title: Identification of torque teno virus in culture-negative endophthalmitis by representational deep DNA sequencing.
abstract: OBJECTIVE
  To test the hypothesis that uncultured organisms may be present in cases of culture-negative endophthalmitis by use of deep DNA sequencing of vitreous biopsies.
  
  
  METHODS
  Single-center, consecutive, prospective, observational study.
  
  
  METHODS
  Aqueous or vitreous biopsies from 21 consecutive patients presenting with presumed infectious endophthalmitis and 7 vitreous samples from patients undergoing surgery for noninfectious retinal disorders.
  
  
  METHODS
  Traditional bacterial and fungal culture, 16S quantitative polymerase chain reaction (qPCR), and a representational deep-sequencing method (biome representational in silico karyotyping [BRiSK]) were applied in parallel to samples to identify DNA sequences corresponding to potential pathogens.
  
  
  METHODS
  Presence of potential pathogen DNA in ocular samples.
  
  
  RESULTS
  Zero of 7 control eyes undergoing routine vitreous surgery yielded positive results for bacteria or virus by culture or 16S polymerase chain reaction (PCR). A total of 14 of the 21 samples (66.7%) from eyes harboring suspected infectious endophthalmitis were culture-positive, the most common being Staphylococcal and Streptococcal species. There was good agreement among culture, 16S bacterial PCR, and BRiSK methodologies for culture-positive cases (Fleiss' kappa of 0.621). 16S PCR did not yield a recognizable pathogen sequence in any culture-negative sample, whereas BRiSK suggested the presence of Streptococcus in 1 culture-negative sample. With the use of BRiSK, 57.1% of culture-positive and 100% of culture-negative samples demonstrated the presence of torque teno virus (TTV) sequences, compared with none in the controls (P=0.0005, Fisher exact test). The presence of TTV viral DNA was confirmed in 7 cases by qPCR. No other known viruses or potential pathogens were identified in these samples.
  
  
  CONCLUSIONS
  Culture, 16S qPCR, and BRiSK provide complementary information in presumed infectious endophthalmitis. The majority of culture-negative endophthalmitis samples did not contain significant levels of bacterial DNA. "Culture negativity" does not seem to be due to failure of growth of fastidious bacteria. The small DNA virus TTV was unexpectedly found in all culture-negative samples and some culture-positive samples. This study cannot distinguish whether TTV is a direct intraocular pathogen, an adjuvant for inflammation, a general marker of inflammation, or a commensal virus but provides a testable hypothesis for a pathogenic mechanism in culture-negative endophthalmitis.
authors.affiliation: Department of Ophthalmology and Visual Science, Washington University, St. Louis, Missouri.
authors.forename: Aaron Y
authors.initials: AY
authors.lastname: Lee
authors.affiliation: Department of Ophthalmology, University of Washington, Seattle, Washington.
authors.forename: Lakshmi
authors.initials: L
authors.lastname: Akileswaran
authors.affiliation: The Retina Service of Wills Eye Hospital, MidAtlantic Retina, Philadelphia, Pennsylvania.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Tibbetts
authors.affiliation: The Retina Service of Wills Eye Hospital, MidAtlantic Retina, Philadelphia, Pennsylvania.
authors.forename: Sunir J
authors.initials: SJ
authors.lastname: Garg
authors.affiliation: Department of Ophthalmology, University of Washington, Seattle, Washington; Department of Biological Structure, University of Washington, Seattle, Washington; Department of Pathology, University of Washington, Seattle, Washington. Electronic address: russvg@u.washington.edu.
authors.forename: Russell N
authors.initials: RN
authors.lastname: Van Gelder
mesh_terms: D000368:Aged; D001082:Aqueous Humor / Q000382:microbiology / Q000821:virology; D001431:Bacteriological Techniques; D004266:DNA Virus Infections / Q000175:diagnosis / Q000382:microbiology / Q000821:virology*; D004269:DNA, Bacterial / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics*; D009877:Endophthalmitis / Q000175:diagnosis / Q000382:microbiology / Q000821:virology*; D015828:Eye Infections, Viral / Q000175:diagnosis / Q000382:microbiology / Q000821:virology*; D005260:Female; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D007621:Karyotyping; D008297:Male; D054892:Metagenome / Q000235:genetics; D011446:Prospective Studies; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D022783:Torque teno virus / Q000235:genetics / Q000302:isolation & purification*; D014776:Virus Cultivation; D014822:Vitreous Body / Q000382:microbiology / Q000821:virology
chemical_list: D004269:DNA, Bacterial; D004271:DNA, Fungal; D004279:DNA, Viral; D012336:RNA, Ribosomal, 16S