The document provides an overview of parasitology and techniques for diagnosing parasitic infections through stool examination. It discusses factors required for reliable diagnosis such as travel history and appropriate specimen collection. It also describes various stool examination techniques including direct wet mounts, concentration methods, and permanent staining. Flotation and sedimentation are outlined as concentration procedures. Considerations for stool collection kits and preservatives are also summarized.

1. Introduction to Parasitology
Required for reliable diagnosis of infection:
 Knowledge of patient - travel history, day care
attendance, refugee?
 Appropriate specimen - number, frequency, time
of collection.
 Use of appropriate procedures - flotation,
sedimentation, staining, etc.
 Adequate training of technologist - college
courses, workshops, continuing education.

2. North Americans Do Not Suffer
From a Multitude of Parasites
 High standards of education - better housing, higher
standard of living.
 General good health - poor health = more susceptible to
disease.
 Nutrition - adequate diet.
 Sanitation - sewers and septic systems keeps raw sewage
out of streams.
 Temperate climate - parasites do better in the warmth of
the tropics.
 Absence of certain vectors - intermediate hosts such as
the tsetse fly, certain snails, etc.

3. Reasons We Have Parasitic
Infections in This Country
 Increased travel - in some areas of the world
parasitic diseases are very common.
 Low understanding about parasitic infections -
results in an increased likelihood of transmission.

4. Definitions
 Parasite - one animal deriving its sustenance from another
without making compensation. The uncompensated animal
is the host.
 Parasitology - the science or study of host-parasite
relationships.
 Medical parasitology - study of parasites which infect
humans.
 Host - the partner providing food and/or protection. Some
parasites require more than one host to complete their life
cycle; Or may not require a host during some stage(s).

5. Definitions, Continued.
Types of Hosts
 Definitive host - the host in which sexual maturity
and reproduction takes place.
 Intermediate host - the host in which the parasite
undergoes essential development.
 Reservoir (carrier) host - the host harboring a
parasite in nature, serving as a source of infection
for other susceptible hosts. Reservoir hosts show
no sign or symptom of disease.
 Paratenic host - an accidental host serving as a
holding place for a parasite.

6. Definitions, Continued.
 Vector - “carrier” of a parasite from one host to another.
Often an insect.
 Symbiosis - “living together,” a close association between
two organisms.
 Mutualism - both organisms are benefited (bacteria in
bowel).
 Commensalism - “eating at the same table;” One organism
is benefited, the other is unaffected.
 Parasitism - one organism is benefited at the expense of
another (the host).

7. A Successful Parasite
 A parasite is successful - when it is in delicate
balance with the host. If the balance is upset, the
host may destroy or expel the parasite; If the host is
overly damaged, it may die - as will the parasite.
 Parasitology is important - because this balance
is not always maintained.

8. Parasitic Damage to Host:
 Trauma - damage to tissues, intestine, liver, eye.
 Lytic action - activity of enzymes elaborated by
organism.
 Tissue response - localized inflammation,
eosinophilia.
 Blood loss - heavy infection with hookworm may
cause anemia.
 Secondary infections - weakened host
susceptible to bacterial infection, etc.

9. Modes of Infection
 Filth-borne or contaminative - where personal hygiene and
community sanitation lacking. Infectious stages remain
viable for long periods in contaminated soil.
 Soil or water-borne - water or dirt which can contain eggs,
etc.; Larvae can penetrate skin of bare feet or enter skin in
infested water.
 Food-borne - inadequately cooked beef, pork, fish, shell
fish.
 Arthropod-borne - the most difficult of all to control.
Mosquitoes transmitting malaria, etc.

10. Collection, Processing, &
Examination of Specimens
 Microscope - objectives must be calibrated in order
to insure accurate measurement of organisms.
 Centrifuges - swinging bucket type is required.

11. Types of specimens which can be examined for
diagnosis of parasites:
 Natural secretions - feces, sputum, and urine are
used to detect lumen dwelling parasites of GI,
pulmonary and genitourinary tracts.
 Blood - usual specimen for detection of blood and
tissue parasites, along with tissue biopsies,
aspirates, etc.
Collection, Processing, &
Examination of Specimens,
continued.

12. Collection, Processing, &
Examination of Specimens,
continued.
Intestinal dwelling parasites - fecal specimens.
 Patient preparation - must avoid substances
which can interfere with stool examination. No anti-
microbial medications should be taken during the
10 days prior to collection of specimens.
 Medications containing antimicrobial agents –
Bismuth, Barium, Mineral oil, Kaolin, Anti-diarrheal
preparations, Laxatives.
 Contaminant free specimens - no urine, water or
dirt - these may destroy organisms, or could
contain confusing free-living organisms.

