Performance evaluation of the high-throughput quantitative Alinity m BK virus assay.

BK virus (BKV) infection or reactivation in immunocompromised individuals can lead to adverse health consequences including BKV-associated nephropathy (BKVAN) in kidney transplant patients and BKV-associated hemorrhagic cystitis (BKV-HC) in allogeneic hematopoietic stem cell transplant recipients. Monitoring BKV viral load plays an important role in post-transplant patient care. This study evaluates the performance of the Alinity m BKV Investigational Use Only (IUO) assay. The linearity of the Alinity m BKV IUO assay had a correlation coefficient of 1.000 and precision of SD ≤ 0.25 Log IU/mL for all panel members tested (2.0-7.3 Log IU/mL). Detection rate at 50 IU/mL was 100%. Clinical plasma specimens tested comparing Alinity m BKV IUO to ELITech MGB Alert BKV lab-developed test (LDT) on the Abbott m2000 platform using specimen extraction protocols for DNA or total nucleic acid (TNA) resulted in coefficient of correlation of 0.900 and 0.963, respectively, and mean bias of 0.03 and -0.54 Log IU/mL, respectively. Alinity m BKV IUO compared with Altona RealStar BKV and Roche cobas BKV assays demonstrated coefficient of correlation of 0.941 and 0.980, respectively, and mean bias of -0.47 and -0.31 Log IU/mL, respectively. Urine specimens tested on Alintiy m BKV IUO and ELITech BKV LDT using TNA specimen extraction had a coefficient of correlation of 0.917 and mean bias of 0.29 Log IU/mL. The Alinity m BKV IUO assay was performed with high precision across the dynamic range and correlated well with other available BKV assays. IMPORTANCE: BK virus (BKV) in transplant patients can lead to adverse health consequences. Viral load monitoring is important in post-transplant patient care. This study evaluates the Alinity m BKV assay with currently available assays.

What is the role of the clinical microbiology laboratory in the care of diagnostically challenging OPAT patients? Illustrative cases and literature review.

Optimal care of patients requiring long-term outpatient parenteral or oral antimicrobial therapy by infectious diseases (ID) specialists is facilitated by an accurate microbiologic diagnosis. Close collaboration between ID specialists and the clinical microbiology laboratory for routine or specialized molecular testing can result in more accurate diagnoses, streamlined antimicrobial regimens, and improved patient outcomes.

Comparison of Analytical Sensitivity (Limit of Detection) of Xpert MTB/RIF and Xpert MTB/RIF Ultra for Non-Sputum Specimens.

Tuberculosis (TB) is a significant public health threat and has remained a leading cause of death in many parts of the world. Rapid and accurate testing and timely diagnosis can improve treatment efficacy and reduce new exposures. The Cepheid Xpert® MTB/RIF tests have two marketed products (US-IVD and Ultra) that are widely accepted for diagnosis of TB but have not yet been approved for non-sputum specimens. Despite numerous studies in the literature, no data for the analytical sensitivity of these two products on the non-sputum samples are available to date. This is the first study that systematically determined the analytical sensitivities of both US-IVD and Ultra tests on cerebrospinal fluid (CSF), tissue, and bronchoalveolar lavage (BAL). The limits of detection (LoDs) on the US-IVD test for both Mycobacterium tuberculosis and rifampin resistance in CFU/mL, respectively, were as follows: CSF (3.3 and 4.6), tissue (15 and 23), and bronchoalveolar lavage (BAL) (45 and 60), and on the Ultra test: CSF (0.16 and 2.7), tissue (0.11 and 12), and BAL (0.65, and 7.5). Overall, the analytical sensitivities of the Ultra test were substantially better than US-IVD for all sample types tested. This study provided a foundation for using either the US-IVD or Ultra test for the early detection of both pulmonary and extrapulmonary (EP) TB. Furthermore, using Ultra could result in higher TB case detection rates in subjects with paucibacillary TB and EP TB, positively impacting WHO goals to eradicate TB.

Multicenter Evaluation of the Cepheid Xpert GBS LB XC Test.

