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Diagnostic Microbiology and Infectious Disease 71 (2011) 366–369 www.elsevier.com/locate/diagmicrobio

Efficacy of and tigecycline in a hamster model of leptospirosis☆,☆☆ Charla C. Tullya, Mary K. Hinkleb, Suzanne McCallc, Matthew E. Griffithb, ⁎ Clinton K. Murrayb, Duane R. Hospenthalb, aDepartment of Medicine, Wilford Hall Medical Center, Lackland Air Force Base, TX 78236, USA bDepartment of Medicine, Brooke Army Medical Center, Fort Sam Houston, TX 78234, USA cDepartment of Clinical Investigation, Brooke Army Medical Center, Fort Sam Houston, TX 78234, USA Received 8 December 2010; accepted 25 August 2011

Abstract

Leptospirosis is a widespread zoonotic infection characterized by acute febrile illness. Severely ill patients may require empiric treatment with broad-spectrum prior to definitive diagnosis. We evaluated the efficacy of minocycline and tigecycline against leptospirosis in a hamster model. Hamsters were treated with either minocycline (5, 10, or 25 mg/kg per day) or tigecycline (5, 10, or 25 mg/kg per day) for 5 days. Controls included untreated animals and -treated animals (5 mg/kg per day). Nine days after infection, all untreated animals were dead. All treated hamsters survived to the end of study (day 21). Study groups showed significantly improved survival compared to the untreated group (P b .01). Minocycline and tigecycline showed survival benefit comparable to the standard treatment, doxycycline. In the absence of doxycycline, minocycline may be considered as an alternative, while tigecycline may be useful in the management of severely ill patients prior to a definitive diagnosis. Published by Elsevier Inc.

Keywords: Leptospirosis; Therapy; Minocycline; Tigecycline; Hamster

1. Introduction in the urine of infected animals. Disease presentation varies widely, from a mild, self-limited febrile illness to a life- Leptospirosis is a zoonotic infection characterized by threatening syndrome involving vasculitis of multiple organ acute febrile illness and caused by the spirochetes of the systems and a mortality of up to 50% (McBride et al., 2005; genus Leptospira. In addition to nonpathogenic serovars, Spichler et al., 2008; World Health Organization, 2003). over 200 pathogenic serovars of Leptospira have been Treatment of leptospirosis is typically based on severity and described (World Health Organization, 2003). Leptospirosis duration of symptoms and usually includes supportive care is a public health threat worldwide, particularly in the tropics in addition to antimicrobials. Currently, antimicrobial and subtropics, and is spread through of leptospires therapy for leptospirosis includes oral or intravenous (IV) doxycycline, IV penicillin G, IV third-generation cephalo- ☆ Disclaimer: The views expressed herein are those of the authors and sporins, or, in milder cases, oral amoxicillin or do not reflect the official policy or position of the Department of the Air (Levett, 2001; World Health Organization, 2003). Diagnosis Force, Department of the Army, Department of the Air Force, Department of of leptospirosis is most often based on serologic testing, Defense, or the US Government. The authors are employees of the US government. This work was prepared as part of their official duties and, as some of which is time and labor intensive and may take such, there is no copyright to be transferred. This work previously was several days to produce results (Effler et al., 2002). Rapid presented in part at the Annual Meeting of the American Society of Tropical tests often lack sensitivity within the first week of illness Medicine and Hygiene, Washington, DC, November 18–22, 2009. ☆☆ (McBride et al., 2005). Therefore, patients who are severely This work is supported by the Global Emerging Infections ill may require empiric treatment with broad-spectrum Surveillance and Response System (GEIS), a division of the Armed Forces Health Surveillance Center. antibiotics prior to definitive diagnosis of leptospirosis. ⁎ Corresponding author. Tel.: +1-210-916-4355; fax: +1-210-916-0388. Minocycline and tigecycline are members of the tetracy- E-mail address: [email protected] (D.R. Hospenthal). clines, a group of broad-spectrum, bacteriostatic antibiotics.

