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

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.

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.

Ribosomal and non-ribosomal PCR targets for the detection of low-density and mixed malaria infections.

BACKGROUND: The unexpected high proportion of submicroscopic malaria infections in areas with low transmission intensity challenges the control and elimination of malaria in the Americas. The current PCR-based assays present limitations as most protocols still rely on amplification of few-copies target gene. Here, the hypothesis was that amplification of different plasmodial targets-ribosomal (18S rRNA) and non-ribosomal multi-copy sequences (Pvr47 for Plasmodium vivax and Pfr364 for Plasmodium falciparum)-could increase the chances of detecting submicroscopic malaria infection. METHODS: A non-ribosomal real-time PCR assay targeting Pvr47/Pfr364 (NR-qPCR) was established and compared with three additional PCR protocols, two of them based on 18S rRNA gene amplification (Nested-PCR and R-qPCR) and one based on Pvr47/Pfr364 targets (NR-cPCR). The limit of detection of each PCR protocol, at single and artificial mixed P. vivax/P. falciparum infections, was determined by end-point titration curves. Field samples from clinical (n = 110) and subclinical (n = 324) malaria infections were used to evaluate the impact of using multiple molecular targets to detect malaria infections. RESULTS: The results demonstrated that an association of ribosomal and non-ribosomal targets did not increase sensitivity to detect submicroscopic malaria infections. Despite of that, artificial mixed-malaria infections demonstrated that the NR-qPCR was the most sensitive protocol to detect low-levels of P. vivax/P. falciparum co-infections. Field studies confirmed that submicroscopic malaria represented a large proportion (up to 77%) of infections among asymptomatic Amazonian residents, with a high proportion of infections (~ 20%) identified only by the NR-qPCR. CONCLUSIONS: This study presents a new species-specific non-ribosomal PCR assay with potential to identify low-density P. vivax and P. falciparum infections. As the majority of subclinical infections was caused by P. vivax, the commonest form of malaria in the Amazon area, 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.

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?

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.

Desenvolvimento de um protocolo de PCR em Tempo Real para diagnóstico de malária subpatente e infecções mistas por Plasmodium Vivax e Plasmodium falciparum / Development of a PCR protocol Real time to diagnosis of malaria and subpatent mixed infections with Plasmodium Vivax and Plasmodium falciparum

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 etal., 2005) – e uma PCR convencional baseada nos alvos Pvr47/Pfr364 (Demas etal., 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 RT-Mangold,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 deagarose 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...

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

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 etal., 2005) – e uma PCR convencional baseada nos alvos Pvr47/Pfr364 (Demas etal., 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 RT-Mangold,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 deagarose 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.