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A case of meningitis in a 38-year old male with pulmonary tuberculosis is described. Cryptococcus neoformans was isolated from the cerebrospinal fluid. The isolate belonged to variety neoformans serotype AD. The patient died before any antimycotic therapy could be instituted.
The paper reports the anti-Cryptococcus activity of combination of ethanolic extracts of leaves of Cassia alata and Ocimum sanctum. The activity of combination of the extracts was heat-stable and worked at acidic pH.
Cryptococcus neoformans var. gattii was isolated for the first time from decaying wood in a hollow of a native jungle tree Guettarda acreana, in a wild area of an Amazon rainforest island, in Brazil. The presence of this variety in a virgin environment without either anthropic action or introduced vegetation is discussed with regard to the common knowledge of Cr. neoformans ecology.
We investigated the in vitro susceptibilities of 213 cerebrospinal fluid isolates of Cryptococcus neoformans isolated from 192 patients through a 10-year period, 1990-99, against fluconazole, voriconazole and posaconazole (SCH56592) by using the NCCLS (National Committee for Clinical Laboratory Standards) macrodilution method, M27-A. The overall MICs50 and MICs90 of fluconazole, voriconazole and posaconazole were found to be 2 and 8 micro g ml-1, <or=0.125 micro g ml-1 (both), and <or=0.015 and 0.06 micro g ml-1, respectively. The MIC ranges, MICs50, and MICs90 of three triazoles were also determined according to the defined year category (1990-94, 1995, 1996, 1997 and 1998-99). The MICs50 and MICs90 of each triazole remained almost unchanged and did not exhibit any sign of an upward shift during a decade (1990-99). However, a tendency for development of possible cross-resistance between the three triazoles was observed only in 1996. Meanwhile, the individual relapsing isolates (n:21) from 20 patients exhibited same, higher or, suprisingly, lower MICs, particularly against fluconazole and posaconazole.
The study compared ground red hot pepper agar (GRHP) and Guizotia abyssinica creatinine agar (GACA), a medium routinely used for isolation of Cryptococcus neoformans. In order to confirm the capacity of GRHP to support the Cr. neoformans growth and pigment production, 15 strains were inoculated onto GRHP and GACA. No significant differences in the growth and pigmentation of the tested strains on the two media were noted. As heavily contaminated specimens, 50 samples of pigeon droppings were examined by plating on GRHP and GACA, which resulted in the isolation of 14 and nine Cr. neoformans strains, respectively. The results indicate that GRHP, as a result of its superior selectivity and significant reduction of contaminant growth, provides better conditions than GACA for isolation and presumptive identification of Cr. neoformans from heavily contaminated specimens.
A total of 64 Cryptococcus neoformans strains, including clinical and environmental Brazilian isolates var. neoformans and var. gattii, were tested for susceptibility to amphotericin B, 5-flucytosine, fluconazole and itraconazole. The tests were performed according to the recommendations of National Committee of Clinical Laboratory Standards and the method of macrodilution in liquid medium of Shadomy et al. [Manual de Microbiologia Clínica, 4th ed. Buenos Aires: Editorial Medica Panamericana, 1987: 1229-38]. For most drugs there was a significant difference between the readings taken at 24 and 48 h with both methods. When the minimum inhibitory concentrations obtained by the two techniques were compared, significant differences were observed for amphotericin B and fluconazole. Overall, differences in drug susceptibility with respect to the origin of the isolates or the variety of the fungus were not observed. As an exception, the gattii variety exhibited a high resistance rate to amphotericin B when the technique of Shadomy et al. was applied, a fact possibly related to the greater difficulty for treatment of the disease caused by this fungal variety.
