Currently, 3D concrete printing technology is not yet able to print ready-mixed concrete with coarse aggregates. Based on an independently developed 3D printing construction equipment system and optimized concrete materials, a 3D concrete printer that can directly print ready-mixed concrete is developed. This paper introduces the whole 3D printing process for one power distribution substation in detail, including the printing equipment, key software, concrete preparation, printing process, and construction inspection. This investigation will provide valuable design and construction experience for the future construction of 3D concrete printing. Architectural 3D printing is an additive-based molding technology based on a computer digital model. It can be quickly formed by printing building materials layer by layer, thus achieving the advantages of high efficiency, low cost, and environmental protection.In 1997, Pegna  developeIn 2017, Panda et al.  studied In 2018, Soltan and Li  conducteHowever, at present, architectural 3D printing technology is still in the research and development stage. From the perspective of application, there are mainly three technical difficulties. First, at the material level, traditional building materials are basically composed of inorganic materials, which makes the molding time very long and makes it difficult to satisfy the requirements of the 3D printing process. It is impossible to change the material properties by hot melt of printing materials such as metal powder and plastic. Second, some building materials that satisfy the requirements of 3D printing technology, such as certain special mortars and special polymers, are difficult to meet the material and structural requirements of buildings, and are also not common building materials. Finally, in the field of equipment application, some institutions and companies have introduced 3D printing components in the factory, and then transport the components to the site for building assembly. This method is more similar to a prefabricated building, and the transportation radius is limited, which does not take advantage of the high efficiency and low cost of building 3D printing. These technical difficulties lead directly to the fact that there are still no proven and fully promoted architectural 3D printing products and technologies in the market.This paper proposes a 3D printing technology and an overall solution from design to on-site, which can directly print commercial ready-mixed concrete. In-filed printing of a power distribution substation will be introduced in detail. Since the traditional commercial ready-mixed concrete is adopted as the printing material, this solves not only the application problem of building materials, but also the structural design for the buildings of this project.At present, various kinds of 3D printers such as polypropylene fiber concrete printers ,20, nyloX-axis track, Y-axis track, Z-axis column track, print head, top stabilization system, and ready-mixed concrete pump truck. The X-axis and Y-axis tracks can control the horizontal movement of the print head, while the horizontal system X-axis and Y-axis can be vertically raised by the trolley on the Z-axis, which satisfies the movement of the print head in the X-, Y-, and Z-axes. The column-type 3D printer has an X-axis width and a Z-axis height of 20 m. It can print buildings with a width and height of no more than 18 m. The length of the Y-axis can be extended indefinitely by the increase in the number of Z-axis columns. Therefore, the column-type concrete 3D printer is simple to install and can print concrete structures of less than six stories. Moreover, the print head and arm of the concrete pump truck are driven independently and each equipped with a positioning sensor that detects the relative position for each other. When the concrete in the print head is continuously printed, the arm of the concrete pump truck itself can perform positioning and feeding work independently.The 3D printer proposed in this study is a column-type mechanical structure, and a four-column frame is set up to implement 3D printing in the frame. As shown in The column-type mechanical structure provides a printing platform for 3D printers, and the main difficulty lies in how to develop a printer suitable for printing ready-mixed concrete. The material composition of ready-mixed concrete is sand, coarse aggregate, cement, and water. The existence of coarse aggregates in ready-mixed concrete poses a huge challenge to the development of 3D printing equipment. The main reason is that it is difficult to develop a print head that can print concrete containing coarse aggregate.The architectural 3D printing system introduced in this study is a commercial ready-mixed concrete building 3D printing system independently developed by the authors. The printing system is characterized in that it comprises a double-assisted print head, which can continuously print uninterrupted ready-mixed concrete with aggregate less than 15 mm in diameter. The working principle of the double-assisted print head is that feed bins A and B can work together at the same time. When one feed bin is extruding and printing concrete, the concrete feeding and slump automatic adjustment starts to work in the other feed bin, in order to achieve the uninterrupted printing of concrete.The reciprocating plugging power system is composed of pistons A and B, as seen in The function of the concrete feeding system is to transport the concrete from the mixing equipment into the feeding bin of the print head through the pipeline and the screw power, and to start and close the feeding by cooperating with switches A1 and B1. It is known that the transportation and feeding processes will introduce uncertain changes to the concrete working performance. As a result, the workability test and adjustment in the feeding bin become important to the concrete printing. The concrete performance test and mix system is equipped with a servo motor that can perform the torque test. As shown in When the concrete slump in the feed bin is between 120 and 130 mm, the printing effect will be the best, that is, the servo motor torque is between 1.25 and 1.75 Nm. If the motor torque is not within this range, the concrete performance adjustment system will be activated. The workability of concrete will be adjusted by adding water, superplasticizer, additives, etc. At the same time, the detection system feeds back the torque data every second. When the torque reaches between 1.25 and 1.75 Nm, the performance adjustment system will be turned off to start printing.It is important to note that the ready-mixed concrete 3D printing system described in this study is equipped with a screw for torque testing in the concrete performance testing system. Therefore, the diameter of coarse aggregate in the concrete is required to be no larger than 15 mm. A V-shape vibrating sieve is designed at the upper end of the concrete feeding system. This sieve has a pore spacing of 15 mm and can filter out aggregates larger than 15 mm in diameter. In addition, if the concrete in the print head is continuously vibrated during the printing process, the mechanical system error caused by the vibration process will be definitely unacceptable. However, it is usually difficult for the printed concrete without any treatment to meet the strength requirement. The squeeze 3D printing system can make the concrete compact by pressure, so that the concrete strength can meet the requirements. The running process for the software system of this 3D printer is shown in The most critical properties for the printing concrete in the fresh state are extrudability and buildability. Extrudability relates to the ability of delivering fresh concrete through a hopper and pumping system to a nozzle where the concrete must be extruded as a continuous filament. It is mainly affected by the workability. Moreover, the printed concrete filaments should be formed with minimal deformation under the weight of subsequent layers. Additionally, the lower filaments should bond to the upper ones to build monolithic components. Therefore, this printing ready-mixed concrete requires a buildability which relates to the ability of printing a certain number of layers or height. Buildability also depends on the workability and mix proportions.In this study, a 3D printer was used to optimize the workability range of concrete which was ready to be extruded. It is known that higher slump has a negative effect on the deformation of concrete after extrusion, whereas lower slump may cause the noncontinuous flow of concrete which affects the buildability and hardened properties of concrete. After a series of trials, a slump of 110 mm was recommended as the most suitable workability in application. After several trials, the mix ratio of 3D printing concrete was optimized and is shown in The rheological parameters of the concrete were tested in this study using an International Center for Aggregate Research (ICAR) rheometer , as shown in Based on the above mixing ratio for the printed concrete, concrete was cast into a cube test mold. The Chinese standard for the test method of mechanical properties on ordinary concrete (GB/T50081-2002)  was adopWith the techniques in equipment and materials for 3D printing mentioned above, this paper introduces the world’s first application of a 3D printed power distribution substation by adopting commercial ready-mixed concrete in China. The full picture of the printer is shown in According to the design, the concrete strength of the wall should not be less than 20 MPa. The wall part of the power distribution substation is printed with C25 ready-mixed concrete, with a diameter of the coarse aggregate less than 15 mm. During the printing process of the power distribution substation, the actual strength of the wall concrete is higher than 20 MPa, which meets the structural design requirements.Constructional columns and ring beams were employed in the structural design of the project to meet the seismic requirements. Therefore, the project optimizes the path of the concrete printing process. The concrete wall is printed horizontally at first, and the position of constructional columns is reserved during the printing process. After the concrete printing wall is finished, constructional columns are then built by means of supporting formwork, tying steel bars and casting concrete, as seen in It is worth noting that for the printing of the lintel above the door and window holes, it is still necessary to use the traditional method of constructing the formwork, that is, using the prefabricated wooden formwork to support the lintel. The length of the wooden formwork is the same as the length of the lintel, whereas the width is slightly larger than the lintel width. Using the 3D printing method on the wooden mold support, the construction of the lintel is achieved.During the construction, the 3D design model is first carried out, and then the 3D printing slice technology is used to slice the building model, generating a layer of digitized code. In addition, using the real-time detection and adjustment function of the 3D printing system, the printing speed, the direction of the squeegee, the motor state, and the system temperature are detected and adjusted.In