Evaluation of building washing machines as an extreme environment for potentially pathogenic fungi.

Washing machines are commonly used in households and are considered indispensable appliances for maintaining cleanliness and hygiene. Environmental conditions within household washing machines are ideal for fungal colonization, which may pose risks to human health and contribute to sick building syndrome. This study aimed to investigate the fungal species contamination in the building washing machines. A total of 50 building washing machines were swab-sampled at three locations: the detergent drawer, the inner and outer parts of the rubber door seal. The housekeeping conditions of these appliances were assessed through a questionnaire. The isolated fungi were identified using standard mycological diagnostic procedures and molecular analysis based on the ITS1/ITS4 and ß-tubulin gene regions. The possibility of fungal agents transferring from contaminated washing machines to autoclaved clothes during laundry cycles was investigated. Fungi were detected in 82% of the sampled appliances, with the inner rubber door seal being the most frequently colonized area. Using conventional and molecular techniques, we identified 122 fungal isolates, encompassing 17 diverse genera of molds, yeast-like, and yeast fungi. The mold fungi included 14 genera of hyaline and black genus. Among these, the most frequently identified genera of hyaline and black fungi were Aspergillus (27.7%), and Cladosporium (10.7%), respectively. This study demonstrates that building washing machines may serve as suitable ecological niches for fungal growth and transmission. Therefore, regular cleaning and disinfection of these devices are necessary.

Evaluating the Typing Power of Six Isoenzymatic Systems for Differentiation of Clinical and Standard Isolates of Candida Species.

Background: Due to the increasing prevalence of candidiasis, early detection of the causative agents may pave the way for the management of this infection. The present study aimed to assess the discriminative power of the six isoenzymatic systems for differentiating the Candida species. Materials and Methods: Sixteen standard Candida albicans and Candida dubliniensis strains and 30 fluconazole-sensitive and fluconazole-resistant clinical strains of Candida albicans were analyzed using a Multilocus Enzyme Electrophoresis (MLEE) method, including six enzymatic systems consisting of malate dehydrogenase (MDH), phosphoglucomutase (PGM), glucose-phosphate isomerase (GPI), glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGD), and malic enzyme (ME). Results: Among the six enzymatic systems, ME showed no diagnostic activity, whereas MDH provided the best species-specific pattern for species discrimination. In addition, the MDH and G6PD systems provided a discriminatory pattern for differentiating C. dubliniensis from C. albicans isolates. The same isoenzymatic activity was detected in all 36 standard and clinical isolates. Moreover, the results showed no correlation between the isoenzymatic profiles and drug resistance. Conclusion: Among the investigated MLEE systems, MDH was able to differentiate between Candida albicans and Candida dubliniensis. Although no association was detected between isoenzyme patterns and fluconazole resistance in this investigation, isoenzyme patterns are likely correlated with virulence factors between species and even within species. To answer these questions, additional studies should be done on more strains.

Phenotypes characterization and ABC genotypes distribution of clinical Candida albicans isolates.

BACKGROUND: Candidemia and vaginitis are the most common types of candidiasis mostly caused by Candida albicans species. C. albicans has several genotypes and the potential ability to form different phenotype colonies on specific media. This study aimed to evaluate the genotype distribution of blood and vaginal C. albicans isolates and phenotype characteristics on Spider and yeast peptone dextrose agar medium. METHODS: A total of 40 clinical Candida albicans isolates comprising vagina (20) and blood (20) were used. ABC typing using CA-INT-R and CA-INT-L primers was performed to span the transposable group I intron of the 25S rDNA gene. For colony phenotypic characteristics, the Spider and YPDA media were used. RESULTS: Among the blood and vaginal isolates, genotype A (12/60%) and genotype C (10/50%) were the most common types, respectively. The highest phenotype shape frequency of the colonies in blood and vaginal samples was the ring and the lowest was the hat/ring. The dominant color phenotype in blood and vaginal samples was gray. There was a significant relationship between genotype and phenotype forms in the blood sample on YPDA medium (p = 0.02). In the Spider medium, there were no significant differences between genotypes and phenotypes. CONCLUSION: In this study, genotype A and genotype C were predominant in blood and vaginal samples, respectively. In both groups, YPD agar medium demonstrated the most variety of phenotypes that was related to genotypes A and C. The variety of phenotypes in both groups was the same in genotypes A and C on the Spider medium.

