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Antimicrobial Agents and Chemotherapy, June 2006, p. 1989-1992, Vol. 50, No. 6
0066-4804/06/$08.00+0     doi:10.1128/AAC.01467-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Efficacy of Macrolides and Telithromycin against Leptospirosis in a Hamster Model

James E. Moon, Michael C. Ellis, Matthew E. Griffith, Joshua S. Hawley, Robert G. Rivard, Suzanne McCall, Duane R. Hospenthal, and Clinton K. Murray^*

Department of Medicine and Department of Clinical Investigation, Brooke Army Medical Center, Fort Sam Houston, Texas

Received 11 November 2005/ Returned for modification 7 March 2006/ Accepted 28 March 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
Human studies support the use of ß-lactams and tetracyclines in the treatment of leptospirosis. Additional agents from these and other classes of antimicrobials also have in vitro activity against Leptospira species, though corroborating in vivo data are limited or lacking. We evaluated the therapeutic efficacy of azithromycin, clarithromycin, and telithromycin in a lethal hamster model of leptospirosis using Leptospira interrogans serogroup Canicola serovar Portlandvere. A range of dosages for each antimicrobial was given to the infected animals on days 2 through 7 (5 days) of the 21-day survival model. All untreated control animals survived less than 10 days from infection. Ninety to 100% of doxycycline controls, treated for 5 days with 5 mg/kg of body weight of drug, survived to 21 days. Treatment with azithromycin (daily dose: 6.25, 12.5, 25, 50, 100, or 200 mg/kg) resulted in 100% survival at all evaluated doses. Animals receiving 20 mg/kg or more of clarithromycin (daily dose: 1, 5, 10, 15, 20, 40, 60, or 100 mg/kg) had improved survival. Ninety-eight percent of animals treated with telithromycin (daily dose: 1, 5, 10, 15, 20, or 40 mg/kg) survived. We conclude that all agents tested have demonstrated in vivo efficacy in treating acute leptospirosis. These results provide support for further evaluation of macrolide and ketolide antimicrobial agents in human trials.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Leptospirosis is an acute febrile illness caused by spirochetes of the genus Leptospira whose clinical manifestations can range from asymptomatic infection to death. Current treatment of leptospirosis includes symptomatic support with or without directed antimicrobial administration, depending on the severity and duration of symptoms (12). Specific antimicrobial therapy for leptospirosis has been examined for over 60 years (13, 17). Antimicrobials of choice include penicillin, doxycycline, cefotaxime, and ceftriaxone (7, 13, 16, 20, 22). This is based on randomized, controlled human studies that have demonstrated the efficacy of these drugs in reducing symptoms and leptospiruria but not necessarily in improving survival. Amoxicillin, ampicillin, and erythromycin have been suggested in less-severe disease, although no randomized, controlled human data are available to support this recommendation (23). Additional antimicrobials, primarily ß-lactams and fluoroquinolones, have shown treatment efficacy in various animal models (1, 4, 5, 6, 9, 19, 24). However, to date, neither animal model nor human trial has addressed the efficacy of most other modern antimicrobials in leptospirosis. Such agents might provide equal or superior efficacy, with easier administration, fewer contraindications (e.g., doxycycline's contraindication in pregnancy or restriction of penicillin use due to allergy), and fewer adverse events (e.g., doxycycline's associated phototoxicity and gastrointestinal side effects) than the proven options. This report describes our experience in using macrolides and telithromycin in treating acute leptospirosis in a lethal hamster model.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animal model. The animal model utilized was adapted from previously published work by Alexander and Rule (1). Female golden Syrian hamsters (Mesocricetus auratus) weighing 50 to 100 g (Harlan, Indianapolis, IN) were inoculated intraperitoneally with 0.5 ml of Ellinghausen-McCullough-Johnson-Harris (EMJH) medium (Becton Dickinson, Sparks, MD) containing 10⁵ leptospires. Leptospira interrogans serogroup Canicola serovar Portlandvere (CA-12-029/CDC Nic 1808) was used for all trials (obtained from David Haake, University of California Los Angeles). This strain was maintained by hamster passage and recovered between passages in semisolid EMJH medium. Inoculum was produced by subculture of these stocks in liquid EMJH medium for 7 days at 30°C and quantified by organism count using a Petroff-Hausser counting chamber and dark-field microscopy. Preliminary studies demonstrated that the chosen strain produced 100% mortality in hamsters with inocula as small as 10² leptospires, and 20% mortality with 10¹ leptospires. The selected inoculum size consistently resulted in death in hamsters between 6 and 9 days after injection. Infected hamsters were observed multiple times a day during the 21-day study. Any study animal demonstrating significant pain or distress or characteristics of a moribund state was humanely euthanized. All animal experimentation was conducted under protocols approved by our Institutional Animal Care and Use Committee.

