Reference Population at risk for contracting parasites

Clinical Parasitology Lecture Individuals in underdeveloped areas and

countries
2nd edition, Refugees
Immigrants
Elizabeth A. Zeibig, PhD, MLS (ASCP) Visitors who are immonucompromised
Individuals living in close quarters
Children who attend day care centers

Chapter 1 Mode of transmission

Parasites - Parasite gains entry into an

unsuspecting host vary by specific
- Organism that live on and obtain
parasite species
their nutrients from another
Mode of Parasite Transmission
organisms
- A field known as parasitology. Ingestion of contamination food
- Responsible for the invasion in the or drink
body (Infection) invasion on the Hand-to-mouth transfer
Insect bite
body (Infestation) and disease. Entry via drilling through the skin
Unprotected sexual relations
Parasitic Mouth-to-mouth contact
Droplet contamination
- The escalation of disease caused by Eye contact with infected
the presence of parasites because of swimming water
global travel tends to result in higher
parasite recovery rates. Types of
Parasite
Disease
Obligatory Cannot survive outside of a host
- Characteristics symptoms, emerged, Parasite
determining an effective means of Facultative Capable of existing independently
healing infected persons become a Parasite of a host
priority. Endoparasite Established inside of a host

Vector Ectoparasite Established in or on the exterior

surface of a host
- Responsible for transmission,
parasite control and elimination also
became important.
Types of
Host Parasitism Two different species
Accidental Other than the of organism that is
or incidental normal one that is beneficial to one at the
host harboring a parasite others expense
Commensal The association
between two different
Definitive Adult sexual phase of organism in which one
host parasite development benefit and has a
occurs neutral effect on the
other
Intermediate Larval asexual phase Pathogenic Parasite that has
host of parasite demonstrated the
development occurs ability to cause
disease
Reservoir Harboring parasites
host that are parasitic for
humans and from Symbiosis
which humans may
become infected - When parasite infect a host
- Primary function of the host is to
Transport Responsible for carry on the parasites life cycle.
host transferring a
parasite from one
location to another
*Parasites have an amazing capability to
Carrier Parasite-harboring adapt to their host surrounding
host that is not
exhibiting any *Parasite alter their antigenic makeup so that
clinical symptoms the host will not recognize the modified
but can infect others parasite as foreign and thus the initiation of
an immune response does not occur.
Parasite-Host
Relationship *some parasite require only definitive host
Terms while others require one or more
intermediate host.
Symbiosis Living together of two
living organism *Parasitic Life Cycle have three common
Commensali Association of two component; mode of transmission,
sm different species of
Infective stage, diagnostic stage.
organism that is
beneficial to one and
neutral to the other
Mutualism Two different
organisms that is
beneficial to both
Parasite life cycle consists of two common
phase;
(a) Route a parasite follows when in or on
the human body
(b) The route a parasite follows
independently of the human body provides
crucial information pertinent to
epidemiology, prevention and control.
Infective stage
Stool
- most commonly submitted sample
for such studies
- Typical stool analysis consist of;
macroscopic and microscopic
technique
*A process to remove fecal debris, which is
often resembles parasitic forms, is
performed on a portion of sample after a
preservative is added to it.

- Morphologic form that invades

O&P
humans
- Parasite recovery method
Diagnostic stage - O stands for Ova and P stands for
- Can be detected via laboratory parasite
retrieval methods.

Symptoms associated with parasitic disease *Specimen including blood, tissue biopsies,
processes
CSF, sputum, urine, and genital material
Diarrhea may also be examined for the presence of
Fever parasites
Chills
Abdominal pain
Abdominal cramping
Elephantiasis Giemsa stain
Anemia
Vitamin deficiency - The procedure of choice for blood
Bowel obstruction sample submitted for parasite study
Edema
Enlargement of major organs
Cellophane tape preparation
Skin lesions
blindness - Methodology for recovery of
pinworm eggs, and the enterotest
Parasite treatment options parasite are among the traditional
antiparasitic medication tests.
change in diet
vitamin supplement Artifacts/confusers
fuid replacement
blood transfusion - Commonly encountered
bed rest
 - Often referred to as Pseudoparasites
may be present in the sample
submitted for parasite study

Protozoa

- Single-celled eukaryotic animal

- Measure 12 to 18 microns, a measure
defined as one millionth of a meter

Metazoa

- Multicellular worms

Anthropods

- Animalia

Ascaris Lumbricoides egg

- Member of the subkingdom metazoa

which includes multicellular
organism such as parasitic worm

*Scientific names for parasites are written in

italics and consist of two components; genus
and species.

