Ebola Outbreak 2018: Diagnostics Again are Essential to Minimize Spread and to Control Disease

On April 4^th, 2018, a new outbreak of Ebola Virus Disease (EVD) occurred in Equateur Province in the Democratic Republic of the Congo. As of June 10^th, there have been a total of 55 EVD cases and 28 deaths with a case fatality rate of 50.9%. Although the outbreak remains active, public health authorities have expressed cautious optimism because there have been no new cases in two of the three affected areas (Bikoro and Wangata zones) since May 17^th, 2018 and the rate of new cases in the third affected zone (Iboko) has slowed.¹

There are many new tools to fight the spread of EVD that were not available in the massive 2014-2016 outbreak in Guinea, Liberia and Sierra Leone. While it is too early to claim success in controlling the current outbreak, these tools appear to be having a positive effect. 

• First, experimental therapeutics are available for the first time during an Ebola outbreak. Four investigational therapeutics are in-country and protocols for their use have been approved by appropriate ethics review boards. These drugs are Zmapp, GS-5734, REGN monoclonal antibody combination, and mAb114. GS-5734 is a small molecule directed against the Ebola RNA-dependent RNA polymerase L required for viral replication.² Zmapp, REGN monoclonal antibody combination and mAb114 are immunotherapeutics that bind to the virus and prevent entry.²
• Second, Merck’s rVSV-ZEBOV vaccine is now available and its safety and efficacy has been supported in an open-label, cluster-randomized ring vaccination trial.³ rVSV-ZEBOV is a recombinant, replication competent vesicular stomatitis virus-based vaccine expressing a surface glycoprotein of Ebolavirus. The vaccination program in the Democratic Republic of the Congo started on May 21^st, 2018. So far, a total of 2,295 people have been vaccinated including front-line health professionals, people who have been exposed to confirmed EVD cases and contacts of these contacts. As reported in the NEJM, vaccination is an early access program for compassionate care, with the intent of patient treatment, but protocols are also in place to conduct research alongside the response and gather more data on safety and effectiveness.⁴ 
• Finally, and possibly most importantly, rapid, commercially manufactured Ebola virus molecular diagnostics are more widely available. These tests detect viral RNA by real-time reverse transcription polymerase chain reaction (RT-PCR). During the 2014 – 2016 outbreak, the US FDA gave authorization of emergency use for Ebola virus RT-PCR assays from a number of manufacturers including Roche, BioFire and Cepheid. In the current outbreak, three mobile laboratories have been deployed in the Democratic Republic of the Congo and all became operational in May using predominantly the Cepheid GeneXpert test system.¹ Robust diagnostic testing is essential for controlling the outbreak because it detects EVD cases in new clusters, confirms or excludes suspected cases for effective contact tracing and surveillance, monitors patients in treatment centers, and informs decisions on whether recovered patients are safe for discharge. Without diagnostic testing, vaccination programs would be less effective because it would be unclear which contacts should receive vaccination; without the insights into viral load provided by testing, it would not be possible to monitor patient response to selected therapeutics.

One of the lessons learned from the experiences of mobile testing laboratories during the 2014 – 2016 outbreak was that molecular diagnostic test systems require quality assurance.⁵ Authors from the World Health Organization EVD response team, Liberia Health ministry, and US CDC EVD response team identified lack of External Quality Assurance (EQA) materials as critical need, writing that “EQA panels were not available for proficiency testing during the outbreak and development of low cost panels should be considered for future epidemics.”⁵ In addition to the lack of validation, training, and quality control materials in the labs, developers of diagnostic assays also did not have access to the disease state materials they needed for assay development.

SeraCare responded to the crisis by rapidly developing and manufacturing AccuPlex recombinant Ebola Reference Material in early 2015. AccuPlex RNA controls use Sindbis virus, an RNA-containing enveloped virus which can be engineered to contain target sequences for another virus or to contain a unique custom internal control sequence specific to the assay. The recombinant Sindbis virus system results in viral particles that are fully encapsulated like the real pathogenic virus in patient’s sample, so the product must go through an extraction process before nucleic acid detection, but the viral particles are replication defective for safer handling.

Figure 1: Schematic representation of AccuPlex technology used in the production of recombinant Sindbis virus particles bearing Ebola target sequences.

