Molecular analysis of fungal populations in patients with oral candidiasis using next-generation sequencing.
ABSTRACT: Oral candidiasis is closely associated with changes in oral fungal biodiversity and is caused primarily by Candida albicans. However, the widespread use of empiric and prophylactic antifungal drugs has caused a shift in fungal biodiversity towards other Candida or yeast species. Recently, next-generation sequencing (NGS) has provided an improvement over conventional culture techniques, allowing rapid comprehensive analysis of oral fungal biodiversity. In this study, we used NGS to examine the oral fungal biodiversity of 27 patients with pseudomembranous oral candidiasis (POC) and 66 healthy controls. The total number of fungal species in patients with POC and healthy controls was 67 and 86, respectively. The copy number of total PCR products and the proportion of non-C. albicans, especially C. dubliniensis, in patients with POC, were higher than those in healthy controls. The detection patterns in patients with POC were similar to those in controls after antifungal treatment. Interestingly, the number of fungal species and the copy number of total PCR products in healthy controls increased with aging. These results suggest that high fungal biodiversity and aging might be involved in the pathogenesis of oral candidiasis. We therefore conclude that NGS is a useful technique for investigating oral candida infections.
Project description: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.
Project description:Unlike other forms of candidiasis, vulvovaginal candidiasis, caused primarily by the fungal pathogen Candida albicans, is a disease of immunocompetent and otherwise healthy women. Despite its prevalence, the fungal factors responsible for initiating symptomatic infection remain poorly understood. One of the hallmarks of vaginal candidiasis is the robust recruitment of neutrophils to the site of infection, which seemingly do not clear the fungus, but rather exacerbate disease symptomatology. Candidalysin, a newly discovered peptide toxin secreted by C. albicans hyphae during invasion, drives epithelial damage, immune activation, and phagocyte attraction. Therefore, we hypothesized that Candidalysin is crucial for vulvovaginal candidiasis immunopathology. Anti-Candida immune responses are anatomical-site specific, as effective gastrointestinal, oral, and vaginal immunities are uniquely compartmentalized. Thus, we aimed to identify the immunopathologic role of Candidalysin and downstream signaling events at the vaginal mucosa. Microarray analysis of C. albicans-infected human vaginal epithelium in vitro revealed signaling pathways involved in epithelial damage responses, barrier repair, and leukocyte activation. Moreover, treatment of A431 vaginal epithelial cells with Candidalysin induced dose-dependent proinflammatory cytokine responses (including interleukin 1? [IL-1?], IL-1?, and IL-8), damage, and activation of c-Fos and mitogen-activated protein kinase (MAPK) signaling, consistent with fungal challenge. Mice intravaginally challenged with C. albicans strains deficient in Candidalysin exhibited no differences in colonization compared to isogenic controls. However, significant decreases in neutrophil recruitment, damage, and proinflammatory cytokine expression were observed with these strains. Our findings demonstrate that Candidalysin is a key hypha-associated virulence determinant responsible for the immunopathogenesis of C. albicans vaginitis.
Project description:The fungus Candida albicans colonizes the oral mucosal surface of 30-70% of healthy individuals. Due to local or systemic immunosuppression, this commensal fungus is able to proliferate resulting in oral disease, called oropharyngeal candidiasis (OPC). However, in healthy individuals C. albicans causes no harm. Unlike humans mice do not host C. albicans in their mycobiome. Thus, oral fungal challenge generates an acute immune response in a naive host. Therefore, we utilized C. albicans clinical isolates which are able to persist in the oral cavity without causing disease to analyze adaptive responses to oral fungal commensalism. We performed RNA sequencing to determine the transcriptional host response landscape during C. albicans colonization. Pathway analysis revealed an upregulation of adaptive host responses due to C. albicans oral persistence, including the upregulation of the immune network for IgA production. Fungal colonization increased cross-specific IgA levels in the saliva and the tongue, and IgA+ cells migrated to foci of fungal colonization. Binding of IgA prevented fungal epithelial adhesion and invasion resulting in a dampened proinflammatory epithelial response. Besides CD19+ CD138- B cells, plasmablasts, and plasma cells were enriched in the tongue of mice colonized with C. albicans suggesting a potential role of B lymphocytes during oral fungal colonization. B cell deficiency increased the oral fungal load without causing severe OPC. Thus, in the oral cavity B lymphocytes contribute to control commensal C. albicans carriage by secreting IgA at foci of colonization thereby preventing fungal dysbiosis.
