Trends in the molecular epidemiology and population genetics of emerging Sporothrix species.

Sporothrix (Ophiostomatales) comprises species that are pathogenic to humans and other mammals as well as environmental fungi. Developments in molecular phylogeny have changed our perceptions about the epidemiology, host-association, and virulence of Sporothrix. The classical agent of sporotrichosis, Sporothrix schenckii, now comprises several species nested in a clinical clade with S. brasiliensis, S. globosa, and S. luriei. To gain a more precise view of outbreaks dynamics, structure, and origin of genetic variation within and among populations of Sporothrix, we applied three sets of discriminatory AFLP markers (#3 EcoRI-GA/MseI-TT, #5 EcoRI-GA/MseI-AG, and #6 EcoRI-TA/MseI-AA) and mating-type analysis to a large collection of human, animal and environmental isolates spanning the major endemic areas. A total of 451 polymorphic loci were amplified in vitro from 188 samples, and revealed high polymorphism information content (PIC = 0.1765-0.2253), marker index (MI = 0.0001-0.0002), effective multiplex ratio (E = 15.1720-23.5591), resolving power (Rp = 26.1075-40.2795), discriminating power (D = 0.9766-0.9879), expected heterozygosity (H = 0.1957-0.2588), and mean heterozygosity (Havp  = 0.000007-0.000009), demonstrating the effectiveness of AFLP markers to speciate Sporothrix. Analysis using the program structure indicated three genetic clusters matching S. brasiliensis (population 1), S. schenckii (population 2), and S. globosa (population 3), with the presence of patterns of admixture amongst all populations. AMOVA revealed highly structured clusters (PhiPT = 0.458-0.484, P < 0.0001), with roughly equivalent genetic variability within (46-48 %) and between (52-54 %) populations. Heterothallism was the exclusive mating strategy, and the distributions of MAT1-1 or MAT1-2 idiomorphs were not significantly skewed (1:1 ratio) for S. schenckii (χ2 = 2.522; P = 0.1122), supporting random mating. In contrast, skewed distributions were found for S. globosa (χ2 = 9.529; P = 0.0020) with a predominance of MAT1-1 isolates, and regional differences were highlighted for S. brasiliensis with the overwhelming occurrence of MAT1-2 in Rio de Janeiro (χ2 = 14.222; P = 0.0002) and Pernambuco (χ2 = 7.364; P = 0.0067), in comparison to a higher prevalence of MAT1-1 in the Rio Grande do Sul (χ2 = 7.364; P = 0.0067). Epidemiological trends reveal the geographic expansion of cat-transmitted sporotrichosis due to S. brasiliensis via founder effect. These data support Rio de Janeiro as the centre of origin that has led to the spread of this disease to other regions in Brazil. Our ability to reconstruct the source, spread, and evolution of the ongoing outbreaks from molecular data provides high-quality information for decision-making aimed at mitigating the progression of the disease. Other uses include surveillance, rapid diagnosis, case connectivity, and guiding access to appropriate antifungal treatment.

Lack of acute pathogenicity and toxicity in mice of an isolate of Metarhizium anisopliae var. anisopliae from spittlebugs.

A monospore strain of Metarhizium anisopliae var. anisopliae (EH-479/2), isolated in Mexico from Aeneolamia sp., was tested for oral acute intragastric pathogenicity and toxicity in CD-1 mice, including a thorough histological study. Animals were inoculated by gavage with one dose (10(8) conidia/animal) of viable (72 mice) and nonviable (24 mice) conidia and compared to 18 control mice. Clinical observations were done daily; mycological and histological tests were performed during necropsies at days 3, 10, 17, and 21 after the inoculation. At the end of the study, no mice showed clinical symptoms of illness, and the animals' mean weight corresponded to that of healthy adults. No inflammatory reactions were identified in analyzed organs, suggesting the nonimmunogenic status of this fungal strain. Evidence of fungal germination was noted in two lymph nodes and in liver and lung of one dead mouse, out of 72 viable-conidia treated mice. There was no evidence of toxicity symptoms in mice inoculated with nonviable conidia. The results obtained support the nonpathogenic and nontoxic status of this fungal strain when administered in a sole intragastric dose in mice.

Histoplasma capsulatum yeast cells attach and agglutinate human erythrocytes.

