Antibiotic Overtreatment of Presumed Urinary Tract Infection Among Children with Spina Bifida.

OBJECTIVE: To identify factors associated with overtreatment of presumed urinary tract infection (UTI) among children with spina bifida using such criteria. STUDY DESIGN: A retrospective review of children with spina bifida (age <21 years) evaluated in the Emergency Department (ED) at a single institution was performed. Patients with a urinalysis (UA) performed who were reliant on assisted bladder emptying were included. The primary outcome was overtreatment, defined as receiving antibiotics for presumed UTI but ultimately not meeting spina bifida UTI criteria (≥2 urologic symptoms plus pyuria and urine culture growing >100k CFU/mL). The primary exposure was whether the components of the criteria available at the time of the ED visit (≥2 urologic symptoms plus pyuria) were met when antibiotics were initiated. RESULTS: Among 236 ED encounters, overtreatment occurred in 80% of cases in which antibiotics were initiated (47% of the entire cohort). Pyuria with <2 urologic symptoms was the most important factor associated with overtreatment (OR 9.6). Non-Hispanic White race was associated with decreased odds of overtreatment (OR 0.3). CONCLUSIONS: Overtreatment of presumed UTI among patients with spina bifida was common. Pyuria, which is not specific to UTI in this population, was the main driver of overtreatment. Symptoms are a cornerstone of UTI diagnosis among children with spina bifida, should be collected in a standardized manner, and considered in a decision to treat.

A Novel Propidium Monoazide-Based PCR Assay Can Measure Viable Uropathogenic E. coli In Vitro and In Vivo.

BACKGROUND: Polymerase chain reaction (PCR) is an important means by which to study the urine microbiome and is emerging as possible alternative to urine cultures to identify pathogens that cause urinary tract infection (UTI). However, PCR is limited by its inability to differentiate DNA originating from viable, metabolically active versus non-viable, inactive bacteria. This drawback has led to concerns that urobiome studies and PCR-based diagnosis of UTI are confounded by the presence of relic DNA from non-viable bacteria in urine. Propidium monoazide (PMA) dye can penetrate cells with compromised cell membranes and covalently bind to DNA, rendering it inaccessible to amplification by PCR. Although PMA has been shown to differentiate between non-viable and viable bacteria in various settings, its effectiveness in urine has not been previously studied. We sought to investigate the ability of PMA to differentiate between viable and non-viable bacteria in urine. METHODS: Varying amounts of viable or non-viable uropathogenic E. coli (UTI89) or buffer control were titrated with mouse urine. The samples were centrifuged to collect urine sediment or not centrifuged. Urine samples were incubated with PMA and DNA cross-linked using blue LED light. DNA was isolated and uidA gene-specific PCR was performed. For in vivo studies, mice were inoculated with UTI89, followed by ciprofloxacin treatment or no treatment. After the completion of ciprofloxacin treatment, an aliquot of urine was plated on non-selective LB agar and another aliquot was treated with PMA and subjected to uidA-specific PCR. RESULTS: PMA's efficiency in excluding DNA signal from non-viable bacteria was significantly higher in bacterial samples in phosphate-buffered saline (PBS, dCT=13.69) versus bacterial samples in unspun urine (dCT=1.58). This discrepancy was diminished by spinning down urine-based bacterial samples to collect sediment and resuspending it in PBS prior to PMA treatment. In 3 of 5 replicate groups of UTI89-infected mice, no bacteria grew in culture; however, there was PCR amplification of E. coli after PMA treatment in 2 of those 3 groups. CONCLUSION: We have successfully developed PMA-based PCR methods for amplifying DNA from live bacteria in urine. Our results suggest that non-PMA bound DNA from live bacteria can be present in urine, even after antibiotic treatment. This indicates that viable but non-culturable E. coli can be present following treatment of UTI, and may explain why some patients have persistent symptoms but negative urine cultures following UTI treatment.

Novel urine biomarkers to distinguish UTI from culture-negative pyuria.

BACKGROUND: Emergency departments (EDs) often rely on urinalysis (UA) to rapidly identify urinary tract infections (UTIs) in children. However, the suboptimal test characteristics of UA can lead to false-positive results. Novel urinary biomarkers may increase the diagnostic precision of UA. In this study, we compared the concentrations of 6 pre-selected proteins: BH3 interacting domain death agonist (BID), B-cell lymphoma 6 protein, ras GTPase-activating protein 1, cathepsin S (CTSS), 3-hydroxyanthranilate 3,4-dioxygenase, and transgelin-2. METHODS: In a pediatric ED, we prospectively enrolled 167 children with UA and urine culture collected. Pyuria was defined as either ≥ 5 white blood cells per high-power field on microscopy or positive leukocyte esterase (LE). The urine culture was considered positive if it yielded ≥ 50,000 colony-forming units per milliliter of any single urinary pathogen. Urine protein levels were measured by enzyme-linked immunosorbent assay and normalized to urine creatinine. RESULTS: BID was significantly higher in the UTI group compared to the culture-negative pyuria group with a mean ratio of 1.42 (95% confidence interval (CI), 1.15, 1.76) when uncorrected for creatinine concentration. When corrected for creatinine concentration, CTSS was significantly elevated in the UTI group compared to the culture-negative pyuria group with a mean ratio of 2.11 (95% CI, 1.39, 3.21). CONCLUSIONS: BID and CTSS concentrations were elevated in the urine of children with UTI compared to those with culture-negative pyuria. These proteins deserve further research into their utility to serve as novel biomarkers for UTI.

Metabolic dysfunction and immunometabolism in COVID-19 pathophysiology and therapeutics.

The COVID-19 pandemic has emerged as a public health crisis and has placed a significant burden on healthcare systems. Patients with underlying metabolic dysfunction, such as type 2 diabetes mellitus and obesity, are at a higher risk for COVID-19 complications, including multi-organ dysfunction, secondary to a deranged immune response, and cellular energy deprivation. These patients are at a baseline state of chronic inflammation associated with increased susceptibility to the severe immune manifestations of COVID-19, which are triggered by the cellular hypoxic environment and cytokine storm. The altered metabolic profile and energy generation of immune cells affect their activation, exacerbating the imbalanced immune response. Key immunometabolic interactions may inform the development of an efficacious treatment for COVID-19. Novel therapeutic approaches with repurposed drugs, such as PPAR agonists, or newly developed molecules such as the antagomirs, which block microRNA function, have shown promising results. Those treatments, alone or in combination, target both immune and metabolic pathways and are ideal for septic COVID-19 patients with an underlying metabolic condition.

Optimizing Rapid Sequence Intubation for Medical and Trauma Patients in the Pediatric Emergency Department.

INTRODUCTION: Rapid sequence intubation (RSI) is a critical procedure for severely ill and injured patients presenting to the pediatric emergency department (PED). This procedure has a high risk of complications, and multiple attempts increase this risk. We aimed to increase successful intubation within two attempts, focusing on medical and trauma patients separately to identify improvement barriers for each group. METHODS: A multifaceted intervention was implemented using quality improvement methods. The analysis included adherence to the standardized process, successful intubation within two attempts, and frequency of oxygen saturations <92% during laryngoscopy. Trauma and medical patients were analyzed separately as team composition differed for each. RESULTS: This project began in February 2018, and we included 290 patients between April 2018 and December 2019. Adherence to the standardized process was sustained at 91% for medical patients and a baseline of 55% for trauma patients with a trend toward improvement. In May 2018, we observed and sustained special cause variations for medical patients' successful intubations within two attempts (77-89%). In September 2018, special cause variation was observed and sustained for the successful intubation of trauma patients within two attempts (89-96%). The frequency of oxygen saturation of <92% was 21% for medical patients; only one trauma patient experienced oxygen desaturation. CONCLUSION: Implementation of a standardized process significantly improved successful intubations within two attempts for medical and trauma patients. Trauma teams had more gradual adherence to the standardized process, which may be related to the relative infrequency of intubations and variable team composition.

Natural killer cell functional defects in pediatric patients with severe and recurrent herpesvirus infections.

Natural killer (NK) cells play a critical role in the host defense against herpesviruses. Although herpesviruses are ubiquitous in human populations, only a minority of people experience severe recurrent infections. We hypothesize that uncharacterized NK cell functional deficits predispose individuals to more significant or frequent herpesvirus infections and reactivations. To investigate this hypothesis, we broadly analyzed NK cell phenotype and functional responses in a cohort of predominantly pediatric patients with recurrent and/or severe herpesvirus infections and compared them to a healthy control population. Our results identified no global differences in cytolysis, degranulation, interferon-γ production, or surface receptor upregulation following cytokine stimulation. However, abnormal NK cell functional responses were observed in nearly one-third of patients (including 3 with hyporesponsiveness to activating signals and 1 with markedly decreased CD11b expression associated with reduced cytotoxicity and degranulation), which might contribute to those individuals' susceptibility to herpesvirus infections.

