Preliminary clinical performance of a Cas13a-based lateral flow assay for detecting Neisseria gonorrhoeae in urine specimens
ABSTRACT Nucleic acid amplification testing (NAAT) for N. gonorrhoeae is unavailable in resource-limited settings. We previously developed a CRISPR-based lateral flow assay for detecting N. gonorrhoeae. We aimed to pair that assay with point-of-care DNA extraction, assess performance in clinical uri...
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American Society for Microbiology
2025-01-01
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| Series: | mSphere |
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| Online Access: | https://journals.asm.org/doi/10.1128/msphere.00677-24 |
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| author | Lao-Tzu Allan-Blitz Gordon Adams Gabriela Sanders Palak Shah Krithik Ramesh Jana Jarolimova Kevin L. Ard John A. Branda Jeffrey D. Klausner Pardis C. Sabeti Jacob E. Lemieux |
| author_facet | Lao-Tzu Allan-Blitz Gordon Adams Gabriela Sanders Palak Shah Krithik Ramesh Jana Jarolimova Kevin L. Ard John A. Branda Jeffrey D. Klausner Pardis C. Sabeti Jacob E. Lemieux |
| author_sort | Lao-Tzu Allan-Blitz |
| collection | DOAJ |
| description | ABSTRACT Nucleic acid amplification testing (NAAT) for N. gonorrhoeae is unavailable in resource-limited settings. We previously developed a CRISPR-based lateral flow assay for detecting N. gonorrhoeae. We aimed to pair that assay with point-of-care DNA extraction, assess performance in clinical urine specimens, and optimize assay kinetics. We collected urine specimens among men presenting with urethritis enrolling in a clinical trial at the Massachusetts General Hospital Sexual Health Clinic. We assessed the quantified DNA yield of detergent-based extractions with and without heat. We selected one detergent for extracting all specimens, paired with isothermal recombinase polymerase amplification for 90 minutes and lateral flow Cas13a detection, interpreted via pixel intensity analysis. We also trained a smartphone-based machine-learning model on 1,008 images to classify lateral flow results. We used the model to interpret lateral flow results from the clinical specimens. We also tested a modified amplification chemistry with a second forward primer lacking the T7-promoter to accelerate reaction kinetics. Extraction with 0.02% Triton X resulted in an average DNA yield of 2.6 × 106 copies/µL (SD ± 6.7 × 105). We treated 40 urine specimens (n = 12 positive) with 0.02% Triton X, and using quantified pixel intensity analysis, the Cas13a-based assay correctly classified all specimens (100% agreement; 95% CI 91.2%–100%). The machine-learning model correctly classified 45/45 strips in the validation data set and all 40 lateral flow strips from clinical specimens. Including the second forward primer reduced incubation time to 60 minutes. Using point-of-care DNA extraction, our Cas13a-based lateral flow N. gonorrhoeae assay demonstrated promising performance among clinical urine specimens.IMPORTANCEUsing a CRISPR-based assay we previously developed for Neisseria gonorrhoeae detection, we developed new techniques to facilitate point-of-care use. We then demonstrated the promising performance of that assay in clinical specimens. Furthermore, we developed a smartphone-based machine learning application for assisting interpretation of lateral flow strip results. Such an assay has the potential to transform the care of sexually transmitted infections in low-resource settings where diagnostic tests are unavailable. A point-of-care pathogen-specific assay, paired with the connectivity offered by a smartphone application, can also support public health surveillance efforts in such areas. |
| format | Article |
| id | doaj-art-cdf674f53bb345808457a105f723c94e |
| institution | Kabale University |
| issn | 2379-5042 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | American Society for Microbiology |
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| spelling | doaj-art-cdf674f53bb345808457a105f723c94e2025-01-28T14:00:56ZengAmerican Society for MicrobiologymSphere2379-50422025-01-0110110.1128/msphere.00677-24Preliminary clinical performance of a Cas13a-based lateral flow assay for detecting Neisseria gonorrhoeae in urine specimensLao-Tzu Allan-Blitz0Gordon Adams1Gabriela Sanders2Palak Shah3Krithik Ramesh4Jana Jarolimova5Kevin L. Ard6John A. Branda7Jeffrey D. Klausner8Pardis C. Sabeti9Jacob E. Lemieux10Division of Global Health Equity, Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts, USABroad Institute of Massachusetts Institute of Technology and Harvard, Boston, Massachusetts, USABroad Institute of Massachusetts Institute of Technology and Harvard, Boston, Massachusetts, USABroad Institute of Massachusetts Institute of Technology and