The occurrence of rare mutations in rifampicin-resistant clinical isolates from Kuwait

Ahmad, Suhail; Mokaddas, Eiman

doi:10.1016/j.ijantimicag.2005.06.009

Cited by 54 publications

(27 citation statements)

References 39 publications

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“…For each group of isogenic mutants, the most common single nucleotide mutation sites were codons 526, 531, and 522, and in total these codons' relative frequencies were 40%, 34%, and 13%, respectively. The frequencies of codon 526 and 531 mutations were similar to the distribution reported among RIF-resistant clinical isolates, while codon 522 mutations have rarely been seen clinically (1,4,7,13,21,22). Mutations in the H37Rv-derived mutants showed the narrowest span, with only five different types recorded (three being single nucleotide substitutions) (data not shown).…”

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confidence: 78%

“…Resistance to RIF is almost entirely coupled (Ͼ97%) to mutations within an 81-bp region of the rpoB gene, called the RIF resistance-determining region (RRDR) (5,23). Mutations in the rpoB gene of Mycobacterium tuberculosis clinical isolates were analyzed by Telenti et al (23), and many studies have since followed (1,4,7,13,21,22). A few studies of in vitrogenerated RIF resistance mutations in M. tuberculosis have also been conducted; however, these were limited to the H37Rv reference strain and a single clinical isolate (3,14,16).…”

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confidence: 99%

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Resistance Levels and rpoB Gene Mutations among In Vitro-Selected Rifampin-Resistant Mycobacterium tuberculosis Mutants

Huitric

Werngren

Juréen

et al. 2006

Antimicrob Agents Chemother

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The distribution and resistance levels of 189 in vitro-selected rifampin-resistant Mycobacterium tuberculosis mutants of Beijing and other genotypes were determined. Apart from a higher amount of codon 522 point mutations and large deletions, a spread of mutations similar to that reported for clinical isolates was seen. Most mutations were correlated with high-level resistance; a lower level, or a MIC of <16 mg/liter, was associated with codon 522 mutations. Multiple mutations were detected in two Beijing mutants.Rifampin (RIF) is a key drug in the four-drug treatment regimen of tuberculosis. It interacts with the ␤-subunit of the RNA polymerase and inhibits the early steps of transcription. Resistance to RIF is almost entirely coupled (Ͼ97%) to mutations within an 81-bp region of the rpoB gene, called the RIF resistance-determining region (RRDR) (5, 23). Mutations in the rpoB gene of Mycobacterium tuberculosis clinical isolates were analyzed by Telenti et al. (23), and many studies have since followed (1,4,7,13,21,22). A few studies of in vitrogenerated RIF resistance mutations in M. tuberculosis have also been conducted; however, these were limited to the H37Rv reference strain and a single clinical isolate (3,14,16).Strains of the M. tuberculosis Beijing genotype have been spreading increasingly and are often associated with multidrugresistant (resistance to at least RIF and isoniazid) tuberculosis (6, 15, 17-19, 24, 25). Isolates of this genotype have been shown not to have a higher in vitro mutation rate for the development of RIF resistance (27). A few clinical studies have analyzed RIF-resistant Beijing isolates, for which no clear differences in occurring RRDR mutations compared to those of other genotypes have been seen (8,18,19,24,25). There are, however, no data on in vitro-selected rpoB mutations in Beijing strains.In the present study, we wanted to investigate the frequency and distribution of rpoB mutations occurring in vitro among several independent M. tuberculosis parental strains and subsequently compare these to available data on clinical isolates. Additionally, we determined whether Beijing genotype mutants display differences in the types and/or the frequencies of rpoB mutations compared to those of mutants of non-Beijing genotypes. Finally, we wanted to measure the levels of resistance associated with the various RRDR mutations.By use of the Luria and Delbrück fluctuation assay (12), 189 RIF-resistant mutants were previously selected (27). By setting a series of parallel cultures from a single parent strain, this method selects spontaneously occurring, independent mutational events in nonselective broth. Then, by inoculation of the cultures on selective media (2 mg/liter RIF), the mutants are obtained. In all, nine fully drug-susceptible parent strains with unique DNA fingerprint patterns (26) were used to select RIFresistant mutants. Eight were clinical isolates, of which one was a Dutch Harlingen isolate (11) and one was the H37Rv (ATCC 25618) reference strain. Based on their spoligotype ...

