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Antimicrobial Agents and Chemotherapy, August 2007, p. 3011-3013, Vol. 51, No. 8
0066-4804/07/$08.00+0     doi:10.1128/AAC.00085-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

New Guinea Pig Model of Cryptococcal Meningitis{triangledown}

William R. Kirkpatrick,1* Laura K. Najvar,1 Rosie Bocanegra,1 Thomas F. Patterson,1,2 and John R. Graybill1,2

Department of Medicine, The University of Texas Health Science Center at San Antonio, Texas 78229-3900,1 Audie Murphy Division, South Texas Veterans Health Care System, San Antonio, Texas 782842

Received 19 January 2007/ Returned for modification 13 March 2007/ Accepted 3 June 2007


   ABSTRACT
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We developed a guinea pig model of cryptococcal meningitis to evaluate antifungal agents. Immunosuppressed animals challenged intracranially with Cryptococcus neoformans responded to fluconazole and voriconazole. Disease was monitored by serial cerebrospinal fluid (CSF) cultures and quantitative organ cultures. Our model produces disseminating central nervous system disease and responds to antifungal therapy.


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The contemporary primary model for cryptococcocal infections is the mouse (5), but mice are ineffective when assessing certain azole drugs (2, 6). Obtaining murine cerebral spinal fluid is difficult, and fluid yields are miniscule. We developed a new guinea pig model of cryptococcal meningitis to evaluate the broadest antifungal range and provide additional means of monitoring disease progression.

Male Hartley guinea pigs (0.5 kg) were immunosuppressed with triamcinolone acetonide daily as described previously (3). The day after initiation of immunosuppression, animals were anesthetized (3) and challenged with Cryptococcus neoformans intracranially (ICr) or intracisternally (ICs) at approximately 8 x 106 CFU in 0.1 ml. For ICr infection, animals were briefly anesthetized, their heads were closely clipped, and the area was swabbed with 70% alcohol. The inoculum was delivered through a 26-gauge needle following direct puncture percranially, 25 mm caudal along the midline from the orbit (4). Animals receiving ICs inoculation were anesthetized, clipped, and swabbed with alcohol, and the inoculum was delivered into the lumen of the foramen magnum (8).

Cerebral spinal fluid (CSF) was drawn 1, 3, 7, 10, and 15 days postinfection. Guinea pigs were terminated on day 15. Each group contained at least two untreated control guinea pigs. Controls were pooled and analyzed as a single group at the study's conclusion. Animal research procedures were approved by The University of Texas Health Science Center at San Antonio's Institutional Animal Care and Use Committee.

Antifungal therapy included oral fluconazole at 10 mg/kg twice a day (BID) (Pfizer, Inc., Groton, CT); oral voriconazole at 5, 10, or 20 mg/kg BID (Pfizer); or amphotericin B (Fungizone; Bristol-Myers Squibb Co., Princeton, NJ) at 1 mg/kg of body weight/day intraperitoneally (i.p.) starting 48 h after challenge and continuing for 13 days. Untreated controls received 0.5 ml sterile water orally (p.o.) daily.

Organ cultures were performed after the death of the animal during treatment (n = 6) or after completion of study on day 15 (n = 58) (1, 7).

Cryptococcus neoformans isolate USC-1597, a clinical isolate used previously in our animal studies, was grown on Sabouraud dextrose agar at 35°C for 72 h and then in brain heart infusion broth at 35°C overnight. Yeasts were concentrated and adjusted to approximately 1 x 107 CFU by hemacytometer in 0.1 ml with sterile saline (1, 7). Seventy-two-hour fluconazole, voriconazole, and amphotericin B MICs against USC-1597 were 1, 0.06, and 0.25 µg/ml, respectively.

The Mann-Whitney test was used for comparing treatment groups. Statistical significance was defined as P < 0.05.

Initial studies using a challenge with <104 CFU by either ICr or ICs route showed inconsistent infection of untreated immunosuppressed animals. By day 15, CFU counts of CSF did not surpass the inoculum and CFU were undetectable in most animals; therefore, additional studies at a higher inoculum were chosen (data not shown). When 9 x 106 CFU was used intracranially, untreated guinea pigs had high CSF counts of cryptococci through the 15-day observation period, with dissemination to kidneys (Table 1). Fluconazole at 10 mg/kg BID reduced CSF and tissue burden by >1 log. Voriconazole at 5 mg/kg BID was ineffective in reducing CSF counts but did reduce brain and kidney counts. Challenge with this amount of C. neoformans by either ICr or ICs produced central nervous system (CNS) disease, but ICr inoculation was technically more simple and therefore was chosen as the infecting route for additional experiments.


