Cryptococcosis Research Papers - Academia.edu (original) (raw)

We studied a series of test conditions in a microtiter system to define the optimal method for determining the susceptibility of Cryptococcus neoformans to antifungal agents. Twenty-one isolates of C. neoformans were grown for 24 or 48 h in four chemically defined media: yeast nitrogen base (BYNB 7); RPMI 1640; synthetic amino acid medium--fungal (SAAMF), buffered at pH 7.0 to select the medium that best supported growth of this fastidious yeast; and yeast nitrogen base, pH 5.4 (YNB 5.4). Maximum growth of C. neoformans, at 35 degrees C, was obtained in YNB 5.4, with the next highest growth levels in BYNB 7, SAAMF, and RPMI. Growth at 24 h was uniformly poor in all media and lacked reproducibility. In contrast, incubation for 48 h gave adequate growth with low standard deviations, and 48 h was selected as the optimal incubation period for this study. Comparison of the relationship between growth kinetics and initial inoculum size for eight cryptococcal isolates showed that 10(4) cel...

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- Biology, Medicine, Clinical Microbiology, Biological Sciences

formerly considered to be restricted to tropical and subtropical regions, but several outbreaks of cryptococcosis involving both immunocompetent and immunocompromised individuals have been reported in Canada, the US Pacifi c Northwest, Australia, and South America, indicating its geographic expansion [2,3]. In Brazil, C. neoformans is distributed throughout the country, whereas C. gattii is mainly found in the North and Northeast regions [4]. Due to the increase in the number of immunocompromised individuals over the past several decades, the prevalence of invasive fungal infections has substantially increased worldwide [5]. This has led to high rates of therapeutic and/or prophylactic use of azole derivates and other antifungals, causing selective epidemiological pressure, which may have contributed to the decreased susceptibility to these drugs [6].

- by Delio J Mora
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- Microbiology, Environmental microbiology, Medical Microbiology, Biology

Cryptococcus neoformans var. gattii has been shown to have a strong association with eucalypts frequently used by koalas and, not surprisingly, it has been shown to colonize the nasal cavities of koalas. The progression from nasal colonization to tissue invasion is critical to understanding the pathogenesis of cryptococcosis in this species and provides a model for pathogenesis of cryptococcosis in other species. Cryptococcal antigenaemia was detected in twenty-eight healthy koalas from three different regions. This was interpreted as representing limited subclinical disease. One koala developed cryptococcal pneumonia 6 months after leaving the study, whereas another developed cryptococcal meningoencephalitis during the course of the study. Opportunistic necropsies on ten antigen-positive koalas resulted in discovery of small cryptococcal lesions in two (paranasal sinus and lung, respectively). Our data suggest that cryptococcal antigenaemia occurs commonly in koalas, especially in areas with a high environmental presence of C. n. var. gattii. Subclinical disease appears most likely to manifest as a small focal lesion in the respiratory tract. Possible outcomes include elimination by an effective immune response, quiescence with possibility of later re-activation or direct progression to overt disease. Symptomatic and subclinical cases showed differences in levels of antigenaemia. The data presented have signi cant implications for koalas in captivity.

- by Joanne Connolly
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- Australia, Medical Microbiology, Medical Mycology, Cryptococcus Neoformans

Background. The objective of this study was to evaluate the immediate and long term result of resectional surgery in pulmonary aspergilloma. Methods. Seventy-two patients who underwent pulmonary resectional surgery for symptomatic aspergilloma between 1990 to 2002 were studied. Seventy-nine definitive operations were carried out, including one bilateral lobectomy for recurrent lesions and six thoracoplasties to deal with post-operative complications, besides 21 pneumonectomies and 51 lobectomies. There were 10 bilobectomies as well, included in the lobectomy group. Results. At a mean follow-up of 3.5 years, there were two post-operative deaths and a few complications occurred in 20 cases translating into a morbidity of 28.57% and a mortality of 2.77 percent. Major complications included were persistent air leak, persistent pleural space, empyema, bronchopleural fistula and massive haemorrhage. All events were seen in cases of complex aspergilloma; cases of simple aspergillomas had an uneventful course. Conclusions. Surgery offers definitive and long term symptom-free survival in cases of pulmonary aspergilloma at a negligible risk; though almost one-third of those undergoing surgery develop some complications, these are largely manageable.

- by Himanshu Pratap
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- Adolescent, Lung Function, Aspergillus fumigatus, Clinical Sciences

Cryptococcosis is the third most common invasive fungal infection in organ transplant recipients after candidiasis and aspergillosis. Reports indicate that cryptococcosis usually presents as symptomatic disease, and despite therapy the mortality remains high. In addition, some data suggest that there might be differences in the incidence and clinical manifestations of cryptococcosis, depending on the specific transplant organ. The incidence of cryptococcosis in our transplant center is significantly higher in heart transplant recipients than in other transplant groups (p Ω0.0001). Although the primary risk factor contributing to cryptococcosis in organ transplant recipients is probably the immunosuppressive therapy used to prevent allograft rejection, environmental factors may also play a role. This is indicated by studies that demonstrate differences in the rate of cryptococcosis according to geographic region. Moreover, data point out differences in the isolation of the fungus from soil samples with higher concentrations of Cryptococcus in areas frequented by birds or contaminated by bird droppings. Therefore, it is prudent to recommend that organ transplant recipients avoid birds or areas contaminated with bird droppings. The current review provides an overview of the changes in the incidence, clinical manifestations, and management of cryptococcosis in organ transplant recipients.

