Rapid detection of Ebolavirus using isothermal recombinase‐aided amplification

Ebolavirus disease (EVD) is an often‐lethal disease caused by the genus Ebolavirus (EBOV). Although vaccines are being developed and recently used, outbreak control still relies on a combination of various factors, including rapid identification of EVD cases. This allows rapid patient isolation and control measure implementation. Ebolavirus diagnosis is performed in treatment centers or reference laboratories, which usually takes a few hours to days to confirm the outbreak or deliver a clear result. A fast and field‐deployable molecular detection method, such as the isothermal amplification recombinase‐aided amplification (RAA), could significantly reduce sample‐to‐result time. In this study, a RT‐RAA assay was evaluated for EBOV detection. Various primer and probe combinations were screened; analytical sensitivity and cross‐specificity were tested. A total of 40 archived samples from the 2014 to 2016 Ebola outbreak in West Africa were tested with both the reference method real‐time RT‐PCR and the established RT‐RAA assay. The assay could detect down to 22.6 molecular copies per microliter. No other pathogens were detected with the Ebolavirus RT‐RAA assay. Testing 40 samples yield clinical sensitivity and specificity of 100% each. This rapid isothermal RT‐RAA assay can replace the previous RT‐RPA and continue to offer rapid EBOV diagnostics.


| INTRODUCTION
Ebola virus (EBOV) causes the Ebola virus disease (EVD), a hemorrhagic fever which often shows high fatality rates. 1 Although vaccines are being developed and have recently been used, 2 outbreak control still relies on a combination of several measures.Diagnosis of EVD requires laboratory tests such as real-time reverse transcription polymerase chain reaction (PCR) or rapid diagnostic tests based on immunoassays.EBOV infection diagnosis is mainly implemented in Ebola treatment centers (ETC) or surrounding laboratories using various in-house and commercial tests. 3Ultimately, the aim is to achieve specific and fast detection of EVD cases.Virus detection is crucial to successfully control EVD outbreaks by allowing the implementation of isolation, safe burials, surveillance, and contact tracing. 4[7] Using Twist DX RPA kits for assay development and assay deployment has faced a severe supply shortage since Twist DX was taken over by Abbot.To ensure the sustainability of ongoing projects a need arose to evaluate the alternative RAA kits which are commercially available without supply issues.The RAA is a variation of RPA in which Escherichia coli SSB replaces T4 phage GP32 as the single-strand binding protein. 8,9 test an existing EBOV RT-RPA we describe the screening of various RPA oligonucleotides with the RT-RAA kit using archived clinical samples.

| RNA standard and RT-RAA oligonucleotides
A 591 base-pair long in vitro RNA sequence was synthetized by TIB MOLBIOL GmbH based on the nucleoprotein-gene of Ebolavirus (GenBank accession number: KR013754.2).
After alignment to latest EBOV sequences six primers and one exo-probe were evaluated in this study (Table 1).The primer/probe combination yielding the highest signal in RT-RAA (threshold time [TT] in minutes and fluorescence intensity in Millivolt [mV]) was selected for further assay validation.TIB MOLBIOL GmbH synthesized all oligonucleotides.A real-time RT-PCR for Ebolavirus was used according to a previous study. 10The 7500 Fast Dx Real-Time PCR Instrument (Applied Biosystems) and the QuantiTect RT-PCR kit (QIAGEN GmbH) were used.

| Analytical sensitivity and specificity
Three primer combinations (EBOO; EBOG; EBOGp) were tested using a concentration of 10 5 molecules/reaction of the EBOV RNA standard.The best combination was selected for further testing.To determine the limit of detection, three independent RT-RAA runs with 100, 10, and 1 RNA molecules/µL were performed.Cross reactivity of the RT-RAA assay was determined using nucleic acids of pathogens that are considered as differential diagnosis.

| Clinical sensitivity
In total, 40 archived samples were used in this study.The extracted RNA from samples of the 2014-2016 outbreak in Guinea from a previous study. 11were tested at Institut Pasteur de Dakar (IPD), Senegal.The study was registered at ClinicalTrials.govunder the Identifier: NCT04235361.

| Analytical sensitivity and specificity
Out of three combinations, the EBOO forward and reverse primers showed best results and were selected for further assay validation (Figure 1).

