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