J. Clin. Microbiol. Doi:10.1128/JCM.01249-17 Copyright © 2017 Havlicek Et Al

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JCM Accepted Manuscript Posted Online 6 December 2017 J. Clin. Microbiol. doi:10.1128/JCM.01249-17 Copyright © 2017 Havlicek et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

1 Title: Rapid Microarray-based Detection of Rifampicin-, Isoniazid- and Fluoroquinolone

2 Resistance in Mycobacterium tuberculosis using a Single Cartridge

4 Running title: Melting curve analysis for resistance detection

5 Downloaded from 6 Authors: Juliane Havliceka, Beatrice Dachsela, Peter Slickersa, Sönke Andresb, Patrick

7 Beckertc,d, Silke Feuerriegelc,d, Stefan Niemannc,d, Matthias Merkerc,d#*, Ines Labuggera*

9 * These authors contributed equally to this work. http://jcm.asm.org/

11 Affiliations:

12 a Alere Technologies GmbH, Jena, Germany

13 b National Reference Center for Mycobacteria, Research Center Borstel, Borstel, on January 3, 2018 by guest

14 Germany

15 c Molecular and Experimental Mycobacteriology, Research Center Borstel, Germany

16 d German Center for Infection Research, Partner site Hamburg-Lübeck-Borstel,

17 Germany

19 Corresponding author: Dr. Matthias Merker, email: [email protected]

20 ABSTRACT

21 Rapid and robust identification of mutations in Mycobacterium tuberculosis complex

22 (MTBC) strains mediating multidrug and extensively drug-resistant (M/XDR) phenotypes

23 is crucial to combat the MDR tuberculosis (TB) epidemic. Currently available molecular

24 TB drug susceptibility tests are either restricted to single targets/drugs (i.e. Xpert Downloaded from 25 MTB/RIF) or aid the risk of cross-contaminations due to the design limitations of the

26 open platform (i.e. line probe assays).

27 With a good understanding of the technical and commercial boundaries we designed a

28 test cartridge with an introduction of dried reagents, based on an oligonucleotide array, http://jcm.asm.org/

29 which has the ability to identify MTBC strains resistant to isoniazid, rifampicin and the

30 fluoroquinolones. The melting curve assay interrogates in a closed cartridge system

31 within 90 minutes 43 different mutations in the rifampicin resistance determining region

32 (RRDR) of rpoB, rpoB codon 572, katG codon 315, the inhA promoter region, and the on January 3, 2018 by guest

33 quinolone resistance determining region (QRDR) of gyrA. The assay performance was

34 evaluated with 265 clinical MTBC isolates from Swaziland including MDR/XDR, non-

35 MDR, and fully susceptible isolates from a drug resistance survey performed in

36 2009/2010. In 99.5% of the cases, the results were consistent with the previously

37 acquired data utilizing Sanger sequencing.

38 By means of the closed cartridge system in combination with the battery-powered

39 AlereTM q analyzer and with the potential to extent the current gene target panel the

40 assay could serve as a rapid and robust point-of-care test in settings lacking

41 comprehensive molecular laboratory infrastructure to differentiate MDR and non-MDR

42 TB-patients and to assist clinicians in early treatment decisions.

45 INTRODUCTION

46 With an increase of more than ten million new cases of infections every year and

47 estimated 1.8 million deaths in 2015 tuberculosis (TB) remains one of the central health

48 problems worldwide. One major cause for the global TB situation and challenges in Downloaded from 49 disease elimination is the increasing occurrence of M. tuberculosis complex (MTBC)

50 bacteria resistant to antibiotics. In 2015 alone there were an estimated 480,000 new

51 cases and approximately 250,000 deaths from multidrug-resistant TB (MDR-TB),

52 defined as resistance towards rifampicin and isoniazid. The average proportion of MDR- http://jcm.asm.org/

53 TB cases with additional resistance to at least one fluoroquinolone and a second-line

54 injectable agent (extensively drug-resistant TB, XDR-TB) was 9.5%, similar to estimates

55 in previous years [1, 2]. The increasing occurrence of drug-resistant MTBC strains is

56 associated, inter alia, to a diagnostic gap in many high incidence regions [1]. It is on January 3, 2018 by guest

57 projected that only one out of three MDR-TB patients worldwide is detected and

58 treatment success rates for MDR-TB patients are dramatically low at 48% [1]. A

59 successful treatment outcome is essentially associated with an early administration of

60 adequate drugs and therefore with an individualized drug susceptibility testing [3-5].

61 Culture-based methods are still considered as gold standard for the diagnosis and

62 resistance detection of MTBC strains is not enabling an early treatment decision.

63 Additionally these methods are heavily burdened and require a complex laboratory

64 infrastructure [6, 7]. Molecular tests become additionally important because

65 subsequently the molecular mechanisms of MTBC drug resistance are well

66 characterized, e.g. defined regions in the genes rpoB coding for a RNA polymerase

67 subunit, katG coding for a catalase, the inhA promoter region controlling RNA-transcripts

68 for an enoyl-acyl carrier protein involved in fatty acid biosynthesis, and gyrA coding for a

69 subunit of the DNA gyrase. Approximately 97% of rifampicin resistant MTBC isolates

70 harbor mutations in the 81-bp rifampicin resistance determining region (RRDR) of the

71 rpoB gene (codons 507-533), wherein the most frequent mutations affect the codons

72 516, 526 and 531 (referring to E. coli nomenclature) [8-10]. Several studies showed that Downloaded from 73 mutations at amino acid position 572 in the rpoB gene are also a contributor for

74 rifampicin resistance in MTBC strains [11-13] and are not interrogated by currently

75 available molecular drug susceptibility tests (DSTs). Isoniazid resistant MTBC strains

76 are 70-90% associated with mutations in codon 315 of the katG gene or in the inhA http://jcm.asm.org/

77 promoter region (positions -8 and -15) [8-10]. Mutations in the quinolone resistance

78 determining region (QRDR) of gyrA (codons 88-94) are associated with resistance

79 towards fluoroquinolones, the backbone of MDR-TB treatment, in more than 80% of

80 MTBC clinical isolates [10, 14, 15]. on January 3, 2018 by guest

81 Over the past decade molecular tests (e.g. Line Probe assays or Xpert MTB/RIF test)

82 have been developed to identify resistance related mutations [16]. However, these tests

83 are either restricted by the investigation of a single resistance target or the risk of

84 contamination due to the open platform. Within this manuscript, we describe a rapid,

85 sensitive and simple test which is performed with a single prototype cartridge and the

86 AlereTM q analyzer. The original functional requirement of the AlereTM q platform was for

87 the detection of HIV using the competitive reporter monitored amplification (CMA); the

88 competitive hybridization of Cy5 labeled reporter oligonucleotides onto a probe array

89 [17]. Now a new method based on melting curve analysis was implemented. The special

90 feature of the here described assay is that the fluorescence measurement is performed

91 on the solid phase. On this account just one fluorescence dye is needed for analysis

92 compared to other previous published melting curve assays [18-22]. This enables the

93 simultaneous detection of a great number of different mutations which are associated

94 with resistance against rifampicin, isoniazid and fluoroquinolones.

