Scholarly article on topic 'Detection of fluoroquinolone resistance in Mycobacterium tuberculosis clinical isolates as determined by gyrA/B gene mutation by using PCR technique'

Detection of fluoroquinolone resistance in Mycobacterium tuberculosis clinical isolates as determined by gyrA/B gene mutation by using PCR technique Academic research paper on "Biological sciences"

CC BY-NC-ND
0
0
Share paper
OECD Field of science
Keywords
{Fluoroquinolone / "XDR TB" / "gyrA and gyrB gene"}

Abstract of research paper on Biological sciences, author of scientific article — A. Salah Eldin, N.M. Mostafa, S.I. Mostafa

Abstract Fluoroquinolones are broad-spectrum antimicrobial agents that have been used with increasing frequency over the past decade. Fluoroquinolones have in vitro and in vivo activity against Mycobacterium tuberculosis. However, resistance to fluoroquinolones in cases of tuberculosis is not routinely assessed. Mutations in a small region of gyrA, called quinolone resistance-determining region (QRDR) and, less frequently, in gyrB are the primary mechanism of FQ resistance in M. tuberculosis. PCR-based techniques provide new possibilities for the rapid diagnosis of first- and second-line drug resistance. Results There were 40 consecutive adults, who had culture confirmed pulmonary tuberculosis during the study period. Mutations were observed in the QRDRs of both gyrA and gyrB in 22 isolates (55%). Only gyrA +ve in 7(17.5%) isolates. Only gyrB +ve in 5(12.5%) isolates. Total gyrA +ve in 29(72.5%) and total gyrB +ve in 28(70%) isolates. Both gyrA and gyrB −ve in 6 (15%). Conclusion The incidence of FO-resistant M. tuberculosis is gradually increasing to alarming levels this may be due to wide spread use of this vital groups of drugs in community-acquired pneumonia and urinary tract infections.

Academic research paper on topic "Detection of fluoroquinolone resistance in Mycobacterium tuberculosis clinical isolates as determined by gyrA/B gene mutation by using PCR technique"

Egyptian Journal of Chest Diseases and Tuberculosis (2012) 61, 349-353

The Egyptian Society of Chest Diseases and Tuberculosis Egyptian Journal of Chest Diseases and Tuberculosis

www.elsevier.com/locate/ejcdt www.sciencedirect.com

ORIGINAL ARTICLE

Detection of fluoroquinolone resistance in Mycobacterium tuberculosis clinical isolates as determined by gyrA/B gene mutation by using PCR technique

A. Salah Eldin a *, N.M. Mostafa b, S.I. Mostafa c

a Lecturer of Chest Department, Faculty of medicine, Beni-Suef University, Egypt b Lecturer of Chest Department, Faculty of medicine, Cairo University, Egypt c Lecturer of Clinical Pathology Department, Faculty of medicine, Beni-Suef University, Egypt

Received 1 August 2012; accepted 10 August 2012 Available online 7 March 2013

KEYWORDS

Fluoroquinolone; XDR TB;

gyrA and gyrB gene

Abstract Fluoroquinolones are broad-spectrum antimicrobial agents that have been used with increasing frequency over the past decade.

Fluoroquinolones have in vitro and in vivo activity against Mycobacterium tuberculosis.

However, resistance to fluoroquinolones in cases of tuberculosis is not routinely assessed.

Mutations in a small region of gyrA, called quinolone resistance-determining region (QRDR) and, less frequently, in gyrB are the primary mechanism of FQ resistance in M. tuberculosis.

PCR-based techniques provide new possibilities for the rapid diagnosis of first- and second-line drug resistance.

Results: There were 40 consecutive adults, who had culture confirmed pulmonary tuberculosis during the study period.

Mutations were observed in the QRDRs of both gyrA and gyrB in 22 isolates (55%). Only gyrA + ve in 7(17.5%) isolates. Only gyrB +ve in 5(12.5%) isolates. Total gyrA +ve in 29(72.5%) and total gyrB +ve in 28(70%) isolates. Both gyrA and gyrB —ve in 6 (15%).

Conclusion: The incidence of FO-resistant M. tuberculosis is gradually increasing to alarming levels this may be due to wide spread use of this vital groups of drugs in community-acquired pneumonia and urinary tract infections.

