Cryptococci

Science . 2005 Jan 13; {Epub ahead of print}
The Genome of the Basidiomycetous Yeast and Human Pathogen Cryptococcus Neoformans; Loftus BJ et al.; Cryptococcus neoformans is a basidiomycetous yeast ubiquitous in the environment, a model for fungal pathogenesis and an opportunistic human pathogen of global importance . We have sequenced its ~20 Mb genome, which contains ~6500 intron-rich gene structures and encodes a transcriptome abundant in alternatively spliced and antisense messages . The genome is rich in transposons, many of which cluster at candidate centromeric regions . The presence of these transposons may drive karyotype instability and phenotypic variation . C . neoformans encodes unique genes that may contribute to its unusual virulence properties and comparison of two phenotypically distinct strains reveals variation in gene content in addition to sequence polymorphisms between the genomes.

Oncology (Huntingt), 2004 Nov, 18(13 Suppl 7), 24 - 9
Combination antifungal therapy: where are we now, and where are we going?
Marr K.
Combination therapy with amphotericin B and flucytosine is considered to be the treatment of choice for cryptococcal infections . However, for other infections and combinations of antifungal infections, the data are less clear-cut . The concurrent use of amphotericin B with an azole has elicited controversy, given the potential of antimicrobial antagonism . The results of one recent candidemia study suggest that the potential antagonism may not be an issue; the combination of amphotericin B and fluconazole provided more effective clearance of Candida from the bloodstream than did fluconazole used alone . Several in vitro and animal studies have shown antagonism between the azoles and amphotericin B for aspergillosis . However, introduction of the new class of agents that target beta-glucan synthase (echinocandins) has invigorated the prospects of combination therapy . The echinocandins and polyenes are not antagonistic, and there is evidence that the echinocandins may provide additive to synergistic activity in combination with triazoles . For patients whose aspergillosis is progressing despite monotherapy, the addition of a second agent, such as an echinocandin, may be reasonable.

Pharm Res, 2004 Dec, 21(12), 2207 - 12
Prophylactic role of liposomized chloroquine against murine cryptococcosis less susceptible to fluconazole; Khan MA et al.; PURPOSE: The prophylactic role of liposomized chloroquine (lip-CQ) has been assessed against less susceptible Cryptococcus neoformans infection in murine model . METHODS: In the current study, we investigated the antifungal activity of lip-CQ against C . neoformans in macrophages cell line (J 774) and murine model . Mice were pretreated with free as well as liposomized formulations of CQ at various doses . The anticryptococcal activity of fluconazole was compared in mice with or without CQ pretreatment . The efficacy of CQ prophylaxis was assessed by survival as well as colony forming units (cfu) in brain and lungs of treated mice . RESULTS: Fluconazole alone was not found significantly effective against C . neoformans in both in vitro and in vivo studies . However, the antifungal activity of fluconazole increases in chloroquine-pretreated mice . Lip-CQ was found to be more effective in comparison to the same dose of free chloroquine in reducing fungal burden from macrophages in vitro and lungs and brain of C . neoformans infected mice . CONCLUSIONS: The enhanced prophylactic activity of lip-CQ seems due to rapid uptake of drug-containing liposomes by macrophages . The liposome-mediated accumulation of CQ in macrophages makes the environment unfavorable (alkaline) for the intracellular multiplication of C . neoformans . Moreover, the increased incidence of multi-drug resistance and diversity of pathogenic microorganisms inhibited or killed by CQ makes it the drug of choice for prophylactic therapy.

Can J Microbiol, 2004 Nov, 50(11), 923 - 34
Ecological study of the fungal populations of the acidic Tinto River in southwestern Spain; Lopez-Archilla AI et al.; The characterization of the microbial ecology of the Tinto River, an extreme habitat with an extremely low pH and a high concentration of heavy metals, revealed an unexpected level of microbial richness . A variety of microbial eukaryotes was isolated, among them several fungal strains that were identified and their physiological characteristics studied . Ninety strains of yeast were isolated from the Tinto River . Fifty-two percent of them were capable of growth in vitro using medium amended with river water . They belong to 6 genera of basidiomycetes (Rhodotorula, Cryptococcus, Tremella, Holtermannia, Leucosporidium, and Mrakia) and 2 of ascomycetes (Candida and Williopsis) . In addition, 349 strains of hyphomycetes belonging to 17 genera (most of them ascomycetes) were isolated and studied . Forty-four percent of the isolated filamentous fungi (154 strains) were capable of growing in vitro using medium amended with Tinto River water . Of this percentage, 19% (29 strains) belonged to the genus Penicillium (16 species) and 66% (102 strains) were included in the genera Scytalidium, Bahusakala, Phoma, and Heteroconium or showed dark sterile mycelia, which probably are of dematiaceous hyphomycetes . In addition, we characterized strains of the ascomycete genera Lecythophora and Acremonium and of the zygomycete genus Mortierella, all of them capable of growing in medium amended with river water . Statistical correlation of biological and physicochemical variables suggested a positive relationship between the dematiaceous hyphomycetes and the most extreme physicochemical conditions found in the Tinto River . Principal components analysis confirmed this relationship and also showed that the Acremonium and Lecythophora groups had environmental preferences similar to those of dematiaceous fungi . The spatial positions of the sampling sites were grouped in 2 main clusters: (i) sampling sites in the mine zone in which most of the dematiaceous, Acremonium, and Lecythophora strains were isolated and (ii) sites that were not in the mine zone and sampling station 5 from which were isolated mainly strains of fungi that were not capable of growing in the medium amended with river water and species of the Penicillium genus.

Can J Microbiol, 2004 Oct, 50(10), 853 - 860
Effect of foliar disease on the epiphytic yeast communities of creeping bentgrass and tall fescue; Allen TW et al.; The effect of mechanical wounding or foliar diseases caused by Sclerotinia homoeocarpa or Rhizoctonia solani on the epiphytic yeast communities on creeping bentgrass and tall fescue were determined by leaf washing and dilution plating . Total yeast communities on healthy bentgrass and tall fescue leaves ranged from 7.9 x 103 to 1.4 x 105 CFU.cm–2 and from 2.4 x 103 to 1.6 x 104 CFU.cm–2, respectively . Mechanically wounded leaves (1 of 2 trials) and leaves with disease lesions (11 of 12 trials) supported significantly larger communities of phylloplane yeasts . Total yeast communities on S . homoeocarpa infected or R . solani infected bentgrass leaves were 3.6–10.2 times and 6.2–6.4 times larger, respectively, than the communities on healthy leaves . In general, healthy and diseased bentgrass leaves supported larger yeast communities than healthy or diseased tall fescue leaves . We categorized the majority of yeasts as white-pigmented species, including Cryptococcus laurentii, Cryptococcus flavus, Pseudozyma antarctica, Pseudozyma aphidis, and Pseudozyma parantarctica . The percentage of pink yeasts in the total yeast community ranged from 2.6% to 9.9% on healthy leaves and increased to 32.0%–44.7% on S . homoeocarpa infected leaves . Pink-pigmented yeasts included Rhodotorula glutinis, Rhodotorula mucilaginosa, Sakaguchia dacryoidea, and Sporidiobolus pararoseus . Foliar disease significantly affected community size and composition of epiphytic yeasts on bentgrass and tall fescue.

Eukaryot Cell, 2005 Jan, 4(1), 202 - 8
Distinct Stress Responses of Two Functional Laccases in Cryptococcus neoformans Are Revealed in the Absence of the Thiol-Specific Antioxidant Tsa1; Missall TA et al.; Laccases are thought to be important to the virulence of many fungal pathogens by producing melanin, a presumed oxygen radical scavenger . A laccase in Cryptococcus neoformans has been shown to synthesize melanin and contributes to the virulence and the survival in macrophages of this fungal pathogen . One C . neoformans laccase gene, LAC1, previously called CNLAC1, has been extensively studied, and we describe a homologous gene, LAC2, that is found 8 kb away from LAC1 in the genome . In this study we report a role for both laccases, in addition to the thiol peroxidase, Tsa1, in oxidative and nitrosative stress resistance mechanisms of C . neoformans . With use of real-time PCR, similar changes in expression of the two laccase genes occur in response to oxidative and nitrosative stresses, but only the regulation of the LAC2 gene during stress is influenced by Tsa1 . Both laccases contribute to melanin production using L-dopa as a substrate and are differentially localized in the cell based on green fluorescent protein fusions . A single deletion of either LAC1 or LAC2 alone had no effect on sensitivity to H(2)O(2) or nitric oxide . However, deletion of either LAC1 or LAC2 in combination with a TSA1 deletion resulted in a slight peroxide sensitivity, and a lac2Delta tsa1Delta deletion strain was sensitive to nitric oxide stress . In addition, the deletion of both laccases reduces survival of C . neoformans in primary macrophages . Based on our expression and functional analysis, we propose a novel model for the interaction of these two systems, which are both important for virulence.

Eukaryot Cell, 2005 Jan, 4(1), 190 - 201
Transcriptional Network of Multiple Capsule and Melanin Genes Governed by the Cryptococcus neoformans Cyclic AMP Cascade; Pukkila-Worley R et al.; Cryptococcus neoformans is an opportunistic human fungal pathogen that elaborates several virulence attributes, including a polysaccharide capsule and melanin pigments . A conserved Galpha protein/cyclic AMP (cAMP) pathway controls melanin and capsule production . To identify targets of this pathway, we used an expression profiling approach to define genes that are transcriptionally regulated by the Galpha protein Gpa1 . This approach revealed that Gpa1 transcriptionally regulates multiple genes involved in capsule assembly and identified two additional genes with a marked dependence on Gpa1 for transcription . The first is the LAC1 gene, encoding the laccase enzyme that catalyzes a rate-limiting step in diphenol oxidation and melanin production . The second gene identified (LAC2) is adjacent to the LAC1 gene and encodes a second laccase that shares 75% nucleotide identity with LAC1 . Similar to the LAC1 gene, LAC2 is induced in response to glucose deprivation . However, LAC2 basal transcript levels are much lower than those for LAC1 . Accordingly, a lac2 mutation results in only a modest delay in melanin formation . LAC2 overexpression suppresses the melanin defects of gpa1 and lac1 mutants and partially restores virulence of these strains . These studies provide mechanistic insights into the regulation of capsule and melanin production by the C . neoformans cAMP pathway and demonstrate that multiple laccases contribute to C . neoformans melanin production and pathogenesis.

Eukaryot Cell, 2005 Jan, 4(1), 147 - 55
A Homolog of Ste6, the a-Factor Transporter in Saccharomyces cerevisiae, Is Required for Mating but Not for Monokaryotic Fruiting in Cryptococcus neoformans; Hsueh YP et al.; Fungal pheromones function during the initial recognition stage of the mating process . One type of peptide pheromone identified in ascomycetes and basidiomycetes terminates in a conserved CAAX motif and requires extensive posttranslational modifications to become mature and active . A well-studied representative is the a-factor of Saccharomyces cerevisiae . Unlike the typical secretory pathway utilized by most peptides, an alternative mechanism involving the ATP-binding cassette transporter Ste6 is used for the export of mature a-factor . Cryptococcus neoformans, a bipolar human pathogenic basidiomycete, produces CAAX motif-containing lipopeptide pheromones in both MATa and MATalpha cells . Virulence studies with a congenic pair of C . neoformans serotype D strains have shown that MATalpha cells are more virulent than MATa cells . Characterization of the MATalpha pheromones indicated that an autocrine signaling loop may contribute to the differentiation and virulence of MATalpha cells . To further address the role of pheromones in the signaling loop, we identified a STE6 homolog in the C . neoformans genome and determined its function by gene disruption . The ste6 mutants in either mating-type background showed partially impaired mating functions, and mating was completely abolished in a bilateral mutant cross . Surprisingly, the MATalpha ste6 mutant does not exhibit a defect in monokaryotic fruiting, suggesting that the activation of the autocrine signaling loop by the pheromone is via a Ste6-independent mechanism . MFalpha pheromone itself is essential for this process and could induce the signaling response intracellularly in MATalpha cells . Our data demonstrate that Ste6 is evolutionarily conserved for mating and is not required for monokaryotic fruiting in C . neoformans.

Eukaryot Cell, 2005 Jan, 4(1), 46 - 54
Cryptococcus neoformans Mitochondrial Superoxide Dismutase: an Essential Link between Antioxidant Function and High-Temperature Growth; Giles SS et al.; Manganese superoxide dismutase is an essential component of the mitochondrial antioxidant defense system of most eukaryotes . In the present study, we used a reverse-genetics approach to assess the contribution of the Cryptococcus neoformans manganese superoxide dismutase (Sod2) for antioxidant defense . Strains with mutations in the SOD2 gene exhibited increased susceptibility to oxidative stress as well as poor growth at elevated temperatures compared to isogenic wild-type strains . The sod2Delta mutants were also avirulent in a murine model of inhaled cryptococcosis . Reconstitution of a sod2Delta mutant restored Sod2 activity, eliminated the oxidative stress and temperature-sensitive (ts) phenotypes, and complemented the virulence phenotype . Characterization of the ts phenotype revealed a dependency between Sod2 antioxidant activity and the ability of C . neoformans cells to adapt to growth at elevated temperatures . The ts phenotype could be suppressed by the addition of either ascorbic acid (10 mM) or Mn salen (200 muM) at 30 degrees C, but not at 37 degrees C . Furthermore, sod2Delta mutant cells that were incubated for 24 h at 37 degrees C under anaerobic, but not aerobic, conditions were viable when shifted to the permissive conditions of 25 degrees C in the presence of air . These data suggest that the C . neoformans Sod2 is a major component of the antioxidant defense system in this human fungal pathogen and that adaptation to growth at elevated temperatures is also dependent on Sod2 activity.

Arq Bras Endocrinol Metabol, 2004 Apr, 48(2), 318 - 23 Epub 2004 Jul 07.
{Pseudotumoral pulmonary cryptococcosis in association with Cushing's syndrome.}; Lacativa PG et al.; Opportunistic infections in endogenous Cushing's syndrome are associated with severe cortisol excess and carry a high mortality . Pulmonary cryptococcosis is one of these opportunistic infections and can mimic a lung neoplasm, therefore making its diagnosis difficult . We report a case of a young male with ACTH-dependent Cushing's syndrome and severe hypercortisolism . The patient achieved cure after the transfenoidal surgery, but developed a febrile state . A chest computed tomography showed a pulmonary nodule that did not change in serial chest radiographs . Diagnosis of tuberculosis, fungal and bacterial infections were inconclusive, so the hypothesis of lung neoplasm became more probable . The necropsy, however, disclosed a pseudotumoral cryptococcosis . Opportunistic infections, like Cryptococcus neoformans, should be considered in patients with Cushing's syndrome and a pulmonary infiltrate.

J Assoc Physicians India, 2004 Mar, 52, 242 - 3
Cutaneous cryptococcosis; Joshi S et al.; We report a case of an apparently immunocompetent male, who presented with a painless nodule over the upper abdominal wall . He gave a history of exposure to pigeon droppings . Cryptococcus neoformans was isolated from the lesion and no underlying disorder could be detected . He improved on treatment with oral Fluoconazole.

J Clin Microbiol, 2005 Jan, 43(1), 464 - 7
Cryptococcus neoformans isolates from transplant recipients are not selected for resistance to calcineurin inhibitors by current immunosuppressive regimens; Blankenship JR et al.; The immunosuppressants tacrolimus (FK506) and cyclosporine A inhibit calcineurin and have potent antifungal activity . In this study, 24% of Cryptococcus neoformans isolates from solid-organ transplant patients exhibited altered sensitivity to these drugs, which may have an impact on the infectious course but does not appear to be the consequence of immunosuppressive therapy.

J Immunol, 2005 Jan 15, 174(2), 1027 - 36
Distinct Roles for IL-4 and IL-10 in Regulating T2 Immunity during Allergic Bronchopulmonary Mycosis; Hernandez Y et al.; Pulmonary Cryptococcus neoformans infection of C57BL/6 mice is an established model of an allergic bronchopulmonary mycosis that has also been used to test a number of immunomodulatory agents . Our objective was to determine the role of IL-4 and IL-10 in the development/manifestation of the T2 response to C . neoformans in the lungs and lung-associated lymph nodes . In contrast to wild-type (WT) mice, which develop a chronic infection, pulmonary clearance was significantly greater in IL-4 knockout (KO) and IL-10 KO mice but was not due to an up-regulation of a non-T cell effector mechanism . Pulmonary eosinophilia was absent in both IL-4 KO and IL-10 KO mice compared with WT mice . The production of IL-4, IL-5, and IL-13 by lung leukocytes from IL-4 KO and IL-10 KO mice was lower but IFN-gamma levels remained the same . TNF-alpha and IL-12 production by lung leukocytes was up-regulated in IL-10 KO but not IL-4 KO mice . Overall, IL-4 KO mice did not develop the systemic (lung-associated lymph nodes and serum) or local (lungs) T2 responses characteristic of the allergic bronchopulmonary C . neoformans infection . In contrast, the systemic T2 elements of the response remained unaltered in IL-10 KO mice whereas the T2 response in the lungs failed to develop indicating that the action of IL-10 in T cell regulation was distinct from that of IL-4 . Thus, although IL-10 has been reported to down-regulate pulmonary T2 responses to isolated fungal Ags, IL-10 can augment pulmonary T2 responses if they occur in the context of fungal infection.

J Dermatol, 1976 Aug, 3(4), 139 - 46
Triple fungal infection--chronic mucocutaneous candidiasis, exophytic dermatophytosis and cryptococcal meningitis: a new and unusual combination; Matsumoto T; The clinical and immunological studies of an unusual case of chronic mucocutaneous candidiasis (CMCC) are reported . An 18-year-old male with life-long CMCC was superinfected by a dermatophyte showing exophytic clinical features in his childhood . In addition he has suffered from fatal cryptococcal meningitis since the age of 18 . The combination of three fungi, i.e., Candida albicans, Trichophyton mentagrophytes and Cryptococcus neoformans, has not been found in an examination of the literature . The author's hypothesis, "immune deviation theory" in the pathogenesis of CMCC, is presented in detail . The possible role of hypovitaminosis A anddisorder of iron metabolism in CMCC and treatment of CMCC are reviewed briefly.

J Rheumatol, 2005 Jan, 32(1), 40 - 3
Central nervous system infections in patients with systemic lupus erythematosus; Hung JJ et al.; OBJECTIVE: To evaluate the clinical profiles of patients with systemic lupus erythematosus (SLE) with central nervous system (CNS) infections . METHOD: We retrospectively reviewed patients with SLE with CNS infections from January 1983 to June 2003 . The clinical features, laboratory data, and prognoses of these patients were recorded . RESULTS: During the 20-year review period, 17 SLE patients with CNS infections were identified . The mean age at CNS infection was 29.6 +/- 15.3 years . Cryptococcal infection was identified in 10 patients and bacterial meningitis in 7 . Most patients (94%) had active SLE at the time of CNS infection . Fifteen patients received corticosteroid therapy and of these, 7 received it in conjunction with immunosuppressive agents . The most common presentation was headache, fever, and vomiting . The mortality rate among the 17 patients was high (41.2%) . CONCLUSION: Cryptococcal meningitis played the major role in CNS infection of patients with SLE, and it cannot be ruled out even when the cerebrospinal fluid (CSF) white blood cell count is within normal range . CSF India ink and latex agglutination testing for cryptococcal antigen should be performed and are effective screening tools to establish an early diagnosis.

Diagn Microbiol Infect Dis, 2005 Jan, 51(1), 19 - 29
Evaluation of Mycograb(R), amphotericin B, caspofungin, and fluconazole in combination against Cryptococcus neoformans by checkerboard and time-kill methodologies; Nooney L et al.; This article reported the identification of heat shock protein 90 (hsp90) homologues by immunoblot in Cryptococcus neoformans . Mycograb(R), a genetically recombinant antibody against hsp90, was evaluated against 8 clinical isolates and the National External Quality Assessment Service for Microbiology strain of C . neoformans alone and in combination with amphotericin B, caspofungin, and fluconazole by checkerboard assay . At the end point of an optically clear well, the minimum inhibitory concentration (MIC) 0's ranged from 256 to 1024 mug/mL for Mycograb(R), from 0.5 to 1 mug/mL for amphotericin B, and from 16 to 32 mug/mL for caspofungin . The combination of Mycograb(R) and amphotericin B produced a fractional inhibitory concentration index from 0.27 to 0.56, indicating a mainly synergistic effect, whereas for caspofungin, it varied from 0.5 to 2 . At an end point of >/=50% inhibition, the MIC-2s varied from 16 to 128 mug/mL for Mycograb(R) and from 0.125 to 16 mug/mL for fluconazole . The fractional inhibitory concentration index classified the combination as indifferent for 5 isolates, additive for 3 more isolates, and synergistic in a single isolate . Time-kill analysis on 2 isolates (F/7844 and F/10156), which had synergistic and additive results with amphotericin B, respectively, on checkerboard was performed with 4-16 mug/mL of Mycograb(R), 2-8 mug/mL of fluconazole, and 0.0625-2 mug/mL of amphotericin B . This demonstrated an increasingly static effect with augmenting concentrations of fluconazole and an initial static effect with amphotericin B at lower concentrations, which became fungicidal as the level of drug increased . The addition of either 4 or 8 mug/mL of Mycograb(R) to 0.5 mug/mL of amphotericin B with C . neoformans F/7844 changed a static effect to a fungicidal effect at 8 h with an increased killing of 1.2 logs at 48 h . With C . neoformans F/10156, the addition of 16 mug/mL of Mycograb(R) to 0.25 mug/mL of amphotericin B produced a difference in killing from 1 logarithm after 4 h to 1.5 logarithms after 48 h . These data suggest that the combination of amphotericin B and Mycograb(R) would be worth exploring in the treatment of infection due to C . neoformans.

J Am Vet Med Assoc, 2004 Dec 1, 225(11), 1716 - 22
Clinicopathologic features of an unusual outbreak of cryptococcosis in dogs, cats, ferrets, and a bird: 38 cases (January to July 2003); Lester SJ et al.; OBJECTIVE: To determine clinical and pathologic findings associated with an outbreak of cryptococcosis in an unusual geographic location (British Columbia, Canada) . DESIGN: Retrospective study . ANIMALS: 1 pink-fronted cockatoo, 2 ferrets, 20 cats, and 15 dogs . PROCEDURE: A presumptive diagnosis of cryptococcosis was made on the basis of serologic, histopathologic, or cytologic findings, and a definitive diagnosis was made on the basis of culture or immunohistochemical staining . RESULTS: No breed or sex predilections were detected in affected dogs or cats . Eleven cats had neurologic signs, 7 had skin lesions, and 5 had respiratory tract signs . None of 17 cats tested serologically for FeLV yielded positive results; 1 of 17 cats yielded positive results for FIV (western blot) . Nine of 15 dogs had neurologic signs, 2 had periorbital swellings, and only 3 had respiratory tract signs initially . Microbiologic culture in 15 cases yielded 2 isolates of Cryptococcus neoformans var grubii (serotype A) and 13 isolates of C . neoformans var gattii (serotype B); all organisms were susceptible to amphotericin B and ketoconazole . Serologic testing had sensitivity of 92% and specificity of 98% . CONCLUSIONS AND CLINICAL RELEVANCE: Serologic titers were beneficial in identifying infection in animals with nonspecific signs, but routine serum biochemical or hematologic parameters were of little value in diagnosis . Most animals had nonspecific CNS signs and represented a diagnostic challenge . Animals that travel to or live in this region and have nonspecific malaise or unusual neurologic signs should be evaluated for cryptococcosis.

DNA Seq, 2004 Aug, 15(4), 291 - 8
Primary structure of dihydrofolate reductase and mitochondrial ribosomal protein L36 genes from the basidiomycete Coprinus cinereus; Aimi T et al.; We amplified and sequenced the dihydrofolate reductase (DHFR) gene of the basidiomycete Coprinus cinereus . Downstream of the DHFR coding region, a mitochondrial (mt) ribosomal protein L36 (RPL36) gene was discovered in the opposite orientation to DHFR gene . Putative polyadenylation signals of the two genes overlapped, both containing the 8-bp palindrome 5'-aatatatt-3' . The finding that C . cinereus DHFR gene is closely clustered with a mt protein gene strongly suggests that C . cinereus DHFR is closely related to mt function and evolution . The amino acid sequence of C . cinereus DHFR is most homologous to eukaryotic proteins such as Cryptococcus neoformans and Pneumocystis carinii DHFRs . However, the sequence of C . cinereus mt RPL36 closely resembles RPL36 of bacteria and cyanobacteria such as Synechocystis sp . and Escherichia coli . This result strongly supports the serial endosymbiotic theory of the development of ancestral eukaryotes, and suggests that C . cinereus mt RPL36 gene originated from the ancestral eubacterial genome.

J Ethnopharmacol, 2005 Jan 15, 96(3), 563 - 8 Epub 2004 Nov 25.
Screening of antifungal agents using ethanol precipitation and bioautography of medicinal and food plants; Schmourlo G et al.; In the search for bioactive compounds, bioautography and ethanol precipitation of macromolecules (proteins, polysaccharides, etc.) of plant aqueous extracts were associated in an antifungal screening . Thus, the supernatants, precipitates (obtained by ethanol precipitation) and aqueous extracts were investigated of medicinal and fruit bearing plants used against skin diseases by the Brazilian population . The agar diffusion and broth dilution methods were used to assess the activity against three fungi: Candida albicans, Trichophyton rubrum and Cryptococcus neoformans . The results, evaluated by the diameter of the inhibition zone of fungal growth, indicate that six plant species, among the 16 investigated, showed significant antifungal activity . The minimal inhibitory concentration (MIC) was determined on plant extracts that showed high efficacy against the tested microorganisms . The most susceptible yeast was Trichophyton rubrum and the best antifungal activity was shown by Xanthosoma sagittifolium supernatant . The bioautography was performed only for the aqueous extracts and supernatants of those plants that showed antifungal activity against Candida albicans and Cryptococcus neoformans, using n-butanol/acetic acid/water (BAW) 8:1:1 to develop silica gel TLC plates . Clear inhibition zones were observed for aqueous extracts of Schinus molle (R(f) 0.89) and Schinus terebinthifolius (R(f) 0.80) against Candida albicans, as for supernatant of Anacardium occidentale (R(f) 0.31) against Cryptococcus neoformans . The separation of macromolecules from metabolites, as in the case of Anacardium occidentale, Solanum sp . and Xanthosoma sagittifolium, enhances antifungal activity . In other cases, the antifungal activity is destroyed, as observed for Momordica charantia, Schinus molle and Schinus terebinthifolius.

Infect Immun, 2005 Jan, 73(1), 592 - 8
Opsonic requirements for dendritic cell-mediated responses to Cryptococcus neoformans; Kelly RM et al.; The encapsulated pathogenic yeast Cryptococcus neoformans is poorly recognized by phagocytic cells in the absence of opsonins . Macrophages will bind and internalize complement- or antibody-opsonized C . neoformans; however, less is known about the role of opsonins in dendritic cell (DC)-mediated recognition of the organism . Thus, we studied the opsonic requirements for binding to C . neoformans by cultured human monocyte-derived and murine bone marrow-derived DCs and whether binding leads to antifungal activity and cytokine release . Binding of unopsonized C . neoformans to human and murine DCs was negligible . Opsonization with pooled human serum (PHS) increased binding, while heat treatment of PHS virtually abolished this binding, thus suggesting a role for heat-labile complement components . PHS plus a monoclonal anticapsular antibody, 3C2, had an additive effect on binding for most cryptococcal strains . Human and murine DCs exhibited pronounced anticryptococcal activity in the presence of the antibody at early (2-h) and late (24-h) time points; however, PHS opsonization did not supplement this anticryptococcal activity . Antifungal activity against C . neoformans opsonized in PHS and/or antibody was partially reduced in the presence of inhibitors of the respiratory burst response . Human, but not murine, DCs released modest amounts of tumor necrosis factor alpha when stimulated with C . neoformans opsonized in PHS and/or antibody . However, opsonized C . neoformans failed to stimulate detectable release of interleukin 10 (IL-10) or IL-12p70 from either DC population . Thus, human and murine DCs show maximal binding to and antifungal activity against C . neoformans via a process highly dependent on opsonization.

Infect Immun, 2005 Jan, 73(1), 39 - 49
Transient neutralization of tumor necrosis factor alpha can produce a chronic fungal infection in an immunocompetent host: potential role of immature dendritic cells; Herring AC et al.; The mechanisms underlying induction of immune dysregulation and chronic fungal infection by a transient tumor necrosis factor alpha (TNF-alpha) deficiency remain to be defined . The objective of our studies was to determine the potential contribution of neutropenia and immature dendritic cells to the immune deviation . Administration of an anti-TNF-alpha monoclonal antibody at day 0 neutralized TNF-alpha only during the first week of a pulmonary Cryptococcus neoformans infection . Transient neutralization of TNF-alpha resulted in transient depression of interleukin-12 (IL-12), monocyte chemotactic protein 1 (MCP-1), and gamma interferon (IFN-gamma) production but permanently impaired long-term clearance of the infection from the lungs even after the levels of these cytokines increased and a vigorous inflammatory response developed . Early neutrophil recruitment was defective in the absence of TNF-alpha . However, as demonstrated by neutrophil depletion studies, this did not account for the decrease in IL-12 and IFN-gamma levels and did not play a role in establishing chronic pulmonary cryptococcal infection . Transient TNF-alpha neutralization also produced a deficiency in CD11c(+) MHC II(+) cells and IL-12 in the lymph nodes, potentially implicating a defect in mature dendritic cell trafficking . Transfer of cryptococcal antigen-pulsed immature dendritic cells into naive mice prior to intratracheal challenge resulted in the development of a nonprotective immune response to C . neoformans that was similar to that observed in anti-TNF-alpha-treated mice (increased IL-4, IL-5, and IL-10 levels, pulmonary eosinophilia, and decreased clearance) . Thus, stimulation of an antifungal response by immature dendritic cells can result in an immune deviation similar to that produced by transient TNF-alpha deficiency, identifying a new mechanism by which a chronic fungal infection can occur in an immunocompetent host.

Antimicrob Agents Chemother, 2005 Jan, 49(1), 350 - 7
Effects of antifungal interventions on the outcome of experimental infections with phenotypic switch variants of Cryptococcus neoformans; Fries BC et al.; In cryptococcal infection, phenotypic switching from a smooth to a mucoid variant can occur in vivo, producing variants with enhanced virulence that are subsequently selected and affect the outcome of infection . Here, we demonstrate that antifungal treatment of the chronically infected host can promote this phenomenon . Amphotericin B treatment reduces fungal burden less effectively in mucoid variant-infected than in smooth variant-infected mice . Consequently, amphotericin B treatment resulted in a more pronounced prolongation of survival in smooth variant-infected than in mucoid variant-infected mice (20 versus 42 days; P < 0.05) . Administration of anticapsular monoclonal antibody mediated better protection in smooth variant-infected than in mucoid variant-infected mice, although a protective effect was not consistently observed at all doses . Most interestingly, both antifungal drug therapy and administration of anticapsular monoclonal antibody promoted the selection of mucoid variants in smooth variant-infected mice, a phenomenon manifested by a statistically higher percentage of mucoid colonies in smooth variant-infected mice than in nontreated control mice . This finding suggests that both chemotherapeutic and immunological antifungal interventions may promote the selection of the more virulent mucoid variant, which could affect the outcome of infection in chronically infected hosts.

Antimicrob Agents Chemother, 2005 Jan, 49(1), 57 - 70
Surface-active fungicidal D-peptide inhibitors of the plasma membrane proton pump that block azole resistance; Monk BC et al.; A 1.8-million-member D-octapeptide combinatorial library was constructed in which each member comprised a diversity-containing N-terminal pentapeptide and a C-terminal amidated triarginine motif . The C-terminal motif concentrated the library members at the fungal cell surface . A primary screen for inhibitors of Saccharomyces cerevisiae and Candida albicans growth, together with an in vitro secondary screen with the S . cerevisiae plasma membrane ATPase (Pma1p) as a target, identified the antifungal D-octapeptide BM0 (D-NH(2)-RFWWFRRR-CONH(2)) . Optimization of BM0 led to the construction of BM2 (D-NH(2)-RRRFWWFRRR-CONH(2)), which had broad-spectrum fungicidal activity against S . cerevisiae, Candida species, and Cryptococcus neoformans; bound strongly to the surfaces of fungal cells; inhibited the physiological activity of Pma1p; and appeared to target Pma1p, with 50% inhibitory concentrations in the range of 0.5 to 2.5 microM . At sub-MICs (<5 microM), BM2 chemosensitized to fluconazole (FLC) S . cerevisiae strains functionally hyperexpressing fungal lanosterol 14alpha-demethylase and resistance-conferring transporters of azole drugs . BM2 chemosensitized to FLC some FLC-resistant clinical isolates of C . albicans and C . dubliniensis and chemosensitized to itraconazole clinical isolates of C . krusei that are intrinsically resistant to FLC . The growth-inhibitory concentrations of BM2 did not cause fungal cell permeabilization, significant hemolysis of red blood cells, or the death of cultured HEp-2 epithelial cells . BM2 represents a novel class of broad-spectrum, surface-active, Pma1p-targeting fungicides which increases the potencies of azole drugs and circumvents azole resistance.

Presse Med, 2004 Nov 6, 33(19 Pt 2), 1385 - 8
{Vasculitis secondary to fungal infections}; Zeller V et al.; THE FUNGI INCRIMINATED: Cerebral vascular lesions are frequently observed in the rhino-cerebral localisations of aspergillosis and mucormycosis, and during cerebral coccidioidomycosis, but far more rarely in neuro-meningeal cryptococcosis and histoplasmosis . Other fungi are occasionally at the origin of vasculitis . THE CONTEXT: Diagnosis is difficult to establish . It should be evoked in immunodepressed patients presenting with clinical and radiological manifestations of cerebral vasculitis . AS FAR AS TREATMENT IS CONCERNED: Treatment relies on antifungal therapy . Steroids and/or anticoagulants can be envisaged in some patients.

Presse Med, 2004 Nov, 33(19-C2), 1385 - 1388
{Vasculitis secondary to fungal infections.}; Zeller V et al.; The fungi incriminated Cerebral vascular lesions are frequently observed in the rhino-cerebral localisations of aspergillosis and mucormycosis, and during cerebral coccidioidomycosis, but far more rarely in neuro-meningeal cryptococcosis and histoplasmosis . Other fungi are occasionally at the origin of vasculitis . The context Diagnosis is difficult to establish . It should be evoked in immunodepressed patients presenting with clinical and radiological manifestations of cerebral vasculitis . As far as treatment is concerned Treatment relies on antifungal therapy . Steroids and/or anticoagulants can be envisaged in some patients.

J Infect Dis, 2005 Jan 15, 191(2), 307 - 11 Epub 2004 Dec 09.
Influence of Indinavir on Virulence and Growth of Cryptococcus neoformans; Monari C et al.; Indinavir selectively inhibited production of some virulence factors of Cryptococcus neoformans, such as urease and protease, but not melanin and phospholipase; moreover, it interfered with capsule formation . These effects led to increased susceptibility of C . neoformans to intracellular killing by natural effector cells . Prolonged incubation with indinavir resulted in inhibition of fungal growth . Indinavir can attenuate the virulence of the fungus, thus augmenting its susceptibility to the antimicrobial activity of natural effector cells . The reduction in cryptococcal infections in human immunodeficiency virus-positive patients might also be related to the antifungal activity of highly active antiretroviral therapy.

J Infect Dis, 2005 Jan 15, 191(2), 234 - 7 Epub 2004 Dec 10.
Dual infections of the central nervous system with epstein-barr virus; Weinberg A et al.; We describe clinical and laboratory characteristics of 16 patients with central nervous system (CNS) infection caused by Epstein-Barr virus (EBV) and another pathogen . Seven of 10 immunocompromised patients had coinfection with viruses (3 with cytomegalovirus, 2 with JC virus, and 2 with varicella zoster virus) and 3 with nonviral pathogens (2 with pneumococcus and 1 with Cryptococcus species) . Three of 6 immunocompetent patients had coinfections with viruses (1 each with herpes simplex virus, varicella zoster virus, and West Nile virus), and 3 had coinfections with nonviral pathogens (2 with Ehrlichia chaffeensis and 1 with Mycoplasma pneumoniae) . The EBV load was similar in immunocompromised and immunocompetent patients and in patients with viral and nonviral coinfections . EBV lytic-cycle mRNA was detected in the cerebrospinal fluid of 5 of 6 tested samples, indicating EBV replication in the CNS during coinfection.

Clin Microbiol Infect, 2004 Dec, 10(12), 1092 - 4
Determination of Cryptococcus neoformans var . neoformans mating type by multiplex PCR; Esposto MC et al.; Mating type plays an important role in the epidemiology and virulence of Cryptococcus neoformans . The present study designed a multiplex PCR method to distinguish the six mating type patterns (Aa, Da, Aalpha, Dalpha, Aa/Dalpha, and Aalpha/Da) of C . neoformans var . neoformans . PCR amplification identified one fragment for Aa (860 bp), Dalpha (413 bp) and Da (645 bp) strains, two fragments for Aalpha (320 and 400 bp) and Aa/Dalpha (860 and 413 bp) strains, and three fragments (645, 400, 320 bp) for an Aalpha/Da strain . The method appears to be a valid, simple and relatively inexpensive tool for epidemiological and virulence studies.

Expert Rev Vaccines, 2004 Dec, 3(6), 701 - 9
Preventative and therapeutic vaccines for fungal infections: from concept to implementation; Deepe Jr GS; Many challenges confront the development of fungal vaccines for humans including differences in host susceptibility, varied pathogenic mechanisms employed by the different species of fungi and mechanisms of host resistance . Hence, no single antigen can be expected to serve as a pan fungal vaccine . Instead, it is likely that progress for fungal vaccines will have to be made at the level of each individual organism . In recent years, tremendous strides have been made in understanding the immunopathogenesis of medically important fungal infections and identifying putative vaccine candidates . Such discoveries will facilitate the introduction of fungal vaccines into the therapeutic armamentarium of clinicians . The fungi under discussion in this review include Candida spp., Aspergillus spp., Cryptococcus neoformans, Coccidioides spp., Histoplasma capsulatum, Blastomyces dermatitidis, Paracoccidioides brasiliensis and Pneumocystis jirovecii.

