Importance:COVID-19 vaccine effectiveness (VE) is typically studied in the context of reducing the risk of severe illness and death. Few studies have estimated VE in preventing transmission and infection with current levels of SARS-CoV-2 population immunity. Objective:To estimate COVID-19 VE against transmission and infection within households. Design, Setting, and Participants:This cohort study was a prospective, case-ascertained household transmission study (performed in New York, Tennessee, and Washington) in which the first household member with confirmed SARS-CoV-2 infection (primary case participant) was identified through outpatient settings and enrolled with their household contacts from January 1, 2024, to January 31, 2025. Participants provided demographic information, and COVID-19 vaccination history was verified by study staff. After enrollment, participants were instructed to collect daily nasal swabs for 10 days regardless of symptoms. Nasal swabs were tested for SARS-CoV-2 via reverse transcription-polymerase chain reaction. Exposure:COVID-19 vaccination history in primary case participants and household contacts categorized as time from most recent vaccination to COVID-19 onset in the primary case participant (≤6 months, 7-12 months, >12 months, and unvaccinated [reference group]). Main Outcomes and Measures:Household contacts were considered infected if at least 1 swab tested positive for SARS-CoV-2. Secondary infection risk was calculated as the number of infected contacts divided by the total number of contacts. Adjusted relative risk (ARR) of infection was estimated using a multivariable Poisson regression model, with generalized estimating equations accounting for household-level clustering. Vaccine effectiveness was calculated as 1 minus the ARR of the primary case participant and household contacts' vaccination status to estimate VE against transmission and against infection, respectively. Results:This analysis included 362 primary case participants (median [IQR] age, 35 [10-53] years; 199 female [55.0%]) and 763 household contacts (median [IQR] age, 29 [12-44] years; 399 female [52.3%]). SARS-CoV-2 infection was detected in 476 household contacts during follow-up for a secondary infection risk of 62.4% (95% CI, 58.7%-65.5%). Household contacts of primary case participants vaccinated 6 months or less before onset had a lower infection risk compared with contacts of unvaccinated primary case participants (ARR, 0.57 [95% CI, 0.35-0.93]). There was no statistically significant difference in infection risk based on vaccination status of household contacts. Conclusions and Relevance:In this cohort study, recent COVID-19 vaccination was associated with a reduced risk of SARS-CoV-2 transmission. These findings suggest that COVID-19 vaccination may have an indirect benefit of decreasing transmission and thus reducing overall exposure to SARS-CoV-2.

Individuals with neurogenic bladder are susceptible to chronic bacterial colonization and urinary tract infections. Neurogenic bladder arises from conditions including spinal cord injuries and spina bifida. We established a longitudinal cohort of 77 children and young adults with spina bifida. Using enhanced urine culture, 16S rRNA sequencing, and whole-genome sequencing of bacterial isolates, we characterized the urine microbiota. We also retrieved prior bacterial isolates from Vanderbilt’s MicroVU biobank, enabling 5-year evolutionary comparisons within subjects. Urine samples showed high abundance of pathogens, including E. coli and Klebsiella. Across time points, subjects exhibited either rapid cycling of strains, often after antibiotics, or persistence of a single strain. Neither pattern consistently correlated with increased antibiotic resistance. Instead, mutations in cell envelope genes mediated immune evasion and altered phage susceptibility, highlighting fitness trade-offs induced by niche adaptation to the bladder. This cohort enables the identification of bacterial adaptation mechanisms.

In March 2024, CDC released symptom-based isolation guidance for viral respiratory illnesses including influenza: isolation from others until fever-free for 24 hours and other symptoms are improving, followed by 5 days of post-isolation precautions. This study evaluated how well these recommendations aligned with influenza virus RNA shedding and assessed self-reported isolation in a multi-site household study during 2023-2025. We analyzed data from 1294 participants with influenza, including 627 with a clear peak in viral RNA levels and symptom resolution during follow-up. Participants provided daily symptom diaries and nasal swabs. We used Kaplan-Meier models to estimate recommended isolation duration based on CDC guidelines and compared this to viral RNA shedding measured by cycle threshold values and viral loads. The median recommended isolation duration was 6 days (interquartile range 4-7). Peak viral RNA shedding occurred a median of 4 days post-symptom onset, falling during recommended isolation for 80% (by Ct) and 69% (by viral load) of participants and increasing to 100% and 97% respectively by day 6 post-isolation. However, only 21% reported isolating from household members (median 2 days), and 80% isolated from the community (median 4 days). These findings support symptom-based isolation and emphasize the importance of post-isolation precautions.

