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

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J. Clin. Microbiol. Doi:10.1128/JCM.01249-17 Copyright © 2017 Havlicek Et Al 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 3 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* 8 9 * These authors contributed equally to this work. http://jcm.asm.org/ 10 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 18 19 Corresponding author: Dr. Matthias Merker, email: [email protected] 1 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. 43 2 44 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 3 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 4 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. 95 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 5 116 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.
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