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      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

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          Abstract

          Background

          The 2013–2016 West African Ebola virus disease epidemic was unprecedented in terms of the number of cases and survivors. Prior to this epidemic there was limited data available on the persistence of Ebola virus in survivors’ body fluids and the potential risk of transmission, including sexual transmission.

          Methodology/Principal findings

          Given the urgent need to determine the persistence of Ebola virus in survivors’ body fluids, an observational cohort study was designed and implemented during the epidemic response operation in Sierra Leone. This publication describes study implementation methodology and the key lessons learned. Challenges encountered during implementation included unforeseen duration of follow-up, complexity of interpreting and communicating laboratory results to survivors, and the urgency of translating research findings into public health practice. Strong community engagement helped rapidly implement the study during the epidemic. The study was conducted in two phases. The first phase was initiated within five months of initial protocol discussions and assessed persistence of Ebola virus in semen of 100 adult men. The second phase assessed the persistence of virus in multiple body fluids (semen or vaginal fluid, menstrual blood, breast milk, and urine, rectal fluid, sweat, saliva, tears), of 120 men and 120 women.

          Conclusion/Significance

          Data from this study informed national and global guidelines in real time and demonstrated the need to implement semen testing programs among Ebola virus disease survivors. The lessons learned and study tools developed accelerated the implementation of such programs in Ebola virus disease affected countries, and also informed studies examining persistence of Zika virus. Research is a vital component of the public health response to an epidemic of a poorly characterized disease. Adequate resources should be rapidly made available to answer critical research questions, in order to better inform response efforts.

          Author summary

          The 2013–2016 West African Ebola virus disease epidemic was unprecedented in the numbers of cases, deaths and survivors. Prior to this epidemic, limited data were available about the persistence of Ebola virus in body fluids of Ebola virus disease survivors and the related risk of transmission, including sexual transmission through survivors’ semen. As more data were urgently needed, a study was implemented in Sierra Leone during the epidemic response operation. When establishing this study, many factors were unknown and the findings needed to be urgently translated into practice. This publication summarizes the methodology used for study implementation and the key lessons learned. Strong community engagement helped rapidly initiate this study despite the difficult circumstances of an overwhelming epidemic in a country with few experienced researchers. Implementation was in two phases, first investigating the persistence of Ebola virus in semen only (100 adult men), and secondly, assessing its persistence in multiple body fluids (semen or vaginal fluid, menstrual blood, breast milk; and urine, rectal fluid, sweat, saliva, tears) of an additional 120 men and 120 women. Data from this study immediately informed national and global guidelines and demonstrated the need for semen testing programs. The lessons learned and study tools developed, supported rapid program implementation in Liberia and Sierra Leone.

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          Most cited references21

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          Ebola RNA Persistence in Semen of Ebola Virus Disease Survivors - Preliminary Report.

          Background Ebola virus has been detected in the semen of men after their recovery from Ebola virus disease (EVD), but little information is available about its prevalence or the duration of its persistence. We report the initial findings of a pilot study involving survivors of EVD in Sierra Leone. Methods We enrolled a convenience sample of 100 male survivors of EVD in Sierra Leone, at different times after their recovery from EVD, and recorded self-reported information about sociodemographic characteristics, the EVD episode, and health status. Semen specimens obtained at baseline were tested by means of a quantitative reverse-transcriptase-polymerase-chain-reaction (RT-PCR) assay with the use of the target-gene sequences of NP and VP40. Results A total of 93 participants provided an initial semen specimen for analysis, of whom 46 (49%) had positive results on quantitative RT-PCR. Ebola virus RNA was detected in the semen of all 9 men who had a specimen obtained 2 to 3 months after the onset of EVD, in the semen of 26 of 40 (65%) who had a specimen obtained 4 to 6 months after onset, and in the semen of 11 of 43 (26%) who had a specimen obtained 7 to 9 months after onset; the results for 1 participant who had a specimen obtained at 10 months were indeterminate. The median cycle-threshold values (for which higher values indicate lower RNA levels) were 32.0 with the NP gene target and 31.1 with the VP40 gene target for specimens obtained at 2 to 3 months, 34.5 and 32.3, respectively, for specimens obtained at 4 to 6 months, and 37.0 and 35.6, respectively, for specimens obtained at 7 to 9 months. Conclusions These data showed the persistence of Ebola virus RNA in semen and declining persistence with increasing months since the onset of EVD. We do not yet have data on the extent to which positivity on RT-PCR is associated with virus infectivity. Although cases of suspected sexual transmission of Ebola have been reported, they are rare; hence the risk of sexual transmission of the Ebola virus is being investigated. (Funded by the World Health Organization and others.).
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            Persistence and genetic stability of Ebola virus during the outbreak in Kikwit, Democratic Republic of the Congo, 1995.

