Human parasites of the gut: epidemiology and diagnostic approaches in the molecular era Classical diagnostic methods for detection of parasites Theo Mank ESCMID Online Lecture Library © by author “classical” • relating to the first developed form or system of a science • relating to, or being music in established European styles and forms (as the symphony and opera) • having recognized and of permanent value ESCMID Online Lecture Library © by author “classical” • relating to the first developed form or system of a science • relating to, or being music in established European styles and forms (as the symphony and opera • having recognized and of permanent value ESCMID Online Lecture Library © by author What are we looking for? • adult worms / proglottides • larvae • helminth eggs • cysts • trofozoites / vegetative stages • oöcysts • spores • Adult worms and eggs are to be found in the definitive host only! ESCMID Online Lecture Library © by author • Protozoans • Helminths – Cryptosporidium sp – Ascaris lumbricoides – Cyclospora cayetanensis – Diphyllobotrium sp – Cystoisospora belli – Enterobius vermicularis – Dientamoeba fragilis – Fasciola hepatica – Entamoeba histolytica – Hymenolepis nana – Giardia lamblia – Hymenolepis diminuta – Microsporidium sp – Hookworm – Schistosoma sp – Blastocystis hominis – Strongyloides stercoralis – Trichuris trichiura – Chilomastix mesnili – Taenia sp – Endolimax nana – Clonorchis sinensis – Entamoeba coli – Opisthorchis sp – Entamoeba dispar – Cappilaria phillipinensis ESCMID– Entamoeba hartmanni Online Lecture Library – Iodamoeba bűtschlii © by author Classical diagnostic methods for detection of intestinal parasites • macroscopic examination • microscopic techniques • non microscopic (non morphological) techniques • ELISA / copro-immunochemistry / Ag testing •ESCMID serology Online Lecture Library © by author ESCMID Online Lecture Library © by author Ascaris lumbricoides male and female ESCMID Online Lecture Library © by author Ascaris lumbricoides male and female ESCMID Online Lecture Library © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library 11 © by author Eg,Em & Tsol ESCMID Online Lecture Library © by author Taenia solium cysticercose – CT scan brain laesions < 2cm – X –ray muscles (eg upperleg) ESCMID Online Lecture Library © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library Serodiagnosis 15 Molecular techniques © by author Enterobius vermicularis Cellulose tape (Scotch tape) • Size 10 mm ESCMID Online Lecture Library © by author microscopic diagnosis of intestinal parasites S. stercoralis Ascaris C. cayetanensis G. lamblia T. trichiura E. histolytica/ D. fragilis hookworm dispar ESCMID Online Lecture Library © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library © by author Microscopic techniques • fresh / preserved stool sample • multiple sampling • concentration techniques / Ridley and Hawgood • wet smear / Iodine stained smears / eosin • permanent staining • eg chlorazol black / IHK / Giemsa / Trichrome • Acid fast staining (ZN ) • Autofluorescence • Optical Bright staining / mod Trichrome ESCMID Online Lecture Library © by author cave • well trained and experienced technicians are essential for reliable microscopy (sensitivity and specificity) • anamnestic data are mandatory for a parasitological workup eg travelhistory; complaints, immuunstatus, eosinophilia, elevated IgE • epidemiological knowledge / priori chance ESCMID Online Lecture Library © by author • Question 1 ESCMID Online Lecture Library © by author • Question 2 ESCMID Online Lecture Library © by author Microscopy Improved diagnosis of intestinal parasites T.F.T. = Triple - Faeces – Test T van Gool, R Weijts, E Lommerse T Mank Triple Faeces Test: an effective tool for detection of intestinal parasites in routine clinical practice. Eur J Clin Microbiol & inf diseases 2003:22(5);284-90 ESCMID Online Lecture Library © by author Characteristics of the TFT • Use of fixative: – SAF • Use of multiple sampling: – 3 consecutive days • Use of permanent stain: – Chlorazol black or Iron Haematoxylin Kinyoun ESCMID Online Lecture Library © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library © by author Evaluation of the TFT in routine clinical practice 462 patients AMC, Amsterdam ESCMID Online Lecture Library © by author Increased recovery of intestinal protozoa in TFT (462 patients) Organism One stool TFT Increase with sample(NF) TFT pathogen G. lamblia 18 24 6 E. histolytica 14 18 4 D. fragilis 0 45 45 apathogen E. coli 52 65 13 E. hartmanni 10 23 13 E. nana 42 47 5 C. mesnilii 7 10 3 I. bütschlii 6 12 6 B. hominis 0 124 124 ESCMID Online Lecture Library © by author Results with one, non fixed, sample