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Post-exposure prophylaxis against Ebola virus disease with experimental antiviral agents: a case-series of health-care workers
Dr Michael Jacobs, FRCPcorrespondenceemail, Emma Aarons, FRCPath, Sanjay Bhagani, FRCP, Ruaridh Buchanan, MRCP, Ian Cropley, FRCP, Susan Hopkins, FRCP, Rebecca Lester, MRCP, Daniel Martin, FRCA, Neal Marshall, MRPharmS, Stephen Mepham, FRCPath, Simon Warren, FRCPath, Alison Rodger, FRCP
Published Online: 25 August 2015
Available: DOI: http://dx.doi.org/10.1016/S1473-3099(15)00228-5
Although a few international health-care workers who have assisted in the current Ebola outbreak in west Africa have been medically evacuated for treatment of Ebola virus disease, more commonly they were evacuated after potential accidental exposure to Ebola virus. An urgent need exists for a consensus about the risk assessment of Ebola virus transmission after accidental exposure, and to investigate the use of post-exposure prophylaxis (PEP). Experimental vaccines have occasionally been used for Ebola PEP, but newly developed experimental antiviral agents have potential advantages. Here, we describe a new method for risk assessment and management of health-care workers potentially exposed to Ebola virus and report the use of experimental antiviral therapies for Ebola PEP in people.
We devised a risk assessment and management algorithm for health-care workers potentially exposed to Ebola virus and applied this to eight consecutive individuals who were medically evacuated to the UK from west Africa between January, and March, 2015. PEP with antiviral agents was given to health-care workers assessed to have had substantial risk exposures to Ebola virus. Participants were followed up for 42 days after potential exposure.
Four of eight health-care workers were classified as having had low risk exposures and managed by watchful waiting in the community. None of these health-care workers developed Ebola virus disease. The other four health-care workers had intermediate or maximum risk exposures and were given PEP with antiviral agents. PEP was well tolerated with no serious adverse effects. None of these four health-care workers, including two with maximum risk exposures from penetrating injuries with freshly used hollow-bore needles, developed Ebola virus disease.
Standardised risk assessment should be adopted and consensus guidelines developed to systematically study the efficacy and safety of PEP with experimental agents. New experimental antiviral treatments are a viable option for PEP against Ebola.
Royal Free London NHS Foundation Trust.
Sexual transmission of Ebola virus in Liberia confirmed using genomic analysis
US ARMY MEDICAL RESEARCH INSTITUTE OF INFECTIOUS DISEASES
A suspected case of sexual transmission of Ebola virus disease (EVD) in Liberia was confirmed using genomic analysis, thanks to in-country laboratory capabilities established by U.S. Army scientists in collaboration with the Liberian Institute for Biomedical Research (LIBR). The work, described in today's edition of the New England Journal of Medicine, provides molecular evidence of Ebola virus (EBOV) transmission between an EVD survivor and his female partner. It also demonstrates the value of real-time genomic surveillance during an outbreak, according to senior author Gustavo Palacios, Ph.D., of the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID).
CPT Suzanne Mate, Ph.D., of USAMRIID, said scientists working at the LIBR earlier this year analyzed blood samples from a female patient who tested positive for EBOV in March 2015 when there had been no new documented cases for 30 days. The patient was reported to have had recent sexual intercourse with a male partner who had survived EVD and had been declared EBOV negative in early October 2014. Following the patient's death on March 27, Mate said, public health officials were able to secure the consent of the male survivor to obtain and test a semen sample from him. The semen sample tested EBOV positive by quantitative RT-PCR, but the assay indicated that the level of viral RNA was low and required a different sample preparation method than the one originally deployed to sequence EBOV RNA from acute samples.
"We implemented a new enrichment strategy in collaboration with scientists from Illumina, Inc. that was pivotal in obtaining the required coverage to complete downstream genomic analysis," said Michael Wiley, Ph.D, of USAMRIID. Next-generation sequencing of the enriched EBOV RNA extracted from the male survivor's semen was used to compare the genome for similarity to the virus RNA extracted from the female patient's blood sample. "Ebola virus genomes assembled from the patient's blood and the survivor's semen were consistent with direct transmission," commented Jason Ladner, Ph.D., of USAMRIID. "The samples shared three genetic substitutions that have not been found in any other Ebola virus sequences in Western Africa."
In addition, said Ladner, these three genetic changes were distinct from the last documented transmission chain in Liberia prior to this case. Combined with epidemiologic data, the genomic analysis provides support for sexual transmission of Ebola virus and for the persistence of infective EBOV in semen for more than 179 days after disease onset. This caused both the Centers for Disease Control and Prevention and the World Health Organization to change their recommendations for convalescent patients regarding sexual contact until more definitive information is obtained about how long Ebola virus can persist in semen.
