While repurposed drugs came in handy earlier in the wake of the coronavirus disease 2019 (COVID-19) pandemic, vaccination has been considered a more sustainable approach. The recent spikes have been linked to “double,” “triple,” and even multi-mutant variants, thus renewing calls for deeper structural and functional insights of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as a lead to rationale design of therapeutics, vaccines, and point-of-care diagnostics. There is a repertoire of findings from the earliest SARS-CoV-2 molecular mimicry to evade host immunity cum host immune responses to the role of the viral glycocalyx in modulating the susceptibility and severity of infection through attraction and repulsive interactions. Recently, molecular studies of some viral components that aid infection in the face of vaccination seem unending. In addition, the wave of infections and the attendant case fatality ratios have necessitated the need for emergency use authorizations for COVID-19 vaccines and in vitro diagnostics. This review provides key updates of SARS-CoV-2, current antigenic and formulation strategies, with emergency use authorizations considerations for future vaccine candidates and diagnostics. We also premise that despite the difficulty in modeling and analyzing glycans, understanding and exploiting their roles in the SARS-CoV-2 architecture is fundamental to glycan-based COVID-19 vaccines devoid of inconsistent clinical outcomes. Although the socio-economic and geopolitical impacts of the pandemic are notable, the public health concerns remain at the core. By June 2021, over 180 million confirmed cases with an approximate 2.2% mortality rate have been reported according to a World Health Organization report (https://covid19.who.int/). This report also depicted the Western Pacific and Americas to bear the least and highest burden of COVID-19, respectively. Typically, the clinical manifestations of the disease are fever, cough, dyspnea, malaise, fatigue and sputum/secretion,2 albeit prolonged pulmonary, neurological, and cardiological consequences have also been seen in numerous cases.3 Currently, there is no standard treatment for the disease; however, the earliest interventions involved the use of repurposed drugs ranging from RNA-dependent RNA polymerase inhibitors,4 protease inhibitors,5 and immunomodulatory drugs6 to miscellaneous agents such as lipoglycopeptides,7 Ivermectin,8 and Tocilizumab. Despite the promising clinical outcomes of these therapeutics, a few events have tempered their use and approval, exemplified by little or unfavorable pharmacokinetic profiles, systemic toxicities, lack of randomized trials, small size of the cohort, and in some cases, inconsistencies in the data from case series or clinical trials.The beginning of the decade ushered in the outbreak of the novel coronavirus disease 2019 (COVID-19)12 Indeed, the causative organism of COVID-19, SARS-CoV-2,13 is expectedly profiled as a lead to developing effective therapeutic and prophylactic agents; however, certain structural features remain elusive in the face of a surge in infections. To this end, this review provides key updates on SARS-CoV-2, especially the under-reported place of glycans in the pathogenesis of the disease, and a synopsis of literature on the host responses to immunogenic components of the vaccine candidates. By keeping up with these studies, we aim to provide a basis for broader and informed rational design of anti-COVID-19 agents, while using key EUA submissions as a watchdog for the ideal vaccine candidate.In parallel, vaccination has been considered a reliable route to post-pandemic normalcy, as demonstrated by vaccines currently in use and others in preclinical and late-clinical stages, all at accelerated pace. The urgency of the demand has also necessitated the emergency use authorization (EUA) of some of these interventions. Fundamentally, development of a vaccine against a viral strain or multiple strains is premised on the understanding of host immune responses to infections. Pointedly, some studies have established strong correlations between vaccine-elicited neutralizing antibodies and a reduction of viral loads in non-human primates infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).14A report by Lu et al. on genomic profiling of the Betacoronavirus SARS-CoV-2 established the genomic size to be in the range of 29 kb, with a phylogenetic analysis that showed that SARS-CoV-2 belonged to the subgenus Sarbecovirus, displaying higher similarity to bat-SL-CoVZC45 and bat-SL-CoVZXC21 than to the human SARS-CoV.15 In addition, Cryo-electron microscopy (Cryo-EM) images elucidated the Betacoronaviruses to be spherical particulate matter with a bordering lipid bilayer that encapsulates an opaque viroplasm internally and protruding S trimers externally.17 Contextually, the transmembrane S glycoproteins, representing the antigenic machinery decorated as homotrimers elaborated on the viral surface of CoVs, facilitate viral entry into host cells18 through unique receptor sites such as the human dipeptidyl-peptidase 4 receptor for the Middle East respiratory syndrome coronavirus S,19 the angiotensin-converting enzyme 2 (ACE2) for the severe acute respiratory syndrome (SARS) coronavirus S20 and SARS-CoV-2 S.21 This event informs a highly conserved significant structural change to the post-fusion form from the pre-fusion conformation of the S protein. In addition to the N, S, E, and M proteins, sixteen non-structural proteins (NSP1-NSP16) have also been profiled and by aid of computational studies, NSPs 3, 5, 11, 14, and 15 were found to be ideal targets for three drug candidates .22 Despite the paucity of data on NSPs as targets for anti-COVID-19 agents, these candidates strongly support the need for a multidirectional approach to curb the effects of SARS-CoV-2.As a requisite concept, the structural and biochemical profile of SARS-CoV-2 underpins the pattern of transmission, pathogenesis of COVID-19, and rational development of drugs and vaccines. The coronaviruses (CoVs) are lipid-enveloped single-stranded positive-sense RNA viruses responsible for multiple respiratory disorders of varying severity in humans. Besides representing the largest RNA viruses, another typical feature of all CoVs is their ability to encode the nucleocapsid (N) protein, the structural spike (S) glycoprotein, and two smaller proteins – the envelope (E) protein and the membrane (M) glycoprotein.24 – the two structural subunits of the S glycoprotein. Expectedly bifunctional, the subunits impact both receptor binding and fusogenic functions. The S1 subunit is responsible for adhering to host cell receptors while the S2 ensures the fusion of the host and viral cellular membranes. The distal S1 folds as four independent domains comprising the N-terminal domain, two C-terminal domains, and the receptor-binding domain  whereas the FP cleavage site primes the host membrane for fusion upon penetration.29 Zhou et al. captured a snapshot of the interaction between S and human ACE2 using Cryo-EM,30 a fundamental that enhances our understanding of how the virus engages the S protein to gain entrance into a host cell. Similar studies are all geared to interfering with the Spike-ACE2 interaction. Despite this feat, protein structures elucidated by Cryo-EM or X-ray crystallographic assays often exclude the sugar components (glycans) that cover large bands of the S surfaces.The protein component of the S glycoprotein is depicted as a single polypeptide chain precursor of approximately 1300 amino acids, which can be cleaved by the host furin-like proteases into an amino (N)-terminal S1 subunit and a carboxyl (C)-terminal S2 subunit31 These roles appear to be ignored in the current discussion of SARS-CoV-2 due to difficulties in modeling the complex glycan network and poor sensitivity of analytical instruments, despite being a major composition on the glycoprotein scaffold. For instance, according to Crispin's model of the glycan framework on the viral S protein, each trimer displays 66 N-linked glycosylation sites (22 N-linked glycan sequons per protomer).32 Crispin et al. reported that the higher the mannose content is, the less the glycans resemble the host cell glycans and the less camouflaged the surface S protein is from the host's immune system. It is now well documented that the S protein utilizes this framework to evade the host humoral immune system by molecular mimicry, thus shielding the immunogenic protein epitopes.35 This glycosylation profile has been strongly posited to likely influence the clinical outcomes in the vaccination campaign, especially with the advent of mutant strains.Since the S protein is the main target of neutralizing antibodies, efforts geared towards vaccine and drug development center around the extensively glycosylated S protein. There are heightened conversations among structural biologists and glycobiologists about the structure elucidation of the glycan (polymers of sugar residues) architecture on the S protein as a strategy to enhance anti-COVID-19 interventions, whether as prophylactics or therapeutics. The current evidential basis relating to glycosylation of viral proteins depends on glycan processing enzymes within the virus or the host. These glycans are also key players in the propagation sequence of the virus, for one, the masking of the S protein from the host antibody machinery. The complexity of the SARS-CoV-2 biology is likely to be connected to the delay in developing safe anti-viral interventions, especially considering the emergency and pandemic status of COVID-19. Indeed, glycosylation within the microbial milieu has far-reaching implications due to glycan roles such as mediating protein stability, folding, antigenicity, immunogenicity, and intermolecular interactions.N-linked glycan sequons per protomer of the S protein. Similar results were obtained by Yang's group,36 which used a stepped collision energy mass spectrometry approach that revealed the N-glycosylation outline of the viral S proteins along with the glycan composition and site-specific numbers of glycans. In parallel, Azadi et al. engaged the proficiency of high-resolution liquid chromatography-tandem mass spectrometry to identify not only 17 out of the predicted 22 N-linked glycans sites, but serendipitously also two O-linked glycans out of the four predicted