A program for real-time surveillance of SARS-CoV-2 genetics.

The COVID-19 pandemic brought forth an urgent need for widespread genomic surveillance for rapid detection and monitoring of emerging SARS-CoV-2 variants. It necessitated design, development, and deployment of a nationwide infrastructure designed for sequestration, consolidation, and characterization of patient samples that disseminates de-identified information to public authorities in tight turnaround times. Here, we describe our development of such an infrastructure, which sequenced 594,832 high coverage SARS-CoV-2 genomes from isolates we collected in the United States (U.S.) from March 13th 2020 to July 3rd 2023. Our sequencing protocol ('Virseq') utilizes wet and dry lab procedures to generate mutation-resistant sequencing of the entire SARS-CoV-2 genome, capturing all major lineages. We also characterize 379 clinically relevant SARS-CoV-2 multi-strain co-infections and ensure robust detection of emerging lineages via simulation. The modular infrastructure, sequencing, and analysis capabilities we describe support the U.S. Centers for Disease Control and Prevention national surveillance program and serve as a model for rapid response to emerging pandemics at a national scale.

Antibody titer levels and the effect on subsequent SARS-CoV-2 infection in a large US-based cohort.

Despite a growing amount of data around the kinetics and durability of the antibody response induced by vaccination and previous infection, there is little understanding of whether or not a given quantitative level of antibodies correlates to protection against SARS-CoV-2 infection or reinfection. In this study, we examine SARS-CoV-2 anti-spike receptor binding domain (RBD) antibody titers and subsequent SARS-CoV-2 reverse transcription polymerase chain reaction (RT-PCR) tests in a large cohort of US-based patients. We analyzed antibody test results in a cohort of 22,204 individuals, 6.8% (n = 1,509) of whom eventually tested positive for SARS-CoV-2 RNA, suggesting infection or reinfection. Kaplan-Meier curves were plotted to understand the effect of various levels of anti-spike RBD antibody titers (classified into discrete ranges) on subsequent RT-PCR positivity rates. Statistical analyses included fitting a Cox proportional hazards model to estimate the age-, sex- and exposure-adjusted hazard ratios for S antibody titer, using zip-code positivity rates by week as a proxy for COVID-19 exposure. It was found that the best models of the temporally associated infection risk were those based on log antibody titer level (HR = 0.836 (p < 0.05)). When titers were binned, the hazard ratio associated with antibody titer >250 Binding Antibody Units (BAU) was 0.27 (p < 0.05, 95% CI [0.18, 0.41]), while the hazard ratio associated with previous infection was 0.20 (p < 0.05, 95% CI [0.10, 0.39]). Fisher exact odds ratio (OR) for Ab titers <250 BAU showed OR = 2.84 (p < 0.05; 95% CI: [2.30, 3.53]) for predicting the outcome of a subsequent PCR test. Antibody titer levels correlate with protection against subsequent SARS-CoV-2 infection or reinfection when examining a cohort of real-world patients who had the spike RBD antibody assay performed.

Prevalence and trends in mono- and co-infection of COVID-19, influenza A/B, and respiratory syncytial virus, January 2018-June 2023.

Objectives: This study aimed to determine the impact of the COVID-19 pandemic on the overall prevalence and co-infection rates for COVID-19, influenza A/B, and respiratory syncytial virus in a large national population. Methods: We conducted a retrospective review of 1,318,118 multi-component nucleic acid amplification tests for COVID-19, influenza A/B, and RSV performed at Labcorp® sites from January 2018 to June 2023, comparing positivity rates and co-infection rates by age, sex, and seasonality. Results: In 2021-2023, 1,232 (0.10%) tested positive for COVID-19 and influenza A/B, 366 (0.03%) tested positive for COVID-19 and RSV, 874 (0.07%) tested for influenza A/B and RSV, and 13 (0.001%) tested positive for COVID-19, influenza A/B, and RSV. RSV positivity rates were particularly higher in Q2 and Q3 of 2021 and in Q3 of 2022. Higher influenza A positivity proportions were found in Q4 of 2021 and again in Q2 and Q4 of 2022. Influenza B positivity had been minimal since the start of the pandemic, with a slight increase observed in Q2 of 2023. Conclusion: Our findings highlight the need for adaptability in preparation for upper respiratory infection occurrences throughout the year as we adjust to the COVID-19 pandemic due to the observed changes in the seasonality of influenza and RSV. Our results highlight low co-infection rates and suggest heightened concerns for co-infections during peaks of COVID-19, influenza, and RSV, which may perhaps be reduced.