the 3D printing design of the cable trench, equipment foundation, and oil collection tank below the ground, the extension length of the print head was optimized, and the complex underground structure was printed, as shown in The main advantage of this 3D printing power distribution substation project is the use of commercial ready-mixed concrete as the printing material, thus meeting the requirements of current regulations in materials and structural design. In addition, it is a full-scale concrete building printed by a 3D printer, which is also rare in the world at present.The total construction time of the power distribution substation project using traditional cast-in-place concrete is 54 days, whereas the construction time using 3D printing is less than 35 days, time which is shortened by 30%. The number of working days for the main part of the building using 3D printing is only four days, which is a 60–80% reduction compared with the traditional construction method. Moreover, since 3D printing replaces the traditional construction process, the dust pollution on the site is reduced and ensures a clean and tidy construction site. The construction waste is less than one ton, and the amount of waste generated is reduced by more than 60% in the construction field, which is highly beneficial to the environment.The 3D printing of the building described in this paper uses coarse aggregate with a particle size of 5–15 mm. Compared with the traditional mortar ink used in 3D printing concrete, it is a big step forward. In addition, a power distribution substation was successfully printed, which met the building standards of most countries in terms of structural design and material specifications. However, currently, the construction of 3D printing equipment can only be operated by a single print head. It is difficult to achieve the collaborative work of multi-printing heads simultaneously. If this can be realized in the future, the printing efficiency will be further increased. Moreover, the current print head can only print materials with a diameter of no more than 15 mm, and the degree of cooperation between the print head and the commercial ready-mixed concrete can be further improved. Additionally, although the highly automated 3D printer can replace manual work in a low-temperature state, due to the special nature of the hydration heat reaction of concrete, the concrete material that can be printed at a low temperature has not yet been formulated. Therefore, it is impossible to carry out 3D printing concrete construction in winter, especially in cold regions. Moreover, it should also be noted that construction with concrete printing not only reduces the labor needed, but can also increase the cost of materials, since a relatively higher-class concrete should be used for the practical engineering based on current technology. In terms of design and construction, the 3D printing construction of multi-story buildings must be improved and verified, such as columns that can be assembled using precast elements, which may highly improve the construction efficiency. At present, the bearing capacity of 3D printing concrete components or structures cannot be evaluated. In the future, it will be necessary to establish a structural performance evaluation system for 3D printing concrete buildings.This paper introduces the whole 3D printing process of a power distribution substation completed in Guangzhou, China. The construction period of the project is short and the manual requirements are greatly reduced. Based on the self-developed printer, C25 ready-mixed concrete with 5–15 mm coarse aggregate was adopted as the printing material to construct the power distribution substation.In terms of equipment, with a double-assisted printhead, it is possible to continuously print uninterrupted ready-mixed concrete during the printing process. In addition, the squeeze pressurization method replacing the vibrating system makes the compactness and strength of the concrete meet the material requirements. In terms of materials, for the printability and buildability of concrete, the 3D printing system is optimized which can meet the printing properties of ready-mixed concrete. Currently, construction with concrete printing reduces the labor needed, but can increase the cost of materials, since a relatively higher-class concrete should be used.This project will provide valuable design and construction experiences for the future construction of concrete 3D printing, which has a certain guiding significance.


The risk of a broken scalpel blade during discectomy is considered extremely rare, while no guidelines exist regarding this complication. We report a case of a robotic broken blade removal following lumbar discectomy. A 52-year-old female was subjected to L4-L5 discectomy. During the annulus resection, the scalpel blade broke and was retained within the disc space. The broken blade migrated towards the abdominal cavity and viscera. Emergency CT angiography scan revealed that the main vessels were intact, while the broken surgical knife was located anterior to the lumbar spine at the L4/L5 level, to the left of the aorta and superiorly of the left common iliac artery. At that point, robot-assisted laparoscopy was performed. The broken instrument was located and carefully removed. It seems more proper that such foreign bodies should be removed, while robotic surgery may play a significant role in cases that the foreign body is near major vessels. Robotic, computer-assisted, or robot-assisted surgery are similar terms for the use of robotic systems aiding in various surgical procedures. Robotic surgery has been developed in order to overcome limitations of minimally invasive surgery and to increase the capabilities of open surgery , 2.The main advantages of computer-assisted surgery for the surgeon are as follows: greater visualization, enhanced dexterity in which dissections can be performed, and greater precision. Since its introduction, robotic surgery rapidly expanded in a plethora of surgical subspecialties, as well as operations. It has been estimated that over 3 million patients have been operated since the early 2000s with the introduction of the da Vinci® device .There have been a few cases reported in the literature so far, describing the use of robotic surgery for iatrogenic complications, such as foreign body removals and the cement removal following percutaneous vertebroplasty . The risA case of a foreign body removal , during a lumbar discectomy, with the use of the “da Vinci® Robotic System” is presented, exhibiting the advantages of computer-assisted surgery for the surgeon.A 52-year-old female, with an unremarkable medical history, with the exception of lumbar herniation, was subjected, under general anesthesia and in a genupectoral position, to L4-L5 discectomy. During the annulus resection, the no. 11 scalpel blade broke and it was retained within the disc space. Attempts to remove the foreign body were performed under fluoroscopy. However, the broken blade migrated towards the abdominal cavity and viscera. Immediately, a CT angiography scan was performed, in order to locate the broken instrument . CT angiThe patient remained during this procedure stable, at all times. Urgently, the patient was placed in a supine position and a robot-assisted laparoscopy was initiated. Under general endotracheal anesthesia, the da Vinci® platform was brought to the operating table between the patient's legs. The camera port was inserted inferior and to the right of the umbilicus using the Hasson technique. Under direct vision, two additional robotic arm trocars were inserted at the right and left iliac fossa, respectively. Once the robot was docked, an exploration of the peritoneal cavity was performed. With the patient tilted to the right, the small intestine was transferred to the right abdominal cavity so that the retroperitoneum below the level of the left kidney could be exposed. The retroperitoneum was carefully dissected using bipolar Cadier forceps on the left arm and monopolar scissor and hook on the second arm. After the access to the abdominal aorta was gained, the broken scalpel was identified in close distance to the aorta and the left common iliac artery with no signs of active bleeding. The scalpel was slowly removed with extreme care not to traumatize the vessels . A finalrd postoperative day. At follow-up, 2 years after the operation, she remains without any signs or symptoms of disease.The patient had an uneventful hospitalization and was discharged at the 3Breaking of the surgical scalpel blade during lumbar discectomy has already been described as associated with the procedure risk . Lumbar Amirjamshidi et al. in 1994 was the first one to report 4 cases of a broken scalpel blade within the intervertebral disc space and migration towards the abdominal cavity and viscera . The autComplications due to broken blades and spontaneous migration have already been reported by De Praetere et al. , while aThis may lead to serious complications, depending upon the location of the broken scalpel blade. The risk of intra-abdominal visceral and vascular injuries is high –8. In thDue to lack of data, there are not clear guidelines regarding this iatrogenic complication. There are still many issues that need to be clarified, such as whether conservative treatment is an option for asymptomatic foreign bodies without associated risks and whether it is better to remove the foreign body in a second intervention and not during the initial operation , 7. It sFurthermore, other options for removal of the broken scalpel should be mentioned. In the present case, the part of the broken blade remained in the intervertebral disc space; therefore, if the operating room was equipped with intraoperative CT, CT-guided removal of the broken scalpel blade through disc space could be a feasible option. The main advantage of this approach would be the avoidance of repositioning the patient from prone to supine positions which could lead to surrounding tissue injuries. Additionally, an issue that should be considered is whether insufflation of the abdominal cavity may have risk for injury of tissues surrounding the broken blade. Hence, open procedure should always be kept in mind.Robotic surgery is an advanced form of minimally invasive or laparoscopic  surgery where surgeons use a computer-controlled robot to assist them in certain surgical procedures , 10. TheThe main advantages of computer-assisted surgery over classical surgery regarding the patient are decreased blood loss, smaller incisions, less pain and hospital stay, and quicker healing, while the main criticism regarding this type of surgery is the increased cost per operation , 4, wherThe presented case is the first case reporting the robot-assisted removal of a broken blade following discectomy. The high-quality visualization and the enhanced dexterity were of utmost importance for the removal of the potentially life-threatening foreign body. Such cases should be reported, since no clear guidelines exist for their management. It seems more proper that broken blades should be removed. If the broken scalpel blade is near major vessels or in difficult-to-approach regions, the use of robot-assisted surgery by experienced surgeons could be an option.


