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.     

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.     

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.     

Frequency and Genetic Diversity of the MAT1 Locus of Histoplasma capsulatum Isolates in Mexico and Brazil

The MAT1-1 and MAT1-2 idiomorphs associated with the MAT1 locus of Histoplasma capsulatum were identified by PCR. A total of 28 fungal isolates, 6 isolates from human clinical samples and 22 isolates from environmental (infected bat and contaminated soil) samples, were studied. Among the 14 isolates from Mexico, 71.4% (95% confidence interval [95% CI], 48.3% to 94.5%) were of the MAT1-2 genotype, whereas 100% of the isolates from Brazil were of the MAT1-1 genotype. Each MAT1 idiomorphic region was sequenced and aligned, using the sequences of the G-217B (+ mating type) and G-186AR (− mating type) strains as references. BLASTn analyses of the MAT1-1 and MAT1-2 sequences studied correlated with their respective + and − mating type genotypes. Trees were generated by the maximum likelihood (ML) method to search for similarity among isolates of each MAT1 idiomorph. All MAT1-1 isolates originated from Brazilian bats formed a well-defined group; three isolates from Mexico, the G-217B strain, and a subgroup encompassing all soil-derived isolates and two clinical isolates from Brazil formed a second group; last, one isolate (EH-696P) from a migratory bat captured in Mexico formed a third group of the MAT1-1 genotype. The MAT1-2 idiomorph formed two groups, one of which included two H. capsulatum isolates from infected bats that were closely related to the G-186AR strain. The other group was formed by two human isolates and six isolates from infected bats. Concatenated ML trees, with internal transcribed spacer 1 (ITS1) -5.8S-ITS2 and MAT1-1 or MAT1-2 sequences, support the relatedness of MAT1-1 or MAT1-2 isolates. H. capsulatum mating types were associated with the geographical origin of the isolates, and all isolates from Brazil correlated with their environmental sources.     

Rapid Identification of Histoplasma capsulatum Directly from Cultures by Multiplex PCR

The multiplex PCR developed from a suspension of the yeast fungi correctly identified fifty-one clinical of H. capsulatum var. capsulatum strains isolated from clinical samples and soil specimens. The multiplex PCR was developed by combining two pairs of primers, one of them was specific to the H. capsulatum and the other one, universal for fungi, turned out to be specific to H. capsulatum, regardless of the fungus isolate studied. Primers designed to amplify a region of about 390-bp (Hc I–Hc II) and a region of approximately 600-bp (ITS1–ITS4) were used to identify a yeast isolated as H. capsulatum when both regions could be amplified. Absolute agreement (100?% sensitivity) could be shown between this assay and the cultures of H. capsulatum according to their morphological characteristics. Failure to amplify the target DNA sequence by PCR with primers Hc I–Hc II in the presence of the ITS1–ITS4 amplicon in isolates of P. brasiliensis, Cryptococcus neoformans, Trichosporon spp, Candida glabrata, C. albicans, C. tropicalis, C. parapsilosis, C. krusei, or Penicillium marneffei was an unequivocal sign of the high specificity of this assay. The assay specificity was also found to be 100?%. Incipient yeast forms obtained from clinical samples were identified as H. capsulatum by the PCR assay described before the morphological characteristics were registered shortening the time of diagnosis.     

Immunohistochemical Cross-Reactivity Between <Emphasis Type="Italic">Paracoccidioides</Emphasis> sp. from Dolphins and <Emphasis Type="Italic">Histoplasma capsulatum</Emphasis>

Paracoccidioidomycosis ceti is a cutaneous disease of cetaceans caused by uncultivated Paracoccidioides brasiliensis or Paracoccidioides spp. Serological cross-reactions between paracoccidioidomycosis ceti and paracoccidioidomycosis, paracoccidioidomycosis and histoplasmosis, and paracoccidioidomycosis and coccidioidomycosis have been reported before. The present study aimed to detect immunohistochemical cross-reaction between antibodies to Paracoccidioides sp. and Histoplasma capsulatum, and vice versa. Thirty murine sera, obtained from experimental infections of 6 isolates of H. capsulatum, were reacted with paraffin-embedded yeast-form cells of Paracoccidioides sp. derived from a case of paracoccidioidomycosis ceti in Japan. The murine sera were also reacted with human isolates of H. capsulatum yeast cells, with P. brasiliensis yeast cells, and with fungal cells of Coccidioides posadasii. Three dolphins’ sera from cases of paracoccidioidomycosis ceti, two human sera from patients with paracoccidioidomycosis, and a serum from a healthy person with a history of coccidioidomycosis were used in order to determine that the tested fungal cells reacted properly. Sera derived from mice infected with an isolate of H. capsulatum reacted positively against yeast cells of Paracoccidioides sp., yeast cells of P. brasiliensis, and fungal cells of C. posadasii, while those derived from other strains were negative. The present study recorded for the first time the cross-reaction between the yeast cells of H. capsulatum and antibodies against Paracoccidioides spp., the yeast cells of Paracoccidioides sp. and antibodies against H. capsulatum, the yeast cells of Paracoccidioides sp. and antibodies against Coccidioides sp., and fungal cells of C. posadasii and antibodies against Paracoccidioides spp.     

