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

Echeverry, Diego F., Deason, Nicholas A., Davidson, Jenna, Makuru, Victoria, Xiao, Honglin, Niedbalski, Julie, Kern, Marcia, Russell, Tanya L., Burkot, Thomas R., Collins, Frank H., and Lobo, Neil F. (2016) Human malaria diagnosis using a single-step direct-PCR based on the Plasmodium cytochrome oxidase III gene. Malaria Journal, 15. 128. pp. 1-12.

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Background: Nested 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.

Methods: The 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.

Results: The 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 ovale wallikeri.

Conclusions: The 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.

Item ID: 43425
Item Type: Article (Research - C1)
ISSN: 1475-2875
Keywords: malaria, blood spots, 18s-rRNA, cytochrome oxidase III, diagnostic, PCR, direct PCR, Plasmodium, Plasmodium ovale, submicroscopic infections
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© 2016 Echeverry et al.

This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (, which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated.

Funders: Bill and Melinda Gates Foundation (BMGF)
Projects and Grants: BMGF Grant No. 45114
Date Deposited: 22 Jul 2016 02:58
FoR Codes: 32 BIOMEDICAL AND CLINICAL SCIENCES > 3207 Medical microbiology > 320704 Medical parasitology @ 100%
SEO Codes: 92 HEALTH > 9202 Health and Support Services > 920203 Diagnostic Methods @ 75%
92 HEALTH > 9201 Clinical Health (Organs, Diseases and Abnormal Conditions) > 920109 Infectious Diseases @ 15%
92 HEALTH > 9204 Public Health (excl. Specific Population Health) > 920404 Disease Distribution and Transmission (incl. Surveillance and Response) @ 10%
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