Persistence of Ebola virus in semen among Ebola virus disease survivors in Sierra Leone: A cohort study of frequency, duration, and risk factors

A E Thorson; G F Deen; K T Bernstein; W J Liu; F Yamba; N Habib; F R Sesay; P Gaillard; T A Massaquoi; S L R McDonald; Y Zhang; K N Durski; S Singaravelu; E Ervin; H Liu; A Coursier; J E Marrinan; A Ariyarajah; M Carino; P Formenty; U Ströher; M Lamunu; G Wu; F Sahr; W Xu; B Knust; N Broutet; on behalf of the Sierra Leone Ebola Virus Persistence Study Group

doi:10.1371/journal.pmed.1003273

. 2021 Feb 10;18(2):e1003273. doi: 10.1371/journal.pmed.1003273

Persistence of Ebola virus in semen among Ebola virus disease survivors in Sierra Leone: A cohort study of frequency, duration, and risk factors

A E Thorson ^1,^2,^*,^#, G F Deen ^3,^#, K T Bernstein ^4,^#, W J Liu ^5,^#, F Yamba ³, N Habib ¹, F R Sesay ⁶, P Gaillard ¹, T A Massaquoi ⁶, S L R McDonald ¹, Y Zhang ⁵, K N Durski ⁷, S Singaravelu ⁷, E Ervin ⁴, H Liu ⁵, A Coursier ⁷, J E Marrinan ¹, A Ariyarajah ⁷, M Carino ¹, P Formenty ⁷, U Ströher ⁴, M Lamunu ⁷, G Wu ⁵, F Sahr ^6,^‡, W Xu ^5,^‡, B Knust ^4,^‡, N Broutet ^1,^‡; on behalf of the Sierra Leone Ebola Virus Persistence Study Group^¶

Editor: Jason Kindrachuk⁸

¹UNDP/UNFPA/UNICEF/WHO/World Bank Special Programme of Research, Development and Research Training in Human Reproduction, Department of Sexual and Reproductive Health and Research, World Health Organization, Geneva, Switzerland

²Department of Global Public Health, Karolinska Institutet, Stockholm, Sweden

³Sierra Leone Ministry of Health and Sanitation, Freetown, Sierra Leone

⁴Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America

⁵National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China

⁶Sierra Leone Ministry of Defense, Freetown, Sierra Leone

⁷Department of Health Emergency Interventions, World Health Organization, Geneva, Switzerland

⁸University of Manitoba Faculty of Health Sciences, CANADA

The authors have declared that no competing interests exist.

‡ These authors are joint senior authors on this work.

¶ Membership of the Sierra Leone Ebola Virus Persistence Study Group is provided in the Acknowledgments.

^✉

* E-mail: thorsona@who.int

Contributed equally.

Roles

A E Thorson: Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Writing – original draft, Writing – review & editing

G F Deen: Conceptualization, Investigation, Project administration, Resources, Supervision, Writing – review & editing

K T Bernstein: Conceptualization, Methodology, Project administration, Supervision, Writing – review & editing

W J Liu: Investigation, Supervision, Writing – review & editing

F Yamba: Investigation, Project administration, Supervision, Writing – review & editing

N Habib: Data curation, Formal analysis, Methodology, Software, Writing – review & editing

F R Sesay: Investigation, Project administration, Resources, Supervision, Writing – review & editing

P Gaillard: Data curation, Formal analysis, Project administration, Resources, Supervision, Validation, Writing – review & editing

T A Massaquoi: Investigation, Project administration, Resources, Supervision, Writing – review & editing

S L R McDonald: Investigation, Project administration, Resources, Supervision, Validation, Writing – review & editing

Y Zhang: Investigation, Resources, Supervision, Writing – review & editing

K N Durski: Conceptualization, Investigation, Project administration, Resources, Supervision, Validation, Writing – review & editing

S Singaravelu: Investigation, Project administration, Software, Supervision, Validation, Writing – review & editing

E Ervin: Data curation, Investigation, Project administration, Supervision, Validation, Writing – review & editing

H Liu: Project administration, Resources, Writing – review & editing

A Coursier: Project administration, Resources, Supervision, Validation, Writing – review & editing

J E Marrinan: Data curation, Investigation, Project administration, Supervision, Validation, Writing – review & editing

A Ariyarajah: Project administration, Resources, Supervision, Validation, Writing – review & editing

M Carino: Project administration, Supervision, Writing – review & editing

P Formenty: Conceptualization, Investigation, Supervision, Writing – review & editing

U Ströher: Investigation, Supervision, Writing – review & editing

M Lamunu: Project administration, Supervision, Writing – review & editing

G Wu: Project administration, Supervision, Writing – review & editing

F Sahr: Resources, Supervision, Writing – review & editing

W Xu: Investigation, Supervision, Writing – review & editing

B Knust: Conceptualization, Funding acquisition, Investigation, Project administration, Resources, Supervision, Writing – review & editing

N Broutet: Conceptualization, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Writing – review & editing

Jason Kindrachuk: Academic Editor

PMCID: PMC7875361 PMID: 33566817

Abstract

Background

Sexual transmission chains of Ebola virus (EBOV) have been verified and linked to EBOV RNA persistence in semen, post-recovery. The rate of semen persistence over time, including the average duration of persistence among Ebola virus disease (EVD) survivors, is not well known. This cohort study aimed to analyze population estimates of EBOV RNA persistence rates in semen over time, and associated risk factors in a population of survivors from Sierra Leone.

Methods and findings

In this cohort study from May 2015 to April 2017 in Sierra Leone, recruitment was conducted in 2 phases; the first enrolled 100 male participants from the Western Area District in the capital of Freetown, and the second enrolled 120 men from the Western Area District and from Lungi, Port Loko District. Mean age of participants was 31 years. The men provided semen for testing, analyzed by quantitative reverse transcription PCR (qRT-PCR) for the presence of EBOV RNA. Follow-up occurred every 2 weeks until the endpoint, defined as 2 consecutive negative qRT-PCR results of semen specimen testing for EBOV RNA. Participants were matched with the Sierra Leone EVD case database to retrieve cycle threshold (Ct) values from the qRT-PCR analysis done in blood during acute disease. A purposive sampling strategy was used, and the included sample composition was compared to the national EVD survivor database to understand deviations from the general male survivor population. At 180 days (6 months) after Ebola treatment unit (ETU) discharge, the EBOV RNA semen positive rate was 75.4% (95% CI 66.9%–82.0%). The median persistence duration was 204 days, with 50% of men having cleared their semen of EBOV RNA after this time. At 270 days, persistence was 26.8% (95% CI 20.0%–34.2%), and at 360 days, 6.0% (95% CI 3.1%–10.2%). Longer persistence was significantly associated with severe acute disease, with probability of persistence in this population at 1 year at 10.1% (95% CI 4.6%–19.8%) compared to the probability approaching 0% for those with mild acute disease. Age showed a dose–response pattern, where the youngest men (≤25 years) were 3.17 (95% CI 1.60, 6.29) times more likely to be EBOV RNA negative in semen, and men aged 26–35 years were 1.85 (95% CI 1.04, 3.28) times more likely to be negative, than men aged >35 years. Among participants with both severe acute EVD and a higher age (>35 years), persistence remained above 20% (95% CI 6.0%–50.6%) at 1 year. Uptake of safe sex recommendations 3 months after ETU discharge was low among a third of survivors. The sample was largely representative of male survivors in Sierra Leone. A limitation of this study is the lack of knowledge about infectiousness.

Conclusions

In this study we observed that EBOV RNA persistence in semen was a frequent phenomenon, with high population rates over time. This finding will inform forthcoming updated recommendations on risk reduction strategies relating to sexual transmission of EBOV. Our findings support implementation of a semen testing program as part of epidemic preparedness and response. Further, the results will enable planning of the magnitude of testing and targeted counseling needs over time.

Author summary

Why was this study done?

Evidence of traces of Ebola virus in semen had been reported among survivors for a very long time after their disease.
This study originated out of a need to expand understanding beyond single reported cases, to gain knowledge on how many male survivors have Ebola virus persistence in semen over time, and for how long on average.
We also aimed to analyze factors associated with longer persistence that could provide information on hypotheses on why some men carry virus in their semen for a very long time after the acute disease.

What did the researchers do and find?

In this study, 220 men who had survived Ebola disease in Sierra Leone during 2015–2016 provided semen specimens, and all of those who had traces of Ebola virus detected were followed every other week until their specimen turned negative.
Seventy-five percent of these men still had traces of Ebola virus in their semen specimen at 6 months after being discharged following acute EVD, and 50% at 204 days.
We also found that longer persistence of virus was significantly associated with severe acute EVD and older age.

What do these findings mean?

We show there is an urgent need to organize a national semen testing program as part of Ebola epidemic preparedness and response.
The findings also show that in addition to testing, targeted safe sex counseling and free access to condoms should be a priority from the start of an outbreak.
While we show that most of the survivors had traces of virus in semen at 6 months, more research is needed to understand the impact of viral RNA in semen on transmission of Ebola virus.

Introduction

The 2014–2016, West African Ebola virus disease (EVD) epidemic was unprecedented in size, with 28,616 reported cases, 11,310 deaths [1], and socioeconomic consequences across Guinea, Liberia, and Sierra Leone [2,3]. The recent global public health emergency in the Democratic Republic of the Congo (DRC), with 3,481 Ebola cases reported at the end of the epidemic, further highlights the need for continued and strengthened efforts to combat EVD.

Clusters of EVD cases with sexual transmission being the most likely source of infection have been confirmed [4]. In Liberia, a male EVD survivor infected his sexual partner 179 days after his EVD onset [5,6]. Furthermore, genomic sequencing showed a man in Guinea infected his sexual partner 470 days after his EVD onset [7].

We performed a systematic review prior to study start, finding that evidence of Ebola virus (EBOV) persistence in survivors’ body fluids and evidence of post-acute-disease sexual transmission used to be limited [8]. More recently, case reports from the West African epidemic suggest longer than expected persistence of EBOV ribonucleic acid (RNA) in semen, with detection in 1 male survivor’s semen more than 2 years after Ebola treatment unit (ETU) discharge [9]. Most published reports on semen persistence so far share the limitations of not being designed to provide population inference of semen EBOV RNA persistence rates. Empirical evidence from longitudinal cohort studies with an adequate sample size and frequent and regular follow-up is missing, and the question of duration and population rate of semen persistence among male survivors is an important research gap. Schindell et al. [10] concluded their review on EBOV persistence in semen by stating that existing studies suggest a great variability in the duration of viral persistence. Similarly, very little is known of risk factors associated with longer persistence. Sissoko et al. [11] and Soka et al. [12] raise the hypothesis that men over 40 years have a higher risk of longer persistence, whereas the respective study designs limit full analytical proof of concept. Associations with other risk factors such as characteristics of acute disease or sociodemographics are unknown.

