对临床分离的90株白假丝酵母菌基因分型和药敏试验研究
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摘要
白假丝酵母菌(Candida albicans),是人体正常菌群之一,在口腔、皮肤、肠道、阴道、肛门等部位均可分离到,是引起人体真菌感染的主要条件致病菌。随着医学科学的发展,各种新药、新技术如广谱抗生素、激素、各种导管插管技术等的广泛使用,导致深部真菌病的发病率愈来愈高,且以C.albicans感染为主。因此,对C.albicans感染的诊断和分型在临床和流行病学研究中都具有重要意义。对C.albicans进行基因分型的目的在于:(1)可追踪传染源,明确传播途径,防止暴发流行;(2)确定同一患者反复感染是源于复发或是再感染;(3)明确某一菌株是否与某种临床表现或疾病相关,从而发现其独特的致病机制;(4)了解感染的发生除与宿主全身或局部易感因素有关外,是否与菌本身的遗传因素有关;(5)了解C.albicans耐药株和敏感株之间基因型的差异、表型和基因型之间的关系等。
本研究采用灵敏特异的PCR分型方法,扩增C.albicans的25S rDNA基因的内含子区可转座Ⅰ型内含子插入片段,根据扩增产物的大小和片段数目来分型。A型为野生型C.albicans,无内含子;B型和C型为有379bp转座子的菌株;E型为具有379bp、252bp、341bp三种转座子的菌株,D型为都柏林假丝酵母菌(C.dubliniensis)。
药敏实验采用ATB FUNGUS 3酵母菌药物敏感性检测板条,对两性霉素B(Amphotericin B,AMB)、氟康唑(Fluconazol,FCA)、伊曲康唑(Itraconazole,ITR)、5-氟胞嘧啶(5-flurocytosine,5-FC)和伏立康唑(Voriconazole,VRC)5种抗真菌药物进行药敏分析。
本研究收集了临床79名患者的痰、咽拭子、大便、血液、尿液、伤口分泌物、脑脊液、静脉插管、穿刺液等标本,从中共分离出C.albicans 90株,对其进行了PCR基因分型鉴定,结果共检出A、B、C三种基因型,未见D、E型,其中A型44株(44/90,48.9%),B型33株(33/90,36.7%),C型13株(13/90,14.4%)。药敏实验结果表明C.albicans的基因型与其抗真菌药物的敏感性之间有一定相关性。各基因型C.albicans均未出现AMB耐药株,两性霉素B仍是治疗危重患者酵母菌感染的首选药;A型对5-FC的敏感率为95.5%,有4.5%耐药;A、B、C三型对FCA的敏感率分别为93.2%、90.9%和76.9%,其中C型C.albicans对FCA的耐药率有高于A型和B型的趋势;A、B、C三型对ITR的敏感率分别为90.8%、81.8%和76.9%,B型和C型C.albicans对ITR的耐药率有高于A型的趋势;A、B、C三型对VRC的敏感率分别为97.7%、96.9%和92.3%,C型C.albicans对VRC的耐药率也有高于A型和B型的趋势。
C.albicans PCR分型方法简便、快速、重复性好、特异性强;不同型别的C.albicans耐药谱有差异。弄清C.albicans的基因型与耐药性之间的关系,对Calbicans感染的诊断、耐药性变异、监控医院内真菌感染情况、真菌流行病学研究、指导临床医师合理用药等,具有重要意义。
Candida albicans is one of the normal floras in human bodies. It can be isolated from the samples in oral cavity, skin, intestine, vagina, anus et al, at the same time it is also the most important opportunity pathogen causing Candida infections. With the development of medical science, application of various new drugs, broad spectrum antibiotics, hormones and all kinds of vessels lead to high incidence of deep fungal infections which C.albicans are the leading cause. Therefore, it is of important significance to the diagnosis and genotyping of C. albicans infections in clinical and epidemiological research, the purpose of genotyping includes: (1) to trace the source of C. albicans infection, make clear the route of transmission, avoid prevalence of infection; (2) to determine the reason of repeated infections happened in one patient is either recurrence or re-infection; (3) to clear the relationship between certain strain of C. albicans and some clinical symptoms or diseases and find out the distinct pathogenesis; (4) to understand relationship between the occurrence of C. albicans infections and the genetic factors, as well as host susceptibility; (5) to study the differences between resistant and sensitive strains of C. albicans, particularly the relationships between phenotype and genotypes .
In this study, the sensitive and specific polymerase chain reaction (PCR) was applied to amplify the target DNA fragment spanning the region of the transposable groupⅠintron of 25S rDNA of Candida albicans. The size of insertion segments of the intron in the 25S rDNA were detected by agarose gel electrophoresis. Genotype A which is wild type of C. albicans has no intron; genotype B and C contain 379bp of insertion segment, genotype E contains 379bp, 252bp and 341bp, genotype D belongs to C. Dubliniensis. The drug susceptibility tests were performed by ATB Fungus 3 tip to analyze the amphotericin B (AMB), fluconazole (FCA), itraconazole (ITR), 5-fiucytosine (5-FC), and voriconazole (VRC).
