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Researchers have found that a new test using a CRISPR-Cas12a system (CRISPR-BP34) diagnosed patients with melioidosis, an often-fatal tropical disease caused by the bacteria Burkholderia pseudomallei, within hours, rather than days as in current bacterial culture methods, so patients could receive the correct, life-saving antibiotics faster.

Farmers planting rice in Southeast Asia © MORU. Photo: Gerhard Jørén.

Developed by researchers at the Mahidol-Oxford Tropical Medicine Research Unit (MORU), Chiang Mai University, and Vidyasirimedhi Institute of Science and Technology (VISTEC) in Thailand, and the Wellcome Sanger Institute in the UK, the test uses CRISPR to detect a genetic target specific to B. pseudomallei with 93 per cent sensitivity.

The researchers say the results, published in Lancet Microbe, mean that more lives could be saved from melioidosis, with a rapid, easy-to-use diagnostic test that could be rolled out globally.

“Melioidosis has been neglected despite its high mortality rate and high incidence in many parts of remote, rural Asia. Early diagnosis is essential so that the specific treatment required can be started as soon as possible. The new rapid diagnostic tool developed through this collaboration has the potential to be a game-changer,” said senior study author Prof Nick Day, Director of MORU.

Melioidosis has broad clinical manifestations, which makes diagnosis challenging and time consuming. Symptoms vary from localised abscess or pneumonia to acute septicaemia, or may present as a chronic infection.

Currently, melioidosis is diagnosed in patients after bacterial samples are cultured, which takes three to four days. In Thailand, approximately 40 per cent of patients with melioidosis die, many within the first one to two days following admission to hospital, while waiting for a diagnosis.

In a new study, the team set out to develop a new rapid test to reduce the time taken to correctly diagnose and treat patients with melioidosis.

The researchers identified a genetic target specific to B. pseudomallei by analysing over 3,000 B. pseudomallei genomes, most of which were sequenced at the Sanger Institute. They searched for conserved regions of the genome and screened the targets against other pathogens and human host genomes, to ensure their chosen target was specific to B. pseudomallei.

Their test, called CRISPR-BP34, involves rupturing bacterial cells and using a recombinase polymerase amplification reaction to amplify the bacterial target DNA for increased sensitivity. Additionally, a CRISPR reaction is used to provide specificity, and a simple lateral flow ‘dipstick’ read-out is employed to confirm cases of melioidosis.

The new test showed enhanced sensitivity at 93 per cent, compared to 66.7 per cent in bacterial culture methods. It also delivered results in less than four hours for urine, pus, and sputum samples, and within one day for blood samples. This is a significant improvement over the current bacterial culture diagnostic method, which typically takes three to four days.

"We carefully designed the rapid diagnostic test based on CRISPR-BP34, with a robust algorithm, and tested its performance in vitro. We are thrilled that the CRISPR-BP34 test demonstrates outstanding diagnostic efficacy when tested on clinical samples, showcasing its potential to significantly impact patient outcomes and potentially save lives in the near future," said Dr Somsakul Wongpalee, co-lead author at Chiang Mai University, Thailand.

This new rapid diagnostic test will enable health professionals to prescribe the correct antibiotics faster, meaning fewer patients will die while waiting for a diagnosis. While saving precious time, the new test will also save resources and money, with fewer unnecessary antibiotics prescribed and less time for patients in hospital.

In next steps for the team, they are currently designing randomised clinical trials to show the effectiveness of these tests in hospital settings. Plus, members of the team will begin investigating the role of human genetics in susceptibility and immune response to melioidosis infection.

"The sensitivity, specificity, speed, and window of clinical intervention offered by CRISPR-BP34 support its prospective use as a point-of-care diagnostic tool for melioidosis. Future development should be focused on scalability and cost reduction.. There’s much work to do, but it can potentially be used anywhere in the world to get the right treatments to patients faster, ultimately saving lives," said Dr Claire Chewapreecha, co-lead author at MORU, Thailand, and Wellcome Sanger Institute International Fellow.

Benchmarking CRISPR-BP34 for point-of-care melioidosis detection in low-income and middle-income countries: a molecular diagnostics study. Pakdeerat S, Boonklang P, Angchagun K, Chomkatekaew C, Apichaidejudom N, Dokket Y, Faosap A, Wongsuwan G, Wuthiekanun V, Aramrueung P, Khamnoi P, Thananchai H, Siriboon S, Chamnan P, Peacock SJ, Day NPJ, Thomson NR, Uttamapinant C, Wongpalee SP, Chewapreecha C. Lancet Microbe. 2024 Mar 4:S2666-5247(23)00378-6. doi: 10.1016/S2666-5247(23)00378-6. Epub ahead of print. PMID: 38493790.