Kan Kültürlerinden İzole Edilen Candida'ların Tür Düzeyinde Tanımlanması ve Antifungal Duyarlılıklarının Belirlenmesi
Abstract
Candida'ların yol açtığı kan dolaşımı enfeksiyonları yüksek morbidite ve mortaliteyle seyretmektedir. Candida'ların tür düzeyinde doğru ve hızlı tanımlanarak antifungal duyarlılıklarının belirlenmesi, tedavinin doğru ve erken yapılmasına olanak sağlamaktadır. Çalışmamızda Van YYÜ Dursun Odabaş Tıp Merkezine başvuran hastalardan alınan kan kültür örneklerinden izole edilen Candida izolatlarının geleneksel yöntemler, otomatize sistemler ve PCR yöntemiyle tür düzeyinde tanımlanması ve standart yöntem olan broth mikrodilüsyon, gradient test ve disk difüzyon yöntemleriyle antifungal direnç profillerinin belirlenmesi amaçlanmıştır. Van YYÜ Dursun Odabaş Tıp Merkezi'ne başvuran hastalardan alınan kan kültür örneklerinden izole edilen 63 Candidaizolatı çalışmaya dahil edilmiştir. İzolatların identifikasyonu geleneksel yöntemlerden germ tüp ve CHROMagar Candida(RTA, Türkiye) ile; otomatize sistemlerden BD Phoenix M50 (Becton Dickonson, ABD)ve MALDI-TOF MS (Biomeriux, Fransa) ile ve moleküler yöntemlerden multipleks PCR ile yapılmıştır. Antifungal duyarlılık için Gradiyent Test ve Disk Difüzyon yöntemleri referans yöntem olarak önerilen Broth Mikrodilüsyon Yöntemi ile karşılaştırılmıştır. Toplam 32 suş, germ tüp testinde pozitiflik vermiş ve C. albicansolarak tanımlanmıştır. Sekiz izolatta yalancı germ tüp pozitifliği bulunmuş ve diğer izolatlar germ tüp negatif olarak tespit edilmiş ve non-albicans Candida olarak belirlenmiştir. CHROMagar Candida besiyerinde yaygın olarak görülen 59 suş tür düzeyinde belirlenirken dört suş tür düzeyinde tanımlanamamış, Candida spp. olarakrapor edilmiştir.Multipleks PCR ile C. albicans olarak saptanan bir suş CHROMagar Candida besiyeri ile C. tropicalis olarak adlandırılmıştır. Türe spesifik primerlerin kullanıldığı multipleks PCR yönteminde 32 suş C. albicans, 11 suş C. parapsilosis, sekiz suş C. tropicalis, yedi suş C. glabrata, üç suş C. kefyr, bir suş C. lusitaniae ve bir suş C. krusei olarak tanımlanmıştır. Phoenix M50 otomatize sistemi 63 suşun 61'ini multipleks PCR'la uyumlu bir şekilde tanımlamış; multipleks PCR yöntemiyle C. tropicalis olarak saptanan bir suş BD Phoenix M50 ile S.cereviseae ve C. albicans olarak tanımlanan bir suş da C. parapsilosis olarak tanımlanmıştır. MALDI-TOF MS otomatize sistemi 63 suşun 60'ını multipleks PCR'la uyumlu bir şekilde tanımlamış; multipleks PCR yöntemiyle C.albicans olarak tespit edilen üç ayrı suşuC.kefyr,C.tropicalis veC. lusitaniaeolarak tanımlamıştır. Antifungal duyarlılık testlerine göre; broth mikrodilüsyon yöntemine göre EUCAST'e göre sınır değerleri belli olan tüm izolatlar amfoterisin B'ye duyarlı bulunmuştur. Bir C.albicans, iki C.parapsilosis ve tüm C. glabrata izolatları flukonazole orta duyarlı; kalan izolatlar duyarlı bulunmuştur. Dirençli bir C. albicans suşu hariç tüm izolatlar vorikonazole duyarlı bulunmuştur. C. albicans izolatlarının 15'inde ve C. parapsilosis izolatlarının altısında mikafungin dirençli, tüm C. glabrata suşları duyarlı bulunmuştur. Disk difüzyon yönteminde Amfoterisin B için bulunan zon çapları 10-20 mm arasında değişmekteydi. Vorikonazol için disk difüzyon ve broth mikrodilüsyon yöntemleri arasındaki uyum C. parapsilosis için %90,9 iken C. albicans ve C. tropicalis için %100 olarak bulunmuştur. Flukonazol için iki yöntem arasındaki uyum C. parapsilosis için %81,8 iken C. albicans için %96,8, C. tropicalis için %100,C. glabrata için %42,8 olarak bulunmuştur. Gradient test amfoterisin B içinbroth mikrodilüsyon yöntemiyle %100 uyumlu bulunmuştur. Flukonazol için iki yöntem arasındaki uyum C. albicans için %56, C. parapsilosis için %36, C. tropicalis için %75 ve C. glabrata için %100 olarak bulunmuştur. Vorikonazol için iki yöntem arasındaki uyum C. albicans için %25, C. parapsilosis için %73, C. tropicalis için %62 olarak bulunmuştur.Gradient test mikafungin için, değerlendirilebilen tüm izolatlarda duyarlı bulunmuştur. Mikafungin için iki yöntem arasındaki uyum C. albicans için %50, C. parapsilosis için %45 ve C. glabrata için %100 olarak bulunmuştur. Geleneksel yöntemler ve otomatize sistemlerle Candida türleri yüksek oranda doğru tanımlanmış; multipleks PCR yöntemiyle karşılaştırıldığında,en yüksek uyumluluk oranı BD Phoenix otomatize sistemiyle elde edilmiştir. Bunu MALDI-TOF MS otomatize sistemi takip etmiştir. Antifungal direncini tespit etmede disk difüzyon yöntemi, broth mikrodilüsyona alternatif bir yöntem olarak belirlenmiştir. Gradient test yöntemi amfoterisin B için güvenli bulunmuştur.
