Majid Abrishami1, Behnam Hashemi2, Mojtaba Abrishami3, Khalil Abnous4, Kamal Razavi-Azarkhiavi5, Javad Behravan6. 1. Professor, Retina Research Center, Department of Ophthalmology, Mashhad University of Medical Sciences , Mashhad, Iran . 2. Student, Department of Pharmacodynamy and Toxicology, School of Pharmacy, Mashhad University of Medical Sciences , Mashhad, Iran . 3. Assistant Professor, Eye Research Center, Farabi Eye Hospital, Tehran University of Medical Sciences , Tehran, Iran . 4. Associate Professor, Department of Pharmacodynamy and Toxicology, School of Pharmacy, Mashhad University of Medical Sciences , Mashhad, Iran . 5. PhD Student, Department of Pharmacodynamy and Toxicology, School of Pharmacy, Mashhad University of Medical Sciences , Mashhad, Iran . 6. Professor, Biotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences , Mashhad, Iran .
Abstract
BACKGROUND: Bacterial endophthalmitis is a sight-threatening complication of ocular surgery which requires urgent medical consideration including comprehensive diagnosis. Polymerase chain reaction (PCR) as a sensitive molecular method has been extensively used for detection of microbial species in clinical specimens. AIM: The aim of this study was to identify the causative organisms of endophthalmitis in our patient population using a procedure based on PCR. MATERIALS AND METHODS: Vitreous samples from 32 patients with post-operative endophthalmitis were collected. Total vitreous DNA was extracted and then assessed by agarose gel electrophoresis. Bacterial 16S rRNA gene was amplified from genomic DNA using PCR with a pair of HAD2 universal primers. Library of PCR products from 16S rRNA, cloned into the pTZ57R/T vector. The ligated products were then transformed into E. coli DH5α strain and grown in the LB-ampicillin/X-Gal/IPTG plate. RESULTS: From the total of 32 vitreous samples, 18 specimens were positive, illustrating the presence of bacterial infection (56.4 %). Twelve species including Escherichia coli, Enterobacter cloacae, Bacillus subtilis, Neisseria gonorrhoeae, Streptococcus pneumoniae, Haemophilus influenzae, Chlamydia trachomatis, Staphylococcus aureus, Neisseria meningitides, Staphylococcus epidermidis, Pseudomonas aeruginosa and Bacillus cereus were identified using BLAST for known 16S rRNA sequences. CONCLUSION: Polymerase chain reaction (PCR) accompanied with cloning and sequencing approved to be sensitive and specific. The rapid molecular technique was useful in detection of 12 major microbial species, in infectious endophthalmitis.
BACKGROUND: Bacterial endophthalmitis is a sight-threatening complication of ocular surgery which requires urgent medical consideration including comprehensive diagnosis. Polymerase chain reaction (PCR) as a sensitive molecular method has been extensively used for detection of microbial species in clinical specimens. AIM: The aim of this study was to identify the causative organisms of endophthalmitis in our patient population using a procedure based on PCR. MATERIALS AND METHODS: Vitreous samples from 32 patients with post-operative endophthalmitis were collected. Total vitreous DNA was extracted and then assessed by agarose gel electrophoresis. Bacterial 16S rRNA gene was amplified from genomic DNA using PCR with a pair of HAD2 universal primers. Library of PCR products from 16S rRNA, cloned into the pTZ57R/T vector. The ligated products were then transformed into E. coli DH5α strain and grown in the LB-ampicillin/X-Gal/IPTG plate. RESULTS: From the total of 32 vitreous samples, 18 specimens were positive, illustrating the presence of bacterial infection (56.4 %). Twelve species including Escherichia coli, Enterobacter cloacae, Bacillus subtilis, Neisseria gonorrhoeae, Streptococcus pneumoniae, Haemophilus influenzae, Chlamydia trachomatis, Staphylococcus aureus, Neisseria meningitides, Staphylococcus epidermidis, Pseudomonas aeruginosa and Bacillus cereus were identified using BLAST for known 16S rRNA sequences. CONCLUSION: Polymerase chain reaction (PCR) accompanied with cloning and sequencing approved to be sensitive and specific. The rapid molecular technique was useful in detection of 12 major microbial species, in infectious endophthalmitis.
Authors: Paulo José Martins Bispo; Gustavo Barreto de Melo; Ana Luisa Hofling-Lima; Antonio Carlos Campos Pignatari Journal: Invest Ophthalmol Vis Sci Date: 2011-02-22 Impact factor: 4.799
Authors: Paulo José Martins Bispo; Gustavo Barreto de Melo; Pedro Alves d'Azevedo; Ana Luisa Höfling-Lima; Maria Cecília Zorat Yu; Antonio Carlos Campos Pignatari Journal: Arq Bras Oftalmol Date: 2008 Sep-Oct Impact factor: 0.872