Wei-Lun Huang,* Ting-Lin Chi, Mei-Hua Wu, and Ruwen Jou*

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1 JOURNAL OF CLINICAL MICROBIOLOGY, July 2011, p Vol. 49, No /11/$12.00 doi: /jcm Copyright 2011, American Society for Microbiology. All Rights Reserved. Performance Assessment of the GenoType MTBDRsl Test and DNA Sequencing for Detection of Second-Line and Ethambutol Drug Resistance among Patients Infected with Multidrug-Resistant Mycobacterium tuberculosis Wei-Lun Huang,* Ting-Lin Chi, Mei-Hua Wu, and Ruwen Jou* Reference Laboratory of Mycobacteriology, Research and Diagnostic Center, Centers for Disease Control, Taipei, Taiwan, Republic of China Received 31 January 2011/Returned for modification 4 March 2011/Accepted 29 April 2011 The GenoType MTBDRsl test and DNA sequencing were used to rapidly detect second-line drug- and ethambutol (EMB)-resistant Mycobacterium tuberculosis. The ability of these two assays to detect the presence of mutations associated with resistance to fluoroquinolones (FLQ), aminoglycosides/cyclic peptide (AG/CP), and EMB in the gyra, rrs, and embb genes (for the GenoType MTBDRsl test) and gyra, gyrb, rrs, eis, embc, emba, embb, and embr genes (for DNA sequencing) was compared to that of conventional agar proportion drug susceptibility testing (DST). We evaluated 234 multidrug-resistant (MDR) M. tuberculosis isolates. The two molecular methods had high levels of specificity (95.8 to 100%). The sensitivities for FLQ resistance detection for both methods were 85.1%. For AG (kanamycin [KM] and amikacin [AM]) and CP (capreomycin CAP]), the sensitivities of resistance detection using the GenoType MTBDRsl test were 43.2%, 84.2%, and 71.4%, respectively, while with the inclusion of an extra gene, eis, in sequencing, the sensitivity reached 70.3% for detection of KM resistance. The sensitivities of EMB resistance detection were 56.2% and 90.7% with the GenoType MTBDRsl test and sequencing, respectively. We found that the GenoType MTBDRsl test can rapidly detect resistance to FLQ, CAP, and AM. The accuracy of the GenoType MTBDRsl test for the detection of FLQ and AM resistance was comparable to that of conventional DST; however, the test was less accurate for the detection of KM and EMB resistance and demonstrated a poor predictive value for CAP resistance. We recommend including new alleles consisting of the eis promoter and embb genes in molecular analysis. However, conventional DST is necessary to rule out false-negative results from molecular assays. Multidrug-resistant (MDR) tuberculosis (TB), which is defined as infection with a Mycobacterium tuberculosis complex isolate that is resistant to at least isoniazid (INH) and rifampin (RIF), is a public health concern that threatens the success of global TB control programs. According to the 2008 report of the Anti-Tuberculosis Drug Resistance Surveillance Global Project led by the World Health Organization (WHO) and the International Union Against Tuberculosis and Lung Disease (the Union), the incidence of MDR in new cases ranged from 0% to 22.3%. In addition, the proportion of extensively drugresistant (XDR) TB, defined as MDR M. tuberculosis with additional resistance to fluoroquinolones (FLQ) and at least 1 second-line injectable drug (including kanamycin [KM], amikacin [AM], and capreomycin [CAP]), ranged from 0% to 30% globally. It has been estimated that 40,000 cases of XDR-TB occur annually worldwide (32). In Taiwan, it was estimated that MDR-TB accounted for 1% of new TB cases and 6.2% of retreatment cases in 2007 (6). Treatment for MDR-TB patients requires the use of second-line drugs for at least 18 to 24 * Corresponding author. Mailing address: Reference Laboratory of Mycobacteriology, Research and Diagnostic Center, Centers for Disease Control, Department of Health, 161 Kun-Yang Street, Nan-Kang, Taipei 115, Taiwan, Republic of China. Phone for Wei-Lun Huang: (886) Fax: (886) hwl@cdc.gov.tw. Phone for Ruwen Jou: (886) Fax: (886) rwj@cdc.gov.tw. Published ahead of print on 4 May months (21, 33). Therefore, the WHO has expressed concern over the emergence of virulent drug-resistant strains of M. tuberculosis and is calling for stronger measures to prevent XDR-TB. A survey conducted from 2000 to 2004 revealed that XDR-TB has been identified in all regions of the world and was prevalent in Russia and in the countries of Eastern Europe and Asia (26). Resistance to the second-line drugs has become a major concern for global TB control. To prevent the emergence of XDR-TB, it is important to increase investment in laboratory infrastructure that enables rapid and improved detection of resistance to second-line drugs. The molecular mechanisms of action of several major antituberculosis drugs and of resistance to these drugs have been elucidated (35). In many bacterial species, FLQ inhibit DNA gyrase (topoisomerase II) and topoisomerase IV, causing bacterial death (7). Topoisomerases are essential enzymes for bacterial DNA replication and transcription (36). Resistance to FLQ is caused by mutations affecting DNA gyrase, which consists of the GyrA and GyrB subunits, encoded by the gyra and gyrb genes, respectively. Most mutations conferring FLQ resistance occur in a conserved region, the quinolone-resistance-determining region (QRDR), of the gyra (320-bp) and gyrb (375-bp) genes. The mutations are most often detected at codons 90, 91, and 94; they rarely involve codon 88 of the gyra gene. In contrast, gyrb mutations are seldom found in clinical M. tuberculosis isolates (35). Among aminoglycosides (AG), KM and its derivative, AM, inhibit protein synthesis through 2502

