Identification of cultured isolates of clinically important yeast species using fluorescent fragment length analysis of the amplified internally transcribed rRNA spacer 2 region

Baère, Thierry De; Claeys, Geert; Swinne, D.; Massonet, Caroline; Verschraegen, Gerda; Muylaert, An; Vaneechoutte, Mario

doi:10.1186/1471-2180-2-21

Cited by 61 publications

(9 citation statements)

References 15 publications

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“…Internal transcribed spacer (ITS) sequencing is considered as a rapid and accurate tool for identification of fungal pathogens [26]. Genetic variation within ITS region is considered to be sufficient for identification and typing of different fungal strains [27][28][29]. Azoles and allylamines (terbinafine) act as antifungal agents by inhibition of ergosterol biosynthesis while polyenes (nystatin) disrupt cell functions by binding to ergosterol in plasma membrane [30,31].…”

Section: Discussionmentioning

confidence: 99%

In vitro antifungal resistance profile of Candida strains isolated from Saudi women suffering from vulvovaginitis

et al. 2020

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Background: Vulvovaginal candidiasis (VVC) represents a universal health hazard that contributes to significant morbidity in women. Resistance of Candida to antifungal therapy has been reported as a public health problem. So, the objective of our current study is to detect resistance profile of different candidal strains. Methods: In this study, isolated Candida strains were identified by conventional methods, confirmed by internal transcribed spacer (ITS) sequencing, and phylogenetically analyzed with reference strains in GenBank. Also, sensitivity of different Candida strains to common antifungal agents was evaluated by disc diffusion method. Results: Candida albicans was identified as the most frequent strain (63%) followed by non-albicans strains, such as C. glabrata (20%), C. tropicalis (13%), and C. krusei (4%). Sensitivity of Candida strains (C. albicans, C. tropicalis and C. glabrata) to commonly used antifungal agents was evaluated through the disc diffusion method. C. glabrata was the most resistant strain and considered to be a multidrug-resistant pathogen, while both, C. albicans and C. tropicalis showed high susceptibility to terbinafine. In contrast, C. albicans showed resistance to fluconazole, clotrimazole, and nystatin, while C. tropicalis, considered as the most sensitive strain, was susceptible to all the antifungal agents tested except nystatin. Terbinafine was the most effective antifungal agent against both C. tropicalis and C. albicans, and hence its minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) for C. albicans and C. tropicalis were evaluated. MICs of terbinafine against C. albicans and C. tropicalis were 5 μg/ml and 2.5 μg/ml, while their MFCs were 10 μg/ml and 5 μg/ml, respectively. Conclusion: The emergence of resistant Candida strains necessitates conduction of the antifungal susceptibility test prior to deciding the medication regime.

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Section: Discussionmentioning

confidence: 99%

In vitro antifungal resistance profile of Candida strains isolated from Saudi women suffering from vulvovaginitis

et al. 2020

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“…The supernatant was discarded and the pellet was re‐suspended to create a measured volume of 500 μ L. Due to the high salt content of the catholyte solution, a freeze‐thaw process was applied to the samples instead of using a commercial DNA extraction kit. Freeze‐thaw protocols have been suggested as a method to extract and purify DNA quickly and efficiently, without the need for chemicals . The samples were stored in a –80 °C freezer for three minutes, adjusted to room temperature, and incubated in a 95 °C heated block for two minutes.…”

Section: Methodsmentioning

confidence: 99%

Low Temperature Domestic Wastewater Treatment in a Microbial Electrolysis Cell with 1 m² Anodes: Towards System Scale‐Up

et al. 2017

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The potential benefits of applying microbial electrolysis cell (MEC) technology to wastewater treatment are clear and profound. Previous pilot studies have demonstrated a 'proof of concept' with domestic waste at ambient temperatures, but have not yet treated waste to required discharge standards, and have not reached energy neutrality. In addition, these reactors have been many orders of magnitude smaller than would be needed for full scale wastewater treatment plants. Scale-up affects many of the parameters that underpin performance; understanding its impact will be vital to further progress. Modifying a previously tested cassette-style design, we reduced the internal resistance, and increased the module size by a factor of 16, constructing an MEC with six 1 m 2 anodes. This created an anodic surface area to volume ratio of 34 m 2 m -3 . The system was operated at a hydraulic retention time of 5 hours on settled domestic wastewater for 217 days, producing more current than a scaled-down reactor, which was run in parallel. The large MEC produced 0.8 L of 93% pure H 2 d -1 at ambient winter temperatures (11.4 + 2.5°C). Chemical oxygen demand (COD) removal averaged 63.5% with an average effluent quality of 124.7 mg COD L -1 , achieving the European Urban Wastewater Treatment Directive (1991) consent.