Three genotypes in 90 strains of C. albicans isolated from clinical specimens have been identified. They are genotype A (44/90, 48.9%), genotype B (33/90, 36.7%) and genotype C (13/90,14.4%). None of 90 isolates presented the drug resistance to AMB. Both genotype B and C are susceptible to 5-FC. 95.5% of isolates genotype A were susceptible to 5-FC while 4.5% is resistant; 93.2% of genotype A, 90.9% of genotype B and 76.9% of genotype C are susceptible to FCA, respectively; 90.8% of genotype A is susceptible to ITR, 81.8% for genotype B and 76.9% for genotype C; 97.7% of genotype A is susceptible to VRC, 96.9% for genotype B and 92.3% for genotype C.
C. albicans genotyping by PCR is convenient and rapid method with higher specificity and repeatability. Identifying the distinct drug-resistant spectrum in various genotypes of C. albicans facilitates the analysis of the hospital infections and research on fungal epidemiology and also promotes understanding the relationship between C. albicans infections and their drug resistance. It is helpful for clinical physicians to prescribe anti-fungal agents rationally and effectively monitor and control hospital infections of C. albicans.
本研究采用灵敏特异的PCR分型方法,扩增C.albicans的25S rDNA基因的内含子区可转座Ⅰ型内含子插入片段,根据扩增产物的大小和片段数目来分型。A型为野生型C.albicans,无内含子;B型和C型为有379bp转座子的菌株;E型为具有379bp、252bp、341bp三种转座子的菌株,D型为都柏林假丝酵母菌(C.dubliniensis)。
药敏实验采用ATB FUNGUS 3酵母菌药物敏感性检测板条,对两性霉素B(Amphotericin B,AMB)、氟康唑(Fluconazol,FCA)、伊曲康唑(Itraconazole,ITR)、5-氟胞嘧啶(5-flurocytosine,5-FC)和伏立康唑(Voriconazole,VRC)5种抗真菌药物进行药敏分析。
本研究收集了临床79名患者的痰、咽拭子、大便、血液、尿液、伤口分泌物、脑脊液、静脉插管、穿刺液等标本,从中共分离出C.albicans 90株,对其进行了PCR基因分型鉴定,结果共检出A、B、C三种基因型,未见D、E型,其中A型44株(44/90,48.9%),B型33株(33/90,36.7%),C型13株(13/90,14.4%)。药敏实验结果表明C.albicans的基因型与其抗真菌药物的敏感性之间有一定相关性。各基因型C.albicans均未出现AMB耐药株,两性霉素B仍是治疗危重患者酵母菌感染的首选药;A型对5-FC的敏感率为95.5%,有4.5%耐药;A、B、C三型对FCA的敏感率分别为93.2%、90.9%和76.9%,其中C型C.albicans对FCA的耐药率有高于A型和B型的趋势;A、B、C三型对ITR的敏感率分别为90.8%、81.8%和76.9%,B型和C型C.albicans对ITR的耐药率有高于A型的趋势;A、B、C三型对VRC的敏感率分别为97.7%、96.9%和92.3%,C型C.albicans对VRC的耐药率也有高于A型和B型的趋势。
C.albicans PCR分型方法简便、快速、重复性好、特异性强;不同型别的C.albicans耐药谱有差异。弄清C.albicans的基因型与耐药性之间的关系,对Calbicans感染的诊断、耐药性变异、监控医院内真菌感染情况、真菌流行病学研究、指导临床医师合理用药等,具有重要意义。
Candida albicans is one of the normal floras in human bodies. It can be isolated from the samples in oral cavity, skin, intestine, vagina, anus et al, at the same time it is also the most important opportunity pathogen causing Candida infections. With the development of medical science, application of various new drugs, broad spectrum antibiotics, hormones and all kinds of vessels lead to high incidence of deep fungal infections which C.albicans are the leading cause. Therefore, it is of important significance to the diagnosis and genotyping of C. albicans infections in clinical and epidemiological research, the purpose of genotyping includes: (1) to trace the source of C. albicans infection, make clear the route of transmission, avoid prevalence of infection; (2) to determine the reason of repeated infections happened in one patient is either recurrence or re-infection; (3) to clear the relationship between certain strain of C. albicans and some clinical symptoms or diseases and find out the distinct pathogenesis; (4) to understand relationship between the occurrence of C. albicans infections and the genetic factors, as well as host susceptibility; (5) to study the differences between resistant and sensitive strains of C. albicans, particularly the relationships between phenotype and genotypes .
