Longitudinal immune profiling of a SARS-CoV-2 reinfection in a solid organ transplant recipient

Prior to the emergence of antigenically distinct SARS-CoV-2 variants, reinfections were reported infrequently - presumably due to the generation of durable and protective immune responses. However, case reports also suggested that rare, repeated infections may occur as soon as 48 days following initial disease onset. The underlying immunologic deficiencies enabling SARS-CoV-2 reinfections are currently unknown. Here we describe a renal transplant recipient who developed recurrent, symptomatic SARS-CoV-2 infection - confirmed by whole virus genome sequencing - 7 months after primary infection. To elucidate the immunological mechanisms responsible for SARS-CoV-2 reinfection, we performed longitudinal profiling of cellular and humoral responses during both primary and recurrent SARS-CoV-2 infection. We found that the patient responded to the primary infection with transient, poor-quality adaptive immune responses. The patients immune system was further compromised by intervening treatment for acute rejection of the renal allograft prior to reinfection. Importantly, we also identified the development of neutralizing antibodies and the formation of humoral memory responses prior to SARS-CoV-2 reinfection. However, these neutralizing antibodies failed to confer protection against reinfection, suggesting that additional factors are required for efficient prevention of SARS-CoV-2 reinfection. Further, we found no evidence supporting viral evasion of primary adaptive immune responses, suggesting that susceptibility to reinfection may be determined by host factors rather than pathogen adaptation in this patient. In summary, our study suggests that a low neutralizing antibody presence alone is not sufficient to confer resistance against reinfection. Thus, patients with solid organ transplantation, or patients who are otherwise immunosuppressed, who recover from infection with SARS-CoV-2 may not develop sufficient protective immunity and are at risk of reinfection.

PD-1highCXCR5-CD4+ Peripheral Helper T (Tph) cells Promote Tissue-Homing Plasmablasts in COVID-19

A dysregulated immune response against coronavirus-2 (SARS-CoV-2) plays a critical role in the outcome of patients with coronavirus disease 2019 (COVID-19). A significant increase in circulating plasmablasts is characteristic of COVID-19 though the underlying mechanisms and its prognostic implications are not known. Here, we demonstrate that in the acute phase of COVID-19, activated PD-1highCXCR5-CD4+ T cells, peripheral helper T cells, (Tph) are significantly increased and promote inflammatory tissue-homing plasmablasts in patients with stable but not severe COVID-19. Analysis of scRNA-seq data revealed that plasmablasts in stable patients express higher levels of tissue-homing receptors including CXCR3. The increased Tph cells exhibited "B cell help" signatures similar to that of circulating T follicular helper (cTfh) cells and promoted B cell differentiation in vitro. Compared with cTfh cells, Tph cells produced more IFN{gamma}, inducing tissue-homing chemokine receptors on plasmablasts. Finally, expansion of activated Tph cells was correlated with the frequency of CXCR3+ plasmablasts in the acute phase of patients with stable disease. Our results demonstrate a novel role for Tph cells in acute viral immunity by inducing ectopic, antibody secreting plasmablasts.

Saliva viral load is a dynamic unifying correlate of COVID-19 severity and mortality

While several clinical and immunological parameters correlate with disease severity and mortality in SARS-CoV-2 infection, work remains in identifying unifying correlates of coronavirus disease 2019 (COVID-19) that can be used to guide clinical practice. Here, we examine saliva and nasopharyngeal (NP) viral load over time and correlate them with patient demographics, and cellular and immune profiling. We found that saliva viral load was significantly higher in those with COVID-19 risk factors; that it correlated with increasing levels of disease severity and showed a superior ability over nasopharyngeal viral load as a predictor of mortality over time (AUC=0.90). A comprehensive analysis of immune factors and cell subsets revealed strong predictors of high and low saliva viral load, which were associated with increased disease severity or better overall outcomes, respectively. Saliva viral load was positively associated with many known COVID-19 inflammatory markers such as IL-6, IL-18, IL-10, and CXCL10, as well as type 1 immune response cytokines. Higher saliva viral loads strongly correlated with the progressive depletion of platelets, lymphocytes, and effector T cell subsets including circulating follicular CD4 T cells (cTfh). Anti-spike (S) and anti-receptor binding domain (RBD) IgG levels were negatively correlated with saliva viral load showing a strong temporal association that could help distinguish severity and mortality in COVID-19. Finally, patients with fatal COVID-19 exhibited higher viral loads, which correlated with the depletion of cTfh cells, and lower production of anti-RBD and anti-S IgG levels. Together these results demonstrated that viral load - as measured by saliva but not nasopharyngeal -- is a dynamic unifying correlate of disease presentation, severity, and mortality over time.

