article: The 2013–15 Ebola outbreak was unprecedented due to sustained transmission within urban environments and thousands of survivors. In 2014 the World Health Organization stated that there was insufficient evidence to give definitive guidance about which body fluids are infectious and when they pose a risk to humans. We report a rapid systematic review of published evidence on the presence of filoviruses in body fluids of infected people and survivors. Scientific articles were screened for information about filovirus in human body fluids. The aim was to find primary data that suggested high likelihood of actively infectious filovirus in human body fluids (viral RNA). Eligible infections were from Marburg virus (MARV or RAVV) and Zaire, Sudan, Taï Forest and Bundibugyo species of Ebola. Cause of infection had to be laboratory confirmed (in practice either tissue culture or RT-PCR tests), or evidenced by compatible clinical history with subsequent positivity for filovirus antibodies or inflammatory factors. Data were extracted and summarized narratively. 6831 unique articles were found, and after screening, 33 studies were eligible. For most body fluid types there were insufficient patients to draw strong conclusions, and prevalence of positivity was highly variable. Body fluids taken >16 days after onset were usually negative. In the six studies that used both assay methods RT-PCR tests for filovirus RNA gave positive results about 4 times more often than tissue culture. Filovirus was reported in most types of body fluid, but not in every sample from every otherwise confirmed patient. Apart from semen, most non-blood, RT-PCR positive samples are likely to be culture negative and so possibly of low infectious risk. Nevertheless, it is not apparent how relatively infectious many body fluids are during or after illness, even when culture-positive, not least because most test results come from more severe cases. Contact with blood and blood-stained body fluids remains the major risk for disease transmission because of the known high viral loads in blood.
  The 2013–15 epidemic of Ebola virus disease in West Africa was the largest recorded filovirus outbreak, as well as the first emergence of the Zaire species of Ebola in a high-density urban setting. It is generally accepted that Ebola is a zoonotic infection whose primary reservoir is probably bats [1]. Contact with wildlife generates a small number of index patients [1,2], and widespread and sustained disease transmission can follow in community settings, with a subsequent high mortality rate [3]. The size of the 2013–15 outbreak increased the need for better understanding of all transmission pathways and specific types of exposure. Hence, much of the previous advice and guidelines needs to be critically reviewed, particularly with regard to risks within communities. It is now generally accepted that both Ebola and the closely related Marburg virus diseases, are typically transmitted through direct or indirect contact with body fluids of an infected individual [4]. However uncertainties remain about which body fluids are infectious and when they pose a risk [5]. In order to address these concerns the World Health Organization released a set of “Q and As” on sanitation concerns during the 2013–15 outbreak [6], which also stated that there was insufficient evidence to give definitive guidance. In order to undertake an adequate risk assessment, knowledge on the presence of the virus in various body fluids is essential. We report a rapid systematic review of the available published evidence on the presence of filoviruses in body fluids of infected people and survivors.
  Medline, Scopus and grey literature (S1 Table) were searched through 23 July 2015 with the string filovir*. af. OR ebola. af. OR ebolavir*. af OR Marburg-virus. af, without restrictions for date or language. The aim was to find primary data that suggested high likelihood of actively infectious virus in human body fluids (viral RNA). Eligible infections were from any of the Marburg virus species and Zaire, Sudan, Taï Forest and Bundibugyo species of Ebola. Species of filovirus not known to be dangerous to humans were excluded. Cause of infection had to be laboratory confirmed (culture or polymerase chain reaction/RT-PCR tests), or evidenced by compatible clinical history with subsequent positivity for filovirus antibodies or inflammatory factors. Post-disease markers in body fluids were not deemed eligible by themselves to confirm cause of disease, because antibodies are widespread in the regional population, including in many people with no relevant clinical history [7–11]. Commentaries, editorials, news reports, protocols or conference presentations were excluded. We took it as given that any patient ill with filovirus should have detectable virus in blood. Therefore, studies which only report a single test result on blood for each patient or did not indicate the patient (s) ’ day of illness were ineligible: they did not add to our objectives of describing viral load change over time in blood, or the likelihood of virus in non-blood products. Similarly, articles which reported primary data that were duplicated elsewhere were not included. Titles and abstracts were screened for inclusion criteria by a single reviewer and verified by a second reviewer. Where abstracts were unavailable, the paper was only assessed in full text if the title included at least one of these keywords: survive, survival, fluids, viral load, urine, semen, saliva, plasma or blood. Inclusion of full text papers was assessed in duplicate and decision differences at all stages were resolved by discussion. Articles were grouped where they reported on the same primary data, to ensure patient results were not duplicated. We searched selected papers for further articles relevant to our research questions. Data were extracted from included studies by a single researcher and verified by a second researcher, for patients with confirmed filovirus infection. Details extracted were: bibliographic details, virus species, date and place of outbreak, body fluids tested, number of people tested and number of samples, days following disease onset and assay methods. A PRISMA checklist [12] is provided (S2 Table). Study validation (S3 Table) was primarily concerned with reliable confirmation of disease cause and presence of eligible filovirus RNA. Validity questions were intended to verify that assay methods were appropriate and validated, that specimens were duplicate tested or compared to controls, and that samples were handled and stored correctly for a relatively short period before testing (< 2 weeks, to reduce the risks of specimen degradation). Data on virus concentration in blood samples is presented in graphic form. Presence or absence of virus in non-blood body fluids was presented as proportion positive with 95% confidence intervals. In order to adjust for multiple samples from the same patient, each specimen was weighted by the inverse of the number of samples from that patient: To prevent a large number of test results from a small number of patients skewing the predicted probability of positivity, each test result was weighted by the inverse of the number of samples from that patient. Thus, if ten test results were from one patient, each carried a weight of 0. 1, but if only one test result existed for another patient, the latter test had a weight of 1 in constructing the probability distribution. Proportions and associated confidence intervals (on a Poisson distribution) were calculated using Stats Direct 3 and the results presented using fir tree diagrams, for samples taken in the first 16 weeks after onset of illness only.
