Microbiological characteristics of clinical isolates of Cryptococcus neoformans in Taiwan: serotypes,mating types,molecular types,virulence factors,and antifungal susceptibility

This study investigated the microbiological characteristics of 100 clinical isolates of Cryptococcus neoformans species complex, including serotypes, mating types, molecular types, antifungal susceptibility and virulence. The isolates were collected at National Taiwan University Hospital from 1999 to 2004. Eight isolates of C. neoformans from pigeon droppings were also evaluated. Among these isolates, 99 were C. neoformans var. grubii serotype A and one was C. neoformans var. gattii serotype B. All of these isolates were α mating types. PCR fingerprinting, generated by primers M13 and (GACA)₄, and URA5 gene restriction fragment length polymorphism analysis revealed that C. neoformans var. grubii isolates belonged to the VNI (98 isolates) and the VNII (one isolate) types, and the single C. neoformans var. gattii was VGI type. The similar profiles of clinical and environmental isolates suggest that patients might acquire these yeasts from the environment. The MIC₉₀ for fluconazole, itraconazole, 5-flucytosine, voriconazole and amphotericin B against all C. neoformans isolates were 8, 0.5, 4, 0.125 and 0.5 mg/L, respectively. All clinical isolates produced urease, phospholipase, capsule and melanin, but these activities varied with individual isolates. Analysis of six clinical and two environmental isolates with various levels of phospholipase activity indicated a correlation between phospholipase activity and the ability to adhere to the lung epithelial cell line, A549. The extent of cell damage, as indicated by lactate dehydrogenase release, also paralleled the phospholipase activity of these isolates. In addition, production of melanin contributed significant protection against amphotericin B killing of the isolates tested.     

In vitro activity of antifungal combinations against planktonic and sessile cells of Candida albicans isolated from medical devices in an intensive care department

Background

Invasive fungal infections are an emerging health problem worldwide. They are responsible for a significant rate of morbidity and mortality. Infections caused by Candida albicans involve proliferation of biofilms on biotic or abiotic surface. These adherent communities exhibit characteristics distinct from planktonic cells such as the ability to tolerate high concentrations of antifungal.

In vitro synergistic effect of minocycline combined with antifungals against Cryptococcus neoformans

BackgroundCryptococcus neoformans infections occur in immunocompromised patients, especially those with HIV infection, chemoradiotherapy after cancer, and organ transplantation. Infection can cause pneumonia and meningoencephalitis in severe cases with a high mortality rate if not treated. Although fluconazole and amphotericin B are the first-line treatments for cryptococcosis, the rate of fluconazole resistance has increased significantly due to long-term use. Minocycline is a derivative of tetracycline that exerts its antibacterial effect through inhibition of bacterial protein synthesis. It is also able to pass the blood-brain barrier to act on the central nervous system. The present study investigates the effects of minocycline in combination with antifungals in treating C. neoformans.ObjectiveTo determine in vitro interactions of minocycline combined with itraconazole, voriconazole, posaconazole, fluconazole and amphotericin B against C. neoformans.MethodsThe minimum inhibitory concentrations (MIC) of the antifungals were determined by the CLSI Clinical and Laboratory Standards Institute M27-A3 microdilution method. The in vitro synergistic effects of minocycline combined with itraconazole, voriconazole, posaconazole, fluconazole, and amphotericin B on C. neoformans were detected by the broth microdilution checkerboard technique and disk diffusion testing.Results and ConclusionThe working concentration ranges were 0.125–4 µg/mL for itraconazole, 0.03–0.125 µg/ml for voriconazole, 0.03–1 µg/ml for posaconazole, 0.25–16 µg/ml for fluconazole, and 0.125–2 µg/ml for amphotericin B. The synergistic rates of minocycline combinations against C. neoformans were 55% with itraconazole, 10% with voriconazole, 85% with posaconazole, 20% with fluconazole, and 70% with amphotericin B. The effective MIC value of minocycline in the synergistic combination decreased to 2–32 µg/ml, while the MIC of itraconazole decreased to 0.03–0.125 µg/ml, voriconazole 0.03–0.125 µg/ml, posaconazole 0.03–0.125 µg/ml, 0.125–4 µg/ml fluconazole, and 0.06–0.50 µg/ml amphotericin B. The disk diffusion assay showed that the plates containing minocycline and antifungal drugs produced inhibition zones with diameters larger than the single drug plates. Minocycline showed no antagonistic effect in the combinations. In conclusion, the combination of minocycline and azoles or amphotericin B has synergistic effects against C. neoformans in vitro.     

