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The Lancet Infectious Diseases 2003; 3:432-442
DOI:10.1016/S1473-3099(03)00671-6
Fluoroquinolones, tuberculosis, and resistance
Amy Sarah Ginsburg a, Jacques H Grosset a and William R Bishai
a 
Summary
Fluoroquinolones in tuberculosis
Fluoroquinolones in the treatment of tuberculosis
Fluoroquinolone resistance
Conclusions
Search strategy and selection criteria
References
Summary
Although the fluoroquinolones are presently used to treat tuberculosis primarily in cases involving resistance or intolerance to first-line antituberculosis therapy, these drugs are potential first-line agents and are under study for this indication. However, there is concern about the development of fluoroquinolone resistance in Mycobacterium tuberculosis, particularly when administered as monotherapy or as the only active agent in a failing multidrug regimen. Treatment failures as well as relapses have been documented under such conditions. With increasing numbers of fluoroquinolone prescriptions and the expanded use of these broad-spectrum agents for many infections, the selective pressure of fluoroquinolone use results in the ready emergence of fluoroquinolone resistance in a diversity of organisms, including M tuberculosis. Among M tuberculosis, resistance is emerging and may herald a significant future threat to the long-term clinical utility of fluoroquinolones. Discussion and education regarding appropriate use are necessary to preserve the effectiveness of this antibiotic class against the hazard of growing resistance.
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One-third of the world's population is infected with Mycobacterium tuberculosis.1,2 In the USA, where 16 377 cases of active tuberculosis were reported in 2000,3 tuberculosis has continued to have a serious impact medically, socially, and financially. Rates of tuberculosis are soaring in sub-Saharan Africa and countries of the former USSR.2,4 Fuelled by the HIV epidemic, nations with previously successful tuberculosis-control programmes are witnessing dramatic increases in tuberculosis rates.5 This deterioration of tuberculosis control has prompted some experts to question whether tuberculosis is, in fact, controllable with the existing armamentarium of tools in the era of HIV.6
Appropriate treatment of patients with active tuberculosis is important in limiting the transmission of M tuberculosis and preventing tuberculosis-related mortality. The global prevalence of drug-resistant tuberculosis7 and the impact of HIV worldwide8 make the identification of potent new agents for the treatment of tuberculosis critically important. Multidrug resistance (defined as resistance to at least isoniazid and rifampicin) is on the rise around the world, particularly in eastern Europe, China, and Iran.7 Despite the availability of effective therapy for almost 50 years, future treatment of tuberculosis will depend, at least in part, on the development of new antituberculosis drugs. By contrast with other infectious diseases, there are relatively few antimicrobial agents clinically effective against M tuberculosis. Currently, tuberculosis treatment requires lengthy courses of medication due to the ability of M tuberculosis to enter a dormant, persistent, antimicrobial-resistant phase. Furthermore, adherence to treatment of tuberculosis is limited by a multitude of clinical, social, financial and behavioural factors. As a result, dose supervision of drug intake is often required. If a shorter duration therapy could be developed and/or a more widely spaced intermittent regimen, both compliance and cure may be improved while decreasing cost. Consequently, there is a need for new agents not only to improve the treatment of multidrug-resistant tuberculosis, but also to augment the efficacy of current approaches to both latent and active tuberculosis.4,9 Fluoroquinolones are promising antimicrobials currently being investigated to fill these needs.
Fluorine-containing nalidixic acid derivatives, the fluoroquinolones were introduced into clinical practice in the 1980s. With broad-spectrum antimicrobial activity, fluoroquinolones are recommended and widely used for the treatment of bacterial infections of the respiratory, gastrointestinal, and urinary tracts, as well as sexually transmitted diseases and chronic osteomyelitis.10?13 Accounting for 11% of all antibiotic sales in the USA in 2002,14 fluoroquinolone use is increasing in the USA and throughout the world.
Fluoroquinolones in tuberculosis
In vitro activity
Fluoroquinolones have in vitro activity against M tuberculosis.15,16 The newer fluoroquinolones, sparfloxacin, gatifloxacin, and moxifloxacin, have lower minimum inhibitory concentrations (MICs) than levofloxacin, ciprofloxacin, and ofloxacin (table 1).17?24 Against rifampicin-tolerant persistent organisms?bacilli that survive and persist despite chemotherapy?in an in-vitro model examining sterilising activity, gatifloxacin and moxifloxacin had the greatest bactericidal activities of the fluoroquinolones tested.25
Table 1. Minimum inhibitory concentrations of fluoroquinolones against Mycobacterium tuberculosis
Fluoroquinolones have also been shown to penetrate into macrophages and have bactericidal activity there.26,27 Within the murine macrophage, levofloxacin has bactericidal activity two to three times that of ofloxacin.28 Within the human macrophage, ofloxacin and levofloxacin have MICs of 1-2 and 0-5 mg/L, respectively.29,30 Ofloxacin's killing activity within the macrophage is enhanced when combined with pyrazinamide.31 Fluoroquinolones when combined with various first-line antituberculosis drugs have shown greater reductions in colony forming units (CFUs) of intramacrophage M tuberculosis than the individual drugs alone,32 indicating that fluoroquinolone activity in vitro is a valuable analytic tool that maybe useful in helping to predict in vivo efficacy.
In vivo activity
The later-generation fluoroquinolones have in vivo activity against M tuberculosis and their activity is concentration-dependent. An in vivo comparison found that gatifloxacin, moxifloxacin, and isoniazid had similar activities against M tuberculosis.33 Treatment studies in mice have shown efficacy, with moxifloxacin as the most bactericidal (greatest decline in CFUs in the lungs and/or spleen) followed, in order of decreasing activity, by sparfloxacin, levofloxacin, and ofloxacin.18,34,35 This apparent superiority of moxifloxacin against M tuberculosis has been corroborated in experimental settings, which demonstrate a bactericidal activity against M tuberculosis equal to or greater than that of isoniazid.17,36 Moxifloxacin was more active at the 100 mg/kg dosage in mice than sparfloxacin, and its bactericidal activity was comparable to that of isoniazid given at the 25 mg/kg dose.17 In experiments conducted in mice evaluating the combination of moxifloxacin 100 mg/kg and isoniazid, there was a suggestion of an additive effect.36 Furthermore, in a study looking at the sterilising activity of moxifloxacin in multidrug therapy, moxifloxacin in combination with rifapentine, a long-lasting rifamycin derivative, dosed once weekly had significant activity.37 Of the fluoroquinolones studied, moxifloxacin seems to have bactericidal potency comparable to that of isoniazid and some sterilising activity that may allow achievement of the goals of shortened or more intermittent therapy as well as improved treatment of latent tuberculosis.
The ratios of two pharmacokinetic parameters, the peak serum concentration (Cmax) and the 24-hour area-under-the curve (AUC24), to the MIC are currently considered and generally accepted as pharmacodynamic correlates of fluoroquinolone efficacy.38,39 In vitro and in vivo studies of fluoroquinolones against various bacterial pathogens have shown that the greatest bactericidal effect and a decreased probability of resistance to fluoroquinolones occurs at Cmax MIC ratios of 8-10 or more and AUC24/MIC ratios of 100-125 or more.38,40?45 According to these parameters, moxifloxacin at the recommended daily dose of 400 mg would be the most active fluoroquinolone against tuberculosis (table 2). However, to determine whether these parameters truly correlate with in vivo efficacy against M tuberculosis, animal modelling experiments and more clinical studies are required.
Table 2. Pharmacokinetic/pharmacodynamic parameters of fluoroquinolones
Activity and safety in human beings
Fluoroquinolones have been used in the treatment of pulmonary, extrapulmonary, and disseminated tuberculosis. In human beings, fluoroquinolones are absorbed readily after oral administration with once-daily dosing, and have effective tissue penetration and distribution into lungs and alveolar macrophages (table 2).
Studies measuring the early bactericidal activity (EBA), the log fall in the CFU count in sputum during the first 48 h of therapy, of ciprofloxacin46,47 and ofloxacin48 in pulmonary tuberculosis have shown these two early-generation fluoroquinolones to be less potent (lower EBAs) than isoniazid during early treatment of tuberculosis49,50 (table 3, table 4). However, there is research demonstrating potent activity of the later-generation 8-methoxyquinolones comparable to that of isoniazid,37,63 and numerous additional EBA studies are underway.
