Rolling Circle Amplification for Direct Detection of rpoB Gene Mutations in Mycobacterium tuberculosis Isolates from Clinical Specimens

Rapid and accurate detection of multidrug resistance (MDR) in Mycobacterium tuberculosis is essential to improve treatment outcomes and reduce global transmission but remains a challenge. Rifampin (RIF) resistance is a reliable marker of MDR tuberculosis (TB) since by far the majority of RIF-resistant strains are also isoniazid (INH) resistant. We have developed a rapid, sensitive, and specific method for detecting the most common mutations associated with RIF resistance, in the RIF resistance determining region (RRDR) of rpoB, using a cocktail of six padlock probes and rolling circle amplification (RCA). We used this method to test 46 stored M. tuberculosis clinical isolates with known RIF susceptibility profiles (18 RIF resistant, 28 susceptible), a standard susceptible strain (H37Rv, ATCC 27294) and 78 M. tuberculosis culture-positive clinical (sputum) samples, 59 of which grew RIF-resistant strains. All stored clinical isolates were correctly categorized, by the padlock probe/RCA method, as RIF susceptible or resistant; the sensitivity and specificity of the method, for direct detection of phenotypically RIF-resistant M. tuberculosis in clinical specimens, were 96.6 and 89.5%, respectively. This method is rapid, simple, and inexpensive and has the potential for high-throughput routine screening of clinical specimens for MDR M. tuberculosis, particularly in high prevalence settings with limited resources.     

Detection of rpoB,katG and inhA gene mutations in Mycobacterium tuberculosis clinical isolates from Chongqing as determined by microarray

The emergence of multidrug-resistance Mycobacterium tuberculosis is an increasing threat to tuberculosis control programmes. Susceptibility testing of Mycobacterium tuberculosis complex isolates by traditional methods requires a minimum of 14 days. This can be reduced significantly if molecular analysis is used. DNA sequencing is a good method for detecting mutation, but cannot be used routinely because of its relatively high cost. A sensitive and specific microarray has been designed to detect mutations in the rifampin resistance determining region of rpoB and loci in katG and inhA associated with isoniazid (INH) resistance. A panel of Mycobacterium tuberculosis isolates containing 13 different rpoB genotypes, two mutation genotypes within codon 315 of katG and one mutation genotypes at inhA was used to validate the microarray. The results obtained indicate that 100% of rifampicin-resistant M. tuberculosis strains isolated in Chongqing had rpoB mutations, with 531-Ser and 526-His being the most common positions substituted. Of the total 50 INH resistant isolates, 82% had a katG315 mutation and 18% had an inhA mutation. All the mutations detected by the microarray method were also confirmed by conventional DNA sequencing. It is demonstrated that the microarray is an efficient, specialized technique and can be used as a rapid method for detecting rifampin and isoniazid resistance.     

Magnitude of Gene Mutations Conferring Drug Resistance in Mycobacterium Tuberculosis Isolates from Lymph Node Aspirates in Ethiopia

Objective: Resistance to drugs is due to particular genomic mutations in the specific genes of Mycobacterium tuberculosis. Timely genetic characterization will allow identification of resistance mutations that will optimize an effective antibiotic treatment regimen. We determine the magnitude of gene mutations conferring resistance to isoniazid (INH), rifampicin (RMP) and ethambutol (EMB) among tuberculosis (TB) lymphadenitis patients.Methods: A cross sectional prospective study was conducted among 226 M.tuberculosis isolates from culture positive lymph node aspirates collected from TB lymphadenitis patients between April 2012 and May 2012. Detection of mutations conferring resistance to drugs was carried out using GenoType^® MTBDRplus and GenoType® MTBDRsl assay.Results: Out of the 226 strains, mutations conferring resistance to INH, RMP, multidrug resistance tuberculosis (MDR-TB) and EMB were 8, 3, 2 and 2 isolates, respectively. There was no isolated strain that showed mutation in the inhA promoter region gene. All INH resistant strains had mutations in the katG gene at codon 315 with amino acid change of S315T1. Among rifampicin resistant strains, two isolates displayed mutations at codon 531 in the rpoB gene with amino acid change of S531L and one isolate was by omission of wild type probes at Q513L. According to mutations associated with ethambutol resistance, all of the isolates had mutations in the embB gene with aminoacid change of M306I. All isolates resistant to INH, RMP and MDR using BacT/AlerT 3D system were correctly identified by GenoType® MTBDRplus assay.Conclusion: We observed mutations conferring resistance to INH at S315T1 of the katG gene, RMP at S531L and Q513L in the rpoB genes and EMB at M306I of the embB gene. In the absence of conventional drug susceptibility testing, the effort to develop easy, rapid and cost effective molecular assays for drug resistance TB monitoring is definitely desirable and the GenoType® MTBDRplus assay was found to be a useful method for diagnosis of resistance to INH, RMP and MDR from lymph node aspirates. Further molecular cluster analysis to determine transmission dynamics of mutated strain is required.     

