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

Abstract

Summary 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 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 to 96.9) compared to the culture approach (76 [66.7%] of 114 positive cases, 95% CI 57.2 to 75.2); while maintaining similar specificity (209 [96.8%] of 216 negative cases, 95% CI 93.4 to 98.7) to the culture (216 [100 %] of 216 negative cases, 95% CI 98.3 to 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.