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 Dr Nelesh Govender GDOH Meeting Programme Implementation Department of Health Health facility staff USAID/ CDC NHLS RTC NICD WRHI Pfizer and Diflucan Partnership Program FPD NICD/ NHLS

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.

Annual incidence of cryptococcal disease in South Africa Related important facts in SA Laboratory confirmed cases 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)

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 Specifically mention: WHO : Rapid Advice: Diagnosis, prevention and Management of cryptococcal disease IN HIV infected adults, adolescents and children.2011

Cryptococcal Antigen Screening Principles 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) Not for adolescents or children +   Treatment +ve serum CrAg, no symptoms Meningitis Module 6

IMMUNO-CHROMATOGRAPHY3 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 Diagnosis of CM: india ink, latex agglutination and/or culture Tissue based : cell count, gram stain, india ink, bacterial culture India ink-done well: 80-98% EIA more expensive Anti-fungal susceptibility for fluconazole largely 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

CrAg Lateral Flow Assay (IMMY) Simple quick Results available in 10 minutes Available and effective Highly 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 REVISED 8

Cryptococcal Screening Program: South Africa 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

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.

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

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%)  

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

Range of Flow cytometers in National program

The Cryptococcal Death Prevention Programme Testing Pilot Dr Nelesh Govender GDOH Meeting The Cryptococcal Death Prevention Programme Testing Pilot 3 lab facilities that serve 442 clinics NICD/ NHLS

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

Monitoring and Evaluation 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

Progress Update Stakeholder Action National DOH Dr Nelesh Govender GDOH Meeting Progress Update Stakeholder Action National DOH Letter of recommendation sent from Director-General to GDOH Gauteng DOH Supportive of programme NHLS CD4 labs Ready to start screening NICD Ready to assist with M&E Pfizer Have not yet committed to providing fluconazole through DPP CDC have committed to funding fluconazole for pilot phase District level government Have been informed of programme Local government Gauteng DOH and local government meeting is pending PEPFAR partners Ready to begin clinical training CDC/ USAID Have provided funds and technical support FPD Ready to lead clinical training NICD/ NHLS

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

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

Mobile Testing Options 10 X GX16 in mobile vehicles assessed mobile capability of various POC instruments for HIV and Tb: rapid HIV, CD4 test and GeneXpert : will get data Large mobile bus: in development for further use as a model for servicing several clinics in a clustered area

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).

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