Lessons learned: A look back at the performance of nine COVID-19 serologic assays and their proposed utility.

BACKGROUND: Serologic assays for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been proposed to assist with the acute diagnosis of infection, support epidemiological studies, identify convalescent plasma donors, and evaluate vaccine response. METHODS: We report an evaluation of nine serologic assays: Abbott (AB) and Epitope (EP) IgG and IgM, EUROIMMUN (EU) IgG and IgA, Roche anti-N (RN TOT) and anti-S (RS TOT) total antibody, and DiaSorin (DS) IgG. We evaluated 291 negative controls (NEG CTRL), 91 PCR positive (PCR POS) patients (179 samples), 126 convalescent plasma donors (CPD), 27 healthy vaccinated donors (VD), and 20 allogeneic hematopoietic stem cell transplant (HSCT) recipients (45 samples). RESULTS: We observed good agreement with the method performance claims for specificity (93-100%) in NEG CTRL but only 85% for EU IgA. The sensitivity claims in the first 2 weeks of symptom onset was lower (26-61%) than performance claims based on > 2 weeks since PCR positivity. We observed high sensitivities (94-100%) in CPD except for AB IgM (77%), EP IgM (0%). Significantly higher RS TOT was observed for Moderna vaccine recipients then Pfizer (p-values < 0.0001). A sustained RS TOT response was observed for the five months following vaccination. HSCT recipients demonstrated significantly lower RS TOT than healthy VD (p < 0.0001) at dose 2 and 4 weeks after. CONCLUSIONS: Our data suggests against the use of anti-SARS-CoV-2 assays to aid in acute diagnosis. RN TOT and RS TOT can readily identify past-resolved infection and vaccine response in the absence of native infection. We provide an estimate of expected antibody response in healthy VD over the time course of vaccination for which to compare antibody responses in immunosuppressed patients.

Hidden in plain sight: urinary Cryptococcus neoformans missed by routine diagnostics in a patient with acute leukemia.

Cryptococcuria is a rare manifestation of localized cryptococcal disease. We present a case of Cryptococcus neoformans urinary tract infection in an immunocompromised host missed by routine laboratory workup. The patient had negative blood cultures, a negative serum cryptococcal antigen (CrAg), and "non-Candida yeast" growing in urine culture that was initially dismissed as non-pathogenic. The diagnosis was ultimately made by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) from a repeat urine culture after transfer to a tertiary care center. Cryptococcus should be considered in the differential of refractory urinary tract infections growing non-Candida yeast.

Cluster of Burkholderia cepacia Complex Infections Associated With Extracorporeal Membrane Oxygenation Water Heater Devices.

BACKGROUND: Burkholderia cepacia complex is a group of potential nosocomial pathogens often linked to contaminated water. We report on a cluster of 8 B. cepacia complex infections in cardiothoracic intensive care unit patients, which were attributed to contaminated extracorporeal membrane oxygenation (ECMO) water heaters. METHODS: In December 2020, we identified an increase in B. cepacia complex infections in the cardiothoracic intensive care unit at Brigham and Women's Hospital. We sought commonalities, sequenced isolates, obtained environmental specimens, and enacted mitigation measures. RESULTS: Whole-genome sequencing of 13 B. cepacia complex clinical specimens between November 2020 and February 2021 identified 6 clonally related isolates, speciated as Burkholderia contaminans. All 6 occurred in patients on ECMO. Microbiology review identified 2 additional B. contaminans cases from June 2020 that may have also been cluster related, including 1 in a patient receiving ECMO. All 8 definite or probable cluster cases required treatment; 3 patients died, and 3 experienced recurrent infections. After ECMO was identified as the major commonality, all 9 of the hospital's ECMO water heaters were cultured, and B. contaminans grew in all cultures. Cultures from air sampled adjacent to the water heaters were negative. Water heater touch screens were culture positive for B. contaminans, and the sink drain in the ECMO heater reprocessing room also grew clonal B. contaminans. Observations of reprocessing revealed opportunities for cross-contamination between devices through splashing from the contaminated sink. The cluster was aborted by removing all water heaters from clinical service. CONCLUSIONS: We identified a cluster of 8 B. cepacia complex infections associated with contaminated ECMO water heaters. This cluster underscores the potential risks associated with water-based ECMO heaters and, more broadly, water-based care for vulnerable patients.

