title: Molecular detection of DHFR gene polymorphisms in Pneumocystis jirovecii isolates from Indian patients.
abstract: BACKGROUND
  Pneumocystis pneumonia (PCP) is an opportunistic life-threatening infection, especially for immunocompromised individuals. A trimethoprim-sulfamethoxazole (TMP-SMX) combination is commonly used for the treatment of PCP, targeting both dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) enzymes. Several studies have already shown that polymorphisms in the DHPS gene are associated with drug resistance. The present study analyzed DHFR gene polymorphisms in Pneumocystis jirovecii recovered from clinical samples from patients admitted to a tertiary care health center in New Delhi, India.
  
  
  METHODS
  Detection of P. jirovecii was performed using Gomori methenamine silver staining (GMS) and nested polymerase chain reaction (PCR) assay targeting the mitochondrial large subunit ribosomal RNA (mt LSU rRNA) gene. The DHFR gene was amplified using nested PCR protocol and was sequenced for detection of polymorphisms.
  
  
  RESULTS
  Of 180 clinical samples, only 4% (7/180) were positive by GMS staining, and 10% (18/180) were positive by mt LSU rRNA PCR assay. Of these 18 positive samples, only 77% (14/18) were amplified by the DHFR gene PCR assay. A total of 16 nucleotide substitutions were observed in 42% (6/14) samples targeted for the DHFR gene, of which 8 nucleotide substitutions were synonymous and the rest were non-synonymous.
  
  
  CONCLUSIONS
  The DHFR gene mutations found in this study may possibly indicate an association of process likely to contribute to therapeutic failure or an evolutionary process, and warrant continuous monitoring.
authors.affiliation: All India institute of Medical Sciences, New Delhi, India. yogitasingh1985@gmail.com.
authors.forename: Yogita
authors.initials: Y
authors.lastname: Singh
authors.forename: Bijay Ranjan
authors.initials: BR
authors.lastname: Mirdha
authors.forename: Randeep
authors.initials: R
authors.lastname: Guleria
authors.forename: Shehla
authors.initials: S
authors.lastname: Khalil
authors.forename: Ashutosh
authors.initials: A
authors.lastname: Panda
authors.forename: Rama
authors.initials: R
authors.lastname: Chaudhry
authors.forename: Anant
authors.initials: A
authors.lastname: Mohan
authors.forename: Sushil Kumar
authors.initials: SK
authors.lastname: Kabra
authors.forename: Lalit
authors.initials: L
authors.lastname: Kumar
authors.forename: Sanjay Kumar
authors.initials: SK
authors.lastname: Agarwal
mesh_terms: D000293:Adolescent; D000328:Adult; D002648:Child; D002675:Child, Preschool; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D025141:Drug Resistance, Fungal; D005260:Female; D014644:Genetic Variation; D006801:Humans; D007194:India; D008297:Male; D008828:Microbiological Techniques; D008875:Middle Aged; D008969:Molecular Sequence Data; D009154:Mutation; D016720:Pneumocystis Infections / Q000382:microbiology*; D045363:Pneumocystis carinii / Q000201:enzymology* / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011446:Prospective Studies; D012335:RNA, Ribosomal / Q000235:genetics; D017422:Sequence Analysis, DNA; D013194:Staining and Labeling; D062606:Tertiary Care Centers; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D055815:Young Adult
chemical_list: D004271:DNA, Fungal; D004275:DNA, Ribosomal; D012335:RNA, Ribosomal; C065096:RNA, ribosomal, 26S; D013762:Tetrahydrofolate Dehydrogenase