetanus, botulism, gas gangrene present as characteristic
Selects for gram anaerobic negative bacilli clinical picturesseek Clostridium
Bacteroides bile esculin agar (BBE) Use simple tests to presumptively group most commonly
Selective/differential for Bacteroides fragilis grp. encountered anaerobic organisms
CCFA Selective differential media for recognition of C. difficile Let the Gram stain guide the work up
Cycloserine will inhibit gram-negative bacteria, while cefox- Differential media and resistance to key antibiotics
itin will inhibit both gram-positive and gram-negative organ- Few key biochemical testsCommon anaerobic bacilli
isms. C. difficile ferments the fructose in the medium Bilegrowth in the presence of 20% bile
resulting in yellow colonies Incorporated in BBE agar
Phenylethyl alcohol (PEA)selects for GPC Inhibits most anaerobes other than Bacteroides and
Selects for gram-positive bacilli and cocci Bilophilia
Egg Yolk Agar (EYA)nonselective, differential medium Kanamycin, vancomycin, colisitin susceptibility by disk
Egg yolk suspension allows detection of lecithinase and Antibiotics disks can be placed on blood agar for all three
lipase agents
Lecithin breakdown results in an opaque precipitate Growth or inhibition in the presence of specific concentrations
Lipase enzyme hydrolyzes results in an iridescent sheen on of antibiotic can be used for presumptive identification
the colony surface Catalase
Thioglycolate broth 15% hydrogen peroxide is used to test for the production of
Nonselective, reduced liquid media catase by anaerobes
Incubation of plates Spot Indole
Anaerobes are most sensitive to oxygen during log phase of Ability to metabolize tryptophan by testing for indole in either
growth tubes or a spot test
Bile Kanamycin Vancomycin Colistin Catalase Indole
Prevotella S R R V - -
Porphyromonas S R S R - +
B. fragilis grp R R R R + V
Fusobacterium V S R S - V
Bilophila R S R S + -
Few key biochemical testsanaerobic gram-negative bacilli
Lecithinasesee egg yolk agar
Lecithinase Double zone of hemolysis CCFA yellow colonies Catalase Indole
Clostridium perfringens + + - - -
Clostridium difficile - - + - -
Clostridium septicum - - - - -
Propionibacterium - - - + +
CHAPTER 1 Microbiology 35

Peptostreptococcus Gram-positive bacillus that tends to form branches,


sometimes with a beaded appearance
General characteristics Common member of the mouth flora of humans
Gram-positive anaerobic cocci in chains Aerotolerant anaerobe
Common member of the normal gut flora and Very slow growing and difficult to recover in culture
respiratory tract Responds well to long-term therapy with penicillin
Almost always found, when clinically significant, in
class of antibiotics
coinfections with other anaerobes and facultative Clinical syndromes
anaerobes Cervical-facial
Basically the only genus of the gram-positive anaer-
Most common, causes lumpy jaw
obic cocci involved in disease Slowly developing abscess, chronic infection
may erupt into sinus tracts on face or neck
Veillonella
Dental surgery or facial trauma predisposition
General characteristics Pulmonary
Gram-negative anaerobic cocci (counterpart of the Aspiration of mouth flora to lower respiratory
aerobic Neisseria) tract
Common member of the mouth and intestinal flora Primary pneumonia may result
of humans Radiography not specific to Actinomyces
Low incidence of pathogenicity Chronic infection in which abscess is formed
If clinically significant, found in coinfections Abscess, which can bore its way to surface and
with other anaerobes and facultative anaerobes produce sinus tracts
Sulfur granules can be seen in the fluid
Propionibacterium Granules are actually aggregated microcolonies
General characteristics Gram smear reveals the long filamentous
Gram-positive, nonspore forming, anaerobic Actinomyces
bacillus Abdominal
Resembles Corynebacterium in morphology and Infection follows ingestion of organism
cell arrangement GI lesions can occur, not unlike those seen in pul-
Some strains are aerotolerant, yet yield better monary infection
growth under strictly anaerobic conditions Abscess can rupture through musculature and
Produces propionic acid skin to form tracts
Widely distributed as normal flora on skin and other Genitourinary
body sites, including the respiratory tract, and may A. israelii may colonize the female genital tract as
act as opportunistic pathogen with other copathogens normal flora
Associated with long-term use of intrauterine
Propionibacterium acnes devices
Indistinguishable from pelvic inflammatory
General characteristics
disease
Most common gram-positive, nonspore forming,
Vaginal discharge, abdominal pain, fever, uri-
anaerobic rod encountered in clinical specimens nary discomfort, etc.
Slowly growing in culture
Treated with antibiotics and removal of the device
Common resident of the pilosebaceous glands of the
human skin
Causative agent of acne vulgaris (pimples) GRAM-POSITIVE BACILLUS:
In addition to acne, P. acnes has been implicated in CLOSTRIDIUM
other infectionss General characteristics
Corneal ulcers Gram-positive bacillus
Heart valves Usually large bacilli
Prosthetic devices Produce endospores
CNS shunts May appear terminal or central
Opportunistic mixed infections with other flora
Excellent survival in environment
Highly susceptible to various b-lactam antimicro- Are strictly anaerobic in metabolism
bial agents such as penicillin G Produce variety of potent toxins
Clostridium spp. of clinical importance
Actinomyces israelii Clostridium perfringens
General characteristics Gas gangrene
Most common human pathogenic species Food poisoning
36 CHAPTER 1 Microbiology

Clostridium tetani Food or wound botulism


Tetanus Toxin: Botulism neurotoxin (BoTN)
Clostridium botulinum Preformed in food source or made by organ-
Botulism ism contaminating a wound
Clostridium difficile Blocks acetylcholine release at the neuromus-
Antibiotic associated diarrhea and colitis cular junction and causes an inhibition of
muscle contraction
Symptoms
Clostridium perfringens Blurred vision, dizziness, muscle weakness,
and flaccid paralysis
Nonmotile gram-positive anaerobic bacillus
Boiling food destroys the toxin
Part of human intestinal flora Infant botulism
Minor opportunistic pathogen
Acquired by ingestion of food containing spores
Appears in mixed flora infection Ranges from mild to fatal disease
Found universally in soil
Honey is the most common source of spores,
Potential to cause major myonecrosis called gas
which then germinate in the childs intestinal
gangrene in wounds tract (contraindicated in children younger than
Gas gangrene
1 year of age)
Results from contaminated wounds
Toxin production causes symptoms of few days
Lesions progress from redness and swelling to
duration and then often subside
greenish blackish decoloration Rare need for use of antitoxin
Toxin destroys muscle tissue
Symptoms
Gas bubbles present in blisters and under skin
Inability to suckle, constipation, flaccid paral-
Fatal within 48 hours without antibiotics
ysis, muscle weakness
Treatment is surgical (debridement), antibiotics,
and hyperbaric O2 chamber
Clostridium difficile
Motile gram-positive anaerobic bacillus
Clostridium tetani Source
Environmental bacteria: Soil Soil, air, water, human and animal feces
Motile gram-positive anaerobic bacillus Use of broad-spectrum antibiotics lowers relative
Endospores contaminate puncture wounds, grow amount of other normal gut flora and allows C. diffi-
anaerobically, and produce toxin to cause the disease cile to proliferate and infect large intestine
Disease
tetanus (lockjaw)
Tetanus C. difficile releases two enterotoxins (A and B)
Painful and rapidly fatal syndrome Can cause diarrhea, but often causes pseudo-
Toxin binds to target nerve cells membranous colitis with destruction of intestinal
Inhibitory interneurons that regulate muscle con- lining (also called antibiotic associate colitis)
Symptoms
traction are blocked
Patients undergo single, constant muscle con- Watery diarrhea, abdominal cramps, fever,
traction bloody stools, nausea, dehydration
Treatment: Oral metronidazole or vancomycin
Infection can lead to respiratory failure
Treatment can include use of antitoxin Either or both of the following tests will confirm the
Protection is afforded by vaccination with toxoid to disorder
raise antibody against the toxin Immunoassay of stool extract for C. difficile
toxins
Colonoscopy showing pathologic findings of
pseudomembranous colitis
Clostridium botulinum
Environmental bacteria: Soil
Motile gram-positive anaerobic bacillus
Endospores may become airborne and contaminate ANAEROBIC GRAM-NEGATIVE BACILLI
food preparation followed by anaerobic storage (non-
heated soups, canning of preserves)
Bacteroides fragilis
Spores germinate to produce one of the most lethal General characteristics
toxins in the world and result in the disease Gram-negative anaerobic bacillus
botulism Common member of the normal gut flora
CHAPTER 1 Microbiology 37

Cause of serious infections if the normal GI mucosal Resistant


barrier is breached Strains are not inhibited by the usually achiev-
Can be carried to virtually any organ of the body via able systemic concentrations of the agent with
bloodstream normal dosage schedules
Often found in coinfections with facultative Clinical efficacy is unlikely
anaerobes Standard for all methods
The organism does not have a characteristic gram- All testing is performed from a pure culture
negative endotoxin Bacterial suspensions are made in comparison to a
turbidity standard
5  105 CFU/mL broth dilution
Other Gram-Negative Anaerobic Bacilli 1  104 CFU/mL agar dilution
1  108 CFU/mL diffusion
Fusobacterium Species
Has spindle-shaped morphology
Found in respiratory and GI tracts ANTIMICROBIAL AGENTS
Found in mixed infections
Definitions
Prevotella melaninogenica Antibiotic: A chemical substance produced by a
Regular bacillus morphology
Found in respiratory and GI tracts microorganism that has the capacity to inhibit the
Cause of lung and dental infections growth of or kill other microorganisms
Antimicrobial: An agent that kills or suppresses
Grows a black-pigmented colony
growth of microorganisms
Porphyromonas Species
Antimicrobial effects
Regular bacillus morphology
Bacteriostatic agents prevent replication but do not
Purple-pigmented bacilli on agar
Mouth and genitourinary tract kill their target
Head, neck, and pleuropulmonary infections and peri- Tetracyclines, macrolides, sulfonamides
Bacteriocidal agents result in cell death
odontal disease
b-Lactams, vancomycin, fluoroquinolones
Mechanisms of antimicrobial action
Cell wall inhibitors
Cell membrane inhibitors
ANTIMICROBIAL SUSCEPTIBILITY Protein synthesis inhibitors
TESTING METHODS Ribosomes
Definitions Other metabolic pathway inhibitors
Minimal inhibitory concentration (MIC) Folate metabolism
Quantitative measure of the susceptibility of a Cell wall synthesis inhibitors
bacterial isolate to an antimicrobial agent The b-lactams
The lowest concentration observed to inhibit Penicillin
growth of the isolate in vitro Cephalosporins
Minimal bactericidal concentration (MBC) Carbapenems and monobactams
Not commonly performed Imipenem and meropenem
The lowest concentration of that antibiotic to kill Aztreonam
the bacterial isolate in vitro Vancomycin
Definitions: breakpoints Penicillins: Mechanism of action
Susceptible Binds to penicillin-binding proteins
Organism can be inhibited by achievable serum Stops transpeptidation (cell wall cross-linking)
or tissue levels at the dosage of antimicrobial Cell wall develops weak spots and bursts
agent recommended for that type of infection Bactericidal, synergistic with aminoglycosides
Favorable outcome Works only on growing cells
Intermediate Cephalosporins: Generation determined by the spec-
MICs approach usually attainable blood or tis- trum of activity
sue levels Carbapenems
Strains may be inhibited by certain antimicrobial Imipenem/cilastatin, meropenem, doripenem
agents in body sites where drugs may be Broad spectrum, low MICs, but expensive
concentrated Problem with CNS toxicity in imipenem if overdosed
Macrolides and respiratory tract Mechanism of action
Provides a buffer zone that prevents technical fac- Same as other b-lactams
tors from causing discrepancies in interpretations Stable to many b-lactamases
38 CHAPTER 1 Microbiology

Except metallob-lactamases (e.g., S. malto- Clindamycin


philia and B. cepacia) Quinupristin/dalfopristin
Vancomycin: Mechanism of action Linezolid
Stops gram-positive cell wall peptidoglycan chain Inhibitors of intermediate metabolism
formation Trimethoprim
Bactericidal for staphylococci and streptococci Sulfa compounds
Bacteriostatic for enterococci Trimethoprim/sulfamethoxazole
Mechanism of resistance Mechanism of action
Gram-negative organisms are usually resistant Stops folate synthesis
Enterococci modify the target site
VRE
MICROBIOLOGY LABORATORY SAFETY:
Staphylococci have shown resistance

STANDARD PRECAUTIONS
Cell membrane inhibitors
Polymyxin Assume all patients are infectious for HIV, HBV, or
Colistin other blood-borne pathogens
Amphotericin Limit access to the laboratory to trained
Polymyxin B, Colistin personnel only
Hydrophobic proteins that disrupt the gram- Use barrier precautions at all times
negative cell membrane Gloves, masks, goggles, coats or gowns where
Active only against gram-negative bacteria indicated
Highly toxic: Renal, neurologic, nausea, vomiting, Leave personal protective equipment in laboratory
diarrhea and out of public areas
Rarely used Thoroughly wash hands and other skin surfaces after
Useful for P. aeruginosa and other resistant gram- gloves are removed and immediately after any
negative organisms contamination
DNA/RNA inhibitors Use particular care with handling and disposal of
Fluoroquinolones sharps
Metronidazole Rigorously follow needle stick policies
Nitrofurantoin Refrain from
Rifampin Eating, drinking, smoking, application of cosmetics
Fluoroquinolones Insertion or removal of contact lenses
Ciprofloxacin, levofloxacin Nail biting or pen or pencil chewing
Fluoroquinolones: Mechanism of action Mouth-pipetting
Inhibit DNA gyrase and/or topoisomerase General laboratory safety
Prevents DNA unwinding and blocks DNA Threat: Chemicals; Defense: Active chemical
synthesis hygiene plan, labeling and storage standards, train-
Bactericidal and concentration dependent ing, material data safety sheets (MSDS), disposal,
Not synergistic with other antibiotics use of fume hoods, spill kits and procedures, barrier
Metronidazole: Mechanism of action protection, eye wash stations
Damages DNA and other molecules directly Threat: FireDefense: Training, extinguishers,
Resistance escape plan, elimination of open flames
Reduced uptake and metabolism, increasing in Threat: ElectricalDefense: Active program of
anaerobes checks and maintenance
Rifampin: Mechanism of action Threat: Gas cylindersDefense: Chaining into
Prevents RNA synthesis position in well-ventilated area, transport with
Inhibits the DNA-dependent RNA polymerase secure dollies
Bactericidal Threat: RadiationDefense: Radiation safety pro-
Intracellular activity grams, monitoring exposure among operators
Active gram-positive organisms
Mutation rate of this enzyme is high
Protein-synthesis inhibitors
Macrolides
CERTIFICATION PREPARATION QUESTIONS
Erythromycin, clarithromycin, azithromycin, For answers and rationales, please see Appendix A.
dirithromycin 1. Spores are found in select groups of bacteria. Which of
Aminoglycosides the following statements describes the major advan-
Gentamicin, tobramycin, amikacin, neomycin tage to the bacteria that possess these structures?
Tetracyclines a. Spores are resistant to heat, cold, drying, most
Tetracycline, doxycycline, minocycline chemicals, and boiling
CHAPTER 1 Microbiology 39

b. Spores allow an organism to better control its


local environment
c. Spores allow bacteria to attach or adhere to host
tissues
d. Organisms with spores have a more efficient
exchange of genetic material
2. Choose the binomial name that is correctly written.
a. Staphylococcus Aureus
b. Staphylococcus species aureus
c. Staphylococcus aureus
d. Staphylococcus aureus
3. Fermentation end-products are often used to aid in
the identification of bacteria. Fermentation results
in which of the following?
a. Conversion of glucose to pyruvate FIGURE 1-1 (Courtesy Joel Mortensen, PhD. See also color plate 1.)
b. Lactic acid, mixed acids, alcohols, CO2
production
c. CO2 and water c. Many cells, many gram-positive cocci in pairs and
d. Specific teichoic acids chains
4. The exchange of cellular DNA between two living d. More than 25 epithelial cells, probable oral con-
bacterial cells that involves an intercellular bridge tamination, suggest recollect
is which of the following processes? 9. 85% N2, 10% H2, 5% CO2 is the environmental
a. Transformation condition that best suits which type of organism?
b. Transduction a. Aerobes
c. Plasmidization b. Anaerobes
d. Conjugation c. Capnophiles
5. Transduction is defined as which of the following? d. Microaerophiles
a. The change of the bacterial genotypes through 10. Which medium can be described as containing bile salts
the exchange of DNA from one cell to another and dyes (bromothymol blue and acid fuchsin) to selec-
b. An internal change in the original nucleotide tively slow the growth of most nonpathogenic gram-
sequence of a gene or genes within an organisms negative bacilli found in the gastrointestinal tract
genome and allow Salmonella spp. and Shigella spp. to grow?
c. The process by which genetic elements such as a. Thayer-Martin
plasmids and transposons excise from one geno- b. MacConkey
mic location and insert into another c. PEA (phenylethyl alcohol)
d. A mechanism that is mediated by viruses, by which d. Hektoen
DNA from two bacteria may come together in one 11. Choose the group of bacteria that is described as
cell, thus allowing for recombination catalase-positive, gram-positive cocci that grow fac-
6. A mordant that is applied after the primary stain to ultatively anaerobic and that form grapelike clusters.
chemically bond the alkaline dye to the bacterial cell a. Neisseria spp.
wall is which of the following? b. Rothia (Stomatococcus) spp.
a. Safranin c. Staphylococcus spp.
b. Crystal violet d. Micrococcus spp.
c. Grams iodine 12. The slide coagulase test is a rapid screening test for
d. Grams decolorizer the production of which of the following?
7. Which of the following bacteria should be considered a. Clumping factor
important pathogens when reading gram-stained b. Free coagulase
smears of soft tissue abscess? c. Extracellular coagulase
a. Streptococcus pneumoniae d. Catalase
b. Neisseria gonorrhoeae 13. The first identification test performed on a clinical
c. Pseudomonas aeruginosa isolate of gram-positive, catalase-positive cocci
d. Staphylococcus aureus should be which of the following?
8. The most appropriate interpretation of a gram- a. Penicillin test
stained smear of a sputum specimen would be b. Gram stain
which of the following? (gram-stained smear, c. Oxidase test
400 .) d. Coagulase test
a. Few epithelial cells, many PMNs 14. The Staphylococcus sp. that is more likely to cause
b. Inadequate specimen, do not culture for anaerobes uncomplicated urinary tract infections in
40 CHAPTER 1 Microbiology

nonhospitalized hosts, especially sexually active b. They are a quick way to rule out streptococcal
young women, is which of the following? pharyngitis and avoid giving antibiotics when
a. Staphylococcus saprophyticus not needed
b. Staphylococcus aureus c. They are always very sensitive and specific for
c. Staphylococcus epidermidis streptococcal pharyngitis
d. Staphylococcus intermedius d. They are a quick and accurate way to diagnose
15. The toxic shock syndrome toxin-1 is an important vir- bacterial and viral pharyngitis
ulence factor in staphylococcal disease. This toxin is 22. The hemolysis of this Streptococcus spp. would best
classified into which of the following groups of toxins? be described as which of the following?
a. Cytolytic toxin
b. Leukocidin
c. Phospholipase
d. Enterotoxin
16. Mannitol salt agar is selective and differential for
which group of organisms?
a. Staphylococcus spp.
b. Enterococcus spp.
c. Gram-positive cocci
d. Streptococcus spp.
17. Within 5 hours of returning home from lunch at your
most favorite fast food restaurant you feel very sick
and are vomiting. Which of the following is the most
likely causative organism?
a. Staphylococcus aureus
b. Vibrio parahaemolyticus
c. Shigella sonnei
d. Escherichia coli
18. The bacterial species that can be described as suscep-
tible to bile and optochin, a-hemolytic, a major cause
of bacterial meningitis, and often carrying an anti-
phagocytic capsule is which of the following?
a. Enterococcus faecalis
b. Streptococcus pneumoniae FIGURE 1-2 (Courtesy Joel Mortensen, PhD. See also color plate 2.)
c. Streptococcus pyogenes
d. Streptococcus agalactiae
19. The bacterial species that can be described as suscep- a. b-Hemolysis
tible to penicillin and bacitracin, b-hemolytic, a major b. g-Hemolysis
cause of bacterial pharyngitis, and often carrying an c. a-Hemolysis
antiphagocytic M protein is which of the following? d. k-Hemolysis
a. Enterococcus faecalis 23. Enterococcus spp. can be differentiated from most
b. Streptococcus pyogenes Streptococcus spp. by which of the following tests?
c. Streptococcus agalactiae a. Growth in presence of 6.5% salt
d. Viridans streptococci b. Production of catalase
20. The bacterial species that can be described as able to c. Production of coagulase
hydrolyze hippurate, b-hemolytic, a major cause of d. Growth on PEA medium
neonatal meningitis and sepsis, and producer of the 24. A pure culture of a b-hemolytic Streptococcus sp.
CAMP factor is which of the following? recovered from a leg ulcer gave the following
a. Streptococcus pneumoniae reactions:
b. Streptococcus pyogenes
c. Streptococcus agalactiae CAMP test Negative Hippurate hydrolysis Negative
Bile esculin slant No growth 6.5% Salt No growth
d. Viridans streptococci
PYR Negative Bacitracin Resistant
21. The rapid antigen detection methods for throat Optochin Resistant SXT Sensitive
swabs used for screening patients for streptococcal
pharyngitis can be best described by which of the fol- Which of the following is the most likely identifica-
lowing statements? tion of this organism?
a. They can be useful in quickly identifying most a. Streptococcus pyogenes
cases of streptococcal pharyngitis b. Streptococcus agalactiae
CHAPTER 1 Microbiology 41

c. Enterococcus faecalis 29. A skin lesion was opened and drained in surgery. The
d. Streptococcus sp., not groups A, B, or D culture was positive for a gram-positive bacillus,
25. The ability to grow well at refrigerator temperatures is which gave the following growth characteristics
a characteristic of which of the following organisms? and biochemical reactions:
a. Mycobacterium gordonae
MacConkey agar: No growth Catalase: Negative
b. Listeria monocytogenes H2S on TSI: Positive Growth of blood agar, nonhemolytic
c. Erysipelothrix Nonmotile No spores
d. Bacillus cereus
26. A catalase-positive, gram-positive bacillus that is not These reactions are consistent with which of the
acid-fast, does not branch, and does not form spores following organisms?
could possibly belong to which group of bacteria? a. Listeria spp.
a. Corynebacterium b. Group B b Streptococcus
b. Bacillus c. Erysipelothrix spp.
c. Nocardia d. Corynebacterium spp.
d. Mycobacterium 30. Which of the following sets of tests provide the best
27. A throat culture was taken from a 6-year-old differentiation of Erysipelothrix from Listeria
boy with a gray pseudomembrane covering his oro- monocytogenes?
pharynx. A catalase-positive organism was isolated a. Gram-stained smear, oxidase, and optochin
on cysteine-tellurite medium and subcultured to b. Gram-stained smear, catalase, and motility
Tinsdale medium, where it grew as black colonies c. CAMP test, hydrogen sulfide production, esculin
with brown halos. A Gram stain was performed on hydrolysis
these colonies. Which of the following cellular mor- d. Reverse CAMP, gram-stained smear, b-hemolysis
phologies was most likely seen? 31. Neonatal meningitis is an uncommon but sig-
a. Gram-positive branching bacilli nificant disease. Two important causes of
b. Gram-positive cocci in short chains this disease may be somewhat difficult to diff-
c. Gram-positive bacilli in irregular clublike erentiate on preliminary observation. Which of
shape the following sets of tests provide the best differen-
d. Gram-positive cocci in grapelike clusters tiation of Streptococcus agalactiae from Listeria
28. A blood culture is positive for gram-positive bacilli monocytogenes?
that gave the following growth characteristics and a. Gram-stained smear, oxidase, and optochin
biochemical reactions: b. Gram-stained smear, catalase, and motility
MacConkey agar: No growth Catalase: Positive c. CAMP test, hydrogen sulfide production,
H2S on TSI: negative Growth of blood agar, nonhemolytic b-hemolysis
Nonmotile No spores d. Reverse CAMP, gram-stained smear, b-hemolysis
32. Which of the following tests is important as a part of
These reactions are consistent with which of the the genus identification or as part of a preliminary
following organisms? identification but is not used as a confirmatory iden-
a. Listeria spp. tification of Bacillus anthracis?
b. Group B b Streptococcus a. Demonstration of a capsule
c. Erysipelothrix spp. b. Demonstration of spore formation
d. Corynebacterium spp. c. Positive PCR test
d. Lysis of the strain by specific bacteriophages
33. Bacillus anthracis and Bacillus cereus can be differ-
entiated in the laboratory by a variety of different test
results. Which of the following sets of tests best dif-
ferentiate these two species?
a. Catalase and glucose fermentation
b. Motility and lecithinase production
c. Oxidase and b-hemolysis on 5% sheep blood agar
d. Motility and b-hemolysis on 5% sheep blood agar
34. Which of the following specimens would be best for
identifying Bacillus cereus as the cause of an out-
break of food poisoning?
a. Blood
b. Rectal swabs
c. Stool samples
FIGURE 1-3 (Courtesy Joel Mortensen, PhD. See also color plate 3.) d. Food
42 CHAPTER 1 Microbiology

35. A first morning sputum sample is received for acid- 40. The gram-negative bacillus that can be described as
fast culture. The specimen is centrifuged, and the sed- oxidase-negative, nitrate-positive, indole-negative,
iment is inoculated on two Lowenstein-Jensen slants citrate-positive, methyl redpositive, urease-
that are incubated at 35 C with 5% to 10% CO2. negative, and H2S-positive is most likely which of
After 1 week, the slants show abundant growth over the following?
the entire surface. Stains reveal gram-negative bacilli. a. Klebsiella pneumoniae
Which of the following should be done to avoid this b. Salmonella enteritidis
problem? c. Escherichia coli
a. Use a medium specifically designed for the d. Shigella sonnei
growth of AFB 41. The swarming gram-negative bacillus that can be
b. Dilute out the sediment before inoculation with described as oxidase-negative, nitrate-positive,
saline indole-negative, and H2S-positive is mostly likely
c. Decontaminate the specimen with NALC which of the following?
sodium hydroxide mixture a. Proteus aerogenes
d. Incubate the tubes at room temperature to retard b. Proteus vulgaris
bacterial growth c. Proteus mirabilis
36. A patient recently arrived in the United States from d. Escherichia coli
Africa presents with a long-standing cutaneous 42. Profuse watery diarrhea (rice water stools), lead-
lesion, which is cultured for bacteria, fungi, and ing to dramatic fluid loss, severe dehydration, and
AFB. An AFB smear is made and is reported as pos- hypotension that frequently leads to death, is the
itive for AFB. After 8 weeks of culture on both non- hallmark of which toxin activity?
selective and selective AFB media, no colonies a. Cholera toxin
appear. Which of the following organisms should b. Enteric endotoxin
be suspected? c. Shiga toxin
a. M. kansasii d. Toxin A
b. M. tuberculosis 43. The selective medium thiosulfate citrate bile
c. M. leprae salts sucrose (TCBS) agar is especially formu-
d. M. avium-intracellulare complex lated for isolating which pathogen from stool
37. The mycobacterial species that occur in humans and cultures?
belong to the M. tuberculosis complex include which a. Vibrio spp.
of the following? b. Salmonella spp.
a. M. tuberculosis, nontuberculous Mycobacteria, c. Shigella spp.
M. bovis, and M. africanum d. Plesiomonas spp.
b. M. tuberculosis, M. gordonae, M. bovis BCG, 44. The majority of human infections with Cam-
and M. africanum pylobacter spp. are caused by which of the
c. M. tuberculosis, M. bovis, M. avium, and M. following?
intracellulare a. Direct contact with carriers of the bacterium
d. M. tuberculosis, M. bovis, M. bovis BCG, and M. b. Contamination of food, milk, or water with
africanum animal feces
38. The Runyon system of classification is based on c. Multiplication of the organism in food products
which of the following? d. Direct contact with persons infected with the
a. Colony and microscopic morphology bacterium
b. Biochemical characteristics 45. In the test for urease production, the presence of
c. Growth rate and colonial pigmentation the enzyme hydrolyzes urea to which of the
d. All of the above are correct following?
39. In identification of mycobacterial isolates, the Tween a. Ammonia and CO2
80 test involves which of the following? b. Putrescine
a. An enzyme that is able to produce Tween c. Amines and CO2
80 from certain ingredients found in the d. Amines and water
medium 46. The bacterial isolate on XLD agar shown in the
b. Lipase that is able to hydrolyze polyoxyethylene image was isolated from a routine stool culture.
sorbitan monooleate into oleic acid and polyox- Which of the following genera and species is the most
yethylated sorbitol likely identification for this organism?
c. The metabolism of niacin to nicotinic acid by a. Klebsiella pneumoniae
enzymatic action b. Salmonella enteritidis
d. Testing the isolate for susceptibility to c. Shigella sonnei
Tween 80 d. Serratia marcescens
CHAPTER 1 Microbiology 43

The organism is most likely which of the following?


a. Klebsiella sp.
b. Shigella sp.
c. Salmonella sp.
d. Escherichia coli
50. The best specimen for the isolation of Bordetella per-
tussis is which of the following?
a. Throat swabs
b. Sputum
c. Nasopharyngeal aspirates
d. Anterior nose swab
51. Organisms belonging to the genus Brucella are best
described by which of the following statements?
a. Gram-positive diplococci
FIGURE 1-4 (Courtesy Joel Mortensen, PhD. See also color plate 4.) b. Gram-positive diphtheroid bacilli
c. Gram-negative coccobacilli
47. The bacterial isolate shown below on CIN agar was d. Gram-negative bacilli
isolated from a routine stool culture. Which of the 52. Serum samples collected on a patient with pneumo-
following genera and species is the most likely iden- nia demonstrate a rising antibody titer to Legionella.
tification for this organism? A bronchoalveolar lavage sample was collected and
revealed a positive DFA test for Legionella, but no
organisms were recovered from this specimen when
it was cultured on the appropriate medium and incu-
bated for 2 days at 35 C in CO2. Which of the fol-
lowing is the best explanation?
a. Culture was not incubated long enough
b. Antibody titer
c. Specimen was incubated at the wrong temperature
d. Positive DFA test result is a false positive
53. Of the following media, which provides the NAD
necessary for the growth of Haemophilus spp.?
a. 5% sheep blood agar
b. Brain heart infusion agar
c. Chocolate agar
d. Nutrient agar
54. Performing the factor requirement test for Haemo-
philus involves which of the following processes?
FIGURE 1-5 (Courtesy Joel Mortensen, PhD. See also color plate 5.) a. Inoculation of unsupplemented media with a light
suspension of the organism and placement of fac-
tors X and V disks on the agar surface
a. Shigella flexneri b. Inoculation of liquid media, unsupplemented and
b. Salmonella enteritidis supplemented with factors X and V
c. Yersinia enterocolitica c. Detecting the presence of enzymes that convert a-
d. Escherichia coli aminolevulinic acid (ALA) into porphyrins
48. Decarboxylation of the amino acids lysine, ornithine, d. Growth of the organism in the presence of bacte-
and arginine results in the formation of which of the rial species that produce X and V factors as met-
following products? abolic by-products
a. Ammonia 55. Of the asaccharolytic, oxidase-positive bacilli that do
b. Urea not grow on MacConkey agar, which one is among
c. CO2 the HACEK group of bacteria known to cause sub-
d. Amines acute bacterial endocarditis?
49. An organism was inoculated into a TSI tube and gave a. Eikenella corrodens
the following reactions: b. Weeksella virosa
Alkaline slant c. Pseudomonas maltophilia
Acid butt d. Sphingomonas paucimobilis
H2S: Not produced 56. Which of the following statements best completes the
Gas: Not produced following thought: Presumptive identification of an
44 CHAPTER 1 Microbiology

oxidase-positive, gram-negative diplococcus on c. Fermenter


Thayer-Martin medium from genital sites of a 6- d. Nonviable
year-old female as Neisseria gonorrhoeae? 61. The oxidase test is a critical test when attempting to
a. Provides the physician with quick and reliable identify nonfermenting gram-negative bacilli. This
results at minimal cost test is designed to determine the presence of which
b. May sometimes be incorrect, and a repeat culture of the following?
should be collected a. b-Galactosidase
c. May sometimes be incorrect and should not be b. Cytochrome oxidase
reported until confirmed c. Glucose oxidizing enzymes
d. Should be done only when venereal disease is d. Oxygen
suspected 62. The blood culture of a patient with a central venous
57. The bacterial species that can be described as oxidase- catheter yielded a gram-negative bacillus growing on
positive, glucose-positive, maltose-positive, sucrose- MacConkey agar with the following reactions:
negative, lactose-negative, and a major cause of bac-
Oxidase Negative Motility Positive
terial meningitis is most likely which of the following?
Glucose oxidative- Maltose oxidative-fermentative
a. Neisseria meningitidis fermentative open Positive (strong)
b. Neisseria gonorrhoeae open Positive (weak)
c. Streptococcus pneumoniae Catalase Positive Esculin hydrolysis Positive
d. Viridans group Streptococcus
58. The bacterial species that can be described as oxidase- Which of the following is the most likely identifica-
positive, glucose-positive, maltose-negative, sucrose- tion of this organism?
negative, lactose-negative, and a major cause of a. Burkholderia cepacia
venereal disease is most likely which of the following? b. Pseudomonas aeruginosa
a. Neisseria meningitidis c. Acinetobacter baumannii
b. Neisseria gonorrhoeae d. Stenotrophomonas maltophilia
c. Streptococcus pyogenes 63. Which organism is associated with the disease
d. Viridans group Streptococcus Melioidosis?
59. Organisms belonging to the genus Neisseria are a. Burkholderia ralstonia
described as which of the following? b. Burkholderia pseudomallei
a. Gram-positive diplococci c. Burkholderia mallei
b. Gram-negative diplococci d. Burkholderia cepacia
c. Gram-negative coccobacilli 64. Differentiation of Stenotrophomonas maltophilia
d. Gram-negative bacilli and Burkholderia cepacia is best accomplished by
60. The following were observed when the Hugh-Leifson which of the following tests?
oxidative-fermentative test was performed on a bac- a. Oxidase test
terial isolate. Which of the options below best b. Maltose and glucose medium
describes the organisms reaction? c. Tyrosine-enriched heart infusion agar
a. Oxidizer d. Growth at 42 C
b. Nonoxidizer 65. The respiratory culture of a patient with cystic fibro-
sis yielded a gram-negative bacillus with the follow-
ing reactions:
Oxidation Fermentation
Oxidase Positive Motility Positive
neg neg
Glucose oxidative-fermentative Gelatin
open Positive hydrolysis Positive
Soluble green pigment on TSA slant Arginine
dihydrolase Positive
Growth at 42 C positive

Which of the following is the most likely identifica-


tion of this organism?
a. Burkholderia cepacia
b. Pseudomonas aeruginosa
c. Acinetobacter baumannii
d. Stenotrophomonas xylosoxidans
FIGURE 1-6 (Photograph by Dr. WH Ewing, courtesy the Centers 66. Which test group best differentiates Acinetobacter
for Disease Control and Prevention, Public Health Image Library, baumannii from Pseudomonas aeruginosa?
http://phil.cdc.gov/. See also color plate 6.) a. Oxidase, motility, nitrate reduction
CHAPTER 1 Microbiology 45

b. Growth on MacConkey agar, catalase, nitrate has now spread to include his trunk. The medical team
reduction has identified a list of possible organisms. Which of
c. Growth on blood agar, oxidase, catalase the following is the most likely cause of this infection?
d. TSI, urea, motility a. Q Fever
67. Which of the following sets of results represent the b. Ehrlichiosis
most common reactions for Moraxella catarrhalis c. Rocky Mountain spotted fever
when tested in CTA sugar tubes? d. Cat scratch disease
a. Glucose: Negative; Maltose: Negative; Lactose: 74. Which of the following is a stage of venereal syphilis
Negative; Sucrose: Negative that is characterized by the appearance of a chancre?
b. Glucose: Positive; Maltose: Negative; Lactose: a. Primary syphilis
Negative; Sucrose: Negative b. Secondary syphilis
c. Glucose: Positive; Maltose: Positive; Lactose: c. Late syphilis
Negative; Sucrose: Negative d. Tertiary syphilis
d. Glucose: Positive; Maltose: Negative; Lactose: 75. Which of the following is a nontreponemal serologic
Positive; Sucrose: Negative test in which soluble antigen particles are coalesced
68. A soluble, bright green pigment can be produced by to form larger particles that are visible as clumps
Pseudomonas aeruginosa. This pigment is known as when they are aggregated by antibody?
which of the following? a. Nontreponemal flocculation (NTF)
a. Pyoverdin b. Fluorescent treponemal antibody absorption
b. Pyocyanin (FTA-ABS) test
c. Pyorubin c. Venereal Disease Research Laboratory
d. Pyophena (VDRL) test
69. A small portion of a colony of a gram-negative bacilli d. T. pallidum particle agglutination (TP-PA) test
was smeared onto a filter paper test system. One per- 76. A patient in a rural area of Massachusetts had a 5-cm
cent tetramethyl-p-phenylenediamine dihydrochlor- red rash with an expanding margin on his back. The
ide was added. At 10 seconds, a dark purple color lesion was obvious for approximately a month and
developed where the colony was added to the paper. then resolved. Several weeks later, the patient experi-
Which of the following statements best describes the enced episodes of partial facial paralysis and painful
test results? joints. Which of the following is the most likely infec-
a. Positive indole test tious agent in this case?
b. Positive oxidase test a. Borrelia hermsii
c. Positive urea test b. Borrelia burgdorferi
d. Positive esculin test c. Leptospira interrogans
70. Characteristics of Mycoplasma and Ureaplasma d. Spirillum minor
include which of the following? 77. A 16-year-old, sexually active patient comes to his
a. They exhibit the presence of a thin gram-positive physicians office because of a circular, 1-cm lesion
like cell wall with no cell membrane in the groin area which is ulcerated but not painful.
b. They demonstrate rapid growth on MacConkey A rapid plasma reagin test is performed and is reac-
agar, slow growth on basic nutrient agar tive with a titer of 1:16. Culture and gram-stain
c. The have only a cell membrane with no cell wall smear results from an exudate of the lesion are neg-
d. They exhibit rapid growth on MacConkey ative. Which of the following is the most likely cause
medium and routine blood agar plates of this lesion?
71. Which of the following is a cause of nongonococcal a. Chlamydia trachomatis
urethritis? b. Neisseria gonorrhoeae
a. Mycoplasma hominis c. Treponema pallidum
b. Mycoplasma pneumoniae d. Haemophilus ducreyi
c. Ureaplasma urealyticum 78. The gram-stained smear shows an organism isolated
d. Mycoplasma orale from a blood culture after bowel surgery. Under
72. Which of the following is the most sensitive method anaerobic incubation conditions, it grew as smooth,
for the diagnosis of Chlamydia trachomatis? white, nonhemolytic colonies. The organism was not
a. Cytology inhibited by colistin, kanamycin, or vancomycin and
b. Culture hydrolyzed esculin. The most likely identification of
c. Nucleic acid amplification this isolate is which of the following?
d. Serologic testing a. Fusobacterium nucleatum
73. An 8-year-old boy from Oklahoma presents with a b. Fusobacterium varium
3-day history of fever, headache, and muscle aches. c. Bacteroides fragilis
A rash first noted this morning on his ankles and wrists d. Prevotella melaninogenica
46 CHAPTER 1 Microbiology

c. Expression of results is by MIC for both


d. The cost of the test is similar per drug
84. Which of the following clinical indications would
most benefit from having quantitative (MIC) testing
rather than qualitative (Sensitive, Intermediate,
Resistant catagories) data from the laboratory?
a. Urinary tract infection
b. Bacterial meningitis
c. Pneumonia caused by Mycoplasma
d. Streptococcal pharyngitis
85. When performing antimicrobial susceptibility test-
ing, the following definition of the minimum inhibi-
tory concentration (MIC) is correct:
a. The highest concentration of an antibiotic in a
FIGURE 1-7 (Photograph by Dr. VR Dowell, Jr, courtesy the Centers dilution series that inhibits growth
for Disease Control and Prevention, Public Health Image Library, b. The lowest concentration of an antibiotic in a
http://phil.cdc.gov/. See also color plate 7.) dilution series that inhibits growth
c. The lowest concentration of an antibiotic in a
79. Pseudomembranous colitis caused by Clostridium dilution series that kills the bacteria
difficile is best confirmed by which of the following d. The lowest concentration of the antibiotic obtain-
laboratory findings? able in the patient without toxicity
a. Presence of the toxin in stool 86. In comparing quantitative MIC dilution testing to
b. Isolation of C. difficile from stool qualitative agar disk diffusion testing, the higher
c. Gas production in thioglycolate media the MIC of the drug for that organism:
d. Gram stain of stool showing many gram-positive a. The smaller is the zone of inhibition
bacilli b. The more susceptible the organism will appear on
80. Lecithinase production, double zone hemolysis on disk diffusion
sheep blood agar, and gram-stained morphology c. The larger is the zone of inhibition
are all useful criteria in the identification of which d. The more toxic is the drug to the patient
of the following? 87. In a quality control (QC) procedure on a new
a. Clostridium perfringens batch of Mueller-Hinton plates using a standard
b. Streptococcus agalactiae QC stock strain of Staphylococcus aureus, the disk
c. Escherichia coli inhibition zone sizes for three of the drugs tested
d. Clostridium tetani were too small and fell below the expected QC range.
81. When activating a hydrogen and carbon dioxide gen- Which of the following is the most likely reason for
erator system used for creating an anaerobic atmo- this observation?
sphere, which of the following is an indication that a. These three antibiotic disks were outdated and
the catalyst and generator envelope are functioning had lost potency
properly? b. These three disks were faulty in that the antibiotic
a. A decrease in temperature of the jar content was too high
b. Bubble formation on the surface of the plates c. Bacterial suspension of Staphylococcus was prob-
c. A change in color of the methylene blue indicator ably contaminated with another organism
d. The formation of a visible cloud of gas d. The plates received insufficient incubation time
82. In a clinical specimen, the presence of sulfur granules 88. Which of the following definitions best fit the term
strongly indicates the presence of which anaerobic urethritis?
bacterium? a. Infection and or inflammation of the terminal
a. Bacteroides fragilis portion of the lower urinary tract
b. Actinomyces spp. b. The isolation of a specified quantitative count of
c. Fusobacterium nucleatum bacteria in an appropriately collected urine spec-
d. Clostridium tetani imen obtained from a person without symptoms
83. Which of the following are a common element or signs of urinary infection
between using the E test and agar disk diffusion c. Dysuria, frequency, and urgency but yielding fewer
(Kirby-Bauer) for antimicrobial susceptibility organisms than 105 colony-forming units of bacte-
testing? ria per milliliter (CFU/mL) urine on culture
a. Both establish an antibiotic gradient in agar d. Inflammation of the kidney parenchyma, calices
b. Both create a circular zone of bacterial (cup-shaped division of the renal pelvis), and
inhibition pelvis
CHAPTER 1 Microbiology 47

