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Evaluation of the MTBDRsl test for detection of second line drug resistance in 1

Mycobacterium tuberculosis. 2

Vo Sy Kiet1, Nguyen Thi Ngoc Lan

2, Duong Duy An

1, Nguyen Huy Dung

2, Dai Viet Hoa

2, 3

Nguyen van Vinh Chau4,

Nguyen Tran Chinh4, Jeremy Farrar

1,3 Maxine Caws

1,3* 4

1. Oxford University Clinical Research Unit, Hospital for Tropical Diseases, 190 Ben Ham Tu, 5

District 5, Ho Chi Minh City, Vietnam 6

2. Pham Ngoc Thach Hospital for Tuberculosis and Lung Diseases, 120 Hung Vuong, District 5, Ho 7

Chi Minh City, Vietnam 8

3. Centre for Clinical Vaccinology and Tropical Medicine, Churchill Hospital, Old Road, 9

Headington, Oxford, United Kingdom. 10

4. The Hospital for Tropical Diseases, 190 Ben Ham Tu, District 5, Ho Chi Minh City Viet Nam 11

12

* Corresponding author: Maxine Caws, Oxford University Clinical Research Unit, Hospital for 13

Tropical Diseases, 190 Ben Ham Tu, District 5, Ho Chi Minh City, Vietnam. Tel: 84 8 3923 9211. 14

email: [email protected] 15

16

17

Keywords: tuberculosis, XDR, fluoroquinolone, resistance, MTBDRsl. 18

Running title: Evaluation of MTBDRsl in M.tuberculosis 19

Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Clin. Microbiol. doi:10.1128/JCM.00201-10 JCM Accepts, published online ahead of print on 23 June 2010

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Abstract 20

The MTBDRsl assay (HAIN Lifesciences, Germany) is a new line-probe assay for the 21

detection of extensively drug resistant tuberculosis (XDR TB). The test simultaneously 22

detects resistance to ethambutol, aminoglycosides/cyclic peptides and fluoroquinolones 23

through detection of mutations in the relevant genes. The assay format is identical to the 24

MTBDR HAIN assay. 25

The assay was evaluated for the detection of second line resistance in Vietnamese isolates 26

using 2 sample sets from Pham Ngoc Thach Hospital microbiology department with 27

existing conventional phenotypic drug susceptibility results for second line drugs: 41 28

consecutive fluoroquinolone resistant isolates and 21 consecutive multi-drug resistant but 29

fluorquinolone sensitive isolates. 30

Sensitivity for detection of fluoroquinolone resistance was 75.6% [95% CI 59.7, 87.6], 31

n=31/41, and for kanamycin resistance 100% [95% CI 47.8, 100], n=5/5. The sensitivity of 32

the test for detection of ethambutol resistance was low, consistent with previous reports, at 33

64.2% [95% CI 49.8, 76.9], n=34/53. 34

Specificity of the test was 100% for all three drugs. This data suggests that the MTBDRsl is 35

a rapid, specific test for the detection of XDR TB but should not be used exclusively to 36

‘rule-out’ second-line drug resistance. Further operational evaluation is required and 37

should be integrated with evaluations of the MTBDR test. 38


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39

Introduction: 40

The World Health Organization (WHO) has estimated that 5% of all tuberculosis cases globally 41

are now multidrug resistant tuberculosis [MDRTB, resistance to at least rifampicin (RIF) and 42

isoniazid (INH)], based on data from more than 100 countries acquired since 2000 [16]. Every 43

year, an estimated 490,000 new MDRTB cases occurs causing more than 130,000 deaths [16]. In 44

2006 the documentation of a rapidly fatal TB outbreak among hospitalized HIV patients in Kwa 45

Zulu Natal[12], South Africa led to the definition of extensively drug resistant tuberculosis 46

(XDR TB) as TB resistant to a fluoroquinolone and injectable second line drug (amikacin, 47

capreomycin or kanamycin) in addition to isoniazid and rifampicin. XDR TB has subsequently 48

been reported from over 50 countries by WHO [16]. It is likely that the majority of XDR TB 49

cases worldwide remain undetected due to the lack of second line drug testing in most high 50

burden settings. There are an estimated 40,000 new extensively drug resistant tuberculosis each 51

year[17]. 52

The recognition of XDR TB world wide has made timely identification of XDR TB cases to 53

achieve effective disease management and to prevent their spread a priority [1, 8]. 54

