Neuroinvasive Bacillus cereus Infection in Immunocompromised Hosts: Epidemiologic Investigation of 5 Patients With Acute Myeloid Leukemia.

Background: Bacillus cereus is a ubiquitous gram-positive rod-shaped bacterium that can cause sepsis and neuroinvasive disease in patients with acute leukemia or neutropenia. Methods: A single-center retrospective review was conducted to evaluate patients with acute leukemia, positive blood or cerebrospinal fluid test results for B cereus, and abnormal neuroradiographic findings between January 2018 and October 2022. Infection control practices were observed, environmental samples obtained, a dietary case-control study completed, and whole genome sequencing performed on environmental and clinical Bacillus isolates. Results: Five patients with B cereus neuroinvasive disease were identified. All patients had acute myeloid leukemia (AML), were receiving induction chemotherapy, and were neutropenic. Neurologic involvement included subarachnoid or intraparenchymal hemorrhage or brain abscess. All patients were treated with ciprofloxacin and survived with limited or no neurologic sequelae. B cereus was identified in 7 of 61 environmental samples and 1 of 19 dietary protein samples-these were unrelated to clinical isolates via sequencing. No point source was identified. Ciprofloxacin was added to the empiric antimicrobial regimen for patients with AML and prolonged or recurrent neutropenic fevers; no new cases were identified in the ensuing year. Conclusions: B cereus is ubiquitous in the hospital environment, at times leading to clusters with unrelated isolates. Fastidious infection control practices addressing a range of possible exposures are warranted, but their efficacy is unknown and they may not be sufficient to prevent all infections. Thus, including B cereus coverage in empiric regimens for patients with AML and persistent neutropenic fever may limit the morbidity of this pathogen.

A decade of clinical microbiology: top 10 advances in 10 years: what every infection preventionist and antimicrobial steward should know.

The past 10 years have brought paradigm-shifting changes to clinical microbiology. This paper explores the top 10 transformative innovations across the diagnostic spectrum, including not only state of the art technologies but also preanalytic and post-analytic advances. Clinical decision support tools have reshaped testing practices, curbing unnecessary tests. Innovations like broad-range polymerase chain reaction and metagenomic sequencing, whole genome sequencing, multiplex molecular panels, rapid phenotypic susceptibility testing, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry have all expanded our diagnostic armamentarium. Rapid home-based testing has made diagnostic testing more accessible than ever. Enhancements to clinician-laboratory interfaces allow for automated stewardship interventions and education. Laboratory restructuring and consolidation efforts are reshaping the field of microbiology, presenting both opportunities and challenges for the future of clinical microbiology laboratories. Here, we review key innovations of the last decade.

Cycle threshold dynamics of non-severe acute respiratory coronavirus virus 2 (SARS-CoV-2) respiratory viruses.

OBJECTIVE: Many providers use severe acute respiratory coronavirus virus 2 (SARS-CoV-2) cycle thresholds (Ct values) as approximate measures of viral burden in association with other clinical data to inform decisions about treatment and isolation. We characterized temporal changes in Ct values for non-SARS-CoV-2 respiratory viruses as a first step to determine whether cycle thresholds could play a similar role in the management of non-SARS-CoV-2 respiratory viruses. DESIGN: Retrospective cohort study. SETTING: Brigham and Women's Hospital, Boston. METHODS: We retrospectively identified all adult patients with positive nasopharyngeal PCRs for influenza, respiratory syncytial virus (RSV), parainfluenza, human metapneumovirus (HMPV), rhinovirus, or adenovirus between January 2022 and March 2023. We plotted Ct distributions relative to days since symptom onset, and we assessed whether distributions varied by immunosuppression and other comorbidities. RESULTS: We analyzed 1,863 positive samples: 506 influenza, 502 rhinovirus, 430 RSV, 219 HMPV, 180 parainfluenza, 26 adenovirus. Ct values were generally 25-30 on the day of symptom onset, lower over the ensuing 1-3 days, and progressively higher thereafter with Ct values ≥30 after 1 week for most viruses. Ct values were generally higher and more stable over time for rhinovirus. There was no association between immunocompromised status and median intervals from symptom onset until Ct values were ≥30. CONCLUSIONS: Ct values relative to symptom onset for influenza, RSV, and other non-SARS-CoV-2 respiratory viruses generally mirror patterns seen with SARS-CoV-2. Further data on associations between Ct values and viral viability, transmissibility, host characteristics, and response to treatment for non-SARS-CoV-2 respiratory viruses are needed to determine how clinicians and infection preventionists might integrate Ct values into treatment and isolation decisions.

