Introduction to Parasitology Required for reliable diagnosis of infection: Knowledge of patient - travel history, day care attendance, refugee? Appropriate

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.

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.

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.

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).

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.

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).

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.

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.

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.

Collection, Processing, & Examination of Specimens

Microscope - objectives must be calibrated in order to insure accurate measurement of organisms.

Centrifuges - swinging bucket type is required.

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.

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.

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.

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.

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.

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.

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.

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.

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.”

Commonly Used Preservatives, Continued

Schaudinn's Fixative - used for staining fresh specimens, contains mercury.

10% buffered formalin - for concentration only.

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.

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.

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.

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.

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.

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.


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).


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.


Concentration Techniques - Purpose: Reduce background fecal debris Increase relative number of parasites Preserve morphology of parasites


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.


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.


Types of concentration procedures, Continued:

Sedimentation Procedures - Concentrate diagnostic stages in sediment. Use of ethyl acetate cleans the specimen by dissolving and floating fat.


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.


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).


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.


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.

Special Stains

Pneumocystis carnii - methenamine-silver; Giemsa; Periodic acid Schiff.

Cryptosporidium parvum - modified acid fast stain examined with fluorescent microscopy.

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.

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.

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.

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.

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.

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.

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.


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.


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).


Biopsies: Muscle - Direct microscopic examination for

presence of Trichinella spiralis larvae. Intestinal or bladder mucosa - Direct

microscopic examination for Schistosoma spp. eggs.

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.

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.


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.


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.

Documents

Introduction to Parasitology Required for reliable diagnosis of infection: Knowledge of patient - travel history, day care attendance, refugee? Appropriate