Population structure of Pneumocystis jirovecii isolated from immunodeficiency virus-positive patients Francisco Esteves a , Jorge Gaspar b , Ade ´ lcia Tavares a , Ine ˆs Moser a , Francisco Antunes c , Kamal Mansinho d , Olga Matos a, * a Unidade de Protozoa ´rios Oportunistas/VIH e Outras Protozooses, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisboa, Portugal b Departamento de Gene ´tica, Faculdade de Cieˆncias Me ´dicas, Universidade Nova de Lisboa, Lisboa, Portugal c Clı´nica das Doenc ¸as Infecciosas, Hospital de Santa Maria, Lisboa, Portugal d Servic ¸o de Doenc ¸as Infecciosas, Hospital de Egas Moniz, Lisboa, Portugal 1. Introduction Pneumocystis jirovecii pneumonia (PcP) remains a major cause of respiratory illness among HIV-infected patients in the highly active antiretroviral therapy (HAART) era. Several aspects of the epidemiology of this disease remain obscure, particularly the geographic and frequency distribution of P. jirovecii clones (Hauser et al., 2001; Miller et al., 2003; Esteves et al., 2008). As the organism cannot be cultured reliably, molecular typing has been used to describe differences between isolates. Description of genetic diversity in P. jirovecii, based on identification of genotypes at single or multiple loci, has been achieved by using PCR followed by DNA sequencing, restriction fragment length polymorphism (RFLP), single strand conformation polymor- phism (SSCP) or type-specific oligonucleotide hybridization (Olsson et al., 1998; Beard et al., 2000; Hauser et al., 2001; Wakefield et al., 2003; Costa et al., 2006b). Specific polymorphic loci have been studied with the purpose of understand important properties of isolates like geographical distribution of genotypes, drug-susceptibility, virulence, and modes of transmission (Miller and Wakefield, 1999; Kazanjian et al., 2000; Beard et al., 2000; Matos et al., 2003; Miller et al., 2007; Esteves et al., 2008, 2009). The aims of this study were: (1) to genetically characterize P. jirovecii in specimens from HIV-positive Portuguese patients on the basis of eight independent loci sequences: the mitochondrial large- subunit rRNA (mtLSU rRNA) and cytochrome b (CYB); and the Infection, Genetics and Evolution 10 (2010) 192–199 ARTICLE INFO Article history: Received 18 May 2009 Received in revised form 18 December 2009 Accepted 25 December 2009 Available online 7 January 2010 Keywords: Pneumocystis jirovecii Multilocus genotyping Allelic distribution HIV-positive patients Population structure ABSTRACT Pneumocystis jirovecii pneumonia (PcP) is an important opportunistic infection among immunocom- promised patients. Genetic characterization of P. jirovecii isolated from HIV-positive patients, based on identification of multiple nucleotide sequences at eight distinct loci, was achieved by using PCR with DNA sequencing and RFLP. The present study showed that the mitochondrial large-subunit rRNA (mtLSU rRNA), the cytochrome b (CYB), the superoxide dismutase (SOD), the b-tubulin (b-tub), the dihydrofolate reductase (DHFR) and the dihydropteroate synthase (DHPS) loci sequences were more variable and therefore giving additional information than the thioredoxin reductase (Trr1) and the thymidylate synthase (TS) genes. Genotyping at those six most informative loci enabled the identification of 48 different P. jirovecii multilocus genotypes (MLGs). Significant statistical associations between infecting P. jirovecii genotypes and patients’ age groups or PcP clinical status were found. Also, mtLSU rRNA sequences and specific genotypes from other three loci (CYB, SOD, and DHFR) were statistically associated. The results suggested large recombination between most P. jirovecii MLGs. However, one MLG occurred at a higher frequency than would be expected according to panmictic expectations, suggesting linkage disequilibrium and clonal propagation. The persistence of this specific MLG may be a consequence of clonal reproduction of this successful genotypic array in a P. jirovecii population with epidemic structure. The present study provided the description of multiple genomic regions of P. jirovecii, improving the understanding of genetic variability and frequency distribution of polymorphic genotypes, and exploring the criteria of clonality by testing over-representation of MLGs. ß 2010 Elsevier B.V. All rights reserved. * Corresponding author. Tel.: +351 21 3652638; fax: +351 21 3632105. E-mail addresses: esteves@ihmt.unl.pt (F. Esteves), jgaspar.gene@fcm.unl.pt (J. Gaspar), atavares@ihmt.unl.pt (A. Tavares), minmoser@gmail.com (I. Moser), ip231874@sapo.pt (F. Antunes), udip@hegasmoniz.min-saude.pt (K. Mansinho), omatos@ihmt.unl.pt (O. Matos). Contents lists available at ScienceDirect Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid 1567-1348/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.meegid.2009.12.007