Duped by dumping syndrome: non-endemic Vibrio cholerae bacteremia in an immunocompetent host with gastric bypass surgery, a case report.

Extra-intestinal infection with non-O1/non-O139 strains of Vibrio cholerae (NOVC) is rare, though bacteremia and hepatobiliary manifestations have been reported. Reduced stomach acid, or hypochlorhydria, can increase risk of V. cholerae infection. We describe a 42-year-old woman with hypochlorhydria due to untreated Helicobacter pylori infection, gastric-bypass surgery, and chronic proton pump inhibitors (PPI) exposure, who developed acute diarrhoea following raw oyster consumption. Her symptoms were attributed to rapid gastric emptying (dumping syndrome) after a negative limited stool work-up. She had persistent diarrhoea, weight loss, and after 5 months was admitted with acute cholecystitis and NOVC bacteremia, requiring cholecystectomy. This is the first reported case of NOVC bacteremia and cholecystitis in a patient with gastric bypass. This case highlights the potential for NOVC biliary carriage, the role of hypochlorhydria as a risk factor for Vibrio infection, and the importance of excluding infectious diarrhoea in patients with new onset of symptoms compatible with dumping syndrome and a relevant travel history.

Simultaneous Measurement of IgM and IgG Antibodies to SARS-CoV-2 Spike, RBD, and Nucleocapsid Multiplexed in a Single Assay on the xMAP INTELLIFLEX DR-SE Flow Analyzer.

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.

IgG Against Human Betacoronavirus Spike Proteins Correlates With SARS-CoV-2 Anti-Spike IgG Responses and COVID-19 Disease Severity.

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.

Genomic Analysis of Clostridioides difficile in 2 Regions of the United States Reveals a Diversity of Strains and Limited Transmission.

BACKGROUND: The distribution of Clostridioides difficile strains and transmission dynamics in the United States are not well defined. Whole-genome sequencing across 2 Centers for Disease Control and Prevention Emerging Infections Program C. difficile infection (CDI) surveillance regions (Minnesota and New York) was performed to identify predominant multilocus sequence types (MLSTs) in community-associated (CA) and healthcare-associated (HCA) disease and assess transmission. METHODS: Whole-genome sequencing was performed on C. difficile isolates from patients with CDI over 3 months between 2016 and 2017. Patients were residents of the catchment area without a positive C. difficile test in the preceding 8 weeks. CDI cases were epidemiologically classified as HCA or CA. RESULTS: Of 422 isolates, 212 (50.2%) were HCA and 203 (48.1%) were CA. Predominant MLSTs were sequence type (ST) 42 (9.3%), ST8 (7.8%), and ST2 (8.1%). MLSTs associated with HCA-CDI included ST1 (76%), ST53 (83.3%), and ST43 (80.0%), while those associated with CA-CDI included ST3 (76.9%) and ST41 (77.8%). ST1 was more frequent in New York than in Minnesota (10.8% vs 3.1%). Thirty-three pairs were closely related genomically, 14 of which had potential patient-to-patient transmission supported by record review. CONCLUSIONS: The genomic epidemiology of C. difficile across 2 regions of the United States indicates the presence of a diverse strain profile and limited direct transmission.

AACC Practical Recommendations for Implementing and Interpreting SARS-CoV-2 Emergency Use Authorization and Laboratory-Developed Test Serologic Testing in Clinical Laboratories.

