Species distribution and antifungal susceptibility profiles of yeasts isolated from onychomycosis: a cross-sectional study with insights into emerging species.

Candida onychomycosis is a common fungal infection affecting the nails, primarily caused by Candida (C.) species. Regarding the increasing trend of Candida onychomycosis and the antifungal resistant phenomenon in recent years, this study aims to evaluate the epidemiological characteristics of Candida onychomycosis, the distribution of emerging species, and the antifungal susceptibility profiles of isolates. Onychomycosis caused by yeast species was confirmed through direct examination and culture of nail scraping among all individuals suspected to have onychomycosis and referred to a medical mycology laboratory between June 2019 and March 2022. Species of yeast isolates were identified using the multiplex PCR and PCR-RFLP methods. The antifungal susceptibility of isolates to common antifungal agents and imidazole drugs was evaluated according to the M-27-A3 CLSI protocol. Among 101 yeast strains isolated from onychomycosis, Candida parapsilosis complex (50.49%) was the most common species, followed by C. albicans (20.79%) and C. tropicalis (10.89%). Rare species of yeasts such as C. guilliermondii and Saccharomyces cerevisiae were also identified by molecular methods. Results obtained from antifungal susceptibility testing showed significant differences in MIC values of isoconazole, fenticonazole, and sertaconazole among different species. Overall, a fluconazole-resistant rate of 3% was found among Candida species. Moreover, there was a statistically significant difference in MICs of fenticonazole and clotrimazole between the two most prevalent causative species, C. parapsilosis complex and C. albicans. Correct identification of the causative agents of onychomycosis and performing susceptibility testing could be helpful in choosing the most appropriate antifungal therapy.

Quantitative analysis of in vitro biofilm formation by clinical isolates of dermatophyte and antibiofilm activity of common antifungal drugs.

BACKGROUND: The ability of dermatophytes to develop biofilm, as one of the virulence factors in fungal infections which contribute to antifungal resistance, is an outstanding aspect of dermatophytosis that has been noted recently. Because of the paucity of data about the biofilm formation by dermatophytes and their susceptibility to antifungal drugs, this study evaluated the biofilm formation by clinical isolates of dermatophytes and antibiofilm activity of common antifungals widely used to manage dermatophytosis. METHODS: The ribosomal DNA internal transcribed spacer (ITS) regions sequencing for species identification of 50 clinical dermatophyte isolates was performed. The ability of isolates to form biofilm and inhibitory activity of itraconazole, terbinafine, and griseofulvin against biofilm formation was assayed by the crystal violet staining method. Optical microscopy and scanning electron microscopy (SEM) were applied for the visualization of the biofilm structures. RESULTS: Trichophyton (T.) mentagrophytes (n: 14; 28%) and T. rubrum (n: 13;26%) were included in more than half of the dermatophyte isolates. Biofilm formation was observed in 37 out of 50 (74%) isolates that were classified as follows: nonproducers (n: 13; 26%), weak producers (n: 4; 8%), moderate producers (n: 16; 32%), and strong producers (n: 17; 34%) by comparison of the absorbance of biofilms produced by clinical strains with control. The mean IC50 values for terbinafine, griseofulvin, and itraconazole were 2.42, 3.18, and 3.78 µg/ml, respectively. CONCLUSIONS: The results demonstrated that most of the clinical dermatophyte isolates are capable to form biofilm in vitro with variable strength. Moreover, terbinafine can be suggested as the first-line choice for the treatment of biofilm-formed dermatophytosis.

Time and cost-efficient identification of Trichophyton indotineae.