Pyrazinamide resistance in multidrug-resistant Mycobacterium tuberculosis isolates in Japan

Thirty-six multidrug-resistant (MDR) Mycobacterium tuberculosis isolates collected in Japan were examined for pyrazinamide susceptibility and pyrazinamidase activity, and analysed by pncA sequencing and a hybridization-based line probe assay (LiPA), which was used to detect pncA mutations for the rapid identification of pyrazinamide-resistant isolates. Pyrazinamide resistance was found in 19 (53%) of them. All pyrazinamide-resistant isolates had no pyrazinamidase activity and at least one mutation in pncA. Among the pncA mutations, 11 had not been previously reported. The results of the LiPA were fully consistent with the DNA sequencing results. A majority of MDR M. tuberculosis isolates in Japan were resistant to pyrazinamide.     

Rapid screening of rpoB and katG mutations in Mycobacterium tuberculosis isolates by high-resolution melting curve analysis

Background: Early detection of multidrug-resistant tuberculosis (MDR-TB) is essential to prevent its transmission in the community and initiate effective anti-TB treatment regimen. Materials and Methods: High-resolution melting curve (HRM) analysis was evaluated for rapid detection of resistance conferring mutations in rpoB and katG genes. We screened 95 Mycobacterium tuberculosis clinical isolates including 20 rifampin resistant (RIF-R), 21 isoniazid resistant (INH-R) and 54 fully susceptible (S) isolates determined by proportion method of drug susceptibility testing. Nineteen M. tuberculosis isolates with known drug susceptibility genotypes were used as references for the assay validation. The nucleotide sequences of the target regions rpoB and katG genes were determined to investigate the frequency and type of mutations and to confirm HRM results. Results: HRM analysis of a 129-bp fragment of rpoB allowed correct identification of 19 of the 20 phenotypically RIF-R and all RIF-S isolates. All INH-S isolates generated wild-type HRM curves and 18 out of 21 INH-R isolates harboured any mutation in 109-bp fragment of katG exhibited mutant type HRM curves. However, 1 RIF-R and 3 INH-R isolates were falsely identified as susceptible which were confirmed for having no mutation in their target regions by sequencing. The main mutations involved in RIF and INH resistance were found at codons rpoB531 (60% of RIF-R isolates) and katG315 (85.7% of INH-R isolates), respectively. Conclusion: HRM was found to be a reliable, rapid and low cost method to characterise drug susceptibility of clinical TB isolates in resource-limited settings.     

Utility of GenoType MTBDRplus assay in rapid diagnosis of multidrug resistant tuberculosis at a tertiary care centre in India

Purpose: Molecular methods which allow rapid detection of tuberculosis as well as drug resistance directly from clinical samples have become the most popular diagnostic methodology with the emergence of multidrug resistant tuberculosis. The aim of the present study was to evaluate the performance of a line probe assay, GenoType MTBDRplus for the rapid detection of Mycobacterium tuberculosis and mutations causing rifampicin and INH resistance directly in smear positive pulmonary specimens and also in M. tuberculosis isolates grown from various clinical specimens. Materials and Methods: The MTBDRplus assay was done directly on 37 smear positive pulmonary specimens and also on 69 M. tuberculosis isolates obtained by rapid automated culture using Bact/Alert 3D. The results were compared with phenotypic drug susceptibility testing (1% proportion method) using Bact/Alert 3D. Results: The sensitivity and specificity for detection of resistance to rifampicin was 100% and 97.3%, and to INH was 91.9% and 98.4%, respectively, in comparison with the phenotypic drug susceptibility testing. Conclusion: MTBDRplus assay had good sensitivity and specificity with turn around time of less than 48 hours. It may be a useful tool for rapid detection of multidrug resistant tuberculosis at a tertiary care centre.     

