The mechanisms that maintain sterility in the urinary tract are incompletely understood; however, recent studies stress the importance of antimicrobial peptides in protecting the urinary tract from infection. Ribonuclease 7 (RNase 7), a potent antimicrobial peptide contributing to urinary tract sterility, is expressed by intercalated cells in the renal collecting tubules and is present in the urine at levels sufficient to kill bacteria at baseline. Here, we characterize the expression and function of RNase 7 in the human urinary tract during infection. Both quantitative real-time PCR and ELISA assays demonstrated increases in RNASE7 expression in the kidney along with kidney and urinary RNase 7 peptide concentrations with infection. While immunostaining localized RNase 7 production to the intercalated cells of the collecting tubule during sterility, its expression during pyelonephritis was found to increase throughout the nephron but not in glomeruli or the interstitium. Recombinant RNase 7 exhibited antimicrobial activity against uropathogens at low micromolar concentrations by disrupting the microbial membrane as determined by atomic force microscopy. Thus, RNase 7 expression is increased in the urinary tract with infection, and has antibacterial activity against uropathogens at micromolar concentrations.

Beta defensins (BDs) are cationic peptides with antimicrobial activity that defend epithelial surfaces including the skin, gastrointestinal, and respiratory tracts. However, BD expression and function in the urinary tract are incompletely characterized. The purpose of this study was to describe Beta Defensin-1 (BD-1) expression in the lower urinary tract, regulation by cystitis, and antimicrobial activity toward uropathogenic Escherichia coli (UPEC) in vivo. Human DEFB1 and orthologous mouse Defb1 mRNA are detectable in bladder and ureter homogenates, and human BD-1 protein localizes to the urothelium. To determine the relevance of BD-1 to lower urinary tract defense in vivo, we evaluated clearance of UPEC by Defb1 knockout (Defb1 -/-) mice. At 6, 18, and 48 hours following transurethral UPEC inoculation, no significant differences were observed in bacterial burden in bladders or kidneys of Defb1 -/- and wild type C57BL/6 mice. In wild type mice, bladder Defb1 mRNA levels decreased as early as two hours post-infection and reached a nadir by six hours. RT-PCR profiling of BDs identified expression of Defb3 and Defb14 mRNA in murine bladder and ureter, which encode for mBD-3 and mBD-14 protein, respectively. MBD-14 protein expression was observed in bladder urothelium following UPEC infection, and both mBD-3 and mBD-14 displayed dose-dependent bactericidal activity toward UPEC in vitro. Thus, whereas mBD-1 deficiency does not alter bladder UPEC burden in vivo, we have identified mBD-3 and mBD-14 as potential mediators of mucosal immunity in the lower urinary tract.

Recent studies stress the importance of antimicrobial peptides in protecting the urinary tract from infection. Previously, we have shown that ribonuclease 7 (RNase 7) is a potent antimicrobial peptide that has broad-spectrum antimicrobial activity against uropathogenic bacteria. The urothelium of the lower urinary tract and intercalated cells of the kidney produce RNase 7 but regulation of its antimicrobial activity has not been well defined. Here we characterize the expression of an endogenous inhibitor, ribonuclease inhibitor (RI), in the urinary tract and evaluate its effect on RNase 7’s antimicrobial activity. Using RNA isolated from non-infected human bladder and kidney tissue, quantitative real-time PCR showed that RNH1, the gene encoding RI, is constitutively expressed throughout the urinary tract. With pyelonephritis, RNH1 expression and RI peptide production significantly decrease. Immunostaining localized RI production to the umbrella cells of the bladder and intercalated cells of the renal collecting tubule. In vitro assays showed that RI bound to RNase 7 and suppressed its antimicrobial activity by blocking its ability to bind the cell wall of uropathogenic bacteria. Thus, these results demonstrate a new immunomodulatory role for RI and identified a unique regulatory pathway that may affect how RNase 7 maintains urinary tract sterility.

Rapid detection of group A beta-hemolytic streptococcus (GAS) is used routinely to help diagnose and treat pharyngitis. However, available rapid antigen detection tests for GAS have relatively low sensitivity, and backup testing is recommended in children. Newer assays are more sensitive yet require excessive time for practical point-of-care use as well as laboratory personnel. The Alere i strep A test is an isothermal nucleic acid amplification test designed to offer highly sensitive results at the point of care within 8 min when performed by nonlaboratory personnel. The performance of the Alere i strep A test was evaluated in a multicenter prospective trial in a Clinical Laboratory Improvement Amendments (CLIA)-waived setting in comparison to bacterial culture in 481 children and adults. Compared to culture, the Aleri i strep A test had 96.0% sensitivity and 94.6% specificity. Discrepant results were adjudicated by PCR and found the Alere i strep A test to have 98.7% sensitivity and 98.5% specificity. Overall, the Alere i strep A test could provide a one-step, rapid, point-of-care testing method for GAS pharyngitis and obviate backup testing on negative results. In developed countries, 15% of school-age children and 4% to 10% of adults will have an episode of group A streptococcus (GAS) pharyngitis each year (1). The accurate diagnosis and treatment of GAS pharyngitis is necessary to reduce symptoms, prevent suppurative and nonsuppurative sequelae, reduce transmission, and avoid unnecessary antibiotic use (1). Yet, clinical features and prediction rules alone are not adequate to make a diagnosis (2). Given that current point-of-care rapid antigen detection tests (RADT) for GAS have a limited sensitivity of approximately 85% (3), current guidelines recommend confirming negative results in children with a backup method, most commonly by culture (1). However, this two-step process adds additional time and costs, and it risks that some patients will be lost to follow-up (4). The Alere i strep A test is a new isothermal nucleic acid amplification test using a nicking enzyme amplification reaction (NEAR) that can provide results in under 8 min in a point-of-care (POC) setting. We performed a prospective, multicenter trial to evaluate the performance of the Alere i strep A test in comparison to bacterial culture, as the reference standard, with PCR adjudication for discrepant results. MATERIALS AND METHODSWe prospectively enrolled subjects at 10 clinical sites within the United States (in Florida, Georgia, Nebraska, New Jersey, New York, and Ohio) from 21 January to 14 March 2014. The sites included emergency departments (general and pediatric), an urgent care center, and private practices (clinical and research focused). Subjects were eligible if they presented with complaints of a sore throat and signs of suspected pharyngitis: pharyngeal erythema, tender cervical lymphadenopathy, swollen tonsils, palatal petechiae, or scarlatiniform rash. Subjects were excluded if they had systemic antibiotic use in the past 2 wee...

Objectives: Point-of-care ultrasound (POCUS) has been identified as a critical skill for pediatric emergency medicine (PEM) physicians. The purpose of this study was to profile the current status of PEM POCUS in pediatric emergency departments (EDs).Methods: An electronic survey was distributed to PEM fellows and attending physicians at four major pediatric academic health centers. The 24-item questionnaire covered professional demographics, POCUS experience and proficiency, and barriers to the use of POCUS in pediatric EDs. We used descriptive and inferential statistics to profile respondent's PEM POCUS experience and proficiency and Rasch analysis to evaluate barriers to implementation.Results: Our return rate was 92.8% (128/138). Respondents were attending physicians (68%) and fellows (28%).Most completed pediatric residencies prior to PEM fellowship (83.6%). Almost all had some form of ultrasound education (113/128, 88.3%). Approximately half (46.9%) completed a formal ultrasound curriculum. More than half (53.2%) said their ultrasound education was pediatric-specific. Most participants (67%) rated their POCUS proficiency low (Levels 1-2), while rating proficiency in other professional competencies (procedures 52%, emergency stabilization 70%) high (Levels 4-5). There were statistically significant differences in POCUS proficiency between those with formal versus informal ultrasound education (p < 0.001) and those from pediatric versus emergency medicine residencies (p < 0.05). Participants identified both personal barriers discomfort with POCUS skills (76.7%), insufficient educational time to learn POCUS (65%), and negative impact of POCUS on efficiency (58.5%)-and institutional barriers to the use of ultrasound-consultants will not use ultrasound findings from the ED (60%); insufficient mentoring (64.7%), and POCUS not being a departmental priority (57%).Conclusions: While POCUS utilization continues to grow in PEM, significant barriers to full implementation still persist. One significant barrier relates to the need for dedicated time to learn and practice POCUS to achieve sufficient levels of proficiency for use in practice.