Real-time PCR assay to detect DNA in sera for the diagnosis of deep-seated trichosporonosis

Deep-seated trichosporonosis due to Trichosporon asahii is life-threatening and has high mortality. A real-time PCR assay to detect T. asahii DNA in sera for diagnosis of this fungal infection was developed. The assay showed a higher sensitivity than polysaccharide antigen detection method. Our new real-time PCR assay may be used for diagnosing deep-seated trichosporonosis due to T. asahii.     

A nested PCR assay to detect DNA in sera for the diagnosis of deep-seated trichosporonosis

Deep-seated trichosporonosis caused by Trichosporon asahii has a high mortality rate and a very poor prognosis. New species-specific oligonucleotide primers for T. asahii were developed from a sequence analysis of rRNA genes that included the internal transcribed spacer regions. A nested PCR assay with specific primers was used to examine 11 serum samples from 7 patients, who were diagnosed with deep-seated trichosporonosis histologically at autopsy. In addition, Trichosporon cell wall polysaccharide (PS) was detected by a latex agglutination (LA) test. Of 11 samples, seven had a positive LA test, and T. asahii DNA was also detected with the nested PCR assay. Of the four samples in which PS antigen was not detected, the nested PCR of two samples was positive. Our new nested PCR assay may be used as an adjunct to conventional methods for diagnosing T. asahii infection.     

Genotyping and Antifungal Drug Susceptibility of Trichosporon asahii Isolated from Chinese Patients

As a major pathogenic agent of trichosporonosis, Trichosporon asahii can cause life-threatening infection in immunocompromised patients. In this study, we analyzed the genotypes of the intergenic spacer (IGS) 1 region of the rRNA gene and the antifungal drug susceptibility of eight T. asahii isolates obtained from Chinese patients. Five genotypes were identified from the eight isolates, including three novel genotypes, three genotype 1, and two genotype 4. The eight T. asahii isolates were resistant to amphotericin B, 5-flucytosine, and terbinafine, but were highly sensitive to fluconazole (FLC), itraconazole (ITC), and voriconazole (VRC). The mean minimum inhibitory concentrations (MICs) of FLC and VRC were significantly lower than those reported in most other countries, while that of ITC was slightly higher. Our results suggest that genotypes of the T. asahii isolated from China are different from those of other countries, and azole drugs appeared to be more effective on the Chinese isolates. These results provide new insights into the epidemiology and antifungal treatment for T. asahii.     

Characterization of the skin fungal microbiota in patients with atopic dermatitis and in healthy subjects

Patients with atopic dermatitis (AD) are highly susceptible to viral, bacterial, and fungal skin infections because their skin is dry and this compromises the barrier function of the skin. Therefore, the skin microbiota of patients with AD is believed to be different from that of healthy individuals. In the present study, the skin fungal microbiota of nine patients with mild, moderate, or severe AD and ten healthy subjects were compared using an rRNA clone library. Fungal D1/D2 large subunit analysis of 3647 clones identified 58 species and seven unknown phylotypes in face scale samples from patients with AD and healthy subjects. Malassezia species were predominant, accounting for 63%-86% of the clones identified from each subject. Overall, the non-Malassezia yeast microbiota of the patients was more diverse than that of the healthy individuals. In the AD samples 13.0 ± 3.0 species per case were detected, as compared to 8.0 ± 1.9 species per case in the samples taken from healthy individuals. Notably, Candida albicans, Cryptococcus diffluens, and Cryptococcus liquefaciens were detected in the samples from the patients with AD. Of the filamentous fungal microbiota, Cladosporium spp. and Toxicocladosporium irritans were the predominant species in these patients. Many pathogenic fungi, including Meyerozyma guilliermondii (anamorphic name, Candida guilliermondii), and Trichosporon asahii, and allergenic microorganisms such as Alternaria alternata and Aureobasidium pullulans were found on the skin of the healthy subjects. When the fungal microbiota of the samples from patients with mild/moderate to severe AD and healthy individuals were clustered together by principal coordinates analysis they were found to be clustered according to health status.     