2 VOL. 49, 2011 SECOND-LINE DRUG RESISTANCE DETECTION IN TB PATIENTS 2503 the modification of ribosomal structures in the 16S rrna subunit (2). Mutations at position 1400 of the 16S RNA (rrs) gene (i.e., position 1401 in a study of Maus et al. [17]) are associated with high-level resistance to KM and AM (2). CAP is a polypeptide antibiotic, and resistance to this drug is conferred by mutation A1401G, C1402T, or G1484T in the rrs gene (17). Diversified resistance patterns were reported for three injectable drugs (17, 31). A mutation at position 1401 of the rrs gene was associated with low-level resistance to CAP as well as high-level resistance to KM and AM (17). A C1402T mutation conferred susceptibility to AM, low-level resistance to KM, and high-level resistance to CAP (17). However, a G1484T mutation led to high-level resistance to all three drugs (17). Multiple mutations that were detected in the rrs gene in one strain conferred cross-resistance to AGs/cyclic peptide (CP) (17). A study revealed that promoter mutations of the eis gene conferred resistance to KM but not to AM (34). Ethambutol (EMB) is a bacteriostatic agent that acts on growing bacilli and has no effect on nonreplicating bacilli. EMB inhibits arabinosyl transferase, encoded by embb, by interfering with biosynthesis of the cell wall component arabinogalactan (29). In M. tuberculosis, embb is organized into an embcab operon with embc and emba. The embb codon 306 mutation is the most frequently observed mutation in clinical isolates that are resistant to EMB (12). However, approximately 35% of EMBresistant isolates lack embb mutations (3), and mutations in the embcab operon were suggested to be involved (24, 25). A novel assay, the GenoType MTBDRsl test (Hain Lifescience GmbH, Nehren, Germany), was developed using PCRbased amplification and a reverse blotting assay that employs specific probes hybridized to nitrocellulose strips to detect resistance to second-line drugs and ethambutol. The assay detects mutations in the gyra gene for FLQ resistance, the rrs gene for AG/CP resistance, and the embb gene for EMB resistance. The GenoType MTBDRsl test has been evaluated in Germany (11), France (5), and Vietnam (14) with limited sample sizes. Hillemann et al. evaluated the GenoType MTBDRsl test and reported a high accuracy for the detection of FLQ, AM, and CAP resistance but low sensitivity for the detection of EMB resistance (11). Mechanisms of drug resistance in M. tuberculosis are complicated. The dual purposes of this study were to evaluate existing molecular assays, the GenoType MTBDRsl test and DNA sequencing, and to find out novel genetic information on drug resistance. MATERIALS AND METHODS Mycobacterium tuberculosis isolates. M. tuberculosis complex isolates identified as multidrug resistant on the basis of bacteriological (culture in Löwenstein- Jensen or MGIT medium), biochemical, and molecular identification and drug susceptibility testing (DST) were collected from each of the 234 TB cases in hospitals throughout Taiwan between January 2008 and February Of the 234 MDR isolates, 181 were resistant to at least one of the second-line drugs tested or ethambutol and 53 isolates were susceptible to all of these drugs, as determined by conventional DST (Table 1). Of the 234 MDR isolates, 124 (53%) were of the Beijing family and 110 (47%) were non-beijing family isolates. Drug susceptibility testing. The agar proportion method on either Middlebrook 7H10 or 7H11 agar (Creative Microbiologicals or Sancordon, Taiwan), and Bactec MGIT 960 Sire kits (Becton Dickinson Diagnostic Systems, Sparks, MD) with a liquid culture system were used. The critical first-line drug concentrations for the agar proportion method on 7H10 agar were 0.2 g/ml for INH, 1.0 g/ml for RIF, 5.0 g/ml for EMB, and 2.0 g/ml for streptomycin (SM); on 7H11 agar, the critical concentrations were 0.2 g/ml for INH, 1.0 g/ml for RIF, TABLE 1. Drug resistance profiles of 234 multidrug-resistant Mycobacterium tuberculosis isolates using conventional drug susceptibility testing Susceptibility a No. (%) FLQ KM CAP AM EMB of strains R R R R R 5 (2.1) R R R R S** 3 (1.3) R R R S R 1 (0.4) R R S R R 3 (1.3) R R S S R 6 (2.6) R R S S S 1 (0.4) R S S S R 47 (20.1) R S S S S 8 (3.4) S R R R R 3 (1.3) S R R R S 1 (0.4) S R S R R 4 (1.7) S R S S R 4 (1.7) S R S S S 6 (2.6) S S R S R 1 (0.4) S S S S R 88 (37.6) S S S S S 53 (22.6) a R, resistant; S, susceptible. 