Table 3. Mean early bactericidal activity (EBA) of antimicrobials against Mycobacterium tuberculosis
Table 4. Clinical studies examining the role of fluoroquinolones in treatment of tuberculosis
Reported severe adverse effects of fluoroquinolones include tendonitis and tendon rupture, photosensitivity, seizure, and QT interval prolongation.64 Gastrointestinal reactions (1-5%) including nausea and vomiting (most common), diarrhoea, anorexia, dyspepsia, and abdominal pain, skin disturbances (less than 2-5%), and central nervous system effects (around 1-2%) including headache, dizziness, vertigo, syncope, tinnitus, insomnia, and drowsiness, are the most common adverse effects, and are mainly mild and reversible.64,63 Hepatitis, renal dysfunction, hypoglycaemia, and anaphylactoid reactions are other effects. Fluoroquinolones are restricted for use in children due to the possibility of mutagenesis and cartilage abnormalities.66?68 Fluoroquinolones are not recommended during pregnancy, although are used as second-line therapy for multidrug-resistant tuberculosis in that setting.69,70
Drug interactions are infrequent between fluoroquinolones and other antituberculosis drugs.71?74 Fluoroquinolone absorption may be reduced when co-administered with antacids containing multivalent cations.75 Increased seizure potential following the co-administration of nonsteroidal anti-inflammatory drugs with certain fluoroquinolones may occur. While older fluoroquinolones such as ofloxacin, ciprofloxacin, and levofloxacin have been found to be generally well-tolerated by patients with an excellent safety record in long-term therapy,74,76?79 this safety has yet to be demonstrated for the newer, more potent 8-methoxyfluoroquinolones such as gatifloxacin and moxifloxacin. Encouragingly, preliminary results from one study evaluating the effect of 6 months of therapy with moxifloxacin, isoniazid, and rifampin in ten patients found no toxicity.80
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Fluoroquinolones in the treatment of tuberculosis
There is a need for more clinical trials evaluating fluoroquinolones as part of first-line therapy against M tuberculosis.16,77,81,82 In a clinical trial from India, patients with drug-susceptible and drug-resistant pulmonary tuberculosis were treated for 4?5 months with an ofloxacin-containing regimens under direct observation.62 Patients were randomly assigned to one of four treatment arms: (1) isoniazid, rifampicin, pyrazinamide, and ofloxacin daily for 3 months; (2) isoniazid, rifampicin, pyrazinamide, and ofloxacin daily for 3 months followed by isoniazid and rifampicin twice a week for 1 month; (3) isoniazid, rifampicin, pyrazinamide, and ofloxacin daily for 3 months followed by isoniazid and rifampicin twice a week for 2 months; or (4) isoniazid, rifampicin, pyrazinamide, and ofloxacin daily for 2 months followed by isoniazid and rifampicin twice a week for 2 months. Up to 2 years after completion of treatment, the cure and relapse rates (2?4%) of the regimens including at least 3 months of intensive-phase therapy followed by 1-2 months of continuation-phase therapy were comparable to standard 6-month regimens. Furthermore, there was no increased incidence of adverse reactions. A 5-year follow up for this study is planned and a randomised clinical trial of 4-month fully intermittent ofloxacin-containing regimens during the induction and continuation phases is in progress. Elsewhere, phase 1 and 2 clinical trials are presently underway evaluating later-generation fluoroquinolones such as gatifloxacin and moxifloxacin.
Fluoroquinolones are currently approved as second-line agents for the treatment of multidrug-resistant tuberculosis by the WHO.83 In the recent consensus treatment guidelines from the American Thoracic Society, US Centers for Disease Control and Prevention, and Infectious Diseases Society of America, incorporating a fluoroquinolone is suggested in potential regimens for the management of patients with drug-resistant tuberculosis.9 Thus, presently, fluoroquinolones are used for prophylaxis of those exposed to multidrug-resistant tuberculosis, for treatment of proven multidrug-resistant tuberculosis, for empiric treatment of tuberculosis disease in settings of high rates of multidrug-resistant tuberculosis, and for patients with severe adverse reactions to first-line agents.56,74,77,81,82,84 In most studies thus far, ciprofloxacin,55,57 ofloxacin,54 levofloxacin,58?60 and sparfloxacin61 have been used in combination with other antituberculosis agents (table 4) However, there also have been cases of fluoroquinolone monotherapy (table 4).46,53,85 For the treatment of potentially multidrug-resistant latent tuberculosis infection, guidelines based on expert opinion recommend a two-drug regimen of pyrazinamide plus either ethambutol or a fluoroquinolone.81,86,87 However, patient intolerance to the pyrazinamide and fluoroquinolone combination has been reported.72,73,88
Acknowledging the inherent difficulties in comparing studies different in methodology, drug regimens, location, and implementation, fluoroquinolones have shown variable results. In two different clinical studies, a fluoroquinolone, ofloxacin or ciprofloxacin, in combination with isoniazid and rifampin demonstrated 100% culture conversion at 6 months;54,57 yet, time to conversion among HIV-infected patients receiving ciprofloxacin was notably longer and there was 9% relapse in the ciprofloxacin group.57 Substituting ciprofloxacin for rifampicin in a regimen also containing isoniazid, pyrazinamide, and streptomycin gave 95% of patients smear negative at 6 months with 17% relapsing in the ciprofloxacin group versus 100% smear negative at 6 months with 6% relapsing in the rifampicin group.55 Adding levofloxacin to the standard regimen of isoniazid, rifampicin, pyrazinamide, and ethambutol did not appear to substantially increase conversion with 96% culture negative in the levofloxacin group at 2 months versus 97% culture negative in the standard therapy group.58
Of note, when evaluating non-response in various studies using ofloxacin with multiple other antituberculosis agents in the treatment of pulmonary multidrug-resistant tuberculosis, most non-responders developed ofloxacin resistance.51,53 There was inadequate sterilisation of the sputum and relapse was significant with 23% in one study and 78% in another.53,85
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Fluoroquinolone resistance
Mechanism of fluoroquinolone resistance
As the only presently licensed antibiotics that directly inhibit DNA synthesis, fluoroquinolones target bacterial topoisomerases II and IV. Unlike most other bacterial species, M tuberculosis lacks topoisomerase IV and includes only topoisomerase II or DNA gyrase, a tetramer consisting of two A and two B subunits, encoded by the genes gyrA (2517 bp) and gyrB (2060 bp), respectively.89 The primary target of fluoroquinolones in Staphylococcus aureus is topoisomerase IV, whereas in Escherichia coli and M tuberculosis it is DNA gyrase. Across bacterial species, different fluoroquinolones favour different enzymes: ciprofloxacin preferentially binds to topoisomerase IV whereas moxifloxacin has a predilection for DNA gyrase.90?94 Since M tuberculosis lacks topoisomerase IV, this may explain, at least in part, the better bactericidal activity of moxifloxacin compared with ciprofloxacin against M tuberculosis. In M tuberculosis, fluoroquinolones function by binding to the bacterial enzyme-DNA complex. The precise mechanisms of fluoroquinolone killing are not fully elucidated; however, strand-breakage, SOS-mediated autolysis, and blockade of replication by the gyrase-fluoroquinolone complex, which may enable bacterial inhibition without lethality, have been proposed.95?98
A conserved region, the quinolone resistance-determining region (QRDR), of the gyrA (320 bp) and gyrB (375 bp) genes is an area involved in the interaction between fluoroquinolone and DNA gyrase. Missense mutations within the QRDR have been identified that are associated with fluoroquinolone resistance (figure).99?105 The most common mutation in fluoroquinolone-resistant M tuberculosis isolates involves a substitution at codon 94 of the M tuberculosis gyrA gene.99 Different aminoacid substitutions in the QRDR cause different levels of resistance, and high level resistance to the fluoroquinolones appears to be generated in a stepwise process of additive mutations.102,104,106 Clinically significant resistance (MIC >2 (xg/mL) to ciprofloxacin or ofloxacin may be achieved with a single gyrase mutation.102,104 At least two mutations in gyrA or mutations in gyrA plus gyrB are required for high-level resistance, with double gyrA mutants expressing the highest level of resistance (MIC 20 fxg/mL) to sparfloxacin.102
Click to enlarge image
Figure. Nucleotide sequence and missense mutations within the QRDR of gyrA. *Codon 95 contains a naturally occurring polymorphism. Data from references 99-105.
