Diagnostic Parasitology PDF

30/11/2024 Laboratory Diagnosis PARASITOLOGY -accurate...

30/11/2024 Laboratory Diagnosis PARASITOLOGY -accurate diagnosis of parasitic infections can help PARASITOLOGY decrease the prevalence and incidence of a parasitic infection. 1. Confirm clinical impression 2. Rule out diagnosis 3. Aid a clinician in the choice of proper medication DIAGNOSTIC PARASITOLOGY 4. Help in monitoring the effect of treatment regimen Diagnostic parasitology is done by: A. Demonstration of parasites (e.g., eggs, larvae, adults, cysts, oocysts, trophozoites) B. Detection of host immune response to the 1 parasites (e.g., Abs and Ags) 2 PARASITOLOGY PARASITOLOGY MACROSCOPIC EXAMINATION OF THE STOOL OUTLINE OF TECHINIQUES (stool) PARASITOLOGY PARASITOLOGY A. Consistency 1. Direct Fecal Smear * reflects the level of hydration * gives an indication as to which organisms are present 2. Kato-thick Smear 3. Concentration B. Color a. Sedimentation Techniques C. Gross examination b. Floatation Techniques *tapeworm proglottids 4. Stool Culture * adult nematodes (Ascaris or Enterobius) 5. Egg counting procedures 6. Perianal Swab 7. Staining stool specimen 3 4 1  30/11/2024 MICROSCOPIC EXAMINATION OF THE STOOL PARASITOLOGY PARASITOLOGY MICROSCOPIC EXAMINATION OF THE STOOL A. Ocular micrometer PARASITOLOGY PARASITOLOGY * specially designed ocular piece equipped with measuring scale * Must be calibrated to ensure accurate measurement * Expressed in microns (µ or µm) defined as 0.001 [10-3] millimeter, or 10-6 meter * Calibration is aided with the use of a stage micrometer containing a calibrated scale divided into 0.01-mm units. * The ocular micrometer is a disk equipped with a line evenly divided into 50 or 100 units No. of microns = (no. of stage micrometer units X 1000) / (no. of ocular micrometer units) = (0.4 x 1000) / 60 = 6. 67 um or 7 um Suggested ranges of the micron value per ocular unit by magnification: (Zeibig, 2013) 10x: 7.5-10 um 40x: 2.5-5 um 5 100x: 1 um 6 1. DIRECT FECAL SMEAR (DFS) 2. KATO THICK SMEAR PARASITOLOGY PARASITOLOGY Routine method of stool examination Employs use of 50-60 mg of stool (size of two mongo beans) Employs use of approx. 2 mg of stool and 0. 85% NSS PARASITOLOGY PARASITOLOGY Primarily useful in detection of motile protozoan trophozoites Uses cellophane paper soaked in a mixture of glycerine and Trophozoites are pale and transparent malachite green solution Nair’s buffered methylene blue (BMB) – demonstrate nuclear Simple and economical morphology of trophozoites Very good in detecting eggs with thick shells (e.g., Ascaris a. Entamoeba cytoplasm: light blue and Trichuris) but not thin shells b. Entamoeba nucleus : dark blue Lugol’s iodine – temporary stains nuclei of protozoan cysts a. cytoplasm : golden yellow * Usefulness is limited in diarrheic and watery stools b. nucleus: pale and refractile * Not able to detect protozoan cyst and trophozoite c. glycogen: deep brown * Light infections may not be detected 7 8 2  30/11/2024 3. CONCENTRATION TECHNIQUES PARASITOLOGY PARASITOLOGY Useful in cases of light infections PARASITOLOGY PARASITOLOGY A. Sedimentation Procedures 1. Acid Ether Concentration Technique (AECT) a. 40% HCl dissolves albuminous material b. ether  dissolves neutral fats - recommended for recovery Trichuris, Capillaria and trematode eggs specially Schistosoma. * Drawbacks: loss of parasite to the plug of debris and possible destruction of protozoan cyst 2. Formalin Ether Concentration Technique (FECT) a. 10% Formalin as preservative b. ether  dissolves neutral fats (highly explosive and flammable) *Ethyl acetate as alternative to ether (not as efficient as ether in the extraction of fat or mucoidal material from the stool) - more efficient in recovering cestode eggs and Giardia cysts 9 10 3. CONCENTRATION TECHNIQUES 3. CONCENTRATION TECHNIQUES PARASITOLOGY PARASITOLOGY Useful in cases of light infections Useful in cases of light infections PARASITOLOGY PARASITOLOGY B. Floatation Procedures 3. Sheather’s Sugar Floatation 1. Zinc Sulfate (ZnSO4) Floatation - employs use of boiled sugar solution preserved with - 33% ZnSO4 with specific gravity of 1.18-1.20 phenol - If parasites are exposed to high specific gravity, - best for recovery of coccidian oocysts distortion and shrinkage of protozoan cysts and thin- (e.g., Cryptosporidium, Cyclospora and Isospora) walled nematode eggs may occur. - better visualization can be appreciated through the use of a phase-contrast microscope 2. Brine Floatation - uses Table salt solution - no need for centrifugation since helminth eggs rise from the surface of the solution. -Schistosoma become badly shrunken - NOT useful for operculated eggs like Clonorchis, Opistorchis and heterophyids because these do not float in brine solution. 