ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 2009, p. 1858–1862 0066-4804/09/$08.00⫹0 doi:10.1128/AAC.01538-08 Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 53, No. 5
Comparative Efficacies of Lipid-Complexed Amphotericin B and Liposomal Amphotericin B against Coccidioidal Meningitis in Rabbits䌤 Karl V. Clemons,1,2,3* Javier Capilla,1,2,3 Raymond A. Sobel,4,5 Marife Martinez,1 Ann-Jay Tong,1 and David A. Stevens1,2,3 California Institute for Medical Research, San Jose, California1; Department of Medicine, Division of Infectious Diseases, Santa Clara Valley Medical Center, San Jose, California2; Department of Medicine, Division of Infectious Diseases and Geographic Medicine,3 and Department of Pathology,4 Stanford University, Stanford, California; and Veterans Affairs Health Care System, Palo Alto, California5 Received 18 November 2008/Returned for modification 29 December 2008/Accepted 28 February 2009
In separate previous studies, we have shown that lipid-complexed amphotericin B (Abelcet [ABLC]) and liposomal amphotericin B (AmBisome [AmBi]) are efficacious against coccidioidal meningitis in rabbits. Here, we compared ABLC and AmBi directly in a coccidioidal meningitis model. Male New Zealand White rabbits were infected with 5 ⴛ 104 Coccidioides posadasii arthroconidia by direct cisternal puncture. Therapy with intravenous ABLC or AmBi at 7.5 or 15 mg/kg of body weight or sterile 5% dextrose water (D5W) began 5 days later. Clinical assessments were done daily; cerebrospinal fluid and blood samples were obtained on day 15 and upon euthanasia. Survivors to day 25 were euthanatized, the numbers of CFU in their tissues were determined, and histology analyses of the brains and spinal cords were done. Controls showed progressive disease, whereas animals treated with either dose of either drug showed few clinical signs of infection. All ABLC- or AmBitreated rabbits survived, whereas eight of nine D5W-treated rabbits were euthanatized before day 25 (P < 0.0001). Numbers of CFU in the brains and spinal cords of ABLC- or AmBi-treated animals were 100- to 10,000-fold lower than those in the corresponding tissues of D5W-treated animals (P < 0.0006 to 0.0001). However, only two or fewer given a regimen of ABLC or AmBi were cured of infection in both tissues. Fewer ABLC-treated rabbits (four of eight treated with 7.5 mg/kg and five of eight treated with 15 mg/kg) than controls (nine of nine) had meningitis at any level of severity (P, 0.015 or 0.043 for animals treated with ABLC at 7.5 or 15 mg/kg, respectively). Although groups of rabbits treated with AmBi regimens did not have significantly fewer animals with meningitis than the control group (P > 0.05), ABLC and AmBi were not significantly different. In this model, intravenous ABLC and AmBi were similarly highly effective, with few clinical signs of infection, 100% survival, and significantly reduced fungal burdens among treated animals. There appeared to be little benefit in using the 15-mg/kg dosage of either formulation. There was no significant advantage of one drug over the other for this indication. Further studies are required to determine the lowest effective doses of these formulations. nous lipid-complexed amphotericin B (Abelcet [ABLC]) and liposomal amphotericin B (AmBisome [AmBi]) are efficacious in a rabbit model of coccidioidal meningitis (1, 2). Dosages of amphotericin B as ABLC or AmBi of 7.5 and 15 mg per kg of body weight given three times per week for 3 weeks were most effective and resulted in cures in some animals, whereas 1 mg/kg of conventional amphotericin B was much less effective and noncurative in the rabbit. These results suggest that intravenous therapy with ABLC or AmBi may be effective, whereas no dose of conventional amphotericin B is effective by this route in humans. Although both lipid formulations are effective in the model, the question of whether one formulation was superior to the other when the two were given at equivalent doses was unanswered. Comparing drug efficacies across studies done at different times does not account for possible variations in the inoculum level or the virulence of the organism, induced disease severity, or variation in the experimental animals and may lead to an incorrect conclusion. Slight differences in disease severity may result in a drug’s being efficacious in a moderately severe infection model but ineffective in a highly severe infection model. Therefore, it is important to do comparisons of the efficacies of drugs side by side, with the
