mteb/CovidQA huggingface dataset:
Analyses were conducted in Stata 15 (StataCorp). Descriptive statistics are reported (medians with interquartile ranges [IQRs], means with standard deviations [SDs], numbers with proportions). Baseline characteristics were compared in χ 2 tests (categorical outcomes) or Wilcoxon rank-sum tests (continuous outcomes). Logistic regression analyses of binary outcomes (eg, hospital admission: yes/no) and quantile regression analyses of continuous outcomes (eg, ED LOS) were undertaken. All analyses were adjusted for baseline characteristics.

Sensitivity analyses limited to data for children (under 18 years of age) or older patients (over 60 years of age) were conducted. P < 0.05 (2-tailed) was deemed statistically significant.

Ethics approval for the investigation was granted by the Human Research Ethics Committee of the South Eastern Sydney Local Health District (reference, HREC/16/POWH/412).
We analysed data for 3741 patients presenting to the four EDs during two periods: 1491 consecutive patients during July-December 2016 (standard PCR) and 2250 during July-December 2017 (rapid PCR). Baseline characteristics for the two groups were similar in terms of sex, triage category, and arrival day of the week, but differed significantly for age, arrival time, and mode of arrival (Box 1 

The overall numbers of tests per patient were similar in the standard PCR (mean, 7.2 tests; SD, 3.8) and rapid PCR groups (mean, 7.1 tests; SD, 3.4). The mean number of microbiology tests per patient was significantly lower for the rapid PCR group (1.5 tests; SD, 1.8) than for the standard PCR group (2.0 tests; SD, 2.1; P < 0.001 after controlling for baseline characteristics).
The 16 265 biochemistry/haematology and microbiology tests comprised 71.1% of the 22 876 other tests (that is, not including PCR virus testing) ordered for patients in the study. After adjusting for baseline characteristics, the proportions of patients for whom full blood count, electrolyte/urea/creatinine levels, liver function, or C-reactive protein were assessed were similar, as were the proportions for urine microscopy, culture and sensitivity tests. Significantly fewer blood culture, blood gas, sputum culture, and respiratory bacterial and viral serology tests were ordered for patients in the rapid PCR group (Box 4). Information, figure 1 ).

ED LOS was similar for the standard PCR and rapid PCR groups in both age-based subgroups (Supporting Information, figure 2A ). The differences in test turnaround time identified in the main analysis were also evident for each age-based subgroup (Supporting Information, figure 2B ).
In this before-and-after study, we found that rapid PCR testing of ED patients for major respiratory viruses was associated with significantly fewer admissions to hospital, more rapid delivery of test results, more patients receiving their test results before leaving the ED, and less frequent ordering of some common microbiology tests.
Other studies have also reported that hospital admission numbers were significantly lower when rapid influenza virus testing was used in EDs. An analysis of outcomes for more than 300 adults at a tertiary care centre in New York found that early diagnosis of respiratory infections was associated with significantly fewer hospitalisations of influenza-positive patients. 7 In a small Irish study (73 patients), the hospital admission rate for obstetric patients declined from 88% to 45% after on-site rapid influenza PCR testing was introduced. 10 The differences in clinical setting and patient group may explain the smaller decline in our study (from 78% to 67%). Non-PCR-based rapid diagnostic tests for respiratory viruses have also been associated with lower hospital admission rates. 11, 12 The main reason for fewer hospital admissions of patients tested by rapid PCR may be that the earlier availability of results enables clinicians to quickly diagnose or exclude respiratory infections and
to make timely and informed decisions about whether to discharge the patient or admit them to hospital. When standard 2 Primary outcomes for 3741 patients at four metropolitan emergency departments (EDs) tested for influenza and respiratory syncytial viruses by standard or rapid polymerase chain reaction (PCR)
After adjusting for baseline characteristics (Box 1): * P = 0.012; ** P < 0.001. ◆
MJA 210 (7) ▪ 15 April 2019 PCR was used, in contrast, our findings suggest that these decisions were made before the test results were available. The possible benefits of not admitting patients to hospital, beyond those for individual patient management, include better infection control and outbreak prevention, as well as reduced demands on hospital resources. 13, 14 The impact of rapid PCR testing on outbreak prevention and infection control measures should be evaluated. Rapid influenza virus testing may also have practical implications for hospital bed management. 10, 15 ED LOS was similar in our study before and after the introduction of rapid PCR methods. This finding was not unexpected, as test turnaround time is not the only rate-limiting factor for decision making in EDs. 16 Before rapid PCR methods were introduced, the long turnaround time of multiplex PCR did not necessarily extend a patient's stay in the ED, as they were usually admitted to hospital or discharged home
before the results were available. Consequently, more rapid delivery of test results alone would not reduce ED LOS.
