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Prof W. Stevens

Cryptococcal Screening- Laboratory perspective and considerations in South Africa and sub-Saharan Africa . Prof W. Stevens Head Department Molecular Medicine and Haematology, University of the Witwatersrand Head National Priority Program, NHLS. Programme Implementation. Department of Health.

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Prof W. Stevens

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  1. Cryptococcal Screening- Laboratory perspective and considerations in South Africa and sub-Saharan Africa Prof W. Stevens Head Department Molecular Medicine and Haematology, University of the Witwatersrand Head National Priority Program, NHLS

  2. Programme Implementation Department of Health Health facility staff USAID/ CDC NHLS RTC NICD WRHI Pfizer and Diflucan Partnership Program FPD

  3. Global burden of HIV-related cryptococcal meningitis ~1 million new cases per year and ~ 625,000 deaths per year13-44% of 13-44% MR in HIV in sub-Saharan Africa Module 1 Park BJ, et al AIDS 2009.

  4. Annual incidence of cryptococcal disease in South Africa Laboratory confirmed cases Related important facts in SA CM major cause of death in JHB ~8000 new cases/ year Median in-hospital case fatality 35% Mostly ARV naïve <30% on ARV treatment at diagnosis Prevalence of cryptococcal antigenaemia in SA (13%) Jarvis, CID 2009 Cryptococcal antigenaemia shown to precede CM by a median duration of 22 days (French et al. AIDS 2002) Opportunities for screening for this disease are missed currently Several separate studies have demonstrated cost-effectiveness where CrAg prevalence >3% SA Study,: CrAg screening of stored samples from the time of enrolment to ART proved 100% sensitive and 96% specific for predicting incident CM during the first year of ART (Jarvis et al, Clin Infect Dis 2009)

  5. Difficulties in Resource Limited Settings in sub-Saharan Africa • Case fatality rate: 35-65% as compared to developed country counterparts (10-20%) • Patients present only when meningitis is advanced • Poor availability of preferred anti-fungal medications • Inability to perform lumbar puncture • Access to diagnostics limited • WHO Rapid Advice : 1) Rapid diagnosis is essential for cryptococcal disease and access needed; 2) value of cryptococcal screening prior to ART initiation; targeted fluconazole and disease prevention. Use of POC LFA recommended, Primary prophylaxis not recommended WHO : Rapid Advice: Diagnosis, prevention and Management of cryptococcal disease IN HIV infected adults, adolescents and children.2011

  6. Identify patients at risk (CD4 <100) Test for cryptococcal antigenaemia before symptom onset Treat with oral fluconazole Prevent cryptococcal meningitis deaths 3 Separate studies demonstrated cost-effectiveness of screening and targeted treatment: cost saving when CrAG prevalence above 3%. (Meya et al.CID 2010) WHO: Routine serum or plasma CrAg screening in ART-naïve adults (but not adolescents or children), followed by pre-emptive anti-fungal therapy if CrAg positive may be considered prior to ART initiation in patients with a CD4 count less than 100 cells/mm3, and where this population also has a high prevalence of cryptococcal antigenaemia. ( WHO Rapid Advice; 2011) Cryptococcal Antigen Screening Principles Treatment + +ve serum CrAg, no symptoms Meningitis Module 6

  7. Laboratory Diagnosis • • Sabouraud dextrose agar • • Bird seed agar • • Confirmatory • • CSF, blood • Gold standard • Up to 14 days • Anti-fungal susceptibility • • • Agglutination and ELISA test • • >90% sensitivity • • CSF, Serum • • Early diagnosis in asymptomatic HIV + patients • When india ink –ve and non- CSF specimens • Lateral Flow Assay • • >90% sensitivity • • CSF, Serum, urine • • Early diagnosis in asymptomatic HIV + patients • Stable temp • 5-15 mins • Limited specificity data, paed data • • India ink • • Rapid diagnosis • • “Halo” • • 30-80% sensitivity • • CSF, Sputum, Tissue • -ve test does not exclude diagnosis MICROSCOPY1ICROSCOPY1 CULTURE2 SEROLOGY1 IMMUNO-CHROMATOGRAPHY3 1Heitman J, et al. Cryptococcus. Washington DC, USA: ASM Press, 2011. 2DoctorFungus.org, 3 CDC, unpublished data 1Heitman J, et al. Cryptococcus. Washington DC, USA: ASM Press, 2011. 2DoctorFungus.org, 3 CDC, unpublished data

  8. CrAg Lateral Flow Assay (IMMY) Simple quickResults available in 10 minutes Available and effectiveHighly sensitive and accurate (>95%) Affordable*R50/test (NHLS estimate) ($6) Minimal Infrastructure Gold conjugated antibodies *Actual CrAg strips quoted by supplier at $2 plus shipping from US to SA, excluding local distribution or taxes

