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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 theP. vivaxETRAMP11.2 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 ofpvetramp11.2in 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 againstP. vivaxmalaria.Methods: Thepvetramp11.2ofP. vivaxisolates 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 208pvetramp11.2sequences 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 vivaxpopulations 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 inP. vivaxpopulations 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.Graphical abstract

This report describes the MalariaGEN Pv4 dataset, a new release of curated genome variation data on 1,895 samples of Plasmodium vivax collected at 88 worldwide locations between 2001 and 2017. It includes 1,370 new samples contributed by MalariaGEN and VivaxGEN partner studies in addition to previously published samples from these and other sources. We provide genotype calls at over 4.5 million variable positions including over 3 million single nucleotide polymorphisms (SNPs), as well as short indels and tandem duplications. This enlarged dataset highlights major compartments of parasite population structure, with clear differentiation between Africa, Latin America, Oceania, Western Asia and different parts of Southeast Asia. Each sample has been classified for drug resistance to sulfadoxine, pyrimethamine and mefloquine based on known markers at the dhfr, dhps and mdr1 loci. The prevalence of all of these resistance markers was much higher in Southeast Asia and Oceania than elsewhere. This open resource of analysis-ready genome variation data from the MalariaGEN and VivaxGEN networks is driven by our collective goal to advance research into the complex biology of P. vivax and to accelerate genomic surveillance for malaria control and elimination.

GPS data processing for all participants in the study

This report describes the MalariaGEN Pv4 dataset, a new release of curated genome variation data on 1,895 samples of Plasmodium vivax collected at 88 worldwide locations between 2001 and 2017. It includes 1,370 new samples contributed by MalariaGEN and VivaxGEN partner studies in addition to previously published samples from these and other sources. We provide genotype calls at over 4.5 million variable positions including over 3 million single nucleotide polymorphisms (SNPs), as well as short indels and tandem duplications. This enlarged dataset highlights major compartments of parasite population structure, with clear differentiation between Africa, Latin America, Oceania, Western Asia and different parts of Southeast Asia. Each sample has been classified for drug resistance to sulfadoxine, pyrimethamine and mefloquine based on known markers at the dhfr, dhps and mdr1 loci. The prevalence of all of these resistance markers was much higher in Southeast Asia and Oceania than elsewhere. This open resource of analysis-ready genome variation data from the MalariaGEN and VivaxGEN networks is driven by our collective goal to advance research into the complex biology of P. vivax and to accelerate genomic surveillance for malaria control and elimination.

Low- and middle-income countries (LMICs) face significant challenges in the control of COVID-19, given limited resources, especially for inpatient care. In a parallel effort to that for vaccines, the identification of therapeutics that have near-term potential to be available and easily administered is critical. Using the United States (US), European Union (EU), and World Health Organization (WHO) clinical trial registries, we reviewed COVID-19 therapeutic agents currently under investigation. The search was limited to oral or potentially oral agents, with at least a putative anti-SARS-CoV-2 virus mechanism and with at least five registered trials. The search yielded 1,001, 203, and 1,128 trials, in the US, EU, and WHO trial registers, respectively. These trials covered 13 oral or potentially oral repurposed agents that are currently used as antimicrobials and immunomodulatory therapeutics with established safety profiles. The available evidence regarding proposed mechanisms of action, potential limitations, and trial status is summarized. The results of the search demonstrate few published studies of high quality, a low proportion of trials completed, and the vast majority with negative results. These findings reflect limited investment in COVID-19 therapeutics development compared with vaccines. We also identified the need for better coordination of trials of accessible agents and their combinations in LMICs. To prevent COVID-19 from becoming a neglected tropical disease, there is a critical need for rapid and coordinated efforts in the evaluation and deployment of those agents found to be efficacious.

Background Antibody responses as serological markers of Plasmodium vivax infection have been shown to correlate with exposure, but little is known about the other factors that affect antibody responses in naturally infected people from endemic settings. To address this question, we studied IgG responses to novel serological exposure markers (SEMs) of P. vivax in three settings with different transmission intensity. Methodology We validated a panel of 34 SEMs in a Peruvian cohort with up to three years’ longitudinal follow-up using a multiplex platform and compared results to data from cohorts in Thailand and Brazil. Linear regression models were used to characterize the association between antibody responses and age, the number of detected blood-stage infections during follow-up, and time since previous infection. Receiver Operating Characteristic (ROC) analysis was used to test the performance of SEMs to identify P. vivax infections in the previous 9 months. Principal findings Antibody titers were associated with age, the number of blood-stage infections, and time since previous P. vivax infection in all three study sites. The association between antibody titers and time since previous P. vivax infection was stronger in the low transmission settings of Thailand and Brazil compared to the higher transmission setting in Peru. Of the SEMs tested, antibody responses to RBP2b had the highest performance for classifying recent exposure in all sites, with area under the ROC curve (AUC) = 0.83 in Thailand, AUC = 0.79 in Brazil, and AUC = 0.68 in Peru. Conclusions In low transmission settings, P. vivax SEMs can accurately identify individuals with recent blood-stage infections. In higher transmission settings, the accuracy of this approach diminishes substantially. We recommend using P. vivax SEMs in low transmission settings pursuing malaria elimination, but they are likely to be less effective in high transmission settings focused on malaria control., Author summary Plasmodium vivax still poses a threat in many countries due to its ability to cause recurrent infections. Key to achieving the goal of malaria elimination is the ability to quickly detect and treat carriers of relapsing parasites. Failing to identify this transmission reservoir will hinder progress towards malaria elimination. Recently, novel serological markers of recent exposure to P. vivax (SEM) have been developed and validated in low transmission settings. It is still poorly understood what factors affect the antibody response to these markers when evaluated in contrasting endemic contexts. To determine the factors that influence the antibody response to SEM, we compared the antibody levels in three sites with different transmission intensity: Thailand (low), Brazil (moderate) and Peru (high). In this study, we found that transmission intensity plays a key role in the acquisition of the antibody repertoire to P. vivax. In highly endemic sites, it is likely that immunological memory resulting from a constant and sustained exposure will impact the performance of SEMs to detect individuals with recent exposure to P. vivax. In summary, SEMs that perform well in low transmission sites do not perform as well in high transmission regions.

