Metagenomic sequencing for investigation of a national keratoconjunctivitis outbreak, Israel, 2022.

BackgroundEpidemics of keratoconjunctivitis may involve various aetiological agents. Microsporidia are an uncommon difficult-to-diagnose cause of such outbreaks.AimDuring the third quarter of 2022, a keratoconjunctivitis outbreak was reported across Israel, related to common water exposure to the Sea of Galilee. We report a comprehensive diagnostic approach that identified Vittaforma corneae as the aetiology, serving as proof of concept for using real-time metagenomics for outbreak investigation.MethodsCorneal scraping samples from a clinical case were subjected to standard microbiological testing. Samples were tested by calcofluor white staining and metagenomic short-read sequencing. We analysed the metagenome for taxonomical assignment and isolation of metagenome-assembled genome (MAG). Targets for a novel PCR were identified, and the assay was applied to clinical and environmental samples and confirmed by long-read metagenomic sequencing.ResultsFluorescent microscopy was suggestive of microsporidiosis. The most abundant species (96.5%) on metagenomics analysis was V. corneae. Annotation of the MAG confirmed the species assignment. A unique PCR target in the microsporidian rRNA gene was identified and validated against the clinical sample. The assay and metagenomic sequencing confirmed V. corneae in an environmental sludge sample collected at the exposure site.ConclusionsThe real-time utilisation of metagenomics allowed species detection and development of diagnostic tools, which aided in outbreak source tracking and can be applied for future cases. Metagenomics allows a fully culture-independent investigation and is an important modality for public health microbiology.

Genome-wide association study of resistance/susceptibility to infectious bovine keratoconjunctivitis in Brazilian Hereford cattle.

Genome-wide association studies were conducted to identify the more informative genomic regions and SNPs, as well as to identify candidate genes associated with infectious bovine keratoconjunctivitis (IBK) resistance/susceptibility in Hereford cattle. A Bayes B statistical approach was initially applied in genome-wide association studies by using deregressed estimated breeding values for IBK resistance/susceptibility. To estimate the combined effect of a genomic region that is potentially associated with QTL, 2504 non-overlapping 1-Mb windows that varied in SNP number were defined, with the most informative 24 windows including 427 SNPs and explaining more than 20% of the estimated genetic variance for IBK resistance/susceptibility. These regions were explored with respect to their biological functions through functional analysis to map potential candidate genes. The significant SNPs were mapped on chromosomes 1, 3, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 18, 20, 23, and 28, and candidate genes were detected as related to the IBK. Most informative SNPs in term of genetic variance were located in proximity of genes related to phenotypic expression of lesions and biological processes associated to the IBK. Knowledge about phenotypic and genomic variation generated in the present study can be used to on design selection strategies to improve the resistance to IBK of Hereford cattle herds.

Difference in the plasma level of miR-628-3p in atopic dermatitis patients with/without atopic keratoconjunctivitis.

INTRODUCTION: Some but not all patients with atopic dermatitis (AD) present with allergic conjunctival disease (ACD) including severe types such as atopic keratoconjunctivitis (AKC) with/without giant papillae. We hypothesized that different factors are involved in the severity of ACD and AD. Recently we reported that hsa-miR-628-3p could affect the balance of innate immunity by suppressing pathogen-associated molecular patterns such as toll-like receptor 3 (TLR3), RIG-I, and MDA-5. We also reported that TLR3 positively regulates ocular surface- and skin inflammation such as contact dermatitis and AD. Here we compared the plasma level of miR-628-3p in AD patients with severe AKC with giant papillae and/or shield ulcers, with the level in healthy controls and AD patient without AKC or with very mild AKC. METHODS: We used the plasma from 32 AD patients with severe AKC, from 40 healthy controls, and from 23 AD patient without AKC or with very mild AKC without giant papillae nor shield ulcers. Quantitative microRNA PCR assays were used to measure their plasma level of miR-628-3p. RESULTS: We found that plasma miR-628-3p was upregulated in AD with severe AKC, but not in severe AD without severe AKC, nor in our healthy controls. CONCLUSION: Our new findings suggest that the plasma miR-628-3p level may represent a marker to predict the presence of severe AKC in AD patients.

