title: Detection of Pneumocystis jirovecii dihydropteroate synthase polymorphisms in patients with Pneumocystis pneumonia.
abstract: In the present study, in order to improve the detection of Pneumocystis jirovecii dihydropteroate synthase (DHPS) mutations in pulmonary specimens of HIV-infected patients with P. jirovecii pneumonia, we evaluated a microfiltration procedure for the removal of human cell contamination and a nested-PCR method, for amplification in specimens with low parasite load. In the studied population, PCR amplification of the DHPS gene was more successful in unfiltered than in filtered specimens, with both touchdown-PCR and nested-PCR procedures (p<0.05 and p<0.001, respectively), but the amount of host DNA in the samples analysed seems to be inversely related with the successful PCR parasite detection. Amplification of P. jirovecii DHPS gene with nested-PCR was achieved in 77.5% of the specimens studied, demonstrating that this is a useful method for the identification of mutations in pulmonary specimens, including samples with low parasite loads, and will facilitate the evaluation of the relationship between the P. jirovecii DHPS polymorphisms and clinical resistance to sulfa drugs.
authors.affiliation: Unidade de Protozoários Oportunistas/VIH e outras Protozooses, Unidade de Parasitologia e Microbiologia Médicas (UPMM), Instituto de Higiene e Medicina Tropical, Lisboa, Portugal.
authors.forename: M C
authors.initials: MC
authors.lastname: Costa
authors.forename: J
authors.initials: J
authors.lastname: Gaspar
authors.forename: K
authors.initials: K
authors.lastname: Mansinho
authors.forename: F
authors.initials: F
authors.lastname: Esteves
authors.forename: F
authors.initials: F
authors.lastname: Antunes
authors.forename: O
authors.initials: O
authors.lastname: Matos
mesh_terms: D001992:Bronchoalveolar Lavage Fluid / Q000382:microbiology; D004271:DNA, Fungal / Q000032:analysis; D004094:Dihydropteroate Synthase / Q000235:genetics*; D005374:Filtration / Q000379:methods; D006801:Humans; D009154:Mutation; D045363:Pneumocystis carinii / Q000201:enzymology* / Q000235:genetics; D011020:Pneumonia, Pneumocystis / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D013183:Sputum / Q000382:microbiology
chemical_list: D004271:DNA, Fungal; D004094:Dihydropteroate Synthase