How SARS-CoV-2 Transformed the Clinical Laboratory: Challenges and Lessons Learned.

The COVID-19 pandemic has made a devastating impact on global health and continues to challenge healthcare infrastructure and delivery. The clinical laboratories were no exception as they are responsible for diagnostic testing that dictates many clinical, infection control, and public health decisions. Information technology and laboratory management tools are critical assets for maintaining and adapting operations in response to crises. When utilized effectively, they promote the integration between the clinical laboratory specialties (e.g., chemistry, hematology, microbiology, and molecular pathology). During the COVID-19 pandemic, our systems and processes were strained due to high testing volumes, demand for rapid turnaround times, supply chain constraints, and constantly evolving testing algorithms and result interpretations as our knowledge of the virus and of diagnostics increased over time. In this report, we describe those challenges and subsequent adaptations made by each clinical laboratory section. We hope these details help to provide potential solutions and approaches for other hospitals facing COVID-19 surges or other future pandemics.

Distinct CD1d docking strategies exhibited by diverse Type II NKT cell receptors.

Type I and type II natural killer T (NKT) cells are restricted to the lipid antigen-presenting molecule CD1d. While we have an understanding of the antigen reactivity and function of type I NKT cells, our knowledge of type II NKT cells in health and disease remains unclear. Here we describe a population of type II NKT cells that recognise and respond to the microbial antigen, α-glucuronosyl-diacylglycerol (α-GlcADAG) presented by CD1d, but not the prototypical type I NKT cell agonist, α-galactosylceramide. Surprisingly, the crystal structure of a type II NKT TCR-CD1d-α-GlcADAG complex reveals a CD1d F'-pocket-docking mode that contrasts sharply with the previously determined A'-roof positioning of a sulfatide-reactive type II NKT TCR. Our data also suggest that diverse type II NKT TCRs directed against distinct microbial or mammalian lipid antigens adopt multiple recognition strategies on CD1d, thereby maximising the potential for type II NKT cells to detect different lipid antigens.

Utility of Histologic and Histochemical Screening for 16S Ribosomal RNA Gene Sequencing of Formalin-Fixed, Paraffin-Embedded Tissue for Bacterial Endocarditis.

OBJECTIVES: 16S ribosomal RNA (rRNA) sequencing is a powerful but expensive tool for the identification of bacteria in culture-negative endocarditis. Histologic criteria to screen formalin-fixed, paraffin-embedded (FFPE) specimens for testing are evaluated. METHODS: Sixty-eight cases of infective endocarditis and controls were histologically reviewed and analyzed by 16S rRNA gene sequencing. RESULTS: Sequencing identified a specific pathogenic organism in 33 (49%) of 68 cases with acute inflammation and in 0 of 10 controls (P = .004). Visualization of organisms by Gram or Grocott methenamine silver stains had the strongest association with positive sequencing, while antibiotic treatment effect and acid decalcification decreased sensitivity. Molecular identifications were concordant with blood culture results in 90% of the cases, and a positive sequencing result was obtained in approximately half of the cases with negative valve cultures. CONCLUSIONS: Histologic screening criteria are extremely helpful for identifying cases likely to be positive by molecular testing and can provide significant cost savings in filtering out low-yield specimens.

Identification of a Potent Microbial Lipid Antigen for Diverse NKT Cells.