Coccidioidal meningitis remains a severe and lethal infection (4, 7, 9–11). If left untreated, it is considered uniformly fatal within 2 years. Despite advances in oral azole therapy over the last decade, therapy is prolonged, often considered to be lifelong, and relapses can occur (3, 8). In addition, cure of the disease may not be attainable using oral azoles, regardless of the treatment duration (3). The use of conventional amphotericin B is limited, since the intravenous administration of conventional amphotericin B is ineffective for treatment. The intrathecal administration of amphotericin B is effective in many instances but is suboptimal because of associated side effects and toxicities and because of technical issues of administration. Thus, improvements in therapy are needed for the treatment of this devastating manifestation of coccidioidomycosis. In previous individual studies, we have shown that intrave* Corresponding author. Mailing address: Division of Infectious Diseases, Santa Clara Valley Medical Center, 751 South Bascom Ave., San Jose, CA 95128. Phone: (408) 998-4557. Fax: (408) 998-2723. E-mail:
[email protected]. 䌤 Published ahead of print on 9 March 2009. 1858
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variables of the experimental system constant. Thus, the aim of the present study was to compare ABLC and AmBi directly in the same experiment to determine their efficacies against experimental coccidioidomycosis. MATERIALS AND METHODS Rabbit model. An experimental model of coccidioidal meningitis in 3-kg male New Zealand White rabbits (Myrtles Rabbitry, Inc., Thompson Station, TN) was established as described previously (1, 2, 5, 6, 12–14). In brief, rabbits were anesthetized using a cocktail of ketamine (Ft. Dodge Animal Health, Ft. Dodge, IA), xylazine (The Butler Co., Dublin, OH), and acepromazine (Butler) and infected by direct cisternal puncture with 5 ⫻ 104 Coccidioides posadasii strain Silveira arthroconidia (1, 2, 5, 6, 12–14). All procedures in these studies were done with the approval of the Animal Care and Use Committee of the California Institute for Medical Research. Therapy. Intravenous therapy began 5 days postinfection, with animals receiving ABLC (Abelcet; Enzon Pharmaceuticals Inc., Bridgewater, NJ) or AmBi (AmBisome; Gilead Sciences, Foster City, CA) at 7.5 or 15 mg/kg or sterile 5% dextrose water (D5W). ABLC and AmBi were prepared according to the manufacturers’ instructions and diluted to 2 mg of amphotericin B per ml in D5W. Solutions were infused at a flow rate of approximately 2 ml/min. Each group comprised eight or nine rabbits. The experiment was performed in two separate blocks using four or five animals per group for each block as described previously (1, 2). Clinical evaluation and samples. Clinical assessments of body weight, body temperature, and mobility were done daily (1, 2). Animals showing signs of distress, such as reduced mobility, were given buprenorphine (Reckitt Benckiser Pharmaceuticals, Inc., Richmond, VA) at 0.03 mg per dose twice daily as an analgesic. Animals with a loss of more than 20% of body weight and those showing signs of severe paresis or paralysis were euthanatized, and their deaths were counted in the total deaths for the day. All surviving animals were euthanatized on day 25 or 26 postinfection. Cerebrospinal fluid (CSF) and blood samples were taken while the rabbits were under anesthesia just prior to infection, after the fifth dosage of the drug, and again just prior to euthanasia. Euthanasia was done by administering 1 ml of a saturated solution of sodium pentobarbital (Euthasol; Virbac AH, Inc., Ft. Worth, TX) intravenously while animals were at a surgical plane of anesthesia by using 3.5% isoflurane at a flow rate of 1.5 liter of oxygen per min. Ketamine (20 mg given intravenously) was administered just prior to the administration of the euthanasia solution (1, 2). After