- by Regis Vilchez
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- Kidney transplantation, Organ Transplantation, American, Risk factors
- by Giorgio Barbarini and +1
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- Microscopy, Biopsy, Antifungal Agents, Cryptococcosis

Given the lack of comprehensive molecular epidemiology studies in Reggio Calabria and Messina, Italy, we decided to perform an extensive environmental sampling to describe the current molecular epidemiology of C. neoformans/C. gattii species complex in southern Italy. In this study, we report the occurrence of serotypes, genotypes and mating-types of isolates of the C. neoformans/C. gattii species complex recovered from environmental sources. In addition, a number of environmental C. neoformans var. grubii strains, isolated in 1997 by our laboratory, were also retrospectively examined in order to compare their genotypes with those recently found and to infer the possible epidemiological changes in our country. One hundred and twentytwo isolates were identified as being C. neoformans, whereas only one was found to belong to C. gattii serotype B, genotype VGI and mating-type alpha. Our data revealed that all environmental isolates of C. neoformans recovered here as well as those previously isolated in 1997 belong to serotype A and genotype VNI and posses a mating-type alpha allele.

- by Orazio Romeo
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- Environmental microbiology, Plant Biology, Venezuela, Cryptococcus gattii
- by Lourdes Abarca
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- Zoonoses, Spain, Cats, Dogs
- by Bodo Wanke and +1
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- Microbiology, Medical Microbiology, Fungi, Innate immunity

This monograph include historical, classification, nomenclature and description of Cryptococcus species. epidemiology, pthogenecity and diseases caused by C. neoformans and C. gattii in man, animals and birds and diagnosis of... more

- by Mohamed Refai
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- Cryptococcus, Cryptococcosis, C. neoformans

CITATIONS 0 READS 231 3 authors:

- by Lena Örnberg
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- Engineering, Psychology, Cognitive Science, Artificial Intelligence

The ability of Cryptococcus neoformans to synthesize polymerized melanin in vitro has been associated with virulence, but it is unclear whether this fungus synthesizes polymerized melanin during infection. To study this question, we used two approaches: one involved the generation of monoclonal antibodies (MAbs) to melanin for use in immunohistochemical studies of C. neoformans-infected rodents, and the other sought to isolate fungal melanin from infected tissues. Digestion of in vitro-melanized C. neoformans cells with proteases, denaturant, and hot concentrated acid yields melanin particles that retain the shape of fungal cells and are therefore called melanin ghosts. BALB/c mice were immunized with melanin ghosts, and two immunoglobulin M MAbs to melanin were generated from the spleen of one mouse. Immunofluorescence analyses of lung and brain tissues of rodents infected with wild-type melanin-producing (Mel ؉ ) C. neoformans strains demonstrated binding of the MAbs to the fungal cell wall. No binding was observed when infections were performed with mutant albino (Mel ؊ ) C. neoformans strains. Particles with striking similarity to melanin ghosts were recovered after digestion of lung and brain tissues from Mel ؉ C. neoformans-infected rodents and were reactive with the MAbs to melanin. No particles were recovered from tissues infected with Mel ؊ C. neoformans. A Mel ؉ C. neoformans strain grown on lung or brain homogenate agar became lightly pigmented and also yielded particles similar to melanin ghosts upon digestion, providing additional evidence that lung and brain tissues contain substrate for C. neoformans melanization. These results demonstrate that C. neoformans synthesizes polymerized melanin during infection, which has important implications for pathogenesis and antifungal drug development.

- by Joshua Nosanchuk
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- Immunohistochemistry, Drug development, Polymers, Biological Sciences

Understanding of the taxonomy and phylogeny of Cryptococcus gattii has been advanced by modern molecular techniques. C. gattii probably diverged from Cryptococcus neoformans between 16 million and 160 million years ago, depending on the dating methods applied, and maintains diversity by recombining in nature. South America is the likely source of the virulent C. gattii VGII molecular types that have emerged in North America. C. gattii shares major virulence determinants with C. neoformans, although genomic and transcriptomic studies revealed that despite similar genomes, the VGIIa and VGIIb subtypes employ very different transcriptional circuits and manifest differences in virulence phenotypes. Preliminary evidence suggests that C. gattii VGII causes severe lung disease and death without dissemination, whereas C. neoformans disseminates readily to the central nervous system (CNS) and causes death from meningoencephalitis. Overall, currently available data indicate that the C. gattii...

- by Wieland Meyer
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- Cryptococcus gattii, Cryptococcosis

In 2001, CDC, the National Institutes of Health, and the Infectious Diseases Society of America convened a working group to develop guidelines for therapy of human immunodeficiency virus (HIV)-associated opportunistic infections to serve as a companion to the Guidelines for Prevention of Opportunistic Infections Among HIV-Infected Persons. In recognition of unique considerations related to HIV infection among infants, children, and adolescents, a separate pediatric working group was established. Because HIV-infected women coinfected with opportunistic pathogens might be more likely to transmit these infections to their infants than women without HIV infection, guidelines for treating opportunistic pathogens among children should consider treatment of congentially acquired infections among both HIV-exposed but uninfected children and those with HIV infection. In addition, the natural history of opportunistic infections among HIV-infected children might differ from that among adults. ...

- by Leslie Serchuck
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- Communicable Diseases, Adolescent, Hepatitis C, Biological Sciences

A 6-year-old spayed domestic shorthaired cat (3.7 kg) was presented for 'pain' and stiffness of the hindquarters. The lameness first became evident 3e4 weeks earlier and progressed subsequently. Stiffness became more prominent 2 weeks prior to presentation. Lameness was asymmetrical and worse on the right. There was no history of trauma. The patient was somewhat inappetent. The cat had been taken to another veterinarian 3 weeks previously; no investigations were undertaken and amoxycillin clavulanate and carprofen (doses unrecorded) had been administered empirically, but without benefit.