Cryptococcosis: epidemiology, fungal resistance, and new alternatives for treatment

Cryptococcosis is an important systemic mycosis and the third most prevalent disease in human immunodeficiency virus (HIV)-positive individuals. The incidence of cryptococcosis is high among the 25 million people with HIV/acquired immunodeficiency syndrome (AIDS), with recent estimates indicating that there are one million cases of cryptococcal meningitis globally per year in AIDS patients. In Cryptococcus neoformans, resistance to azoles may be associated with alterations in the target enzyme encoded by the gene ERG11, lanosterol 14α-demethylase. These alterations are obtained through mutations, or by overexpressing the gene encoding. In addition, C. gattii and C. neoformans present a heteroresistance phenotype, which may be related to increased virulence. Other species beyond C. neoformans and C. gattii, such as C. laurentii, have been diagnosed mainly in patients with immunosuppression. Infections of C. albidus have been isolated in cats and marine mammals. Recent evidence suggests that the majority of infections produced by this pathogen are associated with biofilm growth, which is also related with increased resistance to antifungal agents. Therefore, there is a great need to search for alternative antifungal agents for these fungi. The search for new molecules is currently occurring from nanoparticle drugs of plant peptide origin. This article presents a brief review of the literature regarding the epidemiology of cryptococcosis, as well as fungal resistance and new alternatives for treatment.     

Identification of Cryptococcus gattii by Use of l-Canavanine Glycine Bromothymol Blue Medium and DNA Sequencing

Cryptococcus neoformans and Cryptococcus gattii are closely related pathogenic fungi. Cryptococcus neoformans is ecologically widespread and affects primarily immunocompromised patients, while C. gattii is traditionally found in tropical climates and has been reported to cause disease in immunocompetent patients. l-Canavanine glycine bromothymol blue (CGB) agar can be used to differentiate C. neoformans and C. gattii, but there are few reports of its performance in routine clinical practice. Growth of C. gattii on CGB agar produces a blue color, indicating the assimilation of glycine, while C. neoformans fails to cause a color change. Using reference and clinical strains, we evaluated the ability of CGB agar and D2 large ribosomal subunit DNA sequencing (D2 LSU) to differentiate C. neoformans and C. gattii. One hundred two yeast isolates were screened for urease activity, melanin production, and glycine assimilation on CGB agar as well as by D2 sequencing. Seventeen of 17 (100%) C. gattii isolates were CGB positive, and 54 of 54 C. neoformans isolates were CGB negative. Several yeast isolates other than the C. gattii isolates were CGB agar positive, indicating that CGB agar cannot be used alone for identification of C. gattii. D2 correctly identified and differentiated all C. gattii and C. neoformans isolates. This study demonstrates that the use of CGB agar, in conjunction with urea hydrolysis and Niger seed agar, or D2 LSU sequencing can be reliably used in the clinical laboratory to distinguish C. gattii from C. neoformans. We describe how CGB agar and D2 sequencing have been incorporated into the yeast identification algorithm in our laboratory.Cryptococcus gattii (formerly Cryptococcus neoformans var. gattii) differs from the closely related C. neoformans in several ways, including a contrasting host profile and a reduced susceptibility to certain antifungal agents (19, 20). Cryptococcus gattii has traditionally been thought to be geographically restricted to tropical and subtropical climates, although recent reports indicate that it can be found worldwide, including in regions with distinctly nontropical climates, like the Pacific Northwest (2, 4, 8, 12, 14, 17, 19). Clinically, the majority of cryptococcosis cases that occur in AIDS patients and other immunocompromised hosts are caused by C. neoformans var. neoformans or C. neoformans var. grubii. In contrast, C. gattii has been reported to cause meningoencephalitis and pulmonary infections in hosts who are generally immune competent (9, 14, 18).Since C. gattii is recognized as an emerging pathogen, it is important for the clinical microbiology laboratory to accurately differentiate it from C. neoformans. A recent proficiency testing survey administered by the New York State Department of Health indicated that only 7/140 (5%) clinical laboratories participating in the event were able to correctly identify C. gattii, while the remaining 95% of laboratories surveyed misidentified the isolate as C. neoformans (15). The critique that followed this proficiency event indicated that the inability to correctly identify C. gattii was largely due to the failure to routinely use differential agar. In this report, we describe the implementation of two approaches for the routine identification of C. gattii in a high-volume clinical laboratory. The first approach uses the simple, previously reported biochemical test evaluating growth on the selective medium l-canavanine glycine bromothymol blue (CGB) agar (10, 13, 16). The agar method is low complexity and cost-effective and can be easily incorporated into the workflow of clinical laboratories of any size. We describe the ability of CGB to differentiate C. gattii from C. neoformans and other yeast encountered in our clinical practice. The second approach is a molecular method that utilizes DNA sequencing of the D2 region of the fungal 28S large ribosomal subunit to distinguish C. neoformans from C. gattii. Others have reported differentiation of C. neoformans and C. gattii by the use of sequencing of the internal transcribed spacer region (3, 6, 7), but to our knowledge, this is the first report of the use of D2 large ribosomal subunit region sequencing for the identification of C. gattii.(This study was presented in part at the 107th General Meeting of the American Society for Microbiology, Toronto, ON, Canada, 22 to 25 May 2007.)     

