Analytical Performance Characteristics of the Cepheid GeneXpert Ebola Assay for the Detection of Ebola Virus

Benjamin A Pinsky, Malaya K Sahoo, Johanna Sandlund, Marika Kleman, Medha Kulkarni, Per Grufman, Malin Nygren, Robert Kwiatkowski, Ellen Jo Baron, Fred Tenover, Blake Denison, Russell Higuchi, Reuel Van Atta, Neil Reginald Beer, Alda Celena Carrillo, Pejman Naraghi-Arani, Chad E Mire, Charlene Ranadheera, Allen Grolla, Nina Lagerqvist, David H Persing, Benjamin A Pinsky, Malaya K Sahoo, Johanna Sandlund, Marika Kleman, Medha Kulkarni, Per Grufman, Malin Nygren, Robert Kwiatkowski, Ellen Jo Baron, Fred Tenover, Blake Denison, Russell Higuchi, Reuel Van Atta, Neil Reginald Beer, Alda Celena Carrillo, Pejman Naraghi-Arani, Chad E Mire, Charlene Ranadheera, Allen Grolla, Nina Lagerqvist, David H Persing

Abstract

Background: The recently developed Xpert® Ebola Assay is a novel nucleic acid amplification test for simplified detection of Ebola virus (EBOV) in whole blood and buccal swab samples. The assay targets sequences in two EBOV genes, lowering the risk for new variants to escape detection in the test. The objective of this report is to present analytical characteristics of the Xpert® Ebola Assay on whole blood samples.

Methods and findings: This study evaluated the assay's analytical sensitivity, analytical specificity, inclusivity and exclusivity performance in whole blood specimens. EBOV RNA, inactivated EBOV, and infectious EBOV were used as targets. The dynamic range of the assay, the inactivation of virus, and specimen stability were also evaluated. The lower limit of detection (LoD) for the assay using inactivated virus was estimated to be 73 copies/mL (95% CI: 51-97 copies/mL). The LoD for infectious virus was estimated to be 1 plaque-forming unit/mL, and for RNA to be 232 copies/mL (95% CI 163-302 copies/mL). The assay correctly identified five different Ebola viruses, Yambuku-Mayinga, Makona-C07, Yambuku-Ecran, Gabon-Ilembe, and Kikwit-956210, and correctly excluded all non-EBOV isolates tested. The conditions used by Xpert® Ebola for inactivation of infectious virus reduced EBOV titer by ≥6 logs.

Conclusion: In summary, we found the Xpert® Ebola Assay to have high analytical sensitivity and specificity for the detection of EBOV in whole blood. It offers ease of use, fast turnaround time, and remote monitoring. The test has an efficient viral inactivation protocol, fulfills inclusivity and exclusivity criteria, and has specimen stability characteristics consistent with the need for decentralized testing. The simplicity of the assay should enable testing in a wide variety of laboratory settings, including remote laboratories that are not capable of performing highly complex nucleic acid amplification tests, and during outbreaks where time to detection is critical.

Conflict of interest statement

Competing Interests: Johanna Sandlund, Malaya K. Sahoo, Neil Reginald Beer, Alda Celena Carrillo, Pejman Naraghi-Arani, Chad E. Mire, Charlene Ranadheera, Allen Grolla, and Nina Lagerqvist and Benjamin A. Pinsky have no competing interests. Marika Kleman, Medha Kulkarni, Per Grufman, Malin Nygren, Robert Kwiatkowski, Ellen Jo Baron, Fred Tenover, Blake Denison, Russell Higuchi, Reuel Van Atta, and David H. Persing are employees of Cepheid and receive salary and equity interests in Cepheid. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1. Linearity of the Xpert® Ebola…
Fig 1. Linearity of the Xpert® Ebola Assay and Altona RealStar® Ebolavirus RT-PCR Kit 1.0.
Serial dilutions of inactivated EBOV Yambuku-Mayinga was spiked into EDTA-whole blood and tested in triplicate. Linear regression lines are shown for the Xpert® Ebola nucleoprotein (NP) and glycoprotein (GP) targets (NP: y = -3.44x+46.11, R2 = 0.98; GP: y = -3.87x+51.45, R2 = 0.87), and Altona RealStar® Ebolavirus (y = -4.51x+50.17, R2 = 0.93).

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