title: Genetic characterization of the dihydrofolate reductase gene of Pneumocystis jirovecii isolates from Portugal.
abstract: OBJECTIVE
  The aim of the present study was to evaluate the genetic variation of Pneumocystis jirovecii dihydrofolate reductase (DHFR) gene in an immunocompromised Portuguese population and to investigate the possible association between DHFR genotypes and P. jirovecii pneumonia (PcP) prophylaxis with co-trimoxazole.
  
  
  METHODS
  One hundred and thirty-eight P. jirovecii isolates were submitted to DHFR genetic characterization by PCR and sequencing.
  
  
  RESULTS
  In the studied population, 72.7% of the patients presented sequences identical to the wild-type sequence of the P. jirovecii DHFR gene and 27.3% presented point substitutions. A total of nine substitution sites were identified; four synonymous substitutions at nucleotide positions 201, 272, 312 and 381 were detected in 31 patients. Five non-synonymous substitutions were observed, leading to the DHFR mutations Leu-13-->Ser, Asn-23-->Ser, Ser-31-->Phe, Met-52-->Leu and Ala-67-->Val. With the exception of the polymorphism at position 312 and the mutation at codon 52, all polymorphisms were reported in this study for the first time.
  
  
  CONCLUSIONS
  Our results suggest that DHFR gene polymorphisms are frequent in the Portuguese immunocompromised population but do not seem to be associated with PcP prophylaxis failure (P = 0.748 and P = 0.730).
authors.affiliation: Unidade de Protozoários Oportunistas/VIH e outras Protozooses, Instituto de Higiene e Medicina Tropical, Rua da Junqueira 96, 1349-008 Lisboa, Portugal.
authors.forename: Marina C
authors.initials: MC
authors.lastname: Costa
authors.forename: Francisco
authors.initials: F
authors.lastname: Esteves
authors.forename: Francisco
authors.initials: F
authors.lastname: Antunes
authors.forename: Olga
authors.initials: O
authors.lastname: Matos
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D005838:Genotype; D006801:Humans; D016867:Immunocompromised Host; D008969:Molecular Sequence Data; D009154:Mutation; D045363:Pneumocystis carinii / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification; D011020:Pneumonia, Pneumocystis / Q000382:microbiology*; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D011174:Portugal; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*
chemical_list: D004271:DNA, Fungal; D013762:Tetrahydrofolate Dehydrogenase