text: Scientists have learned that upwards of 25 percent of all people who become infected with Ebola show none of the typical symptoms. The finding suggests the recent West African Ebola Epidemic was more widespread than previously thought, and that new methods need to be developed to diagnose and contain the dreaded virus during an outbreak. 
   
   
   
   Typically, Ebola produces a panoply of symptoms, including fever, unexplained bleeding, headache, muscle pain, rash, vomiting, diarrhea, breathing problems, and difficulty swallowing. As is well documented, this virus is particularly deadly, killing between 10 to 80 percent of people who exhibit these symptoms (the statistical spread reflects the divide between those who have access to intensive care and those who don’t). 
   
   But as a new study published in PLOS Neglected Tropical Diseases shows, a significant portion of people who contract Ebola don’t show any of the typical signs, meaning they’re asymptomatic, or their symptoms are relatively mild and not wholly reflective of how the virus affects most people. In other words, they have “minimally symptomatic infections.” 
   
   Prior to the new study, scientists suspected that Ebola, like many other viruses, might not always manifest in the same predictable way. The new study, led by Gene Richardson, a PhD candidate in anthropology at Stanford University and a former fellow in the Division of Infectious Diseases and Geographic Medicine, now confirms this suspicion, while providing an actual estimate for the rate of asymptomatic and minimally symptomatic infections in Ebola patients. 
   
   Following the recent West African Ebola Epidemic, Richardson and his colleagues went back to the rural village of Sukudu in Sierra Leone, a major hotspot for the outbreak. The village, which is home to 900 residents, recorded 34 cases of Ebola during the epidemic, including 28 deaths. Working with a local physician, Richardson’s team tested 187 men, women, and children from Sukudu who had likely been exposed to Ebola, either because they lived in the same household or shared a public toilet with a person known to have had the disease. 
   
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   Of these, 14 were found to carry an antibody to Ebola—a sure sign that they were infected at some point. A dozen of these villagers said they had no symptoms of the disease, while two recalled having a fever at the time of the outbreak. 
   
   It’s now certainly possible that some of these villagers had full-blown symptoms but were fearful of admitting the truth to doctors. Consequently, more research with a larger sample pool will have to be performed. However, Richardson and his colleagues think they’re onto something, and that the villagers were probably telling the truth. 
   
   Importantly, the researchers aren’t sure if an asymptomatic person with Ebola can transmit the virus. “They were not passing it along in the usual way, through vomiting or diarrhea,” noted Richardson in a statement. “It’s unclear if they can pass it along it sexually.” 
   
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   More research is needed to determine if Ebola can be spread by a person who shows no or very few symptoms, but it’s a very distinct possibility. A person with an infection still has Ebola particles in their body, which could find a way to “jump” to another person. But without the typical symptoms, it’s certainly much more difficult. As Richardson noted, the virus might be transmitted sexually, though that still needs to be proven in an asymptomatic individual. 
   
   Richardson and his colleagues are now working in other villages in Sierra Leone to get a better handle on the true number of people affected during the crisis. But if the figure of 25 percent is correct—and if Ebola can spread via asymptomatic people (that’s still a big if)—that presents a massive headache for health officials. 
   
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   Adding insult to injury, the Ebola virus mutated into a deadlier form during the last outbreak, showing an ability to quickly adapt to human biology and become even more virulent. Taken together, these new findings show that Ebola spreads more rapidly and more broadly through a population, highlighting the need for new detection and containment techniques for when the next outbreak hits. 
   
   
   
   [PLOS Neglected Tropical Diseases] ||||| By using ELISA to measure Zaire Ebola virus antibody concentrations, we identified a significant number of individuals with previously undetected EBOV infection in a ‘hotspot’ village in Sierra Leone, approximately one year after the village outbreak. The findings provide further evidence that Ebola, like many other viral infections, presents with a spectrum of clinical manifestations, including minimally symptomatic infection. These data also suggest that a significant portion of Ebola transmission events may have gone undetected during the outbreak. Further studies are needed to understand the potential risk of transmission and clinical sequelae in individuals with previously undetected EBOV infection. 
   
