Monitoring the appropriateness of antibiotic prescriptions with indicators based on reimbursement data is required to guide antibiotic stewardship (AMS) interventions in nursing homes (NHs). Quantity metrics (QMs) monitor the volume of prescriptions while proxy indicators (PIs) reflect the appropriateness of antibiotic use. Our objectives were: (i) to provide a relevant consensual set of indicators to be used in French NHs; and (ii) to assess the feasibility of their implementation at the national and local scale.Nine French professional organizations implicated in AMS in NHs were asked to nominate at least one member to create a national expert panel of 20 physicians. Twenty-one recently published QMs and 11 PIs were assessed by the expert panel. Indicators were evaluated using a RAND-modified Delphi procedure comprising two online surveys and a videoconference meeting. Indicators were kept in the final list if >70% of stakeholders validated their relevance for estimating the volume (QMs) and appropriateness (PIs) of prescriptions.n = 3), broad-spectrum (n = 6) and second-line antibiotics (n = 2). The three remaining QMs evaluated the route of administration (n = 1) and urine culture prescriptions (n = 2). Ten PIs  were selected to assess the appropriateness of prescriptions for urinary tract infections (n = 2), seasonal variations in prescriptions (n = 2), repeated prescriptions of fluoroquinolones (n = 1), cephalosporins’ route of administration (n = 1), duration of treatment (n = 1), rate of second-line antibiotics (n = 1), co-prescriptions with non-steroidal anti-inflammatory drugs (n = 1), and flu vaccine coverage (n = 1). The panel was in favour of using these indicators for regional and facility level AMS programmes (91%), feedback to NH prescribers (82%), benchmarking by health authorities (55%) and public reporting at the facility level (9%).Of the 21 QM indicators submitted to the panel, 14 were selected, describing the consumption of antibiotics overall (This consensual list of indicators, covering a wide range of frequent clinical situations, may be used as part of the French national AMS strategy for monitoring antibiotic prescriptions in NHs at the national and local levels. Regional AMS networks might manage this selected list to guide personalized action plans with concrete objectives of reducing the quantity and improving the quality of antibiotic prescriptions. Two types of indicators are currently used, quantity metrics (QMs) and proxy indicators (PIs). QMs measure the volume of antibiotic delivery and can be easily calculated, but they give poor indication about possible misuse of antibiotics. On the other hand, PIs can reflect the appropriateness of antibiotic prescriptions in the absence of medical data available, and provide concrete targets for improvement.In France, each NH’s resident has their own GP, and antibiotic prescriptions are mostly delivered by community-based pharmacy, since >80% of NHs don’t have on-site pharmacy.The aim of the present study was to select relevant indicators, adapt the definition of the QMs and PIs if necessary, and select the targets for PIs using a Delphi process involving a national expert panel. Finally, we assessed the feasibility and explored the potential use of these indicators in a national AMS monitoring programme.16 in France, most NHs (>80%) are community-based, without in-house pharmacies. In such settings, antibiotics are prescribed by GPs, dispensed by community pharmacies, and reimbursed by the National Health Insurance (NHI). Different residents accommodated in the same NH can have different GPs. The indicators used in the present study for AMS in NHs were based on the NHI reimbursement database (SNDS). This database includes the following information for all prescriptions: age and gender of the patient; identification of the prescriber; information on the antibiotic dispensed; and the facility identification in case of NH residency. Information on diagnoses and the duration of treatment in days is not available in the database. However, the number of packages dispensed is collected. The NHI database does not include hospital-based NHs (with on-site antibiotic delivery), representing less than 20% of French NHs. For each indicator, individual prescriptions were aggregated at the NH level. QMs estimated the volume of antibiotic consumption per resident-days, using number of prescriptions and/or DDDs, with a numerator–denominator combination. PIs estimated the appropriateness of antibiotic prescriptions at the NH level and were associated with targets reflecting compliance with national guidelines.As detailed previously,In November 2020, nine French professional organizations were asked by e-mail to nominate at least one member to take part in the expert panel, favouring people with demonstrated experience and knowledge in the field of AMS in NHs.18 was used to validate a set of indicators for AMS in NHs. Considering the lack of evidence-based medicine in the field of indicators for AMS, RAND studies can be useful to combine the best available scientific data with the collective statement of experts, in order to yield a consensual list of indicators.17 Theses procedures have the advantage to provide: (i) iteration, permitting experts to change their opinion between the different rounds; (ii) controlled feedback, by communicating the results of previous rounds; and (iii) high-quality statistical data, assuring that perspectives of each expert are equally represented in the final set.19 Our study comprised two online surveys separated by a videoconference meeting. For the first round of the consensus procedure, we used an initial list of 21 QMs and 11 PIs, recently published by Simon et al.,16 based on a systematic literature review, including DRIVE-AB (European project Driving Re-InVEstment in R&D and responsible AntiBiotic use) results,20 and adapted to the French NH context. This list of indicators was converted into a digital web-based questionnaire using Google Forms® (JAC-AMR Online). The expert panel received by e-mail an invitation to complete the online survey, accompanied by an explanatory document . From the outset, experts were informed that the conference was recorded for audio transcription.During the third round, a summary report containing all changes validated during the videoconference was sent to the expert panel. Experts were asked to return free-form comments on the report and to suggest final modifications to the indicators, particularly for those that were newly added.22The fourth round relied on a second survey to assess the updated list of indicators. First, for each individual indicator, experts had to state whether they would retain it in the final list or not. Indicators were retained if >70% of experts validated their relevance for estimating the volume (QMs) and appropriateness (PIs) of antibiotic prescriptions, 70% being the threshold usually selected in the literature for RAND studies.16The final list of indicators was tested using the NHI database in two different French areas for the year 2019. We included all community-based NHs in the Pays de la Loire and Grand Est regions (respectively 3 787  000 and 2 338 000 inhabitants) that were active in 2019. The calculation of QMs and PIs was performed according to the method described elsewhere.On an exploratory basis, experts were secondly asked to give their opinion regarding four possible uses of the indicators: (i) comparisons between NHs in a benchmarking process; (ii) design of AMS programmes at the facility level; (iii) feedback to NH prescribers; and (iv) public reporting of facility-level indicators.To calculate the set of indicators, we used the National Health Data System (SNDS), a strictly anonymous database, comprising all mandatory national health insurance reimbursement data. No informed consent was required because data were anonymized.A panel of 20 experts from nine different organizations was created for the structured consensus procedure. Of these, five were AMS physicians, four NH physicians, three infectious diseases specialists, two geriatricians, two infection control specialists, two policymakers, one GP and one pharmacist. Table The first round was conducted from December 2020 to January 2021. Of the 21 potential QMs Table , 7 were 23 Only one PI, related to co-prescriptions of antibiotics with corticosteroid, was removed to consider clinical situations requiring theses associations (i.e. exacerbated COPD). Three additional PIs were suggested by the panel: two PIs related to the route of antibiotic administration  and one PI capturing the switch in antibiotic treatment based on antimicrobial susceptibility testing in urinary tract infections.The videoconference meeting took place on 22 February 2021. During this stage, five QMs were rejected from the initial list. The other QMs were retained without modification. Of the PIs, four were kept while six underwent substantial changes to the numerator, denominator or target. Regarding the PI related to the duration of antibiotic prescriptions, the definition of ‘prolonged antibiotic course’ was modified from >8 to >7 days, to comply with recent guidelines and AMS programmes.In April 2021, the summary report of the videoconference meeting was sent for review, comments and amendment. After reading the report, experts validated the modification made during the second round: suppression of five QMs and one PI, and the amendments made to six PIs. Another modification was made to one PI during this round. Of the three PIs added in the second round, the experts decided to remove the one related to the prescription of parenteral quinolones due to insufficient room for improvement. The PI reflecting the switch in antibiotic treatment for urinary tract infections was also rejected due to poor clinical meaning.The fourth round was completed between May and June 2021. A total of 27 potential QIs (16 QMs and 11 PIs) were included in the second online questionnaire. Two QMs that did not achieve the 70% positive response rate were rejected, with a mean consensus score of 63.6%. One PI reflecting prescriptions of antibiotics that are not indicated was also rejected, with a score of 63.6%. The mean consensus score for the remaining indicators was 90.3% for the final list of QMs and PIs. The final set of indicators was composed of 14 QMs and 10 Pis, presented in Tables The indicators were assessed using the NHI databases from two French administrative areas: the Grand Est and the Pays de la Loire regions. In 2019, there were 417 NHs in the Grand Est and 443 in the Pays de la Loire