Mutations in rpoB gene and rifabutin susceptibility of multidrug-resistant mycobacterium tuberculosis strains isolated in australia

Tuberculosis remains an important disease worldwide. The emergence of multidrug resistance in strains of Mycobacterium tuberculosis (M. tuberculosis) is one of the most important threats to tuberculous control. Despite continued efforts directed to improve tuberculosis control programs at national and international levels, recent surveys reveal that drug-resistant tuberculosis is still ubiquitous and rates of infection are alarmingly high in several countries. According to Espinal et al. (9), multidrug-resistant tuberculosis (MDR-TB) defining as resistance to at least isoniazid (INH) and rifampin (RIF) was present among new cases in 54 of 58 surveyed geographic sites in 1996 to 1999. Among new cases, alarming rates were reported in Estonia (14.1%), Latvia (9.0%), Tomsk Oblast of Russian Federation (6.5%), Ivanovo Oblast of the Russian Federation (9.0%), and Henan of China (10.8%). Overall, in all 58 surveyed sites and among the previously treated cases (9), the median MDR-TB rate was 9.3%, and for 35 geographic sites it was present at rates of 5% or higher.In M. tuberculosis, monoresistance to RIF is rare, and at least 90% of all RIF-resistant clinical isolates are also resistant to isoniazid (7). Hence, a positive result for RIF resistance would be useful as a strong surrogate of MDR-TB. In resistant isolates, it has been shown that up to 95 to 98% RIF resistance is caused by mutations in the rpoB gene encoding the RNA polymerase ␤-subunit. In the great majority of the RIF-resistant isolates, mutations occurred within a 81-bp hotspot region (the rifampin resistance determining region [RRDR], encoding 27 amino acids and corresponding to codons 507 to 533 or cluster I according to Escherichia coli numbering) in the center of the 3,516-bp rpoB gene (24). Within the RRDR, more than 80 distinct variants of single-base mutations or in-frame deletions or insertions have been reported among RIF-resistant isolates. Although several different molecular techniques such as heteroduplex analysis, line probe assay, and PCR singlestrand conformational length polymorphism have been used for analysis of rpoB gene mutations, these techniques were limited in that only the most frequent types of mutations were included.In the present study we report a rapid assay based on PCR and sequencing of the rpoB gene for direct detection of RRDR-associated RIF-resistant M. tuberculosis in clinical isolates and respiratory specimens. A total of 2,138 respiratory specimens and 142 M. tuberculosis isolates were used to evaluate the specificity and sensitivity of this assay. The results were compared to commercial and in-house diagnostic assays for M. tuberculosis and phenotypic drug susceptibility testing. MATERIALS AND METHODS Specimens and isolates.Between March 1999 and October 2002, 2,138 respiratory specimens (1,807 expectorated sputum and 331 bronchoalveolar lavage samples) were collected from 1571 patients suffering from chest symptoms and/or chest radiographic infiltrates of undetermined origin, including 712 pa-

Section: Discussionmentioning

confidence: 99%

Direct Detection of Rifampin-ResistantMycobacterium tuberculosisin Respiratory Specimens by PCR-DNA Sequencing

Yam

Tam²,

Leung³

et al. 2004

“…RIF resistance is rarely observed as monoresistance and for this reason it is often considered a marker for MDR isolates. Published studies underline the relevance of molecular analysis indicating that isolates harboring mutations in codons 526 and 531 show both high-level resistance to RIF and cross-resistance between rifabutin and RIF (31,40,41 ). INH is the drug used for treatment of latent TB infection in close contacts of infectious cases.…”

Section: Discussionmentioning

confidence: 99%

Use of Genotype MTBDR Assay for Molecular Detection of Rifampin and Isoniazid Resistance in Mycobacterium tuberculosis Clinical Strains Isolated in Italy