Chronic Kidney Disease Testing Among At-Risk Adults in the U.S. Remains Low: Real-World Evidence From a National Laboratory Database.

OBJECTIVE: An estimated 37 million Americans have chronic kidney disease (CKD). Nearly 90% do not know about their condition because of low awareness about the importance of CKD testing and diagnosis among practitioners and people at risk for CKD. This study uses data from a national clinical laboratory to identify guideline-recommended CKD testing rates across the U.S. RESEARCH DESIGN AND METHODS: Patients with Laboratory Corporation of America Holdings (Labcorp) testing between 2013 and 2019 were defined as at risk for CKD if they had any testing ordered with diagnosis codes for diabetes and/or hypertension. Guideline-concordant CKD assessment was defined by estimated glomerular filtration rate (eGFR) and urine albumin-to-creatinine ratio (uACR) testing within the study year. RESULTS: We identified 28,295,982 at-risk patients (mean age 60.6 ± 14.8 years; 53.6% women): 16.2% had diabetes, 63.8% had hypertension, and 20.1% had both comorbidities. Of these, 80.3% did not receive guideline-concordant assessment during the study period. Furthermore, only 21.0% had uACR testing versus 89.6% with eGFR. CKD assessment occurred at least once in 28.7% of patients with diabetes, 10.5% of patients with hypertension, and 41.4% of patients with both conditions. In a state-by-state comparison, annual testing rates ranged from 5 to 30%. The nationwide rate increased modestly each year between 2013 and 2018 (from 10.7% to 15.2%). CONCLUSIONS: Despite guideline recommendations, testing for CKD with uACR and eGFR in U.S. adults with diabetes and hypertension is low in routine clinical care. These data highlight the need for strategies to improve routine CKD assessment nationwide.

Association between attendance at an American diabetes camp and improvements in glycaemic control and treatment satisfaction.

INTRODUCTION: Few studies have evaluated glycaemic control using continuous glucose monitoring (CGM) in individuals before and after attendance at a diabetes camp or by comparing control groups at home to control groups at camp. METHODS: Youth (6-17 years) with T1D and receiving insulin therapy were enrolled at a week-long diabetes camp. They participated in three clinic visits: at the start of a week at home, by initiating a Dexcom G6 CGM system; at the start of a week at camp, where the home week G6 was removed and a camp week G6 was inserted; and after camp, where the camp week G6 was removed. We administered Problem Areas in Diabetes (PAID) surveys at the second and third visits. Participants with <80% CGM data coverage or who did not complete all PAID surveys were excluded from analysis. We compared glycaemic control and PAID scores between the week at home and week at camp. RESULTS: Of 76 enrolled campers, 69 completed the study and 52 had results that qualified for analysis. The mean participant age was 12.5 ± 2.2 years. Camp was associated with significantly improved treatment satisfaction, time in desired glucose range and insulin sensitivity. Time in hyperglycaemia and basal insulin requirements decreased significantly. CONCLUSIONS: Diabetes camp is associated with significant improvements in diabetes treatment satisfaction and glycaemic control compared to home care.

Follow-Up SARS-CoV-2 PCR Testing Outcomes From a Large Reference Lab in the US.

