Intraspecific chromosomal variability in human pathogenic fungi, especially in Histoplasma capsulatum

The ploidy, karyotype, and chromosome length polymorphism (CLP) of human pathogenic fungi were revised with emphasis on Histoplasma capsulatum, the causative agent of the systemic mycosis, histoplasmosis. Currently, different systems of gel electrophoresis are being used to determine fungal electrokaryotypes (EK). By renaturation kinetic and genomic reconstruction in H. capsulatum strains (G-186AS and Downs), estimated genome sizes of 23 and 32 Mb were determined for both strains, respectively. The haploid state was proposed for both strains, although aneuploidy was suggested for the Downs strain. Contour-clamped homogeneous electric field (CHEF), field inversion gel electrophoresis (FIGE), and Southern blot using different probes showed the presence of six to seven chromosomes in the Downs strain (low virulence), whereas four chromosomes were identified in the G-186B strain (high virulence). The use of these methods in the three major H. capsulatum reference strains (G-217B and Downs from the United States of America, G-186B from Panama) revealed distinct chromosome sizes, from 0.5 to 5.7 Mb, with CLP associated with chromosomes size and mobility. Recently, by CHEF, using 19 H. capsulatum isolates from Latin-America and the G-186B strain, five to seven chromosomes with 1.1 to 11.2 Mb molecular sizes were revealed, which again suggested CLP in H. capsulatum. However, to elucidate the EKs polymorphism in H. capsulatum and its relationship with the isolates phenotype more studies are needed to understand the mechanisms controlling ploidy variability.     

Genetic diversity of Histoplasma capsulatum strains in Brazil

This study establishes the genetic relatedness among Brazilian Histoplasma capsulatum samples obtained from different sources. A PCR-based random amplified polymorphic DNA (RAPD) assay was used to delineate polymorphisms among isolates in geographically diverse regions in Brazil. RAPD fingerprints revealed distinct DNA profiles and provided a high level of discrimination among H. capsulatum strains from different locations. Cluster I was composed of H. capsulatum isolates from the northeast region. The majority of strains from southeast and south were categorized as major cluster II. The strain 84564 from Rio de Janeiro State showed no genetic correlation to any of the isolates from the same state. The RAPD patterns of H. capsulatum isolates from Goias (Cluster III) were unrelated to DNA fingerprints observed among the other H. capsulatum strains (48% similarity). This study is the first report that stratifies the clusters of H. capsulatum strains from Brazil by molecular typing and associates them with the geographical origin.     

Geographical distribution of genetic polymorphism of the pathogen Histoplasma capsulatum isolated from infected bats, captured in a central zone of Mexico

Fourteen Histoplasma capsulatum isolates recovered from infected bats captured in Mexican caves and two human H. capsulatum reference strains were analyzed using random amplification of polymorphic DNA PCR-based and partial DNA sequences of four genes. Cluster analysis of random amplification of polymorphic DNA-patterns revealed differences for two H. capsulatum isolates of one migratory bat Tadarida brasiliensis. Three groups were identified by distance and maximum-parsimony analyses of arf, H-anti, ole, and tub1 H. capsulatum genes. Group I included most isolates from infected bats and one clinical strain from central Mexico; group II included the two isolates from T. brasiliensis; the human G-217B reference strain from USA formed an independent group III. Isolates from group II showed diversity in relation to groups I and III, suggesting a different H. capsulatum population.     

Diversity among clinical isolates of Histoplasma capsulatum detected by polymerase chain reaction with arbitrary primers.

Clinical isolates of the fungal respiratory and systemic pathogen Histoplasma capsulatum have been placed in several different classes by using genomic restriction fragment length polymorphisms (RFLPs), but in general have not been distinguished further. We report here that a polymerase chain reaction (PCR)-based DNA fingerprinting method that has been termed arbitrary primer or random amplified polymorphic DNA (RAPD) PCR can distinguish among isolates in a single RFLP class. In this method, arbitrarily chosen oligonucleotides are used to prime DNA synthesis from genomic sites that they fortuitously match, or almost match, to generate strain-specific arrays of DNA fragments. Each of 29 isolates of RFLP class 2, the group endemic in the American Midwest, was distinguished by using just three arbitrary primers. In contrast, laboratory-derived S and E colony morphology variants of two strains were not distinguished from their R parents by using 18 such primers. Thus, the clinical isolates of H. capsulatum are quite diverse, but their genomes remain stable during laboratory culture. These outcomes suggest new possibilities for epidemiological analysis and studies of fungal populations in infected hosts.     

