Diagnostic Parasitology

Laboratory diagnosis
macroscopic or microscopic examination decrease the prevalence and incidence of a parasitic condition Laboratories should be able to: a. Confirm a clinical impression b. Rule out a diagnosis c. Aid in proper medication d. monitoring effect of a treatment

Ability of the laboratory to generate reliable results depends on:

Proper collection, handling and processing of specimens prior to examination Skill of the analyst Quality of equipment used in the examination

Diagnosis of parasitic infection is done by:

Definitive/ Direct: demonstration of parasite - active stage of infection Presumptive/ Indirect: detection of host immune response to the parasites Molecular biological methods: DNA Probes, Polymerase Chain Reaction (PCR)

Direct identification
1. direct mounts - helminth eggs and larvae and protozoans - motile trophozoites - aid visualization of parasite - centrifugation of watery or liquid stool

Preparation of Direct Wet Mounts

a. Saline mount

- motility of trophozoites - protozoan cysts appear more refractile - internal structures often poorly delineated -

b. Iodine preparation

- high light the internal structure - 1% solution of iodine c. Nairs buffered methylene blue stain - stain effective in showing nuclear detail in trophozoite stages when used at low pH (3.64.8)

Kato thick smear (KTS)

--advantages simple and economical efficient for thick shelled intestinal schistosoma and intestinal helminth --disadvantages not suitable for examination of larvae, cysts or egg from certain intestinal parasites not satisfactory for hard and watery stool --use cellophane paper soaked in a mixture of glycerine and malachite green

2. Concentrates method a. flotation - eggs and cysts float to the top - modified zinc floatation - formalin-fixed specimen - advantage: most of the interfering background debris is eliminated

b. sedimentation

- use either gravity or centrifugation - care must be taken to decant the supernatant

3. permanent-stained smears - morphology of are better visualized - for: -future study -teaching collections -consultation with experts - visualize intestinal protozoa in fecal smears

 iron hematoxylin stain - exacting definition of the morphology - staining procedure is somewhat difficult - For staining trophozoites of G. lamblia modified (Wheatleys) Gomoris trichrome stains - easy to perform - good results are obtained

other stains
modified acid fast stain - 1% sulfuric acid as decolorizer - oocysts of Cryptosporidium species and Cyclospora species modified trichrome staining methods (Weber stain and Ryan stain) - tiny organisms of Microsporum

 spread a thin film on the surface of a glass slide smears should be prepared from fresh specimens if possible and immediately place in fixative fixative recommended are either PVA or Schaudinns fixative PVA-fixed staining time: longer most effective: has mercuric chloride; copper sulfate as substitute are inferior disadvantage: T. trichiura eggs and G. lamblia cysts do not concentrate well morphology of larval forms of Strongyloides stercoralis is poor Isospora belli may be missed completely

procedure

Other techniques
Harade-Mori filter-paper strip culture technique Filter paper-slant culture technique Charcoal culture technique Baermann procedure for culture of strongyloides larvae Methods for performing egg counts Techniques for hatching schistosome eggs

Examination of Other Specimen

1. Intestinal specimen other than stool a. Duodenal contents - for G. lamblia and S. stercoralis - methods: saline mount and iodine preparation string test

b. rectal canal specimen - for Enterobius vermicularis - time of collection for optimal detection - early morning - method: cellulose tape c. sigmoid biopsy - for Entamoeba histolytica - use when repeated stool examination fail to reveal organisms

2. Sputum - for larval stages of hookworm, A. lumbricoides or S. stercoralis or the eggs of P. wetermani methods direct saline mount is usually sufficient

3. Urine and body fluids samples of large volume should be allowed to settle for 1 or 2 hours about 50 mL bottom samples are taken for centrifugation method: direct wet mount

4. Tissue biopsy and aspirates

samples: cutaneous ulcers skin nodules lymph nodes sample collection: needle aspirate skin snip biopsy

5. Corneal scrapings or biopsy acanthamoeba keratitis corneal scrapping are placed on a slide and fixed in methyl alcohol for 3 to 5 minutes stain using calcofluor white

6. Muscle for spiral larval form of T. spiralis specimen preparation: biopsy material treated with a digestion fluid before examination method: tease mount from a skeletal muscle biopsy 7. Blood a drop of anticoagulated blood can be placed on a microscopic slide, cover slip and examined

Indirect identification
Serology Limitations: - lack of dependable tests - problems in reliability and interpretation - High cost - Cross reaction - Does not indicate the present parasitological status of the host

Indirect test
complement fixation test (CFT) immunodiffusion (ID) indirect haemagglutination (IHA) indirect immunofluorescent antibody test (IFA) enzyme-linked immunosorbent assay (ELISA) radioimmunoassay (RIA) Less frequently used tests: Latex agglutination capillary agglutination card agglutination

NUCLEIC ACID-BASED DIAGNOSTICS

major issues in nucleic acid diagnostics: Sensitivity - abundance of unique sequences in the parasite genome Specificity - ability of DNA to denature and to renature in a highly specific manner

 DNA Probes Polymerase Chain Reaction (PCR)

SAFETY AND QUALITY CONTROL IN THE PARASITOLOGY LABORATORY

INDIVIDUAL HEALTH HAZARDS a. Eating and Drinking b. Smoking c. Handwashing d. Caring for Uniforms e. Eye Safety f. Obtaining Immunizations

DISPOSAL OF CONTAMINATED MATERIALS

a. Slides - container filled to one-third to one-half capacity with a disinfectant b. Specimens - incineration c. Small Items - same container as the slides d. Spillage - soaked with a disinfectant and allowed to stand for a time

HANDLING OF HAZARDOUS REAGENTS

a. b. c. d. e. Mercuric Compounds Corrosives Flammables Poisons Carcinogens

EQUIPMENT IN THE PARASITOLOGY LABORATORY

a. b. c. d. Refrigerator Autoclave Microbiologic Safety Hoods Centrifuges

STAINS AND REAGENTS

Storage Dark amber bottles with a tight cap information included on a reagent label: - Date opened - Complete name of the reagent - Date of manufacture - Expiration date - Initial of the person who prepared it.

a. Stains - checked periodically - check for expiration dates of stains - carryover from one dish to another should be kept at a minimum b. Reagents - Positive and negative controls - Refrigerate and store reagents accordingly

CONTINUING EDUCATION
a. b. c. d. Inservice Seminars Surveys Workshops, Seminars, and Symposia References