Anti-COVID vaccination in Argentina was carried out using different protocols and variations in periods between administrations, as well as combinations of different vaccine platforms. Considering the relevance of the antibody response in viral infections, we analyzed anti-S antibodies in healthy people at different points of time following the Sputnik immunization procedure.We attended the vaccination centers in the city of Rosario, which had shorter versus longer intervals between both doses. A total of (1021) adults with no COVID-compatible symptoms (throughout the study period) were grouped according to the gap between both vaccine doses: 21 , 30 , and 70 days , as well as an additional group of individuals with heterologous vaccination .While there were no between-group differences in baseline levels of specific antibodies, data collected several weeks after administering the second dose showed that group D had the highest amounts of specific antibodies, followed by values recorded in Groups C, B, and A. The same pattern of group differences was seen when measuring anti-S antibodies at 21 or 180 days after the first and second doses, respectively. Delayed between-dose intervals coexisted with higher antibody titers. This happened even more when using a prime-boost heterologous schedule. Towards the end of November 2019, a series of pneumonia cases with no precise etiological diagnosis began to be detected in Wuhan, China Shortly after that, it was identified as a severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus. The disease rapidly spread worldwide to the extent that on March 11, 2020, the WHO declared the COVID-19 pandemic , which employs two different adenovirus vectors, Ad26 and Ad5, expressing the SARS-CoV-2 spike protein. They were initially administered 21 days apart .Evidence from randomized clinical trials or observational studies demonstrated that most anti-COVID-19 vaccines could confer substantial protection against symptomatic and severe diseases with two doses administered 3 to 4 weeks apart . NeverthIn this way, introducing a mRNA or protein vaccine to boost the first dose of an adenovirus vector rendered vaccine programs more responsive to fluctuations. Heterologous prime-boost strategies have been broadly applied to induce protection against several infectious diseases .With regards to the interval between the first and second dose, evidence from clinical trials established that the second dose of the ChAdOx1nCoV-19 vaccine could be given 8–12 weeks after the first dose as it conferred suitable protection levels against COVID-19 infection –18.Considering the relevance of the humoral immune response in viral infections, we aimed to analyze anti-SARS-CoV-2 immunoglobulins titer at different points of time following immunization. For this, we conducted sampling procedures on people who attended the massive vaccination centers designated by the Ministry of Health of the Province of Santa Fe. We conducted these procedures by following the Strategic Plan for Vaccination against COVID-19 in the Argentine Republic. Assuming that the period between the first and second dose was likely to influence the features of the humoral immune response from vaccinated recipients, we studied the levels of anti-S antibodies in individuals with shorter versus longer intervals between both doses.Given the shortage of the second component of the sputnik vaccine, the Argentine Ministry of Health considered the possibility of implementing a heterologous vaccination using the Moderna vaccine as the second dose. Since this schedule change was voluntary for each potential candidate, we were able to study a group of people vaccinated with Sputnik/Moderna. The response elicited in this group was then compared to those who received two Sputnik doses.In light that following the vaccine implementation, some participants may have the chance to develop a breakthrough SARS-CoV-2 infection, the humoral immune response to vaccination in people who also experienced a SARS-CoV-2 infection was also investigated.https://bit.ly/3YqnMqY.This study is a prospective, longitudinal, observational analysis conducted in a single center. The first two groups (A and B) were health workers from different departments of the Ministry of Health of the Santa Fe Province . Those from Group A were given their second dose 21 days from the first one, as recommended initially. In the case of Group B volunteers, the interval between the first and second dose was extended to 30 days. Group C comprised young adults to whom the second dose was administered 70 days after the first one. An additional group of older adults was also included (Group D). They were subjected to a heterologous prime-boost schedule consisting of Sputnik and Moderna doses, separated by a 107-day interval. The volunteers were given their first vaccine dose between January and July 2021. Participation in the study was voluntary, and all participants signed a written informed consent. The Provincial Bioethics Committee approved the protocol (PROVINCIAL REGISTRY No. 1048). The COVID vaccine regimens and recommendations for the study period in Argentina can be found in the following link: Elecsys Anti-SARS-CoV-2-S Assay (Roche), according to the manufacturer´s instructions. Given that the primary measurement range of the test used is 0.40-250 U/ml, those samples with a detected antibody concentration value > 250 U/ml were automatically diluted 1:10 by the analyzer, for which the maximum measured concentrations were reported as >2500/ml. The samples collected at the moment the first dose was given, 21 days after the first dose at the time the second dose was given, and 21 days after the second dose, were all analyzed simultaneously, whereas the samples obtained at month six were analyzed separately.Once the first blood sample was obtained from each volunteer on the day of the first dose, they were scheduled to come back to the CUDAIO Immunogenetics Laboratory 21 days later to collect the second sample. The following sample was also taken at the vaccination facility on the day of administering the second dose. Volunteers had blood drawn 21 days from the second dose and 6 months from the start of the immunization schedule. Serum samples were stored according to our approved protocols (IRB# PRO02737). All samples were identified and registered, and backups of them were kept. Tables containing each individual’s identification and sample tracking numbers were safeguarded. Then, fully anonymous serum samples were obtained by centrifugation, divided into aliquots, frozen, and stored at -80°C until analyzed. All samples were subjected to freezing and thawing before the ELISA analysis. The antibody titer against the receptor binding domain (RBD) of protein S in sera from the blood sample was measured using Immunoassay to quantitatively determine antibodies of the SARS-CoV-2 spike protein using the Comparisons among groups were performed by non-parametric methods, such as the Kruskal-Wallis analysis, followed by a posthoc test for the multiple comparison approach, when applicable. Paired comparisons were made by the Friedman analysis of variance and the Dunn test. The general linear model was used to evaluate the eventual effects of sex and age on the means of antibody levels for each one of the three antigens. This procedure provides regression analysis and analysis of variance for one dependent variable adjusted by one or more factors. Categorical variables were compared by the chi-square test. Data were considered statistically significant if p< 0.05. Data were analyzed with STATA 6.0 and Prism Graph Pad 4.0 software.An outline of the main characteristics of the study groups according to the immunization schedules and blood collection is provided in The age, gender, and number of people enrolled in the present study are depicted in Antibody titer data is presented according to the immunization protocols. Among individuals with a 21-day interval between the first and second doses, the anti-S antibody levels increased from their baseline levels, reaching the highest amounts at T4 (21 days after the second dose). A slight decrease was observed in samples taken 180 days after the first dose (T5) .Data were also analyzed to establish a potential influence of gender and age by applying the general linear model, which provides regression and variance analysis for the antibody titers adjusted by both factors. Data presented in Antibody titers analysis was also done by comparing individuals who experienced COVID-19 infection upon vaccination. As shown in Vaccines against many pathogens, particularly viruses, are thought to mediate protection by generating an antibody response that can neutralize the infecting inoculum and prevent the development of infections . Beyond Within this framework, our study provides evidence that vaccinated individuals with larger intervals between the first and second doses showed the highest levels of anti-S antibodies in assessments carried out at different time point evaluations following the completion of the prime-boost vaccination. Despite some concern that older individuals may develop a poorer response, it is also worth emphasizing that aged individuals from Group D displayed increased antibody levels. This goes without disregard that age may account for some of the reported differences, particularly at T2 and T3 evaluations where in group B, individuals displayed lower amounts of anti-S antibodies.Within the context of prime-boost immunization, the first shot which is addressed to elicit an initial immune response to some pathogen proteins is followed by a booster injection to recall immune memory. It may be that a delayed dose (mainly for vaccines using viruses as vectors) would benefit from a possible decline in the levels of anti-vector antibodies, thus decreasing the blocking of the carrier before delivering its information.Beyond this fact, it is worth emphasizing that antibody concentrations were well preserved regardless of the delay period between the administration of the first and second doses. This lends some support to adenoviral vaccines as a useful immunization approach.Previous intervention studies employing the ChAdOx1nCoV-19 vaccine revealed increased antibody responses and vaccine efficacy provid the prime-boost interval was extended . FlaxmanStudies using mRNA vaccines also indicated that longer dose intervals enhanced immunogenicity . In the Along with the approach of delayed intervals, heterologous prime-boost COVID-19 vaccination was also promoted to alleviate vaccine supply shortages. Thus, individuals initially primed with an adenovector vaccine further received a second dose, mainly consisting of mRNA vaccines . More reSeveral pieces of evidence indicate that this approach is effective for disease protection  and the Our study indicates that the same holds true when employing the Sputnik vaccine, which was reported to be effective against COVID-19 . To the Regarding study limitations, our assessment did not discriminate between non-protective and protective antibodies, whereas findings correspond to a single-center study. We cannot extend our results to what happens in other scenarios regarding antibody titers in vaccinated people. It is also clear that a longer follow-up will help to get a clearer picture of the dynamics of antibody responses, i.e., its durability beyond the classical schedules .The effect of the immunity generated against the vector itself may still be a matter of debate as certain previous seroprevalence studies about adenoviruses may cast some doubts about the usefulness of the vaccine , 28. OurFinally, we observed that group D had higher antibody titers than groups B and C at day 21, even though all groups received the same first vaccine . Among tWhatever the case, our studies provide a stimulating background to assess whether these findings translate into better protection against COVID-19 disease.This study was supported by Roche Diagnostics with an unrestricted grant for the contribution of the reagents used to determine the concentration of Anti-SARS-CoV-2 S antibodies.https://doi.org/10.57715/UNR/ULPO6K, RDA UNR, V1).The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: The datasets generated for this study can be found in the RDA-UNR repository of academic data (Naidich, Gretel; Santucci, Natalia; Pezzotto, Stella Maris; Ceccarelli, Eduardo; Bottasso, Oscar; Perichón, Mario, 2022, “Determinación de inmunoglobulinas totales tras la vacunación anti-SARS-CoV-2 en voluntarios sanos”, The studies involving human participants were reviewed and approved by Comité Provincial de Bioética. Ministerio de Salud de la Provincia de Santa Fe, Argentina. The patients/participants provided their written informed consent to participate in this study.GN designed the study, obtained samples and data, and contributed to the writing of the manuscript. NS Analyzed data, wrote and edited the manuscript, and prepare the figures. SP collaborated in the statistical analysis of data. EC contributed to the study design and critically revised the manuscript. OB contributed to the study design, analyzed data and wrote the manuscript. MP designed and supervised the study. All authors contributed to the article and approved the submitted version.