sites.37 These slight differences in methodologies and results among the glycan mappings allude to the challenges in consistently producing a glycosylated spike protein for vaccine development owing to the diversity in glycosylation which in turn depends on the preparatory conditions of the protein.Stemming from the unreliability of X-ray crystallography and Cryo-EM techniques to spot glycosylated portions of the SARS-CoV-2 framework, the Crispin group adopted a site-specific mass spectrometric approach to reveal all monomeric 38 Using docking studies and in vitro assays, they submitted that (i) by initial binding to heparan sulfate, the spike's receptor-binding domain adoption of an open conformation enhances S-ACE2 interactions; (ii) the eventual SARS-CoV-2 infection is interdependent on heparan sulfate and ACE2; and (iii) based on these correlations, modification of host heparan sulfate can downregulate SARS-CoV-2 infections. This submission aligns with that of Cai et al., which in elucidating the pre-fusion and post-fusion conformations of the S protein, posited that the unconstrained transition from the former to the later conformation is independent of ACE2-rich cells. In addition, the post-fusion conformation with its stable and rigid structure is aided by strategically decorated N-linked glycans to serve functions other than membrane fusion.39 In this context, the glycan architecture serves as a gate that shields and controls the opening of the spike protein – a basic requisite for infections.CoVs typically engage certain domains on the S protein that can interact with different glycoconjugates as attachment factors or secondary entry receptors. Esko et al. reported that S protein binding depends on ACE2 and heparan sulfate.40 Since the pandemic, it is estimated that asymptomatic individuals and symptomatic without life-threatening conditions account for 40%–80% of the infected population,41 while 15% account for patients with infections that progress to severe pneumonia and 5% show signs of acute respiratory distress syndrome, septic shock and multiple organ failure in some cases.42 The degree of severity is doubtless related to the role of host immunity, which might be connected to genetic variations beside factors such as pre-existing medical conditions, age, and sex. Bearing the number of available reports, our understanding of immunogenomics in infected patients is still evolving. For instance, in strengthening the genetic basis of COVID-19 heterogeneity, emerging body of evidence has linked ABO blood groups43 and ACE2 receptor polymorphisms44 to COVID-19 susceptibility. In addition, Leite et al. reported preliminary evidence that linked immunogenetic markers [human leukocyte antigen B (HLA-B) and interleukins ] to death by COVID-19.45 These correlations, although debatable, can only be subject to further investigations in immunogenomic studies.Although host immunity is an essential protection against the disease, precise regulation of innate immunity is rate-limiting to the progression of immunopathogenesis.46 Since the virus principally attacks ACE2-abundant regions such as the respiratory tract richly decorated with alveolar type 2 cells,48 the S-protein is primed by cellular surface proteolytic enzymes, stimulating viral and cellular membrane fusion. The viral neutralization process proceeds with toll-like receptors recognizing the viral RNA, phagocytosis of the virus, and simplification of the structural components. The phagocytes (antigen-presenting cells), such as dendritic cells, migrate to lymphatic organs and display major histocompatibility complex class 1 and 2 molecules to T-cells via T-cell receptors. This influences the defense pathway of T-cells to the viral entry. In parallel, CD-8 cytotoxic T-cells secrete cytokines such as IL-6 that clear infected cells from the host. The CD-4 T helper cells, on the other hand, enhance cytotoxic T-cells influencing B-lymphocyte production.49 This mechanism is crucial for the viral opsonization period and during the post-vaccination period. Once the T-cells activate the B-cells, B-lymphocytes mature and replicate, creating clones of plasma cells which produce antibodies that recognize and neutralize the virus.Substantially, there are unique clinical and biochemical characteristics obtained from patients that depend on the severity of the disease. As a case in point, Chakraborty et al. reported proinflammatory IgG Fc structures, including afucosylated IgG1 as key features in grading the severity of inflammation in infected individuals.50 To enhance the rate of viral recognition and neutralization induced by vaccination, the concept of designing a “wholistic antigenic” vaccine that highlights incorporating the M and N proteins in a single vaccine formulation51 might be an eventual prospect for COVID-19 and future pandemics. In view of other strategies, DNA and mRNA vaccines although aim to serve the same purpose, they differ in the following sense: (i) while mRNA is processed directly in the cytosol, DNA is required in the nucleus to be decoded; (ii) unlike DNA vaccines, the mRNA vaccines are safer since they are not integrated with host genome thus lack long-term effect on the host's genetic pool; (iii) mRNA tends to denature post protein formation, impacting a short half-life. Despite its immunogenicity, the setback of the mRNA vaccine is its instability and inefficient in vivo delivery.52 In addition, to prevent host RNase-mediated degradation before reaching the target site, the vaccines are encapsulated in positively charged lipid nanoparticles as carrier, self-assembling into virus-sized particles that can be administered through different routes.53 Nanotechnology has found possible benefits in the host immunity conditional to size, surface charge, shape, and hydrophobicity in addition to logistic factors. Albeit nanoparticles can activate the immune responses and diminish cytokine toxicity, but they are principally of merit in targeted site-specificity, prompting a resilient immune reaction. Typically, the lipid nature of nanoparticles enhances entry of genetic material through the lipid cell membrane49; however, the current mRNA vaccines are encapsulated with lipid nanoparticles that require long-term sub-zero storage conditions (−20 to −80°C).55 This feature poses a logistical challenge for low-income communities and consequently the global objective of flattening the curve to the barest minimum. In a similar vein, a viable single-dose vaccination strategy was also reported, which engaged spike-functionalized ferritin nanoparticles that elicited an enhanced antibody response against lentiviral SARS-CoV-2 pseudovirus treatment. These functionalized S proteins were obtained by fusing either the full length S ectodomain (residues 1-1213) or the C-terminal free S protein (residues 1-1143) to the Helicobacter pylori ferritin as an immunoadjuvant.56Exploiting the host translation mechanism, viruses insert their genomic material, thus influencing viral replication, albeit the duration of antibody responses to viral entry is essential to minimizing the damage caused by SARS-CoV-2 infections.57 Potential vaccines are also expected to produce robust immunological responses at administered concentrations especially in a population at the highest risk of infections, that is, the elderly and immunocompromised.Vaccines generally trigger an immune response to recognize and build up defences against specific pathogens but there are a few concerns associated with novel vaccines during a public health emergency. Understanding vaccine-induced immunopathology is key to designing effective vaccines for emerging infectious diseases. Like other vaccines, the major concern with SARS-CoV-2 vaccines is the safety profile in a healthy population, therefore the duo considerations of (i) the vaccine should not elicit life threatening adverse effects in patients, and (ii) the vaccine should prevent severe diseases in immunized persons. Pointedly, the efficacy of the COVID-19 vaccine in protecting against multiple/heterologous viral variants is of high importance, especially with the recent emergence of “double” and “triple” mutant strains in different parts of the world. There are views that current vaccines being used under the EUA have been found to be less effective against some newer strains, thereby restricting their use in localities plagued by those latest strains.59 Immunogenic response-triggered antibodies which bind to viral antigens but fail to effectively neutralize the virus can cause enhanced diseases through the enhancement of viral replication or the formation of immune complexes that activate inflammation pathways.60 This is usually the case with ADE when the antibodies triggered by immunogenic responses complex with viral antigens but fail to effectively neutralize the virus due mainly to suboptimal concentrations and inaccurate specificity amongst other reasons,58 thereby enhancing viral replication and facilitating viral infection of cells.61 ADE was first reported with dengue virus (DENV), which has 4 known serotypes. The immune response to a specific DENV serotype can produce neutralizing antibodies which are effective long term against homologous variants and are cross-reactive with heterologous DENV serotypes.63 These cross-reactive antibodies are protective against heterologous serotypes for only a few months before the concentrations drop to suboptimal levels. At this point, the risk of severe dengue symptoms after infection with heterologous DENV is higher in such individuals compared to naive individuals. In the context of COVID-19, it is not unlikely, although not evidential yet, to correlate the sudden spike in infections and mortality (second and third wave) to a cross-reactivity between the native SARS-CoV-2 and the newer variants.The rapid structural elucidation of the SARS-CoV-2 virus, which began in early 2020, aimed at identifying possible targets for vaccine development, and resulted in vaccines generating neutralizing antibodies with adequate protective titers to prevent disease enhancement upon subsequent infections. However, vaccine induction of enhanced diseases could be antibody-mediated, resulting in antibody dependent enhancement (ADE) and vaccine associated enhanced respiratory disease (VAERD), or could be mediated by Th2 cell-based immunopathologic responses causing