Improving the Molecular Diagnosis of Malaria: Droplet Digital PCR-Based Method Using Saliva as a DNA Source

Abstract

Malaria is an acute febrile disease caused by a protozoan of the genus Plasmodium. Light microscopy (LM) is the gold standard for the diagnosis of malaria. Despite this method being rapid and inexpensive, it has a low limit of detection, which hampers the identification of low parasitemia infections. By using multicopy targets and highly sensitive molecular techniques, it is possible to change this scenario. In this study, we evaluated the performance of droplet digital PCR (ddPCR) to detect Plasmodium DNA obtained from saliva samples (whole saliva and buccal swab) of 157 individuals exposed to malaria transmission from the Brazilian Amazon region. We used the highly sensitive ddPCR method with non-ribosomal multicopy targets for Plasmodium vivax (Pvr47) and Plasmodium falciparum (Pfr364). There was good concordance between the quantitative real-time PCR (qPCR) results from the saliva and blood, except for mixed-species infections. The sensitivity of qPCR was 93% for blood, 77% for saliva, and 47% for swabs. Parasite DNA was not detected in saliva samples in low-density infections compared with the detection in blood samples. ddPCR showed increased sensitivity for detecting Plasmodium in the blood and swabs (99% in blood, 73% in saliva, and 59% in swabs). Notably, ddPCR detected more mixed infections in the blood (15%), saliva (9%), and swabs (18%) than qPCR. Our data showed that the differences between ddPCR and qPCR were the result of a higher number of P. falciparum infections detected by ddPCR. Overall, there was a moderate correlation between parasite densities estimated by the different methods in the blood. Our findings highlight the possibility of using non-invasive sample collection methods for malaria diagnosis by targeting multicopy sequences combined with highly sensitive molecular methods.

A novel sensitive hexaplex high-resolution melt assay for identification of five human Plasmodium species plus internal control

“…PCR-based tests have been extensively described to improve the detection sensitivity and specificity for asymptomatic low parasitemia infections ( Tedla, 2019 ) and are also ideal for diagnosing mixed Plasmodium infections ( Snounou et al., 1993a ). Various PCR-based testing methods have been developed for improving the detection of malaria infection, including nested PCR (nPCR) ( Singh et al., 1999 ; Snounou, 2002 ; Snounou and Singh, 2002 ), droplet digital PCR (ddPCR) ( Costa et al., 2022 ; Koepfli et al., 2016 ; Srisutham et al., 2017 ), and quantitative real-time PCR (qPCR) ( Elsayed et al., 2006 ; Mangold et al., 2005 ; Perandin et al., 2004 ; Rougemont et al., 2004 ; Shokoples et al., 2009 ). Nested PCR is considered to be reliable, allowing for a detection limit as low as 1–0.1 parasites/µL of blood ( Wang et al., 2014 ).…”

Urine and Saliva: Relevant Specimens for Malaria Diagnosis?

“…In addition to nPCR and LAMP, a more recent study tried to improve the detection of Pf and Pv in saliva by using droplet digital PCR (ddPCR), with concurrent evaluation using quantitative PCR (qPCR) [ 37 ]. The ddPCR targeting Pf346 and Pvr47 managed to amplify 76% of Pf and 57% of Pv in saliva samples that were ddPCR-positive in blood, while overall sensitivities of ddPCR in saliva was 73% ( Table 1 ).…”

“…Field samples preserved in ethanol showed superior performance in Plasmodium detection by nPCR to those kept on ice without preservatives, suggesting that preservatives may play a pivotal role in preventing microorganism contamination of field samples that may interfere with the Plasmodial DNA extraction [ 26 ], as well as allow for storage of samples at room temperature without extreme degradation of DNA in samples [ 30 ]. Nonetheless, storage duration of saliva kept at −20 °C or −80 °C was found to have no significant effect on qPCR and ddPCR assays [ 37 ].…”

Malaria Diagnostics

“…Costa et al [35] developed a droplet digital PCR (ddPCR) method for the detection of malaria DNA in saliva. They employed a similar technique to qPCR, using the same probes and primers and a droplet generator.…”