To describe the role of microscope integrated optical tomography (Mi-OCT) in removal of intrastromal corneal foreign body. A young male presented with trauma to the right eye with sugarcane stick. Ocular examination revealed two sugarcane particles, approximately 3.5 mm in greatest dimension, embedded in the corneal stroma. For removal of foreign body, Mi-OCT was switched on and the area of interest was focused. Sugarcane particles appeared as hyperreflective structures embedded in the corneal stroma with everted edges of the overlying corneal epithelium and anterior stroma. Both the sugarcane particles were removed successfully under real time images provided by Mi-OCT without causing any inadvertent damage to the corneal stroma. Mi-OCT can be used as an adjunct in emergency surgical procedures like removal of intrastromal corneal foreign bodies with accurate precision. Mi-OCT has emerged as a useful tool in providing surgeons with real-time cross sectional images of ocular tissues intraoperatively , 2], , , 5]. We. We5]. WA 32-year-old male presented with a history of trauma to the right eye (OD) with a sugarcane stick 1 day back. His best corrected visual acuity in OD was 6/18 and in the left eye (OS) was 6/6. By non-contact tonometry, intraocular pressure in OD and OS were 14 and 16 mmHg, respectively. Slit-lamp examination revealed two sugarcane particles, approximately 3.5 mm in greatest dimension, embedded in the corneal stroma. These were lying horizontally in the paracentral cornea, just inferior to the visual axis. The tract through which these sugarcane particles entered the cornea was, however, not evident on slit-lamp examination. The patient was counseled, and after obtaining written informed consent, he was taken up for removal of the intrastromal corneal sugarcane particles under the operating microscope. Mi-OCT was switched on and the area of interest was focused. Sugarcane particles appeared as hyperreflective structures embedded in the corneal stroma with everted edges of the overlying corneal epithelium and anterior stroma (Figure 1a This report highlights the use of Mi-OCT in the retrieval of two organic intrastromal corneal FBs in a patient where the entry tract could not be appreciated on pre-operative examination. Attempts to remove such organic FBs on the slit-lamp can cause collateral damage to the surrounding corneal tissue and compromise visual outcomes. Mi-OCT can help in the successful removal of such intrastromal corneal FBs by providing real-time visualization of corneal planes, thereby avoiding damage to the surrounding healthy tissue and aiding in relatively atraumatic removal of organic FBs.All authors have contributed equally in the preparation of the manuscript.The authors declare that they have no competing interests.


Although there have been many studies on antibody responses to SARS-CoV-2 in breast milk, very few have looked at the fate of these in the infant, and whether they are delivered to immunologically relevant sites in infants.Mother/infant pairs (mothers who breast milk fed and who were SARS-CoV-2 vaccinated before or after delivery) were recruited for this cross-sectional study. Mother blood, mother breast milk, infant blood, infant nasal specimen, and infant stool was tested for IgA and IgG antibodies against SARS-CoV-2 spike trimer.Thirty-one mother/infant pairs were recruited. Breast milk fed infants acquired systemic anti-spike IgG antibodies only if their mothers were vaccinated antepartum . Breast milk fed infants acquired mucosal anti-spike IgG antibodies (in the nose) only if their mothers were vaccinated antepartum . None of the infants in either group had anti-spike IgA in the blood. Surprisingly, 33% of the infants whose mothers were vaccinated antepartum had high titer anti-spike IgA in the nose . Half-life of maternally transferred plasma IgG antibodies in the Antepartum infant cohort was ~70 days.Vaccination antepartum followed by breast milk feeding appears to be the best way to provide systemic and local anti-SARS-CoV-2 antibodies for infants. The presence of high titer SARS-CoV-2-specific IgA in the nose of infants points to the potential importance of breast milk feeding early in life for maternal transfer of mucosal IgA antibodies. Expectant mothers should consider becoming vaccinated antepartum and consider breast milk feeding for optimal transfer of systemic and mucosal antibodies to their infants. Breast milk feeding is strongly recommended given its many associated health benefits, particularly those related to the neonatal immune system in the first months of life when infants are at greater risk of infections . SARS-Co1/2) of IgG in plasma and nasal compartments of the entire cohort and for infants with more than time point, continuous decay (linear regression) was used on log10-transformed data, similar to what has been reported previously [In this cross-sectional study, mother/infant pairs at the University of Maryland Medical Center were recruited. Recritment of mother/infant pairs was done through word of mouth. These included two groups: mothers vaccinated antepartum who breast milk fed (Antepartum group), and mothers who breast milk fed but who were vaccinated postpartum (Postpartum group). The vaccines administered were 2 doses of the Pfizer-BioNTech BNT162b2 or Moderna mRNA-123 (primary series). All sampling was done after maternal completion of the primary vaccine series. Infant breast milk consumption was either from breastfeeding or bottle feeding of expressed breast milk. IgG and IgA SARS-CoV-2 spike trimer antibody levels in maternal blood and milk, as well as infant blood, nose, and stool were determined by ELISA. In addition, ELISA for SARS-CoV-2 nucleocapsid (N) protein IgG was performed on blood samples. Mother/infant pairs testing positive for SARS-CoV-2 N IgG were excluded from our analysis. ELISA was slightly modified from one that was previously published for plasma , and cuteviously .All mothers provided written informed consent; the study was approved by the University of Maryland Baltimore Institutional Review Board. Statistical analysis was performed with GraphPad Prism 5.The Antepartum group was comprised of 18 mother/infant pairs and the Postpartum group was comprised of 13 mother/infant pairs. The median age of mothers was 33 years (range 27–37) in the Antepartum group and 34 years (range 27–40) in the Postpartum group. The median gestational age of infants in the Antepartum group was 40 weeks (range 30–41) versus 39 weeks (range 38–41) in the Postpartum group. Vaccination occurred at a median 161 days (range 20 to 285) prior to birth in the Antepartum group and 146 days (range 3 to 326) after birth in the Postpartum group. The median age of infants at sampling was at a median of 167 days (range 15 to 402) after birth in the Antepartum group and 353 days (range 38 to 545) in the Postpartum group. Four infants had sampling performed at two timepoints. One mother in the Antepartum group reported a remote history of COVID-19 prior to vaccination. None of the infants had a history of COVID-19. All mother/infant pairs tested negative for IgG to SARS-CoV-2 N protein and thus had no serologic evidence of recent infection. Additional demographic and clinical characteristics of our cohort are detailed in Anti-Spike IgG and IgA were detected in the mothers’ blood and breast milk indicating an appropriate immune response to vaccination . There wAll infants had detectable total (non-antigen specific) IgA and IgG in the nasal samples, and no significant differences in total IgG, IgA, or IgA/IgG ratio were noted between the two groups , though 1/2 of plasma maternal anti-spike IgG in the entire infant cohort was 56.9 days, while the cross-sectional t1/2 of nasal maternal anti-spike IgG in the entire infant cohort was 83.6 days . There was a direct correlation between the plasma anti-spike IgG and nasal anti-spike IgG spike endpoint titers   . The nasTransplacental transfer of IgG occurs  via the In some mammals, infants have the ability to absorb antibodies through the GI tract after delivery. However, studies have shown that this does not occur in human infants, with the exception of pre-mature infants, and the newborn during the first day of life . In one A strength of our study was the use of endpoint titers, and evaluating all relevant compartments, so they could be directly compared to one another. In the Postpartum group, we found anti-spike IgG and IgA antibodies in the mother’s breast milk and blood. This is consistent with prior studies which have found antibodies, including neutralizing antibody in the breast milk , 20. HowIn 1976, there was a seminal study that described protection of infants against RSV that was linked to breast milk feeding, and that this was likely due to IgA . This stIn our study, the most unexpected finding was the presence of high-titer of anti-spike IgA in the nares of infants whose mothers were vaccinated antepartum. This may offer insight into why breast milk feeding can protect against respiratory pathogens. However, the timing of breast milk feeding likely matters, as do other factors, as nasal anti-spike IgA antibody was not found in all infants. None of the infants had SARS-CoV-2 infection by history or serology, thus the only way to transfer the antibodies was via breast milk feeding. This is supported by one prior study in which total IgA was noted to occur in the first weeks of life in half of breast milk fed, but not formula fed newborns . AdditioIn light of the studies mentioned above, and the known transfer of IgA by early breast milk feeding, these data are consistent with immune cells being transferred in a percentage of infants early in life. Alternatively, pharyngonasal reflux or direct inhalation could directly deposit the vaccine-specific IgA on nasal surfaces, but another mechanism for concentrating the antibodies (such as binding to mucin or lectin) would need to be involved. Finally, another