Rapid Detection of Isoniazid Resistance in Mycobacterium tuberculosis Isolates by Use of Real-Time-PCR-Based Melting Curve Analysis

The MeltPro TB/INH assay, recently approved by the Chinese Food and Drug Administration, is a closed-tube, dual-color, melting curve analysis-based, real-time PCR test specially designed to detect 30 isoniazid (INH) resistance mutations in katG position 315 (katG 315), the inhA promoter (positions −17 to −8), inhA position 94, and the ahpC promoter (positions −44 to −30 and −15 to 3) of Mycobacterium tuberculosis. Here we evaluated both the analytical performance and clinical performance of this assay. Analytical studies with corresponding panels demonstrated that the accuracy for detection of different mutation types (10 wild-type samples and 12 mutant type samples), the limit of detection (2 × 10³ to 2 × 10⁴ bacilli/ml), reproducibility (standard deviation [SD], <0.4°C), and the lowest heteroresistance level (40%) all met the parameters preset by the kit. The assay could be run on five types of real-time PCR machines, with the shortest running time (105 min) obtained with the LightCycler 480 II. Clinical studies enrolled 1,096 clinical isolates collected from three geographically different tuberculosis centers, including 437 INH-resistant isolates and 659 INH-susceptible isolates characterized by traditional drug susceptibility testing on Löwenstein-Jensen solid medium. The clinical sensitivity and specificity of the MeltPro TB/INH assay were 90.8% and 96.4%, respectively. DNA sequencing analysis showed that, except for the 5 mutants outside the detection range of the MeltPro assay, a concordance rate between the two methods of 99.1% (457/461) was obtained. Among the 26 mutation types detected, katG S315T (AGC→ACC), inhA −15C→T, katG S315N (AGC→AAC), and ahpC promoter −10C→T accounted for more than 90%. Overall, the MeltPro TB/INH assay represents a reliable and rapid tool for the detection of INH resistance in clinical isolates.     

The GenoType® MTBDRplus assay for detection of drug resistance in Mycobacterium tuberculosis in Sweden

Chryssanthou E, Ängeby K. The GenoType^® MTBDRplus assay for detection of drug resistance in Mycobacterium tuberculosis in Sweden. APMIS 2012; 120: 405–9. The performance of the GenoType^®MTBDRplus assay was compared with conventional drug susceptibility testing (DST) in 604 patients with tuberculosis. The study comprised 477 Mycobacterium tuberculosis complex isolates and 127 preparations of DNA from clinical specimens which had been tested positive for M. tuberculosis by COBAS^®TaqMan^® 48. By DST, isoniazid (INH) monoresistance was diagnosed in 56 (9.3%), rifampicin (RMP) monoresistance in 2 (0.3%) and multidrug resistance (MDR) in 21 (3.5%) of the cases. The sensitivity of the MTBDRplus assay was 87.5%, 100% and 95.2% for INH resistance, RMP resistance and MDR respectively. The specificity was 100% for all resistance patterns. The dominating mutations in RMP and INH resistant isolates were in codon 531 of the rpoB gene and codon 315 of the KatG gene. The turnaround time for detection of drug resistance can be shortened from a median of 21 days for DST to 7 days for the MTBDRplus assay. This may have a significant impact on routine work flow of a mycobacteriology laboratory.     

Evaluation of the AID TB Resistance Line Probe Assay for Rapid Detection of Genetic Alterations Associated with Drug Resistance in Mycobacterium tuberculosis Strains