Development of a high-resolution melt-based assay to rapidly detect the azole-resistant Candida auris isolates.

Background and Purpose: Candida auris is a multidrug-resistant yeast that rapidly spreads, making it the leading Candidate for the next pandemic. One main leading cause of emerging resistant C. auris isolates is nonsynonymous mutations. This study aimed to detect the Y132F mutation, one of the most important azole resistance-associated mutations in the ERG-11 gene of C. auris, by developing a reliable high-resolution melt (HRM)-based method. Materials and Methods: Five C. auris isolates from Iran, plus three control isolates from other Clades were used in the study. The antifungal susceptibility testing through micro broth dilution was performed to recheck their susceptibility to three azole antifungals, including fluconazole, itraconazole, and voriconazole. Moreover, the polymerase chain reaction (PCR) sequencing of the ERG-11 gene was performed. Following the bioinformatic analysis and HRM-specific primer design, an HRM-based assay was developed and evaluated to detect ERG-11 mutations. Results: The minimum inhibitory concentrations of fluconazole among Iranian C. auris isolates ranged from 8 to 64 µg/mL. The PCR-sequencing of the ERG-11 gene and bioinformatic analyses revealed the mutation of Y132F, a substitution consequence of A to T on codon 395 in one fluconazole-resistant isolate (IFRC4050). The developed HRM assay successfully differentiated the targeted single nucleotide polymorphism between mutant and wild types (temperature [Tm]: 81.79 ℃ - cycle threshold [CT]: 20.06 for suspected isolate). For both mutant and non-mutant isolates, the mean Tm range was 81.79-82.39 °C and the mean CT value was 20.06-22.93. These results were completely in accordance with the findings of DNA sequencing. Conclusion: The fast-track HRM-based method successfully detected one of the most common mechanisms of resistance in the ERG-11 gene of C. auris within 3 h. Finally, the development of more panels of HRM assays for the detection of all azole resistance mutations in C. auris ERG-11 is recommended to expand the scope of the field and facilitate the elaboration of rapid and accurate methods of antifungal resistance assessment.

Vitamin D3: A promising antifungal and antibiofilm agent against Candida species.

Background and Purpose: Candida species are opportunistic fungal pathogens that cause mild to life-threatening infections in both immunocompetent and immunocompromised populations. The increasing prevalence of drug-resistant Candida species has posed a significant challenge to the management of infections in clinical settings. Therefore, this study aimed to investigate the direct antifungal and antibiofilm effect of vitamin D3 against Candida species. Materials and Methods: The antifungal activity of vitamin D3 was evaluated by broth microdilution method based on the Clinical and Laboratory Standard Institute. Prevention of biofilm formation by Candida albicans was measured using the XTT assay following exposure to different concentrations of vitamin D3. Moreover, expression of Agglutinin-like sequence gene 1 (ALS1), hyphal wall protein gene (HWP1), secreted aspartyl proteinase 6 gene (SAP6), and morphogenesis pathway regulatory gene (EFG1) were analyzed by real-time polymerase chain reaction using the comparative Ct method (ΔΔ Ct) after exposure to vitamin D3. Results: Vitamin D3 showed antifungal activity against Candida species ranging from 1-128 µg/mL. Furthermore, vitamin D3 inhibited biofilm formation in a dose-dependent manner, with IC50 of 7.5 µg/mL. Treatment with vitamin D3 resulted in significant upregulation of the EFG1, ALS1, and SAP6 genes under hypha-inducing conditions to overcome environmental challenges. Conclusion: Results of the current study demonstrated that vitamin D3 has a significant inhibitory effect on Candida growth and biofilm formation. Considering its demonstrated antifungal and antibiofilm properties, vitamin D3 holds promise as a potential agent for medical applications.

