An academic partnership between a university and an upstate New York school serving students with intellectual and developmental disabilities established a research-driven, weekly onsite asymptomatic COVID-19 testing protocol supplemented by symptomatic testing. The research team, including school leadership, met at least weekly to address implementation issues, impacts of changing pandemic and regulatory requirements, and interpretation and dissemination of findings. The dynamic partnership resulted in 380 participants, more than 16 000 polymerase chain reaction tests, and additional research studies. ( Am J Public Health. 2024;114(S5):S384–S387. https://doi.org/10.2105/AJPH.2024.307687 )

[This corrects the article DOI: 10.1371/journal.pone.0291840.].

An increasingly pressing need for clinical diagnostics has required the development of novel nucleic acid-based detection technologies that are sensitive, fast, and inexpensive, and that can be deployed at point-of-care. Recently, the RNA-guided ribonuclease CRISPR-Cas13 has been successfully harnessed for such purposes. However, developing assays for detection of genetic variability, for example single-nucleotide polymorphisms, is still challenging and previously described design strategies are not always generalizable. Here, we expanded our characterization of LbuCas13a RNA-detection specificity by performing a combination of experimental RNA mismatch tolerance profiling, molecular dynamics simulations, protein, and crRNA engineering. We found certain positions in the crRNA-target-RNA duplex that are particularly sensitive to mismatches and establish the effect of RNA concentration in mismatch tolerance. Additionally, we determined that shortening the crRNA spacer or modifying the direct repeat of the crRNA leads to stricter specificities. Furthermore, we harnessed our understanding of LbuCas13a allosteric activation pathways through molecular dynamics and structure-guided engineering to develop novel Cas13a variants that display increased sensitivities to single-nucleotide mismatches. We deployed these Cas13a variants and crRNA design strategies to achieve superior discrimination of SARS-CoV-2 strains compared to wild-type LbuCas13a. Together, our work provides new design criteria and Cas13a variants to use in future easier-to-implement Cas13-based RNA detection applications.

To the Editors: The Paecilomyces genus includes species of fungus not frequently associated with human infection.1 Thirty pediatric cases of Paecilomyces spp. infections have been reported, with most in patients with indwelling devices and immunocompromise; very few involved central nervous system (CNS) infections and all were fatal.2–4 We present a case of a pediatric Paecilomyces spp. ventriculoperitoneal (VP) shunt infection treated with shunt replacement, liposomal amphotericin B and posaconazole. A 14-year-old girl with a history of myelomeningocele and VP shunt, tethered cord release and procedures for neurogenic bowel and bladder (appendicostomy, appendicovesicostomy), presented with 2 weeks of increasing headache without fever or other symptoms and underwent VP shunt externalization. The illness was preceded by a shunt revision 2 months earlier, at which time the cerebrospinal fluid (CSF) laboratory profile was normal and cultures were sterile. During the externalization, catheter tip fenestrations were noted to be filled with proteinaceous debris, and the distal abdominal catheter appeared obstructed. An abdominal computed tomography scan showed a small rim-enhancing fluid collection with surrounding inflammation, possibly representing an abscess. Broad-spectrum antibacterial therapy was begun. Examination of the CSF showed pseudohyphae and septate hyphae on Gram stain, prompting the addition of fluconazole and liposomal amphotericin B (L-AmB) to maximize antifungal activity. Within 3 days, cultures were growing mold from intraoperative peritoneal fluid and CSF. Repeated samples of CSF yielded mold through the first week of antifungal therapy; during the second hospital week, the mold was identified by colonial morphology and matrix-assisted laser desorption ionization–time of flight mass spectrometry as Paecilomyces spp. (Fig. 1). The antifungal regimen was revised to L-AmB and posaconazole, and a left ventriculopleural shunt was placed. After 18 days of L-AmB and 6 months of posaconazole, the child has remained well-appearing. However, she has required several shunt revisions due to malfunctions of uncertain etiology, each with sterile CSF. Due to possible shunt plugging from undetected infection, her current therapy (8 months after the initial diagnosis) has empirically been changed to oral isavuconazole.FIGURE 1.: Photomicographs and photographs of isolate. A: Gram stain (B) calcofluor-white (C) lactophenol cotton blue and (D) colony morphology of the Paecilomyces isolate.The Paecilomyces genus includes many species of fungus (P. varioti, P. marquandii and P. javanicus, etc.) that are commonly plant and insect pathogens in nature and are not frequently associated with human infection.1 One clinically important species recently was reclassified as Purpureocillium lilacinum.2 Most human infections have been associated with indwelling devices or peritonitis among immunocompromised hosts or patients with underlying chronic diseases, including diabetes.2–4 These molds are hyaline hyphomycetes that grow as filamentous molds on numerous media, forming a thin powdery yellow-brown colony (P. lilacinum is notable for pink/lilac colonies) (Fig. 1). Mycelia have broad, branching and septate hyphae, and phialides have broad bases ending in a long slender neck. Speciation is by molecular targeting of the internal transcribed spacer regions within ribosomal DNA.1–5 Our case represents the first documented pediatric CNS Paecilomyces spp. VP shunt infection. Whether peritonitis seeded the VP shunt, or whether the VP shunt infection seeded the abdomen, is unclear. Previous reports have described Paeciliomyces peritonitis in association with peritoneal dialysis catheters; our patient’s prior abdominal procedures for neurogenic bowel and bladder may have caused the primary abdominal infection.2 One prior report of Paeciliomyces spp. VP shunt infection involved an immunocompetent adult with shunted hydrocephalus following mesh placement for a basilar artery aneurysm.5 Several subsequent shunt revisions were required in this adult for catheter occlusions with “fibrous and proteinaceous material.”5 The authors suspected a possible undiagnosed fungal infection may have existed for months to years, with repeated shunt failures due to “recurrent mycelial plugging of the device.”5 This adult ultimately succumbed to a cerebral aneurysm said to be unrelated to infection. Our patient’s recovery and CSF sterilization indicate that amphotericin B and posaconazole, along with source control, is a possible treatment for CNS Paecilomyces spp. infection. With the increasing complexity of abdominal and CSF diversion surgeries, clinicians should be aware of rare environmental mold infections in children and adolescents.

