Performance of the GeneXpert Ebola Assay for Diagnosis of Ebola Virus Disease in Sierra Leone: A Field Evaluation Study

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Abstract

Background

Throughout the Ebola virus disease (EVD) epidemic in West Africa, field laboratory testing for EVD has relied on complex, multi-step real-time reverse transcription PCR (RT-PCR) assays; an accurate sample-to-answer RT-PCR test would reduce time to results and potentially increase access to testing. We evaluated the performance of the Cepheid GeneXpert Ebola assay on clinical venipuncture whole blood (WB) and buccal swab (BS) specimens submitted to a field biocontainment laboratory in Sierra Leone for routine EVD testing by RT-PCR (“Trombley assay”).

Methods and Findings

This study was conducted in the Public Health England EVD diagnostic laboratory in Port Loko, Sierra Leone, using residual diagnostic specimens remaining after clinical testing. EDTA-WB specimens ( n = 218) were collected from suspected or confirmed EVD patients between April 1 and July 20, 2015. BS specimens ( n = 71) were collected as part of a national postmortem screening program between March 7 and July 20, 2015. EDTA-WB and BS specimens were tested with Xpert (targets: glycoprotein [GP] and nucleoprotein [NP] genes) and Trombley (target: NP gene) assays in parallel. All WB specimens were fresh; 84/218 were tested in duplicate on Xpert to compare WB sampling methods (pipette versus swab); 43/71 BS specimens had been previously frozen.

In all, 7/218 (3.2%) WB and 7/71 (9.9%) BS samples had Xpert results that were reported as “invalid” or “error” and were excluded, leaving 211 WB and 64 BS samples with valid Trombley and Xpert results. For WB, 22/22 Trombley-positive samples were Xpert-positive (sensitivity 100%, 95% CI 84.6%–100%), and 181/189 Trombley-negative samples were Xpert-negative (specificity 95.8%, 95% confidence interval (CI) 91.8%–98.2%). Seven of the eight Trombley-negative, Xpert-positive (Xpert cycle threshold [Ct] range 37.7–43.4) WB samples were confirmed to be follow-up submissions from previously Trombley-positive EVD patients, suggesting a revised Xpert specificity of 99.5% (95% CI 97.0%–100%). For Xpert-positive WB samples ( n = 22), Xpert NP Ct values were consistently lower than GP Ct values (mean difference −4.06, 95% limits of agreement −6.09, −2.03); Trombley (NP) Ct values closely matched Xpert NP Ct values (mean difference −0.04, 95% limits of agreement −2.93, 2.84). Xpert results (positive/negative) for WB sampled by pipette versus swab were concordant for 78/79 (98.7%) WB samples, with comparable Ct values for positive results. For BS specimens, 20/20 Trombley-positive samples were Xpert-positive (sensitivity 100%, 95% CI 83.2%–100%), and 44/44 Trombley-negative samples were Xpert-negative (specificity 100%, 95% CI 92.0%–100%). This study was limited to testing residual diagnostic samples, some of which had been frozen before use; it was not possible to test the performance of the Xpert Ebola assay at point of care.

Conclusions

The Xpert Ebola assay had excellent performance compared to an established RT-PCR benchmark on WB and BS samples in a field laboratory setting. Future studies should evaluate feasibility and performance outside of a biocontainment laboratory setting to facilitate expanded access to testing.

Abstract

Nira Pollock and colleagues evaluate the performance of the GeneXpert Ebola assay for diagnosis of clinical and post-mortem specimens submitted to a field laboratory in Sierra Leone.

Editors' Summary

Background

During the recent Ebola virus disease (EVD) outbreak in West Africa, there were more than 28,000 confirmed, probable, and suspected cases of EVD and more than 11,000 deaths from EVD. Ebola virus is transmitted to people from wild animals and spreads in human populations through contact with the bodily fluids (including blood, saliva, and urine) or organs of infected people and through contact with bedding and other materials contaminated with bodily fluids. The symptoms of EVD, which start 2–21 days after infection, include fever, headache, vomiting, diarrhea, and internal and external bleeding. Infected individuals are not infectious until they develop symptoms, but they remain infectious as long as their bodily fluids contain virus, which can be several weeks. There is no proven treatment for EVD, but supportive care—given under strict isolation conditions to prevent the spread of the disease—improves survival. Currently, there are no licensed Ebola vaccines, but two candidate vaccines are being evaluated.

