F-specific filamentous phages, elongated particles withcircularsingle-stranded DNA encased in a symmetric protein capsid, undergoan intermediate step, where thousands of homodimers of a non-structuralprotein, gVp, bind to newly synthesized strands of DNA, preventingfurther DNA replication and preparing the circular genome in an elongatedconformation for assembly of a new virion structure at the membrane.While the structure of the free homodimer is known, the ssDNA-boundconformation has yet to be determined. We report an atomic-resolutionstructure of the gVp monomer bound to ssDNA of fd phage in the nucleoproteincomplex elucidated via magic-angle spinning solid-state NMR. The modelpresents significant conformational changes with respect to the freeform. These modifications facilitate the binding mechanism and possiblypromote cooperative binding in the assembly of the gVp–ssDNAcomplex. Inovirus are a family of viruses that predominantly infectGram-negative bacteria.2 Most of the current biologicalunderstanding of filamentous phages, including the phage mechanismsof infection, replication, and assembly, comes from extensive researchconducted on a group of three closely related phages, known as F-specificfilamentous phages (Ff phages).4 Ff phages all infect Escherichia coli cells bearing F-pili organelles.The members of the Ff group—phages M13, fd, and f1—exhibit98% DNA sequence identity, and therefore have been studied interchangeably.5 Uniquely among other bacteriophages,infection by filamentous phages does not cause lysis of the host cell6 and the host bacterial cells continue to growand divide as new virion structures are produced within their cytoplasm,with about one-half to three-quarters the growth rate of uninfectedcells.8 First isolated from sewage systems in theearly 1960s, Ff phages have since become prominent model systems,used widely in molecular biology research.11Bacteriophages, the most abundant entitieson earth, are virusesthat infect bacteria. Filamentous bacteriophages of the genus 13 The virion genome is structured as circular single-stranded DNA,encased in a symmetric protein capsid. Within the capsid, the ssDNAforms an anti-parallel two-stranded helix, structurally similar todouble-stranded DNA, but it does not exhibit base-pair complementarity.Most of the capsid is composed of approximately 2700 copies of a 50-residue-long,mostly helical single-coat protein, gVIIIp, known as the major coatprotein.6 Four minor coat proteins, withonly a few copies of each, cap both ends of the virion .6 The wild-type Ff phage genome comprises around6400 nucleotides, and it contains nine genes, encoding for a totalof 11 proteins.14Ff phageparticles have an elongated structure, approximately amicron in length and 6–7 nm in diameter.Escherichia coli cell via attachmentof gIIIp to the tip of the F-pilus, a helical surface filament thatextends from the bacterial surface, containing stoichiometric amountsof proteins and phospholipids.16 The pilus retractstoward the cell envelope, leading to the opening of the virion structure17 and entry of the viral ssDNA into the cytoplasmof the host cell.The initial stage of theFf phage life cycle is the infection ofthe 3 In the early stage after infection, RF is usedfor transcription of phage mRNA, followed by translation and expressionof viral proteins.18 RF undergoes the rollingcircle replication mechanism, resulting in the generation of new positivestrands of the phage genome, which are once again utilized for negative-strandsynthesis. Once the expression level of a certain phage protein, gVp,has reached a critical threshold within the cytoplasm of the host,thousands of homodimers of gVp cooperatively bind to positive strandsof phage ssDNA, coating nearly the entire two-stranded helix and forminga superhelical, left-handed nucleoprotein complex,19 known as the premature virion. By disabling synthesis ofthe negative strand and subsequent generation of RF, further viralgenome replication is prevented.18 Formationof this nucleoprotein complex also packages the circular ssDNA inan elongated form, making the strand geometrically viable for assemblyof the mature virion structure.1 Once theintracellular complex reaches the cell membrane, a secretion-assemblyprocess is initiated when an exposed hairpin loop at one end of thepremature virion, serving as a packaging signal, interacts with theinner membrane and with phage proteins.4 Next, in aprocess that has yet to be characterized in detail, the gVp dimersare stripped from the strand and replaced by the coat proteins, formingthe newly assembled mature infectious virion, which is then extrudedfrom the cell.Next, the phage genome undergoes episomalreplication within thehost cell. The positive strand of the genome is used as a templateto synthesize a negative strand, and together, both strands form adouble-stranded phage genome, known as the replicative form (RF).20 and is non-sequence-specific.21 Depending on experimental conditions, severaldifferentbinding modes have been described, with various stoichiometric ratiosin the range of three to five nucleotides per monomer.5 Under physiological in vitro conditions, the predominantstoichiometry reported is of four nucleotides per monomer.18The binding of gVp dimers to ssDNA is highlycooperative22 (PDBID: 1VQB) andanother via solution NMR24 (PDB ID: 2GVB) for a Y41H mutantof M13 phage gVp, which was found to increase protein solubility.23 Both models are nearly identical in secondaryand tertiary structures (the backbone root-mean-square deviation (RMSD)of the monomer, excluding the DNA binding loop, is 1.5 Å; forthe dimer, it is 1.9 Å24) and agreewith biochemical and biophysical data previously collected on thesystem.24 They differ in the location of the N-terminuswith respect to the core, but the main difference involves the orientationof the DNA binding loop with respect to the core. This results ina solution NMR monomer that is slightly more globular than the X-raystructure, and since the former does not seem to have enough vacantvolume available in the putative binding cleft, it has previouslybeen suggested that this loop would be required to assume a conformationmore similar to that of the X-ray structure for ssDNA binding to takeplace.24Two high-quality models for the structureof free gVp were elucidated,one via X-ray crystallography 1 for gVp18 Thehomodimer structure in crystals of gVp  fold, based on a five-strandedbeta-barrel, was identified in several oligonucleotide and oligosaccharidebinding proteins.27 The barrel forms thehydrophobic core of the monomer, from which four main loops protrude.18 The first is the complex loop (residues 36–43),which is thought to be involved in dimer–dimer interactionsrelated to the assembly of the nucleoprotein complex.25 The second is the dyad loop (residues 68–78). Thedyad loop of one monomer is in spatial proximity to that of the othermonomer; therefore, they both comprise most of the inter-monomer contactsurface of the homodimer.28 A third loopregion, previously referred to as a “broad connecting loop”28 , was reported to be in the vicinity of the viral genome inthe complex.18 Here, it will be termedthe “core loop”. The fourth is the DNA-binding loop(residues 13–31), which was shown to be involved in the bindingof the viral genome. Several findings point to the involvement ofboth the DNA-binding loop and the dyad loop in binding to ssDNA. First,the twofold symmetry axis relating both monomers gives rise to twoconcave clefts in the dimer structure, each formed by the DNA-bindingloop of one monomer and the tip of the dyad loop of the other monomer.29 These clefts can accommodate the two anti-parallel strands of ssDNAin the complex. Second, NMR studies have implicated residues in bothloops as being in spatial proximity to bound nucleotides.29 Third, both loops include several amide protonsthat have been shown to exhibit fast exchange with the solvent, whichcan indicate that the orientations of both loops with respect to thehydrophobic core are flexible.30 Fourth, the calculated electrostatic potential at the surface ofthe putative binding domain, between the two loops, is highly positive,while the opposite side of the dimer, located at the outer surfaceof the nucleoprotein complex, exhibits nearly neutral surface charge.31 Such an asymmetric charge distribution can facilitatebinding to ssDNA as well as the formation of the symmetric nucleoproteincomplex. The latter argument is further corroborated by NMR and fluorescencestudies that have indicated that the binding of ssDNA by gVp dimersis mostly facilitated by electrostatic interactions between positivelycharged side-chain residues of the putative binding regions of theprotein and the negatively charged sugar-phosphate ssDNA backbone.32The gVp monomer is 87 residues long. The predominant,stable formin solution, across a range of pH, temperature, protein concentrations,and salt concentrations, is that of a symmetric homodimer, with atotal molecular mass of 19.4 kDa.s of gVp 1 is most33The structure of the superhelical gVp–ssDNA nucleoproteincomplex has yet to be determined in atomic resolution. No X-ray diffractionstructure was reported, and several attempts have been made to computationallycalculate a plausible model for the complex structure using globalparameters collected empirically on the system. These models utilizethe X-ray structure of the free form as a basis for the model, butas of yet, none of the suggested models were proven to be both accurateand in full agreement with available data regarding the complex.34 In work previouslyreported by our lab, the isotropic chemical shifts of both free gVpand ssDNA-bound gVp have been assigned.36 Quantificationand analysis of chemical shift perturbations (CSPs) led to the conclusionthat gVp undergoes significant structural changes upon binding, andregions expected to undergo the most extensive structural modifications—includingthe DNA-binding loop, the core loop, and the C-terminus—wereidentified.36 MAS ssNMR is also usefulfor protein structure elucidation based on acquisition of internucleardistance-dependent interactions and prediction of backbone torsionangles based on chemical shifts,37 as wellas acquisition of additional structural restraints.39 Given a sufficient amount of restraints distributed along differentregions of the sequence, a carefully designed calculation processcan lead to a converged, viable structural model. Several proteinstructures have previously been solved via MAS ssNMR, and beyond crystallinemonomers, more complex systems include the homodimeric Crh protein,40 CAP-Gly bound to microtubules,41α-synuclein42 and additional amyloid aggregates,43 andthe Anabaena sensory rhodopsin membrane protein in lipid bilayers.44Magic-angle spinning solid-stateNMR (MAS ssNMR) is advantageousfor probing systems of nucleotide-bound proteins, since linewidthsare not dependent on the particle mass, long-range crystallinity isnot required, as well as the ability to sensitively detect conformationalchanges, as even minor changes in the fold result in significant changesto the resonance positions.In this research, we describe our calculated,atomic-resolutionmodel for the structure of the gVp monomer in complex with ssDNA offd phage. We compare the structure to that of free gVp and discussthe biological significance of our findings, which provide insightinto the binding process of gVp to Ff phage ssDNA.36 and are available at the Biological MagneticResonance Bank (BMRB accession id: 51391). In order to elucidate informationon internuclear distances from these assignments, we acquired severaltwo-dimensional (2D) 13C–13C dipolar-basedMAS ssNMR experiments, where cross-peak correlations arise from pairsof NMR-active nuclei in spatial proximity. Depending on experimentalparameters and conditions, 13C pairs with internucleardistances up to approximately 8 Å can potentially give rise tospectrally detectable