Four-dimensional computed tomography (4DCT) is one of the preoperative imaging modalities that can be used to localize a parathyroid adenoma in primary hyperparathyroidism patients however, sensitivity differs in literature and could be improved especially for multiglandular hyperplasia or double adenomas. The most robust feature on the 4DCT for the differentiation between parathyroid adenoma and thyroid gland tissue is arterial enhancement. To make this better visible, we have developed a subtraction map that shows arterial enhancement as a color scale to increase sensitivity for 4DCT. In this report of 3 cases, we present the usefulness of this subtraction map in a 54-year-old male, a 57-year-old female and a 51-year-old male. Subtraction maps may increase sensitivity for 4DCT, especially for multiglandular hyperplasia or double adenomas. Primary hyperparathyroidism (PHPT) is caused by excessive secretion of parathyroid hormone, resulting in hypercalcemia Historically, bilateral neck exploration (BNE) was the treatment of choice, without any prior imaging. However, with the introduction of minimally invasive parathyroidectomy (MIP), preoperative imaging has become essential to identify the location of the adenoma before surgery. Several imaging modalities can be used to localize a parathyroid adenoma, including 4-dimensional computed tomography (4DCT). The sensitivity of 4DCT ranges between 48% and 97% The arterial enhancement is the most robust feature present on the 4DCT to differentiate between parathyroid adenomas and thyroid tissue. To make this better visible, we have developed a subtraction map that shows the arterial enhancement as a color scale. In this report of 3 cases, we present the usefulness of this subtraction map. This report of 3 cases was approved by the local ethics review board (Reference number 2022-2154).Patient 1 is a 54-year-old male with PHPT [PTH 45.3 pmol/L  and calcium 2.86 mmol/L ] and severe osteoporosis. Preoperative imaging consisted of a 4DCT on which a parathyroid adenoma was located dorsally of the left upper quadrant see . A thyroThe second patient is a 57-year-old female with PHPT  and osteopenia. A 4DCT, a Tc-99m-Sestamibi SPECT/CT and a F-18 choline (FCH)-PET/CT were acquired as preoperative imaging, all with negative result , Fig. 7.The third patient is a 51-year-old male with PHPT  and a vitamin D deficiency [29-47 nmol/L ]. A 4DCT was performed, on which no parathyroid adenoma was identified with certainty . A subtrThe current principle to differentiate parathyroid adenomas from thyroid tissue is based on the rapid arterial contrast wash-in of parathyroid adenomas. Our subtraction map demonstrates the arterial enhancement of tissues, which is normally done by the radiologist by simultaneous visual evaluation of the different contrast phases, which is obviously subjective and highly dependent on experience. The subtraction map essentially automates this task and is therefore expected to increase the detection rate of parathyroid adenomas on 4DCT. In all 3 cases, the subtraction map was successful, as an adenoma was revealed that was not identified initially using the 4DCT.There is a big variety in sensitivity in literature for 4DCT. Kluijfhout et al. Two parathyroid adenomas were located on the subtraction map in our first case. In contrast, only 1 parathyroid adenoma was detected using 4DCT. Excision of this adenoma did not result in an adequate PTH decrease, while removal of the second adenoma did lead to satisfactory PTH reduction. This suggests superior evaluation of 4DCT when the subtraction map is used in addition to the original CT series, and it can be beneficial in case of possible double adenomas or multiglandular disease in particular. Moreover, the subtraction map may counter satisfaction of search, as the subtraction map clearly reveals possible structures that show increased arterial enhancement, which could improve the detection rate for double adenomas or multiglandular disease.Furthermore, the first case demonstrates that the subtraction map might be superior in differentiation between thyroid nodules and parathyroid adenomas in close relation to the thyroid. In our case, the cystic parathyroid adenoma was mistakenly regarded as cystic thyroid tissue, based on the 4DCT. During surgery, there was no adequate PTH decrease after excising the adenoma on the left and a second adenoma was identified, which was subsequently confirmed by histology. In retrospect, we have created a subtraction map which revealed a clear difference in arterial contrast enhancement between the parathyroid adenoma and the thyroid. This suggests that the addition of a subtraction map may remarkably improve differentiation of cystic adenomas and/or adenomas located closely to the thyroid.The second case shows that a subtraction map might increase detection rates for 4DCT. In this case, the adenoma was located on the subtraction map, while being undetected on other imaging modalities. Because of the subtraction map, the parathyroid adenoma was identified, which in retrospect could then be located on the 4DCT and FCH-PET/CT as well. As a result, the surgery changed from the invasive intervention BNE to MIP.The third case shows using the subtraction map as confirmation tool. Initially, an experienced radiologist could not identify the adenoma with certainty. The subtraction scan confirmed the expected location, which was later confirmed by the SPECT/CT. Therefore, no additional imaging was necessary.This report of 3 cases shows the potential benefit of a subtraction map in addition to the 4DCT. It is a convenient tool for sensitive detection of possible double adenomas or multiglandular disease. Further research is necessary to determine the role of subtraction maps in 4DCT and preoperative imaging for PHPT.Written informed consent for the publication of this report of 3 cases was obtained from the patients.


