The aim was to further characterize immunoglobulin G4-related disease (IgG4-RD) by a large-scale multicenter study of its clinical and laboratory features conducted by multidisciplinary physicians of IgG4-RD in Japan.Various specialists retrospectively evaluated IgG4-RD patients diagnosed between 1996 and 2015 in five hospitals by analyzing their baseline clinical features, laboratory, imaging, and pathological test findings, and treatment.Of the 334 patients listed, 205 were male and median age at diagnosis was 65 years. The mean number of organs involved was 3.2 at diagnosis. The most frequently affected organs were the salivary glands, followed by the lacrimal glands, lymph nodes, pancreas, retroperitoneum/periaorta, kidneys, and lungs. The mean serum level of IgG4 was 755 mg/dl, and more than 95% of patients had elevated serum IgG4 levels. The median serum level of C-reactive protein (CRP) was 0.1 mg/dl and the level was less than 1 mg/dl in 90% of patients. A total of 34.7% of patients had low serum levels of C3. Serum levels of C3 and non-IgG4 IgG, calculated as the total IgG minus IgG4, showed an inverse correlation in patients with kidney lesions, while serum IgG4 levels were not correlated with serum C3 levels. Corticosteroid was administered in 78.0% of patients, and was effective in all.The serum CRP level is generally low and the serum IgG4 level is elevated in most Japanese IgG4-RD patients, in contrast to western patients. These original findings suggest that these two parameters in IgG4-RD differ in some interesting ways from those hitherto reported in western populations. Additional studies, especially international comparative ones, are needed to elucidate the extent and significance of these differences between populations. Attention will also have to be paid to whether the existence of such differences requires consideration when devising international classification criteria.The online version of this article (doi:10.1186/s13075-017-1467-x) contains supplementary material, which is available to authorized users. Immunoglobulin G4 (IgG4)-related disease (IgG4-RD), a widely recognized systemic inflammatory disorder –3, affecWe retrospectively evaluated 334 patients with IgG4-RD who were diagnosed between 1996 and 2015 in Kanazawa University Hospital, Sapporo Medical University Hospital, Nagaoka Red Cross Hospital, University of Toyama Hospital, Shinshu University Hospital, and affiliated hospitals. Data related to patients’ baseline clinical features, laboratory findings, imaging tests, pathological tests, and treatments were derived from their medical records.+/IgG+ > 40% and > 10 IgG4+ plasma cells/HPF). Since the cutoff number of IgG4-positive plasma cells of the CDC is lower than those of the Consensus statement on the pathology of IgG4-RD [+ PC) in the diagnosis of AIP was reported in 2002, and was also confirmed in the diagnosis of Mikulicz disease (MD) in 2005, which is currently known as IgG4-related dacryoadenitis and sialadenitis. Therefore, after 2005, almost all patients in our study were diagnosed as having IgG4-RD with reference to the immunohistochemical findings of IgG4 staining, clinical and imaging features, and/or serum IgG4 levels. In contrast, 30 patients who were diagnosed as having AIP or MD before 2005 were also included in our study. Of these, four patients were diagnosed with AIP before 2002. Reevaluation of histopathological samples was performed in all four of these patients, and the diagnosis of IgG4-RD was confirmed. Similarly, 26 patients were diagnosed with IgG4-related dacryoadenitis and sialadenitis before 2005. Fifteen of these 26 patients were reevaluated histopathologically and the final diagnosis was confirmed. In the remaining 11 patients, eight had bilateral lachrymal and submandibular gland swelling with elevated serum IgG4 levels, with this combination highly specific for the diagnosis of IgG4-RD. The other three patients had only one set of bilateral lacrimal or bilateral submandibular gland swelling, and all of them had an elevated serum IgG4 level. These three patients were diagnosed with IgG4-RD with reference to the clinical and imaging features and elevation of serum IgG4 levels.To minimize bias regarding the organs affected by IgG4-RD, the diagnosis of IgG4-RD was made by specialists of IgG4-RD in each institution, supported by the comprehensive diagnostic criteria (CDC)  and/or c IgG4-RD , we madeThe numbers of patients with definite, probable, and possible IgG4-RD according to CDC and/or organ-specific criteria were 280 (83.8%), 49 14.7%), and 4 (1.2%), respectively. One patient was diagnosed clinically with IgG4-RD , the affected organs, the diagnostic imaging tests including computed tomography (CT), magnetic resonance imaging (MRI), gallium (Ga) scintigraphy, and positron emission tomography (PET), and the prevalence of biopsy of the affected organs. Serum IgG4 levels were measured using the nephelometric assay in all patients in this study. In addition, as a new serological marker, we defined non-IgG4 IgG as total IgG minus IgG4. Non-IgG4 IgG substitutes for the sum of IgG1, IgG2, and IgG3, and an increase of non-IgG4 IgG seems to mean that the serum level of IgGs which can activate the complement pathway is increased. We also evaluated the prevalence of corticosteroid therapy, average initial and maintenance dose of corticosteroid, effectiveness of corticosteroid therapy, and recurrence rate. The improvement of the affected organs was decided as the changes in symptomatic, radiologic, serologic, or histologic features. In IgG4-related kidney disease (IgG4-RKD), improvement of renal function was also considered. The definition of recurrence was the reappearance or worsening of symptomatic, radiologic, serologic, or histologic features of IgG4-RD. In IgG4-RKD, a rapid increase in the serum level of creatinine, after careful exclusion of other renal diseases, was also considered as recurrence. Reelevation of the serum levels of IgG or IgG4 alone was not regarded as recurrence. Furthermore, we determined the prevalence of diabetes mellitus (DM) at diagnosis of IgG4-RD and malignancy both before and after diagnosis of IgG4-RD.U test for linear variables. The analysis of the organs associated with hypocomplementemia was performed using logistic regression analysis, adjusted for age, sex, and presence/absence of DM. Correlation analysis was performed to confirm the relationship between hypocomplementemia and serum levels of IgG4 or non-IgG4 IgG. Explanatory factor analysis followed by Varimax rotation was used to examine the structure of organ coinvolvement. All statistical tests were performed using SPSS software (version 22). Significant differences were defined as p < 0.05.Differences between groups were assessed by chi-square test for categorical variables, and by Mann–Whitney p = 0.005) . The mean and median ages at diagnosis were 63.8 ± 11.5 and 65 years (range 25–91). All patients were Japanese. The average follow-up period was 4.2 years. The prevalence of diabetes mellitus (DM) was 34.4% and was significantly higher in those with rather than without AIP . The most frequently affected organs were the salivary glands (SG) (72.7%) followed by the lacrimal glands (LG) (57.1%), lymph nodes (56.5%), pancreas (25.5%), retroperitoneum (RP)/periaorta (24.9%), kidney (23.7%), and lung (23.4%). Single organ involvement was seen in 38 of 334 patients (11.4%) . Mean serum level of IgE was 611 ± 1198 IU/ml, and elevated serum levels defined as IgE ≥ 250 IU/ml were seen in 158 of 309 patients (51.1%). Elevation of serum levels of CRP defined as CRP ≥ 0.3 mg/dl was seen in 90 of 328 patients (27.4%). Mean and median serum levels of CRP were 0.42 and 0.10 mg/dl, respectively. In 79.9% and 90.2% of the patients, the serum level of CRP was less than 0.5 and 1.0 mg/dl, respectively (Table p < 0.000)  (Table p = 0.001), 1.84 (95% CI 1.04–3.26), and 1.83 (95% CI 1.04–3.21), respectively   Fig. , which ily) Fig. .Table 4FCT was performed in 99.4% of patients and contrast-enhanced CT in 93.1%. MRI, PET, and Ga scintigraphy were also performed in 30.2%, 45.5%, and 35.1% of patients, respectively, to determine the extent of systemic involvement. According to the histopathological evaluations, the most frequently biopsied organ were the SG (49.0%), followed by the kidney (37.5%), LG (35.1%), lung (33.8%), pancreas (12.8%), and RP/periaorta (1.2%) . The mean initial and maintenance dose of prednisolone was 30.5 and 4.1 mg/day, respectively. Corticosteroid was effective in all patients. Recurrence was noted in 67 of 314 patients (21.3%), in whom 23 of 67 (34.3%) were not receiving corticosteroid at the time of recurrence. The average dose of corticosteroid at first recurrence was 7.1 mg/dl.To the best of our knowledge, this is the largest cohort study thus far to collect IgG4-RD patients and analyze their clinical features. The salient findings of our cohort study can be summarized as follows. First, elevated serum levels of IgG4 were seen in more than 95% of patients with IgG4-RD. Second, the median serum CRP level was 0.1 mg/dl, and the serum CRP level was less than 1 mg/dl in 90.2% of the patients, meaning that serum levels of CRP are within the normal range in most IgG4-RD patients in Japan. Third, low C3 was seen in 34.7% of patients, and its frequency differed depending on the organs affected. The affected organs associated with low serum levels of C3 were the kidney, lung, and pancreas. Serum levels of C3 and non-IgG4 IgG calculated as total IgG minus IgG4 showed inverse correlations in patients with kidney lesion, while serum IgG4 levels did not correlate with serum C3 levels. Fourth, the most frequently affected organs were the salivary glands, lacrimal glands, pancreas, retroperitoneum/periaorta, kidneys, and lungs.Major differences have been noted in the prevalence of affected organs in previously published studies –11. WallIn this study, the serum IgG4 level was elevated in 95% of patients. Inoue et al.  analyzedIn our study, elevation of the serum level of CRP was seen only in 27.4% of patients. The median serum level of CRP was 0.1 mg/dl, and values less than 0.5 and 1.0 mg/dl were present in about 80% and 90% of IgG4-RD patients, respectively. These data indicate that serum levels of CRP in most patients with IgG4-RD are normal or low. Elevated serum IgG4 and pathological features mimicking those of IgG4-RD have been noted in other diseases such as multicentric Castleman disease (MCD)  and antiHypocomplementemia is an important serological feature of IgG4-RD, in particular IgG4-RKD , 25, 26.Interestingly, serum complement levels rapidly normalize after successful corticosteroid therapy in parallel with the reduction of the size of mass or enlarged lesions . MoreoveThe rate of malignancies in this study (17.1%) was higher than those in other Japanese cohort studies  but lowThe recurrence rate of our study was lower than those of western countries. Reports from Spain  and ItalOur study has several limitations. First, biopsies of the affected organs were not performed for 18.9% of the patients because the affected organs were not easily accessible. Second, methodological differences in the measurement of laboratory parameters such as serum complement levels among different periods and different institutions is a weak, but unavoidable, aspect of our retrospective cohort study. However, the effect of the difference of the method employed among different periods and different institutions on complement levels is limited and the influence would seem to be quite small. On the other hand, the serum IgG4 level was measured using nephelometry in all institutions. Therefore, we think no methodological difference existed. Third, we used the serum levels of non-IgG4 IgG instead of those of IgG1, IgG2, or IgG3, because we do not measure serum levels of IgG1, IgG2, and IgG3 in daily clinical practice.It remains