Phenotypic switching and beta-N-acetylhexosaminidase activity of the pathogenic yeast Trichosporon asahii

The pathogenic yeast Trichosporon asahii is the major causative agent of deep-seated trichosporonosis in immunocompromised patients. Although infection by this microorganism is becoming increasingly frequent, information related to its pathogenicity and virulence factors is still limited. Therefore, we investigated phenotypic switching in colony morphology, and the production of extracellular enzymes as a virulence factor. Sixty-one clinical isolates of T. asahii produced four different morphological types on Sabouraud dextrose agar (SDA): 69% WF (white farinose), 18% WP (white pustular), 10% Y (yellowish white), and 3% WC (white cerebriform). Strains of the three major types (WF, WP, and Y) produced two to five colony types when cultured on SDA at 37 C. The frequency of switching between colony types was 10(-2) to 10(-4), as in Candida albicans and Cryptococcus neoformans. Notably, most of the colonies switched to the smooth (S) type irreversibly, at frequencies of 10(-2) to 10(-3). No secreted aspartic proteinase or phospholipase activity was detected in T. asahii, while beta-N-acetylhexosaminidase activity, which catalyzes the hydrolysis of terminal nonreducing N-acetyl-D-hexosamine residues in N-acetyl-beta-D-hexosaminides, was found. Furthermore, enzymatic activity of the S type was significantly greater than that of the parent type in all strains. No other clinically relevant Trichosporon species (T. mucoides, T. inkin, and T. ovoides ) produced this enzyme. These results provide basal information for understanding the pathogenic potential of T. asahii.     

Susceptibilities to Amphotericin B, Fluconazole and Voriconazole of Trichosporon Clinical Isolates

A total of 35 Trichosporon isolates were collected from the Taiwan Surveillance of Antimicrobial Resistance of Yeasts (TSARY) project from 1999 to 2006, and their identifications as well as drug susceptibilities were determined. The most frequently isolated species was T. asahii (62.9%), and the most common clinical sample that yielded Trichosporon isolates was urine (37.1%). The etiology of all seven invasive trichosporonosis was T. asahii. For the 22 T. asahii isolates, the MIC(50) and MIC(90) for amphotericin B were 0.25 and 1 μg/mL, respectively. Those for fluconazole were 2 and 4 μg/mL, respectively, and for voriconazole 0.031 and 0.063 μg/mL, respectively. When the intraclass correlation coefficients (ICCs) and agreements were calculated, we found that the MICs of fluconazole obtained from different methods were similar and the inter-method discrepancies were low. Nevertheless, no unanimous MIC of amphotericin B and voriconazole was obtained among different methods.     

Nosocomial infection due to Trichosporon asahii: clinical revision of 22 cases

Twenty two cases of nosocomial infection caused by Trichosporon asahii, detected during a period of six years (1999-2005) is described. The patients were predominantly males with an average age of 47.3 years-old. The predominant diseases in the study group were respiratory insufficiency, cancer, diabetes, chronic renal insufficiency, cirrhosis and AIDS. The main predisposing conditions were antibiotic therapy, mechanical ventilation, urethral catheterization, catheter, corticoids, transplant, immunosuppressive therapy, chemotherapy, granulocytopenia, surgical procedures and continuous ambulatory peritoneal dialysis. The most used antifungal drugs were fluconazole and amphotericin B. In some cases several antifungals were administered. Five patients did not receive antifungal treatment, and one patient received granulocyte colony stimulating factor (G-CSF). Nine patients showed clinical improvement, nine died and the progress of four patients is unknown. T. asahii is an emergent pathogen in patients with immunodeficiency and its presence in these type hosts can not be considered colonization, as there is an important risk of invasive infection. So, in susceptible patients to develop trichosporonosis it is advisable to take into consideration this disease especially in intensive clinical care units.     

Antifungal pharmacodynamic characteristics of amphotericin B against Trichosporon asahii, using time-kill methodology

We determined the MIC of amphotericin B against 45 Trichosporon asahii isolates from various clinical and environmental sources, and used in vitro time-kill methods to characterize the relationship between amphotericin B concentrations and MIC for four representative T. asahii isolates. Amphotericin B had concentration-dependent antifungal activity. MICs ranged from 0.5 to 16 microg/ml, and most T. asahii isolates (76%, 34/45) were inhibited at safely achievable amphotericin B serum concentrations (< or = 2 microg/ml). However, 40% (18/45) of isolates were not killed at these concentrations (MFCs from 1.0 to 32 microg/ml). At concentrations > or = 2 x MIC, amphotericin B exhibited fungicidal activity (< 99.9% reduction in CFU) over a 12-hr time-period; the maximal effect was achieved at > or =4 x MIC. Susceptibility testing confirmed the resistance of T. asahii to amphotericin B, and in vitro pharmacodynamic results also suggest that amphotericin B is not suitable therapy for T. asahii infection.     

