Cryptosporidium Cystoisospora Belli Cyclospora Cayetanensis © by Author

Coccidians

Cryptosporidium Cystoisospora belli Cyclospora cayetanensis © by author

ESCMID Online Lecture Library

ESCMID Online Lecture Library Diagnostic techniques

Cryptosporidium Cyclospora cayetanensis Cystoisospora belli sporulation In host weeks days seize 2-5 μm 5-8 μm flask

JKJ no uptake no uptake no uptake

Acid fast + ©+ by author +

Autofluorescence - + + ESCMID Online Lecture Library

ESCMID Online Lecture Library Auto fluorescence

• Prepare direct smear or a smear from Ridley sediment in saline (not JKJ)

• Screen smear with fluorescence microscope with excitation filter 340-380 nm (20x10; details 40x10)

• oöcyst wall: blue/white© by fluorescent author

• Also for SAF preserved samples ESCMID Online Lecture Library Treatment Cyclospora / Cystoisospora belli

• Co-trimoxazol – 2dd TMP 160 mg SMX 800 mg x 7-10 d

– Children – 2dd TMP 5 mg/kg SMX 25 mg/kg x 7-10 d

ESCMID Online Lecture Library Cryptosporidium

HIV / AIDS Waterborne outbreaks

ESCMID Online Lecture Library Clinical presentation

Immunocompetent individuals: • Self-limiting diarrhea • Vomiting • Nausea, decreased appetite, weight loss, flatulence • Abdominal pain and cramps.

Immunocompromised individuals: CD4 Tcell-count <200 mm3 © by author • persistent diarrheal infection >30 days • severe illness.

ESCMID Online Lecture Library Clinical presentation / species

- Cryptosporidium parvum - Cryptosporidium hominis

- other species (eg C felis) non pathogenic 4 humans

ESCMID Online Lecture Library Treatment

- Metronidazole / Tinidazole - Secnidazole - Clioquinol - Albendazol - Paromomycine - Cotrimoxazole - Doxycycline

- In different dosages /© combinations by author

ESCMID Online Lecture Library Nitazoxanide

- Alinia / Annita. FDA approved 2004 - > 11 jaar 2dd 500 mg 3 dagen

- Nitrothiazolyl- salicylamide derivaat - 2-acetyloxy-N-(5-nitro-2-thiazolyl)benzamide

- Metabolized to tizoxamide - Pyruvaat:ferredoxine oxidoreductase (PFOR) - Anaerobe energie metabolisme© by author (Giardia)

ESCMID Online Lecture Library Placebo controlled trials Nitazoxanide: 3-day course of nitazoxanide significantly improved the resolution of diarrhoea, parasitological eradication, and mortality in HIV- seronegative, but not HIVseropositive,children.

Rossignol JF, Ayoub A, Ayers MS. J Infect Dis. 2001 Jul 1;184(1):103-6.. Amadi B, Mwiya M, Musuku J, Watuka A, Sianongo S, Ayoub A, Kelly P. Lancet. 2002 2;360(9343):1375-80.

In immunocompromised individuals there is no evidence for effective treatment (paromomycine and nitazoxanide) The most effective therapy in patients with AIDS is highly active antiretroviral treatment (HAART)© of by HIV. author

Abubakar I, Aliyu SH, Arumugam C, Hunter PR, Usman NK. Cochrane Database Syst Rev. 2007 Jan 24;(1): Prevention and treatment of cryptosporidiosis in immunocompromised patients.. ESCMID Online Lecture Library Due to the seriousness of the potential outcomes of cryptosporidiosis, the use of nitaxozanide should be considered in immunocompromised patients

Abubakar I, Aliyu SH, Arumugam C, Hunter PR, Usman NK. Cochrane Database Syst Rev. 2007 Jan 24;(1): Prevention and treatment of cryptosporidiosis in immunocompromised patients..

