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BACKGROUND: Filaggrin gene (FLG) mutations have been identified as the cause of ichthyosis vulgaris (IV) and major predisposing factors for atopic dermatitis (AD). The relationship among AD, IV and FLG mutations has not been clarified yet. Mutations 3321delA and K4671X, two of the most common mutations in Chinese patients, were both statistically associated with AD in case-control studies. MATERIALS AND METHODS: A group of 100 family trios (a total of 300 members with one affected AD proband and both parents) were recruited and screened for three filaggrin null mutations (3222del4, 3321delA and K4671X). The subjects' manifestations of AD and IV were assessed by two experienced dermatologists and recorded in detail. The relationship of common mutations to AD were assessed using both case-control and family-based tests of association. Filaggrin expression was measured in skin of 3 subjects with K4671X heterozygote and the normal control using quantitative real-time RT-PCR and immunohistochemistry. RESULTS: Of 100 probands for AD, 22 were carriers for common FLG mutations and only 2 of them were from 40 none-IV family trios (5.00%), consistent with that of the healthy control group (3.99%, P>0.05). Significant statistical associations were revealed between AD and 3321delA (P

Dear Editor,Cryptococcosis is a fungal infection caused by members of the Cryptococcus neoformans/C. gattii species complex,1 which causes approximately one million infections among HIV-infected pa...

Contains fulltext : 109375.pdf (Publisher’s version ) (Open Access) BACKGROUND: Cryptococcus neoformans is a pathogenic yeast that causes cryptococcosis, a life threatening disease. The prevalence of cryptococcosis in Asia has been rising after the onset of the AIDS epidemic and estimates indicate more than 120 cases per 1,000 HIV-infected individuals per year. Almost all cryptococcal disease cases in both immunocompromised and immunocompetent patients in Asia are caused by C. neoformans var. grubii. Epidemiological studies on C. neoformans in pan-Asia have not been reported. The present work studies the genetic diversity of the fungus by microsatellite typing and susceptibility analysis of approximately 500 isolates from seven Asian countries. METHODOLOGY/PRINCIPAL FINDINGS: Genetic diversity of Asian isolates of C. neoformans was determined using microsatellite analysis with nine microsatellite markers. The analysis revealed eight microsatellite complexes (MCs) which showed different distributions among geographically defined populations. A correlation between MCs and HIV-status was observed. Microsatellite complex 2 was mainly associated with isolates from HIV-negative patients, whereas MC8 was associated with those from HIV-positive patients. Most isolates were susceptible to amphotericin B, itraconazole, voriconazole, posaconazole, and isavuconazole, but 17 (3.4%) and 10 (2%) were found to be resistant to 5-flucytosine and fluconazole, respectively. Importantly, five Indonesian isolates (approximately 12.5% from all Indonesian isolates investigated and 1% from the total studied isolates) were resistant to both antifungals. The majority of 5-flucytosine resistant isolates belonged to MC17. CONCLUSIONS: The findings showed a different distribution of genotypes of C. neoformans var. grubii isolates from various countries in Asia, as well as a correlation of the microsatellite genotypes with the original source of the strains and resistance to 5-flucytosine.

Ubiquitination is a reversible protein modification that influences various cellular processes in eukaryotic cells. Deubiquitinating enzymes remove ubiquitin, maintain ubiquitin homeostasis and regulate protein degradation via the ubiquitination pathway. Cryptococcus neoformans is an important basidiomycete pathogen that causes life-threatening meningoencephalitis primarily in the immunocompromised population. In order to understand the possible influence deubiquitinases have on growth and virulence of the model pathogenic yeast Cryptococcus neoformans, we generated deletion mutants of seven putative deubiquitinase genes. Compared to other deubiquitinating enzyme mutants, a ubp5Δ mutant exhibited severely attenuated virulence and many distinct phenotypes, including decreased capsule formation, hypomelanization, defective sporulation, and elevated sensitivity to several external stressors (such as high temperature, oxidative and nitrosative stresses, high salts, and antifungal agents). Ubp5 is likely the major deubiquitinating enzyme for stress responses in C. neoformans, which further delineates the evolutionary divergence of Cryptococcus from the model yeast S. cerevisiae, and provides an important paradigm for understanding the potential role of deubiquitination in virulence by other pathogenic fungi. Other putative deubiquitinase mutants (doa4Δ and ubp13Δ) share some phenotypes with the ubp5Δ mutant, illustrating functional overlap among deubiquitinating enzymes in C. neoformans. Therefore, deubiquitinating enzymes (especially Ubp5) are essential for the virulence composite of C. neoformans and provide an additional yeast survival and propagation advantage in the host.