Mariano Sanchez-Lockhart, Ph.D., another member of the USAMRIID team, explained that some organs, including the testes, are considered to be "immune privileged" sites. This means that immune responses are tightly regulated in those sites to limit or prevent tissue damage. Other examples of "privileged" sites include the eye, central nervous system, and pregnant uterus. "Within these sites, viruses might evade systemic immune responses and persist longer since the 'immune pressure' is more restricted than systemically," said Sanchez-Lockhart, adding that further studies are necessary to fully explore this mechanism. CPT Jeffrey Kugelman, Ph.D., of USAMRIID, said the team's work has implications for development of Ebola virus therapeutics as well, since any potential treatments would need to be designed to reach immune privileged sites. "For example, drugs that clear the virus in the bloodstream may not address the whole infection," he said. Kugelman, who was instrumental in setting up the onsite genomic sequencing capability at LIBR, said the study illustrates why such a capability is needed. "This work allowed for more informed decisions about how to manage and control the spread of the disease," he commented. "To be in the field, during an outbreak, and have the ability to make near real-time sequencing information available to health care providers and public health officials--that's a first."
Also contributing to the work were scientists from the Liberian Institute for Biomedical Research, the Centers for Disease Control and Prevention, the World Health Organization, the National Institutes of Health, and the Liberian Ministry of Health. Ebola virus causes severe hemorrhagic fever in humans and nonhuman primates with high mortality rates and continues to emerge in new geographic locations, including Western Africa, the site of the largest recorded outbreak to date. Over 28,000 confirmed, probable and suspected cases have been reported in Guinea, Liberia and Sierra Leone, with over 11,000 reported deaths, according to the World Health Organization.
USAMRIID's mission is to provide leading-edge medical capabilities to deter and defend against current and emerging biological threat agents. The Institute plays a key role as the lead military medical research laboratory for the Defense Threat Reduction Agency's Joint Science and Technology Office for Chemical and Biological Defense. USAMRIID is a subordinate element of the U.S. Army Medical Research and Materiel Command. For more information, visit http://www.usamriid.army.mil
This work was supported by the Defense Threat Reduction Agency, the Global Biosurveillance Technology Initiative, Global Emerging Infections Surveillance, and Illumina, by a contract between Battelle Memorial Institute and the U.S. National Institute of Allergy and Infectious Diseases (NIAID), and by the Intramural Research Program, NIAID, National Institutes of Health.
S.E. Mate, J.R. Kugelman, T.G. Nyenswah, J.T. Ladner, M.R. Wiley,T. Cordier-Lassalle, A. Christie, G.P. Schroth, S.M. Gross, G.J. Davies-Wayne, S.A. Shinde, R. Murugan, S.B. Sieh, M. Badio, L. Fakoli, F. Taweh, E. de Wit, N. van Doremalen, V.J. Munster, J. Pettitt, K. Prieto, B.W. Humrighouse, U. Ströher, J.W. DiClaro, L.E. Hensley, R.J. Schoepp, D. Safronetz, J. Fair, J.H. Kuhn, D.J. Blackley, A.S. Laney, D.E. Williams, T. Lo, A. Gasasira, S.T. Nichol, P. Formenty, F.N. Kateh, K.M. De Cock, F. Bolay, M. Sanchez-Lockhart, and G. Palacios. Molecular Evidence of Sexual Transmission of Ebola Virus. N Engl J Med 2015 Oct. published online 5 PM ET 14 Oct.
Ebola treatment beds prevented 57,000 Ebola cases and 40,000 deaths in Sierra Leone
But introducing beds just one month earlier may have halved the outbreak, according to new analysis from the London School of Hygiene & Tropical Medicine
LONDON SCHOOL OF HYGIENE & TROPICAL MEDICINE
The introduction of thousands of Ebola treatment beds by the UK and Sierra Leone governments and NGOs prevented an estimated 57,000 Ebola cases and 40,000 deaths in Sierra Leone, according to new research published in the Proceedings of the National Academy of Sciences. The UK played a leading role in the response to the crisis in Sierra Leone. This focused on increasing the number of available treatment beds which isolated the ill to prevent further infections in the community, changing behaviour so people suspected of having Ebola sought treatment early, and making burials safe. Lead author Dr Adam Kucharski, Lecturer in Infectious Disease Epidemiology at the London School of Hygiene & Tropical Medicine, said: "Our findings show the unprecedented local and international response led to a substantial decline in Ebola transmission. Given the rapid growth of the outbreak in Sierra Leone, if those beds hadn't been in place to isolate the ill and avert further infections, the epidemic could have been much worse."
Figures from the World Health Organization state there have been 13,945 reported cases of Ebola in Sierra Leone to date , with an estimated 70% of infections resulting in death during 2014 . With many cases in Sierra Leone potentially going unreported, the real figure is likely to be much higher. Between September 2014 and February 2015, more than 1,500 treatment beds were introduced in Ebola holding centres and community care centres, and a further 1,200 in Ebola treatment units, to support the overstretched health system in Sierra Leone. Using mathematical models, researchers estimated the potential impact additional treatment beds had in bringing about the decline of the Ebola outbreak. They separated out the effect of beds from other factors that reduced transmission, such as behaviour change, community engagement, improved case finding and increase in safe burials.
