Recurrent fungal infections in a Chinese patient with CARD9 deficiency and a review of 48 cases.

Deficiency of CARD9 (caspase recruitment domain-containing protein 9) has been reported in individuals with recurrent and invasive fungal infections. We report on a patient who first had Trichosporon asahii affecting the skin then Candida albicans infections involving the digestive tract and knee joint, along with elevated serum IgE. After stimulation with C. albicans, peripheral blood mononuclear cells of this patient produced less tumour necrosis factor-α, interferon-γ and interleukin-17 than those of healthy controls. Furthermore, the serum IgE levels of this patient were positively correlated with the severity of fungal infection during the course of treatment. Sanger sequencing identified one homozygous frameshift mutation (p.D274fsX60) in CARD9. We further performed a review including 48 cases with CARD9 deficiency. According to the data published previously, CARD9-deficient patients demonstrated obviously elevated IgE in serum (median 1300 IU mL-1 ), which could distinguish them from otherwise healthy people with fungal infections (area under the curve 0·94, P < 0·001). Patients carrying the mutations Q289X and Q295X had a higher mortality rate (24% vs. 0%, P < 0·05). Patients with the mutations R18W, R35Q, R70W, G72S or Y91H in the CARD domain, and the nonsense mutation Q295X in the coiled-coil domain, seemed to be more prone to Candida infections (90% vs. 20%, P < 0·005) and central nervous system infections (60% vs. 12%, P < 0·005).

Cytokines profile in immunocompetent mice during Trichosporon asahii infection.

Trichosporon asahii is an opportunistic yeastlike fungus commonly associated with systemic infections in immunocompromised patients. Neutropenia is recognized as the main risk factor in infections by T. asahii; however, little is known about the cytokine response during trichosporonosis. Here, we evaluated systemic and local cytokine production and histological damage in immunocompetent mice during systemic infection with T. asahii. We found a significant increased presence of G-CSF, TNF-α, IFN-γ, and IL-6 in sera samples. High levels of G-CSF were found in organs (kidney, liver and spleen); meanwhile IL-10, IL-17A, IL-2, IL-4 and TNF-α were found in low levels. Neutrophils and fungal structures were found in early stage in analyzed organs. Our results demonstrated that T. asahii induces a systemic inflammatory response and G-CSF environment in infected organs in immunocompetent mice and neutrophil recruitment in analyzed tissue suggests the importance of these cells for fungal control.

[FAMILIAL SUMMER-TYPE HYPERSENSITIVITY PNEUMONITIS INDUCED IN SUMMER AND WINTER AT THE WORK PLACE].

We report two family members, a 64-year-old woman (patient 1) and her 37-year-old son (patient 2) diagnosed with summer-type hypersensitivity pneumonitis (SHP). Both patients had high serum titers of anti-Trichosporon asahii antibody. The patients lived in the same house and worked in the same barbershop. Patient 1 was diagnosed with SHP in the summer, and she reacted positively to the provocation test at the work place, but not in the house. Patient 2 was diagnosed with SHP in the winter. Generally, SHP develops and is diagnosed in the summer. The home environment is responsible for most cases of familial SHP. Therefore, our cases of familial SHP are unusual and may suggest that the clinical characteristics of SHP have changed, due to alterations in social and environmental conditions.

The Cell Biology of the Trichosporon-Host Interaction.

Fungi of the genus Trichosporon are increasingly recognized as causative agents of superficial and invasive fungal disease in humans. Although most species are considered commensals of the human skin and gastrointestinal tract, these basidiomycetes are an increasing cause of fungal disease among immunocompromised hosts, such as hematological patients and solid organ transplant recipients. The initiation of commensal or pathogenic programs by Trichosporon spp. involves the adaptation to the host microenvironment and its immune system. However, the exact virulence factors activated upon the transition to a pathogenic lifestyle, including the intricate biology of the cell wall, and how these interact with and subvert the host immune responses remain largely unknown. Here, we revisit our current understanding of the virulence attributes of Trichosporon spp., particularly T. asahii, and their interaction with the host immune system, and accommodate this knowledge within novel perspectives on fungal diagnostics and therapeutics.