The FADS mouse: A novel mouse model of atopic keratoconjunctivitis.

BACKGROUND: Atopic keratoconjunctivitis (AKC) is a chronic allergic conjunctival disease. However, a mouse model of AKC to investigate the underlying mechanism of the therapeutic agents and estimate their efficacy has not been established. We recently generated mice in which Ikk2 is specifically deleted in facial skin fibroblasts and found that these mice spontaneously develop atopic dermatitis (AD)-like facial skin inflammation and scratching behaviors; thus, we named them facial AD with scratching (FADS) mice. OBJECTIVE: We sought to evaluate whether the ocular lesions that FADS mice spontaneously develop are similar to those of patients with AKC and to estimate the efficacy of topical treatments with tacrolimus and betamethasone for FADS mice by using tear periostin, a novel biomarker for allergic conjunctival disease. METHODS: FADS mice, in which Ikk2 is deleted in dermal fibroblasts, were generated by crossing female Ikk2Flox/Flox mice to male Nestincre; Ikk2Flox/+ mice. We conducted histologic analysis of the ocular lesions in FADS mice. Furthermore, we measured periostin in the tears collected from FADS mice untreated or treated with tacrolimus or betamethasone. RESULTS: The FADS mice exhibited severe blepharitis and scratch behaviors for their faces. In these mice, corneal epithelium and stroma showed hyperplasia and infiltration of eosinophils, mast cells, and TH2/TC2 cells. Periostin was significantly expressed in the lesions and tear periostin was upregulated. Betamethasone showed more suppressive effects than did tacrolimus on severe corneal lesions and increased tear periostin level. CONCLUSIONS: The FADS mouse is a novel mouse model of AKC and is useful to examine the therapeutic effects of anti-AKC agents.

Adding evidence to the role of NEUROG1 in congenital cranial dysinnervation disorders.

Congenital cranial dysinnervation disorders (CCDDs) are a heterogeneous group of neurodevelopmental phenotypes caused by a primary disturbance of innervation due to deficient, absent, or misguided cranial nerves. Although some CCDDs genes are known, several clinical phenotypes and their aetiologies remain to be elucidated. We describe a 12-year-old boy with hypotonia, developmental delay, sensorineural hearing loss, and keratoconjunctivitis due to lack of corneal reflex. He had a long expressionless face, severe oromotor dysfunction, bilateral agenesis/severe hypoplasia of the VIII nerve with marked atresia of the internal auditory canals and cochlear labyrinth malformation. Trio-exome sequencing identified a homozygous loss of function variant in the NEUROG1 gene (NM_006161.2: c.202G > T, p.Glu68*). NEUROG1 is considered a causal candidate for CCDDs based on (i) the previous report of a patient with a homozygous gene deletion and developmental delay, deafness due to absent bilateral VIII nerves, and severe oromotor dysfunction; (ii) a second patient with a homozygous NEUROG1 missense variant and corneal opacity, absent corneal reflex and intellectual disability; and (iii) the knockout mouse model phenotype which highly resembles the disorder observed in humans. Our findings support the growing compelling evidence that loss of NEUROG1 leads to a very distinctive disorder of cranial nerves development.

Genomic foundations of evolution and ocular pathogenesis in human adenovirus species D.

Human adenovirus commonly causes infections of respiratory, gastrointestinal, genitourinary, and ocular surface mucosae. Although most adenovirus eye infections are mild and self-limited, specific viruses within human adenovirus species D are associated with epidemic keratoconjunctivitis (EKC), a severe and highly contagious ocular surface infection, which can lead to chronic and/or recurrent, visually disabling keratitis. In this review, we discuss the links between adenovirus ontogeny, genomics, immune responses, and corneal pathogenesis, for those viruses that cause EKC.