© 2013 The Egyptian Society of Chest Diseases and Tuberculosis. Production and hosting by Elsevier B.V.

All rights reserved.

* Corresponding author. Tel.: +20 01005240055. E-mail address: Ab_salah1972@yahoo.com (A. Salah Eldin). Peer review under responsibility of The Egyptian Society of Chest Diseases and Tuberculosis.

Introduction

The World Health Organization (WHO) estimated that there were 0.5 million cases of multi-drug resistant (MDR) tuberculosis (TB) in 2007. Only 8.5% of the estimated global total of smear-positive cases of MDR-TB were notified. By the end of 2008, 55 countries and territories had reported at least one case

0422-7638 © 2013 The Egyptian Society of Chest Diseases and Tuberculosis. Production and hosting by Elsevier B.V. All rights reserved. http://dx.doi.Org/10.1016/j.ejcdt.2012.08.003

of extensively drug-resistant TB (XDR-TB) which are defined as MDR strains that are also resistant to a fluoroquinolone (FQ) and at least one second-line injectable agent (amikacin (AM), Kanamycin (KM) and/or capreomycin (CM) [1].

Fluoroquinolones are broad-spectrum antimicrobial agents that have been used with increasing frequency over the past decade. The particular advantages of fluoroquinolones are their high bioavailability, convenient dosing intervals, and efficacy against a wide array of bacterial infections, including community-acquired pneumonia [2,3].

Fluoroquinolones—in particular, levofloxacin, gatifloxacin, and moxifloxacin—have in vitro and in vivo activity against Mycobacterium tuberculosis [4,5]. Fluoroquinolone resistance in patients with newly diagnosed cases of tuberculosis would be important if identified, because of the current role of fluoro-quinolones in the treatment of tuberculosis (i.e., treatment of patients resistant to or intolerant of first-line therapy) and the potential of this class of drugs to become first line therapy [6,7].

However, resistance to fluoroquinolones in cases of tuberculosis is not routinely assessed, particularly in isolates that are susceptible to current first-line agents.

Current Infectious Diseases Society of America/American Thoracic Society guidelines for community acquired pneumonia recommend that fluoroquinolones be used for both inpatient and outpatient treatment of pneumonia [8].

As a result, fluoroquinolones are frequently prescribed to people who are subsequently diagnosed with tuberculosis.

Among a cohort of patients with tuberculosis in Tennessee from 2000 to 2004, 23% received fluoroquinolone monotherapy before diagnosis. The proportion of exposed people increased from 9% in 2000 to 41% in 2004 (P, 0.001) [9].

The cellular target of FQs in M. tuberculosis is DNA gyr-ase, a type II topoisomerase consisting of two A and two B subunits encoded by gyrA and gyrB genes, respectively [10].

Mutations in a small region of gyrA, called quinolone resistance-determining region (QRDR) and, less frequently, in gyrB are the primary mechanism of FQ resistance in M. tuberculosis [11,12].

Analysis of QRDR alone by genotypic tests has been suggested as sufficient for rapid identification of vast majority of FQ-resistant M. tuberculosis strains as additional target of gyrB did not enhance the sensitivity significantly [13,14].

Reports show that the majority (approximately 50-90%) of FQ-resistant MTB isolates carry mutations in the quinolone resistance-determining region (QRDR) of the gyrA gene [14], and that a small number have mutations in the gyrB gene [15,16]. It was previously postulated that efflux pump mechanisms account for FQ resistance in isolates with wild-type gyr-AB genes [17].

Fluoroquinolone resistance in M. tuberculosis can develop after as little as 13 days of fluoroquinolone therapy [10]. Although fluoroquinolone resistance in M. tuberculosis is not routinely assessed, the proportion of newly diagnosed (i.e., previously untreated patients with tuberculosis with fluoro-quinolone resistance has ranged from 0.15% to 3.6% in previous reports [18,19].

Extensive use of fluoroquinolones for treatment of bacterial infections might result in primary fluoroquinolone-resistant tuberculosis. If primary resistance became common, this would negate the potential of fluoroquinolones to become part of first-line tuberculosis treatment. PCR-based techniques

provide new possibilities for the rapid diagnosis of first- and second-line drug resistance, however, not all mycobacterial laboratories have access to DNA sequencing facilities [20].

