Laboratory Division

Introduction:

The laboratory at NTI has been designated as a National Reference Laboratory (NRL) under the National Tuberculosis Elimination Program (NTEP). Three states viz Karnataka, Maharashtra and Rajasthan have been allocated to the National Reference laboratory at NTI, Bangalore for the purpose of quality assurance in sputum smear microscopy and culture & drug susceptibility testing (CDST).

I.       OBJECTIVES:

A.     To Provide Technical Support to Central TB Division:

• Analyse data collected from the states, assess the performance and gives feedback to CTD for effective implementation of NTEP.

• As a member of the National Lab Committee, provides technical expertise various diagnostic tools used in Mycobacteriology and on External Quality Assessment of sputum smear microscopy.

• 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 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 2^nd 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.

B.         Human Resource Development:

• A nodal centre for training Laboratory personnel and Programme Managers 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 1^st and 2^nd Line drugs.

• Preventive Maintenance and Minor Repairs of the Binocular Microscope and also Facilitates for the Laboratory component in the NTEP Modular Training.

C.      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

•       Analysing 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.

CBNAAT:  

•       External Quality Assurance of CBNAAT using dried tube panels. Coordination of the EQA activity, manufacture and validation of the panels is undertaken by NTI, Bangalore.

D.    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 and CBNAAT.

•       To conduct the annual Proficiency testing in Liquid Culture for Extended Second Line DST and in 1^st and 2^nd Line DST in Line Probe Assay and in CBNAAT.

E.    Operational Research:

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

 F.     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

 

II.   FACILITIES AVAILABLE AT LABORATORY DIVISION, NTI :

A) Smear Microscopy Services:

• Ziehl Neelsen method

• Fluorescent microscopy

B) Culture and DST Services:

a.     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 immune-chromatographic test.

•       DST by the proportion method for first Line Drugs Viz.  Pyrazinamide, Rifampicin, and Isoniazid and second line drugs such as Levofloxacin, Moxifloxacin, Linezolid, Amikacin, are routinely being performed at NTI Laboratory.

b.    Solid Culture, Identification and DST (LJ Medium):

•       Clinical samples are processed using the NALC-NaOH method for homogenization and decontamination. The centrifuged deposit is inoculated onto Lowenstein Jensen medium and incubated at 37 Deg C for growth up to 8 weeks.

•       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.

•       Drug Susceptibility testing by the Proportion sensitivity method is routinely being performed. The isolates testing against first line drugs such as, Isoniazid, Rifampicin, Pyrazinamide and Second line agents such as Fluoroquinolones, second line injectable, Linezolid etc, is available for DRTB diagnosis for proficiency testing purposes.

 

C) Molecular Tests for Drug Resistance Detection:

a.     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 GenoType MTBDRsl version 2.0-line probe is used for molecular detection of resistance-conferring mutations of fluoroquinolones (FLQ) (gyrA and gyrB genes) and second-line injectable drugs (SLID) (rrs and eis genes).

•       The test is performed only on smear positive samples.

 b.    Cartridge based Nucleic Acid Amplification Test (CBNAAT) Gene Xpert: 

•       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.

•       The process of Manufacturing and validating the Dried Tube specimen for EQA Panels involves: Culturing the Mycobacterial strain in Liquid Culture system followed by inactivation of the cultures using sample reagents. Further incubating for 84 days for confirmation of inactivation followed by Preparation of Dried Tube Specimens panel cultures and its validation. Once validated, the DTS panels are ready to be dispatched to the CBNAAT sites. Each site would receive a set of 5 tubes/machine.

c.     Next Generation Sequencing:

•       To further strengthen its research capabilities, the Laboratory at NTI is using the MiSeq-Illumina Next Generation Sequencing instruments for evaluating drug-resistant tuberculosis (DR-TB) cases.

III.     ACHIEVEMENTS OF LABORATORY:

• Laboratory conducted National Drug Resistance Survey (NDRS) in 2014-2016 for determining drug resistance patterns in sputum samples received from all states of the country

• The Laboratory is one of the nodal centre for National Prevalence Survey for TB in the state of Karnataka. The samples received from the various clusters is processed and analysed for its anti-tubercular drug resistance patterns.

• The Laboratory is also one of the nodal centre for the Sero-surveillance for Covid 19 samples in the state of Karnataka.

• The NRL and ICELT has manufactured, validated Dried tube specimen panels for CBNAAT EQA and has initiated EQA for CBNAAT sites in the country.

NATIONAL TUBERCULOSIS INSTITUTE
Directorate General of Health Services
‘AVALON’ No.8, Bellary Road,
Bangalore 560 003
India
Phone:+91 80 23441192, 23441193, 23447951
e-mail:nti@ntiindia.org.in