Accompanying a semi-nested PCR assay to support histopathology findings of fungal keratitis in formalin-fixed paraffin-embedded corneal samples.

BACKGROUND: Fungal species are responsible for 40%-50% of all microbial keratitis cases. Due to the low amount of extracted DNA in ocular Formalin-fixed Paraffin-embedded (FFPE) samples, selecting a reliable molecular method is a substantial issue in this field. METHODS: Sixty-six samples were collected via the penetrating keratoplasty (PK) technique. Histopathology assays were performed using hematoxylin-eosin (H&E) and periodic acid Schiff (PAS) staining methods. The ITS1/ITS4 and ITS1/ITS2 primer pairs were used in a semi-nested polymerase chain reaction (PCR) to target the universal internal transcribed spacer (ITS) region. Some PCR results were validated through sequencing. RESULTS: Fungal DNA was detected in 44 of 66 samples (66.7%), and histopathology was positive for 41 of 66 samples (62.1%). Of 41 histopathologically proven fungal-positive cases, 39 were PCR-positive (95%). Moreover, of 44 PCR-positive samples, 39 (88.6%) were histopathology-positive, and 5 (11.3%) were histopathology-negative. Totally in 39 cases (59%), both histopathology and PCR yielded positive results. The Kappa agreement rate between the two diagnostic methods, including histopathology and PCR, was 0.77. Sensitivity, specificity, positive predictive value, and false predictive value were reported as 88.64%, 90.9%, 95.12%, and 80%, respectively. CONCLUSION: As we reached the acceptable Kappa agreement rate, we concluded that applying the semi-nested PCR assay is a promising method for supporting the evidence by histopathology. Finally, we suggest targeting more specific gene regions using primer pairs that amplify smaller amplicon sizes and surveying novel molecular methods such as NGS to achieve higher sensitivity and Kappa agreement rates.

Magnetic chitosan nanoparticles loaded with Amphotericin B: Synthesis, properties and potentiation of antifungal activity against common human pathogenic fungal strains.

Amphotericin B has long been regarded as the gold standard for treating invasive fungal infections despite its toxic potential. The main objective of this research was to develop a novel IONPs@CS-AmB formulation in a cost-effective manner in order to enhance AmB delivery performance, with lowering the drug's dose and adverse effects. The chitosan-coated iron oxide nanoparticles (IONPs@CS) were synthesized afterward, AmB-loaded IONPs@CS (IONPs@CS-AmB) prepared and characterized by AFM, FT-IR, SEM, EDX, and XRD. Biological activity of the synthesized NPs determined and the cytotoxicity of IONPs@CS-AmB evaluated using the MTT and in vitro hemolysis tests. The IONPs@CS-AmB was synthesized using the coprecipitation method with core-shell structure in size range of 27.70 to ∼70 nm. The FT-IR, XRD and EDX pattern confirmed the successful synthesis of IONPs @CS-AmB. The IONPs@CS-AmB exhibited significant antifungal activity and inhibited the metabolic activity of Candida albicans biofilms. The hemolysis and MTT assays showed that IONPs@CS-AmB is biocompatible with high cell viability when compared to plain AmB and fungizone. The IONPs@CS-AmB is more effective, less toxic and may be a suitable alternative to conventional drug delivery. IONPs@CS-AmB may be a viable candidate for use as a microbial-resistant coating on the surfaces of biomedical devices.

COVID-19-associated facial cutaneous mucormycosis superinfection: A potentially life-threatening disease.

A 49-year-old male was involved in an accident and an abdominal computer tomographic examination revealed papillary renal cell carcinoma of the right kidney. During hospitalization, the patient was infected with COVID-19. In the following COVID-19 treatment, a black dot developed on the right side of the head and face. Antifungal therapy and surgical debridement were initiated and gradual improvement was observed.