Significant challenges exist; although standard protocols exist for second line drug susceptibility 55

testing, strong evidence is lacking on many factors such as reproducibility and reliability of 56

results, applicability of MIC to clinical outcomes and intermethod variability. Profiency testing 57

for second-line drug DST has only recently been integrated into the Supranational reference 58

laboratory panel in an effort to improve standardization of second-line drugs across the WHO 59

reference laboratory network.. 60

Conventional drug resistance testing takes more than 2 weeks to return a result even after a 61

positive culture has been isolated. Rapid commercial liquid-based culture systems such as Bactec 62

MGIT 960 testing (Becton Dickinson) for second line drugs are not yet formally FDA/WHO 63

approved but are reported to be accurate, widely used in developed settings and reduce 64

turnaround times to approximately 8 days [2]. In recent years many second line drug 65

susceptibility testing methods have been developed. The most rapid results are achieved by direct 66


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testing of patient specimens by molecular methods, however, in addition to the high cost of such 67

tests, the sensitivity remains sub-optimal and rigorous contamination control is required to 68

maintain accuracy. The majority of high-burden settings currently lack the resources to 69

implement such tests effectively. Two commercial DNA strip assays, INNO-LiPA RifTB 70

(Innogenetics, Zwijndrecht, Belgium) and MTBDRplus (Hain Lifescience, GmbH, Germany), 71

targeting the rpoB plus katG and inhA genes have been extensively evaluated for use with 72

Mycobacterium tuberculosis (M.tuberculosis) culture and directly on sputum to identify MDR 73

TB cases [9, 14]. The Foundation for Innovative Diagnostics (FIND) demonstration projects in 74

South Africa of the genotype MTBDR plus assay resulted in the recommendation of commercial 75

line-probe assays for use in high burden settings by WHO [18]. This assay is based on a 76

multiplex PCR in combination with reverse hybridization. Either the absence of wild type bands 77

or the appearance of bands targeting specific mutations indicates the presence of a resistant 78

strain. MDR TB cases can be detected within 1 or 2 days of sputum sampling using this assay. 79

In order to rapidly detect second line drug resistance, the MTBDRsl (Hain Lifescience, GmbH, 80

Germany) has been developed. This assay can detect mutations in gyrA, rrs and embB genes, 81

detecting resistance to the fluoroquinolones (FQ), aminoglycosides/cyclic peptides and 82

ethambutol (EMB) respectively, with a single assay. A previous evaluation study in Germany 83

showed that this assay has a high accuracy for FQ, and amikacin-capreomycin resistance testing 84

in clinical strains and sputum[3]. EMB detection was specific (100%) but sensitivity (69.2%) 85

was low. 86

The MTBDRsl assay contains 22 probes, including 16 probes for gene mutation detection and 6 87

probes for the control of the test procedure. The 6 control probes include a conjugate control 88

(CC), an amplification control (AC), a Mycobacterium tuberculosis complex control (TUB) and 89

3 locus probes (gyrA, rrs and embB) for gene amplification control. The remaining probes detect 90

FQ resistance (gyrA), amikacin/capreomycin resistance ( rrs) and ethambutol resistance (embB). 91

The probes contained in the assay do not detect all mutations in these genes but are targeted to 92

the most commonly occurring mutations. 93


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The aim of the present study was to determine the accuracy of this assay for detection of FQ, 94

kanamycin and ethambutol resistance against conventional phenotypic testing as the gold 95

standard on Vietnamese isolates of M.tuberculosis. 96

97

98

Materials and methods: 99

Samples: Two sample sets with a high prevalence of second-line resistance were used for evaluation. 41 100

consecutive FQ resistant isolates from individual patients collected from Pham Ngoc Thach 101

hospital between July 2005 and July 2006.[10]. A set of 21 consecutive pulmonary MDR, FQ-102

sensitive isolates from individual patients collected at Pham Ngoc Thach Hospital laboratory 103

between January and March 2005 was used to assess specificity for FQ resistance detection [13]. 104