Many persons with multiple sclerosis (pwMS) desire to learn how health behaviour changes  might help them manage their disease. Previous research has shown that certain health behaviour changes can improve quality of life (QoL), fatigue and other MS outcomes. Digital health applications may be well suited to deliver relevant health behavioural interventions because of their accessibility and flexibility. The digital health application “levidex” was designed to facilitate health behaviour change by offering evidence-based patient information and cognitive-behavioural therapy techniques to pwMS. By doing so, levidex aims to improve QoL and MS symptoms such as fatigue and mental health.A previous study reported on the development of levidex; this non-randomised pilot study examined the feasibility (practicability and acceptability) of levidex in pwMS with moderate to severe disability. Furthermore, the intervention’s impact on empowerment, stress management, and relevant health behaviours  was explored.levidex was originally developed for newly diagnosed pwMS in the first year after diagnosis and eventually modified to offer access to pwMS with moderate to severe disability. Participants (n = 43) with an Expanded Disability Status Scale between 3.5 and 7.5 and a disease duration of more than one year were eligible to participate. The intervention was used over a period of six months with measurement time points at baseline, month 3 and month 6.Out of 38 participants who completed the six-month intervention period, 18 (47.4%) completed all 16 modules and 9 (23.7%) reached modules 13–16, the long-term maintenance part of levidex. Participants rated levidex positively in terms of practicability and acceptability and had only few points of criticism such as to include more physical exercise routine suggestions suitable for participants with severe impairment. Data on secondary endpoints showed no significant changes.This pilot study provided evidence for the practicability and acceptability of levidex, a digital health application designed to facilitate health behaviour change in pwMS with moderate to severe disability. Adequately powered randomised controlled studies with longer follow-up periods are needed to clarify the benefit of levidex in pwMS with moderate to severe disability.German Clinical Trials Register (DRKS) DRKS00032667 (14/09/2023); Retrospectively registered.The online version contains supplementary material available at 10.1186/s12883-023-03434-w. Multiple sclerosis (MS) is a neurodegenerative disease that affects the central nervous system. About 85% of persons with MS (pwMS) are diagnosed with relapsing-remitting MS (RRMS), whereas the remaining 10–15% are diagnosed with a primary-progressive form (PPMS) .While there has been substantial progress in the medical treatment of RRMS, the situation for SPMS and PPMS is not satisfactory . IntervePwMS have a high affinity to web-based information and use of eHealth technologies . Over thDigital behaviour change interventions provided via the internet could help fill the gap between high patient demand and limited clinician resources . Until nThis single-arm prospective pilot study tested feasibility criteria which included the practicability  and the acceptability  of levidex, a complex digital health application that promotes health behaviour change in pwMS with moderate to severe disability.For eligibility, participants had to be aged between 18 and 65 years and diagnosed with a clinically definite MS for more than one year. As we did not want to exclude persons with RRMS with moderate to severe disability, we did not limit inclusion criteria to SPMS and PPMS. In addition, participants had to have an Expanded Disability Status Scale (EDSS) score between 3.5 and 7.5, indicating moderate to severe disability ranging from gait impairment to a loss of ability to walk . Despitewww.gaia-group.com), a small-to-medium enterprise that focuses on research and development of digital health applications. The underlying software used for the development is ‘Broca’, a proprietary software developed by GAIA. It utilizes rule-based algorithms to create “simulated dialogues” in which patients interact with the programme by choosing from predefined response options, which are then used to custom-tailor subsequent content. Broca-based digital health applications for a range of psychiatric and somatic conditions have been shown to be effective in more than 15 RCTs [levidex is a complex behavioural digital health application tailored for pwMS. It is based on a similar programme, termed ‘optimune’, which aims to support health-promoting behaviour change among breast cancer survivors and has been evaluated in an RCT . Both pr 15 RCTs –36. To f 15 RCTs . These i 15 RCTs .For this study, minor adjustments were made to improve the suitability of levidex for more experienced pwMS with moderate to severe disability (EDSS 3.5–7.5). This includes, for example, a change in the tone of the first module, shifting it away from a more cautious and sensitive tone designed for newly diagnosed pwMS to a more seasoned or experienced tone for pwMS with a longer disease duration. Information regarding immunotherapy decision-making was removed, as in later stages of the disease, immunotherapy may be less relevant. Furthermore, the physical activity part was slightly modified to provide more options for participants with physical impairments.Access to new modules is only provided after a predetermined waiting period to allow participants to reflect on provided content as well as to complete different exercises  before starting a new module. The frequency of newly available content starts with two to three weekly modules and is reduced to biweekly modules and eventually to one module per month in the maintenance phase (last four modules), which results in approximately six months required to comfortably complete all 16 levidex modules.Participants were recruited from the registry database of the MS day clinic at the University Medical Centre Hamburg-Eppendorf (UKE). Participants were selected based on their EDSS score from their last consultation. PwMS who fulfilled inclusion criteria and had indicated a general interest in study participation were contacted by mail. After expressing their willingness to participate, they were additionally screened for eligibility by telephone. Eligible participants then signed informed consent and were provided with login details for levidex. Access to levidex was provided for six months. Due to the beginning of the COVID-19 pandemic, in-person meetings could not take place. Therefore, disability was self-reported by the participants at the beginning of the study using the Patient Determined Disease Steps (PDDS) .Data was collected over a period of six months. All outcome measures as well as their measurement timepoints are provided in Table The primary outcome was the feasibility of levidex, measured as its practicability and acceptability, to determine whether participants found the intervention helpful and were able to apply the suggestions and techniques into their daily lives. This was measured by usage data and a self-developed feasibility questionnaire , depressive and anxiety symptoms, dietary behaviour, and physical activity.Patient empowerment was measured with the Patient Activation Measure (PAM) questionnaire . The rawPhysical activity was measured via the Godin Leisure-Time Exercise Questionnaire (GLTEQ) . We usedDietary behaviour was measured with three inventories. A validated healthy diet screener assessing intake of 11 key food groups that are thought to be preventive for common chronic diseases was used to describe dietary habits and to capture changes in general dietary behaviour , 49. TheExcept for myfood24, all questionnaires were paper-based and sent to the participants by regular mail in pseudonymised form. The myfood24 tool is a secure online platform that is accessed through links sent to participants by e-mail by members of the study team. It is managed by the Dietary Assessment Ltd (a Spin-Out-company of the University of Leeds). Data is stored on a server in the Netherlands with a backup in England. The institution acts in accordance with the General Data Protection Regulation of the European Union and uses data in pseudonymised form.The levidex application contacts participants automatically through optional regular e-mail and short text message reminders to improve adherence to the intervention. In addition, the research team monitored participants’ usage through biweekly usage reports and contacted participants via e-mail or telephone in case of non-usage. During the first three months of study participation, corresponding to completion of modules 8–12, non-usage was defined as not logging into levidex for more than two weeks. Towards the end of study participation, no login for longer than one month was considered as non-usage. Regarding the completion of questionnaires, participants were asked to fill out the forms within one month. In case of missing data, participants were contacted by a study centre member by e-mail or telephone.As there are no clear guidelines on how large the sample size of a feasibility study should be , we consFrom February to August 2020, 59 pwMS from the MS day clinic of the UKE were recruited. Forty-three pwMS completed baseline data and registered for levidex. Baseline data collection started in April 2020. From May 2020 to March 2021, 43 participants used the intervention. Out of these, 38 participants completed the six months intervention period and the final assessment. The myfood24 dietary recall was not completed by two participants who perceived it as too time-consuming. Four participants insufficiently completed the diet screener requiring exclusion of these participants from analysis. A visualisation of the participant flow is provided in Fig. Overall, the sample had a mean age of 52 years and 60.5% were female. Participants were moderately to severely impaired as 81.4% of participants experienced gait disability or needed an ambulatory device. In terms of MS forms, 69.8% were diagnosed with progressive forms of MS while 25.6% were diagnosed with RRMS. In total, 44.2% were on immunotherapy regimes and 93% used the internet at least two times per week. All baseline demographics and clinical characteristics are provided in Table At month 3 of study participation, the median login number of all study participants was 13. This increased to a median of 20 logins at month 6. There were some users who logged on significantly more often with login numbers of up to 180 times.After six months, 27 (71.1%) participants reached the long-term maintenance part of levidex and therefore had access to most content. Out of these 27 participants, 18 (47.4%) participants finished module 16 and hence completed levidex. Three (7.9%) participants did not advance beyond module 6 (introduction and important foundations), and eight (21.1%) participants did not advance beyond module 12 (advanced information and integration into daily routines). With more than half of the participants having logged on enough times to finish all 16 levidex modules and around half of the participants having finished all modules, both ways of measuring usage complemented each other.Thirty-three (86.8%) participants were reminded by the study team once or not at all during the intervention period . In five (13.2%) cases, there were up to three contacts. This occurred when participants indicated having experienced stressful life events and wished for an additional reminder to keep up usage of levidex.Participants were asked to rate the practicability of levidex as well as the perceived benefit in form of self-reported stages of health behaviour change and self-reported health behaviour changes. The corresponding data is provided in Fig. Between 65 and 75% of participants agreed or partly agreed to either have set goals to change their health behaviour, to have made concrete plans to change their health behaviour, to have changed their health behaviour or to want to maintain health behaviour changes.Eleven participants stated that they did not change their health behaviour at all, and out of these, nine disagreed or partly disagreed to have set goals. Out of the 27 participants who stated to have changed at least a part of their health behaviour, the mean score on the 11-point Likert-scale ranged from 4.2 for sleeping behaviour to 7.3 for physical activity.Three (8.1%) participants perceived the messages sent via levidex as stressful or pressuring and seven (18.4%) participants perceived the work with levidex as burdensome. With a mean of 8.2 and 8.7, participants agreed that the navigation was easy and information was understandable.Four main categories emerged as barriers to health behaviour change: The intervention itself, individual problems, systemic issues and the COVID-19 pandemic. Most frequently mentioned barriers were lack of motivation and discipline, the COVID-19 pandemic and MS-associated health issues. The results are visualised in Fig. The corresponding data is provided in Table In terms of empowerment, the PAM showed no change over the course of the study. The average score of 72.4 at baseline puts the participants in the highest of the four activation categories at the beginning of the study. QoL as measured by the HAQUAMS as well as anxiety and depression as measured by the HADS showed no change over the course of the study.The evaluation of the summary diet score indicated a trend towards improvement after 3 and 6 months  and social participation due to negative attitudes from others . FurtherAnother valuable insight of our study is that both the food category and micronutrient intake profile that is associated with a Mediterranean dietary pattern mostly changed in a coherent way. Further studies could use similar approaches to objectify dietary changes.It is possible that the pandemic setting supported improvement in dietary behaviour with the reasons for this effect remaining unclear . In contEven though there was no improvement in the average physical activity score, 12 participants managed to successfully increase their physical activity level in pre-defined categories. Several reasons might explain these results. First, the COVID-19 pandemic may have interfered with physical activity as it has been observed that especially activities with moderate and high intensity decreased in comparison to pre-pandemic levels . MoreoveThere was a substantial decrease of physical activity among participants who already had much higher-than-average levels of physical activity in our study, which might be a result of the COVID-19 pandemic and the closing of public sports facilities. It must also be noted that studies revolving around health behaviour change topics might be more prone to selection bias. To conclude, future studies could screen for lower baseline empowerment levels to deliver a better estimate for beneficial effects of the intervention.As mentioned earlier, some participants criticised that the physical activity part was not sufficiently adapted to the needs of participants with more severe impairments. It was noted that levidex included only few exercises that can be performed while sitting and which do not require balance or complex motion sequences. Some participants indicated that being confronted with unfeasible exercises led to disappointment.The lack of change in depression and anxiety, empowerment