In vitro susceptibilities of clinical isolates of Candida species, Cryptococcus neoformans, and Aspergillus species to itraconazole: global survey of 9,359 isolates tested by clinical and laboratory standards institute broth microdilution methods

The in vitro activity of itraconazole was determined against 7,299 isolates of Candida spp., 1,615 isolates of Cryptococcus neoformans, and 445 isolates of Aspergillus spp. obtained from over 200 medical centers worldwide. Itraconazole was active against all Candida spp. (96% of MICs were < or =1 microg/ml) with the exception of C. glabrata (77% of MICs were < or =1 microg/ml). Itraconazole inhibited 94% of C. krusei and 84% of other fluconazole-resistant Candida species, exclusive of C. glabrata, at a MIC of < or =1 microg/ml. Itraconazole was not active against fluconazole-resistant isolates of C. glabrata. Only modest activity was seen against C. neoformans (80% of MICs were < or =1 microg/ml); however, itraconazole showed excellent activity against Aspergillus spp. (94% of MICs were < or =1 microg/ml). These results provide an update on the antifungal activity of itraconazole against major opportunistic fungal pathogens. In light of the new intravenous formulation of itraconazole these data suggest that this agent remains a viable systemically active antifungal agent.     

Development of positive selectable markers for the fungal pathogen Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic fungal pathogen that causes meningitis in approximately 10% of patients with AIDS. New selectable markers which confer resistance to G418 or phleomycin when transformed into C. neoformans were made. A hygromycin-selectable marker was modified to allow selection with a single copy of the marker.     

Production of species-specific murine monoclonal antibodies against Cryptococcus neoformans which recognize a noncapsular exoantigen

Three monoclonal antibodies (MAbs), designated 7C5, 7C9, and 5G8, against a cytoplasmic antigen of Cryptococcus neoformans were produced. MAbs 7C5 and 7C9 recognize culture filtrate antigen (exoantigen) of both encapsulated and nonencapsulated isolates of this pathogen, which suggests that they do not recognize capsular polysaccharide material. This is supported by immunofluorescence data which show reactivity of all 3 MAbs to cytoplasm and cell membranes only. MAb 7C9 also recognized C. neoformans var. gattii antigens but no other fungal pathogens tested in an enzyme-linked immunosorbent assay, while 7C5 and 5G8 recognized antigens of the cross-reactive pathogen Trichosporon beigelii but did not recognize either C. neoformans var. gattii isolates or any other fungal antigens. By Western blot (immunoblot), 7C9 detected antigen at 110 to 120, 65 to 70, 45 to 50, and 36 to 38 kDa; in addition to the latter band, the other two MAbs recognized a band at approximately 30 kDa. All three MAbs were of the immunoglobulin G1 subclass. The two MAbs which are capable of reacting with noncapsular culture supernatant antigen have possible uses in serodiagnosis, particularly in AIDS patients infected with C. neoformans, since in this group the present latex agglutination test has some limitations.     

Isolation of Cryptococcus neoformans var. neoformans serotype D from Eucalypts in South Brazil.

Cryptococcus neoformans causes the second most common opportunistic infection in patients with AIDS. In Brazil, 4.5% of the AIDS-related opportunistic infections are caused by C. neoformans and all varieties are recognized as etiological agents of cryptococcosis. This pathogen is a ubiquitous environmental yeast, commonly associated with avian excreta and decaying wood, especially Eucalypt species. The aim of the present study was to search for C. neoformans in Eucalypts and analyze the genotypic diversity of the obtained isolates by RAPD and PCR fingerprinting. All obtained isolates have been C. neoformans var. neoformans, serotype D molecular type VNIV. Serotype D, was isolated from 3 (37.5%) out of 8 cities surveyed in the South Brazilian state Rio Grande do Sul. Nine (9%) out of 99 environmental samples were obtained from Eucalypt species, Eucalyptus calmadulensis and Eucalyptus tereticornis. Molecular analysis using RAPD and PCR-fingerprinting revealed very little genetic diversity in the obtained cryptococcal serotype D isolates. To our knowledge this is the first report of the isolation of serotype D from Eucalyptus trees in Brazil. More studies are required in order to establish the ecological significance of this finding.     