difficult to clarify the frequency of individual organs affected by IgG4-RD due to various biases related to the criteria employed for diagnosis and differences in the distribution of the specialties of clinicians making the diagnosis of IgG4-RD. In this study, we believe that we were better able to elucidate the baseline clinical features of IgG4-RD by having specialists of IgG4-RD from a variety of fields participate in the evaluations.Another issue highlighted by this study is the finding of differences in various clinical parameters between our population and western ones. This raises the important question of whether the hitherto proposed international consensus criteria are equally applicable to all populations, and whether the more or less universally recognized clinical spectrum of IgG4-RD is the same in all of them. Further exploration of the reasons underlying any differences may also provide intriguing clues to the underlying nature of IgG4-RD and be of use in devising more accurate diagnostic criteria. To best characterize IgG4-RD and assess the universality of various criteria for diagnosis, future studies, including international comparative ones, will be needed with larger numbers of IgG4-RD patients diagnosed according to various criteria.In Japanese individuals, various clinical parameters in IgG4-RD including serum IgG4 and CRP levels appear to differ in some interesting ways from those hitherto reported in western populations. Additional studies, especially international comparative ones, will be needed to elucidate the extent and significance of these differences between populations. Attention will also have to be paid to whether such differences require consideration when devising diagnostic criteria that are meant to be applied internationally.Additional file 1: Table S1.Gender differences in affected organs. (DOC 64.5 kb)Additional file 2: Table S2.Factor analysis of the affected organs. (DOC 33 kb)


Concrete is prone to crack formation in the tensile zone, which is why steel reinforcement is introduced in these zones. However, small cracks could still arise, which give liquids and gasses access to the reinforcement causing it to corrode. Self-healing concrete repairs and seals these small (300 µm) cracks, preventing the development of corrosion. In this study, a vascular system, carrying the healing agent, is developed. It consists of tubes connected to a 3D printed distribution piece. This distribution piece has four outlets that are connected to the tubes and has one inlet, which is accessible from outside. Several materials were considered for the tubes, i.e., polymethylmethacrylate, starch, inorganic phosphate cement and alumina. Three-point-bending and four-point-bending tests proved that self-healing and multiple self-healing is possible with this developed vascular system. Cracks are the initial symptoms of damage on concrete. Concrete is prone to cracking due to its limited tensile resistance. These cracks can be a gateway for liquids and gasses to the steel reinforcement, causing it to corrode . TraditiIn the case of autonomous self-healing, the recovery process is due to engineered additions. The repair agent is a polymer-based or bacterial agent embedded in protective capsules ,6,7. TheEmbedding glass into concrete has two main drawbacks: glass induces alkali–silica reactions and may not survive in the long-term aggressive chemical environment of concrete . AdditioTo overcome the drawbacks of autonomous healing with multiple short capsules, i.e., cost-ineffectiveness, difficulty to place in concrete, current inability to survive the mixing process, uncertainty of enough capsules breaking and no multiple self-healing possiblilty, the vascular system provides the answer. Vascular self-healing is inspired by the blood-vessel system. The advantages are that bigger volumes of healing agent are available and that such a system can be connected to the outside, allowing (re)filling or replacing the healing agent and so taking the lifetime of the healing agent out of the equation. The design of vascular networks was successfully introduced in polymer matrix composites and remains a hot topic of research in material science . Joseph In this study, the commonly used encapsulation material glass is replaced with alternative brittle materials: poly methyl methacrylate (PMMA), starch, alumina or inorganic phosphate cement (IPC). First, the performance of the new encapsulation materials is evaluated by testing small-scale concrete beams, carrying short tubular capsules, under repeatable bending load. Next, a vascular network is accomplished by adding a distribution piece on both sides of long tubular capsules, which connects the system to the outside.For the first time in the literature, the vascular network idea with an internal distribution piece is applied to design concrete with self-healing properties and the limitations of glass capsules are overcome. This new design aims to provide repeatable repair of different cracks formed in concrete. The assessment of several cracks that interact, close and reopen simultaneously is successfully done by using an integrated monitoring system: an optical method that visualizes cracking stages  is combined with the acoustic emission technique that accurately locates in space and in time the damage in concrete. Both techniques are commonly applied for fracture mechanics studies of construction materials and have shown great monitoring performance in previous autonomous healing studies ,20,21,22It is found that the PMMA capsules are not brittle enough to break when cracking occurs. Additionally, after testing, the beams were cut using a diamond cutter and it was visually observed that the PU-based agent polymerized inside the capsules. Moisture was able to get inside the capsule, causing the healing agent to react. Similarly, after testing the beams carrying starch capsules, they are split in two pieces at the cracked section and the fracture surface is visually inspected. It is noted that the capsules, despite the presence of a waterproof coating, have attracted water from the concrete mixture. A circle around the capsules is observed, as shown in In contrast to starch and PMMA, the inorganic phosphate cementitious (covered by PEEK plasticizer) and the alumina ceramic capsules have shown to be compatible with the PU-based healing agent.Under three-point-bending, a macro-crack initiates from the notch and propagates along the height of the beam. The crack propagation along the capsules successfully leads to their rupture. The moment at which the capsules break is detected based on the AE hits energy analysis previously presented in . In FiguIt is shown that both alumina and IPC capsules rupture only in the presence of macro-cracks wider than 80 μm. More ceramic capsules break in comparison to the cementitious ones. The fact that the capsules rupture energy is greater in the case of ceramic capsules may indicate significant resistance of alumina to damage. The excess resistance of alumina to damage can introduce local stress concentrations and interfacial detachment at the capsules’ zone and modify the concrete mechanical damage. This is not the case for the IPC capsules that break and only emit relatively low energy. The high bond strength between IPC and concrete can be the reason for the latter observation. However, few IPC capsule breakages are detected (one or two for each beam). In both cases, the agent is released in time into the crack void and the healing process is successfully activated.The efficiency is measured considering the strength and stiffness regain. In this way, the beams are reloaded up to two times after the initial damage and their mechanical response is summarized in The specimen with alumina capsules achieve significant stiffness recovery but limited strength restoration. At the third loading, i.e., the second reloading, the alumina specimen show a regain in stiffness of 104%, based on Equation (3). This can be explained by a very efficient healing the second time, a lot more healing agent was able to flow into the crack. The DIC deformation (U) map at the end of the second reloading cycle shows that the crack reopens only at the bottom of the sample and does not propagate higher than the capsules level d. The la-Three-point-bending (notched beams) simulates the guided unique cracking.-Four-point-bending, where multiple cracking occurs, simulates more realistic loading conditions.In this section, specimens with IPC or alumina long capsules, connected to a 3D printed distribution piece  are tested. The mechanical performance of the concrete beams after healing is evaluated for two cases:The addition of capsules in the tensile zones of the concrete specimen has a reinforcing effect. This is due to their superior tensile properties with respect to concrete. Depending on the capsule’s material, the reinforcing effect can be bigger. As a result, the ultimate strength of the beam carrying ceramic capsules is greater than there reference one and the one carrying cementitious capsules. The capsules resist cracking formation and enhance the fracture toughness, but as soon as the ultimate strength of the sample is reached, the crack propagates and the capsules instantly fail.In It is shown that several alumina capsules break at the post-peak stage of loading a. The AEThe difference in the emitted energy between ceramic and cementitious capsules can be attributed to two parameters: firstly, the AE hits energy is relevant to the mechanical properties of the capsule material  and the interfacial bonding developed between the concrete matrix and the capsule. Secondly, the AE energy emission depends on the fracture process that occurs locally: the local stress field is limited at the early loading stage before the crack formation and for this reason the cementitious capsule breaks “softly” emitting lower AE energy. At the ceramic capsules case, the local stress field is significant at the zone of the macro-crack that continuously widens at the post-peak stage. Therefore the capsule that resists this stress accumulates strain and as it breaks emits greater fracture energy. In any case, it is shown that the failure process of short capsules is repeated in the case of the piping system.As capsules break, the healing activation process is initiated since the healing agent is released into the crack void. The concrete beams are left at their neutral, unloaded position and the agent polymerizes. Repeatable and efficient healing can be noted for the IPC vascular system, since for the first and second reloading cycle the mechanical features are restored up to 126% and 96%, respectively.On the contrary, the ceramic piping network achieves significant stiffness recovery but limited strength regain after healing (less than 40%). The result is in agreement with the previous findings regarding the alumina short capsules see . The parxx) maps of the IPC piping network concrete beam at the end of the first and second loading cycle is presented in DIC strain (εxx) maps of the ceramic piping network series show that the crack that forms at the first loading cycle is not located at the middle section of the beam  and the VASC IPC (n = 3) a statistical analysis could be performed.Due to the small amount of specimen that survived the production process, only for the reference  piping networks were tested under four-point-bending. The test aims at evaluating the mechanical response of the embedded piping system in the presence of multiple cracks.Following the testing protocol discussed in previous paragraphs, the concrete beams are loaded up to overall deflection equal to 1 mm. DIC-strain profiles shown in The damage propagation is monitored by DIC and AE techniques for both alumina and IPC piping systems and is presented in This is not the case for the cementitious piping system. Once again, embedding the