Resistance to fluoroquinolones in clinical isolates of M tuberculosis occurs primarily due to mutations in the QRDR of gyrA and, thus far, mutation in the QRDR of gyrA is the most important mechanism of fluoroquinolone resistance in M tuberculosis. However, such mutations are not found in all patients with fluoroquinolone resistance, and do not explain a significant proportion of fluoroquinolone resistance in M tuberculosis. In fact, only 42-85% of clinical isolates with fluoroquinolone resistance have gyrA mutations in the QRDR region.52,101,102 To date, no clinical isolates of M tuberculosis have been associated with mutations in gyrB, though gyrB mutations have been seen in some laboratory isolates.102,106 In a study looking at the emergence of fluoroquinolone-resistant multidrug-resistant M tuberculosis in New York City, multiple mutations in the QRDR of gyrA were uniquely associated with resistant strains; however, fluoroquinolone-resistant isolates from four patients lacked mutations in the QRDR of gyrA.101
Therefore, another mechanism likely accounts for the fluoroquinolone resistance in these isolates. Possibilities include mutations in areas of gyrA or gyrB outside of the QRDR, decreased cell wall permeability to the drug, an active drug efflux pump mechanism such as that seen in Mycobacterium smegmatis, sequestration of drug, or drug inactivation.107?109 In M smegmatis, the lfrA gene, which encodes an efflux pump, allows for a low level of resistance to fluoroquinolones and enables mutation to higher-level resistance.107,110 Energy-dependent active efflux is viewed as important in the development of bacterial resistance to fluoroquinolones.111?113 Decreased levels of intracellular drug accumulation and an energy-dependent efflux pump linked with a change in outer membrane proteins have been identified in Gram-negative bacteria and associated with fluoroquinolone resistance.92,114,115 A possible mechanism of fluoroquinolone resistance may involve a pentapeptide protein mfpA, recently implicated in the intrinsic resistance mechanisms employed by M smegmatis.116 Different resistance mechanisms, often interdependent, may explain various degrees of resistance and also may account for the stepwise selection of highly fluoroquinolone-resistant strains.113,117,118 Furthermore, target gene mutations may differ from one geographic region to another.119
Another study found that the frequency of in vitro fluoroquinolone mutational resistance and the distribution of resistance alleles selected were dependent on the fluoroquinolone concentration.120 When allelic diversity was explored in 600 fluoroquinolone-resistant mutants of mycobacteria, many low-level resistance mutants were selected at low fluoroquinolone concentrations but none contained changes in the QRDR of the gyrA protein. As selection pressure increased with the presence of higher fluoroquinolone concentration, several gyrA mutants became more prevalent; thus, increased concentrations of antibiotic appeared to restrict the diversity of mutants to just a few genotypes. When M tuberculosis is sequentially challenged with increasing concentration of fluoroquinolones, stepwise resistance occurs and these mutations appear to map to the gyrase gene; however, eventually a concentration is reached at which no mutant is recovered.
Drlica's group has proposed a method to reduce the selection of fluoroquinolone-resistant M tuberculosis mutants that entails using antibiotic concentrations that require bacteria to acquire two concurrent resistance mutations for growth.121 They have coined the term ?mutant prevention concentration? (MPC) as a new measure of antibiotic activity that is indicative of the drug concentration above which resistant colonies are no longer recoverable when over 1010 cells are plated. A ?mutant selection window? is described in which antibiotic-resistant mutants are selectively amplified within a concentration range that extends from the point where growth inhibition for susceptible organisms begins (the MIC) to the MPC.122 They contend that since MIC-based strategies need only one resistance mutation for bacteria to grow in the presence of an antimicrobial and infections can contain adequate number of bacterial cells for several first-step resistant mutants to be present, resistant mutants become readily selected. A MPC-based strategy, therefore, has been suggested to block the growth of first-step resistant mutants and require wild-type cells to acquire two resistance mutations for growth, a rare event. Thus, at antibiotic concentrations below the MIC, no mutant will be selected because there is no selective pressure and above the MPC, no mutant will be selected because a double mutation is needed for growth.123 Experimental in vitro data confirm that MPC levels of drug do indeed inhibit strains that harbour first-step gyrA mutations.124
Of the first-line antituberculosis agents tested, none achieves human Cmax levels that exceed the MPC.125 Gatifloxacin and moxifloxacin are fluoroquinolones whose Cmax (3-4 μg/mL and 4-5 μg/mL, respectively) exceeds the MPC (1-5 μg/mL and 2-5 μg/mL, respectively).98,125 However, for all its perceived advantages in vitro, the MPC is theoretical and yet to be validated in vivo where such variables as the host immune response, drug pharmacokinetics and bioavailability, and drug toxicity may limit the applicability of these in vitro findings. As yet, none of the current fluoroquinolones, even the most potent by pharmacodynamic parameters, has a pharmacokinetic profile that would reliably keep serum concentrations above the MPC throughout the dosing interval.
Fluoroquinolone use and resistance
The emergence of fluoroquinolone resistance in bacterial pathogens has typically followed widespread and often injudicious use of these drugs. Resistance first emerged in species such as S aureus and Pseudomonas aeruginosa when single mutations were able to cause clinically relevant losses of susceptibility.126 Thereafter, resistance appeared in bacteria such as E coli, Campylobacter jejuni, and Neisseria gonorrhoeae, in which multiple mutations were necessary to enable clinically relevant resistance.126 More recently, there have been growing concerns, and clinical evidence, demonstrating increasing fluoroquinolone resistance in Streptococcus pneumoniae.39,127-129 Fluoroquinolone-resistant isolates are more common among persons aged 65 years or older, who have the highest density of fluoroquinolone use.129
The link between the widespread use of fluoroquinolones and the acquisition and spread of organisms with reduced susceptibility to fluoroquinolones has been documented in a variety of settings and different bacterial species.128?132 Development of resistance during fluoroquinolone therapy and the stepwise selection of mutants exhibiting high-level resistance to fluoroquinolones as a result of exposure of bacteria to these drugs have been reported. Risk factors for nosocomial infections caused by fluoroquinolone-resistant Gram-negative organisms have included a history of previous infection, immunosuppression, and prior receipt of fluoroquinolones.118 Fluoroquinolone use in animals and human-to-human spread also seem to contribute to development of resistance.126 After the introduction of fluoroquinolones for use in animals in the Netherlands and the USA, fluoroquinolone-resistant campylobacter appeared.133,134
Fluoroquinolone resistance in M tuberculosis
Like other antituberculosis agents, the selection of a fluoroquinolone-resistant subpopulation of M tuberculosis requires an actively multiplying bacillary population large enough to contain spontaneous drug-resistant mutants and exposure to a single active drug.85,108,135 Given the burden of organisms in a pulmonary cavity (on average, 1 ?108 CFU/mL),136 and the natural prevalence of spontaneous fluoroquinolone-resistant mutants (2?10−6 to 1?10−8 drug-resistant mutants per replicating organism),51,99,100 an estimated one to 100 fluoroquinolone-resistant mutants may be present at treatment onset. During fluoroquinolone treatment, fluoroquinolone-susceptible wild-type bacteria are killed and there is in vivo selection of fluoroquinolone-resistant mutants.
Whereas there are no reports of cross-resistance between fluoroquinolones and other classes of antituberculosis agents,137 there is cross-resistance within the fluoroquinolone class,21,56,100 such that reduced susceptibility to one fluoroquinolone likely confers reduced susceptibility to all fluoroquinolones.
Fluoroquinolone susceptibility is not routinely assessed in clinical isolates of tubercle bacilli, so the prevalence of fluoroquinolone resistance in M tuberculosis is unknown. In the USA and Canada, among referral sample isolates between 1996 and 2000, resistance to ciprofloxacin at 2 μg/mL was assessed by the proportion method, and occurred in 1-8% (33/1852); of those, 75-8% (25/33) were also multidrug-resistant.138 In the Philippines, where fluoroquinolone use is poorly controlled, among 117 patients, fluoroquinolone resistance occurred in 53-4% of those with tuberculosis resistant to four or five drugs, in 23-3% with resistance to three drugs, and in 18-2% with resistance to two drugs.139 Because of the high prevalence of tuberculosis in the Philippines, fluoroquinolones are not included in the clinical practice guidelines for the treatment of community-acquired pneumonia.140,141
In vivo selection of fluoroquinolone-resistant mutants is a phenomenon that dates back to the very first clinical trial of fluoroquinolones in the treatment of tuberculosis.135 In a study where ofloxacin monotherapy was provided to 19 patients with multidrug-resistant tuberculosis, all patients initially improved but 12 (63%) subsequently relapsed and harboured fluoroquinolone-resistant mutants. In this and other studies, isolates recovered after fluoroquinolone treatment from all non-responders and most of the patients who relapsed demonstrated resistance to fluoroquinolones.53,85,135 It appears, as with other bacteria, that prior fluoroquinolone use is associated with the emergence of fluoroquinolone-resistant M tuberculosis.
Fluoroquinolone resistance is primarily seen in patients with multidrug-resistant tuberculosis treated with a fluoroquinolone as the only active agent in a failing multidrug regimen. The prevalence of fluoroquinolone resistance in non-multidrug-resistant tuberculosis thus far has not been assessed.
How quickly does fluoroquinolone resistance become clinically apparent and after how much exposure?
The emergence of fluoroquinolone resistance in M tuberculosis has developed rapidly when a fluoroquinolone has been added to other second-line drugs or added singly to a failing regimen and essentially functioned as the sole active agent in a multidrug regimen.46,53,76,85,135 The time to acquisition of fluoroquinolone resistance described to date has varied. In one study, serial isolates of M tuberculosis were cultured from a patient who failed to respond to standard antituberculosis therapy; 10 months after the initiation of ofloxacin, susceptibility testing demonstrated ofloxacin resistance, and by 16 months, resistance to ciprofloxacin and sparfloxacin had been documented.142 In the New York City study, 16 patients with multidrug-resistant tuberculosis who received ciprofloxacin or ofloxacin had isolates that developed fluoroquinolone resistance on therapy; the median time between the collection of a documented fluoroquinolone-susceptible isolate and a fluoroquinolone-resistant isolate was 137 days (range 43-398 days) after a period of fluoroquinolone treatment ranging from 23 to 271 days (median 64 days).143
In a cohort study looking at whether empiric fluoroquinolone monotherapy was associated with the development of M tuberculosis with decreased susceptibility to fluoroquinolones, 19 (35%) of 55 patients with newly diagnosed culture-confirmed tuberculosis received fluoroquinolones including ofloxacin, ciprofloxacin, levofloxacin, trovafloxacin, and gatifloxacin before the diagnosis of tuberculosis and the initiation of standard antituberculosis therapy (A S Ginsburg, unpublished observation). Among all patients in the cohort, the incidence of M tuberculosis with decreased susceptibility to fluoroquinolones was 4%. Of the 19 patients who received fluoroquinolones, two (11%) had M tuberculosis isolates with decreased susceptibility to fluoroquinolones. No fluoroquinolone-resistant M tuberculosis was recovered from patients who had not received fluoroquinolones.