Project description:Oral keratinocytes provide the first line of host defense against oral candidiasis. We speculated that interactions of fungal cell wall components with oral keratinocytes regulate the stress response against Candida infection and examined the expression of genes induced by heat-killed Candida albicans in oral immortalized keratinocytes using a cDNA microarray technique. Of 24,000 genes revealed by that analysis, we focused on HO-1, a stress-inducible gene, as its expression was increased by both heat-killed and live C. albicans In histological findings, HO-1 expression in the superficial layers of the oral epithelium following Candida infection was elevated compared to that in healthy epithelium. We then investigated fungal cell wall components involved in induction of HO-1 expression and found that ?-glucan-containing particles (?-GPs) increased its expression. Furthermore, ?-glucan was observed on the surface of both heat-killed C. albicans and Candida cells that had invaded the oral epithelium. Fungal ?-GPs also promoted induction of intracellular reactive oxygen species (ROS), NADPH oxidase activation, and p38 mitogen-activated protein kinase (MAPK) phosphorylation, while those specific inhibitors inhibited the HO-1 expression induced by fungal ?-GPs. Moreover, fungal ?-GPs induced Nrf2 translocation into nuclei via p38 MAPK signaling, while the HO-1 expression induced by fungal ?-GPs was inhibited by Nrf2-specific small interfering RNA (siRNA). Finally, knockdown of cells by HO-1- and Nrf2-specific siRNAs resulted in increased ?-GP-mediated ROS production compared to that in the control cells. Our results show that the HO-1 induced by fungal ?-GPs via ROS/p38 MAPK/Nrf2 from oral keratinocytes may have important roles in host defense against the stress caused by Candida infection in the oral epithelium.
Project description:Oropharyngeal candidiasis, a common fungal infection in people living with HIV/AIDS (PLWHA), arises from Candida species colonizing the oral cavity. Fluconazole is the preferred treatment and is often used empirically. Few studies have investigated the prevalence of fluconazole resistance in Nigeria. This study aimed at determining the burden of fluconazole resistance among Candida species in the oral cavities of PLWHA. We sampled the oral cavities of 350 HIV-infected adults and an equal number of HIV-negative controls. Candida isolates were identified using germ tube tests, CHROMagar Candida (CHROMagar, Paris, France), and API Candida yeast identification system (BioMérieux, Marcy-l'Étoile, France). Fluconazole susceptibility was determined using the Clinical and Laboratory Standards Institute disc diffusion method. Data were analysed using SPSS version 21 (IBM, New York, NY, USA). The significance level was set at p ? 0.05. The isolation rates for Candida amongst HIV-infected subjects and controls were 20.6% and 3.4%, respectively (p < 0.001). In PLWHA, Candida albicans was most frequently isolated (81.3%) and fluconazole resistance was present in 18 (24%) of the 75 Candida isolates. Resistance to fluconazole was present in half of the non-albicans Candida isolates. Fluconazole resistance is prevalent among oral Candida isolates in PLWHA in the study area with a significantly higher rate among non-albicans Candida spp.
Project description:Studies in patients with genetic defects can provide unique insights regarding the role of specific genes and pathways in humans. Patients with defects in the Th17/IL-17 axis, such as patients harboring loss-of-function STAT3 mutations (autosomal-dominant hyper IgE syndrome; AD-HIES) present with recurrent oral fungal infections. Our studies aimed to comprehensively evaluate consequences of STAT3 deficiency on the oral commensal microbiome. We characterized fungal and bacterial communities in AD-HIES in the presence and absence of oral fungal infection compared with healthy volunteers. Analyses of oral mucosal fungal communities in AD-HIES revealed severe dysbiosis with dominance of Candida albicans (C. albicans) in actively infected patients and minimal representation of health-associated fungi and/or opportunists. Bacterial communities also displayed dysbiosis in AD-HIES, particularly in the setting of active Candida infection. Active candidiasis was associated with decreased microbial diversity and enrichment of the streptococci Streptococcus oralis (S. oralis) and S. mutans, suggesting an interkingdom interaction of C. albicans with oral streptococci. Increased abundance of S. mutans was consistent with susceptibility to dental caries in AD-HIES. Collectively, our findings illustrate a critical role for STAT3/Th17 in the containment of C. albicans as a commensal organism and an overall contribution in the establishment of fungal and bacterial oral commensal communities.
Project description:Background: Little is known about the normal range of metal levels in unstimulated saliva, nor whether these might impact Candida carriage in healthy individuals. Both are important in determining which populations are at risk for candidiasis, as the availability of metal ions can influence the growth and pathogenesis of Candida albicans. Objective: We quantified salivary metals of healthy individuals to determine the correlation with C. albicans oral colonization. Design: Unstimulated whole saliva was collected from healthy adults and plated to determine fungal carriage, and metal content was measured using ICP-mass spectrometry (ICP-MS). Results: Zinc was most abundant, followed by iron, copper, manganese, and nickel. Cultivable oral Candida carriage was found in 48% of people. Total protein levels did not differ in salivas from donors with or without carriage. However, innate fungicidal activity was increased in donors with carriage; correlations between levels of several metals were stronger in salivas with fungal carriage, indicating a shift in the oral environment. Concentrations of copper and manganese, as well as age and gender, were significantly predictive of carriage levels in a multiple regression model. Conclusions: Salivary copper and manganese content along with age and gender could be used as a predictive metric for individuals that are more susceptible to Candida overgrowth.