   We conducted a cross-sectional serosurvey in Sukudu, Kono District, Sierra Leone, from October 2015 to January 2016. A blood sample was collected from 187 study participants, 132 negative controls (individuals with a low likelihood of previous exposure to Ebola virus), and 30 positive controls (Ebola virus disease survivors). IgG responses to Ebola glycoprotein and nucleoprotein were measured using Alpha Diagnostic International ELISA kits with plasma diluted at 1:200. Optical density was read at 450 nm (subtracting OD at 630nm to normalize well background) on a ChroMate 4300 microplate reader. A cutoff of 4.7 U/mL for the anti-GP ELISA yielded 96.7% sensitivity and 97.7% specificity in distinguishing positive and negative controls. We identified 14 seropositive individuals not known to have had Ebola virus disease. Two of the 14 seropositive individuals reported only fever during quarantine while the remaining 12 denied any signs or symptoms during quarantine. 
   
   Evidence for minimally symptomatic Ebola virus (EBOV) infection is limited. During the 2013–16 outbreak in West Africa, it was not considered epidemiologically relevant to published models or projections of intervention effects. In order to improve our understanding of the transmission dynamics of EBOV in humans, we investigated the occurrence of minimally symptomatic EBOV infection in quarantined contacts of reported Ebola virus disease cases in a recognized ‘hotspot.’ 
   
   With over 28,000 reported cases, the 2013–16 West African Ebola virus disease epidemic is the largest and longest on record. This study provides further evidence that Ebola, like other viruses, causes a spectrum of clinical manifestations that may include minimally symptomatic infection. The findings also suggest that many episodes of human-to-human transmission of Ebola virus in West Africa may have gone undetected in the recent outbreak. This has implications for the definition of Ebola virus disease survivorship, delineation of transmission chains, and future vaccine studies. 
   
   In order to improve our understanding of the transmission dynamics of EBOV in humans, we investigated the occurrence of minimally symptomatic EBOV infection in a recognized Ebola ‘hotspot’ [ 11 ], which we defined as an area with a reverse transcription polymerase chain reaction (RT-PCR)-confirmed EVD attack rate above 2% in a two-month period. 
   
   Evidence for minimally symptomatic EBOV infection is limited. During the 2013–16 outbreak in West Africa, it was not considered epidemiologically relevant to published models or projections of intervention effects [ 7 – 10 ]. Moreover, it is not known if clinical sequelae seen in survivors of EVD (e.g., uveitis) exist in individuals who had minimally symptomatic EBOV infection. 
   
   Despite over 28,000 reported cases of Ebola virus disease (EVD) in the 2013–16 pandemic as of March 27, 2016 [ 1 ], we are only beginning to trace the complex biosocial processes that have promoted spread of the virus [ 2 , 3 ]. Important questions remain, including how to best use tools such as new vaccines [ 4 ] and rapid diagnostic tests [ 5 ] to contain future outbreaks, the extent to which symptomatic individuals do not present for care, how to identify and manage clinical sequelae of EVD [ 6 ], and the incidence and transmission dynamics of minimally symptomatic Ebola virus (EBOV) infection. 
   
   In addition to the 16 confirmed EBOV infections with a fatal outcome, the 12 probable deaths from EVD, and 6 confirmed EBOV infections who survived (34 total cases), our serosurvey identified an additional 14 individuals who were anti-GP ELISA IgG-positive. Two of these individuals reported fever during the quarantine period, and 12 were classified as minimally symptomatic, contributing 25% (binomial exact 95% CI, 14% to 40%) to the total caseload. After including the 2 undocumented symptomatic cases with the 34 reported by the DERC, we calculated a 78% symptomatic case fatality ratio (binomial exact 95% CI, 61% to 90%). 
   
   Data analyses. We considered participants to have had minimally symptomatic EBOV infection if they 1) had an anti-GP ELISA above the cut-off as determined in the validation sub-study and 2) denied symptoms consistent with EVD during the period of active transmission in the village. 
   