and QoL outcomes might be caused by insufficient sample size. As this was a feasibility study, no power calculations were performed and therefore all statistical findings could be hampered by a lack of power. Nevertheless, in light of possibly elevated psychological stress associated with the COVID-19 pandemic, levidex might have helped to maintain the status quo or prevent deterioration of mental health .This study has limitations that need to be considered. First of all, as this study was designed as a pragmatic pilot study, only patient-reported outcome measures were used. Further, personal reminders were implemented which might not be feasible in a real-world setting due to limited resources in standard care. However, levidex itself already incorporates pre-programmed reminders to enhance adherence which could be further improved in the future. Baseline values for patient empowerment were already high, as there were no exclusion criteria concerning participants’ activation level and general health behaviour. Therefore, the sample was presumably biased to more active pwMS lowering the chance to show behaviour change.The strength of this study was its use of a programmatic digital health application combining EBPI with CBT and BCTs for all major health behaviour areas. Moreover, it showed good adherence to the intervention despite it being originally designed for newly diagnosed pwMS and only slightly adapted to fit pwMS with moderate to severe disability.The intervention was well accepted and used frequently by the participants. Further studies with larger sample sizes and longer follow-up periods need to be conducted to solidify the evidence on the use of digital health applications such as levidex.Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2


The fertilization of agricultural soil by organic amendment that may contain antibiotics, like manure, can transfer bacterial pathogens and antibiotic-resistant bacteria to soil communities. However, the invasion by manure-borne bacteria in amended soil remains poorly understood. We hypothesized that this kind of process is both influenced by the soil properties  and by the presence of contaminants such as antibiotics used in veterinary care. To test that, we performed a microcosm experiment in which four different soils were amended or not with manure at an agronomical dose and exposed or not to the antibiotic sulfamethazine (SMZ). After 1 month of incubation, the diversity, structure, and composition of bacterial communities of the soils were assessed by 16S rDNA sequencing. The invasion of manure-borne bacteria was still perceptible 1 month after the soil amendment. The results obtained with the soil already amended in situ with manure 6 months prior to the experiment suggest that some of the bacterial invaders were established in the community over the long term. Even if differences were observed between soils, the invasion was mainly attributable to some of the most abundant OTUs of manure (mainly Firmicutes). SMZ exposure had a limited influence on soil microorganisms but our results suggest that this kind of contaminant can enhance the invasion ability of some manure-borne invaders.The online version contains supplementary material available at 10.1007/s00248-022-02020-w. Organic amendment enables fertilization of agricultural soils and allows the recycling of a substantial amount of waste produced by livestock such as manure . This enIn this context, it is important to determine what enables or prevents a microbial invasion from manure to soils. In particular, knowledge is needed to better understand what make manure-borne microorganisms more likely to invade a soil, taking into consideration the influence of both the biotic and abiotic components of soil and manure (as well as their interactions). This question relates to the concept of microbial coalescence, which focuses on the outcome of mixing different environments, each presenting different biotic and abiotic characteristics , 13. AmePrevious studies showed that amended soils durably differ from manure-free soils when considering the abiotic part of coalescence , 15. HowSeveral environmental factors might influence the invasion success of manure-borne bacteria in soil. Firstly, based on the concept of coalescence, the invasion capacity is probably soil-dependent since it may vary according to the biotic  and abiotic  characteristics of the amended soil, which determine the potential presence of available ecological niches . It can In this context, the purpose of our microcosm study was to assess soil invasion by manure-borne bacterial OTUs during an agronomical coalescence event of soil fertilization with manure according to the presence or not of antibiotic pressure. We firstly hypothesized that the capacity of manure-borne bacteria to invade the amended soil would be variable according to the intrinsic abiotic and biotic properties of soils. In particular, among the four soils used, one was amended (in situ) 6 months earlier by the same manure to test whether previous amendment had an impact on invasion outcomes. In addition, we also hypothesized that some manure-origin OTUs would be distinguished from the emerging rare soil OTUs in response to the repetition of their invasive pattern in several of the tested soils. Finally, to verify whether antibiotics can interfere with the coalescence process by promoting the invasion of soils by manure-borne OTUs, a modality made of the antibiotic sulfamethazine (SMZ), which is commonly used in veterinary care, was considered , 32. AftThe four soils, named A, B, C, and D, are anthrosols selected for their different textures and physico-chemical characteristics benzenesulfonamide, Sigma-Aldrich, France] was diluted to 100 mg ml−1 in water and filtered (0.2 μm). Before applying treatments, the manure was incubated overnight at room temperature (20 ± 1°C). Treatments were prepared a few minutes before their application on soils to reach nominal concentrations of 100 mg of SMZ kg−1 dry soil and 13 ml of manure kg−1 dry soil. Then, the microcosms were incubated for 1 month in the dark at 20°C (±1°C). Their humidity has been kept stable by regular watering.The experiment consisted of four different soils amended or not with manure and treated or not with SMZ. Considering four conditions and five replicates per condition, 20 microcosms for each soil were prepared as described below. Fifty grams dry weight (DW) of soils were placed in glass bottles closed with air-permeable lids made of gauze and cotton. In order to gently activate the native soil microorganisms, soil microcosms humidified up to 80% of their water content at pF 2.7, were incubated for 1 week at 20°C (±1°C). SMZ sodium salt [SMZ, CAS number: 1981-58-4, 4-amino-DNA was extracted from subsamples of 250 mg of soil for each microcosm using the DNeasy PowerSoil-htp 96-well DNA isolation kit  following the manufacturer’s instructions and stored at −20°C. As previously described by Billet et al. (2021), the V3–V4 hypervariable region of the bacterial 16S rRNA gene was amplified using a two-step PCR and sequenced on MiSeq  and a Jupyter Notebook developed in-house was used to analyze the sequence data . BrieflyStatistical analyses were carried out using RStudio statistical software (version 1.2.5033). For each soil, we used analysis of variance (ANOVA) model to determine the effects of treatments on the alpha diversity indices of bacterial communities. Normality and homogeneity of the residual distribution were inspected and log10-transformations were performed when necessary.Permutational multivariate analysis of variance (PermANOVA) was used to test significant differences in communities’ structure using adonis function implemented in