Important Candida Species Research Articles

Synthesis, molecular modeling studies and evaluation of antifungal activity of a novel series of thiazole derivatives

In the search for new antifungal agents, a novel series of fifteen hydrazine-thiazole derivatives was synthesized and assayed in vitro against six clinically important Candida and Cryptococcus species and Paracoccidioides brasiliensis. Eight compounds showed promising antifungal activity with minimum inhibitory concentration (MIC) values ranging from 0.45 to 31.2 μM, some of them being equally or more active than the drug fluconazole and amphotericin B. Active compounds were additionally tested for toxicity against human embryonic kidney (HEK-293) cells and none of them exhibited significant cytotoxicity, indicating high selectivity. Molecular modeling studies results corroborated experimental SAR results, suggesting their use in the design of new antifungal agents.

Clinical characteristics of the first cases of invasive candidiasis in China due to pan-echinocandin-resistant Candida tropicalis and Candida glabrata isolates with delineation of their resistance mechanisms.

Echinocandin antifungal agents have become the first-line therapy for invasive candidiasis (IC) in many countries. Despite their increasing use, resistance to this class of drug is, overall, still uncommon. Here, we report two patients from the People’s Republic of China with IC, one with infection caused by pan-echinocandin-resistant Candida tropicalis and the other by pan-echinocandin-resistant Candida glabrata. We also describe the mechanisms of drug resistance of these isolates. The echinocandin-resistant C. glabrata isolate was cultured from ascitic fluid of a 46-year-old male patient with intra-abdominal IC developing after surgery in 2012. This patient had had no prior antifungal exposure. The echinocandin-resistant C. tropicalis isolate was cultured from chest drainage fluid of a 60-year-old female patient with severe coronary disease and lung infection. Prior to culture and identification of the isolate, the patient had received micafungin treatment for 19 days. Both isolates were cross-resistant to micafungin, anidulafungin, and caspofungin, with minimum inhibitory concentration values of ≥2 µg/mL. The amino acid substitution E655K was found adjacent to the FKS2 HS1 region of the C. glabrata isolate, while the substitution S80P were found in the FKS1 HS1 region of the C. tropicalis isolate. This report highlights the emergence of echinocandin resistance in two important non-albicans Candida species. Although the overall prevalence of echinocandin resistance is low in the People’s Republic of China, monitoring of antifungal susceptibility trends in all Candida species is warranted.

An Update on Candida tropicalis Based on Basic and Clinical Approaches.

Candida tropicalis has emerged as one of the most important Candida species. It has been widely considered the second most virulent Candida species, only preceded by C. albicans. Besides, this species has been recognized as a very strong biofilm producer, surpassing C. albicans in most of the studies. In addition, it produces a wide range of other virulence factors, including: adhesion to buccal epithelial and endothelial cells; the secretion of lytic enzymes, such as proteinases, phospholipases, and hemolysins, bud-to-hyphae transition (also called morphogenesis) and the phenomenon called phenotypic switching. This is a species very closely related to C. albicans and has been easily identified with both phenotypic and molecular methods. In addition, no cryptic sibling species were yet described in the literature, what is contradictory to some other medically important Candida species. C. tropicalis is a clinically relevant species and may be the second or third etiological agent of candidemia, specifically in Latin American countries and Asia. Antifungal resistance to the azoles, polyenes, and echinocandins has already been described. Apart from all these characteristics, C. tropicalis has been considered an osmotolerant microorganism and this ability to survive to high salt concentration may be important for fungal persistence in saline environments. This physiological characteristic makes this species suitable for use in biotechnology processes. Here we describe an update of C. tropicalis, focusing on all these previously mentioned subjects.

Identification of Medically Important Candida Species by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Analysis of the rDNA ITS1 and ITS2 Regions

