Aspergillus hubkae, a Novel Species Isolated from a Patient with Probable Invasive Pulmonary Aspergillosis.

A 50-year-old man, previously diagnosed with pulmonary tuberculosis and lung cavities, presented with symptoms including fever, shortness of breath, and cough. A pulmonary CT scan revealed multiple cavities, consolidation and tree-in-bud in the upper lungs. Further investigation through direct examination of bronchoalveolar lavage fluid showed septate hyphae with dichotomous acute branching. Subsequent isolation and morphological analysis identified the fungus as belonging to Aspergillus section Nigri. The patient was diagnosed with probable invasive pulmonary aspergillosis and successfully treated with a three-month oral voriconazole therapy. Phylogenetic analysis based on partial ß-tubulin, calmodulin and RNA polymerase second largest subunit sequences revealed that the isolate represents a putative new species related to Aspergillus brasiliensis, and is named Aspergillus hubkae here. Antifungal susceptibility testing demonstrated that the isolate is resistant to itraconazole but susceptible to voriconazole. This phenotypic and genetic characterization of A. hubkae, along with the associated case report, will serve as a valuable resource for future diagnoses of infections caused by this species. It will also contribute to more precise and effective patient management strategies in similar clinical scenarios.

Molecular typing of a collection of Iranian clinical Trichophyton tonsurans isolates based on the non-transcribed spacer region of rDNA and antifungal susceptibility testing of the species.

INTRODUCTION: Wrestling, considered the national sport of Iran, has gained immense popularity among Iranians. Wrestlers frequently encounter skin conditions, with dermatophyte fungal infections, particularly tinea gladiatorum (TG), being a common issue. TG, caused by the Trichophyton genus, has emerged as a major health concern for wrestlers and other contact sport athletes worldwide. This study aimed to assess the genotypic diversity and antifungal susceptibility of Trichophyton tonsurans isolates responsible for TG in Iranian wrestlers from Mazandaran province, northern Iran. MATERIALS AND METHODS: A total of 60 clinical T. tonsurans isolates collected from various cities in Mazandaran, were included in the study. The isolates were identified through PCR-restriction fragment length polymorphism and sequencing methods. Genomic DNA was extracted from these isolates, and the non-transcribed spacer (NTS) region of ribosomal RNA (rRNA) was targeted for genotyping using newly designed primers. Haplotype analysis was performed to explore genetic diversity, and antifungal susceptibility to terbinafine (TRB) and itraconazole (ITC) was assessed. RESULTS: The results revealed five distinct NTS types: NTS-I, NTS-II, NTS-III, NTS-IV and NTS-V, with NTS-IV being the most prevalent. The distribution of NTS types varied across different cities, suggesting potential transmission patterns among wrestlers. Antifungal susceptibility testing showed that all isolates were susceptible to TRB, while one isolate demonstrated resistance to ITC. Genotypic diversity was not correlated with antifungal susceptibility, emphasising the importance of monitoring susceptibility to ensure effective treatment. Haplotype analysis highlighted significant genetic diversity among the T. tonsurans isolates. This diversity may be attributed to factors such as human-to-human transmission, geographic location and lifestyle changes. The study's findings underscore the need for comprehensive genotypic analysis to understand the epidemiology and evolution of T. tonsurans infections in athletes. CONCLUSION: In conclusion, this study provides valuable insights into the genotypic diversity and antifungal susceptibility of T. tonsurans isolates causing TG in Iranian wrestlers. The presence of multiple NTS types and varying susceptibility patterns highlights the complexity of T. tonsurans infections in this population. Further research is warranted to track the transmission routes and genetic evolution of T. tonsurans strains among wrestlers and develop effective control measures.

The growth inhibitory and apoptotic effects of umbelliprenin in a mouse model of systemic candidiasis.