[Association of polymorphisms of the TRIM21 gene with the severity of dry keratoconjunctivitis in rheumatoid arthritis and Sjogren's disease]. / Vzaimosviaz' polimorfizmov gena TRIM21 s tiazhest'iu sukhogo keratokon''iunktivita pri revmatoidnom artrite i bolezni Shegrena.

Ophthalmologic manifestation of Sjogren's disease (SD) and rheumatoid arthritis (RA) is dry keratoconjunctivitis (dry eye disease; DED). PURPOSE: To study the relationship of polymorphic markers rs7947461 (C/T), rs915956 (C/T), rs4144331 (C/A) of the TRIM21 gene with the severity of DED in patients with RA and SD. MATERIAL AND METHODS: The study included 70 patients with RA (n=27) and SD (n=43). The control group consisted of volunteers without a history of RA or SD (n=35). Alleles of the polymorphic marker C660T rs7947461 of the TRIM21 gene were identified using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method; alleles of the polymorphic marker rs915956 (C/T) and rs4144331 (C/A) of the TRIM21 gene were identified by analyzing DNA melting curves. RESULTS: An association was found between the predisposing genotype (TT) of rs7947461 polymorphic marker and the risk of developing severe DED. The AA genotype of rs4144331 polymorphic marker was found only in severe DED (c2=7.74; OR=17.46, CI95%=1.96-318.38, p=0.02). CONCLUSION: An association was established between rs7947461 (rs660) and rs4144331 and the risk of developing severe DED.

Novel mutation in AIRE gene with autoimmune polyendocrine syndrome type 1.

PURPOSE: Autoimmune polyendocrine type 1 (APS-1) is a complex inherited autosomal recessive disorder. Classically, it appears within the first decade of life followed by adrenocortical insufficiency, mucocutaneous candidiasis, Addison's disease, and hypoparathyroidism. The clinical phenotype of APS-1 varies depending upon mutations in the autoimmune regulator gene (AIRE) on chromosome 21q22.3. METHODS: In this study, we performed Sanger sequencing ofAIRE in Iranian patients to identify different variants and probable new mutations corresponding to a clinical diagnosis of APS-1. RESULTS: After analyzing 14AIRE exons, we detected a novel insertion mutation in exon 2 in a patient who presented with severe APS-1, Lys50AsnfsX168. Furthermore, the known mutations in AIRE, including Arg139X, Arg257X, and Leu323SerfsX51, were detected in enrolled patients. DISCUSSION: According to our results, sequencing analysis ofAIRE provides a useful screening method to diagnose patients with incomplete or unusual clinical presentations of APS-1.

Ocular surface pathogenesis associated with precocious eyelid opening and necrotic autologous tissue in mouse with disruption of Prickle 1 gene.

Ocular surface disease is one major type of eye diseases. Different etiologies trigger distinct pathological responses of the ocular surface. We previously reported that genetically engineered mice with ablation of Prickle 1 manifested precocious eyelid opening with ensuing cornea dysplasia. The current study aimed to characterize the molecular traits and the direct cause of ocular pathology associated with precocious eyelid opening in the Prickle 1 mutant mouse. Prickle 1 mutant mice exhibited a slew of ocular surface pathology including cell proliferation, cell fate transformation and inflammatory infiltration coinciding with the timing of the precocious eyelid opening. Forced eyelid opening in wild type mice did not induce cornea pathology comparable to that of the Prickle 1 mutants. Necrotic tissue debris was found associated with the lesioned cornea. RNAseq analysis of the mutant cornea revealed an expression profile shared by a range of dermatological diseases involving immune responses and cancer. Taken together, the data suggest that the necrotic eyelid debris plays an important role in ocular pathogenesis of the Prickle 1 mutant mouse, which may represent a type of non-infectious keratoconjunctivitis caused by damaged autologous tissues. Additionally, Prickle 1 mutant cornea pathogenesis may offer molecular insights into other types of epithelial pathogenesis.

Results of Detailed Investigations Into Stevens-Johnson Syndrome With Severe Ocular Complications.

Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are acute inflammatory vesiculobullous reactions of the mucosa of the ocular surface, oral cavity, and genitals, and of the skin. Severe ocular complications (SOC) are present in about half of SJS/TEN patients diagnosed by dermatologists. We review our group's findings on the genetic predisposition for and the etiology of SJS/TEN with SOC. We suspected that abnormal innate mucosal immunity, resulting in an anomalous response to commensal bacteria that usually do not elicit such a response, contributes to the ocular surface inflammation seen in SJS/TEN with SOC. We found that cold medicines, including multi-ingredient cold medications and nonsteroidal anti-inflammatory drugs, were the main causative drugs especially in patients with SJS/TEN with SOC. Cold medicine-related SJS/TEN (CM-SJS/TEN) with SOC was strongly associated with HLA-A*02:06 in the Japanese populations, and significantly associated with HLA-B*44:03 in the Japanese and in Indian and Brazilian populations. Single nucleotide polymorphism association analysis showed that the Toll-like receptor 3 (TLR3), prostaglandin-E receptor 3 (PTGER3), and IKZF1 gene were significantly associated with CM-SJS/TEN with SOC and that they could regulate mucocutaneous inflammation including that of the ocular surface. As we found several HLA-SNP sets with a high odds ratio, we postulated that they may help to predict the possible development of SJS/TEN with SOC. From our findings we suggest that besides microbial infection and cold medicines, a combination of multiple gene polymorphisms and their interactions contribute strongly to the onset of CM-SJS/TEN with SOC.

Identification of novel adenovirus genotype 90 in children from Bangladesh.

Novel adenovirus genotypes are associated with outbreaks of disease, such as acute gastroenteritis, renal disease, upper respiratory tract infection and keratoconjunctivitis. Here, we identify novel and variant adenovirus genotypes in children coinfected with enterotoxigenic Escherichia coli, in Bangladesh. Metagenomic sequencing of stool was performed and whole adenovirus genomes were extracted. A novel species D virus, designated genotype 90 (P33H27F67) was identified, and the partial genome of a putative recombinant species B virus was recovered. Furthermore, the enteric types HAdV-A61 and HAdV-A40 were found in stool specimens. Knowledge of the diversity of adenovirus genomes circulating worldwide, especially in low-income countries where the burden of disease is high, will be required to ensure that future vaccination strategies cover the diversity of adenovirus strains associated with disease.

Expression of IL-33 in ocular surface epithelium induces atopic keratoconjunctivitis with activation of group 2 innate lymphoid cells in mice.

In a transgenic mouse line hK14mIL33tg, with the expression of interleukin-33 (IL-33) driven by a keratin 14 promoter, keratoconjunctivitis developed spontaneously between 18 and 22 weeks of age under specific-pathogen-free conditions. These mice showed blepharitis and corneal impairments, and the histology revealed epithelial thickening in the conjunctiva and the cornea with infiltration of eosinophils, mast cells and basophils. IL-5, IL-13 and CCL11 were abundant in lacrimal fluid in the mice, and the gene expressions of IL-4, IL-5, IL-13, IL-33, Prg2 and Mmcp8 were significantly increased in the cornea. Furthermore, group 2 innate lymphoid cells (ILC2) producing IL-5 and IL-13 were markedly increased in the cornea. These phenotypes closely resemble human atopic keratoconjunctivitis (AKC). The characteristic ocular phenotype in these mice strongly suggests that IL-33 is crucial for the development of AKC. The mouse line may be useful as a novel model for research and development of therapeutic strategies for AKC.

CD44 and RHAMM hyaluronan receptors in human ocular surface inflammation.

