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.

The Ebola virus disease (EVD) outbreak in Western Africa was unprecedented in size. Recent figures placed the total number of EVD cases at >28 000 with more than 11000 deaths [1]. A large number of EVD-infected patients survived with multiple sequelae [2]. Current statistics report that up to 17 000 patients with EVD survived [3], and this number may underrepresent the true number of survivors. Although the etiology of many of these sequelae are unknown, some researchers suggest that a failure to clear Ebola virus (EBOV) ribonucleic acid (RNA) may play a role in at least some survivors [4]. Results of monitoring convalescing survivors have demonstrated the persistence of EBOV genomic material in semen and other immunologically privileged sites for much longer than originally anticipated [5–7]. The presence of EBOV RNA has also raised concerns that lingering virus in survivors may lead to transmission, sparking new outbreaks. The concern that sexual transmission could lead to new clusters of cases was brought to the forefront in March 2015 when a new case of EVD was detected [8, 9]. The single risk factor for this case was unprotected vaginal intercourse with a survivor who had been discharged >150 days from the Ebola Treatment Unit. Although the number of documented infections through sexual contact remains low, the ongoing detection of EBOV RNA in the semen of male survivors warrants a risk mitigation strategy that includes monitoring for new cases and clusters, offering seminal fluid testing to EBOV survivors, and counseling for safe sex practices.

Currently, laboratory processing procedures, test methods, and interpretation of results vary within and between countries, with little to no standardization between laboratories in the West African Region. By using different methods with varying extraction efficiencies and limits of detection (LODs), the potential exists for discordant results between laboratories. Without standardization, interpretation of results across assays and compilation of data across the region in a meaningful way is not possible. In addition, standardization efforts are confounded by varying requirements for the safe handling of materials and the availability of sufficient laboratory resources and infrastructure. Efforts to move to a single platform have been hampered by the lack of comparative studies between assays and across sample types (eg, whole blood, oral swabs, semen) [10, 11].

To address the ongoing need for testing of samples other than whole blood, investigators and researchers are often repurposing the existing available assays. Unfortunately, many of the assays that are currently in use were developed and made available under Section 564 of the Federal Food, Drug, and Cosmetic Act, Emergency Use Authorization (EUA) for the detection of EBOV viral RNA in whole blood, serum samples, or plasma specimens [12]. Typically, limited or no data are available to support the use of these assays on other sample matrices. The lack of data and experience with these assays for testing semen specimens raised appreciable concerns regarding their use in the screening of survivors, interpretation of results, and declaration of negative or positive EBOV status.

Previously, we evaluated 2 of the assays currently in use at the National Reference Laboratory, Liberian Institute for Biomedical Research (LIBR), Monrovia, Liberia, for suitability in detecting the presence of EBOV RNA in semen. Qiagen buffer AVL RNA extraction protocols, as well as the Ebola Zaire Target 1 (EZ1) and major groove binder (MGB) real-time–polymerase chain reaction (RT-PCR) assays were demonstrated to be suitable for testing semen samples when performance was compared to blood samples [10]. In the summer of 2015, the World Health Organization (WHO) began deploying GeneXpert instruments and the Xpert EBOV assay, CE-IVD, to Western Africa with EUA approval for use on blood samples for ongoing diagnostic and surveillance efforts [13]. The platform and assay were selected for their safety, ease of use, previous experience in the region with the instrument for tuberculosis testing, and the sensitivity of the assay. Compared to the existing EZ1 and MGB assays, the GeneXpert instrument and Xpert EBOV assay require less operational training and involve fewer sample manipulations. In addition, the Xpert EBOV sample buffer inactivates infectious EBOV during the first step [14], reducing the risk to laboratory workers. The GeneXpert GX Dx software automatically determines if both the detection of EBOV RNA and internal quality controls are met before reporting a positive result. Thus, the software eliminates the need to interpret results and reduces the potential for the introduction of bias, reporting errors, or user variability in establishing thresholds and interpretation of results.

Based on the potential advantages and the projected use of the GeneXpert system, we compared the performance of this assay using semen samples spiked with live EBOV and survivor samples collected during the Partnership for Research on Ebola Vaccines in Liberia (PREVAIL) III study to the existing EZ1 and MGB assays to determine if the GeneXpert system would be appropriate for widespread regional distribution. The PREVAIL III study is a 5-year EVD natural history study of 1500 survivors and 6000 close household and sexual contacts, assessing EVD clinical sequelae, viral persistence, and transmission potential [15].

