eMedicineLive

Multi drug resistant tuberculosis is an emerging global problem that may erode the gains obtained by the tuberculosis control programs worldwide. Multi drug resistant TB refers to drug resistance to two principal anti-TB drugs isoniazid and rifampicin. Studies show upto 6 to 10% of the tuberculosis patients have drug resistance, most of them concentrated in the underdeveloped and developing countries of Africa, Asia and Eastern Europe.  The cause of resistance is the indiscriminate use of anti-TB drugs without following the standard regimen recommended by national and international health agencies and non-compliance by patients with recommended regimens.

The problem is compounded by the fact that no new anti-TB drug that can be widely used has been discovered in past few decades. The second line drugs that are used against MDR tuberculosis are toxic, costly and need to be taken for a prolonged time to achieve cure. The cost of treating a multi drug resistant TB is estimated to be greater than 1000 times that of treating non-resistant tuberculosis.
Hence detecting possible drug resistance earlier is of immense importance both to the individual and the community.

Currently two approaches are available for drug susceptibility testing; direct and indirect.

The direct method which is faster takes about 3 to 4 weeks. In the direct method the testing is carried out on the clinical specimen and the results are available with the culture reports.

In the indirect method, the clinical specimen (eg. Sputum) is cultured and then the culture is subjected to additional testing for drug resistance. While smear positive cases can be tested by the direct method, smear negative but culture positive specimens need to be tested with the indirect methods including
1. Absolute concentration Method
2. Resistance Ratio Method
3. Proportion Method

Absolute concentration method
A standard inoculum is placed on the drug free media and media with graded concentrations of anti-TB drugs. The minimum inhibitory concentration of the anti Tb drugs is calculated.

Resistance ratio method
The resistance of the newly identified TB strain is compared with that of standard lab strain (H37Rv). The strains are incubated in media containing drugs of graded concentrations. The resistance of the newly identified strain from the clinical specimen is compared to resistance of standard lab strain

Proportion method.
The proportion of resistant bacilli to susceptible bacilli in the inoculum can be identified using proportion method. The standardized inoculum is diluted 10 fold and divided into multiple equal portions and incubated in media which is drug free and having graded anti Tb drug concentrations. The number of colonies formed in the drug free media is compared to the number of colonies formed in the media with anti-TB drugs. The proportion of the TB bacilli that is resistant to antibiotics among the entire population of TB bacilli is known. The proportion method is currently the most popular and widely used method.

Several newer techniques to find out drug resistance are under research and some of them are available commercially. Some of them are as follows
Newer tests to detect drug resistance in TB
1)  Microscopic observation drug susceptibility assay
2) Phage based assays
3) Line probe assays
4) Molecular beacons
5) E strip test
6) Xpert MTB/RIF

Microscopic Observation Drug Susceptibility (MODS)
It is a direct method and the results are available in 7 days. Here the sputum after processing with N acetyl cysteine and NaOH is incubated in a liquid medium 7H9 in a 24 well plate. To identify resistance, some of the wells contain anti TB drugs. The microcolonies formed can be detected using the microscope and comparison is made between the drug containing and the drug free wells. Studies show MODS has a greater than 95% sensitivity in detecting TB bacilli and resistance to rifampicin/isoniazid. The 7 day turn around period for culture and resistance identification compares favourably with earlier methods MBBacT (22 days) and LJ medium (68 days)

Phage based assays
In phage based assays, mycobacteriophages are employed to detect the TB bacilli and if drug resistance is present. Bacteriophages are viruses that infect bacteria. After they enter the mycobacteria, the detection is made either by multiplying the phage or emitting light. The results are available in 2-3 days but carrying out the test needs advanced lab facilities and trained personnel.  The commercial tests available are FASTPlaque-TB-MDRi  and FASTPlaque-TB-Response ( Biotec laboratories, UK) for detecting rifampicin resistance.

Line probe assays
Line probe assays have oligonucleotide probes that can detect genetic mutations associated with drug resistance. The commercially available tests include INNO-LiPA Rif.TB and GenoType MTBDR

Molecular Beacons
Molecular beacons emit light when a particular biological reaction takes place. Mutations associated with the drug resistance can be identified using the molecular beacons. It is easier to detect rifampicin resistance as only few mutations are involved whereas in case of isoniazid resistance several different mutations can lead to isoniazid resistance.

E strip test
This is a simple test where strips containing impregnated antibiotics are placed on solid culture medium of tuberculosis. Based on the growth inhibition, the minimum inhibitory concentration of the anti Tb drug is calculated. Thus it may give a secondary measure of resistance.  Though the test is easy to perform and interpret, it can be done only after culture results are available.

Xpert MTB/RIF
This is the latest and the most promising test to detect drug resistance in tuberculosis. The results are available within 2 hours and it can be done at the point of care without the need for sophisticated lab. It is a fully automated molecular test for mycobacterium tuberculosis and detecting rifampicin resistance. The results are impressive with greater than 97% detection of rifampicin resistance within 2 hours.