IL-17A-mediated protection against Acanthamoeba keratitis.

Acanthamoeba keratitis (AK) is a very painful and vision-impairing infection of the cornea that is difficult to treat. Although past studies have indicated a critical role of neutrophils and macrophages in AK, the relative contribution of the proinflammatory cytokine, IL-17A, that is essential for migration, activation, and function of these cells into the cornea is poorly defined. Moreover, the role of the adaptive immune response, particularly the contribution of CD4(+) T cell subsets, Th17 and regulatory T cells , in AK is yet to be understood. In this report, using a mouse corneal intrastromal injection-induced AK model, we show that Acanthamoeba infection induces a strong CD4(+) T effector and regulatory T cell response in the cornea and local draining lymph nodes. We also demonstrate that corneal Acanthamoeba infection induces IL-17A expression and that IL-17A is critical for host protection against severe AK pathology. Accordingly, IL-17A neutralization in Acanthamoeba-infected wild-type mice or Acanthamoeba infection of mice lacking IL-17A resulted in a significantly increased corneal AK pathology, increased migration of inflammatory cells at the site of inflammation, and a significant increase in the effector CD4(+) T cell response in draining lymph nodes. Thus, in sharp contrast with other corneal infections such as herpes and Pseudomonas aeruginosa keratitis where IL-17A exacerbates corneal pathology and inflammation, the findings presented in this article suggest that IL-17A production after Acanthamoeba infection plays an important role in host protection against invading parasites.

Failure of molecular diagnostics of a keratitis-inducing Acanthamoeba strain.

An otherwise healthy 49-year-old female patient presented at the local hospital with severe keratitis in both inflamed eyes. She was a contact lens wearer and had no history of a corneal trauma. In our laboratory for medical parasitology Acanthamoebae were detected microscopically from the cornea scraping and from the fluid of the contact lens storage case after xenical culture and showed the typical cyst morphology of Acanthamoebae group II. The diagnosis of "Acanthamoeba keratitis" was established and successful therapy was provided. While the morphological microscopic method led to the correct diagnosis in this case, an in-house multiplex qPCR and a commercial qPCR showed false negative results regarding Acanthamoeba sp. The subsequent sequencing revealed the Acanthamoeba genotype T4. In the present case report, the inability to detect Acanthamoebae using qPCR only is presented. Therefore, we recommend the utilization of combined different assays for optimal diagnostic purposes.

Human Conjunctival Transcriptome in Acanthamoeba Keratitis: An Exploratory Study.

PURPOSE: The purpose of this study was to identify conjunctival transcriptome differences in patients with Acanthamoeba keratitis compared with keratitis with no known associated pathogen. METHODS: The host conjunctival transcriptome of 9 patients with Acanthamoeba keratitis (AK) is compared with the host conjunctival transcriptome of 13 patients with pathogen-free keratitis. Culture and/or confocal confirmed Acanthamoeba in 8 of 9 participants with AK who underwent metagenomic RNA sequencing as the likely pathogen. Cultures were negative in all 13 cases where metagenomic RNA sequencing did not identify a pathogen. RESULTS: Transcriptome analysis identified 36 genes differently expressed between patients with AK and patients with presumed sterile, or pathogen-free, keratitis. Gene enrichment analysis revealed that some of these genes participate in several biologic pathways important for cellular signaling, ion transport and homeostasis, glucose transport, and mitochondrial metabolism. Notable relatively differentially expressed genes with potential relevance to Acanthamoeba infection included CPS1 , SLC35B4 , STEAP2 , ATP2B2 , NMNAT3 , and AKAP12 . CONCLUSIONS: This research suggests that the local transcriptome in Acanthamoeba keratitis may be sufficiently robust to be detected in the conjunctiva and that corneas infected with Acanthamoeba may be distinguished from the inflamed cornea where no pathogen was identified. Given the low sensitivity for corneal cultures, identification of differentially expressed genes may serve as a suggestive transcriptional signature allowing for a complementary diagnostic technique to identify this blinding parasite. Knowledge of differentially expressed genes may also direct investigation of disease pathophysiology and suggest novel pathways for therapeutic targets.

Innate and Adaptive Gene Single Nucleotide Polymorphisms Associated With Susceptibility of Severe Inflammatory Complications in Acanthamoeba Keratitis.

