Corneal Culture in Infectious Keratitis: Effect of the Inoculation Method and Media on the Corneal Culture Outcome.
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ABSTRACT: 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.
SUBMITTER: Sagerfors S
PROVIDER: S-EPMC8122416 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BACKGROUND:A worldwide lack of donor corneas demands the bioengineered corneas be developed as an alternative. The primary objective of the current study was to evaluate the efficacy of acellular porcine corneal stroma (APCS) transplantation in various types of infectious keratitis and identify risk factors that may increase APCS graft failure. METHODS:In this prospective interventional study, 39 patients with progressive infectious keratitis underwent therapeutic lamellar keratoplasty using APCS and were followed up for 12 months. Data collected for analysis included preoperative characteristics, visual acuity, graft survival and complications. Graft survival was evaluated by the Kaplan-Meier method and compared with the log-rank test. RESULTS:The percentage of eyes that had a visual acuity of 20/40 or better increased from 10.3% preoperatively to 51.2% at 12 months postoperatively. Twelve patients (30.8%) experienced graft failure within the follow-up period. The primary reasons given for graft failure was noninfectious graft melting (n = 5), and the other causes included recurrence of primary infection (n = 4) and extensive graft neovascularization (n = 3). No graft rejection was observed during the follow-up period. A higher relative risk (RR) of graft failure was associated with herpetic keratitis (RR = 8.0, P = 0.046) and graft size larger than 8 mm (RR = 6.5, P < 0.001). CONCLUSIONS:APCS transplantation is an alternative treatment option for eyes with medically unresponsive infectious keratitis. Despite the efficacy of therapeutic lamellar keratoplasty with APCS, to achieve a good prognosis, restriction of surgical indications, careful selection of patients and postoperative management must be emphasized. Trial registration Prospective Study of Deep Anterior Lamellar Keratoplasty Using Acellular Porcine Cornea, NCT03105466. Registered 31 August 2016, ClinicalTrails.gov.
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Project description:For the past 150 years, bacteria have been investigated primarily in liquid batch cultures. Contrary to most expectations, these cultures are not homogeneous mixtures of single-cell bacteria, because free-floating bacterial aggregates eventually develop in most liquid batch cultures. These aggregates share characteristics with biofilms, such as increased antibiotic tolerance. We investigated how aggregates develop and what influences this development in liquid batch cultures of Pseudomonas aeruginosa We focused on how the method of inoculation affected aggregation by assessing aggregate frequency and size using confocal laser scanning microscopy. Several traditional methods of initiating an overnight bacterial culture, i.e., inoculation directly from frozen cultures, inoculation using agar-grown cells, or inoculation using cells grown in liquid cultures, were investigated. We discovered a direct link between the inoculation method and the size and frequency of biofilm aggregates in liquid batch cultures, with inoculation directly from a plate resulting in the most numerous and largest aggregates. These large aggregates had an overall impact on the cultures' subsequent tolerance toward tobramycin, indicating that the inoculation method has a profound impact on antibiotic tolerance. We also observed a mechanism whereby preformed aggregates recruited single cells from the surrounding culture in a "snowball effect," building up aggregated biomass in the culture. This recruitment was found to rely heavily on the exopolysaccharide Psl. Additionally, we found that both Escherichia coli and Staphylococcus aureus produced aggregates in liquid batch cultures. Our results stress the importance of inoculation consistency throughout experiments and the substantial impact aggregate development in liquid batch cultures may have on the outcomes of microbiological experiments.IMPORTANCE Pure liquid cultures are fundamental to the field of microbiological research. These cultures are normally thought of as homogeneous mixtures of single-cell bacteria; the present study shows that this is not always true. Bacteria may aggregate in these liquid cultures. The aggregation can be induced by the method chosen for inoculation. The presence of aggregates can significantly change the outcomes of experiments by altering the phenotype of the cultures. The study found a mechanism whereby preformed aggregates are able to recruit surrounding single cells in a form of snowball effect, creating more and larger aggregates in the cultures. Once formed, these aggregates are hard to remove. Aggregates in liquid cultures may be an immense unseen challenge for microbiologists.
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Project description:PurposeTo examine the clinical presentation, management, and outcomes of culture and polymerase chain reaction (PCR) negative cases of infectious keratitis.MethodsIn this retrospective case series, we evaluated the laboratory and medical records of culture- and PCR-negative cases (2016-2020) reported to a tertiary care center, which were presumed to be infectious keratitis on the basis of clinical history and presentation.ResultsA total of 121 cases with culture-negative keratitis were included in this study. The mean age of the patients was 48.42 ± 1.89 years, and 53.72% were female. At presentation, the presumed etiology was viral in 38.01%, bacterial in 27.27%, fungal in 8.26%, Acanthamoeba in 6.61%, and unlisted in 28.92% of cases. The most common risk factors were a previous history of ocular surface diseases (96.69%) and contact lens use (37.19%). In total, 61.98% of the patients were already on antimicrobial medication at presentation. The initial management was altered in 79 cases (65.29%) during the treatment course. Average presenting and final (post-treatment) visual acuities (VA) were 0.98 ± 0.04 (LogMAR) and 0.42 ± 0.03 (LogMAR), respectively. A significantly higher frequency of patients with a final VA worse than 20/40 (Snellen) had worse VA at initial presentation (p < 0.0001). A history of ocular surface disease, cold sores, and recurrent infection (p < 0.05) were more commonly associated with a presumed diagnosis of viral keratitis. The patients with presumed bacterial etiology were younger and had a history of poor contact lens hygiene (p < 0.05).ConclusionsWe observed a distinct difference in clinical features among patients with culture-negative and PCR-negative keratitis managed for presumed viral and bacterial infections. Although there was significant variability in presentation and management duration in this cohort, the visual outcomes were generally favorable.
