Production of culture filtrates of Sporothrix schenckii in diverse culture media.

Mycelial-form Sporothrix schenckii was studied to determine if growth in complex (Sabouraud-dextrose) or defined culture media (minimal medium, Gibco Medium 199 with and without Hepes buffer) with differing initial pH levels would affect expression of antigen components. Cultures were evaluated by continuous monitoring and serial sampling for various parameters. Great variation was seen in the protein and antigenic patterns induced by the different media. The expression of a 55 kDa component accompanied by significant acidification of the culture medium at the beginning of the stationary growth phase was notable in Sabouraud medium. In the chemically defined media, a 90 kDa component was expressed that reacted with sera from patients with sporotrichosis. The pH in these media showed little change during the different growth phases of the fungus. Among the media studied, minimal medium favored the expression of the greatest number of antigenic components. In all of the assays, the stationary growth phase appeared to be optimal for content of antigenic components. Cross reactions were not observed with any of the culture filtrates using sera from patients with other mycoses.

[Isolation, identification and serotyping of yeasts obtained from the vaginal fluid in patients with clinical vaginitis]. / Aislamiento, identificación y serotipificación de levaduras obtenidas del flujo vaginal en pacientes con clínica de vaginitis.

A study was carried out to determine the presence of Candida in 105 patients with clinical vaginitis who consulted in the Infectious Disease Unit of the Vargas Hospital after referral from Gynecology Service. Yeasts were detected in 23 cases (24%), and identified as C. albicans (12), C. tropicalis (5), C. guilliermondii (3), C. glabrata (2) and C. parapsilosis (1). The presence of hyphae was observed in 50% of the direct examinations, in which the isolated species was C. albicans. These structures were not observed in infections with other species of Candida. In this study, there was relatively little difference between the percentages of serotypes A and B, 58 % and 42, respectively. This is in contrast with previous studies reported in clinical material from Venezuela and other countries, in which serotype A presented a greater incidence than serotype B. Our observations suggest an increase in serotype B C. albicans in vaginal candidiasis.

[Serotypes of Candida albicans isolated from HIV positive patients.]. / Algunas consideraciones acerca de la utilización de fuentes carbonadas en la producción de ácido jasmónico.

Twenty-one HIV-positive patients in different stages of the disease were studied to evaluate candidosis in the oral cavity. All patients in clinical category C were infected with Candida. The most frequently observed clinical forms were pseudomembranous and hypertrophic, in contrast to reports by other authors. Candida albicans was the species isolated in these HIV-positive patients. Alterations of cell-mediated immunity were reflected in the negativity of intradermal test. The predominant serotype of C. albicans in these patients was A, in agreement with what has been found in non-immunosuppressed patients in Venezuela. There was no correlation between the serotype of C. albicans and the clinical forms of candidosis. Based in our results and those of other authors, no conclusions can be drawn concerning a particular serotype as an indicator of immunosuppression.

Comparative ultrastructure and immunolabeling of MHC-II antigens of alveolar macrophages obtained from patients with paracoccidioidomycosis and other lung diseases.

Samples of alveolar macrophages (AM) obtained by bronchoalveolar lavage from patients with either paracoccidioidomycosis, silicosis, sarcoidosis, or allergic alveolitis were investigated by electron microscopy and immunocytochemistry to compare cellular ultrastructure and expression of MHC-II antigens in the AM cell surface. All samples of AM obtained from patients with these pathologies showed heterogeneous structural features. Although, this morphological diversity is also present in AM of healthy donors, our observations seem to indicate that in the diseases studied this morphofunctional diversity is associated with additional ultrastructural characteristics inherent to each disease. In paracoccidioidomycosis the proportion of vacuolated macrophages is significantly lower than in other diseases; this might indicate that in paracoccidioidomycosis the proportion of activated AM is smaller. We observed significant differences in the expression of MHC-II antigens. Silicosis, sarcoidosis, and allergic alveolitis do not differ significantly in the quantity of immunolabeled AM or in the distribution of the label. The percentage of AM from paracoccidioidomycosis that exhibit the MHC-II molecule is very low with poor immunolabeling. In this disease the low expression of the MHC-II molecule could be related to a decrease of the antigen presenting function by AM.

Alkaline phosphatase at the cell wall of the yeast phase of Paracoccidioides brasiliensis.

The activity of alkaline phosphatase demonstrated by histochemical techniques was shown at the cell wall of the yeast form of Paracoccidioides brasiliensis at 3, 6, and 9 days of culture. The results showed a very active deposition at the cell wall as early as 9 days of culture of the fungus which made us think an inactive salt precipitate was also present.