euthanasia, the brain and spinal cord were removed aseptically and divided into halves. The numbers of CFU remaining in the brain and spinal cord were determined from one half of each tissue as described previously (1, 2). The lower limit of detection for this assay is approximately 10 CFU per g of tissue. Histology. One half of the brain and one half of the spinal cord were placed in 10% neutral buffered formalin for histological assessment. Microscopic examination of hematoxylin- and eosin-stained sections was performed by a neuropathologist blinded to the treatment each rabbit had received. Meningitis was defined as mild (i.e., characterized by rare foci of small meningeal inflammatory infiltrates), moderate (i.e., characterized by more numerous and larger infiltrates), and severe (i.e., characterized by large numbers of confluent inflammatory infiltrates with numerous granulomas). Parenchymal findings, including arteritis, ischemia, and infarction, were recorded as described previously (1, 2). Statistics. The comparative survival rates were analyzed by a log rank test, and survival curves were presented as Kaplan-Meier plots. A statistical analysis of CFU results was done by a Mann-Whitney U test.
RESULTS Clinical signs. Data on the clinical signs assessed on a daily basis are shown in Fig. 1. Animals given ABLC or AmBi at either dose displayed minimal signs of infection. Body weights showed an initial rise followed by a drop in all groups beginning on day 5 (Fig. 1, top). This pattern was followed by a continued drop in weight in D5W controls, in contrast to a progressive rise in weight in the treated groups. Interestingly, rabbits given ABLC or AmBi at 15 mg/kg showed a greater drop in weight than the animals given the 7.5-mg/kg doses. Although these animals progressively gained weight through
FIG. 1. Clinical assessments of weight, temperature, and mobility are presented. Groups of eight rabbits received ABLC or AmBi at the doses indicated, and a group of nine rabbits were given D5W and served as controls. For the graph showing mobility, a score of 5 was considered to represent normal movement and lower scores represent a loss of normal mobility, beginning with canted body posture and progressing to reduced and/or stiff movements, paresis of a limb (most often a hind limb), and paralysis, usually of the hind limbs, which would be assigned a score of 1 (1, 2, 13).
the end of the experiment, they did not attain the same weights by the end of the experimental period as the animals given the 7.5-mg/kg doses of ABLC or AmBi. The core body temperatures in all animals spiked at day 4 to 5 after infection (Fig. 1, middle). The body temperatures of the D5W control animals remained elevated throughout the experiment, while the temperatures of ABLC- or AmBi-treated animals returned to a normal baseline level of about 39 to 40°C from day 6 on. The mobility scores of ABLC- and AmBi-treated rabbits showed little change during the experiment (Fig. 1, bottom). In contrast, the mobility scores of controls showed a progressive
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decline, reflecting the progression of meningitis affecting motor function. It should be noted that the spikes observed in the graph for the D5W controls during the latter part of the study are due likely to the euthanasia of a severely ill animal and the removal of that animal’s daily score, reflective of severely reduced mobility. The spike in the curve indicates that the surviving rabbits were better able to ambulate but subsequently lost mobility as their disease worsened. Overall, the results of the clinical assessments showed that treatment with ABLC or AmBi at 7.5 or 15 mg/kg was effective in reducing the severity of the signs of coccidioidal meningitis. Survival. The survival curves of the various groups are presented in Fig. 2, top. All rabbits given ABLC or AmBi at 7.5 or 15 mg/kg survived to the termination of the experiment, day 25. In contrast, eight of nine control animals succumbed to infection and were euthanatized prior to day 25. Although the first animal was euthanatized on day 7 postinfection, most were euthanatized between days 15 and 20. Both formulations at both doses significantly