Background

By January, 2016, all known transmission chains of the Ebola virus disease (EVD) outbreak in west Africa had been stopped. However, there is concern about persistence of Ebola virus in the reproductive tract of men who have survived EVD. We aimed to use biostatistical modelling to describe the dynamics of Ebola virus RNA load in seminal fluid, including clearance parameters.

Methods

In this longitudinal study, we recruited men who had been discharged from three Ebola treatment units in Guinea between January and July, 2015. Participants provided samples of seminal fluid at follow-up every 3–6 weeks, which we tested for Ebola virus RNA using quantitative real-time RT-PCR. Representative specimens from eight participants were then inoculated into immunodeficient mice to test for infectivity. We used a linear mixed-effect model to analyse the dynamics of virus persistence in seminal fluid over time.

Findings

We enrolled 26 participants and tested 130 seminal fluid specimens; median follow up was 197 days (IQR 187–209 days) after enrolment, which corresponded to 255 days (228–287) after disease onset. Ebola virus RNA was detected in 86 semen specimens from 19 (73%) participants. Median duration of Ebola virus RNA detection was 158 days after onset (73–181; maximum 407 days at end of follow-up). Mathematical modelling of the quantitative time-series data showed a mean clearance rate of Ebola virus RNA from seminal fluid of −0·58 log units per month, although the clearance kinetic varied greatly between participants. Using our biostatistical model, we predict that 50% and 90% of male survivors clear Ebola virus RNA from seminal fluid at 115 days (90% prediction interval 72–160) and 294 days (212–399) after disease onset, respectively. We also predicted that the number of men positive for Ebola virus RNA in affected countries would decrease from about 50 in January 2016, to fewer than 1 person by July, 2016. Infectious virus was detected in 15 of 26 (58%) specimens tested in mice.

Interpretation

Time to clearance of Ebola virus RNA from seminal fluid varies greatly between individuals and could be more than 13 months. Our predictions will assist in decision-making about surveillance and preventive measures in EVD outbreaks.

Funding

This study was funded by European Union's Horizon 2020 research and innovation programme, Directorate-General for International Cooperation and Development of the European Commission, Institut national de la santé et de la recherche médicale (INSERM), German Research Foundation (DFG), and Innovative Medicines Initiative 2 Joint Undertaking.

The Ebola virus disease (EVD) outbreak in west Africa lasted more than 2 years, affected almost 29 000 people in Guinea, Liberia, and Sierra Leone, and resulted in more than 11 000 deaths. ¹ On Jan 14, 2016, WHO declared the end of the EVD outbreak in Liberia, with all known chains of transmission stopped in west Africa. ² However, there is concern about persistence of the Ebola virus in survivors, specifically in the male reproductive tract. ^{3 and 4} In conjunction with the discovery of filoviruses in 1967 in Marburg, Germany, Marburg virus has been shown to be sexually transmitted by men who shed the virus in seminal fluid after recovery from the disease. ⁵ Reports from EVD patients subsequently confirmed virus persistence in seminal fluid after recovery for Ebola and Sudan viruses. ^{6 , 7 , 8 , 9 , 10 and 11} A cross-sectional study ¹² detected Ebola virus RNA in seminal fluid of EVD survivors of the 2014–16 epidemic up to 9 months after disease onset. However, there are no quantitative virus-load data for seminal fluid to facilitate mathematical modelling of virus clearance.

Research in context

Evidence before this study

Before this study, reports from previous Ebola and Marburg virus disease outbreaks indicated that filoviruses may be sexually transmitted by male survivors and that seminal fluid of survivors may contain filovirus RNA or infectious particles. We did a PubMed search (1960 to March 31, 2015) using the search string (ebola virus OR ebola fever OR marburg virus OR marburg fever OR filovirus) AND (persistent OR persistence OR semen OR seminal fluid OR sexual transmission) [converted by PubMed into a longer string with 43 terms] and retrieved 83 citations. The search was complemented by gathering information from review articles identified through the PubMed search. We identified five original articles published between 1968 and 2007 that were relevant to the design of our study. These studies described sexual transmission of Marburg virus in one case, and detection of Marburg, Ebola, or Sudan virus or corresponding RNA in seminal fluid of seven of eight male survivors (88%) cumulatively tested. The longest time after the onset that a semen specimen tested positive was 101 and 82 days by RT-PCR and virus isolation, respectively. While these studies provided proof-of-concept for filovirus persistence in seminal fluid, we designed a larger longitudinal study to quantitatively describe the dynamics of Ebola virus RNA load in seminal fluid and estimate the parameters of the clearance kinetic using mathematical modelling. Before submission of this manuscript, we undertook another PubMed search using the same terms to consider all evidence that accumulated during implementation of our study (April 1, 2015, to Feb 21, 2016). The search retrieved 40 citations, of which three were relevant to the interpretation of our data. One cross-sectional study, which tested one seminal-fluid specimen per survivor, detected Ebola virus RNA in 46 of 93 specimens (49%). The longest time after the onset that a specimen tested positive was 284 days. Two articles described one case of sexual transmission of Ebola virus.

