Molecular Screening Versus Phenotypic Susceptibility Testing of Multidrug-Resistant Mycobacterium tuberculosis Isolates for Streptomycin and Ethambutol

Abstract

Proper management of multidrug-resistant tuberculosis (MDR-TB) requires accurate drug susceptibility testing (DST) of Mycobacterium tuberculosis isolates to other (ethambutol [EMB], pyrazinamide, and streptomycin [SM]) first-line drugs. This study compared the performance of Mycobacterium Growth Indicator Tube (MGIT) 960 system for DST of MDR-TB isolates with polymerase chain reaction (PCR) sequencing of embB, rpsL, and rrs genes for detecting resistance to EMB and SM. MDR-TB strains (n = 60) and 25 pansusceptible M. tuberculosis isolates collected during 2011–2016 were tested. Phenotypic DST was performed by MGIT 960 system by using SIRE drug kit. EMB and SM resistance-conferring mutations in embB and rpsL+rrs genes, respectively, were detected by PCR sequencing. No mutations were detected in pansusceptible isolates. Among 60 MDR-TB strains, 35 of 40 SM-resistant and none of 20 SM-susceptible isolates contained rpsL and/or rrs mutations (κ = 0.82, very good agreement). However, all 18 EMB-resistant MDR-TB strains and 33 of 42 EMB-susceptible MDR-TB strains contained an embB mutation (κ = 0.14, poor agreement). Thus, 40 of 60 (67%) and 35 of 60 (58%) isolates were resistant to SM (p = 0.451), while 18 of 60 (30%) and 51 of 60 (85%) isolates were resistant to EMB (p = 0.000) by MGIT 960 system and PCR sequencing, respectively. MGIT 960 system showed acceptable performance for DST for SM; however, it performed poorly for EMB as many MDR-TB strains with embB mutations, which confer low-level resistance to EMB, were detected as EMB susceptible. Molecular screening for resistance-conferring mutations in embB gene is thus superior to MGIT 960 system when accurate EMB susceptibility results are needed for proper management of MDR-TB patients.

Introduction

The global burden of tuberculosis (TB) remains high and drug-resistant TB is endemic in many countries. According to the latest World Health Organization (WHO) annual survey, an estimated 10.4 million new active TB disease cases occurred in 2016, which caused nearly 1.7 million deaths, making TB as the ninth leading cause of death across the globe.¹ Furthermore, 490,000 new cases of multidrug-resistant TB (MDR-TB, Mycobacterium tuberculosis strain resistant at least to rifampicin [RIF] and isoniazid [INH], the two most effective first-line drugs) occurred in 2016.¹ Proper management of MDR-TB requires treatment for nearly 2 years with 4–6 drugs that are 10 times costlier and often cause adverse drug reactions resulting in higher rates of clinical failure and/or disease relapse.^1–3 Incomplete treatment of MDR-TB may also lead to the development of extensively drug-resistant TB (XDR-TB, MDR-TB strains additionally resistant to a fluoroquinolone and kanamycin, amikacin, or capreomycin), which is very difficult to treat in poor and developing countries.^3,4 Favorable outcomes for MDR-TB are heavily dependent on rapid laboratory diagnosis and inclusion of effective first-line drugs in therapy regimens.^5,6 Successful treatment is also required to limit further transmission of MDR-TB and prevent the development of XDR-TB.^2–6

The phenotypic drug susceptibility testing (DST) of M. tuberculosis isolates by solid (Lowenstein-Jensen) medium-based proportion method, considered the gold standard, is slow as it takes 4–6 weeks to report results.⁷ The commercial liquid media-based culture systems and molecular assays have been developed and endorsed by WHO for rapid detection of drug resistance in M. tuberculosis strains.^5,6 However, detection of drug resistance for some drugs (such as RIF) by molecular assays other than DNA sequencing should be validated by phenotypic methods and any discordant results should be confirmed by DNA sequencing of the respective resistance-conferring genes due to imperfect DST by some phenotypic as well as some genotypic assays.^8–11 The semiautomated and radiometric BACTEC 460TB system accurately performed DST in the previous three decades, reporting results typically within 14 days.⁸ However, due to concerns for safe disposal of radioactivity, fully automated culture systems such as the Mycobacterium Growth Indicator Tube (MGIT) 960 system with similar turnaround time subsequently replaced BACTEC 460TB system in clinical mycobacteriology laboratories around the world.⁸ Of the first-line (RIF, INH, ethambutol [EMB] pyrazinamide [PZA], and streptomycin [SM]) anti-TB drugs, standard DST of M. tuberculosis is problematic for EMB (a slow-acting bacteriostatic agent) and PZA (a drug, i.e., effective at pH 5.6, which retards growth of tubercle bacilli) by routine automated culture systems.^6,8 These methods also fail to detect low-level, but clinically significant RIF-resistant strains.^9–11 Consequently, molecular tests based on targeted screening of most common resistance-conferring mutations in target genes have been endorsed as rapid screening tests for diagnosing drug-resistant TB and MDR-TB.^{2,5,6,12–14}

EMB interferes with M. tuberculosis growth by inhibition of arabinosyltransferases (encoded by embCAB operon) required for the synthesis of arabinogalactan that is incorporated in the mycobacterial cell wall.¹⁵ Mutations in embCAB operon are the first step in the evolution of resistance to EMB, but only modestly (threefold to eightfold) increase its minimum inhibitory concentration (MIC).^14–18 These mutations occur most frequently (pooled sensitivity of 0.76) at embB codon 306 (embB306), embB406, and embB497.¹³ Drug regimens (including short-course regimens) include EMB for successful treatment of MDR-TB warranting accurate DST of MDR-TB strains to EMB.^2–4 SM, a bactericidal anti-TB drug, is now sparingly used for the treatment of drug-susceptible TB due to higher frequency of resistance of M. tuberculosis isolates to SM and the availability of other active drugs that can be easily incorporated in oral regimens.¹⁹ However, it is a useful drug for the treatment of MDR-TB provided that the isolate is susceptible to SM.² Resistance to SM in M. tuberculosis isolates is mainly due to mutations in ribosomal protein encoded by rpsL and/or 16S rRNA (rrs) and less frequently due to mutations in gidB or due to changes in efflux pumps.^14,19–21 Resistance to SM in MDR-TB strains also adversely affects treatment outcome, and SM susceptibility in pre-XDR-TB (due to second-line injectable drug resistance) is an important predictor of long-term survival and favorable treatment outcome.^2,22