Mycoses, 2004 Dec, 47(11-12), 455 - 64
Follow-up of epidemiological data of cryptococcosis in Austria, Germany and Switzerland with special focus on the characterization of clinical isolates; Tintelnot K et al.; Summary The present survey in Austria, Germany and Switzerland continued the survey of cryptococcosis set up by the European Confederation of Medical Mycology (ECMM) in 1997 . From 2000 to 2003 77 cases have been reported . An HIV infection is still the most important risk factor (68%) . Young HIV+ women from ASIA contributed to the increase of cryptococcosis in females . A total of 129 clinical isolates of both surveys were genotyped by PCR fingerprinting to study the prevalence of different genotypes . The prevalence of Cryptococcus neoformans var . grubii (serotype A) with the genotypes VNA1 and VNA2 was higher in Germany and Austria (74.5%) than in Switzerland (52%), while in Switzerland the Cr . neoformans hybrids AD (26%) and Cr . neoformans var . neoformans (serotype D) (22%) were more prevalent compared with Germany and Austria (8 and 17.5% respectively) . Cryptococcus gattii isolates were studied by FT-IR spectroscopy . DNA in the ITS region was sequenced to get further information about Cr . neoformans serotype AD strains and about the geographical origin of the Cr . gattii isolates . The ITS sequence of the serotype AD isolates of the genotypes VNAD1, VNAD2 and VNAD4 is usually identical to serotype A or serotype D respectively . In the three isolates of the genotype VNAD3 a genotype-specific sequence pattern was detected . Two autochthonous infections due to Cr . gattii could indicate that the genotype VGIV with the ITS type 'Asia 2' might be endemic in Europe . Zusammenfassung Die vorliegende Erhebung in Osterreich, Deutschland und der Schweiz ist eine Fortfuhrung des Surveys zur Cryptococcose, der durch die European Confederation of Medical Mycology (ECMM) 1997 initiiert wurde . Zwischen 2000 und 2003 wurden 77 Kasuistiken mitgeteilt . Eine HIV Infektion ist weiterhin der wichtigste Risikofaktor (68%) . Junge weibliche HIV-Infizierte aus Asien trugen zum Anstieg der Cryptococcus-Infektionen bei Frauen bei . 129 klinische Isolate aus beiden Surveys wurden mittels PCR fingerprinting genotypisiert . Die Pravalenz von Cr . neoformans var . grubii (Serotyp A) mit den Genotypen VNA1 und VNA2 war in Deutschland und Osterreich hoher (74.5%) als in der Schweiz (52%), wahrend in der Schweiz die Cr . neoformans-Hybride AD (26%) und Cr . neoformans var . neoformans (Serotyp D) (22%) pravalenter waren als in Deutschland und Osterreich (8% bzw . 17.5%) . Cr . gattii-Isolate wurden mittels FT-IR-Spektroskopie untersucht . Die DNA-Sequenzierung der ITS-Region wurde bei Cr . neoformans Serotyp AD-Isolaten durchgefuhrt und bei Cr . gattii-Isolaten hinsichtlich ihrer geographischen Herkunft . Die ITS-Sequenz von Serotyp AD-Isolaten der Genotypen VNAD1, VNAD2 und VNAD4 ist in der Regel entweder mit der des Serotyps A oder D identisch . Bei den drei Isolaten des Genotyps VNAD3 wurde eine genotyp-spezifische Sequenz gefunden . Zwei autochthone Cr . gattii-Infektionen durch den Genotyp VGIV vom ITS-Typ ,,Asia 2'' konnen auf das endemische Vorkommen dieses Genotyps in Europa hindeuten.

Indian J Chest Dis Allied Sci, 2000 Oct-Dec, 42(4), 357 - 66
Recent trends in antifungal therapy: focus on systemic mycoses; Ray A et al.; Amphotericin B (AmB) remains the 'gold standard' for systemic antifungal therapy and newer drug delivery systems have been effectively developed for this drug . The newer azoles e.g . itraconazole, voriconazole and their derivatives are viable alternatives to AmB, and are suitable for oral administration . Combination antifungal therapy has been tried successfully . For instance, AmB plus flucytosine is preferred in cryptococcal meningitis and candidemia . A liquid preparation of itraconazole and cyclodextrin is pharmacokinetically superior to the conventional dosage form of itraconazole, and is particularly effective in mycoses following cytotoxic chemotherapy and bone marrow transplants . Newer potential antifungals such as echinocandins, benanomicins/pradimicins, aureobasidins and nikkomycins are under clinical trials . The role of -immunomodulation in systemic anti-fungal therapy has been proposed and recombinant cytokine therapy (G-CSF, GM-CSF, IFN-g, IL-I, TNF-a) either alone or in combination with AmB have shown good results.

Indian J Chest Dis Allied Sci, 2000 Oct-Dec, 42(4), 317 - 21
Occurrence and significance of Cryptococcus neoformans in vegetables and fruits; Misra VC et al.; Cryptococcus neoformans was isolated from three of 437 (0.6%) samples of a wide variety of vegetables collected from a number of markets in Delhi . The vegetables yielding C . neoformans were tomato, Lycopersicon esculentum (1/70), 'vegetable sponge', Luffa cylindrica (1/56) and brinjal, Solanum melongena (1/55) . Only solitary samples of these three vegetables were found to be positive . In vitro studies at 25 degrees C revealed that a reference C . neoformans isolate readily colonized, as did Candida albicans and Candida viswanathii (controls), sterilized slices of almost all the test vegetables and fruits (cut or uncut surface) which included tomato, 'vegetable sponge', brinjal, potato, carrot, radish, apple, guava, pineapple, pear, papaya, banana and mango . Sterilized juices of the same vegetables and fruits also supported adequate growth of the reference C . neoformans isolate as well as of C . albicans and C . viswanathii . However, the growth of C . neoformans on the afore-mentioned vegetables and fruits was rapidly masked by miscellaneous molds and bacteria if the test samples had not been sterilized prior to inoculation . Cryptococcus neoformans was not found in any of the 275 fruit samples investigated from the same markets . The results indicate that vegetables and fruits are unsuitable as natural habitats for C . neoformans although it may be sporadically isolated from these sources.

Indian J Chest Dis Allied Sci, 2000 Oct-Dec, 42(4), 311 - 5
Cryptococcosis in the immunocompromised host with special reference to AIDS; Khanna N et al.; Cryptococcosis is increasing because of an ever rising population of immunocompromised individuals especially those with acquired immune deficiency syndrome (AIDS) . Cryptococcal infection of the central nervous system (CNS) were diagnosed in 149 cases over a period of 19.5 years (January 1978-June 1998) . Culture was positive in all cases except three who were already on antifungal therapy . India ink mounts of cerebrospinal fluid (CSF) revealed encapsulated cryptococci in 134, and cryptococcal antigen was detected in 111 of 114 patients tested . A comparison of laboratory and certain clinical parameters in patients with and without associated HIV infection showed that a poor CSF cell response and culture of cryptococci from extra-neural sites was more often associated with HIV infection and was statistically significant . Further, presence of concomitant infection especially tuberculosis, and mortality were higher in the HIV positive group.

Indian J Chest Dis Allied Sci, 2000 Oct-Dec, 42(4), 289 - 92
The spectrum of respiratory mycoses in a referral hospital in north-western India; Jindal SK et al.; There has been an increasing recognition of respiratory mycoses in this country in the recent past . Candidiasis, aspergillosis and cryptococcosis have been reported from different centres . Occasional cases of histoplasmosis and sporotrichosis have also been reported . Infections with Pneumocystis carinii, which is now classified as a fungus, are being frequently diagnosed, especially in the immuno-compromised patients such as those on prolonged immunosuppressive therapy or with HIV infection . Although the clinical picture of respiratory mycoses resembles that of any other infection, the presentation in several cases is atypical and the diagnosis is delayed . In a review of our patients of respiratory fungal infections seen in the last few years, we found several interesting examples . We report here the general clinical spectrum of respiratory mycoses and some interesting cases seen at our Centre.

Indian J Chest Dis Allied Sci, 2000 Oct-Dec, 42(4), 279 - 87
Invasive fungal infections in Kuwait: A retrospective study; Khan ZU et al.; A retrospective review of a five year period (1994-1998) revealed that opportunistic mycoses caused by ubiquitous fungal pathogens are a serious problem in the immunocompromised patient population of Kuwait . Patients with renal transplantation and diabetes mellitus were most susceptible to aspergillosis, cryptococcosis, and zygomycosis, whereas patients with candidemia/hematogenous candidiasis had multiple risk factors . Basidiobolomycosis of the rectum in a Bangladeshi male, cryptococcosis due to Cryptococcus neoformans var . gatti in an AIDS patient,fungal peritonitis due to Absidia corymbifera in a patient on peritoneal dialysis, and endocarditis due to Aspergillus terreus detected by direct microscopic examination and culture of the blood clot are some of the notable cases diagnosed during the period under review . The predominance of Candida species other than C . albicans as bloodstream pathogens is another noteworthy observation . Although outbreaks of C . parapsilosis candidemia in neonatal intensive care units contributed significantly to this shift in favour of non-albicans Candida species, a surveillance strategy comprising of molecular, epidemiologic and antifungal susceptibility studies is warranted . With the proposed expansion of organ and bone marrow transplantation facilities in Kuwait, the incidence of opportunistic fungal infections is likely to increase.

Indian J Chest Dis Allied Sci, 2000 Oct-Dec, 42(4), 207 - 19
Respiratory and systemic mycoses: an overview; Randhawa HS; Respiratory and systemic mycoses are globally emerging as a problem of increasing importance in infectious diseases . This is attributed to the growing population of immunocompromised patients due to epidemic outbreak of AIDS or to other factors such as use of immunosuppressive drugs in recipients of organ transplantation . The available evidence has unequivocally established the endemic occurrence of blastomycosis, histoplasmosis and penicilliosis mameffei in India . In fact, pencilliosis marneffei has emerged as a major endemic mycosis of AIDS patients in Southeast Asia . It has manifestations simulating those of histoplasmosis capsulati, and it may spread to other regions with enlarging population of AIDS patients . Comprehensive studies are indicated in order to delineate the endemic areas of the afore-mentioned systemic mycoses . Among the other important systemic mycoses reported from India are aspergillosis, cryptococcosis, candidiasis and zygomycosis . Our current knowledge of the global distribution of systemic mycoses does not depict their true prevalence . It largely reflects the geographic distribution of medical mycologists or other investigators engaged in the study of fungal diseases and their research interests . Invasive aspergillosis has emerged as an important disease in patients with neutropenia and bone narrow transplant recipients, cryptoccosis, penicilliosis marneffei and pneumocystosis in patients with AIDS, fusariosis in patients with leukaemia receiving cytotoxic therapy, zygomycosis in diabetic patients and in patients on defroxamine therapy, and Malasseziafurfur infection in patients on total parenteral nutrition: Opportunistic systemic mycoses due to yeasts and yeast-like fungi have become commoner than those due to filamentous fungi, occupying fourth position in the list of bloodstream pathogens in some centers in USA . Also, their incidence, pattern of clinical presentations and species spectrum have significantly changed, largely due to more frequent and prolonged therapeutic or prophylactic use of antifungal drugs and subsequent development of resistance . Consequently, infections with resistant yeast-like fungi such as C . lusitaniae, C . krusei, C . tropicalis, C . glabrata and Trichosporon ovoides (T . beigelii) have recently been reported with greater frequency . Since respiratory and systemic mycoses have no pathognomonic clinical or radiologic syndrome and mycological diagnostic facilities are restricted to only some of the major metropolitan centres, these diseases may be frequently confused with tuberculosis or other diseases of obscure etiology in India and other developing countries . Greater awareness and a high index of clinical suspicion are important pre-requisites for their diagnosis . Also, active collaboration of internists, pathologists, mycologists and microbiologists is advocated for their expeditous diagnosis and successful management . Further studies should focus on the development of rapid techniques for selective isolation and identification of systemic pathogenic fungi . The problem of antifungal resistance is likely to become more serious in the future as more and more patients with AIDS, bone marrow transplantation and neutropenia will require chemoprophylaxis cover against systemic fungal infections . Thus, it would be of vital importance to intensify search for more potent and less toxic antifungal drugs . It is recognized that an increasing number of people whose life is saved or prolonged due to successful treatment of their underlying diseases fall victim to opportunistic, life threatening systemic mycoses . A great majority of the deaths due to these infections occurs because they remain undiagnosed for want of mycological diagnostic services . In order to cope with the challenge of systemic mycoses, the health authorities of the developing countries are called upon to urgently take necessary measures for establishing a network of diagnostic mycology laboratories.

Eur J Immunol, 2005 Jan, 35(1), 273 - 81
Stimulation via Toll-like receptor 9 reduces Cryptococcus neoformans-induced pulmonary inflammation in an IL-12-dependent manner; Edwards L et al.; Cytosine-phosphate-guanosine-containing oligodeoxynucleotides (CpG ODN) are important vaccine adjuvants that promote Th1-type immune responses . Cryptococcus neoformans is a serious human pathogen that replicates in the lung but may disseminate systemically leading to meningitis, particularly in immunocompromised individuals . Immunization of susceptible C57BL/6 mice with CpG ODN deviates the immune response from a Th2- toward a Th1-type response following infection with C . neoformans . CpG also induces IL-12, TNF, MCP-1 and macrophage nitric oxide production . CD4(+) and CD8(+) T cells producing IFN-gamma increase in frequency, while those producing IL-5 decrease . More importantly, pulmonary eosinophilia is significantly reduced, an effect that depends on IL-12 and CD8(+) T cells but not NK cells . CpG treatment also reduces the burden of C . neoformans in the lung, an effect that is IL-12-, NK cell- and T cell-independent and probably reflects a direct effect of CpG on pathogen opsonization or an enhancement of macrophage antimicrobial activity . An equivalent beneficial effect is also observed when CpG ODN treatment is delivered during established cryptococcal disease . This is the first study documenting that promotion of lung TLR9 signaling using synthetic agonists enhances host defense . Activation of innate immunity has clear therapeutic potential and may even be beneficial in patients with acquired immune deficiency.

Microbes Infect, 2004 Dec, 6(15), 1351 - 5
Interactions of Toll-like receptors with fungi; Levitz SM; The role of Toll-like receptors (TLRs) in signaling immune responses to fungal pathogens is reviewed . TLR2 and TLR4, acting via the adapter protein MyD88, signal responses to Cryptococcus neoformans, Aspergillus fumigatus and Candida albicans in vitro, although the relative significance of these TLRs to host defenses in vivo is unclear.

Indian Pediatr, 2004 Nov, 41(11), 1162 - 4
Systemic humicolus cryptococcosis; Shinde SM et al.; We report a 71/2-year-boy with disseminated systemic cryptococcosis . Although other species have been incriminated, this appears to be the first report of Cryptococcus humicolus . The child was HIV negative . He was treated with amphotericin B and fluconazole with intensive supportive care . The child responded after 6 weeks and is now on maintenance fluconazole therapy.

Eukaryot Cell, 2004 Dec, 3(6), 1601 - 8
UGD1, encoding the Cryptococcus neoformans UDP-glucose dehydrogenase, is essential for growth at 37 degrees C and for capsule biosynthesis; Moyrand F et al.; We report the identification and disruption of the Cryptococcus neoformans var . grubii UGD1 gene encoding the UDP-glucose dehydrogenase, which catalyzes the conversion of UDP-glucose into UDP-glucuronic acid . Deletion of UGD1 led to modifications in the cell wall, as revealed by changes in the sensitivity of ugd1Delta cells to sodium dodecyl sulfate, NaCl, and sorbitol . Moreover, two of the yeast's major virulence factors-capsule biosynthesis and the ability to grow at 37 degrees C-were impaired in ugd1Delta strains . These results suggest that the UDP-dehydrogenase represents the major, and maybe only, biosynthetic pathway for UDP-glucuronic acid in C . neoformans . Consequently, deletion of UGD1 blocked not only the synthesis of UDP-glucuronic acid but also that of UDP-xylose . To differentiate the phenotype(s) associated with the UDP-glucuronic acid defect alone from those linked to the UDP-xylose defect, ugd1Delta mutants were phenotypically compared to strains from which the gene encoding UDP-xylose synthase (i.e., that required for synthesis of UDP-xylose) had been deleted . Finally, studies of strains from which one of the four CAP genes (CAP10, CAP59, CAP60, or CAP64) had been deleted revealed common cell wall phenotypes associated with the acapsular state.

Eukaryot Cell, 2004 Dec, 3(6), 1513 - 24
Cas3p belongs to a seven-member family of capsule structure designer proteins; Moyrand F et al.; The polysaccharide capsule is the main virulence factor of the basidiomycetous yeast Cryptococcus neoformans . Four genes (CAP10, CAP59, CAP60, and CAP64) essential for capsule formation have been previously identified, although their roles in the biosynthetic pathway remain unclear . A genetic and bioinformatics approach allowed the identification of six CAP64-homologous genes, named CAS3, CAS31, CAS32, CAS33, CAS34, and CAS35, in the C . neoformans genome . This gene family is apparently specific in a subclass of the basidiomycete fungi . Single as well as double deletions of these genes in all possible combinations demonstrated that none of the CAP64-homologous genes were essential for capsule formation, although the cas35Delta strains displayed a hypocapsular phenotype . The chemical structure of the glucuronomannan (GXM) produced by the CAS family deletants revealed that these genes determined the position and the linkage of the xylose and/or O-acetyl residues on the mannose backbone . Hence, these genes are all involved in assembly of the GXM structure in C . neoformans.

Eukaryot Cell, 2004 Dec, 3(6), 1476 - 91
Adenylyl cyclase-associated protein Aca1 regulates virulence and differentiation of Cryptococcus neoformans via the cyclic AMP-protein kinase A cascade; Bahn YS et al.; The evolutionarily conserved cyclic AMP (cAMP) signaling pathway controls cell functions in response to environmental cues in organisms as diverse as yeast and mammals . In the basidiomycetous human pathogenic fungus Cryptococcus neoformans, the cAMP pathway governs virulence and morphological differentiation . Here we identified and characterized adenylyl cyclase-associated protein, Aca1, which functions in parallel with the Galpha subunit Gpa1 to control the adenylyl cyclase (Cac1) . Aca1 interacted with the C terminus of Cac1 in the yeast two-hybrid system . By molecular and genetic approaches, Aca1 was shown to play a critical role in mating by regulating cell fusion and filamentous growth in a cAMP-dependent manner . Aca1 also regulates melanin and capsule production via the Cac1-cAMP-protein kinase A pathway . Genetic epistasis studies support models in which Aca1 and Gpa1 are necessary and sufficient components that cooperate to activate adenylyl cyclase . Taken together, these studies further define the cAMP signaling cascade controlling virulence of this ubiquitous human fungal pathogen.

J Antimicrob Chemother . 2004 Dec 8; {Epub ahead of print}
Enhanced anticryptococcal activity of chloroquine in phosphatidylserine-containing liposomes in a murine model; Khan MA et al.; OBJECTIVES: The anticryptococcal activity of chloroquine was assessed after incorporation in phosphatidylserine (PS)-containing negatively charged liposomes in a murine model . METHODS: In the present study, we investigated the antifungal activity of chloroquine entrapped in PS liposomes against Cryptococcus neoformans in the macrophage cell line J 774 and in a murine model . Mice were treated with free as well as liposomal formulations of chloroquine before and after challenging with C . neoformans infection . The anticryptococcal activity of chloroquine was also evaluated in combination with fluconazole in the treatment of systemic murine cryptococcosis . The efficacy of chloroquine treatment was assessed by continued survival as well as by colony forming units (cfu) in liver and brain of treated mice . RESULTS: Chloroquine entrapped in PS liposomes shows increased activity against C . neoformans infection both in in vitro and in vivo studies . Moreover, the antifungal activity of fluconazole increases when used in combination with liposomal chloroquine . Chloroquine in PS liposomes was found to be more effective in comparison with the same dose of free chloroquine or chloroquine entrapped in neutral liposomes . CONCLUSIONS: The enhanced anticryptococcal activity of chloroquine in PS liposomes seems to be due to uptake of drug-containing PS liposomes by macrophages . It can be assumed that liposome-mediated delivery of chloroquine to macrophages results in an unfavourable (alkaline) environment for the growth of C . neoformans inside macrophages.

Fungal Genet Biol, 2005 Jan, 42(1), 20 - 9
Cytochrome c peroxidase contributes to the antioxidant defense of Cryptococcus neoformans; Giles SS et al.; Cryptococcus neoformans is an opportunistic pathogen and the leading cause of fungal meningitis . To survive within the host, this organism must be able to protect itself from oxidative stress . Cytochrome c peroxidase (Ccp1) is a mitochondrial antioxidant that catalyzes the degradation of hydrogen peroxide . In the present study, we characterized the contribution of the C . neoformans Ccp1 to antioxidant defense and for virulence . Consistent with studies of Ccp1 function in Sacchromyces cerevisiae, we found that Ccp1 contributes to resistance against exogenous oxidative stress in vitro . However, the oxidative stress phenotype does not diminish the virulence of ccp1 mutant strains in a murine model of C . neoformans disease . These results suggest that Ccp1 is involved in a complex system of protection against exogenous oxidative stress and that the elimination of this component of the antioxidant defense system does not diminish the virulence of C . neoformans.

J Ethnopharmacol, 2005 Jan 4, 96(1-2), 271 - 7
The composition, geographical variation and antimicrobial activity of Lippia javanica (Verbenaceae) leaf essential oils; Viljoen AM et al.; Lippia javanica is widely distributed throughout South Africa where it is used extensively in traditional herbal preparations . An infusion of the leaves is commonly used as a decongestant for colds and coughs . A preliminary study indicated that the essential oil chemistry varies dramatically both within and between natural plant populations . As the antimicrobial activity may be directly related to the specific composition of the oil, the activity may also fluctuate . The aerial parts of Lippia javanica were collected from various localities in southern Africa to study the essential oil composition and the antimicrobial activity thereof . The hydrodistilled essential oils were analysed by GC/MS and a cluster analysis was performed on the essential oil dataset . From 16 samples (representing five natural populations), 5 chemotypes were identified; a myrcenone rich-type (36-62%), a carvone rich-type (61-73%), a piperitenone rich-type (32-48%), an ipsenone rich-type (42-61%) and a linalool rich-type (>65%) . The myrcenone and linalool chemotypes have been mentioned in the literature but the carvone, ipsenone and piperitenone chemotypes have not previously been reported for Lippia javanica . Time kill studies were performed on three microbial respiratory isolates to document the scientific rationale of using Lippia to treat respiratory complaints in traditional herbal medicine . Klebsiella pneumoniae, Cryptococcus neoformans and Bacillus cereus showed reduction in microbial populations with the strongest bacteriostatic effect observed for Klebsiella pneumoniae.

J Immunol, 2004 Dec 15, 173(12), 7513 - 20
O-acetylation of cryptococcal capsular glucuronoxylomannan is essential for interference with neutrophil migration; Ellerbroek PM et al.; The capsular polysaccharide glucuronoxylomannan (GXM) of Cryptococcus neoformans has been shown to interfere with neutrophil migration . Although several receptors have been implied to mediate this process, the structural perspectives are unknown . Here, we assess the contribution of 6-O-acetylation and xylose substitution of the (1-->3)-alpha-d-mannan backbone of GXM, the variable structural features of GXM, to the interference with neutrophil migration . We compare chemically deacetylated GXM and acetyl- or xylose-deficient GXM from genetically modified strains with wild-type GXM in their ability to inhibit the different phases of neutrophil migration . Additionally, we verify the effects of de-O-acetylation on neutrophil migration in vivo . De-O-acetylation caused a dramatic reduction of the inhibitory capacity of GXM in the in vitro assays for neutrophil chemokinesis, rolling on E-selectin and firm adhesion to endothelium . Genetic removal of xylose only marginally reduced the ability of GXM to reduce firm adhesion . In vivo, chemical deacetylation of GXM significantly reduced its ability to interfere with neutrophil recruitment in a model of myocardial ischemia (65% reduction vs a nonsignificant reduction in tissue myeloperoxidase, respectively) . Our findings indicate that 6-O-acetylated mannose of GXM is a crucial motive for the inhibition of neutrophil recruitment.

J Immunol, 2004 Dec 15, 173(12), 7435 - 43
Innate imprinting by the modified heat-labile toxin of Escherichia coli (LTK63) provides generic protection against lung infectious disease; Williams AE et al.; In a healthy individual, the lung contains few lymphoid cells . However, amplified immune responses, as exemplified during lung infection, can cause extensive tissue damage . We have previously demonstrated that one lung infection modulates the immunopathological outcome to a subsequent unrelated pathogen . Mimicking heterologous immunity may provide a means of enhancing both innate and acquired immunity . We now show that prior lung administration of a modified heat-labile toxin from Escherichia coli (LTK63) enhances immunity to respiratory syncytial virus, influenza virus, and the fungus Cryptococcus neoformans . Treatment with LTK63 decreased lung inflammation and tissue damage and improved the ability to resolve the infection . APCs expressing the activation markers MHC class II, CD80, and CD40 increased in number in the lung . LTK63 treatment increased the pathogen-specific IgA response in the nasal mucosa and simultaneously decreased inflammatory cytokine production (IFN-gamma and TNF-alpha) after infection . The number of activated CD8(+)CD44(+) T cells and the respiratory syncytial virus- or influenza-specific CD8-proliferative responses increased, although the total inflammatory infiltrate was reduced . LTK63 treatment matured lung APCs (LTK63 prevented efficient presentation of whole OVA to DO11.10 cells, whereas OVA peptide presentation was unaffected), enhanced immunity in both a Th1 and Th2 environment, was long lasting, and was not pathogen or host strain specific; the protective effects were partially independent of T and B cells . Innate imprinting by toxin-based immunotherapeutics may provide generic protection against infectious disease in the lung, without the need for coadministered pathogen-specific Ag.

J Med Microbiol, 2004 Dec, 53(Pt 12), 1201 - 6
Quantification and assessment of viability of Cryptococcus neoformans by LightCycler amplification of capsule gene mRNA; Amjad M et al.; Cryptococcus neoformans is an opportunistic fungal pathogen . It infects the central nervous system causing meningitis, which is fatal if untreated, especially in AIDS and immunosuppressed patients . In this study a method of quantification and assessment of viability of C . neoformans by LightCycler RT-PCR amplification of the capsule gene mRNA is established . The sequence of primers and probes were derived from C . neoformans capsular CAP10 gene mRNA (GenBank accession number AF144574), and were species specific . Agarose gel electrophoresis analysis of LightCycler RT-PCR product showed a single band of 223 bp in length . In order to develop an internal control a 223 bp exon fragment of capsule mRNA was cloned in the pCR2.1 plasmid vector and RNA was generated by in vitro transcription . To determine the sensitivity of the assay, serial dilutions of in vitro-transcribed RNA with known concentrations and copy numbers, and serially diluted cultures of viable and nonviable C . neoformans were used . Under optimal conditions as little as 0.472 fg of capsule mRNA could be detected, corresponding to 1-10 c.f.u . ml(-1) of the sample . No amplification was observed from up to 10(5) heat/UV radiation-killed yeast cells and RNA of other bacterial and fungal pathogens and human genomic DNA or RNA . The amplification of capsule mRNA represents a sensitive, specific and quantitative means of detection of viable C . neoformans in clinical specimens and can be useful in the evaluation of the therapeutic efficacy of antifungal drugs in the treatment of C . neoformans meningitis.

Bioorg Med Chem, 2005 Jan 3, 13(1), 121 - 30
Facile synthesis of the heptasaccharide repeating unit of O-deacetylated GXM of C . neoformans serotype B; Zhao W et al.; A heptasaccharide, beta-D-Xylp-(1-->2)-alpha-D-Manp-(1-->3)-{beta-D-Xylp-(1-->2)}-alpha-D-Manp-(1-->3)-{beta-D-GlcpA-(1-->2)}{beta-D-Xylp-(1-->4)}-alpha-D-Manp, the repeating unit of the exopolysaccharide from Cryptococcus neoformans serovar B, was synthesized as its methyl glycoside . Thus 2,3,4-tri-O-benzoyl-beta-D-xylopyranosyl-(1-->2)-3,4,6-tri-O-benzoyl-alpha-d-mannopyranosyl trichloroacetimidate (7) and allyl 2,3,4-tri-O-benzoyl-beta-D-xylopyranosyl-(1-->2)-4,6-di-O-benzoyl-alpha-D-mannopyranoside (8), readily obtained from the corresponding monosaccharide derivatives via simple transformation, were coupled to give a (1-->3)-linked tetrasaccharide 9 . Deallylation of 9 followed by trichloroacetimidate formation produced the tetrasaccharide donor 11 . Condensation of methyl 2,3,4-tri-O-benzoyl-beta-d-xylopyranosyl-(1-->4)-2-O-acetyl-6-O-benzoyl-alpha-D-mannopyranoside (18) with 11 followed by selective deacetylation yielded hexasaccharide acceptor 20 . Coupling of 20 with methyl 2,3,4-tri-O-acetyl-alpha-D-glucopyranosyluronate bromide (21) and subsequent deprotection furnished the target heptaoside . A hexasaccharide fragment, alpha-D-Manp-(1-->3)-{beta-D-Xylp-(1-->2)}-alpha-D-Manp-(1-->3)-{beta-D-GlcpA-(1-->2)}{beta-D-Xylp-(1-->4)}-alpha-D-Manp, was also similarly synthesized as its methyl glycoside.

J Antibiot (Tokyo), 2004 Sep, 57(9), 573 - 8
A new nonadride derivative, dihydroepiheveadride, as characteristic antifungal agent against filamentous fungi, isolated from unidentified fungus IFM 52672; Hosoe T et al.; In the screening of searching for new antifungal agents, a new nonaride compound, dihydroepiheveadride (1), was isolated from unidentified fungus IFM 52672 as the most potent antifungal principle from this organism . The structure of 1 was established on the basis of spectroscopic and chemical investigation, as well as detailed comparison of the spectroscopic and physico-chemical data of the oxidized derivative (3) from 1 with those of heveadride (2) . Compound 1 showed strong antifungal activity against various filamentous fungi including human pathogens Aspergillus fumigatus, Penicillium marneffei and Trichophyton spp . It also showed the growth inhibition activity against certain human pathogenic yeasts such as Trichosporon species, while it had weak or no antifungal activity against Candida spp . and Cryptococcus neoformans, and no antibacterial activity against Bacillus subtilis nor against Escherichia coli . The antifungal potencies of compounds 2 and 3 were found to be weaker than that of 1.

Genetics, 2004 Nov, 168(3), 1177 - 88
Genotype-environment interactions of spontaneous mutations for vegetative fitness in the human pathogenic fungus Cryptococcus neoformans; Xu J; Spontaneous mutation is the ultimate source of all genetic variation . By interacting with environmental factors, genetic variation determines the phenotype and fitness of individuals in natural populations . However, except in a few model organisms, relatively little is known about the patterns of genotype-environment interactions of spontaneous mutations . Here I examine the rates of spontaneous mutation and the patterns of genotype-environment interaction of mutations affecting vegetative growth in the human fungal pathogen Cryptococcus neoformans . Eight mutation accumulation (MA) lines were established from a single clone on the nutrient-rich medium YEPD for each of two temperatures, 25 degrees and 37 degrees . Cells from generations 100, 200, 400, and 600 for each of the 16 MA lines were stored and assayed for vegetative growth rates under each of four conditions: (i) 25 degrees on SD (a synthetic dextrose minimal medium); (ii) 25 degrees on YEPD; (iii) 37 degrees on SD; and (iv) 37 degrees on YEPD . Both MA conditions and assay environments for vegetative growth showed significant influence on the estimates of genomic mutation rates, average effect per mutation, and mutational heritability . Significant genotype-environment interactions were detected among the newly accumulated spontaneous mutations . Overall, clones from MA lines maintained at 37 degrees showed less decline in vegetative fitness than those maintained at 25 degrees . The result suggests that a high-temperature environment might be very important for the maintenance of the ability to grow at a high temperature . Results from comparisons between clinical and environmental samples of C . neoformans were consistent with laboratory experimental population analyses . This study calls into question our long-standing view that warm-blooded mammals were only occasional and accidental hosts of this human fungal pathogen.

Curr Drug Targets Infect Disord, 2004 Dec, 4(4), 311 - 22
IPC synthase as a useful target for antifungal drugs; Sugimoto Y et al.; Inositol phosphorylceramide (IPC) synthase is a common and essential enzyme in fungi and plants, which catalyzes the transfer of phosphoinositol to the C-1 hydroxy of ceramide to produce IPC . This reaction is a key step in fungal sphingolipid biosynthesis, therefore the enzyme is a potential target for the development of nontoxic therapeutic antifungal agents . Natural products with a desired biological activity, aureobasidin A (AbA), khafrefungin, and galbonolide A, have been reported . AbA, a cyclic depsipeptide containing 8 amino acids and a hydroxyl acid, is a broad spectrum antifungal with strong activity against many pathogenic fungi such as Candida spp., Cryptococcus neoformans, and some Aspergillus spp . Khafrefungin, an aldonic acid ester with a C22 long alkyl chain, has antifungal activity against C . albicans, Cr . Neoformans, and Saccharomyces cerevisiae . Galbonolide A is a 14-membered macrolide with fungicidal activity against clinically important strains, and is especially potent against Cr . neoformans . These classes of natural products are potent and specific antifungal agents . We review current progress in the development of IPC synthase inhibitors with antifungal activities, and present structure-activity relationships (SAR), physicochemical and structural properties, and synthetic methodology for chemical modification.

Intern Med, 2004 Oct, 43(10), 977 - 81
Pleural cryptococcosis with idiopathic CD4 positive T-lymphocytopenia; Kawabata T et al.; A 19-year-old man was admitted to our hospital because of chest pain . He was diagnosed as having pleural cryptococcosis by pleural biopsy . His CD4 positive T-lymphocyte count was low (< 300 microl) and there was no evidence of human immunodeficiency virus infection . He was successfully treated with fluconazole . However, his CD4 positive lymphocyte counts remained low after the recovery and he was diagnosed as idiopathic CD4 positive T-lymphocytopenia . Pleural cryptococcosis is rare and its predisposing condition is still controversial . To our knowledge, this is the first case of pleural cryptococcosis associated with idiopathic CD4 positive T lymphocytopenia.

Proc Natl Acad Sci U S A, 2004 Dec 7, 101(49), 17258 - 63 Epub 2004 Dec 7.
A rare genotype of Cryptococcus gattii caused the cryptococcosis outbreak on Vancouver Island (British Columbia, Canada); Kidd SE et al.; 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.

Biochem Biophys Res Commun, 2005 Jan 7, 326(1), 233 - 41
Genomic organization and expression of 23 new genes from MATalpha locus of Cryptococcus neoformans var . gattii; Ren P et al.; The pathogenic yeast Cryptococcus neoformans (Cn) causes cryptococcosis, a life-threatening disease of the brain . Molecular studies of Cn variety gattii have lagged behind other two varieties (var . grubii and var . neoformans) although they have distinct biology and disease patterns . We focused on gene discovery in MATalpha locus because it predominates in clinical strains . A var . gattii cosmid library was screened with DNA probes from other two varieties . Two positive clones were sequenced to identify ORFs based on similarities to known proteins, and to ESTs using bioinformatics, and manually by a curator . Approximately 76kb sequenced DNA revealed 23 genes and ORFs . The existence of predicted genes was verified by RT-PCR analyses designed to amplify spliced sequences . The results confirmed that the transcripts were expressed both at 30 and 37 degrees C . The var . gattii MATalpha locus genes showed rearrangements in order and orientation vis-a-vis other two varieties . Mating-specific genes showed higher nonsynonymous mutation rates, and gene trees showed var . gattii strains in a distinct clade . The identification of the largest number, thus far, of var . gattii structural genes should set the stage for future molecular pathogenesis studies.

Mol Biol Evol . 2004 Nov 24; {Epub ahead of print}
Survey of Simple Sequence Repeats in Completed Fungal Genomes; Karaoglu H et al.; The use of simple sequence repeats or microsatellites as genetic markers has become very popular due to their abundance and length variation between different individuals . SSRs are tandem repeat units of one to six base pairs that are found abundantly in many prokaryotic and eukaryotic genomes . This is the first study examining and comparing SSRs in completely sequenced fungal genomes . We analysed and compared the occurrences, relative abundance, relative density, most common and longest SSRs in nine taxonomically different fungal species: Aspergillus nidulans, Cryptococcus neoformans, Encephalitozoon cuniculi, Fusarium graminearum, Magnaporthe grisea, Neurospora crassa, Schizosaccharomyces pombe, and Ustilago maydis . Our analysis revealed that, in all of the genomes studied, the occurrence, abundance, and relative density of SSRs varied, and was not influenced by the genome sizes . No correlation between relative abundance and the genomes sizes was observed but it was shown that N . crassa, the largest genome analysed had the highest relative abundance of SSRs . In most genomes, mono-, di- and trinucleotide repeats were more abundant than the longer repeated SSRs . Generally in each organism, the occurrence, relative abundance and relative density of SSRs decreased as the repeat unit increased . Furthermore, each organism had its own common and longest SSRs . Our analysis showed that the relative abundance of SSRs in fungi is low compared to the human genome, and that longer SSRs in fungi are rare . In addition to providing new information concerning the abundance of SSRs for each of these fungi, the results provide a general source of molecular markers that could be useful for a variety of applications such as population genetics and strain identification of fungal organisms.

Kansenshogaku Zasshi, 2004 Oct, 78(10), 910 - 5
{A case of primary pulmonary cryptococcosis showing lobar pneumonia on chest X-ray}; Kawamura T et al.; A 36-year-old male who had been in good health visited our hospital because of fever, productive cough and lobar pneumonia of the right lower lobe . Bronchoscopic examination revealed pulmonary cryptococcosis . After the initiation of fluconazole the pneumonia started to improve, however, it was aggravated after the discontinuation of fluconazole because of liver dysfunction induced by it . The pulmonary cryptococcosis improved by lobectomy of the right lower lobe . There have been few reports of lobar pneumonia caused by primary pulmonary cryptococcosis . We report this case with fourteen other cases of primary pulmonary cryptococcosis which have been experienced in our hospital.

