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

Abstract

BackgroundThe 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 FindingsThis 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.ConclusionIn 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.

Molecular Diagnostic Field Test for Point-of-Care Detection of Ebola Virus Directly From Blood

“…Most diagnostic assays for EBOV detection target either the polymerase, nucleoprotein, or GP genes [8,10,23] For this RT-LAMP assay, we chose the GP gene because it is an important virulence factor of EBOV [21]. The GP gene is the target for other assays, such as Xpert [8], Idylla [9], and another RT-LAMP [7].…”

“…To date, many diagnostic tests such as GeneXpert [8], Idylla [9], real-time reverse transcription-polymerase chain reaction (RT-PCR) assays [10], and immunoassays [11], have been developed for the detection of EBOV, but most of these assays are meant to be used by trained laboratory personnel and require additional infrastructure, such as cold storage of regents, laboratory equipment, and lengthy sample-preparation steps [12]. While these diagnostic tests are easy to run in countries that have a good healthcare system, they are problematic in many parts of the world, because of limited laboratory capacity and logistical infrastructure [13].…”

Development, Evaluation, and Integration of a Quantitative Reverse-Transcription Polymerase Chain Reaction Diagnostic Test for Ebola Virus on a Molecular Diagnostics Platform

“…The GeneXpert Ebola assay (Cepheid) and FilmArray Ebola test (BioFire) are 2 other platforms providing a fully integrated and automated PCR workflow for EVD testing, but they still require preloading manipulation steps [8,9,19,20]. In addition, we demonstrated that the Idylla™ prototype Ebola virus test can withstand a short exposure up to 40°C (2 days) and humidity of 70% at 30°C and that the cartridge reagents are stable for 4 months at 30°C, while most other PCR tests require a cold chain [18].…”

“…In an epidemic context, access to accurate, rapid, highly sensitive, and easy-to-use assays with minimal manipulation of potentially highly infectious samples would therefore be a great asset [6,7]. The outbreak in West Africa spurred the development and clinical evaluation of several RT-PCR assays implemented on automated molecular detection platforms, such as the GeneXpert Ebola assay (Cepheid) [8] and the FilmArray Ebola test (BioFire) [9].…”

Diagnosis of Ebola Virus Disease: Past, Present, and Future

“…An evaluation of the analytical performance of the Xpert Ebola assay showed a limit of detection of 73 viral genome copies/ml for inactivated virus, 1 PFU/ml for infectious virus, and 232 RNA copies/ml (71). The analytic and clinical performance characteristics of the Xpert Ebola assay were further evaluated at the NICD in South Africa (72).…”