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
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

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
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)

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

6. 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
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7. 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, DoctorFungus.org, 3 CDC, unpublished data
1Heitman J, et al. Cryptococcus. Washington DC, USA: ASM Press, DoctorFungus.org, 3 CDC, unpublished data

8. 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

9. 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

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: CD4 <100 (11%)

13. 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

14. Range of Flow cytometers in National program

15. 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

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. 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

18. 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

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 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

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 ($ ) =
1. NHLS Data Warehouse 2. Preliminary NHLS estimate 3. GERMS Surveillance Haile et al. APHA Conference Atlanta, 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?
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