The rapid accurate detection of drug resistance mutations in Mycobacterium tuberculosis is essential for optimizing the treatment of tuberculosis and limiting the emergence and spread of drug-resistant strains. The TB Resistance line probe assay from Autoimmun Diagnostika GmbH (AID) (Strassburg, Germany) was designed to detect the most prevalent mutations that confer resistance to isoniazid, rifampin, streptomycin, amikacin, capreomycin, fluoroquinolones, and ethambutol. This assay detected resistance mutations in clinical M. tuberculosis isolates from areas with low and high levels of endemicity (Switzerland, n = 104; South Africa, n = 52) and in selected Mycobacterium bovis BCG 1721 mutant strains (n = 5) with 100% accuracy. Subsequently, the line probe assay was shown to be capable of rapid genetic assessment of drug resistance in MGIT broth cultures, the results of which were in 100% agreement with those of DNA sequencing and phenotypic drug susceptibility testing. Finally, the line probe assay was assessed for direct screening of smear-positive clinical specimens. Screening of 98 clinical specimens demonstrated that the test gave interpretable results for >95% of them. Antibiotic resistance mutations detected in the clinical samples were confirmed by DNA sequencing. We conclude that the AID TB Resistance line probe assay is an accurate tool for the rapid detection of resistance mutations in cultured isolates and in smear-positive clinical specimens.     

Unequal distribution of resistance-conferring mutations among Mycobacterium tuberculosis and Mycobacterium africanum strains from Ghana

Isoniazid (INH) and rifampicin (RMP) resistance in Mycobacterium tuberculosis complex (MTC) isolates are mainly based on mutations in a limited number of genes. However, mutation frequencies vary in different mycobacterial populations. In this work, we analyzed the distribution of resistance-associated mutations in M. tuberculosis and M. africanum strains from Ghana, West Africa. The distribution of mutations in katG, fabG1-inhA, ahpC, and rpoB was determined by DNA sequencing in 217 INH-resistant (INH^r) and 45 multidrug-resistant (MDR) MTC strains isolated in Ghana from 2001 to 2004. A total of 247 out of 262 strains investigated (94.3%) carried a mutation in katG (72.5%), fabG1-inhA (25.1%), or ahpC (6.5%), respectively. M. tuberculosis strains mainly had katG 315 mutations (80.1%), whereas this proportion was significantly lower in M. africanum West-African 1 (WA1) strains (43.1%; p < 0.05). In contrast, WA1 strains showed more mutations in the fabG1-inhA region (39.2%, p < 0.05) compared to M. tuberculosis strains (20.9%). In 44 of 45 MDR strains (97.8%) mutations in the 81-bp core region of the rpoB gene could be verified. Additionally, DNA sequencing revealed that 5 RMP-susceptible strains also showed mutations in the rpoB hotspot region. In conclusion, although principally the same genes were affected in INH^rM. tuberculosis and M. africanum strains, disequilibrium in the distribution of mutations conferring resistance was verified that might influence the efficiency of molecular tests for determination of resistance.     

Evaluation of the GenoType MTBDR assay for detection of rifampicin and isoniazid resistance in Mycobacterium tuberculosis complex isolates

Detection of drug resistance plays a critical role in tuberculosis treatment. The aim of this study was to evaluate the performance of GenoType Mycobacteria Drug Resistance (MTBDR) assay (Hain Lifescience, Germany) and to compare it with radiometric BACTEC 460 TB system (Becton Dickinson, USA) for the detection of rifampicin (RIF) and isoniazid (INH) resistance in 84 Mycobacterium tuberculosis complex (MTBC) isolates. RIF resistance was identified in 6 of 7 (85.7%) isolates and INH resistance was identified in 8 of 14 (57.1%) isolates by the GenoType MTBDR assay. Compared with BACTEC system, the sensitivity, specificity, positive predictive value and negative predictive values were 85.7%, 98.7%, 85.7% and 98.7% for RIF resistance; and 57.1%, 100%, 100% and 92.1% for INH resistance, respectively. GenoType MTBDR assay is reliable when tested specimen is resistant to the tested drugs. Although test was more successful in the detection of RIF resistance, it exhibited low sensitivity for the detection of INH resistance.     

