隐球菌性脑膜炎的临床和实验研究
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摘要
本文旨在发展新生隐球菌性脑膜炎（隐脑）疗效监测指标和确立合适的治疗方案。研究工作包括：1、应用NCCLS M27-A微量稀释法检测了19株新生隐球菌临床分离株对三种抗真菌药物的敏感性，并与各自临床疗效进行比较。结果参试菌株对AmB的MIC值范围在0.5～2(g/ml，对5-FC的MIC值范围在0.25～8(g/ml，对FCA的MIC值范围在2～32(g/ml，其中AmB的MIC范围最为狭窄，现有资料尚不能确定MIC值与临床效果间的相关性。2、建立了流式细胞仪法快速检测新生隐球菌药物敏感性，与微量稀释法结果相比符合率为87.5%，实验时间由72小时缩短为4～5小时。3、电镜观察、动物接种和菌体直接染色方法鉴定脑脊液菌体活性，作为隐脑临床疗效监测的重要指标。4、通过对隐脑患者与正常对照组外周血免疫指标进行分析，发现隐脑患者外周血CD4+细胞及CD4+/CD8+均显著下降。5、对35例隐脑回顾分析表明采用分期综合治疗方案治疗隐脑，明显优于其他治疗方案；AmB鞘内注射应成为常规治疗，在静滴AmB出现副作用时仍可继续使用；脑脊液真菌培养可作为初期治疗结束的指标，脑脊液隐球菌菌体计数作为疗效监测的主要指标是合适的；7例经治疗痊愈的儿童隐脑中4例出现严重的后遗症，及时有效地进行抗真菌治疗、必要时应用外科手段降颅压是减少后遗症的关键。
The aim of this paper is to develop new parameters for evaluating clinical therapeutic efficacy and design reasonable strategy for the treatment of cryptococcal meningitis. The work included: (1) The susceptibilities of 19 clinical cryptococcus neoformans isolates to amphotericin B, 5-flucytosine and fluconazole were determined by NCCLS M27-A broth microdilution method. The correlation of MICs and clinical outcome was calculated. Amphotericin B MICs ranged from 0.5 to 2(g/ml, 5-flucytosine MICs ranged from 0.25 to 8(g/ml, while fluconazole MICs ranged from 2 to 32(g/ml. The M27-A-derived MICs did not correlate with clinical outcome. (2) A rapid flow cytometric assay for in vitro antifungal drug susceptibility testing was developed. Good agreement was shown between flow cytometric and broth macrodilution MICs. Compared with the M27-A macrodilution procedure, this assay was more rapid (4-5h versus 72h). (3) Using electron microscope examination, animal inoculation and neutral red staining, a method for evaluating the viability of cryptococcus in cerebrospinal fluid was developed. (4) Analysis of peripheral blood immune parameters showed patients with cryptococcal meningitis had lower CD4 counts and CD4/CD8 ratios than that in health volunteers. (5) By comparing the efficacy of three different antifungal regimens in the treatment of
cryptococcal meningitis, we suggested that the two-step combination therapeutic regimen be well suited to the treatment of cryptococcal meningitis. Intrathecally administered amphotericin B should be recommended, especially when serious toxicity emerged because of systemic administration. Our study also demonstrated that a negative CSF culture for cryptococci as an endpoint for initial treatment was feasible, while CSF cryptococcal count as the most important parameter evaluating clinical outcome was reasonable. Among 7 cases of Cryptococcal meningitis in children, 4 of them had severe sequelae. Management of impending complication combined medical and surgical treatment modalities.
