rpoB S531L Research Articles

MOLECULAR TYPING AND DRUG RESISTANCE IN MYCOBACTERIUM TUBERCULOSIS COMPLEX ISOLATES FROM JAMOT AND MBALMAYO DISTRICT HOSPITALS, CAMEROON

BackgroundCameroon is a country where tuberculosis still remains a major public health problem. The aim of the present research was to evaluate the potential of molecular markers in predicting first-line...

Structural basis for rifamycin resistance of bacterial RNA polymerase by the three most clinically important RpoB mutations found in Mycobacterium tuberculosis

Since 1967, Rifampin (RMP, a Rifamycin) has been used as a first line antibiotic treatment for tuberculosis (TB), and it remains the cornerstone of current short-term TB treatment. Increased occurrence of Rifamycin-resistant (RIFR ) TB, ∼41% of which results from the RpoB S531L mutation in RNA polymerase (RNAP), has become a growing problem worldwide. In this study, we determined the X-ray crystal structures of the Escherichia coli RNAPs containing the most clinically important S531L mutation and two other frequently observed RIFR mutants, RpoB D516V and RpoB H526Y. The structures reveal that the S531L mutation imparts subtle if any structural or functional impact on RNAP in the absence of RIF. However, upon RMP binding, the S531L mutant exhibits a disordering of the RIF binding interface, which effectively reduces the RMP affinity. In contrast, the H526Y mutation reshapes the RIF binding pocket, generating significant steric conflicts that essentially prevent any RIF binding. While the D516V mutant does not exhibit any such gross structural changes, certainly the electrostatic surface of the RIF binding pocket is dramatically changed, likely resulting in the decreased affinity for RIFs. Analysis of interactions of RMP with three common RIFR mutant RNAPs suggests that modifications to RMP may recover its efficacy against RIFR TB.

Clinical Features and Drug-Resistance Profile of Urinary Tuberculosis in South-Western China: A Cross-sectional Study.

To investigate the epidemiology, clinical features, and drug-resistance profile of urinary tuberculosis (UTB) in south-western China to improve UTB diagnostics.After the screening of 1036 cases of suspected UTB, 193 patients with UTB were enrolled during 2009 to 2014. Urine samples were collected for routine urinalysis, smear, tuberculosis DNA (TB-DNA) detection, and drug-resistant analysis, whereas blood samples were collected for erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and renal function evaluation. Clinical features (such as symptoms and outcome) and imageology results (such as B ultrasonic, computerized tomography, intravenous pyelography, and renography) were also collected and analyzed to investigate the epidemiology, clinical features, and drug-resistance profile.The most common presenting symptoms were urinary irritation (61.1%) and lumbago (49.2%). High proportions of microscopic hematuria (63.2%) and microscopic proteinuria (45.6%) were also observed. The positive rate for TB-DNA was 66.3%. The positive rate for culture was 13.1% and for smear it was 9.8%. The abnormal outcome rates of the computerized tomography, ultrasonography, intravenous pyelography, and the nephrogram were 76.9%, 70.1%, 29.8%, and 37.0%, respectively. The total rate of drug-resistant TB (resistant to at least 1 drug) was 39.7%, of which 20.7% was multidrug-resistance TB. The most prevalent mutation sites were katG S315T1, rpoB S531L, and gyrA D94G.We observed a serious epidemic of drug-resistant UTB and a substantial number of new UTB cases with multidrug resistance TB. Molecular diagnostics is crucial in the definite diagnosis of UTB, and our finding is a supplement and further confirmation of polymerase chain reaction usage for TB diagnosis. We recommend real-time polymerase chain reaction for TB-DNA identification instead of culture, and GenoType tests (MTBDRplus and MTBDRsl assay) for drug resistance as routine assays for patients with suspected UTB.

Genotypic assessment of drug-resistant tuberculosis in Baghdad and other Iraqi provinces using low-cost and low-density DNA microarrays.