- by Patricia Martin
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- Decision Making, Pain, Cats, Clinical Decision Making

A large frontonasal bone flap was created to treat diseases of the paranasal sinuses in 14 horses. The bone flap was made as wide as possible within the confines of the nasolacrimal duct so the floor of the frontal sinus and the dorsal and ventral conchae could be opened. These openings exposed the nasal passages, maxillary sinuses, and ventral conchal sinus thereby facilitating removal of diffuse and localized lesions from these sites. Diseases treated were ethmoid hematomas (4 horses), sinus cysts (5 horses), cryptococcal granuloma, osteoma, hemangiosarcoma, pus in the ventral conchal sinus, and periapical infection of a second molar. Four horses were euthanatized during or after surgery, one because of postsurgical pleuritis and pneumonia (horse with osteoma) and three because of their primary problems (cryptococcal granuloma, hemangiosarcoma, pus in the ventral conchal sinus). Skin suture abscesses that responded to treatment developed in four horses. Ten horses returned to their intended uses, the sinus flaps healed without blemish, and the original problems did not recur. The frontonasal flap technique provided greater access to all paranasal sinuses than methods described previously.

- by David Freeman
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- Veterinary, Horses, Veterinary Surgery, Hemangiosarcoma

Opportunistic pathogens have become of increasing medical importance over the last decade due to the AIDS pandemic. Not only is cryptococcosis the fourth-most-common fatal infectious disease in sub-Saharan Africa, but also Cryptococcus is an emerging pathogen of immunocompetent individuals. The interaction between Cryptococcus and the host's immune system is a major determinant for the outcome of disease. Despite initial infection in early childhood with Cryptococcus neoformans and frequent exposure to C. neoformans within the environment, immunocompetent individuals are generally able to contain the fungus or maintain the yeast in a latent state. However, immune deficiencies lead to disseminating infections that are uniformly fatal without rapid clinical intervention. This review will discuss the innate and adaptive immune responses to Cryptococcus and cryptococcal strategies to evade the host's defense mechanisms. It will also address the importance of these strategies in pathogenesis and the potential of immunotherapy in cryptococcosis treatment.

- by Kerstin Voelz
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- Innate immunity, Biological Sciences, Adaptive Immunity, Immune system

Secreted phospholipase B1 (CnPlb1) is essential for dissemination of Cryptococcus neoformans to the central nervous system (CNS) yet essential components of its secretion machinery remain to be elucidated. Using gene deletion analysis we demonstrate that CnPlb1 secretion is dependent on the CnSEC14 product, CnSec14-1p. CnSec14-1p is a homologue of the phosphatidylinositol transfer protein ScSec14p, which is essential for secretion and viability in Saccharomyces cerevisiae. In contrast to CnPlb1, neither laccase 1-induced melanization within the cell wall nor capsule induction were negatively impacted in CnSEC14-1 deletion mutants (CnDsec14-1 and CnDsec14-1CnDsfh5). Similar to the CnPLB1 deletion mutant (CnDplb1), CnDsec14-1 was hypovirulent in mice and did not disseminate to the CNS by day 14 post infection. Furthermore, macrophage expulsion of live CnDsec14-1 and CnDplb1 (vomocytosis) was reduced. Individual deletion of CnSEC14-2, a closely related CnSEC14-1 homologue, and CnSFH5, a distantly related SEC fourteen like homologue, did not abrogate CnPlb1 secretion or virulence. However, reconstitution of CnDsec14-1 with CnSEC14-1 or CnSEC14-2 restored both phenotypes, consistent with functional genetic redundancy. We conclude that CnPlb1 secretion is SEC14dependent and that C. neoformans preferentially exports virulence determinants to the cell periphery via distinct pathways. We also demonstrate that CnPlb1 secretion is essential for vomocytosis.

- by Wieland Meyer
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- Molecular Microbiology, Macrophages, Biological Sciences, Molecular

The polysaccharide capsule surrounding Cryptococcus neoformans comprises manose, xylose and glucuronic acid, of which mannose is the major constituent. The GDP-mannose biosynthesis pathway is highly conserved in fungi and consists of three key enzymes: phosphomannose isomerase (PMI), phosphomannomutase (PMM) and GDP-mannose pyrophosphorylase (GMP). The MAN1 gene, encoding for the PMI enzyme, was isolated and sequenced from C. neoformans, and a disruption of the MAN1 gene was generated. One MAN1 disruption mutant, man1, which showed poor capsule formation, reduced polysaccharide secretion and morphological abnormalities, was chosen for virulence studies. In both the rabbit and the mouse models of invasive cryptococcosis, man1 was shown to be severely impaired in its virulence, with complete elimination of the yeast from the host. A reconstituted strain of man1 was constructed using gene replacement at the native locus. The wild-type and reconstituted strains were significantly more virulent than the knock-out mutant in both animal models. Our findings reveal that PMI activity is essential for the survival of C. neoformans in the host. The fact that the man1 mutant was not pathogenic suggests that blocking mannose synthesis could be fungicidal in the mammalian host and thus an excellent target for antifungal drug development.

- by Johanna Rivera
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- Molecular Microbiology, Biological Sciences, Molecular, Virulence
- by S. Husain
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- Biological Sciences, Aged, Time Factors, Cryptococcus
- by Holman Mcadams
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- Fungi, Lung, Clinical Sciences, Aged