rigid 3D printed distribution piece controls the crack distribution leading to a unique macro-crack shown at the DIC deformation (U) maps in The AE hits and energy remain low for both cementitious and ceramic vascular systems as the load linearly increases at the beginning of the reloading stage b. When tThe AE location analysis confirms the DIC findings. In the graphs of X-Y coordinates c, the evIn Concrete beams are designed to be tested under four-point-bending. Normal strength concrete beams are prepared with the composition presented in The healing agent (MEYCO MP355 1K) is provided by BASF. This agent is a polyurethane pre-polymer, consisting of two components with, respectively, viscosities of 600 mPa·s and 70 mPa·s at 23 °C, which start to foam in moist surroundings. The expanding foaming reaction of this healing agent may lead to an increase in volume .The capsules are manually filled with the agent and immediately sealed by using X60 adhesive. First a layer of concrete is put into the mold. Then three capsules are carefully placed on this concrete layer, parallel to each other and held into place by a fabric wire anchored at the mold’s sides, the distance between the capsules is around 20 mm. This process is illustrated in A layer of concrete is put on top of these capsules and then a new set of three parallel capsules is put into place at a height of 45 mm from the bottom of the beam. Six capsules are used. The rest of the mold is then carefully filled with concrete.The three-point-bending load induces stresses and forms a unique crack that initiates at the pre-crack and propagates across the beam’s height. The loading stops when the crack opening at the section above the pre-crack is equal to 300 µm considering the serviceability stress limitation (EC 2). During loading, the crack widens and the capsules standing on the crack propagation track break due to tension. The healing agent is released from the capsules and fills the crack void.After loading until a crack width of 300 µm, the specimen is left in unloaded condition for 24 h so that the healing agent has sufficient time to polymerize. Two loading cycles, with a 24 h healing period in between, follow the initial bending and subsequently the initial crack repeatedly reopens as shown in Different alternatives to glass are considered for the design of the agent carriers. Their physical and mechanical properties are briefly described in the following paragraphs. The dimensions of the capsules strongly depend on the availability at the manufacturer and the production process. The presence of the capsules slightly affects the ultimate strength with respect to a normal concrete beam. The amount of healing agent in each capsule is sufficient to fill the formed crack void.Polymethylmethacrylate (PMMA) is a thermoplastic material that is able to resist the aggressive chemical environment of concrete, it has a density that is only half of glass and has a higher strength see , therefoStarch-based materials are brittle when dry, and flexible when exposed to humidity or increased temperatures. In a humid environment, the alpha-bonds that are encountered in starch are gradually destroyed and the material becomes flexible. Starch macaroni tubes were bought from the brand Soubry. The short (around 33 mm long) starch-based capsules used have an outer diameter of around 8 mm and an inner diameter of 2.5 mm. A water-repellent wax coating both on the inside and outside of the starch-based capsules was applied a to avoiInorganic Phosphate Cement (IPC) is an inorganic non-alkaline ceramic material developed at the Vrije Universiteit Brussel (VUB) and has comparable properties to cement-based materials see . It has Ceramics are favorable due to their brittle behavior, their great strength and stiffness see  and greaFrom the design perspective, the innovation of this study is the introduction of a vascular system that can repeatedly provide sufficient healing agent at any damage location. The tubes discussed in the previous section are elongated (100 mm) and placed at the same height covering the tensile loaded section of the beam (across the beam’s length). In a first stage these beams are tested under three-point-bending and the performance of the vascular system is evaluated in the case of a unique guided crack. In a second stage, new beams with a vascular system are tested under four-point-bending simulating the realistic loading conditions. For both three-point and four-point-bending, the imposed speed was 0.04 mm/min. In the case of three-point-bending, the crack width is measured by a crack mouth opening displacement (CMOD) sensor and loading is performed until 300 µm. In the case of four-point-bending, the distance between the upper supports is 180 mm.At the first step, it should be verified that the healing agent can efficiently travel through the capsule using capillary forces and potential energy caused by a height difference. It should be highlighted that the healing agent used is the one with the optimal rheological features: the viscosity of the two components of the polyurethane agent is low enough to flow through the vascular network and is high enough so that the healing agent does not flow out of the crack and is not absorbed too much by the matrix.The flowing of the polyurethane-based healing agent was tested in a simple setup as shown in In the next step, a more complex system is designed: the inlet and outlet tubes are replaced by a distribution piece that guides the agent into the capsules, positioned at the damaged zone of the beam—note that healing is limited to the zone within the distribution pieces. This way, a reservoir system of agent is placed out of the area of damage and fills the tubes with agent only if required. This design allows repeatable self-healing capability. The concept is presented in A channel brings the liquid to the bottom where it is distributed over up to four tubes which can be attached to the 3D printed piece. In F is the applied force, and δ is the associated displacement.The strength and stiffness are chosen as the mechanical features that quantify the material’s cracking resistance. Both features can be accurately calculated considering the mechanical performance of a healed concrete beam that is tested before and after the healing agent is released and polymerizes into the crack void. Note that this can be different depending on the used encapsulation material. The stiffness is calculated with the following formula for the elastic  region of the specimens:maxF is the ultimate load, L is the length between the supports, b is the width, and d is the height of the specimen.The strength is determined using the formula below:The self-healing efficiency is defined as the ratio between a property, i.e., strength or stiffness, at reloading  and at initial loading . Note that this definition includes the effect of the remaining strength/stiffness of a specimen.xx, εyy, and εxy) are obtained from the deformation derivatives.Acoustic emission (AE) and digital image correlation (DIC) are used for inspection during the experiments. As can be seen in The AE resonant sensors (resonant frequency 150 kHz) are attached by means of magnetic holders at the concrete surface at all sides of the concrete beam. The signal is received by the sensors and is amplified by 40 dB before reaching the digital storing system (8-channel AE software provided by Mistras). The received hits are filtered considering a magnitude threshold equal to 40 dB. The AE events are located in the three dimensions using the triangulation technique and considering the P-wave velocity on concrete equal to 4000 m/s. Each plastic deformation, i.e., cracking emits an elastic wave, captured by the sensors. The energy of this waveform can be quantified. Depending on the cracked material, the emitted energy is different. In the case of a heterogeneous material, such as a concrete beam with embedded capsules or vascular system, the differentiation in energy can be used to find out which material is cracking, i.e., the cementitious matrix or the capsules or tubes from the vascular system.In In this study, several alternatives  to glass material are studied to develop the optimal encapsulation system for autonomous healing applications. Both ceramic  and cementitious (IPC) were successful in this study. Additionally, inspired by vascular blood vessels in nature, a vascular network system was developed, by implementing multiple vanes  inside the beam, in order to provide repeatable autonomous healing in concrete. The polymer-based healing agent is automatically released into the concrete as soon as cracking occurs through the vascular network. The agent is delivered from a reservoir station at any location between the reservoirs into the concrete. It aims to fill and mechanically restore the damage the same way as the injection technique does traditionally on concrete repair works. It was found that the fracture toughness under bending load of concrete beams carrying ceramic tubes is significantly modified. The presence of ceramic tubes provides local strain concentration zones at the tensile section of the concrete beams and the ultimate load is increased compared to the reference case (no tubes). On the other hand, the presence of inorganic phosphate cement tubes into concrete does not affect the mechanical properties of the concrete beams and permits stable strain-softening phenomena on concrete. For this latter reason the IPC piping system appears the most viable option. In all cases, the tubes break in the presence of a macro-crack. It was proven by means of Acoustic Emission wave energy analysis that the tubes break as the crack forms and widens. The healing activation by means of tubes rupture is successfully done in all cases. At the next step, the well-studied piping system of healing agent carriers is connected with a reservoir station that stands at the beam’s vicinity and carries the polymer agent. The agent was successfully imported into the piping system through a distribution piece (accessible from outside the beam) as soon as the cracks break the capsules. The mechanical repair was observed at the second and third loading cycle proving that healing can be repeatedly achieved. Multiple self-healing phenomena are observed under four-point-bending tests at which several cracks form and propagate simultaneously. The four-point-bending test set-up on small scale concrete beams provides realistic crack patters and simulates the loading response of real-scale concrete beams under continuous load. The main contribution of this study is the design and manufacturing of the piping system by introducing innovative 3D printed agent suppliers and alternative and more efficient tubes.In future research, the distribution piece should be adapted so that it does not cause crack formation at its position. The integrated self-healing system should be implemented in real concrete applications. In this case, the healing design should face two challenges: repair should be provided in complex micro- to macro-scale cracks and the agent should be delivered at different sections of the concrete structure. Concrete beams or slabs should be constructed and evaluated under realistic loading conditions .Monitoring techniques should also be implemented for the self-healing concrete of the future. A sensing system that provides information on crack formation and propagation can be built using optical fibers or piezoelectric transducers. The sensing system should be embedded into the structure, have no impact on the mechanical and physical features of the concrete and be robust and accurate enough to detect cracks in order to activate in time the healing agent release from the reservoir station. The design of effective vascular healing networks and their application on real concrete structures will be the focus of our future research.


