Genotypes analysis of Candida albicans species complex from healthy individual saliva in Ahvaz, Iran.

Although Candida albicans is a part of the mycoflora of healthy individuals, it is causing mild to severe forms of candidiasis in patients with underlying diseases. The recent molecular investigations were classified three genotypes, A, B and C for C. albicans. The aim of the present study was to detect different genotypes of C. albicans complex species in a normal population in Iran. Saliva was randomly collected from a normal population, homogenized and cultured on CHROMagar Candida. Classical and molecular methods were used for the detection of isolates. Candida 25S rDNA gene was amplified using the primer pairs of CA-INT-L and CA-INT-R for ABC genotyping of C. albicans. Candida albicans complex was recovered from 103/194 (53·1%) normal papulation. Genotype A with a frequency of 41·7% was the most common isolate, followed by genotype C (34%), genotype B (20·4%) and genotype D (3·9%). Genotyping of C. albicans species complex from healthy individuals showed the presence of three A, B and C genotypes of C. albicans and one D genotype of C. dubliniensis.

Genotypic diversity and antifungal susceptibility pattern of Candida albicans species isolated from hospitalized paediatric patients with urinary tract infection in Iran.

AIMS: The present study aimed to determine the microsatellite length polymorphism (MLP) genotypic patterns and antifungal susceptibility profiles of Candida albicans isolated from patients with candiduria. METHODS AND RESULTS: DNA of 50 C. albicans isolates was used for molecular identification based on the ITS1 -5.8s-ITS2 region. MLP assays were performed to amplify three loci (EF3, CDC3 and HIS3), and PCR products were used for fragment analysis. Antifungal susceptibility tests were performed according to CLSI M27 4th ed guidelines. In all, 38 different genotypes were detected with the three polymorphic loci among C. albicans isolates, and only one genotype was homozygous. In comparison to other countries, our genotypes were divided into three clusters, two of which were linked to France and a third of which was linked to Austria. The genetic structures of the 50 C. albicans isolates revealed varied heterozygosity and significant Hardy-Weinberg equilibrium at the EF3 locus. Only one (2%) and four (8%) of isolates showed resistance to fluconazole and itraconazole, respectively. In C. albicans genotype G25, one (2%) of the isolates showed cross-resistance and non-wild-type resistance to posaconazole, itraconazole and fluconazole. CONCLUSION: MLP typing is a useful tool to analyse the genetic structure of C. albicans isolates. High genetic diversity (38 genotypes) was detected in the isolates tested here. Compared to isolates in other countries, the ones from our patients had a clear relationship with French and Austrian isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: Iranian isolates of C. albicans have a distinct genotype and show similarities only with French and Austrian isolates.

Rapid Identification of Seven Waterborne Exophiala Species by RCA DNA Padlock Probes.

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.

Correction to: Rapid Identification of Seven Waterborne Exophiala Species by RCA DNA Padlock Probes.

The Editorial Office of Mycopathologia reports that several paragraphs of Najafzadeh et al. were transcribed with only minor edits from previously published material by Najafzadeh M.J.

In vitro activities of eight antifungal drugs against 106 waterborne and cutaneous exophiala species.

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.

The patterns of colonization and antifungal susceptibility of Candida, isolated from preterm neonates in Khorramabad, South West of Iran.

