National Reference Centre for Parasitology. Brian Ward &Momar Ndao Colloque Blue 2012

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1 National Reference Centre for Parasitology Brian Ward &Momar Ndao Colloque Blue 2012

2 Us Why is Diagnostic Testing for Parasites so Complicated? Critical mass of investigators Limited commercial interest Them Change antigens with life-stage Sophisticated avoidance techniques Body cavity location Intravascular location Intracellular location Massive antigen exposure

3 Tropical Disease Diagnostics How Are We Doing in 2012? (1600s) ( ) (1970s) (1990s) Amoebiasis African Tryps Chagas Disease Leishmaniasis Malaria Schistosomiasis Strongyloidiasis Brucellosis Leptospirosis Dengue West Nile Blastomycosis HIV-1 HIV 2 mid-1990s Late 1990s

4 NRCP/CNRP Mandate Provide service assays (1st line & reference) Surveillance for parasitic diseases Provide reagents upon request Improve or develop assays

5 Parasite Serology Laboratory 8 tests offered (4/8 sent to CDC in Atlanta) 1 technician performing ~ tests/year Only serologic assays - Antigens historical No QA/QC - No idea of quality of assays NRCP/CNRP 25+ tests offered (almost all in Montreal) 2.5 technicians, 1 clerk, 3 PhD trainees ELISA, Antigen capture, Immunoblot, RT-PCR Most assays developed in house or validated Regular QA/QC

6 Increasing Demand for NRCP/CNRP Services

7 Positive Test Results by Province And Test Requested Province Individual Tests

8 Distribution of Tests Requested

9 Surveillance: Trichinella

10 Chagas Disease Sensitivity 100% 100% 100% Specificity 80% 86% 90% EXAMPLE OF PCR RESULTS FROM T. CRUZI INFECTED HUMAN SAMPLES M ELISA Development whole organism TESA-based Potential commercialization Routine PCR then Real-Time PCR : Fresh blood 2 : Dried blood 3 : Fresh serum 336 bp 4 : Fresh plasma 168 bp 5 : Fresh CSF 6 : +ve Control : T. cruzi Vinch 7 : Control (Uninfected blood) 8 :Control (Water) 35 Mass-spectrometry Patents awarded Berrizbiettia M et al J Clin Microbiol 2004 Berrizbiettia M et al J Clin Microbiol 2005 Berrizbiettia M et al Transfusion 2006 Berrizbiettia M el al Acta Tropica 2009 Ndao M et al J Clin Microbiol 2010 Ndao M et al Trends in Parasitol 2011

11 Recombinant Cathepsin L5 Fasciola hepatica EIA Sensitivity Cuban EIA 97.2% rfhcatl5 EIA 97.2% O.D. Cuban Assay Specificity 4 5 % 1 0 % 8 0 % 4 4 % T r i c h i n e l l a S t r o n g y l o i d e s F i l a r i a T o x o c a r a P o s C o n t N e g t C o n t O.D NRCP Assay 7 2 % 6 6 % % 6 6 % Vasquez F et al. (submitted)

12 Schistosomiasis No assay offered ELISA based on crude S. mansoni Ag Refined ELISAs S. mansoni adult S. hematobium adult Ag Screening ELISA with both Ag Development of animal model Import infected snails Harvest cercariae Infect mice and harvest adult worms Useful for ELISA & Proteomics assay development

13 Strongyloidiasis 1994 No assay offered 1999 Commercial assay (LMD) assessed 2006 Recombinant Ag sourced (NIH - F Neva) 2007 Expressed rnie Ag - developed ELISA/Immunoblot Exported novel assay to CDC, UK, other countries TDC cases NIH # crude ELISA NRCP crude ELISA NIH Recomb. ELISA NRCP Recomb. ELISA NRCP Recomb. Immunoblot Normals (44) Ss larva + (17) Ss larva + & HTLV1 (4) Filaria (9+1TPE) Schisto (14+3KU) Hydatid (15) Trichinella nativa (20) Eos. Esophag (20) Metorchis (9)