89. The organism most commonly associated with otitis c. Macrophages


media infections is associated with which of the fol- d. Squamous epithelial cells
lowing positive test results? 95. Which of the following terms is used to describe an
a. Coagulase increase of lymphocytes and other mononuclear cells
b. VP (pleocytosis) in the cerebrospinal fluid and negative
c. Optochin bacterial and fungal cultures?
d. Bacitracin a. Meningoencephalitis
90. Which organism is most often responsible for b. Aseptic meningitis
impetigo? c. Encephalitis
a. Staphylococcus epidermidis d. Meningitis
b. Streptococcus pyogenes 96. The culture of which sample routinely uses quantita-
c. Enterococcus faecalis tion or the counting of bacterial cells present to assist
d. Streptococcus agalactiae in the interpretation?
91. How do staphylococci spread so easily when infect- a. Blood
ing the skin? b. Sputum
a. They produce hyaluronidase, which hydrolyzes c. Urine
hyaluronic acid present in the intracellular d. Abscess
ground substance that makes up connective tissue 97. Gram staining and reading a glass slide with a mixed
b. They produce lipase, which melts the fat under smear of Staphylococcus and Escherichia coli along
the skin, making it easier to spread with each Gram staining run of specimens examined
c. The hemolysins kill the white and red blood cells; within the microbiology laboratory that day is an
then the protease liquefies the skin protein, allow- example of which of the following?
ing easy penetration for the bacteria a. Quality assurance (QA) activity
d. All of the above b. Quality control (QC) activity
92. Routine culture media for use with a specimen of c. National regulatory activity
cerebrospinal fluid should include which of the fol- d. Office of Safety and Health Administration activity
lowing sets of media? 98. Tracking the rate of skin organism contamination
a. 5% sheep blood agar, Lowenstein Jensen agar, among a laboratorys blood culture results on a
7H9 agar monthly basis and introducing specific training to
b. 5% sheep blood agar, thioglycolate broth phlebotomists when rates exceed the norm would
c. 5% sheep blood agar, MacConkey agar, be an example of which of the following?
Sabourad dextrose agar a. Good laboratory practice
d. 5% sheep blood agar, chocolate agar, thioglyco- b. Quality control
late broth c. Universal standards
93. A college student is examined at the emergency d. Quality assurance
department; he is disoriented with a fever, intense 99. Which of the following statements best defines
headache, stiff neck, vomiting, and sensitivity infectious substances?
to light. His friends say that he has been sick for a. Articles or substances capable of posing a risk to
about 2 days and that his condition worsened over safety
the last 3 hours. The physician does a complete b. Substances known or reasonably expected to
blood count (CBC) and electrolytes. The electro- contain pathogens
lytes are normal, but the patients white blood c. Patient samples containing bacteria
count (WBC) is 12,000 cells/L. What test should d. Samples with class 3 pathogens
the doctor order next? 100. Which of the following is an example of an inappro-
a. Urine culture priate specimen or condition that would warrant
b. Stool culture rejection for microbiology culture?
c. Cerebrospinal fluid Gram stain and culture a. A nonsterile container for a stool culture
d. Blood culture b. A swab of a skin and soft tissue infection
94. What cells are found in bacterial vaginosis? c. A tissue sample for anaerobic culture
a. Clue cells d. A 24-hour urine sample for bacteriology
b. Lymphocytes culture
48 CHAPTER 1 Microbiology

SELF-ASSESSMENT
Content Area: ______________________________

Score on Practice Questions: ______________________

List the specific topics covered in the missed questions:

List the specific topics covered in the correct questions:


CHAPTER 1 Microbiology 49

NOTES
CHAPTER

2
Mycology, Virology, and
Parasitology
Linda J. Graeter and Joel E. Mortensen

MYCOLOGY Molds: Obligate hyphae


Yeasts: Unicellular, budding
The Fungal Organism Dimorphic: Two bodies or forms
Mycology terms
A group of nonmotile eukaryotic organisms that have
Perfect fungi
definite cell walls, are devoid of chlorophyll, and
Sexual stage is known
reproduce by means of spores (and conidia) Fungi imperfecti
Heterotrophic
No known sexual stage
Hetero means different, and troph means
Conidia
nourishment
Eukaryotic (fungi) versus Prokaryotic (bacteria) Reproductive structures produced by an asexual
mode
Capsule
Spore
Polysaccharide
Reproductive structures produced sexually, and
Much larger than bacterial capsule
the asexual reproductive cells of the zygomycetes
Antiphagocytic, virulence
Conidiophore: Structure that supports conidia
Mostly in yeast
Annelloconidia: Produced by annellids
Cryptococcus neoformans: Encapsulated yeast
Fungal cell wall Phialoconidia: Produced by phialide
Antigenic Poroconidia: Produced from pores
Sporangium: Saclike structure where sporangio-
Multilayered
spores are formed (Zygomycetes)
Polysaccharides (90%)
Asexual reproduction
Chitin
Arthroconidia: Directly from hyphae by modifica-
Proteins and glycoproteins (10%)
Provides shape and rigidity to cell tion of cell wall (barrels)
Blastoconidia: Budding of cell (mother and
Osmotic protection
Cell membrane daughter)
Chlamydoconidia: Directly from hyphae (swelling)
Bilayered phospholipids
Sexual reproduction
Sterols (ergosterol versus cholesterol)
Ascospore
Functions
Sexual spore formed in a saclike structure after
Protects cytoplasm
meiosis
Regulates intake of nutrients Zygospore
Facilitates capsule and cell wall synthesis
Cytoplasm Round, thick-walled spore produced in a saclike
Nucleus, nucleolus, nuclear membrane, endoplas- structure by fusion of two hyphal tips
Basidiospore
mic reticulum, mitochondria, vacuoles
Mycology terms Spore formed in a club-shaped reproductive
Hypha (plural: hyphae): Filamentous, tubular structure after meiosis
Mycosis (mycoses)
growth
Invasive treatments
True hyphae versus pseudohyphae Immunosuppressive therapy
Septate (aseptate): Cross walls in hyphae
Immunocompromising infections
Mycelium (plural: Mycelia)
Human immune deficiency virus/acquired
Vegetative
immunodeficiency virus (HIV/AIDS)
Aerial
50
CHAPTER 2 Mycology, Virology, and Parasitology 51

Rise in common and uncommon mycoses Systemic mycoses


Organisms and tissue infected Any tissue
Five broad categories of fungal infections Four organisms
Opportunistic fungi True or primary pathogens
Immunocompromised patients Endemic to specific geographic areas
Many different tissues Must travel through the area to become infected
Ubiquitous: Environmental saprobes Thermal dimorphs and yeasts
Monomorphs Two bodies based on temperature
Same structural characteristics under all Blastomyces, Coccidioides, Histoplasma,
conditions Paracoccidioides
Aspergillus, Candida, Mucor, Rhizopus
Superficial mycoses
BASIC CLINICAL MYCOLOGY
Infections of outer, dead layers
No host defense stimulation Specimen collection
No pain or discomfort Important factors in isolating and identifying a fun-
Usually treated because the infection is gal pathogen
unsightly Correct type of specimen
Exophiala, Malassezia, Piedraia, Trichosporon Quality of specimen
Dermatophytic mycoses Rapid transport
Skin, hair, nails Use of appropriate culture media
Deeper than the superficial Processed within 2 hours
Still no living skin penetration Specimen transport
Produce secondary metabolites that irritate Sterile, leak-proof container
Host defense causes itching Dermatologic requires dry container
Sometimes cutaneous and superficial grouped No transport media
Epidermophyton, Microsporum, Trichophyton Processed within a few hours
Subcutaneous mycoses Specimens can be refrigerated at 4 C
Muscle, bone, connective tissues Only if processing is delayed
Traumatic inoculation Blood and cerebrospinal fluid (CSF): 30 to 37 C
Thorns, scratch Dermatologic: 15 to 30 C
Usually remain localized Safety in the mycology laboratory
Cladosporium, Exophiala, Pseudallescheria, Standard precautions
Phialophora, Sporothrix No smoking, eating, drinking, or applying
cosmetics
Contact lenses (no removing or cleaning)
TABLE 2 -1 Major Medically Important Fungi No mouth pipetting
Universal precautions
Category Genus Class 2 or 3 biosafety hoods
Opportunistic fungi Aspergillus Disinfectant Phenol based
Candida Biohazard containers
Mucor Specimen processing: Methods
Rhizopus Direct inoculation
Superficial mycoses Exophiala Adding several drops of specimen to media
Malassezia For solid media, the specimen can be streaked
Piedraia
Specimen types: Bronchial brush/wash, aspi-
Trichosporon
Dermatophytic mycoses Epidermophyton
rates, CSF, swabs, body fluids, hairs, scrapings
Microsporum
Concentration
Trichophyton Large volumes can be concentrated by
Subcutaneous mycoses Cladosporium centrifugation
Exophiala Specimen types: Body fluids, CSF, urines
Pseudallescheria Minced (homogenized)
Phialophora Some solid specimens must be destroyed to
Sporothrix expose a buried pathogen to the media
Systemic mycoses Blastomyces Specimen types: Nails, tissues, biopsies
Coccidioides Culture of fungi
Histoplasma Petri dishes or tubes
Paracoccidioides
Oxygen requirements
52 CHAPTER 2 Mycology, Virology, and Parasitology

Humidity Dermatophyte test medium (DTM)


Subculture sometimes necessary Dermatophytes from heavily contaminated spec-
Temperature range imens (pink-to-red color change)
Dimorphism Commonly used in office practices
Sexual and asexual developmental structures Media for subculture
Get rid of bacterial contamination Potato dextrose agar (PDA)
Teasing needles Potato flake agar (PFA)
Used more than bacteriologic loop Incubation
Electric incinerator Obligate filamentous: 25 or 37 C
Flame causes aerosols Dimorphics: 25 and 37 C
Culture is very important Yeast: 25 or 37 C
Can be main identification Aerobic
No or few biochemicals 3 to 4 weeks
Yeasts are the exception Cornmeal agar for yeast morphology
Culture media Recommended for promoting sporulation
Options Pathogen versus contaminant
Test tubes for primary The clinical picture
Less likely to become contaminated, less Are patients symptoms consistent with fungal
drying infection?
Petri dishes for subculture Does this fungus normally cause these
Larger surface area for growth symptoms?
Use of inhibitory substances may be required Laboratory findings
Chloramphenicol, gentamicin, cycloheximide Fungal elements in tissue or other specimen
May encounter some fungal inhibition Fungus grown in culture
Common media More than one culture positive
Sabouraud dextrose agar (SDA) Quality control
Most common, many fungi grow Assessing quality of specimens
Emmons modification: Less glucose Monitoring performance
Blastomyces dermatitidis Tests, reagents, media, instruments
Mycosel and mycobiotic Quality control culture collections
SDA + chloramphenicol + cycloheximide Personnel
Selective recovery of dimorphs and Performance evaluation
dermatophytes Proficiency testing
Brain heart infusion (BHI) agar Laboratory identification
Enriched to enhance recovery C. neoformans of Direct examination of clinical specimens
and dimorphic transitions in Sporothrix and Laboratory methods and tissue stains
Paracoccidioides Macroscopic/microscopic evaluation
Plates or tubes Colony features and hyphae/conidia mor-
Broth + penicillin for Zygomycetes phology
BHI + gentamicin + chloramphenicol Advanced methods
C. neoformans from contaminated specimen Exoantigen, DNA probes, DNA sequencing
Sabouraud dextrose + BHI (SABHI) Microscopic examination
Strengths of both Direct examination can be used on several types
Enriched medium for Cryptococcus spp., ther- of specimens
mally dimorphic fungi, etc. Can identify yeast and filamentous forms
CHROMagar Candida Culture is used regardless
Selective and differential for presumptive Several preparations for direct examination
identification of genus Candida from primary Potassium hydroxide (KOH) preparation
plates Calcofluor white
Morphology and colors of the yeast colonies India ink: Historical
vary by species KOH preparation
Candida albicanslight to medium green; Examine hair, nails, skin scrapings, fluids, exu-
Candida tropicalislight blue to metallic-blue; dates, and biopsy specimens
Candida kruseilight rose with a whitish Can see important fungal elements
border Hyphae, yeast
Inhibitory mold agar (IMA) Need reduced light or phase-contrast
Inorganic salts, chloramphenicol, gentamicin 15% KOH added to specimen
Inhibits bacteria Dissolves specimen quickly (fungi slowly)
CHAPTER 2 Mycology, Virology, and Parasitology 53

Can be modified to include calcofluor white Transferred to microscope slide


Binds to cell wall and fluoresces blue-white Slide culture
under ultraviolet light Organism subcultured to a small piece
India ink of agar
Historically used with CSF specimens Covered with a coverslip
Negative stain Organism grows onto coverslip: Remove
Creates black background to visualize capsu- and examine
lar material Best method
C. neoformans Examination of molds
More specific/sensitive tests are now available Use one of the three methods listed previously,
Cryptococcal antigen test followed by addition of stain
Tissue examination: Stains Lactophenol cotton blue (LPCB)
Giemsa, Wright-Giemsa Dermatophyte identification
Histoplasmosis capsulatum (intracellular) Hair perforation
Hematoxylin and eosin (H&E) 5- to 10-mm sterile hair floated on sterile
Pink to pinkish-blue water and yeast extract
Meyers mucicarmine Conidia or hyphae inoculated onto water
C. neoformans: Rose red surface
Gomori methenamine silver (GMS) Remove hair shafts and observe in LPCB
Black weekly for 1 month
Papanicolaou stain Trichophyton rubrum negative, Trichophyton
Pink to blue mentagrophytes positive
Periodic acidSchiff (PAS) Urease test
Red or purple Tubes of urease agar are lightly inoculated
Macroscopic examination 5 days at room temperature
Growth conditions T. rubrum negative or weak, T. mentagro-
Yeasts: 2 to 3 days phytes positive
Molds Trichophyton agars
Rapid: Less than 5 days Originally numbers 1 to 4
Intermediate: 6 to 10 days Most laboratories use only 1 and 4
Slow: More than 11 (sometimes 8 weeks) Thiamine requirement
Dimorphism Trichophyton agar 1 (without thiamine)
Pigment and Trichophyton agar 4 (with thiamine)
Front versus back of plate 10 to 14 days, observe for growth
Texture Rice grain growth
Dictated by presence and length of aerial hyphae Sterile, nonfortified rice grain media
Glabrous: Leathery, waxy 10 days, observe for growth
Velvety: Suede, plush Microsporum canis versus Microsporum
Yeastlike: Looks like Staphylococcus audouinii
Cottony: Fluffy Summary: Mold identification
Granular: Powdery Specimen source or infection
Topography Growth rate to reproductive structures
Rugose Colony color front and back on plate
Radial grooves, folded Microscopic morphology
Crateriform Septate or aseptate hyphae
Central depression and raised edge Conidiophore structure
Verrucous Microconidia/macroconidia
Rough knobs Other structures
Cerebriform Advanced techniques
Brainlike Exoantigen test
Examination of molds Rapid information of immunoidentity
Three methods Extract soluble antigen from unknown
Tease/cut preparation isolate
Organism removed directly from culture Concentrate
plate React with antiserum specific to known
Teased apart with teasing needles fungi
Scotch tape preparation Positive control necessary for definitive
Scotch tape pressed onto culture plate identification
54 CHAPTER 2 Mycology, Virology, and Parasitology

Test is read at 24 hours Other agents can form germ tubes


Blastomyces, Coccidioides, Histoplasma Not valid if read after 2 hours
DNA probe True germ tube: C. albicans
Rapid kits that use nucleic acid hybridization No constriction at base, where the tube
to identify fungi in culture attaches to the mother cell
Highly specific to each fungus, because it is A constricted base indicates C. tropicalis
based on DNA sequence Other species have germ tubes
Needs to be performed on cultured organisms Candida stellatoidea (Sucrose assimilation
Not from specimens used to differentiate from C. albicans)
Developed for Coccidioides, Blastomyces, Candida dubliniensis (no growth at 45 C)
Histoplasma Positive and negative controls are necessary
Specialized clinical laboratories are using Fermentation/Assimilation
DNA sequencing techniques to establish fun- Fermentation
gal identifications Carbohydrate use in absence of oxygen
Laboratory identification of yeast Assimilation
Macroscopic morphology Which can be used as a sole carbon source?
Colony color and texture Two systems (assimilation)
Color: White, tan, pink, salmon API 20C (others): Strip test
Can have dematiaceous yeasts Vitek: Automated
Texture: Mucoid, butterlike, velvety, wrinkled Urea hydrolysis
Microscopic morphology: Wet preparation Detected on simple urea agar
Hyphae Rapid, easy
Pseudohyphae Differentiates Cryptococcus from Rhodotorula
Blastoconidia Positive: Pink
Cornmeal Tween 80 agar Negative: Little to no change
Encourages development of chlamydospores Temperature studies
Relationships among hyphae, pseudohyphae, Cryptococcus spp.
and others Weak growth at 35 C and no growth at
Clear media: Can be observed under light 42 C
microscope Candida spp.
Specific organisms associated with specific Several can grow well exceeding 45 C
morphology Order of events
Cornmeal agar morphology Most yeasts
Used in conjunction with carbohydrate usage Wet preparation
Four main morphology types Germ tube
Hyphae Germ tube negative and from sterile site
Pseudohyphae Corn meal morphology
Arthroconidia Physiologic/biochemical tests
Chlamydoconidia or blastoconidia Temperature
Pseudohyphae and blastoconidia only
C. krusei
THE OPPORTUNISTIC MOLDS
Candida parapsilosis
Candida kefyr Most frequently isolated fungi
C. tropicalis Opportunistic infections
Blastoconidia only Infect those who are injured or debilitated
Candida glabrata Common inhabitant of soil and organic debris
C. neoformans Laboratory and environmental contaminant
Arthroconidia
Trichosporon beigelii
Physiologic tests
Acremonium Species
Germ tube test No known sexual stage: Fungi imperfecti
Filamentous outgrowth from blastoconidia Filamentous fungus in plant debris and soil
Most basic and easiest to perform Two more common species
Requires the use of serum or plasma Acremonium falciforme
Some commercially made broths (will last Acremonium kiliense
longer) Cause onychomycosis, keratitis, endocarditis, menin-
Overincubation and overinoculation are big- gitis, peritonitis, and osteomyelitis
gest problems Macroscopic
CHAPTER 2 Mycology, Virology, and Parasitology 55

Rapid grower Most strains either do not grow at all or grow


White, cottony colonies weakly at 37 C
Microscopic Microscopic
Hyaline, septate hyphae Arthroconidia and coarse true hyphae are
Unbranched, solitary, erect phialides formed directly observed
on the hyphal tips Blastoconidia, conidiophores, and pseudohyphae
Conidia usually in clusters or fragile chains are absent
Therapy and susceptibility testing Undifferentiated hyphae may be present
In vitro susceptibility Arthroconidia observed
Limited data and minimum inhibitory concentra- Either rectangular or rounded at the ends
tion (MIC) breakpoints have not been defined Do not alternate with normal cells
Newer azoles (voriconazole, posaconazole) exhibit
good in vitro activity
Itraconazole MICs somewhat higher than MICs in Paecilomyces Species
voriconazole
Sexual stage described: Teleomorph
MICs of caspofungin are relatively low
Soil, decaying plants, and food products
Several species
Paecilomyces lilacinus and Paecilomyces variotii
Fusarium Species most common
No known sexual stage: Fungi imperfecti Causes wide range of mycoses
Plants and soil Emerging opportunistic pathogen
Normal mycoflora of commodities (rice) Onychomycosis, sinusitis, otitis media, endocardi-
More than 20 species tis, osteomyelitis, peritonitis, and catheter-related
Fusarium solani, Fusarium oxysporum, Fusarium fungemia
chlamydosporum Macroscopic
Fusariosis Rapid grower
Emerging cause of opportunistic mycoses P. variotii is thermophilic
Disseminated infections have high mortality Colonies are flat, powdery, or velvety
Trauma or inhaled conidia Initially white and becomes yellow, yellow-
Macroscopic green, yellow-brown, olive-brown, pink, or vio-
Rapid grower, woolly to cottony, flat, spreading let, depending on the species
colonies Reverse is dirty white, buff, or brown
Front: White, cream, tan, salmon, cinnamon, yellow, May resemble Penicillium spp. macroscopically and
red, violet, pink, or purple microscopically
Reverse: Colorless, tan, red, dark purple, Microscopic
or brown Septate, hyaline hyphae
Microscopic Conidiophores are often branched
Macroconidia: Two or more cells, thick walled, Phialides are swollen at the base and taper toward
smooth, and cylindrical or sickle (canoe) shaped the apice
Usually grouped in pairs or brushlike clusters
Conidia are unicellular, hyaline to darkly colored,
Geotrichum Species and form long chains
Lack known sexual stage
Found worldwide in soil, water, air, sewage, plants,
cereals, and dairy products
Penicillium Species
Found in normal human flora Teleomorph described
Genus includes several species Penicillium marneffei is thermal dimorph
More common: Geotrichum candidum, Geotrichum (Southeast Asia)
clavatum, Geotrichum fici Numerous species
May cause opportunistic infections in immunocom- More common: Penicillium chrysogenum, Penicil-
promised host lium citrinum
Infections acquired via ingestion or inhalation Particularly virulent in patients with AIDS
Macroscopic Keratitis, endophthalmitis, otomycosis, necrotizing
Produce rapid growing, white, dry, powdery-to- esophagitis, pneumonia, endocarditis, peritonitis,
cottony colonies resembling ground glass and urinary tract infections (UTIs)
Colony may be yeastlike P. marneffei often fatal
Optimal growth temperature is 25 C Macroscopic
56 CHAPTER 2 Mycology, Virology, and Parasitology

Rapid growing; velvety, woolly, or cottony Rate of growth is usually rapid


Initially white and become blue-green, gray-green, Usually matures in 3 days
olive-gray, yellow, or pinkish Some species are slower
Reverse is usually pale to yellowish Temperature
Microscopic A. fumigatus grows well at 45 C
Flask-shaped phialides Macroscopic
Form brushlike clusters First white then yellow, green, brown, or black
Conidia are round, unicellular, and form unbranch- Microscopic
ing chains at the tips of the phialides Hyphae are septate
Unbranched conidiphore from a foot cell
Vesicles
Scopulariopsis Species Phialides cover the surface of the vesicle entirely
Soil, plant material, feathers, and insects (radiate head) or partially only at the upper
Unique in that it contains both hyaline and dematia- surface (columnar head)
Phialides are either uniseriate (attached to the
ceous species
Scopulariopsis brevicaulis (hyaline) vesicle directly) or biseriate (attached to the
Scopulariopsis cinerea (dematiaceous) vesicle via a supporting cell) metula
Onychomycosis, especially of the toe nails Conidia form radial chains
Disseminated infections: High mortality
Macroscopic
Grow moderately rapidly, granular to powdery ZYGOMYCETES
Front color is white initially and becomes light
Zygomycetes is the name of a class of fungi
brown or buff
This class includes three orders: Mucorales, Mortierel-
Reverse color is usually tan with brownish center
Microscopic lales, and Entomophthorales
Most clinically significant are in Mucorales
Septate hyphae
Absidia, Cunninghamella, Mucor, Rhizomucor,
Conidiophores are hyphae-like and simple or
Rhizopus
branched
Zygomycosis
Sometimes incorrectly referred to as mucormycosis
Inhalation of sporangiospores, trauma (inoculation)
Aspergillus Species Can become invasive
More than 185 species Sinus infections are common
Approximately 20 species have been described as Typical presentation
agents of infection in humans Pulmonary, rhinocerebral, cutaneous, renal, or
Most common: Aspergillus fumigatus, Aspergillus meningeal involvement
flavus, Aspergillus niger Risks
Less common: Aspergillus clavatus, Aspergillus Diabetes, leukopenia, immunosuppression, AIDS,
glaucus group, Aspergillus nidulans burns, intravenous drug
Clinical disease Rapid growth
Three clinical settings More tissue damage, almost always fatal
Opportunistic infections Rapidly fill plate in culture: Lid lifters
Allergic states Differentiation
Toxin production Presence (or absence) and location of rhizoids
Opportunistic infections rootlike structures
Local infections Branched or unbranched nature of
Local colonization in previously developed lung sporangiophore
cavity Size and shape of sporangium
Infection of every organ system has been Macroscopic of similar
described
Disseminated infections
Allergic reactions
Mucor Species
Allergic bronchopulmonary aspergillosis Several pathogenic species
Toxins Many do not grow at 37 C
Aflatoxin Rapid growth, fluffy (cotton candy), white initially
Veterinary diseases and becomes grayish-brown in time
Laboratory Identification Reverse is white
CHAPTER 2 Mycology, Virology, and Parasitology 57

Aseptate or sparsely septate, broad hyphae, sporangio- Tineas: Skin


phores long and branched with terminal sporangia Piedras: Hair
No rhizoids Nonliving layer of the skin and extrafollicular hair
Lack of systemic immune response
Specimens are cultured onto SDA
Rhizopus Species Sometimes with antibiotics
Several important species Diagnosis
50% of all zygomycoses Appearance of lesion
90% of rhinocerebral infections Skin scrapings
Grow very rapidly, cotton-candy white initially and Hair shafts
turns gray to yellowish-brown in time Four main infections
Reverse side is white Tinea versicolor
Pathogenic species of Rhizopus can grow well at 37 C Tinea nigra
Broad, aseptate hyphae, long unbranched White piedra
sporagiophores Black piedra
Rhizoids are produced Tinea versicolor
Malassezia furfur
Superficial infection of the keratinized layers
Rhizomucor Species of skin
Rare cause of zygomycosis Normal flora of skin (90% asymptomatic)
Normally fatal No known reason for predisposition
Colony similar to that of Mucor Clinical picture
Microscopic Patches of hypopigmented or hyperpigmented
Intermediate to Mucor and Rhizopus lesions
Short rhizoids and branched sporangiophores Brown or fawn, scaling, redness
Chest, back, shoulders, arms, abdomen
(itch, burn)
Absidia Species Specimen: Skin
21 species Direct examination: KOH will show yeastlike
and hyphal forms
Absidia corymbifera is the only clinically significant
Culture: Lipophilic organism
species
Add oil overlay and incubate at 37 C
Rapid growth, woolly to cottony, and olive-gray
Microscopic
colonies
Reverse side uncolored Thick-walled hyphae and yeast, some
Broad aseptate hyphae, sporangiophores branched budding
Spaghetti and meatballs
and arise in groups of two to five
Tinea nigra
Sporangiospores are one-celled and round to oval
Hortaea werneckii
Exophilia and Cladosporium werneckii
Cunninghamella Species obsolete names
Central and North America, Southeast Asia, Africa,
Seven species
Europe
Cunninghamella bertholletiae is the only known Most likely environmental
human and animal pathogen Clinical disease
Rapid growing, cottony, and white to tannish-gray
Synonyms: Pityriasis nigra, tinea nigra
Reverse is pale palmaris
Aseptate or sparsely septate broad hyphae, sporan-
Infection of keratinized skin layers of hand
giophores long and branched More common in those under 25 years of age and
Vesicles
females
Sporangiophores are erect and form short lateral
Dark skin on one hand (usually only one)
branches, each of which terminates in a swollen Flat, brown lesion
vesicle Possibility of melanoma must be ruled out
Direct examination: KOH preparation
SUPERFICIAL MYCOSES Culture
Required to differentiate from melanoma
Among the most prevalent of human infectious diseases H. werneckii
Mycotic infections of hair, skin, and nails Macroscopic
58 CHAPTER 2 Mycology, Virology, and Parasitology

Colonies grow slowly and mature within Not usually required


21 days Slow grower (25 C)
Initially pale in color, moist, shiny, and Does not penetrate hair shaft
yeastlike Piedraia hortae
Colonies become velvety, olive black, and cov- Macroscopic
ered with a thin layer of mycelium Colonies are slow growing
The reverse side is black Small, folded, dark brown to black
Does not grow at 37 C May produce a reddish-brown diffusible
Microscopic features pigment
Septate hyphae, yeastlike conidia, and Reverse side is black
chlamydospores Microscopic
Hyaline initially and become olive colored Septate hyphae, asci, and ascospores
Annellides present Asci are ellipsoid, solitary, or in clusters
Annelloconidia are intercalary and lateral and contain eight ascospores
Septate, thick-walled hyphae formed Hyphae pigmented
White piedra
Caused by Trichosporon spp.
SUBCUTANEOUS MYCOSES
Most commonly T. beigelii
T. beigelii may not have taxonomic status Four major infections caused by several fungi
infection of hair of beard and mustache Mycetoma
Environmental (soil and air) Chromoblastomycosis
South and North America, Far East, Europe Phaeohyphomycosis
Clinical disease Sporotrichosis
Soft white to tan nodules Common to all
Surround hair shaft, separated easily from hair Lesion develops at site of inoculation (localized)
Hair breaks at nodule Soil saprophytes that are moderately slow growers
Can become systemic in immunocompromised Most commonly accepted
Direct examination: KOH preparation Cladophialophora, Exophiala, Fonsecaea, Phialo-
Culture phora, Wangiella, Pseudallescheira/Scedosporium,
Not normally required Sporothrix schenckii
SDA: Yeastlike colonies Most infections are due to traumatic inoculation
Microscopic Common in tropics and subtropics
Nodule surrounding hair Some of these fungi cause more than one type of sub-
Trichosporon spp. cutaneous infection
Macroscopic Most are dematiaceous fungi
Colonies are rapid growing Dematiaceous versus hyaline
Yeastlike, may be smooth, wrinkled, Conidiation of dematiaceous fungi
raised, folded Cladosporium type
White to cream colored Resembles a tree, in which conidiophore is the
Urease production characteristic trunk and branched chains of conidia form the
Microscopic branches
Many pseudohyphae and hyphae Phialophora type
Blastoconidia are unicellular and variable in Short conidiophores + phialide, vase shaped, con-
shape idia extruded from phialide and then cluster
Arthroconidia produced Rhinocladiella type
Black piedra Stalked conidiophores that become knobby as
Piedraia hortae conidia are produced, conidia produced sequen-
Fungal infection on hair (scalp) tially until a Cladosporium type of conidiation is
Forms black, stony, hard nodules reached
Central and South America, Southern Asia, Africa Laboratory identification of subcutaneous fungi
Swimming in rivers and stagnant waters Specimens collected by aspiration
Clinical picture Large amount of material, reduces chances that
Nodules firmly attached, can be microscopic to specimen will dry out
visible by naked eye, hair feels rough Granules observed and noted
Direct examination SDA with and without antibiotics
KOH preparation PDA for subculture
Culture Biochemicals are available, but rarely done
CHAPTER 2 Mycology, Virology, and Parasitology 59

Mycetoma Cladisporium carrionii obsolete name


Very slow grower (up to 30 days)
General Colonies are gray-green to black on surface and
Chronic granulomatous disease of feet (lower
reverse, cottony
extremities) Pigmented, septate hyphae
Madura foot or maduromycosis Cladosporium type of conidiation
Enlarged nodules, sinus drainage, bone destruction
Fonsecaea pedrosoi
Exudate contains granules Causes chromoblastomycosis and phaeohyphomycosis
No lymphatic system involvement (remain Traumatic injury
localized) Gray-green to black, cottony colony within 21 days
Two types: Eumycotic and actinomycotic Pigmented, septate hyphae
Laboratory procedures All three types of conidiation
Direct examination: KOH preparation Phialophora, Cladosporium, Rhinocladiella
Identification granules, colorless or pigmented sep-
Fonsecaea compacta
tate hyphae Conidial heads of Cladosporium type of conidiation
Actinomycotic granules: Mycelium with hyphae are more compact
1 mm in diameter Phialophora verrucosa
Eumycotic granules: Wide hyphae (2-4 mm) Causes chromoblastomycosis and phaeohyphomycosis
terminating in chlamydoconidia Autoinoculation and lymphatic system
Macroscopic
Olive-green to black, velvety
Chromoblastomycosis Microscopic
General Pigmented, septate hyphae
Localized disease of skin and subcutaneous tissue Only Phialophora type of conidiation
Verrucoid (wartlike) lesions on feet, legs, hands, Pseudallescheria boydii
and buttocks Scedosporium apiospermum: Name for alternate
Soil saprophytes that are introduced by trauma asexual stage
(worldwide), dematiaceous Major etiologic agent of mycetoma in the United States
Spreads through the body lymphatics or by and Europe
autoinoculation Different from other subcutaneous
Laboratory procedures Grows rapidly, hyaline, has a sexual form
Direct examination: KOH exudate, crusts from Macroscopic
lesion White to brownish-gray, fluffy colonies
Microscopic: Single-celled or clusters of single cells, Microscopic
dark pigment Hyaline, septate hyphae
Culture: SDA at room temperature, hold for Single anelloconidia produced on an anellophore
6 weeks (conidiophore)
Looking for three types of conidiation Exophiala jeanselmei
Cladosporium type Cause of mycetoma and phaeohyphomycosis
Rhinocladiella type Minor trauma and contaminated fomites
Phialophora type Young cultures
Phaeohyphomycosis Appear as black yeasts
Infection of subcutaneous tissue Mature cultures
Classically: Infection with a dematiaceous Velvety colonies
fungus Sticklike conidiophores with clustered conidia
The others have become distinct Wangiella dermatitidis
Mycetoma Causes pheohyphomycosis
Chromoblastomycosis Macroscopic
Sporotrichosis Initially resemble black yeast
Miscellaneous dematiaceous fungi: Introduced Longer 10 days, olive-gray to black velvety or gla-
through trauma brous colony
Systemic infection is a disease of the Wangiella spp. grow better at 40 to 42 C
immunocompromised Microscopic
KOH preparation shows pigmented hyphae Pigmented, septate hyphae
Cladophialophora carrionii Conidiophores are indistinguishable from vegetative
Cause of chromoblastomycosis hyphae, except that conidia are clustered at ends
No shoes, trauma Similar morphology to E. jeanselmei
60 CHAPTER 2 Mycology, Virology, and Parasitology

Acremonium Species Microsporum


Etiologic agent of mycetomas, corneal infections, and Macroconida numerous, thick-walled, rough
nail infections Microconida usually present
See Opportunistic Fungi section Epidermophyton
Macroconida numerous, thin and thick
walled, smooth
Dermatophytes Microconida not formed
Dermatophytosis: Infections of keratinized tissue (hair, Microsporum audouinii
skin, nails) Anthropophilic: Person to person
Most common: Ringworm Children
Three major genera Positive Woods lamp fluorescence
Trichophyton Rare distorted macroconidia, rare microconidia
Microsporum Light-tan front
Epidermophyton Reverse salmon to colorless
Intermediate to slow growers No growth on sterile rice media
Worldwide distribution Microsporum canis
Routes of infection Zoophilic
Defined in three ways Woods lamp fluorescence
Geophilic: Soil to man Skin and hair
Zoophilic: Animal to human Bright yellow colony reverse
Anthropophilic: Person to person Especially on PDA
Approximately 43 accepted species Large spindle-shaped macroconidia
Types of infections 3 to 15 cells, tapering ends
Tinea barbae: Facial hair Many microconidia
Tinea capitis: Scalp Microsporum gypseum
Tinea corporis: Arms, legs, and trunk Geophilic
Tinea cruris: Jock itch affects the groin area Rapid grower
Tinea faciei: Face Not commonly infective
Tinea manuum: Hands Skin and hair
Tinea pedis: Athletes foot Powdery/granular buff-to-brown colony
Tinea unguinum: Fingernails and toenails Rowboat-shaped macroconidia
Hair and hair follicles Six or fewer septa
Favic: Hair follicle, crusty lesions Microsporum nanum
Ectothrix: Colonizes outside of shaft Zoophilic
Endothrix: Hair follicle first, growth Flat beige, brown, or white colony
inside shaft Small macroconidia
Nail and nail bed One or two septa
Onychomycosis Rare cause of tinea corporis in humans
Skin Epidermophyton floccosum
Laboratory diagnosis Anthropophilic
Specimens: Hair, skin scraping, nail scraping or Tinea cruris (+ pedis, + unguium)
clipping Does not infect hair
KOH preparation (+ calcofluor) No microconidia
Hair infections Smooth-walled, club-shaped, groupings of macroconi-
Endothrix dia (beavers tail)
Ectothrix Colony: Khaki-yellow
Skin and nail infections Trichophyton mentagrophytes
Septate hyphae Anthropophilic and zoophilic
Woods lamp Infects all three keratinized tissues
Culture Most common cause of athletes foot
SDA with and without inhibitory agents Buff and powdery to white, cottony colony
30 C for 4 weeks Reverse side may be yellow, brown, colorless,
Colony morphology or red
Microconidia and macroconidia Spiral hyphae
Trichophyton Round clustering microconidia and cigar-shaped
Macroconida rare, thin-walled, smooth macroconidia
Microconida numerous Urease positive, perforates hair
CHAPTER 2 Mycology, Virology, and Parasitology 61

Trichophyton rubrum Clusters of blastoconidia along pseudohyphae, ter-


Anthropophilic: Person to person minal chlamydoconidia
Ectothrix, if infect hair C. albicans CMT morphology
Most commonly infects skin and nails Germ tube positive, sucrose positive
White fluffy colony C. stellatoidea (sucrose negative)
Reverse side red Macroscopic morphology
Tear-drop shaped microconidia Candida tropicalis
Can produce macroconidia Second most common Candida spp.
Urease negative, does not perforate hair Vaginitis, intestinal disease, systemic infections,
Trichophyton tonsurans meningitis
Anthropophilic Infections are aggressive and very difficult to treat with
Endothrix traditional antifungals
Most common endothrix dermatophyte in the Macroscopic
United States Creamy, glabrous with mycelial fringe
Black dot tinea capitis Microscopic
Beige-to-olive granular colony with brown rust edge Blastoconidia are single or small random clusters
Size and shape variation in microconidia along pseudohyphae
Requires thiamine C. tropicalis CMT morphology
Trichophyton schoenleinii Candida parapsilosis
Anthropophilic Major cause of nosocomial infections
Slow growing Indwelling catheter
Endothrix Macroscopic
Colonies orange/brown and wrinkled when young, flat Creamy, glabrous
when mature Microscopic
No macroconidia, rare microconidia Relatively short, crooked or curved pseudohyphae
Antler-shaped hyphae C. parapsilosis CMT morphology
Candida kreusi
Rarely isolated as a cause of endocarditis and vaginitis
THE CLINICALLY SIGNIFICANT YEASTS Macroscopic
Significant part of the normal flora Creamy, flat colonies
Skin and mucous membranes Microscopic
Infections are often endogenous Pseudohyphae and elongated blastoconidia,
Opportunists branch like trees
Greater immune suppression results in a greater vari- C. krusei CMT morphology
ety of yeast infections Torulopsis glabrata
Yeasts are most frequently isolated fungi Also referred to as Candida glabrata
Most commonly found as fungemia
Endocarditis, meningitis, UTI
Candida Species Macroscopic
Common normal flora of skin, mucosa, and Creamy, smooth, moist
digestive tract Microscopic
Can cause many infections Blastoconidia only (on CMT), no pseudohyphae
Vulvovaginitis, thrush, pulmonary infections, eye Saccharomyces cerevisiae
infections, meningitis, endocarditis, and dissemi- The working yeast
nated infections Bread, beer, wine
Opportunist Can occasionally be normal flora
Causative agent of thrush Increasingly isolated from immunocompromised
Indicator of immunosuppression Macroscopic
HIV, prolonged antimicrobial therapy, and chemo- Creamy, smooth, moist
therapy: Can be serious and become disseminated Microscopic
Candida albicans Yeast cells and short pseudohyphae
Most common cause of yeast infection Cryptococcus Species
Can cause disease in any site when host defense is Causative agent of meningitis and pulmonary
altered disease
Macroscopic C. neoformans
Creamy Major cause of opportunistic infection in patients
Microscopic with AIDS
62 CHAPTER 2 Mycology, Virology, and Parasitology