VAERD.60 This was reported as the cause of inflammation and airway obstruction in some participants of the clinical trials who were immunized with the formalin-inactivated respiratory syncytial virus vaccine.64 VAERD is also mediated by Th2-biased immune responses, which cause allergic inflammation through cytokine production and eosinophilic infiltration, resulting in aggravated airway dysfunction and delayed viral clearance.63 Potential concerns of ADE and VAERD following the use of SARS-CoV-2 vaccines have arisen from animal models used in assessing safety and efficacy of some SARS-CoV vaccines after enhanced diseases were observed in some animals following viral challenge,68 but the efficacy data of phase 3 trials of current EUA SARS-CoV-2 vaccines do not report enhanced diseases in the months following the use of the vaccines.70 Recent clinical trials have also shown the reduced effectiveness of some EUA vaccines against some newer variants of SARS-CoV-2, especially B.1.351; however, ADE and VAERD upon infection with these newer strains of the virus have not been reported57 as at the time of this review. Although concerns associated with vaccine-induced disease enhancement have been allayed, the effect of declining antibody titers after vaccination or infections and the potential long-term safety signals remain unknown since COVID-19 vaccines, including the EUA vaccines, still require the Food and Drug Administration (FDA) approval.While ADE is not specific to respiratory diseases, VAERD refers to clinical symptoms of severe respiratory viral infections due to prior immunological responses induced by vaccination. It was first reported after markedly worse respiratory diseases were observed in study participants of the formalin-inactivated respiratory syncytial virus vaccine group and not in the placebo group.Vaccines currently in use are categorized based on the nature of the antigen used in their production, which also determines the storage and administration protocol. The representative antigenic strategies in use or trialed for development against the SARS-CoV-2 are presented in Table 71 Our search indicated that there is only one live attenuated vaccine currently in the early phase of clinical trials. Whole inactivated vaccines, on the other hand, contain viruses that have been killed through chemical or physical processes, reducing the risk of causing diseases and making these vaccines more stable. They are, however, generally less immunogenic and may require multiple doses or an immunoadjuvant to elicit sufficient immune responses since they do not replicate.72Live attenuated vaccines are prepared from whole virions that have been weakened through repeated passages in a cell culture until virulence is reduced and pathogenicity is lost. This type of vaccine thus induces a mild infection akin to the native infection, leading to a strong immune response and long-term immunity, a desirable feature in vaccine candidates. However, there are safety concerns with the use of attenuated vaccines due to their potential to revert to virulent forms, exacerbating the disease conditions, especially in immunocompromised individuals.73 Their results not only are consistent with ongoing phase 1 and 2 clinical trials but also highlight the low dose-induced immunogenicity, thermostability, and high binding affinity of NVX-CoV2373 nanoparticles to ACE2. Although the histopathological assessment of test subjects indicated no evidence of VAERD, it is our view that extensive blood coagulation studies ought to be carried out alongside the findings. This centers on claims and counterclaims of blood clots with associated mortalities in individuals inoculated with the current EUA-approved vaccines. Virus-like particle vaccines also employ the technology of subunit vaccines but present many copies of the antigen in a three-dimensional virus-like structure, so the antigens may be more immunogenic and not require the addition of adjuvants.74 New generation of vaccines include but not limited to recombinant viral vector vaccines and nucleic acid vaccines. They are developed using a classic technology where specific antigens from a pathogen are isolated and advanced vaccine platforms are used to make the antigens elicit immune responses, thus providing a rapid vaccine development opportunity as well as a better safety profile because of its specificity.Protein subunit vaccines host the antigenic portions of the pathogen responsible for eliciting a protective immune response. These antigens are purified from the virus and devoid of live components of the pathogen. Therefore, formulations of this type of vaccines require the inclusion of an adjuvant to stimulate stronger immune responses and produce enhanced immunological memories. Smith et al. recently disclosed the development of a SARS-CoV-2 subunit vaccine (NVX-CoV2373) from stable pre-fusion S protein with immunoadjuvant Matrix-M (NCT04368988) using mice and baboons as test subjects.75 although most of the viral vector vaccines that are in clinical trials require multiple doses. Human and chimpanzee adenoviruses have been used as sources for viral vectors to elicit good immunologic responses; however, there are concerns about pre-existing immunity against the human adenovirus, which reduces its immunogenicity relative to the chimpanzee adenovirus that has lower seroprevalence in most populations.76Viral vector vaccines contain a viral vector that has been genetically modified to reduce its virulence; the vector carries the genetic code of antigens of the target pathogen, which have been copied using recombinant DNA methods. Recombinant viral-vectored vaccines mimic natural infections with the vector to elicit targeted immune responses against the genetically encoded viral antigen. Vector vaccines can be replicating or non-replicating. Replicating vector vaccines infect the cells where vaccine antigens are produced and replicate the viral vector to infect new cells, consequently producing more antigens. On the other hand, the non-replicating vector vaccines access the cells to produce vaccine antigens without replicating new virus particles. Since antigens are produced within the host, both humoral and cellular immune responses are stimulated, raising the possibility of a single dose to achieve adequate protection,74 DNA vaccines require electroporation following injection for effective cellular uptake and must still be transcribed to mRNA before uptake,72 while mRNA vaccines, on the other hand, are quite unstable and readily removed by host cells upon injection. To this end, delivery techniques, which modify the nucleosides-like encapsulation of RNA in liposomes, have been developed to prevent degradation of the vaccine. Lipid nanoparticles are the carrier molecules often used in aiding the uptake of mRNA into cells immediately after injection.77 Although nucleic acid-based vaccines induce both humoral and cellular immune responses, multiple doses are still required for both DNA and RNA vaccines to provide long-term immunity. There are several nucleoside vaccines currently in clinical trials, but none have been licensed for use in humans except mRNA vaccines, which were granted EUA in early 2021.In nucleic acid vaccines, specific antigens are isolated and delivered as DNA or RNA constructs encoding the viral antigens. As the nucleic acid accesses the cell, it initiates the synthesis of the antigenic proteins, which in turn elicits immune responses like those produced by natural infections.78 Despite the impressive indices, these assays still require highly trained professionals to deliver an extremely low error margin. In a similar vein, the authors reported a comparative review of the FDA-EUA serological assays as: Autobio Diagnostics (99%/99.04%), Cellex (93.8%/96%), Orth clinical diagnostics (100%/100%), DiaSorin (97.56%/99.3%), Abbott (100%/99.63%), Bio-Rad (92.2%/99.6%), Roche (100%/99.81%), and Siemens Healthcare assay (100%/99.8%). The likelihood ratios were not reported for both assays, but the likelihood ratios of the IVDs might be needless at this stage since most assays are basically qualitative and the urgency for effective therapeutics overshadows the need for viral load determination or antibody titers in an infected patient. Current evidence suggests that clinicians’ judgment should be based on a combination of both RT-PCR and serological assays for COVID-19 diagnosis79 although the cost implications for low-resource communities can be a motivation for the development of cheap, affordable, and user-friendly point-of-care IVDs.At the onset of the pandemic, a major problem in monitoring and managing the spread of the pandemic was the lack of efficient diagnostic resources, often typified by false results, consequently resulting in risks of delayed or lack of supportive care, exposure of the healthy populace to the disease and possible adverse drug reactions in the case of false positive outcomes. On this premise, the United States FDA adopted EUA to several diagnostic tests for COVID-19. So far, nucleic acid testing for detection of SARS-CoV-2 RNA using the RT-PCR, and serological assays for detection of SARS-CoV-2 specific IgG and IgM antibodies are the two main types of diagnostics with EUA approval for COVID-19. Wang et al. profiled different FDA-EUA approved molecular diagnostics with their sensitivity-specificity ratios. The Centers for Disease Control and Prevention, QIAGEN, PerkinElmer, Thermo Fisher Scientific, and Roche demonstrated a sensitivity-specificity ratio of 1.0, whereas Abbott's Alinity m and BD's BioGx SARS CoV-2 assays depicted sensitivity-specificity ratios of 100%/96.5% and 95%/100%, respectively.80 Although the first viral vector vaccine, which demonstrated 82% efficacy after a second dose, was approved for emergency supply in the UK,81 the exigency of curbing the disease has called for other vaccine strategies that can afford a one-dose administration, efficient logistics, low costs, and availability in low-resource communities. While it is highly likely that more vaccine candidates will gain EUA approval, COVID-19 vaccine hesitancy might pose the next major challenge to achieving herd immunity globally.82As shown in Table 83 One trial reported two cases of adverse events by recipients of an mRNA vaccine, facial edema in one recipient with a history of injection dermatological