The complete genome of Kalamiella piersonii clinical isolate URMC-2103A041 from human bacteremia was determined using the hybrid assembly of short- and long-read sequencing chemistry. The genome contains a 3.93 Mb chromosome, three circular plasmids, and one linear plasmid.

Background This study examined the correlation of classroom ventilation (air exchanges per hour (ACH)) and exposure to CO2 ≥1,000 ppm with the incidence of SARS-CoV-2 over a 20-month period in a specialized school for students with intellectual and developmental disabilities (IDD). These students were at a higher risk of respiratory infection from SARS-CoV-2 due to challenges in tolerating mitigation measures (e.g. masking). One in-school measure proposed to help mitigate the risk of SARS-CoV-2 infection in schools is increased ventilation. Methods We established a community-engaged research partnership between the University of Rochester and the Mary Cariola Center school for students with IDD. Ambient CO2 levels were measured in 100 school rooms, and air changes per hour (ACH) were calculated. The number of SARS-CoV-2 cases for each room was collected over 20 months. Results 97% of rooms had an estimated ACH ≤4.0, with 7% having CO2 levels ≥2,000 ppm for up to 3 hours per school day. A statistically significant correlation was found between the time that a room had CO2 levels ≥1,000 ppm and SARS-CoV-2 PCR tests normalized to room occupancy, accounting for 43% of the variance. No statistically significant correlation was found for room ACH and per-room SARS-CoV-2 cases. Rooms with ventilation systems using MERV-13 filters had lower SARS-CoV-2-positive PCR counts. These findings led to ongoing efforts to upgrade the ventilation systems in this community-engaged research project. Conclusions There was a statistically significant correlation between the total time of room CO2 concentrations ≥1,000 and SARS-CoV-2 cases in an IDD school. Merv-13 filters appear to decrease the incidence of SARS-CoV-2 infection. This research partnership identified areas for improving in-school ventilation.

