64 research outputs found
Ocular surface immune transcriptome and tear cytokines in corneal infection patients
BackgroundMicrobial keratitis is one of the leading causes of blindness globally. An overactive immune response during an infection can exacerbate damage, causing corneal opacities and vision loss. This study aimed to identify the differentially expressed genes between corneal infection patients and healthy volunteers within the cornea and conjunctiva and elucidate the contributing pathways to these conditions’ pathogenesis. Moreover, it compared the corneal and conjunctival transcriptomes in corneal-infected patients to cytokine levels in tears.MethodsCorneal and conjunctival swabs were collected from seven corneal infection patients and three healthy controls under topical anesthesia. RNA from seven corneal infection patients and three healthy volunteers were analyzed by RNA sequencing (RNA-Seq). Tear proteins were extracted from Schirmer strips via acetone precipitation from 38 cases of corneal infection and 14 healthy controls. The cytokines and chemokines IL-1β, IL-6, CXCL8 (IL-8), CX3CL1, IL-10, IL-12 (p70), IL-17A, and IL-23 were measured using an antibody bead assay.ResultsA total of 512 genes were found to be differentially expressed in infected corneas compared to healthy corneas, with 508 being upregulated and four downregulated (fold-change (FC) <−2 or > 2 and adjusted p <0.01). For the conjunctiva, 477 were upregulated, and 3 were downregulated (FC <−3 or ≥ 3 and adjusted p <0.01). There was a significant overlap in cornea and conjunctiva gene expression in patients with corneal infections. The genes were predominantly associated with immune response, regulation of angiogenesis, and apoptotic signaling pathways. The most highly upregulated gene was CXCL8 (which codes for IL-8 protein). In patients with corneal infections, the concentration of IL-8 protein in tears was relatively higher in patients compared to healthy controls but did not show statistical significance.ConclusionsDuring corneal infection, many genes were upregulated, with most of them being associated with immune response, regulation of angiogenesis, and apoptotic signaling. The findings may facilitate the development of treatments for corneal infections that can dampen specific aspects of the immune response to reduce scarring and preserve sight
Understanding clinical and immunological features associated with Pseudomonas and Staphylococcus keratitis
Reducing oral contamination during corneal scrapes
Aims: To identify potential contaminants of the corneal sampling procedure and examine the effect of wearing surgical face masks on the rate of contamination. Methods: Ten surgeons recited out loud a 30 s standardised script for corneal scraping with blood agar plates positioned 30 cm away from them. Three groups were identified: in group 1 a surgical mask was worn; group 2 had no mask worn; and group 3 had no mask but used agar plates pretreated with 5% povidone-iodine as a negative control. Each surgeon repeated the process 10 times for all groups, totalling 30 plates per surgeon and 300 plates for the experiment. All plates were masked and incubated aerobically at 37°C for 24 hours, and the number of colony forming units (CFUs) was determined. Results: At 24 hours, group 1 had a mean of 0.3 CFUs per surgeon; group 2 had 6.4 CFUs per surgeon and group 3 had 0.1 CFUs per surgeon. The difference between group 1 and group 2 was significant (p<0.001) whereas the difference between group 1 and group 3 was non-significant (p=0.4). Use of face masks decreased the number of plates with CFUs by 93% (from 29 to 2 plates) and decreased the total number of CFUs by 95% (from 63 to 3 CFUs). The most common microbiota identified was Streptococcus species. Conclusions: Oral bacterial microbiota may contaminate the slides and media used to collect samples during corneal sampling. Use of a face mask can significantly decrease the rate of contamination of such samples
Proteomic analysis of protein deposits on worn daily wear silicone hydrogel contact lenses
Purpose: Previous studies have demonstrated deposition of tear proteins onto worn contact lenses. In this study, we used proteomic techniques to analyze the protein deposits extracted from worn daily wear silicone hydrogel contact lenses in combination with different lens care solutions. Methods: Worn lenses were collected and protein deposits extracted using urea and surfactant. Protein extracts were desalted, concentrated, and then separated using one-dimensional gel electrophoresis. Individual protein components in extracts were identified using liquid chromatography combined with tandem mass spectrometry (LC-MS-MS) after trypsin digestion. Results: One-dimensional gel electrophoresis revealed that lysozyme and other small proteins (around 20 kDa) were the most abundant proteins in the extracts. LC-MS-MS revealed a wide array of proteins in lens extracts with lysozyme and lipocalin 1 being the most commonly identified in deposit extracts. Conclusions: Worn contact lenses deposit a wide array of proteins from tear film and other sources. Protein deposit profiles varied and were specific for each contact lens material.9 page(s