1 Hajjeh RA, Conn LA, Stephens DS et al. Cryptococcosis in the United States: population-based multistate active surveillance and risk factors in HIV-infected persons. J Infect Dis 1999; 179 : 449 -454. 2 Gaddoni D, Resta F, Baldassari L et al. Criptococcosi cutanea in corso di AIDS. G Ital Dermatol Venereol 1993; 128 : 129 -132. 3 Vandersmissen G, Meuleman L, Tits G et al. Cutaneous cryptococcosis in corticosteroid-treated patients without AIDS. Acta Clin Belg 1996; 51 (2): 111 -117. 4 Nampoory MR, Khan ZU, Johny KV et al. Invasive fungal infections in renal transplant recipients. J Infect 1996; 33 (2): 95 -101. 5 Krcmery V Jr, Kunova A, Mardiak J. Nosocomial Cryptococcus laurentii fungemia in a bone marrow transplant patient after prophylaxis with ketoconazole successfully treated with oral fluconazole. Infection 1997; 25 (2): 130. 6 Bangert RL, Cho BR, Widders PR et al. A survey of aerobic bacteria and fungi in the feces of healthy psittacine birds. Avian Dis 1988; 32 : 46 -52. 7 Slavikova E, Vadkertiova R. Yeasts and yeast-like organisms isolated from fish-pond waters. Acta Microbiol Pol 1995; 44 (2): 181-189. 8 Bellosta M, Gaviglio MR, Mosconi M et al. Primary cutaneous cryptococcosis in an HIV-negative patient. Eur J Dermatol 1999; 9 (3): 224 -226. 9 Dimino-Emme L, Gurevitch AW. Cutaneous manifestations of disseminated cryptococcosis. J Am Acad Dermatol 1995; 32 (5, Part 2): 844 -850. 10 Kordossis T, Avlami A, Velegraki A et al. First report of Cryptococcus laurentii meningitis and a fatal case of Cryptococcus albidus cryptococcaemia in AIDS patients. Med Mycol 1998; 36 (5): 335 -339. 11 Johnson LB, Bradley SF, Kauffman CA. Fungaemia due to Cryptococcus laurentii and a review of non-neoformans cryptococcaemia. References 1 Carlson JA, Murphy M. Androgen receptors and lichen sclerosus (letter). J Am Acad Dermatol 2000; 43: 559. 2 Clifton M, Smoller B. Androgen receptors and lichen sclerosus (reply). J Am Acad Dermatol 2000; 43: 559. 3 Kohlberger PD, Joura EA, Bancher D, Gitsch G, Breitenecker G, Kieback DG. Evidence of androgen receptor expression in lichen sclerosus: an immunohistochemical study. J Soc Gynecol Invest 1998; 5: 331-333. 4 Powell JJ, Wojnarowska F. Lichen sclerosus. Lancet 1999; 353: 1777-1783. 5 Carlson JA, Ambros R, Malfetano J et al. Vulvar lichen sclerosus and squamous cell carcinoma: a cohort, case control, and investigational study with historical perspective; implications for chronic inflammation and sclerosis in the development of neoplasia. J Am Board Fam Pract 1999; 12: 473 -476. 6 Tidy JA, Soutter WP, Luesley DM, MacLean AB, Buckley CH, Ridley CM. Management of lichen sclerosus and intraepithelial neoplasia of the vulva in the UK.

- by Milena Petrovska and +3
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- Differential Diagnosis, Clinical Sciences, Human immunodeficiency virus, Cryptococcus

Pulmonary cryptococcosis may occur in both immunocompromised and immunocompetent hosts. The purpose of the present study was to review the radiologic findings of pulmonary cryptococcosis in King Chulalongkorn Memorial Hospital. The radiographs and computed tomography of the chests of the patients who had a diagnosis of pulmonary cryptococcosis, between 1998-2001, were retrospectively reviewed. Seven patients were included. Five were HIV infected, three had diffuse reticulonodular opacities, two (of the three) patients also had accompanying cavities; two had solely pleural effusion. Two patients were immunocompetent; one had a pulmonary nodule and another one had an endobronchial lesion and multiple pulmonary masses in the collapsed lung seen on CT scan, which were consistent with cryptococcoma. None had adenopathy. There was a difference in the radiologic manifestations between immunocompromised and immunocompetent hosts. Knowledge in radiographic features in pulmonary cryptococcosi...

- by Nitra Piyavisetpat
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- Lung Diseases, Risk factors, Immunocompromised host, Time Factors

Cryptococcus neoformans strains isolated from patients with AIDS secrete acid phosphatase, but the identity and role of the enzyme(s) responsible have not been elucidated. By combining a one-dimensional electrophoresis step with mass spectrometry, a canonically secreted acid phosphatase, CNAG_02944 (Aph1), was identified in the secretome of the highly virulent serotype A strain H99. We created an APH1 deletion mutant (⌬aph1) and showed that ⌬aph1-infected Galleria mellonella and mice survived longer than those infected with the wild type (WT), demonstrating that Aph1 contributes to cryptococcal virulence. Phosphate starvation induced APH1 expression and secretion of catalytically active acid phosphatase in the WT, but not in the ⌬aph1 mutant, indicating that Aph1 is the major extracellular acid phosphatase in C. neoformans and that it is phosphate repressible. DsRed-tagged Aph1 was transported to the fungal cell periphery and vacuoles via endosome-like structures and was enriched in bud necks. A similar pattern of Aph1 localization was observed in cryptococci cocultured with THP-1 monocytes, suggesting that Aph1 is produced during host infection. In contrast to Aph1, but consistent with our previous biochemical data, green fluorescent protein (GFP)-tagged phospholipase B1 (Plb1) was predominantly localized at the cell periphery, with no evidence of endosome-mediated export. Despite use of different intracellular transport routes by Plb1 and Aph1, secretion of both proteins was compromised in a ⌬sec14-1 mutant. Secretions from the WT, but not from ⌬aph1, hydrolyzed a range of physiological substrates, including phosphotyrosine, glucose-1-phosphate, ␤-glycerol phosphate, AMP, and mannose-6-phosphate, suggesting that the role of Aph1 is to recycle phosphate from macromolecules in cryptococcal vacuoles and to scavenge phosphate from the extracellular environment. IMPORTANCE Infections with the AIDS-related fungal pathogen Cryptococcus neoformans cause more than 600,000 deaths per year worldwide. Strains of Cryptococcus neoformans isolated from patients with AIDS secrete acid phosphatase; however, the identity and role of the enzyme(s) are unknown. We have analyzed the secretome of the highly virulent serotype A strain H99 and identified Aph1, a canonically secreted acid phosphatase. By creating an APH1 deletion mutant and an Aph1-DsRedexpressing strain, we demonstrate that Aph1 is the major extracellular and vacuolar acid phosphatase in C. neoformans and that it is phosphate repressible. Furthermore, we show that Aph1 is produced in cryptococci during coculture with THP-1 monocytes and contributes to fungal virulence in Galleria mellonella and mouse models of cryptococcosis. Our findings suggest that Aph1 is secreted to the environment to scavenge phosphate from a wide range of physiological substrates and is targeted to vacuoles to recycle phosphate from the expendable macromolecules.

