María del Rocío Reyes-Montes

Sporothrix schenckii complex and sporotrichosis, an emerging health problem.

Sporothrix schenckii, now named the S. schenckii species complex, has largely been known as the etiological agent of sporotrichosis, which is an acute or chronic subcutaneous mycosis of humans and other mammals. Gene sequencing has revealed the following species in the S. schenckii complex: Sporothrix albicans, Sporothrix brasiliensis, Sporothrix globosa, Sporothrix luriei, Sporothrix mexicana and S. schenckii. The increasing number of reports of Sporothrix infection in immunocompromised patients, mainly the HIV-infected population, suggests sporotrichosis as an emerging global health problem concomitant with the AIDS pandemic. Molecular studies have demonstrated a high level of intraspecific variability. Components of the S. schenckii cell wall that act as adhesins and immunogenic inducers, such as a 70-kDa glycoprotein, are apparently specific to this fungus. The main glycan peptidorhamnomannan cell wall component is the only O-linked glycan structure known in S. schenckii. It contains an α-mannobiose core followed by one α-glucuronic acid unit, which may be mono- or di-rhamnosylated. The oligomeric structure of glucosamine-6-P synthase has led to a significant advance in the development of antifungals targeted to the enzymes catalytic domain in S. schenckii.

Phenotyping and Genotyping of Sporothrix schenckii Isolates According to Geographic Origin and Clinical Form of Sporotrichosis

ABSTRACT Sporothrix schenckii isolates of fixed and lymphocutaneous clinical forms from Mexico (MX), Guatemala (GT), and Colombia (CO) as well as environmental isolates from MX were studied by analyzing their phenotypic characteristics (conidial length, thermotolerance by percent growth inhibition [GI] at 35 and 37°C, median lethal dose [LD50]) and genotypic characteristics (by random amplified polymorphic DNA [RAPD] analysis-PCR). A significant difference (P < 0.01) in the mean conidial length of S. schenckii clinical isolates from CO (x̅ = 4.03 ± 1.04 μm) compared with those of clinical isolates from MX (x̅ = 2.06 ± 0.53 μm) and GT (x̅ = 2.68 ± 0.83 μm) was observed. The lowest thermotolerance, as determined by measurement of percent GI, was exhibited by isolates from CO at 35°C (x̅ = 50.1% ± 15.9%) and 37°C (x̅ = 72.7% ± 10.9%). In general, the highest virulence, as determined by measurement of the LD50 for mice, was observed for the MX environmental isolates. RAPD analysis-PCR with 10-mer primers OPBG-01, OPBG-14, and OPBG-19 generated 52 reproducible bands. The 44 Sporothrix isolates fell into four major groups by hierarchical cluster analysis. The first group (group I), formed by 25 (of 27) isolates from MX, had two subgroups: subgroup Ia with 10 environmental isolates and subgroup Ib with 14 clinical isolates. The second group (group II) had two subgroups: subgroup IIa, formed by isolates from CO, and subgroup IIb, formed by isolates from GT. Groups III and IV each had only one clinical isolate from MX. A principal-component analysis of the same data yielded three distinct groups, depending on the geographical origins of the isolates, including the isolates in groups III and IV from MX, which were grouped with the isolates from MX by principal-component analysis. This study revealed that isolates from CO had low thermotolerances at 35 and 37°C and could be associated with superficial skin lesions in patients with fixed clinical forms of sporotrichosis, the most frequent form of the disease in CO. Distinct patterns dependent on geographical origins were also revealed by RAPD analysis-PCR, but these had no relation to the clinical form of the disease.

Population structure and diversity of the pathogenic fungus Aspergillus fumigatus isolated from different sources and geographic origins

Fifty-five clinical and environmental Aspergillus fumigatus isolates from Mexico, Argentina, France and Peru were analyzed to determine their genetic variability, reproductive system and level of differentiation using amplified fragment length polymorphism markers. The level of genetic variability was assessed by measuring the percentage of polymorphic loci, number of effective alleles, expected heterozygocity and by performing an association index test (I(A)). The degree of genetic differentiation and variation was determined using analysis of molecular variance at three levels. Using the paired genetic distances, a dendrogram was built to detect the genetic relationship among alleles. Finally, a network of haplotypes was constructed to determine the geographic relationship among them. The results indicate that the clinical isolates have greater genetic variability than the environmental isolates. The I(A) of the clinical and environmental isolates suggests a recombining population structure. The genetic differentiation among isolates and the dendrogram suggest that the groups of isolates are different. The network of haplotypes demonstrates that the majority of the isolates are grouped according to geographic origin.