compared to TFT (no. patients = 462) ESCMID Online Lecture Library One, non fixed, sample TFT © by author Conclusions TFT in routine clincial practice: • High sensitivity and specificity • Multiple sampling with high compliance • Low cost sampling and laboratory material • Additional labour-time acceptable ESCMID Online Lecture Library T van Gool, R Weijts, E Lommerse TMank Triple Faeces Test: an effective tool for detection of intestinal parasites in routine ©clinical bypractice. Eur author J Clin Microbiol & inf diseases 2003:22(5);284-90 ELISA (copro-immunochemistry) • 65kD Giardia Specific Antigen (GSA) monoclonal • Single sample (fresh or SAF preserved) • Less intermittent shedding • “excreted” during encystation • 96 wells format • Immunocards (ICT test) – often combined with Cryptosporidium sp & E hist/dispar • DipsticksESCMID Online Lecture Library © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library © by author Name Company Remarks Triage parasite Biosite Giardia lamblia Cryptosporidium spp E. histo/dispar X/pect Remel Giardia solo Giardia / Crypto combi ImmunoCardSTAT Meridian Giardia lamblia Cryptosporidium spp RidaESCMID Quick Online r-biopharm Lecture Giardia soloLibrary Giardia / Crypto combi © by author Litt + comparative studies with TFT Naam Species Triage parasite Giardia lamblia sens: >93% Cryptosporidium sp spec: >98% X/pect Giardia lamblia sens: >95% Cryptosporidium sp spec: >98% ImmunoCardSTAT Giardia lamblia sens: >95% Cryptosporidium sp spec: >98% Rida Quick Giardia lamblia sens: >95% ESCMID Online Cryptosporidium Lecture sp Libraryspec: >98% © by author cave • well trained and experienced technicians are essential for reliable microscopy (sensitivity and specificity) • anamnestic data are mandatory for a parasitological workup eg travelhistory; complaints, immuunstatus, eosinophilia, elevated IgE • epidemiological knowledge / priori chance ESCMID Online Lecture Library © by author Diarrhea I presume ESCMID Online Lecture Library © by author Diarrheal complaints • Protozoans • In case of immuundeficiency – Cryptosporidium! – Microsporidium sp ESCMID Online Lecture Library © by author • Protozoans • Helminths – Cryptosporidium sp – Ascaris lumbricoides – Cyclospora cayetanensis – Diphyllobotrium sp – Cystoisospora belli – Enterobius vermicularis – Dientamoeba fragilis – Fasciola hepatica – Entamoeba histolytica – Hymenolepis nana – Giardia lamblia – Hymenolepis diminuta – Microsporidium sp – Hookworm – Schistosoma sp – Blastocystis hominis – Strongyloides stercoralis – Trichuris trichiura – Chilomastix mesnili – Taenia sp – Endolimax nana – Clonorchis sinensis – Entamoeba coli – Opisthorchis sp – Entamoeba dispar – Cappilaria phillipinensis ESCMID– Entamoeba hartmanni Online Lecture Library – Iodamoeba bűtschlii © by author E. histolytica and E. dispar ESCMID Online Lecture Library © by author Entamoeba histolytica trophozoite ESCMID Online Lecture Library © by author Entamoeba histolytica trophozoite fagositized Red blood cells ESCMID Online Lecture Librarynucleus © by author E. histolytica and E. dispar • E. histolytica/dispar • High priori chance of E. histolytica – Travelling endemic country – Blood in feces – Specific E. histolytica serology – copro-ELISA no discrimination / low sensitivity – PCR ESCMID Online Lecture Library © by author Cryptosporidium sp • Oocysts are small 3-5 μm • Are easily missed in standard O&P • Specific methods are necessary • Many Medical doctors are unfamiliar with the parasite ESCMID Online Lecture Library © by author Cryptosporidium sp • Acid fast staining – Mod ZN, Kinyoun, IHK – Ridley sediment • Auramin staining • IFA • ELISA ESCMID Online Lecture Library © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library © by author Travelhistory (sub) tropics • Helminth infections • Schistosoma sp – prepatency! it takes several weeks after possible exposition / infection before eggs can be found – serology! • Strongyloides stercoralis - Fresh stool sample (<12 hrs) is mandatory ESCMID- serology! Online Lecture Library © by author Nematodes • Strongyloides stercoralis – Fresh stool sample – Baermann – Culture (filariform larvae) – PCR ESCMID Online Lecture Library © by author Differentiation Strongyloides stercoralis / hookworm Filariform larvae (after culture) short mouth long mouth S. stercoralis hookworm ESCMIDopening opening Online Lectureforked tail Librarytapered tail large genital small/no genital unsheathed sheated primordium© by primordium author New developments • Introduction of rt-multiplex PCR as a screening tool for parasitological stoolexaminations ESCMID Online Lecture Library © by author PCR • rt-multiplex PCR – Giardia lamblia Short Subunit