mharakurwa experiences from field lab 1
TRANSCRIPT
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