prolonged survival versus that of the controls (P ⬍ 0.0001). No significant differences among the treatment groups were found (P ⬎ 0.05). Thus, the two formulations were equally effective in prolonging survival. Burden of Coccidioides in the tissues. The recoveries of CFU of C. posadasii from the brains and spinal cords are shown in Fig. 2, middle and bottom panels, respectively. The median burdens in the brains of treated rabbits were 100- to 1,000-fold lower than that recovered from the D5W-treated control animals. Rabbits given ABLC or AmBi at 15 mg/kg had the lowest mean burdens. All treatments significantly reduced the fungal burden in the brain compared to that in controls, with P values as follows: ABLC at 7.5 mg/kg, P ⫽ 0.001; ABLC at 15 mg/kg, P ⬍ 0.0001; and AmBi at 7.5 or 15 mg/kg, P ⫽ 0.0002. There were no significant differences among the treatment groups. ABLC at 7.5 mg/kg cleared three of eight, ABLC at 15 mg/kg cleared five of eight, AmBi at 7.5 mg/kg cleared four of eight, and AmBi at 15 mg/kg cleared five of eight animals of detectable infection in the brain; no controls were free of infection. Similarly, the CFU recovered from the spinal cords (Fig. 2, bottom) showed that all treatments were effective, with 1,000to 10,000-fold reductions in median burdens in treated animals compared to that in controls. Animals given AmBi at 15 mg/kg did not, however, have a lower median fungal burden than those given AmBi at 7.5 mg/kg, whereas the median fungal burden recovered from those given ABLC at 15 mg/kg was lower than that recovered from animals given ABLC at 7.5 mg/kg (the difference between ABLC groups was not significant). All treatments significantly reduced the fungal burden in the spinal cord compared to that in controls, with P values as follows: ABLC at 7.5 or 15 mg/kg, P ⫽ 0.0002; AmBi at 7.5 mg/kg, P ⫽ 0.0002; and AmBi at 15 mg/kg, P ⫽ 0.0006. There were no significant differences in efficacy among the various treatments (P ⬎ 0.05). Fewer animals were free of detectable infection in the spinal cord than were cleared of infection in the brain, with one and three of eight cleared by ABLC at 7.5 and 15 mg/kg, respectively, and four and two of eight cleared by AmBi at 7.5 and 15 mg/kg, respectively; no control animals were free of detectable infection in the spinal cord. Few animals were free of detectable infection in both the brain and the spinal cord. Such results were found in none of the nine controls, none of the eight animals given ABLC at 7.5
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 2. The cumulative mortality is shown in the top panel, and the numbers of CFU recovered from each animal’s brain and spinal cord are shown in the middle and bottom panels, respectively. For the CFU graphs, a value of 0 indicates that no CFU were recovered and that infection, if any, was below the level of detection for the assay. Animals with values of 0 are considered to have been cured.
mg/kg, one of eight given ABLC at 15 mg/kg, two of eight given AmBi at 7.5 mg/kg, and one of eight given AmBi at 15 mg/kg. CSF parameters. White blood cell (WBC) counts were done at the CSF sampling times, on days 0, 15, and 25, and the results are shown in Fig. 3. On day 0, CSF samples had no cells. On day 15, samples from D5W controls generally had the highest counts, ranging from 12.5 ⫻ 105 to 50 ⫻ 105 WBC/ml of CSF, whereas the counts for the treatment group receiving ABLC at 7.5 mg/kg ranged from 2.5 ⫻ 105 to 17.5 ⫻ 105 WBC/ml, those for the group receiving ABLC at 15 mg/kg ranged from 2.5 ⫻ 105 to 12.5 ⫻ 105 WBC/ml, those for the
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degree of meningitis than the number among the controls (P, 0.015 or 0.043 for ABLC at 7.5 or 15 mg/kg, respectively); AmBi treatment (either dosage) did not result in fewer animals with any degree of meningitis than the number among the controls (P ⬎ 0.05). Comparisons among the different treatment groups showed no significant differences. Summary of results. A summary of the results of these studies is presented in Table 2. Overall, both ABLC and AmBi prolonged survival, reduced the number of CFU in the central nervous system, and resulted in reduced histological severity of disease.
