Malaria is the most widespread serious parasitic disease worldwide, with over 2 billion people at risk of infection, over 300 million cases yearly, and over 1 million deaths every year. The primary vector in the Peruvian Amazon, Anopheles darlingi, is a highly anthropophilic rural mosquito. This thesis research focuses primarily on Plasmodium vivax malaria in the Peruvian Amazon, for three reasons: it is the most common form of malaria in the Peruvian Amazon; in comparison with Plasmodium falciparum, Plasmodium vivax malaria is understudied in this region; and the current malaria elimination call to action leads the need for new knowledge on this disease in the Peruvian Amazon. The goal of this thesis research is to contribute with the elimination of malaria transmission by means of testing the overarching central hypothesis that sociodemographics, relapsing Plasmodium vivax malaria, and the use of protein microarray analysis, are current aspects/tools that will help to stopping transmission of Plasmodium vivax malaria within rural villages near Iquitos, and eventually within the Amazon Region of Loreto, Peru. Three studies were conducted and published for this purpose. The first published manuscript analyzes socio- demographical and occupational activities of people at risk for malaria. This analysis allowed us to identify the main risk group among subjects working in the forest or agricultural activities and their partners at home. The second manuscript demonstrated new tools for malaria epidemiology studies: tandem repeat markers and restriction fragment length polymorphism msp3[alpha] markers that have high discriminatory capacity for determining the micro-geography of malaria transmission in the Peruvian Amazon. The third manuscript used genome- level protein microarray analysis to identify new antigens as serological tool to monitor infection status and transmission dynamics, and that proteins recognized by human IgG antibodies are more likely to have amino acid changes or single nucleotide polymorphisms than proteins coded by the genome as a whole, suggesting selection arising from host-pathogen interactions, immune or otherwise. The new P. vivax protein antigens have important potential as tools for sero-epidemiological analysis of surveillance, control and elimination strategies. More generally, these research in the Peruvian Amazon contribute to a better understanding of biological and socio-demographical characteristics of malaria transmission and fundamental properties of the human immune response to Plasmodium vivax

AbstractBackgroundMalaria caused by Plasmodium vivax is an experimentally neglected severe disease with a substantial burden on human health. Because of technical limitations, little is known about the biology of this important human pathogen. Whole genome analysis methods on patient-derived material are thus likely to have a substantial impact on our understanding of P. vivax pathogenesis and epidemiology. For example, it will allow study of the evolution and population biology of the parasite, allow parasite transmission patterns to be characterized, and may facilitate the identification of new drug resistance genes. Because parasitemias are typically low and the parasite cannot be readily cultured, on-site leukocyte depletion of blood samples is typically needed to remove human DNA that may be 1000X more abundant than parasite DNA. These features have precluded the analysis of archived blood samples and require the presence of laboratories in close proximity to the collection of field samples for optimal pre-cryopreservation sample preparation.ResultsHere we show that in-solution hybridization capture can be used to extract P. vivax DNA from human contaminating DNA in the laboratory without the need for on-site leukocyte filtration. Using a whole genome capture method, we were able to enrich P. vivax DNA from bulk genomic DNA from less than 0.5% to a median of 55% (range 20%-80%). This level of enrichment allows for efficient analysis of the samples by whole genome sequencing and does not introduce any gross biases into the data. With this method, we obtained greater than 5X coverage across 93% of the P. vivax genome for four P. vivax strains from Iquitos, Peru, which is similar to our results using leukocyte filtration (greater than 5X coverage across 96% ).ConclusionThe whole genome capture technique will enable more efficient whole genome analysis of P. vivax from a larger geographic region and from valuable archived sample collections.

Anopheles darlingi Root is the most important malaria vector in the Amazonia region of South America. However, continuous propagation of An. darlingi in the laboratory has been elusive, limiting entomological, genetic/genomic, and vector-pathogen interaction studies of this mosquito species. Here, we report the establishment of an An. darlingi colony derived from wild-caught mosquitoes obtained in the northeastern Peruvian Amazon region of Iquitos in the Loreto Department. We show that the numbers of eggs, larvae, pupae, and adults continue to rise at least to the F6 generation. Comparison of feeding Plasmodium vivax ex vivo of F4 and F5 to F1 generation mosquitoes showed the comparable presence of oocysts and sporozoites, with numbers that corresponded to blood-stage asexual parasitemia and gametocytemia, confirming P. vivax vectorial capacity in the colonized mosquitoes. These results provide new avenues for research on An. darlingi biology and study of An. darlingi-Plasmodium interactions.

Large scale antibody responses in Plasmodium vivax malaria remains unexplored in the endemic setting. Protein microarray analysis of asexual-stage P. vivax was used to identify antigens recognized in sera from residents of hypoendemic Peruvian Amazon. Over 24 months, of 106 participants, 91 had two symptomatic P. vivax malaria episodes, 11 had three episodes, 3 had four episodes, and 1 had five episodes. Plasmodium vivax relapse was distinguished from reinfection by a merozoite surface protein-3α restriction fragment length polymorphism polymerase chain reaction (MSP3α PCR-RFLP) assay. Notably, P. vivax reinfection subjects did not have higher reactivity to the entire set of recognized P. vivax blood-stage antigens than relapse subjects, regardless of the number of malaria episodes. The most highly recognized P. vivax proteins were MSP 4, 7, 8, and 10 (PVX_003775, PVX_082650, PVX_097625, and PVX_114145); sexual-stage antigen s16 (PVX_000930); early transcribed membrane protein (PVX_090230); tryptophan-rich antigen (Pv-fam-a) (PVX_092995); apical merozoite antigen 1 (PVX_092275); and proteins of unknown function (PVX_081830, PVX_117680, PVX_118705, PVX_121935, PVX_097730, PVX_110935, PVX_115450, and PVX_082475). Genes encoding reactive proteins exhibited a significant enrichment of non-synonymous nucleotide variation, an observation suggesting immune selection. These data identify candidates for seroepidemiological tools to support malaria elimination efforts in P. vivax-endemic regions.