PCR-based detection of Plasmodium falciparum in saliva using mitochondrial cox3 and varATS primers

Abstract

BackgroundSampling of saliva for diagnosing Plasmodium falciparum infections is a safe, non-invasive alternative to sampling of blood. However, the use of saliva presents a challenge because lower concentrations of parasite DNA are present in saliva compared to peripheral blood. Therefore, a sensitive method is needed for detection of parasite DNA in saliva. This study utilized two recently reported “ultra-sensitive” PCR assays based on detection of the P. falciparum mitochondrial cox3 gene and the multi-copy nuclear varATS gene. The ultra-sensitive assays have an advantage over standard 18S rRNA gene-based PCR assay as they target genes with higher copy numbers per parasite genome. Stored saliva DNA samples from 60 Cameroonian individuals with infections previously confirmed by 18S rRNA gene PCR in peripheral blood were tested with assays targeting the cox3 and varATS genes.ResultsOverall, the standard 18S rRNA gene-based PCR assay detected P. falciparum DNA in 62% of the stored saliva DNA samples, whereas 77 and 68% of the samples were positive with assays that target the cox3 and varATS genes, respectively. Interestingly, the ultra-sensitive assays detected more P. falciparum infections in stored saliva samples than were originally detected by thick-film microscopy (41/60 = 68%). When stratified by number of parasites in the blood, the cox3 assay successfully detected more than 90% of infections using saliva when individuals had > 1000 parasites/μl of peripheral blood, but sensitivity was reduced at submicroscopic parasitemia levels. Bands on electrophoresis gels were distinct for the cox3 assay, whereas faint or non-specific bands were sometimes observed for varATS and 18S rRNA that made interpretation of results difficult. Assays could be completed in 3.5 and 3 h for the cox3 and varATS assays, respectively, whereas the 18S rRNA gene assays required at least 7 h.ConclusionsThis study demonstrates that a PCR assay targeting the cox3 gene detected P. falciparum DNA in more saliva samples than primers for the 18S rRNA gene. Non-invasive collection of saliva in combination with the proposed cox3 primer-based PCR assay could potentially enhance routine testing of P. falciparum during disease surveillance, monitoring, and evaluation of interventions for malaria elimination.Electronic supplementary materialThe online version of this article (10.1186/s41182-018-0100-2) contains supplementary material, which is available to authorized users.

Ribosomal and non-ribosomal PCR targets for the detection of low-density and mixed malaria infections

“…As the sensitivity of any PCR protocol depends largely on the molecular target used [40], the high copy number of Pfr364 (around 20 copies of “subfamily 1” targeted by specific primers) probably facilitated the detection of low levels of P. falciparum in co-infections as compared to 18S rRNA (around 5–8 copies). Although different multi-copy targets have been described as sensitive for molecular diagnosis of malaria [23, 25, 36], those studies did not investigate the reliability of these targets in mixed-malaria infections, which precludes any potential comparison with results described here. In addition, most of the studies have been carried-out in endemic areas, such as Papua New Guinea, that currently does not represent an unstable and low-transmission endemic area [23].…”

“…Although major advances have been reached for the molecular detection of malaria parasites [25, 26, 36, 37], most sensitive PCR-based assays require high-volume of venous blood and complex sample processing [8, 23, 38, 39], being not feasible in the context of malaria routine surveillance. The current study involved investigate the hypothesis that the amplification of both ribosomal and non-ribosomal multi-copy PCR targets could increase the chances of detecting low parasite density and mixed P. falciparum and P. vivax infection.…”

Detection of malaria parasites in dried human blood spots using mid-infrared spectroscopy and logistic regression analysis

“…It is however capable of identifying low-level parasitaemia that are otherwise undetectable by other methods, and distinguishing between individual parasite species. Recent developments in PCR applications have signaled the potential of non-invasive malaria diagnostic options, such as those relying on DNA detection in saliva, urine, sweat, and even faecal excreta [19–21]. Besides, real time PCR assays enable quantitative assessments and comparison of infection loads [15], but are expensive for most laboratories.…”

Detection of malaria parasites in dried human blood spots using mid-infrared spectroscopy and logistic regression analysis

“…It is however capable of identifying low-level parasitaemia that are otherwise undetectable by other methods, and distinguishing between individual parasite species. Recent developments in PCR applications have signaled the potential of non-invasive malaria diagnostic options such as those relying on DNA detection in saliva, urine, sweat and even fecal excreta [19][20][21]. Besides, real time PCR assays enable quantitative assessments and comparison of infection loads [15], but are expensive for most laboratories.…”