regions. The mean number (±SD) of residents per NH was 91.5 ± 36.2 in Grand Est and 93.8 ± 32.2 in Pays de la Loire. The mean age of residents was, respectively, 87.3 ± 7.3 and 88.3 ± 1.8 years, and 75.2% and 74.6% were women. QM and PI results (median and IQR), for the year 2019 in both areas, are presented in Tables On an exploratory basis, the 20 experts were asked to give their opinion regarding possible uses of the indicators, and 11 completed the survey. Most of the experts who answered (91%) were in favour of sharing the indicators within the French regional AMS network for local programmes at the facility level  was lowered to >7 days. This choice was made to be in accordance with the guidelines, provide a more understandable message for AMS programmes, and consider the recent actualization of French guidelines for antibiotic duration,23 which reduced treatment courses to 5 days in some upper respiratory tract infections. Increased antibiotic prescription during winter is a reality, especially for respiratory tract infections,25 which are more frequent in winter but mostly viral.27This list of indicators includes a wide range of frequent clinical situations in NHs, from overuse to misuse of antibiotics. The overuse of antibiotics was approached by 14 indicators, 11 QMs and 3 PIs. Monitoring the prescription of urine cultures is a way of addressing the treatment of asymptomatic bacteriuria. Regarding upper respiratory tract infections in primary care, 80% of treatment courses may exceed the recommended duration.28 repeated prescriptions of quinolones within a 6 month period29 and the choice of drugs for first-line regimens have all been associated with an increased risk of AMR. An indicator related to flu vaccination falls outside the scope of antibiotic reimbursement, but is also available in SNDS database, and is indirectly correlated with antibiotic prescriptions, since flu vaccination might prevent one in 25 antibiotic prescriptions among acute respiratory infections during the influenza season.30 Monitoring and benchmarking these practices may lead to improved use of antibiotics during winter.One QM and five PIs focused on the misuse of antibiotics. Beyond the monitoring of broad-spectrum antibiotics, it is essential that the duration, route of administration and recurrence of prescriptions be addressed. The use of oral cephalosporins that may generate suboptimal concentrations,31 The experts were asked to give their opinion about possible uses for the indicators. Unfortunately, only 11 of the 20 experts completed the survey, so the results have to be taken with caution. Regarding PIs, 91% of our experts supported transmitting them to regional AMS networks for local programmes at the facility level. Several stakeholders, such as the Regional and National Health Insurance organization and the regional antibiotic stewardship networks, might be able to use these data to help NHs develop personalized action plans, with concrete objectives and a monitoring plan. Moreover, 82% were inclined to transmit them to NH prescribers. Almost real-time personalized feedback for prescribers, or integration into a pay-for-performance mechanism, which exists in France for GPs at the individual level but not collectively at the NH level, are strategies worth considering in coming years. Only 55% of experts voted for transmission to Regional Health Authorities for benchmarking purposes. One reason lies in France’s hierarchical health governance, with Regional Health Authorities appearing to be organizations responsible more for regulations, inspections and controls than advice.32 Despite common transparent approaches open to public scrutiny in France, only 9% of experts supported public reporting of the indicators. Public reporting has been associated with increased quality-improvement activity.33 However, this strategy is exposed to the risk of the indicators reported being misunderstood by the public or media.This list of indicators developed and validated for French NHs is easy to generate from NHI databases and may be of use at the international scale for benchmarking purposes. These outputs will contribute to the national monitoring of antibiotic use in French NHs, in line with the 2022–25 national strategy for preventing antibiotic resistance.In this study, 14 QMs and 10 PIs based on the reimbursement database were selected and validated to monitor the use of antibiotics in French NHs. This consensual list of indicators may improve practices at the local, regional and national scale.dlad037_Supplementary_DataClick here for additional data file.


The steel box arch bridge in this study will be subjected to various temperature effects from the construction to the operation stage, including the cement hydration heat effect and the sunshine temperature effect caused by an ambient temperature change. Therefore, it is very important to control the temperature effect of steel box arch bridges. In this study, the newly built Dafeng River Bridge is selected as the steel box arch bridge. This study aims to investigate the temperature effect including hydration heat and the sunshine temperature effect of the construction process of a rigid frame-tied steel box arch bridge. The manuscript presents that the heat dissipation performance of concrete decreases with the increase in the thickness of a mass concrete structure. The average maximum temperature values of layer No. 3 are about 1.3, 1.2, and 1.1 times the average maximum temperature value of layer No. 1 for the mass concrete of the cushion cap, main pier and arch abutment, respectively. The higher the molding temperature is, the higher the maximum temperature by the hydration heat effect is. With each 5 °C increase in the molding temperature, the maximum temperature at the core area increases by about 4~5 °C for the mass concrete. The pipe cooling method is conducive to the hydration heat control effect of mass concrete. Based on the monitored temperature change and displacement change, the influences of daily temperature change on the steel lattice beam and arch rib are analyzed. A temperature rise will cause the structure to have a certain camber in the longitudinal direction, and the longitudinal or transverse displacement caused by the sunshine temperature change is no less than the vertical displacement. Due to the symmetrical construction on both sides of the river, the arch rib deformation on both sides presents symmetrical synchronous changes. Based on 84 h of continuous temperature monitoring on-site, the changing trends of the arch back temperature and ambient temperature are consistent and their difference is small during 1:00~4:00 in the morning, which is determined as the appropriate closure time for the newly built Dafeng River Bridge. In recent decades, with the continuous development of highway and bridge construction in China, the number of bridges built has also increased with different spans and types . The steMass concrete structures first appeared in water conservancy and hydropower engineering, and there are a series of specifications from the temperature field theory to construction methods and temperature and crack control. With the increasing application of mass concrete in engineering, the relevant research is also more comprehensive and in-depth. Lawrence et al.  used numA temperature change will make the structure show the deformation trend of “thermal expansion and cold contraction”. However, for statically indeterminate structures, because the free deformation is subject to boundary conditions, there will be great temperature stress in the structure, which may bring risks to the construction and use of the bridge structure ,21,22. IMost authors of the previous literature have conducted research on mass concrete at a certain location on bridges. Although a large number of temperature effect studies have been conducted, there is a lack of related applications of decision-making in actual bridge construction. Aiming at closing the above gap, this study investigates the cement hydration heat effect on the cushion cap, main pier and arch abutment and the appropriate closure timing based on the sunshine temperature effect in the construction process of the Dafeng River steel box arch bridge. For the mass concrete in the cushion cap, main pier and arch abutment, the pipe cooling method is used to control the hydration heat of cement, and the hydration heat control effect of mass concrete is evaluated based on the arrangement and monitoring results of temperature monitoring points. According to the collected temperature change and displacement change, the influences of daily temperature change on the steel lattice beam and arch rib are analyzed and identified, and the appropriate closure temperature of the arch back is determined for the newly built Dafeng River Bridge.Dafeng River Bridge, located in Qinzhou City, Guangxi, as the main bridge in the Qinzhou–Beihai section of the Lanhai Expressway, crosses the Dafeng River at the bend of the river. The design scheme of the bridge is to demolish and rebuild the upper part and bent cap of the existing bridge without changing its existing navigation, which will be used as the left part of the whole bridge after reconstruction. The newly built Dafeng River Bridge  will be built on the downstream side. The appearance of the reconstructed whole bridge is shown in The newly built Dafeng River Bridge will be a rigid frame-tied steel box arch bridge, with a clear span of 120.0 m, a clear rise of 27.0 m, and a clear rise to span ratio of 1/4.44. As shown in the elevation layout in sφ15.24 will be adopted as a suspender and tie rod.Before construction, various preparations shall be carried out, such as site cleaning. Then, the approach bridge construction will be carried out, and the main bridge pile foundation, cushion cap, main pier and arch rib arch pedestal will be constructed at the same time. Then, the temporary construction trestle of the main bridge will be constructed. The trestle is a steel pipe pile and has a Bailey beam structure. The arch rib section shall be installed in sections to construct the main arch until the construction of the main arch rib and the transverse brace is completed and the closure of the main arch is completed. The supporting support system of the main arch will be dismantled symmetrically and orderly, and the tie rods N2, N3, N10 and N11 will be tensioned at the same time. Then, the arch rib suspenders N4~N11, and the tie rods N5, N8, N13 and N16 will be tensioned. Then, the arch rib suspenders N1–N3 and N12–N14, and tie rods N6, N7, N14 and N15 