0732-8893/$ – see front matter. Published by Elsevier Inc. doi:10.1016/j.diagmicrobio.2011.08.018 C.C. Tully et al. / Diagnostic Microbiology and Infectious Disease 71 (2011) 366–369 367 have activity against many Gram-positive and °C in the dark between uses and allowed to come to room Gram-negative microorganisms, including anaerobes, as temperature prior to administration. Previous in vitro work in well as rickettsiae, chlamydiae, mycoplasmas, and some our institution showed median MICs of 0.125 μg/mL for protozoa. Susceptibility patterns produced by most tetracy- both minocycline and tigecycline on repeated broth micro- cline drugs are similar. However, tigecycline, a dilution susceptibility testing against this strain (Hospenthal, derivative of minocycline, is not affected by some of the D.R., unpublished results). Doxycycline was demonstrated common mechanisms of resistance and therefore to have a median MIC of 0.06 μg/mL (Moon et al., 2007). remains effective against a wider range of bacteria that may be resistant to older tetracyclines, including doxycycline and 2.3. Therapeutic trials minocycline (Chambers and Deck, 2009; Chopra and After infection, animals were divided into 8 groups: 1 Roberts, 2001). Minocycline is an older tetracycline group of 5 untreated controls and 7 groups of 10 treated compound that is typically available and inexpensive animals, including a treated control group of 10 animals. throughout the world, making it a potential substitute for Treated control animals received doxycycline 5 mg/kg IP doxycycline when that drug is unavailable. If found to be once daily on days 2 to 6 after infection. The doxycycline effective against leptospires, tigecycline may be useful in dose for the treated control group was chosen based on empiric treatment of acute febrile illnesses of unknown previously determined effective dose in a hamster model of etiology when leptospirosis is included in the differential leptospirosis (Moon et al., 2006). Groups of 10 animals diagnosis, allowing coverage of a wide range of other received 1 of 3 doses of minocycline (5, 10, or 25 mg/kg) IP infectious diseases. In the study reported herein, we evaluate once daily or 1 of 3 tigecycline doses (5, 10, or 25 mg/kg) IP the efficacy of minocycline and tigecycline in a hamster once daily on days 2 to 6 after infection. model of acute leptospirosis. 2.4. Blood cultures

2. Materials and methods Sterile conical tubes containing 10 mL of semi-solid 0.2% Ellinghausen-McCullough-Johnson-Harris media were in- 2.1. Animal model oculated with 2–3 drops of whole blood obtained at the time of death of each animal. Culture tubes then were incubated at All animal experimentation was conducted under a 30 °C. At 1–2-week intervals, a 0.1-mL sample of each protocol approved by the local Institutional Animal Care culture was obtained approximately 1 cm from the top of and Use Committee. Female Golden Syrian hamsters each culture surface (in positive cultures this location (Mesocretius auratus), 4-6 weeks old and 75-100 g in commonly has a cloudy area of Leptospira growth termed weight (Harlan Sprague Dawley, Indianapolis, IN, USA) the Dinger zone) and examined for the presence of were employed. All animals were infected with 105 Lep- leptospires by dark field microscopy. Cultures were deemed tospira interrogans serovar Portlandvere by intraperitoneal negative if no leptospires were visualized by 6 weeks after (IP) injection as previously described (Moon et al., 2006). inoculation of each culture. All animals were monitored at least twice daily for 21 days, and those exhibiting signs of significant pain or distress or 2.5. Statistical analysis characteristics of a moribund state were humanely eutha- nized. All animals surviving to day 21 were euthanized at SPSS version 16.0 (SPSS, Chicago, IL, USA) was used to that time. Blood samples for culture were obtained via create Kaplan–Meier plots for each study group. Survival cardiac puncture at the time of death to determine the differences between study groups were compared by the log presence of spirochetemia. rank test. P values of b .05 were considered significant.