HIV infection remains of major public health importance in all world and in Romania. In Romania there are a large number of long-term survivors coming from the 1987–1990 generation, the circumstances are due to the fact that an increased number of HIV-infected persons receive a specific therapy.The study was conducted using sociological analysis methods such as comparative analysis, statistical analysis, and with the help of the method of regression analysis, to capture the dynamics of the HIV / AIDS phenomenon in Romania, as well as forecasting the dynamics of the phenomenon over the next decade.This longitudinal analysis of the statistical data provided by the Matei Bals Institute of Romania during the period 2004–2016, shows a progressive increase 1.4 times higher in 2016 compared to 2004, the prediction of the extension of the HIV / AIDS phenomenon for the period 2017–2027, in Romania being almost constant. Regarding the prognosis of the number of persons affected by HIV / AIDS in Romania, for the next 10 years an upward dynamic is predicted with an increasing annual rate of 506 new persons taken into evidence. As the prediction function shows, the trend the access to treatment is increasing for the next decade. After 2006 in Romania, the deaths dynamics is an exponential decreasing one, keeping the same rate of decrease, until 2015.The HIV/AIDS pandemic has changed considerably in the past 27 years. AIDS incidence and mortality in industrialised countries have fallen, and paediatric HIV disease has almost been eliminated as a public health issue, largely through antiretroviral drugs. In Europe, Romania detained until recently among the first place in terms of the number of AIDS-infected children, while, in terms of the total number of cases, it was closer to the Western countries than to the countries in Central and Eastern Europe, during the period 1987–1990  in Romania; I2 Total AIDS deaths (cumulative) in Romania; I3 Total HIV + AIDS lost from evidence; I4 Total HIV + AIDS patients in active evidence.Analysis of the dynamics of the HIV / AIDS phenomenon in Romania and the prognosis of this phenomenon in the period 2017–2027In the following discussion, we highlight the HIV / AIDS depending on each distinct in Romania:2=0,727).From the longitudinal analysis of the data provided by the Matei Bals Institute of Romania, regarding the situation in Romania, for the period 2004–2016 there is a progressive increase 1.4 times higher in 2016 (21702 persons) compared to 2004 (15471 persons ). The prediction of the spread of the HIV / AIDS phenomenon for the period 2017–2027 is almost constant, as it results from the analysis of the linear regression of the data shown in From the official data of the National Board of Fight Against AIDS, “Prof. Dr. Matei Balş” Institute of Infectious Diseases we can see in We find that the share of total cumulative HIV / AIDS in relation to the number of AIDS patients is relatively constant at a rate of 1.67 in 2004, 1.38 in 2010 and 1.46 in 2016 respectively.With regard to the prognosis of AIDS dynamics over the next decade, with the help of the linear regression analysis one foresees a 1.49-fold increase  with a similar growth rate to the HIV / AIDS phenomenon cumulatively predicted over the same period.At the end of 2016, as a result of the analysis of the data recorded in the Matei Bals Institute's national database - over the past 25 years, respectively 21702 cumulated cases, Romania continues to have a large number of “long-term survivors” aged between 20–24 years, 46% being women.2=0,993).In terms of predicting the dynamics of HIV / AIDS deaths over the next decade , the linIn Romania, the monitoring of people affected by HIV / AIDS is extremely important, especially as there are often situations when, despite the fact that a significant number of cases registered in the national surveillance network are highlighted, these cases are subsequently lost from the evidence for more or less objective reasons.It is certain that those cases whose “trace” has been lost can no longer have access to investigation, medication and treatment, and the progression of their disease cannot be known. For this reason, one of the important national indicators highlighted in official statistics refers to the number of people affected by HIV / AIDS lost from records. From the statistical analysis of the official data provided by the Matei Balş Institute it is found that in 2004, 3.26% of the total number of registered persons were lost from the records, there being a constant rate of these losses of 3.50% in 2010, respectively 3, 15% in 2016 .In order to provide treatment and care for HIV / AIDS, Romania has a well-organised system at present.Medical advances over the past 15 years in the field of HIV / AIDS and the introduction of appropriate antiretroviral therapies have enabled a considerable number of affected people from 1987–1990 to survive and reach maturity. Most HIV-infected patients are between the ages of 26–30. Pediatric AIDS cases are currently low due to a number of factors.2=0,898).Based on the longitudinal analysis of statistical data from Matei Balş Institute there is an increasing dynamics of the number of persons with active evidence of HIV / AIDS in the period 2004–2016, registering a gradual increase 1.55 times higher  in 2016 The HIV/AIDS pandemic has tremendously spread at a global level with serious implications upon population .One of the Central and Southeast European countries with a significant number of HIV-AIDS cases and with the highest survival rate in Central and Eastern Europe is Romania. More than 50% of the cases detected in the 1990s are alive due to access to modern treatment schemes, since the life expectancy was only three months in the 1990s . For thiThe emergence and spread of HIV infection in Romania had as its main causes the poor endowments in the hospitals with poor sanitary instruments in the period before 1989 (December 1989 post-communist revolution in Romania), which generated the inappropriate and repeated use of syringes and unsterilized needles in the communities and medical centres, due to transfusions with infected blood associated with the lack of reagents needed to HIV screening diagnosis in blood collection centres due to socioeconomic factors  infections can be reduced through antiretroviral treatment interventions.In Romania, the longitudinal analysis of the statistical data provided by Matei Bals Institute for the period 2004–2016 shows a progressive increase 1.4 times higher in 2016 compared to 2004, the prediction of the extension of the HIV / AIDS phenomenon for the period 2017–2027 being almost constant, as is shown by the linear regression analysis of the data, resulting in a 1.45-fold increase over the next 10 years. Likewise, this prognosis is worrying and warns about the imminence of the phenomenon spread on national level in Romania over the next 10 years.A favourable aspect in Romania is the access to treatment with an increasing trend from 2000 to 2015. As the prediction function shows, the trend is increasing for the next decade, which means there is hope that in 10 years, the increase in access to treatment shall be more than 10 times higher.Ethical issues  have been completely observed by the authors.


The availability of antibiotics presents medical practitioners with a prescribing dilemma. On the one hand, antibiotics provide a safe and effective treatment option for patients with bacterial infections, but at a population level, over-prescription reduces their effectiveness by facilitating the evolution of bacteria that are resistant to antibiotic medication. A game-theoretic investigation, including analysis of equilibrium strategies, evolutionarily stability, and replicator dynamics, reveals that rational doctors, motivated to attain the best outcomes for their own patients, will prescribe antibiotics irrespective of the level of antibiotic resistance in the population and the behavior of other doctors, although they would achieve better long-term outcomes if their prescribing were more restrained. Ever-increasing antibiotic resistance may therefore be inevitable unless some means are found of modifying the payoffs of this potentially catastrophic social dilemma. Antibiotic medication is the treatment of choice for patients who present with symptoms consistent with bacterial infection, but it is difficult for a doctor to be certain that the treatment is appropriate for the condition, because the symptoms typically have other possible causes. Broad-spectrum antibiotics, given their effectiveness against a wide range of bacteria, provide especially useful treatments that can be begun even before specific pathogens have been identified in symptomatic patients. By enabling rapid commencement of treatment, they reduce the risk of serious morbidity and mortality—illness and death in the population. However, whenever antibiotics are prescribed, they facilitate the evolution and spread of antibiotic-resistant bacteria, and antibiotic over-prescription accelerates this process. Antibiotic resistance could lead to the emergence of communicable diseases impervious to all currently available medications, and this may ultimately trigger destructive pandemics. At a special meeting of the United Nations in 2016, the President of the 71st session of the General Assembly declared that antimicrobial resistance has the capacity to kill millions of people each year and that “ultimately, the future of humanity may depend on our ability to respond to the great challenges of antimicrobial resistance” . Players make repeated decisions—this is an indefinitely repeated game. We use the symbol q for the proportion of symptomatic patients in the population who are currently medicated with antibiotics.We begin by specifying a symmetric multiplayer population gamep = ,pi∈P, a strategy profile. To denote the incomplete strategy profile excluding Player i, we writei who chooses a specified strategy pi while the remaining players choose strategies pj≠i.We call pi∈P and for all i ∈ N . This payoff is maximized when q = 1/2, for ϕ > 0 and 0 < q > 1. It follows that, although T is a dominant strategy, every doctor receives a better payoff from choosing any other strategy pi (0 < pi < 1), and every doctor receives the best possible payoff if all doctors choose the strategy pi = 1/2.The payoff i for the pure strategy T (treat with antibiotics with certainty) can be derived from pi = 1:pi = 0:q < 1 and all values of ϕ (0 < ϕ ≤ 1), we have πi>πi, confirming that T is a dominant strategy. With the assumptions of our model, the Nash strategy is p* = 1. For all ϕ ∈  and turns out to beThe replicator equation indicating the increase or decrease of antibiotic treatment decisions T, given the current population relative frequency ϕ = 0, q = 0, and q = 1. For all other values of q, and for all values of ϕ > 0, ϕ increases from 0 to 1, and increasing then falling as q increases from 0 to 1, indicating increasing rate of increase of T choices. We have assumed that ϕ > 0, and this implies that, except when every symptomatic patient within the population has been prescribed antibiotic medication, the decisions to prescribe antibiotics become increasingly frequent until, in equilibrium, all treatment decisions for symptomatic patients involve antibiotics. The role that the infection probability ϕ plays is to accelerate the drift to T at a constant rate as ϕ increases.Values of ϕ ∈  represent rates of increase in antibiotic prescribing. Antibiotic prescribing increases most rapidly when ϕ  approaches 1 and when q (the proportion of patients receiving antibiotics) is 1/3. The state q = 1, with all patients receiving antibiotic treatment, is a strict Nash equilibrium and is asymptotically stable and a global attractor, meaning not only that it is an evolutionarily stable Nash equilibrium, but also that there is a dynamic attraction from all other states to the outcome in which all symptomatic patients receive antibiotic treatment. The state q = 0, where no symptomatic patients receive antibiotic treatment, is an unstable rest point and hence an unstable equilibrium.