Drug susceptibility testing is not routine in the Vietnamese national TB programme and is done 105

only on request from the treating clinician, usually following treatment failure or relapse. 106

Outcome and follow-up data were not available for any of the patients. 107

Drug susceptibilitytesting (DST): Conventional 1% proportion phenotypic DST on 108

Lowenstein Jensen (LJ) media was performed at Pham Ngoc Thach Hospital (PNT) Laboratory. 109

All samples were tested for isoniazid (0.2µg/ml), rifampicin(40 µg/ml), streptomycin (STR, 4 110

µg/ml), ofloxacin(2ug/ml), ethambutol(2ug/ml), kanamycin (20µg/ml), thioacetone (10µg/ml), 111

pyrazinamide (200µg/ml), ethionamide (40 µg/ml), cycloserine (30g/ml) and para-aminosalicylic 112

acid (PAS) (0.5g/ml) resistance. Pham Ngoc Thach Laboratory is the national reference 113

laboratory for southern Vietnam and participates in WHO supranational proficiency testing for 114

DST for all first line drugs, FQN and kanamycin. Performance is consistently higher than the 115

minimum requirement. 116

MTBDRsl assay: PCR and hybridization was done according to the manufacturer’s instructions. 117

For amplification, 35 µl of primer nucleotide mix (PNM, provided in the kit), 5µl of 10X 118

amplification buffer (Quiagen), 1.2 µl of MgCl2 2.5M (Quiagen), 5µl of DNA (at the 15ng/ul 119

concentration) and water was added to a final volume of 50µl. The PCR products were 120

denatured by denaturing solution at room temperature, followed by hybridization with 121


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hybridisation buffer at 450C for 30 min in shaking water bath. After stringent washing, the 122

hybridiation was detected by colorimetric reaction. To control for cross contamination, water in 123

lieu of DNA template was added to one negative control included in each run. 124

gyrA sequencing: All isolates were sequenced in gyrA quinolone resistance determining region 125

(QRDR) to evaluate discrepancies between MDRTBsl and phenotypic testing. All samples were 126

cultured on LJ media and DNA extracted for sequencing using standard methods ([10]. Primers 127

GYRATBF 5’- CAG CTA CAT CGA CTA TGC GA -3’ and GYRATBR 5’- GGG CTT CGG 128

TGT ACC TCA T-3’ were used to amplify a 320bp fragment of gyrA gene ([10]. Sequencing of 129

PCR product was carried out with CEQ 8000 gentic analysis system (Beckman Coulter, USA), 130

and the results were analyzed on the CEQ8000 software. 131

132

Statistics 133

Data was analysed on Stata 9 ( Statacorp, USA). McNemar’s test was used to estimate agreement 134

between the tests. Sensitivity, specificity and 95% confidence intervals were calculated for each 135

drug. 136

Results: 137

Phenotypic DST results: Table 1 summarizes drug susceptibility patterns of FQ-resistant and 138

FQ-sensitive isolates included in the study. Of the 41 FQ-resistant isolates, 43.9% (n=18/41) 139

were resistant to INH, RIF, STR, EMB, PZA but sensitive to all second line drugs. Three isolates 140

(7.3%) were MDR strains resistant to kanamycin in addition to ofloxacin, and therefore XDR 141

TB. One of these XDR isolates showed resistance to all second line drugs tested except PAS. 142

Among the FQ-sensitive isolates, all isolates were resistant to INH, RIF, STR and EMB. 57.1% 143

(n=12/21) isolates were resistant to INH, RIF, STR and EMB but sensitive to PZA and all 144

second line drugs tested. The remaining isolates showed a range of second-line DST profiles . 145

MDRTBsl Assay: Representative hybridisation patterns obtained are shown in figure 1. 146

Interpretable results were obtained for all cultures tested. 147


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FQ-resistance: Concordance between phenotypic testing and MDRTBsl assay was 83.9% 148

(n=52/62) for FQ resistance (table 2), P=0.002. Sensitivity of the MTBDRsl assay for FQ-149

resistance was 75.6% [95% CI 59.7, 87.6], n=31/41. Specificity was 100% [95% CI 83.9, 100]. 150

