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https://www.arca.fiocruz.br/handle/icict/55148
RIBOSOMAL AND NON-RIBOSOMAL PCR TARGETS FOR THE DETECTION OF LOW-DENSITY AND MIXED MALARIA INFECTIONS
Author
Affilliation
Fundação Oswaldo Cruz. Instituto René Rachou. Belo Horizonte, MG, Brazil
Fundação Oswaldo Cruz. Instituto René Rachou. Belo Horizonte, MG, Brazil/Universidade Federal de Minas Gerais. Belo Horizonte, MG, Brazil
Fundação Oswaldo Cruz. Instituto René Rachou. Belo Horizonte, MG, Brazil
Universidade Federal de Uberlândia. Uberlândia, MG, Brazil
Universidade Federal do Mato Grosso. Hospital Júlio Müller. Cuiabá, MT, Brazil
Centro de Pesquisas em Medicina Tropical de Rondônia. Porto Velho, RO, Brazil
Centro de Pesquisas em Medicina Tropical de Rondônia. Porto Velho, RO, Brazil
Fundação Oswaldo Cruz. Instituto René Rachou. Belo Horizonte, MG, Brazil
Fundação Oswaldo Cruz. Instituto René Rachou. Belo Horizonte, MG, Brazil/Universidade Federal de Minas Gerais. Belo Horizonte, MG, Brazil
Fundação Oswaldo Cruz. Instituto René Rachou. Belo Horizonte, MG, Brazil
Fundação Oswaldo Cruz. Instituto René Rachou. Belo Horizonte, MG, Brazil/Universidade Federal de Minas Gerais. Belo Horizonte, MG, Brazil
Fundação Oswaldo Cruz. Instituto René Rachou. Belo Horizonte, MG, Brazil
Universidade Federal de Uberlândia. Uberlândia, MG, Brazil
Universidade Federal do Mato Grosso. Hospital Júlio Müller. Cuiabá, MT, Brazil
Centro de Pesquisas em Medicina Tropical de Rondônia. Porto Velho, RO, Brazil
Centro de Pesquisas em Medicina Tropical de Rondônia. Porto Velho, RO, Brazil
Fundação Oswaldo Cruz. Instituto René Rachou. Belo Horizonte, MG, Brazil
Fundação Oswaldo Cruz. Instituto René Rachou. Belo Horizonte, MG, Brazil/Universidade Federal de Minas Gerais. Belo Horizonte, MG, Brazil
Fundação Oswaldo Cruz. Instituto René Rachou. Belo Horizonte, MG, Brazil
Abstract
BackgroundThe 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 targetsribosomal (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.MethodsA 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.ResultsThe 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 (similar to 20%) identified only by the NR-qPCR.ConclusionsThis 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.
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