Desenvolvimento de um protocolo de PCR em Tempo Real para diagnóstico de malária subpatente e infecções mistas por Plasmodium Vivax e Plasmodium falciparum.

Submitted by Nuzia Santos ([email protected]) on 2015-04-10T19:15:34Z No. of bitstreams: 2 Dissertacao_LaraCottaAmaral.pdf: 1571356 bytes, checksum: d96cdf3ef9b085b1c5f6bb55657eaaac (MD5) Dissertacao_LaraCottaAmaral.pdf: 1571356 bytes, checksum: d96cdf3ef9b085b1c5f6bb55657eaaac (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2015-04-10T19:15:44Z (GMT) No. of bitstreams: 2 Dissertacao_LaraCottaAmaral.pdf: 1571356 bytes, checksum: d96cdf3ef9b085b1c5f6bb55657eaaac (MD5) Dissertacao_LaraCottaAmaral.pdf: 1571356 bytes, checksum: d96cdf3ef9b085b1c5f6bb55657eaaac (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2015-04-10T19:15:54Z (GMT) No. of bitstreams: 2 Dissertacao_LaraCottaAmaral.pdf: 1571356 bytes, checksum: d96cdf3ef9b085b1c5f6bb55657eaaac (MD5) Dissertacao_LaraCottaAmaral.pdf: 1571356 bytes, checksum: d96cdf3ef9b085b1c5f6bb55657eaaac (MD5)Made available in DSpace on 2015-04-10T19:15:54Z (GMT). No. of bitstreams: 2 Dissertacao_LaraCottaAmaral.pdf: 1571356 bytes, checksum: d96cdf3ef9b085b1c5f6bb55657eaaac (MD5) Dissertacao_LaraCottaAmaral.pdf: 1571356 bytes, checksum: d96cdf3ef9b085b1c5f6bb55657eaaac (MD5) Previous issue date: 2014Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, Brasil.O diagnóstico adequado de malária permanece como um dos pilares dos programas de controle da doença no mundo, já que um diagnóstico eficiente permite a identificação precoce de casos e definição do esquema terapêutico, contribuindo para interrupção do ciclo biológico do parasito. A microscopia óptica (MO), atual diagnóstico de referência para malária, tem apresentado limitações, principalmente em casos de co-infecções e baixas parasitemias. Assim sendo, busca-se por técnicas mais sensíveis e específicas para auxiliar a MO, sendo os métodos baseados na reação em cadeia da polimerase (PCR) considerados mais adequados para identificação de indivíduos com infecção submicroscópica. Entretanto, os vários protocolos de PCR apresentados até o momento tem se baseado no gene da subunidade menor do RNA ribossomal 18S dos plasmódios (18S rRNA), que se encontra em poucas cópias no genoma destes parasitos. Recentemente, foram descritas sequências não ribossomais para identificação de Plasmodium vivax (Pvr47) e Plasmodium falciparum (Pfr364), sendo estas sequências promissoras para o diagnóstico molecular de malária. Baseando-se nestes achados, este trabalho propôs o desenvolvimento de um protocolo de Real-Time PCR para validar os alvos Pvr47 e Pfr364 para o diagnóstico de malária vivax e falciparum, respectivamente, com ênfase em infecções mistas e baixas parasitemias. Após padronização com sucesso da técnica de Real-Time PCR (RT-LAMAL), a mesma foi comparada a três outros protocolos moleculares, sendo duas técnicas baseadas no gene 18S rRNA – Nested-PCR (Snounou et al., 1993) e Real-Time PCR (Mangold et al., 2005) – e uma PCR convencional baseada nos alvos Pvr47/Pfr364 (Demas et al., 2011). Para avaliar os protocolos quanto aos seus limites de detecção de infecções únicas e mistas por P. vivax e P. falciparum, foram realizadas titulações de misturas artificiais com diferentes concentrações dos parasitos. Os resultados obtidos nesta etapa revelaram que a PCR-Demas e RT-LAMAL apresentaram os menores limites de detecção para P. vivax e P. falciparum, tanto em infecções únicas quanto mistas, e que o protocolo de RT-Mangold foi incapaz de detectar coinfecções. Posteriormente, os protocolos foram avaliados para o diagnóstico de malária em amostras de campo, incluindo área não endêmica (n=117) e área endêmica para