METHODS

Generation of Laboratory Semen Samples Spiked With Ebola Virus

Individual normal semen and whole blood samples were procured commercially (Lee Biosolutions; Maryland Heights, MO) from donors and through a Research Donor Protocol. Whole blood samples were collect in 9-mL tubes containing tripotassium and ethylenediaminetetraacetic acid and stored at 2°C–8°C until use. Commercially procured semen was stored at −20°C until used, while semen from the Research Donor Protocol was collected using urine collection cups and stored at 2°C–8°C until use. Samples were from healthy donors deemed immunocompetent and had not ejaculated in the last 48–120 hours.

Ebola virus/H.sapiens-tc/GIN/2014/Makona-C05 isolate was kindly provided by Dr Gary P. Kobinger, Public Health Agency of Canada, Winnipeg, Canada. Stocks were produced and titrated on Vero E6 cells as previously described [10]. The resulting EBOV/Mak virus stock (EBOV/H.sapiens-tc/GIN/2014/Mak-C05, GenBank accession no. KP096420) was used for the generation of all mock clinical samples. All laboratory-generated samples containing live virus were handled and processed in Biosafety Level 4 conditions.

Semen and whole blood samples were spiked with logarithmic dilutions of EBOV/Mak isolate ranging from 10 000 to 10 plaque-forming units (PFUs)/mL. Such samples had a negative matrix control for each donor. Spiked Roswell Park Memorial Institute (RPMI) 1640 medium and negative RPMI 1640 medium were also used as cell-free controls. For determination of the LODs, quarter-log dilutions from 24 unique commercial semen samples were generated and tested.

Xpert Testing of EBOV-Spiked Semen and Whole Blood Samples

Initially, semen samples were tested using the Xpert CE-IVD EBOV assay (Cepheid, Sunnyvale, CA) using the recommended procedure for whole blood samples [14]. Subsequently, minor variations of the recommended protocol were evaluated to address invalid test results, including reducing the input volume of semen from 100 μL to 25 µL and adding dithiothreitol (DTT). Spiked semen (25 µL) was either added directly to the provided guanidinium thiocyanate lysis buffer containers or first mixed with 75 µL RPMI cell culture media and then added to the lysis buffers. Subsequently, all samples were mixed thoroughly, incubated for 10 minutes, and then run as recommended by the manufacturer. As an alternative to adjusting the input volume, 100 µL semen was added directly to the lysis buffer and mixed thoroughly. After at least 10 minutes of incubation at room temperature (RT), 100 μL of 1M DTT (Teknova, Hollister, CA) was added and mixed for 5 minutes at RT. The lysed sample (1 mL) was removed and pipetted into an Xpert Ebola cartridge. All assays were run using the GeneXpert IV system (Cepheid) within 30 minutes of the addition of sample to cartridge, and results were generated using GX Dx software (Cepheid).

Ebola Zaire Target 1 and Major Groove Binder RT-PCR Assays

Total RNA was extracted from aliquots of spiked whole blood or semen using the QIAamp Viral RNA Mini Kit (cat# 52906, Qiagen, Valencia, CA) as directed by the manufacturer. An input volume of 70 μL was used for each extraction, and RNA was eluted in a 70-µL volume. The eluted RNA was run with the EZ1 and the MGB RT-PCR assay kits (Critical Reagents Program, Ft. Detrick, MD) on the LightCycler 96 instrument (Roche Diagnostics, Indianapolis, IN) as outlined in the approved EUA instruction booklet [16].

To determine if 1M DTT would significantly enhance the detection of EBOV RNA in semen samples extracted with Qiagen AVL, 1M DTT was added to semen samples in AVL after a 10-minute incubation period. Following a 5-minute incubation period at RT, the samples were extracted according to the Qiagen manufacturer’s directions.