Giardiasis

- Refers to the disease or condition

associated with giardia internalis

*Group of parasites in each classification

table are organized by; kingdom and
subkingdom, phylum and subphylum.
Bolongon, Marjorie B. MLS 3-3
Parasitology Lecture Aug. 29, 2017
Chapter 2
O&P
- Most common procedure performed
in the area of parasitology is the
examination of a stool specimen for
Ova and parasite
Ova
- Refers to the egg stage of select
parasites
Parasite
- Encompasses the other morphologic
forms that may be present.
*Two general components associated with
this routine parasitology procedure;
Macroscopic and microscopic
examination
Microscopic examination
- Consist of three possible
components, each which is detailed
in the selections that follow a
discussion of collection, transport
and fixative for preservation.
*Morphologic forms of protozoa and
helminths may be detected from a properly
collected and prepared stool specimen
*When present, the protozoa forms known
as trophozoites and cyst may be recovered
from these samples.
*Parasite are often shed enter and
subsequently passed in the stool, they may
not appear in a stool specimen on a daily
basis; therefore, multiple specimens are
recommended for adequate detections.
*Typical stool collection protocol consist of
three specimens, one specimen collected
every other day or a total of three collected
in 10 days.
*One exception is in the diagnosis of
amebiasis, in which up to six specimens in
14 days is acceptable.
*stool sample from patients whose therapy
includes barium, bismuth or mineral oil
should be collected in a clean watertight
container with a tight-fitting
2 to 5g
- The acceptable amount of stool
required for parasite study, often
referred to as the size of a walnut
*Urine should not be allowed to contaminate
the stool specimen because it has been
known to destroy some parasite.
*Stool should not be retrieved from the
toilet bowl water because free-living
protozoa and nematodes may be confused
with human parasites
*To demonstrate the motility of Stool preservatives and applicable
protozoan trophozoites, a fresh specimen laboratory procedures
is required Preservative Concentration Permanent Antigen
stain Tests
10% formalin + - +
SAF + + (Iron +
Trophozoite stage hematoxylin)
SVA + + (trichrome or
- iron -
- Sensitive to environmental
hematoxylin)
changes and on release from the Modified PVA + + (trichrome or +
body, disintegrates rapidly (zinc) - iron -
hematoxylin)
- Usually found in liquid stool, it Single-vial + (trichrome or
+ +
its recommended that liquid system iron -
specime be examined within 30 hematoxylin)
minutes of passage.

Formed stool *some laboratory prefer to use a two-vial

- Specimens are not likely to contain
trophozoites; therefore, they can be
held for 24hrs following collection
Freshly collected stool sample
- Which is immediately submitted to
the laboratory, is the ideal specimen
for parasitic examination. When this
is not possible, the sample must be
preserved to maintain its integrity.
Fixative
fixative system; others use a single vial
system.
*If other tests are ordered, such as fecal
immunoassay, the laboratory must ensure
that the fixative is compatible for use with
these techniques.
*Some fixatives contain mercury and
disposal regulations for these compound
could affect the laboratorys decision of
which fixatives to use in ther testing
protocols.

