Development of a Saliva Test for Malaria.

1Mharakurwa S., 1Siame M., 2Sullivan D., 2Shiff C.J., 1Thuma P., 3Geva E., 3Abrams W., 3Malamud D.

1Malaria Institute at Macha, Namwala Road, Choma, Zambia2Johns Hopkins Bloomberg School of Public Health, Baltimore MD21205 USA3Department of Basic Sciences, College of Dentistry, New York University, NY10010, USA

BACKGROUNDNew campaign for curbing the malaria scourge

• WHO, RBM, PMI, PATH, MACEPA public-private partnerships

• Global scale-up of effective malaria intervention ACT’s, ITN’s, IRS

• Possible local or regional elimination

Formidable disease resilience necessitates:• efficient epidemiological surveillance

oresurgence/drug resistance• accurate screening for asymptomatic reservoirs essential • accurate diagnosis pivotal

2007N=330

OR (95% CI)

2008 and 2009N=1151

OR (95% CI)

Comparison at the initial study visita

Crude model 1.00 (0.89, 1.12) 1.12 (0.46, 2.73)

Adjusted for seasonb 0.64 (0.34, 1.22) 0.74 (0.23, 2.42)

Adjusted for season and other participant and household characteristicsc

0.68 (0.33, 1.39) 0.54 (0.15, 1.93)

Comparison during follow-upa

Crude model 0.48 (0.24, 0.98) 0.14 (0.05, 0.37)

Adjusted for seasonb 0.43 (0.20, 0.94) 0.12 (0.05, 0.31)

Adjusted for season and other participant and household characteristicsc

0.41 (0.20, 0.88) 0.12 (0.05, 0.30)

Test-and-treat depletes malaria infection, Macha communities

Sutcliffe et al (2012) PlosOne 7(2): e31396

Waning P. falciparum genetic diversity

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

2005 2006 2008 2009Patent Submicroscopic

2

(0.9 – 4.8)

*** 30

(11.0 - 82.0)

*** 24

(9.2 – 63.3)

2005 2008 P

GMPD (/µl)

[95% CI]

384

[255 - 576]

64

[30 – 131]

< 0.001

Selection for sub-microscopic parasitaemia

Current Malaria Testing

Current P. falciparum Screening Constraints

Necessitates drawing blood Finger-prick Venipuncture

Limitations Obligatory use of needles/sharps in remote settings

oAdequately trained personnel –not readily available VHW level Home level

oBiohazard Community blood taboos, beliefs etc Repeated testing -drug/vaccine efficacy trials/monitoring Low access to potential reservoirs for research/control

programmes

Macha Experiment, 2005

Persistent Community pressure/rumours

Saliva, Urine, filter paper samples

DNA Extraction

Saliva and urine yielded same expected PCR amplicon as blood samples

3D7/IC

(470-700bp)FC27

(290-420bp)

173Qs 173uD 173b M Qs - Qu - uD - b -173Qu

Is the infection in saliva the same as in blood sample?

P. falciparum Detection in Human Saliva

Mharakurwa et al (2006) Malar J 5: 103

FC27

290 - 427bp

3D7

(470 -700 bp)

216QS 34QS 34Cu 34b 210QS 210Qu 210b QS- Cu- b- 3D7M216Qu 216b

Is P. falciparum DNA detection repeatable?

MSP2 –chr 2PfDHFR – chr 4PfDHPS –chr 8Pfmdr1 –chr 5Pfcrt –chr 7TA81 –chr 518S rRNA gene (chr 1, 5, 7, 11)

Chelex on filter paper Whatman FTA classic

card Whatman 903 Qiagen DNEasy kit Oragene kit Polyethylene glycol

(PEG)

Yes, by range of genomic primers and extraction methods, machines…

Yes, extracted at different time points with different methods Yes, other investigators, using Real-time platform:

Nwakanma et al (2007) Am J Trop Med Hyg 77 (Suppl.): 233. Nwakanma et al (2009) J Infect Dis 199 (11) 1567-74. Buppan et al (2010) Malar. J. 9: 72

What are the determinants of amplicon yield?FACTOR SUMMARYExtraction method Higher amplicon with Qiagen than Chelex

Urine: 2.24X higher Saliva: 2.25X higherCurrent saliva optimal

•sensitivity 96%•specificity 98%

Sample type Saliva extracts:1.6 fold higher amplicon yield than urine

Parasite density For each unit increase in log parasite density:1.82-fold increase in amplicon yield

Primer set U1-U4 (370bp, 229bp) 18.5X more likely to amplify than FC27 (750bp, 290-420bp)

Parasitaemia detection thresholds

Detection threshold: 0.1 parasites/µl

Ongagna -Yhombi et al (2013) Malar. J. 12 (1) 74

What is the source of P. falciparum material in the saliva? In process

Migrate bench-top assay to P.O.C. test -NYU collaboration In process

Follow-up Work

Nucleic Acid Detection PfHRP II Antigen detection

PCR LAMP

Microfluidic POC device “Dipstick”/card POC test

Saliva Screening Advantages

Non-invasive, simpler, safer Greater community participation/co-operation Wider access to potential reservoirs for

research/control programmes Strengthen research/surveillance Reduced sample collection cost/workload 1000X more sensitive than RDT/microscopy

Acknowledgements

Gift Moono, Choolwe M. Nachibbatu

Patience Cheelo, Cliff Singanga

Petros Moono

Harry Hamapumbu

Communities, headmen & chiefs of Macha, Mapanza, Muchila and Chikanta areas

MOH Zambia

Thank YOU