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Background: ETRAMP11.2 (PVX_003565) is a well-characterized protein with antigenic potential. It is considered to be a serological marker for diagnostic tools, and it has been suggested as a potential vaccine candidate. Despite its immunological relevance, the polymorphism of the P. vivax ETRAMP11.2 gene (pvetramp11.2) remains undefined. The genetic variability of an antigen may limit the effectiveness of its application as a serological surveillance tool and in vaccine development and, therefore, the aim of this study was to investigate the genetic diversity of pvetramp11.2 in parasite populations from Amazonian regions and worldwide. We also evaluated amino acid polymorphism on predicted B-cell epitopes. The low variability of the sequence encoding PvETRAMP11.2 protein suggests that it would be a suitable marker in prospective serodiagnostic assays for surveillance strategies or in vaccine design against P. vivax malaria., Methods: The pvetramp11.2 of P. vivax isolates collected from Brazil (n = 68) and Peru (n = 36) were sequenced and analyzed to assess nucleotide polymorphisms, allele distributions, population differentiation, genetic diversity and signature of selection. In addition, sequences (n = 104) of seven populations from different geographical regions were retrieved from the PlasmoDB database and included in the analysis to study the worldwide allele distribution. Potential linear B-cell epitopes and their polymorphisms were also explored., Results: The multiple alignments of 208 pvetramp11.2 sequences revealed a low polymorphism and a marked geographical variation in allele diversity. Seven polymorphic sites and 11 alleles were identified. All of the alleles were detected in isolates from the Latin American region and five alleles were detected in isolates from the Southeast Asia/Papua New Guinea (SEA/PNG) region. Three alleles were shared by all Latin American populations (H1, H6 and H7). The H1 allele (reference allele from Salvador-1 strain), which was absent in the SEA/PNG populations, was the most represented allele in populations from Brazil (54%) and was also detected at high frequencies in populations from all other Latin America countries (range: 13.0% to 33.3%). The H2 allele was the major allele in SEA/PNG populations, but was poorly represented in Latin America populations (only in Brazil: 7.3%). Plasmodium vivax populations from Latin America showed a marked inter-population genetic differentiation (fixation index [Fst]) in contrast to SEA/PNG populations. Codon bias measures (effective number of codons [ENC] and Codon bias index [CBI]) indicated preferential use of synonymous codons, suggesting selective pressure at the translation level. Only three amino acid substitutions, located in the C-terminus, were detected. Linear B-cell epitope mapping predicted two epitopes in the Sal-1 PvETRAMP11.2 protein, one of which was fully conserved in all of the parasite populations analyzed., Conclusions: We provide an overview of the allele distribution and genetic differentiation of ETRAMP11.2 antigen in P. vivax populations from different endemic areas of the world. The reduced polymorphism and the high degree of protein conservation supports the application of PvETRAMP11.2 protein as a reliable antigen for application in serological assays or vaccine design. Our findings provide useful information that can be used to inform future study designs., (© 2023. The Author(s).)

Malaria continues to wreak havoc in the Peruvian Amazon. Lengthy research efforts have brought important lessons on its particular epidemiology: the heterogeneous levels of transmission, the large reservoir of both asymptomatic and submicroscopic infections, the co-transmission of Plasmodium vivax and Plasmodium falciparum in the same areas, and the limitations of current diagnostics. Based on these features, the national elimination program could greatly benefit from simplified standard treatment, with the use of artemisinin-based combination therapy and even shorter schemes of primaquine maintaing the total dosing. It is acknowledged that there is some uncertainty regarding the true prevalence of glucose-6-phosphate dehydrogenase deficiency (G6PD) and genetic polymorphisms related to cytochrome P-450 isozyme 2D6 functioning. Once we have a better understanding, tafenoquine, whether or not in combination with a rapid G6PD enzyme test, may become a future pathway to eliminate the otherwise hidden reservoir of the P. vivax hypnozoite through one standard Plasmodium treatment., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

