Improving gonorrhoea molecular diagnostics: Genome mining-based identification of identical multi-repeat sequences (IMRS) in Neisseria gonorrhoeae
Background: Curable sexually transmitted infections (STIs), such as Neisseria gonorrhoeae (N. gonorrhoeae), are a major cause of poor pregnancy outcomes. The infection is often asymptomatic in pregnant women, and a syndrome-based approach of testing leads to a missed diagnosis. Culture followed by m...
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| Format: | Article |
| Language: | English |
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Elsevier
2024-03-01
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| Series: | Heliyon |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2405844024033759 |
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| author | Clement Shiluli Shwetha Kamath Bernard N. Kanoi Racheal Kimani Michael Maina Harrison Waweru Moses Kamita Ibrahim Ndirangu Hussein M. Abkallo Bernard Oduor Nicole Pamme Joshua Dupaty Catherine M. Klapperich Srinivasa Raju Lolabattu Jesse Gitaka |
| author_facet | Clement Shiluli Shwetha Kamath Bernard N. Kanoi Racheal Kimani Michael Maina Harrison Waweru Moses Kamita Ibrahim Ndirangu Hussein M. Abkallo Bernard Oduor Nicole Pamme Joshua Dupaty Catherine M. Klapperich Srinivasa Raju Lolabattu Jesse Gitaka |
| author_sort | Clement Shiluli |
| collection | DOAJ |
| description | Background: Curable sexually transmitted infections (STIs), such as Neisseria gonorrhoeae (N. gonorrhoeae), are a major cause of poor pregnancy outcomes. The infection is often asymptomatic in pregnant women, and a syndrome-based approach of testing leads to a missed diagnosis. Culture followed by microscopy is inadequate and time-consuming. The gold standard nucleic acid amplification tests require advanced infrastructure settings, whereas point-of-care tests are limited to immunoassays with sensitivities and specificities insufficient to accurately diagnose asymptomatic cases. This necessitates the development and validation of assays that are fit for purpose. Methods: We identified new diagnostic target biomarker regions for N. gonorrhoeae using an algorithm for genome mining of identical multi-repeat sequences (IMRS). These were then developed as DNA amplification primers to design better diagnostic assays. To test the primer pair, genomic DNA was 10-fold serially diluted (100 pg/μL to 1 × 10−3 pg/μL) and used as DNA template for PCR reactions. The gold standard PCR using 16S rRNA primers was also run as a comparative test, and both assay products were resolved on 1% agarose gel. Results: Our newly developed N. gonorrhoeae IMRS-PCR assay had an analytical sensitivity of 6 fg/μL representing better sensitivity than the 16S rRNA PCR assay with an analytical sensitivity of 4.3096 pg/μL. The assay was also successfully validated using clinical urethral swab samples. We further advanced this technique by developing an isothermal IMRS, which was both reliable and sensitive for detecting cultured N. gonorrhoeae isolates at a concentration of 38 ng/μL. Combining isothermal IMRS with a low-cost lateral flow assay, we were able to detect N. gonorrhoeae amplicons at a starting concentration of 100 pg/μL. Conclusion: Therefore, there is a potential to implement this concept within miniaturized, isothermal, microfluidic platforms, and laboratory-on-a-chip diagnostic devices for highly reliable point-of-care testing. |
| first_indexed | 2024-04-24T13:50:35Z |
| format | Article |
| id | doaj.art-93f522691f294b85894c9ec82269a299 |
| institution | Directory Open Access Journal |
| issn | 2405-8440 |
| language | English |
| last_indexed | 2024-04-24T13:50:35Z |
| publishDate | 2024-03-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Heliyon |
| spelling | doaj.art-93f522691f294b85894c9ec82269a2992024-04-04T05:04:40ZengElsevierHeliyon2405-84402024-03-01106e27344Improving gonorrhoea molecular diagnostics: Genome mining-based identification of identical multi-repeat sequences (IMRS) in Neisseria gonorrhoeaeClement Shiluli0Shwetha Kamath1Bernard N. Kanoi2Racheal Kimani3Michael Maina4Harrison Waweru5Moses Kamita6Ibrahim Ndirangu7Hussein M. Abkallo8Bernard Oduor9Nicole Pamme10Joshua Dupaty11Catherine M. Klapperich12Srinivasa Raju Lolabattu13Jesse Gitaka14Centre for Research in Infectious Diseases, College of Graduate Studies and Research, Mount Kenya University, Thika, KenyaDivision of Research and Development, Jigsaw Bio Solutions Private Limited, Bangalore, IndiaCentre for Research in Infectious Diseases, College of Graduate Studies and Research, Mount Kenya University, Thika, KenyaCentre for Research in Infectious Diseases, College of Graduate Studies and Research, Mount Kenya University, Thika, KenyaCentre for Research in Infectious Diseases, College of Graduate Studies and Research, Mount Kenya University, Thika, KenyaCentre