Pigment production by Cryptococcus neoformans and other Cryptococcus species from aminophenols and diaminobenzenes.

Cryptococcus neoformans and other Cryptococcus species can produce pigment(s) from many aminophenol and diaminobenzene compounds. Pigment production from these compounds is similar to the conversion of diphenols to melanin by C. neoformans. Several pigmentation patterns (resulting in the identification or grouping of Cryptococcus species) have been observed by using diaminobenzene and aminophenol compounds as substrates. The most common pigmentation pattern observed was pigment production by both C. neoformans and C. terreus. In contrast to the diphenols, only two aminophenols (4-hydroxymetanilamide and 3-aminotyrosine) were found to be highly specific as substrates. They allowed only C. neoformans to produce pigment. When 4-aminosalicylic acid was the substrate, a unique pattern was observed because only C. terreus, C. diffluens, and C. albidus produced pigment. Finally, a pattern was observed in which C. neoformans produced large amounts of pigment from aminophenol and diaminobenzene compounds, whereas the other Cryptococcus species produced smaller amounts. A simplified scheme with three substrates resulted in the identification of C. terreus and C. neoformans as well as two groups of other Cryptococcus species, group I (C. albidus and C. diffluens) and group II (C. laurentii and C. luteolus).     

First case of cryptococcosis in a new species of bandicoot (Bandicota indica) caused by Cryptococcus neoformans var. grubii.

The first case of cryptococcosis caused by Cryptococcus neoformans var. grubii in a new species of bandicoot (Bandicota indica) is described. The animal was trapped in a bamboo thicket in a park located in the city of Jabalpur, India. On necropsy, pathological lesions were seen in the lungs and liver and C. neoformans var. grubii was isolated from the lungs, liver, kidneys, spleen and brain but not the heart or intestine. The soil of the animal's burrow and bamboo debris around it also revealed the presence of C. neoformans var. grubii. We hypothesize that the bandicoots may potentially act as sentinel animals for environmental human pathogenic Cryptococcus species.     

In-vitro antifungal activities of sulfa drugs against clinical isolates of Aspergillus and Cryptococcus species.

In vitro susceptibilities of ten clinical isolates, including five strains of Cryptococcus neoformans var. grubii and five strains of Aspergillus fumigatus, were determined against nine sulfa drugs using a microdilution method. Among the five tested media, minimum inhibitory concentration (MIC) values were observed only in YNB medium: no detectable level MIC value of less than 125 microg/ml was observed in the four remaining media against Cryptococcus species. Of the nine sulfa drugs, of which sulfaphenazole showed the highest antifungal activity, the MIC values for A. fumigatus and C. neoformans var. grubii were, respectively, 64 microg/ml and 4-8 microg/ml, suggesting high susceptibility of C. neoformans to sulfa drugs.     

新型隐球菌与肺泡上皮细胞的体外相互作用

目的研究新型隐球菌与肺泡上皮细胞的体外相互作用,探讨隐球菌肺部感染的发病机制。方法体外培养Ⅱ型肺泡上皮细胞A549(ATCC CCL-185),检测新型隐球菌2种变种对细胞的时间/浓度黏附率、通过率;检测新型隐球菌对细胞的损伤作用;透射电镜观察相互作用的超微结构。结果2种变种的新型隐球菌可以对A549细胞产生黏附与侵袭,黏附率与侵袭率呈现时间依赖性;同时还可以使A549细胞凋亡率升高,对其造成损伤,这与菌体的活力相关。超微结构可见隐球菌与肺泡上皮细胞的黏附与侵袭过程。2种变种之间在黏附率、通过率及对细胞的损伤作用方面差异无统计学意义。结论活的隐球菌黏附与侵袭肺泡上皮细胞是隐球菌感染肺部的重要条件,不同变种对肺部的易感性可能不存在差异。进一步明确二者的作用机制对隐球菌的发病机制研究具有重要意义。     