Genetic diversity of Taenia solium cysticerci from naturally infected pigs of central Mexico.

This study was designed to explore if each individual case of naturally acquired porcine cysticercosis, living in different geographic rural areas of central Mexico, is caused by one or more different specimens of Taenia solium tapeworm. The genetic variability among cysticerci from the same pig and that from different pigs was assessed by random amplified polymorphic DNA markers (RAPDs), through the percentage of polymorphic loci, the number of effective alleles, the expected heterozygosity and the Shannon index. The parasite populations reproductive structure was estimated through the association index (I(A)), and the degree of genetic differentiation and variation was determined using AMOVA. Using six different random primers, and a total of 181 cysticerci from 14 pigs, 88 different loci were amplified: 85% were polymorphic between pigs and 24% within pigs. The phenogram grouped the cysticerci into eight major clusters, with differences in the genetic distances among all cysticerci from 14 pigs ranging from 0.78 to 1. Most of the cysticerci grouped in accord with their different geographical origin and with their pig of origin. The similarity matrix produced from the phenogram (obtained by UPGMA) and the original similarity matrix yielded a good cophenetic correlation (r=0.82317, P=0.0004), which suggests that the phenogram accurately represents the original genetic similarities between isolates. The combination of I(A) (0.0-0.089) with the genetic diversity index (0.009-0.073) supports the idea that DNA diversity in T. solium cysticerci of naturally infected pigs is within the range expected from a recombination process occurring during sexual reproduction. The small genetic diversity found within the cysticerci of each pig (33.81%), when compared with that between pigs (66.19%), indicates that pigs are rarely infected by different tapeworms. It would then appear that porcine cysticercosis courses with effective concomitant immunity, as occurs in ovine cysticercosis.

Development of Specific Sequence-Characterized Amplified Region Markers for Detecting Histoplasma capsulatum in Clinical and Environmental Samples

ABSTRACT Sequence-characterized amplified region (SCAR) markers, generated by randomly amplified polymorphic DNA (RAPD)-PCR, were developed to detect Histoplasma capsulatum selectively in clinical and environmental samples. A 1,200-bp RAPD-PCR-specific band produced with the 1281-1283 primers was cloned, sequenced, and used to design two SCAR markers, 1281-1283220 and 1281-1283230. The specificity of these markers was confirmed by Southern hybridization. To evaluate the relevance of the SCAR markers for the diagnosis of histoplasmosis, another molecular marker (M antigen probe) was used for comparison. To validate 1281-1283220 and 1281-1283230 as new tools for the identification of H. capsulatum, the specificity and sensitivity of these markers were assessed for the detection of the pathogen in 36 clinical (17 humans, as well as 9 experimentally and 10 naturally infected nonhuman mammals) and 20 environmental (10 contaminated soil and 10 guano) samples. Although the two SCAR markers and the M antigen probe identified H. capsulatum isolates from different geographic origins in America, the 1281-1283220 SCAR marker was the most specific and detected the pathogen in all samples tested. In contrast, the 1281-1283230 SCAR marker and the M antigen probe also amplified DNA from Aspergillus niger and Cryptococcus neoformans, respectively. Both SCAR markers detected as little as 0.001 ng of H. capsulatum DNA, while the M antigen probe detected 0.5 ng of fungal DNA. The SCAR markers revealed the fungal presence better than the M antigen probe in contaminated soil and guano samples. Based on our results, the 1281-1283220 marker can be used to detect and identify H. capsulatum in samples from different sources.

Molecular Findings of Disseminated Histoplasmosis in Two Captive Snow Leopards (Uncia uncia)

Abstract This paper reports two cases of disseminated histoplasmosis in captive snow leopards (Uncia uncia). Histoplasmosis was diagnosed based on histopathology, immunohistochemistry, transmission electron microscopy, and molecular findings.

