Rapid antibiotic susceptibility testing of Mycobacterium tuberculosis: its utility in resource poor settings

Purpose: To compare the rapid colorimetric nitrate reductase based antibiotic susceptibility (CONRAS) test performed on Mycobacterium tuberculosis isolates with the conventional method i.e., the proportion method. Methods: One hundred clinical isolates of M. tuberculosis were tested for susceptibility to isoniazid (INH) and rifampicin (RIF) by the conventional proportion method and CONRAS in Middlebrook 7H9 liquid medium enriched with growth supplements (MB7H9S). Results: The performance of the CONRAS test was evaluated using proportion method as the gold standard. The sensitivity (ability to detect true drug resistance) and specificity (ability to detect true drug susceptibility) of the CONRAS test to INH was 93.75 and 98.52% and for RIF it was 96.10 and 100% respectively. The mean time for reporting was 6.3 days and the test showed excellent reproducibility. The kappa (κ) value for INH was 0.92 and for RIF was 0.99, indicating excellent agreement between the two methods. Conclusions: CONRAS test is a rapid and reliable method of drug susceptibility for M. tuberculosis.     

The use of E-test for the drug susceptibility testing of Mycobacterium tuberculosis – A solution or an illusion?

Aim: To evaluate E-test as a tool for rapid determination of drug susceptibility against the conventional LJ method focusing on reliability, expense, ease of standardization and performance of the technique in low resource settings. Materials and Methods: A total of 74 clinical isolates (2004-2005) of Mycobacterium tuberculosis were tested using E-test for susceptibility to streptomycin (STM), isoniazid (INH), rifampicin (RIF) and ethambutol (EMB) by E-strip and LJ (LJPM) proportion methods. Results: The LJPM method, the gold standard, detected resistance against STM in 16.2%, INH in 40.5%, RIF in 18.9% and EMB in 27% cases. In comparison, the resistance values showed by E-test was 66.67% for STM, 57.14% for INH 71.43% for RIF and 80% for EMB. The susceptible correlation was 90.32% for STM, 73.91% for INH, 93.33% for RIF and 59.26% for EMB. E-test correctly identified only eight of the 12 (66.6%) MDR isolates and wrongly identified four isolates which were not MDR. The overall agreement between the two methods was only 48.6%. Resistant isolates showed false positive resistance observed while using E-strip towards all the drugs. Conclusion: E-strips are not quite feasible as a replacement for LJ-proportion method on a large scale due to high risk of cross contamination, laboratory infection, expense associated with it and high false positive resistance observed to all first line drugs. However, the good correlation observed for RIF between the two methods indicates that E-test could contribute to the role in rapid screening of MDR TB isolates as rifampicin mutations are invariably observed in MDR TB isolates.     

Discordance across Several Methods for Drug Susceptibility Testing of Drug-Resistant Mycobacterium tuberculosis Isolates in a Single Laboratory

Given the increases in drug-resistant tuberculosis, laboratory capacities for drug susceptibility testing are being scaled up worldwide. A laboratory must decide among several endorsed methodologies. We evaluated 87 Mycobacterium tuberculosis isolates for concordance of susceptibility results across six methods: the L-J proportion method, MGIT 960 SIRE AST, Gene/Xpert MTB/RIF, GenoType MTBDRplus line probe assay, MycoTB MIC plate, and a laboratory-developed mycobacteriophage quantitative PCR (qPCR)-based method. Most (80%) isolates were multidrug resistant. Of the culture-based methods, the mycobacteriophage qPCR method was fastest, the L-J proportion method was the slowest, and the MGIT method required the most repeat testing (P < 0.05). For isoniazid (INH), 82% of isolates were susceptible by all methods or resistant by all methods, whereas for rifampin (RIF), ethambutol (EMB), and streptomycin (STR), such complete concordance was observed in 77%, 50%, and 51% of isolates, respectively (P < 0.05 for INH or RIF versus EMB or STR). The discrepancies of EMB and STR stemmed largely from diminished concordance of the MGIT EMB results (kappa coefficient range, 0.26 to 0.30) and the L-J STR result (kappa range, 0.35 to 0.45) versus other methods. Phage qPCR and the MycoTB MIC plate were the only methods that yielded second-line susceptibilities and revealed significant quantitative correlations for all drugs except cycloserine, as well as moderate to excellent kappa coefficients for all drugs except for para-aminosalicylic acid. In summary, the performance of M. tuberculosis susceptibility testing differs by platform and by drug. Laboratories should carefully consider these factors before choosing one methodology, particularly in settings where EMB and STR results are clinically important.     

