Few studies address the appropriate duration of post-surgical antibiotic therapy for orthopedic infections; with or without infected residual implants. We perform two similar randomized-clinical trials (RCT) to reduce the antibiotic use and associated adverse events.Two unblinded RCTs in adult patients  with the primary outcomes “remission” and “microbiologically-identical recurrences” after a combined surgical and antibiotic therapy. The main secondary outcome is antibiotic-related adverse events. The RCTs allocate the participants between 3 vs. 6 weeks of post-surgical systemic antibiotic therapy for implant-free infections and between 6 vs. 12 weeks for residual implant-related infections. We need a total of 280 episodes (randomization schemes 1:1) with a minimal follow-up of 12 months. We perform two interim analyses starting approximately after 1 and 2 years. The study approximatively lasts 3 years.Both parallel RCTs will enable to prescribe less antibiotics for future orthopedic infections in adult patients.ClinicalTrial.gov NCT05499481. Registered on 12 August 2022.Protocol version: 2 (19 May 2022)The online version contains supplementary material available at 10.1186/s13063-023-07141-2. The optimum duration of postoperative, systemic antibiotic treatment for orthopedic infections, with or without implant removal, or immune suppression, is unknown , 2.Because of the potential for poor outcomes, many clinicians treat bone and implant infections with a long course of antibiotic therapy. While there are few data supporting the need for prolonged therapy for bone and implant infections, such an approach certainly increases the risk of side effects, development of antibiotic resistance, and costs . MoreoveWe can do better. In a world where the emerging problem of antibiotic resistance gains momentum and policymakers and Infectious Diseases Physicians perform many prospective trials in all sorts of human infections to improve an antibiotic stewardship and reduce the unnecessary burden of antibiotic consumption, we do not understand why these efforts should not be made for the management of orthopedic infections. The study team of the SALATIO trials believes in antibiotic stewardship in orthopedic surgery –12, provRetrospective studies suggest that a maximum duration of 6 weeks is not inferior to longer durations , 13, 14;We are convinced that in orthopedic bone and implant infections, the shorter systemic antibiotic durations are as good, or as bad, as longer durations after surgical debridement. The duration of postsurgical antibiotic prescription is probably less important than other clinical variables such as the surgical approach , 7 or thwww.balgrist.ch). The SALATIO trials start at the Balgrist, but are expandable to other  centers. Supplementary File The Balgrist University Hospital in Zurich is a tertiary, referral center for orthopedic surgery (including for orthopedic infections) and is affiliated to the University of Zurich. It resumes a multi-disciplinary team composed of orthopedic surgeons, internist physicians, specialized nurses and physiotherapists, musculoskeletal expert radiologists, and infectious diseases physicians who are specialized in orthopedic infections. Moreover, this team is supported by a research campus  with BioBanking and a Unit for Clinical and Applied Research with experience in biostatistics and investigative designs  during the first surgical debridement. Here, we evaluate if 6 weeks of systemic and targeted antibiotic therapy postoperatively is not inferior to 12 weeks  after a follow-up of 12 months. For infections with complete implant removal , the second RCT investigates if 3 weeks of post-surgical antibiotic therapy is not inferior to 6 weeks after a follow-up of 12 months. The primary outcomes are always the remission at 1 year postoperatively and the proportion of recurrent infections due to the same pathogens as in the index episode. The secondary outcomes are adverse events, with an emphasis on antibiotic-related events during the intervention and the length of hospital stay in an acute care setting. We do not evaluate costs.An “orthopedic infection” is the concordant microbiological evidence of bacteria in at least two deep intraoperative tissue samples together with radiological  and/or clinical evidence of infection . Histological proof is facultative for this study. Implants are defined as any foreign material; except for allografts, transient wires, or fixator pins outside of the infected bone. Remission of infection is the absence of clinical, and/or radiological, and/or laboratory signs of the (former) infection after the minimal follow-up time of 12 months. Inversely, a clinical failure is any problem leading to unplanned revision surgeries, including for persistent infection during antibiotic therapy, non-infectious reasons , or new infection at the same site. A microbiological recurrence is a recurrence with the same pathogen(s) after the end of the antibiotic therapy.When surgeons, infectious diseases physicians, and internists are consulted for orthopedic infections, they will screen the patients. If inclusion and no exclusion criteria are met, the patient will be informed about the study by the investigators. Upon his/her written consent, we perform two parallel RCTs, depending on the presence of osteosynthesis material at enrolment: SALATIO 1: Infected implant not removed : Randomization 6 vs. 12 weeks (+/− 5 days) of total antibiotic therapy counted since the first debridement for infection. SALATIO 2: Infected implant without residual material : Randomization 3 vs. 6 weeks (+/− 5 days) of total antibiotic therapy counted since the first debridement for infection. Figure Patient’s characteristics: age, sex, known immune suppression , American Society of Anesthesiologists (ASA)-ScoreSurgery and infection data: number and type of surgeries for the actual problem, agent, dose, and duration of pre-surgical antibiotic therapy, local antibiotics used in the bone, cell count (if any), initial serum C-reactive protein (CRP) level, presence of initial bacteremia, implants, microbiological results, histology (facultative)Treatment and outcomes: number of surgeries to treat infection, total duration of antibiotic therapy, duration, agent, and dose of intravenous and oral antibiotic therapy, wound healing problems, presence and duration of vacuum-assisted negative pressure therapy, all adverse events, clinical or microbiological recurrence, date and reasons for re-hospitalization and re-treatment, follow-up data, fatalitiesAdministrative data: total hospitalization length, eventual BioBanking of infected tissuesAt study entry, we assess the following variables:Visit 1 - enrollment (day 1)Visit 2 - day 21 (+/− 5 days)Visit 3 - day 42 (+/− 5 days) Visit 4 - day 84 (+/− 5 days) (if not in the short antibiotic arm)After randomization, the study participants will be actively followed-up for a minimum of 12 months. We will equally review the medical charts of all patients to seek for unscheduled visits. This “passive follow-up” can reach up to 4 years and terminates at the date of database closure. The scheduled study visits take place as follows Fig. .Visit 1 End of treatment (EOT) visit - day 21, 42, or 84 (+/− 5 days) (only if still receiving treatment after visit 2). Test-of-cure  − 12 months (+/− 2 months).www.compendium.ch).During the study visits, we assess the history, adverse events, and the surgical  status. We examine the patients according to the orthopedic standard and add supplementary laboratory exams and radiology only if clinically indicated. Only visit 2 can be by telephone alone. If the patient is currently hospitalized for rehabilitation under medical supervision, the visits can be replaced by the medical assessment during the reeducation/rehabilitation. A microbiologically effective antibiotic therapy beyond 96 h prior to screening is not permitted. However, a 72-h window before debridement is permitted, independently of the duration of prior antibiotic administration. If the patient requires a new antibiotic agent based on microbiological results; independently of the duration of prior ineffective antibiotic therapy, there are no minimal windows or maximal pre-debridement antibiotic durations. Initially, antibiotic therapy is either empiric or targeted to the results of preoperative information. After 2–4 days, antibiotic therapy becomes targeted to the susceptibility profile. The choice of the agent, and its administration route , is at the discretion of the treating clinicians. Nonetheless, for both RCTs, and to achieve a minimal homogeneity, we establish a list of “allowed antibiotics” Table . The invPrincipally, the study protocol and all patients’ follow-ups are oriented to the usual surgical procedures of our clinic. All study-relevant data are part of normal clinical management. The study visits correspond to the usual surgical controls. Only the duration of the postsurgical antibiotic course is new and determined by the study allocation. Moreover, the Principal Investigator is the main Infectious Diseases Physician and all surgeons in the study team have a long experience in orthopedic infections. In our small region, the general practitioners, or other colleagues in other hospitals, do not change a study regimen without contacting us. This is also what we do with the study of patients of other hospitals. We contact the clinician in charge. If a patient does appear to the clinical controls, the secretary calls him/her by phone and/or by e-mail. For all these reasons, we think that the possibility of a major protocol violation or a loss to follow-up is unlikely when conducting our SALATIO trials.All patients can witness adverse events related to surgical and anaesthesiologic procedures and antibiotic administrations, which, however, are related to the therapy itself, and not to the protocol. A theoretical risk could be a higher incidence of microbiological recurrences in the short-duration arms. In contrast, future patients might benefit from shorter therapies, shorter hospital stays, and a reduced risk of adverse events. Our project is a contribution to the professional efforts of antibiotic stewardship that is gaining momentum worldwide.After written informed consent has been given (up to 5 days after surgery), participants will be randomized with a 1:1 ratio by predefined study nurses drawing a sealed envelope that contains a randomization card. Study nurses with experience in clinical trials will generate these cards. They will monitor their use and keep them locked in another office outside of the clinic. Upon randomization, the study team asks the study nurses to randomize. Then, the study nurse randomizes within 24 h without knowing the identity of the new study entry. Importantly, these nurses are separated from the treating clinicians. The investigators will inform the patients and the treating clinicians about the randomization arm. After the assignment, we purposely lack any firm criteria for discontinuing or modifying the allocated interventions for a given trial study participant. Principally, the patient may withdraw his/her consent or is dropped out by the study team. Upon the detection of unintended protocol violations, the Principal Investigators decide if the patient continues the SALATIO trial in the intention-to-Treat population (by exclusion from