malária (n=163). Os resultados revelaram que os protocolos de RTMangold, PCR-Demas e RT-LAMAL foram mais eficientes na detecção de parasitemias submicroscópicas, porém, o protocolo de RT-Mangold novamente mostrou ser incapaz de detectar infecções mistas. Em conjunto, os dados obtidos demonstraram que os alvos Pvr47/Pfr364 foram mais adequados para o diagnóstico molecular de malária vivax e falciparum do que o gene 18S rRNA. Contudo, o protocolo aqui desenvolvido (RT-LAMAL) se mostra em vantagem à PCR-Demas, com maior rapidez na obtenção de resultados, dispensando a revelação em gel de agarose e uso de brometo de etídeo, além de diminuir a possibilidade de contaminação de reagentes e amostras. Portanto, conclui-se que a RT-LAMAL possui grande potencial para o diagnóstico molecular de certeza de pacientes com infecções mistas e baixas parasitemias.Accurate diagnosis of malaria remains as one of the pillars for prevention and control of the disease. An efficient diagnostic test may allow early case detection and appropriate treatment, therefore contributing to the interruption of malaria transmission cycle. Because the optical microscopy (OM) – the gold standard of malaria diagnosis – presents significant limitations, mainly in cases of co-infections and low parasitemias, it seems to be essential to develop a more sensitive and specific diagnostic tool. In this context, methods based on the polymerase chain reaction (PCR) seem to be more suitable for detecting individuals with submicroscopic malaria infections. Unfortunately, the majority of PCR-based methods still rely on the 18S rRNA gene targets, present in few copies in the parasite genome. Recently, new target DNA sequences were described for the identification of Plasmodium vivax (Pvr47) and Plasmodium falciparum (Pfr364). Due to the importance of these findings, the goal of the present study was to validate the Pvr47 and Pfr364 targets for the diagnosis of vivax and falciparum malaria, focusing on the development of a real-time PCR for detection of mixed infection and sub-microscopic parasitemia. After successful standardization of the Real-Time PCR (RT-LAMAL), this-PCR protocol was compared with three well-established PCR protocols, two of them relied on the gene 18S rRNA – Nested-PCR (Snounou et al., 1993) and Real-Time PCR (Mangold et al., 2005) -- and a third protocol based on the Pvr47/Pfr364 as target for a conventional PCR assay (Demas et al., 2011). In order to evaluate these different PCR-protocols in terms of their limit of detection, titrations of artificial mixtures of DNA plasmodial were performed. The results revealed that the PCRDemas and RT-LAMAL presented the lowest limit of detection for either single or mixed P. vivax and P. falciparum infections. In addition, the RT-Mangold protocol was unable to detect co-infections. To further evaluate the performance of these four PCR protocols for diagnosis of malaria in the field, we analyzed samples from malaria-endemic areas (n=163) as well as from non-endemic area (n = 117). While the results confirmed that PCR-Demas and RT-LAMAL protocols as being more appropriate for the diagnosis of submicroscopic infection, the data demonstrated again that the RT-Mangold protocol was unable to detect mixed infections. Together, the data confirmed Pvr47/Pfr364 targets as more suitable for molecular diagnosis of P. vivax and P. falciparum. Of interest, the Real-time PCR developed here (RTLAMAL) offers several advantages over the traditional PCR-Demas, including faster processing time and decreased risk of contamination. In conclusion, that RT-LAMAL has great potential for molecular diagnosis of patients with mixed infections and low levels of parasitemia