Reports on the effect of rapid influenza virus testing and LOS have been conflicting. While evidence for an association between rapid testing and shorter overall inpatient LOS has been reported, 6,11 findings regarding ED LOS are inconsistent. 7, 17, 18 For example, a Cochrane review based on three randomised controlled trials did not find a statistically significant association of rapid viral diagnosis with lower mean ED LOS. 18 In a study in children, ED LOS was actually 26 minutes longer with rapid PCR; inpatient LOS did not differ between the two groups, but was significantly shorter when the analysis was limited to patients with positive test results. 6 We found that ordering of some other microbiology tests, including blood culture, sputum culture, and respiratory bacterial and virus serology, was significantly less frequent for patients tested by rapid PCR. The effect of PCR-based rapid testing on the ordering of other laboratory tests has not previously been reported, although
some studies of antigen-based pointof-care testing have examined this outcome. 12 Consistent with our finding, several investigators have reported fewer blood culture tests 19, 20 and basic biochemistry and haematology tests, including full blood count, 20,21 C-reactive protein testing, 21 and urinalysis, 20,21 when point-of-care testing was used.
The higher rate of positive results for patients tested by rapid PCR than for those tested by standard PCR may reflect a higher prevalence of influenza during 2017 than in 2016.

The rapid PCR system in our study accurately detects influenza viruses A/B and RSV but, unlike the standard multiplex PCR, cannot detect other clinically relevant respiratory viruses, such as rhinovirus, coronavirus, human metapneumovirus, parainfluenza virus, adenovirus, enterovirus, and bocavirus. If infection with other respiratory viruses is suspected, patients may therefore need further investigations. Standard multiplex PCR can provide broader information, as it can detect multiple respiratory viruses in a single run, although the long turnaround time restricts its suitability for urgent clinical decision making. Improving the turnaround time of multiplex PCR analysis may achieve better outcomes.
The strengths of our study include its relatively large sample size; further, unlike many similar investigations, ours was a multicentre study in four hospital EDs, enhancing the generalisability of our findings. However, our analyses were not adjusted for comorbid conditions, as this information was not available. Because our study was an uncontrolled before-and-after study, our results cannot be interpreted as indicating causal relationships. As with all non-randomised trials, we could not fully account for all potential confounding variables, nor for temporal trends and other unmeasured factors, such as changes in clinician testing practices or differences in the prevalence and severity of disease during the two study periods. 22 For example, a shortage of inpatient beds caused by a high prevalence of influenza could influence decisions in a busy ED about admitting patients to hospital. However, we attempted to reduce seasonal effects by selecting similar time frames for the two
study periods, to reduce selection bias by including all ED patients tested for influenza virus and RSV, and to control for differences in baseline patient characteristics by applying multivariate modelling. As medications data were not available to us, we were unable to assess the impact of rapid PCR testing on antibiotic and antiviral drug use. Similarly, the cost-benefit balance of rapid testing was not evaluated because relevant data were not available. The cost per patient of rapid PCR testing is generally higher than for central laboratory testing, but our findings suggest potential savings through lower numbers of hospital admissions and reduced resource use. This question could be evaluated in a further study.