To investigate antibiotic use in poultry farms in Thailand and estimate the total amount of antibiotics used annually in Thai production of chicken meat.In a single province, we surveyed eight farms in which chickens were raised for meat and interviewed the farms’ owners in 2016. The antibiotic use for each chicken was defined as the amount of antibiotic given to the chicken over its entire lifetime divided by the target weight of the chicken at the time of its slaughter. Assuming that the results were nationally representative, we estimated annual antibiotic use on all Thai chickens raised for meat.No use of antibiotics for growth promotion was reported. Five farms raised 1-kg chickens for company A and reportedly used no antibiotics unless the chickens were sick. The other three farms raised 3-kg chickens for company B and reported routine use of antibiotics for prophylaxis. Per kg final weight, each chicken raised for company B was reportedly routinely given a mean of 101 mg of antibiotics – that is, 33 mg of amoxicillin, 29 mg colistin, 19 mg oxytetracycline, 18 mg doxycycline and 2 mg tilmicosin. The total amount of antibiotic used on all Thai chickens raised for meat in 2016 was estimated to be 161 tonnes.Each year in Thailand, many tonnes of antibiotics are probably routinely used in raising chickens for meat. Labels on retail packs of meat should include data on antibiotic use in the production of the meat. The so-called antibiotic footprint has been proposed as a global tool to communicate the total magnitude of antibiotic use in humans and livestock affecting the ecological system.In livestock production, farmers use antibiotics for growth promotion, prophylaxis or therapy – although their use in growth promotion is being heavily discouraged worldwide.Antibiotic use in livestock production is generally suspected to be much higher in low- and middle-income countries than in high-income countries; however, little published evidence exists to support this suspicion. For example, there are few if any published quantitative data on antibiotic use in Thai livestock production,Here, we determined the antibiotic use for farming Thai chicken meat and estimate the total amount of antibiotics used annually in the production of such meat. We anonymized the data in terms of the province and chicken farms surveyed, the farm owners who were interviewed and the companies that the owners were supplying with chicken meat.2 and had an estimated population of 66 million.In 2016, Thailand consisted of 77 provinces, covered 513 000 kmIn a survey in 2016, we visited rural chicken farms and interviewed the farms’ owners in a single province in Thailand. We asked the owner of each surveyed farm about the farm, the chickens raised, the animal feed used and antibiotic usage on the farm. To encourage accurate reporting, we told the interviewees that the data collected would be anonymized and that any resultant reports would not allow the study farms, province and subjects or the companies supplied by the farms to be identified.The main aim of the study was to estimate the antibiotic use for farming chicken meat on the surveyed farms. The antibiotic use for each chicken was defined as the amount of antibiotic given to the chicken over its entire lifetime divided by the target weight of the chicken at the time of its slaughter.,Critically important antimicrobials for human medicine – a report published by the World Health Organization (WHO) in 2017.We used descriptive statistics and no statistical tests were performed. To illustrate a possible use of data on antibiotic footprints on meat packaging, we developed an example based on our findings and the similar concept of providing carbon-footprint information on meat products.We assumed that our results were nationally representative. That is, we assumed that: (i) the proportion of surveyed farms reportedly using antibiotics routinely, for prophylaxis, was the same as the proportion of all Thai farms raising chickens for meat that used antibiotics in this way; and (ii) the prophylactic antibiotic regimen reported by our interviewees was that same as that used across Thailand. We then estimated the amount of antibiotic used annually in all the Thai farms in which chickens are raised for meat. We ignored any non-prophylactic use of antibiotics because the data we recorded on such use were relatively poor.The study protocol was approved by the Ethics Committee of Mahidol University’s Faculty of Tropical Medicine and by the University of Oxford’s Oxford Tropical Research Ethics Committee. Written informed consent was obtained from each of the interviewed farm owners.Although the owners of ten farms were invited to participate, the only owners who agreed to be interviewed were those who had had previous contact with the interviewer and, presumably in consequence, appeared to trust the interviewer. Before giving their consent, each of the eight farm owners who were interviewed wanted to be certain that any reports resulting from the interviews would hide the identity of owner, their farm and the company that the farm supplied. The owners feared that, if any of the information released led to any negative impacts on the reputations of the companies they supplied or on the wider meat market, their contracts with the companies and their relationships with their farming friends and communities could be jeopardized.Of the farms run by the interviewed owners, five (62%) raised 1-kg meat chickens for one company – recorded as company A – and reportedly did not use any antibiotics unless the chickens were sick. The other three surveyed farms (38%) raised 3-kg meat chickens for a different company – recorded as company B – and reportedly routinely used an antibiotic regimen for disease prevention . None ofThe antibiotic regimen used, for routine prophylaxis, to raise 14 000 of the 3-kg meat chickens for company B was observed . In thisAccording to the owners of the farms using routine antibiotic prophylaxis, by the time that a chicken was ready to be transported to company B, it would have been given 101 mg antibiotics – that is, 33 mg of amoxicillin, 29 mg colistin, 19 mg oxytetracycline, 18 mg doxycycline and 2 mg tilmicosin, per kg final weight. In Each year, over eight cycles, a farm with a capacity of 14 000 chickens could raise 112 000 chickens for company B and use 34 kg of antibiotics . If 62% ,,Amoxicillin, colistin, doxycycline, oxytetracycline and tilmicosin were used routinely, for prophylaxis, on three of the farms we surveyed. For human medicine, according to WHO, amoxicillin is a critically important antimicrobial, colistin is one of the highest priority critically important antimicrobials and doxycycline and oxytetacycline are highly important antimicrobials.mcr-1 gene by Escherichia coli.,,Colistin is currently considered to be the last defence against multidrug-resistant bacteria, especially those resistant to carbapenem antibiotics.There appear to be no published data on antibiotic use in European chickens that could be validly compared with the routine antibiotic use, of about 101 mg per kg final weight, that we recorded, in Thailand, in the farms producing chickens for company B. The antibiotic use we recorded does, however, fall within the wide range of corresponding values previously reported for meat, of all types, produced in 30 European countries, i.e. 2.9–434.2 mg per population correction unit.,,–Thai meat and meat products labelled “antibiotic-free” should contain no – or, at least, no detectable, residual antibiotics. Consumers may believe, often incorrectly, that meat so labelled comes from animals that were raised without any antibiotics. Although the use in livestock production of antibiotics that are important in human medicine may pose a particular challenge to human health, the definitions of an antibiotic that is important in human medicine vary from country to country.Our study has several limitations. By ignoring therapeutic use of antibiotics, we will have underestimated the antibiotic use on our study farms. We were pleased to see no use of antibiotics as growth promoters. However, it is possible that antibiotic use in animal agriculture in low- and middle-income countries has been shifting from growth promotion to routine prophylactic use and therapeutic use. Even if successful, attempts to discourage the use of antibiotics as growth promoters may not have led to an overall decline in antibiotic use in livestock production. In the Netherlands, use of antibiotics as growth promoters fell markedly between 1999 to 2006, but multiple measures were later needed to lower the total antibiotic use in animals, from the peak of about 550 tonnes in 2007 to about 250 tonnes in 2012.,We propose that the extent and nature of antibiotic use in animals should be officially and openly reported by each farm, sector and country. Also, the labels on animal products should contain information on antibiotic footprints that is similar to that already given, on some products, on carbon footprints.