BACKGROUNDSerological tools for the accurate detection of recent malaria exposure are needed to guide and monitor malaria control efforts. IgG responses against Plasmodium vivax and P. falciparum merozoite surface protein-10 (MSP10) were measured as a potential way to identify recent malaria exposure in the Peruvian Amazon.METHODSA field-based study included 470 participants in a longitudinal cohort who completed a comprehensive evaluation: light microscopy and PCR on enrollment, at least 1 monthly follow-up by light microscopy, a second PCR, and serum and dried blood spots for serological analysis at the end of the follow-up. IgG titers against novel mammalian cell-produced recombinant PvMSP10 and PfMSP10 were determined by ELISA.RESULTSDuring the follow-up period, 205 participants were infected, including 171 with P. vivax, 26 with P. falciparum, 6 with infections by both species but at different times, and 2 with mixed infections. Exposure to P. vivax was more accurately identified when serological responses to PvMSP10 were obtained from serum (sensitivity, 58.1%; specificity, 81.8%; AUC: 0.76) than from dried blood spots (sensitivity, 35.2; specificity, 83.5%; AUC: 0.64) (PAUC < 0.001). Sensitivity was highest (serum, 82.9%; dried blood spot, 45.7%) with confirmed P. vivax infections occurring 7-30 days before sample collection; sensitivity decreased significantly in relation to time since last documented infection. PvMSP10 serological data did not show evidence of interspecies cross-reactivity. Anti-PfMSP10 responses poorly discriminated between P. falciparum-exposed and nonexposed individuals (AUC = 0.59; P > 0.05).CONCLUSIONAnti-PvMSP10 IgG indicates recent exposure to P. vivax at the population level in the Amazon region. Serum, not dried blood spots, should be used for such serological tests.FUNDINGCooperative agreement U19AI089681 from the United States Public Health Service, NIH/National Institute of Allergy and Infectious Diseases, as the Amazonian International Center of Excellence in Malaria Research.

Despite efforts made over decades by the Peruvian government to eliminate malaria, Plasmodium vivax remains a challenge for public health decision-makers in the country. The uneven distribution of its incidence, plus its complex pattern of dispersion, has made ineffective control measures based on global information that lack the necessary detail to understand transmission fully. In this sense, population genetic tools can complement current surveillance. This study describes the genetic diversity and population structure from September 2012 to March 2015 in three geographically distant settlements, Cahuide (CAH), Lupuna (LUP) and Santa Emilia (STE), located in the Peruvian Amazon. A total 777 P. vivax mono-infections, out of 3264, were genotyped. Among study areas, LUP showed 19.7% of polyclonal infections, and its genetic diversity (Hexp) was 0.544. Temporal analysis showed a significant increment of polyclonal infections and Hexp, and the introduction and persistence of a new parasite population since March 2013. In STE, 40.1% of infections were polyclonal, with Hexp = 0.596. The presence of four genetic clusters without signals of clonal expansion and infections with lower parasite densities compared against the other two areas were also found. At least four parasite populations were present in CAH in 2012, where, after June 2014, malaria cases decreased from 213 to 61, concomitant with a decrease in polyclonal infections (from 0.286 to 0.18), and expectedly variable Hexp. Strong signals of gene flow were present in the study areas and wide geographic distribution of highly diverse parasite populations were found. This study suggests that movement of malaria parasites by human reservoirs connects geographically distant malaria transmission areas in the Peruvian Amazon. The maintenance of high levels of parasite genetic diversity through human mobility is a critical barrier to malaria elimination in this region., Competing Interests: The authors have declared that no competing interests exist.

