Comparison of corneal culture results obtained using the ESwab™ and Amies charcoal swab in canine and feline patients.

OBJECTIVE: The objective of the study was to compare corneal culture results using the ESwab™ and Amies charcoal swab. ANIMALS STUDIED: One hundred fourteen canine and fifteen feline eyes. PROCEDURES: Retrospective analysis of Dick White Referrals bacterial and fungal corneal culture data was conducted. Results were included from canine and feline patients, which presented with suspected infectious keratitis that had samples taken using an Amies charcoal swab followed by an ESwab™ in the same eye. In respect to positive and negative cultures, a McNemar test was conducted comparing instances of disagreement between swab types, and the Kappa coefficient (κ) was calculated to assess the level of agreement between swab types. RESULTS: The ESwab™ produced more positive corneal cultures (71/129 [55.0%]) than the Amies charcoal swab (41/129 [31.8%]). 2/129 eyes produced positive fungal cultures. Considering 37/129 eyes in which both swab types detected a positive corneal culture, the same bacterial species were cultured from each swab type in 34/37 (91.9%) eyes, and an additional bacterial species was cultured by the ESwab™ in 3/37 (8.1%) eyes. In 34/38 (89.5%), instances of disagreement between swab types, the ESwab™ showed a positive culture, and the Amies charcoal swab showed a negative culture from the same eye, and this difference was significant (p < 0.0001). There was a moderate level of agreement between results from both swab types (κ = 0.432). CONCLUSIONS: ESwab™ sampling alone may be superior to Amies charcoal swabs for detecting presence of bacteria in suspected infectious keratitis in cats and dogs.

Effect of topical application of 0.5% proparacaine on corneal culture results from 33 dogs, 12 cats, and 19 horses with spontaneously arising ulcerative keratitis.

OBJECTIVE: To investigate the effect of topically applied proparacaine on bacterial and fungal culture results and to compare cytologic and culture results in patients with ulcerative keratitis. PROCEDURE: Corneal samples were collected from 33 dogs, 19 horses, and 12 cats with spontaneously arising ulcerative keratitis. Samples for bacterial (dogs, cats, horses) and fungal (horses) cultures were collected prior to and following application of 0.5% proparacaine or saline. All patients then received a topical anesthetic, and samples were collected for cytology. Frequency of cultivatable bacteria before (Swab 1) and after (Swab 2) application of proparacaine or saline was compared using Fisher's exact test. Homogeneity of culture and cytology results was assessed using McNemar's test. RESULTS: No difference was detected in number of animals from which bacteria were isolated from Swab 1 or Swab 2 for proparacaine (21/37 and 17/37, respectively) or saline (10/27 and 12/27, respectively). Small numbers prevented analysis of fungal culture results in horses between Swab 1 and Swab 2 for proparacaine (2/12 and 1/12, respectively) or saline (both, 1/8). Bacteria were isolated from 10 of 20 horses and detected cytologically in 3 of these; fungi were isolated from 3 of 20 horses and detected cytologically in 2 of these. Bacteria were detected more frequently using culture (31/64) than cytology (19/64). CONCLUSION: Proparacaine did not significantly alter bacterial or fungal culture results in cats, dogs, or horses; however, clinical significance warrants investigation. Culture and cytology provided complementary data; both should be performed to maximize organism detection in patients with ulcerative keratitis.

A two-centre validation study of sterility test of corneal storage media with elimination of interfering antimicrobials in compliance with the European Pharmacopoeia.