Aim: We aimed to identify the distribution of species in candidal strains isolated from clinical samples and restriction fragment length polymorphism (RFLP) method based on Msp I and Bln I restrictive enzyme cuts of polymerase chain reaction (PCR) products after the amplification of ITS1 and ITS2 regions of rDNA genotypically. Materials and Methods: One hundred and fifty candidal strains isolated from various clinical samples were studies/included. Phenotypic species assessment was performed using automated VITEK-2 system and kit used with the biochemical tests. Common genomic region amplification peculiar to candidal strains was carried out using ITS1 and ITS2 primer pairs. After the amplification, PCR products were cut with Msp I and Bln I restriction enzymes for species identification. Results: The majority of Candida isolates were isolated from urine (78.6%) while other isolates were composed of strains isolated from swab, wound, blood and other samples by 11.3%, 3.3%, 2% and 4.7%, respectively. The result of RFLP analysis carried out with Msp I and Bln I restriction enzymes showed that candidal strains were Candida albicans by 45.3%, Candida glabrata by 19.3%, Candida tropicalis by 14.6%, Candida parapsilosis by 5.3%, Candida krusei by 5.3%, Candida lusitaniae by 0.6% and other candidal strains by 9.3%. Conclusion: When the ability to identify Candida to species level of phenotypic and PCR-RFLP methods was assessed, a great difference was found between these two methods. It may be argued that Msp I and Bln I restriction enzyme fragments can be used in the identification of medically important Candida species. Further studies are needed to develop this kind of restriction profile to be used in the identification of candidal strains.

Evaluation of CHROMagar Candida in the rapid identification of medically important species of Candida

Evaluation of CHROMagar Candida in the rapid identification of medically important species of Candida

Unexpected effects of azole transporter inhibitors on antifungal susceptibility in Candida glabrata and other pathogenic Candida species.

The pathogenic fungus Candida glabrata is often resistant to azole antifungal agents. Drug efflux through azole transporters, such as Cdr1 and Cdr2, is a key mechanism of azole resistance and these genes are under the control of the transcription factor Pdr1. Recently, the monoamine oxidase A (MAO-A) inhibitor clorgyline was shown to inhibit the azole efflux pumps, leading to increased azole susceptibility in C. glabrata. In the present study, we have evaluated the effects of clorgyline on susceptibility of C. glabrata to not only azoles, but also to micafungin and amphotericin B, using wild-type and several mutant strains. The addition of clorgyline to the culture media increased fluconazole susceptibility of a C. glabrata wild-type strain, whereas micafungin and amphotericin B susceptibilities were markedly decreased. These phenomena were also observed in other medically important Candida species, including Candida albicans, Candida parapsilosis, Candida tropicalis, and Candida krusei. Expression levels of CDR1, CDR2 and PDR1 mRNAs and an amount of Cdr1 protein in the C. glabrata wild-type strain were highly increased in response to the treatment with clorgyline. However, loss of Cdr1, Cdr2, Pdr1, and a putative clorgyline target (Fms1), which is an ortholog of human MAO-A, or overexpression of CDR1 did not affect the decreased susceptibility to micafungin and amphotericin B in the presence of clorgyline. The presence of other azole efflux pump inhibitors including milbemycin A4 oxime and carbonyl cyanide 3-chlorophenylhydrazone also decreased micafungin susceptibility in C. glabrata wild-type, Δcdr1, Δcdr2, and Δpdr1 strains. These findings suggest that azole efflux pump inhibitors increase azole susceptibility but concurrently induce decreased susceptibility to other classes of antifungals independent of azole transporter functions.

Prevalence of Candida albicans, Candida dubliniensis and Candida africana in pregnant women suffering from vulvovaginal candidiasis in Argentina

BackgroundVulvovaginal candidiasis (VVC) is a vulvovaginitis commonly diagnosed in gynecology care. In recent years, the taxonomy of the most important pathogenic Candida species, such as Candida albicans have undergone significant changes. AimsThis study examined the prevalence of C. albicans, Candida africana, and Candida dubliniensis in vaginal specimens from 210 pregnant women suffering from vulvovaginitis or having asymptomatic colonization. MethodsPhenotypic and molecular methods were used for the identification of the species. ResultsDuring the studied period, 55 isolates of Candida or other yeasts were obtained from specimens collected from 52 patients suffering from vulvovaginitis (24.8%). C. albicans was the predominant Candida species in 42 isolates (80.7%), either alone or in combination with other species of the genus (5.7%, n=3). Additionally, nine isolates of C. albicans (50%) were obtained from asymptomatic patients (n=18). C. dubliniensis was the causative agent in 2 (3.8%) cases of VVC, and was also isolated in one asymptomatic patient. Molecular assays were carried out using specific PCR to amplify the ACT1-associated intron sequence of C. dubliniensis. The amplification of the HWP1 gene also correctly identified isolates of the species C. albicans and C. dubliniensis. No C. africana was isolated in this work. Some C. albicans isolates were either homozygous or heterozygous at the HWP1 locus. The distribution of heterozygous and homozygous C. albicans isolates at the HWP1 locus was very similar among patients suffering from VVC and asymptomatic patients (p=0.897). ConclusionsThe presence of C. albicans and C. dubliniensis, and the absence of C. africana in pregnant is noteworthy.

