Evidence of persisting Ebola virus in semen

Evidence suggestive of persistent Ebola viruses in semen has been found in five different Ebola outbreaks, including the current epidemic.

EBOV (Ebola virus—Zaire) in semen has been isolated from two men, with infections stemming from two earlier epidemic settings, Kikwit, Congo, in 1995, and Gabon in 1997. The case from Kikwit had the longest semen positivity described, with a positive virus isolation of EBOV on day 82 post-onset. Follow-up sampling was not performed until day 700 post-onset.8 ,9 ,11

Isolation of SUDV (Ebola virus—Sudan) is reported from a man infected and treated in London, but the infection had stemmed from the Sudanese epidemic in 1976; there was also a report from the epidemic in Gulu, Uganda, in 2000, where one man had a positive semen sample with virus isolation on day 40 after his symptoms started12 ,13 (figure 1).

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Figure 1

Depiction of the duration of measured persistence of Ebola virus and Ebola virus RNA in semen (VI, virus isolation).

Detection of EBOV RNA by RT-PCR in semen specimens has further been reported from three other men infected during the epidemic in Kikwit, Congo.8 ,9 During the recent EBOV outbreak in 2014–2015, a suspect case of sexual transmission led to the analysis of semen from one man in Liberia. The semen specimen was positive with RT-PCR on day 199.14 ,15 A man travelling from Liberia to New Delhi, India, following his confirmed recovery from acute Ebola in November 2014, was found to still have presence of EBOV RNA in his semen until some 140 days after symptom onset (Formenty P. Personal communication with National Institute of Virology, Pune, India 2015).

The longest measured duration of persisting EBOV in semen is 284 days, reported from a man in Sierra Leone, infected during the current epidemic.16 The evidence describing duration of viral persistence in semen is summarised in figure 1.

Evidence of persisting EBOV in vaginal secretions

Within the outbreak of EBOV in Kikwit, Congo (DRC), in 1995, 20 female convalescent patients provided 44 vaginal swab specimens at different intervals, ranging from days 12 to 57 post-onset of symptoms.8 All the samples were negative for virus isolation and for viral antigen detection by ELISA.

Six female convalescent patients provided vaginal swab specimens for additional virus isolation and RT-PCR testing, ranging from days 20 to 33 post-onset of symptoms.8 ,9 Only one woman had EBOV RNA detected in her vaginal swab specimen, the latest positive test for detection was performed on day 33 following the start of symptoms, and no further follow-up was carried out. Figure 2 displays an overview of the women tested and the results.

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Figure 2

Vaginal swab specimen examined for viral shedding (Days, days post-symptom onset; RT-PCR, reverse transcriptase PCR; VI, virus isolation).

Evidence of persisting Ebola virus in faeces, saliva, sweat and urine

Faeces analyses or rectal swabs have been performed in 30 patients across different epidemic situations, and analysed with either detection by RT-PCR or virus isolation. Only one patient had a positive examination for viral RNA in faeces, with positive rectal swabs on days 22 and 29. The same woman was the only one to have a positive RT-PCR in vaginal secretions8 (table 1).

Analyses of persisting Ebola virus in saliva have been performed on a total of 35 convalescent patients, including RT-PCR analyses in 13 of these cases. Virus was detected neither by RT-PCR nor by virus isolation in the saliva and throat specimens from convalescent patients.8 ,9 ,12 ,13 ,17 In a study of Ebola detection in Congo during the outbreaks in 2003, oral fluid samples of acutely sick patients were analysed for both antigen and antibody detection.18 While RT-PCR was positive in all specimens from patients with acute Ebola disease, no antigen was detected from the same specimens. The authors conclude that this may be the result of minimal passage of antigen from blood to saliva18 (table 1).

Evidence from one male patient, infected in Sierra Leone in 2014 and treated in Germany, shows viable Ebola virus was found in urine until day 26 post-symptom onset, while blood was negative for EBOV from day 18. EBOV RNA was detected by RT-PCR until day 30 in urine and >day 40 in sweat (no further follow-up yet published), but quantified RNA levels were decreasing with time. Ebola virus was not isolated from his sweat17 (table 1).