By analyzing COVID-19 sequential COVID-19 test results of patients across the United States, we herein attempt to quantify some of the observations we've made around long-term infection (and false-positive rates), as well as provide observations on the uncertainty of sampling variability and other dynamics of COVID-19 infection in the United States. Retrospective cohort study of a registry of RT-PCR testing results for all patients tested at any of the reference labs operated by Labcorp® including both positive, negative, and inconclusive results, from March 1, 2020 to January 28, 2021, including patients from all 50 states and outlying US territories. The study included 22 million patients with RT-PCR qualitative test results for SARS-CoV-2, of which 3.9 million had more than one test at Labcorp. We observed a minuscule <0.1% basal positive rate for follow up tests >115 days, which could account for false positives, long-haulers, and/or reinfection but is indistinguishable in the data. In observing repeat-testing, for patients who have a second test after a first RT-PCR, 30% across the cohort tested negative on the second test. For patients who test positive first and subsequently negative within 96 h (40% of positive test results), 18% of tests will subsequently test positive within another 96-h span. For those who first test negative and then positive within 96 h (2.3% of negative tests), 56% will test negative after a third and subsequent 96-h period. The sudden changes in RT-PCR test results for SARS-CoV-2 from this large cohort study suggest that negative test results during active infection or exposure can change rapidly within just days or hours. We also demonstrate that there does not appear to be a basal false positive rate among patients who test positive >115 days after their first RT-PCR positive test while failing to observe any evidence of widespread reinfection.

A population-based analysis of the longevity of SARS-CoV-2 antibody seropositivity in the United States.

BACKGROUND: This cross-sectional study aimed to track population-based SARS-CoV-2 antibody seropositivity duration across the United States using observational data from a national clinical laboratory registry of patients tested by nucleic acid amplification (NAAT) and serologic assays. Knowledge of antibody seropositivity and its duration may help dictate post-pandemic planning. METHODS: Using assays to detect antibodies to either nucleocapsid (N) or spike (S) proteins performed on specimens from 39,086 individuals with confirmed positive COVID-19 by reverse transcription-polymerase chain reaction (RT-PCR) from March 2020 to January 2021, we analyzed nationwide seropositivity rates of IgG up to 300 days following patients' initial positive NAAT test. Linear regression identified trends in seropositivity rates and logistic regression tested positive predictability by age, sex, assay type and days post-infection. FINDINGS: Seropositivity of IgG antibodies to both SARS-CoV-2 S and N-proteins followed a linear trend reaching approximately 90% positivity at 21 days post-index. The rate of N-protein seropositivity declined at a sharper rate, decaying to 68·2% [95% CI: 63·1-70·8%] after 293 days, while S-antibody seropositivity maintained a rate of 87·8% [95% CI: 86·3-89·1%] through 300 days. In addition to antigen type and the number of days post-positive PCR, age and gender were also significant factors in seropositivity prediction, with those under 65 years of age showing a more sustained seropositivity rate. INTERPRETATION: Observational data from a national clinical laboratory, though limited by an epidemiological view of the U.S. population, offer an encouraging timeline for the development and sustainability of antibodies up to ten months from natural infection and could inform post-pandemic planning.

Use of hemoglobin A1c to identify dysglycemia in cystic fibrosis.

BACKGROUND: Cystic fibrosis (CF) leads to pancreatic endocrine dysfunction with progressive glycemic disturbance. Approximately 30%-50% of people with CF eventually develop CF-related diabetes (CFRD). Pre-CFRD states progress from indeterminant glycemia (INDET) to impaired fasting glucose (IFG) or impaired glucose tolerance (IGT). Screening guidelines recommend inconvenient annual 2-hour oral glucose tolerance tests (OGTTs), beginning at age 10 years. More efficient methods, such as hemoglobin A1C (HbA1c), have been evaluated, but only limited, relatively small studies have evaluated the association between HbA1c and pre-CFRD dysglycemic states. OBJECTIVE: To determine whether HbA1c is an appropriate screening tool for identifying patients with pre-CFRD dysglycemia to minimize the burden of annual OGTTs. METHODS: This retrospective review evaluated medical records data of all University of Massachusetts Memorial Health System CF patients with an HbA1c result within 90 days of an OGTT between 1997 and 2019. Exclusion criteria were uncertain CF diagnosis, other forms of diabetes, or incomplete OGTT. In total, 56 patients were included and categorized according to OGTT results (American Diabetes Association criteria): normal glucose tolerance, INDET, IFG, or IGT. Associations were evaluated between HbA1c and OGTT results and between HbA1c and pre-CFRD dysglycemic states. RESULTS: Mean HbA1c was not significantly different between patients with normal glucose tolerance and those in the INDET (p = 0.987), IFG (p = 0.690), and IGT (p = 0.874) groups. Analysis of variance confirmed the lack of association between HbA1c and glycemia, as mean HbA1c was not significantly different amongst the four categories (p = 0.250). CONCLUSION: There is increasing awareness of the impact of pre-CFRD states, including reduced pulmonary function and nutritional status. Unfortunately, our results do not support using HbA1c as a screening tool for pre-CFRD dysglycemia, specifically INDET, IFG, and IGT. Further studies are warranted to evaluate more efficient screening methods to reduce the burden of annual OGTTs.

