Laboratory diagnosis of Ebola virus disease (EVD) plays a critical role in outbreak response efforts; however, establishing testing strategies for this high-biosafety-level pathogen in resource-poor environments remains extremely challenging. Since the discovery of Ebola virus in 1976 via traditional viral culture techniques and electron microscopy, diagnostic methodologies have trended toward faster, more accurate molecular assays. The unparalleled scope of the 2014-2015 West Africa Ebola epidemic spurred remarkable innovation in the field.
In this article, we describe the evolution of Ebola virus disease diagnostic testing in relation to its past, present and future. As new diagnostic technologies become available, it will be increasingly important for clinicians to understand both the analytic and practical strengths and limitations of these tools. Ultimately, the optimal diagnostic approach for a particular setting will depend upon multiple factors, including population characteristics and disease prevalence, the healthcare setting type, training requirements, regional laboratory capacity, regulatory status, and cost.
Methods for detecting Ebola Virus infection
Testing tools for Ebola virus fall into three basic categories: (i) serologic tests that detect host antibodies generated against the virus, (ii) antigen tests that detect viral proteins, and (iii) molecular tests that detect viral Ribonucleic acid (RNA) sequences. Specific antiviral antibodies can persist for years but the variable onset of antibody responses during acute illness makes serology a minimally useful diagnostic tool. On the contrary, antigen detection and molecular tests have proven very effective for acute diagnosis, as virus levels in the blood typically rise to high levels within the first few days of symptoms.
The incubation period following Ebola virus infection typically ranges between 3 and 13 days, no tests have yet demonstrated the ability to detect Ebola virus prior to the onset of symptoms. Some diagnostic tests have been designed to broadly detect Ebola virus infection, while others distinguish among the five known Ebola virus species (Zaire/Ebola [EBOV], Sudan [SUDV], Tai Forest [TAFV], Reston [RESTV], and Bundibugyo [BDBV]). Major outbreaks of Ebola virus disease in humans have been attributable to EBOV, SUDV, and BDBV; prior to the 2014-2015 epidemic, the origins of EVD outbreaks were restricted to five African countries: Democratic Republic of Congo (formerly Zaire), Sudan, Gabon, Uganda, and Republic of Congo.
The traditional gold standard method to confirm the presence of Ebola virus is viral isolation in cell culture, by using Vero E6 African Green monkey kidney cells. While detection of Ebola virus by these methods is definitive, these methods require biosafety level 4 (BSL-4) containment and are typically restricted to research and public health laboratories.
Serologic assays for the detection of specific antiviral antibodies have been used to demonstrate current or prior infection with Ebola virus since the first outbreak investigations of this virus in 1976. Indirect fluorescent antibody detection test (IFAT), considered to have suboptimal sensitivity and specificity, and the requirement for BSL-4 bio containment rendered this method unsuitable for large-scale diagnostic efforts.
The development of enzyme-linked immunosorbent assay (ELISA) tests for the detection of Ebola virus-specific IgM and IgG antibodies offered a faster, higher-throughput system for serologic testing. ELISAs that utilise recombinant viral proteins have also been developed, but to date they do not appear to have been validated for clinical use. Limited data are available to assess the sensitivity or specificity of these ELISAs.
The detection of viral protein antigens circulating in blood provides a reliable method for diagnosing acute EVD in symptomatic patients. An ELISA for the detection of Ebola virus antigens was first developed at US Army Medical Research Institute of Infectious Diseases (USAMRIID). By this method, viral antigen can be detected in the serum as early as the first day of symptoms. ELISA antigen detection tests utilising monoclonal antibodies has not been reported clinically, as real time reverse transcription-polymerase chain reaction (RT-PCR) techniques have now replaced these tests. During the recent outbreak, lateral flow immunoassays (LFIs) emerged as powerful tools at the point of care.
These assays used polymerase chain reaction (PCR) to amplify the L polymerase, glycoprotein GP, and nucleoprotein NP genes. An important advantage of this method is that RT-PCR detected viral RNA in two specimens collected on the day of symptom onset were negative for antigen at this time point. Early application of conventional RT-PCR demonstrated that it also performed well for detection of virus in other body fluids, such as saliva and seminal fluid.
Real time RT-PCR
This was first developed and tested in early 2000s. It has been seen that lower threshold (Ct) values with higher viral copy numbers correlate with higher mortality. Because existing data indicate that detection of RNA by real-time RT-PCR is variable in the first 72 hours of illness, current guidelines recommend that suspected EVD patients who test negative in this period should be retested after 72 hours of symptoms. Compared to conventional RT-PCR, it yields more rapid results (typically 2 to 3 h); real-time RTPCR assays are now employed by public health reference laboratories.
Automated real time RT-PCR
Several fully automated PCR platforms have been developed in recent years that yields results in 90 minutes or less. The Xpert Ebola assay (Cepheid), is an automated, cartridge based system for RNA extraction and real-time RT-PCR detection of EBOV, NP and GP genes.
The BioFire Defense FilmArray assays (FilmArray Biothreat-Etest and FilmArray NGDS BT-E assay) are automated real-time RT-PCR tests for detection of the EBOV L gene. The Film Array Biothreat-E test, is for the approved use of whole blood and paired urine specimens, while the FilmArray NGDS BT-E test can be used with whole blood, plasma, and serum specimens.
Rapid antigen detection tests
Three EVD rapid diagnostic tests (RDTs) are lateral flow immunoassays (LFIs). ReEBOV (Corgenix, Inc) detects VP40 matrix protein (EBOV, SUDV, BDBV) and is 100% sensitive and 92% specific. Ora Quick Ebola Rapid Antigen Test (OraSure Technologies, Inc) can be done in cadaveric fluid in addition to whole blood while sensitivity is 84% and specificity is 98%. Another rapid antigen detection test is SD Q (SD Biosensor) for which sensitivity is 84.9% and specificity 99.7%.
Viral Sequencing in the 2014-2015 Epidemic
Viral sequencing tools hold the potential to benefit diagnostic efforts in the setting of an emerging outbreak, identification of viral strains responsible for new transmission chains in an ongoing outbreak, estimation of viral mutation rate, and analysis of the impact of viral mutations on the performance of molecular diagnostic. During the 2014-2015 epidemic, next-generation sequencing (NGS) platforms enabled the characterisation of large numbers of viral genomes in a relatively short time frame.
Summary and future directions
Novel diagnostic platforms, such as automated NAATs and rapid antigen detection tests (RDT) that can be used to decentralised healthcare setting with minimal laboratory infrastructure will likely play a major role moving forward. WHO recommends RDT when RT-PCR testing is not immediately available. As more clinical validation data become available and practical experience is compiled, local regulatory agencies, in collaboration with the WHO, will be responsible for developing updated EVD testing algorithms specific for different healthcare settings in both high-prevalence outbreak and low prevalence surveillance scenarios.