Genetic diversity of Histoplasma capsulatum isolated from infected bats randomly captured in Mexico, Brazil, and Argentina, using the polymorphism of (GA)(n) microsatellite and its flanking regions

The genetic diversity of 47 Histoplasma capsulatum isolates from infected bats captured in Mexico, Brazil, and Argentina was studied, using sequence polymorphism of a 240-nucleotides (nt) fragment, which includes the (GA)(n) length microsatellite and its flanking regions within the HSP60 gene. Three human clinical strains were used as geographic references. Based on phylogenetic analyses of 240-nt fragments achieved, the relationships among H. capsulatum isolates were resolved using neighbour-joining and maximum parsimony methods. The tree topologies obtained by both methods were identical and highlighted two major clusters of isolates. Cluster I had three sub-clusters (Ia, Ib, and Ic), all of which contained Mexican H. capsulatum samples, while cluster II consisted of samples from Brazil and Argentina. Sub-cluster Ia included only fungal isolates from the migratory bat Tadarida brasiliensis. An average DNA mutation rate of 2.39 × 10(-9) substitutions per site per year was estimated for the 240-nt fragment for all H. capsulatum isolates. Nucleotide diversity analysis of the (GA)(n) and flanking regions from fungal isolates of each cluster and sub-cluster underscored the high similarity of cluster II (Brazil and Argentina), sub-clusters Ib, and Ic (Mexico). According to the genetic distances among isolates, a network of the 240-nt fragment was graphically represented by (GA)(n) length haplotype. This network showed an association between genetic variation and both the geographic distribution and the ecotype dispersion of H. capsulatum, which are related to the migratory behaviour of the infected bats studied.     

Molecular typing of Histoplasma capsulatum isolated from infected bats, captured in Mexico

The present paper represents data on the genetic polymorphism of 13 Histoplasma capsulatum isolates recovered from infected bats randomly captured in the Mexican states of Morelos, Puebla, and Oaxaca. The polymorphic DNA patterns were analyzed by two-primer RAPD-PCR (random amplified polymorphic DNA-polymerase chain reaction) method. To amplify the fungal genome by PCR, the following primer arrangements were used: 5'-AACGCGCAAC-3' and 5'-AAGAGCCCGT-3'; 5'-AACGCGCAAC-3' and 5'-GTTTCCGCCC-3'; or 5'-AACGCGCAAC-3' and 5'-GCGATCCCCA-3'. A common polymorphic DNA pattern of H. capsulatum was revealed in different assays. This pattern is shared by 7 H. capsulatum isolates recovered from different specimens of nonmigratory bats (Artibeus hirsutus) captured in a cave in Morelos, by 5 isolates recovered from infected migratory bats (Leptonycteris nivalis) captured in Morelos and Puebla, and by 1 isolate from another migratory bat (L. curasoae) captured in Oaxaca. This polymorphic DNA pattern of H. capsulatum could represent fungal markers for the geographic areas studied, and considering its distribution in three different states of the Mexican Republic, the role of bats as responsible for H. capsulatum spreading in nature, in relation to their movements and migrations besides their shelter habits, is suggested. Analyses of DNA patterns of H. capsulatum isolated from infected bats, from clinical cases, and from blackbird excreta, have shown a major relatedness between bats and clinical isolates, in contrast to those isolates from bird excreta.     

Expression and interstrain variability of the YPS3 gene of Histoplasma capsulatum

The YPS3 locus of the dimorphic fungus Histoplasma capsulatum encodes a secreted and surface-localized protein specific to the pathogenic yeast phase. In this study we examined this locus in 32 H. capsulatum strains and variants. Although protein production is limited to a select group of strains, the North American restriction fragment length polymorphism class 2/NAm 2 isolates, the locus was present in all the strains we examined. The YPS3 gene is well conserved in its 5' and 3' regions but displays an intragenic hypervariable region of tandem repeats that fluctuates in size between strains. This feature is similar to that seen with genes encoding several cell surface proteins in other fungi.     

Identification of the infectious source of an unusual outbreak of histoplasmosis, in a hotel in Acapulco, state of Guerrero, Mexico

Three isolates of Histoplasma capsulatum were identified from mice lung, liver, and spleen inoculated with soil samples of the X hotel's ornamental potted plants that had been fertilized with organic material known as compost. The presence of H. capsulatum in the original compost was detected using the dot-enzyme-linked immunosorbent assay. Nested-PCR, using a specific protein Hcp100 coding gene sequence, confirmed the fungal identification associated with an unusual histoplasmosis outbreak in Acapulco. Although, diversity between the H. capsulatum isolate from the hotel and some clinical isolates from Guerrero (positive controls) was observed using random amplification of polymorphic DNA based-PCR, sequence analyses of H-anti and ole fragment genes revealed a high homology (92-99%) between them.     