96 RESULTS Downloaded from 97 Our melting curve assay comprised of two parts, the amplification of the selected target

98 regions rpoB RRDR, rpoB 572, katG 315, gyrA QRDR and the inhA promoter region by

99 a multiplex PCR and the detection of mutations by a melting curve analysis. The

100 efficiency of the multiplex amplification was determined by a standard curve [50]. It was http://jcm.asm.org/

101 calculated to be excellent for all targets as judged by efficiencies of ≥ 0.97 (data not

102 shown). Melting curve analysis was done with a DNA array carrying several immobilized

103 hybridization probes. Initially several different variants of probe sequences were

104 screened to find pairs which yield reliable discrimination between wildtype and mutant on January 3, 2018 by guest

105 strains (data not shown).

106

107 Sensitivity and Specificity

108 The sensitivity was determined using genomic DNA from M. tuberculosis reference

109 strain H37Rv in a concentration range of 5 to 60 copies per reaction. An increase in the

110 detection rate was observed with increasing DNA concentrations. The detection limit of

111 the melting curve assay was 24 copies per reaction (95% confidence interval 21-27

112 copies per reaction) (FIG. 1).

113 In addition to the resistance targets a probe for highly specific detection of MTBC strains

114 was applied. DNA isolated from different non-tuberculosis mycobacteria such as M.

115 intracellulare, M. malmoense, M. asiaticum, M. fortuitum and M. marinum was tested. No

116 unspecific reaction could be observed. Contrary, other species of the MTBC like M.

117 bovis BCG showed signals comparable to M. tuberculosis (FIG. 2).

118

119 Detection of mutations in rpoB, katG, gyrA and in the inhA promoter region

120 A set of 30 different sequence-verified MTBC isolates were analyzed. Overall these Downloaded from 121 isolates are carrying 18 mutations in the rpoB RRDR, one mutation in codon rpoB 572,

122 five different SNPs in codon katG 315, 9 mutations in the gyrA QRDR and two SNPs in

123 the inhA promoter region (Table S1). The results of all isolates are summarized in Table

124 1. If genomic wild type DNA was tested on all probes a discrimination factor < 1 was http://jcm.asm.org/

125 observed, i.e. based on the ratio of the temperature values of the wild type and the

126 mutant probes at a signal level of 0.4, whereas a mutation within the respective target

127 region could be identified by a discrimination factor > 1 at its corresponding probe. In

128 addition there were also discrimination factors > 1 at non-corresponding probes (Table on January 3, 2018 by guest

129 1); however the mutation could be correctly identified as the highest value of the

130 discrimination factor was always measured at the matching probe. With exception of the

131 mutations rpoB Leu511Arg in isolate 853/07 and rpoB Ile569Val in isolate 10523/05 all

132 mutations were identified correctly.

133

134

135

136 Clinical isolates

137 To evaluate the clinical value of our melting curve assay 265 isolates of different MTBC

138 strains from Swaziland were investigated (Table 2). One hundred forty four of the tested

139 isolates showed a wild type genotype within the rpoB RRDR as well as in codon 572. All

140 these isolates could be correctly identified. The other 121 isolates carried mutations in

141 these targets determining rifampicin resistance. Rifampicin resistance mediating

142 mutations could be identified in 99.2% (120/121) of the samples. One isolate exhibiting a

143 rpoB deletion (del 513-514) was misclassified by our assay as mutant carrying

144 Ser513Leu: C(A/T)A. For three isolates with linked mutations, i.e. Asp516Phe: Downloaded from 145 (G/T)(A/C)C and Asn518Asp: (A/G)AC, we could detect the variant in codon 516 and

146 observed a missing mutant probe for codon 518. The most common mutations in our

147 strain collection were Ser531Leu: T(C/T)G (44.63%) and Ile572Phe: (A/T)TC (33.06%).

148 Additionally the following mutations were detected: His526Asp: (C/G)AC (4.96%), http://jcm.asm.org/

149 His526Leu: C(A/T)C (4.96%), His526Tyr: (C/T)AC (4.96%), Asp516Phe: (G/T)(A/C)C

150 (3.31%), Asp516Tyr: (G/T)AC (0.83%), Asp516Val: G(A/T)C (0.83%) and Ser531Trp:

151 T(C/G)G (0.83%).

152 Isoniazid resistance-associated mutations in the target region of katG and in the inhA on January 3, 2018 by guest

153 promoter region were identified in 99.2% (122/123) of all isolates. One isolate with a rare

154 mutation in katG (Met275Ile) that is not interrogated by our assay was not identified.

155 Detected mutations were katG Ser315Thr: A(G/C)C (91.06%) and Ser315Gly: (A/G)GC

156 (4.07%) and -15C→T (26.83%) in the inhA promoter region.

157 The gene gyrA is the major target for mutations mediating resistance against

158 fluoroquinolones. Six of the investigated isolates showed such mutations: Asp94Gly:

159 G(A/G)C (50.00%), Asp94Tyr: (33.33%) and Asp94Asn: (G/A)AC (16.67%) which were

160 all correctly identified. The phylogenetic SNP gyrA Ser95Thr: A(G/C)C (specific for

161 clinical isolates phylogenetically related to the H37Rv reference strain) was correctly

162 identified in 242/242 (100%) of the isolates.

163 Overall, our assay detected for 963/968 (99.5%) known resistance targets (four gene

164 regions in 265 isolates) the correct wild type or mutant sequence, as previously

165 confirmed by Sanger sequencing.

166

167 Assay performance using crude culture material Downloaded from 168 The potential of our melting curve assay for use in a non-laboratory environment was

169 characterized by the investigation of crude culture material. For this purpose four

170 different MTBC strains carrying six different mutations in the genes rpoB, katG, gyrA and

171 in the inhA promoter region were selected. Crude cell culture extracts as well as http://jcm.asm.org/

172 genomic DNA of these strains were tested in comparison with the melting curve assay.

173 All mutations could be clearly identified and no difference in assay performance between

174 genomic DNA and crude culture material was observed with regard to their calculated

175 discrimination factors (Table S2). on January 3, 2018 by guest

176

177 DISCUSSION

178 The melting curve assay utilizing the AlereTM q analyzer platform can be performed with

179 patient derived (sub-cultured) MTBC DNA isolates as well as with crude cell material.

180 The test is able to rapidly and reliably detect clinical relevant mutations that mediate

181 resistance to three major anti-TB drugs, i.e. rifampicin, isoniazid and fluoroquinolones.

