Validation of a new real-time PCR based kit for detection and typing of 5 human pathogenic Plasmodium species, including P. knowlesi in whole blood specimen

Abstract

Purpose: Malaria remains an important cause of morbidity and mortality worldwide. Even in settings in which malaria is not endemic, infections can be seen in individuals who have travelled to or emigrated from regions with ongoing malaria transmission. Human infection is caused by five Plasmodium species including P. knowlesi, which has emerged as a widespread cause of zoonotic human malaria in Southeast Asia, associated with a high risk of severe disease. Clinically, Plasmodium species are managed differently and it is important to identify the causal agent at species level. The purpose of this study was the validation of a new real-time PCR based Kit for detection and typing of human pathogenic Plasmodium species. Methods & Materials: A collection of 105 whole blood samples from individual patients was included in the study. All samples were previously analyzed and typed by an established in-house workflow at the Centro National de Microbiologia, Madrid, Spain. The collection comprised 5 times 15 specimen pre-typed as P. falciparum, P. vivax, P. malariae, P. ovale, P. knowlesi, respectively, 15 specimen previously tested negative for human pathogenic Plasmodium species and 15 specimen previously tested positive for other parasites causing diseases with similar symptoms like malaria. The specimen were thawed, nucleic acid was extracted using the QIAamp DNA Blood Mini QIAcube kit (Qiagen) and eluates were analyzed with the RealStar® Malaria Screen&Type Kit (altona Diagnostics) on an Rotor-Gene 6000 (Corbett-Research). The Internal Control (IC) of the RealStar® kit was added during the nucleic acid extraction process. Results: All 30 specimens pre-tested negative for human pathogenic Plasmodium species were tested negative with the RealStar® Malaria Screen&Type Kit. The IC showed a valid result in all samples. 74 out of 75 specimen pre-tested positive for a human pathogenic Plasmodium species were tested positive and typed identically to the pre-typing results with the RealStar® Malaria Screen&Type Kit. Conclusion: Our study shows, that the RealStar® Malaria Screen&Type Kit provides a reliable assay with high diagnostic sensitivity and specificity for the detection and typing of human pathogenic Plasmodium species. It can be a useful tool in the management of malaria patients.