This review aims to present and discuss the mechanical and environmental properties of two different type of recycled aggregates obtain from construction and demolition waste (CDW): (1) Recycled Concrete Aggregates (RCA) and (2) Mixed Recycled Aggregates (MRA). In addition, the properties of the concrete in the fresh  and hardened state , as well as the environmental impact of the concrete produced with the two types of recycled aggregates, are presented and discussed. Due to the heterogeneous composition of recycled aggregates, the concrete properties can be significantly variable. The systematic review concerns scientific papers published from 2010 to 2020 and it shows the importance of the selection process in order to obtain high quality CDW as well as of the type of recycled aggregates on concrete properties. In particular, recycled concrete aggregates show a better quality and homogeneity than mixed recycled aggregates that make them more suitable for concrete. This work presents an overview on the influence of recycled aggregate quality on the physical, mechanical and environmental properties of concrete. The construction sector in Europe uses about 50% of the total available raw materials, thus consuming huge quantities of natural resources and soil from which this material is extracted . At the This situation is also reflected at Italian level, where the construction sector in 2019 produced about 69 million tons of construction and demolition wastes (CDW) , represeDuring the last few years, many European Countries, no longer having the possibility to dispose of this type of waste in landfills, and having a lack of extractable quarry material, have encouraged the development of recovery processes to transform these wastes into secondary raw materials that can be reused in the construction sector .The practice of reuse has also been promoted in Europe by the Directive 2008/98/EC, which required Member States to reach a minimum percentage of recovery of CDW of 70% by 2020.Many European countries have exceeded this minimum target, even reaching levels of 100% as in the case of The Netherlands, or 98% as the United Kingdom . Italy fThe Italian legislation is trying to promote the use of recycled materials in the production of concrete by means of the Legislative Decree 11 October 2017 which has made mandatory the use of at least 5% of recycled material in concrete structures for public procurements. Furthermore, the use of RA as total or partial substitute of natural aggregate is also allowed by the Italian structural code, which limits the percentage of natural aggregates replacement, depending on the concrete grade and its possible structural applications .The final use of RAs is related to their specific characteristics. According to Silva et al. , two maiThe fears of end users, nowadays, make it difficult to apply these materials in new construction, therefore, increasing their knowledge, and demonstrating how the required technical and environmental criteria are respected, how it can be used and what are the results obtained is an important objective.Assuming that characteristics are worse, in this research, we wish to analyze the results obtained in the numerous studies proposed in the last 10 years, showing how concrete produced with these types of aggregates still has good applicability and how the quality of these aggregates allows higher value uses compared to fillings and road foundations. Many literature studies on this topic are presented, but given the high heterogeneity of the RAs, the results on the mechanical properties of the concrete are very variable.This article aims to perform a statistical analysis of a large amount of data, examining the variation of the data and find correlations between RA type and concrete properties. Furthermore, it aims to analyze how the quality of the RA can affect the mechanical end environmental properties of concrete and the importance of applying a suitable preliminary treatment that allows one to obtain selected RCAs will be discussed.The analysis presented herein was developed on the basis of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement . The proStudies were eligible if they met specified criteria. In particular, the paper selection was carried out in relation to the selected keywords presented in To be eligible for inclusion, studies must be published in scientific papers or review papers in order to analyze only reliable and verified results, thereby conference papers, book chapters, conference review books and editorials were excluded. Moreover, the selected papers must have been published in English between 2010 and 2020, which corresponds to 66% of the total studies found when placing no limitations.The search for eligible studies was conducted using Scopus as a search engine with the combination of keywords listed in Different methodologies for graphical representation were applied for the analysis of the results of this research. All the results were analyzed and processed using the IBM SPSS software . For all the results represented, an overview of the amount of data used in the study was initially given. This graphical representation has been carried out by using histograms representing the frequency of the analyzed values. This allows a first analysis of the data by evaluating which are the most representative cases.The second methodology was based on a box plot according to the representation of Lastly, the third and last method used is the scatterplot, which is used for the graphical representation of the mechanical and physical properties analyzed in the research. Scatterplots are used to determine the intensity of a relationship between two numerical variables. The x-axis represents the independent variable, and the y-axis represents the dependent variable.Initially a total of 5122 studies were identified. After the first screening and deletion of studies falling into the categories of book chapter, conference reviews and articles and keeping only published papers and reviews, 3720 studies remained.After applying the second eligibility criterion, which provides for the sole inclusion of studies published from 2010 to 2020, the number of eligible studies was reduced from 3720 to 3406. Finally, after excluding non-English published studies, the final number of eligible studies was 3085. After removing duplicates, 1682 scientific articles remained. Of these, about 1000 studies were discarded after reviewing the titles and abstracts as these papers did not match the target of the study.Regarding the 1000 discarded papers, they either did not meet the requirements or did not refer to the keywords used to find them. In fact, most of the studies were not related to concrete produced with Ras at all, but rather to other types of aggregates obtained from other recycled materials. Other studies were discarded because the main topic was a statistical analysis and not actual tests on concrete cubes or cylinders. The last inclusion criterion led to all the papers having as topic road and rail construction being discarded.The full text of the remaining 680 publications was examined in detail. A total of 640 studies did not meet the previously described inclusion criteria, while 42 studies did meet the inclusion criteria and were analyzed in this review. The corresponding PRISMA flow chart is presented in Water absorption of RAs is a fundamental characteristic that must be considered in the mix-design of concrete-containing RAs. Several characteristics directly depend on this factor. In particular, compared to natural aggregates, the high water absorption of RAs strongly affects the concrete workability and everything that is directly related to it, such as the water/cement ratio (w/c ratio), the compressive strength and durability properties of concrete ,21,22,23In both cases, 161 specimens were analyzed. As shown in This confirms the significant difference in water absorption between the two types of aggregates. Generally, this increase in water absorption is due to the adherent mortar which is responsible for the porous structure of the RAs ,41,42,43In order to decrease the water absorption, Pedro et al.  stated tMoreover, the water absorption data  were compared between MRAs and RCAs, as shown in Ras’ water absorption depends on the aggregates’ size as well as the strength of the initial concrete. In fact, a lower aggregates size leads to an increase of porosity on the aggregate surfaces and, consequently, to an increase in the water absorption. The same occurs with the strength class of the original concrete; in fact, RCAs from high strength concrete have a less porous structure on the surface of the aggregates which leads to a decrease of water absorption ,49,50,51The water/cement (w/c) ratio is an important parameter for the concrete mix design, and it is strictly related to the water absorption of RA. It can be observed that the w/c values vary from a minimum of 0.29 up to a maximum of 0.97, and the most studied values are 0.40, 0.45 and 0.50, corresponding to the most representative values for construction applications.It can be noticed that, for w/c ratios of 0.4, 0.45 and 0.5, the concrete produced with RCAs (green boxes) has a lower reduction of compressive strength than the corresponding concrete manufactured with MRAs.For these three w/c values, the decrease of compressive strength for concrete made with RCAs is about 10% , while for MRAs it varies from 10 to 20%. Data for a w/c ratio of 0.6 refer to RCAs only; it can be noticed that the average value of the compressive strength decrease is around 20%.In summary, the relationship between the compressive strength and w/c shows high variability in the case of concrete with MRAs, probably due to their heterogeneous characteristics, especially for water adsorption.The experimental scatter can be due also to the addition of superplasticizers used for concrete workability, which enhances the mechanical properties. For instance, Wagih et al.  stated tAn important property for fresh concrete, that has a strong influence on mechanical characteristics and durability, directly connected also to the water absorption and water/cement ratio, is the workability of concrete ,28. FiguA total of 259 workability values according to UNI EN 206)  were anaThe analysis of the literature shows that, among the several factors that affect the workability of concrete, water absorption is the most important one ,55,62. IDue to the high-water absorption of the RAs, RAs are often pre-saturated ,29,31 inThe easiest way to enhance concrete workability is the addition of allowed superplasticizers, even for large levels of substitution, achieving slump values close to those of the reference concrete ,62.As stated by Sri et al.  and VeriDifferent factors can affect the