182 Rifampicin resistance is mainly caused by mutations in the rpoB RRDR and in the rpoB

183 codon 572 [8-13]. Until now more than 50 mutations have been identified within the rpoB

184 RRDR. Mutations in codon 526 and 531 are well-known to be markers for high-level

185 resistance to rifampicin and their detection is therefore of high importance in predicting

186 the resistance profile [52-54]. In addition the molecular detection of the mutations

187 Leu533Pro: C(T/C)G, Leu511Pro: C(T/C)G, Asp516Tyr: (G/T)AC and His526Leu:

188 C(A/T)C have also been described to be linked to observed treatment failures using

189 standard regimens with 600 mg/day rifampicin [55, 56]. Individual RRDR mutations can

190 confer either low- or high-level resistance depending on the amino acid change [57] and

191 are further controversially discussed with regard to the possibilities of high-dose Downloaded from 192 rifampicin treatment or the substitution with rifabutin. This highlights its importance of

193 differentiating between the various resistance markers for more individualized treatment

194 options that could improve treatment outcomes [58, 59]. Furthermore we implemented

195 corresponding probes for the detection of the mutation Phe572Ile: (A/T)TC due to its http://jcm.asm.org/

196 resistance shown in recent studies in relation to rifampicin [11-13].

197 Studies have also shown that resistance to isoniazid is very common in high-TB burden

198 countries, and may not necessarily carry co-resistance to rifampicin [54]. Thus there is a

199 need for testing isoniazid resistance in MTBC strains [60, 61]. For the proof of isoniazid on January 3, 2018 by guest

200 resistance, we applied probes representing the wild type and mutant variants within

201 katG codon 315 as well as at the positions -15 and -8 in the inhA promoter region.

202 Specific mutations in the katG gene are associated with a high-level isoniazid

203 resistance, whereas mutations in the inhA promoter region, especially -15C→T, are the

204 cause for a high-level resistance to ethionamide and prothionamide and low-level

205 resistance to isoniazid [Error! Reference source not found.]. Thus the mutation -

206 15C→T could also lead to an exclusion of ethionamide and prothionamide from an

207 MDR-TB treatment regimen.

208 Resistance to fluoroquinolones is mainly caused by mutations in the gyrA gene [10, 14,

209 15]. The mutations Ala90Val: G(C/T)G, Asp94Asn: (G/A)AC und Asp94Val: G(A/T)C

210 were observed to be related to high-level resistance [63, 64]. In addition several

211 mutations in the gyrB gene have been detected, however not frequently and usually in

212 association with gyrA mutations [9, 47, 65-68]. Fluoroquinolones play an important role

213 and are widely used in the treatment of MDR-TB due to their relatively low side-effects

214 and their high bactericidal activity compared to other TB drugs [10]. Currently new

215 generations of fluoroquinolones are evaluated in clinical trials with the effort to shorten Downloaded from 216 long-term treatment and the adjunctive burden for TB patients [69, 70]. All in all our

217 assay can be used for the detection of the most important mutations which are

218 associated with resistance to rifampicin and isoniazid as well as to fluoroquinolones.

219 This could possibly help to decide on a more effective treatment option by individualized http://jcm.asm.org/

220 mutation assessment. The prototype cartridge was designed with the focus on MDR-TB

221 detection and cannot detect resistance to second-line injectable drugs, however it will be

222 easy to adjust the assay with the respective probes on the array. The same is true in the

223 event that the focus swings to other mutations or drugs, yet the maximum probe on January 3, 2018 by guest

224 number has not been achieved, and an extension of the analysis spectrum is possible.

225 Overall, we tested 30 genomic DNAs from different MTBC strains for the initial

226 performance evaluation of the melting curve assay. With one exception all isolates

227 carrying mutations in one of the respective target regions were tested correctly. The

228 isolate 853/07 with the uncommon mutation Leu511Arg: C(T/G)G could not be identified

229 as the corresponding mutation probe was not applied on the array. Furthermore the

230 rpoB mutation Ile569Val: (A/G)TC outside the rpoB RRDR (isolate 10523/05) was not

231 detected which is likewise a rare mutation and in most cases connected to mutations in

232 the rpoB RRDR [71].

233 For a performance test in a high-incidence setting we tested 265 isolates from a recent

234 drug resistance survey in Swaziland, a country with one of the highest burdens of HIV

235 and TB globally [1]. Sanchez-Padilla et al. observed among 116 multidrug-resistant

236 strains, included in the same drug resistance survey from 2009/2010, 38 (32.8%) strains

237 that carried the rpoB Ile572Phe: (A/T)TC mutation, which confers resistance to

238 rifampicin [23, 72]. Although this mutation was previously reported with low frequency in

239 clinical isolates in Hong Kong and Australia [73], we decided to implement the detection Downloaded from 240 of the rpoB Ile572Phe: (A/T)TC mutation in our melting curve assay. Overall, resistance

241 mediating mutations, previously identified by Sanger sequencing, were confirmed by our

242 assay in all fluoroquinolones resistant isolates, and in 99.2% of the isoniazid and

243 rifampicin resistant isolates. The few exceptions were the mutation rpoB del 513-514 in http://jcm.asm.org/

244 one isolate that was misinterpreted as Ser513Leu: C(A/T)A. Three isolates carrying

245 linked mutations in the rpoB RRDR in codons 516 and 518, were identified with the

246 correct mutation in codon 516 and a missing mutant probe in codon 518. The mutation

247 rpoB Ala633Cys: (G/T)(C/G)C of the isolate 4723/09 is not associated with resistanceIn on January 3, 2018 by guest

248 this case rifampicin resistance was predicted correctly by the mutation rpoB Ile572Phe:

249 (A/T)TC. The rare proposed isoniazid resistance marker katG Asn275Ile: A(C/T)C could

250 not be identified in isolate 1057/10 because this mutations is, similarly to rpoB 633,

251 located outside the defined detection areas. Moreover a clear association between

252 these rare mutations and their impact on the resistance phenotype is still missing [74,

253 75]. These examples of rare and partially unknown mutations also show the conceptual

254 limitations of target based molecular tests in general. The pre-defined set of clinical and

255 epidemiological relevant mutations will need to be carefully selected, is limited, and the

256 evaluation of particular mutations might differ even in adjacent geographic regions, as

257 observed in KwaZulu-Natal and Swaziland in South Africa, both with distinct MDR/XDR

258 strain types and mutation profiles (72, 76).

259 The gold standard for the detection of drug resistance is still the culture-based

260 proportion method that uses Lowenstein-Jensen medium, which is recommended by

261 WHO and has been used for over 50 years [77]. However, due to a generation time of

262 MTBC strains of approximately 24 hours, culture-based methods are very slow,

263 technically challenging and require a well-established laboratory infrastructure. Downloaded from 264 Consequently these aspects delay the detection of drug resistance, lead to inappropriate

265 treatment and spread of drug-resistant strains [78]. Fast culture methods such as Bactec

266 MGIT 960/BactTAlert and phage technique are also tedious and require a BSL 3

267 laboratory environment and specialized staff [79, 80]. Therefore and because drug http://jcm.asm.org/

268 resistance is associated with mutations which are coding for the drug target or for

269 enzymes involved in the inactivating process, molecular test systems are now widely

270 used in (supra-)national TB reference laboratories [81]. Several commercial assays that

271 test for rifampicin, isoniazid and fluoroquinolones resistance are available. Line Probe on January 3, 2018 by guest

272 assays as the GenoType MTBDRplus (v1.0 and v2.0) and the Geno Type MTBDRsl

273 assay (v2.0) which detect resistance mediating mutations for rifampicin, isoniazid,

274 fluoroquinolones, ethambutol as well as for injectable drugs have shown a high

275 concordance with standard diagnostic methods (culture, real-time PCR) and DNA

276 sequencing [82, 83]. Despite its high sensitivity and specificity, the open platform design

277 harbors a risk for a potential amplicon contamination and its performance is still labor-

278 intensive [84]. The Xpert MTB/RIF assay is the first point-of-care system that enables a

279 sensitive and specific detection of rifampicin resistance by direct use of primary sputum

280 samples. In summary, after preparation of the sputum specimen, all subsequent steps,

281 including PCR amplification and results interpretation, are fully automated [85, 86]. So

282 far, the Xpert system is limited to the detection of rifampicin resistance and also other

283 proposed molecular test systems are suitable for detection of multi-drug resistance, but

284 require several manual handling steps and specialized equipment [87-89].