Variation in Lipid Content of Strains of Histoplasma capsulatum Exhibiting Different Virulence Properties for Mice

Nielsen, H. S., Jr. (Duke University Medical Center, Durham, N.C.). Variation in lipid content of strains of Histoplasma capsulatum exhibiting different virulence properties for mice. J. Bacteriol. 91:273-277. 1966.-Lipid content and virulence were studied in six isolates of Histoplasma capsulatum in an attempt to determine whether or not the two factors could be correlated in this fungus. Virulence was evaluated by injecting dba line 1 male mice intracerebrally with 2.8 x 10(4) infective yeast-phase units and recording organ involvement and spontaneous deaths occurring in a 20-day period. Yeast cells were extracted with mixtures of ethyl alcohol-diethyl ether (3:1, v/v), and the total extractable lipid, as determined by solubility in petroleum ether, was separated into acetone-soluble and phospholipid fractions by acetone precipitation. Neutral lipids were measured directly by weighing, whereas total phospholipids were calculated after the colorimetric determination of phosphorus. The mixed phosphatides of two isolates, differing in virulence, were separated into five fractions by use of a column of silicic acid and Hyflo Super-Cel. In the six isolates studied, neither total extractable lipid, acetone-soluble lipid, nor phospholipid showed a quantitative correlation with virulence. Phosphatidylserine, cephalin, phosphoinositides, and sphingolipids were present in essentially the same amounts in the two strains investigated; however, a lecithin fraction was absent in the less virulent form. These data suggest that the quantity of phosphatidylcholine demonstrated for a given isolate of H. capsulatum may provide some insight as to its virulence, although such a relationship is lacking for total lipid, the acetone-soluble fraction, and the combined phospholipids of yeast-phase growth.     

Fatal Histoplasma capsulatum Mitral Endocarditis in a French Patient Treated for Rheumatoid Arthritis

Histoplasmosis is an infectious disease caused by the inhalation of Histoplasma capsulatum spores, a fungus encountered in many diverse areas around the world. Although this infection is often asymptomatic, it may become dramatic in immunocompromised patients. In November 2005, an endocarditis due to Histoplasma capsulatum was diagnosed in a French woman treated for rheumatoid arthritis and who had traveled to South America 2 years earlier. We confirmed the biological diagnosis by mycological, serological, and histological methods. In spite of receiving the appropriate treatment, the patient died 3 months later of cardiac insufficiency. We report here this additional case of Histoplasma endocarditis, by hoping to help rapid and accurate diagnosis of such infections in their early stages of development, in non-endemic areas.     

Maintaining network security: how macromolecular structures cross the peptidoglycan layer

Histoplasma capsulatum is the leading cause of endemic mycosis in the world. Analyses of clinical isolates from different endemic regions show important diversity within the species. Recent molecular studies of two isolates, the Chemotype I NAm2 strain G217B and the Chemotype II Panamanian strain G186A, reveal significant genetic, structural, and molecular differences between these representative Histoplasma strains. Some of these variations have functional consequences, representing distinct molecular mechanisms that facilitate Histoplasma pathogenesis. The realization of Histoplasma strain diversity highlights the importance of characterizing Histoplasma virulence factors in the context of specific clinical strain isolates.     

Characterization of the genetic diversity of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> in São Paulo city,Brazil

Background

Tuberculosis is a major health problem in São Paulo, Brazil, which is the most populous and one of the most cosmopolitan cities in South America. To characterize the genetic diversity of Mycobacterium tuberculosis in the population of this city, the genotyping techniques of spoligotyping and MIRU were applied to 93 isolates collected in two consecutive years from 93 different tuberculosis patients residing in São Paulo city and attending the Clemente Ferreira Institute (the reference clinic for the treatment of tuberculosis).

