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Phenotypic drug resistance and genome sequencing based identified mutations linked to resistance in Mycobacterium tuberculosis isolated from extrapulmonary clinical specimens
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  • Published: 15 February 2026

Phenotypic drug resistance and genome sequencing based identified mutations linked to resistance in Mycobacterium tuberculosis isolated from extrapulmonary clinical specimens

  • Hilina Mollalign1,2,
  • Dawit Hailu Alemayehu3,
  • Kalkidan Melaku3,
  • Abaysew Ayele3,
  • Dawit Chala1,
  • Getu Diriba1,
  • Bazezew Yenew1,
  • Muluwork Getahun1,
  • Bethlehem Adnew3,
  • Shewki Moga1,
  • Jeffrey Michael Collins4,
  • Arash Ghodousi5,6,
  • Dereje Beyene2,
  • Kidist Bobosha3 &
  • …
  • Liya Wassie3 

Scientific Reports , Article number:  (2026) Cite this article

We are providing an unedited version of this manuscript to give early access to its findings. Before final publication, the manuscript will undergo further editing. Please note there may be errors present which affect the content, and all legal disclaimers apply.

Subjects

  • Diseases
  • Microbiology

Abstract

Globally, drug-resistant tuberculosis (DR-TB) is responsible for 13% of mortality attributable to antimicrobial resistance. In Ethiopia, extrapulmonary tuberculosis (EPTB) is a significant public health challenge, and drug resistance (DR) in EPTB is often overlooked. In a cross-sectional study conducted between August 2022 and October 2023, we aimed to explore the magnitude of phenotypic drug resistance and identify genetic mutations linked to resistance using 189 Mycobacterium tuberculosis (MTB) isolates cultured from extrapulmonary clinical specimens. Additionally, we assessed the agreement between phenotypic and whole genome sequencing (WGS) based genotypic drug resistance detection. We performed phenotypic drug sensitivity testing (pDST) using the liquid culture BD BACTEC™ MGIT™ 960 system and WGS using Illumina NextSeq500/550. The genomic data analysis pipelines MTBSeq and TBProfiler were used to predict drug resistance-conferring mutations. The agreement between the pDST and WGS based drug resistances was analyzed using SPSS version 29.0. Our study results showed that the prevalence of any forms of phenotypic resistance to at least one anti-TB drug was 16.9% (95%: CI, 11.9% – 23.1%). Isoniazid-resistant rifampicin-susceptible-TB (Hr-TB) accounted for 2.6% (95%: CI, 0.9% − 6.1%) and multi-drug-resistant TB (MDR-TB) accounted for 4.2% (95%CI: 1.8% − 8.2%). The prevalence of MDR-TB was 2.4% (95%CI: 0.6% − 5.9%) among newly diagnosed and, 21.1% (95%CI: 6.1% − 45.6%) among previously treated cases. More rifampicin-resistances were detected using WGS (8.75%) than the pDST (4.2%). We identified a putative compensatory mutation for rifampicin resistance (rpoBSer450Leu, rpoC Asp747Ala) and a previously unreported mutations on the hotspot rifampicin resistance determining region (rpoB_p. Asn438Thr). There were 3.75% rifampicin mono-resistant-TB (RMR-TB) cases detected through WGS and represented nearly half of the total RR/MDR-TB cases. Mutations conferring rifampicin resistance-interim (rpoB.Ser450Ala) represented most of these RMR-TB. Changes in ethA genes associated with ethionamide resistance were the most common second-line resistance among MDR-TB isolates. There was a substantial to strong agreement between the pDST and WGS for the detection of resistance to first line anti TB drugs. In conclusion, MDR-TB, Hr-TB, and interim-RMR-TB are public health challenges in the realm of EPTB in Ethiopia. Given, most RMR-TB were detected as borderline RR-TB and were missed by both pDST and the rapid molecular diagnostics currently in place, integrating NGS into the national guidelines is highly relevant. This would help for comprehensive detection of mutations including rare and novel mutations missed by the conventional approaches and planning tailored therapy. Investigation of the role of the identified compensatory mutation and the new mutation we reported on the target gene of the drug rifampicin is warranted.

Data availability

All data associated with the main findings are provided in tables and figures. The raw sequence data generated in this study have been deposited in the National Center for Biotechnology Information (NCBI) under BioProject number [PRJNA1174701] (https:/www.ncbi.nlm.nih.gov/bioproject/PRJNA1174701).

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Acknowledgements

This work was supported, in part by the NIH Fogarty International Center Global Infectious Diseases grant D43TW009127, the Ethiopian Public Health Institute (EPHI), the core support from the Armauer Hansen Research Institute (AHRI), and Addis Ababa University. The supporting institutes had no role in the study design, data collection and analyses, decision for publication, or manuscript preparation. We thank Ashleigh Nicole Cox from Georgia State University for providing us with an English Language edit to this research paper.

Funding

This study did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Author information

Authors and Affiliations

  1. Infectious Diseases Research Directorate, Ethiopian Public Health Institute, Addis Ababa, P.O. Box 1242, Addis Ababa, Ethiopia

    Hilina Mollalign, Dawit Chala, Getu Diriba, Bazezew Yenew, Muluwork Getahun & Shewki Moga

  2. Department of Microbial Sciences and Genetics, Addis Ababa University, Addis Ababa, Ethiopia

    Hilina Mollalign & Dereje Beyene

  3. Armauer Hansen Research Institute, Addis Ababa, Ethiopia

    Dawit Hailu Alemayehu, Kalkidan Melaku, Abaysew Ayele, Bethlehem Adnew, Kidist Bobosha & Liya Wassie

  4. Rollins School of Public Health, Emory University, Atlanta, GA, USA

    Jeffrey Michael Collins

  5. Vita-Salute San Raffaele University, Milan, Italy

    Arash Ghodousi

  6. Division of Immunology, Transplantation, and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy

    Arash Ghodousi

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  1. Hilina Mollalign
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  2. Dawit Hailu Alemayehu
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Contributions

HM designed the study, analyzed data and wrote the manuscript including comments from all authors. BY and GD conducted phenotypic drug sensitivity tests. DHA, KM and DC conducted molecular characterization. AG, AA and BA performed bioinformatics analysis. DB, SM, JMC, MG, LW, KB and AG reviewed the manuscript. DB, KB and LW supervised the study. All authors read and approved of the final manuscript.

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Correspondence to Hilina Mollalign.

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Mollalign, H., Alemayehu, D.H., Melaku, K. et al. Phenotypic drug resistance and genome sequencing based identified mutations linked to resistance in Mycobacterium tuberculosis isolated from extrapulmonary clinical specimens. Sci Rep (2026). https://doi.org/10.1038/s41598-026-40253-8

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  • Received: 21 October 2024

  • Accepted: 11 February 2026

  • Published: 15 February 2026

  • DOI: https://doi.org/10.1038/s41598-026-40253-8

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Keywords

  • Extrapulmonary tuberculosis
  • Whole genome sequencing
  • Ethiopia
  • Compensatory mutation
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