fillers, and intractable nausea and vomiting in another recipient with a history of severe headache with nausea that required hospitalization. By adopting the Brighton Collaboration case definition criteria for adverse events following immunizations, Centers for Disease Control and Prevention received 66 case reports typified by two mRNA doses administered in different instances over a 4-week period following EUA approval, reporting at rates of 2.5–4.7 cases/million.83 A randomized study in the UK demonstrated the safety of an EUA viral vector vaccine administered to old-aged participants,84 although in recent time there are reports of blood clotting in individuals who have been vaccinated. These claims are yet to be verified. There are also reports of adverse events in recipients with underlining health conditions, thus suggesting the need for such individuals to be vaccinated under close monitoring.Thus far, most COVID-19 vaccines have been reported to be minimal reactogenic. The commonest among the population are injection site pain, fatigue, headache, muscle pain, and joint pain, and close clinical monitoring is highly recommended in certain populations.Considering the recent SARS-CoV-2 variants, efficient vaccine design is an ongoing venture, and this review highlights key components that are pertinent to flattening the curve in subsequent post-first wave COVID-19 spikes while also submitting our expert views herein. In context, emerging results from host immune responses to SARS-CoV-2 are certain to enhance our understanding of the variations in disease susceptibility and clinical outcomes and how to deal with potential variants or other zoonotic pathogens best. It is now established that clinical outcomes correlate with host immunity. Another recent update is the role of the glycocalyx  in modulating the likelihood of a viral infection through attraction and repulsive interactions. In parallel, in the face of the highly efficacious vaccines currently administered globally is the indistinct “breakthrough SARS-CoV-2 infections” concerns that account for at least 5% non-protection in a fully vaccinated population. While the mechanism is still unclear, we hypothesized with similar reported infections of dengue fever that featured cross-reactivity between antibodies from homologous strains with heterologous DENV serotypes, resulting in devastating events in the naive population as possible cause of potential spikes in COVID-19 cases post-vaccination. A potentially convoluted scenario is the challenge of establishing cross-reactivities between the alpha, beta, gamma, and delta SARS-CoV-2 strains with the immunogenic components of the current vaccines. It, therefore, calls for a renewed approach to vaccine design where all possible SARS-CoV-2 variants of concern and variants of interest are considered in the formulation of vaccines devoid of cross-reactivity tendencies.Pointedly, the glycan architecture is seldom indexed in vaccine design considering their role in the infection sequence. While glycans are resistant to structural changes, the enzymes for the glycan assembly (transferases and glycosidases) remain key influencers to compositional variations in the SARS-CoV-2 and in turn linked to disease pathogenesis. Although no SARS-CoV-2 glycan-specific enzyme has been reported so far, their elucidation and status as anti-COVID-19 targets will expand our therapeutic options. Analytical advancements to the tune of Cryo-EM microcrystal electron diffraction are yet to be engaged to unambiguously resolve the complex viral glycans. Results from such assays will provide broader insights into the pathophysiological implications and deepen our understanding of host responses to these pathogens. In a similar vein, the objective of COVID-19 diagnosis should also extend to the viral glycans as the target epitopes to detect infections. In transitioning from EUA to FDA-approved IVDs, the development of point-of-care IVDs, which hold merits such as sensitivity, specificity, result reproducibility, quick and low cost will come in handy in the event of pandemics where frontline health workers are either limited or overstretched.The authors are grateful for the anonymous reviewers.


Background: This study assesses the perceptions and acceptance of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) vaccination. It also examines its influencing factors among the healthcare workers (HCWs) in the Eastern Cape, South Africa. Methods: In this cross-sectional study performed in November and December 2020, a total of 1308 HCWs from two large academic hospitals participated in the Eastern Cape Healthcare Workers Acquisition of SARS-CoV-2 (ECHAS) study. Validated measures of vaccine hesitancy were explored using a questionnaire. Logistic regression was used to identify the determinants of vaccine hesitancy. Results: The majority were nurses (45.2%), and at risk for unfavourable Covid-19 outcome, due to obesity (62.9%) and having direct contact with individuals confirmed to have Covid-19 (77.1%). The overall acceptance of SARS-CoV-2 vaccine was 90.1%, which differed significantly by level of education. Individuals with lower educational attainment (primary and secondary education) and those with prior vaccine refusal were less likely to accept the SARS-CoV-2 vaccine. However, positive perceptions about the SARS-CoV-2 vaccine were independently associated with vaccine acceptance. Conclusions: The high level of acceptance of SARS-CoV-2 vaccine is reassuring; however, HCWs with a lower level of education and those with prior vaccine refusal should be targeted for further engagements to address their concerns and fears. Since the identification of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causing Coronavirus disease 2019 (COVID-19) and the global pandemic, multiple groups began the race to produce safe and effective vaccines. To date, several vaccines have received approval by the regulating authorities and are being rolled-out in most countries (including South Arica) as a strategy to end the global pandemic. While experts agree that large scale vaccination of populations is the best strategy to gain control of the pandemic through sufficient ‘herd immunity’, convincing citizens that vaccines are safe, effective and necessary is an essential component to any effective vaccination programme.Vaccine hesitancy refers to delay in acceptance or refusal of vaccination despite its availability, and has been encountered since the invention of vaccines in 1796 by Edward Jenner . The rolIn a recent global online poll of around 20,000 people by the Institut de Publique Sondage d’Opinion Secteur (IPSOS), 74% of adults surveyed prior to SARS-CoV-2 vaccine rollouts agreed to get vaccinated. Out of the South Africans surveyed (approximately 500 participants), only 64% reported that they would agree to get vaccinated against SARS-CoV-2, being the fifth lowest vaccine acceptance of the 27 countries sampled ,9,10. ReSouth Africa has suffered through two waves of COVID-19 with over 1.5 million people infected, and over 40,000 recorded deaths to date. Health care workers have suffered high infection rates, especially in the Eastern Cape province, with 11,262 HCWs infected and 262 deaths by the 18 February 2021 . GettingThis cross-sectional survey was a component of a larger Eastern Cape Healthcare Workers Acquisition of SARS-CoV-2 (ECHAS) Study, which studied SARS-CoV-2 antibody seroprevalence among HCWs . Two larEligible participants were doctors, nurses, allied health workers, administrative and support staff aged 18 years or older that were permanently employed by either of the hospitals. All staff were given an opportunity to participate in the study over a period of six weeks between November and December 2020. This study adopted a multi-stage cluster sampling technique in order to ensure a representative sample of HCWs recruited across the various domains within the hospitals. In order to ensure inclusivity, exposure areas were pre-defined as clusters in accordance with the risk assessment by Iversen et al. as high risk  wards and intensive care units (ICU)); Intermediate risk  and other clinical areas) and low risk  . ParticiEach of the HCWs completed a self-administered structured questionnaire . Few participants received support from the research assistants with the completion of the questionnaire. Data were subsequently captured into Research Electronic Data Capture (Redcap) online software with a pre-installed questionnaire. This electronic software is housed by the South African Medical Research Council Server for privacy and confidentiality of data.We obtained relevant demographic data, which included age, sex, race, type of residence, educational level and number of household members. In addition, clinical conditions that have been shown to increase the risks for severe COVID-19 disease or mortality  were self-reported by the participants ,18,19. WExposure to SARS-CoV-2 was assessed by documenting the domain of work within the hospital and additional questions on perceived exposure were elicited: “Have you had direct contact with anyone with COVID-19 at hospital?”, “Have you had direct contact with individuals diagnosed with COVID-19 outside of the hospital?” and “Have you ever been diagnosed with COVID-19?”. It was postulated that HCWs who perceived themselves to be highly vulnerable to contract Covid-19 might be more receptive to accept vaccine. This hypothesis was further tested in the models.Acceptance of SARS-CoV-2 vaccine was the main outcome measure and assessed with the question: “When COVID-19 vaccine becomes available; will you personally receive the vaccine? Responses were categorised as ‘yes’ or ‘no’.General vaccine attitudes of the HCWs were assessed with a set of questions: “Do you believe that a vaccine is needed to end the COVID-19 pandemic?”, “Do you think every HCW should get the COVID-19 vaccine when it becomes available?” and ‘Do you think vaccines are generally safe?” Responses to these questions were categorised as ‘yes’ or ‘no’ options. The HCWs’ attitudes toward vaccines were also elicited with two sets of questions: “Have you ever refused vaccines in the past?” and “Have you ever experienced adverse effects from vaccines before?”