In the two patients with fluoroquinolone-resistant M tuberculosis, fluoroquinolone resistance became clinically detectable rapidly (16 days and 2 months) after relatively short durations of fluoroquinolone therapy (13 days of levofloxacin and then ciprofloxacin and at least 5 days of gatifloxacin). The minimum time required for the selection of fluoroquinolone resistance is not known, but a case report demonstrated that resistance can occur with exposures as brief as 13 days, in this case with levofloxacin and then ciprofloxacin (A S Ginsburg, unpublished observation). These studies suggest that M tuberculosis with decreased susceptibility to fluoroquinolones can develop after short courses of fluoroquinolone therapy.
Fluoroquinolone resistance and HIV/AIDS
Infection with HIV has been associated with increased rates of single-drug and multidrug-resistant tuberculosis in the New York City area and other geographic areas.144?146 However, other studies have shown no association between HIV status and drug-resistant tuberculosis.147?149 The relation between HIV infection and drug-resistant tuberculosis is unclear, but the association seems to be strongest in persons with advanced AIDS. The development of drug resistance at low CD4+ lymphocyte counts may be related to the rapid proliferation of M tuberculosis for longer periods of time in the setting of diminished immune containment in advanced AIDS.150,151 Nosocomial outbreaks of tuberculosis among AIDS patients frequently advance rapidly from known exposure to serious active disease.152 Replication of tubercle bacilli in immunocompromised hosts may lead to a higher burden of mycobacteria, which may then permit higher numbers of resistant organisms to emerge with improper therapy. Fluoroquinolone monotherapy in the setting of HIV may predispose to increased opportunities for the development and selection of resistance mutations. Of note, the absorption of fluoroquinolones appears to be normal in patients with HIV infection, and is not affected by concurrent antiretroviral medication or gastrointestinal changes in the absence of infectious gastroenteritis and severe diarrhoea.153,154
Reports suggest that resistance to specific antituberculosis drugs may be more abundant in people infected with HIV.155?160 Acquired rifamycin resistance has been reported in HIV-infected patients, and appears to be associated with intermittent therapy in the continuation phase of treatment, low CD4+ lymphocyte count, or the use of rifabutin preventive therapy against Mycobacterium avium intracellulare complex.158,161?164 In Tuberculosis Trials Consortium Study 22, four of 30 (13%) HIV-seropositive tuberculosis patients who received once-weekly directly observed isoniazid and rifapentine during the continuation phase of treatment relapsed with rifamycin-monoresistant tuberculosis; the median baseline CD4+ count of these patients was 16 lymphocytes/μL.163,165 In Tuberculosis Trials Consortium Study 23, five of 147 (3-4%) HIV-seropositive tuberculosis patients treated with twice-weekly isoniazid and rifabutin directly observed during the continuation phase of treatment relapsed with rifamycin-monoresistant tuberculosis; the median CD4+ lymphocyte count of these five patients was 23/μL.164 Among immunocompetent patients, rifamycin monoresistance is uncommon,166,167 especially during supervised therapy;146 thus the cause of acquired rifamycin monoresistance among immunocompromised patients is not well understood. It is likely associated with pharmacokinetic mismatch with the companion drug leading to unopposed exposure to rifamycin; the mismatch may be due to altered absorption and/or metabolism of one drug.163
Fluoroquinolone resistance in HIV-seropositive and AIDS patients with M tuberculosis susceptible to isoniazid and rifampin has been reported. A levofloxacin-monoresistant M tuberculosis pulmonary isolate was identified in an AIDS patient with CD4+ lymphocytes under 50/μL who had received ofloxacin for 8 months before the diagnosis of tuberculosis.105 In a study looking at fluoroquinolone resistance in newly diagnosed tuberculosis patients, two of 1373 (0-15%) M tuberculosis isolates from Tuberculosis Trials Consortium patients enrolled between 1995 and 2000 were resistant to ciprofloxacin, and both isolates were from patients co-infected with HIV.138 In the cohort study noted above, isolates of M tuberculosis with decreased susceptibility to fluoroquinolones were identified in two of three patients who had received fluoroquinolone monotherapy before the diagnosis of tuberculosis and had AIDS with CD4+ lymphocyte counts under 50/μL (A S Ginsburg, unpublished observation). It has been shown that mycobacteraemia and positive acid-fast smears are more common with low CD4+ lymphocyte counts.150 Concerns have, therefore, been raised as to whether the propensity to develop fluoroquinolone-resistant M tuberculosis is higher in patients with more advanced immunosuppression from AIDS.
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Conclusions
Fluoroquinolones are rapidly emerging as important drugs for the treatment of tuberculosis. With most of their current use in multidrug-resistant tuberculosis, fluoroquinolones are also under investigation for first-line treatment of pulmonary tuberculosis. There is concern about the rapid development of resistance particularly when fluoroquinolones are administered as the only active agent in a failing multidrug regimen, and treatment failures as well as relapses have been documented under such conditions. Preventing the selection of fluoroquinolone resistance is difficult in patients whose M tuberculosis is susceptible to only a few active drugs.
Resistance leads to increased cost, morbidity, and mortality. Because fluoroquinolones are gaining importance in tuberculosis treatment, the prevention of fluoroquinolone resistance in M tuberculosis is a growing priority. The primary mechanism of fluoroquinolone resistance in M tuberculosis thus far is thought to be mediated by missense mutations within the QRDR of gyrA and/or gyrB. However, this mechanism does not account for all resistance and additional possibilities include mutations in areas of gyrA or gyrB outside of the QRDR, decreased cell wall permeability to the drug, an active drug efflux pump mechanism, sequestration of drug, or drug inactivation. Warning markers for fluoroquinolone resistance include a progressive increase in MIC and resistance to another agent.
When the diagnosis of tuberculosis is considered, avoidance of fluoroquinolone monotherapy and use of effective companion drugs can help prevent the rapid selection of fluoroquinolone-resistant mutants in tuberculosis patients. Choice of fluoroquinolone is also important; currently available later-generation fluoroquinolones such as gatifloxacin and moxifloxacin are more potent antituberculosis agents and appear to be less likely to cause resistance. Furthermore, the selection of fluoroquinolone should include consideration of those with the best pharmacodynamic and safety profiles.
Fluoroquinolones are routinely given alone for the empiric treatment of numerous outpatient infections including community-acquired pneumonia and other respiratory tract complaints. By the time these infections are revealed to be due to M tuberculosis, administration of fluoroquinolones has sometimes resulted in the development of fluoroquinolone-resistant M tuberculosis.100 Patients empirically treated with fluoroquinolones for another presumed infectious process may display clinical improvement, masking the diagnosis of tuberculosis while potentially allowing for the development of fluoroquinolone resistance. The risk of selecting fluoroquinolone-resistant M tuberculosis before a diagnosis of tuberculosis is established is of great concern. Testing M tuberculosis for fluoroquinolone susceptibility should be considered, particularly in patients with prior fluoroquinolone exposure.
Fluoroquinolones are a powerful class of agents and effort should be made to preserve their efficacy and utility. Combination therapy thus far has proved successful in the prevention of resistance in tuberculosis, and as resistance to fluoroquinolones results primarily?if not only?from the selection of resistant mutants, there has been support for the application of combination therapy to prevent development of resistance in other infections. Some investigators argue that fluoroquinolones may be better reserved for specific serious infections such as tuberculosis rather than becoming the workhorse of antimicrobial treatment, thereby helping to slow the ready emergence of resistance which may compromise their long-term clinical utility. The appropriate use of fluoroquinolones is an ongoing debate that requires informed discussion and education; without this, fluoroquinolones may lose their potential value in the treatment of tuberculosis.
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Search strategy and selection criteria
Data for this review were identified by searches of PubMed and references from relevant articles. Search terms were ?tuberculosis?, ?Mycobacterium tuberculosis?, ?quinolones?, ?fluoroquinolones?, ?activity?, ?safety?, ?use?, ?treatment?, ?resistance?, ?mechanism?, ?gyrase?, ?quinolone resistance-determining region?, ?human immunodeficiency virus?, and ?acquired immunodeficiency syndrome?. English language papers were reviewed.
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Conflicts of interest
None declared.
