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Lab Contributions


The Objective of the National Reference Laboratory

1. To Provide Technical Support to Central TB Division

  • Analyses data collected from the states, assesses the performance and gives feedback to CTD for effective implementation of RNTCP.
  • As a member of the National Lab Committee, provides technical expertise on External Quality Assessment of sputum smear microscopy.
  • Provides technical inputs as member of the DOTS Plus committee.
  • Undertakes DOTS Plus appraisals of the states as a committee member.
  • As member of the National Air borne & Infection Control committee it helps in formulating guidelines for infection control.
  • By carrying out External Quality assurance of sputum smear microscopy through on site evaluation of the state level laboratories.
  • Training in Culture and DST by Solid / Liquid culture / Molecular methods.
  • Diagnosis of Multi Drug Resistant Tuberculosis for the states which are in the process of certification for Culture & DST Laboratory.
  • Diagnosis of Extensively Drug Resistant Tuberculosis by doing 2nd line DST for the states supervised by the NTI.
  • To provide National data on the prevalence of drug resistance among new and previously treated cases of tuberculosis by undertaking periodic drug resistance survey.

2. Human Resource Development

  • A nodal centre for training Laboratory personnel in
  • External Quality Assessment of sputum smear microscopy
  • Culture and Drug Susceptibility testing for detection of Drug Resistant Tuberculosis by Solid (Lowenstein Jensen) and Liquid Culture (MGIT 960).
  • Training in Geno Type MTBDR Plus assay, Line Probe Assay.
  • Preventive Maintenance and Minor Repairs of the Binocular Microscope and also Facilitates for the Laboratory component in the RNTCP Modular Training.

3. External Quality Assurance

  • Sputum smear microscopy

To supervise and monitor the External Quality Assessment of sputum smear microscopy in the states allocated to NTI by:

  • performing periodic On-Site Evaluation (OSE) of IRLs as per guidelines
  • Analyzing the recommendations made by the state EQA team during their visit to the districts.
  • Assessing the quality of OSEs performed by the IRLs to the districts.
  • Conducting panel testing for IRL Microbiologists and Lab technicians.
  • Reviewing the Random Blinded Re-checking activities conducted by the DTOs.
  • Culture & DST

    • To conduct supervisory visit to laboratories for strengthening the capacity building and subsequently monitor the proficiency in various technologies.
    • To conduct the annual Proficiency testing in Line Probe Assay.

4. Technical support to States

  • To conduct proficiency testing for accreditation of state level laboratories i.e. the Intermediate Reference Laboratories, Medical College laboratories and other private laboratories for the culture and drug susceptibility test by solid culture (Lowenstein Jensen), Liquid culture (MGIT 960) and Molecular techniques such as Line Probe assay.
  • To conduct proficiency testing for certification of IRLs in LPA.
  • To perform 2nd line DST on the samples received from the state laboratories.

5. Operational Research

  • To Coordinate and conduct Operational Research for the Revised National Tuberculosis Control Programme.

6. To provide technical support to WHO , UNION and SAARC countries

  • By organizing and conducting training programmes in the Quality Assurance for sputum smear microscopy, Culture & Drug Susceptibility testing for the SAARC countries
  • Training in LED FM and Operational Research workshops for the UNION

Facilities Available

I) Smear Microscopy Services:

Ziehl Neelson method

Fluorescent microscopy

II) Culture and DST Services

Solid Culture, Identification and DST (LJ Medium)

  • Clinical samples are processed using the NALC NaOH method or the modified Petroff’s method of homogenization and decontamination. The centrifuged deposit is inoculated onto Lowenstein Jensen medium and incubated at 37Deg C for growth up to 8 weeks. Colonies of Mycobacterium tuberculosis are typically rough, tough and buff in color and appear within 8 weeks of incubation.
  • The isolate is confirmed to be Mycobacterium tuberculosis by performing biochemical tests such as Niacin Production, Nitrate Reductase test and no growth on Para nitro Benzoic acid containing media (500gms).
  • Drug Susceptibility testing by the Proportion sensitivity method is routinely being performed. The isolates are tested against four first line drugs such as Streptomycin, Isoniazid, Rifampicin and Ethambutol for MDRTB diagnosis and against four second line drugs viz Amikacin, Kanamycin, Capreomycin and Ofloxacin for XDRTB diagnosis. DST reading is taken at 28th and 42nd day of incubation.
  • The absolute concentration method or the Minimal inhibitory concentration is performed routinely for confirmation of discrepant DST results.

