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

Date Published: April 30, 2019

Publisher: Public Library of Science

Author(s): Sarah E. Schmedes, Dhruviben Patel, Julia Kelley, Venkatachalam Udhayakumar, Eldin Talundzic, Georges Snounou.


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.

Partial Text

Malaria is a significant public health problem, with over 3.3 billion individuals at risk of infection worldwide [1]. In 2016, there were 216 million reported cases and 445,000 deaths in 91 countries [2]. Malaria infections in humans are caused by the parasites Plasmodium falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi [1]. One of the greatest challenges to malaria control and elimination is the rapid spread of drug-resistant infections around the world [2]. In particular, significant effort is now focused on preventing P. falciparum parasites resistant to first-line artemisinin-combination therapies (ACTs) from further spreading from the Greater Mekong Region [3]. Molecular surveillance of P. falciparum drug resistance markers greatly facilitates tracking of drug-resistant parasites.

Here, we describe the utility of using the MaRS targeted-amplicon deep sequencing assay [4] to sequence indiscriminately the full Plasmodium species mitochondrial genome. One of the advantages of deep sequencing the Plasmodium mitochondrial genome is that is allows for species identification, including mixed-species infections. Targeted-amplicon deep sequencing provides a more comprehensive approach for molecular characterization of Plasmodium species infection by providing a high-throughput, quantitative method for species and mixed-species infection identification and improved detection of minor alleles present within a sample [4].