Conventional phenotypic DST for EMB and SM is time-consuming and often reports false susceptibility of M. tuberculosis to EMB.^23–25 False susceptibility occurs for various reasons, particularly due to the presence of specific mutations in EMB resistance-conferring genes that increase MIC close to the critical concentration of the drug.^{16–18,24,25} Performance comparisons of various DST methods have been extensively performed in many developed and developing countries; however, such studies are nearly absent from Middle Eastern countries. In our previous study based on 70 M. tuberculosis strains (including 59 MDR-TB strains) isolated from TB patients during 2006–2010, we showed that compared to molecular methods, the performance of BACTEC 460TB system was superior to the MGIT 960 system for the detection of EMB resistance, but not for the detection of SM resistance.²⁶ The BACTEC 460TB system was discontinued and phenotypic DST has been performed only by the MGIT 960 system since 2011 in Kuwait. Thus, it was of interest to compare the performance of molecular screening of mutations in main target genes conferring resistance to EMB and SM with phenotypic DST, by the MGIT 960 system, among MDR-TB strains collected during 2011–2016 (after BACTEC 460TB system was discontinued) in Kuwait.

Methods

Clinical specimens and M. tuberculosis isolates

Sixty MDR-TB isolates were tested. These included 48 isolates collected from 48 TB patients (representing all individual patient MDR-TB strains collected during 2011–2016) and 12 repeat isolates from 11 patients. In addition, 25 fully susceptible (pansusceptible) isolates collected during the same period from 25 TB patients were also used. For MDR-TB strains, 6 of 48 (13%) TB patients were Kuwaiti nationals, 1 patient was from Saudi Arabia, while the remaining 33 patients were expatriate workers or their family members mostly (35 of 41) originating from 4 (India, Ethiopia, Philippines, and Nepal) countries. The 60 MDR-TB isolates were grown from 44 pulmonary (sputum, n = 40, and bronchoalveolar lavage, n = 4) and 16 extrapulmonary (aspirate, n = 7; pus, n = 6; tissue biopsy, n = 2; and lymph node, n = 1) clinical specimens, collected after obtaining verbal consent from suspected TB patients as part of routine diagnostic procedures, at Kuwait National TB Reference Laboratory (KNTRL). Collection of data and analyses was carried out without revealing patient identity. The KNTRL has biosafety level (BSL) 2 facilities and successfully participates in periodic drug susceptibility proficiency testing, employing both phenotypic and genotypic DST, and M. tuberculosis isolates with well-characterized resistance-conferring mutation(s) in drug targets are reported as drug resistant even when the phenotypic method reports susceptibility to an anti-TB drug. The study was approved by the Ethics Committees of the Faculty of Medicine, Health Sciences Center, Kuwait University, and the Ministry of Health, Kuwait (Approval No. VDR/EC/2 dated 9-2-2015).

Clinical samples from sterile sites were directly processed while nonsterile specimens were processed by N-acetyl-l-cysteine and sodium hydroxide for the growth of mycobacteria, as described previously.^26,27 All specimens were cultured on solid (Lowenstein-Jensen) and MGIT 960 system media according to the manufacturer's instructions (Becton Dickinson, Sparks, MD,) and as described previously.^26,27 Genomic DNA was obtained from MGIT 960 system culture tubes of M. tuberculosis isolates as described previously.²⁸ All MGIT 960 system cultures were tested for the presence of M. tuberculosis complex DNA by AccuProbe DNA probe assay and an inhouse multiplex polymerase chain reaction (PCR) assay, as described previously.^27,29 All isolates were subjected to DST against first-line drugs by the MGIT 960 system, PCR sequencing of embB306, embB406, and embB497 for detection of resistance to EMB, and PCR sequencing of rpsL and rrs genes for detection of resistance to SM. Molecular detection of resistance to RIF and INH (for confirming MDR-TB) was also performed by using GenoType MTBDRplus line probe assay.

DST by Bactec MGIT 960 system

Phenotypic DST with the automated MGIT 960 system was performed with MGIT 960 system cultures that tested positive for M. tuberculosis by using the SIRE drug kit by following the manufacturer's instructions and as described previously.^26,27 Phenotypic DST was performed at KNTRL and the personnel performing these tests were not informed of genotypic susceptibility test results.

Susceptibility testing for EMB and SM by PCR sequencing

Molecular screening for drug resistance-associated mutations for EMB and SM was performed by PCR sequencing of gene loci most commonly implicated in conferring resistance to these two drugs.^2,12,13 For this purpose, embB306, embB406, and embB497 regions were sequenced for EMB and the complete rpsL gene and rrs gene 500 and 900 regions were sequenced for SM.^{12,13,19–21} The embB306 and embB406 regions were simultaneously amplified by using EMBB1F (5′-CCGACGCCGTGGTGATATTCGGCT-3′) and EMBB1R (5′-CAGTGTGAATGCGGCGGTAACGAC-3′) primers. The embB497 region was amplified by using EMBB2F (5′-GGATCTTGGTGCGCCGTCATCG-3′) and EMBB2R (5′-GTGAACAGGCATAGCGCGGTGA-3′) primers. The rpsL gene was amplified by using RPSLF (5′-CGCAAGGGTCGTCGGGACAAGA-3′) and RPSLR (5′-GGTGACCAACTGCGATCCGTAGA-3′) primers. The 500 and 900 regions of rrs were simultaneously amplified by using RRSF (5′-GGCCTTCGGGTTGTAAACCTCTT-3′) and RRSR (5′-GGTTGCGCTCGTTGCGGGACTTA-3′) primers. The PCR amplification was performed by using the appropriate forward and reverse primer pair described above with the reaction and cycling conditions of hot-start touchdown PCR and the amplicons were detected on 2% agarose gels, as described in detail previously.²⁸ PCR amplicons were purified by using the PCR product purification kit (Qiagen, Hilden, Germany) according to kit instructions.