BACKGROUND: CD44 and RHAMM hyaluronan (HA) receptors have been studied in several systemic diseases such as osteoarthritis and cancer. However, not too much is known about their role in ocular surface disorders. The purpose of this research was to determine if CD44 and RHAMM are implicated in human ocular surface inflammation. METHODS: Upper tarsal conjunctival epithelial samples from patients with active ocular surface inflammation (n = 17) and healthy controls (n = 14) were recovered by brush cytology. Patients were evaluated by an ophthalmologist and classified in different groups according to the etiology (immune atopic diseases or immune non-atopic diseases) and inflammation intensity (mild/moderate or severe). CD44, RHAMM, and p53 mRNAs were measured using real-time PCR. RESULTS: CD44, RHAMM, and p53 mRNAs were detected in all samples. In immune atopic diseases, higher levels of CD44 and RHAMM mRNAs were present, reaching a 300 % increase for RHAMM in severe inflammation (p < 0.001). In contrast, in immune non-atopic diseases, the HA receptors were downregulated. CD44 tended to decrease up to 30 % in severe patients (p = 0.06), and RHAMM decreased 40 % in severe inflammation (p = 0.021). CONCLUSIONS: RHAMM may be implicated in severe ocular surface inflammation affecting the upper tarsal conjunctiva.

Anti-inflammatory effects of rebamipide eyedrop administration on ocular lesions in a murine model of primary Sjögren's syndrome.

BACKGROUND: Topical therapy is effective for dry eye, and its prolonged effects should help in maintaining the quality of life of patients with dry eye. We previously reported that the oral administration of rebamipide (Reb), a mucosal protective agent, had a potent therapeutic effect on autoimmune lesions in a murine model of Sjögren's syndrome (SS). However, the effects of topical treatment with Reb eyedrops on the ocular lesions in the murine model of SS are unknown. METHODS AND FINDING: Reb eyedrops were administered to the murine model of SS aged 4-8 weeks four times daily. Inflammatory lesions of the extraorbital and intraorbital lacrimal glands and Harderian gland tissues were histologically evaluated. The direct effects of Reb on the lacrimal glands were analyzed using cultured lacrimal gland cells. Tear secretions of Reb-treated mice were significantly increased compared with those of untreated mice. In addition to the therapeutic effect of Reb treatment on keratoconjunctivitis, severe inflammatory lesions of intraorbital lacrimal gland tissues in this model of SS were resolved. The mRNA expression levels of IL-10 and mucin 5Ac in conjunctival tissues from Reb-treated mice was significantly increased compared with those of control mice. Moreover, lactoferrin production from lacrimal gland cells was restored by Reb treatment. CONCLUSION: Topical Reb administration had an anti-inflammatory effect on the ocular autoimmune lesions in the murine model of SS and a protective effect on the ocular surfaces.

Polymorphism in THBS1 gene is associated with post-refractive surgery chronic ocular surface inflammation.

PURPOSE: To determine the association of single nucleotide polymorphisms (SNPs) of the thrombospondin 1 (THBS1) gene with development of chronic ocular surface inflammation (keratoconjunctivitis) after refractive surgery. DESIGN: Retrospective cohort study. PARTICIPANTS: Active duty U.S. Army soldiers (n = 143) who opted for refractive surgery. METHODS: Conjunctival impression cytology samples collected from participants before the surgery were used to harvest DNA for genotyping 5 THBS1 SNPs (rs1478604, rs2228262, rs2292305, rs2228262, and rs3743125) using the Sequenom iPLEX Gold platform (Sequenom, San Diego, CA). Samples collected after surgery were used to harvest RNA for gene expression analysis by real-time polymerase chain reaction (PCR). Participants were followed for 1 year after surgery to monitor the status of keratoconjunctivitis. MAIN OUTCOME MEASURES: Genetic basis of the development of chronic keratoconjunctivitis after refractive surgery. RESULTS: Carriers of minor alleles of 3 SNPs each were found to be more susceptible to developing chronic keratoconjunctivitis (rs1478604: odds ratio [OR], 2.5; 95% confidence interval [CI], 1.41-4.47; P = 2.5 × 10(-3); rs2228262 and rs2292305: OR, 1.9; 95% CI, 1.05-3.51; P = 4.8 × 10(-2)). Carriers of the rs1478604 minor allele expressed significantly reduced levels of thrombospondin 1 (TSP1) (P = 0.042) and increased levels of an inflammatory cytokine associated with keratoconjunctivitis, interleukin-1ß (P = 0.025), in their ocular surface epithelial cells compared with homozygous major allele controls. CONCLUSIONS: Genetic variation in the THBS1 gene that results in decreased expression of the encoded glycoprotein TSP1 in ocular surface epithelial cells significantly increases the susceptibility to develop chronic ocular surface inflammation after refractive surgery. Further investigation of THBS1 SNPs in a larger sample size is warranted.

