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Open Access Highly Accessed Methodology

A novel, single-amplification PCR targeting mitochondrial genome highly sensitive and specific in diagnosing malaria among returned travellers in Bergen, Norway

Christel G Haanshuus1*, Stein C Mohn1, Kristine Mørch1, Nina Langeland2, Bjørn Blomberg12 and Kurt Hanevik12

Author Affiliations

1 National Centre for Tropical Infectious Diseases, Department of Medicine, Haukeland University Hospital, Bergen, Norway

2 Institute of Medicine, University of Bergen, Bergen, Norway

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Malaria Journal 2013, 12:26  doi:10.1186/1475-2875-12-26

Published: 22 January 2013

Abstract

Background

Nested PCR is a commonly used technique in diagnosis of malaria owing to its high sensitivity and specificity. However, it is time-consuming, open to considerable risk of contamination and has low cost-efficiency. Using amplification targets presented in multiple copies, such as rRNA 18S, or mitochondrial targets with an even higher copy number, might increase sensitivity.

Methods

The sensitivity and specificity of two newly designed Plasmodium genus-specific single-round amplification PCR programmes, based on previously published primers targeting 18S and mitochondrial genome, were compared with a widely used nested 18S PCR. Analyses of dilution series from Plasmodium falciparum reference material were performed, as well as retrospective analyses of 135 blood samples, evaluated by routine microscopy, from 132 fever patients with potential imported malaria. Sequencing of the 220 bp mitochondrial PCR products was performed.

Results

At the threshold dilution 0.5 parasites/μl, the sensitivity of the mitochondrial PCR was 97% (29/30 parallels), that of the single-round 18S PCR 93% and the reference nested 18S PCR 87%. All three assays detected as low as 0.05 p/μl, though not consistently. In the patient cohort, malaria was diagnosed in 21% (28/135) samples, defined as positive by at least two methods. Both single-round amplification assays identified all malaria positives diagnosed by nested PCR that had sensitivity of 96% (27/28). The mitochondrial PCR detected one additional sample, also positive by microscopy, and was the only method with 100% sensitivity (28/28). The sensitivity and specificity of the mitochondrial PCR were statistically non-inferior to that of the reference nested PCR. Microscopy missed two infections detected by all PCR assays. Sequencing of the genus-specific mitochondrial PCR products revealed different single nucleotide polymorphisms which allowed species identification of the 28 sequences with following distribution; 20 P. falciparum, six Plasmodium vivax, one Plasmodium ovale and one Plasmodium malariae.

Conclusions

In this study, design of PCR programmes with suitable parameters and optimization resulted in simpler and faster single-round amplification assays. Both sensitivity and specificity of the novel mitochondrial PCR was 100% and proved non-inferior to that of the reference nested PCR. Sequencing of genus-specific mitochondrial PCR products could be used for species determination.

Keywords:
Malaria; Diagnostics; PCR; Amplification; Sequencing; Mitochondrial DNA; 18S; Sensitivity; Gametocytes; Returned travellers