Acknowledgments
Funding support was provided by the Global Alliance for Tuberculosis Drug Development and NIH grant AI43846
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DOI:10.1016/S1473-3099(03)00671-6
Fluoroquinolones, tuberculosis, and resistance
Amy Sarah Ginsburg a, Jacques H Grosset a and William R Bishai
a Summary
Fluoroquinolones in tuberculosis
Fluoroquinolones in the treatment of tuberculosis
Fluoroquinolone resistance
Conclusions
Search strategy and selection criteria
References
Summary
Although the fluoroquinolones are presently used to treat tuberculosis primarily in cases involving resistance or intolerance to first-line antituberculosis therapy, these drugs are potential first-line agents and are under study for this indication. However, there is concern about the development of fluoroquinolone resistance in Mycobacterium tuberculosis, particularly when administered as monotherapy or as the only active agent in a failing multidrug regimen. Treatment failures as well as relapses have been documented under such conditions. With increasing numbers of fluoroquinolone prescriptions and the expanded use of these broad-spectrum agents for many infections, the selective pressure of fluoroquinolone use results in the ready emergence of fluoroquinolone resistance in a diversity of organisms, including M tuberculosis. Among M tuberculosis, resistance is emerging and may herald a significant future threat to the long-term clinical utility of fluoroquinolones. Discussion and education regarding appropriate use are necessary to preserve the effectiveness of this antibiotic class against the hazard of growing resistance.
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One-third of the world's population is infected with Mycobacterium tuberculosis.1,2 In the USA, where 16 377 cases of active tuberculosis were reported in 2000,3 tuberculosis has continued to have a serious impact medically, socially, and financially. Rates of tuberculosis are soaring in sub-Saharan Africa and countries of the former USSR.2,4 Fuelled by the HIV epidemic, nations with previously successful tuberculosis-control programmes are witnessing dramatic increases in tuberculosis rates.5 This deterioration of tuberculosis control has prompted some experts to question whether tuberculosis is, in fact, controllable with the existing armamentarium of tools in the era of HIV.6
Appropriate treatment of patients with active tuberculosis is important in limiting the transmission of M tuberculosis and preventing tuberculosis-related mortality. The global prevalence of drug-resistant tuberculosis7 and the impact of HIV worldwide8 make the identification of potent new agents for the treatment of tuberculosis critically important. Multidrug resistance (defined as resistance to at least isoniazid and rifampicin) is on the rise around the world, particularly in eastern Europe, China, and Iran.7 Despite the availability of effective therapy for almost 50 years, future treatment of tuberculosis will depend, at least in part, on the development of new antituberculosis drugs. By contrast with other infectious diseases, there are relatively few antimicrobial agents clinically effective against M tuberculosis. Currently, tuberculosis treatment requires lengthy courses of medication due to the ability of M tuberculosis to enter a dormant, persistent, antimicrobial-resistant phase. Furthermore, adherence to treatment of tuberculosis is limited by a multitude of clinical, social, financial and behavioural factors. As a result, dose supervision of drug intake is often required. If a shorter duration therapy could be developed and/or a more widely spaced intermittent regimen, both compliance and cure may be improved while decreasing cost. Consequently, there is a need for new agents not only to improve the treatment of multidrug-resistant tuberculosis, but also to augment the efficacy of current approaches to both latent and active tuberculosis.4,9 Fluoroquinolones are promising antimicrobials currently being investigated to fill these needs.
Fluorine-containing nalidixic acid derivatives, the fluoroquinolones were introduced into clinical practice in the 1980s. With broad-spectrum antimicrobial activity, fluoroquinolones are recommended and widely used for the treatment of bacterial infections of the respiratory, gastrointestinal, and urinary tracts, as well as sexually transmitted diseases and chronic osteomyelitis.10?13 Accounting for 11% of all antibiotic sales in the USA in 2002,14 fluoroquinolone use is increasing in the USA and throughout the world.
Fluoroquinolones in tuberculosis
In vitro activity
Fluoroquinolones have in vitro activity against M tuberculosis.15,16 The newer fluoroquinolones, sparfloxacin, gatifloxacin, and moxifloxacin, have lower minimum inhibitory concentrations (MICs) than levofloxacin, ciprofloxacin, and ofloxacin (table 1).17?24 Against rifampicin-tolerant persistent organisms?bacilli that survive and persist despite chemotherapy?in an in-vitro model examining sterilising activity, gatifloxacin and moxifloxacin had the greatest bactericidal activities of the fluoroquinolones tested.25
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Table 1. Minimum inhibitory concentrations of fluoroquinolones against Mycobacterium tuberculosis
Fluoroquinolones have also been shown to penetrate into macrophages and have bactericidal activity there.26,27 Within the murine macrophage, levofloxacin has bactericidal activity two to three times that of ofloxacin.28 Within the human macrophage, ofloxacin and levofloxacin have MICs of 1-2 and 0-5 mg/L, respectively.29,30 Ofloxacin's killing activity within the macrophage is enhanced when combined with pyrazinamide.31 Fluoroquinolones when combined with various first-line antituberculosis drugs have shown greater reductions in colony forming units (CFUs) of intramacrophage M tuberculosis than the individual drugs alone,32 indicating that fluoroquinolone activity in vitro is a valuable analytic tool that maybe useful in helping to predict in vivo efficacy.
In vivo activity
The later-generation fluoroquinolones have in vivo activity against M tuberculosis and their activity is concentration-dependent. An in vivo comparison found that gatifloxacin, moxifloxacin, and isoniazid had similar activities against M tuberculosis.33 Treatment studies in mice have shown efficacy, with moxifloxacin as the most bactericidal (greatest decline in CFUs in the lungs and/or spleen) followed, in order of decreasing activity, by sparfloxacin, levofloxacin, and ofloxacin.18,34,35 This apparent superiority of moxifloxacin against M tuberculosis has been corroborated in experimental settings, which demonstrate a bactericidal activity against M tuberculosis equal to or greater than that of isoniazid.17,36 Moxifloxacin was more active at the 100 mg/kg dosage in mice than sparfloxacin, and its bactericidal activity was comparable to that of isoniazid given at the 25 mg/kg dose.17 In experiments conducted in mice evaluating the combination of moxifloxacin 100 mg/kg and isoniazid, there was a suggestion of an additive effect.36 Furthermore, in a study looking at the sterilising activity of moxifloxacin in multidrug therapy, moxifloxacin in combination with rifapentine, a long-lasting rifamycin derivative, dosed once weekly had significant activity.37 Of the fluoroquinolones studied, moxifloxacin seems to have bactericidal potency comparable to that of isoniazid and some sterilising activity that may allow achievement of the goals of shortened or more intermittent therapy as well as improved treatment of latent tuberculosis.
The ratios of two pharmacokinetic parameters, the peak serum concentration (Cmax) and the 24-hour area-under-the curve (AUC24), to the MIC are currently considered and generally accepted as pharmacodynamic correlates of fluoroquinolone efficacy.38,39 In vitro and in vivo studies of fluoroquinolones against various bacterial pathogens have shown that the greatest bactericidal effect and a decreased probability of resistance to fluoroquinolones occurs at Cmax MIC ratios of 8-10 or more and AUC24/MIC ratios of 100-125 or more.38,40?45 According to these parameters, moxifloxacin at the recommended daily dose of 400 mg would be the most active fluoroquinolone against tuberculosis (table 2). However, to determine whether these parameters truly correlate with in vivo efficacy against M tuberculosis, animal modelling experiments and more clinical studies are required.
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Table 2. Pharmacokinetic/pharmacodynamic parameters of fluoroquinolones
Activity and safety in human beings
Fluoroquinolones have been used in the treatment of pulmonary, extrapulmonary, and disseminated tuberculosis. In human beings, fluoroquinolones are absorbed readily after oral administration with once-daily dosing, and have effective tissue penetration and distribution into lungs and alveolar macrophages (table 2).
Studies measuring the early bactericidal activity (EBA), the log fall in the CFU count in sputum during the first 48 h of therapy, of ciprofloxacin46,47 and ofloxacin48 in pulmonary tuberculosis have shown these two early-generation fluoroquinolones to be less potent (lower EBAs) than isoniazid during early treatment of tuberculosis49,50 (table 3, table 4). However, there is research demonstrating potent activity of the later-generation 8-methoxyquinolones comparable to that of isoniazid,37,63 and numerous additional EBA studies are underway.