Evaluation of Agar-Based Medium with Sheep Sera for Testing of Drug Susceptibility of Mycobacterium tuberculosis to Isoniazid,Rifampin, Ethambutol,and Streptomycin

The performance of sheep sera instead of sheep blood in agar-based media was investigated for susceptibility testing of Mycobacterium tuberculosis against primary drugs. The levels of agreement between agar-based medium supplemented with sheep sera and the proportion method on Middlebrook 7H11 agar as the reference method for determining susceptibility to isoniazid (INH), rifampin (RIF), ethambutol (EMB), and streptomycin (STR) were 98.4, 98.4, 95.3, and 100%, respectively.     

Evaluation of the BD Bactec MGIT 320 System for Detection of Mycobacteria and Drug Susceptibility Testing of Mycobacterium tuberculosis

Two different laboratories evaluated growth and detection of mycobacteria and drug susceptibility testing of Mycobacterium tuberculosis by the BD Bactec MGIT 320 using the BD Bactec MGIT 960 (BD Diagnostics, Sparks, MD) as a reference method. Out of 359 processed sputum specimens for detection of mycobacteria, 99.7% were in agreement between the MGIT 320 and MGIT 960. Streptomycin (STR), isoniazid (INH), rifampin (RIF), ethambutol (EMB) (collectively known as SIRE), and pyrazinamide (PZA) drug susceptibility testing was performed on 89 clinical strains, prepared from both liquid and solid inocula. The results of SIRE and PZA were 100% reproducible between the two instruments tested at both laboratories.     

Multicenter Evaluation of the Mycobacteria Growth Indicator Tube for Testing Susceptibility of Mycobacterium tuberculosis to First-Line Drugs

In a multicenter study involving three reference centers for mycobacteria, the reliability of the Mycobacteria Growth Indicator Tube (MGIT) for rapid antimicrobial susceptibility testing (AST) of Mycobacterium tuberculosis was evaluated and compared to the radiometric method (BACTEC 460TB). Test cultures for which the results of the MGIT and BACTEC 460TB tests were discordant were checked by the conventional proportion method on solid medium. Four hundred forty-one isolates have been tested for susceptibility to isoniazid (INH), rifampin (RMP), ethambutol (EMB), and streptomycin (SM). Discrepant results were obtained for three isolates (0.7%) with INH (susceptible by MGIT, resistant by BACTEC 460TB), for four isolates (0.9%) with RMP (susceptible by MGIT, resistant by BACTEC 460TB), for six isolates (1.9%) with EMB (four susceptible by MGIT, resistant by BACTEC 460TB; two resistant by MGIT, susceptible by BACTEC 460TB), and for four isolates (0.9%) with SM (two susceptible by MGIT, resistant by BACTEC 460TB; two resistant by MGIT, susceptible by BACTEC 460TB). When cultures with discordant results were tested by the conventional proportion method, about half of the cultures yielded results similar to the BACTEC 460TB results, while the other half yielded results similar to the MGIT results. Turnaround times were 3 to 14 days (median, 8.8 days) for MGIT and 3 to 15 days (median, 7.8 days) for BACTEC 460TB. There was no statistically significant difference between the susceptibility testing results of the two methods (P > 0.05). These data demonstrate that the MGIT system is an accurate, nonradiometric alternative to the BACTEC 460TB method for rapid susceptibility testing of M. tuberculosis.     

Evaluation of Etest strips for rapid susceptibility testing of Mycobacterium tuberculosis

In this study, our objective was to evaluate Etest strips containing exponential gradients of isoniazid (INH), rifampin (RIF), and streptomycin (STR) for susceptibility testing of Mycobacterium tuberculosis. M. tuberculosis isolates were tested for antimicrobial susceptibilities by the standard proportion method using L?wenstein-Jensen (LJ) medium and by the Etest. The MICs determined by the Etest were obtained at 5, 7, or 10 days. In some strains with Etest-discrepant results, radiometric susceptibility testing (BACTEC) was performed to determine a consensus result. M. tuberculosis concordance between the two methods was 97% (86 of 89 isolates) for RIF, 96% for INH (84 of 87 isolates), and 80% (61 of 76 isolates) for STR. Most of the MICs determined by the Etest were easy to interpret and readable within 5 days. Results correlated well with those obtained by the LJ proportion and BACTEC methods for INH and RIF. However, a high proportion of false-sensitive and false-resistant results were observed, most often for STR. We also observed that variations in the inoculum size of M. tuberculosis isolates affected the MICs to a substantial degree. These discrepancies, along with the expense of the media, the Etest strips, and the specialized equipment required (CO2 incubator), make this method less useful in developing countries.     

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.     