Bloodstream infections caused by Candida have high morbidity and mortality. Accurate and rapid identification of Candida at the species level and determination of their antifungal susceptibility allows accurate and early treatment. In our study, we aimed to identify Candida isolates isolated from blood culture samples taken from patients admitted to Van YYÜ Dursun Odabaş Medical Center at the species level using traditional methods, automated systems and PCR methods, and to determine their antifungal resistance profiles using the standard methods of broth microdilution, gradient test and disk diffusion methods. 63 Candida isolates isolated from blood culture samples taken from patients admitted to Van YYÜ Dursun Odabaş Medical Center were included in the study. Identification of isolates was carried out using traditional methods such as germ tube and CHROMagar Candida (RTA, Turkey); It was performed with automated systems BD Phoenix M50 (Becton Dickonson, USA) and MALDI-TOF MS (Biomeriux, France) and with molecular methods multiplex PCR. For antifungal susceptibility, Gradient Test and Disk Diffusion methods were compared with the Broth Microdilution Method, which is recommended as a reference method. A total of 32 strains were positive in the germ tube test and were identified as C. albicans. False germ tube positivity was found in eight isolates and the other isolates were detected as germ tube negative and determined as non-albicans Candida. While 59 commonly seen strains in CHROMagar Candida medium were identified at the species level, four strains could not be identified at the species level, indicating Candida spp. It was reported as. A strain detected as C. albicans by multiplex PCR was named C. tropicalis with CHROMagar Candida medium. In the multiplex PCR method using species-specific primers, 32 strains were identified as C. albicans, 11 strains as C. parapsilosis, eight strains as C. tropicalis, seven strains as C. glabrata, three strains as C. kefyr, one strain as C. lusitaniae and one strain as C. krusei. BD Phoenix M50 automated system identified 61 of 63 strains compatible with multiplex PCR; A strain identified as C. tropicalis by the multiplex PCR method was identified as S. cereviseae by BD Phoenix M50, and a strain identified as C. albicans was identified as C. parapsilosis. The MALDI-TOF MS automated system identified 60 of 63 strains compatible with multiplex PCR; He identified three separate strains detected as C. albicans by multiplex PCR method as C.kefyr, C. tropicalis and C. lusitaniae. According to antifungal sensitivity tests; According to the broth microdilution method, all isolates with known breakpoints according to EUCAST were found to be sensitive to amphotericin B. One C. albicans, two C. parapsilosis and all C. glabrata isolates were moderately susceptible to fluconazole; the remaining isolates were found to be susceptible. All isolates except one resistant C. albicans strain were susceptible to voriconazole. 15 of the C. albicans isolates and six of the C. parapsilosis isolates were found to be resistant to micafungin, and all C. glabrata strains were sensitive. Zone diameters found for amphotericin B in the disc diffusion method varied between 10-20 mm. The agreement between disk diffusion and broth microdilution methods for voriconazole was found to be 90.9% for C. parapsilosis and 100% for C. albicans and C. tropicalis. For fluconazole, the agreement between the two methods was 81.8% for C. parapsilosis, 96.8% for C. albicans, 100% for C. tropicalis, and 42.8% for C. glabrata.The gradient test was found to be 100% compatible with the broth microdilution method for amphotericin B. For fluconazole, the agreement between the two methods was found to be 56% for C. albicans, 36% for C. parapsilosis, 75% for C. tropicalis and 100% for C. glabrata. For voriconazole, the agreement between the two methods was found to be 25% for C. albicans, 73% for C. parapsilosis, and 62% for C. tropicalis. The gradient test was sensitive to micafungin in all isolates that could be evaluated. For micafungin, the agreement between the two methods was found to be 50% for C. albicans, 45% for C. parapsilosis and 100% for C. glabrata. Candida species have been identified with a high rate of accuracy by traditional methods and automated systems; Compared to the multiplex PCR method, the highest compatibility rate was obtained with the BD Phoenix automated system. This was followed by the MALDI-TOF MS automated system. The disc diffusion method has been identified as an alternative method to broth microdilution in detecting antifungal resistance. Gradient testing method was found to be safe for amphotericin B.