DISCUSSION The aim of this study was to compare ABLC and AmBi directly for the treatment of coccidioidal meningitis. The results show that both formulations are highly effective as therapy when they are administered intravenously (Table 2 gives a summary of the experimental results). Among treated mice, there were few clinical signs of infection, 100% survival, and significantly reduced burdens of Coccidioides in the brains or spinal cords. Overall, the results of the CFU recovery from the brains and spinal cords indicate that both lipid formulations were effective in reducing the fungal burden, which is very similar to previous results (1, 2). Furthermore, at the dosages tested, 7.5 or 15 mg of amphotericin B as ABLC or AmBi per kg, there were no significant differences between doses or formulations. Thus, based on our present results, there appears to be little benefit to the use of the 15-mg/kg dosage of either formulation. AmBi at 7.5 mg/kg did, however, appear to be slightly more effective (but not statistically superior) in reducing the fungal burden than AmBi at 15 mg/kg, with lower median burdens and more animals cleared of detectable CFU in the tissues. These data are similar to those from our previous studies with ABLC but somewhat in contrast to our previous results with AmBi, which indicated the 15-mg/kg dosage to be superior to the 7.5-mg/kg dosage (1, 2). This difference may be due in part to experimental variability, but even in the previous study, AmBi at 7.5 mg/kg was highly effective in prolonging survival and reducing levels of CFU and meningitis (2). The overall cure rates for both amphotericin B formulations showed that the brain was more readily cleared of infection than the spinal cord, which is also similar to our previous results (1, 2). However, in the present study, fewer animals were cleared of infection in both tissues than in the previous studies. It is possible that additional days of dosing or a change in the schedule of daily dosing would improve this result.
FIG. 3. Counts of WBC in the CSF samples from treated rabbits on days 0, 15, and 25 postinfection. Results shown are the mean and standard deviation for the group on each sampling day. The three bars for each group correspond to days 0, 15, and 25, in that order.
animals receiving AmBi at 7.5 mg/kg ranged from 3.75 ⫻ 105 to 17.5 ⫻ 105 WBC/ml, and those for the group treated with AmBi at 15 mg/kg ranged from 2.5 ⫻ 105 to 24 ⫻ 105 WBC/ml. At the day 25 time point, counts of WBC in the CSF samples from the surviving treated animals showed no significant change from those at the day 15 time point, similar to the results in previous studies (1, 2). Coccidioides is often not recovered from the CSF during meningitis. No treated animal had recoverable CFU of Coccidioides in the CSF at day 15 or 25, whereas three controls at each time point were positive for CFU (⬍10 per ml of CSF). Histological evaluation. The histological findings are summarized in Table 1. As expected, control animals had more extensive and severe disease than the treated animals. The severity of the infection was indicated by the findings of arteritis, ischemia, and infarction associated with severe meningitis. ABLC and AmBi at either dosage effectively reduced the histological severity of disease, with only mild or minimal meningitis present. Interestingly, four of eight animals given ABLC at 7.5 mg/kg and three of eight given ABLC at 15 mg/kg had no histological evidence of meningitis. In comparison, zero of nine D5W controls, one of eight animals given AmBi at 7.5 mg/kg, and two of eight given AmBi at 15 mg/kg showed no meningitis. A statistical evaluation by Fisher’s exact test showed that ABLC treatment produced significantly fewer animals with any
TABLE 1. Histopathological findings in NZW rabbits infected intracisternally with C. posadasiia No. of animals with meningitis assessment of:
No. of animals with:
Treatment (dose 关mg/kg兴)
Absent
Mild
Moderate
Severe
Ischemia
Abscess
Arteritis
Infarction
D5W control ABLC (7.5) ABLC (15) AmBi (7.5) AmBi (15)
0 4 3 1 2
1 4 5 6 6
0 0 0 1 0
8 0 0 0 0
7 0 0 0 0
5 0 0 0 0
8 0 0 1 0
2 0 0 0 0
a The table shows the number of animals that had ischemia, abscesses, arteritis, or infarction in brain or spinal cord tissues of the central nervous system. Meningitis was classified as mild, moderate, or severe. Treatment groups consisted of eight animals each, and the control group included nine animals.