- by Ben Crossett
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- Microbiology, Proteomics, Mice, Cryptococcus Neoformans
- by Johanna Rivera
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- Flow Cytometry, Cytokines, Biological Sciences, Infection and immunity

In previous studies we showed that the replication of Cryptococcus neoformans in the lung environment is controlled by the glucosylceramide (GlcCer) synthase gene (GCS1), which synthesizes the membrane sphingolipid GlcCer from the C9-methyl ceramide. Here, we studied the effect of the mutation of the sphingolipid C9 methyltransferase gene (SMT1), which adds a methyl group to position 9 of the sphingosine backbone of ceramide. The C. neoformans Δsmt1 mutant does not make C9-methyl ceramide and, thus, any methylated GlcCer. However, it accumulates de-methylated ceramide and de-methylated GlcCer. The Δsmt1 mutant loses more than 80% of its virulence compared to the wild-type and the reconstituted strain. Interestingly, growth of C. neoformans Δsmt1 in the lung was decreased and C. neoformans cells were contained in lung granulomas, which significantly reduced the rate of their dissemination to the brain reducing the onset of meningoencephalitis. Thus, using fluorescent spectroscopy and atomic force microscopy we compared the wild-type and Δsmt1 mutant and found that the altered membrane composition and GlcCer structure affects fungal membrane rigidity, suggesting that specific sphingolipid structures are required for proper fungal membrane organization and integrity. Therefore, we propose that the physical structure of the plasma membrane imparted by specific classes of sphingolipids represents a critical factor for the ability of the fungus to establish virulence.

- by Atul Singh
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- Microbiology, Medical Microbiology, Atomic Force Microscopy, Cellular microbiology

Background: Detection of subclinical cryptococcal disease using cryptococcal antigen screening among HIV-positive individuals presents a potential opportunity for prevention of both clinical disease and death if patients with detectable cryptococcal antigen are identified and treated pre-emptively. Recently developed point-of-care cryptococcal antigen tests may be useful for screening, particularly in resource-limiting settings, but few studies have assessed their utility. Methodology: The objectives of this study were to determine the prevalence and factors associated with cryptococcal antigenemia in HIV-positive patients with CD4 ' T-cell counts 5200 cells/mL who were initiating ART, and also to evaluate the utility of the point-of-care urine lateral flow assay (LFA) cryptococcal antigen test using two different diluents, compared to gold standard serum antigen testing, as a screening tool. Urine and serum of outpatients initiating antiretroviral therapy at two hospitals in Mwanza were tested for cryptococcal antigen, and demographic and clinical characteristics were obtained using structured questionnaires and patients' files. Patients with asymptomatic cryptococcal antigenemia received oral fluconazole in accordance with World Health Organization recommendations. Results: Among 140 patients screened, 10 (7.1%) had asymptomatic cryptococcal antigenemia with a positive serum cryptococcal antigen. Four of these ten patients had CD4 counts between 100 and 200 cells/mL. The prevalence of cryptococcal antigen detected in urine using a standard (older) and a test (newer) diluent were 44 (31.4%) and 19 (13.6%), with Kappa coefficients compared to serum of 0.28 and 0.51 (pB0.001 for both). Compared to the new LFA diluent for urine cryptococcal antigen, the standard diluent had higher sensitivity (100% versus 80%) but lower specificity (74% versus 92%) using serum cryptococcal antigen as a gold standard. Conclusions: Our findings suggest that HIV-positive outpatients with CD4 counts B200 cells/mL, rather than 100, should be screened for asymptomatic cryptococcal antigenemia given its association with mortality if untreated. Agreement of the urine LFA with the serum LFA was not sufficient to recommend routine screening with urine LFA.

- by Kinanga Magambo and +1
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- Tanzania, Urine, Risk factors, Prevalence

A rapid method to evidence urease activity is described. Urea hydrolysis and consequent production ammonia are detected by a chemical reaction producing a blue phenol compound (indophenol blue). Three hundred and three yeast were tested. Out of 107 urease-positive organisms detected by Christensen's Urea Agar Test (CUAT) 102 were positive by our method. No false negatives were observed by this method when testing 87 Cryptococcus strains. Ths practical screening test for presumptive identification of Cryptococcus neoformans is simple, unaffected by pH changes and requires 15 minutes to be performed,

- by Cristina Canteros
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- Plant Biology, Cryptococcus Neoformans, Phenolic compound, Time Factors

Cryptococcus gattii-induced cryptococcosis is an emerging infectious disease of humans and animals worldwide, with rare descriptions of this infection in domestic animals from Brazil. This study presents the findings associated with C. gattii in dogs from Londrina, Paraná, Southern Brazil. Two dogs, a 3-year-old, female German shepherd and a 6-year-old, male Boxer, were evaluated by a combination of pathological, mycological, and molecular diagnostic techniques. Significant pathological alterations included cryptococcal lymphadenitis, meningoencephalitis, tonsillitis, and rhinitis with nasal cryptococcomas in the German shepherd dog, while cryptococcal lymphadenitis and pneumonia were observed in the Boxer; both dogs had pseudocystic cryptococcosis. The mucicarmine histochemical stain readily identified the intralesional cryptococcal budding organisms in all affected tissues. Mycological culture and isolation confirmed the yeasts as C. gattii due to positive reaction with the L-cana...

- by Selwyn Headley
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- Plant Biology, Brazil, Cryptococcus gattii, Dogs

The pathogenic fungus Cryptococcus neoformans generally initiates infection in mammalian lung tissue and subsequently disseminates to the brain. We performed serial analysis of gene expression (SAGE) on C. neoformans cells recovered from the lungs of mice and found elevated expression of genes for central carbon metabolism including functions for acetyl-CoA production and utilization. Deletion of the highly expressed ACS1 gene encoding acetyl-CoA synthetase revealed a requirement for growth on acetate and for full virulence. Transcripts for transporters (e.g. for monosaccharides, iron, copper and acetate) and for stress-response proteins were also elevated thus indicating a nutrient-limited and hostile host environment. The pattern of regulation was reminiscent of the control of alternative carbon source utilization and stress response by the Snf1 protein kinase in Saccharomyces cerevisiae. A snf1 mutant of C. neoformans showed defects in alternative carbon source utilization, the r...

- by Guanggan Hu
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- Carbon, Molecular Microbiology, Biological Sciences, Virulence

Introduction. Incidence of cryptococcosis of the central nervous system has risen sharply since AIDS became pandemic; from early 1998, the Instituto de Neurología y Neurocirugía in Havana has beaten its own record in the number of cases attended. Aim. To describe the clinical-epidemiological characteristics of patients with this disease who were hospitalised in this centre between 1991 and 2000. Patients and methods. We present a descriptive study of 16 adult individuals who were admitted for this reason. Data on variables related with aspects concerning their epidemiology, clinical features, treatment and evolution were collected from a review of the clinical records and a survey conducted by post. Results. It was found that in no cases did the disease
appear as an epidemic outbreak, in 75% of the patients there was some kind of link with pigeons, none of the patients were HIV positive, and 50% displayed other causes of immunosuppression. Clinical behaviour varied and forms of meningitis and meningoencephalitis were prevalent; 37.5% of the patients displayed mild forms of the disease and 62.5% had more serious forms. The initial symptom in most cases was headache. The most constant CSF pattern was a raised protein level in the cerebrospinal fluid with scarce
cellularity. 87.5% of the patients were cured of the disease by treatment involving amphotericin B, in some cases associated with fluconazole. Death and the presence of post-treatment sequelae were observed in patients with serious clinical forms and late diagnoses.

- by Rick White
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- Genetics, Calcium, Transcription Factors, Biological Sciences

Clinical protocols of 28 cases of cryptococcemia studied between April 1995 and November 2002 were reviewed. The varieties of Cryptococcus neorformans, the underlying disease, and the severity and outcome of the disease were emphasized. Most patients were immunossupressed (89.3% with AIDS) and Cryptococcus neoformans var. grubii was the main recovered variety (92.8%). Regardless of antifungal treatment, in-hospital mortality was 41% strongly associated with APACHE II score, >14 (p<0.01).

- by Flávio de Oliveira
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- Argentina, Adolescent, Disease susceptibility, Child

Cryptococcus neoformans is the causative agent of cryptococcal meningoencephalitis. There is accumulating evidence that C. neoformans is a facultative intracellular pathogen, residing in macrophages and endothelium. The molecular mechanism conferring resistance to phagolysosomal killing in these cells is a key unresolved issue. To gain insight into the fungal adaptive strategies, serial analysis of gene expression was used to map genes differentially expressed in an intraphagocytic environment. By comparing transcript profiles of C. neoformans serotype D B3501 cells recovered from endothelial cells with those from free-grown cryptococci, we identified the cryptococcal homologue of the SKN7 two-component stress response regulator gene from Saccharomyces cerevisiae. Studies with C. neoformans cells disrupted for SKN7 revealed an increased susceptibility to t-butyl hydroperoxide (100% lethality at 0.7 mM, vs. 1.0 mM for wild type) and significantly lower survival rates in endothelial infection experiments. Mice experiments revealed that SKN7 disruption strongly attenuates cryptococcal virulence in vivo. We propose that Skn7 (co-)regulates the fungal adaptive strategy, allowing intraphagocytic survival by conferring resistance to phagolysosomal killing in endothelial cells.

- by Frank Coenjaerts and +2
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- Engineering, Technology, Oxidative Stress, Transcription Factors
- by Michael Price
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- Microbiology, Virulence, Glucose, Mice

Cryptococcus neoformans is a fungal pathogen causing pulmonary infection and a life-threatening meningoencephalitis in human hosts. The fungus infects the host through inhalation, and thus, the host response in the lung environment is crucial for containment or dissemination of C. neoformans to other organs. In the lung, alveolar macrophages (AMs) are key players in the host lung immune response, and upon phagocytosis, they can kill C. neoformans by evoking an effective immune response through a variety of signaling molecules. On the other hand, under conditions not yet fully defined, the fungus is able to survive and proliferate within macrophages. Since the host sphingosine kinase 1 (SK1) regulates many signaling functions of immune cells, particularly in macrophages, in this study we determined the role of SK1 in the host response to C. neoformans infection. Using wild-type (SK1/2 ؉/؉ ) and SK1-deficient (SK1 ؊/؊ ) mice, we found that SK1 is dispensable during infection with a facultative intracellular wild-type C. neoformans strain. However, SK1 is required to form a host lung granuloma and to prevent brain infection by a C. neoformans mutant strain lacking the cell wall-associated glycosphingolipid glucosylceramide (⌬gcs1), previously characterized as a mutant able to replicate only intracellularly. Specifically, in contrast to those from SK1/2 ؉/؉ mice, lungs from SK1 ؊/؊ mice have no collagen deposition upon infection with C. neoformans ⌬gcs1, and AMs from these mice contain significantly more C. neoformans cells than AMs from SK1/2 ؉/؉ mice, suggesting that under conditions in which C. neoformans is more internalized by AMs, SK1 may become important to control C. neoformans infection. Indeed, when we induced immunosuppression, a host condition in which wild-type C. neoformans cells are increasingly found intracellularly, SK1 ؊/؊ survived significantly less than SK1/2 ؉/؉ mice infected with a facultative intracellular wild-type strain, suggesting that SK1 has an important role in controlling C. neoformans infection under conditions in which the fungus is predominantly found intracellularly.