Semi-invariant/type I NKT cells are a well-characterized CD1d-restricted T cell subset. The availability of potent Ags and tetramers for semi-invariant/type I NKT cells allowed this population to be extensively studied and revealed their central roles in infection, autoimmunity, and tumor immunity. In contrast, diverse/type II NKT (dNKT) cells are poorly understood because the lipid Ags that they recognize are largely unknown. We sought to identify dNKT cell lipid Ag(s) by interrogating a panel of dNKT mouse cell hybridomas with lipid extracts from the pathogen Listeria monocytogenes. We identified Listeria phosphatidylglycerol as a microbial Ag that was significantly more potent than a previously characterized dNKT cell Ag, mammalian phosphatidylglycerol. Further, although mammalian phosphatidylglycerol-loaded CD1d tetramers did not stain dNKT cells, the Listeria-derived phosphatidylglycerol-loaded tetramers did. The structure of Listeria phosphatidylglycerol was distinct from mammalian phosphatidylglycerol because it contained shorter, fully-saturated anteiso fatty acid lipid tails. CD1d-binding lipid-displacement studies revealed that the microbial phosphatidylglycerol Ag binds significantly better to CD1d than do counterparts with the same headgroup. These data reveal a highly potent microbial lipid Ag for a subset of dNKT cells and provide an explanation for its increased Ag potency compared with the mammalian counterpart.

The Extended Family of CD1d-Restricted NKT Cells: Sifting through a Mixed Bag of TCRs, Antigens, and Functions.

Natural killer T (NKT) cells comprise a family of specialized T cells that recognize lipid antigens presented by CD1d. Based on their T cell receptor (TCR) usage and antigen specificities, CD1d-restricted NKT cells have been divided into two main subsets: type I NKT cells that use a canonical invariant TCR α-chain and recognize α-galactosylceramide (α-GalCer), and type II NKT cells that use a more diverse αß TCR repertoire and do not recognize α-GalCer. In addition, α-GalCer-reactive NKT cells that use non-canonical αß TCRs and CD1d-restricted T cells that use γδ or δ/αß TCRs have recently been identified, revealing further diversity among CD1d-restricted T cells. Importantly, in addition to their distinct antigen specificities, functional differences are beginning to emerge between the different members of the CD1d-restricted T cell family. In this review, while using type I NKT cells as comparison, we will focus on type II NKT cells and the other non-invariant CD1d-restricted T cell subsets, and discuss our current understanding of the antigens they recognize, the formation of stimulatory CD1d/antigen complexes, the modes of TCR-mediated antigen recognition, and the mechanisms and consequences of their activation that underlie their function in antimicrobial responses, anti-tumor immunity, and autoimmunity.

Recognition of microbial and mammalian phospholipid antigens by NKT cells with diverse TCRs.

CD1d-restricted natural killer T (NKT) cells include two major subgroups. The most widely studied are Vα14Jα18(+) invariant NKT (iNKT) cells that recognize the prototypical α-galactosylceramide antigen, whereas the other major group uses diverse T-cell receptor (TCR) α-and ß-chains, does not recognize α-galactosylceramide, and is referred to as diverse NKT (dNKT) cells. dNKT cells play important roles during infection and autoimmunity, but the antigens they recognize remain poorly understood. Here, we identified phosphatidylglycerol (PG), diphosphatidylglycerol (DPG, or cardiolipin), and phosphatidylinositol from Mycobacterium tuberculosis or Corynebacterium glutamicum as microbial antigens that stimulated various dNKT, but not iNKT, hybridomas. dNKT hybridomas showed distinct reactivities for diverse antigens. Stimulation of dNKT hybridomas by microbial PG was independent of Toll-like receptor-mediated signaling by antigen-presenting cells and required lipid uptake and/or processing. Furthermore, microbial PG bound to CD1d molecules and plate-bound PG/CD1d complexes stimulated dNKT hybridomas, indicating direct recognition by the dNKT cell TCR. Interestingly, despite structural differences in acyl chain composition between microbial and mammalian PG and DPG, lipids from both sources stimulated dNKT hybridomas, suggesting that presentation of microbial lipids and enhanced availability of stimulatory self-lipids may both contribute to dNKT cell activation during infection.