Additional file 1: Supplemental Figure 4. Age and date of clinic visit differences between cases (0,0 on graph) and their matched-controls (depicted as described in legend).

Introducción: En la Amazonía Peruana, la transmisión de la malaria por Plasmodium vivax se mantiene debido a que 1) las recurrencias en esta región son extremadamente comunes; contribuyendo al incremento de los casos submicroscópicos y 2) porque estas infecciones no son detectadas por el Ministerio de Salud, perpetuando así el ciclo de transmisión silente. En este contexto, entender las características y duración de estas infecciones recurrentes submicroscópicas nos permitiría comprender su impacto en la persistencia de la malaria. Métodos: Se realizó un análisis retrospectivo de datos clínicos y epidemiológicos mensuales de dos comunidades de Loreto (Cahuide y Lupuna) desde el año 2013 al 2015. Los participantes proporcionaron muestras de sangre para el diagnóstico de la malaria a través de microscopía y PCR (método molecular). Para determinar la probabilidad de permanecer libre de recurrencias se realizó análisis de supervivencia Kaplan-Meier. Asimismo, se buscó la asociación de factores sociodemográficos con el tiempo de detección a través de análisis de regresión de Cox. Resultados: Durante los tres años de seguimiento, cerca del 57% (195/343) de los individuos presentó al menos una recurrencia detectada por PCR, mientras que a través de microscopía este número se reduce al 42% (143/343). Alrededor del 30% (93/343) de los participantes lo presentó en los tres primeros meses luego de una infección microscópica. La probabilidad de permanecer libre de recurrencias, luego de 6 meses después de una infección, fue 66% por microscopia, y de 62.6% por PCR. A su vez, el tiempo de detección se encontró asociado a factores como comunidad, edad, número de episodios de malaria en su vida y tipo de mosquiteros utilizados. Conclusión: Esta información es relevante para que el actual programa “Plan Malaria Cero” del Ministerio de Salud pueda diseñar mejores estrategias enfocando sus recursos en la búsqueda activa de infecciones submicroscópicas de malaria. 2021-09-15

Additional file 1: Table S1. Knowledge of household heads on malaria. Table S2. Attitudes of household heads on malaria. Table S3. Malaria prevention practices among household heads. Table S4. Malaria episodes and household by basin. Table S5. Direct cost from the provider perspective for uncomplicated malaria by P. vivax in Mazan district 2017 (US$). Table S6. Direct cost from the provider perspective for uncomplicated malaria by P. falciparum in Mazan district 2017 (US$). Table S7. Direct and indirect costs from the patient perspective for uncomplicated malaria by P. vivax in Mazan district 2017 (US$). Table S8. Direct and indirect costs from the patient perspective for uncomplicated malaria by P. falciparum in Mazan district 2017 (US$).

Background: Plasmodium vivax remains a challenge in Peru. The high heterogeneity of its transmission has limited the intervention measures based on global and generalized epidemiological information at medium and long term. Population genetic tools may provide information on the spatial heterogeneity of transmission by linking parasite genetic patterns with epidemiological information. Methods: The genetic diversity and population structures from three settlements located in the Peruvian Amazon, Cahuide (CAH), Lupuna (LUP) and Santa Emilia (STE) were measured on samples collected from September 2012 to March 2015. Results: A total of 777 P. vivax mono- infections were genotyped. LUP showed the lowest proportion of polyclonal infections (0.197) and genetic diversity (Hexp 0.544 ± 0.0012). Moreover, in this study area results showed a significant increment of polyclonal infections and Hexp (p < 0.005), and the introduction and persistence of a new parasite population from March 2014 onward. STE showed the highest proportion of polyclonal infections (0.401) and Hexp (0.596 ± 0.0028), the presence of 4 genetic clusters without signals of clonal expansion and infections with lower parasite densities compared to the other two areas. Even though CAH showed the introduction of at least 4 parasite populations in 2012, a clear reduction of cases (from 213 to 61), a subtle reduction of polyclonal infections (from 0.286 to 0.18), and an unpredicted behavior of Hexp were seen since June 2014. Conclusion: These results suggest that LUP and mainly STE are two areas that maintain basal levels of transmission, and that there are factors that allow the reintroduction of P. vivax. In contrast, malaria cases in CAH are circumstantial, thus, receptivity to malaria transmission and vulnerability to the importation of new parasite population vary drastically among the different study areas analyzed, this information must be considered in the design of current control strategies NIH-NIAID 2019-09-05