            Ebola virus persistence was examined in body fluids from 12 convalescent patients by virus isolation and reverse transcription-polymerase chain reaction (RT-PCR) during the 1995 Ebola hemorrhagic fever outbreak in Kikwit, Democratic Republic of the Congo. Virus RNA could be detected for up to 33 days in vaginal, rectal, and conjunctival swabs of 1 patient and up to 101 days in the seminal fluid of 4 patients. Infectious virus was detected in 1 seminal fluid sample obtained 82 days after disease onset. Sequence analysis of an RT-PCR fragment of the most variable region of the glycoprotein gene amplified from 9 patients revealed no nucleotide changes. The patient samples were selected so that they would include some from a suspected line of transmission with at least three human-to-human passages, some from 5 survivors and 4 deceased patients, and 2 from patients who provided multiple samples through convalescence. There was no evidence of different virus variants cocirculating during the outbreak or of genetic variation accumulating during human-to-human passage or during prolonged persistence in individual patients.
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              ELISA for the detection of antibodies to Ebola viruses.

              EIAs for IgG and IgM antibodies directed against Ebola (EBO) viral antigens have been developed and evaluated using sera of animals and humans surviving infection with EBO viruses. The IgM capture assay detected anti-EBO (subtype Reston) antibodies in the sera of 5 of 5 experimentally infected animals at the time they succumbed to lethal infections. IgM antibodies were also detected in the serum of a human who was infected with EBO (subtype Reston) during a postmortem examination of an infected monkey. The antibody was detectable as early as day 6 after infection in experimentally infected animals and persisted for 400 days in 3 animals who survived infection, and it persisted for approximately 10 years after infection in the sera of 2 humans. Although these data are limited by the number of sera available for verification, the IgM assay seems to have great promise as a diagnostic tool. Furthermore the long-term persistence of the IgG antibodies measured by this test strongly suggests that the ELISA will be useful in field investigations of EBO virus.
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                Author and article information