Why Was This Study Done?

EVD diagnosis during the recent epidemic relied on multi-step reverse transcription polymerase chain reaction (RT-PCR) assays (for example, the Trombley assay) performed in field biocontainment laboratories on blood obtained from a vein using a needle (venipuncture) or on samples of cells and saliva collected from the mouth lining using a swab (buccal swab); buccal swabs are mainly used for surveillance, particularly postmortem screening. Prior to RT-PCR, the sample must be inactivated and nucleic acid extracted. An accurate, fully automated “sample-to-answer” assay that is capable of testing both whole blood and buccal swabs and that minimizes the potential exposure of laboratory personnel to the Ebola virus could greatly improve EVD diagnosis. Here, the researchers evaluate the performance of the Cepheid GeneXpert Ebola assay on whole blood samples and buccal swabs sent to a field laboratory in Sierra Leone for routine testing using the Trombley assay. The Xpert assay is an automated RT-PCR system that integrates all the steps needed to determine the presence of Ebola virus; once the test sample has been inactivated and added to a proprietary cartridge, no further operator action is necessary to generate the result.

What Did the Researchers Do and Find?

The researchers tested 218 whole blood specimens collected from patients with suspected or confirmed EVD using both the Trombley and the Xpert assay. After excluding a few samples that gave Xpert results that were reported as “invalid” or “error,” 22 out of 22 Trombley-positive samples were Xpert-positive, and 181 out of 189 Trombley-negative samples were Xpert-negative. That is, the Xpert assay had a sensitivity (true positive rate) of 100% and a specificity (true negative rate) of 95.8% compared to the benchmark assay. Notably, seven of the eight Trombley-negative but Xpert-positive blood samples were follow-up samples obtained from previously Trombley-positive patients, which suggests that the specificity of the Xpert test was actually 99.5%. When the Xpert assay was used to test 71 buccal swabs, the sensitivity and specificity of the Xpert assay were both 100%. Finally, Xpert results obtained using a pipette versus a swab to pick up a portion of blood for testing were concordant in 78 out of 79 samples; this test was done in part to get an indication of whether the Xpert Ebola assay will work on fingerstick samples—blood samples obtained by using a sterile lancet to pierce the fingertip and then collecting the blood with a swab; this collection method is more easily done in the field at point of care than venipuncture.

What Do These Findings Mean?

These findings show that, compared to an established RT-PCR Ebola virus assay, the Xpert Ebola assay performed well on both whole blood samples and buccal swabs in a field laboratory setting. Although sampling of blood with a swab partly simulated the performance of the Xpert assay on fingerstick samples collected at point of care, fingerstick sample collection will need to be tested directly before the Xpert assay can be used to test individuals for Ebola virus by this method at point of care. Further studies are also needed to evaluate the feasibility and performance of the Xpert assay in a range of clinical settings to determine where and when this assay can be deployed; the need for an uninterrupted power supply and, in some settings, for refrigeration of reagents may prevent its deployment in some resource-limited settings. Ultimately though, these findings suggest that the use of the Xpert Ebola assay could facilitate expanded access to Ebola virus testing.

Additional Information

This list of resources contains links that can be accessed when viewing the PDF on a device or via the online version of the article at http://dx.doi.org/10.1371/journal.pmed.1001980.

The World Health Organization (WHO) provides information about EVD, information about potential EVD vaccines and therapies, and regular updates on the West African EVD epidemic; the WHO website also provides information about efforts to control Ebola in the field and personal stories from people who have survived EVD
The UK National Health Service Choices website provides detailed information on EVD
The US Centers for Disease Control and Prevention also provides information about EVD
More information on the Xpert Ebola test and authorization for its emergency use is available from the US Food and Drug Administration and from WHO

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Most cited references 11

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Measuring agreement in method comparison studies.