阿萨希毛孢子菌生物膜构建及水杨酸干预策略研究

目的在医院内常用生物材料聚氯乙烯（PVC）表面构建阿萨希毛孢子菌的生物膜,评估该生物膜对几种临床抗真菌药物的耐药性,并观察水杨酸是否对阿萨希毛孢子菌生物膜的形成有干预作用。方法菌株鉴定采用API20CAUX并经PCR鉴定复核;使用PVC于RPM11640-MOPS中培养进行阿萨希毛孢子菌生物膜构建;MIC测定采用法国生物梅里埃公司ATB Fungus-3真菌药敏试剂条以及微量液体稀释法,并观察水杨酸对生物膜构建的影响。结果阿萨希毛孢子菌可以通过几个不连续阶段在聚氯乙烯表面形成生物膜,且已使用PVC块上附着的生物膜细胞比未使用PVC块上黏附的生物膜细胞明显密集;固着相即生物膜细胞的MIC比浮游相成倍提高;24h两性霉素B的MIC〉512μ/ml,且经两性霉素B的药物刺激后,阿萨希毛孢子明显可见芽管延长,菌丝交织;水杨酸作用后阿萨希毛孢子菌的菌丝明显变短,孢子短小。结论介入性器械可以作为阿萨希毛孢子菌生物膜构建的黏附基质,使微生物群体黏附于细胞外多聚材料表面而造成持续播散感染,因此生物膜干预对阿萨希毛孢子菌深部感染的治疗有很重要的意义。     

Successful Treatment of Breakthrough Trichosporon asahii Fungemia by the Combination Therapy of Fluconazole and Liposomal Amphotericin B in a Patient with Follicular Lymphoma

Mycopathologia - Invasive trichosporonosis is a rare and lethal fungal infection that occurs in immunocompromised patients. Breakthrough trichosporonosis can occur in patients treated with...     

Update on the Genus <Emphasis Type="Italic">Trichosporon</Emphasis>

Trichosporon spp. are widely distributed in nature and can occasionally belong to the human microbiota. For many years, the unique species of the genus, Trichosporon beigelli, was only known as an environmental and saprophytic fungus occasionally found as the etiological agent of white piedra. However, case reports of invasive trichosporonosis have been frequently published and the genus is currently considered the second most common agent of yeasts disseminated infections. Based on molecular analysis, the taxon T. beigelli was replaced by several species and the taxonomy of the genus was progressively modified. Despite the reported increase of Trichosporon infections refractory to conventional antifungal drugs, there are only a few studies investigating in vitro susceptibility of Trichosporon spp. to new compounds. Difficulties on different species identification as well as the lack of standardized sensitivity tests in vitro, contribute to the limited information available on epidemiology, diagnosis and therapeutics of trichosporonosis.     

Cloning of the lanosterol 14-α-demethylase ( ERG11 ) gene in Trichosporon asahii: a possible association between G453R amino acid substitution and azole resistance in T. asahii

Lanosterol 14-α-demethylase ( Erg11 protein; Erg11p ), encoded by the ERG11 gene, is the primary target of azoles. Recently, a change in affinity of this enzyme for azoles has been reported as a resistance mechanism in several fungal species. Trichosporon asahii ( T.?asahii) is susceptible to fluconazole (FLC). This report identified the ERG11 gene of T.?asahii (NCBI accession; HQ176415). The Erg11p of T.?asahii, presumed from the DNA sequence, was closely related to the Erg11p of Cryptococcus neoformans. Furthermore, a FLC-susceptible strain was cultured in medium containing FLC at concentrations from 4.0 to 16?μg?mL(-1) in order to analyze the development of FLC resistance in T.?asahii. The degree of resistance was related to the FLC concentration of the growth medium. One highly resistant strain that was cultured in the medium containing 16?μg?mL(-1) FLC contained 1 point mutation (G1357C) that caused a single amino acid substitution at G453R. This amino acid is highly conserved among major fungal pathogens, and it is in a region very close to the heme-binding domain, which is characteristic of the cytochrome P450 superfamily, the primary target for the azole class of antifungal agents. This amino acid substitution may have caused the high resistance to azoles in T.?asahii.     