285 In conclusion, our melting curve assay is suitable to detect rifampicin, isoniazid and

286 fluoroquinolones resistance in a prototype cartridge from the application of reaction mix

287 to detection within 90 min. The assay detects reliably MTBC DNA down to 24 copies per Downloaded from 288 reaction and works with genomic DNA as well as with crude cell extracts. The prototype

289 cartridge has the advantage to be run with dried reagents, which has been confirmed

290 with wild type genomic DNA (FIG. S1). Furthermore, the array system is very flexible

291 and resistance markers as well as the maximum amount of spotted probes can be http://jcm.asm.org/

292 straightforwardly adapted on future diagnostic requirements.

293

294 MATERIALS AND METHODS

295 Strains of mycobacteria on January 3, 2018 by guest

296 The MTBC strains used for clinical studies (Table S1) have been isolated under

297 standard routine conditions at the German National Reference Research Center,

298 Borstel, Germany. The tested non-tuberculosis mycobacteria were incubated in tubes

299 with Middlebrook and Cohn 7H10 agar (BD Diagnostics, Franklin Lakes, USA) at 37°C

300 for multiple days at Alere Technologies GmbH, Jena, Germany. To obtain inactivated

301 cell culture material the strains were treated by ultrasound and heated at 99°C for 15

302 minutes each.

303 For a clinical evaluation we tested 265 clinical MTBC isolates which were previously

304 analyzed by Sanger sequencing in the respective resistance associated gene regions.

305 The isolates were derived from a national survey of drug resistance in Swaziland, a

306 region with high MDR-TB incidence [23]. The selection covered MDR-TB strains

307 (n=116), XDR-TB strains (n=1) and non-MDR strains (n=19), and a random selection of

308 fully susceptible strains (n=129).

309

310 Isolation of genomic DNA

311 For isolation of the bacterial genomic DNA inactivated culture material (from Lowenstein Downloaded from 312 Jensen slants) were re-suspended in 1 ml TET buffer (Tris, ETDA, Tween® 20) and

313 filtered through a 5 µm filter pore size (Omnifix® Braun, Melsungen, Germany) to obtain

314 single cells. The bacteria solution was washed twice with TET buffer, centrifuged at

315 5,000  g for 5 min and finally adjusted to an optical density of 0.01 in TET. http://jcm.asm.org/

316 Subsequently 400 µL of the bacteria solution were disrupted using a bead beating

317 device (VWR, Erlangen, Germany) and 300 mg of glass beads (<106 µm, Sigma

318 Aldrich, St. Louis, USA) at 5,000  g for 80 s. The obtained cell lysate was centrifuged at

319 5,000  g for 10 s, the supernatant filtered through a 10 µm pore size filter (Mobitec, on January 3, 2018 by guest

320 Eupen, Belgium) and centrifuged at 5,000  g for 1 min. From 100 µL of this cell lysate

321 the genomic DNA was extracted using the QIAamp Blood Mini Kit (Qiagen, Hilden,

322 Germany) according to manufacturer’s instructions. DNA quantification was performed

323 using the Quant-iTTM PicoGreen® dsDNA Kit (Invitrogen, Carlsbad, USA). Finally DNA

324 sequences of the MBTC strains were determined by GATC Biotech AG (Konstanz,

325 Germany) and analyzed using the GENtle software (http://gentle.magnusmanske.de).

326

327 Primers, TaqMan® probes and array probes

328 Sequence information for all primers, array probes and TaqMan® probes are available

329 in Table S3. The array probes had a C7-amino linker for immobilization on the array

330 surface. Forward primers for melting curve analysis were Cy5 labeled at the 5´end; 14

331 reverse primers for melting curve analysis were unlabeled as well as primers for

332 TaqMan® analysis. All oligonucleotides were synthesized by Eurogentec, Cologne,

333 Germany.

334

335 Efficiency of multiplex target amplification using Real-Time PCR Downloaded from 336 Real-Time PCR was performed with primers and fluorescence labeled TaqMan® probes

337 specific for the targets . Due to limitation in fluorescence channels of the used 7500

338 Real-Time PCR system (Applied Biosystems, Foster City, USA), the multiplex

339 amplification was analyzed in two reactions. Sequence-specific primers were added in http://jcm.asm.org/

340 both reactions, whereas the respective TaqMan® probes were added in one or the other

341 reaction. MTBC strains are characterized by a high GC content whereby an adaption of

342 the used PCR buffer was necessary [24]. To improve the PCR amplification in

343 connection with an increased specificity and reaction yield, betaine and on January 3, 2018 by guest

344 tetramethylammonium chloride were added to the reaction [25-27]. Therefore the

345 amplification was performed in a final reaction volume of 100 µL containing TET (Tris,

346 ETDA, Tween® 20), 75 mM Tris-HCl (pH 8.5, Alere Technologies GmbH, Jena,

347 Germany), 3 mM magnesium chloride (Sigma-Aldrich, St. Louis, USA), one betaine

348 tablet (resulting in 2 M betaine, Friedrich-Alexander University, Erlangen, Germany),

349 100 mM tetramethylammonium chloride (Alere Technologies GmbH, Jena, Germany),

350 0.2 mM of deoxynucleoside triphosphates (Thermo Fisher Scientific, Waltham, USA),

351 0.6 µM of each forward primer (Table S3), 0.2 µM of each reverse primer (Table S3),

352 0.2 µM of corresponding TaqMan® probes (Table S3), 12.5 U of BTR hotstart taq

353 (Biotech rabbit, Henningsdorf, Germany), genomic DNA from M. tuberculosis H37Rv

354 reference strain (102 to 104 copies per reaction, tebu-bio, Offenbach, Germany) and

355 104 copies per reaction of the internal process control (Ionian Technologies, San Diego,

356 USA). For negative controls the same reaction mix was used without any template. The

357 reaction was performed using the 7500 Real-Time PCR system with the subsequent

358 conditions: 95°C for 2 min following 40 cycles at 95°C for 10 s, 64°C for 30 s and 72°C

359 for 30 s. Obtained fluorescence values were used to determine a standard calibration Downloaded from 360 curve as well as to calculate the efficiency of the multiplex amplification of MTBC

361 specific targets.

