Purpose
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.
Design
Single center consecutive prospective observational study.
Participants and Controls
Aqueous or vitreous biopsies from 21 consecutive patients presenting with presumed infectious endophthalmitis, and seven vitreous samples from patients undergoing surgery for non-infectious 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.
Main Outcome Measures
Presence of potential pathogen DNA in ocular samples.
Results
None of 7 control eyes undergoing routine vitreous surgery yielded positive results for bacteria or virus by culture or 16S PCR. Fourteen 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, while BRiSK suggested presence of Steptococcus in one culture-negative sample. Surprisingly, using BRiSK, 57.1% of culture-positive and 100% of culture-negative samples demonstrated presence of Torque Teno Virus (TTV) sequences, compared to none in the controls (Fisher exact, p = 0.0005). Presence of TTV viral DNA was confirmed in seven cases by qPCR. No other known viruses or potential pathogens were identified in these samples.
Conclusion
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 appear 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. The current 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.