R. P. Kowalski

A 5-year evaluation of the adenoclone test for the rapid diagnosis of adenovirus from conjunctival swabs.

The rapid diagnosis of adenoviral ocular infections affords the opportunity to limit the transmission of virus within the community and avoid expensive, unnecessary, and ineffective therapy. This study evaluated the results of a 5-year experience with the Adenoclone test (Cambridge Biotech, Worcester, MA), an enzyme immunoassay, applied directly to conjunctival swabs obtained from infected eyes. The sensitivity of this test was determined on 372 consecutive adenovirus culture-positive ocular specimens. A subset of 106 specimens was evaluated, including a retrospective chart review to determine the relationship between the Adenoclone result and the time to viral cytopathic effect (CPE) in A549 cell culture, ocular titers (90% tissue culture infectious dose; TCID90), serotype, and associated clinical parameters. Overall, the sensitivity for Adenoclone was 38% (142 of 372), which improved to 65% (129 of 199) for samples positive in culture during the first week. A positive Adenoclone test result was associated with a shorter time to CPE in cell culture (p=0.0001). The mean ocular titers (log TCID90) associated with a positive test result were found to be at a significantly higher dilution than a negative result (-1.70 ± 0.93 vs. -0.88 ± 1.00, p<0.0001). A positive Adenoclone outcome was independent of the serotype but directly associated with a recent visit to an ophthalmologists office, follicular conjunctivitis, and conjunctival chemosis. For the rapid diagnosis of adenoviral ocular infections, the Adenoclone test remains useful, but a more sensitive test based on nonradioactive amplification is eagerly anticipated.

Adenoviral Ocular Isolates Demonstrate Serotype-Dependent Differences in In Vitro Infectivity Titers and Clinical Course

The purpose of this study was to evaluate clinical ocular adenoviral isolates for differences among and within serotypes with respect to in vitro infectivity titers and clinical course. The study design included a retrospective chart review and the determination of in vitro infectivity titers (TCIDCKJS) of 90 clinical ocular isolates of various adenoviral serotypes. Adenovirus serotype 8 (AD8) was recovered in significantly greater numbers of patients in the second week of infection compared to all other serotypes (p<.002). AD3 and AD4 presented with the highest infectivity titers during the first week of acute infection. Up to 4 logs of variation was demonstrated in TCIDgoS among isolates of the same serotype. Among the clinical parameters studied, eyelid edema was significantly more common among AD8-infected patients as compared to all other serotypes (p<0.04). For the first time, specific, but limited serotype differences with respect to infectivity titers and clinical course were demonstrated for adenoviral ocular isolates. Important variations in isolate virulence within a given serotype were also observed.

Diphosphonium Ionic Liquids as Broad Spectrum Antimicrobial Agents

Purpose: One of the most disturbing trends in recent years is the growth of resistant strains of bacteria with the simultaneous dearth of new antimicrobial agents. Thus, new antimicrobial agents for the use on the ocular surface are needed. Methods: We synthesized a variety of ionic liquid compounds, which possess 2 positively charged phosphonium groups separated by 10 methylene units in a “bola”-type configuration. We tested these compounds for antimicrobial activity versus a variety of ocular pathogens, as well as their cytoxicity, in vitro in a corneal cell line and in vivo in mice. Results: The ionic liquid Di-Hex C10 demonstrated broad in vitro antimicrobial activity at low micromolar concentrations versus gram-negative and gram-positive organisms, including methicillin-resistant Staphylococcus aureus strains and ocular fungal pathogens. Treatment with Di-Hex C10 resulted in bacterial killing in as little as 15 minutes in vitro. Di-Hex C10 showed little cytotoxicity at 1 &mgr;M versus a corneal epithelial cell line or at 10 &mgr;M in a mouse corneal wound model. We also show that this bis-phosphonium ionic liquid structure is a key because a comparable monophosphonium ionic liquid is cytotoxic to both bacteria and corneal epithelial cells. Conclusions: Here, we report the first use of dicationic bis-phosphonium ionic liquids as antimicrobial agents. Our data suggest that diphosphonium ionic liquids may represent a new class of broad-spectrum antimicrobial agents for the use on the ocular surface.

Lomefloxacin is an effective treatment of experimental bacterial keratitis.

Purpose. Lomefloxacin was evaluated as a potential topical therapy for bacterial keratitis. Methods. Lomefloxacin was compared with ciprofloxacin in different rabbit keratitis models. A total of 216 corneas were infected with Staphylococcus aureus (ciprofloxacin-susceptible and -resistant), Streptococcus viridans, Streptococcus pneumoniae, Pseudomonas aeruginosa, and Serratia marcescens and were treated with lomefloxacin (0.3%), ciprofloxacin (0.3% Ciloxan), and the control phosphate-buffered saline (PBS), respectively. The data were analyzed statistically comparing the decrease in the number of recovered viable bacteria. Results. Compared with PBS-treated control corneas, the colony counts for all bacterial isolates were significantly reduced (p < 0.05) after topical treatment with either lomefloxacin or ciprofloxacin. For Gram-positive bacteria, lomefloxacin and ciprofloxacin were equally effective. For Gram-negative bacteria, lomefloxacin, while effective, was less so than ciprofloxacin under experimental conditions (p < 0.05). Conclusion. Our data, using multiple bacterial keratitis models, suggest that lomefloxacin is promising for therapy of bacterial keratitis. Further clinical studies are needed to expand its use for keratitis therapy.