Therefore, we evaluated the rate of fluoroquinolone resistance among M. tuberculosis isolates.

Materials and methods

Patient population

We conducted a cohort study of adult patients (age >18 years) with sputum positive, culture confirmed pulmonary tuberculosis from both El Abassia & El Omrania Chest hospitals in the period between September 2011 and February 2012. This study was approved from ministry of health tuberculosis control program.

Early-morning sputum specimens were collected and transported to the clinical laboratory for smear examination.

Detailed history taking was obtained from patients as regards previous history of anti tuberculous treatment, recent antibiotic used especially fluroquinolone.

Sample collection & transport

The patient was instructed to cough deeply and expectorated sputum specimens were collected from all patients in a screw-cap, leak-proof sterile containers. Specimens were handled with care as regards collection and transportation.

Decontamination and concentration

The initial concentration of NaOH is 4%. This 4% NaOH solution is mixed with an equal quantity of sodium citrate solution (2.9%) to make a working solution (NaOH concentration in this solution is 2%). When an equal quantity of NaOH-NALC-citrate and sputum are mixed, the final concentration of NaOH in the specimen is 1% [21]. Transfer the specimen to a 50 ml centrifuge tube with a screw cap.

Add NaOH-NALC-sodium citrate solution in a volume equal to the quantity of specimen. Tighten the cap.

Vortex lightly or hand mix for about 15-30 s. Invert the tube so the whole tube is exposed to the NaOH-NALC solution.

Wait 15-20 min (up to 25 min maximum) after adding the NaOH-NALC solution. Vortex lightly or hand mix/invert every 5-10 min or put the tubes on a shaker and shake lightly during the whole time.

Make sure the specimen is completely liquefied. If still mu-coid, add a small quantity of NALC powder (30-35 g) directly to the specimen tube. Mix well.

At the end of 15-20 min, add phosphate buffer (pH 6.8) up to the top ring on the centrifuge tube (plastic tube has a ring for 50 ml mark). Mix well (lightly vortex or invert several times). Addition of sterile water is not a suitable alternative for the phosphate buffer.

Centrifuge the specimen at a speed of 3000g or more for 1520 min. Use of refrigerated centrifugation at a higher speed is known to increase recovery of mycobacteria 2, 62.

After centrifugation, allow tubes to sit for 5 min to allow aerosols to settle. Then carefully decant the supernatant into

a suitable container containing a mycobactericidal disinfectant. Make sure the sediment is not lost during decanting of the supernatant fluid. Add a small quantity (1-2 ml) phosphate buffer (pH 6.8) and resuspend the sediment with the help of a pipette or vortex mixer.

Use the resuspended pellet for making smears and for inoculation of MGIT tubes [22]. Ziehl-Neelsen staining of M. tuberculosis was done according to standards [23].

Culture

Culture was done using Mycobacteria Growth Index Test (MGIT).[24].

Molecular detection of gyrA & gyrB was done according to Soudani et al. [25].

DNA extraction

Colony was taken from MGIT Template DNA was prepared by QIAGEN QIAmp DNA mini kit. Positive control was used Escherichia coli, Staphylococcus aureus resistant to ciprofloxacin.

Amplification

Using Perkin Elmer 9700 for gyrA, a DNA fragment of 216 bp, corresponding to the QRDR, was generated by PCR with the primer pair Pri9 (50-CGCCGGGTGCTCTATG-CAATG-30) and Pri8 (50-CGGTGGGTCATTGCCTGGC-GA-30), used at 4 lM. Amplification reactions were performed as previously described by [26]. For gyrB, a 322-bp fragment was amplified using the primer pair gyrBA (50-GAGTTGGTGCGCGCTAAGAGC-30) and gyrB E (50-CGGCCATCAGCACGATCTTG-30) at 0.4 lM. Amplification reactions were performed as previously described by Dauendorffer et al. [27].

Detection of (QRDR) by PCR

Figure 1 The above gel is for gyrA the (ladder) used is from 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 till 2000 and gyrA is at 216 bp, LAN 1 from the right side is for the marker (ladder), LAN 2-9, 11 and 12 are positive for gyraA, and the gel below is for gyrB. gyrB is at 322 bp, the marker (ladder) used is from 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 till 2000 from the right side LAN 1-6, 8 and 9 are positive for gyra B, LAN 10 is for the ladder. (This photo is taken by gel documentation system).

