Management of Tuberculosis: Diagnostic Approach

Accurate and timely diagnosis of Tuberculosis (TB) is a prerequisite for the treatment and control of spread of infection to other family members of the patient and health professionals dealing with patients. In the recent past several advances in the diagnosis and management of Tuberculosis (TB) have come out. Tools and technology are of great help in understanding of pathogenesis, demonstration of Mycobacterium, drug sensitivity testing and evaluation of prophylaxis.

Tuberculosis is a major health problem requiring early management at diagnostic and treatment level to bring down the mortality rate. In the developing countries the current mortality rate is 1 death per 100,000 population. The global incidence rate of Tuberculosis (TB) reported in the year 2010 was 128/100,000/year. To sustain the control and elimination of Tuberculosis (TB) there is a need for efficient testing and treatment regimens.

Diagnosis of Tuberculosis (TB) has conventionally been relied upon sputum microscopy of Micobacterium tuberculosis (an Acid Fast Bacilli) by Ziehl-Neelsen Staining Technique. The technique is very specific but has a poor sensitivity (around 50%). More sensitive technique used for demonstrating Acid Fast Bacilli (AFB) is by culture on Lowenstein Jensen Medium. Isolation of mycobacteria by culture method is considered to be gold standard in-spite of being time consuming.

The scenario of multi drug resistant TB (MDR-TB) has given a challenge to biomedical scientists to develop new drugs and diagnostic methods. Advances in molecular techniques for the diagnosis of TB have revolutionized diagnostic approach to this public health problem. Various new diagnostic modalities are based on the DNA extraction from the mycobacterial isolates. The nucleotide sequences of DNA are amplified and multiplied millions of times by polymerase chain reaction (PCR) for comparative diagnosis through detection of amplified DNA.

The tests that detect Mycobacterium tuberculosis antigens in clinical specimens could provide rapid and direct evidence of infection. The major targeted antigen of Mycobacterium tuberculosis is Lipo-arabino-mannan (LAM). LAM is detected in the urine of patients suspected of having pulmonary TB (Tuberculosis of lungs) as well as extra-pulmonary TB (Tuberculosis of organs other than lungs).

Molecular Tests are very sensitive and specific for diagnosis of infection and monitoring of treatment as well as for evaluating drug resistance. Uniplex-PCR (single insertion sequence IS6110 of 38 kDa)) or Multiplex-PCR (for multiplex targeting like IS6110 and MPB 64) are of great help for detecting drug resistance. Multiplex-PCR is more sensitive than Uniplex-PCR. Multiplex-PCR is useful in early detection, species differentiation (Mycobacterium tuberculosis or Mycobacterium avium) and epidemiology.

Two Molecular Assays used for rapid diagnosis of a case of TB and drug-resistance testing are:  i) X-pert MTB/RIF, and ii) Line Probe Assay (LPA)

i)                    X-pert MTB/RIF: X-pert MTB/RIF detects Mycobacterium tuberculosis (MTB) and resistance to Rifampicin (RIF) using Real-Time PCR (RT-PCR) Assay by amplifying MTB-specific sequence of the rpoB gene (inherent of MTB genome) that is probed with molecular beacons for mutations within the RIF-resistance determining region. Diagnosis of TB can be determined within 2 hours from the sputum samples with minimal health hazard. X-pert MTB/RIF test has 99% sensitivity and 100% specificity.

ii)                  Line Probe Assay (LPA):  Rapid detection of anti-TB drug resistance by Mycobacterium tuberculosis is the need of the hour for effective treatment and management of patient care. Line Probe Assays have been developed for rapid detection of rifampicin resistance and/or MTB-DR (especially rifampicin in combination with isoniazid). The Line Probe Assay (LPA) employs the hybridization of labeled PCR products with oligonucleotide probe on a strip and reading by colorimeter. The genotype MTB-DRplus Assay also simultaneously detects specific mutations in the katG gene conforming high level isoniazid resistance as well as in the inhA gene conforming low level isoniazid resistance.

The Molecular Assays are labeled for use on isolates from solid and liquid culture as well as directly on sputum smear positive pulmonary specimens. Mycobacterium tuberculosis ‘strain typing’ is very important for the analysis of the spread of tuberculosis as well as for monitoring the evolution of antibiotic resistance. These assays are also used to assess the bacterial load for monitoring of anti-TB treatment (ATT).

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