Both strands of purified amplicons were sequenced. The embB306 and embB406 region amplicons were sequenced by using EMBB1FS (5′-CCGTGGTGATATTCGGCTTT-3′) and EMBB1R (5′-CAGTGTGAATGCGGCGGTAACGAC-3′) primers. The embB497 region was sequenced by using EMBB2FS (5′-GTGCGCCGTCATCGCCTGGT-3′) and EMBB2RS (5′-AACCGTCGACGGTGGGCAGGAT-3′) primers. The rpsL gene was sequenced by using RPSLFS (5′-GGACAAGATCAGTAAGGTCAA-3′) and RPSLRS (5′-CGTTGACCAACGGACGCTTGG-3′) primers. The 500 and 900 regions of rrs were sequenced by using RRSFS (5′-ACCTCTTTCACCATCGACGAA-3′) and RRSRS (5′-GACACGAGCTGACGACAGCCAT-3′) primers. The sequencing reactions with the BigDye terminator (version 3.1) cycle sequencing kit (Applied Biosystems, Inc.) were run and processed according to kit instructions and as described previously.^26,30

Reverse compliments of DNA sequence data were obtained after checking confidence levels with an ABI sequence scanner and aligned with forward sequences using Clustal Omega (www.ebi.ac.uk/Tools/msa/clustalo). The nucleotide and deduced amino acid sequences were compared with the corresponding sequences from susceptible strain M. tuberculosis H₃₇Rv by using Clustal Omega. PCR sequencing was performed at the Department of Microbiology, Faculty of Medicine, Kuwait University, and the tests were repeated for isolates classified as drug resistant by molecular screening, but the MGIT 960 system scored them as drug susceptible.

Molecular detection of resistance to RIF and INH

All isolates were tested by the commercial GenoType MTBDRplus line probe assay kit (Hain Lifesciences, Nehren, Germany) and by PCR sequencing of rifampicin resistance-determining region (RRDR) of rpoB, katG codon 315 region (katG315), and inhA regulatory region (inhARR), as described previously.³⁰ Water was used instead of DNA for negative controls with each run of GenoType MTBDRplus assay. The assay profiles were interpreted according to kit instructions and as described in detail previously.³⁰

Statistical analyses

Statistical analysis was performed by using Fisher's exact test or chi-square test as appropriate and probability levels <0.05 by the two-tailed test were considered significant. The strength of agreement between results by different tests was assessed by using kappa statistics. A kappa coefficient (κ) value of 0–0.2, 0.21–0.4, 0.41–0.6, 0.61–0.8, and 0.81–1.0 indicated poor (or slight) agreement, fair agreement, moderate agreement, good agreement, and very good agreement, respectively. Statistical analyses were performed by using WinPepi software ver. 11.65 (PEPI for Windows; Microsoft, Inc., Redmond, WA) and GraphPad software (GraphPad, La Jolla, CA).

Results

M. tuberculosis isolates

A total of 60 multidrug-resistant M. tuberculosis isolates obtained from 44 pulmonary and 16 extrapulmonary specimens of 48 TB patients were tested for susceptibility to EMB and SM by the MGIT 960 system and PCR sequencing methods. None of the expatriate MDR-TB patients had a previous history of treatment with anti-TB drugs in Kuwait and resistance was detected in M. tuberculosis isolated from all patients before initiation of treatment with anti-TB drugs. One M. tuberculosis isolate was tested from 37 patients, 2 strains were collected from 10 patients, and 3 strains were used from 1 patient. Repeat isolates in each case were obtained within 1 week of isolation of the first isolate. Twenty-five pansusceptible isolates collected from 25 different TB patients were also used. All 85 isolates were identified as M. tuberculosis complex by the AccuProbe DNA probe assay and by the multiplex PCR assay, as expected. The multidrug-resistant status of 60 MDR-TB isolates was confirmed by the GenoType MTBDRplus assay and by PCR sequencing of RRDR of rpoB, katG315, and inhARR. While resistance to both RIF and INH was detected by the absence of hybridization with a wild-type probe and positive reaction with a specific mutant probe for 54 isolates, resistance to 1 of the 2 (RIF or INH) anti-TB drugs was indicated by lack of hybridization with a wild-type probe for 6 isolates by the GenoType MTBDRplus assay. PCR sequencing of RRDR of rpoB, katG315, and inhARR confirmed the presence of resistance-conferring mutations. All 25 pansusceptible M. tuberculosis isolates yielded wild-type sequence patterns for RRDR of rpoB, katG315, and inhARR by the GenoType MTBDRplus assay.

Concordance of DST between MGIT 960 system and molecular screening

Based on DST by the MGIT 960 system, 18 isolates were resistant to RIF and INH only, 2 isolates were additionally resistant to EMB, 24 isolates were additionally resistant to SM, and 16 isolates were resistant to all 4 (RIF, INH, EMB, and SM) drugs (Table 1). When molecular screening based on detection of resistance-conferring mutations in embB306, embB406, and embB497 regions was performed, 51 MDR-TB isolates were detected as EMB resistant (Table 1). Thus, the MGIT 960 system and PCR sequencing detected EMB resistance in 18 of 60 (2 isolates with INH+RIF+EMB and 16 isolates with INH+RIF+EMB+SM resistance pattern) and 51 of 60 (13 isolates with INH+RIF+EMB and 38 isolates with INH+RIF+EMB+SM resistance pattern) MDR-TB strains, respectively, (Table 1) and this difference was statistically significant (p = 0.000). On the contrary, detection of SM resistance by the 2 methods (40 of 60 by MGIT 960 system vs. 35 of 60 by PCR sequencing of rpsL and rrs genes) was not significantly different (p = 0.451). In total, 55 of 60 (92%) isolates were susceptible or resistant to SM by both the MGIT 960 system and molecular screening, while only 27 of 60 (45%) isolates yielded concordant results for EMB and again, this difference was statistically significant (p = 0.000 for SM vs. EMB). The concordance between the MGIT 960 system and molecular screening was studied further by comparing kappa coefficient (κ) values. Most M. tuberculosis isolates yielded concordant results and exhibited very good agreement (κ = 0.82) for resistance to SM, while poor agreement (κ = 0.14) was apparent for resistance to EMB by the two methods. Interestingly, five isolates with discordant results for SM were resistant by the phenotypic method, but were susceptible by molecular screening. On the contrary, 33 of 60 isolates yielded discordant results for EMB and in each case, the isolate was susceptible to EMB by the phenotypic method, but was resistant by molecular screening. Thus, molecular screening identified only 7 of 18 M. tuberculosis isolates with resistance pattern of RIF+INH, while 11 of 18 MDR-TB isolates were scored as additionally resistant to EMB. Furthermore, molecular screening resulted in the identification of 38 isolates as resistant to all four drugs, while phenotypic DST detected only 16 isolates with this resistance pattern (Table 1). No mutation was detected in embB306, embB406, and embB497 regions and rpsL and rrs 500 and 900 regions in any of the 25 pansusceptible M. tuberculosis isolates.

Table 1.