Peru is an upper-middle-income country, but a large part of the population is still unable to benefit from such economic growth, with poverty rates exceeding 40% in rural areas, mainly in the Andean and the Amazon Regions. Malaria remains a significant public health problem in the Peruvian Amazon, mainly in Loreto department where both P.vivax and P. falciparum coexist (ratio: 5/1), accounting for more than 95% of Peru’s malaria cases in 2017 (52,280 cases). 2019-09-05

Background: Faced with the resurgence of malaria in the Peruvian Amazon since 2012, malaria surveillance was enhanced by adding a local active case detection (ACD) strategy to the routine passive case detection (PCD) as a reactive measure in selected communities with unusual number of cases during high transmission season (HTS). We assessed the short-effectiveness of this local ACD-based strategy in four Peruvian Amazonian riverine communities. Methods: A cohort study was conducted in June-July 2015 in Mazan, Loreto. Four consecutive ACD interventions (using light microscopy (LM) and treatment of confirmed infections) at intervals of 10 days were conducted in four riverine communities (Gamitanacocha-GAM, Primero de Enero-PRI, Libertad-LIB, and Urco Miraño-URC) that previously reported unusual number of cases. Blood samples were also collected on filter paper for later PCR. The effectiveness of the local strategy was calculated by dividing the number of infections detected in ACD visits using LM by the total number infections identified by PCR. Results: Of the total 933 censused people in the area, 88.1% were found in at least one of the four ACD visits. 54.2% had confirmed infections by PCR; among them 5 had two infections during the study period. Most infections were asymptomatic (270/451, 59.9%) and due to P. vivax (360/451, 79.8%). P. falciparum infections and P. vivax-P. falciparum co-infections accounted for 15.3% and 4.9% of total infections. Only 22.8% of the total 451 PCR confirmed infections were accurately detected using LM during the four ACD visits. Children aged 5-14 years, and farming as main economic activity were associated with P. vivax infections. Conclusion: Although repeated and consecutive ACD interventions using LM increase the opportunity of detecting and treating malaria infections during HTS, the number of detected infections would be far from the “true” number of infections detected by a highly sensitive diagnostic test. 2019-09-05

Supplementary Methods and Results. (DOCX 24 kb)

Although there were a reduction in malaria burden, with 30% fewer cases in 2018 than in 2014, malaria remains a major public health problem in Peru, mainly in the Amazon region where over 44,000 cases were reported in 2018 (96% of cases in the country). Plasmodium vivax is the predominant species, representing 75.5% of malaria cases, similar to the rest of the region of the Americas. P. vivax infections are characterized by frequent relapses due to persistent liver dormant parasites. Previous studies of our research group in the Peruvian Amazon have shown high proportion of recurrence after radical cure (~75% in one year of follow-up [1]). This study aimed to describe the evolution of parasitemia after a supervised radical cure treatment in an endemic rural village in the Peruvian Amazon follow-up by both, molecular tools (qPCR) and thick smear, during one month. Instituto Hipólito Unanue 2019-09-05

Figure S1. Log transformed virulence factor RNA transcript expression in baseline cultures analyzed by grouping strains according to LRV-1 status compared by Mann-Whitney for the following targets: pooled expression (A), cpb (B), gp63 (C), mpi (D), hsp23 (E), hsp70 (F), hsp90 (G) and hsp100 (H). Figure S2. Log transformed virulence factor RNA transcript expression in supernatants post-macrophage infectivity at 24 and 48 h compared by LRV-1 status using t-test for the following targets: cpb-24 h (A), mpi-24 h (B), hsp70–24 h (C), pooled-48 h (D), cpb-48 h (E), and mpi-48 h (F). Figure S3. Log transformed virulence factor RNA transcript expression in baseline cultures analyzed by grouping strains according to species (L. V. panamensis versus other) compared by Mann-Whitney for the following targets: pooled expression (A), cpb (B), gp63 (C), mpi (D), hsp23 (E), hsp70 (F), hsp90 (G) and hsp100 (H). Figure S4. Log transformed virulence factor RNA transcript expression in supernatants post-macrophage infectivity at 24 and 48 h compared by species using t-test for the following targets: pooled VF-24 h (A), cpb-24 h (B), pooled VF-48 h (C), and cpb-48 h (D). Figure S5. Log transformed virulence factor RNA transcript expression in baseline cultures analyzed by grouping strains according to source of cultured isolate (ATCC® versus clinical) compared by Mann-Whitney for the following targets: pooled expression (A), cpb (B), gp63 (C), mpi (D), hsp23 (E), hsp70 (F), hsp90 (G) and hsp100 (H). Figure S6. Log transformed virulence factor RNA transcript expression in supernatants post-macrophage infectivity at 24 and 48 h compared by source of cultured isolate using t-test for the following targets: pooled VF-24 h (A), pooled VF-48 h (B), cpb-48 h (C), and mpi-48 h (D). (DOCX 1229 kb)

