BackgroundPeru is one of the Latin American countries with the highest malaria burden, mainly due to Plasmodium vivax infections. However, little is known about P. vivax transmission dynamics in the Peruvian Amazon, where most malaria cases occur. The genetic diversity and population structure of P. vivax isolates collected in different communities around Iquitos city, the capital of the Peruvian Amazon, was determined.MethodsPlasmodium vivax population structure was determined by multilocus genotyping with 16 microsatellites on 159 P. vivax infected blood samples (mono-infections) collected in four sites around Iquitos city. The population characteristics were assessed only in samples with monoclonal infections (n = 94), and the genetic diversity was determined by calculating the expected heterozygosity and allelic richness. Both linkage disequilibrium and the genetic differentiation (θ) were estimated.ResultsThe proportion of polyclonal infections varied substantially by site (11% - 70%), with the expected heterozygosity ranging between 0.44 and 0.69; no haplotypes were shared between the different populations. Linkage disequilibrium was present in all populations (IAS 0.14 - 0.61) but was higher in those with fewer polyclonal infections, suggesting inbreeding and a clonal population structure. Strong population differentiation (θ = 0.45) was found and the Bayesian inference cluster analysis identified six clusters based on distinctive allele frequencies.ConclusionThe P. vivax populations circulating in the Peruvian Amazon basin are genetically diverse, strongly differentiated and they have a low effective recombination rate. These results are in line with the low and clustered pattern of malaria transmission observed in the region around Iquitos city.

Background: Considering increasing reports on human infections by Plasmodium knowlesi in Southeast Asian countries, blood samples collected during two large cross-sectional malariometric surveys carried out in a forested area of central Vietnam in 2004 and 2005 were screened for this parasite.

Plasmodium vivax is the geographically most widespread human malaria parasite. To analyze patterns of microsatellite diversity and population structure across countries of different transmission intensity, genotyping data from 11 microsatellite markers was either generated or compiled from 841 isolates from four continents collected in 1999–2008. Diversity was highest in South-East Asia (mean allelic richness 10.0–12.8), intermediate in the South Pacific (8.1–9.9) Madagascar and Sudan (7.9–8.4), and lowest in South America and Central Asia (5.5–7.2). A reduced panel of only 3 markers was sufficient to identify approx. 90% of all haplotypes in South Pacific, African and SE-Asian populations, but only 60–80% in Latin American populations, suggesting that typing of 2–6 markers, depending on the level of endemicity, is sufficient for epidemiological studies. Clustering analysis showed distinct clusters in Peru and Brazil, but little sub-structuring was observed within Africa, SE-Asia or the South Pacific. Isolates from Uzbekistan were exceptional, as a near-clonal parasite population was observed that was clearly separated from all other populations (F ST>0.2). Outside Central Asia F ST values were highest (0.11–0.16) between South American and all other populations, and lowest (0.04–0.07) between populations from South-East Asia and the South Pacific. These comparisons between P. vivax populations from four continents indicated that not only transmission intensity, but also geographical isolation affect diversity and population structure. However, the high effective population size results in slow changes of these parameters. This persistency must be taken into account when assessing the impact of control programs on the genetic structure of parasite populations.

BackgroundCharacterizing the parasite dynamics and population structure provides useful information to understand the dynamic of transmission and to better target control interventions. Despite considerable efforts for its control, vivax malaria remains a major health problem in Peru. In this study, we have explored the population genetics of Plasmodium vivax isolates from Iquitos, the main city in the Peruvian Amazon, and 25 neighbouring peri-urban as well as rural villages along the Iquitos-Nauta Road.Methodology/ ResultsFrom April to December 2008, 292 P. vivax isolates were collected and successfully genotyped using 14 neutral microsatellites. Analysis of the molecular data revealed a similar proportion of monoclonal and polyclonal infections in urban areas, while in rural areas monoclonal infections were predominant (p = 0.002). Multiplicity of infection was higher in urban (MOI = 1.5–2) compared to rural areas (MOI = 1) (p = 0.003). The level of genetic diversity was similar in all areas (He = 0.66–0.76, p = 0.32) though genetic differentiation between areas was substantial (PHIPT = 0.17, p<0.0001). Principal coordinate analysis showed a marked differentiation between parasites from urban and rural areas. Linkage disequilibrium was detected in all the areas ( = 0.08–0.49, for all p<0.0001). Gene flow among the areas was stablished through Bayesian analysis of migration models. Recent bottleneck events were detected in 4 areas and a recent parasite expansion in one of the isolated areas. In total, 87 unique haplotypes grouped in 2 or 3 genetic clusters described a sub-structured parasite population.Conclusion/SignificanceOur study shows a sub-structured parasite population with clonal propagation, with most of its components recently affected by bottleneck events. Iquitos city is the main source of parasite spreading for all the peripheral study areas. The routes of transmission and gene flow and the reduction of the parasite population described are important from the public health perspective as well for the formulation of future control policies.

