Comparison of different DNA-based methods for molecular typing of Histoplasma capsulatum

Abstract

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.

FIG. 1.

(A) M13 fingerprints of 51 H. capsulatum isolates generated by BioloMICS version 7.5.80 (BioAware, Hannut, Belgium). (B) Electrophoretic profiles of 51 H. capsulatum isolates subjected to M13 PCR fingerprinting. (C) DNA banding patterns obtained for H. capsulatum isolates by PCR-RFLP analysis.

FIG. 2.

Evolutionary relationships of 51 taxa. (A) NJ tree based on analysis of combined data for the four loci. The branch lengths are proportional to distance. The evolutionary distances were computed using the maximum composite likelihood method. (B) Strict consensus for 176 MP trees derived from analysis the same data for the four loci. The MP tree was obtained using the close-neighbor interchange algorithm. The numbers at the nodes are percentages and indicate the levels of support based on 500 bootstrap replications of the parsimony procedure; only values greater than 70% are shown. There were a total of 1,432 positions in the final data set. Phylogenetic analyses were conducted using MEGA4.

FIG. 3.

Evolutionary relationships of 70 taxa (linearized) based on analysis of ITS1-5.8S-ITS2 of the rDNA gene. The evolutionary history was inferred using the neighbor-joining method. The percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are indicated at the nodes. The phylogenetic tree was linearized by assuming that the evolutionary rates in all lineages were equal. The tree is drawn to scale, and the branch lengths are in the same units as the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the maximum composite likelihood method and were in expressed as the number of base substitutions per site. There were a total of 418 positions in the final data set. Abbreviations: RJ, Rio de Janeiro; SP, São Paulo; PE, Pernambuco; ES, Espírito Santos; CE, Ceará; RS, Rio Grande do Sul. All Brazilian H. capsulatum sequences are indicated by normal type, and sequences of isolates from other countries are indicated by bold type. Phylogenetic analyses were conducted using MEGA4 (4).