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.     

Agentes de micosis endémicas en un área rural de Argentina: estudio seroepidemiológico en perros

BackgroundThree fungal species causing human disease, namely Paracoccidioides brasiliensis, Histoplasma capsulatum and Coccidioides sp., are endemic in different areas of Argentina. Rates of infection in domestic dogs have been used in other Latin American countries as indicators of the presence of these pathogens in a given area. We used such an approach to investigate the epidemiological relevance of paracoccidiodomycosis, histoplasmosis and coccidioidomycosis in our country.AimTo investigate the presence of P. brasiliensis, H. capsulatum and Coccidioides sp. in a rural area of Argentina called Interfluvio Teuco-Bermejito, located in Chaco province.MethodsWe applied Western Blotting to determine the presence of specific antibodies in sera from 89 domestic dogs inhabiting the area. Antibodies against the following extra-cellular fungal antigens were investigated: gP43 of P. brasiliensis, H/M of H. capsulatum and 120, 82 and 48 kDa antigen bands of Coccidioides sp.ResultsSpecific antibodies against H. capsulatum were found in 9/89 (10%) sera: 8 reacted against both H and M antigens and 1 only reacted against antigen M. Of these 9 sera, one showed additional anti-gp43 activity and another reacted against all the fungal antigens tested.ConclusionsThis is the first study using dog infection to assess the presence of endemic fungal pathogens in Argentina. Our results suggest that H. capsulatum is the main dimorphic fungal pathogen in the Interfluvio Teuco-Bermejito area. Therefore, the diagnosis of histoplasmosis should be taken into account in patients living in this geographic region who show pulmonary or mucocutaneous symptoms compatible with the disease.     

Molecular detection of Histoplasma capsulatum indoors: A public health approach

BackgroundHistoplasmosis, caused by the dimorphic fungus Histoplasma capsulatum, represents an important public health problem, especially in urban environments where bats and humans cohabit indoors.AimsTo detect the presence of H. capsulatum indoors, using samples of bat droppings collected in roost sites inside houses.MethodsA Real-Time TaqMan PCR assay targeting the ITS1 region of the ribosomal DNA of H. capsulatum was carried out.ResultsFifty-nine sampling points in the municipality of São Paulo were inspected, all of them located at inhabited places. H. capsulatum was isolated from nine samples.ConclusionsThe rapid identification and monitoring of sites where the fungus is present may contribute to make a more reliable database of H. capsulatum distribution.     

A natural focus ofHistoplasma capsulatum var.duboisii is a bat cave

The natural reservoir ofHistoplasma capsulatum var.duboisii, the etiological agent of histoplasmosis duboisii (African histoplasmosis) is not yet known. We report the isolation ofH. capsulatum var.duboisii from soil admixed with bat guano and from the intestinal contents of a bat in a sandstone cave in a rural area, Ogbunike in Anambra State of Nigeria. Eight of 45 samples of soil admixed with bat guano yieldedH. capsulatum var.duboisii. Of the 35 bats belonging to the speciesNycteris hispida andTadirida pumila examined, only one (N. hispida) yielded this fungus from its intestinal contents. Identification of the isolates asHistoplasma was confirmed by exoantigen tests and by mating with tester strains ofH. capsulatum. In vitro conversion to large yeast from suggestive ofH. capsulatum var.duboisii was obtained on brain heart infusion agar supplemented with sheep blood and glutamine or cysteine. Pathogenicity tests with mice for all the isolates confirmed their identity by the demonstration of large yeast forms (8–15 µm in diameter) within giant cells in the infected tissues. Investigations on the possible occurrence of human infections in the area are in progress.A poster based on this work was presented at the 11th ISHAM Congress in Montreal, Canada (22–28 June 1991), La-Hoffman Roche, Basel, Switzerland kindly financed the trip of one of us (H.C.G) for the Congress.     

