Mycobacterium Tuberculosis Diagnosis

A policy must be decided for determining which specimens of sputum are to be examined for tubercle bacilli. Pulmonary tuberculosis is both a threat to the life of the patient and a dangerous source of infection to others. It is therefore that cases should be correctly diagnosed at the earliest possible stage and that drug therapy should be based on sensitivity tests made on a culture grown from the patient.
In communities where tuberculosis is moderately or very common, every specimen of sputum received in the laboratory should be screened for tubercle bacilli, regardless of whether the physician requests the examination. At least microscopy of a Ziehl-Neelsen or auramine-stained smear should be done and the positive specimens then cultured.
Dangerous suspected patients are all patients with unexplained cough continuing for more than 4-6 weeks, elderly persons with supposed ‘smoker’s cough’, coughing patients with haemoptysis or cachexia, persons who have been in close contact with patients diagnosed as tuberculous, and patients with illnesses that make them specially vulnerable (e.g. AIDS).
Laboratory staff must be protected against the risk of becoming infected from specimens containing tubercle bacilli. If, therefore, there nay be cases of pulmonary tuberculosis in the patient population served by the laboratory, all specimens of sputum should be regarded as possibly dangerous and the procedures of making smears and seeding cultures should be Performed in a safety cabinet.
Methods
-- Samples processing: All samples ARE subjected to decontamination by NaOH -NALC (N-acetyl-L-cysteine) method and then concentrated by centrifugation for 20 min at 10,000 g . Then the sediments were resuspended in sterile normal saline and subjected to Ziehl Neelsen (Z.N) smear and culture. Sterile samples like CSF, blood samples for Mycobacterium tuberculosis culture are not subjected to decontamination process.
- Loewenstein-Jensen (LJ) culture: Ready to use bottles of Loewenstein-Jensen media (Hispan Lab, Medicopharmatrade. Co, Madrid, Aspen) are inoculated with 0.l ml of concentrated sputum sediments and will be kept in CO2 incubator for 8 weeks. Bottles are inspected twice a week for visible colonies and suspicion growth is subjected to Z.N staining. Negative culture is discarded after 8 weeks (biohazard wate handling). Resulting growth is left in light for 2 hours and examined for yellow pigments to identify photo-chromogen species.
-Culture using radiometrie BACTEC 460 TB system: BACTEC 460 TB system and BACTEC 12B vials (Becton Dickinson Microbiology system, Cockeysville, Md.) were used for radiometrie culture of MTB . BACTEC 12B vials are prepared by addition of antimicrobial agents "PANTA" which contains polymyxin, amphotericin B, nalidixic acid, trimethoprim, and azlocillin. One half ml of concentrated sputum sediments are inoculated into each BACTEC 12B vial and kept at 37°C and then tested 3 times a week. Positive cultures as indicated by BACTEC460 are subjected to further identification by NAP test (P-Nitro-acetyl amino hydroxyl-Proionophenone)for the presence of typical Myucobacterium tuberculosis. The system is obsealate now
The usually used system now is MGIT Bactec 960

-Antimicrobial Drug susceptibility testing (AST): Isolated TB colonies from BACTEC 12B media and from LJ media will be subjected to AST using both Agar proportion method and BACTEC 460 TB system .
a) Agar proportion method: The modified agar proportion method using Middlebrook 7H10 agar plates are used to determine the susceptibility of MTB isolates(Inderlied1996). Briefly, antibiotic stock solutions are diluted and added to Middlebrook 7H10 agar containing 10% oleic acid-albumin-dextrose (OADC) to give the following critical concentrations in quadrant plates: isoniazid (INH) 0.2 µg/ml; rifampicin (RIF) 1µg/ml; ethambutol (EMB) 5.0 µg/ml; streptomycin (SM) 2.0 µg/ml. One hundred microliter aliquots of diluted bacterial samples are inoculated to quadrants of drug containing or drug free media. Drug resistance in 7H10 method of proportion was defined as 1% or more growth of colonies on the drug containing agar quadrant compared to growth on the drug free quadrant. Results were recorded at 21 days after inoculation.
b) BACTEC 460 TB system: The recommendation of the manufacture for sensitivity testing, the modified critical concentrations of provided drugs (SIRE, Becton Dickinson) are adopted: 2 µg/ml for SM; 0.1 µg/ml for INH; 2.0 µg/ml for RIF; and 2.5 µg/ml for EMB.
-Bacteriophage methods or PCR using primers for the target sequences.
-Direct PCR to detect Mycobacterium tuberculosis in clinical samples.
-Tuberculin skin test. Inject 5-10 Iu/ml PPD reagent intradermal in forearm by insulin syringe an surround the area of injection by pen. Read result after 24-48 hours. Interpretate positive if diameter if induration is 10mm or more.