In this study, the sensitive and specific polymerase chain reaction (PCR) was applied to amplify the target DNA fragment spanning the region of the transposable groupⅠintron of 25S rDNA of Candida albicans. The size of insertion segments of the intron in the 25S rDNA were detected by agarose gel electrophoresis. Genotype A which is wild type of C. albicans has no intron; genotype B and C contain 379bp of insertion segment, genotype E contains 379bp, 252bp and 341bp, genotype D belongs to C. Dubliniensis. The drug susceptibility tests were performed by ATB Fungus 3 tip to analyze the amphotericin B (AMB), fluconazole (FCA), itraconazole (ITR), 5-fiucytosine (5-FC), and voriconazole (VRC).
Three genotypes in 90 strains of C. albicans isolated from clinical specimens have been identified. They are genotype A (44/90, 48.9%), genotype B (33/90, 36.7%) and genotype C (13/90,14.4%). None of 90 isolates presented the drug resistance to AMB. Both genotype B and C are susceptible to 5-FC. 95.5% of isolates genotype A were susceptible to 5-FC while 4.5% is resistant; 93.2% of genotype A, 90.9% of genotype B and 76.9% of genotype C are susceptible to FCA, respectively; 90.8% of genotype A is susceptible to ITR, 81.8% for genotype B and 76.9% for genotype C; 97.7% of genotype A is susceptible to VRC, 96.9% for genotype B and 92.3% for genotype C.
C. albicans genotyping by PCR is convenient and rapid method with higher specificity and repeatability. Identifying the distinct drug-resistant spectrum in various genotypes of C. albicans facilitates the analysis of the hospital infections and research on fungal epidemiology and also promotes understanding the relationship between C. albicans infections and their drug resistance. It is helpful for clinical physicians to prescribe anti-fungal agents rationally and effectively monitor and control hospital infections of C. albicans.
引文
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[1]Reed BD,Zazove P,Pierson CL,et al.Candida transmission and sexual behaviors as risks for a repeat episode of candida vulvovaginitis.J Worn Health 2003;12:979-989.
[2]Jackson ST,Mullings AM,Rainford L,et al.The epidemiology of mycotic vulvovaginitis and the use of antifungal agents in suspected mycotic vulvovaginitis and its implications for clinical practice.West Indian Med J,2005;54:192-195.
[3]Wilton L,Kollarova M,Heeley E,et al.Relative risk of vaginal candidiasis after use of antibiotics compared with antidepressants in women:postmarketing surveillance data in England.Drug Saf,2003;26:589-597.
[4]Syed TS,Braverman PK.Vaginitis in adolescents.Adolesc Med Clin,2004;15:235-251.
[5]Richter SS,Galask RP,Messer SA,et al.Antifungal susceptibilities of Candida species causing vulvovaginitis and epidemiology of recurrent cases.J Clin Microbiol,2005;43:2155-2162.
[6]Ferrer J.Vaginal candidosis:epidemiological and etiological factors.Int J Gynecol Obstet,2000;71:21-27.
[7]陈文彬。深部真菌感染病原学诊断概述。中国实用内科杂志,2002.22(1):5-6.
[8]Rees JR,Pinner RW,Hajjeh RA,et al.The epidemiological features of invasive mycotic infections in the San Francisco Bay area,1992-1993:results of population-based laboratory active surveillance.Clin Infect Dis,1998,27:1138-1147.
[9]De Waele JJ,Vogelaers D,Blot S,et al.Fungal infections in patients with severe acute pancreatitis and the use of prophylactic therapy[J].Clin Infect Dis,2003,37(2):208-213.
[10]陈佰义.深部真菌感染的诊断与治疗[J].辽宁医学杂志,2004,18(3):114-115.
[11]Kullberg BJ.Oude Lashof AM.Epidemiology of opportunistic invasive mycoses [J]. Eur J Med Res, 2002,7(5): 183-191.
[12] Ostrosky-Zeichner L, Rex JH, Bennett J, et al. Deeply invasive candidiasis [J]. Infect Dis Clin North Am, 2002,16(4):821-835.
[13] Odabasi Z, Mattiuzzi G, Estey E, et al. Beta-D-glucan as a diagnostic adjunct for invasive fungal infections:Validation, cutoff development,and performance in patients this acute myelogenous leukemia anmyelodysplastic syndrome [J]. Clin Infect Dis, 2004,39(2): 199-205.
[14] Zaas, Aimee K, Alexdander, Barbara D. Galactomannan and advances in fungal diagnostics [J]. Current Opinion In Organ Transplantation, 2005,10(4):307-311.
[15] Jang-jih Lu, Shih-Yi Lee, Tzong-Shi Chiueh. In virto antifungal susceptibility testing of Candida blood isolates and evaluation of the E-test method [J]. J Microbiol Immunol Infect, 2004,37(6):335-342.