BackgroundThere is an increasing body of literature reporting treatment failure of the currently recommended radical treatment of Plasmodium vivax infections. As P. vivax is the main malaria species outside the African continent, emerging tolerance to its radical treatment regime could have major consequences in countries like Peru, where 80% of malaria cases are due to P. vivax. Here we describe the results of a 1-year longitudinal follow up of 51 confirmed P. vivax patients living around Iquitos, Peruvian Amazon, and treated according to the Peruvian national guidelines.MethodologyEach month a blood sample for microscopy and later genotyping was systematically collected. Recent exposure to infection was estimated by detecting antibodies against the P. vivax circumsporozoite protein (CSP) and all PCR confirmed P. vivax infections were genotyped with 16 polymorphic microsatellites.ResultsDuring a 1-year period, 84 recurrent infections, 22 positive also by microscopy, were identified, with a median survival time to first recurrent infection of 203 days. Most of them (71%) were asymptomatic; in 13 patients the infection persisted undetected by microscopy for several consecutive months. The genotype of mostly recurrent infections differed from that at day 0 while fewer differences were seen between the recurrent infections. The average expected heterozygosity was 0.56. There was strong linkage disequilibrium (IAs = 0.29, p<1.10−4) that remained also when analyzing only the unique haplotypes, suggesting common inbreeding.ConclusionIn Peru, the P. vivax recurrent infections were common and displayed a high turnover of parasite genotypes compared to day 0. Plasmodium vivax patients, even when treated according to the national guidelines, may still represent an important parasite reservoir that can maintain transmission. Any elimination effort should consider such a hidden reservoir.

Fourteen published and three newly identified polymorphic microsatellites were used to genotype 69 Plasmodium vivax samples obtained from 39 patients detected over a period of two years who lived in a rural community of central Vietnam. All samples were polyclonal with an average expected heterozygosity of 0.86. Among the 39 patients, 16 experienced 1–5 recurrent episodes of P. vivax malaria, most of them (83%) with a different genotype profile compared with previous infections. The minimal set of microsatellites required for differentiating the genotype profiles of the recurrent infections compared with the full set of 17 microsatellites was explored. A combination of five markers was sufficient to identify all recurrent infections with an unrelated or different genotype profile compared with all previous episodes.

BackgroundDespite the large burden of Plasmodium vivax, little is known about its transmission dynamics. This study explored the population structure and spatio-temporal dynamics of P. vivax recurrent infections after radical cure in a two-year cohort study carried out in a rural community of the Peruvian Amazon.MethodsA total of 37 P. vivax participants recruited in San Carlos community (Peru) between April and December 2008 were treated radically with chloroquine and primaquine and followed up monthly for two years with systematic blood sampling. All samples were screened for malaria parasites and subsequently all P. vivax infections genotyped using 15 microsatellites. Parasite population structure and dynamics were determined by computing different genetic indices and using spatio-temporal statistics.ResultsAfter radical cure, 76% of the study participants experienced one or more recurrent P. vivax infections, most of them sub-patent and asymptomatic. The parasite population displayed limited genetic diversity (He = 0.49) and clonal structure, with most infections (84%) being monoclonal. Spatio-temporal clusters of specific haplotypes were found throughout the study and persistence of highly frequent haplotypes were observed over several months within the same participants/households.ConclusionsIn San Carlos community, P. vivax recurrences were commonly observed after radical treatment, and characterized by asymptomatic, sub-patent and clustered infections (within and between individuals from a few neighbouring households). Moreover low genetic diversity as well as parasite inbreeding are likely to define a clonal parasite population which has important implications on the malaria epidemiology of the study area.

The development of a system for the continuous culture of Plasmodium vivax in vitro would benefit from the use of reticulocytes derived from differentiated hematopoietic stem cells (HCS). At present, the need to use both fresh reticulocytes and fresh P. vivax isolates represents a major obstacle towards this goal, particularly for laboratories located in non-endemic countries. Here, we describe a new method for the cryopreservation of HSC-derived reticulocytes to be used for both P. falciparum and P. vivax invasion tests. Cryopreserved P. falciparum and P. vivax isolates could invade both fresh and cryopreserved HSC-derived reticulocytes with similar efficiency. This new technique allows the storage of HSC-derived reticulocytes which can be used for later invasion tests and represents an important step towards the establishment of a continuous P. vivax culture.