During the West African epidemic, in response to anecdotal reports of suspected sexual transmission, we implemented a prospective, longitudinal cohort study with comprehensive follow-up of EVD survivors in Sierra Leone in 2015. The aim was to study duration and population rates of EBOV persistence in body fluids over time, and associations with risk factors. The cross-sectional baseline analysis was presented earlier [13]. Here we present a survival analysis of EBOV RNA semen persistence rates over time from the full cohort analysis, where participants were followed prospectively over time until specimens were negative for EBOV RNA. We also present a multivariate analysis of risk factors for persistence in semen.

Methods

Study design

This prospective, longitudinal cohort study included a convenience sample of 220 consenting male EVD survivors from Sierra Leone, aged 18 years or above, recruited at Freetown urban and Lungi semi-rural Ebola treatment sites between 27 May 2015 and 12 May 2016, and followed up until April 2017. Study participants were interviewed, and semen tested by quantitative reverse transcription PCR (qRT-PCR) for the presence of EBOV RNA at baseline [14]. Reaching study endpoint was defined as having 2 consecutive EBOV RNA negative specimens by qRT-PCR. Follow-up visits with interviews and semen testing occurred every 2 weeks, until reaching the endpoint. Methods for study implementation, data collection, and counseling, as well as baseline results, have been presented earlier [13–15]. Sample composition and representativity was analyzed in relation to the national EVD survivor database. Individual participant identifications were matched with the Sierra Leone EVD case and laboratory database to retrieve data on blood cycle threshold (Ct) values during acute disease.

Ebola virus detection

Two different qRT-PCR assays were used for EBOV detection in this cohort: NP and VP40 target gene sequences [16–18] until 16 October 2015 at the US Centers for Disease Control and Prevention laboratory in Bo, Sierra Leone, then the GP and NP target gene sequences until study end at the Chinese Center for Disease Control and Prevention (China CDC) laboratory in Freetown using the Ebola Virus Real Time PCR Diagnostic Kit (Chinese FDA Registration Number: 20143402058; China CDC and Daan Gene, Guangzhou, China). The methods were evaluated and compared using the WHO EBOV proficiency test panel, and the sensitivity and specificity of the 2 methods were assessed as similar, above 95% [19]. Further, individual semen specimens from study participants were analyzed to determine viral persistence versus latency, as presented in Whitmer et al. [20].

A housekeeping gene sequence assessed specimen quality, including the presence of cellular beta 2 microglobulin (B2M) mRNA (Invitrogen), which served as an RNA extraction and sample authenticity control.

Outcome variable

The outcome event of analysis was a confirmed negative result, defined as 2 consecutive EBOV RNA negative semen specimens. A cutoff Ct value of 40 was used to define positive versus negative detection in both assays.

Algorithms of interpretation, including for specimen quality, were developed to categorize results into positive, negative, or missing. PCR results were considered (1) missing in cases of no detection of housekeeping gene and target gene sequences, in which case they were handled as missing values in the analyses; (2) EBOV RNA negative when the housekeeping gene was detected but none of the 2 target gene sequences; (3) EBOV RNA positive when both target gene sequences were detected; and (4) EBOV RNA indeterminate when only one target was detected and the other one not. In the dichotomized outcome analyses, indeterminate results were included in the positive category.

Representativity of sample—link to the Sierra Leone national Ebola database

To assess the representativeness of our convenience sample and the possibility of selection bias, the study population was compared to the Sierra Leone national EVD survivor database. The database has been compiled through active searches in Port Loko and Kambia, and mass registrations at survivor conferences in all other districts, and includes 919 adult male survivors. Variables for patient demographics, including age at the time of EVD, occupation, and marital status, were extracted for the comparison.

Variables

Sociodemographic characteristics, symptoms during acute infection, and EVD sequelae

Independent variables included age, education, marital status, household size, relationship to other household members, whether any other household members had EVD, sexual behavior, and adherence to preventive sexual advice after ETU discharge.

Participants reported on the presence of the following during acute EVD: vomiting, diarrhea, being unable to get up to go to the toilet, and being unable to drink by themselves. EVD sequelae (involving eyes, joints, neurological system, abdomen, skin, mental health, and sexual function) were reported at baseline and at every follow-up visit, and any sequelae reported in the 24 months after ETU discharge were used.

Acute EVD Ct values

Associations between persistence duration of EBOV RNA in semen and viremia during acute EVD were analyzed by abstracting acute blood qRT-PCR results from the WHO global laboratory database in Sierra Leone, including multiple blood Ct value tests over time for the same patient [21]. A variable was constructed using the matched individual’s lowest Ct value available as a proxy for maximum viral load: 121 participants could be matched and were included in this subset. The Ct value variable was dichotomized, based on the group mean acute Ct value of 27.9 with a threshold set at Ct value ≥ 27. Higher values indicate lower quantities of circulating EBOV RNA during acute disease, i.e., milder disease, and lower Ct values indicate more EBOV RNA present, i.e., more severe disease [22].

Survival analysis

The Kaplan–Meier interval censoring method was used to calculate the rate of EBOV persistence in semen at different time points starting from ETU discharge. Participants negative at baseline were interval-censored (IC) between the ETU discharge date and date of first baseline testing. Participants who were positive at recruitment were followed every 2 weeks, and considered IC between the dates of their last positive and first confirmed negative result. Survivors who did not reach the endpoint were right-censored, as in leaving the cohort before the event of interest occurred.

A parametric Weibull model was fitted using the IC data and superimposed on the Kaplan–Meier IC curve for the overall sample. The Weibull persistence rates were based on expected times from ETU discharge to EBOV negativity. Interval censoring within the univariate and multivariable semi-parametric IC proportional hazard models was estimated by the SAS ICPHREG procedure [23]. The multivariate model building strategy that gave rise to the final multivariate model, including the considerations for possible interaction effects, is discussed in S1 Statistical Analysis Plan based on methodologies in [24]. Interaction terms were only included if they were highly significant, with p-value < 0.01. The 95% confidence intervals were based on the complementary log–log transformation. Hazard ratio (HR) > 1, HR < 1, and HR = 1 denoted a higher probability, a lower probability, and no difference in the probability of being confirmed negative for EBOV RNA (i.e., a relatively lower, higher, and similar risk of EBOV persistence in semen), respectively. A 2-sided p-value < 0.05 was considered statistically significant.

The prospective study protocol and the prospective statistical analysis plan are provided as S1 Protocol and S1 Statistical Analysis Plan, respectively. The study benefitted from an independent data monitoring committee advising on the conduct, analyses, and dissemination of the study.

Ethics

Ethical permission was granted from the Sierra Leone Ethics and Scientific Review Committee and the WHO Ethical Review Committee (No. RPC736).

This study is reported as per the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guideline (S1 STROBE Checklist).

Results

Study participants and retention

Out of the 220 men enrolled, 203 were included in the analyses while 17 men were excluded because of: no semen sample collected (n = 10), only one sample collected (n = 3), found not infected with Ebola virus (n = 4). Fifty-seven participants presented with at least 2 EBOV RNA positive semen specimens; 37 had, at least once prior to the endpoint, a negative test consecutively followed by a positive test. Of the 7 who were right-censored, 4 had at least once a single negative result that was consecutively followed by a positive result.

Baseline participants’ characteristics and uptake of safe sex advice

The median time between ETU discharge and study admission was 258 days (range: 40–610 days). Baseline characteristics are summarized in Table 1 and have been presented in detail elsewhere [13].

Table 1. Baseline characteristics of EVD survivors enrolled in the study.

Characteristics	Pooled (N = 220)
Days from ETU discharge to study admission
Mean (SD)	299 (141)
Median (IQR)	258 (187, 422)
Minimum, maximum	40, 610
Age (years)
Mean (SD)	31.5 (9.5)
Median (IR)	29.0 (25.0, 36.0)
Minimum, maximum	18.0, 66.0
≤25	67/219 (30.6)
26–35	91/219 (41.6)
>35	61/219 (27.8)
Missing	1
Highest level of education
No education	43/220 (19.6)
Primary/1–8 years of education	70/220 (31.8)
Secondary or higher/8+ years of education	107/220 (48.6)
Marital status
Single/divorced/widowed/separated	98/220 (44.6)
Married/long-term relationship	122/220 (55.4)
Cohabitants in household
Spouse or partner	76/219 (34.7)
Parents	52/219 (23.7)
Extended family	131/219 (59.8)
Household size (number of persons including self)
≤4	56/216 (25.9)
5–8	73/216 (33.8)
9–12	43/216 (19.9)
>12	44/216 (20.4)
Missing	4
Number of household members with EVD (excluding self)
None	58/220 (26.4)
1–2	61/220 (27.7)
3–4	42/220 (19.1)
5+	59/220 (26.8)
Missing
Symptoms during EVD
Vomited	174/219 (79.5)
Too sick to relieve self on toilet	109/219 (49.8)
Diarrhea	174/219 (79.5)
Too sick to drink for more than a day	102/219 (46.6)
Overall health and well-being now as compared to before EVD
Back to normal or same as before	81/218 (37.2)
Worse than before	72/218 (33.0)
Better than before	65/218 (29.8)
New health problems since recovering from EVD	134/220 (60.9)
Symptoms experienced any time from discharge to 24 months
Eye/vision problems	58/220 (26.4)
Joint pain	116/220 (52.7)
Psychological problems (including anxiety and depression)	9/220 (4.1)
Sexual problems/sexual desire not same as before	41/220 (18.6)
Tested for HIV	143/216 (66.2)
Missing	4
Sexual behavior during the first 3 months after recovering from EVD
Had sex	65/220 (29.5)
Sexual desire following EVD recovery was the same as before	167/220 (75.9)
Condom use
Never	20/219 (9.1)
Sometimes	15/219 (6.8)
All the time	28/219 (12.8)
Not sexually active	156/219 (71.2)
Missing	1
Since EVD recovery, difficulty getting or maintaining erection or ejaculating	56/220 (25.5)
Blood Ct value during ETU admission
<27	73/127 (57.5)
≥27	54/127 (42.5)

Open in a new tab

Data are n/N (%), unless otherwise indicated.