Isolates were sequenced in the gyrA gene to evaluate discrepancies. 33/41 (80.4%) of isolates 151

had identical results by sequencing and MTBDRsl (table 3). Of those isolates with discrepant 152

sequencing/MTBDRsl results, all but one isolate (FQ1319) had the same resistant/sensitive 153

classification by both tests. This isolate had no mutation detected by sequencing but showed the 154

presence of D94N gyrA mutation (MUT3B) on the MTBDRsl assay. For the 7 remaining 155

discrepant isolates, one sample (FQ1150) hybridized to the D94G mutation probe (MUT3C) on 156

MDRTBsl but sequencing showed a D94N mutation, which should hybridise at probe MUT3B 157

on MTBDRsl. One isolate (FQ1155) was recorded as resistant by MTBDRsl assay because no 158

hybridization was detected for the WT3 probe (D94X) but this isolate did not hybridise to any 159

mutant probe. Mutation D94Y was shown by sequencing which should have given hybridization 160

for probe MUT3B by MTBDRsl assay. 161

2 samples (FQ1162 and FQ1281) had D94G mutations only on the MTBDRsl assay but both 162

D94G and A90V mutations were detected by sequencing, however this was probably a 163

consequence of a mixed wild-type:resistant population, as detected by sequencing. In both cases, 164

the A90V mutant peak was only 30% of the wild-type height, indicating that the wild-type 165

population appeared to be predominant. 166

The remaining 3 isolates (FQ1283, FQ1297 and FQ1298) showed multiple mutations with 167

MTBDRsl assay which were not all detected by sequencing. All of these samples recorded a 168

resistant result by both tests. These discrepant results may have been due to cross contamination 169

but the 7 discrepant isolates were repeated by MTBDRsl and gave identical results and no 170

contamination was observed in negative controls or results of FQ-sensitive isolates. The 171

difference may therefore be due to differential amplification of targets by the PCR reactions for 172

MTBDRsl and sequencing. 173

Aminogylcoside resistance: Sensitivity (n=5/5) [95% CI 47.8, 100], specificity (n= 57/57) [95% 174

CI 93.7, 100] and concordance were all 100 % for kanamycin resistance, (P=1). Mutations 175


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detected in rrs by MTBDRsl assay were A1401G (n=4/5, 80%) and G1484T (n=1/5, 20%) (table 176

4). 177

Ethambutol resistance: Concordance for EMB resistance was 69.4% (n=43/62), P

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short-course use as broad spectrum antibiotics for the treatment of respiratory infections, 205

allowing resistant populations to emerge but not be fully selected. 206

The low detection rate of ethambutol resistance is consistent with reports from other settings, 207

with embB mutations accounting for between 30 and 70% of ethambutol resistant isolates ([4, 208

15]. This underlines the need to identify other mutations for ethambutol resistance in order to 209

improve the sensitivity of molecular tests, including line-probe assays, for ethambutol resistance 210

detection. 211

It was previously thought that cross-resistance among the aminoglycosides amikacin, and 212

kanamycin was total, however, substantial recent data has demonstrated that this is not the case 213

[5-7]. Specific mutations appear to confer complete cross-resistance while others do not. For 214

example eis promoter mutations confer low-level resistance to kanamycin but not amikacin [7]. 215

Line probe assays may offer the further advantage of identifying specific mutations conferring 216

resistance which may allow the tailoring of optimised individual treatment regimens for patients 217

based on MIC/cross-resistance patterns which would be a particular advantage in XDR TB 218

patients where effective treatment options are severely restricted. However, further research is 219

required to elucidate the nature of such cross-resistance patterns before clinical recommendations 220

can be made. 221

The WHO recommends the use of commercial line probe assays (INNO-LiPA and HAIN) for the 222

rapid detection of MDR TB within National TB Programs with the capacity to implement 223

appropriate laboratory infrastructure, staff training, contamination control, external quality 224

assessment schemes, standardised operating procedures, rapid reporting of results and 225

availability of quality assured second-line drugs. Preliminary economic analysis suggests that 226

line probe assays may offer cost savings over conventional practices: the cost per valid 227

MTBDRplus assay result in South Africa was 19.56 USD when performed directly on primary 228

specimens compared to 31.12 USD for MGIT culture and conventional DST (7H11 media). 229