Apoptotic cell death of macrophage has become recognized as a significant mechanism responsible for the resolution of inflammation. The purpose of this study was to examine how the apoptotic cell death involves the formation and resolution of granulomas in rats intravenously inoculated with Cryptococcus neoformans (Cr. neoformans) and Mycobacterium bovis-derived bacillus Calmette-Guérin (BCG) vaccine. The number and size of granulomas in the livers obtained on days 5, 10, 15, 20 and 25 after inoculation were examined by morphometric image analysis, as well as the occurrence of apoptotic cell death quantitatively analyzed by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL) procedure on tissue sections. In both groups the number and size of granulomas were maximized on day 10, then the granulomas were almost resolved until day 25 when the inoculated Cr. neoformans and BCG almost disappeared. From the induction to the resolving stages of granulomatous inflammation, TUNEL-positive cells constantly appeared in granulomas, and the highest frequency of apoptotic cells in granulomas was observed in the earlier stage of granuloma formation. These results indicate that the maintenance and resolution of infectious granulomas are regulated by the balance between the influx of newly recruited macrophages and the apoptotic elimination of granuloma macrophages. The apoptosis of granuloma macrophages actively involves the cellular turnover in both granuloma formation and resolution.
The human pathogenic fungus Cryptococcus neoformans exhibits the phenomenon of phenotypic switching, a process that generates variant colonies that can differ in morphology, virulence and other characteristics such as capsular glucuronoxylomannan (GXM) size and structure. A previous study established that mucoid colony (MC) variants of C. neoformans were more virulent and elicited a different inflammatory response than smooth colony (SM) variants. In this study, we investigated the interaction of cells from MC and SM variants and their respective GXMs with human T cells and monocytes. Specifically, we measured CD40, CD80 and CD86 expression, lymphoproliferation and interleukin (IL)-4, IL-10, interferon (IFN)-gamma and IL-12Rbeta2 expression in the presence and absence of variant cells and their GXMs. For some immune parameters, both MC and SM strains produced similar results, in particular no differences were observed in IL-4 induction. However, for other critical parameters, including CD86 expression, lymphoproliferation and IL-10 production, the MC variant had effects that can be expected to impair the immune response. Hence, a single C. neoformans strain can elicit several different immune responses depending on the colony type expressed, and this is unlikely to be accounted for by differences in phagocytosis only. The results provide a potential explanation for the higher virulence of the MC variant based on the concept that these cells inhibit the development of a vigorous immune response. Furthermore, the results suggest a mechanism by which phenotypic switching can generate variants able to evade the immune response.
OBJECTIVE: To study the influence of gender and age on the course of infection and the cytokine response in a murine model of disseminated cryptococcosis. METHODS: The course of the infection (survival and fungal load in blood and tissues) as well as pro-inflammatory and anti-inflammatory cytokine responses in plasma and organs were compared according to gender and age in outbred mice previously infected with Cryptococcus neoformans NIH52D. RESULTS: Although survival and fungal load were similar in male and female mice, the expression of all cytokines in plasma and of tumour necrosis factor-alpha and interferon-gamma in spleen was significantly increased in female mice compared to male mice in two independent experiments. Young male mice had a significantly shortened survival, were significantly more infected and had predominant tumour necrosis factor-alpha and interferon-gamma responses in comparison with older male mice. CONCLUSION: Host factors should be taken into account when studying the immune response to experimental C. neoformans infection. Our data support epidemiological and clinical data showing differences in susceptibility to cryptococcosis according to gender and age.
Cases of fluconazole-resistant Cryptococcus neoformans have been reported in AIDS patients previously treated with fluconazole. We report a case of fluconazole-resistant cryptococcal meningitis in an HIV-negative patient not previously exposed to fluconazole. The patient experienced a clinical relapse after discontinuation of therapy with amphotericin B and subsequent initiation of fluconazole therapy. In vitro resistance was initially verified by Etest and tablet diffusion and later confirmed by NCCLS broth microdilution.
OBJECTIVES: The serological response of five patients with AIDS and cryptococcosis to non capsular antigens from Cryptococcus neoformans var. neoformans has been investigated. METHODS: Pressates of different isolates of C. neoformans were used as antigenic preparation for immunoblotting of patient samples. RESULTS: Multiple sera and cerebrospinal fluids sequentially collected from five AIDS patients with cryptococcosis showed a wide heterogeneity in antibody response with bands at 48. 43, 38 and 26 kD being present in all clinical samples of all five patients. The variation in banding patterns of the sequential samples from three patients was correlated with a decrease of the antigen titre and with an amelioration of the cryptococcal infection. CONCLUSIONS: We identified antibodies to four immunodominant non-capsular antigens, which might represent major target molecules of the humoral response of patients with cryptococcosis.
