Melville Matheson

In vitro antibiotic resistance in bacterial keratitis in London.

AIM To document changes in the profile of bacterial isolates from cases of keratitis and changes in their susceptibility to first line antibiotic therapies. METHODS A retrospective review was performed of all bacterial isolates from cases of keratitis seen between 1984 and 1999. In vitro laboratory susceptibilities to antibiotics were determined by the Kirby–Bauer disc diffusion method. The number of isolates, changes in the proportion of bacterial types, and the number that were fully resistant to monotherapy (ofloxacin), dual therapy (gentamicin and cefuroxime), and prophylactic treatment (chloramphenicol) were calculated. RESULTS There were 1312 bacterial isolates over 16 years. Gram positive bacteria accounted for 54.7% of isolates and Staphylococcusspecies (33.4%) were the most frequently isolated organisms. During the study period there has been an increase in the proportion ofPseudomonas species isolates but no overall increase in the proportion of Gram negative isolates. There has not been an increase in the proportion of isolates resistant to ofloxacin since 1995 or an increase in resistance to the combination of gentamicin and cefuroxime. However, since 1984 there has been a significant increase in proportion of Gram negative organisms resistant to chloramphenicol (p=0.0019). CONCLUSIONS An increase in the in vitro resistance of organisms to first line therapies for bacterial keratitis has not been observed. An increased resistance to chloramphenicol indicates that this drug is unlikely to provide prophylactic cover when Gram negative infection is a risk. Continued monitoring for the emergence of antibiotic resistance is recommended.

Persistently culture positive acanthamoeba keratitis: In vivo resistance and in vitro sensitivity

PURPOSE To characterize the risk factors, clinical course, treatment outcome and the association between in vivo resistance and in vitro sensitivity for subjects with persistently culture-positive Acanthamoeba keratitis. DESIGN Retrospective noncomparative case series. PARTICIPANTS Eleven subjects with repeatedly positive cultures for Acanthamoeba treated between January 1990 and December 2000, were reviewed. Only subjects with 2 or more positive cultures, availability of the clinical data, and availability of the last Acanthamoeba isolate were included in this study. METHODS The medical records were analyzed, and the last isolate from each case was tested in vitro for the antiamoebic drugs used clinically: polyhexamethylene biguanide (PHMB), chlorhexidine, propamidine and hexamidine. MAIN OUTCOME MEASURES Risk factors, the clinical outcome and in vitro cysticidal drug sensitivity assay. RESULTS Eleven subjects (11/180, 6.1%) had 2 or more positive cultures of whom 8 eyes of 8 subjects (8/180, 4.45%) were included in this study. Seven of eight (87%) subjects were diagnosed over 1 month from onset (late diagnosis). The most common presenting findings were diffuse stromal infiltrate (5/8, 62.5%), ring infiltrate (5/8, 62.5%), and corneal ulceration (3/8, 37.5%). The clinical course of the disease in all subjects consisted of recurrent episodes of corneal and scleral inflammation, with a mean duration of 13.4 +/- 9 months. All subjects received PHMB, and 5/8 (62.5%) chlorhexidine too; hexamidine was used in combination in 6/8 (75%), and propamidine in 1/8 (12.5%). All subjects had topical steroids, and 5/8 (62.5%) systemic immunosuppression. The disease resolved with corneal scarring in 3/8 (37.5%) subjects, corneal (or impending) perforation treated with therapeutic keratoplasty in 4/8 (50%), and enucleation in 1/8 (12.5%). Final visual acuity was 0.43 +/- 0.37. In vitro most isolates were resistant to propamidine, hexamidine was cysticidal in high concentrations, and PHMB and chlorhexidine had excellent sensitivity profiles. CONCLUSIONS In our large series of Acanthamoeba keratitis with a positive microbiologic diagnosis at presentation, nearly 5% developed recurrent episodes of corneal and scleral inflammation with viable Acanthamoeba in the cornea despite prolonged treatment with biguanides and/or diamidines. There was no correlation between in vitro drug sensitivities and the in vivo response for biguanides.