AIMS: Umbelliprenin has shown promising biological activities, including immunoregulatory, anti-inflammatory, and anti-cancer effects. The present study investigated the growth inhibitory and apoptotic effects of umbelliprenin against Candida albicans in a BALB/c mice model of disseminated candidiasis. METHODS AND RESULTS: First, an antimicrobial assay via microdilution sensitivity test was performed. Then, twenty-five 6-week-old female BALB/c mice (20 ± 12 g) were divided into five groups of five mice, including one control group (no umbelliprenin treatment) and four experimental groups: C. albicans-infected mice treated with umbelliprenin at the doses of 5, 10, 20, and 40 mg kg -1. The brain, lung, kidney, spleen, and liver tissues were examined for fungal infection and histological lesions, and TUNEL staining was performed to assess apoptosis. The ß-1, 3-glucan synthase assay was used to evaluate enzymatic activity, and gene expression analysis was also performed to investigate the transcriptional changes of ERG11, CDR1, ALS1, and HWP1 genes. The MIC of umbelliprenin was 1.5 mg mL-1. Our results showed that at the 40 mg kg -1 dose, umbelliprenin was able to eradicate fungal infection in BALB/c mice. The percentage of apoptotic cells in umbelliprenin-treated groups increased in a concentration-dependent manner. Umbelliprenin (40 mg kg -1) also inhibited the expression of ß-1, 3-glucan synthase, and the genes involved in antifungal resistance (CDR1 and ERG11), as well as the expression of the genes encoding adhesins (ALS1 and HWP1). CONCLUSION: Our results showed that umbelliprenin could promote antifungal effects, partly via inducing apoptosis.

Sheep serve as a reservoir of Trichophyton mentagrophytes genotype V infection.

Human infections by Trichophyton mentagrophytes occur mainly due to contact with diseased animals. In Iran, T. mentagrophytes genotype V is the most prevalent variant of the fungus. We aimed to determine the animal reservoir of T. mentagrophytes genotype V infection. The study was done on a total of 577 dermatophyte strains obtained from animals with signs of dermatophytosis and human patients. The list of extensively sampled animals included sheep, cows, cats and dogs. For human cases, epidemiological data were collected. All dermatophyte isolates from animals along with 70 human isolates morphologically similar to T. verrucosum and T. mentagrophytes genotype V were identified by rDNA internal transcribed spacer region restriction fragment length polymorphism analysis and DNA sequencing. A total of 334 animal dermatophyte strains were identified as Microsporum canis, T. mentagrophytes genotype V, T. verrucosum, Nannizzia gypsea, T. mentagrophytes genotype II*, T. mentagrophytes genotype VII, T. quinckeanum, and N. fulva. All clinical isolates identified as T. mentagrophytes genotype V originated from skin and scalp infections. Almost all veterinary isolates of T. mentagrophytes genotype V were cultured from sheep, but epidemiological data on animal-to-human transmission of T. mentagrophytes genotype V infection were limited and we found evidence in favor of interhuman transmission. In Iran, sheep maintain T. mentagrophytes genotype V population and therefore serve as animal reservoir of respective infections. The role of sheep as the source of human dermatophytosis due to T. mentagrophytes genotype V isolates is yet to be proven.

In this study, we sampled a variety of animals to determine a reservoir of Trichophyton mentagrophytes genotype V infection. With the use of molecular identification techniques, we show that this infection reservoir is represented by sheep.

Potential Inhibitory Effect of Miltefosine against Terbinafine-Resistant Trichophyton indotineae.

Several prolonged and significant outbreaks of dermatophytosis caused by Trichophyton indotineae, a new emerging terbinafine-resistant species, have been ongoing in India in recent years, and have since spread to various countries outside Asia. Miltefosine, an alkylphosphocholine, is the most recently approved drug for the treatment of both visceral and cutaneous leishmaniasis. Miltefosine in vitro activity against terbinafine-resistant and susceptible T. mentagrophytes/T. interdigitale species complex, including T. indotineae, is limited. The current study aimed to assess miltefosine's in vitro activity against dermatophyte isolates, which are the most common causes of dermatophytosis. Miltefosine, terbinafine, butenafine, tolnaftate, and itraconazole susceptibility testing was performed using Clinical and Laboratory Standards Institute broth microdilution methods (CLSI M38-A3) against 40 terbinafine-resistant T. indotineae isolates and 40 terbinafine-susceptible T. mentagrophytes/T. interdigitale species complex isolates. Miltefosine had MIC ranges of 0.063-0.5 µg/mL and 0.125-0.25 µg/mL against both terbinafine-resistant and susceptible isolates. In terbinafine-resistant isolates, the MIC50 and MIC90 were 0.125 µg/mL and 0.25 µg/mL, respectively, and 0.25 µg/mL in susceptible isolates. Miltefosine had statistically significant differences in MIC results when compared to other antifungal agents (p-value 0.05) in terbinafine-resistant strains. Accordingly, the findings suggest that miltefosine has a potential activity for treating infections caused by terbinafine-resistant T. indotineae. However, further studies are needed to determine how well this in vitro activity translates into in vivo efficacy.

