Molecular characterisation of resistant Mycobacterium tuberculosis ...

Original Research: Resistant genotypes of M. tuberculosis in South Africa

Molecular characterisation of resistant Mycobacterium tuberculosis isolates from Dr George Mukhari Hospital, Pretoria, South Africa E Green, LC Obi, M Nchabeleng, BE de Villiers, PP Sein, T Letsoalo, AA Hoosen, PO Bessong

E Green, PO Bessonga, School of Mathematics and Natural Sciences, Department of Microbiology, University of Venda, Thohoyandou a AIDS Virus Research laboratory, Department of Microbiology, University of Venda LC Obi, Research and Academic Directorate, Walter Sisulu University, Mthatha M Nchabeleng, BE de Villiers, PP Sein, T Letsoalo, Department of Microbiological Pathology, University of Limpopo, Medunsa Campus AA Hoosen, Department of Medical Microbiology, Faculty of Health Science, University of Pretoria Correspondence to: Ezekiel Green, Department of Biochemistry and Microbiology, University of Fort Hare, Private Bag X1314, Alice, 5700, South Africa E-mail: [email protected] or [email protected]

Drug-resistant tuberculosis is a serious problem throughout the world. Resistance to rifampicin (RIF) and isoniazid (INH) is due to mutations in the rpoB and katG genes, respectively. The distribution of rpoB and katG gene mutations in RIF- and INH-resistant clinical Mycobacterium tuberculosis (MTB) isolates from Dr George Mukhari Hospital, Garankuwa, South Africa was determined. The rpoBand katG genes were amplified using PCR and sequenced. Among the 240 resistant MTB isolates obtained, 143/240 (59.6%) were multidrug-resistant (MDR), defined as resistance to INH and RIF, 44/240 (18.3%) were resistant to INH and 4.6% (11/240) to RIF while 17.5% (42/240) isolates were resistant to a combination of drugs. A total of 67.1% (161/240) isolates had mutations in the rpoB region. The most frequent mutations encountered were at codon 516 GAC to GTC 42.2% (68/161), codon 526 CAC to GAC 37.3% (60/161), and the least, at codon 531 TCG to TTG 0.6% (1/161). Codon 315 in the katG gene showed mutations in 70% (168/240) isolates resistant to INH. Of these, 58.3% (98/168) isolates showed a mutational change from AGC to ACC, 23.8% (40/168) from AGC to AAC, AGC to ATC in 10.1% (17/168), AGC to CGC in 4.8% (8/168) and AGC to ACA 3% (5/168) while mutations in codon 314 contributed 9.6% (20/209) with the change from ACC to CCC. This study provided information on the genetic diversity of multidrugresistant MTB strains as well as the effects it can take on the clinical management of patients. South Afr J Epidemiol Infect 2008;23(3):11-14

Introduction

nine provinces.8 A study conducted by the South African Medical Research Council (MRC) reported an increase from 1.5% of MDR-TB in new patients in Mpumalanga in South Africa during 1997 to 2.6% during 2001, while in retreated patients the MDR-TB prevalence increased from 8.1% to 13.9%.8 Well-documented outbreaks of MDR-TB have been amply reported in different settings.9 However, the incidence of MDR-TB is still not known in most settings.

Worldwide, an estimated 8-10 million individuals develop tuberculosis (TB) and three million die from the disease each year.1 More than 80% of all TB patients live in sub-Saharan Africa and Asia.1 The incidence of TB in sub-Saharan Africa was almost 400 cases per 100,000 population in 2005. In South Africa, as in the rest of the world, TB remains a major problem2 and accounts for over three in every 1,000 deaths.3 TB was reported as the leading cause of death among human immunodeficiency virus (HIV)-positive adults living in developing countries4.