Cooperative regulation of the host defense to cryptococcal infection by innate immune lymphocytes]

Cryptococcus neoformans is an opportunistic fungal infectious pathogen in immunocompromised patients with acquired immunodeficiency syndrome and hematological malignancies. Recently, innate immune cells, such as NK, NKT and ganmma delta T cells, have been found critical for determining the quality of acquired immunity by affecting the direction of Th1-Th2 balance. Th1-type immune response is important for the host defense against C. neoformans, and innate immunity may involve this process. In the present review, the accumulated knowledge including our own data on the role of innate immune lymphocytes in the host defense to this fungal pathogen are summarized, focusing on NKT and ganmma delta T cells.     

Microbiology of systemic fungal infections

The increased incidence of systemic fungal infections in the past two decades has been overwhelming. Earlier, it was pathogenic dimorphic fungi, which were known to cause systemic infections. However, starting from the 1960s, opportunistic fungi started causing more number of infections, especially in the immunocompromised host. More recently, newer and less common fungal agents are being increasingly associated with infection in immunosuppressed hosts. Amongst dimorphic fungi, infections due to Histoplasma capsulatum and Penicillium marneffei are increasingly reported in patients with AIDS in India. H. capsulatum is found country wide, but P marneffei remains restricted to Manipur state. Although both varieties of C. neoformans, C. neoformans var. neoformans (serotypes A & D), and C. neoformans var. gattii (serotypes B & C) are reported in India, most of the cases reported are of serotype A. Increased incidence of cryptococcosis is reported from all centers with the emergence of AIDS. Systemic infection due to species under Candida, Aspergillus and zygomycetes is widely prevalent in nosocomial setting, and outbreaks due to unusual fungi are reported occasionally from tertiary care centers. This global change in systemic fungal infections has emphasized the need to develop good diagnostic mycology laboratories in this country and to recognize this increasingly large group of potential fungal pathogens.     

Origin of Cryptococcus neoformans var. neoformans Diploid Strains

The basidiomycetous yeast Cryptococcus neoformans is an important human fungal pathogen. Two varieties, C. neoformans var. neoformans and C. neoformans var. gattii, have been identified. Both are heterothallic with two mating types, MATa and MATalpha. Some rare isolates are self-fertile and are considered occasional diploid or aneuploid strains. In the present study, 133 isolates, mostly from Italian patients, were investigated to detect the presence of diploid strains in the Igiene Università Milano culture collection. All of the diploid isolates were further investigated by different methods to elucidate their origins. Forty-nine diploid strains were identified by flow cytometry. PCR fingerprinting using the (GACA)(4) primer showed that the diploid state was associated with two specific genotypes identified as VN3 and VN4. Determination of mating type on V8 juice medium confirmed that the majority of the strains were sterile. PCR and dot blotting using the two pheromone genes (MFa and MFalpha) as probes identified 36 of the 49 diploid isolates as MATa/alpha. The results of pheromone gene sequencing showed that two allelic MFalpha genes exist and are distinct for serotypes A and D. In contrast, the MFa gene sequence was conserved in both serotype alleles. Amplification of serotype-specific STE20 alleles demonstrated that the diploid strains contained one mating locus inherited from a serotype A parent and one inherited from a serotype D parent. The present results suggest that diploid isolates may be common among the C. neoformans population and that in Italy and other European countries serotype A and D populations are not genetically isolated but are able to recombine by sexual reproduction.     

Interleukin-17 is not required for classical macrophage activation in a pulmonary mouse model of Cryptococcus neoformans infection

