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

Malaria Journal

Dhelio Batista Pereira¹  Cor Jesus Fernandes Fontes²  Luiz Felipe Ferreira Guimarães³  Cristiana Ferreira Alves de Brito³  Flora Satiko Kano³  Lara Cotta Amaral³  Taís Nóbrega de Sousa³  Luzia Helena Carvalho³  Daniela Rocha Robortella³  Jean Ezequiel Limongi⁴ 

[1] Centro de Pesquisas em Medicina Tropical de Rondônia (CEPEM);Hospital Júlio Müller, Universidade Federal do Mato Grosso;Instituto René Rachou, Fundação Oswaldo Cruz, FIOCRUZ-MINAS;Universidade Federal de Uberlândia;

关键词: Malaria;    Molecular diagnosis;    PCR;    Submicroscopic;    Mixed-malaria infections;   

DOI  :  10.1186/s12936-019-2781-3

来源: DOAJ

【 摘 要 】

Abstract 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.

【 授权许可】

Unknown