the per-protocol population), or is dropped out. The patient never switches from one allocation arm to the other.The Unit for Clinical and Applied Research will assign an independent monitor (with experience in RCTs) to the study. All patient files, notes, and copies of laboratory and medical test results must be available for monitoring. The monitor will verify all, or a part of the Case Report Forms (CRF), data, and written informed consents. One monitoring visit at the investigator’s site prior to the start and twice  will be organized by the Sponsor. Furthermore, there will be a close-out visit at the study end.A quality assurance audit/inspection of this study may be conducted by the competent authority. The quality assurance auditor/inspector will have access to all medical records, the investigator’s study-related files and correspondence, and the Informed Consent Form. The investigator will allow the persons being responsible for the audit or the inspection to have access to the source data/ documents and to answer any questions arising. All involved parties will keep the patient data strictly confidential.For both SALATIO trials, we need 36 months each; starting in September 2022 . Likewise, a 3 week’s course of targeted systemic antibiotic therapy after the first debridement for orthopedic infection is not inferior to 6 weeks (if there are no implants).We anticipate the corresponding short durations of post-debridement antibiotics reveal a similar remission incidence than the long ones. Provided that there is an adequate surgical strategy, the duration of antibiotic therapy does not modify remission nor microbiological recurrences, and antibiotic-related adverse events will occur in 10% of all treatment arms.Both RCTs are non-inferiority trials. Remissions (at the first therapeutic approach) are set at 94% (6% recurrences in both arms). The maximum acceptable difference  is arbitrarily fixed at 10% regarding the primary outcome “remission.” Assuming a risk of alpha at 0.05 and a power of 80%, it will be necessary to recruit 70 patients in each antibiotic duration arm (short or long). Together with the distinction of the RCT into implant-related and implant-free bone infections, we would finally need 2 × 2 × 70 episodes, equaling a total of 280 episodes. With only very few anticipated dropouts, we would probably need 300 episodes. Our hospital approximately treats 600 episodes of different community-acquired and nosocomial infection episodes per year, of which roughly 150 are orthopedic infections. According to conservative estimates, we will see approximately 100 infection episodes per year that are eligible for the SALATIO trials. The interventional part of the trials will last 3 years (300 different episodes).χ2-test, the Fisher exact, or the Wilcoxon-rank sum tests, as appropriate. We will also perform separate multivariate analysis using a Cox regression model [p value ≤0.2 in the univariate analysis will be included in a stepwise forward selection process for multivariate analysis. Key variables will be checked for co-linearity and interaction. The number of variables in the final model is limited to the ratio of 1 variable to 5 to 8 outcome events [All analyses will be performed for the entire study population and stratified upon the presence of an infected implant. In the second step, we will stratify the analyses basing on the orthopedic specialty. We will use descriptive statistics and perform group comparisons (using the Pearson-on model . Variable events . If the The intent-to-treat (ITT) population will consist of all randomized patients. The per-protocol (PP) population will consist of all randomized patients who complete the study without important deviations from the protocol.Important missing data will lead to patient dropout of the study. Drop-outs will be reported in the Methods section of the publication. Drop-out data will be archived for a minimum of 10 years after study termination or in case of premature termination of the clinical trial.The SALATIO trials will possess very complete data and perform stratified analyses . Formal adjustments for multiple primary outcomes will not be a major problem. Principally, we have only two primary outcomes , while the “microbiological recurrences” are part of the larger group of “clinical failures.” For this adjustment regarding both outcomes, we will formally use the “classical” Bonferroni correction that we consider as the most common approach to account for multiplicity due to its simplicity , 25.p-value limit of 0.025. Formal non-inferiority assessment for “microbiological recurrence” will not be necessary, because of the few number of events anticipated. Likewise, we do not predefine a non-inferiority margin for secondary outcomes adverse events, or the length of hospital stay in acute care settings.For assessment of the formal non-inferiority requirement (regarding the primary outcome “remission”), we will compute with a unidirectional We will perform two interim analyses, after 1 and 2 years, following the inclusion of the first patient. If the differences in the primary outcomes between the short and long antibiotic courses are important, or statistically significant , the indClinicalTrials.gov (Number NCT05499481).The study is registered in the Swiss Federal Complementary Database (BASEC 2022-01012) and in the international registry Trials [This study only makes use of antibiotics that are already authorized in Switzerland for orthopedic infections. The indication and the dosage are used in accordance with the prescribing information and international guidelines. The study protocol will not be changed without prior Sponsor’s and Ethical Committee’s approval. Premature interruption is reported within 30 days. As with the other RCTs we are currently conducting, and of which the protocols have been published in Trials , 10, we Our institution has a standardized procedure for recruiting participants, based on the in- and exclusion criteria Fig. . The invThe investigators uphold the principle of the participant’s right to privacy and that they shall comply with applicable privacy laws. Subject confidentiality will be further ensured by code numbers corresponding to the computer files. For verification, the Ethics Committee and regulatory authorities may require access to medical records, including the medical history.The Sponsor may terminate the study prematurely in certain circumstances, e.g., ethical concerns, insufficient recruitment, when the safety of the participants is at risk, respectively, alterations in accepted clinical practice making the continuation unwise, and early evidence of benefit or harm of the experimental intervention. All patients are free to withdraw from participation in this study at any time, for any reason, and without prejudice. The reason for withdrawal should be documented wherever possible. The withdrawal will not affect the actual medical assistance or future treatments. On rare occasions, the investigators may terminate a patient’s participation to protect his/her best interest. After study termination, the evaluations required at the next scheduled clinical visits will remain.All surgeries will be performed in the participation of an experienced surgeon. The antibiotic therapy is ordered and supervised by internists and infectious diseases physicians with experience in orthopedic infections.An Adverse Event (AE) is any medical occurrence in a study participant, which does not necessarily have a causal relationship with the study procedure. An AE can therefore be any unfavorable and unintended symptom. A Serious Adverse Event (SAE) is classified as any untoward medical occurrence that results in death, is life-threatening, requires in-patient hospitalization or a significant prolongation of hospitalization, persistent or significant disability. Participants with ongoing SAEs at study termination will be followed until recovery or stabilization after termination. The investigators make a causality assessment. All SAEs are reported within 24 h to the Sponsor-Investigator. SAEs resulting in death are reported to the Ethics Committee within 7 days. The Sponsor-Investigator will report the safety signals within 7 days to the local Ethics Committee. Patients who will AE and leave the study will be treated off-study, without restriction, at the study sites.An annual safety report on the participant is submitted once a year to the local Ethics Committee. Moreover, we perform annual interim (futility) analyses.Data is only saved using the secured software REDCap®. When the study is terminated, it will be saved in the same system. Data can only be accessed by defined persons that are investigators of the project.An electronic Case Report Form will be generated for every participant and all data relevant to the study is going to be recorded by authorized persons. The participant ID numbers are automatically assigned in consecutive ascending form by the REDCap® system. Corrections can only be made by authorized persons.For data analysis, subject-related data from REDCap will be exported and analyzed in a statistical software (IBM-SPSS and/or STATA). All health-related data will be archived in the REDCap®. Before data export, all patient identifiers are removed. All data will be stored for a minimum of 10 years. Collection, disclosure, and storage of data are carried out in accordance with Swiss data protection regulations and the Human Research Act. The BioBanking stores the eventual intraoperative samples in accordance with laboratory guidelines as standard.The SALATIO trials shall demonstrate a clinically relevant non-inferiority (10% margin) “in favor” of a significantly shorter antibiotic therapy, compared to the usual long durations in adult patients with various orthopedic infections. This reduction shall concern infections with and without implants ; indepenIn the SALATIO trials, we principally keep a longer antibiotic duration for implant infections, even if we ignore if implant-related infections really must be treated longer. Basically, there are no firm clinical data suggesting that the prolongation of antibiotic prescription should exactly be doubled for implants compared to implant-free bone infections. In the theory and according to in vitro studies, the foreign body nature of the implant makes every infection difficult to treat, even for pathogens not usually considered as virulent , UnsurprNocardia spp. [In contrast, our approach is different from the tradition for implant-free bone infections. In the entire field of orthopedic bone infections, there are very few and inconsistent scientific data proving the benefit of a systemic antibiotic therapy beyond 6 weeks; compared to 4 to 6 weeks or even less. Exceptions are by nature expert opinions in book chapters or past publications without own database analyses; or the therapy of special microorganisms requiring long-lasting antibiotic therapies such as mycobacteria , Nocardidia spp. , actinomdia spp. . To citedia spp. , long bodia spp. , fracturdia spp. , 30, prodia spp. , 4, diabdia spp. . Tone etdia spp. . Chaussadia spp. . Farhad dia spp. ; togethedia spp. . These rdia spp.  of orthoThere are also studies with less than 6 weeks of total antimicrobial therapy, especially in the pediatric literature for hematogenous osteomyelitis and/or septic arthritis , 35. In In our era of critical shortages of effective antibiotics, a shorter treatment duration could decrease antibiotic-related adverse events, costs, and possibly the emergence of resistance. We do not expect major difficulties performing in the SALATIO trials, provided that patients and surgeons agree to participate. Despite two prospective-randomized designs (for infections with and without implants), and only 280 different episodes anticipated, the patients’ voluntary participation might be low. Likewise, patients who are continued to be treated outside of our hospital may have been lost, or have their treatment changed, because the following clinicians might not agree or may lack interest. However, our center is the largest public hospital for orthopedic infections in the region; so, this is unlikely to be a major bias. Lastly, the SALATIO trials exclude episodes with a strictly conservative  treatment of orthopedic infections or management using systemic antibiotics in conjunction with local, intraosseous antibiotic deliveries . Hence, In conclusion, we are confident to finish the SALATIO trials, reveal some answers to frequent questions regarding the post-surgical duration of systemic antibiotic use in orthopedic infections, and avoid unnecessary antibiotic excesses, all of which we consider as a personal mission and commitment .Additional file 1: Supplementary File 1. Protocol SALATIO Trials.Additional file 2: Supplementary File 2. Consent Form in English language. SALATIO Trials.Additional file 3: Supplementary File 3. Internal Document and Proof of Funding.