Rev Argent Microbiol, 2004 Jul-Sep, 36(3), 113 - 7
{Successful discontinuation of antifungal secondary prophylaxis in AIDS-related cryptococcosis}; Negroni R et al.; The clinical and laboratory data of 22 patients with AIDS related cryptococcosis who were able to interrupt antifungal secondary prophylaxis after HAART administration, are presented . They were 14 males and 8 females, between 15 and 50 years old (X: 34 years old) . All patients presented fever and severe deterioration of their general health status, and 19 exhibited a meningeal syndrome . At the start of antifungal treatment, 59% of the cases presented < 50 CD4+ cells/microl, the median viral burden was 134,804 RNA copies/ml and the median titer of serum cryptococcal antigen was 1/3,000 . Amphotericin B by intravenous route, (0.7 mg/kg/day) or fluconazole (600 to 800 mg/day) were given as a treatment of the initial episode, up to CSF cultures negativization . Oral fluconazole (200 mg/day) or intravenous amphotericin B, 50 mg twice a week, were given as a secondary prophylaxis . The secondary prophylaxis was interrupted when the patients had received HAART for an average lapse of 19 months (6 to 36 months) and the median CD4+ cell count was 249/microl . The follow up after secondary prophylaxis discontinuation lasted for a median lapse of 22 months . These data seem to show that secondary prophylaxis is not necessary when the patient are clinically asymptomatic and the CD4+ cell counts are above 150/microl.

Bioorg Med Chem, 2004 Dec 15, 12(24), 6461 - 4
Crystallographic and NMR studies of antiinfective tricyclic guanidine alkaloids from the sponge Monanchora unguifera; Hua HM et al.; Three tricyclic guanidine alkaloids, including 1,8a;8b,3a-didehydro-8beta-hydroxyptilocaulin (1), 1,8a;8b,3a-didehydro-8alpha-hydroxyptilocaulin (2) and mirabilin B (3), were identified from the marine sponge Monanchora unguifera . 1,8a;8b,3a-Didehydro-8alpha-hydroxyptilocaulin (2) is a new stereoisomer of 1, the structure of which was elucidated by spectroscopic analysis, comparison of its spectral data with those of 1, and confirmed by X-ray analysis . Compounds 1 and 2 co-crystallized in an unusual perfect order and packed around an approximate inversion center . A mixture of 1 and 2 is active against the malaria parasite Plasmodium falciparum with an IC50 value of 3.8 microg/mL while mirabilin B (3) exhibited antifungal activity against Cryptococcus neoformans with an IC50 value of 7.0 microg/mL and antiprotozoal activity against Leishmania donovani with an IC50 value of 17 microg/mL.

FEMS Yeast Res, 2004 Dec, 5(3), 253 - 61
Application of temperature gradient gel electrophoresis to the study of yeast diversity in the estuary of the Tagus river, Portugal; Gadanho M et al.; Temperature gradient gel electrophoresis (TGGE) was employed for the assessment of yeast diversity in the estuary of the Tagus river (Portugal) . The molecular detection of yeasts was carried out directly from water samples and, in parallel, a cultivation approach by means of an enrichment step was employed . A nested PCR was employed to obtain a fungal amplicon containing the D2 domain of the 26S rRNA gene . For identification the TGGE bands were extracted, re-amplified, and sequenced . Fourteen fungal taxa were detected and all except one were yeasts . Most yeast sequences corresponded to members of the Ascomycota and only three belonged to the Basidiomycota . Five yeasts (four ascomycetes and one basidiomycete) could not be identified to the species level due to the uniqueness of their sequences . The number of species detected after enrichment was higher than the number of taxa found using the direct detection method . This suggests that some yeast populations are present in densities that are below the detection threshold of the method . With respect to the analysis of the yeast community structure, our results indicate that the dominant populations belong to Debaryomyces hansenii, Rhodotorula mucilaginosa, Cryptococcus longus, and to an uncultured basidiomycetous yeast phylogenetically close to Cr . longus . The combined analysis of direct detection and cultivation approaches indicates a similar community structure at the two sampled sites since nine species were present at both localities.

FEMS Yeast Res, 2004 Dec, 5(3), 247 - 52
Characterization of an antifungal glycolipid secreted by the yeast Sympodiomycopsis paphiopedili; Kulakovskaya TV et al.; An antifungal glycolipid was purified from the culture liquid of the ustilaginomycetous yeast Sympodiomycopsis paphiopedili by column and thin-layer chromatography . According to nuclear magnetic resonance and mass-spectroscopy experiments it was a cellobioside containing 2,15,16-trihydroxypalmitic acid as an aglycon . The minimal effective concentrations leading to ATP leakage and growth inhibition were 45 and 160 microg ml(-1) for Cryptococcus terreus and Candida albicans, respectively.

BMC Infect Dis . 2004 Nov 22;4(1):52.
Spectrum of clinical disease in a series of 135 hospitalised HIV-infected patients from north India; Sharma SK et al.; BACKGROUND: Literature on the spectrum of opportunistic disease in human immunodeficiency virus (HIV)-infected patients from developing countries is sparse . The objective of this study was to document the spectrum and determine the frequency of various opportunistic infections (OIs) and non-infectious opportunistic diseases, in hospitalised HIV-infected patients from north India . METHODS: One hundred and thirty five consecutive, HIV-infected patients (age 34 +/- 10 years, females 17%) admitted to a tertiary care hospital in north India, for the evaluation and management of an OI or HIV-related disorder between January 2000 and July 2003, were studied . RESULTS: Fever (71%) and weight loss (65%) were the commonest presenting symptoms . Heterosexual transmission was the commonest mode of HIV-acquisition . Tuberculosis (TB) was the commonest OI (71%) followed by candidiasis (39.3%), Pneumocystis jiroveci pneumonia (PCP) (7.4%), cryptococcal meningitis and cerebral toxoplasmosis (3.7% each) . Most of the cases of TB were disseminated (64%) . Apart from other well-recognised OIs, two patients had visceral leishmaniasis . Two cases of HIV-associated lymphoma were encountered . CD4+ cell counts were done in 109 patients . Majority of the patients (82.6%) had CD4+ counts <200 cells/microL . Fifty patients (46%) had CD4+ counts <50 cells/microL . Only 50 patients (37%) received antiretroviral therapy . Twenty one patients (16%) died during hospital stay . All but one deaths were due to TB (16 patients; 76%) and PCP (4 patients; 19%) . CONCLUSIONS: A wide spectrum of disease, including both OIs and non-infectious opportunistic diseases, is seen in hospitalised HIV-infected patients from north India . Tuberculosis remains the most common OI and is the commonest cause of death in these patients.

Med Mycol, 2004 Oct, 42(5), 449 - 60
Retrospective study of feline and canine cryptococcosis in Australia from 1981 to 2001: 195 cases; O'Brien CR et al.; A retrospective study of 155 cats and 40 dogs diagnosed with cryptococcosis between 1981 and 2001 was undertaken . Age, sex, breed, clinical findings, feline immunodeficiency virus and feline leukaemia virus status (in cats), species of Cryptococcus causing disease and region of domicile were recorded . Associations between variables were tested . Male and female cats were affected equally . Age ranged from 1 to 16 years, with a preponderance of cats aged between 2 and 3 years . Siamese, Himalayan and Ragdoll breeds were over-represented . Rural cats were more frequently infected with Cryptococcus gattii . Retroviral infection was not identified as a predisposing condition and was not correlated with either species of Cryptococcus or physical findings . Most cats had signs of nasal cavity infection, which was typically localised for a substantial period before invasion of adjacent structures or dissemination . Male and female dogs were affected equally . A marked preponderance of young, large breed dogs was noted . Border Collies, Boxers, Dalmatians, Dobermann Pinschers, Great Danes and German Shepherds were over-represented . Cryptococcus species involved was not affected by place of domicile . Although nasal cavity involvement was important, the canine cohort had a greater propensity to develop secondary central nervous system involvement and disseminated disease than feline cases . There were no clinical findings in either cats or dogs which could be reliably used to distinguish disease caused by Cryptococcus neoformans variety grubii from disease caused by Cryptococcus gattii . Both Cryptococcus species appear to be primary pathogens of cats and dogs, with the upper respiratory tract presumed to be the predominant primary site of inoculation in most but not all cases.

Biomedica, 2004 Sep, 24(3), 324 - 31
{Cryptococcus spp . DNA extraction from environmental samples}; Castaneda A et al.; The genus Cryptococcus encompasses 38 species, but only 3 are associated with disease in humans and animals, Cryptococcus laurentii, Cryptococcus albidus and Cryptococcus neoformans . The last one is the most frequently reported . The disease is acquired by the inhalation of infectious propagules present in the environment . The habitat has been established using extraction techniques with buffer supplemented with antibiotics and plating in selective media . The aim of this work was to evaluate several DNA extraction techniques for Cryptocococus spp . from environmental samples . The control isolates were C . neoformans, C . albidus, C . laurentii and Paracoccidiodes brasiliensis . We also used vermiculita and soil samples contaminated with different yeast concentrations (10 to 10(6) cells/g) and samples naturally contaminated with C . neoformans . DNA was extracted with physical and chemical methods and with a commercial kit, and the DNA was purified with agarose blocks and silica columns . For the PCR amplification we used the CN4-CN5 primers, which are specific for C . neoformans . Only the commercial kit allowed DNA extraction and amplification from contaminated soil samples up to a concentration of 10 cells/g and from one sample naturally colonized . With this work we extracted and amplified DNA from Cryptococcus spp . from environmental samples with appropriate PCR specificity, it will be a tool to establish the ecological areas of C . neoformans in our country.

J Feline Med Surg, 2004 Dec, 6(6), 383 - 90
Infections and some other conditions affecting the skin and subcutis of the naso-ocular region of cats--clinical experience 1987-2003; Malik R et al.; Infections of the skin or subcutis of the naso-ocular region develop through two mechanisms . Cases with lesions but without concomitant signs of nasal disease probably result from cat scratch injuries . Under certain circumstances, such lacerations result in the introduction of saprophytic microorganisms in such large numbers that host defence mechanisms are overwhelmed . This results in localised, variably invasive, disease in an otherwise immunocompetent host . An unpredictable range of organisms can give rise to such infections including a variety of fungal and bacterial genera . Causal organisms will likely vary from one geography to another as a result of differences in soil type and related environmental factors . Accordingly, procurement of appropriate tissue specimens for culture and susceptibility testing is essential to guide therapy, as these cases require medical and sometimes surgical intervention in order to effect a favourable outcome . In contrast, patients with naso-ocular lesions and concurrent signs of nasal disease have a different pathogenesis . Primary infection of the sinonasal region likely results from the inhalation of infectious propagules, with the infection subsequently penetrating overlying bones to invade the subcutaneous space . These lesions are typically the result of cryptococcosis or aspergillosis and must be distinguished from invasive nasal malignancies . An approach to the investigation and treatment of these patients is presented together with photographs of representative cases.

Clin Infect Dis, 2004 Oct 15, 39 Suppl 4, S224 - 7
Combination therapy for invasive mycoses: evaluation of past clinical trial designs; Sobel JD; BACKGROUND: Despite the availability of new antifungals, single-agent therapy frequently falls short of high cure rates . Combination therapy offers potentially higher cure rates, especially for resistant organisms . In vitro studies and experimental animal models have provided conflicting data . METHODS: Retrospective, randomized, controlled clinical studies were reviewed . RESULTS: Results indicate a clear advantage for polyene and flucytosine combination therapy in cryptococcal meningitis and a possible advantage for combination amphotericin B and fluconazole for candidemia . Unfortunately, the few studies published have been flawed by design problems that have compromised the determination of outcome . Study review allows investigators the opportunity to design future studies to ensure optimal evaluation of efficacy . CONCLUSION: Combination antifungal therapy is advantageous in cryptococcal diseases and may have a role in the treatment of invasive candidiasis . The greatest potential exists for combination therapy against aspergillosis and resistant fungi in patients with refractory mycotic disease who experience failure of monotherapy.

Clin Infect Dis, 2004 Oct 15, 39 Suppl 4, S218 - 23
Design of efficacy trials of cytokines in combination with antifungal drugs; Kullberg BJ et al.; Resolution of invasive fungal infections is often dependent on recovery from an immunocompromised state, which indicates that host defense mechanisms are extremely important in the clearance of fungal pathogens . Immunotherapy aimed at enhancement of host defense mechanisms may improve clinical outcome of invasive mycoses . The design of trials of immunotherapy against fungal pathogens requires profound knowledge of the host defense mechanisms that are involved in invasive fungal infections . Prospective phase II studies with recombinant granulocyte colony-stimulating factor and interferon-gamma have been done . Recombinant interferon-gamma is a candidate for phase III trials of adjunctive immunotherapy for cryptococcal meningitis, invasive aspergillosis, and candidemia, but the proper design of future trials will be crucial to establish whether immunotherapy is of clinical value in the treatment of invasive fungal infections.

Rev Iberoam Micol, 2004 Jun, 21(2), 87 - 9
A new caffeic acid minimal synthetic medium for the rapid identification of Cryptococcus neoformans isolates; Vidotto V et al.; Melanin production is one of the most important criteria for rapid identification of Cryptococcus neoformans . Most of the media described in the literature for identifying C . neoformans are very complex; they contain many organic or inorganic compounds and are difficult to prepare and store . The new minimal synthetic caffeic acid medium described in this paper is simpler to prepare, convenient and constitutes an interesting new medium for the rapid identification of C . neoformans isolates.

Rev Iberoam Micol, 1997 Dec, 14(4), 181 - 3
First report of isolation of Cryptococcus neoformans var . neoformans from avian excreta in Kathmandu, Nepal; Pal M; This paper delineates the first report on the saprophytic distribution of Cryptococcus neoformans var . neoformans in the city of Kathmandu, Nepal . Twenty-eight samples of old and dry pigeon droppings collected from different sites in Kathmandu were investigated for the presence of C . neoformans by employing a dilution technique . The organism was isolated from seven (25%) of the specimens, representing four of the ten collection sites . All of the isolates were recovered on Pal's medium (sunflower seed agar) by observing light to dark brown coloured colonies of C . neoformans . However, no isolation could be acheived on Sabouraud medium as all the plates were badly contaminated with rapidly growing moulds . The microscopic morphology of the cultures in PHOL stain revealed circular to val, single or budding yeast cells with thin capsules . Detailed typing of all environmental strains indicated that they belonged to the variety neoformans and a mating type of Filobasidiella neoformans . The results of this study demonstrated that Pal's medium is an excellent differential medium for the screening of environmental specimens and C . neoformans var neoformans is prevalent in the environment of Kathmandu.

Rev Iberoam Micol, 1997 Dec, 14(4), 160 - 3
Study of some lymphocyte subset counts and cytokine levels in cryptococcosis associated with AIDS; Arechavala A et al.; Approximately 100 new cases per year of cryptococcosis in HIV+ patients are observed in Muniz Hospital, 35% of them suffer a fatal outcome within the first four weeks after diagnosis in spite of treatment . Apparently there is not a useful parameter that allows a clear prediction of this early fatal outcome of the disease . The aim of this study is to determine some cytokine levels and several lymphocyte subpopulations counts in order to correlate these results with the evolution of the disease . Forty HIV+ patients suffering culture confirmed cryptococcosis were enrolled in this study, 8 HIV+ patients without cryptococcosis and 8 healthy individuals with negative serology for HIV were included as controls . The following determinations were done in all cases: CD3+, CD4+, CD8+, CD16+CD3-, CD19+ cell counts, IL-1; IL-12, TNFalpha in serum and TNFalpha in CSF . Ten cases with cryptococcosis and AIDS were controlled three months after treatment . The average of CD4+ and NK cell counts in patients before treatment were 22/mul and 90/mul respectively; IL-1 levels were higher in the patients than in the healthy control group, conversely IL-12 levels did not show significant differences in the three studied groups . Serum concentrations of TNFalpha were higher in patients than in the control group and were not modified after treatment, conversely antifungal medication diminished IL-1 concentration and remarkably increased NK cell counts . At the same time antigen levels in serum and CSF decreased . The results obtained seem to show that the immunological alterations observed in these patients are those characteristically exhibited in severe HIV disease and that some parameters such as CD8+ cell counts lower than 200/mul, less than 50 CD4+/mul, more than 50 pg/ml of TNF-alpha and serum capsular antigen titer higher than 1:5000 seem to predict a rapidly fatal course of infection.

PLoS Biol . 2004 Dec;2(12):e384 . Epub 2004 Nov 09.
Convergent evolution of chromosomal sex-determining regions in the animal and fungal kingdoms; Fraser JA et al.; Sexual identity is governed by sex chromosomes in plants and animals, and by mating type (MAT) loci in fungi . Comparative analysis of the MAT locus from a species cluster of the human fungal pathogen Cryptococcus revealed sequential evolutionary events that fashioned this large, highly unusual region . We hypothesize that MAT evolved via four main steps, beginning with acquisition of genes into two unlinked sex-determining regions, forming independent gene clusters that then fused via chromosomal translocation . A transitional tripolar intermediate state then converted to a bipolar system via gene conversion or recombination between the linked and unlinked sex-determining regions . MAT was subsequently subjected to intra- and interallelic gene conversion and inversions that suppress recombination . These events resemble those that shaped mammalian sex chromosomes, illustrating convergent evolution in sex-determining structures in the animal and fungal kingdoms.

Int J STD AIDS, 2004 Nov, 15(11), 758 - 61
Opportunistic infections in hospitalized HIV-infected adults in Ho Chi Minh City, Vietnam: a cross-sectional study; Louie JK et al.; The HIV epidemic is emerging rapidly in Vietnam . We studied the prevalence of opportunistic infections by performing clinical and microbiological investigations in 100 hospitalized HIV-infected adults in Ho Cho Minh City, Vietnam . The median CD4 count was 20 cells/mm(3) and in-hospital mortality was 28% . The most frequent diagnoses were oral candidiasis (54), tuberculosis (37), wasting syndrome (34), lower respiratory tract infection (13), cryptococcosis (9), and penicilliosis (7) . Bacterial (other than tuberculosis) and parasitic infections were uncommon . Regional differences should be considered when deciding which diagnostic procedures and prophylactic measures to implement . In Vietnam, routine mycobacterial blood cultures do not provide greater yield than chest radiography and sputum and lymph node aspirate smears . Prophylactic trimethoprim/sulphamethoxazole against Pneumocystis jiroveci pneumonia may confer little benefit, and high rates of isoniazid resistance may affect the efficacy and feasibility of tuberculosis chemoprophylaxis . However, the usefulness of itraconazole prophylaxis for cryptococcosis and penicilliosis merits further consideration.

Eur J Neurol, 2004 Nov, 11(11), 755 - 9
Neurological complications in AIDS patients: the 1-year retrospective study in Chiang Mai University, Thailand; Subsai K et al.; The first case of AIDS patient in the northern part of Thailand was reported in 1987 (Vithayasai et al., 1996), marking the outbreak of an epidemic . In our experience, the neurological involvement in AIDS patients seems to have changed in pattern and incidence during the last 8 years . We have conducted a retrospective study to review the incidence of AIDS-defining diseases in the patients admitted to Chiang Mai University Hospital, Thailand during the period September 2001 to August 2002 . There were 155 AIDS patients admitted during this specified period, 118 of which were male and 37 female, aged between 16 and 60 . The incidence of neurological complications was 50.3 per 100 person-years, in which central nervous system involvement account for 46.5 per 100 person-years and peripheral nervous system involvement account for 3.8 per 100 person-years . The incidence of cryptococcal meningitis appeared to be decreasing since 1994 whereas the incidence of cerebral toxoplasmosis appeared to be increasing . The incidence of cryptococcal meningitis, cerebral toxoplasmosis and cytomegalovirus (CMV) infection was 18.0, 14.8 and 7.0 per 100 person-years, respectively . Other common non-neurological AIDS-defining illnesses in northern Thailand include pulmonary tuberculosis (15.4 per 100 person-years), extra-pulmonary tuberculosis (9.6 per 100 person-years), and disseminated penicilliosis (12.2 per 100 person-years) . In this way and summarizing, in northern Thailand, the three most common neurological involvements before the era of highly active anti-retroviral therapy are cryptococcal meningitis, cerebral toxoplasmosis and CMV infection . The incidence of cryptococcal meningitis appeared to be decreasing whereas the incidence of cerebral toxoplasmosis seemed to be increasing.

Bioorg Med Chem, 2004 Dec 1, 12(23), 6255 - 69
Carboxylic acid and phosphate ester derivatives of fluconazole: synthesis and antifungal activities; Nam NH et al.; Two classes of fluconazole derivatives, (a) carboxylic acid esters and (b) fatty alcohol and carbohydrate phosphate esters, were synthesized and evaluated in vitro against Cryptococcus neoformans, Candida albicans, and Aspergillus niger . All carboxylic acid ester derivatives of fluconazole (1a-l), such as O-2-bromooctanoylfluconazole (1g, MIC=111 microg/mL) and O-11-bromoundecanoylfluconazole (1j, MIC=198 microg/mL), exhibited higher antifungal activity than fluconazole (MIC > or = 4444 microg/mL) against C . albicans ATCC 14053 in SDB medium . Several fatty alcohol phosphate triester derivatives of fluconazole, such as 2a, 2b, 2f, 2g, and 2h, exhibited enhanced antifungal activities against C . albicans and/or A . niger compared to fluconazole in SDB medium . For example, 2-cyanoethyl-omega-undecylenyl fluconazole phosphate (2b) with MIC value of 122 microg/mL had at least 36 times greater antifungal activity than fluconazole against C . albicans in SDB medium . Methyl-undecanyl fluconazole phosphate (2f) with a MIC value of 190 microg/mL was at least 3-fold more potent than fluconazole against A . niger ATCC 16404 . All compounds had higher estimated lipophilicity and dermal permeability than those for fluconazole . These results demonstrate the potential of these antifungal agents for further development as sustained-release topical antifungal chemotherapeutic agents.

Mycopathologia, 2004 Aug, 158(2), 195 - 9
Cryptococcus neoformans varieties from material under the canopies of eucalyptus trees and pigeon dropping samples from four major cities in Jordan; Hamasha AM et al.; To our best knowledge, any study related to the ecological distribution of Cryptococcus neoformans in Jordan does not exist in the medical literature . In order to determine the environmental occurrence of both varieties of Cryptococcus neoformans in Jordan, pigeon droppings and material under the canopies of eucalyptus trees were collected from four major cities of this country . For the isolation of Cryptococcus neoformans variety gattii from environmental sources, 500 samples of the mixed soil debris, including tree materials, under the eucalyptus trees from cities of Amman, Irbid, Jerash, and Ajlun were collected . Also, 509 samples of pigeon droppings were collected from the same cities for the isolation of Cryptococcus neoformans variety neoformans . After inoculating the samples onto modified Staib agar medium in Petri dishes, a total of 336 melanoid yeast colonies were picked up during screening process . At the end of serial mycological studies, none of these isolates was identified as Cryptococcus neoformans, but all were Cryptococcus species other than C . neoformans . For determining the exact status, more extensive environmental studies need to be done in the future.

Mycopathologia, 2004 Aug, 158(2), 165 - 71
Voriconazole proves effective in long-term treatment of a cerebral cryptococcoma in a chronic nephropathic HIV-negative patient, after fluconazole failure; Sabbatani S et al.; Although being a rare occurrence, brain cryptococcoma may represent an emerging issue, because of its relationship with a broadening range of risk factors, including malignancies, neutropenia, end-organ failure, bone marrow and solid-organ transplantation, and multiple underlying causes of primary-secondary immunodeficiency . A cerebral cryptococcoma in a chronic nephropathic HIV-negative subject with homocystinuria, completely cured with neurosurgery and voriconazole after fluconazole failure, is described.

J Food Prot, 2004 Oct, 67(10), 2132 - 42
Survey of yeasts for antagonistic activity against Salmonella Poona in cantaloupe juice and wounds in rinds coinfected with phytopathogenic molds; Richards GM et al.; Application of yeasts as biocontrol agents to prevent mold decay of fruits and vegetables has been described . We examined 10 yeasts for potential antagonistic activity against survival and growth of Salmonella Poona in cantaloupe juice and decay by Cladosporium cladosporioides and Geotrichum candidum in wounds on cantaloupe rind . Cantaloupe juice was inoculated using five schemes: Salmonella Poona only (1.10 log CFU/ml), high (3.93 to 5.21 log CFU/ml) or low populations (1.79 to 3.26 log CFU/ml) of yeasts only, and Salmonella Poona combined with high or low populations of yeasts . High initial populations of Debaryomyces hansenii, Pichia guilliermondii, and Pseudozyma sp . were antagonistic to Salmonella Poona in cantaloupe juice stored at 20 degrees C for 48 h . Wounds in cantaloupe rinds were inoculated with yeast and mold or yeast, mold, and Salmonella Poona, and cantaloupes were stored at 4 degrees C for 14 days or 20 degrees C for 7 days . The pH of rind tissue inoculated with C . cladosporioides and yeasts increased significantly (P < or = 0.05) at 20 degrees C . Wounds that were inoculated with P . guilliermondii, together with C . cladosporioides or G . candidum, did not show mold growth at 4 and 20 degrees C . Populations of Salmonella Poona (6.40, 7.26, and 7.98 log CFU per sample) were lower in wounds coinoculated with G . candidum and three of the test yeasts (D . hansenii, P . guilliermondii, and Cryptococcus albidus, respectively) compared to coinoculation with G . candidum or the other seven yeasts . Candida oleophila and Rhodotorula glutinis showed the most promise in reducing the population of Salmonella Poona in wounds in rinds of cantaloupes coinoculated with G . candidum and stored at 4 degrees C.

Jpn J Infect Dis, 2004 Oct, 57(5), 203 - 5
Cryptococcuria as a manifestation of disseminated cryptococcosis and isolated urinary tract infection; Kiertiburanakul S et al.; Fungal infection of the genitourinary system is a relatively uncommon presentation . Cryptococcuria has rarely been recognized in clinical practice . Patients with positive urine culture for Cryptococcus neoformans from 1992 to 2003 were retrospectively reviewed . Sixteen patients were identified . Nine (56%) patients were male, with a mean age of 44 +/- 21 (range, 16-88) years . Fifteen (94%) patients had underlying conditions such as HIV infection, diabetes mellitus, hypertension, and/or systemic lupus erythematosus . Thirteen (81%) patients had cryptococcuria as a manifestation of disseminated cryptococcosis, and the rest had only isolated cryptococcuria . Urinary analysis revealed proteinuria (75%), pyuria (31%), and budding yeast (13%) . Nine (56%) patients received antifungal therapy . Other patients were misdiagnosed or died before treatment . The mortality rate was 64% . In conclusion, cryptococcuria is not extremely rare and can present as a manifestation of disseminated cryptococcosis or isolated urinary tract infection.

Exp Mol Pathol, 2004 Dec, 77(3), 246 - 8
Fatal fat embolism following amphotericin B lipid complex injection; Tolentino LF et al.; A case of amphotericin B lipid complex induced fatal fat embolism is described . A 41-year-old Caucasian man with AIDS was undergoing treatment for cryptococcal meningitis with amphotericin B . His course was complicated by renal failure necessitating a change in therapy to amphotericin B lipid complex (Abelcet) . At approximately 48 h, the patient developed tachycardia, tachypnea, respiratory failure, decline in hematocrit, thrombocytopenia, and alteration in mental status . Autopsy findings included fat emboli involving heart, lungs, kidney, and brain . To our knowledge, this is the first case report of a fatal fat embolism caused by intravenous liposome drug delivery.

J Alzheimers Dis, 2004 Oct, 6(5), 503 - 8
Reversible dementia: a case of cryptococcal meningitis masquerading as Alzheimer's disease; Ala TA et al.; A 70-year-old man presented to us in 1994 with a three-year history of worsening dementia . With the exceptions of a Mini-Mental State exam score of 20 and an inability to tandem walk, his physical and neurological examinations were normal . His past medical history revealed that in 1992 he had been evaluated at another institution for memory impairment and bifrontal headaches . A spinal tap had been done in 1992 showing elevated protein, reduced glucose, and a pleocytosis; his CSF fungal culture and cryptococcal antigen test were negative . He subsequently was lost to follow-up, and although his headaches had resolved, his mental status had continued to worsen . In 1994 his CSF cryptococcal antigen was positive, and his CSF fungal culture grew C . neoformans . He gradually improved with treatment for cryptococcal meningitis (CM) . With the exception of mild memory impairment, in 2003 he and his family thought that his mental status had returned to normal . This case emphasizes that: 1) CM should always be kept in the differential diagnosis of dementia; 2) CM may be extremely insidious and difficult to diagnose; and 3) if one is to rule out unequivocally all possible reversible causes of dementia, one should perform a spinal tap.

Mycoses, 2004 Oct, 47(9-10), 431 - 4
Detection of Cryptococcus neoformans var . grubii in honeybee (Apis mellifera) colonies; Ergin C et al.; The plant flora has an important role in the ecology of Cryptococcus neoformans . It is estimated that the environmental spreading and contamination of human beings with this yeast occurs via contaminated particles of plants . Cultivation of canopy parts of plants in selective media is the most widely used isolation method of this yeast . Cryptococcus neoformans var . grubii was isolated from honeybee colonies in Eucalyptus forests but was not isolated from the places where this flora did not exist . Our results indicate that the occurrence of C . neoformans in honeybee colonies during the flowering season of Eucalyptus spp . trees can be an important bioindicator for environmental yeast presence . The screening of honeybee colonies is a practical and a rapid method for the monitoring of the C . neoformans presence in flowering plants.

Curr Genet . 2004 Oct 21; {Epub ahead of print}
A Sch9 protein kinase homologue controlling virulence independently of the cAMP pathway in Cryptococcus neoformans; Wang P et al.; The polysaccharide capsule is one of the established virulence factors in Cryptococcus neoformans that provides a barrier against the host-mediated immune response . Mutation of the gene encoding the Saccharomyces cerevisiae Sch9 protein kinase homologue resulted in cells with enlarged capsules in C . neoformans . Capsule production was abrogated in sch9 pka1 double mutants, indicating that protein kinase A (PKA) signaling is still necessary for capsule formation in sch9 mutants . The sch9 mutant also exhibited increased thermal tolerance, a phenotype similar to sch9 mutant strains of S . cerevisiae . In addition, the sch9 mutant was attenuated in mating and the highly encapsulated cells were attenuated in virulence, in contrast to the pkr1 mutant, lacking the regulatory subunit of protein kinase A, that produced similarly enlarged capsules yet was increased in virulence . Interestingly, the virulence for the sch9 mutant strain could be restored by introduction of a pkr1 mutation; and the sch9 pkr1 mutant strain was dramatically increased in size and capsule thickness, suggesting that Sch9 and PKA function via different targets involved in virulence . Our findings support a model in which Sch9 modulates capsule formation and contributes to the virulence of C . neoformans both independently of and in conjunction with the cAMP-PKA pathway.

Microbiol Immunol, 2004, 48(10), 747 - 53
Comparative evaluation of standard dilution method and commercial kit for frozen plate antifungal susceptibility testing of yeasts using 200 clinical isolates; Makimura K et al.; A comparative evaluation of standard microdilution methods and a commercial kit for frozen plate antifungal susceptibility testing of yeasts was performed using amphotericin B, flucytosine, fluconazole, miconazole, and itraconazole on 200 yeast isolates . The isolates included 100 strains of Candida albicans, eight of C . tropicalis, twelve of C . parapsilosis, eight of C . glabrata, five of Cryptococcus neoformans, thirteen of Trichosporon asahii, and 54 other strains of seven other species of ascomycotic yeasts . Microdilution testing was performed according to the standard method for antifungal susceptibility testing published by the Japanese Society for Medical Mycology (JSMM), which are a modification of the method developed by the National Committee for Clinical Laboratory Standards (NCCLS) M27-P . The commercial kit was prepared according to the manufacturer's instructions . The degree of agreement within +/-1 dilution for 200 clinical isolates against five antifungal agents was excellent with values for amphotericin B, flucytosine, fluconazole, miconazole, and itraconazole of 100%, 99.0%, 97.5%, 97.0%, and 97.0%, respectively . Overall, the frozen plate antifungal susceptibility testing kit provided convenient and reproducible results comparable to those obtained with the JSMM standard method.

Infect Immun, 2004 Nov, 72(11), 6367 - 72
Glucuronoxylomannan, the major capsular polysaccharide of Cryptococcus neoformans, inhibits the progression of group B streptococcal arthritis; Tissi L et al.; Glucuronoxylomannan (GXM), the principal constituent of the Cryptococcus neoformans capsule, modulates the inflammatory response of human monocytes in vitro . Here we examine the efficacy of GXM as a novel anti-inflammatory compound for use against experimental septic arthritis . Arthritis was induced in mice by the intravenous injection of 8 x 10(6) CFU of type IV group B streptococcus (GBS) . GXM was administered intravenously in different doses (50, 100, or 200 microg/mouse) 1 day before and 1 day after bacterial inoculation . GXM treatment markedly decreased the incidence and severity of articular lesions . Histological findings showed limited periarticular inflammation in the joints of GXM-treated mice, confirming the clinical observations . The amelioration of arthritis was associated with a significant reduction in the local production of interleukin-6 (IL-6), IL-1beta, macrophage inflammatory protein 1alpha (MIP-1alpha), and MIP-2 and an increase in systemic IL-10 levels . Moreover, peritoneal macrophages derived from GXM-treated mice and stimulated in vitro with heat-inactivated GBS showed a similar pattern of cytokine production . The present study provides evidence for the modulation of the inflammatory response by GXM in vivo and suggests a potential therapeutic use for this compound in pathologies involving inflammatory processes.

Pharmacol Res, 2004 Dec, 50(6), 605 - 10
Interaction between saquinavir and antimycotic drugs on C . albicans and C . neoformans strains; Casolari C et al.; Candidiasis and cryptococcosis are the most common fungal diseases among patients suffering from HIV infection . In the present work we assess whether the combined therapies, proteinase inhibitors and antimycotic drugs, could modify the therapeutic effect of antimycotics . An in vitro study to evaluate the antifungal effect of saquinavir and antimycotic drugs combination on yeast growth was performed . Strains of C . albicans and C . neoformans from HIV-seropositive patients were used . Susceptibility tests of yeasts to amphotericin B, 5-fluorocytosine, miconazole and fluconazole, singly and in combination with saquinavir, were performed in two different media . In the combinations the antimycotic agents and saquinavir were tested at sub-inhibitory concentrations: 0.1-10 microg ml(-1) and 12.50 microg ml(-1), respectively . The fractionary inhibitory concentration (FIC) index was also calculated . The results show that the interaction between saquinavir and all the antimycotic drugs never resulted in antagonism . Fluconazole acts in more synergistic way, no matter which medium is used . The combined therapy miconazole/saquinavir results in synergism, especially in Sabouraud . The total absence of antagonism and the presence of synergism suggest that a combined therapy could be proposed in the treatment of HIV-seropositive patients to reduce side effects, thanks to the use of lower doses of antimycotic drugs.

Immunology, 2004 Nov, 113(3), 392 - 400
Immunosuppression, interleukin-10 synthesis and apoptosis are induced in rats inoculated with Cryptococcus neoformans glucuronoxylomannan; Chiapello LS et al.; Glucuronoxylomannan (GXM) is the major Cryptococcus neoformans capsular polysaccharide and represents the main virulence factor of this fungus . In in vitro studies we have demonstrated previously that this acidic and high-molecular-weight polysaccharide suppresses lymphoproliferation, modulates cytokine production and promotes apoptosis in spleen mononuclear (Spm) cells from rats . In this study we demonstrate that these phenomena also occur in vivo after the intracardiac inoculation of GXM into normal Wistar rats . The results of this study show suppression of the proliferative response Spm cells to concanavalin A (Con A) or heat-killed C . neoformans (HKCn) in the first 2 weeks after polysaccharide administration . In addition, increased levels of interleukin (IL)-10 were produced by Con A-stimulated Spm cells, coinciding with immunohistochemical GXM detection in the white pulp of spleen . In particular, high production of IL-10 with diminution of IL-2, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha synthesis were detected 14 days after GXM administration . In situ cell death detection by TdT-mediated biotin-dUTP nick-end labelling (TUNEL) reaction in sections of spleen, lung and liver demonstrates apoptosis in tissues with deposits of GXM . These data demonstrate the in vivo ability of GXM to modify cytokine synthesis by Spm cells and to promote host cell apoptosis.

Nihon Kokyuki Gakkai Zasshi, 2004 Sep, 42(9), 854 - 8
{A case of pulmonary aspergillosis with bone invasion and destruction in an AIDS patient}; Hara H et al.; A 59-year-old HIV-infected man who had been treated for pulmonary cryptococcosis in another hospital was suffering from pulmonary tuberculosis . He was admitted to our hospital for treatment of tuberculosis . The chest radiograph on admission showed a large cavity in the left lower lung field . Chest CT showed a mass like a fungus ball in the cavity . Pulmonary aspergillosis was diagnosed from the sputum mycology and serum immunoprecipitins . During the treatment of pulmonary tuberculosis and cryptococcosis, thickening of the wall of the cavity was seen together with adjacent bone destruction . In an autopsy, Aspergillus spp . Were found to have invaded the bone tissue and caused bone destruction . Pulmonary aspergillosis in an AIDS patient is infrequent, and is rare in additional association with bone invasion and destruction.

Cutis, 2004 Sep, 74(3), 165 - 70
Cryptococcal cellulitis in a patient on prednisone monotherapy for myasthenia gravis; Lafleur L et al.; The development of cutaneous cryptococcosis is extremely rare in the human immunodeficiency virus-negative population . The cutaneous manifestations vary greatly, with the rarest presenting as cellulitis . We report a unique case of a 64-year-old woman who had been on prednisone monotherapy for the treatment of myasthenia gravis and subsequently developed cryptococcal cellulitis . This case provides an opportunity to discuss the importance of including cryptococcal cellulitis in the differential diagnosis of cellulitis in a patient on low-dose prednisone who is not responding to empiric antibiotic therapy . Early recognition is crucial as cutaneous manifestations are usually the first sign of disseminated cryptococcosis.

