Assessment of the effect of oral clarithromycin on visual outcome following presumed bacterial endophthalmitis.

PURPOSE: The use of clarithromycin was assessed as a biofilm reducing agent in the management of bacterial endophthalmitis. METHODS: 84 eyes of 83 patients presenting with clinical signs highly suggestive of bacterial endophthalmitis were treated using a standard regimen of intraocular vancomycin, amikacin and systemic steroids, which in addition included oral clarithromycin. Ocular penetration of oral clarithromycin in healthy and inflamed eyes was also assessed. RESULTS: Comparing visual acuities at presentation and 6 months, 66% of patients demonstrated an improvement. Intraocular samples were culture positive in 58% of eyes. As compared to culture positive cases, more culture negative cases achieved a visual acuity of 6/12 or better (p = 0.0047). As compared to patients receiving the standard protocol but without clarithromycin, a greater number of culture negative cases demonstrated an improvement in vision of > or = + 6 Snellen lines (p = 0.023). The ocular penetration of clarithromycin into the anterior chamber of inflamed eyes appears sufficient to allow anti-biofilm activity against bacteria at the basic pH encountered in eyes with endophthalmitis. CONCLUSIONS: The ocular penetration of clarithromycin appears adequate for anti-biofilm activity in inflamed eyes. The beneficial effects of oral clarithromycin on visual outcome has been demonstrated in culture negative eyes with clinical signs highly suggestive of bacterial endophthalmitis. The final visual outcome for culture positive cases remains poor.

Risk factors for development of post-trabeculectomy endophthalmitis.

BACKGROUND/AIMS: Although adjunctive use of antiproliferative agents improves the success rate of glaucoma filtration surgery it profoundly alters the morphology of the filtering bleb. In view of these structural changes, which have been suggested to predispose to bleb infection, the relative importance of potential risk factors in the development of post-trabeculectomy endophthalmitis was investigated. METHODS: A case-control study was performed on patients with post-trabeculectomy endophthalmitis presenting to a single academic centre over a 6 1/2 year period. Cases were diagnosed by the combination of vitreous and aqueous inflammation occurring 4 or more weeks postoperatively with control patients chosen by selecting the three patients undergoing trabeculectomy immediately following each index case. RESULTS: Analysis of these data, derived from 23 cases and 69 controls, demonstrated that an episode of blebitis and the presence of diabetes mellitus were statistically significantly associated with subsequent endophthalmitis (odds ratios (OR) 11.8, 95% CI: 2.21-88.31, p = 0.003 and OR 4.51, 95 % CI 1.02-20.29, p = 0.04 respectively). The data also suggest an association exists between antiproliferative use and endophthalmitis (OR 3.3, 95% CI 0.95-15.19, p = 0.07) as the time interval between filtration surgery and development of endophthalmitis was significantly shorter in patients treated with antiproliferative agents (p = 0.001). CONCLUSIONS: These results provide strong evidence of an increased risk of late endophthalmitis in patients who have diabetes mellitus or have had an episode of blebitis and suggest antiproliferative agents may also have an important role.

PCR-based evidence of bacterial involvement in eyes with suspected intraocular infection.

PURPOSE: To assess the usefulness of polymerase chain reaction (PCR) in detection of bacteria in ocular samples. METHODS: Thirty-seven samples (aqueous and vitreous) were collected from 25 eyes showing typical symptoms and clinical signs of bacterial endophthalmitis. Ocular samples were also collected from 38 eyes that underwent routine surgery and from 15 eyes with intraocular inflammation due to nonbacterial causes. Panbacterial PCR was performed with a nested pair of 16S rRNA gene primers. Subsequent bacterial identification was completed for 18 paired samples (nine eyes) using restriction fragment length polymorphism (RFLP) and DNA sequencing. RESULTS: A 100% concordance was obtained between PCR and culture-positive samples. A PCR product was amplified from all 37 intraocular samples from eyes with suspected infection, whereas only 15 of 22 vitreous samples and 5 of 15 aqueous samples were culture positive. Culture-negative PCR-positive samples contained a preponderance of gram-negative bacterial sequences. Cloning and DNA analysis revealed 30 DNA sequences and included eight bacterial 16S rDNA, which currently remain unidentifiable. The presence of bacterial DNA was associated with an inflammatory response suggestive of infection and not colonization. All 15 samples from inflamed eyes with diverse uveitis diagnoses were PCR negative. The false-positive rate, due to contamination during sampling, was 5%. CONCLUSIONS: Bacterial DNA was detected in all patients with typical clinical signs of endophthalmitis. Gram-negative organisms seem to play a much more important role in the pathogenesis of this disease than previously thought. PCR-based techniques have great value in the confirmation of the diagnosis of bacterial endophthalmitis especially in culture-negative eyes.

