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

Lara Cotta Amaral,Luzia Helena Carvalho,Taís Nóbrega De Sousa,Luiz Felipe Ferreira Guimarães,Jean Ezequiel Limongi,Cristiana Ferreira Alves De Brito,Dhelio Batista Pereira,Flora Satiko Kano,Daniela Rocha Robortella,Cor Jesus Fernandes Fontes

doi:10.1186/s12936-019-2781-3

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 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.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 (~ 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.

Highlights

The unexpected high proportion of submicroscopic malaria infections in areas with low transmission intensity challenges the control and elimination of malaria in the Americas
Since most malaria polymerase chain reaction (PCR)-based protocols still relies on amplification of 18S rRNA gene, which has low sensitivity to detect low-density infections, we evaluated here how useful Pvr47/Pfr364 targets are to detect single and mixed P. vivax and P. falciparum infections in clinical and subclinical malaria
Limit of detection of Pvr47 and Pfr364 NR‐qPCR To determine the limit of detection (LOD) of non-ribosomal real-time PCR (NR-qPCR) protocols, standard curves were constructed for each DNA plasmid carrying the target gene (Pvr47 or Pfr364) through serial dilution ranging from 20,000 to 0.05 copies/μL (Additional file 3)

Summary

Introduction

In order to progress towards malaria control and elimination, it is critical to understand the sources of transmission (the infectious reservoir) and those at risk of infection at the population level [4] In this context, the molecular detection of Plasmodium infections in endemic areas have confirmed previous finding of high frequencies of malaria infections at densities below the limit of conventional microscopic diagnostics [5,6,7,8,9]. The molecular detection of Plasmodium infections in endemic areas have confirmed previous finding of high frequencies of malaria infections at densities below the limit of conventional microscopic diagnostics [5,6,7,8,9] It is relevant as data from systematic reviews have shown that across different geographic areas lowdensity infections may represent the majority of malaria infections [10, 11]. Beyond the practical value of using molecular tools to identify submicroscopic carriers and mixed-species infections, malaria infections at any density may have significant health and socioeconomic consequences [15]

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Results

Conclusion