Kinetics of antibody responses dictate COVID-19 outcome

Recent studies have provided insights into innate and adaptive immune dynamics in coronavirus disease 2019 (COVID-19). Yet, the exact feature of antibody responses that governs COVID-19 disease outcomes remain unclear. Here, we analysed humoral immune responses in 209 asymptomatic, mild, moderate and severe COVID-19 patients over time to probe the nature of antibody responses in disease severity and mortality. We observed a correlation between anti-Spike (S) IgG levels, length of hospitalization and clinical parameters associated with worse clinical progression. While high anti-S IgG levels correlated with worse disease severity, such correlation was time-dependent. Deceased patients did not have higher overall humoral response than live discharged patients. However, they mounted a robust, yet delayed response, measured by anti-S, anti-RBD IgG, and neutralizing antibody (NAb) levels, compared to survivors. Delayed seroconversion kinetics correlated with impaired viral control in deceased patients. Finally, while sera from 89% of patients displayed some neutralization capacity during their disease course, NAb generation prior to 14 days of disease onset emerged as a key factor for recovery. These data indicate that COVID-19 mortality does not correlate with the cross-sectional antiviral antibody levels per se, but rather with the delayed kinetics of NAb production.

Diverse Functional Autoantibodies in Patients with COVID-19

COVID-19 manifests with a wide spectrum of clinical phenotypes that are characterized by exaggerated and misdirected host immune responses1-8. While pathological innate immune activation is well documented in severe disease1, the impact of autoantibodies on disease progression is less defined. Here, we used a high-throughput autoantibody discovery technique called Rapid Extracellular Antigen Profiling (REAP) to screen a cohort of 194 SARS-CoV-2 infected COVID-19 patients and healthcare workers for autoantibodies against 2,770 extracellular and secreted proteins (the "exoproteome"). We found that COVID-19 patients exhibit dramatic increases in autoantibody reactivities compared to uninfected controls, with a high prevalence of autoantibodies against immunomodulatory proteins including cytokines, chemokines, complement components, and cell surface proteins. We established that these autoantibodies perturb immune function and impair virological control by inhibiting immunoreceptor signaling and by altering peripheral immune cell composition, and found that murine surrogates of these autoantibodies exacerbate disease severity in a mouse model of SARS-CoV-2 infection. Analysis of autoantibodies against tissue-associated antigens revealed associations with specific clinical characteristics and disease severity. In summary, these findings implicate a pathological role for exoproteome-directed autoantibodies in COVID-19 with diverse impacts on immune functionality and associations with clinical outcomes.

Multiscale PHATE Exploration of SARS-CoV-2 Data Reveals Multimodal Signatures of Disease

1The biomedical community is producing increasingly high dimensional datasets, integrated from hundreds of patient samples, which current computational techniques struggle to explore. To uncover biological meaning from these complex datasets, we present an approach called Multiscale PHATE, which learns abstracted biological features from data that can be directly predictive of disease. Built on a continuous coarse graining process called diffusion condensation, Multiscale PHATE creates a tree of data granularities that can be cut at coarse levels for high level summarizations of data, as well as at fine levels for detailed representations on subsets. We apply Multiscale PHATE to study the immune response to COVID-19 in 54 million cells from 168 hospitalized patients. Through our analysis of patient samples, we identify CD16hi CD66blo neutrophil and IFN{gamma}+GranzymeB+ Th17 cell responses enriched in patients who die. Further, we show that population groupings Multiscale PHATE discovers can be directly fed into a classifier to predict disease outcome. We also use Multiscale PHATE-derived features to construct two different manifolds of patients, one from abstracted flow cytometry features and another directly on patient clinical features, both associating immune subsets and clinical markers with outcome.

