Analysis of the strain relatedness of oral Candida albicans in patients with diabetes mellitus using polymerase chain reaction-fingerprinting

To increase our understanding of Candida pathogenicity, the identification of those strains most frequently associated with infections is of paramount importance. Polymerase chain reaction (PCR)-based methods are extremely effective in differentiating and determining reproducibility, they require minimum starting material and are rapid and simple to perform. In this study, the genetic relatedness of Candida albicans was assessed for two geographically different patient groups (London, UK and Parma, Italy) affected by diabetes mellitus. C. albicans samples from the oral cavities of non-diabetic healthy subjects were also examined by PCR fingerprinting to evaluate the possible genetic differences among endogenous strains in individuals with and without diabetes mellitus. PCR fingerprinting, with subsequent phylogenetic analysis of C. albicans isolates from the diabetic patients from London and Italy and from the non-diabetic subjects, revealed that there were significant differences (P < 0.0001) between C. albicans isolates indicative of the distinct ecological niches that occur in the oral cavities of these patient cohorts. The most diverse group comprised the isolates from the diabetic patients in the UK, possibly reflecting the antifungal treatment that these patients had received. Further studies that include isolates from patient cohorts with systemic diseases other than diabetes mellitus, and from more diverse geographic localities are required to explain the relatedness of C. albicans isolates in the mouth.     

Distribution and hydrolytic enzyme characteristics of Candida albicans strains isolated from diabetic patients and their non‐diabetic consorts

Introduction: The aim of this study was to investigate the oral colonization profile of Candida albicans strains isolated from diabetic patients and their non‐diabetic consorts. In addition hydrolytic enzyme activity of these isolates was analysed. Methods: The genetic diversity of C. albicans oral isolates from 52 couples was established using isoenzyme marker and cluster analysis. Hydrolytic enzyme characteristics, namely secreted aspartyl proteinases (SAPs) and phospholipases (PLs) were also analysed. Results: Simultaneous colonization by C. albicans was observed in the consorts of 12 couples (23.1%). Patterns of monoclonal and polyclonal oral colonization by C. albicans strains were identified and the coexistence of identical or highly related strains was observed in both members of eight couples. The genetic diversity observed in the total yeast population revealed four large, genetically distinct groups (A to D) and the coexistence of strains in couples or consorts conjugally unrelated. SAP and PL activity was observed in the majority of C. albicans isolates without any association to particular strain, strain clusters (highly related isolates), or clinical characteristics of the consorts (diabetic, non‐diabetic, and gender). Conclusion: Possible sources of transmission and oral propagation of groups (clusters) of strains of C. albicans can occur between diabetic and non‐diabetic consorts. A conjugal genotypic identity exists in most C. albicans‐positive couples, that is, both consorts share identical or highly related strains; however, this identity is not couple‐specific as seen by the coexistence of clusters in couples and unrelated consorts.     

The isolation,identification and molecular analysis of Candida spp. isolated from the oral cavities of patients with diabetes mellitus

Previous studies have shown a high incidence (77%) of isolation of Candida spp. from the oral cavities of patients with type 1 diabetes mellitus. The aim of the present study was to assess the prevalence of yeast in the oral cavities of patients suffering from type 1 and type 2 diabetes mellitus. The patients were classified according to the level of diabetic control (HbA1_c), and further stratified on the presence or absence of dental prosthesis. Oral rinse samples were assessed for the growth of yeast and the degree of colonization. Oral isolates were defined to the species level by both phenotypic and novel molecular methods. The overall proportion (60%) of diabetic patients who had Candida spp. isolated from the oral cavity was similar to that previously reported. Local oral factors, such as the presence of dentures, seemed to have a greater influence than diabetic status on the amount and species of Candida isolated from the oral cavities of diabetic patients. Diabetic patients with dentures had more non‐albicans Candida isolated from their mouths than dentate diabetic patients. Candida dubliniensis was isolated from diabetic patients and may have a predilection for dentate patients.     