Understanding the mechanism of action of antimycobacterial agents and the basis of resistance to these compounds has led to the possibility of predicting drug resistance in M. tuberculosis.10 The mechanism of resistance to RIF involves missense mutations in a well-characterised region of the rpoB gene. Resistance to INH is associated with a variety of mutations, affecting one or more genes.11 Mutation at codon 315 in the gene encoding the catalase-peroxidase (katG) is found in 60% to 70% of clinical isolates.11

Control of the TB epidemic depends on adequate treatment. Although chemotherapy has been an effective tool in the management and control of TB previously, development of drug resistance poses a serious challenge in the control of the disease. Potential factors contributing to development of drug resistance include inadequate treatment regimens prescribed by health staff, poor case holding of TB patients, poor drug supply, poor drug quality, patient error in following prescribed regimens and misuse of TB drugs including non-compliance.5 TB is treated using three front line agents, isoniazid (INH), rifampicin (RIF) and pyrazinamide (PZA), and one of the second line anti-TB drugs, streptomycin (SM) and ethambutol (EMB).6 Multidrug-resistant TB (MDR-TB), defined as resistance to at least INH and RIF with or without resistance to other drugs, has been reported in different parts of the world.7 However, the true magnitude of drug resistance worldwide is still unknown.

The inhA gene is associated with INH resistance; however, this resistance is not frequently reported in clinical isolates.11 The inhA promoter mutations seen are present at codon 24(G-T), and 16(A-G), or 8(T-G/A) and 15(C-T).11 The two gene operon (inhA locus) encoding the enoyl-acyl carrier protein reductase mutations results in over expression of the inhA gene leading to low level INH resistance in 30% of clinical isolates.11 The alkyl hydroperoxide reductase (ahpC) form part of the inhA gene but does not provide resistance of the organism to INH.11 The kasA gene which is important in fatty acid elongation has been shown not to cause INH resistance as mutations in these genes were also observed in INH susceptible strains.11 Codon 463 of the katG gene was also found not to be associated with INH resistance.11

The knowledge of development and spread of resistant Mycobacterium tuberculosis (MTB) is important in guiding TB control strategies. South Africa has a surveillance system for MDR MTB (MDR-TB) that covers

South Afr J Epidemiol Infect

11

2008;23(3)


Antimycobacterial susceptibility profiles

Better understanding of the geographic distribution of strains with these resistant genes and dynamics of dissemination and identification of populations at risk are facilitated by genotyping.12,13 Detection of mutations conferring resistance and the epidemiology of such mutants may reflect the extent of MDR-TB transmission and will assist TB control programmes.

Susceptibility profiles for INH, RIF, ethambutol (EMB) and streptomycin (SM) were obtained (Table 2) using the MGIT 960 instrument (Becton Dickinson Microbiology Systems, Sparks, MD, USA) according to the manufacturer’s instructions.

DNA extraction, PCR, and sequencing

This study therefore investigated the mutations in the rpoB and katG genes responsible for resistance to RIF and INH, respectively, in MTB isolates obtained from Dr George Mukhari Hospital, South Africa, using DNA sequencing. The data will assist in the identification of molecular epidemiological markers of isolates recovered from this particular geographical region, and may assist in understanding the spread of MDR-TB in the area.

DNA was extracted with the Qiagen MinElute PCR purification kit (QIAgen, Valencia, CA, USA), according to the manufacturer’s instructions, and stored at 4°C. Amplification of the katG gene was performed using primers mentioned in Table 1, in a MyCycler thermal cycler (BioRad, South Africa) according to a previously described method15 with slight modifications on cycling conditions (Table 1). The PCR reaction mixture of 50 μl, contained 0.2 μg of genomic DNA, 20 pmol of each primer, 0.2 µM of dNTPs, 1X PCR buffer (10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 50 mM KCl) and 2.5 U of Taq DNA polymerase (White Sci, South Africa). The PCR mixture for rpoB gene15 amplification contained 50 ng of template DNA, 20 pmol of each primer, 2 U of Taq DNA polymerase, 10 mM Tris-HCl (pH 8.4), 2 mM MgCl2, and 200 µM of dNTPs (White Sci, South Africa) in a final reaction volume of 50 µl. The PCR was performed in a MyCycler thermal cycler (BioRad, South Africa) using the conditions mentioned in Table 1.

