Zhe Wan

Identification of Aspergillus fumigatus and Related Species by Nested PCR Targeting Ribosomal DNA Internal Transcribed Spacer Regions

ABSTRACT Aspergillus fumigatus is the most common species that causes invasive aspergillosis. In order to identify A. fumigatus, partial ribosomal DNA (rDNA) from two to six strains of five different Aspergillus species was sequenced. By comparing sequence data from GenBank, we designed specific primer pairs targeting rDNA internal transcribed spacer (ITS) regions ofA. fumigatus. A nested PCR method for identification of other A. fumigatus-related species was established by using the primers. To evaluate the specificities and sensitivities of those primers, 24 isolates of A. fumigatus and variants, 8 isolates of Aspergillus nidulans, 7 isolates ofAspergillus flavus and variants, 8 isolates ofAspergillus terreus, 9 isolates of Aspergillus niger, 1 isolate each of Aspergillus parasiticus, Aspergillus penicilloides, Aspergillus versicolor, Aspergillus wangduanlii, Aspergillus qizutongii, Aspergillus beijingensis, and Exophiala dermatitidis, 4 isolates ofCandida, 4 isolates of bacteria, and human DNA were used. The nested PCR method specifically identified the A. fumigatus isolates and closely related species and showed a high degree of sensitivity. Additionally, four A. fumigatusstrains that were recently isolated from our clinic were correctly identified by this method. Our results demonstrate that these primers are useful for the identification of A. fumigatus and closely related species in culture and suggest further studies for the identification of Aspergillus fumigatus species in clinical specimens.

The Sho1 Sensor Regulates Growth, Morphology, and Oxidant Adaptation in Aspergillus fumigatus but Is Not Essential for Development of Invasive Pulmonary Aspergillosis

ABSTRACT Aspergillus fumigatus is an important opportunistic fungal pathogen. This organism must be able to adapt to stress changes in the microenvironment during host invasion and systemic spread. The high-osmolarity-glycerol (HOG) mitogen-activated protein kinase (HOG-MAPK) signaling pathway plays an important role in regulating morphology, growth, and adaptation to stress and virulence in a number of fungal pathogens. The Sho1 adaptor protein is one important element of the two upstream branches of the HOG-MAPK pathway in Saccharomyces cerevisiae, a signal transduction cascade involved in adaptation to stress. We constructed a sho1 mutant of A. fumigatus, MA21. Both the growth and germination rates of the mutant were reduced, and the MA21 strain had an irregular hyphal morphology characterized by reduced production of phialides and conidia. This gene deletion mutant was sensitive to 2.5 mM hydrogen peroxide and 15 μM menadione, but it appeared to be minimally sensitive to diamide compared to the wild-type strain. In an immunosuppressed mouse model, the mutant was as virulent as the wild-type or complemented strains. These data support the idea that the loss of sho1, a highly conserved gene among fungi, regulates radial hyphal growth and delays germination of A. fumigatus conidia. In addition, the sho1 gene has a visible effect in the adaptation to oxidative stress in A. fumigatus similar to that in S. cerevisiae.

Molecular typing study of the Microsporum canis strains isolated from an outbreak of tinea capitis in a school

Tinea capitis is a dermatophyte infection of the scalp that occurs most often in prepubescent children. Tinea capitis may be transmitted by shared use of contaminated hairbrush, by contact with fomites or by direct physical contact with an infected person. Occasionally, outbreak of tinea capitis would happen under some special conditions. Last year, we found an outbreak of tinea capitis in a school due to Microsporum canis. In epidemiological study, we performed the prevalence survey to all of the exposed persons by physical examinations and mycological laboratory tests, including KOH preparation and fungal cultures. We also investigated the environment in the school. In molecular typing study of the M. canis isolated from patients and the environment, random primer amplification polymorphic DNA (RAPD) method, the specific amplification of subrepeat element in the ribosomal DNA nontranscribed spacer (NTS), and the analysis of DNA sequence in the intertranscribed spacer (ITS) of rDNA were performed. The total number of exposed children was seventy-one , among them forty-two were attacked by tinea capitis. The ratio between boy and girl was 13 : 1. The ages of the patients was ranged from 3.5 years old to 10 years old. Four patients bred cat or dog as pet. Most patients appeared noninflammatory type of tinea capitis and several patients were inflammatory type. Under microscopic examination the invaded hair were all ectothrix. The pathogens isolated from these patients were M. canis. And we also isolated M. canis from the carpet and the pillowcase in the school. The patterns of total strains of M. canis in the RAPD method and PCR amplification of the rDNA NTS region study were identical, and the isolates from patients and the environment contained the same DNA sequences in the ITS region. The outbreak of tinea capitis was caused by M. canis. The M. canis isolated from patients and from the environment were probably the same origin.

