Using the Plasmodium mitochondrial genome for classifying mixed-species infections and inferring the geographical origin of P. falciparum parasites imported to the U.S.

Abstract

The ability to identify mixed-species infections and track the origin of Plasmodium parasites can further enhance the development of treatment and prevention recommendations as well as outbreak investigations. Here, we explore the utility of using the full Plasmodium mitochondrial genome to classify Plasmodium species, detect mixed infections, and infer the geographical origin of imported P . falciparum parasites to the United States (U.S.). Using the recently developed standardized, high-throughput Malaria Resistance Surveillance (MaRS) protocol, the full Plasmodium mitochondrial genomes of 265 malaria cases imported to the U.S. from 2014–2017 were sequenced and analyzed. P . falciparum infections were found in 94.7% (251/265) of samples. Five percent (14/265) of samples were identified as mixed- Plasmodium species or non- P . falciparum , including P . vivax , P . malariae , P . ovale curtisi , and P . ovale wallikeri . P . falciparum mitochondrial haplotypes analysis revealed greater than eighteen percent of samples to have at least two P . falciparum mitochondrial genome haplotypes, indicating either heteroplasmy or multi-clonal infections. Maximum-likelihood phylogenies of 912 P . falciparum mitochondrial genomes with known country origin were used to infer the geographical origin of thirteen samples from persons with unknown travel histories as: Africa (country unspecified) (n = 10), Ghana (n = 1), Southeast Asia (n = 1), and the Philippines (n = 1). We demonstrate the utility and current limitations of using the Plasmodium mitochondrial genome to classify samples with mixed-infections and infer the geographical origin of imported P . falciparum malaria cases to the U.S. with unknown travel history.

A powerful qPCR-high resolution melting assay with taqman probe in Plasmodium species differentiation

“…The hydrolysis probe designed for P. falciparum was included to double differentiate the predominant and potentially most virulent parasite ( P. falciparum ) from the other Plasmodium species: P. ovale wallikeri, P. ovale curtisi, P. vivax, P. malariae , and P. knowlesi . In an attempt to increase the assay specificity and sensitivity, the mitochondrial genome was targeted because it is more conserved within each of the Plasmodium species and exists in multiple copies (up to ~ 22 copies) within each parasite [ 13 – 16 ]. The developed assay’s specificity (98.2 % at 95 % CIs) and sensitivity (LODs of 21.47–46.43 copies/µl, equivalent to 1–2.11 parasites/µl) are comparable to those of other studies like Joste et al [ 27 ] and Murillo et al [ 28 ] with 100 % specificity and 1 parasite/µl sensitivity.…”

“…That makes it a desirable candidate for pathogens surveillance and for Plasmodium species diagnosis. Additionally, the mitochondrial genome exists in multiple copies (up to ~ 22 copies) defining it as a good target for Plasmodium species differentiation [ 11 – 16 ].…”

Malaria molecular surveillance in the Peruvian Amazon with a novel highly multiplexed Plasmodium falciparum Ampliseq assay

“…Similar dhfr/dhps/crt/mdr1 haplotypes have also been reported in Suriname, with a high level of gene flow between neighbouring countries in the Guiana shield (Venezuela, Guyana, Brazil) (48), thought to be driven by mobile populations such as gold miners that have access to non-recommended treatments (116,117) and RDTs for self-diagnosis. Predicting the origin of imported infections based on SNP barcodes is technically possible (118)(119)(120), however, for this study samples from neighboring countries were not available to fully investigate the spatial dynamics within the continent.…”

Progress in Parasite Genomics and Its Application to Current Challenges in Malaria Control

“…The cytochrome c oxidase III (COX3) gene plays a very important role in cellular respiration [67]. It is a mitochondrial gene that inherited solely from the female gametocyte and less likely to undergo genetic recombination, making it an ideal candidate for identifying the origin and transmission of the parasites [68]. -Denotes that the sensitivity or specificity is not reported.…”