Background: Treatment of Plasmodium vivax malaria requires the clearing of asexual parasites, but relapse can be prevented only if dormant hypnozoites are cleared from the liver (a treatment termed "radical cure"). Tafenoquine is a single-dose 8-aminoquinoline that has recently been registered for the radical cure of P. vivax., Methods: This multicenter, double-blind, double-dummy, parallel group, randomized, placebo-controlled trial was conducted in Ethiopia, Peru, Brazil, Cambodia, Thailand, and the Philippines. We enrolled 522 patients with microscopically confirmed P. vivax infection (>100 to <100,000 parasites per microliter) and normal glucose-6-phosphate dehydrogenase (G6PD) activity (with normal activity defined as ≥70% of the median value determined at each trial site among 36 healthy male volunteers who were otherwise not involved in the trial). All patients received a 3-day course of chloroquine (total dose of 1500 mg). In addition, patients were assigned to receive a single 300-mg dose of tafenoquine on day 1 or 2 (260 patients), placebo (133 patients), or a 15-mg dose of primaquine once daily for 14 days (129 patients). The primary outcome was the Kaplan-Meier estimated percentage of patients who were free from recurrence at 6 months, defined as P. vivax clearance without recurrent parasitemia., Results: In the intention-to-treat population, the percentage of patients who were free from recurrence at 6 months was 62.4% in the tafenoquine group (95% confidence interval [CI], 54.9 to 69.0), 27.7% in the placebo group (95% CI, 19.6 to 36.6), and 69.6% in the primaquine group (95% CI, 60.2 to 77.1). The hazard ratio for the risk of recurrence was 0.30 (95% CI, 0.22 to 0.40) with tafenoquine as compared with placebo (P<0.001) and 0.26 (95% CI, 0.18 to 0.39) with primaquine as compared with placebo (P<0.001). Tafenoquine was associated with asymptomatic declines in hemoglobin levels, which resolved without intervention., Conclusions: Single-dose tafenoquine resulted in a significantly lower risk of P. vivax recurrence than placebo in patients with phenotypically normal G6PD activity. (Funded by GlaxoSmithKline and Medicines for Malaria Venture; DETECTIVE ClinicalTrials.gov number, NCT01376167 .).

Background: Tafenoquine, a single-dose therapy for Plasmodium vivax malaria, has been associated with relapse prevention through the clearance of P. vivax parasitemia and hypnozoites, termed "radical cure.", Methods: We performed a phase 3, prospective, double-blind, double-dummy, randomized, controlled trial to compare tafenoquine with primaquine in terms of safety and efficacy. The trial was conducted at seven hospitals or clinics in Peru, Brazil, Colombia, Vietnam, and Thailand and involved patients with normal glucose-6-phosphate dehydrogenase (G6PD) enzyme activity and female patients with moderate G6PD enzyme deficiency; all patients had confirmed P. vivax parasitemia. The patients were randomly assigned, in a 2:1 ratio, to receive a single 300-mg dose of tafenoquine or 15 mg of primaquine once daily for 14 days (administered under supervision); all patients received a 3-day course of chloroquine and were followed for 180 days. The primary safety outcome was a protocol-defined decrease in the hemoglobin level (>3.0 g per deciliter or ≥30% from baseline or to a level of <6.0 g per deciliter). Freedom from recurrence of P. vivax parasitemia at 6 months was the primary efficacy outcome in a planned patient-level meta-analysis of the current trial and another phase 3 trial of tafenoquine and primaquine (per-protocol populations), and an odds ratio for recurrence of 1.45 (tafenoquine vs. primaquine) was used as a noninferiority margin., Results: A protocol-defined decrease in the hemoglobin level occurred in 4 of 166 patients (2.4%; 95% confidence interval [CI], 0.9 to 6.0) in the tafenoquine group and in 1 of 85 patients (1.2%; 95% CI, 0.2 to 6.4) in the primaquine group, for a between-group difference of 1.2 percentage points (95% CI, -4.2 to 5.0). In the patient-level meta-analysis, the percentage of patients who were free from recurrence at 6 months was 67.0% (95% CI, 61.0 to 72.3) among the 426 patients in the tafenoquine group and 72.8% (95% CI, 65.6 to 78.8) among the 214 patients in the primaquine group. The efficacy of tafenoquine was not shown to be noninferior to that of primaquine (odds ratio for recurrence, 1.81; 95% CI, 0.82 to 3.96)., Conclusions: Among patients with normal G6PD enzyme activity, the decline in hemoglobin level with tafenoquine did not differ significantly from that with primaquine. Tafenoquine showed efficacy for the radical cure of P. vivax malaria, although tafenoquine was not shown to be noninferior to primaquine. (Funded by GlaxoSmithKline and Medicines for Malaria Venture; GATHER ClinicalTrials.gov number, NCT02216123 .).

