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[肽核酸荧光原位杂交(PNA FISH)法在鉴定血培养分离的念珠菌属中的应用评估]

[Evaluation of peptide nucleic acid fluorescent in situ hybridization (PNA FISH) method in the identifi cation of Candida species isolated from blood cultures].

作者信息

Aydemir Gonca, Koç Ayşe Nedret, Atalay Mustafa Altay

机构信息

Erciyes University Faculty of Medicine, Department of Medical Microbiology, Kayseri, Turkey.

出版信息

Mikrobiyol Bul. 2016 Apr;50(2):293-9. doi: 10.5578/mb.22092.

Abstract

In recent years, increased number of patients who are hospitalized in intensive care units, received immunosuppressive therapy and treated with broad-spectrum antibiotics that can lead an increase in the incidence of systemic candidiasis. In these patients, the most common clinical manifestation is candidemia. Since the identification of Candida species isolated from blood cultures is time consuming by conventional (morphological and biochemical) methods, rapid, reliable and accurate methods are needed. For this purpose novel systems have been developed to identify the agent directly. The aim of this study was to evaluate the peptide nucleic acid fluorescent in situ hybridization (PNA FISH) method for the identification of Candida species by comparing with the conventional methods. A total of 50 patients who were admitted to Erciyes University Medical Faculty Hospital clinics and followed with prediagnosis of systemic fungal infections whose blood cultures were positive for the yeasts between July 2011 and July 2012 were included in the study. The conventional identification of Candida isolates was performed by considering macroscopic and microscopic morphology, germ tube test, cycloheximide sensitivity, urease activity and carbohydrate assimilation patterns with API 20C AUX (bioMerieux, France) test. PNA FISH method was conducted by the use of a commercial kit namely Yeast Traffic Light(®) PNA FISH (AdvanDx, USA). According to morphological and biochemical characteristics (conventional methods), 19 (38%) out of 50 Candida isolates were identified as C.albicans, 12 (24%) as C.glabrata, five (10%) as C.parapsilosis, five (10%) as C.kefyr, four (8%) as C.krusei, two (4%) as C.guilliermondii, two (4%) as C.tropicalis and one (2%) as C.lusitaniae. On the other hand, 24 (48%) of the isolates were identified as C.albicans/C.parapsilosis (with green fluorescence), 16 (32%) as C.glabrata/C.krusei (with red fluorescence) and one (%2) as C.tropicalis (with yellow fluorescence) properly, however one C.tropicalis strain was misidentified as C.albicans by PNA FISH method. Other eight (16%) strains which were not presented in the evaluation panel of PNA FISH kit (5 C.kefyr, 2 C.guillermondii and 1 C.lusitaniae), gave no fluorescence and determined as other Candida spp. According to this, when the species that could be detected with the kit (C.albicans, C.parapsilosis, C.glabrata, C.krusei and C.tropicalis) were considered, the concordance rate with the conventional methods was determined as 97.6% (41/42) and the total evaluation rate for all the species was 84% (41/50). In conclusion, the most frequent isolated species from blood cultures in our hospital was C.albicans, followed by C.glabrata and C.parapsilosis. Since PNA FISH testing is a practical, reliable and rapid (resulted in 90 minutes) method for the identification of Candida strains at species level isolated from blood cultures, it was thought to be useful in routine laboratories. However, further comparative studies are required with large number of strains with the consideration of cost-effectiveness.

摘要

近年来,入住重症监护病房的患者数量不断增加,他们接受免疫抑制治疗并使用广谱抗生素,这可能导致系统性念珠菌病的发病率上升。在这些患者中,最常见的临床表现是念珠菌血症。由于通过传统(形态学和生化)方法从血培养物中鉴定念珠菌属物种耗时较长,因此需要快速、可靠且准确的方法。为此,已开发出新型系统以直接鉴定病原体。本研究的目的是通过与传统方法比较,评估肽核酸荧光原位杂交(PNA FISH)方法对念珠菌属物种的鉴定能力。本研究纳入了2011年7月至2012年7月期间入住埃尔西耶斯大学医学院附属医院门诊、经预诊断为系统性真菌感染且血培养酵母阳性的50例患者。念珠菌分离株的传统鉴定通过考虑宏观和微观形态、芽管试验、环己酰亚胺敏感性、脲酶活性以及使用API 20C AUX(法国生物梅里埃公司)试验的碳水化合物同化模式来进行。PNA FISH方法使用一种名为酵母交通信号灯(®)PNA FISH(美国AdvanDx公司)的商业试剂盒进行。根据形态学和生化特征(传统方法),50株念珠菌分离株中,19株(38%)被鉴定为白色念珠菌,12株(24%)为光滑念珠菌,5株(10%)为近平滑念珠菌,5株(10%)为克柔念珠菌,4株(8%)为克鲁斯念珠菌,2株(4%)为季也蒙念珠菌,2株(4%)为热带念珠菌,1株(2%)为葡萄牙念珠菌。另一方面,24株(48%)分离株被正确鉴定为白色念珠菌/近平滑念珠菌(绿色荧光),16株(32%)为光滑念珠菌/克鲁斯念珠菌(红色荧光),1株(2%)为热带念珠菌(黄色荧光),然而有1株热带念珠菌菌株被PNA FISH方法误鉴定为白色念珠菌。PNA FISH试剂盒评估面板中未出现的其他8株(16%)菌株(5株克柔念珠菌、2株季也蒙念珠菌和1株葡萄牙念珠菌)未产生荧光,被确定为其他念珠菌属物种。据此,当考虑试剂盒能够检测的物种(白色念珠菌、近平滑念珠菌、光滑念珠菌、克鲁斯念珠菌和热带念珠菌)时,与传统方法的一致性率为97.6%(41/42),所有物种的总评估率为84%(41/50)。总之,我院血培养中最常分离出的物种是白色念珠菌,其次是光滑念珠菌和近平滑念珠菌。由于PNA FISH检测是一种用于从血培养物中分离的念珠菌菌株进行种水平鉴定的实用、可靠且快速(90分钟出结果)的方法,故认为其在常规实验室中有用。然而,考虑到成本效益,还需要对大量菌株进行进一步的比较研究。

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