ϕ and q, typical trajectories determined by the replicator dynamics, an unstable rest point at ϕ = q = 0, and a global attractor at ϕ = q = 1, where antibiotic prescribing is maximized. Because T is a dominant strategy, the basin of attraction from which all trajectories converge on the global attractor includes all states in which ϕ > 0 and 0 < q < 1.Every mathematical model depends on simplifying assumptions, and our antibiotic prescribing game is no exception. It is worth drawing attention to a few of the assumptions in our own model to avoid overgeneralized interpretations or applications of the game. One important assumption, alluded to earlier, is that prescribing doctors are motivated solely to minimize morbidity and mortality in their own patients when they present for treatment. If doctors are instead motivated in some other way, for example, if they strive to maximize the welfare of all patients in the population, present and future, then the antibiotic prescribing game fails to provide an accurate model of their behavior.q = 1), then antibiotic resistance will have reduced the therapeutic efficacy of antibiotics to zero. However, it is possible to imagine a situation in which, for a limited time, antibiotics might retain their efficacy even while all symptomatic patients are receiving them. In those circumstances, a doctor’s payoff for choosing T (treat with antibiotics) decreases as q increases but remains larger than the payoff for U (do not treat with antibiotics) even when q = 1. πi = −λq(0<λ<ϕ). The average payoff to a doctor in the game is then πi = q(−λq)−ϕ(1−q), and the payoff is maximized when q = 1, for λ < ϕ/2. However, it is immediately obvious from y, and it follows that the outcome in which every doctor chooses T is a unique and stable Nash equilibrium, but the game is no longer a tragedy of the commons, because this outcome is not worse for every doctor than the outcome when every doctor chooses U or a mixed strategy pi.Second, we assumed that, if all symptomatic patients are treated with antibiotics  that symptomatic patients are infected with pathogenic bacteria. From any initial pattern of prescribing behavior, doctors will tend over time to increase antibiotic prescribing. This is true despite the fact that any more restrained prescribing strategy, apart from never prescribing antibiotics, yields a better payoff to every doctor, provided that all doctors choose it. The payoff to every doctor is maximized when antibiotics are prescribed to half the symptomatic patients in the population, in practice according to their judgments of the probability of severe bacterial infection. The strategy of prescribing antibiotics to all symptomatic patients may lead to the evolution of antibiotic resistance, ultimately rendering antibiotics useless for treating even the most dangerous infections and also reducing the doctors’ own payoffs. Thus, doctors’ decision making leads inexorably to an outcome that is worse for all of them and their patients than the outcome that would have resulted had they deviated from the game-theoretic rational strategy and exercised restraint. We have therefore proved that the antibiotic prescribing game is a social dilemma of the tragedy of the commons type.This proof has significant implications for the design of interventions to reduce over-use of antibiotics in healthcare settings. The importance of drawing on theories of behavior in the design and implementation of antibiotic stewardship interventions is increasingly recognized , with evManaging this social dilemma requires changing the payoffs of the game, and one way of achieving that is through coercive strategies that promote the good of society over individual interests, although there are both ethical and practical challenges to the restriction and control of antibiotics, particularly in the context of low-income countries where lack of access to antibiotics is a significant threat to health at a societal level . We needChanging behavior in the context of a tragedy of the commons problem is notoriously difficult. However, drawing on theory and evidence from the field of research into social dilemmas in a systematic way to inform intervention development will be critical as part of efforts to protect the antibiotic commons for the future. As Daniel Bernoulli commented in a paper presented to the French Academy of Sciences in 1760: “I simply wish that, in a matter which so closely concerns the wellbeing of the human race, no decision shall be made without all the knowledge which a little analysis and calculation can provide” .Here we provide a proof of the well-known proposition that Inequalities 1 and 2, taken together, imply Inequality 3, and that Inequality 3 implies Inequalities 1 and 2. First, we prove that Inequality 1, when it holds strictly, implies Inequality 3.pi ∈ Pi. If this inequality is strict, and we let ϵ → 0 in Inequality 3, reproduced here as Inequality A1,According to Inequality 1, Suppose now that Inequality 1 holds with equality, so thatπi is continuous at 0, we can rewrite Inequality 3 as follows:First, because To prove that Inequality 3 implies Inequalities 1 and 2, taken together, we note first that if we examine the limit as ϵ → 0, thenNow suppose thatπ = −ϕq, and the payoff associated with U is π = −ϕ. The replicator equation is defined as the current relative frequency of T multiplied by the payoff associated with T relative to the average payoff from both strategies.We assume that prescribing decisions are made repeatedly over time, and the replicators in this analysis are pure strategies T (treat with antibiotics) and U (do not treat with antibiotics). From Eqs q, is given byThe average payoff from both strategies, when the current relative frequency is S1 Table(XLSX)Click here for additional data file.


Inappropriate antibiotic use is implicated in antibiotic resistance and resultant morbidity and mortality. Overuse is particularly prevalent for outpatient respiratory infections, and perceived patient expectations likely contribute. Thus, various educational programs have been implemented to educate the public.We systematically identified public-directed interventions to promote antibiotic awareness in the United States. PubMed, Google Scholar, Embase, CINAHL, and Scopus were queried for articles published from January 1996 through January 2016. Two investigators independently assessed titles and abstracts of retrieved articles for subsequent full-text review. References of selected articles and three review articles were likewise screened for inclusion. Identified educational interventions were coded for target audience, content, distribution site, communication method, and major outcomes.N = 24, 83%) and community sites . The majority included clinician education. Antibiotic prescription rates were assessed for 22 interventions (76%). Patient knowledge, attitudes, and beliefs (KAB) were assessed for 10 interventions (34%). Similar rates of success between antibiotic prescription rates and patient KAB were reported . Patient interventions that did not include clinician education were successful to increase KAB but were not shown to decrease antibiotic prescribing. Three interventions targeted reductions in Streptococcus pneumoniae resistance; none were successful.Our search yielded 1,106 articles; 34 met inclusion criteria. Due to overlap in interventions studied, 29 distinct educational interventions were identified. Messages were primarily delivered in outpatient clinics  contains supplementary material, which is available to authorized users. Antibiotic resistance is a worldwide threat fueled in large part by the inappropriate use of antibiotics. According to the Centers of Disease Control and Prevention (CDC), over 2 million people are affected by antibiotic-resistant infections in the United States each year.  AnnuallyTo reduce antibiotic resistance and adverse effects for patients, it is essential to reduce inappropriate antibiotic use. The evolution of antibiotic resistance is multifactorial, and there is no single intervention that can solve this public health threat. However, evidence suggests that antibiotic stewardship programs can curtail excessive antibiotic use, reduce antibiotic-associated events, decrease antibiotic resistance, and improve patient outcomes. , 12 ImplVarious educational interventions have been implemented with the goal of increasing antibiotic awareness. The objective of this study was to systematically identify, characterize, and evaluate the messaging approaches used in these interventions. We were interested in interventions in the United States, specifically, for a variety of reasons. For one, antibiotic use and resistance rates varies widely between countries.  