This document describes research on developing a saliva test for detecting malaria. Key points: - Current malaria testing requires blood samples, which has limitations in remote settings. A saliva-based test could improve screening. - Previous studies found Plasmodium falciparum DNA can be detected in saliva using PCR, matching what is found in blood. Detection is repeatable with different extraction methods. - Factors like extraction method, parasite density, and primer set influence the yield of detectable DNA in saliva. Detection thresholds can reach 0.1 parasites/μL. - Future work aims to develop the benchtop saliva screening assay into a point-of-care test and explore detecting

2. Development of a Saliva Test for Malaria.
1
Mharakurwa S., 1
Siame M., 2
Sullivan D., 2
Shiff C.J., 1
Thuma P., 3
Geva E., 3
Abrams W., 3
Malamud D.
1
Malaria Institute at Macha, Namwala Road, Choma, Zambia
2
Johns Hopkins Bloomberg School of Public Health, Baltimore MD21205 USA
3
Department of Basic Sciences, College of Dentistry, New York University, NY10010, USA

3. BACKGROUND
New 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

4. 2007
N=330
OR (95% CI)
2008 and 2009
N=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

5. Waning P. falciparum genetic diversity

6. 0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
2005 2006 2008 2009
Patent 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

7. Current Malaria Testing

8. 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

9. 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

10. 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

11. FC27
290 - 427bp
3D7
(470 -700 bp)
216QS 34QS 34Cu 34b 210QS 210Qu 210b QS- Cu- b- 3D7M216Qu 216b

12. Is P. falciparum DNA detection
repeatable?
MSP2 –chr 2
PfDHFR – chr 4
PfDHPS –chr 8
Pfmdr1 –chr 5
Pfcrt –chr 7
TA81 –chr 5
18S 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

13. What are the determinants of amplicon
yield?
FACTOR SUMMARY
Extraction method Higher amplicon with Qiagen than Chelex
Urine: 2.24X higher
Saliva: 2.25X higher
Current 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)

14. Parasitaemia detection thresholds

15. Detection threshold: 0.1 parasites/µl
Ongagna -Yhombi et al (2013) Malar. J. 12 (1)
74

16.  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

17. 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

18. 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