Transmission bottlenecks are defined by the number of unique virions or genotypes that establish an infection, and they restrict viral diversity that passes from one infected host to another. Previous work identified a tight transmission bottleneck for seasonal influenza A virus (IAV) based on analysis of 43 household pairs, largely from a single A(H3N2) predominant season. While many viral and host factors are known to influence IAV transmission in households, their impact on the transmission bottleneck is not clear. Nasal swabs were collected daily from IAV infected individuals enrolled in two case-ascertained U.S. household transmission studies, FluTES (2017/2018-2019/2020 seasons) and RVTN (2021/2022 season). Viruses were sequenced in duplicate, and intrahost single nucleotide variants (iSNV) were identified at a 0·5% frequency threshold using a benchmarked pipeline with >99·99% specificity for mutations present in both replicates. Transmission pairs were defined based on co-residence, test date, and genetic distance. For each possible transmission pair, the bottleneck was estimated using a beta binomial and a clonal mutation model. We sequenced 567 samples from 319 individuals and 102 households in duplicate. Based on epidemiologic linkage and a sequence-based cut-off, we defined 59 transmission pairs for the beta binomial model and 56 transmission pairs for the clonal mutation model. Across all pairs, we identified a transmission bottleneck of 1 both using the beta-binomial model (CI 1, 1) and the clonal mutation model (CI: 1.00, 1.22). In our cohort, influenza season, subtype, and host factors (influenza vaccination status, sex, and age) did not alter the transmission bottleneck. IAV is subject to a tight genetic bottleneck during transmission, which limits onward propagation of newly arising nucleotide variants. Tight bottlenecks appear to be intrinsic to the transmission process, as host and viral factors within households do not affect its size.

Pooled testing represents a resource-conserving diagnostic strategy, and we propose enterovirus-D68 (EV-D68) as an attractive target for pooling (especially locally). To validate an RT-qPCR-based approach, retrospective pools were contrived from EV-D68-positive nasopharyngeal specimens and non-EV-D68-enterovirus-positive specimens. Five/nine-fold pools demonstrated 90.9 %/88.6 % sensitivity versus-unpooled testing (no false-positives), with commensurate results observed prospectively.

Background Understanding protection against SARS-CoV-2 infection by vaccine and hybrid immunity is important for informing public health strategies as new variants emerge.Methods We analyzed data from 3 cohort studies spanning 1 September 2022 to 31 July 2023 to estimate COVID-19 vaccine effectiveness (VE) against SARS-CoV-2 infection and symptomatic COVID-19 among adults with and without prior infection in the United States. Participants collected weekly nasal swabs irrespective of symptoms, participated in annual blood draws, and completed periodic surveys, which included vaccination status and infection history. Swabs were tested molecularly for SARS-CoV-2. VE was estimated by Cox proportional hazards models for the hazard ratios of infections, adjusting for covariates. VE was calculated considering prior infection and recency of vaccination.Results Among 3344 adults, the adjusted VE of a bivalent vaccine against infection was 37.2% (95% CI, 12.3%-55.7%) within 7 to 59 days of vaccination and 21.1% (95% CI, -0.5% to 37.1%) within 60 to 179 days of vaccination when compared with participants who were unvaccinated or had received an original monovalent vaccine dose >= 180 days prior. Overall, the adjusted VE of a bivalent vaccine against infection, in conjunction with prior infection, was 62.2% (95% CI, 46.0%-74.5%) within 7 to 179 days of vaccination and 39.4% (95% CI, 12.5%-61.6%) at >= 180 days when compared with naive participants who were unvaccinated or had received a monovalent vaccine dose >= 180 days prior.Conclusions Adults with prior infection and recent vaccination had high protection against infection and symptomatic illness. Recent vaccination alone provided moderate protection. According to data from 3 prospective cohort studies where participants collected weekly nasal swabs, protection from COVID-19 vaccination against SARS-CoV-2 infection and symptomatic illness was highest among adults with prior infection and recent vaccination. Recent vaccination alone provided moderate protection.