Rapid PCR testing for influenza virus and RSV infections in patients attending EDs was associated with significant improvements in a range of patient and laboratory outcomes, suggesting potential benefits for both the patients and the health care system. A cost-benefit analysis could examine the impact of rapid PCR testing on bed management and antimicrobial drug prescribing.
Techniques to Study Antigen-Specific B Cell Responses

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6667631/

SHA: ee632fa425607e8ff91fc3730bc0782d43ce9c0c

Authors: Boonyaratanakornkit, Jim; Taylor, Justin J.
Date: 2019-07-24
DOI: 10.3389/fimmu.2019.01694
License: cc-by
Abstract: Antibodies against foreign antigens are a critical component of the overall immune response and can facilitate pathogen clearance during a primary infection and also protect against subsequent infections. Dysregulation of the antibody response can lead to an autoimmune disease, malignancy, or enhanced infection. Since the experimental delineation of a distinct B cell lineage in 1965, various methods have been developed to understand antigen-specific B cell responses in the context of autoimmune diseases, primary immunodeficiencies, infection, and vaccination. In this review, we summarize the established techniques and discuss new and emerging technologies for probing the B cell response in vitro and in vivo by taking advantage of the specificity of B cell receptor (BCR)-associated and secreted antibodies. These include ELISPOT, flow cytometry, mass cytometry, and fluorescence microscopy to identify and/or isolate primary antigen-specific B cells. We also present our approach
to identify rare antigen-specific B cells using magnetic enrichment followed by flow cytometry. Once these cells are isolated, in vitro proliferation assays and adoptive transfer experiments in mice can be used to further characterize antigen-specific B cell activation, function, and fate. Transgenic mouse models of B cells targeting model antigens and of B cell signaling have also significantly advanced our understanding of antigen-specific B cell responses in vivo.
Text: In his Nobel lecture in 1908, Paul Ehrlich likened the antibody-antigen interaction to a lock and key. He reasoned that antitoxins (antibodies) contained in a solution in the serum of immunized animals must be identical to a cellular receptor "for a really well-made key will not open different locks at the same time" (1) . It took almost five decades before immunofluorescence microscopy was used to confirm the cellular origin of antibodies (2) . Major strides in the B cell and antibody field followed in the 1970s with the development of hybridoma technology to produce monoclonal antibodies and the discovery that somatic rearrangement during B cell differentiation was responsible for antibody diversification (3, 4) . The subsequent explosion of available monoclonal antibodies led to revolutionary diagnostic, therapeutic, and research reagents to distinguish different types of immune cells (5) . Together, these discoveries have allowed us to probe humoral immunity at the level of
the antigen-specific B cell.
Methods to probe the antigen-specific B cell response have advanced our understanding of how to harness the remarkable breadth of the B cell repertoire and the exquisite specificity of the individual B cell in developing (1) vaccine candidates that elicit protective antibodies; (2) antibodies that prevent disease when given prophylactically; and (3) antibodies that can be given as therapy after the onset of disease. Many of the vaccines currently available were originally developed empirically either by inactivating, attenuating, or administering a subunit of the pathogen. However, vaccine development against pathogens that are traditionally difficult to vaccinate against may rely on a deeper investigation of the B cell response to the antigens exposed on the surface of these pathogens.
