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Human parasites of the gut: epidemiology and diagnostic approaches in the molecular era
Classical diagnostic methods for detection of parasites
Theo Mank
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“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 Lectu
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“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 Lectu
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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 Lectu
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• Protozoans – Cryptosporidium sp – Cyclospora cayetanensis – Cystoisospora belli – Dientamoeba fragilis – Entamoeba histolytica – Giardia lamblia – Microsporidium sp
– Blastocystis hominis
– Chilomastix mesnili – Endolimax nana – Entamoeba coli – Entamoeba dispar – Entamoeba hartmanni – Iodamoeba bűtschlii
• Helminths
– Ascaris lumbricoides – Diphyllobotrium sp – Enterobius vermicularis – Fasciola hepatica – Hymenolepis nana – Hymenolepis diminuta – Hookworm – Schistosoma sp – Strongyloides stercoralis – Trichuris trichiura – Taenia sp – Clonorchis sinensis – Opisthorchis sp – Cappilaria phillipinensis
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Classical diagnostic methods for detection of intestinal parasites
• macroscopic examination
• microscopic techniques
• non microscopic (non morphological) techniques • ELISA / copro-immunochemistry / Ag testing
• serology
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Ascaris lumbricoides male and female
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Ascaris lumbricoides male and female
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Eg,Em & Tsol 11
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Taenia solium cysticercose
– CT scan brain laesions < 2cm
– X –ray muscles (eg upperleg)
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Serodiagnosis Molecular techniques
15
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Enterobius vermicularis Cellulose tape (Scotch tape) • Size 10 mm
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D. fragilis
Ascaris
T. trichiura
C. cayetanensis
E. histolytica/ dispar hookworm
G. lamblia
S. stercoralis
microscopic diagnosis of intestinal parasites
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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
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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 Lectu
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• Question 1
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• Question 2
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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 Lectu
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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
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Evaluation of the TFT in routine clinical practice
462 patients
AMC, Amsterdam
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Increased recovery of intestinal protozoa in TFT (462 patients)
Organism One stool sample(NF)
TFT Increase with 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
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Results with one, non fixed, sample compared to TFT (no. patients = 462)
One, non fixed, sample TFT
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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 T van Gool, R Weijts, E Lommerse TMank 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
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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 • Dipsticks
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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 Rida Quick r-biopharm Giardia solo Giardia / Crypto combi
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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% Cryptosporidium sp spec: >98%
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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 Lectu
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Diarrhea I presume
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Diarrheal complaints
• Protozoans
• In case of immuundeficiency
– Cryptosporidium! – Microsporidium sp
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• Protozoans – Cryptosporidium sp – Cyclospora cayetanensis – Cystoisospora belli – Dientamoeba fragilis – Entamoeba histolytica – Giardia lamblia – Microsporidium sp
– Blastocystis hominis
– Chilomastix mesnili – Endolimax nana – Entamoeba coli – Entamoeba dispar – Entamoeba hartmanni – Iodamoeba bűtschlii
• Helminths
– Ascaris lumbricoides – Diphyllobotrium sp – Enterobius vermicularis – Fasciola hepatica – Hymenolepis nana – Hymenolepis diminuta – Hookworm – Schistosoma sp – Strongyloides stercoralis – Trichuris trichiura – Taenia sp – Clonorchis sinensis – Opisthorchis sp – Cappilaria phillipinensis
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E. histolytica and E. dispar
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Entamoeba histolytica trophozoite
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Entamoeba histolytica trophozoite
fagositized Red blood cells
nucleus ESCMID Online Lecture Library
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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
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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
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Cryptosporidium sp • Acid fast staining
– Mod ZN, Kinyoun, IHK – Ridley sediment
• Auramin staining
• IFA
• ELISA
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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 - serology!
