Author summaryClinical signs and symptoms of leptospirosis are similar to those of other infectious diseases such as dengue, sepsis, and malaria, making it difficult to diagnose. In this study, we developed an RPA-CRISPR/Cas12a -based detection platform to identify the lipL32 gene of pathogenic Leptospira spp. The results showed that the limit of detection (LOD) was approximately 102 cells/mL without cross-reactivity against other infectious diseases. The platform was validated using 110 patients from 15 hospitals in Sisaket province, Thailand. The sensitivity, specificity, and accuracy was found to be 85.2%, 100% and 92.7%, respectively, for the diagnosis of leptospirosis. Assay sensitivity increased at 4–6 d post-onset of fever, with a consistent specificity every day after the onset of fever. We also developed a lateral flow detection assay combined with RPA-CRISPR/Cas12a, which also had a LOD of 102 cells/mL and could correctly distinguish known positive and negative clinical samples in a pilot study. Findings from this study demonstrate the potential effectiveness of the RPA-CRISPR/Cas12a platform in improving speed and accuracy of leptospirosis diagnosis especially in limited-resource settings.
| Abstract
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 findingsA 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/SignificanceThe 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. |
RPA-CRISPR/Cas12a-FBDA/LFDA 문의 논문
Author summary
Clinical signs and symptoms of leptospirosis are similar to those of other infectious diseases such as dengue, sepsis, and malaria, making it difficult to diagnose. In this study, we developed an RPA-CRISPR/Cas12a -based detection platform to identify the lipL32 gene of pathogenic Leptospira spp. The results showed that the limit of detection (LOD) was approximately 102 cells/mL without cross-reactivity against other infectious diseases. The platform was validated using 110 patients from 15 hospitals in Sisaket province, Thailand. The sensitivity, specificity, and accuracy was found to be 85.2%, 100% and 92.7%, respectively, for the diagnosis of leptospirosis. Assay sensitivity increased at 4–6 d post-onset of fever, with a consistent specificity every day after the onset of fever. We also developed a lateral flow detection assay combined with RPA-CRISPR/Cas12a, which also had a LOD of 102 cells/mL and could correctly distinguish known positive and negative clinical samples in a pilot study. Findings from this study demonstrate the potential effectiveness of the RPA-CRISPR/Cas12a platform in improving speed and accuracy of leptospirosis diagnosis especially in limited-resource settings.
Abstract
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.