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“…Capillary electrophoresis has been previously described in some studies (31,32,33,36) as a promising method for clinically important fungi detection, identification, and differentiation of closely related species. Turenne et al (31) first reported the application of this approach for a large number of commercially available strains and approximately 26 clinical strains, including 12 different Candida species.…”

Section: Discussionmentioning

confidence: 99%

“…Another approach is based on PCR with panfungal-or genusspecific primers targeting conserved rRNA regions, followed mostly by sequencing (23) or also by other techniques like restriction analysis (restriction fragment length polymorphism [RFLP]) (24), high-resolution melting (HRM) analysis (25,26,27), microarray-based detection (28, 29), pyrosequencing (30), or determination of amplicon size by using capillary electrophoresis (31,32,33). Capillary electrophoresis has been proven to be superior to classical electrophoresis for separating DNA due to its higher efficiency, higher speed, better sensitivity, and better suitability for automation (34).…”

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confidence: 99%

Evaluation of Fluorescent Capillary Electrophoresis for Rapid Identification of Candida Fungal Infections

et al. 2016

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dEarly diagnosis of fungal infection is critical for initiating antifungal therapy and reducing the high mortality rate in immunocompromised patients. In this study, we focused on rapid and sensitive identification of clinically important Candida species, utilizing the variability in the length of the ITS2 rRNA gene and fluorescent capillary electrophoresis (f-ITS2-PCR-CE). The method was developed and optimized on 29 various Candida reference strains from which 26 Candida species were clearly identified, while Candida guilliermondii, C. fermentati, and C. carpophila, which are closely related, could not be distinguished. The method was subsequently validated on 143 blinded monofungal clinical isolates (comprising 26 species) and was able to identify 88% of species unambiguously. This indicated a higher resolution power than the classical phenotypic approach which correctly identified 73%. Finally, the culture-independent potential of this technique was addressed by the analysis of 55 retrospective DNA samples extracted directly from clinical material. The method showed 100% sensitivity and specificity compared to those of the combined results of cultivation and panfungal PCR followed by sequencing used as a gold standard. In conclusion, this newly developed f-ITS2-PCR-CE analytical approach was shown to be a fast, sensitive, and highly reproducible tool for both culture-dependent and culture-independent identification of clinically important Candida strains, including species of the "psilosis" complex. During the last several decades, the impact and frequency of fungal infections have gained importance mainly due to an increasing number of immunocompromised patients (1, 2). Fungemia cases are being caused mainly by Candida species, which are the fourth most common microorganisms isolated from the blood samples (3, 4, 5). Sepsis due to Candida spp. is a very serious condition and has a higher mortality rate that for bacterial pathogens; reaching 54 to 64% in Candida-associated septic shock (6, 7). Moreover, the current changes in the epidemiology of invasive mycoses has highlighted a shift in the Candida species involved with a reduced proportion of Candida albicans and an increase in non-albicans species, which can show different susceptibility to various antifungal therapies (8,9,10).Early initiation of antifungal therapy is a critical step in the treatment of fungal infections. Therefore, quick, successful detection and identification of the etiological agents is crucial for early targeted therapy and favorable clinical patient outcome (11). Correct species identification is mostly based on phenotypic features and is usually time-consuming because a typical diagnostic workflow takes up to several days. Moreover, the phenotypic methods may lead to misidentification, particularly in the case of closely related species (12, 13). A significant improvement occurred when matrix-assisted laser desorption ionizationϪtime of flight mass spectroscopy (MALDI-TOF MS) was introduced as a routine laboratory procedure, enab...

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Identification of cultured isolates of clinically important yeast species using fluorescent fragment length analysis of the amplified internally transcribed rRNA spacer 2 region

Cited by 61 publications

References 15 publications

In vitro antifungal resistance profile of Candida strains isolated from Saudi women suffering from vulvovaginitis

In vitro antifungal resistance profile of Candida strains isolated from Saudi women suffering from vulvovaginitis

Low Temperature Domestic Wastewater Treatment in a Microbial Electrolysis Cell with 1 m² Anodes: Towards System Scale‐Up

Evaluation of Fluorescent Capillary Electrophoresis for Rapid Identification of Candida Fungal Infections

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Identification of cultured isolates of clinically important yeast species using fluorescent fragment length analysis of the amplified internally transcribed rRNA spacer 2 region

Cited by 61 publications

References 15 publications

In vitro antifungal resistance profile of Candida strains isolated from Saudi women suffering from vulvovaginitis

In vitro antifungal resistance profile of Candida strains isolated from Saudi women suffering from vulvovaginitis

Low Temperature Domestic Wastewater Treatment in a Microbial Electrolysis Cell with 1 m2 Anodes: Towards System Scale‐Up

Evaluation of Fluorescent Capillary Electrophoresis for Rapid Identification of Candida Fungal Infections

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Low Temperature Domestic Wastewater Treatment in a Microbial Electrolysis Cell with 1 m² Anodes: Towards System Scale‐Up