ADLM Guidance Document on Laboratory Diagnosis of Respiratory Viruses.

Respiratory viral infections are among the most frequent infections experienced worldwide. The COVID-19 pandemic has highlighted the need for testing and currently several tests are available for the detection of a wide range of viruses. These tests vary widely in terms of the number of viral pathogens included, viral markers targeted, regulatory status, and turnaround time to results, as well as their analytical and clinical performance. Given these many variables, selection and interpretation of testing requires thoughtful consideration. The current guidance document is the authors' expert opinion based on the preponderance of available evidence to address key questions related to best practices for laboratory diagnosis of respiratory viral infections including who to test, when to test, and what tests to use. An algorithm is proposed to help laboratories decide on the most appropriate tests to use for the diagnosis of respiratory viral infections.

Corrigendum: NGS implementation for monitoring SARS-CoV-2 variants in Chicagoland: an institutional perspective, successes and challenges.

[This corrects the article DOI: 10.3389/fpubh.2023.1177695.].

NGS implementation for monitoring SARS-CoV-2 variants in Chicagoland: An institutional perspective, successes and challenges.

Identification of SARS-CoV-2 lineages has shown to provide invaluable information regarding treatment efficacy, viral transmissibility, disease severity, and immune evasion. These benefits provide institutions with an expectation of high informational upside with little insight in regards to practicality with implementation and execution of such high complexity testing in the midst of a pandemic. This article details our institution's experience implementing and using Next Generation Sequencing (NGS) to monitor SARS-CoV-2 lineages in the northern Chicagoland area throughout the pandemic. To date, we have sequenced nearly 7,000 previously known SARS-CoV-2 positive samples from various patient populations (e.g., outpatient, inpatient, and outreach sites) to reduce bias in sampling. As a result, our hospital was guided while making crucial decisions about staffing, masking, and other infection control measures during the pandemic. While beneficial, establishing this NGS procedure was challenging, with countless considerations at every stage of assay development and validation. Reduced staffing prompted transition from a manual to automated high throughput workflow, requiring further validation, lab space, and instrumentation. Data management and IT security were additional considerations that delayed implementation and dictated our bioinformatic capabilities. Taken together, our experience highlights the obstacles and triumphs of SARS-CoV-2 sequencing.

Clinical Impact of Metagenomic Next-Generation Sequencing of Bronchoalveolar Lavage in the Diagnosis and Management of Pneumonia: A Multicenter Prospective Observational Study.

Rapid and accurate pathogen identification is necessary for appropriate treatment of pneumonia. Here, we describe the use of shotgun metagenomic next-generation sequencing (mNGS) of bronchoalveolar lavage for pathogen identification in pneumonia in a large-scale multicenter prospective study with 159 patients enrolled. The results of mNGS were compared with standard methods including culture, staining, and targeted PCR, and the clinical impact of mNGS was evaluated. A positive impact was defined by a definitive diagnosis made using the mNGS results, or change of management because of the mNGS results, leading to a favorable clinical outcome. Overall, mNGS identified more organisms than standard methods (117 versus 72), detected 17 pathogens that consistently were missed in all cases by standard methods, and had an overall positive clinical impact in 40.3% (64 of 159) of cases. mNGS was especially useful in identification of fastidious and atypical organisms causing pneumonia, contributing to detection of definitive pathogens in 45 (28.3%) cases in which standard results were either negative or insufficient. mNGS also helped reassure antibiotic de-escalation in 19 (11.9%) cases. Overall, mNGS led to a change of treatment in 59 (37.1%) cases, including antibiotic de-escalation in 40 (25.2%) cases. This study showed the significant value of mNGS of bronchoalveolar lavage for improving the diagnosis of pneumonia and contributing to better patient care.

Investigation of Phylogeny and Drug Resistance Mechanisms of Elizabethkingia anophelis Isolated from Blood and Lower Respiratory Tract.

Elizabethkingia species are environmental bacteria associated with opportunistic infections in vulnerable populations. Traditionally, Elizabethkingia meningoseptica was considered the predominant pathogenic species. However, commercial identification systems have routinely misidentified Elizabethkingia anophelis as E. meningoseptica, leading to a mischaracterization of clinical strains and an underestimation of the role of E. anophelis in human disease. Elizabethkingia spp. harbor multidrug resistance (MDR) genes that pose challenges for treatment. Differentiation between Elizabethkingia spp. is particularly important due to differences in antimicrobial resistance (AMR) and epidemiological investigation. In this study, we describe a case of MDR E. anophelis isolated from the blood and lower respiratory tract of a patient who was successfully treated with minocycline. These isolates were initially misidentified by matrix assisted laser desorption ionization-time of flight as E. meningoseptica, whereas whole genome sequencing (WGS) confirmed the isolates as E. anophelis with the closest related strain being E. anophelis NUHP1, which was implicated in a 2012 outbreak in Singapore. Several AMR genes (blaBlaB, blaBlaGOB, blaCME, Sul2, erm(F), and catB) were identified by WGS, confirming the mechanisms for MDR. This case emphasizes the utility of WGS for correct speciation, elucidation of resistance genes, and relatedness to other outbreak strains. As E. anophelis is associated with a high mortality and has been found in hospital system sinks, WGS is critically important for determining strain relatedness and tracking outbreaks in the hospital setting.

Recognition of Diagnostic Gaps for Laboratory Diagnosis of Fungal Diseases: Expert Opinion from the Fungal Diagnostics Laboratories Consortium (FDLC).

Fungal infections are a rising threat to our immunocompromised patient population, as well as other nonimmunocompromised patients with various medical conditions. However, little progress has been made in the past decade to improve fungal diagnostics. To jointly address this diagnostic challenge, the Fungal Diagnostics Laboratory Consortium (FDLC) was recently created. The FDLC consists of 26 laboratories from the United States and Canada that routinely provide fungal diagnostic services for patient care. A survey of fungal diagnostic capacity among the 26 members of the FDLC was recently completed, identifying the following diagnostic gaps: lack of molecular detection of mucormycosis; lack of an optimal diagnostic algorithm incorporating fungal biomarkers and molecular tools for early and accurate diagnosis of Pneumocystis pneumonia, aspergillosis, candidemia, and endemic mycoses; lack of a standardized molecular approach to identify fungal pathogens directly in formalin-fixed paraffin-embedded tissues; lack of robust databases to enhance mold identification with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; suboptimal diagnostic approaches for mold blood cultures, tissue culture processing for Mucorales, and fungal respiratory cultures for cystic fibrosis patients; inadequate capacity for fungal point-of-care testing to detect and identify new, emerging or underrecognized, rare, or uncommon fungal pathogens; and performance of antifungal susceptibility testing. In this commentary, the FDLC delineates the most pressing unmet diagnostic needs and provides expert opinion on how to fulfill them. Most importantly, the FDLC provides a robust laboratory network to tackle these diagnostic gaps and ultimately to improve and enhance the clinical laboratory's capability to rapidly and accurately diagnose fungal infections.

Novel Use of Rapid Antigen Influenza Testing in the Outpatient Setting To Provide an Early Warning Sign of Influenza Activity in the Emergency Departments of an Integrated Health System.

Seasonal influenza virus is associated with high morbidity and mortality especially in vulnerable patient populations. Here, we demonstrate the novel use of Sofia influenza A+B fluorescent immunoassay (FIA), a rapid antigen-based influenza point-of-care test (POCT), combined with Virena software for automatic deidentified tracking of influenza activity across the Los Angeles area and for predicting surges of influenza cases in the emergency department (ED). We divided outpatient clinics into 6 geographic zones and compared weekly influenza activity. In the outpatient setting, there were 1,666 and 274 influenza A and influenza B positives, respectively, across the 2018 to 2019 influenza season and 1,857 and 1,449 influenza A and influenza B positives, respectively, during the 2019 to 2020 influenza season, with zone-specific differences observed. Moreover, we found that a rapid increase in outpatient influenza was followed by an influx in influenza-positive cases in the ED, offering a 1- to 3-week warning sign for ED influx of triple or quadruple the number of influenza cases compared to the prior week. Sofia influenza A+B FIA allows for surveillance of real-time deidentified influenza activity. Tracking of such data may serve as a valuable region-specific influenza indicator and predictor to guide infection prevention measures in both the outpatient and hospital settings. High-impact interventions include designating areas for waiting rooms for influenza-like illnesses, altering staff scheduling in anticipation of surges, and securing sufficient personal protective equipment and antivirals during the height of influenza season.

Amplicon-Based Next-Generation Sequencing for Detection of Fungi in Formalin-Fixed, Paraffin-Embedded Tissues: Correlation with Histopathology and Clinical Applications.

Invasive fungal infections are increasing in prevalence because of an expanding population of immunocompromised individuals. To reduce morbidity and mortality, it is critical to accurately identify fungal pathogens to guide treatment. Current methods rely on histopathology, fungal culture, and serology, which are often insufficient for diagnosis. Herein, we describe the use of a laboratory-developed internal transcribed spacer-targeted amplicon-based next-generation sequencing (NGS) assay for the identification of fungal etiology in fungal stain-positive formalin-fixed, paraffin-embedded tissues by using Illumina MiSeq. A total of 44 specimens from 35 patients were included in this study, with varying degrees of fungal burden from multiple anatomic sites. NGS identified 20 unique species across the 54 total organisms detected, including 40 molds, 10 yeasts, and 4 dimorphic fungi. The histopathologic morphology and the organisms suspected by surgical pathologist were compared with the organisms identified by NGS, with 100% (44/44) and 93.2% (41/44) concordance, respectively. In contrast, fungal culture only provided an identification in 27.3% (12/44) of specimens. We demonstrated that NGS is a powerful method for accurate and unbiased fungal identification in formalin-fixed, paraffin-embedded tissues. A retrospective evaluation of the clinical utility of the NGS results also suggests this technology can potentially improve both the speed and the accuracy of diagnosis for invasive fungal infections.

A Collaborative Tale of Diagnosing and Treating Chronic Pulmonary Aspergillosis, from the Perspectives of Clinical Microbiologists, Surgical Pathologists, and Infectious Disease Clinicians.

Chronic pulmonary aspergillosis (CPA) refers to a spectrum of Aspergillus-mediated disease that is associated with high morbidity and mortality, with its true prevalence vastly underestimated. The diagnosis of CPA includes characteristic radiographical findings in conjunction with persistent and systemic symptoms present for at least three months, and evidence of Aspergillus infection. Traditionally, Aspergillus infection has been confirmed through histopathology and microbiological studies, including fungal culture and serology, but these methodologies have limitations that are discussed in this review. The treatment of CPA requires an individualized approach and consideration of both medical and surgical options. Most Aspergillus species are considered susceptible to mold-active triazoles, echinocandins, and amphotericin B; however, antifungal resistance is emerging and well documented, demonstrating the need for novel therapies and antifungal susceptibility testing that correlates with clinical response. Here, we describe the clinical presentation, diagnosis, and treatment of CPA, with an emphasis on the strengths and pitfalls of diagnostic and treatment approaches, as well as future directions, including whole genome sequencing and metagenomic sequencing. The advancement of molecular technology enables rapid and precise species level identification, and the determination of molecular mechanisms of resistance, bridging the clinical infectious disease, anatomical pathology, microbiology, and molecular biology disciplines.

Large granular lymphocyte leukemia serum and corresponding hematological parameters reveal unique cytokine and sphingolipid biomarkers and associations with STAT3 mutations.

Large granular lymphocyte (LGL) leukemia is a rare hematological disorder with expansion of the T-cell or natural killer (NK) cell lineage. Signal transducer and activator of transcription 3 (STAT3) exhibits somatic activating mutations in 30%-40% of LGL leukemia cases. Transcriptional targets of STAT3 include inflammatory cytokines, thus previous studies have measured cytokine levels of LGL leukemia patients compared to normal donors. Sphingolipid metabolism is a growing area of cancer research, with efforts focused on drug discovery. To date, no studies have examined serum sphingolipids in LGL leukemia patients, and only one study compared a subset of cytokines between the T-LGL and NK-LGL subtypes. Therefore, here, we included both LGL leukemia subtypes with the goals of (a) measuring serum sphingolipids for the first time, (b) measuring cytokines to find distinctions between the subtypes, and (c) establishing relationships with STAT3 mutations and clinical data. The serum analyses identified cytokines (EGF, IP-10, G-CSF) and sphingolipids (SMC22, SMC24, SMC20, LysoSM) significantly different in the LGL leukemia group compared to normal donors. In a mixed STAT3 mutation group, D661Y samples exhibited the highest mean corpuscular volume (MCV) values. We explored this further by expanding the cohort to include larger groups of single STAT3 mutations. Male D661Y STAT3 samples had lower Hgb and higher MCV compared to wild type (WT) or Y640F counterparts. This is the first report examining large groups of individual STAT3 mutations. Overall, our results revealed novel serum biomarkers and evidence that D661Y mutation may show different clinical manifestation compared to WT or Y640F STAT3.

Discovery of a Novel Hypervirulent Acinetobacter baumannii Strain in a Case of Community-Acquired Pneumonia.

PURPOSE: Acinetobacter baumannii is associated with both hospital-acquired infections and community-acquired pneumonia (CAP). Here, we describe a novel strain of A. baumannii in a case of CAP in a previously healthy rural villager from Central Eastern China. MATERIALS AND METHODS: A. baumannii isolated from the patient (LS01) was compared to well-characterized pathogenic strain (AB5075), nosocomial circulating strain in China (ZJ06), and wild-type strain (ATCC17978). Growth rate studies were conducted under different environmental stressors, and virulence studies were performed using Galleria mellonella larvae. Whole genome sequencing (WGS) was performed using MinIon and MiSeq. Center for Genomic Epidemiology, CLCbio, Geneious, and Virulence Factors of Pathogenic Bacteria database were used for genomic analysis. RESULTS: LS01 grew significantly faster at 37°C and 42°C and in the presence of zinc compared to other strains. LS01 was more virulent in G. mellonella, killing all larvae within 8 h. Although WGS revealed 44 virulence genes, these genes were also present in the other strains. While two chromosomally encoded ß-lactamases were identified, there were no plasmids identified and LS01 was pan-susceptible to all antibiotics tested. Phylogenetic analysis revealed that the closest related strains were only 72.552% identical, supporting a novel strain. CONCLUSION: LS01 is a novel strain of hypervirulent yet pan-drug susceptible A. baumannii isolated from a patient with no prior hospitalizations, sick contacts, or any of the typical risk factors. This raises concerns for an emerging pathogen, and more epidemiological studies should be conducted to assess the prevalence of this A. baumannii strain.

An Unusual Carbapenem Resistant Escherichia coli Carrying Plasmid-mediated AmpC and Mutated ompC in A Patient with Recurrent Urinary Tract Infections.

We describe a case of carbapenem resistant E. coli isolated from urine in an 87-year-old woman with recurrent urinary tract infections. Using whole genome sequencing (WGS), we identified the carbapenem resistance mechanism to be a combination of ompC porin loss and plasmid-mediated AmpC gene blaCMY-2 , which was not detected by routine molecular and phenotypic carbapenemase assays. Our case raises a concern for the limitation of current CRE screening tools for emerging resistance mechanisms and demonstrates the utility of WGS as a better tool for characterization of CRE in the clinical setting.