mechanical compressive strength, including water absorption, the strength of the original concrete used to produce RAs, the type of RAs used in the mixture, the quality of these as well as the w/c ratio.ck) and transformed into cubic compressive strength (Rck), assuming the following conventional equation:All the results refer to the compressive strength at 28 days, determined on cubic samples, despite the fact some tests were carried out on cylindrical samples , the median reduction of compressive strength of concrete with RCAs is lower than that of concrete with MRAs.The high variability of values for MRAs concrete, with respect to RCAs concrete is noteworthy. This is due to a lower water absorption of RCAs as compared to MRAs. This statement is confirmed by Guo et al.  who statIt should be also observed that, during mixing, RAs from CDW tend to crumble into finer particles, with higher water absorption, and make the gradation of the cement mixture finer, which, subsequently, provokes a further decrease of concrete strength ,77.As mentioned above, there are several factors that influence the decrease of compressive strength. As Thomas et al.  stated, Bidabadi et al.  and RashAccording to Eckert et al. , in ordeBy considering the difference between high and low-quality RAs, a possible explanation is the different distribution of particles coming from concrete with higher strength, which are less brittle and coarser at the end of the crushing process ,64. AnotIt can be observed that, with 50% substitution, for MRAs the mean value of compressive strength decrease is about 15% while it is lower than 10% when RCAs are used. With 100% substitution of RA, the difference increases to about 22% and 13%, respectively. A total number of 11 papers were considered for the analysis of tensile and flexural strength of concrete containing recycled aggregates . ReferriAs expected, tensile strength also decreases for increasing values the w/c ratio ,28,55, aComparing recycled concrete with the natural one, the average decrease of tensile strength for concrete containing mixed recycled aggregate increases from 5% to 25% with increasing the recycled aggregate amount up to the total substitution. Some extreme values, represented by circles and asterisks, are present and represent values which rarely are found in the selected studies.The average decrease of tensile strength for concrete containing MRAs increases from 5% to 25% when the amount of recycled aggregate increases up to total substitution. Conversely, a lower decrease of tensile strength is observed if aggregates from recycled concrete are used, showing a 5% decrease with total substitution.Similar results are observed for the elastic modulus since these mechanical properties are closely related to each other. Also in this case, by increasing the percentage of replacement of natural aggregate with recycled aggregate, the modulus of elasticity generally decreases ,35,58. TAlso in this case, due to the characteristics of the RAs, some authors found that increasing w/c ratio from 0.4 to 0.5 leads to a decrease in elasticity modulus up to 12%, due to an excess of water after the hydration phase that can reduce the stiffness of the mortar phase in concrete. The latter does not occur if there was an increase of cement in the mixture ,54.Durability of concrete is the ability to maintain its integrity and its characteristics during the whole service life. As compared to concrete made with natural aggregates, due to the attached mortar, shrinkage, chloride penetration and freeze and thaw of RAs concrete can be reduced .Referring to shrinkage, some studies demonstrate a correlation between the increase of shrinkage and the replacement percentage of natural aggregates with RAs ,34,78, p3 while the average density of the natural aggregates is about 2660 kg/m3 [3.Density of concrete with RAs was also analyzed in 16 papers. 60 kg/m3 ,31,43, t60 kg/m3 . MoreoveLavano et al.  studied As far as chloride penetration is concerned, some authors observed that, by increasing the replacement ratio, the resistance of concrete to chloride penetration decreases due to the higher porosity of RAs ,58,79. DReferring to the freeze and thaw resistance of concrete, contrasting results were found. Guo et al.  stated tEnvironmental behavior of concrete containing RAs was evaluated according to both leaching of pollutants from the recycled concrete and the whole sustainability of recycled concrete production. As concerns the leaching of pollutants, Diotti et al. ,81 foundAdditionally, the analysis of the life cycle and the environmental impacts generated by the use or production of RA for concrete was investigated. Marinkovic et al.  found thThe paper presents a systematic review of literature published within the past 10 years (from 2010 to 2020) to provide an overview on the main characteristics of concrete with RAs. The results found in the literature survey presented in this paper show that a sustainable concrete requires specific rules.First of all, the difference in properties of natural aggregates with respect to recycled aggregates from concrete only (RCA) and from mixed construction and demolition waste (MRA) is mainly driven by the presence of attached mortars on the surfaces of the aggregates. Mortar is responsible for the higher absorption, lower density and lower mechanical properties of RAs as compared to the natural one. The removal of attached mortar can increase the characteristics of recycled concrete quality, thus reducing the cement content for reaching the target strength; a possible solution is the increase of the crushing steps without creating further cracks on the surface. Due to the reduced strength of RA concrete, the compressive strength should be related to the environmental requirements; therefore, structural elements requiring lower strength can be made with RAs concrete while structural elements with higher strength should be made with concrete with natural aggregates.From an environmental point of view, beside structural safety, designers should carry out LCA analyses in order to choose the best compressive strength to be adopted for structural elements. The maximum percentage of aggregate substitution should consider the availability of RAs in the surroundings of the construction site, in order to avoid inconvenient environmental costs. If RAs are not enough for a total replacement of natural aggregates, a lower replacement helps in increasing the concrete strength and reducing the cement content.The use of pre-saturated RAs allows to increase concrete workability but this requires special attention to avoid changing the w/c ratio of the concrete with a consequent reduction of mechanical properties. A new generation of superplasticizers could be developed for concretes with RAs.In any case, the quality of the initial CDW for the production of RAs is fundamental and it is possible through a selective demolition. It would allow to obtain higher quality aggregates that enhance the concrete quality which can be hardly reached with MRAs .Leaching test on RAs lead to an increase of pollutants, but under the regulatory limits. In particular, the two types of RAs considered lead to a release of different pollutants, especially chloride from RCA and sulphates from MRAs. Leaching test from monolithic concrete blocks produced with RAs gives no significant results in terms of pollutants.Referring to the economic impact from the use of RAs in construction, results show the difference between revenues and total costs that come into play throughout the life cycle of the product, concluding that downcycling is preferable to landfill. Furthermore, recycling after selective demolition presents a higher income even if demolition is more expensive. Finally, referring to the production of concrete, the most cost-efficient path is mobile recovery, in which cost savings are mainly achieved through a reduction in the transport and production of higher value-added and lower heterogeneity materials. In this context, results show that RAs are sustainable both from the environmental and economic point of view.In a future perspective Xu et al.  demonstrThe 42 selected papers evidence that, by increasing the substitution percentage of natural aggregates, concrete properties become worse. The studies, also demonstrate the importance of the quality of the type of RAs.Regarding the water absorption of the two types of RAs analyzed (RCAs and MRAs), the experimental results highlight the importance of the selection during demolition for reducing water absorption. Experiments also demonstrate the importance of checking the attached mortar on the surface of the RAs; by increasing the number of crushing processes, the attached mortar reduces, thus enhancing the mechanical properties of concrete.Referring to the water/cement ratio, results show the importance of RCAs, in order to enhance workability and limit the compressive strength decrease.For concrete mechanical properties (in particular compressive strength), the importance of the quality of the RAs is observed and discussed since the reduction of compressive strength for concrete produced with RCAs is lower than that of concrete with MRAs. The attached mortar, the size and shape of MRAs are the main reason of the compressive strength decrease. Average compressive strength values of 35 MPa and 45 MPa are obtained using MRAs and RCAs respectively.From the environmental point of view, the release of pollutants from concrete blocks is very low. However, concrete with RAs has a higher environmental impact evaluated by the life cycle assessment analysis, due to the higher cement dosage and RAs transportation