7.5 g/ml for EMB, and 2.0 g/ml for SM. For the MGIT 960 system, the critical drug concentrations were 0.1 g/ml for INH, 1.0 g/ml for RIF, 5.0 g/ml for EMB, and 1.0 g/ml for SM. Isolates resistant to at least INH and RIF were considered MDR and were subjected to DST with second-line drugs. The critical concentrations of second-line drugs for the agar proportion method on 7H11 agar were 2 g/ml for ofloxacin (OFX), 6 g/ml for AM, 6 g/ml for KM, and 10 g/ml for CAP. Growth on the control medium was compared to growth on the drug-containing medium to determine susceptibility. The DST results were categorized resistant or susceptible. The test results were validated when the result for M. tuberculosis H37Rv, included in the same DST, indicated susceptibility. MDR isolates that were resistant to at least ofloxacin and one of three secondline injectable drugs (kanamycin, capreomycin, or amikacin) were defined as XDR. The proficiency of DST was monitored by the WHO supranational reference laboratory network. DST with second-line drugs was performed by two senior medical technologists. Molecular methods. The two genotypic assays were performed blindly by four medical technologists in parallel. The extracted DNA samples were prepared according to the protocol provided by the manufacturer of the GenoType MTBDRsl kit (Hain Lifescience GmbH, Nehren, Germany) (8) and were also used for sequencing. GenoType MTBDRsl test. The GenoType MTBDRsl assay was performed according to the instructions provided by the manufacturer (Hain Lifescience GmbH, Nehren, Germany) (8). For a given isolate, if all of the wild-type (WT) probes showed positive staining and the mutant probes produced no staining, the isolate was considered susceptible. In contrast, the isolate was considered resistant if at least one WT probe was absent or if any mutant probe was present. DNA sequencing of gyra, rrs, and embb. For FLQ resistance, a 426-bp fragment of the gyra gene was analyzed by PCR amplification and sequenced with the oligonucleotide primers gyra-f (5 -GAT GAC AGA CAC GAC GTT GC- 3 ) and gyra-r (5 -AGC ATC TCC ATC GCC AAC G-3 ). For AG/CP resistance, the rrs gene was analyzed; the primers TBrrs1250-F (5 -TTA AAA GCC GGT CTC AGT TC-3 ) and TBrrivs38-R (5 -TAC GCC CCA CCA GTT GGG GC-3 ) were used to amplify a 300-bp fragment of the rrs gene, and the primers TBrrs1250-F and TBrrs0406-R(5 -ACC AGT TGG GGC GTT TTC GT-3 ) were used for sequencing. For EMB resistance, a 344-bp embb fragment was analyzed with primers OG240-F (5 -CGT TCC GGC CTG CAT-3 ) and OG243-R (5 - CAC CTC ACG CGA CAG CA-3 ) (11). The PCRs were performed as follows: 35 cycles at 96 C for 1 min; annealing for 30 s at 65 C for gyra, 40 s at 65 C for rrs, or 1 min at 59 C for embb; and elongation at 72 C for 1 min. Thereafter, the PCR products were analyzed with an ABI 3730 automated sequencer (Applied Biosystems), and the sequence data were assembled and edited using Sequencing Analysis (version 5.2.0) software (Applied Biosystems). Analysis of discordant results between GenoType MTBDRsl test and conventional drug susceptibility testing. For FLQ-resistant isolates without gyra mutations, a 609-bp fragment of the gyrb gene was analyzed by PCR amplification and sequenced with the primers gyrb-f (5 -AAG ACC AAG TTG GGC AAC

3 2504 HUANG ET AL. J. CLIN. MICROBIOL. TABLE 2. GenoType MTBDRsl assay and sequencing results for 234 multidrug-resistant Mycobacterium tuberculosis isolates a No. (%) of isolates with the following result by conventional DST: GenoType MTBDRsl result Sequencing result FLQ AG/CP KM CAP AM EMB R S R S R S R S R S R R 63 (85.1) 0 16 (43.2) 0 10 (71.4) 6 (2.7) 16 (84.2) 0 91 (56.2) 0 R S S R (27) (34.6) 3 (4.2) S S 11 (14.9) 160 (100) 11 (29.7) 197 (100) 4 (28.6) 214 (97.3) 3 (15.8) 215 (100) 15 (9.3) 69 (95.8) a R, resistant; S, susceptible. AC-3 ) and gyrb-r (5 -CTG CCA CTT GAG TTT GTA CA-3 ). In addition, for KM-resistant but AM-susceptible isolates without rrs mutations, the eis gene was analyzed. The primers eis-f (5 -ATT CAG GGC CGA TGA AAT C-3 ) and eis-r (5 -GAT GAT CGA CCG GGT TTG-3 ) were used to amplify a 460-bp fragment, and the primers eis_ f (5 -GGG CCG ATG AAA TCG GT-3 ) and eis-r were used for sequencing. For EMB-resistant isolates without embb codon 306 mutations, four fragments (880, 914, 1,176, and 635 bp) of embc, a 1,223-bp fragment of emba, a 1,306-bp fragment of embb, and two fragments (896 and 898 bp) of embr genes were amplified and sequenced with the primers embc-1f (5 -CCC AAC CAG CCC AAT GTT C-3 ) and embc-1r (5 -GGC GGT GTC CAG GAT GTG-3 ), embc-2f (5 -GCT GCA CAT CCT GGA CAC-3 ) and embc-2r (5 -ACG ACA TTG CCA CCG ATA C-3 ), embc-3f (5 -GTA TCG GTG GCA ATG TCG T-3 ) and embc-3r (5 -CGG GAT GGC GGA CAG TGG T-3 ), and embc-4f (5 -ACC ACT GTC CGC CAT CCC G-3 ) and embc-4r (5 -GAC GAC GGC TGC TAG GCG TG-3 ) for the four fragments of embc, respectively; emba-f (5 -GTG ACT CGC AGC GGG CTG TG-3 ) and emba-r (5 -CGG TGA ACA CAG CGA CCC GG-3 ) for emba; Rv3795b-F (5 -TGG ACG GGC GGG GCT CAA T-3 ) and Rv3795bc-R (5 - GCA AAC AGG GCG AAA AAG A-3 ) for embb; and embr-1f (5 -CGA TCA CCA CAG CGG GCA GC-3 ) and embr-1r (5 -GTT CGA ATG TCA GAG CCT CG-3 ) and embr-2f (5 -CGA GGC TCT GAC ATT CGA AC-3 ) and embr-2r (5 -GCC GAC ACT ATC AAC AAC GG-3 ) for the two fragments of embr, respectively (24, 25). The PCRs were performed as follows: 35 cycles at 96 C for 1 min; annealing for 30 s at 62 C for gyrb, 15 s at 55 C for eis, 1 min at 64 C for embc, 1 min at 67 C for emba, 1 min at 65 C for embb, or 1 min at 61 C for embr; and elongation at 72 C for 2 min. The PCR products were analyzed as described above in the section on DNA sequencing. Data analysis. The performance characteristics of the GenoType MTBDRsl test and DNA sequencing were compared to those of conventional DST for the detection of resistance to second-line drugs and ethambutol. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the molecular assays were calculated. The sensitivities of the GenoType MTBDRsl test and DNA sequencing were compared using a binomial test. P values of 0.05 were considered statistically significant. Nucleotide sequence accession numbers. The nucleotide sequences obtained in this study have been submitted to GenBank under the following accession numbers: JF266640, JF266636, JF266637, JF266641, JF266638, JF266635, and JF for embb gene mutants P404S, Q497P, E504D, A505V, S565G, V602A, and N624D, respectively. RESULTS The drug resistance patterns of 234 MDR isolates are listed in Table 1. The 53 MDR M. tuberculosis isolates that exhibited no resistance to any second-line drugs or to EMB also contained no mutations, as determined using the GenoType MTBDRsl test. GenoType MTBDRsl testing and sequencing of gyra and gyrb mutations conferring FLQ resistance. Of the 234 isolates, including 160 FLQ-susceptible and 74 FLQ-resistant isolates, 95.3% (223/234) had concordant phenotypic and genotypic assay results. Of the 74 FLQ-resistant isolates, 63 (85.1%) revealed mutations conferring FLQ resistance, based on the GenoType MTBDRsl test and DNA sequencing results (Table 2). The other 11 isolates, consisting of 9 Beijing genotype isolates and 2 EAI2_MANILLA genotype isolates, could not be identified as FLQ resistant using the GenoType MTBDRsl test and gyra and gyrb sequencing. The distribution of gene mutations in the 63 FLQ-resistant isolates identified by the GenoType MTBDRsl test is shown in Table 3. The predominant mutations of the GenoType MTBDRsl test identified as conferring FLQ resistance were gyra MUT3C (D94G and D94G/D94Y) (36.5%), gyra MUT1 (A90V) (10.8%), missing WT1 (G88C) (8.1%), missing WT3 (D94Y) (8.1%), and gyra MUT3B (D94N) (6.8%). No MUT3D (D94H) mutations were found in our study. Dual mutations were identified in 5 isolates using the GenoType MTBDRsl test and in six isolates using DNA sequencing (Table 3), while 73% (46/63) of the 63 FLQ-resistant isolates using the GenoType MTBDRsl test harbored at least one mutation at codon 94 of the gyra gene. However, of the 63 FLQ-resistant isolates, 6 (8.1%) had a G88C mutation with no corresponding mutation probe included in the GenoType MTBDRsl test. Representative patterns of the GenoType MTBDRsl test are shown in Fig. 1. Four mixed banding patterns were observed, indicating the coexistence of both susceptible and resistant organisms, including gyra WT and MUT1 (Fig. 1, lane 1), gyra WT and MUT3C (Fig. 1, lane 2), gyra WT, MUT3A, and MUT3C (Fig. 1, lane 3), and gyra WT, MUT1, and MUT3C (Fig. 1, lane 4). In addition, three dual mutations were found in gyra MUT3A and MUT3B (Fig. 1, lane 5), gyra MUT1 and MUT3A (Fig. 1, lane 6), and gyra MUT1 and MUT3C (Fig. 1, lane 7). Sequencing data revealed that seven MDR isolates (one with an extra D94G mutation) harbored a D94Y mutation; however, only the gyra WT3 deletion-staining band was observed on the basis of the GenoType MTBDRsl test (Fig. 1, lane 8). GenoType MTBDRsl testing and sequencing of rrs and eis mutations conferring AG/CP drug resistance. The concordance between the phenotypic and the two genotypic assays for AG/CP resistance was 91% (213/234) in the GenoType MTBDRsl test and 95.3% (223/234) in sequencing for KM resistance, 95.7% (224/234) in sequencing for CAP resistance, and 98.7% (231/234) in sequencing for AM resistance (Table 2). Using the GenoType MTBDRsl test, the A1401G mutation in the rrs gene was identified in 16 isolates, including 1 with an extra WT1 band (Fig. 1, lane 9). No C1402T (i.e., with only the WT1 band deletion and no MUT1-staining band) or G1484T (i.e., with both the WT2 band deletion and a MUT2-staining band) mutations were found in this study (Table 3). Using rrs

4 VOL. 49, 2011 SECOND-LINE DRUG RESISTANCE DETECTION IN TB PATIENTS 2505 TABLE 3. Gene mutations in 74 FLQ-resistant, 38 AG/CPresistant, and 162 EMB-resistant Mycobacterium tuberculosis isolates based on GenoType MTBDRsl testing and sequencing Drug gene(s) and mutation probe(s) of GenoType MTBDRsl test Mutation(s) or codon(s) analyzed No. (%) of isolates FLQ (gyra) MUT1 A90V 8 (10.8) MUT2 S91P 2 (2.7) MUT3A D94A 3 (4.1) MUT3B D94N 5 (6.8) MUT3C D94G 26 (35.1) MUT3C D94G and D94Y 1 (1.4) MUT3D D94H 0 (0) MUT1 MUT3A A90V and D94A 1 (1.4) MUT1 MUT3C A90V and D94G 2 (2.7) MUT3A MUT3B D94A and D94N 1 (1.4) MUT3A MUT3C D94A and D94G 1 (1.4) Missing WT1 G88C 6 (8.1) Missing WT1, WT2 D89N 1 (1.4) Missing WT3 D94Y 6 (8.1) gene sequencing, we identified the A1401G mutation in 10 (83.3%, 10/12) isolates resistant to all three AG/CP drugs and in 6 (85.7%, 6/7) isolates resistant to both KM and AM (Table 1). Furthermore, results of eis gene sequencing revealed that 27% (10/37) of KM-resistant but AM-susceptible isolates harbored C 14T, C 12T, G 10A, and G 10C mutations (Table 3). The sensitivity of DNA sequencing (70.3%) was significantly higher than that of the GenoType MTBDRsl test (43.2%) (P 0.05). GenoType MTBDRsl testing and sequencing of emba, embb, embc, and embr mutations conferring EMB resistance. Of the 234 isolates, of which 162 were EMB resistant and 72 were EMB susceptible, the rates of concordance between the phenotypic assay and the two genotypic assays for EMB resistance were 68.4% (160/234) in the GenoType MTBDRsl test and FLQ (gyrb) WT 11 (14.9) AG/CP (rrs) MUT1 A1401G 16 (42.1) MUT2 G1484T 0 (0) AG/CP (eis) None C 14T 1 (2.6) None C 12T 4 (10.5) None G 10A 4 (10.5) None G 10C 1 (2.6) None WT 12 (31.6) EMB (embb) MUT1A M306I a 18 (11.1) MUT1B M306V 54 (33.3) Missing WT1 M306I b /M306I c /M306L 19 (11.7) EMB (embb, embc, emba, embr) None embb Y319C 7 (4.3) None embb Y319D 2 (1.2) None embb P404S 2 (1.2) None embb G406A 3 (1.9) None embb Q497K 5 (3.1) None embb Q497P 9 (5.6) None embb Q497R 