OBJECTIVE: Usually, 7-20% of preterm neonates colonized by Candida species present invasive candidiasis. Candida albicans, and several non-albicans species cause invasive infection with C. albicans being the most dominant agent. In the last two decades, infection due to non-albicans have been increased dramatically due to their low sensitivity to antifungal drugs such as fluconazole. The aim of present study was to evaluate Candida colonization pattern and antifungal susceptibility among preterm neonates from Khorramabad, South west of Iran. MATERIALS AND METHODS: Samples were collected from 80 preterm neonates, cultured on CHROMagar Candida and incubated at 37°C. All recovered isolates were primarily screened based on classical methods and identified by PCR-RFLP targeting the ITS-rDNA regions. Antifungal susceptibility testing of all isolates was performed according to the CLSI method against amphotericin B, caspofungin, itraconazole, fluconazole and voriconazole. RESULTS: Totally 23 isolates of Candida species were recovered from 20 patients (female: male, 50:50) including, C. albicans (18), C. parapsilosis (2) and C. glabrata (1). Furthermore, the blood cultures from two patients were yielded C. albicans and C. parapsilosis so that patient with C. albicans died after five days. Generally, in this study, 9 (39.1%) isolates were resistant to amphotericin B including; 7 (30.4%) C. albicans and 2 (8.7%) C. parapsilosis. In addition, 2 (8.7%) and 4 (17.4%) isolates were also resistant to itraconazole and caspofungin, respectively. CONCLUSION: In conclusion, Candida colonization among preterm neonates is still an important issue in hospitals. In addition, in spite of a significant amphotericin B resistant Candida, voriconazole, fluconazole, and itraconazole are valuable antifungals, due to fully sensitivity to Candida.

Erratum for Mirhendi et al., the first case of onychomycosis in a koala (Phascolarctos cinereus) due to atypical isolates of Microsporum gypseum, a diagnostic challenge.

[This corrects the article DOI: 10.18869/acadpub.cmm.2.2.2.].

Luliconazole, a new antifungal against Candida species isolated from different sources.

OBJECTIVE: Luliconazole is an inhibitor for sterol 14-α-demethylase in fungal cells with a broad-spectrum antifungal activity against dermatophytes, Candida albicans, Malassezia species, dematiaceous and hyaline hyphomycetes. Furthermore, luliconazole has been clinically used for the treatment of pityriasis versicolor, dermatophytosis, onychomycosis, cutaneous and mucocutaneous candidiasis. In the present study, we aimed to evaluate in vitro antifungal activity of luliconazole against several strains of Candida species recovered from different clinical materials. MATERIALS AND METHODS: In the present study, 104 strains of Candida species including, 34 isolates from vaginitis, 23 isolates from AIDS patients with vaginal candidiasis, 24 isolates from neutropenic patients and 24 isolates from tracheal tubes, were examined for susceptibility tests. A serial dilution of luliconazole (4-0.008µg/mL) was tested against different strains of Candida species recovered from different sources. RESULTS: The minimum inhibitory concentration (MIC) range and MIC90 of vaginal isolates (HIV-) were 1-0.063 and 1µg/mL. Furthermore, the most of strains (50%) had a MIC of 0.5µg/mL. The MIC ranges were similar (2-0.016µg/mL) for both vaginal (HIV+) and neutropenic patients isolates, whereas, MIC90 for them were 0.5 and 1µg/mL, respectively. All tracheal tubes strains were inhibited at the range of 2-0.008µg/mL with MIC90=1µg/mL. Totally, the lowest MIC50 (MIC=0.015µg/mL), MIC90 (MIC=1µg/mL) and MICGM (MIC=0.05µg/mL) are correlated to C. glabrata, a non-albicans species. CONCLUSION: It is concluded that, luliconazole could be an alternative anti-Candida agent, however, in vivo studies must be confirmed usefulness of drug for clinical usage.

Mucormycosis in Iran: A six-year retrospective experience.

Mucormycosis is a devastating infection caused by Mucoralean fungi (Mucormycotina, Mucorales). Data concerning the global epidemiology of mucormycosis are scarce and little is known about the characteristics of mucormycosis in Iran. In this study, we aimed to understand the distribution of this infection in Iran retrospectively and to ascertain whether the patterns of infection are associated with specific host factors or not. A total of 208 cases were included in this study occurring during 2008-2014 and were validated according to (EORTC/MSG) criteria. A rising trend as significant increase from 9.7% in 2008 to 23.7% in 2014 was observed. The majority of patients were female (51.4%) with median age of 50 and the infections were seen mostly in autumn season (39.4%). Diabetes mellitus (75.4%) was the most common underlying condition and sinus involvement (86%) was the mostly affected site of infection. Amphotericin B (AmB) was the drug of choice for the majority of cases. Sixty four isolates did not show any growth in the lab and only 21 cases were evaluated by ITS sequencing, among them; Rhizopus arrhizus var. arrhizus was the dominant species. Considering the high mortality rate of mucormycosis, early and accurate diagnosis, with the aid of molecular methods may provide accurate treatments and improve the survival rate. Therefore, increased monitoring and awareness of this life-threatening disease is critical.

