Human malaria diagnosis using a single-step direct-PCR based on the Plasmodium cytochrome oxidase III gene.

Diego F Echeverry,Frank H Collins,Marcia Kern,Jenna Davidson,Julie Niedbalski,Thomas R Burkot,Victoria Makuru,Nicholas A Deason,Tanya L Russell,Neil F Lobo,Honglin Xiao

doi:10.1186/s12936-016-1185-x

Abstract

BackgroundNested PCRs based on the Plasmodium 18s-rRNA gene have been extensively used for human malaria diagnosis. However, they are not practical when large quantities of samples need to be processed, further there have been challenges in the performance and when interpreting results, especially when submicroscopic infections are analysed. Here the use of “direct PCR” was investigated with the aim of improving diagnosis in the malaria elimination era.MethodsThe performance of the Plasmodium cytochrome oxidase III gene (COX-III) based novel malaria detection strategies (direct nested PCR and direct single PCR) were compared using a 18s-rRNA direct nested PCR as a reference tool. Evaluations were based on sensitivity, specificity and the ability to detect mixed infections using control blood spot samples and field collected blood samples with final species diagnosis confirmation by sequencing.ResultsThe COX-III direct PCR (limit of detection: 0.6–2 parasites/μL) was more sensitive than the 18s-rRNA direct nested PCR (limit of detection: 2–10 parasites/μL). The COX-III direct PCR identified all 21 positive controls (no mixed infections detected) while the 18s-rRNA direct nested PCR identified 18/21 (including four mixed infections). Different concentrations of simulated mixed infections (Plasmodium vivax and Plasmodium falciparum) suggest that the COX-III direct PCR detects only the predominant species. When the 18s-rRNA direct nested PCR was used to detect Plasmodium in field collected bloods spots (n = 3833), there was discrepancy in the results from the genus PCR (16 % positive) and the species-specific PCR (5 % positive). Further, a large portion of a subset of these positive samples (93 % for genus and 60 % for P. vivax), did not align with Plasmodium sequences. In contrast, the COX-III direct PCR clearly identified (single bands confirmed with sequencing) 2 % positive Plasmodium samples including P. vivax, P. falciparum, Plasmodium malariae and Plasmodium ovalewallikeri.ConclusionsThe COX-III single direct PCR is an alternative method for accurate detection of Plasmodium microscopic and submicroscopic infections in humans, especially when a large number of samples require screening. This PCR does not require DNA isolation, is sensitive, quick, produces confident/clear results, identifies all the Plasmodium species infecting humans, and is cost-effective.

Highlights

Nested PCRs based on the Plasmodium 18s-rRNA gene have been extensively used for human malaria diagnosis
The addition of extra blood spots from negative malaria donors into a reaction did not affect the performance of the single direct COXIII PCR at the lowest P. falciparum detected parasitaemia (Fig. 2b)
When the 18s-rRNA nested PCR was validated with the 21 known positive blood samples, the test showed that all samples were positive with the genus malaria PCR (Fig. 2c) and with the species diagnosis as follows: P. falciparum (n = 7), P. vivax (n = 5), P. malariae (n = 1), P. ovale (n = 1) and mixed infection (n = 4) (Fig. 2d), and three samples did not amplify with any of the speciesspecific primers (Table 3)

Summary

Introduction

Nested PCRs based on the Plasmodium 18s-rRNA gene have been extensively used for human malaria diagnosis They are not practical when large quantities of samples need to be processed, further there have been challenges in the performance and when interpreting results, especially when submicroscopic infections are analysed. The histidine-rich protein 2 (HRP2) based RDT is specific for Plasmodium falciparum [6] These RDTs have a limit of detection around 200 parasites/μL and are influenced by other factors such as deletion of target genes, the method of storage and handling of RDTs, concentration of proteins in blood, the manufacturer and subjectivity in interpretation of results which may affect the accuracy of the tests [7,8,9,10,11]

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