A new assay based on terminal restriction fragment length polymorphism of homocitrate synthase gene fragments for Candida species identification

Candida sp. have been responsible for an increasing number of infections, especially in patients with immunodeficiency. Species-specific differentiation of Candida sp. is difficult in routine diagnosis. This identification can have a highly significant association in therapy and prophylaxis. This work has shown a new application of the terminal restriction fragment length polymorphism (t-RFLP) method in the molecular identification of six species of Candida, which are the most common causes of fungal infections. Specific for fungi homocitrate synthase gene was chosen as a molecular target for amplification. The use of three restriction enzymes, DraI, RsaI, and BglII, for amplicon digestion can generate species-specific fluorescence labeled DNA fragment profiles, which can be used to determine the diagnostic algorithm. The designed method can be a cost-efficient high-throughput molecular technique for the identification of six clinically important Candida species.

Antifungal effects of Lavandula binaludensis and Cuminum cyminum essential oils against Candida albicans strains isolated from patients with recurrent vulvovaginal candidiasis

Recurrent vulvovaginal candidiasis (RVVC), which affects approximately 5% of women of reproductive age, is defined as 4 or more episodes of symptomatic Candida vaginitis within a year. The purposes of this study were to determine the chemical compositions and antifungal susceptibility of Cuminum cyminum (C.cyminum) and Lavandula binaludensis (L.binaludensis) essential oils and their combination against Candida albicans (C.albicans) strains isolated from patients with RVVC. C.albicans isolates were identified via germ tube test, CHROMagar and RapID Yeast Plus System. The essential oils were obtained by hydrodistillation in a Clevenger apparatus and analyzed by gas chromatography-mass spectroscopy (GC-MS). The broth microdilution method was used as antifungal susceptibility test (CLSI-M27-A3). The GC-MS analysis allowed 13 components to be determined; the main components of C.cyminum and L.binaludensis essential oils were γ-terpinene (21.07%) and 1,8-cineole (71.56%), respectively. L.binaludensis and C.cyminum oils were effective in inhibiting C.albicans growth at mean concentrations of 7.91±1.61μg/mL and 8.00±1.89μg/mL, respectively. In addition, the combination of C.cyminum with L.binaludensis oils were more active causing inhibition in all C.albicans isolates, with concentrations varying from 3.90 to 11.71μg/mL (mean value: 7.22±1.69μg/mL). The results suggested the potential substitution of the antifungal chemicals by C.cyminum and L.binaludensis essential oils as natural inhibitors to control the growth of the most important pathogenic Candida species and alternative therapies for RVVC.

A high yield DNA extraction method for medically important Candida species: A comparison of manual versus QIAcube-based automated system

The prognosis of infected individuals with candidemia depends on rapid and precise diagnosis which enables optimising treatment. Three fungal DNA extraction protocols have been compared in this study for medically important Candida species. The quality and quantity of the DNA extracted by physical, chemical and automated protocols was compared using NanoDrop ND-2000 spectrophotometer. It was found that the yield and purity (260/230) ratio of extracted DNA was significantly high in the physical treatment-based protocol as compared to chemical based or automated protocol. Extracted DNA-based real time-polymerase chain reaction showed an analytical sensitivity of 103 cfu/mL. The result of this study suggests physical treatment is the most successful extraction technique compared to other two protocols.

Synthesis and evaluation of thiophene-based guanylhydrazones (iminoguanidines) efficient against panel of voriconazole-resistant fungal isolates

