Rapid diagnosis of Extensively Drug Resistant tuberculosis using a ...

Rapid diagnosis of Extensively Drug Resistant tuberculosis using a Reverse Line 1

Blot Hybridization assay. 2

Kanchan Ajbani, 3

PhD student, Dept of Research, P.D. Hinduja National Hospital & Medical Research 4

Centre, Mumbai, India 5

Anjali Shetty, 6

Consultant Microbiologist, Dept of Microbiology, P.D. Hinduja National Hospital & 7

Medical Research Centre, Mumbai, India 8

Ajita Mehta Late*, (Passed away on 6th Feb 2010) 9



Camilla Rodrigues 12



Conflict of interest: There are no conflicts of interest for this manuscript. 15

Source of financial support: The study was funded by National 20 Health and 16

Education Society, P. D. Hinduja National hospital and Medical Research Centre, 17

Mumbai, India. 18

Address for Correspondence: 19

Dr. Camilla Rodrigues 20

Consultant Microbiologist, 21

Dept of Microbiology, Research Laboratories, P. D. Hinduja National Hospital and 22

Medical Research Centre, Veer Savarkar Marg, Mahim, Mumbai – 400016, India. 23

Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Clin. Microbiol. doi:10.1128/JCM.02511-10 JCM Accepts, published online ahead of print on 25 May 2011

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Tel: 0091 – 22 – 24447795 24

Fax No.; 099 – 22 – 24442318 25

E-mail address: [email protected] 26

Short title: Rapid diagnosis of XDR-TB 27

Article type: Major article 28

Key words: XDR-TB, fluoroquinolones, aminoglycosides, RLBH 29

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ABSTRACT 47

BACKGROUND 48

Drug resistance in Tuberculosis (TB) is a matter of grave concern for TB control 49

programs as there is currently no cure for some extensively drug resistant strains. There is 50

concern that this resistance could transmit, stressing the need for additional control 51

measures, as rapid diagnostic method, and newer drugs for treatment. We developed an 52

in house assay that can rapidly detect resistance to drugs involved in the definition of 53

extensively drug resistant tuberculosis (XDR-TB) directly from smear positive 54

specimens. 55

Methods 56

215 phenotypically XDR-TB and 50 pan susceptible isolates were analyzed using 57

the Reverse Line Blot Hybridization (RLBH) assay. The assay was also successfully 58

applied to 73 smear positive clinical specimens. 59

Results and discussion 60

The RLBH assay exhibited good sensitivity for the detection of resistance to 61

isoniazid (99%), rifampicin (99%), fluoroquinolones (95.3%) and second line 62

aminoglycosides (94.8%). The application of this assay on direct smear positive clinical 63

specimen revealed 93% concordance with the phenotypic drug susceptibility test (DST) 64

for the above mentioned drugs. Time to accurate DST results was significantly reduced to 65

3 days from weeks. 66

Conclusion: 67

This molecular assay is a highly accurate screening tool for XDR-TB, which achieves a 68

substantial reduction in diagnostic delays. 69


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INTRODUCTION 70

The estimate of tuberculosis (TB) infections continues to increase globally and in 71

1993 the WHO declared TB a global public health emergency, being the only disease so 72

far to warrant that designation. In many countries where the incidence of TB is high, the 73

major driving force of the epidemic is transmission. The foremost reason for this is that 74

there are prolonged delays from the onset of the TB disease in individuals to the time of 75

actual diagnosis. During this period an active TB case may infect numerous other people 76

thereby contributing to the increase of the epidemic [20]. The emergence of multidrug 77

resistant tuberculosis (MDR-TB), defined as resistance to both rifampicin (RIF) and 78

isoniazid (INH) which is a compounding factor for the control of the disease. Patients 79

harboring MDR-TB need alternative treatment regimens involving second-line drugs that 80

are more expensive, toxic, and less effective. Moreover, extensively Drug Resistant 81

tuberculosis (XDR-TB) (resistance to INH, RIF, fluoroquinolones (FQ) and any one 82

second line injectable i.e. kanamycin (KAN), amikacin (AM), capreomycin (CM)) 83

constitutes an emerging threat for the control of the disease over the period [4]. Accurate 84

diagnosis and initiation of early treatment are essential to reduce transmission and 85

improve the outcome for patients with tuberculosis. Timely identification of drug 86

resistance is especially important to avoid subjecting patients with resistance to 87

inadequate first line treatment for months while awaiting drug susceptibility testing 88

(DST) results. This exposure to inadequate therapy results in amplified drug resistance 89

[21]. 90

Drug resistance in M. tuberculosis is attributed to random mutations in the 91

mycobacterium genome. Mutations in the specific codons can therefore be exploited to 92