the vegan package (permutations = 999).Y follows a Poisson law of parameter Λ as For each soil, the OTU table was filtered (relative abundance >0.05%) and to detect the OTUs significantly impacted by treatments (antibiotic treatment and manure amendment) a generalized linear mixed model was developed. Considering that, the OTU abundance oik is the offset calculated as the log of the sample read sum. μ is the intercept. αi is the fixed effect of manure amendment . βj is the effect of SMZ treatment . (aβ)ij is the interaction effect between the manure amendment and SMZ treatment. Ik is the random effect of the individuals. Zijk is the residual error.p < 0.05). Subsequently, we performed a post hoc Tukey test with the emmeans function of the emmeans package (version 1.6.1) to detect significant differences caused by manure amendment depending of treatment with SMZ (significance threshold was set at p < 0.05). A Bonferroni correction was used to decrease substantially the probability of detecting false positives (significance threshold was set at 0.05).The analysis was performed using the glmer function of the lme4 package (version 1.1-27). Chi-squared test was performed to detect significant pairwise differences for each treatment . Only the community structure of soil D, which received in situ manure from the same pig nursery 6 months before the experiment, was not impacted by manure amending. Therefore, after the 1-month incubation period, the structure of bacterial communities in three of the four soils differed depending on manure amendment modality. Regarding alpha diversity, manure amendment significantly increased the PD whole tree index in all the soils, including soil D . This indicates that phylogenetic diversity of bacterial community was higher in soils amended with manure than in those not amended.Principal component analysis (PCoA) performed on unweighted unifrac matrices showed that after 1 month, the composition of the bacterial community in soils A, B, and C amended with manure was distinct from those not amended : 10 OTUs related to Proteobacteria, 11 to Firmicutes, 2 to Actinobacteria, 1 to Synergitetes, and 1 to Bacteroidetes . Interestingly, the comparison of the four manure-amended soils showed that 10 of these 15 OTUs had a redundant pattern in at least two of them. Among them, 1 Pseudomonodales was significantly impacted by manure in all four soils without exception. It was the most abundant in manure explaining by itself 11.4% of its composition while it was only detected in soil D and not in soil A, B, and C under manure-free conditions. Seven other OTUs  were significantly impacted in all four soils except in soil D. It is noteworthy that these OTUs were always more abundant in soil D under manure-free conditions than in others (Supplementary Tab p<0.001). This indicates that in the soil A SMZ did not significantly affect the presence and absence of OTUs but only the relative abundance of some of them. Eleven OTUs responded to manure spreading depending on SMZ . They all were related to the 25 OTUs whose abundance was increased by manure. Two response patterns were observed. For 4 OTUs not detected in manure, SMZ exposure inhibited the stimulating effect of manure as compared to soil not exposed to SMZ. For the other 7 OTUs, SMZ exposure enhanced the increasing effect of manure on their abundance in a non-negligible way. Indeed, the sum of their relative abundance increased from 2.4% to 4.5% with SMZ addition (data not shown). For this latter pattern, it corresponded to OTUs always detected in manure, one being even only detected in soil amended with manure.SMZ treatment had a significant impact on bacterial community structure only for the soil A when considering the weighted unifrac matrix  and possibly undetected ones . Changes in the composition of microbial communities due to rare OTUs during coalescence processes are in line with the observation of the emergence of the rare-biosphere microorganisms following environmental disturbances . The leaThe communities of soils amended with manure exhibited OTUs with a wider distribution along the phylogenetic tree. This increase in diversity within the soil bacterial community could either correspond to the emergence of endogenous rare OTUs with functional abilities better adapted to this new environment than the original one or to the invasion of soil community. While previous studies provided evidence suggesting soil invasion by manure-borne bacteria, they could not distinguish surely allochthonous invaders from autochthonous OTUs that emerge because of environmental disturbance caused by the adjunction of exogenous matter , 22. In However, through the use of different soils, including one already amended in situ with pig manure 6 months before its sampling , we provided additional evidences supporting the hypothesis of invasion by manure-borne OTUs. First, in response to manure amendment, some of these OTUs presented the same increase pattern in different soils. The extreme case involved the most abundant OTU in the manure, a Pseudomonodales, which was favored in each of the four soils and was not detected in control soils A, B, and C not amended with manure. This response pattern suggests a common manure origin since all the soils presented different biotic and abiotic properties, limiting the chance that they hosted the same rarest OTUs. Secondly, all these OTUs were detected in the non-experimentally amended soil D, suggesting that they were successful in colonizing this soil following previous in situ manure amendment events (the last amendment having taken place 6 months before the experiment).Considering this set of arguments, we assumed that all or almost all the OTUs detected in both manure and soil D and which increased in several soils were from manure origin. All of them were among the most abundant OTUs in manure. Their high abundance in manure bacterial community composition may explain the success of their invasion, since a high inoculation rate is more likely to be successful , 45. OneInterestingly, when examining the abundance of the OTUs that are suspected to be invaders established in soil D during previous in situ amendment of manure (see above), two kinds of establishment patterns can be differentiated, depending on their taxonomic affiliation: the first one relates to the Firmicutes that are quite abundant in soil D, while the other relates to the Proteobacteria that are rare OTUs , who suggested that previous invasions generate legacy effects in soil communities facilitating future invasion attempts . HoweverFinally, the impact of the antibiotic SMZ on the structure of autochthonous soil bacterial communities was relatively low as it was significant in only one of the four soils (soil A). Previous results obtained from the same microcosms may explain this fact . After 1Manure-borne OTUs can colonize soils during the coalescence event of manure amendment. Changes in the composition of bacterial community were mainly attributable to the most abundant OTUs of manure (mainly Firmicutes) with some variations due to soil type. At times, their establishment in bacterial community of soils was still perceptible 1 month after manure amendment. The idea of their long-term establishment was reinforced by their detection in the soil that was amended in situ 6 months prior its sampling for this experiment. SMZ treatment had a limited influence on soil bacterial composition, but in one of the four soils, it enhanced the invasion potential of some manure-borne invaders. It would be interesting to clarify their antibiotic resistant ability.Supplementary file1 Supplementary Fig 1 Comparison of the bacterial diversity in the four soils  amended or not with manure and exposed or not to SMZ. Principal coordinates analysis (PCoA) of the weighted Unifrac distance matrices of 16S rDNA amplicon sequences showing changes in bacterial community structure. The first two axes and the percent of variation explained by each are indicated. Significant effects of amendment of manure and exposure to SMZ are represented by ellipse (P<0.001). (PNG 234 kb)High resolution image (TIF 10023 kb)Supplementary file2 Supplementary Tab 1 GPS coordinates and physical-chemical properties of the four different soils used in the study. (PDF 128 kb)Supplementary file3 Supplementary Tab 2 Phylogeny and relative abundance of the 10 OTUs that were significantly increased in several soils amended with manure (expressed per 10 000 OTUs counted). (PDF 282 kb)