Demonstration of biofilm in infectious crystalline keratopathy using ruthenium red and electron microscopy

OBJECTIVE Bacterial biofilm formation has been implicated in the pathogenesis of infectious crystalline keratopathy. Biofilm cannot be visualized by electron microscopy without the addition of a fixative that stabilizes the polysaccharide-rich bacterial extracellular matrix that surrounds the bacterial colonies in a biofilm. We used ruthenium red as a fixative to evaluate corneal biopsy specimens for the presence of bacterial biofilm in three cases of infectious crystalline keratopathy (ICK) and five cases of chronic microbial keratitis without crystalline changes. DESIGN Case series with clinicopathologic correlation. PARTICIPANTS Eight patients underwent corneal biopsy or therapeutic keratoplasty as part of their management for chronic unresponsive microbial keratitis. METHODS The corneal specimens removed were trisected for microbiology, pathology, and transmission electron microscopy (TEM). The TEM specimens were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer with 0.05% ruthenium red. MAIN OUTCOME MEASURES Demonstration of bacterial biofilm with TEM. RESULTS TEM demonstrated organisms with a surrounding extracellular matrix consistent with a bacterial biofilm in the three cases of ICK but not in the five other cases of chronic microbial keratitis. CONCLUSIONS The presence of biofilm in ICK can be demonstrated with TEM with appropriate fixation techniques that stabilize the bacterial extracellular matrix. Biofilm stains intensely with periodic acid-Schiff because of the polysaccharide-rich extracellular matrix and weakly with Gram stain because of the high proportion of nonviable organisms. Biofilm formation occurs in ICK but probably not in chronic bacterial keratitis without crystalline changes. Secretion of an extracellular matrix by bacteria to form a biofilm is a response to a nutrient-deprived environment in which growth and replication is depressed. The extracellular matrix of the biofilm may mask bacterial antigens, explaining the relative lack of inflammatory response in these infections. It may also be one of the mechanisms explaining the resistance to in vivo antimicrobial therapy when in vitro sensitivities have been proven.

Long-term follow-up after submandibular gland transplantation in severe dry eyes secondary to cicatrizing conjunctivitis

PURPOSE To evaluate the long-term results of autologous submandibular gland transplantation in eyes with cicatrizing conjunctivitis and to determine biomechanical and biochemical features of the resulting salivary tear film. DESIGN Prospective, observational case series. METHODS Fifteen eyes with cicatrizing conjunctivitis with a viable autologous submandibular gland transplantation were compared with 10 eyes with cicatrizing conjunctivitis and a failed submandibular gland transplantation or no submandibular gland transplantation. Best-corrected visual acuity, frequency of tear substitute instillation, severity of dry eye discomfort, lid margin erythema, conjunctival hyperemia, corneal epithelial edema, tear film break-up time, Schirmer test results, and corneal fluorescein and conjunctival Rose Bengal staining were evaluated. In a subgroup central corneal thickness and sensitivity, corneal epithelial barrier function, conjunctival and lid margin flora, and conjunctival impression cytologic analysis results were evaluated. In 3 patients, preoperative and postoperative tear samples were analyzed for viscosity, surface tension, and presence of mucins. RESULTS Submandibular gland autotransplantation resulted in long-term improvement of subjective, objective, and some ocular surface parameters. Salivary mucins were detectable in salivary tears after submandibular gland transplantation. The viscosity of salivary tears was more similar to normal saliva and the surface tension was intermediate between the 2 original secretions. CONCLUSIONS Submandibular gland autotransplantation provides long-term relief from pain and reduces the need for frequent installation of lubricants.

Persistence of acanthamoeba antigen following acanthamoeba keratitis

AIM To investigate the hypothesis that persistent corneal and scleral inflammation following acanthamoeba keratitis is not always caused by active amoebic infection but can be due to persisting acanthamoebic antigens METHODS 24 lamellar corneal biopsy and penetrating keratoplasty specimens were obtained from 14 consecutive patients at various stages of their disease and divided for microscopy and culture. Histological sections were immunostained and screened for the presence ofAcanthamoeba cysts by light microscopy. Cultures were carried out using partly homogenised tissues on non-nutrient agar seeded with E coli. Clinical data were obtained retrospectively from the case notes of these patients. RESULTS Of the 24 specimens, 20 were obtained from eyes that were clinically inflamed at the time of surgery. Acanthamoeba cysts were present in 16 (80%) of these 20 specimens, while only five (25%) were culture positive. Acanthamoeba cysts were found to persist for up to 31 months after antiamoebic treatment. CONCLUSION These findings support the hypothesis thatAcanthamoeba cysts can remain in corneal tissue for an extended period of time following acanthamoeba keratitis and may cause persistent corneal and scleral inflammation in the absence of active amoebic infection. In view of these findings, prolonged intensive antiamoebic therapy may be inappropriate when the inflammation is due to retained antigen rather than to viable organisms

Biofilm formation in infectious crystalline keratopathy due to Candida albicans.

