Improved NAAT assay for the diagnosis of onchocerciasis and its use for detection of circulating cell free DNA.
| Autor: | Bennuru S; Laboratory of Parasitic Diseases, NIAID, NIH, Bethesda, MD USA., Kodua F; Laboratory of Parasitic Diseases, NIAID, NIH, Bethesda, MD USA.; Howard University Hospital, Department of Internal Medicine, Washington DC, USA., Dahlstrom E; Genomics Unit, Research Technologies Branch, NIAID, NIH, Hamilton, USA., Nutman TB; Laboratory of Parasitic Diseases, NIAID, NIH, Bethesda, MD USA. |
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| Jazyk: | angličtina |
| Zdroj: | The Journal of infectious diseases [J Infect Dis] 2024 Sep 23. Date of Electronic Publication: 2024 Sep 23. |
| DOI: | 10.1093/infdis/jiae462 |
| Abstrakt: | Background: The co-endemicity of onchocerciasis with other filariae warrants a better diagnostic tool for elimination efforts that are highly sensitive and specific for use in surveillance and for xenomonitoring. Methods: Utilizing NGS data, qPCR assays were designed for 15 highly repeated targets from O. volvulus (Ov) and 11 from O. ochengi (Oo). The two most promising repeats Ov15R and Ov16R from Ov and OoR1 and OoR5 from Oo were selected for further testing. Results: The analytical sensitivity of both Ov15R and Ov16R were similar with limits of detection (LOD) at 1 fg, and with specificity approaching 100%. Using DNA obtained previously from skin snips form Ov-infected subjects, Ov16R identified 17 additional samples as positive for Ov infections when compared to the "gold standard" O-150. Although Ov16R failed to detect circulating cell-free DNA (ccfDNA) in plasma of Ov-infected individuals, 1ml urine samples from Ov-infected individuals were variably positive for ccfDNA. Interestingly plasma levels of ccfDNA were shown to be easily measurable as early as 12-24 following treatment. To enable processing of larger volumes of urine for better sensitivity, a chitosan-based filter technique was developed that efficiently captured ccfDNA from 1-15ml of urine. Interestingly, Ov15R, Ov16R and O-150 map to the same region(s) of the Ov genome, prompting a redesign of the standard O-150 qPCR. This resulted in a new O-150R assay that performs on par with Ov15R/Ov16R. Conclusion: Each of these assays improve dramatically detection of Ov DNA and can easily be configured to field-friendly isothermal formats. (Published by Oxford University Press on behalf of Infectious Diseases Society of America 2024.) |
| Databáze: | MEDLINE |
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