Comparison of enzymatic activities in different Candida species isolated from women with vulvovaginitis.

INTRODUCTION: Comparing the activities of secreted enzymes in different fungal species can improve our understanding of their pathogenic role. Secretion of various enzymes by Candida species has been considered for determination of their virulence in different Candida infections including vulvovaginitis. AIMS: The aim of this study was to determine and compare the activity of secreted enzymes in Candidia strains isolated from women suspected to vulvovaginal candidiasis (VVC) and referred to some health centers in Khuzestan, Southwestern Iran. PATIENTS AND METHODS: The vaginal secretion samples were taken by swap from 250 suspected women with symptoms of vulvovaginal candidiasis and cultured on CHROMagar Candida medium. Identification of the isolated Candida from culture positive samples performed by the color of colonies and some standard mycological procedures. Activities of phospholipase, hemolysin-α, hemolysin-ß, esterase and proteinase were measured in vitro by standard laboratory protocols. The enzymatic activity index (EAI) was calculated for each enzyme in accordance to relevant protocols. RESULTS: Totally in eighty cases (32%), a Candida strain was isolated which found to be as 52 (65%) Candida albicans; 12 (15%) C. glabrata; 10 (12.5%) C. dubliniensis; 4 (5%) C. krusei, C. tropicalis and C. parapsilosis species (each=1; 1.3%). Among C. albicans strains, 89.1% produced all studied enzymes, while 86% of C. glabrata strains failed to produce proteinase and phospholipase. The EAIs in decreasing order were as hemolysin-ß=0.2895, hemolysin-α=0.5420, esterase=0.5753, proteinase=0.7413, and phospholipase=0.7446, respectively. Activity of phospholipase, esterase and proteinase secreted by C. albicans and C. dubliniensis were significantly more than those released by C. glabrata and C. krusei, while 86% of C. glabrata strains did not show esterase activity. On the other hand, the activity rates of hemolysin α and ß among all studied isolates were almost similar. CONCLUSION: In the present study, the prevalence of VVC among investigated women was higher than the previous report from Khuzestan but C. albicans has yet remained the predominant agent of VVC in this area. Given to the EAI, the virulence of C. albicans in VVC can be mediated by phospholipase, esterase and proteinases.

Epidemiological status of dermatophytosis in Guilan, north of Iran.

BACKGROUND AND PURPOSE: The epidemiological features of dermatophytoses have been characterized in many geographical locations of Iran, but not in Guilan, North of Iran. This study was carried out to determine the distribution pattern of dermatophytoses and their relevant agents in Guilan, North of Iran, over a period of one year, from April 2010 to April 2011. MATERIALS AND METHODS: The clinical samples of skin, hair, and nail from 889 outpatients (317 men vs. 572 women) were used for direct microscopy and culture. All the culture-positive samples were then subjected to amplification of the internal transcribed spacer (ITS) of the nuclear rDNA followed by a restriction fragment length polymorphism (RFLP) assay to verify the causative agents. RESULTS: The infection was confirmed in 90 (44.3%) males and 113 (55.7%) females. The most common type of dermatophytoses was tinea cruris (42.9%), followed by tinea pedis (20.2%), tinea corporis (11.3%), tinea unguium (7.4%), tinea faciei (6.9%), tinea manuum (6.4%), and tinea capitis (4.9%). ITS-RFLP based of the identification of isolates, showed that the infections were significantly associated with anthropophilic species, of Trichophyton rubrum (41.9%), Epidermophyton floccosum (19.7%), T. tonsurans (5.4%), and T.violaceum (2%). Other causative agents were T. interdigitale (22.6%), Microsporum canis (4.9%), T. verrucosum (2.5%), and M. gypseum (1%). CONCLUSION: The higher prevalence of T. rubrum, as the agent of dermatophytoses, than other species has never been reported from Iran and is of public health concern because of the chronic nature of infections with anthropophilic species. To thoroughly investigate the epidemiological trend of dermatophytoses in Iran, further periodical and molecular-based studies are necessary.