The extensive use of antibiotics leads to antibiotic residues in frequently consumed foods. Generally, the main use of antibiotics in animals is to treat and prevent diseases and growth promotion. However, the residues and their breakdown products have several side effects on the human body and, in a broader sense, on the environment. In relation to the human body, the frequency of mutations is increased, the bone marrow is damaged (chloramphenicol), and the reproductive organs of humans are affected. Carcinogenic effects have been found with antibiotics such as sulfamethazine, oxytetracycline, and furazolidone. We summarized data from 73 scientific studies reporting antimicrobial residues in animal products that were freely available for sale. The studies were published in English starting from 1999 till 2021 and identified through the Pubmed search engine. The aims were to find out which antibiotics, legal or illegal, could be found in animal foods worldwide. Which are stable to get into the food chain and exceed the maximum residue limits (MRL) regarding the EU guidelines as a comparison. Reducing antimicrobial residues in food from animal origin and, in addition to this, fighting the tremendous growth and spread of antimicrobial resistance will undoubtedly be one of the most difficult food safety challenges in the coming years. The discovery of antibiotics and their use in animals and humans was indisputably one of the greatest achievements of the 20th century . HoweverOur review aims were as follows: (a) Which antibiotics were detected in foods of animal origin and which test methods were used? (b) How often do consumers get in contact with food contaminated with antibiotics? What country differences are there based on the published studies regarding food animal species and antimicrobial residues? (c) How often have the MRLs (EU guidelines) been exceeded in the case of positive evidence? We extracted all raw data and metrics reported in these studies, discussed the limitations of the methodologies used, and documented data gaps. We hope that this review will help regarding that. The detection of antibiotic residues in animal foods is increasingly investigated and documented, and thus, more data is made available to the scientific community.The number of first search results was huge. So, the summaries of the scientific texts had to be sorted out by hand based on the question in order to include as many papers as possible. Antibiotic residues in animal products that reached consumers had to be neatly separated from antibiotic degradation tests in tissues and methods for detecting these compounds. This resulted in the data after the first review, which was already much smaller as can be seen in As can be seen in For a clearer evaluation, all studies with salmon, other fish from aquaculture, and shrimp were combined as seafood in the evaluation, as can be seen in In Vietnam, China, and Iran, most of the studies related to antibiotic residues in animal products have been carried out, as can be seen in Compared to Asia, the picture in Europe is entirely different, as can be seen in In the data from Africa, as can be seen in The situation on the American continent is entirely different, as can be seen in The determination of antibiotic residues and the determination of their concentration in animal products were carried out in the 73 studies using different test procedures. ®test technology , Copan test kit , Delvo SP® test kit , Equinox test, SNAP tetracycline test kit, SNAPbeta-lactam test kit, the SNAPgentamicin test kit , Ridascreen chloramphenicol, and streptomycin kit ,. Microbial Inhibitor Tests and test kits were used, such as the microbial inhibition test using Bacillus subtilis ATCC-6633 .In the evaluated scientific studies, as can be seen in The use of ready-made test kits was prevalent in African countries. This may also be due to the fact that other laboratory facilities were not available. In some cases, the test kits were used to detect antibiotic residues in the samples initially and then subject them to a more detailed examination method or determine the residues more precisely using this method.Sparus aurata) and sea bass (Dicentrarchus labrax) were found in 18.3% of the samples (n = 82) [Considering all 73 scientific papers evaluated, tetracycline was detected in 39 cases, as can be seen in (n = 82) . ScientiThe maximum residue limit (MRL) is the maximum allowed concentration of residue in a food product obtained from an animal that has received a veterinary medicine or that has been exposed to an antibiotic for use in animal husbandry. The substances found include a wide range of antibiotics used in agriculture. The most critical question in this context, however, is how many substances exceeded the upper limit values. This is displayed in In this work, we reviewed 73 studies on antimicrobial residues in animal products published in English since 1999. In relation to the importance of this topic, the number of publications found was relatively small. This finding can have several causes. In the USA, the European Union, and some Asian countries, the legal guidelines regarding antibiotic residues in food are relatively strict and are also checked by state authorities. A scientific study by non-governmental research groups, therefore, does not seem worthwhile. Unfortunately, there are often rumors among the population in industrialized countries that the foods available there are full of antibiotics, pesticides, and hormones, which would cause serious health problems. It must be noted here that it is precisely in these countries that food safety is highest in terms of residues of various chemicals and pathogenic germs or parasites. Many European countries have already banned the addition of antibiotics to animal feed as a preventive measure in relation to the avoidance of diseases in livestock farms ,93. AddiPenaeus indicus) that contained chloramphenicol residues, this drug gets destroyed or degraded during heat processing treatments [Products that exceed the maximum allowed residue limits pose a serious problem. Heat treatments that occur in cooking processes can reduce the risk of ingestion of sulfonamides, tetracyclines ,96, and eatments . FurtherApart from that, concentrations of antibiotic residues vary between different edible muscle tissues . HoweverThe previous findings of this work were able to locate most of the problems with antibiotic residues in developing countries and emerging economies. It must not be forgotten that due to world trade, many of these animal products also end up on the plates of western consumers. Seafood and fish from aquaculture should be mentioned here. Due to the many positive properties in relation to a balanced diet, the consumption of these animal products is also increasing sharply ,103. ThiIn many European countries, as well as in the USA, there is a trend in animal breeding and husbandry to keep them in their natural habitat, away from factory farming in large stables. In addition, consumers want more and more meat from animals that have been treated with as few antibiotics as possible during rearing and until slaughter. However, can this desire of consumers be implemented without any problems? In a Danish study regarding consumer preferences for meat with reduced antibiotic use in pig production, two-thirds of the consumers stated a positive will to pay more for this product. However, only one in five were willing to pay more than 10% for pork produced using antibiotics, 20% less than average . While cThe presence of antibiotic residues in meat products that are above the maximum permitted limits is a serious problem. Based on the values of the maximum levels of residues in animal tissue and in relation to the daily consumption calculated on the bodyweight of the person, these residues should not pose a health risk. Exceptions are allergic reactions to antibiotics. In this context, however, a possible connection must be mentioned.There appears to be a relationship between antibiotic use and the increase in obesity in people. The number of publications on this topic is increasing steadily. Some authors point out that there is a direct link between the increased use of antibiotics in animal breeding and the increase in the number of obese people in our society over the past 70 years . It woulhttps://pubmed.ncbi.nlm.nih.gov/, the website was accessed on 15 March 2021) was used to search for original scientific papers published in English from January 1999 to March 2021. The following terms were used to search scientific papers using the following keywords:  AND . All retrieved records were saved for further review. Publications not reporting original research data or written in languages other than English were further excluded. Publications containing antimicrobial residues data in the abstract were selected for a first investigation. All scientific papers that dealt with residues of antibiotics were excluded, but in the sense of detection methods or residues in the environment, as well as pollution of animal manure, the rest that contained only research data regarding antimicrobial residues in animal food were reviewed with full content. In addition to the techniques used to detect antibiotic residues, the size of the sample was also an important quality criterion for us. We divided the sample sizes into 6 categories. Up to 50 samples were documented in 16 publications. Up to 100 samples were documented in 19 papers. Up to 200 samples were documented in 20 papers. Up to 300 samples were documented in 12 papers. Up to 400 papers were documented in 6 papers. Twelve papers were summarized for a sample size of over 400 up to 25,000. All work with a sample number below 50 was subjected to a thorough analysis regarding sampling and analytical methods. If this was correct or meaningful, this data was included in our evaluation. Publications were broadly classified by the country where the research took place, year of publishing, and further categorization regarding the animal food product.The PubMed engine : (1) country of study, (2) year, (3) type of animal product, (4) sample size, (5) animal production type, (6) antibiotics found, (7) detection method, and (8) over maximum residue limits.All data were entered as single records (‘datapoints’) in Excel (Microsoft Office). Antimicrobials and antimicrobial classes listed were those included in the World Organisation for Animal Health (OIE) classification: veterinary critically important antimicrobial (VCIA) agents (10 classes); veterinary highly important antimicrobial (VHIA) agents (8 classes); and important veterinary antimicrobial (VIA) agents (8 classes) .The problem of antimicrobial residues in animal products is not new. However, due to the globalization of the food trade, we are constantly facing new challenges. Reduction of the antimicrobial volumes currently used by humans and livestock and limiting the use of broad-spectrum and critically important antimicrobials would be essential. Educational work regarding antimicrobial residues in animal products and the associated development of multi-resistant germs must be stepped up in developing countries. This can only be reduced through increased monitoring of the use of antibiotics in animal breeding. In any case, users must be informed about the health consequences for their customers regarding antibiotic residues in animal products and the development of multi-resistant germs. The ban on the disposal of rubbish, sewage, and manure containing antibiotics must also be mentioned in this context. Alternatives to antibiotics, such as various vaccinations, the use of phages, and phage therapy, must be promoted. The use of prebiotics and probiotics in animal feed and the use of traditional medicinal herbs must also be further researched to ultimately reduce the use of antibiotics in animal breeding to a realistic amount.