Invariant natural killer T cells: an innate activation scheme linked to diverse effector functions.

Invariant natural killer T (iNKT) cells exist in a 'poised effector' state, which enables them to rapidly produce cytokines following activation. Using a nearly monospecific T cell receptor, they recognize self and foreign lipid antigens presented by CD1d in a conserved manner, but their activation can catalyse a spectrum of polarized immune responses. In this Review, we discuss recent advances in our understanding of the innate-like mechanisms underlying iNKT cell activation and describe how lipid antigens, the inflammatory milieu and interactions with other immune cell subsets regulate the functions of iNKT cells in health and disease.

Shared and distinct transcriptional programs underlie the hybrid nature of iNKT cells.

Invariant natural killer T cells (iNKT cells) are innate-like T lymphocytes that act as critical regulators of the immune response. To better characterize this population, we profiled gene expression in iNKT cells during ontogeny and in peripheral subsets as part of the Immunological Genome Project. High-resolution comparative transcriptional analyses defined developmental and subset-specific programs of gene expression by iNKT cells. In addition, we found that iNKT cells shared an extensive transcriptional program with NK cells, similar in magnitude to that shared with major histocompatibility complex (MHC)-restricted T cells. Notably, the program shared by NK cells and iNKT cells also operated constitutively in γδ T cells and in adaptive T cells after activation. Together our findings highlight a core effector program regulated distinctly in innate and adaptive lymphocytes.

Invariant natural killer T cells recognize lipid self antigen induced by microbial danger signals.

Invariant natural killer T cells (iNKT cells) have a prominent role during infection and other inflammatory processes, and these cells can be activated through their T cell antigen receptors by microbial lipid antigens. However, increasing evidence shows that they are also activated in situations in which foreign lipid antigens would not be present, which suggests a role for lipid self antigen. We found that an abundant endogenous lipid, ß-D-glucopyranosylceramide (ß-GlcCer), was a potent iNKT cell self antigen in mouse and human and that its activity depended on the composition of the N-acyl chain. Furthermore, ß-GlcCer accumulated during infection and in response to Toll-like receptor agonists, contributing to iNKT cell activation. Thus, we propose that recognition of ß-GlcCer by the invariant T cell antigen receptor translates innate danger signals into iNKT cell activation.

Innate recognition of cell wall ß-glucans drives invariant natural killer T cell responses against fungi.

iNKT cells are innate T lymphocytes recognizing endogenous and foreign lipid antigens presented in the MHC-like molecule CD1d. The semi-invariant iNKT cell TCR can detect certain bacterial and parasitic lipids and drive iNKT cell responses. How iNKT cells respond to fungi, however, is unknown. We found that CD1d-deficient mice, which lack iNKT cells, poorly control infection with the fungal pathogen Aspergillus fumigatus. Furthermore, A. fumigatus rapidly activates iNKT cells in vivo and in vitro in the presence of APCs. Surprisingly, despite a requirement for CD1d recognition, the antifungal iNKT cell response does not require fungal lipids. Instead, Dectin-1- and MyD88-mediated responses to ß-1,3 glucans, major fungal cell-wall polysaccharides, trigger IL-12 production by APCs that drives self-reactive iNKT cells to secrete IFN-γ. Innate recognition of ß-1,3 glucans also drives iNKT cell responses against Candida, Histoplasma, and Alternaria, suggesting that this mechanism may broadly define the basis for antifungal iNKT cell responses.

Innate and cytokine-driven signals, rather than microbial antigens, dominate in natural killer T cell activation during microbial infection.