We estimated the burden of RSV-associated hospitalizations in US adults 1 year prior to RSV vaccine introduction. The overall annual incidence rate of RSV-associated hospitalization was 31.47 (95% CI: 21.89-43.97) per 100,000 adults. Rates were 10-fold and 17-fold higher among adults 60 to 74 years and ≥ 75 years compared with adults 18 to 49 years old. This prospective assessment demonstrated the burden of RSV-associated hospitalizations among adults, with the highest hospitalization rates among adults ≥ 60 years old, in the year prior to RSV vaccine introduction.

Individuals with neurogenic bladder are particularly susceptible to both chronic bacterial colonization of the bladder and urinary tract infections (UTIs). Neurogenic bladder can arise from a variety of diseases such as diabetes, spinal cord injuries, and spina bifida. To study the ecological and evolutionary dynamics of the microbiome in neurogenic bladder, we developed a longitudinal cohort of 77 children and young adults with spina bifida from two medical centers. We used enhanced urine culture, 16S rRNA sequencing, and whole genome sequencing to characterize the microbial composition of urine and fecal samples. In addition to prospective sample collection, we retrieved prior bacterial isolates from enrolled patients from Vanderbilt's clinical microbial biobank, MicroVU. This allowed us to compare bacterial isolates from the same patients over a period of five years. Urine samples were characterized by high abundance of urinary pathogens, such as E. coli and Klebsiella. From longitudinal isolates from individual patients, we identified two common patterns of urinary tract colonization. We observed either the rapid cycling of strains and/or species, often following antibiotic treatment, or we observed the persistence of a single strain across timepoints. Neither persistence of a strain nor colonization with a new strain or species was associated with increased antibiotic resistance. Rather, in paired longitudinally collected strains from the same patients, mutations were identified in genes that code for cell envelope components associated with immune or phage evasion. Experimental testing revealed that O-antigen/LPS biosynthesis mutations confer protection from the immune system while altering susceptibility to phage predation, reflecting a fitness trade-off. We argue that this unparalleled cohort offers the opportunity to identify mechanisms of bacterial adaptation to the urinary tract that can be exploited in future therapeutic approaches.

Background Human adenoviruses (HAdV) elicit diverse infections, most notably within the respiratory tract. While HAdV is a target in clinical-use PCR assays to diagnose respiratory infections, techniques are not widely available to determine individual HAdV species within clinical specimens. An initial model was previously developed to predict HAdV species from BIOFIRE® Respiratory Pathogen Panels (RPP), evaluated in silico and through contrived specimens. This model was based on melt-curve data of 5 individual amplification reactions underlying the adenoviral result. Methods In this study, the initial model is updated to better reflect prior knowledge of HAdV respiratory epidemiology and applied to a dataset of clinical HAdV-detected RPP samples independently subtyped via PCR. Revised model performance was further assessed through application to clinical proficiency testing events in the BIOFIRE Syndromic Trends database (Trends), a near-real-time network of clinical-use BIOFIRE testing results. The revised model was applied to >100 000 HAdV-detected results in Trends from the United States since 2019. Results Among the independently typed specimens, the revised model accuracy was 95.2% (180/189): 79/82 for HAdV-B, 97/102 for HAdV-C, and 4/5 for HAdV-E. In the Trends dataset, these analyses indicated dynamic epidemiology for HAdV species, including a shift of B-vs-C prevalence at the onset of the COVID-19 pandemic, which has more recently returned to prepandemic ratios, along with low-level prediction of species less typically associated with respiratory infection. Conclusion In silico modeling of melt-curve data from the BIOFIRE RPP can enhance HAdV species surveillance efforts and define viral epidemiology at local, regional, and national levels.

SARS-CoV-2 has undergone repeated and rapid evolution to circumvent host immunity. However, outside of prolonged infections in immunocompromised hosts, within-host positive selection has rarely been detected. Here we combine daily longitudinal sampling of individuals with replicate sequencing to increase the accuracy of and lower the threshold for variant calling. We sequenced 577 specimens from 105 individuals in a household cohort during the BA.1/BA.2 variant period. Individuals exhibited extremely low viral diversity, and we estimated a low within-host evolutionary rate. Within-host dynamics were dominated by genetic drift and purifying selection. Positive selection was rare but highly concentrated in spike. A Wright Fisher Approximate Bayesian Computational model identified positive selection at 14 loci with 7 in spike, including S:448 and S:339. This detectable immune-mediated selection is unusual in acute respiratory infections and may be caused by the relatively narrow antibody repertoire in individuals during the early Omicron phase of the SARS-CoV-2 pandemic.