Elliot Bland, Michael Altman (1999)

Agreement between two methods of clinical measurement can be quantified using the differences between observations made using the two methods on the same subjects. The 95% limits of agreement, estimated by mean difference +/- 1.96 standard deviation of the differences, provide an interval within which 95% of differences between measurements by the two methods are expected to lie. We describe how graphical methods can be used to investigate the assumptions of the method and we also give confidence intervals. We extend the basic approach to data where there is a relationship between difference and magnitude, both with a simple logarithmic transformation approach and a new, more general, regression approach. We discuss the importance of the repeatability of each method separately and compare an estimate of this to the limits of agreement. We extend the limits of agreement approach to data with repeated measurements, proposing new estimates for equal numbers of replicates by each method on each subject, for unequal numbers of replicates, and for replicated data collected in pairs, where the underlying value of the quantity being measured is changing. Finally, we describe a nonparametric approach to comparing methods.

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ReEBOV Antigen Rapid Test kit for point-of-care and laboratory-based testing for Ebola virus disease: a field validation study

Mara Jana Broadhurst, John Daniel Kelly, Ann Miller … (2015)

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.

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Is Open Access

The Case for Improved Diagnostic Tools to Control Ebola Virus Disease in West Africa and How to Get There

Arlene C. Chua, Jane Cunningham, Francis Moussy … (2015)

