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Introduction: Although, Rhizopus species, are the main causative agents of mucormycosis, we know a little about these pathogens. We investigated the virulence potential of Rhizopus arrhizus and R. microsporus strains, obtained from a wide selection of clinical and environmental sources, in Galleria mellonella larvae and a mice model. Methods: In this experiment, a total of 26 strains, 13 for each species of R. microsporus, R. arrhizus were tested in larvae with final concentration of 106/ml per strain. Eight strains were tested in mice model. Inoculated samples were monitored in insect larvae for 6 days and in mice for 14 days. Statistical data were performed using Graphpad Prism software (GraphPad Software, San Diego, CA). Survival data of the samples were plotted using Kaplen-Meyer diagrams and analyzed using log rank test P value was considered 0.05%. Results: R. arrhizus showed higher virulence compare to R. microsporus in both models. No specific difference was seen in pathogenicity between the two varieties of R. arrhizus. Virulence was not affected by source of isolation or production of toxin in some strains of R. microsporus. Virulence pattern was similar in both models. Conclusion: Considering the mortality rate which was happened with strains from environmental sources, we conclude that these fungi have an opportunistic nature, which make them pathogen in susceptible hosts in favorite conditions. Larvae model showed reliable results compare to mice model.

Aspergillus species are emerging causative agents of non-dermatophyte mold onychomycosis. In this study, 48 Aspergillus isolates were obtained from patients with onychomycosis in Mashhad, Iran, during 2015–2018. The aim is to identify the Aspergillus isolates to the species level by using partial calmodulin and beta-tubulin gene sequencing and MALDI-TOF MS, and to evaluate their in vitro susceptibility to ten antifungal drugs: terbinafine, itraconazole, voriconazole, posaconazole, ravuconazole, isavuconazole, caspofungin, micafungin, anidulafungin and amphotericin B according to CLSI M38-A3. Our results indicate that A.flavus (n = 38, 79%) is the most common Aspergillus species causing onychomycosis in Mashhad, Iran. Other detected species were A. terreus (n = 3), A. tubingensis (n = 2), A. niger (n = 1), A. welwitschiae (n = 1), A. minisclerotigenes (n = 1), A. citrinoterreus (n = 1) and A. ochraceus (n = 1). Aspergillus flavus, A. terreus and A. niger isolates were correctly identified at the species level by MALDI-TOF MS, while all cryptic species were misidentified. In conclusion, A. flavus is the predominant Aspergillus species causing onychomycosis due to Aspergillus spp. in Mashhad, Iran. MALDI-TOF MS holds promise as a fast and accurate identification tool, particularly for common Aspergillus species. It is important that the current database of reference spectra, representing different Aspergillus species is expanded to increase the precision of the species-level identification. Terbinafine, posaconazole and echinocandins were in vitro most active against the studies Aspergillus isolates and terbinafine could be the first choice for treatment of onychomycosis due to Aspergillus.

Background: Poly-L-malic acid (PLMA) comprises aliphatic polyester polymers with broad applications in pharmaceutical industries. The fungal microorganisms are among the best natural sources recruited to supply L-malic acid (MA) as a precursor of PLMA. In this study, we investigated MA production ability of 7 clinical isolated of the fungus Aureobasidium pullulans. Materials and Methods: Seven clinical isolates of A. pullulans acquired from Westerdijk Fungal Biodiversity Institute were studied, and the isolate with the highest total MA production was selected for the optimization process. We tried to optimize the output by applying different concentrations of CaCO3 in fungus medium (1.5%, 3%, and 6%) and various incubation temperatures (27°C, 32°C, and 37°C) during 3, 7, and 14 days. Results: Intra-strains variation was significantly strong (P0.05). Conclusion: Overall, we obtained the highest MA production in Sabouraud dextrose agar (SDA)medium with 3% CaCO3 at 27°C after 7 days of incubation. Our study indicated that the fermentation period and CaCO3 concentration significantly alter MA production in A. pullulans var. melanigenum.

Introduction: Little is known about the pathogenicity and virulence properties of the Rhizopus species used in food fermentation. This study aimed to investigate the virulence potential of Rhizopus arrhizus and R. microsporus strains obtained from a wide selection of clinical and environmental sources in an embryonated chicken egg model. Materials & Methods: In total, 26 strains (13 strains from each species) were inoculated in embryonated eggs with final concentration of 106 spore/ml/egg. The eggs were inoculated (0.1 ml) through chorio-allantoic membrane in day 10 and incubated at 37.6 °C and 60% humidity and monitored for 7 days. Ethics code: IR.MUMS.fm.REC.1396.457 Findings: The mortality rate for R. microsporus was slightly higher than that of R. arrhizus. The ability of R. arrhizus and R. microsporus to cause infection was strain-specific but source-independent in the embryonated chicken eggs. The presence of endosymbiont bacteria (Burkhulderia) in some strains of R. microspores did not show any effect on their virulence. Discussion & Conclusions: The occurrence of virulence in strains isolated from fermented food indicates the opportunistic nature of these fungi and possibility of a public health risk for consumers.

Background and Purpose: Keratinophilic fungi play an important role in the decomposition of keratinous substances in nature. This capacity induces dermatomycosis in both humans and livestock. The soil of livestock stables can be a reservoir of keratinophilic fungi. Therefore, the present study was conducted to isolate and identify keratinophilic fungi in the soil of the livestock stables located in Qayen, South Khorasan Province, Iran. Materials and Methods: This study was conducted on 62 soil samples collected from livestock stables. The samples were cultured by means of hair bait technique (HBT). The identification of the isolates was performed based on their morphological characteristics and then confirmed by polymerase chain reaction and sequencing of the ITS regions of ribosomal DNA. Results: A total of 118 isolates of 7 species from 5 genera were identified. Aphanoascus verrucosu (n=70, 59.36%) was detected as the dominant keratinophilic fungus, followed by Arthroderma quadrifidum (n=13, 11.01%), Aphanoascus terreus (n=12, 10.16%), Acremonium (n=12, 10.16%), Arthroderma gertleri (n=5, 4.23%), Fusarium equiseti (n=3, 2.54%), and Uncinocarpus reesii (n=3, 2.54%). Conclusion: Different keratinophilic fungi were isolated from the soil of livestock stables; however, Aphanoascus verrucosu was found to be the dominant species.

The incidence of infections caused by uncommon Chaetomiaceae (Chaetomium and related species) in humans has increased in the recent years. The in vitro activity of eight antifungal drugs (amphotericin B, five azoles, two echinocandins) against 42 morphologically identified Chaetomium strains was determined according to the Clinical and Laboratory Standards Institute (CLSI) guideline. The strains were subsequently identified based on sequences of the internal transcribed spacer 1 and 2 including the intervening 5.8S nrDNA region (ITS) and the partial β tubulin gene (tub2). Chaetomium globosum (n = 24), was the most frequently isolated species, followed by Amesia atrobrunnea (syn. Chaetomium atrobrunnea, n = 6), Dichotomopilus dolichotrichus (syn. Chaetomium dolichotrichum, n = 2) and Acrophialophora jodhpurensis, Chaetomium coarctatum, C. elatum, C. gracile, C. subaffine, C. tarraconense, C. unguicola, Dichotomopilus sp., Dichotomopilus variostiolatus, Ovatospora brasiliensis (all represented by a single strain). The geometric means of the minimum inhibitory concentrations/minimum effective concentrations (MICs/MECs) of the antifungals across all strains were (in increasing order): micafungin 0.12 µg/ml, itraconazole and posaconazole 0.21 µg/ml, amphotericin B 0.25 µg/ml, voriconazole 0.45 µg/ml, isavuconazole 0.54 µg/ml, caspofungin 2.57 µg/ml, and fluconazole 45.25 µg/ml. Micafungin had the lowest geometric mean followed by amphotericin B which had the largest range against tested isolates. All examined C. globosum strains had similar antifungal susceptibility patterns. Fluconazole and caspofungin could not be considered as an option for treatment of infections caused by Chaetomium and chaetomium-like species. Lay summary Infections caused by uncommon fungi such as Chaetomium have increased in the recent years. Chaetomium globosum has been reported from onychomycosis and phaeohyphomycosis. This species often induces superficial infections in immunocompetent patients. The taxonomy of Chaetomium spp. has changed dramatically in the last years. Antifungal treatment is a crucial step for managing these kinds of infections. Therefore, the in vitro activity of eight antifungal drugs against Chaetomium strains was determined and β-tubulin (tub2) sequencing was applied to identify the strains. Chaetomium globosum was the most frequent species in our dataset. Based on the results of susceptibility testing, micafungin had the lowest geometric mean followed by amphotericin B. Fluconazole and caspofungin cannot be considered a proper treatment option for infections caused by Chaetomium and chaetomium-like species.