Background: Faced with the resurgence of malaria, malaria surveillance in the Peruvian Amazon incorporated consecutive active case detection (ACD) interventions using light microscopy (LM) as reactive measure in communities with an unusual high number of cases during high transmission season (HTS). We assessed the effectiveness in malaria detection of this local ACD-based strategy. Methods: A cohort study was conducted in June⁻July 2015 in Mazan, Loreto. Four consecutive ACD interventions at intervals of 10 days were conducted in four riverine communities (Gamitanacocha, Primero de Enero, Libertad and Urco Miraño). In each intervention, all inhabitants were visited at home, and finger-prick blood samples collected for immediate diagnosis by LM and on filter paper for later analysis by quantitative real-time polymerase chain reaction (qPCR). Effectiveness was calculated by dividing the number of malaria infections detected using LM by the number of malaria infections detected by delayed qPCR. Results : Most community inhabitants (88.1%, 822/933) were present in at least one of the four ACD interventions. A total of 451 infections were detected by qPCR in 446 participants (54.3% of total participants); five individuals had two infections. Plasmodium vivax was the predominant species (79.8%), followed by P. falciparum (15.3%) and P. vivax - P. falciparum co-infections (4.9%). Most qPCR-positive infections were asymptomatic (255/448, 56.9%). The ACD-strategy using LM had an effectiveness of 22.8% (detection of 103 of the total qPCR-positive infections). Children aged 5⁻14 years, and farming as main economic activity were associated with P. vivax infections. Conclusions: Although the ACD-strategy using LM increased the opportunity of detecting and treating malaria infections during HTS, the number of detected infections was considerably lower than the real burden of infections (those detected by qPCR).

Background: The incidence of malaria due both to Plasmodium falciparum and Plasmodium vivax in the Peruvian Amazon has risen in the past 5 years. This study tested the hypothesis that the maintenance and emergence of malaria in hypoendemic regions such as Amazonia is determined by submicroscopic and asymptomatic Plasmodium parasitaemia carriers. The present study aimed to precisely quantify the rate of very-low parasitaemia carriers in two sites of the Peruvian Amazon in relation to transmission patterns of P. vivax and P. falciparum in this area., Methods: This study was carried out within the Amazonian-ICEMR longitudinal cohort. Blood samples were collected for light microscopy diagnosis and packed red blood cell (PRBC) samples were analysed by qPCR. Plasma samples were tested for total IgG reactivity against recombinant PvMSP-10 and PfMSP-10 antigens by ELISA. Occupation and age 10 years and greater were considered surrogates of occupation-related mobility. Risk factors for P. falciparum and P. vivax infections detected by PRBC-qPCR were assessed by multilevel logistic regression models., Results: Among 450 subjects, the prevalence of P. vivax by PRBC-PCR (25.1%) was sixfold higher than that determined by microscopy (3.6%). The prevalence of P. falciparum infection was 4.9% by PRBC-PCR and 0.2% by microscopy. More than 40% of infections had parasitaemia under 5 parasites/μL. Multivariate analysis for infections detected by PRBC-PCR showed that participants with recent settlement in the study area (AOR 2.1; 95% CI 1.03:4.2), age ≥ 30 years (AOR 3.3; 95% CI 1.6:6.9) and seropositivity to P. vivax (AOR 1.8; 95% CI 1.0:3.2) had significantly higher likelihood of P. vivax infection, while the odds of P. falciparum infection was higher for participants between 10 and 29 years (AOR 10.7; 95% CI 1.3:91.1) and with a previous P. falciparum infection (AOR 10.4; 95% CI 1.5:71.1)., Conclusions: This study confirms the contrasting transmission patterns of P. vivax and P. falciparum in the Peruvian Amazon, with stable local transmission for P. vivax and the source of P. falciparum to the study villages dominated by very low parasitaemia carriers, age 10 years and older, who had travelled away from home for work and brought P. falciparum infection with them.