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Table 3. Mean early bactericidal activity (EBA) of antimicrobials against Mycobacterium tuberculosis
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Table 4. Clinical studies examining the role of fluoroquinolones in treatment of tuberculosis
Reported severe adverse effects of fluoroquinolones include tendonitis and tendon rupture, photosensitivity, seizure, and QT interval prolongation.64 Gastrointestinal reactions (1-5%) including nausea and vomiting (most common), diarrhoea, anorexia, dyspepsia, and abdominal pain, skin disturbances (less than 2-5%), and central nervous system effects (around 1-2%) including headache, dizziness, vertigo, syncope, tinnitus, insomnia, and drowsiness, are the most common adverse effects, and are mainly mild and reversible.64,63 Hepatitis, renal dysfunction, hypoglycaemia, and anaphylactoid reactions are other effects. Fluoroquinolones are restricted for use in children due to the possibility of mutagenesis and cartilage abnormalities.66?68 Fluoroquinolones are not recommended during pregnancy, although are used as second-line therapy for multidrug-resistant tuberculosis in that setting.69,70
Drug interactions are infrequent between fluoroquinolones and other antituberculosis drugs.71?74 Fluoroquinolone absorption may be reduced when co-administered with antacids containing multivalent cations.75 Increased seizure potential following the co-administration of nonsteroidal anti-inflammatory drugs with certain fluoroquinolones may occur. While older fluoroquinolones such as ofloxacin, ciprofloxacin, and levofloxacin have been found to be generally well-tolerated by patients with an excellent safety record in long-term therapy,74,76?79 this safety has yet to be demonstrated for the newer, more potent 8-methoxyfluoroquinolones such as gatifloxacin and moxifloxacin. Encouragingly, preliminary results from one study evaluating the effect of 6 months of therapy with moxifloxacin, isoniazid, and rifampin in ten patients found no toxicity.80
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Fluoroquinolones in the treatment of tuberculosis
There is a need for more clinical trials evaluating fluoroquinolones as part of first-line therapy against M tuberculosis.16,77,81,82 In a clinical trial from India, patients with drug-susceptible and drug-resistant pulmonary tuberculosis were treated for 4?5 months with an ofloxacin-containing regimens under direct observation.62 Patients were randomly assigned to one of four treatment arms: (1) isoniazid, rifampicin, pyrazinamide, and ofloxacin daily for 3 months; (2) isoniazid, rifampicin, pyrazinamide, and ofloxacin daily for 3 months followed by isoniazid and rifampicin twice a week for 1 month; (3) isoniazid, rifampicin, pyrazinamide, and ofloxacin daily for 3 months followed by isoniazid and rifampicin twice a week for 2 months; or (4) isoniazid, rifampicin, pyrazinamide, and ofloxacin daily for 2 months followed by isoniazid and rifampicin twice a week for 2 months. Up to 2 years after completion of treatment, the cure and relapse rates (2?4%) of the regimens including at least 3 months of intensive-phase therapy followed by 1-2 months of continuation-phase therapy were comparable to standard 6-month regimens. Furthermore, there was no increased incidence of adverse reactions. A 5-year follow up for this study is planned and a randomised clinical trial of 4-month fully intermittent ofloxacin-containing regimens during the induction and continuation phases is in progress. Elsewhere, phase 1 and 2 clinical trials are presently underway evaluating later-generation fluoroquinolones such as gatifloxacin and moxifloxacin.
Fluoroquinolones are currently approved as second-line agents for the treatment of multidrug-resistant tuberculosis by the WHO.83 In the recent consensus treatment guidelines from the American Thoracic Society, US Centers for Disease Control and Prevention, and Infectious Diseases Society of America, incorporating a fluoroquinolone is suggested in potential regimens for the management of patients with drug-resistant tuberculosis.9 Thus, presently, fluoroquinolones are used for prophylaxis of those exposed to multidrug-resistant tuberculosis, for treatment of proven multidrug-resistant tuberculosis, for empiric treatment of tuberculosis disease in settings of high rates of multidrug-resistant tuberculosis, and for patients with severe adverse reactions to first-line agents.56,74,77,81,82,84 In most studies thus far, ciprofloxacin,55,57 ofloxacin,54 levofloxacin,58?60 and sparfloxacin61 have been used in combination with other antituberculosis agents (table 4) However, there also have been cases of fluoroquinolone monotherapy (table 4).46,53,85 For the treatment of potentially multidrug-resistant latent tuberculosis infection, guidelines based on expert opinion recommend a two-drug regimen of pyrazinamide plus either ethambutol or a fluoroquinolone.81,86,87 However, patient intolerance to the pyrazinamide and fluoroquinolone combination has been reported.72,73,88
Acknowledging the inherent difficulties in comparing studies different in methodology, drug regimens, location, and implementation, fluoroquinolones have shown variable results. In two different clinical studies, a fluoroquinolone, ofloxacin or ciprofloxacin, in combination with isoniazid and rifampin demonstrated 100% culture conversion at 6 months;54,57 yet, time to conversion among HIV-infected patients receiving ciprofloxacin was notably longer and there was 9% relapse in the ciprofloxacin group.57 Substituting ciprofloxacin for rifampicin in a regimen also containing isoniazid, pyrazinamide, and streptomycin gave 95% of patients smear negative at 6 months with 17% relapsing in the ciprofloxacin group versus 100% smear negative at 6 months with 6% relapsing in the rifampicin group.55 Adding levofloxacin to the standard regimen of isoniazid, rifampicin, pyrazinamide, and ethambutol did not appear to substantially increase conversion with 96% culture negative in the levofloxacin group at 2 months versus 97% culture negative in the standard therapy group.58
Of note, when evaluating non-response in various studies using ofloxacin with multiple other antituberculosis agents in the treatment of pulmonary multidrug-resistant tuberculosis, most non-responders developed ofloxacin resistance.51,53 There was inadequate sterilisation of the sputum and relapse was significant with 23% in one study and 78% in another.53,85
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Fluoroquinolone resistance
Mechanism of fluoroquinolone resistance
As the only presently licensed antibiotics that directly inhibit DNA synthesis, fluoroquinolones target bacterial topoisomerases II and IV. Unlike most other bacterial species, M tuberculosis lacks topoisomerase IV and includes only topoisomerase II or DNA gyrase, a tetramer consisting of two A and two B subunits, encoded by the genes gyrA (2517 bp) and gyrB (2060 bp), respectively.89 The primary target of fluoroquinolones in Staphylococcus aureus is topoisomerase IV, whereas in Escherichia coli and M tuberculosis it is DNA gyrase. Across bacterial species, different fluoroquinolones favour different enzymes: ciprofloxacin preferentially binds to topoisomerase IV whereas moxifloxacin has a predilection for DNA gyrase.90?94 Since M tuberculosis lacks topoisomerase IV, this may explain, at least in part, the better bactericidal activity of moxifloxacin compared with ciprofloxacin against M tuberculosis. In M tuberculosis, fluoroquinolones function by binding to the bacterial enzyme-DNA complex. The precise mechanisms of fluoroquinolone killing are not fully elucidated; however, strand-breakage, SOS-mediated autolysis, and blockade of replication by the gyrase-fluoroquinolone complex, which may enable bacterial inhibition without lethality, have been proposed.95?98
A conserved region, the quinolone resistance-determining region (QRDR), of the gyrA (320 bp) and gyrB (375 bp) genes is an area involved in the interaction between fluoroquinolone and DNA gyrase. Missense mutations within the QRDR have been identified that are associated with fluoroquinolone resistance (figure).99?105 The most common mutation in fluoroquinolone-resistant M tuberculosis isolates involves a substitution at codon 94 of the M tuberculosis gyrA gene.99 Different aminoacid substitutions in the QRDR cause different levels of resistance, and high level resistance to the fluoroquinolones appears to be generated in a stepwise process of additive mutations.102,104,106 Clinically significant resistance (MIC >2 (xg/mL) to ciprofloxacin or ofloxacin may be achieved with a single gyrase mutation.102,104 At least two mutations in gyrA or mutations in gyrA plus gyrB are required for high-level resistance, with double gyrA mutants expressing the highest level of resistance (MIC 20 fxg/mL) to sparfloxacin.102
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Figure. Nucleotide sequence and missense mutations within the QRDR of gyrA. *Codon 95 contains a naturally occurring polymorphism. Data from references 99-105.