PAPPA-mediated adipose tissue remodeling mitigates insulin resistance and protects against gestational diabetes in mice and humans.

Pregnancy is a physiological state of continuous adaptation to changing maternal and fetal nutritional needs, including a reduction of maternal insulin sensitivity allowing for appropriately enhanced glucose availability to the fetus. However, excessive insulin resistance in conjunction with insufficient insulin secretion results in gestational diabetes mellitus (GDM), greatly increasing the risk for pregnancy complications and predisposing both mothers and offspring to future metabolic disease. Here, we report a signaling pathway connecting pregnancy-associated plasma protein A (PAPPA) with adipose tissue expansion in pregnancy. Adipose tissue plays a central role in the regulation of insulin sensitivity, and we show that, in both mice and humans, pregnancy caused remodeling of adipose tissue evidenced by altered adipocyte size, vascularization, and in vitro expansion capacity. PAPPA is known to be a metalloprotease secreted by human placenta that modulates insulin-like growth factor (IGF) bioavailability through prolteolysis of IGF binding proteins (IGFBPs) 2, 4, and 5. We demonstrate that recombinant PAPPA can stimulate ex vivo human adipose tissue expansion in an IGFBP-5- and IGF-1-dependent manner. Moreover, mice lacking PAPPA displayed impaired adipose tissue remodeling, pregnancy-induced insulin resistance, and hepatic steatosis, recapitulating multiple aspects of human GDM. In a cohort of 6361 pregnant women, concentrations of circulating PAPPA are inversely correlated with glycemia and odds of developing GDM. These data identify PAPPA and the IGF signaling pathway as necessary for the regulation of maternal adipose tissue physiology and systemic glucose homeostasis, with consequences for long-term metabolic risk and potential for therapeutic use.

Treatment of Facial Fractures at a Level 1 Trauma Center: Do Medicaid and Non-Medicaid Enrollees Receive the Same Care?

BACKGROUND: Timing of surgical treatment of facial fractures may vary with the patient age, injury type, and presence of polytrauma. Previous studies using national data sets have suggested that trauma patients with government insurance experience fewer operations, longer length of hospital stay (LOS), and worse outcomes compared with privately insured patients. The objective of this study is to compare treatment of facial fractures in patients with and without Medicaid insurance (excluding Medicare). METHODS: All adults with mandibular, orbital, and midface fractures at a Level 1 Trauma Center between 2009 and 2018 were included. Statistical analyses were performed to assess the differences in the frequency of surgery, time to surgery (TTS), LOS, and mortality based on insurance type. RESULTS: The sample included 1541 patients with facial fractures (mandible, midface, orbital), of whom 78.8% were male, and 13.1% (208) were enrolled in Medicaid. Mechanism of injury was predominantly assault for Medicaid enrollees and falls or motor vehicle accidents for non-Medicaid enrollees (P < 0.001). Patients with mandible and midface fractures underwent similar rates of surgical repair. Medicaid enrollees with orbital fractures underwent less frequent surgery for facial fractures (24.8% versus 34.7%, P = 0.0443) and had higher rates of alcohol and drug intoxication compared with non-Medicaid enrollees (42.8% versus 31.6%, P = 0.008). TTS, LOS, and mortality were similar in both groups with facial fractures. CONCLUSIONS: Overall, the treatment of facial fractures was similar regardless of the insurance type, but Medicaid enrollees with orbital fractures experienced less frequent surgery for facial fractures. Further studies are needed to identify specific socioeconomic and geographic factors contributing to these disparities in care.