   Laboratory analyses. Of the 187 exposed individuals tested, none of whom were previously known to have EVD, we identified 14 (7.5%) who tested positive by anti-GP ELISA ( Fig 3 ). Two (14.3%) of these 14 reported fever during quarantine while the remaining 12 denied any signs or symptoms during quarantine. 
   
   Table 1 shows that 57.8% of participants were male, 35.8% were less than 15 years of age, 48.6% worked outside the home (mostly as farmers or miners), and 79.1% had completed primary school or less. Among exposed participants who were quarantined, 50.2% shared a public latrine with a confirmed case, and 49.8% slept in the house of a confirmed case. 
   
   Survey. We collected the following data from study participants: age, gender, occupation, education level, whether or not they shared a room with a confirmed case of EVD, and whether they had signs or symptoms consistent with EVD that were severe enough to prompt seeking of care during the time of their village outbreak. Specifically, these included fever or unexplained bleeding or any three of the following: headache, myalgia, rash, vomiting, diarrhea, hiccups, breathing problems, or difficulty swallowing [ 13 ]. Other household members verified signs and symptoms. 
   
   Of 193 individuals from Sukudu who were quarantined for sharing a public latrine or living quarters with a confirmed case, only 4 declined to participate. Ten individuals were initially excluded because of fever at the time of initial contact; none of these met the case definition for EVD. On reassessment one week later, all but one had defervesced and were able to provide blood samples. Two participants had failed venipuncture attempts for blood draw. We thus sampled a total of 187 individuals for anti-GP in 24 of 25 previously quarantined houses as defined above (the members of 1 quarantined household could not be located). 
   
   We recruited healthy children and adults age 4 and older who had been placed under quarantine during the period of active Ebola transmission in Sukudu. None of these individuals had participated in an Ebola vaccine trial. Each potential participant was screened for fever or symptoms consistent with EVD, and he/she was excluded if any were present. Medical referral to a nearby Partners In Health-supported clinic (which provides free medical care to those unable to pay) was provided. 
   
   Study population. We conducted a serosurvey in Sukudu, a village of approximately 900 inhabitants (100 households) that was one of three major ‘hotspots’ in Kono District. We obtained a list of quarantined houses and EVD cases from the Kono DERC and verified it with the town chiefs and youth leaders. We also interviewed members of all of the affected households and discussed the identification of EVD cases with them to triangulate DERC records. We defined potential participants as having been exposed to EBOV if they stayed in a house that was quarantined based on sharing a public latrine with a confirmed case or based on a confirmed case living there. All homes were geolocated using a remotely sensed image ( Fig 2 ). 
   
   Fig 1 provides ROC curves and AUCs for the individual and combined ELISA assays. AUCs for the anti-GP, anti-NP, and combined assays were 0.992 (SE = 0.005, 95% CI = 0.983–1.000), 0.975 (SE = 0.010, 95% CI = 0.956–0.995), and 0.997 (unable to determine CIs as the combined ROC was generated through logistic regression), respectively, suggesting that these three tests were statistically equivalent. The cutoff for the anti-GP ELISA that yielded optimal sensitivity (96.7%) and specificity (97.7%) was 4.7 U/mL. 
   
   To determine overall accuracies for the two ELISAs in combination and individually, we generated receiver operating characteristic (ROC) curves and compared the areas under the curve (AUCs) for the individual and combined anti-GP and anti-NP tests [ 12 ]. The cutoff for a positive test was established by ROC analysis, defined as the point along the ROC curve that gave the highest sum of sensitivity (%) and specificity (%). 
   
   We assessed anti-glycoprotein (anti-GP) and anti-nucleoprotein (anti-NP) responses to EBOV by means of commercial ELISA kits (AE 320620–1 and AE 320520–1, respectively) from Alpha Diagnostic International (ADI), San Antonio, TX, USA. These were processed according to the manufacturer’s instructions, with plasma diluted at 1:200. Optical density (OD) was read at 450 nm (subtracting OD at 630nm to normalize well background) on a ChroMate 4300 microplate reader (Awareness Technologies, Marina Del Rey, CA, USA). Values are reported in units per milliliter (U/mL) with the use of reference antibody provided by the manufacturer. 
   
   We recruited 30 EVD survivors as positive controls by contacting the director of the Kono Survivors’ Group and requesting that she identify individuals interested in participating in a serosurvey. Using Kono DERC records, we verified that survivors had been admitted to an Ebola Treatment Unit (ETU) and had a documented RT-PCR positive result for EBOV. To identify a group of people whom we considered at very low risk of previous EBOV infection to serve as negative controls, we used Kono DERC records to locate three villages in Kono District in which no Ebola cases had been reported: In the villages of Tombodu, Wardu, and Njagbwema, we recruited 132 individuals who denied contact with Ebola cases both inside and outside of their communities. 
   
   Kono is a diamond-rich district in eastern Sierra Leone which reported 301 cases of EVD (that is, RT-PCR confirmed individuals who presented for isolation or were identified by surveillance teams) between August 2014 and February 2015 (Kono District Ebola Response Centre (DERC), personal communication). Several of the authors worked closely with the Kono DERC, British military, and International Federation of the Red Cross on containment, isolation, and surveillance during the time of active transmission in the district. Parts I and II of this study were conducted from October 2015 to January 2016 by a team consisting of two physicians, a physician-anthropologist, a laboratory technician, and two community health workers. 
   
   The study protocol was approved by the Sierra Leone Ethics and Scientific Review Committee and the Stanford University Institutional Review Board (Protocol ID: 33882). We held meetings with Paramount, Sectional, and Town Chiefs to discuss the proposal and supply them with written information. We then held town meetings to describe the project and answer community questions. Individuals provided written informed consent or placed a thumbprint after hearing a consent script read in the Krio or Kono languages (both ethics committees approved the use of an oral script and thumbprint for those participants who could not read or write); parents signed for children under the age of 18, and children and adolescents provided verbal assent. Subjects received 50,000 Leones (~$10 US) for their participation in the study. 
   
   Discussion 
   
   One year after the peak of the Ebola epidemic in Sierra Leone, we identified positive IgG responses to Zaire-EBOV in 14 individuals not known to have had EBOV infection from a village classified as a hotspot for Ebola transmission. Of these, the majority reported having had no symptoms consistent with EVD during the epidemic while two reported only fever. Thus, we provide further evidence that Ebola, like many other viral infections, presents with a spectrum of clinical manifestations, including minimally symptomatic infection. In addition, our data suggest that a significant portion of Ebola transmission events may have gone undetected during the epidemic. 
   
   Although it was difficult to verify symptoms through our retrospective interviews (given the considerable denial of EVD during the outbreak due to stigma and the fear of being admitted to an ETU where those admitted were seldom discharged), our data indicate that 25% of EBOV infections may have been minimally symptomatic, which is similar to the data from empiric studies and to recent modeled estimates [14]. 
   
   The phenomenon of previously undetected, minimally symptomatic EBOV infection was evident around the discovery of the virus in 1976. Using an immunofluorescence assay, the World Health Organization/International Study Team found that 19% of contacts of EVD cases—very few of whom gave any history of illness—had antibodies to the virus [15]. In 2000, Leroy and colleagues published a study (based on ELISA/Western blot) and found that of 24 asymptomatic close contacts of Gabonese patients with EVD, 11 developed both IgM and IgG responses to Ebola Zaire antigens, indicating viral infection [16]. Other investigators have found evidence of seropositive individuals in areas without large outbreaks using ELISA and postulate that there may have been active circulation of filovirus without apparent clinical manifestations [17,18]. Heffernan and colleagues also used ELISA in Gabon and found that 1% of individuals in an epidemic zone had IgG antibodies to Ebola Zaire virus, yet no history of exposure [19]. In another study in Gabon, Becquart and colleagues found a 15.3% Ebola Zaire IgG seroprevalence in 220 randomly selected villages and concluded that most of the seropositive persons identified “probably had mild or asymptomatic infection” [20]; however, they used uninfected individuals in France as negative controls. We found that unexposed expatriates (not included as negative controls) had a significantly lower mean log anti-GP (M = 5.25 U/mL, SD = 0.54, N = 12) than unexposed Sierra Leoneans (M = 6.40 U/mL, SD = 1.06, N = 132) using the two-sample t-test for unequal variances, t(20.05) = 6.40, P< = 0.001 (see Fig 3), potentially due to cross-reactivity of our assay with closely related pathogens circulating in the region. 
   