Zooming in on the intracellular microbiome composition of bacterivorous <i>Acanthamoeba</i> isolates
Acanthamoeba, a free-living amoeba in water and soil, is an emerging pathogen causing severe eye infection known as Acanthamoeba keratitis. In its natural environment, Acanthamoeba performs a dual function as an environmental heterotrophic predator and host for a range of microorganisms that resist digestion. Our objective was to characterize the intracellular microorganisms of phylogenetically distinct Acanthamoeba spp. isolated in Australia and India through directly sequencing 16S rRNA amplicons from the amoebae. The presence of intracellular bacteria was further confirmed by in situ hybridization and electron microscopy. Among the 51 isolates assessed, 41% harboured intracellular bacteria which were clustered into four major phyla: Pseudomonadota (previously known as Proteobacteria), Bacteroidota (previously known as Bacteroidetes), Actinomycetota (previously known as Actinobacteria), and Bacillota (previously known as Firmicutes). The linear discriminate analysis effect size analysis identified distinct microbial abundance patterns among the sample types; Pseudomonas species was abundant in Australian corneal isolates (P < 0.007), Enterobacteriales showed higher abundance in Indian corneal isolates (P < 0.017), and Bacteroidota was abundant in Australian water isolates (P < 0.019). The bacterial beta diversity of Acanthamoeba isolates from keratitis patients in India and Australia significantly differed (P < 0.05), while alpha diversity did not vary based on the country of origin or source of isolation (P > 0.05). More diverse intracellular bacteria were identified in water isolates as compared with clinical isolates. Confocal and electron microscopy confirmed the bacterial cells undergoing binary fission within the amoebal host, indicating the presence of viable bacteria. This study sheds light on the possibility of a sympatric lifestyle within Acanthamoeba, thereby emphasizing its crucial role as a bunker and carrier of potential human pathogens
Assessment of genotypes, endosymbionts and clinical characteristics of <i>Acanthamoeba</i> recovered from ocular infection
INTRODUCTION: Acanthamoeba is an emerging pathogen, infamous for its resilience against antiprotozoal compounds, disinfectants and harsh environments. It is known to cause keratitis, a sight-threatening, painful and difficult to treat corneal infection which is often reported among contact lens wearers and patients with ocular trauma. Acanthamoeba comprises over 24 species and currently 23 genotypes (T1-T23) have been identified. AIMS: This retrospective study was designed to examine the Acanthamoeba species and genotypes recovered from patients with Acanthamoeba keratitis (AK), determine the presence of endosymbionts in ocular isolates of Acanthamoeba and review the clinical presentations. METHODOLOGY: Thirteen culture-confirmed AK patients treated in a tertiary eye care facility in Hyderabad, India from February to October 2020 were included in this study. The clinical manifestations, medications and visual outcomes of all patients were obtained from medical records. The Acanthamoeba isolates were identified by sequencing the ribosomal nuclear subunit (rns) gene. Acanthamoeba isolates were assessed for the presence of bacterial or fungal endosymbionts using molecular assays, PCR and fluorescence in situ hybridization (FISH). RESULTS: The mean age of the patients was 33 years (SD ± 17.4; 95% CI 22.5 to 43.5 years). Six (46.2%) cases had AK associated risk factors; four patients had ocular trauma and two were contact lens wearers. A. culbertsoni (6/13, 46.2%) was the most common species, followed by A. polyphaga and A. triangularis. Most of the isolates (12/13) belonged to genotype T4 and one was a T12; three sub-clusters T4A, T4B, and T4F were identified within the T4 genotype. There was no significant association between Acanthamoeba types and clinical outcomes. Eight (61.5%) isolates harboured intracellular bacteria and one contained Malassezia restricta. The presence of intracellular microbes was associated with a higher proportion of stromal infiltrates (88.9%, 8/9), epithelial defect (55.6%, 5/9) and hypopyon (55.6%, 5/9) compared to 50% (2/4), 25% (1/4) and 25% (1/4) AK cases without intracellular microbes, respectively. CONCLUSIONS: Genotype T4 was the predominant isolate in southern India. This is the second report of T12 genotype identified from AK patient in India, which is rarely reported worldwide. The majority of the Acanthamoeba clinical isolates in this study harboured intracellular microbes, which may impact clinical characteristics of AK. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12879-022-07741-4
Comparison of culture, confocal microscopy and PCR in routine hospital use for microbial keratitis diagnosis.