13. Types of Stool Specimens.
Liquid specimens - trophozoite stages are more likely to be
present.
Procedures - direct wet mounts for detecting motility;
permanent stains exhibit the best morphology.
 Must be examined within 30 minutes of passage or placed
into an appropriate preservative.
 Freshly passed specimens are necessary in order to recover
motile trophozoites.
 Cyst formation will not occur once the organism is outside
the body; trophozoites disintegrate rapidly after passage.

14.  Semi-formed specimens (soft specimens) - all
stages may be present.
 Procedures - one should perform all procedures
(direct examinations, concentration, acid fast, &
permanent stain).
Types of Stool Specimens,
Continued.

15. Types of Stool Specimens,
Continued.
 Formed specimens - cysts of protozoa and
eggs/larvae of helminths may be present.
 Procedures - direct wet mounts will serve to detect
those organisms which do not concentrate well.
Concentration is necessary to detect light
infections, but permanent stains are equivocal. If
blood or mucous is present on the specimen, it
should be removed and stained with a permanent
staining procedure.

16. Collection Methods
 Submit fresh specimens directly to lab - these
must be examined within 30 minutes to one hour.
Store these at room temperature if examined within
30 minutes of collection. Refrigerate them
otherwise, but never incubate these specimens.
 Commercially prepared preservation kits - use a
kit if not able to examine within 30 minute frame.
Preservation will insure the integrity of the
specimen.

17. Fixatives and Preservatives
Introduction.
 An ideal preservative would preserve all diagnostic
stages, and not interfere with concentration &
staining techniques.
 If a specimen cannot be processed immediately, at
least prepare a slide for permanent staining on the
day it is received. Then it can be stained during the
next work day, avoiding delaying results.

18. Commonly Used
Preservatives
 MIF (merthiolate - iodine - formalin) - for wet
smear & concentration only. Cannot permanent
stain.
 SAF (sodium acetate - acetic acid) - OK for
concentration, can permanent stain with iron
hemotoxylin only, trichrome stain will not produce
satisfactory results.

19. Commonly Used
Preservatives, Continued
 PVA (polyvinyl alcohol)/Formalin kit - a popular method
using two vials.
 Vial #1: 5 -10% formalin - preserves helminth eggs and
larvae, and protozoan cysts. Specimen can be used for
direct wet mounts and in a concentration procedure.
 Vial #2: polyvinyl alcohol fixative - preserves protozoan
trophozoites (and cysts) for permanent staining. There are a
variety of “fixing agents” in PVA including mercury, zinc, and
copper. Zinc is the best alternative to mercury, which
remains the “gold standard.”

20. Commonly Used
Preservatives, Continued
 Schaudinn's Fixative - used for staining fresh
specimens, contains mercury.
 10% buffered formalin - for concentration only.

21. Stool Collection Kits
Ideally, a kit should have three (3) containers:
 Formalin (5% or 10%) - for concentration.
 PVA-Fixative - for permanent staining.
 Clean vial - for unpreserved portion culture and
assessment.

22. Considerations in the
Selection of a Kit
 Vial size - should be of adequate size to allow for a
representative sample.
 Child proof - children should not be able to open the vials.
 Stirrers, scoops, etc. - should be provided to assist in
getting a specimen into the vial and mixed with the
preservative.
 Labels - that clearly indicate presence of poison; should be
present in several languages.
 Patient label information - name, date, & time of collection.

23.  Collection instructions - in several languages
representing local dialects.
 Mailers - must meet postal regulations (triple
barrier).
 Cost - must be affordable.
Considerations in the
Selection of a Kit, Continued.

24. Specimen Collection
 Collect in a clean container - without urine or water (these
may be damaging to trophozoites).
 Minimum number of specimens - due to irregular shedding
patterns of parasites, a series of three normally passed
specimens is preferred.
 Frequency of collection - collect on alternate days. Never
on same day.
 No laxatives permitted - these can mask infections or
damage organisms.
 Date and time of collection - important, should be required
information.

25. Techniques of Stool
Examination
 Gross examination - grade consistency of the
specimen; decide on best methods of examination
to allow for detecting most likely stages/parasites.
Look for worms, segments of worms. Remove these
for separate identification. If present, blood and/or
mucous should be examined with wet mounts &
permanent stains.

26. Techniques of Stool
Examination, Continued.
Direct wet mounts:
 Used primarily to detect motility - fresh, unpreserved
specimens are examined for motility; preserved specimens
are examined for organisms which do not concentrate well.
 Procedure - use large slide & coverslip. For motility, mix
small amount of specimen with physiological saline and add
coverslip. For a stained preparation, in place of saline, use
iodine stain to reveal nuclear morphology. Density - should
be able to be read newsprint through the smear.