For HIV-1, the discovery of broadly neutralizing antibodies (bnAbs) that protect against infection across diverse viral isolates has intensified efforts to understand the developmental pathway of the rare B cells that produce these antibodies (6) (7) (8) (9) . Insights into the ontogeny of these rare B cells could allow the design of a step-wise vaccine regimen that stimulates the germ-line precursor to expand and mature to produce circulating bnAbs which could protect against HIV acquisition (10, 11) . For RSV, stabilized versions of the fusion (F) protein in the pre-fusion conformation have led to insights in the B cell's response to infection and has generated potentially safer and more efficacious vaccine candidates (12, 13) . Influenza also performs fusion through the stem region of the hemagglutinin protein, and the identification of B cells that target this relatively conserved site has spurred research on the development of a universal influenza vaccine (14) (15) (16) . Like
RSV, HIV, and influenza, the fusion proteins of EBV and CMV exist in a pre-fusion conformation, and stabilization in their pre-fusion states could greatly accelerate vaccine development against these pathogens (17-19). Rare memory B cells producing antibodies specific for the EBV fusion machinery have been isolated; these can neutralize both B cell and epithelial cell infection (20). A new paradigm in malaria vaccine development is also emerging with the discovery of IgM+ and IgD+ memory B cells targeting the Merozoite Surface Protein 1, that rapidly respond to malaria re-infection (21). Further, highly potent neutralizing antibodies targeting a novel and conserved site on the Circumsporozoite Protein have been isolated from B cells (22). Together, these examples demonstrate the importance of studying antigen-specific humoral responses to infectious diseases. The solutions to the crystal structures of surface proteins for a variety of pathogens, the conformational stabilization of
these antigens, and the application of the methods summarized in this review, to probe antigen-specific B cell responses, have created new opportunities for systematic and rational vaccine design for HIV, RSV, EBV, malaria, and many other pathogens.
The study of B cell responses has not only informed vaccine design but has also advanced our understanding of antibodymediated autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus (23, 24). Up to 20% of mature, naïve B cells have receptors with the capacity to bind self-antigens (25). Although these cells are potentially pathogenic, the deletion of B cells with high affinity to self-antigen through apoptosis, anergy of B cells with low affinity to self-antigen, and the absence of T cell help combine together to protect against autoimmune disease in mice (26). The study of autoantigen-specific B cells and a detailed analysis of B cell subsets with pathogenic potential in humans could lead to a better understanding of how to prevent and treat autoimmune diseases.
Although the term antigen-specific B cell is used throughout this mini-review to denote the analysis of B cells based on binding between the B cell receptor (BCR) and a specific antigen used as bait, it is important to keep in mind that BCRs within the polyclonal B cell repertoire exhibit a spectrum of polyreactivity. On one end of the spectrum, a highly polyreactive BCR is able to bind multiple structurally unrelated antigens with physiologically relevant affinities. The frequency of polyreactivity in the normal adult human B cell repertoire has been estimated to be 4% of naïve B cells, 23% of IgG+ memory B cells, and 26% of intestinal IgA+ and IgG+ plasmablasts (27-29). On the other end of the spectrum, a mono reactive BCR is activated only when it encounters a single cognate antigen. Although there are exceptions, the accumulation of somatic hypermutations within the variable regions of the BCR during the process of affinity maturation is generally thought to lead to increased
affinity and specificity for the cognate antigen (30, 31).
Several general techniques are commonly used to identify antigen-specific B cells ( Table 1 ). The B cell enzyme linked immunospot (ELISPOT) technique relies on the principle of capturing the secreted antibody in the vicinity of each cell. In the B cell ELISPOT, antibody secreting B cells (ASCs) present in a sample or differentiated in vitro are added to plates coated with the antigen of interest. Antigen-specific antibodies will bind in close proximity to the location of the individual B cells producing those antibodies. Enzyme or fluorescent labeled secondary antibodies are then used to visualize spots of antibody secretion and binding to plate-bound antigen at the location of the ASCs. Each spot corresponds to antibody produced from a single antigen-specific B cell and therefore the technique is extremely sensitive. Secondary antibodies conjugated to combinatorial colored beads can also be used to detect the antibodies secreted from individual B cells with the advantage of
multiplexing the assay (32). One limitation of the assay is its requirement for antibody secretion by B cells thereby limiting the assay to only a subset of B cells in the repertoire, namely ASCs (33). Memory B cells can be stimulated in vitro to differentiate into ASCs prior to addition to the antigen-coated plate (34) . Further, the antigenspecific B cells identified by ELISPOT are generally not available for downstream analysis.
