A nanopore DNA sequencer is used for real-time genomic surveillance of the Ebola virus epidemic in the field in Guinea; the authors demonstrate that it is possible to pack a genomic surveillance laboratory in a suitcase and transport it to the field for on-site virus sequencing, generating results within 24 hours of sample collection. This paper reports the use of nanopore DNA sequencers (known as MinIONs) for real-time genomic surveillance of the Ebola virus epidemic, in the field in Guinea. The authors demonstrate that it is possible to pack a genomic surveillance laboratory in a suitcase and transport it to the field for on-site virus sequencing, generating results within 24 hours of sample collection. The Ebola virus disease epidemic in West Africa is the largest on record, responsible for over 28,599 cases and more than 11,299 deaths1. Genome sequencing in viral outbreaks is desirable to characterize the infectious agent and determine its evolutionary rate. Genome sequencing also allows the identification of signatures of host adaptation, identification and monitoring of diagnostic targets, and characterization of responses to vaccines and treatments. The Ebola virus (EBOV) genome substitution rate in the Makona strain has been estimated at between 0.87 × 10−3 and 1.42 × 10−3 mutations per site per year. This is equivalent to 16–27 mutations in each genome, meaning that sequences diverge rapidly enough to identify distinct sub-lineages during a prolonged epidemic2,3,4,5,6,7. Genome sequencing provides a high-resolution view of pathogen evolution and is increasingly sought after for outbreak surveillance. Sequence data may be used to guide control measures, but only if the results are generated quickly enough to inform interventions8. Genomic surveillance during the epidemic has been sporadic owing to a lack of local sequencing capacity coupled with practical difficulties transporting samples to remote sequencing facilities9. To address this problem, here we devise a genomic surveillance system that utilizes a novel nanopore DNA sequencing instrument. In April 2015 this system was transported in standard airline luggage to Guinea and used for real-time genomic surveillance of the ongoing epidemic. We present sequence data and analysis of 142 EBOV samples collected during the period March to October 2015. We were able to generate results less than 24 h after receiving an Ebola-positive sample, with the sequencing process taking as little as 15–60 min. We show that real-time genomic surveillance is possible in resource-limited settings and can be established rapidly to monitor outbreaks.

The level of evidence for HIV transmission risk through condomless sex in serodifferent gay couples with the HIV-positive partner taking virally suppressive antiretroviral therapy (ART) is limited compared with the evidence available for transmission risk in heterosexual couples. The aim of the second phase of the PARTNER study (PARTNER2) was to provide precise estimates of transmission risk in gay serodifferent partnerships. The PARTNER study was a prospective observational study done at 75 sites in 14 European countries. The first phase of the study (PARTNER1; Sept 15, 2010, to May 31, 2014) recruited and followed up both heterosexual and gay serodifferent couples (HIV-positive partner taking suppressive ART) who reported condomless sex, whereas the PARTNER2 extension (to April 30, 2018) recruited and followed up gay couples only. At study visits, data collection included sexual behaviour questionnaires, HIV testing (HIV-negative partner), and HIV-1 viral load testing (HIV-positive partner). If a seroconversion occurred in the HIV-negative partner, anonymised phylogenetic analysis was done to compare HIV-1 pol and env sequences in both partners to identify linked transmissions. Couple-years of follow-up were eligible for inclusion if condomless sex was reported, use of pre-exposure prophylaxis or post-exposure prophylaxis was not reported by the HIV-negative partner, and the HIV-positive partner was virally suppressed (plasma HIV-1 RNA <200 copies per mL) at the most recent visit (within the past year). Incidence rate of HIV transmission was calculated as the number of phylogenetically linked HIV infections that occurred during eligible couple-years of follow-up divided by eligible couple-years of follow-up. Two-sided 95% CIs for the incidence rate of transmission were calculated using exact Poisson methods. Between Sept 15, 2010, and July 31, 2017, 972 gay couples were enrolled, of which 782 provided 1593 eligible couple-years of follow-up with a median follow-up of 2·0 years (IQR 1·1-3·5). At baseline, median age for HIV-positive partners was 40 years (IQR 33-46) and couples reported condomless sex for a median of 1·0 years (IQR 0·4-2·9). During eligible couple-years of follow-up, couples reported condomless anal sex a total of 76 088 times. 288 (37%) of 777 HIV-negative men reported condomless sex with other partners. 15 new HIV infections occurred during eligible couple-years of follow-up, but none were phylogenetically linked within-couple transmissions, resulting in an HIV transmission rate of zero (upper 95% CI 0·23 per 100 couple-years of follow-up). Our results provide a similar level of evidence on viral suppression and HIV transmission risk for gay men to that previously generated for heterosexual couples and suggest that the risk of HIV transmission in gay couples through condomless sex when HIV viral load is suppressed is effectively zero. Our findings support the message of the U=U (undetectable equals untransmittable) campaign, and the benefits of early testing and treatment for HIV. National Institute for Health Research.