Cryptococcus neoformans is an opportunistic fungal pathogen that causes disease in individuals with suppressed cell-mediated immunity. Recent studies in our laboratory have shown that increases in pulmonary Th1-type and interleukin-17A (IL-17A) cytokine production, classical macrophage activation, and sterilizing immunity are elicited in response to infection with a gamma interferon (IFN-γ)-producing C. neoformans strain, H99γ. IL-17A-treated macrophages, compared to IL-4-treated macrophages, have been demonstrated to exhibit increased microbicidal activity in vitro, a characteristic consistent with classical macrophage activation. The purpose of these studies is to determine the role of IL-17A in the induction of classically activated macrophages following infection with C. neoformans. Immunohistochemistry and real-time PCR were used to characterize the macrophage activation phenotype in lung tissues of mice treated with isotype control or anti-IL-17A antibodies and given an experimental pulmonary infection with C. neoformans strain H99γ. The pulmonary fungal burden was resolved, albeit more slowly, in mice depleted of IL-17A compared to the fungal burden in isotype control-treated mice. Nonetheless, no difference in classical macrophage activation was observed in IL-17A-depleted mice. Similarly, classical macrophage activation was evident in mice deficient in IL-17A or the IL-17 receptor A, which mediates IL-17A signaling, following pulmonary infection with wild-type C. neoformans strain H99 or H99γ. These studies suggest that IL-17A may play a role in the early immune response to C. neoformans but is not required for classical macrophage activation in mice experimentally infected with C. neoformans.     

Candida krusei, a multidrug-resistant opportunistic fungal pathogen: geographic and temporal trends from the ARTEMIS DISK Antifungal Surveillance Program, 2001 to 2005

Candida krusei is well known as a fungal pathogen for patients with hematologic malignancies and for transplant recipients. Using the ARTEMIS Antifungal Surveillance Program database, we describe geographic and temporal trends in the isolation of C. krusei from clinical specimens and the in vitro susceptibilities of 3,448 isolates to voriconazole as determined by CLSI (formerly NCCLS) disk diffusion testing. In addition, we report the in vitro susceptibilities of bloodstream infection isolates of C. krusei to amphotericin B (304 isolates), flucytosine (254 isolates), anidulafungin (121 isolates), caspofungin (300 isolates), and micafungin (102 isolates) as determined by CLSI broth microdilution methods. Geographic differences in isolation were apparent; the highest frequency of isolation was seen for the Czech Republic (7.6%) and the lowest for Indonesia, South Korea, and Thailand (0 to 0.3%). Overall, 83% of isolates were susceptible to voriconazole, ranging from 74.8% in Latin America to 92.3% in North America. C. krusei was most commonly isolated from hematology-oncology services, where only 76.7% of isolates were susceptible to voriconazole. There was no evidence of increasing resistance of C. krusei to voriconazole from 2001 to 2005. Decreased susceptibilities to amphotericin B (MIC at which 90% of isolates were inhibited [MIC₉₀], 4 μg/ml) and flucytosine (MIC₉₀, 16 μg/ml) were noted, whereas 100% of isolates were inhibited by ≤2 μg/ml of anidulafungin (MIC₉₀, 0.06 μg/ml), micafungin (MIC₉₀, 0.12 μg/ml) or caspofungin (MIC₉₀, 0.25 μg/ml). C. krusei is an uncommon but multidrug-resistant fungal pathogen. Among the systemically active antifungal agents, the echinocandins appear to be the most active against this important pathogen.     

Galleria mellonella as a model system to study Cryptococcus neoformans pathogenesis

Evaluation of Cryptococcus neoformans virulence in a number of nonmammalian hosts suggests that C. neoformans is a nonspecific pathogen. We used the killing of Galleria mellonella (the greater wax moth) caterpillar by C. neoformans to develop an invertebrate host model system that can be used to study cryptococcal virulence, host immune responses to infection, and the effects of antifungal compounds. All varieties of C. neoformans killed G. mellonella. After injection into the insect hemocoel, C. neoformans proliferated and, despite successful phagocytosis by host hemocytes, killed caterpillars both at 37 degrees C and 30 degrees C. The rate and extent of killing depended on the cryptococcal strain and the number of fungal cells injected. The sequenced C. neoformans clinical strain H99 was the most virulent of the strains tested and killed caterpillars with inocula as low as 20 CFU/caterpillar. Several C. neoformans genes previously shown to be involved in mammalian virulence (CAP59, GPA1, RAS1, and PKA1) also played a role in G. mellonella killing. Combination antifungal therapy (amphotericin B plus flucytosine) administered before or after inoculation was more effective than monotherapy in prolonging survival and in decreasing the tissue burden of cryptococci in the hemocoel. The G. mellonella-C. neoformans pathogenicity model may be a substitute for mammalian models of infection with C. neoformans and may facilitate the in vivo study of fungal virulence and efficacy of antifungal therapies.     