- Substances that preserve the

morphology of protozoa and prevent
further development of certain
helminth eggs and larvae.
*The ratio of fixative to stool is important
for the successful recovery of parasite and,
whatever fixative is used, the recommended
ratio is three parts fixative to one part stool
*The specimen must be fixed in the
preservative for at least 30 minutes before
processing begin.
Formalin
- Has been used for many years as an
all-purpose fixative for the recovery
of protozoa and helminthes
- Two concentrations are commonly
used; 5% concentration preserves
protozoan cysts and a 10%
concentration preserves helminthes
eggs and larvae.
 - May be routinely used for direct
examination and concentration
procedures, but not for permanent
smear
- There are three primary advantages
for use of formalin; (1) it is easy to
prepare; (2) It preserves specimens
for up to several years; and (3) it
has a long shelf life
*Disadvantage of formalin is that it does not
preserve parasite morphology adequately for
permanent smears.
Polyvinyl alcohol (PVA)
- Compromised of a plastic powder
that acts as an adhesive for the stool
specimen when preparing slides for
staining
- Most often combined with schaudinn
solution, which usually contains zinc
sulfate, copper sulfate, or mercuric
chloride as a base.
Advantages
Trophozoites and cysts of the
protozoa, as well as most helminthes
eggs, may be detected using this
fixatives
It can be used for preparation of a
permanent stained smear PVA-
preserved specimens have a long shelf
life when stored at room temperature
Disadvantages
Although concentration techniques can
also be performed from a PVA-
preserved specimen, the recovery of
certain parasites is not as effective as
when formalin is used.
Schaudinn solution contains mercury
chloride
*Many laboratories choose to use a two-vial
system a formalin vial for the
concentration technique and a PVA Vial for
the stained slide.
Sodium Acetate Formalin (SAF)
- A viable alternative to the use of
PVA and schaudinn fixative
- Can be used for performing
concentration techniques and
permanent stained smears.
- some laboratories have adopted this
fixative because it only requires a
single vial, and it is mercury-free
- Easy to prepare, has a long shelf life,
and can be used for preparing smears
for staining with the modified acid-
fast stain to detect coccidian oocysts
- Another limiting factor of SAF is in
the choice of permanent stains made
from this fixative.
*Disadvantages
Because of the adhesive properties of
SAF are not good, the addition of
albumin to the microscope slide may
be necessary to ensure adhesion of
the specimen to the slide
Protozoa morphology from SAF-
preserved specimen is not as clear in
permanent stain as when mercury-
containing preservatives are used.
Modified Polyvinyl Alcohol
- Other alternatives to mercury-base
PVA are the use of substitute
compounds containing copper
sulfate or zinc sulfate
Advantage
They can be used for concentration
methods and permanent stained
smear.
*Zinc sulfate fixatives
- Provide better results than copper
sulfate reagents.
Alternative Single-Vial systems
- Free of formalin and mercury and
can be used for concentration
techniques and permanent stained
smears
- Can also be used for performing
fecal immunoassay
- Do not provide the same quality of
preservation as mercury-based
fixatives and organism identification
will be more difficult from
permanent stained slides.
Processing
*Samples are examined from two
perspectives, macroscopic and microscopic
* Color of stool is important because it may
indicate the condition of the patient, such as
whether a patient has recently had a special
procedure or if the patient is on antibiotic
therapy.
*The range of colors varies including black
to green to clay and color in between
*The color of normal stool is brown and
unusual color, such as purple, red or blue
typically suggest that a patient is on
particular medication
*The surface should be examine first for
parasite such as pinworms, tapeworms
proglottids and adult worms
*Sample containing adult worms may be
carefully washed through wire screen. This
process allows for the retrieval and
examination of the parasites for
identification purposes.
*Blood and/or mucus in loose or liquid stool
may suggest the presence of amebic
ulcerations in the large intestine. Bride red
blood on the surface of formed stool usually
associated with irritation and bleeding.
Macroscopic
- Should be examined for the presence
of gross abnormalities
- To perform must receive a fresh,
unpreserved stool specimen.
- The consistency or degree of
moisture in stool specimen may
serve as an indication of the types of
potential parasite present.
*Soft or liquid stool may suggest the
presence of protozoan trophozoites.
*protozoan cysts are most likely to be
found in fully formed stools
*helminthes eggs and larvae may be found Microscope
in liquid or formed stools
- Most important piece of equipment
Macroscopic examination of stool specimens; in the parsitology laboratory
Possible Descriptive terms
Consistency Possible Gross Ocular micrometer
term color Appearance
Terms - Special designed ocular piece
Hard Dark Conspicuously equipped with a measuring scale
Brown fibrous - The diagnostic stages of parasites
Soft Black Fiber scanty to
detected microscopically are
moderate
Mushy Brown Colloidal measured in units known as microns
(homogenous) defined as a unit measuring 0.001
Loose Pale brown Scanty mucus [10] millimeter.
Diarrheic Clay Much mucus - Use to measure object observed
Watery, Yellow Mucus with microscopically accurately
liquid scanty blood - Is a disk that is inserted into the
Formed Red- Others eyepiece of the microscope. The disk
brown (blood,
equipped with a line evenly divided
barium)
Semiformed Green, into 50 to 100 units
other
Calibration