possibility is the transfer of SARS-CoV-2 antigen from mother to child. Although immune complexes in breastmilk and oral IgA in neonates have been shown in peripartum SARS-CoV-2 infection , it is l1/2 ~70 days) and by analyzing the cross-sectional data set as a whole (t1/2 = 56.9 days). However, the calculated cross-sectional t1/2 of the nasal IgG appeared to be even higher (83.6 days). Although we are not confident in the actual t1/2 derived from the plasma and nasal data cross-sectionally, what is interesting is the differing numbers and strength of association. One potential reason for this lack of correlation could be differential regulation of the nasal IgG, with a replenishing source of nasal IgG antibodies after birth, as opposed to plasma IgG which is expected to undergo a steady decay after lack of continuing transfer of antibody after birth. Thus, the findings of high titers of nasal IgG in the infants in the Antepartum group could also be due to the same mechanisms of the transfer of IgA; however, transfer of circulating IgG to mucosal surfaces is also contributing, which could explain the higher percentages of Antepartum infants with detectable nasal IgG compared to IgA, and higher titers of nasal IgG compared to IgA.As mentioned above the half-life of plasma IgG antibodies in infants is known to be much longer than in adults. This was demonstrated in the 4 infants we had repeated measurements (tLimitations of this study include difference in ages of the infants in the two groups (partly due to vaccine schedule), cross-sectional design, small study population, and small sample volume that precluded functional  testing. Although we did look at multiple sites, it is possible that results may be different in other potentially relevant ones not studied (such as lower respiratory tract). Finally, protection of the infant from SARS-CoV-2 was not studied, and postpartum vaccination and protection of the mother could also lead to indirect protection of the infant through cocooning.These data suggest that antepartum vaccination followed by breast milk feeding provides the most reliable source of systemic and mucosal antibodies against SARS-CoV-2 for infants. Breast milk fed infants only acquired systemic anti-SARS-CoV-2 antibodies if their mothers were vaccinated antepartum. While breast milk feeding brings many benefits for the infant, there does not appear to be any meaningful transfer of SARS-CoV-2 specific antibody from mothers to their infants if vaccinated postpartum. Expectant mothers should consider becoming vaccinated antepartum and consider breast milk feeding if they wish to transfer SARS-CoV-2 specific antibodies to their infants.S1 Data(XLSX)Click here for additional data file.


We plan to enrol 60 outpatient women with COVID-19 irrespective of vaccination status diagnosed by PCR swab or RAT (rapid antigen test) within five days and randomised to 14 days of oral broccoli sprout extract (42 mg of sulforaphane daily) or identical microcrystalline cellulose placebo. The primary outcome of this pilot trial will be to assess the feasibility of conducting a larger trial investigating the duration (days) of COVID-19-associated symptoms using a broccoli sprout supplement for COVID-19-affected pregnancies. Pregnant patients remain an at-risk group for severe disease following infection with SARS-CoV-2 and currently unclear consequences for the offspring. Therefore, this study will assess feasibility of using a broccoli sprout supplement, whilst providing important safety data for the use of sulforaphane in pregnancy.Since its discovery in late 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been estimated to be responsible for at least 769.3 million infections and over 6.95 million deaths. Despite significant global vaccination efforts, there are limited therapies that are considered safe and effective for use in the management of COVID-19 during pregnancy despite the common knowledge that pregnant patients have a much higher risk of adverse outcomes. A bioactive compound found in broccoli sprout—sulforaphane—is a potent inducer of phase-II detoxification enzymes promoting a series of potentially beneficial effects notably as an antioxidant, anti-inflammatory, and anti-viral. A pilot, double-blinded, placebo-controlled randomised trial is to be conducted in Melbourne, Australia, across both public and private hospital sectors. We will assess a commercially available broccoli sprout extract in pregnant women between 20 The global pandemic for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) began in late 2019. SARS-CoV-2 is the cause of the coronavirus disease (COVID-19) and, as of 10 August 2023, has been responsible for over 769.3 million infections and at least 6.95 million deaths globally . Whilst Brassica oleracea var. italica) sprouts [A naturally occurring organosulphur and Isothiocyanate, sulforaphane (1-isothiocyanato-4-methylsulfinylbutane), is found within numerous cruciferous vegetables including kale and cabbage but is particularly concentrated within the aerial component of broccoli , its various subvariants BA.2-5, and newer recombinant variants such as XBB1.5 and XBF display an ability to escape the majority of existing SARS-CoV-2-neutralising antibodies . This reSulforaphane has an established safety profile in both adult and paediatric human clinical trials  but withThe decision to complete this study as a pilot is primarily aimed at establishing feasibility, identifying challenges, and outlining future requirements for a larger randomised controlled trial. This is important, given the study design relies on home-visits with active COVID-19-infected pregnant patients in a large metropolitan area of Melbourne. Whilst establishing feasibility for future larger clinical trials, this study will simultaneously provide important initial safety data for use of sulforaphane during pregnancy.+0 to 36+0 weeks gestation infected with SARS-CoV-2 and improve biochemical markers of inflammation. However, prior to establishing a larger clinical trial adequately powered to answer the clinical primary outcome, this study will investigate the unique challenges associated with an interventional outpatient study of COVID-19 pregnant participants investigating feasibility, recruitment, and patient identification strategies, patient acceptability, and work-flow logistics of patient and trial staff activities.We hypothesise that a broccoli sprout supplement containing sulforaphane will reduce the duration of COVID-19-associated symptoms for pregnant women at 20Aim 1. To assess the feasibility of conducting an outpatient study on COVID-19-infected pregnant patients requiring home-visits.Aim 2. To assess if a broccoli sprout supplement containing sulforaphane can reduce COVID-19-associated symptom duration in pregnant women.Aim 3. To investigate the effect of a broccoli sprout supplement on various biochemical markers of inflammation as well as pregnancy and neonatal outcomes.This study is a pilot, double-blinded, parallel arm, randomised, placebo-controlled trial and has been summarised in a trial flow-chart below .n = 60 with 30 participants in each arm receiving either two broccoli sprout powder capsules GeneActive Formulation E (700 mg whole broccoli sprout powder) yielding sulforaphane (21 mg) twice daily or matching placebo capsule containing microcrystalline cellulose. Given that this is a pilot feasibility study, our convenience sample size is informed by predicted community COVID-19 infection rates during protocol design and predicted time until completion, allowing analysis of data to inform future work.A convenience sample size has been set to a total Recruitment of pregnant women will occur at Monash Health Hospitals including Monash Medical Centre, Casey Hospital, Dandenong Hospital and Jessie McPherson Private Hospital, Victoria, Australia. These services cover a large catchment area of southeast Melbourne, Australia, caring for over 12,000 pregnant women and their babies each year. A centralised pilot trial steering committee will be established from the trial investigators for the oversight and management of the trial across the joint health networks.≥18 years of age;+0 to 36+0 weeks;Pregnant with singleton gestation at 20Positive COVID-19 test either via viral PCR or RAT for SARS-CoV-2 within the preceding five days;Any vaccination status against SARS-CoV-2;Signs or symptoms of COVID-19 for ≤seven days before recruitment including but not limited to shortness of breath, anosmia, fevers, sore throat, headache, and myalgia;Able and willing to tolerate oral supplementation with a broccoli sprout extract for the full 14-day course;Able to understand the information provided in the participant information and consent form (PICF), and able to give written informed consent (with interpreter use as required).A study participant must meet the following inclusion criteria to take part in this study:Currently using a broccoli sprout extract or supplement;Contraindications to use of a broccoli sprout extract ;Significant uncertainty of gestational age;Unwillingness or inability to follow the procedures outlined in the PICF;Mentally, cognitively, or legally incapacitated or ineligible to provide informed consent;Currently recruited in another clinical trial using a pharmaceutical, herbal, or nutritional intervention ;Currently on an antibiotic, antiviral, or monoclonal antibody treatment related to acute illness.A study participant will be deemed ineligible to take part in this study if they meet any of the following:Identification of potential trial participants will be predominantly via the local public health unit database of active COVID-19 cases from the community. Additionally, health staff and internally through the Monash Health Women’s and Newborn Department and Jessie McPherson obstetric services. Additionally, patients will be able to self-refer to the study team after testing positive to COVID-19 through the Monash Women’s and Newborn COVID-19 information webpage and through advertisement with flyers and informational posters in patient facing areas. Potential trial recruits will be contacted initially via Short Message Service (SMS) with links to the Monash Women’s website and patient information video outlining the trial. Trial staff will then contact patients via telehealth or phone. If interested in participation, patients will be provided the PICF via secure email . Electronic consent will be used where possible, allowing the study team to obtain consent remotely in an effort to reduce transmission of SARS-CoV-2 virus towards clinical trial staff. Additionally, participants can optionally consent to keeping remaining biological samples for further research studies via the PICF. Clinical trial staff will describe the trial, the voluntary nature of participation, and participation or not in the study will not in any way affect their routine clinical care. If patients consent to trial participation with signed electronic consent, they will be visited at home by the study team and provided study drug capsules, a study diary to record daily symptoms, and have collections of urine, blood, and respiratory viral PCR with an optional faecal sample.