BACKGROUND: During the past 5 years, an outbreak of recalcitrant dermatophytosis due to a novel Trichophyton species generally resistant to terbinafine, T. indotineae, has spread out from South Asia to many countries around the World. These isolates cannot be reliably differentiated from other Trichophyton spp. on the basis of morphological traits and the current laboratory diagnostics relies on sequencing of ribosomal DNA ITS region. OBJECTIVES: In this study, we aimed to introduce two inexpensive and rapid PCR-based assays for differentiation between T. indotineae and other dermatophytes. METHODS: The first introduced assay is based on PCR-restriction fragment length polymorphism (PCR-RFLP) analysis, involving the amplification of TOP2 sequences and the digestion of PCR products by Cfr13I restriction enzyme. The second assay is proposed as conventional endpoint species-specific PCR amplification of the C120-287 intergenic locus. To validate the assays, a total of 191 Trichophyton spp. and 2 Microsporum canis strains with known ITS region sequences were used. From the T. mentagrophytes / T. interdigitale species complex (TMTISC), strains with 18 different ITS genotypes were tested. The sample of TMTISC isolates included 41 T. indotineae strains. RESULTS: TOP2 PCR-RFLP and T. indotineae-specific PCR were positive with testing on DNA of all 41 T. indotineae isolates and two strains of T. mentagrophytes belonging to ITS Types XIII and XVI, but negative with other species and other TMTISC ITS genotypes (n = 152). Therefore, the specificity of both new assays was 99%. CONCLUSION: The two developed diagnostic assays provide accurate and cost-effective means of identifying cultured T. indotineae isolates.

Iranian National Survey on Tinea Capitis: Antifungal Susceptibility Profile, Epidemiological Characteristics, and Report of Two Strains with a Novel Mutation in SQLE Gene with Homology Modeling.

BACKGROUND: The data on the epidemiological and antifungal susceptibility profile of tinea capitis (TC) in Iran has not been updated in recent decades. This report presents the Iranian epidemiological and drug susceptibility data regarding the distribution of dermatophytes species isolated by six national mycology centers for a period of one year (2020-2021). MATERIAL AND METHODS: A total of 2100 clinical samples from individuals suspeted to TC were subjected to mycological analysis of direct microscopy and culture. For definite species identification, the culture isolates were additionally subjected to PCR-RFLP and PCR-sequencing of the ITS ribosomal DNA (ITS-rDNA) region. Antifungal susceptibility profiles for eight common antifungal drugs were determined by CLSI M38-A3 guidelines. The SQLE gene was partially amplified and sequenced in two terbinafine-resistant and two susceptible T. mentagrophytes isolates to elucidate probable substitutions involved in resistance. RESULTS: TC (n = 94) was diagnosed in 75 children (79.8%) and 19 adults (20.2%) by direct microscopy and culture. Frequency of TC was significantly more among males (66 males = 70.2% vs 28 females = 29.8%). The prevalent age group affected was 5-9 years (39.36%). Thirty-two (34.04%) T. mentagrophytes, 27 (28.7%) T. tonsurans, 14 (14.9%) M. canis, 13 (13.8%) T. violaceum, 5 (5.32%) T. indotineae, 2 (2.1%) T. benhamiae, and 1 (1.1%) T. schoenleinii were identified as the causative agents. MIC values of isolates showed susceptibility to all antifungal agents, except for fluconazole and griseofulvin with GM MIC of 11.91 µg/ml and 2.01 µg/ml, respectively. Terbinafine exhibited more activity against isolates, with GM MIC 0.084 µg/ml followed by ketoconazole (0.100 µg/ml), econazole (0.107 µg/ml), itraconazole (0.133 µg/ml), butenafine (0.142 µg/ml), and miconazole (0.325 µg/ml). Two resistant T. mentagrophytes isolates harbored missense mutations in SQLE gene, corresponding to amino acid substitution F397L. Remarkably, one unique mutation, C1255T, in the SQLE sequence of two terbinafine-susceptible T. mentagrophytes strains leading to a change of leucine at the 419th position to phenylalanine (L419F) was detected. CONCLUSIONS: T. mentagrophytes, T. tonsurans, and M. canis remained the main agents of TC in Iran, however less known species such as T. indotinea and T. benhamiae are emerging as new ones. Terbinafine could still be the appropriate choice for the treatment of diverse forms of TC.

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.

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.

Characterization of beta-tubulin DNA sequences within Candida parapsilosis complex.