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  		TABLE 1. Model development and mean log10 CFU of C. neoformans recovered from guinea pig tissues and CSF 15 days after ICr infection

 
Subsequently, guinea pigs were infected with approximately 107 CFU C. neoformans ICr and monitored for 15 days. Serial lumbar punctures, sampling up to 0.25 ml CSF, were done during treatment, and animals were sacrificed following 13 days of therapy. Figure 1 shows the course of infection by quantitative cultures from serial CSF samples. C. neoformans in the CSF of the control animals rose steadily by 2 logs, as shown in Fig. 1A. Figure 1B shows the course in guinea pigs treated with fluconazole at 10 mg/kg BID, with which yeast counts also increased by 1/2 log during the first week of therapy, stabilized, and then ultimately declined by 1 log due to therapy by day 15. A similar pattern was seen with voriconazole therapy at 5, 10, and 20 mg/kg BID, as depicted in Fig. 1C, D, and E. With voriconazole, CSF yeast counts increased up to 1 log within the first week and then, in the second week of therapy, CSF yeast counts decreased by 0.7 log (5 mg/kg, Fig. 1C), 1.6 log (10 mg.kg, Fig. 1D), and 1 log (20 mg/kg, Fig. 1E). Figure 1F shows the yeast growth pattern in CSF with amphotericin B therapy. Yeast counts gradually increased through day 10, and counts were slightly reduced by day 15. Therapy with fluconazole at 10 mg/kg BID or voriconazole at 10 or 20 mg/kg BID was effective in reducing CFU by day 15 compared to the level in controls (P < 0.05).


Figure 1
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  		FIG. 1. Quantitative cultures of CSF, expressed in log10, from guinea pigs challenged ICr with 107 CFU C. neoformans in (A) untreated animals and animals treated with (B) oral fluconazole (FLC) at 10 mg/kg BID, (C) oral voriconazole (VRC) at 5 mg/kg BID, (D) oral voriconazole at 10 mg/kg BID, (E) oral voriconazole at 20 mg/kg BID, and (F) amphotericin B (AMB) at 1 mg/kg i.p. Treated animals received therapy for 12 days. Median counts are indicated by horizontal bars. *, P < 0.05 versus controls.

 
Therapy with either fluconazole or voriconazole decreased the brain tissue burden. Fluconazole at 10 mg/kg BID or voriconazole at 5, 10, or 20 mg/kg BID effectively reduced brain burden by approximately 2 logs, 1 log, 1.5 log, and 1.5 log, respectively, versus time-matched controls (P < 0.05), yet amphotericin B therapy was indistinguishable from that in controls. None of the treatment regimens prevented dissemination to extracerebral organs with the higher inoculum. Only fluconazole at 10 mg/kg BID was effective in reducing kidney tissue burden (P < 0.05).

This model of cryptococcal meningitis produces disseminating primary CNS disease. Dissemination, which occurs within the first 2 weeks is not prevented by the therapies used in this study. Serial CSF counts confirmed the effects of fluconazole and voriconazole. Infection progressed for the first week, stabilized by day 10, and only on day 15 showed decreasing counts, consistent with fluconazole's slowly fungicidal action in this disease, often requiring over 2 months to sterilize CSF (4, 9).

The relative ease of serial blood and CSF sampling in guinea pigs due to size and their ability to metabolize voriconazole more slowly than do mice may translate pharmacokinetic and dose-response analysis data into clinical utility more favorably from this model than from a murine model of cryptococcal meningitis. This chronic infection model should be useful for other trials of antifungal therapy.


   ACKNOWLEDGMENTS
 
This work was supported by NIH NIAID contract no. NO1-AI-25475.

We thank Chris Lambros for assistance in implementing these studies and improving the manuscript. We are indebted to Brent J. Coco and Steve Hernandez for technical assistance.


   FOOTNOTES
 
* Corresponding author. Mailing address: The University of Texas Health Science Center at San Antonio, Department of Medicine, Division of Infectious Diseases, 7703 Floyd Curl Drive, Mail Code 7881, San Antonio, TX 78229-3900. Phone: (210) 567-4823. Fax: (210) 567-3303. E-mail: kirkpatrick{at}uthscsa.edu Back

{triangledown} Published ahead of print on 11 June 2007. Back


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  3. Kirkpatrick, W. R., R. K. McAtee, A. W. Fothergill, M. G. Rinaldi, and T. F. Patterson. 2000. Efficacy of voriconazole in a guinea pig model of disseminated invasive aspergillosis. Antimicrob. Agents Chemother. 44:2865-2868.[Abstract/Free Full Text]
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Antimicrobial Agents and Chemotherapy, August 2007, p. 3011-3013, Vol. 51, No. 8
0066-4804/07/$08.00+0     doi:10.1128/AAC.00085-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




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