Materials and methods Ethical clearance This study was approved by the Research and Ethical Committees of the University of Venda, University of Limpopo (Medunsa Campus) and the Department of Health and Welfare, Limpopo province, South Africa.

Sample sources, processing and staining of specimens The present study was conducted in the TB laboratory at the Dr George Mukhari (DGM) Hospital and 21,913 sputum specimens were included for investigation. If more than one culture-positive specimen per patient were processed, only one positive result was registered for the patient; similarly, if several negative cultures per patient were encountered, without a positive finding, only one negative result was recorded. Specimens were received from DGM Hospital and the clinics in the surrounding area between January 2005 and December 2006. DGM Hospital is a 1,200-bed tertiary referral hospital attached to the Medunsa Campus of the University of Limpopo. Sputum specimens are routinely sent to DGM Hospital microbiology laboratory for TB analysis. Inclusion criteria for sending sputum specimen is from patients were according to the Department of Health guidelines which include clinically suspected TB, treatment failure and retreatment among patients. Processing and staining for microscopy was performed as previously described.14

The amplified DNA fragments were visualised on the UV transilluminator. PCR products were sequenced with a SCE2410 capillary sequencer (Spectrumedix). Sequenced amplicons were examined and compared with the corresponding sequences of the standard H37Rv strain.

Results Antibiotic susceptibility profiles of isolates A total of 9.9% (2,168/21,913) MTB isolates was obtained from sputum specimens cultured in the laboratory during the study period. Of the 11.1% (240/2,168) resistant isolates, 59.6% (143/240) were MDR, 18.3% (44/240) showed resistance to INH and 4.6% (11/240) to RIF alone (Table 2).

PCR and sequencing DNA was isolated from 240 resistant isolates and the control strain H37RV that was susceptible to all four drugs. Sequencing of the PCR products on the rpoB gene hotspot region showed nucleotide substitution at codon 516 (GAC to GTC) in 42.2% (68/161) and codon 526 causing a codon change at CAC to GTC in 37.3% (60/161) of 67.1% (161/240) RIFresistant isolates studied. One isolate 0.6% (1/161) showed the mutation in codon 512 AGC to ACC, 515 ATG to CAT, 516 GAC to GTC, 517 CAG to

Culture, isolation and identification All specimens were processed for culture in the BacT/Alert 3D (BA; bioMérieux, Durham, USA) system. Positive cultures were stained with Ziehl-Neelsen staining and confirmed using the AccuProbe DNA hybridisation assay (GenProbe) according to the manufacturer’s protocol. Isolates were then advanced for susceptibility testing.

Table 1: Primers used for amplification of rpoB and katG genes from resistant Mycobacterium tuberculosis isolated from Dr George Mukhari Hospital Gene

Position

Base pair size Cycling conditions

Reference number

rpoB

Forward Reverse

5'- GGG AGC GGA TGA CCA CCC-3' 2266-2283 350bp 5'-GCG GTA CGG CGT TTC GAT GAA C-3' 2616-2592

Initial denaturation at 94°C for 3 min; 30 cycles of denaturation at 94°C for 45 sec, annealing at 65°C for 30 sec, and extension at 72°C for 30 sec, and a final extension at 72°C for 5 min.

katG

Forward Reverse

5'-AGC TCG TAT GGC ACC GGA AC-3' 5'-TTG ACC TCC CAC CCG ACT TG-3'

35 cycles of 94°C for 1 min, 45 to 60°C for 1 min, and 72°C for 2 min and extension of 5 minutes at 72°C.