A Novel Cell-Associated Protection Assay Demonstrates the Ability of Certain Antibiotics To Protect Ocular Surface Cell Lines from Subsequent Clinical Staphylococcus aureus Challenge

ABSTRACT In vivo effectiveness of topical antibiotics may depend on their ability to associate with epithelial cells to provide continued protection, but this contribution is not measured by standard antibiotic susceptibility tests. We report a new in vitro method that measures the ability of test antibiotics azithromycin (AZM), erythromycin (ERY), tetracycline (TET), and bacitracin (BAC) to associate with mammalian cells and to protect these cells from destruction by bacteria. Mammalian cell lines were grown to confluence using antibiotic-free medium and then incubated in medium containing a single antibiotic (0 to 512 μg/ml). After incubation, the cells were challenged with Staphylococcus aureus ocular isolates, without antibiotics added to the culture medium. Epithelial cell layer integrity was assessed by gentian violet staining, and the minimum cell layer protective concentration (MCPC) of an antibiotic sufficient to protect the mammalian cells from S. aureus was determined. Staining was also quantified and analyzed. Bacterial viability was determined by culture turbidity and growth on agar plates. Preincubation of Chang and human corneal limbal epithelial cells with AZM, ERY, and TET at ≥64 μg/ml provided protection against AZM-susceptible S. aureus strains, with increasing protection at higher concentrations. TET toxicity was demonstrated at >64 μg/ml, whereas AZM displayed toxicity to one cell line at 512 μg/ml. BAC failed to show consistent protection at any dose, despite bacterial susceptibility to BAC as determined by traditional antibiotic susceptibility testing. A range of antibiotic effectiveness was displayed in this cell association assay, providing data that may be considered in addition to traditional testing when determining therapeutic dosing regimens.

Zymar (Gatifloxacin 0.3%) shows excellent Gram-negative activity against Serratia marcescens and Pseudomonas aeruginosa in a New Zealand White rabbit keratitis model.

Purpose: Whereas gatifloxacin, a newer fluoroquinolone, was engineered to increase its Gram-positive potency, we assessed whether it still retained significant Gram-negative activity in vivo. Specifically, we compared the efficacy of Zymar (gatifloxacin 0.3%), Ciloxan (ciprofloxacin 0.3%), and fortified tobramycin (14 mg/mL) in the treatment of experimental Gram-negative bacterial infections of Serratia marcescens (SM) and Pseudomonas aeruginosa (PA) in the New Zealand White (NZW) rabbit keratitis model. Methods: A total of 30 NZW rabbits each were intrastromally inoculated in both eyes with ∼1000 CFU of SM and PA. By E-test, the minimum inhibitory concentrations (MICs; μg/mL) for SM were gatifloxacin (0.125), ciprofloxacin (0.047), and tobramycin (1.5), and for PA were gatifloxacin (0.125), ciprofloxacin (0.19), and tobramycin (0.5). After 16 hours, the rabbits were divided into 4 treatment groups: (1) Zymar, (2) Ciloxan, (3) fortified tobramycin, and (4) saline control. One drop was instilled in both eyes every 15 minutes for 5 doses and then every 30 minutes for 14 doses. One hour after the final treatment, the animals were euthanized, and bacterial colony counts from the corneas were determined. Results: For SM, Zymar and Ciloxan significantly reduced (P < 0.001, ANOVA) the colony counts compared with tobramycin and saline control. Zymar was more effective than Ciloxan (P < 0.001, ANOVA). For PA, all antibiotics reduced equivalently the colony counts compared with the saline control (P = 0.005, ANOVA). Conclusions: The enhanced Gram-positive activity of gatifloxacin is not associated with any decreased Gram-negative activity in vivo. Zymar may prove useful for SM and PA keratitis.

An antimicrobial peptide can enhance the activity of a fluoroquinolone in reducing the colony counts of fluoroquinolone-resistant MRSA in the NZW rabbit keratitis model

Purpose Future therapy of ocular infections may depend on enhancing current drugs if no new drugs are developed. We tested whether an antimicrobial peptide, Nisin (NIS) could enhance the activity of ciprofloxacin (CIP) in a NZW rabbit keratitis model.

Animal models for the treatment of bacterial keratitis

Rabbit models of bacterial keratitis have been used to evaluate the efficacy of anti-infectives in the clinical treatment of bacterial keratitis. These models can determine: 1) ocular toxicity and tolerance of anti-infectives to ocular tissue, 2) penetration of anti-infectives into the cornea, and 3) anti-bacterial efficacy of the anti-infectives to corneal bacterial pathogens. The current presentation will cover the structure and limitations of rabbit bacterial keratitis modeling using published data. Topics will include statistical design, the choice of bacterial pathogens, and positive aspects for possible systemic anti-infective development.