To evaluate the sensitivity and specificity of polymerase chain reaction (PCR), in vivo confocal microscopy (IVCM) and culture for microbial keratitis (MK) diagnosis. Retrospective review of PCR, IVCM and culture results for MK diagnosis at Moorfields Eye Hospital between August 2013 and December 2014. Results PCR results were available for 259 MK patients with concurrent culture for 203/259 and IVCM for 149/259. Sensitivities and specificities with 95% confidence intervals [95% CI] were calculated for Acanthamoeba keratitis (AK) and fungal keratitis (FK), by comparison with culture, for both IVCM and PCR. For AK, FK and bacterial keratitis (BK) sensitivities were calculated, for each diagnostic method, by comparison with a composite reference standard (a positive result for one or more of culture, PCR or IVCM having a specificity of 100% by definition). For the latter, sensitivities with [95% CI] were: for AK, IVCM 77.1% [62.7-88.0%], PCR 63.3% [48.3-76.6%], culture 35.6 [21.9-51.2]; for FK, IVCM 81.8% [48.2-97.7%], PCR 30.8% [9.09-61.4%], culture 41.7% [15.2-72.3%]; for BK, PCR 25.0% [14.7-37.9%], culture 95.6% [87.6-99.1%]. Conclusion IVCM was the most sensitive technique for AK and FK diagnosis but culture remains our gold standard for BK. These findings reflect results to be expected from service providers to UK ophthalmology units and demonstrates the need at our centre for ongoing diagnostic result audit leading to the potential to improve PCR diagnosis. Both FK and AK are now common in the UK; ophthalmology units need to have all these techniques available to optimise their MK management
Identification and quantification of <i>Acanthamoeba</i> spp. within seawater at four coastal lagoons on the east coast of Australia
Acanthamoeba is an opportunistic free-living heterotrophic protist that is the most predominant amoeba in diverse ecological habitats. Acanthamoeba causes amoebic keratitis (AK), a painful and potentially blinding corneal infection. Major risk factors for AK have been linked to non-optimal contact lens hygiene practices and Acanthamoeba contamination of domestic and recreational water. This study investigated the incidence and seasonal variation of Acanthamoeba spp. within coastal lagoons located on the eastern coast of Australia and then examined the association between Acanthamoeba and water abiotic factors and bacterial species within the water.Water samples were collected from four intermittently closed and open lagoons (ICOLLs) (Wamberal, Terrigal, Avoca and Cockrone) every month between August 2019 to July 2020 except March and April. qPCR was used to target the Acanthamoeba 18S rRNA gene, validated by Sanger sequencing. Water abiotic factors were measured in situ using a multiprobe metre and 16S rRNA sequencing (V3-V4) was performed to characterise bacterial community composition. Network analysis was used to gauge putative associations between Acanthamoeba incidence and bacterial amplicon sequence variants (ASVs).Among 206 water samples analysed, 79 (38.3%) were Acanthamoeba positive and Acanthamoeba level was significantly higher in summer compared with winter, spring, or autumn (p = 0.008). More than 50% (23/45) water samples of Terrigal were positive for Acanthamoeba which is a highly urbanised area with extensive recreational activities while about 32% (16/49) samples were positive from Cockrone that is the least impacted lagoon by urban development. All sequenced strains belonged to the pathogenic genotype T4 clade except two which were of genotype clades T2 and T5. Water turbidity, temperature, intl1 gene concentration, and dissolved O2 were significantly associated with Acanthamoeba incidence (p < 0.05). The ASVs level of cyanobacteria, Pseudomonas spp., Candidatus spp., and marine bacteria of the Actinobacteria phylum and Acanthamoeba 18S rRNA genes were positively correlated (Pearson's r ≥ 0.14). The presence of Acanthamoeba spp. in all lagoons, except Wamberal, was associated with significant differences in the composition of bacterial communities (beta diversity).The results of this study suggest that coastal lagoons, particularly those in urbanised regions with extensive water recreational activities, may pose an elevated risk to human health due to the relatively high incidence of pathogenic Acanthamoeba in the summer. These findings underscore the importance of educating the public about the rare yet devastating impact of AK on vision and quality of life, highlighting the need for collaborative efforts between public health officials and educators to promote awareness and preventive measures, especially focusing lagoons residents and travellers
- …