Genetic relatedness of a non-motile variant O157 enteropathogenic Escherichia coli (EPEC) strain and E. coli strains belonging to pathogenic related groups

The study was undertaken to determine the clonal relationship and the genetic diversity among Escherichia coli isolates by comparing a non-motile O157 variant with three O157:H7 EHEC isolates and one O55:H7 enteropathogenic E. coli (EPEC) strain. E. coli strains were characterized by sorbitol phenotype, multilocus enzyme electrophoresis, pulsed-field gel electrophoresis, random amplification polymorphic DNA, and the presence of specific virulence genes (stx, E-hly and LEE genes). Sorbitol fermentation was observed in O157:H- (strain 116I), O55:H7 and O157:H7 (strain GC148) serotypes. stx1 or stx2 and E-hly genes were only detected among O157:H7 isolates. LEE typing revealed specific allele distribution: eaegamma, tirgamma, espAgamma, espBgamma associated with EPEC O55:H7 and EHEC O157:H7 strains (B1/1 and EDL 933), eaealpha, tiralpha, espAalpha, espBalpha related to the 116I O157:H- strain and the GC148 strain presented non-typable LEE sequences. Multilocus enzyme profiles revealed two main clusters associated with specific LEE pathotypes. E. coli strains were discriminated by random amplification of polymorphic DNA-polymerase chain reaction and pulsed-field gel electrophoresis methodologies. The molecular approaches used in this study allowed the determination of the genetic relatedness among E. coli strains as well as the detection of lineage specific group markers.     

Multilocus Sequence Typing of Listeria monocytogenes by Use of Hypervariable Genes Reveals Clonal and Recombination Histories of Three Lineages

In an attempt to develop a method to discriminate among isolates of Listeria monocytogenes, the sequences of all of the annotated genes from the fully sequenced strain L. monocytogenes EGD-e (serotype 1/2a) were compared by BLASTn to a file of the unfinished genomic sequence of L. monocytogenes ATCC 19115 (serotype 4b). Approximately 7% of the matching genes demonstrated 90% or lower identity between the two strains, and the lowest observed identity was 80%. Nine genes (hisJ, cbiE, truB, ribC, comEA, purM, aroE, hisC, and addB) in the 80 to 90% identity group and two genes (gyrB and rnhB) with approximately 97% identity were selected for multilocus sequence analysis in two sets of L. monocytogenes isolates (a 15-strain diversity set and a set of 19 isolates from a single food-processing plant). Based on concatenated sequences, a total of 33 allotypes were differentiated among the 34 isolates tested. Population genetics analyses revealed three lineages of L. monocytogenes that differed in their history of apparent recombination. Lineage I appeared to be completely clonal, whereas representatives of the other lineages demonstrated evidence of horizontal gene transfer and recombination. Although most of the gene sequences for lineage II strains were distinct from those of lineage I, a few strains with the majority of genes characteristic of lineage II had some that were characteristic of lineage I. Genes from lineage III organisms were mostly similar to lineage I genes, with instances of genes appearing to be mosaics with lineage II genes. Even though lineage I and lineage II generally demonstrated very distinct sequences, the sequences for the 11 selected genes demonstrated little discriminatory power within each lineage. In the L. monocytogenes isolate set obtained from one food-processing plant, lineage I and lineage II were found to be almost equally prevalent. While it appears that different lineages of L. monocytogenes can share habitats, they appear to differ in their histories of horizontal gene transfer.     

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.     

Use of Random Amplification of Polymorphic DNA (RAPD) and PCR-Fingerprinting for Genotyping a Scedosporium prolificans (inflatum) Outbreak in Four Leukemic Patients

Four isolates of the pathogenic fungus Scedosporium prolificans (inflatum), causing a previously reported nosocomial outbreak in four leukemic patients, were typed by random amplification of polymorphic DNA (RAPD) with two different 10-mer primers and PCR-fingerprinting with the core sequence of phage M13 as a single primer. Both techniques allowed 10 additional clinical isolates of Scedosporium prolificans from different areas of Spain, including Scedosporium prolificans NCPF 2884, to be classified into 10 different molecular types. The four outbreak isolates consisted of three molecular types with two patients sharing a similar strain, and the remaining two patients infected by two different strains. Received: 28 January 1996 / Accepted: 28 March 1997     

