Excellence Center for Critical Care Nephrology, King Chulalongkorn Memorial Hospital, Bangkok, Thailand.
Faculty of Medicine, Center of Excellence in Critical Care Nephrology, Chulalongkorn University, Bangkok, Thailand.
PLoS Negl Trop Dis. 2023 Aug 25;17(8):e0011596. doi: 10.1371/journal.pntd.0011596. eCollection 2023 Aug.
Lack of available sensitive point-of-care testing is one of the primary obstacles to the rapid diagnosis of leptospirosis. The purpose of this study was to test the performance of two point-of-care tests, a clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 12a (CRISPR/Cas12a) fluorescence-based diagnostic assay (FBDA), a Leptospira immunoglobulin M (IgM) rapid diagnostic test (RDT), and the two tests combined.
METHODOLOGY/PRINCIPAL FINDINGS: For the diagnosis of 171 clinical samples, a recombinase polymerase amplification (RPA)-CRISPR/Cas12a FBDA for whole blood and Leptospira IgM RDT (Medical Science Public Health, Thailand) for serum were used. The confirmed cases were determined by using any positive qPCR, microscopic agglutination test (MAT), and culture results. Diagnostic accuracy was assessed on the first day of enrollment and stratified by the day after symptom onset. The overall sensitivity of the Leptospira IgM RDT and RPA-CRISPR/Cas12a FBDA was 55.66% and 60.38%, respectively. When the two tests were combined, the sensitivity rose to 84.91%. The specificity of each test was 63.08% and 100%, respectively, and 63.08% when combined. The sensitivity of the Leptospira IgM RDT rose on days 4-6 after the onset of fever, while the RPA-CRISPR/Cas12a FBDA continued to decrease. When the two tests were combined, the sensitivity was over 80% at different days post-onset of fever.
CONCLUSIONS/SIGNIFICANCE: The combination of Leptospira IgM RDT and RPA-CRISPR/Cas12 FBDA exhibited significant sensitivity for the detection of leptospires at various days after the onset of fever, thereby reducing the likelihood of misdiagnosis. The combination of these assays may be suitable for early leptospirosis screening in situations with limited resources.
缺乏可用的即时检测是快速诊断钩端螺旋体病的主要障碍之一。本研究旨在测试两种即时检测的性能，一种基于成簇规律间隔短回文重复序列（CRISPR）/CRISPR 相关蛋白 12a（CRISPR/Cas12a）荧光诊断检测法（FBDA），一种钩端螺旋体免疫球蛋白 M（IgM）快速诊断检测法（RDT），以及这两种检测法的联合应用。
方法/主要发现：对于 171 份临床样本的诊断，使用了重组酶聚合酶扩增（RPA）-CRISPR/Cas12a FBDA 检测全血和泰国 Medical Science Public Health 的 Leptospira IgM RDT（血清）。确诊病例是通过任何阳性 qPCR、显微镜凝集试验（MAT）和培养结果来确定的。在入组的第一天评估诊断准确性，并按发病后第几天进行分层。Leptospira IgM RDT 和 RPA-CRISPR/Cas12a FBDA 的总灵敏度分别为 55.66%和 60.38%。当两种检测法联合使用时，灵敏度提高到 84.91%。每种检测法的特异性分别为 63.08%和 100%，联合使用时为 63.08%。Leptospira IgM RDT 的灵敏度在发热后第 4-6 天上升，而 RPA-CRISPR/Cas12a FBDA 则持续下降。当两种检测法联合使用时，在发热后不同的天数，灵敏度均超过 80%。
结论/意义：Leptospira IgM RDT 和 RPA-CRISPR/Cas12 FBDA 的联合应用在发热后不同的天数对钩端螺旋体的检测具有显著的灵敏度，从而降低了误诊的可能性。在资源有限的情况下，这些检测方法的联合应用可能适用于早期钩端螺旋体病的筛查。