Comparison of Drug Susceptibility Results by Mycobacterium Growth Indicator Tube 960 System-Based Phenotypic Method and Correction of Susceptibility Patterns to Ethambutol Following Detection of Resistance-Conferring Mutations in embB

Resistance of M. tuberculosis to	No. of isolates detected by MGIT 960 system	No. of isolates predicted with same resistance pattern after embB sequencing
None	25	25
INH+RIF	18	7
INH+RIF+SM	24	2
INH+RIF+EMB	2	13
INH+RIF+EMB+SM	16	38

EMB, ethambutol; INH, isoniazid; MGIT, Mycobacterium Growth Indicator Tube; RIF, rifampicin; SM, streptomycin.

embB gene mutations in MDR-TB isolates

The results of PCR sequencing of embB306, embB406, and embB497 regions among MGIT 960 system-based EMB-susceptible and EMB-resistant MDR-TB isolates are presented in Table 2. Of 18 EMB-resistant isolates, 11, 0, 7, and 0 isolates contained a mutation at embB306, embB319, embB406, and embB497, respectively. On the other hand, among 42 EMB-susceptible isolates, 24, 2, 5, and 2 isolates contained a mutation at embB306, embB319, embB406, and embB497, respectively. In addition, four other isolates from two Filipino and an Egyptian patient contained a synonymous (CTG355CTA, L355L) and a nonsynonymous (GAG378GCG, E378A) mutation in the embB gene. Since alanine at embB378 represents an ancestral amino acid found in lineage 1, while glutamic acid is found in all modern lineages (lineages 2–4) of M. tuberculosis, E378A mutation in embB represents a phylogenetic polymorphism that is not associated with EMB resistance.^12,25,31 Thus, E378A mutation was not considered further in this study in the context of EMB resistance. No other mutation was detected at other codon positions within the embB306, embB406, and embB497 regions sequenced, and no isolate contained more than one mutation at embB306, embB319, embB406, and embB497. The frequency of embB306 (11 of 18 in EMB-resistant isolates vs. 24 of 42 in EMB-susceptible isolates), embB319 (0 of 18 in EMB-resistant isolates vs. 2 of 42 in EMB-susceptible isolates), and embB497 (0 of 18 in EMB-resistant isolates vs. 2 of 42 in EMB-susceptible isolates) mutations was nearly same, while the frequency of embB406 (7 of 18 in EMB-resistant isolates vs. 5 of 42 in EMB-susceptible isolates) mutation was significantly associated with EMB resistance (p = 0.031). The frequency of ATG306GTG (M306V) mutation was higher in MGIT 960 system-based EMB-resistant strains compared to EMB-susceptible isolates (8 of 18 vs. 9 of 42, p = 0.116), while the frequency of ATG306ATA/ATC (M306I) mutation was lower in EMB-resistant strains compared to EMB-susceptible isolates (3 of 18 vs. 13 of 42, p = 0.346); however, these differences were not statistically significant.

Table 2.

Occurrence of embB Mutations in Mycobacterium Growth Indicator Tube 960 System-Based Ethambutol-Susceptible and Ethambutol-Resistant Multidrug-Resistant Mycobacterium tuberculosis Isolates Analyzed in This Study

	No. of isolates with embB306 as					No. of isolates with embB319 as		No. of isolates with embB406 as			No. of isolates with embB497 as
MGIT 960 system-based result for ethambutol susceptibility	WT ^a	ATA	ATC	GTG	CTG	WT ^a	TCT	WT ^a	GCC	GAC	WT ^a	CAC	CGG	No. of isolates with discordant results
Susceptible, n = 42	18	7	6	9	2	40	2	37	0	5	40	1	1	33
Resistant, n = 18	7	1	2	8	0	18	0	11	5	2	18	0	0	0

WT sequences at embB306, embB319, embB406, and embB497 correspond to ATG (methionine), TAT (tyrosine), GGC (glycine), and CAG (glutamine), respectively.

WT, wild type.

rpsL and rrs gene mutations in MDR-TB isolates

The results of PCR sequencing of rpsL and rrs 500 and 900 regions among MGIT 960 system-based SM-susceptible and SM-resistant MDR-TB isolates are presented in Table 3. All 20 SM-susceptible isolates contained wild-type sequences in rpsL and rrs 500 and 900 regions. Among 40 SM-resistant isolates, 32 isolates contained a mutation in rpsL codon 43 or codon 88, 3 isolates contained a mutation in rrs 500 region (including 1 isolate containing a mutation at rpsL codon 88), 1 isolate contained a mutation in rrs 900 region, while 5 isolates contained wild-type sequences in these genes (Table 3). One isolate contained a double mutation (AAG88ACG in rpsL and C517T in rrs 500 region).

Table 3.

Occurrence of Mutations in rpsL and 500 and 900 Regions of rrs in Mycobacterium Growth Indicator Tube 960 System-Based Streptomycin-Susceptible and Streptomycin-Resistant Multidrug-Resistant Mycobacterium tuberculosis Isolates Analyzed in This Study

	No. of isolates with rpsL as					No. of isolates with rrs 500 as			No. isolates with rrs 900 as
MGIT 960 system-based result for streptomycin	WT	43AGG	88AGG	88ACG	88ATG	WT	517 C/T	527 C/T	WT	906 A/G	No. of isolates with discordant results
Susceptible, n = 20	20	0	0	0	0	20	0	0	20	0	0
Resistant, n = 40	8	24	4	3^a	1	37	2^a	1	39	1	5

One isolate contained double mutation (AAG88ACG in rpsL and 517 C/T in rrs 500 region).

Reproducibility testing and resolution of discrepant results

Repeat isolates yielded the same result as the first isolate from the same patient by the MGIT 960 system as well as by PCR sequencing-based methods. The same DST pattern was obtained by the MGIT 960 system when repeat testing was performed for MDR-TB isolates yielding discrepant results by the two methods. Similarly, the same mutation was detected in the target gene when repeat PCR sequencing was performed for MDR-TB isolates yielding discrepant (susceptible to EMB by the MGIT 960 system, but resistant to EMB by molecular screening) results. The results for all MDR-TB isolates yielding discrepant results by the MGIT 960 system and PCR sequencing methods, and the final susceptibility result for the 33 discrepant isolates are presented in Table 4. In the final analysis, molecular screening showing the presence of well-known resistance-conferring mutations in target genes^{12–14,16–21} was used as indicative of resistance for the resolution of discrepant results. However, for isolates with discrepant results where molecular screening did not detect a well-characterized resistance-conferring mutation in target gene(s) analyzed, DST data by the MGIT960 system was retained as final result since all loci conferring resistance to EMB and SM were not analyzed in this study.

Table 4.