Kynurenic acid underlies sex-specific immune responses to COVID-19

Coronavirus disease-2019 (COVID-19) has poorer clinical outcomes in males compared to females, and immune responses underlie these sex-related differences in disease trajectory. As immune responses are in part regulated by metabolites, we examined whether the serum metabolome has sex-specificity for immune responses in COVID-19. In males with COVID-19, kynurenic acid (KA) and a high KA to kynurenine (K) ratio was positively correlated with age, inflammatory cytokines, and chemokines and was negatively correlated with T cell responses, revealing that KA production is linked to immune responses in males. Males that clinically deteriorated had a higher KA:K ratio than those that stabilized. In females with COVID-19, this ratio positively correlated with T cell responses and did not correlate with age or clinical severity. KA is known to inhibit glutamate release, and we observed that serum glutamate is lower in patients that deteriorate from COVID-19 compared to those that stabilize, and correlates with immune responses. Analysis of Genotype-Tissue Expression (GTEx) data revealed that expression of kynurenine aminotransferase, which regulates KA production, correlates most strongly with cytokine levels and aryl hydrocarbon receptor activation in older males. This study reveals that KA has a sex-specific link to immune responses and clinical outcomes, in COVID-19 infection.

Single-Cell Omics Reveals Dyssynchrony of the Innate and Adaptive Immune System in Progressive COVID-19

A dysregulated immune response against the SARS-CoV-2 virus plays a critical role in severe COVID-19. However, the molecular and cellular mechanisms by which the virus causes lethal immunopathology are poorly understood. Here, we utilize multiomics single-cell analysis to probe dynamic immune responses in patients with stable or progressive manifestations of COVID-19, and assess the effects of tocilizumab, an anti-IL-6 receptor monoclonal antibody. Coordinated profiling of gene expression and cell lineage protein markers reveals a prominent type-1 interferon response across all immune cells, especially in progressive patients. An anti-inflammatory innate immune response and a pre-exhaustion phenotype in activated T cells are hallmarks of progressive disease. Skewed T cell receptor repertoires in CD8+ T cells and uniquely enriched V(D)J sequences are also identified in COVID-19 patients. B cell repertoire and somatic hypermutation analysis are consistent with a primary immune response, with possible contribution from memory B cells. Our in-depth immune profiling reveals dyssynchrony of the innate and adaptive immune interaction in progressive COVID-19, which may contribute to delayed virus clearance and has implications for therapeutic intervention.

Longitudinal immunological analyses reveal inflammatory misfiring in severe COVID-19 patients

Recent studies have provided insights into the pathogenesis of coronavirus disease 2019 (COVID-19)1-4. Yet, longitudinal immunological correlates of disease outcome remain unclear. Here, we serially analysed immune responses in 113 COVID-19 patients with moderate (non-ICU) and severe (ICU) disease. Immune profiling revealed an overall increase in innate cell lineages with a concomitant reduction in T cell number. We identify an association between early, elevated cytokines and worse disease outcomes. Following an early increase in cytokines, COVID-19 patients with moderate disease displayed a progressive reduction in type-1 (antiviral) and type-3 (antifungal) responses. In contrast, patients with severe disease maintained these elevated responses throughout the course of disease. Moreover, severe disease was accompanied by an increase in multiple type 2 (anti-helminths) effectors including, IL-5, IL-13, IgE and eosinophils. Unsupervised clustering analysis of plasma and peripheral blood leukocyte data identified 4 immune signatures, representing (A) growth factors, (B) type-2/3 cytokines, (C) mixed type-1/2/3 cytokines, and (D) chemokines that correlated with three distinct disease trajectories of patients. The immune profile of patients who recovered with moderate disease was enriched in tissue reparative growth factor signature (A), while the profile for those with worsened disease trajectory had elevated levels of all four signatures. Thus, we identified development of a maladapted immune response profile associated with severe COVID-19 outcome and early immune signatures that correlate with divergent disease trajectories.