Harvard, Boston, Massachusetts, USAMassachusetts Institute of Technology, Boston, Massachusetts, USADivision of Infectious Diseases, Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts, USADivision of Infectious Diseases, Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts, USADepartment of Pathology, Massachusetts General Hospital, Boston, Massachusetts, USADepartment of Population and Public Health Sciences, Keck School of Medicine, University of Southern California, Los Angeles, California, USABroad Institute of Massachusetts Institute of Technology and Harvard, Boston, Massachusetts, USABroad Institute of Massachusetts Institute of Technology and Harvard, Boston, Massachusetts, USAABSTRACT Nucleic acid amplification testing (NAAT) for N. gonorrhoeae is unavailable in resource-limited settings. We previously developed a CRISPR-based lateral flow assay for detecting N. gonorrhoeae. We aimed to pair that assay with point-of-care DNA extraction, assess performance in clinical urine specimens, and optimize assay kinetics. We collected urine specimens among men presenting with urethritis enrolling in a clinical trial at the Massachusetts General Hospital Sexual Health Clinic. We assessed the quantified DNA yield of detergent-based extractions with and without heat. We selected one detergent for extracting all specimens, paired with isothermal recombinase polymerase amplification for 90 minutes and lateral flow Cas13a detection, interpreted via pixel intensity analysis. We also trained a smartphone-based machine-learning model on 1,008 images to classify lateral flow results. We used the model to interpret lateral flow results from the clinical specimens. We also tested a modified amplification chemistry with a second forward primer lacking the T7-promoter to accelerate reaction kinetics. Extraction with 0.02% Triton X resulted in an average DNA yield of 2.6 × 106 copies/µL (SD ± 6.7 × 105). We treated 40 urine specimens (n = 12 positive) with 0.02% Triton X, and using quantified pixel intensity analysis, the Cas13a-based assay correctly classified all specimens (100% agreement; 95% CI 91.2%–100%). The machine-learning model correctly classified 45/45 strips in the validation data set and all 40 lateral flow strips from clinical specimens. Including the second forward primer reduced incubation time to 60 minutes. Using point-of-care DNA extraction, our Cas13a-based lateral flow N. gonorrhoeae assay demonstrated promising performance among clinical urine specimens.IMPORTANCEUsing a CRISPR-based assay we previously developed for Neisseria gonorrhoeae detection, we developed new techniques to facilitate point-of-care use. We then demonstrated the promising performance of that assay in clinical specimens. Furthermore, we developed a smartphone-based machine learning application for assisting interpretation of lateral flow strip results. Such an assay has the potential to transform the care of sexually transmitted infections in low-resource settings where diagnostic tests are unavailable. A point-of-care pathogen-specific assay, paired with the connectivity offered by a smartphone application, can also support public health surveillance efforts in such areas.https://journals.asm.org/doi/10.1128/msphere.00677-24Neisseria gonorrhoeaepoint of care diagnosticlateral flowclinical specimensCRISPRCas13a |
| spellingShingle | Lao-Tzu Allan-Blitz Gordon Adams Gabriela Sanders Palak Shah Krithik Ramesh Jana Jarolimova Kevin L. Ard John A. Branda Jeffrey D. Klausner Pardis C. Sabeti Jacob E. Lemieux Preliminary clinical performance of a Cas13a-based lateral flow assay for detecting Neisseria gonorrhoeae in urine specimens mSphere Neisseria gonorrhoeae point of care diagnostic lateral flow clinical specimens CRISPR Cas13a |
| title | Preliminary clinical performance of a Cas13a-based lateral flow assay for detecting Neisseria gonorrhoeae in urine specimens |
| title_full | Preliminary clinical performance of a Cas13a-based lateral flow assay for detecting Neisseria gonorrhoeae in urine specimens |
| title_fullStr | Preliminary clinical performance of a Cas13a-based lateral flow assay for detecting Neisseria gonorrhoeae in urine specimens |
| title_full_unstemmed | Preliminary clinical performance of a Cas13a-based lateral flow assay for detecting Neisseria gonorrhoeae in urine specimens |
| title_short | Preliminary clinical performance of a Cas13a-based lateral flow assay for detecting Neisseria gonorrhoeae in urine specimens |
| title_sort | preliminary clinical performance of a cas13a based lateral flow assay for detecting neisseria gonorrhoeae in urine specimens |
| topic | Neisseria gonorrhoeae point of care diagnostic lateral flow clinical specimens CRISPR Cas13a |
| url | https://journals.asm.org/doi/10.1128/msphere.00677-24 |
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