Plasmodium vivax molecular diagnostics in community surveys: pitfalls and solutions

Abstract

A distinctive feature of Plasmodium vivax infections is the overall low parasite density in peripheral blood. Thus, identifying asymptomatic infected individuals in endemic communities requires diagnostic tests with high sensitivity. The detection limits of molecular diagnostic tests are primarily defined by the volume of blood analysed and by the copy number of the amplified molecular marker serving as the template for amplification. By using mitochondrial DNA as the multi-copy template, the detection limit can be improved more than tenfold, compared to standard 18S rRNA targets, thereby allowing detection of lower parasite densities. In a very low transmission area in Brazil, application of a mitochondrial DNA-based assay increased prevalence from 4.9 to 6.5%. The usefulness of molecular tests in malaria epidemiological studies is widely recognized, especially when precise prevalence rates are desired. Of concern, however, is the challenge of demonstrating test accuracy and quality control for samples with very low parasite densities. In this case, chance effects in template distribution around the detection limit constrain reproducibility. Rigorous assessment of false positive and false negative test results is, therefore, required to prevent over- or under-estimation of parasite prevalence in epidemiological studies or when monitoring interventions.Electronic supplementary materialThe online version of this article (10.1186/s12936-018-2201-0) contains supplementary material, which is available to authorized users.

Utility of ultra-sensitive qPCR to detect Plasmodium falciparum and Plasmodium vivax infections under different transmission intensities

“…For quanti cation of P. falciparum parasites in the PNG sample set, a serial dilution of cultured 3D7 parasites was used. P. vivax samples were quanti ed using a standard curve generated from dilutions of P. vivax control plasmids containing inserts corresponding to the respective qPCR or us-qPCR target sequences [2,10].…”

“…The most widely used qPCR assays for detection of Plasmodium species target genus-or species-speci c regions within the 18S rRNA genes [2,[7][8][9]. The genomes of Plasmodium falciparum (Pf) and P. vivax (Pv) harbour 5 and 3 copies of 18S rRNA genes, however, some copies show substantial sequence variation so that primers and probes for P. vivax target only one copy of this gene family [10]. Comparison of the widely used 18S rRNA assays with ultrasensitive detection methods uncovered a 2-4 times higher parasite prevalence of low-density malaria infections at medium and low transmission settings that previously remained undiagnosed by less sensitive detection tools [6,11,12].…”

“…Enhanced detection sensitivity by qPCR can be achieved in three ways: rstly, by processing larger volumes of blood and concentrating the DNA during the extraction procedure [11,13], secondly, by using ultra-sensitive-qPCR (us-qPCR) assays that target multi-copy sequences in the parasite genome [10,11,[14][15][16] instead of a single-copy or low-copy gene, and thirdly, by detecting highly abundant 18S rRNA transcripts by quantitative reverse-transcriptase PCR (qRT-PCR) instead of rDNA. Collecting a large volume of blood by venipuncture instead of nger prick is mostly not feasible for large surveys.…”

“…RNA should be preserved using speci c buffers and stored at -80°C to avoid degradation. Compared to DNAbased tests, RNA extraction and qRT-PCR are more costly and the handling and ampli cation of the high copy 18S rRNA transcripts are more prone to contamination [2,10]. Both these methods are, therefore, less practicable for malaria surveillance.…”

“…For ultra-sensitive detection of low-density P. falciparum infections, the Pf_varATS us-qPCR was developed that targets the acidic terminal sequences of multi-copy var genes [11]. For low-density P. vivax infections, Pv_mtCOX1 us-qPCR targeting the cytochrome oxidase 1 gene was designed [10]. COX1 is encoded in the mitochondrial genome and is present in at least 20 copies per cell [17].…”