Frequency of the Mating-Type (MAT1) in Histoplasma capsulatum Isolates from Buenos Aires,Argentina

Sex is genetically determined in Histoplasma capsulatum, governed by a sex-specific region in the genome called the mating-type locus (MAT1). We investigate the distribution of isolates of two H. capsulatum mating types in the clades circulating in Buenos Aires, Argentina. Forty-nine H. capsulatum isolates were obtained from the culture collection of the Mycology Center. The MAT1 locus was identified by PCR from the yeast suspension. The analysis of forty-eight isolates from clinical samples exhibited a ratio of 1.7 (MAT1-1:MAT1-2) and the only isolate from soil was MAT1-1. Forty-five H. capsulatum isolates belonged to the LAm B clade (H. capsulatum from Latin American group B clade) and showed a ratio of 1.8 (MAT1-1:MAT1-2). These results suggest an association between the mating types in isolates belonging to the LAm B clade. It remains to be defined whether a greater virulence should be attributed to the differences between the strains of the opposite mating type of the LAm B clade.

     

橡胶树体细胞胚发生过程中DNA甲基化分析

为探讨巴西橡胶树(Hevea brasiliensis)自根幼态无性系与供体间差异产生的原因,应用甲基化敏感扩增多态性扩增技术,对巴西橡胶树体细胞胚发生过程中基因组DNA 胞嘧啶甲基化程度和模式进行了分析。结果表明,在巴西橡胶树体细胞胚发生过程中不同阶段的DNA 甲基化程度不同,以花药的DNA 甲基化程度最高,体细胞胚的DNA 甲基化水平最低。在体细胞胚发生过程中出现了I、Ⅱ和Ⅲ 3 种类型的甲基化多态性带型的改变,包括他们的出现与消失。因此,橡胶树体细胞胚发生过程中可能通过DNA 甲基化甲基化和去甲基化来调控基因的表达。     

Draft Genome Comparison of Representatives of the Three Dominant Genotype Groups of Dairy Bacillus licheniformis Strains

The spore-forming bacterium Bacillus licheniformis is a common contaminant of milk and milk products. Strains of this species isolated from dairy products can be differentiated into three major groups, namely, G, F1, and F2, using random amplification of polymorphic DNA (RAPD) analysis; however, little is known about the genomic differences between these groups and the identity of the fragments that make up their RAPD profiles. In this work we obtained high-quality draft genomes of representative strains from each of the three RAPD groups (designated strain G-1, strain F1-1, and strain F2-1) and compared them to each other and to B. licheniformis ATCC 14580 and Bacillus subtilis 168. Whole-genome comparison and multilocus sequence typing revealed that strain G-1 contains significant sequence variability and belongs to a lineage distinct from the group F strains. Strain G-1 was found to contain genes coding for a type I restriction modification system, urease production, and bacitracin synthesis, as well as the 8-kbp plasmid pFL7, and these genes were not present in strains F1-1 and F2-1. In agreement with this, all isolates of group G, but no group F isolates, were found to possess urease activity and antimicrobial activity against Micrococcus. Identification of RAPD band sequences revealed that differences in the RAPD profiles were due to differences in gene lengths, 3′ ends of predicted primer binding sites, or gene presence or absence. This work provides a greater understanding of the phylogenetic and phenotypic differences observed within the B. licheniformis species.     

Comparison of Different DNA-Based Methods for Molecular Typing of Histoplasma capsulatum