Rapid detection and identification of Candida, Aspergillus, and Fusarium species in ocular samples using nested PCR.

A protocol for the rapid detection of fungal DNA in ocular samples, derived from three species, Candida albicans, Aspergillus fumigatus, and Fusarium solani, has been developed. Two novel panfungal primers complementary to 18S rRNA sequences present in all three species were designed. Panfungal PCR was followed by three nested PCRs utilizing species-specific primers. PCR sensitivity ranged from 50 to 100 fg of free DNA and between one and two C. albicans organisms. In addition, we also developed a rapid and reliable DNA extraction protocol. This protocol minimized DNA loss during extraction, whilst removing compounds from vitreous and aqueous fluids that have previously been shown to have inhibitory effects on PCR. Preliminary results obtained after testing the protocol on three patient samples support culture results and medical history. However, one patient was PCR positive but culture negative, suggesting that the sensitivity of this protocol may exceed that of traditional culture techniques. This system, therefore, constitutes an additional protocol that may significantly aid patient management in cases where fungal endophthalmitis is suspected.

PCR-RFLP-mediated detection and speciation of bacterial species causing endophthalmitis.

PURPOSE: To determine the usefulness of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in the identification and speciation of bacteria causing endophthalmitis. METHODS: PCR-RFLP was performed on 53 strains of 14 bacterial species (eight Gram positive and five Gram negative) collected from both keratitis and endophthalmitis patients. Two pairs of oligonucleotide primers based on the 16S rDNA gene were used to PCR-amplify 1.2- and 1.0-kb fragments of bacterial genomic DNA. RFLPs within the PCR product were used to speciate the organisms. RESULTS: The sensitivity of the nested PCR amplification reaction was one organism. All bacteria tested could be identified and speciated using RFLP analysis except for Escherichia coli and Serratia marcescens, which could not be interdifferentiated using RFLP. Molecular analysis of two vitreous samples from two eyes with typical signs of bacterial endophthalmitis confirmed the presence of E. coli in the vitreous from a culture-positive case with E. coli endophthalmitis and revealed the presence of Staphylococcus epidermidis in the vitreous of a culture-negative case. CONCLUSIONS: It is expected that this technique will provide a useful laboratory tool for future microbiologic diagnosis of patients presenting with endophthalmitis, especially for those eyes that prove culture negative.

Detection of and discrimination between gram-positive and gram-negative bacteria in intraocular samples by using nested PCR.

A nested PCR protocol has been developed for the detection of and discrimination between 14 species of gram-positive and -negative bacteria in samples of ocular fluids. First-round PCR with pan-bacterial oligonucleotide primers, based on conserved sequences of the 16S ribosomal gene, was followed by a gram-negative-organism-specific PCR, which resulted in a single 985-bp amplification product, and a multiplex PCR which resulted in two PCR products: a 1,025 bp amplicon (all bacteria) and a 355 bp amplicon (gram-positive bacteria only). All products were detected by gel electrophoresis. The sensitivity of the assay was between 10 fg and 1 pg of bacterial DNA, depending on the species tested, equivalent to between 24 and 4 live bacteria spiked in water. The identification was complete in 3.5 h. The molecular techniques were subsequently applied to four samples of intraocular fluid, (three vitreous and one aqueous) from three patients with clinical signs of bacterial endophthalmitis (test samples) and two samples of vitreous from a patient with chronic intraocular inflammation (control samples). In all culture-positive samples (two of three vitreous and one of one aqueous), a complete concordance was observed between molecular methods and culture results. PCR correctly identified the gram stain classification of the organisms. The bacterial etiology was also identified in a culture-negative patient with clinical history and signs highly suggestive of bacterial endophthalmitis. Furthermore, control samples from a patient with chronic intraocular inflammation remained PCR negative. In summary, this protocol has demonstrated potential as a rapid diagnostic test in confirming the diagnosis of infection and also determining the Gram status of bacteria with high specificity and sensitivity.

Polymerase chain reaction analysis of corneal epithelial and tear samples in the diagnosis of Acanthamoeba keratitis.