The aim of this paper is to develop new parameters for evaluating clinical therapeutic efficacy and design reasonable strategy for the treatment of cryptococcal meningitis. The work included: (1) The susceptibilities of 19 clinical cryptococcus neoformans isolates to amphotericin B, 5-flucytosine and fluconazole were determined by NCCLS M27-A broth microdilution method. The correlation of MICs and clinical outcome was calculated. Amphotericin B MICs ranged from 0.5 to 2(g/ml, 5-flucytosine MICs ranged from 0.25 to 8(g/ml, while fluconazole MICs ranged from 2 to 32(g/ml. The M27-A-derived MICs did not correlate with clinical outcome. (2) A rapid flow cytometric assay for in vitro antifungal drug susceptibility testing was developed. Good agreement was shown between flow cytometric and broth macrodilution MICs. Compared with the M27-A macrodilution procedure, this assay was more rapid (4-5h versus 72h). (3) Using electron microscope examination, animal inoculation and neutral red staining, a method for evaluating the viability of cryptococcus in cerebrospinal fluid was developed. (4) Analysis of peripheral blood immune parameters showed patients with cryptococcal meningitis had lower CD4 counts and CD4/CD8 ratios than that in health volunteers. (5) By comparing the efficacy of three different antifungal regimens in the treatment of
cryptococcal meningitis, we suggested that the two-step combination therapeutic regimen be well suited to the treatment of cryptococcal meningitis. Intrathecally administered amphotericin B should be recommended, especially when serious toxicity emerged because of systemic administration. Our study also demonstrated that a negative CSF culture for cryptococci as an endpoint for initial treatment was feasible, while CSF cryptococcal count as the most important parameter evaluating clinical outcome was reasonable. Among 7 cases of Cryptococcal meningitis in children, 4 of them had severe sequelae. Management of impending complication combined medical and surgical treatment modalities.
引文
1. Chuck SL, Sande MA. Infections with Cryptococcus neoformans in the acquired immunodeficiency syndrome. N Engl J Med, 1989, 321(12): 794-799
2. 翁心华. 隐球菌病. 见：秦启贤主编. 临床真菌学. 上海：复旦大学出版社，2001. 348-50
3. Hay RJ, Mackenzie DW, Campbell CK，et al. Cryptococcosis in the United Kingdom and the Irish Republic: an analysis of 69 cases. J Infect, 1980, 2(1):13-22
4. Rex JH, Cooper CR Jr, Merz WG, et al. Detection of amphotericin B-resistant Candida isolates in a broth-based system. Antimicrob Agents Chemother, 1995, 39(4):906-9
5. Ghannoum MA, Ibrahim AS, Fu Y, Shafiq MC, et al. Susceptibility testing of Cryptococcus neoformans: a microdilution technique. J Clin Microbiol, 1992, 30(11):2881-6.
6. National Committee for Clinical Laboratory Standards. 1998. Reference method for broth dilution antifungal susceptibility testing of yeasts. M27-A. National Committee for Clinical Laboratory Standards, Wayne, Pa.
7. Revankar SG, Kirkpatrick WR, McAtee RK, et al. Interpretation of trailing endpoints in antifungal susceptibility testing by the National Committee for Clinical Laboratory Standards method. J Clin Microbiol, 1998, 36(1):153-6
8. Wenisch C, Moore CB, Krause R, et al. Antifungal susceptibility testing of fluconazole by flow cytometry correlates with clinical outcome. J Clin Microbiol, 2001, 39(7):2458-62
9. Ramani R, Ramani A, Wong SJ. Rapid flow cytometric susceptibility testing of Candida albicans. J Clin Microbiol, 1997, 35(9):2320-4.
10. Powderly WG, Keath EJ, Sokol-Anderson M, et al. Amphotericin B-resistant Cryptococcus neoformans in a patient with AIDS. Infect Dis Clin Pract, 1992, 1:314-316
11. Naka W, Hanyaku H, Tajima S, et al. Application of neutral red staining for evaluation of the viability of dermatophytes and Candida in human skin scales. J Med Vet Mycol, 1994, 32(1):31-5.
金伯泉. 白细胞分化抗原. 见：金伯泉主编. 细胞和分子免疫学. 第1版. 西安：世界图
12. 书出版社，1995. 1-32
13. Casadevall A, Perfect JR. Cryptococcus neoformans. ASM Press. Washington, D.C. 1998
14. 姚志荣，廖万清，温海. 隐球菌性脑膜炎治疗方法的比较. 第二军医大学学报, 1998, 19(6): 575-576
15. Zhang T, Kawakami K, Qureshi MH, et al. Interleukin-12 (IL-12) and IL-18 synergistically induce the fungicidal activity of murine peritoneal exudate cells against Cryptococcus neoformans through production of gamma interferon by natural killer cells. Infect Immun, 1997, 65(9):3594-3599.
16. 廖万清，吴绍熙 主编. 真菌病学研究进展. 上海：第二军医大学出版社，1998. 62-68
17. 郭宁如，吴绍熙. 两性霉素B脂质体的研究与应用. 中国新药杂志，1996，5(4): 261
18. 李志刚, 廖万清, 温海，等. 高颅压下鞘内注射两性霉素B治疗隐球菌性脑膜炎. 第二军医大学学报, 1998, 19(6): 579-580.