Isolation and antifungal activity evaluation of Satureja khuzestanica Jamzad extract against some clinically important dermatophytes.

OBJECTIVE: Among the fungi, dermatophytes are the major cause of spectrum of superficial mycoses medically known as dermatophytosis (tinea) in human and animal. Treatment of these infections has still remained difficult. The aim of this survey was to evaluate in vitro anti-dermatophytic activity of ethanolic extract (EtOH) from Satureja khuzestanica leaf (SKLE) against some clinically important dermatophyte species from the genera of Trichophyton, Microsporum and Epidermophyton. Minimal inhibition concentration (MIC) of SKLE was tested against 14 dermatophyte strains of 5 species by using agar dilution method. Phytochemical screening of SKLE was carried out by High Performance Thin Layer Chromatography (HPTLC). The results of in vitro anti-dermatophytic activity of SKLE showed with MIC values between 1.250 and 10mg/mL. MIC90 and MIC50 values were as 0.625-1.250 and 0.156-0.312mg/mL, respectively. The MFC values of SKLE were in the range of 1.250-2.50mg/mL and possessed biological activity against dermatophytes. Morever, phytochemical analysis by HPTLC revealed that the ethyl acetate (EtOAc) extracts of SKL contain triterpenes which are known to have biological activity and it seems that this compound be responsible for the anti-dermatophytic activity of this plant. In conclusion, the results of in vitro antifungal susceptibility testing and phytochemical screening revealed that SKLE had both fungistatic and fungicidal activities against dermatophytes and can potentially be helpful as a supplementary or alternative for treatment of dermatophytosis.

Luliconazole, an alternative antifungal agent against Aspergillus terreus.

Aspergillus terreus is the fourth leading cause of invasive and non-invasive aspergillosis and one of the causative agents of morbidity and mortality among immunocompromised and high-risk patients. A. terreus appears to have increased as a cause of opportunistic fungal infections from superficial to serious invasive infections. Although, invasive aspergillosis is often treated empirically with amphotericin B, most A. terreus isolates are resistant both in vivo and in vitro to some antifungal drugs. In this study, we aimed to evaluate antifungals susceptibility profiles of the different strains of A. terreus against amphotericin B, caspofungin, fluconazole, voriconazole, posaconazole and luliconazole. Forty A. terreus strains originating from environmental sources (air and soil) were identified using by macroscopic and microscopic features. Six antifungals including, amphotericin B, caspofungin, fluconazole, voriconazole, posaconazole and luliconazole were applied for susceptibility tests. Our results show that tested isolates had different susceptibility to antifungals. The lowest MICGM related to luliconazole (0.00236µg/ml), followed by posaconazole (0.18621µg/ml), voriconazole (0.22925µg/ml), caspofungin (0.86µg/ml), fluconazole (8µg/ml) and amphotericin B (11.12µg/ml). This study demonstrated that luliconazole had an excellent in vitro activity against all tested isolates of A. terreus, with MICGM 0.00236µg/mL than other tested antifungals. As a result, luliconazole could be a possible alternative antifungal for the treatment of aspergillosis due to A. terreus.

The first case of onychomycosis in a koala (Phascolarctos cinereus) due to atypical isolates of Microsporum gypseum, a diagnostic challenge.