Ct, cycle threshold; ETU, Ebola treatment unit; EVD, Ebola virus disease; IQR, interquartile range; SD, standard deviation.

Sixty-five of the 220 men (30%) reported having had sexual contact during the 3 months following ETU discharge, and more than half of those (54%) reported condom use only sometimes or not at all (Table 1).

EBOV persistence detected in semen over time

At 180 days (6 months) after ETU discharge, the EBOV RNA semen positive rate was 75.4% (95% CI 66.9%–82.0%). The median persistence duration was 204 days, with 50% of men having cleared their semen of EBOV RNA by this time. After 270 days, persistence was 26.8% (95% CI 20.0%–34.2%) and at 360 days, 6.0% (95% CI 3.1%–10.2%) (Table 2; Fig 1). The maximum duration of persistence in semen observed in this cohort was 696 days following ETU discharge.

Table 2. Kaplan–Meier non-parametric interval-censored probability estimates of Ebola virus RNA persistence in semen.

Time from ETU discharge (days)	EBOV persistence probability (95% CI)
0	—
60	96.0 (90.2, 98.4)
90	92.1 (85.2, 95.8)
180	75.4 (66.9, 82.0)
270	26.8 (20.0, 34.2)
360	6.0 (3.1,10.2)
450	5.1 (2.5, 9.0)
540	2.0 (0.6, 4.9)

Open in a new tab

EBOV, Ebola virus; ETU, Ebola treatment unit.

Fig 1 — ETU, Ebola treatment unit; IC, interval-censored; KM, Kaplan–Meier.

Severe acute disease and older age associated with EBOV RNA persistence in semen

Severe disease, measured by lower acute EVD blood Ct value, was significantly correlated with longer EBOV RNA persistence. The probability of persistence at 1 year after ETU discharge was 10.1% (95% CI 4.6%–19.8%) and approached 0% only after 720 days. Among participants with mild acute disease, the decay curve fell steeply to approach 0% already at 1 year (Fig 2).

Fig 2 — ct, cycle threshold; EBOV, Ebola virus; ETU, Ebola treatment unit.

Further, age was significantly associated with EBOV RNA persistence in the multivariate regression model. Showing a dose–response pattern, the youngest men (≤25 years) were 3.4 times (95% CI 2.0, 5.8; p < 0.001) more likely to be EBOV RNA negative in semen, and men aged 26–35 were 1.7 times (95% CI 1.1, 2.6; p = 0.013) more likely, as compared to men aged >35 years, at any given point in time (Table 3).

Table 3. Multivariate model with living with extended family × education interaction term.

Factors	Adjusted HR (95% CI)	Chi-squared p-value
Age (years)
≤25	3.44 (2.04, 5.81)	<0.001
26–35	1.70 (1.12, 2.59)	0.013
>35	1.00
Five or more household members with EVD (excluding self)	0.56 (0.37, 0.84)	0.005
Diarrhea symptoms during EVD	0.49 (0.30, 0.80)	0.004
Education × living with extended family interaction term		0.001
Level 1: Not living with extended family—secondary or higher/8+ years of education (versus primary level or lower/<8 years of education)	2.62 (1.50, 4.58)
Level 2: Living with extended family—secondary or higher/8+ years of education (versus primary level or lower/<8 years of education)	0.77 (0.49, 1.20)

Open in a new tab

HR > 1 implies higher probability of being confirmed EBOV RNA negative in semen (lower likelihood of EBOV persistence); HR < 1 implies a lower probability of being confirmed EBOV RNA negative in semen (increased likelihood of EBOV persistence).

CI, confidence interval; EBOV, Ebola virus; EVD, Ebola virus disease; HR, hazard ratio.

The age-stratified survival models revealed an effect modification of age with acute blood Ct value and persistence, where in the group of survivors who had severe disease at admission (blood Ct value < 27), age older than 25 years was associated with longer persistence (compared to age ≤ 25 years). Twenty percent remained positive at 1 year in this group (generalized log-rank p = 0.001) (Table 4; Fig 3). For those with milder acute disease (blood Ct value ≥ 27), persistence was not significantly different between the 2 age groups (generalized log-rank p = 0.79) (Fig 4). Also, reporting diarrhea during acute disease was significantly associated with longer persistence in semen (Table 3).

Table 4. Multivariate model in a subset of 121 participants with available ETU blood Ct value.

Factors	Adjusted HR (95% CI)	Chi-squared p-value
Age, (years)
≤25	3.17 (1.60, 6.29)	0.001
26–35	1.85 (1.04, 3.28)	0.036
>35	1.00
ETU blood Ct value < 27	0.61 (0.38, 0.98)	0.043
Five or more household members with EVD (excluding self)	0.60 (0.35, 1.03)	0.063
Diarrhea symptoms during EVD	0.48 (0.26, 0.88)	0.018
Education × living with extended family interaction term		0.006
Level 1: Not living with extended family—secondary or higher/8+ years of education (versus primary level or lower/<8 years of education)	2.63 (1.30, 5.34)
Level 2: Living with extended family—secondary or higher/8+ years of education (versus primary level or lower/<8 years of education)	0.71 (0.39, 1.27)

Open in a new tab

Ct, cycle threshold; EBOV, Ebola virus; ETU, Ebola treatment unit; EVD, Ebola virus disease; HR, hazard ratio.

EVD co-morbidity and EVD sequelae

None of the men self-reported co-morbidity for tuberculosis, diabetes, malaria, or sexually transmitted infections at the time of their acute infection with EVD. Among the 143 men tested for HIV at enrollment, 1 tested HIV positive; his semen specimen tested negative for EBOV RNA.

More than half of the participating survivors (60%) reported new health problems following their acute EVD. Symptoms involving the joints, followed by symptoms of the eye/vision and lack of sexual desire or impotency, were most commonly reported (Table 1).

Representativity of study sample

A comparison of study participant demographics with those of registered male EVD survivors 18 years or above in Sierra Leone (n = 919) indicated minimal differences across comparable demographic indicators. In particular, there was a slightly higher proportion of men 26–35 years old recruited to the study (40%) compared to men of the same age group in the national EVD survivor database (34%). Additionally, a greater proportion of study participants were married or in a long-term relationship (56% versus 44%), and a smaller proportion reported not working (2% versus 22%), compared to those in the national registry. Due to limitations in the use of the national EVD survivor database, further statistical comparisons could not be conducted.

Discussion

Our findings show that in a cohort of 220 male EVD survivors from Sierra Leone, 75% of men were still semen positive for EBOV RNA 6 months after being discharged following acute EVD, and 50% at 204 days. At 1 year, less than 10% of study participants overall, but 20% of men aged >25 years who had had severe acute disease, had detectable EBOV RNA in semen. Longer persistence was significantly associated with increased age and severe acute EVD. The EBOV RNA positive rate in semen was considerably higher over time than what has previously been described. Earlier estimates of persistence rates among survivors based on longitudinal observations and modeling show lower estimates of EBOV RNA persistence rates. One study found persistence of 50% at 3.8 months [11] as compared to 50% remaining positive at close to 7 months in our cohort. Modeled estimates from the Postebogui cohort in Guinea, involving 27 men who tested positive in semen at least once, suggested a median duration of 45 days. In a later estimate by Keita et al. [11], the same population was estimated to have a 13%–60% probability of persistence at 6 months, but the probability was highly dependent on variations between different RT-PCR methods involved [11,25,26]. These studies provide important insights into findings from individual cases, followed up with different and sometimes long intervals. Our study involves a larger sample and does not rely on modeling outside of the survival analysis to estimate the population rates. Our finding of a 75% probability of persistence at 6 months hence represents an empirical longitudinal cohort analysis including regular, frequent follow-up and a large sample, which can explain the significant differences to earlier estimates.

The maximum duration of EBOV RNA positivity observed, 696 days, confirms published findings on EBOV RNA presence in semen from analyses of cross-sectional sample composition or case reports of maximum duration of EBOV RNA detection, including the baseline analyses of this study [9,12,13,27–29]. The higher than expected persistence over time we find in this large cohort of survivors, with 75% of men having EBOV RNA present at 6 months, sheds light on hypotheses raised by these studies, by here adding analyses of persistence rates over time from a representative male survivor sample.

We identified low adherence to initial safe sex counseling among 30% of the participants. In combination with our findings of higher than expected rates of EBOV RNA persistence in semen over time, the risk of residual sexual transmission is present. These results represent critical information to inform Ebola epidemic preparedness and response. The impact of a single case of sexually transmitted EBOV infection can be devastating, to both an individual and to public health, as evidenced by the case of sexual transmission 470 days after ETU discharge reported from Guinea [7]. Inconsistent condom use and limited compliance with the initial ETU recommendation of abstinence are identified challenges that merit immediate action to understand barriers to and facilitators of sustained risk reduction.

Our results further highlight the urgent need to ensure efforts to organize a national response providing semen testing, safe sex counseling, and free access to condoms, as a priority from the start of an outbreak. The here included results will inform forthcoming WHO updated guidelines on semen testing and will contribute to raising awareness of semen program testing needs in the context of the ongoing outbreak in DRC. Most semen testing needs would occur during the first year following ETU discharge, with half of the survivors expected to remain qRT-PCR positive up until close to 7 months. Based on our results, early risk identification and targeted counseling on long-term persistence risks among survivors, especially among those >25 years and with higher quantities of EBOV RNA in blood during acute disease, could be part of efforts to motivate safe sex and retain survivors in the testing program. These efforts will also need to be sustained, together with vigilance and responsiveness in the aftermath of the acute epidemic phase. The risk of new clusters of EVD igniting through sexual transmission demands a thorough and complex epidemic response over time, where a sustainable semen testing program is a crucial component.

Participants in our study who were older and those who had severe acute disease, defined as acute blood Ct value < 27, and those with diarrhea during acute EVD had a significantly higher probability of having persistent EBOV RNA in semen over time. Twenty percent of men with severe disease and >25 years had detectable EBOV RNA in semen at 1 year. Overall, younger age seemed to reduce the time to semen EBOV RNA clearance by dose–response pattern. This confirms hypotheses raised from other studies of survivors relating to men >40 years; we show how the risk increase of longer persistence starts at an earlier age than earlier anticipated [9,12]. Several mechanisms, such as immune response to acute viremia, natural aging of the immune system, or inherent co-morbidity, may explain these associations.

