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Recent Advances in the Diagnosis and Management of Ebola Virus Disease
Clinical Microbiology N e w s l e t
Vol. 41, No. 21 November 1, 2019 www.cmnewsletter.com I n Th is
Issu e
185 Recent Advances in the Diagnosis and Management of Ebola Virus Disease 189 An Unusual Case of Acute Invasive Pansinusitis Due to Laceyella sp. in a Febrile Neutropenic Patient with Myelodysplastic Syndrome A case report
Caitlin N. Murphy, Ph.D., University of Nebraska Medical Center, Department of Pathology and Microbiology, 985900 Nebraska Medical Center Omaha, NE 68198. Tel.: 402-552-3305. Email: [email protected] 0196-4399/©2019 Elsevier Inc. All rights reserved
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t e r
Recent Advances in the Diagnosis and Management of Ebola Virus Disease Caitlin N. Murphy, Ph.D., Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska
Abstract Outbreaks of Ebola virus, a causative agent of hemorrhagic fever, in West Africa and the Democratic Republic of the Congo have highlighted the need for assays that can rapidly identify individuals infected with Ebola virus. These assays need to accurately detect and rule-out Ebola virus disease (EVD) and, ideally, not require advanced laboratory techniques. Over the course of the most recent outbreaks, field studies have evaluated the performance of rapid antigen and automated molecular tests, many of which were given emergency use authorization by the Food and Drug Administration. Overall, molecular assays demonstrated high sensitivity, while antigen testing had the highest sensitivity when the viral load was high. Specificity was high across the evaluated test options. In addition to the evaluation of diagnostic test methods, newly developed treatment options and the efficacy of ring vaccination to prevent disease transmission have been investigated. Findings from these field studies have demonstrated that many of the newly developed diagnostic assays, as well as experimental treatments and vaccination, are promising tools for controlling EVD.
Introduction
Corresponding author:
CMN
Ebolavirus was first identified as an etiological agent of hemorrhagic fever when an outbreak occurred in southern Sudan and northern Zaire in 1976 [1]. Since that time, there have been over 20 documented outbreaks that occurred predominantly in rural communities that were geographically isolated [2]. In West Africa, the largest Ebola outbreak occurred from 2014 to 2016, with over 20,000 cases and 11,000 deaths. The West African outbreak has been controlled, but a separate outbreak in the Democratic Republic of the Congo (DRC) continues into its second year. As of 6 August 2019, 2,781 cases and 1,866 deaths have been reported [3]. Prior to these recent outbreaks, the maximum death toll for any one outbreak was 280. As ebolavirus has moved into more urban, mobile communities, it has become more difficult to control the outbreaks. Another unfortunate barrier in the current DRC outbreak is the political instability that exists and has led to ongoing violence, making it very
difficult for humanitarian aid workers to control the outbreak. This unrest has led to distrust in the government and, by extension, the health care workers struggling to make inroads to stop the outbreak. In order to control the transmission of ebolavirus, appropriate standards for diagnosis, treatment, and prevention are needed. In the last 5 years, significant advances have been made in these areas, but the efficacy of experimental treatment and prevention strategies requires further study. This review focuses on the data that have been gathered during the most recent outbreaks. This work has provided valuable data and demonstrated the need for continued development of resources against emerging infections.
General Information Ebolaviruses are negative-stranded RNA viruses that belong to the family Filoviridae. To date, there are six known species of ebolaviruses. Zaire ebolavirus (EBOV) and Sudan ebolavirus are the
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most significant causes of human infection and have case fatality rates of 80 and 50%, respectively. Bundibugyo ebolavirus and Tai Forest ebolavirus are rarer causes of infections in humans. Finally, Reston ebolavirus and Bombali ebolavirus, are considered non-pathogenic to humans.
Transmission and Clinical Presentation EBOV is acquired when mucous membranes or abrasions on the skin come in contact with the blood or body fluids of infected individuals. Following acquisition, EBOV has a tropism for a wide variety of cell types, with early replication occurring primarily in dendritic cells, macrophages, and monocytes. Dissemination to the liver, lymph nodes, and spleen occurs as the infection progresses. As the virus disseminates, it disables the immune system and dysregulates the coagulation cascade, leading to organ failure.
field study investigating its efficacy was performed at two clinical sites in Sierra Leone. A total of 105 patients with suspected EVD were successfully tested with the ReEBOV test and a comparator molecular assay. Twenty-eight patients were positive by both the rapid antigen assay and a comparator real-time reverse transcription (RT)-PCR assay. The test had an overall sensitivity of 100% and specificity of 92% when two readers independently evaluated the results [5]. The ReEBOV assay requires only finger stick blood samples, but the test reagents require refrigeration and reconstitution of control materials, which may limit implementation in resource-limited settings. The ReEBOV assay has a high limit of detection that does not make it suitable for the detection of patients early in disease progression and is therefore not considered ideal for the management of suspect cases [6].