Limiting dilution is another technique that has been used to isolate antigen-specific B cells. In this approach, primary cells can be diluted serially until individual B cells are separated in microwell plates (36) . The B cells can then be cultured and expanded ex vivo and/or immortalized using EBV such that each well contains a monoclonal antibody (3, 37, 38) . Antigen-specific B cells can be selected by screening the culture supernatants for monoclonal antibodies that bind an antigen of interest. Although antibodies can be sequenced and cloned, the requirement for an ex vivo culture prior to selection precludes determination of the transcriptional profile of the original B cell in this approach. This technique can potentially be time-consuming and laborious, but the use of microfluidics and robotics has greatly improved the throughput for selecting antigen-specific B cells (39) . Advances in single cell next generation sequencing technology have allowed high throughput
transcriptional profiling and sequencing of paired immunoglobulin heavy and light chains (40) . In this approach, antigen specificity can be tested after monoclonal antibodies are cloned and produced using the sequencing data. This method can be useful in identifying antigen-specific B cells that have undergone clonal expansion after vaccination or acute infection (41) . Flow cytometry is the most common method used for single cell analysis and isolation (39) . Flow cytometry-based analysis of antigen-specific B cells is dependent on labeling antigen with a fluorescent tag to allow detection. Fluorochromes can either be attached covalently via chemical conjugation to the antigen, expressed as a recombinant fusion protein, or attached non-covalently by biotinylating the antigen. After biotinylation, fluorochrome-conjugated streptavidin is added to generate a labeled tetramer of the antigen. Biotinylation of the antigen at a ratio ≤1 biotin to 1 antigen is important, since each
streptavidin has the potential to bind four biotins. If the ratio of biotin to antigen is >1:1, then clumping and precipitation of the antigen out of solution can occur as soon as streptavidin is added. Alternatively, site directed biotinylation can be accomplished by adding either an AviTag or BioEase tag to the recombinant antigen prior to expression (77, 78) . When site-specific biotinylation is utilized, researchers must keep in mind that the tag may occlude an epitope from recognition by B cells which can be problematic for vaccine antigens. Further, for proteins that oligomerize, multiple tags may be incorporated, possibly resulting in aggregation.
Another important consideration is the potential for confounding by B cells in the repertoire that bind to the fluorochrome, streptavidin, or any linkers rather than to the antigen of interest. Binding between fluorochromes, linkers, or streptavidin and BCRs from humans and mice never exposed to these antigens are generally of low affinity, and these BCRs are generally expressed by naïve and potentially polyreactive B cells (62, 79, 80) . Dual labeling, in which the same antigen is separately labeled with two different fluorochromes, can be used to identify double positive B cells and remove confounding by B cells that bind the fluorochrome (12, 42) . However, even when tetramers are utilized for dual labeling, streptavidin-specific B cells will contaminate the double positive population. To fully remove confounding from the fluorochrome, streptavidin, and linkers, a "decoy" tetramer can be used to identify these contaminating B cells (21, 26). In this approach, the same fluorochrome
used to identify antigen-specific B cells is conjugated to a different fluorochrome such that the emission spectrum is altered by fluorescence resonance energy transfer (FRET) (26). Decoy-binding B cells can therefore be excluded from the true antigen-specific B cells. Notably, it is critical to use the same source of fluorochrome conjugated streptavidin in the tetramer and decoy reagent, because conjugation methods, recombinant streptavidin, and protein fluorochromes like R-phycoerythrin vary enough from company to company to alter some of the epitopes available for B cells to bind.
One weakness of the flow cytometric approach is the reliance on antigens that can be readily conjugated to a fluorochrome or biotinylated. In addition to recombinant proteins and synthesized peptides, labeled polysaccharides, lipids, haptens, virus-like particles, and pseudo viruses have also been used to identify antigen-specific cells by flow cytometry (33, [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] . Further, epitope-specific B cells have been identified by screening bacteriophage-displays or microarray peptide libraries with polyclonal antibodies targeting the native antigen to select conformational epitopes that can be fused to fluorescent proteins for use in flow cytometry (47, 60) .