Early transcriptional response of human monocyte-like THP-1 cells in response to Trichosporon asahii infection.

Trichosporon asahii is the major cause of invasive trichosporonosis, but little is known about the host immune response to this pathogen. In this study, the early transcriptional response of human monocyte-like THP-1 cells to T. asahii infection was evaluated using cDNA microarray and 1,315 differentially expressed genes were identified. The up-regulated genes were mostly involved in both innate and adaptive immune responses, as well as apoptosis and anti-apoptosis processes. Genes encoding the pro-inflammatory cytokines TNF-α, IL-1ß, IL18 and IL-23α, along with the both C-C motif and C-X-C motif chemokines were strongly up-regulated, suggesting that THP-1 cells can mount a powerful inflammatory response to T. asahii infection. Genes encoding pattern recognition receptors were found up-regulated, such as dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin, cluster of differentiation 36 and the long pentraxin 3. Genes encoding members of the dual-spasticity phosphates family were up-regulated, and these genes were considered as a negative feedback mechanism to prevent excessive inflammatory response. The down-regulated genes in T. asahii-infected THP-1 cells were predominantly associated with cell cycle, mitosis, cell division and DNA repair. Thus, our study defines the early transcriptional response of monocyte-like THP-1 cells to T. asahii infection and provides a foundation for further investigations into the pathogenesis of T. asahii infection.

Differentiation of the emerging human pathogens Trichosporon asahii and Trichosporon asteroides from other pathogenic yeasts and moulds by using species-specific monoclonal antibodies.

The fungal genus Trichosporon contains emerging opportunistic pathogens of humans, and is the third most commonly isolated non-candidal yeast from humans. Trichosporon asahii and T. asteroides are the most important species causing disseminated disease in immunocompromised patients, while inhalation of T. asahii spores is the most important cause of summer-type hypersensitivity pneumonitis in healthy individuals. Trichosporonosis is misdiagnosed as candidiasis or cryptococcosis due to a lack of awareness and the ambiguity of diagnostic tests for these pathogens. In this study, hybridoma technology was used to produce two murine monoclonal antibodies (MAbs), CA7 and TH1, for detection and differentiation of Trichosporon from other human pathogenic yeasts and moulds. The MAbs react with extracellular antigens from T. asahii and T. asteroides, but do not recognise other related Trichosporon spp., or unrelated pathogenic yeasts and moulds including Candida, Cryptococcus, Aspergillus, Fusarium, and Scedosporium spp., or the etiologic agents of mucormycosis. Immunofluorescence and Western blotting studies show that MAb CA7, an immunoglobulin G1 (IgG1), binds to a major 60 kDa glycoprotein antigen produced on the surface of hyphae, while TH1, an immunoglobulin M (IgM), binds to an antigen produced on the surface of conidia. The MAbs were used in combination with a standard mycological growth medium (Sabouraud Dextrose Agar) to develop an enzyme-linked immunosorbent assay (ELISA) for differentiation of T. asahii from Candida albicans and Cryptococcus neoformans in single and mixed species cultures. The MAbs represent a major advance in the identification of T. asahii and T. asteroides using standard mycological identification methods.

Identification of fungal DNA in BALF from patients with home-related hypersensitivity pneumonitis.

BACKGROUND: In Japan, a major type of home-related hypersensitivity pneumonitis (HP) is summer-type HP, which is caused by Trichosporon asahii (T. asahii) or Trichosporon mucoides. Some patients with home-related HP test negative for antibodies against Trichosporon; yet, a causative mold antigen cannot be identified. METHODS: We analyzed 19 patients with home-related HP, 8 healthy volunteers, and 35 patients with other diseases. We extracted DNA from cell pellets of bronchoalveolar lavage fluid (BALF), amplified the DNA by PCR using Trichosporon-specific primers or other fungus-specific primers, and cloned as well as sequenced the PCR amplicon. Other primers used were specific for Acremonium chrysogenum, Aspergillus fumigatus, Aspergillus niger, Fusarium napiforme, Humicola fuscoatra, Penicillium corylophilum, and Pezizia domiciliana. RESULTS: We detected Trichosporon DNA (n = 17) and F. napiforme DNA (n = 2) by PCR in 19 patients with home-related HP; however, these species were not identified in healthy volunteers. After sequencing of the PCR amplicon for Trichosporon species, we identified T. asahii (n = 11), Trichosporon japonicum (n = 1), and Cryptococcus uzbekistanesis (n = 4). CONCLUSION: We could detect fungal DNA in BALF cell pellets from patients with home-related HP. These data suggest that this method might be useful to detect antigens responsible for home-related HP.