EBOV is one of the viral etiologies of viral hemorrhagic fever (VHF), but VHF can be caused by four different families of singlestranded RNA viruses. As these agents have a nonspecific presentation and high risk for nosocomial transmission, rapid diagnosis to facilitate cohorting is needed in outbreak settings. Ebola virus disease (EVD) has an incubation period that ranges from 2 to 21 days. At the time of symptom onset, patients are considered contagious. Symptoms at disease onset are initially nonspecific—fever, headache, and myalgia, followed by gastrointestinal symptom onset around day 3 to 5. The third stage of illness is either recovery or deterioration with progression to bleeding, neurological symptoms, and multiorgan failure.
The OraQuick Ebola Rapid Antigen Test (OraSure Technologies, Inc.) offers a rapid antigen test that also detects the EBOV VP40 antigen. It can be utilized to test whole blood from patients with suspected EVD or by using oral fluid from cadaveric specimens. Two hundred forty-four archived oral-fluid specimens were tested with the OraQuick and compared to the Xpert Ebola Test (described below) in a Sierra Leone field laboratory and performed with an overall sensitivity of 94.12% and specificity of 100% [7]. In addition, 75 whole-blood specimens were tested at the CDC and compared to an RT-PCR assay. The overall sensitivity and specificity were 84% and 98%, respectively. Of note, when the PCR-based assay had low threshold cycle (CT) ranges (15 to 24) (suggesting a high viral burden), the sensitivity was 100% [7].
Despite ongoing efforts to identify the animal reservoir, the modes of zoonotic transmission are not well understood, but based on current information, fruit bats are the suspected reservoir of EBOV. Evidence suggests that the West African outbreak was caused by a single transmission event to a 2-year-old boy in Guinea. It was shown that the child most likely acquired EBOV from exposure to Mops condylurus, an insectivorous free-tailed bat [4], suggesting the reservoir for EBOV might be wider than previously thought. Recognition of the outbreak was delayed, as EBOV had not previously been seen in West Africa, while Lassa fever, another VHF, is endemic to West Africa.
Two additional assays were approved by the WHO and/or FDA, the SD Q Line Ebola (SD BioSensor, Inc.) and the DPP Ebola Antigen (Chembio). To date, only the OraQuick assay has been available for use in the DRC outbreak. A new assay, the QuickNavi-Ebola, was evaluated using 928 whole-blood samples collected from patients with suspected EVD. The samples were tested by the Xpert Ebola assay as the comparator assay. The assay demonstrated a sensitivity of 85% and a specificity of 99.8%. As with the other lateral-flow assays described above, a majority of the false negatives (10/12) were in samples with CT values of >30 (suggesting a low viral burden) on the Xpert Ebola assay [8].
Laboratory Diagnosis
Molecular
Control of EBOV outbreaks requires a rapid response based on the recognition of cases early in the course of disease. The primary methodologies currently being investigated for the rapid and accurate diagnosis of EVD rely on detection of either circulating viral RNA or viral antigens. Serological options are available but are not considered useful for timely diagnosis of acute infections. Discussed below are diagnostic options that were granted emergency use authorization (EUA) by the World Health Organization (WHO) and/or the U.S. Food and Drug Administration (FDA).
RT-PCR is considered the gold standard for the diagnosis of EVD, as it is a highly sensitive and specific technique. A drawback of many RT-PCR assays for EBOV is that they can require manual extraction and technical expertise and can be subject to longer turnaround times than other options. Two automated RT-PCR assays that require minimal hands-on time were given EUA by the WHO and FDA.
Antigen During the West African outbreak, the WHO and FDA approved two lateral-flow immunoassays, ReEBOV and OraQuick. The ReEBOV Antigen Rapid Test kit (Corgenix, Inc.), which has since been delisted, detects the VP40 matrix protein of EBOV. A
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The FilmArray system (BioFire Diagnostics and BioFire Defense) BioThreat-E (FA BT-E) test consists of a pouch that contains lyophilized reagents for sample extraction and RT-PCR. The user needs only to rehydrate the pouch and add the patient sample to an injection vial containing lysis reagents. The sample can then be loaded into the pouch. Results are then available within 90 minutes.