The habitat of Coccidioides spp. and the role of animals as reservoirs and disseminators in nature

BackgroundCoccidioidomycosis, a potentially fatal fungal infection, is considered an emergent mycotic disease because of the increased incidence of fungal infections registered over recent years. Infection occurs through the inhalation of arthroconidia from two main species of Coccidioides: Coccidioides immitis and C. posadasii, which are both endemic to arid and semi-arid regions of North America. Coccidioides species not only infect humans but can also infect other mammals (land, aquatic, wild or domestic), reptiles and birds.ObjectiveTo obtain information regarding the habitat of Coccidioides spp. and the animals infected by this fungus and to identify the role that infected animals play as reservoirs and disseminators of this fungus in nature.MaterialsA literature review was conducted to identify the habitat of Coccidioides spp. and the infected non-human animal species targeted by this fungus.Results and conclusionsThis review allows us to suggest that Coccidioides spp. may be classified as halotolerant organisms; nevertheless, to perpetuate their life cycle, these organisms depend on different animal species (reservoirs) that serve as a link with the environment, by acting as disseminators of the fungi in nature.

Phenotypic characteristics of isolates of Aspergillus section Fumigati from different geographic origins and their relationships with genotypic characteristics

BackgroundEpidemiological studies worldwide have shown that A. fumigatus exhibits important phenotypic and genotypic diversity, and these findings have been of great importance in improving the diagnosis and treatment of diseases caused by this fungus. However, few studies have been carried out related to the epidemiology of this fungus in Latin America. This study´s aim is to report on the epidemiology of the fungus by analyzing the phenotypic variability of Aspergillus section Fumigati isolates from different Latin American countries and the relationship between this variability, the geographical origin and genotypic characteristics.MethodsWe analyzed the phenotypic characteristics (macro- and micromorphology, conidial size, vesicles size, antifungal susceptibility and thermotolerance at 28, 37 and 48°C) of A. section Fumigati isolates from Mexico (MX), Argentina (AR), Peru (PE) and France (FR). The results were analyzed using analysis of variance (ANOVA) and Tukeys multiple comparison test to detect significant differences. Two dendrograms among isolates were obtained with UPGMA using the Euclidean distance index. One was drawn for phenotypic data, and the other for phenotypic and genotypic data. A PCoA was done for shown isolates in a space of reduced dimensionality. In order to determine the degree of association between the phenotypic and genotypic characteristics AFLP, we calculated the correlation between parwise Euclidean distance matrices of both data sets with the nonparametric Mantel test.ResultsNo variability was found in the macromorphology of the studied isolates; however, the micromorphology and growth rate showed that the PE isolates grew at a faster rate and exhibited the widest vesicles in comparison to the isolates from MX, AR and FR. The dendrogram constructed with phenotypic data showed three distinct groups. The group I and II were formed with isolates from PE and FR, respectively, while group III was formed with isolates from MX and AR. The dendrogram with phenotypic and genotypic data showed the same cluster, except for an isolate from FR that formed a separate cluster. This cluster was confirmed using PCoA. The correlation between the phenotypic and genotypic data of the isolates revealed a statistically significant association between these characteristics.ConclusionsThe PE isolates showed specific phenotypic characteristics that clearly differentiate them from the rest of the isolates, which matches the genotypic data. The correlation between the phenotypic and genotypic characteristics showed a statistically significant association. In conclusion, phenotypic and genotypic methods together increase the power of correlation between isolates.

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 the 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 or phosphate buffer 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 in 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.

An Isaria fumosorosea SCAR marker for evaluation of soil, insect, and airborne samples

Abstract Molecular detection markers are needed for ecological studies of entomopathogenic fungi. In this study, a sequence-characterized amplified region (SCAR) marker of Isaria fumosorosea was used to detect the fungus in soil, insects, and airborne samples. These were artificially added with different fungal conidial concentrations. Specificity and sensitivity were tested with semi-nested PCR using oligonucleotides E-AA/M-CTA124 F and E-AA/M-CTA124 R for the first amplification and E-AA/M-CTA124 F and E-AA/M-CTA103 R for the second amplification. Specificity assays showed a specific band of 103 bp for DNA samples from 10 I. fumosorosea strains used. Negative results were observed for DNA samples from other species of Isaria, including I. amoene-rosea, I. farinosa, and Paecilomyces carneus as well as with other entomopathogens such as Metarhizium acridum, M. anisopliae, M. majus, M. flavoviride Type E, and Lecanicillium lecanii. Sensitivity assays showed that the specific SCAR marker detected 104 conidia from I. fumosorosea EH-511/3 that were artificially mixed with soil, from 1 to 104 conidia artificially mixed with Galleria mellonella (Lepidoptera: Pyralidae), and from 10 to 105 conidia in Melinex tape for airborne samples. The marker was also able to detect conidia in airborne samples from cotton wicks in a mini wind tunnel. These SCAR markers for I. fumosorosea had excellent specificity and sensitivity and are relevant tools for ecological studies of this fungus.