Identificação molecular de infecções maláricas subclínicas e mistas por alvos não ribossomais de Plasmodium vivax e Plasmodium falciparum

Submitted by Nuzia Santos ([email protected]) on 2019-07-16T16:07:23Z No. of bitstreams: 1 T_2019_LaraCottaAmaral.pdf: 3226498 bytes, checksum: 5db9484406cde9c89328cd0c378be37f (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2019-07-16T16:15:43Z (GMT) No. of bitstreams: 1 T_2019_LaraCottaAmaral.pdf: 3226498 bytes, checksum: 5db9484406cde9c89328cd0c378be37f (MD5)Made available in DSpace on 2019-07-16T16:15:43Z (GMT). No. of bitstreams: 1 T_2019_LaraCottaAmaral.pdf: 3226498 bytes, checksum: 5db9484406cde9c89328cd0c378be37f (MD5) Previous issue date: 2019CNPq, FAPEMIG e Programa de Excelência em Pesquisa (PROEP) do IRR/FIOCRUZ, CAPESFundação Oswaldo Cruz. Instituto René Rachou. Belo Horizonte, MG, Brasil.Em áreas endêmicas para malária, o diagnóstico molecular de infecções por Plasmodium tem identificado altas frequências de infecções de baixa densidade, abaixo do limite de detecção da microscopia convencional. Muitas são as implicações destes achados, já que os portadores de malária submicroscópica podem transmitir os parasitos, atuando como reservatórios da doença. Historicamente, a maioria dos protocolos de PCR para malária se baseia na amplificação das poucas cópias do gene 18S rRNA, que apresenta baixa sensibilidade e reprodutibilidade. Na última década, a mineração de dados genômicos dos parasitos do gênero Plasmodium permitiu a descoberta de novos alvos espécie-específicos múlticópias – tais como Pvr47 em P. vivax e Pfr364 em P. falciparum – promissores para o diagnóstico molecular da malária. Neste trabalho, nós desenvolvemos um protocolo de PCR em tempo real não ribossomal (NR-qPCR) para amplificar os alvos Pvr47/Pfr364. A NR-qPCR foi comparada a três protocolos de PCR bem estabelecidos, dois deles baseados no gene 18S rRNA (Nested-PCR e R-qPCR) e um terceiro, o ensaio de PCR original que possui como alvos Pvr47/Pfr364 (NR-cPCR). Curvas de titulação de infecções mistas artificiais por P. vivax e P. falciparum demonstraram um aumento de sensibilidade da NR-qPCR em comparação com os outros ensaios de PCR, mesmo quando o DNA de uma das espécies esteve presente em uma proporção cem vezes menor do que da outra espécie. A avaliação dos quatro ensaios de PCR em amostras de campo, incluindo indivíduos sintomáticos ou assintomáticos, confirmou que as infecções maláricas submicroscópicas não foram prevalentes entre os pacientes sintomáticos, mas representaram uma grande proporção (até 77%) entre os indivíduos assintomáticos, expostos à malária na Amazônia. De relevância, a amplificação de diferentes alvos plasmodiais (Pvr47/Pfr364 e gene 18S rRNA) não aumentou a chance de detecção de infecções maláricas submicroscópicas. Como a maioria das infecções subclínicas foi causada por P. vivax, a forma mais comum de malária na floresta amazônica, futuros estudos devem investigar o potencial de Pvr47/Pfr364 para detectar infecções de malária mista em campo.In malaria endemic areas, the molecular diagnosis of Plasmodium infections has identified high frequencies of low-density infections, beneath the limit of detection of the conventional microscopy. Many are the implications of these findings as submicroscopic malaria carriers may be able to transmit the parasites, acting as reservoirs of the disease. Historically, most malaria PCR-based protocols rely on amplification of the few copies 18S rRNA gene, which presents low sensitivity and reproducibility. In the last decade, the genomic data mining of Plasmodium parasites allowed the discovery of new species-specific multi-copy targets – such as Pvr47 for P. vivax and Pfr364 for P. falciparum – which seem promising for malaria molecular diagnosis. Here, we developed a non-ribosomal real-time PCR protocol (NR-qPCR) to amplify the Pvr47/Pfr364 targets. The NR-qPCR was compared with three wellestablished PCR protocols, two of them based on the 18S rRNA gene (Nested-PCR and R-qPCR) and the third one, the original PCR assay targeting Pvr47/Pfr364 (NRcPCR). End-point titration curves of artificial mixed P. vivax and P. falciparum infections demonstrated an increase sensitivity of NR-qPCR as compared with the other PCR assays, even when the DNA of one of the species was present in a ratio of hundred-times lower than of the other species. Evaluation of the four PCR assays in field samples, including from individuals symptomatic or asymptomatic, confirmed that submicroscopic malaria infections were not prevalent among symptomatic patients, but represented a large proportion (up to 77%) among asymptomatic Amazonian malaria-exposed individuals. Of relevance, the amplification of different plasmodial targets (Pvr47/Pfr364 and 18S rRNA gene) did not increase the chances of detecting submicroscopic malaria infections. As the majority of subclinical infections were caused by P. vivax, the commonest form of malaria in the Amazon rain forest, future studies should investigate the potential of Pvr47/Pfr364 to detect mixed-malaria infections in the field