Resistance to fluoroquinolones in clinical isolates of M tuberculosis occurs primarily due to mutations in the QRDR of gyrA and, thus far, mutation in the QRDR of gyrA is the most important mechanism of fluoroquinolone resistance in M tuberculosis. However, such mutations are not found in all patients with fluoroquinolone resistance, and do not explain a significant proportion of fluoroquinolone resistance in M tuberculosis. In fact, only 42-85% of clinical isolates with fluoroquinolone resistance have gyrA mutations in the QRDR region.52,101,102 To date, no clinical isolates of M tuberculosis have been associated with mutations in gyrB, though gyrB mutations have been seen in some laboratory isolates.102,106 In a study looking at the emergence of fluoroquinolone-resistant multidrug-resistant M tuberculosis in New York City, multiple mutations in the QRDR of gyrA were uniquely associated with resistant strains; however, fluoroquinolone-resistant isolates from four patients lacked mutations in the QRDR of gyrA.101
Therefore, another mechanism likely accounts for the fluoroquinolone resistance in these isolates. Possibilities include mutations in areas of gyrA or gyrB outside of the QRDR, decreased cell wall permeability to the drug, an active drug efflux pump mechanism such as that seen in Mycobacterium smegmatis, sequestration of drug, or drug inactivation.107?109 In M smegmatis, the lfrA gene, which encodes an efflux pump, allows for a low level of resistance to fluoroquinolones and enables mutation to higher-level resistance.107,110 Energy-dependent active efflux is viewed as important in the development of bacterial resistance to fluoroquinolones.111?113 Decreased levels of intracellular drug accumulation and an energy-dependent efflux pump linked with a change in outer membrane proteins have been identified in Gram-negative bacteria and associated with fluoroquinolone resistance.92,114,115 A possible mechanism of fluoroquinolone resistance may involve a pentapeptide protein mfpA, recently implicated in the intrinsic resistance mechanisms employed by M smegmatis.116 Different resistance mechanisms, often interdependent, may explain various degrees of resistance and also may account for the stepwise selection of highly fluoroquinolone-resistant strains.113,117,118 Furthermore, target gene mutations may differ from one geographic region to another.119
Another study found that the frequency of in vitro fluoroquinolone mutational resistance and the distribution of resistance alleles selected were dependent on the fluoroquinolone concentration.120 When allelic diversity was explored in 600 fluoroquinolone-resistant mutants of mycobacteria, many low-level resistance mutants were selected at low fluoroquinolone concentrations but none contained changes in the QRDR of the gyrA protein. As selection pressure increased with the presence of higher fluoroquinolone concentration, several gyrA mutants became more prevalent; thus, increased concentrations of antibiotic appeared to restrict the diversity of mutants to just a few genotypes. When M tuberculosis is sequentially challenged with increasing concentration of fluoroquinolones, stepwise resistance occurs and these mutations appear to map to the gyrase gene; however, eventually a concentration is reached at which no mutant is recovered.
Drlica's group has proposed a method to reduce the selection of fluoroquinolone-resistant M tuberculosis mutants that entails using antibiotic concentrations that require bacteria to acquire two concurrent resistance mutations for growth.121 They have coined the term ?mutant prevention concentration? (MPC) as a new measure of antibiotic activity that is indicative of the drug concentration above which resistant colonies are no longer recoverable when over 1010 cells are plated. A ?mutant selection window? is described in which antibiotic-resistant mutants are selectively amplified within a concentration range that extends from the point where growth inhibition for susceptible organisms begins (the MIC) to the MPC.122 They contend that since MIC-based strategies need only one resistance mutation for bacteria to grow in the presence of an antimicrobial and infections can contain adequate number of bacterial cells for several first-step resistant mutants to be present, resistant mutants become readily selected. A MPC-based strategy, therefore, has been suggested to block the growth of first-step resistant mutants and require wild-type cells to acquire two resistance mutations for growth, a rare event. Thus, at antibiotic concentrations below the MIC, no mutant will be selected because there is no selective pressure and above the MPC, no mutant will be selected because a double mutation is needed for growth.123 Experimental in vitro data confirm that MPC levels of drug do indeed inhibit strains that harbour first-step gyrA mutations.124
Of the first-line antituberculosis agents tested, none achieves human Cmax levels that exceed the MPC.125 Gatifloxacin and moxifloxacin are fluoroquinolones whose Cmax (3-4 μg/mL and 4-5 μg/mL, respectively) exceeds the MPC (1-5 μg/mL and 2-5 μg/mL, respectively).98,125 However, for all its perceived advantages in vitro, the MPC is theoretical and yet to be validated in vivo where such variables as the host immune response, drug pharmacokinetics and bioavailability, and drug toxicity may limit the applicability of these in vitro findings. As yet, none of the current fluoroquinolones, even the most potent by pharmacodynamic parameters, has a pharmacokinetic profile that would reliably keep serum concentrations above the MPC throughout the dosing interval.
Fluoroquinolone use and resistance
The emergence of fluoroquinolone resistance in bacterial pathogens has typically followed widespread and often injudicious use of these drugs. Resistance first emerged in species such as S aureus and Pseudomonas aeruginosa when single mutations were able to cause clinically relevant losses of susceptibility.126 Thereafter, resistance appeared in bacteria such as E coli, Campylobacter jejuni, and Neisseria gonorrhoeae, in which multiple mutations were necessary to enable clinically relevant resistance.126 More recently, there have been growing concerns, and clinical evidence, demonstrating increasing fluoroquinolone resistance in Streptococcus pneumoniae.39,127-129 Fluoroquinolone-resistant isolates are more common among persons aged 65 years or older, who have the highest density of fluoroquinolone use.129
The link between the widespread use of fluoroquinolones and the acquisition and spread of organisms with reduced susceptibility to fluoroquinolones has been documented in a variety of settings and different bacterial species.128?132 Development of resistance during fluoroquinolone therapy and the stepwise selection of mutants exhibiting high-level resistance to fluoroquinolones as a result of exposure of bacteria to these drugs have been reported. Risk factors for nosocomial infections caused by fluoroquinolone-resistant Gram-negative organisms have included a history of previous infection, immunosuppression, and prior receipt of fluoroquinolones.118 Fluoroquinolone use in animals and human-to-human spread also seem to contribute to development of resistance.126 After the introduction of fluoroquinolones for use in animals in the Netherlands and the USA, fluoroquinolone-resistant campylobacter appeared.133,134
Fluoroquinolone resistance in M tuberculosis
Like other antituberculosis agents, the selection of a fluoroquinolone-resistant subpopulation of M tuberculosis requires an actively multiplying bacillary population large enough to contain spontaneous drug-resistant mutants and exposure to a single active drug.85,108,135 Given the burden of organisms in a pulmonary cavity (on average, 1 ?108 CFU/mL),136 and the natural prevalence of spontaneous fluoroquinolone-resistant mutants (2?10−6 to 1?10−8 drug-resistant mutants per replicating organism),51,99,100 an estimated one to 100 fluoroquinolone-resistant mutants may be present at treatment onset. During fluoroquinolone treatment, fluoroquinolone-susceptible wild-type bacteria are killed and there is in vivo selection of fluoroquinolone-resistant mutants.
Whereas there are no reports of cross-resistance between fluoroquinolones and other classes of antituberculosis agents,137 there is cross-resistance within the fluoroquinolone class,21,56,100 such that reduced susceptibility to one fluoroquinolone likely confers reduced susceptibility to all fluoroquinolones.
Fluoroquinolone susceptibility is not routinely assessed in clinical isolates of tubercle bacilli, so the prevalence of fluoroquinolone resistance in M tuberculosis is unknown. In the USA and Canada, among referral sample isolates between 1996 and 2000, resistance to ciprofloxacin at 2 μg/mL was assessed by the proportion method, and occurred in 1-8% (33/1852); of those, 75-8% (25/33) were also multidrug-resistant.138 In the Philippines, where fluoroquinolone use is poorly controlled, among 117 patients, fluoroquinolone resistance occurred in 53-4% of those with tuberculosis resistant to four or five drugs, in 23-3% with resistance to three drugs, and in 18-2% with resistance to two drugs.139 Because of the high prevalence of tuberculosis in the Philippines, fluoroquinolones are not included in the clinical practice guidelines for the treatment of community-acquired pneumonia.140,141
In vivo selection of fluoroquinolone-resistant mutants is a phenomenon that dates back to the very first clinical trial of fluoroquinolones in the treatment of tuberculosis.135 In a study where ofloxacin monotherapy was provided to 19 patients with multidrug-resistant tuberculosis, all patients initially improved but 12 (63%) subsequently relapsed and harboured fluoroquinolone-resistant mutants. In this and other studies, isolates recovered after fluoroquinolone treatment from all non-responders and most of the patients who relapsed demonstrated resistance to fluoroquinolones.53,85,135 It appears, as with other bacteria, that prior fluoroquinolone use is associated with the emergence of fluoroquinolone-resistant M tuberculosis.
Fluoroquinolone resistance is primarily seen in patients with multidrug-resistant tuberculosis treated with a fluoroquinolone as the only active agent in a failing multidrug regimen. The prevalence of fluoroquinolone resistance in non-multidrug-resistant tuberculosis thus far has not been assessed.
How quickly does fluoroquinolone resistance become clinically apparent and after how much exposure?
The emergence of fluoroquinolone resistance in M tuberculosis has developed rapidly when a fluoroquinolone has been added to other second-line drugs or added singly to a failing regimen and essentially functioned as the sole active agent in a multidrug regimen.46,53,76,85,135 The time to acquisition of fluoroquinolone resistance described to date has varied. In one study, serial isolates of M tuberculosis were cultured from a patient who failed to respond to standard antituberculosis therapy; 10 months after the initiation of ofloxacin, susceptibility testing demonstrated ofloxacin resistance, and by 16 months, resistance to ciprofloxacin and sparfloxacin had been documented.142 In the New York City study, 16 patients with multidrug-resistant tuberculosis who received ciprofloxacin or ofloxacin had isolates that developed fluoroquinolone resistance on therapy; the median time between the collection of a documented fluoroquinolone-susceptible isolate and a fluoroquinolone-resistant isolate was 137 days (range 43-398 days) after a period of fluoroquinolone treatment ranging from 23 to 271 days (median 64 days).143
In a cohort study looking at whether empiric fluoroquinolone monotherapy was associated with the development of M tuberculosis with decreased susceptibility to fluoroquinolones, 19 (35%) of 55 patients with newly diagnosed culture-confirmed tuberculosis received fluoroquinolones including ofloxacin, ciprofloxacin, levofloxacin, trovafloxacin, and gatifloxacin before the diagnosis of tuberculosis and the initiation of standard antituberculosis therapy (A S Ginsburg, unpublished observation). Among all patients in the cohort, the incidence of M tuberculosis with decreased susceptibility to fluoroquinolones was 4%. Of the 19 patients who received fluoroquinolones, two (11%) had M tuberculosis isolates with decreased susceptibility to fluoroquinolones. No fluoroquinolone-resistant M tuberculosis was recovered from patients who had not received fluoroquinolones.
