Monoamine oxidase-A (MAO-A) low-expression variants and increased risk of Plasmodium vivax malaria relapses.

OBJECTIVES: Primaquine is essential for the radical cure of Plasmodium vivax malaria and must be metabolized into its bioactive metabolites. Accordingly, polymorphisms in primaquine-metabolizing enzymes can impact the treatment efficacy. This pioneering study explores the influence of monoamine oxidase-A (MAO-A) on primaquine metabolism and its impact on malaria relapses. METHODS: Samples from 205 patients with P. vivax malaria were retrospectively analysed by genotyping polymorphisms in MAO-A and cytochrome P450 2D6 (CYP2D6) genes. We measured the primaquine and carboxyprimaquine blood levels in 100 subjects for whom blood samples were available on the third day of treatment. We also examined the relationship between the enzyme variants and P. vivax malaria relapses in a group of subjects with well-documented relapses. RESULTS: The median carboxyprimaquine level was significantly reduced in individuals carrying low-expression MAO-A alleles plus impaired CYP2D6. In addition, this group experienced significantly more P. vivax relapses. The low-expression MAO-A status was not associated with malaria relapses when CYP2D6 had normal activity. This suggests that the putative carboxyprimaquine contribution is irrelevant when the CYP2D6 pathway is fully active. CONCLUSIONS: We found evidence that the low-expression MAO-A variants can potentiate the negative impact of impaired CYP2D6 activity, resulting in lower levels of carboxyprimaquine metabolite and multiple relapses. The findings support the hypothesis that carboxyprimaquine may be further metabolized through CYP-mediated pathways generating bioactive metabolites that act against the parasite.

Cooperation for malaria control and elimination in the Guiana Shield.

The Guiana Shield, a small region of South America, is currently one of the main hotspots of malaria transmission on the continent. This Amazonian area is characterised by remarkable socioeconomic, cultural, health, and political heterogeneity and a high degree of regional and cross-border population mobility, which has contributed to the increase of malaria in the region in the past few years. In this context, regional cooperation to control malaria represents both a challenge and an indispensable initiative. This Viewpoint advocates for the creation of a regional cooperative mechanism for the elimination of malaria in the Guiana Shield. This strategy would help address operational and political obstacles to successful technical cooperation in the region and could contribute to reversing the regional upsurge in malaria incidence through creating a functional international control and elimination partnership.

Delayed gametocyte clearance in Plasmodium vivax malaria is associated with polymorphisms in the cytochrome P450 reductase (CPR).

Primaquine (PQ) is the main drug used to eliminate dormant liver stages and prevent relapses in Plasmodium vivax malaria. It also has an effect on the gametocytes of Plasmodium falciparum; however, it is unclear to what extent PQ affects P. vivax gametocytes. PQ metabolism involves multiple enzymes, including the highly polymorphic CYP2D6 and the cytochrome P450 reductase (CPR). Since genetic variability can impact drug metabolism, we conducted an evaluation of the effect of CYP2D6 and CPR variants on PQ gametocytocidal activity in 100 subjects with P. vivax malaria. To determine gametocyte density, we measured the levels of pvs25 transcripts in samples taken before treatment (D0) and 72 hours after treatment (D3). Generalized estimating equations (GEEs) were used to examine the effects of enzyme variants on gametocyte densities, adjusting for potential confounding factors. Linear regression models were adjusted to explore the predictors of PQ blood levels measured on D3. Individuals with the CPR mutation showed a smaller decrease in gametocyte transcript levels on D3 compared to those without the mutation (P = 0.02, by GEE). Consistent with this, higher PQ blood levels on D3 were associated with a lower reduction in pvs25 transcripts. Based on our findings, the CPR variant plays a role in the persistence of gametocyte density in P. vivax malaria. Conceptually, our work points to pharmacogenetics as a non-negligible factor to define potential host reservoirs with the propensity to contribute to transmission in the first days of CQ-PQ treatment, particularly in settings and seasons of high Anopheles human-biting rates.