We report on a molecular investigation carried out to ascertain the prevalence of drug-resistant tuberculosis (TB) and the specific gene mutations responsible for resistance to rifampicin (RIF) and/or isoniazid (INH) in Iraq. In total, 110 clinical isolates from category II TB cases from Baghdad (58%) and several Iraqi provinces (42%) were analysed using colorimetric, low-cost and low-density (LCD) microarrays (MYCO-Direct and MYCO-Resist LCD array kits) to identify the point mutations responsible for resistance in Mycobacterium tuberculosis isolates. We found 76 patients (69.1%) had resistant strains, of which 40 (36%) were multidrug-resistant (MDR)-TB. Where mono-resistance was identified, it was found to be predominantly to RIF (83%). The most common mutations were rpoB S531L (50%), inhA C15T (25%) and katG S315T (15%). The most common MDR-TB genotypes were rpoB S531L with inhA C15T (60%) and rpoB S531L with katG S315T (20%). Where phenotypic analysis of clinical isolates was also performed, genotypic data were found to show excellent correlation with phenotypic results. Correlation was found between the MYCO-Resist LCD array and GenoType MTBDRplus for detection of resistance to RIF. Our study shows MDR-TB in 36% of category II TB cases in Baghdad and surrounding Iraqi provinces, which reflects the World Health Organization findings based on phenotypic studies. Diagnosis of TB and MDR-TB using culture-based tests is a significant impediment to global TB control. The LCD arrays investigated herein are easy to use, sensitive and specific molecular tools for TB resistance profiling in resource-limited laboratory settings.

Genome sequence comparisons of serial multi-drug-resistant Mycobacterium tuberculosis isolates over 21 years of infection in a single patient.

We report a case of chronic pulmonary multi-drug-resistant tuberculosis. Despite 14 years of treatment, Mycobacterium tuberculosis was persistently isolated from sputum. Following treatment cessation the patient remained well, although M. tuberculosis was isolated from sputum for a further 8 years. Genome sequencing of eight serial M. tuberculosis isolates cultured between 1991 and 2011 revealed 17 mutations (0.8 mutations per genome year− 1). Eight of these were persisting mutations and only two mutations were detected in the 7 years following cessation of treatment in 2004. In four isolates there were mixed alleles, suggesting the likely presence of bacterial subpopulations. The initial 1991 isolate demonstrated genotypic resistance to isoniazid (katG W91R), rifampicin (rpoB S531L), ethambutol (embB M306V), streptomycin (gidB L16R), quinolones (gyrA S95T) and P-aminosalicylic acid (thyA T202A). Subsequent resistance mutations developed for pyrazinamide (pncA I31F) and ethionamide (ethA frameshift). Such information might have been instructive when developing a treatment regimen. In retrospect and with the benefit of high-resolution genomic hindsight we were able to determine that the patient received only one or two active anti-tuberculous agents for most of their treatment. Additionally, mutations in bacA and Rv2326c were detected, which may have contributed to the persistent but mild disease course. BacA is likely to be associated with maintenance of chronic infection and Rv2326c with a decreased bacterial metabolic state. These results expand our understanding of M. tuberculosis evolution during human infection and underline the link between antibiotic resistance and clinical persistence.

Evidence for the critical role of a secondary site rpoB mutation in the compensatory evolution and successful transmission of an MDR tuberculosis outbreak strain.

MDR Mycobacterium tuberculosis clinical strains that cause large outbreaks, particularly among HIV-negative patients, are likely to have undergone the most successful compensatory evolution. Hence, mutations secondary to the acquisition of drug resistance are worthy of consideration in these highly transmissible strains. Here, we assessed the role of a mutation within rpoB, rpoB V615M, secondary to the rifampicin resistance-conferring mutation rpoB S531L, which is associated with a major MDR tuberculosis outbreak strain that evolved in an HIV-negative context in northern Tunisia. Using BCG as a model organism, we engineered strains harbouring either the rpoB S531L mutation alone or the double mutation rpoB S531L, V615M. Individual and competitive in vitro growth assays were performed in order to assess the relative fitness of each BCG mutant. The rpoB V615M mutation was found to be invariably associated with rpoB S531L. Structural analysis mapped rpoB V615M to the same bridge helix region as rpoB compensatory mutations previously described in Salmonella. Compared with the rpoB single-mutant BCG, the double mutant displayed improved growth characteristics and fitness rates equivalent to WT BCG. Strikingly, the rpoB double mutation conferred high-level resistance to rifampicin. Here, we demonstrated the fitness compensatory role of a mutation within rpoB, secondary to the rifampicin resistance mutation rpoB S531L, which is characteristic of an MDR M. tuberculosis major outbreak strain. The finding that this secondary mutation concomitantly increased the resistance level to rifampicin argues for its significant contribution to the successful transmission of the MDR-TB strain.