cross-peaks.46 Dipolar-assisted rotational resonance (DARR47) and combined R2nv-driven(CORD48) experiments were conducted atvarious mixing times . In addition, we conducted CHHC49 experimentsthat entail the advantage of direct transfer of polarization betweenprotons that are close in space in folded regions of the polypeptideand have stronger dipolar couplings than 13C spin pairs.51 The 2D experiments were conducted on both fully and sparsely 13C-labeled samples of gVp in complex with unlabeled full-lengthssDNA extracted directly from fd phage . Sparsely labeled samples, where only a subsetof carbon sites are isotopically labeled, give rise to simplified,less crowded spectra with reduced effects of dipolar truncation andrelayed polarization transfer, allowing better detection of long-rangecontacts.53 The resulting spectra alsoentail lower ambiguity levels on average, and therefore fewer assignmentsmay be attributed to each cross-peak.Chemical shiftassignments of the ssDNA-bound form of gVp were reported previously54) was used in order to attribute all possibleassignments to each cross-peak, resulting in a set of both ambiguousdistance restraints (ADRs55) and unambiguousrestraints (a single possible assignment). All restraints were setto a range of 2–8 Å.56Cross-peak lists generatedfrom eight spectra of the fully labeled sample, differing in experimenttype, mixing time, and processing parameters, were concatenated intoa single list. Similarly, 30 spectra of the sparsely labeled samplewere combined. In order to generate a list of distance restraints,home-written Python scripts were provided with the two peak listsand the assigned chemical shifts of ssDNA-bound gVp as input. A chemicalshift tolerance window of 0.3 ppm  while giving precedence to restraints arisingfrom the inherently less ambiguous, more informative sparse data,in spectral regions where the two peak-lists overlapped (see detailsregarding generation of distance restraints in the SI).Bothlists were further simplified by discarding restraints thathad more than 20 possible assignments, as this was shown, in the contextof solution NMR, to have the potential to improve the quality of thecalculated model.36 we can safely assume that the complex assembly process does notentail a complete rearrangement of the overall fold.33 (3) We removed contacts with a high probability of associationwith the homodimer interface. NMR structure calculations of multimersare challenging due to the inability to a priori distinguish betweencross-peaks arising from intra- and inter-monomer correlations.60 When attempting to calculate the monomer structure,any inter-monomer correlation arising from the dimer interface thatwill be erroneously designated as an intra-monomer contact would introduceconformational errors and therefore distort the elucidated fold ofthe monomer model. We therefore ruled out (using a home-written Pythonscript) all restraints that included possible assignments correspondingto an inter-monomer distance shorter than 7 Å in the X-ray structureof the free gVp homodimer under the assumption that the dimer interfacesof the free and ssDNA-bound gVp dimer structures bear similarity.This assumption is further justified by findings previously reportedby our lab, which demonstrated that the CSPs upon binding are relativelylow in the dimer interface.36We applied several filters on the aggregated setof distance restraintsin an attempt to discard restraints that do not arise from carbon–carboncorrelations of structural significance, or those that are in significantviolation of prior knowledge on the system. (1) We removed restraintsthat included a single-bond contact as one or more of the possibleassignments, since such correlations are assumed to be much strongerthan long-range contacts, and do not report on the overall fold ofthe protein. (2) We assumed that any pair of carbon sites furtherapart than 16 Å in the X-ray structure of free gVp will not beclose enough in the ssDNA-bound form of the protein to result in aspectrally detectable cross-peak. While evidence previously reportedby our lab has demonstrated that the structure of the gVp proteinundergoes significant changes upon binding,Theresulting set of restraints, after the application of the threefilters, was the initial set provided as input to the calculationprocess. A total of 1901 distance restraints were provided as inputto the initial step of the structural calculations, including 251unambiguous restraints.61 A total of142 torsion angle restraints were deemed consistent by the softwareand were used as input for the subsequent calculation process.The softwaretool TALOS+ was used in order to generate restraints on the ψ–ϕtorsion angles of the protein backbone.62 software package iteratively to calculate theprotein structure. Both the filtered distance restraints and the predictedangle restraints were provided as input, along with the protein sequence.We used the Xplor-NIHN structures were generated. Afterward, these structureswere sorted according to the energy score, and the k lowest energy structures  were further analyzed in order to updatethe restraints to be provided as input to the subsequent iteration.After each iteration, we used two filtration methods in order to betterinform the set of restraints to be provided as input to the next Xplor-NIHrun. First, distance and angle restraints that were significantlyviolated in a large subset of the top k lowest energystructures were removed (see SI). Second, the averagestructure of the k lowest energy structures was calculatedby Xplor-NIH and any possible assignments corresponding to carbonpairs further apart than a specific cutoff distance (16 Å downto 8.5 Å) in the average structure were ruled out. In doing so,we treated the average structure of the previous run as a low-resolutionmodel of the protein structure and accordingly modified the distancerestraints for the next iteration. We conducted a total of 12 iterationsof Xplor-NIH, with home-written Python scripts used intermittentlyfor the restraint input modification described above (see Xplor scriptsin the SI). Each iteration started froman arbitrary, extended conformation of the protein sequence, withideal covalent geometry. Starting from an initial cutoff distanceof 16 Å for distance restraint filtration from the 25-structure-averageoutput of the first run, we gradually and iteratively decreased thisfiltration cutoff down to 8.5 Å (with k = 10),so that with each passing iteration, this filter on distance restraintswas more strictly reliant on the outcome of the previous run. Revisionsof the distance restraints list throughout the calculation processresulted in an iterative decrease to both their overall amount andto ambiguity levels . The final set of restraints included 112 torsion angle restraintsand 1247 distance restraints, including 593 unambiguous restraints. During each iteration, a total of 63Finally, the lowest energy structure of the 12thiteration wasprovided as an initial structure to a refinement process, conductedin implicit solvent.α-RMSD of the final 10-structure ensemble with respect to their averagestructure is 1.2 Å across all 87 residues . The backbone RMSD of the well-defined regionsof the ensemble  is 0.37 Å, well below the valueof 2.0 Å, often used as the preliminary criterion for structuralconvergence .The average C° for distance and angle restraints, respectively)in some or all members of the ensemble (see SI Tables S6–S9). Therefore, the refined ensemble is ingood agreement with the final set of input restraints, with the vastmajority of internuclear distances and torsion angles within the boundsof the experimentally derived and iteratively filtered constraints.Statistics based on the Ramachandran plot65 of the ψ–ϕ torsion angles of the ensemble indicatedthat none of the analyzed, well-defined residues  lie in thedisallowed regions. This indicates that the majority of the proteinbackbone torsion angles are geometrically feasible in terms of sidechainsteric hindrance. This finding is further corroborated by the MolProbityclash score,66 depicting the average numberof steric atomic clashes larger than 0.4 Å per 1000 atoms (seedetails in the SI). The calculated ensembleclash score  is 26, which is on par with MolProbityclash scores previously reported for other protein structures solvedvia MAS ssNMR .The8ACZ. The Stride secondarystructure determination algorithm68 detectsfour regions as beta strands in the ensemble-averaged structure—residues2–4, 33–35, 43–46, and 61–63, locatedat the hydrophobic core. Both the complex loop (residues 36–43)and the dyad loop (residues 68–78) are relatively well-definedacross the ensemble, with Cα-RMSD values of 1.3 Å  RMSD of 6.4 Å between the ensemble-averaged structure of boundgVp and the X-ray structure of free gVp. Such significant conformationalchanges accompanying the nucleoprotein complex assembly process areexpected given the CSP analysis previously reported by our lab36 and agree well with the results reported here,as will be shown in detail.There are several significant structural changesdetected throughoutthe sequence, reflected overall by a backbone , corroborating this aspect of the calculated structure.Also, this result agrees well with the findings from the previousCSP results.36 The motional pathway forthe transition between the free and bound gVp molecular conformationsremains puzzling in terms of the change to the location of the C-terminuswith respect to the core, and further research is required in orderto decipher the trajectory of such a structural change.Relative to the X-ray structure,the location of the C-terminusundergoes a significant structural change 5C,D. Thi18 As can be seen in our ensemble-averagedbound gVp structure, the available space between the DNA-binding loopand the dyad loop of the monomer is significantly decreased upon binding, we calculated that the dyad loop–core loop inter-loopdistance decreases by 6.6 Å (from 25.2 to 18.6 Å) upon binding,and the DNA-binding loop–core loop inter-loop distance decreasesby 4.4 Å (from 18.4 to 14.0 Å). Once again, this findingis corroborated by the fact that both the DNA-binding loop and thecore loop are expected to undergo significant structural changes,according to previously reported CSP analysis.36 While the results shown here indicate a narrowing of thecleft of a single monomer, if the twofold symmetry axis is preservedupon binding, then the observed approach of the DNA-binding loop andthe dyad loop in an intra-monomer sense is expected to give rise toa shortening of two inter-monomer distances, each defined by the dyadloop of one monomer and the DNA-binding loop of the other monomer.This observation is significant, since it has long been reported thatthe binding of the dimer to the two anti-parallel strands of ssDNAinvolves the approach of two inter-monomer pairs of loops (DNA-bindingloop and dyad loop) toward one another, with each such pair bindingto one of the two strands forming the helical structure within thecomplex.18Anothermajor structural change relates to the concave clefts ofeach dimer structure, hypothesized to accommodate for the bound strandsof the ssDNA. As mentioned, upon dimerization of two gVp monomers,two concave clefts are formed in the homodimer structure, each locatedbetween the dyad loop of one monomer and the DNA-binding loop of theother. binding. This ch68 algorithm, the two short 310 helices reportedin the free X-ray structure are no longer detected in the bound structure.Ramachandran analysis comparing the free and bound forms indicatesthat this discrepancy arises due to slight shifts of the correspondingresidues in ψ–ϕ space . Of the five beta strands comprising the beta barrelin the free form, four are partially detected in the bound model.These strands, recognized at the region most central to the hydrophobiccore, form a short anti-parallel beta sheet, which is a subset ofthe beta barrel motif reported