Plasmodium vivax es un desafío para la eliminación de la malaria. Esta especie cuenta con un estadío hepático latente (Hipnozoíto) que mantiene la transmisión de la enfermedad. El proyecto SEROVIVAX tiene como objetivo contribuir a la eliminación de la malaria en la región amazónica a través del desarrollo y validación de herramientas serológicas que permitan identificar la exposición individual reciente a P. vivax y monitorear los cambios en la intensidad de transmisión. Usando microarreglos de proteínas se evaluó las respuestas de anticuerpos a 507 proteínas de P. vivax en 207 individuos infectados en los últimos 36 meses y confirmados por qPCR (P. vivax positivos). El área bajo la curva (AUC), sensibilidad (SE), especificidad (SP), razones de probabilidad positiva (LR+) y negativa (LR-) fueron calculados a partir de un modelo de clasificación validados, a fin de evaluar la precisión de la respuesta antígeno-anticuerpo que discriminen entre infecciones recientes y antiguas. Se identificaron tres grupos de proteínas que funcionaron mejor individualmente y en combinación; con un alto AUC, alto LR+ y bajo LR-. Individualmente, destacaron dos proteínas: PVX_082680_1o1 (AUC: 0.65, LR +: 1.42, LR-: 0.49, SE: 0.77, SP: 0.45) y PVX_000930_1o1 (AUC: 0.64, LR +: 1.23, LR- : 0,52, SE: 0,83, SP: 0,33). En combinación, dos proteínas que formaron parte de las diez principales proteínas identificadas con mayor frecuencia en cada grupo (alto AUC: 0.82, alto LR+: 2.9, SE: 0.75, SP: 0.74 y bajo LR-: 0.24, SE: 0.85, SP: 0.62). Otras proteínas destacadas fueron PVX_097625_1o1 (proteína de superficie de merozoito 8, MSP-8), PVX_092070_1o1 y PVX_082655_1o1 y PVX_096280_1o1 y PVX_123810_2o2_S2. Se lograron identificar 6 proteínas con un gran potencial que serán producidas y validadas para el uso en herramientas serológicas que permitan el monitoreo del control y eliminación de la malaria en la Amazonía peruana y brasileña. 2019-09-05

Plasmodium vivax is the main cause of malaria in the Peruvian Amazon and Mazan is considered one of its districts with high risk of malaria transmission. In this site, previous studies has described P. vivax transmission as heterogenic and has suggested that its prevalence is influenced by factors linked to human mobilization. However, the contribution of this factor have not been assessed yet. Population genetic tools provide information on the individual contribution of the factors that favor the transmission and evaluate the demographic patterns of dispersion of the parasite populations which are influenced by the human mobilization. In order to evaluate contribution of human mobilization to malariatransmission in Mazan, in this work we use a Bayesian framework to identify genetic sub-populations in each basin and to discriminate between endogenous and imported cases. Socio-demographic and clinical- epidemiological factors associated with the expansion of specific genetic sub- populations, the spatial distribution at a micro-geographical level of different genetic population parameters and the effects of the introduction of new genetic parasite populations on the clinical manifestations of the susceptible human population are also described. Hacia la eliminación de la malaria en el Perú UCSD - UPCH Global Infectious Diseases Training Programme: Endemic Infectious Diseases of the Peruvian Amazon AMAZONIA-International Center of Excellence for Malaria Research Joint project to strengthen research skills on molecular epidemiology and to uncover malaria transmission features relevant for its control in the Peruvian Amazon 2019-09-05

Summary Background DSM265 is a novel, long-duration inhibitor of plasmodium dihydroorotate dehydrogenase (DHODH) with excellent selectivity over human DHODH and activity against blood and liver stages of Plasmodium falciparum. This study aimed to assess the efficacy of DSM265 in patients with P falciparum or Plasmodium vivax malaria infection. Methods This proof-of-concept, open-label, phase 2a study was conducted at the Asociación Civil Selva Amazónica in Iquitos, Peru. Patients aged 18–70 years, weighing 45–90 kg, who had clinical malaria (P falciparum or P vivax monoinfection) and fever within the previous 24 h were eligible. Exclusion criteria were clinical or laboratory signs of severe malaria, inability to take oral medicine, and use of other antimalarial treatment in the preceding 14 days. Patients were divided into cohorts of those with P falciparum (cohort a) or P vivax (cohort b) infection. Two initial cohorts received single oral doses of 400 mg DSM265. Patients were followed up for efficacy for 28 days and safety for 35 days. Further cohorts received escalated or de-escalated doses of DSM265, after safety and efficacy assessment of the initial dose. The primary endpoints were the proportion of patients achieving PCR-adjusted adequate clinical and parasitological response (ACPR) by day 14 for patients infected with P falciparum and the proportion of patients achieving a crude cure by day 14 for those infected with P vivax. Cohort success, the criteria for dose escalation, was defined as ACPR (P falciparum) or crude cure (P vivax) in at least 80% of patients in the cohort. The primary analysis was done in the intention-to-treat population (ITT) and the per-protocol population, and safety analyses were done in all patients who received the study drug. This study is registered at ClinicalTrials.gov (NCT02123290). Findings Between Jan 12, 2015, and Dec 2, 2015, 45 Peruvian patients (24 with P falciparum [cohort a] and 21 with P vivax [cohort b] infection) were sequentially enrolled. For patients with P falciparum malaria in the per-protocol population, all 11 (100%) in the 400 mg group and eight (80%) of ten in the 250 mg group achieved ACPR on day 14. In the ITT analysis, 11 (85%) of 13 in the 400 mg group and eight (73%) of 11 in the 250 mg group achieved ACPR at day 14. For the patients with P vivax malaria, the primary endpoint was not met. In the per-protocol analysis, none of four patients who had 400 mg, three (50%) of six who had 600 mg, and one (25%) of four who had 800 mg DSM265 achieved crude cure at day 14. In the ITT analysis, none of five in the 400 mg group, three (33%) of nine in the 600 mg group, and one (14%) of seven in the 800 mg group achieved crude cure at day 14. During the 28-day extended observation of P falciparum patients, a resistance-associated mutation in the gene encoding the DSM265 target DHODH was observed in two of four recurring patients. DSM265 was well tolerated. The most common adverse events were pyrexia (20 [44%] of 45) and headache (18 [40%] of 45), which are both common symptoms of malaria, and no patients had any treatment-related serious adverse events or adverse events leading to study discontinuation. Interpretation After a single dose of DSM265, P falciparum parasitaemia was rapidly cleared, whereas against P vivax, DSM265 showed less effective clearance kinetics. Its long duration of action provides the potential to prevent recurrence of P falciparum after treatment with a single dose, which should be further assessed in future combination studies. Funding The Global Health Innovative Technology Fund, the Bill & Melinda Gates Foundation, the National Institutes of Health (R01 AI103058), the Wellcome Trust, and the UK Department of International Development.

