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Laboratory diagnosis of Tuberculosis caused by Mycobacterium tuberculosis
Specimen and processing
- sputum, bronchial washings, brushings or biopsies or early morning gastric aspirates, Cerebospinal Fluid (CSF), urine
- Specimens from sputum and other nonsterile sites should be liquefied with N-acetyl-L-cysteine decontaminated with NaOH (kills many other bacteria and fungi), neutralized with buffer, and concentrated by centrifugation.
- Specimens from sterile sites, such as cerebrospinal fluid, do not need the decontamination procedure but can be directly centrifuged, examined, and cultured.
Direct detection methods
- Detection of acid-fast property of mycobacteria to detect them in sputum and other clinical material by the Ziehl–Neelsen (ZN) staining technique.
- Red bacilli are seen against the contrasting background colour.
- Slender, straight or slightly curved rods with barrel or beaded appearance.
- Fluorescence microscopy, based on the same principle of acid-fastness, is increasingly used and is much less tiring.
- As sputum and certain other specimens frequently contain many bacteria and fungi that would rapidly overgrow any mycobacteria on the culture media, deecontamination methods make use of the relatively high resistance of mycobacteria to acids, and certain disinfectants.
- The deposit is used to inoculate Lowenstein Jensen (LJ) medium.
- Specimens such as cerebrospinal fluid and tissue biopsies, which are unlikely to be contaminated,are inoculated directly on to culture media.
- Dry, rough, raised, wrinkled, off white to buff coloured colonies on LJ medium,commonly called as rough, tough and buff colonies.
- The use of liquid medium with radiometric growth detection such as BACTEC 460 TB has simplified culture method.
Figure: Colony morphology of Mycobacterium tuberculosis on Lowenstein Jensen (LJ) agar after 8 weeks of medium
3. Biochemical analysis
- Niacin accumulation test: positive
- Arylsulphatase test: positive
- Neutral red test: positive
- Catalase perioxidase test: perioxidase positive and weakly catalase positive
- Amidase test: positive
- Nitrate reduction test: positive
- Tween 80 hydrolysis: variable result
- Susceptibility to pyrazinamide: sensitive
- Susceptible to thiophen -2-carboxylic acid hydrazide(TCH): not susceptible
4. Animal inoculation
- For many years, guinea pig inoculation had been a popular method of diagnosis of tuberculosis, but it is now regarded as obsolete.
- ELISA techniques have been attempted for rapid diagnosis of different clinical forms of tuberculosis by estimating specific IgM, IgA and IgG antibody titers ewitmploying use of various mycobacterial antigens.
- Latex agglutination test with latex particle coated with rabbit antibody to M tuberculosis has sensitivity of 94.4% and specificity of 2%.
6. Antigen- protein detection
- The detection of microbial products or components has been used in recent years to diagnose infections caused by M. tuberculosis.
- Tuberculostearic acid is a fatty acid that can be extracted from the cell wall of mycobacteria and detected by gas chromatography or mass spectrometry in clinical samples containing few mycobacteria.
7. Molecular techniques
- Subsequent to the introduction of commercially available hybridization assays, commercially available and inhouse– developed nucleic acid amplification tests were used successfully for early identification of M. tuberculosis complex grown in liquid cultures.
- PCR-based sequencing for mycobacterial identification consists of PCR amplification of mycobacterial DNA with genusspecific primers and sequencing of the amplicons.
- The organism is identified by comparison of the nucleotide sequence with reference sequences.
- The most reliable sequence for identification of mycobacteria is the approximately 1500 bp 16S rRNA gene.
Indirect detection methods
1. Tuberculin test
- A purified protein derivative (PPD) is obtained by chemical fractionation of old tuberculin.
- PPD is standardized in terms of its biologic reactivity as tuberculin units (TU).
- A large amount of tuberculin injected into a hypersensitive host may give rise to severe local reactions and a flare-up of inflammation and necrosis at the main sites of infection (focal reactions).
- For this reason, tuberculin tests in surveys use 5 TU in 0.1 mL solution; in persons suspected of extreme hypersensitivity.
- After the tuberculin skin test is placed, the area is examined for the presence of induration no later than 72 hours after placement.
The result interpretation is given as:
- For patients at the highest risk of developing active disease (eg, HIV-infected persons, people who have had exposure to persons with active tuberculosis) 5 mm or larger of induration isconsidered positive.
- Larger than 10 mm is considered positive for persons with increased probability of recent infection. This category might include individuals such as recent immigrants from high-prevalence countries, injection drug users, and health care workers with exposure to tuberculosis.
- For persons at low risk for tuberculosis, 15 mm or larger of induration is considered a positive test result.
- Positive test results tend to persist for several days. Weak reactions may disappear more rapidly.
2. Interferon gamma release assay (IGRA)
- Sometimes the results of the tuberculin skin test are equivocal, particularly in persons who have been vaccinated with BCG or who live in areas where NTM are highly prevalent in the environment.
- In an effort to improve diagnostic accuracy, whole-blood interferon release assays (IGRAs) have been commercially developed.
- These assays are based on the host’s immune responses to specific M tuberculosis antigens ESAT-6 (early secretory antigenic target-6), CFP-10 (culture filtrate protein-10), and TB7.7.
- The tests detect interferon gamma that is released by sensitized CD4 T cells in response to these antigens.
Two commercial assays generally used to detect the interferon are:
- The Quantiferon-Gold In-Tube test (QFT-GIT) is an enzyme-linked immunosorbent assay (ELISA) that detects interferon gamma in whole blood.
- The T-SPOT-TB is an ELISA ImmunoSpot assay that uses purified peripheral blood mononuclear cells.
Treatment of tuberculosis
The first line of anti TB agents that form the core of treatment regimens are
- Isoniazid (INH)
- Rifampin (RIF)
- Pyrazinamide (PZA)
- Ethambutol (EMB)
- The current World Health Organization recommendations are that all new patients with tuberculosis, irrespective of site or severity of disease, and in the absence of evidence of drug resistance, should receive a 6-month course of therapy, consisting of a 2-month intensive phase of rifampicin, isoniazid, pyrazinamide and ethambutol followed by 4-month phase of rifampicin and isoniazid.
- Isoniazid may cause mild psychiatric disturbances and peripheral neuropathy, particularly in alcoholics, but these are usually preventable by prescribing pyridoxine (vitamin B6).
- Resistance may develop during therapy (acquired resistance) with poor quality drugs or inadequate supervision, or patients may be infected with resistant strains (initial or primary resistance).
- Multidrugresistant strains are defined as those resistant to isoniazid and rifampicin, with or without resistance to additional drugs.
- The newer category of extensively drug resistant tuberculosis (XDR-TB) is defined as resistance to, at least, isoniazid, rifampicin, any fluroquinolone and any injectable agent.
The second line drugs include
- A later generation of fluoroquinolones such as moxifloxacin, levofloxacin or gatifloxacin
- An injectable agent such as amikacin, kanamycin or capreomycin
- Two or more core second line agents include ethionamide, prothionamide, cycloserine, terizidone, clofazimine or linezolid
Prevention of tuberculosis
Human tuberculosis is preventable:
- by the early detection and effective therapy of the open or infectious individuals in a community
- by lowering the chance of infection by reducing overcrowding as the most important factors affecting the incidence of tuberculosis are socio-economic ones, particularly those leading to a reduction of overcrowding in homes and workplaces.
- to a limited extent, by vaccination which include BCG vaccination that is given by intracutaneous injection after the birth.
- neonatal vaccination is recommended as prior exposure of the human population to environmental mycobacteria confer some protection, but in others induce inappropriate immune reactions that antagonize protection.