Molecular Analysis of Fungal Populations in Patients with Oral Candidiasis Using Internal Transcribed Spacer Region

PLOS ONE, Dec 2019

Oral candidiasis is closely associated with changes in the oral fungal flora and is caused primarily by Candida albicans. Conventional methods of fungal culture are time-consuming and not always conclusive. However, molecular genetic analysis of internal transcribed spacer (ITS) regions of fungal rRNA is rapid, reproducible and simple to perform. In this study we examined the fungal flora in patients with oral candidiasis and investigated changes in the flora after antifungal treatment using length heterogeneity-polymerization chain reaction (LH-PCR) analysis of ITS regions. Fifty-two patients with pseudomembranous oral candidiasis (POC) and 30 healthy controls were included in the study. Fungal DNA from oral rinse was examined for fungal species diversity by LH-PCR. Fungal populations were quantified by real-time PCR and previously-unidentified signals were confirmed by nucleotide sequencing. Relationships between the oral fungal flora and treatment-resistant factors were also examined. POC patients showed significantly more fungal species and a greater density of fungi than control individuals. Sixteen fungi were newly identified. The fungal populations from both groups were composed predominantly of C. albicans, though the ratio of C. dubliniensis was significantly higher in POC patients than in controls. The diversity and density of fungi were significantly reduced after treatment. Furthermore, fungal diversity and the proportion of C. dubliniensis were positively correlated with treatment duration. These results suggest that C. dubliniensis and high fungal flora diversity might be involved in the pathogenesis of oral candidiasis. We therefore conclude that LH-PCR is a useful technique for diagnosing and assessing the severity of oral candidal infection.

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Molecular Analysis of Fungal Populations in Patients with Oral Candidiasis Using Internal Transcribed Spacer Region

et al. (2014) Molecular Analysis of Fungal Populations in Patients with Oral Candidiasis Using Internal Transcribed Spacer Region. PLoS ONE 9(6): e101156. doi:10.1371/journal.pone.0101156 Molecular Analysis of Fungal Populations in Patients with Oral Candidiasis Using Internal Transcribed Spacer Region Shinsuke Ieda 0 Masafumi Moriyama 0 Toru Takashita 0 Takashi Maehara 0 Yumi Imabayashi 0 Shoichi Shinozaki 0 Akihiko Tanaka 0 Jun-Nosuke Hayashida 0 Sachiko Furukawa 0 Miho Ohta 0 Yoshihisa Yamashita 0 Seiji Nakamura 0 Joy Sturtevant, Louisiana State University, United States of America 0 1 Section of Oral and Maxillofacial Oncology, Division of Maxillofacial Diagnostic and Surgical Sciences, Faculty of Dental Science, Kyushu University , Fukuoka , Japan , 2 Section of Preventive and Public Health Dentistry, Division of Oral Health, Growth and Development, Faculty of Dental Science, Kyushu University , Fukuoka , Japan Oral candidiasis is closely associated with changes in the oral fungal flora and is caused primarily by Candida albicans. Conventional methods of fungal culture are time-consuming and not always conclusive. However, molecular genetic analysis of internal transcribed spacer (ITS) regions of fungal rRNA is rapid, reproducible and simple to perform. In this study we examined the fungal flora in patients with oral candidiasis and investigated changes in the flora after antifungal treatment using length heterogeneity-polymerization chain reaction (LH-PCR) analysis of ITS regions. Fifty-two patients with pseudomembranous oral candidiasis (POC) and 30 healthy controls were included in the study. Fungal DNA from oral rinse was examined for fungal species diversity by LH-PCR. Fungal populations were quantified by real-time PCR and previouslyunidentified signals were confirmed by nucleotide sequencing. Relationships between the oral fungal flora and treatmentresistant factors were also examined. POC patients showed significantly more fungal species and a greater density of fungi than control individuals. Sixteen fungi were newly identified. The fungal populations from both groups were composed predominantly of C. albicans, though the ratio of C. dubliniensis was significantly higher in POC patients than in controls. The diversity and density of fungi were significantly reduced after treatment. Furthermore, fungal diversity and the proportion of C. dubliniensis were positively correlated with treatment duration. These results suggest that C. dubliniensis and high fungal flora diversity might be involved in the pathogenesis of oral candidiasis. We therefore conclude that LH-PCR is a useful technique for diagnosing and assessing the severity of oral candidal infection. - In recent days, interest in oral hygiene in elderly and immunocompromised individuals is increasing because of the associations between oral candidiasis and increased morbidity and mortality from aspiration pneumonia in these patients [1,2]. Although systemic candidiasis caused by Candida albicans is an opportunistic infection of the oral mucosa, the widespread use of empiric and prophylactic antifungal drugs during the last 20 years has caused a shift in fungal flora towards other yeast species such as C. glabrata, C. tropicalis, C. krusei, C. dubliniensis and non-Candida species [3]. More recently, invasive infections caused by yeasts with intrinsic or acquired resistance to antifungal drugs have become a major public health problem [4,5]. It has been reported that the delayed initiation of antifungal drugs for candidal infection leads to poor clinical outcomes and increased mortality [6,7] which has underlined the necessity for early identification and appropriate antifungal treatment. However, the conventional identification kits for oral fungi are based on cultivation methods which are often time consuming and not always conclusive [8]. In contrast, recent genetic approaches using length heterogeneitypolymerase chain reaction (LH-PCR) analysis of internal transcribed spacer (ITS) regions of fungal nuclear rRNA have enabled more exhaustive surveys of fungal communities including the difficult-to-cultivate species [9,10]. The ITS1-5.8S rRNA-ITS2 region is the most widely-sequenced DNA region in fungal species, and can be rapidly amplified using universal fungal primers. These amplicons provide phylogenetic sequence information, especially at lower taxonomic levels [11]. However, this technique has not yet been applied to serial clinical assessments including the effects of treatment on fungal flora. We therefore examined the diversity of fungal species in patients with pseudomembranous oral candidiasis (POC) by LHPCR, and identified the factors associated with resistance to antifungal therapy. Saxons test (g/2 min) Spitting method (ml/15 min) ,Lesion location of oral candidiasis. POC, pseudomembranous oral candidiasis; *p,0.05, **p,0.01 (Students t-test); SWS, stimulated whole salivary flow rate; UWS, unstimulated whole salivary (...truncated)


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Shinsuke Ieda, Masafumi Moriyama, Toru Takashita, Takashi Maehara, Yumi Imabayashi, Shoichi Shinozaki, Akihiko Tanaka, Jun-Nosuke Hayashida, Sachiko Furukawa, Miho Ohta, Yoshihisa Yamashita, Seiji Nakamura. Molecular Analysis of Fungal Populations in Patients with Oral Candidiasis Using Internal Transcribed Spacer Region, PLOS ONE, 2014, Volume 9, Issue 6, DOI: 10.1371/journal.pone.0101156