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

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.

Application of nanodiagnostics in point-of-care tests for infectious diseases

“…30 In the early stages, active tuberculosis was diagnosed by culturing the tuberculosis bacteria; 32 however, it may take several weeks to get a result. Recently, people have also developed blood-based diagnostic methods to detect tuberculosis disease using immunology techniques that have higher sensitivity and specificity, 36 but the requirement of skilled technicians and expensive equipment makes it unsuitable in developing countries.…”

“…35 Several approaches have been developed to diagnose malaria, including microscopy, immunology-based, and PCR. 36,37 For a long time, microscopy was the most popular approach to diagnose malaria, especially in developing countries. 34 Only a microscope and a drop of blood to check the malaria-induced parasites are required.…”

Comparison of Nested PCR and Conventional Analysis of <i>Plasmodium</i> Parasites in Kano, Nigeria

“…A study showed that the critical success behind the identification of Plasmodium in the blood stems from the primers used which amplifies the dihydrofolate reductase (DHFR) and cytochrome oxidase III genes, peculiar to this genus [21], [32]. Precise detection should thus be achieved via recombinant DNA techniques.…”

A novel metabarcoded DNA sequencing tool for the detection of Plasmodium species in malaria positive patients

“…Significant progress has been made in the global fight against malaria through high throughput rapid diagnosis. Sensitive diagnostic tools are needed to detect clinically and subclinically infected patients (Echeverry et al, 2016). High-throughput genomic resources have potential applications in the surveillance, diagnosis, control, and treatment of haemoprotozoan diseases, as well as to study the parasite biology (Chaudhry et al, 2019; Shaukat et al, 2019).…”

“…Determination of sequence variations in the hypervariable 18S rDNA cistron can discriminate between Plasmodium species (Agudelo et al, 2013; Haanshuus et al, 2013; Lee et al, 2015; Lefterova et al, 2015), and overcome limitations of traditional microscopic and immunochromatographic methods for the diagnosis of this group of parasites at species level. Molecular methods including qPCR, species-specific PCR, nested PCR, and multiplex PCR have been described (Canier et al, 2013; Cunha et al, 2009; Das et al, 1995; Echeverry et al, 2016; Haanshuus et al, 2013; Steenkeste et al, 2009), but these are low throughput, hence relatively expensive (Chaudhry et al, 2019). These methods depend on the use of species-specific probes and primers, meaning that only the tested species are identified, hence are limited in their ability to describe true circulating species diversity (Moody, 2002).…”