Buffer AVL Alone Does Not Inactivate Ebola Virus in a Representative Clinical Sample Type

Abstract

Rapid inactivation of Ebola virus (EBOV) is crucial for high-throughput testing of clinical samples in low-. In response to this outbreak, the international community has deployed an increasing number of Ebola diagnostic laboratories into the main West African countries affected (Guinea, Liberia, and Sierra Leone). Rapid diagnosis of EVD in humans is critical in the management of this disease in outbreak situations, as it allows prompt isolation and the chance to provide the best supportive care to patients, which helps reduce the overall infection rate and break the transmission chain.The preferred clinical sample for testing for Ebola virus (EBOV), an enveloped negative-sense single-strand RNA virus, is EDTA-blood, serum, or plasma with the primary diagnostic technology being real-time PCR (2). Other sample types, such as swabs or urine, may also be received by a laboratory. EBOV is designated in the United Kingdom by the Advisory Committee on Dangerous Pathogens (ACDP) as a hazard group 4 pathogen that must be handled under containment level (CL) 4 standards (biosafety level 4 [BSL4] in other countries). As such stringent laboratory infrastructure and containment procedures are required to handle viable EBOV material, only a few laboratories in Europe and elsewhere are suitably equipped (3). Within the timelines and budgets available, it has been impractical to create this laboratory infrastructure in West Africa, and therefore diagnostic laboratories have relied on methods that rapidly inactivate EBOV prior to routine processing and testing of samples by PCR.Laboratory methods of EBOV inactivation include gamma irradiation (4), nanoemulsion (5), photoinducible alkylating agents (6), and UV radiation (7), but these methods are primarily used for research purposes and may not be practicable in an outbreak situation that is likely to involve a high number of samples but reduced capability for handling and manipulation. In this context, any inactivation method must also be compatible with the EBOV PCR diagnostic approach.The CDC recommends Triton X-100 and heat treatment for 1 h for diagnostic samples containing hemorrhagic fever viruses (8), and this method has been adopted by many laboratories for handling of samples that may contain EBOV (9). Heating (alone or with acetic acid) for 1 h at 60°C has also been shown to reduce the titer of EBOV (10). Other guidelines can be nonspecific, specifying only the need for inactivation but not suggesting how (11) or suggesting generic use of denaturing/lysis buffers and/or heat (12). In the United Kingdom, the Advisory Committee on Dangerous Pathogens guidelines state that samples from confirmed cases may be processed in a containment level 2 laboratory using routine autoanalyzers if a containment level 4 laboratory is not available and provided specific procedures are followed (13). Within these guidelines, which encompass the application of multiple clinical tests, there is no specific requirement to inactivate EBOV (or other viral hemorrhagic fever agents) within a sa...

Rapid Bedside Inactivation of Ebola Virus for Safe Nucleic Acid Tests

“…A recent report by Smither et al, in 2015, showed that the frequently used AVL buffer alone did not inactivate EBOV. EBOV-spiked mouse blood treated with AVL buffer needed to be combined with either heat or ethanol to ensure complete EBOV inactivation over the time of three passages (26). We showed that the MPLB buffer inactivated EBOV over the time of three passages without the need for additional treatment.…”

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

“…A separate study (11) has indicated that a combination of Buffer AVL (containing a chaotropic guanidine salt) and heat is required to inactivate EBOV in blood samples-with individual Buffer AVL or heat treatments not inactivating EBOV in samples. In Sierra Leone, the EZ1 RNA extraction and SmartCycler PCR platforms were operated outside biological containment.…”

“…We have not experimentally evaluated the EBOV virucidal efficacy of FilmArray sample buffer (which, like Buffer AVL, contains a guanidine salt), with or without an additional heat treatment. Without any relevant experimental evidence, we included the heat treatment, as a previous study (11) indicated that a dual treatment (guanidine-based buffer and heat) was required to ensure EBOV inactivation. This provided confidence to allow operation of the Sierra Leone FilmArray outside containment.…”

“…In a fresh 2-ml screw cap microtube, 80 l of plasma was mixed with 320 l of Buffer AVL (Qiagen); the tube surface was decontaminated by wiping with 5% chlorine solution, and the tube was left to stand for 10 min. The tube was removed from the isolator, and the sample was heated for 15 min to 60°C, the temperature required to inactivate EBOV in a blood sample mixed with Buffer AVL (11). RNA extraction was performed using the entire heat-treated 400-l plasma/Buffer AVL sample, Qiagen EZ1 virus minikit v2.0, and a Qiagen EZ1 Advanced XL platform to generate a final eluted RNA extract volume of 60 l. Whole MS2 bacteriophage was incorporated during the extraction procedure as an internal and inhibition control.…”

Biosafety standards for working with Crimean-Congo hemorrhagic fever virus

“…Several publications have shown that chaotropic guanidineisothiocyanate in commercial nucleic acid extraction buffers efficiently inactivates most enveloped viral agents including pox-, alpha-, bunya-, flavi-and filoviruses (Blow et al, 2004;Smither et al, 2015;Vinner & Fomsgaard, 2007).…”