A Genomic Approach to Investigating Ocular Surface Microorganisms: Monitoring Core Microbiota on Eyelid Margin with a Dot hybridization Assay.

A sound ocular surface microbiota has been recognized as a part of ocular surface health following a growing body of evidence from next-generation sequencing technique and metagenomic analysis. However, even from the perspective of contemporary precision medicine, it is difficult to directly apply these new technologies to clinical practice. Therefore, we proposed a model based on dot hybridization assay (DHA) to bridge conventional culture with a metagenomic approach in investigating and monitoring ocular surface microbiota. Endophthalmitis, mostly caused by bacterial infection, is the most severe complication of many intraocular surgeries, such as cataract surgery. Hazardous microorganisms hiding and proliferating in the ocular surface microbiota not only increase the risk of endophthalmitis but also jeopardize the effectiveness of the preoperative aseptic procedure and postoperative topical antibiotics. The DHA model enables the simultaneous assessment of bacterial bioburden, detection of target pathogens and microorganisms, and surveillance of methicillin/oxacillin resistance gene mecA in the ocular surface microbiota. This assay revealed heavier bacterial bioburden in men, compatible with a higher risk of endophthalmitis in male patients who underwent cataract surgery. No occurrence of endophthalmitis for these patients was compatible with non-hazardous microorganisms identified by specific dots for target pathogens. Moreover, the mecA dot detected oxacillin-resistant strains, of which culture failed to isolate. Therefore, the DHA model could provide an alternative genomic approach to investigate and monitor ocular surface microorganisms in clinical practice nowadays.

Epidemiology and clinical features of viral anterior uveitis in southern Taiwan-diagnosis with polymerase chain reaction.

BACKGROUND: To report the epidemiology and clinical features of viral anterior uveitis in patients in southern Taiwan. METHODS: A retrospective, case series study. HLA-B27 negative anterior uveitis patients with increased intraocular pressure or corneal edema seen at Kaohsiung Chang Gung Memorial Hospital from January 1, 2007 to January 31, 2018 had their aqueous sent for polymerase chain reaction analysis. Their records were reviewed for demographic data, ocular findings, and laboratory results. RESULTS: In the aqueous samples obtained from 102 eligible eyes, 42 eyes were herpesviridae-positive, which included 9 with herpes simplex virus (8.8%), 5 with varicella-zoster virus (4.9%), 27 with cytomegalovirus (26.5%), and 1 with Epstein-Barr virus (1%). Herpesviridae-positive patients were more likely to be male, and have glaucoma. Glaucoma and pseudophakic eyes were significantly associated with CMV-positive eyes. CONCLUSION: PCR analysis of the anterior chamber fluid is important for the confirmation of the diagnosis of viral anterior uveitis. Cytomegalovirus anterior uveitis is not uncommon in patients in southern Taiwan, and it may follow an uneventful cataract extraction in immunocompetent patients.

Tear Proteomics Approach to Monitoring Sjögren Syndrome or Dry Eye Disease.

Sjögren syndrome (SS) or dry eye disease (DED) is one of the most complicated ocular surface diseases. The goal of this study is to elucidate the relationship of the changes in clinical indices of tear film (TF) homeostasis with respect to tear components to allow for SS-DED monitoring and avoid stably controlled SS-DED patients from re-entering a vicious cycle. This prospective case-control study compared stable SS-DED patients with non-SS-DED control from several aspects, including clinical indices for TF homeostasis, 2 DED diagnostic biomarkers (MMP-9 and lactoferrin), and the proteome of flush tears. Compared with non-SS-DED controls, stably controlled SS-DED subjects had less tear secretion and higher ocular surface inflammation, a higher concentration ratio of tear MMP-9/lactoferrin, a more diverse tear proteome, and lower spectral intensities of lipocalin-1, lacritin, and prolactin-inducible protein among the abundant tear proteins. For stable SS-DED patients, the concentration ratio of tear MMP-9/lactoferrin and the corrected lipocalin-1 signal was positively correlated with ocular inflammation and TF stability, respectively. MMP-9 released from stressed ocular surface epithelium and lipocalin-1 secreted from the energetic lacrimal gland are two tear biomarkers responding well to TF homeostasis. The tear proteomics approach through flush tears is a promising method for monitoring SS-DED patients with a standardized sampling procedure and lactoferrin-corrected analysis.