Further,We included articles investigating a public campaign or patient-directed messaging program to promote awareness of appropriate antibiotic use within the United States.We developed independent literature search strategies for Pubmed, Google Scholar, Embase, CINAHL, and Scopus. These search protocols utilized combinations of relevant keywords or phrases appropriate for each database regarding antibiotics, messaging, and public awareness to retrieve articles published from January of 1996 through January of 2016  covered appropriate use of antibiotics; 2) covered risk of inappropriate use; or 3) relied on CDC materials or principles. “Appropriate use” was defined as either education about differences between viruses and bacteria or the indications for antibiotics. “Risks of inappropriate use” was defined as information regarding possible antibiotic adverse events or antibiotic resistance. Reliance on CDC included the stated use of CDC principles or CDC employees in the development of materials or the direct use of CDC materials.Educational interventions were assessed for the following location(s) of message distribution: emergency department, outpatient clinical setting , community setting, and/or personal residence. “Community settings” was defined to include childcare centers, local pharmacies, community fairs, restaurants, churches, and schools. Messages distributed by public media, such as television or radio, were classified under “community site.” Correspondence mailed directly to specific households was classified separately.Educational messages were analyzed for their mode of communication, including the use of print media , media directly mailed to individuals, public media , video messaging, Short Message Service (SMS) messaging, and/or presentations.All outcomes targeted were coded within these categories: patient or parent knowledge, attitude, and/or beliefs; physician knowledge, attitude, and/or beliefs; antibiotic prescribing; appropriateness of antibiotic prescriptions; adherence to recommended treatment; drug resistance patterns; drug costs and/or healthcare utilization. Outcome effectiveness was reported as defined by the original articles, and the percent effective was determined for both the total number of studies as well as for randomized-controlled trials alone.If an intervention included messages for the medical community, these messages were also characterized. We coded for the presence or absence of messages targeting physicians, nurses, physician assistants, and nurse practitioners. Messaging content was described as risks of inappropriate use; pathogen resistance surveillance data; algorithms, pathways, and/or guidelines; audit and feedback; making use of CDC principles; and/or “cold kit” distribution . The location of distribution was similarly described. Modes of message distribution were analyzed for the use of computer-decision support, group sessions or presentations, email, mailings, and/or continuing medical education (CME).Intervention characteristics were analyzed using descriptive statistics.Interrater reliability for inclusion/exclusion of candidate articles based on title and abstract using Cohen’s Kappa was high ; cases of discordance were resolved by a third investigator. A full-text review was then performed for final selection (κ = 1.0).Articles localized via our secondary search strategy were likewise assessed for further review based on title and abstract (κ = 0.65 95% CI 0.45 to 0.85), and discordance was resolved by a third investigator. Reviewers independently performed a full-text review of these articles for final selection (κ = 1.0).Fourteen of the 34 included articles (41%) were randomized controlled trials, the gold standard for intervention effects. Another 18 articles were prospective studies (53%). Of these non-randomized trials, 14 included a comparison population to distinguish the intervention-attributable effect from secular trends, while 4 studies simply compared post- and pre-intervention measurements. One study used multilevel logistical regression of intercept interview data to analyze changes in knowledge associated with the intervention. Lastly, one article used reports from 3 separate organizations to describe changes in antibiotic use, but no statistics were provided to indicate the significance of these results.The 29 educational interventions were stratified based on primary audience, content of messages, location of distribution, mode of communication, and outcomes studied  specified parents as the primary target. In total, 31% of studies included materials in the Spanish language; only one exclusively targeted Latinos residing the United States. Not all articles described the content of their materials. However, the majority of interventions (83%) explicitly specified educational messages regarding the appropriate use of antibiotics, such as the difference between viruses and bacteria and the specific illnesses for which antibiotics are indicated. Risks of inappropriate use, such as antibiotic resistance and potential side effects, were also commonly communicated (72%). Most interventions noted the use of Center for Disease Control (CDC) principles or materials (69%).Educational messages were distributed in a variety of settings. On average, messages were distributed in 1.6 coded locations per intervention . The majority of interventions distributed some or all their content in an office or clinic setting (83%). A substantial proportion (41%) of the interventions distributed messages within the community . Interventions used on average 1.9 methods of communication . About a quarter of interventions used public media to reach their audience. However, handouts and posters were the most commonly used mode of distribution (90% of interventions).Interventions were varied in their desired outcomes, and many had several major outcomes. Overall, 76% (22/29) of interventions observed favorable changes for at least one of the major outcomes studied.Antibiotic prescription rates were most commonly studied (22 of 29 interventions). This was often measured by prescriptions per visit, prescriptions per visit for respiratory illnesses, prescriptions per physician, or prescriptions per person of community or health organization. Of these interventions, 73% reported reductions in antibiotic prescribing. However, several studies observed significant changes in antibiotic prescribing that were attributable to secular trends. Thus, Tables Appropriateness of antibiotic prescribing was measured for three interventions. , 34, 45 Knowledge, attitudes, and/or beliefs of the non-medical community were also commonly studied (10 interventions). Surveys were used to measure knowledge scores, expectations for treatment with antibiotics, or sense of communication efficacy. Statistically significant improvements in these measures were reported for 7 of these interventions (70%). When restricting to randomized controlled trials (5 of the 10 interventions), 60% showed statistically significant benefits. These studies frequently stratified on health insurance status and education level. Croft et al. found that the overall increase in knowledge was attributable to college-educated parents; there was no significant change in knowledge scores for non-college graduates.  In contrStreptococcus pneumoniae pre- and post- intervention. [Three interventions (10%) measured rates of penicillin non-susceptible vention. , 36, 43 vention. , 36, 43 vention. Cost savings were estimated for two interventions (7%), , 30 attrWhile all interventions were required to have messages to the non-medical community to be included in our review, 19 of the 29 interventions (66%) additionally included an educational intervention for the medical community. These interventions are subanalyzed here.Every intervention that included messages for the medical community targeted physicians, with some interventions also specifying nurses (11%), physician assistants (16%), and nurse practitioners (21%).Of the 19 interventions targeting health professionals, 42% of included information about risks of inappropriate use. The vast majority of interventions employed tools for clinical decision making, such as algorithms, pathways, and guidelines for antibiotic prescribing (90%). Other commonly used methods were pathogen resistance surveillance data (42%) and audit/feedback specific for the physician or site (53%). CDC materials or principles of judicious use were frequently used (58%), like the non-medical community-only interventions. One intervention included financial incentives for physicians. Most of the messages to medical personnel were distributed in the office or clinic setting (84%). The mode of distribution for these messages included group sessions or presentations, mailings, computer decision support systems, continuing medical education, and email.All 19 interventions were directed to parents, though one also catered to children. Seven included information for Spanish speakers (37%).Sixteen of these interventions included information regarding appropriate use of antibiotics (84%). Twelve highlighted the risks of inappropriate use (63%). Lastly, 15 noted the use of CDC principles (79%).An average of 1.8 coded settings were utilized for distribution of these messages to the non-medical community. Most used the clinic or office setting (95%). Nine also distributed materials in community sites (47%). Seven interventions used direct mail (37%). Only one used the emergency department setting (5%). These interventions included an average of 1.9 coded modes of communication. These included print media (90%), mailed media (42%), public media (32%), presentations (26%), and videos (5%).Antibiotic prescription rates were measured in all interventions that targeted both the medical and non-medical community. Most of these interventions reported decreased antibiotic use following the intervention (84%). Six of these interventions were randomized-controlled trials, five of which were effective (83%). Samore et al. compared a community-intervention alone to a community intervention plus clinical decision support; the community-only intervention had no significant impact on antibiotic prescribing rates, while the combined interventions led to a significant decline. Knowledge, attitudes, and/or beliefs of the lay community were analyzed in 3 of these interventions (16%), with 67% reporting a positive effect. However, only 1 of these was a randomized controlled trial; this trial was not effective.Knowledge, attitude, and/or beliefs of the medical community were analyzed in 2 of these interventions, with one being effective (50%). However, the randomized controlled trial was not effective.Appropriateness of physician antibiotic choice was analyzed in 3 of these interventions (16%), one of which was a randomized controlled trial. All were effective.Of the 29 interventions, 10 were strictly public or patient directed. The results specific to these interventions are reported.All primarily targeted adults. Two interventions included materials for Spanish speakers (20%).Most included information about appropriate antibiotic use (80%) and risks of inappropriate use (90%). Five interventions noted use of CDC content (50%)An average of 1.2 coded settings were used. The majority used the clinic or office setting (60%). Other settings included community sites (30%), the emergency department (20%), or personal residence (10%). These interventions included an average of 1.8 coded modes of distribution, including print media (90%), video (40%), presentations (20%), public media (10%), SMS messaging (10%), and direct mail (10%).Knowledge, attitudes, and/or beliefs of the non-medical community were most commonly studied (7 interventions). Of these, 5 were effective (71%). Four of these interventions were randomized controlled trials, 3 of which were effective (75%).Prescription rates were measured for 3 of these interventions (30%). One of these was randomized controlled. None of these were effective at reducing prescription rates.Adherence to prescription was measured for one of these interventions (10%). This