Performance Testing on Clinical Samples

Male survivors enrolled in the PREVAIL III study can voluntarily have their semen tested after 6 months in the study; the samples used here were collected between 9–24 months after EVD symptom onset, with a median of 18 months. Samples were obtained and used with participant consent under the Partnership for Research on Ebola Virus in Liberia’s Ebola Natural History Study (ID NCT02431923). The PREVAIL III semen samples with sufficient volume were tested with the EZ1, MGB, and the modified Xpert Ebola assays. All samples were stored at 37°C at the collection site and during transport to the laboratory. Samples received at night were refrigerated at 4°C overnight. Samples were placed at 37°C as needed to improve sample viscosity. Laboratory staff were blinded to the identity of the donor and the time since discharge from the Ebola Treatment Unit. RNA extractions were performed using the Qiagen AVL protocol as outlined in the EUA instruction booklet and per the manufacturer’s recommendations. Extracted RNA was stored at −80°C for up to 5 days prior to RT-PCR testing. The EZ1, MGB, and ribonuclease P (RNase P) assays were performed as outlined in the approved Emergency Use Authorization (EUA) instruction booklet with minor modifications to the plate layout [16]. Samples tested at LIBR were performed in appropriate personal protective equipment under approved engineering controls.

Interpretation of RT-PCR Results

For the Xpert Ebola assay, samples are positive for EBOV RNA if either the glycoprotein (GP) or nucleoprotein (NP) target is detected by the GeneXpert GX Dx software. If either of the internal controls, specimen processing control (SPC), or Cepheid internal control (CIC) fails, the sample is considered to be invalid and requires retesting. The GeneXpert IV instrument (Cepheid) uses an internal algorithm to report RNA detection, removing potential introduction of bias by the operator. EBOV RNA negativity or positivity and the cycle threshold (Ct) values are reported.

For the EZ1 and RNase P assays, the samples were run as outlined by the developers in the EUA information booklet for use on blood samples; the MGB assays were performed using the identical procedures. Sample runs would be considered invalid or failed if a signal was detected in the negative controls or if no signal was detected in either the positive control or the RNase P control. EBOV RNA negativity or positivity and the Ct values were recorded [16].

Statistical Analysis

Statistical analysis was conducted using open-source R software (https://cran.r-project.org/) or RStudio software (https://www.rstudio.com/) and GraphPad software (GraphPad Software, La Jolla, CA). Comparisons among assays were done using McNemar’s test or the modified McNemar’s test for clustered data when appropriate [17]. Correlation is summarized using Spearman’s ρ, and confirmed via bootstrapped sampling to account for multiple samples from the same individual donors. Exact binomial 95% confidence intervals (CIs) are given throughout.

RESULTS

Optimization of the Xpert EBOV Assay

Initially, invalid results were obtained from the Xpert EBOV assay when semen samples were substituted for whole blood samples using standard input volume, 100 µL. The invalid results were due to the failure of the SPC/CIC. Reducing the amount of the starting sample volume to 25 µL or diluting the semen in media substantially reduced the number of failing SPCs observed, but the reduction in input volume could impact the ability of the assay to detect the presence of low quantities of viral RNA (data not shown). As an alternative, the addition of 100 µL of 1M DTT eliminated the observed issues with the SPC and CIC producing reproducible results (Table 1). This method will be referred to as the modified Xpert EBOV assay from this point forward. The addition of 1M DTT to samples placed in Qiagen AVL failed to provide any detectable change in the detection of EBOV RNA (data not shown).

Table 1.

Comparison of Cycle Threshold Values Obtained from Semen and Whole Blood Samples Spiked With Ebola Virus Using the Modified Xpert EBOV Assay

Donor	Sample Matrix a	EBOV Concentration PFUs/mL	Specimen Processing Control	Cepheid Internal Control	Glycoprotein Target	Nucleoprotein Target
1	semen	1000	29.9	32.4	39.1	32.5
	semen	100	29.5	32.2	UD	37.5
	semen	10	28.8	31.9	UD	41.4
	semen	0	29.6	32	UD	UD
2	semen	1000	25.4	31.1	38.9	32.4
	semen	100	26.3	31.5	43.6	36.2
	semen	10	26.7	31.7	UD	41.7
	semen	0	26.5	31.4	UD	UD
3	semen	1000	24.8	31.1	37.7	31.6
	semen	100	25.2	30.6	UD	36
	semen	10	25.6	31.2	UD	UD.
	semen	0	25.2	31	UD.	UD
4	semen	1000	26.9	31.4	33.7	27.9
	semen	100	26.6	31	42.6	34.9
	semen	10	27.1	30.6	UD	38.6
	semen	0	26.7	31.1	UD	UD
1	blood	1000	29	31.3	36.2	30.5
	blood	100	28.7	31.2	38.9	33.4
	blood	10	28.8	31.4	43.2	36.1
	blood	0	29.8	31.4	UD	UD

Abbreviations: EBOV, Ebola virus; PFUs, plaque-forming units; UD, undetectable.

