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Semen studies on Ebola survivors include PCR positives 9 months post infection.

Edited by niman

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Preliminary study finds that Ebola virus fragments can persist in the semen of some survivors for at least nine months

Preliminary results of a study into persistence of Ebola virus in body fluids show that some men still produce semen samples that test positive for Ebola virus nine months after onset of symptoms.

The report, published today in the New England Journal of Medicine, provides the first results of a long-term study being jointly conducted by the Sierra Leone Ministry of Health and Sanitation, Sierra Leone Ministry of Defence, the World Health Organization and the U.S. Centers for Disease Control and Prevention.

“Sierra Leone is committed to getting to zero cases and to taking care of our survivors, and part of that effort includes understanding how survivors may be affected after their initial recovery,” said Amara Jambai, M.D., M.Sc., Deputy Chief Medical Officer for the Sierra Leone Ministry of Health and Sanitation. “Survivors are to be commended for contributing to the studies that help us understand how long the virus may persist in semen.”

The first phase of this study has focused on testing for Ebola virus in semen because of past research showing persistence in that body fluid.  Better understanding of viral persistence in semen is important for supporting survivors to recover and to move forward with their lives.

“These results come at a critically important time, reminding us that while Ebola case numbers continue to plummet, Ebola survivors and their families continue to struggle with the effects of the disease. This study provides further evidence that survivors need continued, substantial support for the next 6 to 12 months to meet these challenges and to ensure their partners are not exposed to potential virus,” said Bruce Aylward, WHO Director-General’s Special Representative on the Ebola Response.

Ninety three men over the age of 18 from Freetown, Sierra Leone, provided a semen sample that was tested to detect the presence of Ebola virus genetic material. The men enrolled in the study between two and 10 months after their illness began. For men who were tested in the first three months after their illness began, all were positive (9/9; 100 percent). More than half of men (26/40; 65 percent) who were tested between four to six months after their illness began were positive, while one quarter (11/43; 26 percent) of those tested between seven to nine months after their illness began also tested positive. The men were given their test results along with counseling and condoms.

“EVD survivors who volunteered for this study are doing something good for themselves and their families and are continuing to contribute to the fight against Ebola and our knowledge about this disease,” said Yusuf Kabba, National President of the Sierra Leone Association of Ebola Survivors.

Why some study participants had cleared the fragments of Ebola virus from semen earlier than others remains unclear. The U.S. Centers for Disease Control and Prevention in Atlanta is conducting further tests of the samples to determine if the virus is live and potentially infectious.

“Ebola survivors face an increasing number of recognized health complications,” said CDC Director Tom Frieden, M.D., M.P.H. “This study provides important new information about the persistence of Ebola virus in semen and helps us make recommendations to survivors and their loved ones to help them stay healthy."

Until more is known, the more than 8000 male Ebola survivors across the three countries need appropriate education, counseling and regular testing so they know whether Ebola virus persists in their semen; and the measures they should take to prevent potential exposure of  their partners to the virus. Until a male Ebola survivor’s semen has twice tested negative, he should abstain from all types of sex or use condoms when engaging in sexual activity. Hands should be washed after any physical contact with semen. For more information: Interim advice on the sexual transmission of the Ebola virus disease

In the current West African outbreak, continued vigilance to identify, provide care for, contain and stop new cases, are key strategies on the road to achieving zero cases. 


For more information, please contact:

The Sierra Leone Ministry of Health and Sanitation media:
Jonathon Kamara
+076 678 021

WHO Ebola Communications:
Dr. Margaret Harris
+41 227911646 (o)
+41796036224 (m)

CDC Media Relations
(404) 639-3286                                             

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Ebola RNA Persistence in Semen of Ebola Virus Disease Survivors — Preliminary Report