Susceptibility testing of Cryptococcus neoformans: a microdilution technique.

We studied a series of test conditions in a microtiter system to define the optimal method for determining the susceptibility of Cryptococcus neoformans to antifungal agents. Twenty-one isolates of C. neoformans were grown for 24 or 48 h in four chemically defined media: yeast nitrogen base (BYNB 7); RPMI 1640; synthetic amino acid medium--fungal (SAAMF), buffered at pH 7.0 to select the medium that best supported growth of this fastidious yeast; and yeast nitrogen base, pH 5.4 (YNB 5.4). Maximum growth of C. neoformans, at 35 degrees C, was obtained in YNB 5.4, with the next highest growth levels in BYNB 7, SAAMF, and RPMI. Growth at 24 h was uniformly poor in all media and lacked reproducibility. In contrast, incubation for 48 h gave adequate growth with low standard deviations, and 48 h was selected as the optimal incubation period for this study. Comparison of the relationship between growth kinetics and initial inoculum size for eight cryptococcal isolates showed that 10(4) cells per ml yielded optimal growth in BYNB 7 and YNB 5.4, whereas 10(5) cells per ml was optimal in RPMI and SAAMF. Furthermore, variation of inocula from 10(3) to 10(5) cells per ml showed small but significant inoculum effects in determining MICs of fluconazole, amphotericin B, and flucytosine for C. neoformans. Therefore, 10(4) cells per ml was chosen as the optimal inoculum for susceptibility testing in this study. Mean MICs of fluconazole, amphotericin B, and flucytosine for 21 crytococcal isolates in RPMI and BYNB 7 were low (for example, fluconazole had mean MICs of 1.2 and 1.3 micrograms/ml in RPMI and BYNB 7, respectively) and differed significantly from medium to medium. In contrast, the MICs obtained in SAAMF were significantly higher (e.g., fluconazole had a mean MIC of 2.2 micrograms/ml). Variance in MICs was large with fluconazole and flucytosine but small with amphotericin B, irrespective of the medium used. A microtiter system employing BYNB 7 as the medium, 48 h as the incubation period, and 10(4) cells per ml as the final inoculum is a simple, accurate, and reproducible method for the testing of C. neoformans susceptibility to fluconazole, amphotericin B, and flucytosine.     

CRYPTOCOCCAL INFECTION AND TH1-TH2 CYTOKINE BALANCE

Cytokines have been recognized as key factors in determining host resistance to infectious pathogens. In particular, Th1-Th2 cytokine balance in hosts is profoundly associated with the outcome of infection caused by intracellular microbes. In a murine model of pulmonary and disseminated infection with Cryptococcus neoformans, an opportunistic fungal pathogen that frequently leads to fatal meningoencephaltis in severly immunocompromised hosts, expression of cytokine mRNA in the lungs from infected animals revealed Th2-dominant profiles, while administration of IL-12, which rescued mice from fatal infection, converted such balance toward Th1-dominant states in a drastic fashion. Thus, commitment of Th phenotypes critically determines host sensitivity to cryptococcal infection. In this review, we described how Th1-Th2 cytokine balance influences host protective responses to C. neoformans, and we identify the host and pathogen factors that regulate such balance.     

Cryptococcal infection and Th1-Th2 cytokine balance

Cytokines have been recognized as key factors in determining host resistance to infectious pathogens. In particular, Th1-Th2 cytokine balance in hosts is profoundly associated with the outcome of infection caused by intracellular microbes. In a murine model of pulmonary and disseminated infection with Cryptococcus neoformans, an opportunistic fungal pathogen that frequently leads to fatal meningoencephalitis in severely immunocompromised hosts, expression of cytokine mRNA in the lungs from infected animals revealed Th2-dominant profiles, while administration of IL-12, which rescued mice from fatal infection, converted such balance toward Th1-dominant states in a drastic fashion. Thus, commitment of Th phenotypes critically determines host sensitivity to cryptococcal infection. In this review, we described how Th1-Th2 cytokine balance influences host protective responses to C. neoformans, and we identify the host and pathogen factors that regulate such balance.     