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.

Trichophyton indotineae-An Emerging Pathogen Causing Recalcitrant Dermatophytoses in India and Worldwide-A Multidimensional Perspective.

Trichophyton (T.) indotineae is a newly identified dermatophyte species that has been found in a near-epidemic form on the Indian subcontinent. There is evidence of its spread from the Indian subcontinent to a number of countries worldwide. The fungus is identical to genotype VIII within the T. mentagrophytes/T. interdigitale species complex, which was described in 2019 by sequencing the Internal Transcribed Spacer (ITS) region of ribosomal DNA of the dermatophyte. More than 10 ITS genotypes of T. interdigitale and T. mentagrophytes can now be identified. T. indotineae causes inflammatory and itchy, often widespread, dermatophytosis affecting the groins, gluteal region, trunk, and face. Patients of all ages and genders are affected. The new species has largely displaced other previously prevalent dermatophytes on the Indian subcontinent. T. indotineae has become a problematic dermatophyte due to its predominantly in vitro genetic resistance to terbinafine owing to point mutations of the squalene epoxidase gene. It also displays in vivo resistance to terbinafine. The most efficacious drug currently available for this terbinafine-resistant dermatophytoses, based on sound evidence, is itraconazole.

In vitro activities of 8 antifungal agents against geophilic dermatophyte isolates.

BACKGROUND: Members of the Nannizzia gypsea complex are globally the most common geophilic dermatophytes which cause infection in animals and human. Although the susceptibility patterns of anthropophilic or zoophilic dermatophyte species to antifungal agents are well documented, the effectiveness of such drugs against geophilic species have rarely been explored. OBJECTIVES: This study was aimed to evaluate the in vitro antifungal activity of common and new antifungals against a set of environmental and clinical geophilic dermatophyte isolates. METHODS: 108 soil and clinical geophilic isolates from two genera Nannizzia (N. fulva n = 59; N. gypsea n = 43) and Arthroderma (A. quadrifidum n = 4; A. gertleri n = 1; A. tuberculatum n = 1) were included in the study. The in vitro antifungal susceptibility patterns of eight common and new antifungals against the isolates were determined according to broth microdilution method and by CLSI M38-A3 (3rd edition) protocol. RESULTS: MIC values across all isolates from five species ranged as: luliconazole: 0.0002-0.002 µg/ml, terbinafine: 0.008-0.125 µg/ml, efinaconazole: 0.008-0.125 µg/ml, ciclopirox olamine: 0.03-0.5 µg/ml, itraconazole: 0.125-1 µg/ml, amorolfine hydrochloride: 0.125-4 µg/ml, griseofulvin: 0.25-2 µg/ml and tavaborole: 1-8 µg/ml, respectively. CONCLUSION: Luliconazole, terbinafine and efinaconazole exhibited the highest in vitro efficacy, regardless of the dermatophyte species. Further surveillance studies are recommended to confirm the implication of such in vitro data for the clinical recovery rate of dermatophytosis with geophilic species following antifungal therapy.

Pediatric candiduria, epidemiology, genotype distribution and virulence factors of Candida albicans.

The presence of Candida species in urine may be due to colonization of this species in the bladder, urinary catheter, and perineum. Candida albicans has been the most commonly isolated from urine samples in patients with candiduria. Several virulence factors include adhesion to host cells, secreted extracellular enzymes, phenotype switching, and biofilm formation are contributing to the pathogenicity of C. albicans. ABC genotyping is the method based on the determination of 25s rDNA and C. albicans is divided into four genotypes include A, B, C, and E. We aimed to identify Candida species from pediatrics and evaluate extracellular enzyme activities, phenotype switching, biofilm formation, and genotyping in isolates. Urine samples collected, cultured, and yielded yeasts were identified. Phenotype switching, biofilm formation, enzymatic patterns, and genotyping of 50 isolates of C. albicans were evaluated. The Genotyping pattern was compared with extracellular enzymes, biofilm formation, and phenotype switching pattern. 16.2% of urine cultures were positive for the different Candida species. The most common species was C. albicans, followed by C. glabrata. Out of 50 isolates of C. albicans, 72% and 28% isolates were recognized as genotypes A and C. All isolates were produced extracellular enzymes and biofilm formation. In conclusion, candiduria with high colony counts is still a challenge in Iranian pediatrics. Genotype A was the predominant genotype among C. albicans strains. There is a statistical difference between esterase and genotypes of C and A C. albicans.

