cryptococcal screening- laboratory perspective and considerations in south africa and sub-saharan...
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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
NHLS
Department of Health
USAID/ CDC
RTC
Pfizer and Diflucan Partnership Program
Health facility staff
WRHI
FPD
NICD
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 AfricaPark BJ, et al AIDS 2009.
Module 1
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)
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
i. Identify patients at risk (CD4 <100)
ii. Test for cryptococcal antigenaemia before symptom onset
iii. Treat with oral fluconazole
iv. Prevent cryptococcal meningitis deaths
v. 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
Module 6
Treatment
+
+ve serum CrAg, no symptoms
Meningitis
Laboratory Diagnosis
• India ink• Rapid diagnosis• “Halo”• 30-80% sensitivity• CSF, Sputum,
Tissue• -ve test does
not exclude diagnosis
MICROSCOPY1I
CROSCOPY1
• Sabouraud dextrose agar
• Bird seed agar• Confirmatory • CSF, blood• Gold standard• Up to 14 days• Anti-fungal
susceptibility
CULTURE2
• Agglutination and
ELISA test• >90% sensitivity• CSF, Serum• Early diagnosis in
asymptomatic HIV + patients
• When india ink –ve and non- CSF specimens
SEROLOGY1 IMMUNO-CHROMATOGRAPHY3
Lateral Flow Assay• >90% sensitivity• CSF, Serum, urine• Early diagnosis in
asymptomatic HIV + patients
• Stable temp• 5-15 mins• Limited specificity
data, paed data
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 quickResults available in 10 minutesAvailable 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
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
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%)
Average CD4<100 (11%)
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
3 lab facilities that serve 442 clinics
Proposed laboratory work-flow planCD4 Requested
CD4 Test Performed
CD4<100Yes
No
Reflex to CrAg testing
Run CrAg worklist
Find CrAg Samples in CD4 Storage
Run CrAg Test
S tandard name fo rmat
U se o f F7 to change W ork A rea and G roup
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 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 DPPCDC 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
CrAg Test: Estimated Total Cost Per Test in SA
EquipmentPipette 100ulBlood MixerRacks
R 0
R 5
R 10
R 15
R 20
R 25
Test
Costs
Labo
r per
test
Test
Cons
umab
les
Fixed
Costs
Was
te di
spos
al %
Trans
port a
nd lo
gistic
s %
Over
head
s %
CrAg Test Cost Components
Costs [R]Test Costs R 20.75Labor per test R 19.10Test Consumables R 1.49Fixed Costs R 1.06sub-total costs [R] R 41.35Waste disposal % 0.41 Transport and logistics % 3.31 Overheads % 4.96 sub-total % additional 8.68 TOTAL average recurrent cost R 50.03
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 requestedVenipuncture 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 Options10 X GX16 in mobile vehicles
National Programme Cost Evaluation
Number of CD4 specimens <100
per year1 (480,000)
XCost of crypto LFA
test2
(R 50) =
Estimated annual cost of national screening programme
R 24,000,000
Number of CM cases per year3
(8,330)X
Cost of hospitalization for
CM4
(R 20,080)
=Estimated annual cost of current CM management
R 167,266,400
Number of preventable CM cases per year5
(4800)
XCost of
hospitalization for CM
(R 20,080)
=Estimated annual savings from national screening programmeR 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