In the two patients with fluoroquinolone-resistant M tuberculosis, fluoroquinolone resistance became clinically detectable rapidly (16 days and 2 months) after relatively short durations of fluoroquinolone therapy (13 days of levofloxacin and then ciprofloxacin and at least 5 days of gatifloxacin). The minimum time required for the selection of fluoroquinolone resistance is not known, but a case report demonstrated that resistance can occur with exposures as brief as 13 days, in this case with levofloxacin and then ciprofloxacin (A S Ginsburg, unpublished observation). These studies suggest that M tuberculosis with decreased susceptibility to fluoroquinolones can develop after short courses of fluoroquinolone therapy.
Fluoroquinolone resistance and HIV/AIDS
Infection with HIV has been associated with increased rates of single-drug and multidrug-resistant tuberculosis in the New York City area and other geographic areas.144?146 However, other studies have shown no association between HIV status and drug-resistant tuberculosis.147?149 The relation between HIV infection and drug-resistant tuberculosis is unclear, but the association seems to be strongest in persons with advanced AIDS. The development of drug resistance at low CD4+ lymphocyte counts may be related to the rapid proliferation of M tuberculosis for longer periods of time in the setting of diminished immune containment in advanced AIDS.150,151 Nosocomial outbreaks of tuberculosis among AIDS patients frequently advance rapidly from known exposure to serious active disease.152 Replication of tubercle bacilli in immunocompromised hosts may lead to a higher burden of mycobacteria, which may then permit higher numbers of resistant organisms to emerge with improper therapy. Fluoroquinolone monotherapy in the setting of HIV may predispose to increased opportunities for the development and selection of resistance mutations. Of note, the absorption of fluoroquinolones appears to be normal in patients with HIV infection, and is not affected by concurrent antiretroviral medication or gastrointestinal changes in the absence of infectious gastroenteritis and severe diarrhoea.153,154
Reports suggest that resistance to specific antituberculosis drugs may be more abundant in people infected with HIV.155?160 Acquired rifamycin resistance has been reported in HIV-infected patients, and appears to be associated with intermittent therapy in the continuation phase of treatment, low CD4+ lymphocyte count, or the use of rifabutin preventive therapy against Mycobacterium avium intracellulare complex.158,161?164 In Tuberculosis Trials Consortium Study 22, four of 30 (13%) HIV-seropositive tuberculosis patients who received once-weekly directly observed isoniazid and rifapentine during the continuation phase of treatment relapsed with rifamycin-monoresistant tuberculosis; the median baseline CD4+ count of these patients was 16 lymphocytes/μL.163,165 In Tuberculosis Trials Consortium Study 23, five of 147 (3-4%) HIV-seropositive tuberculosis patients treated with twice-weekly isoniazid and rifabutin directly observed during the continuation phase of treatment relapsed with rifamycin-monoresistant tuberculosis; the median CD4+ lymphocyte count of these five patients was 23/μL.164 Among immunocompetent patients, rifamycin monoresistance is uncommon,166,167 especially during supervised therapy;146 thus the cause of acquired rifamycin monoresistance among immunocompromised patients is not well understood. It is likely associated with pharmacokinetic mismatch with the companion drug leading to unopposed exposure to rifamycin; the mismatch may be due to altered absorption and/or metabolism of one drug.163
Fluoroquinolone resistance in HIV-seropositive and AIDS patients with M tuberculosis susceptible to isoniazid and rifampin has been reported. A levofloxacin-monoresistant M tuberculosis pulmonary isolate was identified in an AIDS patient with CD4+ lymphocytes under 50/μL who had received ofloxacin for 8 months before the diagnosis of tuberculosis.105 In a study looking at fluoroquinolone resistance in newly diagnosed tuberculosis patients, two of 1373 (0-15%) M tuberculosis isolates from Tuberculosis Trials Consortium patients enrolled between 1995 and 2000 were resistant to ciprofloxacin, and both isolates were from patients co-infected with HIV.138 In the cohort study noted above, isolates of M tuberculosis with decreased susceptibility to fluoroquinolones were identified in two of three patients who had received fluoroquinolone monotherapy before the diagnosis of tuberculosis and had AIDS with CD4+ lymphocyte counts under 50/μL (A S Ginsburg, unpublished observation). It has been shown that mycobacteraemia and positive acid-fast smears are more common with low CD4+ lymphocyte counts.150 Concerns have, therefore, been raised as to whether the propensity to develop fluoroquinolone-resistant M tuberculosis is higher in patients with more advanced immunosuppression from AIDS.
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Conclusions
Fluoroquinolones are rapidly emerging as important drugs for the treatment of tuberculosis. With most of their current use in multidrug-resistant tuberculosis, fluoroquinolones are also under investigation for first-line treatment of pulmonary tuberculosis. There is concern about the rapid development of resistance particularly when fluoroquinolones are administered as the only active agent in a failing multidrug regimen, and treatment failures as well as relapses have been documented under such conditions. Preventing the selection of fluoroquinolone resistance is difficult in patients whose M tuberculosis is susceptible to only a few active drugs.
Resistance leads to increased cost, morbidity, and mortality. Because fluoroquinolones are gaining importance in tuberculosis treatment, the prevention of fluoroquinolone resistance in M tuberculosis is a growing priority. The primary mechanism of fluoroquinolone resistance in M tuberculosis thus far is thought to be mediated by missense mutations within the QRDR of gyrA and/or gyrB. However, this mechanism does not account for all resistance and additional possibilities include mutations in areas of gyrA or gyrB outside of the QRDR, decreased cell wall permeability to the drug, an active drug efflux pump mechanism, sequestration of drug, or drug inactivation. Warning markers for fluoroquinolone resistance include a progressive increase in MIC and resistance to another agent.
When the diagnosis of tuberculosis is considered, avoidance of fluoroquinolone monotherapy and use of effective companion drugs can help prevent the rapid selection of fluoroquinolone-resistant mutants in tuberculosis patients. Choice of fluoroquinolone is also important; currently available later-generation fluoroquinolones such as gatifloxacin and moxifloxacin are more potent antituberculosis agents and appear to be less likely to cause resistance. Furthermore, the selection of fluoroquinolone should include consideration of those with the best pharmacodynamic and safety profiles.
Fluoroquinolones are routinely given alone for the empiric treatment of numerous outpatient infections including community-acquired pneumonia and other respiratory tract complaints. By the time these infections are revealed to be due to M tuberculosis, administration of fluoroquinolones has sometimes resulted in the development of fluoroquinolone-resistant M tuberculosis.100 Patients empirically treated with fluoroquinolones for another presumed infectious process may display clinical improvement, masking the diagnosis of tuberculosis while potentially allowing for the development of fluoroquinolone resistance. The risk of selecting fluoroquinolone-resistant M tuberculosis before a diagnosis of tuberculosis is established is of great concern. Testing M tuberculosis for fluoroquinolone susceptibility should be considered, particularly in patients with prior fluoroquinolone exposure.
Fluoroquinolones are a powerful class of agents and effort should be made to preserve their efficacy and utility. Combination therapy thus far has proved successful in the prevention of resistance in tuberculosis, and as resistance to fluoroquinolones results primarily?if not only?from the selection of resistant mutants, there has been support for the application of combination therapy to prevent development of resistance in other infections. Some investigators argue that fluoroquinolones may be better reserved for specific serious infections such as tuberculosis rather than becoming the workhorse of antimicrobial treatment, thereby helping to slow the ready emergence of resistance which may compromise their long-term clinical utility. The appropriate use of fluoroquinolones is an ongoing debate that requires informed discussion and education; without this, fluoroquinolones may lose their potential value in the treatment of tuberculosis.
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Search strategy and selection criteria
Data for this review were identified by searches of PubMed and references from relevant articles. Search terms were ?tuberculosis?, ?Mycobacterium tuberculosis?, ?quinolones?, ?fluoroquinolones?, ?activity?, ?safety?, ?use?, ?treatment?, ?resistance?, ?mechanism?, ?gyrase?, ?quinolone resistance-determining region?, ?human immunodeficiency virus?, and ?acquired immunodeficiency syndrome?. English language papers were reviewed.
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Conflicts of interest
None declared.
Acknowledgments
Funding support was provided by the Global Alliance for Tuberculosis Drug Development and NIH grant AI43846
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