SARS-CoV-2 Pandemic Non-Pharmacologic Interventions Temporally Associated with Reduced Pediatric Infections Due to Mycoplasma pneumoniae and Co-Infecting Respiratory Viruses in Arkansas (preprint)

Introduction: Non-pharmacologic interventions (NPIs), such as universal masking, implemented during the SARS-CoV-2 pandemic have reduced respiratory infections among children. This study focuses on evaluating the impact of NPIs on Mycoplasma pneumoniae infections in children, analyzing data from two hospitals in Arkansas, and examining age-related differences and coinfections with other viruses. Methods: The study was approved by the Institutional Review Board and included patients aged [≤]18 years with upper respiratory tract symptoms. Data from the FilmArray(R) Respiratory Panel (FARP) were collected and divided into pre-NPI and NPI periods for analysis. Total test positivity rate and interval change in the positivity rate were evaluated. Statistical differences were determined by Chi-square ({chi}-independence) analysis. Results: A total of 68,949 tests were performed with a statistical increase in testing during the NPI period. The overall test positivity rate for M. pneumoniae decreased by 74% (0.86% to 0.03%) during the NPI period, and the preschool age group had the highest number of positive tests in the pre- and NPI periods (Pre-NPI: n=40, NPI: n=12 positive tests, p=<0.001). The reduction in M. pneumoniae infections was consistent across age groups. Coinfections with other respiratory viruses, particularly human rhinovirus/enterovirus, were observed at much lower levels. Conclusions: NPIs effectively reduced M. pneumoniae in pediatric patients in Arkansas, and coinfections with specific viruses still occurred, albeit at lower levels during the SARS-CoV-2 pandemic. As NPIs are relaxed and the pandemic ends, we expect M. pneumoniae infections to return to pre-pandemic levels within the next 1-2 years.

Genomic Surveillance of SARS-CoV-2 Using Long-Range PCR Primers (preprint)

Whole Genome Sequencing (WGS) of the SARS-CoV-2 virus is crucial in the surveillance of the COVID-19 pandemic. Several primer schemes have been developed to sequence the ~30,000 nucleotide SARS-CoV-2 genome that use a multiplex PCR approach to amplify cDNA copies of the viral genomic RNA. Midnight primers and ARTIC V4.1 primers are the most popular primer schemes that can amplify segments of SARS-CoV-2 (400 bp and 1200 bp, respectively) tiled across the viral RNA genome. Mutations within primer binding sites and primer-primer interactions can result in amplicon dropouts and coverage bias, yielding low-quality genomes with 'Ns' inserted in the missing amplicon regions, causing inaccurate lineage assignments, and making it challenging to monitor lineage-specific mutations in Variants of Concern (VoCs). This study uses seven long-range PCR primers with an amplicon size of ~4500 bp to tile across the complete SARS-CoV-2 genome. One of these regions includes the full-length S-gene by using a set of flanking primers. Using a small set of long-range primers to sequence SARS-CoV-2 genomes reduces the possibility of amplicon dropout and coverage bias.

An ex vivo human precision-cut lung slice platform provides insight into SARS-CoV-2 pathogenesis and antiviral drug efficacy (preprint)

COVID-19 has claimed millions of lives since the emergence of SARS-CoV-2, and lung disease appears the primary cause of the death in COVID-19 patients. However, the underlying mechanisms of COVID-19 pathogenesis remain elusive, and there is no existing model where the human disease can be faithfully recapitulated and conditions for the infection process can be experimentally controlled. Herein we report the establishment of an ex vivo human precision-cut lung slice (hPCLS) platform for studying SARS-CoV-2 pathogenicity and innate immune responses, and for evaluating the efficacy of antiviral drugs against SARS-CoV-2. We show that while SARS-CoV-2 continued to replicate during the course of infection of hPCLS, infectious virus production peaked within 2 days, and rapidly declined thereafter. Although most proinflammatory cytokines examined were induced by SARS-CoV-2 infection, the degree of induction and types of cytokines varied significantly among hPCLS from individual donors, reflecting the heterogeneity of human populations. In particular, two cytokines (IP-10 and IL-8) were highly and consistently induced, suggesting a role in the pathogenesis of COVID-19. Histopathological examination revealed focal cytopathic effects late in the infection. Transcriptomic and proteomic analyses identified molecular signatures and cellular pathways that are largely consistent with the progression of COVID-19 in patients. Furthermore, we show that homoharringtonine, a natural plant alkaloid derived from Cephalotoxus fortunei, not only inhibited virus replication but also production of pro-inflammatory cytokines, and ameliorated the histopathological changes of the lungs caused by SARS-CoV-2 infection, demonstrating the usefulness of the hPCLS platform for evaluating antiviral drugs.