Specific digestive deficiency of phagocytes in paracoccidioidomycosis. Its absence in peripheral blood neutrophils of members of the nuclear family of patients. An initial report.

Paracoccidioidomycosis (PARA) affects only a minority of individuals, who have presumably been exposed to the causative fungus (Paracoccidioides brasiliensis). Neutrophils (PMNs) from patients with PARA show a significant and specific digestive deficiency phagocytosed P. brasiliensis in vitro. It is not known whether the defect is acquired after contact with the fungus, or precedes it. We studied the spouses and offspring of three patients with PARA. Individuals studied stayed in the same house as their husband or father. None of the relatives had evidence of PARA, and their PMNs showed no defect in their ability to digest or kill the fungus. Relatives showed no indication of sensitization against P. brasiliensis. These results are compatible with the view that effective contact with P. brasiliensis may occur only under restricted conditions and that the defect in digestive ability is an infrequent and specific occurrence.

Relationship between digestive and killing abilities of neutrophils against Paracoccidioides brasiliensis.

Peripheral blood neutrophils (PMN) from patients with paracoccidioidomycosis killed and digested Paracoccidioides brasiliensis much less than did PMN from normal individuals or from patients with other diseases. However, deficiency in killing ability was less specific than digestive deficiency and correlated poorly with it. We conclude that the capacities of PMN to digest and kill P. brasiliensis are not intimately related phenomena, and that in paracoccidioidomycosis the key deficiency of neutrophil function is that of digestion of P. brasiliensis.

[Serotyping of 48 isolates of Candida albicans: predominance of serotype A over B in Venezuela]. / Serotipificación de 48 aislados de Candida albicans: predominio del serotipo A sobre el B en Venezuela.

The study of serotyping of isolates of Candida albicans of clinical material obtained from different geographic areas of Venezuela suggest that serotype A is predominant over type B. These results are in relation with results obtained in other countries. Type A serotype is predominant in 57 to 100% in the areas studied. Only in 2 cities serotype B was isolated. Both serotypes of Candida albicans were observed in intertriginous, mucosal, inguinal and lung forms. These studies have allowed us to have information about the predominance of serotypes in certain areas of the country.

Influence of serum on in vitro digestion of Paracoccidioides brasiliensis by neutrophils.

Serum from patients with paracoccidioidomycosis (PARA) did not block digestive abilities of neutrophils (PMNs) from healthy individuals against Paracoccidioides brasiliensis. Conversely, serum from healthy donors did not enhance digestive capacities of PMNs from patients with PARA vis á vis the causative organism. We conclude that the specific digestive defect present in PMNs from patients with PARA is not mediated by serum factors.

Functions of polymorphonuclear leukocytes and individuality of Jorge Lobo's disease: absence of the specific leukocyte digestive defect against Paracoccidioides brasiliensis.

Peripheral blood neutrophils (PMNs) from a patient with Jorge Lobo's disease (JLD) digested well phagocytosed Paracoccidioides brasiliensis. We found no circulating antibodies against P. brasiliensis in the patient's serum. Such neutrophils showed myeloperoxidase activity and also digested normally phagocytosed Candida albicans. We had previously reported the presence of a specific digestive deficiency of PMNs from patients with paracoccidioidomycosis (PARA) vis à vis P. brasiliensis. Current findings provide new information about leukocyte functions in JLD and bolster the view that JLD, PARA and their respective causative microorganisms are distinct.

A novel method for estimating killing ability and digestion of Paracoccidioides brasiliensis by phagocytic cells in vitro.

We describe a novel method by which phagocytosis, digestion and killing of Paracoccidioides brasiliensis yeast cells by polymorphonuclear leukocytes or other phagocytic cells may be estimated simultaneously. Suspensions of P. brasiliensis (yeast-like phase) were sonicated, counted and incubated at 37 degrees C with known numbers of phagocytes. Control preparations contained no phagocytic cells. At given intervals samples were incorporated into gelatin nutrient medium and droplets of the mixtures were incubated at room temperature. Live yeast-like P. brasiliensis germinate in vitro and produce filaments. After incubation, droplets may be melted and examined under phase contrast optics, or the cells may be washed and stained by a variation of Papanicolaou's method. Digested P. brasiliensis, intact but non-germinating yeasts and filamented (viable) yeasts may be identified and counted. Killing and digestive abilities of phagocytes may be estimated by the difference between values obtained from phagocyte-containing and control preparations.

Chemical changes in cell wall structure of five strains of Paracoccidioides brasiliensis.

Differences in cell wall structure of Paracoccidioides brasiliensis strains isolated from localized or disseminated cases of paracoccidioidomycosis and from soil emphasize the importance of standardization if results obtained in different laboratories are to be compared.