Sclerosing mesenteritis (SM) is a rare inflammatory and fibrosing disease primarily involving the small-bowel mesentery. Recently, SM was reported to be closely related to IgG4-related disease (IgG4-RD). This report describes a patient with SM associated with IgG4-RD. A 77-year-old woman with a history of surgery for ectopic pregnancy and wound dehiscence presented with intestinal obstruction. Abdominal enhanced computed tomography (CT) revealed an enhanced, radially shaped, oval mass, 3 cm in diameter, with an unclear rim in the mesentery of the distal ileum, which may have involved the distal ileum. To remove the cause of bowel obstruction, the SM was resected completely and the ileum was resected partially. Histologic examination showed that the mass was composed of spindle cells arranged in a fascicular or storiform pattern; moreover, fibrous stroma was observed, with dense lymphoplasmacytic infiltration and lymphoid follicles. Immunohistochemically, numerous IgG4-positive plasma cells were observed, at a density of 253 per high-powered field, and the IgG4/IgG ratio was about 50 %. Elastica van Gieson (EVG) staining also showed obstructive phlebitis. These findings indicated IgG4-related SM. Although the accurate diagnosis of SM remains difficult without histological analysis, IgG4-RD should be included in the differential diagnosis of unknown mesenteric tumors. Identification of IgG4-RD may prevent unnecessary surgery because corticosteroids may be effective in these patients. Sclerosing mesenteritis (SM) is a rare inflammatory and fibrosing disease of unknown etiology that primarily involves the small-bowel mesentery, most frequently observed in middle-aged and older men –6. SM, aSM was recently reported to be closely related to IgG4-related disease (IgG4-RD) , 10–13, A 77-year-old woman, who had a history of surgery for ectopic pregnancy and wound dehiscence 28 years earlier, presented to another hospital with intermittent abdominal pain. She was diagnosed with an intestinal obstruction and admitted to the hospital. Computed tomography (CT) revealed an irregularly shaped mass, 3 cm in diameter, in the mesentery of the ileum, which was suspected of causing her bowel obstruction. Medical treatment, including long-tube decompression, improved her symptoms, and she was referred to our hospital for further examination and treatment.On admission to our hospital, she had no symptoms and a physical examination showed no abnormalities. Laboratory tests, including those for tumor markers such as CEA and CA19-9, showed no abnormalities. Her serum IgG4 concentration was not measured. Abdominal enhanced CT imaging revealed an enhanced, radially shaped, oval mass, 3 cm in diameter, with an unclear rim in the mesentery of the distal ileum, which may have involved the distal ileum . (3) Histopathologic examination should show marked lymphoplasmacytic infiltration, lymphoid follicles, obstructive phlebitis, dense fibrosis, and infiltration of IgG4-positive plasma cells.SM may be an IgG4-RD –13, dise>40 to 50 %, with >60 to 100 IgG4-positive cells present per high-powered field (HPF) [The rate of IgG4- and IgG-positive cells diagnostic of IgG4-RD has been defined as ld (HPF) . The degld (HPF) . The posA search of PubMed using “IgG4” and “sclerosing mesenteritis” as key words resulted in seven cases that seemed to be IgG4-related sclerosing mesenteritis , 17–19. Most patients with SM are symptomatic, with abdominal pain or a palpable abdominal mass being the most common clinical manifestations , 2, 8. SThe histological differential diagnosis in our patient included leiomyosarcoma (desmin+), inflammatory myofibroblastic tumor , desmoid fibromatosis (β-catenin nuclear+), liposarcoma , follicular dendritic tumor , and malignant lymphoma . A definitive diagnosis of SM requires histological examination of biopsy or surgically excised tissue specimens, unless other organs are apparently affected by IgG4-RD or percutaneous needle biopsy can be easily performed.No consensus has yet been reached for the treatment for SM. Asymptomatic or mildly symptomatic SM may be left untreated , 2. SurgSM may be associated with IgG4-RD in some patients. Although the accurate diagnosis of SM remains difficult in the absence of histological examination, IgG4-RD should be included in the differential diagnosis of unknown mesenteric tumors. Corticosteroids may be effective in these patients, thereby avoiding unnecessary surgery.


To evaluate whether introducing rapid diagnostic testing in conjunction with implementing a stratification algorithm for testing eligibility would be an appropriate clinical and cost saving approach. An internal concurrent 4-month observational study was performed. Positive blood cultures continued to be worked up in accordance with standard of care. An additional call to the infectious disease (ID) pharmacy service occurred for all positive blood cultures with Gram-positive cocci in clusters (GPCC). The ID pharmacy service investigated each case using a prespecified stratification algorithm to minimize unnecessary use of rapid identification testing. 43 patients with GPCC were screened. Only nine patients met inclusion criteria for QuickFISH™ testing. The average expected time avoided to optimize antibiotic therapy is 35 ± 16 hours. If the QuickFISH test had been indiscriminately implemented for all cases, the cost for performing this test would have been $5,590. However, using the prespecified algorithm, only 9 patients were tested for a projected cost of $1,170. Introducing rapid diagnostic testing in conjunction with implementing patient stratification algorithm for rapid identification of GPCC from blood cultures in addition to the ID pharmacy intervention will provide a positive impact on the clinical and economic outcomes in our health care setting.  Staphylococcus aureus (S. aureus) is responsible for a serious bacteremia requiring immediate antibiotic treatment. Delay in optimal therapy is associated with prolonged hospitalization and higher rates of mortality [ Staphylococcus aureus (MRSA) bacteremia with minimum inhibitory concentrations of ≤1.5 mcg/mL. However, this agent has been found to be inferior to antistaphylococcal beta-lactams for methicillin-susceptible Staphylococcus aureus (MSSA) strains [ S. aureus bacteremia on average were hospitalized for 12.3 days and received 29.7 days of antibiotic therapy.Coagulase-positive and coagulase-negative staphylococci (CoNS) are commonly isolated from blood cultures.ortality . Vancomy strains , 3. A prCoNS are common blood culture contaminants of which only about 20% are considered true opportunistic pathogens . A typicConventional methods of bacterial identification which differentiate MRSA from MSSA and CoNS typically require 24–48 hours from the time blood cultures turn positive . Several S. aureus and CoNS directly from positive blood cultures with a sensitivity and specificity exceeding 98% [There are several molecular FDA approved methods for rapid blood culture identification. Testing platforms include GeneXpert, MALDI TOF mass spectrometry, and QuickFISH. Molecular methods range in testing sophistication, need for equipment acquisition, and reagent cost. QuickFISH GPCC BC (QF GPCC) is a peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) test capable of differentiating staphylococci in positive blood cultures. QuickFISH targets the 16S rRNA ofding 98% , 11. FluOur teaching hospital uses culture based technology to identify Staph species from positive blood cultures. The goal of this study is to evaluate whether introducing rapid diagnostic testing in conjunction with implementing a stratification algorithm for testing eligibility would be an appropriate clinical and cost saving approach. Staphylococcus species would impact the use of antibiotics. If the ID pharmacy service deemed there is a need to continue empiric vancomycin, the rapid test would not be performed. However, if the Staph bacteremia was considered to be a monomicrobic infection or a potential contaminant and the patient was on vancomycin, the blood culture isolate would be rapidly identified.An internal concurrent 4-month observational study was performed. Due to platform availability at the time, QuickFISH was selected for this quality improvement project. For the purpose of performing this validation study, test kits and other equipment were provided by the manufacturer free of charge. This study was approved by the Hospital Integrity Board. During the study period, positive blood cultures continued to be worked up in accordance with the standard of care. An additional call to the ID pharmacy service occurred for all positive blood cultures with GPCC during the hours of 0700-1530. The ID pharmacy service investigated each case to determine if rapid identification of theThe target population for testing was identified using a stratified approach  and incl S. aureus identification including mecA gene determination. We calculated the expected time that could have been avoided in hours to optimize antibiotic therapy using the new technology by subtracting 2 hours (if CoNS) or 4 hours (if MSSA) from the time to optimal antibiotic therapy using culture based methodology for microbiological identification.We have defined optimal antibiotic therapy as the switch from empiric vancomycin to cefazolin or nafcillin in patients with MSSA bacteremia or discontinuation of unnecessary vancomycin in patients with CoNS if deemed to be a contaminant. Time to optimal antibiotic therapy for culture based methods was defined as the time microbiology reported the GPCC after performing the Gram stain to the time of antibiotic optimization. For rapid diagnostic testing, the expected time to optimal treatment for CoNS was defined as 2 hours from the time when GPCC was identified by Gram stain and 4 hours forN = 28), 5 patients had MRSA, and 1 patient had MSSA. Of the 6 patients who were excluded due to presence of MRSA risk factors, 4 cultures were deemed to be contaminated and therefore we would have benefited from knowing the identification rapidly to prevent unnecessary antibiotic exposure. Projected hospital cost avoidance in the 9 cases that met the inclusion criteria for testing if rapid testing implemented is as follows: one patient with MSSA bacteremia: using primary literature cost data by Bauer and