The purpose of this study was to validate the sterility test of corneal culture and deswelling/transport media using a device for removal of antimicrobials before incubation in BACTEC™ automated system in two Italian Eye Banks. Corneal culture medium, TISSUE-C, and deswelling/transport medium, CARRY-C, were inoculated with 10-100 cfu of six European Pharmacopoeia (EP) reference strains and either treated with medical device RESEP for removal of antimicrobials (RESEP+ group) or left untreated (RESEP- group) before injection into the BACTEC Plus bottles. The same steps were repeated in the absence of inocula with tryptone soy broth samples as negative controls, and the inocula were also directly injected in the BACTEC™ bottles as growth controls. All the samples were incubated in BACTEC™ automated system for 7 days, and the time to detection of microbial growth was recorded automatically. At both the Eye Banks, in the RESEP+ groups, microbial growth was detected in 100% of samples. In the RESEP- group, the method sensitivity ranged from 66.7 ± 21.1 to 88.9 ± 6.4% for TISSUE-C samples while for CARRY-C samples the method sensitivity ranged from 94.5 ± 5.1 to 100%. The method specificity corresponded to 100% for all the groups at both Eye Banks. This two-centre validation study showed that the use of RESEP increased the sensitivity of sterility test using BACTEC™ automated system up to 100% and, consequently, allowed validation of the method for sterility testing of corneal storage and deswelling/transport media according to the EP requirements. The test could not be validated without the use of RESEP.

Microbial Profile and Clinical Outcomes of Fungal Keratitis at a Single-Center Tertiary Care Hospital.

PURPOSE: To evaluate baseline characteristics, microbiological spectrum, management, and outcomes of patients with culture-proven fungal keratitis. METHODS: Retrospective review of all patients with culture-proven fungal keratitis seen over 6 years at a tertiary referral center. RESULTS: The present study included 62 eyes from 62 patients. Infection with filamentous organisms was more common than with yeast (66.1% vs 27.4%). The most common filamentous organisms were Fusarium (17.7%) and Aspergillus (16.1%), while the most common yeast was Candida (24.2%). The main predisposing factor for filamentous keratitis was contact lens use. Yeast keratitis is most associated with an immunocompromised host and ocular surface disease. Corneal perforation (20.0%) and surgical interventions (46.8%) were common, with 27.4% of eyes requiring at least one penetrating keratoplasty. Filamentous keratitis is more likely than yeast keratitis to require urgent penetrating keratoplasty or enucleation and to receive more than one topical and systemic antifungal agent. Visual outcomes were poor with nearly half of the eyes remaining at 20/200 or worse upon resolution of infection. Worse visual outcomes were associated with poor vision at presentation and a history of ocular surface disease. Antifungal susceptibility testing was not routinely performed, but it demonstrated a relatively high minimum inhibitory concentration for at least one antifungal drug in 90% of cases when performed (16.1%) and guided the direction of treatment for 80% of the cases. CONCLUSION: Fungal keratitis is visually devastating. Infections with filamentous fungi predominated over yeast and were generally treated more aggressively both medically and surgically. Filamentous and yeast keratitis had similar durations of infections and visual outcomes. Antifungal susceptibility testing influenced treatment in 80% of cases in which it was performed.

Corneal Culture in Infectious Keratitis: Effect of the Inoculation Method and Media on the Corneal Culture Outcome.

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: The standard method resulted in a significantly higher proportion of positive culture outcomes among the 94 included episodes of infectious keratitis (61%; 57/94) than the indirect inoculation method (44%; 41/94) (p = 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.

Assessment of Cidofovir for Treatment of Ocular Bovine Herpesvirus-1 Infection in Cattle Using an Ex-Vivo Model.