Malaria has steadily increased in the Peruvian Amazon over the last five years. This study aimed to determine the parasite prevalence and micro-geographical heterogeneity of Plasmodium vivax parasitaemia in communities of the Peruvian Amazon. Four cross-sectional active case detection surveys were conducted between May and July 2015 in four riverine communities in Mazan district. Analysis of 2785 samples of 820 individuals nested within 154 households for Plasmodium parasitaemia was carried out using light microscopy and qPCR. The spatio-temporal distribution of Plasmodium parasitaemia, dominated by P. vivax, was shown to cluster at both household and community levels. Of enrolled individuals, 47% had at least one P. vivax parasitaemia and 10% P. falciparum, by qPCR, both of which were predominantly sub-microscopic and asymptomatic. Spatial analysis detected significant clustering in three communities. Our findings showed that communities at small-to-moderate spatial scales differed in P. vivax parasite prevalence, and multilevel Poisson regression models showed that such differences were influenced by factors such as age, education, and location of households within high-risk clusters, as well as factors linked to a local micro-geographic context, such as travel and occupation. Complex transmission patterns were found to be related to human mobility among communities in the same micro-basin.

Malaria in Peru, dominated by Plasmodium vivax, remains a public health problem. The 1990s saw newly epidemic malaria emerge, primarily in the Loreto Department in the Amazon region, including areas near to Iquitos, the capital city, but sporadic malaria transmission also occurred in the 1990s-2000s in both north-coastal Peru and the gold mining regions of southeastern Peru. Although a Global Fund-supported intervention (PAMAFRO, 2005-2010) was temporally associated with a decrease of malaria transmission, from 2012 to the present, both P. vivax and Plasmodium falciparum malaria cases have rapidly increased. The Peruvian Ministry of Health continues to provide artemesinin-based combination therapy for microscopy-confirmed cases of P. falciparum and chloroquine-primaquine for P. vivax Malaria transmission continues in remote areas nonetheless, where the mobility of humans and parasites facilitates continued reintroduction outside of ongoing surveillance activities, which is critical to address for future malaria control and elimination efforts. Ongoing P. vivax research gaps in Peru include the following: identification of asymptomatic parasitemics, quantification of the contribution of patent and subpatent parasitemics to mosquito transmission, diagnosis of nonparasitemic hypnozoite carriers, and implementation of surveillance for potential emergence of chloroquine- and 8-aminoquinoline-resistant P. vivax Clinical trials of tafenoquine in Peru have been promising, and glucose-6-phosphate dehydrogenase deficiency in the region has not been observed to be a limitation to its use. Larger-scale challenges for P. vivax (and malaria in general) in Peru include logistical difficulties in accessing remote riverine populations, consequences of government policy and poverty trends, and obtaining international funding for malaria control and elimination., (© The American Society of Tropical Medicine and Hygiene.)

Unlabelled: Prevention of relapse of Plasmodium vivax infection is a key treatment goal in malaria. Use of P. vivax genotyping in a multicenter, double-blind, randomized, placebo-controlled phase 2b study in Peru, India, Thailand, and Brazil allowed determination of genetically heterologous or homologous P. vivax infection recurrence following receipt of chloroquine plus one of 4 doses of tafenoquine (50, 100, 300, or 600 mg) or chloroquine plus primaquine, compared with receipt of chloroquine alone. The antihypnozoite efficacy of tafenoquine was evident as a reduction in homologous recurrences of P. vivax infection as drug doses were increased. No clear dose-response pattern was evident for heterologous recurrences of P. vivax infection. Rates of homologous recurrence of P. vivax infection appear to be clinically useful for comparing drug efficacy for the prevention of P. vivax infection relapse., Clinical Trials Registration: NCT01376167., (© The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America.)

Large scale antibody responses in Plasmodium vivax malaria remains unexplored in the endemic setting. Protein microarray analysis of asexual-stage P. vivax was used to identify antigens recognized in sera from residents of hypoendemic Peruvian Amazon. Over 24 months, of 106 participants, 91 had two symptomatic P. vivax malaria episodes, 11 had three episodes, 3 had four episodes, and 1 had five episodes. Plasmodium vivax relapse was distinguished from reinfection by a merozoite surface protein-3α restriction fragment length polymorphism polymerase chain reaction (MSP3α PCR-RFLP) assay. Notably, P. vivax reinfection subjects did not have higher reactivity to the entire set of recognized P. vivax blood-stage antigens than relapse subjects, regardless of the number of malaria episodes. The most highly recognized P. vivax proteins were MSP 4, 7, 8, and 10 (PVX&#95;003775, PVX&#95;082650, PVX&#95;097625, and PVX&#95;114145); sexual-stage antigen s16 (PVX&#95;000930); early transcribed membrane protein (PVX&#95;090230); tryptophan-rich antigen (Pv-fam-a) (PVX&#95;092995); apical merozoite antigen 1 (PVX&#95;092275); and proteins of unknown function (PVX&#95;081830, PVX&#95;117680, PVX&#95;118705, PVX&#95;121935, PVX&#95;097730, PVX&#95;110935, PVX&#95;115450, and PVX&#95;082475). Genes encoding reactive proteins exhibited a significant enrichment of non-synonymous nucleotide variation, an observation suggesting immune selection. These data identify candidates for seroepidemiological tools to support malaria elimination efforts in P. vivax-endemic regions., (© The American Society of Tropical Medicine and Hygiene.)