In vitro antifungal susceptibility to six antifungal agents of 229 Candida isolates from patients with diabetes mellitus

The most common antifungal drugs in current clinical use for the treatment of oral candidosis are polyenes and azoles, mainly used topically. Poor glycaemic control in association with other local factors, such as the presence of oral dental prostheses, salivary pH, salivary flow rate and tobacco habits, may lead to the development of oral candidosis. Topical antifungal agents are frequently used to prevent the development of candidal infections in patients with poor metabolic control, particularly in the elderly wearing dentures. The aim of this study was to assess the antifungal susceptibility of Candida isolates to six antifungal agents using a commercially available kit, Fungitest. The isolated were collected from patients affected by diabetes mellitus from two different geographic localities (London, UK, and Parma, Italy) and from a group of healthy non-diabetic subjects. No differences in antifungal susceptibility to the six agents tested were observed between Candida isolates from diabetic and non-diabetic subjects. However, differences were observed between the two geographically different diabetes mellitus populations. Oral yeast isolates from diabetes mellitus patients in the UK more often displayed resistance or intermediate resistance to fluconazole (P=0.02), miconazole (P<0.0001), and ketoconazole (P=0.01) than did isolates from diabetes mellitus patients in Italy. In addition, more C. albicans isolates were found in diabetic and non-diabetic subjects that were susceptible to fluconazole (P=0.0008 and P=0.01, respectively) than non-albicans isolates. The difference in the antifungal resistance of isolates from the two populations of diabetes mellitus patients may be related to differences in the therapeutic management of candidal infections between the two centres.     

In vitro activity of a monoclonal killer anti‐idiotypic antibody and a synthetic killer peptide against oral isolates of Candida spp. differently susceptible to conventional antifungals

Background/aims: A monoclonal killer anti‐idiotypic antibody (mAbK10) and a synthetic killer peptide, acting as internal images of a microbicidal, wide‐spectrum yeast killer toxin (KT) have been recently shown to express candidacidal in vitro and an in vivo therapeutic activity against experimental mucosal and systemic candidosis models caused by a reference strain of Candida albicans (10S). Material and methods: The in vitro candidacidal activity of mAbK10 and synthetic killer peptide was compared using a colony forming unit assay against a large number of isolates of different Candida spp., obtained from oral saliva of adult diabetic (type 1 and 2) and nondiabetic subjects from Parma (Italy) and London (UK). Results: Both the KT‐mimics exerted a strong dose‐dependent candidacidal activity, probably mediated by the interaction with β‐glucan KT receptors on target yeast cells, against all the tested strains, regardless of their species and pattern of resistance to conventional antifungal agents. Conclusions: These observations open new perspectives in the design and production of candidacidal compounds whose mechanism reflects that exerted in nature by killer yeasts.     

Prevalence and antifungal resistance profile of Candida spp. oral isolates from patients with type 1 and 2 diabetes mellitus

Objective

The goal of the study was to measure the prevalence of Candida spp. in the oral cavity of patients with diabetes types 1 and 2 when compared to healthy individuals and to study antifungal resistance profile of the isolates.

Design

There were 162 subjects in the study: diabetes type 1 (n = 39); control group 1 (n = 50): healthy individuals matched in gender, age, and oral conditions to diabetes type 1 patients; diabetes type 2 (n = 37); control group 2 (n = 36) who were matched to each patient of the diabetes type 2 group. Stimulated saliva was collected and isolates were identified with phenotypic tests. The presence of C. dubliniensis was determined by multiplex PCR.

Results

There were no statistically significant differences in Candida spp. frequency between the diabetes 1 group and its control (p = 0.443) nor between the diabetes 2 group and its control (p = 0.429). C. albicans was the most frequently isolated yeast in all groups. In the diabetes groups, C. stellatoidea, C. parapsilosis, C. tropicalis, C. lipolytica, C. glabrata, and C. krusei were also identified. Additionally, in control groups, C. kefyr was also detected. None of the isolates were resistant to amphotericin B and flucytosine. A low percentage of the isolates were resistant to ketoconazole.

Conclusions

No differences were detected in colonization of Candida spp. oral isolates from type 1 and type 2 diabetes when compared to matched controls. The antifungal resistance of Candida spp. isolates for ketoconazole from type 1 diabetes patients was significantly higher than that of its matched control.     