Findings

Spoligotyping generated 53 different spoligotype patterns. Fifty-one isolates (54.8%) were grouped into 13 spoligotyping clusters. Seventy- two strains (77.4%) showed spoligotypes described in the international databases (SpolDB4, SITVIT), and 21 (22.6%) showed unidentified patterns. The most frequent spoligotype families were Latin American Mediterranean (LAM) (26 isolates), followed by the T family (24 isolates) and Haarlem (H) (11 isolates), which together accounted for 65.4% of all the isolates. These three families represent the major genotypes found in Africa, Central America, South America and Europe. Six Spoligo-International-types (designated SITs by the database) comprised 51.8% (37/72) of all the identified spoligotypes (SIT53, SIT50, SIT42, SIT60, SIT17 and SIT1). Other SITs found in this study indicated the great genetic diversity of M. tuberculosis, reflecting the remarkable ethnic diversity of São Paulo city inhabitants. The MIRU technique was more discriminatory and did not identify any genetic clusters with 100% similarity among the 93 isolates. The allelic analysis showed that MIRU loci 26, 40, 23 and 10 were the most discriminatory. When MIRU and spoligotyping techniques were combined, all isolates grouped in the 13 spoligotyping clusters were separated.

Conclusions

Our data indicated the genomic stability of over 50% of spoligotypes identified in São Paulo and the great genetic diversity of M. tuberculosis isolates in the remaining SITs, reflecting the large ethnic mix of the São Paulo city inhabitants. The results also indicated that in this city, M. tuberculosis isolates acquired drug resistance independently of genotype and that resistance was more dependent on the selective pressure of treatment failure and the environmental circumstances of patients.

     

Acidipila rosea gen. nov., sp. nov., an acidophilic chemoorganotrophic bacterium belonging to the phylum Acidobacteria

Two strains of aerobic acidophilic chemoorganotrophic bacteria designed strains AP8(T) and AP9 were isolated from acid mine drainage and acidic soil, respectively. These isolates were gram-negative, nonmotile cocci and coccobacilli measuring 0.5-0.8 μm in diameter. Cells were capsulated. Colonies on solid media were pink colored. The pH range for growth was 3.0-6.0 (optimum pH 4.5). Sugars, gluconate, and some amino acids were good carbon and energy sources for growth. The main components of cellular fatty acids were C(15:0) iso and C(16:1) ω7c. Menaquinone-8 was the major quinone. The G+C content of genomic DNA was 59.5%. Both strains had identical sequences of 16S rRNA genes that were most closely related to that of the type strain of Acidobacterium capsulatum (96% similarity). There were major differences between the isolates and A. capsulatum in cell morphology, carbon nutrition, and fatty acid profiles. Based on these phylogenetic and phenotypic data, we propose the name Acidipila rosea gen. nov., sp. nov. to accommodate the novel isolates. The type strain is AP8(T) (NBRC 107607(T), KCTC 23427(T)).     

Surface localization of the Yps3p protein of Histoplasma capsulatum

The YPS3 gene of Histoplasma capsulatum encodes a protein that is both resident in the cell wall and also released into the culture medium. This protein is produced only during the pathogenic yeast phase of infection and is also expressed differently in H. capsulatum strains that differ in virulence. We investigated the cellular localization of Yps3p. We demonstrated that the cell wall fraction of Yps3p was surface localized in restriction fragment length polymorphism class 2 strains. We also established that Yps3p released into the G217B culture supernatant binds to the surface of strains that do not naturally express the protein. This binding was saturable and occurred within 5 min of exposure and occurred similarly with live and heat-killed H. capsulatum. Flow cytometric analysis of H. capsulatum after enzymatic treatments was consistent with Yps3p binding to chitin, a carbohydrate polymer that is a component of fungal cell walls. Polysaccharide binding assays demonstrated that chitin but not cellulose binds to and extracts Yps3p from culture supernatants.     

Evidence that the potential for dissimilatory ferric iron reduction is widespread among acidophilic heterotrophic bacteria.

Nineteen characterized strains and isolates of acidophilic heterotrophic bacteria were screened for their abilities to catalyse the reductive dissolution of the ferric iron mineral schwertmannite, under oxygen-limiting conditions. Acidocella facilis, Acidobacterium capsulatum, and all of the Acidiphilium, Acidocella and Acidobacterium-like isolates that grew in liquid cultures were able to reduce iron. In contrast, neither Acidisphaera rubrifaciens nor three Acidisphaera-like isolates tested were found to have the capacity for dissimilatory iron reduction. One of two iron-oxidizing Frateuria-like isolates also reduced iron under oxygen-limiting conditions. Microbial dissolution of schwertmannite was paralleled with increased concentrations of soluble ferrous iron and sulfate in microbial cultures, together with increased pH values and decreased redox potentials. While dissimilatory ferric iron reduction has been described previously for Acidiphilium spp., this is this first report of this capacity in Acidocella and the moderate acidophile Acidobacterium. The finding has significant implications for understanding of the biogeochemistry of acidic environments.     