904-923 620bp 1523-1502

16

16

Table 2: Trends in resistance of M. tuberculosis strains from sputum specimen sent to the TB laboratory at the Dr George Mukhari Hospital INHa N (%)

RIFa N (%)

44(18.3%) 11(4.6%)

EMBa N (%)

SMa N (%)

INH + RIFb N (%)

4(1.6%)

7(3.8%)

29(12%)

EMB + SMc N (%)

SM + RIFc N (%)

INH + EMBc N (%)

INH + SMc N (%)

2(0.8%)

7(3%)

4(1.6%)

10(4.2%)

INH + RIF + EMBc N (%)

INH + EMB + SMc N (%)

INH + SM + RIFc N (%)

INH + RIF + EMB + SMd N (%)

TOTAL RESIST

24(9.2%)

8(3.3%)

19(8%)

71(29.6%)

240(100)

Resistance to a single drug. MDR, (resistant to isoniazid and rifampicin). Combinations, (other combinations of resistance patterns). Polydrug resistance, (resistance to isoniazid, INH, rifampicin, RIF, ethambutol, EMB, and streptomycin, SM).

a

b

c


d

12

2008;23(3)


CCA, 518 AAC to GAA, 519 AAC to CAA, 525 ACC to CAC, 526 CAC to GAC, 529 CGA to CCC, 530 CTG to CCG, 531 TCG to TTG, 532 GCG to CGG and 533 CTG to CGC in the rpoB region. Overall, 17 different mutations were observed, 29.4% (5/17) single, 64.7% (11/17) double and 5.9% (1/17) triple mutations (Figure 1).

Drug resistance in MTB develops as a result of random genetic mutations in genes conferring resistance.17 These genes include the rpoB, katG, embA, rrs, rpsL and pncA which confer resistance to RIF, INH, EMB, SM and PZA, respectively. RIF and INH form the backbone of treatment against MTB.18

A total of 87.1% (209/240) MTB isolates was resistant to INH. Sequencing of the INH-resistant isolates showed a nucleotide substitution at codon 315 in 70% (168/240) of the resistant isolates, of which 95/168 (56.5%) had a codon change from AGC to ACC, 70/168 (41.7%) had a change from AGC to AAC, and 3/168 (1.8%) changed from AGC to ACA. A mutation at codon 314 (ACC to CCC) of 20/209 isolates contributed 9.6% of INH resistance (Figure 2).

The rpoB gene-coding region of MTB was originally analysed by DNA sequencing in 1993.17 RIF resistance is rarely observed as monoresistance and for this reason it is often considered a marker for MDR.18 The nucleotide sequences of the rpoB gene isolated from clinical strains were analysed for mutations on the 81 bp core region. The most prevalent mutation among the RIF-resistant isolates in our study was at codon 516 with 42.2%. However, it was more frequently detected than those reported in Latvia (11%)19 and in Taiwan20 (20.3%), and less frequent than that reported in Egypt15 (42%). Codon 526 was the second highest mutation observed at the DGM Hospital (37%). Compared to other reports, this rate was higher than reported in Greece (19%) and much higher than in the United States21 (7%), Germany22 (4%) and Japan23 (1%).

Discussion Detection of MDR-TB is of primary importance for both patient management and infection control. Different resistant patterns were observed. Of the 240 resistant MTB isolates, 59.6% (143/240) were MDR-TB. This value is higher than the one obtained in Mashhad, Iran, where 80 isolates were tested and only one (0.95%) of the isolates was MDR.16 In our study, resistance to INH alone was found in 18.3% (44/240) isolates and 4.6% (11/240) isolates were resistant to RIF alone.

Codon 530 showed mutation in 15% of the isolates in RIF-resistant isolates from DGM Hospital. Codon 531 is known to be a hot spot for rpoB gene mutations in MTB.21 However, only 3.1% of isolates in this study showed low frequency of mutation in codon 531 as compared

Figure 1: Summary of mutations at codons 512 to 533 in the rpoB gene. The wild-type sequence and amino acids are shown in the middle frame. Nucleotide changes are marked with arrows in the top frame, and the corresponding amino acid changes are denoted in the bottom frame. The nucleotide changes are subscripted with numbers that indicate the percentage of isolates harboring the change. Codons 516, 521, 526, and 530, 531 show novel mutations identified in different strains that were isolated from the TB laboratory at the Dr. George Mukhari Hospital.

