Morphological atypia and molecular profile of Plasmodium vivax: Findings from an outbreak in the Brazilian Amazon.

This study aimed to perform morphological and molecular analyses of parasites isolated from the blood of malaria-infected individuals during an outbreak in the Microregion of Cametá, State of Pará, Brazilian Amazon. A total of 260 positive samples were identified by microscopy as Plasmodium vivax; however, in three samples, forms considered unusual for the species were found and defined as morphological atypia of P. vivax. Single P. vivax infection was confirmed by qPCR in all samples. Among 256 genotyped samples, the VK247 genotype alone was identified in 255 samples, and the VK210 genotype was found in only one. The study showed that this malaria outbreak was caused by the etiological agent P. vivax, and for the first time, morphological atypia was described in isolates circulating in Brazil. Likewise, for the first time, the VK247 genotype was detected predominantly in single infections in an area of the State of Pará, which may suggest a greater circulation of the genotype in the region.

Title: Atypie morphologique et profil moléculaire de Plasmodium vivax : résultats issus d'une épidémie en Amazonie brésilienne. Abstract: Cette étude visait à effectuer des analyses morphologiques et moléculaires de parasites isolés du sang d'individus infectés par le paludisme lors d'une épidémie dans la microrégion de Cametá, État du Pará, Amazonie brésilienne. Au total, 260 échantillons positifs ont été identifiés par microscopie comme Plasmodium vivax mais dans trois échantillons, des formes considérées comme inhabituelles pour l'espèce ont été trouvées et définies comme des atypies morphologiques de P. vivax. Une infection simple à P. vivax a été confirmée par qPCR dans tous les échantillons. Sur 256 échantillons génotypés, le génotype VK247 seul a été identifié dans 255 échantillons, et le génotype VK210 a été trouvé dans un seul échantillon. L'étude a montré que cette épidémie de paludisme était causée par l'agent étiologique P. vivax et, pour la première fois, une atypie morphologique a été décrite dans des isolats circulant au Brésil. De même, pour la première fois, le génotype VK247 a été détecté principalement dans des infections uniques dans une zone de l'État de Pará, ce qui peut suggérer une plus grande circulation du génotype dans la région.

Evaluation of Histidine-Rich Proteins 2 and 3 Gene Deletions in Plasmodium falciparum in Endemic Areas of the Brazilian Amazon.

Histidine-rich proteins 2 and 3 gene (pfhrp2 and pfhrp3) deletions affect the efficacy of rapid diagnostic tests (RDTs) based on the histidine-rich protein 2 (HRP2), compromising the correct identification of the Plasmodium falciparum species. Therefore, molecular surveillance is necessary for the investigation of the actual prevalence of this phenomenon and the extent of the disappearance of these genes in these areas and other South American countries, thus guiding national malaria control programs on the appropriate use of RDTs. This study aimed to evaluate the pfhrp2 and pfhrp3 gene deletion in P. falciparum in endemic areas of the Brazilian Amazon. Aliquots of DNA from the biorepository of the Laboratory of Basic Research in Malaria, Evandro Chagas Institute, with a positive diagnosis for P. falciparum infection as determined by microscopy and molecular assays, were included. Monoinfection was confirmed by nested-polymerase chain reaction assay, and DNA quality was assessed by amplification of the merozoite surface protein-2 gene (msp2). The pfhrp2 and pfhrp3 genes were amplified using primers for the region between exons 1 and 2 and for all extension of exon 2. Aliquots of DNA from 192 P. falciparum isolates were included in the study, with 68.7% (132/192) from the municipality of Cruzeiro do Sul (Acre) and 31.3% (60/192) from Manaus (Amazonas). Of this total, 82.8% (159/192) of the samples were considered of good quality. In the state of Acre, 71.7% (71/99) showed pfhrp2 gene deletion and 94.9% (94/99) showed pfhrp3 gene deletion, while in the state of Amazonas, 100.0% (60/60) of the samples showed pfhrp2 gene deletion and 98.3% (59/60) showed pfhrp3 gene deletion. Moreover, 79.8% (127/159) of isolates displayed gene deletion. Our findings confirm the presence of a parasite population with high frequencies of pfhrp2 and pfhrp3 gene deletions in the Brazilian Amazon region. This suggests reconsidering the use of HRP2-based RDTs in the Acre and Amazonas states and calls attention to the importance of molecular surveillance and mapping of pfhrp2/pfhrp3 deletions in this area and in other locations in the Amazon region to guarantee appropriate patient care, control and ultimately contribute to achieving P. falciparum malaria elimination.

