Molecular techniques in fungal detection and identification represent an efficient complementary diagnostic tool which is increasingly used to overcome limitations of routinely used culture techniques. The aim of this study was to characterize Candida sp. representation in samples from urine, urinary catheter, and ureteral stent biofilm using ITS2 ribosomal DNA (rDNA) amplification followed by fluorescent capillary electrophoresis (f-ITS2-PCR-CE) and to compare the results with those obtained by culture. A total of 419 samples were analyzed, and 106 (25.2%) were found positive, out of which 17 (16%) were polyfungal. The positivity rate did not differ between samples from catheters and stents (23.6% versus 20.9%) or between catheter and stent corresponding urine samples (40.2% versus 30.2%). Ten different Candida species were detected, with Candida parapsilosis (31.4%), Candida albicans (26.5%), and Candida tropicalis (12.4%) predominating. f-ITS2-PCR-CE was evaluated as substantially less time-consuming and 8.3 times more sensitive than the routinely applied culture technique with 1 µl of urine/sonicated fluid inoculated, detecting 67 (19.9%) versus 8 (2.4%) positive samples out of 337 initially analyzed samples. The culture sensitivity considerably improved to 1.7 times lower than that of f-ITS2-PCR-CE after the inoculation volume was increased to 100 µl in the additional 82 samples. Moreover, the molecular technique, unlike routine cultivation, enabled precise pathogen composition determination in polymicrobial samples. In conclusion, the f-ITS2-PCR-CE method was shown to be a quick and efficient tool for culture-independent detection and identification of fungi in urinary tract-related samples, demonstrating a higher sensitivity than culture.

• Klíčová slova
• Candida, biofilms, capillary electrophoresis, fungi, panfungal PCR, polyfungal sample, ureteral stents, urinary catheters,
• MeSH
• biofilmy růst a vývoj   MeSH
• Candida albicans izolace a purifikace   MeSH
• Candida parapsilosis izolace a purifikace   MeSH
• Candida tropicalis izolace a purifikace   MeSH
• Candida klasifikace   izolace a purifikace   MeSH
• diagnostické techniky molekulární přístrojové vybavení   metody   MeSH
• DNA fungální genetika   MeSH
• elektroforéza kapilární metody   MeSH
• fluorescence MeSH
• kandidóza mikrobiologie   moč   MeSH
• lidé MeSH
• močové katétry mikrobiologie   MeSH
• počet mikrobiálních kolonií normy   MeSH
• ribozomální DNA genetika   MeSH
• senioři MeSH
• senzitivita a specificita MeSH
• stenty mikrobiologie   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA fungální MeSH
• ribozomální DNA MeSH

Urinary or ureteral catheter insertion remains one of the most common urological procedures, yet is considered a predisposing factor for urinary tract infection. Diverse bacterial consortia adhere to foreign body surfaces and create various difficult to treat biofilm structures. We analyzed 347 urinary catheter- and stent-related samples, treated with sonication, using both routine culture and broad-range 16S rDNA PCR followed by Denaturing Gradient Gel Electrophoresis and Sanger sequencing (PCR-DGGE-S). In 29 selected samples, 16S rRNA amplicon Illumina sequencing was performed. The results of all methods were compared. In 338 positive samples, from which 86.1% were polybacterial, 1,295 representatives of 153 unique OTUs were detected. Gram-positive microbes were found in 46.5 and 59.1% of catheter- and stent-related samples, respectively. PCR-DGGE-S was shown as a feasible method with higher overall specificity (95 vs. 85%, p < 0.01) though lower sensitivity (50 vs. 69%, p < 0.01) in comparison to standard culture. Molecular methods considerably widened a spectrum of microbes detected in biofilms, including the very prevalent emerging opportunistic pathogen Actinotignum schaalii. Using massive parallel sequencing as a reference method in selected specimens, culture combined with PCR-DGGE was shown to be an efficient and reliable tool for determining the composition of urinary catheter-related biofilms. This might be applicable particularly to immunocompromised patients, in whom catheter-colonizing bacteria may lead to severe infectious complications. For the first time, broad-range molecular detection sensitivity and specificity were evaluated in this setting. This study extends the knowledge of biofilm consortia composition by analyzing large urinary catheter and stent sample sets using both molecular and culture techniques, including the widest dataset of catheter-related samples characterized by 16S rRNA amplicon Illumina sequencing.

• Klíčová slova
• PCR-DGGE, biofilm, double-J catheter, polymicrobial biofilm, stent, ureteral catheter, urinary catheter, urine culture,
• Publikační typ
• časopisecké články MeSH