Studies evaluating the performance of the assay have been conducted in Sierra Leone, Guinea, the United Kingdom, and the United States. In all cases, the performance of the BT-E test was compared to that of an RT-PCR being utilized at the reference laboratory. A majority of testing was performed on whole blood, with the FA BT-E test demonstrating sensitivities ranging from 75 to 95.7% and specificities approaching 100% [9-11]. The study in Guinea took place over the course of 4 months in 2015. The study tested 135 total patients and compared the results of the FA BT-E test to those of two RT-PCR assays in routine use in the diagnostic laboratory. Forty-five of 47 patients with EVD confirmed by both alternative methods were detected, for an overall sensitivity of 95.7% and specificity of 100%. Preliminary findings with a small subset of alternative specimens (35 saliva and 3 urine specimens) also showed high agreement with comparator assays, indicating that testing may be successful using noninvasively collected specimens [9]. The GeneXpert (Cepheid, Sunnyvale, CA) also produces a sample-to-answer RT-PCR-based system designed for the detection of EBOV in whole blood and has the advantage of being a higher-throughput instrument commonly used in many areas for the detection of Mycobacterium tuberculosis. A study performed in South Africa utilizing 281 plasma and serum specimens collected from suspected EVD cases in 2014 and 2015 showed close to 100% sensitivity and specificity when the CT value of the comparator assay was low (£34.99) [12]. A second study using specimens from Sierra Leone demonstrated a sensitivity of 100% and specificity of 99.5% using whole-blood specimens and an alternative comparator assay [13]. During the same study, evaluations of the Xpert Ebola assay for the detection of EBOV in semen and buccal swabs has also shown promise for follow-up testing or testing using less invasively collected specimens. Both assays require minimal specimen handling, and result interpretation is performed by the instrument. A potential advantage of the FA BT-E test is that BioFire assays are often multiplexed and can recognize other agents of VHF or other sequelae that can mimic VHF early in the course of disease. Clinical trials are ongoing to examine the performance of a multi-target panel [14]. In the United States, a confirmed case of EVD still requires confirmation at the CDC, and results of tests with FDA EUA status are considered to be only presumptive. A challenge of comparing the evaluations done in the field on both lateral-flow immunoassays and automated RT-PCR assays discussed in this review is that they were performed in geographically disparate locations with variable study populations and comparator assays. These publications have also expressed concerns that logistical considerations or biosafety issues may limit their utility [15,16].
Treatment ZMapp is an experimental treatment option for EVD. ZMapp is a combination of three monoclonal antibodies that target the surface glycoprotein of EBOV. A randomized trial comparing ZMapp
plus the existing standard of care to the standard of care alone was performed with 72 patients across Liberia, Sierra Leone, Guinea, and the United States. ZMapp was administered by intravenous infusion once every 3 days over the course of 9 days. In the ZMapp group the overall mortality rate was 15% lower than with the standard of care alone, and the posterior probability of ZMapp being superior to the standard of care alone was 91.2% [17]. Most of the deaths in the ZMapp group (7/8) occurred before the patients were able to receive the second infusion. Another factor influencing the efficacy of this treatment during thetrial was the delay from symptom onset to treatment. Many treated patients were in more advanced stages of disease and were therefore more likely to have a poor outcome. Studies have shown in nonhuman primates that therapy initiated in a 5-day window after symptom onset had a ≥90% survival rate [18]. Patients in Guinea were also provided experimental treatment with favipiravir, an influenza treatment that has shown efficacy against EBOV [19]. In this study, no randomization was performed, given the severity of the illness and desire to provide treatment to all patients suffering from EVD. Preliminary evidence from the study indicates that further investigation is warranted for favipiravir as a therapeutic option for patients with intermediate to high viremia, but it does not demonstrate utility when used as monotherapy for a patient with very high viremia associated with the late stages of disease [20]. It has also been shown that the concentrations of favipiravir in plasma did not hit the desired target during this trial, which may have influenced efficacy [21]. As with most studies discussed in this review, enrollment targets fell short of the anticipated goals due to the initiation of these studies occurring as the number of EBOV cases were in decline. With the ongoing outbreak of EBOV in the DRC, four investigational treatment options are being compared: ZMapp; mAb114, which is a single monoclonal antibody; REGNEB3, a cocktail of monoclonal antibodies; and Remdesivir [22].
Vaccination The WHO and many local governments and organizations supported the use of experimental vaccination in an attempt to interfere with disease transmission. The recombinant vesicular stomatitis virus (rVSV)-ZEBOV vaccine was chosen by WHO for trial. The vaccine is a live attenuated rVSV that expresses EBOV glycoprotein in place of the VSV glycoprotein. To evaluate the vaccine, a “ring vaccination” strategy was selected. Ring vaccination works on the basis of identifying EBOV-infected patients and then vaccinating their contacts and contacts of contacts. This was the strategy that was employed to help in the eradication of smallpox. During the initial trial, groups were randomized to either immediate vaccination or delayed vaccination administered 21 days later. Each group consisted of >2,000 individuals who met enrollment criteria. There were no cases of EVD in the immediately vaccinated group compared to 23 cases in the cluster of individuals who received delayed vaccination. The vaccine efficacy was determined to be 100% [23]. Concerns about this study and vaccine were the care differences between the groups
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and the vaccine stability outside of −70°C storage [24-26]. For vaccines to be successful in strategies of ring vaccination, they need to generate a rapid immune response. Trials of rVSV-ZEBOV and another glycoprotein-based vaccine, ChAd3-EBO-Z, have demonstrated robust immune response at 14 days with the peak antibody titer observed at 1 month [27]. Additional experimental vaccines have been developed, but at this time, rVSV-ZEBOV is the only vaccine in use in the DRC. There is concern that varied vaccination strategies among the vaccines may lead to confusion in the affected populations and also that other vaccine formulations do not generate a rapid enough immune response [28].