Antimicrobial agents. Doxycycline was purchased as a powder from Sigma-Aldrich (St. Louis, MO). Azithromycin was purchased in its commercial parenteral formulation from Pfizer (Groton, CT). Clarithromycin (Abbott Laboratories, Abbott Park, IL) and telithromycin (Aventis, Bridgewater, NJ) were obtained directly from their manufacturers in powder formulations. Drug solutions were prepared and stored (if necessary) in accordance with manufacturer recommendations, typically immediately before use or prior to the experiment.

Therapeutic trials. After infection, animals were divided into groups of 10. In each experiment one such group received no antimicrobial therapy, serving as untreated controls, and one group received doxycycline, serving as treated controls. The dose of doxycycline selected for use in treated controls was 5 mg/kg of body weight. Antimicrobial therapy in all experiments was begun on day 2 (infection was on day 0) and continued for 5 days. Six concentrations of azithromycin (6.25, 12.5, 25, 50, 100, and 200 mg/kg), eight concentrations of clarithromycin (1, 5, 10, 15, 20, 40, 60, and 100 mg/kg), and six concentrations of telithromycin (1, 5, 10, 15, 20, and 40 mg/kg) were tested. The efficacy of various doxycycline concentrations (1, 5, 10, 15, 50, and 100 mg/kg) was also examined in the model for further comparison. All drugs were administered once daily via intraperitoneal injection. Each experiment was performed once.

Statistical analysis. Kaplan-Meier curves were compared by the log rank test. P values of 0.01 were considered significant.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Doxycycline improved survival at all doses studied compared to untreated controls. In the independent evaluation of doxycycline dosages (Fig. 1) survival ranged from 80 to 100%. The highest two doses (50 and 100 mg/kg) produced lower survival than that seen with 5, 10, or 15 mg/kg, suggesting possible toxicity at these dosages. Nearly all animals receiving 100 mg/kg were noted to develop diarrhea for 4 to 5 days following the final day of antimicrobial treatment. Diarrhea was not observed in any other treatment group. Among the other experiments, only one animal treated with the selected doxycycline control dose of 5 mg/kg did not survive the 21-day study period.

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FIG. 1. Efficacy of doxycycline in treating fatal leptospirosis.

No deaths occurred in animals treated with azithromycin (Fig. 2). Nearly all animals treated with the two highest doses of azithromycin appeared to poorly tolerate their injections, developing temporary agitation or other signs of physical discomfort. In one animal, a small area of superficial necrosis was noted to develop on the abdomen at an injection site. Secondary to these observations, treatment in these groups (100 and 200 mg/kg) was halted after 3 days. Not receiving the fourth and fifth days of therapy did not impact negatively the overall survival of the animals in these groups.

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FIG. 2. Efficacy of azithromycin in treating fatal leptospirosis.

