title: Multicentre study highlighting clinical relevance of new high-throughput methodologies in molecular epidemiology of Pneumocystis jirovecii pneumonia.
abstract: Pneumocystis jirovecii causes severe interstitial pneumonia (PcP) in immunosuppressed patients. This multicentre study assessed the distribution frequencies of epidemiologically relevant genetic markers of P. jirovecii in different geographic populations from Portugal, the USA, Spain, Cuba and Mozambique, and the relationship between the molecular data and the geographical and clinical information, based on a multifactorial approach. The high-throughput typing strategy for P. jirovecii characterization consisted of DNA pooling using quantitative real-time PCR followed by multiplex-PCR/single base extension. The frequencies of relevant P. jirovecii single nucleotide polymorphisms (mt85, SOD110, SOD215, DHFR312, DHPS165 and DHPS171) encoded at four loci were estimated in ten DNA pooled samples representing a total of 182 individual samples. Putative multilocus genotypes of P. jirovecii were shown to be clustered due to geographic differences but were also dependent on clinical characteristics of the populations studied. The haplotype DHFR312T/SOD110C/SOD215T was associated with severe AIDS-related PcP and high P. jirovecii burdens. The frequencies of this genetic variant of P. jirovecii were significantly higher in patients with AIDS-related PcP from Portugal and the USA than in the colonized patients from Portugal, and Spain, and children infected with P. jirovecii from Cuba or Mozambique, highlighting the importance of this haplotype, apparently associated with the severity of the disease and specific clinical groups. Patients from the USA and Mozambique showed higher rates of DHPS mutants, which may suggest the circulation of P. jirovecii organisms potentially related with trimethoprim-sulfamethoxazole resistance in those geographical regions. This report assessed the worldwide distribution of P. jirovecii haplotypes and their epidemiological impact in distinct geographic and clinical populations.
authors.affiliation: NOVA Medical School/Faculdade de Ciências Médicas, Centro de Toxicogenómica e Saúde Humana (ToxOmics), Departamento de Genética, Universidade Nova de Lisboa, Lisbon, Portugal.
authors.forename: F
authors.initials: F
authors.lastname: Esteves
authors.affiliation: Faculdade de Psicologia e de Ciências da Educação, Coimbra, Portugal.
authors.forename: B
authors.initials: B
authors.lastname: de Sousa
authors.affiliation: CIBER de Epidemiologia y Salud Publica and Instituto de Biomedicina de Sevilla, Hospital Universitario Virgen del Rocio/CSIC/Universidad de Sevilla, Seville, Spain.
authors.forename: E J
authors.initials: EJ
authors.lastname: Calderón
authors.affiliation: Division of Pulmonary and Critical Care Medicine & HIV/AIDS Division, San Francisco General Hospital, Department of Medicine, University of California San Francisco, San Francisco, CA, USA.
authors.forename: L
authors.initials: L
authors.lastname: Huang
authors.affiliation: Centro Hospitalar de Lisboa Norte, Hospital de Santa Maria, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal.
authors.forename: R
authors.initials: R
authors.lastname: Badura
authors.affiliation: Centro Hospitalar de Lisboa Central, Hospital de Curry Cabral, Serviço de Doenças Infecciosas, Lisbon, Portugal.
authors.forename: F
authors.initials: F
authors.lastname: Maltez
authors.affiliation: ISGlobal, Barcelona Ctr. Int. Health Res. (CRESIB), Hospital Clínic Universitat de Barcelona, Barcelona, Spain; Centro de Investigação em Saúde de Manhiça (CISM), Maputo, Mozambique.
authors.forename: Q
authors.initials: Q
authors.lastname: Bassat
authors.affiliation: Laboratory of Molecular Diagnostic, Pedro Kourí Hospital, Havana, Cuba.
authors.forename: Y
authors.initials: Y
authors.lastname: de Armas
authors.affiliation: Instituto de Saúde Ambiental, Faculdade de Medicina da Universidade de Lisboa, Lisbon, Portugal.
authors.forename: F
authors.initials: F
authors.lastname: Antunes
authors.affiliation: Unidade de Parasitologia Médica, Grupo de Protozoários Oportunistas/VIH e Outros Protozoários, Global Health and Tropical Medicine, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisbon, Portugal. Electronic address: omatos@ihmt.unl.pt.
authors.forename: O
authors.initials: O
authors.lastname: Matos
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D016000:Cluster Analysis; D005260:Female; D006239:Haplotypes; D057166:High-Throughput Screening Assays; D006801:Humans; D008297:Male; D008875:Middle Aged; D017720:Molecular Epidemiology / Q000379:methods; D058889:Molecular Typing / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods; D016533:Mycological Typing Techniques / Q000379:methods*; D045363:Pneumocystis carinii / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D011020:Pneumonia, Pneumocystis / Q000453:epidemiology* / Q000382:microbiology*; D020641:Polymorphism, Single Nucleotide; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D055815:Young Adult
keywords: DNA pools; Pneumocystis jirovecii; high-throughput molecular epidemiology; immunosuppressed patients; multilocus genotyping survey; pneumonia