Immunoglobulin G4-related disease (IgG4-RD) is a systemic fibro-inflammatory and idiopathic autoimmune disorder. IgG4-RD can be characterized by the presence of pseudotumors. Inflammatory pseudotumors may involve any part of a human organ. There are few reports of sinus lesions in IgG4-RD. An 82-year-old man has a history of chronic sinusitis for the last several years and no remarkable family history. Two years before disease presentation, the patient experienced intermittent nasal bleeding, stuffy nose, dizziness, and fatigue. Blood test revealed positive (160X) antinuclear antibody with a mixed speckled and nucleolar pattern, IgG level of 1370 mg/dL, and IgG4 level of 99.7 mg/dL. Computed tomography (CT) of the sinus revealed several calcifications in the sphenoid sinus. Surgical findings revealed tumor-like materials. Pathological examination of the soft tissues revealed acute and chronic granulomatous inflammation. Immunohistochemical analysis demonstrated high levels of positive-affinity markers of IgG, IgG4, and CD138 and a IgG4/IgG ratio > 40%. IgG4-RD with pseudotumor was diagnosed. The initial treatment was intravenous methylprednisolone 120 mg daily for three days and oral prednisolone 10 mg twice a day and azathioprine 50 mg daily. The efficacy of the treatment was insufficient, and nasal bleeding did not decrease. Subsequently administered intravenous rituximab 1000 mg monthly for 2 months. Following this treatment, nasal bleeding stopped. CT revealed reduction in nasal mucosal swelling compared with that in a previous scan. This report highlights that in cases with an inflammatory mass mimicking malignancy, IgG4RD should always be considered, and rituximab treatment is recommended upon failure of steroid and azathioprine therapy. Immunoglobulin G4-related disease (IgG4-RD) is a systemic fibro-inflammatory and idiopathic autoimmune disorder. Although its pathogenesis and underlying mechanism remain unclear, IgG4-RD is characterized by the presence of pseudotumors with storiform fibrosis, obliterative phlebitis and a lymphoplasmacytic infiltrate with predominant IgG4+ plasma cells, and elevated serum IgG4 levels . Inflamm3 , hemoglobin level of 5.3 g/dL (reference range: 13–18 g/dL), platelet count of 530,000 cells/mm3 , C-reactive protein level of 10.0 mg/dL (reference range: < 0.5 mg/dL), erythrocyte sedimentation rate of 16.0 mm/h (reference range: 2–15 mm/h), international normalized ratio of 1.03 (reference range: 0.85–1.15), activated partial thromboplastin time of 25.7 s (reference range: 23.9–35.5 s), positive(160X) antinuclear antibody with a mixed speckled and nucleolar pattern, IgG level of 1370 mg/dL (reference range: 635–1741 mg/dL), and IgG4 level of 99.7 mg/dL (reference range: 3–201 mg/dL). Computed tomography (CT) of the sinus revealed diffuse mucoperiosteal thickening in the bilateral maxillary, ethmoid, and sphenoid sinuses (The patient was an 82-year-old man with a history of chronic sinusitis for the last several years and no remarkable family history. Two years before disease presentation, the patient experienced intermittent nasal bleeding, stuffy nose, dizziness, and fatigue. He was treated in several hospitals and underwent functional endoscopic sinus surgery; however, the severity of these symptoms increased gradually. The patient visited our hospital in 2021. Physical examination revealed an enlarged right parotid gland and neck lymphadenopathy. No petechia or ecchymoses were observed in the body. Blood tests revealed a white blood cell count of 7900 cells/mm sinuses b. Severa sinuses a. Based  sinuses . The fun sinuses . Immunoh sinuses . IgG4RD  sinuses c.IgG4-RD is a systemic autoimmune disease with unknown etiology that frequently affects middle-aged men; it is characterized by involvement of a wide variety of organs and pseudotumor formation . InflammDiagnosis of IgG4-RD is based on clinicopathological findings and needs to incorporate features from patient history and clinical examination, as well as serological, radiological, and histopathological investigations . HoweverThus, histopathological and immunohistochemical analyses of biopsy specimens remain cornerstones in the diagnosis of IgG4-RD . HistopaNo standardized treatment has been established for IgG4-RD. Treatment includes surgery, steroids, immunosuppressants, radiotherapy, or a combination of these modalities . SteroidKeiko Ohno et al. reported two cases of IgG4-RD with nasal manifestations, for which oral steroid administration was effective . In addiHerein, we report a rare case of IgG4RD with sinonasal pseudotumors. This report highlights that in cases with an inflammatory mass mimicking malignancy. IgG4RD should always be considered, and rituximab treatment is recommended upon failure of steroid and azathioprine therapy.


We find a decreased abundance of Bifidobacterium spp. and increased abundance of Klebsiella and Enterococcus spp. in the antibiotic treated infants compared to controls. Amoxicillin + cefotaxime shows the largest effects on both microbial community composition and antimicrobial resistance gene profile, whereas penicillin + gentamicin exhibits the least effects. These data suggest that the choice of empirical antibiotics is relevant for adverse ecological side-effects.Broad-spectrum antibiotics for suspected early-onset neonatal sepsis (sEONS) may have pronounced effects on gut microbiome development and selection of antimicrobial resistance when administered in the first week of life, during the assembly phase of the neonatal microbiome. Here, 147 infants born at ≥36 weeks of gestational age, requiring broad-spectrum antibiotics for treatment of sEONS in their first week of life were randomized 1:1:1 to receive three commonly prescribed intravenous antibiotic combinations, namely penicillin + gentamicin, co-amoxiclav + gentamicin or amoxicillin + cefotaxime . Average antibiotic treatment duration was 48 hours. A subset of 80 non-antibiotic treated infants from a healthy birth cohort served as controls . Rectal swabs and/or faeces were collected before and immediately after treatment, and at 1, 4 and 12 months of life. Microbiota were characterized by 16S rRNA-based sequencing and a panel of 31 antimicrobial resistance genes was tested using targeted qPCR. Confirmatory shotgun metagenomic sequencing was executed on a subset of samples. The overall gut microbial community composition and antimicrobial resistance gene profile majorly shift directly following treatment (R Here, in a randomized trial of 147 infants receiving distinct antibiotic regimens for early-onset neonatal sepsis, Reyman et al. characterize the gut microbiome and resistance profiles, finding differential effects of antibiotic combinations on microbial community composition and antimicrobial resistance genes. The various causes of disturbances are also becoming evident. Among others, the effects of caesarean section (CS) delivery, formula feeding (as opposed to breastfeeding) and antibiotics on the developing neonatal gut microbiome have been described8. Antibiotic treatment, in particular, alters species diversity (α-diversity) and community composition, i.e. the ecology, of the gut microbiome for a prolonged period of time in both children and adults10. Antibiotic-induced shifts in gut microbiome composition subsequently result in the selection of antimicrobial resistance (AMR)11. The ecological side effects of antibiotics may be even more pronounced and persistent when administered in the early assembly phase of the neonatal gut microbiome in the first weeks of life. However, in this phase of life, broad-spectrum antibiotics are prescribed in up to 10% of all neonates because of (suspected) early-onset neonatal sepsis (sEONS)13.The importance of the human gut microbiome in health and disease is becoming increasingly clear. Disturbances of the gut microbial community composition after birth are associated with a broad scale of health problems in early infancy and later in life, such as infantile colic, wheezing, allergies, functional gastrointestinal disorders, obesity and generally an altered immune developmentListeria monocytogenes and Escherichia coli. These organisms are susceptible to antibiotic regimens involving combinations of penicillin with gentamicin or amoxicillin combined with cefotaxime, making these treatments equally effective14. However, already two decades ago, a Dutch study performed on a neonatology intensive care unit highlighted the issue of amoxicillin driving the overgrowth of β-lactamase producing bacteria, such as Klebsiella species (spp.)15. Another concern raised was that third-generation cephalosporins, such as cefotaxime, select for resistant Enterobacter spp. strains15. The full ecological impacts of empiric antibiotic treatment administered in early-life on microbiome and AMR gene composition are still not well-known.The most common pathogens causing EONS are group B streptococcus, 16. On average, the antibiotic treatment duration was 48 h. In this work, we find that antibiotic-treated infants show temporarily reduced gut microbial diversity, and major and prolonged ecological perturbations detected using 16S rRNA-based sequencing, compared with healthy term-born controls, as well as shifts in AMR gene profile, as determined by quantitative polymerase chain reaction (qPCR) of a panel of 31 clinically relevant AMR genes. The compositional and AMR gene findings are confirmed by metagenomic shotgun sequencing (MGS) of a subset of samples. Also, we find marked differences in ecological perturbation between the three different antimicrobial regimens, suggesting that, next to adequate treatment, the choice of empirical antibiotics is also relevant for adverse ecological side-effects.To identify the antibiotic regimen with the least ecological and AMR gene selection effects, we performed the ZEBRA study in which we enrolled 147 infants born at term, either by natural delivery or by secondary CS (SCS), for whom broad-spectrum antibiotics were indicated in the first week of life because of sEONS, and randomized them over three most commonly prescribed intravenous antibiotic combinations for this indication in the Netherlands, namely penicillin + gentamicin, co-amoxiclav + gentamicin or amoxicillin + cefotaxime17. All infants were born in hospital except for three spontaneous home births in the antibiotic-treated group (one in each regimen) and five in the control group. From two infants in the amoxicillin + cefotaxime group, no samples were available for analysis, so these infants were excluded from further analyses : penicillin + gentamicin, co-amoxiclav + gentamicin or amoxicillin + cefotaxime. As controls, 80 age-matched term-born infants, born between 19 December 2012 and 2 November 2014 were included from a previous healthy birth cohort study conducted in two of the same hospitalsIn line with sEONS characteristics, neonates treated with antibiotics were more often born with SCS, had a slightly shorter gestational age, a lower Apgar score at 5 min, and their mothers had on average a longer period of ruptured membranes before delivery (Supplementary Table p = 0.01), which was most outspoken immediately after antibiotic treatment . When stratified, we observed that the co-amoxiclav + gentamicin group had the lowest α-diversity immediately after treatment . In inter-regimen comparisons, no significant difference in α-diversity was observed.Antibiotic treatment led to a decrease in α-diversity . Also, at age 1 month, the difference in composition for the amoxicillin + cefotaxime group versus controls was most outspoken, though subsequently, similar recovery patterns were observed as for the other two regimens , showing similar results with even larger effect sizes compared to the primary comparison, underwriting that initial antibiotic treatment was explaining the differences found between groups .The stability of microbial community development within individuals over time was lower in antibiotic-treated infants compared to controls during the first 4 months of life , covariates significantly associated with overall gut microbial community composition in the overarching cohort were: age (R2 4.1%), day care attendance (R2 1.0%), breastfeeding at the time of sampling (R2 0.7%) and mode of delivery . Covariates that differed at baseline between the antibiotic-treated infants and controls were: gravidity of mothers, gestational age, duration of ruptured membranes, duration of hospital stay after birth, presence of siblings <5 years of age and inhouse smoking. As of these variables mode of delivery was the only variable that was associated with microbial community composition over the first year of life, this variable was corrected for in all downstream microbiota analyses.Besides antibiotic treatment , when compared with antibiotic regimen (R2 0.9%), age (R2 5.2%), day care attendance (R2 1.0%), and breastfeeding at time of sampling . Given only two infants showed a culture-proven sepsis, further sub-analyses for this variable were not possible. However, to account for the likelihood of neonatal sepsis in our analyses, we further categorized our treated group according to the duration of antibiotic treatment into short (1–4 days) and long (>4 days) and studied the effect on overall microbial community composition in a cross-sectional manner (Supplementary Table R2 0.4%).No significant association was found between overall microbial community composition and antibiotic treatment duration in the first week of life nor with maternal antepartum antibiotics when analyzed per time point . In a temporal, multivariable analysis (sEONS group only), we found that both variables were, although significantly, only modestly associated with composition  between controls and antibiotic-treated infants. Among others, Bifidobacterium spp. were less abundant in the antibiotic-treated infants compared to the controls from day 1 until day 36, also after adjusting for mode of delivery  and Enterococcus spp. .The colonization patterns in the control group over time were similar to those described in previous healthy infant studies Fig. 7,18. Facn = 20; median age: 6 days) and week 1 samples from controls  confirmed these significant differences in abundance of species. These data also confirmed the annotation of B. adolescentis, B. breve and B. longum strongly correlating with the Bifidobacterium OTU, E. coli correlating with the Escherichia coli OTU, S. epidermidis correlating with the Staphylococcus epidermidis OTU, K. pneumoniae and K. oxytoca correlating with the Klebsiella OTU, and E. faecium correlating with the Enterococcus faecium OTU  as found with the 16S-based sequencing data (R2 9.5%).MGS of a subset of samples from infants directly following antibiotic treatment , but not in the co-amoxiclav + gentamicin regimen. Akkermansia spp. were generally only reduced in the amoxicillin + cefotaxime group. In contrast, Klebsiella abundance was higher in both the co-amoxiclav + gentamicin and penicillin + gentamicin groups, relative to the amoxicillin + cefotaxime group in early life (day 3–17 and 2–16 respectively), whereas this reversed at a later age , we sought to test the potential modifying effect of breastfeeding on our results. We performed a post-hoc analysis to test for differentially abundant taxa between the amoxicillin + cefotaxime group and the controls, with breastfeeding added as a covariate , as the difference in duration of breastfeeding was the greatest between these two groups. The results can be found in Supplementary Tables Bifidobacterium OTU was still less abundant in the infants treated with amoxicillin + cefotaxime versus controls when correcting for breastfeeding, with a similar interval and area of differential abundance identified compared to the analysis without correcting for breastfeeding.Though we did not find a significant difference in duration of breastfeeding between the antibiotic-treated infants and controls, we did identify an effect of breastfeeding duration on microbiota development. Though this effect was smaller than antibiotic exposure itself (aph(2'')-I(de) and the vancomycin resistance gene vanA, which were both present in only 6/939 samples, while the gene that was most commonly present was the multidrug efflux pump unit acrA, found in Gram-negative bacteria (708/939 samples)19. Other commonly observed genes, present in >80% of all infants, were the aminoglycoside resistance genes aac(3')-Ii(acde), aac(6')-Ii, aph(3')-III, and the aadE-like gene, the beta-lactamase encoding genes blaampC and blaCTX-M, the macrolide resistance gene ermB and the tetracycline resistance gene tetQ.All 31 AMR genes included in the Fluidigm qPCR assay were detected in this study. The AMR genes least often detected were the aminoglycoside resistance gene p = 0.02). Temporal analysis showed no significant difference in the diversity of AMR genes between the antibiotic-treated group and controls over the first year of life. Also, no significant inter-regimen differences were found in cross-sectional nor temporal analyses.The AMR gene diversity, i.e. number of observed AMR genes detected within a sample, only differed significantly between the antibiotic-treated infants and controls at 1 month of age , and did not show a significant difference at 12 months of age . Consequently, as mode of delivery and the presence of siblings <5 years of age were the only variables that both differed between the groups at baseline and had a significant effect on AMR gene composition, these variables were corrected for in all downstream resistome analyses when comparing antibiotic-treated infants and controls. In the inter-regimen comparisons, we adjusted for antepartum maternal antibiotics. In line with microbial community composition, we decided to perform stratified analyses into the effect of antibiotic treatment duration categorized as short (1–4 days) or long (>4 days) on AMR gene composition, and again confirmed no significant difference in AMR gene composition at any time point (Supplementary Table Covariates that were significantly associated with AMR gene profile during follow-up are summarized in Supplementary Table aac(6')-aph(2''), aac(6')-Ib, aac(6')-Ii and aph(3')-III, the beta-lactamases blaCMY-2, blaTEM, the penicillin binding protein 2A gene mecA, macrolide resistance genes ermB and ermC and the colistin resistance gene mcr-1. The time intervals in which the differences existed varied from 68 days (aac(6')-Ib) to 371 days (blaCMY-2). Six AMR genes, such as the commonly present tetQ gene, were more abundant in conrols20.In a temporal analysis, 16 out of 32 AMR genes were found to be significantly differentially abundant between the antibiotic-treated infants and controls (Supplementary Table aac(6')-aph(2), blaCMY-2, ermB, ermC and mecA were consistently more abundant in each of the three regimens compared to the controls. In the inter-regimen comparisons, again the penicillin + gentamicin regimen resulted in the enrichment of the lowest number of AMR genes compared to the other two regimens -Ii and aph(3)-Ia, -Ic compared to the penicillin + gentamicin group and of the aadE gene compared to the co-amoxiclav + gentamicin group.When comparing each separate antibiotic regimen with the control group, we found that the penicillin + gentamicin group had, out of the tested panel, the lowest amount of enriched AMR genes (five), versus ten in both the amoxicillin + cefotaxime and co-amoxiclav + gentamicin groups (Supplementary Tables aac(6')-Ii had the strongest positive correlation with E. faecium , which was highly abundant in the infants treated with amoxicillin + cefotaxime . In this way, we could also positively correlate the aac(6')-aph(2) gene, which was consistently more abundant in all three regimens compared to controls, to E. faecium . As expected, mecA was positively correlated with S. epidermidis . E. coli was most strongly positively correlated with blaampC and acrA , all of which were consistently more abundant in the controls.When studying the correlation between the relative abundance of OTUs and AMR genes (Supplementary Table aac(6')-aph(2), ermB, ermC and mecA genes being enriched in the antibiotic-treated infants : 8.56, p.adj < 0.001; 6.10, p.adj = 0.001; 4.95, p.adj = 0.001; 6.49, p.adj < 0.001). Additionally, MGS identified statistically significant enrichment of AMR genes in antibiotic-treated infants not part of our Fluidigm panel, including a broad set of genes coding for resistance against rifampin, fluoroquinolones, aminocoumarins, daptomycin, elfamycins, trimethoprim, sulfonamides, fusidic acid and bacitracin  milk oligosaccharides32, which in the first 4–6 months of life are the sole food source for infants. Hence, potential effects on infant growth and development are imaginable when the abundance of these bacteria is decreased. Also, the extensive outgrowth of potential pathogenic bacteria such as Klebsiella and Enterococcus spp. warrants further studies into the susceptibility to infections following early-life antibiotic treatment. In a previous study, antibiotics in early life have been associated with increased rates of diarrhea in early childhood, which we now hypothesize might be a consequence of persistent