Evaluation of multiplex real-time polymerase chain reaction for the detection of herpes simplex virus-1 and 2 and varicella-zoster virus in corneal cells from normal subjects and patients with keratitis in India.

Purpose: To determine the presence of herpes simplex virus and varicella zoster virus (HSV 1 and 2, VZV) in the cornea of normal subjects by multiplex real time quantitative (qPCR) assay and evaluate its utility in the diagnosis of viral keratitis. Methods: Corneal epithelial cells from 33 eyes of 22 patients undergoing photorefractive keratectomy surgery (controls) and 50 corneal scrapings from 50 patients with suspected HSV keratitis were analyzed for the presence of HSV1 by conventional PCR and for presence of HSV1 and 2 and/or VZV by multiplex real-time PCR. Corneal scrapings of patients were also tested for HSV1 antigen by immunofluorescence assay (IFA). The results were compared and clinical records reviewed. Results: HSV1 and VZV DNA were detected in 8/33 controls (mean-14.3 ± 7.96, range: 3-29.1 copies/mL) and 2/33 controls (mean-10.7 ± 10.9, range 3-18.5 copies/ml) respectively. HSV2 was not detected in any of the controls. Copy numbers above the mean + 1SD of controls were considered significant for viral load in patient samples. Significantly higher number of corneal scrapings (39/50, 78%) from patients were positive for HSV1 (1.2 × 106 copies/mL ± 3.7 × 106 copies/mL) by real time qPCR compared to IFA (11/48, 23%, P value 0.0001) and conventional PCR (20/50, 40%, P value 0.0002). Double infection with HSV-1 (1.5 × 107 copies/ml) and HSV-2 (3.57 × 104 copies/ml) in one case and VZV infection (1.03 × 102 copies/ml) in another was also detected by the multiplex real-time PCR. Conclusion: Multiplex real-time PCR reliably detects HSV1 and 2 and VZV DNA and is ideal for the diagnosis of HSV and VZV keratitis in an ocular microbiology laboratory.

Trends in microbiological spectrum of endophthalmitis at a single tertiary care ophthalmic hospital in India: a review of 25 years.

PURPOSE: To determine trends in the microbial spectrum of endophthalmitis over the past 25 years and to review its antibiotic susceptibility patterns over the last 10 years. METHODS: Microbiology records of culture-positive endophthalmitis cases from 1991 to 2015 were reviewed. Additionally, data between 2005 and 2015 was also analyzed for trends in antibiotic susceptibility. RESULTS: Of the total of 9278 patients, 3319 (35.7%) were culture positive and included bacteria (2840/3319, 85.56%), fungi (387/3319, 11.66%), and mixed cultures (92/3319, 2.7%). Gram-positive bacteria accounted for 67.68% (1922/2840) of the total bacteria seen, with the most prevalent pathogen being Streptococcus pneumoniae and Staphylococcus epidermidis. Among the gram-negative organisms Pseudomonas aeruginosa was the most prevalent while. Aspergillus flavus was the most common fungus isolated and Candida sp. accounted for 6.9% of the total fungi isolated. There was no significant change in the trends of bacteria isolated during the study period. Overall susceptibility patterns showed that gram-positive bacteria were most susceptible to vancomycin (96%) and fluoroquinolones (89%). The resistance to ceftazidime increased from 31% in 2005 to 62% in 2015 (P = 0.006) and amikacin decreased from 36% in 2005 to 33% in 2015 (P = 0.782). Although a significant trend (P < 0.001) toward increasing microbial resistance against cephalosporins and fluoroquinolones was observed, decreasing microbial resistance against glycopeptides and aminoglycosides was also detected. CONCLUSION: The spectrum of pathogens causing endophthalmitis at our institute remained similar over the study period. These findings impact the empiric treatment and choice of antibiotics in patients with endophthalmitis.

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.