13 (8.0) None embb Q497R, embb S565G 1 (0.6) None embb Q497R, embb N624D 1 (0.6) None embb Q497R, embb S565G 1 (0.6) None embb E504D 1 (0.6) None embb A505V 1 (0.6) None embb V602A 1 (0.6) None embc V451I 1 (0.6) None emba G 43C, embb Q497R 1 (0.6) None emba G 43C 1 (0.6) None emba C 16A 1 (0.6) None emba C 16G 1 (0.6) None emba C 11A 2 (1.2) None emba del 3C, embb Q497K 1 (0.6) None embr G245D 1 (0.6) None WT 15 (9.3) a Codon 306 ATG 3 ATA. b Codon 306 ATG 3 ATC. c Codon 306 ATG 3 ATT. FIG. 1. Representative pattern obtained by the GenoType MTBDRsl test for mutations in the gyra, rrs, and embb genes based on sequencing. The control (conjugate, amplification, and M. tuberculosis complex-specific controls), targeted gene (gyra, rrs, and embb), wildtype, and mutation bands are shown from left to right, as follows: CC, conjugate control; AC, amplification control; TUB, M. tuberculosis complex-specific control; gyra, control for gyra amplification; gyra WT1 to WT3, gyra wild-type probes located at codons 85 to 97; gyra MUT1 to MUT3D, gyra mutant probes in codons A90V, S91P, D94A, D94N/Y, D94G, and D94H, respectively; rrs, control for rrs amplification; rrs WT1 and WT2, rrs wild-type probes located at nucleotides 1401/1402 and 1484, respectively; rrs MUT1 and MUT2, rrs mutant probes for A1401G and G1484T, respectively; embb, control for embb amplification; embb WT1, embb wild-type probe located at codon 306; embb MUT1A and MUT1B, embb mutant probes in codons M306I and M306V, respectively. For lanes 1 to 4 and lanes 9 to 10, the mixtures of isolates are shown as follows: lane 1, gyra WT and MUT1; lane 2, gyra WT and MUT3C; lane 3, gyra WT, MUT3A, and MUT3C; lane 4, gyra WT, MUT1, and MUT3C; lane 9, rrs WT and MUT1; lane 10, embb WT1 and MUT1B. Lanes 5 to 7 show dual mutations in gyra: lane 5, MUT3A and MUT3B; lane 6, MUT1 and MUT3A; lane 7, MUT1 and MUT3C; lane 8, gyra WT3 (sequenced mutation in gyra D94Y).

5 2506 HUANG ET AL. J. CLIN. MICROBIOL. TABLE 4. Performance of the GenoType MTBDRsl assay and sequencing results for 234 multidrug-resistant Mycobacterium tuberculosis isolates % a Parameter FLQ AG/CP KM CAP AM EMB G S G S G S G S G S Sensitivity Specificity PPV NPV a G, GenoType MTBDRsl assay; S, sequencing. 92.3% (216/234) in sequencing. Of the 162 EMB-resistant isolates, 91 (56.2%) isolates had mutations conferring EMB resistance that were detected by the GenoType MTBDRsl test and 147 (90.8%) had mutations detected by sequencing (Table 2). The sensitivity of DNA sequencing (90.8%) was significantly higher than that of the GenoType MTBDRsl test (56.2%) (P 0.001). Of the 71 isolates that had no mutation at codon 306 in embb, 7 isolates had the Y319C mutation (TAT 3 TGT), 2 had the Y319D mutation (TAT 3 GAT), 2 had the P404S mutation (CCG 3 TCG), 3 had the G406A mutation (GGC 3 GCC), 6 had the Q497K mutation (CAG 3 AAG), 17 had the Q497R mutation (CAG 3 CCG), 9 had the Q497P mutation (CAG 3 CCG), 2 had the S565G mutation (AGC 3 CGC), and the remaining 5 isolates each had a different mutation in the embb gene; 2 had the G 43C mutation, 1 had the C 16A mutation, 1 had the C 16G mutation, 2 had the C 11A mutation, and 1 had the del 3C deletion in the emba gene; 1 had the V451I mutation (GTC 3 ATC) in the embc gene; and 1 had the G245D mutation (CCG 3 CTG) in the embr gene. Nevertheless, 15 EMBresistant isolates (9.3%) still exhibited a WT pattern both on the GenoType MTBDRsl test and with DNA sequencing. Among the 72 EMB-susceptible isolates, 3 isolates each had mutations at codon S297A (TCG 3 GCG), D328Y (GAT 3 TAT), or D328G (GAT 3 GGT) in the embb gene. In Table 3, the predominant embb gene mutations identified by the two genotypic methods were embb MUT1B (M306V; 33.3%, 54/162) and embb MUT1A (M306I, ATG 3 ATA; 11.1%, 18/162). Of the 54 isolates with the mutation in MUT1B, 4 had WT and MUT1B mixed patterns (Fig. 1, lane 10). Of the 19 EMB-resistant isolates with a missing WT band, 13 had either an ATG 3 ATC (n 9) or an ATG 3 ATT (n 4) M306I base change and 6 had an M306L mutation (ATG 3 CTG). Performance of GenoType MTBDRsl test and sequencing. The sensitivity, specificity, PPV, and NPV for detection of resistance to each drug assessed using the GenoType MTBDRsl test and gene sequencing are shown in Table 4. In this study, discrepancies were found in 10 KM-resistant, 56 EMB-resistant, and 3 EMB-susceptible isolates using the two genotypic methods (Table 2). In addition, six CAPsusceptible isolates were identified to be resistant by both genotypic methods. DISCUSSION Timely and reliable DST is crucial for prompt and effective treatment and for interrupting TB transmission. To initiate quality care for MDR-TB patients, it is crucial to rapidly obtain the results of DST for second-line drugs. However, conventional DST results for second-line drugs are often inaccurate and difficult to interpret (15). Molecular assays are recognized to provide rapid and accurate diagnosis of TB and drug resistance and are considered to be more specific than phenotypic DST. The importance of this study is to demonstrate that the current line probe assay for second-line DST still posed some limitations, while drug resistance gene sequencing is more accurate in detecting second-line drug and EMB resistance. The findings of new mutations together with existing mutations can be used to design diagnostic tests utilizing other detection platforms or technologies, such as microarrays or