Global distribution patterns of Cladophialophora carrionii, agent of human chromoblastomycosis in arid climates of Africa, Asia, Australia, Central-and South-America, were compared with similar data of the vicarious Fonsecaea spp., agents of the disease in tropical rain forests. Population diversities among 73 C. carrionii strains and 60 strains of three Fonsecaea species were analyzed for rDNA ITS, partial β-tubulin, and amplified fragment-length polymorphism (AFLP) fingerprints. Populations differed significantly between continents. Lowest haplotype diversity was found in South American populations, while African strains were the most diverse. Gene flow was noted between the African population and all other continents. The general pattern of Fonsecaea agents of chromoblastomycosis differed significantly from that of C. carrionii and revealed deeper divergence among three differentiated species with smaller numbers of haplotypes, indicating a longer evolutionary history., Author Summary Chromoblastomycosis is a severely mutilating disease caused commonly by two fungal species groups in Cladophialophora and Fonsecaea, which occur in arid climates or in the tropical rain forest, respectively. Understanding the population structure and evolution of chromoblastomycosis is important to reconstruct the epidemiological history and to identify evolutionary process and environmental drivers of disease spread. We used two sequence markers; internal transcribed spacer (ITS) region of rDNA and the partial ß-tubulin (BT2) gene, as well as AFLP markers, respectively, to investigate the population structure and differentiation of 73 C. carrionii strains and 60 strains of three Fonsecaea species. These agents of human disease have diverse distribution patterns and population dynamics even though they share a common ancestor within the order Chaetothyriales. It may be speculated that they have taken advantage of and adapted to human hosts as a new ecological niche.

Cryptococcal meningitis is mainly caused by Cryptococcus neoformans and Cryptococcus gattii, but occasionally other Cryptococcus species and phylogenetically related species are involved. Herein, we present a case of cryptococcal meningitis from China, which was caused by an azole and flucytosine resistant Filobasidium uniguttulatum. In addition, we present an overview of the literature of meningitis caused by Cryptococcus species other than C. neoformans and C. gattii. Eight cases were related to infections of the central nervous system. Leukaemia and cancer were important risk factors in HIV-negative patients. Molecular identification and susceptibility testing are important for proper management of patients because the species involved may differ in susceptibility to antifungal drugs.

Here we present a new and simple medium made by Perilla frutescens seed as a tool for identification of the Cryptococcus species complex, namely Cryptococcus neoformans and Cryptococcus gattii. Its usefulness was evaluated for all major molecular types within the Cryptococcus species complex. As a result, this medium is better for identification of the species complex compared with Guizotia abyssinica or Helianthus annuus seed medium.

VGII/AFLP6 genotype, also considered to be a cryptic species within Cryptococcus gattii, is an emerging pathogen and always related to higher incidence of C. gattii infection. Here, a polymerase chain reaction-based method with specific primers was developed to rapid differentiation of this emerging pathogen.

We employed a nested PCR assay to detect Sporothrix schenckii DNA of 38 strains (including all the 24 mtDNA types) collected from different areas of the world, in tissue of eight mice infected with ATCC10268 strain of the fungus, and the skin biopsies of nine patients with sporotrichosis. In addition, the same procedures were used with two strains of Ceratocystis minor and isolates of 10 species of other pathogenic fungi. The outer primers SS(1) and SS(2) and inner primers SS(3) and SS(4) of the 18S rRNA gene of S. schenckii were employed. A 152 bp fragment was detected in all 38 strains of S. schenckii, eight animal specimens and nine human skin biopsies, but not samples of C. minor and the other fungal species. The detection limit of 50 fg of S. schenckii DNA extract was determined with ethidium bromide staining. In summary, we demonstrated that nested PCR assay could identify S. schenckii of all the mtDNA types and in isolates recovered from different areas of the world. The nested PCR assay seems to be highly sensitive and specific and provides a rapid method for diagnosis of sporotrichosis under conditions of contamination avoidance.