Early-onset neonatal sepsis due to Streptococcus agalactiae (group B Streptococcus [GBS]) infection is one of the leading causes of newborn mortality and morbidity. The latest guidelines published in 2019 recommended universal screening of GBS colonization among all pregnant women and intrapartum antibiotic prophylaxis for positive GBS. The updated procedures allow rapid molecular-based GBS screening using nutrient broth-enriched rectovaginal samples. Commercially available molecular assays for GBS diagnosis target mainly the cfb gene, which encodes a hemolysin protein responsible for producing the Christie-Atkins-Munch-Petersen (CAMP) factor. cfb is considered a conserved gene in essentially all GBS isolates. However, false-negative GBS results on Cepheid Xpert GBS and GBS LB tests due to deletions in or near the region that encodes cfb were reported recently. Therefore, the new Xpert GBS LB XC test was developed. This study is a multicenter evaluation of the new test for GBS identification from nutrient broth-enriched rectal/vaginal samples from antepartum women. A total of 621 samples were prospectively enrolled. The samples were tested with the Xpert GBS LB XC test, the composite comparator method, which included the Hologic Panther Fusion GBS test combined with bacterial culture, followed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) identification, and bacterial culture alone, followed by MALDI-TOF MS identification. The respective sensitivity and specificity of the Xpert GBS LB XC test were 99.3% and 98.7% compared to the composite comparator method and 99.1% and 91.8% compared to bacterial culture alone with MALDI-TOF MS identification. Overall, the Xpert GBS LB XC test performed comparatively to the composite comparator method and is equivalent to traditional bacterial culture followed by MALDI-TOF MS.

Analysis of sputum/tracheal aspirate and nasopharyngeal samples for SARS-CoV-2 detection by laboratory-developed test and Panther Fusion system.

In this study, 127 sputum/tracheal aspirate specimens were evaluated by a laboratory-developed real-time RT-PCR method and Fusion SARS-CoV-2 assay. These specimens were collected from the patients who have nasopharyngeal swab (NPS) samples being used for SARS-CoV-2 detection previously or simultaneously. The overall agreement was 96% between the lower respiratory tract (LRT) and NPS samples, suggesting that LRT specimens could be an option for patients who develop a productive cough or those receiving invasive mechanical ventilation.

Comparison of Respiratory Variations of Subclavian Vein and Inferior Vena Cava in Hospitalized Patients with Kidney Disease.

BACKGROUND: Accurate assessment of relative intravascular volume is critical for appropriate volume management of patients with kidney disease. Respiratory variations of inferior vena cava (IVC) diameter have been used and may correlate with those of subclavian vein (SCV) by bedside ultrasound. The purpose of this study was to assess the relationship between SCV and IVC respiratory variations by bedside ultrasound in a large group of hospitalized patients with acute and/or chronic kidney disease. METHODS: We compared 160 paired SCV and IVC bedside ultrasound studies from 102 semi-recumbent hospitalized adult patients with kidney disease. Patient encounters in which the SCV or IVC could not be clearly visualized were excluded. Collapsibility index=(Dmax-Dmin)/Dmax*100%; D=venous diameter. RESULTS: Relationships between SCV collapsibility index and IVC collapsibility index were not different for longitudinal and transverse views of the SCV. Correlation of SCV collapsibility index with IVC collapsibility index was 0.75 for mechanical ventilation (n=65, P<0.0001) and 0.67 for spontaneous breathing (n=95, P<0.0001). IVC collapsibility index cut-offs <20% for hypervolemia and >50% for hypovolemia corresponded to SCV collapsibility index cut-offs of <22% and >39%, respectively, for both mechanical ventilation and spontaneous breathing encounters. Using these cut-offs for SCV collapsibilities, assessment as hypervolemia versus not-hypervolemia had maximal sensitivity and specificity for predicting respective IVC collapsibility cut-offs of 88% for mechanical ventilation and 74% for spontaneous breathing, and assessment as hypovolemia versus not-hypovolemia had maximal sensitivity and specificity of 91% and 70%, respectively. Concordance, defined as agreement between assessment using SCV CI and assessment using IVC CI, was 85% for mechanical ventilation and 72% for spontaneous breathing when differentiating hypervolemia versus not-hypervolemia and was 89% and 71% respectively when differentiating hypovolemia versus not-hypovolemia. CONCLUSION: Assessment using SCV collapsibility index in the semi-recumbent position has a reasonable concordance with assessment using IVC collapsibility index for both spontaneous breathing and mechanical ventilation, in a wide range of hospitalized patients with concurrent kidney disease, and may be a useful adjunct to assess relative intravascular volume in patients with kidney disease.