| DISCUSSION
In this study, a RT-RAA assay for Ebolavirus was evaluated and yielded excellent clinical sensitivity and specificity of 100% respectively for West African EBOV sequence targets.
EBOV is characterized by high infectivity, and high lethality of EVD, and effective disease treatment options are often accesslimited, especially in low-resource settings. 14,15Efficient diagnostics are restricted to equipped laboratories or treatment facilities.The mean time from the onset of symptoms to hospitalization is approximately 5 days. 16Furthermore, the turn-around time between sample collection and confirmed results is usually up to 3 days depending on the method used. 17Various diagnostic methods based on antigen or antibody capture and RT-PCR are available and approved by the World Health Organization (WHO). 17The RT-PCR is highly sensitive, but needs well-trained personnel, is time-costly and rather expensive due to heavier equipment.Rapid detection of EVD is pivotal to manage treatment centers efficiently, implement control measures, and break the transmission chains, due to the high viral particle shed and its high infectivity. 18int-of-care tests (POCT) can offer significant advantages and fill this diagnostic gap.The WHO-FIND collaboration has highlighted the need of new and rapid molecular POCT for a variety of communicable diseases, including EVD. 19 These are needed in lowand middle-income countries, where outbreaks occur, especially in The RT-recombinase-aided amplification (RAA) amplification curve with the possible primer combinations tested with 5 × 10 5 Ebolavirus (EBOV) molecular standard.The EBOO primers showed the highest fluorescence signal and lowest time threshold.
remote areas. 20,21Rapid antigen-based POCT have been developed and validated.Sensitivities and specificities vary from 62% and 73% onwards, respectively. 22The viral load in samples from an acute Ebola case is high, enhancing the probability of a true positive antigen test. 23However, high background signal can hinder the accuracy of lateral flow tests.Here, specific interactions between particles depend on various biochemical factors, which often need to be optimized to avoid nonspecific binding, leading to false positive results. 24Thus, these tests can be generally used as "rule-out" screening tests. 22,23,25In contrast, molecular POCT have experienced high demand in research and development in recent years, ultimately due to its simplicity and accuracy. 26Additionally, the RPA/RAA amplification technique is tolerant against various inhibitors. 27During the large EVD outbreak in West Africa 2014-2015, a mobile suitcase laboratory was combined with a RT-RPA assay for EBOV.This pointof-care set-up was successfully implemented in Guinea and local healthcare workers were trained.It was shown that the RPA could efficiently test suspected EVD cases. 11The deployment to a hospital and a morgue in Matoto in Conakry actually improved the collaboration between safe and dignified burial times and the population when it became known that test reults were available in 30-40 min. 28th this study, it was shown that the EBOO assay, which combines the EBOO primers with the updated probe, is as sensitive with RT-RAA kits as the previous RT-RPA assay with Twist Exo kits. 11,29This allows to close the supply gap for Twist RPA kits.Pathogens causing similar clinical signs and considered as differentials were not detected with the EBOO assay, confirming excellent analytical specificity.
F I G U R E 2 The limit of detection (LOD) of the EBOO RT-recombinase-aided amplification (RAA) was determined with three independent RT-RAA runs.The assay's LOD is 22.6 copies per microliter (red mark).
T A B L E 2 List of pathogens used for the cross-specificity testing.
False positive patients transferred to an ETC to await confirmatory test could pick up nosocomial Ebola infection.Therefore, the ideal EBOV-POCT needs to be highly sensitive, but most of all highly specific.The EBOO RT-RAA assay was successfully tested with 40 clinical samples and shown to yield an excellent high clinical sensitivity and specificity delivering results in 15 min.
Ideally, a POCT delivers results under 30 min, involves about three procedural steps and is suited for field deployment in remote areas without complex laboratory infrastructure and allows biosafe procedures. 17 Real-time RT-RAA assays were performed in a 50 µL volume with the RT-RAA nucleic acid amplification kit (fluorescent method) (Jiangsu Qitian Gene Biotechnology Co. Ltd).The reaction mix comprised of 25 µL rehydration Buffer, 12.7 µL nuclease-free water, 2.5 µL magnesium acetate (280 mM), 2.1 µL of each primer (10 µM), 0.6 µL probe (10 µM), and 5 µL template, which was added into the lid of the reaction tube containing the freeze-dried pellet.Water was used instead of the DNA template for the negative control.The tube was closed, centrifuged, mixed, centrifuged, and placed immediately into the T8-ISO Instrument (Axxin).The incubation temperature was 42°C for 15 min.A mixing and centrifuging step was performed at 320 s after the test start.The FAM fluorescence signal was recorded in real time.The time threshold (TT) was determined using the T8 Desktop Application (version 2.8.0.0,Axxin) based on the first derivative values.
Twenty clinical samples were real-time RT-PCR and EBOO RT-RAA negative.Twenty samples were real-time RT-PCR and EBOO RT-RAA positive (Supporting Information S1: File S1).Clinical specificity and sensitivity were both 100%.No correlation was found between TT ,20 These aspects are addressed with the EBOO RT-RAA used in the suitcase lab, altogether offering rapid and accurate EBOV diagnosis at the point of need.This is essential for infectious diseases such as EVD, where sample-to-result time is crucial for outbreak management implementing effective counter measures to avoid a recurrence of the large outbreaks, such as in West Africa (2014-2015) and Democratic Republic of the Congo (2018-2020). 30-32F I G U R E 3 Comparison between the threshold time (TT) value of the EBOO RT-recombinase-aided amplification (RAA) and Ct value of the real-time RT-PCR.No correlation was found (r = 0.23), as the RT-RAA reaction is faster than the real-time PCR reaction.The cut-off value for RT-PCR was 35.