+0 to 27+6 and 28+0 to 36+0) will be used. Stratification by site has been utilised to minimise potential differences in population groups across different locations of study sites. Similarly, stratifying by gestational age has been undertaken in case of gestation specific risks from COVID-19 infection variably impacting on pregnancy complications such as preeclampsia, spontaneous pre-term birth, and foetal growth restriction. Patients will be allocated to the broccoli sprout supplement or placebo in a 1:1 ratio. A secure, centralised, web-based system for managing the study database (REDCap) will be used. This will allow for enrolment and randomised allocation to occur at any time of the day. A computer software program embedded into the REDCap system will generate a random allocation sequence for each patient, which will not be accessible to investigators, research coordinators, or study participants during the trial. PICF electronic copies will be uploaded into the password-protected secure REDCap database and other documents maintained on a password protected trial computer. Study participants will be randomised to either intervention. A clinical trial pharmacy has packed the investigational product and matched placebo capsules into sealed bottles with allocation blinded to study team and trial participants. The clinical trials pharmacy will keep a record of study participant identification number as well as treatment group allocation. At completion of the trial, unblinding of study drug interventions will occur by an independent data analyst not involved in data collection.Eligible patients will be randomised immediately after consenting to study enrolment. A permuted block randomisation with blocks of random size and stratification by study site and gestational age  of either 700 mg broccoli sprout powder (each capsule yielding 10.5 mg of sulforaphane), providing a total daily dose of 42 mg of sulforaphane (GeneActiv Formula E) or identical placebo-containing microcrystalline cellulose for 14 days. The study drug will be delivered to the trial participants’ homes along with a drug diary for recording capsule adherence, daily symptoms, and any adverse symptoms experienced during the trial from the intervention. Bottles will be collected after the 14-day intervention and the remaining capsules counted to measure adherence to the study protocol. In the event of severe disease requiring hospitalisation, patients will continue the allocated treatment remaining blinded unless concerns are raised by the treating medical team or patients are unable to continue oral therapies. Additional therapies following randomisation for treatment of COVID-19 including but not limited to monoclonal antibody or anti-viral medication will be documented and will not alter patient participation.The primary aim of this study is to serve as a pilot feasibility study for a larger future randomised controlled trial and as such will be underpowered to answer any clinical outcome related to the duration of COVID-19-associated symptoms. Several limitations, however, are noted in the study design for this trial. At current, the COVID-19 vaccination status of an individual does not preclude nor stratify them during the enrolment process. The study will report on vaccination rate, type of vaccination, number of vaccinations, and recency of vaccination in the reported outcomes. Furthermore, the primary outcome of this study is the duration of patient reported COVID-19-associated symptoms in a patient’s reported daily diary. It is appreciated that a more objective measure of SARS-CoV-2 viral load which is associated with COVID-19 symptom severity may be quantifiable by using a surrogate measure of viral load by reporting on cycle-threshold values  or by daily PCR results across the duration of the study. Given that the primary outcome of the study is patient-reported duration of symptoms, this is what formed the primary study design of biological sample collection and the collection of samples prior to study intervention commencement (Day 1) and then a collection of further samples 7 days later. This will allow a review of CT-values between groups after randomization and 7 days of study intervention and, hence, an analysis of broccoli sprout extract on SARS-CoV-2 viral loads through a surrogate measure of the CT-value. Finally, as a nutritional supplement clinical trial, it may be possible to tightly regulate the nutritional status and dietary intake of study participants during the intervention period to limit dietary confounders in the study outcomes. This would only realistically be achieved by providing a 14-day complete nutritional diet to the study participant and additional household members, which has not been built into this study design. This study does not aim to control all dietary sources of sulforaphane-yielding vegetables and appreciate that possibly inflammatory diets such as those with a high-saturated-fat content will not be controlled for. Furthermore, the study will not be collecting patient nutritional intake though tools such as 24 h food-recall diaries, which we appreciate would provide a more complete overview of the study participants nutritional intake and, hence, address some potential confounders in the study such as alternative dietary sources of glucurophanin and, hence, sulforaphane.As this trial is a pilot feasibility study, the primary goal is to establish future feasibility, challenges, and limitations of an outpatient COVID-19 study design in the South-East Melbourne region. COVID-19 and the public-health approach to managing the pandemic has shifted significantly in the last 12 months, particularly within Australia, with the reduction in morbidity and mortality seen with SARS-CoV-2 infection during Omicron-dominant periods. Identifying COVID-19-positive outpatient pregnant patients in an efficient and timely manner for recruitment to an interventional therapeutic study will be challenging in an era of “COVID-normal” and the reduction in patient-initiated queries to the health network. Additionally, this study will also assess the acceptability of a nutritional supplement for COVID-19 in the pregnant population living within South-East Melbourne. Finally, as this study will be the first to provide broccoli sprout powder yielding sulforaphane as a potential investigational therapeutic during pregnancy, this will also provide important initial maternal and neonatal safety information.Our clinical trial primary outcome is the duration (days) of COVID-19-associated symptoms. These symptoms are to be reported in the patient self-reported study diary and include sore throat, hoarse voice, joint pain, muscle pain, nausea, vomiting, diarrhoea, irregular heartbeat, chills/shivers, abdominal pain, cough, shortness of breath, chest pain, fatigue, headache, runny nose, altered smell, and loss of smell. These symptoms will be reported as present/absent for each day of reporting without severity scales. Self-reporting up to day 14 of the study should be of sufficient duration, as reported from the ZOE COVID study finding Omicron-associated symptom duration to have a mean of 6.9 days and median of 5.0 days .The secondary outcomes will be exploratory only and will broadly investigate the safety of broccoli sprout supplementation during pregnancy.Birth outcomes including mode of delivery;Obstetric complications including post-partum haemorrhage (PPH), pre-eclampsia, preterm pre-labour rupture of membranes (PPROM), and stillbirth;Maternal death;Unplanned hospital presentation within 28 days;Admission to hospital for any reason with >24 h within 28 days;Total duration of hospital admissions within 28 days;Maternal biochemical, inflammatory, and anti-angiogenic markers;Medication related adverse events;Placental cellular and structural abnormalities ;Gastrointestinal microbiome population species as measured through optional faecal microbiome testing .11.Birthweight and birthweight percentile;12.COVID-19-positive polymerase chain reaction (PCR) swab in the neonate if testing performed as part of standard care;13.Admission to neonatal unit and duration ;14.Need for antibiotic therapy and duration of treatment;15.Diagnosis of early-onset sepsis (EOS);16.Diagnosis of late-onset sepsis;17.Bronchopulmonary dysplasia;Hypoxic-ischemic encephalopathy;Sepsis;Anaemia requiring transfusion;Patent ductus arteriosus;Intraventricular haemorrhage;Necrotizing enterocolitis;Retinopathy of prematurity.At least 1 of the following morbidities:Severe neonatal morbidity index (SNMI);18.a.Intrauterine foetal death;Neonatal death;Neonatal intensive care unit admission > 7 days.Includes any of the indicators