Gibrilla F. Deen, M.D., Barbara Knust, D.V.M., Nathalie Broutet, M.D., Ph.D., Foday R. Sesay, M.D., Pierre Formenty, D.V.M., Christine Ross, M.D., Anna E. Thorson, M.D., Ph.D., Thomas A. Massaquoi, M.D., Jaclyn E. Marrinan, M.Sc., Elizabeth Ervin, M.P.H., Amara Jambai, M.D., Suzanna L.R. McDonald, Ph.D., Kyle Bernstein, Ph.D., Alie H. Wurie, M.D., Marion S. Dumbuya, R.N., Neetu Abad, Ph.D., Baimba Idriss, M.D., Teodora Wi, Ph.D., Sarah D. Bennett, M.D., Tina Davies, M.S., Faiqa K. Ebrahim, M.D., Elissa Meites, M.D., Dhamari Naidoo, Ph.D., Samuel Smith, M.D., Anshu Banerjee, Ph.D., Bobbie Rae Erickson, M.P.H., Aaron Brault, Ph.D., Kara N. Durski, M.P.H., Jorn Winter, Ph.D., Tara Sealy, M.P.H., Stuart T. Nichol, Ph.D., Margaret Lamunu, M.D., Ute Ströher, Ph.D., Oliver Morgan, Ph.D., and Foday Sahr, M.D.



Edited by niman

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The number of new cases of Ebola virus disease (EVD) in western Africa has declined from a peak of 1063 cases in the week of October 9, 2014, to fewer than 10 confirmed cases per week for 11 consecutive weeks as of October 7, 2015.1. The main mode of transmission is direct contact with the blood or body fluids of a person with EVD or from the body of a person who died from EVD.2,3However, Ebola virus can persist in the body fluids of survivors during convalescence,4,5 which may result in transmission of the virus. The potential for the persistence of Ebola virus in the semen of male survivors raises concern regarding the possible transmission of the virus to sexual partners.6

Previously, survivors of EVD were told to practice sexual abstinence or to use a condom for 3 months after recovery. These recommendations were based on virus-isolation results from semen specimens obtained from eight survivors of EVD or Marburg virus disease in previous epidemics,5,7-10 in which the longest period that infectious virus was found in semen after the onset of symptoms was 82 days.

In March 2015, a woman in Liberia received a diagnosis of EVD and her only potential exposure that could be ascertained was sexual contact with a male survivor of EVD. Further investigation found Ebola virus RNA in the survivor’s semen 199 days after the onset of his symptoms, with a genetic sequence that matched the sequence from the case patient.11 Although no infectious virus was detected in this semen specimen, the possibility that infectious Ebola virus could persist in the semen of survivors approximately 6 months after the onset of illness prompted the World Health Organization (WHO) and the Centers for Disease Control and Prevention (CDC) to revise their guidelines regarding the length of time that survivors of EVD should avoid unprotected sexual activity.12,13

There are thousands of survivors of EVD in western Africa, and many are sexually active men. Sexual transmission of the Ebola virus could possibly result in new outbreaks several weeks or months after all known chains of transmission in the region have stopped. Although the epidemiologic observations to date suggest that sexual transmission is a rare event, the Sierra Leone Ministry of Health and Sanitation, in collaboration with the Sierra Leone Ministry of Defense, the Sierra Leone Ministry of Social Welfare, Gender, and Children’s Affairs, the WHO, and the CDC initiated a study of the duration of virus persistence in the body fluids of survivors in Sierra Leone. We report initial findings from the pilot phase of the study, which investigated the persistence and viability of Ebola virus in the semen of male survivors of EVD.

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Study Design and Oversight

The Sierra Leone Ministry of Health and Sanitation, the Sierra Leone Ministry of Social Welfare, Gender, and Children’s Affairs, the WHO, and the CDC designed the study, and the Sierra Leone Ministry of Defense, the WHO, and the CDC gathered the data. The data analysis was performed and supervised by the CDC and the WHO. Manuscript planning and drafting were also overseen and performed by the Ministry of Health and Sanitation, the CDC, and the WHO. All these activities were performed in accordance with all applicable laws, regulations, and policies related to the protection of human participants and animals. A complete list of the members of the steering committee and the technical working group is provided in the Supplementary Appendix, available with the full text of this article at NEJM.org.

Study Population, Sampling, and Eligibility Criteria

We recruited a convenience sample of 100 male survivors from the Western Area District of Sierra Leone, which includes the capital of Freetown. We identified study participants who, at informational events that were held in conjunction with survivor associations, indicated interest in participation, as well as persons who were referred from Ebola treatment centers.