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Nematodes • Strongyloides stercoralis
– Fresh stool sample – Baermann – Culture (filariform larvae)
– PCR
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Differentiation Strongyloides stercoralis / hookworm
Filariform larvae (after culture)
S. stercoralis hookworm forked tail tapered tail unsheathed sheated
short mouth long mouth opening opening large genital small/no genital primordium primordium
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New developments
• Introduction of rt-multiplex PCR as a screening tool for parasitological stoolexaminations
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PCR
• rt-multiplex PCR – Giardia lamblia Short Subunit
ribosomal RNA gen (62 bp) – C parvum/hominis DNAJ like
protein gen (138 bp) – E. histolytica SSU rRNA gen
(172 bp) • Verweij et al 2004
• rt-multiplex PCR – Dientamoeba fragilis 5.8S
rRNA (98 bp) – PhHV
• Verweij et al 2007 ESCMID Online Lecture Library
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rt-PCR Giardia: Verweij et al 2004 microscopy: TFT
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rt-PCR Giardia: Verweij et al 2004 Microscopy: TFT Giardia:ELISA: Ridaquick Giardia Crypto Entamoeba combi R-biopharm
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Molecular techniques
• PCR – multiplex rt PCR – Giardia lamblia, Cryptosporidium, D fragilis, E
histolytica
- microscopic confirmation of positive PCR result is mandatory since the clinical relevance of unconfirmed positive PCR results is (still) unclear
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• Protozoans – Cryptosporidium sp – Cyclospora cayetanensis – Cystoisospora belli – Dientamoeba fragilis – Entamoeba histolytica – Giardia lamblia – Microsporidium sp
– Blastocystis hominis
– Chilomastix mesnili – Endolimax nana – Entamoeba coli – Entamoeba dispar – Entamoeba hartmanni – Iodamoeba bűtschlii
• Helminths
– Ascaris lumbricoides – Diphyllobotrium sp – Enterobius vermicularis – Fasciola hepatica – Hymenolepis nana – Hymenolepis diminuta – Hookworm – Schistosoma sp – Strongyloides stercoralis – Trichuris trichiura – Taenia sp – Clonorchis sinensis – Opisthorchis sp – Cappilaria phillipinensis
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• Protozoans – Cryptosporidium sp – Cyclospora cayetanensis – Cystoisospora belli – Dientamoeba fragilis – Entamoeba histolytica – Giardia lamblia – Microsporidium sp
– Blastocystis hominis
– Chilomastix mesnili – Endolimax nana – Entamoeba coli – Entamoeba dispar – Entamoeba hartmanni – Iodamoeba bűtschlii
• Helminths
– Ascaris lumbricoides – Diphyllobotrium sp – Enterobius vermicularis – Fasciola hepatica – Hymenolepis nana – Hymenolepis diminuta – Hookworm – Schistosoma sp – Strongyloides stercoralis – Trichuris trichiura – Taenia sp – Clonorchis sinensis – Opisthorchis sp – Cappilaria phillipinensis
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Parasitic species detected in 2000 TFT sets by rt-PCR and microscopy
Species rt-PCR Giardia/ Crypto/ E. histo/ D.fragilis
TFT microscopy
Cryptosporidium sp 85 62 D. fragilis 245 186 E. histolytica 1 1 (E his/dis)
Giardia lamblia 202 184
Blastocystis hominis 483 Non pathogenic protozoal species like E.coli, E. nana
63
Ascaris lumbricoides 1 Diphyllobotrium latum 1 Hymenolepis nana 2 Taenia saginata 3 (2x proglottides)
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“the best of both worlds”
- Molecular screening rt-PCR e.g.Giardia lamblia, Cryptosporidium parvum/hominis, E. histolytica en D. fragilis TFT-2 - Confirmation of positive PCR results
- Additional test based on request of physician
- Additional test based anamnestic data (lab)
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Laboratory form
Parasitology Questionnaire parasites -Intestinal parasites -foreign travel / tropics? -Schistosoma spp -specific skin disorders? -Strongyloides -eosinofilia? -Amebic abces -immunocompromised / deficient? -Leishmania spp -helminths seen? -Pneumocystis (PCP) -therapy? -Malaria -other: -other: ESCMID Online Lectu
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“the best of both worlds”
DFT plus Duo Feces Test 1 day stool sample 1 part in tube 1 (SAF) 1 part in tube 2 (fresh)
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Conclusions
• It is of major importance to be familiar with the possibilities / impossibilities of the different diagnostic methods used in parasitological stoolexaminations
• anamnestic data are mandatory for a parasitological workup (eg travelhistory; complaints, immuunstatus, eosinophilia, elevated IgE)
• well trained and experienced technicians are essential
for reliable microscopy (sensitivity and specificity)
• epidemiological knowledge on parasitic infections is mandatory
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Conclusions
• parasitological stoolexaminations based on molecular techniques only will result in loss of microscopy skills of well trained and experienced technicians and would be to the detriment of parasitology services worldwide
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