The human gut microbiome, which develops and stabilizes during the early stages of infant life, plays an essential role in host health through the production of metabolic resources and the stimulation and training of the immune system. To study colonization and community functional dynamics of the microbiota based on responses to host immune processes during the normal and dysbiotic establishment of the gut, metaproteomics was conducted on 91 fecal samples collected over the first 90 days of life from 17 hospitalized premature infants. Microbial responses to antibiotic administration and host-imposed metal bactericidal control correlated with community assembly and resiliency of microbes in the developing preterm gut. Specifically, proteins related to antibiotic resistance and metal homeostasis mechanisms were predominant in persisting members in the infant gut environment over the first several weeks of life. Overall, this metaproteomics study provides a unique approach to examine the temporal expansion and resilience of microbial colonization, as it allows simultaneous examination of both host and microbial metabolic activities. Understanding the interplay between host and microbes may elucidate the microbiome’s potential immunomodulatory roles relevant to necrotizing enterocolitis and other dysbiotic conditions in preterm infants. Early-life microbial colonization of mucosal surfaces in the gut during infancy is critical for balanced priming and education of the host immune system. Recent studies are defining the period in early development, a so-called “window of opportunity,” where disruption of critical host-commensal interactions have lasting and sometimes irreversible impacts on the training of specific immune subset . StudiesIn addition to the still poorly understood interplay between the host immune system and microbiota, other external factors, such as antibiotic administration impact the developing gut environment. Preterm infants commonly receive antibiotics early and frequently in life due to their vulnerability to infection . PreviouIn vivo characterization of metal homeostasis in the gut environment is challenging due to the technical limitations of most traditional microbiome research methods. Nevertheless, this is an understudied area that needs to be considered for its unrecognized role in shaping the early life gut.Several studies have shown that antibiotic resistance is linked to tolerance of high environmental concentrations of metals such as copper . The pheTo this end, this study was designed to examine the gut microbiota response to human host-imposed immune control during establishment of the developing gut microbiome over the first few months of life, specifically utilizing a high-resolution LC–MS/MS approach that enables simultaneous investigation of host and microbial functions that impact this colonization process. This was based on a combined, longitudinal metagenomics and metaproteomics study of fecal microbiome samples collected from hospitalized premature infants, including a subset of infants who developed NEC during the study. The developing infant gut is an ideal model system because infant microbiomes are far less complex than adult microbiomes. The early-life period is critical in the maturation of both the immune system and microbial communities. Earlier investigations on a subset of the samples used in the present analysis predominantly focused on microbial metabolic functions and community composition . These iSamples were collected during the first 3 months of life from hospitalized premature infants, including six infants that went on to develop necrotizing enterocolitis (NEC) during the study. In total, 91 stool samples from 17 infants were measured and analyzed . The samp <0.01). Other factors such as delivery mode, feeding type, and disease onset did not significantly influence the functional β-diversity in this sample cohort. To complement the analysis using LMMs, PERMANOVA and homogeneity of multivariate dispersion testing were conducted on the Jaccard and Bray-Curtis distances for each of the metadata categories investigated , many of the observed trends were largely infant-specific. Based on this level of global analysis, it was clear that the approach of evaluating all samples from the infants in the cohort together could lead to a confounded interpretation of the data, as host-microbial interactions are highly dependent on the environmental context at the time of sample collection. In addition, grouping samples without accounting for sampling time washed out some observations of highly dynamic, time-dependent processes.The ability to interrogate these temporal dynamics was a key strength of this sample set, which led us to the conclusion that an alternative approach needed to be taken to analyze the data further. In light of this conclusion, we set out to investigate some of the unanswered questions that arose in previous analyses of this dataset related to antibiotic resistance and shifts in community abundance that were not associated with times of disease onset.E. faecalis within some of the infants in this cohort and the replication rates of selected microbes during periods of antibiotic administration indicated antibiotic resistance might be highly prevalent in organisms dominating the early-life gut environment. However, protein expression of the specific antibiotic resistance mechanisms used by any microbes in this dataset was not interrogated. Here, we set out to determine the extent of antibiotic resistance in the gut microbiome for all infants in this cohort and to characterize the diversity of antibiotic resistance mechanisms being utilized by the microbiota. All quantified microbial proteins were searched against the comprehensive antibiotic resistance database (CARD). In total, 241 protein sequences were identified across all 17 infants that mapped to 93 antibiotic resistance ontology (ARO) terms  terms . These pO) terms .On average, 17 AROs were found in each timepoint across infants. In general, while the overall abundances of antibiotic resistance proteins changed over time, the relative contribution of individual resistance mechanisms utilized by microbiota remained unchanged across time in a specific individual unless perturbation of the system by administration of antibiotics. In addition, the overall ARO composition was highly variable among infants. Citrobacter sp. were expressing CMY-type beta lactamases, which confer resistance to oxyimino-cephalosporins, including claforan. While the susceptibility to antibiotics and associated resistance mechanisms expressed by microbes played an essential role in establishment dynamics, several dramatic changes in organismal abundance were not explained by the expression, or lack thereof, of antibiotic resistance-related proteins in individual microbes. This indicates there must be other drivers of community establishment dynamics, and we hypothesized that microbial responses to host immune mechanisms might explain some shifts in relative organismal abundance across time in each infant.Upon examining the relative summed protein abundance of each organism in the community across the longitudinal series for each infant, some changes in community composition were observed, as would be expected based on antibiotic treatment of the infants over the first several weeks of life. In samples collected after the antibiotics were administered, there was a dramatic shift in the relative community composition based on this perturbation. The expression of some AROs with known resistance to the specific antibiotics administered at disease onset corresponded to the persistence of the microbiota expressing those genes at the time of antibiotic treatment. Several infants were treated with claforan upon diagnosis of sepsis or NEC. Among the infants with samples collected before and after antibiotic treatment, the expression of antibiotic inactivation related proteins by a microbe was indicative of that microbe’s persistence in the gut during periods of antibiotic administration. For example, in infants 61 and 39 , 5A, claLooking at samples across time within each individual yielded unique patterns of community dynamics in response to antibiotic administration and host processes when patient medical information was considered. Leveraging patient metadata regarding disease onset and the timing and types of antibiotics administered to each infant, functional shifts of individual microbiota could be differentiated between microbial responses to known and external perturbations on the gut environment caused by antibiotic administration and unknown and internal perturbations caused by interactions with host processes and other community members. While this type of analysis is highly individual-specific, it provides insights into community establishment dynamics that would otherwise be missed if samples from infants were generalized to broad analysis classes without accounting for the heterogeneity of all longitudinal and environmental factors which have a non-negligent impact on the colonization of the gut environment. To highlight this type of health-status-based longitudinal analysis, selected infants will be highlighted and discussed that have a wide range of sampling time points leading up to disease diagnosis and subsequent antibiotic treatment and samples collected during and after antibiotic administration. The observed protein expression profiles are not unique to these selected example infants but are found in multiple infants across the cohort.Enterococcus faecalis, Klebsiella pnuemoniae, and Enterobacter cloacae), all three are typically susceptible to the types of antibiotics delivered during the course of the study. E. faecalis was the dominant organism, based on summed protein abundance, leading