Abstract
The worldwide dissemination of Mycobacterium tuberculosis strains has led to the study of their genetic diversity. One of the most used genotyping methods is spoligotyping, based on the detection of spacers in the clustered regularly interspaced short palindromic repeats (CRISPR) locus. This study assessed the performance of a microbead-based spoligotyping assay using samples extracted from Ziehl-Neelsen-stained smear-microscopy preparations and described the genetic diversity of Mycobacterium tuberculosis among new TB patients in Southern Nations, Nationalities and Peoples’ Region (SNNPR) in Ethiopia. Among the 91 samples analysed, 59 (64.8%) generated spoligotyping patterns. Fifty (84.7%) samples were classified into 12 clusters (mostly Lineage 4 or 3) comprising 2–11 samples and nine had unique spoligotyping patterns. Among the 59 spoligotyping patterns, 25 belonged to the T1 sublineage, 11 to the T3-ETH, 5 to the URAL, 4 to the H3 and 14 to other L4 sublineages. There was a remarkable variation in genetic distribution in SNNPR compared to other regions of the country. Microbead-based spoligotyping is an easy-to-perform, high-throughput assay that can generate genotyping information using material obtained from smear microscopy preparations. The method provides an opportunity to obtain data of the M. tuberculosis genetic epidemiology in settings with limited laboratory resources.
Highlights
The integrity of sputum for direct extraction
Few studies have reported it is possible to genotype M. tuberculosis from DNA recovered from Ziehl-Neelsen (ZN) stained microscopy preparations[5]
We performed microbead-based spoligotyping directly from ZN-stained microscopy preparations to assess the proportion of samples that could be genotyped when specimens are collected under programmatic conditions in a remote population of Ethiopia, and analysed the genetic distribution of M. tuberculosis among new TB cases
Summary
A total of 91 consecutive smear 2+ or 3+ samples were selected. Interpretable spoligotyping patterns (see Material and Methods) were obtained for 59 (64.8%) of the 91 DNA samples extracted. Another study of 954 M. tuberculosis complex strains from all Ethiopia showed that the most common lineage was Euro-American (lineage 4) (71%), which was spread all over the country[8,13]. DNA extracted from ZN-stained slides has been used previously for spoligotyping, resulting in identical patterns as those obtained with cultured M. tuberculosis strains[14,15]. The participants for this study were diagnosed solely using smear microscopy and the genetic distribution of the strains is partially different to that reported in other studies These differences may be due to the different geographical location and the different classifications used, but may reflect the less selected nature of the participants and highlight the need to develop methods that do not rely on highly selected patients in referral centres to obtain a better representation of the most common circulating genotypes in specific settings. Microbead-based spoligotyping from microscopy preparations is an easy-to-perform and high-throughput assay that could be useful to provide genotyping information in locations without culture facilities and further studies are warranted
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
