Plasmodium vivax infects a hundred million people annually and endangers 40% of the world's population. Unlike Plasmodium falciparum, P. vivax parasites can persist as a dormant stage in the liver, known as the hypnozoite, and these dormant forms can cause malaria relapses months or years after the initial mosquito bite. Here we analyze whole genome sequencing data from parasites in the blood of a patient who experienced consecutive P. vivax relapses over 33 months in a non-endemic country. By analyzing patterns of identity, read coverage, and the presence or absence of minor alleles in the initial polyclonal and subsequent monoclonal infections, we show that the parasites in the three infections are likely meiotic siblings. We infer that these siblings are descended from a single tetrad-like form that developed in the infecting mosquito midgut shortly after fertilization. In this natural cross we find the recombination rate for P. vivax to be 10 kb per centimorgan and we further observe areas of disequilibrium surrounding major drug resistance genes. Our data provide new strategies for studying multiclonal infections, which are common in all types of infectious diseases, and for distinguishing P. vivax relapses from reinfections in malaria endemic regions. This work provides a theoretical foundation for studies that aim to determine if new or existing drugs can provide a radical cure of P. vivax malaria.

Abstract. Plasmodium infection in pregnancy causes substantial maternal and infant morbidity and mortality. In Colombia, both P. falciparum and P. vivax are endemic, but the impact of either species on pregnancy is largely unknown in this country. A cross-sectional study was carried out with 96 pregnant women who delivered at their local hospital. Maternal, placental, and cord blood were tested for malaria infection by microscopy and real-time quantitative polymerase chain reaction (qPCR). A high frequency of infection was detected by qPCR (45%). These infections had low concentrations of parasite DNA, and 79% were submicroscopic. Submicroscopic infections were associated with placental villitis and intervillitis. In conclusion, the overall frequency of Plasmodium infection at delivery in Colombia is much higher than previously reported. These data prompt a re-examination of the local epidemiology of malaria using molecular diagnostics to establish the clinical relevance of submicroscopic infections during pregnancy as well as their consequences for mothers and newborns.

BackgroundThe frequency of pregnancy-associated malaria is increasingly being documented in American countries. In Colombia, with higher frequency of Plasmodium vivax over Plasmodium falciparum infection, recent reports confirmed gestational malaria as a serious public health problem. Thick smear examination is the gold standard to diagnose malaria in endemic settings, but in recent years, molecular diagnostic methods have contributed to elucidate the dimension of the problem of gestational malaria. The study was aimed at exploring the prevalence of gestational, placental and congenital malaria in women who delivered at the local hospitals of north-west Colombia, between June 2008 and April 2011.MethodsA group of 129 parturient women was selected to explore the prevalence of gestational, placental and congenital malaria in a descriptive, prospective and transversal (prevalence) design. Diagnosis was based on the simultaneous application of two independent diagnostic tests: microscopy of thick blood smears and a polymerase chain reaction assay (PCR).ResultsThe prevalence of gestational malaria (thick smear /PCR) was 9.1%/14.0%; placental malaria was 3.3%/16.5% and congenital malaria was absent. A history of gestational malaria during the current pregnancy was significantly associated with gestational malaria at delivery. Plasmodium vivax caused 65% of cases of gestational malaria, whereas P. falciparum caused most cases of placental malaria.ConclusionsGestational and placental malaria are a serious problem in the region, but the risk of congenital malaria is low. A history of malaria during pregnancy may be a practical indicator of infection at delivery.

Abstract. Studies on gestational malaria and placental malaria have been scarce in malaria-endemic areas of the Western Hemisphere. To describe the histopathology of placental malaria in Colombia, a longitudinal descriptive study was conducted. In this study, 179 placentas were studied by histologic analysis (112 with gestational malaria and 67 negative for malaria). Placental malaria was confirmed in 22.35%, 50.0% had previous infections, and 47.5% had acute infections. Typical malaria-associated changes were observed in 37%. The most common changes were villitis, intervillitis, deciduitis, increased fibrin deposition, increased syncytial knots, mononuclear (monocytes/macrophages and lymphocytes), polymorphonuclear cell infiltration, and trophozoites in fetal erythrocytes. No association was found between type of placental changes observed and histopathologic classification of placental malaria. The findings are consistent with those reported for placental malaria in other regions. Plasmodium vivax was the main parasite responsible for placental and gestational malaria, but its role in the pathogenesis of placental malaria was not conclusive.