14 Mass Spectrometry A: Q - an i on i c e xchange WCX ph 9 ( 4-10k D a) 6 chronic Chagas Subjects 6 appropriate controls 2 malaria subjects H H 9500 p H9, C o n tr ol 1 p H9, C o n tr ol 2 p H9,P a t i e n t1 p H9,P a t i e n t2 Four Markers ( kda) 100% sensitive 100% specific B : Q - ca ti on i c exchange I MAC - cu ph 7 ( 4-10kD a ) H H p H7, C o n tr ol 1 p H7, C o n tr ol 2 p H7,P a t i e n t1 p H7,P a t i e n t2

15 Chagas Chronic Infection : Potential biomarker N terminus of Apolipoprotein A1

16 Decision Tree Classification for Training Data Set: Chagas Diseases (CD) vs Healthy Control (HC) M: 24.7 I.1.83 N = 31 CD = HC = M: 13.6 I 5.6 N = 71 CD = 34 HC= 37 M: 28.7 I 2.47 N = 40 CD = HC = N = 24 CD = 3 HC =21 M: 9.3 I 16.5 N = 7 CD = HD = N = 33 CD = 33 HC = 0 M: 8.1 I 0.48 N = 7 CD = HC = N = 5 CD = 0 HC = 5 N = 2 CD = 2 HC = 0 N = 1 CD = 1 HC = 0 N = 6 CD = 0 HC = 6

17 Visceral Leishmaniasis First pass Analysis (p<.001)

18 Visceral Leishmaniasis - II 3379 kda A) Mass/Charge Scatter Plot B) Peak View C) Gel View Intensity P-value ROC 0.983

19 Parasite Serology Interpretation of Results Many antigens heterogenous - not well defined cross-reactions (false positives) inadequate sensitivity (false negatives) Sensitivity & specificity change with context Beware of multiple tests (beware of fishing trips) Many commercial assays poorly characterized

20 Parasite Diagnostics The Future Improved antigen preparation Phase specific diagnosis Recombinant antigens Nucleic acid-based assays Proteomics-based assays

21 Serology tests available at the NCPS (I) Disease Test Sensitivity & Specificity Amoebiasis ELISA Sens. 95% extra intestinal 70% invasive intestinal 10% asympt. cyst. carriage Spec. 97% Echinococcosis ELISA Sens. 85% liver cysts 66% lung cysts 50% E. multilocularis Spec. 93% WB Sens. 84% liver cysts 33% lung cysts 39% E. multilocularis Spec. 99%

22 Serology tests available at the NCPS (II) Disease Test Sensitivity & Specificity Trichinellosis ELISA Sens. 85% Spec. 93% Toxocariasis ELISA Sens. 86% Spec. 98% Filariasis ELISA Sens. ~ 90% Spec. cross-reactions with nematodes IgG4 better specificity Schistosomiasis ELISA Sens. ~ 90% (screening test) WB Sens. S. mansoni 99% S. haematobium 95% S. japonicum 95% Spec. species specific 95%

23 Serology tests available at the NCPS (III) Disease Test Sensitivity & Specificity Malaria IFA Sens./Spec. > 90% Cysticercosis WB Sens. 98% (multiple lesions) <72% (single lesion) <50% (subcutaneous) Spec. 100% Trypanosomiasis IFA Sens % cross-reactions with leishmaniasis Leishmaniasis IFA Sens. > 90% (visceral) < 90% (cutaneous) cross-reactions with Chaga s Paragonimiasis WB Sens % Spec. 99%

24 Serology tests available at the NCPS (IV) Disease Test Sensitivity & Specificity Strongyloidiasis ELISA Sens. 85% Spec. 80% cross-reactions with filariasis Babesiosis IFA Sens. 100% (except AIDS) Spec. 99% Gnathostomiasis WB Sens. 98% Spec. 99% Toxoplasmosis EIA IgG Confirmatory assays under EIA IgM evaluation by the LSPQ immunocapture ISAGA IgM