- by Travis McQuiston
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- Survival Analysis, Biological Sciences, Infection and immunity, Mice

TGF-␤1 (TGF) has been implicated in the pathogenesis of several chronic infections and is thought to promote microbial persistence by interfering with macrophage function. In rats with experimental pulmonary cryptococcosis, increased lung levels of TGF were present at 12 mo of infection. Within the lung, expression of TGF localized to epithelioid cells and foamy macrophages in areas of inflammation. Increased TGF expression was also observed in the lungs of experimentally infected mice and a patient with pulmonary cryptococcosis. TGF reduced Ab and serum-mediated phagocytosis of Cryptococcus neoformans by rat alveolar macrophages (AM) and peripheral blood monocytes, and this was associated with decreased chemokine production and oxidative burst. Interestingly, TGF-treated rat AM limited both intracellular and extracellular growth of C. neoformans. Control of C. neoformans growth by TGF-treated rat AM was due to increased secretion of lysozyme, a protein with potent antifungal activity. The effects of TGF on the course of infection were dependent on the timing of TGF administration relative to the time of infection. TGF treatment of chronically infected rats resulted in reduced lung fungal burden, while treatment early in the course of infection resulted in increased fungal burden. In summary, our studies suggest a dual role for TGF in persistent fungal pneumonia whereby it contributes to the local control of infection by enhancing macrophage antifungal efficacy through increased lysozyme secretion, while limiting inflammation by inhibiting macrophage/monocyte phagocytosis and reducing associated chemokine production and oxidative burst.

- by Johanna Rivera
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- Immunology, Immunohistochemistry, Immunology of the Gut, Cell line
- by Ernesto Nakayasu
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- Mass Spectrometry, Proteomics, Western blotting, Biological Sciences

The fungal pathogen Cryptococcus neoformans has several virulence factors, among which the most important is a polysaccharide capsule. The size of the capsule is variable and can increase significantly during infection. In this work, we investigated the relationship between capsular enlargement and the cell cycle. Capsule growth occurred primarily during the G 1 phase. Real-time visualization of capsule growth demonstrated that this process occurred before the appearance of the bud and that capsule growth arrested during budding. Benomyl, which arrests the cells in G 2 /M, inhibited capsule growth, while sirolimus (rapamycin) addition, which induces G 1 arrest, resulted in cells with larger capsule. Furthermore, we have characterized a mutant strain that lacks a putative G 1 /S cyclin. This mutant showed an increased capacity to enlarge the capsule, both in vivo (using Galleria mellonella as the host model) and in vitro. In the absence of Cln1, there was a significant increase in the production of extracellular vesicles. Proteomic assays suggest that in the cln1 mutant strain, there is an upregulation of the glyoxylate acid cycle. Besides, this cyclin mutant is avirulent at 37°C, which correlates with growth defects at this temperature in rich medium. In addition, the cln1 mutant showed lower intracellular replication rates in murine macrophages. We conclude that cell cycle regulatory elements are involved in the modulation of the expression of the main virulence factor in C. neoformans. IMPORTANCE Cryptococcus neoformans is a pathogenic fungus that has significant incidence worldwide. Its main virulence factor is a polysaccharide capsule that can increase in size during infection. In this work, we demonstrate that this process occurs in a specific phase of the cell cycle, in particular, in G 1 . In agreement, mutants that have an abnormal longer G 1 phase show larger capsule sizes. We believe that our findings are relevant because they provide a link between capsule growth, cell cycle progression, and virulence in C. neoformans that reveals new aspects about the pathogenicity of this fungus. Moreover, our findings indicate that cell cycle elements could be used as antifungal targets in C. neoformans by affecting both the growth of the cells and the expression of the main virulence factor of this pathogenic yeast.

- by Radames Cordero
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- Microbiology, Cell Cycle, Macrophages, Mice

The fungal pathogen Cryptococcus neoformans is a major cause of illness in immunocompromised individuals such as AIDS patients. The ability of the fungus to acquire nutrients during proliferation in host tissue and the ability to elaborate a polysaccharide capsule are critical determinants of disease outcome. We previously showed that the GATA factor, Cir1, is a major regulator both of the iron uptake functions needed for growth in host tissue and the key virulence factors such as capsule, melanin and growth at 37uC. We are interested in further defining the mechanisms of iron acquisition from inorganic and host-derived iron sources with the goal of understanding the nutritional adaptation of C. neoformans to the host environment. In this study, we investigated the roles of the HAP3 and HAPX genes in iron utilization and virulence. As in other fungi, the C. neoformans Hap proteins negatively influence the expression of genes encoding respiratory and TCA cycle functions under low-iron conditions. However, we also found that HapX plays both positive and negative roles in the regulation of gene expression, including a positive regulatory role in siderophore transporter expression. In addition, HapX also positively regulated the expression of the CIR1 transcript. This situation is in contrast to the negative regulation by HapX of genes encoding GATA iron regulatory factors in Aspergillus nidulans and Schizosaccharomyces pombe. Although both hapX and hap3 mutants were defective in heme utilization in culture, only HapX made a contribution to virulence, and loss of HapX in a strain lacking the high-affinity iron uptake system did not cause further attenuation of disease. Therefore, HapX appears to have a minimal role during infection of mammalian hosts and instead may be an important regulator of environmental iron uptake functions. Overall, these results indicated that C. neoformans employs multiple strategies for iron acquisition during infection.