Rev Saude Publica, 2004 Oct, 38(5), 657 - 63 Epub 2004 Oct 18.
{Epidemiologic surveillance and evaluation of meningitis hospital care}; Escosteguy CC et al.; OBJECTIVE: To analyze the clinical-epidemiological profile and in-hospital death predictors of infectious meningitis patients admitted to a public hospital . METHODS: There were studied 694 cases of meningitis notified and investigated by a public hospital's epidemiology service from 1986 to 2002, using the National Information System of Notifiable Diseases (SINAN) as part of the local routine of epidemiologic surveillance . Statistics analysis included multivariate logistic regression . RESULTS: The most frequent etiologies were: cryptococcal (12.3%; case-fatality =37.7%); meningococcal (8.7%; fatality =13.3%); pneumococcal (7.2%; fatality =46%); tuberculous (6.1%; fatality =40.5%); staphylococcal (5.2%; fatality =38.9%), viral (5.5%; fatality =7.9%); Haemophilus (2.9%; fatality =20%) . The proportion of cases of non-specified etiology was 38.8% (fatality =36%) and 17.3% were associated to HIV infection . It was found that 27.1% were nosocomial meningitis and 9.2% of the surviving cases had sequelae . The logistic regression model identified the following death predictors of infectious meningitis: etiology (reference: viral category) -- tuberculous, cryptococcal, staphylococcal, meningococcal, non-specified, other Gram-negative, Candida and pneumococcal; HIV co-infection; coma . Fever, vomiting and neck stiffness were associated to a lower odds of death . CONCLUSIONS: The high proportion of non-specified etiology and high case-fatality may reflect problems in the hospital care process and/or case selection . The epidemiologic surveillance system operating at the hospital level was able to feedback the services with clinical indicators . The use of SINAN at the local level was considered useful and pertinent.

Zhonghua Bing Li Xue Za Zhi, 2004 Oct, 33(5), 424 - 8
{Pathological diagnosis and ultrastructure features of primary pulmonary cryptococcosis: a study of 27 cases.}; Yi XH et al.; OBJECTIVE: To investigate diagnostic histopathology and ultrastructure features of primary pulmonary cryptococcosis (PC) . METHODS: Clinical data and pathologic findings of 27 cases of PC were retrospectively reviewed, light and electron microscopic evaluations and histochemistry stain studies were performed . RESULTS: The specimens consisted of 2 fine-needle aspiration lung biopsies and 25 cases of open lung biopsies . Cryptococcosis granuloma formation was identifiable by histopathological examination in 25 of 27 cases, with gum-like lesion and fungi in the remaining 2 cases . The detection rates of cryptococcus neoformans (CN) by mucicarmine (MC), periodic acid-Schiff (PAS), alcian blue (AB) and Grocott methenamine-silver (GMS) were 87.0% (20/23), 100% (27/27), 66.7% (18/27), and 100% (23/23) respectively . Under the electron microscope, most CN had a simple structure with a few organelles . The capsule was seen in all organisms . A percentage of the organisms showed nuclei, nucleoli, mitochondria and vacuoles . The detection rate of CN by EM was 91.7% (11/12) . CONCLUSIONS: The clinical manifestation and imaging of PC are nonspecific for PC . Lung biopsy is the major diagnostic modality . The detection rate by electron microscopy was quite high . Therefore, a correct diagnosis of pulmonary cyrptococcosis should rely on the combination of histopathological evaluation, histochemistry staining and/or electron microscopic examination.

Pathol Res Pract, 2004, 200(9), 591 - 7
Bone marrow pathology in AIDS; Calore EE et al.; Bone marrow morphology is frequently abnormal in patients with AIDS . In this study, we reviewed 97 bone marrow biopsies of AIDS patients performed between 1998 and 2000 in the Emilio Ribas Institute of Infectology, which is the reference department for HIV . Specific diagnoses were performed in 33 cases . Fungi were observed in eight cases . Five of them were Histoplasma capsulatum, two were Cryptococcus neoformans, and one probably Candida albicans . Acid-fast bacilli were observed in 12 bone marrow biopsies, three of which were diagnosed to have no mycobacteriosis clinically . Foci of necrosis with clusters of macrophages without any well-formed granuloma were observed in nine cases and well-formed granuloma in three cases . Lymphomatous infiltration was observed in four cases of non-Hodgkin's lymphoma and in two Hodgkin's diseases (mixed cellularity) . Extensive necrosis of bone marrow was observed in one case of Burkitt's lymphoma . In conclusion, bone marrow biopsy should be performed to elucidate the etiology of cytopenias, secondary infections, and fever of undetermined origin in AIDS patients.

Rev Iberoam Micol, 2001 Dec, 18(4), 200 - 1
Cutaneous cryptococcosis due to Cryptococcus neoformans var . gattii; Severo LC et al.; A case of cutaneous cryptococcosis due to Cryptococcus neoformans var . gattii in an immunocompetent host is presented . In addition a review of the literature on this subject was carried out and a brief comment made on occurrence of the variety gattii in Brazil.

Rev Iberoam Micol, 2001 Dec, 18(4), 174 - 9
Chromosome length polymorphism in Cryptococcus neoformans clinical and enviromental isolates; Garcia-Bermejo MJ et al.; A protocol for intact DNA preparation from the basidiomycetous yeast Cryptococcus neoformans has been developed and applied to karyotyping C . neoformans isolates displaying different degrees of capsule formation . A total of 46 strains have been analyzed: 23 (50%) isolated from environmental samples (pigeon droppings), all of them belonging to C . neoformans var . neoformans; and 23 (50%) from clinical samples (human and veterinarian) including 10 isolates of C . neoformans var . neoformans and 13 isolates of C . neoformans var . gattii . Our results showed a global genome size ranging from 14.2 to 20.9 Mb for variety neoformans and from 7.9 to 16.8 Mb for variety gattii . The karyotype diversity was very high for variety neoformans (29 different patterns for the 33 analyzed strains) and lower for variety gattii (six different patterns for 13 strains) . No grouping among variety neoformans strains from the same origin was found indicating very high genome diversity for this variety, irrespectively of the origin of the strains.

Clin Infect Dis, 2004 Nov 1, 39(9), e83 - 7 Epub 2004 Oct 05.
Two patients with cryptococcal meningitis and idiopathic CD4 lymphopenia: defective cytokine production and reversal by recombinant interferon- gamma therapy; Netea MG et al.; BACKGROUND: Although Cryptococcus neoformans is a fungal pathogen that causes human disease predominantly in the immunocompromised host, severe cryptococcal infections are occasionally encountered in apparently immunocompetent individuals . Activation of cellular immunity by proinflammatory cytokines plays a central role in anticryptococcal defense . METHODS: We describe 2 patients with severe cryptococcal meningitis who appeared to have idiopathic CD4 lymphopenia . For these patients and for 4 healthy volunteers, ex vivo stimulation of whole blood with microbial stimuli was used to investigate putative defects in cytokine production capacity . RESULTS: Assessment of the cytokine released from the 2 patients with CD4 lymphopenia revealed a defective production of the proinflammatory cytokines interferon (IFN)- gamma and tumor necrosis factor (TNF) but not of the anti-inflammatory cytokine interleukin-10 (IL-10) . One patient with disease progression despite receipt of antifungal treatment was administered immunotherapy with recombinant IFN- gamma . Administration of recombinant IFN- gamma resulted in both restoration of immunological parameters and a sustained clinical recovery . CONCLUSIONS: Refractory meningitis may be due to defective TNF and IFN- gamma production, and IFN- gamma treatment may be useful in patients with an impaired cellular immune response and refractory cryptococcal meningitis.

Syst Appl Microbiol, 2004 Sep, 27(5), 558 - 64
Two new ballistoconidium-forming yeast species, Bullera melastomae and Bullera formosana, found in Taiwan; Nakase T et al.; Two yeast strains, the cells of which contained xylose and Q-10 as the major ubiquinone, were isolated from a plant leaf collected in Taiwan . These yeasts were found to represent two new species of the genus Bullera in the Hymenomycetes . Identification was based on the sequence analysis of the 18S rDNA, the internal transcribed spacer (ITS) regions and the D1/D2 domain of 26S rDNA . The yeasts are named Bullera melastomae sp . nov . and Bullera formosana sp . nov . In the phylogenetic trees based on 18S rDNA and D1/D2 domain of 26S rDNA sequences, these two species constitute a cluster connected with Dioszegia cluster in the Cryptococcus luteolus lineage.

Beijing Da Xue Xue Bao, 2004 Oct, 36(5), 536 - 9
{Application of molecular biology techniques in the identification of pathogenic fungi and the diagnosis of fungal infection.}; Li RY et al.; With the increasing incidence and mortality of fungal infection, the requirements for strict diagnostic approaches became a very urgent issue . Because of the traditional detective techniques, such as culture, gave poor diagnostic outcomes, the molecular biological techniques are expected to develop the potential diagnostic approaches . During the past decades, we have carried out serial studies on the molecular properties of pathogenic fungi, and we would like to review as following . Firstly, we applied several molecular tools in classification and identification of pathogenic fungi . We performed random amplification of polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP) and other techniques in studying the typing, to classify and identify the properties of Dermatophytes, Candida spp., Cryptococcus neoformans, Dematiaceous fungi, and Aspergillus spp . Interestingly, we found the same T . rubrum strain might infect different sites of the host, while a site-specificity displayed in T . mentagrophytes . This finding indicated the genetic discrepancies among the fungi . Beside, we also found that the E . dermatitis strains with different virulences possessed some discrepancies at gene level . We then developed a PCR-based molecular procedure to identify the novel species in Exophiala spp . As the applicable strategy, we also investigated the rDNA sequence properties in several fungi . And as a result, we submitted for the first time to GenBank the complete sequence of Aspergillus fumigatus rDNA/ITSI/ITSII, which provided the basis for designing the species-specific probes and for its further clinical applications . Secondly, we have tried to develop the molecular diagnostic approaches based on our DNA sequence data which were used for identification studies previously . By analyzing the DNA sequence of Aspergillus fumigatus rDNA/ITSI/ITSII, we developed a nested PCR method to detect Aspergillus fumigatus genes . Our preliminary results indicated that this PCR-based molecular approach has great importance in the diagnosis of invasive aspergillosis . We also designed the species-specific probes and then established several in situ hybridization procedures . We found these hybridization methods could get the positive rate up to 81% (13/16), which suggests that these methods have potential diagnostic value for invasive candidiasis and aspergillosis . Based on our experiences, we would conclude that the molecular biological techniques possess great value to investigate the biological properties of pathogenic fungi, and we are looking forward to see more and more molecular tools will be used in the pathogenic mechanisms of fungal infections and antifungal activity studies.

Br J Ophthalmol, 2004 Nov, 88(11), 1455 - 9
Neuro-ophthalmological disorders in HIV infected subjects with neurological manifestations; Mwanza JC et al.; AIMS: To determine the frequency and features of neuro-ophthalmological manifestations in neurologically symptomatic HIV infected patients and to assess whether or not the visual evoked potential (VEP) features in these patients differ from those of neurologically asymptomatic HIV infected patients . METHODS: Neuro-ophthalmological evaluation was performed in 166 neurologically symptomatic confirmed HIV positive patients, of whom 75 with normal ophthalmological examination were further studied by means of VEPs . The VEPs values were compared to those obtained from 53 other confirmed HIV positive subjects with neither ophthalmological nor neurological manifestations, who served as a comparison group and to the references values of our laboratory . RESULTS: An abnormal neuro-ophthalmological examination was noted in 99/166 patients (60%) . Eye movement disorders were present in 99 patients (51%) . Visual field defects were detected in 39% of the patients . Optic neuropathy was noted in 31%, papilloedema in 27% and ocular motor nerve palsies in 26% of the patients . Toxoplasmosis and cryptococcosis were the most frequent associated pathologies, though in some patients the HIV itself was the presumed cause . VEPs were abnormal in 57% and 42% of patients with and without neurological manifestations, respectively . Compared to asymptomatic patients, symptomatic patients had a significantly increased mean latency; however, both groups had significant increase in mean latency compared to reference values . CONCLUSION: Neuro-ophthalmological manifestations are common in neurologically symptomatic HIV infected patients . Subclinical dysfunction in the visual pathways is a common phenomenon in both HIV infected patients with and without neurological symptoms, but neurologically symptomatic patients seem to have more damage in their visual pathways.

Rev Iberoam Micol, 2001 Sep, 18(3), 131 - 2
Experimental infection of almond trees seedlings (Terminalia catappa) with an environmental isolate of Cryptococcus neoformans var . gattii, serotype C; Huerfano S et al.; Recently, our laboratory reported the isolation of Cryptococcus neoformans var . gattii, serotype C for the first time from almond trees (Terminalia catappa) detritus . The aim of the present study was to establish the survival of C . neoformans in almond trees seedlings . Thirty seedlings were infected in the stems and samples were taken and processed at different times and by different techniques . No morphological alterations (macro or microscopic) were observed in the infected plants . However, C . neoformans was found to be viable for at least 100 days after infection . These data constitute our first approach towards the understanding of the yeast interactions with a host-plant.

Rev Iberoam Micol, 2001 Sep, 18(3), 128 - 30
Varietes and serotypes of Cryptococcus neoformans clinical isolates in Colombia; Ordonez N et al.; The aim of this study was to contribute to the knowledge of the geographic distribution of Cryptococcus neoformans varieties, in Colombian patients, and to determine the mating types of such varieties . A total of 370 clinical isolates were studied . C . neoformans var . neoformans was identified in 95.2% of them . C . neoformans var . gattii in 4.8%, 4.5% being serotype B and 0.3%, serotype C . Fifty-five of the 74 (74%) isolates studied were mating type "alpha", all of them C . neoformans var . neoformans . Serotype C had been previously reported only once in South American countries.

Rev Iberoam Micol, 2001 Sep, 18(3), 105 - 8
{Evaluation of Chromalbicans Agar for presumptive identification of Candida albicans.}; Carrillo-Munoz AJ et al.; The utility of Chromalbicans Agar (Biolife Italiana, Milano, Italy) was evaluated with 723 clinical isolates and type culture collection strains from different genera including Candida, Cryptococcus, Pichia, Rhodotorula, Saccharomyces, Trichosporon y Zygosaccharomyces . Presumptive identification was confirmed by germ tube test and carbohydrate assimilation on API-ATB ID 32C (bioMerieux, France) . Growth on Chromalbicans Agar was very useful for the presumptive identification of C . albicans isolates, and sensitivity and specificity values were significantly high (>97%), since a very low number of isolates were found to be false negative or false positive.

Rev Iberoam Micol, 2001 Sep, 18(3), 99 - 104
{Epidemiological study of cryptococcosis in Spain: first results.}; Colom MF et al.; The study constitutes an approach to the knowledge of the epidemiology of cryptococosis in Spain . For detection of cases 167 Spanish hospitals were contacted . All cases included were accompanied by the correspondent isolate of Cryptococcus neoformans, together with clinical, demographic and mycological data . Results obtained from January 1998 to end of December 1999 are analysed and presented here . Fifty-six Spanish hospitals reported 58 cases of cryptococcosis; only 43 of them were adequately documented and accompanied by the clinical isolate . The results showed a higher incidence in males (88.4%) than in females (11.6%); being most frequently affected those between 30 and 40 years old (48.8%) . The 84.6% (33) corresponded to new cases and 15.4% (6) to relapses of the disease . The HIV infection was the most frequent risk factor reported (86%) and, for 29.7% (11) of them, cryptococcosis was the AIDS defining disease . For the diagnosis, CSF analysis showed the best results (India ink; culture and antigen detection) . All strains collected (100%) corresponded to C . neoformans variety neoformans . Serotypes distribution was 45.5% for serotype A and 22.7% for each of serotypes D and AD.

Rev Iberoam Micol, 2001 Jun, 18(2), 65 - 9
{Optimization of the conditions for RAPD-PCR of Candida spp . and Cryptococcus spp.}; Sansinforiano ME et al.; In this study, we determined the optimal RAPD amplification conditions to obtain genetic molecular markers for the rapid and accurate identification of Cryptococcus spp . and Candida spp . The following parameters are modified: template DNA, DNA polymerase, magnesium cloride and primer concentration; denaturation, annealing and extension time, temperature of annealing and thermal cycles . After the optimization, reliable and reproducible RAPD patterns are obtained.

Mycopathologia, 2004 Jul, 158(1), 43 - 7
The first isolation of Cryptococcus neoformans from Eucalyptus trees in South Aegean and Mediterranean Regions of Anatolia in Turkey despite Taurus Mountains alkalinity; Ergin C et al.; Eucalyptus trees are widespread in subtropical parts of Turkey that have alkaline environments due to the soil structure of Taurus Mountains . In this study, the existence of Cryptococcus neoformans in eucalyptus trees in the South Aegean and Mediterranean Regions of Anatolia, Turkey, was screened between March 1998 and September 2002 . Only one strain of Cryptococcus neoformans var . grubii (Serotype A) was isolated from 1175 eucalyptus samples including debris and flowers in culture by Guizotia abyssinica agar . The environmental niche of the isolate was Eucalyptus camaldulensis Dehn in the Gokova Region, in the western part of the Taurus Mountains . In this study, the existence of Cryptococcus neoformans was shown in the eucalyptus flora of Turkey despite the alkaline soil condition.

Mycopathologia, 2004 Jul, 158(1), 25 - 31
Serotype and PCR-fingerprints of clinical and environmental isolates of Cryptococcus neoformans in Chiang Mai, Thailand; Sriburee P et al.; From May 1999 to April 2000, serotypes of clinical and environmental isolates of Cryptococcus neoformans were studied in Chiang Mai province, northern Thailand . Three hundred and eighty-five environmental samples, of which 100 were dove droppings, 55 pigeon droppings and 230 eucalyptus flower, were collected from 7 Amphoes in Chiang Mai . C . neoformans was isolated from 45 of 100 (45.0%) dove dropping samples, 9 of 55 (16.4%) pigeon dropping samples and 2 of 230 (0.9%) eucalyptus flower samples . Serotypes of 56 environmental isolates and 75 clinical isolates of C . neoformans,obtained during the same period, were determined by the slide agglutination test . Fifty-six environmental and 74 clinical isolates belonged to C . neoformans serotype A (C . neoformans var . grubii), and only one clinical isolate belonged to C . neoformans serotype AD . The isolation of C . neoformans var . grubii from eucalyptus flower samples suggests contamination of avian droppings . PCR-fingerprinting, using (GACA)4 as a primer, discriminated 131 clinical and environmental isolates into 2 groups (group I and II) . Seventy-five clinical and 54 environmental isolates were of group I, which had two major specific bands of approximately 1,250 and 960 base pairs . Two environmental isolates, one from pigeon excreta and the other from a eucalyptus flower sample were of group II, which had two major specific bands of approximately 1,180 and 500 base pairs.

Clin Infect Dis, 2004 Oct 15, 39(8), e78 - 82 Epub 2004 Sep 27.
Paradoxical inflammatory reaction during treatment of Cryptococcus neoformans var . gattii meningitis in an HIV-seronegative woman; Einsiedel L et al.; A human immunodeficiency virus (HIV)-seronegative woman was admitted to the hospital with postpartum onset Cryptococcus neoformans var . gattii meningitis and markedly increased intracranial pressure . A poor initial response to antifungal therapy was followed, 2 months after hospital admission, by severe acute meningeal and cerebral inflammation and clearance of yeast cells from cerebrospinal fluid . This first reported case of paradoxical inflammatory reaction to C . neoformans illustrates important aspects of the host-pathogen interaction and highlights possible effects of immunomodulatory therapies.

Genome Res, 2004 Nov, 14(11), 2330 - 5 Epub 2004 Oct 12.
Gene prediction and verification in a compact genome with numerous small introns; Tenney AE et al.; The genomes of clusters of related eukaryotes are now being sequenced at an increasing rate, creating a need for accurate, low-cost annotation of exon-intron structures . In this paper, we demonstrate that reverse transcription-polymerase chain reaction (RT-PCR) and direct sequencing based on predicted gene structures satisfy this need, at least for single-celled eukaryotes . The TWINSCAN gene prediction algorithm was adapted for the fungal pathogen Cryptococcus neoformans by using a precise model of intron lengths in combination with ungapped alignments between the genome sequences of the two closely related Cryptococcus varieties . This approach resulted in approximately 60% of known genes being predicted exactly right at every coding base and splice site . When previously unannotated TWINSCAN predictions were tested by RT-PCR and direct sequencing, 75% of targets spanning two predicted introns were amplified and produced high-quality sequence . When targets spanning the complete predicted open reading frame were tested, 72% of them amplified and produced high-quality sequence . We conclude that sequencing a small number of expressed sequence tags (ESTs) to provide training data, running TWINSCAN on an entire genome, and then performing RT-PCR and direct sequencing on all of its predictions would be a cost-effective method for obtaining an experimentally verified genome annotation.

Biochem Biophys Res Commun, 2004 Nov 12, 324(2), 719 - 28
Cloning and characterization of the lanosterol 14alpha-demethylase (ERG11) gene in Cryptococcus neoformans; Revankar SG et al.; The ergosterol pathway in fungal pathogens is an attractive antimicrobial target because it is unique from the major sterol (cholesterol) producing pathway in humans . Lanosterol 14alpha-demethylase is the target for a major class of antifungals, the azoles . In this study we have isolated the gene for this enzyme from Cryptococcus neoformans . The gene, ERG11, was recovered using degenerate PCR with primers designed with a novel algorithm called CODEHOP . Sequence analysis of Erg11p identified a highly conserved region typical of the cytochrome P450 class of mono-oxygenases . The gene was present in single copy in the genome and mapped to one end of the largest chromosome . Comparison of the protein sequence to a number of major human fungal pathogen Erg11p homologs revealed that the C . neoformans protein was highly conserved, and most closely related to the Erg11p homologs from other basidiomycetes . Functional studies demonstrated that the gene could complement a Saccharomyces cerevisiae erg11 mutant, which confirmed the identity of the C . neoformans gene.

Diagn Microbiol Infect Dis, 2004 Oct, 50(2), 113 - 7
Clinical evaluation of a dried commercially prepared microdilution panel for antifungal susceptibility testing of five antifungal agents against Candida spp . and Cryptococcus neoformans; Pfaller MA et al.; A commercially prepared dried-broth microdilution panel (Sensititre, TREK Diagnostic Systems, Cleveland, OH) was compared with a reference frozen-broth microdilution panel for antifungal susceptibility testing of 728 clinical isolates of Candida spp . and 98 clinical isolates of Cryptococcus neoformans . The antifungal agents tested were amphotericin B, fluconazole, 5-fluorocytosine (5FC), itraconazole, and voriconazole . Microdilution testing was performed according to NCCLS recommendations . Minimum inhibitory concentration (MIC) endpoints were read visually after 48 hours of incubation (72 hours for C . neoformans) and were assessed independently for each microdilution panel . Discrepancies among MIC endpoints of no more than 2 log(2) dilutions were used to calculate the percentage of agreement . Overall levels of agreement between the study and reference panels were 98% for Candida spp . and 93% for C . neoformans . The agreement for each antifungal agent ranged from 96.6% for voriconazole to 99.4% for amphotericin B . The TREK dried microdilution panel appears to be a viable alternative to frozen-broth microdilution panels for testing of Candida spp . and C . neoformans.

Med Mycol, 2004 Aug, 42(4), 333 - 9
Invasive fungal infections in Chile: a multicenter study of fungal prevalence and susceptibility during a 1-year period; Silva V et al.; 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.

Med Mycol, 2004 Aug, 42(4), 325 - 31
Oxy2 as a transcriptional activator gene for copper uptake in Cryptococcus neoformans; Nyhus K et al.; Cryptococcus neoformans is subject to oxidative attack by host immune cells; consequently, oxidant-resistant mechanisms may be important in pathogenesis . Mutations at the OXY2 locus confer decreased laccase and increased sensitivity to hyperbaric oxygen in the background of the oxyl mutation, but, alone, do not confer sensitivity to oxidants . Because metal deficiency can potentiate or ameliorate sensitivity to oxidants, and because the melanin-synthesizing laccase contains copper, we investigated copper acquisition in an oxy2 mutant . We found that its external Cu/Fe reductase activity was lower than that of wild type, and although copper deprivation induced the reductase in the wild type, it did not do so in oxy2 . Oxy2 is sensitive to copper chelation but resistant to high copper, suggesting that copper transport is decreased . The strain expresses large amounts of alternate oxidase in response to Cu-chelation, perhaps in response to defective, Cu-deprived cytochrome oxidase, and is resistant to the oxidant, plumbagin, under this condition, perhaps due to the high alternate oxidase . These phenotypes are similar to those of the mac1- mutant of Saccharomyces cerevisiae and the melanin-deficient grisea mutant of Podospora anserina, in which homologous transcriptional activators for the reductase and copper transporter genes are mutated . They constitute physiologic evidence that oxy2 is mutated in a homologous copper-related transcriptional activator of C . neoformans.

J Clin Microbiol, 2004 Oct, 42(10), 4873 - 5
Antigen assay with the potential to aid in diagnosis of blastomycosis; Durkin M et al.; We report results of an immunoassay for Blastomyces dermatitidis antigenuria . Sensitivity was 92.9%, and specificity was 79.3% . Cross-reactions occurred in 96.3% of patients with histoplasmosis, 100% of patients with paracoccidioidomycosis, 70% of patients with penicilliosis marneffei, 2.9% of patients with cryptococcosis, and 1.1% of patients with aspergillosis . Reproducibility was 96.3% . These findings support a potential role for antigen testing in blastomycosis.

J Clin Microbiol, 2004 Oct, 42(10), 4815 - 7
In vitro antifungal susceptibility of Cryptococcus gattii; Trilles L et al.; We have determined the in vitro susceptibilities of 57 strains of Cryptococcus gattii to nine antifungal agents and have compared the MICs for these strains with those for C . neoformans . MICs were determined by a microdilution reference method . Albaconazole and ravuconazole (MICs of 0.04 and 0.05 microg/ml, respectively) showed the best activities . Micafungin showed no activity (MIC of >128 microg/ml) . In general, C . gattii was less susceptible than C . neoformans to all drugs tested, with the exception of amphotericin B and flucytosine.

Eukaryot Cell, 2004 Oct, 3(5), 1249 - 60
Identification of Cryptococcus neoformans temperature-regulated genes with a genomic-DNA microarray; Kraus PR et al.; The ability to survive and proliferate at 37 degrees C is an essential virulence attribute of pathogenic microorganisms . A partial-genome microarray was used to profile gene expression in the human-pathogenic fungus Cryptococcus neoformans during growth at 37 degrees C . Genes with orthologs involved in stress responses were induced during growth at 37 degrees C, suggesting that a conserved transcriptional program is used by C . neoformans to alter gene expression during stressful conditions . A gene encoding the transcription factor homolog Mga2 was induced at 37 degrees C and found to be important for high-temperature growth . Genes encoding fatty acid biosynthetic enzymes were identified as potential targets of Mga2, suggesting that membrane remodeling is an important component of adaptation to high growth temperatures . mga2Delta mutants were extremely sensitive to the ergosterol synthesis inhibitor fluconazole, indicating a coordination of the synthesis of membrane component precursors . Unexpectedly, genes involved in amino acid and pyrimidine biosynthesis were repressed at 37 degrees C, but components of these pathways were found to be required for high-temperature growth . Our findings demonstrate the utility of even partial-genome microarrays for delineating regulatory cascades that contribute to microbial pathogenesis.

Eukaryot Cell, 2004 Oct, 3(5), 1088 - 100
Introns and splicing elements of five diverse fungi; Kupfer DM et al.; Genomic sequences and expressed sequence tag data for a diverse group of fungi (Saccharomyces cerevisiae, Schizosaccharomyces pombe, Aspergillus nidulans, Neurospora crassa, and Cryptococcus neoformans) provided the opportunity to accurately characterize conserved intronic elements . An examination of large intron data sets revealed that fungal introns in general are short, that 98% or more of them belong to the canonical splice site (ss) class (5'GU...AG3'), and that they have polypyrimidine tracts predominantly in the region between the 5' ss and the branch point . Information content is high in the 5' ss, branch site, and 3' ss regions of the introns but low in the exon regions adjacent to the introns in the fungi examined . The two yeasts have broader intron length ranges and correspondingly higher intron information content than the other fungi . Generally, as intron length increases in the fungi, so does intron information content . Homologs of U2AF spliceosomal proteins were found in all species except for S . cerevisiae, suggesting a nonconventional role for U2AF in the absence of canonical polypyrimidine tracts in the majority of introns . Our observations imply that splicing in fungi may be different from that in vertebrates and may require additional proteins that interact with polypyrimidine tracts upstream of the branch point . Theoretical protein homologs for Nam8p and TIA-1, two proteins that require U-rich regions upstream of the branch point to function, were found . There appear to be sufficient differences between S . cerevisiae and S . pombe introns and the introns of two filamentous members of the Ascomycota and one member of the Basidiomycota to warrant the development of new model organisms for studying the splicing mechanisms of fungi.

Mol Microbiol, 2004 Oct, 54(2), 407 - 19
Cryptococcus neoformans Kin1 protein kinase homologue, identified through a Caenorhabditis elegans screen, promotes virulence in mammals; Mylonakis E et al.; Cryptococcal infections are a global cause of significant morbidity and mortality . Recent studies support the hypothesis that virulence of Cryptococcus neoformans may have evolved via survival selection in environmental hosts, such as amoebae and free-living nematodes . We used killing of the nematode Caenorhabditis elegans by C . neoformans as an assay to screen a library of random C . neoformans insertion mutants . Of 350 mutants tested, seven were identified with attenuated virulence that persisted after crossing the mutation back into a wild-type strain . Genetic analysis of one strain revealed an insertion in a gene homologous to Saccharomyces cerevisiae KIN1, which encodes a serine/threonine protein kinase . C . neoformans kin1 mutants exhibited significant defects in virulence in murine inhalation and haematogenous infection models and displayed increased binding to alveolar and peritoneal macrophages . The kin1 mutant phenotypes were complemented by the wild-type KIN1 gene . These findings show that the C . neoformans Kin1 kinase homologue is required for full virulence in disparate hosts and that C . elegans can be used as a substitute host to identify novel factors involved in fungal pathogenesis in mammals.

AIDS Patient Care STDS, 1998 Sep, 12(9), 681 - 6
Candidal meningitis in HIV-infected patients; Casado JL et al.; Candida meningitis is considered a rare event in HIV-infected patients, and little is known about risk factors, clinical presentation, therapy of choice, or outcome in this population . In a review of 14 cases, we observed a low frequency of the disease, a strong association to other well-known risk factors for systematic candidiasis, such as intravenous drug use, and a chronic course and clinical features that mimic those of cryptococcal or tuberculous meningitis . Although the role of fluconazole treatment remains to be defined, the combination of amphotericin B with flucytosine offers a survival rate similar to non-HIV-infected patients . In addition, the use of suppressive therapy with fluconazole for a prolonged period after clinical improvement seems recommendable.

Microbiol Res, 2004, 159(3), 263 - 75
Molecular identification of yeasts from soils of the alluvial forest national park along the river Danube downstream of Vienna, Austria ("Nationalpark Donauauen"); Wuczkowski M et al.; We analysed the diversity of yeasts from different soils in a river-floodplain landscape at the river Danube downstream of Vienna, Austria ("Nationalpark Donauauen") . 136 strains were isolated, identification of species was done with molecular methods . Partial sequencing of the 26S rRNA gene resulted in 36 different sequences, they could be assigned to 16 genera, apart from two sequence types (from three isolates), which were not clearly assigned to any genus . 18 species were identified and confirmed by means of PCR fingerprinting . The most frequently isolated genus was Cryptococcus (61 isolates and 12 sequence types) . Basidiomycetes dominated with about 60% above the members of the Ascomycetes . About half the yeasts was isolated from the litter, the quantity decreased with soil depth.

Med Oncol, 2004, 21(3), 263 - 7
Primary non-Hodgkin's lymphoma of the bone: a single institution experience; Deshmukh C et al.; Primary non-Hodgkin's lymphoma of the bone is an unusual entity . Twenty-five patients with diffuse large cell lymphoma of the bone were registered at the Tata Memorial Hospital (TMH) from August, 1991, to May, 2002 . Pain at the local site and soft tissue swelling were the commonest symptoms . Involvement of the bones in the lower half of the body was more frequent than the bones in the upper half . Osteolytic lesions and an associated soft tissue mass were the common radiological findings . Nineteen patients received CHOP chemotherapy and five received COP chemotherapy . Twenty-three patients received involved field radiotherapy . The overall response to therapy was 96% . On follow-up, two patients had a nodal relapse . One patient died of progressive disease, and one patient died of cryptococcal meningitis . There were no deaths due to treatment-related toxicity . The mean progression free survival was 9.39 yr and the overall survival was 11.66 yr . The median overall survival has not been reached . At last follow-up, 21 patients were being following up at TMH and are free of disease . CONCLUSION: Primary bone lymphoma is a malignancy that is highly curable with a combination of chemotherapy and radiotherapy.

Rev Iberoam Micol, 2003 Dec, 20(4), 121 - 36
In vitro antifungal activities of anidulafungin and micafungin, licensed agents and the investigational triazole posaconazole as determined by NCCLS methods for 12,052 fungal isolates: review of the literature; Espinel-Ingroff A; The echinocandins anidulafungin and micafungin and the triazole posaconazole are currently undergoing phase III clinical trials . Caspofungin and voriconazole have recently been licensed for the treatment of aspergillosis (both agents), other less common mould (voriconazole) and candidal (caspofungin) infections . This review summarizes the published in vitro data obtained by NCCLS or NCCLS modified methods on the in vitro fungistatic and fungicidal activities of these five agents for yeasts and moulds in comparison to the established agents, amphotericin B, fluconazole, itraconazole, and flucytosine . Among the yeasts, the echinocandins have less activity for Candida parapsilosis and Candida guilliermondii, no activity for Cryptococcus neoformans and Trichosporon spp., but good fungistatic and fungicidal activity in vivo and in vitro for most of the other Candida spp.; this fungicidal activity has been reported by minimum fungicidal concentrations (MFCs) or time kill curve results . The new triazoles exhibit good fungistatic activity (but not fungicidal) for most Candida spp., C . neoformans, and Trichosporon spp . For the Aspergillus spp . evaluated, the echinocandins have similar or better fungistatic activity than those of amphotericin B and the triazoles, but fungicidal activity has been demonstrated only with amphotericin B and the triazoles, with the exception of fluconazole . Most studies showed posaconazole and voriconazole minimum inhibitory concentrations (MICs) ranging from 0.25 to 8 microg/ml for non-solani Fusarium spp., while MIC and minimum effective concentration (MEC) endpoints of the echinocandins were >8 microg/ml . The fungistatic activity of the triazoles is also superior to that of the echinocandins for most of the dimorphic fungi and the Zygomycetes . However, micafungin has activity for the mould phase of most dimorphic fungi, but not for the parasitic or yeast phase of Paracoccidioides brasiliensis . The echinocandins appear to have variable and species dependent fungistatic activity for the dematiaceous fungi, but all agents have poor or no activity against most isolates of Scedosporium prolificans . Only amphotericin B exhibit good fungistatic activity against the Zygomycetes . The combination of caspofungin with some triazoles, amphotericin B or liposomal amphotericin B has been synergistic in vitro, in animal models and in patients . Breakpoints are not available for any mould and antifungal agent combination . In vitro/in vivo correlations should aid in the interpretation of these results, but standard testing conditions are needed for the echinocandins, especially for mould testing, to obtain reliable results.

FEMS Microbiol Lett, 2004 Oct 1, 239(1), 87 - 93
Characterisation and expression of phospholipases B from the opportunistic fungus Aspergillus fumigatus; Shen DK et al.; The phospholipase B family (PLB) are enzymes sharing phospholipase (PL), lysophospholipase (LPL) and lysophospholipase-transacylase (LPTA) activities . They have been shown to be important virulence factors in several human fungal pathogens including Candida albicans and Cryptococcus neoformans . Aspergillus fumigatus, a human opportunistic fungal pathogen leading to a high rate of mortality in immunosuppressed patients is known to possess an extracellular phospholipase B activity . In this paper, we report the molecular characterisation of three PLB genes from A . fumigatus (afplb) using degenerate primers in PCR amplification and data from the A . fumigatus genome project . They are expressed at 37 degrees C, and two of them (afplb1 and afplb3) are induced by lecithin . They encode proteins of 633, 588 and 630 amino acids, respectively, presenting together a T-Coffee score of 81 . They also possess the amino acid triad responsible for enzymatic activity in the mammalian cytosolic PLA2 and other fungal PLBs . AfPLB1 and afPLB3 are secreted with a cleaved signal peptide . The complete cDNA sequences were obtained by RACE-PCR for the two secreted afPLBs and probably account for the extracellular phospholipase activity previously reported in the culture media of A . fumigatus.

FEMS Yeast Res, 2004 Sep, 4(8), 865 - 77
Application and evaluation of denaturing gradient gel electrophoresis to analyse the yeast ecology of wine grapes; Prakitchaiwattana CJ et al.; The performance of denaturing gradient gel electrophoresis (DGGE) for analysing yeasts associated with wine grapes was compared with cultural isolation on malt extract agar (MEA) . After optimisation of PCR and electrophoretic conditions, the lower limit of yeast detection by PCR-DGGE was 10(2) cfuml(-1), although this value was affected by culture age and the relative populations of the species in mixed culture . In mixed yeast populations, PCR-DGGE detected species present at 10-100-fold less than other species but not when the ratio exceeded 100-fold . Aureobasidium pullulans was the main species isolated from immature, mature, and both damaged and undamaged grapes . It was not detected by PCR-DGGE when present at populations less than 10(3) cfug(-1) . When approaching maturity, damaged grapes gave a predominance of Metschnikowia and Hanseniaspora species (10(5)-10(7) cfug(-1)), all detectable using PCR-DGGE . However, various species of Rhodotorula, Rhodosporidium and Cryptococcus were not detected by this method, even when populations were as high as 10(4) cfug(-1) . PCR -DGGE was less sensitive than culture on MEA for determining the yeast ecology of grapes and could not reliably detect species present at populations less than 10(4) cfug(-1) . However, this method detected a greater diversity of species than agar plating.