. A “yes” or “no” response option was provided to elicit the underlying attitude of the HCWs to vaccines in general that could impact on their willingness to accept future COVID-19 vaccines.The Walter Sisulu University Faculty of Health Sciences Ethics Committee granted approval for the implementation of the study protocol (Project Identification Code: 087/2020). Permission was obtained from the Eastern Cape Department of Health as well as the clinical governance of the two hospitals. Information sharing sessions about the study were disseminated through the clinical managers, nursing managers and union representatives. In addition, participants received group information as well as an information sheet detailing the purpose and process of the study prior to signing an informed consent indicating their voluntary participation. Participants’ right to privacy and confidentiality of medical information was respected during and after the study. Unique patient identifying numbers (PTID) were used for coding in order to ensure privacy of medical information. The study was conducted in accordance with the Helsinki Declaration and Good Clinical Practice Guidelines and principles governing human research.The construct and criterion validity of the instrument have been established by selecting variables that have been used successfully in measuring the outcome measures of this study in previous studies ,22,23,24Complete data were captured on Redcap and analysed by using the IBM SPSS Statistics for Windows, Version 27.0 . All data entry errors were corrected after running a simple frequency for all the variables. The baseline characteristics of the study participants are presented as categorical variables with frequencies and percentages.p-value of less than 0.05 was considered statistically significant.The relationship between the main outcome measure (acceptance of Covid-19 vaccine) and participants’ characteristics (baseline characteristics and perception about Covid-19 vaccine) were assessed using chi-square analysis. The direction of the association was analysed using bivariate logistic regression analysis (stepwise forward L-R Method) with a 95% confidence interval (95% CI). Selection of variables into the bivariate logistic regression model was based on factors that were previously reported in the literature and found to be significant in the chi-square analysis. Using a stepwise forwards L-R method, level of education was selected as the most influential factor. Therefore, the final model was composed of the following variables: “Level of education”, “Is the vaccine needed to end the pandemic?”, “Should healthcare workers receive the vaccine?”, “Are vaccines safe?”, “Past vaccine refusal?” and “Experienced adverse reactions from a vaccine”. A Of the total participants (N = 1308) included in the study, nursing staff were the majority (45.2%), followed by support staff (28.7%) and pharmacy staff accounted for the lowest proportion (4.7%). The two hospitals contributed almost equal number of participants (ratio 1:1). The majority of the participants were females (81.5%), aged 26–55 years (79.1%), obese (62.9%) and were of black ethnicity (78.8%). Most of the participants reside in urban areas (90.1%), have attained a tertiary education (71.4%), have never smoked cigarettes (91.1%), have at least one co-morbidity (65.6%) and had not at the time been diagnosed with Covid-19 (69.7%) .p < 0.001) and professional category (p = 0.021). However, there was no significant difference in the acceptance of Covid-19 vaccine by sex, BMI categories, race, smoking status, presence of co-morbidities and prior diagnosis of Covid-19 (p > 0.05)  .A large proportion of the participants (77.1%) reported direct contact with individuals diagnosed with Covid-19 within the hospital while fewer participants (26.5%) confirmed direct COVID-19 contact outside of the hospital. The majority of the participants believed that vaccine will be needed to end the pandemic (90.1%) and that HCWs should receive the vaccine (92.7%). A slightly lower proportion considered vaccines to be safe (86.9%) and had not refused vaccine in the past (86.5%). Most had not experienced adverse events from previously administered vaccines (90.2%). There were no significant associations between perceived risk of exposure  and vaccine acceptance. However, all of the attitude questions regarding vaccines  were significantly associated with vaccine acceptance .In the bivariate logistic regression analysis, individuals with primary (OR 0.14 95% CI 0.33–0.64) or secondary education  were less likely to accept the vaccine in comparison to those with tertiary education. Participants who had refused a vaccine in the past were less likely to accept the vaccine  in comparison to those who had not.However, positive attitudes toward the SARS-CoV-2 vaccine were all significantly associated with vaccine acceptance, including belief that vaccines are needed to end the pandemic , belief that vaccines are safe  and belief that vaccine should be given to every HCW  .There is consensus among experts that large scale vaccination against SARS-CoV-2, leading to attainment of population level “herd immunity” is the best strategy to control the pandemic. However, hesitancy against SARS-CoV-2 vaccine is mounting globally. Therefore, it is essential to convince people at the community level that vaccines are safe, effective and necessary. Health care workers are uniquely positioned to lend their voices and actions in promoting this biomedical strategy to their patients and the community at large, especially in South Africa, which has experienced significant devastation from the COVID-19 pandemic. However, there is currently no published data on the perceptions and acceptance of SARS-CoV-2 vaccine among HCWs in South Africa. This study reports the perceptions and level of acceptance of SARS-CoV-2 vaccine, and further examines the influencing factors of vaccine acceptance among HCWs in the Eastern Cape, South Africa.This survey of 1308 hospital-based HCWs revealed a reassuringly high level of acceptance of the SARS-CoV-2 vaccine (90.1%) in the region. This is higher than previous reports of HCWs’ vaccine acceptance rates of 27% to 75% in other countries, and the general South African public survey of 64% . Due to Factors such as older age, raised BMI, presence of co-morbidities and prior diagnosis of Covid-19, which may influence an individual’s perceived risk of COVID-19 (and hence need for vaccination) were not associated with vaccine acceptance in this study. This is in contrast to other studies that have reported higher vaccine acceptance with HCWs who were male, of older age and with co-morbid illnesses ,10,12. UThis study was performed between November and December 2020, when the first SARS-CoV-2 vaccine trial results had been released , and these vaccines were being registered in a few countries. Hence, these results need to be interpreted in the context of the SARS-CoV-2 vaccine information that was available at that time. With the rapid vaccine development and deployment globally to date, as well as subsequent concerns raised by regulatory bodies over rare but severe adverse events such as vaccine-induced immune thrombotic thrombocytopaenia (VITT), an individual’s perception of vaccine safety may change daily . More soA potential general distrust in vaccines was assessed by reporting refusal of vaccination in the past, which was associated with SARS-CoV-2 vaccine refusal. Vaccines routinely offered to adult HCWs in the South African state sector are Hepatitis B and an annual influenza vaccination, but these are voluntary. Having experienced adverse events from vaccines in the past also correlated with vaccine refusal in this study. These two vaccine questions gave some insight into the reasons for HCWs’ vaccine refusal in this context. Rather than specific concerns with the SARS-CoV-2 vaccine safety, efficacy or need, this sub-group appears to have a general distrust or fear of vaccines, partly informed by past vaccine adverse events. Adverse effects of vaccines have been reported previously to lead to vaccine refusal ,32.An individual vaccine acceptance decision represents a complex sum of vaccine confidence, disease complacency (or fear) and convenience to access vaccination (as per the WHO advisory group) . The firThese findings represent a snapshot of the perceptions, attitudes and acceptance of SARS-CoV-2 vaccine at the time of the study, given that these phenomena are dynamic in nature and may be influenced easily by safety and efficacy data. In addition, the binary nature (yes/no) of the main outcome (vaccine acceptance) did not allow for better understanding of the timing of acceptance of the vaccine. While the majority of the staff will probably accept the vaccine immediately whenever it becomes available others might choose to defer till more people have received the vaccine in the country. Perhaps, a five-point Likert scale showing varying degrees of opinions on vaccine acceptance would have shed more light on the overall perceptions and attitudes of the staff. Though, the investigators provided extended access to all eligible participants, the potential bias due to the convenience sampling and voluntary participation of the HCWs cannot be ignored. Notwithstanding the limitations, the findings are reassuring that HCWs were willing to accept and recommend the vaccine to other HCWs. The staff who chose not to accept the vaccine need further engagement targeting their fears about adverse events and myths surrounding the SARS-CoV-2 vaccine. It should also be noted that this study was implemented in November and December 2020 prior to the roll-out of the SARS-CoV-2 vaccine in South Africa. Therefore, uptake of the SARS-CoV-2 vaccine by the HCWs should be monitored in light of the findings reported in this study.The high level of acceptance of SARS-CoV-2 vaccine among the HCWs (90.1%) in the region is reassuring. Findings that healthcare workers were willing to accept and believed that others should receive SARS-CoV-2 vaccine highlight a positive signal towards the success of a mass vaccination programme in the region. However, healthcare workers with lower levels of education and those with prior vaccine refusal should be targeted for further engagements to address their fears and concerns. It will be crucial to monitor the uptake of the SARS-CoV-2 vaccines whenever they become available and correlate the refusal categories with the findings in this study.