Ebola virus disease (EVD) is a highly lethal condition for which no specific treatment has proven efficacy. In September 2014, while the Ebola outbreak was at its peak, the World Health Organization released a short list of drugs suitable for EVD research. Favipiravir, an antiviral developed for the treatment of severe influenza, was one of these. In late 2014, the conditions for starting a randomized Ebola trial were not fulfilled for two reasons. One was the perception that, given the high number of patients presenting simultaneously and the very high mortality rate of the disease, it was ethically unacceptable to allocate patients from within the same family or village to receive or not receive an experimental drug, using a randomization process impossible to understand by very sick patients. The other was that, in the context of rumors and distrust of Ebola treatment centers, using a randomized design at the outset might lead even more patients to refuse to seek care. Therefore, we chose to conduct a multicenter non-randomized trial, in which all patients would receive favipiravir along with standardized care. The objectives of the trial were to test the feasibility and acceptability of an emergency trial in the context of a large Ebola outbreak, and to collect data on the safety and effectiveness of favipiravir in reducing mortality and viral load in patients with EVD. The trial was not aimed at directly informing future guidelines on Ebola treatment but at quickly gathering standardized preliminary data to optimize the design of future studies. Inclusion criteria were positive Ebola virus reverse transcription PCR (RT-PCR) test, age ≥ 1 y, weight ≥ 10 kg, ability to take oral drugs, and informed consent. All participants received oral favipiravir (day 0: 6,000 mg; day 1 to day 9: 2,400 mg/d). Semi-quantitative Ebola virus RT-PCR (results expressed in “cycle threshold” [Ct]) and biochemistry tests were performed at day 0, day 2, day 4, end of symptoms, day 14, and day 30. Frozen samples were shipped to a reference biosafety level 4 laboratory for RNA viral load measurement using a quantitative reference technique (genome copies/milliliter). Outcomes were mortality, viral load evolution, and adverse events. The analysis was stratified by age and Ct value. A “target value” of mortality was defined a priori for each stratum, to guide the interpretation of interim and final analysis. Between 17 December 2014 and 8 April 2015, 126 patients were included, of whom 111 were analyzed (adults and adolescents, ≥13 y, n = 99; young children, ≤6 y, n = 12). Here we present the results obtained in the 99 adults and adolescents. Of these, 55 had a baseline Ct value ≥ 20 (Group A Ct ≥ 20), and 44 had a baseline Ct value < 20 (Group A Ct < 20). Ct values and RNA viral loads were well correlated, with Ct = 20 corresponding to RNA viral load = 7.7 log₁₀ genome copies/ml. Mortality was 20% (95% CI 11.6%–32.4%) in Group A Ct ≥ 20 and 91% (95% CI 78.8%–91.1%) in Group A Ct < 20. Both mortality 95% CIs included the predefined target value (30% and 85%, respectively). Baseline serum creatinine was ≥110 μmol/l in 48% of patients in Group A Ct ≥ 20 (≥300 μmol/l in 14%) and in 90% of patients in Group A Ct < 20 (≥300 μmol/l in 44%). In Group A Ct ≥ 20, 17% of patients with baseline creatinine ≥110 μmol/l died, versus 97% in Group A Ct < 20. In patients who survived, the mean decrease in viral load was 0.33 log₁₀ copies/ml per day of follow-up. RNA viral load values and mortality were not significantly different between adults starting favipiravir within <72 h of symptoms compared to others. Favipiravir was well tolerated. In the context of an outbreak at its peak, with crowded care centers, randomizing patients to receive either standard care or standard care plus an experimental drug was not felt to be appropriate. We did a non-randomized trial. This trial reaches nuanced conclusions. On the one hand, we do not conclude on the efficacy of the drug, and our conclusions on tolerance, although encouraging, are not as firm as they could have been if we had used randomization. On the other hand, we learned about how to quickly set up and run an Ebola trial, in close relationship with the community and non-governmental organizations; we integrated research into care so that it improved care; and we generated knowledge on EVD that is useful to further research. Our data illustrate the frequency of renal dysfunction and the powerful prognostic value of low Ct values. They suggest that drug trials in EVD should systematically stratify analyses by baseline