The sociodemographic links to persistence merit further in-depth bio-behavioral research.

We found an unexpectedly low frequency of co-morbidity associated with acute EVD, which may be related to higher EVD case fatality rates in patients with additional diagnoses. Similar to other reports on Ebola survivors, the men in our study reported a high frequency (>60%) of post-EVD sequelae including symptoms of sexual fatigue [27]. These results highlight that survivors need continued care efforts, also addressing sexual health.

In this study we opted to use 2 consecutive negative qRT-PCR results, collected 2 weeks apart, as the definition of negativity, but keeping the “time of event” to the first test and applying interval censoring as needed, to accurately reflect time to event. For semen positive participants found positive at baseline, we assumed continuous positivity from ETU discharge until the first test at baseline.

ETU discharge date per certificate was used as the starting point for semen positivity. This date is a proxy for when viremia waned and will likely have underestimated persistence time. Acute Ct values in our study were not available for all participants, were taken at different points in time during acute disease, and were analyzed by different laboratories, which may have diluted the associations observed. The lowest available acute Ct value was consequently retrieved, to mitigate misclassification.

It should also be noted in all interpretations that the qRT-PCR detection of EBOV RNA does not distinguish between viable virus and RNA fragments. In an analysis by Whitmer et al. [20], including positive semen specimens from study participants in this cohort as well as from survivors in the US, it was shown that active EBOV replication occurred, and they concluded that “EBOV persistence within EVD survivors may act as a viral reservoir,” supporting the relevance of a positive qRT-PCR finding. [20].

Our study applied purposive sampling to recruit participants. The comparison of study participants with registered male survivors 18 years or above in Sierra Leone indicated differences across comparable demographic indicators of marriage and unemployment. These differences could reflect selection bias, influencing the generalizability of the results, but these traits were adjusted for as covariates in the multivariate analysis of associations with the outcome.

Recall bias may influence validity, specifically in the questions about the acute disease episode and sexual behavior. This and social desirability in answering may have lowered estimates of risky sexual behavior.

Conclusions

Our findings showed probabilities of semen persistence of EBOV RNA to be 75% at 6 months after ETU discharge, with persistence of 50% at 204 days and less than 10% at 1 year after ETU discharge. Persistence however remained at more than 20% at 1 year among participants >25 years with higher quantities of EBOV RNA in blood during acute disease. Uptake of safer sex recommendations 3 months after ETU discharge was low among a third of survivors. The study population was largely representative of the male EVD survivor population in Sierra Leon, apart from noted differences in marriage status and employment. These variables were adjusted for in the multivariate analysis, and we conclude that our results can be generalized to the wider male survivor population in Sierra Leone, and can also inform management and response to survivors’ needs following EVD in other contexts.

Our results highlight the immediate needs of planning for increased vigilance and efforts to support male survivors with safe sex counseling, including free provision of condoms at discharge, together with implementation of a semen testing program, as part of epidemic preparedness and primary and sustained epidemic response. Evidence exists showing sexual transmission chains after recovery; however, more research is needed to better understand the contribution of sexual transmission at different points in an epidemic. Emerging EVD cases in the aftermath of an epidemic, as was the case in DRC, also merit in-depth analysis to understand the role of EBOV RNA semen persistence in survivors [30].

Supporting information

S1 STROBE Checklist

(DOCX)

Click here for additional data file.^{(46.7KB, docx)}

S1 Protocol. Study protocol for the EBOV persistence study version 1.7.

25 January 2016.

(DOC)

Click here for additional data file.^{(1.7MB, doc)}

S1 Statistical Analysis Plan

(PDF)

Click here for additional data file.^{(295.8KB, pdf)}

Acknowledgments

This work has been developed on behalf of the Sierra Leone Ebola Virus Persistence Study Group (for listing please revert to https://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0005723#ack). The investigators are grateful for advice provided by Ian Crozier, Johan Giesecke, Michael Hughes, Nicolas Meda, Janus Paweska, Donna Spiegelman, and Edith Tarimo in their role as expert advisors of the study’s independent data monitoring committee.

Abbreviations

Ct: cycle threshold
DRC: Democratic Republic of the Congo
EBOV: Ebola virus Zaire
ETU: Ebola treatment unit
EVD: Ebola virus disease
HR: hazard ratio
IC: interval-censored
qRT-PCR: quantitative reverse transcription PCR

Data Availability

Contractual agreements between the study parties (HRP/WHO, US CDC, China CDC, and the MoH SL) exists and data rights reside with MOH-SL, the material owner. Inquiries related to the data, may be directed to Study Data Oversight Committee (SIS@who.int) with Subject: “Inquiry on Ebola Semen persistence study data”.

Funding Statement

NB received funding for this study from the Paul Allen family foundation. Award nr NA. https://pgafamilyfoundation.org/. The study team acknowledges the contributions of the WHO Ebola Response Program, the Paul G. Allen Family Foundation, and the UNDP (United Nations Development Program)–UNFPA (United Nations Population Fund)–UNICEF–WHO–World Bank Special Program of Research, Development and Research Training in Human Reproduction (HRP), a cosponsored program executed by the WHO; the US CDC, the China CDC, the Sierra Leone Ministry of Health and Sanitation and the Ministry of Defence, and the Joint United Nations Program on HIV/AIDS in support of the Sierra Leone Ebola Virus Persistence Study. NO - The PAF had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. YES- individuals employed by the WHO and HRP have contributed to this work.

References

1.World Health Organization. Ebola situation report—30 March 2016 Geneva: World Health Organization; 2016. [cited 2021 Jan 11]. Available from: https://apps.who.int/ebola/current-situation/ebola-situation-report-30-march-2016. [Google Scholar]
2.United Nations Development Group–Western and Central Africa. Socio-economic impact of Ebola virus disease in West African countries New York: United Nations Development Group; 2015. [cited 2021 Jan 6]. Available from: https://www.undp.org/content/dam/rba/docs/Reports/ebola-west-africa.pdf. [Google Scholar]
3.United Nations Economic Commission for Africa. Socio-economic impacts of Ebola on Africa. Revised edition Addis Ababa: United Nations Economic Commission for Africa; 2015. [Google Scholar]
4.Den Boon S, Marston BJ, Nyenswah TG, Jambai A, Barry M, Keita S, et al. Ebola virus infection associated with transmission from survivors. Emerg Infect Dis. 2019;25(2):249–55. 10.3201/eid2502.181011 [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Christie A, Davies-Wayne GJ, Cordier-Lassalle T, Blackley DJ, Laney AS, Williams DE, et al. Possible sexual transmission of Ebola virus—Liberia, 2015. MMWR Morb Mortal Wkly Rep. 2015;64(17):479–81. [PMC free article] [PubMed] [Google Scholar]
6.Mate SE, Kugelman JR, Nyenswah TG, Ladner JT, Wiley MR, Cordier-Lassalle T, et al. Molecular evidence of sexual transmission of Ebola virus. N Engl J Med. 2015;373(25):2448–54. 10.1056/NEJMoa1509773 [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Diallo B, Sissoko D, Loman NJ, Bah HA, Bah H, Worrell MC, et al. Resurgence of Ebola virus disease in Guinea linked to a survivor with virus persistence in seminal fluid for more than 500 days. Clin Infect Dis. 2016;63(10):1353–6. 10.1093/cid/ciw601 [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Thorson A, Formenty P, Lofthouse C, Broutet N. Systematic review of the literature on viral persistence and sexual transmission from recovered Ebola survivors: evidence and recommendations. BMJ Open. 2016;6(1):e008859 10.1136/bmjopen-2015-008859 [DOI] [PMC free article] [PubMed] [Google Scholar]
9.Fischer WA, Brown J, Wohl DA, Loftis AJ, Tozay S, Reeves E, et al. Ebola virus ribonucleic acid detection in semen more than two years after resolution of acute Ebola virus infection. Open Forum Infect Dis. 2017;4(3):ofx155 10.1093/ofid/ofx155 [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Schindell BG, Webb AL, Kindrachuk J. Persistence and sexual transmission of filoviruses. Viruses. 2018;10(12):683 10.3390/v10120683 [DOI] [PMC free article] [PubMed] [Google Scholar]
11.Sissoko D, Duraffour S, Kerber R, Kolie JS, Beavogui AH, Camara AM, et al. Persistence and clearance of Ebola virus RNA from seminal fluid of Ebola virus disease survivors: a longitudinal analysis and modelling study. Lancet Glob Health. 2017;5(1):e80–8. 10.1016/S2214-109X(16)30243-1 [DOI] [PubMed] [Google Scholar]
12.Soka MJ, Choi MJ, Baller A, White S, Rogers E, Purpura LJ, et al. Prevention of sexual transmission of Ebola in Liberia through a national semen testing and counselling programme for survivors: an analysis of Ebola virus RNA results and behavioural data. Lancet Glob Health. 2016;4(10):e736–43. 10.1016/S2214-109X(16)30175-9 [DOI] [PubMed] [Google Scholar]
13.Deen GF, Broutet N, Xu W, Knust B, Sesay FR, McDonald SLR, et al. Ebola RNA persistence in semen of ebola virus disease survivors—final report. N Engl J Med. 2017;377(15):1428–37. 10.1056/NEJMoa1511410 [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Deen GF, McDonald SLR, Marrinan JE, Sesay FR, Ervin E, Thorson AE, et al. Implementation of a study to examine the persistence of Ebola virus in the body fluids of Ebola virus disease survivors in Sierra Leone: Methodology and lessons learned. PLoS Negl Trop Dis. 2017;11(9):e0005723 10.1371/journal.pntd.0005723 [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Abad N, Malik T, Ariyarajah A, Ongpin P, Hogben M, McDonald SLR, et al. Development of risk reduction behavioral counseling for Ebola virus disease survivors enrolled in the Sierra Leone Ebola Virus Persistence Study, 2015–2016. PLoS Negl Trop Dis. 2017;11(9):e0005827 10.1371/journal.pntd.0005827 [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Centers for Disease Control and Prevention. 2014 Ebola virus emergency use authorizations: CDC Ebola virus VP40 real-time RT-PCR assay (CDC)—March 2, 2015. Atlanta: Centers for Disease Control and Prevention; 2021. [cited 2021 Jan 6]. Available from: https://www.fda.gov/MedicalDevices/Safety/EmergencySituations/ucm161496.htm#ebola. [Google Scholar]
17.Centers for Disease Control and Prevention. Ebola virus NP real-time RT-PCR assay. Atlanta: Centers for Disease Control and Prevention; 2016. [cited 2021 Jan 6]. Available from: https://www.fda.gov/downloads/medicaldevices/safety/emergencysituations/ucm436307.pdf. [Google Scholar]
18.Erickson BR, Sealy TK, Flietstra T, Morgan L, Kargbo B, Matt-Lebby VE, et al. Ebola virus disease diagnostics, Sierra Leone: analysis of real-time reverse transcription-polymerase chain reaction values for clinical blood and oral swab specimens. J Infect Dis. 2016;214(Suppl 3):S258–62. 10.1093/infdis/jiw296 [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Ellerbrok H, Jacobsen S, Patel P, Rieger T, Eickmann M, Becker S, et al. External quality assessment study for ebolavirus PCR-diagnostic promotes international preparedness during the 2014–2016 Ebola outbreak in West Africa. PLoS Negl Trop Dis. 2017;11(5):e0005570 10.1371/journal.pntd.0005570 [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Whitmer SLM, Ladner JT, Wiley MR, Patel K, Dudas G, Rambaut A, et al. Active Ebola virus replication and heterogeneous evolutionary rates in EVD survivors. Cell Rep. 2018;22(5):1159–68. 10.1016/j.celrep.2018.01.008 [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Durski KN, Singaravelu S, Teo J, Naidoo D, Bawo L, Jambai A, et al. Development, use, and impact of a global laboratory database during the 2014 Ebola outbreak in West Africa. J Infect Dis. 2017;215(12):1799–806. 10.1093/infdis/jix236 [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Faye O, Andronico A, Faye O, Salje H, Boelle PY, Magassouba N, et al. Use of viremia to evaluate the baseline case fatality ratio of Ebola virus disease and inform treatment studies: a retrospective cohort study. PLoS Med. 2015;12(12):e1001908 10.1371/journal.pmed.1001908 [DOI] [PMC free article] [PubMed] [Google Scholar]
23.SAS system for Windows, release 9.4. Cary (NC): SAS Institute; 2014. [Google Scholar]
24.Collett D. Modelling survival data in medical research. London: Chapman & Hall; 1994. [Google Scholar]
25.Subtil F, Delaunay C, Keita AK, Sow MS, Toure A, Leroy S, et al. Dynamics of Ebola RNA persistence in semen: a report from the Postebogui Cohort in Guinea. Clin Infect Dis. 2017;64(12):1788–90. 10.1093/cid/cix210 [DOI] [PubMed] [Google Scholar]
26.Keita AK, Vidal N, Toure A, Diallo MSK, Magassouba N, Baize S, et al. A 40-month follow-up of Ebola virus disease survivors in Guinea (PostEbogui) reveals long-term detection of Ebola viral ribonucleic acid in semen and breast milk. Open Forum Infect Dis. 2019;6(12):ofz482 10.1093/ofid/ofz482 [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Group PIS, Sneller MC, Reilly C, Badio M, Bishop RJ, Eghrari AO, et al. a longitudinal study of ebola sequelae in Liberia. N Engl J Med. 2019;380(10):924–34. 10.1056/NEJMoa1805435 [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Sow MS, Etard JF, Baize S, Magassouba N, Faye O, Msellati P, et al. New evidence of long-lasting persistence of Ebola virus genetic material in semen of survivors. J Infect Dis. 2016;214(10):1475–6. 10.1093/infdis/jiw078 [DOI] [PubMed] [Google Scholar]
29.Uyeki TM, Erickson BR, Brown S, McElroy AK, Cannon D, Gibbons A, et al. Ebola virus persistence in semen of male survivors. Clin Infect Dis. 2016;62(12):1552–5. 10.1093/cid/ciw202 [DOI] [PubMed] [Google Scholar]
30.World Health Organization. Ebola virus disease Democratic Republic of Congo: external situation report 89. Geneva: World Health Organization; 2020. [cited 2021 Jan 6]. Available from: https://www.who.int/publications-detail/ebola-virus-disease-democratic-republic-of-congo-external-situation-report-89-2019. [Google Scholar]