Galectin-9 expands immunosuppressive macrophages to ameliorate T-cell-mediated lung inflammation.

Galectin-9 (Gal-9) plays pivotal roles in the modulation of innate and adaptive immunity to suppress T-cell-mediated autoimmune models. However, it remains unclear if Gal-9 plays a suppressive role for T-cell function in non-autoimmune disease models. We assessed the effects of Gal-9 on experimental hypersensitivity pneumonitis induced by Trichosporon asahii. When Gal-9 was given subcutaneously to C57BL/6 mice at the time of challenge with T. asahii, it significantly suppressed T. asahii-induced lung inflammation, as the levels of IL-1, IL-6, IFN-gamma, and IL-17 were significantly reduced in the BALF of Gal-9-treated mice. Moreover, co-culture of anti-CD3-stimulated CD4 T cells with BALF cells harvested from Gal-9-treated mice on day 1 resulted in diminished CD4 T-cell proliferation and decreased levels of IFN-gamma and IL-17. CD11b(+)Ly-6C(high)F4/80(+) BALF Mphi expanded by Gal-9 were responsible for the suppression. We further found in vitro that Gal-9, only in the presence of T. asahii, expands CD11b(+)Ly-6C(high)F4/80(+) cells from BM cells, and the cells suppress T-cell proliferation and IFN-gamma and IL-17 production. The present results indicate that Gal-9 expands immunosuppressive CD11b(+)Ly-6C(high) Mphi to ameliorate Th1/Th17 cell-mediated hypersensitivity pneumonitis.

Structural and functional properties of the Trichosporon asahii glucuronoxylomannan.

The virulence attributes of Trichosporon asahii are virtually unknown, despite its growing relevance as causative agent of superficial and invasive diseases in humans. Glucuronoxylomannan (GXM) is a well described virulence factor of pathogenic species in the Cryptococcus genus. GXM is also produced by species of the Trichosporon genus, and both polysaccharides share antigenic determinants, but unlike cryptococcal GXM, relatively little work has been done on trichosporal GXMs. In this study, we analyzed structural and functional aspects of GXM produced by T. asahii and compared them to the properties of the cryptococcal polysaccharide. Trichosporal and cryptococcal GXM shared antigenic reactivity, but the former polysaccharide had smaller effective diameter and negative charge. GXM anchoring to the cell wall was perturbed by dimethylsulfoxide and required interactions of chitin-derived oligomers with the polysaccharide. GXM from T. asahii supernatants are incorporated by acapsular mutants of Cryptococcus neoformans, which renders these cells more resistant to phagocytosis by mouse macrophages. In summary, our results establish that despite similarities in cell wall anchoring, antigenic and antiphagocytic properties, trichosporal and cryptococcal GXMs manifest major structural differences that may directly affect polysaccharide assembly at the fungal surface.

[Familial summer-type hypersensitivity pneumonitis in a husband and wife].

We encountered a family in which two of four members, the husband and his wife, had summer-type hypersensitivity pneumonitis at the same time, about two months after they moved to the residence. A 45-year-old man had cough, fever and exertional dyspnea. Chest computed tomography showed diffuse centriloblar ground-glass attenuation in both lung fields. His 43-year-old wife had chest small nodular shadows and similar symptoms to his husband. Serum anti-Tricosporon cutaneum (T. asahi: serotype II and T. mucoides: serotype I) antibodies of both patients were at the positive level. They were given diagnosis as summer-type hypersensitivity pneumonitis by radiological, serological and histological examinations. The symptoms in both cases were improved immediately after administration of systemic corticosteroid. Summer-type hypersensitivity pneumonitis was assumed to be caused for about two months duration of expousure to antigen.