Molecular characterization and antifungal activity against non-dermatophyte molds causing onychomycosis.

Onychomycosis is a fungal disease that caused by different types of fungi. Non-dermatophyte molds are a large saprophytic fungi group that live in nature and could affect traumatic nails. The aim of this study was to identify non-dermatophyte molds causing onychomycosis and evaluation of several antifungal activities against the isolates. The samples consisted of 50 non-dermatophyte molds isolated from patients with onychomycosis confirmed by direct and culture examination fungal. DNA was extracted, amplified, and sequenced. Disk diffusion method was used to evaluate itraconazole, fluconazole, ketoconazole, terbinafine, posaconazole, and econazole activity against the isolates. The species identified as: Aspergillus flavus 22 (44%), A. niger 12 (24%), A. fumigates, 3 (6%), A. sydowii 3 (6%), A. terreus 1 (2%), Penicillium commune 2 (4%), P. glabrum 2 (4%), P. chrysogenum, 1 (2%), Fusarium solani 3 (6%) and F. thapsinum 1 (2%). Most of the samples were sensitive to terbinafine, itraconazole, and econazole and 94% of the isolates were resistant to fluconazole. This study showed that Aspergillus species were the most common cause of non-dermatophyte mold onychomycosis and fluconazole was the most resistant antifungals. Care must be taken to choose the appropriate antifungal drug for a better cure.

Evaluation of fungal contamination and ochratoxin A detection in different types of coffee by HPLC-based method.

BACKGROUND: Mycotoxins are secondary fungal metabolites that are produced by some toxigenic fungi on foodstuffs which are poisoning and potentiate for human's health hazards. In coffee samples, ochratoxin A and fungal contamination were examined. METHODS: Immunoaffinity columns were used for treating of all 50 samples from four types of coffee, after that high-performance liquid chromatography was used for determining the amount of ochratoxin. For the identification of fungi, all coffee samples were cultured in appropriated media. RESULTS: The results showed that all samples were contaminated by ochratoxin A but only up to 50% of them had toxins higher than acceptable level as detected in black beans (47%), green beans (33.3%), torch (33.3%), and espresso (25%). Black coffee had a higher mean concentration of ochratoxin A than green coffee. CONCLUSION: Predominant fungi isolated from coffee samples were Aspergillus species. Finally, careful monitoring of mycotoxins in coffee samples is essential to improve the quality of this favorable beverage in future.

Potential Pathogenicity of Candida Species Isolated from Oral Cavity of Patients with Diabetes Mellitus.

INTRODUCTION: In the recent decade, the increased immunocompromised population such as diabetic patients makes a high incidence of invasive Candida infections. Diabetes mellitus is the most common endocrine metabolic disorder, and diabetic patients are more susceptible to oral candidiasis infection. Candidiasis is an opportunistic fungal infection caused by many species of Candida. Secretion of exoenzymes plays an important role in the virulence and pathogenesis of Candida species. The aim of this study was to evaluate the potential role of phospholipase, esterase, and hemolytic activity of Candida species isolated from oral cavity lesions of diabetic patients. METHODS: A total of 108 Candida species including 75 Candida albicans and 33 non-Candida albicans species were recovered from the oral cavity of diabetic patients included in our study. Egg yolk agar, Tween 80 opacity medium, and blood agar plate assays were used for determining phospholipase, esterase, and hemolytic activities, respectively. RESULTS: Candida albicans species had the most exoenzyme activity in comparison to non-albicans isolates. Candida albicans isolates showed 97.3%, 100%, and 77.3% phospholipase, hemolysin, and esterase activities, respectively. The difference between Candida albicans and non-Candida albicans was significant in phospholipase (P < 0.001) and hemolytic activity (P = 0.027), but not significant in esterase activity (P = 0.076). CONCLUSION: This study showed that most of the isolates had different enzymatic patterns, and Candida albicans isolates had the most exoenzyme activity. So due to the potential effects of these enzymes in pathogenesis and virulence effects of Candida species, we can conclude that the severity of extracellular enzymes may play a role in the severity of signs and symptoms of Candida oral cavity infections in diabetic patients.