Summary of Discrepant Results for Ethambutol and Streptomycin in 33 Mycobacterium tuberculosis Isolates by Mycobacterium Growth Indicator Tube 960 System in Comparison with the Results of GenoType MTBDRplus and Polymerase Chain Reaction-DNA Sequencing-Based Methods

		Resistance patterns obtained by testing with		Mutations detected by PCR-DNA sequencing in
Serial No.	Isolate No.	MGIT 960 system	GenoType MTBDRplus^a	embB	rpsL	rrs	Final resistance pattern
1	10471/11	INH+RIF+SM	INH+RIF	Q497H	None	None	INH+RIF+EMB+SM
2	11185/11	INH+RIF	INH+RIF	M306I	None	None	INH+RIF+EMB
3	11502/11	INH+RIF+SM	INH+RIF	M306I	K43R	None	INH+RIF+EMB+SM
4	932/11	INH+RIF+SM	INH+RIF	M306I	K43R	None	INH+RIF+EMB+SM
5	2550/11	INH+RIF	INH+RIF	M306L	None	None	INH+RIF+EMB
6	3762/11	INH+RIF+SM	INH+RIF	G406D	K43R	None	INH+RIF+EMB+SM
7	1031/12	INH+RIF	INH+RIF	Q497R	None	None	INH+RIF+EMB
8	1670/12	INH+RIF	INH+RIF	M306V	None	None	INH+RIF+EMB
9	1751/12	INH+RIF	INH+RIF	M306I	None	None	INH+RIF+EMB
10	3301/12	INH+RIF+SM	INH+RIF	M306V	K88R	None	INH+RIF+EMB+SM
11	5608/12	INH+RIF+SM	INH+RIF	G406D	K43R	None	INH+RIF+EMB+SM
12	13075/12	INH+RIF+SM	INH+RIF^b	M306V	None	None	INH+RIF+EMB+SM
13	4777/12	INH+RIF	INH+RIF	Y319S	None	None	INH+RIF+EMB
14	S566/12	INH+RIF	INH+RIF	Y319S	None	None	INH+RIF+EMB
15	493/13	INH+RIF+SM	INH+RIF	M306V	None	None	INH+RIF+EMB+SM
16	3608/13	INH+RIF+SM	INH+RIF	M306V	K43R	None	INH+RIF+EMB+SM
17	12495/13	INH+RIF+SM	INH+RIF	M306I	None	None	INH+RIF+EMB+SM
18	463/14	INH+RIF+SM	INH+RIF	G406D	K43R	None	INH+RIF+EMB+SM
19	S57/14	INH+RIF+SM	INH+RIF	G406D	K43R	None	INH+RIF+EMB+SM
20	9422/14	INH+RIF	INH+RIF	M306I	None	None	INH+RIF+EMB
21	12412/14	INH+RIF	INH+RIF	M306I	None	None	INH+RIF+EMB
22	16676/14	INH+RIF	INH+RIF	M306I	None	None	INH+RIF+EMB
23	12848/14	INH+RIF+SM	INH+RIF	M306V	K43R	None	INH+RIF+EMB+SM
24	16684/15	INH+RIF+SM	INH+RIF^b	M306I	None	A-906-G	INH+RIF+EMB+SM
25	2723/15	INH+RIF+SM	INH+RIF	M306V	K43R	None	INH+RIF+EMB+SM
26	4177/15	INH+RIF+SM	INH+RIF	M306L	None	None	INH+RIF+EMB+SM
27	12735/15	INH+RIF	INH+RIF	M306I	None	None	INH+RIF+EMB
28	10246/15	INH+RIF+SM	INH+RIF	G406D	K43R	None	INH+RIF+EMB+SM
29	7868/15	INH+RIF+SM	INH+RIF^b	M306I	K43R	None	INH+RIF+EMB+SM
30	7866/15	INH+RIF+SM	INH+RIF^b	M306I	K43R	None	INH+RIF+EMB+SM
31	4784/16	INH+RIF+SM	INH+RIF^b	M306I	K43R	None	INH+RIF+EMB+SM
32	5089/16	INH+RIF+SM	INH+RIF	M306V	K43R	None	INH+RIF+EMB+SM
33	5873/16	INH+RIF+SM	INH+RIF	M306V	K43R	None	INH+RIF+EMB+SM

This test detects susceptibility to INH and RIF only.

Mutations were detected only by lack of hybridization with a WT probe by GenoType MTBDRplus assay and were confirmed by PCR sequencing of the respective loci.

PCR, polymerase chain reaction.

Discussion

Until 2011, phenotypic DST of M. tuberculosis isolates was performed simultaneously with the BACTEC 460TB system and MGIT 960 system in Kuwait. We previously showed that M. tuberculosis strains collected during 2006–2010 exhibited only 76% agreement for EMB between phenotypic and genotypic methods, while the agreement for other first-line drugs was >93%.²⁶ From January 2011, BACTEC 460TB system was discontinued and phenotypic DST was performed only by the MGIT 960 system. Data on MDR-TB strains collected during 2011–2016 have shown that 40 of 60 (67%) and 35 of 60 (58%) MDR-TB isolates were resistant to SM by the MGIT 960 system and PCR sequencing, respectively (p = 0.451). Furthermore, none of 20 SM-susceptible isolates, but 35 of 40 SM-resistant MDR-TB isolates, contained a mutated rpsL and/or rrs (κ = 0.82, very good agreement with MGIT 960 system) genes. Five MGIT 960 system-based SM-resistant isolates lacked a mutation in rpsL/rrs genes, which is not surprising as resistance to SM may also involve mutations in efflux pumps, gidB, and other genes, which were not analyzed in this study.^2,12,19–21 Our results thus reinforce previous observations that the MGIT 960 system is an acceptable alternative to the BACTEC 460TB system for accurate DST of M. tuberculosis isolates for SM.^6,20,26