Anti-Cancerous Effect of Inonotus taiwanensis Polysaccharide Extract on Human Acute Monocytic Leukemia Cells through ROS-Independent Intrinsic Mitochondrial Pathway.

Acute leukemia is one of the commonly diagnosed neoplasms and causes human death. However, the treatment for acute leukemia is not yet satisfactory. Studies have shown that mushroom-derived polysaccharides display low toxicity and have been used clinically for cancer therapy. Therefore, we set out to evaluate the anti-cancerous efficacy of a water-soluble polysaccharide extract from Inonotus taiwanensis (WSPIS) on human acute monocytic leukemia THP-1 and U937 cell lines in vitro. Under our experimental conditions, WSPIS elicited dose-dependent growth retardation and induced apoptotic cell death. Further analysis showed that WSPIS-induced apoptosis was associated with a mitochondrial apoptotic pathway, such as the disruption of mitochondrial membrane potential (MMP), followed by the activation of caspase-9, caspase-3, and PARP (poly(ADP-ribose) polymerase) cleavage. However, a broad caspase inhibitor, Z-VAD.fmk, could not prevent WSPIS-induced apoptosis. These data imply that mechanism(s) other than caspase might be involved. Thus, the involvement of endonuclease G (endoG), a mediator arbitrating caspase-independent oligonucleosomal DNA fragmentation, was examined. Western blotting demonstrated that WSPIS could elicit nuclear translocation of endoG. MMP disruption after WSPIS treatment was accompanied by intracellular reactive oxygen species (ROS) generation. However, pretreatment with N-acetyl-l-cysteine (NAC) could not attenuate WSPIS-induced apoptosis. In addition, our data also show that WSPIS could inhibit autophagy. Activation of autophagy by rapamycin decreased WSPIS-induced apoptosis and cell death. Taken together, our findings suggest that cell cycle arrest, endonuclease G-mediated apoptosis, and autophagy inhibition contribute to the anti-cancerous effect of WSPIS on human acute monocytic leukemia cells.

Discordance of Mutation Statuses of Epidermal Growth Factor Receptor and K-ras between Primary Adenocarcinoma of Lung and Brain Metastasis.

Mutations on epidermal growth factor receptor (EGFR) of adenocarcinomas of lung have been found to be associated with increased sensitivity to EGFR tyrosine kinase inhibitors and K-ras mutations may correlate with primary resistance. We aimed to explore the discordant mutation statuses of EGFR and K-ras between primary tumors and matched brain metastases in adenocarcinomas of lung. We used a sensitive Scorpion ARMS method to analyze EGFR mutation, and Sanger sequencing followed by allele-specific real-time polymerase chain reaction to analyze K-ras mutation. Forty-nine paired tissues with both primary adenocarcinoma of lung and matched brain metastasis were collected. Thirteen patients (26.5%) were discordant for the status of EGFR between primary and metastatic sites. K-ras gene could be checked in paired specimens from 33 patients, thirteen patients (39.6%) were discordant for the status of K-ras. In primary lung adenocarcinoma, there were 14 patients of mutant EGFR had mutant K-ras synchronously. This study revealed that the status of EGFR mutation in lung adenocarcinomas is relatively consistent between primary and metastatic sites compared to K-ras mutation. However, there are still a few cases of adenocarcinoma of lung showing discordance for the status of EGFR mutation. Repeated analysis of EGFR mutation is highly recommended if tissue from metastatic or recurrent site is available for the evaluation of target therapy.

The incidence of resistance-associated variants to NS5A in HCV subtypes 1a and 1b in Taiwan.