Found in soil contaminated with pigeon excreta Pneumocystis jiroveci


Meningitis: Predilection for central nervous system Group is contested
All species are surrounded by a capsule Yeast-protozoa-fungus
Gives the mucoid colony appearance Opportunistic
India ink detects capsule: Negative stain AIDS
Being replaced by latex agglutination for cryptococ- Cellular immunity
cal antigen Pneumocystis pneumonia
India ink has low detection rate Fever, nonproductive cough, shortness of breath
Do not produce true hyphae or pseudohyphae on corn- Destroys alveolar cells
meal agar, blastoconidia only Laboratory diagnosis
All species are urease positive Must demonstrate the organism in tissue, lavage, or
Nitrate variable sputum
Phenol oxidase Cannot culture except in animal
C. neoformans GMS commonly used stain
Causes melanin production on caffeic acid agar or Deflated ball
bird seed agar Fluorescent antibody available
Dark colony color
Sugar assimilation also varies
Rhodotorula Species
DIMORPHIC FUNGI
Bright, salmon-colored colonies Mycoses that involve major body systems or more
Closely related to Cryptococcus than one kind of tissue
Capsule production Some include the opportunists in this category
Urease positive These do not need situational help
Some are nitrate positive Thermal dimorphism
Not common agents of disease Blastomyces dermatitidis
Do cause some opportunistic infections Coccidioides immitis
Geotrichum candidum Histoplasma capsulatum
Normal flora in intestinal tract Paracoccidioides brasiliensis
Causes rare infections in immunocompromised Sporothrix schenckii
Macroscopic Penicillium marneffei
White, moist, yeast-like Clinical disease
Microscopic Cause multiple kinds of infections, varying
True hyphae, segment into arthroconidia, no severities
blastoconidia Primary infection is pulmonary
Trichosporon beigelii Incidence of benign infection is far greater than
Cause of white piedra fatal disseminated disease
Personal hygiene disease Immunocompetent: Asymptomatic and resolves
Emerging agent of disseminated infection spontaneously
Mostly in cancer patients Common cold or flu symptoms
Produces arthroconidia and blastoconidia on Can progress to acute or chronic disease
cornmeal agar Granulomatous lesions in lungs
Malassezia furfur Granuloma: Collection of macrophages, giant
Normal skin flora in 90% of humans cells, and proteinaceous material (wall off
Tinea versicolor infection)
Catheter-related infections in patients on long-term Yeast forms can enter lymph through macrophage
intravenous lipids and disseminate to other organ systems
Macroscopic Disseminated infections are fast moving and
Cream/brown wrinkled normally fatal
Microscopic Specimen processing
Yeastlike cells Tissue specimens: Minced
Sporobolomyces Species Pleural fluid and CSF: Concentrated
Most often recovered from environmental samples Mucus or pus: Mucolytic agent
Rare cause of infection in immunocompromised patients When dimorph is suspected
Macroscopic Quick transport
Salmon-colored smooth colonies Do not hold at room temperature (bacteria may
Microscopic overgrow)
Oval, elongate yeast cells, projectile spores Do not refrigerate
CHAPTER 2 Mycology, Virology, and Parasitology 63

Microscopic Laboratory identification


KOH preparation: Easiest, quickest Specimen sources
Calcofluor Sputum
H&E, PAS, and GMS for in situ tissue staining Aspirated pus from lymph node and subcutane-
Culture media ous tissue
Primary isolation Skin scrapings and biopsies
SDA or SABHI with and without Blood, urine, CSF (systemic)
antimicrobials Collection and handling
Incubation at 30 C Aseptic, plated promptly
BHI is sometimes recommended for better recov- Macroscopic morphology
ery on primary culture 25 C: Slow growth of mold on SDA, white-to-
PDA for subculture beige waxy colony
Laboratory 37 C: Yeast appears after 10 to 15 days, on
Risk factors enriched media
CAN CAUSE LABORATORY-ACQUIRED Microscopic morphology
INFECTIONS Mold: Fine, septate, hyaline hyphae
Extreme caution must be taken when these Conidia directly on hyphae or on lateral
organisms are handled conidiophores
Transmitted by respiratory route Yeast: Hyaline, large cells, budding
All cultures handled in biosafety hood
Slide cultures should not be performed
Diagnosis: Immunologic methods
Coccidioides immitis
Most are not strong antigens Endemic in hot, semiarid climates
Cellular not humoral responses Southwestern United States and northern Mexico
Antibody detection Valley fever
Positive: Exposure (not necessarily infection) Saprobe in mold form (desert soil)
Those in endemic area are positive Small threat to immunocompetent
Most patients with AIDS patients will be Occupational hazard
negative (no Ab) Most virulent of all agents of human mycoses
Laboratory identification Causes mild infection in everyone who inhales it
Exoantigen test Clinical disease
Detects cell free extracts of the fungus Primary pulmonary coccidiodomycosis
Material extracted from mold phase is reacted Asymptomatic and self-limiting
with known antisera Disseminated rate in immunocompromised much
More definitive identification than colony higher than that of other fungal agents
morphology alone Specimen sources
Molecular methods Sputum
Skin scraping
Blood, urine
Blastomyces dermatitidis Laboratory identification
Endemic to North America Macroscopic morphology
Mississippi River valley 3 to 5 days on SDA/SABHI
Gilchrists disease Arthroconidia in 7 to 10 days
Most likely a soil saprobe Colonies are white and cottony (cobwebs)
Found in wood, tree bark, rotting vegetation, Yeast form not found in laboratory
river banks Spherules can be experimentally formed
Biggest threat to immunocompromised Microscopic morphology
12 cases of laboratory-acquired disease Septate hyaline hyphae
Blastomycosis Wide arthroconidia: Barrel shaped
Chronic granulomatous disease affecting lungs, Disjuncture cells
skin, and mucous membranes Cultures are extremely hazardous because of
Chronic cutaneous blastomycosis many arthroconidia
Ulcerated lesions
Exposed or mucocutaneous tissues
Systemic blastomycosis Histoplasma capsulatum
Involves any organ: Bone lesions and osteomye- Worldwide
litis are often encountered Endemic to Mississippi River and Ohio River valleys
64 CHAPTER 2 Mycology, Virology, and Parasitology

Soil saprobe with high nitrogen content (chicken, bird, Laboratory identification
and bat guano) Specimen sources
Spelunkers disease Exudates and pus from lesions
Mostly occupational hazard Tissue biopsy
Clinical disease Macroscopic
Histoplasmosis Mold in 3 to 5 days at 25 C
Chronic granulomatous lung disease Mature colonies are dark and flat
5% progress to an acute fulminating, rapidly Yeast at 37 C (white or tan)
fatal disease (mostly in children) Microscopic
Organism found in macrophages Mold: Delicate thin hyphae, septate, frequently
Patients with AIDS are at high risk found as ropes, conidiophores produce multiple
First found in histiocytes (histo) conidia in flowerets arrangements
No actual capsule (capsulatum) Two types of conidia
Laboratory identification Small oval, unicellular conidia
Macroscopic Large, dark walled spheres
Slow growing mold Yeast: Cigar shaped at 37 C
Tan, fluffy colonies
Yeast form in 10 to 15 days on enriched media FUNGUS-LIKE BACTERIA
Microscopic
Fine septate hyphae, microconidia and Actinomycetes
macroconidia Three major genera
Macroconidia become tuberculate with age Actinomyces, Nocardia, and Streptomyces
Yeast cells bud at narrow neck Others: Rhodococcus, Actinomadura, and
Nocardiopsis
All higher bacteria:
Paracoccidioides brasiliensis Thought to be fungi for years
Endemic to northwest, central, and southern South Some species form aerial mycelia in culture
Clinical manifestations are similar to those of systemic
America, Central America, and southern Mexico
Soil saprobe of acid soil fungal infection
Causes paracoccidioidomycosis Actinomyces are anaerobic, Nocardia and Streptomy-
Asymptomatic and self-limiting ces are aerobic
Can disseminate to other tissues Nocardia stain partially acid-fast, Actinomyces and
Can cause cutaneous disease Streptomyces are not acid-fast
Specimen: Same All genera may produce granules, Actinomyces almost
Laboratory identification always produce granules
Macroscopic
Mold colony mature in 2 to 3 weeks Actinomyces Species
Flat, white colonies Gram-positive obligate anaerobes
Yeast will form on enriched media at 37 C Reside in the mouth and in the intestinal tract
Microscopic Form abscesses and swelling at site of infection
Fine, septate, hyaline hyphae Diagnosis can be made by direct microscopy
Conidiation absent on modified SDA Yellow sulfur granules: Bacterium and its waste
Yeast form: Multiple thin-necked buds (mariner Actinomyces israelii (most common), but several other
wheel) bacteria in this genus are capable of causing disease

Sporothrix schenckii Nocardia Species


Found worldwide (soil saprobe) Nocardiosis
Sometimes grouped with subcutaneous mycoses Ubiquitous soil saprophytes
Organism is also a thermal dimorph Route: Inhalation, direct inoculation
Occupational risks Nocardia asteroides most common
Gardening: Rose gardener disease Nocardia brasiliensis
Clinical disease Most important in tropical areas
Sporotrichosis Cutaneous infection with normal immune function
Chronic cutaneous and subcutaneous mycosis 70% of cases are seen in immunocompromised
characterized by ulcers and abscesses along lym- Nocardiosis
phatic channels Generally: Immunocompromised population
CHAPTER 2 Mycology, Virology, and Parasitology 65

Nocardia may colonize the respiratory tract Sometimes partially acid-fast


Immunocompetent individuals with compro- Does not hydrolyze
mised pulmonary function Casein, xanthine, tyrosine
Pneumonia can disseminate No branching on tap water agar
Kidney, skin, gastrointestinal (GI) tract, and
brain are common targets
Laboratory identification Streptomyces Species
Gram-variable/modified acid-fast bacilli positive Streptomyces griseus (found in soil)
Strictly aerobic Musty smell
Filamentous and branching Nonpathogenic
May be isolated on routine media Forms colony in 3 to 5 days at 35 C
Colonies usually form within 4 days Waxy, white powdery top
May require up to 2 to 4 weeks Gram-positive filamentous bacilli
Nocardia spp. can be difficult to isolate by culture Nonacid-fast
Faster growing organisms may overgrow Aerial, tertiary branching on tap water agar
Colony morphology Hydrolyzes casein, xanthine, and tyrosine
Colonies smooth and moist or have a moldlike,
gray-white, waxy or powdery appearance
Distinct, strong mildew odor Actinomadura Species
Microscopic Eight reported species, two more common
Usually gram-variable or beaded appearance Actinomadura madurae
Alternating gram-positive and gram-negative Actinomadura pelletieri
segments along a filament Causes mycetoma
Nocardia under suboptimal conditions appears uni- Found only in tropics
formly gram-negative Gram-positive filamentous bacilli
Modified Ziehl-Neelsen or Kinyoun acid-fast stain Nonacid-fast
Nocardia organisms are acid-fast with these Aerial, tertiary branching on tap water agar
modified staining procedures Hydrolyzes casein and tyrosine
Tests based on acid-fastness alone are not reliable
for differentiation
Differentiation of Nocardia spp. Nocardiopsis Species
Tap water agar morphology Soil saprophyte
Differentiate Nocardia spp. and other aerobic Similar to Streptomyces and Actinomadura
actinomycetes Thermotolerant
Nocardia spp. have recursively branching Grows at higher temperatures
hyphae with aerial hyphae Very rare case of mycetoma
Biochemical characteristics
Hydrolysis of casein, tyrosine, or xanthine
N. asteroides
VIROLOGY
Does not hydrolyze casein, xanthine, or tyrosine
N. brasiliensis Properties of viruses
Hydrolyzes casein and tyrosine Small
Molecular identification DNA or RNA, not both
Replicate or multiply on their own
Intracellular
Rhodococcus equi Replication is directed by viral nucleic acid
Soil saprophyte Lack genes and enzymes necessary for energy
Associated with domestic farm animals production
Causes pulmonary infection that resembles tuberculosis They depend on the machinery of the host cell for
Pneumonia that spreads to brain, liver, spleen protein and nucleic acid production
Opportunistic Some are capable of inducing cancerous growth in
AIDS, transplants, Hodgkins lymphoma, lym- animals and culture
phoma, and leukemia Hepatitis B: Liver cancer
Laboratory identification Characteristics of a typical virion
Colony forms in 2 to 4 days Either DNA or RNA, single or double stranded
Glistening, smooth, pink to red In contrast to eukaryotes and prokaryotes,
Gram-positive coccobacillus viruses do not contain both
66 CHAPTER 2 Mycology, Virology, and Parasitology

A capsid or protein coat Clinical Virology: Culture Method


Enveloped viruses have an outer envelope com-
1937: Propagated yellow fever virus in chick embryos
posed of lipids and polysaccharides
Successfully produced an attenuated vaccine
Important definitions
Capsid: Protein shell Influenza vaccine still produced in eggs
Nucleocapsid: Nucleic acid + capsid Growth of viruses
Capsomeres: Structural units of capsid Chicken embryos: Historical
Envelope: Lipid membrane around nucleocapsid in Tissue explants: Research use only
Cell culture
some viruses
Stolen from cell: Essential for infectivity Primary cell culture
Virion: Complete virus particle Diploid cell lines
Characteristics used to classify viruses Continuous cell lines
Type of genetic material (either DNA or RNA) they Primary cell cultures
Derived directly from donor (animal or human)
contain
Size and shape of the assembled virus Most common are kidney cells
Presence or absence of an envelope Rhesus monkey, rabbit kidney
Type of host that it infects One or two passages
Type of disease produced Diploid cell line
Prepared from animal tissues
Taxonomy/nomenclature
International Committee on Taxonomy of Viruses Usually fibroblasts from lung or foreskin
Terminally differentiated, postmitotic
divides all viruses to families (-viridae)
Subfamilies (-virinae) Limited to 20 to 50 passages
Genera (-virus) Continuous cell lines
Species or virus name Single cell type that can be propagated indefinitely
Above the family level, orders (-virales) may be used Do not resemble the cell of origin
http://www.virustaxonomyonline.com Often abnormal in chromosome morphology
Other viruslike things and number
Prion Derived from tumors or mutagenic treatment of pri-
Proteinaceous infectious particle mary cell culture
Unlimited passages
Structures that replicate through conversion of
Care and feeding of cells
other host proteins
Cells are usually in tubes or in shell vials
Exact mechanisms of action and reproduction
are unknown Flasks are used for some applications
In tubes, the cells are on the down side
Transmissible spongiform encephalopathy
Cells need to be fed or refed with appropriate
Scrapie
Kuru medium
Tubes and vials are read using an inverted
Creutzfeldt-Jakob disease (CJD)
Bovine spongiform encephalopathy (BSE): microscope
Cytopathic effects (CPEs)
Mad cow disease
CPEs can take a variety of forms
Replication of virus particles
Attachment Rounding up and detachment
Specific cell receptor Cell lysis
Responsible for varying cell tropism Swelling of nuclei
Penetration Formation of fused cells termed syncytia
Induction time varies among viral agents
Virus passing through cell membrane
Hemadsorption
May take cell membrane as protection
Uncoating Certain viruses produce hemagglutinin
Hemagglutinin binds erythrocytes
Removes all or part of the capsid
Exposes the nucleic acid Human type O
Biosynthesis Chicken
Proteins, nucleic acids, and other components Guinea pig
Hemadsorption performed on cell cultures from
Some made in tremendous excess
Morphogenesis respiratory specimens
Fluorescent antibody tests
Components assembled
Used for confirmation of CPEs
Often uses enzymes encoded by virus
Release Some can be used on patient samples
Examples include
Budding through membrane
Lysis of membrane Cytomegalovirus (CMV)
CHAPTER 2 Mycology, Virology, and Parasitology 67

Respiratory viruses Vesicle fluid


Herpes simplex virus (HSV) HSV, varicella-zoster virus (VZV)
Enterovirus groups Skin scrapings: Papillomavirus, molluscum
Shell vial cultures contagiosum
Cells grown on coverslips in the bottom of a vial Disadvantages with electron microscopy
(shell vial) Expensive equipment
Patient specimens added to vial and centrifuged to Expensive maintenance
drive the virus into the cell Require experienced observer
Incubated and stained Sensitivity often low
Time to positive usually much less than with con- Nucleic acid amplification
ventional tube cultures Allows the amplification of specific target DNA
Used for several different agents sequences by a factor of approximately 106
CMV PCR is the most common method but other varia-
Herpes simplex and varicella-zoster tions are being developed and used
Respiratory viruses Detection of the PCR product usually has been by
Chlamydia agarose gel electrophoresis, probe hybridization,
Some systems contain mixed cell types or DNA sequencing
Real-time PCR has streamlined the amplification
and the detection processes
Advantages of PCR
Clinical Virology: Nonculture Methods Extremely high sensitivity
Direct examination Fast turnaround time for real-time PCR
Antigen detection Nucleic acid amplification
Immunofluorescence, enzyme-linked immuno- Disadvantages of PCR
sorbent assay (ELISA), etc. Extremely liable to contamination
Electron microscopy High degree of operator skill required
Morphology of virus particles Not easy to set up a quantitative assay
Immune electron microscopy A positive result may be difficult to interpret
Light microscopy Serologic tests for viral infections
Histologic appearance Classic techniques
Inclusion bodies Complement fixation tests
Limited to pathology Hemagglutination inhibition tests
Antigen detection Immunofluorescence techniques
Enzyme immunoassay (EIA) Counter-immunoelectrophoresis
Plate-based assays Older techniques
Lateral flow technology Radioimmunoassay
Nasopharyngeal aspirate Newer techniques
Respiratory syncytial virus (RSV) Enzyme-linked immune assay (EIA)
Influenza Particle agglutination
Stool Western blot
Rotavirus Recombinant immunoblot assay
Adenovirus Lateral flow rapid devices
Advantages and disadvantages Serology
Advantages Primary infection
Result available quickly, point-of-care testing Fourfold rise titer of immunoglobulin G (IgG) or
Potential problems total antibody between acute and convalescent
Reduced sensitivity compared to cell culture or sera
PCR: 40% to 80% Presence of IgM
Labor intensive A single high titer of IgG
Not well suited to a core laboratory Reinfection
Electron microscopy Fourfold or greater rise in titer of IgG or total
106 virus particles/mL required for visualization antibody between acute and convalescent sera
Approximately 50,000 to 60,000 magnification Absence or slight increase in IgM
normally used Use of serologic results
Original method to find many viruses Disease dependent
Viruses may be detected in the following specimens Rubella and hepatitis A
Feces: Rotavirus, adenovirus, noroviruses, astro- Clinical symptoms coincide with antibodies
virus, calicivirus Detection of IgM or rise in IgG: Disease
68 CHAPTER 2 Mycology, Virology, and Parasitology

Respiratory and diarrhea viruses may cause dis-


T A B L E 2- 2 Medically Important Viruses
ease before antibody rise
RSV or influenza Groups Virus Families
Serologic diagnosis would be retrospective
Major groups of DNA viruses Adenoviridae
HIV produces clinical disease months or years Hepadnaviridae
after seroconversion Herpesviridae
Antibody: Definitive diagnosis Papillomaviridae
Some infections can be detected only by serology Parvoviridae
Bartonella Polyomaviridae
Limitations of serology Poxviridae
Long period of time required using paired sera Major groups of RNA viruses Arenaviridae
Extensive antigenic cross-reactivity Astroviridae
HSV and VZV Bunyaviridae
Japanese B encephalitis and dengue Caliciviridae
Coronaviridae
CMV and Epstein-Barr virus
Filoviridae
Immunocompromised patients often have reduced
Flaviviridae
or absent immune response Orthomyxoviridae
Late AIDS Paramyxoviridae
Patients with infectious mononucleosis or diseases Picornaviridae
such as systemic lupus erythromatosus may react Reoviridae
nonspecifically Retroviridae
Transfusion may give a false positive result because Rhabdoviridae
of the transfer of antibody Togaviridae
Guidelines for selecting and collecting specimens for
virology
Culture only infected sites Polyomaviridae
Collect and send tissue or fluid Poxviridae
Do not use swabs Adenoviruses
Send fluid in its original container/syringe Double-stranded DNA (dsDNA), replicate in nucleus
Collect and send as much specimen as possible Icosahedral, nonenveloped
Tools of the trade 51 serotypes
Swabs Causes respiratory disease, eye infections, and GI
Swab for skin lesion disease
Nasopharyngeal swabs from the nasopharynx Can cause cancer in animals
All must go into viral transport medium Hepadnavirus
Viral/chlamydial culture transport media dsDNA with a short single-stranded region
Supplied by the laboratory Icosahedral core with envelope
Usually stored refrigerated Human: Hepatitis B
Inoculate and send on ice Two important antigens
Nasal wash kit Surface Ag
Sterile saline included Core Ag
Use for rapid virus tests and nucleic acid Virus not isolated in culture
amplification test Serologic test
Use for culture by transfer to vial transport Hepatitis
medium or directly Cirrhosis
Specimen labeling Hepatocelluar carcinoma
The specimen must be labeled accurately and Transmission through blood
completely Transfusion
Indicate exactly what the specimen is Drug abuse
Sexual contact
Vaccine
Major Groups of DNA Viruses Engineered
Adenoviridae Infants at birth, health care workers
Hepadnaviridae Family Herpesvidae
Herpesviridae Herpesviruses
Papillomaviridae dsDNA, replicate in nucleus
Parvoviridae Icosahedral nucleocapsid, envelope
CHAPTER 2 Mycology, Virology, and Parasitology 69

120- to 200-nm diameter Transient in normal individuals


Most prominent feature: Latency In immunocompromised
Herpes simplex virus Can be severe
Two types Blood hemoglobin decreases and cannot recover
Cold sores Not isolated in culture
Genital Detected by molecular assays
Skin and mucous membrane infections Poxviruses
Encephalitis dsDNA, replicate in cytoplasm of cell
Isolated easily in culture Largest viruses (400  250 nm)
Varicella-Zoster Virus Complex construction
Chickenpox and shingles Brick shape, lipid envelope
Vaccine important in controlling outbreaks Resistant to environmental factors
Shingles return Smallpox: Variola
Tzanck stain: Giant cells Eradicated by the World Health Organization
Isolation more difficult than HSV Stocks in Atlanta and Moscow
Cytomegalovirus Good vaccine and no animal reservoir
Isolated from blood, urine, throat Vaccinia
In adults: Syndrome similar to mononucleosis, may Used to immunize against smallpox
infect kidney (shed in urine) Rare zoonotic
In immunocompromised: Kidney, eye, lung, often fatal Africa
Laboratory tests Others
Shell vial culture
Serology
Epstein-Barr Virus
Major Groups of RNA Viruses
Heterophile-positive infectious mononucleosis (85%) Arenaviridae
Can produce tumors Astroviridae
Not isolated in culture Bunyaviridae
Serologic diagnosis Caliciviridae
Early antigen Coronaviridae
Viral capsid antigen: IgM and IgG Filoviridae
Nuclear antigen Flaviviridae
Human Herpesvirus 6, 7, and 8 Orthomyxoviridae
Human herpesvirus6 Paramyxoviridae
Exanthema subitum/roseola infantum Picornaviridae
Sixth disease Reoviridae
Spread by respiratory route Retroviridae
Molecular assays used for detection Rhabdoviridae
Human herpesvirus7 Togaviridae
Cause a small percentage of roseola Arenaviruses
Human herpesvirus8 ssRNA, enveloped
Found in Kaposis sarcoma Helical capsid symmetry
Papillomavirus Lymphocytic choriomeningitis virus
dsDNA Benign aseptic meningitis
Over 100 types of human (HPV) Lassa fever virus: Africa
Infect skin and mucous membranes Junin and Machupo viruses: South America
Causes Rodent reservoir, greater than 50% mortality
Warts Biosafety level 4 containment
Cervical cancer Bunyaviruses
Laryngeal carcinoma ssRNA, enveloped
No culture: Molecular assays are used Helical capsid symmetry
Parvoviruses La Crosse encephalitis virus
Single-stranded DNA (ssDNA), nucleus replication Mouse host, mosquito vector
Small nonenveloped Encephalitis
Human parvovirus B-19 Hantaviruses
Erythema infectiosum (fifth disease) Mouse host
Infects bone marrow cells (erythrocyte) Respiratory infection
Causes aplastic crisis Not isolated in the laboratory
70 CHAPTER 2 Mycology, Virology, and Parasitology

Calicivirus Can be isolated in culture


ssRNA, nonenveloped Subtle CPE
Icosahedral symmetry Hemadsorption
Family Caliciviridae Paramyxoviruses
Sapporo Helical and enveloped, larger than myxoviruses
Norovirus Have only one long ssRNA genome
Prototype strain is Norwalk virus No reassortment
Outbreaks of diarrhea Replicate in both the nucleus and cytoplasm
Genetically and antigenically diverse Five genera: Mumps, parainfluenza 1 to 4, measles,
Noncultivatable RSV, metapneumovirus
Coronaviruses Parainfluenza virus
ssRNA, enveloped Four antigenic types
Pleomorphic/spherical capsid Respiratory infections
Large club-shaped spikes on surface gives corona Isolated from throat
effect Grow in cell culture
Filoviruses Hemadsorption for identification
ssRNA Measles virus
Helical symmetry, long and slender One serologic type
Marburg and Ebola viruses Maculopapular rash, fever, respiratory disease
Monkey reservoir but transmitted to humans Can be isolated in culture
More than 80% mortality Hemadsorption for identification
Pan-organ effects Mumps virus
Contact with blood Infects parotid salivary glands
Biosafety level 4 containment Can infect testis, ovaries, kidneys
Flaviviruses Isolated from throat swab or urine
ssRNA, enveloped Identified by hemadsorption
Icosahedral symmetry RSV
St. Louis encephalitis, West Nile virus, yellow fever, Bronchiolitis, pneumonia in infants
dengue, hepatitis C Labile virus
Not isolated by culture Produces typical CPE
Hepatitis C Virus Monoclonal antibody to confirm
Blood or sexual contact Rapid testing available
No other vector Metapneumovirus
Chronic liver infection Acute respiratory tract infections worldwide in
Serology most common diagnosis children and adults
Screen with EIA Annual epidemics in winter and spring
Western blot confirmation months
Also detected in blood by molecular assays Two distinct human metapneumovirus groups
Orthomyxoviruses with subgroups
ssRNA: Eight segments PCR for diagnosis, for now
Helical symmetry capsid, enveloped Picornaviruses
Replicate in cytoplasm ssRNA
Influenza viruses Very small: Approximately 27 nm
Two important surface Ag No envelope, icosahedral symmetry
Neuraminidase Replicate in cytoplasm of cell
Hemagglutinin Enteroviruses
Segmented genomes: Heavy reassortment Fecal-oral transmission
Antigenic shift: Large Polioviruses: 3 types
Antigenic drift: Small Coxsackievirus A: 24 types
Animal strains Coxsackievirus B: 6 types
Three antigenic groups Echoviruses: 34 types
A, B, C Many cultivatable
Severe in elderly, immunocompromised Rhinoviruses
Pandemics Common cold virus
1918 More than 100 serotypes
Avian flu Can be isolated in culture
Swine flu Acid-sensitive
CHAPTER 2 Mycology, Virology, and Parasitology 71

Limited to upper respiratory tract Can be isolated in culture and mice


Limited growth at 37 C Diagnosis by immunofluorescence of brain tissue
Hepatitis A Virus Togaviruses
25% of hepatitis ssRNA
Usually fecal-oral transmission Enveloped, icosahedral symmetry
Food Family Togaviridae
Water Genus alphavirus: Arboviruses
Needles Genus rubivirus: Rubella
No growth in cell culture Arboviruses
Serology for diagnosis Eastern and Western equine encephalitis
Reovirus Bird reservoir
Respiratory enteric orphan Mosquito vectors
dsRNA, 60 to 80 nm Symptoms include fever, encephalitis, rash
Nonenveloped, icosahedral symmetry Cell culture possible, but serology most commonly used
Replicates in the cytoplasm Rubella virus
Rotaviruses Transmitted by droplets
Fecal-oral Infection in children mild
Causes infantile diarrhea Congenital disease serious
Not isolated in laboratory, ELISA Mother contracts rubella in first trimester
At least six serotypes Vaccine developed for children because of congenital
Retroviruses disease
ssRNA (may have two copies) Cell culture possible but serology most common
Enveloped, icosahedral symmetry
All have reverse transcriptase
DNA made from RNA
Integrates into genome
PARASITOLOGY
Many can cause tumors in animals Techniques
Replicate in nucleus and cytoplasm Specimen collection and processing
HIV 1 and 2, human T-lymphotropic virus 1 and 2 Stool: Routine is three specimens, every other
HIV day within 10 days
100 to 400 nm, cylindrical or conical core Clean, watertight container with a tight lid
Two broad types: 1 and 2 5 g, not contaminated with water, urine, bar-
Several subtypes in type 1 ium, or other substances
Type 1, clade (subtype) B in United States Liquid or near-liquid specimens examined
Type 2 mainly in Africa within 30 minutes of collection to preserve
Does not form tumors motile trophozoites
Infects CD4 + cells Soft specimens examined within 60 minutes of
Lymphocytes, macrophage, brain cells, and collection
dendritic cells Formed specimens processed within 24 hours,
Destroys immune system may be refrigerated
Characteristic secondary diseases Fixatives: Two-vial system, usually formalin
Pneumocystis pneumonia, CMV, Kaposis and polyvinyl alcohol (PVA) or one-vial sys-
sarcoma tem of sodium acetate formalin (SAF)
ELISA and Western blot analysis 3 parts fixative to 1 part stool
Rhabdoviruses 5% to 10% formalin, concentration
ssRNA, bullet shaped, enveloped methods, and iodine-stained mounts
Replicate in cytoplasm PVA, concentration methods, and tri-
Vesicular stomatitis virus of horses chrome and other permanent stains
Rabies virus SAF: Concentration methods and perma-
One serologic type nent stains
Encephalitis PVA contains mercury, SAF is a mercury-
Bite of infected animal free alternative
Travels up sensory nerves to the central nervous Concentration methods
system (CNS) Fresh or preserved stools
Incubation of 2 to 16 weeks Sedimentation: Formalin-ethyl acetate
Allows time for vaccine Floatation, zinc sulfate with specific grav-
100% fatal if untreated ity of 1.18 to 1.20
72 CHAPTER 2 Mycology, Virology, and Parasitology

Macroscopic Major Medically Important


Consistency TABLE 2-3 Parasitescontd
Appearance, color
Contaminants Classification Genus and Species
Larva, proglottids Protozoa: Coccidia Microsporidia spp.
Microscopic Isospora belli
Calibrated ocular micrometer Cyclospora cayetanensis
Direct saline wet mount for motile Cryptosporidium parvum
trophozoites Protozoa: Misc Toxoplasma gondii
Iodine-stained wet mount from processed Blastocystis hominis
specimen Cestodes: Tapeworms Diphyllobothrium latum
Permanent stains from processed specimen Hymenolepis nana
Trichrome: Cytoplasm is blue-green, purple; Hymenolepis diminuta
Taenia spp.
nuclear structures, red to pink
Taenia saginata
Iron hematoxylin: Primarily for intestinal pro-
Taenia solium
tozoa, cytoplasm is blue to purple, nuclear Dipylidium caninum
structures are blue to black Echinococcus granulosus
Modified acid-fast: For coccidian protozoa, Trematodes: Flukes Clonorchis sinensis
oocysts are red Fasciolopsis buski
Other specimen types Fasciola hepatica
Specimen of choice depending on organism in ques- Heterophyes heterophyes
tion and clinical situation Metagonimus yokogawa
Duodenal contents Paragonimus westermani
Sigmoidoscopy specimens Schistosoma haematobium
Enterotest Schistosoma japonicum
Schistosoma mansoni
Blood
Blood parasites: Babesia Babesia microti in United
Urine
States
CSF and other fluids Babesia divergens in Europe
Sputum Blood parasites: Malaria Plasmodium falciparum
Tissue specimens Plasmodium malariae
Plasmodium ovale
Plasmodium vivax
Plasmodium knowlesi
Blood parasites: Filariae Brugia malayi
T A B L E 2- 3 Major Medically Important Parasites Loa loa
Wuchereria bancrofti
Classification Genus and Species Mansonella ozzardi
Nematodes: Intestinal Ascaris lumbricoides Onchocerca volvulus
Enterobius vermicularis Blood parasites: Leishmania braziliensis
Necator americanus HemoflagellatesLeishmania Leishmania donovani complex
Ancylostoma duodenale Leishmania tropica complex
Strongyloides stercoralis Leishmania mexicana complex
Trichuris trichiura Blood parasites: Trypanosoma cruzi
Nematodes: Nonintestinal Dracunculus medinensis Hemoflagellates Trypanosoma brucei
Trichinella spiralis Trypanosomes rhodesiense
Protozoa: Amoeba Entamoeba histolytica Trypanosoma brucei
Entamoeba coli gambiense
Entamoeba hartmanni Arthropods Pediculus humanus humanus
Endolimax nana Pediculus humanus capitis
Iodamoeba butschlii Phthirus pubis
Acanthamoeba spp. Ixodes scapularis
Naeglaria fowleri Dermacentor andersoni
Protozoa: Flagellates Giardia lamblia Dermacentor variabilis
Trichomonas vaginalis Ornithodoros spp.
Chilomastix mesnili Cimex lectularius
Trichomonas hominis Ctenocephalides canis
Dientamoeba fragilis Ctenocephalides felis
Protozoa: Ciliate Balantidium coli Sarcoptes scabei
Continued
CHAPTER 2 Mycology, Virology, and Parasitology 73

Nematodes Adult larvae 9 to 12 mm, hook at end of tail; male


possesses copulatory bursa
Intestinal Filariform larvae penetrate skin, lymphatic system, to
Ascaris lumbricoides bloodstream, to lung; penetrate alveoli, coughed up,
Roundworm swallowed; migrate to large intestine
Worldwide; most common intestinal helminth infection Buccal cavity has cutting plates
Ova in stool Asymptomatic if light infection; GI symptoms, ane-
Ova are the infective stage mia, weight loss, breathing difficulty, bloody sputum,
Ova are diagnostic cough; GI symptoms more severe if worm burden
Ova 85 to 95  38 to 45 mm. Corticated or decorti- heavy; eosinophilia
cated. Unfertilized ovoid, 40 to 74  30 to 50 mm Repeated infection can cause dermal irritation,
Embryonate in soil, resist environmental conditions ground itch
Adult larva largest intestinal nematode, 22 to 35 cm Ova considered to be indistinguishable from those of
in length Ancyclostoma duodenale
Larva emerge in small intestine, migration to blood- Ancyclostoma duodenale
stream, liver, lung, to pharynx; swallowed, return to Old World hookworm
intestine Europe, Far East, Asia, Africa typically
GI symptoms, fever, pulmonary or asymptomatic, Ova considered to be indistinguishable from those of
eosinophilia N. americanus, although A. duodenale ova are 55 to
250,000 ova per day, so worm burden can be high 60  40 mm
Enterobius vermicularis Buccal cavity has teeth
Pinworm Other details are same as for N. americanus
Worldwide; most common intestinal helminth infec- Strongyloides stercoralis
tion in the United States Threadworm
Scotch tape preparation is specimen of choice, ova or Worldwide
adult larva Larva in stool at rhabditiform stage are the diagnostic
Ova are infective stage stage, ova rarely seen
Ova and larva are the diagnostic stages Infection caused by third-stage filariform larval pene-
Ova 48 to 60  20 to 35 mm; oval, thick shell; flat on tration of skin, typically the foot
one side; developing larva folded inside Ova 48  35 mm, advanced cleavage state, indistin-
Adult larva female 7 to 14 mm, male 2 to 4 mm, white guishable from hookworm
to light yellow Rhabditiform larvae 15  220 mm, short buccal cavity
Hatch in small intestine, adults in colon; migrate to and prominent genital primordium
anus to deposit ova Filariform larvae is third stage, long esophagus,
Severe anal itching, inflammation notched tail
Ova infective in 4 to 6 hours, deposit in clothes, bed Adult female 2 mm, short buccal cavity, long esophagus
linens, toys Unique lifecycle, three mechanisms
Highly communicable Direct: Same as that of hookworm
Retroinfection: Ova hatch in anus but migrate back Indirect: Larva freely living in environment, pro-
into colon to reproduce duce infective rhabditiform larva
Autoinfection: Infective ova are ingested, hand Autoinfection: Filariform larva develop in hosts
to mouth intestine, invade bloodstream
Human is only known host Asymptomatic if light infection; GI symptoms,
Ova may carry Dientamoeba fragilis, dual infections malabsorption, weight loss, breathing difficulty,
are seen bloody sputum, cough, eosinophilia
Necator americanus Repeated infection can cause dermal irritation,
New World hookworm ground itch
North and South America typically
Ova are the diagnostic stage, larva can also be found Trichuris trichiura
in stool
Whipworm
Infection caused by third-stage filariform larval pene-
Ova in stool
tration of skin, typically the foot
Ova are infective stage
Ova 60 to 75  40 mm, cell cleavage can be seen,
Ova are diagnostic
thin shell
Ova 50 to 55  25 mm, barrel shaped with bipolar
Rhabditiform larvae 15  270 mm, long buccal cavity hyaline plugs
and small genital primordium, cutting plates
Adult larva 2 to 5 cm, male smaller with curled tail; pos-
Filariform larvae is third stage, short esophagus, terior end is large, resembles a whip handle; anterior end
pointed tail is smaller, resembles whip; can be found in stool
74 CHAPTER 2 Mycology, Virology, and Parasitology

Larva emerge in small intestine, migrate to cecum Asymptomatic carrier state or can cause dysentery,
then to colon which can be severe, and abscesses in liver, spleen,
Asymptomatic with light infection; heavier worm bur- lung, brain
den includes GI symptoms, weight loss, weakness, Entamoeba coli
eosinophilia; childrens symptoms can include GI, Commensal
anemia, and, if untreated, prolapsed rectum Cysts or trophozoites in stool are diagnostic
Cysts: 10 to 30 mm, round, one to eight nuclei, more
Nonintestinal
than four to differentiate from Entamoeba histolytica,
Trichinella spiralis
eccentric karyosome, uneven peripheral chromatin,
Trichina worm
splintered end chromatoid bar
Worldwide
Trophozoites: 15 to 50 mm, slow motility, one nucleus
Laboratory diagnosis
with eccentric karyosome, coarse cytoplasm
Histologic preparation of encysted tissue, typically
Entamoeba hartmanni
skeletal muscle Commensal
Serologic methods
Cysts or trophozoites in stool are diagnostic
Elevated muscle enzymes
Cysts: Less than 10 mm, resemble those of E.
Ingesting undercooked contaminated meat: Pork, deer,
histolytica
bear, walrus
Trophozoites: Less than 12 mm, resemble those of E.
Encysted larva 100  5 mm, coil in cyst in muscle
Ingestion of infected meat, larva excysts, and develops histolytica
Endolimax nana
in intestine; adult female deposits larva, which migrate
Commensal
through bloodstream to skeletal muscle and encyst,
Cysts or trophozoites in stool are diagnostic
which ends lifecycle
Cysts: 5 to 10 mm, one to four nuclei with blot-like
Asymptomatic or flulike symptoms; heavier infec-
karyosome
tion includes GI symptoms, weakness, fever, pain, Trophozoites: 5 to 12 mm, one nucleus with blot-like
edema, muscular pain; can be fatal during migra-
karyosome
tory phase
Iodamoeba butschlii
Dracunculus medinensis
Commensal
Guinea worm
Cysts or trophozoites in stool are diagnostic
Africa, India, Asia, Middle East
Cysts: 5 to 20 mm, one nucleus, large glycogen vacuole
Laboratory diagnosis
Trophozoites: 8 to 20 mm, one nucleus
Examination of emerging larva from the skin ulcer
Ingestion of infected copepods; larva emerge in intes- Acanthamoeba Species
Pathogen
tine of host, develop and migrate to connective tissue
Granulomatous amoebic encephalitis (GAE)
and body cavities, and to subcutaneous tissues, where
Worldwide
adult female deposits larva; ulcer forms, from which Trophozoites and cysts in CSF, in brain tissue, or at
larva can emerge
Allergic reaction, skin ulcer autopsy
Trophozoites: 12-45 mm, one nucleus, slow motility
Cysts: 8-25 mm, double cell wall, one nucleus
Environmental organism, enters nasal passages,
Protozoa bloodstream to CNS; swimming in ponds, lakes in
Amoeba warm months
Entamoeba histolytica GAE: Stiff neck, headaches, seizures, can progress
Pathogenic rapidly
Amoebic dysentery, amoebic abscess Eye involvement, contact lens fluid contamination,
Worldwide, a leading cause of parasitic death keratitis
Cysts are infective form Keratitis: Ocular pain, vision impairment
Cysts or trophozoites in stool are diagnostic, or amoe- Naeglaria fowleri
bic abscess fluid or sigmoidoscopy specimen Pathogen
Cysts: 10 to 20 mm, round, one to four nuclei, central Worldwide
karyosome, fine and even peripheral chromatin, Primary amoebic meningoencephalitis (PAM)
rounded chromatoid bar; young cysts can contain PAM: Stiff neck, headaches, seizures, can progress
glycogen vacuole rapidly
Trophozoites: 12 to 60 mm, rapid and directional Trophozoites in CSF, in brain tissue, or at autopsy
motion, one nucleus with central karyosome; ingested Trophozoites: 8-25 mm, one nucleus, slow motility
red blood cells (RBCs) are diagnostic Cysts: No known cyst form
CHAPTER 2 Mycology, Virology, and Parasitology 75