microbial dysbiosis33.On the level of individual taxa, we found especially various beneficial 34. While this regimen might clinically be the least popular due to the frequency of penicillin administration and the need for gentamicin serum level monitoring, we think this regimen deserves reconsideration for the treatment of sEONS on neonatal wards given the uniformly lower ecological side-effects observed, and considering the three regimens compared are equally adequate treatments for this indication14. Interestingly, some AMR genes were more present in controls. While this may be counterintuitive, it is known that many commensals carry AMR genes, which we confirmed by correlating the AMR genes enriched in controls with the presence and abundance of commensal bacteria, such as the blaAMPC gene in E. coli35.Importantly, when comparing the antibiotic regimens, we found that all three showed significant ecological side effects. However, the amoxicillin + cefotaxime regimen showed the most outspoken ecological effects, especially on overall microbial community composition, stability of microbiota development, and AMR gene profile shifts directly following treatment. The co-amoxiclav + gentamicin regimen showed the most obvious reduction in microbial diversity and most persistent effects on AMR gene profile. Importantly, the penicillin + gentamicin regimen showed the least detrimental effects on all these parameters. In part, this can be explained by the minimal penetration into the gut lumen of aminoglycosides given intravenously36. For this reason, more emphasis on improving the diagnostic accuracy of EONS is crucial, as the principle of when in doubt, there’s no harm in treating appears far from true. Reducing the number of neonates in whom we initiate broad-spectrum antibiotic treatment is feasible, given the fact that prescription rates vary widely even between European countries with similar infection statistics38. This strongly suggests that guidelines underpinning these national differences should be compared to identify factors that could lead to more reserved prescription practices. Moreover, ongoing efforts to improve the prediction of EONS, for instance through the application of the EONS calculator, could further help to reduce unnecessary antibiotic treatment, even in countries with already low prescription rates39. Altogether, this would ideally result in—first and foremost—safe treatment guidelines, whilst simultaneously preserving early-life microbiome development at this critical stage of life.There is a rapid increase in awareness regarding the side-effects of antibiotics on selection and development of AMR in pathogenic bacteria on a population level. Most hospitals currently enforce antimicrobial stewardship programs that ensure appropriate antibiotic therapy. However, these programs do not consider ecological side-effects yet, largely because the information is lacking. Presently, the main focus is to shorten the duration of broad-spectrum antibiotic treatment as much as possible. However, in our study, although a modest effect was observed for antibiotic treatment duration in association with microbiota composition, the effect was negligible compared to the initiation of antibiotics in the first place. This held true when testing the effect of antibiotic treatment duration in a categorized way, better reflecting the clinical setting (0–4 days versus >4 days). Currently, antibiotics are prescribed in 4–10% of all neonates, whereas only an estimated 1 in 1000 will develop a proven infection, likely resulting in unnecessary treatment of >90% of all treated children14.Strengths of this study include analyzing samples before the start of antibiotic treatment to eliminate baseline differences as potential confounders. Importantly, albeit modest differences in overall microbiota composition and AMR gene profiles existed in our study between neonates with sEONS and controls at enrolment (before antibiotics), these were both non-significant and of limited relevance compared to the observed antibiotic effects. Furthermore, we used a randomized study design to compare the impact of three commonly used broad-spectrum regimens. Additionally, we validated our results through an independent MGS technique. The results of this study are highly applicable to other hospitals and countries, as generally common antibiotic regimens were comparedA potential weakness of this study is its molecular epidemiological nature, and as such, causality between antibiotic treatment and observed ecological effects is not fully proven. However, the likelihood that our observations are a direct biological consequence of antibiotics is high, as we see no significant differences in microbiota composition or AMR gene profiles before antibiotic exposure, but marked and slowly waning effects following the exposure. Our findings are also in line with known microbial susceptibility patterns of observed bacterial species. Furthermore, because of ethical reasons, we had to include a separate control group, instead of integrating controls in our randomized design. Though the control group was enrolled from the same hospitals and using the same methodology, the slight difference in enrolment years may have caused minor unidentified potential confounding, though the microbiota composition and AMR gene profiles before antibiotic treatment look reassuringly similar. Also, due to clinical, practical and safety reasons we could not perform blinding. A potential selection bias may have occurred as a consequence of an above-average number of parents with a higher level of education having opted to participate in our study. This may also have affected the low percentage of inhouse smoking observed, and may therefore affect the generalizability of our results to some extent. Finally, we cannot rule out that parents of sEONS infants were more aware of possible side-effects of antibiotic treatment due to the participation in a trial studying precisely these effects, and therefore this may have affected their hesitance for antibiotic treatment at a later stage.In conclusion, we found significant long-term effects of broad-spectrum antibiotic treatment for sEONS. We believe our data suggest that more emphasis should be put on reducing the number of neonates that receive broad-spectrum antibiotics for sEONS, and if needed, to preferably prescribe penicillin + gentamicin, as this regimen causes the least ecological side-effects.17. We included 80 out of 120 infants from this birth cohort that were born vaginally or by SCS, had not received antibiotics in the first week of life and whose samples could be age-matched to those of the sEONS infants. Written informed consent was obtained from both parents. For pediatric studies, the Netherlands has a strict two-parent consent policy, and a deferred approval from the second parent is only granted in special circumstances, such as in acute illness where enrolment is required to occur in a short timeframe. After a thorough review procedure, the regional medical ethical committee granted approval to start enrolment in early January 2015. At that time, the medical ethics committee still had to reach a final decision on our request to allow enrolment of sEONS infants with a single parent’s consent before randomization and collection of samples, and a deferred consent from the second parent . The ethical committee requested additional information to come to a weighted decision on this, and allowed the start of enrolment in the meantime, as long as we used the strict parental consent rules. Permission for deferred consent was granted after the following committee meeting. Our trial was then registered in the Netherlands Trial Registry, leading to March 2015 being noted as date of registry. By that time six patients had been enrolled using the strict parental consent rules. Both ZEBRA and MUIS studies were approved by the Dutch National Ethics Committee . Participants did not receive compensation. The randomized trial conformed to the Consolidated Standards of Reporting Trials (CONSORT) guidelines (checklist uploaded during submission). In four cases a switch to a different antibiotic was made after blood/urine culture results became known and in 13 cases a switch to a different antibiotic was made in order for the infant to finish the treatment orally. These infants were also included in the intention to treat analysis as these practices reflect the clinical setting.We performed a randomized study in 147 infants who required broad-spectrum antibiotics for treatment of sEONS in their first week of life. Infants were recruited at the Spaarne Gasthuis Hoofddorp and Haarlem, Diakonessenhuis Utrecht and Tergooiziekenhuis Blaricum in the Netherlands . Inclusion criteria were indication for broad-spectrum antibiotic treatment due to sEONS in the first seven days of life, birth by vaginal delivery or SCS, gestational age of ≥36 weeks, absence of prenatally established underlying morbidity, parental age of ≥18 years and the ability of parents to understand the Dutch or English language. A subset of healthy, term-born infants from the Dutch Microbiome Utrecht Infant Study (MUIS), served as controls40. For power calculations, we used data of OTUs with high and low variability and abundance, and varying effect sizes. Our power calculation was verified by the online (HMP-based) tool as soon as this became available41. We initially aimed to enrol 132 infants, 44 infants per antibiotic regimen, allowing a drop-out of 10%. Due to the accidental loss of a set of samples from 11 participants, approval was granted by the ethical committee to prolong enrolment in order to replace the lost samples, to ensure the power of the study. Eventually, 147 infants were enrolled, 49 per antibiotic regimen.The study was initially powered by making use of previously published infant microbiota data, ensuring a power of 0.8 to detect at least significant differences in α- and -diversity between groups, and at least two-fold differences in abundance of the 25 most important OTUsInfants were randomly allocated 1:1:1 to three most commonly prescribed intravenous antibiotic combinations for sEONS in the Netherlands, namely penicillin + gentamicin, co-amoxiclav + gentamicin or amoxicillin + cefotaxime. Blinding was not performed, as this was not deemed relevant in our observational study with shifts in the gut microbiome and AMR gene composition as primary study outcomes. The sequence with which participants were allocated to the groups was generated with the Research Manager software (Cloud9 Software BV) by an