Our data also showed that molecular testing was superior to phenotypic DST for EMB as 18 of 60 (30%) and 51 of 60 (85%) MDR-TB isolates were resistant to EMB by the MGIT 960 system and PCR sequencing, respectively (p = 0.000). Not only all 18 (100%) EMB-resistant strains but also 33 of 42 (79%) EMB-susceptible MDR-TB strains contained a nonsynonymous resistance-conferring mutation in embB gene (κ = 0.14, poor agreement with MGIT 960 system). Thus, the MGIT 960 system falsely reported 55% (33 of 60) of all EMB susceptibility results for MDR-TB strains isolated from TB patients during 2011–2016 in Kuwait. This is the largest discrepancy between phenotypic DST by the MGIT 960 system and genotypic DST by PCR sequencing of embB reported so far for EMB. It is pertinent to mention here that, unlike our previous study,²⁶ data from pansusceptible isolates were not included for determining the strength of agreement between EMB susceptibility results by PCR sequencing and the MGIT 960 system. In our previous study based on 70 M. tuberculosis isolates (including 4 polydrug-resistant, 7 pansusceptible, and 59 MDR-TB strains) recovered from TB patients during 2006–2010, 17 isolates yielded discrepant results (15 isolates as EMB resistant by PCR sequencing, but EMB susceptible by MGIT 960 system, and 2 isolates as EMB susceptible by PCR sequencing, but EMB resistant by MGIT 960 system; κ = 0.53).²⁶ However, the agreement for MDR-TB strains alone was lower (κ = 0.428) since all pansusceptible strains (similar to this study) and polydrug-resistant strains yielded concordant EMB susceptibility results. Furthermore, compared to this study, the higher frequency of M306V mutation and lower frequency of M306I/L mutation in embB gene among MDR-TB strains in our previous study also contributed to slightly higher concordance since isolates with M306V mutation are more likely to be detected as EMB resistant by the MGIT 960 system (explained in more detail below).²⁶ A recent study from Bangladesh reported that up to 49% of all EMB-susceptible findings by the MGIT 960 system were false and the authors concluded that another methodology should be used for more accurate DST of M. tuberculosis isolates (specially for MDR-TB strains) for EMB.²⁴ Two studies from China have also reported that 45% MDR-TB isolates susceptible to EMB contained a nonsynonymous mutation in embB gene with majority of isolates containing a mutation at embB306, embB406, or embB497.^32,33 Few other studies have also shown discordant results between phenotypic DST methods for EMB and/or genotypic testing by PCR sequencing of embB gene, and the discordance was linked to false susceptibility testing by phenotypic methods, particularly the MGIT 960 system.^23,25

Our data further showed that 11, 0, 7, and 0 isolates among 18 EMB-resistant MDR-TB strains and 24, 2, 5, and 2 isolates among 40 EMB-susceptible MDR-TB strains contained a mutation at embB306, embB319, embB406, and embB497, respectively. As expected, mutations at embB306 occurred most frequently (35 of 60, 58%), but were also found at relatively higher frequency (12 of 60, 20%) at embB406 among MDR-TB strains in Kuwait. Furthermore, the frequency of mutations only at embB406 (p = 0.031), but not at embB306, embB319, and embB497, was significantly associated with EMB resistance. Mutations in embB (including embB306, embB319, embB406, and embB497) gene in EMB-susceptible isolates have been described previously.^32–36 The frequency of mutations at embB306, embB406, and embB497 in EMB-resistant and EMB-susceptible MDR-TB isolates has also shown variations in different studies. For instance, the presence of an embB306 mutation was shown to be strongly associated (p = 0.000) with EMB resistance in some studies^{26,32,33,36–38}; however, it was nonsignificant (p > 0.05) in this study and another study from Poland.³⁹ On the contrary, the frequency of mutations at embB406 and embB497 in EMB-resistant and EMB-susceptible MDR-TB isolates has shown much wider variations. While the frequency of embB406 mutations was significantly associated with EMB resistance in some studies,^37,38 it was nonsignificantly associated^33,36 or inversely associated³² with EMB resistance in some other studies. Similarly, the frequency of embB497 mutations was significantly associated with EMB resistance in some studies^32,37,38; however, it was nonsignificantly associated^33,36 in some other studies. These contrasting observations either reflect regional variations in the frequency of mutations at different codon positions in embB gene among drug-resistant M. tuberculosis isolates or they are due to the presence of different genotypes in different settings. In this context, differences in the frequency of specific mutations in resistance-conferring genes in different geographical locations have also been noted for RIF, INH, and other first-line and second-line drugs.^2,19

Previous studies have shown that mutations in embB develop first during evolution of resistance of M. tuberculosis to EMB, while high-level resistance develops later due to acquisition of additional mutations in embCAB operon or other genes.^{14,16–18,40,41} Furthermore, some (M306I/L) mutations at embB306 increase MIC near or just above the breakpoints that define EMB resistance and are likely to be detected as EMB susceptible or EMB resistant, while others (M306V) increase MICs by fourfold to eightfold and are more likely to be detected as EMB resistant by phenotypic DST methods.^14,16–18 Consistent with these observations, the occurrence of M306I/L mutations was higher in EMB-susceptible strains (15 of 42) compared to EMB-resistant strains (3 of 18), while the occurrence of M306V mutation was higher in EMB-resistant strains (8 of 18) compared to EMB-susceptible strains (9 of 42); however, these differences did not reach statistical significance (p > 0.05). Furthermore, in the mouse model of TB, infection with M. tuberculosis with M306V mutation required a much higher dose of EMB to restrict bacterial growth in the lung compared to wild-type tubercle bacilli.¹⁸ Based on these observations, it has been suggested that patients infected with embB mutants should be considered having EMB-resistant TB even if the isolates appear to be EMB susceptible by phenotypic DST methods to avoid evolution of secondary mutations and selection of fully drug-resistant strains.^{16–18,40,41} Since the WHO and CDC recommend to test all first-line drugs with rapid phenotypic DST methods,^1,7 our results show that further work is needed to determine whether a different EMB concentration in the MGIT 960 system will improve agreement with molecular screening approaches for detection of EMB resistance in M. tuberculosis.

Our study has a few limitations. (1) Additional phenotypic tests such as broth dilution or proportion method were not performed to correlate the susceptibility with the kind of mutations in EMB-susceptible and EMB-resistant isolates; (2) the MIC values of MDR-TB isolates to EMB were not determined; (3) only mutations in embB306, embB406, and embB497 regions were determined, but mutations in other regions of embB or other genes were not determined; and (4) the linkage of M. tuberculosis genotype with the frequency of specific mutations was not studied in this investigation.

In conclusion, our results demonstrate that compared to molecular screening for resistance-conferring mutations, an automated liquid culture-based MGIT 960 system is suitable for DST of M. tuberculosis for SM, but its performance is markedly lower for determining susceptibility to EMB. Many M. tuberculosis isolates with well-defined embB mutations that confer low level, but clinically significant resistance to EMB were erroneously detected as EMB susceptible by the MGIT 960 system. Our data show that molecular screening for resistance-conferring mutations in embB is superior to the MGIT 960 system when accurate EMB susceptibility results are needed for proper management of patients with drug-resistant TB and MDR-TB. Further work is urgently required to determine whether a different EMB concentration in the MGIT 960 system will improve agreement with molecular testing for the detection of EMB resistance in M. tuberculosis.

Footnotes

Acknowledgment

The study was supported by Research Sector, Kuwait University (Grant No. YM08/14).

