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 ...

University of Rochester Medical Center

New Delhi metallo-beta-lactamases (NDMs) are an uncommon but emerging cause of carbapenem resistance in the United States. Genomic factors promoting their domestic spread remain poorly characterized. A prospective genomic surveillance program among Boston-area hospitals identified multiple new occurrences of NDM-carrying strains of  and  complex in inpatient and outpatient settings, representing the first occurrences of NDM-mediated resistance since initiating genomic surveillance in 2011. Cases included domestic patients with no international exposures. PacBio sequencing of isolates identified strain characteristics, resistance genes, and the complement of mobile vectors mediating spread. Analyses revealed a common 3,114-bp region containing the  gene, with carriage of this conserved region among unique strains by diverse transposon and plasmid backbones. Functional studies revealed a broad capacity for  transmission by conjugation, transposition, and complex interplasmid recombination events. NDMs represent a rapidly spreading form of drug resistance that can occur in inpatient and outpatient settings and in patients without international exposures. In contrast to Tn-based spread of  carbapenemases (KPCs), diverse transposable elements mobilize NDM enzymes, commonly with other resistance genes, enabling naive strains to acquire multi- and extensively drug-resistant profiles with single transposition or plasmid conjugation events. Genomic surveillance provides effective means to rapidly identify these gene-level drivers of resistance and mobilization in order to inform clinical decisions to prevent further spread.

Diverse Vectors and Mechanisms Spread New Delhi Metallo-β-Lactamases among Carbapenem-Resistant  in the Greater Boston Area.

 is a nosocomial pathogen associated with drug resistance and outbreaks in intensive care units. In a 5-month period in 2017, we experienced an increased incidence of cultures for carbapenem-resistant  (CR-KA) from an adult cardiothoracic intensive care unit (CICU) involving 15 patients. Phylogenomic analysis following whole-genome sequencing (WGS) identified the outbreak CR-KA isolates to group together as a tight monoclonal cluster (with no more than six single nucleotide polymorphisms [SNPs]), suggestive of a protracted intraward transmission event. No clonal relationships were identified between the CICU CR-KA strains and additional hospital CR-KA patient isolates from different wards and/or previous years. Carbapenemase-encoding genes and drug-resistant plasmids were absent in the outbreak strains, and carbapenem resistance was attributed to mutations impacting AmpD activity and membrane permeability. The CICU outbreak strains harbored an integrative conjugative element (ICE) which has been associated with pathogenic  lineages (ICE10). Comparative genomics with global  genomes showed our outbreak strains to group closely with global sequence type 4 (ST4) strains, which, along with ST93, likely represent dominant  lineages associated with human infections. For poorly characterized pathogens, scaling analyses to include sequenced genomes from public databases offer the opportunity to identify emerging trends and dominant clones associated with specific attributes, syndromes, and geographical locations.

Next-Generation-Sequencing-Based Hospital Outbreak Investigation Yields Insight into Klebsiella aerogenes Population Structure and Determinants of Carbapenem Resistance and Pathogenicity.

The genome sequence of a  strain isolated from the urine of a patient with acute cystitis and sepsis is reported. The genome contains  and (M) genes, consistent with the isolate's phenotypic resistance to macrolides and tetracycline.

Genome Sequence of a Facklamia hominis Isolate from a Patient with Urosepsis.

Extended-spectrum β-lactamase (ESBL)-producing  pose significant treatment and infection prevention challenges.  sequence type (ST) 131 associated with the  gene has been the dominant lineage of ESBL-producing  in the US and worldwide. In this study, our objective was to determine the β-lactamase profile, means of dissemination, prevalence, and the clonal identity of ESBL-producing  in our region of Western New York. Whole-genome SNP-based phylogenomics was used to assess 89 ceftriaxone-resistant (CTR) . Isolates were collected from both inpatients and outpatients and from urine and sterile-sites over a 2 month period in 2017 or throughout the year, respectively. ST131 was the predominant ST (46.0%), followed by ST38 (15.7%). The  gene was commonly found in 53.7% of ST131 isolates, whereas the  gene was found in 26.8% of ST131, though was significantly associated with ST38, and was found in 71.4% of those strains. When compared to ST131, ST38  exhibited increased frequency of resistance to nitrofurantoin and decreased frequency of resistance to ciprofloxacin and ampicillin-sulbactam. Using Nanopore long-read sequencing technology, an analysis of the ESBL genetic context showed that the  gene was chromosomal in 68.2% of ST131, whereas the  gene was plasmid-borne in all ST131 and 90% of ST38 isolates. Notably, the  gene in ST38 resided on highly-related (99.0-100.0% identity and 65.0-98.0% query coverage) conjugative IncF plasmids of distinct plasmid multi-locus sequence types (pMLSTs) from those in ST131. Furthermore, ST131 and ST38 were found to harbor different antibiotic resistance gene and virulence factor profiles. These findings raise the possibility of an emerging ESBL-producing  lineage in our region.

Genomic Surveillance of Ceftriaxone-Resistant  in Western New York Suggests the Extended-Spectrum β-Lactamase  Is Emerging on Distinct Plasmids in ST38.

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.

Clinical Pathogen Genomics.

The entire spectrum of diagnostic testing, from reagent supply to test performance, has been a major focus during the coronavirus disease 2019 (COVID-19) pandemic. The hope for serologic testing is that it will provide both epidemiologic information about seroprevalence as well as individual information about previous infection. This information is particularly helpful for high-risk individuals who may be outside of the viral shedding window, such as children with suspected multisystem inflammatory syndrome. It is not yet understood whether serologic testing can be interpreted in terms of protective immunity. These concerns must be addressed using highly sensitive and specific tests.

Measuring the Serologic Response to Severe Acute Respiratory Syndrome Coronavirus 2: Methods and Meaning.

2020: A Year for Clinical Microbiology.

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 (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 (= 534) of discarded sera was profiled from patients (= 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.

Extraction-Free Methods for the Detection of SARS-CoV-2 by Reverse Transcription-PCR: a Comparison with the Cepheid Xpert Xpress SARS-CoV-2 Assay across Two Medical Centers.

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.

Nicole D. Pecora

Departments of Pathology and Laboratory Medicine,
Clinical Microbiology,
Department of Microbiology and Immunology,
Departments of Pathology and Laboratory Medicine, Clinical Microbiology

University of Rochester Medical Center

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

Nicole D. Pecora

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Departments of Pathology and Laboratory Medicine,Clinical Microbiology,Department of Microbiology and Immunology,Departments of Pathology and Laboratory Medicine, Clinical Microbiology

University of Rochester Medical Center

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

Departments of Pathology and Laboratory Medicine,
Clinical Microbiology,
Department of Microbiology and Immunology,
Departments of Pathology and Laboratory Medicine, Clinical Microbiology