Although amphotericin B remains the drug of choice for systemic fungal infections, its use is limited by considerable side effects. In The Peoples' Republic of China, commercial Allium sativum derived compounds are widely used as an antifungal drug to treat systemic fungal infections. To evaluate the scientific merit of using A. sativum derived compounds as antifungal agents, we studied a Chinese commercial preparation, allitridium. This preparation contained mainly diallyl trisulfide as confirmed by high performance liquid chromatography. Allitridium, with and without amphotericin B, was tested to determine its efficacy in killing three isolates of Cryptococcus neoformans. The minimum inhibitory concentration of the commercial preparation was 50 micrograms/ml and the minimum fungicidal concentration was 100 micrograms/ml against 1 x 10(5) organisms of C. neoformans. In addition, the commercial preparation was shown to be synergistic with amphotericin B in the in vitro killing of C. neoformans. This study demonstrates that diallyl trisulfide and other polysulfides possess potent in vitro fungicidal effects and their activity is synergistic with amphotericin B. These observations lend laboratory support for the treatment of cryptococcal infections with both amphotericin B and the Chinese commercial preparation.
Allium sativum (garlic) derived preparations are used alone or with amphotericin B in Asia to treat human systemic fungal infections and cryptococcal meningitis. To evaluate the scientific merit of using allicin-derived compounds as an anti-fungal drug, we prepared a concentrated A. sativum extract that contained 34% allicin, 44% total thiosulfinates, and 20% vinyldithiins. We found that the concentrated extract possessed potent in vitro fungistatic and fungicidal activity against 3 different isolates of Cryptococcus neoformans. The minimum inhibitory concentration of the concentrated garlic extract against 1 x 10(5) organisms of C. neoformans ranged from 6 to 12 micrograms/ml. In addition, in vitro synergistic fungistatic activity with amphotericin B was demonstrated against all isolates of C. neoformans. This study lends laboratory support for the treatment of cryptococcal infections with concentrated garlic extracts.
We reviewed the records of 106 patients with cryptococcal infections and the acquired immunodeficiency syndrome (AIDS) treated at San Francisco General Hospital. We examined four issues: the efficacy of treatment with amphotericin plus flucytosine as compared with amphotericin alone, the efficacy of suppressive therapy, the prognostic clinical characteristics, and the course of nonmeningeal cryptococcosis. In 48 of the 106 patients (45 percent), cryptococcosis was the first manifestation of AIDS. Among the 89 patients with cryptococcal meningitis confirmed by culture, survival did not differ significantly between those treated with amphotericin plus flucytosine (n = 49) and those treated with amphotericin alone (n = 40). Flucytosine had to be discontinued in over half the patients because of cytopenia. Long-term suppressive therapy with either ketoconazole or amphotericin was associated with improved survival, as compared with survival in the absence of suppressive therapy (median survival, greater than or equal to 238 vs. 141 days; P less than 0.004). The only clinical features independently associated with a shorter cumulative survival were hyponatremia and a positive culture for cryptococcus from an extrameningeal source. The 14 patients with nonmeningeal cryptococcosis had a median survival (187 days) and rate of relapse (20 percent) similar to those in the patients with meningitis (165 days and 17 percent, respectively). From this retrospective study of cryptococcal infections in patients with AIDS we conclude that the addition of flucytosine to amphotericin neither enhances survival nor prevents relapse, but long-term suppressive therapy appears to benefit these patients.
Cryptococcus gattii causes life-threatening infection of the pulmonary and central nervous systems in hosts with normal immunity and traditionally has been considered to be restricted geographically to tropical and subtropical climates. The recent outbreak of C. gattii in the temperate climate of Vancouver Island, BC, Canada, led to a collaborative investigation. The objectives of the current study were to ascertain the environmental source of the outbreak infections, survey the molecular types of the outbreak and environmental cryptococcal isolates, and determine the extent of genetic diversity among the isolates. PCR-fingerprinting and amplified fragment length polymorphism (AFLP) were used to examine the genotypes, and mating assays were performed to determine the mating type of the isolates. All outbreak and environmental isolates belonged to C. gattii. Concordant results were obtained by using PCR-fingerprinting and AFLP analysis. The vast majority of clinical and veterinary infections were caused by isolates of the molecular type VGII/AFLP6, but two were caused by molecular type VGI/AFLP4. All environmental isolates belonged to molecular type VGII/AFLP6. Two or three subtypes were observed within VGII/AFLP6 among outbreak and environmental isolates. All mating-competent isolates were of the alpha-mating type. The emergence of this usually tropical pathogen on Vancouver Island highlights the changing distribution of this genotype and emphasizes the importance of an ongoing collaborative effort to monitor the global epidemiology of this yeast.
