Indian Journal of Medical Microbiology Medknow Publications on behalf of Indian Association of Medical Microbiology ISSN: 0255-0857 EISSN: 1998-3646 Vol. 29, Num. 3, 2011, pp. 280-282

Indian Journal of Medical Microbiology, Vol. 29, No. 3, July-September, 2011, pp. 280-282

Original Article

Assessment of trends of ofloxacin resistance in Mycobacterium tuberculosis

JS Verma¹, D Nair¹, D Rawat¹, N Manzoor²

¹ Department of Microbiology, VMMC and Safdarjung Hospital, New Delhi, India
² Department of Bio-Sciences, Jamia Millia Islamia University, New Delhi, India
Correspondence Address: D Nair, Department of Microbiology, VMMC and Safdarjung Hospital, New Delhi, India, Deepthinair2@gmail.com

Date of Submission: 06-Dec-2010 
Date of Acceptance: 06-Mar-2011

Code Number: mb11067

PMID: 21860110

DOI: 10.4103/0255-0857.83913

Abstract

Keywords: Fluoroquinolone, MDR-TB, ofloxacin

Introduction

Despite all the advances made in tuberculosis (TB) treatment and management, India accounts for one-fifth of the global incidence ad two-thirrd of the cases in South-East Asia.^[1] The increasing global burden of this disease and the escalating problem of multidrug resistant (MDR) and extensive drug-resistant tuberculosis (XDR-TB) have led to increase pressure on using second-line drugs (SLDs).^[2] Several studies have shown that MDR-TB can be cured by a combination of second-line drugs. Among them, Ofloxacin (OFX) is one of the potent fluoroquinolone (FQ) recommended for use as prophylaxis treatment of patients exposed to MDR-TB (defined as simultaneously resistant to at least isoniazid and rifampicin), for treatment of proven MDR-TB, for empirical treatment of TB disease in settings with high rates of MDR-TB and for patients with severe adverse reaction to first-line agents.^[3],[4] Clinical trials have demonstrated their efficacy in combination with other drugs for treatment of MDR-TB strains.^[5]

They have excellent pharmacokinetic profiles, achieve good tissue and cellular distribution with minimal adverse effects.^[6],[7] But over the past decade, the increased use of FQ in the community, especially to treat other respiratory infectious diseases caused their decreased susceptibility in Mycobacterium tuberculosis (M. tuberculosis) strains.^[8],[9]

As SLDs susceptibility testing is performed only in few reference laboratories in India, therefore the data on OFX resistance trends is limited. There were some data ^{[6],[10],[11],[12],[13]} on OFX susceptibility which showed variable performance of the drug and highlighted significant increase in OFX resistance in last 5 years. It is therefore critically important to determine the potential of OFX before they would be used as second-line anti-TB agent in the management of drug-resistant TB. This is especially important for those patients with recurrent TB after treatment, those with MDR-TB as well as those who have previously received FQ therapy. With this objective, a study was undertaken to assess the status and extent of OFX resistance in M. tuberculosis strains in a defined population.

Materials and Methods

This study was undertaken in the tertiary care center (approx. 1700 bedded) of New Delhi (India) during April 2008 - September 2009. A total of 102 (n=102) fully characterized M. tuberculosis strains (47 susceptible to first-line drugs and 55 resistant to at least Isoniazid (INH) and Rifampicin (RIF) irrespective to other drugs), isolated from individual patients clinical samples from 2006 to 2009, were studied for susceptibility testing of OFX with a critical concentration of 2 μg/ml by Lowenstein Jensen (LJ) proportion method. Stock solution of OFX (Sigma Chemical Co., USA) was prepared in distilled water acidified with minimal volume of 1N HCL to facilitate the dissolution.^[14]

Bacterial suspension of approx. 4mg/ml (2-3 loopful growth) was prepared. ^[15] This suspension was inoculated on drug-free (control) as well as drug-containing LJ slant media with the help of loop. These slants were incubated at 37⁰C and first reading were taken after 14 days then on 28^th day and final reading was on 42^nd day. The reading was done by counting the colony-forming units (CFU) and compared with control culture. An isolate was considered resistant if it yielded a growth of 20 colonies or more on drug-containing media. The standard strain of M. tuberculosis H37Rv was also tested which were found to be sensitive at <1 μg/ml levels for OFX.

Results

Out of the 47 (n=47) susceptible isolates studied, 40 (85.1%) isolates were showed inhibition at OFX at 2 μg/ml while in 55 (n=55) MDR isolates, 34 (61.8%) isolates showed susceptibility to OFX. The results are summarized in [Table - 1]. On performing Fisher`s exact test [Table - 2], MDR patients had significant (P-value: 0.0136) 1.377-fold increased risk to (95% confidence interval) become resistant to OFX than susceptible isolates.

