Effect of digital ocular massage on intraocular pressure and Schlemm's canal dimensions.

Digital ocular massage has been reported to temporarily lower intraocular pressure (IOP). This could be related to an enhanced aqueous humor outflow; however, the mechanism is not clearly understood. Using anterior segment optical coherence tomography, the Schlemm's canal (SC) and trabecular meshwork (TM) can be imaged and measured. Here, 66 healthy adults underwent digital ocular massage for 10 min in their right eyes. The IOP and dimensions of the SC and TM were measured before and after ocular massage. All subjects demonstrated IOP reduction from 15.7 ± 2.5 mmHg at baseline to 9.6 ± 2.2 mmHg immediately after, and median of 11.6 mmHg 5-min after ocular massage (Friedman's test, p < 0.001). There was significant change in SC area (median 10,063.5 µm2 at baseline to median 10,151.0 µm2 after ocular massage, Wilcoxon test, p = 0.02), and TM thickness (median 149.8 µm at baseline to 144.6 ± 25.3 µm after ocular massage, Wilcoxon test, p = 0.036). One-third of the subjects demonstrated collapse of the SC area (-2 to -52%), while two-thirds showed expansion of the SC area (2 to 168%). There were no significant changes in SC diameter (270.4 ± 84.1 µm vs. 276.5 ± 68.7 µm, paired t-test, p = 0.499), and TM width (733.3 ± 110.1 µm vs. 733.5 ± 111.6 µm, paired t-test, p = 0.988). Eyes with a higher baseline IOP demonstrated a greater IOP reduction (Pearson correlation coefficient r = -0.521, p < 0.001). Eyes with smaller SC area at baseline showed greater SC area expansion (Pearson correlation coefficient = -0.389, p < 0.001). Greater IOP reduction appeared in eyes with greater SC area expansion (Pearson correlation coefficient r = -0.306, p = 0.01). Association between change in IOP and change in TM thickness was not significant (Spearman's ρ = 0.015, p = 0.902). Simple digital ocular massage is an effective method to lower IOP values, and change in the SC area was significantly associated with IOP changes.

In Silico Serotyping Based on Whole-Genome Sequencing Improves the Accuracy of Shigella Identification.

Bacteria of the genus Shigella, consisting of 4 species and >50 serotypes, cause shigellosis, a foodborne disease of significant morbidity, mortality, and economic loss worldwide. Classical Shigella identification based on selective media and serology is tedious, time-consuming, expensive, and not always accurate. A molecular diagnostic assay does not distinguish Shigella at the species level or from enteroinvasive Escherichia coli (EIEC). We inspected genomic sequences from 221 Shigella isolates and observed low concordance rates between conventional designation and molecular serotyping: 86.4% and 80.5% at the species and serotype levels, respectively. Serotype determinants for 6 additional serotypes were identified. Examination of differentiation gene markers commonly perceived as characteristic hallmarks in Shigella showed high variability among different serotypes. Using this information, we developed ShigaTyper, an automated workflow that utilizes limited computational resources to accurately and rapidly determine 59 Shigella serotypes using Illumina paired-end whole-genome sequencing (WGS) reads. Shigella serotype determinants and species-specific diagnostic markers were first identified through read alignment to an in-house curated reference sequence database. Relying on sequence hits that passed a threshold level of coverage and accuracy, serotype could be unambiguously predicted within 1 min for an average-size WGS sample of ∼500 MB. Validation with WGS data from 380 isolates showed an accuracy rate of 98.2%. This pipeline is the first step toward building a comprehensive WGS-based analysis pipeline of Shigella spp. in a field laboratory setting, where speed is essential and resources need to be more cost-effectively dedicated.IMPORTANCEShigella causes diarrheal disease with serious public health implications. However, conventional Shigella identification methods are laborious and time-consuming and can be erroneous due to the high similarity between Shigella and enteroinvasive Escherichia coli (EIEC) and cross-reactivity between serotyping antisera. Further, serotype interpretation is complicated for inexperienced users. To develop an easier method with higher accuracy based on whole-genome sequencing (WGS) for Shigella serotyping, we systematically examined genomic information of Shigella isolates from 53 serotypes to define rules for differentiation and serotyping. We created ShigaTyper, an automated pipeline that accurately and rapidly excludes non-Shigella isolates and identifies 59 Shigella serotypes using Illumina paired-end WGS reads. A serotype can be unambiguously predicted at a data processing speed of 538 MB/min with 98.2% accuracy from a regular laptop. Once it is installed, training in bioinformatics analysis and Shigella genetics is not required. This pipeline is particularly useful to general microbiologists in field laboratories.

Comparison of IMS Platforms for detecting and recovering Escherichia coli O157 and Shigella flexneri in foods.

Two automated platforms using immunomagnetic separation technology were compared for detecting and recovering Escherichia coli O157 in ground beef and sprouts and Shigella flexneri in green onions. The foods were inoculated with <20 CFU/25 g and tested at 5 and 24 h postincubation. Immunomagnetic beads were mixed with food enrichments, processed through the Pathatrix Auto or KingFisher Flex, and tested by real-time PCR (qPCR) and recovery on selective agars. At 5 h, the Pathatrix Auto detected E. coli O157 in 90% and 60% of the ground beef and sprouts samples and S. flexneri in all of the green onion samples. It also recovered E. coli O157 in all the samples but could not recover S. flexneri in any of the green onion samples. In comparison, the KingFisher Flex detected E. coli O157 in 80% and 30% of the ground beef and sprouts samples and S. flexneri in all of the green onion samples. It also recovered E. coli O157 in 90% of the ground beef samples but none of the sprouts samples and S. flexneri in 20% of the green onion samples. At 24 h, both platforms detected and recovered the target bacteria in all of the samples.

O serogroup specific real time PCR assays for the detection and identification of nine clinically relevant non-O157 STECs.

TaqMan™ real time PCR assays were designed for each of the non-O157 STEC O serogroups most commonly associated with human illness: O26, O45, O91, O103, O111, O113, O121, O128, and O145. The nine RT-PCR assays can be run as single assays when a known pathogen is of concern, or multiplexed in three reactions, to quickly screen for the most clinically relevant O serogroups. All assays included an internal amplification control constructed from the green fluorescent protein gene as an indicator of PCR inhibition. Of 103 strains tested, the inclusive tests accurately identified the O serogroup for 101 strains. The exclusive tests for each assay yielded no false positives for over 120 Escherichia coli strains and 23 non-E. coli bacteria tested. Furthermore, the RT-PCR assays were tested by inoculating four food matrices, milk, apple juice, lettuce, and ground beef, at ≤30 CFU/25 g or mL. Following a 24h selective enrichment, the RT-PCR assays detected STECs in all foods except for one ground beef sample inoculated with O111, and all apple juice samples inoculated with O113. The assays could also detect each O serogroup in human stool specimens inoculated with STECs at 1000 CFU/0.5 g of stool following 24 h enrichment.

Recirculating immunomagnetic separation and optimal enrichment conditions for enhanced detection and recovery of low levels of Escherichia coli O157:H7 from fresh leafy produce and surface water.

The objective of this study was to develop a rapid, simple method for enhanced detection and isolation of low levels of Escherichia coli O157:H7 from leafy produce and surface water using recirculating immunomagnetic separation (RIMS) coupled with real-time PCR and a standard culture method. The optimal enrichment conditions for the method also were determined. Analysis of real-time PCR data (C(T) values) suggested that incubation of lettuce and spinach leaves rather than rinsates provides better enrichment of E. coli O157:H7. Enrichment of lettuce or spinach leaves at 42 degrees C for 5 h provided better detection than enrichment at 37 degrees C. Extended incubation of surface water for 20 h at 42 degrees C did not improve the detection. The optimized enrichment conditions were also employed with modified Moore swabs, which were used to sample flowing water sites. Positive isolation rates and real-time PCR results indicated an increased recovery of E. coli O157:H7 from all samples following the application of RIMS. Under these conditions, the method provided detection and/or isolation of E. coli O157:H7 at levels as low as 0.07 CFU/g of lettuce, 0.1 CFU/g of spinach, 6 CFU/100 ml of surface water, and 9 CFU per modified Moore swab. During a 6-month field study, modified Moore swabs yielded high isolation rates when deployed in natural watershed sites. The method used in this study was effective for monitoring E. coli O157:H7 in the farm environment, during postharvest processing, and in foodborne outbreak investigations.