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

Background Melioidosis 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. Methods We 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. Findings A window of 3-4 days was required for gold-standard culture diagnosis, which resulted in delayed treatment. 199 [22·7%] of 875 patients died prior to diagnosis results while 114 [26·3%] 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 [93·0%] of 114 positive cases, 95% CI 86·6 - 96·9) compared to the culture approach (76 [66·7%] of 114 positive cases, 95% CI 57·2 - 75·2); while maintaining similar specificity (209 [96·8%] of 216 negative cases, 95% CI 93·4-98·7) to the culture (216 [100 %] of 216 negative cases, 95% CI 98·3-100·0). Interpretation The 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. Funding Chiang Mai University Thailand and Wellcome Trust UK Evidence before this study Melioidosis 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 = 98·6%, IQR 94·0 - 100·0), but low sensitivity (median = 57·1%, IQR = 44·3 - 82·5). 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 = 94·5%, IQR = 88·6 - 96·2) but poor sensitivity (median = 80·2%, IQR = 71·0 - 88·1). 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 = 99·8%, IQR = 91·6-100·0), but an observed low sensitivity for melioidosis (median = 77·1%, IQR = 20·8-97·8), 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 study To date, no study has measured the impact of delayed diagnosis on melioidosis. We assessed the number of deaths occurring prior to culture-confirmed diagnosis (22·7%) and those after diagnosis but within 28 days post-admission (26·3%), 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 93·0% and 96·8%, 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 evidence The 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. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement SPW was funded by TSRI Fundamental Fund 2022, Chiang Mai University (grant number 65A104000009) and Faculty of Medicine Research Fund, Chiang Mai University (grant number MIC-2562-06372). CCho was funded by Wellcome International Master Fellowship (221418/Z/20/Z). CChe was funded by Wellcome International Intermediate Fellowship (216457/Z/19/Z) and Sanger International Fellowship. This research was funded in whole, or in part, by the Wellcome Trust. The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Two related studies were conducted and reported in the manuscript. Study 1 which quantified the time taken to diagnose melioidosis based on culture and patient outcome was approved by Sunpasitthiprasong Hospital Ethical Review Board (015/62C) and the Oxford Tropical Research Ethics Committee (OxTREC, 25-19). Study 2 was a diagnostic evaluation of the sensitivity and specificity of a CRISPR-BP34 prototype assay and was approved by the Ethical Review Board of Sunpasitthiprasong Hospital (029/65C). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors