Sequential Treatment Failure With Aztreonam-Ceftazidime-Avibactam Followed by Cefiderocol Due to Preexisting and Acquired Mechanisms in a New Delhi Metallo-ß-lactamase-Producing Escherichia coli Causing Fatal Bloodstream Infection.

We report a fatal case of New Delhi metallo-ß-lactamase (NDM)-producing Escherichia coli in a bacteremic patient with sequential failure of aztreonam plus ceftazidime-avibactam followed by cefiderocol. Acquired resistance was documented phenotypically and mediated through preexisting and acquired mutations. This case highlights the need to rethink optimal treatment for NDM-producing organisms.

Accuracy of QuantiFERON in active tuberculosis suspects with comorbidities and nontuberculous mycobacterial infection in Northern California.

The QuantiFERON-TB Gold (QFT) is routinely utilized in North American health systems to detect a cellular immune response to Mycobacterium tuberculosis antigens in symptomatic and asymptomatic patients. The sensitivity of QFT in tuberculosis (TB) patients with comorbidities is not well established and the specificity of QFT in patients with nontuberculous mycobacteria (NTM) infections is incompletely understood. Between 2012 and 2023, all patients with culture-positive TB and patients with NTM infection per the expert diagnostic guidelines or biopsy-proven NTM infection who had a concurrent QFT test were included in this study. The sensitivity and specificity of QFT were measured in TB and NTM patients, respectively. In 109 patients with active TB, the overall sensitivity of QFT was 78.0% (85/109; 95% CI: 70.1, 85.7). The sensitivity was 86.0% (49/57; 95% CI: 76.6, 94.8) and 69.2% (36/52; 95% CI: 56.7, 81.8) in immunocompetent and immunocompromised patients, respectively. The overall specificity of QFT in 88 patients with NTM infection was 76.1% (67/88; 95% CI: 67.2, 85.0). After the exclusion of 17 NTM patients with risk factors for latent TB infection, the specificity was 94.4% (67/71; 95% CI: 89.1, 99.7). Two patients had NTM species known to cross-react with QFT. In two NTM patients infected with species (Mycobacterium intracellulare subsp. intracellulare and Mycobacterium intracellulare subsp. chimaera) not known to cross-react, whole genome sequencing did not detect ESAT-6 or CFP-10. In Northern California, the QFT assay demonstrated moderately low to moderately high sensitivity in TB patients and very high specificity in NTM patients, thus ruling out concerns for cross-reactivity with NTM.

Dynamic Epidemiological Networks: A Data Representation Framework for Modeling and Tracking of SARS-CoV-2 Variants.

The large-scale real-time sequencing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes has allowed for rapid identification of concerning variants through phylogenetic analysis. However, the nature of phylogenetic reconstruction is typically static, in that the relationships between taxonomic units, once defined, are not subject to alterations. Furthermore, most phylogenetic methods are intrinsically batch mode in nature, requiring the presence of the entire data set. Finally, the emphasis of phylogenetics is on relating taxonomical units. These characteristics complicate the application of classical phylogenetics methods to represent relationships in molecular data collected from rapidly evolving strains of an etiological agent, such as SARS-CoV-2, since the molecular landscape is updated continuously as samples are collected. In such settings, variant definitions are subject to epistemological constraints and may change as data accumulate. Furthermore, representing within-variant molecular relationships may be as important as representing between variant relationships. This article describes a novel data representation framework called dynamic epidemiological networks (DENs) along with algorithms that underpin its construction to address these issues. The proposed representation is applied to study the molecular development underlying the spread of the COVID-19 (coronavirus disease 2019) pandemic in two countries: Israel and Portugal spanning a 2-year period from February 2020 to April 2022. The results demonstrate how this framework could be used to provide a multiscale representation of the data by capturing molecular relationships between samples as well as those between variants, automatically identifying the emergence of high frequency variants (lineages), including variants of concern such as Alpha and Delta, and tracking their growth. Additionally, we show how analyzing the evolution of the DEN can help identify changes in the viral population that could not be readily inferred from phylogenetic analysis.

Comparative genomics of Enterobacter cloacae complex before and after acquired clinical resistance to Ceftazidime-Avibactam.

Resistance to Ceftazidime-Avibactam in Enterobacter cloacae is poorly understood. Whole genome sequencing identified 6 variants in isolates collected from a patient before and after acquiring Ceftazidime-Avibactam resistance. This included a Phe396Leu mutation in acrB, a component of the AcrAB-TolC efflux pump, possibly mediating enhanced efflux of Ceftazidime and/ or Avibactam.

A dual-caged resorufin probe for rapid screening of infections resistant to lactam antibiotics.

The alarming increase of antimicrobial resistance urges rapid diagnosis and pathogen specific infection management. This work reports a rapid screening assay for pathogenic bacteria resistant to lactam antibiotics. We designed a fluorogenic N-cephalosporin caged 3,7-diesterphenoxazine probe CDA that requires sequential activations to become fluorescent resorufin. A series of studies with recombinant ß-lactamases and clinically prevalent pathogens including Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae and Serratia marcescens demonstrated that CDA possessed superior sensitivity in reporting the activity of ß-lactamases including cephalosporinases and carbapenemases. After a simple filtration, lactam-resistant bacteria in urine samples could be detected at 103 colony-forming units per milliliter within 2 hours.

Clinical Accuracy and Impact of Plasma Cell-Free DNA Fungal Polymerase Chain Reaction Panel for Noninvasive Diagnosis of Fungal Infection.

BACKGROUND: Invasive fungal infection (IFI) is a growing cause of morbidity and mortality in oncology and transplant patients. Diagnosis of IFI is often delayed due to need for invasive biopsy and low sensitivity of conventional diagnostic methods. Fungal cell-free DNA (cfDNA) detection in plasma is a novel testing modality for the noninvasive diagnosis of IFI. METHODS: A novel bioinformatic pipeline was created to interrogate fungal genomes and identify multicopy sequences for cfDNA polymerase chain reaction (PCR) targeting. A real-time PCR panel was developed for 12 genera and species most commonly causing IFI. Sensitivity and specificity of the fungal PCR panel were determined using plasma samples from patients with IFI and non-IFI controls. Clinical impact of the fungal PCR panel was evaluated prospectively based on the treating team's interpretation of the results. RESULTS: Overall, the sensitivity and specificity were 56.5% (65/115; 95% confidence interval [CI], 47.4-65.2) and 99.5% (2064/2075; 95% CI, 99.0-99.7), respectively. In the subset of patients with an optimized plasma volume (2 mL), sensitivity was 69.6% (48/69; 95% CI, 57.9-79.2). Sensitivity was 91.7% (11/12; 95% CI, 62.5-100) for detection of Mucorales agents, 56.3% (9/16; 95% CI, 33.2-76.9) for Aspergillus species, and 84.6% (11/13; 95% CI, 56.5-96.9) for Candida albicans. In a prospective evaluation of 226 patients with suspected IFI, cfDNA testing was positive in 47 (20.8%) patients and resulted in a positive impact on clinical management in 20 of 47 (42.6%). CONCLUSIONS: The fungal cfDNA PCR panel offers a noninvasive approach to early diagnosis of IFI, providing actionable results for personalized care.

Diversity of resistance mechanisms in carbapenem-resistant Enterobacteriaceae at a health care system in Northern California, from 2013 to 2016.

The mechanism of resistance in carbapenem-resistant Enterobacteriaceae (CRE) has therapeutic implications. We comprehensively characterized emerging mechanisms of resistance in CRE between 2013 and 2016 at a health system in Northern California. A total of 38.7% (24/62) of CRE isolates were carbapenemase gene-positive, comprising 25.0% (6/24) blaOXA-48 like, 20.8% (5/24) blaKPC, 20.8% (5/24) blaNDM, 20.8% (5/24) blaSME, 8.3% (2/24) blaIMP, and 4.2% (1/24) blaVIM. Between carbapenemases and porin loss, the resistance mechanism was identified in 95.2% (59/62) of CRE isolates. Isolates expressing blaKPC were 100% susceptible to ceftazidime-avibactam, meropenem-vaborbactam, and imipenem-relebactam; blaOXA-48 like-positive isolates were 100% susceptible to ceftazidime-avibactam; and metallo ß-lactamase-positive isolates were nearly all nonsusceptible to above antibiotics. Carbapenemase gene-negative CRE were 100% (38/38), 92.1% (35/38), 89.5% (34/38), and 31.6% (12/38) susceptible to ceftazidime-avibactam, meropenem-vaborbactam, imipenem-relebactam, and ceftolozane-tazobactam, respectively. None of the CRE strains were identical by whole genome sequencing. At this health system, CRE were mediated by diverse mechanisms with predictable susceptibility to newer ß-lactamase inhibitors.

Precision identification of diverse bloodstream pathogens in the gut microbiome.

A comprehensive evaluation of every patient with a bloodstream infection includes an attempt to identify the infectious source. Pathogens can originate from various places, such as the gut microbiota, skin and the external environment. Identifying the definitive origin of an infection would enable precise interventions focused on management of the source1,2. Unfortunately, hospital infection control practices are often informed by assumptions about the source of various specific pathogens; if these assumptions are incorrect, they lead to interventions that do not decrease pathogen exposure3. Here, we develop and apply a streamlined bioinformatic tool, named StrainSifter, to match bloodstream pathogens precisely to a candidate source. We then leverage this approach to interrogate the gut microbiota as a potential reservoir of bloodstream pathogens in a cohort of hematopoietic cell transplantation recipients. We find that patients with Escherichia coli and Klebsiella pneumoniae bloodstream infections have concomitant gut colonization with these organisms, suggesting that the gut may be a source of these infections. We also find cases where typically nonenteric pathogens, such as Pseudomonas aeruginosa and Staphylococcus epidermidis, are found in the gut microbiota, thereby challenging the existing informal dogma of these infections originating from environmental or skin sources. Thus, we present an approach to distinguish the source of various bloodstream infections, which may facilitate more accurate tracking and prevention of hospital-acquired infections.

Ultrasensitive Detection of Clostridioides difficile Toxins A and B by Use of Automated Single-Molecule Counting Technology.

Current tests for the detection of Clostridioides (formerly Clostridium) difficile free toxins in feces lack sensitivity, while nucleic acid amplification tests lack clinical specificity. We have evaluated the Singulex Clarity C. diff toxins A/B assay (currently in development), an automated and rapid ultrasensitive immunoassay powered by single-molecule counting technology, for detection of C. difficile toxin A (TcdA) and toxin B (TcdB) in stool. The analytical sensitivity, analytical specificity, repeatability, and stability of the assay were determined. In a clinical evaluation, frozen stool samples from 311 patients with suspected C. difficile infection were tested with the Clarity C. diff toxins A/B assay, using an established cutoff value. Samples were tested with the Xpert C. difficile/Epi assay, and PCR-positive samples were tested with an enzyme immunoassay (EIA) (C. Diff Quik Chek Complete). EIA-negative samples were further tested with a cell cytotoxicity neutralization assay. The limits of detection for TcdA and TcdB were 0.8 and 0.3 pg/ml in buffer and 2.0 and 0.7 pg/ml in stool, respectively. The assay demonstrated reactivity to common C. difficile strains, did not show cross-reactivity to common gastrointestinal pathogens, was robust against common interferents, allowed detection in fresh and frozen stool samples and in samples after three freeze-thaw cycles, and provided results with high reproducibility. Compared to multistep PCR and toxin-testing procedures, the Singulex Clarity C. diff toxins A/B assay yielded 97.7% sensitivity and 100% specificity. The Singulex Clarity C. diff toxins A/B assay is ultrasensitive and highly specific and may offer a standalone solution for rapid detection and quantitation of free toxins in stool.

Clostridium difficile PCR Cycle Threshold Predicts Free Toxin.

There is no stand-alone Clostridium difficile diagnostic that can sensitively and rapidly detect fecal free toxins. We investigated the performance of the C. difficile PCR cycle threshold (CT ) for predicting free toxin status. Consecutive stool samples (n = 312) positive for toxigenic C. difficile by the GeneXpert C. difficile/Epi tcdB PCR assay were tested with the rapid membrane C. Diff Quik Chek Complete immunoassay (RMEIA). RMEIA toxin-negative samples were tested with the cell cytotoxicity neutralization assay (CCNA) and tgcBIOMICS enzyme-linked immunosorbent assay (ELISA). Using RMEIA alone or in combination with CCNA and/or ELISA as the reference method, the accuracy of CT was measured at different CT cutoffs. Using RMEIA as the reference method, a CT cutoff of 26.35 detected toxin-positive samples with a sensitivity, specificity, positive predictive value, and negative predictive value of 96.0% (95% confidence interval [CI], 90.2% to 98.9%), 65.9% (95% CI, 59.0% to 72.2%), 57.4% (95% CI, 52.7% to 62%), and 97.1% (95% CI, 92.8% to 98.9), respectively. Inclusion of CCNA in the reference method improved CT specificity to 78.0% (95% CI, 70.7% to 84.2%). Intercartridge lot CT variability measured as the average coefficient of variation was 2.8% (95% CI, 1.2% to 3.2%). Standardizing the input stool volume did not improve CT toxin specificity. The median CT values were not significantly different between stool samples with Bristol scores of 5, 6, and 7, between pediatric and adult samples, or between presumptive 027 and non-027 strains. In addition to sensitively detecting toxigenic C. difficile in stool, on-demand PCR may also be used to accurately predict toxin-negative stool samples, thus providing additional results in PCR-positive stool samples to guide therapy.