Assessment of copy number variation in genes related to drug resistance in Plasmodium vivax and Plasmodium falciparum isolates from the Brazilian Amazon and a systematic review of the literature

Abstract Background Parasite resistance to anti-malarials represents a great obstacle for malaria elimination. The majority of studies have investigated the association between single-nucleotide polymorphisms (SNPs) and drug resistance; however, it is becoming clear that the copy number variation (CNV) is also associated with this parasite phenotype. To provide a baseline for molecular surveillance of anti-malarial drug resistance in the Brazilian Amazon, the present study characterized the genetic profile of both markers in the most common genes associated with drug resistance in Plasmodium falciparum and Plasmodium vivax isolates. Additionally, these data were compared to data published elsewhere applying a systematic review of the literature published over a 20-year time period. Methods The genomic DNA of 67 patients infected by P. falciparum and P. vivax from three Brazilian States was obtained between 2002 and 2012. CNV in P. falciparum multidrug resistance gene-1 (pfmdr1), GTP cyclohydrolase 1 (pfgch1) and P. vivax multidrug resistance gene-1 (pvmdr1) were assessed by real-time PCR assays. SNPs in the pfmdr1 and pfcrt genes were assessed by PCR–RFLP. A literature search for studies that analysed CNP in the same genes of P. falciparum and P. vivax was conducted between May 2014 and March 2017 across four databases. Results All analysed samples of P. falciparum carried only one copy of pfmdr1 or pfgch1. Although the pfcrt K76T polymorphism, a determinant of CQ resistance, was present in all samples genotyped, the pfmdr1 N86Y was absent. For P. vivax isolates, an amplification rate of 20% was found for the pvmdr1 gene. The results of the study are in agreement with the low amplification rates for pfmdr1 gene evidenced in the Americas and Africa, while higher rates have been described in Southeast Asia. For P. vivax, very low rates of amplification for pvmdr1 have been described worldwide, with exceptions in French Guiana, Cambodia, Thailand and Brazil. Conclusions The present study was the first to evaluate gch1 CNV in P. falciparum isolates from Brazil, showing an absence of amplification of this gene more than 20 years after the withdrawal of the Brazilian antifolates therapeutic scheme. Furthermore, the rate of pvmdr1 amplification was significantly higher than that previously reported for isolates circulating in Northern Brazil

Submicroscopic malaria parasite carriage: how reproducible are polymerase chain reaction-based methods?

The polymerase chain reaction (PCR)-based methods for the diagnosis of malaria infection are expected to accurately identify submicroscopic parasite carriers. Although a significant number of PCR protocols have been described, few studies have addressed the performance of PCR amplification in cases of field samples with submicroscopic malaria infection. Here, the reproducibility of two well-established PCR protocols (nested-PCR and real-time PCR for the Plasmodium 18 small subunit rRNA gene) were evaluated in a panel of 34 blood field samples from individuals that are potential reservoirs of malaria infection, but were negative for malaria by optical microscopy. Regardless of the PCR protocol, a large variation between the PCR replicates was observed, leading to alternating positive and negative results in 38% (13 out of 34) of the samples. These findings were quite different from those obtained from the microscopy-positive patients or the unexposed individuals; the diagnosis of these individuals could be confirmed based on the high reproducibility and specificity of the PCR-based protocols. The limitation of PCR amplification was restricted to the field samples with very low levels of parasitaemia because titrations of the DNA templates were able to detect < 3 parasites/µL in the blood. In conclusion, conventional PCR protocols require careful interpretation in cases of submicroscopic malaria infection, as inconsistent and false-negative results can occur

Submicroscopic malaria parasite carriage: how reproducible are polymerase chain reaction-based methods?