362

363 Melting curve analysis for detection of MTBC drug resistance http://jcm.asm.org/

364 The melting curve assay was performed in a prototype cartridge (Alere Technologies

365 GmbH, Jena, Germany) on the AlereTM q analyzer. After the reaction mix was applied to

366 the cartridge all steps from the amplification to the detection were performed

367 automatically. In the first step a multiplex amplification of the defined targets was on January 3, 2018 by guest

368 performed using Cy5 labeled forward primers. The fluorescence-labeled amplicons were

369 denatured and the single strands hybridized to the spotted array probes which carry

370 either the wild type or the the mutant genotype sequence (FIG. 3, A). At the beginning of

371 the melting curve profile non-matching amplicon-probe bindings were dissolved (FIG. 3,

372 B) and with further increase in temperature also the amplicons complementary to the

373 probe sequence at their specific melting points (FIG. 3, C).

374 Our assay used 61 several immobilized probes representing the wild type or mutation

375 genotypes for the detection of 43 different mutations mediating resistance towards

376 rifampicin, isoniazid and fluoroquinolones. With the melting curve assay SNPs in codons

377 511 (Leu511Pro: C(T/C)G), 513 (Ser513Leu: C(A/T)A, Ser513Lys: (C/A)AA, Ser513Pro:

378 C(A/C)A), 516 (Asp516Phe: (G/T)(A/T)C, Asp516Val: G(A/T)C, Asp516Tyr: (G/T)AC),

379 518 (Asn518Ser: A(A/G)C), 522 (Ser522Gln: (T/C)(C/A)G, Ser522Leu: T(C/T)G,

380 Ser522Trp: T(C/G)G), 526 (His526Asp: (C/G)AC His526Arg: C(A/G)C, His526Asn:

381 (C/A)AC, His526Leu: C(A/T)C, His526Tyr: (C/T)AC, His526Cys: (C/T)(A/G)C,

382 His526Gln: CA(C/A), His526Pro: C(A/C)C, His526Ser: (C/A)(A/G)C), 531 (Ser531Leu:

383 T(C/T)G, Ser531Trp: T(C/G)G), 533 (Leu533Pro: C(T/C)G) and 572 (Ile572Phe: Downloaded from 384 (A/T)TC) could be precisely identified [28-38]. For the detection of isoniazid resistance

385 probes carrying SNPs in the katG codon 315 (Ser315Asn: A(G/A)C, Ser315Ile: A(G/T)C,

386 Ser315Gly: (A/G)GC, Ser315Thr: A(G/C)C, Ser315Thr: A(G/C)(C/A)) and within the inhA

387 promoter region -8(-8T→A, -8T→C) and -15(-15C→T) were applied [39-44]. Resistance http://jcm.asm.org/

388 towards fluoroquinolones is mostly associated with mutations in the target region gyrA

389 [10, 14, 15]. Therefore specific probes carrying SNPs in the codons 88 (Gly88Cys:

390 (G/T)GC), 89 (Asp89Asn: (G/A)AC), 90 (Ala90Val: G(C/T)G), 91 (Ser91Pro: (T/C)CG)

391 and 94 (Asp94Ala: G(A/C)C, Asp94Asn: (G/A)AC, Asp94Gly: G(A/G)C, Asp94His: on January 3, 2018 by guest

392 (G/C)AC, Asp94Tyr: (G/T)AC, Asp94Val: G(A/T)C) were spotted onto the array [45-48].

393 In addition a specific probe at position 95 defining the phylogenetic Ser95Thr: A(G/C)C

394 mutation, which is not associated with resistance to fluoroquinolones [49], was also

395 implemented.

396 The melting curve assay was performed in a reaction volume of 100 µL comprising 75

397 mM TRIS hydrochloride pH 8.5 (Alere Technologies GmbH, Jena, Germany), 3 mM

398 magnesium chloride (Sigma-Aldrich, St. Louis, USA), 1 betaine tablet (resulting in 2 M

399 betaine, university Friedrich-Alexander, Erlangen, Germany), 100 mM

400 tetramethylammonium chloride (Alere Technologies GmbH, Jena, Germany), 0.2 mM

401 deoxynucleoside triphosphate mixture (Thermo Fisher Scientific, Waltham, USA), 0.6

402 µM of each Cy5 labeled forward primer (Table S3), 0.2 µM of each reverse primer

403 (Table S3), 25 U of BTR hotstart taq (Biotech rabbit, Henningsdorf, Germany), 104

404 copies per reaction of the internal process control (Ionian Technologies, San Diego,

405 USA) and 1 µL of genomic MTBC DNA or crude cell culture material. The genomic

406 DNAs with known genotypes were tested with threefold of the LoD concentration. The

407 DNA isolates of the clinical validation study as well as the crude cell material were Downloaded from 408 diluted 1:1000 with TRIS-EDTA buffer (Sigma-Aldrich, St. Louis, USA). Negative

409 controls were also performed. The amplification was performed at 95°C for 2 min

410 following 40 cycles at 95°C for 10 s, 64°C for 30 s and 72°C for 30 s. A magnesium

411 chloride pellet stored in the cartridge (resulting in 200 mM magnesium chloride, Alere http://jcm.asm.org/

412 Technologies GmbH, Jena, Germany) was dissolved with the reaction mix containing

413 the generated Cy5 labeled amplicons. After a denaturation at 95°C for 2 min the

414 amplicons were hybridized on the array probes at 30°C for 2 min. Subsequently a

415 washing step using buffer WB2.10 (Alere Technologies GmbH, Jena, Germany) and a on January 3, 2018 by guest

416 magnesium chloride pellet (resulting in 200 mM magnesium chloride, Alere

417 Technologies GmbH, Jena, Germany) was performed. Finally an image of the array was

418 acquired during the melting curve analysis in a temperature range from 51°C to 71°C

419 with 2°C increments.

420

421

422 Data processing and analysis

423 The acquired image series was analyzed using the Iconoclust software (Alere

424 Technologies GmbH, Jena, Germany). The array spots were identified using a defined

425 grid. The raw fluorescence signal of each array spot was calculated by subtraction of the

426 non-specific background from its absolute value. Afterwards an interpolation was

427 performed to receive a continuous signal. If the raw signal exceeded 0.05, 0.1 or 0.2

428 (depending on the considered probe) it was declared as detected and the signal was

429 normalized. For each probe the temperature value at a signal level of 0.4 was

430 determined. For corresponding probe pairs representing wild type and mutant genotype

431 the ratio of the temperature values were calculated, the so-called discrimination factor. A Downloaded from 432 discrimination factor < 1 represented the wild type and a discrimination factor > 1 the

433 mutant genotype.