ABSTRACT Urinary tract infections (UTIs) impose a substantial burden on patient quality of life and urine testing accounts for the majority of workload in many clinical microbiology laboratories. Traditional UTI diagnosis relies on symptoms, urinalysis, and culture which are interpreted based on historical guidelines. This approach, while foundational, presents limitations, particularly in complex cases. Low-level bacteriuria and the presence of fastidious organisms are often overlooked or entirely missed in standard urine culture, stressing the need for novel diagnostic methods and technologies. This mini-review summarizes the existing state of UTI diagnostics in 2024 and covers current and upcoming technologies including rapid molecular-based pathogen identification, next-generation sequencing, and advanced antimicrobial susceptibility testing. However, these methods represent unique challenges, and as they are implemented, they will require the field to adapt to new concepts to avoid misdiagnosis and overtreatment.

Abstract Background By fall 2023, RSV immunizations to protect older adults, pregnant people, and infants were recommended in the United States. Our aim was to describe uptake of and barriers to RSV immunization among community-based individuals during the first season of availability.Table 1:Targeted RSV immunization populations, ACIP recommendations, and participant eligibility criteria for this analysis, April 20241. Due to limitations in how data were collected in the parent study, the eligibility criteria used for the analysis may differ from exact ACIP recommended criteria.2. Nirsevimab, a single injection during the infant’s first respiratory season, was recently recommended by ACIP and preferred by American Academy of Pediatrics over palivizumab, which requires monthly injections during the respiratory season and is recommended for a subset of high-risk infants. Palivizumab has been available in the United States since 1998.3. Shared clinical decision-making allows flexibility for providers and patients to consider individual risk for RSV disease, while considering patient preferences.RSV = respiratory syncytial virus; ACIP = Advisory Committee for Immunization Practices Methods Starting January 2024, participants were enrolled in a household transmission study of COVID-19 and RSV. All participants completed questionnaires capturing demographics, household characteristics, vaccination and medical history, and their knowledge, attitudes, and behaviors towards RSV and RSV immunizations. Participants included in this analysis were those eligible for RSV immunizations based on Advisory Committee for Immunization Practices guidelines (Table 1). Participants were considered immunized if they had a verified RSV immunization or plausible self-report i.e., reported an RSV immunization with the date of the immunization and a location or manufacturer (Figure 1). We compared characteristics, knowledge, attitudes, and behaviors stratified by immunization uptake using chi-squared or Fishers exact tests.Figure 1:Participant RSV immunization eligibility and receipt11. Immunization history was self-reported by participants and verified by study staff using medical and pharmacy records. Participants were considered immunized if they had a verified RSV immunization or plausible self-report meaning they self-reported an RSV immunization with the date of the immunization and a location or manufacturer.RSV = respiratory syncytial virus; KAP = Knowledge, Attitudes, and Practices survey Results Among participants enrolled and eligible for this analysis as of April 2024 (n=63), 15 (24%) received RSV immunization: 14/44 (32%) among older adults, 1/16 (6%) among infants/young children, and 0/3 (0%) among pregnant people (Figure 1). Older adults who did not receive the vaccine were more likely to be younger (67 vs 71 years, p=0.009) and less likely to have received an updated 2023-24 COVID-19 vaccine (40% vs 79%, p=0.02; Table 2). Among older adults who replied they would not or were not sure if they would get vaccinated, reasons included that they needed more information (43%; 9/21) or were not sure how/where to get the vaccine (14%, 3/21; Table 3).Table 2:Individual and household characteristics of participants eligible for RSV immunization1 by RSV immunization status21 RSV immunization for older adults is a vaccine and for infants/young children is a monoclonal antibody2 Immunization history was collected self-reported by participants and verified by study staff using medical and pharmacy records. Participants were considered immunized if they had a verified RSV immunization or plausible self-report meaning they self-reported an RSV immunization with the date of the immunization and a location or manufacturer.3 Wilcoxon rank sum test; Fisher's exact test; Pearson's Chi-squared testmAb = monoclonal Antibodies; RSV = respiratory syncytial virus Conclusion A quarter of individuals who were eligible for RSV immunization had received it during the 2023-24 RSV season. Older adults on the younger end of the recommendation were less likely to be vaccinated; it is unclear if this is due to limited awareness of RSV disease, perceived lower risk, or other reasons. Better education and awareness of RSV disease and vaccine safety, effectiveness, and availability may increase uptake among older adults. Ongoing enrollment may allow insight into barriers for infants and pregnant people. Table 3: Individual beliefs and attitudes towards RSV and RSV immunizations of participants eligible for RSV immunization1 by RSV immunization status2 1 RSV immunization for older adults is a vaccine and for infants/young children is a monoclonal antibody 2 Immunization history was collected self-reported by participants and verified by study staff using medical and pharmacy records. Participants were considered immunized if they had a verified RSV immunization or plausible self-report meaning they self-reported an RSV immunization with the date of the immunization and a location or manufacturer. 3 Fisher's exact test 4 Responses provided by a parent/caregiver of infant/young child Disclosures Carlos G. Grijalva, MD, MPH, AHRQ: Grant/Research Support|CDC: Grant/Research Support|FDA: Grant/Research Support|Merck: Advisor/Consultant|NIH: Grant/Research Support|SyneosHealth: Grant/Research Support Jonathan Schmitz, MD, PhD, D(ABMM), Biofire/Biomerieux: Grant/Research Support|Genmark/Roche: Grant/Research Support|Pfizer: Honoraria|Vela: Grant/Research Support Helen Y. Chu, MD, MPH, Abbvie: Advisor/Consultant|Merck: Advisor/Consultant|Vir: Advisor/Consultant Janet A. Englund, MD, Abbvie: Advisor/Consultant|AstraZeneca: Advisor/Consultant|AstraZeneca: Grant/Research Support|GlaxoSmithKline: Advisor/Consultant|GlaxoSmithKline: Grant/Research Support|Meissa Vaccines: Advisor/Consultant|Merck: Advisor/Consultant|Pfizer: Board Member|Pfizer: Grant/Research Support|Pfizer: Speaker at meeting|SanofiPasteur: Advisor/Consultant|Shinogi: Advisor/Consultant

