Typing of Histoplasma capsulatum isolates based on nucleotide sequence variation in the internal transcribed spacer regions of rRNA genes

Abstract

The nucleotide sequences of internal transcribed spacer (ITS) regions of rRNA genes of 24 isolates of Histoplasma capsulatum were examined. The results indicate that the sequences of ITS regions in different isolates are not identical. Sequence variations were found at 20 positions in the 496 bp that were sequenced. Ten different sequence patterns, designated types A through H, were observed when the sequences from the 24 isolates were aligned. Twelve isolates from Indianapolis were classified into four different types. Two isolates from New York belonged to type G. Three isolates from different cities were type F. The remaining six isolates were of different types.

FIG. 1

Nested PCR for H. capsulatum ITS regions. The first PCR is performed with primers P3 and 2R8, and the second PCR is done with primers F5 and 2R5. The regions amplified and the sizes of PCR products are as indicated. Primer sequences are shown in the lower portion of the diagram.

FIG. 2

Sequences of ITS1, 5.8S rRNA gene, and ITS2 of H. capsulatum. U18363 is the A. capsulatus sequence deposited in the GenBank database. Sequence #16 (positions 101 to 596) is the first H. capsulatum sequence obtained in this study. Sequences written in lowercase letters are the first PCR primers (P3 and 2R8). Double-underlined sequences are the second PCR primers. Bases that are underlined are the ones found to be variable among the 24 isolates; the position numbers of most of these bases are indicated. Underlined position numbers denote bases that are not variable among the 24 isolates but are different between the two sequences compared. Bases in sequence #16 that are the same as for U18363 are represented with asterisks. Missing bases in either sequences are marked by hyphens.

FIG. 3

Alignments of ITS sequences from 24 isolates. The consensus sequences of nucleotides found to be variable among the 24 isolates are shown with position numbers indicated. Bases of each isolate that are the same as for the consensus sequence are represented with asterisks, and the missing bases are denoted by hyphens. Isolate numbers are shown on the left. The type of each isolate is shown in parentheses next to the specimen numbers.