Synergistic effects have previously been observed for a natural compound, tetrandrine (TET), with fluconazole (FLC) in vitro and in the treatment of Candida albicans-infected mice. To investigate the mechanisms of these synergistic effects, 16 strains of C. albicans from the same parent but with different FLC sensitivities were examined using flow cytometry and fluorescent spectrophotometry. Rhodamine 123 (Rh123)-positive cells and intracellular Rh123 fluorescence intensity were determined in accumulation/efflux experiments involving no or a noncytotoxic dose of TET. Total RNA extracted from each strain was used to compare the expressions of drug efflux pump genes in FLC-susceptible, -susceptible dose-dependent, and -resistant strains before and 24 h after TET administration. Accumulation experiments determined that mean percentages of Rh123-positive cells were 26.65% (TET-free) and 70.99% (TET 30 microg/ml), and mean respective intracellular Rh123 fluorescence intensities were 11.34 and 18.00. Efflux experiments showed that percentages of Rh123-positive cells were 1.79% (TET free) and 42.57% (TET 30 microg/ml), respectively, and respective mean intracellular Rh123 fluorescence intensities were 0.74 and 2.19. Differences in MDR1, FLU1, CDR1, and CDR2 expression levels in the absence of TET were statistically significant (p0.05) between FLC-susceptible, -susceptible dose-dependent, and -resistant strains. Compared with TET-free conditions, 24 h TET-treated strains showed statistically different (p0.05) expression of MDR1 (FLC-resistant strain), FLU1 (FLC-susceptible dose-dependent and -resistant strains), and CDR1 and CDR2 (FLC-susceptible, -susceptible dose-dependent, and -resistant strains). Thus TET can inhibit the C. albicans drug efflux system and reduce drug efflux. Its mechanism of action is related to the inhibition of expression of the drug efflux pump genes MDR1, FLU1, CDR1, and CDR2.

Sebaceous gland diseases are a group of common dermatological diseases with multiple causes. To date, a systematic report of the risk factors for sebaceous gland diseases in adolescents has not been published. The aim of this study was to assess the prevalence and risk factors for certain sebaceous gland diseases (seborrhea, seborrheic dermatitis, acne, androgenetic alopecia and rosacea) and their relationship to gastrointestinal dysfunction in adolescents. From August-October, 2002-2005, a questionnaire survey was carried out to obtain epidemiological data about sebaceous gland diseases. Using random cluster sampling, 13 215 Han adolescents aged 12-20 years were recruited from four countries or districts (Macau; Guangzhou, China; Malaysia; and Indonesia). The statistical software SPSS ver. 13.0 was used to analyze the data. The prevalence of seborrhea, seborrheic dermatitis, acne, androgenetic alopecia and rosacea was 28.27%, 10.17%, 51.03%, 1.65% and 0.97%, respectively. Based on multivariate logistic regression analysis, the risk factors for sebaceous gland diseases included: age; duration of local residency; halitosis; gastric reflux; abdominal bloating; constipation; sweet food; spicy food; family history of acne; late night sleeping on a daily basis; excessive axillary, body and facial hair; excessive periareolar hair; and anxiety. There was a statistically significant difference in the prevalence of gastrointestinal symptoms (halitosis; gastric reflux; abdominal bloating; constipation) between patients with and without sebaceous gland diseases (chi(2) = 150.743; P = 0.000). Gastrointestinal dysfunction is an important risk factor for diseases of the sebaceous glands and is correlated with their occurrence and development.