Dosages of clarithromycin above and including 20 mg/kg resulted in improved survival (Fig. 3). Initial experimentation with clarithromycin tested dosages of 1, 5, 10, 15, 20, and 40 mg/kg. Two additional treatment groups (60 and 100 mg/kg) were added as a second experiment when initial treatment response fell short of that seen with doxycycline. A proportional dose response was noted between 20 and 60 mg/kg, with survival improving from 30 to 90% with increasing dosage. However, at the highest dosage (100 mg/kg), survival was inferior to that of the 60-mg/kg group (70% compared to 90%), suggesting possible toxicity. However, no abnormal physical findings (including diarrhea) were observed with clarithromycin treatment.

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FIG. 3. Efficacy of clarithromycin in treating fatal leptospirosis.

Only 1 of 60 animals treated with telithromycin did not survive to the end of the study (Fig. 4). Similar to the azithromycin trial, some animals treated with the three highest doses of telithromycin (15 mg/kg, 20 mg/kg, and 40 mg/kg) appeared to experience a brief period of agitation and, in several animals, a slight amount of nasal discharge following the first series of injections. All noted symptoms resolved within minutes. During the second and remaining injections only a few animals demonstrated this brief agitation and little to no nasal discharge was observed. As in the azithromycin trial, animals with observed symptoms did not have a reduced survival. Azithromycin and telithromycin produced statistically significant improvement in survival when compared to no treatment (P < 0.001); however, at the lower dose of clarithromycin decreased survival was noted.

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FIG. 4. Efficacy of telithromycin in treating fatal leptospirosis.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Azithromycin, clarithromycin, and telithromycin all demonstrated efficacy in preventing mortality in our animal model of leptospirosis. The observed efficacy of doxycycline was consistent with that reported in previous leptospiral animal research (1, 8). The efficacy of the two macrolide antimicrobial agents azithromycin and clarithromycin supports earlier research that demonstrated in vivo efficacy of erythromycin in treating various rodent models of leptospirosis (2, 5, 9). These results, excluding the lower doses of clarithromycin, are also consistent with our more recent in vitro susceptibility studies which demonstrated that these same agents have superior potency compared to doxycycline and other traditional agents tested across a large series of Leptospira strains (11, 14, 15).

The reasons for the inefficacy of the lower doses of clarithromycin (1, 5, 10, and 15 mg/kg), compared to the other study agents, are unknown at this time. In our prior in vitro work clarithromycin had MICs for various strains of Leptospira equal or superior to either azithromycin or telithromycin (15). Thus, this result was against expectation. Pharmacokinetically, clarithromycin has been clearly shown to have slower intracellular uptake and faster efflux than either azithromycin or telithromycin (3). However, the importance of such differences in regards to antimicrobial efficacy against leptospiral infection is unknown.

With two of the drugs studied, doxycycline and clarithromycin, some deaths were noted in the highest-dosage groups in contrast with lower concentrations of drug. In nearly all cases the deaths in these higher-concentration groups occurred later than those in untreated control groups (range, 6 to 17 days following infection). Most of the observed symptoms, including the diarrhea seen with the 100-mg/kg dose of doxycycline, did not occur in any of the untreated controls. These facts suggest that this increased mortality may have resulted from treatment rather than infection. During the design of this study the doses for each drug were chosen to encompass a range of values surrounding a weight-based human-comparable dose for the particular antimicrobial. In consideration of the inherent difficulties in determining true dose correspondence between humans and animals, and in order not to miss a therapeutic effect by setting too narrow of a range, high and low extremes were chosen from well outside the expected normal human dose range. Each dose was confirmed for safety and lack of toxicity in rodents by a literature review of comparable studies in which the same dose and route of administration were used. While no matching events were found for the study drugs, antimicrobial toxicity in hamsters is well documented among many of the earliest studies of leptospirosis. In experiments in the 1950s hamsters treated orally or subcutaneously with various antimicrobial agents, most notably penicillin and erythromycin, developed diarrhea after several days of treatment, followed rapidly by emaciation and death (5, 9). These older studies were unable to fully explain the link between the antibiotics and their observed toxicity. Likewise, in the experiments reported here, the exact nature of the drug-related toxicity, if any, remains uncertain. Necropsies performed on hamsters which perished after exhibiting symptoms were unrevealing, and efforts to collect samples for Clostridium difficile toxin assay were unsuccessful. However, it must be noted that in at least one similar study performed at our institution (unpublished data) C. difficile toxin was isolated in a stool sample taken from a hamster exhibiting diarrhea after treatment with imipenem. This suggests at least one possible explanation for some of the observed toxicity.