A series of new thiophene-based guanylhydrazones (iminoguanidines) were synthesized in high yields using a straightforward two-step procedure. The antifungal activity of compounds was evaluated against a wide range of medicaly important fungal strains including yeasts, molds, and dermatophytes in comparison to clinically used drug voriconazole. Cytotoxic properties of compounds were also determined using human lung fibroblast cell line and hemolysis assay. All guanylhydrazones showed significant activity against broad spectrum of clinically important species of Candida spp., Aspergillus fumigatus, Fusarium oxysporum, Microsporum canis and Trichophyton mentagrophytes, which was in some cases comparable or better than activity of voriconazole. More importantly, compounds 10, 11, 13, 14, 18 and 21 exhibited excellent activity against voriconazole-resistant Candida albicans CA5 with very low minimal inhibitory concentration (MIC) values <2μgmL−1. Derivative 14, bearing bromine on the phenyl ring, was the most effective compound with MICs ranging from 0.25 to 6.25μgmL−1. However, bis-guanylhydrazone 18 showed better selectivity in terms of therapeutic index values. In vivo embryotoxicity on zebrafish (Danio rerio) showed improved toxicity profile of 11, 14 and 18 in comparison to that of voriconazole. Most guanylhydrazones also inhibited C. albicans yeast to hyphal transition, essential for its biofilm formation, while 11 and 18 were able to disperse preformed Candida biofilms. All guanylhydrazones showed the equal potential to interact with genomic DNA of C. albicans in vitro, thus indicating a possible mechanism of their action, as well as possible mechanism of observed cytotoxic effects. Tested compounds did not have significant hemolytic effect and caused low liposome leakage, which excluded the cell membrane as a primary target. On the basis of computational docking experiments using both human and cytochrome P450 from Candida it was concluded that the most active guanylhydrazones had minimal structural prerequisites to interact with the cytochrome P450 14α-demethylase (CYP51). Promising guanylhydrazone derivatives also showed satisfactory pharmacokinetic profile based on molecular calculations.

Development of Candida-Specific Real-Time PCR Assays for the Detection and Identification of Eight Medically Important Candida Species.

Culture-based identification methods have been the gold standard for the diagnosis of fungal infection. Currently, molecular technologies such as real-time PCR assays with short turnaround time can provide desirable alternatives for the rapid detection of Candida microbes. However, most of the published PCR primer sets are not Candida specific and likely to amplify DNA from common environmental contaminants, such as Aspergillus microbes. In this study, we designed pan-Candida primer sets based on the ribosomal DNA-coding regions conserved within Candida but distinct from those of Aspergillus and Penicillium. We demonstrate that the final two selected pan-Candida primer sets would not amplify Aspergillus DNA and could be used to differentiate eight medically important Candida pathogens in real-time PCR assays based on their melting profiles, with a sensitivity of detection as low as 10 fg of Candida genomic DNA. Moreover, we further evaluated and selected species-specific primer sets covering Candida albicans, Candida glabrata, Candida tropicalis, and Candida dubliniensis and show that they had high sensitivity and specificity. These real-time PCR primer sets could potentially be assembled into a single PCR array for the rapid detection of Candida species in various clinical settings, such as corneal transplantation.

Characterization and Biofilm Detection among Clinically Important Candida Species

Background: Candida is the fourth most common cause of blood stream infection in hospitalized patients. Candidiasis is associated with intravenous lines and prosthetic lines are problematic, as they can act as substrates for biofilm production, the propensity of forming this is more in immuno suppressed patients. Materials and methods: A total of 20 clinical isolates of Candida spp were obtained from different clinical conditions of different immuno compromised patients and were processed for its characterization followed by assessing the biofilm production. Results: Of 20 isolates, 40% were C. albicans, 35% were C. tropicalis, 15% were C. krusei and each of 5% were C. kefyr and C. parapsilosis respectively. 5% of isolates were strong biofilm formers whereas, 65% were moderate biofilm producers in our Candida isolates. Conclusion: The present study reveals there was an increase in degree of isolation of non-albicans Candida spp from immuno compromised individuals. These isolates were moderately producing biofilm. Hence, prompt identification and detection of biofilm are essential in clinical setup where more number of Candida have been encountered.

Active immunizations with peptide-DC vaccines and passive transfer with antibodies protect neutropenic mice against disseminated candidiasis

We previously report that peptide-pulsed dendritic cell (DC) vaccination, which targeting two peptides (Fba and Met6) expressed on the cell surface of Candida albicans, can induce high degree of protection against disseminated candidiasis in immunocompetent mice. Passive transfer of immune sera from the peptide immunized mice or peptide-related monoclonal antibodies demonstrated that protection was medicated by peptide-specific antibodies. In this study the efficacy of active and passive immunization against disseminated candidiasis was tested in mice with cyclophosphamide-induced neutropenia. Peptide-DC vaccines were given to mice prior to induction of neutropenia. We show active immunization with either Fba or Met6 peptide-DC vaccine significantly improved the survival and reduced the fungal burden of disseminated candidiasis in those immunocompromised mice. Importantly, we show that administration of two protective monoclonal antibodies also protect neutropenic mice against the disease, implying possibility of developing a successful passive immunotherapy strategy to treat the disease and protect against disseminated candidiasis. The results of this study are crucial as they address the fundamental questions as to whether the synthetic peptide vaccine induced immunity protects the host during a neutropenic episode. We anticipate that this peptide-vaccine study will serve as the foundation of future investigations into new peptide vaccines comprised of cell surface peptides from other medically important Candida species, as well as other fungi.