Disclosure Statement

No competing financial interests exist.

References

World Health Organization. 2017. Global tuberculosis report 2017. WHO/HTM/TB/2017.23. WHO, Geneva.

Ahmad

, and Mokaddas

. 2014. Current status and future trends in the diagnosis and treatment of drug-susceptible and multidrug-resistant tuberculosis. J. Infect. Pub. Health, 7:75–91.

Dheda

, Chang

K.C.

, Guglielmetti

, Furin

, Schaaf

H.S.

, Chesov

, Esmail

, and Lange

. 2017. Clinical management of adults and children with multidrug-resistant and extensively drug-resistant tuberculosis. Clin. Microbiol. Infect., 23:131–140.

Matteelli

, Roggi

, and Carvalho

A.C.

. 2014. Extensively drug-resistant tuberculosis: epidemiology and management. Clin. Epidemiol., 6:111–118.

Drobniewski

, Nikolayevskyy

, Balabanova

, Bang

, and Papaventsis

. 2012. Diagnosis of tuberculosis and drug resistance: what can new tools bring us? Int. J. Tuberc. Lung Dis., 16:860–870.

Schön

, Miotto

, Köser

C.U.

, Viveiros

, Böttger

, and Cambau

. 2017. Mycobacterium tuberculosis drug-resistance testing: challenges, recent developments and perspectives. Clin. Microbiol. Infect., 23:154–160.

World Health Organization. 1998. Guidelines for surveillance of drug resistance in tuberculosis. WHO and the International Union Against Tuberculosis and Lung Disease. Int. J. Tuberc. Lung Dis., 2:72–89.

Van Deun

, Martin

, and Palomino

J.C.

. 2010. Diagnosis of drug-resistant tuberculosis: reliability and rapidity of detection. Int. J. Tuberc. Lung Dis., 14:131–140.

Van Deun

, Barrera

, Bastian

, Fattorini

, Hoffmann

, Kam

K.M.

, Rigouts

, Rüsch-Gerdes

, and Wright

. 2009. Mycobacterium tuberculosis strains with highly discordant rifampin susceptibility test results. J. Clin. Microbiol., 47:3501–3506.

10.

Rigouts

, Gumusboga

, de Rijk

W.B.

, Nduwamahoro

, Uwizeye

, de Jong

, and Van Deun

. 2013. Rifampin resistance missed in automated liquid culture system for Mycobacterium tuberculosis isolates with specific rpoB mutations. J. Clin. Microbiol., 51:2641–2645.

11.

Mokaddas

, Ahmad

, Eldeen

H.S.

, and Al-Mutairi

. 2015. Discordance between Xpert MTB/RIF assay and Bactec MGIT 960 culture system for detection of rifampin-resistant Mycobacterium tuberculosis isolates in a country with a low tuberculosis (TB) incidence. J. Clin. Microbiol., 53:1351–1354.

12.

Campbell

P.J.

, Morlock

G.P.

, Sikes

R.D.

, Dalton

T.L.

, Metchock

, Starks

A.M.

, Hooks

D.P.

, Cowan

L.S.

, Pilkaytis

B.B.

, and Posey

J.E.

. 2011. Molecular detection of mutations associated with first- and second-line drug resistance compared with conventional drug susceptibility testing of Mycobacterium tuberculosis. Antimicrob. Agents Chemother., 55:2032–2041.

13.

Cheng

, Cui

, Li

, and Hu

. 2014. Diagnostic accuracy of a molecular drug susceptibility testing method for the antituberculosis drug ethambutol: a systematic review and meta-analysis. J. Clin. Microbiol., 52:2913–2924.

14.

Nebenzahl-Guimaraes

, Jacobson

K.R.

, Farhat

M.R.

, and Murray

M.B.

. 2014. Systematic review of allelic exchange experiments aimed at identifying mutations that confer drug resistance in Mycobacterium tuberculosis. J. Antimicrob. Chemother., 69:331–342.

15.

Telenti

, Philipp

, Sreevatsan

, Bernasconi

, Stockbauer

K.E.

, Wieles

, Musser

J.M.

, and Jacobs

W.R.

Jr . 1997. The emb operon, a unique gene cluster of Mycobacterium tuberculosis involved in resistance to ethambutol. Nat. Med., 3:567–570.

16.

Safi

, Sayers

, Hazbon

M.H.

, and Alland

. 2008. Transfer of embB codon 306 mutations into clinical Mycobacterium tuberculosis strains alters susceptibility to ethambutol, isoniazid and rifampin. Antimicrob. Agents Chemother., 52:2027–2034.

17.

Safi

, Fleischmann

R.D.

, Peterson

S.N.

, Jones

M.B.

, Jarrahi

, and Alland

. 2010. Allelic exchange and mutant selection demonstrate that common clinical embCAB gene mutations only modestly increase resistance to ethambutol in Mycobacterium tuberculosis. Antimicrob. Agents Chemother., 54:103–108.

18.

Plinke

, Walter

, Aly

, Ehlers

, and Niemann

. 2011. Mycobacterium tuberculosis embB codon 306 mutations confer moderately increased resistance to ethambutol in vitro and in vivo. Antimicrob. Agents Chemother., 55:2891–2896.

19.

Ahmad

, and Mokaddas

. 2009. Recent advances in the diagnosis and treatment of multidrug-resistant tuberculosis. Resp. Med., 103:1777–1790.

20.

Spies

F.S.

, da Silva

P.E.

, Ribeiro

M.O.

, Rossetti

M.L.

, and Zaha

. 2008. Identification of mutations related to streptomycin resistance in clinical isolates of Mycobacterium tuberculosis and possible involvement of efflux mechanism. Antimicrob. Agents Chemother., 52:2947–2949.

21.

Sun

, Zhang

, Xiang

, Pi

, Guo

, Zheng

, Li

, Zhao

, Tang

, Luo

, Rastogi

, Li

, and Sun

. 2016. Characterization of mutations in streptomycin-resistant Mycobacterium tuberculosis isolates in Sichuan, China and the association between Beijing-lineage and dual-mutation in gidB. Tuberculosis (Edinb.), 96:102–106.

22.

Kim

D.H.

, Kim

H.J.

, Park

S.K.

, Kong

S.J.

, Kim

Y.S.

, Kim

T.H.

, Kim

E.K.

, Lee

K.M.

, Lee

S.S.

, Park

J.S.

, Koh

W.J.

, Lee

C.H.

, Kim

J.Y.

, and Shiml

T.S.

. 2010. Treatment outcomes and survival based on drug resistance patterns in multidrug-resistant tuberculosis. Am. J. Respir. Crit. Care Med., 182:113–119.