Discovery of New Trichophyton Members, T. persicum and T. spiraliforme spp. nov., as a Cause of Highly Inflammatory Tinea Cases in Iran and Czechia.

Pathogens from the Trichophyton benhamiae complex are one of the most important causes of animal mycoses with significant zoonotic potential. In light of the recently revised taxonomy of this complex, we retrospectively identified 38 Trichophyton isolates that could not be resolved into any of the existing species. These strains were isolated from Iranian and Czech patients during molecular epidemiological surveys on dermatophytosis and were predominantly associated with highly inflammatory tinea corporis cases, suggesting possible zoonotic etiology. Subsequent phylogenetic (4 markers), population genetic (10 markers), and phenotypic analyses supported recognition of two novel species. The first species, Trichophyton persicum sp. nov., was identified in 36 cases of human dermatophytosis and one case of feline dermatophytosis, mainly in Southern and Western Iran. The second species, Trichophyton spiraliforme sp. nov., is only known from a single case of tinea corporis in a Czech patient who probably contracted the infection from a dog. Although the zoonotic sources of infections summarized in this study are very likely, little is known about the host spectrum of these pathogens. Awareness of these new pathogens among clinicians should refine our knowledge about their poorly explored geographic distribution. IMPORTANCE In this study, we describe two novel agents of dermatophytosis and summarize the clinical manifestation of infections. These new pathogens were discovered thanks to long-term molecular epidemiological studies conducted in Czechia and Iran. Zoonotic origins of the human infections are highly probable, but the animal hosts of these pathogens are poorly known. Further research is needed to refine our knowledge about these new dermatophytes.

In vitro antifungal susceptibility patterns of Trichophyton benhamiae complex isolates from diverse origin.

BACKGROUND: Species from the Trichophyton benhamiae complex are mostly zoophilic dermatophytes which cause inflammatory dermatophytosis in animals and humans worldwide. OBJECTIVES: This study was purposed to (a) to identify 169 reference and clinical dermatophyte strains from the T benhamiae complex species by molecular method and adhering to the newest taxonomy in the complex (b) to evaluate the in vitro antifungal susceptibility profile of these strains against eight common and new antifungal agents that may be used for the treatment of dermatophytosis. METHODS: All isolates, mainly originated from Europe but also from Iran, Japan and USA, were subjected to ITS-rDNA sequencing. The in vitro antifungal susceptibility profiles of eight common and new antifungal drugs against the isolates were determined by CLSI M38-A2 protocol and according to microdilution method. RESULTS: Based on the ITS-rDNA sequencing, T benhamiae was the dominant species (n = 102), followed by T europaeum (n = 29), T erinacei (n = 23), T japonicum (n = 10), Trichophyton sp (n = 4) and T eriotrephon (n = 1). MIC ranges across all isolates were as follows: luliconazole: 0.0002-0.002 µg/ml, terbinafine: 0.008-0.125 µg/ml, efinaconazole: 0.008-0.125 µg/ml, ciclopirox olamine: 0.03-0.5 µg/ml, itraconazole: 0.06-2 µg/ml, griseofulvin: 0.25-4 µg/ml, amorolfine hydrochloride: 0.125-4 µg/ml and tavaborole: 1-16 µg/ml. CONCLUSION: Luliconazole, efinaconazole and terbinafine were the most potent antifungals against T benhamiae complex isolates, regardless of the geographic locations where strains were isolated. These data might help dermatologists to develop effective therapies for successful treatment of infections due to T benhamiae complex species.

Molecular identification and antifungal susceptibility profiles of Candida dubliniensis and Candida africana isolated from vulvovaginal candidiasis: A single-centre experience in Iran.