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rapidly detect drug resistance [8, 9, 12, 19, 22]. Resistance to first line drugs (INH, RIF) 93

is attributed to mutations in inhA and katG for INH and rpoB for RIF [18, 19, 22, 23]. 94

Resistance to fluoroquinolones and second line aminoglycosides is most frequently 95

associated with mutations in gyrA, gyrB and rrs respectively. Studies have demonstrated 96

that by targeting mutations in codon 90, 91 and 94 in gyrA approximately 70- 90% of all 97

fluoroquinolone resistance can be accurately predicted [2, 5, 17]. Similarly, mutations in 98

the rrs gene particularly the hot spot region from 1401- 1484 can accurately predict 99

resistance to second line aminoglycosides [10, 16]. The reverse line blot hybridization 100

(RLBH) [11, 24] assay is a probe based method where multiple oligonucleotide probes 101

carrying the mutant sequence and wild type sequence are immobilized on nitrocellulose 102

strips and hybridized with biotin labeled PCR products. This technique had been 103

successfully applied for the identification of mutations related to the resistance of 104

isoniazid, rifampicin and streptomycin at our centre. The assay was optimized to 105

additionally detect mutations conferring FQ and second line aminoglycosides resistance 106

(KAN, AM, CM), with the same hybridization conditions such that a single blot could be 107

used to detect resistance to the anti tubercular drugs included in the XDR-TB definition. 108

This enabled us to reduce the time to accurate DST. 109

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MATERIALS AND METHODS 118

Setting :- 119

The study was carried at a mycobacteriology laboratory at P. D. Hinduja National 120

Hospital & MRC (PDHNH) a tertiary care centre in Central Mumbai with a referral bias 121

towards complicated and non- responding cases. The study was approved by the internal 122

institutional review board (IRB). The laboratory receives about 10,000 cultures annually 123

with a culture positivity rate of 40%. Being private tertiary laboratory susceptibility is 124

only done on request wherein of the 3609 requests for DST from Jan 08-Dec 09, 65% of 125

isolates are MDR-TB and of these 9.1% are XDR-TB. 126

A] Bacterial isolates 127

Two hundred and fifteen consecutive XDR-TB isolates tested by Mycobacterial Growth 128

Indicator Tube (MGIT 960) TB system were collected over a period of 24 months (Jan 08 129

- Dec 09). 50 consecutive pan susceptible isolates tested by MGIT 960 were also tested 130

using RLBH assay. All the collected strains were stored at -700C. DNA extraction from 131

mycobacterial culture isolates was carried out by the cetyl trimethyl ammonium bromide- 132

sodium chloride (CTAB-NaCl) method [15]. 133

B] Clinical specimens 134

73 consecutive acid fast bacilli (AFB) smear positive clinical specimens (1+ and above) 135

collected over a period of 5 weeks were also tested by RLBH method. Of the 73 136

specimens, 31 specimens were 1+ on acid fast bacilli smear, 15 were 2+ and 27 were 3+ 137

AFB smear. Smear positive specimens were subjected to decontamination by NaLC- 138

NaOH [6] method and the sediment was used for DNA extraction that was carried out 139

using Qiagen DNA extraction kit. 140


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Amplification of all targets in multiplex PCR 141

Enlisted in table 1 are the PCR primer sequences wherein the primers for inhA, 142

katG and rpoB are the same as previous [14]. Novel primers were designed for 143

amplifying gyrA, gyrB and rrs that are also listed in table1. The cycling condition for the 144

touch down multiplex for the isolates and direct clinical specimen are provided as 145

supplemental material. 146

Designing the specific sequence probes for the fluoroquinolones and second line 147

aminoglycosides 148

The initial 150 XDR-TB isolates were amplified as single targets and sequenced 149

[1]. Based on the mutations specific amino labeled oligonucleotide probes for 150

fluoroquinolones and second line aminoglycosides were designed so that they can be 151

processed under same hybridization and washing conditions as the MDR blot and 152

covalently linked to a nylon membrane in parallel lines. 6 mutant probes and 2 wild type 153

probes were designed for gyrA and 1 wild and 1 mutant probe for gyrB. For 154

aminoglycosides the rrs region was targeted with 2 wild type probes and 3 mutant probes 155

correlating with high level resistance to KAN, AM and CM (Table 2). The uniqueness 156

and cross reactivity of the all newly designed and modified probes was analyzed with the 157

BLAST search (http://www.ncbi.nlm.nih.gov). 158

Hybridization with amplified products 159

Heat denatured PCR products were applied on the membrane in the miniblotter and 160

hybridized at 53 0C for 60 min. The membrane was washed twice with gentle shaking in 161