PLoS Med. doi: 10.1371/journal.pmed.1003273.r001

Decision Letter 0

Helen Howard

10 Feb 2020

Dear Dr Thorson,

Thank you for submitting your manuscript entitled "Persistence of Ebola virus in semen - A cohort study among survivors in Sierra Leone" for consideration by PLOS Medicine.

Your manuscript has now been evaluated by the PLOS Medicine editorial staff and I am writing to let you know that we would like to send your submission out for external peer review.

However, before we can send your manuscript to reviewers, we need you to complete your submission by providing the metadata that is required for full assessment. To this end, please login to Editorial Manager where you will find the paper in the 'Submissions Needing Revisions' folder on your homepage. Please click 'Revise Submission' from the Action Links and complete all additional questions in the submission questionnaire.

Please re-submit your manuscript within two working days, i.e. by .

Once your full submission is complete, your paper will undergo a series of checks in preparation for peer review. Once your manuscript has passed all checks it will be sent out for review.

Feel free to email us at plosmedicine@plos.org if you have any queries relating to your submission.

Kind regards,

Helen Howard, for Clare Stone PhD

Acting Editor-in-Chief

PLOS Medicine

plosmedicine.org

PLoS Med. doi: 10.1371/journal.pmed.1003273.r002

Decision Letter 1

Adya Misra

27 Mar 2020

Dear Dr. Thorson,

Thank you very much for submitting your manuscript "Persistence of Ebola virus in semen - Risk factors, magnitude and duration, results from a longitudinal cohort study of survivors in Sierra Leone" (PMEDICINE-D-20-00300R1) for consideration at PLOS Medicine.

Your paper was evaluated by a senior editor and discussed among all the editors here. It was also discussed with an academic editor with relevant expertise, and sent to independent reviewers, including a statistical reviewer. The reviews are appended at the bottom of this email and any accompanying reviewer attachments can be seen via the link below:

[LINK]

In light of these reviews, I am afraid that we will not be able to accept the manuscript for publication in the journal in its current form, but we would like to consider a revised version that addresses the reviewers' and editors' comments. Obviously we cannot make any decision about publication until we have seen the revised manuscript and your response, and we plan to seek re-review by one or more of the reviewers.

In revising the manuscript for further consideration, your revisions should address the specific points made by each reviewer and the editors. Please also check the guidelines for revised papers at http://journals.plos.org/plosmedicine/s/revising-your-manuscript for any that apply to your paper. In your rebuttal letter you should indicate your response to the reviewers' and editors' comments, the changes you have made in the manuscript, and include either an excerpt of the revised text or the location (eg: page and line number) where each change can be found. Please submit a clean version of the paper as the main article file; a version with changes marked should be uploaded as a marked up manuscript.

In addition, we request that you upload any figures associated with your paper as individual TIF or EPS files with 300dpi resolution at resubmission; please read our figure guidelines for more information on our requirements: http://journals.plos.org/plosmedicine/s/figures. While revising your submission, please upload your figure files to the PACE digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at PLOSMedicine@plos.org.

We expect to receive your revised manuscript by Apr 17 2020 11:59PM. Please email us (plosmedicine@plos.org) if you have any questions or concerns.

***Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.***

We ask every co-author listed on the manuscript to fill in a contributing author statement, making sure to declare all competing interests. If any of the co-authors have not filled in the statement, we will remind them to do so when the paper is revised. If all statements are not completed in a timely fashion this could hold up the re-review process. If new competing interests are declared later in the revision process, this may also hold up the submission. Should there be a problem getting one of your co-authors to fill in a statement we will be in contact. YOU MUST NOT ADD OR REMOVE AUTHORS UNLESS YOU HAVE ALERTED THE EDITOR HANDLING THE MANUSCRIPT TO THE CHANGE AND THEY SPECIFICALLY HAVE AGREED TO IT. You can see our competing interests policy here: http://journals.plos.org/plosmedicine/s/competing-interests.

Please use the following link to submit the revised manuscript:

https://www.editorialmanager.com/pmedicine/

Your article can be found in the "Submissions Needing Revision" folder.

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see http://journals.plos.org/plosmedicine/s/submission-guidelines#loc-methods.

Please ensure that the paper adheres to the PLOS Data Availability Policy (see http://journals.plos.org/plosmedicine/s/data-availability), which requires that all data underlying the study's findings be provided in a repository or as Supporting Information. For data residing with a third party, authors are required to provide instructions with contact information for obtaining the data. PLOS journals do not allow statements supported by "data not shown" or "unpublished results." For such statements, authors must provide supporting data or cite public sources that include it.

We look forward to receiving your revised manuscript.

Sincerely,

Adya Misra, PhD

Senior Editor

PLOS Medicine

plosmedicine.org

-----------------------------------------------------------

Requests from the editors:

Title-Please revise your title according to PLOS Medicine's style. Your title must be nondeclarative and not a question. It should begin with main concept if possible. "Effect of" should be used only if causality can be inferred, i.e., for an RCT. Please place the study design ("A randomized controlled trial," "A retrospective study," "A modelling study," etc.) in the subtitle (ie, after a colon).

Abstract

Please structure your abstract using the PLOS Medicine headings (Background, Methods and Findings, Conclusions).

Background- Provide the context of why the study is important. The final sentence should clearly state the study question.

Please ensure that all numbers presented in the abstract are present and identical to numbers presented in the main manuscript text. * Please include the study design, population and setting, number of participants, years during which the study took place, length of follow up, and main outcome measures. * Please quantify the main results (with 95% CIs and p values). * Please include the important dependent variables that are adjusted for in the analyses. * Please include the actual amounts and/or absolute risk(s) of relevant outcomes (including NNT or NNH where appropriate), not just relative risks or correlation coefficients. (example for absolute risks: PMID: 28399126). * Please include a summary of adverse events if these were assessed in the study. * In the last sentence of the Abstract Methods and Findings section, please describe the main limitation(s) of the study's methodology/study design.

Abstract Conclusions:

* Please address the study implications without overreaching what can be concluded from the data; the phrase "In this study, we observed ..." may be useful.

* Please interpret the study based on the results presented in the abstract, emphasizing what is new without overstating your conclusions.

* Please avoid vague statements such as "these results have major implications for policy/clinical care". Mention only specific implications substantiated by the results.

* Please avoid assertions of primacy ("We report for the first time....")