unaffiliated research nurse using 11 blocks of 12 (4:4:4) and one block of 15 (5:5:5) to enable a balanced randomization over the three regimens in the four hospitals. Allocation concealment was achieved using sealed, opaque envelopes which were delivered to the hospitals. Initial enrolment was performed by trained physicians who assigned the groups by selecting one of the sealed envelopes. Neither the research nurse who generated the allocation sequence nor the enrolling physicians were involved with the rest of the trial.42. All materials were directly stored at −20 °C before being transferred (<2 weeks) to a −80 °C freezer until further laboratory processing. Perinatal characteristics of the participants were obtained at baseline and parents filled in online questionnaires about health characteristics at age 1, 4, 6, 8, 10 and 12 months (for the control infants a questionnaire was filled in at 9 months instead of at 8 and 10 months). Breastfeeding at time of sampling was defined as an infant receiving any breastfeeding at that time point . Exclusive formula feeding was defined as an infant not having received any breastfeeding at all.The duration of antibiotic treatment was not specified in the protocol, but was prescribed by the responsible physician depending on the clinical signs, reflecting the clinical setting. Rectal swabs and/or faeces were collected strictly before the start of antibiotic treatment  and 24–48 h after treatment cessation  and at 1, 4 and 12 months of age . The sample moments before and immediately after antibiotic treatment corresponded with 1 day  and 1 week of life  of the controls  and 500 μl of phenol saturated with Tris-HCl  in a 96-wells plate, and performed an extra phenol/chloroform step. Mechanical disruption of samples was achieved using a Mini-BeadBeater-96  for 2 min at 2100 oscillations per min. The extracted DNA was eluted in a final volume of 60 μl of elution buffer . Further adaptations were applied to the samples collected at the first time point44, being expected to be of low bacterial abundance and diversity, with the additional changes of using 150 μl of faeces  and implementing an extra step with wash buffer 1. DNA blanks and a positive control consisting of a mix of up to six random faecal samples were used for quality control. The amount of bacterial DNA was determined by quantitative polymerase chain reaction (qPCR) as previously described using universal primers specifically designed to amplify the bacterial 16S rRNA genes  on the 7500 Fast Real-Time qPCR system (Applied Biosystems)44.Bacterial DNA was isolated from faecal material using the Mag Mini DNA Isolation Kit  according to the manufacturer’s recommendations as previously described46. Primers included Illumina adapters and a unique 8-nt sample index sequence key45. After amplification, quantification of the amount of amplified DNA per sample was executed with the dsDNA 910 Reagent Kit on the Fragment Analyzer . Samples yielding insufficient DNA after amplification, defined as <0.5 ng/μl, were repeated with a higher concentration of template DNA. A mock control, positive control, DNA isolation blank and up to three PCR blanks were included in each PCR plate. Amplicons were pooled equimolarly and purified from 1.2% agarose gel using the Gel Extraction Kit . The library was quantified using the Quant-iT™ PicoGreen® dsDNA Assay Kit . 16S rRNA sequencing was performed on the Illumina MiSeq platform .For the sequencing of the V4 hypervariable region of the 16S rRNA gene, ~500 pg of DNA was amplified using F515/R806 primers and 30 amplification cycles47. In short, we applied an adaptive, window-based trimming algorithm  setting the quality threshold to q = 30 and length threshold to 150 nucleotides48. Error correction was conducted with BayesHammer 49. Each set of paired-end sequence reads was assembled using PANDAseq (version 2.10) and demultiplexed with QIIME (version 1.9.1)51. Singleton and chimeric reads (UCHIME) were removed. OTU picking was performed using VSEARCH abundance-based greedy clustering with a 97% identity threshold52. OTU annotation was established using the Naïve Bayesian RDP classifier (version 2.2) and the SILVA reference database54. This resulted in an OTU-table containing 18,951 taxa in total. We created an abundance-filtered dataset selecting OTUs present at a confident level of detection (0.1% relative abundance) in at least two samples, hereafter referred to as our raw OTU-table55. The raw OTU-table consisted of in total 730 taxa (0.49% sequences excluded with filtering). Next, we used both the prevalence and frequency methods of the decontam package with the default threshold of 0.1 to exclude possible contaminants, discarding 35 taxa, and thus retaining 695 taxa in total56.The samples and their sequences described in this manuscript are part of a larger dataset existing of 2176 faecal/rectal swab samples and controls, and together were processed using our in-house bioinformatics pipelineFor the purpose of this manuscript, 989 samples from 225 infants (90.7% of obtained samples from 145 antibiotic-treated and 80 control infants) were eligible for microbiota analysis  maintaining a quality threshold of 30 and a minimum read length of 35 base pairs. Per-sample and per-run SAM files were generated using Bowtie2 and the MetaPhlAn2 and MEGARes databases60, while adhering to recommended parameters, including suppressing unaligned reads. Each SAM file was assigned a read group and SAM files from different runs were merged sample-wise using Picard61. Merged SAM files for each sample were sorted and indexed using SAMtools62. Raw reads and counts of reads mapped to each AMR gene were generated using SAMtools idxstats. To calculate counts per million in order to normalize read counts for library size, counts were divided by a normalization factor of the library size divided by 1,000,000.To validate our 16S rRNA sequencing and qPCR data, we performed MGS on a randomly selected subset of 32 faecal samples collected from 20 antibiotic treated infants post antibiotics and 12 controls at 1 week of age. Sample libraries were prepared using the Truseq Nano gel free library preparation kit. Using a NovaSeq instrument, 150 base paired-end sequence data was generated yielding 1500 M reads (two runs). Reads were trimmed using Cutadapt63. Of the 81 genes covered in these studies, we selected a total of 31 that were detected in ≥1 sample in either study and were deemed clinically relevant for our pediatric population and the antibiotic regimens being compared.The primer set used in the qPCR assays covered 31 AMR genes, including genes encoding extended-spectrum β-lactamases, carbapenemases and proteins involved in vancomycin resistance  and Non-Template Controls (NTCs). We standardized the within-individual variation across time points by including all time points from a given individual on a single chip. Individuals were then randomized across chips. Ct values were extracted using the BioMark Real-Time PCR analysis software. NTCs and positive controls were checked within and between chips for errors and consistency. The detection limit on the Biomark system was set to a Ct value of 20 for AMR targets and 34 (because of lack of pre-amplification) for the 16S RNA reference gene. Sample-primer combinations that were failed by the software were checked manually and either passed or failed according to the suitability of the melting and amplification curves. Melting curve analysis was performed for all reactions within the Biomark Real-Time PCR analysis software to ensure the validity of the measured biological signals. Melting curves of the reactions were compared to those of the positive controls, and reactions showing unreliable S-curves were discarded. A sample was only included when a minimum of two of its triplicate reactions resulted in a Ct value below the detection limit, and for each included sample we calculated an average Ct value. Delta Ct values were calculated using the formula CtAMR—Ct16S rRNA. To account for the preamplification step of the AMR primers and to avoid negative delta Ct values, we added 20 to this value. Depending on the downstream statistical analysis, we transformed the dataset to include binary information or continuous Ct values with an arbitrary value of 32 appointed to negative sample-primer reactions.For the identification of AMR genes through qPCR we used the 96.96 BioMark™ Dynamic Array for Real-Time PCR . The concentration of each sample was measured using a specific 16S qPCR and all samples were normalized to 0.1 ng per ul. Any sample below this threshold was included in an undiluted state. Samples were pre-amplified with all primers for a total of 14 cycles, according to the manufacturer’s instructions, excluding the 16S rRNA primer, due to the high abundance of 16S rRNA present in the samples, compared to the low abundance of the AMR targets. Primers were used at a concentration of 500 nM. The multiplex qPCR was run using the manufacturer’s recommended settings, with the exception of the melting temperature being held at 56 °C and the total number of cycles set to 35, which were adjusted according to the melting temperatures of the primers used, previous optimization and our own internal validationOf the 989 samples that were included in the microbiota analyses, 939 samples passed the Fluidigm BioMark™  quality check and were included in the resistome analyses . Ordinations were based on the BC dissimilarity matrix of relative abundance data with parameter trymax 10,000.The overall gut microbial community composition of faecal samples and rectal swabs was visualized using non-metric multidimensional scaling plots , based on PERMANOVA-tests per time point and across all time points using 1999 permutations using relative abundance data. To test for multivariate spread, we used the function betadisper (vegan package69). The dispersion of the data of the compared groups was significantly heterogenous immediately after antibiotic treatment onwards for the antibiotic-treated versus non-treated comparisons , while this was only the case immediately after antibiotics and at 1 month of age for the comparisons between the separate regimens and controls. In the inter-regimen comparisons, the dispersion of the data was homogenous at all time points. In the multivariable, temporal analyses all variables were included that showed a significant association with microbiota composition when tested individually, and age and subject were added to control for repeated measures.Associations between clinical outcome and microbiota composition were analyzed with