How many are we missing with ID NOW COVID-19 assay using direct nasopharyngeal swabs? Findings from a mid-sized academic hospital clinical microbiology laboratory.

Here, we retrospectively analyzed the comparative results of 182 paired dry nasopharyngeal swabs tested by Abbott ID NOW and nasopharyngeal swabs in viral transport medium by real-time RT-PCR methods. While the overall agreement was 96.2%, we found that of 15 samples that were tested positive with RT-PCR methods, 7 were missed by ID NOW, resulting in a false-negative rate of 47%.

Unexpected Cholera Bacteremia in a 91 Year Old Caucasian Male Patient.

Cholera is an illness caused by Vibrio cholerae; its main symptom is acute watery diarrhea. Some infections are asymptomatic or result in patients presenting with mild diarrhea, but complications, such as bacteremia, can be fatal. Being endemic in Africa, Southeast Asia, and Haiti, V. cholerae infection cases in the United States are primarily considered travel-related. Herein, we report a case of a 91 year old Caucasian man, a Texas Gulf Coast resident, who developed bacteremia due to V. cholerae despite having no international travel history. Culture workup by mass spectrometry, automated biochemical system, and 16S ribosomal RNA (rRNA) gene sequencing confirmed V. cholerae. This case conveys an important reminder to clinicians and laboratory professionals regarding potentially serious cholera illnesses due to the domestic prevalence of V. cholerae in the coastal regions of the United States.

Changes in cardiac output with hemodialysis relate to net volume balance and to inferior vena cava ultrasound collapsibility in critically ill patients.

Cardiac output may increase after volume administration with relative intravascular volume depletion, or after ultrafiltration (UF) with relative intravascular volume overload. Assessing relative intravascular volume using respiratory/ventilatory changes in inferior vena cava (IVC) diameters may guide volume management to optimize cardiac output in critically ill patients requiring hemodialysis (HD) and/or UF.We retrospectively studied 22 critically ill patients having relative intravascular volume assessed by IVC Collapsibility Index (IVC CI) = (IVCmax-IVCmin)/IVCmax*100%, within 24 h of cardiac output measurement, during 37 intermittent and 21 continuous HD encounters. Cardiac output increase >10% was considered significant. Net volume changes between cardiac outputs were estimated from "isonatremic volume equivalent" (0.9% saline) gains and losses.Cardiac output increased >10% in 15 of 42 encounters with IVC CI <20% after net volume removal, and in 1 of 16 encounters with IVC CI ≥20% after net volume administration (p = 0.0136). All intermittent and continuous HD encounters resulted in intradialytic hypotension. Net volume changes between cardiac output measurements were significantly less (median +1.0 mL/kg) with intractable hypotension or vasopressor initiation, and net volume removal was larger (median -22.9 mL/kg) with less severe intradialytic hypotension (p < 0.001). Cardiac output increased >10% more frequently with least severe intradialytic hypotension and decreased with most severe intradialytic hypotension (p = 0.047).In summary, cardiac output may increase with net volume removal by ultrafiltration in some critically ill patients with relative intravascular volume overload assessed by IVC collapsibility. Severe intradialytic hypotension may limit volume removal with ultrafiltration, rather than larger volume removal causing severe intradialytic hypotension.

Syk-dependent glycolytic reprogramming in dendritic cells regulates IL-1ß production to ß-glucan ligands in a TLR-independent manner.

Dendritic cells (DCs) activated via TLR ligation experience metabolic reprogramming, in which the cells are heavily dependent on glucose and glycolysis for the synthesis of molecular building blocks essential for maturation, cytokine production, and the ability to stimulate T cells. Although the TLR-driven metabolic reprogramming events are well documented, fungal-mediated metabolic regulation via C-type lectin receptors such as Dectin-1 and Dectin-2 is not clearly understood. Here, we show that activation of DCs with fungal-associated ß-glucan ligands induces acute glycolytic reprogramming that supports the production of IL-1ß and its secretion subsequent to NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome activation. This acute glycolytic induction in response to ß-glucan ligands requires spleen tyrosine kinase signaling in a TLR-independent manner, suggesting now that different classes of innate immune receptors functionally induce conserved metabolic responses to support immune cell activation. These studies provide new insight into the complexities of metabolic regulation of DCs immune effector function regarding cellular activation associated with protection against fungal microbes.

Analysis of glycogen metabolic pathway utilization by dendritic cells and T cells using custom phenotype metabolic assays.

In the field of immunology, there is an increasing interest in cellular energy metabolism and its outcome on immune cell effector function. Activation of immune cells leads to rapid metabolic changes that are central to cellular biology in order to support the effector responses. Therefore, the need for user-friendly and dependable assay technologies to address metabolic regulation and nutrient utilization in immune cells is an important need in this field. Redox-dye reduction-based Phenotype MicroArray (PM) assays, which measure NADH reduction as a readout, developed by Biolog Inc., provide a wide screening of metabolites both in bacteria and mammalian cells. In this study, we delineate a detailed protocol of a customized Biolog assay for investigation of a specific metabolic pathway of interest. The option to be able to easily customize this technology offers researchers with a convenient assay platform to methodically examine specific nutrient substrates or metabolic pathways of interest in a rapid and cost effective manner.

The role of nitric oxide in metabolic regulation of Dendritic cell immune function.

Dendritic cells (DCs) are canonical antigen presenting cells of the immune system and serve as a bridge between innate and adaptive immune responses. When DCs are activated by a stimulus through toll-like receptors (TLRs), DCs undergo a process of maturation defined by cytokine & chemokine secretion, co-stimulatory molecule expression, antigen processing and presentation, and the ability to activate T cells. DC maturation is coupled with an increase in biosynthetic demand, which is fulfilled by a TLR-driven upregulation in glycolytic metabolism. Up-regulation of glycolysis in activated DCs provides these cells with molecular building blocks and cellular energy required for DC activation, and inhibition of glycolysis during initial activation impairs both the survival and effector function of activated DCs. Evidence shows that DC glycolytic upregulation is controlled by two distinct pathways, an early burst of glycolysis that is nitric oxide (NO) -independent, and a sustained commitment to glycolysis in NO-producing DC subsets. This review will address the complex role of NO in regulating DC metabolism and effector function.

Cell-Intrinsic Glycogen Metabolism Supports Early Glycolytic Reprogramming Required for Dendritic Cell Immune Responses.

Dendritic cell (DC) activation by Toll-like receptor (TLR) agonists causes rapid glycolytic reprogramming that is required to meet the metabolic demands of their immune activation. Recent efforts in the field have identified an important role for extracellular glucose sourcing to support DC activation. However, the contributions of intracellular glucose stores to these processes have not been well characterized. We demonstrate that DCs possess intracellular glycogen stores and that cell-intrinsic glycogen metabolism supports the early effector functions of TLR-activated DCs. Inhibition of glycogenolysis significantly attenuates TLR-mediated DC maturation and impairs their ability to initiate lymphocyte activation. We further report that DCs exhibit functional compartmentalization of glucose- and glycogen-derived carbons, where these substrates preferentially contribute to distinct metabolic pathways. This work provides novel insights into nutrient homeostasis in DCs, demonstrating that differential utilization of glycogen and glucose metabolism regulates their optimal immune function.

Necroptosis of Dendritic Cells Promotes Activation of γδ T Cells.

γδ T cells function at the interface between innate and adaptive immunity and have well-demonstrated roles in response to infection, autoimmunity and tumors. A common characteristic of these seemingly disparate conditions may be cellular stress or death. However, the conditions under which ligands for γδ T cells are induced or exposed remain largely undefined. We observed that induction of necroptosis of murine or human dendritic cells (DC) by inhibition of caspase activity paradoxically augments their ability to activate γδ T cells. Furthermore, upregulation of the stabilizer of caspase-8 activity, c-FLIP, by IL-4, not only greatly reduced the susceptibility of DC to necroptosis, but also considerably decreased their ability to activate γδ T cells. Collectively, these findings suggest that the induction of necroptosis in DC upregulates or exposes the expression of γδ T cell ligands, and they support the view that γδ T cells function in the immune surveillance of cell stress.