Evidence of sexual transmission from convalescent patients

A case of sexual transmission in humans was established for the Marburg virus, in the 1967 epidemic.19 In Kikwit, in 1995, of 81 household-contacts followed for some 21 months, one 20-year-old woman had a weakly positive IgM response 52 days after sexual exposure from a convalescent partner. She had a later IgG negative serum sample. The ambiguous antibody results make interpretations, as to whether she may have had an asymptomatic Ebola infection or not, difficult.9 A case–control study from Kikwit additionally showed that household contacts who had had direct physical contact with the acutely ill or with a dead patient, or who were exposed to their body fluids, were at risk of acquiring infection, whereas none of the 78 household members without physical contact with the case during the acute illness got infected.20

Molecular evidence suggestive of suspect sexual transmission from an Ebola convalescent has been reported from the current epidemic in Liberia.21 A male Ebola survivor was discovered to have a positive EBOV RNA in his semen 199 days post-symptom onset when his wife fell ill with acute Ebola, without having a reported exposure to acute EVD.14 Matching of genetic sequences strongly suggest that the route of transmission was sexual.14 ,21

Furthermore, a doctor at a clinic in Monrovia, Liberia, reported three cases of suspect sexual transmission from male survivors to their female partners. For one of the female partners surviving her acute Ebola, a concurrent timeline could be established between sexual exposure and symptom onset. She reported no other known exposure to the Ebola virus.22 Recent reports from Bombali, Koinadougou and Port Loko districts, in Sierra Leone, similarly suggest suspected sexual transmission from several men to their female partners, while no in-depth investigations have yet been reported.23 ,24

Male and female latex condom efficacy

Procured male latex condoms are regularly quality controlled against ISO (International Organisation of Standardisation) standards. These include tests for stability, leakage and breakage, to be fulfilled by every manufactured lot and/or individual condom.25 A female latex condom has been approved by the US Food and Drug Administration (FDA) and the WHO/UNFPA, with manufacturing standards and efficacy that, to a large extent, match the male latex condom.26 ,27 As EBOV has a diameter of 80 nm, the virus may be transmitted sexually by leaking through small pores in latex condoms. In vitro testing has been performed to examine the possibility of penetration of small viruses, by using test viruses and bacteriophages.28–31 These studies showed that, during tests mimicking real-life use, and when using a surrogate challenge virus of 27 nm, a minority of condoms deemed intact may still leak through pores, risking passage of virus. The small size of the challenge virus allowed for conservative interpretations of condom efficacy. The study found that 12 of 470 (2.6%) tested latex condoms leaked despite being intact in a regular water leakage test. They allowed virus penetration of an average 0.0007 mL, which would correspond to about 0.00001 mL of semen in users, after correcting the value for viscosity and pressure. The worst performing condom did leak an estimated corrected value of some 0.05 mL semen.31 While condoms are interpreted as having a high enough efficacy as a barrier method against viral sexually transmitted disease (STD), for diseases where an infectious dose is present in <0.1 mL of semen, pores in intact condoms may still be of relevance to transmission.29

The FDA approved female latex condom FC2 was tested for viral permeability in vitro by a similar bacteriophage as was used for male condom testing. The sample size was small, but found rates of pore leakage of 5% for the FC2, the same rate as the male latex condom. The pore leakage rate was deemed acceptable for providing adequate protection against viral STD (as opposed to the earlier polyurethane model FC1, which showed a higher frequency of pore leakage).26

Male and female latex condom effectiveness

No studies on condom effectiveness in relation to EBOV were identified. A Cochrane review examined the effectiveness of male condom use on HIV transmission in sero-discordant heterosexual couples, and estimated that HIV-negative partners were 80% less likely to become HIV infected than persons in similar relationships in which condoms were not used.32 Effectiveness rates for prevention of HIV are similar for female condoms.26 ,33 ,34 The effectiveness of condoms in reducing transmission of HIV between discordant men who have sex with men is found to be lower, 61%.35

Survival outside the host, implications to transmission

In a laboratory study, dried EBOV did survive in the dark for several hours at 20–25°C.36 EBOV can also survive in liquid media for more than 46 days in room temperature, which would be of relevance to condom handling and risk of infectious virus prevailing.37 This may be pertinent in view of oral sex and masturbation, as well as vaginal and anal sex. However, a recent study that confirms EBOV survives longer in liquids than in dried blood, also suggests that hospital conditions, as opposed to warmer and more humid conditions, seem to enhance survival outside the host.38 Hypothetically, climate conditions involving high humidity and high temperatures would hence not be promoting outside-host survival. No studies were identified indicating transmission by way of a convalescent patient shedding virus outside the host and then causing transmission.