Thymus vulgaris essential oil and thymol inhibit biofilms and interact synergistically with antifungal drugs against drug resistant strains of Candida albicans and Candida tropicalis

Role of biofilm in disease development and enhance tolerance to antifungal drugs among Candida species has necessitated search for new anti-fungal treatment strategy. Interference in pathogenic biofilm development by new antifungal compounds is considered as an attractive anti-infective strategy. Therefore, the objective of this study was to evaluate Thymus vulgaris essential oil and its major active compound, thymol for their potential to inhibit and eradicate biofilms alone and in combination with antifungal drugs against Candida spp. with especial reference to Candida tropicalis. Anti-candidal efficacy of T. vulgaris and thymol in terms of minimum inhibitory concentration (MIC) was first determined to select the sub-MICs against C. albicans and C. tropicalis. Biofilm formation in the presence and absence of test agents was determined in 96-well microtiter plate by XTT reduction assay and effect of essential oils at sub-MICs of the test agents on biofilm development on glass surface was analysed by light and scanning electron microscopy. Synergistic interaction between essential oils and antifungal drugs were studied by checkerboard method. Effect of sub-MIC of T. vulgaris (0.5 × MIC) and thymol (0.5 × MIC) on biofilm formation showed a significant reduction (P < 0.05) in biofilms. Light microscopy and SEM studies revealed disaggregation and deformed shape of C. albicans biofilm cells and reduced hyphae formation in C. tropicalis biofilm cells at sub-MICs of thymol. Significant effect of T. vulgaris and thymol was also recorded on pre-formed biofilms of both C. albicans and C. tropicalis. T. vulgaris and thymol also showed synergy with fluconazole against both in planktonic and biofilm mode of growth of C. albicans and C. tropicalis. However, synergy with amphotericin B is clearly evident only in planktonic Candida cells. Thyme oil and thymol alone or in combination with antifungal drugs can act as promising antibiofilm agent against drug resistant strains of Candida species and needs further in vivo study to synergise its therapeutic efficacy.     

In Vitro Susceptibility of the Yeast Pathogen Cryptococcus to Fluconazole and Other Azoles Varies with Molecular Genotype