FEMS Yeast Res, 2004 Sep, 4(8), 765 - 71
Cryptococcus neoformans capsule biosynthesis and regulation; Janbon G; The capsule is certainly the most prominent virulence factor in Cryptococcus neoformans: acapsular strains are avirulent, and capsular polysaccharides have a deleterious effect on the immune system . Until very recently, very few genes involved in capsule biosynthesis had been identified - and this despite the existence of a detailed body of work concerning the capsule's composition, structure and their regulation by environmental factors . The tremendous development of experimental tools and techniques suited to the study of C . neoformans biology together with the sequencing of three complete genomes have, over the last three years, enabled the identification of a number of proteins which participate directly in biosynthesis of the capsule or which regulate its size . Even though this knowledge is still preliminary, it gives us a clearer picture of the various events needed for biosynthesis of this fascinating structure.

Cornea, 2004 Oct, 23(7), 730 - 1
Scleral ulceration caused by Cryptococcus albidus in a patient with acquired immune deficiency syndrome; Garelick JM et al.; PURPOSE: To describe a case of a scleral ulceration caused by Cryptococcus albidus in a patient with acquired immune deficiency syndrome (AIDS) . DESIGN: Interventional case report . A 16-year-old girl with AIDS was admitted to the hospital with a 1-week history of cough, fever, and sudden onset of a painless scleral lesion on her left eye . Culture of the lesion revealed the fungus Crypotococcus albidus . RESULTS: The patient was treated with topical amphotericin B and systemic itraconazole, and she recovered fully over 4 weeks without sequelae . CONCLUSION: Cryptococcus albidus rarely causes disease in humans . It should be considered as a potential cause of ocular and systemic disease in patients with AIDS.

Bioorg Med Chem, 2004 Oct 15, 12(20), 5465 - 83
Synthesis, antimicrobial activity and molecular modeling studies of halogenated 4-{1H-imidazol-1-yl(phenyl)methyl}-1,5-diphenyl-1H-pyrazoles; Menozzi G et al.; During the course of our studies in the azole antifungals area, we synthesized a number of 1,5-disubstituted 4-{1H-imidazol-1-yl(phenyl)methyl}-1H-pyrazoles, analogues of bifonazole . 1,5-Diphenyl-1H-pyrazole 3 showed weak antimycotic and antibacterial activities in vitro against Candida albicans, Cryptococcus neoformans and Staphylococcus aureus . In order to increase these properties, given that the halo substitution was found to be capable of enhancing antifungal effects, we prepared a series of fluoro and chloro derivatives of 3 . The microbiological evaluation carried out on newly synthesized compounds included in vitro assays for antifungal, antibacterial and antimycobacterial activities . Among the tested compounds, some dichloro and trichloro-derivatives showed interesting antimicrobial properties . In particular, compounds 10j,k,l produced inhibitory effects against pathogen representatives of yeast (C . albicans, C . neoformans) and Gram positive bacteria (S . aureus) similar or superior to those of bifonazole . In addition, their activity against Mycobacterium tuberculosis was superior to that of clotrimazole and econazole, which were used as reference drugs . The replacement, in these compounds, of chlorine with fluorine atoms led to inactive derivatives . Docking studies were carried out on the most active compounds, in order to rationalize the pharmacological results.

Mol Microbiol, 2004 Sep, 53(5), 1385 - 96
Regulatory roles for the homeodomain and C2H2 zinc finger regions of Cryptococcus neoformans Ste12alphap; Chang YC et al.; The STE12alpha gene of Cryptococcus neoformans encodes a protein containing both homeodomain and zinc finger regions . As homeodomains and zinc finger regions are important domains for the function of many transcription factors, we used site-specific mutagenesis to delineate the roles of these two domains . The homeodomain and zinc finger regions are each important for the function of Ste12alphap . DNA binding ability, mating frequency, and haploid fruiting capability were reduced in strains with mutations in the homeodomain, whereas virulence and capsule size in the mouse brain were increased . In contrast, mutations in the zinc fingers region resulted in decreased virulence, reduced capsule size in the mouse brain and decreased gene expression of capsule associated genes . In addition, phospholipase activity was increased in the zinc finger mutants . Taken together, most of the phenotypes previously observed in the ste12alpha deletion strains were reproduced in these two types of mutants . However, unlike mutations in the homeodomain/zinc finger region, complete deletion of STE12alpha caused a severe reduction in virulence and a decrease in phospholipase activity . These data suggest that region(s) other than the homeodomain and zinc finger regions of Ste12alphap contribute to the variable influences on the different phenotypes observed in C . neoformans .

J Biol Chem, 2004 Dec 3, 279(49), 51669 - 76 Epub 2004 Dec 3.
UDP-glucose dehydrogenase plays multiple roles in the biology of the pathogenic fungus Cryptococcus neoformans; Griffith CL et al.; Cryptococcus neoformans is a pathogenic fungus surrounded by an elaborate polysaccharide capsule that is strictly required for its virulence in humans and other mammals . Nearly half of the sugar residues in the capsule are derived from UDP-glucuronic acid or its metabolites . To examine the role of these nucleotide sugars in C . neoformans, the gene encoding UDP-glucose dehydrogenase was disrupted . Mass spectrometry analysis of nucleotide sugar pools showed that the resulting mutant lacked both UDP-glucuronic acid and its downstream product, UDP-xylose, thus confirming the effect of the knockout and indicating that an alternate pathway for UDP-glucuronic acid production was not used . The mutant was dramatically affected by the lack of specific sugar donors, demonstrating altered cell integrity, temperature sensitivity, lack of growth in an animal model of cryptococcosis, and morphological defects . Additionally, the polysaccharide capsule could not be detected on the mutant cells, although the possibility remains that abbreviated forms of capsule components are made, possibly without proper surface display . The capsule defect is largely independent of the other observed changes, as cells that are acapsular because of mutations in other genes show lack of virulence but do not exhibit alterations in cell integrity, temperature sensitivity, or cellular morphology . All of the observed alterations were reversed by correction of the gene disruption.

FEMS Yeast Res, 2004 Oct, 5(1), 1 - 10
Role of laccase in the biology and virulence of Cryptococcus neoformans; Zhu X et al.; Laccase is an important virulence factor for the human pathogen, Cryptococcus neoformans . In this review, we examine the structural, biological and genetic features of the enzyme and its role in the pathogenesis of cryptococcosis . Laccase is expressed in C . neoformans as a cell wall enzyme that possesses a broad spectrum of activity oxidizing both polyphenolic compounds and iron . Two paralogs, CNLAC1 and CNLAC2, are present in the fungus, of which the first one expresses the dominant enzyme activity under glucose starvation conditions . Regulation of the enzyme is in response to various environmental signals including nutrient starvation, the presence of multivalent cations and temperature stress, and is mediated through multiple signal transduction pathways . Study of the function and regulation of this important virulence factor has led to further understanding of mechanisms of fungal pathogenesis and the regulation of stress response in the host cell environment.

Eur J Clin Invest, 2004 Sep, 34(9), 631 - 40
Cryptococcal capsular glucuronoxylomannan reduces ischaemia-related neutrophil influx; Ellerbroek PM et al.; BACKGROUND: The capsular polysaccharide glucuronoxylomannan (GXM) of Cryptococcus neoformans interferes with the chemotaxis and transendothelial migration of neutrophils . Intravenous administration of purified GXM has been shown to reduce the influx of inflammatory cells in an animal model of bacterial infection . Here we show that isolated GXM can also interfere with neutrophil migration in a model of inflammation not related to infection . We assessed the effects of intravenous GXM on neutrophil infiltration in a rat model of myocardial ischaemia, where neutrophil infiltration has been shown to contribute to postischaemic reperfusion injury . MATERIALS AND METHODS: Rats were subjected to coronary artery ligation followed by a 3-h reperfusion period . Myeloperoxidase-activity was measured in the ischaemic tissues as a marker of neutrophil infiltration . RESULTS: Intravenous administration of GXM markedly reduced the influx of neutrophils in the ischaemic myocardium as measured by a 65% reduction of tissue MPO activity . This reduction of MPO activity was clearly correlated to the serum concentration of GXM . As complement activation by GXM was minimal at the doses applied in vivo, it is unlikely that generation of chemotactic C5a in the circulation by GXM caused the observed reduction in leucocyte migration . CONCLUSION: Purified cryptococcal GXM has the ability to reduce neutrophil influx even outside the scope of infection.

J Antimicrob Chemother, 2004 Nov, 54(5), 854 - 69 Epub 2004 Sep 16.
Combinations of antifungal agents in therapy-what value are they?
Cuenca-Estrella M.
Concurrent or sequential antifungal treatment for invasive mycoses has been typically considered as an option to improve results of monotherapy . However, data on the efficacy of combination therapy are sparse and consist largely of results from studies in vitro and experimental animal models . These studies have yielded controversial results depending on the criteria used to evaluate the antifungal interaction . Several combinations that showed synergy in vitro failed to do so in animal models . Overall, apart from cryptococcal infections, combined antifungal therapy is not significantly better than monotherapy in terms of clinical efficacy . It is questionable whether combination therapy should be used in most cases as there is a lack of evidence from well-designed clinical trials . However, combination therapy could be an alternative to monotherapy for patients with invasive infections that are difficult to treat, such as those due to multi-resistant species and for those who fail to respond to standard treatment.

Vaccine, 2004 Sep 28, 22(29-30), 4062 - 8
Efficacy of immune sera from human immunoglobulin transgenic mice immunized with a peptide mimotope of Cryptococcus neoformans glucuronoxylomannan; Maitta RW et al.; The efficacy of antibody mediated immunity against Cryptococcus neoformans has not been established experimentally for human antibodies . Our group has previously shown that immunization with a conjugate consisting of a peptide mimotope of the C . neoformans capsular polysaccharide glucuronoxylomannan (GXM), P13, and diphtheria toxoid (P13-DT) prolonged survival of transgenic mice with human immunoglobulin loci, XenoMouse mice, which were challenged with a lethal dose of C . neoformans . In the study reported herein, we determined the efficacy of human antibodies in the sera of immunized XenoMouse mice against C . neoformans in passive transfer experiments in naive BALB/c mice . Survival studies were performed with sera from XenoMouse mice expressing human IgG2/kappa (G2/k mice) or IgG4/kappa (G4/k mice) that had been immunized with P13-tetanus toxoid (TT)/Alhydrogel with or without CpG, and G2/k mice that had been immunized with P13-DT/Alhydrogel/CpG or Alhydrogel/CpG, obtained on day 7 (early sera) and days 30 or 35-59 (late sera) after primary immunization . Compared to mice receiving sera from G2/k-PBS-treated mice, the survival of naive mice was prolonged by both early and late sera from G2/k-P13-DT/Alhydrogel/CpG-immunized mice, but only late sera from G2/k-P13-TT/Alhydrogel/CpG-immunized mice . Late, but not early sera from G2/k-Alhydrogel/CpG-immunized mice also prolonged survival . For all sera, prolongation of survival was associated with GXM-specific serum IgM . Sera from G2/k mice that received P13-TT without CpG, and all groups of G4/k mice had low to undetectable levels of antibody to GXM and were not protective . Our findings suggest that GXM-specific human IgM may be a functional mediator of protection against C . neoformans.

FEMS Immunol Med Microbiol, 2004 Oct 1, 42(2), 187 - 95
The human immunodeficiency virus (HIV) protease inhibitor indinavir directly affects the opportunistic fungal pathogen Cryptococcus neoformans; Blasi E et al.; Highly active antiretroviral therapy (HAART), that includes human immunodeficiency virus (HIV) protease inhibitors (PIs), has been remarkably efficacious including against some opportunistic infections . In this report we investigated the effect(s) of the PI indinavir on protease activity by Cryptococcus neoformans, an opportunistic fungal pathogen responsible for recurrent meningoencephalitis in AIDS patients . Indinavir was also tested for potential effects on other parameters, such as fungal viability, growth ability and susceptibility to immune effector cells . It was found that indinavir impaired cryptococcal protease activity in a time- and dose-dependent fashion . The phenomenon was similarly detectable in ATCC/laboratory strains and clinical isolates . C . neoformans growth rate was also significantly reduced upon exposure to indinavir, while fungal viability was not affected and mitochondrial toxicity not detected . Furthermore, as assessed by an in vitro infection model, indinavir significantly and consistently augmented C . neoformans susceptibility to microglial cell-mediated phagocytosis and killing . Overall, by providing the first evidence that indinavir directly affects C . neoformans, these data add new in vitro insights on the wide-spectrum efficacy of PIs, further arguing for the clinical relevance of HAART against opportunistic infections in AIDS.

Braz J Infect Dis, 2004 Apr, 8(2), 175 - 9 Epub 2004 Sep 08.
Cerebral infarction related to cryptococcal meningitis in an HIV-infected patient: case report and literature review; Leite AG et al.; Neurological dysfunction as the first manifestation of AIDS has been found in 10 to 20% of symptomatic human immunodeficiency virus infections . However, stroke has rarely been reported in AIDS patients . The most common causes of cerebral infarction in AIDS are central nervous system infections: toxoplasmosis, cryptococcal meningitis and tuberculosis . Potential vascular mechanisms for cerebral infarction and transient neurological deficits among AIDS patients include deposition of antigen-antibody complexes with vasculitis and infarction, and a direct toxic effect of a viral antigen or infectious agent on vascular endothelium . The role of cryptococcal meningitis in vasculopathy is still not clear . We report a case of cerebral infarction in an HIV-infected patient, with cryptococcal meningitis as the first manifestation of AIDS.

Rev Inst Med Trop Sao Paulo, 2004 Jul-Aug, 46(4), 203 - 7 Epub 2004 Sep 03.
Neurocryptococcosis: diagnosis by PCR method; Paschoal RC et al.; Cryptococcus neoformans detection was optimized using PCR technique with the objective of application in the clinical laboratory diagnosis . The amplification area was ITS and 5,6S which encodes the ribosomal RNA (rRNA) . A total of 72 cerebrospinal fluid (CSF) samples were used, obtained from cases with and without AIDS . The patients had cryptococcal meningitis (n = 56) and meningitis caused by other agents (n = 16) . The results demonstrated that PCR test had the highest sensitivity rates, superior to culture (85.7%) and to India ink test (76.8%) . PCR was found to be sensitive in detecting 1 cell/mL and highly specific since it did not amplify other fungal DNA . The comparative analysis of the methods showed that PCR is more sensitive and specific and is applicable as an important laboratorial resource for neurocryptococcosis diagnosis.

Ren Fail, 2004 May, 26(3), 333 - 8
Hypercalcemia and elevated serum 1.25-dihydroxyvitamin D in an end-stage renal disease patient with pulmonary cryptococcosis; Wang IK et al.; Hypercalcemia occurs relatively often in dialysis patients . The most common cause of hypercalcemia in dialysis patients is the conventional therapy with calcium and calcitriol . Besides, secondary hyperparathyroidism, low turnover bone diseases, and immobilization are also common causes of hypercalcemia in dialysis patients . Fungal infection associated with hypercalcemia has been infrequently reported . We describe a 71-year-old female woman with end-stage renal disease and diabetes mellitus, who developed severe hypercalcemia . Pulmonary cryptococcosis, with increased concentration of serum 1,25-dihydroxyvitamin D (1,25(OH)2D), was diagnosed . Her serum concentration of calcium and 1,25(OH)2D returned to normal after antifungal treatment . Thus, hypercalcemia was mediated by extrarenal overproduction of 1,25(OH)2D in this patient.

Biochem J, 2005 Jan 1, 385(Pt 1), 225 - 32
The Nramp orthologue of Cryptococcus neoformans is a pH-dependent transporter of manganese, iron, cobalt and nickel; Agranoff D et al.; Cryptococcus neoformans is an important human opportunistic pathogen and a facultative intracellular parasite, particularly in HIV-infected individuals . Little is known about metal ion transport in this organism . C . neoformans encodes a single member of the Nramp (natural resistance-associated macrophage protein) family of bivalent cation transporters, known as Cramp, which we have cloned and expressed in Xenopus laevis oocytes and Spodoptera frugiperda Sf 21 insect cells . Cramp induces saturable transport of a broad range of bivalent transition series cations, including Mn2+, Fe2+, Co2+ and Ni2+ . Maximal cation transport occurs at pH 5.5-6.0, consistent with the proton gradient-based energetics of other Nramp orthologues . Mn2+ transport is diminished in the presence of 140 mM Na+, compatible with a Na+ slippage mechanism proposed for the Saccharomyces cerevisiae Nramp orthologue Smf1p . Cramp resembles Smf1p with respect to predicted membrane topology, substrate specificity and pH dependence, but differs in terms of its apparent affinity for Mn2+ and negligible inhibition by Zn2+ . Cramp is the first Nramp orthologue from a fungal pathogen to be functionally characterized . Insights afforded by these findings will allow the formulation of new hypotheses regarding the role of metal ions in the pathophysiology of cryptococcosis.

Microbiology, 2004 Sep, 150(Pt 9), 3013 - 23
Cryptococcus neoformans methionine synthase: expression analysis and requirement for virulence; Pascon RC et al.; This paper describes (i) the expression profile of the methionine synthase gene (MET6) in the human pathogenic fungus Cryptococcus neoformans and (ii) the phenotypes of a C . neoformans met6 mutant . In contrast to the MET3 gene, which showed no significant change in expression in any environmental condition tested, the MET6 gene showed a substantial induction in response to methionine and a dramatic transcriptional induction in response to homocysteine . Like a met3 mutant, the met6 mutant was a methionine auxotroph . However, relative to a met3 mutant, the met6 mutant grew very slowly and was less heat-shock resistant . In contrast to a met3 mutant, the met6 mutant lost viability when starved of methionine, and it was deficient in capsule formation . Like a met3 mutant, the met6 mutant was avirulent . In contrast to a met3 mutant, the met6 mutant was hypersensitive to fluconazole and to the calcineurin inhibitors FK506 and cyclosporin A . A synergistic fungicidal effect was also found between each of these drugs and met6 . The phenotypic differences between the met3 and met6 mutants may be due to the accumulation in met6 mutants of homocysteine, a toxic metabolic intermediate that inhibits sterol biosynthesis.

J Infect Dis, 2004 Oct 1, 190(7), 1310 - 7 Epub 2004 Aug 18.
Intrathecal production and secretion of vascular endothelial growth factor during Cryptococcal Meningitis; Coenjaerts FE et al.; BACKGROUND: Patients with cryptococcal meningitis (CM) show elevated intracranial pressure (ICP) and blood-brain barrier (BBB) disruption in most cases . Elevated ICP is an important contributor to mortality . Vascular endothelial growth factor (VEGF) might be the mediator of BBB disruption during CM . METHODS: We measured VEGF levels in serum, plasma, and cerebrospinal fluid (CSF) of 95 patients and 63 control subjects, and we analyzed the required trigger and cellular source of VEGF secretion in vitro . RESULTS: Cryptococcus neoformans and its capsular antigens dose-dependently induced VEGF secretion by polymorphonuclear neutrophils, monocytes, and peripheral blood mononuclear cells (PBMCs) . VEGF production by PBMCs induced by antigens strongly exceeded production by monocytes (P<.001) . The addition of major histocompatibility complex class II antibody inhibited this production of VEGF (P=.005) . Confirming the in vitro data, patients with CM showed significantly elevated VEGF levels in CSF (P<.001), plasma (P=.028), and serum (P<.001), compared with healthy control subjects . Calculated VEGF indices demonstrated that VEGF was produced intrathecally . CONCLUSIONS: Our findings suggest that VEGF plays a role in the pathophysiology of CM . We propose that CD4(+) T lymphocytes--stimulated by monocytes acting as antigen-presenting cells--are the cells that produce VEGF in response to cryptococcal antigens.

Cell Microbiol, 2004 Oct, 6(10), 953 - 61
A synthetic peptide as a novel anticryptococcal agent; Cenci E et al.; An engineered, killer decapeptide (KP) has been synthesized based on the sequence of a recombinant, single-chain anti-idiotypic antibody (KT-scFv) acting as a functional internal image of a yeast killer toxin . Killer decapeptide exerted a strong fungicidal activity against Candida albicans, which was attributed to peptide interaction with beta-glucan . As this polysaccharide is also a critical component of the cryptococcal cell wall, we wondered whether KP was also active against Cryptococcus neoformans, a human pathogen of increasing medical importance . We found that KP was able to kill both capsular and acapsular C . neoformans cells in vitro . Furthermore, KP impaired the production of specific C . neoformans virulence factors including protease and urease activity and capsule formation, rendering the fungus more susceptible to natural effector cells . In vivo treatment with KP significantly reduced fungal burden in mice with cryptococcosis and, importantly, protected the majority of immunosuppressed animals from an otherwise lethal infection . Given the relevance of cryptococcosis in immunocompromised individuals and the inability of conventional drugs to completely resolve the infection, the results of the present study indicate KP as an ideal candidate for further studies on novel anticryptococcal agents.

J Infect, 2004 Oct, 49(3), 253 - 5
Cryptococcus neoformans var . gattii meningitis in an HIV-positive patient: first observation in the United Kingdom; Bodasing N et al.; We present a case of meningitis due to Cryptococcus neoformans var . gattii in an HIV-infected man recently returned from South Africa . We believe this is the first imported case of meningitis secondary to Cryptococcus neoformans var . gattii in an HIV-infected patient to be reported in the UK.

J Clin Neurosci, 2004 Sep, 11(7), 765 - 7
Intracranial and dermatological cryptococcal infection in an immunocompetent man; Krishnan AV et al.; We report the case of a 72-year-old man with a history of Parkinson's Disease who presented with a history of falls, cognitive impairment and depressed mood . Neurological examination revealed moderate rigidity and bradykinesia . Alterations to his anti-Parkinsonian medication resulted in improvements in his mobility but no change in mental state . He was also noted to have developed multiple raised skin lesions . One of these lesions was biopsied and grew Cryptococcus neoformans . He subsequently had a cerebral MRI scan which showed enhancing lesions, in the left parietal lobe and right superior cerebellar peduncle, consistent with intracranial cryptococcal infection . Images of one of the skin lesions and the cerebral MRI scan are shown.

Yeast, 2004 Aug, 21(11), 919 - 26
An efficiently regulated promoter system for Cryptococcus neoformans utilizing the CTR4 promoter; Ory JJ et al.; Cryptococcus neoformans is an opportunistic fungal pathogen responsible for serious meningitis . Although many useful molecular tools have been developed for its study, there are currently few inducible promoters available for general use . To address this need, we have constructed expression plasmids incorporating upstream elements of the C . neoformans copper transporter gene CTR4, and tested them in C . neoformans serotypes A and D . In response to copper deprivation, these plasmids mediate high-level expression of a reporter protein . This expression can be completely repressed using physiologically low concentrations of copper . Notably, this new family of copper-sensing promoters demonstrates excellent expression in serotype A, contrasting with other available promoters . These plasmids therefore offer efficient and regulated expression for both serotypes A and D, and should be valuable tools for the C . neoformans research community .

Ophthalmologica, 2004 Sep-Oct, 218(5), 328 - 32
The HIV RNA Levels of Plasma and Ocular Fluids in AIDS Patients with Ophthalmic Infections; Hsu WM et al.; The aim of the present study was to evaluate the impact of highly active antiretroviral therapy (HAART) on HIV viral load of plasma and intraocular fluids in AIDS patients with ophthalmic opportunistic infections . We further compared the treatment effect of HAART on these patients . From June 1997 to July 2003, we examined and followed up the ophthalmic conditions of 49 patients receiving HAART with ophthalmic diseases during this period . The method of reverse-transcriptase polymerase chain reaction was used to detect and monitor HIV load in plasma and/or aqueous humor of AIDS patients . Before HAART, the HIV levels in the plasma and aqueous humor in 8 AIDS patients with ophthalmic opportunistic infections were significantly higher than those in 6 patients with HIV-related retinopathy (p < 0.05) . Compared to the eye findings and clinical improvement, HIV loads of aqueous humor in 10 of 14 AIDS patients (6 with HIV-related retinopathy, 5 with cytomegalovirus retinitis, 2 with toxoplasmic retinitis and 1 with cryptococcal chorioretinitis) declined to undetectable levels (< 400 copies/ml) after 4-8 months of HAART . HIV virus levels in the plasma of AIDS patients were significantly decreased, and the CD4 counts of these patients were significantly increased (Wilcoxon test) after initiation of HAART .

Indian J Gastroenterol, 2004 Jul-Aug, 23(4), 145 - 6
Hepatobiliary dysfunction as initial manifestation of disseminated cryptococcosis; Kothari AA et al.; Disseminated cryptococcosis presenting as biliary obstruction is rare . We report a 35-year-old HIV-negative man who presented with clinical features suggestive of obstructive jaundice, and radiological features suggestive of Klatskin's tumor, but who ultimately was found to have cryptococcal involvement of the liver and biliary tract as part of disseminated cryptococcosis . The patient responded to antifungal therapy.

Jpn J Infect Dis, 2004 Aug, 57(4), 137 - 45
Regulation by innate immune T lymphocytes in the host defense against pulmonary infection with Cryptococcus neoformans; Kawakami K; Recently, innate immune lymphocytes, such as natural killer (NK) T cells and gamma/delta antigen receptor-bearing T (gamma delta T) cells, have garnered much attention, and their biological significance in the tumor immunity, allergic diseases and infectious diseases is extensively exploited . We have addressed the role of these cells in the host defense using a mouse model of pulmonary infection with Cryptococcus neoformans, which frequently causes fatal meningoencephalitis in AIDS patients . Host defense to this fungal pathogen is largely mediated by cellular immunity, and type-1 helper T (Th1) cells play a central role in this process . This infection causes a prompt accumulation of both NKT and gamma delta T cells in the lung tissues in a monocyte chemoattractant protein (MCP)-1-dependent or -independent manner, respectively . Genetic deletion of V alpha 14+ NKT cells ameliorates the Th1 response and clearance of microorganisms in the lungs, whereas these host protective responses are rather enhanced in mice lacking gamma delta T cells . Thus, in some aspect, these innate immune lymphocytes may co-regulate the Th1-mediated response for induction of the moderate host defense . gamma delta T cells may act to keep the balance of Th1-Th2 responses in a proper manner by suppressing the exaggerated Th1 response caused by NKT cells . In this review, I describe the recent research development in the innate immune host defense against cryptococcal infection in respiratory organs with emphasis on our data in the regulatory role of NKT cells and gamma/delta T cells.

Antimicrob Agents Chemother, 2004 Sep, 48(9), 3312 - 6
Posaconazole and amphotericin B combination therapy against Cryptococcus neoformans infection; Barchiesi F et al.; To investigate the effects of posaconazole (POS) and amphotericin B (AMB) combination therapy in cryptococcal infection, we established an experimental model of systemic cryptococcosis in CD1 mice by intravenous injection of three distinct clinical isolates of Cryptococcus neoformans . Therapy was started 24 h after the infection and continued for 10 consecutive days . POS was given at 3 and 10 mg/kg of body weight/day, while AMB was given at 0.3 mg/kg/day . Combination therapy consisted of POS given at a low (combo 3) or at a high (combo 10) dose plus AMB . Survival studies showed that combo 3 was significantly more effective than POS at 3 mg/kg for two isolates tested (P value, < or = 0.001), while combo 10 was significantly more effective than POS at 10 mg/kg for all three isolates (P values ranging from <0.001 to 0.005) . However, neither combination regimen was more effective than AMB alone . For two isolates, combination therapy was significantly more effective than each single drug at reducing the fungal burden in the brain (P values ranging from 0.001 to 0.015) but not in the lungs . This study demonstrates that the major impact of POS and AMB combination therapy is on brain fungal burden rather than on survival.

Neurology, 2004 Aug 24, 63(4), 713 - 4
Steroid responsive late deterioration in Cryptococcus neoformans variety gattii meningitis; Lane M et al.; The authors describe the clinical course of Cryptococcus neoformans variety gattii infection in a young immunocompetent man who had a late deterioration characterized by headaches, subarachnoid inflammation, hydrocephalus, and stroke that reproducibly responded to steroids . These findings, in combination with declining markers of CSF infection, are consistent with the late deterioration being caused by sterile arachnoiditis rather than ongoing infection.

J Antibiot (Tokyo), 2004 Jun, 57(6), 379 - 82
YM-215343, a novel antifungal compound from Phoma sp . QN04621; Shibazaki M et al.; Through our screening for novel antifungal compounds, YM-215343 was found in the culture extract of Phoma sp . QN04621 . The structure of YM-215343 was determined by several spectroscopic experiments as a novel compound closely related to apiosporamide and fischerin . YM-215343 exhibited antifungal activity against the pathogenic fungi, Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus with MIC values of 2 to approximately 16 microg/ml . It also showed cytotoxicity against HeLa S3 cells with an IC50 of 3.4 microg/ml.

Eur J Clin Microbiol Infect Dis, 2004 Sep, 23(9), 672 - 6 Epub 2004 Aug 18.
Recognition of fungal pathogens by Toll-like receptors; Netea MG et al.; Toll-like receptors (TLRs) have been identified as a major class of pattern-recognition receptors . Recognition of pathogen-associated molecular patterns by TLRs, either alone or in heterodimerization with other TLR or non-TLR receptors, induces signals responsible for the activation of the innate immune response . Recent studies have demonstrated a crucial involvement of TLRs in the recognition of fungal pathogens such as Candida albicans, Aspergillus fumigatus, and Cryptococcus neoformans . Through the study of fungal infection in knock-out mice deficient in either TLRs or TLR-associated adaptor molecules, it became apparent that specific TLRs such as TLR2 and TLR4 play differential roles in the activation of the various arms of the innate immune response . Recent data also suggest that TLRs offer escape mechanisms to certain pathogenic microorganisms, especially through TLR2-driven induction of anti-inflammatory cytokines . These new data have substantially increased our knowledge of the recognition of fungal pathogens, and the study of TLRs remains one of the most active areas of research in the field of fungal infections .

J Immunol, 2004 Sep 1, 173(5), 3357 - 65
NK cells use perforin rather than granulysin for anticryptococcal activity; Ma LL et al.; Cytotoxic lymphocytes have the capacity to kill microbes directly; however, the mechanisms involved are poorly understood . Using Cryptococcus neoformans, which causes a potentially fatal fungal infection in HIV-infected patients, our previous studies showed that granulysin is necessary, while perforin is dispensable, for CD8 T lymphocyte fungal killing . By contrast, the mechanisms by which NK cells exert their antimicrobial activity are not clear, and in particular, the contribution of granulysin and perforin to NK-mediated antifungal activity is unknown . Primary human NK cells and a human NK cell line YT were found to constitutively express granulysin and perforin, and possessed anticryptococcal activity, in contrast to CD8 T lymphocytes, which required stimulation . When granulysin protein and mRNA were blocked by granulysin small interfering RNA, the NK cell-mediated antifungal effect was not affected in contrast to the abrogated activity observed in CD8 T lymphocytes . However, when perforin was inhibited by concanamycin A, and silenced using hairpin small interfering RNA, the anticryptococcal activities of NK cells were abrogated . Furthermore, when granulysin and perforin were both inhibited, the anticryptococcal activities of the NK cells were not reduced further than by silencing perforin alone . These results indicate that the antifungal activity is constitutively expressed in NK cells in contrast to CD8 T lymphocytes, in which it requires prior activation, and perforin, but not granulysin, plays the dominant role in NK cell anticryptococcal activity, in contrast to CD8 T lymphocytes, in which granulysin, but not perforin, plays the dominant role in anticryptococcal activity.

Infect Immun, 2004 Sep, 72(9), 5373 - 82
Involvement of CD14, toll-like receptors 2 and 4, and MyD88 in the host response to the fungal pathogen Cryptococcus neoformans in vivo; Yauch LE et al.; The major capsular polysaccharide of Cryptococcus neoformans, glucuronoxylomannan (GXM), is recognized by Toll-like receptor 2 (TLR2), TLR4, and CD14 . In these studies, mice deficient in CD14, TLR2, TLR4, and the TLR-associated adaptor protein, MyD88, were utilized to investigate the contribution of TLRs and CD14 to in vivo host defenses against C . neoformans . MyD88(-/-) mice had significantly reduced survival compared with wild-type C57BL/6 mice after intranasal (i.n.) and intravenous (i.v.) infection with live C . neoformans . CD14(-/-) mice had reduced survival when infected i.v., while TLR2(-/-) mice died significantly earlier after i.n . infection . Mortality was similar comparing TLR4 mutant C3H/HeJ mice and control C3H/HeOuJ mice following i.v . or i.n . challenge with C . neoformans . The course of pulmonary cryptococcosis was studied in more detail in the CD14(-/-), TLR2(-/-), and MyD88(-/-) mice . MyD88(-/-) mice infected i.n . had higher numbers of CFU in the lungs as well as higher GXM levels in the sera and lungs 7 days after infection than wild-type mice did . Surprisingly, there were no major differences in the levels of tumor necrosis factor alpha, interleukin-4 (IL-4), IL-10, IL-12p70, or gamma interferon in the lungs of C . neoformans-infected knockout mice compared with wild-type mice . Histopathologic analysis of the lungs on day 7 postinfection revealed minimal inflammation in all mouse groups . These studies demonstrate a major role for MyD88 and relatively minor roles for CD14 and TLR2 in the response to cryptococcal infection, with the decreased survival of MyD88(-/-) mice correlating with increased numbers of lung CFU and serum and lung GXM levels.

Infect Immun, 2004 Sep, 72(9), 5175 - 80
Age-related resistance of C57BL/6 mice to Cryptococcus neoformans is dependent on maturation of NKT cells; Blackstock R et al.; Conflicting results have been reported regarding the ability of C57BL/6 mice to clear infections due to Cryptococcus neoformans . Examination of the various experimental protocols used suggested that C57BL/6 mice might develop the ability to resist infection as they mature . We analyzed the ability of C57BL/6 mice of different ages to respond to immunization with cryptococcal antigen or to clear a cryptococcal infection . Mice were immunized with a soluble cryptococcal culture filtrate antigen (CneF) emulsified in complete Freund's adjuvant (CneF-CFA) . Delayed-type hypersensitivity (DTH) reactions elicited by the immunization were significantly stronger in 15-week-old C57BL/6 mice than in 7-week-old mice . Analysis of cryptococcal CFU 8 weeks following intratracheal infection of 7-week-old mice or 15-week-old mice revealed a relative inability of the younger animals to control the infection . Six-week-old immunized and infected mice cleared cryptococci from brain, spleen, and liver in a manner similar to that of immunized and infected 15-week-old mice . However, the older mice cleared cryptococci much more efficiently from the lungs . The possible role for NKT cells was determined by passive transfer of thymocytes from 10-week-old mice (containing mature NKT cells) or 2-week-old mice (containing immature NKT cells) to 6-week-old mice . The 10-week-old thymocytes significantly enhanced the ability of the mice to develop a DTH response after immunization with CneF-CFA, while animals treated with 2-week-old thymocytes did not improve their DTH response after immunization . The cells in the 10-week-old thymocyte population responsible for improvement of DTH responses were identified as being NK1.1 positive.

Infect Immun, 2004 Sep, 72(9), 4985 - 95
Cryptococcal yeast cells invade the central nervous system via transcellular penetration of the blood-brain barrier; Chang YC et al.; Cryptococcal meningoencephalitis develops as a result of hematogenous dissemination of inhaled Cryptococcus neoformans from the lung to the brain . The mechanism(s) by which C . neoformans crosses the blood-brain barrier (BBB) is a key unresolved issue in cryptococcosis . We used both an in vivo mouse model and an in vitro model of the human BBB to investigate the cryptococcal association with and traversal of the BBB . Exposure of human brain microvascular endothelial cells (HBMEC) to C . neoformans triggered the formation of microvillus-like membrane protrusions within 15 to 30 min . Yeast cells of C . neoformans adhered to and were internalized by the HBMEC, and they crossed the HBMEC monolayers via a transcellular pathway without affecting the monolayer integrity . The histopathology of mouse brains obtained after intravenous injection of C . neoformans showed that the yeast cells either were associated with endothelial cells or escaped from the brain capillary vessels into the neuropil by 3 h . C . neoformans was found in the brain parenchyma away from the vessels by 22 h . Association of C . neoformans with the choroid plexus, however, was not detected during up to 10 days of observation . Our findings indicate that C . neoformans cells invade the central nervous system by transcellular crossing of the endothelium of the BBB.

Scand J Immunol, 2004 Sep, 60(3), 226 - 32
Tolerability and anti-inflammatory effects of glucuronoxylomannan in collagen-induced arthritis; Mirshafiey A et al.; This investigation was planned to assess the therapeutic efficacy of glucuronoxylomannan (GXM) in collagen-induced arthritis (CIA) . GXM was isolated from culture filtrate of Cryptococcus neoformans var . gattii, serotype C . CIA was induced by the immunization of Dark Agouti rats with bovine type II collagen in incomplete Freund's adjuvant . GXM solution at two doses, 25 and 50 mg/kg, was administered intraperitoneally . Onset of i.p . injections of GXM to prevention and treatment groups was days 0 and 10 postimmunization, respectively . The WEHI-164 cell line was used for assaying tolerability, matrix metalloproteinase type 2 (MMP-2) activity and apoptosis . MMP-2 activity was assessed using zymography . For assessment of apoptosis, the terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling method was used . The results of this experiment showed that the treatment of CIA with GXM at a dose of 50 mg/kg could suppress disease development both prophylactically and therapeutically . This beneficial effect of GXM was associated with a significant decrease in the anti-CII antibody response compared with untreated rats . Moreover, GXM therapy could diminish MMP-2 activity, but it had no notable effect on apoptosis . GXM also showed a high tolerability compared with certain steroidal and non-steroidal anti-inflammatory drugs . We conclude that GXM suppresses the development of disease in CIA and it could be recommended as a new immunosuppressive and anti-inflammatory agent for further investigations.

