To Örebro University

PURPOSE: To describe the bacterial findings by a targeted sequencing approach from corneal samples of patients with microbial keratitis and factors influencing culture outcome of indirectly inoculated corneal specimen.

METHODS: Prospective inclusion of patients fulfilling predefined criteria of microbial keratitis. Samples from the corneal lesion were collected and dispensed in liquid transport medium, from which both culture and targeted amplification and sequencing of the V3-V4 region of the 16S rRNA gene were carried out. Additional standard corneal culture from the corneal lesions was also performed. Factors influencing culture outcome of indirectly inoculated corneal samples were identified by a multivariate regression model incorporating quantitative data from sequencing.

RESULTS: Among the 94 included patients with microbial keratitis, contact lens wear (n = 69; 73%) was the most common risk factor. Contact lens wearers displayed significant differences in the bacterial community composition of the corneal lesion compared to no lens wearers, with higher abundance of Staphylococcus spp., Corynebacterium spp., and Stenotrophomonas maltophilia. Targeted sequencing detected a potential corneal pathogen in the highest proportional abundance among 9 of the 24 (38%) culture-negative patients with microbial keratitis. Age, bacterial density in the sample, and prior antibiotic treatment significantly influenced culture outcome of indirectly inoculated corneal samples.

CONCLUSION: Targeted sequencing may provide insights on pathogens in both culture negative episodes of microbial keratitis and among subgroups of patients with microbial keratitis as well as factors influencing culture outcome of indirectly inoculated corneal samples.

Purpose: To describe the corneal microbiome in infectious keratitis in relation to contact lens wear or not, and culture outcome. To explore if targeted sequencing may provide information concerning: i) culture negative episodes, ii) variables influencing culture outcome of corneal samples dispensed in transport medium.

Methods: Prospective inclusion of patients fulfilling predefined criteria of infectious keratitis. Corneal samples were dispensed in liquid Amies medium, from which both culture and targeted sequencing of the V3-V4 region of the 16S rRNA gene were carried out. Additional standard corneal culture was also performed. Main outcome measures were bacterial findings by targeted sequencing in relation to contact lens wear and culture outcome, and identification of variables influencing corneal culture outcome of indirectly inoculated corneal samples, using quantitative data from the PCR.

Results: In all, 94 episodes of infectious keratitis were included, of which 70 (74%) had bacterial growth on corneal culture. In median, 15 (range 8-30) different bacterial genera per episode of infectious keratitis were detected by targeted sequencing. The contact lens wearers (69/94; 73%) displayed significant (p=0.01) differences in the bacterial community composition of the corneal lesion compared to non-wearers, with higher abundance of Staphylococcus spp. Corynebacterium spp. and Stenotrophomonas maltophilia. Among the culture negative episodes (n=24) Brevundimonas was found to be significantly (adjusted p<0.05) enriched. Sequencing detected a potential corneal pathogen such as Clostridium, Staphylococcus, Brevundimonas, Pseudomonas and Veillonella, with a relative abundance of at least 20% in more than half of the culture negative episodes (14/24; 58%). Bacterial density in the sample had the highest impact on culture outcome (OR 6.3; p=0.009) but also age increased the odds for a positive culture outcome (OR 1.04; p=0.034), while prior antibiotic treatment significantly reduced the odds of a positive corneal culture to a fifth (OR 0.2; p=0.031).

Conclusions: Targeted sequencing can provide a potential corneal pathogen in case of a negative culture outcome in patients with infectious keratitis, as well as providing insights on the corneal microbiome of infectious keratitis and factors influencing its composition.

Infectious keratitis is a potentially sight threatening ophthalmological emergency. Contact lens wear is a common risk factor. Diagnostic advances such as MALDI-TOF MS provides new insights into the spectrum of corneal pathogens and on microbes previously considered as commensals. Corynebacterium macginleyi was described in 1995, and in 2018, the genomic features of three isolates were reported after whole-genome sequencing. Here we describe the clinical characteristics of patients with infectious keratitis (n = 29) presumably caused by Corynebacterium macginleyi, and analyze the genomic features of C. macginleyi (n = 22) isolated from the corneal ulcers of these patients. The disease course was uneventful apart from minor interventions such as corneal cross-linking and amniotic membrane transplant. Genome sequencing and comparison revealed a highly conserved core genome of C. macginleyi. Based on the analyses of single nucleotide polymorphisms, the population could be divided into two main clades that also differed in a few clade-specific genomic islands. Patients infected with an isolate belonging to the minor clade (n = 7) presented a more severe disease. Comparisons with other corynebacterial species clearly separated C. macginleyi. C. macginleyi may be considered a corneal pathogen; genomic analysis provided insights into its population structure and disease-causing potential.

BACKGROUND: To compare two different methods of corneal culture in infectious keratitis: multiple sampling for direct inoculation and enrichment (standard method) and a single sample via transport medium for indirect inoculation (indirect inoculation method).

METHODS: Prospective inclusion of patients fulfilling predefined criteria of infectious keratitis undergoing corneal culture according to both studied methods in a randomized order.

