In response to the 2022 global outbreak of the monkeypox virus (MPXV), researchers have harnessed cutting-edge genetic technology to develop Australia’s first genetic assay for the detection of the virus.
In a collaborative study published in The Lancet Microbe, the team from the Peter Doherty Institute for Infection and Immunity and Walter and Eliza Hall Institute of Medical Research (WEHI), detailed the development of MPXV-CRISPR – a CRISPR-based diagnostic method in Australia specifically designed to target genetic sequences found only in MPXV.
While the CRISPR technology is most known for its genome editing capability, new applications have emerged, including leveraging it for the design of powerful and highly sensitive diagnostic tools, and the new device is able to detect MPXV in clinical samples with acute precision and at a faster rate than any other method.
The University of Melbourne’s Dr Soo Jen Low, a Research Officer at the Doherty Institute and co-author of the study, said several in-house and commercial tests have been developed for the diagnosis of mpox; however, these were predominantly PCR-based, designed for use in a centralised laboratory setting.
“In this study, we aimed to develop a rapid, sensitive, and specific point-of-care test (PoCT) to detect MPXV in clinical samples using isothermal amplification coupled with CRISPR and CRISPR-associated protein (Cas),” Dr Low explained.
“To work, MPXV-CRISPR has to be ‘programmed’ to recognise the virus. We used a database of 523 MPXV genomes to carefully engineer ‘guides’ to bind to the specific part we are looking for on the viral DNA. Getting this right was crucial for the success of our diagnostic tool.
“In essence, when viral DNA is present in a clinical sample, the CRISPR system is guided to the target and subsequently emits a signal to indicate the presence of the virus. Our testing method can achieve sensitivity and precision levels comparable to the gold-standard PCR methods, but in a fraction of the time.”
Using the primer–gRNA set determined to produce the best signal, the assay was applied to a large set of clinical samples and compared with a gold-standard MPXV qPCR assay. Overall, the MPXV-CRISPR assay with a fluorescence readout demonstrated 99·5% concordance with qPCR, with the only discrepancy being a sample with a very low viral load.
“Further, our assay showed no cross-reactivity against other viral pathogens tested, including those with similar clinical presentations such as varicella zoster virus, probably due to our comprehensive in-silico primer and gRNA design, which was subsequently optimised in vitro for increased specificity,” Dr Low said.
“Importantly, our assay was able to detect MPXV across a range of clinical specimen types (skin lesions, anal swabs, and oropharyngeal samples). This finding is relevant given that many individuals might present with lesions in the oropharynx or anus.”
Dr Matthew O’Neill, a Research Assistant at WEHI and co-first author of the paper, explained that the speed at which this new technology can provide a diagnosis is one of the groundbreaking features of MPXV-CRISPR.
“Currently, mpox test results might not be available for up to several days after sample collection, depending on geographical and logistical considerations. In parallel, MPXV-CRISPR can detect the virus in just 45 minutes,” he said.
“Compared with the gold-standard qPCR, our assay with fluorescence readout had a sensitivity of 100% and specificity of 99·3% at 4 min, with an analytical sensitivity at 40 min of 1 genome copy per µL.”
In line with the WHO standards, where diagnostic tests should be accurate, accessible, and affordable, the team is working on adapting MPXV-CRISPR into a portable device, that could be deployed at points of care around the country for rapid, on-site detection of monkeypox virus.
Dr Shivani Pasricha, a Senior Research Officer at WEHI, Junior Laboratory Head at the Doherty Institute, and co-senior author of the paper, said MPXV-CRISPR has the potential to revolutionise the way Australia managed mpox, making a meaningful impact on public health.
“By improving access to quick and reliable diagnoses around Australia, including in places with limited resources and in remote areas, this decentralised approach to testing could enable faster treatment and improve patient outcomes, while fast-tracking our capacity to manage future outbreaks,” Dr Pasricha said.
“Of note, a recent study using qPCR suggested that viral loads in lesions from patients with mpox generally have Ct values of 30 or less, while another study found a high correlation between viral DNA concentration and virus infectivity in clinical specimens, suggesting a qPCR threshold of Ct 35 or less for predicting a poorly infectious or non-infectious specimen.
“In our study, applying a qPCR threshold of Ct 35 or less or of Ct 30 or less did not result in a change in sensitivity, suggesting that our MPXV-CRISPR assay is capable of detecting MPXV in individuals during their infectious period.”