^aN = 4 replications.

Comparison of EZ1, MGB, and Modified Xpert Ebola Assays Using EBOV-Spiked Semen

Identical aliquots of spiked semen samples from the same parent sample were tested using the EZ1, MGB, and modified Xpert EBOV assays. The Xpert assay was able to detect lower quantities of viral RNA more consistently than the EZ1 and MGB assays (Table 2).

Table 2.

Comparison of Detection of EBOV in Spiked Semen Samples Using Modified Xpert EBOV Assay or Existing MGB and EZ-1 Assays

Percent Consensus in Detection of GP and NP Targets from Semen Samples by Assay a
EBOV Concentration (PFUs/mL)	EZ1 and MGB % Consensus	GeneXpert % Consensus	P Value b
100	2/6 (33)	6/6 (100)	.125
10	0/6 (0)	3/6 (50)	.25
5	0/6 (0)	0/6 (0)	1.000
GP Percent-Positive Semen Samples by Assay
EBOV Concentration (PFUs/mL)	EZ1 and MGB GP + (%)	GeneXpert GP + (%)	P Value
100	2/6 (33)	6/6 (100)	.125
10	0/6 (0)	3/6 (50)	.25
5	0/6 (0)	0/6 (0)	1.000
NP Percent-Positive Semen Samples by Assay
EBOV Concentration (PFUs/mL)	EZ1 and MGB NP + (%)	GeneXpert NP + (%)	P Value
100	5/6 (83)	6/6 (100)	1.000
10	5/6 (83)	6/6 (100)	1.000
5	4/6 (67)	4/6 (67)	1.000

Abbreviations: EBOV, Ebola virus; EZ1, Ebola Zaire target 1; GP, glycoprotein; MGB, major groove binder; NP, nucleoprotein; PFUs, plaque-forming units.

^aConsensus is defined here as both assays (GP target and NP target), resulting in a positive outcome. Analysis was performed on the separate GP and NP targets.

^bN = 6 replicates.

Modified Xpert Ebola Assay Sensitivity, Specificity, and Determination of the Limit of Detection in Semen

To evaluate the specificity and potential for false positives, a panel of 30 unique EBOV-naive semen samples was tested. All of these samples tested negative (data not shown), giving an exact binomial 95% CI for the specificity of (0.88, 1.0). In addition, a total of 290 semen samples from 130 Liberian males who had close contact with Liberian EBOV survivors was also screened; all samples tested negative.

A pilot study was performed using the modified Xpert assay on spiked samples from 4 commercial donors (4/4), suggesting an LOD of 56 and 10 PFUs/mL for the GP and NP assays, respectively (Figure 1A and 1B). Subsequently, a larger panel of normal semen donors was used to validate these findings. The GP assay detected EBOV RNA in 71% (17/24) of samples at 56 PFUs/mL (95% CI: .46, .88) for the sensitivity, and 96% (23/24) of samples at 100 PFUs/mL (95% CI: .75, 1.0), indicating 100 PFUs/mL is a more accurate estimate of the LOD for the this assay for semen samples (Figure 1C). From the NP assay, 96% of samples at 10 PFUs/mL concentration were detected (Table 3). Of note, PFUs/mL calculations were extrapolated from a titer obtained from a plaque assay of a single viral stock. Although the stock used in these experiments has been well characterized with established acceptance criteria for the assays used, the ratio of viral genome to PFUs may vary between viral stocks.

Figure 1.

Determination of the LODs for all EBOV-spiked semen samples using the modified Xpert assay. Preliminary and expanded quarter-log dilution of EBOV was spiked into semen donor samples. Results of exploratory LOD experiments determined an LOD for both modified Xpert GP (A) and NP targets (B) over 2 days with multiple donors. Asterisks indicate the last dilution point in which all spiked donor samples returned a positive outcome (4/4). C, The LODs for each gene target were tested in an additional 20 donors. A number of samples were unexpectedly undetected at the preselected GP LOD (A; asterisk); the previous quarter-log concentration was 96% positive at 100 PFUs/mL.

Abbreviations: EBOV, Ebola virus; GP, glycoprotein; LODs, limits of detection; NP, nucleoprotein; PFUs, plaque-forming units.

Table 3.