Figure 2: Summary of mutations at codons 309 to 329 in the KatG gene. The wild-type sequence and amino acids are shown in the middle frame. Nucleotide changes are marked with arrows in the top frame, and the corresponding amino acid changes are denoted in the right. The amino acids are subscripted with numbers that indicate the percentages of isolates harboring the change. Amino acid change to threonine, asparagine, and isoleucine exhibit high degrees of polymorphism.


13

2008;23(3)


to the 54.9% reported in Taiwan.20 This mutation was more frequently observed in Germany19 (71%), Italy 20 (59%), Greece24 (56%), Japan23 (43%), the United States23 (32%) and Mozambique25 (21%). Published studies attest to the fact that isolates harboring mutations in codons 526 and 531 show high-level resistance to RIF as well as cross-resistance between rifabutin and RIF.26

8. Weyer K, Lancaster J, Van der Walt M, the DOTS-Plus Study group of SA. DOTS-Plus for multidrugresistant (MDR) tuberculosis in South Africa: results from the first cohort of patients treated with a standardised regimen under tuberculosis control programme conditions (abstract). Int J Tubercl Lung Dis 2002; 6(10): S138 9. Filliol I, Driscoll JR, Van Soolingen, et al. Snapshot of moving and expanding clones of Mycobacterium tuberculosis and their global distribution assessed by spoligotyping in an international study. J Clin Microbiol 2003; 41: 1963-1970 10. Torres MJ, Criado A, Palomares JC. Use of real-time PCR and fluorimetry for rapid detection of rifampin and isoniazid resistance associated mutations in Mycobacterium tuberculosis. J Clin Microbiol 2000; 38: 3194-3199

INH is a pro-drug that requires activation by the catalase-peroxidase enzyme produced by the MTB organism.27 The katG gene encodes the catalase peroxidase enzyme and is present in the variable regions of the MTB genome and contains a repetitive DNA sequence. It was reported that the MTB W (New York City) strain and its progeny may belong to the Beijing family.28 Specific features of this strain include a rare mutation in the katG codon 315 AGC to ACA (Ser to Thr) mutation.28 In this study, 3% of INH-resistant strains had mutation of 315 AGC to ACA. This percentage is lower than that observed in the St Petersburg area of Russia (92%), Lithuania (85.7%) and the Netherlands (89%).28,29 Other mutations shown in our isolates include 24% (AGC to AAC) described previously in isolates from Spain,30 10% (AGC to ATC) described in Turkey31 and 5% (AGC to CGC) previously described in Turkey.32 However, wider spectrums of other mutations were observed in the USA and other European countries.33

11. Johnson R, Streicher EM, Louw GE, Warren RM, Van Helden PD, Victor TC. Drug resistance in Mycobacterium tuberculosis. Curr Issues Mol Biol 2006; 8: 97-112 12. Sola C, Ferdinand S, Sechi LA, et al. Mycobacterium tuberculosis molecular evolution in western Mediterranean Islands of Sicily and Sardinia. Infect Genet Evol 2005; 5: 145-156 13. Kent PT, Kubica GP. Public health mycobacteriology: a guide for the level III laboratory. Atlanta (GA): Centers for Disease Control, 1985 14. Fujiki A. Bacteriology examination to stop TB manual. The research institute tuberculosis Japan, 2001 15. Siddiqi N, Shamim M, Hussain S. et al. Molecular characterization of multidrug-resistant isolates of Mycobacterium tuberculosis from patients in North India. Antimicrob Agents Chemother 2002; 46(2): 443-450 16. Namaei MH, Sadeghian A, Naderinasab M, Ziaee M. Prevalence of primary drug resistant Mycobacterium tuberculosis in Mashhad, Iran. Indian J Med Res 2006; 124: 77-80 17. Zhang Y, Telenti A. Genetics of drug resistance in Mycobacterium tuberculosis. In: Hatfull GF, Jacobs WR. Jr. (eds). Molecular genetics of mycobacteria. Washington DC: ASM Press; 2000: 235-251