ESCMID Online Lecture Library Additional techniques 4 diagnosis of intestinal parasites

ESCMID Online Lecture Library

ESCMID Online Lecture Library Quality control

• External quality proficiency panels

• Internal quality program – Internal standard – Other test eg G lamblia ELISA – Inter observer testing – Spot test © by author – QC fixatives – Prevalence rates in own population ESCMID Online Lecture Library

ESCMID Online Lecture Library Schistosomiasis

• Glycerin sedimentation

• Serology (time lag between possible exposition – testing minimal 8 weeks!) © by author

ESCMID Online Lecture Library Strongyloides stercoralis

Immunocompromised / transplantation

• Baermann technique for rabditiform larvae – Hookworm!! genital premordium

• Culture for filariform larvae – Hookworm: tapered© tail by Stongyloides: author forked tail

• Serology (timelag!!) ESCMID Online Lecture Library High probability of helminth infection • eg refugees

• Ridley concentration from all 3 fecal samples ( 2 SAF fixed, 1 fresh)

ESCMID Online Lecture Library Microsporidium

• Profound diarrhoea in immunocompromised patient in particular HIV/AIDS (CD4 < 100)

• Saline Ridley / no wooden sticks

• Calcofluor staining

• Trichrome acc weber

• PCR © by author

• Spores are very small ESCMID Online Lecture Library Non- morfological techniques

• Copro ELISA

• PCR © by author

ESCMID Online Lecture Library Potential pro’s and cons

• No specialized personnel in parasite morfology is needed anymore • Expensive training program

• For ELISA every laboratory can do it • For PCR trained personnel and molecular facilities are mandatory © by author • Antigens and DNA are shedded continuously; so single sample testing is sufficient ESCMID Online Lecture Library Frequently found intestinal parasites in the Netherlands

Protozoans Helminths

Giardia lamblia Ascaris lumbricoides Entamoeba histolytica/dispar Enterobius vermicularis Cryptosporidium spp Hymenolepis nana Cyclospora cayetanensis Hymenolepis diminuta Dientamoeba fragilis Hookworm Cytoisospora belli Schistosoma spp Microsporidium spp Strongyloides stercoralis Trichuris trichiura Blastocystis hominis Taenia spp Endolimax nana Entamoeba hartmanni Entamoeba coli © by author Enteromonas hominis Iodamoeba bütschlii Chilomastix mesnili ESCMID Online Lecture Library Frequently found intestinal parasites in the Netherlands

Protozoans Helminths

• Copro ELISA

– Giardia / Crypto: from SAF and fresh – E. histolytica: only from fresh • Sens higher if performed from sediment after saline Ridley © by author • PCR – fresh stoolsample (day 2) ESCMID Online Lecture Library Sensitivity of the Triple-Faeces-Test (T.F.T.) versus Enzyme Immuno Assay in the laboratory diagnosis of Giardiasis

ESCMID Online Lecture Library Methods

• Prospective study

• TFT sets from routine clinical practice

• Microscopy (AMC)

• JKJ direct smear, Chlorazol Black stained smear formalin ether sedimentation according Ridley

• EIA testing (Haarlem) • Three stool samples© by (day author 1, 2, and 3) in three EIA’s Prospect Giardia (Alexon, Remel), Techlab test Biotech Trinity test ESCMID Online Lecture Library Methods

• Microscopy according to standardized protocol (magnification, time)

• EIA observer blind

• Discrepancy study © by author

ESCMID Online Lecture Library Results

T.F.T. sets (patients) included: 171

• Microscopy positive: 9 (5,3%)

• Remel ProsPect Giardia positive: 10 (+1) • Giardia test Techlab positive: 26 (+19) • Giardia Trinity Biotech positive: 52 (+43) discrepancy study:© false by negativeauthor microscopy 1 x

ESCMID Online Lecture Library Results of the microscopic examination of the TFT set of Giardia lamblia infected cases (N=9)

Case day 1 day2 day3

1 - cysts - 2 - cysts - 3 cyst + trof cysts cyst + trof 4 cysts - - 5 cysts cysts - 6 - cysts cysts 7 - - cysts 8 - © bycyst authors cyst + trof 9 cysts cysts -

