Can mutation detection accurately predict XDR- TB: study from a tertiary care centre India

JCM Accepts, published online ahead of print on 2 February 2011 J. Clin. Microbiol. doi:10.1128/jcm.00113-11 Copyright 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. Can mutation detection accurately predict XDR- TB: study from a tertiary care centre India Kanchan Ajbani, PhD student, Dept of Research, P.D. Hinduja National Hospital & Medical Research Centre, Mumbai, India Camilla Rodrigues, Consultant Microbiologist, Dept of Microbiology, P.D. Hinduja National Hospital & Medical Research Centre, Mumbai, India Shubhada Shenai, Dept of Research, P.D. Hinduja National Hospital & Medical Research Centre, Mumbai, India Ajita Mehta, Late* Consultant Microbiologist, Dept of Microbiology, P.D. Hinduja National Hospital & Medical Research Centre, Mumbai, India * Passed away on 6 th Feb 2010. Conflict of interest: There are no conflicts of interest for this manuscript. Kanchan Ajbani: No Conflict Camilla Rodrigues: No Conflict Shubhada Shenai: No Conflict

Source of financial support: The study was funded by National Health and Education Society, P. D. Hinduja National hospital and Medical Research Centre, Mumbai, India. Address for Correspondence Dr. Camilla Rodrigues Consultant Microbiologist, Dept of Microbiology, Research Laboratories, P. D. Hinduja National Hospital and Medical Research Centre; Veer Savarkar Marg, Mahim, Mumbai 400016, India. Tel: 0091 22 24447795 Fax No.; 099 22 24442318 E-mail address: dr_crodrigues@hindujahospital.com

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 ABSTRACT We screened and spoligotyped 150 consecutive phenotypically confirmed XDR-TB isolates (Jan 08 to Mar 09) for rifampicin, isoniazid, fluoroquinolones and aminoglycoside resistance targeting rpob, inha, katg, gyra, gyrb and rrs. Mutations predominant amongst XDR- TB were 100% S315T (katg), 97% S531L (rpob), 53% D94G(gyrA), 71% A1401G(rrs). Spoligotyping revealed 62% to be Beijing. Key words: XDR-TB, fluoroquinolone, aminoglycoside. Running title: Extensively Drug Resistant tuberculosis 22 23 24 25

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 The worldwide emergence of Extensively Drug Resistant tuberculosis (XDR-TB) is a major setback to tuberculosis (TB) control (9, 11, 12, 15). XDR-TB, defined as Multi Drug Resistant (MDR) TB [i.e. resistant to rifampicin and isoniazid] with additional resistance to any fluoroquinolone (FQ) and to any one of three second line injectables i.e. capreomycin, kanamycin or amikacin (21). The mycobacteriology laboratory of P D Hinduja National Hospital (PDHNH), a tertiary care centre in central Mumbai received 7482 clinical specimens for TB culture using Mycobacterial Growth Indicator Tube (MGIT) and Lowenstein Jensen (L.J) from Jan 2008- Mar 2009. Our laboratory has a referral bias towards non responders as we receive mainly treatment failures and complicated TB cases and drug susceptibility testing (DST) is performed only on request. 3899 samples were positive for Mycobacterium tuberculosis complex and 2522 patients requested DST of which 1640 (65%) were MDR and 150 (9.1%) were XDR-TB by MGIT (19). We studied mutations targeting the genes included in definition of XDR-TB [inha, katg, gyra (1, 5, 9, 14, 20, 27), gyrb and rrs (2, 16, 28)] and fingerprinted these patients isolates by spoligotyping (6). DNA extraction from pure culture was performed by the Cetyl trimethyl ammonium bromide (CTAB NaCl) method (23). A single tube touchdown multiplex PCR for three target genes associated with resistance to isoniazid (inha, katg) and rifampicin (rpob) was done and screened for mutations using an in House Reverse Line Blot Hybridisation (RLBH) assay (22). The screening of katg, resulted in the identification of G315C (100%) the S315T mutation in katg (Table 1) conferring high-level resistance to isoniazid with an altered catalase- peroxidase activity (24). 21(14%) of these isolates showed C-15T mutation in the promoter region of inha gene in addition to katg mutation. The promoter mutation was also seen among XDR-TB isolates in Portugal (18) and China (25). S531L mutation in rpob was more prevalent; observed in 146(97%) of these isolates, whereas H526Y mutation was observed only in 4 (3%) isolates. A