Bloodstream infections caused by Candida have high morbidity and mortality. Accurate and rapid identification of Candida at the species level and determination of their antifungal susceptibility allows accurate and early treatment. In our study, we aimed to identify Candida isolates isolated from blood culture samples taken from patients admitted to Van YYÜ Dursun Odabaş Medical Center at the species level using traditional methods, automated systems and PCR methods, and to determine their antifungal resistance profiles using the standard methods of broth microdilution, gradient test and disk diffusion methods. 63 Candida isolates isolated from blood culture samples taken from patients admitted to Van YYÜ Dursun Odabaş Medical Center were included in the study. Identification of isolates was carried out using traditional methods such as germ tube and CHROMagar Candida (RTA, Turkey); It was performed with automated systems BD Phoenix M50 (Becton Dickonson, USA) and MALDI-TOF MS (Biomeriux, France) and with molecular methods multiplex PCR. For antifungal susceptibility, Gradient Test and Disk Diffusion methods were compared with the Broth Microdilution Method, which is recommended as a reference method. A total of 32 strains were positive in the germ tube test and were identified as C. albicans. False germ tube positivity was found in eight isolates and the other isolates were detected as germ tube negative and determined as non-albicans Candida. While 59 commonly seen strains in CHROMagar Candida medium were identified at the species level, four strains could not be identified at the species level, indicating Candida spp. It was reported as. A strain detected as C. albicans by multiplex PCR was named C. tropicalis with CHROMagar Candida medium. In the multiplex PCR method using species-specific primers, 32 strains were identified as C. albicans, 11 strains as C. parapsilosis, eight strains as C. tropicalis, seven strains as C. glabrata, three strains as C. kefyr, one strain as C. lusitaniae and one strain as C. krusei. BD Phoenix M50 automated system identified 61 of 63 strains compatible with multiplex PCR; A strain identified as C. tropicalis by the multiplex PCR method was identified as S. cereviseae by BD Phoenix M50, and a strain identified as C. albicans was identified as C. parapsilosis. The MALDI-TOF MS automated system identified 60 of 63 strains compatible with multiplex PCR; He identified three separate strains detected as C. albicans by multiplex PCR method as C.kefyr, C. tropicalis and C. lusitaniae. According to antifungal sensitivity tests; According to the broth microdilution method, all isolates with known breakpoints according to EUCAST were found to be sensitive to amphotericin B. One C. albicans, two C. parapsilosis and all C. glabrata isolates were moderately susceptible to fluconazole; the remaining isolates were found to be susceptible. All isolates except one resistant C. albicans strain were susceptible to voriconazole. 15 of the C. albicans isolates and six of the C. parapsilosis isolates were found to be resistant to micafungin, and all C. glabrata strains were sensitive. Zone diameters found for amphotericin B in the disc diffusion method varied between 10-20 mm. The agreement between disk diffusion and broth microdilution methods for voriconazole was found to be 90.9% for C. parapsilosis and 100% for C. albicans and C. tropicalis. For fluconazole, the agreement between the two methods was 81.8% for C. parapsilosis, 96.8% for C. albicans, 100% for C. tropicalis, and 42.8% for C. glabrata.The gradient test was found to be 100% compatible with the broth microdilution method for amphotericin B. For fluconazole, the agreement between the two methods was found to be 56% for C. albicans, 36% for C. parapsilosis, 75% for C. tropicalis and 100% for C. glabrata. For voriconazole, the agreement between the two methods was found to be 25% for C. albicans, 73% for C. parapsilosis, and 62% for C. tropicalis. The gradient test was sensitive to micafungin in all isolates that could be evaluated. For micafungin, the agreement between the two methods was found to be 50% for C. albicans, 45% for C. parapsilosis and 100% for C. glabrata. Candida species have been identified with a high rate of accuracy by traditional methods and automated systems; Compared to the multiplex PCR method, the highest compatibility rate was obtained with the BD Phoenix automated system. This was followed by the MALDI-TOF MS automated system. The disc diffusion method has been identified as an alternative method to broth microdilution in detecting antifungal resistance. Gradient testing method was found to be safe for amphotericin B.
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Mikrobiyoloji, Microbiology
Turkish CoHE Thesis Center URL
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