in the free structure. The fifth strandthat is not detected at all in the bound form is located at the C-terminusof the free structure, where our model exhibits a significant changein tertiary structure . Since some of the beta-strands offree gVp are short, the residues that exhibit larger changes to theirtorsion angles, due to the tertiary changes throughout the structure,suffice to prevent determination of several regions as beta-strands.The free and bound gVp structures also differ inthe recognitionof secondary structure elements 7. Accordpace see S6. Of thThe free form X-ray structure also includes a shortanti-parallelbeta ladder at the dyad loop (residues 68–70 and 76–78in the free structure), which is not detected in the bound form bythe Stride algorithm. In the bound structure, the conformation ofboth sections of the protein backbone with respect to one anotheris no longer viable for the formation of a set of hydrogen bonds necessaryfor formation of beta strands 5B. Yet, Another interestingstructural difference involves the alignmentof the core loop with respect to the DNA-binding loop and the dyadloop 8. IntheWe reported the first high-resolution 3Dmodel for the structureof the gVp monomer in its conformation when bound to full-length ssDNAof the fd bacteriophage virus. Our model is directly based on restraintselucidated from experimental magic-angle spinning NMR data. The structurecalculation protocol makes minimal use of the known free structurein order to improve the quality of the restraint input to the calculationof the bound form. It also takes measures in order to avoid conformationaldistortions that may arise from inter-monomer contacts of the homodimer.This structure joins a relatively short list of non-crystallineprotein structures that have been determined via MAS ssNMR in thecontext of their entire high-molecular-weight biological assembly,further demonstrating the power of this method in structural studies.As the use of AI methods for accurate determination of protein structurebased solely on sequence is becoming prevalent, MAS ssNMR presentsa tool to study specific structural changes involved in processesof DNA binding or complex assembly.Results previously reportedby our group have presented spectralevidence that gVp undergoes significant structural changes upon bindingto ssDNA in the intracellular nucleoprotein complex. CSP analysishas detected regions of interest along the protein sequence, wheresuch modifications to the conformation were expected to be most prominent.The structure reported here confirms the three main regions that werepredicted to be most conformationally altered in the bound form: TheDNA-binding loop protrudes from the core in a different angular conformationwhen compared to the free structure; the location of the C-terminuswith respect to the hydrophobic core is changed, posing a large structuralchange with respect to the free protein: The core loop, which protrudesin a different direction from the DNA-binding loop and the dyad loopin the free form, is aligned with both loops in the bound form. Theseconformational changes seem to facilitate two processes: the bindingto viral ssDNA, as the concave cleft where the ssDNA is hypothesizedto be bound to the protein assumes a narrower conformation, and thecooperative binding interactions of dimers forming the capsid of thecomplex,as the entire protein assumes a more compact conformation.


Post-COVID conditions in children and adolescents were mostly investigated as the incidence of individual or clusters of symptoms. We aimed to describe the findings of studies assessing key outcomes related to global wellbeing and recovery in children and adolescents from a public health perspective. We searched the Cochrane COVID-19 Study Register and WHO COVID-19 Global literature on coronavirus disease database on 5 November 2021 and tracked ongoing studies published after this date. We included observational studies on children and adolescents with a follow-up greater than 12 weeks and focused on the outcomes of quality of life, recovery/duration of symptoms, school attendance and resource use/rehabilitation. We assessed their methodological quality, and we prepared a narrative synthesis of the results. We included 21 longitudinal and 4 cross-sectional studies (6 with a control group) with over 68 thousand unvaccinated children and adolescents with mostly asymptomatic or mild disease. Study limitations included convenience sampling, a poor description of their study population and heterogeneous definitions of outcomes. Quality of life was not largely affected in adolescents following COVID-19, but there might be greater impairment in young children and in those with more severe forms of the disease (4 studies). There might also be an impairment in daily activities and increased school absenteeism following COVID-19, but the findings were heterogeneous (5 studies). A total of 22 studies provided highly variable estimates based on heterogeneous definitions of overall persistence of symptoms (recovery), ranging from 0 to 67% at 8–12 weeks and 8 to 51% at 6–12 months. We found limited data on resource use and the need for rehabilitation. One controlled study indicated that the quality of life of infected children and adolescents might not substantially differ from controls. All controlled studies found a higher burden of persistent symptoms in COVID-19 cases compared with test-negative controls or cases of influenza. There is limited evidence on the short and long-term well-being of children following SARS-CoV-2 infection. High-quality longitudinal studies with control groups are needed to describe the outcomes in this population, especially in vaccinated children and those affected by new variants of the virus. The spread of the novel coronavirus designated as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) triggered a pneumonia and systemic disease outbreak called Coronavirus disease 2019 (COVID-19) which spread throughout the world in early 2020 .Beyond the acute phase, Long COVID is usually used as a term to describe the persistence or recurrence of health symptoms beyond the acute phase of infection, considered to extend to four weeks ,5. The WDespite the favourable outcomes for children and adolescents during the acute stage of the disease, long-term consequences of the infection have been reported . Lopez-Lwww.rki.de/post-covid-evimaps, accessed on 25 September 2022). Up to November 2021, we found few studies that assessed the long-term course and prognosis of symptoms related to Long COVID in children and adolescents. Most studies focused on the prevalence of individual symptoms . In this systematic review, we aim to describe the findings of studies assessing key outcomes related to wellbeing and recovery in children and adolescents using the evidence derived from our evidence map [Our team has developed an evidence map of observational studies analysing long-term symptoms and sequelae following SARS-CoV-2 infection, which is available on the website of the Robert Koch Institute , including those embedded in randomised controlled trials with 12 or more weeks of follow-up time. We excluded case reports, case series and those only focusing on people with sequelae or persistent symptoms.Type of participants: We included children and adolescents with documented SARS-CoV-2 infection following clinical, imaging or laboratory criteria with an assessment of symptoms or sequelae four weeks after infection, including those with asymptomatic or mildly symptomatic infection.Health-related Quality of Life: including measurements of physical-mental-social functioning Changes in work/occupational and study (school attendance)Survival related to Long COVID Recovery/duration of symptomsNeed for rehabilitation/resource useWe included studies assessing the following outcomes:Timing of the outcomes: Since the acute phase of COVID-19 is usually defined by the first four weeks, persistent symptoms were defined beyond this period up to 12 weeks. Symptoms that might be attributable to post-COVID-19 condition (WHO definition) as a syndrome were included as those lasting 12 weeks or more. These symptoms were further divided into short-term (12 weeks or more up to 6 months), medium-term (more than 6 months up to 12 months) and long-term: more than 12 months.https://covid-19.cochrane.org, accessed on 25 September 2022; comprising PubMed, Embase, Cochrane CENTRAL, ClinicalTrials.gov (accessed on 25 September 2022), WHO International Clinical Trials Registry Platform and medRxiv) and the WHO COVID-19 Global literature on coronavirus disease database . The full methods for the search design and selection process of the evidence map are detailed elsewhere [Two independent researchers (LG and JVAF) identified the studies from our evidence map. To produce this evidence map we performed a comprehensive, systematic search with no restrictions on the language of publication or publication status on November 5th, 2021. Databases searched for the evidence map were the Cochrane-COVID-19 Study Register (lsewhere . We seleWe developed a dedicated data abstraction form that we pilot tested using Google Spreadsheets. Two independent reviewers (LG and GO) extracted outcome data relevant to this review as needed for calculating summary statistics and measures of variance. For prevalence estimates (dichotomous data), we extracted natural frequencies or percentages with confidence intervals when available. We attempted to obtain the means and standard deviations or data necessary to calculate this information for continuous outcomes. We resolved any disagreements by discussion or, if required, by consultation with a third review author (JVAF). We contacted the authors of included studies to obtain key missing data as needed. We did not perform imputations. For studies that fulfil the inclusion criteria, two review authors (LG and GO) independently extracted the following information: bibliographic details, study design, country, setting, age , vaccination status, diagnosis and severity of COVID-19, gender, socioeconomic status, prognostic factors and outcomes relevant to this review.We assessed the risk of bias in each study using the JBI tool for prevalence studies . This toDifferences in the measurements of the outcomes as well as in the clinical features of study populations included in this body of research precluded the conduct of a meta-analysis. Therefore, we described the results narratively (for three of the outcomes) and produced summary tables (for one of the outcomes) with the prevalence estimates for each outcome, including proportions and mean scores . We descWe screened 9768 results from de-duplicated retrieved records to populate our evidence map. After excluding those that focused on adults and those not meeting our eligibility criteria for this review, we identified 25 studies, including over 68 thousand children and adolescents . For those studies with an incomplete description of participants, severity and comorbidities were the most frequent missing details. Most studies analysed the data with sufficient coverage of all identified subgroups. In almost half of the studies, some issues arose related to the methods used to identify the outcomes of interest, although all included studies assessed all participants following the same criteria. None of the studies presented serious concerns related to the statistical analyses as it was mostly simple descriptive statistics . Problems related to loss of follow-up or low response rates were identified in only a minority of studies (See Four studies (two controlled) assessed this outcome ,36,38,44Five studies reported this outcome ,34,36,40One study with 430 mostly non-severe cases, with an estimate of fewer than 58 adolescents (based on the age distribution), highlighted that 57% of all participants presented some restriction in daily activities at a mean follow-up of 176 ± 35.1 days . AnotherTwenty-two studies assessed this outcome. As we found substantial heterogeneity in study design, inclusion criteria, follow-up time and definition of recovery, we summarised the data on the overall duration of symptoms and the lack of recovery following COVID-19  in Four studies reported some data on this outcome. A large controlled study using insurance data with 57,763 children and adolescents reported an incidence rate of 436 new diagnoses/1000 persons-year, following COVID-19 compared to 335 among controls   [One smaller school-based retrospective study included 109 COVID-19 seropositive and 1246 seronegative children and adolescents and highlighted a similar proportion of symptoms lasting >4 and >12 weeks (ailable) .p-value < 0.05) [Two controlled studies presented relevant data but were not disaggregated for the subgroup of children and adolescents. One study reported the persistence of symptoms in a cohort of 293 participants (16 children) and highlighted that those with confirmed infection had a higher persistence of symptoms compared to seronegative exposed controls ( < 0.05) . The oth < 0.05) .The LongCOVIDKidsDK study identified that more female participants had persistent symptoms in the case and in the control group for those 12–18 years, but not for younger age groups  ,30. The One uncontrolled study focusing exclusively on children and including 518 participants performed multivariable logistic regression to investigate the association of demographic characteristics with persistent symptoms . The stuhttps://osf.io/b7dwy/, accessed on 25 September 2022).We identified six ongoing studies. One cohort study aims to describe the follow-up of children presenting at emergency departments at 14 and 90 days ,51. FiveMost of the studies were conducted in high-income countries, and over half (52%) included a case mix of unvaccinated children and adults in the earlier phases of the pandemic. Data on recovery rates are highly heterogeneous, ranging from 54% to 95% at six-month follow-ups and 49% to 92% at 12 months. Most children with persistent symptoms reported mild or no impairment in their quality of life at a 6-month follow-up. Most children with persistent symptoms reported no substantial impairment in their school functioning at 3–6 month follow-ups, although their parents indicated their opinion that their children may be spending less time in school as a consequence of the pandemic rather than the illness itself. Hospital readmission and interventions due to persistent dysphagia following PIMS were the most frequently reported rehabilitation interventions related to resource use. The certainty of the evidence for these outcomes is very limited. Most studies presented some concerns related to study design, including the recruitment of participants, the sample size  and the description of study subjects and setting. Heterogeneity in study populations and how outcomes were measured and reported prevented us from conducting a meta-analysis.Two previous systematic reviews on Long COVID in children and adolescents reported a wide range of symptoms following acute SARS-CoV-2 infection, including respiratory, neurological or cognitive symptoms ,17. In tSeveral large ongoing studies aim to provide insight into the long-term health impacts on children and adolescents following SARS-CoV-2 infection. Implementing follow-up questionnaires, the COVID-19 Schools Infection Survey, England provides regular estimates of the proportions of children and adolescents 3–16 years who have symptoms persisting for at least 12 months after testing positive for SARS-CoV-2 infections well as proportions who are impacted by these symptoms in everyday life . The BriOur findings highlight the need for continued research in order to understand and quantify the health impact of COVID-19 among children and adolescents and to assure appropriate health care and social services. Including control groups is essential in order to identify health impacts directly related to Sar-CoV-2 infection rather than other contextual factors. However, given the increasing numbers of children and adolescents getting infected and a higher chance of asymptomatic or paucisymptomatic COVID-19 among children, in particular small children than among adults, it will be increasingly challenging to choose appropriate control groups. Above all, the choice and definition of health endpoints will need harmonisation, even more so with regard to research on Post-COVID-19 conditions among children and adolescents than among adults .We empirically derived a search strategy with high sensitivity to retrieve reports on post-COVID-19 conditions, but it might be less sensitive for reports focused on more specific sets of complications. Moreover, smaller studies and poorly described ones might not have been picked up by the search. However, many Cochrane reviews used this set of comprehensive resources ,59,60, aThere is limited evidence on the short and long-term well-being of children following SARS-CoV-2 infection. The findings from these first studies indicate that long-term symptoms may be present in children, but health impact estimates were heterogeneous. High-quality longitudinal studies with defined health outcomes and valid control groups are needed to understand and quantify the direct and indirect health impacts of the COVID-19 pandemic on children and adolescents and to provide appropriate health care and counselling services. This is particularly important as original research studies need to cover the later stages of the pandemic following the implementation of vaccination and the emergence of new variants as well as reinfections.


Around half (50.7%) of the selected genes do not have any strong trans-associations and are not detected by standard methods. We provide further evidence for causal basis of the target genes through a series of follow-up analyses. These results show ARCHIE is a powerful tool for identifying sets of genes whose trans-regulation may be related to specific complex traits.Large scale genetic association studies have identified many trait-associated variants and understanding the role of these variants in the downstream regulation of gene-expressions can uncover important mediating biological mechanisms. Here we propose ARCHIE, a summary statistic based sparse canonical correlation analysis method to identify sets of gene-expressions trans-regulated by sets of known trait-related genetic variants. Simulation studies show that compared to standard methods, ARCHIE is better suited to identify “core”-like genes through which effects of many other genes may be mediated and can capture disease-specific patterns of genetic associations. By applying ARCHIE to publicly available summary statistics from the eQTLGen consortium, we identify gene sets which have significant evidence of  Large-scale genetic association studies have identified many trait-associated variants that influence gene expression. Here, the authors present ARCHIE, a tool for identifying sets of genes whose regulation may be related to specific complex traits. It has been shown that these GWAS identified variants have a substantial overlap with expression quantitative trait loci (eQTL) i.e., variants that are associated with the expression levels of genes7. A number of tools11 have been developed to identify potential target genes through which genetic associations may be mediated by investigating the effect of variants on local genes (cis-eQTL), typically within 1 Mb region around the variant, but underlying causal interpretation remains complicated due to linkage disequilibrium and pleiotropy. A recent study has shown that a modest fraction of trait-heritability can be explained cis-mediated bulk gene-expressions12, but future studies with more cell-type specific information has the potential to explain further.Genome-wide association studies (GWAS) have identified tens of thousands of common variants associated with a variety of complex traitscis-eQTL, studies of trans-eQTL have received less attention though they have the potential to illuminate downstream genes and pathways that would shed light on disease mechanism. A major challenge has been the limited statistical power for detection of trans-eQTL effects due to much weaker effects of SNPs on expressions of distal genes compared to those in cis-regions and a very large burden of multiple testing. However, trans-effects, when detected, has been shown to be more likely to have tissue-specific effects14 and are more enriched than cis-eQTLs among disease loci15. Trans-eQTLs are, in general, known to act on regulatory circuits governing broader groups of genes16 and thus have the potential to uncover gene networks and pathways consequential to complex traits18. Limited studies of trans-eQTL effect of known GWAS loci have identified complex downstream effects on known consequential genes for diseases19. In fact, an “omnigenic” model of complex traits has been hypothesized under which a large majority of genetic associations is mediated by cascading trans-effects on a few “core genes”21. Thus, given the increasing scope of eQTL studies, it has become even more important to comprehensively identify trait-specific trans-associations to highlight biological processes and mechanisms underlying phenotypic change. However, to the best of our knowledge, no framework has been developed to detect such trait-specific trans-association patterns and sets of trait-relevant genetically regulated genes, specifically leveraging summary statistics from transcriptomic studies, which are more readily available than individual-level genotype data.Compared to 27 framework, termed Aggregative tRans assoCiation to detect pHenotype specIfic gEne-sets (ARCHIE), which identifies sets of distal genes whose expression levels are trans-associated to (or regulated by) groups of GWAS SNPs associated to a trait. The method requires summary statistics from standard SNP-gene expression trans-eQTL mapping, estimates of linkage disequilibrium (LD) between the variants and co-expression between genes, which can be estimated using publicly available datasets. Together, the selected variants and genes (jointly termed ARCHIE components) reflect significant trait-specific patterns of trans-association trans-association summary statistics for SNPs associated with 29 traits reported by the eQTLGen consortium19. The results show that ARCHIE can identify trait-specific patterns of trans-associations and relevant sets of variants and co-regulated target genes. The majority (50.7%) of the target genes we detect, are novel, meaning they would not have been identified by standard trans-eQTL mapping alone. We provide independent evidence supporting our results, using a series of downstream analysis to show that the selected target genes are enriched in known trait-related pathways and define directions of associations for the SNPs that are more enriched for underlying trait heritability than expected by chance. The proposed methods can be further applied in the future to association statistics data on other types of high throughput molecular traits, such as proteins and metabolites, to understand their mediating role in genetic architecture of complex trait.Here we apply the proposed method to analyze large-scale Z values and p values) available for a set of variants identified through large-scale GWAS of a given trait of interest, from standard trans-eQTL analysis across large number of distal genes. We further assume that we have additional reference datasets to estimate correlation (linkage disequilibrium) among the SNPs and among gene expressions in the underlying population of interest. ARCHIE uses these datasets to employ a sCCAs24 which produces sparse linear combinations of the trait-related variants (termed variant-component) that is associated with sparse linear combination of genes (termed gene-component) where