This study aimed to estimate the direct medical costs and out-of-pocket (OOP) expenses associated with inpatient and outpatient care for IHD, based on types of health insurance. Additionally, we sought to identify time trends and factors associated with these costs using an all-payer health claims database among urban patients with IHD in Guangzhou City, Southern China.Data were collected from the Urban Employee-based Basic Medical Insurance (UEBMI) and the Urban Resident-based Basic Medical Insurance (URBMI) administrative claims databases in Guangzhou City from 2008 to 2012. Direct medical costs were estimated in the entire sample and by types of insurance separately. Extended Estimating Equations models were employed to identify the potential factors associated with the direct medical costs including inpatient and outpatient care and OOP expenses.P < 0.05]. The direct medical costs and OOP expenses for all patients increased from 2008 to 2009, and then decreased during the period of 2009–2012. The time trends of direct medical costs between the UEBMI and URBMI patients were different during the period of 2008-2012. The regression analysis indicated that the UEBMI enrollees had higher direct medical costs (P < 0.001) but had lower OOP expenses (P < 0.001) than the URBMI enrollees. Male patients, patients having percutaneous coronary intervention operation and intensive care unit admission, patients treated in secondary hospitals and tertiary hospitals, patients with the LOS of 15–30 days, 30 days and longer had significantly higher direct medical costs and OOP expenses .The total sample included 58,357 patients with IHD. The average direct medical costs per patient were Chinese Yuan (CNY) 27,136.4  in 2012. The treatment and surgery fees were the largest contributor to direct medical costs (52.0%). The average direct medical costs of IHD patients insured by UEBMI were significantly higher than those insured by the URBMI [CNY 27,749.0  vs. CNY 21,057.7, The direct medical costs and OOP expenses for patients with IHD in China were found to be high and varied between two medical insurance schemes. The type of insurance was significantly associated with direct medical costs and OOP expenses of IHD. Ischemic heart disease (IHD) is a major public health concern worldwide, encompassing acute myocardial infarction, chronic stable angina, chronic IHD, and associated heart failure, . AccordiIn China, the incidence of IHD was ~59.1/100,000 in 2012 , and it In order to improve access to healthcare services and provide financial protection for patients, China has launched two main basic health insurance schemes for the urban Chinese population: the Urban Employee-based Basic Medical Insurance (UEBMI) and Urban Resident-based Basic Medical Insurance (URBMI) , 17. TheSeveral studies have estimated the direct medical costs of IHD in both developed and developing countries –27. A stOnly a few studies have examined the direct medical costs of IHD in China –30. Le eDifferent from previous studies, this research aimed to analyze data collected from all different levels of hospitals to estimate the direct medical costs of both inpatient and outpatient care related to IHD by different type of health insurance and figure out the time trends and the factors associated with such direct medical costs and out-of-pocket (OOP) expenses among urban patients with IHD in Guangzhou City, Southern China.This study collected data from the UEBMI and URBMI claims databases in Guangzhou City between 2008 and 2012, which contained sociodemographic information, medical conditions, hospitalization costs and outpatient costs based on actual amounts paid to all different levels of hospitals. Guangzhou, the capital city of Guangdong province, is one of the largest municipalities in Southern China. Until the year of 2012, there were 93.4% citizens covered by these two urban health insurance schemes in Guangzhou City . This stWe conducted a retrospective observational study with a cross-sectional design to evaluate the direct medical costs of IHD. This study collected all insurance claims on hospitalization care from January 1, 2008, to December 31, 2012 with the primary diagnosis of IHD according to the International Classification of Disease Tenth Revision  . BesidesDirect medical costs referred to the annual medical costs per patient with IHD, including outpatient costs and inpatient costs according to different types of health services. From the payer perspective, the direct medical costs, outpatient and inpatient costs all included individual OOP expenses and the reimbursement amounts. Individual OOP expenses were the costs that were not covered by health insurance and must be paid by patients themselves. In terms of cost composition, direct medical costs consisted of western drug fees, traditional Chinese drug fees, treatment and surgery fees, bed fees, laboratory and diagnostic fees and other fees. The western drug fees were expenses for western medicines while the traditional Chinese drug fees were spending on Chinese patent drugs and Chinese herbal medicines. Treatment and surgery fees were total spending of surgical procedures, medical consumable materials, radiation therapy, oxygen therapy, anesthesia, blood transfusion, and the