Sex differences in immune responses to SARS-CoV-2 that underlie disease outcomes

A growing body of evidence indicates sex differences in the clinical outcomes of coronavirus disease 2019 (COVID-19)1-4. However, whether immune responses against SARS-CoV-2 differ between sexes, and whether such differences explain male susceptibility to COVID-19, is currently unknown. In this study, we examined sex differences in viral loads, SARS-CoV-2-specific antibody titers, plasma cytokines, as well as blood cell phenotyping in COVID-19 patients. By focusing our analysis on patients with mild to moderate disease who had not received immunomodulatory medications, our results revealed that male patients had higher plasma levels of innate immune cytokines and chemokines including IL-8, IL-18, and CCL5, along with more robust induction of non-classical monocytes. In contrast, female patients mounted significantly more robust T cell activation than male patients during SARS-CoV-2 infection, which was sustained in old age. Importantly, we found that a poor T cell response negatively correlated with patients age and was predictive of worse disease outcome in male patients, but not in female patients. Conversely, higher innate immune cytokines in female patients associated with worse disease progression, but not in male patients. These findings reveal a possible explanation underlying observed sex biases in COVID-19, and provide important basis for the development of sex-based approach to the treatment and care of men and women with COVID-19.

Saliva is more sensitive for SARS-CoV-2 detection in COVID-19 patients than nasopharyngeal swabs

Rapid and accurate SARS-CoV-2 diagnostic testing is essential for controlling the ongoing COVID-19 pandemic. The current gold standard for COVID-19 diagnosis is real-time RT-PCR detection of SARS-CoV-2 from nasopharyngeal swabs. Low sensitivity, exposure risks to healthcare workers, and global shortages of swabs and personal protective equipment, however, necessitate the validation of new diagnostic approaches. Saliva is a promising candidate for SARS-CoV-2 diagnostics because (1) collection is minimally invasive and can reliably be self-administered and (2) saliva has exhibited comparable sensitivity to nasopharyngeal swabs in detection of other respiratory pathogens, including endemic human coronaviruses, in previous studies. To validate the use of saliva for SARS-CoV-2 detection, we tested nasopharyngeal and saliva samples from confirmed COVID-19 patients and self-collected samples from healthcare workers on COVID-19 wards. When we compared SARS-CoV-2 detection from patient-matched nasopharyngeal and saliva samples, we found that saliva yielded greater detection sensitivity and consistency throughout the course of infection. Furthermore, we report less variability in self-sample collection of saliva. Taken together, our findings demonstrate that saliva is a viable and more sensitive alternative to nasopharyngeal swabs and could enable at-home self-administered sample collection for accurate large-scale SARS-CoV-2 testing.

Analytical sensitivity and efficiency comparisons of SARS-COV-2 qRT-PCR assays

The recent spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exemplifies the critical need for accurate and rapid diagnostic assays to prompt clinical and public health interventions. Currently, several quantitative reverse-transcription polymerase chain reaction (qRT-PCR) assays are being used by clinical, research, and public health laboratories. However, it is currently unclear if results from different tests are comparable. Our goal was to evaluate the primer-probe sets used in four common diagnostic assays available on the World Health Organization (WHO) website. To facilitate this effort, we generated RNA transcripts to be used as assay standards and distributed them to other laboratories for internal validation. We then used (1) RNA transcript standards, (2) full-length SARS-CoV-2 RNA, (3) pre-COVID-19 nasopharyngeal swabs, and (4) clinical samples from COVID-19 patients to determine analytical efficiency and sensitivity of the qRT-PCR primer-probe sets. We show that all primer-probe sets can be used to detect SARS-CoV-2 at 500 virus copies per reaction, except for the RdRp-SARSr (Charite) confirmatory primer-probe set which has low sensitivity. Our findings characterize the limitations of currently used primer-probe sets and can assist other laboratories in selecting appropriate assays for the detection of SARS-CoV-2.

A Prospective Audit of Airway Code Activations and Adverse Events in Two Tertiary Hospitals

