Development of a novel PCR assay for the identification of the black yeast, Exophiala (Wangiella) dermatitidis from adult patients with cystic fibrosis (CF)

Cystic fibrosis (CF) patients may suffer increased morbidity and mortality through colonisation, allergy and invasive infection from fungi. The black yeast, Exophiala dermatitidis (synonym Wangiella dermatitidis) has been found with increasing frequency in sputum specimens of CF patients, with reported isolation rates ranging from 1.1 to 15.7%. At present, no diagnostic PCR exists to aid with the clinical laboratory detection and identification of this organism. A novel species-specific PCR-based assay was developed for the detection of E. dermatitidis, based on employment of rDNA operons and interspacer (ITS) regions between these rDNA operons. Two novel primers, (designated ExdF & ExdR) were designed in silico with the aid of computer-aided alignment software and with the alignment of multiple species of Exophiala, as well as with other commonly described yeasts and filamentous fungi within CF sputum, including Candida, Aspergillus and Scedosporium. An amplicon of approximately 455 bp was generated, spanning the partial ITS1 region – the complete 5.8S rDNA region – partial ITS2 region, employing ExdF (forward primer [16-mer], 5′-CCG CCT ATT CAG GTC C-3′ and ExdR (reverse primer [16-mer], 5′-TCT CTC CCA CTC CCG C-3′, was employed and optimised on extracted genomic DNA from a well characterised culture of E. dermatitidis, as well as with high quality genomic DNA template from a further 16 unrelated fungi, including Candida albicans, C. dubliniensis, C. parapsilosis, C. glabrata, Scedosporium apiospermum, Penicillium sp., Aspergillus fumigatus, Aspergillus versicolor, Pichia guilliermondii, Rhodotorula sp., Trichosporon sp., Aureobasidium pullulans, Fusarium sp., Mucor hiemalis, Bionectria ochroleuca, Gibberella pulicaris. Results demonstrated that only DNA from E. dermatitidis gave an amplification product of the expected size, whilst none of the other fungi were amplifiable. Subsequent employment of this primer pair detected this yeast from mycological cultures from 2/50 (4%) adult CF patients. These two patients were the only patients who were previously shown to have a cultural history of E. dermatitidis from their sputum. E. dermatitidis is a slow-growing fungus, which usually takes up to two weeks to culture in the microbiology laboratory and therefore is slow to detect conventionally, with the risk of bacterial overgrowth from common co-habiting pan- and multiresistant bacterial pathogens from sputum, namely Pseudomonas aeruginosa and Burkholderia cepacia complex organisms, hence this species-specific PCR assay may help detect this organism from CF sputum more specifically and rapidly. Overall, employment of this novel assay may help in the understanding of the occurrence, aetiology and epidemiology of E. dermatitidis, as an emerging fungal agent in patients with CF.     

Decreasing Incidence of Trichophyton mentagrophytes in Korea: Analysis of 6,250 Cases during the Last 21-Year-Period (1992-2012)

Trichophyton mentagrophytes is the second common dermatophyte in Korea. However, few reports have been issued on the epidemiological and mycological characteristics of T. mentagrophytes in Korea based on long-term, large-scale study. The purpose of this study was to elucidate the epidemiological and mycological characteristics of T. mentagrophytes in Korea. During the 21-yr-period from 1992 to 2012, 6,250 patients with T. mentagrophytes were surveyed to determine annual incidence and the distribution of subjects by age, sex, season, involved sites, and place of residence. T. mentagrophytes infections were confirmed by fungal culture. In addition, the colony appearance of T. mentagrophytes was classified as granular, persicolor, powdery, or downy. Epidemiological analysis showed that annual incidence reached a peak in 2005, and then gradually decreased. T. mentagrophytes infection was most common in July, and was found predominantly in middle-aged adults, especially in those in their forties. Mycological analysis showed a powdery colony appearance was the most common, followed by persicolor and granular colonies. Toewebs were most frequently involved. This investigation on T. mentagrophytes provides insights into its incidence and characteristics.

Graphical Abstract

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Evaluation of murine lung epithelial cells (TC-1 JHU-1) line to develop Th2-promoting cytokines IL-25/IL-33/TSLP and genes Tlr2/Tlr4 in response to Aspergillus fumigatus

Objective

The aims of this study were to determine the role of live and heat-killed Aspergillus fumigatus conidia in releasing interleukin (IL)-25, IL-33 and thymic stromal lymphopoietin (TSLP) and to express Toll-like receptor (Tlr)2 and Tlr4 genes.