Project description:Oral candidiasis is a common side effect of cancer chemotherapy. To better understand predisposing factors, we followed forty-five subjects who received 5-fluorouracil- or doxorubicin-based treatment, during one chemotherapy cycle. Subjects were evaluated at baseline, prior to the first infusion, and at three additional visits within a two-week window. We assessed the demographic, medical and oral health parameters, neutrophil surveillance, and characterized the salivary bacteriome and mycobiome communities through amplicon high throughput sequencing. Twenty percent of all subjects developed oral candidiasis. Using multivariate statistics, we identified smoking, amount of dental plaque, low bacteriome and mycobiome alpha-diversity, and the proportions of specific bacterial and fungal taxa as baseline predictors of oral candidiasis development during the treatment cycle. All subjects who developed oral candidiasis had baseline microbiome communities dominated by Candida and enriched in aciduric bacteria. Longitudinally, oral candidiasis was associated with a decrease in salivary flow prior to lesion development, and occurred simultaneously or before oral mucositis. Candidiasis was also longitudinally associated with a decrease in peripheral neutrophils but increased the neutrophil killing capacity of Candida albicans. Oral candidiasis was not found to be associated with mycobiome structure shifts during the cycle but was the result of an increase in Candida load, with C. albicans and Candida dubliniensis being the most abundant species comprising the salivary mycobiome of the affected subjects. In conclusion, we identified a set of clinical and microbiome baseline factors associated with susceptibility to oral candidiasis, which might be useful tools in identifying at risk individuals, prior to chemotherapy.
Project description:Background:Bronchial asthma is a chronic inflammatory disease of airways. The disease itself along with the principal medication used makes the oral cavity susceptible to most common opportunistic infection, i.e., oral candidiasis. There are many species of Candida causing oral candidiasis, but the most prevalent among them is Candida albicans. Hence, assessing C. albicans count in response to disease and its treatment is necessary. This enables us to educate asthma patients about side effects of medication and highlight the necessity for oral health care, thereby improving their quality of life. Aims:The present study aims to evaluate the effects of asthma and its medication on C. albicans count in saliva samples of asthmatic adult patients taking medication for 3-5 years and compare C. albicans count in saliva samples among cases and controls. Materials and Methods:Thirty asthmatic adults taking medication for asthma since 3-5 years' age ranging from 20 to 50 years and equal number of age- and sex-matched healthy participants were included in the study. In both groups, saliva was collected and inoculated on Sabouraud Dextrose Agar culture plates for estimation of C. albicans counts. C. albicans counts were assessed in colony-forming unit/milliliter. Statistical Analysis:Mann-Whitney U-test and Fisher's exact t-test were used. Results:The C. albicans count is significantly higher among asthmatics than healthy individuals. Conclusions:The present study concludes that there is increased candidal growth among asthmatics as compared to their normal healthy counterpart.
Project description:Oral microbiota contribute to health and disease, and their disruption may influence the course of oral diseases. Here, we used pyrosequencing to characterize the oral bacteriome and mycobiome of 12 HIV-infected patients and matched 12 uninfected controls. The number of bacterial and fungal genera in individuals ranged between 8-14 and 1-9, among uninfected and HIV-infected participants, respectively. The core oral bacteriome (COB) comprised 14 genera, of which 13 were common between the two groups. In contrast, the core oral mycobiome (COM) differed between HIV-infected and uninfected individuals, with Candida being the predominant fungus in both groups. Among Candida species, C. albicans was the most common (58% in uninfected and 83% in HIV-infected participants). Furthermore, 15 and 12 bacteria-fungi pairs were correlated significantly within uninfected and HIV-infected groups, respectively. Increase in Candida colonization was associated with a concomitant decrease in the abundance of Pichia, suggesting antagonism. We found that Pichia spent medium (PSM) inhibited growth of Candida, Aspergillus and Fusarium. Moreover, Pichia cells and PSM inhibited Candida biofilms (P?=?.002 and .02, respectively, compared to untreated controls). The mechanism by which Pichia inhibited Candida involved nutrient limitation, and modulation of growth and virulence factors. Finally, in an experimental murine model of oral candidiasis, we demonstrated that mice treated with PSM exhibited significantly lower infection score (P?=?.011) and fungal burden (P?=?.04) compared to untreated mice. Moreover, tongues of PSM-treated mice had few hyphae and intact epithelium, while vehicle- and nystatin-treated mice exhibited extensive fungal invasion of tissue with epithelial disruption. These results showed that PSM was efficacious against oral candidiasis in vitro and in vivo. The inhibitory activity of PSM was associated with secretory protein/s. Our findings provide the first evidence of interaction among members of the oral mycobiota, and identifies a potential novel antifungal.