Molecular and phenotypic analysis of Candida dubliniensis: A recently identified species linked with oral candidosis in HIV-infected and AIDS patients

The discovery and characterisation of a novel species of Candida, termed Candida dubliniensis, associated with oral candidosis in HIV-infected individuals is described. These organisms share several phenotypic characteristics in common with Candida albicans and Candida stellatoidea, including the ability to produce germ tubes and chlamydospores. However, in contrast to these latter two species, C. dubliniensis isolates produce abundant chlamydospores, which are often arranged in contiguous pairs, triplets and other multiples suspended from a single suspensor cell. They belong to C. albicans serotype A and exhibit atypical substrate assimilation profiles. Genomic DNA fingerprinting analysis with the C. albicons-specific probe 27A and five different oligonucleotide probes consisting of short repeat sequence-containing motifs, demonstrated that C. dubliniensis has a distinct genomic organisation relative to C. albicans and C. stellatoidea. This was confirmed by karyotype analysis and random amplified polymorphic DNA (RAPD) analysis. Comparison of 500 bp of the V3 variable region of the large ribosomal subunit genes from 14 separate C. dubliniensis isolates and the corresponding sequences from C. albicans, C. stellatoidea, C. tropicalis, C. glabrata, C. parapsilosis, C. kefyr and C. krusei demonstrated that the C. dubliniensis isolates formed a homogenous cluster (100% similarity), representing a discrete taxon within the genus Candida that was significantly different from the other species analysed.     

Genetic diversity and exoenzyme activities of Candida albicans and Candida dubliniensis isolated from the oral cavity of Brazilian periodontal patients

Objective

Mucosal surfaces are the primary oral reservoirs of Candida species, but these species can also be found in subgingival biofilm. The present study investigated the genetic diversity and production of exoenzymes of C. albicans and C. dubliniensis isolated from the oral cavity of systemically healthy patients with periodontitis.

Design

Fifty-three patients were analysed. Samples were collected from three oral cavity sites (periodontal pocket, gingival sulci and oral mucosa), plated and, after isolation, suspect strains of C. albicans and C. dubliniensis were identified by PCR. The genetic diversity of the isolates was evaluated by RAPD and the activities of the secreted aspartyl proteinases and phospholipases were evaluated by the agar plate method.

Results

Twenty-one patients showed positive results for Candida spp. There were no statistically significant differences between genders, or between sites. C. albicans was the most frequently found specie, while C. dubliniensis was isolated from the periodontal pocket of only one patient. Sixteen genotypes were detected among the C. albicans isolates, and one among the C. dubliniensis isolates. The similarity coefficient (S_SM) values among the C. albicans genotypes ranged from 0.684 to 1.0 with an average of 0.905 ± 0.074. All isolates produced high levels of Saps and most of them produced high levels of phospholipases. No relationship was found between the genotypes and the pattern of enzymatic production. There was no association between specific genotypes and their site of isolation.

Conclusions

The results of the present study suggest that genetically homogeneous strains of C. albicans are present in the oral cavity of patients with periodontitis and that these strains are capable of producing high levels of exoenzyme.     

Selection and pathogenicity of Candida albicans in HIV infection

The progression of immune-suppression due to infection with HIV is mirrored by an increase in the severity and prevalence of oral candidosis. The transition of C. albicans from harmless commensal to unrelenting pathogen in the susceptible host is a fine line attributable to an extensive repertoire of selectively expressed virulence determinants, including hyphal formation, thigmotropism, protease secretion, adherence and phenotypic switching. The transition from commensal to pathogen has been linked to the formation of elongated hyphae which may more readily penetrate epithelial surfaces. Invasion may be enhanced by thigmotropism which enables hyphae to sense intercellular junctions and surface discontinuities in order to find areas that may be more readily breached. Electron micrograph studies suggest that C. albicans are able to penetrate mucosal tissues by secreting hydrolytic enzymes at the tip of the invading hyphae. Recent studies have shown that protease secretion is upregulated in C. albicans isolated from AIDS patients. Similarly, the propensity for C. albicans to adhere to oral mucosa appears to be enhanced in isolates from HIV-infected subjects. These two virulence determinants may be coordinately regulated and DNA fingerprinting studies suggest that these phenotypic alterations may be associated with the selection of C. albicans with altered genotypes. The mechanisms by which hypervirulent C. albicans may be selected in HIV-infected patients is likely to be a multifactorial process associated with the pathobiological effects of HIV infection and to the increased candidal proliferation so evident in these patients.     