Ct value, as a surrogate of viral load. They also suggest that favipiravir monotherapy merits further study in patients with medium to high viremia, but not in those with very high viremia. ClinicalTrials.gov NCT02329054 In 2014 and 2015, an Ebola virus outbreak larger than any known before occurred in West Africa. Ebola virus disease (EVD) is highly contagious, and many infected people die. Central to the emergency response to the recent outbreak were local Ebola treatment centers where patients were diagnosed, were isolated, and received supportive care. With thousands of patients dying and many health workers contracting the disease, fear was ubiquitous and distrust abundant. While conducting research in this environment was extremely challenging, the urgent need for treatments and the opportunity to conduct studies that could bring such treatments closer to reality was also recognized. In September 2014, WHO released a short list of existing drugs that were candidates for clinical trials among patients infected in the outbreak. Favipiravir, an antiviral drug developed in Japan for patients with severe influenza, was on the list. Because of the urgent need to find drugs that could reduce deaths caused by Ebola, the researchers decided to conduct a clinical trial using favipiravir in patients with EVD in Guinea. In view of the circumstances, they decided against a randomized controlled trial and instead designed a study where all participants would receive the same treatment. In randomized controlled trials only some participants receive the treatment in addition to standard care, while others serve as a control group and receive standard care only, or standard care plus a placebo. Such studies allow stronger conclusions to be drawn about whether a treatment is safe and whether it works or not. The researchers had two main reasons for this decision. First, patients from the same family or village often sought EVD treatment at the same time, and the researchers felt that it was ethically unacceptable to randomize such groups, with only some of them receiving the experimental drug. Second, the strict isolation procedures imposed to interrupt virus transmission had intensified fear in affected communities and fueled rumors of illicit drug experimentation and organ theft at the treatment centers. In this context, the researchers worried that a randomized study might increase distrust among the community and the reluctance of patients to seek care. Rather than seeking definitive answers about the safety and efficacy of favipiravir in patients with EVD, the objectives of the study as it was designed were to test the feasibility and acceptability of an emergency trial in the context of a large Ebola outbreak and to learn lessons from the experience. In addition, the researchers planned to collect data on the safety and effectiveness of favipiravir in reducing mortality and viral load in patients with EVD in the hope that their preliminary findings could improve the design of subsequent trials and the chance to provide conclusive answers. After 13 weeks of preparation, the trial took place from December 2014 to April 2015 at four separate Ebola treatment centers, three in rural areas and one in an urban setting. In addition to standard care (which included rehydration, antimalarial and antibacterial therapies, and medication to reduce fever, pain, and nausea), all participants were given favipiravir by mouth for ten days, at doses substantially higher than those recommended for patients with influenza. Outcomes measured were mortality, viral load changes over time (based on blood samples), and adverse events. EVD was confirmed with an assay that used patient blood and provided an estimate of the viral load, that is, of how much virus the blood contained. Because viral load was known to influence the course of EVD, the researchers analyzed the participants in two groups, namely, those with a viral load estimate above a certain threshold and those with viral load estimate below the threshold. They also used existing data from Guinean patients diagnosed with Ebola earlier in the outbreak who had received only standard care and calculated an expected mortality rate for patients above and below the viral load threshold. The researchers were able to enroll 126 participants in the trial. Of these, 111 were included in the final analysis. Of 99 adult and adolescent participants 13 years and older, 55 were in the lower viral load group and 44 in the higher viral load group. Mortality was 20% in the former and 91% in the latter. Neither