Detection of EBOV Genomic Material in Laboratory-generated Semen Samples and Sensitivity in Two Gene Targets using the Modified Xpert EBOV Assay^a

EBOV Concentration (PFUs/mL)	% Positive (95% CI) Number of Positive/Total Tested
	NP	GP
100	100 (85.8, 100) 24/24	95.8 (78.8, 99.9) 23/24
56	100 (85.8, 100) 24/24	70.8 (48.9, 87.4) 17/24
10	95.8 (78.8, 99.9) 23/24	25.0 (9.8, 46.7) 6/24
Negative	0 (0, 14.2) 0/24	0 (0, 14.2) 0/24

Abbreviations: CI; confidence interval; EBOV, Ebola virus; GP, glycoprotein; NP, nucleoprotein; PFUs, plaque-forming units.

^aLimit of detection was observed at an EBOV concentration of 100 PFUs/mL for the GP target and 10 PFUs/mL for the NP target. No known negative sample tested (n = 24) resulted in a positive outcome.

Direct Comparison of EZ1, MGB, and Modified Xpert Ebola Assays With West African Clinical Samples

Semen samples from EBOV survivors collected under the PREVAIL III study were tested using the EZ1, MGB, and modified Xpert EBOV assays. An initial study was performed on samples (total of 402 samples from 145 participants) received by the laboratory with sufficient volume for testing. Of these samples, 94 samples from 54 individuals had detectable EBOV RNA by at least 1 test (Table 4). The modified Xpert assay detected EBOV RNA more frequently than did the EZ1 and MGB assays. Of the 94 positive samples, 90 were detected by the modified Xpert assay. In addition, RNA encoding the NP gene was more commonly detected than RNA encoding the GP gene, regardless of the assays used. For the modified Xpert EBOV assay, 87 of the 94 positive samples were NP positive, while 45 samples were GP positive. GP was detected only 3 times in the absence of NP using the modified Xpert assay. The correlation between the 2 targets is shown in Figure 2.

Table 4.

Field Evaluation of Semen Samples From EBOV Survivors Using Modified Xpert Ebola, MGB, and EZ1 Assays

Type of Assay	Outcome	Modified Xpert Ebola GP		MGB NP
		Outcome
		Positive	Not Detected	Positive	Not Detected
Modified Xpert Ebola NP	Positive	42	45	10	77
	Not detected	3	312	3a	312
EZ1 GP	Positive	7	1a	6	2
	Not detected	38	356	7	387

Abbreviations: EBOV, Ebola virus; EZ1, Ebola Zaire target 1; GP, glycoprotein; MGB, major groove binder; RT-PCR, real-time–polymerase chain reaction; NP, nucleoprotein.

^aFour samples were labeled as discordant in that EBOV was detected on the EZ1 GP and/or MGB NP but not detected on the modified Xpert EBOV assay.

Figure 2.

Ct correlation of NP versus GP targets from modified Xpert CE-IVD assays. Plot consists of 145 unique survivors tested several times (402 samples in total) for the prevalence of EBOV RNA in semen. Correlation of Ct values between NP and GP targets was strong (Spearman’s ρ = 0.68, both unadjusted and boot-strapped). Dotted sloping line represents the line of best fit from all samples for which both targets were detected. Horizontal and vertical dashed lines represent the assay cut-off point and only serve as delineation between tests resulting in a Ct value versus undetected.

Abbreviations: Ct, cycle threshold; EBOV, Ebola virus; GP, glycoprotein; NP, nucleoprotein; RNA, ribonucleic acid.

Of the 94 positive samples, 15 were detected using conventional RT-PCR. Similar to the modified Xpert CE-IVD assay, the NP target detected RNA in 13 samples, while the GP target detected RNA in 8 samples. When comparing platforms, the conventional RT-PCR only detected EBOV RNA in the absence of GeneXpert detection in 4 samples (Table 4, gray cells). To test the null hypothesis that the frequency of positive samples is the same across the assays, we used a modification of McNemar’s test, which accounts for multiple samples coming from the same individual [17].

DISCUSSION

The need to maintain real-time EBOV testing of EVD survivors and contacts in Western Africa are ongoing components of surveillance efforts and survivor care. Simultaneously, efforts are underway in the 3 Ebola-affected countries to support male survivors’ concerns about seminal viral persistence, fears of infecting loved ones, and uncertainties regarding conceiving children. Based on molecular and epidemiologic data, the WHO considers that at least 12 cluster outbreaks have come from the survivor community [18]. Yet, variability in testing methods and differential interpretation of results preclude an accurate assessment of prevalence of regional seminal carriage in male EVD survivors, making risk mitigation challenging. To address these needs, a safe, sustainable, reliable, and systematic approach to seminal testing in the West African subregion that provides sensitive and reproducible results is critical.