                Contributors
                Role: MethodologyRole: Project administrationRole: SupervisionRole: VisualizationRole: Writing – review & editing
                Role: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: SupervisionRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: SoftwareRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: InvestigationRole: Project administration
                Role: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: Validation
                Role: ConceptualizationRole: Funding acquisitionRole: MethodologyRole: Project administrationRole: ResourcesRole: SupervisionRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Funding acquisitionRole: InvestigationRole: Project administrationRole: ResourcesRole: SupervisionRole: Validation
                Role: ConceptualizationRole: InvestigationRole: MethodologyRole: ResourcesRole: SoftwareRole: SupervisionRole: ValidationRole: Writing – review & editing
                Role: MethodologyRole: SupervisionRole: Writing – review & editing
                Role: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: Writing – review & editing
                Role: InvestigationRole: Project administrationRole: ResourcesRole: Validation
                Role: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: Writing – review & editing
                Role: Funding acquisitionRole: InvestigationRole: Project administrationRole: ResourcesRole: SoftwareRole: SupervisionRole: Validation
                Role: ConceptualizationRole: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: Writing – review & editing
                Role: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: SoftwareRole: SupervisionRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: SoftwareRole: SupervisionRole: ValidationRole: VisualizationRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: MethodologyRole: Project administrationRole: SupervisionRole: VisualizationRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: MethodologyRole: Project administrationRole: SoftwareRole: SupervisionRole: Validation
                Role: ConceptualizationRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: SoftwareRole: SupervisionRole: ValidationRole: VisualizationRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: MethodologyRole: Project administrationRole: ResourcesRole: SoftwareRole: SupervisionRole: VisualizationRole: Writing – review & editing
                Role: MethodologyRole: Project administrationRole: Supervision
                Role: Editor
                Journal
                PLoS Negl Trop Dis
                PLoS Negl Trop Dis
                plos
                plosntds
                PLoS Neglected Tropical Diseases
                Public Library of Science (San Francisco, CA USA )
                1935-2727
                1935-2735
                11 September 2017
                September 2017
                : 11
                : 9
                : e0005723
                Affiliations
                [1 ] Clinical Studies, Internal Medicine, Connaught Hospital, Ministry of Health and Sanitation, Freetown, Sierra Leone
                [2 ] Comprehensive Care & Support for EVD Survivors, EVD Research, World Health Organization, Freetown, Sierra Leone
                [3 ] 34 Military Hospital, Sierra Leone Ministry of Defence, Freetown, Sierra Leone
                [4 ] Viral Special Pathogens Branch, Division of High Consequence Pathogens and Pathology, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America
                [5 ] Department of Reproductive Health and Research, Family, Women's and Children's Health, World Health Organization, Geneva, Switzerland
                [6 ] National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, People's Republic of China
                [7 ] Sierra Leone-China Friendship Biological Safety Laboratory, Chinese Center for Disease Control and Prevention, Freetown, Sierra Leone
                [8 ] Strategic information, Joint United Nations Programme on HIV/AIDS, Freetown, Sierra Leone
                [9 ] Social & Behavioral Research and Evaluation Branch, Division of Sexually Transmitted Disease Prevention, National Center for HIV/AIDS, Viral Hepatitis, STD, & TB Prevention, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America
                [10 ] Office of Global Activities, Division of Sexually Transmitted Disease Prevention, National Center for HIV/AIDS, Viral Hepatitis, STD, & TB Prevention, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America
                [11 ] HIV Care and Treatment Branch, Division of Global HIV/AIDS and Tuberculosis, Center for Global Health, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America
                [12 ] Pandemic and Epidemic Disease Department, Outbreaks and Health Emergencies, World Health Organization, Geneva, Switzerland
                [13 ] Division of Global Health Protection, Center for Global Health, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America
                The University of Kansas, UNITED STATES
                Author notes

                The authors have declared that no competing interests exist.

                ‡ These authors also contributed equally to this work.

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

                Author information
                http://orcid.org/0000-0003-2363-2812
                Article
                PNTD-D-17-00141
                10.1371/journal.pntd.0005723
                5593174
                28892501
                b7ee3612-8f86-4538-88ef-5f4da85e1331

                This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

                History
                : 31 January 2017
                : 18 June 2017
                Page count
                Figures: 1, Tables: 1, Pages: 18
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/100000952, Paul G. Allen Family Foundation;
                Award Recipient :
                Funding was provided by the World Health Organization (PF NB), the United States Centers for Disease Control and Prevention (BK OM STN), the Chinese Center for Disease Control and Prevention (WX HL WJL YZ GW ML), and the Paul G. Allen Family Foundation ( http://www.pgafamilyfoundation.org/) (PF NB AET). The World Health Organization gratefully acknowledges the financial contribution of the WHO's Ebola Response Programme (PF), The Paul G. Allen Family Foundation (NB PF AET), and the UNDP-UNFPA-UNICEF-WHO-World Bank Special Programme of Research, Development and Research Training in Human Reproduction (HRP) (NB) in support of the Sierra Leone Ebola Virus Persistence Study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The views expressed in this article are those of the authors and do not necessarily represent the official positions, decisions, policy or views of the Government of Sierra Leone Ministry of Health and Sanitation, Ministry of Defence, the World Health Organization, the United States Centers for Disease Control and Prevention, or the Chinese Center for Disease Control and Prevention.
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