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

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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

re 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 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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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


• 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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• Helminths



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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 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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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

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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


• 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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Human parasites of the gut - ESCMID

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