The ability of yeast cells of Histoplasma capsulatum to attach and agglutinate human erythrocytes has been described. This is the first report involving these yeasts in the hemagglutination phenomenon. Results revealed that the yeast cells were able to bind to erythrocytes irrespective of blood groups and to agglutinate them when a high density of yeast cells was used. Assays on the inhibition of yeast attachment to erythrocytes were also performed, using sugar-treated yeast cells. Results indicate that galactose (Gal), mainly the beta-anomer, specially inhibited yeast attachment. Disaccharides (Gal-derivatives) and glycosaminoglycans containing Gal residues, mainly chondroitin sulfate C, promote this type of inhibition. In addition, preliminary data of inhibition assays also involved a probable ionic strength driven mechanism mediated by sialic acid and heparan sulfate, suggesting that yeast binding to erythrocytes could be associated with negative charges of both molecules.

Environmental conditions favoring bat infection with Histoplasma capsulatum in Mexican shelters.

Histoplasma capsulatum was isolated from gut, lung, liver, and spleen of 17 of 208 captured bats belonging to 6 different genera and species. Three of the 17 infected bats were from the State of Guerrero and 14 were from the State of Morelos. All were adult bats: 6 males (1 Pteronotus parnellii, 2 Natalus stramineus, 2 Artibeus hirsutus, and 1 Leptonycteris nivalis) and 11 females (1 Myotis californicus, 1 Mormoops megalophylla, 8 A. hirsutus, and 1 L. nivalis). High rates of bat infection with H. capsulatum were found in the monitored sites of the State of Morelos. Histoplasma infection of N. stramineus, A. hirsutus, and L. nivalis should be considered as the first records in the world. The fungus isolated from infected bats was identified by its typical mycelial-phase morphology and by its yeast-phase conversion. Exoantigen production confirmed the fungal identification by the presence of specific precipitation lines in double immunodiffusion assays using human immune serum. Histopathologic studies showed intracellular yeast-like cells compatible with H. capsulatum yeast-phase in tissues of several bats, especially in pulmonary (intra-alveolar and septal) macrophages, with none or minimal tissue reaction. In contrast to past reports, present data support a high risk of bat infection with H. capsulatum in Mexican cave environments.

[Production of fungal antigens from local strains for the immunodiagnosis of mycoses in Mexico]. / Producción de antígenos fúngicos autóctonos en el inmunodiagnóstico de micosis en México.

Fungal antigens with good reactivity and specificity are essential for the immunodiagnosis of systemic mycoses. We give here data on crude and purified antigens of Histoplasma capsulatum, Coccidioidis immitis and Paracoccidioides brasiliensis from local strains and which are the more prevalent in Mexico. The crude antigens had good reactivity in precipitating and skin testing whereas the purified antigens (DPPC: deproteinized polysaccharide protein complex) had a higher specificity in more sensitive tests such as ELISA and complement fixation. Our efficiency analysis showed that the crude antigens are best for epidemiologic studies due to their low cost, easy handling and fast detection. The purified ones are suited to establish with more precision the diagnosis of systemic mycoses.

Biological and sociocultural approaches of histoplasmosis in the State of Guerrero, Mexico.

Histoplasmosis is a deep mycosis with a high prevalence in America. Its aetiological agent, Histoplasma capsulatum, is found in Mexico, mainly in confined spaces, where it grows on bat guano or bird droppings. A research project has been developed in the State of Guerrero, Mexico, where many contaminated caves and mines are visited by individuals and epidemic outbreaks have been recorded. Data concerning human skin test response to histoplasmin antigen, host genetic predisposition to Histoplasma infection, sociocultural, socioeconomical and ethnobiological aspects of the disease in Guerrero are summarized in this paper.

[Infectivity of the entomopathogenic fungus Verticillium lecanii in mice and guinea pigs]. / Infectividad del hongo entomopatógeno Verticillium lecanii en ratones y cobayos.

The resistance of plague insects to chemical insecticides as well as the importance of a healthy environment demands an alternative for agricultural plagues. Among others, biological control seems an alternate strategy with fungal entomopathogens playing a relevant role. The hyphomycete Verticillium lecanii is a natural bioregulator of aphids, scales and white-flies that attack different agricultural plantations. Its use in biological control programs must be assessed previously by safety procedures such as its innocuity in mammals and useful animals and plants. The aim of this study was pointed at demonstrating the innocuity of V. lecanii in mice and guinea pigs. Two strains of the fungus were injected intraperitoneally (10(8) conidia/kg of animal weight) to 130 mice and 66 guinea pigs. Two control groups were included, one injected with heat-killed fungi and the other with sterile physiological saline. The animals were killed at 8, 30 and 70 days after infection, and mycological and histopathological studies performed in their organs. Negative results obtained with the live fungus in the same manner as the two control groups, suggest the innocuity of V. lecanii in mice and guinea pigs.