Chronic summer-type hypersensitivity pneumonitis initially misdiagnosed as idiopathic interstitial pneumonia.

The clinical features of chronic hypersensitivity pneumonitis (HP) are similar to idiopathic interstitial pneumonias (IIPs) including idiopathic pulmonary fibrosis (IPF). We report 2 cases of chronic summer-type HP with insidious onset. They were misdiagnosed as having IIPs before referral to our hospital. Anti-trichosporon antibodies were positive in these cases. Their disease progressed due to the intermittent or continuous exposure to the antigen. Chronic summer-type HP should be included in the list of differential diagnosis of chronic interstitial lung diseases. Environmental investigation for an accurate diagnosis is important to convince the patient of the necessity to strictly avoid any future exposure to antigen.

[Summer-type hypersensitivity pneumonitis-immunological examinations in onset and non-onset members].

We examined immunological differences by non-specific and specific stimuli, using Trichosporon asahii (Ta), in 5 subjects in one family. The mother and her eldest daughter, among the 5 subjects in the family, were given diagnoses of summer-type hypersensitivity pneumonitis (HP) as a result of imaging, serological and histological examinations. The symptoms in both cases improved immediately after systemic corticosteroid therapy. Their home environment was cleaned up, and there has been no recurrence. All family subjects showed a positive reaction for the precipitin of Ta crude antigen, in addition four subjects, but not the father, revealed positive reactions for Type II Ta antigen in agglutination tests. In patients with HP, the concentrations of IFN-gamma by anti-CD3 + and anti-CD28 antibody stimulation were lower than those of healthy subjects. The concentrations of IFN-gamma and TNF-alpha for Ta antigen stimulation revealed no differences between the patients and healthy subjects in the family. In conclusion, we could not find any differences between the patients and non-patients in the family from the results of the serological and cytokine production measurement.

[Familial summer-type hypersensitivity pneumonitis--case report and review of literature].

A 37-year-old-woman was admitted to our hospital because of chest bilateral reticular shadow with fever, cough, general malaise and exertional dyspnea in the summer. A diagnosis of summer-type hypersensitivity pneumonitis (SHP) was made by radiological, serological and histological examinations. Her 10-year-old daughter had chest reticular shadows and similar symptoms. These two patients were positive for serum anti-Trichosporon cutaneum (T. asahii, T. mucoides) antibodies and T. asahii was identified from cultured samples obtained from their house. They recovered spontaneously after hospitalization or isolation from the antigen. We reviewed the clinical features in sixteen families with familial SHP reported in Japan. Children aged under 15 years old accounted for 34% and there was no gender difference among patients. This finding differs from the conventionally defined features of patients with SHP. Measurements of serum KL-6, SP-D and SP-A seem to be useful for auxiliary diagnosis and monitoring the disease activity of SHP, especially in pediatric cases who cannot undergo invasive evaluation.

[A familial case of summer-type hypersensitivity pneumonitis possibly associated with bird breeder's lung diagnosed by bronchoalveolar lavage fluid].

Case 1: A 32-year-old woman had cough and exertional dyspnea in August 2002, and chest computed tomographic scan revealed diffuse centrilobular nodules. Bronchoalveolar lavage fluid (BALF) showed a high proportion of lymphocytes with a decreased CD 4/CD 8 ratio. Transbronchial lung biopsy (TBLB) specimens showed alveolitis. Summer-type hypersensitivity pneumonitis was diagnosed on the basis of positive findings of anti-Trichosporon antibodies in the serum. Case 2: A 64-year-old man, the father of Case 1, also had cough and exertional dyspnea in August 2003. He had been in close contact with pigeons. Chest computed tomographic scan revealed bilateral map-like ground-glass opacities predominantly in the upper lobes. BALF showed a high proportion of lymphocytes with a decreased CD 4/CD 8 ratio. TBLB specimens showed alveolitis, granuloma and Masson body in the air spaces. Specific IgG and IgA antibodies against Trichosporon asahii, IgA antibodies against Trichosporon mucoides, and IgA antibodies against pigeon dropping extracts were found only in the BALF but not in the serum. Although a positive finding of returning-home provocation test was definitive in diagnosing summer-type hypersensitivity pneumonitis, he was also suspected of having bird fancier's lung.