Cryptococcosis is primarily caused by Cryptococcus neoformans and Cryptococcus gattii. These two pathogenic species each divide into four distinct molecular genotypes. In this study, we examined whether genotype influenced susceptibility to antifungal drugs used to treat cryptococcosis using the broth microdilution method described by the Clinical and Laboratory Standards Institute. C. gattii isolates belonging to molecular genotype VGII had significantly higher MIC values for flucytosine and all azole antifungal agents tested, particularly fluconazole, than isolates of other C. gattii genotypes. In an extended analysis of fluconazole susceptibility, VGII isolates from the north and west of Australia required higher drug levels for inhibition than those from Vancouver Island, Canada. Within C. neoformans, genotype VNII had significantly lower geometric mean MICs for fluconazole than genotype VNI. These results indicate that cryptococcal species, molecular genotype, and region of origin may be important when deciding treatment options for cryptococcosis.Cryptococcosis, caused by the encapsulated yeasts Cryptococcus neoformans and Cryptococcus gattii, is a fungal disease of humans and animals (10). C. neoformans is a worldwide, usually opportunistic pathogen that typically infects immunosuppressed patients, including those with HIV/AIDS (38, 41). In contrast, C. gattii is a primary pathogen that affects immunocompetent people and has caused significant outbreaks in animals in Australia and in humans and animals in Canada (18, 40).The two pathogenic Cryptococcus species divide into eight major molecular types: VNI to VNIV for C. neoformans and VGI to VGIV for C. gattii. There is increasing evidence that these molecular types may represent cryptic species (4, 36), with differences found in important traits, including virulence, geographic range, epidemiology, and population genetics (3, 8). VNI and VGI molecular types are widespread and cause most of the disease attributed to C. neoformans and C. gattii, respectively. C. neoformans VNII to VNIV and C. gattii VGIII and VGIV are less common, and VGIII, VGIV, and VNIV appear to be geographically restricted (15, 21). C. gattii VGII, until recently considered to be rare, has received increasing attention due to its link with a large, ongoing outbreak of cryptococcosis that originated on Vancouver Island, Canada, and has now extended into the Pacific Northwest of the United States (5, 7, 21). VGII also predominates in some parts of Australia and South America and has likely been endemic in these regions for a substantial period (31, 45).Clinical data suggest that the response to antifungal therapy is slower in C. gattii infection than in C. neoformans infection (41, 42) and that more prolonged treatment may be required (13). This is corroborated by various in vitro studies that have found that C. gattii may be less susceptible than C. neoformans to antifungal agents, in particular, fluconazole (13, 17, 44); however, other studies report no significant differences in susceptibility (9, 43). A possible reason for these contradictory findings is that most studies have not considered genetic or geographic differences within the two Cryptococcus species. Iqbal et al. (19) found significant differences in susceptibility between VGII subgenotypes VGIIa, VGIIb, and VGIIc that occur in the Pacific Northwest of Canada and the United States, suggesting that genotype and origin may influence MICs to antifungal agents.The aim of the present study was to determine whether the most common genotypes of C. gattii and C. neoformans differ in their in vitro susceptibility to common antifungal agents. Reduced susceptibility to fluconazole was evident in C. gattii VGII, and this was further explored in an extended set of isolates from Australia and Vancouver Island, Canada, where C. gattii VGII accounts for a substantial proportion of infection.     

Differentiation of Cryptococcus neoformans varieties and Cryptococcus gattii using CAP59-based loop-mediated isothermal DNA amplification

Members of the Cryptococcus species complex (C. neoformans and C. gattii) are opportunistic pathogens responsible for frequently fatal cases of meningoencephalitis. These yeasts have been classified into five serotypes. Serotypes A andDare assigned to C. neoformans var. grubii and C. neoformans var. neoformans, respectively, Serotype AD strains are hybrids and serotype B and C strains are considered to belong to the related but distinct species C. gattii. Previous studies have identified ‘serotype-associated' alleles of several genes in the Cryptococcus species complex. We developed a loop-mediated isothermal DNA amplification method using CAP59 allele-specific primers to identify the serotypes A, D and B/C of the Cryptococcus species complex.     

Phenotypic Differences of Cryptococcus Molecular Types and Their Implications for Virulence in a Drosophila Model of Infection

Compared to Cryptococcus neoformans, little is known about the virulence of the molecular types in Cryptococcus gattii. We compared in vitro virulence factor production and survival data using a Drosophila model of infection to further characterize the phenotypic features of different cryptococcal molecular types. Forty-nine different isolates were inoculated into wild-type flies and followed for survival. In vitro, isolates were assessed for growth at 30 and 37°C, melanin production, capsule size, resistance to H₂O₂, and antifungal susceptibility. A mediator model was used to assess molecular type and virulence characteristics as predictors of survival in the fly model. VGIII was the most virulent molecular type in flies (P < 0.001). At 30°C, VGIII isolates grew most rapidly; at 37°C, VNI isolates grew best. C. gattii capsules were larger than those of C. neoformans (P < 0.001). Mediator model analysis found a strong correlation of Drosophila survival with molecular type and with growth at 30°C. We found molecular-type-specific differences in C. gattii in growth at different temperatures, melanin production, capsule size, ability to resist hydrogen peroxide, and antifungal susceptibility, while growth at 30°C and the VGIII molecular type were strongly associated with virulence in a Drosophila model of infection.     