Pyrosequencing-based analysis of the mucosal microbiota in healthy individuals reveals ubiquitous bacterial groups and micro-heterogeneity

This study used 16S rRNA-based pyrosequencing to examine the microbial community that is closely associated with the colonic mucosa of five healthy individuals. Spatial heterogeneity in microbiota was measured at right colon, left colon and rectum, and between biopsy duplicates spaced 1 cm apart. The data demonstrate that mucosal-associated microbiota is comprised of Firmicutes (50.9% ± 21.3%), Bacteroidetes (40.2% ± 23.8%) and Proteobacteria (8.6%± 4.7%), and that interindividual differences were apparent. Among the genera, Bacteroides, Leuconostoc and Weissella were present at high abundance (4.6% to 41.2%) in more than 90% of the studied biopsy samples. Lactococcus, Streptococcus, Acidovorax, Acinetobacter, Blautia, Faecalibacterium, Veillonella, and several unclassified bacterial groups were also ubiquitously present at an abundance <7.0% of total microbial community. With the exception of one individual, the mucosal-associated microbiota was relatively homogeneous along the colon (average 61% Bray-Curtis similarity). However, micro-heterogeneity was observed in biopsy duplicates within defined colonic sites for three of the individuals. A weak but significant Mantel correlation of 0.13 was observed between the abundance of acidomucins and mucosal-associated microbiota (P-value = 0.04), indicating that the localized biochemical differences may contribute in part to the micro-heterogeneity. This study provided a detailed insight to the baseline mucosal microbiota along the colon, and revealed the existence of micro-heterogeneity within defined colonic sites for certain individuals.     

Analysis of Malassezia microbiota in healthy superficial human skin and in psoriatic lesions by multiplex real-time PCR

Yeasts from the genus Malassezia are members of the normal biota of human skin, and may play a role in dermatopathology. Our previous study of the fungal microbiota from healthy subjects and from patients with psoriasis using clone library analysis revealed the presence of five Malassezia species and four uncharacterized phylotypes. We now compared the Malassezia microbiota from six healthy body locations and two psoriatic lesions, and evaluated its stability over time using multiplex real-time PCR. Samples from each body location were obtained monthly, for 4 months. Dual-labeled probes were designed to recognize four Malassezia sp. and two uncharacterized groups, and a genus-specific probe was also developed. A good correspondence was obtained between real-time PCR data and clone library analyses. Malassezia restricta was the most abundant species in the majority of samples, and high amounts of Malassezia globosa were also detected. The uncharacterized phylotype 1 was usually detected in lower proportions, nevertheless it was present in most samples. The microbiota was host-specific and relatively stable over time. In accordance with our previous observations, no significant dichotomy between samples from healthy skin and from psoriatic lesions was found; the samples clustered according to the subject, rather than health status.     

Vaccinia virus induces strong immunoregulatory cytokine production in healthy human epidermal keratinocytes: a novel strategy for immune evasion

Iatrogenic cutaneous infection with vaccinia virus (VV) and naturally occurring systemic infection with variola virus both lead to the characteristic skin "pox" lesions. Despite significant medical experience with both viruses, surprisingly little is understood about the interactions between these poxviruses and healthy resident skin cells. In recent years, it has become clear that skin plays an essential role in modulating both innate and adaptive immune responses, in part by producing and responding to a variety of cytokines and chemokines upon stimulation. Antagonists of many of these compounds are encoded in poxvirus genomes. Infection of skin cells with poxvirus may lead to a unique pattern of cytokine and chemokine production that might alter the cutaneous immune surveillance function. In this study, we infected primary cultures of human skin cells with VV and monitored antigen expression, virus replication, and cytokine production from the infected cells. While T cells, Langerhans cells, and dermal dendritic cells were infected abortively, keratinocytes, dermal fibroblasts, and dermal microvascular endothelial cells (HMVEC-d) all supported the complete virus life cycle. In contrast to the robust viral replication in fibroblasts and HMVEC-d, only limited viral replication was observed in keratinocytes. Importantly, VV infection of keratinocytes led to up-regulation of immunoregulatory and Th2 cytokines, including transforming growth factor beta, interleukin-10 (IL-10), and IL-13. We propose that the rapid induction of keratinocyte Th2 and immunoregulatory cytokines represents a poxvirus strategy to evade immune surveillance, and the limited viral multiplication in keratinocytes may be a protective mechanism to help the immune system "win the race."     