from SNMI and additionally:Severe perinatal morbidity and mortality index (SPMMI).19.2 < 94%;Documented SpO20.Admission to intensive care unit (ICU);21.2);Requirement for any oxygen therapy  in a ventilated patient with positive end-expiratory pressure (PEEP) of at least 5 cm H2 and bilateral opacities not fully explained by heart failure or volume overload that occurs within 7 days of a clinical insult (37);Diagnosis of maternal acute respiratory distress syndrome (ARDS) as defined by the European Society of Intensive Care Medicine as new or worsening respiratory symptoms that includes a combination of acute hypoxaemia  .Electronic medical records will be utilised to record maternal and neonatal outcomes, as well as to collect maternal demographics including body mass index (BMI), ethnicity, drug and alcohol use, smoking status, and maternal comorbidities.g for ten minutes and both plasma and serum stored at −80 °C. The plasma and serum will be analysed for angiogenic and antiangiogenic markers including sFlt-1, sEng (soluble endoglin), and PlGF . Urine samples will be collected in sterile specimen containers and then transferred on ice to the laboratory and stored in collection tubes at −80 °C. Faecal specimen collection will be undertaken using sterile self-contained faecal sample collection kits  and stored at −80 °C. Faecal samples have been collected for possible future analysis of microbiome alpha and beta diversity following exposure to broccoli sprout or placebo. Respiratory viral PCR swabs (Kangjian Virus Collection and Preservation System) for SARS-CoV-2 genotyping and for the calculation of CT (cycle threshold) values as a surrogate marker of viral load will also be collected. Placental samples collected at the time of birth will be transported on ice to the laboratory. Placental samples will include four 1 cm3 sections of placenta that will have the maternal and foetal sides removed and washed in phosphate-buffered saline (PBS). Samples will then be snap-frozen in liquid nitrogen and prior to storage at −80 °C. Full sections of the placenta both peripheral and central as well as sections of umbilical cord will be preserved in 10% neutral buffered formalin (NBF) and then fixed in paraffin wax. The placenta will also be sent for formal histology reporting by the site’s anatomical pathology department.Biological specimens collected on days 1 and 7 of the trial during planned home visits include blood, urine, oro-pharyngeal nasal PCR swab, and an optional faecal sample. At the time of birth, optional maternal and umbilical cord blood, placenta, and faecal sample will be collected. Participant maternal peripheral blood and umbilical cord blood will be placed into both ethylenediaminetetraacetic acid (EDTA) and clot-activating blood tubes for both plasma and serum collection. Biochemical and inflammatory bloodwork collected from trial participants will be analysed through Monash Health Pathology and include full blood examination (FBE), electrolytes, urea and creatinine (EUC), liver function testing (LFTs), C-reactive peptide (CRP), ferritin and coagulation studies. Once blood has been collected, EDTA tubes will be placed on ice, the others will remain at room temperature for transport to hospital pathology for routine processing. Additional EDTA and silica (clot-activator) tubes will be taken to the laboratory and centrifuged at 1200× p-values of <0.05 will be the cut-off for statistically significance. We do not plan to undertake any interim analysis for this pilot trial.In addition to primarily investigating feasibility for a larger broccoli sprout supplement clinical trial, we will also investigate the ability of sulforaphane to reduce COVID-19-associated symptom duration for pregnant patients with COVID-19. This will be reported as mean duration (days) of symptoms of participants between the investigational product (broccoli sprout powder) and placebo to a maximum of 14 days of symptom recording. Data will be collated and analysed according to the intention-to-treat principle. The primary outcome analysis will be performed using a Fine–Gray competing risk model with death before the event (resolution of symptoms) treated as a competing risk. Appreciably, with the reduction in severe morbidity and mortality, it is unlikely that death before the event (resolution of COVID-19-associated symptoms) will occur. Regardless, in the unfortunate setting of a new variant associated with a more severe disease phenotype, the Fine–Gray model will provide a more accurate method for the analysis when compared to other statistical models, as it will accommodate the competing risk of death in the situation it occurs. Additionally, results will be reported as sub-distribution hazard ratios (SHR) and 95% confidence intervals. In the competing risk models, subjects who experience death remain in the risk set and the SHR denotes the instantaneous rate of the event of interest in subjects who are still alive  and also those who have died. Sulforaphane, provided as a broccoli sprout extract, is a naturally occurring bioactive compound that has been investigated in many human clinical trials . Based oIn the event of adverse events occurring, the assessment and reporting of such events will occur as outlined by the Sponsor  and the National Health and Medical Research Council (NHMRC). Adverse events and serious adverse events (SAE) will be recorded and reported in detail within the participants’ electronic medical record and submitted to Monash Health and Monash Health Human Research and Ethics Committee (HREC) for further assessment. Study participant data will be collected electronically and stored securely on the web-based REDCap trial database allowing retrieval of information for audit or review as required. The Sponsor, Monash Health, is responsible for the provision of insurance for the trial through the Victorian Managed Insurance Agency (VMIA).The sponsor Monash Health has had no ultimate authority over the design of the clinical trial, trial management, patient recruitment, data or sample collection, analysis or the interpretation of data, or the writing of the manuscript for publication. The principal investigators NJF, KRP, DLR, and SAM will form a trial steering committee to oversee trial management. Any amendments to trial protocol will be registered with ANZCTR and submitted to the publishing journal. As this is a pilot phase I study, there will not be a data safety management board (DSMB) convened, given the exploratory nature of the outcomes. Instead, a trial group will oversee the trial and review any safety reports and overall quality of the data including participant retention and protocol adherence.Participant welfare and safety;The conduct of the trial in accordance with the Monash Health HREC-approved protocol;A failure to adhere to Monash Health Research Directorate and HREC conditions of approval;A recommendation from the trial group that the trial should cease completely due to safety concerns.Prematurely, permanent, or temporary ceasing of recruitment will occur if the chief principal investigator or the sponsor  believes there are issues relating to any, or all of:n = 60);The researchers have recruited, as per protocol, all trial participants  has been completed and acknowledged.The trial will conclude as per study protocol when:The trial cannot be modified after recruitment has commenced, unless approved by HREC.To inform clinical management or under conditions of an SAE where disclosure of the intervention is necessary;At the conclusion of the trial to determine the effect of the intervention.The trial can only be unblinded in the following circumstances:This trial was reviewed and approved (RES-21-0000-708A) by the Monash Health Human Research Ethics Committee (HREC) with registration on the Australian and New Zealand Clinical Trials Registry (ANZCTR:12622000173796) prior to commencement. This trial will be conducted in compliance with local and NHMRC guidelines. The trial will occur as stipulated by the clinical trial protocol and amendments or alterations in the clinical trial protocol will only occur after written approval by the Monash Health HREC unless there is an apparent and immediate patient safety risk. Study participants will be able to optionally request to be informed of their allocation and study outcomes at trial completion. Clinical trial outcomes will be disseminated locally, nationally, and internationally at meetings and conferences in peer-reviewed journals.The CO-Sprout study is the first randomised controlled trial to investigate a broccoli sprout extract as a potential therapeutic in pregnancy targeting excessive inflammation and oxidative stress via the active ingredient sulforaphane. COVID-19 in pregnancy results in a state of inflammation and oxidative stress despite largely limiting vertical transmission to the foetus . Severe Sulforaphane has potent antioxidant and anti-inflammatory effects, implying it may have other potential therapeutic indications throughout pregnancy, both stabilising the maternal system but also through mitigation of some of the harmful effects to the developing foetus and placenta as seen with maternal immune activation. CO-Sprout will investigate important safety questions for use of sulforaphane in pregnancy, appreciating that this study will be underpowered for most rare safety outcomes. Further investigational studies will be required, particularly to investigate the short and long-term infant outcomes for mothers exposed to sulforaphane in pregnancy. However, given our previous work on broccoli sprout supplementation in pregnant individuals and the pre-existing evidence base, both laboratory and animal, we do not envisage any adverse outcomes for either mother or baby from exposure to broccoli sprout powder. Regardless, this study will collect both maternal and neonatal outcomes to further establish the evidence base and inform future trials and studies.