In vitro activity of anidulafungin,caspofungin, fluconazole and amphotericin B against biofilms and planktonic forms of Candida species isolated from blood culture in patients with hematological malignancies

ObjectiveThe aim of the present study was to evaluate the in vitro susceptibility of anidulafungin, caspofungin, fluconazole and conventional amphotericin B against biofilms and planktonic forms of Candida species isolated from blood culture in patients with hematological malignancies.Materials and methodsAntifungal susceptibility for planktonic forms and biofilms of Candida was determined by broth microdilution method as described by Clinical and Laboratory Standards Institute M27 methodology and metabolic XTT-based [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] reduction assay, respectively.ResultsA total of 75 Candida isolates were evaluated between 2006–2018 yy at the National Research Center for Hematology, Russia, Moscow. Biofilm production was detected in 34 (45.3%) Candida species. Antifungal susceptibility was tested for 27 common species of Candida forming biofilms (8 C.krusei, 7 C.tropicalis, 7 C.albicans, 5 C.parapsilosis).MICs below the susceptibility breakpoints were found for 100% of planktonic forms of Candida species for anidulafungin, 85.2% for caspofungin, and 66.7% for fluconazole. Amphotericin B MIC₉₀ for Candida species were less than or equal to 1 μg/ml. Candida biofilms were susceptible in vitro for both tested echinocandins, but MIC₈₀ of anidulafungin were lower compared to caspofungin. The highest MIC₈₀ against Candida biofilms was found for fluconazole (>1,024 μg/ml for all tested isolates) and for conventional amphotericin B (range 4–16 μg/ml).ConclusionThe majority of Candida isolates grown as planktonic forms were susceptible to anidulafungin, caspofungin, conventional amphotericin B and fluconazole. Anidulafungin displayed higher activity against Candida biofilms than caspofungin. All Candida biofilms were resistant to fluconazole and conventional amphotericin B.     

Anticryptococcal activity of hexane extract from Spondias tuberosa Arruda and associated cellular events

Cryptococcosis is an opportunistic systemic mycosis whose treatment is limited to three drugs. In this work, we evaluated the antifungal activity of a hexane extract (HE) from Spondias tuberosa leaves against Cryptococcus neoformans and Cryptococcus gattii. Minimal inhibitory concentrations (MIC) were determined, and putative mechanisms were evaluated by flow cytometry. In addition, an in vivo infection assay was performed using Tenebrio molitor larvae. Treatment with HE inhibited the growth of standard and clinical isolates of C. neoformans and C. gattii (MICs ranging from 0.78 to 3.12 mg/mL), significantly (P < 0.05) increased mitochondrial superoxide anion levels, and induced mitochondrial membrane depolarization, loss of lysosomal membrane integrity, and phosphatidylserine externalization. The mean survival time of C. gattii-infected T. molitor larvae significantly (P < 0.05) increased from 1.225 days in control to 3.067 and 3.882 days in HE-treated groups (78 and 156 mg/kg, respectively). In conclusion, HE showed anticryptococcal activity, induced mitochondrial and lysosomal damage in yeast cells, and exhibited anti-infective action against C. gattii in T. molitor larvae.     

Impact of iron chelators on growth and expression of iron-related genes of Cryptococcus species

IntroductionIron chelator has previously demonstrated fungicidal effects. This study aimed to investigate the antifungal activity of the iron chelators deferoxamine (DFO) and deferasirox (DSX) against Cryptococcus.Materials and methodsCryptococcus neoformans and Cryptococcus gattii were used to determine the minimal inhibitory concentrations (MICs) of DFO and DSX, and the fractional inhibitory concentration index (FICI) of DFO and DSX when combined with amphotericin B (AMB). Expression of cryptococcal CFT1, CFT2, and CIR1 genes was determined using real-time polymerase chain reaction (PCR).ResultsNeither DFO nor DSX alone showed antifungal activity against Cryptococcus strains. When combined with AMB, the MICs of DFO and DSX decreased from > 200 μg/mL to 6.25 or 12.5 μg/mL. The MIC of AMB decreased one-fold dilution in most strains when combined with iron chelators. The FICI of DFO + AMB and DSX + AMB was 0.5 and 1, respectively. C. neoformans showed significant growth retardation when incubated with a combination of sub-MIC concentrations of AMB and DFO; whereas, C. gattii demonstrated lesser growth retardation in DFO + AMB. No cryptococcal growth retardation was observed when DSX was combined with AMB. When C. neoformans was grown in DFO, the CFT1, CFT2, and CIR1 proteins were expressed 1.7, 2.0, and 0.9 times, respectively. When C. neoformans was grown in DSX, the CFT1, CFT2, and CIR1 genes were expressed 0.5, 0.6, and 0.3 times, respectively.ConclusionSynergistic antifungal activity of combination DFO and AMB was observed in Cryptococcus. Relatively increased CFT1 and CFT2 expression may be associated with the effect of DFO that inhibits the growth of fungi.     

