Isoniazid (INH) is one of the primary chemotherapeutic and prophylactic drugs used against Mycobacterium tuberculosis, the causative agent of tuberculosis, which remains the leading single cause of death due to an infectious agent throughout the world. Recent studies indicate that the median rate of primary resistance to INH is 7.3% (range, 1.5 to 32%) and that the rates of acquired resistance range from 5.3 to 70% globally (9, 45). The overall rate of resistance to INH is 8.4% in the United States and has remained relatively stable in the last decade (23). Global reports of clusters of tuberculosis cases caused by drug-resistant strains together with the emergence and dissemination of multidrug-resistant tuberculosis have underscored the need for research into the mechanisms of drug resistance and the design of more effective antituberculous agents. Despite the use of INH for several decades, the molecular basis for its bactericidal action and the mechanisms by which INH resistance evolves in M. tuberculosis are only beginning to be understood.INH has a simple chemical structure consisting of a hydrazide group attached to a pyridine ring, but its mode of action is very complex (8). It is proposed that INH enters M. tuberculosis as a prodrug by passive diffusion and is activated by catalase-peroxidase, encoded by katG, to generate free radicals, which then attack multiple targets in the cells (6). Recent studies have shown that an NADH-dependent enoyl acyl carrier protein (ACP) reductase, encoded by inhA, and a -ketoacyl ACP synthase, encoded by kasA, are two potential intracellular enzymatic targets for activated INH; and both of these enzymes are involved in the biosynthesis of mycolic acids (4,19,20). Resistance-associated amino acid substitutions have been identified in the katG, inhA, and kasA genes of INHresistant clinical isolates of M. tuberculosis (7,20,24,26,29). In addition, mutations in the oxyR-ahpC intergenic region have been identified in INH-resistant isolates (36). Additional genetic and biochemical studies have shown that certain promoter mutations of alkylhydroperoxide reductase, encoded by ahpC, in INH-resistant isolates result in overexpression of ahpC as a compensatory mechanism for the loss of catalase activity due to katG mutations (15, 32). Recently, missense mutations were identified in ndh, a gene encoding NADH dehydrogenase, which is an essential respiratory chain enzyme that regulates the NADH/NAD ϩ ratio in cells (18,22). The molecular mechanism by which mutations in ndh confer INH resistance in M. tuberculosis is poorly understood. In addition, low-level INH resistance in mycobacteria has been shown to be
Denaturing gradient gel electrophoresis (DGGE) was used to probe for mutations associated with rifampin (RIF) resistance in the rpoB gene of Mycobacterium tuberculosis. DGGE scans for mutations across large regions of DNA and is comparable to DNA sequencing in detecting DNA alterations. Specific mutations are often recognized by their characteristic denaturation pattern, which serves as a molecular fingerprint. Five DGGE primer sets that scanned for DNA alterations across 775 bp of rpoB were developed. These primer sets were used to scan rpoB for DNA alterations in 296 M. tuberculosis patient isolates from the United States-Mexico border states of Texas and Tamaulipas. The most useful primer set scanned for mutations in the rifampin resistance-determining region (RRDR) and detected mutations in 95% of the RIF-resistant isolates compared to 2% of RIF-susceptible isolates. Thirty-four different alterations were observed within the RRDR by DGGE. In addition, isolates harboring mixtures of DNA within rpoB were readily detected by DGGE. A second PCR primer set was used to detect the V146A mutation in 5 to 7% of RIF-resistant isolates. A third primer set was used to detect mutations in 3% of RIF-resistant isolates, some of which also harbored mutations in the RRDR. Only 1 of 153 RIF-resistant isolates did not have a detectable rpoB mutation as determined by DGGE and DNA sequencing. These results demonstrate the power and usefulness of DGGE in detecting mutations associated with drug resistance in M. tuberculosis.Drug resistance is a major concern in the global tuberculosis epidemic. Multidrug resistance, defined as resistance to at least isoniazid and rifampin (RIF), is greater than 10% in many countries and appears to be more widespread than previously documented (25,26). Resistance to RIF is almost exclusively associated with mutations in the rpoB gene that encodes the -subunit of RNA polymerase (17, 28). Over 70 distinct rpoB mutations have been reported for RIF-resistant Mycobacterium tuberculosis isolates worldwide (7,17,20,28). Approximately 95% of RIF-resistant isolates harbor mutations in the rifampin resistance-determining region (RRDR), an 81-bp region within rpoB that spans codons 507 to 533. Mutations at the serine 531, histidine 526, and aspartate 516 codons have been observed in approximately 86% of RIF-resistant isolates and therefore represent hot spots within the RRDR (17, 28). Alterations outside of the RRDR have also been reported for the N-terminal, CII, and CIII regions of rpoB (6, 28) (Fig. 1).Molecular techniques are receiving increased scrutiny as alternatives to traditional drug susceptibility testing for M. tuberculosis (22). Molecular techniques can detect DNA alterations in hours or days, while assaying for drug resistance by culture methods takes weeks. Although the clustering of most mutations in the RRDR simplifies molecular analysis, the heterogeneity of mutations represents a challenge in detecting DNA alterations as a predictor of RIF resistance. These genetic considerations affect b...
