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Abay Atnafu,1 Liya Wassie,1 Melaku Tilahun,1,&ast; Selfu Girma,1 Yohannes Zenebe,2 Muluye Abebe Beyene,1 Ashenafi Alemu,1 Emnet Fisseha,1 Haymanot Agze,1 Tigist Desta,1 Kassu Desta,3,&ast; Kidist Bobosha1,&ast; 1Armauer Hansen Research Institute, Addis Ababa, Ethiopia; 2Department of Medical Laboratory Sciences, College of Medicine and Health Sciences, Bahir Dar University, Bahir Dar, Ethiopia; 3Department of Medical Laboratory Sciences, College of Health Sciences, Addis Ababa University, Addis Ababa, Ethiopia&ast;These authors contributed equally to this workCorrespondence: Abay Atnafu, Tel +251912205822, Email ayatab.ayele7@gmail.comBackground: Extrapulmonary tuberculosis (EPTB), particularly tubercular lymphadenitis (TBLN), remains to pose a huge public health problem in Ethiopia. A significant number of TBLN patients who completed a full course anti-TB treatment regimen were reported to have enlarged lymph nodes and other TB-like clinical presentations. This could either be from a paradoxical reaction or microbiological relapse, possibly due to mono/multi-drug resistance.Objective: To investigate the rate of mono and multidrug resistance patterns of Mycobacterium tuberculosis as a cause of the observed treatment failures in clinically diagnosed and anti-TB treatment (newly or previously)-initiated LN patients.Methods: A cross-sectional study was conducted on 126 TBLN-suspected and previously treated patients between March and September 2022. Data were analyzed using SPSS (Version 26.0). Descriptive statistics were used to determine the frequency, percentage, sensitivity, specificity, and positive and negative predictive values. The level of agreement was determined using Cohen’s kappa and a Chi-square test was used to measure the association between risk factors and laboratory test outcomes. A P-value < 0.05 was considered statistically significant.Results: Mycobacterium tuberculosis was confirmed in 28.6% (N=36) of the 126 cases using BACTEC MGIT 960 culture detection method. Approximately, 13% (N=16) of the samples were collected from previously treated TBLN patients, of which 5/16 (31.3%) were multi-drug resistant, 7/16 were drug-sensitive and 4/16 were culture negative. To rule out other non-tuberculous agents, all samples were grown on blood and Mycosel agar plates, and no growth was detected.Conclusion: The emergence of drug resistant (DR) TB seems to not just be limited to pulmonary form but also to TBLN. In this study we observed a considerable number of microbiologically confirmed relapses among previously treated cases, possibly indicating the need for confirmation of drug resistance using rapid molecular methods or phenotypical methods during treatment follow up.Keywords: tubercular lymphadenitis, relapse, drug sensitivity testing, DST, FNA