Molecular Techniques in the Diagnosis of Mycobacterium tuberculosis and the Detection of Drug Resistance

Abstract

Early diagnosis of Mycobacterium tuberculosis disease is crucial in initiating treatment and interrupting the train of transmission. The increasing incidence of MDR TB worldwide has also placed emphasis on the need for early detection of drug resistance, particularly to isoniazid and rifampicin. Molecular diagnostic techniques and automated culture systems have reduced turnaround times in the modern mycobacteriology laboratory, and the continuing evaluation and development of such techniques is increasing the use of molecular technology in developed nations. Simple phenotypic methods for the detection of resistance to first-line drugs and genotypic kit-form assays for detection of rifampicin resistance have been developed that have become key tools in the containment of MDR TB.

The resumption of consumption: a review on tuberculosis

“…The mutation rate responsible for isoniazid resistance is 100 times greater than the one responsible for rifampicin resistance, and is usually the first modification in the susceptibility of wild-type M. tuberculosis (Petrini & Hoffner 1999). Since, in the United Kingdom, 90% of the rifampicin-resistant isolates are also isoniazidresistant, a single positive resistance result to the former can be considered a strong indicator of MDR-TB (Caws & Drobniewski 2001). Streptomycin acts as an inhibitor of prokaryotic protein synthesis initiation.…”

“…However, one of the main problems here relies on the cost of these methods, restricting their use to developed nations. Molecular detection methods usually initiate with genomic sequence amplification, generally through polymerase chain reaction (PCR), which uses specific repetitive or DNA single copy sequences, and can give a specific and sensitive diagnosis in a few hours (Bloom & Murray 1992, Caws & Drobniewski 2001. In vitro amplification of specific sequences in the pathogen's genome allows a rapid diagnosis with a greater sensitivity and specificity degree than standard traditional methods that have been established along the past years.…”

“…Other rapid detection systems have recently been developed as an alternative based on phenotypic methods, which can be adapted for use in susceptibility tests. One of these techniques uses mycobacteriophages with the lux gene inserted in the genome, for example; other methods include flow cytometry and reverse transcriptase PCR (Caws & Drobniewski 2001).…”

A Rifampin-Hypersensitive Mutant Reveals Differences between Strains of Mycobacterium smegmatis and Presence of a Novel Transposon, IS 1623

“…Mutations at codons 531, 526, and 516 are the most common (13). A variety of assays that detect these sequence polymorphisms have been developed (3,23) and allow the rapid determination of the drug susceptibilities of clinical M. tuberculosis isolates. In addition, an estimated 90% of rifampin-resistant clinical isolates are also isoniazid resistant, such that rifampin resistance is a positive indicator of MDR TB.…”

“…In addition, an estimated 90% of rifampin-resistant clinical isolates are also isoniazid resistant, such that rifampin resistance is a positive indicator of MDR TB. However, about 4% of rifampin-resistant clinical isolates of M. tuberculosis have no mutations in the 81-bp core region or elsewhere in the rpoB gene (3,8,14,20). In addition, some mycobacteria, particularly atypical mycobacteria, such as Mycobacterium smegmatis, Mycobacterium avium, and Mycobacterium intracellulare, are resistant to rifampin, even though they possess sensitive RNA polymerase (9,14,20).…”