Cryptococcus neoformans (CN), an encapsulated, ubiquitous environmental yeast, is pathogenic for humans, primarily those with compromised immune function. CN is believed to be a facultative intracellular pathogen. Time-lapsed video microscopy revealed that yeast began to replicate and divide 2 hours after ingestion by J774.16 macrophage cells, with the average cell hosting 10-40 organisms of varying morphologies before ultimately lysing and releasing organisms, either singly or in clumps. Intracellular growth was accompanied by the accumulation of polysaccharide-filled vesicles in the macrophage. Studies with fluorescently labeled dextran revealed that the phagolysosomal compartment became leaky during the course of intracellular infection. Consistent with this observation, phagosomes containing CN had an increased pH relative to similar phagosomes containing inert magnetic beads, as indicated by a colorimetric change in the pH-sensitive Lysosensor dye. Immunocytochemistry revealed differences in the reactivity of polysaccharide elaborated by CN inside macrophages relative to that expressed in vitro. Taken together these results are suggestive of a novel mechanism of intracellular survival by an encapsulated organism, whereby ingestion is followed by damage to the phagosomal membrane resulting in continuity with the cytoplasm, accumulation of polysaccharide-containing vesicles, and possibly, production of a structurally different polysaccharide.
The subcellular localization of the cluster of differentiation 63 (CD63) tetraspanin and its interaction with the class II MHC antigen presentation pathway were examined in the context of phagocytosis by live cell imaging, by using monomeric red fluorescent protein-tagged mouse CD63 expressed in primary bone marrow-derived cell cultures. Upon phagocytosis of Cryptococcus neoformans and polystyrene beads, CD63 was recruited selectively to C. neoformans-containing phagosomes in a MyD88-independent acidification-dependent manner. Bead-containing phagosomes, within a C. neoformans-containing cell, acidified to a lesser extent and failed to recruit CD63 to a level detectable by microscopy. CD63 recruitment to yeast phagosomes occurred independently of class II MHC and LAMP-1. These observations indicate that the composition of distinct phagosomal compartments within the same cell is determined by phagosomal cargo and may affect the outcome of antigen processing and presentation.
Cryptococcus neoformans STE12alpha, a homologue of Saccharomyces cerevisiae STE12, exists only in MATalpha strains. We identified another STE12 homologue, STE12a, which is MATa specific. As in the case with Deltaste12alpha, the mating efficiency for Deltaste12a was reduced significantly. The Deltaste12a strains surprisingly still mated with Deltaste12alpha strains. In MATalpha strains, STE12a functionally complemented STE12alpha for mating efficacy, haploid fruiting, and regulation of capsule size in the mouse brain. Furthermore, when STE12a was replaced with two copies of STE12alpha, the resulting MATa strain produced hyphae on filament agar. STE12a regulates mRNA levels of several genes that are important for virulence including CNLAC1 and CAP genes. STE12a also modulates enzyme activities of phospholipase and superoxide dismutase. Importantly, deletion of STE12a markedly reduced the virulence in mice, as is the case with STE12alpha. Brain smears of mice infected with the Deltaste12a strain showed yeast cells with a considerable reduction in capsule size compared with those infected with STE12a strains. When the disrupted locus of ste12a was replaced with a wild-type STE12a gene, both in vivo and in vitro mutant phenotypes were reversed. These results suggest that STE12a and STE12alpha have similar functions, and that the mating type of the cells influences the alleles to exert their biological effects. C. neoformans, thus, is the first fungal species that contains a mating-type-specific STE12 homologue in each mating type. Our results demonstrate that mating-type-specific genes are not only important for saprobic reproduction but also play an important role for survival of the organism in host tissue.