Further Considerations The diagnostic assays discussed above have shown promise for the detection of EBOV in whole blood and alternative specimens. No existing assays can detect EBOV prior to symptom onset, and many, like those described here, have decreased sensitivity when viremia is low, which occurs early in in the course of disease. As effective treatment will likely depend on initiation of therapy early in disease progression, continued development of assays with increased analytical sensitivity is warranted. A majority of the work to date has been done with EBOV, as that is the most predominant species and has been shown to have the highest associated mortality. However, it is probable that future outbreaks or sporadic cases of EVD will occur with other species, and it is unclear how most of the diagnostic, treatment, and management options would perform for individuals infected with other species of ebolavirus. Like most infectious diseases, EVD can produce a diagnostic conundrum. Many VHFs and endemic illnesses, such as malaria, typhoid, and cholera, may present with similar symptoms of vomiting and diarrhea with concurrent fever early in disease presentation. While the scientific community has made advances in the diagnosis of EBOV, it is important to remember that improvements in the diagnosis of other pathogens is important for outbreak response. It is also imperative to maintain the availability of these testing options. As has been discussed elsewhere, assays that were evaluated and previously approved for EUA are not readily available for the ongoing outbreak [29].
Acknowledgements I thank all of those in the scientific and health care communities who have worked tirelessly to control past and ongoing outbreaks. Highlighted here is just a small subset of the work that has been done to further this cause.
References [1] Johnson KM, Lange JV, Webb PA, Murphy FA. Isolation and partial characterisation of a new virus causing acute haemorrhagic fever in Zaire. Lancet 1977;1:569-71. [2] Centers for Disease Control and Prevention. Years of Ebola virus disease outbreaks. Available from: https://www.cdc.gov/vhf/ebola/ history/chronology.html [Accessed 30 August 2019].
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[3] World Health Organization. Ebola situation reports: Democratic Republic of the Congo. Available from: https://www.who.int/ebola/ situation-reports/drc-2018/en [Accessed 30 August 2019]. [4] Mari Saez A, Weiss S, Nowak K, Lapeyre V, Zimmermann F, Dux A, et al. Investigating the zoonotic origin of the West African Ebola epidemic. EMBO Mol Med 2015;7:17-23. [5] Broadhurst MJ, Kelly JD, Miller A, Semper A, Bailey D, Groppelli E, et al. ReEBOV Antigen Rapid Test kit for point-of-care and laboratory-based testing for Ebola virus disease: a field validation study. Lancet 2015;386:867-74. [6] Su S, Wong G, Qiu X, Kobinger G, Bi Y, Zhou J. Diagnostic strategies for Ebola virus detection. Lancet Infect Dis 2016;16:294-5. [7] World Health Organization. Emergency use assessment and listing for Ebola virus disease IVDs. Available from: https:// www.who.int/diagnostics_laboratory/160324_final_public_report_ ea_0023_021_00.pdf [Accessed 30 August 2019]. [8] Makiala S, Mukadi D, De Weggheleire A, Muramatsu S, Kato D, Inano K, et al. Clinical evaluation of QuickNavi(TM)-Ebola in the 2018 outbreak of Ebola virus disease in the Democratic Republic of the Congo. Viruses 2019;11. [9] Gay-Andrieu F, Magassouba N, Picot V, Phillips CL, Peyrefitte CN, Dacosta B, et al. Clinical evaluation of the BioFire FilmArray® BioThreat-E test for the diagnosis of Ebola virus disease in Guinea. J Clin Virol 2017;92:20-4. [10] Weller SA, Bailey D, Matthews S, Lumley S, Sweed A, Ready D, et al. Evaluation of the Biofire FilmArray BioThreat-E Test (v2.5) for rapid identification of Ebola virus disease in heat-treated blood samples obtained in Sierra Leone and the United Kingdom. J Clin Microbiol 2016;54:114-9. [11] Southern TR, Racsa LD, Albarino CG, Fey PD, Hinrichs SH, Murphy CN, et al. Comparison of FilmArray and Quantitative Real-Time Reverse Transcriptase PCR for detection of Zaire ebolavirus from contrived and clinical specimens. J Clin Microbiol 2015;53:2956-60. [12] Jansen van Vuren P, Grobbelaar A, Storm N, Conteh O, Konneh K, Kamara A, et al. Comparative evaluation of the diagnostic performance of the prototype Cepheid GeneXpert Ebola assay. J Clin Microbiol 2016;54:359-67. [13] Semper AE, Broadhurst MJ, Richards J, Foster GM, Simpson AJ, Logue CH, et al. Performance of the GeneXpert Ebola Assay for diagnosis of Ebola virus disease in Sierra Leone: a field evaluation study. PLoS Med 2016;13:e1001980. [14] United States National Library of Medicine. Clinical evaluation of the FilmArray® Global Fever (GF) Panel. Available from: https:// clinicaltrials.gov/ct2/show/NCT02968355. [Accessed 30 August 2019]. [15] Wonderly B, Jones S, Gatton ML, Barber J, Killip M, Hudson C, et al. Comparative performance of four rapid Ebola antigen-detection lateral flow immunoassays during the 2014-2016 Ebola epidemic in West Africa. PLoS One 2019;14:e0212113. [16] Van den Bergh R, Chaillet P, Sow MS, Amand M, van Vyve C, Jonckheere S, et al. Feasibility of Xpert Ebola Assay in Medecins Sans Frontieres Ebola program, Guinea. Emerg Infect Dis 2016;22:210-6. [17] PREVAIL II Writing Group, Multi-National PREVAIL II Study Team, Davey RT, Jr., Dodd L, Proschan MA, Neaton J, et al. A randomized, controlled trial of ZMapp for Ebola virus infection. N Engl J Med 2016;375:1448-56. [18] Qiu X, Wong G, Audet J, Bello A, Fernando L, Alimonti JB, et al. Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp. Nature 2014;514:47-53. [19] Oestereich L, Ludtke A, Wurr S, Rieger T, Munoz-Fontela C, Gunther S. Successful treatment of advanced Ebola virus infection with T-705 (favipiravir) in a small animal model. Antiviral Res 2014;105:17-21.