Mutations at embB306 codon and their association with multidrug resistant M. tuberculosis clinical isolates

Purpose: The presence of embB306 mutation in ethambutol (EMB)-susceptible (EMBs) clinical isolates questions the significance of these mutations in conferring resistance to EMB. The present study was carried out to determine the occurrence of embB306 mutation in EMB-resistant (EMBr) and EMBs strains of M. tuberculosis. One hundred and four multidrug-resistant tuberculosis (MDR-TB) strains were also included to establish the relevance of excessive use of rifampicin (RIF) and isoniazid (INH) in occurrence of embB306 mutations in EMBs M. tuberculosis isolates. Materials and Methods: Deoxyribonucleic acid (DNA) from M. tuberculosis clinical strains was isolated by cetyltrimethylammonium bromide (CTAB) method. Phenotypic and genotypic drug susceptibility testing (DST) was performed on 354 M. tuberculosis isolates by using standard proportion method and multiplex-allele-specific polymerase chain reaction assay, respectively. Results: The overall frequency of embB306 mutations in EMBr isolates was found to be five times higher than its occurrence in EMB-susceptible isolates (50% vs 10%). Further, the association between embB306 mutation and EMB-resistance was observed to be statistically significant (P = 0.000). Conclusion: The embB306 is not only the main causative mutation of EMB resistance, but is a sensitive applicant marker for EMB-resistance study.     

GenoType MTBDRplus assay for molecular detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis strains and clinical samples

The purpose of this study was to evaluate the GenoType MTBDRplus assay (Hain Lifescience GmbH, Nehren, Germany) for its ability to detect resistance to rifampin (RIF) and isoniazid (INH) in Mycobacterium tuberculosis clinical strains and directly in clinical samples. A total of 62 clinical strains characterized with the Bactec 460TB system were included. For the INH-resistant strains, the MIC was measured and sequencing was performed. Sixty-five clinical samples from 28 patients (39 smear-positive samples and 26 smear-negative samples) were also tested directly. The corresponding isolates of the clinical specimens were studied with the Bactec 460TB system. The overall rates of concordance of the MTBDRplus assay and the Bactec 460TB system for the detection of RIF and INH susceptibility in clinical strains were 98.3% (61/62) and 79% (49/62), respectively. The rate of concordance between the Bactec 460TB system and the MTBDRplus test for the detection of INH resistance in the group of 27 strains with low-level resistance was 62.9% (17/27), and that for the detection of INH resistance in the group of 21 strains with high-level resistance was 85.71% (18/21). Valid test results were obtained for 78.45% (51/65) of the clinical samples tested. The rates of concordance between both assays for the detection of drug resistance in these samples were 98% (50/51) for RIF and 96.2% (49/51) for INH. Taking into account only one sample per patient, the overall rate of concordance between both tests was 92.85% (26/28). The GenoType MTBDRplus assay is easy to perform and is a useful tool for the management of tuberculosis, as it allows the detection of resistance to RIF and INH in M. tuberculosis strains and also in clinical samples.     

Performance Assessment of the GenoType MTBDRplus Test and DNA Sequencing in Detection of Multidrug-Resistant Mycobacterium tuberculosis