Effects of combination of Cryptococcus gattii and IFN-γ, IL-4 or IL-27 on human bronchial epithelial cells

BackgroundAirway epithelial cells are crucial for the establishment of cryptococcosis. In experimental cryptococcosis, the Th2 immune response is associated with host susceptibility, while Th1 cells are associated with protection. The absence of IL-27 receptor alpha in mice favor the increase Cryptococcus neoformans burden in the lung. Here, we evaluated the effects of the combination of IL-4, IFN-γ or IL-27 with C. gattii on human bronchial epithelial cells (BEAS-2B).MethodsBEAS-2B were stimulated with IL-4, IFN-γ or IL-27 (100 ng/mL) and/or live yeast forms of C. gattii (multiplicities of infection (MOI) of 1–100) and vice-versa, as well as with heat-killed cells of C. gattii for 24 h.ResultsNone of the C. gattii MOIs had cytotoxic effects on BEAS-2B when compared to control. The cells stimulated by cytokines (IL-4, IFN-γ or IL-27) followed by live yeast forms of C. gattii (MOI of 100) infection and vice-versa demonstrated a reduction in IL-6, IL-8 and/or CCL2 production and activation of STAT6 (induced by IL-4) and STAT1 (induced by IL-27 or IFN-γ) when compared to cells stimulated with C. gattii, IL-4, IFN-γ or IL-27. In the combination of cytokines and heat-killed cells of C. gattii, no inhibition of these inflammatory parameters was observed. The growth of C. gattii was increased while the phagocytosis of live yeast forms of C. gattii in the BEAS-2B were reduced in the presence of IL-4, IFN-γ or IL-27.ConclusionThe association of live yeast forms, but not heat-killed yeast forms, of C. gattii with IL-4, IFN-γ or IL-27 induced an anti-inflammatory effect.     

Cryptococcus gattii Isolates from the British Columbia Cryptococcosis Outbreak Induce Less Protective Inflammation in a Murine Model of Infection than Cryptococcus neoformans