Antimicrobial action of sodium hypochlorite and castor oil solutions for denture cleaning – in vitro evaluation

The objective of this in vitro study was to evaluate the antimicrobial action of sodium hypochlorite (0.25% and 0.50%) and 10% castor oil solutions against specific microorganisms, by counting Colony Forming Units (CFU) of clinically important bacteria and Candida species. Acrylic resin specimens (n = 320; Lucitone 550) were obtained from square metal matrices (10 x 10 x 2 mm), sterilized by microwave (650W, for 6 minutes) and contaminated by Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, Bacillus subtilis, Escherichia coli, Streptococcus mutans, Enterococcus faecalis and Candida glabrata. The specimens were immersed for 20 minutes in one of the following hygiene solutions (n = 10/each): A - 0.25% Sodium hypochlorite; B - 0.5% Sodium hypochlorite; C - 10% Castor oil solution; and D (Control) - saline. Adhered cells were suspended and inoculated into a selective solid medium (37ºC for 24 h). The Student's t-test (α = 0.05) was performed to compare log10(CFU+1)/mL between Groups C and D. The results showed that sodium hypochlorite (0.25% and 0.5%) completely eliminated all detectable microorganisms. The castor oil solution eliminated B. subtilis and reduced counts for other strains. Differences between C and D were significant (p < 0.05) for all species except for E. faecalis. Both sodium hypochlorite solutions (0.25% and 0.5%) were effective in eliminating all microorganisms evaluated, and may be useful as cleaning solutions for complete dentures. The castor oil solution provided moderate efficacy and performed differently on the tested species, with the strongest effect on B. subtilis and with non-significant action on E. faecalis.

