Spatio-temporal analysis of malaria incidence in the Peruvian Amazon Region between 2002 and 2013.

Malaria remains a major public health problem in the Peruvian Amazon where the persistence of high-risk transmission areas (hotspots) challenges the current malaria control strategies. This study aimed at identifying significant space-time clusters of malaria incidence in Loreto region 2002-2013 and to determine significant changes across years in relation to the control measures applied. Poisson regression and purely temporal, spatial, and space-time analyses were conducted. Three significantly different periods in terms of annual incidence rates (AIR) were identified, overlapping respectively with the pre-, during, and post- implementation control activities supported by PAMAFRO project. The most likely space-time clusters of malaria incidence for P. vivax and P. falciparum corresponded to the pre- and first two years of the PAMAFRO project and were situated in the northern districts of Loreto, while secondary clusters were identified in eastern and southern districts with the latest onset and the shortest duration of PAMAFRO interventions. Malaria in Loreto was highly heterogeneous at geographical level and over time. Importantly, the excellent achievements obtained during 5 years of intensified control efforts totally vanished in only 2 to 3 years after the end of the program, calling for sustained political and financial commitment for the success of malaria elimination as ultimate goal.

Population Genetics of Plasmodium vivax in the Peruvian Amazon.

BACKGROUND: Characterizing 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/ RESULTS: From 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 ([Formula: see text] = 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/SIGNIFICANCE: Our 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.

Hotspots of Malaria Transmission in the Peruvian Amazon: Rapid Assessment through a Parasitological and Serological Survey.

BACKGROUND: With low and markedly seasonal malaria transmission, increasingly sensitive tools for better stratifying the risk of infection and targeting control interventions are needed. A cross-sectional survey to characterize the current malaria transmission patterns, identify hotspots, and detect recent changes using parasitological and serological measures was conducted in three sites of the Peruvian Amazon. MATERIAL AND METHODS: After full census of the study population, 651 participants were interviewed, clinically examined and had a blood sample taken for the detection of malaria parasites (microscopy and PCR) and antibodies against P. vivax (PvMSP119, PvAMA1) and P. falciparum (PfGLURP, PfAMA1) antigens by ELISA. Risk factors for malaria infection (positive PCR) and malaria exposure (seropositivity) were assessed by multivariate survey logistic regression models. Age-specific seroprevalence was analyzed using a reversible catalytic conversion model based on maximum likelihood for generating seroconversion rates (SCR, λ). SaTScan was used to detect spatial clusters of serology-positive individuals within each site. RESULTS: The overall parasite prevalence by PCR was low, i.e. 3.9% for P. vivax and 6.7% for P. falciparum, while the seroprevalence was substantially higher, 33.6% for P. vivax and 22.0% for P. falciparum, with major differences between study sites. Age and location (site) were significantly associated with P. vivax exposure; while location, age and outdoor occupation were associated with P. falciparum exposure. P. falciparum seroprevalence curves showed a stable transmission throughout time, while for P. vivax transmission was better described by a model with two SCRs. The spatial analysis identified well-defined clusters of P. falciparum seropositive individuals in two sites, while it detected only a very small cluster of P. vivax exposure. CONCLUSION: The use of a single parasitological and serological malaria survey has proven to be an efficient and accurate method to characterize the species specific heterogeneity in malaria transmission at micro-geographical level as well as to identify recent changes in transmission.

Modelling the potential of focal screening and treatment as elimination strategy for Plasmodium falciparum malaria in the Peruvian Amazon Region.