Evaluation of genetic mutations associated with mycobacterium tuberculosis resistance to isoniazid, rifampicin (MTBDR plus), fluoroquinolone and injectable second-line drugs (MTBDRsl)

Aims and objectivesTuberculosis (TB) is one of the major public health problems in Armenia. In 2009, TB incidence in Armenia was reported to be as high as 45.5 per 100,000 population, and TB mortality was 3.9 per 100,000 population. Only 35% of estimated new sputum smear-positive pulmonary TB patients are notified annually. From 1729 TB cases notified in 2010, only 329 patients were sputum smear-positive. The treatment success rate of new sputum smear-positive pulmonary TB patients was 76%, which is below the WHO target of 85%. Poor treatment outcome is partially explained by the high prevalence of drug resistant forms of TB. According to the 2007 drug resistance survey, MDR-TB among never-treated patients was 9.4% and 43.2% among previously treated cases with 4% of XDR cases. This represents an enormous public health challenge for Armenia; early identification and effective treatment of patients with MDR-TB is crucial to prevent further spread of the disease. MethodsA total of 583 specimens are sent to the NRL Armenia being either acid-fast bacterium positive or negative, but culture positive. Rifampin and isoniazid resistance was performed on 77 out of the 583 specimens of DR TB clinical isolates to analyze resistance for FQL, AM/CP and EMB.The DNA preparation method used was as described and recommended by the manufacturer (MTBDR plus version 1.0 and MTBDR sl version 2.0 Hain Lifescience, Nehren).For 99 (88.4%) and 160 (76.2%) strains, the mutations causing rifampicin and isoniazid resistance were located in the codon of rpoB S531L and katG S315T 1, respectively. Regarding isoniazid, 14 (6.6%) strains have a mutation in the inhA regulatory region (C15T) and 31 (14.7%) in both katG and InhA.Mutations detected in the FQ, EMB and rrs-resistant respectively are: 13 (16.9%) strains with the majority of mutations (10 [13%]) in the codon of 94 of the gyrA gen; 20 (26%) strains, with the majority of mutations (7 [9.1%]) in the codon of 306 of the EMB gen; and 13 (16.8%) strains with the majority of mutations (9 [11.6%]) in the codon of 1401 of the rrs gen.Comparing with phenotypically DST sensitivity and specificity for RMP was 97.6% (69 strains) and 100%, respectively. False resistance was detected for 2 strains containing missed bands of rpoB (W6, 7, 8) and 1 strain W7. Sensitivity and specificity for INH was 98.33%, 87.5% (69 strains). From 2 strains, 1 was false resistant (missing of WT 1, 2 bands) and 1 false sensitive results for INH. The sensitivity and specificity for AM/CM was 80%, 100%, and for the FQL 85.7%, 96.72%, respectively (69 strains). ConclusionRapid and accurate detection of resistance to first- and second-line anti-TB drugs is the key to successful therapy and interruption of the transmission chain of MDRTB strains. In summary, this data represent an important addition to the rare epidemiological data concerning resistance patterns of MTB in Armenia and showed that the application of the GenoType MTBDRplus and GenoType MTBDRsl assays might be useful additional tools to allow for a rapid and safe diagnosis of MDR and XDR TB. In mutations associated with katG, high dosages of isoniazid in future will be considered for treatment.

Multidrug-Resistant Tuberculosis in Panama Is Driven by Clonal Expansion of a Multidrug-Resistant Mycobacterium tuberculosis Strain Related to the KZN Extensively Drug-Resistant M. tuberculosis Strain from South Africa

