Multilocus genotyping of Pneumocystis jirovecii from adult HIV-infected patients with Pneumocystis pneumonia.

Abstract

The human fungal pathogen Pneumocystis jirovecii causes severe pneumonia in patients with impaired immune systems. As the organism cannot be cultured reliably it is not possible to describe differences among isolates using conventional techniques and so instead molecular typing has been used. Description of genetic diversity in P. jirovecii, based on identification of single or multiple nucleotide polymorphisms at several loci, has been achieved by using PCR with DNA sequencing, single strand conformation polymorphism (SSCP) or type-specific oligonucleotide hybridization. Loci studied include the large subunit mitochondrial rRNA (mt LSU rRNA) [2,13,14,15], small subunit mitochondrial rRNA (mt SSU rRNA) [13,15], the nuclear 5S rRNA [8] and 26S rRNA [5], the intron 6 region of b tubulin (b TUB) [5], cytochrome b [16] arom [1], superoxide dismutase (SOD) [3] dihydropteroate synthase (DHPS) [2], thymidylate synthase [8,11] and the internal transcribed spacer regions 1 and 2 (ITS 1 and ITS 2) of the nuclear rRNA operon [9,12]. An alternative method for identifying differences among isolates of P. jirovecii, which uses identification of the number of tandem repeats in the Upstream Conserved Region of the Major Surface Antigen has been described recently [10]. Identification of single or multiple polymorphisms at individual loci enables description of � 5 genotypes and so provide limited information for discrimination between isolates. In contrast, the ITS of P. jirovecii exhibits high genetic diversity; 87 of a potential total of 810 genotypes have been described [9,12]. This suggests that these genes are evolving faster than other genes and this may render this locus less useful for genotyping in longitudinal studies. Furthermore, as an increasing number of ITS genotypes have been described, attempts at correlating specific genotypes with clinical parameters (e.g. disease severity) have been unsuccessful [6]. Multilocus genotyping enables simultaneous description of several loci providing information that may be used to attempt strain characterization. The aim of this study was to use multilocus genotyping at four independent loci, the mt LSU rRNA, the mt SSU rRNA, the DHPS (which is under selective pressure from sulfa drug exposure) [7], and the SOD, in order to describe differences among isolates of P. jirovecii.