will be tensioned. Finally, the temporary trestle will be dismantled orderly and symmetrically, the tie rods N2~N8 and N10~N16 will be tensioned, and the construction of the whole bridge will be completed. The tie rods will be tensioned two times. The first tensioning control force will 1400 kN, the second tensioning control force of tie rods N2~N8 will 2200 kN, and the tensioning control force of tie rods N10~N16 will be 2250 kN. For the main material parameters of the newly built Dafeng River Bridge, Q355C will be used as an arch rib, transverse brace, and lattice beam, and Q235C will be used as a steel deck. C40, C35 and C50 will be used for the main pier, deck, sidewalk and access slab, cushion cap concrete and arch abutment concrete. The length, width and height of the cushion cap of the newly built Dafeng River Bridge will be 13.2 m, 8.4 m and 3.0 m, respectively. The length, width and height of the main pier will be 8.5 m, 4.4 m and 11.0 m, respectively, of which the length, width and height of the lower section will be 2.5 m, 4.4 m and 6.0 m, respectively, and the length, width and height of the upper section will be 8.5 m, 4.4 m and 5.0 m, respectively. The length, width and height of the arch abutment will be 7.5 m, 3.4 m and 4.923 m, respectively. Therefore, the cushion cap, main pier including the lower and upper sections, and arch abutment will be made of typical mass concrete.The temperature problem of the hydration heat of mass concrete is very complex, and the external temperature, construction conditions and raw material changes will cause temperature changes, which could be more accurately understood and grasped only through monitoring. During the performance of mass concrete temperature control, in order to test the construction quality and temperature control effect, the temperature control measures will be adjusted in a timely manner and improved by mastering the temperature control information. During the concrete pouring process, it will be necessary to monitor the concrete pouring temperature. During the curing process, it will also be necessary to monitor the temperature rise and fall of the concrete pouring block, the temperature difference between the inside and outside, the cooling rate and the ambient temperature. In this study, the temperature monitoring system of mass concrete is composed of a temperature sensor, data acquisition system and data transmission system. The monitoring system has the functions of displaying, storing and processing temperature and time parameters, and can draw the temperature change curve of measuring points in real-time. The number of temperature measuring points should not be less than 50. The allowable error of the temperature monitoring system shall not be greater than 0.5 °C. The test range of temperature shall be −30 °C~125 °C. Before the installation of a temperature sensor, it shall be soaked 1 m underwater for 24 h together with the transmission wire without damage. In this study, the pipe cooling method was adopted to control the thermal cracking of the massive concrete structures of the newly built rigid frame-tied steel box arch bridge. The digital temperature sensor and wireless temperature collector shown in  are usedmoldingT) shall be measured at least twice per shift. The maximum temperature of concrete in the temperature measuring area is defined as the maximum temperature of concrete (maxT). The concrete surface temperature (surfT) refers to the temperature at 50 mm from the concrete outer surface. The concrete bottom temperature (botT) refers to the temperature at 50 mm from the bottom of the concrete pouring block. The difference between the maximum temperature of concrete and the surface temperature is defined as the temperature difference between the surface and interior of the concrete (ΔsurfTint-). The temperature rise peak (TRP) is the highest temperature rise inside the concrete pouring body. The cooling rate (CR) refers to the temperature reduction value every day or every 4 h after the internal temperature of the concrete reaches the peak temperature under the condition of heat dissipation. The temperature value at the shady ventilation outside the structure is the concrete ambient temperature (airT).In the process of temperature measurement, the following test items need to be defined. At the same time, the layout of measuring points is closely related to that of the test items. The temperature of the concrete mixture when pouring it into the mold Arrangement of Temperature Monitoring Points and Cooling PipeDuring the construction process of the project, a detailed temperature monitoring scheme of hydration heat will be developed to determine a comprehensive temperature measuring point arrangement, and the hydration heat temperature of mass concrete will be monitored in real-time to guide the actual construction. (b)Analysis of Hydration Heat and Cracking Based on Temperature MonitoringmoldingT), temperature rise peak (TRP), the maximum temperature of concrete (maxT), maximum cooling rate (maxCR), the maximum temperature difference between the surface and interior (ΔsurfTint-max-), the maximum temperature difference between the concrete surface and air (ΔsurfTair-max-), and the maximum temperature when monitoring is stopped (maxTstop-) are selected for the statistics of the temperature monitoring of a mass concrete cushion cap.According to the requirements of the Chinese specification “Technical Code for Temperature Measurement and Control of Mass Concrete” (GB/T 51028-2015) , the molmoldingT is strictly controlled at about 35 °C in this study, and the moldingT value of each layer is basically stable, in which the relatively low moldingT value also avoids an accelerating hydration heat reaction. The TRP value is the difference between the maxT and moldingT values; therefore, the TRP and maxT show a similar change trend, increasing first and then decreasing. This is because the hydration heat reaction of layered concrete is a cumulative process, and the TRP increases with the construction’s progress, leading to an increase in the maxT. Generally, the temperature of the core area of layer No. 3  of layered concrete is higher than that of other layers, gradually decreasing to the outward surfaces. The heat dissipation performance of concrete decreases with the increase in the thickness of the mass concrete structure, and the layered pouring of concrete has little impact on the temperature change of the mass concrete. For the mass concrete cushion cap at four different locatitons, the average maxT value of layer No. 3 is about 1.3 times the average maxT value of layer No. 1. At the same time, the TRP value is less than 45 °C. For the pouring of mass concrete, due to the large volume of concrete, the temperature generated by the hydration heat effect in the concrete increases rapidly, while the heat dissipation is slow, resulting in a high temperature inside the concrete and a large temperature difference between the surface and the inside of the concrete, which makes it very easy for concrete cracking to occur. In this study, it could be found that the temperature difference between the surface and interior (ΔsurfTint-) and the temperature difference between the concrete’s surface and air (ΔsurfTair-) will be within the control range. With the help of the cooling pipe layout of the cushion cap, the cooling rate inside the mass concrete will be well controlled. The pipe cooling method is conducive to the hydration heat control effect of mass concrete, and can effectively reduce the central temperature of concrete structures. The cooling rate of mass concrete fluctuates greatly due to many influencing factors. Due to the strong heat dissipation capacity of the surface layer, the cooling rate of layer No. 5 is much higher than that of the other layers. The maxT value of layer No. 5 decreases by an average of 9% compared to the lowest maxT value of the other layers. When the monitoring is stopped, the maximum temperature (maxTstop-) of each layer will be basically near the molding temperature (moldingT). At the same time, the difference between the maximum temperature of concrete and the minimum temperature of the environment for three consecutive days can be controlled at 20 °C when the monitoring is stopped. By performing temperature control through cooling water, there will be no visible cracks in the mass concrete of the cushion cap at the upstream and downstream of the Nanning and Beihai sides.(c)Arrangement of Temperature Monitoring Points and Cooling PipeTemperature monitoring points will be arranged at the halfway point of the symmetry axis in the longitudinal and transverse directions of the main pier concrete, as shown in (d)Analysis of Hydration Heat and Cracking Based on Temperature MonitoringmoldingT of the lower and upper main piers was strictly controlled below 35 °C in this study, and the moldingT value of each layer was basically stable, in which the relatively low moldingT value also avoided accelerating the action of hydration heat after pouring. According to the comparative temperature analysis of the upstream and downstream with different moldingT values, it can be seen that the ambient temperature of concrete has a certain influence on its temperature field. The higher the molding temperature is, the higher the maximum temperature value by the hydration heat effect is. With each 5 °C increase in the moldingT value, the maxT value at the core area of layer No. 3 increased by about 4.5 °C for the mass concrete in the lower main pier, and the maxT value of the core area of layer No. 3 could even be raisedx by about 5.1 °C for the mass concrete in the upper main pier. Considering the stable moldingT value and TRP = (maxT − moldingT), the TRP and maxT showed basically the same change trend, increasing first and then decreasing. This is because the hydration heat reaction of layered concrete is a cumulative process, and the TRP increases with the construction progress, leading to the increase in maxT. The temperature of the core area at layer No. 3  of layered concrete was higher than that of the other layers, gradually decreasing to the outward surfaces. For the four different locations, the average maxT value of layer No. 3 was about 1.2 times the average maxT value of layer No. 1 for the lower main pier. Meanwhile, the maximum TRP value was less than 55 °C and its average value was less than 45 °C. For the pouring of mass concrete, due to the large volume of concrete, the temperature generated by the hydration heat effect in the concrete increased rapidly, while the heat dissipation