Laccase and melanization in clinically important Cryptococcus species other than Cryptococcus neoformans

The laccase enzyme and melanin synthesis have been implicated as contributors to virulence in Cryptococcus neoformans. Since isolations of Cryptococcus species other than C. neoformans from clinical specimens have been increasing, we examined the laccase activities of C. albidus, C. laurentii, C. curvatus, and C. humicola. Incubation of cells with epinephrine produced adrenochrome color in C. albidus, C. laurentii, and C. curvatus but not in C. humicola. Activity was always less than in C. neoformans. Laccase was detected in the soluble fractions of disrupted C. albidus, C. laurentii, and C. curvatus cells. Activity staining of partially purified enzyme after nondenaturing polyacrylamide gel electrophoresis revealed that laccases from C. albidus, C. laurentii, and C. curvatus migrated more slowly than that from C. neoformans. One strain of C. curvatus exhibited two melanin bands. Thus, several clinically emerging Cryptococcus species express laccase and can synthesize melanin.     

Rapid identification of Cryptococcus neoformans var. grubii, C. neoformans var. neoformans, and C. gattii by use of rapid biochemical tests, differential media, and DNA sequencing

Rapid identification of Cryptococcus neoformans var. grubii, Cryptococcus neoformans var. neoformans, and Cryptococcus gattii is imperative for facilitation of prompt treatment of cryptococcosis and for understanding the epidemiology of the disease. Our purpose was to evaluate a test algorithm incorporating commercial rapid biochemical tests, differential media, and DNA sequence analysis that will allow us to differentiate these taxa rapidly and accurately. We assessed 147 type, reference, and clinical isolates, including 6 other Cryptococcus spp. (10 isolates) and 14 other yeast species (24 isolates), using a 4-hour urea broth test (Remel), a 24-hour urea broth test (Becton Dickinson), a 4-hour caffeic acid disk test (Hardy Diagnostics and Remel), 40- to 44-hour growth assessment on l-canavanine glycine bromothymol blue (CGB) agar, and intergenic spacer (IGS) sequence analysis. All 123 Cryptococcus isolates hydrolyzed urea, along with 7 isolates of Rhodotorula and Trichosporon. Eighty-five of 86 C. neoformans (99%) and 26 of 27 C. gattii (96%) isolates had positive caffeic acid results, unlike the other cryptococci (0/10) and yeast species (0/24). Together, these two tests positively identified virtually all C. neoformans/C. gattii isolates (98%) within 4 h. CGB agar or IGS sequencing further differentiated these isolates within 48 h. On CGB, 25 of 27 (93%) C. gattii strains induced a blue color change, in contrast to 0 of 86 C. neoformans isolates. Neighbor-joining cluster analysis of IGS sequences differentiated C. neoformans var. grubii, C. neoformans var. neoformans, and C. gattii. Based on these results, we describe a rapid identification algorithm for use in a microbiology laboratory to distinguish clinically relevant Cryptococcus spp.     

High-resolution melting analysis for identification of the Cryptococcus neoformans-Cryptococcus gattii complex

We have developed a two-step method based on high-resolution melting (HRM) that reliably identifies species from the Cryptococcus species complex (Cryptococcus neoformans var. grubii, Cryptococcus neoformans var. neoformans, and Cryptococcus gattii). Our results indicate that HRM can provide a fast protocol to identify and distinguish among the main Cryptococcus species.     

Typing and patterns of cellular morphological alterations in Cryptococcus neoformans and Cryptococcus gattii isolates exposed to a panel of killer yeasts.