Miotto

Piana

Penati³

et al. 2006

105

Drug-resistant Mycobacterium tuberculosis strains are a real threat to tuberculosis (TB) control worldwide, as strains resistant to major anti-TB drugs have emerged. Multidrug-resistant TB (MDR-TB; resistant at least to rifampin and isoniazid) requires prolonged and expensive chemotherapy, with low cure and high fatality rates (8). In several Eastern European countries economic crisis and health system weaknesses have led to an increase in the numbers of TB cases, together with the establishment of hot spots for MDR-TB. Laboratory surveillance of drug resistance and early identification of resistant strains are critical steps for the beginning of appropriate treatment. Any delay in resistant strain identification jeopardizes the efforts to control the transmission of the disease.Rifampin (RIF) resistance is due to mutation in a relatively small fragment (81 bp) of the rpoB gene encoding for the ␤-subunit of the RNA polymerase (12, 29, 35); isoniazid (INH) resistance is caused by mutations in one of several regions of the katG gene, the inhA regulatory and coding region, and the ahpC-oxyR, ndh, and kasA genes (3,4,18,19,26,29,32,33,36,39). Analysis of strains collected in different countries shows different prevalences of the mutations (24).Easy-to-perform, rapid, and cost-effective assays based on molecular techniques that are suitable for application in clinical mycobacteriology laboratories are necessary to evaluate the presence of genomic mutations conferring resistance. Detection of resistance by conventional methods is inadequate due to the slow growth rate of M. tuberculosis; in addition, direct detection of known mutations could be more reliable in predicting the response to therapy.The Genotype MTBDR (Hain Lifescience, Nehren, Germany) is a new commercial and easy-to-perform assay developed for the detection of RIF and/or INH resistance in TB strains. The test is based on reverse hybridization between amplicons derived from a multiplex PCR and nitrocellulosebound wild-type and mutated probes for the mutations of interest.The aim of the present study was to evaluate Genotype MTBDR as a rapid diagnostic tool to detect rpoB and katG gene mutations that are associated with RIF and INH resistance, respectively, in TB isolates.We tested 206 M. tuberculosis strains isolated in Italy during the past 3 years, including 139 MDR strains and 30 fully susceptible ones. A total of 69 strains were collected from Italian patients, and 137 were obtained from foreign-born people. In order to show that the test is suitable for the direct detection on clinical specimens, we also performed the test on respiratory samples collected from active TB patients. (Table 1). A total of 85% of the MDR strains were isolated from retreatment cases. MATERIALS AND METHODS Strains

“…It was demonstrated that more than 90% of Mycobacterium tuberculosis isolates with the rifampin-resistant (Rif r ) phenotype possess single-nucleotide substitutions within an 81-bp fragment, the so-called hot spot region, of the rpoB gene (24,33). Similar information on the frequency of occurrence of rpoB mutations among isolates from different geographic origins has accumulated in recent years (9,11,17,22,(25)(26)(27)29).…”

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confidence: 99%

“…Although the genotype of rpoB, as a marker for MDR and epidemiology of M. tuberculosis from different geographic regions has been reported by several groups in the last decade (4,5,11,12,16,17,22,(25)(26)(27)(28)(29)32), we have expanded this information by adding data from China, Japan, Korea, and Taiwan. Our analysis was also influenced by the dominance of the Beijing family in these regions (Qian et al, Abstr.…”

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confidence: 99%

rpoB Genotypes of Mycobacterium tuberculosis Beijing Family Isolates from East Asian Countries

Qian

Abe

Lin³

et al. 2002

The 81-bp region of the rpoB gene in 66 Rif r Mycobacterium tuberculosis isolates from China, Japan, Korea, and Taiwan was analyzed. Twelve single-nucleotide substitutions in the rpoB gene were detected. The most prevalent mutations were at Ser-531 (52%), Asp-516 (17%), and His-526 (11%). Mutations were not found in seven (11%) of the isolates. Higher mutation rates in 50 Beijing family isolates were found than in other isolates for mutations at Asp-516 (18 and 12.5%, respectively) and His-526 (12 and 6.3%, respectively). The different rates of mutation may reflect the choice of rifamycin analogs.