Table 1 Clinical data of patients selected.

Sex 34 Male 6 Female

Age 18-72 years old Mean age 47.1

Cases 28 New cases (70%) 12 Relapse (30%)

Risk factors 9 Diabetics

4 addicts one on

steroids (IPF)

Amplified products were subjected to electrophoresis in 2% agarose gels in Tris-borate-EDTA buffer and visualized under UV light. The gyrA band was visualized at 216 bp and gyrB at 322 bp.

Results

There were 40 consecutive adults, who had culture confirmed pulmonary tuberculosis during the study period. Six Female (15%) & 34 (85%) males Mean age 47.1 years (32-72 years)

Twenty eight (70%) newly diagnosed (patients were deemed to have newly diagnosed tuberculosis if they had not received standard antituberculosis treatment before developing culture-confirmed tuberculosis). Twelve relapse (30%)

Twelve patients were associated with risk factors for lower immunity (9 patients (22.5%) were diabetics, and 3 patients (7.5%) were drug addict).

All cases had chest X-ray findings (infiltration, cavitation). (See Fig. 1, Tables 1 and 2).

Table 2 Laboratory findings.

Zeil Neelsen for Sputum —ve and MGIT —ve 3

Zeil Neelsen for Sputum — ve and MGIT +ve 1

Zeil Neelsen for Sputum +ve and MGIT +ve 34

Zeil Neelsen for Sputum +ve and MGIT —ve 3

Only gyrA +ve 7(17.5%)

Only gyrB +ve 5(12.5%)

Both gyrA and gyrB +ve 22(55%)

Both gyrA and gyrB — ve 6(15%)

gyrA +ve 29(72.5%)

gyrB + ve 28(70%)

Mutations were observed in the QRDRs of both gyrA and gyrB in 22 isolates (55%). Only gyrA +ve in 7(17.5%) isolates. Only gyrB + ve in 5(12.5%) isolates. Total gyrA +ve in 29(72.5%) and total gyr B +ve in 28(70%) isolates. Both gyrA and gyrB —ve in 6(15%).

Discussion

This study on fluoroquinolone resistance among smear-positive pulmonary TB cases is the first of its kind in Egypt.

Fluoroquinolones (FQs) are the most promising antituber-culous therapeutic agents to be developed in 40 years [28].They are widely used for the treatment of multidrug-resistant (MDR) tuberculosis (TB) despite the lack of clinical trials evaluating optimal doses, duration, and combinations [29]. There is concern about levels of preexisting FQ-resistant TB in regions with high drug resistance rates because these drugs are often available over the counter and are additionally prescribed as broad-spectrum antibiotics for the treatment of undiagnosed respiratory infections [30].

Mutations in short regions of gyrA, known as QRDR, have been associated with FQ resistance in MTB [31]. Several studies [32] have analyzed the mutations in the gyrA gene in clinical isolates of MTB. Most of these studies focused on the frequency of the mutations in gyrA/gyrB genes in FQ-resistant MTB strains. Thereare, however, no data on the association of mutations in gyrA/gyrB and FQ resistance levels in MTB isolates.

Only Yin et al. have shown conclusively that different substitutions of amino acid 94 resulted in different levels of levo-floxacin resistance [33].

Although the sample size in our study was substantially small, the incidence of infection with fluoroquinolone-resistant M. tuberculosis appeared to be high. This may have been due at least in part to increased use of fluoroquinolones for the treatment of lower respiratory tract infection. One rather important mechanism for the development of fluoroquino-lone-resistant TB is the suboptimal use of second line drug regimens, especially in the presence of a poorly functioning program in the treatment of multidrug-resistant TB.

In our study 55% of the clinical isolates were positive for both gyrA and gyrB, this agree with Yin and Yu [32] as they detect 44 of 60 (73.3%) levofloxacin-resistant MTB clinical isolates, including 17 of 18 (94.4%) high-level resistant strains and 27 of 42 (64.3%) low-level resistant strains, had mutation in QRDR of gyrA gene.

The mutation patterns involved six patterns of single codon mutation (H70R, A90V, S91A, D94G, D94A or D94N) and one pattern of double codons mutation (A90V with D94A). Of 60 levofloxacin-resistant MTB clinical isolates, only one (1.6%) mutated in gyrB gene (T511N) [31].