11 12 3  30/11/2024 4. STOOL CULTURE METHODS PARASITOLOGY PARASITOLOGY BAERMANN TECHNIQUE Useful for hookworm identification PARASITOLOGY PARASITOLOGY Stools positive for hookworm ova and/or Strongyloides rhabditiform larva can be cultured until the filariform larvae develop A. Coproculture - positive stools mixed with moistened soil and granulated charcoal - Larvae are harvested using Baermann procedure 13 14 4. STOOL CULTURE METHODS 5. EGG COUNTING PROCEDURES PARASITOLOGY PARASITOLOGY Used to correlate the severity of clinical disease with the PARASITOLOGY PARASITOLOGY B. Harada-Mori or the Test tube Culture Method intensity of infection or worm burden - employs use of test tubes and filter paper strips - also used for cultivation of intestinal protozoan A. Kato-Katz Method or Cellophane Covered Thick Smear - Filariform larva will move downwards against the B. Stoll Egg Count upward capillary movement of water and can be recovered from the water at the bottom of the tube WHO classification of intensity of infections with STH and - Strongyloides larvae move upwards and accumulate at Schistosomiasis the upper end of the filter paper strip Organism Light Intensity Moderate Intensity Heavy Intensity A. lumbricoides 1-4,999 epg 5,000-49,999 epg ≥ 50,000 epg T. trichiura 1-999 epg 1,000-9,999 epg ≥ 10,000 epg Hookworm 1-1,999 epg 2,000-3,999 epg ≥ 4,000 epg S. japonicum and 1-99 epg 100-399 epg ≥ 400 epg S. mansoni 7cc of boiled water or distilled water 15 16 4  30/11/2024 5. EGG COUNTING PROCEDURES PARASITOLOGY PARASITOLOGY A. KATO-KATZ METHOD or CELLOPHANE COVERED THICK PARASITOLOGY PARASITOLOGY SMEAR Uses a measured amount of stool which has been sieved through a wire mesh and pressed under cellophane paper soaked in malachite green solution Uniform amount of stool is examined using a template with a uniform sized hole in the middle Consistency is the main determinant of the sensitivity of this technique For ID of Schistosoma ova, 1% eosin can be layered over the cellophane paper 17 18 5. EGG COUNTING PROCEDURES PARASITOLOGY PARASITOLOGY PARASITOLOGY PARASITOLOGY B. STOLL EGG COUNT 0.1 N NaOH and a stool displacement flask calibrated at 56 mL and 60 mL Sodium hydroxide serve as stool diluent, it saponifies fat and free eggs from fecal debris Uses Stoll pipettes calibrated at 0.075 mL and 0.15 mL to measure amount of diluted stool 19 20 5  30/11/2024 6. PERIANAL SWAB (Cellulose Tape or Scotch Tape Method) PARASITOLOGY PARASITOLOGY Used to recover eggs of E. vermicularis and Taenia spp. PARASITOLOGY PARASITOLOGY In some laboratories, a drop of toluene or xylene solution helps in the visualization of eggs 21 22 7. STAINING OF STOOL SAMPLES REFERENCES PARASITOLOGY Performed specifically for the examination of the nuclear Belizario, V. and De Leon, W. (2004). Philippine Textbook of Medical PARASITOLOGY characteristics of amoeba. Parasitology. Second Edition. University of the Philippines Manila. Ermita, Also useful for ID of other intestinal protozoans such as Manila. Balantidium coli and Giardia spp. a. Iron- hematoxylin Belizario, V et al. (2013). Lecture Notes for PH 201 (Medical b. Trichome Parasitology Module). University of the Philippines - Manila, Pedro Gil St., c. Chlorazol Black E Ermita, Manila. Kinyoun staining for coccidians Manahan, E. (2007). Lecture Notes for Parasitology. University of Santo Tomas. Espana, Manila. McPherson, Richard A et. al (2011). “Henry’s Clinical Diagnosis and Management by Laboratory Methods”. Philadelphia, USA. Zeibig, Elizabeth A. (2013). Clinical Parasitology: A Practical Apporach 2nd Edition. Singapore. 23 Center for Disease Control and Prevention (CDC) 24 6