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ANTIMICROB. AGENTS CHEMOTHER. TABLE 2. Summary of results
Treatment and dose (mg/kg)
a
No. in group
No. surviving at day 25 (P value)a
Mean log10 CFU per sample (no. of CFU-free samples) 关P value兴a of: Brain tissue
No. of rabbits cured
Spinal cord tissue
Histological severity of disease (P value)a
D5W
9
1
3.58 (0)
3.86 (0)
0
Severe
ABLC 7.5 15
8 8
8 (⬍0.0001) 8 (⬍0.0001)
0.92 (3) 关0.001兴 0.32 (5) 关⬍0.0001兴
1.17 (1) 关0.0002兴 0.76 (3) 关0.0002兴
0 1
Mild (⬍0.05) Mild (⬍0.05)
AmBi 7.5 15
8 8
8 (⬍0.0001) 8 (⬍0.0001)
0.51 (4) 关0.0002兴 0.54 (5) 关0.0002兴
0.58 (4) 关0.0002兴 1.18 (2) 关0.0006兴
2 1
Mild (⬎0.05) Mild (⬎0.05)
P values for comparison with results for the D5W control group.
Interestingly, as assessed by histology, ABLC at 7.5 mg/kg appeared to have reduced the severity of infection to a greater degree than AmBi at an equivalent dosage of amphotericin B. Although this difference between the two regimens was not statistically significant, ABLC treatment was significantly better than D5W control treatment, while AmBi treatment resulted in the same number of animals with no apparent meningitis as that among the D5W controls. Thus, these results would suggest that there may be some slight benefit to the use of ABLC over AmBi with respect to the reduction of the pathological severity during infection. In this comparison, neither ABLC nor AmBi had a significant advantage in the treatment of coccidioidal meningitis. Each formulation was highly effective against coccidioidal meningitis at the doses tested. The mechanism by which ABLC or AmBi is more highly effective than conventional amphotericin B given intravenously against coccidioidal meningitis is undetermined. Low measured CSF drug concentrations are not relevant where organisms free in CSF are not a feature of the disease. Low reported parenchymal drug concentrations may reflect dilution, by the uninvolved brain, of the drug present in a critical and unmeasured site. Such a site may be the meninges and adjacent brain. It is also possible that more of the lipid-formulated amphotericin B reaches the sites of infection by carriage in infiltrating monocytic cells, thus raising the localized concentration of amphotericin B to effective levels. If true, this would be effectively a targeting of the drug to the site of infection, which may not occur with conventional amphotericin B. Additional studies to evaluate the localization of the drug to the site of infection would be illuminating in this respect. It also would be of interest to determine whether a longer duration of dosing or a more intensive regimen of dosing (e.g., daily dosing) would result in a cure. In addition, further studies would be required to determine whether the doses of 3 to 5 mg/kg of the ABLC or AmBi formulations used most often in the clinical setting would be effective in the rabbit model and what the lowest effective dose of either lipid formulation of amphotericin B may be, thus facilitating clinical trials and possibly enabling the demonstration of differences between drugs, on a milligram-per-kilogram basis, that may not be apparent when higher, fully effective doses of both are compared.
ACKNOWLEDGMENT These studies were funded in part by Enzon Pharmaceuticals, Inc.