Clinical manifestations and management of cryptococcal infection

Cryptococcus neoformans is an important fungal pathogen causing invasive infection, especially of the central nervous system in this era of the HIV/AIDS epidemic. The choice of treatment depends on site(s) of infection and the patient's immune status. Use of appropriate antifungal agents decreases mortality significantly, but requires continued therapy and long-term maintenance to prevent relapses. The use of liposomal amphotericin B (L Amp B) has overcome some of the difficulties usually found in this setting. The major advantage of these liposomal formulations are faster clearance of C. neoformans [cerebrospinal fluid (CSF) negative] and a reduction in amphotericin toxicity. The majority of clinical efficacy data related to L Amp B are derived from compassionate use studies and case series. Use of liposomal amphotericin has also shown to be a cost effective approach.     

Characterization of heteroresistance to fluconazole among clinical isolates of Cryptococcus neoformans

Strains of Cryptococcus neoformans expressing heteroresistance to fluconazole have been described previously. The present study was conducted to investigate the prevalence of heteroresistance among clinical isolates of C. neoformans and to characterize the heteroresistant phenotypes. A total of 107 clinical isolates of C. neoformans for which the MICs of fluconazole ranged from 0.25 to 32 microg/ml were selected. The isolates were chosen to represent a broad geographic distribution. Of the 107 C. neoformans isolates tested, 4 grew on medium containing fluconazole at concentrations that were four to eight times higher than the MICs for each strain. A fifth isolate, for which the fluconazole MIC was 32 microg/ml, grew on agar with 64 microg of fluconazole per ml. These five isolates (4.7% of the total number) were confirmed to exhibit heteroresistant compositions by population analysis. The degree and frequency of resistance varied among the isolates. Stepwise selection by exposure to fluconazole resulted in subclones of all five strains for which the fluconazole MIC was >64 microg/ml. Subclones of three strains demonstrated a homogeneous population of resistant cells on medium containing 64 microg of fluconazole/ml. The resistance was sensitive to incubation temperature, that is, heteroresistance was demonstrable only at 30 degrees C by agar-based tests, and was reversible through serial transfers on fluconazole-free medium over a period of 8 days. These results suggest that the fluconazole-heteroresistant phenotype of C. neoformans exists in a significant proportion of clinical isolates and that fluconazole resistance can be developed by selection from heteroresistant clones and induction by exposure to fluconazole.     

Comparison study of broth macrodilution and microdilution antifungal susceptibility tests.

An evaluation of broth dilution antifungal susceptibility tests was performed by determining both the micro- and macrodilution MICs of amphotericin B, flucytosine, fluconazole, ketoconazole, and cilofungin against 38 isolates of Candida albicans, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Cryptococcus neoformans, and Torulopsis glabrata. The following preliminary antifungal working group recommendations of the National Committee for Clinical Laboratory Standards for broth macrodilution tests with antifungal agents were used: inocula standardized to 1 x 10(4) to 5 x 10(4) CFU/ml with a spectrophotometer, RPMI 1640 medium buffered with morpholinopropanesulfonic acid (pH 7.0), incubation at 35 degrees C for 24 to 48 h, and an additive drug dilution procedure. Broth microdilution MICs were higher (two or more dilutions) than broth macrodilution MICs for all isolates tested with amphotericin B and for most isolates tested with ketoconazole, fluconazole, and cilofungin. MICs of flucytosine were the same by both techniques or lower by the broth microdilution test except in tests with C. neoformans. However, the only statistically significant differences between the two tests were observed with amphotericin B against all isolates (P = 0.01 to 0.07), ketoconazole against C. neoformans (P = 0.01 to 0.02), and cilofungin against C. albicans (P = 0.05 to 0.14). Tests performed with less dense inocula (1 x 10(3) to 5 x 10(3] produced similar results.     