Environmental organism, enters nasal passages, then Asymptomatic in men or urethritis, vaginitis with yel-
bloodstream to CNS; swimming in ponds, lakes in low discharge
warm months
Ciliate
Flagellates Balantidium coli
Giardia lamblia Pathogen
Pathogenic Worldwide
Worldwide Cysts or trophozoites in stool are diagnostic
Giardiasis, travelers diarrhea, Cysts: 50 to 75 mm, round, two nuclei, small micronu-
Worldwide, natural water sources cleus, large kidney beanshaped macronucleus,
Cysts are infective form cytosome, cilia
Cysts or trophozoites in stool are diagnostic or on sig- Trophozoites: 50 to 100 mm, round, two nuclei, small
moidoscopy specimen, shed irregularly micronucleus, large kidney beanshaped macronu-
Immunologic methods, PCR cleus, cytosome, cilia
Cysts: 8 to 14 mm, one to four nuclei, four axonemes, Asymptomatic or GI symptoms, mild dysentery,
four median bodies, oval abscesses in intestinal mucosa, anemia
Trophozoites: 9 to 21 mm, two nuclei, two axonemes,
eight flagella, sucking disk, tear-drop shape Coccidia
Asymptomatic or GI symptoms, can be severe, light- Cryptosporidium parvum
colored stools, incubation period is 10 to 36 days, Pathogen
can be self-limiting Worldwide
Dientamoeba fragilis Oocyst is infective stage
Pathogenic Oocysts in stool are diagnostic, immunologic
Thought to be worldwide methods, modified acid-fast stain
Trophozoite is infective form Oocysts: 4 to 6 mm, round, no sporocysts
Trophozoites in stool are diagnostic Food-borne and water-borne illness
Cysts: No known cyst form Mild GI symptoms, self-limiting, in compromised
Trophozoites: 7 to 12 mm, one or two nuclei, 80% patients more severe, can be fatal
binucleate Isospora belli
Asymptomatic or GI symptoms, mild dysentery Pathogen
Possible association with Enterobius vermicularis Worldwide
infection Oocyst is infective stage
Chilomastix mesnili Oocysts in stool are diagnostic, immunologic
Commensal methods, modified acid-fast stain
Worldwide Oocysts: 30  12 mm, ovoid, one or two sporocysts,
Cysts and trophozoites in stool are diagnostic thin shell
Cysts: 5 to 10 mm, lemon shape, one nucleus, cytosome Human is definitive host
with fibrils Asymptomatic or GI symptoms, mild, self-limiting
Trophozoites: 10 to 20 mm, one nucleus, four flagella, Cyclospora cayetanensis
cytosome with fibrils Pathogen
Trichomonas hominis Thought to be worldwide
Commensal Oocysts in stool are diagnostic, modified acid-
Thought to be worldwide fast stain
Trophozoites in stool are diagnostic Oocyst is infective form
Cysts: No known cyst form Oocysts: 7 to 10 mm, round, two sporocysts
Trophozoites: 7 to 15 mm, one nucleus, four flagella, Linked to water-borne and food-borne illness
undulating membrane, axostyle Mild GI symptoms
Trichomonas vaginalis Microsporidia Species
Pathogenic Pathogen
Worldwide Thought to be worldwide
Trophozoite is infective form Spores in stool are diagnostic, modified acid-fast stain,
Trophozoites in urine, vaginal secretions, urethral serologic methods
secretions are diagnostic Spore is infective form
Cysts: No known cyst form Spores: 1 to 5 mm, round, two sporocysts
Trophozoites: 5 to 15 mm, one or two nuclei, four fla- Enterocytozoon bieneusi is most commonly isolated
gella, undulating membrane, axostyle, jerky motility species, seen in patients with HIV
in fresh specimens Mild GI symptoms, peritonitis, hepatitis
76 CHAPTER 2 Mycology, Virology, and Parasitology

Miscellaneous Protozoa Ova are infective stage


Blastocystis hominis Ova in stool are diagnostic
Pathogen although pathogenicity is unclear Ova: 45  40 mm, typically round, hexacanth embryo
Thought to be worldwide with three pairs of hooklets, shell with bipolar thicken-
Cyst is infective stage ings and filaments in clear embryophore
Cysts in stool are diagnostic, immunologic methods Scolex: Four suckers, short rostellum with hooks
Cysts: 8 to 30 mm, round, central vacuole surrounded Proglottids: Rectangular
by several peripheral nuclei Ingestion of infective ova. Cysticercoid larva develop
Mild GI symptoms in intestine, scolex emerges and attaches to intestinal
Toxoplasma gondii mucosa and further develops. Ova from the adult larva
Pathogen can pass in feces as an infective ovum or autoreinfect
Toxoplasmosis the human host
Worldwide, many animals harbor the organism No intermediate host required
Oocyst is infective stage Can be asymptomatic or GI symptoms, weight loss,
Immunologic methods abdominal pain
Oocysts: 10 to 15 mm, oval, two sporocysts Hymenolepis diminuta
Tachyzoites: 5  3 mm, crescent shape, one central Rat tapeworm
nucleus Worldwide
Bradyzoites: 5  3 mm, crescent shape, one central Ova are infective stage
nucleus, form a packet in host cell that contains many Ova in stool are diagnostic
bradyzoites Ova: 55  85 mm, typically round, hexacanth embryo
Cat is definitive host, humans are accidental hosts with three pairs of hooklets, shell with bipolar thicken-
by four mechanisms: Ingest oocyst from cat feces; ings but no filaments in clear embryophore
ingest contaminated meat from cattle, pig, sheep; Scolex: Four suckers, small projecting rostellum
transplacental to fetus; blood transfusion without hooks
Asymptomatic or flulike symptoms in mild cases, Proglottids: Rectangular
but can be chronic; congenital results in fetal death, Human is accidental host, primary host is rat. Inges-
mental retardation, blindness, severe brain damage, tion of infective ova. Cysticercoid larva develop in
or neonate is asymptomatic at birth intestine, scolex emerges and attaches to intestinal
mucosa and further develops. Ova from the adult larva
can pass in feces as an infective ovum or autoreinfect
Cestodes: Tapeworms the human host
Diphyllobothrium latum No intermediate host required
Broadfish tapeworm Can be asymptomatic or GI symptoms, weight loss,
Great Lakes area, Alaska, South America, Asia, Africa, abdominal pain
Scandinavia Taenia Species
Ova are infective stage Worldwide
Ova in stool are diagnostic, more rarely proglottids Two species
Ova: 55 to 75  40 to 55 mm, operculated, abopercular Ova indistinguishable, 35  25 mm, round, hexacanth
knob, not a hexacanth embryo, coracidium sur- embryo with three pairs of hooklets, surrounded
rounded by dark shell by striated embryophore, sunburst appearance,
Scolex: Almond shaped with two long sucking grooves nonoperculated
Proglottids wider than long, central uterine structure Ova in stool are diagnostic, more rarely proglottids
Two intermediate hosts. Infection caused by ingestion Infection caused by ingesting undercooked beef or
of pleurocercoid in undercooked or raw fish. Scolex pork that contains cysticercus larva, larva emerge in
emerges and attaches to intestinal mucosa. Ova passed small intestine where scolex attaches to intestinal
in stool, further develops if deposited in water. Coraci- mucosa
dium hatches, larva ingested by copepod. Develops Taenia saginata
to procercoid in copepod, which is ingested by fresh- Beef tapeworm
water fish; procercoid develops to pleurocercoid larva Scolex: 1 to 2 mm, four suckers
in fish Proglottids: greater than 1000, rectangular, 15 to 30
Can be asymptomatic or GI symptoms, weight loss, uterine branches
abdominal pain, vitamin B12 deficiency, pernicious Taenia solium
anemia Pork tapeworm
Hymenolepis nana Scolex: 1 to 2 mm, four suckers, rostellum, and hooks
Dwarf tapeworm Proglottids: less than 1000, square, 7 to 15 uterine
Worldwide, most common tapeworm in United States branches
CHAPTER 2 Mycology, Virology, and Parasitology 77

Can cause cysticercosis after ingesting infective ovum. Ova 30  15 mm with miracidium, shoulders are large,
Onchosphere migrates to organs, muscle; can invade operculated, terminal knob
brain, potentially fatal Metacercariae mature in liver, migrate to bile duct
Dipylidium caninum Can be asymptomatic or GI symptoms, weight loss,
Dog or cat tapeworm abdominal pain
Worldwide Fasciolopsis buski
Egg packets in stool, or proglottids are diagnostic Large intestinal fluke
Larva are infective stage Far East, India, Indonesia
Egg packets each containing 10 to 30 ova, ova Ova in stool
40  60 mm, oncosphere with three pairs of hooklets Ova are infective stage
Scolex: Four suckers, small rostellum with several cir- Ova in the stool are diagnostic
cles of spines Ova 128 to 140  80 mm, oval, contain miracidium,
Proglottids: Seed shaped operculated
Human is accidental host, primary host is dog or Adult larva: 5  1.5 cm, ovoid
cat. Intermediate host is flea ingestion of larval stage, Ingestion of infected water plants; miracidium
develops to adult larva. Egg packets or proglottids develops, adult resides in small intestine of host
passed in stool. Cycle continues if these are ingested Abdominal pain, GI symptoms, jaundice, malabsorp-
by flea tion syndrome, intestinal obstruction in severe cases
Flea is intermediate host Fasciola hepatica
Can be asymptomatic or GI symptoms, weight loss, Sheep liver fluke
abdominal pain Worldwide, areas cattle and sheep ranching
Echinococcus granulosus Ova are infective stage
Hydatid tapeworm, or dog tapeworm Ova in stool are diagnostic
West and southwest United States and Alaska, South Ova 128 to 150  80 mm, oval, contain miracidium,
America, Africa, Asia, Australia, Middle East, areas operculated
where dogs and sheep or cattle coexist Adult larva: 3  1 cm, ovoid, possesses shoulders that
Laboratory diagnosis by examining hydatid cyst fluid, distinguish it from F. buski
hydatid sand under microscope, scolices floating in Human is accidental host, sheep definitive host. Inges-
fluid; serological methods tion of infected water plants, miracidium develops,
Ova are not the diagnostic phase but are identical to adult resides in bile ducts of host
ova of Taenia spp. Abdominal pain in liver area, GI symptoms, jaundice
Hydatid cyst consists of daughter cysts surrounded by Heterophyes heterophyes
a capsule. Brood capsules can form in the germinal Heterophid fluke
layer of the cyst. Daughter cysts and brood capsule Africa, Far East, Near East, Egypt
contain scolices, which can develop into adult larva Ova are infective stage
Scolex: Four suckers and hooks Ova in the stool are diagnostic
Larva: 5 mm, scolex, neck, and three proglottids Ova 15 to 30 mm, oval, flask shaped, contain miracid-
Human is accidental intermediate and end host. Inges- ium, operculated, small shoulders, thick shell, can lack
tion of infected ova. Larvae penetrate intestinal mucosa terminal knob
and migrate to organs, usually lung, liver. Hydatid cyst Ova of Heterophyes heterophyes, C. sinensis, and
develops in the organ. Dog is definitive host, sheep Metagonimus yokogawa very similar
intermediate host. Cysts form in sheep, dog ingests Adult larva: 1  0.5 mm, pyriform, spines cover outside
infected sheep viscera. Cyst develops into adult larva, Ingestion of undercooked fish, miracidium develops,
which reside in dogs intestine, ova passed in feces adults reside in small intestine
Symptoms depend on location and size of cyst. Asymp- Asymptomatic or heavier infections cause GI symp-
tomatic until cyst enlarges. Pain in area. Can be fatal if toms, abdominal pain, eosinophilia
cyst ruptures, because fluid can cause anaphylactic Metagonimus yokogawa
shock. New cyst can form from ruptured cyst if scolex Heterophid fluke
is extruded Far East, Europe, Siberia
Ova are infective stage
Trematodes: Flukes Ova in stool are diagnostic
Clonorchis sinensis Ova 15 to 30 mm, oval, flask shaped, contain miracid-
Chinese liver fluke ium, operculated, small shoulders, thin shell, can lack
Far East terminal knob
Ingesting undercooked fish containing infective Adult larva: 1  1.5 mm, piriform, spines cover outside
metacercariae Ingestion of undercooked fish; miracidium develops,
Ova in stool are diagnostic adults reside in small intestine
78 CHAPTER 2 Mycology, Virology, and Parasitology

Asymptomatic or heavier infections cause GI symp- Schistosoma mansoni


toms, abdominal pain, eosinophilia Mansons blood fluke
Paragonimus westermanni Central and South America, Puerto Rico, West Indies
Oriental lung fluke Ova in stool or rectal biopsy
Asia, Africa, India, South America Cercariae penetrate skin to infect human host
Ova are infective stage Ova 100 to 185  40 to 75 mm, oblong, contain devel-
Ova in stool or in sputum are diagnostic oped miracidium, large lateral spine
Ova 78 to 120  45 to 60 mm, oval, contain undeve- Adult larva: 2 cm, oblong, male and female organisms
loped miracidium, thin shell, operculated with shoul- Cercariae penetrate skin to infect human host, migrate
ders, thickening at terminal end to bloodstream to develop. Adult resides in blood ves-
Adult larva: 1  0.5 mm, oval, spines sels around intestinal tract. Ova from adult females are
Ingestion of undercooked crayfish or crab. Developing excreted in urine. If deposited in water, miracidium
larva migrate to small intestine, into peritoneal cavity, infects snail where it develops into cercariae
into diaphragm, then to lung tissue Snail is intermediate host
Pulmonary symptoms, cough, bloody sputum, eosino- Reservoir hosts include cattle, sheep, dog, cat, mon-
philia, other symptoms if larvae migrate to other keys, rodents
organs Asymptomatic or skin irritation at penetration site,
Schistosoma haematobium swimmers itch, abdominal pain, cough, fever,
Bladder fluke eosinophilia
Africa, Middle East, Iran, Iraq, Saudi Arabia
Ova in concentrated urine sample, immunodiagnostic
methods
Cercariae penetrate skin to infect human host Blood Parasites
Ova 110 to 170  40 to 70 mm, oblong, contain devel- Babesia
oped miracidium, large terminal spine Babesiosis
Adult larva: 2 cm, oblong, male and female Babesia microti in United States
organisms Babesia divergens in Europe
Cercariae penetrate skin to infect human host, migrate Giemsa-stained thick and thin blood smears, serologic,
to bloodstream to develop. Adult resides in blood ves- PCR, blood smears collected at the patients bedside
sels around urinary bladder. Ova from adult females Small delicate ring form trophozoites, 1 to 2 mm
are excreted in urine. If deposited in water, miracidium Rings in single, double, or classic tetrad
infects snail, where it develops into cercariae Tetrad: Maltese cross
Snail is intermediate host Fever, chills, sweating, myalgias, fatigue, hepatosple-
Reservoir hosts include cattle, sheep, dog, cat, mon- nomegaly, and hemolytic anemia
keys, rodents Malaria
Asymptomatic, skin irritation at penetration site, Tropical and subtropical worldwide
swimmers itch, abdominal pain, cough, fever, eosino- Five Plasmodium spp. infect humans
philia, painful urination, hematuria Lifecycle summary
Schistosoma japonicum Sporozoites injected during mosquito feeding
Blood fluke Invade liver cells
Far East Liver replication ! merozoites
Ova in stool or rectal biopsy Merozoites invade RBCs
Cercariae penetrate skin to infect human host Repeated erythrocytic schizogony
Ova 50 to 85  40 to 60 mm, slightly oblong, contain Gametocytes infect mosquito
developed miracidium, small lateral spine Fusion of gametes in gut
Adult larva: 2 cm, oblong, male and female organisms Sporogony on gut wall sporozoites invade salivary
Cercariae penetrate skin to infect human host, migrate glands
to bloodstream to develop. Adult resides in blood ves- Giemsa-stained thick and thin blood smears, serologic,
sels around intestinal tract. Ova from adult females are PCR; blood smears are collected at the patients
excreted in urine. If deposited in water, miracidium bedside.
infects snail, where it develops into cercariae Anopheles mosquito is vector
Snail is intermediate host Plasmodium falciparum
Reservoir hosts include cattle, sheep, dog, cat, mon- RBC normal size
keys, rodents Schuffners stippling: No
Asymptomatic or skin irritation at penetration site, Merozoites in schizonts: 8 to 36, average 24
swimmers itch, abdominal pain, cough, fever, Ring forms single or double chromatin dots
eosinophilia Multiple ring forms: Common
CHAPTER 2 Mycology, Virology, and Parasitology 79

Banana-shaped gametocyte Onchocerca volvulus


Accole forms Blinding filaria, river blindness
Plasmodium vivax Tropical Africa and Central America
RBC normal size Giemsa-stained skin snips
Schuffners stippling: Yes No periodicity
Merozoites in schizonts: 12 to 24, average 16 Simulium blackfly is vector
Ring forms single chromatin dot No sheath, no nuclei in tail tip
Multiple ring forms: Occasional Subcutaneous fibrous nodules
Ameboid trophozoites Blindness if eye is affected
Plasmodium malariae Mansonella ozzardi
RBC normal size New World filaria
Schuffners stippling: No North, Central, and South America
Merozoites in schizonts: 6 to 12, average 8, rosette Giemsa-stained blood
Single chromatin dot No periodicity
Multiple ring forms: Rare Simulium blackfly is vector, or Culicoides midge
Band form trophozoite No sheath, nuclei in tail but not to tip
Plasmodium ovale Asymptomatic, lymphadenopathy
RBC oval Blindness if eye is affected
Schuffners stippling: Yes Hemoflagellates
Merozoites in schizonts: 4 to 12, average 8 Leishmaniae
Single chromatin dot Leishmania braziliensis
Multiple ring forms: Occasional Mucocutaneous leishmaniasis
Plasmodium knowlesi Mexico, Central and South America
Simian malaria form thought to be rarely found in Giemsa-stained slides of the affected body sites,
humans amastigotes
Forms resemble P. falciparum or P. malariae Sandfly is vector, bite transfers promastigotes to the
Filariae human host, promastigotes migrate to reticuloendo-
Brugia malayi thelial cells and develop to amastigotes
Malayan filaria Lesions in mucocutaneous tissues
Tropical and subtropical worldwide Can be self-limiting
Giemsa-stained blood, Knotts technique Leishmania donovani Complex
Nocturnal periodicity Visceral leishmaniasis, dumdum fever, kala-azar
Aedes, Anopheles, Mansonia mosquitos are vectors, Africa, India, Middle East, Far East
intermediate host Giemsa-stained slides of the affected body sites, amas-
Sheath, two nuclei in tail tip tigotes, serologic methods, Montenegro screening
Asymptomatic for months, years; granulomatous skin test
lesions, fever, chills, lymphadenopathy Affects visceral tissue
Elephantiasis Sandfly is vector, incubation period of weeks to months
Loa loa Flulike symptoms resembling malariae, GI symptoms,
Eyeworm abdominal pain, hepatosplenomegaly
Africa Darkening of the skin, black fever or kala-azar
Giemsa-stained blood, Knotts technique Can be fatal
Diurnal periodicity Leishmania mexicana Complex
Chrysops fly is vector New World cutaneous leishmaniasis
Sheath, continuous nuclei in tail tip Central and South America, Mexico
Inflammation at bite site, Calabar swellings at any Giemsa-stained slides of the lesions, amastigotes
body site Affects skin
Wuchereria bancrofti Sandfly is vector
Bancrofts filaria Skin lesion, ulcer
Tropical and subtropical worldwide Can be self-limiting
Giemsa-stained blood, Knotts technique Leishmania tropica Complex
Nocturnal periodicity Old World cutaneous leishmaniasis, Baghdad boil,
Aedes, Anopheles, Culex mosquito are vectors, Delhi boil
intermediate host Middle East, Northern Africa
Sheath, no nuclei in tail tip Giemsa-stained slides of the lesions or fluid, amasti-
Fever, chills, lymphadenopathy gotes, serologic testing
Elephantiasis Affects skin
80 CHAPTER 2 Mycology, Virology, and Parasitology

Sandfly is vector Vector for typhus (Rickettsia prowazekii), trench fever


Skin lesion, ulcer (Bartonella quintana), and relapsing fever (Borrelia
Can be self-limiting recurrentis)
Trypanosomes Spread from human to human
Trypanosoma brucei gambiense Body lice usually on the body and head
West African sleeping sickness Crab lice usually in pubic region, spread to the arm-
West and Central Africa pits, facial hair, eyebrows, and eyelashes
Giemsa-stained slides blood or lymph nodes, CSF Pediculus humanus humanus
studies Body louse
Tsetse fly is vector; bite transfers trypomastigotes to Pediculus humanus capitis
the human host, migrate to lymphatic system, eventu- Head louse
ally to CNS Phthirus pubis
Asymptomatic for a period. Chancre develops at bite Crab or pubic louse
site, flulike symptoms, rash, lymphadenopathy, Win- Ticks
terbottoms sign in neck area. Kerandels sign can Hard Ticks
develop, delayed sensation to pain. CNS symptoms Ixodes scapularis
in final stages, coma, and death Deer tick
Trypanosoma brucei rhodesiense Main vector of Lyme disease
East African sleeping sickness Ixodes pacificus in the U.S. West Coast states also able
East and Central Africa to transmit Lyme disease
Giemsa-stained slides of blood, CSF studies Dermacentor andersoni
Tsetse fly is vector; bite transfers trypomastigotes to Rocky Mountain wood tick, western United States
the human host, which migrate to lymphatic system, Vector of many diseases, including Rocky Mountain
eventually to CNS spotted fever, tularemia, Colorado tick fever, and
Virulent. Asymptomatic for a short period. CNS Q fever
involvement early, weight loss, lethargy, confusion, Dermacentor variabilis
Winterbottoms sign in neck area may be present. In American dog tick, eastern United States
final stages, glomerulonephritis, myocarditis, coma, Soft Ticks
and death Ornithodoros spp.
Trypanosoma cruzi Parasitize mammals
Chagas disease Transmit relapsing fever
Central and South America, Mexico, southern United Fleas
States Cat flea: Ctenocephalides felis
Giemsa-stained slides to blood, serologic methods Dog flea: C. canis can be found on cats and dogs
Reduviid bug is vector; defecates near bite, transfers Serve as intermediate host for tapeworms
trypomastigotes to the human host. Amastigotes and Feed on humans as well as pets
trypomastigotes facilitate cell damage throughout Cause a localized skin reaction
body. Liver, brain, and heart muscle involved Mites
Can also be transmitted by blood transfusion, placenta Sarcoptes scabei is the cause of scabies worldwide
Chagas can be asymptomatic, chronic, or acute. Cha- Transmitted by contact
goma develops at bite site, typically face. Eye area Organisms burrow into the skin on the webbing
swelling is Romanas sign. Acute flulike symptoms. side of fingers, later spreading to the wrists, elbows
Chronic disease early or years later. Myocarditis, and beyond
mega colon, hepatosplenomegaly, brain damage, Bedbugs
death True insect
Children most at risk Cimex lectularius
Arthropods Preferential feeding host is human
Nocturnal blood meals
Symptoms occur days after bite
Ectoparasites and Vectors of Disease Mosquitos
Lice, ticks, fleas, mites, bedbugs, mosquitos True insect
Structures used in arthropod identification Blood meals
Body parts, legs, wings, antenna, mouth parts Transmit malariae, filariasis, dengue fever, yellow
Lice fever, West Nile virus
Lice occur worldwide and in all socioeconomic classes Species include Culex, Anopheles, Aedes, Mansonia
CHAPTER 2 Mycology, Virology, and Parasitology 81

a. Sporangium
CERTIFICATION PREPARATION QUESTIONS b. Blastoconidia
For answers and rationales, please see Appendix A. c. Ascospores
1. Which of the following terms is best described as the d. Conidiophore
process of reproduction in yeast that begins with a 6. A patient with a Woods lamppositive, dermatophytic
weakening and outpouching of the yeast cell wall infection has a skin scraping taken for culture. The
and then formation of a cell wall septum between organism grows on SDA agar with a light-tan front
the mother and daughter yeast cells? and salmon-colored reverse. Microscopically the
a. Binary fission organism produces rare distorted macroconidia and
b. Unisexual division rare microconidia. Additionally, there was no growth
c. Budding on sterile rice media. What is the most likely organism?
d. Outpouch germing a. Microsporum canis
2. The loose intertwined network of basic structural b. Microsporum gypseum
units of the molds that penetrates the substrate from c. Microsporum audouinii
which it obtains the necessary nutrients for growth is d. Epidermophyton floccosum
called which of the following? 7. A KOH preparation of respiratory secretions of a 78-
a. Hyphae year-old man reveals large, spherical, thick-walled
b. Germ tubes yeast cells 8 to 15 mm in diameter, usually with a single
c. Pseudohyphae bud that is connected to the parent cell by a broad base.
d. Mycelium Which fungus will likely be isolated from the culture?
3. The term hyaline molds is used to describe which of a. Coccidioides immitis
the following? b. Blastomyces dermatitidis
a. Molds that have septate hyphae c. Histoplasma capsulatum
b. Molds that have septate, nonpigmented hyphae d. Paracoccidioides brasiliensis
c. The presence of pigmentation within the hyphae 8. Which of the following is a key characteristic of Coc-
or the spores cidioides immitis?
d. Molds with intercalated hyaline chlamydoconidia a. Has a higher dissemination rate in white females
4. Large, usually multiseptate and club-shaped or spindle- b. Is endemic in the northeastern United States
shaped spores are called which of the following? c. Produces endosporulating spherules in tissue
a. Microconidia d. Forms foot cells
b. Macroconidia 9. Using PAS to stain a respiratory specimen from a
c. Conidiophores patient with lung disease, the technologists observed
d. Phialides the organisms in the image. Based on the microscopic
5. A Scotch tape preparation is made from a mold morphology shown in the image, the most likely iden-
growing on solid media in the mycology laboratory. tification of the dimorphic fungi is which of the
The structure shown in the image is best described as following?
which of the following?

FIGURE 2-2 (Courtesy Joel Mortensen, PhD. See also color


plate 9.)

a. Blastomyces dermatitidis
FIGURE 2-1 (Courtesy Joel Mortensen, PhD. See also color plate 8.) b. Coccidioides immitis
82 CHAPTER 2 Mycology, Virology, and Parasitology

c. Histoplasma capsulatum 14. An immunocompromised patient exhibited fever,


d. Sporothrix schenckii nonproductive cough, and shortness of breath. Rou-
10. A landscaper noticed a hard, unmovable lump under tine and fungal cultures did not grow. The respira-
the skin of his index finger but decided to ignore it. A tory specimen was stained with a silver stain in
month later, the lump ulcerated to present a necrotic anatomic pathology. Based on the microscopic mor-
appearance, and two more lesions developed further phology in the image, what is the most likely identi-
up the wrist and forearm. A histologic stain of mate- fication of this organism?
rial from deep in the lesions showed elongated yeast
cells resembling cigars. What disease is suspected?

FIGURE 2-4 (From Public Health Photo Library [PHL 960]. See also
color plate 11.)
FIGURE 2-3 (Courtesy Joel Mortensen, PhD. See also color
plate 10.) a. Pneumocystis jiroveci
b. Saccharomyces sp.
c. Candida albicans
a. Mycetoma d. Cryptococcus sp.
b. Sporotrichosis 15. A significant amount of yeast was isolated from a
c. Chromoblastomycosis vaginal culture of a patient in the teen clinic of your
d. Blastomycosis hospital. It exhibited the following characteristics:
11. A germ tubenegative yeast is isolated in the labora- Microscopic: Clusters of blastoconidia along pseu-
tory. The isolate is found to be negative for urease dohyphae, terminal chlamydoconidia
and unable to assimilate dextrose, maltose, or Positive germ tube
sucrose. CMT agar morphology showed blastoconi- Positive sucrose
dia only. The organism is most likely: Which of the following is the most likely identifica-
a. Candida albicans tion of this fungi? (Image from primary plate,
b. Candida parapsilosis gram-stained smear, 40 .)
c. Torulopsis glabrata
d. Geotrichum candidum
12. Which of the following is a key characteristic by
which an unknown Cryptococcus spp. can be identi-
fied as Cryptococcus neoformans?
a. Appearance of yellow colonies
b. Positive urease test
c. Presence of a capsule
d. Positive niger seed agar test
13. Which of the following statements concerning the
germ tube test is true?
a. Using a heavy inoculum enhances the rapid pro-
duction of germ tubes
b. Germ tubes should be read after 2 hours of incu-
bation at 25 C
c. Candida albicans and Candida tropicalis can be
FIGURE 2-5 (Courtesy Joel Mortensen, PhD. See also color
used as positive and negative controls, plate 12.)
respectively
d. Serum will be stable for 1 year if stored at room a. Rhodotorula rubra
temperature b. Candida albicans
CHAPTER 2 Mycology, Virology, and Parasitology 83

c. Geotrichum candidum 18. A mold isolated in the laboratory displays a white


d. Trichosporon beigelii cottony macroscopic morphology. On microscopic
16. The pharmacy at your hospital was concerned about evaluation, hyaline, septate hyphae, and tooth-
the hyperalimentation fluid they were preparing. The brush-like conidiophres are seen. The most likely
high lipid contact was a concern for contamination. organism is which of the following?
A PAS stain of the suspect fluid is shown. Which of a. Aspergillus sp.
the following organism would most likely demon- b. Acremonium sp.
strate this morphology? c. Gliocladium sp.
d. Scopulariopsis sp.
19. A mold is isolated in the laboratory that displays a
velvety, gray-green colony morphology. On micro-
scopic evaluation, flask-shaped conidiophores
arranged in a brushlike formation are seen. The most
likely organism is which of the following?
a. Penicillium sp.
b. Acremonium sp.
c. Paecilomyces sp.
d. Scopulariopsis sp.
20. A patient who underwent solid organ transplant
appears to have systemic fungemia. The organism
that has grown from the blood cultures macroscopi-
cally had a blue-green color to the colony, matured in
3 days, and grew well at 45 C. Microscopically, foot
FIGURE 2-6 (Courtesy Joel Mortensen, PhD. See also color cells were seen and the phialides were uniserate with
plate 13.) a round vesicle and columnar conidia. Which of the
following is the most likely identification of this
mold?
a. Candida albicans
b. Malassezia furfur
c. Trichosporon cutaneum
d. Scedosporium apiospermum
17. Several important types of conidiation of dematiac-
eous fungi exist. The image is an example of which
one of these forms? (Lactophenol cotton blue stain.)

FIGURE 2-8 (Courtesy Joel Mortensen, PhD. See also color


plate 15.)

a. Aspergillus fumigatus
b. Aspergillus niger
c. Scopulariopsis sp.
d. Fusarium sp.
21. The protein coat that surrounds the nucleic acid of a
virion is called which of the following?
a. Capsomere
FIGURE 2-7 (Courtesy Joel Mortensen, PhD. See also color b. Capsid
plate 14.)
c. Capsule
d. Nucleocapsid
a. Cladosporium type 22. During viral assembly, how are viral envelopes
b. Phialophora type acquired?
c. Rhinocladiella type a. By production of envelope constituents by host
d. Rinderpest type cellular DNA
84 CHAPTER 2 Mycology, Virology, and Parasitology

b. As the virion buds from a host cell membrane


c. Through replication of viral nucleic acid
d. As host cell lysis produces many membrane
fragments
23. Prions are best described by which of the
following?
a. Infectious viral RNA without capsid proteins
b. Infectious protein with no associated nucleic
acid
c. Infectious viral DNA without capsid proteins
d. Nonenveloped virus highly resistant to heat and
chemical inactivation
24. The viral nucleocapsid always contains which of the
following?
a. Viral genome
b. Virus-encoded glycoprotein
c. Virus-encoded polymerase FIGURE 2-9 (Courtesy Joel Mortensen, PhD. See also color
plate 16.)
d. Viral envelope
25. Which of the following viruses are thought to
predominately cause gastroenteritis? a. Herpes simplex virus
a. Hepadnaviruses b. Adenovirus
b. Filoviruses c. Cytomegalovirus
c. Noroviruses d. Epstein-Barr virus
d. Arboviruses 31. Trophozoite forms of amoebae are found in what
26. Which of the following groups contains the SARS type of stool specimen?
virus? a. Formed
a. Calicivirus b. Loose
b. Coronavirus c. Soft
c. Flavivirus d. Watery
d. Filovirus 32. Which preservation method is most suitable and
27. Which of the following groups of virus is best the most widely used for subsequent fixed smear
described as: preparation?
ssRNA, enveloped, a. Formalin-ethyl acetate
Pleomorphic/spherical capsid b. PVA
Large club-shaped spikes on surface gives corona c. Trichrome
effect d. MIF
Causes approximately 15% of coldlike illness 33. If the ova of this parasite are ingested by humans, the
a. Influenza A oncosphere form can migrate through the body via
b. Influenza B the bloodstream, resulting in the condition known
c. Coronaviruses as cysticercosis. Which of the following is correct?
d. Pneumovirus a. Taenia solium
28. Which of the following is the specimen of choice for b. Entamoeba histolytica
detecting rotavirus? c. Hymenolepis nana
a. Throat swab d. Clonorchis sinensis
b. Urine sample 34. Ova recovered from the stool are routinely used to
c. Bronchoalveolar wash diagnose infections caused by all of the following
d. Feces sample except?
29. The test of choice and most sensitive assay for use a. Necator americanus
with CSF to diagnose aseptic meningitis caused by b. Ascaris lubricoides
enterovirus is which of the following? c. Trichuris trichiura
a. Cell culture d. Strongyloides stercoralis
b. PCR 35. An MLS finds an E. coli cyst on a wet mount of a
c. Antigenemia immunoassay fresh stool specimen. Which of the following should
d. Shell vial assay be done?
30. A specimen from a genital lesion was inoculated into a. Request a second specimen
a standard set of cells for virus isolation. On day 1 the b. Look for additional E. coli cysts
human foreskin fibroblasts exhibited the CPE shown c. Examine the remaining area of the wet preparation
in the figure. d. Generate a final report
CHAPTER 2 Mycology, Virology, and Parasitology 85

36. Which of the following parasites have migration a. Ascaris lumbricoides


through the lungs as part of their lifecycle? b. Ancyclostoma duodenale
a. Necator americanus, Ancylostoma duodenale, c. Necator americanus
Strongyloides stercoralis d. Trichuris trichiura
b. Giardia lamblia, Wuchereria bancrofti, Brugia 39. The eggs in the figure below were found in the urine
malayi of a Peace Corp worker who had just returned to the
c. Enterobius vermicularis, Trichuris trichiura, Tri- United State after spending 2 years in the Middle
chinella spiralis East. The eggs measured about 160 mm long  60 mm
d. Toxocara canis, Toxoplasma gondii, Blastocystis wide. Which of the following organisms is the most
hominis likely identity?
37. The image below is of a suspected parasite seen on
direct examination of material taken from a corneal
scraping in an ophthalmology clinic. The most likely
identification of the parasite in this specimen is
which of the following?

FIGURE 2-12 (Courtesy the Centers for Disease Control and Pre-
vention. See also color plate 19.)

a. Diphyllobothrium latum
b. Schistosoma haematobium
FIGURE 2-10 (Courtesy the Centers for Disease Control and
Prevention. See also color plate 17.) c. Schistosoma japonicum
d. Schistosoma mansoni
a. Acanthamoeba sp. 40. A patient was diagnosed with cysts in his liver. He is
b. Enterobius sp. originally from Australia, where he was involved in
c. Paragonimus sp. a sheep herding operation. The adult parasite shown
d. Naegleria sp. below was passed by his pet dog. It measured 5 mm.
38. The organism shown below was recovered from the What is the most likely identification of this organism?
stool of a patient who resides in rural Texas. The
most likely identification is which of the following?

FIGURE 2-11 (Photograph by Dr. Mae Melvin, courtesy the Cen-


ters for Disease Control and Prevention, Public Health Image Library, FIGURE 2-13 (Courtesy the Centers for Disease Control and Pre-
http://phil.cdc.gov/. See also color plate 18.) vention. See also color plate 20.)
86 CHAPTER 2 Mycology, Virology, and Parasitology

a. Diphyllobothrium latum a. Chilomastix mesnili


b. Dipylidium caninum b. Cyclospora cayetanensis
c. Echinococcus granulosus c. Giardia lamblia
d. Taenia solium d. Iodamoeba butschlii
41. These trophozoites were found in a trichrome- 43. The image below is a cyst found in a human fecal
stained slide of a stool sample, measuring an average smear. The cyst measured about 12 mm in length
of 25 microns in diameter. Which of the following is and contained four nuclei and a rounded chromatoid
the most likely identity of this organism? bar. The patient had severe diarrhea and some blood
in the stool. What is the most likely identification of
this organism?

FIGURE 2-14 (Courtesy the Centers for Disease Control and Pre-
vention. See also color plate 21.)

a. Endolimax nana FIGURE 2-16 (Courtesy the Centers for Disease Control and Pre-
b. Entamoeba coli vention. See also color plate 23.)
c. Balantidium coli
d. Dientamoeba fragilis
42. The cyst in the image below was observed in a stool
sample of a child at a daycare center. The ovoid cyst a. Endolimax nana
measures approximately 10  8 microns. Which b. Entamoeba coli
organism is the most likely cause of the childs c. Entamoeba histolytica
diarrhea? d. Iodamoeba butschlii
44. Match the parasite with the most appropriate
description.
_____ Plasmodium falciparum
_____ Plasmodium malariae
_____ Plasmodium ovale
_____ Plasmodium vivax
a. RBC enlarged, oval, Schuffners dots, gameto-
cytes seen by day 4 to 18
b. Large RBC, troph irregular, multiple phases seen,
gametocytes appear early
c. Delicate ring forms, multiple rings per cell,
crescent-shaped gametocytes after 7 to 10 days
d. RBC normal in size and color, troph compact,
band forms may be seen, gametocytes seen
after weeks
45. Microfilariae found in the blood that have a sheath,
demonstrate nocturnal periodicity and exhibit nuclei
FIGURE 2-15 (Courtesy the Centers for Disease Control and Pre- that do not extend to the tail tip are which of the
vention. See also color plate 22.) following?
CHAPTER 2 Mycology, Virology, and Parasitology 87

a. Blastocystis hominis
b. Cyclospora cayetanensis
c. Isospora belli
d. Balantidium coli
48. Match the scientific name with the corresponding
common name.
_____ Sarcoptes scabei
_____ Ixodes scapularis
_____ P. humanus humanus
_____ Cimex lectularius
a. Body louse
b. Bedbug
c. Scabies
d. Lyme disease
49. The only known human tapeworm with an opercu-
lum is:
FIGURE 2-17 (Courtesy the Centers for Disease Control and Pre-
vention. See also color plate 24.) a. Diphyllobothrium latum
b. Hymenolepis nana
c. Giardia lamblia
a. Brugia malayi d. Schistosoma haematobium
b. Onchocerca volvulus 50. Identify the following organism as it appears in this
c. Loa loa peripheral blood smear.
d. Wuchereria bancrofti
46. Necator americanus rhabditiform larvae can be dif-
ferentiated from Strongyloides stercoralis rhabditi-
form larvae by:
a. Length of the notched tail
b. Length of the head region
c. Segmentation
d. Size of the genital primordium
47. The image below is from a fecal smear of an individ-
ual complaining of diarrhea and intestinal discom-
fort. The parasites were numerous and quite
variable in size, but the majority measured about
15 to 20 mm in diameter. What is the most likely iden-
tification of this organism?

FIGURE 2-19 (Courtesy the Centers for Disease Control and Pre-
vention. See also color plate 26.)

a. Trypanosoma sp.
b. Leishmania sp.
c. Wuchereria bancrofti
d. Loa loa

FIGURE 2-18 (Courtesy the Centers for Disease Control and Pre-
vention. See also color plate 25.)
88 CHAPTER 2 Mycology, Virology, and Parasitology

SELF-ASSESSMENT
Content Area: ______________________________

Score on Practice Questions: ______________________

List the specific topics covered in the missed questions:

List the specific topics covered in the correct questions:


CHAPTER 2 Mycology, Virology, and Parasitology 89

NOTES
CHAPTER

3
Hematology
Sandy Cook

HEMATOPOIESIS
Skull
Regulated process of blood cell production
Hematopoietic stem cells give rise to red blood cells
(RBC), white blood cells (WBC), and platelets
Most hematopoiesis occurs in the bone marrow of
adults; however, some changes in cell production Proximal end of
occur from conception to adulthood large bones
Bone marrow Sternum
Tissue present in the cavities of cortical bones Vertebrae
Red marrow: Hematopoietically active
marrow located in most bones in early fetal
Axial skeleton
and childhood development but transitions
to fewer locations as an adult Iliac crest
Adults have red marrow in the proximal
ends of long bones, sternum, skull, scapu-
lae, ribs, and pelvis, with approximately
equal amounts of red and yellow marrow
(Figure 3-1)
Yellow marrow: Hematopoietically inactive
marrow, consisting primarily of fat cells
Sites of hematopoiesis (Figure 3-2 and Table 3-1)

Cell Lines Produced


The hematopoietic stem cells give rise to the different cell
lines. Progenitor cells commit to various states of matu-
ration, with the common lymphoid progenitor producing
lymphoid cells and the common myeloid progenitor
leading to the production of the neutrophil, monocyte,
erythrocytic, and megakaryocytic cell lines (Figure 3-3) FIGURE 3-1 The adult skeleton, in which darkened areas depict
active red marrow hematopoiesis. (From Rodak BF, Fritsma GA,
Keohane E: Hematology: clinical principles and applications, ed 4,
RED BLOOD CELL PRODUCTION AND St Louis, 2012, Saunders.)
DESTRUCTION
Pronormoblasts divide and progress to form mature
erythrocytes (1) Pronormoblast, (2) basophilic normoblast,
Production of RBCs from the multipotential stem cell (3) polychromatophilic normoblast, (4) ortho-
BFU-E: Burst-forming unitErythroid chromic normoblast, (5) reticulocyte, (6)
CFU-E: Colony-forming unitErythroid mature erythrocyte
Pronormoblast dividing and progressing to As cells divide and mature, cell size gradually
become a mature erythrocyte decreases and the nucleus is eventually extruded
Stages from most immature to mature at maturity (Table 3-2)

90
CHAPTER 3 Hematology 91

Cellularity (%)

100

Bone marrow
80
Vertebra
Yolk sac Liver
60
Sternum
1 2 3
40 Femur Sites of hematopoiesis
Tibia Rib 1 Mesoblastic
20 2 Hepatic
Spleen Lymph nodes
3 Myeloid
0
1 2 3 4 5 6 7 8 9 10 20 30 40 50 60
Fetal months Birth Age in years
FIGURE 3-2 Sites of hematopoiesis. (From Rodak BF, Fritsma GA, Keohane E: Hematology: clinical principles and applications, ed 4, St Louis,
2012, Saunders.)