pyrosequencing. The line probe technology, mainly the GenoType MTBDRplus test, has been adopted by only the Reference Laboratory at the Taiwan Centers for Disease Control for MDR confirmation since In this study, we evaluated the performance of a rapid molecular assay, the GenoType MTBDRsl test, to detect resistance to fluoroquinolones, aminoglycosides/cyclic peptide, and ethambutol of 234 confirmed MDR M. tuberculosis isolates. Of the 234 MDR isolates, 124 (53%) were Beijing family and 110 (47%) were non-beijing family. We did not observe significant differences in drug resistance patterns, nor did we identify unique resistance mutations in the Beijing and non- Beijing families in this study. Previous studies demonstrated the accuracy of the GenoType MTBDRsl test to be 75.6% to 90.6% for detecting FLQ resistance, 77% to 100% for detecting KM resistance, 80% to 86.7% for detecting CAP resistance, 84.8% to 100% for detecting AM resistance, and 57% to 69.2% for detecting EMB resistance (5, 11, 14). In our study, the GenoType MTBDRsl test identified 85.1% of FLQ-resistant isolates, 43.2% of KM-resistant isolates, 71.4% of CAPresistant isolates, 84.2% of AM-resistant isolates, and 56.2% of EMB-resistant isolates. The test can be used for the rapid detection of resistance to FLQ, CAP, and AM but not to KM or EMB. The sensitivity of detection of FLQ resistance in our study (85.1%, n 74) was lower than that reported in Germany (90.6%, n 32) (11) and France (87.5%, n 24) (5) but higher than that reported in Vietnam (75.6%, n 41) (14). On

6 VOL. 49, 2011 SECOND-LINE DRUG RESISTANCE DETECTION IN TB PATIENTS 2507 the basis of the DNA sequencing, the frequency of gyra mutations conferring FLQ resistance detected by the GenoType MTBDRsl test was comparable to the range in previous reports (71% to 92%) (4, 20, 30). We observed that the mutation frequency of D94G (47.7%) in the gyra gene was higher than that reported in other studies, whose results ranged from 33.3% to 41.5% (5, 11, 14). Furthermore, heteroresistant isolates might result from the coexistence of wild-type and mutant alleles of the gyra gene at the preliminary stage of full drug resistance. Heteroresistance might also result from specific treatment regimens, host fitness, differently mutated strains, or hypermutable alleles of the DNA repair genes (19, 20). High rates of heteroresistance to FLQ-resistant isolates were reported in Germany (21.9%) (11), Russia (16.6%) (20), and Vietnam (21.4%) (14); however, the rates were low in our study (7.9%) and in France (4.2%) (5). Isolates with the D94Y (9.5%) mutation were expected to give both the gyra WT3 deletion and the MUT3B hybridization using the GenoType MTBDRsl test. However, only the gyra WT deletion was observed, as described by Kiet et al. (14). Previously, studies reported a less frequent occurrence of gyrb mutations than gyra mutations. Although we sequenced the gyrb gene from codon 410 to codon 606 to cover all of the previously reported mutations (5, 20), no mutation was found among the 11 FLQ-resistant isolates without gyra mutations. This finding suggests that mutations in other genes, such as mfpa (Rv3361c) (10) or the active efflux pump Rv2686c- Rv2687c-Rv2688c operon (20, 23), might be responsible. Only a few studies have focused on the genes responsible for AG/CP resistance (2, 13, 16, 17, 31). It was found that A1401G, C1402T, and G1484T mutations in the rrs gene are associated with AG/CP resistance. Specifically, the A1401G mutation in the rrs gene is associated with resistance to KM and AM (13). In our study, the A1401G mutation appeared in 16 of the KMand AM-resistant isolates with 100% specificity and a 100% PPV using the GenoType MTBDRsl test. A mutation at position 1401 seems to provide a better marker for AM resistance than for KM resistance. In addition, some KM-resistant M. tuberculosis strains isolated either in France (23.1%, 3/13) (5) or in Georgia (16.7%, 13/78) (13) did not possess the rrs mutation; this finding also applied to 56.8% (21/37) of KM-resistant M. tuberculosis isolates in our study. It is possible that KM resistance is caused by a mutation in another gene, such as the eis promoter region (34). In fact, we observed that 27% of our KM-resistant but AM-susceptible isolates harbored mutations in the eis gene. Therefore, the eis gene was recommended to be included in our routine molecular analysis for KM resistance. Cross-resistance to KM, AM, and CAP was reported to be associated with the A1401G mutation of the rrs gene (5, 17). However, we did not observe the same result. We identified six CAP-susceptible isolates with the A1401G mutation in the rrs gene. These data are concordant with those of the study by Alangaden et al. (2). These authors found 13 isolates that harbored the A1401G mutation in the rrs gene, including 6 CAP-resistant isolates, 6 susceptible isolates, and 1 isolate with unknown susceptibility. Furthermore, Jugheli et al. reported that position 1402 in the rrs gene should be the major determinant of CAP resistance rather than position 1401 (13). Low detection rates of embb gene mutations in EMB-resistant isolates were reported in Germany (69.2%) (11), Vietnam (64.2%) (14), France (57.1%) (5), Kuwait (30%) (1), and this study (56.2%). The molecular basis of the EMB resistance included in the GenoType MTBDRsl test is insufficient, even though the codon 306 mutations of the embb gene were significantly