250 ml 2X SSPE/0.5% SDS for 10 min at 600C. The membrane was subsequently 162

incubated at 420C with 1:4000 diluted streptavidin-peroxidase conjugate in 2X 163


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SSPE/0.5% SDS for 60 min, washed twice with 250 ml 2X SSPE/0.5% SDS at 420C for 164

10 min, rinsed twice with 2X SSPE at room temperature (RT) for 5 min, and subjected to 165

luminescent detection of hybrids with enhanced chemiluminescence (ECL) detection 166

system followed by exposure to the ECL Hyperfilm (Amersham Biosciences). The 167

presence of a clearly visible black signal was considered as a positive hybridization 168

reaction. The results were easy to interpret (Figure 1). For reuse, the membranes were 169

stripped in 1% SDS solution at 800C (twice for 40 min) and rinsed in 20 mM EDTA, pH 170

8.0 at RT. The membrane can be stripped and re-used up to 7 times without 171

compromising the results. 172

Reproducibility of the RLBH assay was validated by testing 215 clinical XDR-TB 173

isolates (of these 150 were also sequenced and results were compared with sequencing 174

[1] as well as RLBH), 50 susceptible isolates and 73 smear positive clinical specimens 175

(+1 and above). The results of the clinical specimen were compared with phenotypic 176

susceptibility performed on MGIT. 177

Statistical analysis: 178

A 2 × 2 table was used to calculate Sensitivity, specificity of the RLBH assay in 179

comparison with the phenotypic DST i.e. MGIT. A p value (significance) of < 0.05 is 180

deemed statistically significant. To compare two tests, e.g. RLBH & MGIT, kappa was 181

used as a measure of agreement with 95% confidence interval (CI). 182

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RESULTS 188

Probe Designing: 189

Initial 150 XDR- TB isolates and 50 pan susceptible isolates were sent for sequencing for 190

screening mutations in gyrA, gyrB and rrs and based on the mutations seen; the 191

respective wild and mutant probes were designed for the fluoroquinolones and second 192

line aminoglycosides that were used for standardizing the RLBH assay conditions. 193

Evaluation of the RLBH assay: 194

The 150 sequenced isolates were then used as known controls for the respective 195

mutations. The latter 65 XDR- TB isolates were only screened by RLBH assay. 196

Performance of the assay: 197

A] Rifampicin resistance: 198

Of the total 215 XDR-TB and 50 pan susceptible isolates screened by RLBH assay, one 199

isolate showed RIF resistance by MGIT (phenotypic assay) but did not show any 200

mutation with the RLBH assay. The diagnostic sensitivity of the assay to detect RIF 201

resistance was calculated to be 99% with the specificity being 100% as none of the 50 202

pan susceptible isolates showed any mutation with RLBH. The kappa value was 0.99 203

(95% CI) indicating good agreement between RLBH and phenotypic susceptibility. 204

B] Isoniazid resistance: 205

Of the total 215 XDR-TB and 50 pan susceptible isolates, one isolate showed INH 206

resistance by MGIT but did not bind with any mutant probes on the RLBH membrane. 207

The diagnostic sensitivity of the assay to detect INH resistance was calculated to be 99% 208

with the specificity being 100% as none of the 50 pan susceptible isolates showed any 209


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mutation with RLBH. The kappa value was 0.99 (95% CI) indicating good agreement 210

between RLBH and phenotypic susceptibility. 211

C] Fluoroquinolone resistance: 212

Of the total 215 XDR-TB and 50 pan susceptible isolates, 10 isolates phenotypically 213

resistant to fluoroquinolones did not reveal any mutation by the RLBH assay. These 214

samples were sent for sequencing to check for the mutations in gyrA and gyrB. 215

Sequencing did not show any mutations, indicating mutations outside the targeted region 216

or other mechanisms i.e. efflux mechanism. The diagnostic sensitivity of the RLBH assay 217

to detect fluoroquinolone resistance was calculated to be 95.34% with a specificity of 218

100% as none of the 50 pan susceptible isolates showed any mutation with RLBH. The 219

kappa value was 0.89 (95% CI) indicating good agreement between RLBH and 220

phenotypic susceptibility. 221

D] Second line aminoglycosides (kanamycin & amikacin): 222

Of the 215 XDR-TB and 50 pan susceptible isolates 11 isolates were phenotypically 223

resistant to KAN and AM but did not reveal any mutation by the RLBH assay. These 224

samples were sent for sequencing for the rrs region to check for the mutations. 225

