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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: mcaws@hotmail.com 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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250
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
255
256
257
258
259
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261
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
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278
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280
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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
FQ1094 R ∆WT3 + MUT3C D94G D94G Yes
FQ1095 R WT + MUT3A D94A D94A* Yes
FQ1150 R WT + MUT3C D94G D94N No
FQ1151 R ∆WT3 + MUT3C D94G D94G Yes
FQ1152 R WT + MUT3C D94G D94G* Yes
FQ1153 R ∆WT2 + MUT1 A90V A90V Yes
FQ1154 S WT WT NONE Yes
FQ1155 R ∆WT3 D94X D94Y Partial
FQ1156 R ∆WT2 + MUT1 A90V A90V Yes
FQ1157 S WT WT NONE Yes
FQ1158 R ∆WT2 + MUT1 A90V A90V Yes
FQ1159 R ∆WT3 + MUT3A D94A D94A Yes
FQ1160 S WT WT NONE Yes
FQ1161 S WT WT NONE Yes
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
FQ1284 S WT WT NONE Yes
FQ1286 R ∆WT3 + MUT3C D94G D94G Yes
FQ1288 S WT WT NONE Yes
FQ1289 R WT+ MUT1 + MUT3A A90V/D94A A90V*/D94A* Yes
FQ1290 R ∆WT3 + MUT3A D94A D94A Yes
FQ1291 R ∆WT3 + MUT3C D94G D94G Yes
FQ1292 R ∆WT3 + MUT3A + MUT3C D94A/D94G D94G*/D94A* Yes
FQ1293 R ∆WT3 + MUT3A D94A D94A Yes
FQ1294 S WT WT NONE Yes
FQ1295 R WT + MUT3C D94G D94G* Yes
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FQ1296 R ∆WT3 + MUT3C D94G D94G Yes
FQ1297 R ∆WT3 + MUT3B + MUT3C D94N/D94Y and
D94G D94G* Partial
FQ1298 R ∆WT3 + MUT3B + MUT3C D94N/D94Y and
D94G D94G* Partial
FQ1318 S WT WT NONE Yes
FQ1319 R WT + MUT3B D94N/D94Y NONE No
FQ1320 R ∆WT2 + MUT1 A90V A90V Yes
FQ1321 R ∆WT2 + MUT1 A90V A90V Yes
FQ1322 R ∆WT3 + MUT3C D94G D94G Yes
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
294
295
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296
297
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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