Bovine herpesvirus-1 (BoHV-1) infection contributes to keratoconjunctivitis, respiratory disease, and reproductive losses in cattle. The objective of this study was to determine the most appropriate ophthalmic antiviral agent for BoHV-1 inhibition using in-vitro culture and novel ex-vivo bovine corneal modeling. Half-maximal inhibitory concentrations of BoHV-1 were determined for cidofovir, ganciclovir, idoxuridine, and trifluridine via in-vitro plaque reduction assays. In-vitro cytotoxicity was compared amongst these compounds via luciferase assays. Trifluridine and cidofovir were the most potent BoHV-1 inhibitors in vitro, while trifluridine and idoxuridine were the most cytotoxic agents. Therefore, cidofovir was the most potent non-cytotoxic agent and was employed in the ex-vivo corneal assay. Corneoscleral rings (n = 36) from fresh cadaver bovine globes were harvested and equally divided into an uninfected, untreated control group; a BoHV-1-infected, untreated group; and a BoHV-1-infected, cidofovir-treated group. Virus isolation for BoHV-1 titers was performed from corneal tissue and liquid media. Histologic measurements of corneal thickness, epithelial cell density, and tissue organization were compared between groups. Substantial BoHV-1 replication was observed in infected, untreated corneas, but BoHV-1 titer was significantly reduced in cidofovir-treated (1.69 ± 0.08 × 103 PFU/mL) versus untreated (8.25 ± 0.25 × 105 PFU/mL, p < 0.0001) tissues by day 2 of culture. No significant differences in histologic criteria were observed between groups. In conclusion, cidofovir warrants further investigation as treatment for BoHV-1 keratoconjunctivitis, with future studies needed to assess in-vivo tolerability and efficacy.

Stability of Growth Factors in Autologous Serum Eyedrops After Long-Term Storage.

PURPOSE: The purpose of this study is to assess the stability of the growth factors (GF) in autologous serum eyedrops under different storage conditions. METHODS: The concentration of epidermal growth factor (EGF), transforming growth factor-ß (TGF-ß1), platelet-derived growth factor AB (PDGF-AB), and albumin was measured in fresh and defrosted samples of autologous serum under different storage conditions. The fresh and defrosted samples were cooled at 4 °C, and they were studied immediately after preparation, or after defrosting, and after 1, 2, 3, and 4 weeks. The concentration of GF was also assessed after 1, 3, 6, and 9 months at -20 °C. We also investigated how the different storage conditions influence the biological effects of autologous serum on conjunctival and corneal cell cultures. RESULTS: The concentration of EGF, TGF-ß1, PDGF-AB, and albumin remained stable over the 4 weeks at 4 °C, both in fresh and in defrosted samples. Likewise, no statistically significant differences were found between the GF concentration in fresh samples and after 1, 3, 6, and 9 months of freezing at -20 °C. Moreover, no differences were found on the cell proliferation and differentiation between cultured cells with fresh or defrosted samples after 4 weeks at 4 °C or after 1, 3, 6, or 9 months at -20 °C. CONCLUSIONS: Long-term storage of autologous serum eyedrops at -20 °C does not affect the concentration of GF, simplifies clinical logistics, and reduces the frequency of blood extractions from the patients.

TLR2 activation in corneal stromal cells by Staphylococcus aureus-induced keratitis.

The Toll-Like Receptor 2 (TLR2) plays an active and important role in Staphylococcus aureus-induced chronic ocular inflammation. The aim of this study was to investigate the expression and function of TLR2 of corneal stromal cells in ex vivo rabbit model of S. aureus keratitis. Corneal buttons with sclera rims placed in an ex vivo air-interface organ culture were assigned to two groups: corneas with epithelial and stromal abrasions. Each group was then divided into two sub-groups exposed to UV-killed S. aureus ATCC 6538P and S. aureus ATCC 29213, respectively. TLR2 and IL-8 mRNA expressions were analyzed by quantitative real-time RT-PCR. TLR2 localization was visualized by immunofluorescence analysis. The results demonstrated that TLR2 and IL-8 mRNA were significantly expressed in the stromal cells of the groups exposed to S. aureus strains. Moreover, it has been demonstrated that, after corneal injury, keratocytes differentiated into myofibroblasts became able to express TLR2 only when exposed to S. aureus. Identification of mechanisms regulation of corneal TLRs may lead to development of therapeutic interventions aimed at controlling corneal inflammation. This ex vivo model can be used to clarify the molecular events of bacterial-corneal tissue interactions and their inflammatory consequences.