Background: Currently in the U.S. it is recommended that tuberculosis screening and treatment programs be targeted at high-risk populations. While a strategy of targeted testing and treatment of persons most likely to develop tuberculosis is attractive, it is uncertain how best to accomplish this goal. In this study we seek to identify geographical areas where on-going tuberculosis transmission is occurring by linking Geographic Information Systems (GIS) technology with molecular surveillance.
BackgroundDirectly observed therapy (DOT) is a widely recommended and promoted strategy to manage tuberculosis (TB), however, there is still disagreement about the role of DOT in TB control and the impact it has on reducing the acquisition and transmission of drug resistant TB. This study compares the portion of drug resistant genotype clusters, representing recent transmission, within and between communities implementing programs differing only in their directly observed therapy (DOT) practices.MethodsGenotype clusters were defined as 2 or more patient members with matching IS6110 restriction fragment length polymorphism (RFLP) and spoligotype patterns from all culture-positive tuberculosis cases diagnosed between January 1, 1995 and December 31, 2001. Logistic regression was used to compute maximum-likelihood estimates of odds ratios (ORs) and 95% confidence intervals (CIs) comparing cluster members with and without drug resistant isolates. In the universal DOT county, all patients received doses under direct observation of health department staff; whereas in selective DOT county, the majority of received patients doses under direct observation of health department staff, while some were able to self-administer doses.ResultsIsolates from 1,706 persons collected during 1,721 episodes of tuberculosis were genotyped. Cluster members from the selective DOT county were more than twice as likely than cluster members from the universal DOT county to have at least one isolate resistant to isoniazid, rifampin, and/or ethambutol (OR = 2.3, 95% CI: 1.7, 3.1). Selective DOT county isolates were nearly 5 times more likely than universal DOT county isolates to belong to clusters with at least 2 resistant isolates having identical resistance patterns (OR = 4.7, 95% CI: 2.9, 7.6).ConclusionsUniversal DOT for tuberculosis is associated with a decrease in the acquisition and transmission of resistant tuberculosis.
We have previously described the development of a live, fully attenuated Mycobacterium tuberculosis (Mtb) vaccine candidate strain with two independent attenuating auxotrophic mutations in leucine and pantothenate biosynthesis. In the present work, those studies have been extended to include testing for protective efficacy in a long-term guinea pig survival model and safety testing in the highly tuberculosis susceptible Rhesus macaque. To model the safety of the ΔleuD ΔpanCD strain in HIV-infected human populations, a Simian Immunodeficiency Virus (SIV)-infected Rhesus macaque group was included. Immunization with the non-replicating ΔleuD ΔpanCD conferred long-term protection against challenge with virulent M. tuberculosis equivalent to that afforded by BCG as measured by guinea pig survival. In safety studies, clinical, hematological and bacteriological monitoring of both SIV-positive and SIV-negative Rhesus macaques immunized with ΔleuD ΔpanCD, revealed no vaccine-associated adverse effects. The results support the further development of the ΔleuD ΔpanCD strain as a viable tuberculosis (TB) vaccine candidate.
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