associated with EMB resistance (12). In this study, the predominant (59.3%) mutation in embb M306V was comparable to the mutations reported from Vietnam (68.6%) (14), Germany (72.2%) (11), and France (87.5%) (5). To resolve the low sensitivity of EMB resistance detection, we identified several novel mutations in the embb gene at codons other than codon 306. The codon 319 mutation was found in nine EMBresistant isolates, including seven that had the Y319C mutation and two that had the Y319D mutation. Sugawara et al. also found Y319C and Y319D mutations in two EMB-resistant isolates (28). In addition, we observed that 19.8% (32/162) of our EMB-resistant isolates harbored mutations at codon 497 in the embb gene. It is worth mentioning that seven novel mutations, P404S, Q497P, E504D, A505V, S565G, V602A, and N624D, in the embb gene were identified. However, using DNA sequencing, we found three isolates with mutations in codon 297 and in codon 328 of the embb gene that were susceptible to EMB. In addition to the embb gene, other genomic regions, such as the emba, embc, and embr genes, might contribute to EMB resistance (27). Since 43.8% (71/162) of our EMB-resistant isolates exhibited WT patterns when using the GenoType MTBDRsl test alone, 9 isolates harbored mutations in the emba, embc, and embr genes. In addition, some reports have suggested that embb codon 306 mutations do not confer resistance to EMB but rather constitute a common polymorphism of the susceptibility to develop any type of drug resistance or MDR (9, 18, 22, 27). Although those reports offer speculation on the biological role of this mutation (3), further studies are needed to identify actual mechanisms of EMB resistance. We found several limitations on applying the GenoType tests in routine clinical diagnosis. First, the high costs of the GenoType MTBDRplus ($35 per test) and GenoType MTBDRsl ($80 per test) tests make routine screening of any suspected TB and/or MDR-TB cases impractical. Currently, we use those two tests for confirmation of MDR M. tuberculosis isolates and for screening of high-risk populations, including retreated cases, MDR-TB contacts, and suspected cases from regions in Taiwan where MDR-TB is prevalent. Second, due to the lower sensitivities of detection of KM and EMB resistance using the GenoType MTBDRsl test, the following algorithm for screening an MDR-TB patient is suggested: decontaminated sputum is initially tested with the GenoType MTBDRplus test, and the remaining sample of this MDR patient is retested using the GenoType MTBDRsl. Subsequently, DNA sequencing of the eis and emb genes has to be done to exclude false drug susceptibility. Third, these two GenoType tests require DNA amplification and staining steps, diverse amplification devices, and varied staining times, which might lead to different interpretations of results. Fourth, since the banding patterns of the GenoType tests are not always obvious, a well-trained medical technician is needed to interpret test results. In addition, quality control has to be well implemented before such services are provided. Therefore, GenoType tests are recommended to be used in the designated clinical laboratories for targeted testing. In conclusion, the present study demonstrated that the

7 2508 HUANG ET AL. J. CLIN. MICROBIOL. GenoType MTBDRsl test can be applied to the rapid detection of FLQ, AG/CP, and EMB resistance. The accuracy of the GenoType MTBDRsl test for the detection of FLQ and AM resistance was satisfactory compared to DST results obtained using conventional culture-based methods, but the results were less accurate for the detection of KM and EMB resistance and carried a lower positive predictive value for the detection of CAP resistance. The A1401G mutation of the rrs gene was a marker for resistance to both KM and AM. Given the lower sensitivity of KM and EMB resistance detection, this test should not be used to rule out resistance when a wild-type result is obtained. We suggest that new alleles of the eis promoter in the KM resistance gene and embb codon 497 in the EMB resistance gene be included to improve the sensitivity of a molecular test for genetically diverse M. tuberculosis strains isolated from different geographical areas. Although molecular techniques could be used for rapid detection of resistance to second-line drugs and EMB, we find that they still cannot replace conventional DST. ACKNOWLEDGMENTS This work was supported by grant DOH98-DC-2025 from the Centers for Disease Control, Department of Health, Taiwan. We thank the clinical mycobacteriology laboratories for providing Mycobacterium tuberculosis isolates, as well as Huang-Yao Chen, Rai- Che Chien, Yung-Lin Chang, and Yuh-Min Kuo for their technical support. REFERENCES 1. Ahmad, S., A. A. Jaber, and E. Mokaddas Frequency of embb codon 306 mutations in ethambutol-susceptible and -resistant clinical Mycobacterium tuberculosis isolates in Kuwait. Tuberculosis 87: Alangaden, G. J., et al Mechanism of resistance to amikacin and kanamycin in Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 42: Alcaide, F., G. E. Pfyffer, and A. Telenti Role of embb in natural and acquired resistance to ethambutol in mycobacteria. Antimicrob. Agents Chemother. 