[16] Weissman Z, Berdicevsky I, Cavan B, et al. Molecular Identificallon of Candida albicans [J]. J Med Vet Mycol, 1995,33(3):205-207.
[17] Buchman TG, Rossier M, Merz WG, et al. Detection of surgical pathogens by in vitro DNA amplification. part 1. Rapid identification of Candida albicans by in vitro amplification of a fungus-specific gene [J]. Surgery, 1990,108(2):338.
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[1]Reed BD,Zazove P,Pierson CL,et al.Candida transmission and sexual behaviors as risks for a repeat episode of candida vulvovaginitis.J Worn Health 2003;12:979-989.
[2]Jackson ST,Mullings AM,Rainford L,et al.The epidemiology of mycotic vulvovaginitis and the use of antifungal agents in suspected mycotic vulvovaginitis and its implications for clinical practice.West Indian Med J,2005;54:192-195.
[3]Wilton L,Kollarova M,Heeley E,et al.Relative risk of vaginal candidiasis after use of antibiotics compared with antidepressants in women:postmarketing surveillance data in England.Drug Saf,2003;26:589-597.
[4]Syed TS,Braverman PK.Vaginitis in adolescents.Adolesc Med Clin,2004;15:235-251.
[5]Richter SS,Galask RP,Messer SA,et al.Antifungal susceptibilities of Candida species causing vulvovaginitis and epidemiology of recurrent cases.J Clin Microbiol,2005;43:2155-2162.
[6]Ferrer J.Vaginal candidosis:epidemiological and etiological factors.Int J Gynecol Obstet,2000;71:21-27.
[7]陈文彬。深部真菌感染病原学诊断概述。中国实用内科杂志,2002.22(1):5-6.
[8]Rees JR,Pinner RW,Hajjeh RA,et al.The epidemiological features of invasive mycotic infections in the San Francisco Bay area,1992-1993:results of population-based laboratory active surveillance.Clin Infect Dis,1998,27:1138-1147.
[9]De Waele JJ,Vogelaers D,Blot S,et al.Fungal infections in patients with severe acute pancreatitis and the use of prophylactic therapy[J].Clin Infect Dis,2003,37(2):208-213.
[10]陈佰义.深部真菌感染的诊断与治疗[J].辽宁医学杂志,2004,18(3):114-115.
[11]Kullberg BJ.Oude Lashof AM.Epidemiology of opportunistic invasive mycoses [J]. Eur J Med Res, 2002,7(5): 183-191.
[12] Ostrosky-Zeichner L, Rex JH, Bennett J, et al. Deeply invasive candidiasis [J]. Infect Dis Clin North Am, 2002,16(4):821-835.
[13] Odabasi Z, Mattiuzzi G, Estey E, et al. Beta-D-glucan as a diagnostic adjunct for invasive fungal infections:Validation, cutoff development,and performance in patients this acute myelogenous leukemia anmyelodysplastic syndrome [J]. Clin Infect Dis, 2004,39(2): 199-205.
[14] Zaas, Aimee K, Alexdander, Barbara D. Galactomannan and advances in fungal diagnostics [J]. Current Opinion In Organ Transplantation, 2005,10(4):307-311.
[15] Jang-jih Lu, Shih-Yi Lee, Tzong-Shi Chiueh. In virto antifungal susceptibility testing of Candida blood isolates and evaluation of the E-test method [J]. J Microbiol Immunol Infect, 2004,37(6):335-342.
[16] Weissman Z, Berdicevsky I, Cavan B, et al. Molecular Identificallon of Candida albicans [J]. J Med Vet Mycol, 1995,33(3):205-207.
[17] Buchman TG, Rossier M, Merz WG, et al. Detection of surgical pathogens by in vitro DNA amplification. part 1. Rapid identification of Candida albicans by in vitro amplification of a fungus-specific gene [J]. Surgery, 1990,108(2):338.
[18] Miyakawa Y, Mabuchi T, Kagaya K, et al. Isolation and characterization of a species-specific DNA fragment for detection of Candida albicans by polymerase chain reaction. J Clin Microbiol, 1992,30(4):894-900.
[19] Tamura M, Watanabe K, Mikami Y, et al. Molecular characterization of new clinical isolates of Candida albicans and C.dubliniensis in Japan: analysis reveals a new genotype of C.albicans with group I intron [J]. J Clin Microbiol, 2001,39(12):4309-4315.
[20] Michael J, McCullough MJ, Karl V, et al. Molecular and phenotypic characterization of genotypic Candida albicans subgroups and comparison with Candid a dubliniensis and Candid a stellatoidea [J]. J Clin Microbiol, 1999,37(2):417-421.