Taking into account both reported and presumed unreported cases, the researchers say 57,000 Ebola cases were prevented up to February 2015 as a direct result of the introduction of treatment beds. Given that the Ebola case fatality rate in Sierra Leone was near 70%, the researchers estimate this averted 40,000 deaths. However, they estimate that had the beds been introduced just one month earlier, an additional 12,500 reported and unreported cases could have prevented. If only 60% of Ebola cases were reported (as has been stated in field studies), this figure equates to 7,500 reported cases  - more than half of the total number of cases recorded by the World Health Organization. Study co-author Professor John Edmunds of the London School of Hygiene & Tropical Medicine, said: "There has been much criticism of the international community's slow response to the Ebola outbreak. Our analysis suggests putting treatment beds in place just one month earlier could have further reduced the size of the outbreak and potentially saved thousands of more lives. The way we prepare for, and respond to, future outbreaks of Ebola and other infectious diseases needs to be strengthened."
Beginning in December 2013, the world's largest ever Ebola epidemic primarily affected Liberia, Guinea and Sierra Leone in West Africa. Liberia has recently been declared Ebola free, while Guinea and Sierra Leone have very few cases left. The study authors have played an active role throughout the Ebola crisis, providing data to assist governments and NGOs in planning their response. The London School of Hygiene & Tropical Medicine is involved in many aspects of the ongoing Ebola response, including clinical trials of vaccines and convalescent plasma treatment, and work to strengthen global response to infectious disease epidemics.
This new research by Dr Kucharski and colleagues was funded by the Research for Health in Humanitarian Crises (R2HC) Programme, managed by Enhancing Learning and Research for Humanitarian Assistance (ELRHA). The £8 million R2HC programme is funded equally by the Wellcome Trust and DFID. The researchers chose to focus on the impact of beds due to limited available data on other control measures in some districts of Sierra Leone. They note that with better data on the timing and role of different interventions - both clinical and non-clinical - it would be possible to obtain more accurate estimates about the precise contribution of different factors to the decline of the epidemic.
For more information or to request interviews, please contact the London School of Hygiene & Tropical Medicine press office on firstname.lastname@example.org or +44(0)2079272802.
Notes to Editors:
Adam J. Kucharski, Anton Camacho, Stefan Flasche, Rebecca E. Glover, W. John Edmunds, and Sebastian Funk. Measuring the impact of Ebola control measures in Sierra Leone. Proceedings of the National Academy of Sciences. DOI: 10.1073/pnas.1508814112
An embargoed copy of the article is available through EurekAlert. Journalists should register at http://www.eurekalert.org/register.php and request access to Proceedings of the National Academy of Sciences (PNAS) materials. If you are already registered with EurekAlert, you can request access to PNAS at http://www.eurekalert.org/account.php.
If you wish to provide a link to this paper for your readers, please use the following, which will go live when the embargo lifts: http://www.pnas.org/cgi/doi/10.1073/pnas.1508814112
 Figures as of 7 October 2015: http://apps.who.int/ebola/ebola-situation-reports
 West African Ebola Epidemic after One Year -- Slowing but Not Yet under Control. New England Journal of Medicine. DOI:10.1056/NEJMc1414992 http://www.nejm.org/doi/full/10.1056/NEJMc1414992
 7,500 figure calculated separately by researchers and does not appear in PNAS paper.
About the London School of Hygiene & Tropical Medicine
The London School of Hygiene & Tropical Medicine is a world-leading centre for research and postgraduate education in public and global health, with 3,900 students and more than 1,000 staff working in over 100 countries. The School is one of the highest-rated research institutions in the UK, and among the world's leading schools in public and global health. Our mission is to improve health and health equity in the UK and worldwide; working in partnership to achieve excellence in public and global health research, education and translation of knowledge into policy and practice. http://www.lshtm.ac.uk
About the Research for Health in Humanitarian Crises (R2HC) programme
The R2HC programme aims to improve health outcomes by strengthening the evidence base for public health interventions in humanitarian crises. Visit http://www.elrha.org/work/r2hc for more information. In response to the Ebola outbreak in West Africa, the R2HC launched an emergency Ebola Health Research Call in August 2014, aiming to fund research which will help to strengthen interventions to tackle this and future outbreaks.
About the Wellcome Trust
The Wellcome Trust is a global charitable foundation dedicated to improving health. We provide more than £700 million a year to support bright minds in science, the humanities and the social sciences, as well as education, public engagement and the application of research to medicine. Our £18 billion investment portfolio gives us the independence to support such transformative work as the sequencing and understanding of the human genome, research that established front-line drugs for malaria, and Wellcome Collection, our free venue for the incurably curious that explores medicine, life and art. http://www.wellcome.ac.uk
Latest technology could help curb repeat Ebola crisis, experts say
UNIVERSITY OF EDINBURGH
Recent developments in surveillance technology could enable a swifter, more effective response to potentially deadly outbreaks of disease, a study has found. The Ebola crisis has highlighted a need to bolster global surveillance and enhance the capability to react appropriately to further outbreaks, experts say. This should include making use of modern technologies for detecting disease, sharing information in real time and analysing data.
A team of infectious disease experts from the University of Edinburgh and the Wellcome Trust Sanger Institute reviewed the global response to recent outbreaks, including Ebola, swine flu and Middle East Respiratory Syndrome (MERS). They found that shortcomings in the response to Ebola highlight the need to adopt state-of-the-art techniques to detect and monitor potential infectious disease outbreaks anywhere in the world. Technological advances such as rapid testing of patients and fast genetic analysis of viruses could help experts deal more effectively with emergency situations as they unfold, thereby saving lives.
