The Prevalence and Association of Different Uropathogens Detected by M-PCR with Infection-Associated Urine Biomarkers in Urinary Tract Infections.

Background: Many emerging uropathogens are currently identified by multiplex polymerase chain reaction (M-PCR) in suspected UTI cases. Standard urine culture (SUC) has significantly lower detection rates, raising questions about whether these organisms are associated with UTIs and truly cause inflammation. Objective: To determine if microbes detected by M-PCR were likely causative of UTI by measuring inflammatory biomarkers in the urine of symptomatic patients. Design Setting and Participants: Midstream voided urine was collected from subjects ≥60 years presenting to urology clinics with symptoms of UTI (n = 1132) between 01/2023 and 05/2023. Microbe detection was by M-PCR and inflammation-associated biomarker (neutrophil gelatinase-associated lipocalin, interleukin 8, and interleukin 1ß) was by enzyme-linked immunosorbent assay. Biomarker positivity was measured against individual and groups of organisms, E. coli and non-E. coli cases, emerging uropathogens, monomicrobial and polymicrobial cases. Outcome Measurements and Statistical Analysis: Distributions were compared using 2-sample Wilcoxon Rank Sum test with 2-tailed p-values < 0.05 considered statistically significant. Results and Limitations: M-PCR was positive in 823 (72.7%) specimens with 28 of 30 (93%) microorganisms/groups detected. Twenty-six of twenty-eight detected microorganisms/groups (93%) had ≥2 biomarkers positive in >66% of cases. Both non-E. coli cases and E. coli cases had significant biomarker positivity (p < 0.05). Limitations were that a few organisms had low prevalence making inferences about their individual significance difficult. Conclusion: The majority of microorganisms identified by M-PCR were associated with active inflammation measured by biomarker positivity, indicating they are likely causative of UTIs in symptomatic patients. This includes emerging uropathogens frequently not detected by standard urine culture.

Urine biomarkers individually and as a consensus model show high sensitivity and specificity for detecting UTIs.

BACKGROUND: Current diagnoses of urinary tract infection (UTI) by standard urine culture (SUC) has significant limitations in sensitivity, especially for fastidious organisms, and the ability to identify organisms in polymicrobial infections. The significant rate of both SUC "negative" or "mixed flora/contamination" results in UTI cases and the high prevalence of asymptomatic bacteriuria indicate the need for an accurate diagnostic test to help identify true UTI cases. This study aimed to determine if infection-associated urinary biomarkers can differentiate definitive UTI cases from non-UTI controls. METHODS: Midstream clean-catch voided urine samples were collected from asymptomatic volunteers and symptomatic subjects ≥ 60 years old diagnosed with a UTI in a urology specialty setting. Microbial identification and density were assessed using a multiplex PCR/pooled antibiotic susceptibility test (M-PCR/P-AST) and SUC. Three biomarkers [neutrophil gelatinase-associated lipocalin (NGAL), and Interleukins 8 and 1ß (IL-8, and IL-1ß)] were also measured via enzyme-linked immunosorbent assay (ELISA). Definitive UTI cases were defined as symptomatic subjects with a UTI diagnosis and positive microorganism detection by SUC and M-PCR, while definitive non-UTI cases were defined as asymptomatic volunteers. RESULTS: We observed a strong positive correlation (R2 > 0.90; p < 0.0001) between microbial density and the biomarkers NGAL, IL-8, and IL-1ß for symptomatic subjects. Biomarker consensus criteria of two or more positive biomarkers had sensitivity 84.0%, specificity 91.2%, positive predictive value 93.7%, negative predictive value 78.8%, accuracy 86.9%, positive likelihood ratio of 9.58, and negative likelihood ratio of 0.17 in differentiating definitive UTI from non-UTI cases, regardless of non-zero microbial density. NGAL, IL-8, and IL-1ß showed a significant elevation in symptomatic cases with positive microbe identification compared to asymptomatic cases with or without microbe identification. Biomarker consensus exhibited high accuracy in distinguishing UTI from non-UTI cases. CONCLUSION: We demonstrated that positive infection-associated urinary biomarkers NGAL, IL-8, and IL-1ß, in symptomatic subjects with positive SUC and/or M-PCR results was associated with definitive UTI cases. A consensus criterion with ≥ 2 of the biomarkers meeting the positivity thresholds showed a good balance of sensitivity (84.0%), specificity (91.2%), and accuracy (86.9%). Therefore, this biomarker consensus is an excellent supportive diagnostic tool for resolving the presence of active UTI, particularly if SUC and M-PCR results disagree.

Emerging and Fastidious Uropathogens Were Detected by M-PCR with Similar Prevalence and Cell Density in Catheter and Midstream Voided Urine Indicating the Importance of These Microbes in Causing UTIs.