With the ongoing evolution of the SARS-CoV-2 virus updated vaccines may be needed. We fitted a model linking immunity levels and protection to vaccine effectiveness data from England for three vaccines  and two variants . Our model reproduces the observed sustained protection against hospitalisation and death from the Omicron variant over the first six months following dose 3 with the ancestral vaccines but projects a gradual waning to moderate protection after 1 year. Switching the fourth dose to a variant-matched vaccine against Omicron BA.1/2 is projected to prevent nearly twice as many hospitalisations and deaths over a 1-year period compared to administering the ancestral vaccine. This result is sensitive to the degree to which immunogenicity data can be used to predict vaccine effectiveness and uncertainty regarding the impact that infection-induced immunity (not captured here) may play in modifying future vaccine effectiveness. Evaluation of the effectiveness of COVID-19 vaccines is increasingly challenging due to high levels of exposure to infection and vaccination. Here, the authors use a model-based approach incorporating these factors and estimate that using a variant-matched rather than ancestral booster could prevent nearly twice as many hospitalisations and deaths over one year. Despite several vaccines showing high initial efficacy against infection with the ancestral virus, the sequential emergence of variants of concern has substantially reduced the effectiveness of vaccines in blocking infection and onward transmission, although efficacy against severe outcomes has been more durable6. The emergence and global spread of the Omicron variant and its subtypes has resulted in repeated infection due to waning and reduced effectiveness of vaccine- and infection-induced immunity8. Omicron has now replaced prior variants globally and has been the dominant variant circulating for over 1 year, albeit with several emerging sub-variants9. Two Omicron-specific bivalent vaccines, that include antigens representing both the ancestral Wuhan virus and Omicron subtypes, are now available10. These have demonstrated higher immunogenicity against the Omicron BA.1 subvariant and against the BA.4/BA.5 subvariants than the ancestral vaccines12.The rapid development and roll-out of SARS-CoV-2 vaccines has had a major effect on the health impacts of the global pandemic, substantially reducing COVID-19 cases, hospitalisations, and deaths13) will also influence the effectiveness of vaccine booster programmes.As SARS-CoV-2 continues to evolve, it is likely that both existing and updated variant-specific vaccines will lag viral antigenic evolution. Moreover, as is currently the case for influenza vaccines, decisions regarding investment in, or introduction of, new vaccines, as well as assessment of the need for further boosting, will likely be based on immunogenicity and safety data rather than clinical trials. Obtaining reliable data on vaccine efficacy will be hampered by the high degree of infection-induced, broad-based antiviral immunity among most of the world’s population, making the identification of appropriate comparator groups challenging. This ongoing interaction between infection-induced immunity and vaccination  could act as a correlate of protection across a range of SARS-CoV-2 vaccines. In this, a non-linear dose–response model is estimated to relate NAT to protection against different clinical endpoints—capturing the more rapid decline in protection against mild disease that occurs as NAT declines over time compared to the slower decline in protection against more severe endpoints. Using this model, they subsequently predicted the loss of efficacy against emerging variants15, as well as more recently the potential benefit of introducing variant-specific vaccines against a range of circulating variants16. One of the limitations with a model based on NAT alone is that it does not capture the broader immune responses generated by vaccination (or infection) and how this may differ between vaccines17. For example, studies have demonstrated that the adenovirus-vectored AstraZeneca AZD1222 vaccine induced broad T-cell responses even though the level of NAT induced following vaccination is lower than that of the mRNA vaccines18. It is also suggested that inactivated whole virus vaccines  should induce even more broadly based immune responses, which are in turn predicted to be both more durable and less susceptible to viral immune escape19.A method for estimating vaccine efficacy from immunogenicity data was proposed by Khoury et al.14 we infer the underlying immune dynamics by fitting to national-level vaccine effectiveness estimates from England. Although the immunological mechanisms of protection from severe disease are not entirely clear, NAT are a well-established mechanistic correlate of protection from infection. Whilst cellular immunity is likely to play an additional role in protection from severe COVID-19, for simplicity we assume that a single immunological marker providing a surrogate for protection against infection can capture patterns of protection from severe disease. In so doing, we obtain estimates of vaccine effectiveness against three endpoints—symptomatic mild disease, hospitalisation, and death—for combinations of three widely used vaccines. By incorporating follow-up through 2021 and 2022, we estimate the vaccine effectiveness against both the Delta variant (dominant in 2021) and the Omicron BA.1/BA.2 variants . We use the fitted model to provide short-term projections of vaccine effectiveness, noting the considerable uncertainty associated with these, and to estimate the potential effectiveness of variant-adapted vaccines.Here we infer a simple model of immune waning and boosting directly from clinical endpoints. Using a similar model framework to that developed by Khoury et al.Figure 14 is adjusted based on immunogenicity data alone . The dashed lines in Fig. 15 and 9.7-fold reduction from the Wuhan virus to the Omicron BA.1 variant16, respectively. For both the Delta and the Omicron variants, applying the fold reductions estimated from immunogenicity data to the relationship inferred against the Wuhan virus results in a more pessimistic prediction of vaccine effectiveness than was inferred from fitting directly to vaccine effectiveness data against the Delta and Omicron variants.We compared this relationship to that expected if the original relationship between NAT and protection inferred from clinical trial data against the Wuhan virus by Khoury et al.20. The alternative boosting model produced a poorer fit to the data compared to our main model  provided the best overall fit to the data used in the fitting Table . The relel Table  but geneel Table .14 for neutralisation titre decay following infection although our model structure includes a more gradual transition to the slow delay over a 75-day period. The estimate of the half-life for the subsequent longer period of decline