up to NEC diagnosis on day nine and decreased in relative abundance following subsequent initiation of antibiotic treatment. After 6 days of continuous antibiotics, it was drastically reduced in relative abundance, corresponding to its susceptibility to the antibiotics administered and the lack of resistance mechanisms. E. cloacae increased in relative abundance after the administration of antibiotics on day nine and persisted as the dominant organism in the community until the measurement on day 82. K. pnuemoniae was the dominant organism with protein expression relating to antibiotic resistance mechanisms , observed across the entire infant cohort. LCN2 is known for its ability to bind bacterial siderophores, microbial proteins that assist in iron harvesting, such as enterobactin and salmochelin, to limit the bacterial iron acquisition and subsequently hamper the growth of microbes .Based on the indications that some infants were mounting an active immune response utilizing copper and iron trafficking-related antimicrobial activities, we focused on related metal trafficking activities in the microbiota. In each infant with increased host expression of proteins related to copper toxicity as an immune response, including infant 61, some microbes expressed proteins associated with copper toxicity resistance or tolerance, including CueR, CueO, and CopA. These proteins are specifically involved in the efflux of Cu (I) from the cell and the transformation of Cu (I) into Cu (II). In addition, there was expressional evidence of resistance-nodulation-division (RND) family transporters which many confer both metal and antibiotic resistance across the infant cohort. For example, proteins that comprise the cusABCF system were expressed by Gram negative bacteria in 11 of the 17 infants in the cohort. These resistance mechanisms may explains how some organisms within these infants might be persisting despite pressure from both host defences and antibiotic administration.E. cloacae expressed many proteins related to copper efflux. While copper efflux-related proteins were expressed by some other bacteria in this infant during the sample collection period, fewer proteins related to this function were observed and they were in reduced abundance compared to E. cloacae. This suggests copper efflux and resistance were not as prominent in these organisms, even if there was an active response to excess copper. In addition, some of the proteins involved in the copper response identified in the two other dominant organisms do not have definitive physiological roles, unlike the proteins observed in E. cloacae, where there is a definitive path of efflux and detoxification of copper. Among the other dominant organisms in infant 61, E. faecalis was also found to express copper homeostasis-related proteins , but at lower levels than E. cloacae. In addition to proteins involved in mitigating intracellular copper toxicity, only E. cloacae showed expression of multiple proteins related to the biosynthesis of the siderophore enterobactin among the organisms in this infant. As only E. cloacae was the only organism with observed expressional evidence of producing and importing enterobactin in this infant due to the elevated demand for iron-based on high levels of Cu (I) ions produced through host-defense mechanisms, the other species were compromised by their inability to offset Cu (I) levels intracellularly with iron and the lack of different mechanisms to export Cu (I).Comparing the functional activities of the individual microbes in this infant, we found that C. freundii expressed the majority of the antibiotic resistance-related proteins in this infant and dramatically increased in abundance after administration of these antibiotics. This supports the idea that organisms with antibiotic resistance mechanisms may persist and expand while the organisms lacking these mechanisms become dramatically reduced in abundance after antibiotic treatment. While the expression of antibiotic resistance genes by organisms during periods of antibiotic administration plays a role in shaping overall community composition, there are also changes in organismal abundance that are not correlated to microbial response to antibiotic treatment.Infant 39 had multiple time points sampled across the first several weeks of life, with samples collected before and after NEC diagnosis and subsequent antibiotic administration. There was a shift in community structure at the time of NEC diagnosis on day 24 and the subsequent treatment, which we presume was predominantly caused by the administration of antibiotics . For exaLactobacillus rhamnosus. We hypothesized that the dramatic increase and later decrease of calprotectin might be influencing the relative abundance of L. rhamnosus over the first several weeks of life. As calprotectin inhibits bacterial growth through chelation of manganese and zinc, we looked to associated mechanisms, such as membrane transporters, in L. rhamnosus related to manganese and zinc import that helped this organism import these metals into the cell to overcome the host-imposed metal limitation. This organism expressed four key proteins related to manganese trafficking; mntA, mntB, mntC, and mntH. The MntABC protein complex has been demonstrated to import manganese during low Mn supply, and mntH specifically competes with calprotectin for luminal manganese (. L. rhamnosus expressed non-specific zinc/manganese transport system substrate-binding proteins and a manganese-dependent inorganic pyrophosphatase (ppaC), which indicates the microbe was actively importing manganese and utilizing it within the cell. Notably, L. rhamnosus was also expressing a putative drug exporter of the RND superfamily, which may also contribute to the persistence of this organism during periods of antibiotic administration.There was also an unexplained shift in community functional composition between samples on DOL 13 and 20. During this time period, there was a corresponding host expression of host immune proteins related to manganese and zinc trafficking. Two host proteins, S100A8 and S100A9, can be involved in metal sequestration by antimicrobial peptides and were found in high abundance across all 91 samples in the dataset. These two proteins form the heterodimer calprotectin, which is often used as a fecal biomarker of inflammation and sequesters both zinc (Zn) and manganese (Mn) when it is released by neutrophils at infection sites . In infaanganese . In addiAcross the entire dataset, there were 1,000s of microbial and human peptides measured per sample, which equated to an average of 3,000 human proteins and 9,000 microbial proteins identified per infant. This proteomic coverage achieved allowed in-depth elucidation of both host and microbiota function in the early-life preterm gut. Among the host proteins related to immune function, which represented 10–15% of human proteins quantified in the dataset, 40–50% of these quantified proteins were unique to specific immune pathways, which led to the confident interpretation of the host functional data related to the research questions addressed in this study. The functional β-diversity analysis showed that this intra-and inter-individual functional variability is not restricted to the microbiota. Host protein expression related to immune function was variable and dependent on the environmental context each infant faced during this early-life period. In spite of the inter-individual variability, the longitudinal measurements collected in this study enabled an in-depth investigation of intra-individual variability of microbial and host immune function simultaneously. Intra-individual variability corresponded with microbial responses to both external perturbations of the gut environment, through the administration of antibiotics, and also to host immune functions. This large variability in preterm human infants makes it difficult to generalize results to a larger cohort, as other environmental factors  can have non-negligent influences on the composition and functionality of gut microbiota and host. However, the findings in this study should drive interest in additional longitudinal studies with the recruitment of larger cohorts and a more balanced experimental design in terms of the timing of sample collection and control of environmental factors, including antibiotic administration.Microbial mechanisms, such as antibiotic resistance, play a pivotal role in shaping the early-life preterm gut. The present study indicates that, in addition to antibiotic resistance mechanisms, metal homeostasis mechanisms correlate with community establishment dynamics. In fact, metal homeostasis may play a more prominent role in early-life community establishment than previously recognized. Metals are involved in many reactions and are essential to life. They are essential cofactors required for many reactions for both the host and microbes in the intestinal environment, and lack of these metals leads to organismal damage and death . Howevervia iron displacement from solvent-exposed iron–sulfur clusters  is then delivered to copper pumps which undergoes trafficking to the phagolysosome compartment where copper ions are loaded. Accumulation of Cu (I) within the phagolysosome contributes to bacterial killing through membrane damage, displacement of iron by copper in iron–sulfur (Fe-S) clusters, and formation of reactive oxygen species  in cytoplasmic vesicles that partially fuse with the phagolysosome, flooding invading microbes with toxic levels of copper ions . The accE. faecalis) or copper efflux proteins (K. pnuemoniae) relates to a decrease in relative abundance, while expression of proteins related to both of these mechanisms (E. cloacae) relates to increased relative abundance in the community after environmental stress. Overall, the observation that a microbe’s ability to circumvent the host’s attempt to limit some microbial growth through copper-mediated toxicity and limitation of iron harvesting contributed to its persistence and establishment in the environment.The longitudinal measurements of microbial functional dynamics in infant 61 indicate that following the increase in the apparent host-imposed copper toxicity on day eight, the relative abundance of the microbiota is driven by the ability to respond to this host immune response. In summary, the observed organismal abundance trends in this infant during periods of antibiotic administration and host immune-mediated metal toxicity show that microbial resistance and homeostasis mechanisms to both of these perturbations are critical for persistence and maintaining establishment in the developing gut. For example, lacking either antibiotic resistance mechanisms  in manganese and zinc-limited environments.Some microbes have overcome this strategy by utilizing additional membrane transporters such as the ones shown in the figure that enhance their ability to import manganese into the cell. Based on the presence of manganese import-related transporters and the expression of several manganese-dependent proteins in microbes who increased in abundance during periods of high host expression of calprotectin, we can infer host sequestration of this metal did not impact the growth of As both manganese and zinc are cofactors for many enzymes involved in critical processes required for bacterial growth, including central