Introduction: This study compared microbial compositions of midstream and catheter urine specimens from patients with suspected complicated urinary tract infections to determine if emerging and fastidious uropathogens are infecting the bladder or are contaminants. Methods: Urine was collected by in-and-out catheter (n = 1000) or midstream voiding (n = 1000) from 2000 adult patients (≥60 years of age) at 17 DispatchHealth sites across 11 states. The two groups were matched by age (mean 81 years), sex (62.1% female, 37.9% male), and ICD-10-CM codes. Microbial detection was performed with multiplex polymerase chain reaction (M-PCR) with a threshold for "positive detection" ≥ 10,000 cells/mL for bacteria or any detection for yeast. Results were divided by sex. Results: In females, 28 of 30 microorganisms/groups were found by both collection methods, while in males 26 of 30 were found by both. There were significant overlaps in the detection and densities of classical uropathogens including Escherichia coli, Enterococcus faecalis, and Klebsiella pneumoniae, as well as emerging uropathogens including Actinotignum schaalii and Aerococcus urinae. In females, detection rates were slightly higher in midstream voided compared to catheter-collected (p = 0.0005) urine samples, while males showed the opposite trend (p < 0.0001). More polymicrobial infections were detected in midstream voided compared to catheter-collected samples (64.4% vs 45.7%, p < 0.0001) in females but the opposite in males (35.6% vs 47.0%, p = 0.002). Discussion: In-and-out catheter-collected and midstream voided urine specimens shared significant similarities in microbial detections by M-PCR, with some differences found for a small subset of organisms and between sexes. Conclusion: Non-invasive midstream voided collection of urine specimens for microbial detection and identification in cases of presumed UTI does not result in significantly more contamination compared to in-and-out catheter-collected specimens. Additionally, organisms long regarded as contaminants should be reconsidered as potential uropathogens.

Comparison Shows that Multiplex Polymerase Chain Reaction Identifies Infection-associated Urinary Biomarker-positive Urinary Tract Infections That Are Missed by Standard Urine Culture.

Background: Multiplex polymerase chain reaction (M-PCR) has increased sensitivity for microbial detection compared with standard urine culture (SUC) in cases diagnosed as urinary tract infections (UTIs), leading to questions whether detected microbes are likely causative of UTIs or are incidental findings. Objective: To compare infection-associated biomarker levels against M-PCR and SUC results in symptomatic cases with a presumptive diagnosis of a UTI by a urologist. Design setting and participants: Participants were ≥60 yr old and presented to urology clinics between January and April 2023 with symptoms of UTIs (n = 583). Urine microbial detection was by M-PCR and SUC. Three infection-associated biomarkers (neutrophil gelatinase-associated lipocalin, interleukin-8, and interleukin-1ß) were measured by enzyme-linked immunosorbent assay. Symptomatic cases with elevated biomarkers, detection of uropathogens, and a specialist clinical diagnosis of a UTI were considered definitive UTI cases. Outcome measurements and statistical analysis: Distributions were compared using two-sample Wilcoxon rank sum test, with two-tailed p values of <0.05 considered statistically significant. Results and limitations: In cases with M-PCR-positive/SUC-negative results (n = 80), all median biomarker levels were significantly higher (p < 0.0001) than in cases with M-PCR-negative/SUC-negative results (n = 107). Two or more biomarkers were positive in 76% of M-PCR-positive/SUC-negative specimens. Limitation was an inability to examine associations between each individual organism and inflammation. Conclusions: A significant number of M-PCR-positive/SUC-negative cases had elevated levels of infection-related urinary biomarkers, especially when infection was caused by organisms other than Escherichia coli. This is a strong indication that microbes detected by M-PCR, which would be missed by SUC, are associated with UTIs. Patient summary: We compared infection-associated biomarkers in patients diagnosed with urinary tract infections (UTIs) against the detection of microorganisms by standard urine culture (SUC) and multiplex polymerase chain reaction (M-PCR). We found that most patients with microorganisms detected by M-PCR, which were missed by SUC, had elevated markers of inflammation, indicating that these organisms were likely causative of UTIs.

Improving Patient Outcomes While Reducing Empirical Treatment with Multiplex-Polymerase-Chain-Reaction/Pooled-Antibiotic-Susceptibility-Testing Assay for Complicated and Recurrent Urinary Tract Infections.

This study compared rates of empirical-therapy use and negative patient outcomes between complicated and recurrent urinary tract infection (r/cUTI) cases diagnosed with a multiplex polymerase chain reaction or pooled antibiotic susceptibility testing (M-PCR/P-AST) vs. standard urine culture (SUC). Subjects were 577 symptomatic adults (n = 207 males and n = 370 females) presenting to urology/urogynecology clinics between 03/30/2022 and 05/24/2023. Treatment and outcomes were recorded by the clinician and patient surveys. The M-PCR/P-AST (n = 252) and SUC (n = 146) arms were compared after patient matching for confounding factors. The chi-square and Fisher's exact tests were used to analyze demographics and clinical outcomes between study arms. Reduced empirical-treatment use (28.7% vs. 66.7%), lower composite negative events (34.5% vs. 46.6%, p = 0.018), and fewer individual negative outcomes of UTI-related medical provider visits and UTI-related visits for hospitalization/an urgent care center/an emergency room (p < 0.05) were observed in the M-PCR/P-AST arm compared with the SUC arm. A reduction in UTI symptom recurrence in patients ≥ 60 years old was observed in the M-PCR/P-AST arm (p < 0.05). Study results indicate that use of the M-PCR/P-AST test reduces empirical antibiotic treatment and negative patient outcomes in r/cUTI cases.

Elevated UTI Biomarkers in Symptomatic Patients with Urine Microbial Densities of 10,000 CFU/mL Indicate a Lower Threshold for Diagnosing UTIs.

The literature lacks consensus on the minimum microbial density required for diagnosing urinary tract infections (UTIs). This study categorized the microbial densities of urine specimens from symptomatic UTI patients aged ≥ 60 years and correlated them with detected levels of the immune response biomarkers neutrophil gelatinase-associated lipocalin (NGAL), interleukin-8 (IL-8), and interleukin-1-beta (IL-1ß). The objective was to identify the microbial densities associated with significant elevation of these biomarkers in order to determine an optimal threshold for diagnosing symptomatic UTIs. Biobanked midstream voided urine samples were analyzed for microbial identification and quantification using standard urine culture (SUC) and multiplex-polymerase chain reaction (M-PCR) testing, while NGAL, IL-8, and IL-1ß levels were measured via enzyme-linked immunosorbent assay (ELISA). NGAL, IL-8, and IL-1ß levels were all significantly elevated at microbial densities ≥ 10,000 cells/mL when measured via M-PCR (p < 0.0069) or equivalent colony-forming units (CFUs)/mL via SUC (p < 0.0104) compared to samples with no detectable microbes. With both PCR and SUC, a consensus of two or more elevated biomarkers correlated well with microbial densities > 10,000 cells/mL or CFU/mL, respectively. The association between ≥10,000 cells and CFU per mL with elevated biomarkers in symptomatic patients suggests that this lower threshold may be more suitable than 100,000 CFU/mL for diagnosing UTIs.

Structures of the TRPM5 channel elucidate mechanisms of activation and inhibition.

The Ca2+-activated TRPM5 channel plays essential roles in taste perception and insulin secretion. However, the mechanism by which Ca2+ regulates TRPM5 activity remains elusive. We report cryo-EM structures of the zebrafish TRPM5 in an apo closed state, a Ca2+-bound open state, and an antagonist-bound inhibited state. We define two novel ligand binding sites: a Ca2+ site (CaICD) in the intracellular domain and an antagonist site in the transmembrane domain (TMD). The CaICD site is unique to TRPM5 and has two roles: modulating the voltage dependence and promoting Ca2+ binding to the CaTMD site, which is conserved throughout TRPM channels. Conformational changes initialized from both Ca2+ sites cooperatively open the ion-conducting pore. The antagonist NDNA wedges into the space between the S1-S4 domain and pore domain, stabilizing the transmembrane domain in an apo-like closed state. Our results lay the foundation for understanding the voltage-dependent TRPM channels and developing new therapeutic agents.

Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy.

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium homeostasis, particularly store-operated calcium entry, which is critical to maintaining proper function of synaptic vesicle release and intracellular signaling pathways. Accordingly, TRPC channels have been implicated in a variety of human diseases including cardiovascular disorders such as cardiac hypertrophy, neurodegenerative disorders such as Parkinson's disease, and neurologic disorders such as spinocerebellar ataxia. Therefore, TRPC channels represent a potential pharmacologic target in human diseases. However, the molecular mechanisms of gating in these channels are still unclear. The difficulty in obtaining large quantities of stable, homogeneous, and purified protein has been a limiting factor in structure determination studies, particularly for mammalian membrane proteins such as the TRPC ion channels. Here, we present a protocol for the large-scale expression of mammalian ion channel membrane proteins using a modified baculovirus gene transfer system and the purification of these proteins by affinity and size-exclusion chromatography. We further present a protocol to collect single-particle cryo-electron microscopy images from purified protein and to use these images to determine the protein structure. Structure determination is a powerful method for understanding the mechanisms of gating and function in ion channels.

Prospective protocol for lymphedema education and surveillance in a breast health center.

Participation in the ONS Foundation-supported Breast Cancer Survivorship Quality Measures Set pilot study indicated less than optimal assessment and educational practices for lymphedema surveillance at one author's institution. This finding led staff to expand on nursing practice associated with lymphatic dysfunction and to propose a surveillance tool that is easy to use in a busy clinical setting. Based on current trends in lymphatic surveillance, arm measurements were used to assess for relative percentage differences compared to baseline. The referral threshold for lymphedema diagnosis was set at a 10% relative difference in circumferential measurements, pre- and postoperatively, as well as during the survivorship period. Enhanced education efforts, inclusive of staff and patients, will be established throughout the continuum of care. As lymphedema research and standard of care consensus become established, this surveillance model will be adapted in accordance with evidence-based practice guidelines.