Sequencing did not show any mutations, indicating mutations outside the targeted region 226

or other mechanisms of resistance. The diagnostic sensitivity for the detection of 227

resistance to second line aminoglycosides by RLBH assay was 94.8% with specificity of 228

100%. The kappa value was 0.87 (95% CI) indicating good agreement between RLBH 229

and phenotypic susceptibility. 230

The various mutations seen in the 215 XDR-TB isolates are listed in table 3. Of all the 231

XDR-TB isolates 213/215 (99%) showed katG S315T substitution correlating with 232


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isoniazid resistance. 199/215 (92%) showed S531L substitution in the rpoB gene. The 233

mutations in the gyrA were seen on the 90th

, 91st and 94

th codon. The frequency of the 234

D94G substitution was seen in 101/215 (46%) of XDR-TB isolates. The other 235

substitutions seen on the 94th

codon were D94A and D94N seen in 31/215 (14%) and 236

27/215(12.5%) respectively. 106/215 XDR-TB isolates showed A1401G mutation in rrs 237

region and were phenotypically resistant to KAN and AMK but susceptible to CM. A 238

further 56/215 XDR-TB isolates with the same mutation showed phenotypic resistance to 239

KAN, AMK and CM. The G1484T mutation in rrs resulted in resistance to all three 240

second line injectables by phenotypically method. 241

Applicability on Direct smear positive clinical specimen: 242

The results obtained on isolates were promising but they were time consuming, thus the 243

assay was tested on direct clinical specimens. 73 specimens were tested, of these 8 244

culture proven to be mycobacteria other than tuberculosis (MOTT) showed only binding 245

with Mycobacterial complex and no binding with Mycobacterium tuberculosis probe on 246

the membrane indicating atypical infection which was in concordance with the 247

phenotypic MGIT method. There were 7 samples that showed inhibition with RLBH. The 248

percent of agreement for susceptibility testing between the phenotypic test and RLBH 249

assay was calculated to be 93% for all the targeted drugs. With the application of the 250

assay on direct clinical specimens the turn around time to results was reduced to 3 days 251

(assay takes about 4-5 hours but DNA extraction and PCR amplification totals the time to 252

results as 3 days) as compared to 2- 4 weeks as in case of the phenotypic liquid culture 253

system. 254


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The RLBH membrane was used 7 times without compromising the signal strength but the 255

strength of the signal was reduced from 8th

use thereby causing difficulty to accurately 256

read the results. 257

Time to DST 258

On an average the time to culture positivity for a smear 3+ positive is within 7 days [13] 259

and thereafter another 10-14 days for DST results. The total time to DST for a smear 3+ 260

positive specimen would therefore be 17- 21 days. This is increased with smear 2+ 261

positive specimen wherein time to identification is 8-10 days and thereafter another 10-14 262

days for DST total time being 18- 24 days. Similarly for smear 1+ positive specimen the 263

time to identification is 11 days and thereafter 10-14 days for DST the total time being 264

21- 25 days. In comparison the RLBH assay takes only 3 days from direct 1+ smear 265

positive specimen (including the DNA extraction, PCR amplification and assay 266

procedure) as compared to phenotypic method. 267

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DISCUSSION 278

The present study is a retrospective study that shows the rapidity of the RLBH assay 279

using a probe based technique over the phenotypic methods with a sensitivity for 280

detection of INH, RIF, FQ and second line aminoglycosides resistance as 99%, 99%, 281

95.34% and 94.8% respectively. These performance characteristics are indicative of 282

equivalence with conventional DST. In most developing countries, DST is usually 283

performed on solid media, such as Lowenstein Jensen wherein results are available after 284

8-18 weeks during which time many patients with MDR-TB or XDR-TB may have died, 285

transmitted their disease or both. Conventional DST for second-line drug testing is far 286

less standardized than first-line DST. The critical concentrations are not as clear cut, 287

often as that the minimum inhibitory concentration (MIC) values are close to the assumed 288

critical concentrations. The RLBH assay detects the presence of MTB with resistant 289

pattern to INH, RIF, FQ, KAN, AM and CM within a short span of 3 days directly from a 290

smear positive clinical specimen by detect point mutations present in rpoB, katG, inhA, 291

rrs, gyrA and gyrB. This could help clinicians in initiating appropriate therapy and 292

implement infection control measures to curb the spread of resistance. Compared with 293

PCR based sequencing and DNA microarray techniques, RLBH can be performed 294

without expensive devices. Though mutations to the first line drugs are well studied and 295

among the Indian XDR-TB isolates in a study conducted at Delhi [7] the key mutations 296

observed in the rpoB were S531L and S315T in katG which is similar to our findings. In 297

a previous study from this centre [14] done on MDR isolates revealed an array of point 298

mutations from 511- 531 in rpoB, which seems to have been selected out in case of 299