Treatment of Acute Cholecystitis: Do Medicaid and Non-Medicaid Enrolled Patients Receive the Same Care?

BACKGROUND: Nationally, Medicaid enrollees with emergency surgical conditions experience worse outcomes overall when compared with privately insured patients. The goal of this study is to investigate disparities in the treatment of cholecystitis based on insurance type and to identify contributing factors. METHODS: Adults with cholecystitis at a safety-net hospital in Central Massachusetts from 2017-2018 were included. Sociodemographic and clinical characteristics were compared based on Medicaid enrollment status (Medicare excluded). Univariate and multivariate analyses were used to compare the frequency of surgery, time to surgery (TTS), length of stay (LOS), and readmission rates between groups. RESULTS: The sample (n = 203) included 69 Medicaid enrollees (34%), with a mean age of 44.4 years. Medicaid enrollees were younger (p = 0.0006), had lower levels of formal education (high school diploma attainment, p < 0.0001), were more likely to be unmarried (p < 0.0001), Non-White (p = 0.0012), and require an interpreter (p < 0.0001). Patients in both groups experienced similar rates of laparoscopic cholecystectomy, TTS, and LOS; however, Medicaid enrollees experienced more readmissions within 30 days of discharge (30.4% vs 17.9%, p < 0.001). CONCLUSION: Despite anticipated population differences, the treatment of acute cholecystitis was similar between Medicaid and Non-Medicaid enrollees, with the exception of readmission. Further research is needed to identify patient, provider, and/or population factors driving this disparity.

Treatment of appendicitis: Do Medicaid and non-Medicaid-enrolled patients receive the same care?

BACKGROUND: Studies using national data sets have suggested that insurance type drives a disparity in the care of emergency surgery patients. Large databases lack the granularity that smaller, single-institution series may provide. The goal of this study is to identify factors that may account for differences in care between Medicaid and non-Medicaid enrollees with appendicitis in central Massachusetts. METHODS: All adult patients with acute appendicitis in an academic medical center between 2010 and 2018 were included. Sociodemographic and clinical characteristics were compared according to Medicaid enrollment status. Analyses were performed to assess differences in the frequency of operative treatment, time to surgery, length of stay, and rates of readmission. RESULTS: The sample included 1,257 patients, 10.7% of whom (n = 135) were enrolled in Medicaid. The proportions of patients presenting with perforated appendicitis (28.9% vs 31.2%, P = .857) and undergoing laparoscopic appendectomy (96.3% vs 90.7%, P = .081) were similar between the 2 groups, as were length of stay (20 hours 30 minutes versus 22 hours 38 minutes, P = .109) and readmission rates (17.8% vs 14.5%, P = .683). Medicaid enrollees did experience somewhat greater time to surgery (6 hours 47 minutes versus 4 hours 49 minutes, P < .001). CONCLUSION: Despite anticipated differences in population, the treatment of appendicitis was similar between Medicaid and non-Medicaid enrollees. Medicaid enrollees experienced greater time to surgery; however, further studies are needed to explain this disparity in care.

Global mapping of transcription factor motifs in human aging.

Biological aging is a complex process dependent on the interplay of cell autonomous and tissue contextual changes which occur in response to cumulative molecular stress and manifest through adaptive transcriptional reprogramming. Here we describe a transcription factor (TF) meta-analysis of gene expression datasets accrued from 18 tissue sites collected at different biological ages and from 7 different in-vitro aging models. In-vitro aging platforms included replicative senescence and an energy restriction model in quiescence (ERiQ), in which ATP was transiently reduced. TF motifs in promoter regions of trimmed sets of target genes were scanned using JASPAR and TRANSFAC. TF signatures established a global mapping of agglomerating motifs with distinct clusters when ranked hierarchically. Remarkably, the ERiQ profile was shared with the majority of in-vivo aged tissues. Fitting motifs in a minimalistic protein-protein network allowed to probe for connectivity to distinct stress sensors. The DNA damage sensors ATM and ATR linked to the subnetwork associated with senescence. By contrast, the energy sensors PTEN and AMPK connected to the nodes in the ERiQ subnetwork. These data suggest that metabolic dysfunction may be linked to transcriptional patterns characteristic of many aged tissues and distinct from cumulative DNA damage associated with senescence.