each non-zero element of the variant (or gene)-component indicates that the respective variant (or gene) is selected. . Subsequently, we found that at approximately level 9 × 10−04, ARCHIE achieves similar type-I error rate as the FWER of trans-eQTL mapping at level 1  × 10−06. We use these levels for subsequent empirical power estimations.We first compare ARCHIE with standard trans analysis using a series of simulation studies. ARCHIE addresses a composite null hypothesis that there is no association between a group of variants and genes, while standard trans-eQTL mapping tests a series of individual null hypotheses of no association across variant-gene pairs. Thus, these two methods are not directly comparable in terms of power unless the type-I error rates are calibrated with respect to a common benchmark. We first evaluate the type-I error rate for each method under a global null hypothesis of no association between a group of SNPs and a gene network Fig. . Type-I te Table  at all lN = 1000 and 30,000) reflecting the approximate sample sizes of GTEx v8 and eQTLGen studies respectively.We next compared the power of ARCHIE to identify gene-sets associated to trait-related SNPs in comparison to standard trans-eQTL mapping, for several different causal models . For a given trait, we used summary statistics for 100 variants across 5000 genes of which a certain proportion (δ) of the variants were related to the given trait and rest were variants randomly sampled from different traits reflecting the general background of trans-associations expected for GWAS variants , consistently across the four different traits. In particular, at δ = 0, the results would correspond to the type-I error of ARCHIE under the competitive null hypothesis. We found that at a level of 1 × 10−04, the competitive type-I error of ARCHIE is conservative for all the traits under consideration indicating that ARCHIE produces reduced false positives  from standard trans-eQTL mapping for more than 10,000 variants across numerous loci in the genome, curated from external databases, that have been identified to be associated to traits and diseases in large scale GWAS. For each of the 29 traits, we extracted the trans-association summary statistics for the variants associated with the trait and only the genes that were either more 5 Mb away from each of the variants or on a different chromosome. Thus, by design, all the genes in the analysis for a trait were distal to all the variants under consideration. We then applied ARCHIE to this trans-eQTL summary statistics and selected the trait-specific target genes via the significant gene components  with any variant associated to that traits. The remaining 50.7% genes (termed “novel genes”) harbors only weaker  associations and hence cannot be detected by standard trans-eQTL mapping alone; these genes display a similar pattern of trans-association with corresponding selected trait-related variants and are detectable only via the significant ARCHIE components. In fact, a large majority of the novel genes (89%) harbor multiple weaker trans-associations with the variants selected in the variant component. We made the list of target genes and variants selected by ARCHIE for each phenotype publicly available through an openly accessible database (https://github.com/diptavo/ARCHIE). Here, we focus on results for three different phenotypes, reflecting three different classes of diseases, their corresponding trans-association patterns, the selected target gene-sets, and the novel genes detected by ARCHIE  across 7,756 genes. Of these, 7047 genes were expressed in whole blood of Genotype-Tissue Expression (GTEx)28 v8 individuals. Testing against the competitive null hypothesis (See previous section), we identified one significant ARCHIE component capturing trans-association patterns significantly related to SCZ  and possibly multiple weaker  association as reported by eQTLGen. The remaining 59 genes (78.6%) only had weaker trans-associations with SCZ-related variants (Table trans-eQTL mapping and were not reported as significant findings by the eQTLGen consortium. We further investigated the robustness of the results using coexpression estimates from gene expressions levels reported in Whole Blood by the Depression Genes and network (DGN) study29. The results show that out of 75 genes and 59 novel genes identified by ARCHIE, 62 and 45 were also selected in the replication analysis using data from DGN, demonstrating the potential robustness of the results by ARCHIE.Schizophrenia is a neuropsychiatric disorder that affects perception and cognition. The eQTLGen consortium reports complete (non-missing) SCZ Fig.  consistip value < 0.001) than expected by chance , we estimated the approximate heritability of SCZ explained by the trans-associations between the selected genes and variants, using individual level data from UK Biobank. We compared the estimate to (1) the expected distribution of heritability explained by the selected SNPs and randomly chosen genes (excluding the selected genes) for SCZ and (2) the expected distribution of heritability explained by the selected SNPs and genes for a randomly chosen trait. The results showed that the selected SNPs and genes are significantly enriched in trait heritability , a gene that underlies interneuron migration and several neurodegenerative diseases30, was identified by aggregating weaker associations from 20 SCZ-related SNPs in the variant component, but does not have any significant trans-associations. Similarly, caveolin-1 (CAV1), which is a known regulator of a SCZ risk gene (DISC1)31, aggregates 13 weaker association to SCZ-related variants in the variant component. Notably, the target genes identified by ARCHIE include genes such as HSPA5 and AP5S1, which not only harbor multiple trans-associations from SCZ-related variants but have also been reported to have cis-variants associated with psychiatric disorders33. We investigated whether in general the genes selected by ARCHIE had have evidence of association with SCZ through cis variants. Aggregating results from several large-scale cis-eQTL studies across tissues34, we found that 12 of the 59 of the  novel genes have nominally significant  evidence of cis-regulatory SNPs to be associated with SCZ or other different neuropsychiatric diseases. Further, eQTLGen consortium reports the association of gene expressions with several publicly available polygenic risk scores (PRS) of SCZ at different p value thresholds, curated from external databases. We found that out of the 59 novel genes identified, 38 (64.4%) had a nominal association with at least one reported PRS for SCZ.Several of the 59 identified novel genes have previously been reported to be associated with neurological functions. For example, chemokine receptor 4 (35 (https://fuma.ctglab.nl) and ShinyGO36 (http://bioinformatics.sdstate.edu/go), we investigated if the selected genes represented known SCZ-related biological mechanisms (See Methods for details). Among the significantly enriched pathways, the majority (51.3%) were immune related. In particular, we identified 42 GO pathways37, 36 canonical pathways41 and 4 hallmark pathways42 to be strongly enriched  for the selected genes with 73 (89.0%) of them containing at least one novel gene  for the selected genes , GO-055065 metal ion homeostasis44 , GO-0006915: apoptotic process45 , GO-0043005: neuron projection  have previously been suggested to be linked to SCZ  for targets of ten TFs  indicating that the corresponding proteins may physically interact. Although the analysis is performed using trans-associations in whole blood, we found several of the selected target genes were differentially expressed in tissues directly related with SCZ. For example, three novel genes , were highly upregulated in several brain tissues  enrichment analysis using STRING (v11.0)61. The eQTLGen consortium reports complete (non-missing) trans-association summary statistics for 163 SNPs associated with Ulcerative Colitis, curated from multiple large-scale GWAS, across 12,010 genes. Of these, 10,307 genes were expressed in Whole Blood from GTEx v8 individuals. Using ARCHIE, we detected two significant variant-gene components comprising of 74 SNPs and 148 genes in total  were novel, meaning they did not have any strong trans-association  for heritability of UC than expected by chance alone and also the selected trans-associations explained heritability of UC more than expected for a random trait or disease  is a form of inflammatory bowel disease, affecting the innermost lining of colon and rectum, causing inflammation and sores in the digestive tract and can lead to several colon-related symptoms and complications including colon cancerase Fig. .Fig. 4AnGPA33) is known to impact intestinal permeability62 and is an established colon cancer antigen63. Recent research using mouse models have reported a connection between the regulation of GPA33 and the development of colitis and other colon-related inflammatory syndromes64. We also identify spermine oxidase (SMOX) through its weaker association with 9 UC-related variants. SMOX is significantly upregulated in individuals with inflammatory bowel diseases65 and has been implicated in gastric and colon inflammations as well as carcinogenesis66.Several of the novel target genes detected have been previously linked to intestinal inflammations and diseases. For example, glycoprotein A33  for the selected target genes  has previously been reported to be associated to development of UC via suppression of immune response67. Various GO pathways related to endocytosis, lymphocyte activation, T-cell activation are found to be overrepresented in the selected target genes as well  for targets of 18 different TFs, majority of which have been previously reported to be involved in mucosal inflammation, inflammation of the intestine and epithelial cells and in immune-related responses   Fig. . Additiocis mediation15. In one example from our analysis, we observe that, among the 41 variants selected by variant-component 1, one UC-related variant rs3774959 is a cis-eQTL of NFKB1 . The Nuclear factor κB (NF-κB) family of transcription factors (TF) including NFKB1, has been extensively reported to be involved in immune68 and inflammatory responses69. In particular, mutations in the promoter region of Nuclear factor κB1 (NFKB1) have been strongly implicated to be associated with UC70, although the downstream target genes of NFKB1 that are associated with UC, are largely unknown. Among 106 target genes selected in the first gene component, there are 6 genes  that are reported targets for NFKB1  in TRRUST v2.050. Thus, it can be conceptualized that the selected UC-related variant may regulate the expression levels of the six selected targets of NFKB1 via cis-regulation of NFKB1 expression levels, influencing UC-status downstream.We further investigated if any known mechanism can explain how the selected genes are associated with the selected variants, including mechanisms reflecting trans-association summary statistics for 122 SNPs associated with prostate cancer, curated from multiple large-scale GWAS, across 12,951 genes. Of these, 11,385 genes were expressed in Whole Blood from GTEx v8 individuals. Using ARCHIE, we detected two significant variant-gene components comprising 33 SNPs, spanning 14 different loci across the genome, and 53 genes in total  were novel .Prostate cancer (PC) is one of the most common types of cancers in middle-aged and older men, having a high public health burden with more than 3 million new cases in USA per year. The eQTLGen consortium reports complete (non-missing) el Table , but theTP53 aggregates weaker trans-associations with 9 PC-related variants in ARCHIE component 1  and mRNA 3’-UTR binding . Notably, we found the selected genes to be enriched for targets of several transcription factors many of which have been associated