Diversity of fungal communities on lesions of the face, upper limbs and back in patients with atopic dermatitis / 中华皮肤科杂志

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.

Clinical Observation of Chemoradiotherapy Combined with Thermotherapy in the Treatment of Middle and Advanced Esophageal Cancer / 中国药房

OBJECTIVE:To observe short-term efficacy and ADR of nedaplatin concurrent chemoradiotherapy combined with thermotherapy in the treatment of middle and advanced esophageal cancer. METHODS:64 patients with middle and advanced esophageal cancer were randomly divided into control group and treatment group,with 32 cases in each group. Control group was given nedaplatin concurrent chemoradiotherapy,nedaplatin 30 mg/m2,ivgtt,every week during chemotherapy;treatment group re-ceived thermotherapy by high frequency heating machine before chemoradiotherapy,60 min/time,twice a week;received chemora-diotherapy 30 min after thermotherapy. Short-term efficacy and ADR were observed in 2 groups. RESULTS:The short-term total effective rate of treatment group(84.4%)was higher than that of control group(62.5%),with statistical significance(P<0.05). The incidence of bone marrow suppression,radioactive esophagitis and gastrointestinal reactions in treatment group vs. control group were(21.9%)vs.(46.9%),(18.8%)vs.(56.3%),(31.3%)vs.(59.4%),with statistical significance(P<0.05). CONCLU-SIONS:Nedaplatin concurrent chemoradiotherapy combined with thermotherapy is better than concurrent chemoradiotherapy in the treatment of middle and advanced esophageal cancer with low incidence of ADR.

Diagnosis of primary herpes simplex in the upper lip with PCR and transmission electron microscopy: a case report / 中华皮肤科杂志

A 66-year-old man was admitted for a 7-day history of painful blisters and erosions in the upper lip.Real-time PCR with herpes simplex virus (HSV) type-specific primers showed that the blister fluid and crusts were positive for HSV-1,but negative for HSV-2.Observation of the blister wall with transmission electron microscopy (TEM) revealed 3 types of nucleocapsid in the karyoplast of epithelia cells,including the electron-translucent core,granular core and electron-dense core.Numerous matured viral particles with envelope were found in the cytoplasm,which were identified as HSV.The diagnosis was made as herpes simplex in the upper lip based on the above findings.PCR based molecular typing combined with observation of HSV particles via TEM may be an effective approach to the definite diagnosis of primary herpes simplex.

Cutaneous and subcutaneous coinfection by Lichtheimia corymbifera and Candida parapsilosis: a case report / 中华皮肤科杂志

To report a case of cutaneous and subcutaneous coinfection caused by Lichtheimia corymbifera and Candida parapsilosis.A 67-year-old female peasant consulted about proliferative granuloma developing on her left forearm after topical application of a Chinese herbal drug and splint fixation for the treatment of suspected fracture of the wrist.Direct microscopic examination showed gram positive budding yeast cells in lesion secretions.Pathological study with periodic acid-Schiff (PAS) and gormori methenamine silver (GMS) staining revealed broad non-separate hyphae in the corneum and dermis.Fungal culture of lesional tissue at 35℃ grew both mould and yeast.The mould was identified as Lichtheimia corymbifera based on morphological findings and sequences of the internal transcribed space (ITS) 1-4 regions.Thermal tolerance study revealed that the isolate grew fast at 37℃ but slowly at 40℃.Under a scanning electron microscope,the acrogenous sporangia were pear-shaped with conical sporangiophores originating from the top of stolon,which were among but not opposite to the rhizoids.The yeast was identified as Candida parapsilosis by Chromagar test and D1/D2 region sequencing.As antimicrobial susceptibility test indicated,the Lichtheimia corymbifera isolate was most sensitive to terbinafine and itraconazole.The proteolytic activity of Lichtheimia corymbifera was higher than that of Candida parapsilosis.The granuloma completely subsided after surgical resection and 6-week treatment with oral itraconazole 200 mg twice a day.No recurrence was observed during a 4-year follow-up.

Black-dot ringworm caused by Trichophyton tonsurans and analysis of its extracellular enzymatic activity / 中华皮肤科杂志

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.

Random amplified polymorphic DNA analysis of Malassezia isolates from cutaneous lesions of pityriasis versicolor / 中华皮肤科杂志

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.