Testing of susceptibility of Mycobacterium tuberculosis to isoniazid and rifampin by mycobacterium growth indicator tube method.

We tested isolates of Mycobacterium tuberculosis recovered from 117 patients for their susceptibilities to isoniazid (INH) and rifampin (RIF) by the Centers for Disease Control and Prevention's disk modification of the indirect method of proportions (MOP) test and a three-tube mycobacteria growth indicator tube (MGIT; BBL) antimycobacterial susceptibility test (AST). Sixty-seven of the M. tuberculosis isolates were recovered from Lowenstein-Jensen (BBL) subcultures, and 50 of the isolates were recovered from MGIT cultures of samples from various body sites. For the MGIT AST method, 0.5 ml of test organism suspension was inoculated into an MGIT with 0.1 micrograms of INH per ml, an MGIT with 1.0 micrograms of RIF per ml, and growth control MGIT. The tubes were incubated at 37 degrees C and were examined daily. The MGIT AST results were interpreted as follows: susceptible if the tubes containing INH or RIF did not fluoresce within 2 days of the time that the positive growth control fluoresced and resistant if the tubes containing INH or RIF did fluoresce within 2 days of the time that the positive growth control fluoresced. The mean time fluorescence for the positive growth control was 5.5 days. The two methods were in agreement for 114 of the 117 isolates from patients, while for 3 isolates there were minor discordant results.     

Rapid detection of Mycobacterium tuberculosis resistance to second-line drugs by use of the manual mycobacterium growth indicator tube system

The objective of this study was to evaluate the manual mycobacterium growth indicator tube (MGIT) system for the testing of Mycobacterium tuberculosis susceptibility to second-line drugs compared to the proportion method. One hundred eighty-eight M. tuberculosis isolates were tested for susceptibility to ofloxacin, kanamycin, ethionamide, and capreomycin by the manual MGIT, and results were compared to those obtained with the proportion method on 7H11 agar, considered a reference method. Results for ofloxacin and capreomycin were excellent, with 100% accuracy, and a result of 99.4% accuracy was achieved for kanamycin. For ethionamide, accuracy was lower, with a result of 86.7% compared to that of the proportion method. We proposed the following critical concentrations for the drugs: for ofloxacin, 2.0 μg/ml; for kanamycin, 2.5 μg/ml; for ethionamide, 5 μg/ml; and for capreomycin, 2.5 μg/ml. The time required to obtain results was an average of 8 days by the manual MGIT and 3 weeks by the reference method. Our results show that the manual MGIT is an accurate method for the rapid susceptibility testing of M. tuberculosis to second-line drugs. There is no need for a machine when using the manual MGIT, and results can be read with a simple UV lamp or with a semiquantitative reader, which considerably reduces the cost of the method.     

Performance of BACTEC 960 Mycobacteria Growth Indicator Tube in the Susceptibility Testing of Genetically Characterized<Emphasis Type="Italic"> Mycobacterium tuberculosis</Emphasis> Isolates

In this study, the 7H10 agar proportion method was compared with the BACTEC TB-460 and BACTEC MGIT 960 systems (BD Biosciences, USA) for the susceptibility testing of 22 genetically characterized Mycobacterium tuberculosis isolates for isoniazid, rifampin, streptomycin, and ethambutol. The 7H10 agar proportion method agreed with the resistant genotype in 87.3%, BACTEC TB-460 in 92.7%, and the MGIT in 96.4% of the cases, showing the high sensitivity of MGIT in the detection of resistant isolates.     

Comparative study for determination of Mycobacterium tuberculosis susceptibility to first- and second-line antituberculosis drugs by the Etest using 7H11, blood, and chocolate agar

We investigated the performance of blood and chocolate agar as alternatives to Middlebrook 7H11 agar for testing the susceptibility of Mycobacterium tuberculosis to first-and second-line drugs by the Etest method. A total of 39 strains of M. tuberculosis including 22 multidrug-resistant M. tuberculosis strains and 17 susceptible strains were tested. In conclusion, our results showed that chocolate agar gave insufficient growth, needing up to 21 days of incubation, while results on blood agar were comparable to those on Middlebrook 7H11 agar and can be further explored as an alternative for Etest-based susceptibility testing of M. tuberculosis.     