  Twenty-nine studies reported on filovirus detection in blood either in a time series for individual patients or for a group of five or more patients. 26 articles were concerned with Zaire Ebola, two with Marburg virus [42] and one with SUDV Ebola [45]. Many reports [n = 14; 17,21–23,27,30,31,33,35–37,40,42,44] merely reported presence or absence of viral RNA. Six papers gave viral load/concentration in blood as CT values [19,24,28,32,34,39] and two articles [7,18] stated tissue-culture infectious dose (TCID50). A 1978 paper reported infectious units [20] and five studies gave results in copies per ml [15,25,26,38,45]. Plaque-forming units (PFU) were also reported [14]. There was also a mix of detection methods (RT-PCR, culture and NAAT), and some papers gave results for duplicated samples by two different assay methods with different units (eg. , CT values by RT-PCR and presence/absence by culture [19]). Sometimes for reporting purposes, authors converted their observed metrics (such as CT values) to another reporting unit (eg. , TCID50, [18]). Standardising these results to a single measurement unit was problematic. Multiplying TCID50 by 0. 7 for adults converts to PFU [46] and conversion curves from CT values to TCID50 have been produced for Ebola virus [18], but translating a mere presence/absence test to other units is less straightforward. Viral loads were often presented in imprecise graphic forms [bar charts or scatterplots, 14,25,28,32,38,45]. Confidence intervals or ranges for viral load estimates were provided (graphically) in only one paper [14]. As a result, most viral loads were somewhat inexact, lacked confidence intervals and are not equivalent between patient groups. The blood measurements should still indicate when viral loads are probably highest and when they are likely to approach undetection. Fig 2 shows viral load information in blood or blood products from culture methods (left side panels) or RT-PCR (right side), in the units reported by the stated authors, plotted against corresponding day of illness. The limit of detection (LoD) is the bottom value on each chart, either stated or implicit in the reporting methods. It merits mention that the true LoD for CT values (Panel d) may be uncertain. Most authors follow a rule of ≥ 40 cycles but it has been argued that for asymptomatic EBOV convalescents, the true LoD may be ≥ 36 cycles. [19] Presence/absence data in other articles [17,21,23,27,30,31,33,35,37] are not shown in Fig 2 but can be used to observe that overall, only 7 of the 145 individually identified patients in our extracted data had detectable virus in blood after day 16 of illness (using LoD ≤ 40). The latest positivity was day 29 of illness, using the criterion that CT value ≤ 40. With a revised CT value detection threshold of 36, the latest date for virus detected in blood among patients in our extracted data would be day 20 [18]. Twenty two papers reported on the presence of filovirus in non-blood body fluids, three of which concerned Marburg virus [41–43], one SUDV [13] and the remainder Zaire Ebola. The proportions of positive samples are illustrated in Figs 3–6, grouped by testing method or date (before day 113 after onset of illness). The numbers in parenthesis after each fluid type indicate the number of patients who provided samples for each body fluid. The numbers on the right side are the mean probability for positivity (also shown as a cross mark) with 95% confidence interval in parentheses (shown as lines both sides of the cross). We distinguish between results from culture (Fig 3) or by RT-PCR (Fig 4). RT-PCR results are further divided into all early samples (Fig 5, <17 days after onset of illness) or all late samples (Fig 6, >16 days after onset of illness). Days 16/17 are an imperfectly identified typical transition point from active illness to convalescence, because our included papers often did not identify the last date of viraemia or active illness. We observe that blood samples are rarely positive on days 17–112 (Fig 2; virus was detected in blood of just 7 of 145 individuals who can be individually identified in our extracted data). Most deaths from Ebola virus disease occur by day 16 of illness [47,48]; the latest individually identified death in our dataset was on day 16 of illness [23]. Overall, body fluid samples tested by RT-PCR (Fig 4) are about four times more likely to test positive than those tested by culture (Fig 3). This approximate discrepancy also exists among the six studies that provided duplicated results by both culture and RT-PCR [13,16,17,25,30,40]. Results for saliva and other oral fluids are particularly in disagreement (less than 5% positive by culture and almost 50% positive by RT-PCR). Virus was detected in most breastmilk samples, but the dataset is too small (three samples from two patients) to be conclusive. Data on seminal fluid are the most consistent between assay methods, with a mean weighted probability of 21% positivity for culture before day 113, and 28% for RT-PCR. Virus was detected in 73% of seminal fluid samples from 26 samples provided by 18 survivors before day 113. The latest positive result (in seminal fluid) was 203 days after onset of illness, but no results were available for days 111 to 202 or days 204 to 696 after onset of disease. Filovirus was not detected in seminal fluid of six survivors at 697–707 days post-illness [16,17]. Probability of detection by RT-PCR in different body fluids is shown before day 17 (Fig 5) and after day 16 (Fig 6). The small number of patients providing