was slow, resulting in a high temperature inside the concrete and a large temperature difference between the surface and the inside of the concrete, which very easily results in concrete cracking. In this study, it could be found that the temperature difference between surface and interior (ΔsurfTint-) and temperature difference between concrete surface and air (ΔsurfTair-) were within the control range. The average value of the ΔsurfTint- values for all layers was controlled at less than 30 °C. With the help of the cooling pipe layout of the main pier, the cooling rate inside the mass concrete was well-controlled. There were little significant increases in the maxT and TRP values of layer No. 3, indicating that the pipe cooling method is conducive to the hydration heat control effect of mass concrete, and can effectively reduce the central temperature of concrete structures. The cooling rate of mass concrete fluctuates greatly due to many influencing factors. Due to the strong heat dissipation capacity of the surface layer, the cooling rate of layer No. 5 was much higher than that of the other layers. For the lower main pier, the maxT value of layer No. 5 decreased by an average of 9% compared to the lowest maxT value of the other layers. For the upper main pier, the maxT value of layer No. 5 decreasesd by an average of about 19% compared to the lowest maxT value of the other layers. When the monitoring is stopped, the maximum temperature (maxTstop-) of each layer woill be basically lower or near the molding temperature (moldingT). At the same time, the difference between the maximum temperature of concrete and the minimum temperature of the environment for three consecutive days can be controlled at 20 °C when the monitoring is stopped. By performing temperature control through cooling water, there will be no visible cracks in the mass concrete in the lower upper main piers at the upstream and downstream of Nanning and Beihai sides.(e)Arrangement of Temperature Monitoring Points and Cooling PipeTemperature monitoring points will be arranged at the halfway point of the symmetry axis in the longitudinal and transverse directions of the concrete of the arch abutment constructed first, as shown in (f)Analysis of Hydration Heat and Cracking Based on Temperature MonitoringmoldingT of the arch abutment was controlled at about 30 °C in this study, and the moldingT value of each layer was basically stable, in which the relatively low moldingT value also avoided accelerating the action of the hydration heat after pouring. Considering the stable moldingT value and TRP = (maxT − moldingT), the TRP and maxT showed basically the same change trend, increasing first and then decreasing. This is because the hydration heat reaction of layered concrete is a cumulative process, and the TRP increases with the construction’s progress, leading to the increase in the maxT. Generally, the temperature of layer No. 3  of the layered concrete was higher than that of other layers. For the four different locations, the average maxT value of layer No. 3 was about 1.1 times the average maxT value of layer No. 1. At the same time, the maximum TRP value was less than 55 °C and its average value was less than 45 °C. For the pouring of mass concrete, due to the large volume of concrete, the temperature generated by the hydration heat effect in the concrete increased rapidly, while the heat dissipation was slow, resulting in a high temperature inside the concrete and a large temperature difference between the surface and the inside of the concrete, which very easily causes concrete cracking. In this study, it Was found that the temperature difference between the surface and interior (ΔsurfTint-) and the temperature difference between the concrete’s surface and air (ΔsurfTair-) were within the control range. With the help of the cooling pipe layout of the arch abutment, the cooling rate inside the mass concrete was well-controlled. The cooling rate of mass concrete fluctuated greatly due to many influencing factors. When the monitoring is stopped, the maximum temperature (maxTstop-) of each layer will be basically lower or near the molding temperature (moldingT). At the same time, the difference between the maximum temperature of concrete and the minimum temperature of the environment for three consecutive days can be controlled at 20 °C when the monitoring is stopped. By performing temperature control through cooling water, there will be no visible cracks in the mass concrete in the arch abutment at the upstream and downstream of the Nanning and Beihai sides.The bridge structure is located in the outdoor environment, and its internal temperature field is affected by many factors such as solar radiation, ambient temperature, etc. Due to the good thermal conductivity of steel, steel box arch bridges are more sensitive to the ambient temperature compared to ordinary reinforced concrete bridges. Ambient temperature change includes seasonal temperature change and daily temperature change. The influence of seasonal temperature changes on a structure is relatively simple, and the change is uniform. However, daily temperature change is more complex, especially under the effect of sunlight, which causes temperature differences, thus causing the displacement of the structure, and also produces the temperature’s secondary internal force for a statically indeterminate structure. The positioning of the lattice beam is the foundation of the arch rib’s assembly, and it is important to analyze the influence of temperature on the displacement of the lattice beam in construction monitoring, as it directly affects the assembly accuracy of the arch rib and the verticality of the suspender.Continuous temperature and deformation monitoring of the lattice beam was carried out to study the relationship between the position change of the suspender beam for the lattice beam and the temperature change in the outdoor environment. On the day of monitoring, it was sunny with a certain temperature difference between morning and noon. The detailed temperature–time curve relationship is shown in As one of the most important factors affecting the deflection of arch bridges, the ambient temperature change includes the seasonal temperature change and daily temperature change. The daily temperature change is more complex, especially under the effect of sunlight, which will cause the deflection of the arch rib, which will also lead to longitudinal, transverse and vertical deformations. The influence of seasonal temperature change on the structure is relatively simple, and its change is uniform. In order to study the relationship between the position change of segment N4 for the arch rib and temperature change in the outdoor environment, continuous temperature and deformation monitoring of the arch rib was carried out for 24 h. On the day of monitoring, it was sunny with a certain temperature difference between morning and noon, and the maximum temperature change was 9.6 °C. The detailed temperature–time curve relationship is shown in After the whole bridge is closed, the structure will become statically indeterminate, and the temperature stress generated by the bridge will be able to exceed the stress even under a live load. According to the above analysis, the structural temperature effect under the action of sunshine temperature change will be large, which will mainly have a large effect on the deflection during the bridge construction process and the stress state after the transformation of the bridge system, with a small cycle and rapid change. In order to better determine the appropriate time for arch rib closure, this study monitored the arch back temperature at the final closure and ambient temperature for 84 h before the arch rib’s closure, and the specific monitoring results are shown in (1)maxT value of layer No. 3 was about 1.3, 1.2, and 1.1 times the average maxT value of layer No. 1 for the mass concrete in the cushion cap, main pier and arch abutment, respectively.The heat dissipation performance of concrete decreases with the increase in the thickness of a mass concrete structure. The temperature of the core area of the middle layer was the highest for the mass concrete structures, gradually decreasing to the outward surfaces. The average (2)moldingT value, the maxT value at the core area of layer No. 3 increased by about 4~5 °C for mass concrete. The pipe cooling method is conducive to the hydration heat control effect of mass concrete, and can effectively reduce the central temperature of concrete structures.The ambient temperature of concrete has a certain influence on its temperature field. The higher the molding temperature, the higher the maximum temperature value by the hydration heat effect. With each 5 °C increase in the (3)A temperature rise will cause the structure to have a certain camber in the longitudinal direction, and the longitudinal or transverse displacement value caused by the temperature change (temperature rise or drop) under the sunshine will be no less than the vertical displacement value. Due to the symmetrical construction on both sides of the river, the arch rib deformation on both sides will basically present symmetrically synchronous changes.(4)In view of the large structural temperature effect under the action of sunshine temperature change, the appropriate closure time of the arch rib is determined as 1:00~4:00 in the morning, during which the changing trends of arch back temperature and ambient temperature will be consistent, and the temperature difference is relatively small.This study investigates the temperature effect of the construction process of a rigid frame-tied steel box arch bridge. For the mass concrete in the cushion cap, main pier and arch abutment, the hydration heat control effect of mass concrete by the pipe cooling method is evaluated. Based on the monitored temperature change and displacement change, the influences of daily temperature change on the steel lattice beam and arch rib were analyzed and identified, and the appropriate closure temperature of the arch back was determined. From the experimental and analytical results, the following conclusions can be drawn:This study investigated the cement hydration heat effect of the cushion cap, main pier and arch abutment and determined the appropriate closure timing based on the sunshine temperature effect in the Dafeng River steel box arch bridge construction. However, the influence of the pipe cooling method on the hydration heat as well as the sunshine temperature effect of steel in bridge construction is worthy of further exploration.


