AUTHOR=Costa Gabriel Luíz , Alvarenga Denise Anete Madureira de , Assis Gabriela Maíra Pereira de , Aguiar Anna Caroline Campos , Louzada Jaime , Pereira Dhélio Batista , Pina-Costa Anielle de , Hirano Zelinda Maria Braga , Moreira Sílvia Bahadian , Pissinatti Alcides , Brasil Patrícia , Daniel-Ribeiro Cláudio Tadeu , Sousa Taís Nóbrega de , Alves de Brito Cristiana Ferreira TITLE=Malaria mitochondrial diagnosis: challenges and pitfalls JOURNAL=Frontiers in Tropical Diseases VOLUME=4 YEAR=2023 URL=https://www.frontiersin.org/journals/tropical-diseases/articles/10.3389/fitd.2023.1204195 DOI=10.3389/fitd.2023.1204195 ISSN=2673-7515 ABSTRACT=Background

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.

Methods

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.

Results

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.

Conclusions

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.