PURPOSE: Acanthamoeba is an uncommon cause of corneal infection in which the best visual outcome follows prompt diagnosis and a long course of appropriate antimicrobial therapy. Because conventional detection techniques for Acanthamoeba have certain limitations, we investigated the ability of the polymerase chain reaction (PCR) to confirm the clinical diagnosis of Acanthamoeba keratitis, with the ultimate aim of achieving early diagnosis. METHODS: Using two different pairs of primers, PCR was performed on representative cultured Acanthamoeba isolates to confirm the assay's ability to amplify Acanthamoeba DNA from a wide range of acanthamoebae. Subsequently, corneal epithelial samples from 19 patients and tear samples from 12 patients with Acanthamoeba keratitis were analyzed by PCR for the presence of Acanthamoeba DNA. RESULTS: Acanthamoeba DNA was amplified by PCR from 16 (84%) of 19 corneal epithelial samples, whereas Acanthamoeba was cultured from 10 samples (53%), all of which were PCR positive. Tear samples from 8 (66%) of 12 patients were positive on PCR testing, and one tear sample was PCR positive, whereas the corresponding epithelial biopsy had yielded a negative PCR result. Samples from culture-positive patients were positive on PCR testing more frequently than those from culture-negative patients (10/10 culture-positive corneal epithelial and 5/7 [71%] culture-positive initial tear samples versus 6/9 [66%] culture-negative corneal epithelial and 2/5 [40%] culture-negative tear samples). All control epithelial (n = 15) and tear (n = 15) samples yielded negative results. CONCLUSIONS: PCR was a more sensitive diagnostic test than a culture for Acanthamoeba keratitis, and the use of two different primers achieved better sensitivity than a single set. A PCR of a tear sample also may be a useful complementary test and, in combination with PCR of epithelial samples, would prove particularly helpful in confirming the clinical diagnosis in culture-negative cases.

Polymerase chain reaction and restriction fragment length polymorphism mediated detection and speciation of Candida spp causing intraocular infection.

PURPOSE: To determine the usefulness of polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis in the identification and speciation of Candida spp that causes ocular infection. METHODS: Oligonucleotide primers based on the cytochrome P450 L1 A1 demethylase gene were used to successfully amplify by PCR a single 1.0-kb and a single 500-bp DNA fragment from C. albicans, C. tropicalis, C. krusei, C. glabrata, C. parapsilosis, and C. pelliculosa genomic DNA. RFLPs within the PCR product were identified after restriction enzyme digestion. RESULTS: The sensitivity of the amplification reaction after two rounds of PCR was 10 fg genomic C. albicans DNA or one copy of the gene. No amplification product was obtained when DNA from C. guilliermondii, Aspergillus fumigatus, Fusarium solani, human leukocytes, or 10 species of bacteria was used as a template. Experiments with spiked normal vitreous demonstrated equal sensitivity as long as the volume of vitreous did not exceed 20% of the total PCR volume. RFLP analysis of the PCR product generated from each species obtained from the first- and second-round amplification products enabled species identification after digestion with specific endonucleases. Application of the technique to four clinical samples was successful. CONCLUSIONS: It is expected that the simplicity of the DNA extraction technique allied with the broad specificity of the outer primers for all ophthalmically relevant Candida spp and the sensitivity of the second-round PCR will aid in the detection of fungal DNA in small intraocular samples. PCR-RFLP analysis has great potential in the rapid detection and identification of Candida spp and in the provision of a useful laboratory tool for the future.

Enterobacter cloacae endophthalmitis: report of four cases.

Members of the genus Enterobacter are commensal organisms of the gastrointestinal tract and are considered pathogenic only for patients with lowered resistance to infection (e.g., chronic infection, cancer, or diabetes mellitus) or those with impaired immunity (congenital, acquired, or impaired immunity secondary to therapy). We report on four cases of endophthalmitis caused by Enterobacter cloacae: two in patients with acute postoperative endophthalmitis, one in a patient with delayed bleb-related endophthalmitis, and one in a patient presenting with presumed posttraumatic endophthalmitis. Each patient presented with severe disease many days after the onset of ocular symptoms, and two patients had systemic risk factors accounting for a reduced resistance to infection. Endophthalmitis caused by gram-negative bacilli is characterized by acute onset, rapid progression, and poor final visual outcome. Each of these patients was treated by a standard protocol with intravitreal, systemic, and topical antibiotics and systemic steroids. Despite treatment, the final visual outcomes for three of these patients was no perception of light, and that for one patient remained perception of hand movements only. In common with endophthalmitis caused by other gram-negative organisms, intraocular infection secondary to Enterobacter cloacae infection is a devastating disease which, despite treatment, results in extensive ocular damage and severe visual loss. Since 1966, only four cases of endophthalmitis secondary to infection with members of this genus have been reported. This report presents four cases which occurred over a period of 14 months and, to the best of our knowledge, the first case of bleb-related endophthalmitis secondary to E. cloacae infection.

Corneal infiltration after recurrent corneal epithelial erosion.