TABLE 3 -1 Sites of Hematopoiesis

Age Location Product Produced Other


Embryo Aorta-gonad-mesonephros Hematopoietic stem cells,
(AGM) region/yolk sac primitive erythroblasts, and
fetal hemoglobins
Fetal Liver Erythroblasts, granulocytes, Liver is the primary site through first trimester
Other sites (spleen, kidney, monocytes Bone marrow becomes active around the fifth month of
thymus, lymph nodes) gestation
Bone marrow
Birth through Bone marrow All hematopoietic cell lines Bone marrow is the primary site
adult Some involvement from lymph Babies and children have more red marrow activity, whereas
nodes, spleen, liver, kidney, normal adult marrow has an approximately equal
thymus composition of red and yellow marrow
Thymus activity decreases after childhood

RBC synthesis is stimulated by erythropoietin, Each heme molecule can combine reversibly with
which is produced primarily in the kidney in one molecule of oxygen
response to hypoxia The globin portion contains two pairs of
RBCs normally have a lifespan of approximately globin chains, two a chains and two non-a chains
120 days. At the end of their life, they are a and z chain production is controlled by genes
removed by intravascular or extravascular on chromosome 16
hemolysis, and the contents of the cell are b, g, d, and e chain production is controlled by
recycled or excreted. genes on chromosome 11
Chain production is turned on and off through
stages of development, leading to production
HEMOGLOBIN (HGB) PRODUCTION
of mainly a and b chains with maturity
Occurs from pronormoblast to reticulocyte stage of (Table 3-4)
RBCs Hgb is used for the transport of gases
Heme is synthesized in the mitochondria, so mature O2 affinity of Hgb is low at a low O2 tension in the
RBCs are unable to produce heme because of loss of body and affinity is high at a high O2 tension. This
mitochondria with maturation is demonstrated by the O2 dissociation curve
Normal Hgb is composed of four heme molecules (Figure 3-4)
nested in four globin molecules Oxyhemoglobin is the primary Hgb for gas
Heme is composed of a protoporphyrin IX ring with transport in the body, but other Hgb variants may
Fe+2 at its center be seen
92 CHAPTER 3 Hematology

Long-term self- Short-term self-


renewing stem cell renewing stem cell

Multipotent progenitor
hematopoietic stem cell

Common Common
myeloid progenitor lymphoid progenitor

Granulocyte-monocyte Eosinophil-basophil Megakaryocyte-erythrocyte Dendritic Pre-B Pre-T Natural


progenitor progenitor progenitor cell killer cell

Myeloblast Monoblast Myeloblast Myeloblast Pronormoblast Megakaryoblast B lymphoblast T lymphoblast

Neutrophil Monocyte Eosinophil Basophil Erythrocyte Megakaryocyte B cell T cell

Macrophage Mast cell Platelets Plasma cell


FIGURE 3-3 Diagram of hematopoiesis shows derivation of cells from the multipotent stem cell. (From Rodak BF, Fritsma GA, Keohane E:
Hematology: clinical principles and applications, ed 4, St Louis, 2012, Saunders.)

T A B L E 3- 2 Red Blood Cell Maturation

Nucleus-to- % in Bone Bone Marrow Transit


Cell or Stage Diameter Cytoplasm Ratio Nucleoli Marrow Time (hr)
Pronormoblast 12-20 mm 8:1 1-2 1 24
Basophilic normoblast 10-15 mm 6:1 0-1 1-4 24
Polychromatic normoblast 10-12 mm 4:1 0 10-20 30
Orthochromic normoblast 8-10 mm 1:2 0 5-10 48
Shift (stress) reticulocyte 8-10 mm No nucleus 0 1 48-72*
(polychromatic erythrocyte)
Polychromatic erythrocyte 8-8.5 mm No nucleus 0 1 24-48*
From Rodak BF, Fritsma GA, Keohane E: Hematology: clinical principles and applications, ed 4, St Louis, 2012, Saunders.
*Transit time in peripheral blood.
CHAPTER 3 Hematology 93

Maintain the RBC membrane flexibility


100
Supply offshoot pathways

in
Hexose monophosphate shunt

lob

7.6
Protection of the RBC from oxidant damage
og
80
My

pH
by production of reduced glutathione
B Methemoglobin reductase pathway
% O2 saturation

7.2
60
Maintaining iron in the ferrous form for Hgb
pH
7.4
P50 to limit the production of methemoglobin
pH

40 C Rapoport-Luebering pathway
A
Regulation of O2 delivery to tissues by the
production of 2,3 bisphosphoglycerate (2,3
20 Hemoglobin
BPG) (Figure 3-5)

WHITE BLOOD CELLS


0 20 40 60 80 100
Neutrophils
PO2 (mm Hg)
Phagocytic cells present in the peripheral circula-
FIGURE 3-4 Oxygen dissociation curve. A, Normal dissociation tion destroy foreign substances and microorganisms
curve. B, Left-shifted curve with reduced P50 caused by a
Constitute the majority of circulating WBCs in adults
decrease in 2,3-bisphosphoglycerate (2,3-BPG), partial pressure of
carbon dioxide (PCO2), temperature, and H+ ions (raised pH). A Development (Table 3-3)
left-shifted curve is also seen with hemoglobin variants that have Stages from most immature to mature include
increased oxygen affinity. C, Right-shifted curve with increased 1. myeloblast 2. promyelocyte 3. myelocyte 4.
P50 caused by an elevation in 2,3-BPG, PCO2, temperature, and H+ metamyelocyte 5. band 6. segmented neutrophil
ions (lowered pH). A right-shifted curve is also seen with
hemoglobin variants that have decreased O2 affinity. (From Rodak
(Table 3-3).
BF, Fritsma GA, Keohane E: Hematology: clinical principles and Normal neutrophil function
applications, ed 4, St Louis, 2012, Saunders.) Cells move to a site of inflammation, and gran-
ules are released to assist in travel and adhesion.
Hemoglobin Variants Once at their target site, cells work to eliminate
the foreign material by phagocytosis Granule
Methemoglobin Hgb containing Fe+3 instead of the normally contents and neutrophil extracellular traps
reduced Fe+2 are used to trap and kill microorganisms
Formed normally as a result of oxidation; (Boxes 3-1 to 3-3)
however, it is usually kept from Location
overproduction because of the Present in a circulating pool in which neutrophils
methemoglobin reductase pathway, an
travel throughout the peripheral circulation
offshoot of the Embden-Meyerhof pathway
Sulfhemoglobin Hgb formed as a result of oxidation of Hgb by
and a marginating pool in which neutrophils
materials containing sulfur line the walls of the vasculature, waiting to be
Process is irreversible, so once Hgb has become called into use. In the bone marrow, before
sulfhemoglobin, it will remain for the life of release to the peripheral vasculature, a storage
the cell pool and mitotic pool are present
Unable to transport O2, leading to cyanosis Eosinophils
Carboxyhemoglobin Hgb resulting from heme iron-binding carbon Maturation is similar to neutrophil maturation,
monoxide although granule contents are different; eosinophils
CO has a high O2 affinity and does not give up have a variety of substances in their granules (see
O2 to the tissues easily Box 3-3)
Small amounts of carboxyhemoglobin are Normal function
produced within the cells, but most problems
occur as a result of environmental exposures
Cells serve in immune regulation, from antigen
presentation to initiation of immune response
Primarily increased in parasitic infection (especially
RED BLOOD CELL METABOLISM AND
helminths) and allergic disorders
PHYSIOLOGY Basophils
Mature RBCs have no mitochondria, so rely on anaerobic Maturation is similar to eosinophil and neutrophil
glycolysis for energy via the Embden-Meyerhof pathway maturation. Basophils have several different types
Energy is needed for the main Embden-Meyerhof of granules (Box 3-4)
pathway Cells have immunoglobulin E (IgE) receptors that
Maintain cation gradients lead to their effectiveness in allergic and hypersen-
Keeps potassium inside and sodium outside sitivity reactions. They also play a role in initiating
the RBC the immune response
Embden-Meyerhof Pathway
(Anaerobic Pathway of Glucose Metabolism)
H2O2 H2O Hexose Monophosphate Pathway

Glutathione peroxidase

GSH GSSG

Glucose Glutathione reductase


ATP
Hexokinase (1 ATP)
ADP NADP NADPH

Glucose 6-phosphate 6-phospho-gluconate


Glucose-6-phosphate dehydrogenase
Glucose phosphate
isomerase

Fructose 6-bisphosphate Pentose phosphate


ATP
Phosphofructokinase (1 ATP)
ADP
Fructose 1,6-bisphosphate
aldolase
Dihydroxyacetone Glyceraldehyde
phosphate 3-phosphate
Triose phosphate isomerase
Methemoglobin
Glyceraldehyde NAD reductase +H Methemoglobin
3-phosphate
dehydrogenase NADH Methemoglobin Hemoglobin
reductase

1,3-Bisphosphoglycerate Methemoglobin
Bisphosphoglycerate Reductase Pathway
mutase
ADP
(+2 ATP)
Phosphoglycerate ATP
Rapaport-Luebering
Pathway kinase

2,3-Bisphosphoglycerate
(2,3-BPG) 3-Phosphoglycerate
Bisphosphoglycerate
phosphatase

Phosphoglyceromutase

2-Phosphoglycerate

Enolase

Phosphoenolpyruvate

ADP
Pyruvate (+2 ATP)
kinase ATP

Pyruvate

NADH

NAD

Lactate
FIGURE 3-5 Glucose metabolism in the erythrocyte. ADP, Adenosine diphosphate; ATP, adenosine triphosphate; G6PD, glucose-6-phosphate
dehydrogenase; NAD, nicotinamide adenine dinucleotide; NADH, nicotinamide adenine dinucleotide (reduced form); NADP, nicotinamide
adenine dinucleotide phosphate (oxidized form). (From Rodak BF, Fritsma GA, Keohane E: Hematology: clinical principles and applications,
ed 4, St Louis, 2012, Saunders.)
CHAPTER 3 Hematology 95

TABLE 3 -3 Neutrophil Development

Stage Description Image

Myeloblast Earliest recognizable stage; cells are large with


large nuclei and loose chromatin, some
primary granules may be seen

(From Rodak BF, Fritsma GA, Keohane E: Hematology:


clinical principles and applications, ed 4, St Louis, 2012,
Saunders. See Color Plate 27.)

Promyelocyte Cell begins to decrease in size, and chromatin


begins to compact, with nucleus often
appearing as eccentric; primary granules are
prominent

(From Rodak BF, Fritsma GA, Keohane E: Hematology: clinical


principles and applications, ed 4, St Louis, 2012, Saunders.
See Color Plate 28.)

Myelocyte Cell continues to decrease in size, and chromatin


continues to compact into a round nucleus;
last stage capable of mitosis; secondary
granules are formed

(From Rodak BF, Fritsma GA, Keohane E: Hematology: clinical


principles and applications, ed 4, St Louis, 2012, Saunders.
See Color Plate 29.)

(Continued)
96 CHAPTER 3 Hematology

TABLE 3-3 Neutrophil Developmentcontd

Stage Description Image

Metamyelocyte Cell continues to decrease in size, chromatin


continues to compact into a kidney bean
shape; secondary and tertiary granules are
formed

(From Rodak BF, Fritsma GA, Keohane E: Hematology: clinical


principles and applications, ed 4, St Louis, 2012, Saunders.
See Color Plate 30.)

Band neutrophil Nucleus shows compact chromatin that is shaped


into a horseshoe form; tertiary and secretory
granules are formed

(From Rodak BF, Fritsma GA, Keohane E: Hematology: clinical


principles and applications, ed 4, St Louis, 2012, Saunders.
See Color Plate 31.)

Segmented Nucleus begins to segment into three to four


neutrophil lobes, each attached by a threadlike nuclear
filament; secretory granules are formed

(From Rodak BF, Fritsma GA, Keohane E: Hematology: clinical


principles and applications, ed 4, St Louis, 2012, Saunders.
See Color Plate 32.)
CHAPTER 3 Hematology 97

NOTE: Mast cells are somewhat related to baso- Monocytes


phils; however, they are tissue cells used in allergic Maturation starts with a monoblast and con-
reactions and inflammation tinues to the promonocyte and mature monocyte
stages
Cell appearance shows large cells, the largest
in peripheral circulation, with finely granular cyto-
Normal Hemoglobins and Globin plasm (ground-glass appearance) and a nucleus
TABLE 3 -4 Chains with relatively loose, lacy chromatin, with the occa-
sional presence of folding or indentation. Cyto-
Embryonic Fetal Adult
plasm may show vacuolization
Gower 1: z2e2 Hgb F: a2g2 Hgb A: a2b2 Functions
50%-90% 97% Cells are used in both innate and adaptive immu-
Gower 2: a22e2 Hgb A: a2b2 Hgb A2: a2d2
nity. They can recognize and phagocytize foreign
 10%-40% 1.5%-3.5%
materials; in addition they can serve as antigen-
Portland: z2g2 Hgb A2: a2d2 < 2% Hgb F: a2g2 < 2%
presenting cells to initiate T and B cells they

BOX 3 -1 Neutrophil Granules

Primary (Azurophilic) Granules Tertiary Granules


Formed during the promyelocyte stage Formed during metamyelocyte and band stages
Last to be released (exocytosis) Second to be released
Contain Contain
Myeloperoxidase Gelatinase
Acid b-glycerophosphatase Collagenase
Cathepsins Lysozyme
Defensins Acetyltransferase
Elastase b2-Microglobulin
Proteinase-3 Secretory Granules (Secretory Vesicles)
Others Formed during band and segmented stages
Secondary (Specific) Granules First to be released (fuse to plasma membrane)
Formed during myelocyte and metamyelocyte stages Contain (attached to membrane)
Third to be released CD11b/CD18
Contain Alkaline phosphatase
b2-Microglobulin Vesicle-associated membrane-2
Collagenase CD10, CD13, CD14, CD16
Gelatinase Cytochrome b558
Lactoferrin Complement 1q receptor
Neutrophil gelatinaseassociated lipocalin Complement receptor-1
Others

From Rodak BF, Fritsma GA, Keohane E: Hematology: clinical principles and applications, ed 4, St Louis, 2012, Saunders.

BOX 3 -2 Phagocytosis

Recognition and attachment Oxygen independent


Phagocyte receptors recognize and bind to certain foreign molec- The pH within the phagosome becomes alkaline and then neutral,
ular patterns and opsonins such as antibodies and complement the pH at which digestive enzymes work.
components. Primary and secondary lysosomes (granules) fuse to the phago-
Ingestion some and empty hydrolytic enzymes and other bacteriocidal mol-
Pseudopodia are extended around the foreign particle and enclose ecules into the phagosome.
it within a phagosome (engulfment). Formation of neutrophil extracellular traps
The phagosome is pulled toward the center of the cell by polymer- Nuclear and organelle membranes dissolve, and activated cyto-
ization of actin and myosin and by microtubules. plasmic enzymes attach to DNA.
Killing and digestion The cytoplasmic membrane ruptures and DNA with attached
Oxygen dependent enzymes is expelled, so that the bacteria are digested in the exter-
Respiratory burst through the activation of nicotine adenine nal environment.
diphosphate oxidase (reduced form). H2O2 and hypochlorite are
produced.
From Rodak BF, Fritsma GA, Keohane E: Hematology: clinical principles and applications, ed 4, St Louis, 2012, Saunders.
98 CHAPTER 3 Hematology

B O X 3- 3 Eosinophil Granules

Primary Granules Others


Formed during promyelocyte stage Small lysosomal granules
Contain Acid phosphatase
Charcot-Leyden crystal protein Arylsulfatase B
Secondary Granules Catalase
Formed throughout remaining maturation Cytochrome b558
Contain Elastase
Major basic protein (core) Eosinophil cationic protein
Eosinophil cationic protein (matrix) Lipid bodies
Eosinophil-derived neurotoxin (matrix) Cyclooxygenase
Eosinophil peroxidase (matrix) 5-Lipoxygenase
Lysozyme (matrix) 15-Lipoxygenase
Catalase (core and matrix) Leukotriene C4 synthase
b-Glucuronidase (core and matrix) Eosinophil peroxidase
Cathepsin D (core and matrix) Esterase
Interleukins-2, -4, and -5 (core) Storage vesicles
Interleukin-6 (matrix) Carry proteins from secondary granules to be released into the
Granulocyte-macrophage colony-stimulating factor (core) extracellular medium

From Rodak BF, Fritsma GA, Keohane E: Hematology: clinical principles and applications, ed 4, St Louis, 2012, Saunders.

B O X 3- 4 Basophil Granules T A B L E 3- 5 Normal Range Values in Adults

Secondary Granules Adult Adult


Histamine Male Female
Platelet activating factor WBC (109/L) 4.5-11.5 4.5-11.5
Leukotriene C4 RBC (1012/L) 4.60-6.00 4.00-5.40
Interleukin-4 Hgb (g/dL) 14.0-18.0 12.0-15.0
Interleukin-13 Hct (%) 40-54 35-49
Vascular endothelial growth factor A MCV (fL) 80-100 80-100
Vascular endothelial growth factor B MCH (pg) 26-32 26-32
Chondroitin sulfates (e.g., heparin) MCHC (g/dL) 32-36 32-36
RDW (%) 11.5-14.5 11.5-14.5
Platelet (109/L) 150-450 150-450
are and can be used for housekeeping purposes to MPV (fL) 6.8-10.2 6.8-10.2
remove dead cells and debris Neutrophils (%) 50-70 50-70
NOTE: Once monocytes migrate to tissues, they Lymphocytes (%) 18-42 18-42
serve as tissue macrophages with similar functions Monocytes (%) 2.0-11 2.0-11
Lymphocytes Eosinophils (%) 1.0-3 1.0-3
The maturation stages are 1. lymphoblast 2. pro- Basophils (%) 0-2 0-2
Segmented neutrophils (109/L) 2.3-8.1 2.3-8.1
lymphocyte 3. mature lymphocyte. There are three
Band neutrophils (109/L) 0-0.6 0-0.6
major subgroups of lymphocytes: T cells, B cells,
Lymphocytes (109/L) 0.8-4.8 0.8-4.8
and natural killer (NK) cells. Monocytes (109/L) 0.45-1.3 0.45-1.3
Lymphocytes can be produced in both the bone mar- Eosinophils (109/L) 0-0.4 0-0.4
row and the lymphoid tissues. Cells can return from Basophils (109/L) 0-0.1 0-0.1
an inactive/resting form into active blasts, as needed
Functions Hct, Hematocrit; Hgb, hemoglobin; MCH, mean corpuscular hemoglobin;
MCV, mean corpuscular volume; MPV, mean platelet volume; RBC, red blood
B cells produce antibodies and also play a role cell; RDW, red cell distribution width; WBC, white blood cell.
in antigen presentation to the T cells
T cells mediate the immune response
are relatively similar, some slight variations may occur
based on a specific laboratorys population (Table 3-5)
LABORATORY CONSIDERATIONS
Normal Ranges Staining of Blood and Bone Marrow Samples
Patient values are compared against established normal Smears are made of blood or bone marrow to provide
ranges, which can vary based on age, gender, population, smears with the best possible distribution of cellular
and geographic distribution. Although normal ranges elements, leaving a critical area or examination area
CHAPTER 3 Hematology 99

where a single layer of cells is evenly dispersed, allowing Measured by placing whole blood in capillary tubes
visualization of individual cellular elements and centrifuging to read the packed cell volume on a
Wright or Wright-Giemsa stains (Romanowsky-type manual microhematocrit reading device
stains) are polychrome stains used to stain slides Hct values are available on automated general hematol-
of peripheral blood and bone marrow, which gives ogy cell counters; however, values are derived by using a
elements their characteristic colors calculation as opposed to a physical measurement
Slides are fixed with a methanol fixative, followed
by staining with a solution or solutions containing
eosin and methylene blue to impart color to cellu- Reticulocyte Count
lar elements
Eosin is acidic, so it will stain basic elements, such as Reticulocytes are the final stage before an RBC
Hgb and basic proteins found in some cell granules reaches maturity. Reticulocytes may be counted
Methylene blue is basic and will stain acidic elements, manually to determine the erythrocyte production
such as cell nuclei and immature cell cytoplasm and release from the bone marrow
Stained slides can be used for counting the 100 cell Manual counts are performed by incubating ethylene-
count WBC differential and examining RBC and diametetraacetic acid (EDTA) whole blood with a
platelet morphology. In the case of bone marrow, supravital stain, usually new methylene blue. If any
aspirate and core biopsy slides can be stained for RNA or residual organelles are present, they will take
a bone marrow differential, myeloid-to-erythroid up the supravital stain and are visible microscopically
Various methods are used for the manual reticulo-
(M/E) ratio, or sample cellularity
cyte count, including the Miller ocular and other
Red Blood Cell Morphology techniques to determine the total percentage of
reticulocytes present
See Tables 3-6 and 3-7 Automated reticulocyte counts are now commonly
performed
Red Blood Cell Indices Various methods are used for automated counts,
including treating RBCs with a stain or fluorescent
Red blood cell indices may be calculated manually or
dye to identify the reticulocyte by optical methods
derived from automated instrumentation.
or flow cytometry. Automation allows for a larger
See Table 3-8 number of cells to be examined.

Manual and Semiautomated Techniques


Most hematologic testing is performed using auto- Sickle Cell Testing
mated instrumentation; however, a few manual tech- Samples may be screened for the presence of abnormal
niques may be used on occasion Hgb because of the different solubility properties
of various Hgb
Hemacytometer Hgb S exhibits decreased solubility when deoxygen-
ated, as opposed to the more soluble Hgb A and A2
Counting chamber used for manual cell counting, cur- Screening tests use a lysing agent and a dithionite
rently most frequently used for body fluid cell counting solution, or other reducing agent, to induce deoxy-
Based on the use of a known counting area of
genation of Hgb. Abnormal Hgbs have decreased
nine squares each with an area of 1 mm2 and a total solubility and will precipitate in the solution,
volume of 0.9 mm3 for a total area of 9 mm3. A stan- appearing cloudy on observation
dard formula is used to determine a total cell count/m Positive sickle cell screens require follow-up with a
L (1 mm3 1 mL) (Figure 3-6) more definitive test, such as Hgb electrophoresis,
high-performance liquid chromatography (HPLC),
Total
0 cell count cells=mL 1 or isoelectric focusing
B Cells counted C
B C
@ Number of squares counted A  Reciprocal dilution
area of squares counted  depth Erythrocyte Sedimentation Rate (ESR)
Screening test used to screen or monitor for various
Manual hematocrit (Hct, also known as inflammatory states. The ESR looks at how much
microhematocrit) RBC settling will occur in a well-mixed whole blood
sample over a 1-hour period
Hct is the volume of packed RBCs occupying a specific RBCs normally have a net negative charge, causing
volume of whole blood, expressed as a percentage or in them to repel each other in a whole blood sample,
liters per liter leading to slow settling of the RBCs over time
100 CHAPTER 3 Hematology

T A B L E 3- 6 Description of Red Blood Cell Abnormalities and Commonly Associated Disease States

RBC Abnormality Cell Description Commonly Associated Disease States


Anisocytosis Abnormal variation in RBC volume or diameter Hemolytic, megaloblastic, iron-deficiency anemia
Macrocyte Large RBC (>8 mm in diameter), MCV >100 fL Megaloblastic anemia
Myelodysplastic syndrome
Chronic liver disease
Bone marrow failure
Reticulocytosis
Oval macrocyte Large, oval RBC Megaloblastic anemia
Poikilocytosis Abnormal variation in RBC shape Severe anemia
Certain shapes helpful diagnostically
Spherocyte Small, round, dense RBC with no central pallor Hereditary spherocytosis
Immune hemolytic anemia
Extensive burns (along with schistocytes)
Elliptocyte, ovalocyte Elliptical (cigar shaped), oval (egg shaped), RBC Hereditary elliptocytosis or ovalocytosis
Iron-deficiency anemia
Thalassemia major
Myelophthisic anemias
Stomatocyte RBC with slitlike area of central pallor Hereditary stomatocytosis
Rh deficiency syndrome
Acquired stomatocytosis (liver disease, alcoholism)
Artifact
Sickle cell Thin, dense, elongated RBC pointed at each end; may be curved Sickle cell anemia
Sickle cellb-thalassemia
Hgb C crystal Hexagonal crystal of dense Hgb formed within the RBC membrane Hgb C disease
Hgb SC crystal Fingerlike or quartzlike crystal of dense Hgb protruding from the Hgb SC disease
RBC membrane
Target cell (codocyte) RBC with Hgb concentrated under and around the periphery Liver disease
resembling a target Hemoglobinopathies
Thalassemia
Schistocyte (schizocyte) Fragmented RBC resulting from rupture in the peripheral circulation Microangiopathic hemolytic anemia* (along with
microspherocytes)
Traumatic cardiac hemolysis
Extensive burns (along with microspherocytes)
Helmet cell (keratocyte) RBC fragment in shape of a helmet Same as schistocyte
Folded cell RBC with membrane folded over Hb C disease
Hb SC disease
Acanthocyte (spur cell) Small, dense RBC with few irregularly spaced projections of varying Severe liver disease (spur cell anemia)
length Neuroacanthocytosis (abetalipoproteinemia,
McLeods syndrome)
Burr cell (echinocyte) RBC with blunt or pointed, short projections that are usually evenly Uremia
spaced over the surface of cell; present in all fields of blood film Pyruvate kinase deficiency
but in variable numbers per field{
Teardrop cell RBC with a single pointed extension resembling a teardrop or pear Primary myelofibrosis
(dacryocyte) Myelophthisic anemia
Thalassemia
Megaloblastic anemia

From Rodak BF, Fritsma GA, Keohane E: Hematology: clinical principles and applications, ed 4, St Louis, 2012, Saunders.
*Such as thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, disseminated intravascular coagulation.
{
Cells with similar morphology that are unevenly distributed in a blood film (not present in all fields) are likely the result of a drying artifact in blood film
preparation; these artifacts are sometimes referred to as crenated RBCs.
Hgb, Hemoglobin; MCV, mean cell volume; RBC, red blood cell; SC, sickle cell.

When a change to the charge occurs, usually resulting


from increases in plasma proteins, the cells become
Hematology Instrumentation
attracted to each other, leading to increased settling Cell counters
speeds of the RBCs Automated method for performing complete blood
Tests are performed with manual Westergren and cell counts
Wintrobe procedures or automated analyzers to Count cells using impedance and optical
allow for a faster reading measurements
CHAPTER 3 Hematology 101

Erythrocyte Inclusions: Description, Composition, and Selected Commonly Associated


TABLE 3 -7 Disease States

Appearance in Inclusion Associated Diseases


Inclusion Supravital Stain Appearance in Wright Stain Composition and Conditions
Diffuse basophilia Granules and Bluish tinge throughout cytoplasm; RNA Hemolytic anemia
filaments also referred to as After treatment for iron,
polychromasia vitamin B12, or folate
deficiency
Basophilic Granules and Blue-purple granules distributed Precipitated RNA Lead poisoning
stippling filaments throughout cytoplasm Thalassemia
(punctate Hemoglobinopathies
basophilia) Abnormal heme synthesis
Howell Jolly body Dense, round, granule Dense, round, blue or purple DNA (nuclear Hyposplenism
granule; usually one per cell; fragment) After splenectomy
occasionally multiple Megaloblastic anemia
Hemolytic anemia
Heinz body Round granule Not visible Denatured Glucose-6-phosphate
attached to inner hemoglobin dehydrogenase
membrane deficiency
Unstable hemoglobins
Oxidant drugs/chemicals
Pappenheimer Clusters of small Clusters of small, light blue Iron Sideroblastic anemia
bodies* granules granules, often near periphery Hemoglobinopathies
of cell Hyposplenism
Megaloblastic anemia
Cabot ring Rings or figure-eights Blue rings or figure-eights Remnant of mitotic Megaloblastic anemia
spindle Myelodysplastic syndromes
Hgb H Fine, evenly dispersed Not visible Precipitate of b Hgb H disease
granules chains of
hemoglobin

From Rodak BF, Fritsma GA, Keohane E: Hematology: clinical principles and applications, ed 4, St Louis, 2012, Saunders.
*Blue (siderotic) granules observed in Prussian blue stain.
Hgb, Hemoglobin.

TABLE 3 -8 Red Blood Cell Indices

Normal
Definition Manual Calculation Range
Mean corpuscular volume (MCV) Average volume of an individual RBCAnalyzer: (Hct [%]/RBC)  10 80-100 fL
Measures directly
or
Mean corpuscular hemoglobin (MCH) Average weight of hemoglobin in an individual (Hgb [g/dL]/RBC)  10 28-34 pg
RBCAnalyzer: Measures directly
or
Mean corpuscular hemoglobin Ratio of hemoglobin mass to the cell volume (Hgb [g/dL]/Hct [%])  100 32-36 g/dL
concentration (MCHC)
Red cell distribution width (RDW) Variation of RBC volume used to help identify 11.5%-14.5%
the presence of anisocytosis
Analyzer calculation
Standard Deviation of MCV  100
Mean MCV

Impedance: Uses changes in electrical charge as Pulses formed by electrical resistance are
cells, which have low conductivity, move counted
through an electrically conductive fluid to deter- The number of pulses recorded is propor-
mine cell count tional to the cell count
102 CHAPTER 3 Hematology

1 mm

1 mm
R R

R R

side view

cover slip 0.1 mm


moat moat

FIGURE 3-6 Hemacytometer and close-up view of the counting areas as seen under the microscope. The areas for the standard white blood cell
count are labeled W, and the areas for the standard red blood cell count are labeled R. The entire center square is used for counting platelets.
(From Rodak BF, Fritsma GA, Keohane E: Hematology: clinical principles and applications, ed 4, St Louis, 2012, Saunders.)

Pulse size is proportional to cell size NOTE: If a corrected WBC count is needed,
Can be used to count WBC, RBC, and platelets it should be lower than the original WBC
Optical counts: Use of optical light scatter to deter- count from the analyzer
mine the size and complexity of cells present as a
single cell moves through a focused light source
Bone Marrow Collection
Forward and side scatter are used to deter-
mine an automated differential count Bone marrow samples may be needed for evaluation of
Hgb measurement the patients hematologic condition. Bone marrow
Determined using a cyanmethemoglobin reagent samples, including a core biopsy and aspirate, are col-
to quantitate the amount of Hgb spectrophoto- lected by a physician, and the samples obtained are
metrically within the instrument processed in the laboratory
Calculations Samples are collected and analyzed for various reasons,
Automated calculations performed from mea- including diagnosis and staging hematopoietic malig-
sured parameters provide values for Hct, mean nancies, diagnosis and evaluation of unexplained
corpuscular volume (MCV), mean corpuscular cytopenias or systemic disorders, follow-up after patients
hemoglobin (MCH), mean corpuscular hemo- undergo various treatments (chemotherapy, radiation,
globin concentration (MCHC), and red blood transplants), determining sources of otherwise undiag-
cell distribution width (RDW) nosed infections, and other miscellaneous purposes
Corrected WBC count Bone marrow samples are collected from sites
In the case of elevated numbers of nucleated RBCs, where active red marrow is more prevalent, usually
the nucleated red blood cells (NRBCs) should the posterior superior iliac crest; however, the ster-
be counted independently of the 100 cell count num or anterior iliac crest may be used in adult
WBC differential. Some analyzers automatically patients. Sometimes tibia samples are obtained in
correct for the presence of NRBCs; however, young children
sometimes the WBC count must be corrected Aspirate samples are used to assess morphology and
manually perform a differential count and M:E ratio
Calculated WBC Correction for NRBCs The M:E ratio looks at the number of myeloid
Corrected WBC count Analyzer WBC cells compared to the numbers of nucleated ery-
count  100/100 + number of NRBCs per throid precursors
100 WBCs counted Lymphocytes are not included in this ratio
CHAPTER 3 Hematology 103

A normal M:E ratio is usually in the range of


Lineage-Associated Markets
2:1 to 4:1 Commonly Analyzed in Routine Flow
Core biopsy samples are used to determine bone mar- T A B L E 3- 9 Cytometry
row cellularity
Helps assess if the sample is normocellular, hypocel- Lineage Markers
lular, or hypercellular Immature CD34
Cytochemical stains, flow cytometry, and other spe- CD117
cialized testing may be performed on bone marrow Terminal deoxynucleotidyl transferase
samples Granulocytic/monocytic CD33
CD13
CD15
Flow Cytometry CD14
Erythroid CD71
Flow cytometry is an automated method of sorting Glycophorin A
cells. It uses a cell suspension injected into a stream Megakaryocytic CD41
sheath fluid to determine specific characteristics of CD42
the cell, including size, complexity, immunopheno- CD61
type, and cytochemistry B lymphocytes CD19
Hydrodynamic focusing: Cells in suspension CD20
move through sheath fluid in a single-file line, allow- CD22
k Light chain
ing each to individually pass through a laser light
Light chain
Allows specific characterization of each cell T lymphocytes CD2
Forward scatter for size determination CD3
Side scatter for complexity of the cell CD4
Gating: Electronic boundaries created to separate a CD5
specific population of interest CD7
Performed during or after initial analysis CD8
Fluorescently tagged antibodies can be used to
determine cell lineage and aid in characterizing
patient immunophenotype (Table 3-9) Glossitis
Splenomegaly
Neurologic symptoms
Special Stains and Cytochemistry Common tests for initial anemia evaluation
Special staining techniques may be performed to Complete blood count (CBC) with peripheral smear
help differentiate cell types, particularly in cases of leu- review
kemia (Table 3-10) Determines RBC, Hgb, Hct, and RBC indices
Flow cytometry has decreased the use of cytochem-
Normal Range Resulting Morphology
istry in differentiating leukemias; however, ele-
(If Abnormal)
ments are incorporated into flow cytometry or MCV 80-100 fL <80 fL: Microcytic
still performed as stand-alone testing to assist in a >100 fL: Macrocytic
definitive diagnosis MCH 28-34 pg
MCHC 32-36 g/dL <32 g/dL: Hypochromic
36-40 g/dL: Normochromic
>40 g/dL: Results may be invalid
ANEMIA AND RED BLOOD CELL
DISORDERS
Smear examination will reveal the appearance
Usually defined as decreased ability of blood to carry
of RBCs (anisocytosis and poikilocytosis and
O2 or as a decrease in RBCs/Hgb from an established
inclusions)
reference range Reticulocyte count
Common physical symptoms
Shows the bone marrow response to decreases
Fatigue
in RBCs
Shortness of breath
Pallor Anemias may be classified by combinations of differ-
Cardiac issues ent criteria
Other symptoms may be more related to specific Morphology
causes of anemia RBC indices are used to gauge size and
Jaundice hemoglobinization
Pica Normocytic/normochromic
104 CHAPTER 3 Hematology

T A B L E 3- 1 0 Staining Techniques

Negative
Cytochemical Stain Element Stained Positive Reaction Reaction Diagnostic Utility
Myeloperoxidase (MPO) Enzyme found in the Myeloblasts and promyelocytes, Lymphoblasts Differentiating AML versus ALL
primary granules although some weak activity
of granulocytic may be seen in monocytes
cells
Sudan black B (SBB) Lipids found in Myeloblasts and promyelocytes, Lymphoblasts Differentiating AML versus ALL
primary and although some weak activity
secondary may be seen in monocytes
granules and in
lysosomal
granules of
monocytes
Specific esterase (naphthol Esterase enzymes Myeloblasts Lymphoid cells Identifying cells of myeloid
AS-D chloroacetate found in origin
esterase) neutrophils
Nonspecific esterase Esterase enzymes Monoblasts and monocytes Granulocytes AML of myeloid versus
(a-naphthyl acetate or found in and lymphoid monocytic origin
butyrate esterase) monocytes cells
Tartrate-resistant acid Isoenzymes of acid Hairy cell lymphocytes (in hairy All cells except Positive diagnosis of hairy cell
phosphatase (TRAP) phosphatase cells, isoenzyme 5 of acid hairy cell leukemia
phosphatase is not inhibited lymphocytes
with the addition of tartrate,
leaving a positive reaction)
Periodic acidSchiff (PAS) Glycogen, Multiple cell types Normal AML of erythroid or
mucoproteins erythroblasts megakaryoblastic origin
and and some cases of ALL
high-molecular- based on staining pattern
weight (leukemic lymphoblasts
carbohydrates may have a coarse blocklike
pattern, whereas erythroid
precursors have a coarse
and granular staining
pattern
Leukocyte alkaline Enzyme in the Neutrophils (activity is scored in Differentiation of CML (low
phosphatase (LAP) secondary mature bands and activity) and leukemoid
granules of segmented neutrophils on a reaction (high activity)
mature 0-4 rating scale)
neutrophils
Terminal deoxynucleotidyl DNA polymerase in Lymphoblasts Myeloblasts and Identification of lymphoblasts
transferase (TdT) cell nuclei monoblasts in ALL

ALL, Acute lymphoblastic leukemia; AML, acute myelogenous leukemia; CML, chronic myelogenous leukemia.

Microcytic/hypochromic IRON AND HEME DISORDERS


Macrocytic/normochromic
Iron-deficiency anemia (IDA): Lack of iron to make
Function adequate heme
Defects leading to RBC decreases Sideroblastic anemia: Adequate/excess iron that is not
Proliferation: RBCs are not produced at able to be effectively incorporated into heme
normal rates Anemia of chronic disease/inflammation: Adequate
Maturation: RBCs are produced in the mar- iron stores that have impaired release for incorpora-
row but may not mature appropriately tion into heme/RBCs
Survival: RBCs are produced appropriately Hemochromatosis: Iron disorder that is not anemia,
but are lost/destroyed prematurely with excess iron absorption and stores
CHAPTER 3 Hematology 105

Iron Depletion

Normal Iron Status Stage 1 Stage 2 Stage 3

Storage Iron Transport Iron Functional Iron


Depletion Depletion Depletion
(Iron Deficiency Anemia)

Iron Storage Compartment

Iron Transport Compartment

Functional Iron Compartment

Laboratory test values


Hemoglobin N N N

Serum iron N N

TIBC N N

Ferritin N

FIGURE 3-7 Development of iron deficiency anemia. , Increased; #, decreased; N, normal; TIBC, total iron-binding capacity. (Modified from
Suominen P, Punnonen K, Rajamaki A, et al: Serum transferrin receptor and transferrin receptorferritin index identify healthy subjects with
subclinical iron deficits, Blood 92:2934-2939, 1998; reprinted with permission.)