Two-dimensional immunoelectrophoresis of histoplasmin and a purified polysaccharide-protein antigen of Histoplasma capsulatum.

Crude histoplasmin and a polysaccharide-protein complex (PPC-histo) antigens obtained from culture filtrates of Histoplasma capsulatum were analyzed by single and tandem two-dimensional immunoelectrophoresis (TD-IEP) using a rabbit hyperimmune anti-histoplasmin polyvalent serum. Single TD-IEP showed 14 arc precipitates for histoplasmin. Continuity of arcs 2, 6, and 7, and 9 and 10 was observed, suggesting a different polymeric configuration of the same antigen. This was also confirmed in tandem TD-IEP of histoplasmin with homologous (PPC-histo) and heterologous PPC's from Blastomyces dermatitidis, Paracoccidioides brasiliensis and Coccidioides immitis. Tandem TD-IEP of histoplasmin and PPC-histo displayed a similar antigenic pattern to histoplasmin alone, being arcs 1 and 3 more evident and apparently present only in histoplasmin and PPC-histo. Tandem TD-IEP showed common antigens among the other heterologous fungal purified antigens, and seems useful to observe the multiplicity of antigens present in fungal preparations and to identify those precipitates (arcs 1 and 3) that are predominant in the purified preparation.

Retrospective serological study of histoplasmosis in Mexico.

Mycotic immunodiagnosis was performed in 325 patients with clinical evidence of systemic mycoses, over a 5-year period, from different hospitals of Mexico City. Results showed 168 individuals that presented one positive serological test to Histoplasma capsulatum antigens. From these, only 27 patients were serologically positive to two or more tests, such as tube precipitin, immunodiffusion, complement fixation, and ELISA, and developed signs and symptoms of a histoplasmosis clinically classified as primary pulmonary. Four of them presented an underlying disease including one positive HIV patient. Twenty-two came from endemic histoplasmosis zones of the country and most of them acquired the disease in caves or uninhabited houses. The diagnosis of histoplasmosis should be based on reliable laboratory data which could raise more significant information of its incidence in Mexico.

Efficiency of crude and purified fungal antigens in serodiagnosis to discriminate mycotic from other respiratory diseases.

Mycotic immunodiagnosis was performed in 186 hospitalized patients with different respiratory diseases, mostly considered as tuberculosis and others with a doubtful diagnosis. Crude histoplasmin, coccidioidin, paracoccidioidin, blastomycin, candidin, aspergillin, and sporotrichin, as well as purified polysaccharide-protein complexes (PPC) of Histoplasma capsulatum, Coccidioides immitis, and Paracoccidioides brasiliensis were used as antigens. Immune tests used included skin test (ST), gel immunodiffusion (ID), counterimmunoelectrophoresis (CIE), complement fixation (CF), and ELISA. A possible association with candidosis was observed in 17% of patients with tuberculosis and diabetes; one presumptive paracoccidioidomycosis, one confirmed aspergillosis, and six cases of active histoplasmosis were determined. Candidin ST showed 29% of positive reactions with an increased frequency in patients between 31 and 55 years of age. CF test showed the highest positivity percentages with crude antigens, specially for Candida antigen (26.3%) and histoplasmin (18.2%). Cross reactions were evident with crude antigens but decreased when PPC's were used in ELISA.

Immunological separation of Entomophthorales genera.

Exoantigens from Erynia neoaphidis, Conidiobolus major, C. thromboides, C. obscurus, Zoophthora radicans, and Basidiobolus ranarum were obtained from culture filtrates of fungal material grown in a yeast extract, peptone dialysate, dextrose medium and were tested against specific hyperimmune antisera prepared from E. neoaphidis, C. major, C. thromboides, and B. ranarum by the immunodiffusion technique. Specific precipitins were observed for E. neoaphidis and B. ranarum, while cross-reactions were detected among C. major, C. thromboides, and C. obscurus. The results suggest that genera of Entomophthorales can be easily separated by this simple immunological procedure.