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.

This is a report of established invasive Aspergillus rhinosinusitis in a patient diagnosed with COVID‐19 and afflicted by AML, which was initially considered to be rhinocerebral mucormycosis.

Background Exophiala is the main genus of black fungi comprising numerous opportunistic species. Data on antifungal susceptibility of Exophiala isolates are limited, while infections are potentially fatal. Materials and methods In vitro activities of eight antifungal drugs (AMB, five azoles, two echinocandins) against 126 clinical (n = 76) and environmental (n = 47) isolates from around the world were investigated. E. oligosperma (n = 58), E. spinifera (n = 33), E. jeanselmei (n = 14) and E. xenobiotica (n = 21) were included in our dataset. Results The resulting MIC90 s of all strains were as follows, in increasing order: posaconazole 0.063 μg/ml, itraconazole 0.125 μg/ml, voriconazole and amphotericin B 1 μg/ml, isavuconazole 2 μg/ml, micafungin and caspofungin 4 μg/ml, and fluconazole 64 μg/ml. Posaconazole, itraconazole and micafungin were the drugs with the best overall activity against Exophiala species. Fluconazole could not be considered as a treatment choice. No significant difference could be found among antifungal drug activities between these four species, neither in clinical nor in environmental isolates. Conclusion Antifungal susceptibility data for Exophiala spp. are crucial to improve the management of this occasionally fatal infection and the outcome of its treatment.

Background and Purpose: Pneumocystis pneumonia (PCP) is one of the most common and life-threatening fungal diseases in patients with human immunodeficiency, treated with immunosuppressive medications. Immunocompetent people can also be a spreading agent for PCP. Regarding this, the aim of the present study was to diagnose and identify Pneumocystis jirovecii in bronchoalveolar lavage (BAL) samples obtained from patients with pulmonary disorder using a molecular method. Materials and Methods: For the purpose of the study, BAL samples (n=138) were collected from patients, undergoing bronchoscopy at the different departments of university hospitals affiliated to Mashhad University of Medical Sciences, Mashhad, Iran, during a period of one year (i.e., April 2014 until May 2015). Giemsa staining and molecular identification were carried out for each sample. The samples were also subjected to nested polymerase chain reaction (PCR), sequencing, and genotyping based on mitochondrial ribosomal large subunit (mtLSU rRNA) of P. jirovecii. The phylogenic tree was constructed by MEGA6 software. Results: The results of direct microscopic examination revealed the presence of P. jirovecii in 3 (2.2%) out of 138 samples; in addition, nested PCR and sequencing led to the detection of species in 17 (12.3%) samples. Out of patients with positive results, 10 (25%) and 7 (7.1%) cases were immunosuppressed and immunocompetent, respectively. The most common clinical symptoms among patients with pneumocystis were fever, dyspnea, and dry cough. In addition, genotypes III and II were the dominant genotypes in our dataset. Conclusion: Nested PCR and sequencing methods showed higher sensitivity and specificity as compared with a direct staining technique. Genotype III was identified as the most dominant type in patients with pulmonary disorder in Mashhad.

Chronic rhinosinusitis (CRS) is known by the inflammation of the sinonasal mucosa. Limited data are available on the fungal biofilms in CRS. This study aimed to investigate the role of fungal agents in sinonasal polyposis, and also to identify the dominant fungal pathogens by PCR and mycological studies. Sixty sinonasal biopsy specimens were examined; 31 sinonasal polyposis (SNP) as the study group and 29 concha bullosa without any history of chronic rhinosinusitis as the control. The specimens underwent histopathological examination for bacterial and fungal biofilms; routine mycological methods and then PCR using the internal transcribed spacer (ITS) region. Four (12.9%) specimens of the SNP group had a positive KOH preparation for fungal elements. The positive culture for Aspergillus flavus was obtained in 5 (16%) cases and one control. The identified isolates included one Cryptococcus magnus and two Penicillium chrysogenum in the study group and one P. chrysogenum in the control group. The fungal agents didn’t have a significant role in the etiopathology of the SNP. Moreover, the results of mycological and histopathological methods had not any agreement with the molecular technique in CRS.

Background and Purpose: Herein, we report the first case of fungal keratitis due to Aspergillus minisclerotigenes in a 68-year-old rural woman admitted to the Ophthalmology Center of Khatam-Al-Anbia Hospital in Mashhad, northeast of Iran. Case report: The patient presented with severe pain, burning, foreign body sensation, and reduced vision in her right eye. She had long-term uncontrolled diabetes and was not able to close her eye due to an anatomical problem with the eyelid. The cornea smear sample was cultured, and the fungus was initially identified as Aspergillus flavus. The isolated strain was further identified by sequencing a part of the calmodulin gene as A. minisclerotigenes. The patient did not respond to any antifungal treatments (e.g., amphotericin B and voriconazole drops, and fluconazole 300 mg/day); therefore, she was eventually subjected to corneal transplantation surgery. Conclusion: Fungal keratitis can be caused by the less common species. The reliable identification of the causative agents can be accomplished by the implementation of molecular methods.

Comision Nacional de Investigacion Cientifica y Tecnologica (CONICYT)Comision Nacional de Investigacion Cientifica y Tecnologica (CONICYT); Fondo Nacional de Desarrollo Cientifico y Tecnologico (FONDECYT), ChileComision Nacional de Investigacion Cientifica y Tecnologica (CONICYT)CONICYT FONDECYT [11140562]; Iranian National Science Foundation (INSF)Iran National Science Foundation (INSF) [96003337]

Background and Purpose: Dermatophytes as the causative agents of dermatophytosis (ringworm) are widely spread around the world. Accurate identification of dermatophytes in one area can be particularly important for epidemiological studies. Regarding this, the aim of the present study was to describe the species spectrum of dermatophytes, isolated from patients in Mashhad city, Iran, using the molecular-based method. Materials and Methods: This study was conducted on 79 dermatophyte isolates obtained from the human skin, hair, and nail specimens. Species identification was performed by the polymerase chain reaction-restriction fragment length polymorphism analysis of ribosomal DNA internal transcribed spacer regions using MvaI restriction enzyme. Results: The identified species included Trichophyton mentagrophytes/T. interdigitale species complex (n=37, 46.8%), Epidermophyton floccosum (n=12, 15.2%), T. rubrum (n=8, 10.1%), Microsporum canis (n=8, 10.1%), T. violaceum (n=5, 6.3%), T. tonsurans (n=4, 5.1%), Nannizzia gypsea (n=3, 3.8%), T. benhamiae (n=1, 1.3%), and T. verrucosum (n=1, 1.3%). The clinical forms of infection were tinea corporis (n=26, 32.8%), tinea cruris (n=22, 27.8%), tinea capitis (n=10, 12.6%), tinea unguium (n=7, 9%), tinea manuum (n=6, 8%), tinea pedis (n=5, 6.3%), and tinea faciei (n=3, 3.5%). Conclusion: As the findings indicated, T. mentagrophytes/T. interdigitale species complex had the highest prevalence, and T. benhamiae appeared to be a new emerging agent of dermatophytosis in Mashhad, northeastern Iran.