Several studies have reported associations between COVID-19 vaccination and risk of cardiac diseases, especially in young people; the impact on mortality, however, remains unclear. We use national, linked electronic health data in England to assess the impact of COVID-19 vaccination and positive SARS-CoV-2 tests on the risk of cardiac and all-cause mortality in young people (12 to 29 years) using a self-controlled case series design. Here, we show there is no significant increase in cardiac or all-cause mortality in the 12 weeks following COVID-19 vaccination compared to more than 12 weeks after any dose. However, we find an increase in cardiac death in women after a first dose of non mRNA vaccines. A positive SARS-CoV-2 test is associated with increased cardiac and all-cause mortality among people vaccinated or unvaccinated at time of testing. Rare but serious cardiac disease side effects have been linked to COVID-19 vaccinations, especially in young people. Here, the authors find very little evidence of an association between vaccination and mortality, except for in females after a non mRNA vaccine, but show an increased risk of death following COVID-19 infection While the randomised clinical trials focused on short-term efficacy against symptomatic infection, which was subsequently found to wane and be escaped by SARS-CoV-2 variants7, real-world evidence has indicated stronger and more sustained effectiveness against severe disease and death due to COVID-199. However, it is also important to consider their safety, which can be difficult to assess in randomised clinical trials that are not powered to detect rare adverse events10.On the 8 December 2020, the United Kingdom (UK) began administering vaccines against COVID-19 according to the priority groups determined by the Joint Committee on Vaccination and Immunisation (JCVI)11, and an increased risk of thrombotic and other rare cardiovascular events after the ChAdOx1 nCoV-19 vaccine (Oxford-AstraZeneca)13. There is also evidence of a range of other rare neurological complications14. However, the absolute risk of severe complications is low and needs to be assessed against the increased risks associated with SARS-CoV-2 infection if unvaccinated16. The balance of risk and benefit is particularly important to determine in younger people, due to the lower risk of COVID-19 hospitalisation and death in this age group17.There have been rare cases of serious adverse events reported with the COVID-19 vaccines. Previous studies have shown an increase in the risk of myocarditis and myopericarditis associated with mRNA vaccines including BNT162b2 (Pfizer-BioNTech) and mRNA-1273 (Moderna)18. For comparison purposes, we assessed the impact of a positive test for SARS-CoV-2 on the same outcomes in vaccinated and unvaccinated individuals.Comparisons of the risk of death in vaccinated and unvaccinated young people are subject to confounding due to the vaccine prioritisation of, and higher vaccination rates among, those with underlying health conditions. To minimise potential confounding, we used a self-controlled case series (SCCS) design, where each participant acts as their own control, to compare the risk of death in the twelve weeks after vaccination (‘risk period’) to a subsequent reference periodHere, we show that among the population of England aged 12–29, mortality is not significantly increased in the first twelve weeks after COVID-19 vaccination compared with more than 12 weeks after any dose. Observed reductions in all-cause mortality are consistent with a time-varying healthy vaccinee effect due to postponement of vaccination during periods of poor health. However, in subgroup analyses we find a significant elevation in the risk of cardiac death in women after a first dose of non-mRNA vaccines, and a smaller, non-significant increase in cardiac death after second dose of mRNA vaccines in men. By contrast, a positive SARS-CoV-2 test is associated with increased cardiac and all-cause mortality among both vaccinated and unvaccinated individuals.There were 3807 deaths of 12 to 29 year-olds in England that occurred between 8 December 2020 and 25 May 2022 and were registered by 8 June 2022  of which were linked to a vaccination record (629 from NIMS and 1 from the supplementary NHS point of care extract). 63.3% (399) of first doses, 53.5% (228) of second doses and 98.2% (108) of third doses in the hospital deaths dataset were mRNA based. Of those who received both the first and second vaccination (422), 12.0% (51) received a different type of vaccine for each dose  were due to a cardiac event, and 353 (11.0%) were linked to a preceding positive SARS-CoV-2 test (297 (9.2%) occurred unvaccinated at the date of test registration and 56 (1.7%) vaccinated at the date of test registration). There were 1123 corresponding hospital deaths recorded in HES between 8 December 2020 and 31 December 2021 of individuals who were not infected on admission to hospital, of which 181 (16.1%) were linked to a preceding positive SARS-CoV-2 test (150 (13.4%) unvaccinated on date of test registration and 31 (2.8%) vaccinated). Participant characteristics were again similar across the three case series  Fig. . There wWe found a significant decrease in the incidence of all-cause registered death, driven by the first two weeks after vaccination  for the first dose and all doses combined, there were significant increases in the incidence of cardiac death , which, if valid, would correspond to one additional cardiac registered death for every 359,294  men aged 12-29 who received a second dose of an mRNA vaccine. This result was sensitive to variation of the risk period, with no increase observed with a 6 week risk period ). , driven by the first week  individuals aged 12–29 and unvaccinated on date of positive test registration, and 1 additional death for every 55,661  individuals aged 12–29 and vaccinated on date of positive test registration.For all three analyses of death after vaccination, varying defined lengths of risk period were consistent with the main findings in remaining below or not significantly different to 1  would have also biased the vaccination results. A further distinction between the US-based study and our current publication, is the vaccination type; in our study, mRNA vaccination primarily represents the BNT162b2 Pfizer-BioNTech vaccine and non-mRNA vaccination the ChAdOx1 Oxford-AstraZeneca vaccine, whereas in the US, mRNA vaccination represents a larger proportion of the mRNA-1273 Moderna vaccine and non-mRNA primarily represents the Ad26.COV2.S Johnson & Johnson vaccine, and that dosing intervals were shorter in the USA compared to in the UK.A recent analysis of data from Florida (United States) found increased risk of cardiac death in the first four weeks after mRNA vaccination in people aged 18–39 years22, Denmark11, and England23, with higher increased risk generally found among young males24. We found little evidence of increased risk of death due to cardiac events after an mRNA vaccine for either sex. Whilst there was some indication of a potential increase in risk for men after a second dose of a mRNA vaccine, the result was sensitive to the choice of the risk period.Several other studies have highlighted the association between COVID-19 vaccination and the risk of myocarditis and other cardiac events. Vaccination with mRNA vaccines is associated with an increased risk of myocarditis or myopericarditis, especially in young people, in investigation using data from the US25.While vaccinees often have higher rates of comorbidity  compared with those who are unvaccinated, early negative effect estimates are frequently observed for safety outcomes in studies estimating vaccine effectiveness—a pattern that is consistent with a time-varying healthy vaccinee effect due to postponement of vaccination during periods of illness or recovery16 however, ours are based on one of the largest studies of mortality in such a young cohort, where deaths are extremely rare.Our findings of elevated cardiac mortality following positive tests for SARS-CoV-2 are also consistent with evidence from Sweden, showing elevated risk of myocardial infarction and ischaemic stroke following COVID-19;26.While the risk of mortality after positive SARS-CoV-2 tests was most pronounced in the first few weeks, it was not clear from our results whether the excess risk had fully resolved by 12 weeks. A recent matched cohort analysis in an older but geographically similar population, found that the excess risk of cardiovascular disease resolved at around 12 weeks after positive SARS-CoV-2 testOur study has several strengths. First, we used death registration records and deaths that had occurred in hospital for the whole of England, linked to all vaccination records, including those which were not registered in the NIMS vaccination database because people died shortly after vaccination. Using a self-controlled case series, our estimates account for between-person differences, which is crucial because young people who were clinically extremely vulnerable were prioritised for vaccination.The main limitation in our analysis of death registration data is delay due to coroner referrals. Not all deaths that occurred in the period had been registered. Deaths of young people are more likely to be referred to the coroner and registration delays can be substantial. Adjustment for calendar time can account for the fact that more recent deaths are more likely to be under coroner investigation and therefore less likely to have been registered. But this adjustment cannot account for any increase in coroner referral related to time since vaccination – for instance, if deaths occurred very soon after vaccination they are more likely to be investigated by a coroner than deaths occurring at any other time. Our analysis using deaths recorded in hospital records, which are not affected by registration delays, was consistent with the analysis of registered deaths, suggesting that differential coroner referral in the period immediately postvaccination did not substantially affect analysis of registered deaths. However, a further limitation of the HES analysis is that sudden cardiac deaths, which would have occurred out of hospital, would not be captured.27. Sensitivity tests showed that while this does not significantly affect the results in the main analysis for deaths after vaccination, the estimates for death after a positive SARS-CoV-2 test are inflated, particularly in the first week, due to this bias. Therefore, individuals whose positive SARS-CoV-2 test was on admission to hospital were excluded from the hospital dataset for the analysis of positive tests for SARS-CoV-2.Bias could also have been introduced in the HES analyses due to routine testing for SARS-CoV-2 on admission to hospital21.The SCCS design examines variation in risk over time within individuals and so correct specification of the risk period is crucial. Our sensitivity analyses exploring varying lengths of risk period were all consistent with the main analyses but a very long-lasting increase in risk postvaccination would not have been detectable with this SCCS approach. Conversely, it is also possible that the time-limited healthy vaccinee effect masks a smaller, short-term increase in risk due to vaccination; myocarditis tends to appear very soon after vaccination, with evidence of the median time from vaccination to symptom onset of 2 daysAnother implication of the SCCS design is that comparisons cannot be made between the different case series, such as those vaccinated and unvaccinated at time of positive SARS-CoV-2 test. These groups can be expected to differ in important ways such as comorbidity and strain of SARS-CoV-2. Importantly, the difference cannot be interpreted as an estimate of vaccine efficacy.Lastly, our ability to analyse longer risk periods was limited by the spacing between doses. The negative effects observed in sensitivity analysis with risk periods of more than twelve weeks suggest a negative bias with risk periods that exceeded dosing intervals.28. This provides reassurance that mRNA vaccines pose minimal risk of increased mortality in the first twelve weeks post-vaccination in young individuals. However, it is important to continue to monitor mortality after vaccination as more deaths are being registered, and extend the surveillance to other age groups and deaths from other causes.Whilst