The impact of COVID-19 disruptions on global Bacillus Calmette-Guérin (BCG) coverage and paediatric tuberculosis (TB) mortality is still unknown. To fill this evidence-gap and guide mitigation measures, we estimated the impact of COVID-19 disruptions on global BCG coverage and paediatric TB mortality. First, we used data from multiple sources to estimate COVID-19-disrupted BCG vaccination coverage. Second, using a static mathematical model, we estimated the number of additional paediatric TB deaths in the first 15 years of life due to delayed/missed vaccinations in 14 scenarios—varying in duration of disruption, and magnitude and timing of catch-up. We estimated a 25% reduction in global BCG coverage within the disruption period. The best-case scenario  resulted in an additional 886 (0.5%) paediatric TB deaths, and the worst-case scenario (6-month disruption with no catch-up) resulted in an additional 33,074 (17%) deaths. The magnitude of catch-up was found to be the most influential variable in minimising excess paediatric TB mortality. Our results show that ensuring catch-up vaccination of missed children is a critical priority, and delivery of BCG alongside other routine vaccines may be a feasible way to achieve catch-up. Urgent action is required to support countries with recovering vaccination coverages to minimise paediatric deaths. On 22 May 2020, Gavi (the Vaccine Alliance), the United Nations Children’s Fund (UNICEF) and the World Health Organisation (WHO) warned of approximately 80 million children at risk of missing their childhood vaccination ,2. TransDuring prior epidemics such as Ebola, BCG has been particularly impacted due to stockouts in affected regions . As a reMissed or delayed BCG vaccination leaves children vulnerable to TB disease. Previous studies have estimated that even briefly delayed BCG vaccinations may increase the burden of paediatric TB mortality ,9 and coCurrently, there are limited data on the global reduction of BCG coverage caused by COVID-19-related disruptions, and the potential impact of such disruptions on paediatric TB mortality is still unknown. To fill this evidence gap, we conducted a two-stage analysis (1) to estimate the impact of COVID-19 disruptions on global BCG coverage, and (2) repurposed an existing model to estimate the additional paediatric TB mortality in LMICs with a universal BCG policy. The magnitude of these disruptions needs to be urgently quantified to prompt swift and effective remedial action by the global health community. While we focus on the impact of the COVID-19 pandemic on BCG disruptions, the method presented in this study may also be relevant for other childhood vaccines disrupted during this pandemic period.We collated available data from peer-reviewed papers, newspaper articles, published country level reports, and conversations with experts to estimate the magnitude of global reduction in BCG coverage. Specifically, we searched for information on whether BCG was interrupted in-country due to COVID-19, and if so, the period of disruption and the percentage reduction compared to 2019 values. Where data indicated some disruption to BCG, but specific data were not available, we used estimates of disruption to other routinely delivered paediatric vaccines as a proxy.2020) within the disruption period in 2020 was estimated as follows:i2020 (percentage of BCG given in the disruption period in 2020 compared to the same period in 2019) in each country (i) was multiplied by a weighting Wi. The weighting was calculated as the number of BCG doses (N i2019) each country delivered in 2019 divided by the global number of doses of BCG provided in 2019 (G2019). We assumed that reduction estimates from countries where data were available, and our estimate of R2020, were representative and generalisable to all 152 LMIC with a policy for universal neonatal vaccination. R2020 was the final estimate of relative reduction in BCG coverage applied in the model. The global estimate of relative BCG coverage reduction  catch-up would occur linearly within 3 months following the end of the disruption period, (ii) catch-up would occur linearly within 6 months following the end of the disruption period (iii) catch-up was timed with the MMR vaccine (approximately 19 months) as a proxy for co-delivering BCG with other routine paediatric immunisations. The global MMR timeliness data were estimated by Clark et al., where 60% of eligible children receive the vaccine within 1 year and 90% by 2 years of age [Pulse survey data showed that vaccinations at birth were least affected by COVID-19-related disruptions, and that outreach programmes were more affected than healthcare facilities .Therefore, we assumed that vaccinations usually administered at birth in healthcare facilities were not disrupted, but vaccinations usually given at birth outside healthcare facilities, and all vaccinations usually given after birth could be disrupted. Using this, we estimated the proportion of the BCG-eligible cohort affected by COVID-19 disruption was 69% from UNICEF maternal and neonatal health data and Clark et al vaccination timing data .t 2020) was estimated as:t 2019 was the number of neonates who would have received the vaccine each week t in 2019 (i.e. no disruption), P2020 was the proportion of the BCG eligible cohort affected by COVID disruption (69%), B2020 was the percentage of neonates born during the disruption period (i.e. 25% of the birth cohort in the 3-month disruption scenarios and 50% of the birth cohort born in the 6-month disruption scenarios), and R2020 was relative reduction in BCG coverage level during disruption (25%).Applying this, the number of vaccinated children each week in 2020  to estimate the additional number of global TB deaths in the affected birth cohort in the first 15 years of life due to missed and delayed vaccinations, between the baseline (no disruption) and each of the 14 scenarios of disruption. We used a static mathematical model in Microsoft Excelt is the number of unvaccinated children at each week of age (t), Vt is the number of vaccinated children at week t, and VE is the vaccine efficacy against TB death. The estimate for vaccine efficacy against TB death (VE) was based upon published meta-analysis [t and Ut) was calculated using data from Clark et al [The full model has been described in detail elsewhere . In summ1 − VE)).where Utanalysis , and therk et al . Backgrork et al . Estimatrk et al . The estRisk of TB death was calculated as an age-specific weekly individual risk of tuberculosis death in unprotected children aged 0–4 years and 5–14 years. This risk was based on the number of tuberculosis deaths in the age groups (estimated by WHO ) dividedt) in each scenario to estimate the number of TB deaths in the baseline  and in each disrupted scenario. The difference was calculated between each disruption scenario and the baseline scenario. Uncertainty analysis was conducted using Oracle Crystal Ball®, with 100,000 parameter sets generated assuming a log normal distribution on the parameters outlined in This age-specific risk was applied to the number of children at risk  number of excess deaths averted in all scenarios with that characteristic, compared to the average (mean) number of excess deaths averted in all scenarios without that characteristic. For example, the benefit of reducing the duration of disruption from 6 to 3 months, was calculated as the mean of the median excess deaths in scenarios B1 to B7 (6 months disruption), compared to the mean of the median excess deaths in scenarios A1 to A7 (3 months disruption).We obtained disruption data from 29 countries . We obta2020) was 25% compared to the same period in 2019. Few countries reported the duration of disruption, and where duration was reported the disruption began in March or April 2020, continuing for 3 to 6 months [Using these data, we estimated that the relative global reduction in BCG coverage within the disruption period , was estimated to avert an average of 21,930 excess deaths, when compared to scenarios without catch-up (scenarios A1 and B1). The duration of disruption was found to be the second most influential characteristic assessed in our model. A three-month disruption period was found to avert an average of 7295 excess deaths (scenarios B1 to B7), when compared to scenarios with a six-month disruption period (scenarios A1 to A7).Reducing the length of time taken to meet the catch-up target was found to be the least influential characteristic. Taking 3 months to catch-up was found to avert an average of 1612 excess deaths , when compared to scenarios with catch-up over 19 months (as defined by the MMR schedule) .We estimated a global average of 25% reduction in BCG coverage within the COVID-19-related disruption period, with countries reporting a reduced coverage of between 0 and 100% compared to BCG coverage in the same period in 2019. We estimated this could result in an additional 886 (UR: 41–2151) paediatric TB deaths, for a 3-month disruption period followed by catch-up to previous BCG coverage within 3 months, to an additional 33,074  paediatric TB deaths if disruption continued for 6 months with no catch-up.We explored a variety of scenarios for the duration of disruption, and the timing and coverage of catch-up. The results indicate that maximising the coverage of BCG catch-up is especially important for averting paediatric TB deaths. Vaccinating all children who have missed their BCG vaccines could prevent an average of 21,930 paediatric TB deaths. Little is currently known about the duration of COVID-19-related disruptions as few countries have reported on this. Our analysis suggests that minimising the duration of disruption by even 3 months can avert an average of 7295 additional paediatric TB deaths. Rapid action is required to mitigate and minimise the burden of paediatric TB mortality that will otherwise inevitably take place in many settings. Ensuring that BCG vaccination coverage is recovered following periods of disruption is an important intervention and will minimise the excess paediatric TB mortality in the coming years. However, catch-up campaigns can be costly and logistically challenging. We used the timing of MMR vaccination as an example of providing BCG catch-up alongside other routine