The species belonging to the genus Fonsecaea are the main causative agents of chromoblastomycosis. The invasive potential of Fonsecaea differs significantly among its various sibling species. Moreover, the lack of clarity on the virulence and availability of precise markers to distinguish and detect Fonsecaea species is attributed to the different ways of dissemination and pathogenicity. Therefore, the present study aimed to propose new molecular tools to differentiate between sibling species causing chromoblastomycosis. We used an infection model of chromoblastomycosis in BALB/c to study species-specific molecular markers for the in vivo detection of Fonsecaea species in biological samples. Specific primers based on the CBF5 gene were developed for Fonsecaea pedrosoi, Fonsecaea monophora, Fonsecaea nubica, and Fonsecaea pugnacius. In addition, a padlock probe was designed for F. pugnacius based on ITS sequences. We also assessed the specificity of Fonsecaea species using in silico, in vitro, and in vivo assays. The results showed that markers and probes could effectively discriminate the species in both clinical and environmental samples, enabling bioprospecting of agents of chromoblastomycosis, thereby elucidating the infection route of the disease.

Aureobasidium pullulans and A. melanogenum are black-yeast-like surface colonisers and are commonly encountered as contaminants in the hospital. The species are able to produce melanin which play a role in protection against environmental stress and irradiation. Aureobasidium melanogenum shows higher frequency in opportunistic infections compared to A. pullulans. Comparative pathogenicity of opportunistic black yeasts between Aureobasidium pullulans and A. melanogenum to explain the observed differences in frequency in infection. Degrees of melanisation and thermotolerance were measured, and virulence of strains from different sources was examined in Galleria mellonela and murine infection models. Aureobasidium melanogenum responds with increased melanisation to temperature stress and generally survives at 37°C, A. pullulans on average scored less on these parameters. In the murine model, differences between species were not significant, but the melanised A. melanogenum group showed the highest virulence. This result was not reproducible in Galleria mellonella larvae at 25°C. The A. melanogenum black group showed higher pathogenicity in murine model, indicating that the combination of melanisation and thermotolerance rather than species affiliation is instrumental. Galleria larvae did not survive very well at 37°C, and hence, this model is judged insufficient to detect the small virulence differences observed in Aureobasidium.

As a novel risk factor, COVID-19 has led to an increase in the incidence of candidemia and an elevated mortality rate. Despite being of clinical importance, there is a lack of data regarding COVID-19-associated candidemia (CAC) among Iranian patients. Therefore, in this retrospective study, we assessed CAC epidemiology in the intensive care units (ICUs) of two COVID-19 centers in Mashhad, Iran, from early November 2020 to late January 2021. Yeast isolates from patients’ blood were identified by 21-plex polymerase chain reaction (PCR) and sequencing, then subjected to antifungal susceptibility testing according to the CLSI M27-A3 protocol. Among 1988 patients with COVID-19 admitted to ICUs, seven had fungemia (7/1988, 0.03%), among whom six had CAC. The mortality of the limited CAC cases was high and greatly exceeded that of patients with COVID-19 but without candidemia (100% (6/6) vs. 22.7% (452/1988)). In total, nine yeast isolates were collected from patients with fungemia: five Candida albicans, three C. glabrata, and one Rhodotorula mucilaginosa. Half of the patients infected with C. albicans (2/4) were refractory to both azoles and echinocandins. The high mortality of patients with CAC, despite antifungal therapy, reflects the severity of the disease in these patients and underscores the importance of rapid diagnosis and timely initiation of antifungal treatment.

The Aspergillus species are main causative agents of various infections such as invasive aspergillosis (IA) in immunocompromised patients and these infections have high mortality rates. In this study, we provide insight in the species causing aspergillosis in Iran based on morphology and sequence data. Clinical (n = 117) and environmental isolates (n = 54) collected during 2010–2016 from University hospitals in Mashhad and Tehran (Iran) were identified both morphologically and molecularly using partial calmodulin (CaM) gene sequences. Clinical cases were identified based on EORTC/MSG criteria. Aspergillus flavus (n = 96, 55%) was the most prevalent species among the clinical and environmental isolates while A. fumigatus (n = 13, 7.5%) ranked fourth after A. tubingensis (n = 23, 13%) and A. welwitchiae (n = 18, 10%). Species such as A. tubingensis, A. welwitschiae, A. fumigatus, A. sydowii, A. neoniger and A. terreus were present in both clinical and environmental samples indicating the possible environmental source of infections. Interestingly, A. niger was isolated only once. Furthermore, 13 other rare and cryptic Aspergillus species were detected. Pulmonary and respiratory disorders (n = 33), followed by transplantation (n = 23), invasive fungal rhinosinusitis (n = 14), and haematological malignancies (n = 12) were major predisposing factors. According to EORTC/MSG criteria, there were 43 probable cases identified followed by 36 cases for each of proven and possible ones. Correct molecular identification will be useful for further epidemiological studies.

Candida tropicalis is one of the major candidaemia agents, associated with the highest mortality rates among Candida species, and developing resistance to azoles. Little is known about the molecular mechanisms of azole resistance, genotypic diversity, and the clinical background of C. tropicalis infections. Consequently, this study was designed to address those questions. Sixty-four C. tropicalis bloodstream isolates from 62 patients from three cities in Iran (2014-2019) were analyzed. Strain identification, antifungal susceptibility testing, and genotypic diversity analysis were performed by MALDI-TOF MS, CLSI-M27 A3/S4 protocol, and amplified fragment length polymorphism (AFLP) fingerprinting, respectively. Genes related to drug resistance (ERG11, MRR1, TAC1, UPC2, and FKS1 hotspot9s) were sequenced. The overall mortality rate was 59.6% (37/62). Strains were resistant to micafungin [minimum inhibitory concentration (MIC) ≥1 μg/ml, 2/64], itraconazole (MIC > 0.5 μg/ml, 2/64), fluconazole (FLZ; MIC ≥ 8 μg/ml, 4/64), and voriconazole (MIC ≥ 1 μg/ml, 7/64). Pan-azole and FLZ + VRZ resistance were observed in one and two isolates, respectively, while none of the patients were exposed to azoles. MRR1 (T255P, 647S), TAC1 (N164I, R47Q), and UPC2 (T241A, Q340H, T381S) mutations were exclusively identified in FLZ-resistant isolates. AFLP fingerprinting revealed five major and seven minor genotypes; genotype G4 was predominant in all centers. The increasing number of FLZ-R C. tropicalis blood isolates and acquiring FLZ-R in FLZ-naive patients limit the efficiency of FLZ, especially in developing countries. The high mortality rate warrants reaching a consensus regarding the nosocomial mode of C. tropicalis transmission.

