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Clinical and Laboratory Standards Institute guideline M48 – Laboratory Detection and Identification of Mycobacteria covers topics related to laboratory diagnosis of mycobacterial infections, including safety and risk assessment, referrals, clinical significance, acceptable specimen types, specimen collection, transport, and storage, specimen processing methods, microscopy for direct detection, molecular methods for directly detecting mycobacteria in patient specimens, culture methods, contamination issues, reporting, quality control, and conventional identification methods as they relate to mass spectrometry and genotypic procedures. Recommendations for managing the unique challenges associated with the increasing incidence of Mycobacterium tuberculosis and nontuberculous mycobacteria infections are included.