27. Techniques of Stool
Examination, Continued.
Direct wet mounts, Continued:
 Advantages - will reveal helminth eggs and larvae; may
reveal motile trophozoite and nonmotile cysts; will reveal
other cells indicative of an inflammatory process,
(macrophages, leucocytes).
 Disadvantages - does not lend itself to oil immersion
examination; may not reveal adequate morphology causing
misinterpretation; if preparation is too thick, organisms will
be missed; cannot be used on PVA-preserved specimens
(iodine stain coagulates the PVA).

28. Techniques of Stool
Examination, Continued.
Stained Wet Mounts:
 Liquid specimens - more likely to contain
trophozoites, buffered methylene blue may be
used. Iodine is too harsh for trophozoites - often
damages morphology of nucleus.
 Formed specimens - more likely to contain only
cysts, popular stains include Dobell, Lugol, or
D'Antoni iodine stains. Do not make smears too
thick; examine them systematically.

29. Techniques of Stool
Examination, Continued.
Concentration Techniques - Purpose:
 Reduce background fecal debris
 Increase relative number of parasites
 Preserve morphology of parasites

30. Techniques of Stool
Examination, Continued.
Types of concentration procedures:
 Flotation Procedures - Floats parasites free of
fecal debris by using a solution having a specific
gravity greater than the parasites, and less than
background fecal matter. Can lose operculated
eggs, and other larger eggs since they are too
heavy to float.

31. Techniques of Stool
Examination, Continued.
Flotation Procedures, Continued:
 Advantages - can provide clean concentrate;
reagents have long shelf life, & are available
commercially; morphology is adequate.
 Disadvantages - specific gravity must be checked
frequently; the larger helminth eggs will not float;
must be examined immediately or organisms will
begin to settle.

32. Techniques of Stool
Examination, Continued.
Types of concentration procedures, Continued:
 Sedimentation Procedures - Concentrate
diagnostic stages in sediment. Use of ethyl acetate
cleans the specimen by dissolving and floating fat.

33. Techniques of Stool
Examination, Continued.
Sedimentation Procedures, Continued:
Formalin - Ethyl Acetate Procedure - Most
commonly used sedimentation procedure (good for
either fresh or preserved specimens).
 Reagents Used:
5% or 10% Formalin - kills organisms & preserves
morphology.
Ethyl Acetate - dissolves fat & floats artifacts.

34. Techniques of Stool
Examination, Continued.
Formalin - Ethyl Acetate Procedure, Continued.
 Advantages - allows the recovery of all helminth eggs,
larvae, and protozoan cysts; easy to perform; can be read
anytime following concentration; several stopping places
exist in the procedure.
 Disadvantages - must exercise caution since ethyl acetate
is flammable; preparations not as clean as with floatation;
amount of specimen used in relation to reagents must be
carefully monitored (too much specimen can interfere with
efficiency of cleansing & concentration).

35. Techniques of Stool
Examination, Continued.
Permanently Stained Smears - aimed at trophozoite stages,
and are most useful in the identification of organisms.
Factors Affecting Permanent Staining:
 Age of specimen - organisms deteriorate with time.
 Consistency - specimens with significant mucus difficult to
stick to the slide.
 Fixation - must thoroughly mix specimen with preservatives.
 Smear preparation - must not be too thick or thin.
 Reagents - must be replaced every 40 slides or weekly.

36. Techniques of Stool
Examination, Continued.
Permanent Staining Procedures:
 Iron Hematoxylin Procedure - provides excellent
morphology; time consuming and difficult to
perform.
 Wheatley’s Trichrome Stain Procedure - rapid
procedure and technically easier to perform; stable
reagents; more reproducible results.

37. Special Stains
 Pneumocystis carnii - methenamine-silver;
Giemsa; Periodic acid Schiff.
 Cryptosporidium parvum - modified acid fast
stain examined with fluorescent microscopy.

38. Immunoserologic Detection
(Parasitic Serology Tests)
 Test kits detect presence of antigen (organisms) or
antibody - Those detecting antigen are satisfactory but do
not concentrate the amount of antigen present. Other
procedures may demonstrate the antigen as well, and less
expensively.
 Tests that measure antibody include - Enzyme
Immunoassay (EIA), Complement Fixation (CF), Latex
Agglutination (LA), Direct & Indirect Immunofluorescence
(DIF & IIF), Indirect hemagglutination (IHA), Bentonite
flocculation (BF), Immunoblot (IB).
 Procedures & testing could improve - Standardization of
antigens, reference reagents, and procedures would
improve interpretation of results.

39. Identification by Molecular
Methods
 Nucleic acid probes and molecular techniques
to aid in detection of malaria, toxoplasmosis,
amebiasis and leishmaniasis are available for
diagnostic and epidemiologic purposes.