Researchers say such tools are already available, and should be brought into public health planning around the world in order to help combat future disease outbreaks. Their work, published in Science Translational Medicine, was supported by the Wellcome Trust and the EU Horizon 2020 programme (COMPARE). Professor Mark Woolhouse, of the University of Edinburgh's Centre for Immunity, Infection and Evolution, said: "We cannot afford to wait for the next outbreak of infectious disease before putting effective systems in place to safeguard public health. Global surveillance would be costly, but in our highly connected world, early detection and rapid action against outbreaks are to everyone's benefit."
Professor Paul Kellam, Group Leader of Virus Genomics at the Wellcome Trust Sanger Institute, said: "Real-time analysis of virus genomes such as Ebola is an important addition to our toolkit for investigating disease outbreaks. When combined with the date and location of the sample, we can determine how the virus spreads. These technologies can improve the management of an outbreak, thereby saving lives."
Ebola and Its Global Research Architecture—Need for an Improvement
The current Ebola outbreak poses a threat to individual and global public health. Although the disease has been of interest to the scientific community since 1976, an effective vaccination approach is still lacking. This fact questions past global public health strategies, which have not foreseen the possible impact of this infectious disease. To quantify the global research activity in this field, a scientometric investigation was conducted. We analyzed the research output of countries, individual institutions and their collaborative networks. The resulting research architecture indicated that American and European countries played a leading role regarding output activity, citations and multi- and bilateral cooperations. When related to population numbers, African countries, which usually do not dominate the global research in other medical fields, were among the most prolific nations. We conclude that the field of Ebola research is constantly progressing, and the research landscape is influenced by economical and infrastructural factors as well as historical relations between countries and outbreak events.
For the first time in the history of the disease, the Ebola virus left its local setting and affected people not only in isolated rural areas, but reached larger towns and cities leading to worldwide repercussions. This development prompted a joint global response to this health threat. This encompassed not only immediate relief efforts, but also an up search in global research work. In this study, the scientific output in Ebola research available in one of the mayor medical search platforms was characterized. We studied among others the origin of research, the collaboration between countries and the research topics. Partly, the obtained data was weighted against economic parameters. We attained a detailed map of the research activities from the discovery of Ebola in 1976 up to today. Our research provides the first overview of the worldwide Ebola research output. It might help stakeholders in Ebola research to better plan investigations with a global perspective.
Chip-based technology enables reliable direct detection of Ebola virus
Hybrid device integrates a microfluidic chip for sample preparation and an optofluidic chip for optical detection of individual molecules of viral RNA
UNIVERSITY OF CALIFORNIA - SANTA CRUZ
A team led by researchers at UC Santa Cruz has developed chip-based technology for reliable detection of Ebola virus and other viral pathogens. The system uses direct optical detection of viral molecules and can be integrated into a simple, portable instrument for use in field situations where rapid, accurate detection of Ebola infections is needed to control outbreaks. Laboratory tests using preparations of Ebola virus and other hemorrhagic fever viruses showed that the system has the sensitivity and specificity needed to provide a viable clinical assay. The team reported their results in a paper published September 25 in Nature Scientific Reports.
An outbreak of Ebola virus in West Africa has killed more than 11,000 people since 2014, with new cases occurring recently in Guinea and Sierra Leone. The current gold standard for Ebola virus detection relies on a method called polymerase chain reaction (PCR) to amplify the virus's genetic material for detection. Because PCR works on DNA molecules and Ebola is an RNA virus, the reverse transcriptase enzyme is used to make DNA copies of the viral RNA prior to PCR amplification and detection. "Compared to our system, PCR detection is more complex and requires a laboratory setting," said senior author Holger Schmidt, the Kapany Professor of Optoelectronics at UC Santa Cruz. "We're detecting the nucleic acids directly, and we achieve a comparable limit of detection to PCR and excellent specificity."
In laboratory tests, the system provided sensitive detection of Ebola virus while giving no positive counts in tests with two related viruses, Sudan virus and Marburg virus. Testing with different concentrations of Ebola virus demonstrated accurate quantification of the virus over six orders of magnitude. Adding a "preconcentration" step during sample processing on the microfluidic chip extended the limit of detection well beyond that achieved by other chip-based approaches, covering a range comparable to PCR analysis. "The measurements were taken at clinical concentrations covering the entire range of what would be seen in an infected person," Schmidt said.
Schmidt's lab at UC Santa Cruz worked with researchers at Brigham Young University and UC Berkeley to develop the system. Virologists at Texas Biomedical Research Institute in San Antonio prepared the viral samples for testing. The system combines two small chips, a microfluidic chip for sample preparation and an optofluidic chip for optical detection. For over a decade, Schmidt and his collaborators have been developing optofluidic chip technology for optical analysis of single molecules as they pass through a tiny fluid-filled channel on the chip. The microfluidic chip for sample processing can be integrated as a second layer next to or on top of the optofluidic chip.