Invasive Fungal Infections: Diagnosis and Treatments in China / 대한의진균학회지

The body location and clinical appearance of fungal infections depends on the fungal virulence, infectious route and host immunological state. The result being that patients with mycoses consult with different clinical departments. The diagnosis of mycoses is based on the detection of fungal elements such as hyphae and/or yeast cells from the involved tissues. Isolation of the fungus is the precondition for species identification and antifungal treatment. To think clinically and to emphasize the mycology is the basic consideration of medical mycology research. Mycologists play a key role in the collaboration between the clinical and laboratory aspects. The clinician always wants to know what the fungus is and how to treatment the mycosis. Fungal pathogens are often stealthy and difficult to detect in infected patients during the early stages of the diseases and this is when therapies would be the most effective. Routine techniques commonly employed in the detection of fungal diseases including microscopic examination, culturing and serology are seriously hampered by lengthy waits of times for results and low accuracy. The clinician may want prophylaxis or to use empirical antifungal treatment to see if it does/does not work. The problem is that some of the patients do not respond to the antifungal treatment, because the doctor lacked sufficient evidence of fungus infection to give the doctor confidence to continue treatment. Accurate and early diagnosis of fungal diseases is critical for managing mycotic diseases. This is usually done by direct microscopic examination (DME) of KOH preparations. Good specimens are the key point that directly affects the quality of microscopic evidence and culture. The most important aspect is culturing samples on different media with or without chloramphenicol and cycloheximide and incubated at room temperature and 37degrees C. Early treatment could save a patient's life. We start treatment at the time we have the proof of fungal infection, i.e., KOH positive. Itraconazole, fluconazole, terbinafine, amphotericin B or its liposome form, can be used alone or in combination based on the fungal species involved and the site of infection.

Identification of a strain Prototheca zopfii var. hydrocarbonea by analyzing the sequence of ribosome RNA gene / 中国人兽共患病学报

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.

Isolation of Pathogenic Fungi from Patients with Onychomycosis by Multipoint Inoculation Method / 中华皮肤科杂志

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

Malassezia globosa is the Main Species Found in Hair Follicles of Patients with Malassezia Folliculitis / 中华皮肤科杂志

Objective To examine the distribution of Malassezia species in follicular contents and perifollicular superficial skin in patients with Malassezia folliculitis and search for its causative agent. Methods A total of 120 patients with Malassezia folliculitis were investigated. Follicular lesions at three different anatomic sites were selected in each patient. Perifolliclar superficial skin specimens were taken by sterile adhesive tape, and the follicular contents of the same follicle were taken by sterile haemostatic forceps. The above specimens were cultured respectively on media containing rapeseed oil. The isolated colonies were identified by their physiological and morphological characteristics. Results Out of 319 isolates obtained from the perifollicular superficial skin, 247 isolates (77.43%) were identified as M. sympodialis, 40 isolates (12.54%) as M. furfur, 27 isolates(8.46%) as M. globosa and 5 isolates(1.57%) as M. obtusa. Out of 314 isolates obtained from follicular contents, 252 isdates(80.25%) were identified as M. globosa, 57 isolates(18.15%) as M. sympodialis, 4 isolates(1.27%) as M. furfur, and 1 isolate(0.32%) as M. obtusa. There was statistical difference in species distribution between the follicular contents and the perifolliclar superficial skin (P

Isolation and Identification of Malassezia Species from Lesional and Non-lesional Sites of Patients with Pityriasis Versicolor / 中华皮肤科杂志

Objective To investigate the distribution of Malassezia species in lesional and non-lesion-al sites of patients with pityriasis versicolor(PV),species-variation in different anatomic sites and in lesions with different pigmentation,and the relationship between various Malassezia species and severity and age of PV patients.Methods A total of629skin specimens taken by sterile adhesive tape from the lesions and non-lesional skin were inoculated on media containing rapeseed oil in113patients with PV.Isolated colonies were identified to species based on physiological and morphological characteristics.Results The isolation rates of Malassezia spp.were not significantly different from both lesions and corresponding non-lesional skin.Among non-lesional sites,the isolation rate was significantly higher in forehead and trunk than that in upper and lower extremities.Five species were identified out of565strains obtained from the patients,including M.sympodialis(44.78%),M.furfur(32.94%),M.globosa(11.68%),M.obtusa(5.84%)and M.restricta(4.76%).Two dif-ferent species were isolated simultaneously from27sites.There was no obvious difference in species distribu-tion patterns between lesions and non-lesional sites.M.restricta was isolated from forehead exclusively.Species-variation was closely linked to lesions with different pigmentation and the age of patients,not to the severity of disease.Conclusion There is neither statistical difference of Malassezia isolation rate and species distribution between lesions and non-lesional skin,nor correlation between disease severity and species-varia-tion.The anatomic sites,the diversity of pigmentation pattern and the age of patients seem to be associated with different Malassezia species.