with different types and subtypes of cancer  which has been identified to be associated with breast cancer, ovarian cancer, colorectal cancer and others76. We also found enrichment for targets of SSRP1 , which is differentially regulated in a wide spectrum of malignant tumors77 along with enrichment for targets of MYC and TP73 which are well-established cancer-related genes  enriched for the selected genes are immune related as observed in the previous examples as well. This might have been driven by the fact that eQTLGen reports summary trans-associations with groups of coregulated genes. ARCHIE involves estimating LD and coexpression from publicly available reference datasets. However, we found that the results can be reasonably robust to the choice of reference data and estimation errors arising due to sampling variations  and key tumor suppressor genes (PC). Further, using a trans-expression imputation approach, we demonstrated that the selected genes can significantly mediate heritability-associated trait-related variants. All of these analyses point out that the trans-association patterns we detect are likely to have trait-specific biological basis.Application of the method to eQTLGen consortium trans-heritability analysis. However, it is likely that incorporation of the GWAS effect sizes  of trait association for the SNPs in the sCCA itself will lead to improved power for detection of the trait-specific target genes. One of the immediate ways to incorporating that might be to weight the variants with weights proportional to squared  of the GWAS effect size. However, incorporating the direction of the GWAS effect with the sCCA framework remains an interesting problem and merits further research. Additionally, incorporation of information on cis-genes and known functional annotation of genetic variants can improve the power of the analysis as well.There are several limitations of the proposed method and current analysis. First, in the current version ARCHIE, we begin with a set of genetic variants associated with a trait, but we do not incorporate the underlying association directions and effect sizes in the analysis. This approach allowed us to independently investigate identified target genes through testing for consistency of directions of association of the SNPs with the trait and those with the expressions of the target through the 78. In fact, in presence of correlation between SNPs as well gene expressions, elastic net regularization approach can be effective in accurate selection. In the future, usefulness of alternative types of penalty functions in selecting the genes and variants merits further research. Further, due to the lack of existing methods to identify trait-specific trans associations, we have compared the performance against the standard trans-eQTL mapping. Although the eQTLGen data analysis shows that ARCHIE can identify a broader range of gene-sets trans-regulated by GWAS variants as compared to standard trans-eQTL mapping, the goals of ARCHIE and the standard analysis are different and therefore not directly comparable—the goal of the standard analysis is to identify association in individual variant-gene pair irrespective of trait specificity. In contrast, ARCHIE can identify trait-specific trans-regulated target genes harboring multiple weaker associations with trait-related variants. As newer methods are developed to detect trait-relevant trans-regulated genes, more comprehensive analyses comparing alternative approaches will allow us to understand their benefits and limitations.Although the estimation of the ARCHIE components is computationally efficient, in the current implementation, the resampling-based testing method is computationally intensive. In the future, further research is merited to develop analytical approximation techniques to reduce the computational burden of ARCHIE. Since variable selection is a major goal in ARCHIE, we have introduced regularization via the L1 penalty due to its proven theoretical selection consistencyCurrently, not many transcriptomic studies have made the summary statistic from trans-eQTL mapping available. However, as these studies grow in size and with improved methods of data sharing, we expect more studies to make the summary statistics from trans-eQTL mapping available, allowing researchers to investigate a broader range of diseases and traits. Further, ARCHIE can be broadly applicable to understand role of other types of molecular traits, such as proteins and metabolites, in mediating complex trait genetic associations. In the future, as data on molecular biomarkers become increasingly available in large biobanks, tools like ARCHIE will be increasingly needed to understand common pathways through which genes and biomarkers interact to cause specific diseases.trans-association patterns that will be present only in specific disease-relevant tissues, cell types or/and dynamic stages79. In the future, we will seek applications for ARCHIE in various types of emerging eQTL databases to provide a more complete map of networks of genetic variants and trans-regulated gene expressions and relevant contexts.In this article, we have analyzed summary statistics reported by eQTLGen in the whole blood. This is primarily because of the substantial effective sample size of eQTLGen. While the approach can be applied to eQTL results from other tissues, the underlying sample sizes may be too limited to yield sufficient power. Although blood might not be the most relevant tissue for a number of traits, our analyses did detect trans association patterns that appear to have a broader biological basis in the disease genetics, from multiple independent lines of evidence. Nevertheless, it is likely that our analysis has missed many trans-associations from multiple trait-related variants. ARCHIE is a powerful tool for identifying target gene sets through which the effect of genetic variants on a complex trait may be mediated. In the future, applications of the methods to a variety of existing and new data on association between genetic variants with high-throughput molecular traits can provide insights to biological mechanisms underlying genetic basis of complex traits.In summary, in this article we have developed a summary-based method, ARCHIE, to detect trait-specific gene-sets by aggregating 19 is a large-scale multi-study effort to identify to study the downstream effects of trait-related variants via their effects on gene expression in whole blood. The consortium consists of 37 individual studies with a collective sample size of 31,684 participants. With this sample size, the study has relatively higher power to detect moderate to weaker effects of variants on gene expression. 10,317 variants related to complex traits, compiled from several GWAS databases, were tested for trans-associations with the expression levels of 19,964 genes in whole blood. The authors have made summary statistics  for these trans-eQTL mapping analyses freely available to public.eQTLGen: The eQTLGen consortium28 aims to study tissue-specific gene expression and regulation. We used individual-level data from GTEx (v8) whole blood to construct the co-expression matrix (GTEx: The Genotype-Tissue Expression (GTEx) projectUK Biobank: UK Biobank is a large biobank study with above 500,000 participants. Among several data resources available, the genotype data, hospitalization records, and health-records data are available. We used individual-level genotype data from UK Biobank to construct LD matrix (trans-heritability explained by the selected target genes (See Methods). We included the individuals with European ancestry in the analysis. For example, in the analysis of SCZ, we used a sample of 366,326 participants from UK Biobank to construct the imputed gene-expression levels and evaluate the corresponding regression r2 as an estimate of trans-heritability on SCZ as a binary phenotype.The phenotype data constructed from hospitalization and health-data records were used in the quantification and testing of enrichment in p variants and h norm of a vector x; 1 penalty. The subsequent pairs of sCCA components are obtained by similarly maximizing the correlation between Our proposed method, Aggregative trans association to detect phenotype-specific gene-sets (ARCHIE), can select target genes trans-associated with trait-related variants using summary statistics in a sparse canonical correlation framework. To introduce the details of ARCHIE, first, we briefly describe sCCA. For n individuals, let mentclass2pt{minimmentclass2pt{minimtrans-eQTL mapping . Given the trans-association summary statistics across the variants related with the trait and all the corresponding distant genes (variant > 5 Mb away from the transcription start site of the gene), we first adjust for the correlation within the variants and genes through appropriate LD and co-expression matrices, estimated from reference panels with individual-level genotype (or dosage) and gene expression data respectively, as follows:Z values from the standard trans-eQTL mapping across all pairs of variants and gene-expressions. It is important to adjust for the dependence within the variants and gene expression levels using the LD and co-expression matrices respectively, since we aim to identify trait-relevant gene-sets and variants through independent trans-associations. Furthermore, gene-expression levels in bulk tissues may appear to be correlated due to cell composition effects as well, which needs to be adjusted for by incorporating the estimated co-expression between the genes.To apply ARCHIE, thus, we start with the summary statistics from mentclass2pt{minim24 which produces a sparse linear combination of the variants , which may bias the competitive null distribution towards the trans-association cc-values for that phenotype.Then we use ARCHIE with the same sparsity levels as the original analysis, to extract the gene and variant components and calculate corresponding cc values. We repeat this step multiple (M) times to generate a competitive null distribution of cc values. We then evaluate the observed cc-values from the original analysis against the corresponding competitive null distributions to calculate the p-value. In particular, the N individuals . First, we randomly sampled N unrelated individuals with genotypes at 50 independent and randomly selected SNPs from the UK Biobank with minor allele frequencies ranging from 10 to 40%. The SNPs were then arranged in sets of five each. Each set of five SNPs was then used to simulate the cis-gene expression of eight genes in total marked in red . Any ARCHIE component with p-value less than the chosen level was declared significant and the corresponding selected genes were noted  across for p variants and g genes. Out of the p variants, we set δ proportion of them to be related to a particular trait. For high values of δ we expect that the trans-summary statistics matrix would reflect trans-association patterns pertaining to the trait and hence should be captured by the ARCHIE components. We then applied ARCHIE on this matrix and evaluate the significance of the components. We replicate this experiment multiple times in a given setting, to estimate the empirical probability of at least one ARCHIE component to be significant . In line with the consortium, we defined any gene to be trans to a variant if the variant was located at least 5 Mb from the transcription start site of the gene or on another chromosome. The data contains multiple variants associated with the same trait analyzed for trans-eQTL mapping. Our analysis was restricted to phenotypes that had at least 100 associated variants tested for trans-mapping in the consortium, producing 29 phenotypes. Figure trans-eQTL association statistics  and removed all genes that were in within 5 Mb of any of the trait-related variants. In the preprocessing step, we filtered for any missing data