Biochem J, 2004 Dec 1, 384(Pt 2), 377 - 84
Cryptococcal phospholipases: a novel lysophospholipase discovered in the pathogenic fungus Cryptococcus gattii; Wright LC et al.; The pathogenic fungus Cryptococcus neoformans produces an extracellular PLB1 (phospholipase B1), shown previously to be a virulence factor . A novel phospholipase (LPL1) with only LPL (lysophospholipase) and LPTA (transacylase) activities has now been characterized in C . gattii, and found to be a 66-kDa glycoprotein (by SDS/PAGE), with a native molecular mass of 670 kDa . The pI was 6.3, and it was active at high temperatures (to 70 degrees C), as well as at both acidic and neutral pH values . It was stimulated by calcium and palmitoyl carnitine at pH 7.0, but not at pH 5.0, and palmitoyl lysophosphatidylcholine was the preferred substrate . Sequencing indicated that LPL1 is a novel cryptococcal lysophospholipase, and not the gene product of CnLYSO1 or PLB1 . A protein with only LPL and LPTA activities was subsequently isolated from two strains of C . neoformans var . grubii . A PLB1 enzyme was isolated from both C . gattii and a highly virulent strain of C . neoformans var . grubii (H99) . In both cases, all three enzyme activities (PLB, LPL and LPTA) were present in one 95-120 kDa glycoprotein (by SDS/PAGE) with pI 3.9-4.3 . Characterization of PLB1 from C . gattii showed that it differed from that of C . neoformans in its larger native mass (275 kDa), high PLB activity relative to LPL and LPTA, and preference for saturated lipid substrates . Differences in the properties between the secreted phospholipases of the two cryptococcal species could contribute to phenotypic differences that determine their respective environmental niches and different clinical manifestations.

Pediatr Surg Int, 2004 Aug, 20(8), 633 - 5 Epub 2004 Aug 13.
Cryptococcal mesenteric lymphadenitis: an unusual cause of acute abdomen; Karaguzel G et al.; Cryptococcal infection of intraabdominal organs or tissues is extremely rare . Herein we report a child with mesenteric cryptococcal lymphadenitis who presented with an acute abdomen misdiagnosed as acute appendicitis . Definitive diagnosis was established with 2nd look and lymph node biopsy . Clinicians should remember that cryptococcal infection of mesenteric lymph nodes may rarely mimic an acute abdomen and cause delay in diagnosis.

J Med Microbiol, 2004 Sep, 53(Pt 9), 935 - 40
Cryptococcus neoformans infections at Vancouver Hospital and Health Sciences Centre (1997-2002): epidemiology, microbiology and histopathology; Hoang LM et al.; An outbreak of infections due to a rare subspecies of Cryptococcus neoformans (var . gattii) was recognized on Vancouver Island (VI), British Columbia, in 2002, which had affected 59, mostly immunocompetent, individuals since 1999 . The objectives of this study were to: (1) determine if the outbreak had spread to Vancouver and its surrounding communities and (2) review the epidemiological, clinical and pathological features of all cryptococcal infections in patients admitted to the Vancouver Hospital and Health Sciences Centre (VHHSC) over a 5 year period . VHHSC microbiology and pathology databases were searched for cryptococcal infections from 1 June 1997 to 31 December 2002 . Hospital charts of all identified patients were reviewed . Available cryptococcal isolates and histopathological specimens were reviewed . Twenty-six cases of cryptococcosis were identified in both HIV-positive (n = 15) and HIV-negative (n = 11) patients . C . neoformans var . grubii was cultured from 13 patients, of whom 10 were HIV-positive . The outbreak strain, C . neoformans var . gattii, was detected in three patients; all had travelled to VI . C . neoformans var . neoformans was cultured from two patients, Cryptococcus laurentii was cultured from one, and seven patients had cryptococcosis based on histopathology alone, without cultures . The majority (10/15) of the HIV-positive patients developed systemic disease whilst HIV-negative patients (8/11) presented with pulmonary cryptococcosis . Lung biopsies revealed necrotizing and/or fibrosing granulomas, with cryptococcal cells in 5 of 10 specimens . Brain biopsies showed cryptococcal organisms within leptomeninges and deeper structures with minimal associated inflammation . This retrospective study demonstrated a sharp increase in the total number of C . neoformans infections in both immunocompromised and immunocompetent patients at the VHHSC in 2002 . There was no evidence of spread of the outbreak strain to the Greater Vancouver area . This is the first correlation of clinical and investigational findings of cryptococcosis in a region in North America where C . neoformans varieties gattii and grubii are endemic.

Mycoses, 2004 Aug, 47(7), 319 - 25
Effects of several antifungal drug combinations against clinical and environmental isolates of Cryptococcus neoformans from China; Zhu LP et al.; The in vitro interactions of caspofungin (CSP) with terbinafine (TRB) and ravuconazole (RVC) with 5-fluorocytosine (5-FC) were tested against 82 clinical and environmental isolates of Cryptococcus neoformans from China . The interaction of CSP with TRB proved synergistic against those isolates with a CSP MIC < or =2 microg ml-1 (5% of the isolates), additive against 42% of the isolates and indifferent against 53% . The effects of RVC with 5-FC were synergistic, additive or indifferent against 8%, 26% and 67% of the isolates, respectively . No antagonistic effects were found among any of the drugs . The combinations of CSP with TRB and RVC with 5-FC may display beneficial effects in a strain-dependent manner, while in no case showed antagonistic effects . These data might be of use to design safer and more efficient treatments for patients with cryptococcosis and warrant further evaluation.

Mycoses, 2004 Aug, 47(7), 310 - 4
Prevalence, serotypes and mating patterns of Cryptococcus neoformans in the pellets of different avifauna in Madras, India; Gokulshankar S et al.; A total of 887 pellets of different avifauna were screened for the presence of Cryptococcus neoformans . One hundred and six of 887 samples (12%) yielded Cr . neoformans in culture . The report on the isolation of Cr . neoformans from the pellets of the crow appears to be new and of greater significance because of the ubiquitous prevalence of this bird in India . The prevalence of both MAT a and MAT alpha mating types were recorded . The serotype D was predominant over serotype A . The findings of the present study reveal the growing diverse ecological niche of Cr . neoformans in a the pellets of various avifauna in India.

Curr Genet, 2004 Oct, 46(4), 193 - 204 Epub 2004 Aug 07.
Cryptococcus neoformans mitochondrial genomes from serotype A and D strains do not influence virulence; Toffaletti DL et al.; Cryptococcus neoformans is an encapsulated pathogenic yeast producing meningoencephalitis . Two primary strains in genetic studies, serotype A H99 and serotype D JEC21, possess dramatic differences in virulence . Since it has been shown that mitochondrial gene expression is prominent at the site of the infection and there are significant differences between mitochondrial gene structure and regulation between the serotype A and D strains, this study used AD hybrids to move serotype A and D mitochondria under different genomic influences . When the serotype D MATa strain is involved in the mating crosses, there is uniparental transmission of mitochondrial DNA, but with the serotype A MATa strain, mitochondrial DNA can be inherited from either parent and recombination in the mitochondrial genome may also occur . In virulence studies between serotype A and D strains, it was found that the primary genetic control of the virulence composite for growth in the central nervous system is encoded in the nuclear DNA and not through mitochondrial DNA.

Proc R Soc Lond B Biol Sci, 2004 Aug 7, 271(1548), 1557 - 64
Major histocompatibility complex controls the trajectory but not host-specific adaptation during virulence evolution of the pathogenic fungus Cryptococcus neoformans; McClelland EE et al.; Genes of the major histocompatibility complex (MHC) play a critical role in immune recognition and are the most genetically diverse loci known . One hypothesis to explain this diversity postulates that pathogens adapt to common MHC haplotypes and thus favour selection of new or rare alleles . To determine whether the pathogenic yeast Cryptococcus neoformans adapts to MHC-dependent immune responses, it was serially passaged in two independent replicate lines of five B10 MHC-congenic strains and Balb/c mice . All passaged lines increased in virulence as measured by reduced host survival . MHC influenced the rate (trajectory) of virulence increase during passages as measured by significant differences in mortality rate (p < 0.001) . However, when the post-passage strains were tested, no MHC differences in mortality rate remained and only minor differences in titres were observed . Also contrary to expectations, increased virulence in three lines passaged in B10 mice had a larger effect in Balb/c mice, and the evolution of virulence in lines passaged in alternating hosts was not retarded . To our knowledge, these data represent the first experimental test of MHC-specific adaptation in a non-viral pathogen . The failure to observe MHC effects despite dramatically increased virulence and host-genotype-specific adaptation to non-MHC genes suggests that escape of MHC-dependent immune recognition may be difficult for pathogens with unlimited epitopes or that other virulence factors can swamp MHC effects .

Parassitologia, 2004 Jun, 46(1-2), 183 - 6
{Update on the diagnosis of dermatomycosis}; Tampieri MP; Dermatomycosis are mycotic diseases of skin caused by a few mycetes: dermatophytes, and some opportunistic fungi as Malassezia, Candida (not C . albicans), Trichosporon, Rhodutorula, Cryptococcus or Aspergillus, Geotrichum, Alternaria, etc . Dermatophytes are a group of closely related filamentous fungi that invade keratinized tissue (skin, hair, nails) of humans and other animals and produce infection called dermatophytosis or ringworm or "tinea" . The etiological agents of dermatophytosis are classified in three genera: Microsporum, Trichophyton and Epidermophyton (Deuteromycetes) . On the basis of their primary habitat dermatophytes are divided in Anthropophilic dermatophytes (parasitic organisms that infect humans), Zoophilic dermatophytes (parasitic organisms that infect animals, but also humans: agents of zoonosis) and Geophilic dermatophytes (saprobic fungi associated with keratinous materials in soil) . In the soil there are also structure associated with contagion, ("spore", "arthroconidium", or "clamydospore") of anthropophilic and zoophilic dermatophytes that may persist for years, in the environment, in hair or skin scales . Since on the skin of animals there are many saprobic organisms (Malassezia) and many fungi may infect the fur, it is important to make an accurate diagnosis . Dermatophytosis are communicable diseases acquired from infected animals or from fomites . Infections caused by dermatophytes is a ringworm . These infections may range from mild and superficial, almost subclinical, to a few areas of scaling to a highly inflammatory reaction with extensive areas of scarring and alopecia . Granuloma formations (mycetoma-like) may occur especially in cats . Dermatophytes, as filamentous fungi, undergo radial fungi: collection of skin material is best made by collecting the scales near the edges of the rings . Hairs are best sampled by plucking; a scalpel may be used to scrape scales; brushes have also been used . Sample materials are best transported in dry packet . The Wood's light may be used to identify infected fluorescent hairs . Direct microscopy, although false negative up to 50% of cases, is a highly efficient screening technique . Scraping and hairs should mixed to 10-15% KOH . Culture is a valuable adjunct to direct microscopy and is essential to identify more dermatophytes . A medium selective against most nondermatophytic moulds and bacteria is used as a primary isolation medium . Many typical isolates of common dermatophytes can be identified directly from primary isolation media . Identification characters include: colony pigmentation, texture, morphological structure (macroconidia, microconidia, spirals, pectinate branches, etc) . Nutritional requiment, growth in special media, "in vitro" perforation, mating studies are procedures used to identify atypical isolates . Serological approaches have revealed difficulties . Many kinds of molecular biologic techniques have been made available for clinical diagnosis recently; almost all of these techniques involve the polymerase chain reaction (PCR).

FEBS Lett, 2004 Aug 13, 572(1-3), 46 - 50
Prp8 intein in fungal pathogens: target for potential antifungal drugs; Liu XQ et al.; Inteins are self-splicing intervening sequences in proteins, and inteins of pathogenic organisms can be attractive drug targets . Here, we report an intein in important fungal pathogens including Aspergillus fumigatus, Aspergillus nidulans, Histoplasma capsulatum, and different serotypes of Cryptococcus neoformans . This intein is inside the extremely conserved and functionally essential Prp8 protein, and it varies in size from 170 aa in C . neoformans to 819 aa in A . fumigatus, which is caused by the presence or absence of an endonuclease domain and a putative tongs subdomain in the intein . Prp8 inteins of these organisms were demonstrated to do protein splicing in a recombinant protein in Escherichia coli . These findings revealed Prp8 inteins as attractive targets for potential antifungal drugs to be identified using existing selection and screening methods.

Eukaryot Cell, 2004 Aug, 3(4), 1028 - 35
Mutation of the regulator of G protein signaling Crg1 increases virulence in Cryptococcus neoformans; Wang P et al.; The regulator of G protein signaling homolog Crg1 was found to be a key regulator of pheromone-responsive mating in the opportunistic human fungal pathogen Cryptococcus neoformans . A mutation in the CRG1 gene has greatly increased virulence in the prevalently distributed MATalpha strains of the fungus . Mouse survival time was shortened by 40%, and the lethal dosage was 100-fold less than that of wild-type strains . In addition, the increased virulence of crg1 mutant strains was dependent on the transcription factor homolog Ste12alpha but not on the mitogen-activated protein kinase homolog Cpk1 . The enhanced mating due to CRG1 mutation, however, was still dependent on Cpk1 . Interestingly, crg1 mutants of MATalpha cells produced dark melanin pigment under normally inhibitory conditions, which may relate to the mechanism for increased virulence.

Clin Pharmacokinet, 2004, 43(11), 725 - 32
Effect of rifampicin on the pharmacokinetics of fluconazole in patients with AIDS; Panomvana Na Ayudhya D et al.; OBJECTIVE: To study the effect of rifampicin on the pharmacokinetics of fluconazole and on clinical outcomes of fluconazole treatment in patients with AIDS-related cryptococcal meningitis . PATIENTS: Forty Thai patients with AIDS and cryptococcal meningitis, of whom 20 had been receiving oral rifampicin for at least 2 weeks to treat tuberculosis . METHODS: Patients were treated for cryptococcal meningitis with amphotericin 0.7 mg/kg/day for 14 days followed by fluconazole 400 mg/day, which was reduced to 200 mg/day once culture of cerebrospinal fluid (CSF) became negative . Patients with tuberculosis received oral rifampicin 600 mg/day at night . Blood samples were collected from the first 12 patients in each group and pharmacokinetic parameters for fluconazole were calculated . CSF samples were collected by lumbar puncture and monitored for eradication of Cryptococcus neoformans . RESULTS: Concomitant administration of rifampicin with fluconazole resulted in significant changes in the pharmacokinetic parameters of fluconazole, including a 39% increase in elimination rate constant, 28% shorter elimination half-life, 22% decrease in area under the concentration-time curve, 17% decrease in maximum concentration and 30% increase in clearance (p < 0.05) . Different fluconazole regimens did not affect the extent of change in the elimination rate constant . Although serum concentrations of fluconazole during the time that patients received rifampicin concomitantly with fluconazole 200 mg/day were generally lower than the minimum inhibitory concentration for C . neoformans, there were no significant differences in clinical outcomes between the two groups to date (p = 0.792) . CONCLUSIONS: Coadministration of rifampicin with fluconazole caused significant changes in the pharmacokinetic parameters of fluconazole . Long-term monitoring for recurrence rates of cryptococcal meningitis is required to assess the clinical significance of this interaction.

South Med J, 2004 Jul, 97(7), 692 - 5
Primary cutaneous cryptococcosis in a lung transplant recipient; Baumgarten KL et al.; Cryptococcal skin lesions are found in 10 to 15% of patients with disseminated cryptococcosis . Primary skin inoculation by Cryptococcus neoformans is rare but has been reported . We report the first known case of primary cutaneous cryptococcosis in a lung transplant recipient . Our patient, a 57-year-old man, underwent left single-lung transplantation and presented with a nonhealing ulcer 50 months later . Skin histopathology and culture confirmed C . neoformans . Serum and cerebrospinal fluid cryptococcal antigen tests were negative . The lesion healed after treatment with fluconazole . To date, disseminated disease is not evident . Primary cutaneous cryptococcosis has been reported in kidney and liver recipients but not in lung transplantation recipients . Nonhealing ulcers in immunocompromised patients mandate aggressive diagnostic procedures . Differential diagnosis of these cutaneous lesions should consider fungi, including C . neoformans.

Yeast, 2004 Jul 30, 21(10), 809 - 12
Environmental isolation of Cryptococcus neoformans from an endemic region of HIV-associated cryptococcosis in Thailand; Kuroki M et al.; We successfully isolated Cryptococcus neoformans from chicken faeces in suburban areas of Thailand . C . neoformans was isolated from 36/150 houses (24.0%) in the dry season and 6/150 (4.0%) in the rainy season . All environmental isolates were of serotype A . The high isolation rate of 24% from chicken faeces has never been reported previously . Our environmental study could probably explain the high incidence of cryptococcal meningitis in HIV patients in Thailand.

J Clin Microbiol, 2004 Aug, 42(8), 3865 - 8
Cryptococcus infection in tropical Australia; Jenney A et al.; Eighteen cases of disease caused by the saprophytic fungi Cryptococcus neoformans and Cryptococcus bacillisporus are described from the Northern Territory of Australia . The majority of infections were with Cryptococcus bacillisporus and in the rural Aboriginal population, often causing pulmonary mass lesions.

J Clin Microbiol, 2004 Aug, 42(8), 3789 - 94
Scytalidium dimidiatum causing recalcitrant subcutaneous lesions produces melanin; Morris-Jones R et al.; Scytalidium dimidiatum is a pigmented dematiaceous coelomycete that typically causes chronic superficial skin diseases and onychomycosis, as well as deeper infections, such as subcutaneous abscesses, mycetoma, and even fungemia in immunocompromised patients . A second species, Scytalidium hyalinum, has hyaline hyphae and arthroconidia and is considered by some authors to be an albino mutant of S . dimidiatum . This study aimed to confirm the presence of melanin or melanin-like compounds (which have been previously implicated in the virulence of other fungal pathogens) in S . dimidiatum from a patient with multiple subcutaneous nodules . Treatment of the hyphae and arthroconidia with proteolytic enzymes, denaturant, and concentrated hot acid yielded dark particles, which were stable free radicals, consistent with their identification as melanins . Extracted melanin particles from S . dimidiatum cultures were labeled by melanin-binding monoclonal antibodies (MAbs) from Sporothrix schenckii, Aspergillus fumigatus, and Cryptococcus neoformans . Lesional skin from the patient infected with S . dimidiatum contained fungal cells that were labeled by melanin-binding MAbs, and digestion of the tissue yielded dark particles that were also reactive . S . hyalinum was also subjected to the melanin extraction protocol, but no dark particles were yielded.

J Pediatr Hematol Oncol, 2004 Aug, 26(8), 526 - 8
Cryptococcal sepsis diagnosed by bone marrow examination; Abdul-Rahman OA et al.; Disseminated cryptococcal disease is often associated with immunodeficient states . The diagnosis is usually made using standard antigen tests on serum and cerebrospinal fluid in patients with known immunodeficiency . Often, blood and cerebrospinal fluid cultures also yield Cryptococcus neoformans . The authors describe a child whose diagnosis remained elusive until a bone marrow aspiration, performed as part of an evaluation for suspected neoplasm, revealed the offending organism.

Med Mycol, 2004 Jun, 42(3), 229 - 38
Molecular epidemiology of Cryptococcus neoformans isolates from AIDS patients of the Brazilian city, Rio de Janeiro; Igreja RP et al.; A high biodiversity of Cryptococcus neoformans isolates is known to exist in some Brazilian urban areas, raising the possibility that patients may encounter multiple inoculum sources in their daily life . C . neoformans isolates from two groups of AIDS patients with cryptococcosis from Rio de Janeiro were studied by polymerase chain reaction (PCR) fingerprinting and randomly amplified polymorphic DNA (RAPD) analysis . The first group contained 60 serial isolates obtained from 19 patients over periods ranging from 18 to 461 days; the intent was to determine whether the original strain persisted or whether reinfection with a new strain occurred . The second group was made up of 22 isolates from 11 patients, and consisted of a pair of isolates collected from blood and cerebrospinal fluid from each patient either before or shortly after treatment was initiated . The aim was to determine if the patient was infected by different strains simultaneously . All isolates were subtyped by PCR fingerprinting, using minisatellite (M13), and microsatellite {(GACA)4 and (GTG)5} specific primers, and RAPD analysis employing the combined primers 5SOR and CN1 . The majority of isolates were C . neoformans var . grubii, specifically, molecular types VNI or VNII, but numerous distinguishable subtypes were found . Only three isolates were C . n . var . gattii (molecular types VGI or VGII) . Except in two cases, all isolates obtained from the same patient showed identical PCR profiles independent of time of isolation or body site . Almost all patients, however, carried unique genotypes not found in any other patient . Our results confirm that persistent cryptococcal infection is caused by relapse rather than reinfection, but they also show that in exceptional cases, patients may be infected with more than one C . neoformans strain.

Mol Biol Cell, 2004 Oct, 15(10), 4476 - 89 Epub 2004 Jul 28.
PAK kinases Ste20 and Pak1 govern cell polarity at different stages of mating in Cryptococcus neoformans; Nichols CB et al.; Sexual identity and mating are linked to virulence of the fungal pathogen Cryptococcus neoformans . Cells of the alpha mating type are more prevalent and can be more virulent than a cells, and basidiospores are thought to be the infectious propagule . Mating in C . neoformans involves cell-cell fusion and the generation of dikaryotic hyphae, processes that involve substantial changes in cell polarity . Two p21-activated kinase (PAK) kinases, Pak1 and Ste20, are required for both mating and virulence in C . neoformans . We show here that Ste20 and Pak1 play crucial roles in polarized morphogenesis at different steps during mating: Pak1 functions during cell fusion, whereas Ste20 fulfills a distinct morphogenic role and is required to maintain polarity in the heterokaryotic mating filament . In conclusion, our studies demonstrate that PAK kinases are necessary for polar growth during mating and that polarity establishment is necessary for mating and may contribute to virulence of C . neoformans.

AIDS, 2004 Aug 20, 18(12), 1615 - 27
Immune restoration disease after antiretroviral therapy; French MA et al.; Suppression of HIV replication by highly active antiretroviral therapy (HAART) often restores protective pathogen-specific immune responses, but in some patients the restored immune response is immunopathological and causes disease {immune restoration disease (IRD)} . Infections by mycobacteria, cryptococci, herpesviruses, hepatitis B and C virus, and JC virus are the most common pathogens associated with infectious IRD . Sarcoid IRD and autoimmune IRD occur less commonly . Infectious IRD presenting during the first 3 months of therapy appears to reflect an immune response against an active (often quiescent) infection by opportunistic pathogens whereas late IRD may result from an immune response against the antigens of non-viable pathogens . Data on the immunopathogenesis of IRD is limited but it suggests that immunopathogenic mechanisms are determined by the pathogen . For example, mycobacterial IRD is associated with delayed-type hypersensitivity responses to mycobacterial antigens whereas there is evidence of a CD8 T-cell response in herpesvirus IRD . Furthermore, the association of different cytokine gene polymorphisms with mycobacterial or herpesvirus IRD provides evidence of different pathogenic mechanisms as well as indicating a genetic susceptibility to IRD . Differentiation of IRD from an opportunistic infection is important because IRD indicates a successful, albeit undesirable, effect of HAART . It is also important to differentiate IRD from drug toxicity to avoid unnecessary cessation of HAART . The management of IRD often requires the use of anti-microbial and/or anti-inflammatory therapy . Investigation of strategies to prevent IRD is a priority, particularly in developing countries, and requires the development of risk assessment methods and diagnostic criteria.

Eur J Clin Microbiol Infect Dis, 2004 Aug, 23(8), 638 - 41 Epub 2004 Jul 23.
Cavitating pneumonia after treatment with infliximab and prednisone; Arend SM et al.; Tumor necrosis factor (TNF)-alpha antagonists constitute a novel class of immunomodulating drugs that are used for the treatment of an increasing number of inflammatory disorders . These agents are associated with an increased risk of tuberculosis, but the risk of other infections is less clear . Reported here is the case of a patient who developed cavitary pneumonia after treatment with infliximab (monoclonal TNF-alpha antibodies) and corticosteroids for rheumatoid arthritis . Cryptococcus neoformans was the only pathogen isolated from bronchoalveolar lavage fluid . The patient responded well to fluconazole . The risk of infection after treatment with TNF-alpha antagonists and the possible causative microorganisms are discussed.

Cell Microbiol, 2004 Sep, 6(9), 883 - 91
An early imbalance of interleukin 12 influences the adjuvant effect of mannoproteins of Cryptococcus neoformans; Pietrella D et al.; Mannoprotein from Cryptococcus neoformans induces protective response against a lethal challenge with this fungus or with Candida albicans . This phenomenon is largely related to early production of interleukin 12 (IL-12) and induction of T helper 1 response . Our study assesses whether the early absence of this critical cytokine could account for the incomplete activation of cellular response and whether the immune system compensates this imbalance . The results show that the neutralization of early IL-12 enhanced IL-18 production but decreased IFN-gamma secretion and IL-12R expression by splenic CD4 T cells . In contrast, IL-18R was not augmented despite an increase in IL-18 production . The co-stimulatory pathway was partially dysregulated because splenic macrophages showed unmodified B7-2, and a decrease of B7-1 expression . This dysregulation led to incomplete proliferative response of T cells in response to Cryptococcus neoformans and to increased fungal load in the brain 21 days post infection . The inability to dispose early IL-12, forced the immune system to compensate the imbalance and produced a series of long-lasting dysregulations involving the co-stimulatory pathway and T cell activation.

Infect Immun, 2004 Aug, 72(8), 4810 - 8
Protective and nonprotective human immunoglobulin M monoclonal antibodies to Cryptococcus neoformans glucuronoxylomannan manifest different specificities and gene use profiles; Maitta RW et al.; The features of protective murine antibodies to the Cryptococcus neoformans capsular polysaccharide glucuronoxylomannan (GXM) have been rigorously investigated; however, the characteristics of protective human antibodies to GXM have not been defined . We produced monoclonal antibodies (MAbs) from XenoMouse mice (transgenic mice that express human immunoglobulin M {IgM}, IgG2, and kappa) which were immunized with a C . neoformans serotype D strain 24067 GXM-diphtheria toxoid conjugate . This study reports the specificity and efficacy of three human IgM MAbs, G14, G15, and G19, generated from these mice . Each MAb was specific for GXM, but G14 and G19 had different specificity based on their binding to serotype A strain H99 and SB4 GXMs, to which G15 did not bind . Nucleic acid sequence analysis revealed that G15 uses V(H)3-64 in the germ line configuration . G14 and G19 use V(H)6-1, which has somatic mutations . All of the MAbs use V kappa DPK22/A27 . Studies of MAb efficacy in BALB/c mice showed that administration of 0.1 mg, but not 1 or 0.01 mg, of G15 prolonged survival against lethal C . neoformans strain 24067 challenge, whereas G14 and G19 were not protective at any dose . This panel of MAbs illustrates that serotype D GXM has epitopes that elicit human antibodies that can be either protective or nonprotective . Our findings suggest that V(H) gene use may influence GXM specificity and efficacy, and they provide insights into the possible contribution that V(H) gene use may have in resistance and susceptibility to cryptococcosis.

J Assoc Physicians India, 2003 Nov, 51, 1102 - 4
Human immunodeficiency virus infection in a patient with systemic lupus erythematosus; Wanchu A et al.; We describe a 47 years lady with systemic lupus erythematosus (SLE) who was infected with human immunodeficiency virus (HIV), due to transfusion either by blood or platelet concentrate . There was a near remission in the disease and during the course of follow up she developed cryptococcal meningitis . The approach to the diagnosis of HIV infection in a patient with SLE, the effect of SLE on the virus and vice versa and some management issues in this setting are discussed.

Haematologica, 2004 Jul, 89(7), 852 - 6
Cryptococcosis in patients with hematologic malignancies . A report from GIMEMA-infection; Pagano L et al.; BACKGROUND AND OBJECTIVES: Cryptococcosis is an important cause of morbidity and death in immunocompromised patients . The aim of this study was to evaluate clinical and laboratory characteristics, and outcome of patients with cryptococcosis complicating hematologic diseases . DESIGN AND METHODS: This was a retrospective study, conducted over a ten-year period (1993-2002) in 21 hematology divisions, in tertiary care or university hospitals . RESULTS: This study evaluated 17 patients with hematologic diseases who developed cryptococcosis . Possible risk factors recognized before the onset of the infection were: administration of steroids for the underlying malignancy (6 patients), diabetes mellitus (4 patients), cutaneous lesions (2 patients) and autoimmune disease, hepatic cirrhosis, chronic renal failure and exposure to pigeons (1 patient each) . Five patients received prophylaxis, consisting of fluconazole in 2 cases . Fever, neurological and respiratory signs developed according to the primary sites of infection (5 blood, 5 central nervous system, 4 lung, and 1 each in gut, skin and mouth) . Diagnosis was made by positive microbiological culture, antigen detection in serum or cerebrospinal fluid, or polymerase chain reaction . All patients started specific treatment (fluconazole, 7 patients; amphotericin-B deoxycolate or liposomal amphotericin-B, 10 patients) . Two patients died from cryptococcosis within 30 days after diagnosis . INTERPRETATION AND CONCLUSIONS: Cryptococcosis in patients with hematologic malignancies is a rare complication . In neutropenic patients, it is less fatal than other fungal infections (i.e . aspergillosis or candidemia) . Specific treatment, started promptly, positively influences the outcome.

Bull Soc Pathol Exot, 2004 May, 97(2), 119 - 21
{Cryptococcal meningitis in HIV-infected patients at Bobo-Dioulasso hospital (Burkina Faso)}; Millogo A et al.; Cryptococcus neoformans is an important fungal pathogen in immunocompromised patients . A retrospective study was conducted to investigate the occurrence of Cryptococcus neoformans infection in patients admitted to Bobo-Dioulasso Hospital over a 3 year-period . During this period, cryptococcal meningo-encephalitis was diagnosed in 36 individuals . The median age of the patients under study was 34.25 years . There was a male preponderance (24 males/12 females) in our report . Typical presentations were persistent headaches (27 cases/36), neck stiffness (16/36), altered consciousness (14/36), fever (12/36) and convulsions (9/36) . Oral candidiasis coexisted with cryptococcal meningitis in 7 patients . HIV serology was positive in all patients . At diagnosis, lymphocytes counts were < 1500/mm3 in 66.66% patients . CSF examination with India ink helped to the diagnosis of cryptococcosis in all cases . Cryptococcus neoformans was associated with Streptococcus pneumoniae in 4 patients . 15/36 patients died within 1 to 29 days after admission . High mortality was related to delayed diagnosis . Cryptococcal meningitis highly contributes to mortality in HIV-infected patients in Burkina Faso and it may occur in patients not severely immunocompromised patients . A need exists to improve strategies for clinical management of AIDS patients in poor African countries.

J Antimicrob Chemother, 2004 Aug, 54(2), 563 - 5 Epub 2004 Jul 14.
Increasing in vitro resistance to fluconazole in Cryptococcus neoformans Cambodian isolates: April 2000 to March 2002; Sar B et al.; OBJECTIVES: Cryptococcal meningitis is the third-most-common opportunistic infection in HIV patients in Cambodia . Hospitalized patients were given amphotericin B for initial therapy followed by fluconazole for maintenance therapy . The antifungal drug susceptibility of Cryptococcus neoformans isolated from cerebrospinal fluid (CSF) was determined . METHODS: Isolates of C . neoformans were collected during active laboratory-based surveillance, the first batch from April 2000 to March 2001 (134 new cases), the second batch from April 2001 to March 2002 (268 new cases) . Etest strips were used to determine the MICs of amphotericin B and fluconazole . The antigenic agglutination slide test was used for serotyping . RESULTS: The MIC(50)s and MIC(90)s of fluconazole changed significantly from year 2000 to 2002; the MIC(50)s increased from 4 to 12 mg/L, and the MIC(90)s from 12 to 96 mg/L . For amphotericin B, the MIC(50)s and MIC(90)s remained stable . Moreover, in the second batch, fluconazole MICs were >/=256 mg/L for 20 isolates . By serotyping, it was found that 98.5% of the isolates were serotype A . CONCLUSIONS: C . neoformans strains isolated from CSF of AIDS patients in Cambodia remain susceptible in vitro to amphotericin B . These strains are less susceptible in vitro to fluconazole, 2.5% being resistant in the first year and 14% in the second year of study . Nevertheless, in vitro resistance of C . neoformans to fluconazole appeared to be linked to extended maintenance treatments.

J Gen Appl Microbiol, 2004 Apr, 50(2), 65 - 9
Cryptococcus paraflavus sp . nov . (Tremellales), isolated from steppe plants in Russia; Golubev WI et al.; Three strains related to Cryptococcus flavus were isolated from plants collected in the Prioksko-terrasny biosphere reserve (Russia) . Physiological characterization, mycocinotyping, sequencing of the D1/D2 domain of the 26S rDNA and the ITS region revealed their separate taxonomic position . The name Cryptococcus paraflavus is proposed to accommodate these isolates (type strain VKM Y-2923).

Clin Exp Dermatol, 2004 Jul, 29(4), 366 - 8
A granulomatous response to tribal medicine as a feature of the immune reconstitution syndrome; Farrant P et al.; Summary Immune reconstitution is a well recognized phenomenon associated with the use of highly active antiretroviral therapy (HAART) for HIV infection . After the administration of HAART there is a rise in CD4 T-cell count in the circulation brought about by cessation of HIV replication . This allows the body to respond to antigens that it previously ignored . This manifests itself most commonly as an overt illness to previously ignored pre-existing infections such as Mycobacterium tuberculosis, herpes simplex virus, varicella zoster virus, hepatitis B and C viruses, cytomegalovirus, cryptococcal infection, human papilloma virus and molluscum contagiosum . There are further reports of reactions to sarcoid and tattoo pigment and one previous case reported of a granulomatous reaction to a foreign body . We report another case of a foreign body granuloma reaction, to tribal medicine implanted in tribal marks made in childhood in a Zimbabwean woman . This reaction is part of the immune reconstitution syndrome.

Thyroid, 2004 Jun, 14(6), 471 - 4
Cryptococcal thyroiditis and hyperthyroidism; Avram AM et al.; We report a case of cryptococcal thyroiditis presenting with hyperthyroidism that evolved through a transient euthyroid phase to hypothyroidism and finally recovered to normal function . This four-phase clinical presentation is similar to that of subacute thyroiditis, and it is unusual in the setting of infectious nonviral thyroiditis . Cryptococcal thyroiditis is rare; only three cases have been reported . Our patient is the first who survived the disseminated cryptococcal infection with thyroid involvement, thus enabling longitudinal clinical and endocrinologic follow-up.

Genetics, 2004 Jun, 167(2), 619 - 31
A genetic linkage map of Cryptococcus neoformans variety neoformans serotype D (Filobasidiella neoformans); Marra RE et al.; To construct a genetic linkage map of the heterothallic yeast, Cryptococcus neoformans (Filobasidiella neoformans), we crossed two mating-compatible strains and analyzed 94 progeny for the segregation of 301 polymorphic markers, consisting of 228 restriction site polymorphisms, 63 microsatellites, two indels, and eight mating-type (MAT)-associated markers . All but six markers showed no significant (P < 0.05) segregation distortion . At a minimum LOD score of 6.0 and a maximum recombination frequency of 0.30, 20 linkage groups were resolved, resulting in a map length of approximately 1500 cM . Average marker density is 5.4 cM (range 1-28.7 cM) . Hybridization of selected markers to blots of electrophoretic karyotypes unambiguously assigned all linkage groups to chromosomes and led us to conclude that the C . neoformans genome is approximately 20.2 Mb, comprising 14 chromosomes ranging in size from 0.8 to 2.3 Mb, with a ratio of approximately 13.2 kb/cM averaged across the genome . However, only 2 of 12 ungrouped markers hybridized to chromosome 10 . The hybridizations revealed at least one possible reciprocal translocation involving chromosomes 8, 9, and 12 . This map has been critical to genome sequence assembly and will be essential for future studies of quantitative trait inheritance.

Eur J Med Chem, 2004 Jul, 39(7), 579 - 92
Synthesis of novel substituted tetrazoles having antifungal activity; Upadhayaya RS et al.; In an effort to find potent antifungal agents, a variety of triazole derivatives with a 5-substituted tetrazole structure 6, 7, 12 and 14 were prepared and evaluated for antifungal activity against Candida spp., Cryptococcus neoformans, and Aspergillus spp . in vitro . The location of the methyl group at the C-3 of compounds 12 and 14 has been demonstrated to be a key structural element of antifungal potency.

HIV Med, 2004 Jul, 5(4), 289 - 95
Causes of death among patients with HIV in Singapore from 1985 to 2001: results from the Singapore HIV Observational Cohort Study (SHOCS); Bellamy R et al.; OBJECTIVES: To investigate the major primary and contributory causes of death among HIV patients in Singapore . DESIGN: A retrospective observational cohort study of all adult patients seen at the national referral centre for HIV in Singapore between 1985 and 2001 . METHODS: Data were extracted from the patients' records by 10 trained health care workers . AIDS-defining conditions were established using predefined criteria . For each case, a single principal cause of death and up to three contributory causes were identified . RESULTS: A total of 1504 patients aged 17 years or over were seen before the end of 2001, of whom 504 have died . The most frequent principal causes of death were Mycobacterium avium (17.5%), Mycobacterium tuberculosis (9.7%), pneumonia (cause unknown) (6.5%) and Cryptococcus neoformans (6.7%) . Three hundred and eighteen patients (63.1%) died from an AIDS-defining condition . CONCLUSIONS: The causes of death were similar to those found in Western cohorts, except that disseminated M . avium was a more frequent cause of death.

HIV Med, 2004 Jul, 5(4), 253 - 7
Hepatic pathology in AIDS: a pathological study from Mumbai, India; Lanjewar DN et al.; OBJECTIVES: To assess the spectrum of hepatic disorders in AIDS, liver specimens from 171 patients (155 autopsies and 16 biopsies) were reviewed . METHODS: A retrospective and prospective study of 171 autopsy and biopsy specimens was carried out at a tertiary level hospital in Mumbai, India . RESULTS: Of the patients included in the study, 127 (74%) were male and 44 (26%) were female . The heterosexual route was the predominant mode of HIV transmission, identified in 163 (95%) patients . A total of 99 of 171 patients (58%) showed significant pathological lesions, and the most common pathological processes involving the liver appeared to be secondary to infections . None of our patients showed isolated infectious diseases of the liver . The spectrum of liver diseases identified was as follows: tuberculosis in 70 patients (41%), cryptococcosis in eight (5%), cytomegalovirus infection in six (3%), hepatitis B infection in five (3%), candidiasis in one (0.5%), malaria in one (0.5%), cirrhosis in six (3%), amyloidosis in one (0.5%) and primary hepatic lymphoma in one (0.5%) . CONCLUSIONS: AIDS patients were found to have a high prevalence of underlying hepatic abnormalities . The spectrum of disease among patients with AIDS in India differs from that in developed countries . Our results suggest that hepatic tuberculosis is more common in AIDS than previously recognized, and that liver specimens should be examined routinely for the presence of acid-fast bacilli.