Prevalence of superficial-cutaneous fungal infections in Shiraz, Iran: A five-year retrospective study (2015-2019).

BACKGROUND: Superficial and cutaneous fungal infections are common in tropical areas. The aim of this study was to provide a basic database of superficial and cutaneous mycoses and the most common etiological agents among patients. METHODS: Between 2015 and 2019, a total of 1807 patients suspected of superficial and cutaneous mycosis referring to the mycology laboratory of Shiraz medical school, Fars, Iran were evaluated. Specimens were taken from the patients' affected area, and clinical samples were examined by direct microscopy and culture. The epidemiological profile of the patients was collected. RESULTS: A total of 750 patients were confirmed with mycoses. Positive samples totaled 750 cases consisting of the nail (373/49.7%), skin (323/43%), head (47/6.26%), and mucosal membrane (4/0.5%). The yeasts group included 304 Candida spp. (70.3%), 123 Malassezia spp. (28.47%), and 5 Rhodotorula spp. (1.1%). The filamentous fungi were distributed as 34.8% dermatophytes and 7.5% non-dermatophyte. The clinical types of dermatophytosis were tinea unguium (110/261), tinea capitis (50/261), tinea pedis (48/261), tinea corporis (37/261), and tinea cruris (16/261). Non-dermatophyte molds included A. flavus 17, A. niger 4, Aspergillus spp. 15, Penicillium. 10, Fusarium 6, Mucor 2, Stemphylium 1, and Alternaria 1. CONCLUSION: This study provides useful data for the study trends of superficial and cutaneous fungal infections in a specific area. The mycological data confirmed higher incidence of candidiasis (mainly onychomycosis) and dermatophytosis in patients affected by fungal pathogens, which helped to better understand the epidemiological aspects of these mycoses.

Survey of aflatoxins and ochratoxin A contamination in spices by HPLC-based method in Shiraz, Southern of Iran.

Among food and agricultural products, spices play important roles in the diets of millions of people worldwide. These products may be colonized by fungi genus and subsequently mycotoxin production. Due to the large demand and supply of spice for cooking, preservative effects, or medicine purpose, it is essential that further investigation is designed to examine mycotoxins in spice. In the present study, the possible contamination of spices by aflatoxins (AFTs) and ochratoxin A (OTA) were analyzed. A total of 80 spice samples (curry, sumac, ginger, and saffron) were purchased and cultured on appropriate medium. Simultaneously mycotoxins from spices were extracted with immunoaffinity columns (IAC), and the occurrence of AFTs (B1 + B2 + G1 + G2) and OTA was then determined using high-performance liquid chromatography (HPLC) with a fluorescence detector (FD). The results depicted that 62 (77.5%) and 58 (72.5%) spice samples were contaminated with AFTs and OTA, respectively. Out of the 80 analyzed spices samples, the mean concentration of AFTs and OTA was higher in the curry samples. Among spices that contaminated with mycotoxins, 5 (6.25%) and 2 (10%) of the samples were above the acceptable limit of AFTs (≥ 10 µg/kg) and OTA (≥ 15 µg/kg), respectively. Aspergillus species were the predominant species isolated, followed by Penicillium, and finally Mucor species.Among the examined samples, only few curry samples were contaminated with mycotoxins above acceptable limit. Despite this low level of contamination, this spice is used daily in the cuisine of this region of the world, and consequently, even the small amount of these heat stable toxins for a long time may cause many adverse effects. Hence, it is recommended to monitor the toxicogenous fungi contamination and level of mycotoxins in the spices.

Molecular identification of Malassezia species isolated from neonates hospitalized in Neonatal intensive care units and their mothers.

