human parasites of the gut - escmid

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

“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

re Library

“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

re Library

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

Classical diagnostic methods for detection of intestinal parasites

• macroscopic examination

• microscopic techniques

• non microscopic (non morphological) techniques • ELISA / copro-immunochemistry / Ag testing

• serology

Ascaris lumbricoides male and female

Eg,Em & Tsol 11

Taenia solium cysticercose

– CT scan brain laesions < 2cm

– X –ray muscles (eg upperleg)

Serodiagnosis Molecular techniques

Enterobius vermicularis Cellulose tape (Scotch tape) • Size 10 mm

D. fragilis

Ascaris

T. trichiura

C. cayetanensis

E. histolytica/ dispar hookworm

G. lamblia

S. stercoralis

microscopic diagnosis of intestinal parasites

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

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

• Question 2

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

Evaluation of the TFT in routine clinical practice

462 patients

AMC, Amsterdam

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

Results with one, non fixed, sample compared to TFT (no. patients = 462)

One, non fixed, sample TFT

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

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

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

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%

cave • well trained and experienced technicians are essential

• 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

Diarrheal complaints

• Protozoans

• In case of immuundeficiency

– Cryptosporidium! – Microsporidium sp

• Helminths

E. histolytica and E. dispar

Entamoeba histolytica trophozoite

fagositized Red blood cells

nucleus ESCMID Online Lecture Library

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

Cryptosporidium sp • Acid fast staining

– Mod ZN, Kinyoun, IHK – Ridley sediment

• Auramin staining

• IFA

• ELISA

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!

Nematodes • Strongyloides stercoralis

– Fresh stool sample – Baermann – Culture (filariform larvae)

– PCR

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

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

• Verweij et al 2007 ESCMID Online Lecture Library

rt-PCR Giardia: Verweij et al 2004 microscopy: TFT

rt-PCR Giardia: Verweij et al 2004 Microscopy: TFT Giardia:ELISA: Ridaquick Giardia Crypto Entamoeba combi R-biopharm

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

• Helminths

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

Ascaris lumbricoides 1 Diphyllobotrium latum 1 Hymenolepis nana 2 Taenia saginata 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)

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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)

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

• epidemiological knowledge on parasitic infections is mandatory

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

human parasites of the gut - escmid

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