metabolism, mechanisms for bacteria to compete with the host for these metals are essential for the survival of the organisms . Both main vivo clinical context, as this approach allows simultaneous measurement of both host and microbial function. In this study, community assembly and persistence of microbiota in the early-life developing preterm gut were correlated with individual microbial responses to antibiotic administration and host-imposed metal bactericidal control. Specifically, microbial protein expression of mechanisms related to antibiotic resistance and metal homeostasis were predominant in community members that persisted in the highly dynamic gut environment over the first several weeks of life. A deeper understanding of the functional interplay at the host-microbe interface may clarify the potential immunomodulatory and defense roles of microbiota that are relevant to dysbiotic conditions, such as necrotizing enterocolitis, in premature infants.Metaproteomics studies provide a unique opportunity to study microbial colonization, establishment, and persistence in an , and 50ug of digested peptides were analyzed in technical duplicate via two-dimensional nanospray liquid chromatography–tandem mass spectrometry (LC–MS/MS) on an LTQ-Orbitrap Elite mass spectrometer (Thermo Scientific).Sample collection, processing, and metaproteomic measurements by LC–MS/MS were already described . BrieflyMatched metagenome-derived protein databases for each sample were compiled into infant-specific databases containing any predicted proteins for that individual. Metagenomic data used for the construction of the predicted protein databases used in the present study was conducted previously . In addiAnnotation of immune-related proteins was performed using the Reactome Database (version 77). Proteins that mapped to several immune pathways were counted for each pathway. Further analysis was performed at the pathway level with the requirement that at least one protein in the pathway is unique. For annotation of microbial proteins, taxonomy assignments were performed as previously described . KO termp <0.05) and an insignificant betadisper result  after Benjamini-Hochberg correction.The samples’ dissimilarities were assessed using Jaccard and Bray-Curtis distances for NMDS ordinations using the metaMDS  function in the vegan R package. Trends in functional dissimilarity between samples were assessed using a linear mixed-effects model (LMM) with the lmer function in the lme4 package in R. Fixed effects analyzed included day of life, gestational age, birth age, delivery mode, feeding type, NEC diagnosis, infection diagnosis, sepsis diagnosis, and birth weight. Infant ID was included as a random effect in the model to account for repeated measures over time. Before the Jaccard distance calculations, the microbial protein groups were summed to the KO term level. Permutational multivariate analysis of variance (PERMANOVA), applying 999 permutations, was used to assess statistical significance in beta diversity between categorical variables of interest—related to patient health metadata, including delivery mode, antibiotic usage, health/disease status, etc. The betadisper package in R was used as a complementary assessment to the PERMANOVA testing to test for homogeneity of multivariate dispersion to ensure the within-group dispersion was not significant using 999 permutations. A category was considered to be significant in the analysis if there was a significant PERMANOVA result .This manuscript has been authored by UT-Battelle, LLC under Contract No. DE-AC05-00OR22725 with the U.S. Department of Energy. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a non-exclusive, paid-up, irrevocable, world-wide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes. The Department of Energy will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan (The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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IgG4-related hypophysitis (IgG4-RH) is a rare chronic inflammatory condition of the pituitary gland. This study reports the presentation, management and outcomes for patients with histologically proven IgG4-related hypophysitis.A prospectively maintained electronic database was searched over a 14-year period from 1 January 2007 to 31 December 2020 at a single academic centre to identify all patients with a histological diagnosis of IgG4-RH. A retrospective case note review from electronic health records was conducted for each case to extract data on their presentation, management and outcomes.A total of 8 patients  with a median age of 51 years were identified. The most common presenting symptoms were headache , fatigue  and visual impairment . Three patients were initially treated with high-dose steroids aiming for reduction of the pituitary mass. However, ultimately all patients underwent transsphenoidal surgery. Post-operative changes included radiological reduction in pituitary mass in all patients that had imaging , improvement in vision , residual thick pituitary stalk , persistent anterior hypopituitarism  and panhypopopituitarism including diabetes insipidus .IgG4-RH is an increasingly recognised entity presenting with a variety of symptoms and signs. Clinical presentation is similar to other forms of hypophysitis. It is therefore important to consider IgG4-RH as a differential and to have a low threshold for pituitary biopsy, the diagnostic gold standard. The diagnosis of IgG4-RH will guide decisions for additional workup for IgG4-related disease, multi-disciplinary team involvement and follow-up. IgG4-related disease (IgG4-RD) is an incompletely understood immunological condition that can affect various organs in the body . A relatInflammation of the pituitary gland (hypophysitis) is rare with a reported incidence of around 1 per 9 million population  and a prHypophysitis can be classified in multiple ways: by anatomical location of pituitary involvement, by aetiology and by pathological examination. The anatomical classification serves to inform the extent of involvement of the pituitary gland and its associated hormone deficiencies. It either involves the adenohypophysis, neurohypophysis including the stalk or both. Aetiological classification is either primary or secondary hypophysitis. Primary hypophysitis includes cases with a distinct autoimmune mechanism or idiopathic in nature. Secondary causes include those with a clear aetiopathogenesis often due to inflammation secondary to other sellar diseases or part of multi-organ systemic involvement. Pathological variants include lymphocytic or lymphoplasmacytic with or without sclerosis, non-necrotizing granulomatous and necrotizing granulomatous.IgG4-RH is reported in fewer than 5% of all cases of hypophysitis . HoweverThis is a retrospective cohort study including all patients with IgG4-RH confirmed on histopathology. The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement was used in the preparation of this section of the manuscript . This stThe study was conducted at the National Hospital for Neurology and Neurosurgery (NHNN), which is a tertiary referral centre and the largest pituitary centre in the UK . ManagemA retrospective case note review from electronic health records was conducted for each case to extract data on patients’ demographics, clinical presentation, endocrine assessment, radiological morphology, histology and treatment outcome.We included only patients with histological evidence of IgG4-RH. All patients therefore had undergone transsphenoidal surgery. Routine screening for IgG4-RH at NHNN started in 2011, when assays to measure serum IgG4 were available to the pathology service and all hypophysitis cases going back to 2007 have been subsequently reviewed, screened and pathological specimens re-stained by immunophenotyping for IgG4-related disease.Data collection on presentation included age, sex, ethnicity, visual and endocrinological outcomes, imaging features and serum IgG4 levels. Radiological data were recorded from pre-operative pituitary magnetic resonance imaging (MRI) scans including size of the pituitary mass, enhancement of mass, thickness of stalk and optic chiasmal compression. Data included pituitary hormone profile, levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), thyroid-stimulating hormone (TSH), free thyroxine (fT4), prolactin, insulin-like growth factor-1 (IGF-1) and 9 am cortisol. Patients were considered to have adult growth hormone deficiency if there was failure of GH response (< 3 μg/L) on dynamic testing by an insulin tolerance test or glucagon stimulation test post-operatively. Adrenocorticotropic hormone (ACTH) deficiency or secondary adrenal insufficiency was defined as low morning cortisol (< 100 nmol/L), an insufficient serum cortisol response during standard dynamic testing as above or if the patient was receiving glucocorticoid replacement therapy pre-operatively. Gonadotrophin deficiency was considered in men with low morning serum testosterone levels, pre-menopausal women with low serum estradiol with inappropriately normal or low LH and FSH levels and post-menopausal women with inappropriately normal or low LH and FSH levels. TSH deficiency was considered with low or normal TSH levels and low fT4 levels. Central diabetes insipidus was diagnosed biochemically in the context of polyuria and polydipsia or in those in those cases where desmopressin was started for their osmotic symptoms pre-operatively. Data on treatment included high-dose steroids, surgical resection and the use of immunosuppressive drugs. Data of pathology included histological examination to look for IgG4 plasma cells per high power frame, their ratio with total IgG cells and correlation with biochemical serum IgG4 levels.Outcome data reported endocrine and radiological outcome. Endocrine outcome was based on baseline pituitary profile testing and where applicable on dynamic functional testing. Radiological follow-up was based on MDT recommendation and included serial interval scans. The focus of the scans was to look for any residual mass, reduction in size, change in thickness of stalk and persistence of optic chiasmal compression.No formal power calculation was performed. Due to limited incidence of the disease, we included all cases that were histologically diagnosed at our centre between 2007 and 2020. Basic data was evaluated using descriptive statistics. Mean and standard deviation (SD) were used to describe continuous variables. Median and interquartile range (IQR) were used to describe data not normally distributed.Over the 14-year span, we identified 40 cases that had a surgical biopsy to determine the cause of hypophysitis. IgG4-RH was detected in a total of eight patients  at diagnosis. The male-to-female ratio was 1.66. Majority of patients were Caucasian , followed by Asian  and Black  ethnicity. The most common symptom at presentation was headache , followed by fatigue  and clinical visual impairment . Symptoms related to endocrine dysfunction included sexual dysfunction  and osmotic symptoms of polyuria and/or polydipsia  , ACTH , FSH and LH . Only one case (case 3) had an inadequate response of GH on insulin tolerance testing. Case 1 was not assessed for GH deficiency as was lost to follow-up. The other six cases did not have dynamic testing for GH deficiency but had low–normal IGF-1 levels