BACKGROUND: Focal screening and treatment (FSAT) of malaria infections has recently been introduced in Peru to overcome the inherent limitations of passive case detection (PCD) and further decrease the malaria burden. Here, we used a relatively straightforward mathematical model to assess the potential of FSAT as elimination strategy for Plasmodium falciparum malaria in the Peruvian Amazon Region. METHODS: A baseline model was developed to simulate a scenario with seasonal malaria transmission and the effect of PCD and treatment of symptomatic infections on the P. falciparum malaria transmission in a low endemic area of the Peruvian Amazon. The model was then adjusted to simulate intervention scenarios for predicting the long term additional impact of FSAT on P. falciparum malaria prevalence and incidence. Model parameterization was done using data from a cohort study in a rural Amazonian community as well as published transmission parameters from previous studies in similar areas. The effect of FSAT timing and frequency, using either microscopy or a supposed field PCR, was assessed on both predicted incidence and prevalence rates. RESULTS: The intervention model indicated that the addition of FSAT to PCD significantly reduced the predicted P. falciparum incidence and prevalence. The strongest reduction was observed when three consecutive FSAT were implemented at the beginning of the low transmission season, and if malaria diagnosis was done with PCR. Repeated interventions for consecutive years (10 years with microscopy or 5 years with PCR), would allow reaching near to zero incidence and prevalence rates. CONCLUSIONS: The addition of FSAT interventions to PCD may enable to reach P. falciparum elimination levels in low endemic areas of the Amazon Region, yet the progression rates to those levels may vary substantially according to the operational criteria used for the intervention.

Patterns of selection on Plasmodium falciparum erythrocyte-binding antigens after the colonization of the New World.

Pathogens, which have recently colonized a new host species or new populations of the same host, are interesting models for understanding how populations may evolve in response to novel environments. During its colonization of South America from Africa, Plasmodium falciparum, the main agent of malaria, has been exposed to new conditions in distinctive new human populations (Amerindian and populations of mixed origins) that likely exerted new selective pressures on the parasite's genome. Among the genes that might have experienced strong selective pressures in response to these environmental changes, the eba genes (erythrocyte-binding antigens genes), which are involved in the invasion of the human red blood cells, constitute good candidates. In this study, we analysed, in South America, the polymorphism of three eba genes (eba-140, eba-175, eba-181) and compared it to the polymorphism observed in African populations. The aim was to determine whether these genes faced selective pressures in South America distinct from what they experienced in Africa. Patterns of genetic variability of these genes were compared to the patterns observed at two housekeeping genes (adsl and serca) and 272 SNPs to separate adaptive effects from demographic effects. We show that, conversely to Africa, eba-140 seemed to be under stronger diversifying selection in South America than eba-175. In contrast, eba-181 did not show any sign of departure from neutrality. These changes in the patterns of selection on the eba genes could be the consequence of changes in the host immune response, the host receptor polymorphisms and/or the ability of the parasite to silence or express differentially its invasion proteins.

Population structure and spatio-temporal transmission dynamics of Plasmodium vivax after radical cure treatment in a rural village of the Peruvian Amazon.

BACKGROUND: Despite 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. METHODS: A 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. RESULTS: After 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. CONCLUSIONS: In 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.

Assessing malaria transmission in a low endemicity area of north-western Peru.

BACKGROUND: Where malaria endemicity is low, control programmes need increasingly sensitive tools for monitoring malaria transmission intensity (MTI) and to better define health priorities. A cross-sectional survey was conducted in a low endemicity area of the Peruvian north-western coast to assess the MTI using both molecular and serological tools. METHODS: Epidemiological, parasitological and serological data were collected from 2,667 individuals in three settlements of Bellavista district, in May 2010. Parasite infection was detected using microscopy and polymerase chain reaction (PCR). Antibodies to Plasmodium vivax merozoite surface protein-119 (PvMSP119) and to Plasmodium falciparum glutamate-rich protein (PfGLURP) were detected by ELISA. Risk factors for exposure to malaria (seropositivity) were assessed by multivariate survey logistic regression models. Age-specific antibody prevalence of both P. falciparum and P. vivax were analysed using a previously published catalytic conversion model based on maximum likelihood for generating seroconversion rates (SCR). RESULTS: The overall parasite prevalence by microscopy and PCR were extremely low: 0.3 and 0.9%, respectively for P. vivax, and 0 and 0.04%, respectively for P. falciparum, while seroprevalence was much higher, 13.6% for P. vivax and 9.8% for P. falciparum. Settlement, age and occupation as moto-taxi driver during previous year were significantly associated with P. falciparum exposure, while age and distance to the water drain were associated with P. vivax exposure. Likelihood ratio tests supported age seroprevalence curves with two SCR for both P. vivax and P. falciparum indicating significant changes in the MTI over time. The SCR for PfGLURP was 19-fold lower after 2002 as compared to before (λ1 = 0.022 versus λ2 = 0.431), and the SCR for PvMSP119 was four-fold higher after 2006 as compared to before (λ1 = 0.024 versus λ2 = 0.006). CONCLUSION: Combining molecular and serological tools considerably enhanced the capacity of detecting current and past exposure to malaria infections and related risks factors in this very low endemicity area. This allowed for an improved characterization of the current human reservoir of infections, largely hidden and heterogeneous, as well as providing insights into recent changes in species specific MTIs. This approach will be of key importance for evaluating and monitoring future malaria elimination strategies.