Although this cost assessment did not include the costs of establishing laboratory infrastructure 230

and staff training for molecular assays and will vary in different settings, these data suggest that 231

commercial line-probe assays are economically viable in some high burden settings [18]. 232


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233

This study evaluated the MTBDRsl on DNA extracted from cultural isolates and did not evaluate 234

performance of the test directly on sputum samples. Hilleman et al. evaluated 64 sputum samples 235

and achieved good sensitivity but further data is required on performance directly on sputum. 236

However, initial evaluations of the MTBDRsl test suggest that it may meet similar performance 237

characteristics to the line probe assays currently available for the detection of MDR TB, and 238

could be implemented for the timely identification of XDR TB cases in settings already 239

performing the MDR TB assays. The rapid identification of XDR TB cases will allow early 240

initiation of appropriate therapy, infection control and prevent further amplification of drug 241

resistance. Operational studies should be conducted to evaluate the performance of the test in 242

high-burden settings and determine the optimal algorithms for implementation of the test by 243

national TB Programs in settings of high drug resistance.244


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245

Table 1. Phenotypic drug susceptibility profiles of 41 isolates resistant to ofloxacin and .21 ofloxacin control isolates 246

Drug susceptibility pattern Ofloxacin resistant

isolates

Ofloxacin sensitive

isolates

INH RIF STR EMB PZA KANA ETHIO CYCLO TB1 PAS No. (%) N0 (%)

1 R R R R S S S R S S 0 3 (14.3%)

2 R R R R S R R S S S 0 2 (9.5)

3 R R R R S S R R R S 0 2 (9.5)

4 R R R R S S R S S R 0 1 (4.8%)

5 R R R R S S S S R S 0 1 (4.8%)

6 R R R R S S S S S S 6 (14.6) 12 (57.1%)

7 R R R R R S S S S S 18 (43.9) 0

8 R R R S R S S S S S 3 (7.3) 0

9 R R R S S S S S S S 2 (4.9) 0

10 R S R R R S S S S S 2 (4.9) 0

11 R R R R R R R R R S 1 (2.4) 0

12 R R R R R R S S S R 1 (2.4) 0

13 R R R R R S R S R S 1 (2.4) 0


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14 R R R R R S R S S S 1 (2.4) 0

15 R R R R R S S S S R 1 (2.4) 0

16 R R R S S R S S S S 1 (2.4) 0

17 R R R S S S R S S S 1 (2.4) 0

18 R R S R S S S S S R 1 (2.4) 0

19 S S R S S S S S S S 1 (2.4) 0

20 S S S S S S S S S S 1 (2.4) 0

Total 41 (100%) 21 (100%)

247

Note: INH: isoniazid,, RIF: rifampicin, STR: streptomycin, EMB: ethambutol, PZA: pyrazinamide, KANA: kanamycin, ETHIO: ethionamide, 248

CYCLO: cycloserine, TB1: thioacetazone, PAS: p-aminosalicylic acid, R: resistant, S: sensitive 249


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Table 2: Concordance between MTBDRsl and phenotypic testing for ethambutol, ofloxacin and 251

Kanamycin resistance. 252

253

Phenotypic Result (gold standard) MTBDRsl assay

Drug Resistant n (%) Sensitive n(%)

Ethambutol

Resistant n=53 34 (64.2%) 19 (35.8%)

Sensitive n=9 0 9 (100%)

Total n=62 34 (54.8%) 28 (45.2%)

Ofloxacin

Resistant n=41 31 (75.6%) 10 (24.4%)0

Sensitive n=21 0 21 (100%)

Total n=62 31 (50%) 31 (50%)

Kanamycin

Resistant n=5 5 (100%) 0

Sensitive n=57 0 57 (100%)

Total n=62 5 (8.8%) 57 (91.9)

254

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Figure 1. Representative patterns obtained by the MTBDRsl assay. Lane 1: negative control. 262

Lane 2 and 3: FQ, EMB and KAN susceptible strains. Lane 4,5 6 and 7: resistant strains (lane 4: 263

resistant to FQ and EMB; lane 5: resistant to FQN, AMP/CMP and EMB; lane 6: resistant to 264

