Abstract
Increasing numbers of travelers returning from endemic areas, migrants, and refugees have led to a significant rise in the number of imported malaria cases in non-endemic countries. Real- time PCR serves as an excellent diagnostic tool, especially in regions where experience in microscopy is limited. A novel fluorescence resonance energy transfer-based real-time PCR (FRET-qPCR) was developed and evaluated using 56 reference samples of the United Kingdom National External Quality Assessment Service (UK NEQAS) for molecular detection of malaria, including P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi. Species identification is based on single nucleotide polymorphisms (SNPs) within the genome where the MalLC640 probe binds, lowering the melting temperature in the melting curve analysis. The novel FRET-qPCR achieved 100% (n = 56) correct results, compared to 96.43% performing nested PCR. The high sensitivity, with a calculated limit of detection of 199.97 parasites/mL blood for P. falciparum, is a significant advantage, especially if low-level parasitemia has to be ruled out. Even mixed infections of P. falciparum with P. vivax or P. ovale, respectively, were detected. In contrast to many other real-time PCR protocols, this novel FRET-qPCR allows the quantitative and species-specific detection of Plasmodium spp. in one single run. Solely, P. knowlesi was detected but could not be differentiated from P. vivax. The turnaround time of this novel FRET-qPCR including DNA extraction is less than two hours, qualifying it for routine clinical applications, including treatment monitoring.
Highlights
Malaria is still one of the most important infectious diseases worldwide and remains endemic in many low and middle income countries
According to the Tm value in the fluorescence resonance energy transfer (FRET)-qPCR, the samples were assigned to P. falciparum (Tm 63.5–66 ̊C), P. malariae (Tm 63.0–63.5 ̊C), P. ovale (Tm 58–60 ̊C), and P. vivax/knowlesi (Tm 56–57.5 ̊C), respectively (Fig 1)
Based on the determined Tm values, all positive samples were assigned to the correct Plasmodium species, except that P. knowlesi could not be differentiated from P. vivax because of their identical Tm value (S1 Fig)
Summary
Malaria is still one of the most important infectious diseases worldwide and remains endemic in many low and middle income countries. Considering the apparent advantages of these methods, not all of the published Plasmodium real-time PCR protocols have been fully evaluated: e.g. some cannot differentiate between species [3, 10, 11], some do not detect all species [4, 5, 12] and for others, a genus-specific PCR has to be performed first and has to be followed by additional reactions [8]. These shortcomings are diminishing the apparent benefits of real-time PCR and are prolonging the time to obtain the final result. The aim of the current study was to develop a novel real-time PCR assay that allows a rapid, highly sensitive, quantitative, and species-specific diagnosis of malaria and to validate this PCR for routine diagnostics by evaluating its performance with international references samples
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