Iron-Deficiency Anemia Parasite related


Laboratory diagnosis of IDA (Figure 3-7)
Iron intake and stores do not meet the bodys needs for CBC
RBC production Varies with the severity of depletion
Inadequate intake
RBC, Hgb, Hct may be decreased
Daily intake does not meet daily loss MCV and MCH often are decreased, resulting in
Nutritional deficiencies microcytic/hypochromic cells
Increased requirements
RBCs tend to be small with increased
Rapid growth periods central pallor because of the lack of avail-
Menstruating women able Hgb
Pregnancy and lactation Iron studies
Absorption issues
Serum iron: Decreased (may be normal in early
Enterocytes are unable to absorb iron stages)
Celiac disease Ferritin: Decreased
Bariatric surgeries Total iron-binding capacity (TIBC): Increased
Medications % Saturation: Decreased
Other diseases impairing absorption Treatment and follow-up
Loss of gastric absorption with age Determine and treat any underlying condition
Parasitic infections Oral iron supplements
Chronic RBC loss RBC transfusions only if Hgb is critically low
Chronic GI bleeding Response to therapy regimen monitored by
Prolonged menorrhagia
Reticulocyte counts increase within the 2 weeks
Other chronic bleeds
of supplementation
Aspirin related CBC and Hgb increases 2 to 3 weeks after
Alcohol related supplementation
106 CHAPTER 3 Hematology

Sideroblastic Anemia CBC


Anemia is usually mild
Anemia characterized by the presence of normal or
RBC, Hgb, Hct may be slightly decreased (Hgb
increased iron that is not effectively incorporated into 9-11 g/dL)
heme MCV and MCH are usually normal
Hereditary
Reticulocytes are normal to decreased
X-Linked or autosomal Iron studies
Acquired
Serum iron: Increased
Refractory anemia (as seen in myelodysplastic
Ferritin: Increased (or may be normal)
syndromes) TIBC: Decreased
Drugs and toxins
% Saturation: Decreased
Lead Bone marrow will show increased iron stores in
Alcohol macrophages
Other varied drugs or toxins Treatment and follow-up
Laboratory diagnosis of sideroblastic anemia Underlying condition may be treated
CBC In some cases erythropoietin or iron may be admin-
Varies istered for the patient
RBC, Hgb, or Hct may be decreased
MCV and MCH may be decreased (microcytic
and hypochromic cells)
Cells are often normocytic/normochromic in Hemochromatosis
cases of lead poisoning.
Occasionally, siderotic granules are seen Iron problem that does not involve anemia
Coarse basophilic stippling is seen in lead poi- Increased iron stores (absorption greater than loss)
soning, although it can also be seen in other Stored as ferritin and hemosiderin
conditions Often stored around organs (heart, liver,
Iron studies pancreas)
Serum iron: Increased Acquired
Ferritin: Increased Transfusion related
TIBC: Decreased In cases of chronic transfusion, the body recycles
% Saturation: Decreased or normal the iron from transfused RBCs, in addition to its
Bone marrow own senescent RBCs
Sometimes performed to reveal presence of Chronic liver disease
increased iron/sideroblasts Alcoholism
Treatment and follow-up Supplemental or dietary iron overload
Hereditary forms may require medications to Inherited
stimulate heme synthesis Relatively high frequency in people of northern
Acquired forms may require the removal of the European descent (1/200)
offending toxin or problem Several known mutations
Classic hereditary hemochromatosis, associated
with the HFE gene
Anemia of Chronic Inflammation (Disease) Hepcidin mutations, associated with the
Acquired anemia characterized by abundant iron HAMP gene
stores, yet iron cannot be readily incorporated into Hemojuvelin mutations, associated with the
serum or RBCs for use HJV gene
Occurs as a result of increases in various acute phase
reactants present with inflammation which slows iron
release that is needed by developing cells
Hepcidin decreases iron release from macrophages MACROCYTIC ANEMIAS
and hepatocytes Megaloblastic anemia results from defective DNA
Lactoferrin competes with transferrin for plasma synthesis
iron, but RBCs cannot incorporate this because of Vitamin B12 and folic acid deficiencies
lack of lactoferrin receptors Nonmegaloblastic macrocytic anemia results from
Ferritin binds iron, but developing RBCs lack other causes
ferritin receptors and cannot incorporate into the Liver disease
erythroid precursors Alcoholism
Laboratory diagnosis of anemia of chronic Hypothyroidism
inflammation Reticulocytosis
CHAPTER 3 Hematology 107

Megaloblastic Anemia Increases in need


Pregnancy
Impairment of DNA synthesis leads to large, abnormal
Lactation
cells. Growing children
Most commonly caused by lack of vitamin B12 Impaired absorption and use
and/or folic acid, although some other conditions Inability to obtain vitamin B12 from food in the
may show megaloblastic changes stomach
All rapidly dividing, nucleated cells, including Gastric issues
RBCs, are affected Inability to produce hydrochloric acid, gas-
Ineffective erythropoiesis, with cells showing tric bypass, drugs for lowering gastric
nuclear-cytoplasmic asynchrony as they mature acidity
Vitamin B12 and folic acid are needed for DNA Lack of intrinsic factor
synthesis (Figure 3-8) Autoimmune disease, Helicobacter
Causes for folate deficiency
pylori infection, gastrectomy
Poor dietary intake
Competition for vitamin B12
Increases in need
Diphyllobothrium latum
Pregnancy Intestinal bacteria in blind loop
Lactation syndrome
Growing children Excessive loss
Impaired absorption and use
May occur in renal dialysis patients, so
Intestinal diseases, including celiac disease and patients are supplemented with folic acid (see
sprue Figure 3-8)
Intestinal surgery Physical examination
Medications General anemia symptoms
Excessive loss Glossitis
May occur in renal dialysis patients, so patients Gastrointestinal (GI) symptoms
are often supplemented with folic acid Vitamin B12 deficiency also may result in neurologic
Causes for vitamin B12 deficiency
symptoms
Poor diet
Memory loss, balance, and gait abnormalities,
Lack of dietary vitamin B12 or folic acid personality changes
Uridine Thymidine dATP dCTP dGTP

3
dUMP dTMP dTTP DNA

5,10-methylene THF DHF

Glycine
2 4
Serine THF*
*Polyglutamation of THF (addition of 1-6 more glutamic
acid residues) is essential for its retention in cell
Methionine

Vitamin B12 1 Site of


folate trap
Homocysteine
5-methyl THF 1 Methionine synthase and vitamin
B12 (in methylcobalamin form)
Cytoplasm
2 Serine hydroxymethyl transferase
Plasma and vitamin B6
5-methyl THF 3 Thymidylate synthetase
4 Dihydrofolate reductase

FIGURE 3-8 Role of folate and vitamin B12 in DNA synthesis. Folate enters the cell as 5-methyltetrahydrofolate (5-methyl THF). In the cell, a
methyl group is transferred from 5-methyl THF to homocysteine, converting it to methionine and generating tetrahydrofolate (THF). This reaction
is catalyzed by methionine synthase and requires vitamin B12 as a cofactor. THF is then converted to 5,10-methylene THF by the donation of a
methyl group from serine. The methyl group of 5,10-methylene THF is then transferred to deoxyuridine monophosphate (dUMP), which converts
it to deoxythymidine monophosphate (dTMP) and converts 5,10-methylene THF to dihydrofolate (DHF). This reaction is catalyzed by thymidylate
synthetase. dTMP is a precursor of deoxythymidine triphosphate (dTTP), which is used to synthesize DNA. THF is regenerated by the conversion of
DHF to THF by the enzyme DHF reductase. A deficiency of vitamin B12 prevents the production of THF from 5-methyl THF; as a result, folate
becomes metabolically trapped as 5-methyl THF. This constitutes the folate trap. (From Rodak BF, Fritsma GA, Keohane E: Hematology:
clinical principles and applications, ed 4, St Louis, 2012, Saunders.)
108 CHAPTER 3 Hematology

In vitamin B12 deficiencies, prolonged/severe Although a classic means of determining the


cases may have demyelination of the neurons; cause of vitamin B12 deficiency, the Schilling
folic acid deficiency does not have neurologic test is no longer performed regularly in the
involvement United States. Homocysteine and MMA are
Laboratory diagnosis of megaloblastic anemias replacing this test, because they are better
CBC indicators of the deficiency
Decreases in RBC count, WBC, platelet, Hgb, Treatment of megaloblastic anemia
and Hct; elevated MCV Once the underlying cause is established, specific
RBCs include oval macrocytes, and inclusions treatment can be determined
may be present (Howell-Jolly bodies, NRBCs, Remove/repair underlying problem
Cabot rings) Supplement with appropriate deficient vitamin,
Neutrophils may appear hypersegmented either vitamin B12 or folic acid
Bone marrow In cases of intrinsic factor problems,
May be used to confirm presence of megaloblas- intramuscular injection of vitamin B12 can
tic anemia be used
Megaloblastic changes are apparent (nucleus- If neurologic issues occur with vitamin B12
to-cytoplasm [N:C] asynchrony) deficiency, they may be irreversible based on
Hypercellularity with increased, abnormal the extent of the damage
RBC precursors Follow-up testing
Giant bands and metamyelocytes CBC and reticulocyte count
Other laboratory testing Reticulocyte response may be seen within a week
Serum vitamin B12 and folic acid assays of treatment
Decreases appear with deficiencies Hypersegmented neutrophils are replaced by
Methylmalonic acid (MMA) normal neutrophils in about 2 weeks
Increased in vitamin B12 deficiency, because it CBC may return to overall normal state in 3 to
is needed for conversion of methylmalonyl 6 weeks
coenzyme A (CoA) in the pathway
Homocysteine
Nonmalignant Macrocytic Anemias
Increased in vitamin B12 and folic acid defi-
ciency, because vitamin B12 is needed for con- Macrocytosis without megaloblastic changes can
verting homocysteine to methionine in the occur
pathway Normal babies
Intrinsic factor antibodies After delivery, macrocytosis and reticulocytosis
Parietal cell antibodies are normal in newborns
Ova and parasite examination in cases of sus- MCV may increase up to 123 fL
pected vitamin B12 deficiency resulting from D. Liver disease
latum infection Alcoholism
Schilling test Hypothyroidism
Classic two-part test used to determine if the
cause of vitamin B12 deficiency is malabsorption,
dietary deficiency, or a lack of intrinsic factor
HYPOPROLIFERATIVE DISORDERS
Part I: Patients are dosed orally with radi- Disorders occurring as a result of decreased or
olabeled vitamin B12, followed by a flush- absent production of hematopoietic cells in the bone
ing dose of unlabeled vitamin B12. Excess marrow
vitamin B12 is filtered by the kidney, and Includes aplastic anemia, both inherited and
urine is measured for radioactivity. If radi- acquired, in addition to other disorders resulting
olabeled vitamin B12 levels are elevated, the from decreases in bone marrow production
patient is likely deficient in vitamin B12 and Other disorders resulting from decreases in bone
unable to absorb the vitamin marrow production include
Part II: If the vitamin B12 excretion in part I Pure red cell aplasia
is decreased, the patient receives an oral Congenital dyserythropoietic anemia
dose of radiolabeled vitamin B12 and a dose Myelophthisic anemia
of intrinsic factor. If the radiolabeled vita- Anemia resulting from chronic kidney disease
min B12 levels are increased from those in
part I, the patient is lacking intrinsic factor
and has pernicious anemia. If the levels are
Aplastic Anemia
abnormal, the patient may have another Rare disorders characterized by pancytopenia in the
defect leading to malabsorption peripheral circulation. Result from decreased bone
CHAPTER 3 Hematology 109

marrow production of RBC, WBC, and platelets Hypocellular with increased fat cells
because of deficiency or damage of hematopoietic Biopsy is needed for accurate diagnosis
stem cells Decreased granulocytes, RBCs, and platelets
Acquired aplastic anemia Treatment
Most cases identified are acquired, the majority of Eliminate the problem causing bone marrow
which are idiopathic failure, if it can be identified
Cause of the marrow failure in idiopathic cases is Transfusions (RBCs and platelets) can be
currently unknown given, as needed
Other acquired cases have been linked to various Immunotherapy may be used
drugs/chemicals, radiation, viral infections, and Bone marrow/stem cell transplant can be per-
other miscellaneous causes formed for severe cases, if a suitable donor is
Inherited aplastic anemia available and the recipient meets certain
A smaller number of cases are inherited, including criteria
Fanconis anemia, dyskeratosis congenita, and Treatments vary based on the specific case,
Shwachman-Diamond syndrome disorder, and suspected cause
Fanconis anemia
Chromosomes are susceptible to breakage and
Pure Red Cell Aplasia
the cell may not be able to repair DNA damage.
Cells also show accelerated telomere shortening Bone marrow exhibits decreased production of RBCs
and apoptosis. and RBC precursors, whereas other cell lines are pre-
This property is used to help diagnose sent and produced normally
Fanconis anemia Acquired
Genetic mutations in one of 13 genes Primary is idiopathic or autoimmune
FANCA mutations occur most frequently. Secondary is usually associated with tumors, infec-
Inheritance is autosomal recessive in all of tions, drugs or chemicals, other disorders
the associated genes except FANCB, which Transient erythroblastopenia of childhood (TEC)
is an X-linked gene. Pure red cell aplasia (PRCA) seen in children,
Appears at birth or in early childhood often related to viral infection
Symptoms resulting from pancytopenia may Treated by transfusion, as needed, although
be apparent early on or manifest later in life most patients eventually restore their ability
Skeletal abnormalities may be seen in many, in to produce RBCs
addition to abnormalities in skin pigmentation, Congenital
and organ problems Diamond-Blackfan anemia
Patients may have a higher risk for malignancies, Mutations are usually autosomal dominant;
in addition to the bone marrow failure however, they also may occur sporadically
Dyskeratosis congenita Many patients show symptoms before 1 year of
Very rare disorder in which chromosomes have age, although some are asymptomatic
short telomeres Many patients have physical issues, including
Inheritance is autosomal dominant, X-linked bone malformations
recessive, and autosomal recessive, with various Congenital dyserythropoietic anemia (CDA)
mutations present Group of inherited rare disorders leading to inef-
Patients may show abnormalities in skin pigmen- fective erythropoiesis
tation and nails, in addition to a variety of Patients have varying degrees of refractory
abnormalities anemia and symptoms resulting from the inef-
Shwachman-Diamond syndrome fective erythropoiesis, in addition to variable
Autosomal recessive disorder leading to neutro- physical effects
penia and/or anemia and thrombocytopenia Mutations are usually autosomal recessive; how-
Patients have decreased pancreatic enzymes, ever, rare cases of an autosomal dominant inher-
skeletal anomalies, and increased risk of itance have been reported
infection Symptoms usually appear in childhood or
General CBC findings in aplastic anemia adolescence
Pancytopenia, with decreases in one or more cell lines Anemia of chronic kidney disease
Anemia with normocytic, normochromic RBCs Individuals are unable to produce erythropoie-
Decreased granulocytes with normal to slightly tin, leading to decreased production of RBCs
decreased lymphocytes Therapy for PRCA
Decreased platelets Therapy is variable based on each specific anemia,
Decreased reticulocyte production including supportive therapy, transfusion therapy,
Bone marrow corticosteroids, treating the underlying problem
110 CHAPTER 3 Hematology

Patients are usually normal and asymptomatic


HEMOGLOBINOPATHIES
unless in extreme conditions of hypoxia
Disorders resulting from genetic mutations that lead to Laboratory diagnosis
structural changes in the Hgb molecule CBC
Most occur because of amino acid substitutions in Generally normal, although some target cells
the b-globin chains may be present
Numerous Hgb variants exist; however, several are Sickle screen
more common than others Positive
Common abnormal Hgbs Hgb Electrophoresis and HPLC
Hgb S Hgb A and Hgb S are present
Usually affects African patients or patients of
African descent Hemoglobin C
Mutation: b6(Glu!Val)
Usually affects patients of West Africa or West African
Hgb S polymerizes into long, thin polymers
descent
to form sickles when O2 saturation decreases
Mutation: b6(Glu ! Lys)
Hgb C polymerizes into thick crystals when O2 sat-
Sickle Cell Disease uration decreases
RBC shape alteration is less extreme than in Hgb S
Homozygous for Hgb S
Majority of Hgb present is Hgb S Crystals look like bars of gold or the Washington
Clinical symptoms Monument
Diagnosis
Chronic hemolytic anemia, autosplenectomy,
CBC
vasoocclusion, vasoocclusive crises, susceptibility
Mild-to-moderate anemia
to bacterial infections, acute chest syndrome, pul-
Increased target cells and Hgb C crystals
monary hypertension, myocardial infarction, and
may appear
numerous other complications
Some polychromasia and NRBCs may be present
Laboratory diagnosis of sickle cell disease
Solubility testing is negative
CBC
Hgb electrophoresis and HPLC
Normocytic, normochromic anemia
Positive for Hgb C if homozygous and positive
Various poikilocytes present
for Hgb A and C if heterozygous for the mutation
Drepanocytes (sickle cells)
Treatment is usually not needed and prognosis
Target cells, NRBC, Howell-Jolly bodies,
is good
polychromasia are commonly seen
NOTE: Hgb C-Harlem is characterized by a double
WBC count usually elevated, with neutrophils
mutation (b6(Glu!Val) and b73(Asn!Asp) and is clinically
predominating
significant if inherited in combination with Hgb S
Reticulocyte count is elevated
Bone marrow is not indicated
Other tests
Hemoglobin E
Dithionite solubility (sickle screen) is positive Usually affects patients from Southeast Asian, particu-
Hgb electrophoresis is positive for Hgb S and larly Thailand
NO Hgb A is present Mutation: b26(Glu!Val)
HPLC or isoelectric focusing may also be used to Diagnosis
demonstrate Hgb S CBC
Treatment and follow-up Mild anemia
Supportive therapy and prognosis Microcytes and target cells
Transfusions, prophylactic antibiotics, avoidance Solubility testing is negative
of situations leading to low O2 saturation, and Hgb electrophoresis and HPLC
dehydration Positive for Hgb E
Hydroxyurea to help retain Hgb F production Therapy is not usually needed and prognosis is good
Bone marrow transplant may be an option if a
matched donor is available Hemoglobin SC
Appropriate therapy can lead to a life span of Combination disorder with inheritance of mutations
approximately 50 years for both Hgb S and Hgb C
Most common of the compound disorders
Sickle Cell Trait Similar clinical picture to sickle cell disease; however, it
Heterozygous for Hgb S does not usually manifest clinically until teenage years
Majority of Hgb present is Hgb A and tends to be slightly less severe than Hgb S
CHAPTER 3 Hematology 111

Laboratory diagnosis Hgb H disease


CBC Three gene deletion (/a)
Mild anemia Unpaired b chains will form tetramers of
Target cells and Hgb SC crystals may be present Hgb H
Hgb SC polymerizes into crystalline struc- Other Hgb present are Hgb A2, some
tures that have features of both Hgb S and Hgb Bart
Hgb C, often described to look like birds or Clinical symptoms
fingers Mild-to-moderate chronic hemolytic anemia
Solubility testing is positive because of the pres- Splenomegaly
ence of Hgb S Other variable findings may occur
Hgb electrophoresis and HPLC CBC
Hgb S and Hgb C are present Decreased RBC and Hgb
Therapy and prognosis is similar to sickle cell Microcytic, hypochromic RBCs, target cells,
disease, although expected life span is longer and other poikilocytes may be present
Hgb H inclusions (precipitated Hgb H) may
be seen when using a supravital stain, such
Combination Disorders
as new methylene blue
Mutations may occur in combination because of inher- a-Thalassemia minor
itance of different genetic mutations from each parent Two gene deletion (/aa) or (a/a)
Each compound disorder has variable symptoms and Asymptomatic presentation
severities Usually no therapy is needed
Some examples include Hgb SC disease, Hgb Sb thal- CBC
assemia, Hgb CHarlem Mildly decreased Hgb and Hct, with micro-
cytic, hypochromic cells
Silent carrier
THALASSEMIAS One gene deletion (a/aa)
Disorders caused by genetic mutations that lead to No clinical symptoms, because globin chain ratio
is almost normal
quantitative changes in the amount of globin chains
No therapy is needed
produced, resulting in an imbalance of globin chain
CBC
synthesis
Types and severity of thalassemia depends on the No hematologic abnormalities
Diagnosis
globin gene mutated (a or b) and the number of
Diagnosis is by genetic analysis
genes affected by the mutation
a-Globin is coded for on chromosome 16
A normal genotype is designated as aa/aa b-Thalassemia
b-Globin is coded for on chromosome 11
Thalassemias caused by mutations or deletions in the
A normal genotype is designated as b/b
b-globin genes
b+ Designates decreased production of b Four main groups with varying clinical
chains; b0 shows the absence of b chains
presentations
a-Thalassemia major
a-Thalassemia
Occurs with genotypes (b+/b+), (b+/b0),(b0/b0)
Thalassemias caused by mutations or deletions in the Also called Cooleys anemia
a-globin genes Severe hemolytic anemia, usually diagnosed at
Four main groups approximately 6 months of age when Hgb F levels
Hydrops fetalis/Hgb Bart normally decrease (the patient cannot make Hgb A)
Four gene deletion (/) so no a chains are Clinical picture shows hepatosplenomegaly and
produced distinct bone changes, if untreated, in addition
Hgbs present are Bart (g4) with small amounts to other physical issues
of Hgb Portland and Hgb H (b4) Pathologic fractures and abnormalities of the
Not compatible with life as the blood is unable to skull are frequently seen as a result of ery-
oxygenate tissues because of high O2 affinity of throid hyperplasia in the bone marrow
Hgb Bart CBC
Fetus will either be delivered prematurely and Severe anemia
die shortly after birth or will be delivered still- RBC count is slightly elevated, but pro-
born in the third trimester nounced hypochromic/microcytic cells and
Clinically exhibit severe anemia, cardiac target cells are present, in addition to other
defects, and hepatosplenomegaly varied morphologies and NRBCs
112 CHAPTER 3 Hematology

Bone marrow
HEMOLYTIC ANEMIA
Usually not performed, but would show ery-
throid hyperplasia and ineffective erythropoiesis Disorders of premature RBC destruction, leading to
Hgb electrophoresis and HPLC anemia
Increased Hgb F, slightly increased Hgb A2, Classified in several different ways
little or no Hgb A Intrinsic versus extrinsic defects
Therapy and prognosis Acquired versus hereditary defects
Supportive therapy with regular transfusions Intravascular versus extravascular hemolysis
Iron chelation therapy to help avoid iron General features
overload Clinical presentation
Bone marrow transplant if a good match is General anemia symptoms when hemolysis leads
available to anemia
b-Thalassemia intermedia Jaundice may be present, variable depending
Occurs with genotypes (bsilent/bsilent), (db0/db0), on cause
(b0/db0) Splenomegaly in cases of extravascular
Clinical symptoms and CBC hemolysis
Varies because of multiple genotypic Gallstones in cases of chronic hemolysis
presentations Laboratory testing
Usually not transfusion dependent Increased bilirubin: RBCs are breaking down
Anemia varies between that of b-thalassemia Decreased haptoglobin: Free Hgb from intravascu-
minor and b-thalassemia major lar hemolysis may exceed haptoglobins binding
b-Thalassemia minor capacity
Occurs with genotypes (b+/b), (b0/b) Increased reticulocyte count
Clinical symptoms and CBC Variable anemia depending on degree and fre-
Usually mild anemia quency of hemolysis
Normal to elevated RBC with decreased Anisocytosis and poikilocytosis on the CBC, usu-
Hgb and Hct ally including some macrocytosis and polychro-
Poikilocytosis (target cells and some masia (reticulocyte production) and
others) spherocytes and/or schistocytes depending on
Hgb electrophoresis and HPLC the cause of the anemia
Elevated Hgb A2 and F
Silent carrier
Occurs with genotypes (bsilent/b)
Hemolytic Anemias Resulting from Intrinsic
Defects
No clinical symptoms, because globin chain
ratio is almost normal Abnormalities in Red Blood Cell Membrane
No therapy is needed Hereditary Spherocytosis
CBC does not show hematologic Incidence is 1/3000 of northern European ancestry
abnormalities Autosomal dominant inheritance in most cases
No hematologic abnormalities Mutations affect genes coding for membrane pro-
Diagnosis is by genetic analysis teins, leading to changes in the membrane skeleton
and decreased survival because of decreased
deformability
Other Related Disorders Symptoms are variable, but a symptomatic clinical
Hereditary persistence of fetal hemoglobin (HPFH) picture shows anemia, jaundice, and splenomegaly
Deletion in the b-globin gene leading to increased CBC
production of Hgb F (g chains) Decreased RBC, Hgb, and Hct, increased
Patients are usually asymptomatic; abnormali- MCHC and RDW
ties will show up in Hgb electrophoresis/HPLC Spherocytes, polychromasia
where increases in Hgb F are present Other testing
Hgb Lepore Family history to look for evidence of
db fusion gene inheritance
Shows anemia similar to b-thalassemia Direct antiglobulin test (DAT) is negative,
minor which rules out immune-mediated cause
Combination disorders Osmotic fragility is increased because of
Hgb Sthalassemia decreased membrane deformability
Hgb Cthalassemia Autohemolysis tests and membrane protein
Hgb Ethalassemia studies may be performed
CHAPTER 3 Hematology 113

Treatment and prognosis Cells lack glycoslyphosphatidlyinositol-anchored pro-


Many are asymptomatic, but those with teins, including CD55 and CD59
severe hemolysis may require splenectomy RBCs are susceptible to complement lysis, because
or transfusion therapy CD55 and CD59 inhibit complement and are absent,
Hereditary Elliptocytosis cells may lyse spontaneously
Mutations in genes coding for spectrin or band 4.1, Clinical presentation
leading to disruption of the cell shape in circulation Usually manifests in young adults, but can occur at
Incidence is 1 in 2000 to 4000, although it is any age
more common in Africa and the Mediterranean Variable symptoms related to the hemolysis, throm-
and those descended from the area bosis resulting from thrombophilia, and bone mar-
Several variants occur, including hereditary row failure may occur
pyropoikilocytosis Laboratory findings
Many cases are asymptomatic, and disorder is discov- General signs of intravascular hemolysis, including
ered incidentally; however, some exhibit more pro- hemoglobinuria
nounced hemolysis Reticulocytes show a slight increase
CBC Bone marrow examination may be done to look for
Normal (in asymptomatic cases) to decreased RBC, underlying marrow failure or cytogenetic
Hgb, and Hct (in hemolytic cases) abnormalities
Increased elliptocytes, although morphology is Flow cytometry shows deficiencies of CD55 and
more extreme in hereditary pyropoikilocytosis, CD59
which also may show schistocytes and micro- CD24 and CD15 also may be deficient
spherocytes Therapy and prognosis
Other testing Eculizumab can be used for hemolysis to decrease
Thermal sensitivity may be increased in hereditary complement activity
elliptocytosis with spectrin mutations Supportive transfusion, prophylactic antibiotics,
Molecular testing may be done to look for vitamin supplementation to counter loss through
mutations kidneys, anticoagulants are used if there are clotting
Treatment and prognosis complications, and stem cell transplant may be an
Symptomatic cases with anemia may require trans- option if a suitable donor is available
fusion therapy and sometimes splenectomy Abnormalities in Enzymes
Usually asymptomatic cases require no treatment Glucose-6-Phosphate Dehydrogenase Deficiency
and have a good prognosis RBCs are unable to reduce glutathione, which is
Acanthocytosis needed to battle oxidant damage in the cell, leading
Spur cell anemia to oxidation of Hgb into Heinz bodies, which are then
Defects in RBC membrane lipid balance, often removed from circulation
resulting from liver issues X-linked disorder
Seen in severe liver disease as a result of excess Multiple mutations and enzyme a presentation of
free plasma cholesterol that accumulates on chronic hemolytic anemia to patients with few or
the RBC membrane, leading to deformation of no abnormalities.
the cell within the spleen, if cells are not Tends to present in patients in Africa and the Mid-
hemolyzed East, and their descendants
CBC Most common RBC enzyme deficiency, with a prev-
Moderate anemia with acanthocytes alence of up to 5% worldwide
Clinical presentation Clinical presentation
Splenomegaly, jaundice Most patients are asymptomatic unless exposed
Therapy and prognosis to something that will trigger hemolytic episodes
Prognosis is poor unless a patient can success- Oxidative drugs, including antimalarial medica-
fully undergo a liver transplant tions, infections, and fava beans are main trig-
Neuroacanthocytosis gers of hemolysis
Rare inherited disorders with neurologic symptoms Leads to transient hemolytic episodes within sev-
and acanthocytosis eral hours of exposure and may begin the return
Abetalipoproteinemia, McLeods syndrome to normal once the offending trigger is removed
Paroxysmal Nocturnal Hemoglobinuria or resolved
Rare acquired disorder resulting from stem cell muta- Hemoglobinuria may be one of the first clinical
tion in the PIGA gene clues
Defect in platelets and WBCs, as well Laboratory testing
Severity is variable depending on the phenotype CBC is normal unless patient has an episode
114 CHAPTER 3 Hematology

With hemolytic episodes, patients exhibit a nor- Disseminated Intravascular Coagulation (DIC)
mocytic or normochromic anemia and Hgb may Activation of all parts of the hemostatic systems lead-
drop quickly, but response can be variable ing to the production of fibrin clots, the consumption
Bite and blister cells may occur on Wrights stain, of platelets and coagulation proteins, and degradation
and Heinz bodies are often visualized when using of fibrin. Clotting and bleeding both occur
a Heinz body stain or other supravital stain. Acute or chronic, both secondary to other underlying
Other morphologic findings may be present conditions
Glucose-6-phosphate dehydrogenase (G6PD) enzyme Clinical presentation
activity screens and quantitative assays Variable presentation, because patients show
May be used to screen or to assess the degree of symptoms consistent with the underlying disorder
severity that has prompted disseminated intravascular
Therapy and prognosis coagulation
Usually involves avoiding or removing the trigger Underlying causes may include infections, malig-
for hemolysis nancies, obstetric complications, venom exposure,
Most cases require no treatment and resolve on and chronic inflammation, among others
their own, but some severe cases may require trans- CBC
fusion therapy Anemia and decreased platelet count, WBC may be
Pyruvate Kinase Deficiency elevated
Autosomal recessive disorder, relatively rare Schistocytes and apparent thrombocytopenia
Leads to adenosine triphosphate (ATP) depletion and Laboratory findings
Coagulation tests are abnormal
increase in 2,3 BPG
Clinical findings Elevated prothrombin time (PT), activated par-
Variable from asymptomatic to chronic hemolytic tial thromboplastin time (aPTT)
D-dimer/fibrin degradation products (FDPs), and
episodes
Laboratory findings decreased fibrinogen
CBC Therapy and prognosis
Underlying disorder should be treated, in addition
Variable RBC and Hgb
to supportive therapies. In cases of acute DIC, ther-
Increased echinocytes with other variable mor-
phologic findings apies may be more aggressive to try to stem organ
Pyruvate kinase enzyme activity can be measured failure. Heparin can be used carefully to try to stop
activation of the coagulation cascade. Blood prod-
spectrophotometrically. Activity is decreased.
Treatment and prognosis ucts may also be used, including frozen plasma to
Supportive treatment with transfusions, as replace consumed coagulation proteins and replace
indicated blood volume, platelet transfusions may be admin-
Some severe cases may require splenectomy istered if thrombocytopenia is severe
Thrombotic Thrombocytopenic Purpura (TTP)
Patients have long von Willebrand factor (vWF) multi-
mers that bind vascular endothelium and platelets, trig-
Hemolytic Anemias Resulting from gering platelet aggregation. Platelets are used up in this
Extrinsic Defects process and microclots block small blood vessels, which
leads to shearing of the RBCs in circulation
A variety of different conditions can cause mechanical Disorder is acquired or inherited
destruction of the circulating RBCs; these are not Several subtypes are present
immune mediated hemolytic anemias Most patients have a decrease or mutation in the
Microangiopathic Hemolytic Anemia ADAMTS 13 gene, which is normally used to cleave
Group of disorders characterized by intravascular long vWF multimers into smaller fractions, helping
fragmentation of RBCs as they move through blood to avoid excess platelet adhesion to the endothelium
vessels obstructed by microclots or endothelial Clinical findings
damage Usually characterized by a combination of symp-
CBC shows decreases in Hgb and Hct with schisto- toms, including microangiopathic hemolytic ane-
cytes usually appearing on the peripheral smear, in mia, thrombocytopenia, neurologic symptoms,
addition to possible other morphologies renal dysfunction, and fever
Other laboratory testing CBC
Bilirubin (unconjugated) is increased Characterized by decreased Hgb (usually <10 g/dL)
Haptoglobin is decreased and platelet count (<20  109/L), with schistocytes
Hemostasis testing varies based on the specific on the peripheral smear
disorder Laboratory testing
CHAPTER 3 Hematology 115

Coagulation tests (PT, APTT) are usually normal Mild hemolysis resulting from RBCs flowing
vWF multimer analysis is abnormal around the implanted valves
Therapy and prognosis Patients are often asymptomatic, but severe cases
Plasma exchange therapy to remove large vWF mul- may present with noticeable anemia
timers and providing the missing ADAMTS 13 pro- CBC may show the presence of schistocytes
tease can lead to favorable prognosis If anemia is severe, patients may require transfusion
Immunosuppressive therapy may also be used therapy and surgical repair of the prosthetic valve
Hemolytic Uremic Syndrome (HUS) March hemoglobinuria (exercise induced)
Microangiopathic hemolytic anemia with thrombocy- Condition occasionally seen in long-distance run-
topenia and renal involvement as a result of clots form- ners or others who engage in intense exercise
ing in the microvasculature of the kidney Although hemolysis may occur, patients usually do
Acquired disorder, usually found in young children not have anemia unless hemolysis is recurrent
with a history of hemorrhagic Escherichia coli or Shi- Hemoglobinuria may be present, in addition to
gella dysenteriae infections, although it may be found decreased haptoglobin levels
in adults after exposure to immunosuppressive agents Therapy includes minimizing physical trauma or
or chemotherapy discontinuing the activity that leads to hemolysis
Clinical presentation Infectious Agents
Children often present with a bloody diarrhea, CBC Malaria (Plasmodium spp.)
abnormalities, and renal issues, whereas adults tend Caused by infection with one of the major species of
to present with renal issues and CBC abnormalities Plasmodium
without bloody diarrhea Malarial parasites may lyse RBCs as they use Hgb,
CBC in addition to the destruction of infected cells by
Decreased RBC, Hgb (<10 g/dL), Hct, and platelets extravascular hemolysis; additionally, inflamma-
Smear shows schistocytes and decreased platelets tory response can lead to inhibited and ineffective
Laboratory testing erythropoiesis
Blood urea nitrogen and creatinine are elevated Clinical symptoms vary but often include fever,
Culture results may be positive for E. coli or S. chills, headache, and other physical manifestations
dysenteriae Organism presence can be confirmed by visualiza-
Urinalysis shows elevated protein, blood, and casts tion of organism, intracellular or extracellular, on
Therapy and prognosis a peripheral smear
Supportive therapy, as needed; prognosis is usually Treatment using chloroquine drugs is administered
favorable unless patient harbors organism from areas known
Other Causes for chloroquine resistance, where other drugs need
HELLP Syndrome to be used. Transfusion therapy may be used, too, if
Hemolysis, elevated liver enzymes, and low platelet anemia is severe
count Babesia
Relatively uncommon complication of pregnancy, Babesia microti is the most common cause of infection
although it is more likely to affect patients with pre- Some patients are asymptomatic, whereas others
eclampsia toward the end of their pregnancy show mild-to-severe anemia and generalized flulike
Vascular insufficiency in the placenta can lead to dys- symptoms
function in the maternal endothelium, causing platelet Diagnosis is confirmed by the visualization of
activation and fibrin deposition in the small vessels organism (ring forms or tetrads) on peripheral
Laboratory testing smear, in addition to antibody testing for organism
CBC shows decreased platelet count and lactate Bacteria
dehydrogenase is elevated Toxin-producing microorganisms can occasionally
Therapy and prognosis lead to hemolysis
Relatively good with supportive therapy and deliv- Clostridium perfringens
ery of the fetus and placenta a-Toxin is produced and can hydrolyze mem-
Hypertensive Crisis and Malignant Hypertension brane phospholipids, rendering changes in RBC
Severe increase in blood pressure, leading to acute shape and deformability
organ damage Hemolysis is severe and can lead to DIC and
Endothelial cells are damaged, leading to activation of renal failure; prognosis is poor
the hemostatic system Additional Causes of Mechanical Hemolysis
Platelets return to normal once the blood pressure is Drugs
controlled Chemicals
Mechanical Damage Venoms
Prosthetic heart valves Thermal injury
116 CHAPTER 3 Hematology

Immune-Mediated Hemolytic Anemia CBC


RBC life span is shortened because of presence of anti- Mild-to-severe anemia with polychromasia and
bodies, usually IgG or IgM, on RBC surfaces spherocytes
Autoimmune or alloimmune causes Laboratory testing
IgM antibodies usually activate complement, leading DAT is positive in majority of cases
to intravascular and extravascular hemolysis Treatment and prognosis
IgG antibodies can occur with or without the pres- In symptomatic cases, prednisone therapy may be
ence of complement, and most removal attempts used
are extravascular, leading to hemolysis or the Immunosuppressive therapy may be used, but
increased presence of spherocytes leaves the risk for side effects
General laboratory findings in immune-mediated Transfusion therapy can be used in cases with severe
hemolytic anemia anemia
CBC Cold Agglutinin Disease
Decreased RBC, Hgb, and Hct with macrocytes, Usually caused by IgM antibodies that react best at 4 C
spherocytes, and polychromasia; increased Usually do not react at temperatures above 30  C
reticulocyte count IgM antibodies bind to the RBC after exposure to
Laboratory testing colder temperatures, and they can activate the comple-
Increased bilirubin and lactate dehydrogenase, ment cascade as they move through the cooler extrem-
decreased haptoglobin ities. When cells return to warmer areas, the IgM is no
DAT is positive and can further be tested for IgG longer a factor; however, complement remains and is
and C3d removed via extravascular hemolysis or in some cases,
intravascularly
Autoimmune Hemolytic Anemia Can occur in acute or chronic state
Caused by autoantibodies that attach to the RBC sur- Chronic cold agglutinin disease (CAD) is rare and
face (Table 3-11) may be idiopathic or secondary to lymphoid
Warm Autoimmune Hemolytic Anemia malignancy
Warm autoimmune hemolytic anemia is the most Clinical presentation
common autoimmune anemia (AIHA), occurring as Symptoms are variable, because patients have vari-
idiopathic or secondary disease able anemia (mild to severe). Depending on the
Secondary particularly in B-cell lymphoid malig- severity, patients may show general anemia symp-
nancies, such as chronic lymphocytic leukemia toms and acrocyanosis
(CLL), solid tumors, autoimmune disorders, and Acute CAD can occur secondary to Mycoplasma
viral infections pneumoniae and viral infections
Usually caused by IgG autoantibodies that react the CBC
best at 37 C If blood has cooled before analysis, values will not
Usually extravascular hemolysis occur in their normal proportions, leading to

T A B L E 3- 1 1 Characteristics of Autoimmune Hemolytic Anemias

Mixed-Type
Warm Autoimmune Cold Aggultinin Paroxysmal Cold Autoimmune Hemolytic
Hemolytic Anemia Disease Hemoglobinuria Anemia
Immunoglobulin class IgG (rarely IgM, IgA) IgM IgG IgG, IgM
Optimum reactivity 37 C 4 C; reactivity extends 4 C 4 -37 C
temperature of to >30 C
autoantibody
Sensitization detected by IgG or IgG + C3d; only C3d C3d C3d IgG and C3d
direct antiglobulin test uncommon
Complement activation Variable Yes Yes Yes
Hemolysis Extravascular primarily Extravascular; rarely Intravascular Extravascular and
intravascular intravascular
Autoantibody specificity Panreactive or Rh complex; I (most), i (some), Pr P Panreactive; unclear
rarely specific Rh or other (rare) specificity
antigen
From Rodak BF, Fritsma GA, Keohane E: Hematology: clinical principles and applications, ed 4, St Louis, 2012, Saunders.
Ig, Immunoglobulin.
CHAPTER 3 Hematology 117

decreases in the RBC and Hct, with a dispropor- Drug is discontinued and should be avoided in the
tional (normal) Hgb and grossly abnormal future
increases in the MCV, MCH, and MCHC. The If anemia is severe, the patient may require transfu-
Hgb value is normal because the method of mea- sion therapy
surement requires lysis of cells before analyzing
Hgb. Agglutinates may be seen on peripheral Alloimmune Hemolytic Anemias
Hemolytic anemia resulting from immune incompati-
smear in high-titer cold agglutinins. Warming sam-
bility of donor and recipient (or immune incompatibil-
ple before reanalyzing may resolve the agglutina-
ity of mom and baby)
tion or keeping sample warm until the time of
Antibodies can be IgM or IgG with intravascular and/
analysis may also help
Laboratory testing or extravascular hemolysis
Onset can be immediate or delayed
Cold agglutinin titer is increased
Therapy and prognosis Transfusion Reactions
Patients do not often require transfusions unless Acute Hemolytic Reaction
hemolysis leads to a severe anemia. Patients are usu- Occurs within hours of transfusion of incompatible
ally instructed to avoid cold temperatures blood products
Paroxysmal Cold Hemoglobinuria Most commonly caused by ABO incompatibility,
Acute cold AIHA associated with the Donath- because recipients produce natural IgM antibodies to
Landsteiner antibody (anti-P autoantibody) the incompatible antigen, leading to complement-
Donath-Landsteiner antibody is a biphasic anti- mediated intravascular hemolysis.
Clinical presentation
body that can bind to RBCs and partially activate
Various symptoms occur, including fever,
complement at low temperatures (optimal binding
at 4  C) but full-blown complement activation chills, urticaria, chest pain, back pain, shock,
and hemolysis occur at 37 C cardiac symptoms, and bleeding (if DIC is
Clinical presentation present)
Mainly affects children, although it can occur in CBC
Hgb is decreased
adults
Symptoms include fever, malaise, and extremity and Laboratory tests
Hemoglobinuria, hemoglobinemia
back pain that usually manifests 1 to 2 weeks after a
DAT is usually positive on posttransfusion specimen
respiratory infection. Patients often have a rapid
Haptoglobin is decreased
onset of hemolysis and hemoglobinuria, leading
Coagulation tests may be abnormal if DIC occurs
to a severe anemia
CBC Therapy and prognosis
Severe anemia after hemolytic episodes (Hgb often Transfused unit must be stopped as soon as
<5 g/dL) with morphology showing polychromasia possible, and treatment to minimize or correct the
and spherocytes, in addition to various other clinical symptoms is undertaken quickly
morphologies Delayed Hemolytic Transfusion Reaction
Laboratory tests Reaction may occur days to weeks after initial transfu-
DAT is positive for C3d (autoanti-P usually disso- sion, because the recipients antibody titer may take
ciates from RBCs at 37 C) time to increase
Donath-Landsteiner test is positive Antibodies implicated are usually IgG and can bind to
Therapy and prognosis transfused RBCs, which are then removed by extravas-
Transfusion therapy is needed with severe hemo- cular hemolysis
lytic episodes; however, the disorder is usually CBC
self-limiting and has a favorable prognosis May not show adequate posttransfusion increase
in Hgb
Drug-Induced Hemolytic Anemia Laboratory tests
Characterized by a sudden onset of anemia with hall-
DAT is positive
marks of hemolytic anemia after a patient is exposed to Bilirubin is usually indirect fraction may be
a medication
increased
Patients produce antibodies to the medication, which
are either drug dependent or drug independent Hemolytic Disease of the Fetus and Newborn
Laboratory testing Rhesus (Rh) hemolytic disease of the fetus and new-
DAT is positive born (HDFN) occurs when maternal IgG antibodies
CBC cross the placenta and enter fetal circulation, binding
Anemia of varying severity to fetal RBCs positive for the corresponding antigen,
Therapy and prognosis leading to extravascular hemolysis
118 CHAPTER 3 Hematology