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Project description:PurposeTo investigate the development of corneal neovascularization, the corneal expression of vascular endothelial growth factor (VEGF), and the antiangiogenic effects of a VEGF-inhibitory antibody during experimental keratomycosis.MethodsScarified corneas of BALB/c mice were topically inoculated with Candidaalbicans and monitored daily for corneal neovascularization. A murine gene microarray compared infected corneas to controls 1 day after inoculation. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) determined levels of genes encoding VEGF-A, VEGF-B, VEGF-C, and VEGF-D and placental growth factor in infected, mock-inoculated, and normal corneas. Immunostaining localized VEGF-A in corneal sections. An anti-VEGF-A antibody that binds to murine VEGF was evaluated for effects on corneal neovascularization and fungal recovery.ResultsEyes with C. albicans keratitis manifested limbal capillary budding on the second postinoculation day, and intrastromal neovascular tufts subsequently grew at a mean rate of 250+/-80 microm/day. One day after the onset of C. albicans keratitis, VEGF-A was upregulated 12.5 fold (p=0.01) by microarray and 8.8 fold (p=0.004) by real-time RT-PCR, followed by a measured decline toward baseline over one week. VEGF-A was present in the epithelium and stroma of infected corneas. Scarification alone did not alter VEGF expression compared to the normal cornea. Anti-VEGF-A antibody significantly (p<0.01) decreased the formation of new corneal blood vessels during experimental keratomycosis without adversely affecting the fungal load of C. albicans keratitis.ConclusionsUntreated C. albicans keratitis induces VEGF-A and leads to progressive corneal neovascularization that is preventable by a VEGF-blocking antibody.
| S-EPMC2756518 | biostudies-literature
Project description:MicroRNAs (miRNAs) are small, stable non-coding RNA molecules with regulatory function and marked tissue specificity that post-transcriptionally regulate gene expression, however their role in fungal keratitis remain unknown. Our purpose was to identify the miRNAs in human cornea from fungal keratitis patients and understand their key role in regulation of pathogenesis. Corneal samples from normal cadaver (n=3) and fungal keratitis (n=5) patients were pooled separately and total RNA was extracted. Deep sequencing was done using Illumina HiSeq1000 platform to identify miRNA profile. We identified seventy five differentially expressed miRNAs in fungal keratitis corneas. Select miRNAs were validated by real-time RT-PCR (Q-PCR). We predicted their role in regulating target genes in several pathways by combining miRNA target genes and pathway analysis, and mRNA expression of select target genes were further analysed by Q-PCR. MiR-21-5p, miR-223-3p, miR-146b-5p, miR-155-5p, miR-511-5p were found to be involved in inflammatory and immune responses, regulating Toll like receptor signaling pathways, which is of particular interest. MiR-451a with an increased expression in keratitis may have a role in wound healing by targeting Macrophage Migration Inhibitory Factor (MIF). Further, we highlighted that Neurotrophin signaling pathway may play a role in wound healing process. One novel miRNA was also detected in cornea. In conclusion, several miRNAs with high expression in fungal keratitis corneas point towards their role in regulation of pathogenesis. Further insights in understanding miRNAs role in wound healing and inflammation may help design new therapeutic strategies.
2016-07-28 | GSE64843 | GEO
Project description:Bacterial keratitis results in corneal scarring and subsequent visual impairment. The long-term evolution of corneal scars has not been well described. In this case series, we identified patients who had improvement in corneal scarring and visual acuity from a clinical trial for bacterial keratitis.We searched the records of the Steroids for Corneal Ulcers Trial (SCUT) for patients who had improvement in vision between the 3-month and 12-month visits and reviewed their clinical photographs.Of the 500 patients enrolled in SCUT, five patients with large central corneal scars due to bacterial keratitis are presented. All experienced improvement in rigid contact lens-corrected visual acuity from months 3 to 12. All patients also had marked improvement in corneal opacity during the same time period. None of the patients opted to have penetrating keratoplasty.Corneal scars may continue to improve even many months after a bacterial corneal ulcer has healed. The corneal remodeling can be accompanied by considerable improvement in visual acuity, such that corneal transplantation may not be necessary.
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