BACKGROUND AND PURPOSE: Candida parapsilosis is a common cause of candidemia in children and patients with onco-hematological diseases, septic arthritis, peritonitis, vaginitis, and nail and skin infections. Regarding this, the present study was condcuted to evaluate intra- and inter-species variation within beta-tubulin DNA sequence of C. parapsilosis complex in order to establish the utilization of this gene in the identification and phylogenetic analysis of the species. MATERIALS AND METHODS: A total of 23 isolates representing three different species of C. parapsilosis complex were used in this study, all of which were identifed by ITS-sequencing. For the successful amplification of beta-tubulin gene, a newly designed set of pan-Candida primers was used, followed by bilaterally sequence analysis for pairwise comparisons, determination of multiple alignments, evaluation of sequence identity levels, counting sequence difference, and construction of phylogenetic tree. RESULTS: The multiple alignment of 623-629 bp-long nucleotide (nt) sequences reflecting the beta-tubulin gene indicated an inter-species divergence ranging within 0-68 nt in C. parapsilosis, C. orthopsilosis, and C. metapsilosis with a mean similarity of 84.7% among the species. Meanwhile, the intra-species differences of 0-20 and 0-6 nt were found between the strains of C. parapsilosis and C. orthopsilosis, respectively. The phylogenetic tree topology was characterized by a clade made up by C. parapsilosis and C. orthopsilosis, while C. metapsilosis formed a related but separate lineage. CONCLUSION: Our data provided the basis for further discoveries of the relationship between the species belonging to C. parapsilosis complex. Furthermore, the findigns of the prsent study revealed the efficiency of beta-tubulin DNA sequence data in the identification and taxonomy of C. parapsilosis and other pathogenic yeasts.

Clinical evaluation of ß-tubulin real-time PCR for rapid diagnosis of dermatophytosis, a comparison with mycological methods.

Following our previous report on evaluation of the beta tubulin real-time PCR for detection of dermatophytosis, this study aimed to compare the real-time PCR assay with conventional methods for the clinical assessment of its diagnostic performance. Samples from a total of 853 patients with suspected dermatophyte lesions were subjected to direct examination (all samples), culture (499 samples) and real-time PCR (all samples). Fungal DNA was extracted directly from clinical samples using a conical steel bullet, followed by purification with a commercial kit and subjected to the Taq-Man probe-based real-time PCR. The study showed that among the 499 specimens for which all three methods were used, 156 (31.2%), 128 (25.6%) and 205 (41.0%) were found to be positive by direct microscopy, culture and real-time PCR respectively. Real-time PCR significantly increased the detection rate of dermatophytes compared with microscopy (288 vs 229) with 87% concordance between the two methods. The sensitivity, specificity, positive predictive value, and negative predictive value of the real-time PCR was 87.5%, 85%, 66.5% and 95.2% respectively. Although real-time PCR performed better on skin than on nail samples, it should not yet fully replace conventional diagnosis.

Enumeration and identification of dust fungal elements from the weather inversion phenomenon in Isfahan, Iran.

BACKGROUND: Fungi are the major pathogens or allergens for which the air is the natural medium of their dispersal. Since the air pollution is associated with a wide range of adverse health outcomes, then identification of the type and population of fungi in these conditions will help the management of hygienic and control of fungal disease. MATERIALS AND METHODS: A total of 103 dust samples were collected from glass surfaces of different places by sedimentation method. Pollution standard indexes were provided by Environmental Protection Agency in Isfahan. All dust samples were mixed and homogenized in distilled water containing antibacterial agents. Serial cultures were done in 5 times experiments on two standard culture media. Isolated fungal colonies were identified by their standard morphologic and physiologic criteria. The analysis was performed by Mann-Whitney test calculating by SPSS version 20 (SPSS Inc., Chicago, IL, USA). RESULTS: The real mean of total culture-able fungi in 1 g of sedimentation dust were account about 44800 colonies of different fungi. More than half of the viable fungi (62.8%) could grow out of 1 g of dust on Mycosel agar were the genera of Aspergillus, Penicillium and Cladosporium with 28.8%, 23.4% and 10.6% respectively. The dominant genus could grow on Sabouraud dextrose agar with chloramphenicol medium were the genera of Aspergillus, Cladosporium and Penicillium with 23.7%, 21.1% and 14.5% respectively. CONCLUSIONS: Our data show the amount and variety of viable colony-forming fungi, which we are faced with in Isfahan during the air pollution condition. The real abundance of fungal particles and non-cultivable fungi in dust are still poorly understood and remain for further study in the future.

Analysis of beta-hemolysis in human blood agars by Streptococcus pyogenes.

The aim of the study was to assess the reliability of human blood agar media (HuBA) in identifying Streptococcus pyogenes by hemolysis analysis. We analyze several factors that might affect the accuracy of HuBA media for microbial analysis, including incubation time, blood group, Rh factor and presence of antistreptolysin-o.