ESCMID Online Lecture Library Results of the microscopic examination and EIA of the TFT set of Giardia lamblia infected cases (N=11)

Case day 1 day2 day3 1 neg cysts neg 2 neg cysts neg 3 cysts + trof cysts cyst + trof 4 cysts neg neg 5 cysts cysts neg 6 neg cysts cysts 7 neg neg cysts 8 neg © bycyst authors cyst + trof 9 cysts cysts neg 10 neg neg neg 11 ESCMIDneg Online neg Lecture Libraryneg Testcharacteristics of the different diagnostic strategies compared with the yield of the ‘optimal test’ for laboratory diagnosis of giardiasis

Diagnostic Strategy Optimal test false negatives sensitivity NPV (%) (%) microscopy day 1 7 36 95 microscopy day 2 4 64 97 microscopy day 3 7 36 96

TFT 2 82 99

Prospect day 1 © by4 author 64 98 Prospect day 2 3 73 98 Prospect day 3 3 73 98 ProspectESCMID TFT Online Lecture1 Library91 99 Conclusions

Although to a lesser extent than cysts and trofozoites, the shedding of G. lamblia specific antigens is intermittent in patients infected with Giardia lamblia.

The T.F.T. set has a higher sensitivity as compared to a “single EIA”

The Remel (Alexon) ProsPect Giardia test is a powerful diagnostic tool in the laboratory diagnosis of giardiasis © by author

ESCMID Online Lecture Library New developments / trends 1

New tests

R-Biopharm – ridascreen ridascreen Cryptosporidium ridascreen Giardia lamblia

Multi parasite testing Giardia + Cryptosporidium

ESCMID Online Lecture Library Name Firm Remarks

Triage parasite Biosite Giardia lamblia Cryptosporidium spp E. histo/dispar

X/pect Remel Giardia solo (Oxoid) Giardia / Crypto combi

Rida Quick r-biopharm Giardia solo ESCMID Online(Bipharma) Lecture Giardia Library / Crypto combi

ESCMID Online Lecture Library

ESCMID Online Lecture Library Comparative studies in the Netherlands

Name Species

Triage parasite Giardia lamblia sens: >93% spec: >98%

X/pect Giardia lamblia sens: >95% spec: >98%

ImmunoCardSTAT Giardia lamblia sens: >95% © by authorspec: >98% Rida Quick Giardia lamblia sens: >95% spec: >98% ESCMID Online Lecture Library

ESCMID Online Lecture Library Design and study population

• Study A - known samples Entamoeba histolytica/dispar (N=50) Giardia lamblia (N=25) Cryptosporidium parvum (N=19) Negatieve controls (N=50)

• Study B - Prospective study © by author November 1999 - November 2000 Samples from routine clinical practice N=548 ESCMID Online Lecture Library Giardia lamblia

Study A Prospective study Combined (N=75) (N=548) (N=627)

N positives (microscopy) 25 23 48 Prevalence (%) 33,3 4,2 7,7 False Negatives 1 1 2 Sensitivity (%) 96,0 95,6 95,8 Neg. Pred. Value (%) 98,0 99,8 99,7 False Positives 0 6 6 Specificity (%) 100© by author 98,8 99,0 Pos. Pred. Value (%) 100 78,6 88,5

ESCMID Online Lecture Library Parasitic species found in stool samples from patients in routine clinical practice in Haarlem, the Netherlands. (N=548)

Species N Triage Prevalence (%)

Giardia lamblia 23 22 4.2 Cryptosporidium spp 6 4 1.1 Entamoeba histolytica/dispar 7 3 1.3 Dientamoeba fragilis 45 8.2

C. cayetanensis © by author 1 Blastocystis hominis 126 23.0 non pathogenic protozoans 42 7.7 Helminth eggsESCMID Online Lecture22 Library4.0 Non morfological techniques

• Can be very helpful

– Negative / positive predictive value largely depends on the population you work for!

– What would be missed in case you use ELISA / PCR as a screening tool?

– How to monitor treatment effects? ESCMID Online Lecture Library