Submitted by Nuzia Santos ([email protected]) on 2015-02-02T11:09:58Z No. of bitstreams: 1 2014_013.pdf: 612202 bytes, checksum: 0b0dfc01b7f9ecdf1fd8f04eac043720 (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2015-02-02T11:10:06Z (GMT) No. of bitstreams: 1 2014_013.pdf: 612202 bytes, checksum: 0b0dfc01b7f9ecdf1fd8f04eac043720 (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2015-02-02T11:15:52Z (GMT) No. of bitstreams: 1 2014_013.pdf: 612202 bytes, checksum: 0b0dfc01b7f9ecdf1fd8f04eac043720 (MD5)Made available in DSpace on 2015-02-02T11:15:52Z (GMT). No. of bitstreams: 1 2014_013.pdf: 612202 bytes, checksum: 0b0dfc01b7f9ecdf1fd8f04eac043720 (MD5) Previous issue date: 2014Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, BrasilUniversidade Federal de São João Del Rei. Departamento de Bioengenharia. São João Del Rey, MG, BrasilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, BrasilUniversidade Federal de Mato Grosso. Cuiabá, MT, BrasilUniversidade Federal de Mato Grosso. Cuiabá, MT, BrasilUniversidade Federal de Mato Grosso. Cuiabá, MT, BrasilCentro de Controle de Zoonoses de Uberlândia. Uberlândia, MG, BrasilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, BrasilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, BrasilThe polymerase chain reaction (PCR)-based methods for the diagnosis of malaria infection are expected to accurately identify submicroscopic parasite carriers. Although a significant number of PCR protocols have been described, few studies have addressed the performance of PCR amplification in cases of field samples with submicroscopic malaria infection. Here, the reproducibility of two well-established PCR protocols (nested-PCR and real-time PCR for the Plasmodium 18 small subunit rRNA gene) were evaluated in a panel of 34 blood field samples from individuals that are potential reservoirs of malaria infection, but were negative for malaria by optical microscopy. Regardless of the PCR protocol, a large variation between the PCR replicates was observed, leading to alternating positive and negative results in 38% (13 out of 34) of the samples. These findings were quite different from those obtained from the microscopy-positive patients or the unexposed individuals; the diagnosis of these individuals could be confirmed based on the high reproducibility and specificity of the PCR-based protocols. The limitation of PCR amplification was restricted to the field samples with very low levels of parasitaemia because titrations of the DNA templates were able to detect < 3 parasites/µL in the blood. In conclusion, conventional PCR protocols require careful interpretation in cases of submicroscopic malaria infection, as inconsistent and false-negative results can occur

The effectiveness of mobile application for monitoring diabetes mellitus and hypertension in the adult and elderly population: systematic review and meta-analysis

Abstract Context Arterial Hypertension (AH) and Diabetes Mellitus (DM) are diseases that are getting worse all over the world. Linked to this advance, is the growing digital health market with numerous mobile health applications, which aim to help patients and professionals in the proper management of chronic diseases. The aim of this study was to analyze, through a systematic review and meta-analysis, the effectiveness of using mobile health applications in monitoring AH and/or DM in the adult and elderly population. Methods The systematic review and meta-analysis was carried out in accordance with the Preferred Reporting Items for Systematic Reviews and Metanalyses guidelines and involved searching five databases – Medline/PubMed, Embase, CINAHL, Virtual Library in Health and Cochrane Library. The review included randomized and cohort clinical trials testing the effects of the intervention on changing biochemical parameters and clinical efficacy in people treated for AH and/or DM. The quality of the selected studies was assessed based on the evaluation criteria of the Joanna Briggs Institute tool. The random effects meta-analysis method was used to explain effect distribution between studies, by Stata® software (version 11.0) and publication bias was examined by visual inspection of graphs and Egger test. Results We included 26 studies in the systematic review and 17 in the meta-analysis. These studies were published between 2014 to 2022 in 14 countries. Were reported improvement in knowledge and self-management of AH and DM, social motivation with treatment and behavioral change, reduction in glycated hemoglobin values, fasting glucose and blood pressure, improvement in adherence to drug treatment, among others. The result of the meta-analysis showed that there is evidence that the use of mobile applications can help reduce glycated hemoglobin by 0.39% compared to the usual care group. Conclusions Monitoring and self-monitoring of behaviors and health care related to AH and DM in adults and the elderly through mobile applications, has clinically significant effectiveness in reducing glycated hemoglobin levels. Future studies should provide more evidence and recommendations for best practices and development of digital health interventions. Trial registration PROSPERO. International Prospective Registry of Systematic Reviews. CRD42022361928

Prospective assessment of malaria infection in a semi-isolated Amazonian indigenous Yanomami community: Transmission heterogeneity and predominance of submicroscopic infection.

