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To determine the magnitude of Plasmodium vivax relapsing malaria in rural Amazonia, we carried out a study in four sites in northeastern Peru. Polymerase chain reaction-restriction fragment length polymorphism of PvMSP-3α and tandem repeat (TR) markers were compared for their ability to distinguish relapse versus reinfection. Of 1,507 subjects with P. vivax malaria, 354 developed > 1 episode during the study; 97 of 354 (27.5%) were defined as relapse using Pvmsp-3α alone. The addition of TR polymorphism analysis significantly reduced the number of definitively defined relapses to 26 of 354 (7.4%) (P < 0.05). Multivariate logistic regression modeling showed that the probability of having > 1 infection was associated with the following: subjects in Mazan (odds ratio [OR] = 2.56; 95% confidence interval [CI] 1.87, 3.51), 15–44 years of age (OR = 1.49; 95% CI 1.03, 2.15), traveling for job purposes (OR = 1.45; 95%CI 1.03, 2.06), and travel within past month (OR = 1.46; 95% CI 1.0, 2.14). The high discriminatory capacity of the molecular tools shown here is useful for understanding the micro-geography of malaria transmission.

Infection of mosquitoes by humans is not always successful in the setting of patent gametocytemia. This study tested the hypothesis that pro- or anti-inflammatory cytokines are associated with transmission of Plasmodium vivax to Anopheles darlingi mosquitoes in experimental infection. Blood from adults with acute, non-severe P. vivax malaria was fed to laboratory-reared F1 An. darlingi mosquitoes. A panel of cytokines at the time of mosquito infection was assessed in patient sera and levels compared among subjects who did and did not infect mosquitoes. Overall, blood from 43 of 99 (43%) subjects led to mosquito infection as shown by oocyst counts. Levels of IL-10, IL-6, TNF-α, and IFN-γ were significantly elevated in vivax infection and normalized 3 weeks later. The anti-inflammatory cytokine IL-10 was significantly higher in nontransmitters compared with top transmitters but was not in TNF-α and IFN-γ. The IL-10 elevation during acute malaria was associated with P. vivax transmission blocking.

Molecular tools to distinguish strains of Plasmodium vivax are important for studying the epidemiology of malaria transmission. Two sets of markers-tandem repeat (TR) polymorphisms and MSP3α-were used to study Plasmodium vivax in patients in the Peruvian Amazon region of Iquitos. Of 110 patients, 90 distinct haplotypes were distinguished using 9 TR markers. An MSP3α polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using HhaI and AluI revealed 8 and 9 profiles, respectively, and 36 profiles when analyzed in combination. Combining TR and PCR-RFLP markers, 101 distinct molecular profiles were distinguished among these 110 patients. Nine TR markers arrayed along a 100 kB stretch of a P. vivax chromosome containing the gene for circumsporozoite protein showed non-linear linkage disequilibrium (I(SA) = 0.03, P = 0.001). These findings demonstrate the potential use of TR markers for molecular epidemiology studies.

Polymerase chain reaction (PCR) detection of Plasmodium DNA is highly sensitive in diagnosing malaria. The specimen of choice for this assay has been whole blood samples from malaria patients. To retrospectively determine malaria infection rates in populations or cohorts for whom stored serum samples are available, we determined the ability of a nested PCR assay to detect Plasmodium DNA in stored serum samples. The PCR result was positive in 20 of 23 serum samples from patients with microscopy-confirmed malaria and negative in 8 of 8 healthy controls, resulting in a sensitivity of 87% and specificity of 100%. In all positive samples, species were correctly identified by PCR except for one case where a mixed infection was detected. The PCR is able to detect Plasmodium DNA in serum samples frozen up to 2.5 years and has the potential for the retrospective identification of malaria parasitemia in patient cohorts to determine potential interactions of malaria and other diseases such as human immunodeficiency virus/acquired immunodeficiency syndrome.