To facilitate the management of multidrug-resistant (MDR) tuberculosis, two nucleic acid sequence-based methods, the GenoType MTBDRplus test and DNA sequencing, were assessed for the rapid detection of drug-resistant Mycobacterium tuberculosis for the first time in the Asia-Pacific region. The performances of these two assays in detecting the presence of rifampin (rifampicin) (RIF) and isoniazid (INH) resistance-associated mutations in the rpoB, katG, inhA regulatory region, inhA, and oxyR-ahpC genes were compared to that of a conventional agar proportion drug susceptibility test. A total of 242 MDR and 30 pansusceptible M. tuberculosis isolates were evaluated in this study. The sensitivities obtained for RIF-resistant detection by the GenoType MTBDRplus test and by resistance gene sequencing were 95.5% and 97.9%, respectively. The sensitivities for INH resistance detection by the GenoType MTBDRplus test and by resistance gene sequencing were 81.8% and 93.4%, respectively. Together, the sensitivity for MDR tuberculosis detection was 78.5% with the GenoType MTBDRplus test and 91.3% by resistance gene sequencing. The specificity for RIF resistance, INH resistance, and MDR detection was 100% by both methods. The GenoType MTBDRplus test has the advantage of a short turnaround time for drug-resistant M. tuberculosis detection. Overall, the two assays performed equally well in detecting RIF resistance (P = 0.13). However, DNA sequencing demonstrated superior performance in detecting INH resistance (P < 0.001) and MDR tuberculosis (P < 0.001). We suggest that new alleles of INH resistance genes should be evaluated to improve the sensitivity of the GenoType MTBDRplus test, especially for different geographic areas with genetically diverse M. tuberculosis strains.The emergence of multidrug-resistant tuberculosis (MDR-TB), defined as infection with a Mycobacterium tuberculosis complex isolate resistant to at least isoniazid (INH) and rifampin (rifampicin) (RIF), is a public health concern and threatens global TB control programs (22). In Taiwan, approximately 15,000 new TB cases are diagnosed annually, of which an estimated 4% are MDR-TB (12). Therefore, the Taiwan Centers for Disease Control (CDC) not only has strengthened directly observed treatment in the management of TB as of 2006, to prevent MDR generation, but also has implemented a DOTS-Plus (directly observed treatment, short-course) strategy for the management of MDR-TB patients as of 2007 (8). However, this program can be hampered by delayed laboratory diagnosis. The completion of diagnosis by conventional methods and drug susceptibility testing (DST) of M. tuberculosis normally take months.The World Health Organization and partners have endorsed the use of the molecular test GenoType MTBDRplus (Hain Lifescience GmbH, Nehren, Germany) for rapid detection of high-risk MDR-TB cases, even directly from certain clinical specimens (1, 4, 6, 10, 15, 21). The GenoType MTBDRplus test is a PCR-based amplification and reverse blotting assay that employs specific probes hybridized to nitrocellulose strips to detect RIF and INH resistance. The assay detects mutations in the rpoB gene for RIF resistance, in the katG gene for high-level INH resistance, and in the inhA regulatory region gene for low-level INH resistance. To evaluate the reliability of the assay, DNA sequencing analyses of rpoB for RIF and katG, the inhA regulatory region gene, inhA, or oxyR-ahpC for INH were conducted in parallel.Our previous study demonstrated the genetic diversity of MDR M. tuberculosis isolates with novel alleles in the rpoB gene in Taiwan (11). Likewise, the distribution of M. tuberculosis isolates differs in different geographic regions (5, 11). The GenoType MTBDRplus test has been assessed in Europe (6, 10, 15, 21), South Africa (4), and the Caribbean (1), but not in the Asia-Pacific region, where there is a high prevalence of Beijing family M. tuberculosis isolates. Here we report the performance of the revised GenoType MTBDRplus test compared to that of DNA sequencing using a culture-based phenotypic DST, which is considered the gold standard for routine clinical practice.     

Concordance between Molecular and Phenotypic Testing of Mycobacterium tuberculosis Complex Isolates for Resistance to Rifampin and Isoniazid in the United States

Multidrug-resistant (MDR) isolates of Mycobacterium tuberculosis complex (MTBC) are defined by resistance to at least rifampin (RMP) and isoniazid (INH). Rapid and accurate detection of multidrug resistance is essential for effective treatment and interruption of disease transmission of tuberculosis (TB). Overdiagnosis of MDR TB may result in treatment with second-line drugs that are more costly, less effective, and more poorly tolerated than first-line drugs. CDC offers rapid confirmation of MDR TB by the molecular detection of drug resistance (MDDR) for mutations associated with resistance to RMP and INH along with analysis for resistance to other first-line and second-line drugs. Simultaneously, CDC does growth-based phenotypic drug susceptibility testing (DST) by the indirect agar proportion method for a panel of first-line and second-line antituberculosis drugs. We reviewed discordance between molecular and phenotypic DST for INH and RMP for 285 isolates submitted as MTBC to CDC from September 2009 to February 2011. We compared CDC''s results with those from the submitting public health laboratories (PHL). Concordances between molecular and phenotypic testing at CDC were 97.4% for RMP and 92.5% for INH resistance. Concordances between CDC''s molecular testing and PHL DST results were 93.9% for RMP and 90.0% for INH. Overall concordance between CDC molecular and PHL DST results was 91.7% for RMP and INH collectively. Discordance was primarily attributable to the absence of known INH resistance mutations in isolates found to be INH resistant by DST and detection of mutations associated with low-level RMP resistance in isolates that were RMP susceptible by phenotypic DST. Both molecular and phenotypic test results should be considered for the diagnosis of MDR TB.     