REFERENCES 1. Capilla, J., K. V. Clemons, R. A. Sobel, and D. A. Stevens. 2007. Efficacy of amphotericin B lipid complex in a rabbit model of coccidioidal meningitis. J. Antimicrob. Chemother. 60:673–676. 2. Clemons, K. V., R. A. Sobel, P. L. Williams, D. Pappagianis, and D. A. Stevens. 2002. Efficacy of intravenous liposomal amphotericin B (AmBisome) against coccidioidal meningitis in rabbits. Antimicrob. Agents Chemother. 46:2420– 2426. 3. Dewsnup, D. H., J. N. Galgiani, J. R. Graybill, M. Diaz, A. Rendon, G. A. Cloud, and D. A. Stevens. 1996. Is it ever safe to stop azole therapy for Coccidioides immitis meningitis? Ann. Intern. Med. 124:305–310. 4. Johnson, R. H., and H. E. Einstein. 2006. Coccidioidal meningitis. Clin. Infect. Dis. 42:103–107. 5. Sorensen, K. N., R. A. Sobel, K. V. Clemons, L. Calderon, K. J. Howell, P. R. Irani, D. Pappagianis, P. L. Williams, and D. A. Stevens. 2000. Comparative efficacies of terbinafine and fluconazole in treatment of experimental coccidioidal meningitis in a rabbit model. Antimicrob. Agents Chemother. 44: 3087–3091. 6. Sorensen, K. N., R. A. Sobel, K. V. Clemons, D. Pappagianis, D. A. Stevens, and P. L. Williams. 2000. Comparison of fluconazole and itraconazole in a rabbit model of coccidioidal meningitis. Antimicrob. Agents Chemother. 44:1512–1517. 7. Stevens, D. A. 2006. Coccidioidal meningitis. Clin. Infect. Dis. 43:385. 8. Stevens, D. A., and K. V. Clemons. 2007. Azole therapy of clinical and experimental coccidioidomycosis. Ann. N. Y. Acad. Sci. 1111:442–454. 9. Williams, P. L. 2007. Coccidioidal meningitis. Ann. N. Y. Acad. Sci. 1111: 377–384. 10. Williams, P. L. 2001. Vasculitic complications associated with coccidioidal meningitis. Semin. Respir. Infect. 16:270–279. 11. Williams, P. L., R. Johnson, D. Pappagianis, H. Einstein, U. Slager, F. T. Koster, J. J. Eron, J. Morrison, J. Aguet, and M. E. River. 1992. Vasculitic and encephalitic complications associated with Coccidioides immitis infection of the central nervous system in humans: report of 10 cases and review. Clin. Infect. Dis. 14:673–682. 12. Williams, P. L., S. L. Leib, P. Kamberi, D. Leppert, R. A. Sobel, Y. D. Bifrare, K. V. Clemons, and D. A. Stevens. 2002. Levels of matrix metalloproteinase-9 within cerebrospinal fluid in a rabbit model of coccidioidal meningitis and vasculitis. J. Infect. Dis. 186:1692–1695. 13. Williams, P. L., R. A. Sobel, K. N. Sorensen, K. V. Clemons, L. M. Shuer, S. S. Royaltey, Y. Yao, D. Pappagianis, J. E. Lutz, C. Reed, M. E. River, B. C. Lee, S. U. Bhatti, and D. A. Stevens. 1998. A model of coccidioidal meningoencephalitis and cerebrospinal vasculitis in the rabbit. J. Infect. Dis. 178: 1217–1221. 14. Zucker, K. E., P. Kamberi, R. A. Sobel, G. Cloud, D. N. Meli, K. V. Clemons, D. A. Stevens, P. L. Williams, and S. L. Leib. 2006. Temporal expression of inflammatory mediators in brain basilar artery vasculitis and cerebrospinal fluid of rabbits with coccidioidal meningitis. Clin. Exp. Immunol. 143:458– 466.