Younger and Rural Children are More Likely to be Hospitalized for SARS-CoV-2 Infections. (preprint)

Purpose: To identify characteristics of SARS CoV2 infection that are associated with hospitalization in children initially evaluated in a Pediatric Emergency Department (ED). Methods: We identified cases of SARS CoV2 positive patients seen in the Arkansas Childrens Hospital (ACH) ED or hospitalized between May 27, 2020, and April 28, 2022 using ICD10 codes within the Pediatric Hospital Information System (PHIS) Database. We compared infection waves for differences in patient characteristics, and used logistic regressions to examine which characteristics led to a higher chance of hospitalization. Findings: We included 681 preDelta cases, 673 Delta cases, and 970 Omicron cases. Almost 17% of patients were admitted to the hospital. Compared to Omicron infected children, preDelta and Delta infected children were twice as likely to be hospitalized (OR=2.2 and 2.0, respectively; p<0.0001). Infants less than 1 year of age were >3 times as likely to be hospitalized than children ages 5 to 14 years regardless of wave (OR=3.42; 95%CI=2.36 to 4.94). Rural children were almost 3 times as likely than urban children to be hospitalized across all waves (OR=2.73; 95%CI=1.97 to 3.78). Finally, those with a complex condition had nearly a 15 fold increase in odds of admission (OR=14.6; 95%CI=10.6 to 20.0). Conclusions: Children diagnosed during the preDelta or Delta waves were more likely to be hospitalized than those diagnosed during the Omicron wave. Younger and rural patients were more likely to be hospitalized regardless of wave. We suspect lower vaccination rates and larger distances from medical care influenced higher hospitalization rates.

The Serological Sciences Network (SeroNet) for COVID-19: Depth and Breadth of Serology Assays and Plans for Assay Harmonization (preprint)

Background: In October 2020, the National Cancer Institute (NCI) Serological Sciences Network (SeroNet) was established to study the immune response to COVID-19, and to develop, validate, improve, and implement serological testing and associated technologies. SeroNet is comprised of 25 participating research institutions partnering with the Frederick National Laboratory for Cancer Research (FNLCR) and the SeroNet Coordinating Center. Since its inception, SeroNet has supported collaborative development and sharing of COVID-19 serological assay procedures and has set forth plans for assay harmonization. Methods: To facilitate collaboration and procedure sharing, a detailed survey was sent to collate comprehensive assay details and performance metrics on COVID-19 serological assays within SeroNet. In addition, FNLCR established a protocol to calibrate SeroNet serological assays to reference standards, such as the U.S. SARS-CoV-2 serology standard reference material and First WHO International Standard (IS) for anti-SARS-CoV-2 immunoglobulin (20/136), to facilitate harmonization of assay reporting units and cross-comparison of study data. Results: SeroNet institutions reported development of a total of 27 ELISA methods, 13 multiplex assays, 9 neutralization assays, and use of 12 different commercial serological methods. FNLCR developed a standardized protocol for SeroNet institutions to calibrate these diverse serological assays to reference standards. Conclusions: SeroNet institutions have established a diverse array of COVID-19 serological assays to study the immune response to SARS-CoV-2 virus and vaccines. Calibration of SeroNet serological assays to harmonize results reporting will facilitate future pooled data analyses and study cross-comparisons.

Estimated pediatric SARS-CoV-2 seroprevalence in Arkansas over the first year of the COVID-19 pandemic (preprint)

BackgroundSARS-CoV-2 seroprevalence studies have largely focused on adults but little is known about spread in children. We determined SARS-CoV-2 seroprevalence in children and adolescents from Arkansas over the first year of the COVID-19 pandemic. MethodsWe tested remnant serum samples from children from 1-18 years who visited Arkansas hospitals or clinics for non-COVID19-related reasons from April, 2020 through April, 2021 for SARS-CoV-2 antibodies. We used univariable and multivariable regression models to determine association between seropositivity and participant characteristics. ResultsAmong 2400 participants, seroprevalence rose from 7.9% in April/May 2020 (95% CI, 4.9-10.9%) to 25.8% in April 2021 (95% CI, 22.2-29.3%). Hispanic and black children had a significantly higher association with antibody positivity than white children in multiple sampling periods. ConclusionsBy spring 2021, most children in Arkansas had not been infected with SARS-CoV-2. With the emergence of SARS-CoV-2 variants, recognition of long-term effects of COVID-19, and the lack of an authorized pediatric SARS-CoV-2 vaccine, these results highlight the importance of including children in SARS-CoV-2 public health, clinical care, and research strategies. These findings are important for state and local officials as they consider measures to limit SARS-CoV-2 spread in schools and daycares for the 2021-2022 school year.

Temporal Variations in Seroprevalence of SARS-CoV-2 Infections by Race and Ethnicity in Arkansas. (preprint)

Objective: Our objective is to estimate CoV-2 infection rates in a rural state using seroprevalence of antibodies to CoV-2 as an indicator of infection. Study Design and Setting: This is a single-site study within an academic center and regional programs within the state of Arkansas. We obtained residual serum samples from a convenience sample of adults who were outpatients and came to the hospital or regional clinic for non-COVID-related reasons. We collected remnant in three time periods (August 15 to September 5, September 12 to October 24, and November 7 to December 19). Results: In 2020, the overall age, gender, and race standardized prevalence of CoV-2 antibodies was 2.6% (August to September), 4.1% (September to October), and 7.4% (November to December). There was no difference in seroprevalence between urban compared to rural areas. Positive tests were not uniformly distributed across racial and ethnic minorities. Higher seroprevalence rates were found in Hispanics and Blacks or African Americans compared to whites across all time periods. Conclusions: In a state with a large rural population, 2.6-7.4% of people experienced CoV-2 infection by December 2020. Blacks and Hispanics had disproportionately higher rates of CoV-2 infections than whites.