BACKGROUND: Vulvovaginal candidiasis (VVC) is a common and debilitating long-term illness affecting million women worldwide. This disease is caused mainly by Candida albicans and a lesser extent by other species, including the two phylogenetically closely related pathogens Candida africana and Candida dubliniensis. OBJECTIVES: In this study, we report detailed molecular epidemiological data about the occurrence of these two pathogenic yeasts in Iranian patients affected by VVC, or its chronic recurrent form (RVVC), and provide, for the first time, data on the antifungal activity of two new drugs, efinaconazole (EFN) and luliconazole (LUL). METHODS: A total of 133 vaginal yeast isolates, presumptively identified as C albicans by phenotypic and restriction analysis of rDNA, were further analysed by using a specific molecular method targeting the HWP1 gene. All C africana and C dubliniensis isolates were also tested for their in vitro susceptibility to a panel of modern and classical antifungal drugs. RESULTS AND CONCLUSIONS: Based on the molecular results, among 133 germ-tube positive isolates, we identify 119 C albicans (89.47%), 11 C africana (8.27%) and 3 C dubliniensis (2.26%) isolates. C africana and C dubliniensis showed low MIC values for most of the antifungal drugs tested, especially for EFN and LUL, which exhibited a remarkable antifungal activity. High MIC values were observed only for nystatin and terbinafine. Although C albicans remains the most common Candida species recovered from Iranian VVC/RVVC patients, our data show that its prevalence may be slightly overestimated due to the presence of difficult-to-identify closely related yeast, especially C africana.

Diversity of Geophilic Dermatophytes Species in the Soils of Iran; The Significant Preponderance of Nannizzia fulva.

A molecular epidemiology study was conducted between 2016 and 2017 by a network of collaborators from 12 provinces in the Islamic Republic of Iran. A total of 1484 soil samples from different habitats were screened for the presence of dermatophytes by using the hair baiting technique. The primary identification of isolates was carried out by amplification and MvaI restriction fragment length polymorphism (RFLP) of the internal transcribed spacers regions of ribosomal DNA (ITS-rDNA). The identifications, especially in the cases of isolates with unknown RFLP patterns, were confirmed by sequencing of the ITS-rDNA region. As a result, 256 isolates were recovered. The isolation rate was higher in soils with pH range 7.1-8.0, collected from animal habitats (n = 78; 34%) and parks and gardens (n = 75; 32%), geographically from Mazandaran Province (n = 115; 49.5%) and seasonally in the spring (n = 129; 50.4%), all of which were statistically significant (p < 0.05). The dermatophytes comprising five species of the two genera, viz., Nannizzia fulva (n = 214), N. gypsea (n = 34), Arthroderma quadrifidum (n = 5), A. gertleri (n = 2) and A. tuberculatum (n = 1), were isolated. The geophilic dermatophytes occurred in various soils from different parts of Iran; however, surprisingly, N. fulva emerged as the dominant species, outnumbering the common geophilic species of N. gypsea. For the definitive identification of soil inhabitant dermatophytes, DNA-based identification is strongly recommended.

Familial Cases of Trichophyton benhamiae Infection Transmitted from a Guinea Pig in Iran.

Trichophyton benhamiae is a zoophilic dermatophyte mainly transmitted to humans from guinea pigs. This zoophilic species can also cause dermatophytosis as reported by human contact with other animals, such as rabbit, cat, and fox. Here, we report the tinea faciei and tinea corporis cases: a 12-year-old girl and her 53-year-old father, with no history of immunodeficiency and underlying disease, caused by T. benhamiae transmitted from a guinea pig in Iran. Dermatological examination revealed several erythematous, round, scaly, and approximately 1-4-cm-diameter lesions in both patients. The girl had seven skin lesions, and her father presented two skin lesions on the front side of his neck. The girl's lesions had started 3 weeks before and her father's lesions appeared 7 days after the first clinical appearance of the lesions in the daughter. The girl had daily close contact with a guinea pig, while her father did not have any direct exposure to the pet. Examination of the lesions scraping with 10% potassium hydroxide (KOH 10%) revealed hyaline septate hyphae and arthroconidia. The dermatophyte isolated in culture was identified as T. benhamiae using molecular analysis. The patients were successfully treated using topical sertaconazole nitrate 2% cream twice a day for 4 weeks.

