4/2/2013

M265 Medical Microbiology

Specimen Collection & Handling for the Microbiology Laboratory Ref: Burtons Microbiology for the Health Sciences, 9th Ed Ch 13

Lecture # 17

Dr. Samer Swedan

Chapter 13 Outline
Introduction Clinical Specimens Types of Clinical Specimens Usually Required to Diagnose Infectious Diseases The Pathology Department (The Lab) Anatomical Pathology Clinical Pathology

Role of Healthcare Professionals in the Submission of Clinical Specimens

Importance of High-Quality Clinical Specimens Proper Selection, Collection, and Transport of Clinical Specimens Contamination of Clinical Specimens with Indigenous Microflora

The Clinical Microbiology Laboratory Organization Responsibilities

4/2/2013

Introduction
The proper diagnosis of an infectious disease requires: 1. Taking a complete patient history 2. Conducting a thorough physical examination of the patient

3. Carefully evaluating the patients signs and symptoms

4. Implementing the proper selection, collection, transport, and processing of appropriate clinical specimens

Steps involved in the diagnosis of infectious diseases

4/2/2013

Clinical Specimens
Specimens collected from patients such as blood, urine, feces, and cerebrospinal fluid (CSF), are known as clinical specimens. Specimens commonly submitted to the hospitals Clinical Microbiology Laboratory (CML) include: blood, bone marrow, bronchial washings, sputum, CSF, cervical and vaginal swabs, feces, hair and nail clippings, pus, skin scrapings, synovial fluid, throat swabs, tissue specimens, urethral discharge material, urine, and urogenital secretions. All specimens should be of the highest possible quality!

Clinical Specimens, cont.

The Role of Healthcare Professionals in the Submission of Clinical Specimens: There should be a close working relationship among the members of the healthcare team to ensure a proper diagnosis of infectious diseases. Healthcare professionals who collect and transport specimens should exercise extreme caution during the collection and transport of specimens. In the laboratory, all specimens are handled carefully, exercising Standard Precautions.

4/2/2013

Role of Healthcare Professionals in the Submission of Clinical Specimens, cont.

Although laboratory professionals do not themselves make diagnoses, they make laboratory observations and generate test results that assist clinicians to correctly diagnose infectious diseases and initiate appropriate therapy.

Importance of High-Quality Clinical Specimens

High-quality clinical specimens are required to achieve accurate, clinically relevant laboratory results. The 3 components of specimen quality are: Proper specimen selection Proper specimen collection Proper transport of the specimen to the laboratory The laboratory must provide the person collecting the specimen with written guidelines regarding the selection, collection and transport of specimens (e.g. Laboratory Policies and Procedures Manual). The person who collects the specimen is ultimately responsible for its quality.

4/2/2013

Proper Selection, Collection, and Transport of Clinical Specimens

Specimens must be properly selected. Specimens must be collected properly. Material (i.e., specimens) should be collected from a site where the suspected pathogen is most likely to be found. Specimens should be obtained before antimicrobial therapy, if possible. The acute stage of the disease is the most appropriate time to collect a specimen. Specimen collection should be performed with care to avoid harming the patient. A sufficient quantity of the specimen must be obtained to provide enough material for all required diagnostic tests.

Proper Selection, Collection, and Transport of Clinical Specimens, cont.

All specimens should be placed or collected into a sterile container to prevent contamination. Whenever possible, a sterile, disposable specimen container should be used. The specimen container must be properly labeled and accompanied by an appropriate request slip with adequate instructions. Specimens should be collected and delivered to the lab as early in the day as possible to allow sufficient processing time.

Specimens should be protected from heat and cold and promptly delivered to the laboratory.
Hazardous specimens must be handled with even greater care to avoid contamination of couriers, patients, and healthcare professionals.

4/2/2013

Proper method of packaging cultures, specimens, and other biological hazardous materials for the purposes of transport.

Below: Shipping Label

Source: Appendix4 from CD

Types of Clinical Specimens Usually Required to Diagnose Infectious Diseases

Blood Usually sterile The presence of bacteria in the bloodstream is known as bacteremia. Septicemia is a serious disease involving chills, fever, prostration, and the presence of bacteria or their toxins in the bloodstream. To prevent contamination of a blood specimen with indigenous skin flora, extreme care must be taken to use aseptic technique.

4/2/2013

Proper Method of Preparing the Venipuncture Site When Obtaining Blood for Culture

Types of Clinical Specimens Usually Required to Diagnose Infectious Diseases, cont.

Urine Normally sterile in the bladder; becomes contaminated by indigenous microflora of the distal urethra during voiding. Contamination is reduced by collecting a clean-catch, midstream(CCMS) urine. Urine culture involves 3 parts: A colony count (using a calibrated loop) Isolation and identification of the pathogen Antimicrobial susceptibility testing

4/2/2013

Urine Colony Count

The colony count is a way of estimating the number of viable bacteria that are present in a urine specimen. A calibrated loop, either 0.01 mL or 0.001 mL, is used to inoculate the entire surface of a blood agar plate. After incubation at 37oC overnight, the colonies are counted and the number is multiplied by the dilution factor (either 100 for the 0.01 mL loop, or 1000 for the 0.001 mL loop) to determine the number of colonyforming units (CFUs). # Colonies x dilution factor = # CFUs/mL

Obtaining a Urine Colony Count

Incubate 37C Overnight

4/2/2013

Types of Clinical Specimens Usually Required to Diagnose Infectious Diseases, cont.