of 581 days (95% CrI 354–872 days) was consistent with the 500 days assumed by Khoury et al.14, and with the wide uncertainty bounds indicating a remaining degree of uncertainty in the longer-term durability of protection.The fitted model parameters are summarised in Table Table 20. One year after vaccination, these levels are predicted to decline further, resulting in relatively low protection against infection or mild disease, and moderate protection against hospitalisation . This is also in line with the more recent data which show 51.1% vaccine effectiveness (95% CI: 45.7–56.0%) between 9 and 12 months following dose 320. Fitting to the younger age-group results in similar estimates of vaccine effectiveness, albeit with a slightly steeper decline in vaccine effectiveness against hospitalisation and death  for AZD1222, 70.3% (95% CrI 68.0–71.8%) for mRNA-1273 and 64.1% (95% CrI 61.7–65.7%) for BNT162b2. These are in line with recent estimates of vaccine effectiveness  in England across all vaccines which show 65.3% vaccine effectiveness (95% CI 61.7–68.6%) between 3 and 6 months following the 3rd doseth Table .16 to explore the potential benefit of variant-adapted vaccines. They estimated a 1.61-fold (95% CI 1.5-1.8) increase in NAT against across all strains compared to the ancestral vaccines, with a higher increase  against homologous strains and lower increase  against heterologous strains. We applied this increase to the immunogenicity generated by the mRNA.1273 vaccine to illustrate the value of variant-matched vaccines . The resulting estimates of vaccine effectiveness are shown in Table 16. However, this pattern is not sustained over time with a predicted more rapid drop in efficacy against both mild disease and hospitalisation endpoints during 1 year of follow-up with the ancestral vaccine compared to the variant-adapted vaccine, such that effectiveness against hospitalisation reduces to 59.5% (95% CrI 53.9–62.7%) 365 days following the fourth dose with the ancestral vaccine but remains substantially higher  with the variant-adapted vaccine. Across a 1-year period, the overall impact is such that just over half of the additional protection is predicted to occur from delivering a 4th booster dose compared to no booster, and the other half from switching to the variant-adapted vaccine rather than continuing with the ancestral vaccine  develops against SARS-CoV-2 and given the ongoing uncertainty in the evolution of the virus.14. However, our results show that doing so results in a more pessimistic prediction of vaccine effectiveness than was observed in the English population data. This may be in part due to the uncertainty in the fold reduction given the widespread variation reported across the different laboratory studies. However, it may also indicate that immune responses other than NAT - from both vaccination and through ongoing exposure to the virus in the community - are providing a higher degree of cross-protection against new variants than would be predicted based on NATs alone.One of the main challenges with planning future vaccine booster strategies is assessing the potential effectiveness of future variant-specific vaccines. Given the speed with which new variants continue to emerge, coupled with the difficulty in identifying appropriate comparator groups to directly estimate vaccine effectiveness, it is likely that decisions will need to be made based on immunogenicity data. One method for doing this is to apply the fold reductions estimated from immunogenicity data to the relationship inferred against the Wuhan virus in the original paper by Khoury et al.21.Our results suggest that the value of the ancestral vaccines, whilst providing initial high levels of protection, has gradually been diminished through both waning of protection following the 3rd and subsequent doses and the substantial immune escape presented by the Omicron variant. In combination, these two effects combine to generate a substantial additional estimated benefit of switching 4th and subsequent doses to variant-adapted vaccines—which we estimate to prevent nearly double the number of episodes of severe disease over a 1-year period compared to delivering the 4th dose with the ancestral vaccine. It should be noted that this estimate is sensitive to the underlying shape of our inferred relationship between immune levels and protection, and in particular to the precise point at which there is a rapid drop in protection compared to the “plateau” at high immunity levels. It also depends on the degree to which the new sub-variants that have subsequently emerged exhibit immune escape from the variant-adapted vaccines. However, early follow-up of the impact of bivalent boosters in the US have demonstrated an added benefit over monovalent boostersOne of the major limitations of our work is that it was not possible to distinguish the combined effects of infection- and vaccine-induced immunity. The vaccine effectiveness data to which we fit our model is based on the full population of England with effectiveness estimates obtained by comparing outcomes according to vaccine dose against those with no prior vaccination. Given the widespread circulation of the SARS-CoV-2 virus in the community in England throughout the latter half of 2021 and first half of 2022 (from which these data derive), it is likely that a substantial proportion of both the vaccinated and unvaccinated cohorts will have experienced one or more episodes of infection. This will generate potential biases in vaccine effectiveness as has been recognised across studies attempting to use population level data. This bias will be propagated through to our short-term projections of vaccine effectiveness beyond the observation period. Thus, these patterns of vaccine effectiveness may not hold in other countries with different background levels of infection-induced immunity, and different levels of vaccine mix and uptake over time. Furthermore, there may be additional biases in the data due to other differences in the populations who were not vaccinated, those that received the first two doses but chose not to receive the 3rd dose, and those who received the 3rd dose. These could act to bias the vaccine effectiveness estimates in either direction.A second limitation in understanding the immune dynamics driving these patterns of vaccine effectiveness was the lack of associated immunological measurements. To overcome this, we inferred immunity levels by treating them as an unobserved process by utilising the parametric forms that have previously been developed to relate NAT to clinical protection. In doing so, we capture the effect of all aspects of the immune response—including both antibody-mediated immunity and potential T-cell responses – in our inferred immunity levels. However, this simple approach, whilst appropriate for short-term parametric projections, may fail to fully capture longer-term immune dynamics. In particular, more complex immune dynamics could generate patterns of decay in immunity levels that diverge from the simple bi-phasic pattern we have assumed here. Furthermore, it does not allow us to gain any further mechanistic insight into the underlying immune dynamics driving the observed vaccine effectiveness against the different clinical endpoints. In particular, we are unable to determine in our analysis whether any single immune marker can be considered a correlate of protection against both mild and severe infection. Further research in this area will require careful analysis of large population cohorts containing both immunological and clinical measurements.22. However, this switch in endpoint also means that it is difficult for us to retrospectively fit all datasets given the changing