   Our study has several possible limitations. First, if asymptomatic EBOV infection is a common occurrence, it is possible that some of the “true negatives” used for the validation of our ELISA assays could have been infected with EBOV. We did not use a microneutralization assay for comparison purposes, nor did our pool of EVD survivors represent all of the possible antibody titers in the regional population. Although the serologic assay conferred sensitivity and specificity greater than 95%, the assay protocol produced antibody titers that were considered qualitative in nature. Second, we relied on study participants and their household contacts’ memories of events that had taken place up to a year prior when we classified them as symptomatic or asymptomatic (potential recall bias); however, during the outbreak, most quarantined households were monitored daily by surveillance teams that conducted symptom screens and measured temperatures, and individuals were brought to treatment facilities for EBOV testing if they screened positive. Third, our IgG assays indicate previous infection but provide no information on when that infection took place. The 3 negative controls with positive tests either represent false positives due to cross-reactive antibodies or previously infected individuals. (We did not perform IgM ELISAs, as other investigators have demonstrated that Ebola IgM titers largely diminish within 60 to 90 days of symptom onset [21,22].) Fourth, we did not ascertain whether there were non-quarantined individuals who had minimally symptomatic infection. 
   
   Our study focused on the quarantined population of one village. Extrapolation of our findings to other villages and generalizability to the epidemic should be approached with caution. Although many of the study participants were followed by surveillance teams during the time of active EBOV transmission in their village, the timing of the possible infection cannot be known from IgG data, and it is improbable that surveillance teams followed each individual throughout the duration of the outbreak in Sierra Leone. Thus, the concern that IgG positive persons indeed had but denied symptoms cannot be excluded. 
   
   Despite these limitations, our serosurvey provides a deeper perspective on EBOV transmission, and more village-level serosurveys could enhance our understanding of undetected EBOV transmission at the epidemic level. Furthermore, our findings suggest there would be value in exploring the interaction of seropositive persons and EVD cases to improve our understanding of exposure risk. As a result, we may learn more about how efforts at containment can be improved. The data also have important implications for future vaccine studies that rely on detecting antibody to EBOV. Lastly, the findings support the World Health Organization’s interim guidance on clinical care for survivors of EVD, which defines a survivor as a person: 
   
   With a confirmed positive result on RT-PCR testing for EBOV on any body fluid who subsequently recovered; 
   
   And/or 
   
   And/or Who is IgM and/or IgG positive on serological testing for EVD and has not been vaccinated against EBOV [23]. 
   
   There is ongoing discussion in West Africa over the definition of survivorship, usually specified by having a positive EBOV RT-PCR result and discharge from an ETU. Plans are underway to develop national registries and provide ID cards to such survivors, so as to delineate individuals eligible for free social and medical support services. Should the notion of survivorship be extended to all those who are IgG positive, including those who had minimally symptomatic infection or who were sick but were never tested at the time of illness? How we define a community of suffering is always problematic and should be revisited given that this definition has implications for identity, stigma, and access to social and medical services. 
   
   In conclusion, by using ELISA to measure Zaire-EBOV antibody concentrations, we identified a significant number of individuals with previously undetected minimally symptomatic EBOV infection in a ‘hotspot’ village in Sierra Leone, approximately one year after the village outbreak. Further studies are needed to understand the potential risk of transmission and clinical sequelae in individuals with minimally symptomatic EBOV infection. ||||| Between 2013 and 2016, tens of thousands of people in West Africa contracted Ebola virus disease, making the epidemic the largest and longest on record. But the number of reported cases -- roughly 28,000 -- may be an underestimate of the total number of people infected, since it includes only people who showed symptoms of disease. Researchers reporting in PLOS Neglected Tropical Diseases have discovered individuals in Sierra Leone who showed no signs of the disease but have evidence of prior Ebola infection in their immune systems. 
   