colleagues, the cost avoidance would have been $21,387; two ED patients with contaminated blood culture bottles: cost avoidance for ED call back would have been $718 × 2 = $1436 ; one patient had a contaminated blood culture bottle and discharge held by one day with a cost avoidance for extra day being approximately $1,024; and 5 patients for which vancomycin was initiated due to the panic call of GPCC in blood culture. The average drug acquisition cost of vancomycin per day is approximately $20. Therefore, $100 in drug acquisition cost could have been avoided . If we apply Gander and colleagues' estimates to all 8 contaminated blood cultures, the expected cost avoidance would total  $69,760 [A total of 43 patients with GPCC were screened for QuickFISH test eligibility. Nine patients met the inclusion criteria for testing and therefore underwent QuickFISH . Time to $69,760 . During Time to optimal antibiotic therapy may be decreased by approximately 1–3 days if interventions have been made based on the results of the rapid diagnostic assays for the 9 cases that met the inclusion criteria for testing. Additionally, earlier discharge may be achieved and call back to the ED following discharge may be avoided. Stratification based on eligibility for the rapid identification test minimizes the use of this test for which early identification of GPCC is not likely to impact empiric antibiotic therapy. Therefore, a stratification algorithm for rapid molecular identification of GPCC from blood cultures was created accordingly . Among t S. aureus. This time included the expected microbiology workup time following the Gram stain and did not include the lag time between the microbiology reporting and the antibiotic optimization. A two-arm, prospective study design could overcome this limitation which is the potential future direction of this trial. Fourth, the presence of MRSA risk factors was not an appropriate exclusion criteria and was subsequently removed from the hospital's proposed rapid diagnostic stratification algorithm. Finally, some of the cost avoidance data were extrapolated from previously published cost avoidance trials.Even though molecular assays have been shown to reduce the time to optimal antibiotic therapy as well as length of hospital stay and cost of stay in previously published literature, in this observational study, we tested the appropriateness of a stratified approach to minimize the use of the rapid diagnostics to include only cases where early identification of GPCC is likely to impact empiric antibiotic therapy. However, this study has several limitations. First, it represented a small sample size because the ID PharmD is not available 24 hours per day for 7 days to receive all the notifications for GPCC. Second, it is a single center, 4-month observational trial. Third, rapid diagnostic was tested for validation only and the intervention was not allowed. Therefore, actual time to optimal antibiotic therapy for rapid diagnostic test was unable to be measured which could otherwise be longer than 2 hours for CoNS and 4 hours forIntroducing rapid molecular testing in conjunction with implementing patient stratification algorithm for rapid molecular identification of GPCC from blood cultures in addition to the ID pharmacy intervention will provide a positive impact on the clinical and economic outcomes in our health care setting.


Antibiotic exposure and specimen volume are known to affect pathogen detection by culture. Here we assess their effects on bacterial pathogen detection by both culture and polymerase chain reaction (PCR) in children.PERCH  is a case-control study of pneumonia in children aged 1–59 months investigating pathogens in blood, nasopharyngeal/oropharyngeal (NP/OP) swabs, and induced sputum by culture and PCR. Antibiotic exposure was ascertained by serum bioassay, and for cases, by a record of antibiotic treatment prior to specimen collection. Inoculated blood culture bottles were weighed to estimate volume.Streptococcus pneumoniae from NP culture was approximately 30% in cases and approximately 32% in controls. Several bacteria had significant but marginal reductions (by 5%–7%) in detection by PCR in NP/OP swabs from both cases and controls, with the exception of S. pneumoniae in exposed controls, which was detected 25% less frequently compared to nonexposed controls. Bacterial detection in induced sputum by PCR decreased 7% for exposed compared to nonexposed cases. For every additional 1 mL of blood culture specimen collected, microbial yield increased 0.51% , from 2% when volume was ≤1 mL to approximately 6% for ≥3 mL.Antibiotic exposure ranged by specimen type from 43.5% to 81.7% in 4223 cases and was detected in 2.3% of 4863 controls. Antibiotics were associated with a 45% reduction in blood culture yield and approximately 20% reduction in yield from induced sputum culture. Reduction in yield of Antibiotic exposure and blood culture volume affect detection of bacterial pathogens in children with pneumonia and should be accounted for in studies of etiology and in clinical management. Identifying the microbiological cause of pneumonia can help guide treatment and prevention strategies. However, this remains challenging for a variety of reasons, including suboptimal diagnostic accuracy of testing methods, difficulties obtaining adequate blood specimen volumes in young children, and the administration of antibiotics prior to collection of diagnostic specimens . TreatmeBlood volume has been reported as the most important factor influencing the sensitivity of blood cultures among patients with sepsis , 9, and Here we aim to quantify the effect of antibiotic exposure and specimen volume on bacterial pathogen recovery from blood and respiratory specimens taken from children hospitalized with severe and very severe pneumonia, and to investigate the effect of key covariates of interest including age, human immunodeficiency virus (HIV) infection status, chest radiograph (CXR) finding, disease severity, and research site on these estimates.The Pneumonia Etiology Research for Child Health (PERCH) study was a multicountry, case-control study of the etiology of severe pneumonia in children from developing countries . EnrollmBlood, nasopharyngeal, and oropharyngeal (NP/OP) swabs, induced sputum (IS), and urine were collected from cases at study enrollment. A CXR was collected from cases as soon as possible after clinical evaluation and study enrollment and was reviewed by a trained panel using standardized guidelines for interpretation . CXRs weStreptococcus pneumoniae by polymerase chain reaction (PCR). Respiratory specimens were tested by multiplex PCR including 33 different bacterial and viral targets ), an aminoglycoside (2023 [49.6%]), and penicillin (1401 [34.3%]). Nearly half of cases (1833 [44.9%]) received combination therapy containing aminoglycoside with amoxicillin and/or penicillin. Among the 464 treated cases who did not receive amoxicillin or penicillin, 410 (88.4%) received ceftriaxone instead. With the exception of amoxicillin and amoxicillin/clavulanate, antibiotics were administered parenterally >95% of the time. Routine cotrimoxazole prophylaxis was reported in 79 of 902 (8.8%) cases and 138 of 940 (14.7%) controls in South Africa, and 76 of 611 (12.4%) of cases and 139 of 686 (20.3%) controls in Zambia, the 2 sites with highest HIV prevalence.Among cases, 18.3% of 4211 blood specimens, 25.3% of 4223 NP/OP swabs, 71.7% of 3800 IS specimens, and 68.6% of 2388 urine specimens were collected after antibiotic administration at the study facility. Blood was the first specimen obtained for 91.3% of 4176 cases who had respiratory or urine samples collected in addition to blood. The proportion of blood samples collected after antibiotic administration at the study facility varied by site, from 4.3% in Mali to 47.9% in Zambia .There was significant variation in the proportion of children receiving antibiotics at referral facilities prior to PERCH enrollment. Three sites  had no facility that formally referred cases. In Zambia, 92.7% of cases visited another facility before coming to the study hospital and 83.6% received antibiotics at that facility. At the 3 remaining sites, 1.3% (The Gambia), 11.9% , and 19.4% (Thailand) of cases received antibiotics at a referral facility .Serum antibiotic bioassay was performed on 94.1% of cases and 90.6% of controls; antibiotics were detected in 25.6% and 2.3%, respectively, with the highest frequency of detection in South African cases (50.0%) . Paired Of the 96.0% of cases and 91.3% of controls  with data available to define “antibiotic exposure” prior to blood collection, 43.5% of cases and 2.3% of controls met the composite exposure definition  after adjusting for site, blood volume, age category, abnormal CXR, and very severe (compared to severe) pneumonia . AssociaS. pneumoniae from NP swabs was significantly reduced for both cases and controls pretreated with antibiotics, and this relationship was consistent when restricted to the CXR-positive, HIV-uninfected cases and HIV-uninfected controls (The culture yield of controls . Among CP < .001); this did not differ by CXR or HIV status. In contrast, controls that were antibiotic exposed had fewer bacterial targets detected by NP/OP PCR than unexposed controls , which also did not differ by HIV status. Cases and controls with antibiotic exposure before specimen collection were less likely to be positive by NP/OP PCR for S. pneumoniae, Haemophilus influenzae, or Moraxella catarrhalis  associated with microbiologically confirmed pneumococcal pneumonia threshold [S. pneumoniae, H. influenzae, and M. catarrhalis in IS specimens was significantly lower in exposed cases . Among cases with IS positive for S. pneumoniae, H. influenzae, or M. catarrhalis by PCR, reduced bacterial density was associated with antibiotic exposure (Detection of pneumococcus in whole blood by PCR was not significantly affected by antibiotic exposure (ed cases . The samBlood culture volumes were available for 3553 of 4232 (84.0%) specimens. Of the 679 specimens with missing volumes, 558 (82.2%) were from South Africa. Mean blood culture volume was 2.05 mL ; only 12.4% of all blood culture specimens met the target volume of ≥3.0 mL  in children <13 years of age reduced blood culture yield by 62%–73% in patients with