Itraconazole has a broad spectrum of activity against the most common fungal pathogens. Prior problems with absorption in severely ill patients have been overcome with the introduction of an oral solution and an intravenous preparation. An open-labeled, non-competitive, multicenter phase IV study was conducted to investigate the efficacy and safety of itraconazole administered intravenously for the treatment of invasive fungal infections in Chinese patients. Patients were treated with itraconazole intravenously for 2 weeks (200 mg twice daily for 2 days, then 200 mg once daily for 12 days) followed by 28 days of oral capsules (200 mg twice daily). Efficacy evaluation included an assessment of the clinical efficacy, fungal efficacy and total efficacy on days 14 and 42. Of 156 evaluable patients, 35 patients had proven and 62 suspected invasive fungal infections, and 59 patients were treated empirically. On day 14 the total efficacy rate in patients with proven infection was 54.3% (19/35; 95% confidence interval [CI], 37-71%) and on day 42 it was 65.7% (23/35; 95% CI, 48-81%). The most common adverse events were hypokalemia (13.5%), gastrointestinal disorders (12.8%), elevation of liver enzymes (10.9%) and increase of bilirubin (8.3%). Itraconazole intravenously followed by oral capsules is thus tolerated and effective in severely ill patients with proven invasive fungal infection.

A urease-negative serotype A strain ofCryptococcus neoformans(B-4587) was isolated from the cerebrospinal fluid of an immunocompetent patient with a central nervous system infection. TheURE1gene encoding urease failed to complement the mutant phenotype. Urease-positive clones of B-4587 obtained by complementing with a genomic library of strain H99 harboured an episomal plasmid containing DNA inserts with homology to thesudAgene ofAspergillus nidulans. The gene harboured by these plasmids was namedURE2since it enabled the transformants to grow on media containing urea as the sole nitrogen source while the transformants with an empty vector failed to grow. Transformation of strain B-4587 with a plasmid construct containing a truncated version of theURE2gene failed to complement the urease-negative phenotype. Disruption of the nativeURE2gene in a wild-type serotype A strain H99 and a serotype D strain LP1 ofC. neoformansresulted in the inability of the strains to grow on media containing urea as the sole nitrogen source, suggesting that theURE2gene product is involved in the utilization of urea by the organism. Virulence in mice of the urease-negative isolate B-4587, the urease-positive transformants containing the wild-type copy of theURE2gene, and the urease-negative vector-only transformants was comparable to that of the H99 strain ofC. neoformansregardless of the infection route. Virulence of theURE2disruption stain of H99 was slightly reduced compared to the wild-type strain in the intravenous model but was significantly attenuated in the inhalation model. These results indicate that the importance of urease activity in pathogenicity varies depending on the strains ofC. neoformansused and/or the route of infection. Furthermore, this study shows that complementation cloning can serve as a useful tool to functionally identify genes such asURE2that have otherwise been annotated as hypothetical proteins in genomic databases.

Purpose of review Fungal respiratory disease is associated with a high mortality in immunocompromised patients. This review aims to describe the recent advances in the aetiology, clinical presentation, diagnosis and management of fungal respiratory disease. Recent findings Invasive aspergillosis is an uncommon complication of hematopoietic stem cell transplants and solid-organ transplants but continues to be associated with poor outcome. Isolation of Aspergillus spp. from respiratory samples preceded acute rejection in lung-transplantation recipients. Molecular typing suggested that there was a very weak correlation between the environmental and patient isolates of Aspergillus fumigatus in invasive pulmonary aspergillosis. Serological and molecular detection of Aspergillus antigens or fungal DNA may improve the diagnosis of pulmonary aspergillosis, but the sensitivity is variable and more studies are needed. Voriconazole and caspofungin are choice to Aspergillus and Candida infection, especially to azole-resistant Candida spp. Treatment combining amphotericin B preparations, early surgical resection of infected tissue and discontinuation of immunosuppressive treatment may improve prognosis of mucormycosis. Summary Invasive filamentaous fungal infections of lung remain important causes of death in immunocompromised patients. Development of new early diagnostic tools and well-designed multicenter evaluations of diagnostic methods and therapeutic regimens available at present are the important work in the next 2-3 years.