mortality rate was significantly different from that of earlier patients who had received only standard care. The researchers also found that favipiravir was well tolerated. None of the patients stopped the course of treatment, vomiting following drug intake was rare, and no severe adverse events were attributed to the drug. The researchers did not see a difference in mortality between patients who reported onset of symptoms less than three days before the start of treatment and those whose symptoms had started more than three days the start of treatment. The report shows that it is possible to conduct an emergency trial during an outbreak in a low-resource setting. In fact, at the time of its acceptance, this paper reported on an Ebola treatment trial larger than any other yet published. The experience described should be useful for similar undertakings in the future. The following conditions contributed to the success of the trial: close collaboration between researchers, local health officials, and affected communities on one hand, and flexibility in design, conduct, and analysis based on close monitoring and interim assessments on the other. Besides using interim results to influence the conduct and analysis of their own trial, the researchers also shared these results with the scientific community in real time, and this feedback influenced other research during the outbreak. The trial could not answer definitively whether favipiravir treatment was safe or reduced mortality in patients with EVD. The results suggest that the drug is unlikely to be beneficial for patients with very high viral loads, at least when given by itself. They also suggest that favipiravir is safe in patients with lower viral loads, and that in such patients additional efficacy studies are warranted. Intermediate analysis of various measurements in trial participants showed that the estimate of viral load from the field EVD diagnosis test is a good proxy for the actual viral load (determined after the samples were shipped to and analyzed in a reference laboratory in France) and suitable as a surrogate marker. The results also confirm that viral load is a strong predictor of mortality. This list of resources contains links that can be accessed when viewing the PDF on a device or via the online version of the article at http://dx.doi.org/10.1371/journal.pmed.1001967.

Analysis of 179 new Ebola virus sequences from patient samples collected in Guinea between March 2014 and January 2015 shows how different lineages evolved and spread in West Africa. Miles Carroll and colleagues report describe the genetic evolution of Ebola virus circulating in West Africa, based on 179 new virus sequences from patient samples collected in Guinea between March 2014 and January 2015. Their analysis shows how different lineages evolved and spread in West Africa between Sierra Leone, Guinea and Liberia. West Africa is currently witnessing the most extensive Ebola virus (EBOV) outbreak so far recorded1,2,3. Until now, there have been 27,013 reported cases and 11,134 deaths. The origin of the virus is thought to have been a zoonotic transmission from a bat to a two-year-old boy in December 2013 (ref. 2). From this index case the virus was spread by human-to-human contact throughout Guinea, Sierra Leone and Liberia. However, the origin of the particular virus in each country and time of transmission is not known and currently relies on epidemiological analysis, which may be unreliable owing to the difficulties of obtaining patient information. Here we trace the genetic evolution of EBOV in the current outbreak that has resulted in multiple lineages. Deep sequencing of 179 patient samples processed by the European Mobile Laboratory, the first diagnostics unit to be deployed to the epicentre of the outbreak in Guinea, reveals an epidemiological and evolutionary history of the epidemic from March 2014 to January 2015. Analysis of EBOV genome evolution has also benefited from a similar sequencing effort of patient samples from Sierra Leone. Our results confirm that the EBOV from Guinea moved into Sierra Leone, most likely in April or early May. The viruses of the Guinea/Sierra Leone lineage mixed around June/July 2014. Viral sequences covering August, September and October 2014 indicate that this lineage evolved independently within Guinea. These data can be used in conjunction with epidemiological information to test retrospectively the effectiveness of control measures, and provides an unprecedented window into the evolution of an ongoing viral haemorrhagic fever outbreak.