severe or fatal disease, and greater blood volume yielded higher bacterial detection by culture . StaphylFor NP culture specimens, antibiotic exposure reduced detection of pneumococcus by culture by approximately 30% in cases and controls, with a more modest reduction in detection (by approximately 5%–7%) of bacterial pathogens by PCR. These findings are similar to those in a study of an elderly population in Finland where detection of pneumococcus was significantly reduced for NP culture and showed a nonsignificant trend toward reduction by PCR . The redOur analyses have several limitations. Most importantly, there was potential for misclassification of antibiotic exposure status. Antibiotic type, dose, and timing relative to specimen collection could all be expected to impact serum antimicrobial activity, potentially resulting in misclassification, and could not be taken into account in our definition. Although the timing between administration and specimen collection at the study facility was available, we felt that the application of a time cutoff for exposure would be arbitrary and therefore considered any specimen that was collected following antibiotic administration to be exposed. Serum bioassay was selected over urine to evaluate presence of antibiotics because urine was >4 times more likely than serum to be collected after antibiotic administration. However, serum has been shown to be less sensitive than urine for this application , and theSpecimens identified as “not exposed to antibiotics” may also have been misclassified as antibiotics administered by the parents were not captured in our definition. If so, our findings would again be biased toward the null, and the true impact of exposure would therefore be of a greater magnitude than what is reported here. The level of misclassification might vary by study site, which may explain some site differences, as might the exclusion of participants missing antibiotic exposure status, which also varied by site. Finally, small numbers of positive blood cultures limited our ability to analyze pathogen-specific associations with antibiotic exposure, and the large proportion of missing blood culture volumes from South Africa reduced the precision of the estimate of the impact of blood culture volume on positivity for this site.S. pneumoniae, H. influenzae, and M. catarrhalis, to pneumonia etiology.Despite these limitations, our data reveal consistent and statistically significant reductions in the detection of bacterial pathogens in blood and respiratory specimens of children associated with exposure to antibiotics prior to specimen collection and low blood volume. Because few bacterial pneumonias are bacteremic, a reduction in the sensitivity blood culture worsens the already existing challenge of isolating an etiologic agent from blood. Clinicians managing the care of pneumonia patients and researchers evaluating the relative contribution of bacterial pathogens to the etiology of these syndromes should make efforts to draw blood culture volumes ≥3 mL, collect specimens prior to antibiotic administration, obtain accurate measurements of antibiotic exposure, and interpret test results in accordance with blood volume and prior antibiotic exposure. Failure to do so may overestimate the proportion of antibiotic-resistant organisms and underestimate the relative contributions of antibiotic-sensitive pathogens, such as Clinical Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. Supplementary materials are available at DAP_7_15_Supplemental_tables_23Nov16Click here for additional data file.


Lactobacillus species and increased the abundance of potentially pathogenic Streptococcus suis in the lumen of the stomach and small intestine. Interestingly, the intervention increased the abundance of Treponema only in the colonic lumen and that of Faecalibacterium only in the ileal mucosa. Furthermore, the antibiotic intervention exerted location-specific effects on the functional potential involved in the phosphotransferase system (decreased sucrose phosphotransferase in the stomach) and antibiotic-resistance genes (increased in the colon). These results point to an early-life antibiotic-induced dramatic and location-specific shift in the gut microbiota, with profound impact in the foregut and less impact in the hindgut. Collectively, these findings provide new insights into the membership of the microbiota along the gastrointestinal tract of piglets and highlight the importance of considering the foregut microbiota in health management of piglets at early life.Early-life antibiotic interventions can change the predisposition to disease by disturbing the gut microbiota. However, the impact of antibiotics on gut microbiota in the gastrointestinal tract is not completely understood, although antibiotic-induced alterations in the distal gut have been reported. Here, employing a piglet model, the microbial composition was analyzed by high-throughput 16S rRNA gene sequencing and PICRUSt predictions of metagenome function. The present study showed clear spatial variation of microbial communities in the stomach and intestine, and found that the administration of antibiotics  in early life caused markedly differential alterations in the compartmentalized microbiota, with major alterations in their spatial variation in the lumen of the stomach and small intestine. In piglets fed an antibiotic-free diet, most of the variation in microbial communities was concentrated in gut segments and niches (lumen/mucosa). The microbial diversity was higher in the lumen of stomach and duodenum than that in ileum. The early-life antibiotic intervention decreased the abundance of some  Gut microbiota are important to the metabolism and health of their hosts. The composition and distribution of the microbiota of the intestine of pigs have been extensively investigated . RelativProteobacteria and Firmicutes dominated in the digesta of ileum while Bacteroidetes and Firmicutes dominated in the digesta of colon (Anaerobacter and lower Prevotella in the lumen than mucosa in ileum (11–1012 CFU/mL), stomach and small intestine have relatively low numbers of bacteria, with 101–103 CFU/mL in the lumen of stomach and duodenum, and 104–107 CFU/mL in the lumen of jejunum and ileum  of the pig varied between small intestine and large intestine and between lumen and mucosa . In growof colon . The micin ileum . In addiin ileum . These snd ileum . Thus, tntestine . Thus, tFaecalibacterium prausnitzii and Eubacterium in the jejunal content at day 8 of age in piglets . The hypothesis was that early-life antibiotics may differentially affect the microbial community structure in the foregut. We focused on the early-life antibiotic intervention as in-feed antibiotics are regularly used for disease prevention and growth promotion during the early-life period (from day 7 to 42 of age), when piglets are sensitive to external pathogens. The results showed that although not as diverse as in the colon, the stomach and duodenum of piglets also had a highly diverse microbiota. This finding provides new insight into the membership of the microbiota along the GIT, with its function yet to be explored. Importantly, early-life in-feed antibiotics predominantly altered the microbial composition in the foregut rather than hindgut. These findings also implicate the importance to consider the foregut microbiota in health management of piglets.This study was carried out in accordance with the recommendations of the Guidelines for the Care and Use of Animals of Nanjing Agricultural University. The protocol was approved by the Ethical Committee of Nanjing Agricultural University, Nanjing (authorization number SYXK (Su) 2011–0036).A total 16 litters of newborn piglets  were allotted to control group and in-feed antibiotic group, with 8 litters per group. At day 7, while staying with their respective sows and sucking sow milk, the piglets started to receive creep feed containing either no antibiotics (control group) or antibiotics (antibiotic group). At day 23, all the piglets were weaned and transported to a nursery house in a controlled environment with 1.8 × 2.5 m pens (16 pens) fitted with a hard plastic fully slotted floor, and fed the same diet as creep feed with or without antibiotics until day 42 of age. For the antibiotic group, a mixture of antibiotics  was added to the creep feed. Oxytetracycline calcium, an antibiotic that directly targets both Gram-positive and Gram-negative bacteria, and kitasamycin, a macrolide antibiotic that directly targets Gram-positive bacteria, and olaquindox, an antibiotic that improves feed efficiency, were chosen because a mixture of them are regularly used in piglets . This coThe creep feeding diet was based on corn and soybean-meal and designed to meet the requirement of At day 42, after fasting overnight, one piglet from each litter was randomly selected for sampling. The GIT of each piglets was removed immediately after slaughter and segments  were identified and ligated before separation. Digesta of the stomach, duodenum, jejunum, ileum, and colon were collected and stored at -70°C until analysis. The mucosa was rinsed with sterile ice-cold phosphate buffered saline several times to remove the remains of free-floating bacteria. Mucosal samples from duodenum, jejunum, and ileum were collected by scraping off the mucosa using a sterile glass microscope slide. Mucosal scrapings were kept on ice until being stored at -70°C. A total of 128 samples from 8 gut locations of 16 piglets, were collected.2O. Amplicons were extracted from 2% agarose gels and purified using the AxyPrep DNA Gel Extraction Kit  according to the manufacturer’s instructions and quantified using QuantiFluor -ST . Purified amplicons were pooled in equimolar amounts and paired-end sequenced (2 × 250) on an Illumina MiSeq platform according to the standard protocols  and mucosal scrapings  were extracted from 0.3 g of sample using a bead-beating and phenol-chloroform extraction as previously described . The V3–rotocols .Raw sequence data were trimmed, filtered, aligned, and classified using Mothur software package (Ver 1.32.0) . To imprAfter trimming the primer, barcode, and chimeras, the unique sequences were identified and aligned against high quality 16S rRNA sequences from the Greengenes reference alignment . After sHere we