Multi-drug-resistant tuberculosis pathophysiology

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Editor-In-Chief: C. Michael Gibson, M.S., M.D. [1]

Overview

Tuberculosis is a granulomatous infection tansmitted mainly through droplets and can have pulmonary and extra pulmonary manifestations. Multi drug resistant strains of tuberculosis have been emerging at an alarming rate and they might be developed due to primary resistance or acquired resistance. These type of resistance are mainly through genetic mutations in genes like inhA, katG and rpob genes. These molecular pathophysiology can be detected through pyrosequencing, DNA sequencing and electrophoresis.

Pathophysiology

Tuberculosis is a granulomatous infection which is chiefly transmitted through droplets. The granuloma prevents the dissemination of mycobacteria and provides a pathway for immune cell communication. Within the granuloma, T lymphocytes (CD4) secrete cytokines, such as interferon gamma, which activate local macrophages to kill the bacteria with which they are infected It is asymptomatic in 90% of immunocompetent individuals. In symptomatic patients it can present as pulmonary or extra pulmonary manifestations. The primary infection can progress to certain complications like disseminated infection. Tuberculosis can influence the progression of HIV if concomitantly present. Depending on the age of the patient, tuberculosis may have different clinical manifestations, progression, and prognosis.

Further information: Tuberculosis pathophysiology

MDR-TB can develop in the course of the treatment of fully sensitive TB and this is always the result of patients missing doses or failing to complete a course of treatment but MDR-TB strains appear to be less fit and less transmissible. This is known as acquired resistance to to anti tubercular drugs. Usually MDR strains do not dominate and isoniazid INH resistant tuberculosis is less potent. Also it is usually the people who are immunocompromised who are more susceptible to catching tuberculosis.

Drug resistance in Mycobacterium tuberculosis bacteria arises mainly through genetic mutations in the following aspects.

• Produce an activity of gene that binds or destroys the drug (e.g., mutations in inhA increase the amount of InhA protein which interferes with the activity of isoniazid by binding sufficient isoniazid to reduce its effective concentration in the bacterium to below an inhibitory level) .
• Block activation of a prodrug (e.g., mutations in katG lead to loss of the ability of catalase to activate the prodrug isoniazid to its active form),
• Block the activity of a drug (mutations in rpoB prevent binding of rifampicin to RNA polymerase and inhibition of transcription)

Test to detect the molecular pathophysiology

• The mutations that cause resistance to many of the antituberculosis drugs have been established, though much work remains to be done to identify the molecular basis of resistance for some of the drugs and to determine the predictive value of finding a particular mutation in a strain of M. tuberculosis . For example, approximately 95% of rifampin-resistant M. tuberculosis strains carry mutations within the rifampin-resistance determining region (RRDR), an 81-bp region encoding codons 507 through 533 of the rpoB gene.
• PCR is used to amplify a target sequence followed by a second assay to determine if the sequence contains a mutation associated with resistance. Methods that have been described for the latter include the following

• Pyrosequencing
• DNAsequencing
• Finding mismatches in heteroduplexes (e.g., branch migration inhibition or temperature gradient HPLC analysis ), and hybridization assays (e.g., membrane hybridization, molecular beacons,microarrays, or line-probe assays).
• Electrophoretic detection methods (e.g., single strand conformation polymorphism)

• Kits for detecting mutations associated with rifampicin resistance that are commercially available in Europe and elsewhere include line-probe assays . Some also detect mutations associated with isoniazid resistance. In-house PCR-based tests using molecular beacons have also been used for diagnostic purposes in a few clinical laboratories.
• For the assays that are subjected to hybridization, target gene causing resistance is amplified by PCR, and the PCR labeled products hybridized to oligonucleotide probes that are immobilized in a microarray or on a nitrocellulose strip. Mutations are detected by lack of binding to wild-type probes and/or by binding to probes specific for commonly occurring mutations. The performance of the line-probe assays relative to culture-based DS tests was evaluated in meta-analyses
• Hybridization probes which emit fluorescence only when hybridized to their target is called molecular beacons. Molecular beacons can discriminate between targets differing by a single nucleotide. Because molecular beacons can use different fluorophores, real-time [[PCR] assays can be designed in which different DNA fragments or mutations can be amplified and detected simultaneously in the same tube. For example, a single-well assay has been developed that uses five molecular beacons to detect mutations associated with rifampicin resistance in M. tuberculosis bacteria and appears to perform similarly as the line-probe assays. In the California Microbial Diseases Laboratory, molecular beacons were designed to detect mutations in rpoB, katG, and inhA promoter region genes and directly applied to clinical specimens or to cultures. Comparison of molecular beacons results with results of culture-based drug-susceptibility testing showed 96% to 97% agreement in a series of approximately 1,000 clinical specimens and cultures

• An approach that combines rapid (< 2hrs) DNA sequencing (K. Musser, personal communication) with PCR-amplification of the RRDR with was conducted at the Wadsworth Center . Two primers was developed to sequence the 81-bp RRDR of the rpoB gene and to gain a clear and accurate pyrogram. Evaluation of the pyrosequencing approach was in primary specimens positive for M. tuberculosis complex DNA by real-time PCR. Final results were compared with conventional susceptibility testing results or DNA sequencing. This test has a detection limit of <1 colony forming unit,99% agreement in the 188 cultures and specimens tested and 100% specificity.

• Molecular genetic tests for the other antituberculosis drugs are much less developed and studied than the tests for rifampicin resistance. A meta-analysis of the performance of the Hain MTBDR(plus) assay for detecting isoniazid revealed a pooled sensitivity of 0.85 (95%CI 0.77– 0.90) which ranged from 57%–100% and a pooled specificity of 0.99 (95%CI 0.98–1.00) which was fairly consistent across studies. Validation studies conducted in the California Microbial Diseases Laboratory that used archived cultures revealed that the molecular beacon test displayed 82.7% sensitivity, 100% specificity, 100% positive predictive value, and 98.1% negative predictive value for detecting isoniazid resistance . Experiments for detetcting the XDR TB defining resistances, are in various stages of development from discovery of the mutations associated with resistance to development of prototype assays and laboratory-based evaluations.