Benchmarking CRISPR-BP34 for point-of-care melioidosis detection in LMIC: a molecular diagnostics study

BackgroundMelioidosis is a grossly neglected but often-fatal tropical disease. The disease is named "a great mimicker" after its broad clinical manifestations, which makes disease diagnosis challenging and time-consuming. To improve diagnosis, we developed and evaluated the performance of the CRISPR-Cas12a system called "CRISPR-BP34" to detect Burkholderia pseudomallei DNA across clinical specimens from patients suspected to have melioidosis. MethodsWe documented time taken for diagnosis, antibiotics prescribed during the waiting period, and infection outcomes in 875 melioidosis patients treated in a hospital in northeast Thailand between October 2019 and December 2022. In the last six months, we performed CRISPR-BP34 detection on clinical specimens (blood, urine, respiratory secretion, pus and other body fluids) collected from 330 patients with suspected melioidosis and compared its performance to the current gold-standard culture-based method. Discordant results were validated by three independent qPCR tests. FindingsA window of 3-4 days was required for gold-standard culture diagnosis, which resulted in delayed treatment. 199 [22{middle dot}7%] of 875 patients died prior to diagnosis results while 114 [26{middle dot}3%] of 433 follow-up cases had been diagnosed, treated, but died within 28 days of admission. A shorter sample-to-diagnosis time of less than 4 hours offered by CRISPR-BP34 technology could lead to faster administration of correct treatment. We demonstrated an improved sensitivity of CRISPR-BP34 (106 [93{middle dot}0%] of 114 positive cases, 95% CI 86{middle dot}6 - 96{middle dot}9) compared to the culture approach (76 [66{middle dot}7%] of 114 positive cases, 95% CI 57{middle dot}2 - 75{middle dot}2); while maintaining similar specificity (209 [96{middle dot}8%] of 216 negative cases, 95% CI 93{middle dot}4-98{middle dot}7) to the culture (216 [100 %] of 216 negative cases, 95% CI 98{middle dot}3-100{middle dot}0). InterpretationThe sensitivity, specificity, speed, window of clinical intervention, and ease of operation offered by the CRISPR-BP34 support its use as a point-of-care diagnostic for melioidosis. FundingChiang Mai University Thailand and Wellcome Trust UK Research in context Evidence before this studyMelioidosis is an often-severe infectious disease caused by the bacterium Burkholderia pseudomallei. It is estimated to affect 165,000 individuals annually worldwide, of which 89,000 cases are fatal. The disease diagnosis is challenging due to diverse clinical presentations, low awareness, limited diagnostic options, or even a lack of diagnostic tests. A PubMed search conducted from the database inception to 6 May 2023, using the terms "melioidosis" AND "diagnosis test," yielded 207 results, 40 of which presented clinical evaluations of rapid melioidosis diagnostic tests. Antigen-based diagnostic tests, which detect the presence of B. pseudomallei, reported high specificity (median = 98{middle dot}6%, IQR 94{middle dot}0 - 100{middle dot}0), but low sensitivity (median = 57{middle dot}1%, IQR = 44{middle dot}3 - 82{middle dot}5). The test sensitivity suffers from the often-low concentration of the bacterial antigens in patients samples, which can vary by specimen type and stage of infection. Antibody-based diagnostic tests that detect host antibodies against B. pseudomallei typically exhibit satisfactory specificity (median = 94{middle dot}5%, IQR = 88{middle dot}6 - 96{middle dot}2) but poor sensitivity (median = 80{middle dot}2%, IQR = 71{middle dot}0 - 88{middle dot}1). These tests are often impacted by variations in antibody responses to B. pseudomallei and the duration required for antibody production. Furthermore, standardisation remains challenging due to the influence of different serum titres on sensitivity and background of the tests. Likewise, quantitative PCR exhibits a high degree of specificity (median = 99{middle dot}8%, IQR = 91{middle dot}6-100{middle dot}0), but an observed low sensitivity for melioidosis (median = 77{middle dot}1%, IQR = 20{middle dot}8-97{middle dot}8), which is likely attributed to the genetic heterogeneity of B. pseudomallei genomes. Additionally, these studies consistently reported a demand for improved speed and ease of implementation in resource-limited settings where melioidosis is endemic. With the limitations of current diagnostic methods, a culture-confirmed approach with 60% sensitivity, 100% specificity, and a diagnosis time of 2-7 days still stands as the gold standard for melioidosis diagnosis. Added value of this studyTo date, no study has measured the impact of delayed diagnosis on melioidosis. We assessed the number of deaths occurring prior to culture-confirmed diagnosis (22{middle dot}7%) and those after diagnosis but within 28 days post-admission (26{middle dot}3%), highlighting the urgent need for prompt action. To address this, we developed the CRISPR-BP34 test, which utilises isothermal amplification of a nucleic acid target followed by site-specific detection using a CRISPR-Cas12a enzyme. We successfully implemented this assay in a resource-limited setting in northeast Thailand, where the disease prevalence is among the highest in the world. The assay achieved a diagnostic sensitivity and specificity of 93{middle dot}0% and 96{middle dot}8%, respectively, with a limit of detection ranging from 50-250 cfu/mL. Early diagnosis can be achieved within four hours of patient admission, which is significantly faster than the gold-standard test that typically takes several days. Moreover, the ultrasensitivity of the CRISPR-BP34 assay enabled the detection of low levels of B. pseudomallei in hemoculture bottles, which could be missed due to mixed infections, poor aseptic technique, or other causes, leading to undiagnosed melioidosis. Implications of all available evidenceThe CRISPR-BP34 assay holds great promise for the management and control of melioidosis. Its minimal setup and shallow learning curve make it well-suited for resource-limited settings. Additionally, its speed and high sensitivity enable early diagnosis and treatment, which are crucial for saving patients lives.