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

Transcription

1 JCM Accepts, published online ahead of print on 2 February 2011 J. Clin. Microbiol. doi: /jcm 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 Conflict of interest: There are no conflicts of interest for this manuscript. Kanchan Ajbani: No Conflict Camilla Rodrigues: No Conflict Shubhada Shenai: No Conflict

2 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 , India. Tel: Fax No.; address:

3 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

4 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 Mar 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 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

5 previous study from this centre (22) on MDR isolates revealed an array of point mutations from 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 Novel primers were designed to amplify a 350bp region of gyra, 550bp region for gyrb for fluoroquinolone resistance and 500bp region for rrs (codon ) 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.

6 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.

7 96 97 Thus mutational analysis could conclusively predict 100% resistance to isoniazid, rifampicin, fluoroquinolone and 98% for aminoglycosides amongst our XDR- TB cohort

8 114 ACKNOWLEDGEMENT: The study was funded by the National Health and Education Society, P. D. Hinduja National Hospital and Medical Research Centre, Mumbai. India 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

9 135 REFERECES: Alangaden G, Manavathu E, Vakulenko S, Zvonok N, Lerner S Characterization of fluoroquinolone resistant mutant strains of Mycobacterium tuberculosis selected in the laboratory and isolated from patients. Antimicrobial agents and Chemotherapy. 39: Alangaden G, Kreiswirth B, Aouad A, et al Mechanism of resistance to Amikacin and Kanamycin in Mycobacterium tuberculosis. Antimicrobial agents and Chemotherapy. 42: Almeida, D., C. Rodrigues, T. F. Ashavaid, A. Lalvani, Z. F. Udwadia, and A. Mehta High incidence of the Beijing genotype among multidrugresistant isolates of Mycobacterium tuberculosis in a tertiary care center in Mumbai, India. Clin. Infect. Dis. 40: Arora, J., U. B. Singh, N. Suresh, T. Rana, C. Porwal, A. Kaushik, and J. N. Pande Characterization of predominant Mycobacterium tuberculosis strains from different subpopulations of India. Infect. Genet. Evol. 9: Aubry A, Veziris N, Cambau E, Pernot C, et al Novel gyrase mutation in quinolone resistant and hypersusceptible clinical isolates of Mycobacterium tuberculosis: Functional Analysis of mutant enzymes. Antimicrobial agents and Chemotherapy. 50: Boer M, Zanden A, Soolingen D Simultaneous detection and typing of Mycobacterium tuberculosis complex bacteria. CLI. 7. Brudey K, Driscoll J, RigoutsL,et al Mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology. BMC Microbiology. 1471:6-23.

10 Cheng A, Yew W, Chan E, Chin M, et al Multiplex PCR amplimer conformation analysis of rapid detection of gyra mutations in fluroquinolone resistant Mycobacterium tuberculosis clinical isolates. Antimicrobial agents and Chemotherapy. 48: Editorial XDR-TB TB a global threat. Lancet. 368: Eldholm V, Matee M, Mfinanga S, et al A first insight into the genetic diversity of Mycobacterium tuberculosis in Dar es Salaam, Tanzania, assessed by spoligotyping. BMC Microbiology. 76 (6): Gandhi N R, Moll A, Strum W, et al Extensively drug resistant tuberculosis as a cause of death in patients co infected with tuberculosis and HIV in a rural area of South Africa. Lancet. 368: Gupta A, Espinal M, Raviglione M Should tuberculosis programmes invest in second- line treatments for multi drug resistant tuberculosis (MDR-TB). International Journal of tubercle and lung disease. 12: Kai Man Kam, Chi Wai yip, Tze Leung Cheung, Hei Sing Tang, Oi Chi Leung, and Mei Yuk Chan Stepwise Decrease in Moxifloxacin susceptibility amongst clinical isolates of Multidrug resistant Mycobacterium tuberculosis: Correlation with Ofloxacin susceptibility. Microbial Drug Resistance. 12: Kocagoz T, Hackbarth C, Unsal I, Rosenberg H, et al Gyrase mutations in laboratory selected fluoroquinolone resistant mutants of mycobacterium tuberculosis H37Ra. Antimicrobial agents and chemotherapy. 40: LoBue Extensively drug- resistant tuberculosis. Current Opinion in Infectious Disease. 22:

11 Maus C, Plikaytis B, Shinnick T Molecular analysis of cross resistance to Capreomycin, Kanamycin, Amikacin and Viomycin in Mycobacterium tuberculosis. Antimicrobial agents and Chemotherapy. 49: Maus C, Plikaytis B, Shinnick T Mutation of tlya Confers Capreomycin Resistance in Mycobacterium tuberculosis. Antimicrobial agents and Chemotherapy. 49: Perdigao J. Macedo R, Malaquias A, Ferreira A, Brum L, Portugal I Genetic analysis of extensively drug- resistant Mycobacterium tuberculosis strains in Lisbon, Portugal. J Antimicrob Chemother. 65: Rodrigues C., Jani J, Shenai S, Thakkar P, Siddiqi S, Mehta A Drug susceptibility testing of Mycobacterium tuberculosis against second line drugs using the Bactec MGIT 960 System. Int J Tuberc Lung Dis. 12: Ruiru S, Zhang J, Li C, Kazumi Y, Sugawara I Emergence of Ofloxacin resistance in Mycobacterium tuberculosis Clinical Isolates from China as determined by gyra mutation analysis using Denaturing High- Pressure Liquid Chromatography and DNA Sequencing. JCM.44: Shah. S, Wright. A, Bai. G, Barrera. L et al Worldwide emergence of Extensively Drug- Resistant Tuberculosis. Emerging Infectious Disease. 13: Shenai. S, Rodrigues. C and Mehta. A Rapid speciation of 15 clinically relevant mycobacteria with simultaneous detection of resistance to rifampicin, isoniazid and streptomycin in M.tuberculosis complex. Int J Infect Dis. 13:46-58.

12 Somerville W, Thibert L, Schwartzman K, Behr M Extraction of Mycobacterium tuberculosis DNA: a question of containment. Journal of clinical microbiology. 43: Somoskovi A, Parsons L, Salfinger M The molecular basis of resistance to Isoniazid, Rifampicin, and Pyrazinamide in Mycobacterium tuberculosis. Respir Res. 2: Sun Z, Chao Y, Zhang X, Zhang J, Li Y, Qiu Y, Nie L, Guo A, Li C Characterization of Extensively drug resistant Mycobacterium tuberculosis clinical isolates in China. JCM. 46: Suzuki et al Detection of Kanamycin resistant Mycobacterium tuberculosis by identifying mutations in the 16S rrna gene. Journal of clinical microbiology. 36: Takiff H, Salazar L, Guerrero C, Philipp W, Huang W, et al Cloning and Nucleotide Sequence of Mycobacterium tuberculosis gyra and gyrb Genes and detection of quinolone resistance mutations. Antimicrobial agents and Chemotherapy. 38: Tsukamura M, Mizuno S Cross resistant relationships among the aminoglycoside antibiotics in Mycobacterium tuberculosis. J. Gen. Microbiol. 88: Van Soolingen D, Hermans PW, Dehaas PE, Soll DR, van EmbdenJD Occurrence and stability of insertion sequences in Mycobacterium tuberculosis complex strains: evaluation of an insertion sequence-dependent DNA polymorphism as a tool in the epidemiology of tuberculosis. J Clin Microbiol. 29:

13 227 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

14 229 Table 2: Spoligotyping pattern of the 150 XDR-TB isolates No Octal code Spoligotype No. of isolates Beijing CAS T EAI EAI family 35 with the % genotype Downloaded from Haarlem T on August 27, 2018 by guest 233