Simple Real-Time PCR and Amplicon Sequencing Method for Identification of Plasmodium Species in Human Whole Blood

Abstract

Malaria is the leading identifiable cause of fever in returning travelers. Accurate Plasmodium species identification has therapy implications for P. vivax and P. ovale, which have dormant liver stages requiring primaquine. Compared to microscopy, nucleic acid tests have improved specificity for species identification and higher sensitivity for mixed infections. Here, we describe a SYBR green-based real-time PCR assay for Plasmodium species identification from whole blood, which uses a panel of reactions to detect species-specific non-18S rRNA gene targets. A pan-Plasmodium 18S rRNA target is also amplified to allow species identification or confirmation by sequencing if necessary. An evaluation of assay accuracy, performed on 76 clinical samples (56 positives using thin smear microscopy as the reference method and 20 negatives), demonstrated clinical sensitivities of 95.2% for P. falciparum (20/21 positives detected) and 100% for the Plasmodium genus (52/52), P. vivax (20/20), P. ovale (9/9), and P. malariae (6/6). The sensitivity of the P. knowlesi-specific PCR was evaluated using spiked whole blood samples (100% [10/10 detected]). The specificities of the real-time PCR primers were 94.2% for P. vivax (49/52) and 100% for P. falciparum (51/51), P. ovale (62/62), P. malariae (69/69), and P. knowlesi (52/52). Thirty-three specimens were used to test species identification by sequencing the pan-Plasmodium 18S rRNA PCR product, with correct identification in all cases. The real-time PCR assay also identified two samples with mixed P. falciparum and P. ovale infection, which was confirmed by sequencing. The assay described here can be integrated into a malaria testing algorithm in low-prevalence areas, allowing definitive Plasmodium species identification shortly after malaria diagnosis by microscopy.M alaria infections are a major cause of morbidity and mortality throughout the world, with 198 million cases and 584,000 deaths globally in 2013 and with the heaviest burden of disease seen in developing countries (1). Malaria is also the single most common etiologic agent of febrile illness in travelers returning to areas where malaria is not endemic (2). In a recent GeoSentinel surveillance study, Plasmodium infections accounted for 21% of 6,957 patients with fever in a cohort of 24,920 ill returned travelers (3). Malaria was most frequently diagnosed in febrile persons returning from areas where malaria is endemic such as Sub-Saharan Africa, South and Central Asia, and Latin America (3). Additionally, 33% of mortality among febrile travelers was attributed to malaria (3). Thus, the ability to promptly identify and treat malaria in nonendemic areas is critical. Furthermore, identification of the infecting species is essential for effective treatment of P. vivax and P. ovale. These Plasmodium species possess a dormant hypnozoite stage in the liver that can reactivate and cause disease months to years after the initial infection (4). Primaquine is the only antimalarial drug that can eradicate the hypnozoites and is, t...

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Development of a deep amplicon sequencing method to determine the proportional species composition of piroplasm haemoprotozoa as an aid in their control

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