BACKGROUND: Serological markers have been proposed as alternative tools to monitor and assess malaria intensity in unstable and low transmission settings. It is recommended to use multiple antigens for optimizing the sensitivity of the antibody detection against Plasmodium species and determining past malaria exposure. This work aimed to evaluate the antibody reactivity against a novel chimeric protein containing the Apical Membrane Antigen 1 (AMA-166) ectodomain and the 19 kDa carboxyl fragment of the Merozoite Surface Protein 1 (MSP119) of. Plasmodium vivax (PvAMA166-MSP119) on dried blood samples (DBS) from individuals living in the Peruvian Amazon. MATERIALS AND METHODS: A total 823 DBS on filter paper were collected from individuals living in the Mazan district, and analyzed using indirect enzyme-linked immunosorbent assay (ELISA) to measure P. vivax immunoglobulin G (IgG) responses. Three antigens were used in the analysis: PvAMA1, PvMSP119, and their chimaera. To ensure a standardization of the sample results across ELISA plates, the percent positivity (PP) of each sample was calculated using the OD of the positive control serum as 100%. Then, the cut-off of PPs for seropositivity (indicating malaria exposure) was generated using a mixture model. Kappa (k) was used to evaluate agreement between serological results. RESULTS AND CONCLUSIONS: The proportion of seropositivity according to the antigen used was: 27.2% (n=224) for PvMSP119, 33.7% (n=277) for PvAMA1, and 33.9% (n= 279) for PvAMA166-MSP119. Seropositivity to any of both independent antigens (PvMSP119 and/or PvAMA1) was 39.6% (n=326). An almost perfect agreement (k=0.867, p

Additional file 2. The percentage of intermediate metabolizers by treatment. The percentage of subjects who were intermediate metabolizers (IM) with error bars representing the standard error of the mean.

Additional file 1. The percentage of intermediate metabolizers (IM) by country. The percentage of subjects who were intermediate metabolizers (IM) is shown with error bars representing the standard error of the mean. The three subjects from India are not shown as all were extensive metabolizers (EMs).

Objectives. Explore a new target for molecular diagnosis of Leishmania. Materials and methods. We evaluated the utility of the gene that encodes the heat shock protein 20-kDa (Hsp20) for detecting Leishmania by polymerase chain reaction (PCR). PCR was normalized and analytical parameters were determined, as well as the validity and diagnostic accuracy, and concordance with the PCR - 18S. PCR-Hsp20 with DNA was obtained from a group of clinical samples from different sources. Results. The analytical parameters were adequate. The sensitivity obtained was 86% and the specificity was 100%. The concordance with the reference method was good (Ƙ = 0.731), which supports its potential use for diagnosis. The possibility of subsequent identification of the species by sequencing the amplified product gives an additional advantage. Conclusions. The usefulness of this gene as a new target for the detection of Leishmania was demonstrated. Because of its potential, it is recommended to improve the sensitivity of the method and to evaluate it in different endemic regions. Objetivos. Explorar una nueva diana para el diagnóstico molecular de Leishmania. Materiales y métodos. Se evaluó la utilidad del gen que codifica la proteína de choque térmico de 20kDa (hsp20) para la detección de Leishmania por medio de la reacción en cadena de la polimerasa (PCR).Se normalizó la PCR y se determinaron los parámetros analíticos, así como la validez y seguridad diagnóstica y la concordancia con la PCR-18S. Se evaluó la PCR-hsp20 con ADN obtenido de un grupo de muestras clínicas de distinta procedencia. Resultados. Los parámetros analíticos resultaron adecuados. La sensibilidad obtenida fue de 86% y la especificidad del 100%, la concordancia con el método de referencia resultó buena (ƙ=0,731), lo que apoya su posible uso para el diagnóstico. La posibilidad de identificación posterior de la especie mediante secuenciación del producto amplificado le confiere una ventaja adicional. Conclusiones. Se demuestra la utilidad de este gen como una nueva diana para la detección del género Leishmania. Debido a su potencial, se recomienda mejorar la sensibilidad del procedimiento y realizar su evaluación en diversas regiones endémicas.