Simulation of Cellular Energy Restriction in Quiescence (ERiQ)-A Theoretical Model for Aging.

Cellular responses to energy stress involve activation of pro-survival signaling nodes, compensation in regulatory pathways and adaptations in organelle function. Specifically, energy restriction in quiescent cells (ERiQ) through energetic perturbations causes adaptive changes in response to reduced ATP, NAD+ and NADP levels in a regulatory network spanned by AKT, NF-κB, p53 and mTOR. Based on the experimental ERiQ platform, we have constructed a minimalistic theoretical model consisting of feedback motifs that enable investigation of stress-signaling pathways. The computer simulations reveal responses to acute energetic perturbations, promoting cellular survival and recovery to homeostasis. We speculated that the very same stress mechanisms are activated during aging in post-mitotic cells. To test this hypothesis, we modified the model to be deficient in protein damage clearance and demonstrate the formation of energy stress. Contrasting the network's pro-survival role in acute energetic challenges, conflicting responses in aging disrupt mitochondrial maintenance and contribute to a lockstep progression of decline when chronically activated. The model was analyzed by a local sensitivity analysis with respect to lifespan and makes predictions consistent with inhibitory and gain-of-function experiments in aging.

Distinct Cell Stress Responses Induced by ATP Restriction in Quiescent Human Fibroblasts.

Quiescence is the prevailing state of many cell types under homeostatic conditions. Yet, surprisingly little is known about how quiescent cells respond to energetic and metabolic challenges. To better understand compensatory responses of quiescent cells to metabolic stress, we established, in human primary dermal fibroblasts, an experimental 'energy restriction' model. Quiescence was achieved by short-term culture in serum-deprived media and ATP supply restricted using a combination of glucose transport inhibitors and mitochondrial uncouplers. In aggregate, these measures led to markedly reduced intracellular ATP levels while not compromising cell viability over the observation period of 48 h. Analysis of the transcription factor (TF) landscape induced by this treatment revealed alterations in several signal transduction nodes beyond the expected biosynthetic adaptations. These included increased abundance of NF-κB regulated TFs and altered TF subsets regulated by Akt and p53. The observed changes in gene regulation and corresponding alterations in key signaling nodes are likely to contribute to cell survival at intracellular ATP concentrations substantially below those achieved by growth factor deprivation alone. This experimental model provides a benchmark for the investigation of cell survival pathways and related molecular targets that are associated with restricted energy supply associated with biological aging and metabolic diseases.

Balancing stealth and echogenic properties in an ultrasound contrast agent with drug delivery potential.

Contrast agents are currently being modified to combine diagnostic and therapeutic capabilities. For ultrasound (US) imaging with polymeric contrast agents, it is necessary to modify the shell to create "stealth" microbubbles but without these modifications sacrificing the agent's ability to interact with the focused US beam. We hypothesize that addition of the classic immune shielding molecule polyethylene glycol (PEG) to a polylactide (PLA) microbubble shell will affect the acoustic and physical properties of the resulting agents. In an effort to determine the best formulation to achieve a balance between stealth and acoustic activity, we compared two PEGylation techniques; addition of increasing amounts of PEG-PLA copolymer and employing incorporation of a PEG lipid (LipidPEG) into the shell. Loss of acoustic enhancement occurred in a dose-dependent manner for both types of PEGylated agents (loss of signal occurred at >5 wt% PEG-PLA and >1 wt% LipidPEG), while immune activation was also reduced in a dose-dependent manner for the PEG-PLA agents. This study shows that the balance between acoustic behavior and improved immune avoidance was scalable and successful to different degrees with both PEGylation methods, and was best achieved using for PEG-PLA at 5 wt% and for LipidPEG at 1 wt%. Studies are ongoing to evaluate the best method for the targeting and drug delivery capabilities of these agents for applications in cancer treatment. This study represents the basis for understanding the consequences of making modifications to the native polymeric shell.