  9. Collaboration with CDC/USAID South Africa, National Health Laboratory System (NHLS), and South Africa NDOH Included in DOH National Strategic Plan 2012 Phased implementation Phase 1 Reflex lab screening at all facilities using 3 pilot NHLS laboratory nodes Reflex lab screening in HIV clinics with on-site private laboratories Phase 2 Nationwide implementation using NHLS laboratory reflex screening Standard training and program monitoring tools Cryptococcal Screening Program: South Africa

  10. Pilot Lab Evaluation to establish feasibility for reflexing CrAg in CD4 laboratories in SA • CD4 testing is received as whole blood EDTA samples in 62 labs • Random routine patient samples received for PLG/CD4 counts • CD4 counts less than 100 cells/µl were selected n=166 • Tested on the CrAg rapid Lateral Flow Assay method for Cryptococcal antigen (IMMY) • Manufacturer supplied Positive control (n=16) and manufacturer recommended Negative control (diluent) (n=16) were analysed with each batch of patient samples tested (contained in the kit). • A positive patient sample and two negative patient samples were tested repeatedly (n=5) to assess reproducibility of dipsticks. • The plasma and whole blood of two CrAg positive patients were tested repeatedly to assess results of whole blood versus plasma. • Whole blood samples (n=60) were tested to assess feasibility of using whole blood instead of plasma.

  11. Results and Conclusion • Of 166 tested, 6 patients tested positive ,i.e. 3.6% positivity. • All 6 patients were known patients who had tested positive previously on the Cryptococcus Latex agglutination method. • The results of the positive and negative control samples included with each batch of samples tested were in keeping with the expected result and no invalid tests were noted. • The reproducibility exercises for both the positive and negative samples confirmed that the same results were noted on at least five repeat dipsticks. • All samples tested positive on plasma also tested positive when whole blood was used (only positive samples were used for this exercise). • Sixty samples that tested negative for CrAg were done using whole blood and no false positives were noted. • Method was simple, required minimal training and therefore could be easily implemented in CD4 Laboratories. • Minimal additional consumables are required: pipette, timer etc • Whole blood testing can be used that will simplify the method further by avoiding centrifuging and separation of samples. • EQA possible with spiked plasma samples

  12. What are the likely number of tests needed in SA? CD4 Numbers Average CD4<100 (11%) In summary: • Total number of CD4 tests performed in 2011 – 3,821,734 • Of these, a total of 437,303 tests were CD4 <100 (11%) • Jan-June 2012: 230 866 CD4 <100 (11%)

  13. Planned Expansion of CD4 Footprint (62 labs) Continuous monitoring to ensure total coverage Green sites: upgrade to include CD4

  14. Range of Flow cytometers in National program

  15. The Cryptococcal Death Prevention Programme Testing Pilot 3 lab facilities that serve 442 clinics

  16. Proposed laboratory work-flow plan CD4 Requested CD4 Test Performed CD4 <100 Yes No Reflex to CrAg testing Run CrAg worklist Find CrAg Samples in CD4 Storage Run CrAg Test Report/ Auth DISA Phone queue to clinic or sms to relevant HCW/Site

  17. Existing data sources NHLS laboratory system DPP Registers National cryptococcal meningitis surveillance (GERMS) ART Cohort data New platforms Sentinel vs. all-site Laboratory vs. facility based Monitoring and Evaluation

  18. Progress Update

  19. CrAg Test: Estimated Total Cost Per Test in SA Actual CrAg strips quoted by supplier at $2 plus shipping from US to SA, excluding local distribution or taxes

  20. Multi-disciplinary POC HIV/TB Project Sites • Protocol initiation (CD4, ALT, Creatinine, HB, lactate) • First urban site (HJH) • N=160 random patients (for ART or monitoring) consented, • Numbers of tests requested at any one time: • 34% = 4 tests at one visit • 25%=3 tests • 21% = 2 tests • 17.8%=1 test • n=1 patient had 5 tests requested Venipuncture based project (CD4 queries around finger-stick emerged in other studies) • Second site activated and study completed(Tshwane district hospital), same protocol applied, data collected and being analysed Addition of CrAg Test to clinic POC project sites

  21. Mobile Testing Options10 X GX16 in mobile vehicles

  22. National Programme Cost Evaluation Number of CD4 specimens <100 per year1 (480,000) Estimated annual cost of national screening programme R 24,000,000 Cost of crypto LFA test2 (R 50) X = Cost of hospitalization for CM4 (R 20,080) Estimated annual cost of current CM management R 167,266,400 Number of CM cases per year3 (8,330) X = Cost of hospitalization for CM (R 20,080) Number of preventable CM cases per year5 (4800) X Estimated annual savings from national screening programme R 96,384,000 ($12 048 000) = 1. NHLS Data Warehouse 2. Preliminary NHLS estimate 3. GERMS Surveillance 2009 4. Haile et al. APHA Conference Atlanta, 2001 5. Based on 3% CrAg+ positivity (Govender et al, unpublished) , 28% CM development among CrAg+ (Jarvis et al. Clin Infect Dis 2009), & 10% unpreventable deaths in pts presenting with overt CM (Meintjes, personal communication).

  23. How could CrAg screening fit into routine care and testing algorithms? Module 6

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