Assessment and Optimization of the GeneXpert Diagnostic Platform for Detection of Ebola Virus RNA in Seminal Fluid

Abstract

Recent studies have suggested that Ebola virus (EBOV) ribonucleic acid (RNA) potentially present in the semen of a large number of survivors of Ebola virus disease (EVD) in Western Africa may contribute to sexual transmission of EVD and generate new clusters of cases in regions previously declared EVD-free. These findings drive the immediate need for a reliable, rapid, user-friendly assay for detection of EBOV RNA in semen that is deployable to multiple sites across Western Africa. In this study, we optimized the Xpert EBOV assay for semen samples by adding dithiothreitol. Compared to the assays currently in use in Liberia (including Ebola Zaire Target 1, major groove binder real-time-polymerase chain reaction assays, and original Xpert EBOV assay), the modified Xpert EBOV assay demonstrated greater sensitivity than the comparator assays. Thus, the modified Xpert EBOV assay is optimal for large-scale monitoring of EBOV RNA persistence in male survivors.

Kikwit Ebola Virus Disease Progression in the Rhesus Monkey Animal Model

“…Samples were tested using the Cepheid GeneXpert. Plasma samples were processed according to the manufacturer’s instructions and semen samples were processed as described previously [ 20 ]. Samples with a failed internal control (SAC, sample adequacy control), but passing internal controls (CIC, Cepheid internal control), were considered negative due to limited cross-reactivity with human and nonhuman primate housekeeping genes.…”

Pan-filovirus one-step reverse transcription-polymerase chain reaction screening assay

“…Therefore, the products of these RT-PCR reactions could serve as appropriate sequencing templates in attempts to identify the filovirus species or strain in a sample. This is important, as for several distinct virus species and lineages of the genus Ebolavirus and Marburgvirus more specific and sensitive tests, such as reverse transcription quantitative real-time PCR (RT-qPCR) assays are available (Gibb et al, 2001, Drosten et al, 2002, Weidmann et al, 2004, Towner et al, 2004, Trombley et al, 2010, U.S. FDA, 2014, Pinsky et al, 2015, Southern et al, 2015, WHO, 2015, TIB MolBiol & Roche, n.d., Cnops et al, 2016, Rieger et al, 2016, Semper et al, 2016, Loftis et al, 2017, Pettitt et al, 2017. Overall, with hundreds of viral genome copies or less, the limit of detection (LOD) of each of these real-time RT-qPCR tests (Clark et al, 2018) is around two orders of magnitude lower than that (~73,600 copies) found for the end-point RT-PCR developed in this study.…”

Recent advances in the development and evaluation of molecular diagnostics for Ebola virus disease

“…The specimen is then added to the reaction cartridge, which is sealed for the duration of the analysis and can be discarded and incinerated after use. The Xpert® Ebola assay is most sensitive at detecting the NP gene, with median cycle thresholds being roughly 5 Cq values lower for NP than for GP [62,64]. The software scores a discrepant result as 'indeterminate', requiring repeat testing and preliminary data suggests the Xpert® Ebola assay is up to a log more sensitive than the Altona assays [32] (Table 1), but the only way to confirm differences in analytical sensitivity between two diagnostic assays is through head-to-head comparison.…”

“…Ebolavirus can persist and be isolated from semen for up to several months after infection [34,67], although documented evidence of transmission from semen is rare. This may be due to ostracizing of Ebola survivors leading to less sexual activity and hence exposure of contacts to seminal fluid or it may suggest the risk of transmission from semen for convalescent patients is far lower than the risk of transmission from those who are acutely ill. Spiking studies have evaluated the Xpert® Ebola assay's performance in semen, which initially flagged internal control failures [64], but this was overcome by reducing the sample volume and addition of dithiothreitol (DTT) to stabilize the RNAdependent DNA polymerase. Conversely, another spiking study (using inactivated Ebola virus) did not report any indeterminate results, and determined LODs of 1000 copies/ml and 275 copies/ml for semen and whole blood, respectively [68].…”