Representação social da malária na perspectiva dos garimpeiros em Boa Vista ­ Roraima / Social representation of malaria from the prespective of miners in Boa Vista ­ Roraima / Representación social de la malaria en la perspectiva de los mineros de Boa Vista ­ Roraima

Objetivo: analisar o núcleo central da representação social acerca da malária na ótica dos garimpeiros e dos indivíduos que exercem atividades laborais em região de garimpo que buscam atendimento no Pronto Atendimento Cosme e Silva em Boa Vista/RR. Método:estudo descritivo exploratório de carácter qualitativo delineado por meio da Teoria das Representações Sociais, formulada por Serge Moscovici em 1961. Foram realizadas 72 entrevistas semiestruturadas com garimpeiros. A análise dos dados ocorreu com o auxílio do software OpenEvoc 0.92 desenvolvido pelo Prof. Dr. Hugo Cristo em 2012. Resultados: foi constatado que as percepções dos garimpeiros acerca da malária está diretamente relacionada com a sintomatologia da doença, principalmente pelos traumas vivenciados devidoa ausência de serviço de saúde disponível no local de trabalho. Enquanto que, as percepções dos garimpeiros sobre o garimpo estão vinculadas a condição econômica deste grupo. Conclusão:observa-se que a busca pelo bem-estar financeiro, faz o garimpeiro se submeter às relações extremamente precárias de trabalho, incluindo a exposição ao vetor da malária.

Objective: to analyze the central nucleus of the social representation about malaria from the perspective of miners and individuals who carry out work activities in a mining region who seek care at the Cosme e Silva Emergency Room in Boa Vista/RR. Method:descriptiveexploratory study of a qualitative nature outlined through the Theory of Social Representations, formulated by Serge Moscovici in 1961. 72 semi-structured interviews were carried out with miners. Data analysis took place with the aid of the OpenEvoc 0.92 software developed by Prof. Dr. Hugo Cristo in 2012. Results:it was found that the prospectors' perceptions about malaria are directly related to the symptoms of the disease, mainly due to the traumas experienced due to the lack of health services available in the workplace. Meanwhile, the prospectors' perceptions about mining are linked to the economic condition of this group. Conclusion:it is observed that the search for financial well-being makes the prospector submit to extremely precarious work relationships, including exposure to the malaria vector.

Objetivo: analizar el núcleo central de la representación social sobre la malaria en la perspectiva de mineros y personas que ejercen actividades laborales en una región minera que buscan atención en el Servicio de Emergencia Cosme e Silva de Boa Vista/RR. Método:estudio descriptivo exploratorio de carácter cualitativo esbozado a través de la Teoría de las Representaciones Sociales, formulada por Serge Moscovici en 1961. Se realizaron 72 entrevistas semiestructuradas con mineros. El análisis de datos se llevó a cabocon la ayuda del software OpenEvoc 0.92 desarrollado por el Prof. Dr. Hugo Cristo en 2012. Resultados:se encontró que las percepciones de los prospectores sobre la malaria están directamente relacionadas con los síntomas de la enfermedad, principalmente debido a los traumas experimentados por la falta de servicios de salud disponibles en el lugar de trabajo. Por su parte, las percepciones de los buscadores sobre la minería están ligadas a la condición económica de este grupo. Conclusión: se observa que labúsqueda de bienestar económico hace que el prospector se someta a relaciones de trabajo extremadamente precarias, incluida la exposición al vector de la malaria.