Antifungal activity of statins against Aspergillus species.

The cholesterol-lowering agents known as statins have in vitro activities against human pathogenic fungi, such as Candida species, Cryptococcus neoformans, and Zygomycetes. Synergy between statins and azoles against these fungi has also been reported. We evaluated the in vitro activities of two statins, lovastatin and simvastatin, alone and in combination with azoles and amphotericin B, against clinical isolates of Aspergillus spp. A disk diffusion assay showed that both statins were active against Aspergillus spp. The minimal inhibitory concentration (MIC) ranges for lovastatin and simvastatin against Aspergillus spp. were 16 to >256 microg/ml and 4 to >256 microg/ml, respectively. Although both statins were fungicidal for A. fumigatus, the MICs were vastly higher than clinically achievable concentrations. The results of a combined agar dilution-Epsilometer test as well as a disk diffusion assay showed that neither statin had any effect on the in vitro activities of itraconazole, voriconazole, or amphotericin B against Aspergillus spp.

In vitro combined activity of amphotericin B, caspofungin and voriconazole against clinical isolates of Trichosporon asahii

Disseminated infections caused by Trichosporon asahii are difficult to resolve. Combination regimens with synergistic drugs could provide additional options for treating trichosporonosis. The aim of this study was to evaluate the antifungal activities of voriconazole (VCZ), caspofungin (CAS) and amphotericin B (AMB) alone or in combination in vitro against clinical isolates of T. asahii. The combined antifungal activities of VCZ, CAS and AMB against 18 clinical isolates were assessed by a chequerboard microdilution method. CAS combined with AMB showed the highest percentage of synergistic effects (89%), much higher than those of the other two combinations (AMB/VCZ and CAS/VCZ both 17%). No antagonistic effect was observed in any case. This study demonstrates that the activity of two combined antifungals, especially the combination of CAS and AMB, against T. asahii is more effective than that of a drug alone against this fungus, suggesting that combined antifungal therapy may be a potential strategy for treating disseminated trichosporonosis.

Afyap1, encoding a bZip transcriptional factor of Aspergillus fumigatus, contributes to oxidative stress response but is not essential to the virulence of this pathogen in mice immunosuppressed by cyclophosphamide and triamcinolone

Aspergillus fumigatus, an important human fungal pathogen, encounters high levels of reactive oxygen species following its ingestion by phagocytes. Reactive oxygen species are important mediators of the fungicidal activities of phagocytes. In yeasts, YAP1 encodes for transcriptional factors that contribute to their oxidative stress response and given the importance of the stress response, we hypothesized that the YAP1 homologue in A. fumigatus plays a similar role in this fungus. In this study, we found that Afyap1, the Yap1 homologue of A. fumigatus, confers protection against oxidative stress. Replacement of Afyap1 with the marker gene pyrG (DeltaAfyap1) resulted in hypersensitivity of A. fumigatus to oxidants such as H(2)O(2) and menadione. In contrast, an A. fumigatus strain harboring multiple-copy Afyap1 was resistant to these two oxidants as well as the oxidant diamide. However, DeltaAfyap1 and strain harboring multiple-copy Afyap1 were comparable in their virulence to a wild-type A. fumigatus strain in a murine model of invasive pulmonary aspergillosis. Taken together, these results demonstrate that Afyap1 is involved in oxidative stress response but is not an essential virulence factor for A. fumigatus.

Microbiological characteristics of medically important Trichosporon species.

Trichosporon species are opportunistic pathogens associated with a high mortality rate in immunocompromised patients. Disseminated trichosporonosis is uncommon but reports are increasing. In this study, using 16 stock clinical isolates of suspected Trichosporon species and 4 known Trichosporon strains, we investigated the morphology, physio-biochemistry, molecular biology and antifungal susceptibility characteristics of these Trichosporon spp. and discovered that ITS sequence-based identification is a rapid and accurate identification alternative to most phenotypic or physio- biochemical methods. In vitro antifungal susceptibility tests showed high amphotericin B, itraconazole and terbinafine MIC value in these Trichosporon strains.