Background and Purpose: Given the important role of Malassezia spp. in skin diseases and other associated infections in neonates, this study aimed to investigate the presence and frequency of Malassezia spp. in the skin of neonates hospitalized in neonatal intensive care units and their mothers using culture and accurate molecular-based methods. Materials and Methods: In total, 205 samples were collected from 130 neonates (>4-day-old) and 75 mothers. Isolation of Malassezia spp. from the skin was performed using Leeming-Notman agar and modified Dixon agar media. To compare the Malassezia microflora on the skin of the neonates and their mothers, a polymerase chain reaction-sequencing method was performed for spp. identification of 92 isolates obtained from neonates and their mothers. Moreover, possible associated risk factors for the colonization of Malassezia spp. on the skin were recorded. Results: Cultures from 62.3% of neonates and 77.3% of mothers were positive for Malassezia spp. growth. Malassezia globosa was the most prevalent isolated spp. found in the skin of the study population. It is noteworthy that a rare Malassezia spp., Malassezia arunalokei, was isolated from the skin of one neonate. There was a 76% similarity between the mother-neonate isolate sequences results. The statistical analysis showed that the type of feeding is a significant (P<0.001) associated factor for Malassezia skin colonization. Conclusion: The findings support the hypothesis that the colonization of Malassezia in neonates is significantly influenced by that of the mother, and this may be associated with breastfeeding.

Chemical composition and antifungal activities of aromatic water of Zataria multiflora Boiss.

Background and Purpose: In Iranian culture, aromatic waters harboring a slight amount of essential oil have been popularly used for many years as a pleasant non-alcoholic drink with various medicinal properties.In this study, chemical composition of Zataria multiflora Boiss. (ZM) aromatic water was determined and its in vitro and in vivo antifungal properties were investigated. Materials and Methods: Chemical composition of the essential oil extracted from aromatic water (AW) of ZM was analyzed by Gas chromatography and gas chromatography-mass spectrometry (GC-MS). The antimicrobial activity of the AW against Candida species was determined by broth micro-dilution methods. Additionally, biofilm formation inhibition and antioxidant activity of the AW were measured using XTT reduction and DPPH methods, respectively. Antifungal activities of the AW in the prevention and/or treatment of gastrointestinal (GI) candidiasis in animal models were also evaluated. Results: The GC-MS analysis revealed that the major constituents of ZM AW were Carvacrol (46.56%) and Thymol (40.67%). The ZM AW inhibited the growth and biofilm formation of Candida species in the range of 0.25-0.5 V/V. Moreover, ZM AW significantly decreased Candida colonization in therapeutic groups (P<0.05). Conclusion: Given the wide therapeutic potential of ZM AW, including antifungal and antioxidant activities, it might be possible to use it in the management of mucocutaneous or alimentary candidiasis.

Epidemiology of candidemia in Shiraz, southern Iran: A prospective multicenter study (2016-2018).

Systematic candidemia studies, especially in southern Iran, are scarce. In the current prospective study, we investigated candidemia in three major healthcare centers of Shiraz, the largest city in southern Iran. Yeast isolates from blood and other sterile body fluids were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and subjected to antifungal susceptibility testing (AFST) using the broth microdilution method. Clinical data were retrieved from patients' medical records. In total, 113 yeast isolates were recovered from 109 patients, over 60% of whom received fluconazole. Antifungal drugs were prescribed without considering species identification or AFST. The all-cause mortality rate was 28%. Almost 30% of the patients were from intensive care units (ICUs). Candida albicans (56/113; 49.5%) was the most prevalent species followed by C. glabrata (26/113; 23%), C. parapsilosis (13/113; 11.5%), C. tropicalis (7/113; 6.2%), and C. dubliniensis (5/113; 4.4%). Only five isolates showed antifungal resistance or decreased susceptibility to fluconazole: one C. orthopsilosis isolate from an azole-naïve patient and two C. glabrata, one C. albicans, and one C. dubliniensis isolates from patients treated with azoles, who developed therapeutic failure against azoles later. Our results revealed a low level of antifungal resistance but a notable rate of azole therapeutic failure among patients with candidemia due to non-albicans Candida species, which threaten the efficacy of fluconazole, the most widely used antifungal in southern regions of Iran. Candidemia studies should not be confined to ICUs and treatment should be administered based on species identification and AFST results.