EMB; lane 7: resist to FQ and EMB). Lane 8, 9, 10 and 11: strains containing wild-type and FQ 265

resistant populations. 266

Specific probes are: CC – conjugate control; AC – amplification control; TUB – Mycobacterium 267

tuberculosis complex control; gyrA – gyrA gene amplification control; gyrA WT 1-3 – gyrA 268

wild type probes; gyrA MUT1, gyrA MUT2, gyrA MUT 3A-D – gyrA mutant probes for A90V, 269

S91P, D94A, D94N, D94Y, D94G and D94H, respectively; rrs – rrs amplification control; rrs 270

WT1-2 – rrs wild type probes; rrs MUT1-2 – rrs mutant probes for A1401G and G1484T, 271

respectively; embB – embB amplification control; embB WT1 – embB wild type probe I codon 272

306; embB MUT1A and embB MUT1B – embB mutant probes for M306I and M306V, 273

respectively. 274

275

276


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Table 3: MTBDRsl assay and sequencing results for gyrA mutation of 42 phenotypically FQN 285

resistant strains included in the study 286

287

288

MTBDRsl results

GyrA

Sequencing

result

concordance

Strain

result hybridisation pattern mutation detected

by MTBDRsl

FQ1091 R WT + MUT3C D94G D94G* Yes

FQ1092 R ∆WT3 + MUT3A D94A D94A Yes

FQ1093 R ∆WT3 + MUT3C D94G D94G Yes


FQ1095 R WT + MUT3A D94A D94A* Yes

FQ1150 R WT + MUT3C D94G D94N No



FQ1153 R ∆WT2 + MUT1 A90V A90V Yes

FQ1154 S WT WT NONE Yes

FQ1155 R ∆WT3 D94X D94Y Partial







FQ1162 R ∆WT3 + MUT3C D94G A90V*/D94G* Partial

FQ1281 R WT + MUT3C D94G A90V*/D94G* Partial

FQ1283 R WT+MUT1+MUT3B + MUT3C A90V/D94N/D84Y

and D94G A90V*/D94G* Partial




FQ1289 R WT+ MUT1 + MUT3A A90V/D94A A90V*/D94A* Yes



FQ1292 R ∆WT3 + MUT3A + MUT3C D94A/D94G D94G*/D94A* Yes





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FQ1297 R ∆WT3 + MUT3B + MUT3C D94N/D94Y and

D94G D94G* Partial

FQ1298 R ∆WT3 + MUT3B + MUT3C D94N/D94Y and

D94G D94G* Partial


FQ1319 R WT + MUT3B D94N/D94Y NONE No




FQ1323 R ∆WT2 + MUT1 A90V A90V yes

FQ1324 S WT WT NONE yes

FQ1325 S WT WT NONE yes

289

Note: R, resistant; S, susceptible; WT, wild type 290

*: hetero peak 291

Partial: At least one identical mutation detected by both tests. 292

∆WT indicates lack of hybridization to wild-type probe 293

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Table 4: MTBDRsl pattern of 53 phenotypically Ethambutol resistant and 5 phenotypically kanamycin 298

resistant isolates. 299

300

Ethambutol

(EmbB gene)

MTBDRsl pattern* Codon mutation No. (%) of isolates

∆WT ATG306ATC/ATT 5 (9.4)

∆WT + MUT1A M306I 5 (9.4)

∆WT + MUT1B M306V 21 (39.6)

WT + MUT1A WT + M306I 1 (1.9)

WT + MUT1B WT + M306V 2 (3.8)

WT+MUT1A+MUT1B WT + M306I + M306V 1 (1.9)

WT WT 18(34.0)

Total 53 (100)

Kanamycin

(rrs gene)

∆WT1 + WT2 + MUT1 A1401G 4 (44.4)

WT1 + WT2 + MUT1 WT + G1484T 1 (11.1)

Total 5(100)

301

*Probe names as given in MTBDRsl manual (see figure 1). 302

∆WT indicates lack of hybridization to wild-type probe 303


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Downloaded from //jcm.asm.org/content/jcm/early/2010/06/23/JCM...2010/06/23 · testing of patient specimens by molecular methods, however, in addition to the high cost of such tests,