Infection and transmission dynamics of plasmodium vivax in Papua New Guinea

Background Plasmodium vivax mainly affects Asia, Central and South America and is responsible for 350-450 millions cases per year, hence 25-40% of annual infections of malaria worldwide. In Papua New Guinea (PNG) P. vivax prevalence is among the highest worldwide. The biggest challenge for the control of P. vivax infections is the formation of dormant liver stages, which have the ability to relapse and cause disease even after successful clearance of asexual stages in the blood circulation. P. vivax strains in PNG relapse frequently and fast, and one of the highest doses of Primaquine is necessary to reduce the relapse rate in this region. Aims and objectives The overall aim of this thesis was to deepen our understanding of P. vivax molecular epidemiology, infections, transmission and its contribution to the infectious reservoir of P. vivax malaria in PNG. The specific objectives pursued can be summarized as follows. First, to assess P. vivax infection dynamics and transmission dynamics in semi-immune children and to contribute to the understanding of the biology of relapses by comparing two treatment arms. Second, to identify the best RNA sampling strategy for field surveys and improve molecular detection and quantification of P. falciparum and P. vivax gametocytes in field samples. Third, to develop genotyping tools to better study the dynamics of gametocyte production in multi-clone infections in consecutive samples. Methods For the above objectives, the laboratory work of this thesis was split into three parts. (i) P. vivax PCR-positive blood samples from a treatment-to-reinfection survey were genotyped by the marker msp1F3 and analyzed for gametocyte carriage by pvs25 qRT-PCR. These samples were collected from 524 children aged 5-10 years and actively and passively followed-up over 8 months in PNG. The children were randomly attributed to two treatment arms consisting of blood-stage clearing drugs and either PQ or placebo. Genotyping data and gametocyte positivity were used to investigate the contribution of relapses to the infectious reservoir of P. vivax malaria. The molecular epidemiology of relapses was assessed by comparing the investment in gametocytogenesis, the molecular force of blood-stage infections (molFOB, number of distinct blood-stage infections per child and year) and the duration of infections in both trial arms using a Bayesian approach that allows for imperfect detection of blood-stage infections. (ii) In a cross-sectional survey in 315 5-10 years old children from Papua New Guinea, the optimal strategy for gametocyte detection in field studies was assessed. Gametocytes need to be detected by amplification of stage-specific transcripts, which requires RNA-preserving blood sampling. The efficiency of sampling and storage on filter paper versus in solution were compared. (iii) 111 archived DNA samples from PNG were genotyped by capillary electrophoresis for 6 length-polymorphic and gametocyte-specific markers and their diversity was determined. Serial dilution of gametocyte enriched culture of P. falciparum 3D7 strain permitted to establish the detection limit of each marker in vitro. The two most promising markers, pfg377 and pfs230, were then tested to simultaneously genotype paired RNA and DNA samples from 46 individuals from Burkina Faso. Results (i) In the randomized treatment-to-reinfection trial, children who received PQ showed 82% lower risk of experiencing at least one P. vivax infection. The estimated duration of infection, the parasite density and the probability of detection of individual infecting clone (detectability) was similar in both arms of the trial. Gametocyte densities and carriage in positive samples also did not differ between trial arms. Durations increased by age, whereas parasite density and detectability decreased by age. Over the 8 months follow-up, molFOB in placebo arm was 9.9 infections per child and year and almost three times as high as molFOB in PQ arm with 3.5. About 2 relapses were observed per each new infection at all villages. The increasing individual exposure of participants, as measured by molFOB, translated proportionally into an increased relapse burden. (ii) In the cross-sectional survey in PNG, RNAprotect resulted in the highest proportion of gametocyte positive samples and gametocyte-specific transcript yields. The RNA-based detection resulted in a P. falciparum positivity of 24.1%; of these 40.8% carried gametocytes. P. vivax positivity was 38.4% with 38.0% carrying gametocytes. Most of the gametocyte carriers were also positive by DNA-based detection. (iii) Analysis of genotyping markers of P. falciparum gametocytes revealed highest discrimination power for pfs230 with 18 alleles, followed by pfg377 with 15 alleles. When assays were performed in parallel on RNA and DNA, only 85% pfs230 samples and 60% pfg377 samples contained at least one matching genotype in DNA and RNA. Conclusion This thesis was the first attempt to fill the gaps in the knowledge of infection dynamics of relapses and new infections in semi-immune children. By comparing two trial arms results demonstrated how relapses and new infections have similar duration, parasite density, detectability and investment in gametocytogenesis. The mathematical model applied to genotyping data from the two treatment arms proved very useful to evaluate the infection dynamics of P. vivax. This was achieved despite a major complication of P. vivax molecular epidemiology, namely the unknown history of infections in our participants giving rise to relapses. Data generated during the course of this thesis was used to highlight that relapses are the major contributors to P. vivax infections and transmission in PNG. The molecular genotyping tools developed to study P. falciparum gametocyte transmission dynamics will open up new investigations of clone interaction, within-host competition, and clonal fitness. So far, very little is known on gametocyte dynamics in natural infections, where concurrent clonal infections might contribute to transmission equally or in competition with each other. This determines parasite recombination in mosquitoes, which in turn has major consequences for development of multi-locus drug resistance phenotypes or antigenic diversity.

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