ABSTRACT
Objective:To investigate the diversity and structural characteristics of fungal communities on lesions of the face, upper limbs and back in patients with atopic dermatitis (AD) .Methods:Samples were collected from the lesions on the face, upper limbs and back of 10 AD patients, who visited the Department of Dermatology, the First Affiliated Hospital of Chengdu Medical College from September to October in 2015, and collected from the corresponding body sites of 10 healthy controls. DNA was extracted from the samples, and subjected to MiSeq high-throughput sequencing for diversity index analysis, species composition analysis and principal component analysis. Statistical analysis was carried out by using two-independent-sample t test for comparisons between two groups, one-way analysis of variance for comparisons among multiple groups, and least significant difference- t test for multiple comparisons. Results:Diversity index analysis showed that Shannon index was significantly higher in the samples from the lesions on the face, upper limbs and back of the AD patient group than in those from corresponding body sites of the healthy control group ( t = 2.67, 2.37, 3.34 respectively, all P < 0.05) . Species composition analysis showed that Malassezia was predominant in the skin samples from the face, upper limbs and back of the AD patient group and healthy control group, and the total abundance of Malassezia globosa and Malassezia restricta was about 80%. The abundance of Candida and Aspergillus in the total samples was significantly higher in the AD patient group than in the healthy control group ( t = 3.515, 2.137 respectively, both P < 0.05) . There was no significant difference in the abundance of major fungal genera on the face between the AD patient group and healthy control group (all P > 0.05) ; the abundance of Candida in the upper limbs was significantly higher in the AD patient group than in the healthy control group ( t = 3.186, P < 0.05) , and the abundance of Aspergillus in the back was significantly higher in the AD patient group than in the healthy control group ( t = 2.736, P < 0.05) . In either the AD patient group or the healthy control group, there was no significant difference in the abundance of major fungal genera among samples from the face, upper limbs and back (all P > 0.05) . Moreover, no significant difference in the abundance of major fungal genera was observed among the mild, moderate and severe AD patient groups (all P > 0.05) . Principal component analysis showed that fungal communities in the samples from the lesions on the face, upper limbs and back of the AD patient group were not clustered by the disease severity. Conclusions:The diversity of fungal communities is significantly higher in the lesions on the face, upper limbs and back of the AD patients than in the normal skin at the corresponding body sites of the healthy controls. Malassezia is the dominant fungal genus in both lesions of the AD patients and normal skin of the healthy controls at the above body sites. The composition of fungal communities in lesional samples may be uncorrelated with the disease severity in AD patients.
ABSTRACT
Objective To report a case of black-dot ringworm caused by Trichophyton tonsurans in a 3-year-old girl. Methods Lesional hair was obtained from the patient and subjected to direct microscopic examination as well as culture. Subsequently, the isolate underwent morphological, biochemical and molecular biology identification. The extracellular enzymatic activity of the isolate was analyzed. Results Microscopy revealed that the hair shaft was filled with fungal spores. Typical colony of the isolate was grayish-white with downy appearance. Slide culture showed centipede-like, lateral, rod-shaped microconidia. Urease test was positive. The amplification of ribosomal DNA (rDNA) ITS domains by PCR produced a 687 bp-sized fragment which had a 100% homology with the sequences of several Trichophyton tonsurans strains in the GenBank database. The extracellular enzymatic activity analysis showed an increase in the activity of alkaline phos-phatase, acid phosphatase, esterase (C4), β-glucosidase, leucine arylamidase, N-acetyl-β-glucosaminidase and a-mannosidase. Conclusions The pathogenic fungus is identified as Trichophyton tonsurans based on morphological and biochemical features as well as sequence of the ITS region of rDNA, and the child was diagnosed with black-dot ringworm.
ABSTRACT
Objective To investigate intraspecific and interspecific variation within Malassezia iso-lates from patients with pityriasis versicolor by random amplified polymorphic DNA (RAPD) analysis, to learn the difference between RAPD analysis and physiological and biochemical methods in the typing of Malassezia species, and to explore the relationship between RAPD patterns and Malassezia species. Methods A total of 47 Malassezia isolates were obtained from 34 patients with pityriasis versicolor, and they were classified into 5 species by morphological, physiological and biochemical features, I.e., M. Fin'fur, M. Obtusa, M. Globosa, M. Restricta and M. Sympodialis. Genomic DNA was extracted from the 47 clinical isolates and 10 reference strains (including 7 species) of Malassezia. PCR was performed using 4 random primers including S22, S24, S25 and S33. RAPD patterns were analyzed by NTSYS software and dendrogram was autogenerated. Results Genomic DNA of most strains was successfully amplified with four primers, espe-cially with primers S22 and S24 that resulted in rather stable and clear DNA bands. A total of 82 fragments were amplified from all tested strains. These strains showed both interspecifie and intraspecific variation. Multiple swains were isolated from different body sites of 4 patients and identified into different species by biochemical and morphological typing; those swains from same hosts occupied contiguous positions in the dendrogram and exhibited a high genetic convergence. Conclusion The phenomenon that different strains from a co-host show a high genetic convergence indicates that species specificity and evolution of Malassezia are closely related to its hosts.
ABSTRACT
A strain of Prototheca species isolated from a case of meningitis was identified by routine morphologic and biochemical methods as well as amplification of the related genes, in which the 28S large-subunit (LSU) region of ribosomal RNA (rRNA) gene and intergenic space (ITS) were amplified with universal fungal primers. The small-subunit (SSU) rRNA gene was amplified with eukaryote-specific primers and Prototheca genus-specific primers. Then, compared the sequences with the ones posted on BLAST (www. ncbi. nlm. nih. gov/BLAST). The organism choice giving the closest match, up to 99%, was considered the most likely correct identification. It was found that this strain of fungus grew well at 25 ℃ or 37 ℃. Smooth,moist colonies with white color were observed on Sabouraud Dextrose Agar (SDA) and Potato Dextrose Agar (PDA). Microscopically, globular or ovoid cells, a number of round, ovoid shaped endospores could be observed. No hypha, ascus or blastic conidia was found upon cultivation on SDA. Based on the morphological characteristics, this isolate could be identified as Prototheca species. The identity with Prototheca wickerhamii was 2.9 % as demonstrated by the API 20C AUX system. Sequence analysis showed that the ITS gene was proved to be a complex structural region which was not suitable for the identification of Prototheca species, but the LSU and SSU rDNA regions showed 94% and 99.9% sequence identities with Prototheca zopfii var. hydrocarbonea (P. zopfii var. hydrocarbonea) respectively, indicating that the SSU rRNA gene sequence might be more reliable on than the LSU rRNA gene sequence for identification of Prototheca species.
ABSTRACT
Objectives To compare the difference between multipoint inoculation and routine method for isolation of pathogenic fungi from nail samples of onychomycosis,and to analyze the epidemiology of pathogenic fungi in those patients.Methods The nail clipping samples from each patient were inoculated onto the plates with Sabouraud's agar,Sabouraud's agar without cycloheximide and medium containing rapeseed oil,respectively,by an approach of at least seven inoculating points in each plate (multipoint inoculation),and onto medium slope in tubes with the same media as above mentioned (routine method).In the multipoint inoculation method,plates with more than 3 colonies were taken for further identification of pathogenic fungi based on morphological and biochemical properties.Results Based on the data from 150 samples of onychomycosis,significant differences were found between multipoint inoculation method and routine method (P