Objectives. To assess the competency of microscopists for malaria diagnosis using standardized slide sets in the Peruvian Amazon. Material and methods. Cross-sectional study carried out in 122 first level health facilities of the Peruvian Amazon, between July and September 2007. Within the frame of the project “Control Malaria in the border areas of the Andean Region: A community approach” (PAMAfRO), we evaluated the malaria diagnosis performance in 68 microscopists without expertise ( 1 year of expertise) and 76 microscopists with expertise ( 1 year) using standardized sets of 20 blood smear slides according to the World Health Organization (WHO) recommendations. A correct diagnosis (correct species identification) was defined as “agreement”, a microscopist was qualified as an “expert” if they have an agreement ≥90% (≥ 18 slides with correct diagnosis), as a “referent” with an agreement between 80% and 90%, “competent” if they are between 70 and 80% and “in training” if they have 70%. Results. Microscopists with expertise (68.6%) had more agreement than those without expertise (48.2%). The competency assessment was acceptable (competent, referent, or experts levels) in 11.8% of the microscopists without expertise and in 52.6% from those with expertise. The agreement was lower using blood smear slides with P. falciparum with low parasitaemia, with P. malariae and with mixed infections. Conclusions. Is the first assessment, we found only one of three microscopists from the Peruvian Amazon is competent fro malaria diagnosis according to the WHO standards. from this baseline data, we have to continue working in order to improve the competency assessment of the microscopists within the frame of a quality assurance system. Objetivos. Evaluar la competencia de los microscopistas en el diagnóstico de la malaria mediante paneles de láminas estandarizados en la Amazonía peruana. Materiales y métodos. Estudio transversal, realizado entre los meses de julio y septiembre de 2007, en 122 establecimientos de salud de primer nivel de atención de la Amazonía peruana. En el marco del Proyecto PAMAfRO, se evaluó las competencias en el diagnóstico de malaria en 68 microscopistas sin experiencia (un año), utilizando paneles estandarizados de 20 láminas siguiendo las recomendaciones de la Organización Mundial de la Salud (OMS). Se definió concordancia como el porcentaje de aciertos que tuvo el microscopista en los diagnósticos (identificación de especie del parásito). Un microscopista con concordancia ≥90% (≥18 láminas acertadas) fue definido como “experto”, 80 a 90% “referente”, 70 a 80% “competente” y 70% “en entrenamiento”. Resultados. Los microscopistas con experiencia (68,6%) tuvieron mayor concordancia que aquellos sin experiencia (48,2%). La competencia fue aceptable (niveles competente, referente o expertos) en 11,8% de los microscopistas sin experiencia y en 52,6% de aquellos con experiencia. La concordancia fue menor para láminas con P. falciparum con parasitemia baja, con P. malariae y con infecciones mixtas. Conclusiones. En esta primera evaluación, se evidenció que solo uno de cada tres microscopistas de la amazonía peruana es competente para el diagnóstico de malaria según estándares de la OMS. A partir de esta línea de base, se debe mejorar las competencias de los microscopistas en el marco de un sistema de aseguramiento de la calidad.

We present a short summary of the root causes that contribute to global warming and a host of evidence of the reality that affects us; such as: raising the temperature of the earth, melting glaciers, rising ocean level, increases the frequency and intensity of weather events, all as a result of the unusual accumulation of greenhouse gases, as product of human activity. There are implications that directly or indirectly, the climate change has to health in particular for Andean countries; such as: disorders linked to the availability and quality of water and food, respiratory disease, vector-borne infections, cancer and pathologies chronic degenerative tables associated with climatic disasters and extreme temperatures. finally we review proposals and courses of action. Se presentan de manera resumida las causas fundamentales que contribuyen al calentamiento global y una serie de evidencias de la realidad que nos afecta: aumenta la temperatura de la tierra, se derriten los glaciares, sube el nivel de los océanos y se incrementa la frecuencia e intensidad de los eventos meteorológicos; todo ello como producto de la acumulación inusitada de gases de efecto invernadero, provenientes de la actividad humana. Se plantea las implicaciones que, de forma directa o indirecta, el cambio climático tiene para la salud, en particular para los países andinos: trastornos vinculados con la disponibilidad y calidad del agua y los alimentos, afecciones respiratorias, infecciones de transmisión vectorial, cáncer y enfermedades crónico degenerativas, cuadros asociados con desastres climáticos y temperaturas extremas. finalmente, se revisa las propuestas y cursos de acción.