To help build sustainable EVD diagnostic capacity during the summer of 2015, the WHO, working with the Foundation for Innovative New Diagnostics (FIND), the respective Ministries of Health, and other organizations began deploying the GeneXpert instrument with the EBOV assays across the 3 affected West African countries [13]. This closed-cartridge system is simple to use and requires minimal training for operation. In addition, the sample lysis buffer used in this platform inactivates EBOV and reduces the risk to laboratory workers [19]. The sensitivity of the Xpert EBOV assay is higher than conventional PCR assay, as indicated by the lower LOD for this assay compared to conventional PCR assays used during the outbreak. During the development of the original Xpert EBOV assay, the specificity and the potential for false positives in blood was extensively assessed by evaluating 500 EBOV-negative blood samples and confirming negativity by the manufacturer (Blake Denison, personal communication). Based on these data, an algorithm was developed and integrated into the Xpert GX Dx software to generate results without the potential for the introduction of bias or variability from user interpretation. Use of the Xpert GX Dx software across laboratories standardizes results. Similar to the other assays in use, a limitation for the Xpert EBOV assay is the lack of development and testing for sample types other than whole blood.

In this paper, we developed a simple modification to the existing Xpert EBOV assay to enable analysis of seminal fluid samples. The data presented using both spiked and clinical samples demonstrate that the GeneXpert EBOV assay is reliable for testing of semen samples. Furthermore, in a head-to-head comparison, the modified Xpert EBOV assay outperformed 2 frequently used EBOV diagnostic PCR assays [20]. Using both the Xpert Ebola assay and conventional PCR tests, a total of 94 EBOV RNA-positive samples were identified from 402 semen samples. Of these 94 positive EBOV samples, less than 17% were detected by the existing conventional PCR, and only 4 samples that tested EBOV-positive using the conventional PCR were not detected by the modified Xpert assay. The discordance in the detection of EBOV RNA between conventional RT-PCR and the modified Xpert EBOV assay may be an indication of primer and probe mismatches due to genetic drift of the EBOV GP and NP genes, or it may be attributed to a low concentration of EBOV RNA in the samples. In addition, given the open nature of conventional PCR assays, there is a greater risk for the introduction of low levels of contaminants. This risk has often led to the use of a “Ct-cutoff” in which samples with Ct values in the upper-30 to lower-40 range are often considered suspect by some (but not all) laboratories. Although the protocols differ by laboratory and assays, often samples with higher Ct values will be flagged for either a repeat test, confirmation by auxiliary testing, or the testing of a subsequent sample collected 24–72 hours later. The use of a closed-cartridge system reduces the potential for introduction of contamination and may increase confidence in the validity of higher Ct values. In addition, testing of large panels of negative samples in both control and real-world settings further builds confidence in the acceptance of positive results at higher Ct values. The analysis of 290 semen samples from close contacts at LIBR and 30 commercially obtained normal donors with the modified Xpert EBOV assay all produced negative results.

The lack of false positives from either the close contacts or the spiked laboratory-generated samples and the determined LODs using spiked samples support the use of this assay as the preferred choice for testing of semen for EBOV RNA. The distribution of the Cepheid platform by WHO across the 3 EVD-affected countries facilitates potential adoption of a single platform for EBOV RNA testing of semen and blood samples. Furthermore, given the enhanced sensitivity, this Xpert EBOV assay should be considered as the gold standard for the assessment of new PCR assays as they become available.

Notes

Acknowledgments. Ebola virus/Makona-C05 isolate was kindly provided by Dr Gary P. Kobinger, Public Health Agency of Canada, Winnipeg, Canada. The authors thank Dr Peter Jahrling, Dr Michael Sneller, and Dr Clifford Lane for their support and scientific input, and Laura Bollinger for critically editing this manuscript.

Financial support. This work was supported in part by Battelle Memorial Institute’s prime contract with the US National Institute of Allergy and Infectious Diseases (Contract # HHSN272200700016I). J. P., E. S., J. M., K. J., B. D.-K., K. T., and J. L. performed this work as employees of Battelle Memorial Institute.

Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.