Malaria mitochondrial diagnosis: challenges and pitfalls

High-copy genomic sequences could be used as PCR targets for the detection of Plasmodium infections, providing increased sensitivity over single- or low-copy genes. Mitochondrial genomes of malaria parasites are present in multiple copies in a single mitochondrion, and each parasite has many mitochondria. Here, we describe the development of seven species-specific qPCR assays for the diagnosis of Plasmodium vivax and Plasmodium falciparum, targeting coding and non-coding mitochondrial genomic regions.The optimization of the qPCR protocols involved a gradient of annealing temperatures and concentrations of primers and probes, as well as the inclusion of PCR additives/enhancers [e.g., dimethyl sulfoxide (DMSO), glycerol, bovine serum albumin (BSA)] to improve the specificity of qPCR amplification.Non-specific amplification of other Plasmodium species and of human targets was observed in different levels for all assays. Regardless of the late Cq values for most non-specific amplifications, the application of a cutoff value did not completely exclude false-positive amplification, compromising the specificity and also the sensitivity of the assays.Therefore, although mitochondrial targets have higher sensitivity, they frequently lose specificity due to their high levels of sequence conservation. A screening to evaluate the cross-reaction between Plasmodium species and the non-specific amplification of human malaria-free samples must be performed for Plasmodium mitochondrial assays.