Participants were eligible for inclusion if they were men 18 years of age or older, could provide an official survivor certificate issued by the Ministry of Health and Sanitation (such certificates are provided to persons with laboratory-confirmed cases of EVD when they are discharged from an Ebola treatment center), and could provide written informed consent to participate in the study. We compensated participants for each visit to the study site. The research protocol was reviewed and approved by the Sierra Leone Ethical Review Board and the WHO Ethical Review Committee.

Data Collection

A member of the study team administered a questionnaire to all the participants at the time of enrollment to gather information about their EVD episode, self-reported health status, sexual behavior, and sociodemographic characteristics. The date of EVD onset was self-reported, and the date of discharge from the Ebola treatment center was ascertained from the participants’ survivor certificates. We asked participants to provide a semen specimen in a private room and provided instructions to ensure that proper infection-control procedures were followed.

We gave participants pretest counseling at the time of enrollment and post-test counseling 2 weeks later when they received their individual reverse-transcriptase–polymerase-chain-reaction (RT-PCR) results. The counseling included information about the test performed, the meaning of the results, and education about sexual risk-reduction practices, including appropriate condom use and disposal. Trained counselors also offered participants a voluntary, confidential rapid test for the human immunodeficiency virus (HIV), according to the national testing algorithm. We referred participants to a clinic for survivors of EVD if it was needed, as determined by the trained medical staff of the study, or requested.

Laboratory Analyses

After the semen specimens were collected, they were refrigerated (at 5 to 8°C) for no longer than 3 days and transported to the CDC field laboratory in Bo District, Sierra Leone. We performed quantitative RT-PCR testing using Ebola virus–specific gene targets (NP and VP40) and the human β2-microglobulin (B2M) gene, as described previously.14,15 We considered a specimen to be positive if the VP40 and NP gene targets were both detected within 40 cycles of replication. The specimen was considered to be negative if neither Ebola virus gene target was detected and the findings with respect to B2M status were positive. The findings were ruled to be indeterminate if either the VP40 or the NP gene target was detected but not both. Amplification of B2M served as an extraction control and RNA quality control.

The cycle-threshold value for each gene target is reported as the number of replication cycles that had occurred when the target was first detected. Cycle-threshold values have an inverse association with virus quantity, such that higher quantities of virus in given specimens have lower cycle-threshold values.16

Statistical Analysis

We report the number of participants who had a positive, indeterminate, or negative result on quantitative RT-PCR at enrollment according to the number of days between the self-reported onset of illness and the date that the semen specimen was obtained, rounded to the nearest whole month. Median cycle-threshold values, according to months after the onset of EVD, are reported, with the range of values observed for the NP and VP40 gene targets. Sociodemographic characteristics at baseline are also presented. Analysis of the data was performed with the use of Stata software, version 13.1 (StataCorp). 

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Study Participants

RT-PCR results were available for 93 of the 100 participants enrolled. Three participants were withdrawn from the study in accordance with the protocol after they were unable to provide a specimen at two consecutive visits. Four participants did not have diagnostic RT-PCR results from semen testing at baseline (i.e., the cycle-threshold value for B2M was above the cutoff value) and were excluded from this analysis.

The mean age of the 93 participants was 30 years (range, 18 to 58). A total of 15% of the participants had no formal education, 22% had less than 6 years of education, and 63% had 6 or more years. When the participants were asked about income, 43% reported not knowing their monthly income, 24% reported earning less than $100 (U.S.) per month, 13% reported earning in the range of $100 to $1,000 per month, 10% reported earning more than $1,000 per month, and 10% did not answer. No participant reported having received a diagnosis of HIV infection, tuberculosis, or diabetes. Among the 93 participants, the time from illness onset to study enrollment was 2 to 3 months (64 to 120 days) for 9 men (10%), 4 to 6 months (121 to 210 days) for 40 men (43%), 7 to 9 months (211 to 300 days) for 43 men (46%), and 10 months (306 days) for 1 man (1%).

Detection of Ebola RNA in Semen

A total of 46 of the 93 men (49%) had a specimen that was positive on quantitative RT-PCR. Ebola virus RNA was detected in the semen of all 9 men from whom a specimen was obtained during the first 3 months after the onset of illness, in the semen of 26 of 40 men (65%) from whom a specimen was obtained at 4 to 6 months, and in the semen of 11 of 43 men (26%) from whom a specimen was obtained at 7 to 9 months (Figure 1FIGURE 1nejmoa1511410_f1.gifResults on Quantitative RT-PCR in Initial Semen Specimens Obtained from Survivors of Ebola Virus Disease, According to Time after Symptom Onset.). The results for 1 participant who had a specimen obtained at 10 months were indeterminate. The proportions of men with semen samples that were negative or indeterminate on quantitative RT-PCR were higher with increasing months after the onset of EVD.