Codon 315 mutations were shown to be associated with high-level resistance to INH.34 Results obtained from this study suggest that serine substitution at codon 315 of the katG gene is a characteristic of local INH-resistant strains and therefore can serve as a genetic marker for INH-resistance in our region. The other mutation observed was in codon 314 8.3% (ACC to CCC). The same mutation was observed in 25% of the isolates from Düzce, Turkey.27

18. Ahmed S, Araj GF, Akbar PK, Fares E, Chugh TD, Mustafa AS. Characterization of rpoB mutations in rifampin-resistant Mycobacterium tuberculosis isolates from the Middle East. J Diag Microb Inf Dis 2000; 38: 227-232 19. Tracevska T, Jansone I, Broka L, Baumanis V. Mutations in the rpoB and KatG genes leading to drug resistance in Mycobacterium tuberculosis in Latvia. J Clin Microb 2002; 40(10): 3789-3792 20. Jou R, Chen HY, Chiang CY, Yu MC, Su IJ. Genetic diversity of multidrug-resistant Mycobacterium tuberculosis isolates and identification of 11 novel rpoB alleles in Taiwan. J Clin Microb 2005; 43(3): 1390-1394

Ongoing investigation of resistance mechanisms involving antimycobacterial drugs should be conducted in South Africa and should include genes, other than the katG gene, known to be involved in INH resistance, especially the inhA gene which, as indicated in the introduction section, accounts for up to 30% of INH resistance, mainly low-level, in some studies.11 The present study provides valuable data on the different kinds of mutations occurring at various target loci in resistant MTB strains isolated at the DGM Hospital in South Africa. According to our knowledge, mutation at codon 314 in INH-resistant isolates has never been reported in Africa.

21. Kapur V, Li L-L, Iordanescu S. Characterization by automated DNA sequencing of mutations in the gene (rpoB) encoding the RNA polymerase b subunit in rifampicin-resistant Mycobacterium tuberculosis strains from New York City and Texas. J Clin Microbiol 1994; 32: 1095-1098 22. Rinder H, Dobner P, Feldmann K. Disequilibria in the distribution of rpoB alleles in rifampicinresistant M. tuberculosis isolates from Germany and Sierra Leone. Microbial Drug Resist 1997; 3: 195-197 23. Ohno H, Koga H, Kohno S. Relationship between rifampicin MICs for and rpoB mutations of Mycobacterium tuberculosis strains isolated in Japan. Antimicrob Agents Chemother 1996; 40: 1053-1056 24. Matsiota-Bernard P, Vrioni G, Marinis E. Characterization of rpoB mutations in rifampicin-resistant clinical Mycobacterium tuberculosis isolates from Greece. J Clin Microbiol 1998; 36: 20-23 25. Cougant DA, Sandven P, Eng J. Detection of rifampicin resistance among isolates of Mycobacterium tuberculosis from Mozambique. Microbial Drug Resist 1999; 4: 321-326

Acknowledgements

26. Ramaswami S, Musser JM. Molecular genetic basis of antimicrobial agent resistance in Mycobacterium tuberculosis: 1998 update. Tuber Lung Dis 1998; 79: 3-29

The authors are thankful to the laboratory management for allowing us to work in their departmental laboratory at the University of Limpopo, Medunsa Campus. We would like to also thank the staff of the TB section, Microbiological Pathology Laboratory, NHLS-DGM Tertiary Laboratory Complex, Pretoria, for technical assistance. This study was supported by the National Research Foundation, South Africa.

