Background and Objectives: This research aimed to explore the genetic diversity and phylogenetic relationships of My- cobacterium tuberculosis (Mtb) strains, as well as to assess their drug susceptibility, specifically in strains isolated from immigrant patients attending the Referral Tuberculosis Laboratory in Mashhad. Materials and Methods: A total of 52 sputum samples isolated from patients were examined utilizing the Mycobacterial Interspersed Repetitive-Unit Variable Number of Tandem Repeats (MIRU-VNTR). Drug-susceptibility testing against ri- fampin (RIF) and isoniazid (INH) was measured utilizing the proportional strategy. Thereafter, for more examination, Xpert MTB/RIF and multiplex allele-specific PCR (MAS-PCR) was performed to determine RIF and INH-resistance within the Mtb strains. Results: Among 52 Mtb isolates, 2 (3.8%) were resistant to rifampin and one isolate was resistant to both INH and RIF and considered as multidrug-resistance (MDR) isolate. According to MIRU-VNTR, the most prominent genetic-variation pat- terns of these samples, were related to NEW-1 (n=18, 34.6%), followed by CAS/Delhi (n=17, 32.7%), Haarlem (n=12, 23%), Uganda I (n=2, 3.8%), S (n=1, 1.9%), Beijing (n=1, 1.9%), and unknown (n=1, 1.9%) genotypes. The statistical analysis showed that the estimated percentage of the recent TB-transmission in this study was 0.21%. Conclusion: The result of this study indicated a great diversity of MTBC circulating among Afghan-immigrants which might be one of the reasons for the infection to become active. The relatively high percentage of resistant isolates in the studied population shows the importance of screening the immigrants especially at the entry borders and treatment and follow up of patients, to control TB-incidence in country.

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