Cryptococcus neoformans and Cryptococcus gattii are encapsulated basidiomycetous yeasts that cause meningoencephalitis. The action of killer yeasts on the growth of one hundred genotypically characterized C. neoformans var. neoformans, C. neoformans var. grubii, and C. gattii clinical and environmental isolates was evaluated. Killer studies were performed on yeast malt-methylene blue (YM-MB) agar Petri dishes, and a dendrogram was obtained based on a quantitative data matrix using the diameter of the inhibition halo. The cellular morphological characteristics of dead cells within the halo were observed by means of optical and scanning electron microscopy. There was no formation of pores on the cell surface of the sensitive cells in contact with the toxins, at least for C. neoformans. The sensitivity patterns of clinical and environmental isolates to the killer toxins demonstrated that there is correlation between killer sensitivity of Cryptococcus species or varieties and some of the killer strains. In this case, the isolates were discriminated using the killer sensitivity patterns, and this could be used as a complementary tool to PCR-fingerprinting in epidemiological studies.     

Feral pigeons as carriers of Cryptococcus laurentii, Cryptococcus uniguttulatus and Debaryomyces hansenii.

We collected fresh droppings and cloaca samples from feral pigeons Columba livia in the southern Swedish city of Malm?, and isolated the following fungi: Debaryomyces hansenii var. hansenii, Cryptococcus laurentii and Cryptococcus uniguttulatus. The first two species are known to be pathogenic to humans. No strains of Cryptococcus neoformans var. neoformans were found. Our results indicate that feral pigeons can be carriers of medically significant fungi other than Cryptococcus neoformans var. neoformans.     

Distribution of Cryptococcus gattii and Cryptococcus neoformans in decayed trunk wood of Syzygium cumini trees in north-western India.

The aim of this study is to report the regional distribution of Cryptococcus. gattii and Cryptococcus. neoformans in decayed wood inside trunk hollows of Syzygium cumini trees (Java plum, Indian black berry) investigated in Amritsar (Panjab), Meerut Cantt. and Bulandshahr (Uttar Pradesh) and Delhi, in north-western India. Two hundred and seventeen wood samples collected from 74 S. cumini trees were investigated. This includes 7 known positive S. cumini trees in Delhi subjected to a mycological surveillance for perennial colonization by C. gattii and C. neoformans. Cryptococcus gattii showed the highest prevalence (89%) in S. cumini trees in Delhi, followed by 27%, 12.5% and 9% prevalence in Bulandshahr, Amritsar City and Meerut Cantt., respectively. In contrast, C. neoformans had the highest prevalence (54%) in Amritsar, followed by 44% in Delhi, 9% in Bulandshahr and 0% in Meerut Cantt. Furthermore, 44% of the S. cumini trees in Delhi, 9% in Bulandshahr and 8% in Amritsar were concomitantly colonized by both C. gattii and C. neoformans. A mycological surveillance over 4.8-5.2 years of 7 selected S. cumini trees in Delhi revealed perennial colonization by both the Cryptococcus species. In addition, air samples taken close to the decayed trunk hollows of 4 of the perennially colonized S. cumini trees contained strains of the C. neoformans species complex. Of a random sample of 48 isolates serotyped, 26 (54%) were C. neoformans, serotype A, and 22 (46%) C. gattii, serotype B. Determination of mating type alleles was done in 44 of the isolates, comprising 31 of C. neoformans, serotype A and 13 of C.gattii, serotype B. All of them proved to be mating type alpha (MATalpha). The data on high prevalence, fungal population density, perennial colonization and aerial isolations indicate that decayed wood in trunk hollows of S. cumini trees is to-date the main well documented primary environmental niche of C. gattii and C. neoformans in north-western India. Attention is drawn to the likely health hazard posed by the environmental reservoirs of C. gattii and C. neoformans occurring in tree trunk hollows in proximity to human and animal habitations.     