Landscape of candidemia is blurred in Iran, and only two studies from Tehran have extensively explored the epidemiology of candidemia. However, candidemia data from the other regions are notoriously scarce, which precludes from reaching a consensus regarding species distribution, the burden of antifungal resistance, and the clinical features of infected patients. Therefore, we conducted the current prospective candidemia study in Shiraz, one of the largest cities located in the south of Iran, from April 2016 to April 2018. More than 63% of the candidemia infections were treated by fluconazole and species identification and antifungal susceptibility testing were not used for decision making regarding the choice of antifungal treatment. Approximately 70% of the candidemia cases occurred in the wards outside of the ICUs. Candida albicans, C. glabrata, C. parapsilosis, C. tropicalis, and C. dubliniensis were the five leading causative agents of candidemia. Antifungal resistance was rare and fluconazole resistance and/or non-wild type phenotypes were noticed in five isolates, only one was C. albicans and the rest were non-albicans Candida (NAC) species, including C. glabrata, C. dubliniensis, and C. orthopsilosis. Except for C. orthopsilosis, which was isolated from an azole-naïve patient, the rest of isolates were recovered from patients treated with azoles and all showed therapeutic failure to azoles. Collectively, our data will complete the candidemia picture in Iran and show that, although the level of resistance was rare, the therapeutic failure was notable among NAC species, which threatens the efficacy of fluconazole, the most widely used antifungal in Southern regions of Iran. Moreover, we showed that candidemia is poorly managed in Iran since species identification tools along with antifungal susceptibility testing were not used to select appropriate antifungal treatment.

High detection of virulence factors by Candida species isolated from bloodstream of patients with candidemia.

OBJECTIVE: Candida species are the normal inhabitants of the skin and mucosa that cause a wide range of debilitating diseases in immunocompromised patients and other susceptible individuals. The present study aimed to evaluate the production of exoenzymes and the biofilm formation capacity of Candida species isolated from candidemia. MATERIALS AND METHODS: In this study, a total of 100 stock Candida species isolates consist of 50 Candida albicans and 50 non-Candida albicans Candida species (24 C. glabrata, 15 C. parapsilosis, 5 C. dubliniensis, 3 C. tropicalis, 2 C. krusei and 1 C. fabianii) which previously were recovered from patients with candidemia were used. The enzymatic activity tests for hemolysin, proteinase, and phospholipase were performed by using blood Sabouraud dextrose agar, bovine serum albumin medium and egg yolk agar, respectively. Biofilm formation was determined by microplate assay method. RESULT: All of the Candida albicans species could produce hemolysin. The predominant enzyme activity of species included strong and very strong levels of phospholipase, proteinase and hemolysin activity were belonged to Candida albicans isolates. There were statistically significant differences in hemolysin (P < 0.001), proteinase (P = 0.003) and phospholipase (P < 0.001) activity between two groups of albicans and non-albicans species. The biofilm formation was seen in 30 (60%) of C. albicans and 49 (98%) of non-C. albicans species. There was significant statistical differences between the two groups of isolates in biofilm formation (P < 0.001). CONCLUSION: It is clear that Candida species have ability to produce several enzymes as virulence factors to contribute its pathogenicity. There were significant differences in virulence factors between the two C. albicans and non- C. albicans group. The ability for biofilm formation and producing exo-enzyme were an important virulence factors in Candida species isolates. This differences found in this report might have role in severity of disease caused by different species.

Comparative Analysis of Virulence Factors of Homozygous and Heterozygous Strains of Candida albicans Vaginal Isolates.