XDR-TB isolates to be Ser531Leu. Among rifampicin resistance isolates our findings are 300


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in concordance with other studies [1] that are dominated by Ser531Leu mutation among 301

the XDR-TB. There are limited mutation studies for the second line drugs, the FQ 302

resistance is correlated with mutations in the Quinolone Resistance Determining Region 303

(QRDR) in gyrA majorly and gyrB and 90% prediction of the FQ resistance was achieved 304

with mutations at 90th

and 94th

codon which is similar to the findings at Delhi [7] 305

XDR-TB isolates. Studies from out of India also show mutation in the QRDR region of 306

gyrA similar to the mutations observed among our XDR-TB isolates [1]. The resistance to 307

second line injectables can be predicted by the nucleotide changes in the 3’ part of the 308

16S rRNA (rrs) gene. With this assay 89% of the second line aminoglycoside resistance 309

could be detected. The key mutations were seen at A1401G and G1484T similar to 310

characterization of XDR-TB isolates from Delhi [7]. This study was dominated by the 311

A1401G and G1484T mutations in the rrs whereas other studies [1] have reported several 312

other point mutations in the rrs gene. 313

Commercial assays MTBDRplus and MTBDRsl (Hain Lifesciences) [8] is strip based 314

and uses two strips per patient that is priced approximately at USD 49 (separate strip for 315

MDR and FQ plus injectible) excluding the equipment cost. In contrast RLBH uses a 316

single membrane to identify mutations for XDR-TB and the membrane once prepared can 317

be stripped and reused for at least 7 times without affecting the signal strength. 40 318

samples along with positive control and negative control can be detected simultaneously 319

at one time and this reduces the cost of the assay making it suitable for application in 320

routine clinical laboratory in developing countries. Though the GeneXpert [3] also 321

evaluated at our centre is the new state of the art technology, it can only detect resistance 322

to rifampicin and the cost of the test at reduced price for India is USD17 excluding the 323


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cost of the instrument whereas our assay targets all the drugs involved in XDR-TB 324

definition. Thus, the present assay is an adequate tool to rapidly detect these point 325

mutations that confer resistance to INH, RIF, FQ, KAN, AM, and CM. Besides being 326

cost effective, this assay enables initiation of early treatment which could curb the spread 327

of resistant strains thus helping in control of tuberculosis. 328

The assay can detect only those mutations that are covered by the wild type or 329

mutant probe. Novel mutations with locations other than the probe region or outside the 330

amplified region would be a shortcoming. But the system is an open system wherein 331

newer probes can be added. Though molecular assays have a short turn around time and are 332

the state of the art they cannot completely replace the conventional methods as there are 333

multiple genes involved in resistance and the molecular assays target only known mutations. 334

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CONCLUSION 348

In this study we have attempted to characterize the genetic mechanism of the XDR 349

isolates wherein we saw good concordance with the phenotypic resistance in case of 350

isoniazid, rifampicin and fluoroquinolones and second line aminoglycosides. As XDR-TB is 351

emerging as a highly threatening pathogen in a pervasive association with the acquired 352

immunodeficiency syndrome epidemic, rapid tests to promptly identify resistance to first- 353

and second-line antitubercular agents are urgently required. It has been observed that point 354

mutations in rpoB, inhA, katG, gyrA, gyrB and rrs can accurately predict resistance to these 355

drugs. RLBH assay proposes to be an adequate tool for direct analysis of XDR-TB from 356

clinical specimens thus fulfilling many attributes of an ideal TB diagnostic test as proposed 357

by the WHO for developing countries. 358

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Table 1: Primer sequences for inhA, katG, rpoB, gyrA, gyrB and rrs and cycling 374

condition. 375

Target region Primer sequences

inhA TB 92M- 5’ CCT CGC TGC CCA GAA AGG GA TCC -3’

TB 93M- 5’ biotin-CCG GGT TTC CTC CGG T-3’

katG KGF-biotin-5 AGA GCT CGT ATG CCG GA-3’

KGR-5’ GCG AAT GAC CTT GCG CAG ATC-3’

rpoB Rpo F - 5’-TCA AGG AGA AGC GCT ACG ACC TGG-3

Rpo R- 5’ biotin- GGG TCT CGA TCG GGC ACA T-3’

gyrA gyAF 5’CGA ACC GGT TGA CAT CGA GCA GGA G 3’

gyA R 5’ biotin CAG CAT CTC CAT CGC CAA CG 3’

gyrB gyrB F 5’ GAA GCC AAC CCC ACC GAC GCG A 3’

gyrB R (5’ biotin CTG CGC TGC CAC TTG AGT TTG 3’)

rrs rrs F 5’CGT TCC CTT GTG GCC TGT GTG CAG 3’

rrs R 5’ biotin GTT GGG GCG TTT TCG TGG TGC 3’