Stable azole drug resistance associated with a substrain of Candida albicans from an HIV-infected patient

Oral candidiasis is one of the earliest and most frequent complications of a failing immune system in HIV-infected individuals. For several years, oral candidiasis has been treated effectively with azole drugs, the one most frequently used is fluconazole. Unfortunately, extensive use of the drug for treatment and prophylaxis has led to treatment failure in an increasing number of patients. In most of these cases, strains of C. albicans isolated from the infection are less susceptible to fluconazole. The development of azole resistance in strains of C. albicans has been studied biochemically and more recently with molecular techniques. One excellent example of the development of azole resistance in C. albicans has been documented in a series of 17 C. albicans isolates from a single patient over a 2-year period. During this time, the patient experienced 14 episodes of oral candidiasis and was treated with increasing doses of fluconazole. Molecular and biochemical analyses confirms that the isolates are the same strain of C. albicans and that the resistance in these isolates is stable over 600 generations, suggesting that the changes in this strain are genetic in nature. In addition, the development of resistance is correlated with the identification of a substrain or variant of the original strain, as identified by restriction fragment length polymorphism (RFLP) analysis with the moderately repetitive probe, Ca3. The analysis of this series of isolates demonstrates that azole drug resistance is associated with several small genetic changes, each of which contributes to the overall resistance of the strain. Clearly, continual use of azole drugs by a patient can select for genetic changes that render oral candidiasis refractory to treatment.     

Antifungal drug susceptibility of oral Candida albicans isolates may be associated with apoptotic responses to Amphotericin B

J Oral Pathol Med (2010) 39 182–187 Background: Candida albicans is the important opportunistic fungal pathogens which can cause oral Candidiasis and even more seriously systemic infection. Apoptosis of C. albicans induced by environmental factor such as weak acid and antifungal drugs were studied recently. Illustrating the phenomenon of apoptosis in C. albicans may help us to discover new antifungal therapy by activating the fungal cells to suicide. Methods: Two oral C. albians clinical isolates which isolated respectively from healthy host [Strain 23C: minimal inhibition concentration (MIC) is 0.125 μg/ml for Amphotericin B (AmB)] and advanced cancer patient (Strain 28A: MIC is 2 μg/ml for AmB), were induced by 1 μg/ml AmB in vitro for 200 min, and then studied the apoptosis markers using terminal deoxynucletidyltransferase‐mediated dUTP nick end labeling (TUNEL) (shown by diaminobenzidine and fluorescent isothiocyanate), and the ultrastructure of cell nuclear using transmission electron microscope (TEM), quantitative analysis using flow cytometry for the rapid exposure of phosphatidylserine at the outer membrane and propodium iodide (PI) double staining. C. albicans conference strain YEM30 was used as the control strain. Results: With TUNEL assay and TEM, we detected the typical characteristics of apoptosis. Strain 23C (with low MIC) showed significantly higher percentage of apoptosis (19.92%) compared with Strain 28A (with high MIC) which was isolated from the cancer patient (7.29%) (P < 0.01). In addition, 7.3% of early apoptosis cells of Strain 23C can form colonies on the plates, while 15% for Strain 28A. None of the PI (+) cells can form colony. Conclusions: Apoptosis of oral C. albicans isolates can be induced by AmB. The feature of antifungal drug susceptibility of the oral C. albicans clinical isolates may associate with the response of apoptosis inducing.     

Identification of Candida dubliniensis among oral yeast isolates from an Italian population of human immunodeficiency virus‐infected (HIV+) subjects

Candida dubliniensis, an emerging oral pathogen, phenotypically resembles Candida albicans so closely that it is easily misidentified as such. The aim of the present study was to evaluate the usefulness of two phenotypic methods, growth at 45°C and 2,3,5‐triphenyltetrazolium chloride (TTC) reduction, for confirming presumptive identification of C. dubliniensis and C. albicans by colony color on CHROMagar Candida (CAC) medium. A combination of these methods was used to establish the prevalence of oral C. dubliniensis in an Italian population of 45 human immunodeficiency virus (HIV)‐infected subjects. Twenty‐two samples (48.9%) were positive for yeasts on CAC medium producing a total of 37 fungal isolates. The colony color and 45°C growth ability test correctly identified all C. dubliniensis and C. albicans isolates (5/37, 13.5%, and 16/37, 43.2%, respectively), while assessment of TTC reduction misidentified one C. albicans isolate. The isolation rate of C. dubliniensis was 11.1% (5/45 patients). All of the C. dubliniensis isolates were highly susceptible to fluconazole (MIC = 0.5 µg/ml). The combination of CAC medium screening with growth at 45°C and TTC reduction tests may represent a simple, reliable and inexpensive identification protocol for C. dubliniensis.     

