Background: Nontuberculosis mycobacterium (NTM) is the emerging group of organisms being recognized as etiological agents for diverse clinical conditions such as lymphadenitis, cutaneous, and pulmonary or disseminated lesions. Diverse background patients can acquire these infections such as immunocompetent, immunocompromised patients, or postoperative settings. Rapid addition of newer strains to this group necessitates heightened suspicion in the clinical settings. Specific requirements for cultures, biochemical testing, and molecular methods are needed to diagnose these organisms. Methods: The prospective study conducted at Nizam's Institute of Medical Sciences from January 2019 to December 2021 using various clinical samples using molecular techniques such as line probe assay and hsp-65 gene sequencing to discover new NTM species. The management is challenging since it requires prolonged treatment, multiple drugs, drug resistance, and individualization of treatment in the combination of surgery if needed. In this article, we describe three different NTM species which were not reported in India and highlight to consider these organisms in adequate clinical situation. Results: Mycobacterium iranicum is a rare strain with quick growth and scotochromogenic colonies that are orange-colored. Eight distinct strains were discovered in clinical samples from six different countries: Two each from Iran, Italy, Greece, the Netherlands, Sweden, and the United States. Two of the strains were recovered from cerebrospinal fluid, which is unusual. Mycobacterium species AW6 is an unidentified and unclassified Mycobacterium according to NCBI taxonomy. Mycobacteria malmoense has been linked to lymphadenitis, notably cervical adenitis in children, and pulmonary infection in the majority of cases. Using Line Probe Assay and hsp-65 gene sequencing, novel and uncommon species of NTM were detected from a clinical samples, including sputum and tissue. Conclusion: We report three unusual species of NTMs: M. iranicum, M. species-AW6, and M. malmoense for the first time in India. Novel and rare emerging species of NTMs need to be considered in diverse clinical situations for appropriate therapy and good clinical outcomes.

Introduction: Non-tuberculous Mycobacteria (NTM) pulmonary disease is often unrecognised or misdiagnosed as Mycobacterium tuberculosis (MTB), Multi-Drug Resistant Tuberculosis (MDRTB), because of similar clinical presentation in counties with high burden of Tuberculosis (TB) including India. In India due to lack of awareness among clinicians and lack of laboratory facilities to diagnose these infections, its prevalence is largely unknown. Aim: To evaluate the efficacy of identification of NTM species by Matrix Assisted Laser Desorption Ionisation Time of Flight (MALDI-TOF) and Heat Shock Protein (hSP65) gene sequencing and to determine their Antimicrobial Susceptibility Testing (AST). Materials and Methods: All the clinical specimens from pulmonary and extra-pulmonary TB suspects at Nizam’s Institute of Medical Sciences, Hyderabad, Telangana, India, over a period of one year i.e., from June 2017 to May 2018 were included in the study. The specimens were subjected to microscopy, culture and GeneXpert. The NTMs isolated in the culture were further characterised genotypically by MALDI-TOF and hsp65 gene sequencing. The identified NTM isolates were subjected to AST. All the methods were followed as per the standard protocols. Data was analysed using SPSS 25. Results: A total of 1085 samples were processed out of which Mycobacteria was detected in 201 cases (18.5%). Among the culture positives, MTB complex was detected in 146 cases (13.5%) and NTM in 55 (5.06%). Mycobacterium abscessus was the predominant isolate. The most common co-morbidities were bronchiectasis and Chronic Obstructive Pulmonary Disease (COPD). Linezolid, clarithromycin, moxifloxacin and amikacin showed high sensitivity. Conclusion: Molecular assay helps in rapid identification which can lead to targeted therapy and can thus combat antimicrobial resistance. The MALDI-TOF and hsp65 gene sequencing also offers quick results at a low cost and is easy to perform hence it can be considered as an alternate diagnostic tool for identification.