BACKGROUND: The clinical laboratory continues to play a critical role in managing the coronavirus pandemic. Numerous US Food and Drug Administration 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 AACC convened a panel of experts from clinical chemistry, microbiology, and immunology laboratories; the in vitro diagnostics 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 LDT assays are described, along with a quality management approach. Four indications for serologic testing are outlined. Recommendations for result interpretation, reporting comments, and the role of orthogonal testing are also presented. 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 to 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.

Fatal Neonatal Sepsis Associated with Human Adenovirus Type 56 Infection: Genomic Analysis of Three Recent Cases Detected in the United States.

BACKGROUND: Human adenovirus (HAdV)-D56 was first described in 2011 by genomics analysis of a strain isolated in France in 2008 from a fatal case of neonatal infection. Since then, it has been reported in cases of keratoconjunctivitis and male urethritis. Three epidemiologically unrelated fatal cases of neonatal sepsis associated with infection by HAdV-D strains with a similar genetic makeup were documented in the United States between 2014 and 2020. METHODS: Whole genome sequences were obtained for the isolated strains, and genomics analyses were conducted to compare them to phylogenetically related HAdV-D genomic sequences available in GenBank. RESULTS: The three new US strains were indistinguishable by in silico restriction enzyme analysis. Their genome sequences were 99.9% identical to one another and to the prototype strain isolated in 2008 from a similar context of disease. The phylogenetic reconstruction revealed a highly supported clustering of all HAdV-D56 strains isolated in various countries since 1982. Our comparison to serologically intermediate strains 15/H9 described in the literature indicated that HAdV-D56-like viruses have circulated worldwide since the late 1950s. CONCLUSION: As with other HAdV-D genotypes with the ability to infect ocular and genital mucosae, the risk of severe prenatal or perinatal HAdV-D56 infection must be considered.

Detection of CTX-M-27 ß-Lactamase Genes on Two Distinct Plasmid Types in ST38 Escherichia coli from Three U.S. States.

Infections caused by extended-spectrum-ß-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 ß-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.

The New York State SARS-CoV-2 Testing Consortium: Regional Communication in Response to the COVID-19 Pandemic.

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2, created an unprecedented need for comprehensive laboratory testing of populations, in order to meet the needs of medical practice and to guide the management and functioning of our society. With the greater New York metropolitan area as an epicenter of this pandemic beginning in March 2020, a consortium of laboratory leaders from the assembled New York academic medical institutions was formed to help identify and solve the challenges of deploying testing. This report brings forward the experience of this consortium, based on the real-world challenges which we encountered in testing patients and in supporting the recovery effort to reestablish the health care workplace. In coordination with the Greater New York Hospital Association and with the public health laboratory of New York State, this consortium communicated with state leadership to help inform public decision-making addressing the crisis. Through the length of the pandemic, the consortium has been a critical mechanism for sharing experience and best practices in dealing with issues including the following: instrument platforms, sample sources, test performance, pre- and post-analytical issues, supply chain, institutional testing capacity, pooled testing, biospecimen science, and research. The consortium also has been a mechanism for staying abreast of state and municipal policies and initiatives, and their impact on institutional and laboratory operations. The experience of this consortium may be of value to current and future laboratory professionals and policy-makers alike, in dealing with major events that impact regional laboratory services.

Genomics of Staphylococcus aureus ocular isolates.

Staphylococcus aureus is a major cause of ocular infections, often resulting in devastating vision loss. Despite the significant morbidity associated with these infections, little is yet known regarding the specific strain types that may have a predilection for ocular tissues nor the set of virulence factors that drive its pathogenicity in this specific biological niche. Whole genome sequencing (WGS) can provide valuable insight in this regard by providing a prospective, comprehensive assessment of the strain types and virulence factors driving disease among specific subsets of clinical isolates. As such, a set of 163-member S. aureus ocular clinical strains were sequenced and assessed for both common strain types (multilocus sequence type (MLST), spa, agr) associated with ocular infections as well as the presence/absence of 235 known virulence factors in a high throughput manner. This ocular strain set was then directly compared to a fully sequenced 116-member non-ocular S. aureus strain set curated from NCBI in order to identify key differences between ocular and non-ocular S. aureus isolates. The most common sequence types found among ocular S. aureus isolates were ST5, ST8 and ST30, generally reflecting circulating non-ocular pathogenic S. aureus strains. However, importantly, ocular isolates were found to be significantly enriched for a set of enterotoxins, suggesting a potential role for this class of virulence factors in promoting ocular disease. Further genomic analysis revealed that these enterotoxins are located on mobile pathogenicity islands, thus horizontal gene transfer may promote the acquisition of enterotoxins, potentially amplifying S. aureus virulence in ocular tissues.