Comparison of two DNA obtainment methods as alternative protocols for the detection of human malária parasites by nested PCR

The correct and precise laboratory diagnosis of human malaria is still a challenge because the reference method, the Giemsa-stained thick blood smear (TS), has limitations that present problems for malaria control. Because of these problems, several studies have attempted to develop alternative methods for malaria diagnosis. Many of these studies focus on molecular diagnosis methods and have led to the development of some alternatives to TS. However, their limitations include high cost, protocol complexity and variable quality of DNA sources and reagents. Nested PCR has been shown to be a good method in this respect and it can be improved by using a high-quality source of DNA. In this study we evaluated two methods for the obtainment of DNA from dried blood samples on filter paper: 1) washing and 2) saponin/chelex-100. The second method showed higher sensitivity and specificity compared to the first, as it detected more infections, whether single or mixed, as well as Plasmodium malariae infections. Based on these results, we present this method as the protocol of choice for DNA obtainment. Nested PCR using saponin/chelex-100 for DNA extraction could be an alternative or complementary diagnosis method for human malaria parasites, but it is not appropriate for routine use...

Um diagnóstico laboratorial correto e preciso de malária humana ainda é considerado um desafio, pois o método de referência, o da gota espessa com colocação pelo Giemsa, apresenta limitações que dificultam o controle da malária. Devido a esses problemas, várias pesquisas têm objetivado desenvolver métodos alternativos para o diagnóstico da malária. Grande parte desses estudos aborda métodos de diagnóstico molecular, que têm acarretado o desenvolvimento de algumas alternativas ao método de coloração pelo Giemsa. No entanto, esses métodos, por sua vez, apresentam suas limitações, que incluem seu alto custo, a complexidade do protocolo e a variação da qualidade das fontes de DNA e de reagentes. Neste aspecto, a técnica de nested PCR tem demonstrado ser um bom método e pode ser melhorado usando uma fonte de DNA de alta qualidade. Neste estudo foram avaliados dois métodos para a obtenção de DNA de amostras de sangue seco colhidas em papel de filtro: 1) lavagem e 2) saponina/Chelex-100. O segundo método apresentou sensibilidade e especificidade mais altas em relação ao primeiro, pois detectou mais infecções, tanto simples como mistas, bem como infecções por Plasmodium malariae. Com base nesses resultados, apresentamos o segundo como o protocolo de escolha para a obtenção de DNA. A técnica de nested PCR usando saponina/Chelex-100 para extração de DNA pode ser um método alternativo ou complementar de diagnóstico de parasitas da malária humana, mas não é considerado adequado para o uso de rotina...

Risco de transmissão de malária humana em área de implantação de projeto de prospecção mineral, município de Juruti, Estado do Pará / Human malaria transmission risk in an implementation area of mineral prospection, Juruti Municipality, Para State, Brazil

Este estudo objetivou avaliar o risco de transmissão de malária humana em áreas de influência do projeto Juruti no período 2006 a 2008. Um total de 976 mosquitos anofelinos foram capturados por atração humana protegida, identificados e processados para determinação de infectividade pelo teste de imunoensaio (ELISA) e, parte deles (10por cento) para taxa de paridade. Para o inquérito hemoscópico, foram feitas duas coletas (setembro/2007 na comunidade Capiranga e invasão Nova Vitória e março/2008 somente na última) utilizando-se o método da gota expressa (GE). Foram identificadas oito espécies de mosquitos anofelinos, com predomínio da espécie An. albitarsis s.l (76,8por cento) cuja taxa de paridade foi de 9,6por cento. A taxa de infecção foi zero para os primeiros dois anos de estudo e de 0,5por cento em 2008, quando um exemplar de An. albitarsis s.l coletado na comunidade de Santa Maria foi positivo para P. vivax-VK247. O índice de picada homem/hora (IPHH) variou de 0,1 a 8,1. Todas as 148 amostras de sangue foram negativas pela GE. Concluiu-se, portanto, que o risco de transmissão de malária na área estudada é baixo, apesar da presença de mosquitos vetores. Contudo, faz-se necessária vigilância permanente por causa, principalmente, do intenso fluxo migratório gerado pelo projeto.