for Research in Infectious Diseases, College of Graduate Studies and Research, Mount Kenya University, Thika, KenyaCentre for Research in Infectious Diseases, College of Graduate Studies and Research, Mount Kenya University, Thika, KenyaCentre for Research in Infectious Diseases, College of Graduate Studies and Research, Mount Kenya University, Thika, KenyaAnimal and Human Health Program, International Livestock Research Institute, Nairobi, KenyaAnimal and Human Health Program, International Livestock Research Institute, Nairobi, KenyaDepartment of Materials and Environmental Chemistry, Stockholm University, SwedenDepartment of Biomedical Engineering, Boston University, Boston, MA, USADepartment of Biomedical Engineering, Boston University, Boston, MA, USADivision of Research and Development, Jigsaw Bio Solutions Private Limited, Bangalore, India; Corresponding author.Centre for Research in Infectious Diseases, College of Graduate Studies and Research, Mount Kenya University, Thika, Kenya; Corresponding author.Background: Curable sexually transmitted infections (STIs), such as Neisseria gonorrhoeae (N. gonorrhoeae), are a major cause of poor pregnancy outcomes. The infection is often asymptomatic in pregnant women, and a syndrome-based approach of testing leads to a missed diagnosis. Culture followed by microscopy is inadequate and time-consuming. The gold standard nucleic acid amplification tests require advanced infrastructure settings, whereas point-of-care tests are limited to immunoassays with sensitivities and specificities insufficient to accurately diagnose asymptomatic cases. This necessitates the development and validation of assays that are fit for purpose. Methods: We identified new diagnostic target biomarker regions for N. gonorrhoeae using an algorithm for genome mining of identical multi-repeat sequences (IMRS). These were then developed as DNA amplification primers to design better diagnostic assays. To test the primer pair, genomic DNA was 10-fold serially diluted (100 pg/μL to 1 × 10−3 pg/μL) and used as DNA template for PCR reactions. The gold standard PCR using 16S rRNA primers was also run as a comparative test, and both assay products were resolved on 1% agarose gel. Results: Our newly developed N. gonorrhoeae IMRS-PCR assay had an analytical sensitivity of 6 fg/μL representing better sensitivity than the 16S rRNA PCR assay with an analytical sensitivity of 4.3096 pg/μL. The assay was also successfully validated using clinical urethral swab samples. We further advanced this technique by developing an isothermal IMRS, which was both reliable and sensitive for detecting cultured N. gonorrhoeae isolates at a concentration of 38 ng/μL. Combining isothermal IMRS with a low-cost lateral flow assay, we were able to detect N. gonorrhoeae amplicons at a starting concentration of 100 pg/μL. Conclusion: Therefore, there is a potential to implement this concept within miniaturized, isothermal, microfluidic platforms, and laboratory-on-a-chip diagnostic devices for highly reliable point-of-care testing.http://www.sciencedirect.com/science/article/pii/S2405844024033759Identical multirepeat sequencesNeisseria gonorrhoeaeIsothermal |
| spellingShingle | Clement Shiluli Shwetha Kamath Bernard N. Kanoi Racheal Kimani Michael Maina Harrison Waweru Moses Kamita Ibrahim Ndirangu Hussein M. Abkallo Bernard Oduor Nicole Pamme Joshua Dupaty Catherine M. Klapperich Srinivasa Raju Lolabattu Jesse Gitaka Improving gonorrhoea molecular diagnostics: Genome mining-based identification of identical multi-repeat sequences (IMRS) in Neisseria gonorrhoeae Heliyon Identical multirepeat sequences Neisseria gonorrhoeae Isothermal |
| title | Improving gonorrhoea molecular diagnostics: Genome mining-based identification of identical multi-repeat sequences (IMRS) in Neisseria gonorrhoeae |
| title_full | Improving gonorrhoea molecular diagnostics: Genome mining-based identification of identical multi-repeat sequences (IMRS) in Neisseria gonorrhoeae |
| title_fullStr | Improving gonorrhoea molecular diagnostics: Genome mining-based identification of identical multi-repeat sequences (IMRS) in Neisseria gonorrhoeae |
| title_full_unstemmed | Improving gonorrhoea molecular diagnostics: Genome mining-based identification of identical multi-repeat sequences (IMRS) in Neisseria gonorrhoeae |
| title_short | Improving gonorrhoea molecular diagnostics: Genome mining-based identification of identical multi-repeat sequences (IMRS) in Neisseria gonorrhoeae |
| title_sort | improving gonorrhoea molecular diagnostics genome mining based identification of identical multi repeat sequences imrs in neisseria gonorrhoeae |
| topic | Identical multirepeat sequences Neisseria gonorrhoeae Isothermal |
| url | http://www.sciencedirect.com/science/article/pii/S2405844024033759 |
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