Diagnostic usefulness of the intradermal test with histoplamin in non-endemic areas of histoplasmosis

The histoplasmosis in Spain is an imported disease presenting in most of case diagnostic difficulties. In this paper, the intradermal skin test with Histoplasma capsulatum antigen as diagnostic method in immunocompetent patients with clinical and radiological signs compatible with histoplasmosis after being visited Central and South American endemic counties, in which this mycosis is endemic. Nine Spanish patients coming from different countries of Latin America with fever and acute respiratory symptoms compatible with histoplasmosis were studied. Other nine accompanying subjects and five controls were also evaluated. Patients underwent mycological cultures and and serological tests for H. capsulatum. Intradermal test with 1% histoplasmine were done in all patients. Serology and skin tests tests were also performed in accompanying people. Intradermal were done in healthy controls. Skin test with histoplasmine were positive in seven of the nine patients. Six of these showed precipitating antibodies against the same antigen. H. capsulatum was only isolated from bone marrow biopsy samples in one patient. The seven patients were given itraconazole by oral route and all symptoms improved after 2 and 4 weeks. In five accompanying subjects the skin test were also positive so that a subclinical histoplasmosis was diagnosed. In the remaining patients and healthy accompanying subjects histoplasmosis infection was excluded. In non endemic geographical areas of histoplasmosis intradermal skin test with histoplasmin when used in immunocompetent individuals is an easy and reliable method for the diagnosis of this mycosis as well as for epidemiological studies.     

Electrophoresis karyotype and chromosome-length polymorphism of Histoplasma capsulatum clinical isolates from Latin America

Intact chromosomes of 19 clinical isolates of Histoplasma capsulatum recently obtained in Argentina, Mexico and Guatemala and the laboratory reference strain G186B from Panama were analyzed using pulsed-field gel electrophoresis. Chromosomal banding patterns of the human isolates revealed 5-7 bands, ranging from 1.3 to 10 Mbp in size. Strain G186B showed five bands of approximately 1.1, 2.8, 3.3, 5.4 and 9.7 Mbp. Thirteen different electrokaryotypes were identified, indicating that the genome of H. capsulatum varies widely in nature, as observed previously in laboratory strains. No definite association was found between electrokaryotype and geographical or clinical source.     

In-vitro studies with SF 86-327, a new orally active allylamine derivative

SF 86-327 (Sandoz Forschungsinstitut) is an orally active allylamine derivative related to naftifine. The antifungal activity of SF 86-327 was compared in vitro with those of naftifine, ketoconazole, and itraconazole (R 51,211, Janssen Pharmaceutica) by agar dilution. 120 fungal isolates were tested. Also, the antifungal activities of SF 86-327 and naftifine against 18 dimorphic pathogens were assayed in vitro by broth dilution. Results of these studies support claims that SF 86-327 is a broad spectrum antifungal agent. Results of the broth dilution studies also revealed that SF 86-327 was both fungistatic and fungicidal in vitro for isolates of Blastomyces dermatitidis, Histoplasma capsulatum and Sporothrix schenckii at concentrations as low as 0.05 micrograms ml-1 (18 isolates tested, MIC90 = 0.39 micrograms ml-1, MFC90 = 12.5 micrograms ml-1).     

Varying Virulence in Rabbits Infected with Different Filamentous Types of Histoplasma capsulatum

Histoplasma capsulatum filamentous primary isolates and their subcultures are separable into two distinct colonial types (A and B) having different microscopic characteristics. Yeast forms of the A and B types and the parent (P) strains from which they are derived are microscopically indistinguishable. Critically standardized inocula of living P, A, and B yeasts from one strain of H. capsulatum (G-184) were injected intravenously into 12 rabbits. Each type produced progressively debilitating disease, but in varying degrees. Of the 12 animals, 6 died within 2 to 14 weeks. A persisting copious nasal exudate, beginning at or before 1 week, was cultured weekly at 26 C on Mycosel (BBL) agar. Pure cultures of A and B filamentous type colonies were recovered from exudates of animals receiving A and B yeasts, respectively, whereas both filamentous types were isolated from rabbits injected with P yeasts, with B predominating. Only A and B yeasts thus maintained their filamentous integrity during animal passage. It was noted that dissemination of H. capsulatum through the nares of infected rabbits represents a possible hazard to laboratory personnel heretofore unrecognized. It is also a possible means of cross-infecting or sensitizing or cross-infecting and sensitizing animals housed in the same room, if A and B yeasts prove not to be antigenically identical.