Added value of this study

This study provides longitudinal data that allows systematic investigation of the dynamics of Ebola virus RNA concentrations in seminal fluid in a cohort of male survivors. The quantitative time-series data revealed considerable variability between participants in the clearance of Ebola virus RNA from seminal fluid. Although rare, survivors can continuously shed Ebola virus RNA for more than 13 months after disease onset. We also describe the clearance kinetic as well as its variability using biostatistical modelling.

Implications of all the available evidence

The longitudinal data described in this study and cross-sectional data are complementary. Biostatistical modelling of these datasets facilitates reliable prediction of the proportion of men with seminal fluid positive for Ebola virus RNA over time after onset of disease and their prevalence during an epidemic. These predictions will assist in decision-making about surveillance and preventive measures in Ebola virus disease outbreaks.

In this longitudinal study, we measured viral load in seminal fluid from a cohort of men discharged from three Ebola treatment units (ETUs) in Guinea between January and July, 2015. We aimed to collect sequential samples of seminal fluid every 3–6 weeks from participants during follow-up to analyse patterns of intensity and duration of Ebola virus shedding in seminal fluid over time.

We recruited men aged 18–65 years who had had a PCR-confirmed EVD diagnosis, and were discharged from the ETUs in Conakry-Gbessia, Forecariah, or Coyah, Guinea, between Jan 20, 2015, and July 6, 2015. We contacted patients who had been discharged between Jan 20 and May 5, 2015, by telephone; and prospectively enrolled patients who had been discharged between May 6 and July 6, 2015, during their routine 1 month follow-up visit.

We followed up participants at the study centre established at the ETU in Coyah. Participants provided a sample of seminal fluid through masturbation in a private environment. Follow-up visits were scheduled every 3–6 weeks until two consecutive samples were negative for Ebola virus RNA on PCR, as recommended by WHO. ³ We telephoned study participants to remind them of follow-up visits 3 days before appointments. At enrolment and each follow-up visit, participants were counselled about safe sexual practices, and received condoms and an expense allowance. The test result was communicated by phone to the participants in conjunction with safe sex counselling.

We collected clinical and demographic data, including date of EVD onset and treatment, from patients' case files. Most study participants had received favipiravir on a compassionate use basis during the acute illness in adherence to the protocol developed for the Jiki trial. ¹³

Participants gave written informed consent and the study was approved by the National Committee for Ethics in Medical Research of Guinea (permit No. 64/CNERS/15).

We processed samples of seminal fluid from participants immediately after collection at the European mobile laboratory (EMLab) unit in Coyah. Workers extracted RNA from 50 μL of seminal fluid using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) after addition of the internal control RNA for the PCR assay. We tested samples for Ebola virus using the RealStar Zaire Ebolavirus RT-PCR Kit (altona Diagnostics, Hamburg, Germany; this kit is derived from the RealStar Filovirus Screen RT-PCR Kit and contains only the reagents for detection of Ebola virus) on a Rotor-Gene Q thermocycler (Qiagen). ¹⁴ We included only the runs with valid internal control in analysis and we used threshold cycle (Ct) as an inverse measure of Ebola virus RNA concentration in the specimen. The high precision of the kit facilitates pooling of Ct values from separate runs for biostatistical analysis ( appendix p 1 ). Relative virus RNA concentration (c_rel, in arbitrary units) was estimated from the Ct values using an equation (equation 1) based on experimentally generated standard curves ( appendix p 1 ).