COVID-19 vaccination has been linked to an increased risk of myocarditis and other cardiac events in young people, we found no evidence of substantially increased mortality risk, either due to cardiac events or overall, from mRNA vaccines, which suggest that cases of myocarditis or myopericarditis due to mRNA COVID-19 vaccines are unlikely to be fatal. We do, however, find evidence of an increased risk of cardiac death after a first dose of a non mRNA vaccine among females. It should also be noted that non mRNA vaccines are no longer used in the UK vaccination programmeEthical approval was obtained from the National Statistician’s Data Ethics Advisory Committee (NSDEC(20)12).29, by applying a more appropriate SCCS model to an updated outcome dataset, an additional, independent outcome dataset, and additional sensitivity analyses, as described further below.This analysis improves and extends a previous analysis published by the Office for National Statistics30 and hospital deaths recorded in Hospital Episode Statistics for England (HES)31. Unlike death registrations, deaths recorded in HES are not subject to delay due to coroner referrals. The deaths datasets included deaths registered by 8 June 2022 that occurred between 8 December 2020 (the start of the vaccine rollout in the England) and 25 May 2022; and HES records where the discharge status indicated deaths between 8 December 2020 and 31 March 2022. Where the same death was recorded in both the registration and hospital datasets, the date of death, date of birth, and sex were taken from the death registration, otherwise these were taken from HES. There were 367 records of deaths in HES  that did not have a linked death registration, likely due to an ongoing coroner inquest.We separately analysed two independent sources of data on death: deaths registered with the Office for National Statistics (ONS)32 and a supplementary extract from NHS point of care data provided by NHS-Digital. The NIMS data includes most COVID-19 vaccinations administered in England since 8 December 2020. However, in rare cases, if the death was recorded on the Personal Demographics Service (PDS) before the vaccination record was sent to NIMS then the patient’s vaccination registration in NIMS is not updated. The supplementary extract of NHS point-of-care data includes all vaccination records affected so. The NIMS and point-of-care extracts include all vaccinations recorded by 14 June 2022.Death data were linked to data on COVID-19 vaccination from the National Immunisation Management Service (NIMS)33 recorded between 9 September 2020 (when mass testing became available) and 31 December 2021. The latter include both laboratory polymerase chain reaction and self-reported rapid antigen tests.To assess the relative incidence of death following a positive test SARS-CoV-2, we linked death records to national testing data from pillar 1  and pillar 2 (tests in the community)The linkage across databases was conducted using NHS number, which was available for 99.96% of NIMS records, 99.7% of ONS death registrations, 99.1% of deaths in HES, and 100% of the point-of-care extract from NHS-Digital.Data linkage and preparation were conducted using R 3.5, Python version 3.6 and Spark version 2.4.34. We separated the analysis for people who were vaccinated or unvaccinated on the day the positive SARS-CoV-2 test was recorded, where vaccinated is having received at least one dose any day before the day of infection.The study population included all people whose deaths were recorded since the start of the vaccination roll-out on 8 December 2020 and who were aged 12–29 years on the date of death. There were insufficient numbers and follow-up to study vaccination in children below 12 years of age. For the analysis of deaths after a positive SARS-CoV-2 test, we further restricted the study population to deaths that occurred up to 31 December 2021 (the last date on which a positive test could be recorded in our dataset) and individuals whose positive SARS-CoV-2 test was on the day of admission to hospital were excluded. Since the Omicron-dominant period is defined as starting on the 20 December 2021, the data covers mainly the Delta dominant (17 May to 19 December 2021) and Pre-delta (pre-16 May 2021) periods35. Episodes were counted from the first positive test but only those occurring on or after 9 September 2020 were included in the analysis.The main exposure was any dose of COVID-19 vaccination within the previous twelve weeks. The comparative exposure was a SARS-CoV-2 positive test within the previous twelve weeks. The date of positive test is defined as the start of the last episode of COVID-19 for each individual, where a new episode was defined as a positive result more than 120 days after the start of any previous episode. This is in line with the time period for reinfection used in the COVID-19 Infection SurveyThree outcomes were analysed: all-cause registered death and cardiac registered death (ICD-10 code I30-I52 mentioned on the death certificate), from ONS death registrations; and all-cause hospital death, recorded in HES. Each analysis included all participants who experienced the outcome of interest (cases).36. Event-dependent exposure occurs when the event of interest influences the likelihood of exposure; death represents an extreme example in which any subsequent exposure is impossible after the event. This specific SCCS approach compares the risk of death during a predefined risk period following exposure (vaccination or positive test for SARS-CoV-2) with a reference period of all time subsequent to the risk period. By comparing time periods within individuals, time-invariant factors such as sex and, in general, age group and comorbidity, are implicitly controlled for; time-varying factors can be controlled by the inclusion of unexposed cases in the analysis and by the inclusion of calendar time as a covariate37.We used a SCCS approach designed to handle multiple event-dependent exposuresParticipants were followed from 8 December 2020 to the 25 May 2022 for death registrations and 31 March 2022 for hospital deaths and were not censored at death. For the analyses of positive SARS-CoV-2 test, follow up ended at the 31 December 2021 when testing data ended. The risk period was defined as the first twelve weeks after vaccination/positive test while the reference period as all the following weeks to the end of study. In addition, all the weeks prior to vaccination/positive test from 8 December 2020, or all weeks throughout the follow up time if the individual was unvaccinated/never positive, were also included in order to adjust for calendar time.The exposure was the week since vaccination/positive SARS-CoV-2 test for each of the first twelve weeks individually, or the twelve weeks together. For vaccination, the risk weeks were further categorised by dose or considered for all doses combined.39 The calendar day of the start of each week was included in the model using a restricted cubic spline. Adjusting for calendar time is a way to capture the impact of increasing registration delays over time (more recent deaths being more likely to be under coroner review), as well as of seasonal mortality trends and changing SARS-CoV-2 infection rates. The length of each week in days was included as an offset in the model, as some weeks are not complete (such as if a vaccination occurs part way through a week). Incidence rate ratios (IRRs) for cardiac and all-cause deaths in risk periods relative to reference periods were estimated using each model. 95% confidence intervals were obtained from the model estimates. For the main estimates, we also calculated 95% confidence using bootstrapping. Similar analyses were conducted focusing on unvaccinated and vaccinated individuals for deaths after positive test for SARS-CoV-2.The SCCS models were fitted using a conditional Poisson regression model using a pseudo-likelihood method on a person-week level dataset40. We used the total number of people who tested positive in pillar 1 and pillar 2 stratified by vaccination status on date of test registration over the period of interest as a measure of exposed cases . Analyses were also stratified by vaccine vector (mRNA or ‘not mRNA or unknown’) for the first two doses. ‘mRNA’ includes the Pfizer BNT162b2 Pfizer-BioNTech and mRNA-1273 Moderna vaccines. ‘Not mRNA or unknown’ includes the ChAdOx1 Oxford-AstraZeneca vaccine, all other recorded vaccine manufacturers and non-recorded vaccination manufacturers. The analysis of each vaccine vector included all unvaccinated people and all people who received at least one dose of the vaccine vector of interest. The doses were renumbered to correspond to the first, second and third doses of the vaccine vector of interest only. We also break down the analysis of vaccine vector by sex.36. We also assessed whether results could be biased by routine testing for SARS-CoV-2 infection in admission to hospital by omitting individuals who had positive test date on the day of admission to hospital for the hospital episode prior to death in the analyses of hospital deaths.We assessed sensitivity to specification of the risk period by exploring a range of different lengths of risk period and to the type of calendar adjustment by adjusting in fortnightly intervals rather than using a restricted cubic splineNo patient or member of the public was involved in this study.Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


The primary outcome was the injection attempt defining one attempt and multiple attempts. 90 patients were recruited and allocated to 30 per group. There were no differences between A, AL, and PAL respectively regarding the success in one attempt rate , VAS during local anesthetic injection , VAS during intra-articular injection , radiation doses , radiation time , and complications. The post-injection mHHS was improved in all three approaches without significant differences.Fluoroscopy-guided injection via the anterior (A), anterolateral (AL), or proximal anterolateral (PAL) approaches are the common hip injection techniques without comparing the efficacy of the three techniques. The prospective randomized controlled trial was conducted from August 2020 to March 2022. Included patients with intra-articular hip disorders indicated an intra-articular steroid injection. Excluded significant spine pathology with radiculopathy or significant neurological deficits, previous hip surgery of the injection side, suspected tumor or infection origins, steroid or contrast media allergy, and body mass index > 35 kg/m Intra-articular steroid injection has a sensitivity of 91.5% and specificity of up to 100% to identify intra-articular pathology if the patient experienced improvement of symptoms after the procedures2.The use of intra-articular steroid injection of the hip helps differentiate between intra-articular hip pathologies and extra-articular hip pathologies as the causes of hip pain and for the therapeutic management of hip osteoarthritis or synovitis8, the anterolateral approach (AL)9, and the proximal anterolateral approach (PAL)10. Despite the large quantities of research surrounding the use of intra-articular steroid injection of the hip, the majority of researchers emphasized the improvement of symptoms, and in comparing the assistive method such as comparing ultrasound-assisted intra-articular hip injection with fluoroscopic assisted intra-articular hip injection. None of the studies focus on comparing the accuracy and efficacy of three common fluoroscopic guidance intra-articular hip injection approaches.Currently, the intra-articular hip injection has been widely used, with the three common approaches being the anterior approach (A)This study aimed to compare the efficacy of anterior, anterolateral, and proximal anterolateral approaches of the fluoroscopy-guided intra-articular hip injection. We hypothesized that there would be a significant difference in efficacy between the three intra-articular steroid injection approaches and that this study would be able to guide the physician in choosing the best approach for their patients.The patient recruitment was performed at Thammasat University Hospital in January 2020 after approval by the ethics committee. All procedures performed in studies involving human participants were approved by the ethics committee of Thammasat University (Registration no. MTU-EC-OT-1-184/62) on 