RESULTS: = 0.002) and a significantly higher proportion of microorganisms than the indirect inoculation method, with a Cohen's kappa of 0.38 (95% CI: 0.28-0.49) for agreement between the methods. Subanalysis of culture results showed that direct inoculation on gonococcal agar only combined with the indirect inoculation method resulted in a similar rate of culture positive patients and proportion of detected microorganisms to the standard method.

CONCLUSION: Indirect inoculation of one corneal sample cannot replace direct inoculation of multiple corneal samples without loss of information. A combination of directly and indirectly inoculated samples can reduce the number of corneal samples by four without statistically significant differences in culture outcome or in the proportion of detected microorganisms.

Objective: This study aimed to compare the efficacy of a cotton tipped applicator and a knife blade in obtaining corneal samples in patients with infectious keratitis.

Methods and analysis: This is a retrospective cohort study of patients with suspected infectious keratitis during 2004-2014. Samples for corneal culture were obtained by a cotton tipped applicator and a knife blade, and directly inoculated on GC agar, blood agar and Sabouraud agar.

Results: In all, 355 patients were included. Corneal sampling by cotton tipped applicator yielded a significantly higher rate of patients with positive corneal culture, 156/355 (43.9%), compared with knife blade, 111/355 (31.3%) (p<0.001). On a patient level, the culture results obtained by the cotton tipped applicator and the knife blade were identical in 269/355 (76%) of the patients. The overall agreement between the two instruments on microbial level was 0.66 (Cohen's kappa 95% CI 0.60 to 0.72).

Conclusion: Corneal sampling by cotton tipped applicator generated a higher rate of positive corneal cultures and a higher proportion of isolated microbes than by knife blade. Future studies with randomised sampling order are needed to establish which instrument, cotton tipped applicator or knife blade, is the most effective in sampling microbes for direct inoculation in patients with infectious keratitis.

PURPOSE: To describe predisposing risk factors, causative microorganisms and their antibiotic susceptibility patterns in infectious keratitis during an 11-year period in Region Örebro County, Sweden.

METHODS: This is a descriptive study conducted as a retrospective audit of clinical records. Patients who received treatment for infectious keratitis at any of the three ophthalmological departments within Region Örebro County, Sweden, between 2004 and 2014 were included if they fulfilled the predefined criteria for infectious keratitis. Data regarding culture results, antibiotic susceptibility pattern and risk factors for infectious keratitis were obtained from medical records and microbiological reports.

RESULTS: In total, 398 episodes of infectious keratitis in 392 patients were included, and 285 were culture positive. The most common predisposing risk factor was contact lens wear (45%). Coagulase-negative staphylococci (39.6%) was the most commonly isolated type of organism. Staphylococcus aureus (15.1%) followed by Moraxella spp. (7.4%) and Pseudomonas aeruginosa (6.7%) were among the most common isolated bacteria not considered to be commensal. Reduced susceptibility to fluoroquinolones was observed in five of 43 S. aureus isolates and in four of nine Streptococcus pneumoniae isolates.

CONCLUSION: The most common predisposing risk factor for keratitis was contact lens wear. Among the most common microbes, not considered to be exclusively commensals, isolated from the cornea in microbial keratitis were S. aureus, Moraxella spp. and P. aeruginosa. The antibiotic susceptibility patterns showed low proportion of resistance. Empiric treatment of suspected infectious keratitis with topical fluoroquinolones and chloramphenicol might be considered in a setting like ours pending culture results.

PURPOSE: To evaluate the relationship between metabolic syndrome and some of its components with the incidence of cataract extraction.

METHODS: A population-based prospective cohort with a total of 45 049 men, aged 45-79 years, from the Cohort of Swedish Men completed in 1997 a self-administered questionnaire concerning anthropometric measurements and lifestyle factors. The men were followed from 1 January 1998 through 31 December 2012, and the cohort was matched with registers of cataract extraction. The main outcome measure was incident cases of age-related cataract extraction.

RESULTS: Over the 15-years of follow-up, 7573 incident cases of cataract extraction were identified. After controlling for potential confounders, the association between single components of metabolic syndrome, abdominal adiposity, diabetes and hypertension and risk of cataract extraction was rate ratio (RR): 1.04; 95% confidence interval (CI): 0.99-1.10, RR: 1.77; 95% CI: 1.64-1.92 and RR: 1.06; 95% CI 1.00-1.13, respectively. The risk of cataract extraction increased with increasing numbers of metabolic syndrome components (p < 0.0001). Men aged 65 years or younger at baseline with all three components of the metabolic syndrome had a relative risk of 2.43 (95% CI: 1.95-3.01) for cataract extraction.

CONCLUSION: In this cohort of middle-aged and elderly men, metabolic syndrome with the combination of abdominal adiposity, diabetes and hypertension was associated with an increased risk for cataract extraction, especially among men aged 65 years or younger. These findings put emphasis on the importance of weight control and healthy lifestyle behaviours in order to prevent cataract.