Histoplasma capsulatum is very prevalent in the environment and is one of the most common causes of mycoses in humans and diverse animals in Brazil. Multiple typing methods have been developed to study H. capsulatum epidemiology; however, there is limited information concerning comparisons of results obtained with different methods using the same set of isolates. To explore the diversity of H. capsulatum in Brazil and to determine correlations between the results of three different molecular typing techniques, we examined 51 environmental, animal, and human isolates by M13 PCR fingerprinting, PCR-restriction fragment length polymorphism (RFLP) analysis of the internal transcribed region 1 (ITS1)-5.8S-ITS2 region of the rDNA locus, and DNA sequencing and phylogenetic analysis of parts of four protein-encoding genes, the Arf (ADP ribosylation factor), H-anti (H antigen precursor), Ole (delta-9 fatty acid desaturase), and Tub1 (alpha-tubulin) genes. Each method identified three major genetic clusters, and there was a high level of concordance between the results of the typing techniques. The M13 PCR fingerprinting and PCR-RFLP analyses produced very similar results and separated the H. capsulatum isolates included in this study into three major groups. An additional approach used was comparison of our Brazilian ITS1-5.8S-ITS2 sequences with the sequences deposited previously in NCBI data banks. Our analyses suggest that H. capsulatum can be divided into different molecular types that are dispersed around the world. Our results indicate that the three methods used in this study are reliable and reproducible and that they have similar sensitivities. However, M13 PCR fingerprinting has some advantages over the other two methods as it is faster, cheaper, and more user friendly, which especially increases its utility for molecular typing of Histoplasma in situations where laboratory facilities are relatively limited.Histoplasmosis is a serious community-acquired infection in the United States (28) and in certain countries of Latin America, where it is an especially significant problem in patients with AIDS (14). This disease is one of the most common systemic mycoses in Brazil, where epidemiological surveys carried out using the histoplasmin skin test have indicated that it is endemic in all areas surveyed (15). Data suggest that the numbers of cases of histoplasmosis in Brazil may be underestimated and that the areas where it is endemic are more widespread than previously thought.Histoplasma capsulatum is a dimorphic fungus that grows as a mold and produces aerial hyphae at 25 to 30°C, but it undergoes morphogenesis to a yeast phase at 37°C. The filamentous phase of this organism is usually found in soil enriched with several compounds, such as nitrogen and phosphate compounds. When conidial or hyphal fragments are inhaled by humans or animals, H. capsulatum changes to the yeast form and continues to replicate as a yeast. Although H. capsulatum has been recognized as an important fungal pathogen in immunocompromised hosts, particularly AIDS patients (27), there are significant gaps in our knowledge of this species'' epidemiology and pathogenesis. For instance, systemic histoplasmosis has been found in patients with AIDS who do not reside in regions where it is endemic (29), leading to the suggestion that the disease can result from reactivation of a previously acquired H. capsulatum infection. The clinical manifestations of histoplasmosis range from asymptomatic infections, mild flu-like symptoms, or pneumonia to a systemic disease involving the skin, brain, intestine, adrenal glands, and/or bone marrow (6). Importantly, diverse strains of H. capsulatum have been identified worldwide, and the strains vary in virulence. In addition to classical biochemical assays, distinctions between strains may be based on colony morphology or polymorphism of the genome (19).Multiple typing methods have been developed to study the epidemiology of H. capsulatum. These methods are based on phenotypic characteristics, such as antigenic profiles (13) and multilocus enzyme electrophoresis results (2), or on DNA-based analysis. Most recently, typing has been accomplished by analysis of fatty acid profiles of H. capsulatum (34). Molecular typing methods are generally considered to have advantages over phenotypic methods in terms of the stability of genomic markers and greater levels of typeability. Several genotype-based methods, such as hybridization of target genes (probes), chromosomal DNA typing, restriction fragment length polymorphism (RFLP) analysis, random amplified polymorphic DNA (RAPD) analysis, and sequencing, have been described for H. capsulatum (4, 5, 7, 8, 11, 17, 19). Despite the abundance of previously developed molecular techniques, there is limited information concerning comparisons of the results obtained with different methods using the same set of isolates. In H. capsulatum, no single approach based on DNA assays has been the dominant method.The current study was done to explore the diversity of H. capsulatum in Brazil and to determine the correlation between the results of three different molecular typing techniques. For this analysis, we used M13 PCR fingerprinting, PCR-RFLP analysis of the internal transcribed spacer 1 (ITS1)-5.8S-ITS2 region of the rDNA gene, and analysis of the nucleotide sequence polymorphism of four partial genes. M13 PCR fingerprinting (25) is based on generation of multiple PCR products with different electrophoretic mobilities. PCR fingerprinting primers are typically designed using repetitive DNA sequences (31), and the products facilitate detection of two types of genetic variations: (i) differences in the length of DNA and (ii) alterations in the sequence of the priming regions. PCR-RFLP analysis of the ITS1-5.8S-ITS2 region of the rDNA gene (9) involves use of a gene-specific PCR in combination with restriction digestion in order to generate highly stable and reproducible markers. Analysis of the nucleotide sequence polymorphism is based on the sequences of four partial protein-encoding genes (Arf, the H-anti gene, Ole, Tub1) (4). Additionally, to assess the utility of an assay to study the global epidemiology of the fungus, we performed a DNA sequencing analysis of the ITS1-5.8S-ITS2 region to compare the Brazilian H. capsulatum ITS1-5.8S-ITS2 sequences with sequences obtained for H. capsulatum strains isolated in other countries.