Campylobacter ureolyticus is an emerging pathogen increasingly appreciated as a common cause of gastroenteritis and extra-intestinal infections in humans. Outside the setting of gastroenteritis, little work has been done to describe the genomic content and relatedness of the species, especially regarding clinical isolates. We reviewed the epidemiology of clinical C. ureolyticus cultured by our institution over the past 10 years. Fifty-one unique C. ureolyticus isolates were identified between January 2010 and August 2022, mostly originating from abscesses and blood cultures. To clarify the taxonomic relationships between isolates and to attribute specific genes with different clinical manifestations, we sequenced 19 available isolates from a variety of clinical specimen types and conducted a pangenomic analysis with publicly available C. ureolyticus genomes. Digital DNA:DNA hybridization suggested that these C. ureolyticus comprised a species complex of 10 species clusters (SCs) and several subspecies clusters. Although some orthologous genes or gene functions were enriched in isolates found in different SCs and clinical specimens, no association was significant. Nearly a third of the isolates possessed antimicrobial resistance genes, including the ermA resistance gene, potentially conferring resistance to macrolides, the treatment of choice for severe human campylobacteriosis. This work effectively doubles the number of publicly available C. ureolyticus genomes, provides further clarification of taxonomic relationships within this bacterial complex, and identifies target SCs for future analysis.

Campylobacter ureolyticusis an emerging pathogen increasingly appreciated as a common cause of gastroenteritis and extra-intestinal infections in humans. Outside the setting of gastroenteritis, little work has been done to describe the genomic content and relatedness of the species, especially regarding clinical isolates.

Abstract Background A protective antibody response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial to decrease morbidity and mortality from severe coronavirus disease 2019 (COVID-19) disease. The effects of preexisting anti-human coronavirus (HCoV) antibodies on the SARS-CoV-2–specific immunoglobulin G (IgG) responses and severity of disease are currently unclear. Methods We profiled anti-spike (S), S1, S2, and receptor-binding domain IgG antibodies against SARS-CoV-2 and 6 HCoVs using a multiplex assay (mPLEX-CoV) with serum samples from SARS-CoV-2 infected (n = 155) and pre–COVID-19 (n = 188) cohorts. Results COVID-19 subjects showed significantly increased anti-S SARS-CoV-2 IgG levels that were highly correlated with IgG antibodies against OC43 and HKU1 S proteins. However, OC43 and HKU1 anti-S antibodies in pre–COVID-19 era sera did not cross-react with SARS-CoV-2. Unidirectional cross-reactive antibodies elicited by SARS-CoV-2 infection were distinct from the bidirectional cross-reactive antibodies recognizing homologous strains RaTG13 and SARS-CoV-1. High anti-OC43 and anti-S2 antibody levels were associated with both a rapid anti–SARS-CoV-2 antibody response and increased disease severity. Subjects with increased sequential organ failure assessment (SOFA) scores developed a higher ratio of S2- to S1-reactive antibodies. Conclusions Early and rapid emergence of OC43 S- and S2-reactive IgG after SARS-CoV-2 infection correlates with COVID-19 disease severity.

The xMAP INTELLIFLEX DR-SE enabled simultaneous and semi-quantitative detection of both IgM and IgG to three different SARS-CoV-2 antigens in a single assay. The assay format is advantageous for rapid and medium-throughput profiling using a small volume of specimen. ABSTRACT The multiplex capabilities of the new xMAP INTELLIFLEX DR-SE flow analyzer were explored by modifying a serological assay previously used to characterize the IgG antibody to infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The goal was to examine the instrument’s performance and to simultaneously measure IgM and IgG antibody responses against multiple SARS-CoV-2 antigens in a single assay. Specific antibodies against the SARS-CoV-2 spike (S), receptor binding domain (RBD), and nucleocapsid (N) proteins were investigated in 310 symptomatic case patients using a fluorescent microsphere immunoassay and simultaneous detection of IgM and IgG. Neutralization potential was studied using the addition of soluble angiotensin-converting enzyme 2 (ACE2) to block antibody binding. A profile extending to 180 days from symptom onset (DFSO) was described for antibodies specific to each viral antigen. Generally, IgM levels peaked and declined rapidly ∼3–4 weeks following infection, whereas S- and RBD-specific IgG plateaued at 80 DFSO. ACE2 more effectively prevented IgM and IgG binding in convalescent cases > 30 DFSO, suggesting those antibodies had greater neutralization potential. This work highlighted the multiplex and multi-analyte potential of the xMAP INTELLIFLEX DR-SE, and provided further evidence for antigen-specific IgM and IgG trajectories in acute and convalescent cases. IMPORTANCE The xMAP INTELLIFLEX DR-SE enabled simultaneous and semi-quantitative detection of both IgM and IgG to three different SARS-CoV-2 antigens in a single assay. The assay format is advantageous for rapid and medium-throughput profiling using a small volume of specimen. The xMAP INTELLIFLEX DR-SE technology demonstrated the potential to include numerous SARS-CoV-2 antigens; future work could incorporate multiple spike protein variants in a single assay. This could be an important feature for assessing the serological response to emerging variants of SARS-CoV-2.

Background: Carbapenem-resistant Enterobacterales (CRE) are difficult to treat and can cause significant morbidity and mortality, however most data reflect carbapenemase-producing infections. Objective: Our objective was to evaluate clinical outcomes of non-carbapenemase-producing CRE (nCP-CRE) compared with carbapenem-susceptible Enterobacterales (CSE) infections. Methods: This was a retrospective, multicenter, observational study (January 1, 2018 to December 31, 2020). The primary outcome was clinical success at 30 days with secondary outcomes, including clinical success at 90 days, clinical success based on treatment for nCP-CRE, persistent bacteremia, intensive care unit (ICU) admission, length of stay, and rate of Clostridioides difficile or multidrug resistant infections. Results: The final analysis included 211 patients: 142 (67%) with CSE and 69 (33%) with nCP-CRE infections. Prior carbapenem exposure was more common with nCP-CRE (15% vs 4%, P = 0.01). Clinical success at 30 days was similar between groups (77% vs 74%, P = 0.73). There were no differences in secondary outcomes. There was an overall low use of carbapenems (empiric 6%, definitive 7%). Most nCP-CRE infections were treated with a monotherapy carbapenem-sparing regimen (empiric 88%, definitive 90%). Limitations include the retrospective design and the high rate of urinary infections. Conclusion and Relevance: Our study found no difference in clinical outcomes between nCP-CRE and CSE infections. Application of this study with future studies would help in determining optimal regimens for these infections.

Abstract Background Klebsiella pneumoniae is a leading cause of nosocomial infections, and hypervirulent K. pneumoniae (hvKp) is an increasingly recognized community-associated pathogen. However, the epidemiology and pathogenicity of hvKp and K. pneumoniae complex infections in the US remains poorly understood. We characterized the tissue tropism, antimicrobial resistance, and virulence factors associated with K. pneumoniae complex members and hvKp using whole genome sequencing. Methods We analyzed all Klebsiella pneumoniae isolates from liver sources from 2017-2020 and isolates from other sterile tissue sites from 2018-2019 in a western New York regional academic microbiology laboratory by whole genome sequencing. Only one isolate per patient was included. Basic clinical and demographic data was obtained from the medical chart. Virulence factors and antibitoic resistance genes were identified using Kleborate version 2.0. Hypervirulence was defined as the presence of rmpA or rmpA2 and the aerobactin gene cluster (iuc). Results We sequenced 25 Klebsiella pneumoniae isolates from liver sources from 2017-2020 and an additional 29 isolates from other non-blood sterile sites from 2018-2019. 80% of the hepatic K. pneumoniae complex isolates were K. pneumoniae sensu stricto (n=20). The remaining isolates were identified as K. quasipneumoniae (n=3) and K. variicola (n=2). Non-hepatic tissue isolates contained only one K. variicola; the remainder were K. pneumoniae sensu stricto. Seven isolates met criteria for hypervirulence; all were K. pneumoniae sensu stricto isolated from liver abscesses (28% of such isolates). All were community onset infections, and only 2 patients with hvKp had prior international travel identified. None had additional sites of infection documented. Four isolates were identified as ESBL phenotypically and all had blaCTX-M-15 detected; one of which was K. quasipneumoniae. Conclusion This study suggests that hypervirulent K. pneumoniae hepatic abscesses may be more prevalent in the US than previously appreciated. Non-sensu stricto K. pneumoniae complex members were infrequently isolated from bodily tissues, less likely to carry hypervirulence genes but may harbor extended antibiotic resistance. Disclosures All Authors: No reported disclosures.