Multidrug-resistant tuberculosis (MDR-TB) is a significant health problem in Panama. The extent to which such cases are the result of primary or acquired resistance and the strain families involved are unknown. We performed whole-genome sequencing of a collection of 66 clinical MDR isolates, along with 31 drug-susceptible isolates, that were isolated in Panama between 2001 and 2010; 78% of the MDR isolates belong to the Latin American-Mediterranean (LAM) family. Drug resistance mutations correlated well with drug susceptibility profiles. To determine the relationships among these strains and to better understand the acquisition of resistance mutations, a phylogenetic tree was constructed based on a genome-wide single-nucleotide polymorphism analysis. The phylogenetic tree shows that the isolates are highly clustered, with a single strain (LAM9-c1) accounting for nearly one-half of the MDR isolates (29/66 isolates). The LAM9-c1 strain was most prevalent among male patients of working age and was associated with high mortality rates. Members of this cluster all share identical mutations conferring resistance to isoniazid (KatG S315T mutation), rifampin (RpoB S531L mutation), and streptomycin (rrs C517T mutation). This evidence of primary resistance supports a model in which MDR-TB in Panama is driven by clonal expansion and ongoing transmission of several strains in the LAM family, including the highly successful MDR strain LAM9-c1. The phylogenetic analysis also shows that the LAM9-c1 strain is closely related to the KwaZulu-Natal (KZN) extensively drug-resistant TB strain identified in KwaZulu-Natal, South Africa. The LAM9-c1 and KZN strains likely arose from a recent common ancestor that was transmitted between Panama and South Africa and had the capacity to tolerate an accumulation of multiple resistance mutations.

Genetic characterization of compensatory evolution in strains carrying rpoB Ser531Leu, the rifampicin resistance mutation most frequently found in clinical isolates

The evolution of rifampicin resistance in Mycobacterium tuberculosis is a major threat to effective tuberculosis therapy. Much is known about the initial emergence of rifampicin resistance, but the further evolution of these resistant strains has only lately been subject to investigation. Although resistance can be caused by many different mutations in rpoB, among clinical M. tuberculosis isolates the mutation rpoB S531L is overwhelmingly the most frequently found. Clinical isolates with rpoB S531L frequently carry additional mutations in genes for RNA polymerase subunits, and it has been speculated that these are fitness-compensatory mutations, ameliorating the fitness cost of the primary resistance mutation. We tested this hypothesis using Salmonella as a model organism. We created the rpoB S531L mutation in Salmonella and then evolved independent lineages with selection for mutants with increased relative fitness. Relative fitness associated with putative compensatory mutations was measured after genetic reconstruction in isogenic strains. Compensatory mutations were identified in genes coding for different subunits of RNA polymerase: rpoA, rpoB and rpoC. Genetic reconstructions demonstrated that each of these secondary mutations reduced the fitness cost of the rpoB S531L resistance mutation. The compensatory mutations identified in Salmonella cluster in similar locations to the additional mutations found in M. tuberculosis isolates. These new data strongly support the idea that many of the previously identified rpoA, rpoB and rpoC mutations in rifampicin-resistant M. tuberculosis (rpoB S531L) are indeed fitness-compensatory mutations.

Molecular patterns of multidrug resistance of Mycobacterium tuberculosis in Georgia

BackgroundTuberculosis (TB) infections caused by multidrug-resistant Mycobacterium tuberculosis (MDR MTB) remain a significant public health concern worldwide. Georgia has a high prevalence of MDR MTB. The genetic mechanisms underlying the emergence of MDR MTB strains in this region are poorly understood and need to be determined for developing better strategies for TB control. This study investigated the frequency of major drug resistance mutations across rpoB, katG and inhA loci of Georgian MDR MTB strains and explored differences between new and previously treated patients. MethodsA total of 634 MTB strains were examined for which an MDR phenotype had been previously determined by the proportions method. The GenoType®MTBDRplus system was applied to screen the strains for the presence of rpoB (S531L, H526D, H526Y, and D516V), katG (S315T) and inhA promoter region (C15T and T8C) mutations. The target loci were amplified by PCR and then hybridized with the respective site-specific and wild type (control) probes. ResultsOut of the 634 isolates tested considered by phenotypic testing to be resistant to RIF and INH, this resistance was confirmed by the GenoType®MTBDRplus assay in 575 (90.7%) isolates. RIF resistance was seen in 589 (92.9%) and INH resistance was seen in 584 (92.1%); 67.2% and 84.3% of MDR strains harbored respectively rpoB S531L and katG S315T mutations (generally known as having low or no fitness cost in MTB). The inhA C15T mutation was detected in 22.6% of the strains, whereas rpoB H526D, rpoB H526Y, rpoB D516V and inhA T8C were revealed at a markedly lower frequency (⩽5.2%). The specific mutations responsible for the RIF resistance of 110 isolates (17.4%) could not be detected as no corresponding mutant probe was indicated in the assay. There was no specific association of the presence of mutations with the gender/age groups. All types of prevailing mutations had higher levels in new cases. ConclusionsA great majority of the Georgian MDR MTB strains have a strong preference for the drug resistance mutations carrying no or low fitness cost. Thus, it can be suggested that MDR MTB strains with such mutations will continue to arise in Georgia at a high frequency even in the absence of antibiotic pressure.