Background: Peru has presented a decreasing malaria trend during the last decade, particularly in areas on northwestern coast; however, a limited number of cases continues to be reported yearly mainly in malaria hotspots., Methods: A two-phase study was conducted to identify spatial and temporal clusters of incident Plasmodium vivax malaria, as well as to determine risk factors associated with households (HH) presenting P. vivax malaria episodes in an urban area of the northwestern Peruvian Coast from June 2008 to May 2010. In the first stage, a full census of the study population was conducted, including geo-referencing of reported P. vivax episodes. In the second stage, a population-based case-control study allowed the identification of risk factors associated with HHs reporting episodes. A total of 117 case HHs with reported P. vivax and 117 control HHs without malaria episodes were assessed. A semi-structured questionnaire was used to interview the head of households and to collect data on HH location and structure, availability of public services, preventive malaria measures, family member with outdoor occupation (farmer, moto-taxi driver), and other HH characteristics. Univariate and multivariate logistic regression analyses were performed to determine case-HH risk factors. SaTScan was used to detect spatial and temporal P. vivax malaria clusters., Results: The most likely spatial cluster of malaria incidence included 1,040 people (22.4% of total population) in 245 HHs (24.6% of total HHs) accounting for 283 malaria episodes (40.1% of total episodes) during the study period (RR = 2.3, p < 0.001). A temporal cluster was also identified from April 12, 2009 to July 4, 2009 accounting for 355 malaria episodes (50.4% of total episodes) (RR = 7.2, p = 0.001). Factors significantly associated with case HHs compared with control HHs were: proximity to water drain < 200 metres (OR = 2.3, 95% CI: 1.3, 4.0); HH size >5 individuals (OR = 1.8, 95% CI: 1.0, 3.2); lack of potable water (OR = 1.8, 95% CI: 1.1, 3.2); and having domestic and peridomestic animals (OR = 3.6, 95% CI: 1.3, 9.5)., Conclusion: Plasmodium vivax malaria incidence is highly heterogeneous in space and time in the urban study area with important geographical and housing risk factors associated with symptomatic episodes.

Anopheles darlingi Root is the most important malaria vector in the Amazonia region of South America. However, continuous propagation of An. darlingi in the laboratory has been elusive, limiting entomological, genetic/genomic, and vector-pathogen interaction studies of this mosquito species. Here, we report the establishment of an An. darlingi colony derived from wild-caught mosquitoes obtained in the northeastern Peruvian Amazon region of Iquitos in the Loreto Department. We show that the numbers of eggs, larvae, pupae, and adults continue to rise at least to the F6 generation. Comparison of feeding Plasmodium vivax ex vivo of F4 and F5 to F1 generation mosquitoes showed the comparable presence of oocysts and sporozoites, with numbers that corresponded to blood-stage asexual parasitemia and gametocytemia, confirming P. vivax vectorial capacity in the colonized mosquitoes. These results provide new avenues for research on An. darlingi biology and study of An. darlingi-Plasmodium interactions.

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.

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.

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.