Clonal identity of Candida albicans in the oral cavity and the gastrointestinal tract of pre‐school children

The clonal relationship between oral and fecal Candida albicans isolated from children of pre‐school age was examined using RAPD analysis. Significantly higher levels of C. albicans were found in saliva, dental plaque, carious specimens and stools of 56 patients with severe caries as compared to 52 healthy control subjects. The highest prevalence was found in carious specimens and a strong correlation was observed between its presence in saliva, dental plaque, carious specimen and feces. RAPD analysis of isolates from 23 patients with simultaneous oral and fecal C. albicans revealed clonal counterparts present in both oral and stool samples in 15 cases; five patients harbored closely related strains; and three patients harbored unrelated strains. Our results demonstrate a strong correlation between oral and gastrointestinal C. albicans colonization. We assume that carious teeth may constitute an ecologic niche for C. albicans potentially responsible for recurrent oral and non‐oral candidiasis.     

Innocent until proven guilty: mechanisms and roles of Streptococcus–Candida interactions in oral health and disease

Candida albicans and streptococci of the mitis group colonize the oral cavities of the majority of healthy humans. While C. albicans is considered an opportunistic pathogen, streptococci of this group are broadly considered avirulent or even beneficial organisms. However, recent evidence suggests that multi‐species biofilms with these organisms may play detrimental roles in host homeostasis and may promote infection. In this review we summarize the literature on molecular interactions between members of this streptococcal group and C. albicans, with emphasis on their potential role in the pathogenesis of opportunistic oral mucosal infections.     

Periodontal disease in gestational and type 1 diabetes mellitus pregnant women

Oral Diseases (2011) 17 , 515–521 Objective: The present study evaluated the relationship between periodontal disease and its clinical variables in Brazilian non‐diabetic pregnant women (C), gestational diabetes mellitus (GDM), or type 1 diabetes mellitus (T1DM). Subjects and methods: A periodontal exam was performed in one hundred and sixty‐one pregnant women (GDM:80; T1DM:31; C:50) by a single‐blinded calibrated examiner who recorded plaque index (PI), gingival index (GI), bleeding index (BI), gingival margin location (GM), probing depth (PD), clinical attachment level (CAL), bleeding on probing (BOP), and tooth mobility index (MI). The medical variables were age, pregestational body mass index (pre‐BMI), fasting plasma glucose (FPG), and glycated hemoglobin (HbA _1c ). Results: The GI, GM, PD, CAL, BOP, and MI were significantly higher ( P < 0.01) among GDM and T1DM than for C. The PI was higher in GDM and similar between C and T1DM. The Adjusted Final Model for medical variables to evaluate the effects of groups on periodontal parameters confirmed these results. Conclusions: The presence of periodontal disease was significantly higher in Brazilian diabetic pregnancies (GDM and T1DM) when compared to non‐diabetic pregnant women (C). The degree of periodontal disease was similar between the GDM and T1DM groups. Age, pregestational BMI, and HbA_1c were factors related to CAL development in these two types of diabetes mellitus.     

Diversity,frequency and antifungal resistance of Candida species in patients with type 2 diabetes mellitus

Objective: To determine number, species of Candida and Candida resistance to antifungal therapy according to the metabolic control state and the associated salivary changes in patients with type 2 diabetes mellitus (DM2).

Materials and methods: Samples of non-stimulated saliva were collected from 52 patients with DM2. Salivary pH was measured and cultured on Sabouraud glucose agar and the values of CFU/ml were calculated. The species were presumptively identified using CHROMagar Candida^® plates, and identification was confirmed by polymerase chain reaction (PCR). C. albicans isolates were cultured on SGA tetracycline agar with nystatin and fluconazole diffusion disks to measure susceptibility.

Results: Sixty six percent of the yeasts isolated were Candida albicans, followed by C. glabrata (20.7%). In patients with decompensated DM2, there was an inverse association between HbA1c value and salivary pH. At higher levels of salivary acidification, a greater diversity and quantity of yeasts of the genus Candida were observed. With nystatin, higher inhibition was observed at lower pH.

Conclusions: The antifungal therapies could be more effective if it consider, qualitative salivary characteristics as pH, that could determine the susceptibility of species of Candida to at least to nystatin, which is the most used antifungal for treatment to oral candidiasis in patients with DM2.     