Clonal expansion of fluconazole resistant (FLZ-R) Candida parapsilosis isolates is increasingly being identified in many countries, while there is no study exploring the antifungal susceptibility pattern, genetic diversity, and clinical information for Iranian C. parapsilosis blood isolates. Candida parapsilosis species complex blood isolates (n = 98) were recovered from nine hospitals located in three major cities, identified by MALDI-TOF MS, and their genetic relatedness was examined by AFLP fingerprinting. Antifungal susceptibility testing followed CLSI-M27-A3 and ERG11, MRR1 and hotspots 1/2 (HS1/2) of FKS1 were sequenced to assess the azole and echinocandin resistance mechanisms, respectively. Ninety-four C. parapsilosis and four Candida orthopsilosis isolates were identified from 90 patients. Only 43 patients received systemic antifungal drugs with fluconazole as the main antifungal used. The overall mortality rate was 46.6% (42/90) and death mostly occurred for those receiving systemic antifungals (25/43) relative to those not treated (17/47). Although, antifungal-resistance was rare, one isolate was multidrug-resistant (FLZ = 16 μg/ml and micafungin = 8 μg/ml) and the infected patient showed therapeutic failure to FLZ prophylaxis. Mutations causing azole and echinocandin resistance were not found in the genes studied. AFLP revealed five genotypes (G) and G1 was the main one (59/94; 62.7%). Clinical outcome was significantly associated with city (P = 0.02, α < 0.05) and Mashhad was significantly associated with mortality (P = 0.03, α < 0.05). Overall, we found a low level of antifungal resistance for Iranian C. parapsilosis blood isolates, but the noted MDR strain can potentially become the source of future infections and challenge the antifungal therapy in antifungal-naïve patients. AFLP typing results warrants confirmation using other resolutive typing methods.

The main purpose of our study was to evaluate multiplex PCR assay targeting novel genes for detection of five fungal and bacterial agents in BAL samples; because many fungi and bacteria that cause respiratory infections have similar clinical symptoms, diagnosing and differentiating them are therefore essential to controlling and treating them. A total of 100 BAL specimens from a mycobacterium and mycology laboratory were collected from patients suspected of having TB or other respiratory diseases. Novel DNA targets for Aspergillus, Nocardia, Cryptococcus, and Streptomyces were found using modified comparative genomic analysis. Afterward, the primers were designed based on novel targets, and the sensitivity and specificity of the newly designed primers were evaluated. These primers, along with specific primers for M. tuberculosis (SDR), were used in a multiplex PCR assay. The results showed the culture test to be more sensitive than the PCR assay in detecting M. tuberculosis. However, in the detection of Aspergillus, the PCR assay was more sensitive than the culture test. We also found one positive culture and two positive PCR assays for Nocardiosis. Cryptococcal infections and Streptomyces associated with lung diseases were not identified by the culture test nor by the PCR assay. The multiplex PCR is one of the cheapest molecular diagnostic tests readily available for BAL samples in clinical laboratories. This assay can be used for early reports of the causative agents and for treating patients with appropriate drugs at an early stage.

Chromoblastomycosis is a chronic, cutaneous or subcutaneous mycosis characterized by the presence of muriform cells in host tissue. Implantation disease is caused by melanized fungi related to black yeasts, which, in humid tropical climates, are mainly members of the genus Fonsecaea. In endemic areas of Brazil, F. pedrosoi and F. monophora are the prevalent species. The current hypothesis of infection is traumatic introduction via plant materials, especially by plant thorns. However, isolation studies have demonstrated a low frequency of the agents in environmental substrates. The present study aimed to detect F. pedrosoi and F. monophora in shells of babassu coconuts, soil, plant debris, and thorns from endemic areas of chromoblastomycosis in Maranhão state, northern Brazil, using Rolling Circle Amplification (RCA) with padlock probes as a new environmental screening tool for agents of chromoblastomycosis. In addition to molecular screening, the environmental samples were analyzed by fungal isolation using mineral oil flotation. The limit of detection of the RCA method was 2.88 × 107 copies of DNA per sample for the used padlock probes, indicating that this represents an efficient and sensitive molecular tool for the environmental screening of Fonsecaea agents. In contrast, with isolation from the same samples using several selective methods, no agents of chromoblastomycosis were recovered.

Chromoblastomycosis is one of the most prevalent implantation fungal infections caused by melanized fungi, affecting individuals with certain risk factors with high morbidity due to its recalcitrant nature. It is difficult to identify the etiological agents and thus a suitable reproductive molecular identification method applicable in developing countries has been investigated. We report the identification of four different fungal causative agents of chromoblastomycosis by reverse line blotting hybridization (RLB) based on biotin-labeled PCR products and amine labeled probes to hybridize. Sixty five reference strains, including type strains, i.e. Fonsecaea pedrosoi, F. monophora, F. nubica, and Phialophora verrucosa, obtained from the CBS-KNAW were included in this study. Internal transcribed spacer 1 (ITS1) regions of relevant species were aligned and adjusted using BIONUMERICS v. 4.61 in order to design four specific probes to identify informative nucleotide polymorphisms. The final identification of these species by RLB assay was concordant with ITS sequencing and showed 100% specificity with no cross hybridization, able to identify all tested strains. The time and cost were less compare to other routine identification methods such as sequencing. This assay allows sensitive and specific simultaneous detection and identification of a different fungal species.

The black yeast genus Exophiala includes numerous potential opportunistic species that potentially cause systematic and disseminated infections in immunocompetent individuals. Species causing systemic disease have ability to grow at 37–40 °C, while others consistently lack thermotolerance and are involved in diseases of cold-blooded, waterborne vertebrates and occasionally invertebrates. We explain a fast and sensitive assay for recognition and identification of waterborne Exophiala species without sequencing. The ITS rDNA region of seven Exophiala species (E. equina, E. salmonis, E. opportunistica, E. pisciphila, E. aquamarina, E. angulospora and E. castellanii) along with the close relative Veronaea botryosa was sequenced and aligned for the design of specific padlock probes for the detection of characteristic single-nucleotide polymorphisms. The assay demonstrated to successfully amplify DNA of target fungi, allowing detection at the species level. Amplification products were visualized on 1% agarose gels to confirm specificity of probe–template binding. Amounts of reagents were reduced to prevent the generation of false positive results. The simplicity, tenderness, robustness and low expenses provide padlock probe assay (RCA) a definite place as a very practical method among isothermal approaches for DNA diagnostics.

Non-fumigatus Aspergillus species are the leading cause of Aspergillus infections in the tropical and subtropical regions of the world. In a prospective study between 2015 and 2016, a total of 150 bronchoalveolar (BAL) specimens was collected from patients suspected to pulmonary aspergillosis (PA) underlying immunodeficiencies in Mashhad, Northeastern Iran, located in the Middle East. All Aspergillus strains were phylogenetically identified at the species level by PCR-sequencing of partial β-tubulin gene. Overall, Aspergillus species were isolated from 20 specimens originating from 10 (50%) patients with cancer, 5 (25%) patients receiving corticosteroid therapy, 3 (15%) organ transplant recipients and 2 (10%) patients admitted to intensive care unit (ICU). A. flavus complex was the predominant 15 (75%) cause of probable invasive pulmonary aspergillosis (IPA), followed by A. tubingensis 3 (15%), and 2 (10%) A. fumigates complex. In conclusion, distribution of clinical Aspergillus species in the tropical region of the Middle East shows predominance of the non-fumigatus Aspergillus spp., which warrants further attention by health care professionals.