20/01/2020. The study protocol was registered and submitted to the Thai Clinical Trials Registry (TCTR) with the TCTR identification number TCTR20200722002. The date of the first registration was 22/07/2020. All methods were performed in accordance with the relevant guidelines and regulations.2.A prospective randomized controlled trial of hip intra-articular injection was started from 01/08/2020 to 31/03/2022 after the protocol had been registered and approved in the clinical trial registry. The inclusion criteria included patients with intra-articular hip disorders from osteoarthritis, femoroacetabular impingement (FAI), synovitis, and labral lesions who indicated an intra-articular steroid injection. The exclusion criteria included significant degenerative spine pathology with radiculopathy or significant neurological deficits, previous hip surgery of the injection side, presence of an osteolytic or osteoblastic lesion on imaging suspected tumor or infection origins, active infection around the hip, allergic to steroid or contrast media, and body mass index (BMI) more than 35 kg/m11.The term “efficacy” in this study was primarily defined as the success of “one attempt of fluoroscopy-guided injection”. The secondary outcomes of efficacy included pain during injection, radiation doses and time, complications, and short-term functional score improvement using the modified Harris hip score (mHHS)From the pilot study, the resulting efficacy defined by “single attempt of injection” for the anterior, anterolateral, and proximal anterolateral approaches in 30 patients were 70%, 70%, and 90% respectively. Based on a non-inferiority trail with a 10% non-inferiority limit, the significance level  was 0.05, and the power was 90%. The output of the sample size calculation from n4Studies was 29 samples per group, with a total of 87 patients.After patient screening and recruitment by eligible criteria, all patients were advised of the procedures, risks, and benefits of the research, thereafter the patients were informed and signed the consent form. The pre-injection mHHS was evaluated on recruitment day. On the injection day, patients stayed in a supine position and put the pillow underneath the patient’s knees to provide about 20 degrees of hip flexion. The injection site was prepared in a standard sterile fashion. The approach of injection was random using computerized randomization with the block of 6 and allocated with a sealed opaqued envelope before injection. The injection was performed by a single fellowship-trained surgeon in hip arthroscopy under fluoroscopic guidance to enhance visual clarity, in the operation room without the pre-medication. The surface landmark of each injection approach on the right hip is shown in Fig. The intra-articular injection was performed using needle No.25, and a local anesthetic solution of 1% xylocaine without adrenaline 3 mL was injected. The Visual analog scale (VAS) during local anesthetic injection was evaluated. Then, using spinal needle No. 20 (Length of 90 mm) to inject the hip joint following protocol for each approach, the mixture of “Triamcinolone acetonide (40 mg) 1 mL plus an OmniplaqueTM (350mg/mL) 1 mL plus the 1% Xylocaine without adrenaline 8 mL” were injected into the hip joint. Fluoroscopic imaging (PHILIPS BV Pulsera) was used in the anteroposterior (AP) view to identify whether the approach was successful. “Success” is defined by the contrast media filled inside the hip capsule.“Single attempt” was defined as the single shot in injection without redirection of the spinal needle . “More than one attempt or multiple attempts” was defined as the need for redirection by pulling a spinal needle out for more than 40 mm . VAS during intra-articular hip injection was recorded.Radiation exposure was recorded as doses and radiation time. Complication such as infection or vasovagal symptoms was recorded (if occurred). The patients were made appointments for subsequence follow-ups at 2 weeks intervals and were evaluated for post-injection mHHS.Descriptive data were reported as frequencies  or mean ± standard deviation (SD). The Fisher exact test was used to compare categorical data between groups e.g. gender, and affected side. The Oneway-ANOVA test was used for comparisons of continuous variables with normal distribution, and the Kruskal-Wallis’s test was used for continuous variables with non-normal distribution including age, BMI, VAS during local anesthetic injection, VAS during intra-articular injection, radiation exposure, pre-injection mHHS, post-injection mHHS. P < 0.05 was statistically significant. Data were analyzed using Stata version 14.2 .A total of 90 patients were included with 30 patients in each group. The CONSORT flow diagram is presented in Fig. Patient characteristics are shown in Table The success in “single attempt” was 80%, 80%, and 90% in A, AL, and PAL approaches respectively. There was no statistically significant difference in single-attempt success between the three injection techniques (p = 0.533). The accuracy rate of intra-articular injection in the study was 100% in all approaches either single or multiple attempts by the fluoroscopic guidance and controlled injection, confirmed by observing the contrast media filled within the hip joint capsule in all cases. There were no statistically significant differences in the VAS during local anesthetic injection (p = 0.5), VAS during intra-articular injection (p = 0.587), radiation doses (p = 0.788), and radiation time p = 0.723). The results are shown in Table 3. The reThe overall complication was 1 patient of 90 (1%) in the PAL injection approach, the patient developed vasovagal symptoms after a single attempt of injection which spontaneously resolved after a rest of about 2–3 minutes without the need for further management. There was no patient with injury of the lateral femoral cutaneous nerve of the thigh or infection after injection. For the post-injection mHHS collected 2 weeks after injection, there was an improvement from pre-injection in all approaches.From this study, using the three approaches, namely anterior, anterolateral, and proximal anterolateral approaches for fluoroscopy-guided intra-articular hip injection, the overall successful intra-articular hip injection in a single attempt is 83%, with the anterior approach in 80%, anterolateral approach in 80%, and proximal anterolateral approach in 90%, we did not find the statistically significant differences between three approaches. The VAS during local anesthetic injection, VAS during intra-articular injection, radiation doses, radiation time of exposure, and the post-injection mHHS were similar in all three approaches without significant differences between groups.8–96.7%9 accuracy for the anterior approach, 94–95% accuracy for the proximal anterolateral approach12, and 100% accuracy for the anterolateral approach9. Our study had a high accuracy rate, we hypothesized that this might result from the difference in the experience of performers and the injection techniques in each study. Furthermore, in our injection technique, the hip was about 20 degrees flexion, we would check the direction of the needle using the fluoroscopic control until reached the desired direction to the anterior or anterolateral site of the femoral head-neck junction and the tip of the needle always need to touch the bone before we injected. We injected the steroid and the mixtures by slightly pushing and pulling the needle backward-forward around the point of injection area and controlling with the sequential fluoroscopy. With this technique, we’ve achieved a standardized procedure with reliable results for hip injection.The accuracy rate in our study is 100%  intra-articular injection in all approaches during the use of fluoroscopic guidance and controlled injection. After the tip of the needle reached the femoral head-neck area, we found the contrast within the hip joint capsule in all patients, we could not observe any extra-articular leakage of the radio-opaque media. Compared to the previous study on the accuracy of each approach which reported a 939 showed anterior and lateral median radiation doses of 1 mGy and 3 mGy, respectively. The radiation doses in our study are within the standard exposure for radiographs of the pelvis (1.31 mGy)13.For the secondary outcome, we report no statistically significant difference in pain using a visual analog scale (VAS), both during local anesthetic injection and intra-articular hip injection between the three approaches, with the mean VAS of about 4 on both procedures. Even though the data focusing on pain during the procedure have not yet been previously reported, from the result of our study which showed a similar level of pain in all approaches, we can conclude that pain should not be a factor when considering the intra-articular hip injection approaches. Additionally, we’ve found no statistically significant difference in radiation exposure between the approaches. The average radiation doses  were less than another study15 related to the injection.Throughout our study, we’ve only experienced one complication from ninety patients, which accounts for 1.1%. Details on the event were as follows, the patient was 29 years 29-year-old man diagnosed with secondary osteoarthritis of the right hip after trauma, he was allocated to the PAL injection group, and during the procedure, he experienced lightheadedness with normal vital signs and oxygen saturation. The symptoms recovered spontaneously after resting for 2 minutes, thus the symptom was considered to be caused by vasovagal symptoms. Following 2 weeks, the patient returned for follow-up with an improvement in mHHS from 58.3 pre-injection to 82.5 post-injection, no further complication was recorded. There were no serious complications18 and we aimed to evaluate the short-term efficacy between the three different hip injection approaches.Comparing the pre-injection mHHS with post-injection mHHS, there was no statistically significant difference between the three groups. However, comparing the pre-injection score with post-injection mHHS in the same group, we reported a significant improvement in post-injection mHHS in all three groups. The results represent the improvement in very short-term outcomes of successful intra-articular steroid injection of the hip despite the approach being used. We did not evaluate the outcomes of longer follow-up because of the natural history of the intra-articular steroid injection that mainly affected only about 6–12 weeks after injection20. This study used the fluoroscopy-guided hip injection, which is common and standard for the diagnostic and therapeutic management of hip disorder patients22.Hip injection using anatomic landmarks without ultrasonographic or radiographic assistance is useful and provides acceptable accuracyOur study is the first study to compare the efficacy between three commonly used techniques for intra-articular hip injection. No study compared the safety and outcomes of each injection approach. Additionally, all operating procedures were performed by a single fellowship-trained surgeon in hip arthroscopy, and the operative setting was standardized in every patient therefore the errors and variations in the injection techniques were minimized in our study.Our study is not without limitations, however. Due to the single well-trained performer in our study, our result may not be generalizable to a general orthopedic surgeon or general practitioners, however in providing detailed instruction on the operative setting and procedure we used in this study, we believed that similar result could be achieved even with less experienced practitioners. Furthermore, our study has a small sample size, which might not represent the general population, and has low power to detect the statistical significance. The very short-term outcomes were evaluated that could not evaluate the effect of each approach in terms of medium- to long-term therapeutic outcomes. Finally, the non-blind nature of our study design could cause bias in the outcome measure.Fluoroscopy-guided hip injection via the anterior (A), anterolateral (AL), or proximal anterolateral (PAL) approaches were comparable in efficacy regarding the number of attempts, VAS during injection, radiation exposure, post-injection mHHS, and complications. The physicians can choose any of their preferred fluoroscopy-guided hip injection approaches.