J Antimicrob Chemother, 2004 Aug, 54(2), 456 - 64 Epub 2004 Jul 01.
A multicentre pharmacoepidemiological study of therapeutic practices in invasive fungal infections in France during 1998-1999; Lortholary O et al.; OBJECTIVE: To study the pharmacoepidemiology of the prescription of systemic antifungal agents in 48 French haematology, intensive care and infectious diseases units . PATIENTS AND METHODS: Cases of invasive fungal infections (IFI) were identified retrospectively over a 1 year period . Data on underlying condition, IFI diagnosis, antifungal treatment and outcome were collected on the last five cases in each centre . Factors associated with first line therapy and with death were identified by multivariate analysis . RESULTS: Two hundred and nine cases were included (102 aspergillosis, 86 candidiasis, 15 cryptococcosis) . Amphotericin B, in different formulations, was the first line therapy in 60%, azoles in 32%, combinations in 8% . Haematological malignancies and neutropenia were associated with less frequent initial prescription of azoles {OR = 0.3 (0.1-0.8) and OR = 0.3 (0.1-0.9), respectively} . In aspergillosis, younger age and neutropenia were associated with less frequent initial prescription of azoles {OR = 0.03 (0.002-0.6) and OR = 0.09 (0.03-0.3), respectively} and previous history of IFI was associated with a higher probability of azole prescription {OR = 17.2 (2.4-124.3)} . In candidiasis, haematological malignancy and co-prescription of nephrotoxic agents were associated with a less frequent initial prescription of azoles {OR = 0.1 (0.04-0.4) and OR = 0.2 (0.06-0.9), respectively} . Three factors were associated with a lower risk of death: cryptococcosis {OR = 0.16 (0.03-0.98)}, hospitalization in infectious diseases units {OR = 0.40 (0.16-0.97)} and recent surgery {OR = 0.26 (0.08-0.80)} . Severe renal insufficiency was associated with a higher probability of death {OR = 8.77 (1.97-38.97)} . CONCLUSIONS: Our results emphasize factors associated with the antifungal therapeutic decision and with the outcome of IFI.

Nippon Yakurigaku Zasshi, 2004 Jul, 124(1), 41 - 51
{Nonclinical studies and clinical studies on fosfluconazole, a triazole antifungal agent (Prodif)}; Kawakami Y et al.; Fosfluconazole is a phosphate prodrug of fluconazole that has been developed to reduce the volume of fluid required to administer fluconazole by the intravenous route . Fosfluconazole is hydrolyzed by alkaline phosphatase to fluconazole and phosphoric acid . Fosfluconazole had no significant antifungal activity in vitro . However, in rat models of acute systemic candidiasis and intracranial cryptococcosis, fosfluconazole retained the antifungal potency and efficacy of fluconazole . This reflects the effective conversion of the prodrug to the parent during the course of the experiments . The 2-day-loading dose regimen led to earlier achievement of target fluconazole steady state plasma concentrations compared to use of the 1-day- or no-loading dose regimen of fosfluconazole . The efficacy and safety of fosfluconazole were investigated with the 2-day-loading dose regimen in patients with deep-seated mycosis caused by Candida and Cryptococcus species . The efficacy rates were 73.8% in the domestic Phase III study and 91.7% in the foreign Phase III study . Adverse events were observed in 31 cases (19.4%) out of 160 in both studies . These results indicate that fosfluconazole is effective for the treatment of deep-seated mycosis and shows no clinically significant adverse events in the Phase III studies

Transpl Infect Dis, 2004 Mar, 6(1), 33 - 6
Hepatic cryptococcosis in a heart transplant recipient; Utili R et al.; Cryptococcosis primarily occurs in patients with impaired immune response . While pulmonary and/or cerebral involvement are more often described, there is limited experience of its presence in other sites . We present a case of hepatic cryptococcosis with possible pulmonary involvement in a 54-year-old male heart transplant recipient . Two months after heart transplantation, he developed a persistent, moderate dyspnea with fever and signs of liver damage . Diagnosis was made with liver biopsy for a concurrent reactivation of chronic hepatitis B virus (HBV) infection already present before transplant . Along with a mild chronic HBV hepatitis with fibrosis, we observed sinusoidal dilation and groups of bright, rounded, colorless cells with a central nucleus suggestive of cryptococci . Periodic acid-Schiff stain clearly showed encapsulated yeasts, which supported the diagnosis . Cryptococcal antigen was positive in serum and negative in the cerebrospinal fluid . Computed tomography scan of the chest demonstrated a mild interstitial infiltrate . The patient promptly responded to reduction of immunosuppressive therapy and antifungal treatment with amphotericin B lipid complex and flucytosine followed by maintenance treatment with fluconazole . Cryptococcosis should always be considered in the differential diagnosis in immunocompromised hosts with dyspnea and signs of extrapulmonary involvement . Diagnosis of extrapulmonary and extraneural cryptococcosis is difficult and often fortuitous; a histopathological examination of tissues involved is probably warranted.

Ann Pathol, 2004 Apr, 24(2), 187 - 91
{Disseminated pulmonary infection due to Cryptococcus neoformans in a non immunocompromised patient}; Hofman V et al.; We report a case of a primary pulmonary infection due to Cryptococcus neoformans developed in a 40 Year old immunocompetent and HIV negative man . Radiologic findings consisted in diffuse, bilateral reticular and nodular opacities . Bronchoscopy was normal and bronchoalveolar lavage showed numerous macrophages without associated pathogens . A thoracovideoscopy with an open lung biopsy showed numerous cryptococci, free in the alveoli or located within the macrophages . They were associated with an inflammatory infiltrate in the interalveolar spaces, predominantly composed of mononuclear cells . Ultrastructural study showed yeasts with numerous intracytoplasmic organites, a nucleus, a wall and a thick capsule . Pulmonary cryptococcosis is rare in immunocompetent hosts and can be difficult to diagnose since clinicoradiologic features observed in this cryptococcal infection mimic other infectious or neoplastic diseases.

Carbohydr Res, 2004 Jul 12, 339(10), 1779 - 86
Synthesis of a hexasaccharide fragment of the O-deacetylated GXM of C . neoformans serotype B; Zhao W et al.; beta-D-Xylp-(1-->4)-alpha-D-Manp-(1-->3)-{beta-D-Xylp-(1-->2)}-alpha-D-Manp-(1-->3)-{beta-D-Xylp-(1-->2)}-alpha-D-Manp, the fragment of the exopolysaccharide from Cryptococcus neoformans serovar B, was synthesized as its methyl glycoside . Thus, acetylation of allyl 3-O-benzoyl-4,6-O-benzylidene-alpha-D-mannopyranoside (1) followed by debenzylidenation and selective 6-O-benzoylation afforded allyl 2-O-acetyl-3,6-di-O-benzoyl-alpha-D-mannopyranoside (4) . Glycosylation of 4 with 2,3,4-tri-O-benzoyl-D-xylopyranosyl trichloroacetimidate (5) furnished the beta-(1-->4)-linked disaccharide 6 . Deallylation followed by trichloroacetimidate formation gave the disaccharide donor 8, and subsequent coupling with allyl 2,3,4-tri-O-benzoyl-beta-D-xylopyranosyl-(1-->2)-4,6-di-O-benzoyl-alpha-D-mannopyranoside (9), produced the tetrasaccharide 10 . Reiteration of deallylation and trichloroacetimidate formation from 10 yielded the tetrasaccharide donor 12 . The downstream disaccharide acceptor 18 was obtained by condensation of 5 with methyl 3-O-acetyl-4,6-O-benzylidene-alpha-D-mannopyranoside, followed by debenzylidenation, benzoylation, and selective 3-O-deacetylation . Coupling of 18 with 12 afforded the hexasaccharide 19, and subsequent deprotection gave the hexasaccharide glycoside 20 . Selective 2"-O-deacetylation of 19 gave the hexasaccharide acceptor 21 . Condensation of 21 with glucopyranosyluronate imidate 22 did not produce the expected heptasaccharide glycoside; instead, a transacetylation product 19 was obtained . Meanwhile, there was no reaction between 21 and the bromide donor 23.

Jpn J Infect Dis, 2004 Jun, 57(3), 113 - 5
Serotypes of clinical cerebrospinal fluid Cryptococcus neoformans isolates from southern Taiwan and their in vitro susceptibilities to amphotericin B, fluconazole, and voriconazole; Chang WN et al.; In this study, 34 clinical cerebrospinal fluid isolates of Cryptococcus neoformans were serotyped, and their in vitro susceptibilities to amphotericin B, fluconazole, and voriconazole were analyzed . Of these 34 isolates, serotype A was found in 29 isolates and serotype B in the other five . The voriconazole geometric mean MIC was significantly lower than the amphotericin B/antibiotic medium 3 geometric mean MIC (P < 0.0001 at both 48 and 72 h), as well as the fluconazole geometric mean MIC (P < 0.0001 at both 48 and 72 h) . Of the three antifungal agents, only fluconazole, with geometric mean MICs at both 48 and 72 h, showed significant difference between the serotypes A and B of C . neoformans.

Antimicrob Agents Chemother, 2004 Jul, 48(7), 2724 - 6
In vitro antifungal susceptibilities of uncommon basidiomycetous yeasts; Serena C et al.; The in vitro activities of eight antifungal drugs against 50 isolates of basidiomycetous yeasts were determined by a microdilution method . In general fluconazole and micafungin were inactive . Terbinafine was active only against Sporobolomyces salmonicolor . The activities of the other antifungals were variable and depended on the species tested . The new triazoles showed the lowest MICs, but amphotericin B and itraconazole were the only drugs active against Cryptococcus albidus.

Infect Immun, 2004 Jul, 72(7), 4271 - 4
Antibodies produced in response to Cryptococcus neoformans pulmonary infection in mice have characteristics of nonprotective antibodies; Zaragoza O et al.; Murine cryptocococcal pulmonary infection elicited serum immunoglobulin M (IgM) and IgG to the capsular polysaccharide, but only IgG stained yeast cells in alveoli . Both isotypes produced punctuate immunofluorescence patterns on yeast cells like those of nonprotective antibodies . The difficulties involved in associating humoral immunity with protection in murine cryptocococcal infection could reflect nonprotective antibody responses.

Mikrobiologiia, 2004 Mar-Apr, 73(2), 226 - 32
{Seasonal dynamics in a yeast population on the Oxalis acetosella L . leaves}; Glushakova AM et al.; Analysis of an epiphytic yeast population on the leaves of the evergreen common wood sorrel Oxalis acetosella L . throughout a year showed that the density and the species composition of this population underwent regular seasonal changes . There were almost no yeasts on the young spring leaves . However, the yeast population on the mature leaves tended to increase in the autumn, reaching a maximum after the formation of continuous snow cover . Then the yeast population on the leaves tended to decrease, reaching a minimum in the spring . The species diversity of the yeasts was maximum in the autumn . The population of the epiphytic yeast species Cystofilobasidium capitatum, Rhodotorula fujisanensis, Leucosporium scottii, and Cryptococcus flavus peaked in the autumn . On the other hand, the population of the widespread epiphytic species Cryptococcus laurentii on the wood sorrel leaves peaked in January . The relative abundance of the red-pigmented phytobionts Rhodotorula glutinis and Sporobolomyces roseus virtually did not change throughout the year . The relative abundance of the euribiotic species Cryptococcus albidus showed irregular monthly variations . The data obtained show that the epiphytic microbial population of various plants can be comprehensively studied only by analyzing this population throughout the vegetative period of the plants.

Clin Microbiol Infect, 2004 Jun, 10(6), 590 - 2
Simplified sunflower (Helianthus annuus) seed agar for differentiation of Candida dubliniensis from Candida albicans; Khan ZU et al.; This study evaluated sunflower (Helianthus annuus) seed agar (SSA) for differentiation of Candida dubliniensis from Candida albicans on the basis of colony characteristics and chlamydospore production . Simplified SSA without creatinine and KH(2)PO(4) was also used . On both media, C . dubliniensis isolates (n = 25) developed rough colonies and formed abundant chlamydospores after incubation for 24-48 h at 28 degrees C, while C . albicans isolates (n = 53) showed smooth colonies with no evidence of chlamydospore formation . Cryptococcus neoformans isolates (n = 10) formed brown colonies on both media . Simplified SSA offers a simple and inexpensive tool for presumptive differentiation of C . dubliniensis from C . albicans in clinical microbiology laboratories.

Respir Med, 2004 Jun, 98(6), 488 - 94
Non-infectious and unusual infectious mimics of community-acquired pneumonia; Alves dos Santos JW et al.; Noninfectious or unusual infectious diseases may present with clinical, radiological and laboratorial characteristics of community-acquired pneumonia (CAP) . Usually their presence is only suspected after treatment failure, leading to inappropriate interventions, unnecessary costs and risks related to the untreated potentially life-threatening disease . The present study aimed to assess the noninfectious or unusual infectious diseases that may be misdiagnosed as CAP that progresses with treatment failure . Sixteen hospitalized patients with presumptive diagnosis of CAP and treatment failure were described . The most prevalent symptoms were fever and cough . Radiological pattern of air-space disease was observed in 10 (62%) patients . The diagnosis was established by autopsy (12%) or invasive procedures (88%), as follows: open lung biopsy (nine), flexible fiberoptic bronchoscopy (two), transthoracic fine needle aspiration (two) and bone marrow aspiration (one) . Eight patients had noninfectious diseases: pulmonary embolism, cryptogenic organizing pneumonia, Wegener's granulomatosis, hypersensitivity pneumonitis, bronchocentric granulomatosis, neoplastic disease and acute leukemia . The unusual infectious diseases were: tuberculosis, cryptococcosis, actinomycosis, histoplasmosis and paracoccidioidomycosis . Patients with noninfectious or unusual infectious diseases may present with symptoms and radiological findings that mimic CAP . These diseases should always be suspected in patients who do not respond to initial empirical antimicrobial treatment, especially young patients or those without comorbidity.

Ann Allergy Asthma Immunol, 2004 May, 92(5), 483 - 91; quiz 492-4, 575
Clinical use of immunoassays in assessing exposure to fungi and potential health effects related to fungal exposure; Trout DB et al.; OBJECTIVE: To review and summarize current evidence regarding the proper role of immunoassays in clinical assessments of exposure to fungi and health effects related to fungal exposure . DATA SOURCES: We reviewed relevant scientific investigations and previously published reviews concerning this topic . STUDY SELECTION: The authors' clinical, laboratory, and public health experiences were used to evaluate relevant data for scientific merit . RESULTS: Testing to determine the presence of IgE to specific fungi may be a useful component of a complete clinical evaluation in the diagnosis of illnesses that can be caused by immediate hypersensitivity such as allergic rhinitis and asthma . Detection of IgG to specific fungi has been used as a marker of exposure to agents that may cause illnesses such as hypersensitivity pneumonitis . However, the ubiquitous nature of many fungi and the lack of specificity of fungal antigens limit the usefulness of these types of tests in the evaluation of potential building-related illness and fungal exposure . Specific serologic tests (such as tests for cryptococcal antigen, coccidioidal antibody, and Histoplasma antigen) have been shown to be useful in the diagnosis of some fungal infections, but these are the exception not the rule . CONCLUSIONS: There is currently not enough scientific evidence to support the routine clinical use of immunoassays as a primary means of assessing environmental fungal exposure or health effects related to fungal exposure . Health care providers who care for persons expressing concerns about the relationship of symptoms to potential exposure to fungi are advised to use immunoassay results with care and only as an adjunct to a comprehensive approach to patient care.

Eukaryot Cell, 2004 Jun, 3(3), 752 - 63
Novel chimeric spermidine synthase-saccharopine dehydrogenase gene (SPE3-LYS9) in the human pathogen Cryptococcus neoformans; Kingsbury JM et al.; The Cryptococcus neoformans LYS9 gene (encoding saccharopine dehydrogenase) was cloned and found to be part of an evolutionarily conserved chimera with SPE3 (encoding spermidine synthase) . spe3-lys9, spe3-LYS9, and SPE3-lys9 mutants were constructed, and these were auxotrophic for lysine and spermidine, spermidine, and lysine, respectively . Thus, SPE3-LYS9 encodes functional spermidine synthase and saccharopine dehydrogenase gene products . In contrast to Saccharomyces cerevisiae spe3 mutants, the polyamine auxotrophy of C . neoformans spe3-LYS9 mutants was not satisfied by spermine . In vitro phenotypes of spe3-LYS9 mutants included reduced capsule and melanin production and growth rate, while SPE3-lys9 mutants grew slowly at 30 degrees C, were temperature sensitive in rich medium, and died upon lysine starvation . Consistent with the importance of saccharopine dehydrogenase and spermidine synthase in vitro, spe3-lys9 mutants were avirulent and unable to survive in vivo and both functions individually contributed to virulence . SPE3-LYS9 mRNA levels showed little evidence of being influenced by exogenous spermidine or lysine or starvation for spermidine or lysine; thus, any regulation is likely to be posttranscriptional . Expression in S . cerevisiae of the full-length C . neoformans SPE3-LYS9 cDNA complemented a lys9 mutant but not a spe3 mutant . However, expression in S . cerevisiae of a truncated gene product, consisting of only C . neoformans SPE3, complemented a spe3 mutant, suggesting possible modes of regulation . Therefore, we identified and describe a novel chimeric SPE3-LYS9 gene, which may link spermidine and lysine biosynthesis in C . neoformans .

J Immunol, 2004 Jun 15, 172(12), 7629 - 34
Accumulation of gammadelta T cells in the lungs and their regulatory roles in Th1 response and host defense against pulmonary infection with Cryptococcus neoformans; Uezu K et al.; The present study was designed to elucidate the role of gammadelta T cells in the host defense against pulmonary infection with Cryptococcus neoformans . The gammadelta T cells in lungs commenced to increase on day 1, reached a peak level on day 3 or 6, and then decreased on day 10 after intratracheal infection . The increase of these cells was similar in monocyte chemoattractant protein (MCP)-1-deficient mice, although that of NK and NKT cells was significantly reduced . The number of live microorganisms in lungs on days 14 and 21 was significantly reduced in mice depleted of gammadelta T cells by a specific mAb compared with mice treated with control IgG . Similarly, elimination of this fungal pathogen was promoted in gammadelta T cell-deficient (TCR-delta(-/-)) mice compared with control littermate mice . Finally, lung and serum levels of IFN-gamma on days 7 and 14 and on day 7 postinfection, respectively, were significantly higher in TCR-delta(-/-) mice than in littermate mice, whereas levels of TGF-beta showed the opposite results . IL-4 and IL-10 were not different between these mice . IFN-gamma production by draining lymph node cells upon restimulation with cryptococcal Ags was significantly higher in the infected TCR-delta(-/-) mice than in control mice . Our results demonstrated that gammadelta T cells accumulated in the lungs in a manner different from NK and NKT cells after cryptococcal infection and played a down-modulatory role in the development of Th1 response and host resistance against this fungal pathogen.

Microbiology, 2004 Jun, 150(Pt 6), 1925 - 35
The RAM1 gene encoding a protein-farnesyltransferase beta-subunit homologue is essential in Cryptococcus neoformans; Vallim MA et al.; Many small G proteins require post-translational modification to allow functional association to the cell membrane . This process often involves the enzymic addition of hydrophobic prenyl groups to a conserved cysteine residue near the C-terminus of the protein . The enzymes that catalyse these reactions include protein farnesyltransferase and protein geranylgeranyltransferases . The human fungal pathogen Cryptococcus neoformans requires functional Ras and Rho proteins in order to undergo normal growth and differentiation . Since farnesylation and geranylgeranylation are likely required for the proper function of these small G proteins, we hypothesized that inhibition of these prenylation events would alter the growth and cellular morphogenesis of this fungus . We cloned the RAM1 gene encoding the single protein-farnesyltransferase beta-chain homologue in C . neoformans . Using a gene-disruption strategy in a diploid C . neoformans strain, we demonstrated that this gene encodes an essential function, in contrast to the case in Saccharomyces cerevisiae, in which the homologous RAM1 gene is not essential for growth . Pharmacological inhibition of farnesyltransferase activity resulted in dose-dependent cytostasis of C . neoformans, as well as prevention of hyphal differentiation . Simultaneous inhibition of farnesylation and calcineurin signalling results in a synthetic effect on growth . Protein farnesylation is required for the growth and cellular differentiation of C . neoformans and may provide novel targets for antifungal therapy.

J Clin Microbiol, 2004 Jun, 42(6), 2786 - 8
Immunoreactivity of cryptococcal antigen is not stable under prolonged incubations in human serum; McFadden DC et al.; The stability of cryptococcal antigen from Cryptococcus neoformans serotype A and D strains at different temperatures in serum and other solvents was studied . Samples stored at -20 or 4 degrees C had equivalent stabilities as measured by the Premier Cryptococcal Antigen kit and the Cryptococcal Antigen Latex Agglutination System (CALAS) kit . However, using the Premier Cryptococcal Antigen kit, there was a 91% loss of reactivity in samples incubated in human serum for 4 weeks at 37 degrees C . A loss of reactivity of more than 99% was observed after incubation at 45 degrees C for 4 weeks . The capsular antigen was not detected by the Premier Cryptococcal Antigen kit after 16 weeks at elevated temperatures . Antigen titers were also reduced in a latex agglutination assay (CALAS) after 4 weeks at 37 and 45 degrees C . The loss of antigen reactivity was a function of pH and temperature.

FEMS Microbiol Lett, 2004 Jun 15, 235(2), 311 - 4
Synthesis and immunogenicity of polysaccharide-protein conjugate composed of galactoglucoxylomannan of Cryptococcus laurentii; Bystricky S et al.; Galactoglucoxylomannan (GalGXMan) antigen of Cryptococcus laurentii was conjugated to protein carrier by a simple one step reaction . The conjugate was immunogenic in rabbits and reinjection elicited booster response with significant increase of serum IgG (H+L) level . Induction of this Ig-isotype was confirmed in experiments with Protein A . Effectiveness of immune serum to inhibit the growth of C . laurentii was demonstrated . The results indicate that the prepared conjugate could be considered as effective immunogen with potential for incorporation in the vaccine.

J Infect Dis, 2004 Jun 15, 189(12), 2185 - 91 Epub 2004 May 24.
Recombinant interferon- gamma 1b as adjunctive therapy for AIDS-related acute cryptococcal meningitis; Pappas PG et al.; We conducted a phase 2, double-blind, placebo-controlled study to evaluate the safety and antifungal activity of adjuvant recombinant interferon (rIFN)- gamma 1b in patients with acquired immunodeficiency syndrome and acute cryptococcal meningitis . Patients received 100 or 200 microg of rIFN- gamma 1b or placebo, thrice weekly for 10 weeks, plus standard therapy with intravenous amphotericin B, with or without flucytosine, followed by therapy with fluconazole . End points included conversion of cerebrospinal fluid fungal cultures from positive to negative at 2 weeks, resolution of symptoms, and survival . Among 75 patients, 2-week culture conversion occurred in 13% of placebo recipients, 36% of rIFN- gamma 1b (100 microg) recipients, and 32% of rIFN- gamma 1b (200 microg) recipients . There was a trend toward improved combined mycologic and clinical success in rIFN- gamma 1b recipients (26% vs . 8%; P=.078) . Therapy with rIFN- gamma 1b was well tolerated, and there was no apparent influence on serial CD4 cell counts and human immunodeficiency virus load measurements . Adjunctive therapy with rIFN- gamma 1b holds promise for patients with acute cryptococcal meningitis and warrants further study.

Mycopathologia, 2004 Apr, 157(3), 263 - 4
First isolation of Cryptococcus magnus from a cat; Kano R et al.; A 6-month-old male Japanese domestic cat with otitis externa due to Aspergillus fumigatus was treated with antifungal agents for 25 days and appeared to be cured . Many yeast colonies however developed from the ear canal samples on Sabouraud's dextrose agar at 27 degrees C for 5 days, instead of A . fumigatus . This yeast colony was cream-colored and slim in texture with smooth and highly glossy surface after 5-day incubation on Sabouraud's dextrose agar at 27 degrees C . The isolate was identified as Cryptococcus magnus by mycological analysis and 28S ribosomal analysis.

Kansenshogaku Zasshi, 2004 Apr, 78(4), 327 - 30
{Primary pulmonary cryptococcosis exhibiting the radiological characteristics of bronchiolitis obliterans organizing pneumonia}; Kishi K et al.; A 31-year-old male was admitted to Toranomon Hospital because of a cough and bilateral patchy infiltrates shown on the chest radiograph . He had been well prior to admission . Chest CT scan revealed patchy areas of air-space consolidation with air-bronchogram and adjacent ground-glass opacities, suggestive of bronchiolitis obliterans organizing pneumonia (BOOP) . Transbronchial lung biopsy specimen confirmed the formation of epithelioid cell granulomas without necrosis and the coexistence of organizing pneumonia . The titer of serum cryptococcal antigen increased to 1:256 . According to these findings, a diagnosis of primary pulmonary cryptococcosis was made, although cryptococci were neither recognized in the specimen nor cultured from the bronchial lavaged fluid . Chest radiograph showed spontaneous regression in a short period of time . However, itraconazole was administered for the prolonged cough, and the symptom disappeared . The titer of serum cryptococcal antigen decreased to 1:16 after the therapy . Pulmonary cryptococcosis should be considered as one of the differential diagnoses when chest CT scan shows combined air-space consolidation and ground-glass opacities.

Lancet, 2004 May 29, 363(9423), 1764 - 7
Combination antifungal therapies for HIV-associated cryptococcal meningitis: a randomised trial; Brouwer AE et al.; BACKGROUND: It frequently takes more than 2 weeks for drug treatments for cryptococcal meningitis to sterilise cerebrospinal fluid (CSF) . In-vitro and animal studies lend support to the use of combinations of amphotericin B, flucytosine, and fluconazole for treatment of cryptococcosis . We compared the fungicidal activity of combinations of these drugs for initial treatment of patients with cryptococcal meningitis . METHODS: 64 patients with a first episode of HIV-associated cryptococcal meningitis were randomised to initial treatment with: amphotericin B (0.7 mg/kg daily); amphotericin B plus flucytosine (100 mg/kg daily); amphotericin B plus fluconazole (400 mg daily); or triple therapy with amphotericin B, flucytosine, and fluconazole . Our primary endpoint was fungicidal activity, measured by the rate of reduction in CSF cryptococcal colony-forming units (CFU) from serial quantitative CSF cultures on days 3, 7, and 14 of treatment . FINDINGS: Baseline CSF CFU counts were an important prognostic factor . Clearance of cryptococci from the CSF was exponential and was significantly faster with amphotericin B plus flucytosine than with amphotericin B alone (p=0.0006), amphotericin B plus fluconazole ( p=0.02), or triple therapy (p=0.02) . INTERPRETATION: At these doses, amphotericin B plus flucytosine is the most rapidly fungicidal regimen . Quantification of CSF cultures provides a powerful new means to accurately assess the fungicidal activity of new treatment regimens for cryptococcal meningitis.

Respir Care, 2004 Jun, 49(6), 606 - 8
Pneumonia due to Cryptococcus neoformans in a patient receiving infliximab: possible zoonotic transmission from a pet cockatiel; Shrestha RK et al.; The use of humanized antibody against tumor necrosis factor alpha (TNF-alpha) may increase the risk of various opportunistic infections, including tuberculosis and fungal infections . We report a case of cryptococcal pneumonia in a patient who was taking infliximab for rheumatoid arthritis . A temporally related exposure history raised the possibility that our patient acquired the infection from his pet cockatiel . It seems prudent to advise patients receiving infliximab to avoid exposure to pet avian excreta.

Int J Food Microbiol, 2004 Jun 15, 93(3), 297 - 304
Sodium bicarbonate enhances biocontrol efficacy of yeasts on fungal spoilage of pears; Yao H et al.; The addition of 2% (w/v) sodium bicarbonate (SBC) in the suspensions of antagonistic yeast Cryptococcus laurentii or Trichosporon pullulans significantly limited spore germination and germ tube elongation of Penicillium expansum and Alternaria alternata in potato dextrose broth (PDB) medium . Biocontrol activity of C . laurentii or T . pullulans against postharvest decay caused by P . expansum and A . alternata in pear fruits was significantly increased when C . laurentii or T . pullulans combined with SBC . Combining C . laurentii or T . pullulans with SBC provided a more effective control on P . expansum and A . alternata than applying the antagonistic yeast or SBC alone . Effects of C . laurentii with and without SBC on controlling P . expansum and A . alternata were better than those of T . pullulans . C . laurentii in combination with SBC showed the best control of disease caused by A . alternata in pear fruits.

Drugs, 2004, 64(11), 1163 - 80
Mucosal and systemic fungal infections in patients with AIDS: prophylaxis and treatment; Ruhnke M; In countries where highly active antiretroviral therapy (HAART) is widely available, a decrease in the incidence of fungal infections has been observed in the last 5 years compared with countries that cannot afford this treatment . Even refractory fungal infections may be controlled when HAART is given to patients, and end-stage AIDS infections, such as aspergillosis, are now only infrequently seen . In contrast, fungal infections in certain regions, such as penicilliosis in Southeast Asia or cryptococcosis in Sub-Saharan Africa, are a growing problem . Antifungal therapy for documented infections has not changed very much during recent years; however, new drugs such as caspofungin and voriconazole may be more effective in the treatment of opportunistic fungal infections, in particular, those involving resistant organisms . Secondary antifungal prophylaxis for many opportunistic pathogens can now be temporarily or even permanently discontinued in many HIV-positive patients who have a marked improvement in immune function parameters, such as CD4(+) cell counts, after initiation of HAART . The link between effective virustatic control of HIV infection and a decreasing incidence of fungal infections has been recognised; and so, despite the availability of very effective new antifungal drugs, the cornerstone of treatment and prevention of opportunistic fungal infections in patients with HIV infection is effective antiretroviral therapy including protease inhibitors.

Seizure, 2004 Jul, 13(5), 358 - 61
Focal nonconvulsive status epilepticus associated to PLEDs and intense focal hyperemia in an AIDS patient; Cury RF et al.; PURPOSE: Periodic lateralised epileptiform discharges (PLEDs) can be seen associated to nonconvulsive status epilepticus (NCSE), although their pathophysiological meaning remains questionable . Functional neuroimaging has suggested that, in this setting, PLEDs may indeed be an ictal pattern . In this report we describe perfusional changes in a patient with AIDS, PLEDs and NCSE . METHODS: A 37-year-old man with AIDS, cryptococcosis and recurrent epileptic seizures was admitted . After initial treatment, he remained comatose, and had MRI and serial EEG recordings performed . Technetium-99m-ethyl cysteinate dimer (99mTc-ECD) SPECT scans were also obtained, before and after continuous benzodiazepine infusion . RESULTS: EEG disclosed PLEDs over the right fronto-polar region while MRI revealed meningeal thickening and scattered unspecific findings . SPECT revealed marked focal hyperperfusion overlapping the areas with PLEDs, both resolved after continuous midazolam infusion and clinical improvement . CONCLUSIONS: This report demonstrates association of PLEDs, NCSE, and focal hyperperfusion on SPECT, additionally supporting the concept of PLEDs as an ictal pattern . Considering that status epilepticus may eventually not be detected by conventional approaches alone, we advocate the use of functional neuroimaging to assess suspected patients with impaired consciousness.

Clin Imaging, 2004 May-Jun, 28(3), 191 - 5
Pulmonary cryptococcosis in immunocompetent patients: HRCT characteristics; Murayama S et al.; We analyzed the high-resolution CT (HRCT) findings of cryptococcosis in immunocompetent patients . The predominant manifestations were multiple nodules (n=6) and a single nodule (n=7) . Regarding the pattern of multiple nodules, two cases of cavities or necrosis, four cases of surrounding centrilobular micronodules and five cases of "acinar" nodules were seen . No "tree-in-bud" appearance was detected . Five of seven cases of a single nodule were classified as polygonal, and two of them were round . Two cases accompanied micronodules and one case cavitation . Although no "tree-in-bud" appearance was observed, pulmonary cryptococcosis mimics tuberculosis.

Infect Immun, 2004 Jun, 72(6), 3674 - 9
Antibody to Cryptococcus neoformans glucuronoxylomannan inhibits the release of capsular antigen; Martinez LR et al.; Cryptococcus neoformans releases capsular polysaccharide in the supernatant of liquid cultures and in tissues . Significantly less glucuronoxylomannan (GXM) was released by C . neoformans in the presence of capsule-binding monoclonal antibody (MAb) . MAb-mediated inhibition of GXM release may be another mechanism by which humoral immunity can mediate protection against this pathogen.

Infect Immun, 2004 Jun, 72(6), 3643 - 5
The alpha-specific cell identity factor Sxi1alpha is not required for virulence of Cryptococcus neoformans; Hull CM et al.; Cryptococcus neoformans is a human fungal pathogen that has two mating types (a and alpha) . Experiments have shown that in some backgrounds alpha strains are more virulent than a strains . Our studies reveal that the only known alpha-specific factor, SXI1alpha, is not necessary for virulence.

Infect Immun, 2004 Jun, 72(6), 3478 - 88
Interaction of Blastomyces dermatitidis, Sporothrix schenckii, and Histoplasma capsulatum with Acanthamoeba castellanii; Steenbergen JN et al.; Several dimorphic fungi are important human pathogens, but the origin and maintenance of virulence in these organisms is enigmatic, since an interaction with a mammalian host is not a requisite for fungal survival . Recently, Cryptococcus neoformans was shown to interact with macrophages, slime molds, and amoebae in a similar manner, suggesting that fungal pathogenic strategies may arise from environmental interactions with phagocytic microorganisms . In this study, we examined the interactions of three dimorphic fungi with the soil amoeba Acanthameobae castellanii . Yeast forms of Blastomyces dermatitidis, Sporothrix schenckii, and Histoplasma capsulatum were each ingested by amoebae and macrophages, and phagocytosis of yeast cells resulted in amoeba death and fungal growth . H . capsulatum conidia were also cytotoxic to amoebae . For each fungal species, exposure of yeast cells to amoebae resulted in an increase in hyphal cells . Exposure of an avirulent laboratory strain of H . capsulatum to A . castellanii selected for, or induced, a phenotype of H . capsulatum that caused a persistent murine lung infection . These results are consistent with the view that soil amoebae may contribute to the selection and maintenance of certain traits in pathogenic dimorphic fungi that confer on these microbes the capacity for virulence in mammals.

Infect Immun, 2004 Jun, 72(6), 3359 - 65
Cryptococcus neoformans capsule structure evolution in vitro and during murine infection; Garcia-Hermoso D et al.; Cryptococcus neoformans capsule structure modifications after prolonged in vitro growth or in vivo passaging have been reported previously . However, nothing is known about the dynamics of these modifications or about their environmental specificities . In this study, capsule structure modifications after mouse passaging and prolonged in vitro culturing were analyzed by flow cytometry using the glucuronoxylomannan-specific monoclonal antibody E1 . The capsule structures of strains recovered after 0, 1, 8, and 35 days were compared by using the level of E1-specific epitope expression and its cell-to-cell heterogeneity within a given cell population . In vitro, according to these parameters, the diversity of the strains was higher on day 35 than it was initially, suggesting the absence of selection during in vitro culturing . In contrast, the diversity of the strains recovered from the brain tended to decrease over time, suggesting that selection of more adapted strains had occurred . The strains recovered on day 35 from the spleen and the lungs had different phenotypes than the strains isolated from the brain of the same mouse on the same day, thus strongly suggesting that there is organ specificity for C . neoformans strain selection . Fingerprinting of the strains recovered in vitro and in vivo over time confirmed that genotypes evolved very differently in vitro and in vivo, depending on the environment . Overall, our results suggest that organ-specific selection can occur during cryptococcosis.

Antimicrob Agents Chemother, 2004 Jun, 48(6), 2014 - 20
Effects of voriconazole on Cryptococcus neoformans; van Duin D et al.; Voriconazole is a broad-spectrum triazole that offers extended activity against molds and yeasts that are not susceptible to earlier azole-type drugs . Recent studies indicate that melanization can severely reduce the susceptibility of certain antifungal drugs, but there is no information as to whether voriconazole is vulnerable to this effect . The activity of voriconazole on C . neoformans was assessed by MIC analysis and time-kill assays for melanized and nonmelanized cells . Cell morphology, capsule release, and phagocytosis of C . neoformans were studied in the presence or absence of subinhibitory concentrations of voriconazole . Voriconazole was fungicidal at concentrations of >/=8 microg/ml in vitro against the strains of C . neoformans examined, and its efficacy was not diminished by melanization . Cells grown in subinhibitory concentrations of voriconazole had smaller cellular and capsular volumes than cells grown in the absence of drug . The induction of the capsule by serum was not affected by voriconazole . Cells grown in subinhibitory concentrations of voriconazole released their capsule and were phagocytosed at rates comparable with yeast grown without the antifungal . The high activity of voriconazole against both melanized and nonmelanized cells results suggest that voriconazole may be a particularly valuable drug for cryptococcosis.

Curr Genet, 2004 Aug, 46(2), 92 - 102 Epub 2004 May 19.
Investigation of the basis of virulence in serotype A strains of Cryptococcus neoformans from apparently immunocompetent individuals; D'Souza CA et al.; Cryptococcus neoformans serotype A strains commonly infect immunocompromised patients to cause fungal meningitis . To understand the basis of serotype A cryptococcal infections in apparently immunocompetent patients, we tested two hypotheses: the strains were naturally occurring hypervirulent pkr1 (PKA regulatory subunit) mutants, or the strains were hybrids with C . neoformans var . gattii strains that normally infect immunocompetent individuals . Analysis of clinical isolates obtained from apparently immunocompetent individuals from three continents revealed that none were pkr1 mutants, but several exhibited phenotypes consistent with perturbations in cAMP signaling . Additionally, none of the strains were unusual hybrids with gattii strains . Except for one strain that was an AD hybrid, all others were serotype A (var . grubii) isolates . Taken together, our findings indicate that the ability of these clinical isolates to infect apparently normal individuals may be attributable to mutations other than pkr1 and/or underlying immune system impairment in patients.