with the presence of three or more pituitary hormone deficiencies to suggest they would have inadequate response to dynamic testing . One casRadiological imaging demonstrated features suggestive of hypophysitis including an enlarged pituitary mass in all patients with extension into the suprasellar space, a thickened pituitary stalk in 5 patients 5/8;62.5%) and one patient (case 5) had optic chiasmal compression  of the patients had initial biochemical evaluation for IgG4 levels as the test was not routinely done prior to 2011. The median level was 152.5 mg/dL , Table .Table 3BPostoperative endocrine status after 1 year was as follows: anterior hypopituitarism , followed by panhypopopituitarism . One patient was lost to follow-up. Three patients were initially treated with high-dose glucocorticoids aiming for a reduction in the size of the pituitary mass. In these three cases  initially, oral glucocorticoid dose ranged between 20 and 40 mg of prednisolone for a minimum of 4 weeks followed by gradual dose reduction over a period of 1 to 6 months. In one patient (case 4), a short course of intravenous methylprednisolone was also trialed. However, despite relatively high-dose glucocorticoids, all patients proceeded to transsphenoidal surgery to make a definitive diagnosis. A persistently enlarged pituitary gland following treatment with oral glucocorticoids is seen in Fig. Following a histological diagnosis of IgG4-RH, three asymptomatic patients from our case series  were screened to determine if their pituitary impairment was secondary to IgG4-RD. Fluorodeoxyglucose-positron emission tomography (FDG-PET) scans were performed to look for any extra-pituitary involvement: one patient (case 3) had a normal scan; one patient had an avid right submandibular gland (case 6), which was resected but with negative IgG4 histology; and the last patient (case 8) had non-specific activity in the pituitary gland and low-grade uptake in their large vessels. The remaining five asymptomatic cases did not have any imaging modality to screen for secondary involvement from IgG4-RD.Postoperative scanning demonstrated radiological reduction in size of the pituitary mass in all patients, except (case 1) the one lost to follow-up. However, a residual thick pituitary stalk was seen in more than two-thirds of the patients . Of the two patients  that presented with clinical visual impairment (cases 4 and 8), there was improvement of visual outcomes in one of them. A residual thick stalk is seen in Fig. All the cases were histologically confirmed to have IgG4-RH by immunophenotyping. The criteria for histopathologic diagnosis are in accordance with the Leporati criteria for diagnosing IgG4-RH, formulated in 2011  scan of the head may show a hypodense pituitary mass with adjacent bony changes, but it may also be completely normal . Hence, We have used the criteria devised by Leporati et al. which are now widely accepted to diagnose IgG4-RH. It is currently the only pituitary specific clinical diagnostic criteria . It inclCriterion 1: pituitary histopathology; mononuclear infiltration of the pituitary gland, rich in lymphocytes and plasma cells, with more than 10 IgG4-positive cells/hpf.Criterion 2: pituitary MRI; sellar mass and/or thickened pituitary stalk.Criterion 3: biopsy-proven involvement in other organs; association with IgG4-positive lesions in other organs.Criterion 4: serology; increased serum IgG4 (> 140 mg/dL).Criterion 5: response to glucocorticoids; shrinkage of the pituitary mass and symptom improvement with steroids.Diagnosis of IgG4-RH is established when any of the following is fulfilled: Criterion 1 OR Criteria 2 and 3 OR Criteria 2, 4 and 5.The ACR and EULAR have jointly set forward a point-based classification criterion for IgG4-related disease . It has Pathogenesis of the disease remains unclear; an autoimmune mechanism or chronic infection has been suggested as possible causes . AutoantThere is no standardised treatment of IgG4-RH due to the rarity of the disease, varied clinical presentation and lack of clinical trials comparing the efficacy of different medical treatment options. The main aims of treatment are to reduce the mass effects and to compensate for pituitary hormone deficiencies. Typically, glucocorticoids are the first line treatment for autoimmune hypophysitis, and the dose is tapered over weeks to months guided by clinical manifestation and imaging . GlucocoWhilst there are established pathological diagnostic criteria for IgG4-RD, small biopsies lacking the characteristic morphological alterations or showing atypical histological features are not uncommon. It may still be difficult to diagnose confidently purely on pathological grounds, and correlation with clinical and serological findings allows a more confident diagnosis . We stroThis study has several limitations. As it represents a retrospective case series for a rare disease, the sample size is small. It is subject to selection bias, and being retrospective, it depends on the accuracy and availability of the data records. The diagnosis remains complex, and none of our patients had extra-pituitary involvement despite asymptomatic screening in several cases. Although this is a monocentric study given the multi-cultural patient base of the centre and uncertain guidance over a 14-year period, the management has not been homogenous. This cohort only includes patients with a confirmed histological diagnosis, and there are certainly more patients with IgG4-RH who did not require a surgical intervention. This limits its generalizability to larger populations of patients.IgG4-RH is an incompletely understood rare disease. As opposed to other causes of hypophysitis, which affect young female patients, IgG4-RH has a male preponderance in the sixth decade of life. It is being increasingly recognised due to greater awareness amongst physicians, greater access to the serum IgG4 assay and repeat staining of histological samples previously labelled with unspecified cause of hypophysitis. Given that there is a clinical overlap with other forms of hypophysitis and that the management is distinct, a low threshold should be kept for making a definitive diagnosis by a biopsy. We suggest that all patients should have a serum IgG4 measurement and discussion at the pituitary MDT and IgG4 MDT (if available) regarding extra-pituitary screening imaging if hypophysitis is suspected at diagnosis. If the serum IgG4 concentration is > 140 mg/dL and a trial of glucocorticoids resolve the presenting symptoms, then IgG4-RH is diagnosed according to the Leporati criteria. In case there is failure of resolution of presenting symptoms with glucocorticoids or if there is any surgical indication or diagnostic uncertainty and serum IgG4 concentrations are not elevated, then an early pituitary biopsy should be sought. If there is extra-pituitary organ involvement found on initial screening, then a biopsy of the affected organ should be sought to make a diagnosis of IgG4-RH. The approach to diagnosing and managing a case of suspected IgG4-RH is outlined in Fig.


Several researchers devoted considerable efforts to partially replace natural aggregates in concrete with recycled materials such as recycled tire rubber. However, this often led to a significant reduction in the compressive strength of rubberized concrete due to the weaker interfacial transition zone between the cementitious matrix and rubber particles and the softness of rubber granules. Thereafter, significant research has explored the effects of supplementary cementitious materials such as zeolite, fly ash, silica fume, and slag used as partial replacement for cement on rubberized concrete properties. In this study, systematic experimental work was carried out to assess the mechanical properties of palm oil fuel ash (POFA)-based concrete incorporating tire rubber aggregates (TRAs) using the response surface methodology (RSM). Based on the findings, reasonable compressive, flexure, and tensile strengths were recorded or up to 10% replacement of sand with recycled tire fibre and fine TRAs. In particular, the reduction in compressive, tensile, and flexural strengths of POFA concrete incorporating fibre rubber decreased by 16.3%, 9.8%, and 10.1% at 365 days compared to normal concrete without POFA and rubber. It can be concluded that utilization of a combination of POFA and fine or fibre rubber could act as a beneficial strategy to solve the weakness of current rubberized concrete’s strength as well as to tackle the environmental issues of the enormous stockpiles of waste tires worldwide. Concrete is a manufactured material that is most widely used since invented; comprises three major elements: binder , aggregates, and water. In suitable proportions, these elements will set and harden over time ,2,3. AggWaste tire aggregate is non-biodegradable in the short term. The main components waste tire aggregate consists of are the natural and synthetic rubber . The styrene-butadiene in the synthetic rubber ratio influences the properties of the polymer: with high styrene content, the rubbers are harder and less rubbery, and takes a longer time to dissolve under normal conditions, which leads to a rise in environmental concern when it ends life service ,19,20,21R2) of 0.9. Similarly, the efficiency of regressions and machine-learning (ML) models such as support-vector machine (SVM), genetic programming (GP), and Artificial neural network (ANN), were also developed to evaluate the strength of rubberized concrete [In addition, the majority of the existing literature relies on the traditional lab work, while the systematic experimental work of rubberized concrete is still in its infant. For example, response surface methodology (RSM) was widely used to not only reduce the number of experimental designs, but also to assess the interaction and relationship between the affecting parameters, as well as to predict and optimize the output effectively ,59,60. Fconcrete . From thIn this study, Ordinary Portland Cement (OPC), Type I, which meets standard EN 197-1-CEM I—42.5 N, was used for this research. The POFA used in this study was obtained from Kahang mill, Kluang Johor, in the south of Malaysia. The POFA, with a particle size less than 200 µm, was collected and transferred to the grounding machine. 2) at 16.4%, and aluminum oxide (Al2O3) at 4.24%. Meanwhile, POFA is rich in SiO2 at 63.7%, which is a percentage similar to that of other pozzolanic materials. The results on the characteristics of POFA and OPC are presented in two parts, namely physical properties, and chemical characterization in The pozzolanic activity of POFA improves with size reduction . TherefoNatural river sand quartz (fine aggregate) and gravel (coarse aggregate) were used in compliance with ASTM C33  requirem1 has a maximum length of 16 mm. Fibre rubber aggregate (R1) was replaced partially as the natural fine aggregate. Fine granular tire rubber aggregate—Type R2 sized was replaced partially as a fine aggregate. Coarse granular tire rubber aggregate—Type R3 particle size was replaced as a conventional coarse aggregate , fine granular crumb rubber (1 to 4 mm), and coarse granular crumb rubber (5 to 8 mm). Fine fibre tire rubber aggregate—Type Rggregate . Figure A high-range water reducer was used to achieve the desired workability. The basic component of super plasticiser (SP) is synthetic polymers base, and it is a chloride-free product. The used SP meets the requirement of ASTM C494 for types A and F. This admixture is compatible with all types of cement. This type of SP is normally dispensed at a rate of 0.8 to 3 litres per 100 kilograms of cementitious material. A 20% level of POFA replacement with 0.38 water–binder ratio and with the introduction of 1% super plasticiser was determined as the optimum SP admixture to satisfy the desired workability from the previous steps of modification mix design. The next phase of modification of the concrete mix design is the introduction of a rubber aggregate to the concrete mixture. There is no standard method or globally accepted method to manage the amount and size of rubber aggregates for rubberized concrete. According to previous experiences, many opinions were given by researchers relative to experimental observations in different studies. Two methods can be used for substituting materials in concrete mixtures: equivalent weight of rubber materials or equivalent absolute volume of rubber materials in the mixture. Both methods have been used in previous studies. Some researchers applied substitute materials based on the weight percentages of the total mixture ,65,66, wVarious tests were conducted to identify the normal and POFA-modified rubberized concrete properties. The workability of freshly mixed POFA-rubberized concrete was evaluated through a slump measurement, as outlined in ASTM C143; a slump test was conducted using a frustum of cone for the mould with the following values: 305 mm high; 102 mm diameter at the top; 203 mm diameter at the base. After filling the container with concrete in three layers, each layer was compacted 25 times using a steel rod with a standard 16 mm diameter. Towards reducing errors, particularly the variation in surface friction’s influence on slump, the interior of the mould as well as its base were moistened at the start of the test. The slump measurement was set to the nearest 5 mm. The pressure method ASTM C231/M231-14 was applied to obtain the air content of fresh POFA-rubberized concrete. Type A air meter was used for the evaluation of the air content of fresh concrete. The test was started within 15 min after concrete mixing. The bowl was filled with three equal layers, and each layer was rodded 25 times. Then, the base was struck with a mallet 12 to 15 times to remove entrapped air. The flange of the measuring bowl and that of the cover were thoroughly cleaned and then clamped. Water was added over the fresh concrete until the water level reached the zero mark on the tube. The vent was closed, and the pressure was provided by a hand pump. Pressure was applied until the pressure gauge reached a calibrated point (1.4 kPa). When the pressure reached the required value, the air content of concrete was computed.The mechanical properties of modified rubberized concrete were conducted by measuring the compressive, indirect tensile, and flexural strength according to ASTM C39/C39M-12, ASTM C496/C496M-11, and BS 1881: Part 118, respectively. The specimens were duly prepared and the surface area of the concrete was elevated by polishing using a lathe machine. Both the top and bottom sides were elevated with a maximum of 0.05 mm tolerance in elevation. The loading machine was cleaned, and the digital unit of the machine was programmed with the required sample information. A constant loading was selected and applied until the failure of the specimen.Additionally, microstructure studies were conducted by using scanning electron microscope (SEM) and X-ray Diffraction analysis (XRD) to provide supplementary data on the physical and chemical properties of POFA and the effect on proposed concrete. SEM test was used to study the microstructure of concrete. A JOEL 7600F SEM versatile ultra-high-resolution device with variable pressure solution, equipped with an energy dispersive X-ray analyzer, was used. The magnification of JOEL 7600F was in range of 25 to 1,000,000. The 7600F was equipped with an SDD type X-ray detector system. This type of detector is a significant advance that can acquire and process more than 100,000 X-ray counts per second. To achieve clear resolution of surface structure, specimens were coated with platinum in an “Auto Fine Coater”. Drying was necessary, as specimens containing a large amount of moisture or oil will often contaminate the inside of the electron optical column. This is normally caused by deposits that evaporate from the vacuum chamber in which the test specimen is positioned. XRD analysis was conducted using a Bruker—D8 Pioneer diffractometer. The X-ray Source was a 2.2 kW X-ray tube. The running conditions for the X-ray tube were 40 kV and 40 mA. The identification of crystalline phase was computer-based using the Xpert Score software. The database is useful for identifying approximately 200,000 metal crystal phases, alloys, oxides, salts, and other compounds.n axial points, and 2n factorial points where n is the number of independent variables. Q) was found to be nine using Equation (1), where m represents the number of centre points [Z and Zo are the real value of independent value and real value of independent variables at the centre point, respectively [L denoted the coded value of the independent variable. The main idea behind deploying response surface methodology is to (1) assess the relationship and interaction between the variables, and (2) predict and obtain optimum values of the influential factors that achieve the best performance of the target output . Therefoe points . Moreoveectively . Furtherβii is the quadratic coefficients, βo corresponds to intercept of the model, and βi denotes to the linear coefficients. It should be noted that three models were developed to optimize the mechanical properties of POFA concrete incorporating rubber. In the first model, X1 and X2 represent the input data involving Fibre rubber and time, while Y is the response . In the second model, the input parameters were fine rubber and time, while coarse rubber and time were represented in the third model. For the purpose of verification of the proposed equation, analysis of variance (ANOVA) was taken into account. In particular, R2 was calculated to evaluate the closeness between the response and real results as shown in Equation (4) [TSS represents the total sum of square error, ESS is the sum of square error based on the predicted results, and denote the mean value of actual value. In addition, PY and AY are the predicted and real values. In the same context, DF is the degree of freedom and RSS represents the sum square of differences error between the actual and predicted values. The predicted W refers to the estimated residual sum of square without the thi. Meanwhile, the signal-to-noise ratio was evaluated using the adequate precision (S/N) as shown in Equation (7) in which the value should be greater than 4 [2 denotes to the residual mean square. In addition, p-value and F-value were also taken into account to validate the significance of the proposed equation. The achievement of high F-value and p-value less than 0.005 led to considering the equation is significant [Second order polynomial equation was considered to obtain the mechanical properties of concrete. The general form of second order polynomial equation is presented in Equation (3) . Where βtion (4) . Where Stion (5) . Where, tion (6) . The difdata set . W referr than 4 , where σnificant .(3)Y=βo1 rubberized concrete. The graph of the slump value exhibited a bell curve, with R210CP20 appearing at the peak of the graph with a slump value of 105 mm. The obtained results indicated that incorporating fine granular rubber aggregates led to the enhancement of workability when the concentration of R2 TRA was lower than 20% of the total natural fine aggregates. The workability of rubberized concrete was decreased when the level of rubber aggregate replacement reached 30%. For instance, the slump value of R220CP20 and R230CP20 was 95 mm and 85 mm, respectively. The slump of concrete containing coarse granular rubber aggregates with 1% SP were obtained as 55, 45, 30 m, and 20 mm for 5%, 10%, 20%, and 30% rubberized concrete, respectively. The slump values exhibited a greater reduction in concrete incorporating coarse rubber aggregates compared with the two other types of fine rubber aggregates. Furthermore, lower workability was observed with higher coarse rubber concentration. Finally, the lower workability of R3 rubberized concrete can be attributed to the roughness of rubber particles and the higher specific surface area than those of natural aggregates. This reduction may also be related to the poor cohesion between rubber aggregates, cement paste, and natural aggregates [Slump test was conducted to evaluate the effect of rubber aggregates on the workability of POFA-modified concrete in terms of the level of rubber aggregate substitution and the types of rubber aggregates. The slump values of rubberized concrete in various amounts and types are presented in gregates ,80.1) showed higher values of air content than concrete incorporating fine granular particles (R2). The shape of the fibre rubber aggregates plays an important role in this behavior, and the number of particles per unit volume of the fine fibre was higher than that of R2. R1 also entrapped more air during the mixing process of concrete because of the shape of fibre, such that it might easily bend during the mixing process and could thus entrap air more than the granular aggregates. Meanwhile, the air content of concrete containing coarse rubber aggregates (R3) showed lower values than concrete with fine rubber particles (R1 and R2). Rubber particles are known to be non-polar by nature (water insoluble). Thus, during mixing, they were able to trap air bubbles at the particle surfaces. Moreover, the specific gravity of coarse granular rubber aggregates (1.364) was higher than that of both types of fine (1.332) and fibre rubber aggregates (0.884), and the number of rubber particles in a unit was less than that for both fine rubber aggregates. Therefore, the amount of air bubbles was reduced, perhaps causing the lower air content in the R3 batches of the concrete mix [rete mix ,81.1) at different curing times. When R1 concentration increased, lower strength development was observed as shown in p-value of fibre rubber was 0.0004, whereas the F-value of CA was greater than 255 for all data sets indicating that the effect of fibre rubber on compressive strength was significant.It should also be noted that the compressive strength values of normal concrete were 34.43, 40.41, 45.08, 48.65, 49.78, and 51.18 MPa at 7, 28, 56, 90, 180, and 360 curing days, respectively, while the compressive strength values for 20% POFA concrete were 33.5, 41.25, 47.57, 51.2, and 54.53 MPa at the aforementioned curing periods. The gain in compressive strength of POFA concrete was higher than that of OPC, and this can be attributed to the pozzolanic reaction of POFA, which continued beyond the age of 28 days, as well as the lack of hydrated POFA particles acting as fine fillers in the concrete. A higher compressive strength reflects the high fineness of the POFA, which promotes the pozzolanic behaviour that is a major factor for strength development in POFA concrete ,82,83.1) were lower than that of normal and 20% POFA concrete at all ages. An