Association of MDR-TB isolates with clinical characteristics of patients from Northern region of India

Purpose: We sought to determine the characteristics and relative frequency of transmission of MDR-TB in North India and their association with the clinical and epidemiological characteristics of TB-patients. Materials and Methods: To achieve the objectives PCR-SSCP, MAS-PCR and direct DNA sequencing were used against 101 Mycobacterium tuberculosis isolates. Results: Multidrug-resistant-TB isolates were found to be significantly higher (P = 0.000) in previously treated patients in comparison to newly diagnosed patients. Further, significant differences (P = 0.003) were observed between different age groups (Mean ± SD, 28.6 ± 11.77) of the TB patients and multidrug resistance. Most frequent mutations were observed at codons 531 and 315 of rpoB and katG genes, respectively, in MDR-TB isolates. Conclusion: Routine surveillance of resistance to anti-TB drugs will improve timely recognition of MDR-TB cases and help prevent further transmission in Northern India.     

Simplified Microarray System for Simultaneously Detecting Rifampin,Isoniazid, Ethambutol,and Streptomycin Resistance Markers in Mycobacterium tuberculosis

We developed a simplified microarray test for detecting and identifying mutations in rpoB, katG, inhA, embB, and rpsL and compared the analytical performance of the test to that of phenotypic drug susceptibility testing (DST). The analytical sensitivity was estimated to be at least 110 genome copies per amplification reaction. The microarray test correctly detected 95.2% of mutations for which there was a sequence-specific probe on the microarray and 100% of 96 wild-type sequences. In a blinded analysis of 153 clinical isolates, microarray sensitivity for first-line drugs relative to phenotypic DST (true resistance) was 100% for rifampin (RIF) (14/14), 90.0% for isoniazid (INH) (36/40), 70% for ethambutol (EMB) (7/10), and 89.1% (57/64) combined. Microarray specificity (true susceptibility) for first-line agents was 95.0% for RIF (132/139), 98.2% for INH (111/113), and 98.6% for EMB (141/143). Overall microarray specificity for RIF, INH, and EMB combined was 97.2% (384/395). The overall positive and negative predictive values for RIF, INH, and EMB combined were 84.9% and 98.3%, respectively. For the second-line drug streptomycin (STR), overall concordance between the agar proportion method and microarray analysis was 89.5% (137/153). Sensitivity was 34.8% (8/23) because of limited microarray coverage for STR-conferring mutations, and specificity was 99.2% (129/130). All false-susceptible discrepant results were a consequence of DNA mutations that are not represented by a specific microarray probe. There were zero invalid results from 220 total tests. The simplified microarray system is suitable for detecting resistance-conferring mutations in clinical M. tuberculosis isolates and can now be used for prospective trials or integrated into an all-in-one, closed-amplicon consumable.     

Profiling of rpoB Mutations and MICs for Rifampin and Rifabutin in Mycobacterium tuberculosis

Resistance to rifampin (RIF) and rifabutin (RFB) in Mycobacterium tuberculosis is associated with mutations within an 81-bp region of the rpoB gene (RIF resistance-determining region [RRDR]). Previous studies have shown that certain mutations in this region are more likely to confer high levels of RIF resistance, while others may be found in phenotypically susceptible isolates. In this study, we sought to determine the relationship between the MICs of RIF and RFB and rpoB RRDR mutations in 32 multidrug-resistant (MDR), 4 RIF-monoresistant, and 5 susceptible M. tuberculosis clinical isolates. The MICs were determined using the MGIT 960 system. Mutations in the rpoB RRDR were determined by Sanger sequencing. RpoB proteins with mutations S531L (a change of S to L at position 531), S531W, H526Y, and H526D and the double mutation D516A-R529Q were associated with high MICs for RIF and RFB. Five isolates carrying the mutations L511P, H526L, H526N, and D516G-S522L were found to be susceptible to RIF. Several mutations were associated with resistance to RIF and susceptibility to RFB (F514FF, D516V, and S522L). Whole-genome sequencing of two MDR isolates without rpoB RRDR mutations revealed a mutation outside the RRDR (V146F; RIF MIC of 50 μg/ml). The implications of the polymorphisms identified in the second of these isolates in RIF resistance need to be further explored. Our study further establishes a correlation between the mutations and the MICs of RIF and, also, RFB in M. tuberculosis. Several rpoB mutations were identified in RIF- and RFB-susceptible isolates. The clinical significance of these findings requires further exploration. Until then, a combination of phenotypic and molecular testing is advisable for drug susceptibility testing.