Purpose: Over a third of patients with Acanthamoeba keratitis (AK) experience severe inflammatory complications (SICs). This study aimed to determine if some contact lens (CL) wearers with AK were predisposed to SICs due to variations in key immune genes. Methods: CL wearers with AK who attended Moorfields Eye Hospital were recruited prospectively between April 2013 and October 2014. SICs were defined as scleritis and/or stromal ring infiltrate. Genomic DNA was processed with an Illumina Low Input Custom Amplicon assay of 58 single nucleotide polymorphism (SNP) targets across 18 genes and tested for association in PLINK. Results: Genomic DNA was obtained and analyzed for 105 cases of AK, 40 (38%) of whom experienced SICs. SNPs in the CXCL8 gene encoding IL-8 was significantly associated with protection from SICs (chr4: rs1126647, odds ratio [OR] = 0.3, P = 0.005, rs2227543, OR = 0.4, P = 0.007, and rs2227307, OR = 0.4, P = 0.02) after adjusting for age, sex, steroids prediagnosis, and herpes simplex keratitis (HSK) misdiagnosis. Two TLR-4 SNPs were associated with increased risk of SICs (chr9: rs4986791 and rs4986790, both OR = 6.9, P = 0.01). Th-17 associated SNPs (chr1: IL-23R rs11209026, chr2: IL-1ß rs16944, and chr12: IL-22 rs1179251) were also associated with SICs. Conclusions: The current study identifies biologically relevant genetic variants in patients with AK with SICs; IL-8 is associated with a strong neutrophil response in the cornea in AK, TLR-4 is important in early AK disease, and Th-17 genes are associated with adaptive immune responses to AK in animal models. Genetic screening of patients with AK to predict severity is viable and this would be expected to assist disease management.

A novel antimicrobial peptide on the ocular surface shows decreased expression in inflammation and infection.

PURPOSE: Antimicrobial peptides (AMPs) are cationic host defense peptides with microbicidal and cell-signaling properties. They show promise as potential therapeutic agents. In the present study, a beta-defensin AMP gene was isolated from the ocular surface for the first time, and its expression was characterized in the presence of ocular inflammation and/or infection. METHODS: Total RNA was obtained from impression cytology samples of the conjunctiva and cornea of normal patients and of those with bacterial, viral, acanthamoeba, or dry eye disease. The expression of the beta-defensin AMP DEFB-109 was determined by using reverse transcription-polymerase chain reaction (RT-PCR). Relative quantification of the gene in the various groups was performed by means of real-time PCR. RESULTS: DEFB-109 was constitutively expressed in all samples. The gene showed significantly decreased expression in the presence of all types of inflammation/infection. Reduced expression featured most prominently in acanthamoeba infection; the least change from normal was in dry eye. CONCLUSIONS: The discovery of DEFB-109 on the ocular surface enhances our knowledge of the profile of AMPs at this important mucosal surface. The fact that its expression is significantly reduced in both inflammatory and infective ocular surface disease reflects not only an intimate balance between this host defense gene and microbes but indicates a role other than purely microbicidal. This discovery will enable the mechanisms behind the intriguing phenomenon of reduced gene expression of an AMP in disease states to be uncovered.

Isolation and genotyping of potentially pathogenic Acanthamoeba strains from dust sources in Iran.

Opportunistic free-living amoebae of the Acanthamoeba genus present genotypes/strains that are causative agents of Acanthamoeba keratitis (AK), with genotype T4 related to most AK cases worldwide. In order to prove that exposure to dirt and dust could be an important risk factor for contact lens wearers in Iran, 13 strains isolated from dust samples in this area were classified at the genotype level. Results revealed the presence of T4, T5 and T11 genotypes within these samples. To our knowledge, this is the first study presenting the identification of pathogenic genotypes of Acanthamoeba in dust samples in Iran and around the world.

Acanthamoeba keratitis update-incidence, molecular epidemiology and new drugs for treatment.

A reliable figure for the expected incidence of Acanthamoeba keratitis of one per 30000 contact lens wearers per year has now been obtained from a combination of three cohort and three Questionnaire Reporting Surveys; 88% of cases wore hydrogel lenses and 12% wore rigid lenses. This figure now provides a basis for the expected number of cases against which to judge either epidemic outbreaks or effects of prevention with disinfecting solutions, better hygiene, or the use of disposable lenses. Molecular biology of Acanthamoeba has advanced considerably in the last 10 years with new automated sequencing technology. This has allowed the construction of a genotype identification scheme with 13 different genotypes against which to compare clinical isolates for epidemiological investigations or pathogenicity markers. So far, only four genotypes have been associated with keratitis of which the majority have been T4 but T3, T6, and T11 have each caused individual cases. Each genotype is heterogenous and can be further subdivided by comparison of sequences of diagnostic fragments of 18S rDNA, riboprinting by PCR-RFLP of 18S rDNA, or by mitochondrial DNA RFLP. Drug therapy has been revolutionised with the introduction of the biguanides-chlorhexidine or polyhexamethylene biguanide-with most but not all infections quickly resolving. Failure can still occur occasionally and further research is needed on more effective combination chemotherapy. A number of guanidines have been identified in this paper that could be usefully pursued as part of combination chemotherapy along with the alkylphosphocholines.