Research Highlights

Treating and diagnosing opportunistic infections in immunosuppressed patients remains a very important aspect for all clinicians working in the field of transplantation. Candidiasis is the most common fungal infection in hospitalized patients, and for this reason, the correct diagnosis of some of the important Candida albicans or non-albicans Candida species is essential, as invasive candidiasis caused by non-albicans is associated with higher resistance rates.1,2 Mylonakis and coworkers estimated mortality rates in patients who received antifungal therapy immediately after blood cultures to be 24% while a delayed treatment starting on days 2 and 3 increased mortality rates to 37% and 41%, respectively. Candida species are traditionally diagnosed by using microscopy, histopathology, culture, plating samples on chromogenic culture media as well as identifying enzyme, metabolite, structural components of the cell wall (1,3-β-D-glucan), antibody (antimannan) or antigen (mannan). PCR testing comes with shortcomings as well and has not yet been accepted as standard of care. Blood cultures and 1,3-β-D-glucan assays have not been specific for Candida leading to potential false-positive results. Rapid Identification of Medically Important Candida Isolates Using High Resolution Melting Analysis Nemcova E, Cernochova M, Ruzicka F, Malisova B, Freiberger T, Nemec P. PloS ONE. 2015;10(2):e0116940 In this report, the authors describe a combination of real-time polymerase chain reaction (PCR) and high-resolution melting analysis (HRMA) for the rapid detection of 25 medically important Candida species. Real-time PCR followed by HRMA showed reproducible melting peaks for 21 of 25 Candida species. Misidentification occurred with C. orthopsilosis, C. metapsilosis, and C. fabianii. Clinically, the correct diagnosis of C. parapsilosis is significant as this strain is less susceptible to antifungals. Notably all C. parapsilosis were clearly distinguishable from other strains using this method. This is the widest range of Candida species ever tested and identified using the HRMA method including a large number of 143 clinical isolates covering 23 species for validation. Real-time PCR followed by HRMA appears as an efficient and simple method diagnosing most candida species. Of note, the diagnosis can be made within 6 hours based on HRMA compared to 24 to 48 hours with the biochemical methods. T2 magnetic resonance assay for the rapid diagnosis of candidemia in whole blood: a clinical trial. Mylonakis E, Clancy CJ, Ostrosky-Zeichner L, et al. Clin Infect Dis. 2015;60(6):892–899 A nanodiagnostic method using a manual application of T2 magnetic resonance (T2MR) was used in this study to diagnose 5 Candida species: C. albicans, C. tropicalis, C. Krusei, C. glabrata and C. parapsilosis. A total of 250 blood culture samples were manually supplemented with clinically relevant titres of the above Candida species and 50 samples were used as negative controls. These samples formed the contrived or control arm of the study, and they were randomly inserted into the 2264 clinical specimens to check how different titres of Candida would react to the methodology. A total of 463 samples were excluded for technical errors or invalid results. The results of the primary analysis showed an overall specificity of 99.4% per assay and 98.1% per patient. Importantly, the mean time to obtain a negative result was only 4.2 hours compared to longer than 120 hours for a blood culture (BCT). There were 31 discordant cases between T2MR and blood culture: 2 had positive BCT and negative T2MR and 29 negative BCT but positive T2MR. This can be related to either false-positive T2MR or false negative BCT. The BCT methods are known to have poor sensitivity, and clinical samples are shown to be positive in only 38% of proven invasive candidiasis.3 Therefore, it is very likely that these cases represent Candida species infections that were missed by BCT. Importantly, this new technology has a high sensitivity in identifying non-albicans Candida like C. glabrata for which this test has an overall sensitivity of 84%. This study represents the first extensive clinical trial using T2MR technology. REFERENCES Wisplinghoff H, Bischoff T, Tallent SM, et al. Nosocomial bloodstream infections in US hospitals: analysis of 24,179 cases from a prospective nationwide surveillance study. Clin Infect Dis. 2004;39(3):309–317. Chi HW, Yang YS, Shang ST, et al. Candida albicans versus non-albicans bloodstream infections: the comparison of risk factors and outcome. J Microbiol Immunol Infect. 2011;44(5):369–375. Avni T, Leibovici L, Paul M. PCR diagnosis of invasive candidiasis: systematic review and meta-analysis. J ClinMicrobiol. 2011;49(2):665–670.

Comparative Study of Esterase and Hemolytic Activities in Clinically Important Candida Species, Isolated From Oral Cavity of Diabetic and Non-diabetic Individuals.

Background:Diabetes mellitus as a chronic metabolic disease occurs in patients with partial or complete deficiency of insulin secretion or disorder in action of insulin on tissue. The disease is known to provide conditions for overgrowth of Candida species. Candida spp. cause candidiasis by many virulence factors such as esterase, hemolysin and phospholipase.Objectives:This study aimed to compare esterase and hemolytic activity in various Candida species isolated from oral cavity of diabetic and non-diabetic individuals.Patients and Methods:Swab samples were taken from 95 patients with diabetes (35 men and 60 women) and 95 normal persons (42 men and 53 women) and cultured on Sabouraud dextrose agar. Identification of isolated yeasts was performed by germ tube test, morphology on CHROMagar Candida medium, corn meal agar and ability to grow at 45°C. Hemolysin activity was evaluated using blood plate assay and esterase activity was determined using the Tween 80 opacity test.Results:Different Candida species were isolated from 57 (60%) diabetic and 24 (25%) non-diabetic individuals. Esterase activity was detected in all Candida isolates. Only 21.6% of C. albicans from patients with diabetes had esterase activity as + 3, while it ranged from + 1 to + 2 in others. Hemolytic activity was determined in C. albicans, C. dubliniensis, C. glabrata and C. krusei as 0.79, 0.58, 0.66 and 0.74, respectively. Hemolytic activity was significantly different in the two groups of diabetics and non-diabetics.Conclusions:Oral carriage of C. albicans in the diabetic group (n = 42; 66.7%) was significantly greater than the control group (n = 16; 57.1%). Esterase activity of C. albicans in diabetic group was higher than non-diabetic group. Although C. albicans remains the most frequently pathogenic yeast for human, but other species are increasing.

Rapid identification of medically important Candida isolates using high resolution melting analysis.