19. Mylonakis E, Merriman NA, Rich JD, et al. Use of cerebrospinal fluid shunt for the management of elevated intracranial pressure in a patient with active AIDS-related cryptococcal meningitis. Diagn Microbiol Infect Dis, 1999, 34(2):111-4
20. 王文莉，王端礼，李世荫，等. NCCLS方案检测氟康唑对酵母菌的最小抑菌浓度. 中华皮肤科杂志，1996，29（5）：374-376
2. 翁心华. 隐球菌病. 见：秦启贤主编. 临床真菌学. 上海：复旦大学出版社，2001. 348-50
3. Hay RJ, Mackenzie DW, Campbell CK，et al. Cryptococcosis in the United Kingdom and the Irish Republic: an analysis of 69 cases. J Infect, 1980, 2(1):13-22
4. Rex JH, Cooper CR Jr, Merz WG, et al. Detection of amphotericin B-resistant Candida isolates in a broth-based system. Antimicrob Agents Chemother, 1995, 39(4):906-9
5. Ghannoum MA, Ibrahim AS, Fu Y, Shafiq MC, et al. Susceptibility testing of Cryptococcus neoformans: a microdilution technique. J Clin Microbiol, 1992, 30(11):2881-6.
6. National Committee for Clinical Laboratory Standards. 1998. Reference method for broth dilution antifungal susceptibility testing of yeasts. M27-A. National Committee for Clinical Laboratory Standards, Wayne, Pa.
7. Revankar SG, Kirkpatrick WR, McAtee RK, et al. Interpretation of trailing endpoints in antifungal susceptibility testing by the National Committee for Clinical Laboratory Standards method. J Clin Microbiol, 1998, 36(1):153-6
8. Wenisch C, Moore CB, Krause R, et al. Antifungal susceptibility testing of fluconazole by flow cytometry correlates with clinical outcome. J Clin Microbiol, 2001, 39(7):2458-62
9. Ramani R, Ramani A, Wong SJ. Rapid flow cytometric susceptibility testing of Candida albicans. J Clin Microbiol, 1997, 35(9):2320-4.
10. Powderly WG, Keath EJ, Sokol-Anderson M, et al. Amphotericin B-resistant Cryptococcus neoformans in a patient with AIDS. Infect Dis Clin Pract, 1992, 1:314-316
11. Naka W, Hanyaku H, Tajima S, et al. Application of neutral red staining for evaluation of the viability of dermatophytes and Candida in human skin scales. J Med Vet Mycol, 1994, 32(1):31-5.
金伯泉. 白细胞分化抗原. 见：金伯泉主编. 细胞和分子免疫学. 第1版. 西安：世界图
12. 书出版社，1995. 1-32
13. Casadevall A, Perfect JR. Cryptococcus neoformans. ASM Press. Washington, D.C. 1998
14. 姚志荣，廖万清，温海. 隐球菌性脑膜炎治疗方法的比较. 第二军医大学学报, 1998, 19(6): 575-576
15. Zhang T, Kawakami K, Qureshi MH, et al. Interleukin-12 (IL-12) and IL-18 synergistically induce the fungicidal activity of murine peritoneal exudate cells against Cryptococcus neoformans through production of gamma interferon by natural killer cells. Infect Immun, 1997, 65(9):3594-3599.
16. 廖万清，吴绍熙 主编. 真菌病学研究进展. 上海：第二军医大学出版社，1998. 62-68
17. 郭宁如，吴绍熙. 两性霉素B脂质体的研究与应用. 中国新药杂志，1996，5(4): 261
18. 李志刚, 廖万清, 温海，等. 高颅压下鞘内注射两性霉素B治疗隐球菌性脑膜炎. 第二军医大学学报, 1998, 19(6): 579-580.
19. Mylonakis E, Merriman NA, Rich JD, et al. Use of cerebrospinal fluid shunt for the management of elevated intracranial pressure in a patient with active AIDS-related cryptococcal meningitis. Diagn Microbiol Infect Dis, 1999, 34(2):111-4
20. 王文莉，王端礼，李世荫，等. NCCLS方案检测氟康唑对酵母菌的最小抑菌浓度. 中华皮肤科杂志，1996，29（5）：374-376