samples leads to large confidence intervals for most body fluids. Saliva is unusual in being provided from over 50 patients in both the early and late monitoring periods which leads to smaller confidence intervals. The probability of virus detection in saliva was fairly similar in both periods, falling only modestly from 55% to 45% before and after 16 days of illness. Specific viral load data were usually not provided for non-blood samples. From the limited available viral load data, it seems that viral loads can be high or may be almost negligible in non-blood body fluids (high viral load is much more likely to be detected in blood). For instance, very low viral loads were reported in urine (CT values = 36–40+) even when patients had high viraemia [28,29]. For stools or rectal swabs from symptomatic patients, virus was not detected in Kreuels et al [25] but viral load in stools was relatively high in two other patients (maximum log 105–105. 5 copies/ml: [26,38]). High viral loads for saliva were found in some studies (on days 1–8, lowest CT value = 21, maximum log 105. 5–106. 3 copies/ml: [15,34,38]), but virus was undetected in saliva of other patients on days 9–16 of illness [17,20,31,38].
  It is very hard for any systematic review to be fully up-to-date so soon after a large outbreak. Our search only covers research published on and before 23 July 2015. It is not desirable, however, to wait until all data from the 2014–2015 epidemic are available because the scientific information may be needed soon to help manage the next outbreak. There were biases in how body fluid data were collected. Surveys exist among convalescents regardless of severity of original disease or possible sequelae [16,17], but many data collected after day 16 were on patients who were not representative of most patients in a filovirus outbreak. Many samples after day 16 came from a small number of patients treated in high resource (western) hospitals; these samples were more likely to have virus detected than samples from patients in Africa. It may be reasonable to assume samples collected in in an outbreak area were less reliably stored or processed, and therefore more likely to incorrectly find no virus detected. However, data were also more likely to be collected from patients still exhibiting symptoms (treated at any location), especially viraemia, whereas all testing stopped on patients who recovered quickly. Thus, test results tend to only be available for those who recently tested positive. This bias in patient sample recruitment falsely elevates the apparent probability of late positivity seen in our results. Further difficulties with the reliability of the detection tests include the ever present possibility of false positives by any method, the contradictory results reported from different test assay methods on identical samples (especially culture vs. RT-PCR), or indeed the uncertainty about the appropriate threshold for correct CT values to use when declaring Limit of Detection [19]. Filovirus has been found in most types of body fluid, but not every sample from every symptomatic patient. Furthermore, with the exception of semen, most non-blood, RT-PCR positive samples are likely to be culture negative and so of low infectious risk. Nevertheless, it is not apparent how relatively infectious many body fluids are, even when culture positive, not least because viral loads are uncertain. Contact with blood and blood-stained body fluids remains the major risk for disease transmission.
summary: The 2013–15 Ebola outbreak has been much larger and lasted longer than any previous Ebola emergence, and is further unusual because of the thousands of survivors left behind. In 2014 the World Health Organization stated that lack of evidence made it impossible to give confident guidance about which body fluids of Ebola patients or survivors are infectious. This review summarises published evidence on presence of Ebola and the closely related Marburg virus (filoviruses) in body fluids of infected people and survivors though end July 2015. Scientific and other reports were searched for tests to detect filovirus in human body fluids on otherwise confirmed cases. 33 reports had relevant test results. Blood products were confirmed as likely to be highly infectious among actively ill cases. In actively ill patients, filovirus was often found in non-blood body fluids. However, (apart from in semen), it is rare for viable virus to be found in survivors. It is not apparent how relatively infectious most body fluids are, due to inconsistencies in testing techniques and lack of data about how much virus is present at different points of illness and recovery.
keywords: reverse transcriptase-polymerase chain reaction
  medicine and health sciences
  body fluids
  pathology and laboratory medicine
  viral transmission and infection
  pathogens
  tropical diseases
  microbiology
  ebola hemorrhagic fever
  organisms
  viruses
  urine
  filoviruses
  rna viruses
  neglected tropical diseases
  molecular biology techniques
  viral load
  research and analysis methods
  viral hemorrhagic fevers
  infectious diseases
  semen
  artificial gene amplification and extension
  medical microbiology
  microbial pathogens
  molecular biology
  hematology
  blood
  anatomy
  ebola virus
  virology
  physiology
  viral pathogens
  biology and life sciences
  viral diseases
  hemorrhagic fever viruses
  polymerase chain reaction
title: Presence and Persistence of Ebola or Marburg Virus in Patients and Survivors: A Rapid Systematic Review