Relationship between pathogenesis and immune regulation mechanisms in histoplasmosis: a hypothetical approach.

The cellular immune response may be the principal mechanism of defense against infection with Histoplasma capsulatum. Subsequently, regulation of cell-mediated immunity could be fundamental to the outcome of disease. Suppressive phenomena-present in the host before infection or brought about by fungal components-would explain the various courses of infection. The fact that high titers of antibodies are associated with a poor cellular response suggests that antibodies may have a role as "regulatory molecules" in histoplasmosis. Antibodies may modulate the expression of the cellular response either by direct inhibition or by activation of the suppressor cell population, thereby interfering with the infectious process.

Antigens from Histoplasma capsulatum and Blastomyces dermatitidis. I. Immunological comparative studies from polysaccharide-protein complexes of both fungi.

Certain groups of fungi share chemical structures which makes difficult isolation and differentiation of specific antigens by the usual methods of extraction and purification. Therefore, we have oriented our studies to the immunological and biochemical characterization of differences and similarities of molecular structures from fungi, etiologic agents of systemic mycoses, hoping to establish criteria for the utilization and handling of these antigens. A deproteinized polysaccharide-protein complex (D-PPC) was isolated from Histoplasma capsulatum and Blastomyces dermatitidis. The immunological studies with humoral tests indicate a variable cross reaction between antigens of both species. In immunodiffussion systems, the reaction was specific for each species using saline solution of phosphate butter solution, while using an agarose veronal system, the cross reactions were very evident. In addition, differences in cross reactions were obtained with immunoelectrophoresis, haemagglutination and complement fixation microtest. This variation to cross reaction responses suggest that these antigens (D-PPC) share common structures but at the same time must have some different component owned by each one of the fungi which makes them more specific than crude antigens.

[Study of the pathogenicity of Conidiobolus coronatus in experimental animals]. / Estudio de la patogenicidad de Conidiobolus coronatus en animales de experimentacion.

The pathogenicity of a strain of Conidiobulus coronatus isolated from Aenolamia postica was investigated in 152 mice, 42 guinea pigs and 47 hamsters. Four routes of inoculation were used: intraperitoneal, subcutaneous, intratracheal and scarification in the oral mucosa. Each animal was inoculated 3 times at 3 week intervals. Animals were sacrificed at 1, 8, 15, 45 of 225 days after the first inoculation. Observations of macroscopic lesions (nodules, abscesses, adhesions, etc.) direct examinations, cultures and histopathological studies of different tissues were performed. The aforementioned lesions of tissue were the most frequently found and all of them were resolved by spontaneous cure. The animals inoculated intraperitoneally and subcutaneouslly presented the highest number of postive results. The histopathological features at the 1st and 8th days after inoculation showed an acute inflamatory infiltrate with well preserved fungus filaments; at the 15th day granulomas with giant cells were observed containing fragmented and disintegrated fungal hyphae; at the 45th and 225th days a fibrosis reaction was observed and no fungal filaments, could be found. In no animal was rhinoentomophthoromycosis never reproduced, and according to these results it is considered that either this strain is not a pathogen or its pathogenicity is very low.

[An endemic area of sporotrichosis in Guatemala]. / Etude d'une zone d'endémie sporotrichosique au Guatémala.

An endemic area of sporotrichosis is described in the Lake of Ayarza District, South Guatemala, where 53 patients have been observed within 3 years. In 45.3% of the cases, the infection appeared after handling fish. The disease was more frequently observed in man (83%) and in patients less than 30 years of age. The most frequent clinical type was the ascending lymphocutaneous sporotrichosis of the limbs. Some cases of ulcerative or verrucous lesions were seen. Almost all the patients cured rapidly either by potassium iodide (46 patients) or spontaneously, or after application of local heat (4 patients). Intradermal tests were performed in healthy population in the endemic area and, for comparison, in Guatemala City. Whole yeast cell antigens of Sporothrix schenckii and Ceratocystis stenoceras were used in these tests. Skin tests to both antigens were more frequently positive in the endemic area; the highest frequency was obtained with the antigen of C. stenoceras. A serological study was performed in 26 patients. The fluorescent antibody staining technique was more sensitive than yeast cell and latex particles agglutinations. C. stenoceras was isolated from bark of some trees, especially Eucalyptus, in the environment, but not S. schenckii.