Malária: A crise silenciosa em Alto Alegre, Roraima e o fantasma do garimpo na Terra Indígena Yanomami / Malaria: The Silent Crisis in Alto Alegre, Roraima and the Specter of Mining in the Yanomami Indigenous Land / Malaria: La crisissilenciosa en Alto Alegre, Roraima y el fantasma de la minería en la Tierra Indígena Yanomami

Objetivo:analisar a evolução dos casos de malária em Roraima, principalmente no município de Alto Alegre, estratificado por aglomerações por área geográfica habitada -rural, urbana e área indígena no período de 2013 a 2022. Método:Estudo ecológico do tipo série temporal baseado em dados secundários dos casos confirmados de malária em Roraima, no período de 2013 a 2022. Resultados:nesse período foram confirmados 72.828 casos de malária em Roraima, dos quais 78,0% foram por Plasmodium vivax. Além disso, a maior parte dos casos se concentrou no município de Alto Alegre, correspondendo a 41,1%. Portanto, este foi o município que mais produziu malária procedente de garimpo, apesar de existirem outros que registraram aumento da doença nesse período. Conclusão:assim, osmunicípios de Alto Alegre, Amajari, Caracaraí, Iracema e Mucajaí, juntos respondem por 77.8% de toda malária produzida em Roraima. Estes municípios fazem parte da rota do garimpo ilegal na terra indígena Yanomami.

Objective: analyze the evolution of malaria cases in Roraima, especially in the municipality of Alto Alegre, stratified by geographical areas of habitation -rural, urban, and indigenous areas, from 2013 to 2022. Method: A time series ecological study based on secondary data of confirmed malaria cases in Roraima from 2013 to 2022. Results:During this period, 72,828 cases of malaria were confirmed in Roraima, of which 78.0% were due to Plasmodium vivax. Furthermore, the majority of cases were concentrated in the municipality of Alto Alegre, accounting for 41.1%. Therefore, this was the municipality that produced the most malariafrom mining activities, despite others experiencing an increase in the disease during this period. Conclusion: Thus, the municipalities of Alto Alegre, Amajari, Caracaraí, Iracema, and Mucajaí together accounted for 77.8% of all malaria cases produced in Roraima. These municipalities are part of the illegal mining route within the Yanomami indigenous land.

Objetivo: analizar la evolución de los casos de malaria en Roraima, principalmente en el municipio de Alto Alegre, marcado por aglomeraciones por área geográfica habitada -rural, urbana, en el área indígena en el período comprendido desde 2013 hasta 2022. Método: Estudio ecológico de tipo serie temporal basado en datos secundarios de los casos confirmados de malaria en Roraima, en el periodo de 2013 hasta 2022. Resultados: Durante este periodo fueron confirmados 72.828 casos de malaria en Roraima, de los cuales 78,0% correspondieron a Plasmodium Vivax. Además, la mayor parte de los casos estaban concentrados en el municipio de Alto Alegre, correspondiendo a 41,1%. Por lo tanto, este fue el condado que más produjo malaria proveniente de la minería, a pesar de existir otros que registraron aumentos de la enfermedad durante este periodo. Conclusión: Siendo así, los condados de Alto Alegre, Amajari, Caracaraí, Iracema y Mucajaí, juntos representan el 77.8% de toda la malaria existente en Roraima. Estos condados hacen parte de la ruta de minería ilegal en tierras indígenas Yanomam.

Improving the Molecular Diagnosis of Malaria: Droplet Digital PCR-Based Method Using Saliva as a DNA Source.

Malaria is an acute febrile disease caused by a protozoan of the genus Plasmodium. Light microscopy (LM) is the gold standard for the diagnosis of malaria. Despite this method being rapid and inexpensive, it has a low limit of detection, which hampers the identification of low parasitemia infections. By using multicopy targets and highly sensitive molecular techniques, it is possible to change this scenario. In this study, we evaluated the performance of droplet digital PCR (ddPCR) to detect Plasmodium DNA obtained from saliva samples (whole saliva and buccal swab) of 157 individuals exposed to malaria transmission from the Brazilian Amazon region. We used the highly sensitive ddPCR method with non-ribosomal multicopy targets for Plasmodium vivax (Pvr47) and Plasmodium falciparum (Pfr364). There was good concordance between the quantitative real-time PCR (qPCR) results from the saliva and blood, except for mixed-species infections. The sensitivity of qPCR was 93% for blood, 77% for saliva, and 47% for swabs. Parasite DNA was not detected in saliva samples in low-density infections compared with the detection in blood samples. ddPCR showed increased sensitivity for detecting Plasmodium in the blood and swabs (99% in blood, 73% in saliva, and 59% in swabs). Notably, ddPCR detected more mixed infections in the blood (15%), saliva (9%), and swabs (18%) than qPCR. Our data showed that the differences between ddPCR and qPCR were the result of a higher number of P. falciparum infections detected by ddPCR. Overall, there was a moderate correlation between parasite densities estimated by the different methods in the blood. Our findings highlight the possibility of using non-invasive sample collection methods for malaria diagnosis by targeting multicopy sequences combined with highly sensitive molecular methods.

