The combination of RPA-CRISPR/Cas12a and Leptospira IgM RDT enhances the early detection of leptospirosis.

BACKGROUND: 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.

Seroprevalence of leptospirosis among blood donors in an endemic area.

Thailand is known to be endemic for leptospirosis. This bacterium may pose a potential risk to transfusion safety. This study was a cross-sectional study examining the seroprevalence of leptospirosis among Thai blood donors. A total of 1053 serum specimens collected from blood donors residing in 5 regions of Thailand during March to September 2020 were included in this study. All samples were tested for the presence of antibodies to 22 leptospiral serovars using the microscopic agglutination test (MAT) and anti-Leptospira IgG antibodies using commercially available enzyme immunoassay. We found no evidence of recent exposure to Leptospira spp. in sera of healthy Thai blood donors by MAT, including those in higher-risk areas. However, in this same group, we did find small numbers of past exposure (1.7%) to Leptospira spp. by IgG ELISA. According to the findings of this study, there is currently no evidence for implementing new blood banking procedures to identify possible carriers in Thailand, however these should be continually monitored and revised according to the infectious disease burden in each country. It should be noted that there was a difference in the occupation rate between the general population reported in Thailand and blood donors in this study; it may not reflect the actual situation in the country.

Rapid and sensitive point-of-care detection of Leptospira by RPA-CRISPR/Cas12a targeting lipL32.

BACKGROUND: One of the key barriers preventing rapid diagnosis of leptospirosis is the lack of available sensitive point-of-care testing. This study aimed to develop and validate a clustered regularly-interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 12a (CRISPR/Cas12a) platform combined with isothermal amplification to detect leptospires from extracted patient DNA samples. METHODOLOGY/PRINCIPAL FINDINGS: A Recombinase Polymerase Amplification (RPA)-CRISPR/Cas12a-fluorescence assay was designed to detect the lipL32 gene of pathogenic Leptospira spp. The assays demonstrated a limit of detection (LOD) of 100 cells/mL, with no cross-reactivity against several other acute febrile illnesses. The clinical performance of the assay was validated with DNA extracted from 110 clinical specimens and then compared to results from qPCR detection of Leptospira spp. The RPA-CRISPR/Cas12a assay showed 85.2% sensitivity, 100% specificity, and 92.7% accuracy. The sensitivity increased on days 4-6 after the fever onset and decreased after day 7. The specificity was consistent for several days after the onset of fever. The overall performance of the RPA-CRISPR/Cas12a platform was better than the commercial rapid diagnostic test (RDT). We also developed a lateral flow detection assay (LFDA) combined with RPA-CRISPR/Cas12a to make the test more accessible and easier to interpret. The combined LFDA showed a similar LOD of 100 cells/mL and could correctly distinguish between known positive and negative clinical samples in a pilot study. CONCLUSIONS/SIGNIFICANCE: The RPA-CRISPR/Cas12 targeting the lipL32 gene demonstrated acceptable sensitivity and excellent specificity for detection of leptospires. This assay might be an appropriate test for acute leptospirosis screening in limited-resource settings.