Antimicrobial stewardship (AMS) interventions are being implemented to optimize antibiotic use including the use of antibiotic prescription charts. This study reviewed the use of antibiotics before and after the introduction of an antibiotic prescription chart in a paediatric medical ward of an academic tertiary hospital in Johannesburg.a cross-sectional retrospective review of patient records was conducted for patients admitted to a paediatric medical ward of an academic tertiary hospital over two study periods; before and after the introduction of an antibiotic prescription chart. Data were captured on a Microsoft® Excel (2010) spreadsheet and analyzed using Stata/IC 15.1 .antibiotic use decreased significantly by 7.04% following the introduction of the antibiotic prescription chart (p=0.027). Fields often left unfilled on the antibiotic prescription chart include age (100%), a record of renal function (GFR/CrCl) (97.46%), time of antibiotic prescribing (83.62%) and a record of culture and sensitivity results (80.17%).the findings of this study show an improvement in antibiotic use, the frequency of culture and sensitivity testing and documentation of relevant parameters after the introduction of the antibiotic prescription chart. The use of an antibiotic prescription chart is a practical way to achieve optimal antibiotic use and to encourage proper detailing of the clinical components necessary for antibiotic selection in a hospital setting in a developing country. The unabating challenge of the development of bacterial resistance coupled with a dry antimicrobial drug pipeline has made antibiotic resistance a global public health challenge . AntibioSetting: this study was conducted in a paediatric medical ward of a public academic tertiary hospital in Johannesburg, South Africa. The hospital has 1088 beds with over 4,000 staff members and functions as a referral hospital for several hospitals in its cluster. The paediatric medical ward consists of 24 beds and admits patients under the age of six with medical and non-infectious diagnoses.Study design: a retrospective record review of antibiotic usage was carried out across two study periods. The first study period was between 1st August and 31st October 2016, which represents the period before the introduction of the antibiotic prescription chart (pre-introduction), while the second study period was between 1st August and 31st October 2019 and represents the period after the introduction of the antibiotic prescription chart (post-introduction).Data collection and statistical analysis: records of all patients  admitted to the study ward, who received an antibiotic throughout the study periods, were included in the study. Data retrieved included patient demographics , diagnosis, source of infection, prescribed antibiotics, antibiotic dosage, frequency of dosing, route of administration as well as microbial culture and sensitivity testing. Data were retrieved and analyzed using Stata/IC 15.1 . Patient characteristics and all outcome measures were quantitatively compared for both study periods and presented using descriptive analysis and comparison tables. Pearson´s Chi-square was used to test associations between selected categorical variables. A p-value not exceeding 0.05 was regarded as significant.Data reliability and validity: data were collected by the same researcher. Unique study numbers were assigned to each patient record for easy tracing. The same variables were collected throughout the study and sorted before data analysis. Other studies have used a similar data collection tool [ion tool ,20. The Ethical considerations: ethical clearance to conduct this study was obtained from the University of the Witwatersrand Human Research Ethics Committee (ref. no. M190711).A total of 336 patient records were reviewed with 225 patients receiving an antibiotic: 109 in the pre-introduction period and 116 in the post-introduction period.Antibiotic utilization: this study found that antibiotics were prescribed to 70.78% of patients in the pre-introduction period and 63.74% in the post-introduction period, with a statistically significant difference in antibiotic prescribing across both study periods (p=0.027). The mean ± SD number of antibiotics prescribed per patient in the pre-introduction period was 1.91 ± 1.14 and 1.96 ± 1.08 in the post-introduction period with no statistically significant difference (p=0.3679). Three or fewer antibiotics per admission were frequently prescribed per patient over both study periods, representing 92.66% and 90.52% of patients in the pre-introduction and post-introduction periods respectively  in the number of tests requested across both study periods.Duration, antibiotics prescribed and routes of administration: antibiotic regimens of one to seven days were frequently administered across both study periods ( periods . On averThe use of the antibiotic prescription chart in the post-introduction period: antibiotics were prescribed on 85.59% of antibiotic prescription charts in the post-introduction period (n period . Half (5n period . Furthern period . The comThe introduction of an antibiotic prescription chart as an AMS strategy is beneficial in improving antibiotic use in healthcare institutions. The antibiotic prescription chart serves as a means of surveillance and enables the proper selection of antibiotics when used optimally. This study sought to review antibiotic use before and after implementing an antibiotic prescription chart in a paediatric medical ward of an academic tertiary hospital.Antibiotic utilization: this study recorded a total of 70.78% and 63.74% of patients being administered at least one antibiotic in the pre-introduction and post-introduction periods respectively. Other studies have reported higher ranges of antibiotic prescribing of 72-84% [f 72-84% -24 per pf 72-84%  and in sf 72-84% , Ghana (f 72-84%  and Nigef 72-84% . Inapprof 72-84% ,6,29. Fuf 72-84% . Howeverf 72-84% . This stf 72-84% ,32,33. Af 72-84% . Thus, aThis is similar to reports from studies conducted in paediatric wards in South African hospitals ,36 and aCulture and sensitivity testing: the need for appropriate culture and sensitivity testing in antibiotic selection cannot be overemphasized. It is essential that samples are appropriately and promptly obtained for testing and requested before initiating antibiotic therapy to optimize an accurate diagnosis [iagnosis . The proiagnosis . In addiiagnosis . Adhereniagnosis . The mosiagnosis . Antibioiagnosis . There iLimitations: in the study ward, adherence to the antibiotic chart was insufficient, and as such, this study may not have entirely reflected the true picture of the public health sector based on the data collected. Another limitation of this study was that patient comorbid, chronic and immunosuppressed conditions were not linked to antibiotic prescribing, including prophylaxis with sulfamethoxazole/trimethoprim in patients with HIV. This may have influenced research findings such as the duration of antibiotic therapy and number of antibiotics prescribed.The outcome of this study indicates a statistically significant decrease in the proportion of patients prescribed antibiotics as well as a non-significant increase in the culture and sensitivity tests requested after the introduction of the antibiotic prescription chart. Evidence for proper implementation of the antibiotic prescription chart in this study is lacking. The antibiotic prescription chart is not being used to its full potential with poor documentation practices evident. Emphasizing the benefit of the antibiotic prescription chart as well as a regular review of antibiotic therapy using the antibiotic prescription chart by a clinical pharmacist and infectious disease physician may ensure optimal antibiotic use.High rate of antibiotic prescribing;Surveillance studies regarding AMS practices and the impact of introducing an antibiotic prescription chart are scarce.An antibiotic prescription chart can positively impact optimal antibiotic use;There is a gap in the effective implementation of the antibiotic prescription chart.


Subjects with elevated IgG4 (IgG4 > 86 mg/dL) had significantly higher ESR, CRP, HAQ-DI, and DAS 28 and a poorer treatment response compared to the group with non-elevated IgG4. After multivariate analysis, only HAQ-DI  and DAS28  remained significantly associated with elevated serum IgG4. The preliminary findings of this study could suggest serum IgG4 to be a potential biomarker of disease activity and functional disability in RA. Immunoglobulin (Ig) G4 accounts for 4–6% of the total IgG in a healthy human. Several evidence-based studies have suggested that the level of IgG4 is significantly elevated in autoimmune diseases, including rheumatoid arthritis (RA). The clinical significance of IgG4 in RA with regard to disease activity, severity, and treatment response remains elusive. We consecutively recruited 174 patients with RA from our rheumatology clinic. All subjects were assessed for their disease activity based on DAS28, radiographic joint damage based on the Modified Sharp Score (MSS), the functional capacity based on the Health Assessment Questionnaire –Disability Index (HAQ-DI), and treatment responsiveness using the European League Against Rheumatism (EULAR) response criteria. The serum IgG4 of the recruited subjects was measured via the ELISA test. The mean serum IgG4 level was 60.23 ± 30.08 mg/dL. We found that serum IgG4 had significant positive correlations with disease activity (r = 0.406;  Rheumatoid Arthritis (RA) is the most common form of symmetrical inflammatory arthritis. The peripheral small  and large  joints may be involved in RA. It affects millions of people in this country, especially women. Adults of all age groups can be affected by this disease . RA is aImmunoglobulins (Ig) are structurally unique proteins that consist of two pairs of heavy and light chains. It plays a pivotal role in autoimmune diseases such as RA, systemic lupus erythematosus (SLE), and myasthenia gravis. These proteins are subclassed into IgA, IgD, IgG, and IgM. IgG is the major immunoglobulin class that makes up about 80% of the total human serum Ig . IgG canThe IgG4-autoimmune disease (IgG4-AIDs) is characterized by autoantibody responses predominantly of the IgG4 subclass against a known antigen. These disorders can affect many organ systems, including the kidneys, nervous system, haematopoietic system, and skin. As IgG4 is predominantly anti-inflammatory, the pathogenic effects are speculated to be from the blocking of essential protein–protein interactions from the target antigen. The pathological consequences of IgG4 autoantibodies may differ from one disease to another .Recent studies have shown how serum IgG4 was significantly elevated in the RA population. However, the precise role of IgG4 in RA and the rest of the autoimmune diseases remains to be elucidated . In RA, To date, there has been a lack of data on the correlation between serum IgG4 levels and the various clinical and biochemical aspects of RA, such as disease activity, joint damage, the responsiveness to diseases modifying anti-rheumatic drugs (DMARDs), and functional capacity. This prompted us to conduct a study to fill the aforementioned knowledge gap. We reckon that IgG4 is a potential therapeutic target in RA. Hence, the purpose of this study was to elucidate the clinical significance of IgG4 in RA with regard to disease activity, joint damage, functional disability, and treatment response.This cross-sectional study was conducted in Hospital Canselor Tuanku Muhriz (HCTM), the National University of Malaysia, from June 2022 to April 2023, with the approval of the Research Ethics Committee of HCTM. Funding was obtained from the Fundamental Research Grant Scheme (FRGS/1/2021/SKK08/UKM/02/1) by the Ministry of Higher Education of Malaysia.Patients were recruited from the rheumatology clinic of HCTM. The inclusion criterion was patients with a confirmed diagnosis of RA based on the American College of Rheumatology 2010 classification criteria of RA [We used convenient sampling; a non-probability sampling method was used whereby patients were approached during their scheduled clinic visits. All patients were counseled, and informed consent was obtained before recruitment into this study. Demographic data such as age, gender, and race were recorded. We obtained data on patient’s disease characteristics, such as seropositivity, disease duration, baseline DAS28, and DMARDs, from their medical records. Disease activity at the time of recruitment was recorded and scored using the 28-joints-based disease activity score and using the erythrocyte sedimentation rate  was used to determine the severity of the radiographic joint damage  by a sinBased on the European League Against Rheumatism (EULAR) response criteria, patients were further divided into good, moderate, and non-responders. Patients with low disease activity or in remission and who had achieved an improvement in their DAS28 score of more than 1.2 from the baseline were considered good responders. Moderate responders were patients who achieved an improvement in their DAS28 scores of between 0.6 and 1.2 from the baseline and with moderate disease activity or an improvement of more than 1.2 in patients with high disease activity. The non-responders were subjects with an improvement of less than 0.6 in DAS28 scores from the baseline regardless of disease activity, with an improvement between 0.6 and 1.2 from the baseline with high disease activity .About 3–5 mL of blood samples were taken from the subjects and processed in a centrifugal separator at 3000 rpm for 15 min. The separated sera were stored at −70 °C prior to the measurement. The serum concentration of IgG4 was tested using a commercially available enzyme-linked immunosorbent assay (ELISA). The kits  were pre-coated with the antibody specific to human IgG4. The sensitivity of the test was 83–97%, while the specificity was 60–85%.p-value of less than 0.05 was considered statistically significant.Data were analyzed using SPSS version 25 software  and GraphPad Prism software version 7.0 . We used Spearman’s rank test to assess the correlation between IgG4 and the other continuous variables, such as DAS 28 scores, MSS scores, and HAQ-DI scores. To compare subjects with elevated IgG4 to those with non-elevated IgG4 levels, the data were analyzed using the Mann–Whitney test for continuous variables, which were expressed as the median (range), whereas Pearson’s Chi-Square test was used for categorical variables and expressed in numbers (percentages). Multivariate analysis was performed using multinomial logistic regression, with serum IgG4 levels  as the independent variable. The data were expressed as the mean ± standard deviation (SD), counts (percentage), and median (range). The correlation coefficient and r-value were interpreted using the Statistic Corner by Mukaka et al. as a reference . A p-valA total of 200 subjects with RA were recruited in this study; however, 26 subjects were excluded from the final analyses as their IgG4 levels were unavailable due to either missing samples or rejected samples. p < 0.001), ESR , DAS28 , MSS , and HAQ-DI . Despite many variables being found to have a significant relationship with IgG4 levels, the correlations were strong (r > 0.40) for only DAS28 and HAQ-DI. Age and disease duration showed no correlations with IgG4 levels. IgG4 levels had a significant linear relationship with DAS28, MSS, and HAQ-DI (p < 0.001).The mean serum IgG4 level was 60.23 ± 30.08 mg/dL. None of the subjects had undetectable levels of IgG4 . Table 2d HAQ-DI . On multp = 0.039), including CRP (p < 0.001), ESR (p = 0.030), DASR28 (p < 0.001), HAQ-DI (p < 0.001), and EULAR treatment response (p < 0.001). In terms of multivariate analysis using a multinomial logistic regression (p = 0.018) and DAS28 remained significantly associated with elevated IgG4.The cut-off IgG4 level to diagnose IgG4-related disease was 135 mg/dL . Otherwigression , only HAOur study demonstrated elevated serum IgG4 levels in 19.5% of the studied RA population, using a cut-off value of 86 mg/dL. Throughout the literature, the frequency of elevated IgG4 in RA has ranged widely from 6% to 46% using different cut-off levels ,18,19,20One of the novel findings of this study was the significant positive correlation between serum IgG4 levels and RA disease activity. Disease activity in RA reflected synovial inflammation due to the upregulation of circulating cytokines such as IL-1, IL-6, and tumor necrosis factor α. The synthesis of IgG4 in vitro was regulated by IL-6. IL-6 could enhance IgG4 production via the IL-21 expressed in CD4+ T cells , which iConsidering multivariate analysis, HAQ-DI had an independent association with elevated IgG4 after adjusting for confounders. The functional disability was greater in the elevated IgG4 group despite comparable median MSS scores, which reflected the severity of joint damage. Functional disability in RA is multifactorial and could be the consequence of several factors, such as joint damage, secondary osteoarthritis, entrapment neuropathies, sarcopenia, and tendinopathies . To dateWe found an association between IgG4 levels and treatment response based on EULAR response criteria. Almost all subjects with elevated IgG4 were non-responders. Studies with conventional DMARDs, as well as advanced therapies, have consistently shown a marked decline in IgG4 levels with treatment ,27,28. CAlthough IgG4 levels showed a significant correlation with radiographic joint damage based on MSS scores, the strength of this relationship was negligible. This explained the comparable median MSS scores between the elevated and non-elevated IgG4 groups. The non-elevated IgG4 group had shorter disease duration and lower disease activity which could account for the above findings. In addition, the MSS assessment included the scoring of only the hands and feet. Many RA patients have radiographic changes in larger joints, such as the knees, elbows, and shoulders, which could have been missed in this study. We acknowledge the limitations of this study. The study was designed without including a control arm comprising non-RA subjects. This was due to budget constraints. We did not perform measurements of the total IgG. The percentage of IgG4 over the total IgG could lend credence to our findings. In conclusion, this study suggests that serum IgG4 is a potential biomarker of disease activity and functional disability in RA. Our findings may be considered preliminary and should be interpreted with caution. Further studies in this regard are much needed to consolidate our findings.


Leishmania parasite in the vertebrate host. Among them, salivary apyrase with anti-hemostatic properties has a crucial role during the blood meal process. In the present study, a Genome-Walking method was used to characterize a full-length nucleotide sequence of Phlebotomus (P.) kandelakii apyrase (Pkapy). Bioinformatics analyses revealed that Pkapy is a ~ 36 kDa stable and hydrophilic protein that belongs to the Cimex family of apyrases. Moreover, recombinant proteins of Pkapy and P. papatasi apyrase (Ppapy) were over-expressed in Escherichia coli BL2 (DE3) and their antigenicity in BALB/c mice was evaluated. Dot-blot and ELISA results indicated that both recombinant apyrases could induce antibodies in BALB/c. Moreover, a partial cross-reactivity between Pkapy and Ppapy was found. In vitro stimulation of splenocytes from immunized mice with the recombinant proteins indicated cross-reactive T cell proliferative responses. Cytokine analysis revealed significant production of IFN-γ (p < 0.001) and IL-10 (p < 0.01) in response to Pkapy. In conclusion, the full-length nucleotide sequence and molecular characteristics of Pkapy were identified for the first time. Immunologic analyses indicated that Pkapy and Ppapy are immunogenic in BALB/c mice and show partial cross-reactive responses. The immunity to Pkapy was found to be a Th1-dominant response that highlights its potential as a component for an anti-Leishmania vaccine.Sand fly salivary proteins have immunomodulatory and anti-inflammatory features; hence, they are proven to perform important roles in the early establishment of  Leishmania (L.) parasites in the Old World. During blood-feeding, the parasites are inoculated at the biting site along with the sand fly saliva. The saliva contains pharmacologically-active components to facilitate the acquisition of blood meals and exhibits immunomodulatory properties2. These compounds also support the survival and multiplication of the parasites and facilitate disease progression4. It has been shown that pre-exposure to non-infected sand flies or pre-immunization with salivary gland lysate (SGL) might protect mice, hamsters, dogs, and non-human primates against a few Leishmania species3. Both humoral and cellular immune responses against different salivary components of sand fly have been detected. Generally, anti-saliva antibody response and DTH/T cell proliferative responses are considered markers of exposure to sand fly bites and protection against leishmaniases, respectively4.Phlebotomine sand flies are blood-feeding insects that act as vectors of different species of Phlebotomus (P.) kandelakii, P. perfilewi transcaucasicus, and P. tobbi have been identified in different visceral leishmaniasis (VL) foci in Iran. P. kandelakii is one of the most prevalent vectors in the North-East and North-West regions of the country where natural infection of this species by L. infantum has been reported6. Although several reports have been published related to the composition and immunogenicity of the salivary proteins of at least 13 sand fly species4, not much information is available about the composition