definition of hospitalisation. This limits our ability to truly assess waning in vaccine effectiveness over time.One of the challenges with estimating vaccine effectiveness from population cohort data is the difficulty with distinguishing hospitalisations or deaths arising due to COVID-19 from those arising from other causes but in which patients also received a positive COVID-19 diagnosis. The vaccine effectiveness estimates that we used to fit our model are based on hospital admission data with a recent positive diagnosis and hence do not allow us to disentangle this. However, analyses by UKHSA have shown similar vaccine effectiveness estimates when restricting the data to those that were admitted with a respiratory diagnosis which, whilst imperfect, may be more representative of those admitted due to COVID-19Regular booster vaccination is expected to be a key part of the ongoing management of COVID-19 over the coming years, especially among older and more clinically vulnerable populations where protection from any degree of SARS-CoV-2 infection may be crucial. As the SARS-CoV-2 virus continues to evolve, validated models that can estimate the effectiveness of modified vaccine products based on immunogenicity data alone will be increasingly important for assessing the benefit of additional doses with either existing or variant-modified vaccines. Our results demonstrate the challenges with doing so, given the more complex immune landscape that has arisen with both past vaccination and ongoing infection with multiple Omicron sub-variants across the world.polymerase chain reaction (PCR) tests including symptomatic cases and asymptomatic infections detected through screening in schools and workplaces), hospitalisations (defined as admission recorded in the Emergency Care Dataset within 14 days of a positive test) and death (within 28 days of a positive test) with the Delta and Omicron BA.1/BA.2 variants from England. Data were available for three vaccines administered in England—the Oxford/AstraZeneca AZD1222 vaccine, the Pfizer-BioNTech BNT162b2 vaccine, and the Moderna mRNA-1273 vaccine – in various combinations. For our primary analysis we use data from all age groups from the two publications by Andrews et al.24 alongside the estimates in the >65 age group from Stowe et al.25 in which only age-stratified estimates were provided. We additionally provide results using the <=65 age group in supplementary material. The original publications did not stratify these estimates by sex or gender. For these population-level estimates, data on prior infection was not available and hence the vaccine effectiveness estimates are representative of patterns of protection against a background of partial (and unknown) infection-induced immunity. These vaccine effectiveness estimates may therefore be biased if there are differences between the unvaccinated and vaccinated groups in terms of exposure to the virus.We used empirical estimates of vaccine effectiveness against what we term mild disease  immunity level (IL) over time, and protection against mild disease, hospitalisation and death14. We first express an individual’s IL over time, i drawn from a log10-normal distribution at dose j. Based on B-cell dynamics, we assume an initial period of fast decay with half-life 28.We followed the approach of Khoury et al.14 we assume a logistic relationship between IL and vaccine effectiveness to capture time-varying vaccine protection against infection or mild disease 14. Under this approach, we estimate different Following Khoury et al.14, our approach differs in two ways. First, rather than explicitly using NAT as a correlate, we backwards infer the IL over time from the estimates of vaccine effectiveness. This allows us to capture broader immunity mechanisms if they are not mediated via NAT directly. Second, we simplified the mathematical function for the decay in IL over time, reducing the number of parameters and using a flexible functional form that is able to fit both a single and bi-phasic data. In doing so we are able to infer the pattern of decay that best explains the vaccine effectiveness estimates over time.Whilst building on the modelling framework proposed by Khoury et al.29. We consider this as an alternative model structure in the Supplementary Appendix.An alternative model is one in which protection against severe disease  conditional upon infection or mild disease is assumed to be constant over time30. As a further exploration of the impact of model structure on our results, we assumed an alternative model in which there is a vaccine- and dose-dependent boost to IL, which therefore restores IL following 3rd and subsequent doses to a level that depends on both the magnitude of the boost and the level of IL achieved post dose 2. Under this model, the IL achieved at 3rd and subsequent doses is therefore dependent on the vaccine regime used for the primary course but by being related to the level achieved post dose 2, does not change according to the time that has elapsed since dose 2. The results from this model are given in the Supplementary Appendix.We considered two different approaches to capture the effect of third and fourth doses. In our main analysis, we consider a vaccine-dependent restoration of IL to a fixed dose-dependent level after each dose. Under this model, the restoration of protection is independent of the past decay in IL. Furthermore, the IL achieved at the 3rd and subsequent doses is independent of the vaccine regime used for the two initial primary course doses, consistent with results from the COV-BOOST trial14 obtained parameters for this model of vaccine-induced protection by fitting the relationship between NAT data following dose 2  to clinical efficacy data from Phase III trials14. In order to incorporate the information from this independent set of clinical data, we used their resulting estimates determining the relationship between NAT and protection as informative priors for our Bayesian model fitting. We used data from Wheatley et al.31 and Pelleau et al.32 as mildly informative priors for the durability of the immune response over time. In addition, we used immunogenicity data on the NAT observed following the second and third doses from clinical trials to generate informative prior distributions for the three vaccines  increase in NAT against across all strains compared to the ancestral vaccines, with a higher increase against  against homologous strains and lower increase  against heterologous strains. These data are not age-stratified16. This scaling of NAT was sampled from the reported estimates assuming a Normal distribution and then applied  to the fitted model to generate estimates of vaccine effectiveness against infection, hospitalisation, and death using our model that relates IL to protection from the BA.1/2 variant. We note that there is considerable uncertainty in this scaling, given the variability in immunogenicity responses generated across different laboratories.As new COVID-19 vaccines will be evaluated based on immunogenicity data, we use NAT to provide a preliminary estimate of the effectiveness of variant-adapted vaccines that are now in widespread use. To estimate the benefits for vaccine effectiveness, we used estimates of the relative neutralisation titres of variant-adapted vaccines compared to ancestral vaccines reported by Khoury et al.This work was undertaken by an international team utilising previously published data from the UK. Ethics permission was not sought as all data are in the public domain. Previous versions of this work were regularly shared with UK and international scientific committees to support local COVID-19 response policies.Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary