Multi-center clinical evaluation of the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay in nasopharyngeal swab specimens from symptomatic individuals.

The symptomology is overlapping for respiratory infections due to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), influenza A/B viruses, and respiratory syncytial virus (RSV). Accurate detection is essential for proper medical management decisions. This study evaluated the clinical performance of the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay in nasopharyngeal swab (NPS) specimens from individuals of all ages with signs and symptoms of respiratory infection consistent with COVID-19, influenza, or RSV. Retrospective known-positive and prospectively obtained residual NPS specimens were collected during two respiratory seasons in the USA. Clinical performance was established by comparing Panther Fusion SARS-CoV-2/Flu assay results to a three-molecular assay composite comparator interpretation for SARS-CoV-2 and to the FDA-cleared Panther Fusion Flu A/B/RSV assay results for all non-SARS-CoV-2 targets. A total of 1,900 prospective and 95 retrospective NPS specimens were included in the analyses. The overall prevalence in prospectively obtained specimens was 20.7% for SARS-CoV-2, 6.7% for influenza A, and 0.7% for RSV; all influenza B-positive specimens were retrospective specimens. The positive percent agreement of the Panther Fusion assay was 96.9% (378/390) for SARS-CoV-2, 98.0% (121/123) for influenza A virus, 95.2% (20/21) for influenza B virus, and 96.6% (57/59) for RSV. The negative percent agreement was ≥98.5% for all target viruses. Specimens with discordant Panther Fusion SARS/Flu/RSV assay results all had cycle threshold values of ≥32.4 (by comparator or by Panther Fusion SARS/Flu/RSV assay). Only five co-infections were detected in the study specimens. The Panther Fusion SARS-CoV-2/Flu/RSV assay provides highly sensitive and specific detection of SARS-CoV-2, influenza A virus, influenza B virus, and RSV in NPS specimens.

Performance of Non-nasopharyngeal Sample Types for Molecular Detection of SARS-CoV-2.

Nasopharyngeal swabs have historically been considered the preferred specimen type for the detection of respiratory viruses, including SARS-CoV-2. However, in response to a global pandemic with shortages of swabs and specimen transport media, limited access to qualified health care personnel, and needs for large-scale testing in nonmedical settings, alternative sample types have been validated for COVID-19 diagnosis. The purpose of this review is to highlight the diagnostic accuracy and clinical utility of non-nasopharyngeal respiratory samples for SARS-CoV-2 molecular diagnostic testing.

Multicenter Evaluation of a Gradient Diffusion Method for Antimicrobial Susceptibility Testing of Helicobacter pylori.

Helicobacter pylori is an important human pathogen associated with peptic ulcer disease, dyspepsia, and gastric malignancy. Antimicrobial susceptibility testing (AST) is often requested for patients who fail eradication therapy. The Clinical and Laboratory Standards Institute (CLSI) reference method, agar dilution (AD), is not performed in most laboratories and maintaining organism viability during transit to a reference laboratory is difficult. We assessed the performance of the Etest (bioMérieux) as a method for H. pylori AST in comparison to AD. Etest MICs were determined for 83 H. pylori isolates at ARUP and Cleveland Clinic (CC). Categorical agreement (CA), very major, major, and minor errors (VME, ME, and mE) were determined for Etest using AD performed at Mayo Clinic Laboratories as the reference method. Testing on isolates with errors was repeated to determine final results summarized below. For clarithromycin, 66.3% of isolates were resistant (R) by AD; Etest results at each laboratory showed 1mE (1.2%) and 1 ME (3.8%). For tetracycline, only 2 isolates were R by AD; a single VME occurred at both sites (98.8% CA, 50% VME) with the same isolate. Applying EUCAST levofloxacin breakpoints to interpret ciprofloxacin results, 60.2% of isolates were R by AD; ARUP CA was 97.6% (1 ME (3%), 1 VME (2%)) and CC CA was 96.3% (1 ME (3%), 2 VMEs (4%)). Despite high error rates, the categorical agreement was acceptable (>90%) for all three antibiotics between AD and Etest. In-house susceptibility testing by gradient diffusion can allow for testing of fastidious organisms that may not survive transport to specialized laboratories; however, the method is not without technical challenges. Characterization of resistance mechanisms, increased AD dilutions, and testing from the same inoculum may determine if the observed errors reflect technical issues or breakpoints that need optimization. IMPORTANCE Routine antimicrobial susceptibility testing (AST) of Helicobacter pylori by agar dilution is difficult to perform and not practical in most clinical microbiology laboratories. The Etest gradient diffusion method can be a reliable alternative for H. pylori AST with the advantage of being a less laborious quantitative method. This work reveals that an optimized Etest method can provide acceptable performance for H. pylori AST and describes the challenges associated with this methodology.

Clinical Laboratory Perspective on Streptococcus halichoeri, an Unusual Nonhemolytic, Lancefield Group B Streptococcus Causing Human Infections.

Streptococcus halichoeri is a relatively newly identified species of pyogenic streptococci that causes zoonotic infection in humans. S. halichoeri was first described in 2004 as indigenous to seals, and only 8 reports of human S. halichoeri infection have been published. S. halichoeri grows as small, white, nonhemolytic colonies and may be strongly catalase-positive on routine blood agar media, which can lead to isolates being misidentified as coagulase-negative staphylococci. S. halichoeri tests positive for Lancefield group B antigen, like S. agalactiae, but can be identified with matrix-assisted laser desorption/ionization time of flight mass spectrometry or partial 16S rRNA sequencing. We describe 3 cases of S. halichoeri bone and joint infections in patients in the United States with underlying health conditions. In addition, we examine the microbiologic characteristics of S. halichoeri and discuss the importance of fully identifying this organism that might otherwise be disregarded as a skin commensal.

SARS-CoV-2 innate effector associations and viral load in early nasopharyngeal infection.

COVID-19 causes severe disease with poor outcomes. We tested the hypothesis that early SARS-CoV-2 viral infection disrupts innate immune responses. These changes may be important for understanding subsequent clinical outcomes. We obtained residual nasopharyngeal swab samples from individuals who requested COVID-19 testing for symptoms at drive-through COVID-19 clinical testing sites operated by the University of Utah. We applied multiplex immunoassays, real-time polymerase chain reaction assays and quantitative proteomics to 20 virus-positive and 20 virus-negative samples. ACE-2 transcripts increased with infection (OR =17.4, 95% CI [CI] =4.78-63.8) and increasing viral N1 protein transcript load (OR =1.16, CI =1.10-1.23). Transcripts for two interferons (IFN) were elevated, IFN-λ1 (OR =71, CI =7.07-713) and IFN-λ2 (OR =40.2, CI =3.86-419), and closely associated with viral N1 transcripts (OR =1.35, CI =1.23-1.49 and OR =1.33 CI =1.20-1.47, respectively). Only transcripts for IP-10 were increased among systemic inflammatory cytokines that we examined (OR =131, CI =1.01-2620). We found widespread discrepancies between transcription and translation. IFN proteins were unchanged or decreased in infected samples (IFN-γ OR =0.90 CI =0.33-0.79, IFN-λ2,3 OR =0.60 CI =0.48-0.74) suggesting viral-induced shut-off of host antiviral protein responses. However, proteins for IP-10 (OR =3.74 CI =2.07-6.77) and several interferon-stimulated genes (ISG) increased with viral load (BST-1 OR =25.1, CI =3.33-188; IFIT1 OR =19.5, CI =4.25-89.2; IFIT3 OR =245, CI =15-4020; MX-1 OR =3.33, CI =1.44-7.70). Older age was associated with substantial modifications of some effects. Ambulatory symptomatic patients had an innate immune response with SARS-CoV-2 infection characterized by elevated IFN, proinflammatory cytokine and ISG transcripts, but there is evidence of a viral-induced host shut-off of antiviral responses. Our findings may characterize the disrupted immune landscape common in patients with early disease.

SARS-CoV-2 Innate Effector Associations and Viral Load in Early Nasopharyngeal Infection.

To examine innate immune responses in early SARS-CoV-2 infection that may change clinical outcomes, we compared nasopharyngeal swab data from 20 virus-positive and 20 virus-negative individuals. Multiple innate immune-related and ACE-2 transcripts increased with infection and were strongly associated with increasing viral load. We found widespread discrepancies between transcription and translation. Interferon proteins were unchanged or decreased in infected samples suggesting virally-induced shut-off of host anti-viral protein responses. However, IP-10 and several interferon-stimulated gene proteins increased with viral load. Older age was associated with modifications of some effects. Our findings may characterize the disrupted immune landscape of early disease.

Clinical evaluation of the BioFire® Respiratory Panel 2.1 and detection of SARS-CoV-2.

We evaluated the performance of the BioFire® Respiratory Panel 2.1 (RP2.1) in the detection of SARS CoV-2 in comparison against three other SARS CoV-2 EUA assays. In these studies, the RP2.1 panel had 98 % positive percent agreement (48/49) and 100 % negative percent agreement (49/49). Since 30 % of nasopharyngeal swab specimens have a SARS CoV-2 Ct >30 and thus detection of virus in low titers is clinically relevant, a sample with a high titer was diluted and each 10 fold dilution was tested in triplicate and compared against 6 other EUA approved SARS CoV-2 assays. These data suggested that the BioFire® RP2.1 panel, along with four other SARS CoV-2 assays (Roche cobas, Cepheid Xpert Xpress, BioFire® Defense COVID19, and NECoV19), consistently detected viral RNA at the 10-7 dilution. Overall, these studies suggest that the BioFire® RP2.1 assay can be used to detect acute cases of SARS CoV2 in addition to patients with low viral titer later in disease presentation.

Evaluation of a Novel High-Definition PCR Multiplex Assay for Simultaneous Detection of Tick-Borne Pathogens in Human Clinical Specimens.

The incidence of tick-borne infections in the United States has risen significantly in the past decade. Ticks can transmit a variety of pathogens, including bacteria, protozoa, and viruses, that can cause serious illnesses. Therefore, the use of rapid, sensitive, and specific multiplex tests is important to identify the pathogen(s) in the acute phase and determine appropriate treatment to minimize the severity of the disease. The purpose of this study was to evaluate ChromaCode's research use only (RUO) nine-target high-definition PCR (HDPCR) tick-borne pathogen (TBP) panel using 379 retrospective, remnant whole-blood and synovial fluid specimens previously submitted to Associated Regional and University Pathologists (ARUP) Laboratories and tested by clinically validated real-time PCR assays for Ehrlichia spp., Anaplasma phagocytophilum, Babesia spp., or Lyme Borrelia spp. The performance characteristics evaluated included positive percent agreement (PPA) and negative percent agreement (NPA) with the ARUP laboratory-developed tests (LDTs). All tested targets had an initial PPA greater than 97.0%, except Ehrlichia ewingii, with a PPA of 88.9%. The NPAs for all targets were between 98.8% and 100%. The TBP panel detected three coinfections, with two of Babesia microti and A. phagocytophilum and one of B. microti and E. chaffeensis, which were confirmed by the LDTs. There were 16 samples with discordant results compared to the LDT results, five of which were resolved by repeat testing on the TBP panel and bidirectional sequencing. Following discrepant resolution, the final PPA and NPA for the TBP panel were 97.7% (95% confidence interval [CI], 95.2% to 99.0%) and 99.6% (95% CI, 99.3% to 99.8%), respectively, with an overall agreement of 99.5% (95% CI, 99.2% to 99.7%) with the LDTs.

Helicobacter canis bacteraemia and cellulitis in a patient with end-stage renal disease.

Introduction. Invasive infections by Helicobacter canis are uncommon and occur primarily in immunocompromised patients. Here, we describe a case of H. canis bacteraemia and cellulitis in a patient with end-stage renal disease (ESRD). Case presentation. A 49-year-old male with ESRD on haemodialysis presented to an emergency department with cellulitis overlying his left upper extremity arteriovenous fistula for 3 days without constitutional symptoms. Mild leucocytosis and thrombocytopenia was noted on initial laboratory work up. The patient received a dose of vancomycin initially, and then transitioned to oral doxycycline prior to discharge 3 days later. Blood cultures drawn on admission were positive with curved Gram-negative rods at day 5. Routine sub-cultures initially failed to isolate the organism; however, small, tan colonies were observed on sheep blood agar incubated under microaerobic conditions. H. canis was identified by 16S rRNA sequencing. Antimicrobial-susceptibility testing was not performed due to poor growth and lack of interpretive guidelines. The patient was ultimately treated successfully with amoxicillin/clavulanic acid. Conclusion. This case illustrates the importance of recognizing H. canis infections in immunocompromised patients, especially in those with recent pet exposure. In addition, this case highlights the need for improved laboratory diagnostics for H. canis as isolation and identification of this fastidious organism is challenging.

Increased cAMP in monocytes augments Notch signaling mechanisms by elevating RBP-J and transducin-like enhancer of Split (TLE).

In cells of the innate immune system, pathological increases in intracellular cAMP attenuate immune responses and contribute to infections by bacteria such as Bacillus anthracis. In this work, cAMP from B. anthracis edema toxin (ET) is found to activate the Notch signaling pathway in both mouse macrophages and human monocytes. ET as well as a cell-permeable activator of PKA induce Notch target genes (HES1, HEY1, IL2RA, and IL7R) and are able to significantly enhance the induction of these Notch target genes by a Toll-like receptor ligand. Elevated cAMP also resulted in increased levels of Groucho/transducin-like enhancer of Split (TLE) and led to increased amounts of a transcriptional repressor complex consisting of TLE and the Notch target Hes1. To address the mechanism used by ET to activate Notch signaling, components of Notch signaling were examined, and results revealed that ET increased levels of recombinant recognition sequence binding protein at the Jκ site (RBP-J), a DNA binding protein and principal transcriptional regulator of Notch signaling. Overexpression studies indicated that RBP-J was sufficient to activate Notch signaling and potentiate LPS-induced Notch signaling. Further examination of the mechanism used by ET to activate Notch signaling revealed that C/EBP ß, a transcription factor activated by cAMP, helped activate Notch signaling and up-regulated RBP-J. These studies demonstrate that cAMP activates Notch signaling and increases the expression of TLE, which could be an important mechanism utilized by cAMP to suppress immune responses.

Glycogen synthase kinase 3 activation is important for anthrax edema toxin-induced dendritic cell maturation and anthrax toxin receptor 2 expression in macrophages.

Anthrax edema toxin (ET) is one of two binary toxins produced by Bacillus anthracis that contributes to the virulence of this pathogen. ET is an adenylate cyclase that generates high levels of cyclic AMP (cAMP), causing alterations in multiple host cell signaling pathways. We previously demonstrated that ET increases cell surface expression of the anthrax toxin receptors (ANTXR) in monocyte-derived cells and promotes dendritic cell (DC) migration toward the lymph node-homing chemokine MIP-3ß. In this work, we sought to determine if glycogen synthase kinase 3 (GSK-3) is important for ET-induced modulation of macrophage and DC function. We demonstrate that inhibition of GSK-3 dampens ET-induced maturation and migration processes of monocyte-derived dendritic cells (MDDCs). Additional studies reveal that the ET-induced expression of ANTXR in macrophages was decreased when GSK-3 activity was disrupted with chemical inhibitors or with small interfering RNA (siRNA) targeting GSK-3. Further examination of the ET induction of ANTXR revealed that a dominant negative form of CREB could block the ET induction of ANTXR, suggesting that CREB or a related family member was involved in the upregulation of ANTXR. Because CREB and GSK-3 activity appeared to be important for ET-induced ANTXR expression, the impact of GSK-3 on ET-induced CREB activity was examined in RAW 264.7 cells possessing a CRE-luciferase reporter. As with ANTXR expression, the ET induction of the CRE reporter was decreased by reducing GSK-3 activity. These studies not only provide insight into host pathways targeted by ET but also shed light on interactions between GSK-3 and CREB pathways in host immune cells.

Adenomatous polyposis coli protein associates with C/EBP beta and increases Bacillus anthracis edema toxin-stimulated gene expression in macrophages.

The production of cAMP from Bacillus anthracis edema toxin (ET) activates gene expression in macrophages through a complex array of signaling pathways, most of which remain poorly defined. In this study, the tumor suppressor protein adenomatous polyposis coli (APC) was found to be important for the up-regulation of previously defined ET-stimulated genes (Vegfa, Ptgs2, Arg2, Cxcl2, Sdc1, and Cebpb). A reduction in the expression of these genes after ET exposure was observed when APC was disrupted in macrophages using siRNA or in bone marrow-derived macrophages obtained from C57BL/6J-Apc(Min) mice, which are heterozygous for a truncated form of APC. In line with this observation, ET increased the expression of APC at the transcriptional level, leading to increased amounts of APC in the nucleus. The mechanism utilized by APC to increase ET-induced gene expression was determined to depend on the ability of APC to interact with C/EBP ß, which is a transcription factor activated by cAMP. Coimmunoprecipitation experiments found that APC associated with C/EBP ß and that levels of this complex increase after ET exposure. A further connection was uncovered when silencing APC was determined to reduce the ET-induced phosphorylation of C/EBP ß at Thr-188. This ET-mediated phosphorylation of C/EBP ß was blocked by glycogen synthase kinase 3 (GSK-3) inhibitors, suggesting that GSK-3 is involved in the activation of C/EBP ß and supporting the idea of APC helping direct interactions between GSK-3 and C/EBP ß. These results indicate that ET stimulates gene expression by promoting the formation of an inducible protein complex consisting of APC and C/EBP ß.

Autoregulatory characteristics of a Bacillus anthracis serine/threonine kinase.

BA-Stk1 is a serine/threonine kinase (STK) expressed by Bacillus anthracis. In previous studies, we found that BA-Stk1 activity is modulated through dephosphorylation by a partner phosphatase, BA-Stp1. In this study, we identified critical phosphorylation regions of BA-Stk1 and determined the contributions of these phosphodomains to autophosphorylation and substrate phosphorylation. The data indicate that BA-Stk1 undergoes trans-autophosphorylation within a regulatory domain, referred to as the activation loop, which carries eight putative regulatory serine and threonine residues. We identified activation loop mutants that impacted kinase activity in three different manners: regulation of autophosphorylation (T162), regulation of substrate phosphorylation (T159 and S169), and regulation of overall kinase activity (T163). Tandem mass spectrometry (MS/MS) analysis of the phosphorylation profile of each mutant revealed a second site of phosphorylation on the kinase that was influenced by the phosphorylation status of the activation loop. This second region of the kinase contained a single phosphorylation residue, S214. Previous work has shown S214 to be necessary for downstream substrate phosphorylation, and we have shown that this residue is subject to dephosphorylation by BA-Stp1. These findings indicate a connection between the phosphorylation status of the activation loop and phosphorylation of S214, and this suggests a previously undescribed model for how a bacterial STK shifts from a state of autophosphorylation to targeting downstream substrates.

Regulatory interactions of a virulence-associated serine/threonine phosphatase-kinase pair in Bacillus anthracis.

In the current study, we examined the regulatory interactions of a serine/threonine phosphatase (BA-Stp1), serine/threonine kinase (BA-Stk1) pair in Bacillus anthracis. B. anthracis STPK101, a null mutant lacking BA-Stp1 and BA-Stk1, was impaired in its ability to survive within macrophages, and this correlated with an observed reduction in virulence in a mouse model of pulmonary anthrax. Biochemical analyses confirmed that BA-Stp1 is a PP2C phosphatase and dephosphorylates phosphoserine and phosphothreonine residues. Treatment of BA-Stk1 with BA-Stp1 altered BA-Stk1 kinase activity, indicating that the enzymatic function of BA-Stk1 can be influenced by BA-Stp1 dephosphorylation. Using a combination of mass spectrometry and mutagenesis approaches, three phosphorylated residues, T165, S173, and S214, in BA-Stk1 were identified as putative regulatory targets of BA-Stp1. Further analysis found that T165 and S173 were necessary for optimal substrate phosphorylation, while S214 was necessary for complete ATP hydrolysis, autophosphorylation, and substrate phosphorylation. These findings provide insight into a previously undescribed Stp/Stk pair in B. anthracis.

Effects of endogenous D-alanine synthesis and autoinhibition of Bacillus anthracis germination on in vitro and in vivo infections.

Bacillus anthracis transitions from a dormant spore to a vegetative bacillus through a series of structural and biochemical changes collectively referred to as germination. The timing of germination is important during early steps in infection and may determine if B. anthracis survives or succumbs to responsive macrophages. In the current study experiments determined the contribution of endogenous D-alanine production to the efficiency and timing of B. anthracis spore germination under in vitro and in vivo conditions. Racemase-mediated production of endogenous D-alanine by B. anthracis altered the kinetics for initiation of germination over a range of spore densities and exhibited a threshold effect wherein small changes in spore number resulted in major changes in germination efficiency. This threshold effect correlated with D-alanine production, was prevented by an alanine racemase inhibitor, and required L-alanine. Interestingly, endogenous production of inhibitory levels of D-alanine was detected under experimental conditions that did not support germination and in a germination-deficient mutant of B. anthracis. Racemase-dependent production of D-alanine enhanced survival of B. anthracis during interaction with murine macrophages, suggesting a role for inhibition of germination during interaction with these cells. Finally, in vivo experiments revealed an approximately twofold decrease in the 50% lethal dose of B. anthracis spores administered in the presence of D-alanine, indicating that rates of germination may be directly influenced by the levels of this amino acid during early stages of disease.