increase in rubber particle content was found to deteriorate compressive strength further. The 28-day compressive strength of R1 rubberized concrete was reduced by 5.3%, 15.9%, 29.8%, and 41.5% compared to the case where the level of rubber replacement was 5%, 10%, 20%, and 30% of fine natural aggregates, respectively. An interesting point is that the rate of strength development in rubberized POFA concrete further increased at 28 days. For instance, R15CP20 showed much closer values of strength to that of normal concrete after 28 days. The compressive strength of rubberized concrete was found to improve with the introduction of 20% POFA to the concrete. Moreover, the values of the reduction in strength were limited to 2.7%, 13.6%, 27.8%, and 39.9% for 5% to 30% rubber replacement when compared to normal concrete.On the other hand, the compressive strengths of concrete containing fibre rubber aggregates (R2) are presented in 2 rubber aggregates. The initial 28-day compressive strength for normal concrete was 40.41 MPa, but the value decreased to 37.6, 30.6, 26.4, and 23.6 MPa when 5%, 10%, 20%, and 30% R2 tire rubber aggregates were introduced to the concrete mixture. The results showed that the strength behaviour of rubberized concrete highly depended on the substitution ratio of rubber aggregates. Furthermore, the compressive strength was found to decrease drastically with increasing tire rubber particle content. For instance, the compressive strength was reduced by 6.9%, 24.2%, 34.6%, and 41.5% when fine granular TRA were replaced at 5%, 10%, 20%, and 30% sand in concrete. The strength behaviour of concrete incorporating R2 rubber particles was in agreement with the common understanding that the replacement of natural aggregates with tire rubber particles results in a reduction in compressive strength. However, accurate design, proper curing of concrete, and pozzolanic material addition, such as POFA, may improve the compressive strength of rubberized concrete. Emiroglu et al. [Apart from fibre rubber to fine rubber, the experimental and theoretical results of the compressive strength of concrete containing fine granular rubber aggregates  are illustrated in 3 replacement, respectively. The strength gain behaviour of concrete incorporating POFA and R3 aggregates is in agreement with the pattern of POFA concrete. However, the rate of gain strength in rubberized POFA concrete was lower than that in POFA concrete alone, but it was observed that the reduction percentages in strength of concrete incorporating 5% R3 aggregates were 19.4%, 19.03%, 18.84%, 18.6%, and 18.4% for 28, 56, 90, 180, and 360 days, respectively, in comparison with normal concrete at the corresponding ages. The trend of lower strength of concrete containing R3 rubber aggregates demonstrated that the size of rubber aggregates significantly influenced the strength of rubberized concrete. A comprehensive analysis and discussion regarding the effect of rubber aggregate size are given in the next section.In addition, both experimental and estimated results of the compressive strength of concrete incorporating granular coarse rubber aggregates (R1) showed better performance than fine granular rubber aggregates (R2) in terms of compressive strength. This better performance significantly continued for 10% rubber aggregate replacement, but the further addition of rubber aggregates beyond 20% resulted in the disappearance of the ascendancy of fine rubber aggregates (R1). For instance, the compressive strength values of R130CP20 were 16.32, 24.28, 28.02, 29.2, 30.53, and 31.6 MPa for 7, 28, 56, 90, 180, and 360 days, respectively. Likewise, the results of R230CP20 were 16.5, 23.6, 27.6, 28.8, 30.23, and 31.4 MPa for the corresponding days. These results indicated that the compressive strength of 30% R1 and R2 rubberized concrete showed similar performance, with the difference in compressive strength being insignificant for all ages of concrete.It can be inferred that, the compressive strength of rubberized POFA concrete was significantly influenced by the size, type, and proportion of rubber aggregates in concrete. The results indicated that fine fibre rubber aggregates (RThis shows that the type (fibre and granular) of rubber aggregate was only important for low levels of replacement, and for medium and high amounts of proportion, the influence of the type of rubber particles on compressive strength was minimal. In other words, the deterioration of compressive strength with 30% replacement of fine natural aggregates by tire rubber particles was more significant in terms of the amount of rubber aggregate in the mixture, fibres, or granular rubber particles. Indeed, the compressive strength behaviour of concrete attributed to the incorporation of fine rubber aggregates was affected and controlled by the type of rubber aggregate for 5% and 10% replacement, but the effect of rubber type on the strength behaviour was reduced for 20% replacement. For 30% replacement, the behaviour was significantly controlled by the rubber aggregate content rather than the type of rubber particles.The maximum amount of coarse replacement aggregate was limited to 30%, and the compressive strength could be marginally acceptable for concrete used for structural application. The strength behaviour of rubberized concrete may be explained by several possible reasons for the reduction in strength with the inclusion of coarse rubber aggregates in the concrete mixture. The TRAs were significantly softer than the surrounding cement paste and natural aggregates. Therefore, rubber aggregates showed a higher elasticity and deformability than other concrete components upon the application of load. In this case, cracks begin forming around the rubber particles. This hastened the failure of rubberized concrete. Another reason may be the non-polar nature of rubber particles, which may significantly reduce the adhesion between rubber aggregates and cement paste, thereupon creating a weak phase in the concrete. The soft rubber aggregates may be considered as voids in the concrete matrix. Furthermore, a higher degree of air contact in concrete may boost the weakness of rubberized concrete when subjected to applied loads. Moreover, some researchers, such as Eldin and Senuci , likened1) achieved the best result among the three types of rubber aggregates. The reduction in splitting tensile strength was more pronounced with the use of coarse rubber aggregates than with fine rubber particles. The percentage of reduction was observed for all levels of replacement. The results indicated that in relation to the size and type of rubber aggregates, the fine fibre rubber particles performed better than granules in both coarse and fine particles. This was in line with the finding of Li et al. [1 and R2 with 30% tire rubber particles were extremely close, such that no significant difference in tensile behaviour was found in both groups. The behaviour and trend of tensile strength in concrete with high-volume rubber content can be concluded to be independent of the type or shape of rubber aggregates in the same grade.The results of the splitting tensile strength of rubberized concrete are presented in i et al. , who reci et al.  supporteA visual inspection of the crushed sample showed different behaviours of rubberized concrete because the rubber particles were never cut or broken under loads; the rubber aggregates were observed in intact conditions . DespiteThe flexural strength of concrete containing 20% POFA and 5% to 30% tire rubber aggregate is presented in 1) aggregates yielded concrete with poorer performance than that with granular particles (R2 and R3), regardless of the gradient particle size for all levels of replacement. This was exactly opposite of the trend of the compressive strength of concrete with these three types of aggregates. The performance of R1 and R2 in terms of flexural strength was very close. For instance, flexural strength was 5.35 (−14.94%) and 5.77 (−9.8%) at 28 and 90 days, respectively, for R15CP20. Meanwhile, these values were 5.61 (−10.81%) and 5.96 MPa (−6.9%) for R25CP20.The reduction in flexural strength was not as significant as observed in terms of the reduction in compressive strength because of the incorporation of TRA. Furthermore, the substitution of fine fibre rubber 2 in a cement paste. Ca(OH)2 acts as an indicator for pozzolanic reactions. Chindaprasirt et al. [2 content decreases while fortifying the sulphate resistance in the concrete. The pozzolanic reaction of high-fineness POFA is denser and more homogenous, enabling it to increase the compressive strength of concrete.The X-ray diffraction patterns of raw materials of OPC and POFA are depicted in t et al.  reported130CP20 specimens (24.3 MPa) presented lower numbers of cracks with smaller size compared to the R230CP20 (23.6 MPa) and R330CP20 (20.5 MPa) specimens; with a wider bond zone, the compressive strength of concrete containing coarse TRAs was lower than that of concrete containing fine TRAs, regardless of whether the shape was fibre or granular. For the fine TRAs, it was concluded that concrete containing fine fibre rubber aggregates performed better than that incorporating fine granular rubber at lower than 30% replacement. A possible explanation for the difference in performance between granular and fibre particles may be attributed to the different load transferring capabilities between granular and fibre rubber aggregates. Once the load is applied to rubberized concrete, granular rubber particles do not have enough length to transfer the applied load through interfacial frictional force, whereas fibres have a longer length to transfer the applied load, thus resulting in higher strength.This section presents the outcomes of the research.The workability of rubberized concrete made with fine and fibre TRAs is much closer to that of conventional concrete if the rubber replacement level is limited to 10%. Increasing the size and percentage of TRAs decreased the workability due to the increased friction by rubber particles.130CP20 was 2.88 times higher than that of concrete containing 20% POFA alone, indicating that the air content increased by increasing the fraction of rubber aggregate, which is attributed to the geometry and specific gravity of rubber particles.The air content in RA significant decrease in strength with a higher level of rubber replacement especially beyond 20% can be ascribed to the softer and non-polar nature of TRAs which leads to reducing the cohesion of the concrete matrix. Meanwhile, the influence of the type of rubber particles on strength was minimal.The compressive strength of POFA-influenced concrete started to exceed that of OPC concrete at 28 days by 2%, confirming that the pozzolanic reaction of POFA is less influential at early ages and increases with time.The pozzolanic reaction of POFA enables the increase in the strength of concrete, as a consequence of denser concrete by generating more (C–S–H) gel and enhancing the bond between the TRA particles and cement paste.R2 > 0.99, R > 0.994), thus confirming both the robustness and reliability of the models.The non-linear equations proposed here proved their ability to predict the compressive, tensile, and flexural strength with minimum error and high correlation between the actual and predicted data (A reasonable difference (less than 0.2) between