The Ebola virus disease (EVD) outbreak in West Africa, with the widest and most intense transmission occurring in Guinea, Liberia, and Sierra Leone, has claimed more than 8,000 lives since it began in December 2013. It has had a massive impact on already fragile health systems and now threatens food security. The latest World Health Organization (WHO) report has shown that there has been a decrease in the cases in January 2015 [1]. Since the identification of Ebola in Guinea in March 2013, rapid deployment of international mobile laboratories through WHO networks—Global Outbreak Alert and Response Network (GOARN) [2] and Emerging and Dangerous Pathogens Laboratory Network (EDPLN) [3]—has been vital to outbreak control operations. Deployable laboratories from multiple international organizations have been established near Ebola treatment centers (ETC) in Guinea, Liberia, and Sierra Leone. The organizations providing laboratories are China Centers for Disease Control Lab, European Union Mobile Laboratory Consortium (EM Lab), Institute Pasteur Dakar, Institute Pasteur Lyon, Institute Pasteur Paris, Institut National de Recherche Bio-Médicale Mobile lab in Democratic Republic of Congo, National Institute for Communicable Diseases in South Africa, Public Health England Mobile lab, Public Health Canada Mobile Lab, Russian Rospotrebnadzor Mobile Lab, United States Centers for Disease Control (CDC), US First Area Medical Laboratory, US National Institutes of Health, and US Naval Medical Research Center Mobile Lab. The primary function of these laboratories has been to confirm disease in patients with suggestive symptoms in order to trigger isolation and contact tracing and to document cure and/or non-infectiousness in survivors. Current diagnostic testing is performed exclusively using reverse transcription polymerase chain reaction (RT-PCR) on RNA extracted from venous blood samples collected by personnel wearing full personal protective equipment. In the absence of simpler methods, diagnosis of EVD in this outbreak has, until now, relied exclusively on testing conducted in these internationally run, mobile laboratories. However, several technical and social factors conspire to delay diagnosis, starting with weak surveillance systems and slow patient access to centralized ETCs. While the mean processing time is 5 hours (time difference from when samples are received in the laboratory to when they are tested), there is a marked difference in the time from when the samples are collected from suspected patients to the time they are received by the laboratory. That time difference varies between 1 to 32 days, with a mean of 1.5, 1.8, and 2.1 days for Guinea, Liberia, and Sierra Leone, respectively. (unpublished, Pierre Formenty, WHO). There is a need to ameliorate transport of samples to the laboratories. Without the possibility of rapid, local confirmed diagnosis, patients are often reluctant to go to testing sites because of fear of contracting the disease during involuntary delays. Partly because of these delays, by the time of diagnosis the majority of patients with confirmed EVD have been symptomatic (and infectious) for 5–6 days [4]. Recent analysis by WHO of data collected from March 2014 to January 2015 from the three most affected countries found that the time difference between the date of onset of symptoms and the date of sample collection from suspected patients varies between 1 and 21 days, with a mean of 7.2 days. (Unpublished, Pierre Formenty, WHO). The lack of proven interventions (treatment and vaccines) makes early identification and isolated management of EVD cases with rapid and accurate diagnostics even more fundamental to interrupting disease transmission and bringing the outbreak under control [5]. Early detection and care is critical for transmission interruption, initiation of contact tracing, and accurate epidemiological surveillance and has been associated with improved prognosis. What strategies can lead to safe and faster diagnosis? Though, clearly, cases are clustered—as of October 14, 90% of cases were reported from 14 districts [6]—new foci of transmission constantly emerge, and the changing distribution of cases suggests that it will be unwise to rely on a limited number of high-throughput laboratories and that more decentralized testing could decrease delays in notification. Delays described above are serious, as multiple studies have shown that in the first 2 days of symptomatology, some patients may have very high loads of circulating virus and thus are prone to spread EVD in the family and community [4,7]. The current centralized diagnostic testing strategy is adequate for case confirmation and proof of cure. But given the EVD geographic spread, the loss of many chains of transmission and the high number of unidentified cases, the current strategy might not be adequate for providing the kind of rapid and flexible response that would help stop Ebola transmission. Certainly, we should continue with current surveillance strategy of active identification of cases and contact tracing. But in addition, we should establish in non-Ebola health care facilities a complementary surveillance system using safely administered point-of-care diagnostic tests to screen patients for non-Ebola infections and to accelerate detection of hidden and unknown chains of Ebola transmission. Indeed, the majority of fevers and other non-specific symptoms seen in the outbreak area are not caused by EVD and may be due to one of various other infections endemic in the region, including malaria, typhoid fever, shigellosis, cholera, leptospirosis, rickettsiosis, relapsing fever, meningitis, hepatitis, and other viral hemorrhagic fevers. Among patients with EVD, co-infections with malaria are not uncommon (internal Medécins Sans Frontières report: Epi Bulletin Ebola Epidemic in West Africa). Having a point-of-care test could facilitate the safe reopening of non-Ebola health facilities that have been closed because of the outbreak. In the last quarter of 2014 in Liberia, 62% of health facilities were closed. In the three countries heavily affected by EVD, there has been a significant drop in outpatient visits, institutional deliveries and childhood immunizations [8]. It is important that health care workers dealing with patients outside ETCs have the capacity to exclude EVD without being exposed to additional risks of acquiring EVD. More than 800 health care workers have reportedly been infected with EVD, and initial analysis shows that a substantial proportion of infections occurred outside the context of Ebola treatment and care centers [1,9]. Although the exact proportion is unknown, the frequency of reports of health care workers becoming infected through their work in routine medical practice outside ETCs is a telling commentary on the need to couple strengthening of infection