In previous reports, we described a PCR cycle control approach in which the hybridization state of optically labeled L-DNA enantiomers of the D-DNA primers and targets determined when the thermal cycle was switched from cooling to heating and heating to cooling. A consequence of this approach is that it also "adapts" the cycling conditions to compensate for factors that affect the hybridization kinetics of primers and targets. It assumes, however, that the hybridization state of the labeled L-DNA analogs accurately reflects the hybridization state of the D-DNA primers and targets. In this report, the Van't Hoff equation is applied to determine the L-DNA concentration and ratio of L-DNA strands required by this assumption. Simultaneous fluorescence and temperature measurements were taken during L-DNA controlled cycling, and the optical and thermal switch points compared as a function of both total L-DNA concentration and ratio of strands. Based on the Van't Hoff relationship and these experimental results, L-DNA best mirrors the hybridization of PCR primers and targets when total L-DNA concentration is set equal to the initial concentration of the D-DNA primer of interest. In terms of strand ratios, L-DNA hybridization behavior most closely matches the behavior of their D-DNA counterparts throughout the reaction when one of the L-DNA strands is far in excess of the other. The L-DNA control algorithm was then applied to the practical case of the SARS-CoV-2 N2 reaction, which has been shown to fail or have a delayed Cq when PCR was performed without nucleic acid extraction. PCR Cq values for simulated "unextracted" PCR samples in a nasopharyngeal background and in an NaCl concentration similar to that of viral transport media were determined using either the L-DNA control algorithm (N = 6) or preset cycling conditions (N = 3) and compared to water background controls run in parallel. For preset cycling conditions, the presence of nasopharyngeal background or a high salt background concentration significantly increased Cq, but the L-DNA control algorithm had no significant delay. This suggests that a carefully designed L-DNA-based control algorithm "adapts" the cycling conditions to compensate for hybridization errors of the PCR D-DNA reactants that produce false negatives.

Herpes simplex viruses are enveloped DNA viruses that cause primary infections with episodic reactivation due to latency within the sensory ganglia. Disease manifestations vary with patient age, immune system status, and antecedent triggering factors, causing a range of human diseases from asymptomatic shedding to multisystem disease with widespread mucocutaneous lesions and meningoencephalitis. HSV envelope glycoproteins are key factors that facilitate binding, fusion, and entry into host cells. Latency-associated transcripts mediate neuronal cell survival and inhibition of apoptosis while maintaining HSV DNA in a lysogenized state. HSV diagnosis involves a repertoire of laboratory techniques, including serology, culture, fluorescent antibody staining of viral antigens, and most recently, nucleic acid amplification testing. Vaccine strategies that exploit the importance of HSV surface glycoproteins have had marginal success to-date but remain under active investigation.