BACKGROUND: Resistance-associated variants (RAVs) to direct-antiviral agents (DAAs) may hamper treatment. There was a lack of data on the natural prevalence of RAVs in Taiwanese HCV-infected patients. We investigated the real-life presence of RAVs in the nonstructural 5A (NS5A) region in HCV genotype 1a and 1b in chronically infected individuals in Taiwan. METHODS: In this single-center cohort study, nested polymerase chain reaction and direct sequencing analysis was used to determine the frequency of RAVs in the HCV NS5A region in patients with HCV genotype 1a (n = 55) and 1b (n = 525). RESULTS: In genotype 1a strains, the incidence of RAVs was 16.4% (9/55) in the NS5A region (M28V/T, n = 6, 10.9%; Q30L, n = 1, 1.8%; Y93N/H, n = 3, 5.5%). In genotype 1b, the incidence of RAVs was 17.5% (92/525) in the NS5A region (L31I/M/V, n = 7, 1.3%; Y93 H/S, n = 87, 16.5%). Patients with RAVs had significantly higher HCV RNA levels (6.1 ± 0.7 vs 5.9 ± 0.8 log IU/mL, p = 0.001) and lower rGT levels (28.9 ± 18.9 vs. 42.9 ± 57.0 U/L, p = 0.001) compared to those without RAVs. Multivariate analysis identified HCV RNA levels (odds ratio = 1.145, 95% CI: 1.060-1.237, p = 0.001) and rGT (OR = 0.989, 95% CI: 0.978-0.999, p = 0.035) as risk factors that are associated with the presence of RAVs. Importantly, there is no association between the presence of RAVs and no SVR (3.8% in patients with RAVs, 15.9% in patients without RAVs, p = 0.32). CONCLUSION: RAVs, especially M28V and Y93H in the NS5A region, were highly prevalent in patients with genotype 1a and 1b HCV, respectively, in Taiwan, and they were linked to high HCV RNA levels and low rGT levels. Before using the NS5A inhibitors, the presence of mutated HCV variants should be taken into consideration.

Determination of Tear Lipid Film Thickness Based on a Reflected Placido Disk Tear Film Analyzer.

This study aims at determining the thickness of the tear lipid layer (LL) observed from a placido-disc-based tear film analyzer. We prospectively collected reflections of placido-disk LL images using a tear film analyzer (Keratograph® 5M, Oculus) from subjects with dry eye symptoms. The LL thickness (LLT) over the inferior half of the cornea was estimated with the use of interference color analysis and the preprocessing of images with and without ring segmentation were obtained and analyzed. Moreover, LLTs before and after 1 h of applying topical ointment (Duratears, Alcon) were compared to validate the estimation of LLT. Our results suggested that the tear LLT can be assessed using a placido-disk-based tear film analyzer and interference color analysis. We verified a high correlation between non-segmented and segmented LL images and estimated LLT increase after applying ointment. In addition, we concluded that LLT can be evaluated by direct interference analysis without segmentation preprocessing.

Increase lipid tear thickness after botulinum neurotoxin A injection in patients with blepharospasm and hemifacial spasm.

The aim of this study was to investigate changes in the tear film lipid layer thickness (LLT) and aqueous tear production after botulinum neurotoxin A (BoNT) injection in patients with benign essential blepharospasm (BEB) and hemifacial spasm (HFS). Eleven and six patients with BEB and HFS, respectively, who received BoNT injection were consecutively enrolled in this prospective study. The blepharospasm disability index (BSDI), blink pattern, dry eye symptoms, Schirmer test 1 findings, LLT, eyelid performance, and corneal integrity were evaluated before and after treatment. Both BEB and HSF patients experienced remarkable relief from spasms and ocular discomfort after BoNT injection. LLT, the partial blink rate, the snap-back time, the lid distraction distance, and lateral canthal laxity were significantly increased at 1 month after treatment. There were no significant changes in Schirmer test 1 findings and meibomian gland dropout. Our findings suggest that LLT, a decisive factor for tear film stability, significantly increases at 1 month after BoNT injection for BEB and HFS. A decrease in BSDI and an increase in the snap-back time may contribute to the increase in LLT; this mechanism is probably responsible for the relief from dryness after BoNT injection in patients with facial movement disorders.

Molecular Bioburden of the Lens Storage Case for Contact Lens-Related Keratitis.