Environmental prevalence of Cryptococcus neoformans and Cryptococcus gattii in India: an update

An overview of work done to-date in India on environmental prevalence, population structure, seasonal variations and antifungal susceptibility of Cryptococcus neoformans and Cryptococcus gattii is presented. The primary ecologic niche of both pathogens is decayed wood in trunk hollows of a wide spectrum of host trees, representing 18 species. Overall, C. neoformans showed a higher environmental prevalence than that of C. gattii which was not found in the avian habitats. Apart from their arboreal habitat, both species were demonstrated in soil and air in close vicinity of their tree hosts. In addition, C. neoformans showed a strong association with desiccated avian excreta. An overwhelming number of C. neoformans strains belonged to genotype AFLP1/VNI, var. grubii (serotype A), whereas C. gattii strains were genotype AFLP4/VGI, serotype B. All of the environmental strains of C. neoformans and C. gattii were mating type α (MATα). Contrary to the Australian experience, Eucalyptus trees were among the epidemiologically least important and, therefore, the hypothesis of global spread of C. gattii through Australian export of infected Eucalyptus seeds is rebutted. Reference is made to long-term colonization of an abandoned, old timber beam of sal wood (Shorea robusta) by a melanin positive (Mel(+)) variant of Cryptococcus laurentii that was pathogenic to laboratory mice.     

Evaluation of a new method for identification of Cryptococcus neoformans which uses serologic tests aided by selected biological tests.

A new method for identifying Cryptococcus neoformans isolates and their serotypes by the slide agglutination test using five kinds of factor sera, with the aid of nitrate reduction, phenol oxidase, and growth at 37 degrees C tests was evaluated by using 36 reference strains and 75 clinical isolates of C. neoformans. The results showed that the reference strains were identified exactly as they were labeled, and clinical isolates were identified as C. neoformans serotypes A, D, and AD. C. neoformans could be distinguished from other Cryptococcus species that cross-reacted with factor sera by their ability to grow at 37 degrees C. These results indicate that the slide agglutination test combined the use of factor sera for isolates which grow at 37 degrees C is a useful method for identification of C. neoformans and their serotypes and that the nitrate reduction test (negative in 100% of the isolates) and the phenol oxidase test (positive in approximately 95% of the isolates) can be used to confirm that the species is C. neoformans.     

Cyclosporin A inhibits the growth of Cryptococcus neoformans in a murine model.

Cryptococcus neoformans is a frequent opportunistic infectious agent in patients with decreased T-lymphocyte-mediated immune function, including those with acquired immune deficiency syndrome. Cyclosporin A (CsA), a potent inhibitor of T-lymphocyte function, was administered subcutaneously to mice to study the pathogenesis of C. neoformans infections in the setting of impaired T-cell function. Surprisingly, survival was prolonged indefinitely in animals that received immunosuppressive doses of CsA following either intratracheal or intravenous inoculations of C. neoformans. Furthermore, following intratracheal inoculation, mice treated with CsA cleared C. neoformans from their lungs more rapidly than did control mice. CsA directly inhibited the growth of C. neoformans when it was added to cultures in vitro at concentrations comparable to the blood levels achieved in experimental mice. Thus, CsA inhibited both in vitro and in vivo growth of C. neoformans. While these results must be extended to studies in humans, these data suggest that patients who now receive CsA-immunosuppressive therapy may be fortuitously protected against infections with C. neoformans. Furthermore, research into cyclosporin derivatives may yield compounds with less immunosuppressive properties and enhanced antifungal activity.     

Antigenic characterization of Cryptococcus neoformans serotypes and its application to serotyping of clinical isolates

Antigenic analysis of the four serotypes of Cryptococcus neoformans was carried out by slide agglutination with reciprocal adsorption methods. With this procedure the antigenic patterns of the serotypes were established. Serotypes A and D had antigenic factors 1, 2, 3, 7 and 1, 2, 3, 8, respectively. Serotypes B and C were found to have antigenic factors 1, 2, 4, 5 and 1, 4, 6, respectively. Factor sera, prepared according to the antigenic patterns demonstrated by adsorption studies, proved to be useful for rapidly and accurately identifying C. neoformans serotypes. Some patterns similar to those of the C. neoformans serotypes were observed in five other Cryptococcus species and two Candida species. The proton magnetic resonance spectra of polysaccharides from the C. neoformans serotypes correlated well with their antigenic characteristics. Phenol oxidase test reactions and growth at 37 degrees C were useful criteria for determining which yeasts should be chosen for clinical application of factor sera for serotyping of C. neoformans. Sixty-two Japanese isolates of C. neoformans were serotyped. Fifty-eight of these isolates were serotype A, three were serotype A-D, and one was serotype D.     

