Development of a Duplex Real Time PCR for Precise Detection of Plasmodium sp. for Clinical Diagnosis in Brazilian Amazon Basin

Molecular methods are more reliable than microscopy and rapid diagnostic tests to accurately diagnose malaria, particularly in situations of low parasitaemia and mixed infections. Among the actions to malaria control, rapid and accurate identification of the species is crucial for a safe and effective treatment, reduction in the risk of transmission and a better understanding of parasites epidemiology, which contribute to the strengthening of malaria control strategies in endemic areas, especially in countries targeting pre-elimination. A TaqMan-based duplex real time PCR qualitative assay for the detection of four species of malaria parasites, Plasmodium vivax, P. falciparum, P. malariae and P. ovale, was evaluated using dried blood spots from patients with fever (axillary temperature >= 37.5 C) or history of fever in last 24h presenting for malaria diagnosis in three localities in Para state, Brazilian Amazon Basin, between 2014 to 2016. Parasite DNA was extracted from dried blood spots using commercial kits. Blood smears were also examined using Giemsa-stained microscopy with two independent readings. The assay was compared to microscopy and to an established nested PCR assay, used as gold-standard test. The specificity of the new assay was confirmed by the lack of cross-reactivity with Trypanosoma cruzi and Leishmania sp. DNA. Out of 312 samples, the sensitivity and specificity (95% CI) of duplex real time PCR were 98.57% (95.3-99.8) and 100.0% (93.4-100), respectively, while the sensitivity and specificity (95% CI) of microscopy were 97.14% (95.5-99.0) and 100.0% (93.0-100), respectively. The correlation between duplex real time PCR and nested PCR showed excellent agreement (kappa=0.98). Further, duplex real time PCR identified a greater number of mixed infections than nested PCR, showing good agreement (kappa=0.87). Our findings highlight that it is possible to implement a duplex real time PCR method for the detection of Plasmodium sp. in clinical samples, being a valuable tool for the accurate diagnosis of malaria, especially in the detection of mixed infections. On the other hand, attempts to evaluate simpler and new molecular methods are needed to investigate the performance of these methods in clinical management at point of care in remote areas of countries such as Brazil