Invariant natural killer T cells (iNKT cells) are critical for host defense against a variety of microbial pathogens. However, the central question of how iNKT cells are activated by microbes has not been fully explained. The example of adaptive MHC-restricted T cells, studies using synthetic pharmacological α-galactosylceramides, and the recent discovery of microbial iNKT cell ligands have all suggested that recognition of foreign lipid antigens is the main driver for iNKT cell activation during infection. However, when we compared the role of microbial antigens versus innate cytokine-driven mechanisms, we found that iNKT cell interferon-γ production after in vitro stimulation or infection with diverse bacteria overwhelmingly depended on toll-like receptor-driven IL-12. Importantly, activation of iNKT cells in vivo during infection with Sphingomonas yanoikuyae or Streptococcus pneumoniae, pathogens which are known to express iNKT cell antigens and which require iNKT cells for effective protection, also predominantly depended on IL-12. Constitutive expression of high levels of IL-12 receptor by iNKT cells enabled instant IL-12-induced STAT4 activation, demonstrating that among T cells, iNKT cells are uniquely equipped for immediate, cytokine-driven activation. These findings reveal that innate and cytokine-driven signals, rather than cognate microbial antigen, dominate in iNKT cell activation during microbial infections.

How invariant natural killer T cells respond to infection by recognizing microbial or endogenous lipid antigens.

Invariant natural killer T (iNKT) cells have evolved to recognize CD1d-presented lipid antigens and are known to play important roles during infection with bacterial, viral, protozoan, and fungal pathogens. The limited antigen specificity and reactivity to self- and foreign antigens distinguish iNKT cells from MHC-restricted T cells and bear similarity to innate-like lymphocytes, such as NK cells, gammadelta T cells, MZB and B1-B cells. This review summarizes how direct recognition of microbial lipids or synergistic stimulation by self-lipids and pro-inflammatory cytokines results in activation of these innate-like iNKT cell during infection. iNKT cell activation in the absence of foreign antigen recognition is unique for cells bearing TCRs and underscores that not only the function but also the activation mechanism of iNKT cells is innate-like, and distinct from adaptive T cells. The different pathways of activation endow iNKT cells with the ability to respond rapidly to a wide variety of infectious agents and to contribute effectively to the early immune response during infection.

Multiple tissue-specific isoforms of sulfatide activate CD1d-restricted type II NKT cells.

The glycosphingolipid sulfatide (SO(3)-3Galbeta1Cer) is a demonstrated ligand for a subset of CD1d-restricted NKT cells, which could regulate experimental autoimmune encephalomyelitis, a murine model for multiple sclerosis, as well as tumor immunity and experimental hepatitis. Native sulfatide is a mixture of sulfatide isoforms, i.e. sulfatide molecules with different long-chain bases and fatty acid chain lengths and saturation. Here, we demonstrate that sulfatide-specific CD1d-restricted murine NKT hybridomas recognized several different sulfatide isoforms. These included the physiologically relevant isoforms C24:1 and C24:0, major constituents of the myelin sheet of the nervous system, and C16:0, prominent in the pancreatic islet beta-cells. The most potent sulfatide isoform was lysosulfatide (lacking a fatty acid). Shortened fatty acid chain length (C24:1 versus C18:1), or saturation of the long fatty acid (C24:0), resulted in reduced stimulatory capacity, and fatty acid hydroxylation abolished the response. Moreover, sulfatide was not responsible for the natural autoreactivity toward splenocytes by XV19 T hybridoma cells. Our results reveal a promiscuity in the recognition of sulfatide isoforms by a CD1d-restricted NKT-cell clone, and suggest that sulfatide, a major component of the myelin sheet and pancreatic beta-cells, is one of several natural ligands for type II CD1d-restricted NKT cells.

Synthesis and biological activity of alpha-galactosyl ceramide KRN7000 and galactosyl (alpha1-->2) galactosyl ceramide.

We herein report a faster and less cumbersome synthesis of the biologically attractive, alpha-galactosyl ceramide (alpha-GalCer), known as KRN7000, and its analogues. More importantly, the use of a silicon tethered intramolecular glycosylation reaction gave easy access to the diglycosyl ceramide Gal(alpha1-->2)GalCer, which has been shown to require uptake and processing to the biologically active alpha-GalCer derivative.