The results of this study, particularly the nearly uniform protection exhibited by azithromycin and telithromycin, both oral drugs without toxicity in children and during pregnancy, bear further investigation. Current treatment options (ß-lactams and tetracyclines) have factors limiting their practical application in many resource-poor areas, where outbreaks of leptospirosis are most likely to occur. Parenteral ß-lactam antimicrobial agents are currently the drugs of choice in severe infection. However, parenteral administration requires significant medical resources, and those patients with severe penicillin allergies may not be able to tolerate these agents at all. In less-severe disease, doxycycline is the only proven agent, although amoxicillin, ampicillin, fluoroquinolones, and erythromycin have also been used. All these agents are inexpensive, but the use of doxycycline in children and pregnancy is avoided due to possible toxicities. Effective drugs without such restrictions or resource requirements would certainly aid treatment in many endemic regions.

Even more clinically interesting is the fact that the effects of azithromycin were seen even in groups in which only 3 days of treatment were given. This suggests that this agent may have potential for use in shortened or even single-dose therapy or as a prophylaxis agent. Historically, doxycycline is the only agent to have been tested and proven in both animals and humans for use in prophylaxis against leptospirosis. Studies have shown that a single 200-mg dose of doxycycline given weekly, or after an acute exposure, is capable of reducing both the development of clinical symptoms and mortality from leptospirosis (8, 10, 18, 21). Given its success in this trial, its long half-life, and its intracellular concentration (3), single-dose azithromycin might prove to be an alternative to single-dose doxycycline for leptospirosis chemoprophylaxis or therapy. An azithromycin prophylaxis protocol using the current hamster model is under way at our facility to evaluate this possibility.

In this study we have documented for the first time the therapeutic efficacy of azithromycin, clarithromycin, and telithromycin in an acute lethal hamster model of leptospirosis. The results of this study indicate that these agents may provide alternative treatment options for leptospirosis. Further studies are warranted to determine if this effect is reproducible in humans.

 
We thank Richard Harris for invaluable veterinary assistance, John Ward for his help in statistical analysis, and Abbott Laboratories and Aventis for gifts of drugs.

The views expressed herein are those of the authors and do not reflect the official policy or position of the Department of the Army, Department of Defense, or the U.S. Government.

 
* Corresponding author. Mailing address: Infectious Disease (MCHE-MDI-7E), Brooke Army Medical Center, 3851 Roger Brooke Drive, Fort Sam Houston, Texas 78234. Phone: (210) 916-4355. Fax: (210) 916-0388. E-mail: Clinton.Murray{at}amedd.army.mil.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Antimicrobial Agents and Chemotherapy, June 2006, p. 1989-1992, Vol. 50, No. 6
0066-4804/06/$08.00+0     doi:10.1128/AAC.01467-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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• Griffith, M. E., Moon, J. E., Johnson, E. N., Clark, K. P., Hawley, J. S., Hospenthal, D. R., Murray, C. K. (2007). Efficacy of Fluoroquinolones against Leptospira interrogans in a Hamster Model. Antimicrob. Agents Chemother. 51: 2615-2617 [Abstract] [Full Text]  
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This Article Abstract Full Text (PDF) An erratum has been published Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Moon, J. E. Articles by Murray, C. K. Search for Related Content PubMed PubMed Citation Articles by Moon, J. E. Articles by Murray, C. K.