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.

Diagnóstico molecular de infecção malárica e maldeias indígenas ianomâmis

Nos últimos anos, a incidência de malária no Brasil vem reduzido significativamente, particularmente, em virtude das medidas de controle. Neste novo cenário, faz-se necessário identificar e tratar a malária submicroscópica, uma vez que estes indivíduos constituem fonte de infecção para o mosquito vetor. Neste contexto, nosso grupo de pesquisa vem aprimorando o diagnóstico molecular de malária e, recentemente, padronizou uma reação em cadeia da polimerase em tempo real (RT-PCR), altamente sensível, para a detecção dealvos não ribossomais (Pvr47 e Pfr364) dos plasmódios. Baseado nestes achados, o objetivo do presente trabalho foi investigar a malária subpatente em tribos indígenas de povos Ianomâmis, onde as baixas parasitemias parecem ser frequentes. A população de estudo foi constituída de indivíduos pertencentes a cinco aldeias ianomâmis do Polo Base Marari, estado do Amazonas. O estudo envolveu dois cortes transversais, sendo realizados nos meses de setembro (primeiro corte transversal) e novembro (segundo corte transversal)de 2014. Foram incluídos no estudo um total de 914 indígenas, com cerca de 1590 amostras de sangue coletadas em papel de filtro. Para o diagnóstico molecular de malária, quatro diferentes protocolos de PCR foram utilizados, sendo três baseados em alvos ribossomais(18S rRNA) e um em alvos não ribossomais (Pvr47/Pfr364) dos plasmódios.

Os resultados aqui obtidos permitiram demonstrar que os alvos Pvr47/Pfr364 foram os mais sensíveis para o diagnóstico de malária submicroscópica, embora não se mostraram adequados para o diagnóstico de espécie. Dentre os protocolos baseados no gene 18S rRNA aqui utilizados(Nested-PCR, RT-Mangold e RT-Rougemont), a Nested-PCR (método molecular dereferência) se mostrou mais apropriada para o diagnóstico específico das infecções maláricas submicroscópicas. Em conjunto, este estudo de infecção malárica em populações ianomâmis permitiu demonstrar que: (i) enquanto 0,9% das amostras foram positivas pelamicroscopia ótica (MO), diagnóstico de rotina, os protocolos moleculares identificaram 7,8% de postividade; (ii) P. vivax foi a espécie predominante, seguido do P. malariae e P. falciparum em proporções similares; (iii) a positividade por malária diminuiu com a idade(crianças>adolescentes>adultos) e foi influenciada pelas variações sazonais(setembro>novembro); (iv) a prevalência de malária variou significativamente nas diferentes aldeias, sugerindo que a doença não está homogeneamente distribuída na área de estudo. Espera-se que os resultados aqui obtidos possam contribuir para o direcionamento e monitoração de medidas de controle em reservas indígenas, bem como estimar a real incidência de malária nas áreas sob vigilância epidemiológica.