Summary. A rare case of pyoderma gangraenosum caused by Rhizopus arrhizus is reported. The patient, a 50-year-old male farmer, was admitted to hospital complaining of gangrening and festering of the right upper arm with severe pain for nearly 2 months. A lesion was found on the inside of right upper arm. The central skin part of the lesion became black dry gangrenous, the periphery was deeply ulcerated with yellow-green pus. The necrotic crust, biceps, triceps and vessels inherited a histopathologically proven fungal infection. The fungus isolated was identified as Rhizopus arrhizus. The infection was successfully treated with itraconazole. Zusammenfassung. Es wird von einem seltenen Fall eines Pyoderma gangraenosum, verursacht von Rhizopus arrhizus, berichtet. Der 50-jahrige Patient, ein Landwirt, wurde in das Krankenhaus eingewiesen mit seit 2 Monaten bestehenden Beschwerden einer Gangran und Eiterungen mit starken Schmerzen im rechten Oberarm. Eine Lasion zeigte sich an der Innenseite des rechten Oberarms. Die Haut im Zentrum der Gangran war schwarz und trocken, die Peripherie war ulzeriert und sonderte gelbgrunen Eiter ab. In der nekrotischen Kruste sowie an Bizeps, Trizeps und Getfasen konnte histopathologisch eine Pilzinfektion nachgewiesen werden. Das Isolat wurde als Rhizopus arrhizus identifiziert. Die Infektion heilte unter Itraconazolbehandlung ab.

To review the characteristics and evolution of the fungal spectrum, and the risk factors causing fungal infection, and to make progress in diagnosing fungal infection after organ transplantation.An English-language literature search (MEDLINE 1990 - 2000) and bibliographic review of textbooks and review articles.Twenty-three articles were selected from the literature that specifically addressed the stated purpose.Fungal infections in organ transplant patients were generally divided into two types: (1) disseminated primary or reactivation infection with one of the geographically restricted systemic mycoses; (2) opportunistic infection by fungal species that rarely cause invasive infection in normal hosts. The risk factors of fungal infection after a transplant can be evaluated and predicted according to the organ recipient's conditions before, during and after the transplant. Progress in early diagnostic methods during the past 10 years has mainly revolved around two aspects, culture and non-culture.It is important to undertake a systemic evaluation on the condition of the organ recipient before, during and after a transplant; should any risk factor for fungal infection be suspected, diagnosis should be made as early as possible by employing mycological techniques including culture and non-culture methods.

To observe the unique DNA profile and the relationship between DNA profile and phenotype of Trichophyton rubrum,and establish an effective molecular method to identify T. rubrum.Three primers, including (GACA)(4), (GTG)(5) and M13 core sequence (5'-GAGGGTG-GCGGTTCT-3'), were used to distinguish variations among 20 clinical isolates of T. rubrum and Trichophyton mentagrophytes.Different PCR-fingerprinting was seen between T. rubrum and T. mentagrophytes using three different primers. 2 stains of T. rubrum were identified among 6 supposed T. mentagrophytes.T. rubrum can be distinguished using PCR, and (GACA)(4) is the most suitable primer.

The purpose of this study was to compare the efficacy of different dosage regimens in the management of onychomycosis with itraconazole and to determine the results of a further 1-week intermittent pulse treatment in non-cured patients. In this study, 153 patients were randomly allocated to four groups. Patients in group A were treated with daily doses of 100 mg for 3 months in the case of fingernail onychomycosis and for 4 months in the case of toenail onychomycosis. Patients in the other groups received a intermittent pulse therapy, in which the drug was taken for 1 week, then discontinued for 3 weeks, three cycles for fingernail and four cycles for toenail infection. The daily doses were 400 mg (group B), 300 mg (group C) and 200 mg (group D). After therapy, non-cured patients were treated further with one cycle in which the daily dose was 400 mg. Patients were subsequently observed for 9 months and efficacy was assessed by mycological examination and the growth of unaffected nails. At the end of the therapy, the cure rates in the four groups were 19.1% (A), 15.2% (B), 18.9% (C) and 17.9% (D), and no significant differences were found between each of B, C, D and A. At the end of the study, the cure rates were 76.2%, 91.3%, 78.4%, 28.6% respectively. The group that received further treatment had a cure rate of 55.6% at the end of the first month and of 83.3% in the second month. Drug tolerability was equally good in the four groups. Intermittent pulse therapy with a daily dose of 400 mg had the highest cure rate. Treatment of improved but non-cured patients with a dose of 400 mg intermittent pulse therapy markedly increased the cure rate. All treatment regimens were well tolerated.