vaccines, that could minimise cost and logistics of catching up BCG coverage. Our analysis showed that the coverage of catch-up may be more important than the timing of the catch-up, where reducing the catch-up period from 19 months (MMR timing) to 3 months only averts 1612 excess deaths. Therefore, children could receive the BCG vaccine when they attend health facilities for other routine check-ups or vaccines. In many BCG-scarce settings a vial of BCG is only opened if enough children are present for vaccination, thus creating missed opportunities for vaccination when few children are present ,24. ProvGiven that COVID-related disruptions may also impact the ability to catch up BCG vaccination through routine visits, other options may also need to be considered to help minimise the length of disruptions and facilitate catch-up vaccination. Examples include increasing access by providing vaccines in clinics, separated in time or location from unwell patients, drive-through clinics, or door-to-door vaccination provision, and by increasing demand through public health messaging on the importance of continuing timely routine vaccination during lockdowns . PoliticCOVID-19 has substantially disrupted general health services, especially in countries where TB is endemic . WHO estA recent survey in South-East Asia and Western Pacific noted fear of infection, restriction of movement, and limited access to healthcare as key reasons for COVID-related disruption to immunisation services in 2020 . As hospGavi identifies 68 countries where immunisation services have been affected due to the COVID-19 pandemic ,33. UNICBCG vaccination supply is already highly sensitive to stockouts, and fluctuations caused by instability in the production market . A largeFurthermore, several studies have explored the potential for BCG to have non-specific beneficial effects against COVID-19 disease . RecentlOne of the key limitations of this study is the lack of good estimates for BCG efficacy against TB death, we used estimates from a meta-analysis on the effectiveness of BCG with wide confidence intervals (8–88%) . ResearcWe chose to use a static model and therefore our model does not account for any potential reduction in transmission due to decrease in disease burden. However, any potential effects are likely to be small as paediatric tuberculosis cases are minimally infectious.Hospitals have struggled to maintain health services during the pandemic, therefore our estimate of births taking place in health facilities may be an overestimate. In addition, stock-outs may affect the number of vaccinations taking place in health facilities at the time of birth. Our results therefore likely underestimate the level of impact on BCG due to COVID-19 related interruptions. Data were only available for 29 countries covering 64% of the BCG-eligible population, but our estimate for BCG disruption carries some uncertainty due to missing data. We found substantial variation in the magnitude and duration of COVID-19-related disruption by country and by month, with disruption continuing periodically through 2020 and into 2021. Therefore, our estimates of the duration of disruption are likely to be an underestimate. It will be necessary to re-evaluate these results when updated and consistent global data on the number of BCG vaccines delivered in 2020 and 2021 are available. In this model, we did not account for the non-specific effects of BCG, and the potential impact of BCG disruptions on excess paediatric mortality may be even larger. A country-specific model estimating the importance of delays in BCG vaccination found that delays in BCG were particularly important in the first period of life, where all-cause mortality is highest . Thus, tEnsuring catch-up vaccination of children who have missed their scheduled vaccines is a critical immunisation priority to minimise excess paediatric TB deaths. Our results suggest that high levels of catch-up vaccination coverage, in addition to minimising the duration of disruption, should be a priority to minimise additional paediatric TB deaths due to COVID-related disruptions. BCG catch-up alongside other routine vaccines or at other health facility contacts may be a feasible way to deliver vaccines without separate campaigns. Global action is required to support the most disadvantaged countries in overcoming the logistical challenges this intervention will entail, and action is required to support countries with recovering vaccination coverage as soon as possible.


The following protocol describes our workflow for processing wastewater with the goal of detecting the genetic signal of SARS-CoV-2. The steps include pasteurization, virus concentration, RNA extraction, and quantification by RT-qPCR. We include auxiliary steps that provide new users with tools and strategies that will help troubleshoot key steps in the process. This protocol is one of the safest, cheapest, and most reproducible approaches for the detection of SARS-CoV-2 RNA in wastewater. Owing to a pasteurization step, it is safe for use in a BSL2 facility. In addition to making the protocol safe for the personnel involved, pasteurization had the added benefit of increasing the SARS-CoV-2 genetic signal. Furthermore, the RNA obtained using this protocol can be sequenced using both Sanger and Illumina sequencing technologies. The protocol was adopted by the New York City Department of Environmental Protection in August 2020 to monitor SARS-CoV-2 prevalence in wastewater in all five boroughs of the city. In the future, this protocol could be used to detect a variety of other clinically relevant viruses in wastewater and serve as a foundation of a wastewater surveillance strategy for monitoring community spread of known and emerging viral pathogens. Tracking SARS-CoV-2 infections often involves detecting SARS-CoV-2 RNA via RT-qPCR in biological samples obtained from patients that develop symptoms associated with COVID-19 . AdditioWith the goal of sequencing SARS-CoV-2 RNA from wastewater, we developed a protocol to extract and quantify viral RNA. The initial step in the development of this protocol was the decision to pasteurize our samples at 60°C for an hour on arrival at the laboratory. Given that SARS-CoV-2 is a risk group 3 (RG3) agent, inactivation of the virus before processing is often required before samples can be processed in a BSL2 laboratory. Happily, as we report here, pasteurization did not impair our ability to detect SARS-CoV-2, but instead, improved it. Our protocol includes the spiking-in with a control virus to determine the efficiency of recovery. Interestingly, while SARS-CoV-2 recovery was not impaired by pasteurization, the two control spike-in viruses tested bovine coronavirus (BCoV)  and bactA second major decision was to employ centrifugation and filtering (0.2 μM) to remove wastewater solids which we made at the beginning of our study. While SARS-CoV-2 may associate with solids, removing solids facilitates the downstream processing steps and may remove genomic contamination that would impair our ability to deep sequence SARS-CoV-2 so we adopted filtering early on the development of our protocol. As a counterpoint, filtration is one of the more expensive steps of the protocol so those desiring to reduce costs may consider eliminating filtration. We were not able to acquire consistent results without filtration so we opted to filter all samples following pasteurization.Since viruses are greatly diluted in wastewater, virion concentration is a significant challenge. We considered three common protocols to concentrate SARS-CoV-2 virus present in the water: ultracentrifugation , skimmedAs we were mindful of the need to find cost effective solutions, we investigated alternative, kit-free approaches to RNA isolation. In our hands, TRIzol (ThermoFisher Inc.) performed better than the QIAamp Viral RNA Mini kit (Qiagen Inc.). As TRIzol is cheaper per sample than column-based kits, we adopted it for the final protocol. An added benefit of TRIzol relevant to downstream sequencing applications is that TRIzol segregates RNA in a separate layer from DNA, unlike column-based isolation kits, which isolate both RNA and DNA.In addition to the RNA isolation method, we compared the performance of different RT-qPCR enzymes, TaqPath 1-Step RT-qPCR enzyme (Thermofisher Inc.) and One Step PrimeScript III enzyme . Enough RNA can be acquired for RT-qPCR and isolated RNA is suitable for whole genome amplification and sequencing. As a general note, wastewater treatment plants indicated in our figures have been deidentified. There is no correspondence between the numerical wastewater treatment plant (WWTP) IDs in different figures. Moreover, experiments described in different figures were performed at different times using different wastewater samples. Our purpose here is not to report regional prevalence, but rather to demonstrate the reliability and consistency of our protocol.Our protocol results in the reproducible isolation and quantification of SARS-CoV-2 RNA from wastewater samples . To determine if longer incubation similarly impacts SARS-CoV-2 recovery, we compared SARS-CoV-2 quantitation for samples incubated in PEG/NaCl for 24 hrs versus 48 hrs. While longer storage resulted in slightly improved virus recovery, the difference was not significant  with TRIzol reagent (ThermoFisher Inc.). Our results showed that TRIzol facilitates significantly better RNA recovery than the kit at a fraction of the cost  and the One Step PrimeScript III enzyme (Takara Bio USA Inc.) Our results indicated that the two enzymes performed equally well  SARS-CoV-2 Panel kit as well as the Qiagen QIAseq® SARS-CoV-2 Primer Panel and QIAseq FX DNA Library kit and have obtained SARS-CoV-2 sequences from several of our wastewater treatment plants. Known and novel variants were identified. We continue to optimize and improve our library preparation methods to increase both length of coverage and depth of coverage for our NYC samples. In addition, we are developing real-time assays for the identification and quantification of additional viruses that circulate among our New York communities including Influenza.We used both the Swift NormalaseS1 File(PDF)Click here for additional data file.S1 Video(M4V)Click here for additional data file.