The genomes of two human monkeypox virus strains from recently reported cases in our local region that were associated with the 2022 global outbreak were sequenced. Genomes from clinical isolates provide valuable information for epidemiological tracking and analysis of strain evolution and can be especially important during the early phases of outbreaks.

BACKGROUND The clinical laboratory continues to play a critical role in managing the coronavirus pandemic. Numerous FDA emergency use authorization (EUA) and laboratory developed test (LDT) serologic assays have become available. The performance characteristics of these assays and their clinical utility continue to be defined in real-time during this pandemic. The American Association for Clinical Chemistry (AACC) convened a panel of experts from clinical chemistry, microbiology, and immunology laboratories, the in vitro diagnostics (IVD) industry, and regulatory agencies to provide practical recommendations for implementation and interpretation of these serologic tests in clinical laboratories. CONTENT The currently available EUA serologic tests and platforms, information on assay design, antibody classes including neutralizing antibodies, and the humoral immune responses to SARS-CoV-2 are discussed. Verification and validation of EUA and LDTs are described along with quality management approach. Four indications for serologic testing are outlined. Result interpretation, reporting comments, and the role of orthogonal testing are also recommended. SUMMARY This document aims to provide a comprehensive reference for laboratory professionals and healthcare workers to appropriately implement SARS-CoV-2 serologic assays in the clinical laboratory and interpret test results during this pandemic. Given the more frequent occurrence of outbreaks associated with either vector-borne or respiratory pathogens, this document will be a useful resource in planning for similar scenarios in the future.

The COVID-19 pandemic has underscored the need for rapid high-throughput methods for sensitive and specific serological detection of infection with novel pathogens, such as SARS-CoV-2. Multiplex serological testing can be particularly useful because it can simultaneously analyze antibodies to multiple antigens that optimizes pathogen coverage, and controls for variability in the organism and the individual host response. Here we describe a SARS-CoV-2 IgG 3-plex fluorescent microsphere-based assay that can detect both IgM and IgG antibodies to three major SARS-CoV-2 antigens-the spike (S) protein, spike angiotensin-converting enzyme-2 (ACE2) receptor-binding domain (RBD), and nucleocapsid (Nc). The assay was shown to have comparable performance to a SARS-CoV-2 reference assay for IgG in serum obtained at ≥21 days from symptom onset but had higher sensitivity with samples collected at ≤5 days from symptom onset. Further, using soluble ACE2 in a neutralization assay format, inhibition of antibody binding was demonstrated for S and RBD.

RT-PCR is the gold-standard for the diagnosis of SARS-CoV-2 infection, however, testing has been complicated by supply shortages and long turn-around times.….

RT-PCR is the gold-standard for the diagnosis of SARS-CoV-2 infection, however, testing has been complicated by supply shortages and long turn-around times.….

Infections caused by extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli are a significant cause of morbidity and health care costs. Globally, the prevailing clonal type is ST131 in association with the blaCTX-M-15 β-lactamase gene. However, other ESBLs, such as blaCTX-M-14 and blaCTX-M-27, can also be prevalent in some regions. We identified ST38 ESBL-producing E. coli from different regions in the United States which carry blaCTX-M-27 embedded on two distinct plasmid types, suggesting the potential emergence of new ESBL lineages.

Recent improvements in next-generation sequencing technologies have enabled clinical laboratories to increasingly pursue pathogen genomics for infectious disease diagnosis. Clinical laboratories can also benefit from whole-genome sequence characterization of cultured isolates, helping to resolve infection prevention questions pertaining to pathogen outbreaks and surveillance. Metagenomic sequencing from primary specimens can also provide laboratories with an unbiased universal test for situations where traditional methods fail to identify infectious etiologies despite, high clinical suspicion. Here, the most useful applications of whole-genome sequence and metagenomic sequencing are summarized, as are the main advantages, limitations, and considerations for building an in-house clinical genomics program.