A simple multiplex polymerase chain reaction assay for rapid identification of the common pathogenic dermatophytes: Trichophyton interdigitale, Trichophyton rubrum, and Epidermophyton floccosum.

BACKGROUND AND PURPOSE: The most common etiological agents of human dermatophytosis in various parts of the world are Trichophyton rubrum, Trichophyton interdigitale, and Epidermophyton floccosum. The main aim of this study was to design and evaluate a simple and straightforward multiplex polymerase chain reaction (PCR) assay for reliable identification/differentiation of these species in clinical isolates. MATERIALS AND METHODS: The reliable sequences of several molecular targets of dermatophytes species were used to design a multiplex PCR for the identification of common pathogenic dermatophytes. The isolates and clinical specimens examined in this study included seven standard strains of dermatophytes, 101 isolates of dermatophytes and non-dermatophyte molds/yeasts which had already been identified by sequencing or PCR-restriction fragment length polymorphism (RFLP), and 155 clinical samples from patients suspected of cutaneous mycoses. RESULTS: Species-specific primer pairs for T. rubrum and T. interdigitale/T. mentagrophytes were designed based on the sequence data of the translation elongation factor 1-alpha gene, and the primers for E. floccosum targeted the specific sequence of the internal transcribed spacer region (ITS). The multiplex PCR successfully detected T. rubrum, T. interdigitale/T. mentagrophytes, and E. floccosum strains that were identified by sequencing or PCR-RFLP. However, the primer pairs selected for T. interdigitale/T. mentagrophytes cross-reacted with Trichophyton tonsurans. In testing the PCR system directly for clinical samples, the proportion of positive multiplex PCR was higher than positive culture (68.1% vs. 55.4%, respectively). CONCLUSION: The multiplex assay could detect three common agents out of several causal agents of dermatophytosis, namely T. rubrum, T. interdigitale, and E. floccosum. Therefore, by adding pan-dermatophyte primers it can be used as a comprehensive detection/identification test.

Multidrug-resistant Trichophyton mentagrophytes genotype VIII in an Iranian family with generalized dermatophytosis: report of four cases and review of literature.

BACKGROUND: The global spread of terbinafine-resistant Trichophyton mentagrophytes with point mutations in the squalene epoxidase (SQLE) gene is a big concern. AIM: The present study presents a series of unusual familial cases of generalized dermatophytosis caused by multidrug-resistant T. mentagrophytes genotype VIII. METHODS: Initially, the skin samples of each patient were taken and then subjected to direct microscopy and culture in Mycosel Agar. The molecular identification of Trichophyton species (spp.) was performed for all family members. In addition, the immunologic tests were requested, and an antifungal susceptibility test was carried out using the broth microdilution protocol based on the Clinical and Laboratory Standards Institute M38, third edition. The SQLE gene for a terbinafine-resistant T. mentagrophytes genotype VIII was sequenced and confirmed its nucleotide sequence to KU242352 as a susceptible strain. RESULTS: Based on the results of mycological examination and ITS rDNA sequencing, the etiologic agent was identified as T. mentagrophytes as a zoophilic dermatophyte. This species showed multiple drug resistance in vitro against terbinafine (minimum inhibitory concentration (MICs ≥8 µg/ml), itraconazole (MIC ≥4), and fluconazole (MIC ≥16). The SQLE gene of the isolate was subjected to sequencing for mutation, which showed a point mutation as TTC/TTA in the gene leading to Phe397Leu amino acid substitution in the enzyme. Only one of the family members responded to itraconazole and was cured after the long-term use of itraconazole. Other family members were treated with oral voriconazole with no recurrence. CONCLUSION: The transmission of this resistant T. mentagrophytes to other countries due to globalization is a serious issue to be considered.

Investigation of in vitro antifungal susceptibility testing and genetic diversity of clinical isolates of Trichophyton benhamiae and Trichophyton eriotrephon in Iran.