The median cycle-threshold values increased as the months after the onset of EVD increased. For specimens obtained at 2 to 3 months, the values were 32.0 with the NP gene target and 31.1 with the VP40 gene target; for those obtained at 4 to 6 months, the values were 34.5 and 32.3, respectively; and for those obtained at 7 to 9 months, the values were 37.0 and 35.6, respectively (Table 1TABLE 1nejmoa1511410_t1.gifProportion of Positive Findings on Quantitative RT-PCR and Cycle-Threshold Values in the Semen of Survivors of Ebola Virus Disease, According to Time after Symptom Onset.).

The longest time after the onset of a participant’s EVD symptoms that a semen specimen obtained at baseline remained positive on quantitative RT-PCR was 284 days (9 months). Conversely, the shortest time after symptom onset in a participant that an initial semen specimen was negative on quantitative RT-PCR was 128 days (4 months). Indeterminate results were encountered in 10 initial specimens in the range of 152 to 273 days after the onset of symptoms.

When we considered the number of days after a participant’s discharge from the Ebola treatment center, the longest time that an initial semen specimen remained positive on quantitative RT-PCR was 272 days (9 months). The shortest time after a participant’s discharge that an initial semen specimen was negative on quantitative RT-PCR was 100 days (3 months).

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We gathered evidence showing that an Ebola virus RNA signal on quantitative RT-PCR was found in the semen of male survivors of EVD at least 9 months after the onset of symptoms. Because the data in this report are cross sectional, we are limited to reporting only the point prevalence among participants rather than describing individual-level persistence and clearance of the RT-PCR signal over time. Among this cross-sectional group of participants, all 9 male survivors who provided a sample during the first 3 months after the onset of illness had positive results on quantitative RT-PCR. During months 4 to 6, more than half the enrolled survivors had positive results on quantitative RT-PCR. The percentage of male survivors with positive results continued to decline over time, with approximately one quarter of the participants having positive findings on quantitative RT-PCR at 7 to 9 months after onset.

We observed that the median cycle-threshold values for the NP and VP40 gene targets increased over time, which indicated that the median quantity of viral RNA in the semen decreased over time. Our study cohort included only survivors whose onset of illness was 10 months or less before enrollment, so we do not yet know how long survivors of EVD may have Ebola RNA detectable on quantitative RT-PCR in semen. Follow-up of this cohort is ongoing, and this report will be finalized when additional data to address the issues of infectivity are available.

The detection of Ebola virus RNA by quantitative RT-PCR does not necessarily indicate that infectious virus is present. The quantitative RT-PCR assay used in this study is highly sensitive, with a detection limit per reaction of 30 median tissue-culture–infective doses (TCID50) for the NP and VP40 gene targets in blood and urine samples to which a known quantity of live virus was added.14,15 However, the targeted RNA sequences detected by quantitative RT-PCR could be detecting the presence of the full genome from an intact replicating virus or from smaller fragments that are unable to replicate and infect a host cell. Virus-isolation assays are under way, in which the specimens will be inoculated onto mammalian cells and the cell cultures will be observed for cytopathic effect as the virus replicates, which is the best available standard to approximate infectivity.

The cycle-threshold value for Ebola RNA has been shown to be a good approximation of the viral load in blood,16 with an increasing cycle-threshold value indicating a decrease in the viral load. A limited study that examined the relationship between cycle-threshold values and virus isolation did not detect infectious virus in blood specimens from patients with EVD when cycle-threshold values were greater than 35.5 with the NP gene target.17 However, experiments have not yet been performed to predict the cycle-threshold value at which viable virus can no longer be cultured in semen. It is possible that even if men provide samples that are positive on quantitative RT-PCR several months after the onset of illness, the higher cycle-threshold values (such as the median values of 37.0 value with the NP gene target and 35.6 with the VP40 gene target at 7 to 9 months observed in the current study) may indicate that their semen is no longer infectious. Ongoing serial testing until the men in this study cohort have two consecutive negative results on quantitative RT-PCR, as defined above, and performing viral culture of the RT-PCR–positive specimens will enable us to address the question of the duration of persistence of potentially infectious virus in semen.

