The revised definition of a new sputum smear positive pulmonary TB case is based on the presence of at least one acid-fast bacillus (AFB) in at least one sputum sample in countries with a well functioning EQA system The number of specimens required for diagnosis of smear positive pulmonary TB is two, with one of them being a morning sputum specimen. Two sputum specimens are collected over one, or two consecutive days. Of the two sputum specimens, one is collected on the spot and the other is an early morning specimen collected at home by the patient

RNTCP has established a nation wide laboratory network, encompassing over 12,500 designated sputum Microscopy Centers (DMCs), which are being supervised by Intermediate reference laboratories (IRL) at state level, and National Reference laboratories (NRL) & Central TB division at the national level. Efforts have been made to consolidate the laboratory network into a well organized one, with a defined hierarchy for carrying out sputum microscopy with external quality assessment (EQA), Drug resistance Surveillance (DRS), mycobacterium culture and Drug susceptibility testing (DST) and DOTS-Plus related activities.

The four NRLs under the program are Tuberculosis Research Centre (TRC), Chennai, National Tuberculosis Institute (NTI), Bangalore, Lala Ram Swarup Institute of Tuberculosis and Respiratory diseases (LRS), Delhi and JALMA Institute, Agra. The NRLs work closely with the IRLs, monitor and supervise the IRL’s activities and also undertake periodic training for the IRL staff in EQA, culture & DST activities.

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