Altered Fatty Acid Metabolism Due to Rifampicin-Resistance Conferring Mutations in therpoBGene ofMycobacterium tuberculosis: Mapping the Potential of Pharmaco-metabolomics for Global Health and Personalized Medicine

Abstract We investigated the use of gas chromatography mass spectrometry (GC-MS) metabolomics to better characterize rifampicin-resistance by comparing the fatty acid metabolomes of two rpoB mutant Mycobacterium tuberculosis strains (S522L and S531L) to that of a fully susceptible wild-type parent strain. Using the generated GC-MS metabolite data, principal component analysis (PCA) showed a clear differentiation between all three sample groups analyzed. We subsequently identified those metabolites contributing most to the variation in the data using PCA and partial least squares discriminant analysis (PLS-DA). The altered metabolite markers detected in the rifampicin-resistant mutants indicate a decreased synthesis of various 10-methyl branched-chain fatty acids and cell wall lipids, and an increased use of the shorter-chain fatty acids as carbon sources. Furthermore, the rpoB S531L mutant, previously reported to occur in well over 70% of all clinical rifampicin-resistant M. tuberculosis strains, potentially showed a better capacity for using these alternative energy sources, compared to the less frequently detected rpoB S522L mutant. This study is the first of its kind to associate rifampicin resistance, rpoB mutations, and the β-subunit of RNA polymerase in M. tuberculosis, with an altered fatty acid metabolism, thereby demonstrating the role that pharmaco-metabolomics can play in identifying new markers associated with drug resistance.

Drug resistance-conferring mutations in Mycobacterium tuberculosis from Madang, Papua New Guinea

BackgroundMonitoring drug resistance in Mycobacterium tuberculosis is essential to curb the spread of tuberculosis (TB). Unfortunately, drug susceptibility testing is currently not available in Papua New Guinea (PNG) and that impairs TB control in this country. We report for the first time M. tuberculosis mutations associated with resistance to first and second-line anti-TB drugs in Madang, PNG. A molecular cluster analysis was performed to identify M. tuberculosis transmission in that region.ResultsPhenotypic drug susceptibility tests showed 15.7% resistance to at least one drug and 5.2% multidrug resistant (MDR) TB. Rifampicin resistant strains had the rpoB mutations D516F, D516Y or S531L; Isoniazid resistant strains had the mutations katG S315T or inhA promoter C15T; Streptomycin resistant strains had the mutations rpsL K43R, K88Q, K88R), rrs A514C or gidB V77G. The molecular cluster analysis indicated evidence for transmission of resistant strain.ConclusionsWe observed a substantial rate of MDR-TB in the Madang area of PNG associated with mutations in specific genes. A close monitoring of drug resistance is therefore urgently required, particularly in the presence of drug-resistant M. tuberculosis transmission. In the absence of phenotypic drug susceptibility testing in PNG, molecular assays for drug resistance monitoring would be of advantage.

Evaluation of the AdvanSure MDR-TB GenoBlot Assay for Detection of Rifampin and Isoniazid ResistantMycobacterium tuberculosisComplex in Respiratory Specimens

Background: We evaluated the performance of the AdvanSure MDR-TB GenoBlot Assay kit (AdvanSure MDR-TB, LG Life Science, Korea) to detect mutations related to rifampin (RFP)and isoniazid (INH)-resistant Mycobacterium tuberculosis complex in respiratory specimens. Methods: From February 2010 to June 2010, a total of 542 M. tuberculosis clinical isolates were collected from pulmonary tuberculosis patients in six university hospitals across Korea. We analyzed the conventional drug susceptibility testing (DST) and compared the results with those of the AdvanSure MDR-TB. Results: Compared with the conventional DST, the overall agreement rates, sensitivity, and specificity were 98.2% (532/542), 84.6% (33/39), and 99.2% (499/503), respectively, for RFP resistance and 96.1% (521/542), 79.7% (59/74), 98.7% (462/468), respectively, for INH resistance. The three common rpoB mutations were rpoB S531L (53.8%), rpoB D516V (15.4%) and rpoB H526R (7.7%) in RFP-resistant strains. For INH resistance, the katG S315T mutation (58.1%) was the most common, followed by inhA C-15T (23.0%) and katG S315N (4.1%). Conclusion: The AdvanSure MDR-TB showed high concordance with the conventional DST and would be helpful for early detection of RFP and INH resistance, although it requires improved sensitivity. (Korean J Clin Microbiol 2012;15:117-124)