- by Guanggan Hu
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- Microbiology, Immunology, Medical Microbiology, Western blotting

Summary. The authors report the clinical and microbiological findings of a 6-month follow-up of nine AIDS patients affected with cryptococcosis. Among these, seven patients suffered from meningoencephalitis and two from disseminated infection. The antifungal therapy during acute illness included the administration of amphotericin B at doses of 0.6 mg kg-1 day-1 i.v. plus flucytosine at doses of 100 mg kg-1 day-1 i.v. during the first 15 days followed by itraconazole at doses of 400 mg day-1 p.o. in the following 15 days. The maintenance treatment included itraconazole at doses of 200 mg day-1 p.o. indefinitely. During the 6-month follow-up, one patient died of hepatic failure related to C virus (HCV) hepatitis reactivation and another patient died of polymicrobial pneumonia. In two patients, the presence of multiple nodular lesions in the cerebral computerized tomography (CT) scan, related to cryptococcal granulomas, was associated with the persistance of fungi in the cerebrospinal fluid. In three patients with meningoencephalitis the three-drugs regimen was effective in eradicating the neurological infection, and relapses were not observed during the maintenance therapy with itraconazole during the 6-month follow-up. The two patients with haematogenous cryptococcosis did not relapse after the 6-month follow-up.Zusammenfassung. Die Studie informiert über einen 6-Monate-Zeitraum einer noch nicht abgeschlossenen klinisch-mikrobiologischen Beobachtung an neun Cryptococcose-AIDS-Patienten, sieben davon mit Cryptococcus-Meningoenzephalitis und zwei mit disseminierter Cryptococcose. Alle Patienten wurden in der akuten Phase mit Amphotericin B (0.6 mg kg-1 d-1 i.v.) und Flucytosin (100 mg kg-1 d-1 i.v.) 15 Tage lang behandelt. Anschließend erhielten die Patienten Itraconazol (400 mg d-1 per os) ebenfalls über 15 Tage. Als Dauernachbehandlung wurde Itraconazol (200 mg d-1) auf unbestimmte Zeit verabreicht. Während der 6-monatigen Beobachtungszeit starb ein Patient an Leberversagen aufgrund der Reaktivierung einer HCV-Hepatitis sowie ein weiterer an polymikrobieller Pneumonie. In zwei Fällen mit multiplen hypodensen Läsionen im Enzephalo-CT, interpretiert als granulomatöse Lokalisation der Cryptococcose, war die Prognose negativ, weil nach 30 Therapietagen die Liquorkultur noch positiv war. Bei drei Patienten erwies sich das Therapieregime als wirksam. Rückfalle wurden in der 6-monatigen Nachbeobachtungs-periode nicht beobachtet, ebenso wenig bei den beiden Patienten mit disseminierter Cryptococcose.

- by paolo sacchi
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- Chronic Disease, Follow-up studies, Clinical Sciences, Antifungal Agents

inmunodeprimidos hasta que el TARGA logra sus efectos, en los que no desean o no pueden tomar TARGA, en aquellos en los que este fracasa y en el pequeño grupo de infectados que son incapaces de recuperar cifras adecuadas de linfocitos T... more

- by josue lopez
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- Medical Microbiology, Drug interactions, Adolescent, Cryptococcus Neoformans

... Dr Graeme Meintjies is supported by the Wellcome Trust. ... 4. Lehloenya R, Meintjes G. Dermatologic manifestations of the immune reconstitution inflammatory syndrome. Dermatol Clin 2006; 24:549-570. ... Bunning RD, Werner FB.... more

- by Paul Bohjanen and +1
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- Treatment Outcome, Biological Sciences, Cryptococcus Neoformans, AIDS
- by Mahendra Pal
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- Veterinary Medicine, Cryptococcus Neoformans, Immunocompromised host, Cryptococcosis

A novel modified India ink technique for the diagnosis of Cryptococcus neoformans in cerebrospinal fluid specimens is described. It employs 2% chromium mercury and India ink. This technique allows a clear identification of some external and internal structures of the organism. Three layers from the outer capsule that have previously been discerned only by electron microscopy are distinguished.

- by Luis Huicho
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- Carbon, Clinical Microbiology, Biological Sciences, Cerebrospinal Fluid

- by Rick White
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- Microbiology, Immunology, Medical Microbiology, Western blotting

We have studied infection of Cryptococcus neoformans in the non-vertebrate host Galleria mellonella with particular interest in the morphological response of the yeast. Inoculation of C. neoformans in caterpillars induced a capsule-independent increase in haemocyte density 2 h after infection. C. neoformans manifested a significant increase in capsule size after inoculation into the caterpillar. The magnitude of capsule increase depended on the temperature, being more pronounced at 37uC than at 30uC, which correlated with an increased virulence of the fungus and reduced phagocytosis at 37uC. Capsule enlargement impaired phagocytosis by haemocytes. Incubation of the yeast in G. mellonella extracts also resulted in capsule enlargement, with the polar lipidic fraction having a prominent role in this effect. During infection, the capsule decreased in permeability. A low proportion of the cells (,5%) recovered from caterpillars measured more than 30 mm and were considered giant cells. Giant cells recovered from mice were able to kill the caterpillars in a manner similar to regular cells obtained from in vivo or grown in vitro, establishing their capacity to cause disease. Our results indicate that the morphological transitions exhibited by C. neoformans in mammals also occur in a non-vertebrate host system. The similarities in morphological transitions observed in different animal hosts and in their triggers are consistent with the hypothesis that the cell body and capsular responses represent an adaptation of environmental survival strategies to pathogenesis.

- by Arturo Casadevall
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- Engineering, Physics, Chemistry, Biology