Rapid direct testing of susceptibility of Mycobacterium tuberculosis to isoniazid and rifampin on nutrient and blood agar in resource-starved settings

In this study, we evaluated the performance of blood agar (by macroscopic growth) and nutrient agar (by a microcolony detection method) for drug susceptibility testing of Mycobacterium tuberculosis against rifampin (RIF) and isoniazid (INH), using 67 smear-positive sputum specimens. The direct proportion method on Lowenstein-Jensen (LJ) medium was used as the "gold standard." Compared with LJ medium, results for both media were in 100% agreement for RIF, while for INH the agreement levels for blood agar and nutrient agar were 98% and 95%, respectively. Within 2 weeks, 100% of specimens yielded results on blood agar, while 96.8% of specimens yielded results on nutrient agar. Our study showed that blood agar and nutrient agar can be used as alternative media for direct susceptibility testing of RIF and INH, especially in resource-poor settings.     

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.     

Use of Mycobacteriophage Quantitative PCR on MGIT Broths for a Rapid Tuberculosis Antibiogram

Phenotypic culture-based drug susceptibility testing (DST) for Mycobacterium tuberculosis is a valuable tool to identify four to six active drugs for individualized multidrug-resistant (MDR) tuberculosis (TB) regimens. Current culture-based methods are slow, however; therefore, we evaluated a rapid mycobacteriophage-based quantitative PCR (qPCR) assay for use directly on M. tuberculosis-positive MGIT broths. We compared phage qPCRs, using a simple cutoff of 3 for the ΔCq value (where Cq is quantification cycle, and ΔCq is calculated as the Cq of starting phage minus the Cq of TB isolates in drug-containing medium), on 325 clinical M. tuberculosis MGIT broth cultures versus the respective subcultured isolates tested by agar proportion. The median accuracy for the 13 drugs/concentrations tested was 98%, with most discrepancies being false-resistant results. Evaluation of phage qPCR on greater numbers of resistant strains of 393 isolates grown on Löwenstein-Jensen medium showed similar findings, with a median accuracy, sensitivity, and specificity of 97%, 90%, and 99%, respectively. This rapid culture-based DST methodology can be performed for any drug on TB-positive MGIT broths, with a specimen-to-antibiogram turnaround time of approximately 23.9 days, compared with waiting 58.6 days for isolate growth on solid medium followed by agar proportion DST.     

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.     

Multicenter Evaluation of Fully Automated BACTEC Mycobacteria Growth Indicator Tube 960 System for Susceptibility Testing of Mycobacterium tuberculosis

The reliability of the BACTEC Mycobacteria Growth Indicator Tube (MGIT) 960 system for testing of Mycobacterium tuberculosis susceptibility to the three front-line drugs (isoniazid [INH], rifampin [RIF], and ethambutol [EMB]) plus streptomycin (STR) was compared to that of the BACTEC 460 TB system. The proportion method was used to resolve discrepant results by an independent arbiter. One hundred and ten strains were tested with an overall agreement of 93.5%. Discrepant results were obtained for seven strains (6.4%) with INH (resistant by BACTEC MGIT 960; susceptible by BACTEC 460 TB), for one strain (0.9%) with RIF (resistant by BACTEC MGIT 960; susceptible by BACTEC 460 TB), for seven strains (6.4%) with EMB (six resistant by BACTEC MGIT 960 and susceptible by BACTEC 460 TB; one susceptible by BACTEC MGIT 960 and resistant by BACTEC 460 TB), and for 19 strains (17.3%) with STR (resistant by BACTEC MGIT 960 and susceptible by BACTEC 460 TB). After resolution of discrepant results, the sensitivity of the BACTEC MGIT 960 system was 100% for all four drugs and specificity ranged from 89.8% for STR to 100% for RIF. Turnaround times were 4.6 to 11.7 days (median, 6.5 days) for BACTEC MGIT 960 and 4.0 to 10.0 days (median, 7.0 days) for BACTEC 460 TB. These data demonstrate that the fully automated and nonradiometric BACTEC MGIT 960 system is an accurate method for rapid susceptibility testing of M. tuberculosis.     

Flow Cytometric Testing of Susceptibilities of Mycobacterium tuberculosis Isolates to Ethambutol, Isoniazid, and Rifampin in 24 Hours

Susceptibility testing of Mycobacterium tuberculosis is seriously limited by the time required to obtain results. We show that susceptibility testing of clinical isolates of M. tuberculosis can be accomplished rapidly with acceptable accuracy by using flow cytometry. The susceptibilities of 35 clinical isolates of M. tuberculosis to various concentrations of isoniazid, rifampin, and ethambutol were tested by the agar proportion method and by flow cytometry. Agreement between the results from the two methods was 95, 92, and 83% for isoniazid, ethambutol, and rifampin, respectively. Only 11 discrepancies were detected among 155 total tests. The results of flow cytometric susceptibility tests were available within 24 h of inoculation of drug-containing medium, while the proportion method required 3 weeks to complete. The flow cytometric method is also simple to perform.     

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.     

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.