Although the epidemiology of pathogenic Candida species is changing due to invasive diseases, Candida albicans has become the common cause of human infections worldwide. Candida albicans is a diploid yeast with a mostly clonal mode of reproduction and without known complete sexual cycle. This species has two heterozygous and homozygous strains at hyphal wall protein 1 gene locus (hwp1). Little is known about virulence factors of these strains. The aim of this study was to evaluate the exoenzyme activity of heterozygous and homozygous C. albicans strains. A total of 60 stock Candida albicans species isolates, which consisted of 30 homozygous and 30 heterozygous strains, were used for exoenzyme activities. We used egg yolk agar, Sabouraud blood agar, and bovine serum albumin agar for evaluation of phospholipase, hemolysin, and proteinase activity, respectively. Homozygous strains of Candida albicans had more phospholipase and proteinase activity than heterozygous strains. However, there were no significant statistical differences between the two strains in the severity of exoenzymes production. Beta hemolysin activity was seen in 100% and 96.7% of the homozygous and heterozygous strains, respectively. The results of this study indicated that both of the strains exhibited exoenzyme activities in different ranges. There were no significant statistical differences in virulence factors between the homozygous and heterozygous strains.

Translation elongation factor 1-alpha gene as a marker for diagnosing of candidal onychomycosis.

BACKGROUND AND PURPOSE: Culture-based identification methods have been the gold standard for the diagnosis of candidal onychomycosis. Molecular technologies, such as polymerase chain reaction (PCR) assays, can provide an alternative for the rapid detection of Candida species. The present study was conducted to investigate a pan-Candida PCR assay based on the translation elongation factor 1-alpha (TEF-1α) gene for the detection of the most prevalent pathogenic Candida species. MATERIALS AND METHODS: For the purpose of the study, an optimized pan-Candida PCR primer pair was designed, and the target was amplified and sequenced. The analytical and clinical diagnostic performance of the designed primers was tested using 17 reference strains, 137 nail scrapings suspected of onychomycosis, and 10 healthy nail specimens. RESULTS: The use of the universal pan-Candida primers designed on TEF-1α gene resulted in the successful amplification of a 270-base pair fragment in all Candida species tested, except for C. glabrata, and reacted neither with other fungi nor with E. coli. The sequence difference count matrix showed poor insertion/deletion differences (0-2 nt) among Candida species. Among 137 nail specimens, 35% (n=48), 30.7% (n=42), and 40.1% (n=55) of the samples were found to be positive by direct microscopy, culture, and pan-Candida PCR, respectively. CONCLUSION: Based on the findings, the PCR-based detection targeting the DNA TEF-1α gene is a rapid and simple procedure for the diagnosis of candidal onychomycosis directly from nail sample.

Emergence of Terbinafine Resistant Trichophyton mentagrophytes in Iran, Harboring Mutations in the Squalene Epoxidase (SQLE) Gene.

INTRODUCTION: Trichophyton mentagrophytes and T. interdigitale are important causative agents of superficial mycoses, demonstrating emergent antifungal drug resistance. We studied the antifungal susceptibility profiles in Iranian isolates of these two species. METHODS: A total of 96 T. interdigitale and 45 T. mentagrophytes isolates were subjected to molecular typing by ribosomal ITS region. Antifungal susceptibility profiles for terbinafine, griseofulvin, clotrimazole, efinaconazole, luliconazole, amorolfine and ciclopirox were obtained by CLSI broth microdilution method. The squalene epoxidase (SQLE) gene was subjected to sequencing for mutations, if any, in isolates exhibiting elevated MICs for terbinafine. RESULTS: Luliconazole and efinaconazole showed the lowest MIC values against T. mentagrophytes and T. interdigitale isolates. There were five isolates with terbinafine MICs ≥32 µg/mL in our sample. They belonged to T. mentagrophytes type VIII and harbored two alternative SQLE gene sequence variants, leading to Phe397Leu and Ala448Thr or Leu393Ser and Ala448Thr substitutions in the enzyme. All terbinafine resistant strains could be inhibited by luliconazole and efinaconazole. CONCLUSION: This study documented a step in the global spread of resistance mechanisms in T. mentagrophytes. However, treatment alternatives for resistant isolates were available.