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[通过两种不同方法对临床样本中分离出的丝状真菌进行鉴定及其药敏结果]

[Identification of filamentous fungi isolated from clinical samples by two different methods and their susceptibility results].

作者信息

Direkel Sahin, Otağ Feza, Aslan Gönül, Ulger Mahmut, Emekdaş Gürol

机构信息

Mersin University Faculty of Medicine, Department of Medical Microbiology, Mersin, Turkey.

出版信息

Mikrobiyol Bul. 2012 Jan;46(1):65-78.

PMID:22399173
Abstract

Molds are widely distributed in nature. Aspergillus spp. represent the most frequently observed causative agents, however less frequent pathogens Fusarium, Scedosporium and Zygomycetes have also been considered the most important causes of morbidity and mortality in profoundly immunosuppressed hosts. The aims of this study were to identify filamentous fungi isolated from clinical specimens by conventional and molecular methods, and to detect their antifungal susceptibilities. A total of 6742 clinical specimens obtained from hospitalized patients at critical units of Mersin University Medical Faculty Hospital and sent to our laboratory between April 2008-January 2010 were included in the study. The isolates were identified by classical mycological methods and polymerase chain reaction-based DNA sequencing. Susceptibilities to fluconazole and voriconazole were tested by disk diffusion method and to fluconazole, voriconazole, amfoterisin B, caspofungin and posaconazole by E-test. Filamentous fungi were isolated from 71 (1.05%) samples (13 sputum, 4 wound, 4 peritoneal fluid, 3 extrenal ear discharge, 3 abscess and one of each cerebrospinal fluid, blood, tissue biopsy, nasal swab and conjunctival swab) which belonged to 32 patients (13 female, 19 male; age range 7 months-77 years, mean age: 46.6 years). Of the patients 62.3% presented one or more risk factors such as chronic renal failure (n= 8), chronic obstructive lung disease (n= 6), malignancy (n= 6), diabetes mellitus (n= 5) and peripheral vascular disease (n= 5). Of the isolates six were identified as Aspergillus niger, six as Aspergillus flavus, five as Aspergillus fumigatus, four as Aspergillus terreus, five as Fusarium spp., two as Bipolaris spp., and one of each as Acremonium spp., Aurebasidium spp., Mucor spp., and Scedosporium spp. By conventional methods. Three isolates exhibited different identities by DNA sequencing. All Aspergillus isolates were correctly identified at species level by both methods, Other fungi were identified at genus level by conventional methods and at species level by DNA sequencing. Fluconazole minimum inhibitory concentration (MIC) values were determined as > 256 mg/L in all strains, except Scedosporium; voriconazole MIC values were < 0.38 mg/L in all Aspergillus spp. Caspofungin MIC values were > 32 mg/L for Fusarium, Scedosporium, Rhizopus and Bipolaris strains and ≤ 0.006-0.125 mg/L in all Aspergillus isolates, In three strains (Fusarium equiseti, Cylindrocarpon lichenicola and Rhizopus oryzae) posaconazole minimum inhibitory concentration (MIC) values were > 32 mg/L, however it was < 1.5 mg/L, for the other strains. Amphotericin B MIC values were > 32 mg/L for Fusarium, Scedosporium, Rhizopus and all A.terreus strains and < 2 mg/L for the others. E-test and disk diffusion test results were compatible with each other for all the antifungal agents tested. In conclusion, the identification of filamentous fungi such as Aspergillus and Fusarium spp. is easily and reliably achieved by conventional methods. Since the rate of invasive fungal infections is increasing currently, filamentous molds should be searched especially in the clinical specimens of immunocompromised patients for accurate and prompt diagnosis of such infections and to decrease the related mortality risk.

摘要

霉菌在自然界中广泛分布。曲霉菌属是最常见的致病病原体,然而,较少见的病原体镰刀菌属、头孢霉属和接合菌纲也被认为是导致深度免疫抑制宿主发病和死亡的最重要原因。本研究的目的是通过传统方法和分子方法鉴定从临床标本中分离出的丝状真菌,并检测它们的抗真菌药敏性。本研究纳入了2008年4月至2010年1月期间从梅尔辛大学医学院医院重症监护病房住院患者处获取并送至我们实验室的6742份临床标本。通过经典真菌学方法和基于聚合酶链反应的DNA测序对分离株进行鉴定。采用纸片扩散法检测氟康唑和伏立康唑的药敏性,采用E试验检测氟康唑、伏立康唑、两性霉素B、卡泊芬净和泊沙康唑的药敏性。从71份(1.05%)样本中分离出丝状真菌(13份痰液、4份伤口样本、4份腹腔积液、3份外耳道分泌物、3份脓肿样本,以及各1份脑脊液、血液、组织活检、鼻拭子和结膜拭子样本),这些样本来自32例患者(13例女性,19例男性;年龄范围7个月至77岁,平均年龄:46.6岁)。62.3%的患者存在一种或多种危险因素,如慢性肾衰竭(n = 8)、慢性阻塞性肺疾病(n = 6)、恶性肿瘤(n = 6)、糖尿病(n = 5)和周围血管疾病(n = 5)。通过传统方法,分离出的菌株中有6株被鉴定为黑曲霉,6株为黄曲霉,5株为烟曲霉,4株为土曲霉,5株为镰刀菌属,2株为双极霉属,各1株为顶孢霉属、 Aurebasidium属、毛霉属和头孢霉属。通过DNA测序,有3株分离株显示出不同的鉴定结果。两种方法均能在种水平正确鉴定所有曲霉菌株,其他真菌通过传统方法鉴定到属水平,通过DNA测序鉴定到种水平。除头孢霉属外,所有菌株的氟康唑最低抑菌浓度(MIC)值均测定为> 256 mg/L;所有曲霉菌属的伏立康唑MIC值均< 0.38 mg/L。镰刀菌属、头孢霉属、根霉属和双极霉属菌株的卡泊芬净MIC值> 32 mg/L,所有曲霉菌株的卡泊芬净MIC值≤ 0.006 - 0.125 mg/L。在3株菌株(木贼镰刀菌、地衣柱孢霉和米根霉)中,泊沙康唑最低抑菌浓度(MIC)值> 32 mg/L,而其他菌株的泊沙康唑MIC值< 1.5 mg/L。镰刀菌属、头孢霉属、根霉属和所有土曲霉菌株的两性霉素B MIC值> 32 mg/L,其他菌株的两性霉素B MIC值< 2 mg/L。对于所有检测的抗真菌药物,E试验和纸片扩散试验结果相互一致。总之,通过传统方法可以轻松、可靠地鉴定曲霉菌属和镰刀菌属等丝状真菌。由于目前侵袭性真菌感染的发生率正在上升,应特别在免疫受损患者临床标本中查找丝状霉菌,以便准确、及时诊断此类感染并降低相关死亡风险。

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