27. Ozturk EC, Sanic A, Kaya D, Ceyhan N. Molecular analysis of isoniazid, rifampin, and streptomycin resistance in Mycobacterium tuberculosis isolates from patients with tuberculosis in Düzce, Turkey. Jpn J Infect Dis 2005; 58: 309-312 28. Van Doorn HR, Kuijper EJ, Van der Ende A, et al. The susceptibility of Mycobacterium tuberculosis to isoniazid and the Arg to Leu mutation at codon 463 of katG are not associated. J Clin Microbiol 2001; 39: 1591-1594 29. Bakonyte D, Baranauskaite A, Cicenaite J, Sosnovskaja A, Stakenas P. Molecular characterization of isoniazid-resistant Mycobacterium tuberculosis clinical isolates in Lithuania. Antimicrob. Agents Chemother 2003; 47(6): 2009-2011

References 1. Global tuberculosis control: surveillance, planning, financing. WHO Report 2006. Geneva: World Health Organization: 2006 (WHO/HTM/TB/2006.362)

30. Herrera-León L, Molina T, Saíz P, Sáez-Nieto JA, Jiménez MS. New multiplex PCR for rapid detection of isoniazid-resistant Mycobacterium tuberculosis clinical isolates. J Antimicrob Agents Chemother 2005; 49(1): 144-147

2. Rampe AK, Hess A, Clay CG, Du Toit D. Characterisation of Mycobacterium tuberculosis strains isolated at Ga-Rankuwa Hospital by fingerprinting with insertion sequence IS6110. South Afr J Epidemiol Infect 2004; 19(3,4): 96-100 3. Lall N, Meyer JJM. In vitro inhibition of drug-resistant strains of Mycobacterium tuberculosis by ethnobotanically selected South African plants. J Ethnopham 1999; 66: 347-354

31. Yang Z, Durmaz R, Yang D, et al. Simultaneous detection of isoniazid, rifampin, and ethambutol resistance of Mycobacterium tuberculosis by a single multiplex allele-specific polymerase chain reaction (PCR) assay. Diag Micro Infec Dis 2005; 53: 201-208

4. Traoré HN, Meyer D. Necrosis of host cells and survival of pathogens following iron overload in an in vitro model of co-infection with human immunodeficiency virus (HIV) and Mycobacterium tuberculosis. Int J Antimicrob Agents 2007; doi:10.1016/j.ijantimicag.2006.11.09

32. Parsons LM, Salfinger M, Clobridge A, et al. Phenotypic and molecular characterization of Mycobacterium tuberculosis isolates resistant to both isoniazid and ethambutol. Antimicrob Chemother 2005; 49(6): 2218-2225

5. Crofton J, Chaulet P, Maher D, et al. Guidelines for the management of drug resistance in tuberculosis. WHO/TB/96.210 (Rev. 1) 1997 6. Arcus VL, Lott JS, Johnston JM, Backer EN. The potential impact of structural genomics on tuberculosis drug discovery. DDT 2006; 11: 28-34

33. Van Soolingen D, De Haas PE, Van Doorn HR, Kuijper E, Rinder H, Borgdorff MW. Mutations at amino acid position 315 of the katG gene are associated with high-level resistance to isoniazid, other drug resistance, and successful transmission of Mycobacterium tuberculosis in the Netherlands. J Infect Dis 2000; 182: 1788-1790

7. Mattila HJ, Soini H, Eerola E, et al. A Ser315Thr substitution in katG is predominant in genetically heterogeneous multidrug-resistant Mycobacterium tuberculosis isolates originating from the St Petersburg area in Russia. J Antimicrob Chemother 1998; 42(9): 2442-2445

34. Ozturk CE, Sanic A, Kaya D, Ceyhan Í. Molecular analysis of isoniazid, rifampin and streptomycin resistance in Mycobacterium tuberculosis isolates from patients with tuberculosis in Düzce, Turkey. Jpn J Infect Dis 2005; 58: 309-312


14

2008;23(3)