The World Health Organization (WHO) estimates that approximately one-third of the world's population is infected with Mycobacterium tuberculosis
, with an estimated 9.27 million new cases reported in 2007 (20
). In that year alone, an estimated 1.77 million people died from this treatable disease. Despite this significant burden, only a limited number of tests have been developed and implemented for the rapid diagnosis of tuberculosis (TB). Further, since the majority of TB disease burden occurs in underdeveloped and resource-limited settings, the need for a cost-efficient method is paramount.
The emergence of drug-resistant strains of M. tuberculosis
is one of the most critical issues facing TB researchers and clinicians today. Multidrug-resistant (MDR) M. tuberculosis
is defined as being resistant to the two best first-line drugs used to treat TB: rifampin (RIF) and isoniazid (INH). Extensively drug-resistant (XDR) M. tuberculosis
is defined as having additional resistance to a fluoroquinolone (ciprofloxacin, moxifloxicin, etc.) and an injectable drug (kanamycin, capreomycin, or amakacin), the two best classes of second-line drugs. The WHO estimates that 5% of new TB cases are MDR, with approximately 10% of those actually being XDR (20
). Compounding this problem is the fact that no new drugs have been developed and approved for the treatment of TB in the past 30 years (16
). The limited number of antibiotics available to treat TB necessitates rapid diagnosis not only to reduce the spread of drug-resistant strains but also to monitor and limit the emergence of newly resistant strains.
While RIF and INH are very effective in the treatment of susceptible strains of M. tuberculosis
, drug resistance can emerge quickly, in part due to patient nonadherence to the multidrug regimen or noncontinuous treatment. The molecular basis of resistance to these drugs is well documented. The target of RIF is the β-subunit of bacterial DNA-dependent RNA polymerase, which is encoded by the rpoB
gene. At the genetic level, the majority of RIF resistance is due to the accumulation of mutations within an 81-bp region of rpoB
, termed the rifampicin resistance determinant region (RRDR). Mutations within this region account for up to 98% of the RIF resistance observed (15
). The strong correlation between genotypic changes in this region resulting in phenotypic resistance makes the RRDR an optimal target for the design of rapid molecular diagnostics.
There are two described mechanisms that account for the majority of INH resistance. The most common mechanism involves mutations within the katG
gene, which encodes a catalase peroxidase whose activity is required for the activation of INH (9
). Nucleotide changes resulting in amino acid substitutions at codon 315 of katG
account for up to 50% of the clinical resistance to INH (15
). Another less common mutation occurs in the promoter region of the inhA
gene, which encodes enoyl-ACP reductase, which is required for mycolic acid biosynthesis (18
). Mutations at this locus account for up to 34% of the clinical INH resistance observed and are typically found in combination with additional mutations in katG
The vast majority of mutations that occur within rpoB
, and the inhA
promoter regions are due to accumulation of single-nucleotide polymorphisms (SNPs), of which there are four classes (8
). Class I SNPs, also called transitions, are changes in which a purine is exchanged for a purine (A/G→G/A) or a pyrimidine is exchanged for a pyrimidine (C/T→T/C) (8
). Class II, III, and IV SNP changes are collectively referred to as transversions, and all involve the change of a purine to a pyrimidine, or vice versa (17
). Class II changes result in A/C→C/A or T/G→G/T transversions, class III changes result in C/G→G/C transversions, and class IV changes result in A/T→T/A changes (8
). These genetic mutations often result in phenotypic changes, such as RIF and INH resistance observed in M. tuberculosis
, and are excellent targets for rapid molecular diagnostics.
A significant obstacle in controlling TB is the amount of time required to reach a diagnosis. Due to the slow growth rate of M. tuberculosis
, the initial diagnosis can take up to 6 weeks, with up to an additional 12 weeks to obtain drug susceptibility profiles for clinical isolates, depending on the techniques available to the laboratory. These labor-intensive methods can cause significant delays in identifying MDR or XDR cases, adjusting treatment regimens, and initiating epidemiological investigations. Recently, attention has shifted toward the development of dependable, molecular-based assays that can rapidly detect drug resistance. The development of new methodologies could potentially reduce the time required to diagnose drug resistance so that effective treatment regimens can be established. Direct sequencing of genes known to have a role in antibiotic resistance is one method that is currently used. However, while reliable, it is costly and may not be readily available. Another rapid method, the GenoType MTB
assay (Hain Lifescience GmbH, Nehren, Germany), has made substantial contributions to the area of rapid diagnostics but still requires approximately 8 h to complete the assay and additional training to ensure that results are interpreted correctly (7
). High-resolution melt (HRM) analysis is a molecular technique that can be used for detecting subtle genetic changes, such as SNPs conferring drug resistance in M. tuberculosis
. By slowly melting the DNA amplicon products of a real-time PCR assay, slight genetic differences can be visualized by changes in dissociation profiles.
The current study describes the use of multiple real-time PCR chemistries and HRM technology to detect RIF, INH, and more importantly, MDR strains of M. tuberculosis. This novel assay design is also capable of distinguishing M. tuberculosis complex bacteria (MTC) from nontuberculous mycobacterium (NTM) strains. This assay provides a rapid, robust, and inexpensive way to identify MDR TB that could result in numerous advantages over current molecular and culture-based techniques.