ESCMID Online Lecture Library © by Author “Classical”

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

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

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

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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

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 Library spec: >98%

• 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

E. histolytica and E. dispar

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

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

• 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 ESCMID• Verweij et al 2007Online Lecture Library © by author ESCMID Online Lecture Library rt-PCR Giardia: Verweij et al 2004 microscopy: TFT © by author

rt-PCRESCMID Giardia: Verweij et al 2004 Online Lecture Library Microscopy: TFT Giardia:ELISA: Ridaquick Giardia Crypto Entamoeba combi R-biopharm © by author 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 ESCMID Online Lecture Library

• Protozoans • Helminths

Parasitic species detected in 2000 TFT sets by rt-PCR and microscopy Species rt-PCR TFT Giardia/ Crypto/ E. histo/ D.fragilis 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 63 species like E.coli, E. nana

Ascaris lumbricoides 1 DiphyllobotriumESCMID latum Online Lecture Library1 Hymenolepis nana 2 Taenia saginata© by author 3 (2x proglottides) “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)

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 Lecture Library © by author ESCMID Online Lecture Library © by author “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)

• 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 ESCMID Online Lecture Library

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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