Studies on plating efficiency and estimation of viability of suspensions of Paracoccidioides brasiliensis yeast cells.

Mild sonication was used to obtain single cell suspensions of Paracoccidioides brasiliensis. These cells were intact by microscopic criteria. Direct cell counts in a given inoculum and colony formation on various media were used to determine plating efficiency. Sonicated and nonsonicated cell suspensions were used to study plating efficiency and to estimated viability by means of vital dyes. Methylene blue Erythrosin B, and Janus green were unreliable when used with P. brasiliensis, but vital dyes were accurate when tested with Candida albicans. Acridine orange gave more meaningful results of viability. Estimates of viability, however, changed significantly as a result of relatively minor alterations in the composition of the suspending medium. In initial experiments, the plating efficiency of P. brasiliensis was dismally low. It descended abruptly with increasing dilution of inoculum. Efficiency was much improved if horse serum was added to brain heart infusion plates or if glucose glycine yeast extract (GGY) plates were incubated at room temperature and mycelial colonies were counted. With the technique we report, current plating efficiency of sonicated suspensions is of the order of 25%. Our results and procedures have an important bearing upon those studies concerned with in vitro killing of P. brasiliensis in suspensions or with isolating this fungus from clinical or environmental specimens.

Defect of in vitro digestive ability of polymorphonuclear leukocytes in paracoccidioidomycosis.

Selected functions of polymorphonuclear leukocytes were studied in patients with paracoccidioidomycosis (South American blastomycosis), in healthy control individuals, and in patients with diseases unrelated to paracoccidioidomycosis. Patients with paracoccidioidomycosis were also evaluated by standard immunological techniques. Phagocytosis and digestion of Paracoccidioides brasiliensis yeastlike cells in vitro was estimated by an original method. It was based on the appearance of phagocytosed P. brasiliensis in preparations stained by a modification of the Papanicolaou method and examined with phase-contrast optics. Interpretation of such findings was confirmed by electron microscopy. Two strains of P. brasiliensis were used. Strain 8506 was freshly isolated from a patient. Strain Pb9 was known to be nonpathogenic and to have a peculiar cell wall composition. Yeastlike cells of the Pb9 strain were digested significantly better than those of strain 8506. A higher number of leukocytes per fungus cells led to a higher proportion of digested P. brasiliensis. Leukocytes from patients with paracoccidioidomycosis phagocytosed the fungus in a normal way, but had a significant lower ability to digest it in vitro. When individual cases were analyzed, there was an excellent correlation between clinical evolution and digestive ability of polymorphonuclear leukocytes. There was good correlation between both of these and immunological parameters. Leukocytes from all groups behaved comparably in tests of general leukocyte function and in their abilities to kill and digest Candida albicans. Our results indicate that, as a group, polymorphonuclear leukocytes from patients with paracoccidioidomycosis had a significant, rather specific, defect in their in vitro digestive capacity against phagocytosed P. brasiliensis. There was also an inverse correlation between strain pathogenicity and its susceptibility to in vitro digestion by polymorphonuclear leukocytes. Our findings are concordant with and relevant to clinical reality.

Demonstration and quantification oomycosis.

Using a radioimmunoassay technique, we have determined the total IgE concentration and specific IgE antibody level against Paracoccidioides brasiliensis in the sera of patients with mycologically confirmed active paracoccidioidcosis. Patients with aspergilloma, or individuals with antibodies against Cadida albicans, and sera of apparently healthy individuals from the endemic area were also examined. The level of total IgE was significantly elevated in 9 out of 10 cases of paracoccidioidomycosis. The mean level of specific IgE against P. brasiliensis was significantly higher in the serum of the patients in comparison with control individuals. The percentage of specific IgE relative to the total varied from 0.38 to 2.28%. The differences between IgE levels in pare other mycoses were statistically significant. Our results suggest a possible involvement of specific IgE antibodies against P. brasiliensis in the immune response of patients with paracoccidioidomycosis. The hyperimmunoglobulinemia E may be related to the T-cell defect that characterizes the severe forms of the disease.

New method for estimating digestion of Paracoccidioides brasiliensis by phagocytic cells in vitro.

We describe a method by which phagocytosis and digestion of Paracoccidioides brasiliensis yeast cells by polymorphonuclear leukocytes or other phagocytic cells may be estimated. Suspensions of P. brasiliensis in its yeastlike phase were sonicated, counted, and incubated with known numbers of peripheral blood polymorphonuclear leukocytes. At given intervals, cytocentrifuge droplets were stained by a variation of Papanicolaou's method. Stained preparations were examined with phase-contrast optics. Digested organisms showed total or partial disappearance of protoplasm. Green-stained cell walls resisted digestion. The proportion of digested cells as a function of time was estimated.