Oral Candida albicans from patients infected with the human immunodeficiency virus and characterization of a genetically distinct subgroup of Candida albicans

Candida albicans has been shown to vary in its phenotypic expression with the progression of human immunodeficiency virus (HIV) infection. This study was designed to investigate whether in Category IV HIV infected patients (CDC, Atlanta, USA) these phenotypic changes were related to changes in the genetic strain of the organism. Isolates of C. albicans were obtained from 45 patients with HIV infection during the progression of their disease as determined by percentage T4 lymphocyte count. Isolates were strain differentiated using two methods. In 67 per cent of the patients a single strain of C. albicans, as determined by the DNA analysis, was isolated from each individual over the experimental period. The phenotypic expression of the genetically identical strains isolated from each patient varied considerably over the experimental period with the morphotype 754 being predominant. These results showed that the genotype of C. albicans isolated persisted in the majority of HIV infected individuals, but that the phenotypical expression of this strain changed. A finding in this study was that 18 strains of C. albicans had DNA which did not hybridize to the probe used. These strains were analysed for the presence of two other C. albicans specific DNA segments using PCR. The probe 27A hybridizing strains yielded PCR products which differed from the non-hybridizing strains. Five of these genetically atypical C. albicans strains and 98 of the C. albicans strains were then analysed for purported virulence factors. The genetically atypical C. albicans strains, by comparison with typical C. albicans strains, produced greater amounts of extracellular proteinase (p = 0.038, Student's t test), adhered to a greater degree to buccal epithelial cells (p = 0.018, Student's nest) and were less susceptible to the anti-fungal drug flucytosine (p = 0.0003, Mann-Whitney test). Analysis of these strains with other common antifungal drugs showed no statistically significant variation in susceptibility. The results of this study indicated that these genetically atypical C. albicans strains possess increased virulence by comparison with typical C. albicans strains.     

The isolation,identification and molecular analysis of Candida spp. isolated from the oral cavities of patients with diabetes mellitus

Previous studies have shown a high incidence (77%) of isolation of Candida spp. from the oral cavities of patients with type 1 diabetes mellitus. The aim of the present study was to assess the prevalence of yeast in the oral cavities of patients suffering from type 1 and type 2 diabetes mellitus. The patients were classified according to the level of diabetic control (HbA1c), and further stratified on the presence or absence of dental prosthesis. Oral rinse samples were assessed for the growth of yeast and the degree of colonization. Oral isolates were defined to the species level by both phenotypic and novel molecular methods. The overall proportion (60%) of diabetic patients who had Candida spp. isolated from the oral cavity was similar to that previously reported. Local oral factors, such as the presence of dentures, seemed to have a greater influence than diabetic status on the amount and species of Candida isolated from the oral cavities of diabetic patients. Diabetic patients with dentures had more non-albicans Candida isolated from their mouths than dentate diabetic patients. Candida dubliniensis was isolated from diabetic patients and may have a predilection for dentate patients.     

The development and validation of a rapid genetic method for species identification and genotyping of medically important fungal pathogens using high‐resolution melting curve analysis

Accurate, rapid and economical fungal species identification has been a major aim in mycology. In this study, our goal was to examine the feasibility of a high‐resolution melting curve analysis (HRMA) of internal transcribed regions ITS1 and ITS2 in ribosomal DNA (rDNA) for a rapid, simple and inexpensive differentiation of eight clinically relevant Candida species (Candida albicans, Candida glabrata, Candida parapsilosis, Candida krusei, Candida tropicalis, Candida guilliermondii, Candida dubliniensis and Candida lusitaniae). In addition, for the first time, we tested the applicability of HRMA to classify C. albicans strains into four previously described genotypes (A, B, C and D) using a primer set that spans the transposable intron region of 25S of rDNA. Type and unknown clinical oral isolates were used in this study and the melting curve analysis was compared with both amplicons' sequencing and agarose gel electrophoresis analysis. Real‐time PCR and subsequent HRMA of the two described rDNA regions generated distinct melting curve profiles that were in accord with sequencing and gel electrophoresis analysis, highly reproducible, and characteristic of each of the eight Candida species and C. albicans genotypes. Moreover, results were obtained in 4 h and without the need for any post‐amplification handling, so reducing time and cost. Owing to its simplicity and speed, this technique is a good fit for genotypic analysis of hundreds of clinical strains in large epidemiological settings.