The etiology of papular urticaria: a systematic review / 中华皮肤科杂志

Objective To investigate the etiology of papular urticaria: is it caused only by arthropod-like insects-bite allergy, or by multiple factors such as food allergy, disturbance of gastrointestinal function and infection? Methods We searched, by computer and manually, the foreign and domestic literature related to the etiology of papular urticaria published since 1950s, and according to the methods of evidence-based medicine, systematically evaluated the evidence supporting either the insect-bite theory or the multiple factor theory. Results Twenty-nine articles ( 22 English and 7 Chinese ) supported the theory of hypersensitivity to bites from certain insects such as mosquitoes, gnats, fleas, mites, bedbugs etc. Two articles in Chinese mentioned the possibilities other than insect-bite, but the reliability was unconvincing, because the authors did not present the source of data or statistical methods used in the articles. The evidence from epidemiology, histopathology, laboratory and clinical researches all supported the insects-bite theory. No proven evidence was found supporting other aetiological hypotheses. Conclusion Our results suggest that papular urticaria is caused only by the allergy to stings or bites of arthropods, and other hypotheses still lack convincing evidence.

The Effect of Candida albicans on the Expression Level of Human ?-defensin-2 mRNA in Keratinocytes in vitro / 中华皮肤科杂志

Objective To investigate the effect of Candida albicans and its components on the expression level of human beta-defensin-2 mRNA (HBD-2) in keratinocytes in vitro. Methods Different components of Candida albicans were isolated by lyticase, repeated freezing and thawing, sonication, and centrifugation. The keratinocytes and HaCaT cell lines were co-cultured with Candida albicans and its cellular components for 24 h. The expression level of HBD-2 mRNA was detected by reverse-transcription polymerase chain reaction (RT-PCR). Results Low expression level of HBD-2 mRNA in the unstimulated keratinocytes and HaCaT cells was detected. The HBD-2 mRNA expression levels in the keratinocytes stimulated by Candida albicans, the extract of its cell wall, and pure mannan were significantly increased (P 0.05). Conclusions Candida albicans, the extract of cell wall of Candida albicans, and commercial mannan can increase the expression level of HBD-2 mRNA in keratinocytes.

Topical Retinoids for Acne Vulgaris: Efficacy and Safety: a Systematic Review / 中华皮肤科杂志

Objective To evaluate the efficacy and safety of topical retinoids in the treatment of acne vulgaris compared with placebo, antibiotics, benzoyl peroxide and sulfur preparation. Methods According to the Cochrane reviewer′s handbook, randomized controlled clinical trials were selected for the systematic review. Results Up to 2002, 15 clinical trials (2,439 patients) that met the inclusion criteria were selected. There were four clinical trials which showed that topical retinoids were more effective than that of placebo (RR=1.87, and 95% CI: 1.13 ~ 3.11),especially for noninflammatory lesions (RR=12.70,and 95% CI : 4.09 ~ 39.40). There were 3 clinical trials which showed that topical retinoids had better efficacy than that of sulfur preparations (RR=1.75, and 95% CI: 1.42 ~ 2.16). For 7 clinical trials of retinoids compared with benzoyl peroxide, and 3 clinical trials of retinoids compared with antibiotics, no conclusion could be drawn. All the clinical trials showed that there were local side effects, including erythema, and scaling etc in the patients using topical retinoids, but no systematic side effects were observed, however, pregnant women had to be very cautious. Conclusions Topical retinoids are effective for acne vulgaris, and has better efficacy than sulfur preparation does, but there is not enough evidence to clarify that the efficacy of topical retinoids is better than that of benzoyl peroxide and antibiotics.