This analysis pushes human-to-human transmission back to mid-October to mid-November of 2019 in Hubei Province, China, with a likely short interval before epidemic transmission was initiated.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may have had a history of abortive human infections before a variant established a productive enough infection to create a transmission chain with pandemic potential. Therefore, the Wuhan cluster of infections identified in late December of 2019 may not have represented the initiating event. Pekar Science, this issue p. 412 SARS-CoV-2 human-to-human transmission likely initiated in mid-October to mid-November 2019 in Hubei, China. Understanding when severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged is critical to evaluating our current approach to monitoring novel zoonotic pathogens and understanding the failure of early containment and mitigation efforts for COVID-19. We used a coalescent framework to combine retrospective molecular clock inference with forward epidemiological simulations to determine how long SARS-CoV-2 could have circulated before the time of the most recent common ancestor of all sequenced SARS-CoV-2 genomes. Our results define the period between mid-October and mid-November 2019 as the plausible interval when the first case of SARS-CoV-2 emerged in Hubei province, China. By characterizing the likely dynamics of the virus before it was discovered, we show that more than two-thirds of SARS-CoV-2like zoonotic events would be self-limited, dying out without igniting a pandemic. Our findings highlight the shortcomings of zoonosis surveillance approaches for detecting highly contagious pathogens with moderate mortality rates. In late December of 2019, the first cases of COVID-19, the disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), were described in the city of Wuhan in Hubei province, China  of all sequenced SARS-CoV-2 genomes to be in late November or early December 2019, with uncertainty estimates typically dating to October 2019 : 6.64 104 to 9.27 104]. The tMRCA of these circulating strains was inferred to fall within a 34-day window with a mean of 9 December 2019 (95% HPD: 17 November to 20 December) (We first explored the evolutionary dynamics of the first wave of SARS-CoV-2 infections in China. We used Bayesian phylodynamics (ecember) . This esIf the tMRCA postdates the earliest documented cases, then the earliest diverged SARS-CoV-2 lineages must have gone extinct . As thesWe find only weak evidence for basal lineage loss between 24 December 2019 and 13 January 2020 (fig. S3A). The root tMRCA is within 1 day of the tMRCA of virus sampled on or after 1 January 2020 in 78.5% of posterior samples (fig. S3B). The tMRCA of genomes sampled on or after 1 January 2020 is 3 days later than the tMRCA of all sampled genomes. By contrast, the mean tMRCA does not change when considering genomes sampled on or after 1 January 2020 versus on or after 13 January 2020. This consistency indicates a stabilization of coalescent processes at the start of 2020, when an estimated total of 1000 people had been infected with SARS-CoV-2 in Wuhan  . The medAs a robustness check, we also simulated epidemics with more densely and more sparsely connected contact networks , rescaling the per-contact transmission rate to maintain empirical epidemic growth dynamics. We also explored the effects of faster  and slower  epidemic doubling times (table S2). Slower transmission rates led to more days between the index case and the stable coalescence, but modifying the density of the contact network had minimal effect on this time interval (fig. S4).To estimate the date of infection for the index case in Hubei province, we combined the retrospective molecular clock analysis with the forward epidemic simulations (fig. S5). We identified the stable tMRCA in the posterior trees as an anchor to the real-world calendar dates and then extended this date back in time according to the number of days between the index case infection and the time of stable coalescence from the compartmental epidemic simulations. However, a random sample of tMRCAs and days from index case infection to coalescence will not produce epidemiologically meaningful results because many of these combinations do not precede the earliest dates of reported COVID-19 cases. Therefore, we implemented a rejection samplingbased approach to generate a posterior distribution of dates of infection for the Hubei index case, conditioning on at least one individual who had progressed past the presymptomatic stage in the simulated epidemic before the date of the first reported COVID-19 case .In our primary analysis, we assume that 17 November represents the first documented case of COVID-19 (ascertained or unascertained in the SAPHIRE model). Under this assumption, the median number of days between index case infection and stable coalescence after rejection sampling is 37 days (95% HPD: 12 to 55 days) . ConsequThis time frame for the Hubei index case is robust (fig. S7). Epidemic simulations with faster or slower transmission rates and more or less densely connected contact networks produce similar date estimates. Furthermore, using the root tMRCA from the sampled posterior trees, rather than adjusting for the shifting coalescence between 24 December 2019 and 1 January 2020, produces a median date of 3 November . Incorporating a relaxed molecular clock or adjusting the coalescent prior assumption (Skyline versus Skygrid) also had minimal effect.If we enforce that there must be at least one ascertained case in our simulations before 17 November 2019, the median date of the Hubei index case is pushed back about a week to 28 October  (fig. S7). However, the distinction between ascertained and unascertained in the original SAPHIRE model was meant to reflect the probability of missed diagnoses in January 2020 of the Wuhan epidemic and does not account for the investigations that resulted in retrospective diagnoses in November and December 2019.If we discount the reported evidence of retrospective COVID-19 diagnoses throughout the end of November and instead take 1 December as representing the first confirmed case of COVID-19, then the median time between index case infection and stable coalescence after rejection sampling is 23 days (95% range: 1 to 47 days) . Under tIt is reasonable to postulate that the variant of SARS-CoV-2 that first emerged was less fit than the variant that spread through China and that evolutionary adaptation was critical to its establishment in humans ; this adThese one-phase and two-phase epidemic simulations both suggest that the tMRCA is not representative of the emergence of SARS-CoV-2. In the primary analysis, the index case remains infected at the tMRCA  that are not characterized by superspreading events (Our results highlight the unpredictable dynamics that characterized the earliest days of the COVID-19 pandemic. The successful establishment of SARS-CoV-2 postzoonosis was far from certain, as more than two-thirds of simulated epidemics quickly went extinct. It is highly probable that SARS-CoV-2 was circulating in Hubei province at low levels in November 2019 and possibly as early as October 2019, but not earlier. Nonetheless, the inferred prevalence of this virus was too low to permit its discovery and characterization for weeks or months. By the time that COVID-19 was first identified, the virus had firmly established itself in Wuhan. This delay highlights the difficulty in surveillance for novel zoonotic pathogens with high transmissibility and moderate mortality rates.The high extinction rates we inferred suggest that spillover of SARS-CoV-2like viruses may be frequent, even if pandemics are rare (Even though all of the earliest documented cases of COVID-19 were found in Hubei province, we cannot discount the possibility that the index case initially acquired the virus elsewhere. Nonetheless, our dating inference is insensitive to geography. Furthermore, our results suggest that if the virus first emerged in a rural community, it would have needed to migrate to an urban setting to avoid extinction. The lack of reports of COVID-19 elsewhere in China in November and early December suggests that Hubei province is the location where human-to-human transmission chains were first established.The circumstances surrounding the emergence of SARS-CoV-2 in Hubei province remain shrouded. Although SARS-CoV-2 is repeatedly adapting to spread among humans (