[20] Sissoko D, Laouenan C, Folkesson E, M’Lebing AB, Beavogui AH, Baize S, et al. Experimental treatment with favipiravir for Ebola virus disease (the JIKI Trial): a historically controlled, single-arm proofof-concept trial in Guinea. PLoS Med 2016;13:e1001967. [21] Nguyen TH, Guedj J, Anglaret X, Laouenan C, Madelain V, Taburet AM, et al. Favipiravir pharmacokinetics in Ebola-infected patients of the JIKI trial reveals concentrations lower than targeted. PLoS Negl Trop Dis 2017;11:e0005389. [22] United States National Library of Medicine. Investigational therapeutics for the treatment of people with Ebola virus disease. Available from: https://clinicaltrials.gov/ct2/show/NCT03719586 [Accessed 30 August 2019]. [23] Henao-Restrepo AM, Camacho A, Longini IM, Watson CH, Edmunds WJ, Egger M, et al. Efficacy and effectiveness of an rVSVvectored vaccine in preventing Ebola virus disease: final results from the Guinea ring vaccination, open-label, cluster-randomised trial (Ebola Ca Suffit!). Lancet 2017;389:505-18.
[24] Arnemo M, Viksmoen Watle SS, Schoultz KM, Vainio K, Norheim G, Moorthy V, et al. Stability of a vesicular stomatitis virus-vectored Ebola vaccine. J Infect Dis 2016;213:930-3. [25] Metzger WG, Vivas-Martinez S. Questionable efficacy of the rVSVZEBOV Ebola vaccine. Lancet 2018;391:1021. [26] Longini IM, Rottingen JA, Kieny MP, Edmunds WJ, HenaoRestrepo AM. Questionable efficacy of the rVSV-ZEBOV Ebola vaccine—authors’ reply. Lancet 2018;391:1021-2. [27] Kennedy SB, Bolay F, Kieh M, Grandits G, Badio M, Ballou R, et al. Phase 2 placebo-controlled trial of two vaccines to prevent Ebola in Liberia. N Engl J Med 2017;377:1438-47. [28] Winslow RL, Milligan ID, Voysey M, Luhn K, Shukarev G, Douoguih M, et al. Immune responses to novel adenovirus type 26 and modified vaccinia virus Ankara-vectored Ebola vaccines at 1 year. JAMA 2017;317:1075-7. [29] Cnops L, De Smet B, Mbala-Kingebeni P, van Griensven J, AhukaMundeke S, Arien KK. Where are the Ebola diagnostics from last time? Nature 2019;565:419-21.
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Vol. 41, No. 21 November 1, 2019 www.cmnewsletter.com I n Th is
Issu e
185 Recent Advances in the Diagnosis and Management of Ebola Virus Disease 189 An Unusual Case of Acute Invasive Pansinusitis Due to Laceyella sp. in a Febrile Neutropenic Patient with Myelodysplastic Syndrome A case report
Caitlin N. Murphy, Ph.D., University of Nebraska Medical Center, Department of Pathology and Microbiology, 985900 Nebraska Medical Center Omaha, NE 68198. Tel.: 402-552-3305. Email: [email protected] 0196-4399/©2019 Elsevier Inc. All rights reserved
Stay Current... Stay Informed.
t e r
Recent Advances in the Diagnosis and Management of Ebola Virus Disease Caitlin N. Murphy, Ph.D., Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska
Abstract Outbreaks of Ebola virus, a causative agent of hemorrhagic fever, in West Africa and the Democratic Republic of the Congo have highlighted the need for assays that can rapidly identify individuals infected with Ebola virus. These assays need to accurately detect and rule-out Ebola virus disease (EVD) and, ideally, not require advanced laboratory techniques. Over the course of the most recent outbreaks, field studies have evaluated the performance of rapid antigen and automated molecular tests, many of which were given emergency use authorization by the Food and Drug Administration. Overall, molecular assays demonstrated high sensitivity, while antigen testing had the highest sensitivity when the viral load was high. Specificity was high across the evaluated test options. In addition to the evaluation of diagnostic test methods, newly developed treatment options and the efficacy of ring vaccination to prevent disease transmission have been investigated. Findings from these field studies have demonstrated that many of the newly developed diagnostic assays, as well as experimental treatments and vaccination, are promising tools for controlling EVD.