Sex differences in murine susceptibility to systemic viral infections.

Increased susceptibility to autoimmunity in females is often viewed as the consequence of enhanced immunoreactivity providing superior protection against infections. We paradoxically observed greater mortality in female compared to male mice during systemic viral infections with three large double-stranded DNA viruses (herpes simplex virus type I [HSV], murine cytomegalovirus [MCMV], and vaccinia virus [VV]). Indeed, female mice were 27-fold more susceptible to infection with HSV than male mice. Elimination of estrogen by ovariectomy in female mice or addition of estrogen to castrated male mice only partially eliminated the observed sex differences following HSV infection. However, the differences observed in survival between female and male mice were nearly abrogated in the absence of type I interferon receptor signaling and substantially mitigated in absence of DAP12 signaling. Interestingly, the sex-specific impact of type I interferon receptor and DAP12 signaling differentially influenced survival during systemic viral infections with type I interferon receptor signaling enhancing male survival and DAP12 signaling increasing the susceptibility of female mice. These results have potential implications for the sex disparities observed in human autoimmune disorders.

Prevalence of antibodies against seasonal influenza A and B viruses during the 2009-2010 and 2010-2011 influenza seasons in residents of Pittsburgh, PA, USA.

Seroprevalence of antibodies against influenza viruses from 1000 people between the ages of 0 to 90 years of age (100 samples for each decade of life) in the Pittsburgh, PA, USA was measured. One year removed from the outbreak of novel H1N1 influenza into the human population in the Northern Hemisphere and following the emergence of a new H3N2 influenza isolate, sera was collected to determine the hemagglutination-inhibition antibodies against influenza A/H1N1, A/H3N2, and B viruses representative of viruses in the vaccine used for the 2010-2011 influenza season. The seroprevalence of antibodies to influenza virus, A/California/7/2009 (H1N1), increased from the previously reported November 2009 samples and the samples collected at the end of the 2010 influenza season (June 2010) during the 2010-2011 season in all age groups, but people the under the age of 20 had the highest rise in the number of positive samples. The number of individuals positive for H1N1 stayed the same through the entire influenza season. In contrast, there were little to no positive serum samples against the H3N2 virus, A/Perth/16/2009, from samples collected during the 2009-2010 influenza season, however, titers against these viruses rose significantly during the early months of the 2010-2011 season with the highest number of positive samples detected in the very young and very old populations. However, these titers waned by May, 2011 in those over the age of 40. There was a rise in adults to the B/Brisbane/60/2008 influenza virus in adults in samples collected in October, 2010, but these titers quickly declined. The highest titers to B influenza were detected in people between the ages of 10-30 years of age. These findings may have implications for the development of vaccination strategies aiming at the protection against seasonal and/or pandemic influenza virus infection and pre-pandemic preparedness activities.

Ly49H engagement compensates for the absence of type I interferon signaling in stimulating NK cell proliferation during murine cytomegalovirus infection.

NK cells vigorously proliferate during viral infections, resulting in an expanded pool of innate lymphocytes that are able to participate in early host defense. The relative contributions of cytokines and activation receptors in stimulating NK cell proliferation during viral infections are not well characterized. In this study, we demonstrated that signaling through the NK cell activation receptor Ly49H was able to compensate for the absence of cytokine stimulation in the preferential phase of viral-induced proliferation during murine cytomegalovirus infection. In the absence of type I IFN stimulation, NK cell proliferation was strongly biased toward cells expressing the Ly49H receptor, even at early time points when minimal preferential Ly49H-mediated proliferation was observed in wild-type mice. In the absence of effective Ly49H signaling or following infection with virus that did not express the ligand for Ly49H, no difference was observed in the proliferation of subsets of NK cells that either express or lack expression of Ly49H, although the overall proliferation of NK cells in IFNalphabetaR(-/-) mice was substantially reduced. These results highlight the contribution of NK cell activation receptors in stimulating proliferation and subsequent expansion of NK cells that are able to recognize virally infected cells.