BackgroundReal-time PCR is a sensitive and specific method for the analysis of Plasmodium DNA. However, prior purification of genomic DNA from blood is necessary since PCR inhibitors and quenching of fluorophores from blood prevent efficient amplification and detection of PCR products.MethodsReagents designed to specifically overcome PCR inhibition and quenching of fluorescence were evaluated for real-time PCR amplification of Plasmodium DNA directly from blood. Whole blood from clinical samples and dried blood spots collected in the field in Colombia were tested.ResultsAmplification and fluorescence detection by real-time PCR were optimal with 40× SYBR® Green dye and 5% blood volume in the PCR reaction. Plasmodium DNA was detected directly from both whole blood and dried blood spots from clinical samples. The sensitivity and specificity ranged from 93-100% compared with PCR performed on purified Plasmodium DNA.ConclusionsThe methodology described facilitates high-throughput testing of blood samples collected in the field by fluorescence-based real-time PCR. This method can be applied to a broad range of clinical studies with the advantages of immediate sample testing, lower experimental costs and time-savings.

BackgroundA large-scale study was set up in order to study the epidemiology, clinical aspects, and immunopathology of gestational and placental malaria in north-west Colombia. In this region, recent reports using a qPCR technique, confirmed frequencies of infection, by Plasmodium falciparum or Plasmodium vivax, up to 45%. Given the high rates of infection observed both in mother and placenta, a first exploratory study was proposed in order to characterize the effect on the inflammation status, tissue damage and hypoxia in Plasmodium spp. infected placentas.MethodsA descriptive, prospective, cross-sectional design was applied to pregnant women with (PM+) and without (PM-) placental malaria. Messenger RNA expression of Fas, FasL; COX-1, COX-2, HIF, VEGF, and the cytokines IL-2, IL-4, IL-10, IFN-γ and TNF, were measured in peripheral and placental blood using a quantitative PCR. The percentage of apoptotic cells was determined with a TUNEL assay.ResultsIn total 50 placentas were studied: 25 were positive for submicroscopic infection and 25 were negative for Plasmodium infection. Expression of IL-4 and IL-10 was observed high in placental tissue of PM+, while IL-2 was high in peripheral blood of the same group. Expression of TNF and IFNγ in peripheral blood of the PM + group was high. Similarly, the apoptotic index and Fas expression were significantly high in PM+. However, FasL expression was observed low in PM + compared to PM-. Inflammation markers (HIF, VEGF) and hypoxia markers (COX-1, COX-2) were high in the PM + group.ConclusionDuring placental malaria expression of some pro-inflammatory cytokines is up-regulated and markers of hypoxia and tissue damage are increased in cases of submicroscopic infection.

iIn pregnancy, parity-dependent immunity is observed in response to placental infection with Plasmodium falciparum. Antibodies recognize the surface antigen, VAR2CSA, expressed on infected red blood cells and inhibit cytoadherence to the placental tissue. In most settings of malaria endemicity, antibodies against VAR2CSA are predominantly observed in multigravid women and infrequently in men, children, and nulligravid women. However, in Colombia, we detected antibodies against multiple constructs of VAR2CSA among men and children with acute P. falciparum and Plasmodium vivax infection. The majority of men and children (>60%) had high levels of IgGs against three recombinant domains of VAR2CSA: DBL5, DBL3X, and ID1-ID2. Surprisingly, these antibodies were observed only in pregnant women, men, and children exposed either to P. falciparum or to P. vivax. Moreover, the anti-VAR2CSA antibodies are of high avidity and efficiently inhibit adherence of infected red blood cells to chondroitin sulfate A in vitro, suggesting that they are specific and functional. These unexpected results suggest that there may be genotypic or phenotypic differences in the parasites of this region or in the host response to either P. falciparum or P. vivax infection outside pregnancy. These findings may hold significant clinical relevance to the pathophysiology and outcome of malaria infections in this region.

Background. In pregnancy, Plasmodium falciparum parasites express the surface antigen VAR2CSA, which mediates adherence of red blood cells to chondroitin sulfate A (CSA) in the placenta. VAR2CSA antibodies are generally acquired during infection in pregnancy and are associated with protection from placental malaria. We observed previously that men and children in Colombia also had antibodies to VAR2CSA, but the origin of these antibodies was unknown. Here, we tested whether infection with Plasmodium vivax is an alternative mechanism of acquisition of VAR2CSA antibodies.Methods. We analyzed sera from nonpregnant Colombians and Brazilians exposed to P. vivax and monoclonal antibodies raised against P. vivax Duffy binding protein (PvDBP). Cross-reactivity to VAR2CSA was characterized by enzyme-linked immunosorbent assay, immunofluorescence assay, and flow cytometry, and antibodies were tested for inhibition of parasite binding to CSA.Results. Over 50% of individuals had antibodies that recognized VAR2CSA. Affinity-purified PvDBP human antibodies and a PvDBP monoclonal antibody recognized VAR2CSA, showing that PvDBP can give rise to cross-reactive antibodies. Importantly, the monoclonal antibody inhibited parasite binding to CSA, which is the primary in vitro correlate of protection from placental malaria.Conclusions. These data suggest that PvDBP induces antibodies that functionally recognize VAR2CSA, revealing a novel mechanism of cross-species immune recognition to falciparum malaria.