Biofilm growth facilitates the interaction of uropathogenic Escherichia coli (UPEC) with the host environment. The extracellular polymeric substances (EPS) of UPEC biofilms are composed prominently of curli amyloid fiber and cellulose polysaccharide. When the organism is propagated as a colony biofilm on agar media, these macromolecules can generate pronounced macroscopic structures. Moreover, curli/cellulose associate tightly with Congo red, generating a characteristic pink-to-red staining pattern when the media is supplemented with this dye. Among different clinical isolates of UPEC, changes in the abundance of curli/cellulose can lead to diverse colony biofilm phenotypes on a strain-by-strain basis. Nevertheless, for any given isolate, these phenotypes are classically homogenous throughout the colony biofilm. Here, we report that a subset of clinical UPEC isolates display heterogenous ‘peppermint’ colony biofilms, with distinct pale and red subpopulations. Through isolation of these subpopulations and whole genome sequencing, we demonstrate various emergent mutations associated with the phenomenon, including within the gene encoding the outer membrane lipoprotein nlpI. Deletion of nlpI within independent strain-backgrounds increased biofilm rugosity, while its overexpression induced the peppermint phenotype. Upregulation of EPS-associated proteins and transcripts was likewise observed in the absence of nlpI. Overall, these results demonstrate that EPS elaboration in UPEC is impacted by nlpI. More broadly, this phenomenon of intra-strain colony biofilm heterogeneity may be leveraged as a tool to identify additional members within the broad collection of genes that regulate or otherwise affect biofilm formation.

As population immunity to SARS-CoV-2 evolves and new variants emerge, the role and accuracy of antigen tests remain active questions. To describe recent test performance, the detection of SARS-CoV-2 by antigen testing was compared with that by reverse transcription-polymerase chain reaction (RT-PCR) and viral culture testing during November 2022-May 2023. Participants who were enrolled in a household transmission study completed daily symptom diaries and collected two nasal swabs (tested for SARS-CoV-2 via RT-PCR, culture, and antigen tests) each day for 10 days after enrollment. Among participants with SARS-CoV-2 infection, the percentages of positive antigen, RT-PCR, and culture results were calculated each day from the onset of symptoms or, in asymptomatic persons, from the date of the first positive test result. Antigen test sensitivity was calculated using RT-PCR and viral culture as references. The peak percentage of positive antigen (59.0%) and RT-PCR (83.0%) results occurred 3 days after onset, and the peak percentage of positive culture results (52%) occurred 2 days after onset. The sensitivity of antigen tests was 47% (95% CI = 44%-50%) and 80% (95% CI = 76%-85%) using RT-PCR and culture, respectively, as references. Clinicians should be aware of the lower sensitivity of antigen testing compared with RT-PCR, which might lead to false-negative results. This finding has implications for timely initiation of SARS-CoV-2 antiviral treatment, when early diagnosis is essential; clinicians should consider RT-PCR for persons for whom antiviral treatment is recommended. Persons in the community who are at high risk for severe COVID-19 illness and eligible for antiviral treatment should seek testing from health care providers with the goal of obtaining a more sensitive diagnostic test than antigen tests (i.e., an RT-PCR test).

IntroductionSARS-CoV-2 has demonstrated that, in targeted circumstances, viral quantification within respiratory specimens can valuably inform patient management, as well as research. Nevertheless, the pandemic has illustrated concomitant challenges for obtaining high-quality (and broadly comparable) respiratory viral loads. This includes a critical need for standardization and calibration, even though the necessary resources may not always be available for emergent pathogens and non-bloodstream specimens.MethodsTo these ends, we describe a novel strategy for implementing quantitative SARS-CoV-2 testing with International Unit-based calibration. Earlier in the course of the pandemic-when analytic resources were far more limited-select residual SARS-CoV-2 positive specimens from routine care in our diagnostic laboratory were pooled to formulate a clinically realistic secondary standard of high volume and analyte concentration, which was cross-calibrated to the primary SARS-CoV-2 standard of the World Health Organization.ResultsThe resultant calibrators were integrated into the original CDC RT-qPCR assay for SARS-CoV-2, whose (now broadened) performance characteristics were defined to generate a test appropriate for both clinical and research use. This test allowed for the quantification of virus in respiratory specimens down to a validated lower limit of quantification of 103.4 IU/ml.ConclusionsBy self-formulating calibrators from this derivative-of-care secondary standard, we successfully validated respiratory viral loads without the commercial availability (at that time) of quantitative assays or calibrators. As the SARS-CoV-2 pandemic continues to decline-and even beyond this pathogen-this strategy may be applicable for laboratories seeking to implement viral load testing for nontraditional specimen types despite limited resources.

OBJECTIVES/GOALS: Antibiograms are used to guide empiric antibiotic selection. However, it is unclear if antibiotic profiles differ between symptomatic urinary tract infections (UTIs) and asymptomatic bacteriuria (ASB). We aimed to compare antibiotic susceptibility profiles of urinary E. coli isolates from patients with a symptomatic UTI to those with ASB. METHODS/STUDY POPULATION: We conducted a cohort study of 1,140 urinary E. coli isolates from unique patients that received care through Vanderbilt University Medical Center (VUMC) from Nov 2020 – Jun 2021. We included any patient that was seen at VUMC as an inpatient, outpatient or at the emergency department with ≥ 105 colony forming units/mL E. coli detected from a clinical urine specimen. Chart abstractions were performed to capture reported UTI symptoms and demographic information. Descriptive statistics were conducted to compare antibiotic susceptibility profiles (i.e., susceptible, intermediate, resistant) between symptomatic and ASB groups. The risk of detection of a multidrug-resistant organism (MDRO) (intermediate, or resistant to at least one antibiotic in three or more classes) was assessed between groups. RESULTS/ANTICIPATED RESULTS: Among 1,140, 1,018 (89%) and 122 (11%) were symptomatic and ASB, respectively. When comparing symptomatic and ASB, the median ages were 50 and 46. Groups had similar proportions of no indwelling catheter (94% v. 95%) and without diabetes (87% v. 88%). The collection setting between inpatient, emergency department, and outpatient were similar with most being outpatient (79% v. 83%). The proportion of patients who were pregnant, immuno compromised, or had a structural/functional urinary tract abnormality were higher in the symptomatic group. The proportion of isolates resistant and susceptible to tested antibiotics were similar between groups, with only ciprofloxacin showing slightly higher resistance among ASB (16% v. 25%). The risk of MDRO detection was similar between groups (RR: 0.858, 95% CI: 0.64, 1.15). DISCUSSION/SIGNIFICANCE: Antibiotic susceptibility comparison demonstrated similar profiles, which suggests antibiogram use as appropriate to guide ASB treatment. Results offer insight on whether traditional methods for assessing antibiotic susceptibility on population-levels could benefit from further refinement by patient-specific clinical parameters.

The declining cost of performing bacterial whole-genome sequencing (WGS) coupled with the availability of large libraries of sequence data for well-characterized isolates have enabled the application of machine-learning (ML) methods to the development of nonlinear sequence-based predictive models. We tested the ML-based model developed by Next Gen Diagnostics for prediction of cefepime phenotypic susceptibility results in Escherichia coli. ABSTRACT The declining cost of performing bacterial whole-genome sequencing (WGS) coupled with the availability of large libraries of sequence data for well-characterized isolates have enabled the application of machine-learning (ML) methods to the development of nonlinear sequence-based predictive models. We tested the ML-based model developed by Next Gen Diagnostics for prediction of cefepime phenotypic susceptibility results in Escherichia coli. A cohort of 100 isolates of E. coli recovered from urine (n = 77) and blood (n = 23) cultures were used. The cefepime MIC was determined in triplicate by reference broth microdilution and classified as susceptible (MIC of ≤2 μg/mL) or not susceptible (MIC of ≥4 μg/mL) using the 2022 Clinical and Laboratory Standards Institute breakpoints. Five isolates generated both susceptible and not susceptible MIC results, yielding categorical agreement of 95% for the reference method to itself. Categorical agreement of ML to MIC interpretations was 97%, with 2 very major (false, susceptible) and 1 major (false, not susceptible) errors. One very major error occurred for an isolate with blaCTX-M-27 (MIC mode, ≥32 μg/mL) and one for an isolate with blaTEM-34 for which the MIC cefepime mode was 4 μg/mL. One major error was for an isolate with blaCTX-M-27 but with a MIC mode of 2 μg/mL. These preliminary data demonstrated performance of ML for a clinically important antimicrobial-species pair at a caliber similar to phenotypic methods, encouraging wider development of sequence-based susceptibility prediction and its validation and use in clinical practice.