Traditional nets interfere with the uptake of long-lasting insecticidal nets in the Peruvian Amazon: the relevance of net preference for achieving high coverage and use.

BACKGROUND: While coverage of long-lasting insecticide-treated nets (LLIN) has steadily increased, a growing number of studies report gaps between net ownership and use. We conducted a mixed-methods social science study assessing the importance of net preference and use after Olyset® LLINs were distributed through a mass campaign in rural communities surrounding Iquitos, the capital city of the Amazonian region of Peru. METHODS: The study was conducted in the catchment area of the Paujil and Cahuide Health Centres (San Juan district) between July 2007 and November 2008. During a first qualitative phase, participant observation and in-depth interviews collected information on key determinants for net preference and use. In a second quantitative phase, a survey among recently confirmed malaria patients evaluated the acceptability and use of both LLINs and traditional nets, and a case control study assessed the association between net preference/use and housing structure (open vs. closed houses). RESULTS: A total of 10 communities were selected for the anthropological fieldwork and 228 households participated in the quantitative studies. In the study area, bed nets are considered part of the housing structure and are therefore required to fulfil specific architectural and social functions, such as providing privacy and shelter, which the newly distributed Olyset® LLINs ultimately did not. The LLINs' failure to meet these criteria could mainly be attributed to their large mesh size, transparency and perceived ineffectiveness to protect against mosquitoes and other insects, resulting in 63.3% of households not using any of the distributed LLINs. Notably, LLIN usage was significantly lower in houses with no interior or exterior walls (35.2%) than in those with walls (73.8%) (OR = 5.2, 95CI [2.2; 12.3], p<0.001). CONCLUSION: Net preference can interfere with optimal LLIN use. In order to improve the number of effective days of LLIN protection per dollar spent, appropriate quantitative and qualitative methods for collecting information on net preference should be developed before any LLIN procurement decision is made.

Multiple independent introductions of Plasmodium falciparum in South America.

The origin of Plasmodium falciparum in South America is controversial. Some studies suggest a recent introduction during the European colonizations and the transatlantic slave trade. Other evidence--archeological and genetic--suggests a much older origin. We collected and analyzed P. falciparum isolates from different regions of the world, encompassing the distribution range of the parasite, including populations from sub-Saharan Africa, the Middle East, Southeast Asia, and South America. Analyses of microsatellite and SNP polymorphisms show that the populations of P. falciparum in South America are subdivided in two main genetic clusters (northern and southern). Phylogenetic analyses, as well as Approximate Bayesian Computation methods suggest independent introductions of the two clusters from African sources. Our estimates of divergence time between the South American populations and their likely sources favor a likely introduction from Africa during the transatlantic slave trade.

True versus apparent malaria infection prevalence: the contribution of a Bayesian approach.

AIMS: To present a new approach for estimating the "true prevalence" of malaria and apply it to datasets from Peru, Vietnam, and Cambodia. METHODS: Bayesian models were developed for estimating both the malaria prevalence using different diagnostic tests (microscopy, PCR & ELISA), without the need of a gold standard, and the tests' characteristics. Several sources of information, i.e. data, expert opinions and other sources of knowledge can be integrated into the model. This approach resulting in an optimal and harmonized estimate of malaria infection prevalence, with no conflict between the different sources of information, was tested on data from Peru, Vietnam and Cambodia. RESULTS: Malaria sero-prevalence was relatively low in all sites, with ELISA showing the highest estimates. The sensitivity of microscopy and ELISA were statistically lower in Vietnam than in the other sites. Similarly, the specificities of microscopy, ELISA and PCR were significantly lower in Vietnam than in the other sites. In Vietnam and Peru, microscopy was closer to the "true" estimate than the other 2 tests while as expected ELISA, with its lower specificity, usually overestimated the prevalence. CONCLUSIONS: Bayesian methods are useful for analyzing prevalence results when no gold standard diagnostic test is available. Though some results are expected, e.g. PCR more sensitive than microscopy, a standardized and context-independent quantification of the diagnostic tests' characteristics (sensitivity and specificity) and the underlying malaria prevalence may be useful for comparing different sites. Indeed, the use of a single diagnostic technique could strongly bias the prevalence estimation. This limitation can be circumvented by using a Bayesian framework taking into account the imperfect characteristics of the currently available diagnostic tests. As discussed in the paper, this approach may further support global malaria burden estimation initiatives.