The fungal pathogen Cryptococcus neoformans causes approximately one million cases of cryptococcosis per year in people with AIDS. In contrast, the related species C. gattii is responsible for a much smaller number of cases, but these often occur in immunocompetent people. In fact, C. gattii has emerged in the last decade as the frequent cause of cryptococcosis in otherwise healthy people in British Columbia. We analyzed the immune responses elicited by three C. gattii strains and one C. neoformans strain in mice as a first step toward understanding why C. gattii is able to cause disease in immunocompetent hosts. The C. gattii strains all induced a less protective inflammatory response in C57BL/6 mice by inhibiting or failing to provoke the migration of neutrophils to sites of infection. The C. gattii strains also failed to elicit the production of protective cytokines, such as tumor necrosis factor alpha, compared to the ability of the C. neoformans strain. Despite these differences, the strain representing the major outbreak genotype from British Columbia showed a virulence equivalent to that of the C. neoformans strain, while two other C. gattii strains had reduced virulence. Taken together, our results indicate that C. gattii strains thrive in immunocompetent hosts by evading or suppressing the protective immune responses that normally limit the progression of disease caused by C. neoformans.Cryptococcus neoformans is an opportunistic fungal pathogen that can cross the blood-brain barrier to cause a life-threatening disease of the central nervous system (5). In the environment, the fungus is found in trees, soil, and avian excreta, where it exists as spores or desiccated yeast cells (9, 16, 26, 50). Infection is initiated primarily by the inhalation of these cells, and the fungus can spread from the lungs to cause systemic cryptococcosis and meningoencephalitis. C. neoformans var. grubii (serotype A) typically infects people with compromised immune systems, and this serotype causes the majority of cryptococcosis cases in AIDS patients (33). In contrast, a related species, C. gattii (serotype B), does not have the same impact on AIDS patients and tends to infect people regardless of their immune status (27, 43). C. gattii infections originally were found primarily in tropical and subtropical regions of the world, such as South America and Australia (34). However, since 1999, an outbreak of C. gattii infections has been occurring in the temperate climate of Vancouver Island in British Columbia. This outbreak has resulted in at least 200 human cases and eight deaths (3, 21). More recently, cases of cryptococcosis caused by C. gattii have occurred on the mainland in British Columbia as well as in Washington and Oregon in the United States. (8, 45).The mechanisms of the immune response to cryptococcal species have been best established for infections caused by C. neoformans (using strains of both the A and D serotypes). Normally, most of the fungal spores or yeast cells that are inhaled do not even reach the lungs and are cleared due to air turbulence and ciliary action (42). The small numbers of Cryptococcus spores or yeast cells that do reach the lung parenchyma are cleared by a protective inflammatory immune response involving the production of cytokines such as tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ), and a leukocyte infiltrate consisting of neutrophils, Th1-associated classically activated macrophages, Th1-lymphocytes, and dendritic cells (DCs) (12, 24, 31, 32). In contrast, a nonprotective response involves the production of high levels of the Th2 cytokine interleukin-4 (IL-4) as well as a diffuse pulmonary infiltrate consisting of immature DCs and alternatively activated macrophages (12, 24, 31, 32, 47). Protective inflammation involving neutrophil recruitment is crucial for the induction of adaptive Th1 immune responses against cryptococcal infections and for an effective response to other respiratory pathogens (1, 10, 15). Furthermore, the timing of the cellular infiltration is important, and early neutrophilia is associated with protective immune responses against C. neoformans (24). Additionally, mice deficient in myeloperoxidase, an enzyme preferentially expressed in neutrophils, produce weak Th1 immune responses and have a significantly reduced survival time upon C. neoformans infection compared to that of wild-type mice (1).Although only a few studies have examined the immune response during C. gattii infection, there is some evidence that C. gattii induces less protective immune responses than C. neoformans. For example, Dong and Murphy showed that culture filtrate antigens from C. gattii strains inhibit neutrophil migration in vitro and in vivo, whereas culture filtrate antigens from C. neoformans stimulate neutrophil migration (17). They proposed that C. gattii strains are able to cause disease in immunocompetent people because they inhibit neutrophil migration into the lungs during the initial stage of infection. A more recent study found that even though a strain of C. gattii inhibited neutrophil migration in vitro, neutrophil infiltration into the lungs of infected rats was similar between C. neoformans and C. gattii infections (52). Thus, it was suggested that C. gattii infections are not cleared efficiently by immunocompetent hosts because they do not induce protective inflammation during infection, and as a result they are not able to elicit the adaptive Th1-type immune responses that are elicited with infection with C. neoformans (52). However, this idea has not been explored beyond the study of differential C. neoformans and C. gattii effects on neutrophil function.In light of these studies, we examined the cytokine profiles and pulmonary infiltrates in the lungs of infected mice to determine whether there were differences in the immune responses for a commonly studied C. neoformans isolate (serotype A) and selected C. gattii strains representing the genotypes causing the current outbreak in British Columbia. Our study revealed lower levels of neutrophil infiltration and reduced inflammatory cytokine production in the lungs of mice infected with C. gattii compared to those of mice infected with C. neoformans. This analysis is an important first step toward understanding the ability of C. gattii strains to cause disease in immunocompetent hosts.     

In vitro effects of ambroxol on Cryptococcus adherence,planktonic cells,and biofilms

The antifungal effects of ambroxol (Amb; the metabolite VIII of bromhexine) against Cryptococcus planktonic cells and mature biofilms were investigated in this study. Amb showed antifungal activity against planktonic cells and mature biofilms. Disk diffusion test similarly showed antifungal profile for planktonic cells. Furthermore, Amb was found to be synergetic with fluconazole against planktonic cells and reduced the adherence of cells to polystyrene. Our results suggest that Amb can inhibit cryptococcal cells and biofilms, indicating its potential role in the prevention and treatment of cryptococcosis.     