Translational medical mycology guides clinical and laboratory practice on fungal diseases

Patients with fungal infection having skin lesions may consult a dermatologist, which is a diagnostic and therapeutic challenge. Dermatologists take samples from the lesion to check the fungal elements under a microscope by KOH preparation and then treat the patient. This model has advanced from bedside to bench and from bench to bedside(B to B to B), which is defined as Translational Medical Mycology. Dermatologists have an advantageous position in finding, isolating and identifying the pathogenic fungi and treating the patient with antifungal drugs. Samples should be cultured in different media with or without chloramphenicol and cycloheximide and incubated at room temperature or 37 ℃. Non-culture techniques such as polymerase chain reaction based molecular identification, transmission electron microscopy, scanning electron microscopy, biochemistry tests and histopathology are also necessary to confirm the identification of the species, especially when the routine culture is negative. We start treatment upon obtaining evidence of fungal infection,i.e., positive KOH examination. Antifungal drugs such as itraconazole, fluconazole, terbinafine and amphotericin B can be used alone or in combination based on the fungal species and the location of the lesion. Practice on fungal infection includes screening of the patient, merging all of the laboratory techniques and methods from the microbiologists, pathologists, molecular researchers, identification of the pathogen and determination of the optimum antifungal drug.     

红色毛癣菌raubitschekii变种致Majocchi肉芽肿一例

目的 报道1例Majocchi肉芽肿,对其病原菌红色毛癣菌变种raubitschekii的形态学、生理学及分子学特征进行研究,并通过基因分型的方法分析其深在感染与浅表皮肤感染的关系.方法 进行临床和病理检查,真菌镜检和培养,尿素酶试验,内转录间隔区(ITS区)序列分析.对来源于该患者病变趾甲及组织的菌株及7株红色毛癣菌的rDNA非转录间隔区(NTS)串联重复亚单位1(TRS-1区)进行PCR.结果 48岁女性患者,背部、臀部、大腿出现红色丘疹、结节2个月.9个月前曾行肝移植术,甲癣病史3年,术后加重.经病理和真菌学检查,确诊为Majocchi肉芽肿.足趾甲和组织真菌培养菌落和显微镜下形态、尿素酶试验阳性,结合ITS区序列分析结果,证实致病菌为红色毛癣菌变种raubitschekii.TRS-1区扩增后显示,病变趾甲和组织来源的菌株基因型完全一致,与其余临床分离株有差异.结论 NTS区的TRS-1区显示基因多态性,病变趾甲和组织基因型完全一致,提示两者来源相同.     

A systematic review and meta-analysis on the epidemiology,casual agents and demographic characteristics of onychomycosis in Iran

Onychomycosis or fungal nail infection is one of the most common fungal infections. Nearly 50% of all nail disorders are caused by fungi. This systematic review and meta-analysis was conducted to determine the prevalence of onychomycosis across Iran. We searched English and Persian databases for studies reporting the epidemiologic features of onychomycosis in Iranian people from January 2000 to December 2018. Literature search revealed 307 studies, of which 24 studies met the eligibility criteria. In order to identifying the existence of publication bias among studies, funnel plots were used. The results of the meta-analysis were visualized as a forest plot representing the prevalence estimates of each study. Heterogeneity was also analyzed using the I², Chi², and Tau² statistics. A high level of I² and Chi² was obtained among studies, which provides evidence of notable heterogeneity between studies. The results of current study revealed that the highest prevalence of onychomycosis was related to Mazandaran and Tehran provinces, respectively. As in the literature hypothesized shift in etiologic agents from yeasts to dermatophytes or molds could not be confirmed. Females were affected more frequently than males and in both sexes the highest incidence of infection occurrence was at the ages of > 50 years. It seems the highest prevalence of onychomycosis in Mazandaran and Tehran provinces is due to the concentration of specialist doctors and research centers in these two provinces compared with others which leads to more detection and more care of the disease. Therefore, further educational strategies in order to accurate diagnosis in other provinces is necessary to reduce the risk of onychomycosis in Iran.     

Comparison of biofilm-producing ability of clinical isolates of Candida parapsilosis species complex

ObjectiveCandida parapsilosis is one of the main emerging non-Candida albicans species leading to superficial and systemic fungal infections in humans. Candida has the ability to produce biofilms associated with pathogenesis. The aim of the study was to estimate biofilm-producing ability of clinical isolates of C. parapsilosis sp. complex.MethodsClinical samples of C. parapsilosis complex have been analyzed. Crystal violet (CV) staining and tetrazolium reduction assay (MTT) have been used to analyze the clinical isolates ability to produce biofilms. The biofilm's structural characteristics have been assessed by using scanning electron microscopy.ResultsAll 65 isolates were able to form biofilm. In addition, no significant difference was found between biofilm quantification based on two assays at different time intervals (24 h, 48 h, 72 h, 96 h) (P > 0.05), with the exception of Candida orthopsilosis, which exhibited higher metabolic activity at 24 h time point (P < 0.05). Moreover, metabolic activity and production of biofilm biomass demonstrated statistically significant correlation (r = 0.685, P < 0.01). According to microscopic observations, the investigated clinical strains formed the similar surface topography with the slight differences in morphology; in addition, there was no statistically significant difference between efficiency of two assays to quantify biofilm.ConclusionIt was shown that, similar to C. parapsilosis sensu stricto, two cryptic identified species (C. orthopsilosis and Candida metapsilosis) obtained from different clinical samples, were biofilm producers, while C. parapsilosis sensu stricto exhibited the highest biofilm production.