J Med Microbiol, 2004 Jun, 53(Pt 6), 509 - 14
ELISA for early diagnosis of histoplasmosis; Guimaraes AJ et al.; An ELISA was developed and evaluated as a method for detecting antibodies against glycosylated and deglycosylated histoplasmin (HMIN) . Sera from patients with histoplasmosis, paracoccidioidomycosis, sporotrichosis, coccidioidomycosis, aspergillosis, cryptococcosis and healthy donors were tested by ELISA against purified, deglycosylated histoplasmin (ptHMIN) and compared with purified, native (i.e . glycosylated) histoplasmin (pHMIN) . Although cross-reactivity was not abolished when ptHMIN was used in the test, it was reduced (pHMIN ELISA 93 % versus ptHMIN ELISA 96 %) . However, there were statistically significant differences between the sensitivities of these two methods for the detection of antibodies (pHMIN ELISA 57 % versus ptHMIN ELISA 92 %; P < 0.001) and between the efficiency of the methods (pHMIN ELISA 83 % versus ptHMIN ELISA 95 %; P < 0.001) . These parameters compare better than previously published data relating to the use of treated HMIN in diagnostic ELISAs . Some of the reactivities of serum samples were compared by immunoblotting using deglycosylated HMIN and by immunodiffusion using the crude antigen . The results demonstrated that cross-reactions with heterologous sera in both ELISAs could also be observed in immunoblotting and arose from shared protein epitopes . These data suggest that ELISA using deglycosylated HMIN is a very sensitive diagnostic method and, by using commercially available antigen, it can be easily standardized and performed faster than previous Western blot-based tests using the same antigen . It provides a useful adjunct to existing methods of diagnosis that could be applied even in situations where laboratory facilities were relatively limited.

J Chin Med Assoc, 2004 Feb, 67(2), 89 - 92
Disseminated cryptococcosis with pulmonary and marrow involvement mimicking radiological features of malignancy; Su CT et al.; The most commonly involved sites of cryptococcosis are the lungs and the central nervous system . Cryptococcal osteomyelitis is a rare complication of disseminated cryptococcosis, and the vertebraes are the most common site of this infection . The most common underlying disease is sarcoidosis, followed by tuberculosis and previous steroid therapy . Conservative treatment alone or treatment with a combination of the medical and surgical curettage is successful in most cases . We report a case of cryptococcal osteomyelitis in a 63-year-old immunocompetent male who presented with lower back pain over the sacral region for several years . Radiologic studies showed a pulmonary mass and a radiolytic lesion involving the left ischial bone, which mimicked pulmonary malignancy with bone metastasis . Biopsy of the lung mass and the bone lesion revealed abundant cryptococcal organisms, and cryptococcal osteomyelitis was diagnosed.

Eur J Neurol, 2004 May, 11(5), 297 - 304
Guidelines for the diagnosis and management of neurological complications of HIV infection; Portegies P et al.; The spectrum of neurological complications of HIV-infection has remained unchanged through the years, but its epidemiology changed remarkably as a result of the introduction of highly active antiretroviral therapy (HAART) . Guidelines for the diagnosis and treatment of cerebral toxoplasmosis, cryptococcal meningitis, progressive multifocal leukoencephalopathy, CMV encephalitis, CMV polyradiculomyelitis, tuberculous meningitis, primary CNS lymphoma, HIV dementia, HIV myelopathy and HIV polyneuropathy are given with a grading of evidence and recommendations.

Eur J Clin Microbiol Infect Dis, 2004 Jun, 23(6), 506 - 8 Epub 2004 May 13.
In vitro activity of amphotericin B, fluconazole and voriconazole against 162 Cryptococcus neoformans isolates from Africa and Cambodia; Chandenier J et al.; In order to determine the potential role that various antifungal agents might have in the management of cryptococcosis in tropical areas, the in vitro susceptibility of Cryptococcus neoformans isolates from Africa ( n=52) and Cambodia ( n=110) to three antifungal agents (amphotericin B, fluconazole and voriconazole) were compared using the E-test method . The results of this study (i) confirm the value of the E-test for testing the in vitro susceptibility of C . neoformans towards voriconazole; (ii) provide the first evidence demonstrating good activity of amphotericin B, fluconazole and voriconazole against Cambodian isolates; and (iii) show there are differences in susceptibility between African and Asian C . neoformans isolates, with Cambodian isolates appearing less susceptible to the agents tested but with amphotericin B maintaining good activity.

Eur J Clin Microbiol Infect Dis, 2004 Jun, 23(6), 471 - 6 Epub 2004 May 13.
Adenosine deaminase activity in cerebrospinal fluid of HIV-infected patients: limited value for diagnosis of tuberculous meningitis; Corral I et al.; Adenosine deaminase activity (ADA) determination in cerebrospinal fluid (CSF) is considered a specific test for the diagnosis of tuberculous meningitis . In order to study the variability of this marker in patients with different neurological disorders associated with HIV infection, and its utility for the diagnosis of tuberculous meningitis in these patients, the ADA levels in 417 CSF samples from HIV-infected patients with neurological symptoms were reviewed . HIV infection, HIV-associated neurological disorders, and progressive multifocal leukoencephalopathy were not associated with elevated ADA in CSF . Among patients with meningitis, receiver operating characteristic curve analysis gave an optimal ADA cut-off point of 8.5 IU/l for the diagnosis of tuberculous meningitis, with 57% sensitivity, 87% specificity, and an area under the curve of 0.747 (similar to that for CSF glucose concentration) . False-positive results were found in patients with neurological CMV disease and cryptococcal, lymphomatous, and probable candidal meningitis . The results of this study indicate that ADA determination in CSF has limited utility for the diagnosis of tuberculous meningitis in HIV-infected patients.

HIV Med, 2004 May, 5(3), 140 - 3
A multicentre, randomized, double-blind, placebo-controlled trial of primary cryptococcal meningitis prophylaxis in HIV-infected patients with severe immune deficiency; Chetchotisakd P et al.; OBJECTIVES: To assess the efficacy and survival benefit of low-dose fluconazole (400 mg weekly) for primary prophylaxis for cryptococcal meningitis in patients with advanced HIV infection . METHODS: A prospective multicentre, randomized, double-blind, placebo-controlled study was carried out in HIV-infected patients with CD4 counts <100 cells/microL . RESULTS: Of 90 patients enrolled, 44 received fluconazole and 46 received placebo . The baseline characteristics were similar in the two groups . On an intent-to-treat basis, 10 cases of cryptococcal meningitis developed, three (6.8%) in the fluconazole group and seven (15.2%) in the placebo group . Patients in the placebo group were more likely to develop cryptococcal meningitis than those in the fluconazole group {hazard ratio=2.23; 95% confidence interval (CI): 0.58-8.63; P=0.245} . The survival benefit of fluconazole was greater than that of the placebo . The number of deaths per 10 000 person-days was 2.7 for the fluconazole group (2/7342) and 11.7 for the placebo group (9/7713) (rate difference=9; 95% CI: 0.4-17.5; P=0.046) . Based on survival analysis, patients in the placebo group were 4.3 times more likely to die than those in the fluconazole group (95% CI: 0.9-19.8; P=0.065) . CONCLUSION: Fluconazole 400 mg once weekly for primary prophylaxis for cryptococcal meningitis in Thailand should be considered in HIV-infected patients, as our study suggested a survival benefit . However, a larger study should be conducted to confirm our findings.

J Feline Med Surg, 2004 Jun, 6(3), 189 - 98
A retrospective analysis of feline bronchoalveolar lavage cytology and microbiology (1995-2000); Foster SF et al.; A retrospective study of 88 bronchoalveolar lavage specimens from 80 cats presenting to the University of Sydney Veterinary Centre between 1995 and 2000 was performed . Bronchoalveolar lavage cytology and microbiology in conjunction with other diagnostic aids and patient records were used to classify cases as lower respiratory tract infections (LTRIs), LRTI revisits, feline bronchial disease, cardiac disease, neoplastic disease and multisystemic disease . Cases for which a definitive diagnosis could not be made were classified as inconclusive . Infectious agents identified were Mycoplasma spp., Pasteurella spp., Bordetella bronchiseptica, Salmonella typhimurium, Pseudomonas sp., Cryptococcus neoformans, Aelurostrongylus abstrusus and Eucoleus aerophilus . The study highlights the importance of bronchoalveolar lavage cytology and microbiology in the evaluation of feline lower respiratory tract disease.

J Feline Med Surg, 2004 Jun, 6(3), 167 - 80
Lower respiratory tract infections in cats: 21 cases (1995-2000); Foster SF et al.; Twenty-one lower respiratory tract infections diagnosed in cats at University of Sydney Veterinary Centre between 1995 and 2000 were identified retrospectively . Patient records were analysed to determine historical, clinical, clinicopathologic and radiographic features of lower respiratory tract infections . Response to therapy was also assessed . Infectious agents identified were Mycoplasma spp., Pasteurella spp., Bordetella bronchiseptica, Salmonella typhimurium, Pseudomonas sp., Mycobacterium thermoresistible, Cryptococcus neoformans, Toxoplasma gondii, Aelurostrongylus abstrusus and Eucoleus aerophilus . The study provides a detailed retrospective analysis of infectious lower respiratory tract disease in this population of cats.

Microbiology, 2004 May, 150(Pt 5), 1547 - 58
Cryptococcus neoformans Ilv2p confers resistance to sulfometuron methyl and is required for survival at 37 degrees C and in vivo; Kingsbury JM et al.; Acetolactate synthase catalyses the first common step in isoleucine and valine biosynthesis and is the target of several classes of inhibitors . The Cryptococcus neoformans ILV2 gene, encoding acetolactate synthase, was identified by complementation of a Saccharomyces cerevisiae ilv2 mutant . C . neoformans is highly resistant to the commercially available acetolactate synthase inhibitor, sulfometuron methyl (SM) . Expression of C . neoformans ILV2 in S . cerevisiae conferred SM resistance, indicating that the SM resistance of C . neoformans is due, at least in part, to C . neoformans Ilv2p . The C . neoformans ILV2 gene was disrupted . The ilv2 mutants were auxotrophic for isoleucine and valine and the auxotrophy was satisfied by these amino acids only when proline, and not ammonium, was the nitrogen source, indicating nitrogen regulation of amino acid transport . ilv2 mutants rapidly lost viability at 37 degrees C and when starved for isoleucine and valine . Consistent with these phenotypes, an ilv2 mutant was avirulent and unable to survive in mice . Because C . neoformans Ilv2p is required for virulence and survival in vivo, inhibitors of branched-chain amino acid biosynthesis may make valuable antifungal agents.

J Clin Microbiol, 2004 May, 42(5), 2209 - 11
Comparison of VITEK 2 with ITS2-fragment length polymorphism analysis for identification of yeast species; Massonet C et al.; A total of 61 clinical yeast isolates of Candida, Cryptococcus, Blastoschizomyces, and Saccharomyces spp . were used to compare two identification techniques, VITEK 2 and ITS2-fragment length polymorphism analysis (ITS2-FLP), with ID32C as the reference method . ID32C identified 58 isolates correctly . ITS2-FLP with Instagene DNA extraction identified 59 isolates . ITS2-FLP combined with boiling-freezing DNA extraction identified 55 isolates . VITEK 2 identified 41 isolates correctly.

Sarcoidosis Vasc Diffuse Lung Dis, 2004 Mar, 21(1), 71 - 6
Cryptococcosis and sarcoidosis: strange bed-fellows . A report of five cases; Riha RL et al.; BACKGROUND AND AIM: Sarcoidosis is known to predispose to cryptococcal infection . In this case series, variations in presentation, diagnostic dilemmas and responses to treatment are highlighted . METHODS: Of several hundred patients referred to the sarcoidosis clinic at The Prince Charles Hospital, Brisbane and to the private clinic of the co-author between 1990 and 2002, five subsequently developed cryptococcal infections as a complication of sarcoidosis . All five subjects were treated by the co-author (RKAA) . A review of the literature was also performed . RESULTS: Cryptococcal infection occurred in 4 patients with sarcoidosis being treated with steroids and one patient who was not on treatment . All responded to antifungal therapy . Cryptococcosis was diagnosed by transbronchial (2), bronchial (1) and cutaneous (2) biopsies . Fluconazole was used in those with pulmonary infections but not in cutaneous disease where excision sufficed . One patient with pulmonary cryptococcosis from immunosuppressants also developed a nocardial brain abscess . CONCLUSIONS: Our series of patients with sarcoidosis and cryptococcal infections is unique in Australia . Although an unusual infection, cryptococcosis should always be considered in patients with sarcoidosis as it may be overlooked particularly in the lungs and can be fatal if untreated . Further immune dysregulation through steroid use may contribute significantly to the disease manifestations . Understanding why cryptococcosis and not other infections is more common in patients with sarcoidosis may reveal more about the mechanisms of granuloma formation and the nature of sarcoidosis itself.

Med Mycol, 2004 Apr, 42(2), 181 - 6
Serotype distribution of Cryptococcus neoformans in patients in a tertiary care center in India; Banerjee U et al.; The prevalence of specific serotypes of Cryptococcus neoformans in a given area bears on regional epidemiological patterns, the expected spectrum of clinical disease, and predicted response to therapy . In this retrospective study we analyzed the serotypes of 45 degrees C neoformans isolates from 36 North Indian patients with varied clinical presentations . The majority of the isolates were serotype A (87%), and surprisingly, a significant number were serotype B (five isolates, 11%), which caused infection in patients both positive and negative for HIV . One unusual isolate was not typable with factor sera . Study of serotype distribution in patients showed serotypes A and B to be present, respectively, in 92% and 8% of 36 patients . In one apparently immunocompetent patient two serotypes, A and B, were isolated simultaneously from two different sites, lung and scalp abscess . This is the first reported case in which an individual was infected with two serotypes at the same time . In one HIV-infected child serotype A was isolated from blood . Our results suggest that the distribution of serotypes in Indian clinical isolates is different than that found in other regions.

Med Mycol, 2004 Apr, 42(2), 149 - 58
Cryptococcus neoformans var . gattii can exploit Acanthamoeba castellanii for growth; Malliaris SD et al.; It has recently been proposed that the origin and maintenance of virulence in certain environmental fungi is influenced by their interactions with non-vertebrate hosts such as amoebae and nematodes . In prior studies we have shown that the interactions of the soil amoebae Acanthamoeba castellanii with Cryptococcus neoformans varieties neoformans and grubii resemble those with macrophages . Here we extend those studies to C . neoformans variety gattii and describe quantitative differences in the type and outcome of the interactions observed relative to the other varieties . C . neoformans var . gattii proliferated in the presence of A . castellanii but the interaction was primarily extracellular with a paucity of phagocytic events . Experiments with acapsular cells coated with polysaccharide suggest that differences in the capsule structure may be responsible for the different interactions between cells of varieties neoformans, grubii, and gattii with amoebae . The ability of C . neoformans var . gattii to exploit amoebae indicates that despite major biological differences between C . neoformans varieties, all retain the ability to be pathogenic for A . castellanii.

Med Mycol, 2004 Apr, 42(2), 135 - 47
Molecular typing of Cryptococcus neoformans by PCR fingerprinting, in comparison with serotyping and Fourier transform infrared-spectroscopy-based phenotyping; Lemmer K et al.; Molecular typing by PCR fingerprinting using the single primer (GACA)4 was performed with 110 isolates of Cryptococcus neoformans . Seventy clinical isolates of C . neoformans var . neoformans from Germany (n = 52) and Africa (n = 18) were included . Of these, serotype A (C . neoformans var . grubii) accounted for 47 isolates, serotype D for 12 and serotype AD for 11 . Fourier transform infrared (FT-IR) spectroscopy was evaluated for its discriminatory power in phenotyping . Molecular types, defined by different PCR fingerprinting patterns, were compared to serotypes, and both sets of results were compared with the results of analysis by FT-IR spectroscopy . PCR fingerprinting revealed genotypic diversity within each serotype; it showed three different genotypes (designated VNA1-VNA3) within serotype A, two within serotype D (VND1 and VND2), and three within serotype AD (VNAD1-VNAD3) . The nomenclature of molecular types within C . n . var . neoformans, as seen in publications to date, is not uniform . In this study, the name assigned to each genotype was based on the 98.6% concordance of genotypes with serotypes, a correspondence that facilitates interlaboratory comparison . This nomenclature is tentatively recommended as a standard . FT-IR spectroscopy combined with hierarchical cluster analysis successfully distinguished C n . var . neoformans from C . n . var . gattii . For C . n . var . neoformans, FT-IR confirmed three distinct genotypes within serotype A and was able to distinguish isolates derived from particular patients as well as isolates differing at the sub-genotype level . Within C . n . var . gattii, the serotypes B and C did not correlate with the four genotypes VGI-VGIV . However, these serotypes could clearly be separated by FT-IR spectroscopy . The molecular profiles were reproducible, and were more stable and more discriminating than serotyping . In connection with a standardized nomenclature, PCR fingerprinting can be a beneficial tool for global epidemiological studies . FT-IR spectroscopy adds an additional level of resolution.

Ann Neurol, 2004 May, 55(5), 687 - 94
The urokinase receptor is overexpressed in the AIDS dementia complex and other neurological manifestations; Cinque P et al.; The urokinase-type plasminogen activator (uPA) and its receptor (uPAR) play an important role in extracellular matrix degradation and cell migration in the central nervous system (CNS) . To investigate the role of the uPA/uPAR system in the pathophysiology of acquired immunodeficiency syndrome dementia complex (ADC), we measured soluble uPAR (suPAR) levels in cerebrospinal fluid (CSF) and plasma from human immunodeficiency virus (HIV)-1-infected patients and controls . CSF suPAR levels were significantly higher in HIV-1-infected patients than in controls and in patients with ADC or opportunistic CNS infections (CNS-OIs) than in neurologically asymptomatic patients, irrespective of HIV-1 disease stage . The highest levels of suPAR were found in patients with ADC, and among those with CNS-OIs in patients with cytomegalovirus encephalitis or cryptococcosis . Plasma suPAR levels were higher in HIV-1-infected patients than in controls and increased with HIV-1 disease stage regardless of the presence of CNS disease . In patients with ADC or CNS-OIs, CSF suPAR levels correlated with CSF HIV-1 RNA, but not with plasma suPAR concentrations . Highly active antiretroviral therapy was associated with a significant and parallel decrease of both CSF suPAR and HIV-1 RNA . In brain tissue from patients with HIV-1 encephalitis, uPAR was highly expressed by microglial and multinucleated giant cells staining positively for HIV-1 . The overexpression of uPAR in the CNS of patients with ADC suggests that the uPA/uPAR system may contribute to the tissue injury and neuronal damage in this disease.

Arq Neuropsiquiatr, 2004 Mar, 62(1), 147 - 9 Epub 2004 Apr 28.
Meningoradiculitis due to Cryptococcus neofermans in an immunocompetent patient; Deus-Silva L et al.; Meningoradiculitis refers to combined involvement of meninges and nerve roots . The most frequent location is the lumbosacral region . Etiology is diverse, including inflammatory, infectious and neoplastic disorders . Meningoradiculitis is a rare form of involvement in cryptococcal infection . We describe a case of subacute lower limbs flaccid paresis diagnosed as lumbosacral meningoradiculitis in view of cerebrospinal fluid (CSF) inflammatory changes and typical enhancement on MRI of lumbar spine . Cryptococcus neoformans was isolated from CSF . Extensive screening yielded no immunodeficiencies.

Mycopathologia, 2004 Feb, 157(2), 185 - 9
Antimicrobial activities of selected Cyathus species; Liu YJ et al.; Twelve selected Cyathus species were tested for their abilities to produce antimicrobial metabolites . Most of them were found to produce secondary exo-metabolites that could induce morphological abnormalities of rice pathogenic fungi Pyricularia oryzae . Some extracts from the cultivated liquid obviously inhibited human pathogenic fungi Aspergillus fumigatus, Candida albicans and Cryptococcus neoformans . Activities against six human pathogenic bacteria were also obtained from some of these extracts.

Medicine (Baltimore), 2004 May, 83(3), 188 - 92
Cryptococcus neoformans Infection in Patients With Cirrhosis, Including Liver Transplant Candidates; Singh N et al.; We reviewed the cases of patients with cirrhosis, including liver transplant candidates, at our institution in the last 3 years (n = 5) and those individually described in the literature (n = 28), to assess unique characteristics and outcome of cryptococcosis in these patients . Sixty-four percent (21/33) of the patients had no other recognized immunosuppression . Peritonitis (in 45%, 15/33 of the patients) with modest pleocytosis in the ascitic fluid, was the most common presenting feature . Median time to detection of Cryptococcus in the ascitic fluid cultures was 6 days . Overall mortality rate was 81% (26/32); death was deemed attributable to cryptococcosis in 24/26 patients who died . Evaluation of culture-negative neutrocytic ascites in febrile cirrhotic patients warrants consideration of cryptococcal peritonitis.

J Biochem (Tokyo), 2004 Apr, 135(4), 533 - 40
Cloning and expression in Escherichia coli of the D-aspartate oxidase gene from the yeast Cryptococcus humicola and characterization of the recombinant enzyme; Takahashi S et al.; The D-aspartate oxidase (DDO) from the yeast Cryptococcus humicola UJ1 (ChDDO) is highly specific to D-aspartate . The gene encoding ChDDO was cloned and expressed in Escherichia coli . Sequence analysis of the ChDDO gene showed that an open reading frame of 1,110 bp interrupted by two introns encodes a protein of 370 amino acids . The deduced amino acid sequence showed an FAD-binding motif and a peroxisomal targeting signal 1 in the N-terminal region and at the C-terminus, respectively, and also the presence of certain catalytically important amino acid residues corresponding to those catalytically important in D-amino acid oxidase (DAO) . The sequence exhibited only a moderate identity to human (27.4%) and bovine (28.0%) DDOs, and a rather higher identity to yeast and fungal DAOs (30.4-33.2%) . Similarly, phylogenetic analysis showed that ChDDO is more closely related to yeast and fungal DAOs than to mammalian DDOs . The gene expression was regulated at the transcriptional level and specifically induced by the presence of D-aspartate as the sole nitrogen source . ChDDO was expressed in an active form in E . coli to an approximately 5-fold greater extent than in yeast . The purified recombinant enzyme was identical to the native enzyme in physicochemical and catalytic properties.

Am J Pathol, 2004 May, 164(5), 1761 - 71
Urease expression by Cryptococcus neoformans promotes microvascular sequestration, thereby enhancing central nervous system invasion; Olszewski MA et al.; Our objective was to determine the role of the cryptococcal virulence factor urease in pulmonary-to-central nervous system, dissemination, invasion, and growth . C . neoformans H99, the urease knockout strain (ure1) derived from H99, and the urease restored strain ure1+URE1-1 were used for the studies . The absence of cryptococcal urease (ure1infection) resulted in significant protection from the high mortality observed in H99-infected mice . All H99-infected mice had extremely high cryptococcal loads in their brains at the time of death, whereas only two of six animals that died of ure1 infection had detectable C . neoformans in the brain . Histological analysis of the blood-to-brain invasion by C . neoformans H99 demonstrated wedging of the yeasts in small capillaries, altered structure of microvessel walls, formation of mucoid cysts initiated in the proximity of damaged microcapillaries, and the absence of an inflammatory response . Direct inoculation of H99, ure1, and ure1+URE1-1 into the brain demonstrated that urease was not required to grow in the brain . However, the dissemination patterns in the brain, spleen, and other organs after intravenous inoculation indicated that cryptococcal urease contributes to the central nervous system invasion by enhancing yeast sequestration within microcapillary beds (such as within the brain) during hematogenous spread, thereby facilitating blood-to-brain invasion by C . neoformans.

Microbiol Immunol, 2004, 48(4), 237 - 42
Phenotypic switching and beta-N-acetylhexosaminidase activity of the pathogenic yeast Trichosporon asahii; Ichikawa T et al.; The pathogenic yeast Trichosporon asahii is the major causative agent of deep-seated trichosporonosis in immunocompromised patients . Although infection by this microorganism is becoming increasingly frequent, information related to its pathogenicity and virulence factors is still limited . Therefore, we investigated phenotypic switching in colony morphology, and the production of extracellular enzymes as a virulence factor . Sixty-one clinical isolates of T . asahii produced four different morphological types on Sabouraud dextrose agar (SDA): 69% WF (white farinose), 18% WP (white pustular), 10% Y (yellowish white), and 3% WC (white cerebriform) . Strains of the three major types (WF, WP, and Y) produced two to five colony types when cultured on SDA at 37 C . The frequency of switching between colony types was 10(-2) to 10(-4), as in Candida albicans and Cryptococcus neoformans . Notably, most of the colonies switched to the smooth (S) type irreversibly, at frequencies of 10(-2) to 10(-3) . No secreted aspartic proteinase or phospholipase activity was detected in T . asahii, while beta-N-acetylhexosaminidase activity, which catalyzes the hydrolysis of terminal nonreducing N-acetyl-D-hexosamine residues in N-acetyl-beta-D-hexosaminides, was found . Furthermore, enzymatic activity of the S type was significantly greater than that of the parent type in all strains . No other clinically relevant Trichosporon species (T . mucoides, T . inkin, and T . ovoides ) produced this enzyme . These results provide basal information for understanding the pathogenic potential of T . asahii.

Antimicrob Agents Chemother, 2004 May, 48(5), 1664 - 9
In vitro interactions between antifungals and immunosuppressants against Aspergillus fumigatus; Steinbach WJ et al.; The optimal treatment for invasive aspergillosis remains elusive, despite the increased efficacy of newer agents . The immunosuppressants cyclosporine (CY), tacrolimus (FK506), and sirolimus (formerly called rapamycin) exhibit in vitro and in vivo activity against Candida albicans, Cryptococcus neoformans, and Saccharomyces cerevisiae, including fungicidal synergy with azole antifungals . We report here that both FK506 and CY exhibit a clear in vitro positive interaction with caspofungin against Aspergillus fumigatus by disk diffusion, microdilution checkerboard, and gross and microscopic morphological analyses . Microscopic morphological analyses indicate that the calcineurin inhibitors delay filamentation, and in combination with caspofungin there is a positive interaction . Our findings suggest a potential role for combination therapy with calcineurin pathway inhibitors and existing antifungal agents to augment activity against A . fumigatus.

Antimicrob Agents Chemother, 2004 May, 48(5), 1561 - 9
In vitro antifungal activities of inhibitors of phospholipases from the fungal pathogen Cryptococcus neoformans; Ganendren R et al.; Secreted phospholipase B is a proven virulence factor for the pathogenic fungus Cryptococcus neoformans and exhibits three phospholipase activities in the one protein . These are phospholipase B (PLB), lysophospholipase (LPL), and lysophospholipase transacylase (LPTA) . Our aim was to investigate the feasibility of using this enzyme as a target for antifungal therapy . We determined in C . neoformans var . grubii strain H99 that 82% of PLB activity was secreted but that 64% of LPL activity and 70% of LPTA activity were cell associated . Cell-associated activities (cytosolic and membrane) were further characterized, since it is likely that any fungicidal effect would depend on inhibition of these enzymes . Four commercially available compounds with structural similarities to phospholipid substrates were tested as inhibitors . These were alexidine dihydrochloride (compound A), dioctadecyldimethylammonium bromide (compound O), 1,12 bis-(tributylphosphonium)dodecane dibromide (compound P), and decamethonium dibromide (compound D) . The best phospholipase inhibitors (compounds A and P) were also the most potent antifungal agents by the standard broth microdilution test . Compound A was highly selective for secreted and cell-associated PLB activities and showed no inhibition of mammalian phospholipase A(2) at 0.25 micro M . Compound O, which was specific for secretory and cytosolic LPL and LPTA and membrane-associated PLB, was not antifungal . We conclude that inhibitors of cryptococcal phospholipases can be selective for fungal enzymes and intrinsically antifungal . They also provide tools for assessing the relative importance of the various enzyme activities in virulence . Our results enable further rational structure-function studies to validate the use of phospholipases as antifungal targets.

Cell Microbiol, 2004 Jun, 6(6), 581 - 92
Cryptococcal glucuronoxylomannan interferes with neutrophil rolling on the endothelium; Ellerbroek PM et al.; The major capsular polysaccharide glucuronoxylomannan (GXM) of the pathogenic fungus Cryptococcus neoformans has been associated with depression of a variety of immunological host responses . For one, GXM has been shown to interfere with the migration of phagocytes to sites of inflammation by interference with both chemokinesis and leucocyte adhesion to the endothelium . We reported previously that GXM blocks the firm adhesion of neutrophils (PMNs) to endothelium in a static adhesion model, most probably by interfering with E-selectin binding pathways . Using a flow model, we now demonstrate that GXM also interferes with the initial rolling phase of PMN adhesion to endothelium (40% decrease) as well as to E-selectin-transfected CHO cells (43% inhibition) . Furthermore, we show that CD14 and TLR4, which are known receptors for GXM, mediate this interference with PMN rolling . However, thus far, we are not able to identify the ligand of E-selectin on the surface of PMNs that is specifically affected by GXM . In conclusion, cryptococcal GXM interferes with both rolling and fixed binding of neutrophils on the endothelium, providing a novel means of contributing to the absence of neutrophil infiltration observed in cryptococcal infections.

Biol Proced Online, 2004, 6, 10 - 15 Epub 2004 Mar 3.
Experimental modulation of capsule size in Cryptococcus neoformans; Zaragoza O et al.; Experimental modulation of capsule size is an important technique for the study of the virulence of the encapsulated pathogen Cryptococcus neoformans . In this paper, we summarize the techniques available for experimental modulation of capsule size in this yeast and describe improved methods to induce capsule size changes . The response of the yeast to the various stimuli is highly dependent on the cryptococcal strain . A high CO(2) atmosphere and a low iron concentration have been used classically to increase capsule size . Unfortunately, these stimuli are not reliable for inducing capsular enlargement in all strains . Recently we have identified new and simpler conditions for inducing capsule enlargement that consistently elicited this effect . Specifically, we noted that mammalian serum or diluted Sabouraud broth in MOPS buffer pH 7.3 efficiently induced capsule growth . Media that slowed the growth rate of the yeast correlated with an increase in capsule size . Finally, we summarize the most commonly used media that induce capsule growth in C . neoformans.

J Med Microbiol, 2004 May, 53(Pt 5), 445 - 9
Cutaneous cryptococcosis in a diabetic renal transplant recipient; Gupta RK et al.; A diabetic renal transplant recipient with cellulitis caused by Cryptococcus neoformans, serotype A, is described . The diagnosis was based on the demonstration of capsulated, budding yeast cells in the aspirated material and tissue from the cellulitic lesion and isolation of the aetiological agent in culture . The isolate formed well-developed capsules in the brain tissue of experimentally infected mice and produced cherry-brown colonies on niger seed medium . The patient's serum was positive for cryptococcal antigen (titre 1 : 4) with no other evidence of systemic infection . He was successfully treated with AmBisome, followed by fluconazole, resulting in the complete resolution of cellulitis and disappearance of the cryptococcal antigen . This report underscores the fact that patients with cutaneous cryptococcosis should be thoroughly evaluated, as it may be the first manifestation of a systemic disease . Prompt diagnosis and treatment are important to improve survival.

FEMS Yeast Res, 2004 May, 4(7), 737 - 44
Isolation of a CDC28 homologue from Cryptococcus neoformans that is able to complement cdc28 temperature-sensitive mutants of Saccharomyces cerevisiae; Takeo K et al.; A partial cDNA fragment of the Cryptococcus neoformans homologue of the main cell cycle control gene CDC28/cdc2 was isolated using degenerate primer RT-PCR . A subsequent search in the C . neoformans genome database identified several sequences similar to CDC28/cdc2 . A part of the sequence which showed the highest similarity to CDC28/cdc2 turned out to be identical to the partial cyclin-dependent kinase (Cdk) cDNA fragment isolated by degenerate RT-PCR . The full-length coding region of this Cdk homologue was amplified by RT-PCR using primers designed to target regions around start and stop codons, and the gene was named CnCdk1 . To determine its function, an analysis of deduced amino acid sequence of the CnCdk1 was performed and its ability to rescue Saccharomyces cerevisiae cdc28-temperature sensitive mutants was tested . S . cerevisiae cdc28-4 and cdc28-1N strains transformed with the pYES2- CnCdk1 construct exhibited growth at 36.5 degrees C in galactose-raffinose medium, but not in glucose medium . Results of the sequence analysis and the fact that CnCdk1 is able to complement the S . cerevisiae cdc28-ts mutation support its assumed role as the CDC28/cdc2 homologue in C . neoformans.

AIDS, 2004 Feb 20, 18(3), 555 - 62
Epidemiology of HIV-associated cryptococcosis in France (1985-2001): comparison of the pre- and post-HAART eras; Dromer F et al.; OBJECTIVE: To analyse the epidemiological evolution of cryptococcosis in France after the introduction of highly active antiretroviral therapy (HAART) . DESIGN: Retrospective study of cryptococcosis cases recorded at the National Reference Center for Mycoses in France since 1985 . METHODS: Using the national surveillance data, we reviewed 1644 cases of HIV-associated cryptococcosis diagnosed in France (population, 59 million) between 1985 and 2001 and compared them to 335 cases recorded in HIV-negative patients . RESULTS: The total number of cryptococcosis cases evolved in parallel to that recorded for HIV-infected patients . Changes occurring after HAART introduction were analysed . A negative binomial regression model established a 46% decrease of the incidence of cryptococcosis during the post-HAART era (1997-2001, n = 292) compared to the pre-HAART era (1985-1996, n = 1352) . According to multivariate analysis, African origin, older age, heterosexual HIV contamination, no previous AIDS-defining illness, and no previous HIV infection diagnosis were variables independently associated with an increased risk of cryptococcosis during the post-HAART era . During the same period, the characteristics of the HIV-negative population did not change . CONCLUSIONS: Our analysis of the national surveillance identified demographic factors associated with an increased risk of cryptococcosis in the post-HAART era suggesting that failure to consult and considering oneself not at risk were determinant in the current epidemiology of HIV-related cryptococcosis in France.

Clin Microbiol Rev, 2004 Apr, 17(2), 413 - 33
Microorganisms resistant to free-living amoebae; Greub G et al.; Free-living amoebae feed on bacteria, fungi, and algae . However, some microorganisms have evolved to become resistant to these protists . These amoeba-resistant microorganisms include established pathogens, such as Cryptococcus neoformans, Legionella spp., Chlamydophila pneumoniae, Mycobacterium avium, Listeria monocytogenes, Pseudomonas aeruginosa, and Francisella tularensis, and emerging pathogens, such as Bosea spp., Simkania negevensis, Parachlamydia acanthamoebae, and Legionella-like amoebal pathogens . Some of these amoeba-resistant bacteria (ARB) are lytic for their amoebal host, while others are considered endosymbionts, since a stable host-parasite ratio is maintained . Free-living amoebae represent an important reservoir of ARB and may, while encysted, protect the internalized bacteria from chlorine and other biocides . Free-living amoebae may act as a Trojan horse, bringing hidden ARB within the human "Troy," and may produce vesicles filled with ARB, increasing their transmission potential . Free-living amoebae may also play a role in the selection of virulence traits and in adaptation to survival in macrophages . Thus, intra-amoebal growth was found to enhance virulence, and similar mechanisms seem to be implicated in the survival of ARB in response to both amoebae and macrophages . Moreover, free-living amoebae represent a useful tool for the culture of some intracellular bacteria and new bacterial species that might be potential emerging pathogens.

Int J Pharm, 2004 May 4, 275(1-2), 61 - 72
Leucinostatin-A loaded nanospheres: characterization and in vivo toxicity and efficacy evaluation; Ricci M et al.; Leucinostatin A (Leu-A) is a nonapeptide exerting a remarkable activity especially against Candida albicans and Cryptococcus neoformans; nevertheless, its employment is limited due its toxicity . Therefore, we recently developed liposomal formulations, as suitable delivery systems, in order to increase its therapeutic index . However, liposomes present disadvantages related to their long-term instability . For this reason poly(lactic-co-glycolic) nanospheres (NS) were chosen as alternative colloidal carriers for Leu-A delivery . NS were formulated by spontaneous emulsification solvent diffusion method . This study investigates the effects of different parameters on drug encapsulation efficiency and particle size as well . The best preparation obtained was also characterized for its in vitro release, in vivo acute toxicity (LD50), and effectiveness against C . albicans in mice . In vitro release was performed over 100 h and resulted sufficiently sustained with more than 93% of the peptide released . Acute toxicity showed that the LD50 was increased more than 18-fold and the study on systemic candidiasis models revealed high effectiveness of the NS in reducing either the growth of fungal colonies in infected mice liver or in the mortality index . In conclusion, we can propose that Leu-A loaded NS could represent a new promising therapeutic system against Candida infection.

Emerg Infect Dis, 2004 Jan, 10(1), 143 - 5
Antifungal susceptibilities of Cryptococcus neoformans; Archibald LK et al.; Susceptibility profiles of medically important fungi in less-developed countries remain uncharacterized . We measured the MICs of amphotericin B, 5-flucytosine, fluconazole, itraconazole, and ketoconazole for Cryptococcus neoformans clinical isolates from Thailand, Malawi, and the United States and found no evidence of resistance or MIC profile differences among the countries.

Mycoses, 2004 Apr, 47(3-4), 168 - 73
Cryptococcal meningitis in two apparently immunocompetent Maltese patients; Zahra LV et al.; Two cases of cryptococcal meningitis occurring in immunocompetent men are described . The first case involves a farmer in whom cryptococcal meningitis was rapidly diagnosed using direct microscopy, latex antigen tests and culture of cerebrospinal fluid (CSF) . In the second case, initial mycological tests on CSF (direct microscopy and culture) were repetitively negative and latex antigen tests gave unconvincing results . The patient was started on triple antituberculosis therapy, on which he improved immediately; therapy was continued for 1 year with the patient remaining well . However, within 1 week of stopping therapy, he had to be readmitted with neurological signs and symptoms . Culture of CSF this time yielded Cryptococcus neoformans and the antigen test was consistently positive . These are the first reported cases of cryptococcal meningitis in immunocompetent patients in Malta.

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