Infectious crystalline keratopathy (ICK) is an uncommon, indolent corneal infection in which the slow clinical course contrasts with the rapid laboratory growth and microbiological sensitivities of the infecting organism. This prospective study aimed to determine whether biofilm production was the cause of this disparity. A case of failed medical management of ICK in a patient with Stevens-Johnson syndrome is presented. A penetrating keratoplasty yielded corneal tissue that was freshly fixed for electron microscopy using 0.05% ruthenium red and 2.5% gluteraldehyde. Candida albicans was grown from 3/3 broths, and fungi with morphology consistent with Candida were seen on histological examination. Electron microscopy revealed microorganisms morphologically typical of Candida surrounded by a polysaccharide-rich glycocalyx consistent with a biofilm. We concluded that Candida albicans is capable of producing a biofilm and is a known cause of ICK. This case is supportive evidence that biofilm production is associated with cases of ICK and may explain the chronic, pauciinflammatory features of ICK and its relative resistance to antibiotic treatment.

Bacterial adherence and glycocalyx formation onunworn hydrogel lenses

Abstract Bacterial adherence and colonisation of hydrogel contact lenses may be a relevant factor in contact lens-related infections,particularly in users of disposable extended-wear contact lenses or of sterile contact lens care materials. The adherence of one strain of Pseudomonas aeruginosa to unworn hydrogel contact lenses was evaluated to elucidate the initial time course of adherence and to examine the effects of material charge. Adherence was evaluated using quantitative bacteriology and scanning electron microscopy (SEM). With 75% water content non-ionic contact lenses, bacterial adherence increased with time and reached a maximum after 30–60min. Organisms adhered in higher numbers to unworn non-ionic contact lenses compared with ionic lenses ( p SEM showed no preferential adherence of bacteria to lathing marks or surface defects. After 30min incubation, a thin fixed film wasvisible with SEM and adherent bacteria were associated with fibrous material. There was little further change in appearance with longer incubation times.

Persistently culture positive acanthamoeba keratitis

PURPOSE To characterize the risk factors, clinical course, treatment outcome and the association between in vivo resistance and in vitro sensitivity for subjects with persistently culture-positive Acanthamoeba keratitis. DESIGN Retrospective noncomparative case series. PARTICIPANTS Eleven subjects with repeatedly positive cultures for Acanthamoeba treated between January 1990 and December 2000, were reviewed. Only subjects with 2 or more positive cultures, availability of the clinical data, and availability of the last Acanthamoeba isolate were included in this study. METHODS The medical records were analyzed, and the last isolate from each case was tested in vitro for the antiamoebic drugs used clinically: polyhexamethylene biguanide (PHMB), chlorhexidine, propamidine and hexamidine. MAIN OUTCOME MEASURES Risk factors, the clinical outcome and in vitro cysticidal drug sensitivity assay. RESULTS Eleven subjects (11/180, 6.1%) had 2 or more positive cultures of whom 8 eyes of 8 subjects (8/180, 4.45%) were included in this study. Seven of eight (87%) subjects were diagnosed over 1 month from onset (late diagnosis). The most common presenting findings were diffuse stromal infiltrate (5/8, 62.5%), ring infiltrate (5/8, 62.5%), and corneal ulceration (3/8, 37.5%). The clinical course of the disease in all subjects consisted of recurrent episodes of corneal and scleral inflammation, with a mean duration of 13.4 +/- 9 months. All subjects received PHMB, and 5/8 (62.5%) chlorhexidine too; hexamidine was used in combination in 6/8 (75%), and propamidine in 1/8 (12.5%). All subjects had topical steroids, and 5/8 (62.5%) systemic immunosuppression. The disease resolved with corneal scarring in 3/8 (37.5%) subjects, corneal (or impending) perforation treated with therapeutic keratoplasty in 4/8 (50%), and enucleation in 1/8 (12.5%). Final visual acuity was 0.43 +/- 0.37. In vitro most isolates were resistant to propamidine, hexamidine was cysticidal in high concentrations, and PHMB and chlorhexidine had excellent sensitivity profiles. CONCLUSIONS In our large series of Acanthamoeba keratitis with a positive microbiologic diagnosis at presentation, nearly 5% developed recurrent episodes of corneal and scleral inflammation with viable Acanthamoeba in the cornea despite prolonged treatment with biguanides and/or diamidines. There was no correlation between in vitro drug sensitivities and the in vivo response for biguanides.