BACKGROUND AND PURPOSE: Superficial mycotic infections have been only poorly described in koalas and there are no reliable mycologically confirmed data regarding clinical isolation of dermatophytes in this animal. We report an 11-year-old female koala, kept in a zoo in Tokyo, Japan, and presenting with hyperkeratotic lesions and scaly plaques on forepaw claws and pads reminiscent of fungal infection. CASE REPORT: Direct microscopy of the scrapings was indicative of a dermatophyte infection. By culture and subsequent repeated subculturing of clinical specimens on Sabouraud dextrose agar, Mycobiotic agar, and potato dextrose agar, two distinct strains with different colony morphotypes (designed as types I and II) were identified. Macroscopic and microscopic characteristics of the strains were suggestive of three different species, i.e. Microsporum canis, M. gypseum, and M. fulvum. However, partial sequencing of internal transcribed spacer (ITS) region of rDNA, translation elongation factor-1α (Tef-1α), and beta-tubulin (BT2) genes confirmed the identity of both isolates as M. gypseum. The animal was treated with a continuous terbinafine regimen (250 mg/kg) once daily for 12 weeks. CONCLUSION: To the best of our knowledge, the present report is the first confirmed case of dermatophytosis in a koala. The genetics underlying a variety of phenotypic traits in most classical dermatophyte species are unknown, and further studies are needed to understand this phenomenon.

Aspergillus species as emerging causative agents of onychomycosis.

BACKGROUND: Onychomycosis is a common nail infection caused by dermatophytes, non-dermatophyte molds (NDM), and yeasts. Aspergillus species are emerging as increasing causes of toenail onychomycosis. The purpose of this study was species delineation of Aspergillus spp. isolated from patients with onychomycosis. METHODS: During a period of one year (2012-2013), nail samples were collected from patients clinically suspected of onychomycosis and subjected to microscopic examination and culture. Species identification was performed based on macro- and micro-morphology of colonies. For precise species identification, PCR-amplification and sequencing of the beta-tubulin gene followed by BLAST queries were performed where required. RESULTS: A total of 463/2,292 (20.2%) tested nails were diagnosed with onychomycosis. Among the positive specimens, 154 cases (33.2%) were identified as saprophytic NDM onychomycosis, 135 (29.2%) of which were attributable to Aspergillus. Aspergillus species isolated from the infected nails included Aspergillus flavus (77.3%, n=119), Aspergillus niger (n=4), Aspergillus tubingensis (n=4), Aspergillus terreus (n=3), Aspergillus sydowii (n=2), Aspergillus spp. (n=2), and Aspergillus candidus (n=1). Among the patients diagnosed with onychomycosis due to Aspergillus (average patient age, 47.4 years), 40 had fingernail and 95 toenail involvement. The large toenails were most commonly affected. CONCLUSIONS: This study identified a markedly high occurrence of A. flavus, and this fungus appears to be an emerging cause of saprophytic onychomycosis in Iran. The study moreover highlights the necessity of differentiating between dermatophytic and non-dermatophytic nail infections for informed decisions on appropriate therapy.

Use of Single-enzyme PCR-restriction Digestion Barcode Targeting the Internal Transcribed Spacers (ITS rDNA) to Identify Dermatophyte Species.

BACKGROUND: Dermatophytes are the most common causative agents of superficial mycoses. Species identification of these fungi is important from therapeutic and epidemiological point of wive. Traditional approaches for identification of dermatophytes at the species level, relying on macroscopic and microscopic features of the colonies, usually are time-consuming and unreliable in many circumstances. Recently a broad varieties of rapid and accurate DNA-based techniques were successfuly utilized for species delineation of dermatophytes. METHODS: The ITS1-5.8S-ITS2 region of rDNA from various reference strains of dermatophyte species were amplified using the universal fungal primers ITS1 and ITS4.The PCR products were digested by a single restriction enzyme, MvaI. The enzyme was evaluated in both in silico and practical PCR-RFLP assay to find the exact differentiating restriction profiles for each species. To validate the standardized PCR-RFLP system, all tested strains were subjected to sequencing and sequence analysis. RESULTS: The obtained RFLP patterns were specific for many species including T. interdigitale, T. rubrum, T. violaceum, M. persicolor, M. audouinii, M. nanum (A. obtusum) and E. floccosum but were similar for some closely related species such as M. canis / M. ferrugineum. Sequencing of the ITS1-5.8S-ITS2 fragment from all type strains affirmed the RFLP findings. CONCLUSION: It was practically revealed that the ITS-PCR followed by MvaI-RFLP is a useful and reliable schema for identification and differentiation of several pathogenic species and can be used for rapid screening of even closely related species of dermatophytes in clinical and epidemiological settings.