Our cross-sectional analysis of baseline data describes the preliminary results in this cohort. Follow-up of this preliminary report continues so that we may investigate the presence and persistence of virus in the semen of survivors of EVD, including studying the relationship among cycle-threshold values, viral isolation, and genome sequencing; assessing how long semen from a survivor of EVD will remain positive; and exploring risk factors for the persistence of Ebola virus in semen.

Although our findings are based on a cohort of 100 male survivors of EVD, the public health implications are still uncertain. The ongoing study of quantitative RT-PCR positivity and virus isolation in semen will provide better estimates of the duration of viral persistence and related probabilities of persistence at various points in time.

We do not yet have sufficient information to assess the risk of transmission through sexual intercourse, oral sex, or other sex acts from men with viable virus in their semen. Before the Ebola epidemic in western Africa, a single case of Marburg virus disease and one case of EVD had been linked to sexual contact with survivors of Marburg virus disease and EVD, respectively.7,10 In western Africa, cases that have been linked to sexual contact with survivors of EVD have not been systematically documented, and fewer than 20 in total have been suspected (Knust B, CDC; Formenty P, WHO: personal communication).

Although the potential contribution of sexual transmission to the scale of the epidemic is largely unknown, the unprecedented number of more than 16,000 survivors of EVD across Sierra Leone, Guinea, and Liberia, roughly half of whom are male, creates the potential for transmission and initiation of new chains of transmission, even months after the outbreak has ended. Even though there have been only rare cases of EVD linked to sexual transmission, research is needed to investigate whether infectious virus may be present in vaginal fluid or other body fluids after recovery, and the testing of additional body fluids in both male and female survivors is planned.

Programs such as semen testing and preventive behavioral counseling are needed in order to help survivors of EVD appreciate and mitigate the possible risk of sexual transmission. Such programs would help men and women understand their individual risk and take appropriate measures to protect their sexual partners, specifically in regard to condom use and disposal, and could provide links to care and counseling programs for survivors. Because semen-testing programs are not yet universally available, outreach activities are needed to provide education regarding recommendations and risks to survivor communities and sexual partners of survivors in a way that does not further stigmatize the community of survivors of EVD.

Persons who survive EVD face myriad challenges. Many survivors have family members and friends who died from EVD. Many are unemployed, face stigma from their communities, and have lingering sequelae in addition to the risk of persisting virus in semen. Due respect and continuing efforts that have strong sustainable support from within the local communities are crucial in mitigating negative effects in terms of further stigma attached to survivors.


Supported by the WHO, the CDC, the Sierra Leone Ministry of Health and Sanitation, and the Joint United Nations Program on HIV/AIDS (UNAIDS).

Disclosure forms provided by the authors are available with the full text of this article at NEJM.org.

The views expressed in this article are those of the authors and do not necessarily represent the official positions of the World Health Organization (WHO) or the Centers for Disease Control and Prevention (CDC).

This article was published on October 14, 2015, at NEJM.org.

We thank all the study participants, without whom this study would not have been possible, and the membership of the Sierra Leone Association of Ebola Survivors.


From the Sierra Leone Ministry of Health and Sanitation (G.F.D., A.J., A.H.W., S.S.), Sierra Leone Armed Forces (F.R.S., T.A.M., M.S.D., B.I., F.S.), and Sierra Leone Ministry of Social Welfare, Gender, and Children’s Affairs (T.D.) — all in Freetown, Sierra Leone; Centers for Disease Control and Prevention, Atlanta (B.K., C.R., E.E., K.B., N.A., S.D.B., E.M., B.R.E., A. Brault, J.W., T.S., S.T.N., U.S., O.M.); World Health Organization, Geneva (N.B., P.F., A.E.T., J.E.M., S.L.R.M., M.S.D., T.W., F.K.E., D.N., A. Banerjee, K.N.D., M.L.); and Karolinska Institutet, Stockholm (A.E.T.).

Address reprint requests to Dr. Deen at Connaught Hospital, Freetown, Sierra Leone, or at.

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