2.2. Antimicrobial agents 3. Results Doxycycline hyclate (Bedford Laboratories, Bedford, OH, USA) and tigecycline (Wyeth Pharmaceuticals, Phila- All of the untreated animals became moribund and were delphia, PA, USA) were purchased in their commercially euthanized by day 9 (Fig. 1). All of the doxycycline-treated available, parenteral formulations. These were reconstituted control animals survived to day 21. All animals in the in normal saline under aseptic conditions prior to use. minocycline and tigecycline treatment groups survived to Minocycline (Sigma-Aldrich, St. Louis, MO, USA) was day 21 without evidence of disease. Overall, survival for purchased in powder form and dissolved in normal saline each of the experimental treatment groups was significantly solution. All were diluted to appropriate concentrations to improved compared to untreated controls (P b .01). On the allow for administration of each dose in a volume of 0.5 mL contrary, there was no difference between the experimental (5 mg/kg of doxycycline or 5, 10, or 25 mg/kg of minocycline- and tigecycline-treated groups and the doxy- minocycline or tigecycline, based on mean animal weight cycline-treated control group. Blood cultures from 4 of 5 of per group). All reconstituted drug solutions were stored at 4 the untreated animals became positive for spirochete growth 368 C.C. Tully et al. / Diagnostic Microbiology and Infectious Disease 71 (2011) 366–369 100 In circumstances when penicillin or another beta-lactam 90 is not a treatment option (such as in the setting of known patient hypersensitivity) or doxycycline is not 80 Untreated available, minocycline may be considered an acceptable 70 Doxycycline 5 mg/kg/day alternative for treatment of leptospirosis once further studies Tigecycline 5 mg/kg/day 60 Tigecycline 10 mg/kg/day confirm its efficacy in humans. Currently, severe cases of 50 Tigecycline 25 mg/kg/day leptospirosis are commonly treated with IV penicillin (World Minocycline 5 mg/kg/day Health Organization, 2003). In severe illness, a broad- 40 Minocycline 10 mg/kg/day spectrum antibiotic may be more appropriate, while serologic Percent survival Minocycline 25 mg/kg/day 30 testing and cultures are pending to confirm the diagnosis, to 20 cover other potential etiologies of acute febrile illness. With 10 its broad spectrum of activity and limited resistance, tigecycline holds the potential to be useful in this setting. 0 13579111315171921 In addition to its use as the drug of choice in mild to Days after infection moderate leptospirosis, doxycycline is also used in prophy- laxis against this infection based on a study performed in US Fig. 1. Survival of hamsters treated 5 days with minocycline (5, 10, or 25 mg/kg once daily) and tigecycline (5, 10, or 25 mg/kg once daily). Controls: military personnel. That study conducted in troops deployed untreated and doxycycline (5 mg/kg once daily). to Panama to attend jungle warfare training showed the rate of infection of leptospirosis to be 4.2% among the placebo group, compared with 0.2% among those receiving doxycy- cline 200 mg by mouth once weekly (Takafuji et al., 1984). by 6 weeks after inoculation. Blood cultures from all In the absence of an available licensed vaccine, prophylaxis doxycycline-treated controls were negative at 6 weeks, as with minocycline may be a viable option when and where were blood cultures from all minocycline- and tigecycline- doxycycline is not available based on our study's results. treated groups. The blood culture results support our outcome data, with 4 of the 5 cultures from our untreated animals becoming positive for leptospiral growth by 6 weeks, supporting 4. Discussion leptospirosis as the cause of death. That the single culture from the one untreated animal did not grow the organism This study is the first to evaluate the efficacy of likely only reflects the difficulty of growing Leptospira in minocycline and tigecycline in an in vivo model of acute culture. The negative culture results in even the lowest doses leptospirosis. Once daily dosing of both minocycline and of our study drugs, minocycline and tigecycline, support the tigecycline improved survivability at all doses compared to likelihood that of even lower doses of these drugs might be untreated controls and was as effective as the current effective against leptospirosis in this model. accepted therapy, doxycycline, in the treatment of leptospi- There are limitations of this study. Although it is not rosis. This may have been suspected based on the known unexpected that all treated animals survived and that all similarities of our study drugs, minocycline and tigecycline, untreated animals did not, the lack of dose–response curves to doxycycline. However, our results are important because, does not allow any estimation of a minimal effective dose of before now, in vivo efficacy trials had not been conducted on minocycline or tigecycline in our study. Further studies minocycline and tigecycline. should be done to ascertain whether lower doses of each of In addition to our study results and its similarity to these medications are equally effective, or perhaps superior doxycycline, use of minocycline in human disease is further to, doxycycline in vivo. Also, the lack of pharmacokinetic supported by 3 case reports of its use in patients with data for minocycline or tigecycline in hamsters makes laboratory-confirmed leptospirosis. Two of these reports estimation of comparable treatment doses in humans (published in Japanese) describe minocycline 100 mg IV difficult. Studies of the of minocycline every 12 h as effective against leptospirosis in the case and tigecycline in hamsters would allow extrapolation of patients (Sakamoto et al., 2001, 2005). The one English case our study's results to determine potentially effective doses report documents the course of a patient diagnosed with in humans. Weil's syndrome (severe leptospirosis) confirmed by micro- Minocycline and tigecycline have been found as effective agglutination testing (the gold standard serologic test). as doxycycline at treating acute leptospirosis in an animal Although initially treated with IV penicillin, the patient model when given at the doses tested. The findings of our was later switched to minocycline 400 mg IV daily because study support the few documented case reports of minocy- of penicillin hypersensitivity. He received a 2-week total cline as an alternative treatment of leptospirosis. Human course of minocycline and fully recovered (Ding et al., studies are needed to determine the potential for addition of 2001). There are no previously described cases of leptospi- minocycline and tigecycline to the list of available treatment rosis being treated with tigecycline. options for leptospirosis. Also, additional studies will be C.C. Tully et al. / Diagnostic Microbiology and Infectious Disease 71 (2011) 366–369 369 beneficial to study the use of minocycline as chemoprophy- against leptospirosis in a hamster model. Antimicrob – laxis against leptospirosis when doxycycline is not an Agents Chemother 50:1989 1992. Moon JE, Rivard RG, Griffith ME, Ressner RA, McCall S, Reitstetter RE, available option. Hospenthal DR, Murray CK (2007) Effect of timing and duration of therapy of leptospirosis in a hamster model. J Antimicrob References Chemother 59:148–151. 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