Clinical presentation Congenital neutropenia may occur in several disor-


Fetus may show erythroid hyperplasia in the mar- ders. The disorders are relatively rare
row and extramedullary hematopoiesis to compen- Acquired neutropenia occurs more commonly. It is
sate for hemolysis usually as a result of decreased production in the
Laboratory testing bone marrow, anti-neutrophil antibodies, chemo-
Maternal samples are tested for ABO and Rh to therapy and radiation, and severe infections. Cell
determine the need for RhIG to help prevent alloim- production is unable to keep up with cell
munization to fetal D antigen consumption
Antibody titers may be used to help monitor the
Eosinophilia
patient and determine if other methods are needed Absolute counts are increased above 0.40  109/L
for monitoring the patient Frequently seen in parasitic infections and allergies
CBC (fetus) May also be increased in some myeloproliferative neo-
Decreased Hgb with polychromasia and NRBCs on
plasms, including chronic myelogenous leukemia
the peripheral smear (CML) and chronic eosinophilic leukemia
Reticulocyte count is increased
Bilirubin is usually increased Basophilia
DAT is positive Absolute counts are increased above 0.15  109/L
Therapy and prognosis Can be seen in some hypersensitivity reactions
In severe cases, intrauterine transfusion may be May be increased in malignancies, particularly myelo-
indicated or exchange transfusions may be adminis- proliferative neoplasms, including CML
tered after delivery Monocytosis
Phototherapy can help reduce bilirubin after delivery Absolute counts are increased above 1.1  109/L
NOTE: ABO HDFN also can occur and is more common Often seen as patients are recovering from infections
than Rh HDFN; however, it is usually asymptomatic May be increased in some solid tumors and hemato-
or produces mild anemia with spherocytes and poly- logic malignancies, including acute monocytic leuke-
chromasia in addition to hyperbilirubinemia. IgM anti- mia, acute myelomonocytic leukemia or chronic
bodies are unable to cross the placenta myelomonocytic leukemia
Lymphocytosis
Absolute counts are increased above 10.0  109/L
(children) or above 4.8  109/L (adults)
NONMALIGNANT WHITE BLOOD Reactive lymphocytes are typically present in infec-
CELL DISORDERS tious monocytosis and other viral infections
A more normal lymphocyte morphology is seen in dis-
Quantitative White Blood Cell Disorders orders such as Bordetella pertussis infection
Neutrophilia Lymphocytopenia
Causes of Neutrophilia Absolute counts are decreased below 2.0  109/L (chil-
Increase in relative and/or absolute numbers of cells dren) or below 1.0  109/L (adults)
(>8.7  109/L or >70%) Decreased counts are often seen in immunodefi-
Neutrophils can move from marginating to circulat- ciencies, particularly human immunodeficiency virus
ing pool or increased need can lead to additional infection and also during steroid treatment
release from bone marrow into circulation
Physiologic neutrophilia, also known as shift or
Changes in White Blood Cell Morphology
transient neutrophilia occurs when the body is
under stress. Cells move from the marginating to Abnormalities (changes) seen in neutrophils
the circulating pool. Counts will return to normal Dohle bodies: Pale bluish inclusions in the cyto-
levels plasm composed of rough endoplasmic reticulum,
Infections, particularly bacterial usually associated with bacterial infections and
Inflammation inflammation
Medications Toxic granulation: Large bluish-black granules
Increases may be caused by some neoplasms, partic- appearing in the cytoplasm, usually present in
ularly myeloproliferative disorders inflammation
Numerous others Vacuolization: Vacuoles within the cytoplasm that
are often indicative of phagocytosis. Vacuoles can
Neutropenia be seen in bacterial or fungal infections, but also
Causes of Neutropenia may appear as artifact in old samples
Decrease in the absolute numbers of cells Hypersegmentation: Nuclei have more than five
(<2.0  109/L) segments, usually seen in infection. This may also
CHAPTER 3 Hematology 119

be seen as a nuclear abnormality without infection granulomatous disease test negative for the ability
in patients with megaloblastic anemia to reduce the substance
Changes seen in lymphocytes Leukocyte adhesion disorder (LAD)
Reactive/variant: Mature lymphocytes showing Mutations in the genes needed to form cell adhesion
nuclear and cytoplasmic changes after stimulation molecules, particularly b integrins
by antigens. Cells tend to be large with abundant Three subtypes of LAD, all affecting neutrophil
cytoplasm with slightly less chromatin clumping adhesion
than resting lymphocytes Patients have difficulties with recurrent infections

Inherited Abnormalities of Neutrophils


Pelger-Huet anomaly HEMATOPOIETIC NEOPLASMS
Autosomal dominant mutation of the lamin B
Myeloproliferative Neoplasms
receptor
Neutrophils are hyposegmented, with nuclei Clonal hematopoietic stem cell disorders that result in
showing mature chromatin. Nuclei shapes are the overproduction and accumulation of cells in the
round/oval, bands, or bilobed and separated by a thin granulocytic, RBC, and platelet cell lines, leading to
filament chronic neoplasms
Cells function normally, as granule function is not Disorders often have an insidious onset and progress
impaired slowly through chronic stages, usually terminating in
May-Hegglin anomaly an aggressive acute stage
Autosomal dominant mutation of the MYH9 gene Disorders can occur at any age, but most patients are
Dohle-like inclusions are found in neutrophils, over 40 years of age
eosinophils, basophils, and monocytes, in addition WHO has classified myeloproliferative neoplasms
to the presence of thrombocytopenia and giant (MPNs) into four major categories, in addition to sev-
platelets eral less common categories
Usually asymptomatic, although patients may CML
exhibit bleeding as a result of thrombocytopenia; Polycythemia vera
WBCs function normally Primary myelofibrosis
Alder-Reilly anomaly Essential thrombocythemia
Autosomal recessive disorder leading to the inabil- Additional, less common categories include chronic
ity to fully degrade mucopolysaccharides neutrophilic leukemia, chronic eosinophilic leuke-
Cells are filled with large, prominent granules com- mia, mast cell disease, and unclassified
posed of mucopolysaccharides Chronic Myelogenous Leukemia
WBCs function normally Characterized by production and accumulation of
Chediak-Higashi syndrome neutrophils in all stages of maturation
Autosomal recessive mutation of the LYST Chronic disorder that can lead to an acute/accelerated
gene that affects all cells with lysosomal phase several years after onset if the disorder is not
organelles treated
All WBCs may show presence of large lysosomal Acute phase usually terminates in an acute leukemia
granules Clinical presentation
Patients tend to die early in life as a result of bacte- Anemia, bleeding, infection, sometimes splenomegaly
rial infections, because cells do not function CBC
normally Elevated WBC, often greater than 100  109/L
Patients also may exhibit bleeding, albinism, and Elevations of all granulocytic cells, showing all
neurologic issues stages of maturation
Chronic granulomatous disease Left shift through the promyelocyte stage
Mutations, either X-linked recessive or autosomal Predominant WBCs tend to be segmented neu-
recessive, in the proteins coding for NADPH trophils, bands, metamyelocytes, and myelocytes
oxidase Myeloblasts and promyelocytes are present in
Phagocytic cells produce superoxide, which is the chronic stage, although they are less than
needed for the kill mechanism that targets many 5% of the differential
bacteria and fungi Eosinophils and basophils tend to be elevated
Patients tend to have frequent infections Bone marrow
Cells look normal but are unable to kill many bac- Hypercellular with an elevated M:E ratio
teria or fungi, leading to frequent infections Erythroid cells are often decreased
Testing for the disorder uses the nitroblue tetrazo- Megakaryocytes are present in normal to increased
lium reduction test, in which patients with chronic numbers
120 CHAPTER 3 Hematology

Laboratory testing Additional chromosomal abnormalities may


Uric acid is increased as a result of elevated cell be present
turnover Patients present as they would with an acute
Leukocyte alkaline peroxidase (LAP) score is leukemia
decreased Polycythemia Vera
A low score can help differentiate CML from leu- Characterized by increased RBCs, granulocytes, and
kemoid reaction, where the LAP score is increased platelets in the peripheral blood, with notable
Cytogenetics and molecular testing increases in RBC and Hgb, while erythropoietin levels
Karyotyping remain normal to decreased
Philadelphia chromosome is present, which is Patients present with a JAK2 V617F mutation, which
required for diagnosis affects the cellular response to erythropoietin, in addi-
Formed by the translocation of the long arms tion to decreasing normal apoptosis
of chromosomes 9 and 22, also leading to the Clinical presentation
production of the BCR-ABL fusion gene. Patients may show symptoms related to increased
BCR-ABL codes for protein 210 (p210), RBC mass, including headaches and ruddy cyanosis
which results in an increase of tyrosine from the increase in circulating RBCs
kinase activity, and prognosis is more CBC
favorable if p210 is present Elevated Hgb and Hct, with normal RBC morphol-
If protein 190 (p190), another protein ogy, increases in WBC and platelets
with increased tyrosine kinase activity, Bone marrow
is present, the prognosis is poor Hypercellular with increases in all three cell lines
Fluorescence in situ hybridization (FISH) testing during the initial chronic phase
Identifies the presence of the BCR-ABL fusion Some patients will progress to a spent phase, in
gene used in diagnosis which bone marrow becomes fibrotic and spleno-
Reverse-transcriptase polymerase chain reaction megaly becomes prominent, often as a result of
(RT-PCR) extramedullary hematopoiesis
Used to monitor cytogenetic and molecular Laboratory testing
remission Erythropoietin level is decreased
Therapy and progression RBC mass is increased
Therapy LAP is normal to increased
Imatinib mesylate (Gleevic) therapy has been Cytogenetics and molecular testing
favorable in leading to remissions JAK2 V617F mutation present in the majority of
Tyrosine kinase inhibitor working to block patients
the tyrosine kinase activity Other JAK2 mutations may be present
Response can lead to complete remission Therapy and progression
Relapse may occur in some patients who Therapeutic phlebotomy is used to decrease RBC
develop imitanib resistance counts and provide relief from symptoms
Previous to imatinib, various therapies were used Myelosuppressive therapy also may be used to con-
to decrease the tumor burden, from chemother- trol cell burden
apy to interferon-a (IFN-a) Progression to an acute leukemia may occur in
Bone marrow transplant from a matched donor, approximately 15% of patients
particularly in younger patients, can also be used Essential Thrombocythemia
Progression Characterized by increased platelets and megakaryo-
Before the use of imitanib therapy, a chronic period poiesis; however, platelets may not function
of the disease would occur and then it would tran- normally
sition to an acute leukemia (blast phase) Clinical presentation
Often, before the blast stage, the disease Patients may present with symptoms related to
would transition through an accelerated stage bleeding and clotting, including neurologic symp-
in which clinical presentation and laboratory toms, myocardial infarction, headache, mucous
values would begin to deteriorate as the blast membrane bleeds, and others
count increased May be asymptomatic but incidental finding of
Blast crisis: Terminal phase of CML increased platelets may lead to further workup
Increased blasts in the peripheral blood and Diagnosis requires the ruling out of reactive throm-
bone marrow (>20%) bocytosis or other MPNs
Blast origin may be myeloid or lymphoid CBC
Extramedullary hematopoiesis also may Increased platelet counts, >450  109/L with sus-
occur tained elevations
CHAPTER 3 Hematology 121

Platelets may be of variable size and granularity Disorder similar to CML; however, the majority of the
Slight decreases in Hgb and Hct cells are mature (>90%), and fewer than 10% are
Bone marrow immature neutrophils
Megakaryopoiesis without increased erythropoiesis Differential diagnosis requires the absence of the Phil-
or granulopoiesis adelphia chromosome, in addition to ruling out any
Megakaryocytes may be larger than normal other causes of reactive neutrophilia
Laboratory testing Chronic Eosinophilic Leukemia
Little additional testing is currently used for Chronic disorder showing an elevated absolute eosin-
diagnosis ophil count (>1.5  109/L) with no evidence of reac-
Cytogenetics and molecular testing tive eosinophilia (parasitic infections, allergic
JAK2 V617F mutation may be present reactions, etc.) or other malignancies that feature
Therapy and progression increased eosinophils
Prevention of bleeding and clotting episodes Abnormalities and immature forms of eosinophils are
Myelosuppression to suppress platelet present in the peripheral circulation and bone marrow
production Mastocytosis
Patients usually survive longer than 10 years Group of chronic disorders with accumulations of
mast cells within the organ systems. Classified into
Primary Myelofibrosis seven subcategories by the WHO
Other names have been used for this disorder, includ- Clinical presentation usually includes urticarial lesions
ing chronic idiopathic myelofibrosis and myelofibrosis and a variety of other symptoms based on the sub-
with myeloid metaplasia group of the disorder
Characterized by bone marrow fibrosis, extramedul- Mast cells may be elevated in the bone marrow or skin
lary hematopoiesis, and increases in megakaryocytes lesions
Clinical presentation KIT mutations occur in many with systemic
Nonspecific symptoms including fatigue, weak- mastocytosis
ness, shortness of breath, in addition to Prognosis is variable from benign to aggressive
splenomegaly Unclassifiable
CBC Catch-all group of MPNs to classify disorders that are
Normal to decreased Hgb and Hct with RBC and consistent with MPN diagnosis, including those that
platelet abnormalities do not meet the WHO diagnostic criteria for diagnosis
Teardrops, other morphologies, polychromasia, or disorders that occur in conjunction with another
and NRBC disorder
Platelet counts are variable, as are platelet mor-
phologies. Circulating micromegakaryocytes
may be seen
MYELODYSPLASTIC SYNDROMES
Bone marrow Group of neoplastic disorders characterized by periph-
Areas of fibrosis with hypercellularity of granulo- eral blood cytopenias and dyspoiesis that occur in one
cytes and megakaryocytes or more cell lines
Cytogenetics and molecular testing Cytopenias do not respond to most usual therapies
JAK2 mutation may be seen Most cases arise de novo or related to other therapy
Therapy and progression (usually chemotherapy or radiation)
Survival is approximately 5 years after diagnosis, Usually affects patients older than 50 years of age, with
although some patients have lived longer a median age of diagnosis of 70 years of age
Multiple therapies may be used, including RBC and Clinical symptoms
chemotherapy and immunotherapy Clinical symptoms parallel the dyspoietic cell line(s)
Treatment for anemia RBC: Anemia symptoms
Radiation or splenectomy may be used to target Platelets: Bruising/bleeding
splenomegaly and spleen pain or hyperfunction Granulocytes: Increased infections
Bone marrow transplant may be used in younger CBC
patients Cytopenias may occur in one or more cell lines.
Dysplasia may be present
Other Myeloproliferative Neoplasms Bone marrow
Chronic Neutrophilic Leukemia Cells present are dyspoietic and have any number
Chronic disorder characterized by an elevated WBC of abnormalities from abnormal nuclei, abnormal
count (>25  109/L) with a proportional increase in granulation of cytoplasm, N/C asynchrony, and
neutrophils and their precursors in the bone other abnormalities, and they may not function
marrow normally
122 CHAPTER 3 Hematology

Examples of dyspoietic cell appearance Abnormalities most frequently occur in chromo-


Dyserythropoieis: Oval macrocytes, multi- somes 5, 7, 8, 11, 13, 20
nucleate NRBCs, nuclear bridging, other Treatment and prognosis
abnormal nuclear shapes, inclusions Treatment varies between the different disorders in
(Howell-Jolly bodies, siderotic granules, baso- the group; however, supportive therapy tends to be
philic stippling) used, often because of patient age and presence of
Dysmyelopoiesis: Abnormal granulation other coexisting diseases
(lacking granules, decreased granule presence, Transfusion therapy, prophylactic antibiotics,
or large granules), N/C asynchrony, abnormal growth factors to stimulate bone marrow
nuclear shape, multinucleate WBCs response, or immunosuppressive therapy
Dysmegakaryopoiesis: Giant platelets, agra- Patients <70 years of age may be candidates for
nular platelets, circulating micromegakaryoc- stem cell transplant
tyes, unusual nuclei Prognosis is variable based on subgroup, because
Laboratory testing levels of risk are based on the types of dyspoiesis,
Chromosomal abnormalities are present in approx- number of blasts present, and cytogenetic
imately half of the cases of MDS findings
Karyotyping can help determine treatments and WHO (2008) Classification (Table 3-12)
their predicted response Refractory Cytopenia with Unilineage Dysplasia
Refractory Anemia with Ringed Sideroblasts

T A B L E 3- 1 2 Peripheral Blood and Bone Marrow Findings in Myelodysplastic Syndromes

Disease Blood Findings Bone Marrow Findings


Refractory cytopenia with unilineage dysplasia Unicytopenia* Unilineage dysplasia:  10% of cells in one myeloid lineage
(RCUD), refractory anemia (RA), refractory No or rare blasts (<1%){ <5% blasts
neutropenia (RN), refractory <15% of erythroid precursors are ringed sideroblasts
thrombocytopenia (RT)
Refractory anemia with ringed sideroblasts (RARS) Anemia  15% of erythroid precursors are ringed sideroblasts
No blasts Erythroid dysplasia only
<5% blasts
Refractory cytopenia with multilineage dysplasia Cytopenia(s) Dysplasia in 10% of cells in two or more myeloid lineages
(RCMD) No or rare blasts (<1%){ (neutrophil and/or erythroid precursors and/or
No Auer rods megakaryocytes)
<1  109/L monocytes <5% blasts in marrow
No Auer rods
15% ringed sideroblasts
Refractory anemia with excess blasts 1 (RAEB-1) Cytopenia(s) Unilineage or multilineage dysplasia
<5% blasts 5%-9% blasts{
No Auer rods No Auer rods
<1  109/L monocytes
Refractory anemia with excess blasts 2 (RAEB-2) Cytopenia(s) Unilineage or multilineage dysplasia
5-19% blasts 10-19% blasts{
 Auer rods{  Auer rods{
<1  109/L monocytes
Myelodysplastic syndrome, unclassified (MDS-U) Cytopenia(s) Unequivocal dysplasia in <10% of cells in one or more myeloid
1% blasts{ cell lines when accompanied by a cytogenetic abnormality
considered as presumptive evidence for a diagnosis of MDS
<5% blasts
MDS associated with isolated del(5q) Anemia Normal to increased megakaryocytes with hypolobulated nuclei
Usually normal or <5% blasts
increased platelet Isolated del(5q) cytogenetic abnormality
count No Auer rods
No or rare blasts (<1%)
From Swerdlow SH, Campo E, Harris NL, et al, editors: WHO classification of tumours of haematopoietic and lymphoid tissues, ed 4, Lyon, France, 2008, IARC
Press.
*Bicytopenia may occasionally be observed. Cases with pancytopenia should be classified as MDS-U.
{
If the marrow myeloblast percentage is <5% but there are 2%-4% myeloblasts in the blood, the diagnostic classification is RAEB-1. Cases of RCUD and RCMD
with 1% myeloblasts in the blood should be classified as MDS-U.
{
Cases with Auer rods and <5% myeloblasts in the blood and <10% myeloblasts in the marrow should be classified as RAEB-2.
CHAPTER 3 Hematology 123

Refractory Cytopenia with Multilineage Dysplasia RAEB-2: 10% to 19% blasts, Auer rods may be
Refractory Anemia with Excess Blasts present
Myelodysplastic Syndrome with Isolated 5q Prognosis
Deletion Disease is more aggressive because more blasts are
Myelodysplastic Syndrome, Unclassifiable present, leading to a higher number of cases that can
Childhood Myelodysplastic Syndromes transform into AML

Refractory Cytopenia with Unilineage Myelodysplastic Syndrome with Isolated


Dysplasia (RCUD)
5q  Deletion (5q-Syndrome)
CBC
CBC
Dysplasia occurs in more than 10% of one cell line
Refractory anemia and other parameters are
Less than 1% blasts in peripheral blood
Bone marrow relatively normal
Other parameters are relatively normal
<5% blasts in bone marrow
Bone marrow
Evidence of dyspoiesis
Less than 5% blasts
Prognosis
Abnormal megakaryocytes and erythroid
Survival of 2 to 5 years after diagnosis
hyperplasia
Other testing
Refractory Anemia with Ringed Cytogenetics showing a 5q  deletion
Sideroblasts (RARS) Prognosis
Survival of approximately 12 years, disease tends to
CBC
be stable
Anemia, possibly dimorphic population of normal
and hypochromic cells, dyserythropoiesis
No blasts in peripheral blood
Bone marrow Myelodysplastic Syndrome, Unclassifiable
<15% ringed sideroblasts Cytopenias and some marrow dysplasia are present;
Prognosis however, it may not meet all criteria to be classified
Survival of 6 to 10 years after diagnosis into one of the specific groups of MDS

Refractory Cytopenia with Multilineage


Dysplasia MYELOPROLIFERATIVE AND
CBC
MYELODYSPLASTIC NEOPLASMS
One or more cytopenias is present Category of disorders with features of both myelopro-
Dysplasia in two or more lines liferative and myelodysplastic neoplasms (MDS/MPN)
Less than 1% blasts in peripheral blood
Bone marrow
Less than 5% blasts, some ringed sideroblasts may Chronic Myelomonocytic Leukemia (CMML)
be present Two subgroups, CMML-1 and CMML-2, can occur in
Prognosis adults
Disease course is more aggressive Elevated WBC count with monocytosis (>1.5  109/L)
and less than 20% blasts and promonocytes in the
peripheral blood and bone marrow
Refractory Anemia with Excess Blasts Dysplasia in one or more myeloid line, particularly
(RAEB) dysgranulopoiesis
Two subtypes occur: RAEB-1 and RAEB-2 Prognosis is variable; however, CMML-1 has fewer
CBC blasts, leading to a more favorable prognosis than
Cytopenias in all three cell lines, in addition to dys- CMML-2, in which more blasts are present
myelopoiesis and/or dysmegakaryopoiesis
RAEB-1: 2% to 4% blasts, decreased monocytes
RAEB-2: 5% to 19% blasts, decreased monocytes
Juvenile Myelomonocytic Leukemia (JMML)
Some blasts may have Auer rods Similar to CMML, but it occurs in children up to age 14
Bone marrow These patients are usually candidates for stem cell
RAEB-1: 5% to 9% blasts transplant
124 CHAPTER 3 Hematology

Atypical Chronic Myeloid Leukemia Small lymphoblasts, which are up to two times
larger than a normal lymphocyte, with small
Blood picture is similar to that of CML, but cells
amounts of cytoplasm
exhibit dysplasia, particularly in the granulocyte line Nucleoli are present but may not be prominent
Philadelphia chromosome BCR/ABL1 negative
Large lymphoblasts, which are 2 to 3 times larger
Prognosis is poor
than a normal lymphocyte with prominent nucle-
oli and abnormalities in the nuclear membrane
Myeloproliferative and Myelodysplastic Other laboratory tests
Neoplasms Unclassifiable Flow cytometry is used to determine specific precur-
sor cells present based on CD markers present
Cytopenias and some marrow dysplasia is present, Breaks ALL into subtypes
along with features of myeloproliferative disease; 1. Early B-cell (pro-B) ALL: TdT +, CD34 +,
however, it may not meet all criteria to be classified CD19+, CD20 , CD22 +
into one of the specific groups of MDS/MPN 2. Intermediate preB-cell ALL: TdT +, CD34
+/, CD19 +, CD20 +/, CD10+
3. PreB-cell ALL: TdT +, CD19+, CD20+,
ACUTE LYMPHOBLASTIC LEUKEMIA (ALL) CD22+, CD10 +/
Acute leukemia characterized by the presence of more 4. PreT-cell ALL: TdT +/, CD34 +/, CD2 +,
than 20% lymphoid blasts in the peripheral blood and/ CD3 +, CD7 +, CD10 +/
or bone marrow Therapy
Primarily affects children, but is also seen in older Treatment varies based on age and risk
adults Low risk: Children 1 to 10 years of age with a
WHO classifies into B lymphoblastic leukemia/lym- lower WBC count, no extramedullary disease
phoma (B-ALL) and T lymphoblastic leukemia/lym- High risk: Adults, children younger than 1 year
phoma (T-ALL). Both are divided into several of age and those with extramedullary disease
subgroups based on recurrent genetic abnormalities. Treatment usually begins with induction therapy,
The subtype can influence treatment and prognosis followed by consolidation therapy, and continua-
Genetic abnormalities in B cell lymphoblastic tion therapy
leukemia/lymphoma include Intrathecal therapy may be used if there is evi-
t(9;22)(q34;q11.2); BCR-ABL1 dence of blasts infiltrating the CNS
t(11q;23); MLL For patients not responding to initial therapy or
t(12;21) (p13;q22); TEL-AML1(ETV6- patients no longer in remission, stem cell trans-
RUNX1) plants can be used if a matched donor is present
Hypodiploidy Prognosis
Hyperdiploidy Varies based on age, number of blasts, immunophe-
t(5;14)(q31;q32); IL3-IGH notype, and genetic mutations
(1;19)(q23;p13.3); E2A-PBX1 (TCF3-PBX1) Prognosis tends to be better in cases in which
T-cell lymphoblastic leukemia/lymphoma may exhibit Patient is younger
abnormal karyotypes, but they are not as defined as Blast counts are lower
seen in B-cell acute lymphoblastic leukemia (B-ALL) Immunophenotype fits the regular criteria
Clinical presentation Chromosomal translocations have variable
Generalized symptoms including fatigue and fever, in prognosis
addition to bleeding from mucous membranes If patient is Philadelphia chromosome pos-
Lymphadenopathy and bone pain may be present itive, they tend to have a poorer prognosis
because of leukemic cells Hyperdiploidy in children is favorable but
Malignant cells may also infiltrate the central ner- not in adults
vous system (CNS), leading to blast presence in Hypodiploidy has a poor prognosis overall
the spinal fluid
CBC ACUTE MYELOID LEUKEMIA (AML)
Variable WBC count; counts can range from low to
elevated numbers Acute myeloid leukemia (AML) is characterized by the
Usually see anemia, thrombocytopenia, and presence of greater than 20% blasts (WHO 2008 Clas-
neutropenia sification System) in the peripheral blood and/or bone
Not all patients have circulating lymphoblasts; marrow
however, the lymphoblasts exhibit two main mor- Most common group of leukemias in adults and chil-
phologies when they are seen dren younger than 1 year of age
Clinical presentation
CHAPTER 3 Hematology 125

Patients often present with nonspecific symptoms, CBC and bone marrow show myeloblasts, mono-
related to the bone marrow takeover by the abnor- blasts, and promonocytes, and may see dysplastic
mal clone of malignant cells, which may crowd out eosinophils in marrow
normal cells Extramedullary involvement may be present, par-
Anemia: The malignant clone decreases space ticularly in the CNS
available for erythroid precursors, leading to Prognosis often shows a good remission rate
typical anemia symptoms, such as fatigue, AML with t(15;17)(q22;q12); (PML/RARA); also
pallor, etc. referred to as acute promyelocytic leukemia (APL)
Thrombocytopenia: The malignant clone All ages, but usually seen in younger adults, often
decreases space available for megakaryocyte females
production, leading to symptoms of bleeding CBC and bone marrow show myeloblasts/abnormal
and bruising promyelocytes, Auer rods usually present singly or
Neutropenia: Fewer mature cells are present in bundles
because of the hyperproliferation of immature Complications can include DIC, because primary
myeloid cells. May lead to infections granules in promyelocytes can serve as procoagulants
and fever Cells have a maturation block at the promy-
CBC elocyte stage, and all-trans retinoic acid (ATRA)
WBC counts are variable, usually between 5 and therapy can be used to make cells continue maturation
30  109/L, but can range from 1 to 200  109/L Remission may be achieved with ATRA therapy
Platelet and RBC counts may be decreased because unless patients do not respond to ATRA.
of heavy leukemic clone involvement in the bone AML with t(9;11)(p22;q23); (MLLT3-MLL)
marrow Rare, usually occurring in children
Blasts may be present on the peripheral smear Clinical signs may involve gingival bleeding, DIC,
Specific smear abnormalities are present in each and skin effects
specific subtype CBC and bone marrow show increased monoblasts
Bone marrow and immature monocytes
Hypercellular marrow with more than 20% blasts Prognosis is moderate to poor
of myeloid origin present AML with t(6;9)(p23;q34); (DEK-NUP214)
Specific smear abnormalities are present in each Rare, usually in teenagers or younger adults
specific subtype CBC often exhibits pancytopenia; blasts often are
Other laboratory tests monocytic or have Auer rods
Uric acid is increased because of high cell turnover Prognosis is usually poor
Lactate dehydrogenase is increased because of high AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2);
cell turnover (RPN1-EVI1)
Calcium may be decreased Rare, usually occurs in adults
Phosphate may be increased CBC shows variable blast morphology, dyspoietic
Potassium may be decreased cells, platelet abnormalities
Therapy and prognosis Bone marrow shows increased and abnormal
Variable, based on specific classifications megakaryoblasts
WHO 2008 classifies acute leukemia subgroups Prognosis is poor
based on molecular features and cytogenetics, in addi- AML (megakaryoblastic) with t(1;22)(p13;q13);
tion to traditional cell counts, morphologic appear- (RBM15-MKL1)
ance, and bone marrow examination. Specific Rare, usually occurs in infants, especially those with
molecular and cytogenetic features can influence treat- Down syndrome
ment and prognosis Clinical symptoms of organomegaly, anemia and
thrombocytopenia, megakaryoblasts and micro-
megakaryocytes
Acute Myeloid Leukemia with Recurrent Marrow may show fibrotic areas
Genetic Abnormalities Prognosis has shown improvement with intense
AML with t(8;21)(q22;q22); (RUNX1-RUNX1T1) chemotherapy regimens
Children and young adults
CBC and bone marrow show myeloblasts with Auer
Acute Myeloid Leukemia with
rods and some maturation; some dysplasia
Prognosis is usually favorable Myelodysplasia-Related Changes
AML with inv(16)(p13.1q22) or t(16:16)(p13.1;q22); Usually affects older adults
(CBFB-MYH11) Patients have a history of MDS or other similar
All ages, although usually seen in younger patients disorders
126 CHAPTER 3 Hematology

More than 20% blasts, dysplastic cell morphology in Acute Erythroid Leukemia
one or more cell lines Divided into acute erythroleukemia or pure erythroid
Usually has a poor prognosis leukemia
Both show more than 50% normoblastic cells
in the bone marrow; however, acute erythroleuke-
Therapy-Related Myeloid Neoplasms mia additionally shows more than 20%
myeloblasts
Myeloid neoplasms associated with treatment with CBC and bone marrow show immature RBCs with
chemotherapy and/or radiation obvious dysplastic changes
Prognosis tends to be poor Cytochemistry shows normoblasts staining with a diffuse
or block pattern with periodic acidSchiff (PAS) stain
Prognosis is poor
Acute Myeloid Leukemia, Not Otherwise Acute Megakaryoblastic Leukemia
Specified Characterized by cytopenias, with some cases showing
Contains disorders that have not yet been recognized thrombocytosis. Dysplastic cells are present in all
to have a common genetic abnormality cell lines
Characterized by morphology, flow cytometry, and CBC and bone marrow shows more than 20% blasts,
cytochemistry system with over 50% megakaryoblasts
Acute Myeloid Leukemia with Minimal Differentiation Flow cytometry is positive for CD41, CD42b, CD61
Usually affects infants and older adults Acute Anemias of Ambiguous Lineage
Flow cytometry is positive for CD13, CD33, CD34, Some leukemias may have characteristics, both by
CD117 morphology and immunophenotyping, of more than
CBC and bone marrow show blasts with no evidence one cell type
of maturation These may be designated as biphenotypic or bilineage
Cytochemistry is negative for myeloperoxidase and acute leukemias
Sudan black B
Acute Myeloid Leukemia Without Maturation CHRONIC LYMPHOID NEOPLASMS
Usually affects adults but can occur at any age
Neoplasms of mature lymphoid cells, both B cells and
Flow cytometry is positive for CD13, CD33, CD117,
T cells
and frequently CD34 Increases in mature lymphoid cells in peripheral circu-
CBC and bone marrow show numerous blasts, many
lation (leukemic) or in masses in the tissues
with Auer rods, and less than 10% of WBCs do not
(lymphoma)
mature beyond promyelocyte stage
Therapies and prognosis vary based on the specific
Cytochemistry is positive for myeloperoxidase and
disorders and mutations present (Table 3-13)
Sudan black B
Acute Myeloid Leukemia with Maturation
May occur in all age groups Chronic Lymphocytic Leukemia (CLL)/Small
CBC and bone marrow show more than 20% blasts, Lymphocytic Lymphoma
more than 10% maturing cells are neutrophilic. Cells Mature B-cell disorder with an indolent course
may show Auer rods and other dysplastic features CBC shows elevated WBC counts with a predomi-
Acute Myelomonocytic Leukemia nance of small lymphoid cells, usually with dense,
Flow cytometry is positive for CD13, CD33, CD14,
hypermature nuclei and little cytoplasm, and smudge
CD4, CD11b, CD11c, CD64, CD36 cell are frequently seen
CBC and bone marrow show increased myeloid and Bone marrow shows decreased M:E ratio because of
monocytoid cells in the blood and bone marrow elevated lymphoid cells.
More than 20% of marrow cells are monocytoid Flow cytometry is positive for CD5, CD19,
Acute Monoblastic and Monocytic Leukemia CD20, CD23
Usually seen in younger patients Usually affects the elderly with a slowly progressing
Clinical symptoms may include skin and gingival
disease
issues, in addition to bleeding problems Survival is currently around 10 years after diagnosis
Flow cytometry is positive for CD14, CD4, CD11b,
CD11c, CD36, CD64, CD68
CBC shows increases in monocytes, and >80% of Prolymphocytic Leukemia
marrow cells are of monocytic origin Rare B-cell disorder that affects both T-cell and B-cell
Cytochemistry is positive for nonspecific esterase lines, looking similar to CLL but with the presence of
positive prolymphocytes
CHAPTER 3 Hematology 127

CBC shows elevated WBC counts with lymphocytosis, may be seen, but they do not tend to circulate in
including prolymphocytes with prominent nucleoli large numbers
Bone marrow Bone marrow
Increased presence of prolymphocytes Often exhibits areas of fibrosis, leading to a dry tap on
Flow cytometry is positive for CD19, CD20, FMC7, aspiration. Hairy cells may be visible on core biopsy
and sometimes CD5 Flow cytometry is positive for CD19, CD20, CD22,
Prognosis CD11c, CD25, CD103
Disease progression is more aggressive than in CLL Cytochemistry (tissue samples, including bone mar-
T-cell varieties are more aggressive than B-cell row core biopsy)
varieties Positive for tartrate-resistant acid phosphatase
(TRAP) stain
Hairy lymphocytes may be seen
Hairy Cell Leukemia (HCL) Annexin A1 is the most specific marker for HCL, in
addition to being positive for DBA-44
Chronic B-cell neoplasm with lymphocytes showing
Prognosis
threadlike or hairy projections
Patients may achieve long remissions with therapy,
Relatively rare, usually occurring in middle-aged
including IFN-a or purines
adults, predominantly males
Clinical presentation
Patients often present with massive splenomegaly
CBC
Plasma Cell Neoplasms
The CBC is often relatively normal, although it B cell disorders leading to increases in plasma cells
may show pancytopenia. Neoplastic hairy cells Typically affects older adults

TABLE 3-13 Morphologic and Immunophenotypic Features of Mature B-Cell Lymphomas

Immunophenotype/ Cell of
Subtype Architectural Features Cytologic Characteristics Cytogenetics Origin
Chronic lymphocytic Diffuse lymphocytic Small lymphoid cells CD20 +, CD19 +, CD5 +, Naive or
leukemia/small proliferation with growth CD23+ memory
lymphocytic lymphoma lefts B cells
B-cell prolymphocytic Diffuse proliferation Medium-sized lymphoid cells with CD20 +, CD19 +, FMC7 Unknown
leukemia distinct punched-out nucleoli +, CD5 +/ mature
and abundant cytoplasm B cell
Mantle cell lymphoma Diffuse, nodular, or mantle Medium-sized lymphocytes with CD20 +, CD19 +, CD5 +, Mantle
zone pattern irregular nuclei FMC7+, cyclin D1 +, zone cell
t(11;14)
Follicular lymphoma Follicular pattern Medium-sized lymphocytes with CD20 +, CD19 +, CD10 Germinal
indented nuclei and variable +, BCL-6 +, BCL-2 + center
numbers of large lymphoid cells t(14;18) cell
Extranodal marginal zone Diffuse lymphoid Medium-sized lymphocytes with CD20 +, CD19 +, CD43 Marginal
lymphoma of mucosa- proliferation, occasionally irregular nuclei and clear +/ zone cell
associated lymphoid marginal zone or nodular abundant cytoplasm
tissue pattern
Plasma cell myeloma, Sheets or large aggregates of Plasma cells, frequently with CD20, CD19 +/, CD38 Plasma cell
plasmacytoma plasma cells cytologic atypia +, CD138+,
cytoplasmic light
chain+
Diffuse large B-cell Diffuse proliferation Large lymphoid cells CD20 +, CD19 +, CD10 Different
lymphoma +/, BCL-6 +/, BCL- stages
2+/, CD5+/ of
mature
B cells
Burkitt lymph Diffuse lymphoid proliferation Medium-sized lymphocytes with CD20 +, CD19 +, CD10 Germinal
^_frp_secowid=85.4046 with starry sky pattern evenly distributed chromatin, +, BCL-6 +, BCL-2, center
oma inconspicuous nucleoli t(8:14) cell
From Rodak BF, Fritsma GA, Keohane E: Hematology: clinical principles and applications, ed 4, St Louis, 2012, Saunders.
128 CHAPTER 3 Hematology

Plasma Cell Myeloma Characterized by clefted cells (butt cells) in the periph-
Clinical presentation eral circulation or cerebrospinal fluid
Variable based on type of myeloma. Multiple mye-
loma is characterized by bone involvement,
CERTIFICATION PREPARATION QUESTIONS
whereas Waldenstroms macroglobulinemia does
not show bone involvement. Plasma cells may infil- For answers and rationales, please see Appendix A.
trate other tissues, such as the CNS 1. A physician wants to obtain a measure of a patients
CBC iron stores. Which of the following tests would be the
WBC counts may appear normal or increased, with most suitable?
circulating plasma cells or plasmacytoid lympho- a. Serum iron
cytes depending on disease progression b. Serum transferrin (TIBC)
Bone marrow c. Serum ferritin
Sheets of plasma cells may be seen in the marrow d. Transferrin saturation
Laboratory testing 2. A 68-year-old woman visited her physician with
Total protein tends to be elevated reports of fatigue and weakness. A CBC was ordered,
Surface Ig is positive and the patients results were as follows:
Protein and immunoelectrophoresis is positive for
the protein(s) present in excess RBC 2.50  1012/L Hct 18.8% MCH 24.8 pg
Flow cytometry is positive for CD 19, CD20, CD138, Hgb 6.2 g/dL MCV 75.2 fL MCHC 33%
CD38, and monoclonal cytoplasmic Ig, but is negative
for surface Ig Which of the following would be a plausible diagno-
Prognosis sis for this patient?
Variable depending on the specific plasma cell mye- a. Iron-deficiency anemia
loma present; however, multiple myeloma is often b. Vitamin B12 deficiency
quickly progressive c. Anemia of chronic inflammation
d. Hemochromatosis
3. A peripheral smear shows a decreased RBC count
Lymphoma with microcytic, hypochromic cells with small grape-
like inclusions in the RBCs on both Wright stain and
Lymphoid Neoplasms Manifesting as Solid Tumors Prussian blue stain. This is consistent with:
Hodgkin Lymphoma a. Iron-deficiency anemia
Localized tumor of the lymph nodes, tumor cells do
b. Sideroblastic anemia
not enter the peripheral blood c. Pernicious anemia
Manifests with the presence of Reed-Sternberg cells in
d. b-Thalassemia minor
the tumor 4. Given the following results of iron studies, which dis-
Reed-Sternberg cells are described as having the
order is the most likely?
appearance of owl eyes or popcorn
Non-Hodgkin Lymphoma # Serum iron TIBC
B-cell disorder is most common, although T-cell or # Ferritin # % Saturation
NK-cell lymphomas may occur
Sezary syndrome (mycosis fungoides) is an example of a. Iron-deficiency anemia
b. Sideroblastic anemia
a T-cell lymphoma. The Sezary cells are small to
c. Anemia of chronic inflammation
medium sized lympoid cells with convoluted or cere-
d. Hemochromatosis
briform nuclei. The disorder has skin involvement
5. Acquired sideroblastic anemia may be present in all
and may disseminate throughout the body
Multiple types of non-Hodgkin lymphoma exist, of the following except:
a. Alcoholism
including follicular, mantle cell, diffuse large B cell,
b. Lead poisoning
among many others
Flow cytometry for immunophenotyping is often c. Malabsorption
d. Myelodysplastic syndromes
used along with cytology for diagnosis
6. A patient has a macrocytic anemia, and the physician
Circulating Lymphoma suspects pernicious anemia. Which test would best
Some cases of non-Hodgkin lymphoma may have rule in a definitive diagnosis of pernicious anemia?
peripheral blood involvement when tumor cells enter a. Homocysteine
the peripheral circulation b. Intrinsic factor antibodies
CHAPTER 3 Hematology 129

c. Ova and parasite examination for D. latum c. Microcytic, normochromic cells with increased
d. Bone marrow examination poikilocytosis
7. Megaloblastic anemias result from which of the d. Macrocytic, hypochromic cells with increased
following? polychromasia
a. Deficiencies in free erythrocyte protoporphyrin 15. Which of the following disorders does not have a
b. Deficiencies in Vitamin B12 and folic acid hemolytic component?
c. Increases in iron and hepcidin a. Sickle cell anemia
d. Decreases in liver function b. Autoimmune hemolytic anemia
8. A patients bone marrow showed erythroid hyperpla- c. Glucose-6-phosphate dehydrogenase deficiency
sia with signs of dysplastic maturation, particularly d. Anemia of chronic disease
in the RBC precursors. This is consistent with which 16. A patient presents with evidence of a hemolytic
of the following? anemia. Spherocytes, polychromasia, and macro-
a. Sickle cell anemia cytosis are observed. Which of the following
b. b-Thalassemia major would best help to distinguish the cause of the
c. Pernicious anemia anemia?
d. G6PD deficiency a. Osmotic fragility
9. The CBC for a 57-year-old man had the b. DAT
following results. Which tests would be best to c. G6PD activity assay
order next? d. Vitamin B12 level
17. Paroxysmal nocturnal hemoglobinuria is character-
RBC 2.50  1012/L Hct 26.0% MCH 34 pg
Hgb 8.5 g/dL MCV 104 fL MCHC 33%
ized by flow cytometry results that are:
a. Negative for CD55 and CD59
a. Iron studies b. Positive for CD55 and CD59
b. Vitamin B12 and folic acid levels c. Negative for CD4 and CD8
c. Bone marrow examination d. Positive for all normal CD markers
d. Intrinsic factor antibodies 18. G6PD deficiency episodes are related to which of the
10. The majority of acquired aplastic anemia cases usu- following?
ally results from which of the following? a. Exposure to oxidant damage
a. Unknown causes b. Defective globin chains
b. Pregnancy c. Antibodies to RBCs
c. Chloramphenicol exposure d. Abnormal protein structures
d. Radiation exposure 19. Which of the following disorders is not classified as a
11. Which of the following values is the most likely to be microangiopathic hemolytic anemia?
normal in a patient with aplastic anemia? a. Disseminated intravascular coagulation
a. RBC count b. Hemolytic uremic syndrome
b. Absolute neutrophil count c. Traumatic cardiac hemolytic anemia
c. Absolute lymphocyte count d. Thrombotic thrombocytopenic purpura
d. Platelet count 20. A previously healthy 36-year-old woman with
12. Fanconis anemia is an inherited aplastic anemia with visited her physician because of a sudden onset of easy
mutations that lead to: bruising and bleeding. Of the following, which is the
a. Increased chromosome fragility most likely cause of her laboratory results?
b. Myelophthisic anemia
c. Pancreatic issues WBC 10.5  109/L RBC 3.00  1012/L Hgb 8.0 g/dL
d. RBC enzymatic defects Hct 25.0% MCV 83 fL MCH 26 pg
MCHC 32% Platelets 18  109/L Differential:
13. Which of the following is decreased in cases of intra-
Normal WBCs
vascular hemolytic anemia? with moderate
a. Bilirubin schistocytes
b. Urine hemosiderin and
c. Haptoglobin polychromasia
d. Plasma hemoglobin PT: 12.8 seconds aPTT: 34 seconds
14. Typical CBC findings in hemolytic anemia include:
a. Microcytic, hypochromic cells with increased a. Sickle cell anemia
poikilocytosis b. Chronic myelogenous leukemia
b. Macrocytic, normochromic cells with increased c. Disseminated intravascular coagulation
polychromasia d. Thrombotic thrombocytopenic purpura
130 CHAPTER 3 Hematology