- Performed with the use of a stage

Microscopic Examination
- To detect presence of parasite in a
stool specimen
- Microscopic examination of stool for
ova and parasites involves three
distinct procedures, direct wet
preparations, concentrated
technique resulting in
concentrated wet preperations, and
a permanently stained smear
*If the specimen received in fixative, the
direct wet preparation can be eliminated
from the O&P procedure; the
concentrate and permanent stain
techniques are performed.
micrometer containing a calibrated
scale divided into 0.01 mm units
- This procedure involves aligning the
eyepiece and stage scale on the
microscope
Direct wet preparation
- Defined as a slide made by mixing a
small portion of unfixed stool (stool
with no added preservatives) with
saline or iodine and subsequent
examination of the resultant mixture
under the microscope
- Other parasite stage that might be
observed in a direct wet preparation
 include protozoan cysts, oocysts,
helminth eggs, and larvae
- Perform only on fresh specimen
Trophozoite mortality
- Can only be demonstrated in fresh
specimens, especially those of a
liquid or soft consistency. If the
specimen is received in the
laboratory in a fixative, this
procedure can be eliminated from the
Q&P assay
Direct Saline wet preparation
- Made by placing a drop of 0.85%
saline on a glass slide and mixing
with small portion of unfixed stool
using a wooden applicator stick or
another mixing tool.
- Oil immersion is not usually
recommended on wet specimen
unless the cover slip is sealed to the
slide.
- A temporary seal can be prepared
using a hot paraffin-petroleum jelly
mixture around the edges of the
cover slip.
Direct iodine wet preparation
- Enhance the detail protozoan cysts
- Is made using a drop of iodine in
place of saline.
Iodine
- Kills any trophozoites present
*It is recommended to use direct saline and
direct iodine wet preparations on each
sample that requires this component of
testing.
Concentration methods
- Examination of concentration of the
fecal specimen
Concentration technique
- Provide the ability to see any
parasites present clearly.
- Can be performed on fresh or
preserved stool
- Two types of concentration methods
available; sedimentation and
floatation. These techniques use
differences in specific gravity and
centrifugation to separate the parasite
from the fecal debris and increase
their recovery.
Sedimentation techniques
- Parasites are concentrated in the
sediment of the tube following
centrifugation and the sediment is
examined microscopically.
Floatation techniques
- Parasite are less dense than the
solutions used and during
centrifugation, they float to the
surface
Formalin-ethyl acetate sedimentation
procedure
- Most widely used sedimentation
technique
 - Base on specific gravit
- Ethyl acetate is added to a saline-
washed formalin-fixed sample and
the tube is then centrifuged.
Advantages
Provides good recovery of most
parasites and is easy to perform
Disadvantages
Is that the preparation contains more
fecal debris than a floatation
technique and is more challenging to
the microscopist
Zinc sulfate flotation technique
- Also based on differences in specific
gravity between the sample debris,
which in this case is heavy and sink
to the bottom of the test tube and
potential parasite, which are lighter
and float towards the top of the tube.
Advantages
More fecal debris is removed and it
yields a cleaner preparation
Disadvantage
Some helminth eggs are very dense
and will not float.
*concentrated wet preparations are referred
to as concentrated saline preparations and
concentrated iodine wet preparations
Permanent stains
- Final procedure in the O&P
examination is the preparation and
examination of permanent stain
smear
- Design to confirm the presence of
protozoa cysts and/or trophozoites
*Permanent stain smear is not the
method use of choice for the
identification of helminths eggs or larvae
because these parasites often stain too
dark or appear distorted.
*Helminth eggs or larvae are best
detected and identified using
concentration technique
*two common stain used for routine
O&P testing include trichrome and iron
hematoxylin
Wheatley trichrome
- Most widely used permanent stain
- Uses reagent with a relatively long
shelf life and the procedure is easy to
perform
Iron hematoxylin
- May be used instead of the trichrome
technique
- Excellent morphology of the
intestinal protozoa
- Detection of acid-fast parasites in
addition to the other protozoa
normally recovered using the iron
hematoxylin stain
Specialized stains
- Important permanent stain procedure
for the detection of the oocysts of
cryptosporidium, as well as those of
Isospora and Cyclospora
 Disadvantage
They do not detect oocysts of the
coccidian parasite or spores of
