Coccidians. Cryptosporidium Cystoisospora belli Cyclospora cayetanensis. by author

Transcription

1 Coccidians Cryptosporidium Cystoisospora belli Cyclospora cayetanensis

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6 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 Autofluorescence - + +

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8 Auto fluorescence Prepare direct smear or a smear from Ridley sediment in saline (not JKJ) Screen smear with fluorescence microscope with excitation filter nm (20x10; details 40x10) oöcyst wall: blue/white fluorescent Also for SAF preserved samples

9 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

10 Cryptosporidium HIV / AIDS Waterborne outbreaks

11 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 persistent diarrheal infection >30 days severe illness.

12 Clinical presentation / species - Cryptosporidium parvum - Cryptosporidium hominis - other species (eg C felis) non pathogenic 4 humans

13 Treatment - Metronidazole / Tinidazole - Secnidazole - Clioquinol - Albendazol - Paromomycine - Cotrimoxazole - Doxycycline - In different dosages / combinations

14 Nitazoxanide - Alinia / Annita. FDA approved > 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 (Giardia)

15 Placebo controlled trials Nitazoxanide: 3-day course of nitazoxanide significantly improved the resolution of diarrhoea, parasitological eradication, and mortality in HIVseronegative, but not HIVseropositive,children. Rossignol JF, Ayoub A, Ayers MS. J Infect Dis Jul 1;184(1): Amadi B, Mwiya M, Musuku J, Watuka A, Sianongo S, Ayoub A, Kelly P. Lancet ;360(9343): 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 HIV. Abubakar I, Aliyu SH, Arumugam C, Hunter PR, Usman NK. Cochrane Database Syst Rev Jan 24;(1): Prevention and treatment of cryptosporidiosis in immunocompromised patients..

16 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 Jan 24;(1): Prevention and treatment of cryptosporidiosis in immunocompromised patients..

17 Additional techniques 4 diagnosis of intestinal parasites

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19 Quality control External quality proficiency panels Internal quality program Internal standard Other test eg G lamblia ELISA Inter observer testing Spot test QC fixatives Prevalence rates in own population

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21 Schistosomiasis Glycerin sedimentation Serology (time lag between possible exposition testing minimal 8 weeks!)

22 Strongyloides stercoralis Immunocompromised / transplantation Baermann technique for rabditiform larvae Hookworm!! genital premordium Culture for filariform larvae Hookworm: tapered tail Stongyloides: forked tail Serology (timelag!!)

23 High probability of helminth eg refugees infection Ridley concentration from all 3 fecal samples ( 2 SAF fixed, 1 fresh)

24 Microsporidium Profound diarrhoea in immunocompromised patient in particular HIV/AIDS (CD4 < 100) Saline Ridley / no wooden sticks Calcofluor staining Trichrome acc weber PCR Spores are very small

25 Non- morfological techniques Copro ELISA PCR

26 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 Antigens and DNA are shedded continuously; so single sample testing is sufficient

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

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

29 Non- morfological techniques Copro ELISA Giardia / Crypto: from SAF and fresh E. histolytica: only from fresh PCR Sens higher if performed from sediment after saline Ridley fresh stoolsample (day 2)

30 Sensitivity of the Triple-Faeces-Test (T.F.T.) versus Enzyme Immuno Assay in the laboratory diagnosis of Giardiasis

31 Prospective study Methods 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 (day 1, 2, and 3) in three EIA s Prospect Giardia (Alexon, Remel), Techlab test Biotech Trinity test

32 Methods Microscopy according to standardized protocol (magnification, time) EIA observer blind Discrepancy study

33 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 negative microscopy 1 x

34 Results of the microscopic examination of the TFT set of Giardia lamblia infected cases (N=9) Case day 1 day2 day3 1 - cysts cysts - 3 cyst + trof cysts cyst + trof 4 cysts cysts cysts cysts cysts cysts 8 - cysts cyst + trof 9 cysts cysts -

35 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 cysts cyst + trof 9 cysts cysts neg 10 neg neg neg 11 neg neg neg

36 Testcharacteristics of the different diagnostic strategies compared with the yield of the optimal test for Diagnostic Strategy laboratory diagnosis of giardiasis Optimal test false negatives sensitivity NPV (%) (%) microscopy day 1 microscopy day microscopy day TFT Prospect day Prospect day Prospect day Prospect TFT

37 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

38 New tests New developments / trends 1 R-Biopharm ridascreen ridascreen Cryptosporidium ridascreen Giardia lamblia Multi parasite testing Giardia + Cryptosporidium Make it easier rapid test / dipstick

39 Name Firm Remarks Triage parasite Biosite Giardia lamblia Cryptosporidium spp E. histo/dispar X/pect Remel Giardia solo (Oxoid) Giardia / Crypto combi ImmunoCardSTAT Meridian Giardia lamblia Cryptosporidium spp Rida Quick r-biopharm Giardia solo (Bipharma) Giardia / Crypto combi

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43 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% spec: >98% Rida Quick Giardia lamblia sens: >95% spec: >98%

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45 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 November November 2000 Samples from routine clinical practice N=548

46 Giardia lamblia Study A Prospective study Combined (N=75) (N=548) (N=627) N positives (microscopy) Prevalence (%) 33,3 4,2 7,7 False Negatives Sensitivity (%) 96,0 95,6 95,8 Neg. Pred. Value (%) 98,0 99,8 99,7 False Positives Specificity (%) ,8 99,0 Pos. Pred. Value (%) ,6 88,5

47 Parasitic species found in stool samples from patients in routine clinical practice in Haarlem, the Netherlands. (N=548) Species N Triage Prevalence (%) Giardia lamblia Cryptosporidium spp Entamoeba histolytica/dispar Dientamoeba fragilis C. cayetanensis 1 Blastocystis hominis non pathogenic protozoans Helminth eggs

48 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? What to do with a positive testresult? Confirmation?? How to monitor treatment effects?