prevention practices and extension of Ebola diagnostic testing more broadly into district or local health centers. Rapid, field-adapted point-of-contact/care tests that are highly predictive of EVD and do not carry extensive biosafety requirements could drastically improve detection of EVD patients through decentralized diagnostic testing. Unfortunately, there is currently no commercially available test with stringent regulatory approval that will meet the needs of most decentralized testing centers. However, this is primarily a financial and logistical problem, not a technical one. For other diseases such as HIV, tuberculosis, and malaria, diagnostic assays with adequate performance based on detection of antigens or nucleic acid sequences have been developed and successfully introduced, with proper shepherding from the public sector [10–12]. A number of semi-portable molecular amplification tests platforms that integrate sample processing and greatly simplify PCR procedures have been designed and developed in recent years, including for diseases prevalent in resource-limited settings. Such devices can potentially be used for EVD detection, if adapted to the appropriate biosafety requirements. Alternatively, antigen detection, which has worked relatively well—albeit in a cumbersome ELISA format, could form the basis for a rapid diagnostic test (RDT) that, if sensitive and specific enough, could be valuable. The type of sample to be used (e.g., blood versus saliva) and the sample collection requirements (finger prick versus phlebotomy) will be key features for the tests to be simple enough for use in disseminated, low-resource locations. In recognition of the urgent need for improved Ebola diagnostics, and to guide diagnostic research and development (R&D), a consensus target product profile (TPP) has been developed by the authors and other key partners (Table 1). The intended use of the profiled product is to distinguish symptomatic patients with acute EVD infection from those with non-Ebola virus infection. The TPP outlines two sets of test characteristics: desired (ideal) and acceptable, both of which would allow for varying degrees of decentralized EVD testing through reduced requirements for laboratory infrastructure and for technical expertise in sample collection and/or running the assay. However, in both cases, the requirement for very high test sensitivity and specificity is maintained, due to the serious individual and public health impacts of both false-positive and false-negative EVD results. 10.1371/journal.pntd.0003734.t001 Table 1 Target product profile for Zaire ebolavirus: rapid, simple test to be used in the control of the Ebola outbreak in West Africa. Key Features Desired Acceptable Priority Features Target population Patients presenting with fever to health care facilities for assessment. Target use setting Decentralized health care facilities with no laboratoryinfrastructure available Decentralized health care facilities with minimum laboratory infrastructure available Intended use In Ebola outbreak setting, distinguish between symptomatic patients with acute Ebola virus infection and non-Ebola virus infection without the need for confirmatory testing In Ebola outbreak setting, distinguish between symptomatic patients with acute Ebola virus infection and non-Ebola virus infection with the need for confirmatory testing Clinical sensitivity a , b >98% >95% Analytical specificity >99% >99% Type of analysis Qualitative or Quantitative Qualitative Sample type Capillary whole blood from finger stick once/if the use of this type of samples has been validated Whole blood from phlebotomy, in particular if collection is simple and automated to reduce biosafety requirements Other, less invasive sample types (e.g., saliva, buccal) once/if their use has also been validated TEST PROCEDURE Number of steps to be performed by operator 1 hour for single use test after opening the pouch >½ hour for single use test after opening the pouch Reagents reconstitution All reagents ready to use Reconstitution acceptable if very simple to do. Need to prepare the reagents prior utilization All liquids, including water, already in kit Training needs Less than half a day for any level health care worker. Job aid provided Less than 2 days for any level of health care worker. Job aid provided Time dedicated to training session for end users Equipment (if needed) Small and portable, handheld instrument Small, table-top device, portable Weight <2 kg Power requirements None required 110–220 V AC current Optional: 110–220 V AC current DC power with rechargeable battery lasting up to 8 hours of testing DC power with rechargeable battery lasting up to 8 hours of testing Need for maintenance/spare parts None 1 annual calibration ideally by operator a Clinical sensitivity in first 10 days of presentation. Allow for repeat testing as per WHO guidelines. b Reference test: Lab-validated quantitative PCR assay on blood sample (whole blood or plasma) drawn by phlebotomy. c Biosafety resources for Ebola: http://www.who.int/csr/disease/ebola/en/; http://www.who.int/csr/resources/publications/ebola/filovirus_infection_control/en/ Concurrently, WHO has also launched an emergency assessment, quality assurance mechanism for EVD diagnostics to inform procurement. Rapidly developing and providing access to diagnostic tests aligned with the proposed TPP will require that research and development move at an accelerated pace. Continued coordination from WHO will be needed in order to achieve this goal, including such actions as: Developing consensus target product profiles to inform diagnostic developers about the specific needs Creating and distributing RNA and/or protein standards for early R&D support and analytical testing Creating and coordinating a virtual specimen bank network, to ensure equitable access to clinical and virological materials for assay development and evaluation Establishing ethical and pragmatic standards for rapid assay endorsement Conducting laboratory studies to assess analytical performance Conducting field studies to demonstrate clinical performance and feasibility of implementation in Ebola settings Deploying teams to support training of local health worker teams to safely use the tests in the early implementation phase There are no good technological reasons why rapid diagnostics for use in decentralized locations cannot be developed to help stop this outbreak. To achieve this goal, the field needs a rapid mobilization of resources and coordination of work to accelerate the development and validation of these urgently needed tools, as well as equitable access. To help achieve this, WHO is providing this coordinating role in partnership with Foundation for Innovative New Diagnostics (FIND), Médecins Sans Frontières (MSF), and the multiple actors involved with the EVD response.