In the Amazon basin, indigenous forest-dwelling communities typically suffer from a high burden of infectious diseases, including malaria. Difficulties in accessing these isolated ethnic groups, such as the semi-nomadic Yanomami, make official malaria data largely underestimated. In the current study, we longitudinally surveyed microscopic and submicroscopic malaria infection in four Yanomami villages of the Marari community in the northern-most region of the Brazilian Amazon. Malaria parasite species-specific PCR-based detection of ribosomal and non-ribosomal targets showed that approximately 75% to 80% of all malaria infections were submicroscopic, with the ratio of submicroscopic to microscopic infection remaining stable over the 4-month follow-up period. Although the prevalence of malaria infection fluctuated over time, microscopically-detectable parasitemia was only found in children and adolescents, presumably reflecting their higher susceptibility to malaria infection. As well as temporal variation, the prevalence of malaria infection differed significantly between villages (from 1% to 19%), demonstrating a marked heterogeneity at micro-scales. Over the study period, Plasmodium vivax was the most commonly detected malaria parasite species, followed by P. malariae, and much less frequently P. falciparum. Consecutive blood samples from 859 out of the 981 studied Yanomami showed that malaria parasites were detected in only 8% of the previously malaria-positive individuals, with most of them young children (median age 3 yrs). Overall, our results show that molecular tools are more sensitive for the identification of malaria infection among the Yanomami, which is characterized by heterogeneous transmission, a predominance of low-density infections, circulation of multiple malaria parasite species, and a higher susceptibility in young children. Our findings are important for the design and implementation of the new strategic interventions that will be required for the elimination of malaria from isolated indigenous populations in Latin America

Detection of Plasmodium simium gametocytes in non-human primates from the Brazilian Atlantic Forest

Abstract Background Plasmodium species of non-human primates (NHP) are of great interest because they can naturally infect humans. Plasmodium simium, a parasite restricted to the Brazilian Atlantic Forest, was recently shown to cause a zoonotic outbreak in the state of Rio de Janeiro. The potential of NHP to act as reservoirs of Plasmodium infection presents a challenge for malaria elimination, as NHP will contribute to the persistence of the parasite. The aim of the current study was to identify and quantify gametocytes in NHP naturally-infected by P. simium. Methods Whole blood samples from 35 NHP were used in quantitative reverse transcription PCR (RT-qPCR) assays targeting 18S rRNA, Pss25 and Pss48/45 malaria parasite transcripts. Absolute quantification was performed in positive samples for 18S rRNA and Pss25 targets. Linear regression was used to compare the quantification cycle (Cq) and the Spearman's rank correlation coefficient was used to assess the correlation between the copy numbers of 18S rRNA and Pss25 transcripts. The number of gametocytes/µL was calculated by applying a conversion factor of 4.17 Pss25 transcript copies per gametocyte. Results Overall, 87.5% of the 26 samples, previously diagnosed as P. simium, were positive for 18S rRNA transcript amplification, of which 13 samples (62%) were positive for Pss25 transcript amplification and 7 samples (54%) were also positive for Pss48/45 transcript. A strong positive correlation was identified between the Cq of the 18S rRNA and Pss25 and between the Pss25 and Pss48/45 transcripts. The 18S rRNA and Pss25 transcripts had an average of 1665.88 and 3.07 copies/µL, respectively. A positive correlation was observed between the copy number of Pss25 and 18S rRNA transcripts. Almost all gametocyte carriers exhibited low numbers of gametocytes (< 1/µL), with only one howler monkey having 5.8 gametocytes/µL. Conclusions For the first time, a molecular detection of P. simium gametocytes in the blood of naturally-infected brown howler monkeys (Alouatta guariba clamitans) was reported here, providing evidence that they are likely to be infectious and transmit P. simium infection, and, therefore, may act as a reservoir of malaria infection for humans in the Brazilian Atlantic Forest