Also agree with Zhenling et al. [34]. Mutations were observed in the QRDRs of gyrA/gyrB in 87 out of 95 (91.6%) OFX-resistant MTB strains [34].

Mutations other than those affecting gyrA and other mechanisms could result in FQ-R, including: decreased cell-wall permeability to drug, efflux pumps, drug sequestration or perhaps even drug inactivation [35]. In a small number of cases, FQ-R could be associated with gyrB mutations and a probable efflux mechanism [36].

Our study was limited by the lack of the patients fluroqui-nolone exposure data. There is also natural polymorphism in the nucleotide sequences of gyrA/gyrB, that is not associated with drug resistance as natural polymorphism occur in both fluoroquinolone sensitive and resistant bacteria, so we recommend performing DNA sequencing and concomitant drug susceptibility testing.

Conclusion

The incidence of FO-resistant M. tuberculosis is gradually increasing to alarming levels this may be due to wide spread

use of this vital groups of drugs in community-acquired pneumonia and urinary tract infections.

Recommendations

We recommend judicious use of fluoroquinolones as a broad spectrum antibiotics and it is ideally to be reserved for treatment of resistant TB or at least limit the use of prolonged or repeated courses of FQ in patients at risk of having active TB.

We recommend further researches as DNA sequencing to exclude natural polymorphisms and detect different kinds of mutations affecting the genes. Also, recommend drug sensitivity testing to correlate the degree of resistance and drug inhibition with gene mutation and the possible methods to overcome this problem.

References

[1] 2010 Wr: Global tuberculosis control. 2010. www.who.int/tb/ publications/global_report/2010/en/index.html.

[2] J.G. Bartlett, S.F. Dowell, L.A. Mandell, T.M. File Jr., D.M. Musher, M.J. Fine, Practice guidelines for the management of community-acquired pneumonia in adults. Infectious Diseases Society of America, Clin. Infect. Dis. 31 (2000) 347-382.

[3] M.S. Niederman, L.A. Mandell, A. Anzueto, et al, Guidelines for the management of adults with community-acquired pneumonia: diagnosis, assessment of severity, antimicrobial therapy, and prevention, Am. J. Respir. Crit. Care Med. 163 (2001) 1730-1754.

[4] B. Ji, N. Lounis, C. Truffot-Pernot, J. Grosset, In vitro and in vivo activities of levofloxacin against Mycobacterium tuberculosis, Antimicrob. Agents Chemother. 39 (1995) 13411344.

[5] B. Ji, N. Lounis, C. Maslo, C. Truffot-Pernot, P. Bonnafous, J. Grosset, In vitro and in vivo activities of moxifloxacin and clinafloxacin against Mycobacterium tuberculosis, Antimicrob. Agents Chemother. 42 (1998) 2066-2069.

[6] S.H. Gillespie, N. Kennedy, Fluoroquinolones: a new treatment for tuberculosis?, Int J. Tuberc. Lung Dis. 2 (1998) 265-271.

[7] K. Tahaoglu, T. Torun, T. Sevim, et al, The treatment of multidrug resistant tuberculosis in Turkey, New Engl. J. Med. 345 (2001) 170-174.

[8] L. Mandell, R. Wunderink, A. Anzueto, J. Bartlett, G. Campbell, N. Dean, S. Dowell, T. File Jr., D. Musher, M. Niederman, et al, Infectious Diseases Society of America/ American Thoracic Society consensus guidelines on the management of community-acquired pneumonia in adults, Clin. Infect. Dis. 44 (2007) S27-S72.

[9] P.D. Gaba, C. Haley, M R. Griffin, E. Mitchel, J. Warkentin, E. Holt, P. Baggett, T.R. Sterling, Increasing outpatient fluoroquinolone exposure before tuberculosis diagnosis and impact on culture-negative disease, Arch. Intern. Med. 167 (2007) 2317-2322.

[10] A.S. Ginsburg, S.C. Woolwine, N. Hooper, W.H. Benjamin Jr., W.R. Bishai, S.E. Dorman, T.R. Sterling, The rapid development of fluoroquinolone resistance in M. tuberculosis, New Engl. J. Med. 349 (2003) 1977-1978.