BACKGROUND: Trichophyton benhamiae is a zoophilic dermatophyte, known as one of the causative agents of dermatophytosis. OBJECTIVES: The purpose of this study was to explore the genotypes of T. benhamiae strains isolated from geographically different areas of Iran and also to evaluate in vitro antifungal susceptibility profile of these strains against seven antifungal drugs. METHODS: Twenty-two strains of T. benhamiae and two strains of T. eriotrephon were isolated from patients with distinct types of dermatophytosis. DNA extraction and amplification of rDNA regions using ITS1 and ITS4 primers were conducted on the isolates. The in vitro antifungal susceptibility of posaconazole (PSC), voriconazole (VRC), itraconazole (ITC), ketoconazole (KET), caspofungin (CAS), terbinafine (TRB) and griseofulvin (GRZ) was evaluated according to CLSI M38-A2 protocol. RESULTS: The multiple alignment of the ITS-rDNA sequences of T. benhamiae indicated a mean similarity of 99.5%, with 0-3 interspecies nucleotide difference. The geometric mean (GM) values of minimum inhibitory concentrations (MICs) and minimum effective concentrations (MECs) across the all isolates were respectively: TRB: 0.025 mg/L, PSC: 0.032 mg/L, ITC: 0.050 mg/L and VRC: 0.059 mg/L with lower values and CAS: 0.31 mg/L, KTZ: 0.56 mg/L and GRZ: 0.76 mg/L with higher values. CONCLUSION: Diverse ITS sequence types of T. benhamiae were shown in different geographical regions of Iran. The TRB, PSC and ITC were the most effective drugs against T. benhamiae strains, respectively. Furthermore, in our study, two strains of T. eriotrephon as a scarce dermatophyte species were described.

Molecular identification and antifungal susceptibility among clinical isolates of dermatophytes in Shiraz, Iran (2017-2019).

Dermatophytosis is a common superficial mycotic infection affecting individual's quality of life worldwide. The present study aimed to perform species-level identification and evaluate the antifungal susceptibility patterns of dermatophytes isolated in Shiraz, Iran. This cross-sectional study was conducted on clinical samples collected during 2017-2019 from 307 patients suspected of having dermatophytosis. The isolates were identified by direct microscopy, culture and internal transcribed spacer ribosomal DNA sequencing, and their antifungal susceptibility patterns were determined by the microdilution method. Among 307 patients, dermatophytosis was diagnosed by microscopy in 190 (61.8%) subjects and confirmed in 130 (42.3%) cases by both microscopy and culture. It was found out tinea pedis was the most common clinical manifestation, and Trichophyton mentagrophytes was the most prevalent species (28.4%), followed by T tonsurans (23.8%), Microsporum canis (11.5%), T interdigitale (10%), T verrucosum (6.9%), T rubrum (6.9%), T benhamiae (4.6%), T violaceum (3%), T simii (3%), Epidermophyton floccosum (0.7%) and M ferrugineum (0.7%). Moreover, it was revealed that luliconazole with a geometric mean (GM) minimum inhibitory concentration (MIC) of 0.03 µg ml-1 was the most effective agent against all tested isolates. Regardless of species, 30% of isolates responded to high MICs of griseofulvin (MIC90  > 2 µg ml-1 ). The increasing prevalence of nonindigenous species of T simii, T benhamiae and M ferrugineum in Shiraz, Iran, was a notable finding. In addition, infections due to zoophilic species showed an increasing trend. These epidemiological data, along with antifungal susceptibility patterns, may have implications for clinical decision-making and successful treatment.

Phylogenetic Analysis of Clinically Relevant Fusarium Species in Iran.

Fungi of the genus Fusarium are well known as major plant pathogens but also cause a broad spectrum of human infections. Sixty-three clinical isolates, collected during 2014-2017, were identified using a part of the TEF1 gene as barcoding marker. Fusarium fujikuroi species complex (FFSC, n = 41, 65%) showed to be the dominant etiological agent, followed by F. solani species complex (FSSC, n = 14, 22%) and F. oxysporum species complex (FOSC, n = 7, 11%). There was one strain belonging to F. lateritium species complex (FLSC, n = 1, 1.5%). For final identification, a phylogenetic tree was constructed including the type strains of each species complex. Most cases of fusariosis were due to nail infection (n = 38, 60.3%), followed by keratitis (n = 22, 34%). Fusarium infections are difficult to be treated due to their intrinsic resistance to different azoles; however, accurate and fast identification of etiological agents may enhance management of the infection. We present the first phylogenetic study on clinical Fusarium spp. from Iran.