The effects of temperature, pH, and salinity on the growth and dimorphism of Penicillium marneffei

Penicillium marneffei is an important thermal dimorphic fungus that is endemic to Southeast Asia and China and causes penicilliosis, an AIDS-defining disease. Dimorphic switching is considered an important growth characteristic associated with its pathogenicity. In recent years, the molecular mechanisms underlying both dimorphic switching and monomorphic growth have been studied. However, little is known about the physical and chemical factors that impact either dimorphic switching or monomorphic growth of this organism. Further, the natural history of the disease is unclear. Our experiments focus upon the effects of temperature, pH and salinity on both growth phases of P. marneffei. We compared 11 isolates of P. marneffei and found that all could grow at a wide temperature range (8.0-39.8 degrees C), but growth was dramatically inhibited at 40 degrees C. The morphological switch from hyphae to yeast growth was initiated at 32 degrees C. However, the sensitivity to elevated temperatures during this transition varied among isolates. Both hyphae and yeast growth forms grew much better at acidic (pH 5, 6) and neutral pH than at alkaline conditions. While similar sensitivities were observed at high concentrations of NaCl and CaCl(2), in general, yeast cells displayed a greater sensitivity to both compounds. Our data demonstrate that isolate differences occur in growth patterns. Importantly, the growth requirements defined in our study may shed light on the environmental conditions that favor its survival, a subject that is not completely resolved in the current literature.

In vitro interaction of terbinafine with itraconazole and amphotericin B against fungi causing chromoblastomycosis in China

The combined effects of terbinafine with itraconazole and amphotericin B against Cladophialophora carrionii, Phialophora verrucosa and Fonsecaea pedrosoi were evaluated in vitro by the checker-board method and expressed as a fractional inhibitory concentration (FIC) index. Synergy was observed with the combination of terbinafine and itraconazole against one isolate of C. carrionii and no antagonism was observed. When amphotericin B was combined with terbinafine or itraconazole, no synergy or antagonism was noted with all isolates included in this investigation.

Antifungal Activity of Antifungal Drugs, as Well as Drug Combinations Against Exophiala dermatitidis

To evaluate the inxa0vitro efficacy of common antifungal drugs, as well as the interactions of caspofungin with voriconazole, amphotericin B, or itraconazole against the pathogenic black yeast Exophiala dermatitidis from China, the minimal inhibitory concentrations (MICs) of terbinafine, voriconazole, itraconazole, amphotericin B, fluconazole, and caspofungin against 16 strains of E. dermatitidis were determined by using CLSI broth microdilution method (M38-A2). The minimal fungicidal concentrations (MFCs) were also determined. Additionally, the interactions of caspofungin with voriconazole, amphotericin B, itraconazole or fluconazole, that of terbinafine with itraconazole, or that of fluconazole with amphotericin B were assessed by using the checkerboard technique. The fractional inhibitory concentration index (FICI) was used to categorize drug interactions as following, synergy, FICIxa0≤xa00.5; indifference, FICIxa0>xa00.5 and ≤4.0; or antagonism, FICIxa0>xa04.0. The MIC ranges of terbinafine, voriconazole, itraconazole, amphotericin B, fluconazole, and caspofungin against E. dermatitidis were 0.06–0.125xa0mg/l, 0.25–1.0xa0mg/l, 1.0–2.0xa0mg/l, 1.0–2.0xa0mg/l, 16–64xa0mg/l, and 32–64xa0mg/l, respectively. The inxa0vitro interactions of caspofungin with voriconazole, amphotericin B, and itraconazole showed synergic effect against 10/16(62.5%), 15/16(93.75%), and 16/16(100%) isolates, while that of caspofungin with fluconazole showed indifference. Besides, the interaction of terbinafine with itraconazole as well as that of fluconazole with amphotericin B showed indifference. Terbinafine, voriconazole, itraconazole, and amphotericin B have good activity against E. dermatitidis. The combinations of caspofungin with voriconazole, amphotericin B or itraconazole present synergic activity against E. dermatitidis. These results provide the basis for novel options in treating various E. dermatitidis infections.