Schmidt's lab designed and built the microfluidic chip in collaboration with coauthor Richard Mathies at UC Berkeley who pioneered this technology. It is made of a silicon-based polymer, polydimethylsiloxane (PDMS), and has microvalves and fluidic channels to transport the sample between nodes for various sample preparation steps. The targeted molecules--in this case, Ebola virus RNA--are isolated by binding to a matching sequence of synthetic DNA (called an oligonucleotide) attached to magnetic microbeads. The microbeads are collected with a magnet, nontarget biomolecules are washed off, and the bound targets are then released by heating, labeled with fluorescent markers, and transferred to the optofluidic chip for optical detection.
Schmidt noted that the team has not yet been able to test the system starting with raw blood samples. That will require additional sample preparation steps, and it will also have to be done in a biosafety level 4 facility. "We are now building a prototype to bring to the Texas facility so that we can start with a blood sample and do a complete front-to-back analysis," Schmidt said. "We are also working to use the same system for detecting less dangerous pathogens and do the complete analysis here at UC Santa Cruz."
The lead authors of the paper are postdoctoral researcher Hong Cai and graduate student Joshua Parks, both in Schmidt's lab at UC Santa Cruz. A team led by Aaron Hawkins at BYU fabricated the silicon-based optofluidic chips. Virologist Jean Patterson led the team at Texas Biomedical Research Institute that prepared viral samples for testing. This research was supported by the W. M. Keck Center for Nanoscale Optofluidics at UC Santa Cruz and grants from the National Institutes of Health and the National Science Foundation.
Prognostic Analysis of Patients with Ebola Virus Disease
Full article link.
The Ebola virus causes an acute, serious illness which is often fatal if untreated. However, factors affecting the survival of the disease remain unclear. Here, we investigated the prognostic factors of Ebola virus disease (EVD) through various statistical models.
Sixty three laboratory-confirmed EVD patients with relatively complete clinical profiles were included in the study. All the patients were recruited at Jui Government Hospital, Sierra Leone between October 1st, 2014 and January 18th, 2015. We first investigated whether a single clinical presentation would be correlated with the survival of EVD. Log-rank test demonstrated that patients with viral load higher than 106 copies/ml presented significantly shorter survival time than those whose viral load were lower than 106 copies/ml (P = 0.005). Also, using Pearson chi-square test, we identified that chest pain, coma, and viral load (>106 copies/ml) were significantly associated with poor survival of EVD patients. Furthermore, we evaluated the effect of multiple variables on the survival of EVD by Cox proportional hazards model. Interestingly, results revealed that patient’s age, symptom of confusion, and viral load were the significantly associated with the survival of EVD cases (P = 0.017, P = 0.002, and P = 0.027, respectively).
These results suggest that age, chest pain, coma, confusion and viral load are associated with the prognosis of EVD, in which viral load could be one of the most important factors for the survival of the disease.
The current outbreak of Ebola virus disease (EVD) in West Africa is the largest and most complex Ebola outbreak since the virus was first discovered in 1976. Factors affecting the survival of the disease remain unclear. Here, we investigated the prognostic factors of EBV from 63 cases with relatively complete clinical profiles in Sierra Leone. Using different statistical models, we found that age, chest pain, coma, confusion and viral load were associated with the prognosis of EVD, in which viral load could be one of the most important factors for the survival of the disease.
Ebola virus mutations may help it evade drug treatment
US ARMY MEDICAL RESEARCH INSTITUTE OF INFECTIOUS DISEASES
Genetic mutations called "escape variants" in the deadly Ebola virus appear to block the ability of antibody-based treatments to ward off infection, according to a team of U.S. Army scientists and collaborators. Their findings, published online this week in the journal Cell Reports, have implications for the continued development of therapeutics to treat Ebola virus disease, which has claimed the lives of over 11,000 people in West Africa since last year.
Ebola virus overruns the immune system, thus overwhelming the host's ability to fight off the infection. One strategy for treatment is based on the administration of a "cocktail" of antibodies that have the ability to neutralize the virus and allow the host to mount an effective immune response. One such cocktail, known as MB-003, has demonstrated efficacy in nonhuman primates infected with the virus. MB-003 and ZMAb were early formulations used in proof-of-principle trials that led to the more advanced formulation, ZMapp, currently in development and authorized for compassionate use in the West African Ebola outbreak response.
To understand the reasons for the improvement, investigators at the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID) Center for Genome Sciences (CGS) examined nonhuman primates that succumbed to Ebola virus despite being treated with MB-003 one or two days post-infection. Viral nucleic acids were isolated from blood samples drawn at multiple time points and were sequenced to provide a description of the viral population. This approach is routinely utilized at USAMRIID to study the development of viral therapeutic resistance against antiviral countermeasures, according to Dr. Gustavo Palacios, director of the center and senior author of the study.
Two clusters of changes in the viral genome were observed in one of the animals that succumbed. Several of those changes corresponded with the viral target sites of two of the cocktail's antibodies, triggering an in-depth molecular analysis of the development and impact of those changes. "The molecular analysis allowed us to see where the cocktails were inducing changes in the genome, and to link those changes to the treatment failure," said co-first author CPT Jeffrey Kugelman, Ph.D., of USAMRIID. Based on these findings, tissues at selected time-points were used for viral isolation, which finally allowed the "rescue" of the mutated virus. "When this rescued virus was sequenced, we observed that the clusters of changes had progressed from affecting a small portion of the viral population to becoming mutations--permanent changes in the genome--without disrupting any major viral functions, including the ability to cause infection."
