Loop-Mediated Isothermal Amplification (LAMP)

LAMP is a novel, rapid and simplified NAA platform developed by Eiken Chemical Co. Ltd., Japan. The platform is not specific to TB and has been used to detect other organisms. Its major advantages are speed, simplicity, visual detection and the lack of requirement for a thermal cycler and highly skilled technicians, thus facilitating high throughput and its use at peripheral diagnostic centres. The technology uses four different primers specifically designed to recognize six distinct regions on the target gene, and the reaction process proceeds at a constant temperature (isothermal) using strand displacement reaction (Figure 1). Amplification and detection of the gene products can be completed in a single step, by incubating the mixture of sample, primers, DMA polymerase with strand-displacement activity and substrates at a constant temperature. The amplification efficiency is high, and DMA can be amplified 109-1010 times in 15-60 min. Due to its high specificity, the presence of amplified product can indicate the presence of target gene.

Immunological Tests & Serological Assays

Immune-based tests for the detection of antibodies, antigens and immune complexes have been attempted for decades. These assays generally detect humoral immune response. A major challenge with immunological diagnosis of TB is that TB presents a wide spectrum ranging from exposure, through latent TB infection and active disease, to severe disease. Furthermore several other factors can affect the performance of immune based tests: BCG vaccination, exposure to non- tuberculosis mycobacteria, M. tuberculosis strain and HIV co-infection. A good immunological test must distinguish between the various states of TB, and also distinguish between TB and other mycobacterial exposures.

Different types of blood tests have been proposed for serologic diagnosis of TB. The first studies using partially purified antigens allowed the detection of anti-mycobacterial antibodies in TB patients, but the tests showed poor specificity. The use of highly purified native or recombinant antigens increased specificity, but decreased sensitivity. It has also been found that the degree of humoral response to TB is heterogeneous; for this reason, the use of serodiagnostic tests based on mixtures of multiple M. tuberculosis antigens has been proposed. However, until now, none of these tests have shown to be predictive enough to warrant their routine use as diagnostic tests for TB.11 A recent meta-analysis of commercial serological antibody detection tests for TB concluded that these tests currently have no role in diagnosis of pulmonary TB.

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Rapid Culture Systems

Mycobacterial culture on selective media remains the most sensitive method for detecting M. tuberculosis bacilli in clinical specimens and allows subsequent strain characterization, including DST. The slow replication time of M. tuberculosis requires that solid media cultures be incubated for 2-8 weeks (depending on the inoculated bacterial concentration) for the growth of the millions of organisms necessary to generate visible colonies. This process can be accelerated by microscopically detecting immature colonies, detecting products of bacterial replication, or using bacteriophages as markers of mycobacterial viability. Several tests have been developed to exploit these principles.

Mycobacteria Growth Indicator Tube (MGIT)

The Mycobacteria Growth Indicator Tube (MGIT) system is based on a glass tube containing a modified Middlebrook 7H9 broth together with a fluorescence quenching-based oxygen sensor embedded at the bottom of the tube. When inoculated with M. tuberculosis, consumption of the dissolved oxygen produces fluorescence when illuminated by a UV lamp. The MGIT system has been thoroughly evaluated in clinical settings for the detection and recovery of mycobacteria." MGIT can be used for the rapid detection of Acid-Fast Bacilli (AFB) as well as for susceptibility testing. It allows for the good growth of most mycobacterial species.

More recently the MGIT system has been fully automated and turned into the BACTEC MGIT 960 system, which is a non-radiometric, noninvasive system with the tubes incubated in a compact system that reads them automatically.".

TK Medium

TK Medium is a novel colorimetric system that indicates growth of mycobacteria by changing its color. Metabolic activity of growing mycobacteria changes the color of the culture medium, and this enables an early positive identification before bacterial colonies appear (Figure 2). This test can distinguish between mycobacteria and contamination. TK medium also enables susceptibility testing for drug resistance. The average time to detection with TK Medium was 2 weeks, as compared with 4 weeks with the conventional LJ medium. Large multicentric studies are ongoing, and they should help to define the accuracy of this new rapid culture system.'