title: Comparative evaluation of uniplex, nested, semi-nested, multiplex and nested multiplex PCR methods in the identification of microbial etiology of clinically suspected infectious endophthalmitis.
abstract: OBJECTIVE
  This study is aimed to determine the utility of various polymerase chain reaction (PCR) methods in vitreous fluids (VFs) for detecting the infectious genomes in the diagnosis of infectious endophthalmitis in terms of sensitivity and specificity.
  
  
  METHODS
  This prospective and consecutive analysis included a total of 66 VFs that were submitted for the microbiological evaluation, which were obtained from 66 clinically diagnosed endophthalmitis patients presented between November 2010 and October 2011 at the tertiary eye care referral centre in South India. Part of the collected VFs were subjected to cultures and smears, and the remaining parts were utilized for five PCR methods: uniplex, nested, semi-nested, multiplex and nested multiplex after extracting DNA, using universal eubacterial and Propionibacterium acnes species-specific primer sets targeting 16S rRNA gene in all bacteria and P. acnes, and panfungal primers, targeting 28S rRNA gene in all fungi.
  
  
  RESULTS
  Of the 66 VFs, five (7.5%) showed positive results in smears, 16 (24%) in cultures and 43 (65%) showed positive results in PCRs. Among the 43 positively amplified VFs, 10 (15%) were positive for P. acnes genome, one for panfungal genome and 42 (62%) for eubacterial genome (including 10 P. acnes positives). Among 42 eubacterial-positive VFs, 36 were positive by both uniplex (first round) and multiplex (first round) PCRs, while nested (second round) and nested multiplex (second round) PCRs produced positive results in 42 and 41 VFs, respectively. Of the 43 PCR-positive specimens, 16 (37%) had positive growth (15 bacterial and one fungal) in culture. Of 50 culture-negative specimens, 27 (54%) were showed positive amplification, of which 10 were amplified for both P. acnes and eubacterial genomes and the remaining 17 were for eubacterial genome alone.
  
  
  CONCLUSIONS
  Nested PCRs are superior than uniplex and multiplex PCR. PCRs proved to be a powerful tool in the diagnosis of endophthalmitis, especially for detecting uncultured microbes.
authors: Department of Microbiology & Molecular Biology, Aravind Eye Hospital & Postgraduate Institute of Ophthalmology, SN High Road, Tirunelveli 627001, Tamil Nadu, India. jayahar@tvl.aravind.org
authors: Madasamy Jayahar
authors: MJ
authors: Bharathi
authors: Nandagopal
authors: N
authors: Murugan
authors: Gunasekaran
authors: G
authors: Rameshkumar
authors: Rengappa
authors: R
authors: Ramakrishnan
authors: Yerahaia Chinna
authors: YC
authors: Venugopal Reddy
authors: Chandrasekar
authors: C
authors: Shivkumar
authors: Srinivasan
authors: S
authors: Ramesh
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002648:Child; D002675:Child, Preschool; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics*; D009877:Endophthalmitis / Q000175:diagnosis* / Q000453:epidemiology / Q000382:microbiology*; D015818:Eye Infections, Bacterial / Q000175:diagnosis* / Q000453:epidemiology / Q000382:microbiology*; D005260:Female; D006801:Humans; D007194:India; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods*; D011446:Prospective Studies; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012307:Risk Factors; D012680:Sensitivity and Specificity; D014822:Vitreous Body / Q000382:microbiology; D055815:Young Adult
chemical_list: D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S