Study on Chemotactic activity of Candida Albicans Proteinase for Neutrophils and Lymphocytes / 中华皮肤科杂志

The chemotactic activity of Candida albicans proteinase toward human peripheral leuko- cytes was examined by a modified Boyden chamber method.It was found that C.albicans proteinase in- duce dose-dependent chemotactic activity for neutrophils with maximal activity at 500 nmol/L(P

Experimental Study of Antifungal Effect of Povidone Iodine Solution on Candida Albicans / 中华皮肤科杂志

The effect of povidone iodine solution(PIS)on the metabolic activity of Candida albi- cans and the effect of PIS treated C.altn'cans to cultured human keratinocytes were studied by using the Alamar blue fluorescence assay.It was found that PIS inhibited the metabolic activity of Candida albi- cans with a dose-dependent manner,and that the LD_(50)of PIS for Candida albicans is 0.075%.After treating for 30 min by 2% PIS/PBS(-),Candida albicans almost lost the adherence and invasion to the cultured human keratinocytes,the wall of yeast cells became uneven and shrunken,the intracellular structures became obviously indistinct,and the yeasts could not develop to hypha form.Candida albi- cans,treated with 2% PIS in PBS(-)for 30 min or with 10% PIS Sabouraud's liquid medium for 33 hr,did not form colony when it was inoculated to Sabouraud's dextrose agar.This result suggests that the fungicidal effective of PIS is influenced by its solvent.

A Case of Disseminated Cryptococcosis with Cutaneous Manifestations and Osteomyelitis: Clinical and Laboratory Investigations / 中华皮肤科杂志

Objective To report a case of disseminated cryptococcosis with cutaneous manifestations and osteomyelitis. Methods and Results A 33 year old female was admitted due to multiple nodules and ulcers on the upper arms, shoulders, buttocks and thighs for one year. The patient was pregnant when admitted, and gave birth to a premature baby during her illness. The nodules increased half a month after delivery, which was suspected to be hematogenously disseminated pulmonary tuberculosis and was given anti tuberculous therapy for three months but failed. Physical examination showed there were 39 nodules or ulcers on the face, gum, trunk, buttocks and extre mities. The bone structure of the left tibia and fibula destroyed and a sinus developed on the left fibula. Microbiologic examination showed that lots of spores were seen in the smear of pus and necrotic tissues, which produced yeast like colonies in culture with positive urease and caffeic acid test. Cryptococcus neoformans, serotype A was identified by API yeast reaction band and serology. Inoculation with mice and rats showed that their brains, lungs and livers were involved easily. Further identification as C.neoformans var.neoformans was obtained based on sequence analysis of ribosomal internal transcribed spacer region 2. The anti tuberculous therapy was stopped and anti fungal therapy was initiated at once. Intravenous and topical amphotericin B in combination with fluconazole were chosen in the initial therapy and itraconazole for maintenance. The nodules disappeared after 30 days and the last ulcer in the left tibia healed completely after 200 days. The anti fungal therapy was discontinued after 277 days and the patient was completely cured.

Interaction between Candida albicans and Cultured Human Keratinocytes / 中华皮肤科杂志

Objective To study the morphological,biologic response of cultured human keratin ocytes to Candida albicans and the molecular mechanism.Methods ①A yeast form of C.albicans was added t o a monolayer of keratinocytes.The adherent organisms were observed by scanning and transmission electron mi-croscope at different incubation periods.Boiled C.albicans and latex beads of a similar size(3.2?m)were also inoculated for comparison.②The effect of supernatant from the co-culture of keratinocytes with inta ct C.albicans on keratinocyte growth was measured by the fluorescence intensity and cell count.③Keratinocytes were incubated with C.albicans or latex beads,and the level of cytok ine in the keratinocytes and supernatant were measured with enzyme -linked im munosorbent assay kits.Results①The number of adherent C.albicans increased with the lapse of time,whi le boiled C.albicans did not adhere at all.Many latex bead s adhered to the keratinocytes,and were then easily phagocytised.Fibril -like structu res stretched from the keratinocyte s adhered to the organisms or latex beads.②The conditioned medium of 50%concen tration significantly promote cell growth,while that with boiled C.albicans or latex beads moderately stimulate d keratinocyte growth.③Ker-atinocytes treated with intact C.albicans had significantly higher level of IL-1?in the supernatant but lower in the cell extract.Both TGF -?and bFGF increased either in the medi a or in the extract.Conclusion These results suggest that keratinocytes have non -specific phagocytic activ ity.C.albicans are able to adhere to ker-atinocytes and stimulate the release of various cytokines from keratinocytes,which may induce an inflammatory reaction and cell growth.