[11] A.S. Ginsburg, J.H. Grosset, W.R. Bishai, Fluoroquinolones, tuberculosis, and resistance, Lancet Infect. Dis. 3 (2003) 432442.

[12] K.C. Chang, W.W. Yew, R.C.Y. Chan, Rapid assays for fluoroquinolone resistance in Mycobacterium tuberculosis: a systematic review and meta analysis', Antimicrob. Chemother. 65 (2010) 1551-1561.

[13] D. Hillemann, S. Rusch-Gerdes, E. Richter, Feasibility of the Geno Type MTBDRsl assay for fluoroquinolone, amikacin-

capreomycin and ethambutol resistance testing of Mycobacterium tuberculosis strains and clinical specimens, J. Clin. Microbiol. 47 (2009) 1767-1772.

[14] I. Mokrousov, T. Otten, O. Manicheva, Y. Potapova, B. Vishnevsky, O. Narvskaya, N. Rastogi, Molecular characterization of ofloxacin-resistant Mycobacterium tuberculosis strains from Russia, Antimicrob. Agents Chemother. 52 (2008) 2937-2939.

[15] D.A. Duong, T.H. Nguyen, T.N. Nguyen, V.H. Dai, T.M. Dang, S.K. Vo, D.A. Do, V.V. Nguyen, H.D. Nguyen, N.S. Dinh, J. Farrar, M. Caws, Beijing genotype of Mycobacterium tuberculosis is significantly associated with high-level fluoroquinolone resistance in Vietnam, Antimicrob. Agents Chemother. 53 (2009) 4835-4839.

[16] S. Feuerriegel, H.S. Cox, N. Zarkua, H.A. Karimovich, K. Braker, S. Riisch-Gerdes, S. Niemann, Sequence analyses of just four genes to detect extensively drug-resistant Mycobacterium tuberculosis strains in multidrug-resistant tuberculosis patients undergoing treatment, Antimicrob. Agents Chemother. 53 (2009)3353-33561.

[17] I. Escribano, J.C. Rodriguez, B. Llorca, E. Garcia-Pachon, M. Ruiz, G. Royo, Importance of the efflux pump systems in the resistance of Mycobacterium tuberculosis to fluoroquinolones and linezolid, Chemotherapy 53 (2007) 397-401.

[18] L. Bozeman, W. Burman, B. Metchok, L. Welch, M. Weiner, Tuberculosis Trials Consortium. Fluoroquinolone susceptibility among mycobacterium tuberculosis isolates from the United States and Canada, Clin. Infect. Dis. 40 (2002) 386-391.

[19] R. Devasia, M. Griffin, A. Blackman, S. May, E. Holt, T. Smith, J. Warkentin, E. Mitchel, Trends in fluoroquinolone resistance and exposure in newly diagnosed tuberculosis: 2002-2006 [abstract], Am. J. Respir. Crit. Care Med. 177 (2008) A19.

[20] A. Somoskovi, J. Dormandy, D. Mitsani, J. Rivenburg, M. Salfinger, Use of smear-positive samples to assess the PCR-based GenoType MTBDR assay for rapid, direct detection of the Mycobacterium tuberculosis complex as well as its resistance to isoniazid and rifampin, J. Clin. Microbiol. 44 (2006) 44594463.

[21] W.K. Aldous, J.I. Pounder, J.L. Cloud, L. Gail, Comparison of six methods of extracting Mycobacterium tuberculosis DNA from processed sputum for testing by quantitative real-time PCR, J. Clin. Microbiol. (2005) 2471-2473.

[22] P.T. Kent, G.P. Kubica, Public Health Microbiology, a Guide for the Level III Laboratory. Centers for Disease Control, Division of Laboratory Training and Consultation, US Department of Health and Human Services, US Government Printing Office, Atlanta, GA, 1985.

[23] Ming-Chih Yu, Huang-Yao Chen, Mei-Hua Wu, Wei-Lun Huang, Yuh-Min Kuo, Fang-Lan Yu, Ruwen Jou, Evaluation of the rapid MGIT TBc identification test for culture confirmation of Mycobacterium tuberculosis complex strain detection, J. Clin. Microbiol. (2011) 802-807.