was a randomized controlled trial that was not effective.We found that most public messaging interventions focused on educating parents of young children through office-based posters and handouts, often produced by the Centers for Disease Control and Prevention. Common rationale for catering to this audience included the likely role of parental expectations in the high rates of inappropriate antibiotic use in children. , 46, 50 Many interventions were multifaceted and distributed educational materials within the community as well as clinical sites. While we aimed to locate interventions targeting the public, over half of the interventions also included a clinician-education component. This was accomplished primarily through prescribing guidelines, audit and feedback of prescribing practices, and pathogen surveillance data, which were distributed through group presentations and mailings. The risks of antibiotic resistance were not as frequently communicated to the medical community, likely due to assumed prior knowledge of this target audience. The majority of interventions observed at least one favorable outcome, and improvements in antibiotic prescribing and patient knowledge, attitudes, and/or beliefs were reported at similar rates. When restricting our analysis to randomized controlled trials, the majority of interventions were still effective in their respective outcomes.Thus, these interventions suggest that patient-directed messages can influence patient attitudes, knowledge, and beliefs about antibiotics, which are important factors in physician overprescribing. The success rate for this outcome was similar for interventions with or without a provider education component. Subanalyses included in some of the campaign evaluations suggested that educational interventions may be best directed to the Medicaid-insured and non-college graduates to ameliorate a lack of access to health education, although one study found the opposite. Most studies that measured antibiotic prescribing reported success. The addition of a clinician-directed component may be imperative for this reduction in antibiotic use. Every intervention that resulted in decreased antibiotic prescribing included a provider intervention. This contrasted with only 3 interventions measuring antibiotic prescribing that did not include a clinician component, none of which were effective. However, interventions that included clinician education were generally more comprehensive, as evidenced by the greater average number of settings and modes of distribution, which could explain that difference in rates of success. Regardless, clinician education may be an effective way to spread antibiotic awareness among the community indirectly. One international intervention evaluated provider communication training and found it to be effective in lowering antibiotic prescribing for both upper and lower respiratory tract infections.  In this Streptococcal pneumoniae rates, but these changes were not sustained. [It is unclear whether these observed changes in antibiotic prescribing are influencing antibiotic resistance rates. One study measured short-term decreases in stained.  LongitudThe measured changes in prescription drug use have the potential to streamline the spending of limited healthcare funds. However, costs were not commonly studied in the analyses of these interventions, representing a general gap of knowledge that could motivate future implementation of similar programs.This systematic review has several strengths, including a rigorous search strategy developed with the assistance of a reference librarian and a highly reliable classification scheme. However, our review has several limitations. It is possible that relevant interventions were missed, though the assistance of a trained librarian in the formation of our search strategy, as well as our secondary search of all references, likely reduced this probability. Secondly, meta-analysis of intervention effectiveness was not performed due to heterogeneity among the interventions in terms of audience, messages, delivery, and outcomes. Additionally, some retrieved articles did not describe the content of the educational materials in much detail; various interventions likely included messages about appropriate antibiotic use and risks of antibiotic overuse that were not included in the calculations. Thus, the descriptive statistics for these measures likely represent an underestimate. Further, the effectiveness of educational interventions reported here is dependent on the quality of the included articles. For example, some quasi experimental studies may have poorly-matched comparison groups, though we hoped to ameliorate this by reporting descriptive statistics for the subset of randomized-controlled trials. Publication bias could also lead to the overestimation of educational intervention effectiveness.With increasing antimicrobial resistance, stimulated in part by overuse of antibiotics in healthcare settings, changing the beliefs and actions of both the lay and the medical communities is important. Previous educational programs and public messaging campaigns have resulted in substantial declines in antibiotic use and should be used as models for succeeding efforts to curb inappropriate antibiotic use and subsequent antibiotic resistance. Future interventions should consider both the lay and medical communities, and target appropriate use as well as potential adverse effects of antibiotics. Furthermore, a multifaceted approach may be most effective in changing patient perspectives and reducing antibiotic prescribing. Resources from the Centers for Disease Control and Prevention appear to be an effective and easily-implemented way to inform the lay community. Among the most effective campaigns, clinician decision-making tools and prescribing profiles are commonly employed. These methods have demonstrated the ability to reduce antibiotic prescribing both within individual healthcare organizations as well as community wide.While existing research is limited, our systematic review provides evidence that patient-directed messaging is sufficient to change antibiotic-related knowledge, attitudes, and/or beliefs. It has not yet been shown whether these changes in perspectives can affect antibiotic prescribing in the absence of medical provider intervention. Given that the most impactful campaigns have included a clinician outreach component, more research is needed to determine how we can best integrate the public awareness and clinician activation strategies in future campaigns. In addition, further research is also needed to evaluate whether the observed changes in antibiotic prescribing are sufficient to impact antibiotic susceptibility long-term.Additional file 1:Appendices A-D. Appendix A delineates the individualized search strategies for PubMed, Google Scholar, Embase, CINAHL, and Scopus. Appendix B includes article localization and article study design. Appendix C lists the antibiotic educational interventions and their associated articles. Appendix D is a table of coded intervention characteristics. (DOCX 141 kb)


Immunoglobulin G4-related disease (IgG4-RD) is the name applied to a corticosteroid and/or B-cell depletion responsive illness, in which patients present with the consequences of usually multiorgan, relapsing and remitting, fibroinflammation.Gut, Hubers et al describe a body of work that identifies the first IgG4 autoantibody against an antigen which appears to be specific to IgG4-RD (IgG4-associated cholangitis (IAC) and autoimmune pancreatitis (AIP)), at the exclusion of its major differential diagnoses: primary sclerosing cholangitis and cholangiocarcinoma.IgG4 molecules have structural and functional characteristics suggesting anti-inflammatory and tolerance-inducing effects,This work is in the context of next-generation sequencing studies—a technique that has enabled identification of immunoglobulin clones within a restricted repertoire—yielding strong evidence to support an antigen-driven process driving the pathology of IgG4-RD. An examination of circulating plasmablasts in those with active IgG4-RD has found the expanded pool of cells to have undergone class switching to IgG4, to be oligoclonally restrictedHelicobacter pylori and self  to drive disease.H. pylori infection and IgG4-RD has since been strongly disputed.Nevertheless, the number and nature (foreign or self) of the antigens that drive the disease remains a subject for ongoing study, collaboration and cross-validation. Dutch and UK patient questionnaires revealed an association with chronic exposure to industrial dusts, gases, oils, solvents and pesticides in ‘blue-collar’ professionals—though further work to elucidate candidate antigens and causality is important.Notwithstanding these shortcomings, there is compelling evidence of extant autoantigens in disease. The passive transfer of purified human immunoglobulins (IgG1 and IgG4) from people with active IgG4-RD into neonatal mice led to binding, and subsequent damage to exocrine organs .Thus, the identification of specific antibodies against a cytosolic target by Hubers is consistent with findings from others.Some caution must remain about the observations because there is a lack of validation in an external cohort, and of the 50 patients with IgG4-RD, only 9 had sera that reacted to Annexin A11—the authors have rightly not presented this as a diagnostic test. Validating this selective finding and understanding whether and how it relates to disease pathogenesis is key to appreciating the long-term impact of the work. The same group have published elegant work demonstrating dominant IgG4+ B-cell receptor clones accurately distinguish patients with IAC and AIP from primary sclerosing cholangitis and cholangiocarcinoma.The causal relationship between the observed immunoglobulin response and pathology remains another hole in our knowledge. Hubers demonstrates that IgG4 from sera of patients diminishes IgG1 binding to Annexin A11.The tolerogenic effects of IgG4 in IgG4-RD remain speculative, although they are well established in other disease settings.Gut lends a significant boost to the evidence base. How this reflects host risk continues to evolve and of note are conference reports now exploring host genetic risk, in robustly collected populations. A genome-wide association study of IgG4-RD, for example, has been performed in a Japanese population , and this reported three susceptibility loci consistent with antigen-driven disease: HLA-DRB1, HLA-A and FCGR2B, the latter encoding a low affinity receptor for IgG.Seemingly, IgG4-RD is, in part, antigen driven, and Hubers’ article in this issue of IgG4-RD is as much related to IgG4, as it is to clonally expanded B-cell populations, and an array of T cell subsets, although it is not classically preneoplastic, with plasmablast expansion being oligoclonal, not polyclonal. New technologies have increased our understanding of the changes in B-cell populations in different stages of the disease, but focus is now shifting additionally to delineating the role of T cells, in particular T follicular helper (Tfh) cells. Tfh cells help B cells and augment germinal centre development. They play a critical role in immunoglobulin somatic hypermutation and class switching of antibodies.IgG4-RD, while very rare, remains an informative disease to study. Therapeutically, it portrays an immune-mediated disease with treatment options beyond corticosteroids, thanks to a greater understanding of the underlying pathophysiology . Scienti