Subsequently, the rescued virus was tested for neutralization against the cocktail and its individual components, demonstrating the inability of the therapeutic to control replication. "At this point, we knew that the mutations were, in fact, 'escape variants' that were cumulatively responsible for reduced efficacy of the MB-003 therapeutic," Palacios said. "However, we were unsure of the frequency of this type of event."
To help answer that question, the scientists analyzed other independently conducted but similarly designed studies of MB-003. "We were able to identify, using the same tools, a second animal with a similar pattern of changes," Kugelman noted. "Strikingly, the two animals had four sites in common. This information leads us to believe that these sites are under 'selection pressure' by the therapeutic, meaning that the antibody cocktail binds and promotes elimination of the original virus, while the escape variants continue the infection." Palacios added that further work is needed to document each individual mutation's effect on each of the three antibodies. "Our research has already established that a few amino acid changes may be sufficient to erode the binding of multiple antibodies in a cocktail," he said. "It would be very important to determine which changes have a direct impact on binding."
The team's findings highlight the importance of selecting different target domains for each component of the therapeutic cocktail to minimize the potential for viral escape, according to the authors. Two of the antibodies in the MB-003 cocktail target a related region of the Ebola virus glycoprotein, and thus are more susceptible to localized changes affecting multiple antibodies. Furthermore, these findings are important for scientists studying Ebola virus to provide a basis for how quickly the virus can adapt to therapeutics. This rate will be important for predictive models of therapeutic resistance.
Ebola virus causes severe hemorrhagic fever in humans and nonhuman primates with high mortality rates and continues to emerge in new geographic locations, including West Africa, the site of the largest outbreak to date. Over 28,000 confirmed, probable and suspected cases have been reported in Guinea, Liberia and Sierra Leone, with more than 11,000 reported deaths, according to the World Health Organization.
The study's authors are Jeffrey R. Kugelman, Johanny Kugelman-Tonos, Jason Ladner, Carolyn M. Keeton, Elyse R. Nagle, Karla Y. Garcia, Jeffrey W. Froude, Ana I. Kuehne, Sina Bavari, John M. Dye, Mariano Sanchez-Lockhart, and Gustavo F. Palacios, USAMRIID; James Pettit, Gene G. Olinger, and Jens H. Kuhn, Integrated Research Facility at Fort Detrick, National Institute of Allergy and Infectious Diseases, National Institutes of Health; and Larry Zeitlin, Mapp Biopharmaceutical, Inc.
USAMRIID's mission is to provide leading-edge medical capabilities to deter and defend against current and emerging biological threat agents. The Institute plays a key role as the lead military medical research laboratory for the Defense Threat Reduction Agency's Joint Science and Technology Office for Chemical and Biological Defense. USAMRIID is a subordinate laboratory of the U.S. Army Medical Research and Materiel Command.
This work was supported by the Defense Threat Reduction Agency.
Reference: Emergence of Ebola Virus Escape Variants in Infected Nonhuman Primates Treated with the MB-003 Antibody Cocktail: Kugelman et al., 2015, Cell Reports 12, 1-10. September 29, 2015.
Link to paper online: http://www.cell.com/cell-reports/pdf/S2211-1247(15)00923-7.pdf
New Ebola test could help curb disease spread
Using magnetic nanoparticles means simpler, cheaper diagnosis
Amsterdam, September 9, 2015 - A new Ebola test that uses magnetic nanoparticles could help curb the spread of the disease in western Africa. Research published in Biosensors and Bioelectronics shows that the new test is 100 times more sensitive than the current test, and easier to use. Because of this, the new test makes it easier and cheaper to diagnose cases, enabling healthcare workers to isolate patients and prevent the spread of Ebola. The authors of the study, from the Chinese Academy of Sciences, say their new technology could be applied to the detection of any biological molecules, making it useful to diagnose other infectious diseases, like flu, and potentially detect tumors and even contamination in wastewater.
The Ebola virus causes an acute illness that is deadly in half of all cases, on average. The current outbreak of Ebola, which started in March 2014, affects countries in west Africa. In the most severely affected countries, like Guinea, Liberia and Sierra Leone, resources are limited, making control of the outbreak challenging. There is no vaccine for Ebola, so detecting the virus is key to controlling the outbreak: with an accurate diagnosis, patients can be isolated and treated properly, reducing the risk of spread. "In west Africa, resources are under pressure, so complicated, expensive tests are not very helpful," said Professor Xiyun Yan, one of the authors of the study from the Chinese Academy of Sciences. "Our new strip test is a simple, one-step test that is cheap and easy to use, and provides a visible signal, which means people don't need training to use it. We think it will be especially helpful in rural areas, where technical equipment and skills are not available."