Cross-border malaria in the triple border region between Brazil, Venezuela and Guyana.

The state of Roraima, in Brazil, has recently seen an increase in the number of reported Plasmodium falciparum infections believed to be imported from neighboring countries. The objective of this study was to determine the prevalence of Plasmodium species among patients attending malaria health posts in Roraima and quantify the infections attributable to imported malaria. This cross-sectional case study was carried out between March 2016 and September 2018. Study participants were recruited as they exited the malaria health post. Information about residence, occupation and travel history was collected using a questionnaire. A dried blood spot was collected and used for malaria diagnosis by PCR. A total of 1222 patients were enrolled. Of the 80% Plasmodium positive samples, 50% were P. falciparum, 34% P. vivax, 8% mixed P. falciparum/P. vivax and 0.2% mixed P. falciparum/P. ovale infections and 8% tested positive for Plasmodium, but the species could not be identified. 80% of the malaria patients likely acquired infections in Venezuela and the remaining 20% acquired in Guyana, Brazil, Suriname and French Guyana. 50% of the study participants reported to be working in a mine. Results from this study support the hypothesis that imported malaria contribute to the bulk of malaria diagnosed in Roraima. These findings are in keeping with previous findings and should be considered when developing malaria control interventions.

Larval habitats, species composition and distribution of malaria vectors in regions with autochthonous and imported malaria in Roraima state, Brazil.

BACKGROUND: Malaria control requires local action. Assessing the vector diversity and abundance provides information on the local malariogenic potential or risk of transmission. This study aimed to determine the Anopheles species composition, habitats, seasonal occurrence, and distribution in areas with autochthonous and imported malaria cases in Roraima State. METHODS: A longitudinal study was conducted from January 2017 to October 2018, sampling larvae and adult mosquitoes in three municipalities of Roraima State: Boa Vista, Pacaraima and São João da Baliza. These areas have different risks of malaria importation. Four to six mosquito larval habitats were selected for larval sampling at each municipality, along with two additional sites for adult mosquito collection. All larval habitats were surveyed every two months using a standardized larval sampling methodology and MosqTent for adult mosquitoes. RESULTS: A total of 544 Anopheles larvae and 1488 adult mosquitoes were collected from the three municipalities studied. Although the species abundance differed between municipalities, the larvae of Anopheles albitarsis s.l., Anopheles nuneztovari s.l. and Anopheles triannulatus s.l. were collected from all larval habitats studied while Anopheles darlingi were collected only from Boa Vista and São João da Baliza. Adults of 11 species of the genus Anopheles were collected, and the predominant species in Boa Vista was An. albitarsis (88.2%) followed by An. darlingi (6.9%), while in São João da Baliza, An. darlingi (85.6%) was the most predominant species followed by An. albitarsis s.l. (9.2%). In contrast, the most abundant species in Pacaraima was Anopheles braziliensis (62%), followed by Anopheles peryassui (18%). Overall, the majority of anophelines exhibited greater extradomicile than peridomicile-biting preference. Anopheles darlingi was the only species found indoors. Variability in biting times was observed among species and municipalities. CONCLUSION: This study revealed the composition of anopheline species and habitats in Boa Vista, Pacaraima and São João da Baliza. The species sampled differed in their behaviour with only An. darlingi being found indoors. Anopheles darlingi appeared to be the most important vector in São João da Baliza, an area of autochthonous malaria, and An. albitarsis s.l. and An. braziliensis in areas of low transmission, although there were increasing reports of imported malaria. Understanding the diversity of vector species and their ecology is essential for designing effective vector control strategies for these municipalities.