INTRODUCTION@#Airway management outside the operating room can be challenging, with an increased risk of difficult intubation, failed intubation and complications. We aim to examine airway practices, incidence of difficult airway and complications associated with airway code (AC) activation.@*METHODS@#We conducted a prospective audit of AC activations and adverse events in two tertiary hospitals in Singapore. We included all adult patients outside the operating room who underwent emergency intubation by the AC team after AC activation. Adult patients who underwent emergency intubation without AC activation or before the arrival of the AC team were excluded. Data were collected and documented by the attending anaesthetists in a standardised survey form shortly after their responsibilities were completed.@*RESULTS@#The audit was conducted over a 20-month period from July 2016 to March 2018, during which a total of 224 airway activations occurred. Intubation was successful in 218 of 224 AC activations, giving a success rate of 97.3%. Overall, 48 patients (21.4%) suffered an adverse event. Thirteen patients (5.8%) had complications when intubation was carried out by the AC team compared with 35 (21.5%) by the non-AC team.@*CONCLUSION@#Dedicated AC team offers better success rate for emergency tracheal intubation. Non-AC team attempted intubation in the majority of the cases before the arrival of the AC team. Increased intubation attempts are associated with increased incidence of adverse events. Equipment and patient factors also contributed to the adverse events. A multidisciplinary programme including the use of supraglottic devices may be helpful to improve the rate of success and minimise complications.

Survey of neuromuscular monitoring and assessment of postoperative residual neuromuscular block in a postoperative anaesthetic care unit

INTRODUCTION@#The use of neuromuscular blocking agents (NMBAs) is common during general anaesthesia. Neuromuscular monitoring with a peripheral nerve stimulator (PNS) is essential to prevent postoperative residual neuromuscular block (PRNB), defined as a train-of-four (TOF) ratio < 0.9. PRNB remains a common complication and may contribute to morbidity in the postoperative anaesthetic care unit (PACU).@*METHODS@#An online survey was sent to anaesthesiologists in our department to assess their knowledge and clinical practices related to neuromuscular blockade. Next, a study was conducted on adult patients scheduled for elective surgery under general anaesthesia requiring NMBAs. Upon admission to the PACU, TOF monitoring was performed.@*RESULTS@#A large proportion of anaesthesiologists showed a lack of knowledge of neuromuscular blockade or non-adherence to the best clinical practices associated with it. The majority (98.7%) stated that they did not routinely use PNS monitoring. In the clinical study, TOF monitoring was only used in 17.9% of the 335 patients who were assessed. The prevalence of PRNB was 33.4% and was associated with the elderly (age ≥ 65 years), a higher dose of NMBA used, a shorter duration of surgery, and a shorter duration between the last dose of NMBA and measurement of PRNB in the PACU. The incidence of adverse symptoms in the PACU was observed to be higher in patients with PRNB.@*CONCLUSION@#PRNB remains a clinically significant problem, but routine PNS monitoring is rare in our institution. This is compounded by inadequate knowledge and poor adherence to best clinical guidelines related to neuromuscular blockade.

Fibreoptic intubation in airway management: a review article

Since the first use of the flexible fibreoptic bronchoscope, a plethora of new airway equipment has become available. It is essential for clinicians to understand the role and limitations of the available equipment to make appropriate choices. The recent 4th National Audit Project conducted in the United Kingdom found that poor judgement with inappropriate choice of equipment was a contributory factor in airway morbidity and mortality. Given the many modern airway adjuncts that are available, we aimed to define the role of flexible fibreoptic intubation in decision-making and management of anticipated and unanticipated difficult airways. We also reviewed the recent literature regarding the role of flexible fibreoptic intubation in specific patient groups who may present with difficult intubation, and concluded that the flexible fibrescope maintains its important role in difficult airway management.

Awake supraglottic airway guided flexible bronchoscopic intubation in patients with anticipated difficult airways: a case series and narrative review / 대한마취과학회지

Awake intubation is indicated in difficult airways if attempts at securing the airway after induction of general anesthesia may lead to harm due to potential difficulties or failure in those attempts. Conventional awake flexible bronchoscopic intubation is performed via the nasal, or less commonly, oral route. Awake oral flexible bronchoscopic intubation (FBI) via a supraglottic airway device (SAD) is a less common technique; we refer to this as ‘supraglottic airway guided’ FBI (SAGFBI). We describe ten cases with anticipated difficult airways in which awake SAGFBI was performed. After sedation and adequate airway topicalization, an Ambu Auragain™ SAD was inserted. A flexible bronchoscope, preloaded with a tracheal tube, was then inserted through the SAD. Finally, the tracheal tube was railroaded over the bronchoscope, through the SAD and into the trachea. The bronchoscope and the SAD were carefully removed, whilst keeping the tracheal tube in-situ. The technique was successful and well tolerated by all patients, and associated complications were rare. It also offered the advantages of performing an ‘awake test insertion’ of the SAD, an ‘awake look’ at the periglottic region, and an ‘awake test ventilation.’ In certain patients, awake SAGFBI offers advantages over conventional awake FBI or awake videolaryngoscopy. More research is required to evaluate its success and failure rates, and identify associated complications. Its place in difficult airway algorithms may then be further established.