23.

Angra

P.K.

, Taylor

T.H.

, Iademarco

M.F.

, Metchock

, Astles

J.R.

, and Ridderhof

J.C.

. Performance of tuberculosis drug susceptibility testing in U.S. laboratories from 1994 to 2008. J. Clin. Microbiol., 50:1233–1239.

24.

Banu

, Rahman

S.M.

, Khan

M.S.

, Ferdous

S.S.

, Ahmed

, Gratz

, Stroup

, Pholwat

, Heysell

S.K.

, and Houpt

E.R.

. 2014. Discordance across several methods for drug susceptibility testing of drug-resistant Mycobacterium tuberculosis isolates in a single laboratory. J. Clin. Microbiol., 52:156–163.

25.

Yakrus

M.A.

, Driscoll

, McAlister

, Sikes

, Hartline

, Metchock

, and Starks

A.M.

. 2016. Molecular and growth-based drug susceptibility testing of Mycobacterium tuberculosis complex for ethambutol in the United States. Tuberc. Res. Treat., 2016:3404860.

26.

Ahmad

, Mokaddas

, Al-Mutairi

, Eldeen

H.S.

, and Mohammadi

. 2016. Discordance across phenotypic and molecular methods for drug susceptibility testing of drug-resistant Mycobacterium tuberculosis isolates in a low TB incidence country. PLoS One, 11:e0153563.

27.

Mokaddas

, Ahmad

, and Samir

. 2008. Secular trends in susceptibility patterns of Mycobacterium tuberculosis isolates in Kuwait, 1996–2005. Int. J. Tuberc. Lung Dis., 12:319–325.

28.

Abal

A.T.

, Ahmad

, and Mokaddas

. 2002. Variations in the occurrence of the S315T mutation within the katG gene in isoniazid-resistant clinical Mycobacterium tuberculosis isolates from Kuwait. Microb. Drug Resist., 8:99–105.

29.

Mokaddas

, and Ahmad

. 2007. Development and evaluation of a multiplex PCR for rapid detection and differentiation of Mycobacterium tuberculosis complex members from non-tuberculous mycobacteria. Jpn. J. Infect. Dis., 60:140–144.

30.

Al-Mutairi

, Ahmad

, and Mokaddas

. 2011. Performance comparison of four methods for rapid detection of multidrug-resistant Mycobacterium tuberculosis strains. Int. J. Tuberc. Lung Dis., 15:110–115.

31.

Feuerriegel

, Köser

C.U.

, and Niemann

. 2014. Phylogenetic polymorphisms in antibiotic resistance genes of the Mycobacterium tuberculosis complex. J. Antimicrob. Chemother., 69:1205–1210.

32.

Shi

, Li

, Zhao

, Jia

, Li

, Coulter

, Jin

, and Zhu

. 2011. Characteristics of embB mutations in multidrug-resistant Mycobacterium tuberculosis isolates in Henan, China. J. Antimicrob. Chemother., 66:2240–2247.

33.

Zhao

L.L.

, Sun

, Liu

H.C.

, Wu

X.C.

, Xiao

T.Y.

, Zhao

X.Q.

, Li

G.L.

, Jiang

, Zeng

C.Y.

, and Wan

K.L.

. 2015. Analysis of embCAB mutations associated with ethambutol resistance in multidrug-resistant Mycobacterium tuberculosis isolates from China. Antimicrob. Agents Chemother., 59:2045–2050.

34.

Mokrousov

, Ottem

, Vyshnevskiy

, and Narvskaya

. 2002. Detection of embB306 mutations in ethambutol-susceptible clinical isolates of Mycobacterium tuberculosis from Northwestern Russia: implications for genotypic resistance testing. J. Clin. Microbiol., 40:3810–3813.

35.

Hazbón

M.H.

, Bobadilla del Valle

, Guerrero

M.I.

, Varma-Basil

, Filliol

, Cavatore

, Colangeli

, Safi

, Billman-Jacobe

, Lavender

, Fyfe

, García-García

, Davidow

, Brimacombe

, León

C.I.

, Porras

, Bose

, Chaves

, Eisenach

K.D.

, Sifuentes-Osornio

, Ponce de León

, Cave

M.D.

, and Alland

. 2005. Role of embB codon 306 mutations in Mycobacterium tuberculosis revisited: a novel association with broad drug resistance and IS6110 clustering rather than ethambutol resistance. Antimicrob. Agents Chemother., 49:3794–3802.

36.

Park

Y.K.

, Ryoo

S.W.

, Lee

S.H.

, Jnawali

H.N.

, Kim

C.K.

, Kim

H.J.

, and Kim

S.J.

. 2012. Correlation of the phenotypic ethambutol susceptibility of Mycobacterium tuberculosis with embB gene mutations in Korea. J. Med. Microbiol., 61:529–534.

37.

Cui

, Li

, Cheng

, Yang

, Lu

, Hu

, and Ge

. 2014. Mutations in the embC-embA intergenic region contribute to Mycobacterium tuberculosis resistance to ethambutol. Antimicrob. Agents Chemother., 58:6837–6843.

38.

, Jia

, Huang

, Sun

, and Zhang

. 2015. Mutations found in embCAB, embR, and ubiA genes of ethambutol-sensitive and -resistant Mycobacterium tuberculosis clinical isolates from China. Biomed. Res. Int., 2015:951706.

39.

Bakuła

, Napiórkowska

, Bielecki

, Augustynowicz-Kopeć

, Zwolska

, and Jagielski

. 2013. Mutations in the embB gene and their association with ethambutol resistance in multidrug-resistant Mycobacterium tuberculosis clinical isolates from Poland. Biomed. Res. Int., 2013:167954.

40.

Lingaraju

, Rigouts

, Gupta

, Lee

, Umubyeyi

A.N.

, Davidow

A.L.

, German

, Cho

, Lee

J.I.

, Cho

S.N.

, Kim

C.T.

, Alland

, and Safi

. 2016. Geographic differences in the contribution of ubiA mutations to high-level ethambutol resistance in Mycobacterium tuberculosis. Antimicrob. Agents Chemother., 60:4101–4105.

41.

Safi

, Lingaraju

, Amin

, Kim

, Jones

, Holmes

, McNeil

, Peterson

S.N.

, Chatterjee

, Fleischmann

, and Alland

. 2013. Evolution of high-level ethambutol-resistant tuberculosis through interacting mutations in decaprenylphosphoryl-β-d-arabinose biosynthetic and utilization pathway genes. Nat. Genet., 45:1190–1197.