A Comprehensive Analysis of the Genetic Diversity of Plasmodium falciparum Histidine-Rich Protein 2 (PfHRP2) in the Brazilian Amazon.

Early diagnosis and treatment are fundamental to the control and elimination of malaria. In many endemic areas, routine diagnosis is primarily performed microscopically, although rapid diagnostic tests (RDTs) provide a useful point-of-care tool. Most of the commercially available RDTs detect histidine-rich protein 2 (HRP2) of Plasmodium falciparum in the blood of infected individuals. Nonetheless, parasite isolates lacking the pfhrp2 gene are relatively frequent in some endemic regions, thereby hampering the diagnosis of malaria using HRP2-based RDTs. To track the efficacy of RDTs in areas of the Brazilian Amazon, we assessed pfhrp2 deletions in 132 P. falciparum samples collected from four malaria-endemic states in Brazil. Our findings show low to moderate levels of pfhrp2 deletion in different regions of the Brazilian Amazon. Overall, during the period covered by this study (2002-2020), we found that 10% of the P. falciparum isolates were characterized by a pfhrp2 deletion. Notably, however, the presence of pfhrp2-negative isolates has not been translated into a reduction in RDT efficacy, which in part may be explained by the presence of polyclonal infections. A further important finding was the discrepancy in the proportion of pfhrp2 deletions detected using two assessed protocols (conventional PCR versus nested PCR), which reinforces the need to perform a carefully planned laboratory workflow to assess gene deletion. This is the first study to perform a comprehensive analysis of PfHRP2 sequence diversity in Brazilian isolates of P. falciparum. We identified 10 PfHRP2 sequence patterns, which were found to be exclusive of each of the assessed regions. Despite the small number of PfHRP2 sequences available from South America, we found that the PfHRP2 sequences identified in Brazil and neighboring French Guiana show similar sequence patterns. Our findings highlight the importance of continuously monitoring the occurrence and spread of parasites with pfrhp2 deletions, while also taking into account the limitations of PCR-based testing methods associated with accuracy and the complexity of infections.

The impact of imported malaria by gold miners in Roraima: characterizing the spatial dynamics of autochthonous and imported malaria in an urban region of Boa Vista.

BACKGROUND The number of malaria cases in Roraima nearly tripled from 2016 to 2018. The capital, Boa Vista, considered a low-risk area for malaria transmission, reported an increasing number of autochthonous and imported cases. OBJECTIVES This study describes a spatial analysis on malaria cases in an urban region of Boa Vista, which sought to identify the autochthonous and imported cases and associated them with Anopheles habitats and the potential risk of local transmission. METHODS In a cross-sectional study at the Polyclinic Cosme e Silva, 520 individuals were interviewed and diagnosed with malaria by microscopic examination. Using a global positional system, the locations of malaria cases by type and origin and the breeding sites of anopheline vectors were mapped and the risk of malaria transmission was evaluated by spatial point pattern analysis. FINDINGS Malaria was detected in 57.5% of the individuals and there was a disproportionate number of imported cases (90.6%) linked to Brazilian coming from gold mining sites in Venezuela and Guyana. MAIN CONCLUSIONS The increase in imported malaria cases circulating in the west region of Boa Vista, where there are positive breeding sites for the main vectors, may represent a potential condition for increased autochthonous malaria transmission in this space.

Molecular Surveillance for Polymorphisms Associated with Artemisinin-Based Combination Therapy Resistance in Plasmodium falciparum Isolates Collected in the State of Roraima, Brazil.

