Molecular analysis of fungal populations in patients with oral candidiasis using internal transcribed spacer region.
ABSTRACT: 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: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:Candida dubliniensis is a recently identified chlamydospore-positive yeast species associated with oral candidiasis in human immunodeficiency virus (HIV)-infected (HIV+) patients and is closely related to Candida albicans. Several recent reports have described atypical oral Candida isolates with phenotypic and genetic properties similar to those of C. dubliniensis. In this study 10 atypical chlamydospore-positive oral isolates from HIV+ patients in Switzerland, the United Kingdom, and Argentina and 1 isolate from an HIV-negative Irish subject were compared to reference strains of C. albicans and Candida stellatoidea and reference strains of C. dubliniensis recovered from Irish and Australian HIV+ individuals. All 11 isolates were phenotypically and genetically similar to and phylogenetically identical to C. dubliniensis. These findings demonstrate that the geographical distribution of C. dubliniensis is widespread, and it is likely that it is a significant constituent of the normal oral flora with the potential to cause oral candidiasis, particularly in immunocompromised patients.
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:Dental caries is one of the most common diseases worldwide. Bacteria and fungi are both commensals in the oral cavity; however, most research regarding caries has focused on bacterial impacts. The oral fungal mycobiome associated with caries is not well characterized, and its role in disease is unclear. ITS1 amplicon sequencing was used to generate taxonomic profiles from site-specific supragingival plaque samples (n?=?82) obtained from 33 children with different caries status. Children were either caries free (CF), caries active with enamel lesions (CAE), or caries active with dentin lesions (CA). Plaque samples were collected from caries-free surfaces (PF) and from enamel (PE) and dentin (PD) lesions. Taxonomic profiles representing the different categorizations (CF-PF, CAE-PF, CAE-PE, CA-PF, CA-PE, and CA-PD) were used to characterize the mycobiome and its change through disease progression. A total of 139 fungal species were identified. Candida albicans was the most abundant species, followed by Candida dubliniensis We found that severely progressed plaque communities (CA-PD) were significantly different from healthy plaque communities (CF-PF). A total of 32 taxa were differentially abundant across the plaque categories. C. albicans, C. dubliniensis, Nigrospora oryzae, and an unclassified Microdochium sp. were correlated with caries, whereas 12 other taxa were correlated with health. C. dubliniensis increased steadily as caries progressed, suggesting that C. dubliniensis may play an important role in caries pathogenicity. In contrast, four health-associated fungal taxa have the potential to antagonize the cariogen Streptococcus mutans via xylitol production, suggesting a possible fungal mechanism that could contribute to maintenance of dental health.IMPORTANCE Early-childhood caries is one of the most prevalent diseases in children worldwide and, while preventable, remains a global public health concern. Untreated cavities are painful and expensive and can lead to tooth loss and a lower quality of life. Caries are driven by acid production via microbial fermentation of dietary carbohydrates, resulting in enamel erosion. While caries is a well-studied disease, most research has focused on bacterial impacts, even though fungi are commensal organisms living within the plaque biofilm. There is very little known about how fungi impact caries pathogenicity. The elucidation of fungal taxa involved in caries disease progression is necessary for a more holistic view of the human oral microbiome. Data from this study will improve our understanding of how the fungal community changes as disease progresses and provide insight into the complex etiology of dental caries, which is necessary for the development of treatment plans and preventative measures.
Project description:Candida dubliniensis is a newly identified species of Candida that is phenotypically similar to but genetically distinct from C. albicans. This organism has been recovered with increasing frequency from the oral cavities of human immunodeficiency virus (HIV)-infected and AIDS patients and has been implicated as a causative agent of oral candidiasis and systemic disease. In the present study we characterized the molecular mechanisms of resistance to fluconazole (FLC) in C. dubliniensis clinical isolates from two different HIV-infected patients with oropharyngeal candidiasis. Isolates were identified to the species level by phenotypic and genotypic tests. DNA-typing techniques were used to assess strain identity. Antifungal susceptibility testing was performed by NCCLS techniques. Northern blotting analysis was used to monitor the expression of genes encoding lanosterol demethylase (ERG11) and efflux transporters (CDR and MDR1) in matched sets of C. dubliniensis-susceptible and -resistant isolates by using probes generated from their homologous C. albicans sequences. In addition, ERG11 genes were amplified by PCR, and their nucleotide sequences were determined in order to detect point mutations with a possible effect in the affinity for azoles. Decreasing susceptibilities to FLC were detected in C. dubliniensis isolates recovered from both patients during the course of treatment. FLC-resistant C. dubliniensis isolates from one patient demonstrated combined upregulation of the MDR1, CDR1, and ERG11 genes. Among the isolates from the second patient, all isolates showing decreased susceptibility to FLC demonstrated upregulation of MDR1, whereas the levels of mRNA for the ERG11 genes remained constant and the expression of CDR genes was negligible. Fourteen point mutations were found in the ERG11 genes of the isolates with decreased susceptibility to FLC. These data demonstrate that the development of azole resistance in C. dublinensis clinical isolates from HIV-infected patients treated with FLC is mediated by multiple molecular mechanisms of resistance, similar to the observations found in the case of C. albicans.