21. Warm autoimmune hemolytic anemia is usually b. This patient has infectious mononucleosis and
caused by which of the following? warm autoimmune hemolytic anemia
a. IgA antibodies c. This patient is likely to have b-thalassemia
b. IgG antibodies minor
c. IgM antibodies d. There is a specimen quality issue because of a cold
d. Complement agglutinin
22. Which of the following conditions is not associated 29. Hemoglobin H disease is described as:
with secondary warm autoimmune hemolytic anemia? a. /a
a. CLL b. a/a
b. Idiopathic onset c. /bb
c. Rheumatoid arthritis d. b/b
d. Viral infections 30. A 3-year-old female patient is seen in the hematology
23. The mutation seen in sickle cell anemia is: clinic to investigate the cause of her persistent ane-
a. b6Glu!Val mia. Hemoglobin electrophoresis was ordered, and
b. b6Glu!Lys results showed an elevation in Hgb F, with a small
c. b26Glu!Lys increase in Hgb A2. What is the most likely disorder
d. b63Glu!Arg based on these results?
24. The majority of hospitalizations associated with a. a-Thalassemia major
sickle cell anemia are due to: b. b-Thalassemia major
a. Cardiomegaly c. a-Thalassemia minor
b. Cholelithiasis d. Hemoglobin H disease
c. Pneumonia 31. A 36-year-old male patient has a CBC performed as
d. Vasoocclusion part of a routine work physical. The WBC count was
25. Patients with sickle cell trait usually have RBC mor- 6.5  109/L with a differential count of 48% neutro-
phology that includes which of the following? phils, 40% lymphocytes, 8% monocytes, 3% eosin-
a. Normocytic, normochromic RBCs with occa- ophils, and 1% basophils. The majority of the
sional target cells neutrophils were mature but hyposegmented, show-
b. Normocytic, normochromic RBCs with rare ing bandlike or single nuclei. What disorder would be
sickle cells suspected?
c. Hypochromic, microcytic RBCs with moderate a. Alder-Reilly anomaly
target cells b. Leukocyte adhesion deficiency
d. Macrocytic, normochromic cells with c. Pelger-Huet anomaly
occasional NRBCs d. Reed Sternberg syndrome
26. Which laboratory test is best used for definitive diag- 32. A 38-year-old male patient has the following CBC
nosis of sickle cell anemia? results:
a. Solubility testing
b. Hemoglobin electrophoresis WBC RBC Hgb 16.0 g/dL
c. Peripheral smear review for sickle cells 32.5  109/L 5.50  1012/L
d. Bone marrow analysis Hct 48.0% Platelet Differential: 49%
27. A peripheral smear review shows mildly anemic sam- 225  109/L segmented
ple with target cells and oblong hexagonal crystal- neutrophils, 9%
loids. What is a possible identity for the crystalloids? bands, 25%
lymphocytes, 9%
a. Hemoglobin S
monocytes, 1%
b. Hemoglobin C eosinophils, 4%
c. Hemoglobin SC metamyelocytes, 3%
d. Hemoglobin E myelocytes; RBC and
28. An 18-year-old man has a CBC done when visiting platelet morphology
his physician for a persistent sore throat. He has appear normal
the following results:
WBC 12.5  109/L RBC 6.00  1012/L Hgb 10.0 g/dL Which of the following conditions is the most likely
Hct 30.0% MCV 60 fL MCH 20 pg
cause of these results?
MCHC 33% Platelet 218  109/L
a. Bacterial infection
Which of the following is most likely? b. CML
a. This patient is normal with a slightly elevated c. Refractory anemia
WBC count because of his sore throat d. Viral infection
CHAPTER 3 Hematology 131

33. Which of the following cytochemical stains is best CD79a(+), TdT(+). Which of the following diagno-
used to distinguish cells of monocytic origin? ses is the most likely?
a. a-Naphthyl acetate esterase a. Intermediate B-cell ALL
b. Naphthol AS-D chloroacetate esterase b. PreB-cell ALL
c. Myeloperoxidase c. T-cell ALL
d. Periodic acidSchiff d. PreT-cell ALL
34. A positive tartrate-resistant acid phosphatase 41. Which of the following may predict a better progno-
(TRAP) stain is indicative of: sis in patients with ALL?
a. Burkitts lymphoma a. The patient is a child
b. Chronic myelogenous leukemia b. Peripheral blood blast counts greater than
c. Hairy cell leukemia 30  109/L
d. Multiple myeloma c. The Philadelphia chromosome is present
35. Which mutation is shared by a large percentage of d. The patient is hypodiploid
patients with polycythemia vera, essential thrombo- 42. A 28-year-old female patient presented to the emer-
cythemia, and primary myelofibrosis? gency department with symptoms suggestive of
a. BCR/ABL DIC. A CBC and coagulation studies were ordered.
b. JAK2 V617F The peripheral smear showed blasts and immature
c. PDGFR cells with heavy granulation and Auer rods. Which
d. RUNX1 of the following disorders would be the most likely?
36. A patient has a CBC and peripheral smear with an a. AML with t(9;11)(p22;q23); MLLT3-MLL
elevated WBC count and left shift, suggestive of a b. AML with t(15;17)(q22;q12); PML-RARa
diagnosis of CML. Which of the following tests c. ALL with t(12;21)(p13;q22); ETV6-RUNX1
would be the most helpful in confirming the sus- d. ALL with t(9;22)(q34;q11.2); BCR-ABL1
pected diagnosis? 43. A patient presents with an elevated WBC count,
a. Cytochemical staining for myeloperoxidase increased monocytes, and blasts present on the
and LAP differential. Flow cytometry is performed with the
b. Karyotyping for the Philadelphia chromosome following results: CD4+, CD11b+, CD11c+, CD13+,
c. Flow cytometry for myeloid cell markers CD14+, CD33+, CD36+, CD64+. Which of the follo-
d. Lymph node biopsies for metastasis wing diagnoses is the most likely?
37. A patient has a splenomegaly, and his CBC shows a a. AML with minimal differentiation
left shift; bizarre RBCs, including dacryocytes; and b. AML with maturation
notable platelet abnormalities. Which of the follow- c. Acute myelomonocytic leukemia
ing would be the most helpful in determining the d. Acute monoblastic leukemia
patients diagnosis? 44. A 75-year-old male patient visits his physician for an
a. Bone marrow biopsy annual checkup. His CBC showed an elevated WBC
b. LAP staining count with numerous small lymphocytes and smudge
c. Karyotyping for the Philadelphia chromosome cells, and a subsequent bone marrow biopsy and
d. Spleen biopsy aspirate showed hypercellularity with increased lym-
38. Which of the following peripheral blood findings phoid cells. What is a presumptive diagnosis based
would not be expected in a patient with a myelodys- on this information?
plastic syndrome? a. Acute lymphoblastic leukemia
a. Hypogranular neutrophils b. Chronic lymphocytic leukemia/small cell lym-
b. Binucleate neutrophils and NRBCs phocytic lymphoma
c. Circulating micromegakaryocytes c. Hairy cell leukemia
d. Decreased vitamin B12 and folic acid d. Therapy-related acute myelogenous leukemia
45. Which of the following is not considered a disorder
39. The WHO system classifies this disorder as a Myelo-
of plasma cells?
proliferative/Myelodysplastic syndrome.
a. Monoclonal gammopathy of undetermined
a. Refractory Anemia with Ringed Sideroblasts
significance
b. 5q  Syndrome
b. Multiple myeloma
c. Chronic Myelomonocytic Leukemia
c. Sezary syndrome
d. Refractory Anemia with Multilineage Dysplasia
d. Waldenstroms macroglobulinemia
40. A 4-year-old male patient presents with a slightly ele- 46. Which of the following sets of CD markers are asso-
vated WBC count, and occasional blasts are present ciated with T lymphocytes?
on the differential. Flow cytometry is performed with a. CD2, CD3, CD4
the following results: CD10(+), CD19 (+), CD22(+), b. CD13, CD14, CD15
132 CHAPTER 3 Hematology

c. CD19, CD20, CD22 c. Immunosuppressed


d. CD34, CD71, CD117 d. A patient with leukemia
47. Bone marrow cellularity is most often estimated by 55. Which of the following cell types exhibit IgE recep-
examining which of the following? tors on their surface membranes?
a. Aspirate a. Basophils
b. Buffy coat b. Eosinophils
c. Core biopsy c. Band neutrophils
d. Crush preparations d. Monocytes
48. A dry tap may be seen in bone marrow aspirations in 56. A 62-year-old female patients CBC showed the fol-
all of the following conditions except: lowing results: total WBC count of 14.0  109/L,
a. Aplastic anemia RBC count of 3.95  1012/L, and platelet count of
b. Hairy cell leukemia 245  109/L. The differential showed 65% seg-
c. Multiple myeloma mented neutrophils, 10% bands, 15% lymphocytes,
d. Primary myelofibrosis and 10% monocytes. Toxic granulation and Dohle
49. The largest hematopoietic cells present in the bone bodies were seen in many of the neutrophils. Which
marrow are: of the following is most likely?
a. Lymphoblasts a. The patient had just finished running a half
b. Megakaryocytes marathon
c. Osteoblasts b. The patient has a bacterial infection
d. Pronormoblasts c. The patient is normal
50. Hemoglobin A contains which of the following con- d. The patient has a helminth infection
figurations of globin chains? 57. A CBC on a patient with Chediak-Higashi syndrome
a. a2b2 is expected to exhibit which of the following?
b. a2d2 a. Giant platelets and Dohle-like inclusions in the
c. a2g2 cytoplasm of all granulocytes
d. a2e2 b. Large, darkly staining cytoplasmic granules in
51. Which of the following locations is not a site of extra- all WBCs
medullary hematopoiesis? c. Giant fused granules and lysosomes in WBC
a. Bone marrow cytoplasm
b. Liver d. Leukocytosis and bilobed eosinophils
c. Spleen 58. Patients with infectious mononucleosis often have
d. Thymus the following CBC results:
52. Patients with renal failure often exhibit compro- a. Lymphocytosis, including increased variant/
mised hematopoietic activity because of which of reactive lymphocytes
the following? b. Lymphocytopenia with numerous small
a. Concurrent depression of thyroid lymphocytes
hormones c. Neutrophilia, including a predominant shift to
b. Decreased production of erythropoietin the left
c. Decreased production of GM-CSF d. Neutropenia with a distinct predominance of
d. Bone marrow suppression caused by medica- toxic granulation
tions 59. Flow cytometry for monitoring a patient with acqui-
53. Which of the following best describes the function of red immunodeficiency syndrome should include
the Rapoport-Luebering pathway? markers for which of the following?
a. It produces ATP to help maintain RBC membrane a. CD30 and CD42
deformability b. CD4 and CD8
b. It results in the reduction of glutathione c. CD34 and CD33
c. It produces 2,3 diphosphoglycerate d. CD21 and CD22
(2,3 DPG) 60. Which of the following disorders is classified as a
d. It produces cytochrome b reductase myelodysplastic/myeloproliferative disease?
54. A 3-year-old male patient visits the pediatrician a. Acute promyelocytic leukemia
for a well-child checkup and routine CBC. He has b. Chronic lymphocytic leukemia
a total WBC count of 5.0  109/L, RBC count of c. Atypical chronic myelogenous leukemia
3.8  1012/L, and platelet count of 225  109/L. The d. Essential thrombocythemia
differential showed 25% segmented neutrophils, 61. All of the following cells are derived from
62% lymphocytes, 10% monocytes, and 3% eosino- CFU-GEMM, common myeloid progenitor cells
phils. This patient is likely: except:
a. A normal child a. Basophils
b. Suffering from an acute bacterial infection b. Lymphocytes
CHAPTER 3 Hematology 133

c. Neutrophils 68. Polycythemia vera can be differentiated from


d. RBCs secondary polycythemia because of polycythemia
62. A patients differential count shows an elevated vera presenting with which of the following?
eosinophil count. This is consistent with which of a. Elevated hemoglobin results
the following? b. Decreased erythropoietin levels
a. Aplastic anemia c. Normal to decreased WBC counts
b. Bacterial infection d. Erythroid hyperplasia in the marrow
c. Parasitic infection 69. The genetic mutation associated with CML is:
d. Viral infection a. t (15;17)(q22;q12)
63. Antibodies are produced by which of the following: b. t(11;14)(p15;q11)
a. Macrophages c. t(9:22)(q34;q11.2)
b. T lymphocytes d. t(8:21)(q22;q22)
c. Plasma cells 70. Which of the following is not classified as a myelo-
d. Basophils proliferative neoplasm?
64. The nitroblue tetrazolium reduction test is used to a. Chronic eosinophilic leukemia
assist in the diagnosis of: b. Essential thrombocythemia
a. Leukocyte adhesion disorders (LADs) c. Mastocytosis
b. Chronic granulomatous disease (CGD) d. Waldenstroms macroglobulinemia
c. May-Hegglin anomaly 71. What is the minimum percentage of ringed sidero-
d. Pelger-Huet anomaly blasts present in the bone marrow for a diagnosis
65. A newly diagnosed patient has an acute leukemia. of refractory anemia with ringed sideroblasts?
Which of the following would initially be the a. 10%
most useful in determining the origin of the blasts seen? b. 15%
a. Leukocyte alkaline peroxidase (LAP) and nonspe- c. 20%
cific esterase (NSE) d. >25%
b. Periodic acidSchiff (PAS) and tartrate-resistant 72. All of the following are considered to be signs of dys-
acid phosphatase (TRAP) erythropoiesis except:
c. Myeloperoxidase (MPO) and terminal dexoynu- a. Multinucleate RBCs
cleotidyl transferase (TdT) b. Basophilic stippling
d. Sudan black B and brilliant cresyl blue c. Dohle bodies
66. Therapy for CML often includes the use of a targeted d. Oval macrocytes
tyrosine kinase inhibitor, such as: 73. Features of dysmyelopoiesis and dysmegakaryopoi-
a. Imatinib mesylate esis seen on a peripheral smear or bone marrow in
b. All-trans retinoic acid cases of myelodysplastic syndromes include all of
c. Ablative chemotherapy the following except:
d. 2-CDA/cladribine a. Pelgeroid neutrophils
67. A 58-year-old female was seen by her physician for b. Neutrophils showing hypogranulation
increasing fatigue. Her CBC shows the following c. Giant abnormal platelets with abnormal gran-
results: ules
d. Siderotic granules
WBC RBC Hgb 17.5 g/dL
15.5  109/L 5.90  1012/L 74. The peripheral blood and bone marrow picture
Hct 53.0% Platelet Differential: 55% sometimes will look similar in myelodysplastic syn-
425  109/L segmented neutrophils, dromes and some RBC disorders. Which of the fol-
3% bands, 30% lowing RBC disorders tends to have a peripheral
lymphocytes, 9% smear appearance similar to cases of myelodysplastic
monocytes, 1% syndromes?
eosinophils, 2% a. Iron deficiency anemia
metamyelocytes; RBC b. a-Thalassemia minor
and platelet c. Megaloblastic anemia
morphology appear
d. Warm autoimmune hemolytic anemia
normal
75. Most of the chromosome abnormalities seen in mye-
lodysplastic syndrome involve which of the follow-
Which of the following conditions is the most likely
ing chromosomes?
cause of these results?
a. 5, 7, 8, 11, 13, 20
a. Chronic myelogenous leukemia
b. 2, 3, 9, 15, 16, 26
b. Polycythemia vera
c. 3, 6, 10, 14, 21
c. Acute bacterial infection
d. 1, 4, 15, 17, 21
d. The patient is normal
134 CHAPTER 3 Hematology

76. Which of the following is not one of the recurrent genetic What is the most likely reason that the physician
abnormalities seen in cases of acute myeloid leukemia? ordered a lumbar puncture after receiving the
a. AML with t(8;21)(q22;q22); AML1(CBFa)/ETO CBC results?
b. AML with t(15;17)(q22;q12); (PML/RARa) a. To rule out an acute case of meningitis
c. AML with inv(16)/p(13;q22); (CBFb/MYH11) b. To look for leukemia cells in the spinal fluid
d. AML with t(1;19)(q23;q13); (E2A/PBX1) c. To rule out infectious mononucleosis
77. AML with 11q23 (MLL) abnormalities are associ- d. To rule out multiple sclerosis
ated with which cell line? 82. A 78-year-old man was previously diagnosed with
a. Eosinophil chronic lymphocytic leukemia (CLL). Periodic
b. Erythrocyte CBCs were ordered, and several months of CBCs
c. Monocyte maintained an appearance consistent with cases
d. Neutrophil of CLL.
78. T-cell ALL most commonly affects which of the WBC 58.5 RBC 3.90  1012/L Hgb 12.0 g/dL
following? 1012/L
a. Infants Hct 36.0% MCV 92 fL MCH 3 pg
MCHC 33% Platelet 132  109/L Differential: 70%
b. Teenaged males
lymphocytes, 8%
c. Adult females segmented
d. Elderly males neutrophils, 2%
79. Which of the following disorders is considered to be monocytes, 20%
classified by WHO as an AML, not otherwise unidentified cells with
classified? lymphoid appearance
a. Acute erythroid leukemia and a prominent
b. Acute megakaryoblastic leukemia nucleolus
c. Acute promyelocytic leukemia Which of the following is most likely?
d. AML without maturation a. The patient has developed Sezary syndrome
80. A 69-year-old female patient presented with symp- b. The patient has developed prolymphocytic leukemia
toms of fatigue and easy bruising. A CBC was ordered. c. The patient has developed multiple myeloma
The peripheral smear showed a large number of d. The patient now has a concurrent case of CLL
blasts, anemia, and thrombocytopenia. A bone mar- and ALL
row examination was performed, revealing hypercel- 83. Multiple myeloma exhibits laboratory features
lularity and a blast appearance similar to that of the except which of the following?
peripheral smear. Flow cytometry revealed cells posi- a. Occasional plasma cells in the peripheral blood
tive for CD 13, CD 33, CD 34, CD 38, CD 117, and b. Rouleaux
HLA-DR. Cells were negative for TdT, myeloperoxi- c. Hypercalcemia
dase, and nonspecific esterase. Based on this informa- d. Decreased immunoglobulin
tion, which of the following is most likely? 84. The diagnostic cell type seen in Hodgkin lymphoma
a. AML with minimal differentiation is:
b. AML without maturation a. Binucleate plasma cell
c. B-cell ALL without maturation b. Reed Sternberg cell
d. Acute monoblastic leukemia c. Bence Jones lymphocyte
81. A 3-year-old female patient was having symptoms of d. Burkitt lymphocyte
lethargy and bruising and reported pain in her legs. 85. Which of the following appearances describes the
Her mother also mentioned noticing several swollen types of cells seen in Sezary syndrome?
lymph nodes when bathing the child. The pediatri- a. Plasma cells containing immunoglobulin deposits
cian ordered a CBC, which had the following results. b. Large circulating micromegakaryocytes
WBC 18.5  1012/L c. Lymphocytes with convoluted, cerebriform
RBC 3.00  1012/L nuclei
Hgb 9.0 g/ d. Prolymphocytes with prominent azurophilic
dL granules
Hct 27.0% MCV 90 fL MCH 30 pg 86. Which of the following best describes the function of
MCHC 33% Platelet 58  109/L the hexose-monophosphate pathway?
Differential: blastocytes, 6% segmented a. It produces ATP to help maintain RBC membrane
80% neutrophils, 8% lymphocytes, deformability
6% monocytes. RBC b. It results in the reduction of glutathione
morphology was normal, and
c. It produces 2,3 diphosphoglycerate (2,3 DPG)
platelets were markedly
decreased
d. It produces cytochrome b reductase
CHAPTER 3 Hematology 135

87. A patient has a reticulocyte count of 3.5%. This c. Increased RBC count with normal RBCs
shows which of the following? d. Increased RBC count with microcytic/
a. Bone marrow response in producing more RBCs hypochromic RBCs
because of increased need 94. Patients with sickle cell anemia and b-thalassemia
b. A normal reticulocyte count major may not show clinical symptoms until the
c. Patient transfusion of whole blood patient is at least 6 months of age because of which
d. Lack of response to vitamin therapy after a diag- of the following?
nosis of iron-deficiency anemia a. The mutations are acquired after the child is born
88. Which of the following cases does not warrant a bone b. The mutations are activated by dietary and
marrow examination? maternal factors
a. Presence of blasts on the peripheral smear c. The mutations may not manifest clinically at birth
b. Postchemotherapy assessment for minimal resid- because the presence of hemoglobin F decreases
ual disease d. The mutations lead to elevations in a genes that
c. Diagnosis of iron-deficiency anemia compensate for the decreased gene expression
d. Diagnosis of suspected systemic fungal infection 95. The thymus is a site used as a maturation compart-
89. A bone marrow sample for a patient with newly diag- ment for:
nosed chronic myelogenous leukemia would often be a. B cells
expected to have an M/E ratio of: b. T cells
a. 1:1 c. Megakaryocytes
b. 2:1 d. Monocytes
c. 1:2 96. A manual hemocytometer count was required to
d. 10:1 check a patients total WBC count. A 1:20 dilution
90. Which of the following is not implicated as a cause of was made and used when the four large W
nonmegaloblastic macrocytic anemia? squares were counted on both sides of the hemacy-
a. Alcoholism tometer. A total of 105 cells were counted between
b. Hemochromatosis the two sides. What was the patients total WBC
c. Hypothyroidism count?
d. Liver disease a. 0.33  109/L
91. Which of the following results is consistent with a b. 2.1  109/L
diagnosis of aplastic anemia? c. 2.6  109/L
a. Hypocellular bone marrow, absolute neutrophil d. 5.3  109/L
count of 0.5  109/L, platelet count of 40  109/L, 97. Hereditary elliptocytosis results from defects in
Hgb 8 g/dL which of the following?
b. Hypocellular bone marrow, absolute neutrophil a. Ankyrin
count of 2.5  109/L, platelet count of 75  109/L, b. Band 3 protein
Hgb 10 g/dL c. Spectrin
c. Hypercellular bone marrow, absolute neutrophil d. Pyruvate
count of 1.5  109/L, platelet count of 98. Primary neutrophil granules contain:
100  109/L, Hgb 14 g/dL a. Acetyltransferase, collagenase, gelatinase, lyso-
d. Hypocellular bone marrow, absolute neutrophil zyme, b2-microglobulin
count of 0.5  109/L, platelet count of 90  109/L, b. Alkaline phosphatase, cytochrome b558, com-
Hgb 11 g/dL plement receptor 1, complement 1q receptor,
92. The following statement is true of mutations in a- vesicle-associated membrane-2
thalassemia compared to those seen in b- c. b2-Microglobulin, collagenase, gelatinase lacto-
thalassemia: ferrin, neutrophil gelatinase-associated lipocalin
a. Mutations in a-thalassemia occur as a result d. Acid b-glycerophosphatase, cathespins, defen-
of reduced or absent expression of the globin gene sins, elastase, myeloperoxidase, proteinase-3
b. Mutations in a-thalassemia occur as a result of 99. A 36-year-old man visited the emergency depart-
the deletion of one or more globin genes ment because of alternating episodes of fever and
c. The a-globin gene is expressed on chromosome 11 chills that persisted over several days. The patient
d. The b-globin gene is expressed on chromosome 16 stated he had not felt well since returning from a
mission trip to Africa. The physician ordered a
93. A patients genotype is  a/a. This patient will have
a CBC that shows which of the following? CBC with the following results.
a. Decreased RBC count with numerous
target cells WBC RBC Hgb 12.0 g/dL
b. Decreased RBC count with microcytic/ 3.5  109/L 3.80  1012/L
hypochromic RBCs Hct 36.0% MCV 95 fL MCH 32 pg
136 CHAPTER 3 Hematology

MCHC 33% Platelet Differential: Normal WBC 100. Patients with suspected paroxysmal cold hemoglo-
145  109/L distribution, normocytic binuria can be confirmed by performing which of
normochromic RBCs the following?
with some inclusions a. Direct antiglobulin test (DAT)
present and several b. Donath-Landsteiner test
abnormal platelet-like c. Osmotic fragility test
structures shaped like d. G6PD activity assay
boomerangs

What should be done with this sample next? REFERENCE


a. Rerun the sample to make sure it is not Rodak BF, Fritsma GA, Keohane E: Hematology: clinical principles
clotted and applications, ed 4, St Louis, 2012, Saunders.
b. Clean the stainer and make another slide to
examine
c. Refer the sample to the pathologist for further
identification
d. Report the results, because the results are
normal
CHAPTER 3 Hematology 137

SELF-ASSESSMENT
Content Area: ______________________________

Score on Practice Questions: ______________________

List the specific topics covered in the missed questions:

List the specific topics covered in the correct questions:


138 CHAPTER 3 Hematology

NOTES
CHAPTER

4
Hemostasis
Charity E. Accurso

STEPS OF HEMOSTATIC RESPONSE DISORDERS OF VASCULATURE


AFFECTING HEMOSTASIS
Injury to endothelium (or vessel)
#
Primary hemostasis (formation of primary hemostatic
Hereditary
plug, platelets have the main role) Hereditary hemorrhagic telangiectasia (also called
# Osler-Weber-Rendu disease): Abnormal formation of
Secondary hemostasis (formation of fibrin clot, coagula- vessels in which arterial blood may flow directly into
tion proteins are the major contributor) a vein without passing through a capillary. The con-
# necting area is often fragile and ruptures easily, result-
Fibrinolysis (removal of clot) ing in bleeding and bruising
Ehlers-Danlos syndrome: Connective tissue disorder
caused by mutation in collagen synthesis; resulting
SYSTEMS OF HEMOSTASIS blood vessels are fragile and easily broken

1. Vasculature
2. Platelets Acquired
3. Clot formation Type of Purpura (More Common Types)
4. Fibrinolytic Decreased connective tissue
Senile purpura: Degeneration of skin matrix result-
ing in weak capillaries
ROLE OF VASCULATURE Excess glucocorticoid: Cushings syndrome and
Hemostasis usually occurs in the arterioles and venules therapeutic glucocorticoids can result in vessel
Endothelial cells line lumen fragility
Luminal side coated by glycocalyx (carbohydrates Vitamin C deficiency (scurvy): Vessel fragility
and proteins) resulting from disruption of collagen production
Abluminal side is attached to basement membrane Paraprotein disorders
Amyloidosis: Deposition of amyloid material in ves-
(type IV collagen and proteins)
Vessels are nonthrombotic under normal sels leading to fragility; thrombosis is also possible
Paraproteins: Many different effects, depending on
circumstances
Negatively charged surfaces repel (endothelium and malignancy
Vasculitis: Inflammation of blood vessels leading
platelets)
Inhibit platelet activation: Prostacyclin (PGI2) and to complement activation; immune complex deposition
nitric oxide (NO) synthesis and secretion, ADPase also leads to activation and aggregation of platelets
Inactivation of thrombin: Heparin sulfate, Henoch-Schonlein purpura: Considered to be
thrombomodulin another form of vasculitis more commonly affecting
Damaged vessels are prothrombotic children
Exposure of subendothelium: Collagenplatelet
activation
Secretion of platelet activating factor ROLE OF PLATELETS
Secretion of von Willebrand factor (vWF): Platelet Characteristics
adhesion Circulate as inert cell fragments
Release of tissue factor: Aids in secondary hemosta- Repel each other and endothelial lining (nonthrom-
sis activation botic property)

139
140 CHAPTER 4 Hemostasis

T A B L E 4- 1 Platelet Components, Functions, and Structure

Major
Zones Location Within Platelet Significant Components and Major Functions
Peripheral Glycocalyx Factor V: Component of prothrombinase complex, attachment site for factor X on platelet
zone Cytoplasmic membrane surface
Open canalicular system vWF: Transports factor VIII, mediates adhesion between platelets via GPIb/IX
Submembranous area Fibrinogen: Converted to fibrin in final clot formation stages
GPIb/IX: Platelet receptor for vWF
GPIIb/IIIa: Platelet receptor for fibrinogen (and others)
Others: Glycolipids, phospholipids, proteins, mucopolysaccharides
Structural Circumferential and throughout the Microtubules
zone platelet Microfilaments
Intermediate filaments
All involved in maintenance of shape and shape change on platelet activation
Organelle Internally located Granules
zone a (50-80 per platelet): See Table 4-2
Dense (3-8 per platelet): See Table 4-2
Lysosomal granules: Hydrolytic
Peroxisomes
Mitochondria
Glycogen particles
Membrane Surface connected open canalicular SCCS: Interior of platelet and connects to platelet surface; allows substances to enter platelet
systems system (SCCS, OCS) and others to exit; important in storage and secretion; serves as source of surface
Dense tubular system (DTS) membrane after activation
DTS: Does not connect to platelet surface, primarily a source of ionized calcium, site of
prostaglandin and thromboxane synthesis

GP, Glycoprotein; vWF, von Willebrand factor.

Become activated after an injury


After activation, platelets interact with other plate-
lets and the damaged vessel wall platelet vWF

GPIb/IX receptor collagen from


Platelet Ultrastructure basement membrane
FIGURE 4-1 Platelet adhesion.
The platelet is divided into arbitrary zones described
by location and function (Table 4-1) vWF: Stored in a-granules in platelets and Weibel-
Palade bodies in endothelial cells
Platelet Functions Important step that triggers several events leading to
platelet activation
Passive surveillance: Monitor vessel lining for small
holes or gaps, platelets plug holes without activation
of coagulation system
Platelet Activation
Formation of primary hemostatic plug Triggered after platelet adhesion or exposure to
Provides phospholipid surface for secondary agonist
hemostasis Results: Shape change, altered orientation of phos-
Promotion of healing by stimulation of smooth muscle pholipids, new receptor expression, changes in
cells and fibroblasts biochemistry
Platelet agonists: Collagen, adenosine diphosphate
(ADP), thrombin, epinephrine, thromboxane A2
PRIMARY HEMOSTASIS (TXA2), arachidonic acid
TXA2: Synthesized from arachidonic acid by
Platelet Adhesion cyclooxygenase and thromboxane synthase,
Major interaction is the binding of platelet receptor stimulates platelet granule secretion, enhances
glycoprotein Ib (GPIb)/IX to vWF, which binds to col- vasoconstriction; if blocked, secretion is
lagen (Figure 4-1) impaired; aspirin blocks cyclooxygenase
CHAPTER 4 Hemostasis 141

Major Components (and Select


TABLE 4 -2 Functions) of Platelet Granules

Dense Bodies a-Granules


FIGURE 4-2 Shape change. ADP: Platelet agonist Factor V: Fibrin formation
positive feedback to
Collagen and thrombin are strong agonists enhance platelet
ADP and epinephrine are weak agonists response and
Required presence of TXA2 and platelet recruitment
aggregation ATP: Activation of Ca2+ Factor XI: Fibrin formation
channel, agonist for
other cells
Shape Change Calcium: Secondary Fibrinogen: Converted to fibrin,
hemostasis platelet aggregation
Occurs after agonist stimulation, appearance of pseu- Serotonin: Platelet Protein S: Regulation of fibrin
dopods, will convert to original shape if stimulus is not agonist, vasoconstriction formation via protein C pathway
sufficient (Figure 4-2) TFPI: Regulation of fibrin formation by
Microtubules, microfilaments, and intermediate fil- inhibiting factor VII/tissue factor
aments reorganize so that organelles are centrally complex
located in the activated platelet vWF: Binding of platelets to collagen
Phospholipid orientation: Large surface avail- PAI-1: Inhibitor of fibrinolysis
able for biochemical reactions in secondary PF4: Heparin neutralizing,
hemostasis chemoattractant
Receptor expression: GPIb/IX on surface, increase -Thromboglobulin: Chemoattractant
in number of GPIIb/IIIa receptors on surface Thrombospondin: Stabilization of
platelets

Platelet Secretion ADP, Adenosine diphosphate; ATP, adenosine triphosphate; PAI-1,


plasminogen activator inhibitor1; PF4, platelet factor4; TFPI, tissue
Requires adenosine triphosphate (ATP); open canalic- pathway factor inhibitor; vWF, von Willebrand factor.
ular system fuses with granular membrane, and con-
tents of a-granules and dense bodies are released to
the outside of the platelet (Table 4-2)
Agonists released further activate platelets
Summary of Major Biochemical
Calcium released for use in secondary hemostasis
TABLE 4 -3 Mediators of Activation
(Table 4-3)
Arachidonic Increased cytoplasmic Ca2+: Results in
Platelet Aggregation pathway phospholipase A2 activation
Phospholipase A2: Hydrolyzes arachidonic acid
vWF binding to GPIb/IX activates an intracellular sig- Cyclooxygenase: Synthesizes thromboxane A2
naling pathway that results in the activation of GPIIb/ from arachidonic acid (thromboxane
IIIa, which then binds to fibrinogen synthesis involved)
TXA2: Platelet agonist, required for secondary
vWF binding GPIb/IX aggregation
#Intracellular signaling Aspirin: Inhibits cyclooxygenase (lifetime of
platelet)
GPIIb/IIIa activation and binding to fibrinogen
Fibrinogen forms bridges to other GPIIb/IIIa recep- Ca2+ Intracellular signaling: Required for several
reactions, including secondary hemostasis,
tors on other activated platelets, resulting in platelet
activation of some cellular enzymes
aggregates; Ca2+ is needed for aggregation
Fibrinogen and Ca2+ are delivered locally from CAMP CAMP: Negative regulator of platelet activation,
production of CAMP inhibits protein kinase
granules and dense tubular system
which inhibits aggregation
Primary aggregation versus secondary aggregation:
ADP: Inhibits adenyl cyclase
In vitro
Phospholipase Activation of several reactions leads to calcium
Primary: Loose aggregation, reversible if stimu-
C mobilization, granule secretion, and
lus is not sufficient
fibrinogen receptor expression
Secondary: Irreversible provided sufficient
G proteins Platelet agonists bind to G protein receptors,
stimulus; occurs after internal ADP release,
intracellular messaging system
TXA2 synthesis and release, further stimulation
then occurs ADP, Adenosine diphosphate; CAMP, cyclic adenosine monophosphate.
142 CHAPTER 4 Hemostasis

Substrate fibrinogen: Acted on by thrombin


SECONDARY HEMOSTASIS Enzymes: Circulate as zymogens
Through a series of enzymatic reactions, the primary Activation: Two routes
platelet plug is reinforced by fibrin Conformational change
Secondary hemostasis is a complex system of procoa- Proteolytic cleavage
gulant activities and control activities to contain and
limit clot formation
Zymogens are inactive precursors of coagulation Intrinsic Pathway
factors Activation of contact factors when they come into con-
Zymogens serve as substrates for previous enzy-
tact with negatively charged surfaces
matic reaction in the coagulation cascade Glass, kaolin, ellagic acid
Vitamin Kdependent factors: II, VII, IX, X, protein C, Not dependent on calcium
and protein S Deficiency of contact factors (XII, PK, and HK)
Vitamin Kdependent factors are not functional
does not lead to in vivo bleeding issues. Deficiency
unless an additional carboxyl group (COOH) is added of XI is associated with bleeding abnormalities in
to the g-carbon of the glutamic acid residues. This approximately 50% of individuals
reaction is called g-carboxylation and is dependent Contact factors are involved in activation of fibri-
on vitamin K. The factors will be formed in the absence nolysis, complement activation, kinin formation,
of Vitamin K but will not be functional because this inflammation, and angiogenesis (Figure 4-3)
modification is required for binding to a negative
phospholipid surface
The coagulation cascade has two pathwaysintrinsic
Extrinsic Pathway
and extrinsicand shares a common final pathway,
the common pathway Damage to the vessel results in the exposure of tissue
The end-point of the common pathway is the forma- factor on the surface of nonvascular cells
tion of a fibrin clot that reinforces the platelet plug VII and VIIa bind to tissue factor in the presence of cal-
The concept of the three pathways was derived from in cium to form the VIIa/tissue factor complex, also
vitro experiments; physiologically, hemostasis occurs called extrinsic Xase, and the extrinsic pathway is thus
through one pathwaythe tissue factor pathway activated. Extrinsic Xase also can activate IX in the
intrinsic pathway (Figure 4-4)
Grouping of Coagulation Factors
See Tables 4-4 and 4-5 Common Pathway
Cofactors enhance activity of enzymes Begins with the activation of X by either the intrinsic
Va is a cofactor for Xa, no enzymatic activity alone or extrinsic pathway (Figure 4-5)
VIIIa is a cofactor for IXa, no enzymatic End result: Formation of fibrin clot (see Figure 4-5)
activity alone Important notes about fibrin formation (Figure 4-6)
High-molecular-weight kininogen (HK) is a cofac- Thrombin cleaves fibrinopeptides from fibrinogen,
tor for XIIa and Xia forming a fibrin monomer. Fibrin monomers associ-
Protein S is a cofactor for activated protein C ate in half-staggered overlap pattern between the D
Tissue factor is a cofactor for VIIa and E domains

T A B L E 4- 4 Grouping of Coagulation Factors

Contact Group Fibrinogen Group Prothrombin Group


Factors HK, PK, XII, XI XIII, VIII, V, I X, IX, VII, II
Consumed in clot No Yes No, except for II
Molecular weight 80,000-173,000 Da 300,000-350,000 Da 50,000-100,000 Da
Critical to hemostasis XI is essential to hemostasis Yes Yes
XII, PK, HK do not play a major role in
hemostasis (in vivo), so deficiency does not
cause bleeding
Other important information Activation of fibrinolytic, kinin, and Thrombin acts on all factors Vitamin K dependent
complement systems; role in inflammation
HK, High-molecular-weight kininogen.
TABLE 4 -5 Important Factors in Hemostasis

Factors Activation and Functions Important Notes


Contact Factors
XII (Hageman factor) Activated to XIIa by kallikrein, plasmin, or autoactivation (from contact Deficiency not associated with bleeding
with negatively charged surface) condition
XIIa cleaves PK to kallikrein
XIIa (+ cofactor HK) converts XI to XIa
Activates fibrinolytic and complement systems
XI Activated by XIIa, thrombin, and XIa Hemophilia C
Activates IX in the presence of calcium Binds to surface of activated platelets
Prekallikrein (PK) Activated to kallikrein by XIIa Majority circulates bound to HK
Cleaves HK in smaller fragmentskinin Chemoattractant
Activates plasminogen to plasmin conversion Deficiency not associated with bleeding
Conversion of scuPA to uPA condition
High-molecular-weight Kinin source Located in endothelial cells, platelets,
kininogen (HK) Accelerates XII and PK activation granulocytes
Deficiency not associated with bleeding
condition
Prothrombin Group: Vitamin K Dependent
II (prothrombin) Cleaved to thrombin by prothrombinase
Thrombin: Many functions (both procoagulant and anticoagulant;
other functions described in section on regulation)
Cleaves fibrinopeptides from fibrinogen to form a fibrin monomer
Further activates Va, VIIIa, and XIIIa
Activates platelets
Stimulates release of vWF and PAI-1 from endothelial cells and
expression of tissue factor
VII Small amount circulates as VIIa, which further activates VII after
binding to tissue factor
Component of extrinsic Xase (with tissue factor and Ca2+)
Activates X and IX as part of complex
IX Activated by XIa in the presence of Ca2+ Binds to activated platelets
Additional activation by VIIa/tissue factor complex Hemophilia B
Component of intrinsic Xase (with VIIIa and Ca2+) X-Linked inheritance
Activates X (as part of Xase)
X Activated by extrinsic Xase (VIIa, VIIIa, tissue factor, Ca2+) or intrinsic
Xase (IXa, VIIIa, Ca2+)
Complexes with Va, Ca2+ and phospholipids to form prothrombinase
Fibrinogen Group
VIII Activated by Xa or thrombin Circulates associated with vWF
Cofactor for IXa Hemophilia A
Component of intrinsic Xase (with IXa and Ca2+)