and retained the genes that were also expressed in GTEx (v8)28 whole blood. This produced a list of approximately 129  variants and 10,219  genes on an average per phenotype. ARCHIE requires two additional matrices representing the correlation among the variants themselves (a linkage-disequilibrium matrix) and among the gene-expression levels (a co-expression matrix), which can be estimated using reference data. We constructed the LD-matrix for the variants from individual-level genotype information using 5,000 randomly selected, unrelated European samples in UK Biobank80. For the correlation between gene-expressions, we used a penalized co-expression matrix81 of the corresponding genes constructed from the covariate-adjusted quantile normalized gene-expression levels for individuals in GTEx v8 data. Subsequently, for the given trait, we extracted the selected variants and genes using the significant components and were evaluated for presence of false-positives due to cross-mapping.To identify phenotype-specific trans-eQTL associations82. With the selected ARCHIE components, we extracted the nearby genes expressed in GTEx v8 whole blood for the selected variants (TSS within ±500 kb of the variant) and evaluated the cross-mapping scores for these genes with the selected target genes. Across the 3 traits analyzed in this article, we found that all such gene pairs were mostly non cross-mappable , indicating that the trans-association patterns were less likely to be affected by false positive arising from alignment errors.Alignment errors due to similarity in sequenced reads can lead to a substantial rise in false positives for detecting Z scores for regression-based trans-eQTL mapping for a set of underlying p variants and g genes. We will assume that, using ARCHIE, we have identified G target genes that capture trait-specific trans-association patterns. To perform the test as proposed above, we require individual-level phenotype and genotype data independent of the samples used in the original analysis. Given genotypes (or dosages) at the p variant sites for an individual k, for each target gene, we define the trans-imputed expression scores (TIES) as the predicted expression value for the jth target gene asith trait-related variant on the jth gene, kth individual at the ith variant and ith variant. We construct the TIES under two different schemes:trans-association statistics reported in eQTLGen.Using all the trait-related variants with complete Using only the trait-related variants selected in the significant components.In the following, we propose a method for quantifying trait heritability explained by the GWAS variants that would be mediated by the identified target-genes and develop a corresponding test for enrichment through comparison of such estimates of mediated heritability associated with that from random genes. For or a particular trait of interest, we start with the 2 from this regression model as a measure of association between the TIES and the phenotype value. The pseudo-r2 would provide an estimate of trans-heritability, meaning it can quantify the variance explained by the trait-related variants that is expected to be mediated via the selected target genes in the context of the trans-associations reported. To test whether the observed r2 is significant in comparison to what is expected at random, we adopt a resampling-based approach. We sampled g genes  from the genome, constructed the corresponding TIES for the individuals and recorded the r2 for the regression model. This would a null estimate of trans-heritability of the SNPs expected to be mediated by a set of g genes. If the observed pseudo-r2 is substantially higher than the null estimates, we can infer that the trans-associations selected by ARCHIE explain higher variance compared to that expected through random trans-associations. We performed resampling multiple (1000) times to generate a control (null) distribution of r2 to reflect the associations expected from random genes. We then calculated the p value of the observed r2 using the originally selected g genes from this control distribution to evaluate whether the TIES have any significant association with the phenotype.To evaluate how strongly the TIES for the G target genes are associated with the phenotype levels, we use the following multiple regression model2 reflects the proportion of trait-variance explained by the TIES. Thus, a significantly higher r2 would imply that the selected genes harbor several trans-associations and mediate the effects of the trait-related variants more than any random set of genes. As the analysis of association between TIES and trait (for both the selected trans genes and random genes) is performed in an independent dataset, and no information on directions or magnitudes of trait association for the SNPs are used in the original ARCHIE analysis, the test for heritability enrichment provides independent validation of the relevance of selected target-genes in explaining genetic associations for the trait. In our application, we used individual-level phenotype and genotype data from UK Biobank participants to estimate association between TIES and traits.Approximately, the observed rWe also performed several other follow-up analyses including PPI enrichment, pathway enrichment, and differentially expressed genes enrichment. These analyses were carried out using pre-established standard pipelines. For full details on these see Supplementary Note Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1-10Reporting Summary


Nations thorughout the world are imposing mandatory quarantine on those entering the country. While such measures may be effective in reducing the importation of COVID-19, the mental health implications remain unclear.This study sought to assess mental well-being and factors associated with changes in mental health in individuals subject to mandatory quarantine following travel.Travellers arriving at a large urban international airport completed online questionnaires on arrival and days 7 and 14 of mandated quarantine. Questionnaire items such as travel history, mental health, attitudes towards COVID-19, and protection behaviours were drawn from the World Health Organization Survey Tool for COVID-19.There was a clinically significant decline in mental health over the course of quarantine among the 10,965 eligible participants. Poor mental health was reported by 5.1% of participants on arrival and 26% on day 7 of quarantine. Factors associated with greater decline in mental health were younger age, female gender, negative views towards quarantine measures, and engaging in fewer COVID-19 prevention behaviours.While the widespread use of quarantine may be effective in limiting the spread of COVID-19, the mental health implications are profound and have largely been ignored in public policy decision-making. Psychiatry has a role to play in contributing to the public policy debate to ensure that all aspects of health and well-being are reflected in decisions to isolate people from others.No significant relationships.


Resonator networks are available to fulfill these promises in multiple platforms using evanescent field coupling between modes of individual high-Q resonators; for example, whispering gallery modes in optical resonators5, photonic crystal cavities8, or femtosecond-laser-written waveguide arrays11. Beyond integrated photonic technologies, perfect quantum state transfer has been discussed in coupled cavity resonator arrays loaded with three-level atoms12, integrated superconducting technologies14, quantum transducers integrating superconducting15 or optical16 systems to mechanical resonators, Rydberg atoms in a lattice17, as well as the coupling of distant resonators via photonic waveguides20.Integrated photonic quantum technologies22 and entangled23 photonic qubits was demonstrated using an array of eleven coupled waveguides. Recent advances on reconfigurable nanoelectronical networks allowed perfect coherent transfer on-chip24. On the theoretical side, there is a recent report of quantum state transfer of two-photon Fock and NOON states but not of squeezed and coherent states for twenty coupled waveguides25. The latter left the question on the feasibility of nonclassical state transfer in such resonator networks open and we aim to answer it.Here, we focus on the transfer of non-classical states of light required, for example, by switching and routing. We draw inspiration from the so-called su(2) symmetry, that includes the so-called In the following, we deal with the idea of normal modes for a completely connected resonator network described by an auxiliary Hermitian coupling matrix feasible of unitary diagonalization. Then, we explore the idea of quantum state transfer in such coupled resonator network to show that it reduces to find the structure of an auxiliary evolution matrix given in terms of orthogonal polynomials. In order to provide a particular example, we study the quantum analog of photonic lattice with an underlying 26 of a Heisenberg picture method proposed by one of us27. Let us start from a completely connected resonator network,28 as well as some penta-diagonal symmetric matrices29. On a case-by-case basis, this may extend to block tri- and penta-diagonal matrices. In these and other cases, there exists a unitary matrix transformation We follow a Schrödinger picture equivalentm-th resonator,n-th resonator at some given transfer time For the sake of providing a practical example, let us focus on transferring an arbitrary initial state at the m-th to the n-th resonator reduces to find a unitary decomposition of the auxiliary Hermitian matrix, whenever it is possible, calculate the auxiliary evolution matrix elements, and find a transfer time At this point, our problem of transferring the localized initial state from the su(2) symmetry that provides coherent transfer of classical light and single-photon states30. In the quantum regime, it has been used to discuss the idea of synthetic dimensions31. We will work with a general form32 that translates into a coupled resonator network with the following Hamiltonian26,m-th resonator are su(2) symmetry with Bargmann parameter su(2) algebra33,34. Again, we must be cautious with the ramifications of the Wei–Norman decomposition parameter In photonics, there exists a well-known photonic lattice with an underlying The Wei–Norman decomposition parameters, Something interesting happens at half the reconstruction time,m-th resonator into the In the classical and single-excitation quantum regime, the so-called mentclass2pt{minimmentclass2pt{minimWe showed that a normal modes approach provides a tractable framework for propagation of non-classical light in networks of coupled resonators. Our approach yields propagation in terms of orthogonal polynomials that provide further insight into the dynamics in the network whenever it is possible to use a unitary diagonalization of the auxiliary Hermitian coupling matrix of the system; for example, resonator networks described by auxiliary tri-diagonal Hermitian coupling matrices and some penta-diagonal real symmetric coupling matrices.su(2) symmetry and were able to identify that it provides full quantum state reconstruction; that is, state transfer to the same initial site. In addition, we showed that it offers both partial and full reconstruction at half the full reconstruction time. In the case of partial reconstruction, the Fock state probability distribution is recovered but the amplitudes for odd Fock state components show a In particular, we studied a network with underlying m-th and the We also explored quantum state transfer in the equivalent of the so-called Our normal modes approach makes it straightforward to calculate correlations and other quantum quantities of interest in terms of orthogonal polynomials whenever a unitary diagonalization for the auxiliary Hermitian matrix exists.