50 51 52 previous study from this centre (22) on MDR isolates revealed an array of point mutations from 511-531 in rpob; however S531L in rpob mutation dominated among XDR-TB isolates. This is similar to a study from Portugal (18) where the S531L mutation dominated in XDR-TB. 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 Novel primers were designed to amplify a 350bp region of gyra, 550bp region for gyrb for fluoroquinolone resistance and 500bp region for rrs (codon 1401-1484) for aminoglycoside resistance. The initial 50 amplified products were analyzed for bidirectional sequencing and after mutation confirmation, subsequent 100 products were sent for unidirectional sequencing (Chromous Biotech Pvt Ltd, Bangalore, India). DNA sequencing of gyra gene showed the presence of D94G mutation in 79 (53%) isolates phenotypically resistant to ofloxacin and moxifloxacin, whilst A90V mutation was observed in 37 (25%) isolates that were phenotypically sensitive to moxifloxacin but resistant to ofloxacin. This needs to be verified with Minimum Inhibitory Concentration (MIC) at higher drug concentrations for ofloxacin (13). The 94 th codon mutation D94G showed a high frequency i.e. 53% amongst our isolates. Other mutations at the 94 th codon D94A and D94N were found in 12% and 9% of isolates respectively. Other studies (25, 18) also show similar mutations in gyra. All isolates were sequenced for gyrb mutations but only one isolate phenotypically resistant to both ofloxacin and moxifloxacin showed a N510D mutation in gyrb in addition to D94G mutation in gyra. Sequencing of rrs gene revealed presence of A1401G mutation in 106 (71%) isolates that were phenotypically resistant to kanamycin and amikacin. Cross resistance between the two aminoglycosides kanamycin and amikacin is believed to exist (2, 16, 26, 28). 42 (28%) isolates phenotypically resistant to capreomycin in addition to kanamycin and amikacin showed presence of a G1484T mutation whereas other studies (18, 25) have reported several point mutations in rrs gene in addition to the 1401 and 1484 mutation.

73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 For capreomycin resistant isolates, the tlya gene (17) that was split in two parts (tlya1 of 500bp and tlya2 of 300bp) was amplified using novel primers. The amplified products were sent for bidirectional sequencing. Of the 42 capreomycin resistant isolates sequenced for tlya in addition to rrs; 13 (31%) isolates showed A205G (K69E) and three isolates showed deletion of nucleotide T at 674 resulting in frameshift mutation. The remaining 26 isolates only showed G1484T mutation in the rrs with no tlya mutation. Two isolates that were phenotypically resistant to kanamycin, amikacin and capreomycin did not show any mutation in the rrs gene. This could be due to mutations in other hot spot regions that were not targeted in this study. Fingerprinting of all isolates was performed by spoligotyping (6) and the pattern was compared to an International Spoligotyping database (SpolDB4) (7, 29). Spoligotype pattern revealed that Beijing genotype was predominant among XDR-TB isolates that accounted for 62% of all the isolates followed by Central- Asian (CAS) (14%), T families i.e. T1 (13%), East- African- Indian (EAI) 5(7%) and EAI 3 (2%). There were three single isolates that each belonged to family 35 (assigned by Spotclust database which uses computer algorithm based on studies from SpolDB3 (10)), T4 and Haarlem genotype (Table 2). The sharing of a single spoligotype by 62% of isolates suggests an important role for transmission of a dominant resistant clone. Spoligotyping studies from Mumbai have shown other genotypes i.e. CAS and Manu1 to be predominant (4) whereas previous study from our centre (3) has reported high proportion of Beijing strains amongst resistant cases. However, results of the present study should not be over interpreted as sampling bias cannot be ruled out due to the non responders referred to our centre. Epidemiological associations between patients could not be ascertained as patients were not residents from the same locality or work place and 119/150 (79%) were on second line drugs for the duration of more than 1 year.

96 97 Thus mutational analysis could conclusively predict 100% resistance to isoniazid, rifampicin, fluoroquinolone and 98% for aminoglycosides amongst our XDR- TB cohort. 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113

114 ACKNOWLEDGEMENT: 115 116 The study was funded by the National Health and Education Society, P. D. Hinduja National Hospital and Medical Research Centre, Mumbai. India. 117 118 119 120 121 122 123 124 125 126 127 128 129 130 The authors have no potential conflict of interest relevant to the contents of this manuscript. The study has been approved by the Institutional Review Board of P. D. Hinduja National Hospital and Medical Research Centre, Mumbai. India. 131 132 133 134

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227 Table 1: Frequency of mutations in the various genes observed among our 150 XDR-TB isolates Drug Gene Mutation seen Amino acid change No. of isolates isoniazid katg G315C S315T 150 (100%) isoniazid inha C-15T 21 (14%) rifampicin rpob C531T S531L 146(97%) rifampicin rpob A526G H526Y 4 (3%) Fluoroquinolone i.e. ofloxacin gyra C90T A90V 37 (25%) Fluroquinolones ofloxacin and gyra T91C S91P 2 (1%) moxifloxacin Fluoroquinolone ofloxacin and gyra A94G D94G 79 (53%) moxifloxacin Fluroquinolones ofloxacin and gyra A94C D94A 18 (12%) moxifloxacin Fluoroquinolone ofloxacin and gyra G94A D94N 14 (9%) moxifloxacin Aminoglycoside i.e. kanamycin, rrs A1401G 106 (71%) amikacin Aminoglycoside i.e. kanamycin, amikacin, capreomycin rrs G1484T 42 (28%) capreomycin (n= 42) tlya+ rrs A205G K69E 13 (31%) capreomycin (n= 42) tlya+ rrs +G1484T del T at nt 674 +G1484T Frameshift 3 (7%) 228

229 Table 2: Spoligotyping pattern of the 150 XDR-TB isolates No Octal code Spoligotype No. of isolates 1 2 3 4 5 6 000000000003771 Beijing 703777740003771 CAS 377777777760771 T1 737777777413371 EAI5 477777777413071 EAI3 703600000000771 family 35 with the % genotype 94 62 21 14 19 13 10 6.7 3 2 1 0.6 Downloaded from http://jcm.asm.org/ 230 231 232 7 8 757777774020771 Haarlem1 777760057760771 T4 1 0.6 1 0.6 on August 27, 2018 by guest 233