Molecular Tools for Malaria Surveillance

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August Bates
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1 Ingrid Felger Molecular Diagnostics Unit Dept. of Medical Parasitology & Infection Biology Molecular Tools for Malaria Surveillance Molecular Detection Quantification (Genotyping)

2 What is the most sensitive assay for parasite detection in a fingerprick blood sample? [ Plasma/protein Serology ] Template RNA extraction DNA extraction PCR Target 18S rrna transcripts 18S rrna genes Copy number highly abundant 3/5 per haploid genome Limit of Detection parasites/ μl 1-2 parasites/μl

3 RNA-based versus DNA-based diagnosis twice as high prevalence rates in PNG * cut-off at 10 Pf 18SrRNA copies/µl * cut-off at 10 Pf 18SrRNA copies/µl (Wampfler et al PLoS ONE)

1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86 91 96 101 106 What method to chose

00 100000.00 Pf 18SrRNA on RNA Pf 18SrRNA on DNA 10000.00 1000.00 100.00 10.00 Decision to use a cut-off as in NASBA P.

00 1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 Field samples

4 What method to chose for field work? DNA-or RNA-based assays? P. falciparum Number of transcripts Pf 18SrRNA on RNA Pf 18SrRNA on DNA Decision to use a cut-off as in NASBA P. vivax Field samples Pv: 8x lower densities than Pf Pv 18SrRNA on RNA Pv 18SrRNA on DNA Field samples (own unpublished results)

5 RNA-based vs. DNA-based parasite & gametocyte detection Marker RNA-based assay DNA-based assay 18S rrna Abundant transcripts 3 copies / genome Extremely high sensitivity Standard sensitivity Disadvantages: Quantification imprecise Cross-contamination (aerosols) during RNA extraction; cut-off necessary Advantages: good correlation with LM No contamination issues

6 Lessons learned from using rrna transcripts as diagnostic marker A major proportion of infections not noticed with standard techniques Beware of ribosomal RNA, only use with greatest caution & tight control Unlikely field applicable, unless enclosed in fully contained system Quantification is not very precise (expression levels/rna degradation) Blood volume matters! Detection limit = 1 parasite in even large volume pooling of samples possible Ultra-low density infections carry gametocytes

7 Development of ultra-sensitive DNA-based qpcr TARE-2: telomere-associated repetitive element kb long blocks of bp repeat units with slightly degenerate sequences approx copies /genome var-ats: acidic terminal segment (semi-conserved) 59 var genes in 3D7 Is a multi-copy PCR target suitable for quantification? YES, good correlation!

8 Implication for prevalence rate: plus 16% Microscopic Submicroscopic Ultra-low density (Hofmann et al PLOS Medicine) P. falciparum prevalence in 498 individuals from Tanzania Proportion of gametocyte carriers by pfs25 qrt-pcr

9 Ignore ultra-low density infections in Malaria Surveillance? 63% gametocyte carriers in submicroscopic infections 40% gametocyte carriers in samples positive only by TARE-2/var qpcr Not known: infectiousness to mosquitoes of ultra-low densities Assays suitable for Malaria Surveillance? yes pooling of 10 blood samples prior to DNA extraction possible without loss of sensitivity by TARE2 and ATS assays applicable in low endemicity regions, e.g. 2% prevalence Primary applications of ultra-low density assays Clinical Trials Controlling quality of other diagnostic methods Generating very precise prevalence data / basic research

10 Entomological incocculation rate molecularforce of Infection: measure of individual exposure Parasite prevalence Gametocyte prevalence Detected on transcript level by qrt-pcr or NASBA Most sensitive molec. marker: pfs25 & pvs25 (own unpublished result)

11 Do we need molecular assays for gametocytes? Detection? positive/negative No All infections produce gametocytes at a steady rate, gametocytes follow asexual densities (Eksi et al.2012 PLOS Pathogen) Epigenetics of sexual commitment (M.Llinas/T. Voss) Asexual & gametocyte densities correlated (Koepfli et al. 2015) Detection is a matter of blood volume processed! Quantification? Yes only for certain purposes Monitoring of drug effects in clinical trials Vaccine effects in comparator groups But: RNA-based quantification is not easy!

12 Parasite prevalence Gametocyte prevalence Infectiousness to mosquitoes Mosquito feeding assays Churcher et al Elife

13 Infectiousness to mosquitoes - Membrane / Skin feeding assays Substantial literature on infectiousness demonstrated experimentally for Symptomatic infections Asymptomatic infections Microscopy-positive infections with submicroscopic gametocytes Submicroscopic infections for both asexual & gametocyte by LM. Review Stone et al Trends in Parasitol Research on-going in several endemic countries to establish infectiousness to mosquitoes. Focus: asymptomatic (normally untreated!) individuals

14 Key questions remain to be experimentally answered: Infectiousness of Microscopy-negative, but PCR-positive blood RDT-negative, but PCR-positive blood Standard qpcr-negative, but positive by ultra-low density assay Wanted: Definition of minimum parasite density resulting in mosquito infection (for different transmission settings) Practical consequence: Either relax or increase sensitivity level of diagnosis for surveillance-response strategies

15 Conclusion human infectious reservoir Well described Low density reservoir Needed: More hard work

16 Research & Development needed Consensus marker for transmission intensity (e.g. molecular prevalence) External Quality Control: Need to QC plus calibrate tests against state-of-the-art detection & quantification EQC, calibration of methods used for surveillance Tools to capture micro-heterogeneity in transmission (hot pops) & migration Strategies for case (infection) detection POC diagnostics for control programmes

17 Considerations for future work Two distinct molecular tools for malaria surveillance needed: 1. High throughput platform centralized lab: cheap and highly sensitive for good quality prevalence data 2. Simple point-of-contact (POC) test : possibly isothermal, selfcontained, microfluidics, but not miniature in blood volume Costs of molecular tests in surveillance: field work is costly! PCR does not inflate budget, but provides high quality data With inclining transmission increasing need for non-malaria fever diagnostics ( regional POC testing)

Leanne Robinson Albinama study team Seif

Stephan Karl The study teams TZ & PNG The

National Science Foundation ICEMR (Internatl.

18 Thanks Natalie Hofmann Rahel Wampfler Felistas Mwingira Sarah Javati Tom Smith Leanne Robinson Albinama study team Seif Shekalaghe Ivo Mueller Cristian Koepfli Stephan Karl The study teams TZ & PNG The study participants Financial support Swiss National Science Foundation ICEMR (Internatl. Centers of Excellence in Malaria Research) Bill and Melinda Gates Foundation

Comparison of light microscopy and nested PCR assay in detecting of malaria mixed species infections in an endemic area of Iran

Comparison of light microscopy and nested PCR assay in detecting of malaria mixed species infections in an endemic area of Iran Aliehsan Heidari, Manizheh Nourian, Hossein Keshavarz Associate Prof. Dept.