A Rapid, Multiplex Dual Reporter IgG and IgM SARS-CoV-2 Neutralization Assay for a Multiplexed Bead-Based Flow Analysis System.

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.

A Multiplex Microsphere IgG Assay for SARS-CoV-2 Using ACE2-Mediated Inhibition as a Surrogate for Neutralization.

The coronavirus disease 2019 (COVID-19) pandemic has highlighted the challenges inherent to the serological detection of a novel pathogen such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Serological tests can be used diagnostically and for surveillance, but their usefulness depends on their throughput, sensitivity, and specificity. Here, we describe a multiplex fluorescent microsphere-based assay, 3Flex, that can detect antibodies to three major SARS-CoV-2 antigens-spike (S) protein, the spike ACE2 receptor-binding domain (RBD), and nucleocapsid (NP). Specificity was assessed using 213 prepandemic samples. Sensitivity was measured and compared to that of the Abbott Architect SARS-CoV-2 IgG assay using serum samples from 125 unique patients equally binned (n = 25) into 5 time intervals (≤5, 6 to 10, 11 to 15, 16 to 20, and ≥21 days from symptom onset). With samples obtained at ≤5 days from symptom onset, the 3Flex assay was more sensitive (48.0% versus 32.0%), but the two assays performed comparably using serum obtained ≥21 days from symptom onset. A larger collection (n = 534) of discarded sera was profiled from patients (n = 140) whose COVID-19 course was characterized through chart review. This revealed the relative rise, peak (S, 23.8; RBD, 23.6; NP, 16.7 [in days from symptom onset]), and decline of the antibody response. Considerable interperson variation was observed with a subset of extensively sampled intensive care unit (ICU) patients. Using soluble ACE2, inhibition of antibody binding was demonstrated for S and RBD, and not for NP. Taking the data together, this study described the performance of an assay built on a flexible and high-throughput serological platform that proved adaptable to the emergence of a novel infectious agent.

A multiplex microsphere IgG assay for SARS-CoV-2 using ACE2-mediated inhibition as a surrogate for neutralization.

The COVID-19 pandemic has highlighted challenges inherent to serological detection of a novel pathogen like SARS-CoV-2. Serological tests can be used diagnostically and for surveillance, but their usefulness depends on throughput, sensitivity and specificity. Here, we describe a multiplex fluorescent microsphere-based assay, 3Flex, that can detect antibodies to three SARS-CoV-2 antigens-spike (S) protein, the spike ACE2 receptor-binding domain (RBD), and nucleocapsid (NP). Specificity was assessed using 213 pre-pandemic samples. Sensitivity was measured and compared to the Abbott™ ARCHITECT™ SARS-CoV-2 IgG assay using serum from 125 unique patients equally binned ( n = 25) into 5 time intervals (≤5, 6 to 10, 11 to 15, 16 to 20, and ≥21 days from symptom onset). With samples obtained at ≤5 days from symptom onset, the 3Flex assay was more sensitive (48.0% vs. 32.0%), but the two assays performed comparably using serum obtained ≥21 days from symptom onset. A larger collection ( n = 534) of discarded sera was profiled from patients ( n = 140) whose COVID-19 course was characterized through chart review. This revealed the relative rise, peak (S, 23.8; RBD, 23.6; NP, 16.7; in days from symptom onset), and decline of the antibody response. Considerable interperson variation was observed with a subset of extensively sampled ICU patients. Using soluble ACE2, inhibition of antibody binding was demonstrated for S and RBD, and not for NP. Taken together, this study described the performance of an assay built on a flexible and high-throughput serological platform that proved adaptable to the emergence of a novel infectious agent.

Clinical Pathogen Genomics.

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.