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Table 2: Probe sequences for fluoroquinolone and aminoglycosides 394

No Probe Description Sequences

gyrA gene

1 90th codon Wild type

5'- ACC ACC CGC ACG GCG ACG CGT C-3'

2 90th codon Mutant type

5'- ACCACC CGC ACG GCG ACG TGT C-3'

3 91st codon Mutant type

5'-ACC CGC ACG GCG ACG CGC C-3'

4 94th codon Wild type

5'-CG ATC TAC GAC AGC CTG GTG-3'

5 94th codon Mutant type I

5'-CG ATC TAC GCC AGC CTG GTG-3'

6 94th codon Mutant type II

5'-CG ATC TAC GGC AGC CTG GTG-3'

7 94th codon Mutant type III

5’- CGA TCT ACT ACA CCC TGG TG

8 94th codon Mutant type IV

5’ CGA TCT ACA ACA CCC TGG TG- 3’

9 95th codon Polymorphism

5'-CGA TCT ACG ACA CCC TGG TG-3'

gyrB gene

10 510 Wild type

5’ GTG CTA AAG AAC ACC GAA G 3’

11 510 Mutant type

5’ CGA CCG GGT GCT AAA GAG CA 3’

rrs gene

12 1401 Wild type

5'-ACC GCC CGT CAC GTC ATG AA-3'

13 1401 Mutant type

5'-ACC GCC CGT CGC GTC ATG AA-3'

14 1402 Mutant type

5’ ACC GCC CGT CAA GTC ATG AA 3

15 1484 Wild type

5'-CGG CGA TTG GGA CGA AGT C-3'

16 1484 Mutant type

5'-CGG CGA TTG GGA CTA AGT C-3'

395

396

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398

399

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402

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Table 3: Mutations seen in XDR patients 406

Drug Gene Mutation seen

Amino

acid

change

No. of

isolates

Isoniazid katG G315C S315T 213

Isoniazid inhA+katG C-15T 44

Rifampicin rpoB C531T S531L 199

Rifampicin rpoB A526G H526Y 7

Rifampicin rpoB G516T +

A526G

D516 T 2

Rifampicin rpoB A516T +

A526G

D516E 2

Rifampicin rpoB A516T D516E 2

Rifampicin rpoB G516T D516T 1

Fluoroquinolone i.e. ofloxacin gyrA C90T+ C95G

C90T

A90V 23

21

Fluoroquinolones ofloxacin &

moxifloxacin

gyrA T91C S91P 2

Fluoroquinolone ofloxacin &

moxifloxacin

gyrA A94G+ C95G

A94G

D94G

83

18

Fluoroquinolones ofloxacin &

moxifloxacin

gyrA A94C + C95G

A94C

D94A

19

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Fluoroquinolone ofloxacin &

moxifloxacin

gyrA G94A + C95G

G94A

D94N

19

8

Aminoglycoside i.e.

kanamycin, amikacin

rrs

A1401G

106

Aminoglycoside i.e.

kanamycin, amikacin,

capreomycin

rrs

A1401G

56

Aminoglycoside i.e.

kanamycin, amikacin,

capreomycin

rrs G1484T 42

407

408

409

410

411

412

413

414

415

416

417

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Figure 1: RLBH performed for rpoB, katG, inhA, gyrA, gyrB and rrs. 419

420

421

422 423

All the different probes are blotted vertically and patient’s sample (PCR product) are applied horizontally. 424

Probe in lane no 1 is common probe for all mycobacteria then there is probe for MTB complex. Then 5 425

wild type probes for rpoB gene to check rifampicin susceptible cases and 16 mutant probes for detecting 426

rifampicin resistance. Similarly we targeted inhA wild type and mutant probe for promoter region followed 427

by katG gene wild type and mutant probes for codons 315. This is further followed by the wild and mutant 428

probes for the gyrA mutations in codon 90, 91, 94 and 95. Similarly we also targeted the gyrB 510 region 429

with wild type and mutant probe. For the second line aminoglycoside rrs region 1401, 1402, 1484 were 430

used. To illustrate an example, patient sample marked by the arrow has mycobacterial infection due to M. 431

1 2 3 4 5 6 7 8 9 10 1112 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 363738 39 40 41 4243

1 - MYC (Common for

Mycobacteria)