Currently there are two ways to test for the Ebola virus: using a method called polymerase chain reaction (PCR), which makes copies of the molecules for detection, and with antibody-capture enzyme-linked immunosorbent assay (ELISA), which gives a visual indication when a given molecule is in a sample. PCR is very sensitive, but is expensive and complicated, requiring special skills and technical equipment. The ELISA - or gold strip test - is cheaper but sensitivity is very low, which means it often gives the wrong results. The new test, called the nanozyme test, uses magnetic nanoparticles, which work like enzymes to make the signal stronger, giving a clearer result you can see with the naked eye. The test can detect much smaller amounts of the virus, and is 100 times more sensitive than the gold strip test. "People have loved the strip test for many years, but it has a major weakness: it's not sensitive enough. We're very excited about our new nanozyme test, as it is much more sensitive and you don't need any specialist equipment to get a quick, accurate result," said Dr. Yan. Strip tests work by attaching molecules called antibodies to gold particles to look for a particular molecule in a sample. When they attach to the molecule you're looking for, in this case a virus, they produce a signal, such as a color change. In order to find the virus, the particles need to be labelled with enzymes, which speed up detection and signalling.
With the new nanozyme test, the researchers applied magnetic nanoparticles as a nanozyme probe in place of gold nanoparticles. After labeling with an antibody that attaches to the Ebola virus, this novel probe is able to recognize and separate the virus in a sample. The nanoparticles are magnetic, so to concentrate the virus particles in a sample, all you need to do is hold the sample against a magnet; no expensive equipment is needed. The nanozyme test is 100 times more sensitive than the gold strip test, detecting molecules called glycoproteins on the surface of the Ebola virus at concentrations as low as 1 nanogram per milliliter. The researchers have applied for a patent for the new test, which is currently being taken to west Africa by the CDC to use in the field. The researchers are also collaborating with clinical teams to apply the technology to other diseases, and with a company that treats wastewater to see if it can help remove environmental contamination.
"Nanozyme-strip for rapid local diagnosis of Ebola" by Demin Duan, Kelong Fan, Dexi Zhang, Shuguang Tan, Mifang Liang, Yang Liu, Jianlin Zhang, Panhe Zhang, Wei Liu, Xiangguo Qiu, Gary P. Kobinger, George Fu Gao, Xiyun Yan (doi: 10.1016/j.bios.2015.05.025). The article appears in Biosensors and Bioelectronics, published by Elsevier.
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Efficacy and effectiveness of an rVSV-vectored vaccine expressing Ebola surface glycoprotein: interim results from the Guinea ring vaccination cluster-randomised trial
Ana Maria Henao-Restrepo, MD, Prof Ira M Longini, PhD, Prof Matthias Egger, MD, Natalie E Dean, PhD, Prof W John Edmunds, PhD, Anton Camacho, PhD, Miles W Carroll, PhD, Moussa Doumbia, MD, Bertrand Draguez, MD, Sophie Duraffour, PhD, Godwin Enwere, FWACP, Rebecca Grais, PhD, Stephan Gunther, MD, Stefanie Hossmann, MSc, Prof Mandy Kader Kondé, PhD, Souleymane Kone, MSc, Eeva Kuisma, PhD, Prof Myron M Levine, MD, Sema Mandal, MD, Gunnstein Norheim, PhD, Ximena Riveros, BSc, Aboubacar Soumah, MD, Sven Trelle, MD, Andrea S Vicari, PhD, Conall H Watson, MFPH, Sakoba Kéïta, MD, Dr Marie Paule Kieny, PhD†correspondenceemail, Prof John-Arne Røttingen, MD†
A recombinant, replication-competent vesicular stomatitis virus-based vaccine expressing a surface glycoprotein of Zaire Ebolavirus (rVSV-ZEBOV) is a promising Ebola vaccine candidate. We report the results of an interim analysis of a trial of rVSV-ZEBOV in Guinea, west Africa.
For this open-label, cluster-randomised ring vaccination trial, suspected cases of Ebola virus disease in Basse-Guinée (Guinea, west Africa) were independently ascertained by Ebola response teams as part of a national surveillance system. After laboratory confirmation of a new case, clusters of all contacts and contacts of contacts were defined and randomly allocated 1:1 to immediate vaccination or delayed (21 days later) vaccination with rVSV-ZEBOV (one dose of 2 × 107 plaque-forming units, administered intramuscularly in the deltoid muscle). Adults (age ≥18 years) who were not pregnant or breastfeeding were eligible for vaccination. Block randomisation was used, with randomly varying blocks, stratified by location (urban vs rural) and size of rings (≤20 vs >20 individuals). The study is open label and masking of participants and field teams to the time of vaccination is not possible, but Ebola response teams and laboratory workers were unaware of allocation to immediate or delayed vaccination. Taking into account the incubation period of the virus of about 10 days, the prespecified primary outcome was laboratory-confirmed Ebola virus disease with onset of symptoms at least 10 days after randomisation. The primary analysis was per protocol and compared the incidence of Ebola virus disease in eligible and vaccinated individuals in immediate vaccination clusters with the incidence in eligible individuals in delayed vaccination clusters. This trial is registered with the Pan African Clinical Trials Registry, number PACTR201503001057193.