Background and purpose Vulvovaginal candidiasis (VVC) is a common problem in women. The purpose of this study was to identify Candida isolates by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) from women with vulvovaginitis that were referred to Ghaem Hospital, Mashhad, Iran. Materials and methods This study was conducted on 65 clinical samples isolated from women that were referred to Ghaem Hospital. All specimens were identified using phenotyping techniques, such as microscopy and culture on Sabouraud dextrose agar and corn meal agar. In addition, all isolates were processed for MALDI-TOF MS identification. Results Out of the 65 analyzed isolates, 61 (94%) samples were recognized by MALDI-TOF MS. However, the remaining four isolates (6%) had no reliable identification. According to the results, C. albicans (58.5%) was the most frequently isolated species, followed by C. tropicalis (16.9%), C. glabrata (7.7%), C. parapsilosis (7.7%), and guilliermondii (3.1%). Conclusion As the findings indicated, MALDI TOF MS was successful in the identification of clinical Candida species. C. albicans was identified as the most common Candida species isolated from the women with VVC. Moreover, C. tropicalis was the most common species among the non-albicans Candida species.

Background and Purpose: Rhinosinusitis is a common disorder, influencing approximately 20% of the population at some time of their lives. It was recognized and reported with expanding recurrence over the past two decades worldwide. Undoubtedly, correct diagnosis of fungi in patients with fungal rhinosinusitis affects the treatment planning and prognosis of the patients. Identification of the causative agents using the standard mycological procedures remains difficult and time-consuming. Materials and Methods: Based on clinical and radiological parameters, 106 patients suspected of fungal rhinosinusitis were investigated in this cross-sectional prospective study from April 2012 to March 2016 at an otorhinolaryngology department. In this study, internal transcribed spacer (ITS) and calmodulin (CaM) sequencing were respectively validated as reliable techniques for the identification of Mucorales and Aspergillus to species level (both agents of fungal rhinosinusitis). Results: Of these, 63 (59.4%) patients were suspected of allergic fungal rhinosinusitis (AFRS), 40 (37.7%) patients suspected of acute invasive fungal rhinosinusitis (AIFRS), and 3 (2.8%) patients suspected of fungus ball. In patients suspected of AFRS, AIFRS, andfungus ball only 7, 29, and 1 had positive fungal culture, respectively. After ITS and CaM sequencing, Aspergillus flavus was the most common species isolated from non-invasive forms, and A. flavus and Rhizopus oryzae were more frequently isolated from invasive forms. Conclusion: Aspergillus flavus is the most common agent of fungal rhinosinusitis in Iran, unlike most other reports from throughout the world stating that A. fumigatus is the most frequent causative agent of this disease.

Occurrence and diversity of dermatophyte mycoflora in 298 soil samples from Ahvaz, Southwest of Iran was investigated by using the hair-baiting technique. The samples were collected during spring (n = 210) and autumn (n = 88) of 2015, and the fungal isolates were identified based on the macro- and micro-morphology of colonies and with further ITS-rDNA RFLP and sequencing. Totally, 60 soil samples (20.1%) were positive for dermatophyte growth whose pH varied from 7.0 to 7.9. The highest (26.6%) and the lowest (14.3%) recovery rates were from the animal resorts and the streets soils samples, respectively. Seasonally, 16.7% of the spring samples and 28.4% of the autumn samples were positive. Based on molecular identification, three species of two genera were identified viz. M. fulvum (n = 57), M. canis (n = 2) and zoophilic Trichophyton interdigitale (n = 1). As a specific goal in the study, differentiation of the species in Microsporum gypseum complex was established by measuring the mean length and width of macroconidia in some strains of M. gypseum, M. fulvum and M. incurvatum. Mean size for macroconidia length and width in three species showed that M. gypseum and M. incurvatum can morphologically be differentiated from M. fulvum but not from each other. M. fulvum was the most abundant species isolated from the soils of Ahvaz; however, to comprehensively specify the distribution pattern of geophilic dermatophytes in the soils of this city further investigations are needed. Identification based on micro-morphometric is not effective for species distinction in M. gypseum complex, while molecular procedures based on sequencing of certain DNA regions are the most reliable and applicable strategies for this purpose.

Gliotoxin, identified from a screen of fungal metabolites, disrupts 7SK snRNP, releases P-TEFb, and reverses HIV-1 latency., A leading pharmacological strategy toward HIV cure requires “shock” or activation of HIV gene expression in latently infected cells with latency reversal agents (LRAs) followed by their subsequent clearance. In a screen for novel LRAs, we used fungal secondary metabolites as a source of bioactive molecules. Using orthogonal mass spectrometry (MS) coupled to latency reversal bioassays, we identified gliotoxin (GTX) as a novel LRA. GTX significantly induced HIV-1 gene expression in latent ex vivo infected primary cells and in CD4+ T cells from all aviremic HIV-1+ participants. RNA sequencing identified 7SK RNA, the scaffold of the positive transcription elongation factor b (P-TEFb) inhibitory 7SK small nuclear ribonucleoprotein (snRNP) complex, to be significantly reduced upon GTX treatment of CD4+ T cells. GTX directly disrupted 7SK snRNP by targeting La-related protein 7 (LARP7), releasing active P-TEFb, which phosphorylated RNA polymerase II (Pol II) C-terminal domain (CTD), inducing HIV transcription.

A leading pharmacological strategy towards HIV cure requires “shock” or activation of HIV gene expression in latently infected cells with Latency Reversal Agents (LRAs) followed by their subsequent clearance. In a screen for novel LRAs we used fungal secondary metabolites (extrolites) as a source of bio-active molecules. Using orthogonal mass spectrometry (MS) coupled to latency reversal bioassays, we identified gliotoxin (GTX) as a novel LRA. GTX significantly induced HIV-1 gene expression in latent ex vivo infected primary cells and in CD4+ T cells from all aviremic HIV-1+ participants. RNA sequencing identified 7SK RNA, the scaffold of the P-TEFb inhibitory 7SK snRNP complex to be significantly reduced upon GTX treatment of independent donor CD4+T cells. GTX disrupted 7SK snRNP, releasing active P-TEFb, which then phosphorylated RNA Pol II CTD, inducing HIV transcription. Our data highlight the power of combining a medium throughput bioassay, mycology and orthogonal mass spectrometry to identify novel potentially therapeutic compounds.

Despite the increasing occurrence of Candida orthopsilosis and Candida metapsilosis in clinical settings, little is known about their microbiological and clinical properties. Herein, we conducted a national retrospective study (2014–2019) from multiple centers in Iran. Among the 1,770 Candida isolates collected, we identified 600 Candida parapsilosis species complex isolates. Isolate identification was performed by 9-plex PCR, matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF MS), and rDNA sequencing, and antifungal susceptibility testing (AFST) followed CLSI M27-A3/S4; genotyping was performed by amplified fragment length polymorphism (AFLP) analysis; and clinical information was mined. Thirty-one isolates of C. orthopsilosis from various clinical sources, one mixed sample (blood) concurrently containing C. orthopsilosis and C. parapsilosis and one isolate of C. metapsilosis from a nail sample were identified. Although both 9-plex PCR and MALDI-TOF successfully identified all isolates, only 9-plex PCR could identify the agents in a mixed sample. For the C. orthopsilosis isolates, resistance (non-wild type) was noted only for itraconazole (n = 4; 12.5%). Anidulafungin and fluconazole showed the highest and voriconazole had the lowest geometric mean values. AFLP analysis showed three main and four minor genotypes. Interestingly, 90% of nail isolates clustered with 80% of the blood isolates within two clusters, and four blood isolates recovered from four patients admitted to a hospital clustered into two genotypes and showed a high degree of similarity (>99.2%), which suggests that C. orthopsilosis disseminates horizontally. Supported by our data and published case studies, C. orthopsilosis and C. metapsilosis can be linked to challenging clinical failures, and successful outcomes are not always mirrored by in vitro susceptibility. Accordingly, conducting nationwide studies may provide more comprehensive data, which is required for a better prognosis and clinical management of patients.