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confidence: 78%

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confidence: 99%

Resistance Levels and rpoB Gene Mutations among In Vitro-Selected Rifampin-Resistant Mycobacterium tuberculosis Mutants

Huitric

Werngren

Juréen

et al. 2006

Antimicrob Agents Chemother

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“…The discrepant result constitutes one sample that was found to be susceptible by LiPA and resistant by culture. Previous studies reported up to 5% of RMP-resistant strains with no mutation in the rpoB core region screened by the LiPA test (1,2,7,11,28). The results for susceptibility to INH were available for 94 of the RMP-resistant specimens by the culture method, and 92.6% of them were also INH resistant.…”

Section: Resultsmentioning

confidence: 99%

Direct Detection of Mycobacterium tuberculosis Complex DNA and Rifampin Resistance in Clinical Specimens from Tuberculosis Patients by Line Probe Assay

et al. 2006

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The INNO-LiPA.Rif TB test (LiPA) has only been applied to a limited number of clinical specimens. To assess the utility of this test for detecting Mycobacterium tuberculosis complex DNA and rifampin (RMP) resistance, 420 sputum samples comprising specimens from untreated (n ‫؍‬ 160) and previously treated (n ‫؍‬ 260) patients from 11 countries in Asia, Africa, Europe, and Latin America were tested. DNA was extracted from sputum samples by using a modification of the Boom's method, while the rpoB Tuberculosis (TB) remains one of the most important diseases worldwide. In recent years, the incidence of TB has been rising, as has the prevalence of drug-resistant cases in many parts of the world (31). A high rate of drug resistance in a community would compromise the effective standardized chemotherapy and jeopardize TB control, especially in regions with high human immunodeficiency virus prevalence where the susceptibility to disease is higher.Rifampin (RMP) is a key component for the effectiveness of the World Health Organization-recommended short-course chemotherapy. Therefore, patients in whom resistance to this drug develops have a poor prognosis, particularly when the resistance to RMP is associated with resistance to other anti-TB drugs (9). Multidrug-resistant (MDR) TB, i.e., resistance to at least RMP and isoniazid (INH), the two most potent anti-TB drugs is a problem of increasing importance in both developed and developing countries (31). Several previous studies suggested that RMP resistance could be a good indicator for MDR in some settings with high MDR prevalence (10,28,30,31). Therefore, early diagnosis of the disease and rapid detection of resistance to this major anti-TB agent are essential for the optimal control of TB.The use of molecular techniques based on PCR amplification of genes involved in resistance mechanisms, followed by the detection of key mutations associated with resistance, provides faster RMP susceptibility results than the classical methods that are based on the growth of bacilli on culture media. Unfortunately, to date, only a few molecular tests have been standardized and extensively evaluated for the rapid detection of resistance to RMP in Mycobacterium tuberculosis when applied to cultured isolates. One such test, the INNO-LiPA.Rif TB (Innogenetics, Belgium) is recommended for application on isolates, and its performance for this purpose has been found to be highly reliable by several studies (10,15,22). However, since M. tuberculosis DNA can be detected in clinical samples, independent studies were conducted to assess the applicability of the test to clinical specimens, which would provide even faster results. Recent reviews on the molecular detection of RMP resistance found only a few studies and small sample sizes (16,17) for the direct detection of RMP resistance in clinical specimens. This necessitates the need for larger studies to assess the reliability of the direct application of LiPA to clinical specimens.Here, we have analyzed an extended number of sputum samples fr...