Introduction
Corresponding author:
CMN
Ebolavirus was first identified as an etiological agent of hemorrhagic fever when an outbreak occurred in southern Sudan and northern Zaire in 1976 [1]. Since that time, there have been over 20 documented outbreaks that occurred predominantly in rural communities that were geographically isolated [2]. In West Africa, the largest Ebola outbreak occurred from 2014 to 2016, with over 20,000 cases and 11,000 deaths. The West African outbreak has been controlled, but a separate outbreak in the Democratic Republic of the Congo (DRC) continues into its second year. As of 6 August 2019, 2,781 cases and 1,866 deaths have been reported [3]. Prior to these recent outbreaks, the maximum death toll for any one outbreak was 280. As ebolavirus has moved into more urban, mobile communities, it has become more difficult to control the outbreaks. Another unfortunate barrier in the current DRC outbreak is the political instability that exists and has led to ongoing violence, making it very
difficult for humanitarian aid workers to control the outbreak. This unrest has led to distrust in the government and, by extension, the health care workers struggling to make inroads to stop the outbreak. In order to control the transmission of ebolavirus, appropriate standards for diagnosis, treatment, and prevention are needed. In the last 5 years, significant advances have been made in these areas, but the efficacy of experimental treatment and prevention strategies requires further study. This review focuses on the data that have been gathered during the most recent outbreaks. This work has provided valuable data and demonstrated the need for continued development of resources against emerging infections.
General Information Ebolaviruses are negative-stranded RNA viruses that belong to the family Filoviridae. To date, there are six known species of ebolaviruses. Zaire ebolavirus (EBOV) and Sudan ebolavirus are the
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185
most significant causes of human infection and have case fatality rates of 80 and 50%, respectively. Bundibugyo ebolavirus and Tai Forest ebolavirus are rarer causes of infections in humans. Finally, Reston ebolavirus and Bombali ebolavirus, are considered non-pathogenic to humans.
Transmission and Clinical Presentation EBOV is acquired when mucous membranes or abrasions on the skin come in contact with the blood or body fluids of infected individuals. Following acquisition, EBOV has a tropism for a wide variety of cell types, with early replication occurring primarily in dendritic cells, macrophages, and monocytes. Dissemination to the liver, lymph nodes, and spleen occurs as the infection progresses. As the virus disseminates, it disables the immune system and dysregulates the coagulation cascade, leading to organ failure.
field study investigating its efficacy was performed at two clinical sites in Sierra Leone. A total of 105 patients with suspected EVD were successfully tested with the ReEBOV test and a comparator molecular assay. Twenty-eight patients were positive by both the rapid antigen assay and a comparator real-time reverse transcription (RT)-PCR assay. The test had an overall sensitivity of 100% and specificity of 92% when two readers independently evaluated the results [5]. The ReEBOV assay requires only finger stick blood samples, but the test reagents require refrigeration and reconstitution of control materials, which may limit implementation in resource-limited settings. The ReEBOV assay has a high limit of detection that does not make it suitable for the detection of patients early in disease progression and is therefore not considered ideal for the management of suspect cases [6].
EBOV is one of the viral etiologies of viral hemorrhagic fever (VHF), but VHF can be caused by four different families of singlestranded RNA viruses. As these agents have a nonspecific presentation and high risk for nosocomial transmission, rapid diagnosis to facilitate cohorting is needed in outbreak settings. Ebola virus disease (EVD) has an incubation period that ranges from 2 to 21 days. At the time of symptom onset, patients are considered contagious. Symptoms at disease onset are initially nonspecific—fever, headache, and myalgia, followed by gastrointestinal symptom onset around day 3 to 5. The third stage of illness is either recovery or deterioration with progression to bleeding, neurological symptoms, and multiorgan failure.
The OraQuick Ebola Rapid Antigen Test (OraSure Technologies, Inc.) offers a rapid antigen test that also detects the EBOV VP40 antigen. It can be utilized to test whole blood from patients with suspected EVD or by using oral fluid from cadaveric specimens. Two hundred forty-four archived oral-fluid specimens were tested with the OraQuick and compared to the Xpert Ebola Test (described below) in a Sierra Leone field laboratory and performed with an overall sensitivity of 94.12% and specificity of 100% [7]. In addition, 75 whole-blood specimens were tested at the CDC and compared to an RT-PCR assay. The overall sensitivity and specificity were 84% and 98%, respectively. Of note, when the PCR-based assay had low threshold cycle (CT) ranges (15 to 24) (suggesting a high viral burden), the sensitivity was 100% [7].