AIMS: To describe the clinical features of patients with a history of recurrent corneal epithelial erosion who develop acute corneal infiltration. METHODS: The records were reviewed of patients who had previously been examined and treated for recurrent corneal epithelial erosion and who presented again with signs suggestive of a microbial keratitis. RESULTS: 11 patients were described; one patient presented with similar signs on two occasions. There was typically a paracentral epithelial defect > 2 mm in diameter with an associated stromal infiltrate and an intense anterior uveitis. Three patients had a hypopyon, and four developed a subepithelial ring infiltrate. Samples were taken for microscopy and bacterial culture, with a positive isolate from two of 12 episodes (16%). Treatment with topical antibiotics and topical corticosteroid resulted in rapid re-epithelialisation and a reduction of inflammation. There was good visual outcome for all eyes, with a recurrence or symptoms of epithelial erosion in only one eye after a mean follow up period of 18 months. CONCLUSIONS: Corneal infiltrates are an uncommon complication of recurrent corneal epithelial erosion. Despite the intensity of the infiltration the majority are culture negative using established techniques. There is typically rapid resolution and a good visual outcome, with a tendency for the episode to mark the end of further symptoms of epithelial erosion.

The diagnosis of delayed post-operative endophthalmitis by polymerase chain reaction of bacterial DNA in vitreous samples.

Delayed post-operative endophthalmitis is a complication of modern cataract extraction and posterior chamber lens implantation. Propionibacterium acnes has been isolated in a few such cases but the majority are culture-negative, compounding surgical and medical management decisions. A method of detecting bacterial, and specifically P. acnes, DNA by the polymerase chain reaction (PCR) directed at 16S rDNA is reported. Nested PCR with universal eubacterial primers complimentary to regions of 16S rDNA conserved sequences detected 50 fg of bacterial DNA spike in normal vitreous. Nested PCR with P. acnes primers detected 10 fg of DNA. Vitreous samples from 29 patients undergoing vitrectomy for reasons unrelated to infection and 23 samples from 19 patients with delayed post-operative endophthalmitis were analysed. Four (14%) of 29 normal individuals and 17 (74%) of 23 delayed cases gave positive results with universal eubacterial primers. None of 29 and eight of 23 samples gave positive results with P. acnes primers. The 14% positive rate with universal primers in non-infected cases may limit their use in delayed post-operative endophthalmitis. PCR detection of bacterial DNA with specific primers from vitreous samples may prove a useful means of diagnosing delayed post-operative endophthalmitis and facilitating management decisions when conventional bacterial culture is negative.

Acanthamoeba keratitis. The value of early diagnosis.

BACKGROUND: The treatment of Acanthamoeba keratitis has been increasingly successful as diagnoses are made earlier. The authors investigated features of the disease and prognosis in a consecutive series of 15 patients who were treated within 1 month of initial symptoms. METHODS: A database of patients with Acanthamoeba infection presenting between March 1984 and March 1992 was analyzed. The recognition, presenting features, culture methods, results, and treatment of the early cases were reviewed to determine the reasons for a good outcome. RESULTS: Recognition depended on perineural infiltrates (11/15), uveitis (10/15), limbitis (14/15), and infiltrated epithelium; 6 of 15 patients had epithelial defects, but only 3 of 15 had ring infiltrates or ulcers. Epithelial biopsy was culture-positive in 12 of 15 patients. Most (11/15) patients needed only two anti-amebal drugs. One patient only required penetrating keratoplasty for uncontrolled disease. The final visual acuity was at least 6/12 in all patients who had been treated within 1 month of first symptoms, whereas only 17 (53%) of 32 eyes of patients who presented after 1 month achieved a visual acuity of 6/12. CONCLUSIONS: Subtle diagnostic signs, supported by comprehensive microbiologic investigation, justify the immediate instigation of specific antiamebal therapy. Treatment within 1 month of onset results in a lower morbidity and a good visual outcome.

Survey of the contamination of eyedrops of hospital inpatients and recommendations for the changing of current practice in eyedrop dispensing.

Topical ophthalmic medications used on the ward and from the outpatient area have been taken and cultured for potential bacterial contamination in the laboratory. We examined 143 bottles used by patients who had had routine cataract surgery and trabeculectomy. We also examined for bacterial contamination 216 bottles of eyedrops used in the outpatient area of the hospital. No contamination was found in the postoperative eyedrops, but five bottles were contaminated from the outpatient area (2.3%). The bacterial growth from outpatient drops was of the same order of magnitude as in previous studies. The practice in the UK for postoperative eyedrops to be discarded and fresh, separate bottles to take home is discussed. We recommend that this practice be changed so that the postoperative drops used for 72 hours or less are taken home.

Warning about bacterial contamination of therapeutic contact lenses.

Three of twenty-six soft therapeutic contact lenses were found to have bacterial contaminants in their original sealed manufacturers' containers. We wish to alert clinicians to this danger.