2 – MTB (M.

tuberculosis) complex

3 - RIF WT1 (codons

509 -514)

4 - RIF WT2 (codons

514 – 520)

5 - RIF WT3 (codons

521 -525)

6 - RIF WT4 (codons

524-529)

7 - RIF WT5 (codons

530-534)

8 - 533 CCG

9 - 531 TTG

10- 531 TGG

11 - 526 GAC

12 - 526 TAC

13 - 526 CGC

14- 526 TGC

15 - 526 AAC

16- 522 TTG

17 - 522 TGG

18 - Del 516

19 - 516 TAC

20 - 516 GAC

21 - 516 GTC

22 - 516 GGC

23- 513 CCA

24 – inh A wt

25- inhA mt

26-katG 315wt

27- katG 315 mt

28- gyA90 wt

29- gyA 90mt

30- gyA 91 mt

31- gyA94 wt

32- gyA 94mt AAC

33-gyA94mt GCC

34- gyA 94 mt GGC

35- gyA94 mt TAC

36- gyA 95 mt

37- gyB wt

38- gyBmt

39-rrs 1401 wt

40-rrs 1401 mt

41-rrs 1402 mt

42-rrs 1484 wt

43-rrs 1484 mt


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tuberculosis complex and shows binding with probes 3-6 which are the rpoB wild type codon 509- 529. It 432

does not show binding with probe 7 but shows binding with probe 9 indicating RIF resistance due to 531 433

TTG mutation. Further on the same sample shows binding with probe 24 which is inhA promoter wild type 434

and shows binding with probe 27 which is katG 315 mutant probe, thereby indicating INH resistance due to 435

katG mutation. Same sample shows binding with 28th

, 31st, 36th

, 37th

, 39th

and 42nd

probe which correspond 436

to gyrA 90 wild type, gyrA 94 wild type, gyrA 95 polymorphism, gyrB wild type, rrs 1401 wild type and rrs 437

1484 wild type respectively, thus giving the result for that particular sample to be resistant to INH, RIF and 438

susceptible to FQ and KAN, AM, CM. 439

440

Acknowledgement: 441

We sincerely thank the National Health Education Society, P D Hinduja National hospital 442

and Medical Research Centre for funding this study and also the entire staff of the 443

Mycobacteriology Department at P D Hinduja National hospital and Medical Research 444

Centre. I would also like to thank Dr. Mankeshwar for his esteemed guidance in the 445

statistical analysis. 446

447

448

449

450

451

452

453

454

455

456


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References: 457

1. Ajbani K, Rodrigues C, Shenai S, Mehta A. Mutation detection and accurate diagnosis 458

of Extensively Drug Resistant Tuberculosis: Report from a tertiary care centre in India. 459

Journal of Clinical Microbiology. 2011; 49(4): 1588- 90. 460

2. Alangaden G, Manavathu E, Vakulenko S, Zvonok N, Lerner S. 461

Characterization of fluoroquinolone resistant mutant strains of Mycobacterium 462

tuberculosis selected in the laboratory and isolated from patients. Antimicrobial 463

agents and Chemotherapy. 1995; 39: 1700- 1703. 464

3. Boehme CC, Nabeta P, Hillemann D, Nicol MP, Shenai S,Krapp F et al. Rapid 465

molecular detection of tuberculosis and Rifampicin resistance. The New England Journal 466

of Medicine. 2010; 363 (11): 1005- 15. 467

4. Gandhi N, Nunn P, Dheda K, Schaaf H, et al. Multidrug resistant and extensively 468

drug resistant tuberculosis: a threat to global control of tuberculosis. Lancet. 2010; 469

375: 1830- 43. 470

5. Ginsburg. A, Grosset. J, Bishai. W. Fluoroquinolones, tuberculosis, and resistance. 471

Lancet Infect Dis, 2003; 3; 432 – 442. 472

6. Kent and Kubina GP. Public health mycobacteriology a guide for level III laboratory. 473

Atlanta, CA: U.S. Department of Health and Human Services, Public health services. 474

Center for disease control. 1985. P 64-8. 475

7. Khanna A, Raj V S, Tarai B, Sood R, et al. Emergence of Extensive Drug resistant 476

(XDR) Mycobacterium tuberculosis and molecular characterization of these clinical 477

isolates from Delhi region, India. Antimicrob. Agents Chemother. 2010; 54 (11): 4789- 478

4793. 479


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/

8. Kiet V S, Lan NT M, An D D, Dung N et al. Evaluation of the MTB DRsl test for 480

detection of second line drug resistance in Mycobacterium tuberculosis. Journal of 481