defined the segments to represent longitudinal locations (from stomach to colon) and niches to represent radial locations (from mucosa to lumen). To determine the impact of gut segments, niches (lumen/mucosa) and antibiotics on the variation observed, Redundancy analysis (RDA) was performed at OTU level using Canoco 5.0 software .1  analysis (q < 0.05).Graphing and statistical analysis were performed using Graphpad Prism  and IBM SPSS Statistics software . For comparisons between 2 groups, unpaired Student’s analysis , limitinSRP102481.The sequence information in this paper has been deposited in the GenBank Sequence Read Archive database under accession number P > 0.05) between the control group (mean ± SEM: 515.6 ± 13.9 g) and antibiotic group . Since the feed intake was similar among individual piglets in the antibiotic group, we assumed that the antibiotic consumption was also similar among the individual piglets.The average daily feed intake per piglet was not statistically different  per sample were identified.P = 0.03), gut location (P = 0.002), and lumen/mucosa (P = 0.002) all had a significant impact on the microbial composition in the piglets . Among these, the gut segment was mainly responsible for shaping the bacterial profile of the piglets (contribution = 57.8%), followed by the niche  and antibiotic intervention (contribution = 11.6%).To determine the impact of the gut segment , niche , and antibiotics on the observed variation, RDA analysis was performed. The RDA analysis of the microbiota structure at the OTU level indicated that the antibiotic intervention . In the control group, there was a significant difference in the microbiota composition of the lumen and mucosa and the microbiota composition of different mucosa sites  than that of the ileum, as measured by the Shannon index . The antibiotic intervention did not alter the microbial structure of the mucosa of the duodenum, jejunum, or ileum . These findings confirmed the varied microbiota composition of segments versus niches. Furthermore, these results indicated that at the end of the experiment, early-life antibiotics had marked effects on the overall structure of gut microbiota in foregut lumen of piglets.To further reveal the impact of the antibiotic intervention on the microbiota composition, PCoA was performed based on unweighted (presence/absence information) Unifrac distances. Different samples clustered primarily by the niche (mucosa/lumen)  was most dominant in the lumen  and Lactobacillus  dominant in the stomach and small intestine and Subdoligranulum  dominant in the colon  was most dominant in the mucosa , hereafter referred to Escherichia, dominant in the jejunum and ileum and Alcaligenes  dominant in the duodenum , with the relative abundance of Prevotella decreasing from 0.357 to 0.041% , especially that of Escherichia and Actinobacillus genera . Among Firmicutes, the relative abundance of Clostridium, Bacillus, and Sharpea decreased in response to the antibiotic intervention in the lumen of the stomach, duodenum, and jejunum . In the colonic lumen, the antibiotic intervention significantly increased the relative abundance of Treponema and decreased the relative abundance of Prevotella compared with the control group . These results suggested that the effects of the antibiotics on the microbial composition in the lumen varied between gut locations.The relative abundance of Spirochaeta 44-fold in the mucosa of the ileum compared with the control group . Among Proteobacteria, the relative abundance of Actinobacillus decreased in response to the antibiotic intervention in the mucosa of the jejunum . Among Tenericutes, the relative abundance of Mycoplasma decreased in response to the antibiotic intervention in the mucosa of the ileum . Collectively, these results indicated that the antibiotic intervention had profound effects on the lumen of the stomach and small intestine but few effects on the mucosa.The antibiotic intervention did not show effects on the relative abundance of dominant bacterial phyla in the mucosa at the end of the experiment but increased the relative abundance of the subdominant phylum Clostridium disporicum (OTU1212), Eubacterium biforme (OTU1865), and L. mucosae (OTU1088) decreased in the antibiotic intervention group in the lumen of the stomach, duodenum, and jejunum (q < 0.05). On the other hand, in response to the antibiotic intervention, the relative abundance of S. porcorum (OTU1762) and S. suis (OTU1070) increased in both the stomach and jejunum, and the relative abundance of Escherichia coli (OTU1800) increased in the jejunum (q < 0.05). The antibiotic intervention also reduced the relative abundance of Gemmiger formicilis (OTU218), Ruminococcus lactaris (OTU570), R. faecis (OTU592), and Coprococcus comes  in the duodenum but markedly increased the relative abundance of S. suis (OTU1070) in the ileum , the relative abundance of the major dominant phylotypes in the lumen and mucosa was identified . The relative abundance of KOs predicted to be involved in glycosyltransferases, and starch and sucrose metabolism was higher in the lumen of both the duodenum and jejunum than in the mucosa (q < 0.05). The relative abundance of KOs predicted to be involved in propionate metabolism, butyrate metabolism, flagellar assembly, and lipopolysaccharide biosynthesis in the mucosa of the jejunum was higher than in the lumen (q < 0.05). These results indicated the different functional potential of the lumen versus the mucosa in the small intestine. Furthermore, the relative abundance of KOs predicted to be involved in the phosphotransferase system, including those that play a role in the phosphorylation of glucose, β-glucoside, and cellobiose, was higher in the lumen of the colon than in the jejunum (q < 0.05), as shown in Figure 5. These results suggested that the functional potential of the gut microbiota varied according to gut locations.The metagenomic prediction showed that the functional capability of the lumen and mucosa differed . We further analyzed the KOs predicted to be involved in carbohydrate-active enzymes. The impact of the antibiotic intervention on the enzymes varied among the gut locations (Supplementary Table Table 2). In the lumen of the colon, the antibiotic intervention increased the relative abundance of KO-related genes emrK and emrY that confer resistance to tetracycline more than 70-fold compared with the control group (q < 0.05). These results showed that antibiotics may have profound effects on KOs associated with antibiotic resistance in the lumen but not the mucosa.The metagenomic prediction also showed that compared to the control group, the antibiotic intervention increased the relative abundance of KOs predicted to be involved in the phosphorylation of trehalose and galactitol, whereas it decreased those involved in the phosphorylation of sucrose and β-glucoside in the lumen of the stomach , but then weakened gradually, with largest effect in the jejunum and less effects in the colon as revealed by the numbers of genera affected. This may be possibly related to the gradual dilution of antibiotics in the gut, which led to the weakened effects. Pharmacokinetic analysis of olaquindox , oxytetre mucosa . In addin mucosa . TherefoL. mucosae, R. lactaris, and C. comes and increased relative abundances of the phylum Proteobacteria, genus Escherichia, and phylotypes S. suis and S. pasteurianus have been associated with disease condition. L. mucosae is regarded as a beneficial microbe and is decreased in humans with Crohn’s disease  . Both inmdtL gene, which confers resistance to chloramphenicol . These increases occurred mainly in the colonic lumen but not in the stomach, duodenum, or jejunum. In growing pigs, in-feed antibiotics using a chlortetracycline, sulfamethazine, and penicillin mixture were reported to augment the abundance of the phenicol , 2014b, lycoside , in the Based on the PICRUSt prediction, the present study revealed alterations in the abundance of potential genes involved in antibiotic resistance. However, the prediction algorithm depended on representative genomes from the sequenced bacteria. Other types of investigations, for example, studies using a metagenomics sequencing-based approach, may allow to determine the function of porcine gut microbiota, which can be studied in future research. Nevertheless, the metagenomic prediction in the present study also provides valuable insights into the effect of antibiotic interventions in early life on microbiota function in piglets.This study simultaneously investigated the spatial variation of gut microbiota and its response to an early-life antibiotics intervention. It is striking that the early-life antibiotic intervention resulted in differential and compartmentalized alterations in the gut microbiota from the stomach to colon. The diversity of the microbiota in the stomach and duodenum was highly similar, but markedly different to that in the jejunum and ileum. The dominant bacteria were similar in the stomach and small intestine, but different to those in the colon. The composition and functional potential of the microbiota in the mucosa differed from those in the lumen. The early-life antibiotic intervention caused a more marked alteration in the microbial composition and functional potential in the foregut versus hindgut, pointing to an antibiotic-induced differential and compartmentalized impact. The antibiotic intervention led to an increase in bacteria groups associated with an increased risk of disease. Furthermore, the increase in potential ARGs may point to collateral antibiotic-induced alterations in the gut microbiota of the piglets. The marked compartmentalized alterations induced by the early-life antibiotics highlights the importance of considering the foregut microbiota in antibiotic-related research. This study provides new insights into the membership of the microbiota along the GIT of piglets and highlights the importance of considering the foregut microbiota in health management of piglets.CM and YY performed the experiments. CM, YY, and YS analyzed the data. CM, YY, and WZ wrote the paper. YS, EZ, and WZ contributed the conception of the work. EZ and WZ revised it critically for important content. WZ had primary responsibility for the final content.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.