Recombinant Sindbis constructs were designed bearing either ~2,000-bp of Ebola Zaire glycoprotein gene (GP) sequence or bearing ~1,500-bp of nucleoprotein gene (NP) sequence. The RNA constructs were introduced into mammalian tissue culture cells, and the recombinant viruses were collected from the culture supernatant. Although they are not replication competent, the recombinant virus particles were heat treated as an added safety precaution. The AccuPlex recombinant Ebola viruses were diluted into defibrinated human plasma and shown to be compatible with the Cepheid® Xpert Ebola Assay as well as with the other EUA assays that target the GP and NP genes for detection. AccuPlex Ebola material, which is US-IVD and CE marked, was used by assay developers to speed up development and authorization of Emergency Use of their diagnostic assays and was also formulated into training panels for use in testing labs. Results from three independently manufactured lots, tested on the Cepheid Xpert are shown in Table 1 and indicate the consistency of the quality control material. 

Table 1:  AccuPlex rEbola GP/NP Reference Material tested on a Cepheid® Xpert Ebola Assay.  Ct is the Cycle threshold value. SAC is the sample adequacy control which verifies human source DNA is in the sample.

Stability of reference materials is critical, especially if they are to be useful to mobile labs deployed near outbreak sites. One of the advantages of the AccuPlex recombinant virus technology is that it produces virus particles that are highly stable at elevated temperatures. AccuPlex Recombinant Ebola Reference Material was stressed through storage at 37 °C to accelerate any degradation of the product. At designated time points, vials were removed from the stress condition, viral RNA was extracted, and testing was performed via a laboratory developed TaqMan quantitative real time PCR assay. There was no loss of signal across 22 days of 37 °C stress indicating that the reference material is appropriate for use in situations where cold chain logistics are not guaranteed.

Figure 2:  Results of TaqMan real time quantitation of AccuPlex Ebola samples unstressed (time = 0) or stressed for 1, 3, 5, 11 or 22 days at 37 ˚C. Test results shown are means value of triplicates and error bars as standard deviations. Shaded band is 95% confidence intervals.

Beyond Ebola, new or re-emerging viruses pose a public health threat and outbreaks lead to tragic loss of human life as well as economic hardship to the communities affected. For example, in May 2018, a Nipah virus disease (NiV) outbreak was reported in Kerala, India and caused 17 deaths. To control outbreaks, WHO recommends the implementation of proven strategies for prevention and control including enhanced surveillance activities such as active case finding, case investigation, and contact tracing.  However, surveillance is not effective without rapid, robust molecular diagnostics to quickly confirm or exclude cases. How can AccuPlex reference materials help ensure laboratory preparedness for the next viral pathogen outbreak? AccuPlex reference materials have optimized and standardized manufacturing processes, which allow target sequences for new pathogens to be quickly incorporated into new reference materials. Recombinant virus technology allows rapid modulation of the sequence contained in reference materials to match outbreak strains and the non-replicative recombinant viruses are non-infectious. The formulation of the recombinant viruses in biological matrices, such as plasma, means that these materials undergo the same extraction challenges as the unknown specimens, and so are able to monitor the full testing process. The stability of the materials at elevated temperatures make these reference materials ideal for training and validation activities. SeraCare has generated pilot reference materials for emerging threats such as Zika virus, Lassa Virus, Marburg Virus, SARS-CoV, and Chikungunya and is collaborating with assay developers to use these materials in the development of future diagnostics.

References:

1. Ebola Virus Disease Democratic Republic of the Congo, External Situation Report 10.  Date of issue: 12 June 2018.  Published by WHO Health Emergency Program.
2. Bixler SL,  Duplantier AJ, and Bavari Discovering Drugs for the Treatment of Ebola Virus. Curr Treat Options Infect Dis. 2017; 9(3): 299–317.
3. Henao-Restrepo AM et al. Efficacy and effectiveness of an rVSV-vectored vaccine in preventing Ebola virus disease: final results from the Guinea ring vaccination, open-label, cluster-randomised trial. The Lancet. 2017; 389(10068): 505-518.
4. Haug, CJ. Keeping Your Cool — Doing Ebola Research during an Emergency. N Engl J Med 2018; 378:2353-2355.
5. Raftery P, Condell O, Wasunna C, Kpaka J, Zwizwai R, Nuha M, et al. (2018) Establishing Ebola Virus Disease (EVD) diagnostics using GeneXpert technology at a mobile laboratory in Liberia: Impact on outbreak response, case management and laboratory systems strengthening. PLoS Negl Trop Dis 12(1): e0006135. https://doi.org/10.1371/journal. pntd.0006135

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AccuPlex^TM Technology for Rapid Development of Molecular Diagnostic Assays in Response to Viral Infectious Diseases

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• Why AccuPlex reference materials can serve as full‑process controls, positive controls, and internal controls
• How AccuPlex reference materials were deployed to fight the 2014 Ebola crisis and today's Zika outbreak
• What enables AccuPlex recombinant virus to accommodate large target sequence for highly multiplexed assays while remaining safe to handle