Liquid Culture, Identification and DST (MGIT 960 Automated System)

  • Clinical samples which are processed by the NALC NaOH method alone can be utilized for liquid culture. Both MDRTB & XDRTB diagnosis are being performed by testing the culture isolates against first line and second line drugs.
  • Isolates are confirmed to be Mycobacterium tuberculosis using an immunochromatographic test.

III) Molecular Tests for Drug Resistance Detection

Genotype MTBDR plus test – Line Probe Assay (LPA)

  • Rapid detection of MDRTB can be performed by the Line Probe Assay.
  • The Genotype MTBDR plus test is based on the DNA STRIP technology and permits the molecular genetic identification of the M.tuberculosis complex and its resistance to rifampicin or isoniazid from culture isolates or directly from smear positive pulmonary sputum samples.
  • The identification of rifampicin resistance is enabled by the detection of the most significant mutations of the rpo B gene. For testing high level of isoniazid resistance the kat G gene is examined and for testing low level isoniazid resistance the inh A gene is examined.
  • The test can be performed only on smear positive sputum samples.

Cartridge based Nucleic Acid Amplification Test (CBNAAT) GeneXpert

  • Is an automated Nucleic Acid Amplification test (NAAT) that is specific for TB and detects resistance to Rifampicin through the rpoB gene on both smear positive and smear negative sputum samples is also being used for MDRTB diagnosis.
  • It is rapid, closed, isothermal system using multiple primers. This test is particularly useful while screening diagnostic cases that are sero-positive for HIV infection and /or are smear negative with radiological signs of pulmonary TB.

Genetic Sequencer

  • Sequencing of the Tuberculosis genome is undertaken to identify and establish resistance to different drugs.

IV) Mycolic Acid Profiling for Non-Tuberculous Mycobacteria (NTM) by High

Performance Liquid Chromatography (HPLC)

  • Identification of suspected non-tuberculous mycobacterial isolates encountered among the samples processed in the laboratory and build a spectral library of those non-tuberculous mycobacteria for which standard chromatograms are not available.

Current activities of NRL


The laboratory has the facility to undertake Solid and MGIT 960 Liquid culture Drug susceptibility testing for MDR & XDRTB Diagnosis. The proportion sensitivity testing method of DST for first line (S, H, R, E) and second line drugs (Am, Cm, Km, Ofx,) are routinely being used. The molecular method such as the Line Probe Assay is also used for rapid diagnosis of MDRTB.


ZN and Fluorescent microscopy are routinely performed for all clinical samples received at the laboratory.

Solid culture

MDR TB Diagnosis of sputum samples received from Jammu and Kashmir are being performed by Solid culture system (LJ). DST for four first line drugs are performed by the proportion sensitivity method.

Liquid culture

XDRTB Diagnosis of culture isolates received from the following laboratories are being performed by the MGIT 960 liquid culture system:

  1. Maharashtra: Mumbai, P.D.Hinduja Hospital and Research Centre, IRL Nagpur, IRL Pune, SRL Mumbai
  2. Madhya Pradesh: Bhopal Memorial Hospital and Research Centre & Choitram Hospital and Research Centre
  3. Orissa : IRL Cuttack
  4. West Bengal : IRL Kolkata
  5. Jharkhand : IRL Ranchi
  6. Karnataka : IRL Karnataka
  7. Rajasthan: IRL Ajmer, SMS Medical College, Jaipur

DST by the proportion method for second line drugs such as Amikacin, Capreomycin, Kanamycin, Ofloxacin, are routinely being used.

Line Probe Assay

The MDR status of the culture isolates which have been sent for XDRTB diagnosis is routinely being confirmed through LPA.