A novel metabarcoded 18S ribosomal DNA sequencing tool for the detection of Plasmodium species in malaria positive patients.

Various PCR based methods have been described for the diagnosis of malaria, but most depend on the use of Plasmodium species-specific probes and primers; hence only the tested species are identified and there is limited available data on the true circulating species diversity. Sensitive diagnostic tools and platforms for their use are needed to detect Plasmodium species in both clinical cases and asymptomatic infections that contribute to disease transmission. We have recently developed for the first time a novel high throughput 'haemoprotobiome' metabarcoded DNA sequencing method and applied it for the quantification of haemoprotozoan parasites (Theleria and Babesia) of livestock. Here, we describe a novel, high throughput method using an Illumina MiSeq platform to demonstrate the proportions of Plasmodium species in metabarcoded DNA samples derived from human malaria patients. Plasmodium falciparum and Plasmodium vivax positive control gDNA was used to prepare mock DNA pools of parasites to evaluate the detection threshold of the assay for each of the two species. The different mock pools demonstrate the accurate detection ability and to show the proportions of each of the species being present. We then applied the assay to malaria-positive human samples to show the species composition of Plasmodium communities in the Punjab province of Pakistan and in the Afghanistan-Pakistan tribal areas. The diagnostic performance of the deep amplicon sequencing method was compared to an immunochromatographic assay that is widely used in the region. The deep amplicon sequencing showed that P. vivax was present in 69.8%, P. falciparum in 29.5% and mixed infection in 0.7% patients examined. The immunochromatographic assay showed that P. vivax was present in 65.6%, P. falciparum in 27.4%, mixed infection 0.7% patients and 6.32% malaria-positive cases were negative in immunochromatographic assay, but positive in the deep amplicon sequencing. Overall, metabarcoded DNA sequencing demonstrates better diagnostic performance, greatly increasing the estimated prevalence of Plasmodium infection. The next-generation sequencing method using metabarcoded DNA has potential applications in the diagnosis, surveillance, treatment, and control of Plasmodium infections, as well as to study the parasite biology.