The Authors analyse the histopathological alterations observed in 378 cases of Mucocutaneous Leishmaniasis from Três Braços, Bahia, of whom 307 (81,4%) were only cutaneous, 54 (14,2%) only mucosae and in 17 (4,4%) both integuments were involved simultaneously. A cellular infiltration of lymphocytes and plasma cells was invariably present in all forms and during healing. In mucosal cases, this reaction may have an important role in maintaining an auto agression in the presence of few or no parasites. The plasma cell is a constant ele-ment in well developed lesions but not present in healing lesions. Its presence almost always denotes a tendancy to relapse. Mast cells were observed in both cutaneous and mucosal lesions but predominate in the former. Its number was significantly greater in those with an exudative and necrotic granulomatous reaction where necrotic phenomena are marked. Eosinophils were significantly associated with mast cells suggesting the existence of association between these cells in the modulation of the inflammatory response. Two types of granulomatous reaction were observed: a disorganized one, often related to tissue necrosis, and an organized, tuberculoid type granuloma. The first type is interpreted as of post necrotic origin resulting in a reduction in antigenic load with maintenance of antibody levels, creating the conditions to establish a granuloma similar to that observed in immunocomplex lesions with excess of antibody. The other type of rection was the granuloma of epithelioid cells, which tend to arise in two groups of patients. In young patients, with short time cutaneous lesions and normal positive leishmanin skin tests, this type of granuloma perhaps similar to the specific hypersensibility granuloma described by EPSTEIN (1977). In the other group of patients, the leishmanin skin test was always exaggerated. In these cases the hypersensitive granuloma is associated with a cellular hypersensivity, perhaps amplified by sequestration of antigen, reforcing the antigenic stimulus of the granuloma. As a result, treat- ment of this group is more difficult and the prognosis worse in consequence. Os A.A. analisam as alterações histopatológicas observadas em 378 casos de Leishmaniose Tegumentar da localidade de Três Braços Estado da Bahia, dos quais 307 eram de portadores de lesões exclusivamente cutâneas, 54 de portadores de lesões exclusivamnte mucosas e 17 de portadores de lesões cutâneo-mucosas. A infiltração histiolinfoplasmocitária, na maioria dos casos, parece desempenhar o papel de resposta celular inespecífica à presença de um irritante tecidual, porém, nos casos de forma mucosa, não se pode afastar a possibilidade de que esse infiltrado esteja participando de uma reação de tipo autoagressivo. O plasmócito constitui um elemento quase constante nas lesões desenvolvidas, mas não tem sido observado nas lesões residuais, quer em via de cura ou já cicatrizadas; sua presença nestes casos denota, quase sempre, tendência à recidiva. Os mastdcitos foram observados em lesões tanto da forma cutânea como da forma mucosa, mas predominavam nas primeiras. Seu número foi significantemente maior no padrão de Reação Exsudativa e Neerótico Granulomatosa, onde os fenômenos necróticos são bem desenvolvidos. Os eosinófilos apresentaram associação significativa com os mastócitos, confirmando a existência de um eixo bidirecional entre estás duas células, o qual deve participar da modulação inflamatória, na Leishmaniose Tegumentar. Dois tipos de reação granulomatosa foram observados: um desorganizado, em relação, muitas vezes, com a necrose tissular, e outro organizado, mais raro, do tipo tuberculóide. O primeiro foi interpretado como de origem pós-necrótica, surgindo com a redução da carga parasitária, propiciada pelos fenômenos necróticos: eliminado o antígeno e mantidos os níveis de anticorpos, surgem as condições necessárias ao estabelecimento do granuloma, semelhante àquele observado nas lesões por imunocomplexo em excesso de anticorpos. O outro tipo de reação foi o granuloma de células epiteliódes, que surgiu em dois grupos de pacientes. Nos pacientes jovens, com doença de curto tempo de evolução e intradermorreação não exacerbada, este tipo de granuloma talvez seja a expressão da Hipersensibilidade Granulomatosa Específica, descrita por EPSTEIN (1977). No outro grupo de pacientes, havia em todos intradermorreação exacerbada. Nestes casos a hipersensibilidade granulomatosa, associando-se ã hipersensibilidade mediada por células — agora ampliada pelo seqüestro do antígeno —, reforçaria o processo granulomatoso, através da reverberação do estímulo antígênico; isso tornaria o tratamento mais difícil e pior o prognóstico para o caso.

The Authors analysed the humoral response in biopsy material from 90 patients with Mucocutaneous Leishmaniasis caused by Leishmania braziliensis braziliensis, utilising the immunoperoxidase method to identify IgA, IgG, IgM, C3 complement fraction and fibrin in the tissue. The presence of IgG, IgA and IgM was found in tissue plasma cells with a predominance of IgG and this was correlated significantly with the number of plasma cells in the lesion. The presence of immunoglobulins in the tissues stimulated the opsonisation of parasites and/or their antigens resulting in necrosis which represents one of the effective mechanisms to reduce parasite load. In ne- crotic areas and the walls of inflammed vessels immunoglobulins were deposited as well as the C3 fraction of complement and fibrin — immunocomplex fractions derived from the host. This tissue necrosis was interpreted as the result of the action of immunocomplexes in a region with equivalent or a smally excess of antigen (Arthus type reaction). The presence of parasite antigens expressed on the macrophage membrane in the initial phase of the lesion when in contact with tissue immunoglobulins, leads to the installation of an antigen-antibody reaction resulting in the appearance of necrosisin Mucocutaneous Leishmaniasis. Os A.A. analisaram a resposta humoral nas lesões de 90 pacientes de Leishmaniose Tegumentar — causada por Leishmania braziliensis brasiliensis —, utilizando o método da imunoperoxidase para identificar nos tecidos a presença de IgA, IgG, IgM, fração C3 do complemento e fibrina. Constataram a presença de IgA, IgC e IgM nos plasmócitos tissulares, com predomínio de IgG. Admitiram aue a passagem dessas imunoglobulinas para os tecidos possibilitando a opsonização do parasites e/ou de seus antígenos, permitiria a ocorrência de fenômenos necróticos que representam um dos mecanismos eficazes de redução da carga parasitária. Efetivamente, nas áreas de necrose e nas paredes dos vasos inflamados identificaram depósito de imunoglobulinas, fração C3 do complemento e fibrina — elementos do hospedeiro que fazem parte dos imunocomplexos. Interpretaram essa necrose tissular como o resultado da ação de imunocomplexos na região de equivalência ou com discreto excesso de antígenos ítipos ABTHTJS). A presença de antígenos parasitários, expressos nas membranas dos macrófagos quando em contato com imunoglobulinas tissulares, na fase inicial da lesão, possibilitaria a instalação de uma reação antígeno-anticorpo, a qual explicaria o aparecimento da necrose na Leishmaniose Tegumentar.