434

435 ACKNOWLEDMENTS http://jcm.asm.org/

436 The authors thank Annette Wagenhaus, Christina Lenhart, Katja Herzog and Sandra

437 Henk (Alere Technologies GmbH, Jena, Germany) for their outstanding technical

438 assistance. We thank also Kerstin Scheubert (Alere Technologies GmbH, Jena,

439 Germany) for the adaption of the evaluation software, Cornelius Middelhoff (Alere on January 3, 2018 by guest

440 Technologies GmbH, Jena, Germany) for the evaluation of the LoD data series as well

441 as Andrew Garfield for proof-reading of this manuscript.

442 This work was supported by a grant from the Bill & Melinda Gates Foundation (grant

443 number OPP1068618).

444

445 Conflict of interest

446 JH, BD, PS and IL are employees at Alere Technologies GmbH. The European Society

447 of Mycobacteriology awarded M. M. the Gertrud Meissner Award, which is sponsored by

448 Hain Lifescience. Company associations and funding sources had no role in the study

449 design, data collection and analysis, decision to publish, or preparation of the

450 manuscript.

451

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725 Swaziland. N Engl J Med. 372(12): 1181-1182.

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728 determining region associated with rifampicin resistance in Mycobacterium

729 tuberculosis. J Antimicrob Chemother. 66: 730-733.

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732 Rifampicin-Resistant Clinical Mycobacterium tuberculosis Isolates. J Clin Microbiol.

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738 BJ, Bantubani N, Almeida DV, Alvarado L, Chapman SB, Mvelase NR, Duffy EY,

739 Fitzgerald MG, Govender P, Gujja S, Hamilton S, Howarth C, Larimer JD, Maharaj

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741 J, Milisana KP, O’Donnell MR, Birren BW, Bishai WR, Pym AS, Earl AM. 2015.

742 Evoluation of Extensively Drug-Resistant Tuberculosis over Four Decades: Whole

743 Genome Sequencing and Dating Analysis of Mycobacterium tuberculosis Isolates

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745 1001880.

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759 isoniazid, rifampicin, and pyrazinamide in Mycobacterium tuberculosis. Respir Res. Downloaded from 760 2(3): 164-168.

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764 2635-2640.

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769 Study. J Clin Microbiol. 53(9): 2961-2969.

770 84. Kim SJ. 2005. Drug-susceptibility testing in tuberculosis: methods and reliability of

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776 363(11): 1005-1015.

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779 MTB/RIF Assay. J Clin Microbiol. 48(7): 2495-2501.

780 87. Pholwat S, Liu J, Stroup S, Gratz J, Banu S, Rahman SMM, Ferdous SS,

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786 Anti-Tuberculosis Drug Resistance Mutations by Allele-Specific Primer Extension

787 on a Microsphere-Based Platform. Ann Lab Med. 35: 487-493.

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789 Comparative Evaluation of Sloppy Molecular Beacon and Dual-Labeled Probe on January 3, 2018 by guest

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792 ONE 10(5): e0126257. doi:10.1371/journal.pone.0126257

793

794 FIGURES

795 FIG. 1. Sensitivity of the melting curve assay for the multiplex amplification of MTBC

796 strain specific targets rpoB RRDR, rpoB 572, katG, gyrA QRDR and the inhA promoter

797 region using the prototype cartridge and the AlereTM q analyzer. The determined

798 sensitivity was 24 copies per reaction with a confidence interval of 95% [21 copies per

799 reaction; 27 copies per reaction]. For each concentration (copies per reaction) 25 tests

800 were performed.

801

802 FIG. 2. Specificity of the melting curve assay. The x-axis shows the tested mycobacterial

803 species including M. tuberculosis, M. bovis BCG and non-tuberculosis mycobacteria. A

804 probe specific for MTBC strains is used as internal process control for verification of

805 species specificity. Hybridization signals > 0.05 at 63°C are defined as positive and Downloaded from 806 indicate the presence of strains belonging to the MTBC; thus M. tuberculosis and M.

807 bovis BCG were measured as positive at the specific spot. Non-tuberculosis

808 mycobacteria (NTM) showed no cross reaction with 106 copies per reaction in a reaction

809 mix (hybridization signal < 0.05). http://jcm.asm.org/

810

811 FIG. 3. Detection principle of the melting curve assay. For the targets rpoB RRDR, rpoB

812 572, katG, gyrA QRDR and the inhA promoter region probes representing the wild type

813 (wt) and mutant (mut) genotypes were spotted onto the array. Cy5 labeled amplicons on January 3, 2018 by guest

814 (e.g. wild type) are generated during the multiplex amplification which bind initially to

815 sequence-specific wild type as well as unspecific mutant probes due to the low

816 temperature profile (A). With increase in temperature the unspecific amplicon bindings

817 dissolve resulting in a loss of signal at the non-matching mutant probes compared to the

818 matching wild type probes (B). At the final temperature of 71°C a very low fluorescence

819 signal is detected on the array spots as almost all bindings are dissolved (C).

820

821 TABLES

822 TABLE 1. Determined discrimination factors on array probes

823 TABLE 2. Clinical evaluation of M. tuberculosis isolates from Swaziland

Downloaded from http://jcm.asm.org/ on January 3, 2018 by guest Downloaded from http://jcm.asm.org/ on January 3, 2018 by guest Downloaded from http://jcm.asm.org/ on January 3, 2018 by guest Downloaded from

rpoB RRDR

Probes 516Val_v03 516Val_v03 516Tyr_v03 526Tyr_v04 518Ser_v02 522Trp_v01 526Ser_v01 531Trp_v05 511Pro_v02 522Gln_v03 526Gln_v03 526Pro_v04 533Pro_v04 513Lys_v02 526Arg_v04 513Leu_v03 522Leu_v02 526Leu_v04 526Cys_v03 516Phe_v03 526Asp_v04 526Asn_v06 513Pro_v04 531Leu_v04 Samples H37Rv 0.93 0.94 0.90 0.95 0.96 0.93 0.93 0.89 0.84 0.88 0.87 0.88 0.89 0.89 0.94 0.93 0.88 0.90 0.96 0.89 0.93 0.89 * 3736/04 1.13 0.94 0.87 0.92 0.96 0.94 0.94 0.89 0.84 0.87 0.88 0.93 0.93 0.93 0.97 0.96 0.91 0.94 0.95 0.93 0.94 0.99 * 698/05 0.87 1.01 0.94 1.09 0.98 0.96 0.94 0.90 0.83 0.87 0.87 0.89 0.90 0.90 0.95 0.94 0.90 0.93 0.97 0.89 0.91 0.89 * 10502/06 0.89 1.04 0.93 1.01 0.97 0.96 0.94 0.89 0.83 0.86 0.86 0.88 0.89 0.89 0.94 0.92 0.86 0.91 0.95 0.92 0.94 0.91 * 6695/04 0.92 0.92 0.92 0.95 0.96 0.93 0.91 1.05 0.85 0.88 0.92 0.89 0.88 0.91 0.93 0.92 0.86 0.90 0.96 0.87 ** ** 1.06