Establishing an effective empirical antifungal therapy requires that national surveillance studies be conducted. Herein, we report the clinical outcome of infections with and the microbiological features of Iranian isolates of Candida glabrata derived from patients suffering from candidemia. C. glabrata isolates were retrospectively collected from four major cities in Iran; identified by a 21-plex PCR, matrix-assisted laser desorption ionization–time of flight mass spectrometry, and large subunit of ribosomal DNA sequencing; and genotyped by amplified fragment length polymorphism (AFLP). Mutations in PDR1, ERG11, and hot spot 1 (HS1) of FKS1 and FKS2 were investigated, and antifungal susceptibility testing (AFST) was performed (by the CLSI M27-A3 and M27-S4 methods). Seventy isolates of C. glabrata were collected from 65 patients with a median age of 58 years. Fluconazole was the most widely used (29.23%) and least effective antifungal agent. The overall crude mortality rate was 35.4%. Only one strain was resistant to fluconazole, and 57.7% and 37.5% of the isolates were non-wild type (non-WT) for susceptibility to caspofungin and voriconazole, respectively. All isolates showed the WT phenotype for amphotericin B, posaconazole, and itraconazole. HS1 of FKS1 and FKS2 did not harbor any mutations, while numerous missense mutations were observed in PDR1 and ERG11. AFLP clustered our isolates into nine genotypes; among them, genotypes 1 and 2 were significantly associated with a higher mortality rate (P=0.034 and P= 0.022, a PDR1, T745A, died despite treatment with fluconazole or caspofungin. Overall, Iranian isolates of C. glabrata were susceptible to the major antifungal drugs. Application of genotyping techniques and sequencing of a specific gene (PDR1) might have prognostic implications.

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.

The fungi Trichophyton mentagrophytes and T interdigitale account for significant amount of dermatophytosis cases worldwide. These two dermatophytes form a species complex and have a number of ribosomal internal transcribed spacer (ITS) region genotypes, allowing simultaneous species identification and strain typing. Our aim was to describe the geographic distribution of T mentagrophytes/T interdigitale ITS region genotypes and find an association between the genotypes and clinical presentations of respective infections. We performed rDNA ITS region sequencing in 397 Iranian T mentagrophytes/T interdigitale isolates and analysed all available in GenBank entries with sequences of this kind. For the study, 515 clinical annotations were available. Statistical analysis was performed by chi-squared test and Spearman rank correlation analysis. A total of 971 sequences belonged to genotypes with at least 10 geographic annotations and were classified on the basis of exclusive occurrence in a particular region or high relative contribution to a regional sample. We discerned Asian and Oceanian ("KU496915" Type V, "KT192500" Type VIII, "KU315316"), European ("FM986750" Type III, "MF926358" Type III*, "KT285210" Type VI) and cosmopolitan ("FM986691" Type I, "JX122216" Type II, "KP132819" Type II* and "AF170453" Type XXIV) genotypes. There was statistically significant difference in the ITS genotype distribution between different affected body sites. Trichophyton mentagrophytes "KT192500" Type VIII correlated with tinea cruris, T mentagrophytes "KU496915" Type V correlated with tinea corporis, T interdigitale "JX122216" Type II correlated with tinea pedis and onychomycosis. Trichophyton mentagrophytes and T interdigitale genotypes can be associated with distinct geographic locations and particular clinical presentations.

Purpose. Identification of the emerging yeast species Candida nivariensis among presumptively identified Iranian Candida glabrata isolates. Methodology. Clinical C. glabrata species complex isolates from blood (n=100; 46.9%), vaginal swabs (n=20; 9.4%), bronchoalveolar lavage (n=10; 4.7%) and sputum (n=12; 5.6%) from 68 patients from Iran were investigated. Isolates were characterized by CHROMagar, multiplex PCRs, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), amplified fragment length polymorphism (AFLP) fingerprinting, internal transcribed spacer (ITS)/large subunit (LSU) rDNA and FKS1/FKS2 sequencing, and the European Committee on Antimicrobial Susceptibility Testing broth microdilution method. A comprehensive literature review was conducted and all the relevant clinical and microbiological data were collected.Results. Four C. nivariensis isolates were recovered from blood samples of three subjects and were all consistently identified by nine-plex PCR, Bruker MALDI-TOF MS, and LSU and ITS rDNA sequencing. AFLP genotyping clustered the isolates into two groups. Sequencing of the FKS1 and FKS2 hotspots showed no accountable amino acid substitutions. All isolates were susceptible to amphotericin B, fluconazole, itraconazole, posaconazole, voriconazole, anidulafungin and micafungin.Conclusion. In total, 4 out of 213 clinical C. glabrata species complex candidemia isolates were C. nivariensis. Improvement of the BioMerieux Vitek MS database is required to accurately identify C. nivariensis and it is advised to alternatively use CHROMagar and/or PCR-based techniques. As other species within the Nakaseomyces clade may cause infection and showed high MIC values for antifungals, inclusion of their spectra into the MALDI-TOF MS database seems relevant. Due to developing resistance to fluconazole and insufficient efficacy of caspofungin, the combination of catheter removal plus treatment with caspofungin, or voriconazole, or micafungin might be effective for patients.

Summary: The spa gene occurs in all strains of Staphylococcus aureus (S. aureus), can function as a genetic marker and might be used distinguish strains at the species level. Hence, due to these advantages, we used spa typing and the Based Upon Repeat Pattern (BURP) to assign the clonal and phylogenetic relationships of S. aureus strains. The sensitivity of S. aureus strains to methicillin was determined using agar disk diffusion. The extracted DNA from 56 isolates of S. aureus was subjected to PCR to detect the spa gene with specific primers. The spa typing method was performed for each of the isolates, and then, BURP was used to cluster spa types (spa-CCs). Finally, using relevant software, the phylogenic tree was drawn. The results of this study showed that 25 out of 56 (44.6%) isolates were resistant to methicillin. The typing of S. aureus isolates revealed 24 different spa types among 56 isolates, and BURP analysis clustered the 24 spa types into 5 spa clonal complexes (CCs) and 12 singletons. The process of spa typing, in combination with BURP analysis, provides an efficient method for investigating phylogenetic and clonal relationships among clinical isolates and can be useful for monitoring bacterial spread between hospitals and communities as well as between and within hospitals. Keywords: Staphylococcus aureus, spa gene, BURP, Phylogenetic relationships

Several species of the genus Exophiala are found as opportunistic pathogens on humans, while others cause infections in cold-blooded waterborne vertebrates. Opportunism of these fungi thus is likely to be multifactorial. Ecological traits [thermotolerance and pH tolerance, laccase activity, assimilation of mineral oil, and decolorization of Remazol Brilliant Blue R (RBBR)] were studied in a set of 40 strains of mesophilic Exophiala species focused on the salmonis-clade mainly containing waterborne species. Thermophilic species and waterborne species outside the salmonis-clade were included for comparison. Strains were able to tolerate a wide range of pHs, although optimal growth was observed between pH 4.0 and 5.5. All strains tested were laccase positive. Strains were able to grow in the presence of the compounds (mineral oil and RBBR) with some differences in assimilation patterns between strains tested and also were capable of degrading the main chromophore of RBBR. The study revealed that distantly related mesophilic species behave similarly, and no particular trend in evolutionary adaptation was observed.