rRNA-based profiling of bacteria in the axilla of healthy males suggests right-left asymmetry in bacterial activity

The activity of the human armpit microbiota triggers the formation of body odor. We used differential 16S rRNA gene (rDNA)- and rRNA-based terminal-restriction fragment length polymorphism fingerprinting in combination with cloning and sequencing to identify active members of the human armpit microbiota. DNA and RNA were isolated from skin scrub samples taken from both armpits of 10 preconditioned, healthy males. The fingerprint profiles indicated pronounced similarities between the armpit microbiota in the right and the left axillae of an individual test person, but larger differences between the axilla microbiota of different individuals. Using 16S rDNA and rRNA sequence data, the majority of peaks in the armpit profiles were assigned to bacteria affiliated with well-known genera of skin bacteria. The relative abundances of all groups were similar among the rDNA and rRNA samples, suggesting that all groups of armpit bacteria were active. Surprisingly, the relative abundance of sequences affiliated with Peptoniphilus sp. was by far and with statistical significance the highest in the rRNA samples of the right armpits. Thus, bacteria affiliated with Peptoniphilus sp. might have been particularly active in the right axillae of the test persons, possibly owing to the handedness of the test persons, which might cause different environmental conditions in the right axillae.     

Staphylococcus epidermidis role in the skin microenvironment

Wound healing is a complex dynamic physiological process in response to cutaneous destructive stimuli that aims to restore the cutaneous’ barrier role. Deciphering the underlying mechanistic details that contribute to wound healing will create novel therapeutic strategies for skin repair. Recently, by using state‐of‐the‐art technologies, it was revealed that the cutaneous microbiota interact with skin immune cells. Strikingly, commensal Staphylococcus epidermidis‐induced CD8+ T cells induce re‐epithelization of the skin after injury, accelerating wound closure. From a drug development perspective, the microbiota may provide new therapeutic candidate molecules to accelerate skin healing. Here, we summarize and evaluate recent advances in the understanding of the microbiota in the skin microenvironment.     

抑制阿萨希毛孢子菌顶体对其增殖及致病性的影响

目的探讨抑制阿萨希毛孢子菌(Trichosporon asahii,T.asahii)顶体(Spitzenkrper)形成后对其细胞增殖及致病性的影响。方法经不同浓度细胞松弛素D(0.5、1.0及2.0μmol/L)处理阿萨希毛孢子菌,以抑制其顶体形成,体外测定生长曲线及生长菌落变化;小鼠体内接种上述菌悬液后,统计小鼠3周内的死亡率,并取内脏进行组织培养和病理检查,统计感染率。结果经细胞松弛素D处理抑制顶体形成后,T.asahii细胞生长明显延迟8~16 h,生长菌落直径明显变小;小鼠死亡率及感染率亦出现不同程度降低,并随着药物浓度的升高,其致病性明显降低。结论抑制阿萨希毛孢子菌顶体形成后,细胞增殖明显受到抑制,且致病性也随细胞松弛素D浓度的增加而降低。提示顶体可以作为研制新型抗真菌药物的新靶点。     

Characterization of Candida Species from Different Populations in Taiwan

The opportunistic Candida species existing as part of commensal microbiota in humans are usually the etiological agents causing infections. We investigated whether isolates collected from different age groups, hospital units, and sources have distinct characteristics. A total of 913 isolates comprising 395 Candida albicans, 230 Candida tropicalis, 202 Candida glabrata, 62 Candida parapsilosis, 13 Candida krusei, and 11 of other six species were analyzed. Urine was the most common source (41.2%), followed by sputum (16.3%), blood (15.2%), and others (27.3%). Candida albicans and C. parapsilosis were more prevalent in the working group [from 19 to 65 years], whereas C. tropicalis and C. glabrata were more prevalent in the elder one (≥ 66 years). We found that the age of patients and the source of isolates affect the distribution of species. On the other hand, the drug susceptibility of isolates was associated with fungal species and whether patients were hospitalized.