Rapid detection and diagnosis of herpetic keratitis using quantitative microfluidic polymerase chain reaction system for herpes simplex and varicella-zoster virus DNA: a case series.

BACKGROUND: A microfluidic real-time polymerase chain reaction (PCR) system can rapidly detect the viral DNA in specimens. Detection of herpes simplex virus (HSV) and varicella-zoster virus (VZV) DNA in tears is a useful diagnostic tool for herpes simplex virus keratitis (HSK) and herpes zoster ophthalmicus (HZO). METHODS: In total, 20 patients were included in this cross-sectional study. Among them, 8 patients with infectious epithelial HSK and 12 patients with HZO were included in HSK and HZO groups, respectively. In addition, 8 patients with non-herpetic keratitis and 4 healthy individuals without keratitis were included in the control group. Numbers of HSV and VZV DNA copies in tears of all patients and individuals were evaluated using a microfluidic real-time PCR system. Regarding HSV/VZV DNA test, tear specimens were collected by filter paper method using Schirmer's test paper, and subsequently, DNA was extracted from the filter paper using an automated nucleic acid extractor. Afterward, quantitative PCR was performed using a microfluidic real-time PCR system. RESULTS: From tear collection to real-time PCR result determination, the HSV/VZV DNA test took approximately 40 min. In the HSK group, the sensitivity and specificity of the HSV DNA tests were 100% each. The median value (range) of number of HSV DNA copies for affected eyes was 3.4 × 105 copies/µL (under a lower detection limit of 7.6). In the HZO group, the sensitivity and specificity of the VZV DNA tests were 100% each. The median value (range) of number of VZV DNA copies for affected eyes was 5.3 × 105 copies/µL (under a lower detection limit of 5.6 × 10-2). CONCLUSION: In conclusion, quantitative PCR for HSV and VZV DNA in tears using a microfluidic real-time PCR system is useful for diagnosing and monitoring HSK and HZO.

Two cases of dupilumab-associated conjunctivitis with high expression of IL-8 mRNA on the ocular surface: a case report.

BACKGROUND: Dupilumab-induced ocular surface disease (DIOSD) has been reported in patients with atopic dermatitis treated with dupilumab, and has been recognized as an adverse event of dupilumab. Our objective was to describe two cases of DIOSD with alterations in eotaxin-2 and interleukin (IL)-8 messenger ribonucleic acid (mRNA) expression on the ocular surface. CASE PRESENTATION: In the ocular surface test, specimens were collected from the patient's ocular surface, and eotaxin-2 and IL-8 mRNA levels in the specimens were measured using real-time polymerase chain reaction. The clinical score of ocular surface findings was quantified using a 5-5-5 exacerbation grading scale for allergic conjunctivitis. The first case was of a 27-year-old man who developed DIOSD 3 months after starting treatment with dupilumab injection for atopic dermatitis. After 5 weeks of topical instillation of tacrolimus ophthalmic suspension, the clinical score of ocular surface findings improved and IL-8 and eotaxin-2 mRNA expression levels gradually decreased. The second patient was a 55-year-old man who developed DIOSD 11 weeks after the start of treatment with dupilumab injection for atopic dermatitis. Four weeks after starting ophthalmological treatment with tacrolimus ophthalmic suspension, his clinical scores on ocular surface findings improved and IL-8 mRNA expression levels decreased. The ocular surface test in this case revealed increased expression levels of IL-8 mRNA on the ocular surface at the onset of DIOSD, which decreased with the improvement of objective findings. CONCLUSIONS: DIOSD, which has been successfully treated with tacrolimus ophthalmic suspension, may involve IL-8-related inflammation in addition to type 2 inflammation.

Dysbiosis of Ocular Surface Microbiota in Patients With Refractive Allergic Conjunctival Diseases.

PURPOSE: We investigated ocular surface microbiota dysbiosis in patients with refractory allergic conjunctival diseases (ACDs; stratified into mild and severe groups) treated with topical tacrolimus. METHODS: Patients (n = 21) with refractory ACDs (including vernal and atopic keratoconjunctivitis) actively treated with topical tacrolimus and 6 healthy controls were evaluated. Based on clinical scores and expression of specific cytokines on the ocular surface, patients with ACDs were divided into mild and severe groups using cluster analysis. The microbial composition of tear specimens collected from patients with mild and severe ACD and control subjects using the Schirmer test paper was determined through next-generation 16S rRNA sequencing analysis. RESULTS: Compared with healthy controls, patients with ACDs exhibited significantly decreased ocular surface microbiota α-diversity. Ocular surface microbiota mainly comprised members of the phyla Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria in all groups. The relative abundance of ocular surface microbiota in patients with ACDs was increased for phylum Firmicutes and decreased for phylum Proteobacteria (compared with control subjects). The genera Blautia (vs. mild ACD group) and Morganella (vs. control group) exhibited significantly increased abundance only in the severe ACD group. CONCLUSIONS: The ocular surface microbiota in patients with severe ACD exhibited decreased diversity and exacerbation of dysbiosis compared with that in patients with mild ACD and control subjects. Patients with mild refractory ACD also exhibited decreased diversity of these microbiota. These alterations in microbiota indicated a change in the ocular surface of patients with refractory ACD (be it because of disease pathogenesis or topical immunomodulatory treatment).

Alterations in Mucin-Associated Gene Expression on the Ocular Surface in Active and Stable Stages of Atopic and Vernal Keratoconjunctivitis.

PURPOSE: To evaluate the presence of ocular surface mucin in patients with atopic and vernal keratoconjunctivitis (AKC/VKC), we investigated the mRNA expression levels of SAM-pointed domain-containing ETS-like factor (SPDEF) and mucin-related genes on the ocular surface. METHODS: Nineteen patients with AKC or VKC were divided into two groups based on the severity of the disease as determined by their clinical scores for AKC/VKC: the stable group and the active group. Impression cytology was performed in all patients using filter paper, and the expression levels of SPDEF, MUC1, MUC4, MUC5AC, MUC16, and eotaxin-2 mRNA were determined by real-time reverse-transcription polymerase chain reaction. RESULTS: The results showed that the expression levels of SPDEF and MUC5AC mRNA in the active group were significantly decreased compared with those in the stable group. Furthermore, clinical scores were significantly negatively correlated with the expression levels of SPDEF mRNA and significantly positively correlated with the expression levels of eotaxin-2, which is a biomarker for eosinophilic inflammation on the ocular surface. Cluster analysis classified the patients with AKC/VKC into three clusters, and the stable group was divided into two clusters according to the condition of ocular surface mucin. CONCLUSIONS: Ocular surface mucin in patients with AKC/VKC is altered in accordance with the clinical severity of the disease.