Background and Purpose: Candidiasis is a major fungal infection, and Candida albicans is the major cause of infections in humans. The Clinical and Laboratory Standards Institute (CLSI) developed new breakpoints for antifungal agents against C. albicans. In this multi-center study, we aimed to determine the drug susceptibility profile of C. albicans, isolated from Iranian population according to new species-specific CLSI. Materials and Methods: Clinical samples were cultured on Sabouraud dextrose agar and were incubated at room temperature for seven days. The isolates were transferred to Professor Alborzi Clinical Microbiology Research Center, Shiraz, Iran. C. albicans were identified by using API 20C AUX system. Broth microdilution method was used to determine the minimum inhibitory concentrations (MICs) of amphotericin B, caspofungin, voriconazole, fluconazole, posaconazole, itraconazole, and ketoconazole, based on CLSI document M27-S4 and new breakpoints for some azoles and caspofungin. Results: Overall, 397 C. albicans were isolated from patients admitted to ten university hospitals in Iran. The MIC90 of the isolates to amphotericin B, caspofungin, voriconazole, fluconazole, posaconazole, itraconazole, and ketoconazole were 0.125, 0.125, 0.125, 1, 0.064, 0.5, and 0.125 μg/ml, and rates of resistance were 0.5%, 0.3%, 3.8%, 2.8%, and 2.5% for amphotericin B, caspofungin, voriconazole, fluconazole, and itraconazole, respectively. Conclusion: According to our data, fluconazole is the drug of choice for management of patients at risk for systemic candidiasis throughout the region, since it is cost-effective with low side effects.

BACKGROUND: Tinea capitis is very common in Western China, with the most widespread aetiological agent being Trichophyton violaceum, while Microsporum canis is prevalent in the remainder of China. Conventional diagnostics and internal transcribed spacer (ITS) sequencing analyses have proven relatively limited due to the close phylogenetic relationship of anthropophilic dermatophytes. Therefore, alternative molecular tools with sufficient specificity, reproducibility and sensitivity are necessary. OBJECTIVES: To evaluate two molecular techniques [multiplex ligation-dependent probe amplification (MLPA) and rolling circle amplification (RCA)] for rapid detection of the aetiological agents of tinea capitis, T. violaceum and M. canis. METHODS: Probes of RCA and MLPA were designed with target sequences in the rDNA ITS gene region. Strains tested consist of 31 T. violaceum, 22 M. canis and 24 reference strains of species that are taxonomically close to the target species. RESULTS: The specificity and reproducibility of RCA and MLPA in detection of T. violaceum and M. canis were both 100% in both species. Sensitivity testing showed that RCA was positive at concentrations down to 1·68 × 10(6) copies of DNA in the TvioRCA probe, and 2·7 × 10(8) copies of DNA in McRCA. MLPA yielded positive results at concentrations of DNA down to 1·68 × 10(1) copies in the TvioMLPA probe and 2·7 × 10(2) in McMLPA. CONCLUSIONS: The two techniques were sufficiently specific and sensitive for discriminating the target DNA of T. violaceum and M. canis from that of closely related dermatophytes. RCA and MLPA are advantageous in their reliability and ease of operation compared with standard polymerase chain reaction and conventional methods.

Incidence of fungal sinusitis due to the genus Fusarium has increased during the last two decades. We report a case of fungus ball sinusitis with multiple sinuses involvement in an Iranian 21-year-old woman. The patient was diagnosed as having a fungus ball-type sinusitis in computed tomography scan. The sinus biopsy revealed fungal structures on histopathological and direct microscopic examinations and a Fusarium species arose in culture. Partial sequencing of the translation elongation factor 1-alpha identified the isolate as F. proliferatum. Removal of all lesions by endoscopic surgery resulted in a favorable outcome. To the best of our knowledge, this is the first case of F. proliferatum-associated fungus ball which involved multi-sinus and highlights the efficiency of molecular methods for discrimination of fungal agents involved.

Background and Purpose: Pneumocystis pneumonia, caused by Pneumocystis jirovecii, is a fatal disease threatening patients with AIDS or immunosuppression. Assessment of colonization in these patients is of great significance, since it can lead to severe pulmonary disorders. Considering the scarcity of published reports on Pneumocystis jirovecii isolates from patients in Mashhad, Iran, we aimed to evaluate pneumocystis colonization in individuals with different pulmonary disorders. Materials and Methods: We used nested polymerase chain reaction (PCR) method to amplify mitochondrial large subunit-ribosomal ribonucleic acid (mtLSU-rRNA) gene in 60 bronchoalveolar lavage (BAL) samples, obtained from patients, referring to the Department of Internal Medicine (Pulmonary Diseases Section) at Imam Reza Hospital, affiliated to Mashhad University of Medical Sciences, Mashhad, Iran. Results: DNA of Pneumocystis jirovecii was detected in 10 out of 60 BAL samples (16.66%) via nested PCR method. Conclusion: According to the present findings, the colonization rate of Pneumocystis jirovecii was similar to the rates reported in other similar studies in Iran.

The aim of this study was to develop and evaluate a padlock probe based on the Rolling Circle Amplific ation (RCA), which targeted to 16S-23S rDNA region of S. mutans . The specificity of developed padlock probe was te sted for DNA within a panel strains, including S. mutans isolated from the saliva and reference strains of the genus Streptococcus , as well as total DNA samples of biofilm and saliv a. The results were positive either for DNA samples of S. mutans or DNA samples recovered from the biofilm and sali va revealing the specificity of designed padlock probe. The padlock probe based on the RCA was prove d to be an effective, reproducible method for S. mutans detection and demonstrated the possibility of a ra pid detection and accurate identification of S. mutans infection.

Members of the family Chaetomiaceae are ubiquitous ascosporulating fungi commonly, which reside in soil enriched with manure or cellulosic materials. Their role as human pathogens is largely ignored. However, the ability of some species to grow at high temperature enables them to play an important role as opportunistic pathogens. The family contains several genera and species that have never been reported to cause human infection. Hereby, three new species are described; two belong to the genus Subramaniula and one represents a Chaetomium species. Subramaniula asteroides was isolated from various sources including eye and skin infections as well as from the natural environment, and S. obscura was isolated from a toe infection. Chaetomium anamorphosum was isolated from a kidney transplant patient suffering from fungal peritonitis. All species described were previously misidentified as Papulaspora spp. due to the formation of cellular clumps or bulbil-like structures, which are characteristic of Papulaspora. The isolates failed to form sexual fruit bodies and ascospores remained absent, which is an unusual feature for the generally ascosporulating genera Chaetomium and Subramaniula; minute conidia from phialides were sometimes observed.