the adonis2 function  allowing not only to detect biomarker OTUs related to antibiotic treatment but also to identify the specific time intervals in which significant differences existed. For this analysis, the raw OTU-table was filtered and OTUs with >10 reads in ≥50 samples were included, resulting in 512 OTUs (of the 692)47. This filtering step was performed separately for each subset of data used for the different comparisons. The SS-ANOVA analysis was adjusted for covariates that both had (1) an effect on overall gut microbiota composition and (2) were unevenly distributed between the antibiotic-treated infants versus controls, namely mode of delivery, and the inter-regimen comparisons were adjusted for antepartum maternal antibiotics. Although gravidity of mothers, gestational age, duration of ruptured membranes, hospital stay duration after birth, presence of siblings <5 years of age and inhouse smoking were all associated with antibiotic-treated infants, we did not find an association between these clinical variables and gut microbiota composition, therefore these six variables were excluded as covariates from downstream fitTimeSeries analyses. While day care attendance and breastfeeding at the time of sampling were associated with gut microbiota composition, these variables did not differ at baseline and therefore were also excluded as covariates from the downstream analyses.Individual bacterial taxa and their succession patterns, and potential differences thereof between groups, were studied at the lowest taxonomic annotated level (OTU). Differential abundance testing was executed with smoothing spline analysis of variance . The diversity of AMR genes was defined as the observed number of different AMR genes present in a sample. Comparisons in AMR diversity were calculated using the Wilcoxon test. The overall AMR gene composition, or profiles, of faecal samples and rectal swabs were visualized as described above, with the exception that the ordination was based on the Jaccard index of the binary data. Associations between clinical outcome and AMR gene profiles were analyzed similarly to the 16S data, but again using binary data and the Jaccard index. Dispersion of data was only significantly heterogeneous in one case, namely at the time point immediately after antibiotic treatment in the comparison of the amoxicillin + cefotaxime regimen versus controls.71), though no filtering was performed to allow for testing of all 31 AMR genes and no normalization was applied in the function. The SS-ANOVA analysis was adjusted for covariates that both had (1) an effect on overall AMR gene composition and (2) were unevenly distributed between the groups compared, so for siblings <5 years of age and mode of delivery in the antibiotic-treated infants versus controls and for antepartum maternal antibiotics in the inter-regimen comparisons.Differential abundance testing of the AMR genes was also executed with SS-ANOVA (fitTimeSeries function, metagenomeSeq packageWe evaluated the correlation between 16S OTUs and AMR genes by calculating the Pearson correlation coefficient for all individual taxa based on their relative abundance. Positive coefficients indicated a negative correlation between OTU and gene abundance, as a high Ct value indicates low abundance of an AMR gene.71 was used to assess the group differences in AMR genes as identified by MGS in a subset of 32 samples, after removing rare features present in <10 samples, resulting in 343 out of 1504 AMR genes included in this analysis.The fitZig function of the metagenomeSeq packageFurther information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


IgG4-related disease (IgG4-RD) is a rare systemic fibroinflammatory disease frequently associated with allergy. The pathogenesis of IgG4-RD is poorly understood, and effective therapies are limited. However, IgG4-RD appears to involve some of the same pathogenic mechanisms observed in allergic disease, such as T helper 2 (Th2) and regulatory T cell (Treg) activation, IgG4 and IgE hypersecretion, and blood/tissue eosinophilia. In addition, IgG4-RD tissue fibrosis appears to involve activation of basophils and mast cells and their release of alarmins and cytokines. In this article, we review allergy-like features of IgG4-RD and highlight targeted therapies for allergy that have potential in treating patients with IgG4-RD. IgG4-related disease (IgG4-RD) is a rare, potentially life-threatening fibro-inflammatory disease of unknown etiology. IgG4-RD is clinically characterized by the presence of fibro-inflammatory masses in various organs, dense lymphoplasmacytic infiltration of IgG4-postitive plasma cells, storiform fibrosis and elevated serum levels of IgG4 , 2. AlthAllergy describes an abnormal adaptive immune response to foreign antigen . Allergens are an important driver of Type 2 immune responses, which likely evolved in response to helminth infections . The halIgG4-RD and allergic disorders share certain immunopathologic features, such as activation of Th2-like pathways, elevated IgE and IgG4 production, peripheral blood eosinophilia, and tissue mast cell infiltration , 16, 17.The pathogenesis of IgG4-RD is not yet fully understood, but it is thought to involve varied mechanisms including autoimmunity-associated and allergy-associated factors Table 1.via secretion of profibrotic cytokines , induction of IgG4 class-switching, expansion of plasmablasts, and production of autoantibodies   and TGF-β, that together may drive fibroblast activation, collagen production, and tissue fibrosis  , 53. IgEfibrosis , 55,  can also be important mediators of allergic disease . TARC isvia activation of effector Tregs and suppression of Th2 immune responses (via an interferon regulatory factor 4 (IRF4) signaling pathway. This differentiation led to secretion of IL-9 from Th9 cells that promoted plasma cell differentiation and immunoglobulin class switch towards IgG4 -DRB1 and Fc gamma receptor 2B (FCGR2B) . Interesoduction .A cross-sectional study of 114 patients with IgG4-RD showed that 19% had been previously diagnosed with allergic diseases, including allergic rhinitis, sinusitis, or bronchial asthma . In a reAspergillus fumigatus IgG4-antibodies were found in cystic fibrosis patients with ABPA  (30/46 or 65.2%) were more prone to allergic manifestations  and more often had eosinophilia compared to patients without CRS . IgG4-reEosinophilic granulomatosis with polyangiitis (EGPA) is a necrotizing vasculitis of small to medium-sized vessels associated with asthma, eosinophilia, paranasal sinusitis, pulmonary infiltrates and mononeuritis multiplex . ElevateMast cells are key effector cells in urticaria and many cases of angioedema . HistamiHigh levels of serum IgG4 and an elevated IgG4/total IgG ratio were observed in one patient with typical HUV . Takao eEven though Type 2 immune responses and allergy are observed in patients with IgG4-RD, they are not necessarily always linked mechanistically . Indeed,Peripheral eosinophilia and elevated serum IgE levels have been reported along with history of allergies and atopic symptoms in 40% of patients with type I autoimmune pancreatitis, consistent with the hypothesis that allergic mechanisms drive IgG4-RD . HoweverIgG4-RD is thought to result from abnormal activation of both innate and adaptive immunity. IgG4-RD might reflect an individual’s propensity towards pathologic Th2 immune responses, as observed in allergic conditions. Thus, like with allergy, targeting signaling pathways that are involved in Th2 immunity could be effective in IgG4-RD Table 2.First-line treatment for IgG4-RD is glucocorticoids. Rituximab and other immunosuppressive agents such as azathioprine, methotrexate or mycophenolate mofetil are recommended in recurrent or refractory disease, but relapses and flares are common even with these agents . Multi-oMonoclonal antibodies (mAbs) targeting IL-4Ra, such as dupilumab , ameliorKasaian et al. created a recombinant bifunctional IL-4/IL-13 antagonist that in a mouse of model of asthma reduced not only IL-4–dependent increase in serum IgE levels but also IL-13–dependent airway hyper-responsiveness and lung inflammation . Dual ILvia stimulation and activation of TGF-β1  or the IL-5 receptor (IL-5R)  reduce peripheral eosinophilia  and therTezepelumab is a fully human anti-TSLP mAb that prevents interaction of TSLP with its receptor, thereby inhibiting downstream inflammatory pathways; tezepelumab has been used for the treatment of severe asthma in phase II trials . CombineIgG4-RD is a rare fibro-inflammatory disease of unknown etiology. Although the adaptive arm of the immune response is thought to play a major role in disease pathogenesis, the specific mechanisms are incompletely understood. In this review article, we discuss clinical and mechanistic overlap between IgG4-RD and allergy in order to better understand IgG4-RD and identify therapeutic targets for which approved agents already exist. A significant subset of patients with IgG4-RD have allergic disease or features of allergy, such as elevated IgE levels, tissue and blood eosinophilia, and infiltration of affected tissues by IgE-primed mast cells. More research into this subset, including genetic studies, is needed to better understand IgG-RD’s relationship to allergy.Recent work in IgG4-RD has identified allergy-like, Th2-related pathways of aberrant immune activation that may contribute to tissue inflammation and fibrosis. These include overproduction of molecules involved in Th2 differentiation , generation of IgG4- and IgE-secreting plasma cells , recruitment/activation of tissue eosinophils (IL-5) and mast cells (IL-9), and tissue fibrosis (TGF-β). Thus, targeting one or more of these molecules with drugs currently available for the treatment of allergy may be beneficial in patients with IgG4-RD, for which no specific therapy has yet been proven effective.DM, DS, TM, and GH substantially contributed to this review with regards to content and structure of the manuscript. All authors contributed to the article and approved the submitted version.DM is supported by Pfizer US Pharmaceuticals Group grant with sponsor award number 53857367. TM is supported by NIH grants R21AR075134, R01 AR074939 and R21 AR077266. DS is funded by NIAID intramural program. The funder bodies were not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.TM has received consulting fees from Cugene, Kiniksa, Miro Bio, and QiLu Pharmaceuticals, has an ownership share in Amdax, and has received research funding from Gilead Sciences.The remaining 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.