microsporidia.
*Alternative test have been developed that
are often referred to as rapid methods, or
stool-screening method. These methods
can be obtained as kits that contain
monoclonal anti body
Duodenal material
*Parasite that reside in the small intestine
may be more difficult to recover in a stool
specimen
*the specimen may be collected by
nasogastric intubation or by the enteric
capsule test
*Parasite that can be observed in this type of
specimen include; giardia intestinalis
trphozoites, cryptosporidium spp.,
Isopora belli, strongyloids stercoralis and
eggs of Fasciola hepatica or clonorchis
sinesis
Enterotest
- Simpler method for collecting
duodenal material without requiring
intubation. The patient swallow a
gelatin capsule that contain a coiled
length of yarn.
Sigmoidoscopy material
- Helpful for detecting E. Histolytica.
Material from ulcer obtained by
aspiration or scraping should be
examined by direct wet preparations
and permanent stains
Cellophane tape preparation
- The specimen of choice for the
detection of Enterobius vermicularis
(pin worm) eggs
Blood
- Parasite that may be recovered in
blood including; leishmania
donovani and trypanosome spp.,
plasmodium and babesia Spp. And
microfilariae.
Collection and handling
- Blood specimens for parasite study
must be collected by aseptic
technique
- Most laboratories use venipuncture
specimens collected with an
anticoagulant.
- Blood specimens should be collected
in tubes containing Ethylene diamine
tetraaetic acid (EDTA).
*If Malaria is suspected, it is best to
prepare smear within 1 hour of
collection, because storage of blood for a
longer period leads to distortion and
possible loss of malaria parasite
 *Once the blood sample has been
collected, two types of smears may be
made; thick and thin.
Thick smears
- Frequently satisfactory for screening
purposes, particularly when malaria
is suspected
- Primarily used when parasite are few
in number or when thin smears are
negative
Advantages
Increase the ability to detect the
malaria parasite
Disadvantages
Is that red blood cells have been
lysed and it is not possible to assess
the morphology of parasite that are
detected.
Thin Smear
- Provide the best view of the malaria
parasite in the RBC
Dehemoglobinized thick smear
- Typically have much higher
concentration of parasite than thin
smear
Permanent stain
- Two permanent stains commonly
used for the detection of blood
parasites; Wrights stain and
Giemsa stain
Wrights stain
- which contains the fixative and stain
in one solution
- typically yields only satisfactory
results
Giemsa stain
- In which the two are separated
- Thus considered the preferred stain
because it allows for the detection of
parasite detail necessary for species
identification
Knot technique
- Designed to concentrate blood
specimen suspected of containing
low numbers of microfilariae
Buffy coat slide
Buffy coat - layer of white blood
cells between the plasma and red
blood cells that results from
extracted from blood specimens,
stained , stained with giemsa stain
and microscopically examined for
leishamania and trpanosoma
Cultures
Leishmania Spp and trypanosome
cruzi uses Novy-MacNeal-Nicolle
(NNN) medium.
NNN slant
- Inoculated by the addition of a single
drop of collected blood or ground
tissue
Cerebrospinal Fluid
- Specimen may be collected for the
diagnosis of amebic condition
associated with select ameba
- A wet preparation can be prepared to
search for the presence of the
characteristics morphologic forms of
Naegleria fowleri and
Acanthamoeba spp. And the
trypomastigote stage of
trpanosoma spp.
CFS sendiment
- Is inoculated to the medium, sealed
and incubated at 35 C
Hepatic abscess
- Material is the specimen of choice
for patients suspected of liver
abscesses caused by E. Histolytica
Sputum
- Typically collected and tested from
patients suspected of being infected
by the fluke
Urine
- Specimen of choice for
demonstrating the motile
trophozoites.
Alternative techniques for the
diagnosis of T. Vaginalis include
antigen detection methods using
latex agglutination and EIA
procedures
Eye specimen
- Acanthamoeba keratitis is best
diagnosed by the collection and
examination of corneal scrapings
- Acanthameoba T, gondii,
micrcosporidia and Loa Loa are
also potential eye pathogens
Mouth scraping
- Sample choice for detection of E.
ginggivalis and trichomonas tenax
are helpful for the recovery of
parasites such as N. Fowleri
Skin Snips
- Useful for detection of onchocerca
volvulus may be made using of two
collection techniques
Culture methods
- Not a common means of detecting
parasites
Xenodiagnosis
- Techinique used for the diagnosis of
changas disease
Immunologic testing
- Usually considered as an adjunct or
supplement to standard laboratory
protocols