Background Antifungal susceptibility testing is a subject of interest in the field of medical mycology. The aim of the present study were the distributions and antifungal susceptibility patterns of various Candida species isolated from colonized and infected immunocompromised patients admitted to ten university hospitals in Iran. Methods In totally, 846 Candida species were isolated from more than 4000 clinical samples and identified by the API 20 C AUX system. Antifungal susceptibility testing was performed by broth microdilution method according to CLSI. Results The most frequent Candida species isolated from all patients was Candida albicans (510/846). The epidemiological cutoff value and percentage of wild-type species for amphotericin B and fluconazole in Candida albicans, Candida tropicalis, Candida glabrata and Candida krusei were 0.5 μg/ml (95%) and 4 μg/ml (96%); 1 μg/ml (95%) and 8 μg/ml (95%); 0.5 μg/ml (99%) and 19 μg/ml (98%); and 4 μg/ml (95%) and 64 μg/ml (95%), respectively. The MIC90 and epidemiological cutoff values to posaconazole in Candida krusei were 0.5 μg/ml. There were significant differences between infecting and colonizing isolates of Candida tropicalis in MIC 90 values of amphotericin B, and isolates of Candida glabrata in values of amphotericin B, caspofungin, and voriconazole (P

Background: Invasive fungal infections without proper treatment could lead to high mortality rate, especially in immunocompromised patients. Candida species distribution and drug susceptibility patterns vary in different areas. Understanding the etiologic agents and drug susceptibility patterns in each region are required for the best management of patients with Candida infections. Objectives: The aim of this study was to identify Candida species isolated from clinical samples of six university hospitals in Iran and detect their susceptibility patterns to seven antifungal agents. Methods: Clinical samples from patients with fungal infections were cultured on Sabouraud dextrose agar. Isolated yeasts were identified by API 20C AUX kit, according to the manufacturer’s instructions. Drug susceptibility patterns to amphotericin B, caspofungin, voriconazole, fluconazole, posaconazole, itraconazole and ketoconazole were determined, according to CLSI M27-A3 and S4. Results: In total, 428 species of Candida were isolated from clinical samples (1950 samples). Most isolated species were Candida albicans, followed by C. tropicalis, C. parapsilosis, C. kefyr, C. famata, C. glabrata, C. krusei, C. dubliniensis, C. guilliermondii and C. lusitaniae. Sensitivity rate of C. albicans to amphotericin B, caspofungin, voriconazole, fluconazole, and itraconazole was 96.6, 99.5, 88.6, 90.6, and 52 with MIC90 values equal to 0.25 µg/mL, 0.125µg/mL, 0.125 µg/mL, 2 µg/mL, and 1 µg/mL, respectively. The MIC 90 values for ketoconazole and posaconazole were 0.125µg/mL and 0.064µg/mL, respectively. Different sensitivity to antifungal agents was present in non-albicans Candida species especially in C. krusei, C. glabrata, and C. tropicalis. Conclusions: According to this study, C. albicans is the most prevalent etiologic agent in infected patients and caspofungin is the most effective antifungal agent. Knowledge about etiologic agents and their susceptibility patterns in each region is helpful for successful treatment of the patients. © 2017, Jundishapur Journal of Microbiology.

In this study, we developed rapid and sensitive assays for the detection of Cladophialophora carrionii , a common agent of human chromoblastomycosis. The isothermal techniques evaluated were rolling-circle amplification (RCA), multiplex ligation-dependent probe amplification (MLPA), and loop-mediated isothermal amplification (LAMP). The probes for RCA and MLPA were designed with target sequences in the rDNA internal transcribed spacer gene ( ITS ) region, and LAMP primers were designed using the elongation factor 1α gene ( EF1 ); these probes and primers specifically amplified DNA of isolates of the species. The three techniques were sufficiently specific and sensitive for discriminating target DNA of C. carrionii from that of related Cladophialophora species and other agents of chromoblastomycosis. RCA, MLPA, and LAMP are advantageous in their reliability and ease of operation compared to standard PCR and conventional methods.

Infectious arthritis due to Candida glabrata is very rare. A 40-year-old Iranian man had developed a painful swelling on the left knee since a year ago. A surgery (meniscectomy) was performed on his knee. However, in follow-up visit after 2 months, the patient's condition was deteriorated. Direct examination of synovial fluid with Gram and hematoxylin-eosin stains were negative for any bacterial or fungal infection or crystal elements; however, inoculation into BACTEC™ Mycosis IC/F and Plus Aerobic/F culture bottles led to the isolation of a yeast strain. The macroscopic examination on CHROMagar™ Candida medium combined with microscopical examination on CMT80 agar made a presumptive identification of the isolate to be considered as C. glabrata, and it was later on confirmed by ITS sequencing. Initial empirical treatment was started with intravenous amphotericin B for 4 weeks followed by oral itraconazole which was unsuccessful. Prescription of an oral 150-mg tablet of fluconazole was considered for a 2-month course. All symptoms completely declined, and no recurrence of infection was detected. Antifungal susceptibility testing (AFST) was performed for this isolate, and the result showed sensitivity to both amphotericin B and itraconazole and less susceptibility to fluconazole while clinical recovery was achieved by fluconazole. In any suspected clinical case caused by infectious agents, application of an effective fungal diagnostic test should be considered to avoid complications due to misdiagnosis. The correlation of AFST result with real in vivo therapeutic responses can be strain or patient dependent, and this should be considered for a successive treatment.

Cyphellophora is a genus of black yeast-like fungi characterised by having simple phialides with multiseptate, curved conidia. Judging from SSU and LSU data, Cyphellophora was found to be located in a well-supported clade within the Chaetothyriales comprising a number of species occurring on human skin and nail. Cyphellophora is phylogenetically close to Phialophora europaea, P. reptans and P. oxyspora, though morphologically these species produce single-celled phialoconidia rather than multiseptate ones. Pseudomicrodochium suttonii and P. fusarioides have dark colonies and phylogenetically fit in with Cyphellophora; the type species of Pseudomicrodochium, P. aciculare, has similar, septate conidia but has a hyaline thallus. In the present study, multilocus phylogenetic analyses were combined with morphology and physiology. Sequences of the internal transcribed spacer region, the DNA dependent RNA polymerase II largest subunit and the partial beta tubulin gene were analysed for a set of 30 strains. Two novel species, Cyphellophora pauciseptata and Phialophora ambigua were discovered. Cyphellophora eucalypti was reduced to synonymy of C. guyanensis. The role of the studied fungi between colonization and infection of human skin was discussed. Putative virulence factors for these black yeast-like fungi were hypothesized to be the ability to assimilate monoaromatic hydrocarbons, to produce melanin pigments, and to tolerate the temperature of epidermal human skin.

We investigated the resolving power of the beta tubulin protein-coding gene (BT2) for systematic study of dermatophyte fungi. Initially, 144 standard and clinical strains belonging to 26 species in the genera Trichophyton, Microsporum, and Epidermophyton were identified by internal transcribe spacer (ITS) sequencing. Subsequently, BT2 was partially amplified in all strains, and sequence analysis performed after construction of a BT2 database that showed length ranged from approximately 723 (T. ajelloi) to 808 nucleotides (M. persicolor) in different species. Intraspecific sequence variation was found in some species, but T. tonsurans, T. equinum, T. concentricum, T. verrucosum, T. rubrum, T. violaceum, T. eriotrephon, E. floccosum, M. canis, M. ferrugineum, and M. audouinii were invariant. The sequences were found to be relatively conserved among different strains of the same species. The species with the closest resemblance were Arthroderma benhamiae and T. concentricum and T. tonsurans and T. equinum with 100% and 99.8% identity, respectively; the most distant species were M. persicolor and M. amazonicum. The dendrogram obtained from BT2 topology was almost compatible with the species concept based on ITS sequencing, and similar clades and species were distinguished in the BT2 tree. Here, beta tubulin was characterized in a wide range of dermatophytes in order to assess intra- and interspecies variation and resolution and was found to be a taxonomically valuable gene.

The in vitro activities of eight antifungal drugs against 106 clinical and environmental isolates of waterborne and cutaneous Exophiala species were tested. The MICs and minimum effective concentrations for 90% of the strains tested ( n = 106) were, in increasing order, as follows: posaconazole, 0.063 μg/ml; itraconazole, 0.25 μg/ml; micafungin, 1 μg/ml; voriconazole, 2 μg/ml; isavuconazole, 4 μg/ml; caspofungin, 8 μg/ml; amphotericin B, 16 μg/ml; fluconazole, 64 μg/ml.