PURPOSE: To elucidate the bioburden of a lens care system in patients with contact lens (CL)-related keratitis. METHODS: In this prospective case-control study, the CL storage case was used as the target of bioburden assessment. Participants were CL wearers with (n = 26) or without (n = 24) keratitis in southern Taiwan. The case fluid sample was consecutively collected and assessed using a dot hybridization assay (DHA) for bioburden assessment. The DHA was composed of 3 universal bacterial probes for detecting all bacteria, genus-specific probes for Pseudomonas and Acinetobacter, and 1 probe for Acanthamoeba. The receiver operating characteristic curve was used to analyze the differentiating performance of a probe. RESULTS: The storage case bioburden was significantly higher in those with CL-related keratitis than in control subjects; patients with CL-related keratitis had stronger standardized signals in all universal bacterial probes. Moreover, in the cases of these patients, the bioburden was significantly heavier in confirmed infectious keratitis than in presumed noninfectious keratitis. Receiver operating characteristic analysis showed that stronger signals in all 3 universal bacterial probes and the Pseudomonas probe might suggest that the wearers have infectious keratitis. Only the storage case of the Acanthamoeba keratitis case showed positive detection by the Acanthamoeba probe. CONCLUSIONS: A heavier bioburden in the lens storage case was associated with a higher risk of CL-related keratitis and infectious keratitis. Inappropriate maintenance of the CL will lead to microbial contamination and transfer the pathogen onto the ocular surface causing keratitis accordingly. The DHA assessment for the lens storage case might provide an alternative way to differentiate infectious from noninfectious CL-related keratitis.

Pulmonary nocardiosis in southern Taiwan.

BACKGROUND/PURPOSE: Nocardiosis mainly affects immunocompromised patients. The objectives of this study were to better understand the epidemiologic, demographic, clinical, and laboratory information in patients with pulmonary nocardiosis in southern Taiwan. METHODS: Retrospective analyzing patients aged ≥18 years with culture-proven pulmonary nocardiosis received treatment at KCGMH between January 2004 and June 2010. Nocardiae were identified by 16S rRNA gene sequence analysis. Patients with pulmonary nocardiosis caused by the mostly commonly encountered Nocardia sp. were compared with those with pulmonary nocardiosis due to other Nocardia spp. RESULTS: Among the 20 patients included, cough (80%) and fever (50%) were the 2 leading symptoms/signs, while lobar consolidation (50%) and pleural effusion (40%) were the most frequent radiographic manifestations. Eighteen patients (90%) had at least one underlying disease/condition. Nocardia cyriacigeorgica was most commonly found. Compared with those whose pathogens were other Nocardia spp., patients with pulmonary nocardiosis caused by N cyriacigeorgica experienced higher clinical severity as measured by APACHE II score (19.8 ± 7.0 vs. 12.8 ± 6.7; p = 0.04) and ICU admission rate (100% vs. 25%; p < 0.01). Thirteen patients (65%) turned out to be fatal. The severity (APACHE II score, 18 ± 6 vs. 10 ± 8; p = 0.02) and the proportion of acute and subacute pulmonary nocardiosis (76% vs. 0%, p = 0.03) between fatal and survived patients differed significantly. CONCLUSIONS: N cyriacigeorgica was the most common pathogen in southern Taiwan. Higher mortality rate in patients with pulmonary nocardiosis was related to disease severity and acute and subacute infection.

Mannose-binding lectin gene polymorphisms and mycobacterial lymphadenitis in young patients.

BACKGROUND: Tuberculosis (TB) has recently re-emerged as a major public health threat worldwide. There is strong evidence that host genetic factors influence individual susceptibility to TB and that, once infected, young children and immunocompromised patients are at increased risk for mycobacterial disease and progression to extrapulmonary lymphadenitis. METHODS: The association between polymorphisms of mannose-binding lectin and the susceptibility of 139 children with cervical mycobacterial lymphadenitis and infected with Mycobacterium tuberculosis was investigated. RESULTS: The frequencies of genotypes A/B and B/B, based on codon 54 polymorphisms, were significantly different in TB-infected versus healthy control subjects. The frequency of the A/B genotype was significantly lower in TB-infected children (odds ratio = 0.56; 95% confidence interval: 0.36-0.87; P = 0.01), and the B/B genotype was significantly higher in TB-infected children (odds ratio = 4.68; 95% confidence interval: 1.35-16.3; P = 0.01) compared with healthy controls. The HYB haplotype appeared significantly more often to be protective in the healthy control population (odds ratio = 0.23; 95% confidence interval: 0.05-0.96; P = 0.046). Ex vitro phagocytosis assays indicated that high-expression mannose-binding lectin genotypes are associated with an increased risk of infection with M. tuberculosis. CONCLUSIONS: The present study suggests that mannose-binding lectin can protect against TB or predispose the host to the disease depending on the haplotype pair of the host. The low-expression genotype A/B and the HYB haplotype may be associated with protection against intracellular mycobacterial infections, whereas the high-expression genotype A/A may confer susceptibility to disease.