of P. kandelakii’s saliva.Several sand fly species including . have reported important differences in the antigen components of the SGL from P. papatasi, P. perniciosus, and P. halepensis. Besides, they have indicated that sera from mice bitten by P. papatasi cross-react with the homologous SGL but not with SGL from the other two species7. Therefore, exploring the salivary antigens from different Leishmania vectors is a main priority for the researchers.The composition and immunogenicity of the salivary proteins are species-specific. Volf et al8. Apyrases of the blood-feeding insects are divided into three families. The Cimex family of apyrases functions exclusively with Ca2+ while the other two families require either Ca2+ or Mg2+ for their actions10. The Cimex family of apyrases is among the most prevalent proteins in sand fly saliva and counteracts the host's hemostatic system by inhibiting the blood coagulation cascade, platelet aggregation, and vasoconstriction12. Apyrases from different sand fly species elicit antibody responses in various hosts including P. papatasi apyrase (Ppapy) in humans and mice14, P. perniciosus apyrase in mice, rabbits, and dogs18, P. tobbi apyrase in rabbit19, and P. orientalis apyrase in domestic animals20. Altogether, the results of these studies have confirmed that apyrases belong to the most antigenic salivary proteins and can be recognized by sera of the repeatedly-bitten hosts16. Therefore, anti-apyrase antibodies can be considered a promising biomarker of sand fly exposure or even a risk factor for Leishmania transmission among the hosts. Besides, cell-mediated immune responses against P. ariasi apyrase and P. papatasi apyrase have been documented23.Apyrases are enzymes that hydrolyze ATP and ADP to AMP and orthophosphate; thereby, can destroy these important stimuli of platelet aggregation and inflammation at the biting site24; however, only partial information has been available on P. kandelakii apyrase (Pkapy) gene. In the present study, we aimed for the first time to identify a full-length nucleotide sequence of Pkapy from Iran. Besides, the amino acid sequence of Pkapy was analyzed by in silico approaches. The recombinant protein was then expressed in Escherichia coli and its antigenicity in BALB/c mice was evaluated. Since P. papatasi is the main and proven vector of L. major in different parts of Iran23, we also compared the in-silico findings of Pkapy and Ppapy and the immunogenicity and potential cross-reactivity of recombinant Pkapy and Ppapy were investigated in BALB/c mice. Finally, the cytokine profile in response to Pkapy was evaluated.The sequences of apyrases from different species of sand flies have so far been reportedP. kandelakii apyrase. During the synthesis stage of ssDNA using gradient PCR, the lowest temperature with no amplicon was 58 °C. Therefore, this temperature was considered the annealing temperature for performing the first step of the procedure (Table Pkapy gene was submitted to GenBank (Accession No.: MN893300).A Genome-Walking procedure was performed to determine the full-length sequence of a partially known part of re Table b. For thre Table a. To evare Table b. Based re Table a, and amre Table b were seN- and O-glycosylation sites in Pkapy revealed that N247 is susceptible to N-glycosylation while there is no O-glycosylation site in this molecule , extended strand (39.81%), and random coil 52.43%). Since 162 of 309 amino acid residues were found to be localized to the random coil, this would probably be the main secondary structure of Pkapy (Table .43%. Sin(H-CAN) demonstrated that the targeted residues are conserved to some extent , 4 of the 13 residues important for binding to nucleotides , and 1 of the 3 residues essential for nucleotidase activity in H-CAN  were conserved in Pkapy  and 68.0% (230/338), respectively.Protein sequence alignment comparison between Pkapy QNG40038.1) with apyrases from a few sand fly species and other related sequences is indicated in Fig.  with apyo.: 2H2N PDB). Thus, H-CAN was considered as the reference molecule in superimposition and to find the counterparts of the structurally-important residues. The structural superimpositions of Pkapy and Ppapy with H-CAN revealed that they are very similar in structure, especially in critical residues and functional domains  of the residues was calculated to show the residues’ solvent accessibility. Higher scores were determined as larger solvent accessibility of the residues. The PI values (above the default 0.5 scores) of discontinuous B cell epitopes for Pkapy and Ppapy are reported in Supplementary Table E. coli and a high level of purification was achieved by Ni–NTA affinity chromatography. SDS-PAGE and Western blotting of the purified recombinant Ppapy and Pkapy indicated distinct bands with ~ 39 kDa as the expected Mw of the recombinant proteins  were tested. Notably, antisera produced against the recombinant apyrases reacted with SGL from P. papatasi . Likewise, sera from Pkapy-immunized mice reacted with Ppapy protein significantly stronger compared to Ppapy and BSA (p < 0.0001). The significantly higher reactivity of anti-Ppapy with Pkapy, and anti-Pkapy with Ppapy compared to reactivity with BSA (p < 0.05) was indicative of the specificity of the antigen–antibody reaction. The mean OD value for reactivity of anti-Ppapy sera with Ppapy protein was 1.4 while for the same amount of Pkapy-coated-wells, the mean OD value was 0.2. On the other hand, the OD values for a reaction between anti-Pkapy sera and Pkapy and Ppapy proteins were 1.6 and 0.3, respectively  induces a specific DTH response to the SGL from P. ariasi21. Furthermore, transfection of PBMC with a plasmid coding for Ppapy has resulted in a Th1 cellular immune response22. This designates the development of cell-mediated immune response by the apyrases and represents them as promising anti-Leishmania vaccine candidates. There are more studies on antibodies against apyrases; in fact, anti-apyrase antibodies have been considered as a potential biomarker of sand fly exposure and a risk factor for Leishmania transmission as has been shown in the case of canine exposure to P. perniciosus bites15.VL is endemic in different parts of Iran and P. kandelakii apyrase was characterized for the first time. Here, in contrast to many other studies that had identified the salivary proteins through the construction of cDNA libraries, we used a Genome-Walking nucleotide sequencing technique to complete the upstream and downstream sequences of a partially available middle part of P. kandelakii apyrase from Iran. The analysis of the coding sequence of Pkapy gene indicated that it belonged to the Cimex family of apyrases. Protein sequence alignments showed considerable homology with the other apyrases of the Cimex family and their vertebrate homologs, namely CAN. Bioinformatics analyses revealed that Pkapy like other known apyrases of Phlebotomus sand flies is a ~ 36 kDa protein. Besides, the protein is hydrophilic and stable with one N-glycosylation susceptible site (N247), which makes it an appropriate candidate for vaccine development.In the present study, a full-length nucleotide sequence of . P. papatasi is a proven vector of L. major in Iran and the most abundant sand fly species in the endemic areas of the country23. In an attempt to identify sand fly proteins that can be used as a global or general anti-Leishmania vaccine, we compared the in-silico characteristics of Ppapy with those of Pkapy. Moreover, the potential antigenic cross-reactivities of these two proteins were explored. Ppapy was found to be a hydrophilic and stable molecule with two N-glycosylation susceptible sites , compared to Pkapy which had only one N-glycosylation susceptible site (N247). The RMSD values of the structurally-important residues of Pkapy and Ppapy and the Ramachandran plots indicated that both proteins were structurally similar to H-CAN as the reference molecule. The superimpositions and comparisons of the critical residues of the active sites revealed that the targeted residues are completely similar to their counterparts.Characterizing the diversity and degrees of homology between salivary proteins from various sand flies is an important issueThe in silico analyses of the B cells epitopes showed that most of the predicted epitopes were conserved between Ppapy and Pkapy; although, Ppapy exhibits two more linear epitopes. The profiles of the antigenic peptides were very similar together since the epitopes were located in the same topological positions, based on the primary structures of both proteins. Seven discontinuous B cell epitopes with similar topological positions for each protein were also predicted.-reactivity between Pkapy and Ppapy, by dot-blot and ELISA analyses. Cross-reactive antibodies and cross-protection have been reported between closely-related species of Phlebotomus subgenus i.e. P. papatasi and P. duboscqi28. However, P. papatasi and P. kandelakii are not closely-related species and this might be the reason for the low cross-reactivity between Pkapy and Ppapy. Our dot-blot data also showed that SGL from P. papatasi could be recognized by anti-recombinant Ppapy and Pkapy antibodies. These findings suggest the conservation of epitopes of the native apyrase in the recombinant proteins expressed in E. coli. This was in line with the findings that antibodies against P. perniciosus saliva reacted with the recombinant apyrases 29.The obtained results also indicated that the recombinant Pkapy and Ppapy proteins were immunogenic in BALB/c mice. Considering the similarities found by the bioinformatics analyses, we examined the antigenic crossLeishmania-specific Th1 response4. Increased production of IL-10 by murine macrophages in response to SP01  is also in line with our finding. Altogether, the immunogenic properties of Pkapy with respect to BALB/c mice highlight its potential as a component for an anti-Leishmania vaccine.We also investigated the cellular immune response to Ppapy and Pkapy by assessment of their proliferative responses. These results indicated that splenocytes from Ppapy- and Pkapy-immunized mice could specifically recall responses to their homologous proteins. In addition, cross-reactivity was documented between Ppapy and Pkapy, since both groups of immunized mice responded to both proteins. Evaluation of the cytokines profile after stimulation with Pkapy showed higher levels of IFN-γ, in mice immunized with Pkapy, compared to the unimmunized mice. These findings indicated that the immunity to Pkapy is a Th1-dominant response. Th1 response to saliva components at the site of inoculation may activate the infected macrophages, leading to the killing of the parasites during the early phase of the infection, and may also promote a faster P. kandelakii, an important vector of L. infantum in Iran. Immunologic studies indicated that Pkapy and Ppapy are immunogenic