Mycobacterium Tuberculosis Genetic Diversity and Drug Resistance Conferring Mutations in the Democratic Republic of the Congo

Background: The Democratic Republic of the Congo (DRC) belongs to the 22 tuberculosis (TB) high-burden countries and to the 27 high-burden multidrug-resistant (MDR)-TB countries. To date, there are no data on the genetic diversity of Mycobacterium tuberculosis in the DRC. Objective: To describe the genetic diversity and the distribution of drug resistance conferring mutations of clinical M. tuberculosis isolates from the DRC. Design: We analysed consecutive M. tuberculosis single patient isolates cultured in 2010 at the laboratory of the National TB Control Programme in Kinshasa. Setting: National TB Control Programme in Kinshasa, DRC. Results: Isolates from 50 patients with pulmonary TB were analysed, including 45 patients (90%) who failed treatment. All isolates belonged to the Euro-American lineage (main phylogenetic Lineage 4). Six different spoligotype families were observed within this lineage, including LAM (20 patients, 40%), T (15 patients; 30%), U (4 patients; 8%), S (3 patients; 6%), Haarlem (2 patients; 4%), and X (1 patient; 2%). No M. africanum strains were observed. The most frequently detected drug resistance-conferring mutations were rpoB S531L and katG S315T1. Various other mutations, including previously unreported mutations, were detected. Conclusions: The Euro-American lineage dominates in the DRC, with substantial variation in spoligotype families. This study fills an important gap on the molecular map of M. tuberculosis in sub-Saharan Africa.

Molecular snapshot of drug-resistant and drug-susceptible Mycobacterium tuberculosis strains circulating in Bulgaria

We report results of the first study on the molecular basis of drug resistance in Mycobacterium tuberculosis strains currently circulating in Bulgaria. The study panel consisted of 133 (including 37 drug-resistant) isolates recovered from newly diagnosed, adult pulmonary TB patients from different regions of Bulgaria in 2005–2006. Three types of the rpoB mutations were found in 20 of 27 RIF-resistant isolates; rpoB S531L was the most frequent. Eleven (48%) of 23 INH-resistant isolates had katG S315T mutation. inhA −15C > T mutation was detected in one INH-resistant isolate (that also had katG315 mutation) and three INH-susceptible isolates. A mutation in embB306 was found in 7 of 11 EMB-resistant isolates. Comparison with spoligotyping and 24-VNTR locus typing data suggested that emergence and spread of drug-resistant and MDR-TB in Bulgaria are not associated with any specific spoligotype or MIRU-VNTR genotype.

Evaluation of the rpoB macroarray assay to detect rifampin resistance in Mycobacterium tuberculosis in Beijing, China

The aim of this study was to evaluate the modified rifampin oligonucleotide (RIFO) macroarray method to detect mutations in the hot-spot region of the rpoB gene, associated with rifampin (RIF) resistance in Mycobacterium tuberculosis. The study sample included 123 strains of M. tuberculosis isolated in the Beijing, China, area in 2002-2005, including 73 RIF-resistant and 40 pansusceptible strains. The genotypic assay successfully identified 91.8% of the RIF-resistant strains, whereas no mutations were found in RIF-susceptible strains. The most frequently detected rpoB mutations were in the codons 516, 526, and 531, together accounting for 74% of RIF-resistant strains. Spoligotyping subdivided all strains into 11 unique profiles and 3 profiles shared by 3, 4, and 103 strains, respectively. The 113 strains belonged to the Beijing family genotypes, defined by the specific spoligotype signature (absence of signals 1-34) and deletion of the RD105 region. The rpoB S531L (TCG-->TTG) mutation was found in 57.4% of the RIF-resistant strains of the Beijing genotype. A mutation in the rpoB hot-spot region was found in 51 of the 55 (92.7%) multidrug-resistant strains (i.e., resistant to at least RIF and isoniazid), thus demonstrating the added utility of the modified RIFO method to predict multidrug resistance. The RIFO method is relatively simple to perform and allows straightforward interpretation of results; consequently, it can be used in clinical diagnostic laboratories as a fast complement to phenotypic methods.