New insights into culture negative endophthalmitis by unbiased next generation sequencing.

The proof-of-concept, study to investigate the presence of microorganisms in presumed infectious endophthalmitis using Next generation sequencing (NGS) was carried out in vitreous biopsies from 34 patients with endophthalmitis, and thirty patients undergoing surgery for non-infectious retinal disorders as controls. Following DNA extraction using the Qiagen mini kit and PCR amplification of the V3-V4 regions of the bacterial 16S rRNA and ITS 2 region of fungus, they samples were sequenced on an Illumina HiSeq 2500 Machine. Paired reads were curated, taxonomically labeled, and filtered. Culture based diagnosis was achieved in 15/34 (44%) patients while NGS diagnosed the presence of microbes in 30/34 (88%) patients (bacteria in 26/30, fungi in 2/30, mixed infections in 2/30 cases). All 30 controls were negative for bacteria or fungus by NGS. There was good agreement between culture and NGS for culture-positive cases. Among culture negative cases, DNA of common culturable bacteria were identified like Streptococcus sp., Staphylococcus sp., Pseudomonas sp., Gemella sp., Haemophilus sp., Acinetobacter sp. The specificity of NGS with culture and clinical diagnosis was found to be 20% and 100% respectively and sensitivity of NGS with culture and clinical diagnosis was found to be 87.5% and 88% respectively. NGS appears to be promising diagnostic platform for the diagnosis of infectious culture negative endophthalmitis.

Candida Species From Eye Infections: Drug Susceptibility, Virulence Factors, and Molecular Characterization.

Purpose: To determine the type of Candida species in ocular infections and to investigate the relationship of antifungal susceptibility profile to virulence factors. Methods: Fifty isolates of yeast-like fungi from patients with keratitis, endophthalmitis, and orbital cellulitis were identified by Vitek-2 compact system and DNA sequencing of ITS1-5.8S-ITS2 regions of the rRNA gene, followed by phylogenetic analysis for phenotypic and genotypic identification, respectively. Minimum inhibitory concentration of six antifungal drugs was determined by E test/microbroth dilution methods. Phenotypic and genotypic methods were used to determine the virulence factors. Results: Phylogenetic analysis showed the clustering of all isolates into eight distinct groups with a major cluster formed Candida parapsilosis (n = 21), which was the most common species by both Vitek 2 and DNA sequencing. Using χ2 test no significant difference was noted between the techniques except that Vitek 2 did not identify C. viswanathii, C. orthopsilosis, and two non-Candida genera. Of 43 tested Candida isolates high susceptibility to amphotericin B (39/43, 90.6%) and natamycin (43/43, 100%) was noted. While none of the isolates produced coagulase, all produced esterase and catalase. The potential to form biofilm was detected in 23/43 (53.4%) isolates. Distribution of virulence factors by heat map analysis showed difference in metabolic activity of biofilm producers from nonbiofilm producers. Conclusions: Identified by Vitek 2 and DNA sequencing methods C. parapsilosis was the most common species associated with eye infections. Irrespective of the virulence factors elaborated, the Candida isolates were susceptible to commonly used antifungal drugs such as amphotericin B and natamycin.