DAP12 Inhibits Pulmonary Immune Responses to Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic fungal pathogen that is inhaled into the lungs and can lead to life-threatening meningoencephalitis in immunocompromised patients. Currently, the molecular mechanisms that regulate the mammalian immune response to respiratory cryptococcal challenge remain poorly defined. DAP12, a signaling adapter for multiple pattern recognition receptors in myeloid and natural killer (NK) cells, has been shown to play both activating and inhibitory roles during lung infections by different bacteria and fungi. In this study, we demonstrate that DAP12 plays an important inhibitory role in the immune response to C. neoformans. Infectious outcomes in DAP12^−/− mice, including survival and lung fungal burden, are significantly improved compared to those in C57BL/6 wild-type (WT) mice. We find that eosinophils and macrophages are decreased while NK cells are increased in the lungs of infected DAP12^−/− mice. In contrast to WT NK cells, DAP12^−/− NK cells are able to repress C. neoformans growth in vitro. Additionally, DAP12^−/− macrophages are more highly activated than WT macrophages, with increased production of tumor necrosis factor (TNF) and CCL5/RANTES and more efficient uptake and killing of C. neoformans. These findings suggest that DAP12 acts as a brake on the pulmonary immune response to C. neoformans by promoting pulmonary eosinophilia and by inhibiting the activation and antifungal activities of effector cells, including NK cells and macrophages.     

Hospital Trichosporon asahii isolates with simple architecture biofilms and high resistance to antifungals routinely used in clinical practice

Infections by Trichosporon spp. are increasing worldwide and its treatment remains a challenge. Colonization of medical devices has been considered as a predisposing factor for trichosporonosis, which is related to fungal biofilm production. Thus, this study aimed to evaluate the ability of six hospital T. asahii isolates to form biofilm on abiotic surface, as well as to investigate the impact of three classic antifungals on both planktonic and biofilm forms. The fungal identification was based on macro and micromorphological characteristics, biochemical tests and confirmation by mass spectrometry assisted by the flight time desorption/ionization matrix (MALDI-TOF MS). Antifungal susceptibility assay of planktonic cells showed inhibitory and fungicidal concentrations ranging from 2.5 to 10 µg/mL for voriconazole, 2 to 8 µg/mL for fluconazole, and 1 to 4 µg/mL for amphotericin B. All T. asahii strains were able to form biofilms on the polystyrene microplates surface within 24 h, showing a simple architecture when compared with Candida spp. biofilm. On the other hand, the same antifungals did not show action in neither the inhibition of biofilm formation nor on the formed biofilm. Concluding, the present study reinforced the relevance of the MALDI-TOF MS methodology for a safe identification of T. asahii. Classic antifungals were active on the planktonic form, but not on the biofilms. All isolates formed biofilms on the polystyrene microplates and showed a simple architecture.     

Decayed wood inside hollow trunks of living trees of Tamarindus indica,Syzygium cumini and Mangifera indica as natural habitat of Cryptococcus neoformans and their serotypes in Jabalpur City of Central India

ObjectiveColonization of Cryptococcus gattii and Cryptococcus neoformans var. grubii inside the decaying wood of hollow trunks of Tamarindus indica, Syzygium cumini and Mangifera indica is reported in Jabalpur City in Central India.MethodsThe decayed wood inside the hollows trunks of 133 trees mostly belonging to the three species T. indica, S. cumini and M. indica was examined. The 108 samples performed during the 1010 days monitoring period were inoculated on Staib's medium. The strains of C. neoformans isolated were identified by morphological, biochemical and serological characters.ResultsFour out of 36 (11.11%) T. indica trees investigated proved to be positive for C. gattii (serotype B) and three out of 36 (8.33%) T. indica trees were positive for C. neoformans var. grubii (serotype A). Two out of 30 (6.66%) M. indica trees were found to be positive for C. gattii serotype B and one of 30 (3.33%) was positive for C. neoformans var. grubii serotype A. Of 42 S. cumini trees, four (9.52%) were found to be positive for C. gattii serotype B and two (4.76%) for C. neoformans var. grubii serotype A. The two varieties never co-occurred in the same hollow of any of the trees investigated. The data strongly support the colonization of T. indica, M. indica and S. cumini trees by both varieties of C. neoformans. Evidence of this was found by repeated isolations and by the high population density in the decayed wood during the 1010 days monitoring period. M. indica is reported, for the first time, as the natural habitat of both varieties of C. neoformans. For the first time T. indica is also reported to harbor C. gattii serotype B.ConclusionOur results further reinforce recently emerging evidence that the natural habitat of C. gattii and C. neoformans var. grubii is more generalized than specific.