Molecular and epidemiological characterization of Plasmodium vivax recurrent infections in southern Mexico

Abstract

Background: In southern Mexico, malaria transmission is low, seasonal, and persistent. Because many patients are affected by two or more malaria episodes caused by Plasmodium vivax, we carried out a study to determine the timing, frequency, and genetic identity of recurrent malaria episodes in the region between 1998 and 2008.

Methods: Symptomatic patients with more than one P. vivax infection were followed up, and blood samples were collected from primary and recurrent infections. DNA extracted from infected blood samples was analyzed for restriction fragment length polymorphism (RFLP) in genes encoding csp and msp3α, as well as size variation in seven microsatellites.

Results: One hundred and forty six parasite samples were collected from 70 patients; of these, 65 patients had one recurrent infection, four had two, and one had three recurrent infections. The majority of recurrent infections occurred within one year of the primary infection, some of which were genetically homologous to the primary infection. As the genetic diversity in the background population was high, the probability of homologous re-infection was low and the homologous recurrences likely reflected relapses. These homologous recurrent infections generally had short (< 6 months) or long (6-12 months) intervals between the primary (PI) and recurrent (RI) infections; whereas infections containing heterologous genotypes had relatively longer intervals. The epidemiological data indicate that heterologous recurrences could be either relapse or re-infections.

Conclusions: Genetic and temporal analysis of P. vivax recurrence patterns in southern Mexico indicated that relapses play an important role in initiating malaria transmission each season. The manifestation of these infections during the active transmission season allowed the propagation of diverse hypnozoite genotypes. Both short- and long-interval relapses have contributed to parasite persistence and must be considered as targets of treatment for malaria elimination programs in the region to be successful.

Figures

Figure 1

Distribution of recurrent intervals for P. vivax infections. All recurrent P. vivax infections documented in the records of the Malaria Control Program (Jurisdiction VII of Chiapas) during 1998–2007 (n=590, comprising 94% of all the samples excluding those with extreme longer intervals. PI, primary infection; RI, recurrent infection).

Figure 2

Plasmodium vivax msp3α genotypes. PCR products were digested with Alu I, and the digested products are separated side-by-side with their undigested products on agarose gels. a) Shows different genotypes indicated by capital letters underneath. b) Lanes 1, 3, 5, 7, 9, and 11 are undigested products of ~2.0 kb, and lanes 2, 4, 6, 8, 10, and 12 were the same products digested with Alu I. Lane 2 presents genotype C; lane 4, 10, and 12 present genotype B; lane 6 and 8 are mixed genotypes of A+C; c) genotype G; d) genotype D; and e) genotype H. m, 100 bp DNA ladder.

Figure 3

Microsatellite diversity and Plasmodium vivax infection rate in southern Mexico during a 10-year period.A. Pattern of allelic variation for 7 microsatellite loci using 474 bloodspot samples collected over 10 years. Grey, mean number of alleles (Na); white, mean number of alleles ≥ 5%; black line, mean effective heterozygosity (He = [n/(n-1)]×1- ∑pi2), shows a slight but persistent decrease over the time frame (R2 = 0.3109). B. Number of malaria cases per 1,000 people during a 10 year period across the entire study area (black bars), and within villages with positive malaria cases only (white bars). API: number of cases per year per 1,000 people.

Figure 4

Concordance between P. vivax cspr-msp3α and microsatellite patterns for primary and recurrent infections with different latency periods (n=39 pair samples). Each data point indicates one sample pair (PI and RI). Circles indicate paired infections with homologous cspr-msp3α genotypes. Brown circles, rare msp3α genotypes (D, E, G); orange circles, common msp3α genotypes (A, B, C). Green triangles indicate paired infections with heterologous cspr-msp3α genotypes.