40. Quantitative Worm Egg Count
 This test is best used to estimate worm burden.
This must be performed on unpreserved
specimens. Preservatives dilute the specimen,
eliminating the ability to calculate “eggs per
gram” of feces.

41. In Vitro Cultivation of
Parasites
 Primarily used for blood & tissue protozoa.
Can culture for intestinal protozoa, but not
generally done due to time and sensitivity of
test issues.

42. Animal Inoculation
 Not routinely done - expensive, time consuming, lacks
sensitivity.
 Use - primarily used for isolation of blood and tissue
parasites (trypanosomes, etc.).
 Xenodiagnosis - may be considered a “special case” of
animal inoculation. The term was originally applied to the
diagnosis of Chagas' disease. After placing uninfected
reduviid bugs on a patient suspected of having the disease,
and allowing them to feed, the bugs are examined for
developmental stages of the parasite Trypanosoma cruzi.

43. Use of Other Specimens
 Anal Swabs / Scotch Tape Preparation for
Enterobius vermicularis - must be collected in
the morning prior to bathing or bowel movement;
used for diagnosis of pinworm infections but other
helminth eggs can be seen also, especially Taenia
spp. eggs.
 Procedure - make impressions with sticky paddle
or clear cellophane tape around the anus of the
patient; examine for eggs under the microscope.

44. Use of Other Specimens,
Continued.
Urogenital Specimens:
 Trichomonas vaginalis - vaginal, urethral, prostatic
exudates are examined via wet mounts, looking for motile
organisms.
Urine Specimens:
 T. vaginalis - often seen in urine of infected individuals.
 Schistosoma hematobium - inhabits blood vessels
around the urinary bladder, eggs “pop” into bladder as result
of expansion and contraction of the bladder along with the
aid of a terminal spine on the egg.

45. Use of Other Specimens,
Continued.
Sputum Specimens:
 Paragonimus westermani (the lung fluke) - the worm lives
in lung tissue; eggs are shed into alveoli, and are present in
the sputum.
 Ascaris lumbricoides, Strongyloides stercoralis, and
Hookworm – larvae can be present in sputum as a result of
lung migration.
 Entamoeba histolytica – trophozoites may be present as a
result of pulmonary amebic abscesses.
 Pneumocystis carinii - organisms may be seen in the
sputum of AIDS patients.

46. Use of Other Specimens,
Continued.
Aspirates and Biopsies:
 Aspiration of duodenal contents - Can be examined for
Giardia lamblia and Strongyloides stercoralis.
 Entero-Test - a capsule containing a free-wheeling piece of
yarn used to sample duodenal contents.
 Sigmoidoscopy/tissue biopsies - material from mucosa
may be examined for parasites.
 Abscess aspirates - usually for extra-intestinal amoebiasis
(wall of abscess is best area to examine).

47. Use of Other Specimens,
Continued.
Biopsies:
 Muscle - Direct microscopic examination for
presence of Trichinella spiralis larvae.
 Intestinal or bladder mucosa - Direct
microscopic examination for Schistosoma spp.
eggs.

48. Procedures for Detecting
Blood Parasites
Collection of Blood Samples:
 Finger, heel or earlobe sticks - preferred for thick and thin
blood smears.
 EDTA samples - best if smear made within one hour.
 Sodium citrate specimens - used for larger amounts of
blood to be used in concentration or cultivation.
 Clot tubes - for serological procedures; lets clot retract.

49. Procedures for Detecting
Blood Parasites, Continued.
Examination of Blood Samples:
 Wet Mounts - screening for motile organisms
(trypanosomes & filariae).
Permanent Stained Smears:
Stains - are methylene blue-eosin based.
 Wright's stain - alcohol based, can not adjust pH.
 Giemsa - water based, can adjust pH; preferred
stain for malaria examination.

50. Procedures for Detecting
Blood Parasites, Continued.
Thick Blood Films:
 Will detect - malaria parasites, trypanosomes, and
microfilariae.
 Preparation - 3 or 4 drops of blood stirred together to size
of a dime; must dehemoglobinize in buffered water prior to
staining with Wright’s stain (not necessary if using Giemsa
stain); newsprint just legible through smear.
 Advantage - concentrates blood, picks up light infections.
 Disadvantage - infected red blood cells are lost; more
experience is needed to recognize organisms. Must dry
overnight before staining.

51. Procedures for Detecting
Blood Parasites, Continued.
Thin Blood Films:
 Will detect - malaria parasites, trypanosomes, and
microfilariae.
 Preparation - same as for CBC differential.
 Advantages - allows for observation of infected red
blood cell. Morphology of organisms is better.
 Disadvantages - must examine for 30 minutes or
100 fields. Light infections may be missed.