Accurate diagnosis of malaria is fundamental for the adequate treatment of patients, prevention of mortality and disease control. Microscopy is the gold standard for diagnosis, however its low limit of dectection may lead to a misdiagnosis of mixed infections and loss of low-density infections of Plasmodium. The molecular diagnostic methods, combined with the detection of multicopy targets, could overcome these limitations. The present study describes the development of molecular diagnosis methods based on the multicopy targets Pvr47 and Pfr364, respectively, for P. vivax and P. falciparum detection, using the Real-Time PCR (qPCR) and Droplet Digital PCR (ddPCR). Blood, saliva and buccal swab samples were collected from 229 patients of the Brazilian Amazon region between 2017 and 2020. The sensitivity of qPCR in saliva was about 78%; however it was only 47% in buccal swab. In general, malaria parasite DNA was successfully detected in parasite density of ≥ 1500 parasites/µL. A smaller proportion of concordant results was obtained by ddPCR in saliva (52% vs 64% by ddPCR and qPCR, respectively). Noticeably, ddPCR detected a higher proportion of mixed infections in blood (22%), saliva (16%) and buccal swab (26%) than qPCR. Our data show that the differences found between ddPCR and qPCR were the result of a higher number of P. falciparum infections being detected by ddPCR. There were moderate correlation between parasite densities estimated by the different methods in blood. Whereas a significant correlation was observed for all analysis performed for P. vivax, only density estimates obtained by qPCR and LM correlate to each other for P. falciparum. Altogether, our findings show the relevance of using multicopy targets and more sensitive molecular methodologies to detect low parasitemia, making possible the use of alternative sources of DNA.
Funding source: RSTMH, CNPq, FAPEMIG, CAPES