The average removal rates of TC and SMX were about 59% and 72%, respectively. The abundance of the main ARGs responsible for resistance to TC and SMX increased obviously after antibiotics addition, especially when TC and SMX in combination (increased 3.20-fold). The tetC and sul1 genes were the predominant genes in the development of TC and SMX resistance, in which gene sul1 had the highest abundance among all the detected ARGs. Network analysis revealed that under antibiotic pressure, the core bacterial groups carrying multiple ARGs formed and concentrated in about 20 genera such as Dechloromonas, Candidatus Accumulibacter, Aeromonas, Rubrivivax, in which intI1 played important roles in transferring various ARGs except sul3.The removal of antibiotics and widespread of antibiotic resistance genes (ARGs) have received continuous attention due to the possible threats to environment. However, little information is available on the evolution of antibiotic resistance and the relationship between ARGs and microbial communities under long-term exposure to sub-inhibitory concentrations of antibiotics. In our study, two laboratory-scale anoxic-aerobic wastewater treatment systems were established and operated for 420 days to investigate the evolution of antibiotic resistance under exposure of 5 mg·L Antibiotics are widely applied to treat infections or employed as prophylactics in human and animal medicine, among which tetracycline (TC) and sulfamethoxazole (SMX) are used most frequently ,3,4,5,6.intI1, ISCR1 and Tn916/1545) [In fact, the occurrence of antibiotics inevitably affects the functional microorganisms in activated sludge and contributes to the proliferation and dissemination of ARGs in wastewater treatment systems ,12,17,1816/1545) . Further16/1545) ,21,22.With the increasing attention on antibiotics and ARGs pollution, many studies have been found to research the impact of antibiotics on the performance of activated sludge systems, as well as the existence of ARGs in activated sludge under antibiotic exposure ,25,26,27Adding a single antibiotic is beneficial for researching under controlled conditions. TC was preferentially selected as the representative antibiotic because this broad-spectrum antibiotic was the most widely used and frequently detected antibiotic around the world ,23,30. HThe objectives of this study were to further investigate the removal of TC and SMX, the evolution of antibiotic resistance and the relationship between the main ARGs and the microbial compositions in the above A-O system. High-throughput genetic sequencing, quantitative polymerase chain reaction (qPCR) analysis, and network analysis were applied to explore the host relationship with ARGs and help clarify the mechanism of ARGs transmission between microbial communities under antibiotics selection pressure. Our report is the first comparative study about the evolution of antibiotic resistance under long-term exposure of microorganisms to sub-inhibition concentrations of a single antibiotic (TC) and combined antibiotics (TC and SMX). The results of this study will provide improved treatment guidance to reduce environmental risk for wastewater treatment plants that receive wastewater containing antibiotic residues, such as wastewater arising from hospitals and livestock farming.The evolution of antibiotic resistance was studied in an anoxic-aerobic system.The average removal rates of TC and SMX were about 59% and 72%, respectively.The ARGs abundance for TC and SMX increased significantly with antibiotic exposure.The ARGs were possibly related to 20 main bacterial genera under antibiotic pressure.intI1 played important roles in horizontal transfer of various ARGs except sul3.Gene −1 TC from day 41 to 200 and a combination of 5 mg·L−1 TC and 1 mg·L−1 SMX from day 241 to 420. Recovery period was from day 201 to 240, in which 50% of the activated sludge in both systems was exchanged with fresh sludge in order to keep the sludge states similar as much as possible. Both systems were operated at room temperature (about 25 °C) in a darkened room that excluded light and prevented photolysis of TC and SMX. The whole operational period (420 days) was divided into four phases: Phase 1 , phase 2 , phase 3 , and phase 4 .Experiments were conducted in two identical laboratory-scale A-O systems (System A and System B) as shown schematically in The intention of the study was to investigate comparatively the evolution of antibiotic resistance associated with their corresponding resistance genes when exposed to a single antibiotic (TC) and a combination of antibiotics (TC and SMX) in a lab-scale A-O wastewater treatment system. Considering that the difference of antibiotics concentration in actual wastewater treatment systems especially in hospital and livestock wastewaters, the selective antibiotics concentrations in our experiment were estimated from published reports ,32,33,34® Soil DNA Kit . The purity and yield of the DNA were determined by spectrophotometric analysis . According to the ratio of light absorbance at 260 (A260) and 280 nm (A280), the purity of nucleic acid was estimated, and the ratio of pure DNA was between 1.8 and 2.0. The extracted DNA was detected using 1% (w/v) agarose gel electrophoresis at 110 V for 40 min and the purified DNA samples were immediately stored at −20 °C until the extracts were sent for high-throughput sequencing.Sludge samples were taken on days 1, 100, 200, 240, 330, and 420 of the system operation time. The total DNA was extracted using the E.Z.N.A.−1 using methanol in a brown flask as the stock solution, which was stored at −20 °C. The calibration standards of TC  and SMX  were prepared by a serial dilution of the stock solution. The analysis of TC and SMX was performed on an Agilent 1260  HPLC system. Agilent ChemStation software integral to the HPLC was used to acquire and process data. The analytical procedures for TC and SMX were performed as previously described [Solid phase extraction and high performance liquid chromatography (HPLC) were used to extract and determine the concentration of TC and SMX in water samples according to the procedure of Wang et al. (2017) . TC and escribed ,37,38, atetA, tetC, tetL, tetO, tetW, tetX), three sulfonamide resistance genes , and the integrase gene of class 1 integrons (intI1). Primer sequences targeting these genes are listed in ® 96 qPCR System  with SYBR® Green PCR master mix  following the protocol reported previously [® Green Master Mix, 0.4 μL forward/reverse primer (10 μM), 2.0 μL template DNA, and 7.2 μL double distilled water. The procedure for qPCR amplification was set according to the kit and the thermo cycling steps were as follows: ① Preincubation: 95 °C for 300 s; ② two-step amplification for 45 cycles: 95 °C for 10 s and 60 °C for 30 s; ③ melting: 95 °C for 15 s, 60 °C for 60 s, and 95 °C for 15 s; and ④ cooling: 37 °C for 30 s. All the processing steps were carried out while the samples were on ice and all samples including the negative controls were quantified in triplicate to guarantee low technical variance.The abundances of genes were analyzed by qPCR, including six TC resistance genes .The figures were plotted using Origin Pro 9.1 software . To investigate the relationship among microbial community members and ARGs, a correlation matrix was constructed by calculating all possible pair-wise Spearman’s rank correlations using SPSS 20.0 statistical software . A −1 in the influent and effluent, respectively, with the removal rate of 58.40% when exposed to TC alone from day 41 to 200. Under exposure to combined TC and SMX (days 241–420), the average TC removal rate was 59.11% with some fluctuations, suggesting that TC removal was similar during these two conditions. However, the mean removal rate of SMX  was higher than that of TC under the combined exposure of TC and SMX, resulting in an average effluent SMX concentration of 1.07 μg·L−1 in system B.The target TC and SMX concentration in the influent and effluent were measured every 15 days from day 41 to 420 to determine the antibiotics removal capacity of system B . The avePrevious reports have indicated that the antibiotic removal mainly depends on biodegradation and adsorption in wastewater treatment systems ,42. The In this study, the similarity of mean TC removal by microorganisms in both conditions  revealed the stable sorption performance of activated sludge under long-term antibiotic pressure. The SMX removal rate was 23.23% higher than that of TC. This was probably because the functional amino group on the aromatic ring of SMX could be oxidized by autotrophic nitrifying bacteria, and some microbial communities probably utilized SMX as energy or carbon sources for growth ,44.tetA, tetC, tetL, tetO, tetW, and tetX), three sulfonamide resistance genes , and the integrase gene of class 1 integron (intI1) were determined using qPCR analysis in both systems. The relative abundances  of ARGs in different phases for system A and system B are shown in To estimate the evolution of ARGs under antibiotic pressure, the relative abundances of six TC resistance genes , two ribosomal protection protein genes (tetO and tetW), and a single enzymatic inactivation gene (tetX) [tetC > tetA > tetX > tetL > tetW > tetO. The most obvious increase occurred in the relative abundance of tetC (p < 0.05), which increased 8.64-fold from an average of 4.28 × 10−3 copies/16S rRNA (system A) to 3.69 × 10−2 copies/16S rRNA (system B) under antibiotic pressure  ,46. The pressure . Meanwhice to TC . Previouibiotics .tetX gene is a unique enzymatic inactivation gene that encodes tetracycline-degrading monooxygenase and can catalyze the inactivation of varieties of tetracyclines [tetX was 2.93 × 10−3 copies/16S rRNA, which was 1.51 times larger (p > 0.05) than the abundance in control system A (1.94 × 10−3 copies/16S rRNA). Moreover, the average abundance of other TC genes  was also higher in system B than in system A during the whole experimental period except for some individual phase. For example, compared to that in system A, the abundance of tetL decreased by 35.87% under exposure to TC alone in phase 2 (days 41–200). Certainly, under the pressure of long-term antibiotics exposure, the proliferation of TC resistance genes increased obviously. This demonstrated that antibiotics addition might promote positively the accumulation and transmission of TC resistance genes during the whole experimental period in wastewater treatment system.The cyclines . With thsul1, sul2, and sul3) all significantly (p < 0.05) increased as the system operation time increased, implying an enrichment of sulfonamide resistance genes in system B when exposed to antibiotics. The trend of relative abundances for the three sulfonamide resistance genes followed the order sul1 > sul2 > sul3, which was consistent with previous studies that showed sul1 was the most prevalent gene among sulfonamide resistance genes [sul1 had the highest average abundance among all the detected ARGs throughout the whole 420-day experiment even in the control system A where no antibiotic was added , sul2 (3.17-fold), and sul3 (1.44-fold) genes, compared to the abundancies when exposed to TC alone in system B. This implied that the sulfonamide resistance genes were mainly driven by the presence of SMX in the wastewater treatment system. A similar result showed that over the course of one year, the concentrations of ARGs encoding tetracycline, sulfamethoxazole, and erythromycin increased, and that the triple effects of antibiotics on proliferation of the ARGs were much greater than the dual effects [Moreover, exposure to the combined TC and SMX caused distinct increases of the three sulfonamide resistance genes as indicated by increases in the average abundance of the  effects . It has  effects .intI1 (one of the most common types of mobile genetic elements for ARG capture) could be considered as playing an important role in the widespread proliferation of antibiotic resistance though horizontal gene transfer. There was a slight increase of 1.18-fold (compared to the abundance in system A) for the average abundance of intI1 in system B when exposed to antibiotics. This increase was mainly due to the exposure of TC and SMX in combination, which increased the abundance of intI1 to 2.50 times than in system A. Exposure to TC alone dramatically decreased the abundance of intI1 by 76.35% in the system from day 41 to 200  reported that the abundance of intI1 increased by 0.6 orders of magnitude after SMX and TC were added [intI1 was reported under pressure of TC [intI1 significantly decreased by 1.005 log in reactors which tetracyclines and sulfonamides were added [The existence of 1 to 200 . The evore added . Similarre of TC , the comre of TC , and there of TC . Howeverre added .intI1, while the combined exposure to TC and SMX stimulated the proliferation of intI1 through horizontal gene transfer. In particular, intI1 exhibited significant correlation with sul1 and sul2, especially for sul1, which may serve as a partial structure in the 3′-conserved segment region of the class 1 integron [intI1.Results from the present study indicated that exposure to a single antibiotic (TC) had only a limited effect on the spread of integron ,50. Thissul1 was the most abundant gene with average abundances of 1.42 × 10−2 copies/16S rRNA (system A) and 1.92 × 10−2 copies/16S rRNA (system B). In phase 2 (days 41–200), the total abundance of ARGs increased about 2.02-fold compared to that in system A. One of the tetracycline efflux pump genes tetA increased remarkably by 4.28-fold from 6.15 × 10−3 to 2.63 × 10−2 copies/16S rRNA in system B. However, the abundances of other tetracycline ARGs were stable or even decreased when microbes were exposed to TC alone, indicating that tetA was the predominant gene in the development of TC resistance. It is noteworthy that the sul1 gene was detected as the dominant ARG in both system A and system B even though there was no sulfonamides exposure during this phase; therefore, sul1 had already been widely distributed in the activated sludge.The total abundance of ARGs was also estimated at different phases to further elucidate the evolution of antibiotics resistance. During the initial phase , the abundances of ARGs were similar in both systems and tetA and sul2) in system B exhibited no such decrease or delay, even though the pressure of antibiotics had been removed for more than one month. The combined exposure to TC and SMX in system B during phase 4 (days 241–420) greatly stimulated propagation and transmission of ARGs with a 3.20-fold increase, compared to the abundances in system A, in which relatively high abundances of tetC and sul1 were also observed. These results suggested that compared to ribosomal protection protein genes (tetO and tetW) or the enzymatic inactivation gene (tetX) in this study, the efflux pump genes (tetA and tetC) were more active in the propagation and transmission for TC resistance genes under antibiotic pressure. For sulfonamide resistance, sul1 was consistently a predominant gene in the presence or absence of antibiotic pressure, while sul2 was obviously induced by the exposure of SMX. These differences should receive more attention, especially during treatment of livestock farming wastewater and hospital wastewater containing high concentrations of antibiotics, because the transferred resistance might pose substantial risks to the environment.During the recovery phase (days 201–240), the abundances of the detected ARGs substantially decreased in system A, while the propagation of most ARGs . The top 50 genera in each sample were selected for comparative analysis with TC and sulfonamide resistance genes, as well as the intI1 gene. As shown in The relative abundances of the dominant genera were listed in order to understand the evaluation of microbial community structure more clearly when it was exposed to antibiotics in the A-O wastewater treatment systems on day 1, 100, 200, 240, 330, and 420 . NetworktetC and sul1), as well as the only enzymatic inactivation gene (tetX) were mainly associated with about 25 genera (the purple module in Ferruginibacter (3.09%), Sediminibacterium (2.80%), Candidatus Accumulibacter (2.74%), Haliscomenobacter (2.67%), Rhodobacter (1.94%), and Runella (1.56%), etc. of which, 14 of the 25 genera were also closely related with tetA, tetO, tetW, sul1, and sul2. These results suggested that bacterial genera already carried more than one antibiotic resistance gene. Another module included 15 possible host bacterial genera (the red module in intI1 and tetL. This group included Dechloromonas (2.29%), Phaeodactylibacter (1.17%), Aeromonas (0.62%), Rubellimicrobium (0.61%), Streptococcus (0.48%), and Hydrogenophaga (0.44%)  .Dechloromonas (11.58%), Candidatus Accumulibacter (4.99%), Aeromonas (2.41%), Rubrivivax (1.07%), Roseomonas (1.06%), and Rhodobacter (0.36%), etc. of which 20 were simultaneously related with all the detected tetracycline resistance genes  and the most abundant sulfonamide resistance gene (sul1). Nine genera in particular  were also found to have relationships with sul2 and int1 under antibiotics pressure. These bacteria possibly carried multiple antibiotic resistance and played major roles in the spread of ARGs. Compared to the 14 possible hosts carrying multiple ARGs in system A, only five genera  appeared in both systems A and B. The other 15 genera most likely were newly developed genera and possibly carried multiple ARGs under the pressure of TC and SMX, such as Candidatus Accumulibacter, Rubrivivax, Rheinheimera, Haliscomenobacter, and Runella, etc.As shown in Dechloromonas, Candidatus Accumulibacter, and Rhodobacter were reported as the global core bacterial community in 1200 samples taken from 269 wastewater treatment plants in more than 20 countries [Dechloromonas and Candidatus Accumulibacter were also reported to be related with tetA and sul1 under the pressure of tetracycline and sulfonamide [Rhodobacter has been known for its excellent adaptability to different living environment and this genus was reported to contain the sul1 gene [Dechloromonas (13.47-fold) and Candidatus Accumulibacter (4.80-fold) compared to their abundance in system A , Candidatus Accumulibacter (4.99%), Ferruginibacter (2.41%), Pseudomonas (2.36%), Flavobacterium (2.25%), Runella (1.94%), and Thauera (0.73%) all increased. Moreover, the sul3 gene was correlated with Dechloromonas (11.58%), Roseomonas (1.06%), Sphaerotilus (0.93%), Sphingopyxis (0.49%), Filimonas (0.37%), and Rhodobacter (0.36%) (The second group included 12 genera (the green module in  (0.36%) .intI1 integron is regarded as the most important and sensitive indicator of bacteria’s ability to incorporate exogenous gene cassettes [intI1 also showed a close relationship with other detected ARGs, except for sul3. This indicated that intI1 was an important component in the widespread of ARGs. The co-occurrence associations among these ARGs  and their potential bacterial hosts may result in the propagation and transmission of antibiotic resistance through vertical and horizontal gene transfer under antibiotics pressure, thereby facilitating the transfer of antibiotics resistance more widely among bacteria.The assettes . Most geThiothrix and Sphaerotilus are both well-known filamentous bacteria that are involved in sludge bulking, and have been validated as the main bulking causative bacteria in the type of A-O wastewater treatment system examined in this study [sul2 gene showed an obvious tendency to increase when exposed to antibiotics in system B, which was possibly related to the higher percentage of Thiothrix that resulted from the exposure of microorganisms to TC and SMX  and Pseudomonas (0.95%) on day 420 in system B , indicating that these genera were not able to endure the biotoxicity of the four antibiotics [e of SMX ,56,57. Hsystem B . Other ribiotics ,26,27,46The relationships between microbes and ARGs under antibiotic pressure were obviously shown by the network analysis. However, these relationships must be further validated using other approaches, such as culture-dependent methods, metagenomics sequencing, and gene chip technology ,58. FurtintI1 had extensive relationships with these bacterial genera. Although further research using other approaches (such as culture-dependent methods) was required to validate the relationships between ARGs and specific bacterial groups, this is the first report about the evolution relationship between antibiotic resistance and microbial community composition in a wastewater treatment system under long-term exposure of sub-inhibition concentrations of combined antibiotics. Considering the antibiotic contaminant facts in most wastewater treatment plants, the influence of lower concentrations of antibiotics exposure, more types of antibiotic combinations, or even the antibiotic metabolites on the evolution of antibiotic resistance genes should be further investigated and clarified in the future research.In conclusion, antibiotics such as TC and SMX could be partially removed or degraded in the A-O wastewater treatment system. Exposure of sub-inhibition concentrations of antibiotics, especially combinations of different antibiotics, could substantially increase the corresponding ARGs. However, with the antibiotic exposure, the ARGs seemed to be more concentrated in some specific core bacterial genera resulting in production and evolution of multiple antibiotic resistant bacteria, in which the