It is difficult to determine the actual sensitivity and specificity of the serological assay kits. For the purposes of our study, the specificities of two kits were presumed to be 100%, though the actual specificities might be lower. Additionally, the sensitivities of the combined results were not determined. Therefore, we cannot exclude the possibility of contamination by false-positive and false-negative results. As Lyu et al mentioned, our results must be interpreted with care. Optimization of cutoff values and/or the combined use with other antibody detection kits is expected to provide greater reliability for the results.2,3 the antibody kinetics against each SARS-CoV-2 protein in asymptomatic infections remain poorly understood. Lei et al reported that the antibody levels in asymptomatic infections were lower than those in symptomatic infections, with the immunoglobulin G (IgG) response declining or becoming undetectable in 2 months.4 Single positive cases may reflect low antibody levels. Further analysis of the antibody kinetics in asymptomatic infections will be needed. Lyu et al also mentioned possible contamination resulting from vaccinated participants. Notably, our study assessed the seroprevalence of anti–SARS-CoV-2 IgG among 23,234 visitors to Tokyo-area hospitals during the interval from September 2020 through March 2021. According to the Tokyo Metropolitan Government, as of April 12, 2021, only 418 residents of Tokyo had been vaccinated.5 We, therefore, believe vaccination would have little influence on seroprevalence in our study.The second issue addressed in the letter is the single positive samples. To detect anti-SARS-CoV-2 antibodies, we used two kits and combined the results. Although the antibody kinetics in symptomatic patients with coronavirus disease 2019 (COVID-19) have been reported in several studies,The third issue addressed in the letter is the method used to detect anti-N protein antibody. We used an iFlash 3000 chemiluminescence immunoassay analyzer  with an iFlash-SARS-CoV-2 IgG-N kit (YHLO Biotech).We thank the authors of this letter for their comments and suggestions.


This study estimates the point prevalence of symptomatic respiratory tract infections (RTIs) among returned Hajj pilgrims and their contacts in 2021. Using the computer-assisted telephone interview (CATI) technique, domestic pilgrims were invited to participate in this cross-sectional survey two weeks after their home return from Hajj. Of 600 pilgrims approached, 79.3% agreed to participate and completed the survey. Syndromic definitions were used to clinically diagnose possible influenza-like illnesses (ILI) and COVID-19. Median with range was applied to summarise the continuous data, and frequencies and proportions were used to present the categorical variables. Simple logistic regression was carried out to assess the correlations of potential factors with the prevalence of RTIs. The majority of pilgrims (88.7%) reported receiving at least two doses of the COVID-19 vaccine before Hajj. Eleven (2.3%) pilgrims reported respiratory symptoms with the estimated prevalence of possible ILI being 0.2%, and of possible COVID-19 being 0.4%. Among those who were symptomatic, five (45.5%) reported that one or more of their close contacts had developed similar RTI symptoms after the pilgrims’ home return. The prevalence of RTIs among pilgrims who returned home after attending the Hajj 2021 was lower compared with those reported in the pre-pandemic studies; however, the risk of spread of infection among contacts following Hajj is still a concern. The Hajj pilgrimage is an annual religious mass gathering (MG) event in Makkah city, Kingdom of Saudi Arabia (KSA). During a pre-pandemic year, up to 3 million Muslims spent extended time at multiple sites in Makkah to perform various religious rituals . This HaSeveral respiratory viruses have emerged over the last several years such as Middle East respiratory syndrome coronavirus (MERS-CoV), which posed a great public health threat to the Hajj pilgrimage; nevertheless, no major outbreaks have been documented to date . For insHajj and other MGs not only amplify the risk of infections among participants but also accelerate the transmission of these infections among their close contacts, making MGs a significant risk to global health security ,12. OnlyData are scarce on the post-Hajj prevalence of RTIs, and to the best of our knowledge, no study has assessed the prevalence of symptomatic RTIs among pilgrims during Hajj amidst the COVID-19 pandemic. A study conducted by our team during the peak period of Hajj in 2021 demonstrates the need for a post-Hajj surveillance study . To thisA cross-sectional study was conducted among returning domestic pilgrims  who attended Hajj (18–23 July) in the year 2021. Pilgrims of both genders aged 18 years or above were eligible to participate in the survey, while those who declined to participate or did not speak Arabic or English were excluded. Ethics approval was obtained from the Biomedical Ethics Committee of Umm Al-Qura University, Makkah, KSA (reference number: HAPO-02-K-012-2021-07-708), and all participants provided voluntary informed consent.The survey was conducted using computer-assisted telephone interviews (CATI). A convenience sample of 600 pilgrims was contacted by phone from a list provided by Hajj hamlahs  two weeks after their return from Hajj. Pilgrims who responded to the calls were interviewed using a structured questionnaire, and their responses were recorded in Microsoft Forms, an electronic survey development cloud-based software . The questionnaire consisted of two major parts: part one captured data about participants’ non-identifiable socio-demographics including the presence of pre-existing comorbidities, smoking status, and receipts of influenza and COVID-19 vaccinations, while part two focused on collecting information related to the occurrence of respiratory symptoms and some constitutional or gastrointestinal symptoms. In addition, pilgrims who developed symptoms were asked about the onset date and duration of these symptoms, their history of contact with room-/tent-mates suffering from RTIs during Hajj and whether any of their close contacts at home or work who were not pilgrims developed similar symptoms following any pilgrims’ home return.A case of RTI was clinically defined as any pilgrim who reported having at least one respiratory symptom, including cough, sore throat, rhinitis, dyspnoea or smell or taste dysfunction at any time between the conclusion of Hajj rituals and the day of recruitment . The syndromic definitions for possible ILI and possible COVID-19 were applied according to criteria described and assessed elsewhere ,17. We up-value less than 0.05 was considered statistically significant.The recorded data were analysed using Statistical Package for Social Sciences (SPSS) . For the descriptive analysis, the median with range was applied to summarise the continuous data, whereas frequencies and proportions were used to present the categorical variables as well as the prevalence of RTIs and possible ILI and possible COVID-19. The correlation of potential factors with the estimated prevalence of RTIs was carried out using simple logistic regression. The potential factors were chosen by reviewing the literature on respiratory infections and COVID-19 in Hajj and community settings ,19. The A total of 600 pilgrims were invited to participate, of whom 476 (79.3%) agreed to participate and completed the survey. The median age of the pilgrims was 50 (range 18–70) years . About oEleven pilgrims (2.3%) reported developing one or more RTI symptoms two weeks after Hajj; of those, nine (81.8%) had rhinitis, six (54.5%) had a cough, five (45.5%) had a sore throat and two (18.2%) had dyspnoea with smell or taste dysfunction. p > 0.05).Only one participant met the definition of possible ILI, which translates to an estimated prevalence of 0.2%, and two met the definition of possible COVID-19, translating to an estimated prevalence of 0.4%; both of them were fully vaccinated and had no known history of COVID-19. In a simple logistic regression analysis, as illustrated in This study estimated the post-Hajj prevalence of symptomatic RTIs among pilgrims who attended the 2021 Hajj in the midst of the ongoing COVID-19 pandemic. The current study identified a low prevalence of possible ILI and possible COVID-19 among pilgrims. Rhinitis, cough and sore throat were the commonest clinical respiratory symptoms presented in our survey, and this was similar to what previous studies documented among Hajj pilgrims ,5,20. HoAlmost half of the symptomatic pilgrims in this survey reported that one or more of their close contacts had developed similar RTI symptoms, which represents a high risk of contact transmission following Hajj. Previous studies that employed molecular diagnosis also showed the role of contact in the propagation of respiratory viral infection among Hajj pilgrims. For instance, a cross-sectional study involving UK pilgrims conducted in 2005 showed that 58% of pilgrims had a history of contact with ill pilgrims, and the frequency of influenza diagnosis was 17% among those who reported contact with URTI compared with 9% who reported no contact . In a coA significant proportion of pilgrims carry respiratory viruses and remain symptomatic upon their arrival to their home countries from Hajj, as was evidenced in several laboratory-based surveillance studies. In 2013, a study conducted among returning Hajj pilgrims at Kotoka International Airport in Ghana concluded that of all participants, 651 (77.6%) had respiratory symptoms, and 179 (21.3%) tested positive for one or more respiratory viruses . In anotThe overall prevalence of RTIs among pilgrims in this Hajj season was markedly lower in comparison with the data reported in the pre-pandemic studies. For instance, a longitudinal survey conducted among domestic pilgrims from Riyadh city  in 2010 showed that 53% developed RTIs ; similarThe extent of COVID-19 related to the Hajj setting presented in our study was much lower than the KSA national data. A nationwide seroprevalence cross-sectional study estimated an overall prevalence of COVID-19 of 11% in KSA . The strThe strength of this study is that to the best of our knowledge it is the only study that surveyed the health of pilgrims after Hajj amidst the COVID-19 pandemic. This study also has some limitations. It was conducted during an exceptional Hajj season when no overseas pilgrims were allowed unless they were already resident in KSA and pilgrims had to be healthy with no pre-existing diseases; therefore, the generalisability of the results is limited. Since we used the CATI method to collect data from participants, we ensured that we would not have missing data. However, some of the non-sensitive variables such as smoking status have a few missing data which are treated by available case analysis. Given that the study was conducted during COVID-19, the primary focus of this manuscript was on viral infection, but bacterial infection is also a possibility. A study conducted over 4 years (2014–2017) involving 485 French pilgrims showed that 33% of pilgrims acquired at least 1 respiratory virus, and 35% acquired at least 1 respiratory bacterium, suggesting that bacterial infections too are important [The results of this survey indicate that although the 2021 Hajj was conducted during COVID-19 challenges, the prevalence of symptomatic RTIs among the returning Hajj pilgrims was low, and only a small fraction of pilgrims met the syndromic definitions of ILI or COVID-19. Therefore, the COVID-19-related public health countermeasures during the Hajj seem to be a success story for conducting future MGs amidst pandemics, and adopting such changes can play an essential role in mitigating the transmission of COVID-19 and other RTIs as well as ensuring the safety and wellness of participants.