7941/01 0.92 0.94 0.91 0.95 0.96 0.94 0.93 0.90 0.92 1.09 0.97 0.88 0.90 0.87 0.94 0.92 0.85 0.90 0.95 0.88 0.94 0.93 * http://jcm.asm.org/ 49 -02 -SR4a 0.93 0.94 0.91 0.95 0.95 0.94 0.93 0.89 0.98 1.04 1.15 0.86 0.88 0.84 0.92 0.90 0.85 0.89 0.93 0.86 0.93 0.89 * 5976/01 0.93 0.94 0.91 0.95 0.96 0.93 0.93 0.89 0.83 0.87 0.86 0.93 0.97 0.93 1.10 1.01 0.99 0.97 1.04 0.96 0.94 0.95 * 4787/03 0.93 0.94 0.91 0.96 0.96 0.94 0.93 0.89 0.93 0.85 0.87 0.95 0.97 0.97 0.99 1.07 1.00 0.91 1.05 0.94 0.92 0.88 * 3307/03 0.93 0.93 0.90 0.94 0.96 0.94 0.93 0.88 0.88 0.85 0.86 0.98 0.92 1.08 0.98 1.03 0.95 0.96 0.99 0.97 0.94 0.89 * 10427/01 0.93 0.95 0.91 0.96 0.96 0.94 0.92 0.89 0.89 0.84 0.85 0.98 1.04 0.98 1.00 1.07 1.14 0.94 0.98 1.05 0.91 0.91 * 2822/06 0.93 0.93 0.91 0.95 0.96 0.94 0.93 0.89 0.86 0.85 0.86 1.15 0.96 1.05 0.98 1.07 0.97 0.93 0.97 0.97 0.92 0.89 * H37Rv -SR4k 0.93 0.94 0.91 0.95 0.96 0.93 0.93 0.89 0.84 0.86 0.89 0.93 0.99 0.97 1.04 1.07 0.94 1.05 1.12 0.92 *** *** * 4724/03 0.90 0.94 0.91 0.95 0.96 0.94 0.93 0.90 0.94 0.87 0.88 0.95 1.04 0.93 1.00 1.02 1.01 0.96 1.02 0.93 0.98 0.98 * 368/01 0.92 0.94 0.91 0.95 0.96 0.94 0.93 0.89 0.86 0.87 0.87 0.90 0.88 0.91 0.94 0.93 0.87 0.90 0.96 0.89 1.07 1.01 * H37Rv -SR8a2 0.93 0.94 0.91 0.95 0.96 0.94 0.93 0.89 0.84 0.87 0.90 0.88 0.92 0.90 0.95 0.93 0.87 0.90 0.96 0.87 *** *** * 4709/09 0.91 0.94 0.91 0.95 0.97 0.94 0.93 0.90 0.86 0.87 0.90 0.91 0.91 0.91 *** 0.96 0.89 0.91 0.99 0.87 0.94 *** * 12401/03 0.90 0.94 0.91 0.95 0.96 0.94 0.93 0.89 0.88 0.88 0.87 0.88 0.90 0.91 0.95 0.93 0.92 0.90 0.97 0.89 1.05 1.01 * 3355/02 0.93 0.94 0.91 0.95 0.96 0.94 0.93 0.89 0.83 0.87 0.88 0.90 0.90 0.90 0.95 0.94 0.88 0.94 0.97 0.89 *** 0.90 * 3429/03 0.92 0.94 0.91 0.96 0.96 0.94 0.93 0.89 0.84 0.87 0.87 0.89 0.90 0.90 0.95 0.93 0.88 0.91 0.97 0.90 0.97 0.92 * on January 3, 2018 by guest 1429/02 0.93 0.94 0.91 0.97 0.96 0.94 0.93 0.88 0.84 0.87 0.91 0.90 0.93 0.90 0.95 0.93 0.90 0.94 0.98 0.88 0.97 0.98 * 8085/03 0.93 0.91 0.88 0.96 0.99 1.07 1.03 0.85 0.83 0.86 0.86 0.90 0.89 0.89 0.94 0.93 0.89 0.91 0.95 0.88 0.94 0.89 * 853/07 0.97 0.94 0.96 0.97 1.08 1.00 1.04 0.84 0.82 0.86 0.84 0.90 0.89 0.89 0.94 0.94 0.88 0.90 0.95 0.87 0.96 0.90 * H37Rv -SO4a 0.92 0.94 0.91 0.95 0.96 0.93 0.93 0.89 0.84 0.86 0.87 0.91 0.92 0.92 0.95 0.94 0.93 0.93 0.98 0.93 0.96 0.93 * 4535/04 0.90 0.94 0.90 0.95 0.96 0.95 0.93 0.89 0.83 0.87 0.86 0.88 0.88 0.90 0.94 0.93 0.87 0.89 0.96 0.90 1.07 0.93 * 464/11 0.93 0.94 0.91 0.95 0.96 0.94 0.93 0.88 0.83 0.87 0.87 0.89 0.89 0.89 0.94 0.92 0.87 0.91 0.96 0.87 1.07 0.97 * 1598/06 0.92 0.94 0.90 0.95 0.96 0.93 0.93 0.88 0.86 0.87 0.88 0.89 0.89 0.90 0.94 0.92 0.89 0.91 0.96 0.92 1.05 0.95 * 10523/05 0.93 0.93 0.90 0.95 0.96 0.94 0.93 0.88 0.84 0.87 0.87 0.89 0.88 0.90 0.94 0.92 0.86 0.90 0.95 0.87 1.08 0.98 * 3075/05 0.93 0.94 0.90 0.95 0.96 0.93 0.92 0.89 0.83 0.87 0.87 0.88 0.88 0.89 0.94 0.92 0.86 0.90 0.95 0.87 1.08 0.96 * 8444/05 0.90 0.94 0.91 0.95 0.96 0.94 0.93 0.88 0.87 0.87 0.87 0.89 0.93 0.91 0.94 0.94 0.88 0.94 0.97 0.89 1.06 0.92 *

Downloaded from

katG inhA gyrA QRDR Controls rpoB rpoB

Probes MTB MTB Msme Msme sp_02 94Val_v02 94Val_v02 90Val_v01 90Val_v01 94His_v04 94His_v04 94Tyr_v02 94Ala_v01 94Ala_v01 94Gly_v02 94Gly_v02 95Thr_v01 95Thr_v01 91Pro_v04 91Pro_v04 315Ile_v01 315Ile_v01 88Cys_v01 88Cys_v01 94Asn_v02 94Asn_v02 89Asn_v04 89Asn_v04 -8T>A_v02 -8T>A_v02 -8T>C_v02 315Gly_v04 572Phe_v04 315Asn_v06 -15C>T_v02 315Thr2_v04 315Thr1_v01 Samples H37Rv 0.97 0.96 0.96 0.93 0.92 0.94 0.96 0.95 0.96 0.93 0.98 0.94 0.96 0.98 0.99 0.93 0.98 0.98 0.97 0.99 x x - 3736/04 0.97 0.96 0.96 0.92 0.91 0.94 0.98 0.96 0.96 0.91 0.99 0.94 0.97 1.01 0.98 0.98 0.99 0.97 0.95 1.02 x x - 698/05 0.97 1.05 1.04 1.11 1.07 1.00 0.96 0.92 0.92 0.93 0.99 0.93 0.97 0.98 0.97 0.95 0.98 0.97 0.95 1.02 x x -

