title: Application of real time polymerase chain reaction targeting kex 1 gene & its comparison with the conventional methods for rapid detection of Pneumocystis jirovecii in clinical specimens.
abstract: OBJECTIVE
  As there are no standard laboratory techniques for the rapid detection of Pneumocystis jirovecii in India, this study was undertaken to evaluate and establish an optimal and rapid technique for the detection of P. jirovecii by comparing three different techniques - staining technique, application of a real time polymerase chain reaction (RT-PCR) targeting kex 1 gene and application of nested PCR targeting mitochondrial large subunit (mtLSU) gene for rapid detection of P. jirovecii in HIV positive patients.
  
  
  METHODS
  One hundred and fifty sputum specimens from HIV positive (n = 75) and HIV negative (n = 75) patients were subjected to three different techniques -KOH/Calcoflour and Grocott methanamine silver staining (GMS), RT-PCR targeting kex1 gene, PCR targeting mtLSU region followed by DNA sequencing and BLAST analysis.
  
  
  RESULTS
  Among the 75 HIV positive patients, P. jirovecii was detected in 19 (25.33%) patients by the staining techniques, and in 23 (30.65%) patients each by PCR targeting mtLSU region and by RT- PCR targeting kex1 gene of P. jirovecii. PCR based DNA sequencing targeting mtLSU region revealed 97-100 per cent sequence homology with P. jirovecii sequences in GenBank.
  
  
  CONCLUSIONS
  Of the three techniques for detection of P. jirovecii evaluated in this study, false negativity was found to be more in staining technique and it also required high technical expertise to interpret the result. Both nested PCR and RT-PCR were reliable and equally sensitive, in rapid detection of P. jirovecii, but RT-PCR technique also generated the copy numbers for knowing the severity of infection.
authors.forename: Mani
authors.initials: M
authors.lastname: Revathy
authors.affiliation: L & T Microbiology Research Center, Vision Research Foundation, Kamal Nayan Bajaj Research Centre, Chennai, India.
authors.forename: Kulandai Lily
authors.initials: KL
authors.lastname: Therese
authors.forename: Radhakishnan
authors.initials: R
authors.lastname: Bagyalakshmi
authors.forename: Chokaliingam
authors.initials: C
authors.lastname: Chandrasekar
authors.forename: Suria
authors.initials: S
authors.lastname: Kumar
authors.forename: Hajib N
authors.initials: HN
authors.lastname: Madhavan
mesh_terms: D000293:Adolescent; D000328:Adult; D001992:Bronchoalveolar Lavage Fluid / Q000382:microbiology; D002648:Child; D002675:Child, Preschool; D005260:Female; D006801:Humans; D007194:India; D007223:Infant; D008297:Male; D008875:Middle Aged; D045363:Pneumocystis carinii / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity; D011020:Pneumonia, Pneumocystis / Q000175:diagnosis* / Q000382:microbiology; D060888:Real-Time Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D012697:Serine Endopeptidases / Q000187:drug effects / Q000235:genetics*; D013183:Sputum / Q000382:microbiology
chemical_list: C470926:Kex1 protein, Pneumocystis jirovecii; D012697:Serine Endopeptidases