Mharakurwa experiences from field lab 1

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  • The Malaria Institute at Macha was set up by missionary doctors struggling to fight against malaria, notably Dr. phil Thuma through his US-based NGO MMRI, BIC, MoH and JHMRI to establish a malaria field research and training centre and officially opened in January 2005. It is full of challenges to run a lab in a rural area and in fact when it all started no prefessional could even contemplate coming here. However, as time would tell this turned out to be such a blessing for the community, for Zambia and indeed beyond. By 2010, the institute had to change its name to Macha Research Trust, because it was fire right in malaria ’s belly and the scourge was fast disappearing. As we speak now this lab now is only one of the labs as we have diversified to other major public health problems. In the next handful of slides I will share with you one of quite a few strategies fashioned at Macha labs.
  • There was also a striking shrink in terms of the mean, median and range overall distribution of expected heterozygosity per bar code locus for infections from 2008 compared to 2005. We are working on current ICEMR samples (accumulating slowly) and will compare with other sites where malaria is unchanged and resurging, respectively.
  • First of all we observed that there was selection for increasingly more submicroscopic infections over time from 2005 through 2009. This appeared like an adaptive response allowing the parasite population to exist in subpatent infections although malaria cases seemed to have almost disappeared. Clearly also the geometric mean parasite density decreased substantially on infections from 2008 compared to 2005.
  • We tracked changes in the Plasmodium parasite population in the community. Our hypothesis was that the parasite undergo genetic changes as it adapted to intervention. also we wanted to detect impact of intervention on the parasite population. I will show you the data we have so far. what we were able to do as part of my training was to employ barcode as well as MOI analysis to document changes in malaria parasite populatoon at Macha where there has been dramatically control.
  • Figure 2 Amplicon from saliva, urine and blood extracts of field samples 216, 34 and 210. Matching MSP2 alleles are seen in saliva (Qs) and blood (b) amplicon from each individual. In contrast, between-patient polymorphic differences are evident, reflecting diverse infections. Urine samples from these patients did not amplify, except that of 210 (210Qu). Qs, Qu denote Qiagen saliva and urine extracts, respectively, by crude lysate approach; Cu denotes Qiagen urine extracts, by cultured animal cells protocol; Du denotes Chelex direct extraction on whole urine. Qs-, Cu-, b- were corresponding extracts of saliva, urine and blood samples from healthy negative control, while 3D7 was positive control laboratory standard
  • Mharakurwa experiences from field lab 1

    1. 1. 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
    2. 2. 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
    3. 3. 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
    4. 4. Waning P. falciparum genetic diversity
    5. 5. 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
    6. 6. Current Malaria Testing
    7. 7. 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
    8. 8. 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
    9. 9. 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
    10. 10. FC27 290 - 427bp 3D7 (470 -700 bp) 216QS 34QS 34Cu 34b 210QS 210Qu 210b QS- Cu- b- 3D7M216Qu 216b
    11. 11. 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
    12. 12. 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)
    13. 13. Parasitaemia detection thresholds
    14. 14. Detection threshold: 0.1 parasites/µl Ongagna -Yhombi et al (2013) Malar. J. 12 (1) 74
    15. 15.  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
    16. 16. 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
    17. 17. 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

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