[Trichosporon antibodies (corrected of antigen) and HLA-antigen in summer-type hypersensitivity pneumonitis in a family].

We encountered a family in which all of the three members (the parents, a 45-year-old woman and 51-year-old man, and their 15-year-old daughter) had Trichosporon cutaneum antibodies (corrected of antigen), and two (the parents) suffered from summer-type hypersensitivity pneumonitis in the late summer. The husband complained of dry cough, fever and dyspnea on exertion from July after severe interstitial pneumonitis and was treated with steroid pulse in September 2004. His wife visited our hospital and complained of a common cold-like symptom which progressed in August 2005. The couple were given diagnoses of summer-type hypersensitivity pneumonitis because they were positive for serum anti-Trichosporon mucoides antigen and asahii antigen. Their asymptomatic daughter was positive for these antigens. Both wife and daughter had HLA-DQ 8 (3) and 9 (3) that are suggested to be important HLA antigens related to the occurrence of summer-type hypersensitivity pneumonitis.

TA-19, a novel protein antigen of Trichosporon asahii, in summer-type hypersensitivity pneumonitis.

The most common form of hypersensitivity pneumonitis in Japan is summer-type hypersensitivity pneumonitis (SHP), which is caused by the inhalation of Trichosporon asahii or Trichosporon mucoides. To seek protein antigens relevant to the immunopathogenesis of SHP, we constructed a cDNA expression library of T. asahii, a major causative yeast species of SHP. Using the immunoscreening method, we identified and cloned a novel gene encoding a 19-kD protein, named TA-19, which proved to be specifically recognized in the bronchoalveolar lavage (BAL) fluids and sera of patients with SHP. IgG, IgA, and IgM antibodies to the recombinant TA-19 protein were significantly elevated in the sera as well as in the BAL fluids from SHP patients compared with those from non-SHP groups. This protein also induced SHP-specific proliferation of the mononuclear cells from both the peripheral blood and BAL. These results reveal that TA-19 derived from T. asahii may play a relevant role in specific cellular and humoral immune responses in patients with SHP.

Increased release of glucuronoxylomannan antigen and induced phenotypic changes in Trichosporon asahii by repeated passage in mice.

Clinically important fungi such as Candida albicans and Cryptococcus neoformans are known to undergo phenotypic changes after repeated subculture or passages in vivo. However, there are no reports describing this phenomenon in Trichosporon species. This study investigated whether in-vivo passages of environmental isolates of Trichosporon asahii in mice changes their phenotype; three environmental isolates and 14 clinical isolates (from deep-seated infections) were used. The shape of the colony and cell type were observed, and the titre of glucuronoxylomannan (GXM) antigen and concentration of (1-->3)-beta-D-glucan were measured for each isolate. Changes in these features were also examined after three passages of the environmental isolates in mice. The shape of colonies and cell types were clearly different in environmental and clinical isolates. Furthermore, the clinical isolates released significantly higher levels of GXM antigen than environmental isolates (titre: log2 9.4 SD 0.7 versus log2 5.4 SD 1.4). The phenotype of passaged isolates was significantly different from the original environmental isolates with respect to the morphology of colonies and cell type and GXM release (titre: log2 10.0 SD 0.7 versus log2 5.4 SD 1.4). These results suggest that the phenotypic changes in T. asahii occur as a result of in-vivo passages. This process may allow a proportion of the fungal population to escape eradication by the host immune system, as GXM antigen is considered to protect the fungi against phagocytosis by polymorphonuclear leucocytes and monocytes in vivo.