Given that the C580Y polymorphism in the Plasmodium falciparum propeller domain of the kelch 13 gene (pfk13) was documented in Guyana, monitoring for mutations associated with antimalarial resistance was undertaken in neighboring Roraima state in Brazil. Polymorphisms in the pfmdr1 and pfk13 genes were investigated in 275 P. falciparum samples. No pfk13 mutations were observed. Triple mutants 184F, 1042D, and 1246Y were observed in 100% of the samples successfully sequenced for the pfmdr1 gene, with 20.1% of these having an additional mutation at codon 1034C. Among them, 2.5% of samples harbored two copies of the pfmdr1 gene. We found no evidence of the spread of C580Y parasites to Roraima state, Brazil. As previously observed, the 184F, 1042D, and 1246Y mutations in the pfmdr1 gene appear to be fixed in this region. Continued molecular surveillance is essential to detect any potential migration or local emergence of artemisinin-resistant mutation.

The impact of imported malaria by gold miners in Roraima: characterizing the spatial dynamics of autochthonous and imported malaria in an urban region of Boa Vista

BACKGROUND The number of malaria cases in Roraima nearly tripled from 2016 to 2018. The capital, Boa Vista, considered a low-risk area for malaria transmission, reported an increasing number of autochthonous and imported cases. OBJECTIVES This study describes a spatial analysis on malaria cases in an urban region of Boa Vista, which sought to identify the autochthonous and imported cases and associated them with Anopheles habitats and the potential risk of local transmission. METHODS In a cross-sectional study at the Polyclinic Cosme e Silva, 520 individuals were interviewed and diagnosed with malaria by microscopic examination. Using a global positional system, the locations of malaria cases by type and origin and the breeding sites of anopheline vectors were mapped and the risk of malaria transmission was evaluated by spatial point pattern analysis. FINDINGS Malaria was detected in 57.5% of the individuals and there was a disproportionate number of imported cases (90.6%) linked to Brazilian coming from gold mining sites in Venezuela and Guyana. MAIN CONCLUSIONS The increase in imported malaria cases circulating in the west region of Boa Vista, where there are positive breeding sites for the main vectors, may represent a potential condition for increased autochthonous malaria transmission in this space.

Field evaluation of malaria malachite green loop-mediated isothermal amplification in health posts in Roraima state, Brazil.

BACKGROUND: Microscopic detection of malaria parasites is the standard method for clinical diagnosis of malaria in Brazil. However, malaria epidemiological surveillance studies specifically aimed at the detection of low-density infection and asymptomatic cases will require more sensitive and field-usable tools. The diagnostic accuracy of the colorimetric malachite green, loop-mediated, isothermal amplification (MG-LAMP) assay was evaluated in remote health posts in Roraima state, Brazil. METHODS: Study participants were prospectively enrolled from health posts (healthcare-seeking patients) and from nearby villages (healthy participants) in three different study sites. The MG-LAMP assay and microscopy were performed in the health posts. Two independent readers scored the MG-LAMP tests as positive (blue/green) or negative (clear). Sensitivity and specificity of local microscopy and MG-LAMP were calculated using results of PET-PCR as a reference. RESULTS: A total of 91 participants were enrolled. There was 100% agreement between the two MG-LAMP readers (Kappa = 1). The overall sensitivity and specificity of MG-LAMP were 90.0% (95% confidence interval (CI) 76.34-97.21%) and 94% (95% CI 83.76-98.77%), respectively. The sensitivity and specificity of local microscopy were 83% (95% CI 67.22-92.66%) and 100% (95% CI 93.02-100.00%), respectively. PET-PCR detected six mixed infections (infection with both Plasmodium falciparum and Plasmodium vivax); two of these were also detected by MG-LAMP and one by microscopy. Microscopy did not detect any Plasmodium infection in the 26 healthy participants; MG-LAMP detected Plasmodium in five of these and PET-PCR assay detected infection in three. Overall, performing the MG-LAMP in this setting did not present any particular challenges. CONCLUSION: MG-LAMP is a sensitive and specific assay that may be useful for the detection of malaria parasites in remote healthcare settings. These findings suggest that it is possible to implement simple molecular tests in facilities with limited resources.