Cryptococcus laurentii fungemia in a premature neonate

Cryptococcus spp. other than Cryptococcus neoformans are generally considered nonpathogenic to humans. There are only 15 case reports of disease in humans caused by Cryptococcus laurentii infection. Underlying diseases and predisposing risk factors seem to play an important role in these cases. Our patient is the first case of an extremely low birth weight infant with C. laurentii fungemia reported in the English literature. In our case, the MIC of amphotericin B for C. laurentii was 0.25 to 1 microg/ml and the patient had a good outcome following the administration of amphotericin B at 10 mg/kg combined with central venous catheter removal. There will undoubtedly be an increasing occurrence of unusual fungal infections accompanying further advances in medicine. A high degree of suspicion and improvements in the techniques for culture and identification will contribute to the earlier diagnosis and treatment of unusual fungal infections.     

Invasive fungal infections in Chile: a multicenter study of fungal prevalence and susceptibility during a 1-year period.

During the first year of an ongoing surveillance program of invasive fungal infections (IFI) a total of 130 patients (56% male) with fungal strains isolated from blood and other sterile sites were reported from 13 hospitals in Chile. Significant yeast isolates were obtained from 118 patients, and molds affected 12 patients. The main patient groups affected were neonates, children less than 1 year old and adults aged 50-79 years. All fungal bloodstream infections (BSI) were due to yeasts; 79 patients (61%) were affected. The main risk factors recorded were antibiotic therapy (76%), stay in the intensive care unit (ICU) (70%) and presence of a central venous catheter (65%). Nosocomial infections were represented in 83.5% of BSI. Overall, Candida albicans (40.8%), C. parapsilosis (13.1%), C. tropicalis (10%) and Cryptococcus neoformans (10%) were the most common species. Aspergillus fumigatus (3.1%) was the most frequent mold. C. albicans (48.1%) and C. parapsilosis (17.7%), were the most frequent agents recovered from blood. Saccharomyces cerevisiae and Trichosporon mucoides, two emerging pathogens, were also isolated. All yeasts tested were susceptible to amphotericin B with minimal inhibitory concentration (MIC) < or = 1 microg/ml. Resistance to itraconazole (MIC > or = 1 microg/ml) and fluconazole (MIC > or = 64 microg/ml) was observed in 4 and 6% of cases, respectively. C. glabrata was the least susceptible species, with 50% of isolates resistant to itraconazole and 33% resistant to fluconazole, with one strain showing combined resistance. Reduction of BSI requires greater adherence to hand-washing and related infection control guidelines.     

Global trends in the antifungal susceptibility of Cryptococcus neoformans (1990 to 2004)

The antifungal susceptibilities of 1,811 clinical isolates of Cryptococcus neoformans obtained from 100 laboratories in 5 geographic regions worldwide between 1990 and 2004 were determined. The MICs of amphotericin B, flucytosine, fluconazole, voriconazole, posaconazole, and ravuconazole were determined by the National Committee for Clinical Laboratory Standards broth microdilution method. Isolates were submitted to a central reference laboratory (University of Iowa) from study centers in Africa (5 centers, 395 isolates), Europe (14 centers, 102 isolates), Latin America (14 centers, 82 isolates), the Pacific region (7 centers, 50 isolates), and North America (60 centers, 1,182 isolates). Resistance to amphotericin B, flucytosine, and fluconazole was < or = 1% overall. Susceptibility to flucytosine (MIC, < or = 4 microg/ml) ranged from 35% in North America to 68% in Latin America. Similarly, only 75% of isolates from North America were susceptible to fluconazole (MIC, < or = 8 microg/ml) compared to 94 to 100% in the other regions. Isolates remained highly susceptible to amphotericin B (99% susceptibility at a MIC of < or = 1 microg/ml) over the entire 15-year period. Susceptibility to flucytosine (MIC, < or = 4 microg/ml) increased from 34% in 1990 to 1994 to 66% in 2000 to 2004. Susceptibility to fluconazole (MIC, < or = 8 microg/ml) increased from 72% in 1990 to 1994 to 96% in 2000 to 2004. Voriconazole, posaconazole, and ravuconazole all were very active (99% of isolates susceptible at MIC of < or = 1 microg/ml) against this geographically diverse collection of isolates. We conclude that in vitro resistance to antifungal agents used in the treatment of cryptococcosis remains uncommon among isolates of C. neoformans from five broad geographic regions and has not increased over a 15-year period.