An increasing trend in non albicans infections and various susceptibility patterns to antifungal agents implies a requirement for the quick and reliable identification of a number of medically important Candida species. Real-time PCR followed by high resolution melting analysis (HRMA) was developed, tested on 25 reference Candida collection strains and validated on an additional 143 clinical isolates in this study. All reference strains and clinical isolates inconclusive when using phenotypic methods and/or HRMA were analysed using ITS2 sequencing. Considering reference and clinical strains together, 23 out of 27 Candida species could be clearly distinguished by HRMA, while the remaining 4 species were grouped in 2 pairs, when applying the mean Tm ± 3 SD values, the shape of the derivative melting curve (dMelt curve) and, in some cases, the normalized and temperature—shifted difference plot against C. krusei. HRMA as a simple, rapid and inexpensive tool was shown to be useful in identifying a wide spectrum of clinically important Candida species. It may complement the current clinical diagnostic approach based on commercially available biochemical kits.

Genetic diversity of medically important and emerging Candida species causing invasive infection.

BackgroundGenetic variation in the ribosomal DNA (rDNA) internal transcribed spacer (ITS) region has been studied among fungi. However, the numbers of ITS sequence polymorphisms in the various Candida species and their associations with sources of invasive fungal infections remain poorly investigated. Here, we characterized the intraspecific and interspecific ITS diversity of Candida spp. strains collected from patients with bloodstream or oroesophageal candidiasis.MethodsWe selected cultures of representative medically important species of Candida as well as some rare and emerging pathogens. Identification was performed by micromorphology and by biochemical testing using an ID32C® system, as well as by the sequencing of rDNA ITS. The presence of intraspecific ITS polymorphisms was characterized based on haplotype networks, and interspecific diversity was characterized based on Bayesian phylogenetic analysis.ResultsAmong 300 Candida strains, we identified 76 C. albicans, 14 C. dubliniensis, 40 C. tropicalis, 47 C. glabrata, 34 C. parapsilosis (sensu stricto), 31 C. orthopsilosis, 3 C. metapsilosis, 21 Meyerozyma guilliermondii (C. guilliermondii), 12 Pichia kudriavzevii (C. krusei), 6 Clavispora lusitaniae (C. lusitaniae), 3 C. intermedia, 6 Wickerhamomyces anomalus (C. pelliculosa), and 2 C. haemulonii strains, and 1 C. duobushaemulonii, 1 Kluyveromyces marxianus (C. kefyr), 1 Meyerozyma caribbica (C. fermentati), 1 Pichia norvegensis (C. norvegensis), and 1 Lodderomyces elongisporus strain. Out of a total of seven isolates with inconsistent ID32C® profiles, ITS sequencing identified one C. lusitaniae strain, three C. intermedia strains, two C. haemulonii strains and one C. duobushaemulonii strain. Analysis of ITS variability revealed a greater number of haplotypes among C. albicans, C. tropicalis, C. glabrata and C. lusitaniae, which are predominantly related to endogenous sources of acquisition. Bayesian analysis confirmed the major phylogenetic relationships among the isolates and the molecular identification of the different Candida spp.ConclusionsMolecular studies based on ITS sequencing are necessary to identify closely related and emerging species. Polymorphism analysis of the ITS rDNA region demonstrated its utility as a genetic marker for species identification and phylogenetic relationships as well as for drawing inferences concerning the natural history of hematogenous infections caused by medically important and emerging Candida species.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-015-0793-3) contains supplementary material, which is available to authorized users.

Detection of medically important Candida species by absolute quantitation real-time polymerase chain reaction.

Background:The number of invasive candidiasis cases has risen especially with an increase in the number of immunosuppressed and immunocom promised patients. The early detection of Candida species which is specific and sensitive is important in determining the correct administration of antifungal drugs to patients.Objectives:This study aims to develop a method for the detection, identification and quantitation of medically important Candida species through quantitative polymerase chain reaction (qPCR).Materials and Methods:The isocitrate lyase (ICL) gene which is not found in mammals was chosen as the target gene of real-time PCR. Absolute quantitation of the gene copy number was achieved by constructing the plasmid containing the ICL gene which is used to generate standard curve. Twenty fungal species, two bacterial species and human DNA were tested to check the specificity of the detection method.Results:All eight Candida species were successfully detected, identified and quantitated based on the ICL gene. A seven-log range of the gene copy number and a minimum detection limit of 103 copies were achieved.Conclusions:A one-tube absolute quantification real-time PCR that differentiates medically important Candida species via individual unique melting temperature was achieved. Analytical sensitivity and specificity were not compromised.