Use of a trans-tracheal rapid insufflation of oxygen device in a “cannot intubate, cannot oxygenate” scenario in a parturient: a case report / 대한마취과학회지

BACKGROUND: The trans-tracheal rapid insufflation of oxygen (TRIO) device is less commonly used and is an alternative to trans-tracheal jet ventilation for maintaining oxygenation in a “cannot intubate, cannot oxygenate” (CICO) scenario. CASE: We report the successful use of this device to maintain oxygenation after jet ventilator failure in a parturient who presented with the CICO scenario during the procedure for excision of laryngeal papilloma. CONCLUSIONS: A stepwise approach to the airway plan and preparation for an event of failure is essential for good materno-fetal outcomes. The TRIO device may result in inadequate ventilation that can lead to hypercarbia and respiratory acidosis. Hence, it should only be used as a temporizing measure before a definitive airway can be secured.

Awake supraglottic airway guided flexible bronchoscopic intubation in patients with anticipated difficult airways: a case series and narrative review / 대한마취과학회지

Awake intubation is indicated in difficult airways if attempts at securing the airway after induction of general anesthesia may lead to harm due to potential difficulties or failure in those attempts. Conventional awake flexible bronchoscopic intubation is performed via the nasal, or less commonly, oral route. Awake oral flexible bronchoscopic intubation (FBI) via a supraglottic airway device (SAD) is a less common technique; we refer to this as ‘supraglottic airway guided’ FBI (SAGFBI). We describe ten cases with anticipated difficult airways in which awake SAGFBI was performed. After sedation and adequate airway topicalization, an Ambu Auragain™ SAD was inserted. A flexible bronchoscope, preloaded with a tracheal tube, was then inserted through the SAD. Finally, the tracheal tube was railroaded over the bronchoscope, through the SAD and into the trachea. The bronchoscope and the SAD were carefully removed, whilst keeping the tracheal tube in-situ. The technique was successful and well tolerated by all patients, and associated complications were rare. It also offered the advantages of performing an ‘awake test insertion’ of the SAD, an ‘awake look’ at the periglottic region, and an ‘awake test ventilation.’ In certain patients, awake SAGFBI offers advantages over conventional awake FBI or awake videolaryngoscopy. More research is required to evaluate its success and failure rates, and identify associated complications. Its place in difficult airway algorithms may then be further established.

Use of a trans-tracheal rapid insufflation of oxygen device in a “cannot intubate, cannot oxygenate” scenario in a parturient: a case report / 대한마취과학회지

BACKGROUND@#The trans-tracheal rapid insufflation of oxygen (TRIO) device is less commonly used and is an alternative to trans-tracheal jet ventilation for maintaining oxygenation in a “cannot intubate, cannot oxygenate” (CICO) scenario.CASE: We report the successful use of this device to maintain oxygenation after jet ventilator failure in a parturient who presented with the CICO scenario during the procedure for excision of laryngeal papilloma.@*CONCLUSIONS@#A stepwise approach to the airway plan and preparation for an event of failure is essential for good materno-fetal outcomes. The TRIO device may result in inadequate ventilation that can lead to hypercarbia and respiratory acidosis. Hence, it should only be used as a temporizing measure before a definitive airway can be secured.

Anaesthetic management of acute airway obstruction

The acutely obstructed airway is a medical emergency that can potentially result in serious morbidity and mortality. Apart from the latest advancements in anaesthetic techniques, equipment and drugs, publications relevant to our topic, including the United Kingdom's 4th National Audit Project on major airway complications in 2011 and the updated American Society of Anesthesiologists' difficult airway algorithm of 2013, have recently been published. The former contained many reports of adverse events associated with the management of acute airway obstruction. By analysing the data and concepts from these two publications, this review article provides an update on management techniques for the acutely obstructed airway. We discuss the principles and factors relevant to the decision-making process in formulating a logical management plan.