Discussion

India is one of the prominent destinations with the highest rate of FQ abuse and resistance in the world. The acquisition of FQ resistance by susceptible M. tuberculosis strains was facilitated due to a selection pressure from their widespread use in the treatment of TB and possibly other infections in the community, ^[8],[9] which is hyperendemic for tuberculosis. Resistance to OFX was considered to be a risk factor related to treatment failure and relapse in MDR-TB. In the present study, we observed 14.9 % OFX resistance in first-line drug susceptible isolates and 38.2% in MDR isolates. Thus, an increase in frequency of OFX resistance in both susceptible as well as MDR isolates was found. These findings were consistent with the data reported by earlier Indian study, ^[13] which showed OFX resistance of 11.2% in non-MDR cases and 54.5% in MDR isolates. Other investigators ^[11],[12] have disputed these findings by finding this increased trend only in MDR isolates. These differences might be explained by when the studies were conducted in relation to the time FQ were introduced for treatment of TB and to differences in the geographical regions from which the strains were isolated. These inclinations of MDR isolates to become OFX resistant could be due to many factors. OFX and other FQ are often part of many other regimens and MDR cases are often belonging to Cat II or relapse/failure category thus has a greater chance of getting exposed to FQs. Also FQs. are bulky molecules and there efficacy is affected by cell permeability and INH is known to reduce cell permeability by blocking cell wall synthesis.

There were also reports of OFX-resistant strains of M. tuberculosis or the acquiring OFX resistance during treatment. ^{[16],[17],[18]} Retrospective studies have shown that empiric antituberculous treatment with FQ or FQ use for misdiagnosed pneumonia delayed diagnosis of tuberculosis in an endemic area and impaired outcomes. ^[12] Research studies ^[6],[10] from Philippines and Taiwan experienced marked increase in resistance to OFX in the later phase as compared to early period of the treatment. Therefore, it was observed that exposure to OFX and other FQ for the treatment of other bacterial diseases does appear to be predominantly responsible for the increasing trend of resistance. Therefore, strong guidelines need to be mentioned in the TB control program to keep FQ as the last line therapy when no other agents are active or tolerable. By delaying the emergence of resistance, the process of transformation from MDR strains into XDR strains could be slow down.

Our finding highlights the general risk of acquiring resistance to OFX, jeopardizing the potential for these drugs to be used as second-line anti-TB agents in the management of drug-resistant TB and creating incurable TB strains. But, it is alarming that the rate of resistance to FQ significantly increased with time, indicating the capability of these isolates to survive under selection pressure.

There also seems to be a great degree of cross-resistance among various derivatives. ^[19],[20] So, there is a scope for further studies with the other quinolones. A potential solution may be replacement of the recommended second line quinolones with C-8 methoxy substituted FQ like moxifloxacin which shown to have good bactericidal activity in murine tuberculosis and in human patients. ^[21]

In conclusion, we recommend that as OFX showed significantly decreased susceptibility in tuberculosis, FQ susceptibility should be routinely assessed for clinical isolates of M. tuberculosis, especially for drug-resistant isolates, and when patients have a prior history of TB or prior use of anti-tuberculous drugs, so that the most optimal agents can be prescribed.

Acknowledgment

We acknowledge the financial grant (Senior Research fellowship) provided by Indian Council of Medical Research (ICMR) to one of our author, Jitender Singh Verma for the study.