Background: The rapid diagnostic tests for malaria (RDT) constitute a fast and opportune alternative for non-complicated malaria diagnosis in areas where microscopy is not available. The objective of this study was to validate a RDT named Parascreen under field conditions in Iquitos, department of Loreto, Peru. Parascreen is a RDT that detects the histidine-rich protein 2 (HRP2) antigen from Plasmodium falciparum and lactate deshydrogenase from all Plasmodium species., Methods: Parascreen was compared with microscopy performed by experts (EM) and polymerase chain reaction (PCR) using the following indicators: sensitivity (Se), specificity (Sp), positive (PV+) and negative predictive values (PV-), positive (LR+) and negative likehood ratio (LR-)., Results: 332 patients with suspected non-complicated malaria who attended to the MOH health centres were enrolled between October and December 2006. For P. falciparum malaria, Parascreen in comparison with EM, had Se: 53.5%, Sp: 98.7%, PV+: 66.7%, PV-: 97.8%, LR+: 42.27 and LR-: 0.47; and for non-P. falciparum malaria, Se: 77.1%, Sp: 97.6%, PV+: 91.4%, PV-: 92.7%, LR+: 32.0 and LR-: 0.22. The comparison of Parascreen with PCR showed, for P. falciparum malaria, Se: 81.8%, Sp: 99.1%, PV+: 75%, PV-: 99.4, LR+: 87.27 and LR-: 0.18; and for non-P. falciparum malaria Se: 76.1%, Sp: 99.2%, PV+: 97.1%, PV-: 92.0%, LR+: 92.51 and LR-: 0.24., Conclusions: The study results indicate that Parascreen is not a valid and acceptable test for malaria diagnosis under the field conditions found in the Peruvian Amazon. The relative proportion of Plasmodium species, in addition to the genetic characteristics of the parasites in the area, must be considered before applying any RDT, especially after the finding of P. falciparum malaria parasites lacking pfhrp2 gene in this region.

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.

Malaria transmission from humans to mosquitoes is modulated by human host immune factors. Understanding mechanisms by which the human host response may impair parasite infectivity for mosquitoes has direct implications for the development of transmission-blocking vaccines. We hypothesized that despite a low transmission intensity of malaria in the Peruvian Amazon region of Iquitos, transmission-blocking immunity against Plasmodium vivax might be common, given an unexpectedly high proportion of asymptomatic parasitemic individuals in this region. To test this hypothesis, the ability of symptomatic P. vivax malaria patients to experimentally infect wild-caught outbred Anopheles darlingi mosquitoes was tested using the indirect membrane feeding technique. Only half (52/102) of P. vivax parasitemic patients successfully infected mosquitoes. Transmitters were more likely to have gametocytes (OR 6.35, P = 0.003), high parasitemia (OR 3.79, P = 0.024), and, in terms of basic clinical parameters, a slower pulse rate (mean +/- SD: 82.3 +/- 12.3 versus 88.7 +/- 13.5, P = 0.016) than non-transmitters. Log(10) gametocytemia and log(10) real-time reverse transcriptase Pvs25 PCR quantifying gametocytes were significantly and positively correlated with oocyst counts (correlation coefficient 0.505, R2 = 0.26, P = 0.001). These experiments are the first to establish a system of determining transmission patterns in experimental infection of outbred natural neotropical malaria vectors in the Amazon region. Patients with P. vivax inefficiently infect outbred An. darlingi mosquitoes, raising the possibility that some degree of naturally occurring transmission-blocking immunity is present on a population basis in the Peruvian Amazon, an area of low intensity of malaria transmission.

Objective: To determine the sensitivity and specificity of the rapid diagnostic test OptiMAL for diagnosis of Plasmodium vivax malaria., Material and Methods: We included all the patients who sought medical attention in the San Martin Pangoa Hospital, Junin, an area endemic for vivax malaria in Peru, between October and December 1998, who had fever during the previous 72 hours and who were older than 12 months. The gold standard for diagnosis was thick blood film microscopy. We determined the parasitemia rate for each of the positive slides. We calculated sensitivity, specificity, positive predictive value and negative predictive value of the test., Results: We included 72 patients; 39 of them were positive for P. vivax by microscopic examination. The sensitivity of the Optimal test was 92.3%, the specificity 100%, the positive predictive value 100% and the negative predictive value 91.6%. The accuracy of the test was 95.8%. The sensitivity of the OptiMAL test progressively decreased when parasitemia was lower than 1,000 parasites/microliter., Conclusions: the OptiMAL test has a high sensitivity and specificity for diagnosis of P. vivax malaria. However, its sensitivity decreased when parasitemia levels were lower. It is a very simple technique, which makes it a good alternative for malaria diagnosis in remote places, although its elevated cost is still a problem.