title: Use of different primer directed sequence amplification by polymerase chain reaction for identification of Pneumocystis jirovecii in clinical samples.
abstract: BACKGROUND
  Pneumocystis carinii pneumonia (PCP), caused by opportunistic agent Pneumocystis jirovecii (formerly, Pneumocystis carinii is one of the most serious respiratory infection in immunocompromised patients.
  
  
  OBJECTIVE
  The present study was conducted to compare polymerase chain reaction (PCR) assays targetting three different genes of Pneumocystis to study their application in its diagnosis.
  
  
  METHODS
  One hundred and eighty (n = 180) clinical samples from 145 immunocompromised patients with clinical suspicion of PCP and 35 samples from control group of 30 immunocompetent individuals with respiratory infections other than PCP were prospectively examined for the presence of Pneumocystis jirovecii (P. jirovecii). All the samples were subjected to microscopic examination, one single [major surface glycoprotein, (MSG)] and two nested [mitochondrial large subunit ribosomal ribonucelic acid, (mtLSU rRNA) and internal transcribed spacer (ITS) region], polymerase chain reaction assays.
  
  
  RESULTS
  Microscopic examination was positive in only six (n = 6) patients, whereas single round MSG PCR detected P. jirovecii deoxyribonucleic acid (DNA) in 16 cases. When the clinical samples were tested by mtLSU rRNA and ITS nested PCR assays, it was possible to detect seven additional cases of PCP, making it to a total of 23 cases. None of the clinical specimens in control group (n = 30) were positive by any of the above-mentioned techniques. Amongst the 81 bronchoalveolar lavage (BAL) samples tested, 16 were positive by MSG PCR, while 20 were positive by both nested, i.e., mtLSU rRNA and ITS PCR assays. Similarly, out of 50 sputum samples, only three were positive by MSG, seven by mtLSU rRNA and six by ITS nested PCR assays.
  
  
  CONCLUSIONS
  It has been observed that MSG is relatively more sensitive when single round PCR assay is used for detection of human Pneumocystosis compared to the first (single) rounds of either ITS or mtLSU rRNA nested PCRs. However, the two nested PCRs using ITS and mtLSU rRNA have been found to be more sensitive. On comparison of two nested PCR assays, the results have been more or less comparable.
authors.affiliation: Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India.
authors.forename: Rashmi
authors.initials: R
authors.lastname: Gupta
authors.forename: Bijay Ranjan
authors.initials: BR
authors.lastname: Mirdha
authors.forename: Randeep
authors.initials: R
authors.lastname: Guleria
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
authors.forename: Kalpana
authors.initials: K
authors.lastname: Luthra
mesh_terms: D000293:Adolescent; D000328:Adult; D002648:Child; D002675:Child, Preschool; D017931:DNA Primers; D005260:Female; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007223:Infant; D008297:Male; D008875:Middle Aged; D045363:Pneumocystis carinii / Q000302:isolation & purification*; D011020:Pneumonia, Pneumocystis / Q000175:diagnosis*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012331:RNA, Fungal / Q000378:metabolism
chemical_list: D017931:DNA Primers; D005656:Fungal Proteins; D012331:RNA, Fungal