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“…Previous studies have shown that 94 to 98% of Rif r M. tuberculosis strains show a mutation in cluster I (10,11,14,15). Resistance-associated mutations have also been described for cluster II (codons 496 and 497) and for codons 176, 486, 558, and 598 (1,6,7).…”

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confidence: 97%

Newly Developed Primers for Comprehensive Amplification of the rpoB Gene and Detection of Rifampin Resistance in Mycobacterium tuberculosis

et al. 2007

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New rpoB gene primers for detecting Rif r in Mycobacterium tuberculosis complex bacteria achieved 100% specificity and 88% (fresh sputa) and 92% (ethanol-preserved sputa) diagnostic sensitivity and detected up to 4 CFU/sample. Of the 99 Rif r isolates examined, 97% had mutations within cluster I, 2% at codon 176, and 1% at codon 497.Molecular detection of rifampin-resistant (Rif r ) Mycobacterium tuberculosis usually relies on amplification of the hotspot zone for resistance-conferring mutations (cluster I, covering codons 432 to 458 according to the M. tuberculosis nomenclature) (7) of the rpoB gene (4,8,10,12,13,17). Previous studies have shown that 94 to 98% of Rif r M. tuberculosis strains show a mutation in cluster I (10,11,14,15). Resistance-associated mutations have also been described for cluster II (codons 496 and 497) and for codons 176, 486, 558, and 598 (1, 6, 7).We describe and evaluate new primers, covering the entire region with all currently known significant mutations in a single assay.Oligonucleotides were designed on the basis of an alignment of the rpoB gene sequence from the H37Rv M. tuberculosis reference strain (NC 000962; NCBI bank), some relevant nontuberculous mycobacteria (NTM), and nonmycobacterial species using ClustalX (version 1.83.1) software. Amplify software (version 1.2; University of Wisconsin-Madison) was used to estimate the stabilities and binding capacities of the selected oligonucleotides and to simulate PCRs. Figure 1 shows the relative locations of the selected primers.A single PCR with primers rpoBgeneSA (5Ј-GGTTCGCCGC GCTGGCGCGAAT-3Ј) and rpoBgeneRB (5Ј-GACCTCCTCG ATGACGCCGCTTTCT-3Ј) was used for bacterial suspensions, whereas a nested PCR with primers rpoBgeneSAnew (5Ј-GCAA AACAGCCGCTAGTCCTAGTCCGA-3Ј) and rpoBgeneRA (5Ј-GCGCCATCTCGCCGTCGTCAGTACAG-3Ј) for the first run, and rpoBgeneSA and rpoBgeneRB as inner primers, was used to amplify clinical specimens.The first run of the nested PCR was performed with a final volume of 50 l containing 10 mM Tris-HCl (pH 8.6), 50 mM KCl, 1.65 mM MgCl 2 , 200 M of each deoxynucleoside triphosphate, 12.5 pmol of each primer, 1.5 U Taq polymerase (Promega, Madison, WI), and 5 l of DNA extract from clinical specimens. PCR was performed using a PTC 100 MJResearch thermocycler (Whaltham, MA) as follows: a hot start (90°C) followed by 5 min at 94°C; 45 cycles of 45 s at 94°C, 1 min 30 s at 66°C, and 45 s at 72°C; and a final extension of 10 min at 72°C. The second run was performed with a final volume of 25 l enzyme mixture with 0.5 U Taq polymerase and 0.25 l of the first PCR amplicon as follows: a hot start (90°C) followed by 5 min at 94°C, 29 cycles of 45 s at 94°C, 1 min 45 s at 72°C (annealing and extension), and a final extension of 10 min at 72°C. For bacterial suspensions, a single PCR was run under similar conditions but using a 50-l enzyme mixture and 45 cycles. Amplicons were analyzed with a 2% (wt/vol) agarose gel.DNA was extracted from sputum by an adapted Boom extraction method (16) and from bacterial suspensions in 1ϫ TE bu...

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The occurrence of rare mutations in rifampicin-resistant clinical isolates from Kuwait

Cited by 54 publications

References 39 publications

Resistance Levels and rpoB Gene Mutations among In Vitro-Selected Rifampin-Resistant Mycobacterium tuberculosis Mutants

Resistance Levels and rpoB Gene Mutations among In Vitro-Selected Rifampin-Resistant Mycobacterium tuberculosis Mutants

Direct Detection of Mycobacterium tuberculosis Complex DNA and Rifampin Resistance in Clinical Specimens from Tuberculosis Patients by Line Probe Assay

Newly Developed Primers for Comprehensive Amplification of the rpoB Gene and Detection of Rifampin Resistance in Mycobacterium tuberculosis

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