Isolation of a variant human adenovirus identified based on phylogenetic analysis during an outbreak of acute keratoconjunctivitis in Chennai.

BACKGROUND & OBJECTIVES: Though several viruses are responsible for conjunctivitis, but human adenovirus (HAdV) is by far the most common cause. Epidemic conjunctivitis causes morbidity and early detection of aetiological agent is essential in preventing spread of disease as some of serotypes of adenoviruses cause a severe form of conjunctivitis. This study was undertaken to identify the causative agent of conjunctivitis outbreak in Chennai in 2010. METHODS: Conjunctival samples collected from 17 patients with conjunctivitis were subjected to virological investigations. Culture and PCR for detection of adenovirus and enterovirus were carried out. PCR positive products were further subjected for DNA sequencing. The nucleotide sequences of the hexons of isolates were analyzed by comparison with all 51 human adenovirus strains. Phylogenetic tree was constructed using DAMBE software. RESULTS: Among 17 patients, seven were positive for adenovirus by PCR on the direct specimen, none was positive for enterovirus. Eleven of 30 conjunctival swabs showed cytopathic effect in HEp-2 cell line and were confirmed as HAdV by PCR. The DNA sequence data of the 11 isolates had equal percentage of homology with HAdV 6 and 2 on blast analysis. On phylogenetic analysis with GeneBank data of 51 adenovirus strains, 11 isolates from patients during the outbreak of conjunctivitis formed a separate clade indicating a new variant strain. INTERPRETATION & CONCLUSIONS: Based on phylogenetic analysis it was concluded that the recent conjunctivitis outbreak that occurred in Chennai was caused by a variant adenovirus strain.

Genetic parameters of infectious bovine keratoconjunctivitis and its relationship with weight and parasite infestations in Australian tropical Bos taurus cattle.

BACKGROUND: Infectious bovine keratoconjunctivitis (IBK) or 'pinkeye' is an economically important ocular disease that significantly impacts animal performance. Genetic parameters for IBK infection and its genetic and phenotypic correlations with cattle tick counts, number of helminth (unspecified species) eggs per gram of faeces and growth traits in Australian tropically adapted Bos taurus cattle were estimated. METHODS: Animals were clinically examined for the presence of IBK infection before and after weaning when the calves were 3 to 6 months and 15 to 18 months old, respectively and were also recorded for tick counts, helminth eggs counts as an indicator of intestinal parasites and live weights at several ages including 18 months. RESULTS: Negative genetic correlations were estimated between IBK incidence and weight traits for animals in pre-weaning and post-weaning datasets. Genetic correlations among weight measurements were positive, with moderate to high values. Genetic correlations of IBK incidence with tick counts were positive for the pre-weaning and negative for the post-weaning datasets but negative with helminth eggs counts for the pre-weaning dataset and slightly positive for the post-weaning dataset. Genetic correlations between tick and helminth eggs counts were moderate and positive for both datasets. Phenotypic correlations of IBK incidence with helminth eggs per gram of faeces were moderate and positive for both datasets, but were close to zero for both datasets with tick counts. CONCLUSIONS: Our results suggest that genetic selection against IBK incidence in tropical cattle is feasible and that calves genetically prone to acquire IBK infection could also be genetically prone to have a slower growth. The positive genetic correlations among weight traits and between tick and helminth eggs counts suggest that they are controlled by common genes (with pleiotropic effects). Genetic correlations between IBK incidence and tick and helminth egg counts were moderate and opposite between pre-weaning and post-weaning datasets, suggesting that the environmental and (or) maternal effects differ between these two growth phases. This preliminary study provides estimated genetic parameters for IBK incidence, which could be used to design selection and breeding programs for tropical adaptation in beef cattle.