   An estimated 10-80 percent of people who show symptoms of Ebola virus disease (EVD) die, depending on their access to intensive care. However, researchers have also seen some evidence that a minority of people may catch the Ebola virus without developing symptoms. During the 2013-2016 outbreak, however, these so-called "minimally symptomatic infections" were not considered epidemiologically relevant to models, projections, or intervention efforts so were not studied or tracked in detail. 
   
   From October 2015 through January 2016, a year after the peak of the Ebola epidemic, Eugene Richardson, of Partners In Health, and colleagues conducted a survey of people living in Sukudu, Kono District, Sierra Leone, a village of 900 inhabitants which had been a major "hotspot" of Ebola. The team identified 193 adults and children over age 4 who had lived with or shared a latrine with a confirmed case of Ebola during the period of active transmission but were never identified as EVD cases. The researchers collected blood from 187 of these individuals. They then tested the blood samples -- as well as positive and negative controls from known Ebola cases and people not exposed to the virus -- for Ebola glycoprotein antibodies, the presence of which indicates a past Ebola infection. 
   
   Of the 187 exposed individuals, none of whom were previously known to have Ebola virus disease, the researchers identified 14 who tested positive for Ebola antibodies. Two of these 14 admitted having a fever while being quarantined, while the other 12 denied any signs or symptoms. However, the study could not pinpoint the timing of any past infection, nor guarantee that any individuals were not prevaricating about a lack of symptoms. The results also can't be extrapolated to other villages affected by the outbreak without more data. 
   
   "The findings provide further evidence that Ebola, like many other viral infections, presents with a spectrum of clinical manifestations, including minimally symptomatic infection," the researchers write. "These data also suggest that a significant portion of Ebola transmission events may have gone undetected during the outbreak." 
   
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   Please contact plosntds@plos.org if you would like more information about our content and specific topics of interest. 
   
   All works published in PLOS Neglected Tropical Diseases are open access, which means that everything is immediately and freely available. Use this URL in your coverage to provide readers access to the paper upon publication: http://dx. plos. org/ 10. 1371/ journal. pntd. 0005087 (Link goes live upon article publication) 
   
   Funding: This study was funded by Stanford University Center for Innovation in Global Health, UCSF Emergency Ebola Response, Abundance Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 
   
   Competing Interests: The authors have declared that no competing interests exist.
target: – Ebola doesn't always show itself through fever, vomiting, and diarrhea. A new PLOS study finds that some people with the virus show mild or no symptoms at all—a potential concern for preventing its spread. Researchers who visited the village of Sukudu in Sierra Leone, a hot spot in the recent West African Ebola outbreak, found up to 25% of Ebola infections "may have been minimally symptomatic," meaning "a significant portion of Ebola transmission events may have gone undetected" in Guinea, Sierra Leone, and Liberia, NBC News reports. At least 28,000 people are known to have been infected during the epidemic, at least 11,000 of whom died. In Sukudu, there were 34 known Ebola cases, including 28 deaths among 900 residents, reports Gizmodo. A year after the worst of the outbreak was over, Stanford University researchers tested the blood of 187 people in Sukudu who'd lived with or shared a toilet with someone infected with Ebola, per a release. They found 14 had Ebola antibodies, suggesting they'd previously been infected, while 12 said they didn't recall feeling sick or having a fever. Believing them to be truthful as health workers kept an eye on villagers during the outbreak, researchers now plan to visit other villages "so we can begin to answer the question of what was the true burden of disease," the study author says. "We expect to find a lot more undocumented survivors." It's not clear if asymptomatic patients are contagious. "They were not passing it along in the usual way, through vomiting or diarrhea," he says. "It's unclear if they can pass it along sexually." (This Ebola-like disease can shut down organs.)