Newer diagnostic techniques for tuberculosis

2.1. Interferon-assays(IFN-γ) 

This test measures the amount of interferon-γ; in the blood. On stimulation by mycobacterial peptides the amount of IFN-gamma increases in the blood. This test is different from conventional BCG test as it uses proteins known as ESAT-6 and CFP-10, which are present in Mycobacterium tuberculosis but absent from all BCG strains and from most non-tuberculosis mycobacteria. It is intended for use as a diagnostic aid for M. tuberculosis infection, including both tuberculosis disease and latent tuberculosis infection (LTBI). The advantages of, Interferon-assays compared with the tuberculin skin test, are that results can be obtained after a single patient visit, and that, because it is a blood test which is performed in a qualified laboratory, the variability associated with skin test reading can be eliminated.

An additional advantage of this newer generation blood test is that the test is not affected by past BCG vaccination, and will eliminate the unnecessary treatment of patients with BCG-related false positive results. Furthermore, the Interferon-assays does not affect the result of future Interferon-assays tests (i.e. no “boosting,” as with the TST).

Various interferon- assays available are-

3.1. Direct methods for detection of Mycobacteria or its products 

3.2. Identification of Mycobacterial species 

Earlier biochemical's were used for identification of mycobacterial species,6 however these were time consuming. To over come this various methods have come up, these are –

a)Lipid profiles – Since mycobacteria have characteristic lipid profile, these can be analyzed by high-pressure liquid chromatography.7

b)SDS-PAGE – The sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles of an easily and rapidly prepared soluble protein fraction were used in conjunction with conventional techniques to identify different strains of Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium. bovis BCG, Mycobacterium africanum, Mycobacterium avium, Mycobacterium kansasii, Mycobacterium marinum, Mycobacterium gastri, Mycobacterium simiae and Mycobacterium malmoense. With the SDS-PAGE technique, all strains of the M. tuberculosis complex were recognized as belonging to one species. By visual analysis of the SDS-PAGE polypeptide profiles, only minor differences between strains of the same species were seen and each species show a characteristic polypeptide profile.8

c)Sherlock Mycobacterial Identification System – The Sherlock Mycobacterial Identification System (SMIS; MIDI, Inc.) has been developed for the rapid, computer-assisted identification of mycobacterial species based on the separation and quantification of mycolic acids using HPLC technology.9

d)GenoType assay – The GenoType assay allows for the identification of 16 different mycobacterial species that are most frequently isolated in the clinical laboratory.10 Isolation is commonly done by PCR amplification of the 16S–23S ribosomal DNA spacer region followed by hybridization of the biotinylated amplified DNA products with 16 specific oligonucleotide probes.10 The specific probes are immobilized as parallel lines on a membrane strip.10

e)PCR restriction enzyme analysis – It identifies 65kDa hsp gene and 16s rRNA gene of mycobacteria.

f)DNA chips – Also called DNA-microarrays. These consists of very large number of evenly spaced spots of DNA fixed to microscopic slide. Each spot is a unique DNA fragment transferred by a gridding robot from 96-well plates on to the slides. DNA extracts from ‘unknown’ isolates yield distinctive patterns of hybridization on the slide.

5. Serological diagnosis of TB.11 

i) Immunochromatographic tests

Five highly purified antigens (including one of 38kDa) secreted by M. tuberculosis during active infection are immobilized in four lines on the test strip. The test detects the presence of immunoglobulin G (IgG) antibodies to these antigens. A total of 30μl of serum is added to a blue pad and then diffuses along the test strip. When the test card is closed, anti-human IgG attached to colloidal gold particles binds to any bound human IgG antibodies, producing one or more pink lines. The presence of one or more pink lines in the strip's test area is considered a positive test result.

The RAPID TEST TB – a one-step colored immunochromatographic assay. It detects antibodies to the recombinant 38-kDa antigen from M. tuberculosis expressed in and purified from Escherichia coli. A total of 100μl of serum is added to the reaction tube, and the test strip is placed into the tube, which is then capped. After 15min of incubation, the presence of two colored bands is considered a positive test result.

ii) Enzyme linked immunosorbent assays

TUBERCULOSIS IgA EIA detects IgA antibody to a mycobacterial Kp90 immuno-cross-reactive antigenic compound (ImCRAC). 100μl of diluted (1:400) serum was distributed in microtiter wells, and incubated at 37°C in a dark humid environment for 60min. The wells were subsequently incubated with 100μl of peroxidase-labeled anti-human IgA conjugate at 37°C in a dark humid environment for 60min. After another wash cycle, 100μl of peroxidase substrate, tetramethylbenzidine containing hydrogen peroxide, was added to the wells. The colorimetric reaction proceeded in the dark for 30min at room temperature (20 to 25°C) until 100μl of stop agent was added. The absorbance values at 450nm were recorded with an automatic reader system. The ratio of the OD for the unknown serum sample to the cutoff OD was used to interpret the results.

The PATHOZYME-TB complex test detects serum IgG antibody to a recombinant 38-kDa antigen from M. tuberculosis expressed in and purified from E. coli. This kit is specific for the diagnosis of disease due to M. tuberculosis complex. The procedures were similar to those described for TUBERCULOSIS IgA EIA, except that the sera were diluted 1:50, the second incubation was for 30min rather than 60min, the first and second incubations were not in a dark humid environment, and the third incubation was in the dark at 37°C for 15min. Three standards (with 2, 4, and 16serounits/ml) were provided for the generation of a semilogarithmic reference curve. Because the sera were diluted 1:50, the units extrapolated from the reference curve were multiplied by 50 to obtain serounits for result interpretation.

The individual PATHOZYME-MYCO lgG, lgA, and lgM assay. The three assays measure the levels in serum of IgG, IgA, and IgM antibodies, respectively, to two antigens; lipoarabinomannan (LAM) and recombinant 38-kDa antigen. These kits detect infection due to mycobacterium species. The procedures were identical to those described for PATHOZYME-TB complex except that the sera were diluted 1:100 rather than 1:50 and all three incubations were at room temperature. For the IgG and IgA assays three standards (with 2, 4, and 16serounits/ml) were provided for generation of a semilogarithmic reference curve. Because the sera were diluted 1:100, the units extrapolated from the reference curve were multiplied by 100 to obtain serounits for result interpretation. For the IgM assay, low- and high-positive control sera were provided. The OD of the low-positive control was used for the interpretation of the results.