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Author and article information

Contributors

Lorenz von Seidlein: Role: Academic Editor

Journal

Journal ID (nlm-ta): PLoS Med

Journal ID (iso-abbrev): PLoS Med

Journal ID (publisher-id): plos

Journal ID (pmc): plosmed

Title: PLoS Medicine

Publisher: Public Library of Science (San Francisco, CA USA )

ISSN (Print): 1549-1277

ISSN (Electronic): 1549-1676

Publication date (Electronic): 29 March 2016

Publication date Collection: March 2016

Volume: 13

Issue: 3

Electronic Location Identifier: e1001980

Affiliations

[1 ]Public Health England, Porton Down, United Kingdom

[2 ]Partners In Health, Boston, Massachusetts, United States of America

[3 ]Public Health England Laboratory, Port Loko, Sierra Leone

[4 ]Mid Essex Hospital Services NHS Trust, Chelmsford, United Kingdom

[5 ]Liverpool School of Tropical Medicine, Liverpool, United Kingdom

[6 ]Wellbody Alliance, Freetown, Sierra Leone

[7 ]Harvard Medical School, Boston, Massachusetts, United States of America

[8 ]Department of Laboratory Medicine, Boston Children’s Hospital, Boston, Massachusetts, United States of America

Mahidol-Oxford Tropical Medicine Research Unit, THAILAND

Author notes

I have read the journal's policy and the authors of this manuscript have the following competing interests: MM is a member of the Editorial Board of PLOS Medicine. The authors declare that no further competing interests exist.

Conceived and designed the experiments: AES MJB JDK AM MM NRP. Performed the experiments: JR GMF. Analyzed the data: AES MJB JR AM NRP. Contributed reagents/materials/analysis tools: AES AJHS CHL TJGB NRP. Wrote the first draft of the manuscript: AES MJB NRP. Contributed to the writing of the manuscript: AES MJB JR AJHS AM TJGB NRP. Agree with the manuscript’s results and conclusions: AES MJB JR GMF AJHS CHL JDK AM TJGB MM NRP. Organization and supervision of study training and testing procedures performed at PHE Porton Down and PHE Port Loko Laboratory: AES JR GMF AJHS CHL TJGB. Study protocol development and institutional approvals: AES MJB CHL JDK AM TJGB MM NRP. All authors have read, and confirm that they meet, ICMJE criteria for authorship.

* E-mail: Nira.pollock@ 123456childrens.harvard.edu

Article

Publisher ID: PMEDICINE-D-15-02390

DOI: 10.1371/journal.pmed.1001980

PMC ID: 4811569

PubMed ID: 27023868

SO-VID: 59a6d984-136d-4400-8dd8-d93fdce08a8c

License:

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

History

Date received : 11 August 2015

Date accepted : 11 February 2016

Page count

Figures: 1, Tables: 2, Pages: 15

Funding

Funding for the study was provided by a gift from the Abundance Foundation. Cepheid provided a platform and test kits for the study but did not provide any monetary support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Performance of the GeneXpert Ebola Assay for Diagnosis of Ebola Virus Disease in Sierra Leone: A Field Evaluation Study

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Abstract

Background

Methods and Findings

Conclusions

Abstract

Editors' Summary

Background

Why Was This Study Done?

What Did the Researchers Do and Find?

What Do These Findings Mean?

Additional Information

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IET: COVID-19 related research

Most cited references 11

Measuring agreement in method comparison studies.

ReEBOV Antigen Rapid Test kit for point-of-care and laboratory-based testing for Ebola virus disease: a field validation study

The Case for Improved Diagnostic Tools to Control Ebola Virus Disease in West Africa and How to Get There

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