Between April 1, 2015, and July 20, 2015, 90 clusters, with a total population of 7651 people were included in the planned interim analysis. 48 of these clusters (4123 people) were randomly assigned to immediate vaccination with rVSV-ZEBOV, and 42 clusters (3528 people) were randomly assigned to delayed vaccination with rVSV-ZEBOV. In the immediate vaccination group, there were no cases of Ebola virus disease with symptom onset at least 10 days after randomisation, whereas in the delayed vaccination group there were 16 cases of Ebola virus disease from seven clusters, showing a vaccine efficacy of 100% (95% CI 74·7–100·0; p=0·0036). No new cases of Ebola virus disease were diagnosed in vaccinees from the immediate or delayed groups from 6 days post-vaccination. At the cluster level, with the inclusion of all eligible adults, vaccine effectiveness was 75·1% (95% CI −7·1 to 94·2; p=0·1791), and 76·3% (95% CI −15·5 to 95·1; p=0·3351) with the inclusion of everyone (eligible or not eligible for vaccination). 43 serious adverse events were reported; one serious adverse event was judged to be causally related to vaccination (a febrile episode in a vaccinated participant, which resolved without sequelae). Assessment of serious adverse events is ongoing.
The results of this interim analysis indicate that rVSV-ZEBOV might be highly efficacious and safe in preventing Ebola virus disease, and is most likely effective at the population level when delivered during an Ebola virus disease outbreak via a ring vaccination strategy.
WHO, with support from the Wellcome Trust (UK); Médecins Sans Frontières; the Norwegian Ministry of Foreign Affairs through the Research Council of Norway; and the Canadian Government through the Public Health Agency of Canada, Canadian Institutes of Health Research, International Development Research Centre, and Department of Foreign Affairs, Trade and Development.
Available free of charge: here.
ReEBOV Antigen Rapid Test kit for point-of-care and laboratory-based testing for Ebola virus disease: a field validation study
Mara Jana Broadhurst, MD, John Daniel Kelly, MD, Ann Miller, PhD, Amanda Semper, DPhil, Daniel Bailey, PhD, Elisabetta Groppelli, PhD, Andrew Simpson, FRCPath, Tim Brooks, FRCPath, Susan Hula, MSc, Wilfred Nyoni, MSc, Alhaji B Sankoh, DLT, Santigi Kanu, DLT, Alhaji Jalloh, Quy Ton, MD, Nicholas Sarchet, RN, Peter George, MD, Mark D Perkins, MD, Betsy Wonderly, BSc, Prof Megan Murray, MD, Dr Nira R Pollock, MD
At present, diagnosis of Ebola virus disease requires transport of venepuncture blood to field biocontainment laboratories for testing by real-time RT-PCR, resulting in delays that complicate patient care and infection control efforts. Therefore, an urgent need exists for a point-of-care rapid diagnostic test for this disease. In this Article, we report the results of a field validation of the Corgenix ReEBOV Antigen Rapid Test kit.
We performed the rapid diagnostic test on fingerstick blood samples from 106 individuals with suspected Ebola virus disease presenting at two clinical centres in Sierra Leone. Adults and children who were able to provide verbal consent or assent were included; we excluded patients with haemodynamic instability and those who were unable to cooperate with fingerstick or venous blood draw. Two independent readers scored each rapid diagnostic test, with any disagreements resolved by a third. We compared point-of-care rapid diagnostic test results with clinical real-time RT-PCR results (RealStar Filovirus Screen RT-PCR kit 1·0; altona Diagnostics GmbH, Hamburg, Germany) for venepuncture plasma samples tested in a Public Health England field reference laboratory (Port Loko, Sierra Leone). Separately, we performed the rapid diagnostic test (on whole blood) and real-time RT-PCR (on plasma) on 284 specimens in the reference laboratory, which were submitted to the laboratory for testing from many clinical sites in Sierra Leone, including our two clinical centres.
In point-of-care testing, all 28 patients who tested positive for Ebola virus disease by RT-PCR were also positive by fingerstick rapid diagnostic test (sensitivity 100% [95% CI 87·7–100]), and 71 of 77 patients who tested negative by RT-PCR were also negative by the rapid diagnostic test (specificity 92·2% [95% CI 83·8–97·1]). In laboratory testing, all 45 specimens that tested positive by RT-PCR were also positive by the rapid diagnostic test (sensitivity 100% [95% CI 92·1–100]), and 214 of 232 specimens that tested negative by RT-PCR were also negative by the rapid diagnostic test (specificity 92·2% [88·0–95·3]). The two independent readers agreed about 95·2% of point-of-care and 98·6% of reference laboratory rapid diagnostic test results. Cycle threshold values ranged from 15·9 to 26·3 (mean 22·6 [SD 2·6]) for the PCR-positive point-of-care cohort and from 17·5 to 26·3 (mean 21·5 [2·7]) for the reference laboratory cohort. Six of 16 banked plasma samples from rapid diagnostic test-positive and altona-negative patients were positive by an alternative real-time RT-PCR assay (the Trombley assay); three (17%) of 18 samples from individuals who were negative by both the rapid diagnostic test and altona test were also positive by Trombley.
The ReEBOV rapid diagnostic test had 100% sensitivity and 92% specificity in both point-of-care and reference laboratory testing in this population (maximum cycle threshold 26·3). With two independent readers, the test detected all patients who were positive for Ebola virus by altona real-time RT-PCR; however, this benchmark itself had imperfect sensitivity.
Article available free of charge here.