The Data Availability Statement (DAS) requires revision. For each data source used in your study:

a) If the data are freely or publicly available, note this and state the location of the data: within the paper, in Supporting Information files, or in a public repository (include the DOI or accession number).

b) If the data are owned by a third party but freely available upon request, please note this and state the owner of the data set and contact information for data requests (web or email address). Note that a study author cannot be the contact person for the data.

c) If the data are not freely available, please describe briefly the ethical, legal, or contractual restriction that prevents you from sharing it. Please also include an appropriate contact (web or email address) for inquiries (again, this cannot be a study author).

Please use Vancouver style for references and place all references within square brackets

Author summary

At this stage, we ask that you include a short, non-technical Author Summary of your research to make findings accessible to a wide audience that includes both scientists and non-scientists. The Author Summary should immediately follow the Abstract in your revised manuscript. This text is subject to editorial change and should be distinct from the scientific abstract. Please see our author guidelines for more information: https://journals.plos.org/plosmedicine/s/revising-your-manuscript#loc-author-summary

Introduction

Please address past research and explain the need for and potential importance of your study. Indicate whether your study is novel and how you determined that. If there has been a systematic review of the evidence related to your study (or you have conducted one), please refer to and reference that review and indicate whether it supports the need for your study.

Please conclude the Introduction with a clear description of the study question or hypothesis.

Methods

Please provide details or citations of all interview guides used in the study. If these are not published, please provide a copy as SI files.

Please report the number of [patients, samples, etc] and dates of recruitment, and account for all methods used in your study.

Ethics approval- please provide details and name of the approving committee in the methods

Please ensure that the study is reported according to the STROBE guideline, and include the completed STROBE checklist as Supporting Information. Please add the following statement, or similar, to the Methods: "This study is reported as per the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guideline (S1 Checklist)."

The STROBE guideline can be found here: http://www.equator-network.org/reporting-guidelines/strobe/

When completing the checklist, please use section and paragraph numbers, rather than page numbers.

Did your study have a prospective protocol or analysis plan? Please state this (either way) early in the Methods section.

a) If a prospective analysis plan (from your funding proposal, IRB or other ethics committee submission, study protocol, or other planning document written before analyzing the data) was used in designing the study, please include the relevant prospectively written document with your revised manuscript as a Supporting Information file to be published alongside your study, and cite it in the Methods section. A legend for this file should be included at the end of your manuscript.

b) If no such document exists, please make sure that the Methods section transparently describes when analyses were planned, and when/why any data-driven changes to analyses took place.

c) In either case, changes in the analysis-- including those made in response to peer review comments-- should be identified as such in the Methods section of the paper, with rationale.Please provide references to databases used in the study

Please provide further details about the housekeeping gene used in this study, along with the sequence as supplementary information

Page 6 footnote-please move this to main text

Is it necessary to mention FAM and VIC reporter dyes on page 7 without any background? Please revise so that the methods can be read and understood by a general audience

Please provide details about when this study was carried out, when were samples collected and details of follow-up.

PLOS does not permit "data not shown.” Please remove this claim, or do one of the following: a) If you are the owner of the data relevant to this claim, please provide the data in accordance with the PLOS data policy, and update your Data Availability Statement as needed. b) If the data not shown refer to a study from another group that has not been published, please cite personal communication in your manuscript text (it should not be included in the reference section). Please provide the name of the individual, the affiliation, and date of communication. The individual must provide PLOS Medicine written permission to be named for this purpose. c) For any other circumstance, please contact me ASAP.

Discussion

Please present and organize the Discussion as follows: a short, clear summary of the article's findings; what the study adds to existing research and where and why the results may differ from previous research; strengths and limitations of the study; implications and next steps for research, clinical practice, and/or public policy; one-paragraph conclusion.

Comments from the reviewers:

Reviewer #1: In the proposed paper, the authors present the follow-up of semen of Ebola survivors. They find that Ebola virus was present in many individuals, even a year after follow-up. This presents an interesting follow-up to the original paper (Deen et al., NEJM) as it includes some longitudinal data not included in the original paper. Nevertheless I was confused by the statistical approach taken.

My major concern/confusion is with the main regression analysis. The authors conduct a multivariable Weibull regression, where they include an interaction term between education and living with extended family. They also dedicate an entire table (Table 3.2) to stratified results for living and not living with extended family. The rationale for this approach was not set out in the introduction or methods and only briefly mentioned in the results. I also struggle to come up with a mechanistic rationale as to why this would be of particular interest. In the discussion there is a mention of reinfection risk over time, but then again, I struggle with why living with extended family is a good marker of this. I think this focus is an unfortunate distraction for the paper and I would consider removing it.

The 95% confidence intervals for the Kaplan-Meier curve (Figure 1) look very narrow, especially as there are only 220 individuals. The authors should clarify how these were calculated. The authors should also include 95% confidence intervals for the Weibull fit in Figure 1.

There are no uncertainty intervals presented in Figure 2 and Figure 3 - these need to be included.

The authors refer to 'data not shown' to say their sample is representative of the wider population. There is sufficient space in the article to set out the comparisons between their cohort and the wider population in a table - and show how the two populations are different and the same.

The authors use two sequential negative tests as a marker of being a true negative. It would be useful to set out how frequently a negative was followed up by a positive test to give readers an idea of how robust this definition is.

Lower CT score is used as a proxy for severe disease. The rationale for this proxy association should be referenced (e.g., Faye et al., PLoS Medicine). Viral load itself is of interest (irrespective if it is linked to symptoms), however, it will depend on when it was tested during acute illness - this weakness should be discussed.

On page 6, I do not understand the sentence 'A housekeeping gene sequence assessed specimens' quality through compatibility with human DNA' - the authors should clarify exactly what was done.

The authors use a simple dichotomization of greater/less that a CT of 27 for high/low disease severity - it would be useful to look at additional bins of CT (or use a spline) to see the full relationship between the two.

Reviewer #2: In this manuscript, Thorson et al analyzed the persistence of EBOV RNA in semen from a longitudinal cohort of survivors in Sierra Leone. Using two different EBOV RT-PCR, they monitored the kinetics of EBOV persistence in semen and established the survival function of EBOV RNA persistence over time according to time after discharge and to initial viremia and age. They demonstrated that the persistence correlated with age and other factors such as the occurrence of diarrhea during acute disease. The study is well designed and managed, the statistical analyses are robust, and the main strength of the study is the longitudinal follow-up in survivors with samples obtained very frequently. Some results obtained with this cohort have previously been published in NEJM by the authors and the current report provides the complete and final follow-up of the cohort.

The main weakness of this study is the lack of real novelty of the results, as a lot of reports describing the persistence of EBOV RNA in semen have already been published, with quite comparable conclusions. For example, the most recent publication (Keita, Open Forum Infect Dis), not cited by the authors, described a similar cohort with analysis of several body fluids including semen. Compared to the previous reports, no new discovery was provided in the current study.

Major concerns.

It would have been interesting and important to perform viral isolation in addition to viral RNA detection to obtain information about the risk of EBOV sexual transmission according to the time after discharge. In addition, viral isolation would have allowed to perform genome sequencing of persisting viruses. These data would have constituted an important advance in the field.

The authors should have cited and discussed more exhaustively the existing literature about EBOV persistence such as Whitmer 2018 (Cell Reports 22, 1159), Keita 2019 (OFID 6,12), Barnes 2017 (CID 65:1400)…

Minor concerns.

In table 1, the information about the sexual desire appear twice, but with different values

Reviewer #3: The study by Thorson et al. addresses a public health problem for Ebola virus disease by providing a thorough, and very well written, analysis of samples and data rather difficult to achieve. I only suggest minor changes.

While the study provides academically sound results, their public health relevance could be improved by addressing some virological issues.

Results of the same study was already published earlier (Deen et al. 2017). Of 220 participants EBOV RNA was detected up to 1.5 years after ETU discharge. They stated, that "These data showed the long-term presence of Ebola virus RNA in semen and declining persistence with increasing time after ETU discharge." An added value of the published data could be achieved by evaluating and concluding directly the risk of sexual transmission of EVD. Barnes et al. (CID 2017:65) describe in their case report the isolation of viable EBOV in semen up to day 32 (after clinical recovery from EVD) and active replication in cells present in semen. Unfortunately, the submitted results does not relate to viable virus in semen but only viral RNA fragments. Although this might be difficult to achieve, it would strengthen any recommendation based on the presented data. By using an internal standard the amount of virus genome copy numbers could be used instead of PCR ct-values for a more informative conclusion and risk analysis. It has been shown by Barnes et al. that low ct-values of EBOV can be detected in semen within the first weeks/months after recovery from EVD. It would be helpful to correlate ct-values with amounts of viable virus (either directly from the clinical samples or by spiking experiments). Further, results are only defined as positive and negative by ct-values below or above 40. Depending on the PCR protocol a more informative outcome and conclusion could be achieved by presenting detailed ct-values or even better viral genome copy numbers.

About the housekeeping gene target as internal control: was RNA being used after DNase treatment to avoid measuring DNA fragments? Please specify in methods section. Quality control for a RNA target should be undertaken by use of a RNA internal control.

Despite recommendations for safe sex, a third of survivors had a low uptake. Did you include testing of sex partners for monitoring of a possible transmission/infection? This could easily be tested by serology? This would further allow calculating or discussing the risk for sexual transmission and resulting infection with EBOV?

As the author's state in their study, a high proportion of male survivors regularly had unprotected sex after recovery from EVD, and obviously without causing another outbreak. How can this be explained?

Reviewer #4: Thorson and colleagues present a thorough analysis of the magnitude and duration of Ebola virus persistence in semen from survivors in Sierra Leone. The authors also include analysis of potential risk factors that influence persistence.

Overall, I had few, if any, concerns with the manuscript. There were a few instances of redundant use of terms in the same or back-to-back sentences; however, these could be rectified with an additional thorough proofread.

The authors should be commended on their analysis. In particular, the authors provide evidence for more thorough behavioral guidance and analysis regarding sexual/reproductive health for EVD survivors. In addition, the authors have identified correlations between age and duration of persistence. As the authors point out, this could be related to a number of biological variables that should be further studied.

Taken together, the authors have provided an informative manuscript that expands on their prior work while also providing guidance for public health practices.