Despite ongoing efforts to identify the animal reservoir, the modes of zoonotic transmission are not well understood, but based on current information, fruit bats are the suspected reservoir of EBOV. Evidence suggests that the West African outbreak was caused by a single transmission event to a 2-year-old boy in Guinea. It was shown that the child most likely acquired EBOV from exposure to Mops condylurus, an insectivorous free-tailed bat [4], suggesting the reservoir for EBOV might be wider than previously thought. Recognition of the outbreak was delayed, as EBOV had not previously been seen in West Africa, while Lassa fever, another VHF, is endemic to West Africa.
Two additional assays were approved by the WHO and/or FDA, the SD Q Line Ebola (SD BioSensor, Inc.) and the DPP Ebola Antigen (Chembio). To date, only the OraQuick assay has been available for use in the DRC outbreak. A new assay, the QuickNavi-Ebola, was evaluated using 928 whole-blood samples collected from patients with suspected EVD. The samples were tested by the Xpert Ebola assay as the comparator assay. The assay demonstrated a sensitivity of 85% and a specificity of 99.8%. As with the other lateral-flow assays described above, a majority of the false negatives (10/12) were in samples with CT values of >30 (suggesting a low viral burden) on the Xpert Ebola assay [8].
Laboratory Diagnosis
Molecular
Control of EBOV outbreaks requires a rapid response based on the recognition of cases early in the course of disease. The primary methodologies currently being investigated for the rapid and accurate diagnosis of EVD rely on detection of either circulating viral RNA or viral antigens. Serological options are available but are not considered useful for timely diagnosis of acute infections. Discussed below are diagnostic options that were granted emergency use authorization (EUA) by the World Health Organization (WHO) and/or the U.S. Food and Drug Administration (FDA).
RT-PCR is considered the gold standard for the diagnosis of EVD, as it is a highly sensitive and specific technique. A drawback of many RT-PCR assays for EBOV is that they can require manual extraction and technical expertise and can be subject to longer turnaround times than other options. Two automated RT-PCR assays that require minimal hands-on time were given EUA by the WHO and FDA.
Antigen During the West African outbreak, the WHO and FDA approved two lateral-flow immunoassays, ReEBOV and OraQuick. The ReEBOV Antigen Rapid Test kit (Corgenix, Inc.), which has since been delisted, detects the VP40 matrix protein of EBOV. A
186
Clinical Microbiology Newsletter 41:21,2019 | ©2019 Elsevier
The FilmArray system (BioFire Diagnostics and BioFire Defense) BioThreat-E (FA BT-E) test consists of a pouch that contains lyophilized reagents for sample extraction and RT-PCR. The user needs only to rehydrate the pouch and add the patient sample to an injection vial containing lysis reagents. The sample can then be loaded into the pouch. Results are then available within 90 minutes.
Studies evaluating the performance of the assay have been conducted in Sierra Leone, Guinea, the United Kingdom, and the United States. In all cases, the performance of the BT-E test was compared to that of an RT-PCR being utilized at the reference laboratory. A majority of testing was performed on whole blood, with the FA BT-E test demonstrating sensitivities ranging from 75 to 95.7% and specificities approaching 100% [9-11]. The study in Guinea took place over the course of 4 months in 2015. The study tested 135 total patients and compared the results of the FA BT-E test to those of two RT-PCR assays in routine use in the diagnostic laboratory. Forty-five of 47 patients with EVD confirmed by both alternative methods were detected, for an overall sensitivity of 95.7% and specificity of 100%. Preliminary findings with a small subset of alternative specimens (35 saliva and 3 urine specimens) also showed high agreement with comparator assays, indicating that testing may be successful using noninvasively collected specimens [9]. The GeneXpert (Cepheid, Sunnyvale, CA) also produces a sample-to-answer RT-PCR-based system designed for the detection of EBOV in whole blood and has the advantage of being a higher-throughput instrument commonly used in many areas for the detection of Mycobacterium tuberculosis. A study performed in South Africa utilizing 281 plasma and serum specimens collected from suspected EVD cases in 2014 and 2015 showed close to 100% sensitivity and specificity when the CT value of the comparator assay was low (£34.99) [12]. A second study using specimens from Sierra Leone demonstrated a sensitivity of 100% and specificity of 99.5% using whole-blood specimens and an alternative comparator assay [13]. During the same study, evaluations of the Xpert Ebola assay for the detection of EBOV in semen and buccal swabs has also shown promise for follow-up testing or testing using less invasively collected specimens. Both assays require minimal specimen handling, and result interpretation is performed by the instrument. A potential advantage of the FA BT-E test is that BioFire assays are often multiplexed and can recognize other agents of VHF or other sequelae that can mimic VHF early in the course of disease. Clinical trials are ongoing to examine the performance of a multi-target panel [14]. In the United States, a confirmed case of EVD still requires confirmation at the CDC, and results of tests with FDA EUA status are considered to be only presumptive. A challenge of comparing the evaluations done in the field on both lateral-flow immunoassays and automated RT-PCR assays discussed in this review is that they were performed in geographically disparate locations with variable study populations and comparator assays. These publications have also expressed concerns that logistical considerations or biosafety issues may limit their utility [15,16].