The aim was to evaluate efficacy and tolerance of a short-schedule treatment regimen using a small dose of itraconazole in pulse intermittently. The open study evaluated the pulse therapy consisting of monthly 1-week cycles of oral itraconazole 200 mg daily for 2-3 consecutive months in 42 patients with onychomycosis. After active therapy, patients were evaluated for a maximum period of 1 year. Twelve of 42 patients were considered as being clinically cured, 17 were markedly improved, 11 were improved and two were failures. A mycological examination at the final visit was performed on all patients. Thirty-five were negative and seven were positive. This short treatment was well tolerated, with no adverse reactions, and may offer a new option for treatment of onychomycosis.

Summary Background Cryptococcosis is a severe fungal infection with a high mortality rate among solid-organ transplant recipients. Today, China is among the countries performing the most kidney transplants worldwide, however data on the association of cryptococcosis with kidney transplantation in mainland China remain scarce and fragmented. Methods We retrospectively analyzed cases of culture-confirmed cryptococcosis following kidney transplantation that have occurred at our hospital and reviewed the published cases in China over the last 30 years. Results Cryptococcosis in kidney transplant recipients was mainly caused by Cryptococcus neoformans var. grubii VNI strains and occurred most frequently in patients aged 41–50 years (37.9%, 11/29). The average time to infection after kidney transplantation was 5.16±3.97 years. The clinical manifestations were found to be diverse, with slight to moderate headache and fever, meningeal irritation, and high cerebrospinal fluid pressure being relatively common. Physicians should be alert to these symptoms among kidney transplant recipients. Conclusions Cryptococcosis is a serious infection among kidney transplant recipients in mainland China. It has unique characteristics, such as a relatively long time to onset after kidney transplantation, and diverse clinical manifestations. Treatment with intrathecal injection of amphotericin B is considered effective for central nervous system involvement. The findings of this study also highlight the urgent need for multicenter, prospective, and multidisciplinary clinical studies and education on cryptococcosis in kidney transplant recipients in China.

Occurrence of non-Candida albicans Candida (NCAC) species that are associated with elevated MIC values and therapeutic failures are increasing. As a result, timely and accurate means of identification to the species level is becoming an essential part of diagnostic practices in clinical settings. In this study, 301 clinically isolated yeast strains recovered from various anatomical sites [Blood (n = 145), other sites (n = 156)] were used to assess the accuracy and practicality of API 20C AUX and 21-plex PCR compared to MALDI-TOF MS and large subunit rDNA (LSU rDNA). MALDI-TOF MS correctly identified 98.33% of yeast isolates, 100% of top five Candida species, 95.7% of rare yeast species, while 1.3% of isolates were misidentified. API 20C AUX correctly identified 83.7% of yeast isolates, 97.2% of top five Candida species, 61.8% of rare yeast species, while 16.2% of yeast isolates were misidentified. The 21-plex PCR, accurately identified 87.3% of yeast isolates, 100% of top five Candida species, 72% of rare yeast species, but it misidentified 1.3% of rare yeast species while 9.9% of whole yeast isolates were not identified. The combination of rapidity of 21-plex PCR and comprehensiveness of API 20C AUX, led to correct identification of 92% of included yeast isolates. Due to expensiveness of MALDI-TOF MS and sequencing, this combination strategy could be the most accurate and inexpensive alternative identification strategy for developing countries. Moreover, by the advent and development of cost-effective, reliable, and rapid PCR machines that cost 130 US dollars, 21-plex could be integrated in routine laboratories of developing and resource-limited countries to specifically identify 95% causative agents of yeast-related infections in human. Databases of MALDI-TOF MS, API 20C AUX, and the number of target species identified by 21-plex require further improvement to keep up with the diverse spectrum of yeast species.

Table S1. CBS reference strains utilized for optimization of 9-plex PCR. (DOCX 20 kb)

Table S1. CBS reference strains utilized for optimization of 9-plex PCR. (DOCX 20 kb)