Any attachments provided with reviews can be seen via the following link:

[LINK]

PLoS Med. 2021 Feb 10;18(2):e1003273. doi: 10.1371/journal.pmed.1003273.r003

Author response to Decision Letter 1

27 Apr 2020

Attachment

Submitted filename: response to reviewers April 2020.docx

Click here for additional data file.^{(40.1KB, docx)}

PLoS Med. doi: 10.1371/journal.pmed.1003273.r004

Decision Letter 2

Clare Stone

30 Jun 2020

Dear Dr. Thorson,

Thank you very much for re-submitting your manuscript "Persistence of Ebola virus in semen among survivors in Sierra Leone: A cohort study of frequency, duration and risk factors" (PMEDICINE-D-20-00300R2) for review by PLOS Medicine.

I have discussed the paper with my colleagues and the academic editor and it was also seen again by reviewers. I am pleased to say that provided the remaining editorial and production issues are dealt with we are planning to accept the paper for publication in the journal.

The remaining issues that need to be addressed are listed at the end of this email. Any accompanying reviewer attachments can be seen via the link below. Please take these into account before resubmitting your manuscript:

[LINK]

Our publications team (plosmedicine@plos.org) will be in touch shortly about the production requirements for your paper, and the link and deadline for resubmission. DO NOT RESUBMIT BEFORE YOU'VE RECEIVED THE PRODUCTION REQUIREMENTS.

In revising the manuscript for further consideration here, please ensure you address the specific points made by each reviewer and the editors. In your rebuttal letter you should indicate your response to the reviewers' and editors' comments and the changes you have made in the manuscript. Please submit a clean version of the paper as the main article file. A version with changes marked must also be uploaded as a marked up manuscript file.

Please also check the guidelines for revised papers at http://journals.plos.org/plosmedicine/s/revising-your-manuscript for any that apply to your paper. If you haven't already, we ask that you provide a short, non-technical Author Summary of your research to make findings accessible to a wide audience that includes both scientists and non-scientists. The Author Summary should immediately follow the Abstract in your revised manuscript. This text is subject to editorial change and should be distinct from the scientific abstract.

We expect to receive your revised manuscript within 1 week. Please email us (plosmedicine@plos.org) if you have any questions or concerns.

We ask every co-author listed on the manuscript to fill in a contributing author statement. If any of the co-authors have not filled in the statement, we will remind them to do so when the paper is revised. If all statements are not completed in a timely fashion this could hold up the re-review process. Should there be a problem getting one of your co-authors to fill in a statement we will be in contact. YOU MUST NOT ADD OR REMOVE AUTHORS UNLESS YOU HAVE ALERTED THE EDITOR HANDLING THE MANUSCRIPT TO THE CHANGE AND THEY SPECIFICALLY HAVE AGREED TO IT.

If you have any questions in the meantime, please contact me or the journal staff on plosmedicine@plos.org.

We look forward to receiving the revised manuscript by Jul 07 2020 11:59PM.

Sincerely,

Adya Misra, PhD

Senior Editor

PLOS Medicine

plosmedicine.org

------------------------------------------------------------

Requests from Editors:

Please ensure that in the resubmitted version the main, revised doc is the only doc submitted in the file. This should be a clean doc with no tracking (I am finding it very difficult to know what is the final version and this is likely to add delays if it comes back again like this.) and also the shaded ‘confidential’ removed. When multiple copies are submitted in the revised file it is confusing as to which is the latest version. Any old or tracked versions can go into a supp file.

Abstract “Leon” correct to Leone

Abstract -we need accurate dates (insert months) where participants were recruited from, how many there were and some summary demographic information including mean age.

Abstract – “EVD Ct-values” on first mention, please define. Also ETU discharge

Abstract and throughout – p values need to be provided with 95% Cis for all quantifiable data

“Longer persistence was significantly associated” – please provide data and stats to support such statements. And again on page 16

Please check spellings (from Author Summary: Eevidence)

Refs should be presented in square not rounded brackets in the main text

PLOS Medicine requires that the de-identified data underlying the specific results in a published article be made available, without restrictions on access, in a public repository or as Supporting Information at the time of article publication, provided it is legal and ethical to do so. Please see the policy at http://journals.plos.org/plosmedicine/s/data-availability and FAQs at http://journals.plos.org/plosmedicine/s/data-availability#loc-faqs-for-data-policy

The email address you provide – who is that for? We cannot allow authors to be points of contact and we need to know what restrictions are in place for those who apply for access. It is not sufficient to say data is private

Page 13 “right-censored” what does this mean?

Page 16 “None of the men reported co-morbidity in the forms of tuberculosis, diabetes, malaria or sexually transmitted infections (STI) in relation to their acute infection with EVD” please state clearly this is self reported and can be inaccurate as such and what do you mean by ‘in relation to’. Arent they independent ?

STROBE – please report using sections and paragraphs as pages change on revision / formatting.

Analysis plan – we asked if you had a prospective analysis plan – this is different to a statistical plan and thank you for providing that, but we still need an analysis plan or a discussion in the methods

As a reminder: did you study have a prospective protocol or analysis plan? Please state this (either way) early in the Methods section. a) If a prospective analysis plan (from your funding proposal, IRB or other ethics committee submission, study protocol, or other planning document written before analyzing the data) was used in designing the study, please include the relevant prospectively written document with your revised manuscript as a Supporting Information file to be published alongside your study, and cite it in the Methods section. A legend for this file should be included at the end of your manuscript. b) If no such document exists, please make sure that the Methods section transparently describes when analyses were planned, and when/why any data-driven changes to analyses took place. c) In either case, changes in the analysis-- including those made in response to peer review comments-- should be identified as such in the Methods section of the paper, with rationale.

Comments from Reviewers:

Reviewer #1: The paper is much improved. I have no further comments.

Reviewer #2: The main weakness of the study, it means the lack of real novelty of the findings according to the existing literature, has been discussed in the rebuttal by the authors. They are right in claiming that their study represents the most complete and statistically robust evaluation of the EBOV persistence in semen to date. However, I am still convinced that this new study only confirms previous reports and does not represent a breakthrough in the field, even if the analysis of the associations between sociodemographic and acute disease severity and persistence constitutes a new finding.

The other main criticism, which is the lack of viral isolation data in semen, is still present and the author's answer unfortunately does not allow to lower this issue. Indeed, viral isolation from semen is not feasible in the current study. Instead, they cite a paper from Whitmer (Cell Reports, 2018) to provide data about viral isolation from semen arguing that data from the survivors included in this cohort were presented in this report. However, unless I am mistaken, viral isolation from semen is only presented for 5 survivors hospitalized in the USA in this manuscript. The impossibility to provide data about infectious virus load within semen is unfortunate, as it would have constituted a substantial added value and novelty to the manuscript

Reviewer #3: The authors have implemented most suggested improvements except for the issue of providing validity of their data base for all follow up analyses.

By only stating the limitations of RT PCR versus virus isolation in the paper and not addressing them will give the reader an insufficient base of their results for the conclusions drawn. PCR ct-values are assay dependent and do not allow a comparison with results from other studies using another PCR assay (also: different assays were applied in the published study). It is understandable, that the test for viable virus in semen can only be achieved with great effort and the described challenges with shipping and laboratory limitations in-country might not be possible to change subsequently. The referencing of the Whitmer paper (2018) is of value but does not allow the reader to get the ct-values of the present study in correlation to virus amounts within the samples. I would like to encourage presenting the data and cut-off rather by means of virus genome copy numbers than by ct-value. Ct-values are only readouts of a machine assay dependent and do not present a functional meaning in contrast to virus genome copy numbers, which is easily obtained by including an internal standard.

Reviewer #4: I have no additional concerns or edits required for the manuscript from Thorson and colleagues and feel this is an important addition to the literature on Ebola virus persistence.

Any attachments provided with reviews can be seen via the following link:

[LINK]

PLoS Med. doi: 10.1371/journal.pmed.1003273.r005

Decision Letter 3

Richard Turner

29 Dec 2020

Dear Dr. Thorson,

I am writing concerning your manuscript submitted to PLOS Medicine, entitled “Persistence of Ebola virus in semen among survivors in Sierra Leone: A cohort study of frequency, duration and risk factors.”

We have now completed our final technical checks and have approved your submission for publication. You will shortly receive a letter of formal acceptance from the editor.

Kind regards,

PLOS Medicine

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 STROBE Checklist

(DOCX)

Click here for additional data file.^{(46.7KB, docx)}

S1 Protocol. Study protocol for the EBOV persistence study version 1.7.

25 January 2016.

(DOC)

Click here for additional data file.^{(1.7MB, doc)}

S1 Statistical Analysis Plan

(PDF)

Click here for additional data file.^{(295.8KB, pdf)}

Attachment

Submitted filename: response to reviewers April 2020.docx

Click here for additional data file.^{(40.1KB, docx)}

Data Availability Statement

[pmed.1003273.ref001] 1.World Health Organization. Ebola situation report—30 March 2016 Geneva: World Health Organization; 2016. [cited 2021 Jan 11]. Available from: https://apps.who.int/ebola/current-situation/ebola-situation-report-30-march-2016. [Google Scholar]

[pmed.1003273.ref002] 2.United Nations Development Group–Western and Central Africa. Socio-economic impact of Ebola virus disease in West African countries New York: United Nations Development Group; 2015. [cited 2021 Jan 6]. Available from: https://www.undp.org/content/dam/rba/docs/Reports/ebola-west-africa.pdf. [Google Scholar]

[pmed.1003273.ref003] 3.United Nations Economic Commission for Africa. Socio-economic impacts of Ebola on Africa. Revised edition Addis Ababa: United Nations Economic Commission for Africa; 2015. [Google Scholar]

[pmed.1003273.ref004] 4.Den Boon S, Marston BJ, Nyenswah TG, Jambai A, Barry M, Keita S, et al. Ebola virus infection associated with transmission from survivors. Emerg Infect Dis. 2019;25(2):249–55. 10.3201/eid2502.181011 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pmed.1003273.ref005] 5.Christie A, Davies-Wayne GJ, Cordier-Lassalle T, Blackley DJ, Laney AS, Williams DE, et al. Possible sexual transmission of Ebola virus—Liberia, 2015. MMWR Morb Mortal Wkly Rep. 2015;64(17):479–81. [PMC free article] [PubMed] [Google Scholar]

