Browsing by Author "Kamita, Moses."

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    Highly Sensitive Molecular Assay for the Detection of Treponema pallidum infection
    (Research square, 2024-05-02) Shiluli, Clement.; Kamath, Shwetha.; Kanoi, Bernard N.; Kimani, Racheal.; Maina, Michael.; Waweru, Harrison.; Kamita, Moses.; Ndirangu, Ibrahim.; Abkallo, Hussein M.; Oduor, Bernard.; Pamme, Nicole.; Dupaty, Joshua.; Klapperich, Catherine M.; Lolabattu, Srinivasa Raju.; Gitaka, Jesse.
    Introduction: According to the World Health Organization (WHO) more than 7 million new Treponema pallidum (T. pallidum) infections were reported among people aged 15–49 years in 2020 globally, the majority of them in developing countries. Syphilis, which is caused by T. pallidum is transmitted through contact with active lesions of a sexual partner or from an infected pregnant woman to her foetus. Gold standard T. pallidum laboratory diagnosis methods include dark-field microscopy, silver staining, direct fluorescence immunoassays and the rabbit infectivity test. However, these tests are associated with false positive or negative results. The gold standard 16S ribosomal (rRNA) gene polymerase chain reaction (PCR) is used for routine amplification of T. pallidum conserved genes. Here we report on an ultrasensitive syphilis diagnostic method, based on de novo genome mining of the T. pallidum DNA to identify identical multi repeat sequences (IMRS) as amplification primers. Methods: We used genome-mining approaches to find IMRS distributed throughout the T. pallidum genome to design a primer pair that target four repeat sequences. Genomic T. pallidum DNA was diluted from 8.14×104 to 8.14×10− 2 genome copies/𝜇l and used as template in the IMRS-based amplification assay. For performance comparison, 16S rRNA PCR assay was employed. Probit analysis was used to calculate the lower limit of detection of the T. pallidum-IMRS PCR and the conventional 16S rRNA PCR assays. Results: Probit analysis confirmed that the T. pallidum-IMRS primers offered higher test sensitivity of 0.03 fg/𝜇L compared to the 16S rRNA PCR (0.714 pg/𝜇L). Using the T. pallidum-IMRS primers, we were able to observe considerable isothermal amplification of genomic DNA at a starting concentration of 0.01 pg/µL. Conclusion: De novo genome mining of T. pallidum IMRS as amplification primers can serve as a platform for developing ultrasensitive diagnostics for Syphilis and potentially a wide range of infectious pathogens.
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    Improving gonorrhoea molecular diagnostics: Genome mining-based identification of identical multi-repeat sequences (IMRS) in Neisseria gonorrhoeae
    (Cell Press, 2024-03-30) Shiluli, Clement.; Kamath, Shwetha.; Kanoi, Bernard N.; Kimani, Racheal.; Maina, Michael.; Waweru, Harrison.; Kamita, Moses.; Ndirangu, Ibrahim.; Abkallo, Hussein M.; Oduor, Bernard.; Pamme, Nicole.; Dupaty, Joshua.; Klapperich, Catherine M.; Lolabattu, Srinivasa Raju.; Gitaka, Jesse.
    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.