Tuberculous bacilli was discovered more than a 100 Years ago, however, it still remains a major health problem. Presently about 33 % of the world population is suffering from tuberculosis. This already complex situation is compounded by increase in the HIV infections. Early diagnosis therefore becomes very essential to curb the spread of disease.
The diagnostic tests for tuberculosis range from simple to complex. The two basic approaches to diagnosis are direct and indirect. The direct approach includes detection of mycobacterium and the indirect approach uses the humoral or cellular response.
Microscopy is the simplest and most rapid procedure currently available to detect acid-fast bacilli (AFB) in clinical specimens by Ziehl-Neelson staining method or its modifications. The limit of detection with this method is that it requires at least 5 X 104 bacilli per ml of sputum. Fluoroscent staining method offers the advantage of screening the smear under low power where large number of slides is screened in less time.
The main advantage of smear microscopy:
It is inexpensive, is relatively easy to perform and read and hence detect transmitters of tubercle bacilli.Results can be reported within hours of receipt of the sample and provides reliable epidemiological indicators needed.
Isolation of mycobacteria from clinical sample by culture still represents the corner stone on which definitive diagnosis of tuberculosis and other mycobacterioses relies. Culture can be performed on conventional egg based solid medium such as Lowenstein- Jensen medium and agar based ones, such as Middle Brook 7H10 or 7H11. The major constraint of culturing mycobacteria in conventional media is its slow growth which necessitates a mean incubation period of at least 4 weeks. The drug susceptibility tests to anti-tuberculosis drugs require additional 4 weeks.
Rapid Identification Of Mycobacterium Isolates
Micro colony detection on solid media
In this method, plates poured with thin layer of middle brook 7H11 agar medium are incubated and examined microscopically on alternate days for the first 2 days and less frequently thereafter. In less than 7 days, micro colonies of slow growing mycobacteria such as M.tuberculosis can be detected, the recovery of mycobacteria is less efficient and it is labour intensive. Since M. tuberculosis grows more rapidly in liquid medium forming strings and tangles, which can be observed under the inverted light microscope with 40x magnification. This method is a better alternative for culturing tubercle bacilli.
Septi-check AFB method
This is a biphasic medium system consist of an enriched selective broth and non selective middle brook agar on one side and two sections on the other side. One with NAP & egg containing agar & second with chocolate agar for detection of contamination.This non-radiometric approach has the potential to expedite processing, obviate CO2 incubation requirements and facilitates early detection of positive cultures. This method requires about 3 weeks of incubation. The unique advantage of this technique is the simultaneous detection of M. tuberculosis, non- tuberculous mycobactera (NTM), other respiratory pathogens and even contaminants.
Radiometric BACTEC 460 TB method
This technique is specific for mycobacterial growth, wherein 14C labeled palmitic acid in 7H12 medium is used. When the 14c labeled substrate is metabolized, 14c02 is prodiced & measured by BACTEC system & reported in terms of growth Index (GI) value.Using the same system, drug susceptibility tests can also be performed for all the anti tuberculosis drugs when sufficient growth is observed. Mycobacteria in clinical samples can be detected in half the time compared to conventional culture methods.
MGIT 960 mycobacteria detection system
It is an automated system for the growth and detection of mycobacteria with a capacity to incubate and continuously monitor 960 mycobacteria growth indicator tube (MGIT) every 60 minutes for increase in fluorescence.
This is a non- radiometric continuous monitoring system with a computerized database management. The system is based on colorimetric detection of CO2
ESP culture system II
This is a fully automated continuous monitoring system based on the detection of pressure changes within the headspace above the broth culture medium in a sealed bottle, i.e. either gas production or gas consumption due to microbial growth. A special detection algorithm is present in this system for the detection of very slowly growing mycobacteria.
D) Demonstration of Mycobacteria directly from clinical samples.
FAST Plaque TB method uses mycobacteriophage to detect the presence of M.TB directly from sputum samples.
Polymerase Chain reaction
The PCR allows sequences of DNA present in only a few copies of mycobacteria to be amplified in vitro such that the amount of amplified DNA can be visualized and identified. If appropriate sequences specific for M. tuberculosis are selected, 10-1000 organisms can be readily identified. The PCR methodology is rapid; results are available within a day of DNA extraction from the sample. A number of target genes of mycobacterial DNA have been evaluated for diagnosis by PCR and various other genotypic methods. Transcription mediated amplification (TMA) and nucleic acid amplification (NAA). This approach identifies the presence of genetic information unique to M. tuberculosis complex directly from pre-processed clinical specimens.
Nucleic acid amplification assays (NAA)
Molecular tests such as NAA assays have been developed for rapid confirmation of suspected TB cases MTD (Gen Probe) and Amplicor (Roche) are two available NAA assays. MTD test has been FDA approved for use in smear negative cases in which the clinical suspicion of TB is high.
Gene X Pert MTB Assay
New test test that control T.B by rapid diagnosis and its drug resistance. The test simultaneously detects M.TB complex and resistance to Rifampicin in less than 2 Hours. It is a Nucleic acid amplification test that uses a disposable cartridge with the Gene Xpert Instrument system. The sputum sample is mixed with the reagent that is provided with assay and a cartridge containing this mixture is placed in the Gene Xpert machine.
Measurement of IFN gamma producing cells
The ESAT 6 is a specific antigen and a strong inducer of IFN gamma production by T cells of TB patients. The M. tuberculosis genome encompasses regions of different RD. These RD may encode potential antigens relevant for protection or diagnosis. The level of IFN gamma increases in treated compared to untreated patients, and is associated with improved immunity against TB. Hence this may be useful for monitoring TB patients.