The Authors describe the histopathological findings observed in biopsies from 378 cases of Mucocutaneous Leishmaniasis, caused by Leishmania braziliensis braziliensis. All the patients lived in the locality of Três Braços, State of Bahia, an endemic area of Leishmaniasis in Brazil. Parasites were found in 63.7% of patients with cutaneous form and in 35.7% of cases with mucous lesions, although always in reduced number. Histopathologic findings allowed the caracterization of five different patterns: 1. Exsudative-cellular reaction, due to infiltraction of histiocytes, lymphocytes and plasm cells; 2. Exsudative-necrotic reaction, characterized by the association of necrosis with cellular infiltration; 3. Exsudative and necrotic-granulomatous reaction, corresponding to pattern described as chronic-granulomatous inflammation with necrosis; 4. Exsudativegranulomatous reaction, characterized a disorganized granulomatous reaction, without tissue necrosis; 5. Exsudative-tuberculoid reaction — in which a typical tuberculoid granuloma is formed. The follow-up study in 49 patients showed, in sucessive biopsies, changes in the histopathologic patterns in 63.2% of cutaneous forms and in 45.4% of the mucous forms. It was concluded that the exsudative-cellular reaction represents both the inicial and final pattern of Mucocutaneous Leishmaniasis lesion. The other four histopathologic patterns characterize intermediate forms found during the evolution of the disease. Os autores analisam material de biópsias de 378 casos de Leishmaniose Tegumentar, causada por Leishmania braziliensis braziliensis, da localidade endêmica de Três Braços (Estado da Bahia). O parásitos, embora escassos, foram encontrados em 63,7% dos casos da forma cutânea e em 37,5% dos casos da forma mucosa. As alterações dérmicas ou do córion da mucosa permitiram identificar cinco padrões histopatológicos: 1) Reação Exsudativa Celular, constituída por um infiltrado histiolinfoplasmocitário; 2) Reação Exsudativa e Necrótica, na qual ocorre uma necrose no seio do infiltrado inflamatório; 3) Reação Exsudativa e Necrótico-Granulomatosa, que corresponde ao quadro descrito como inflamação crônica granulomatosa com necrose; 4) Reação Exsudativa e Granulomatosa, onde se observa uma reação granulomatosa desorganizada, sem que esteja presente necrose tecidual; 5) Reação Exsudativa e Tuberculóide, caracterizada pelo granuloma tuberculóide. O estudo evolutivo realizado em 49 casos, mostrou que houve uma mudança de padrão histopatológico observada, em biópsias sucessivas, em 63,2% dos casos da forma cutânea e em 45,4% dos casos da forma mucosa. Através desse estudo, é possível afirmar-se que o padrão de Reação Exsudativa Celular constitui o quadro inicial e final da lesão, com os demais padrões aparecendo interposto durante a evolução da doença.

Additional file 1: Table S1. Baseline socio-demographic characteristics of study participants by villages. Table S2. Baseline household characteristics of study participants by villages.

Additional file 1: Table S1. Baseline socio-demographic characteristics of study participants by villages. Table S2. Baseline household characteristics of study participants by villages.

Despite the decline in its incidence in the past decade, malaria remains an important public health problem in Peru. A challenge faced by the National Malaria Control Programme (NMCP) is the ability to efficiently target and eliminate the remaining parasite reservoir in endemic areas, where malaria transmission is low, seasonal and increasingly focal. Two cross-sectional surveys that combined parasitological (PCR, microscopy) and serological metrics (seroprevalence, seroconversion rates) were conducted to characterize malaria transmission dynamics in low endemic areas of the Pacific northern coast and the Amazon region in Peru. Besides the serological evidence for high heterogeneity in malaria exposure, both to Plasmodium falciparum and Plasmodium vivax, at micro-geographical level and over time, PCR results confirmed that most malaria infections in the study areas were asymptomatic and sub-microscopic, and therefore unlikely to be detected by the routine passive case detection (PCD). Since the NMCP has recently introduced focal screening and treatment (FSAT) to overcome the limitations of PCD for detecting asymptomatic carriers, a modelling approach was developed to assess the potential impact of this strategy on the P. falciparum transmission in the Amazon region and to provide insights for the best criteria for its implementation. Although model predictions suggest that combined interventions enabled to reach elimination levels, the progression rates to those levels varied substantially according to different operational criteria (timing, duration, diagnostic tool) used for the intervention. The uncovered asymptomatic and sub-patent reservoir calls for an immediate shift in the malaria control policy in Peru from detecting and controlling clinical malaria to targeting and eliminating all malaria infections (both asymptomatic and symptomatic). The combination of molecular and serological tools have repeatedly proved their added value in accurately understanding the local malaria epidemiology, determining risk factors, describing spatial heterogeneity in transmission, detecting malaria hotpots for targeted interventions as FSAT, and identifying recent changes in malaria transmission in the study areas. (SP - Sciences de la santé publique) -- UCL, 2015