[24] D.H. Mueller, L. Mwenge, M. Muyoyeta, M.W. Muvwimi, R. Tembwe, R. McNerney, P. Godfrey-Faussett, H.M. Ayles, Costs and cost-effectiveness of tuberculosis cultures using solid and liquid media in a developing country, Int. J. Tuberc. Lung Dis. 12 (10) (2008) 1196-1202.

[25] A. Soudani, S. Hadjfredj, M. Zribil, T. Messaoud, A. Masmoudi, B. Majed, C. Fendri, First report of molecular characterization of fluoroquinolone-resistant Mycobacterium tuberculosis isolates from a Tunisian hospital, Clin. Microbiol. Infect. 16 (2010) 1454-1457.

[26] I. Guillemin, V. Jarlier, E. Cambau, Correlation between quinolone susceptibility patterns and sequences in the A and B subunits of DNA gyrase in mycobacteria, Antimicrob. Agents Ch. 42 (8) (1998) 2084.

[27] J.N. Dauendorffer, I. Guillemin, A. Aubry, et al, Identification of mycobacterial species by PCR sequencing of quinolone resistance determining regions of DNA gyrase genes, J. Clin. Microbiol. 41 (2003) 1311-1315.

[28] L.E. Ziganshina, S.B. Squire, Fluoroquinolones for treating tuberculosis. Cochrane Database Syst. Rev. 4 (2007) CD004795, http://dx.doi.org/10.1002/14651858.CD004795.pub3.

[29] World Health Organization, Guidelines for the programmatic management of drug-resistant tuberculosis, WHO/HTM/ TB2006, World Health Organization, Geneva, Switzerland, 2006.

[30] Long, R., H. Chong, V. Hoeppner, H. Shanmuganathan, K. Kowalewska-Grochowska, C. Shandro, J. Manfreda, A. Senthilselvan, A. Elzainy, T. Marrie: Empirical treatment of community-acquired pneumonia and the development of fluoroquinolone-resistant tuberculosis. Clin. Infect. Dis. 48 (2009) 1354-136.

[31] H.E. Takiff, H.E. Salazar Takiff, L. Salazar, C. Guerrero, W. Philipp, W.M. Huang, B. Kreiswirth, S.T. Cole, W.R. Jacobs Jr., A. Telenti, Cloning and nucleotide sequence of Mycobacterium tuberculosis gyrA and gyrB genes and detection of quinolone resistance mutations, Antimicrob. Agents Chemother. 38 (1994) 773-780.

[32] X. Yin, Z. Yu, Mutation resistant Mycobacterium tuberculosis clinical isolates from Guangdong Province in China, J. Infect. 61 (2) (2010) 150-154 [Epub May, 2010].

[33] S. Feuerriegel, H.S. Cox, N. Zarkua, H.A. Karimovich, K. Braker, S. Rüsch-Gerdes, S. Niemann, Sequence analyses of just four genes to detect extensively drug-resistant Mycobacterium tuberculosis strains in multidrug-resistant tuberculosis patients undergoing treatment, Antimicrob. Agents Chemother. 53 (2009) 3353-3356.

[34] Zhenling Cui, Jie Wang, Junmei Lu, Xiaochen Huang, Zhongyi Hu, Association of mutation patterns in gyrA/B genes and ofloxacin resistance levels in Mycobacterium tuberculosis isolates from East China in 2009 (Abstract), BMC Infect. Dis. 2011, 11:78. < http://www.biomedcentral.com1471-2334/11/78 >.

[35] M.B. Conde, A. Efron, C. Loredo, G R. De Souza, N.P. Graca, M.C. Cezar, M. Ram, M.A. Chaudhary, W.R. Bishai, A.L. Kritski, et al, Moxifloxacin versus ethambutol n the initial treatment of tuberculosis: a double-blind, randomised, controlled phase II trial, Lancet 373 (9670) (2009) 1183-1189.

[36] R. Rustomjee, C. Lienhardt, T. Kanyok, G.R. Davies, J. Levin, T. Mthiyane, C. Reddy, A.W. Sturm, F.A. Sirgel, J. Allen, et al, A phase II study of the sterilising activities of ofloxacin, gatifloxacin and moxifloxacin in pulmonary tuberculosis, Int. J. Tuberc. Lung Dis. 12 (2) (2008) 128-138.