Treatment ZMapp is an experimental treatment option for EVD. ZMapp is a combination of three monoclonal antibodies that target the surface glycoprotein of EBOV. A randomized trial comparing ZMapp
plus the existing standard of care to the standard of care alone was performed with 72 patients across Liberia, Sierra Leone, Guinea, and the United States. ZMapp was administered by intravenous infusion once every 3 days over the course of 9 days. In the ZMapp group the overall mortality rate was 15% lower than with the standard of care alone, and the posterior probability of ZMapp being superior to the standard of care alone was 91.2% [17]. Most of the deaths in the ZMapp group (7/8) occurred before the patients were able to receive the second infusion. Another factor influencing the efficacy of this treatment during thetrial was the delay from symptom onset to treatment. Many treated patients were in more advanced stages of disease and were therefore more likely to have a poor outcome. Studies have shown in nonhuman primates that therapy initiated in a 5-day window after symptom onset had a ≥90% survival rate [18]. Patients in Guinea were also provided experimental treatment with favipiravir, an influenza treatment that has shown efficacy against EBOV [19]. In this study, no randomization was performed, given the severity of the illness and desire to provide treatment to all patients suffering from EVD. Preliminary evidence from the study indicates that further investigation is warranted for favipiravir as a therapeutic option for patients with intermediate to high viremia, but it does not demonstrate utility when used as monotherapy for a patient with very high viremia associated with the late stages of disease [20]. It has also been shown that the concentrations of favipiravir in plasma did not hit the desired target during this trial, which may have influenced efficacy [21]. As with most studies discussed in this review, enrollment targets fell short of the anticipated goals due to the initiation of these studies occurring as the number of EBOV cases were in decline. With the ongoing outbreak of EBOV in the DRC, four investigational treatment options are being compared: ZMapp; mAb114, which is a single monoclonal antibody; REGNEB3, a cocktail of monoclonal antibodies; and Remdesivir [22].
Vaccination The WHO and many local governments and organizations supported the use of experimental vaccination in an attempt to interfere with disease transmission. The recombinant vesicular stomatitis virus (rVSV)-ZEBOV vaccine was chosen by WHO for trial. The vaccine is a live attenuated rVSV that expresses EBOV glycoprotein in place of the VSV glycoprotein. To evaluate the vaccine, a “ring vaccination” strategy was selected. Ring vaccination works on the basis of identifying EBOV-infected patients and then vaccinating their contacts and contacts of contacts. This was the strategy that was employed to help in the eradication of smallpox. During the initial trial, groups were randomized to either immediate vaccination or delayed vaccination administered 21 days later. Each group consisted of >2,000 individuals who met enrollment criteria. There were no cases of EVD in the immediately vaccinated group compared to 23 cases in the cluster of individuals who received delayed vaccination. The vaccine efficacy was determined to be 100% [23]. Concerns about this study and vaccine were the care differences between the groups
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and the vaccine stability outside of −70°C storage [24-26]. For vaccines to be successful in strategies of ring vaccination, they need to generate a rapid immune response. Trials of rVSV-ZEBOV and another glycoprotein-based vaccine, ChAd3-EBO-Z, have demonstrated robust immune response at 14 days with the peak antibody titer observed at 1 month [27]. Additional experimental vaccines have been developed, but at this time, rVSV-ZEBOV is the only vaccine in use in the DRC. There is concern that varied vaccination strategies among the vaccines may lead to confusion in the affected populations and also that other vaccine formulations do not generate a rapid enough immune response [28].
Further Considerations The diagnostic assays discussed above have shown promise for the detection of EBOV in whole blood and alternative specimens. No existing assays can detect EBOV prior to symptom onset, and many, like those described here, have decreased sensitivity when viremia is low, which occurs early in in the course of disease. As effective treatment will likely depend on initiation of therapy early in disease progression, continued development of assays with increased analytical sensitivity is warranted. A majority of the work to date has been done with EBOV, as that is the most predominant species and has been shown to have the highest associated mortality. However, it is probable that future outbreaks or sporadic cases of EVD will occur with other species, and it is unclear how most of the diagnostic, treatment, and management options would perform for individuals infected with other species of ebolavirus. Like most infectious diseases, EVD can produce a diagnostic conundrum. Many VHFs and endemic illnesses, such as malaria, typhoid, and cholera, may present with similar symptoms of vomiting and diarrhea with concurrent fever early in disease presentation. While the scientific community has made advances in the diagnosis of EBOV, it is important to remember that improvements in the diagnosis of other pathogens is important for outbreak response. It is also imperative to maintain the availability of these testing options. As has been discussed elsewhere, assays that were evaluated and previously approved for EUA are not readily available for the ongoing outbreak [29].
Acknowledgements I thank all of those in the scientific and health care communities who have worked tirelessly to control past and ongoing outbreaks. Highlighted here is just a small subset of the work that has been done to further this cause.
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