Project description:Interactions between colonizing commensal microorganisms and their hosts play important roles in health and disease. The opportunistic fungal pathogen Candida albicans is a common component of human intestinal flora. To gain insight into C. albicans colonization, genes expressed by fungi grown within a host were studied. The EFH1 gene, encoding a putative transcription factor, was highly expressed during growth of C. albicans in the intestinal tract. Counterintuitively, an efh1 null mutant exhibited increased colonization of the murine intestinal tract, a model of commensal colonization, whereas an EFH1 overexpressing strain exhibited reduced colonization of the intestinal tract and of the oral cavity of athymic mice, the latter situation modeling human mucosal candidiasis. When inoculated into the bloodstream of mice, both efh1 null and EFH1 overexpressing strains caused lethal infections. In contrast, other mutants are attenuated in virulence following intravenous inoculation but exhibited normal levels of intestinal colonization. Finally, although expression of several genes is dependent on transcription factor Efg1p during laboratory growth, Efg1p-independent expression of these genes was observed during growth within the murine intestinal tract. These results show that expression of EFH1 regulated the level of colonizing fungi, favoring commensalism as opposed to candidiasis. Also, different genes are required in different host niches and the pathway(s) that regulates gene expression during host colonization can differ from well-characterized pathways used during laboratory growth.
Project description:The causes of fungemia include immunosuppression and neutropenia stemming from diverse factors as well as the placement of central venous catheters. However, the relationship between fungemia and the oral cavity has not been substantiated. In this study, we explored the pathological conditions of Candida albicans-derived oral candidiasis in a mouse model, which always develops oral mucositis as a complication. In oral candidiasis, the hyphae of C. albicans are believed to primarily invade the stratum granulosum, but not the subepithelium, of the mucous membrane. We provide histological evidence that in concomitant oral mucositis, the hyphae infiltrate the subepithelium and blood vessels. Blood cultures and tissue samples revealed the onset of fungemia only in the mucositis-induced groups. Positive numbers of colony-forming units were found in groups A (chemotherapy), B (chemotherapy?+?mucositis) and C (mucositis), but were highest in group B. Some organs revealed positive CFU in groups B and C. The presence of fungal DNA in blood plasma and tissue was confirmed by PCR. The fungal DNA frequency was significantly higher in the mucositis group when compared with the non-mucositis group. The results suggest that fungi first invade the subepithelium and then the blood vessels, from which they disseminate throughout the body, and that oral mucositis is an important risk factor for fungemia. This study clearly demonstrates the relationship between oral mucositis, fungemia, and the potential systemic fungal dissemination, which has not been previously proven. Our findings highlight the importance of oral care for patients at risk of fungemia.
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:Candidiasis is a fungal infection caused by Candida species that have formed a biofilm on epithelial linings of the body. The most frequently affected areas include the vagina, oral cavity and the intestine. In severe cases, the fungi penetrate the epithelium and cause systemic infections. One approach to combat candidiasis is to prevent the adhesion of the fungal hyphae to the epithelium. Here we demonstrate that the endocannabinoid anandamide (AEA) and the endocannabinoid-like N-arachidonoyl serine (AraS) strongly prevent the adherence of C. albicans hyphae to cervical epithelial cells, while the endocannabinoid 2-arachidonoylglycerol (2-AG) has only a minor inhibitory effect. In addition, we observed that both AEA and AraS prevent the yeast-hypha transition and perturb hyphal growth. Real-time PCR analysis showed that AEA represses the expression of the HWP1 and ALS3 adhesins involved in Candida adhesion to epithelial cells and the HGC1, RAS1, EFG1 and ZAP1 regulators of hyphal morphogenesis and cell adherence. On the other hand, AEA increased the expression of NRG1, a transcriptional repressor of filamentous growth. Altogether, our data show that AEA and AraS have potential anti-fungal activities by inhibiting hyphal growth and preventing hyphal adherence to epithelial cells.
Project description:Invasive fungal infections are a major cause of morbidity and mortality among solid organ transplant (SOT) and hematopoietic stem cell transplant (HSCT) recipients. Transplant patients are at risk for such invasive fungal infections. The most common invasive fungal infections are invasive candidiasis in the SOT and invasive aspergillosis in the HSCT. In this article, we will discuss the epidemiology of invasive fungal infections in the transplant recipients and susceptibility patterns of the fungi associated with these infections. Additionally, the pharmacology and clinical efficacy of the new antifungal, isavuconazole, and the new posaconazole formulations will be reviewed. Isavuconazole is a new extended-spectrum triazole that was recently approved for the treatment of invasive aspergillosis and mucormycosis. Advantages of this triazole include the availability of a water-soluble intravenous formulation, excellent bioavailability of the oral formulation, and predictable pharmacokinetics in adults. Posaconazole, a broad-spectrum triazole antifungal agent, is approved for the prevention of invasive aspergillosis and candidiasis in addition to the treatment of oropharyngeal candidiasis. Posaconazole oral suspension solution has shown some limitations in the setting of fasting state absorption, elevated gastrointestinal pH, and increased motility. The newly approved delayed-release oral tablet and intravenous solution formulations provide additional treatment options by reducing interpatient variability and providing flexibility in these set of critically ill patients. This review will detail these most recent studies.