New, special stain for histopathological diagnosis of cryptococcosis.

The Masson-Fontana silver stain for melanin was employed for the differentiation of pathogenic fungal species in human or mouse tissues. The fungi studied were Candida albicans, Candida tropicalis, Candida glabrata (Torulopsis glabrata), Cryptococcus neoformans, Cryptococcus bacillisporus, Coccidioides immitis, Blastomyces dermatitidis, Histoplasma capsulatum, Paracoccidioides brasiliensis, Sporothrix schenckii, Rhizopus rhizopodiformis, and Aspergillus fumigatus. The tissue sections stained with Masson-Fontana silver stain showed a dark brown to black color in the wall of cryptococci, whereas the walls of remaining fungal species were hyaline, except for those of S. schenckii. The yeastlike cells of S. schenckii showed faint brown pigment on the wall. Cultures of these fungi showed staining characteristics identical to those of the in vivo results. Cultures of four nonpathogenic Cryptococcus species, Cryptococcus uniguttulatus, Cryptococcus laurentii, Cryptococcus terreus, and Cryptococcus luteolus, were also tested for staining by the Masson-Fontana procedure. Of these, only C. laurentii stained positively, and the pigment on the cell wall was as intense as that of the cells of C. neoformans. These results indicate that the Masson-Fontana silver stain can be used as a specific stain in the histological diagnosis of cryptococcosis.     

Karyotyping of Cryptococcus neoformans as an epidemiological tool.

Karyotyping of Cryptococcus neoformans var. neoformans can be used as an epidemiological tool for C. neoformans infections. In this study of over 40 isolates from both clinical and environmental sources, 90% had a unique chromosome banding by pulsed-field electrophoresis. There was no conserved pattern associated with body site of infection, geographical location of the isolate, or human immunodeficiency virus status. Karyotypes of individual isolates remained stable during both in vitro passage and in vivo infections. Karyotype was used to exclude the possibility of nosocomial spread of C. neoformans in one clinical situation and supported relapse in two other cases. Because of its variable sizes between isolates, karyotyping of C. neoformans is a convenient method for molecular identification of different strains.     

In vivo role of dendritic cells in a murine model of pulmonary cryptococcosis

Dendritic cells (DC) have been shown to phagocytose and kill Cryptococcus neoformans in vitro and are believed to be important for inducing protective immunity against this organism. Exposure to C. neoformans occurs mainly by inhalation, and in this study we examined the in vivo interactions of C. neoformans with DC in the lung. Fluorescently labeled live C. neoformans and heat-killed C. neoformans were administered intranasally to C57BL/6 mice. At specific times postinoculation, mice were sacrificed, and lungs were removed. Single-cell suspensions of lung cells were prepared, stained, and analyzed by microscopy and flow cytometry. Within 2 h postinoculation, fluorescently labeled C. neoformans had been internalized by DC, macrophages, and neutrophils in the mouse lung. Additionally, lung DC from mice infected for 7 days showed increased expression of the maturation markers CD80, CD86, and major histocompatibility complex class II. Finally, ex vivo incubation of lung DC from infected mice with Cryptococcus-specific T cells resulted in increased interleukin-2 production compared to the production by DC from na?ve mice, suggesting that there was antigen-specific T-cell activation. This study demonstrated that DC in the lung are capable of phagocytosing Cryptococcus in vivo and presenting antigen to C. neoformans-specific T cells ex vivo, suggesting that these cells have roles in innate and adaptive pulmonary defenses against cryptococcosis.