Cerebrospinal Fluid (CSF) Meningitis is inflammation or infection of the membranes (meninges) that surround the brain and spinal column. Encephalitis is inflammation or infection of the brain. Meningoencephalitis is inflammation or infection of both the brain and meninges. CSF is collected by lumbar puncture into a sterile tube; this is a surgically aseptic procedure performed by a physician. CSF is considered a STAT (emergency) specimen in the lab!

Technique of Lumbar Puncture

4/2/2013

Types of Clinical Specimens Usually Required to Diagnose Infectious Diseases, cont.

Sputum Sputum is pus that accumulates deep within the lungs of a patient with pneumonia, tuberculosis, or other lower respiratory infection. Often, specimens labeled sputum are actually just saliva; saliva specimens dont provide clinically relevant information. If TB is suspected, extreme care should be taken! Better specimens can be obtain by bronchial aspiration or transtracheal aspiration.

Types of Clinical Specimens Usually Required to Diagnose Infectious Diseases, cont.

Throat Swabs Routine throat swabs are used to determine whether a patient has strep throat Specific swab types and cultures may be necessary when suspecting certain pathogens Wound Specimens Whenever possible, a wound specimen should be an aspirate (i.e., pus collected by needle and syringe), rather than a swab Specimens collected by swab are frequently contaminated with indigenous microflora

10

4/2/2013

Types of Clinical Specimens Usually Required to Diagnose Infectious Diseases, cont.

Fecal Specimens Ideally, fecal (stool) specimens should be collected at the laboratory and processed immediately to prevent a decrease in temperature, which would allow the pH to drop and cause the death of many Shigella and Salmonella species. Bacteria in fecal flora are obligate-, aerotolerant-, and facultative anaerobes. A combination of direct microscopic examination, culture, biochemical tests, and immunologic tests may be performed to identify Gram-negative and Gram-positive bacteria, fungi, intestinal protozoa, and intestinal helminths isolated from fecal specimens.

The Pathology Department

Clinical specimens are submitted to the Clinical Microbiology Laboratory (CML), which is a part of the Pathology Department.

11

4/2/2013

SKIP

SKIP
The Pathology Department, cont.
Clinical Pathology Consists of several laboratories in addition to the Clinical Microbiology Laboratory: Clinical Chemistry, Urinalysis, Hematology/Coagulation, Blood Bank, and Immunology. Personnel include pathologists, chemists and microbiologists, clinical laboratory scientists (also known as medical technologists - MTs), and clinical laboratory technicians (also known as medical laboratory technicians - MLTs).

12

4/2/2013

The Clinical Microbiology Laboratory

The CML may be under the direction of a pathologist, a microbiologist, or a senior clinical laboratory scientist.

The Clinical Microbiology Laboratory, cont.

Responsibilities Primary mission of the CML is to assist clinicians in the diagnosis and treatment of infectious diseases.

The 4 major daily responsibilities are to:

1. Process various clinical specimens that are submitted to the CML 2. Isolate pathogens from those specimens 3. Identify (speciate) the pathogens 4. Perform antimicrobial susceptibility testing, when appropriate to do so

13

4/2/2013

Isolation and Identification (Speciation) of Pathogens

Bacteriology Section Bacterial pathogens are isolated from specimens, tests are performed to identify the bacterial pathogens, and antimicrobial susceptibility testing is performed whenever appropriate to do so. CML professionals are very much like detectives and crime scene investigators, in that they gather clues about a pathogen until they are able to identify it. Numerous phenotypic characteristics are used to identify the bacteria (e.g., Gram reaction, cell shape, motility, presence and location of spores, presence or absence of various enzymes, etc.)

CML professionals are very much like detectives and crime scene investigators -- gathering clues about a pathogen until they have enough information to identify it.

14

4/2/2013

Minisystems Used to Identify Bacteria

Copyright 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

Copyright 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

API-20E for identification of Enterobacteriaceae

Enterotube II for identification of Enterobacteriaceae

Diagram Ilustrating the 3 types of Hemolysis That Can be Observed on a Blood Agar Plate

(Gamma) hemolysis =
No Hemolysis

15

4/2/2013

Isolation and Identification (Speciation) of Pathogens, cont.

Mycology Section Responsibility is to assist clinicians in the diagnosis of fungal infections (mycoses) The specimens processed here are the same as those that are processed in the Bacteriology Section, with the addition of hair and nail clippings and skin scrapings.

A variety of procedures are used to identify fungal pathogens including special media, KOH preps, biochemical tests (for yeasts), and a combination of microscopic and macroscopic observations (for moulds).

A Colony (Mycelium) of the Mould Aspergillus fumigatus, a Common Cause of Pulmonary Infections in Immunosuppressed Patients

Colonies (Mycelia) of a Penicillium Species

16

4/2/2013

Isolation and Identification (Speciation) of Pathogens, cont.

Parasitology Section Assists clinicians in the diagnosis of parasitic diseases Parasites are identified by observing and recognizing various parasite life cycle stages (e.g., trophozoites, cysts, microfilariae, eggs, larvae, adult worms) in specimens identified primarily by their physical appearance (e.g., size, shape, internal details) Virology Section Assists clinicians in the diagnosis of viral diseases Techniques used in the identification of viruses include immunodiagnostic tests, cytologic or histologic examination, electron microscopy, molecular techniques, virus isolation by cell cultures, and cytopathic effect (CPE)

Isolation and Identification (Speciation) of Pathogens, cont.

Mycobacteriology Section (also called the TB Lab) Assists clinicians in the diagnosis of tuberculosis (TB) Various types of specimens are submitted, but sputum is the most common type Mycobacterium spp. are identified by the acid-fast staining procedure and by using a combination of growth characteristics (e.g., growth rate, colony pigmentation, and morphology) and a variety of biochemical tests

17