[pmed.1003273.ref006] 6.Mate SE, Kugelman JR, Nyenswah TG, Ladner JT, Wiley MR, Cordier-Lassalle T, et al. Molecular evidence of sexual transmission of Ebola virus. N Engl J Med. 2015;373(25):2448–54. 10.1056/NEJMoa1509773 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pmed.1003273.ref007] 7.Diallo B, Sissoko D, Loman NJ, Bah HA, Bah H, Worrell MC, et al. Resurgence of Ebola virus disease in Guinea linked to a survivor with virus persistence in seminal fluid for more than 500 days. Clin Infect Dis. 2016;63(10):1353–6. 10.1093/cid/ciw601 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pmed.1003273.ref008] 8.Thorson A, Formenty P, Lofthouse C, Broutet N. Systematic review of the literature on viral persistence and sexual transmission from recovered Ebola survivors: evidence and recommendations. BMJ Open. 2016;6(1):e008859 10.1136/bmjopen-2015-008859 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pmed.1003273.ref009] 9.Fischer WA, Brown J, Wohl DA, Loftis AJ, Tozay S, Reeves E, et al. Ebola virus ribonucleic acid detection in semen more than two years after resolution of acute Ebola virus infection. Open Forum Infect Dis. 2017;4(3):ofx155 10.1093/ofid/ofx155 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pmed.1003273.ref010] 10.Schindell BG, Webb AL, Kindrachuk J. Persistence and sexual transmission of filoviruses. Viruses. 2018;10(12):683 10.3390/v10120683 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pmed.1003273.ref011] 11.Sissoko D, Duraffour S, Kerber R, Kolie JS, Beavogui AH, Camara AM, et al. Persistence and clearance of Ebola virus RNA from seminal fluid of Ebola virus disease survivors: a longitudinal analysis and modelling study. Lancet Glob Health. 2017;5(1):e80–8. 10.1016/S2214-109X(16)30243-1 [DOI] [PubMed] [Google Scholar]

[pmed.1003273.ref012] 12.Soka MJ, Choi MJ, Baller A, White S, Rogers E, Purpura LJ, et al. Prevention of sexual transmission of Ebola in Liberia through a national semen testing and counselling programme for survivors: an analysis of Ebola virus RNA results and behavioural data. Lancet Glob Health. 2016;4(10):e736–43. 10.1016/S2214-109X(16)30175-9 [DOI] [PubMed] [Google Scholar]

[pmed.1003273.ref013] 13.Deen GF, Broutet N, Xu W, Knust B, Sesay FR, McDonald SLR, et al. Ebola RNA persistence in semen of ebola virus disease survivors—final report. N Engl J Med. 2017;377(15):1428–37. 10.1056/NEJMoa1511410 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pmed.1003273.ref014] 14.Deen GF, McDonald SLR, Marrinan JE, Sesay FR, Ervin E, Thorson AE, et al. Implementation of a study to examine the persistence of Ebola virus in the body fluids of Ebola virus disease survivors in Sierra Leone: Methodology and lessons learned. PLoS Negl Trop Dis. 2017;11(9):e0005723 10.1371/journal.pntd.0005723 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pmed.1003273.ref015] 15.Abad N, Malik T, Ariyarajah A, Ongpin P, Hogben M, McDonald SLR, et al. Development of risk reduction behavioral counseling for Ebola virus disease survivors enrolled in the Sierra Leone Ebola Virus Persistence Study, 2015–2016. PLoS Negl Trop Dis. 2017;11(9):e0005827 10.1371/journal.pntd.0005827 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pmed.1003273.ref016] 16.Centers for Disease Control and Prevention. 2014 Ebola virus emergency use authorizations: CDC Ebola virus VP40 real-time RT-PCR assay (CDC)—March 2, 2015. Atlanta: Centers for Disease Control and Prevention; 2021. [cited 2021 Jan 6]. Available from: https://www.fda.gov/MedicalDevices/Safety/EmergencySituations/ucm161496.htm#ebola. [Google Scholar]

[pmed.1003273.ref017] 17.Centers for Disease Control and Prevention. Ebola virus NP real-time RT-PCR assay. Atlanta: Centers for Disease Control and Prevention; 2016. [cited 2021 Jan 6]. Available from: https://www.fda.gov/downloads/medicaldevices/safety/emergencysituations/ucm436307.pdf. [Google Scholar]

[pmed.1003273.ref018] 18.Erickson BR, Sealy TK, Flietstra T, Morgan L, Kargbo B, Matt-Lebby VE, et al. Ebola virus disease diagnostics, Sierra Leone: analysis of real-time reverse transcription-polymerase chain reaction values for clinical blood and oral swab specimens. J Infect Dis. 2016;214(Suppl 3):S258–62. 10.1093/infdis/jiw296 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pmed.1003273.ref019] 19.Ellerbrok H, Jacobsen S, Patel P, Rieger T, Eickmann M, Becker S, et al. External quality assessment study for ebolavirus PCR-diagnostic promotes international preparedness during the 2014–2016 Ebola outbreak in West Africa. PLoS Negl Trop Dis. 2017;11(5):e0005570 10.1371/journal.pntd.0005570 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pmed.1003273.ref020] 20.Whitmer SLM, Ladner JT, Wiley MR, Patel K, Dudas G, Rambaut A, et al. Active Ebola virus replication and heterogeneous evolutionary rates in EVD survivors. Cell Rep. 2018;22(5):1159–68. 10.1016/j.celrep.2018.01.008 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pmed.1003273.ref021] 21.Durski KN, Singaravelu S, Teo J, Naidoo D, Bawo L, Jambai A, et al. Development, use, and impact of a global laboratory database during the 2014 Ebola outbreak in West Africa. J Infect Dis. 2017;215(12):1799–806. 10.1093/infdis/jix236 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pmed.1003273.ref022] 22.Faye O, Andronico A, Faye O, Salje H, Boelle PY, Magassouba N, et al. Use of viremia to evaluate the baseline case fatality ratio of Ebola virus disease and inform treatment studies: a retrospective cohort study. PLoS Med. 2015;12(12):e1001908 10.1371/journal.pmed.1001908 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pmed.1003273.ref023] 23.SAS system for Windows, release 9.4. Cary (NC): SAS Institute; 2014. [Google Scholar]

[pmed.1003273.ref024] 24.Collett D. Modelling survival data in medical research. London: Chapman & Hall; 1994. [Google Scholar]

[pmed.1003273.ref025] 25.Subtil F, Delaunay C, Keita AK, Sow MS, Toure A, Leroy S, et al. Dynamics of Ebola RNA persistence in semen: a report from the Postebogui Cohort in Guinea. Clin Infect Dis. 2017;64(12):1788–90. 10.1093/cid/cix210 [DOI] [PubMed] [Google Scholar]

[pmed.1003273.ref026] 26.Keita AK, Vidal N, Toure A, Diallo MSK, Magassouba N, Baize S, et al. A 40-month follow-up of Ebola virus disease survivors in Guinea (PostEbogui) reveals long-term detection of Ebola viral ribonucleic acid in semen and breast milk. Open Forum Infect Dis. 2019;6(12):ofz482 10.1093/ofid/ofz482 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pmed.1003273.ref027] 27.Group PIS, Sneller MC, Reilly C, Badio M, Bishop RJ, Eghrari AO, et al. a longitudinal study of ebola sequelae in Liberia. N Engl J Med. 2019;380(10):924–34. 10.1056/NEJMoa1805435 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pmed.1003273.ref028] 28.Sow MS, Etard JF, Baize S, Magassouba N, Faye O, Msellati P, et al. New evidence of long-lasting persistence of Ebola virus genetic material in semen of survivors. J Infect Dis. 2016;214(10):1475–6. 10.1093/infdis/jiw078 [DOI] [PubMed] [Google Scholar]

[pmed.1003273.ref029] 29.Uyeki TM, Erickson BR, Brown S, McElroy AK, Cannon D, Gibbons A, et al. Ebola virus persistence in semen of male survivors. Clin Infect Dis. 2016;62(12):1552–5. 10.1093/cid/ciw202 [DOI] [PubMed] [Google Scholar]

[pmed.1003273.ref030] 30.World Health Organization. Ebola virus disease Democratic Republic of Congo: external situation report 89. Geneva: World Health Organization; 2020. [cited 2021 Jan 6]. Available from: https://www.who.int/publications-detail/ebola-virus-disease-democratic-republic-of-congo-external-situation-report-89-2019. [Google Scholar]

PERMALINK

Persistence of Ebola virus in semen among Ebola virus disease survivors in Sierra Leone: A cohort study of frequency, duration, and risk factors

A E Thorson

G F Deen

K T Bernstein

W J Liu

F Yamba

N Habib

F R Sesay

P Gaillard

T A Massaquoi

S L R McDonald

Y Zhang

K N Durski

S Singaravelu

E Ervin

H Liu

A Coursier

J E Marrinan

A Ariyarajah

M Carino

P Formenty

U Ströher

M Lamunu

G Wu

F Sahr

W Xu

B Knust

N Broutet

Roles

Abstract

Background

Methods and findings

Conclusions

Author summary

Why was this study done?

What did the researchers do and find?

What do these findings mean?

Introduction

Methods

Study design

Ebola virus detection

Outcome variable

Representativity of sample—link to the Sierra Leone national Ebola database

Variables

Sociodemographic characteristics, symptoms during acute infection, and EVD sequelae

Acute EVD Ct values

Survival analysis

Ethics

Results

Study participants and retention

Baseline participants’ characteristics and uptake of safe sex advice

Table 1. Baseline characteristics of EVD survivors enrolled in the study.

EBOV persistence detected in semen over time

Table 2. Kaplan–Meier non-parametric interval-censored probability estimates of Ebola virus RNA persistence in semen.

Fig 1. Survival function (Kaplan–Meier analysis) of Ebola virus RNA persistence in semen over time since ETU discharge, with Weibull fit.

Severe acute disease and older age associated with EBOV RNA persistence in semen

Fig 2. Survival function of EBOV RNA persistence stratified by acute/ETU blood cycle threshold values, within the sub-population with matched values available.

Table 3. Multivariate model with living with extended family × education interaction term.

Table 4. Multivariate model in a subset of 121 participants with available ETU blood Ct value.

Fig 3. Survival function of Ebola virus RNA persistence over time (days) stratified by age, within the sub-population who had severe acute disease (ETU cycle threshold value < 27).

Fig 4. Survival function of Ebola virus RNA persistence over time (days) stratified by age, within the sub-population who had milder acute disease (ETU cycle threshold value ≥ 27).

EVD co-morbidity and EVD sequelae

Representativity of study sample

Discussion

Conclusions

Supporting information

Acknowledgments

Abbreviations

Data Availability

Funding Statement

References

Decision Letter 0

Helen Howard

Roles

Decision Letter 1

Adya Misra

Roles

Author response to Decision Letter 1

Decision Letter 2