in BALB/c mice and show cross-reactive responses. Moreover, cytokine analysis of the immunized mice revealed a Th1-type response to recombinant Pkapy. These results are providing insights into Pkapy as a potential candidate for a vector-based vaccine.This is the first report on the characterization of an apyrase from Phlebotomus kandelakii sand flies were captured in Bojnurd area of North Khorasan Province (North-East of Iran) using CDC traps and sticky papers. The sand flies were identified based on external and internal morphological characteristics of the head and abdominal terminalia . The genomic DNA samples from 5 female sandflies were extracted and pooled. Female P. papatasi (originating from Turkey), was kindly provided by Professor Petr Volf . Salivary glands of P. papatasi were dissected out under a stereo microscope into PBS, and were disrupted by three freeze/thaw cycles. Salivary homogenates were centrifuged at 10,000 × g for 3 min and the supernatants were used for the experiments.Pkapy gene was publicly available, a Genome-Walking sequencing technique was performed to obtain the upstream and downstream sequences of the available segment, known as P. kandelakii clone kandG2a apyrase-like protein (GenBank accession No.: JF899992). As shown in Table Since at the beginning of this study, only a partial middle part of 30. Firstly, to amplify the targeted sequences downstream of the gene, the synthesis of single-stranded DNA (ssDNA) molecules was evaluated using F-GSPa primer at 57–65 °C using a thermocycler . The products were electrophoresed on 1% agarose gel and analyzed. The lowest temperature in which no amplicon was seen was selected as the best temperature for further ssDNA molecule synthesis. The synthesis was performed separately in 7 tubes labeled A to G. Each tube was allocated for one of the GWPs in the following step. Amplification reactions were composed of 7.5 μl ExPrimeTaq Premix 2X , 400 nM F-GSPa primer, and sterilized D.W. up to 15 μl. The PCR program is mentioned in Table Seven Genome-Walking primers , as well as long and short universal tagging primers , were used according to the previous studiesSecondly, after immediately adding 1 μl from each of the 7 GWPs, along with 3 μl ExPrimeTaq Premix and 1 unit of Taq DNA polymerase  individually to the 7 reaction tubes, PCR was carried out according to the program indicated in Table The fragments with acceptable sizes were subjected to gel extraction and were then subcloned into a PTZ57R/T  and sequenced . To identify the upstream sequence of the gene, all the above-mentioned procedures were carried out, except that the specific reverse primers were used.Pkapy gene was subjected to BLASTX <http://blast.ncbi.nlm.nih.gov/Blast.cgi> which indicated that the sequence belonged to the Cimex family of apyrases. The protein sequence of Pkapy was aligned with other apyrase protein sequences from P. papatasi (AAG17637.1), P. orientalis (AGT96454.1), Lutzomyia (Lu.) longipalpis (AAD33513.1), Cimex lectularius (AAD09177.1), Drosophila melanogaster (CAL26008.1), and their mammalian homologous  from Rattus norvegicus (CAC85467.1), Canis lupus familiaris (XP_005624095.1) and Human (Q8WVQ1.1)). Multiple alignments were performed using Clustal Omega  based on the ClustalW method31. The presence and location of signal peptide cleavage sites were predicted by Signal P-5.0 server  based on a combination of several artificial neural networks32. The physicochemical properties such as instability index, isoelectric point, aliphatic index, grand average of hydropathy (GRAVY), molecular weight (Mw), and isoelectric point (pI) were analyzed with ProtParam server (https://web.expasy.org/protparam/). To predict the amino acids that undergo glycosylation, NetNGlyc 1.0 (http://www.cbs.dtu.dk/services/NetNGlyc/) and NetOGlyc 4 servers (http://www.cbs.dtu.dk/services/NetOGlyc/) were used. The secondary structure was predicted by PHD secondary structure server (https://npsa-prabi.ibcp.fr/cgi-bin/secpred_phd.pl).The obtained full sequence of the http://swissmodel.expasy.org) was employed to predict the 3D structure of Pkapy and Ppapy based on homology modeling. According to QMEAN  and GMQE , the best-predicted structures with the highest score for each protein were selected and used for comparing the structural properties. The superimposition and root-mean-square deviation (RMSD) analyses were performed by DeepView/Swiss-PdbViewer v.4.1.0 and UCSF Chimera v.1.11.233.SWISS-MODEL server (http://tools.iedb.org/bcell/). The BepiPred-2.0 server predicts B-cell epitopes from the protein sequence, where the residues with scores above the default value of 0.5 were predicted as epitopes and colored in yellow on the graph. Further, the discontinuous B cell epitopes were predicted using Ellipro method in the IEDB database (http://tools.iedb.org/ellipro/) which is based on solvent accessibility and flexibility34.Linear B cell epitopes of Pkapy and Ppapy were predicted using BepiPred-2.0 of the IEDB analysis resource (E. coli and synthesized by Biomatik Corp. (Canada), subcloned into pET-21b (+) vector for expression with a C-terminal 6 × His-tag. The constructed expression vectors were transformed into E. coli BL21  and verified by restriction digestion and nucleotide sequencing. Protein expression was induced by the addition of Isopropyl β-d-1-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM to a culture of LB with ampicillin (100 μg/ml), containing verified E. coli BL21 with the construct, grown to OD600 ~ 0.5 . The culture was grown for an additional 2 h after the induction as above.Constructs containing regions encoding each protein without the putative secreted signal peptide were optimized for expression in 35.The expressed recombinant proteins were purified by Ni–NTA Superflow resin , following the instructions under denaturing conditions. The proteins were dialyzed overnight against several changes of PBS and endotoxins were removed using a Pierce High-Capacity Endotoxin Removal Resin spin column , according to the manufacturer’s recommendations. The concentrations of the purified recombinant proteins were determined by Bradford assayThe purified recombinant Ppapy and Pkapy proteins were subjected to 10% SDS-PAGE, then electroblotted onto PVDF membrane using a wet Bio-Rad transfer system . The membrane was blocked with 5% (w/v) skimmed milk in PBS and incubated with anti-His antibody  as the primary antibody. Finally, the membrane was incubated with goat anti-mouse IgG HRP antibody  as the secondary antibody. The reactivity was detected using 3, 3-diaminobenzidine tetrahydrochloride (DAB).Female BALB/c mice (4–6 weeks old) were purchased from the animal facility of the Production Complex of the Pasteur Institute of Iran. All animal studies were performed in line with the ARRIVE reporting guidelines. Approval was granted by the Ethics Committee of the Pasteur Institute of Iran (IR.PII.REC.1395.109), and the experiments were performed in accordance with relevant guidelines and regulations.Mice (4 in each group) were immunized three times at 2-week intervals subcutaneously at the base of the tails with 10 µg of either Ppapy or Pkapy along with 15 µg Quil A , as an adjuvant. The optimal dose of proteins for immunization was determined in a preliminary study in which mice were immunized with 5, 10, and 15 µg of Pkapy. When the sera were checked for the presence of anti-apyrase antibody, the results of 15 µg Pkapy were almost similar to 10 µg; hence, the latter amount was selected for the immunization. The control group received Quil A only. Mice were retro-orbitally bled for sera preparation two weeks after the last immunization.P. papatasi, and an unrelated protein  were spotted onto a nitrocellulose membrane. The spots were dried and the membrane was blocked with 5% skimmed milk in PBS. The membrane was then incubated with 1:10 diluted sera collected from the immunized and unimmunized mice for 2 h at RT. For the secondary antibody, an HRP-conjugated goat anti-mouse antibody was added and incubated at RT for 1 h. Finally, spots were developed using DAB substrate solution in presence of hydrogen peroxide. Stained dots on a white background indicated positive results.To detect immunogenicity and the potential cross-reactivity of the recombinant apyrases, dot-blot and ELISA were performed. For the dot-blot assay, 1 μg of each Ppapy, Pkapy, SGL (1 gland/dot) of female Ppapy, Pkapy, and BSA were used in 1 μg/well concentration to coat 96-well ELISA plates  in duplicates and were then incubated at 4 °C overnight. After washing, pre-diluted sera (1:100) from each mouse (4 mice in each group) were added to the wells in a direct and cross manner to evaluate its reactivity with the immunized protein and the counterpart protein. After incubation for 2 h at RT, HRP-conjugated goat anti-mouse antibody  was added to the plates and incubated for 1 h at RT. The plates were developed with 3,3′,5,5′-Tetramethylbenzidine  and read at 450 nm by a microplate reader . The levels of IgG antibody were reported as OD values of each well minus the OD values of the control wells.36. In brief, the splenocytes were stimulated with recombinant Pkapy, Ppapy, or an irrelevant recombinant protein (FHA from Bordetella pertussis). Moreover, the splenocytes of unimmunized mice were stimulated with the recombinant apyrases. Negative control cultures consisted of the medium without any antigen. The cultures were incubated at 37 °C, 5% CO2 for 3 days. Subsequently, 20 μl of Alamar blue reagent  was added to each well and incubated for 4 h. Three replicates of each sample were analyzed on a microplate reader at 570 nm with a reference wavelength of 690 nm  and the percentage of Alamar Blue reduction was calculated for stimulated and unstimulated cells according to Al-Nasiry et al.37. The stimulation index (SI) was calculated by dividing the mean results of the stimulated cells by the mean results of the un-stimulated cells. For cytokine assays, splenocytes from Pkapy-immunized mice were stimulated with recombinant Pkapy for 4 days and subsequently, the culture supernatants were recovered and stored at − 80 °C for determination of IFN-γ, IL-4, and IL-10 levels. Cytokines were assessed by Mabteck ELISA kits , according to the manufacturer’s instructions.Alamar blue assay was used to evaluate the proliferative responses of the lymphocytes to the recombinant proteins. Two weeks after the last immunization, the spleen cells from each mouse (4 mice in each group) were recovered and cultured as previously describedp < 0.05 was considered to be significant, and data were represented as mean + SD.ANOVA followed by Tukey’s multiple comparisons test and Student’s t-test were used to evaluate the statistical significance of the obtained data for ELISA and T cell proliferation, respectively. Statistical analysis was performed with GraphPad Prism software . The Supplementary Information.