References

1.	World Health Organization. Global tuberculosis control. WHO report 2009; WHO/HTM/TB/2009.  Back to cited text no. 1
2.	Blumberg HM, Burman WJ, Chaisson RE, Daley CL, Etkind SC, Friedman LN, et al. Treatment of tuberculosis. Am J Respir Crit Care Med 2003;167:603-62.  Back to cited text no. 2  [PUBMED]  [FULLTEXT]
3.	Berning SE. The role of fluoroquinolones in tuberculosis today. 0 Drugs 2001;61:9-18.   Back to cited text no. 3
4.	Bryskier A, Lowther J. Fluoroquinolones and tuberculosis. Expert Opin Investig Drugs 2002;11:233-58.  Back to cited text no. 4  [PUBMED]  [FULLTEXT]
5.	Alangaden GJ, Lerner SA. The clinical use of fluoroquinolones for the treatment of mycobacterial diseases. Clin Infect Dis 1997;25:1213-21.  Back to cited text no. 5  [PUBMED]  [FULLTEXT]
6.	Grimaldo ER, Tupasi TE, Rivera AB, Quelapio MD, Cardaño RC, Derilo JO, et al. Increased resistance to ciprofloxacin and ofloxacin in multidrug-resistant Mycobacterium tuberculosis isolates from patients seen at a tertiary hospital in the Philippines. Int J Tuberc Lung Dis 2001;5:546-50.  Back to cited text no. 6
7.	Umubyeyi AN, Rigouts L, Shamputa IC, Fissette K, Elkrim Y, Portaels F, et al. Limited fluoroquinolone resistance among Mycobacterium tuberculosis isolates from Rwanda: Results of a national survey. J Antimicrob Chemother 2007;59:1031-3.  Back to cited text no. 7
8.	Wang JY, Lee LN, Lai HC, Wang SK, Jan IS, Yang PC, et al. Fluoroquinolone resistance in Mycobacterium tuberculosis isolates: Associated genetic mutations and relationship to antimicrobial exposure. J Antimicrob Chemother 2007;59:860-5.  Back to cited text no. 8
9.	Ginsburg AS, Grosset JH, Bishai WR. Fluoroquinolones, tuberculosis and resistance. Lancet 2003;3:432-42.  Back to cited text no. 9
10.	Huang TS, Kunin CM, Shin-Jung Lee S, Chen YS, Tu HZ, Liu YC. Trends in fluoroquinolone resistance of Mycobacterium tuberculosis complex in a Taiwanese medical centre: 1995-2003. J Antimicrob Chemother 2005;56:1058-62.  Back to cited text no. 10  [PUBMED]  [FULLTEXT]
11.	Jain NK, Surpal BB, Khanna SP, Fatima T . 0 In vitro activity of ofloxacin against clinical isolates of Mycobacterium tuberculosis. Indian J Tuberc 1996;43:183.   Back to cited text no. 11
12.	Wang JY, Hsueh PR, Jan IS, Lee LN, Liaw YS, Yang PC, et al. Empirical treatment with a fluoroquinolone delays the treatment for tuberculosis and is associated with a poor prognosis in endemic areas. Thorax 2006;61:903-8.   Back to cited text no. 12  [PUBMED]
13.	Singh M, Chauhan DS, Gupta P, Das R, Srivastava RK, Katoch VM, et al. In vitro effect of fluoroquinolones against Mycobacterium tuberculosis isolates from Agra and Kanpur region of north India. Indian J Med Res 2009;129:542-7.  Back to cited text no. 13
14.	Guillemin I, Jarlier V, Cambau E. Correlation between quinolone susceptibility patterns and sequences in the A and B subunits of DNA gyrase in mycobacteria. Antimicrob Agents Chemother 1998;42:2084-8.  Back to cited text no. 14  [PUBMED]  [FULLTEXT]
15.	Gupta P, Jadaun GP, Das R, Gupta UD, Srivastava K, Chauhan A, et al. Simultaneous ethambutol and isoniazid resistance in clinical isolates of Mycobacterium tuberculosis. Indian J Med Res 2006;123:125-30.  Back to cited text no. 15  [PUBMED]
16.	Ginsburg AS, Hooper N, Parrish N, Dooley KE, Dorman SE, Booth J, et al. Fluoroquinolone resistance in patients with newly diagnosed tuberculosis. Clin Infect Dis 2003;11:1448- 52.  Back to cited text no. 16
17.	Lee AS, Tang LL, Lim IH, Wong SY. Characterization of pyrazinamide and ofloxacin resistance among drug resistant Mycobacterium tuberculosis isolates from Singapore. Int J Infect Dis 2002;6:48-51.  Back to cited text no. 17  [PUBMED]
18.	Tuberculosis Research Centre, Chennai. Shortening short course chemotherapy: A randomised clinical trial for treatment of smear positive pulmonary tuberculosis with regimens using ofloxacin in the intensive phase. Indian J Tuberc 2002;49:27- 38.  Back to cited text no. 18
19.	Tada A, Kawata N, Shibavama T, Takahanshi S, Hirano A, Kimura G, et al. In vitro antituberculous activity of ofloxacin and levofloxacin against multidrug-resistant tuberculosis and clinical outcomes.Kekkaku. 2006;81:337-44.  Back to cited text no. 19
20.	Rose AD, Amondrea B, Carolyn M, Svetlana E, Teresa S, Nancy H, et al. Fluoroquinolone resistance in Mycobacterium tuberculosis: An assessment of MGIT 960, MODS and nitrate reductase assay and fluoroquinolone cross-resistance. J Antimicrob Chemother 2009;63:1173-8.  Back to cited text no. 20
21.	Pletz MW, DeRoux A, Roth A, Neumann KH, Mauch H, Lode H. Early bactericidal activity of moxifloxacin in treatment of pulmonary tuberculosis: A prospective randomized study. Antimicrob Agents Chemother 2004;48:780-2.  Back to cited text no. 21

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