Clinical Microbiology. 2010; 48: 2934- 39. 482

9. Martin- Casabona N, Mimo DX, Gonzalez T, Rossello J, Arcalis L. Rapid method 483

for testing susceptibility of Mycobacterium tuberculosis by using DNA probes. 484

Journal of Clinical Microbiology. 1997; 35: 2521- 2525. 485

10. Maus C, Plikaytis B, Shinnick T. Molecular analysis of cross resistance to Capreomycin, 486

Kanamycin, Amikacin and Viomycin in Mycobacterium tuberculosis. Antimicrobial 487

agents and Chemotherapy. 2005; 49: 319-397. 488

11. Mokrousov I, Bhanu VN, Suffys PN et al. Multicentre evaluation of reverse line 489

blot assay for detection of drug resistance in Mycobacterium tuberculosis clinical 490

isolates. Journal of Microbiological Methods. 2004; 57:323-335. 491

12. Musser JM. Antimycobacterial agent resistance in mycobacteria: molecular genetic 492

insights. Clin Microbiol Rev. 1995; 8:496-514. 493

13. Rodrigues C, Shenai S, Sadani M, Sukhadia N, et al. Evaluation of the BACTEC MGIT 494

960 system for the recovery and identification of Mycobacterium tuberculosis complex in 495

a high throughput tertiary care centre. IJMM. 2009; 27(3): 217- 21. 496

14. Shenai. S, Rodrigues. C, Mehta. A. Rapid speciation of 15 clinically relevant 497

mycobacteria with simultaneous detection of resistance to rifampin, isoniazid, and 498

streptomycin in Mycobacterium tuberculosis. International Journal of Infectious 499

Diseases, 2009; 13:46-58. 500


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/

15. Somerville W, Thibert L, Schwartzman K, Behr M. Extraction of Mycobacterium 501

tuberculosis DNA: a question of containment. Journal of Clinical Microbiology. 502

2005; 43: 2996- 2997. 503

16. Suzuki et al. Detection of Kanamycin resistant Mycobacterium tuberculosis by 504

identifying mutations in the 16S rRNA gene. Journal of Clinical Microbiology. 1998; 505

36:1220- 1225. 506

17. Takiff H, Salazar L, Guerrero C, Philipp W, Huang W, et al. Cloning and 507

nucleotide Sequence of Mycobacterium tuberculosis gyrA and gyrB Genes and 508

detection of quinolone resistance mutations. Antimicrobial agents and 509

Chemotherapy. 1994; 38: 773- 780. 510

18. Telenti A, Honore N, Bernasconi C, March J, Ortega A, Heym B, Takiff HE and 511

Cole ST. Genotypic assessment of isoniazid and rifampicin resistance in 512

Mycobacterium tuberculosis: a blind study at reference laboratory level. Journal of 513

Clinical Microbiology. 1997b; 35:719-723. 514

19. Torres MJ, Criado A, Palomares JC and Aznar J. Use of real-time PCR and 515

fluorimetry for rapid detection of rifampin and isoniazid resistance-associated 516

mutations in Mycobacterium tuberculosis. Journal of Clinical Microbiology. 2000; 517

38:3194-3199. 518

20. Uys P, Warren R, Helden P. A threshold value for the time delay to TB diagnosis. 519

PLOS One. 2007; 2(8): 1- 5. 520

21. Yagui M, Perales M, Asencios L, Vergara L et al. Timely diagnosis of MDR- TB 521

under program conditions: is rapid drug susceptibility testing sufficient? Int J Tuberc 522

Lung Dis. 2006; 10(8): 838- 843. 523


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/

22. Yam WC, Tam DM, Leung CC, Tong HI, Chan KH, Leung ET, Wong KC, Yew 524

WW ,et al. Direct detection of rifampicin resistant Mycobacterium tuberculosis in 525

respiratory specimens by PCR-DNA sequencing. Journal of Clinical Microbiology. 526

2004; 42, 4438-4443. 527

23. Zhang M, Yue J, Yang YP, Zhang HM, Lei JQ, Jin RI, Zhang SL and Wang 528

HH. Detection of mutations associated with isoniazid resistance in Mycobacterium 529

tuberculosis isolates from China. Journal of Clinical Microbiology.2005; 43: 5477-530

5482. 531

24. Zhang Y, Coyne MY, Will SG, Levenson CH and Kawasaki ES. Single base 532

mutational analysis of cancer and genetic diseases using membrane bound modified 533

oligonucleotides. Nucleic Acids Res. 2000; 19:3929:3933. 534

535

536

537

538

539

540

541

542

543

544

545

546


http://jcm.asm

.org/D

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