10502/06 0.97 1.05 1.03 1.11 1.05 0.99 0.98 0.96 0.95 0.93 1.00 0.95 0.98 0.98 0.98 0.94 0.98 0.97 0.95 1.01 x x - http://jcm.asm.org/ 6695/04 0.98 1.06 1.04 1.11 1.07 1.00 0.94 0.98 0.97 0.93 0.94 0.93 0.97 0.97 0.98 0.94 0.98 0.97 0.95 1.01 x x - 7941/01 0.97 0.96 0.96 0.92 0.92 0.94 0.94 0.92 0.90 0.90 0.97 0.89 0.93 0.96 0.96 0.95 0.98 0.96 0.94 1.06 x x - 49 -02 -SR4a 0.97 0.95 0.96 0.91 0.91 0.94 0.99 0.97 0.96 0.94 0.99 0.96 0.98 0.99 0.98 0.94 0.99 0.97 0.95 1.01 x x - 5976/01 0.96 1.06 1.04 1.12 1.07 1.00 0.95 0.93 0.92 0.91 0.98 0.90 0.94 0.98 0.97 0.96 0.98 0.97 0.96 1.03 x x - 4787/03 0.96 1.05 1.04 1.11 1.07 1.00 0.93 0.93 0.91 0.93 0.98 0.93 0.97 0.99 0.98 0.93 0.98 0.97 0.95 1.02 x x - 3307/03 0.97 0.96 0.96 0.92 0.91 0.94 0.91 0.88 1.06 0.90 0.97 0.89 0.93 0.97 0.97 0.93 0.98 0.96 0.95 1.05 x x - 10427/01 0.97 1.06 1.04 1.12 1.07 1.00 0.94 0.92 0.90 0.90 0.97 0.90 0.93 0.97 0.97 0.93 0.98 0.96 0.94 1.05 x x - 2822/06 0.97 0.95 0.96 0.92 0.91 0.94 0.89 0.87 1.07 0.90 0.97 0.88 0.93 0.97 0.96 0.95 0.97 0.96 0.94 1.08 x x - H37Rv -SR4k 0.96 0.95 0.96 0.92 0.91 0.94 0.93 0.91 0.90 0.90 0.97 0.89 0.93 0.97 0.97 0.91 0.96 0.97 0.95 0.97 x x - 4724/03 0.96 1.05 1.04 1.11 1.07 1.00 0.94 0.92 0.90 0.90 0.96 0.89 0.93 0.98 0.97 0.96 0.98 0.96 0.95 1.06 x x - 368/01 0.98 1.05 1.04 1.12 1.07 1.01 0.93 0.91 0.91 0.90 0.97 0.90 0.93 0.97 0.97 0.93 0.98 0.96 0.94 1.06 x x - H37Rv -SR8a2 0.97 0.96 0.97 0.93 0.92 0.94 0.93 0.91 0.91 0.91 0.97 0.91 0.93 0.97 0.97 0.90 0.96 0.96 0.95 0.97 x x - 4709/09 1.10 1.05 1.04 1.11 1.06 0.99 0.94 0.93 0.92 0.90 0.97 0.90 0.93 0.98 0.97 0.93 0.98 0.98 0.95 1.05 x x - 12401/03 0.97 1.14 1.05 1.05 1.05 1.01 0.93 0.92 0.91 0.90 0.97 0.89 0.93 0.96 0.97 0.95 0.98 0.97 0.95 1.07 x x - on January 3, 2018 by guest 3355/02 0.97 1.06 1.13 1.03 1.05 1.00 0.92 0.91 0.90 0.90 0.97 0.89 0.93 0.96 0.97 0.95 0.98 0.96 0.95 1.06 x x - 3429/03 0.97 0.98 0.97 0.93 0.94 1.10 0.87 0.85 1.07 0.90 0.97 0.90 0.93 0.96 0.97 0.95 0.98 0.96 0.95 1.04 x x - 1429/02 0.96 1.07 1.05 1.05 1.19 1.03 0.93 0.91 0.90 0.90 0.96 0.88 0.93 0.96 0.97 0.95 0.98 0.97 0.94 1.08 x x - 8085/03 0.97 1.05 1.03 1.11 1.06 1.00 1.06 0.99 0.96 0.90 0.97 0.88 0.93 0.97 0.97 0.93 0.98 0.96 0.94 1.09 x x - 853/07 0.97 1.05 1.03 1.11 1.06 1.00 0.94 0.92 0.89 0.86 1.16 0.97 0.91 0.96 0.96 0.93 0.97 0.96 0.94 1.08 x x - H37Rv -SO4a 0.96 0.96 0.96 0.92 0.91 0.94 0.93 0.91 0.91 0.91 0.97 1.07 0.93 0.93 0.94 0.90 0.93 0.93 0.93 0.97 x x - 4535/04 0.97 1.06 1.04 1.11 1.07 1.00 0.93 0.92 0.91 0.90 0.98 0.96 1.08 *** 0.94 1.00 0.95 0.96 *** 1.04 x x - 464/11 0.97 1.05 1.04 1.12 1.07 1.00 0.93 0.92 0.93 0.90 0.97 0.89 0.93 1.10 1.00 1.02 1.05 1.02 1.01 1.06 x x - 1598/06 0.97 1.06 1.04 1.13 1.07 1.00 0.93 0.93 0.91 0.90 0.98 0.90 0.93 1.03 1.00 1.04 1.02 1.00 1.03 1.06 x x - 10523/05 0.92 1.05 1.04 1.11 1.06 1.00 0.93 0.91 0.89 0.89 0.98 0.89 0.92 1.02 0.98 1.03 1.01 1.04 0.96 1.07 x x - 3075/05 0.97 1.14 1.05 1.04 1.05 1.00 0.92 0.91 0.90 0.89 0.97 0.89 0.91 1.05 1.10 1.05 1.06 1.03 0.99 1.08 x x - 8444/05 0.95 0.96 0.97 0.92 0.92 0.94 0.88 0.87 1.06 0.90 0.97 0.92 0.92 0.97 1.04 0.93 1.12 1.04 0.98 0.95 x x - * no reaction of the mutant probe (strong discrimination) ** cross reaction with mutant 533Pro (probe overlapping); no clear/weak reaction *** clear identification in raw data plain text – wild type detection bold text – mutant genotype detection, highest discrimination factor underlined

RIF INH FQ No. No. No. No. No. No. Total (%) wildtype mutant wildtype mutant wildtype mutant a specimens (%) (%) (%) (%) (%) (%) Melting curve 144 121 143 122 259 6 265 assay (54.3) (45.7) (54.0) (46.0) (97.7) (2.3) (100) 144 121 142 123 259 6 265 Sequencing

(54.3) (45.7) (53.6) (46.4) (97.7) (2.3) (100) Downloaded from a excluding the phylogenetic SNP Ser95Thr

http://jcm.asm.org/ on January 3, 2018 by guest