41: Antonova, O. V., et al Detection of mutations in Mycobacterium tuberculosis genome determining resistance to fluoroquinolones by hybridization on biological microchips. Bull. Exp. Biol. Med. 145: Brossier, F., N. Veziris, A. Aubry, V. Jarlier, and W. Sougakoff Detection by GenoType MTBDRsl test of complex mechanisms of resistance to second-line drugs and ethambutol in multidrug-resistant Mycobacterium tuberculosis complex isolates. J. Clin. Microbiol. 48: Centers for Disease Control, Department of Health, Executive Yuan Taiwan tuberculosis control report. Centers for Disease Control, Department of Health, Executive Yuan, Taipei, Taiwan. /public/data/ pdf. 7. Drlica, K Mechanism of fluoroquinolone action. Curr. Opin. Microbiol. 2: Hain Lifescience GmbH GenoType MTBDRsl: instruction manual. Hain Lifescience GmbH, Nehren, Germany. 9. Hazbón, M. H., et al 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: Hegde, S. S., et al A fluoroquinolone resistance protein from Mycobacterium tuberculosis that mimics DNA. Science 308: Hillemann, D., S. Rusch-Gerdes, and E. Richter Feasibility of the GenoType MTBDRsl assay for fluoroquinolone, amikacin-capreomycin, and ethambutol resistance testing of Mycobacterium tuberculosis strains and clinical specimens. J. Clin. Microbiol. 47: Isola, D., et al A pyrosequencing assay for rapid recognition of SNPs in Mycobacterium tuberculosis embb306 region. J. Microbiol. Methods 62: Jugheli, L., et al High level of cross-resistance between kanamycin, amikacin, and capreomycin among Mycobacterium tuberculosis isolates from Georgia and a close relation with mutations in the rrs gene. Antimicrob. Agents Chemother. 53: Kiet, V. S., et al Evaluation of the MTBDRsl test for detection of second-line-drug resistance in Mycobacterium tuberculosis. J. Clin. Microbiol. 48: Kim, S. J Drug-susceptibility testing in tuberculosis: methods and reliability of results. Eur. Respir. J. 25: Maus, C. E., B. B. Plikaytis, and T. M. Shinnick Mutation of tlya confers capreomycin resistance in Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 49: Maus, C. E., B. B. Plikaytis, and T. M. Shinnick Molecular analysis of cross resistance to capreomycin, kanamycin, amikacin, and viomycin in Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 49: Mokrousov, I., T. Otten, B. Vyshnevskiy, and O. Narvskaya Detection of embb306 mutations in ethambutol-susceptible clinical isolates of Mycobacterium tuberculosis from northwestern Russia: implications for genotypic resistance testing. J. Clin. Microbiol. 40: Mokrousov, I Multiple rpob mutants of Mycobacterium tuberculosis and second-order selection. Emerg. Infect. Dis. 10: Mokrousov, I., et al Molecular characterization of ofloxacin resistant Mycobacterium tuberculosis strains from Russia. Antimicrob. Agents Chemother. 52: Nathanson, E., et al Multidrug-resistant tuberculosis management in resource-limited settings. Emerg. Infect. Dis. 12: Parsons, L. M., et al Phenotypic and molecular characterization of Mycobacterium tuberculosis isolates resistant to both isoniazid and ethambutol. Antimicrob. Agents Chemother. 49: Pasca, M. R., et al Rv2686c-Rv2687c-Rv2688c, an ABC fluoroquinolone efflux pump in Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 48: Plinke, C., et al embcab sequence variation among ethambutolresistant Mycobacterium tuberculosis isolates without embb306 mutation. J. Antimicrob. Chemother. 65: Ramaswamy, S. V., et al Molecular genetic analysis of nucleotide polymorphisms associated with ethambutol resistance in human isolates of Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 44: Shah, N. S., et al Worldwide emergence of extensively drug-resistant tuberculosis. Emerg. Infect. Dis. 13: Srivastava, S., A. Ayyagari, T. N. Dhole, K. K. Nyati, and S. K. Dwivedi emb nucleotide polymorphisms and the role of embb306 mutations in Mycobacterium tuberculosis resistance to ethambutol. Int. J. Med. Microbiol. 299: Sugawara, I., et al The molecular epidemiology of ethambutol-resistant Mycobacterium tuberculosis in Henan Province, China. Jpn. J. Infect. Dis. 58: Telenti, A., et al The emb operon, a gene cluster of Mycobacterium tuberculosis involved in resistance to ethambutol. Nat. Med. 3: van Doorn, H. R., et al Fluoroquinolone resistance detection in Mycobacterium tuberculosis with locked nucleic acid probe real-time PCR. Int. J. Tuberc. Lung Dis. 12: Via, L. E., et al Polymorphisms associated with resistance and crossresistance to aminoglycosides and capreomycin in Mycobacterium tuberculosis isolates from South Korean patients with drug-resistant tuberculosis. J. Clin. Microbiol. 48: World Health Organization Anti-tuberculosis drug resistance in the world. Fourth Global Report. WHO/HTM/TB/ The World Health Organization/International Union against Tuberculosis and Lung Disease (WHO/UNION) Global Project on Anti-Tuberculosis Drug Resistance Surveillance World Health Organization, Geneva, Switzerland. http: //whqlibdoc.who.int/hq/2008/who_htm_tb_ _eng.pdf. 33. World Health Organization Guidelines for the programmatic management of drug-resistant tuberculosis. WHO/HTM/TB/ World Health Organization, Geneva, Switzerland. /WHO_HTM_TB_ _eng.pdf. 34. Zaunbrecher, M. A., R. D. Sikes, Jr., B. Metchock, T. M. Shinnick, and J. E. Posey Overexpression of the chromosomally encoded aminoglycoside acetyltransferase eis confers kanamycin resistance in Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. U. S. A. 106: Zhang, Y., and W. W. Yew Mechanisms of drug resistance in Mycobacterium tuberculosis. Int. J. Tuberc. Lung Dis. 13: Zhao, X., C. Xu, J. Domagala, and K. 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