Plasmodium vivax sub-patent infections after radical treatment are common in Peruvian patients: results of a 1-year prospective cohort study.

BACKGROUND: There 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. METHODOLOGY: Each 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. RESULTS: During 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 (I(A)(s) = 0.29, p<1.10(-4)) that remained also when analyzing only the unique haplotypes, suggesting common inbreeding. CONCLUSION: In 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.

Multilocus genotyping reveals high heterogeneity and strong local population structure of the Plasmodium vivax population in the Peruvian Amazon.

BACKGROUND: Peru 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. METHODS: Plasmodium 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 (theta) were estimated. RESULTS: The 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 (theta = 0.45) was found and the Bayesian inference cluster analysis identified six clusters based on distinctive allele frequencies. CONCLUSION: The 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.

Adherence to 7-day primaquine treatment for the radical cure of P. vivax in the Peruvian Amazon.

Despite being free of charge, treatment adherence to 7-day primaquine for the radical cure of Plasmodium vivax was estimated at 62.2% among patients along the Iquitos-Nauta road in the Peruvian Amazon. The principal reason for non-adherence was the perceived adverse effects related to local humoral illness conceptions that hold that malaria produces a hot state of body, which is further aggravated by the characteristically hot medical treatment. Notably, patients were willing to adhere to the first 3 days of treatment during which symptoms are most apparent and include the characteristic chills. Nevertheless, as symptoms abate, the perceived aggravating characteristics of the medication outweigh the perceived advantages of treatment adherence. Improving community awareness about the role of primaquine to prevent further malaria transmission and fostering a realistic system of direct observed treatment intake, organized at community level, can be expected to improve adherence to the radical cure of P. vivax in this area.

A randomised controlled trial to assess the efficacy of dihydroartemisinin-piperaquine for the treatment of uncomplicated falciparum malaria in Peru.

BACKGROUND: Multi-drug resistant falciparum malaria is an important health problem in the Peruvian Amazon region. We carried out a randomised open label clinical trial comparing mefloquine-artesunate, the current first line treatment in this region, with dihydroartemisinin-piperaquine. METHODS AND FINDINGS: Between July 2003 and July 2005, 522 patients with P. falciparum uncomplicated malaria were recruited, randomized (260 with mefloquine-artesunate and 262 with dihydroartemisinin-piperaquine), treated and followed up for 63 days. PCR-adjusted adequate clinical and parasitological response, estimated by Kaplan Meier survival and Per Protocol analysis, was extremely high for both drugs (99.6% for mefloquine-artesunate and 98.4% and for dihydroartemisinin-piperaquine) (RR: 0.99, 95%CI [0.97-1.01], Fisher Exact p = 0.21). All recrudescences were late parasitological failures. Overall, gametocytes were cleared faster in the mefloquine-artesunate group (28 vs 35 days) and new gametocytes tended to appear more frequently in patients treated with dihydroartemisinin-piperaquine (day 7: 8 (3.6%) vs 2 (0.9%), RR: 3.84, 95%CI [0.82-17.87]). Adverse events such as anxiety and insomnia were significantly more frequent in the mefloquine-artesunate group, both in adults and children. CONCLUSION: Dihydroartemisinin-piperaquine is as effective as mefloquine-artesunate in treating uncomplicated P. falciparum malaria but it is better tolerated and more affordable than mefloquine-artesunate (US$1.0 versus US$18.65 on the local market). Therefore, it should be considered as a potential candidate for the first line treatment of P. falciparum malaria in Peru. TRIAL REGISTRATION: ClinicalTrials.gov NCT00373607.