other forms of treatment excluding medicine therapy. Surgical procedures included catheter-based procedures such as percutaneous coronary intervention (PCI) . Bed feeThe factors associated with total direct medical costs and OOP expenses for IHD patient were selected based on literature and the Andersen's behavioral model of health services use . IndividIn this study, the dependent variable was annual direct medical expenditures and OOP expenses per patient with IHD, including inpatient and outpatient expenses. The primary independent variable was the type of insurance (patients insured by the scheme of UEBMI or URBMI).The other covariates included age, gender, ICU admission, operation of PCI, hospital levels, LOS, and presence of comorbidities. Age was categorized into four groups: 18–59 years old, 60–69 years old, 70–79 years old, and 80 years old and above. Gender was dichotomized as female and male. We chose two dummy variables as proxy measures for the severity of disease—whether having an ICU admission, and having the operation of PCI . Hospitavia R language version 4.1.3 and STATA version 15.0 .Descriptive statistics , median and 25–75th percentiles) were used for demographic characteristics and costs. Due to the skewed distribution of direct medical expenditure and OOP expenses, the Kruskal-Wallis Rank Sum test was used to identify the differences in cost composition between the two types of insurance. We also described the time trends of direct medical costs and OOP expenses across different types of insurance. The Friedman's two-way non-parametric analysis of variance test was performed to figure out the disparities in patient characteristics related to direct medical expenditure and OOP expenses by types of insurance. The Extended Estimating Equations (EEE) model  was seleA large sample of 58,357 patients with IHD were in this study. Most of the patients were insured by the UEBMI scheme (90.8%) and the rest were covered by the URBMI scheme (9.2%) see . More thThe mean direct medical costs per patient with IHD was CNY 27,136.4 , including CNY 26,283.2  for inpatient care and CNY 853.2 (USD 135.2) for outpatient care see . The inpP < 0.001). In terms of cost composition, the treatment and surgery fees accounted for the largest proportion of direct medical costs for both the UEBMI enrollees (52.4%) and the URBMI enrollees (45.9%)  see . The UEBAs shown in The time trends of OOP expenses were similar to the direct medical costs. The OOP expenses of all IHD patients increased from CNY 9,351.1  in 2008 to CNY 10,840.9  in 2009 see  and therWhen comparing two health insurance schemes, the direct medical costs for UEBMI patients initially increased from CNY 24,967.9  in 2008 to CNY 29,070.9  in 2009 and then decreased 2.1% per year during the period of 2009–2012. On the contrary, the direct medical costs of IHD for URBMI patients increased 3.0% per year from CNY 19,325.8  in 2008 to CNY 21,730.9  in 2012. The time trends of OOP expenses among UEBMI subgroup and URBMI subgroup were similar.P < 0.001)  see . For theIn addition, among the overall samples and all subgroups, the mean direct medical costs and OOP expenses of IHD patients recruited in tertiary hospitals were far more than those of patients staying in primary hospitals. Patients with LOS longer than 30 days had higher direct medical costs and OOP expenses than patients with shorter LOS. Patients having diabetes mellitus had higher direct medical cost than those with hypertension.P < 0.001), after controlling for other covariates (P < 0.001).This study found that the types of insurance, PCI operation, gender, age, hospital levels, ICU admission, LOS, and comorbidities (hypertension and diabetes mellitus) were significantly associated with direct medical costs and OOP expenses of IHD see . CompareP < 0.001). Compared with patients aged 18–59, patients 60–69 and 70–79 had CNY 918.6 (USD 145.5) and CNY 1,460.3 (USD 231.3) lower OOP expenses (P < 0.001), and patients aged 80 and above had CNY 1,159.3 (USD 183.7) lower direct medical costs and CNY 2,349.4 (USD 372.2) lower OOP expenses (P < 0.001). Patients having PCI operation and ICU admission incurred significantly higher direct medical costs and OOP expenses (P < 0.001). Compared to patients treated in primary hospitals, IHD patients staying in secondary hospitals had CNY 7,093.3  higher direct medical costs and CNY 1,192.9 (USD 189.0) higher OOP expenses (P < 0.001), and patients staying in tertiary hospitals had CNY 19,006.6  higher direct medical costs and CNY 5,505.5 (USD 872.2) higher OOP expenses (P < 0.001). In comparison with LOS of <15 days, patients with the LOS between 15 and 30 days had CNY16,432.5  higher direct medical costs and CNY 3,705.0 (USD 586.9) higher OOP expenses (P < 0.001), and patients with the LOS of more than 30 days had CNY53,216.7  higher direct medical costs and CNY 10,763.1  higher OOP expenses(P < 0.001). Also, IHD patients with hypertension incurred lower direct medical costs and lower OOP expenses (P < 0.001), but patients with diabetes mellitus had higher direct medical costs and lower OOP expenses (P < 0.001).Male patients had significantly higher direct medical costs and OOP expenses than their female counterparts , which included CNY 26,283.2  for inpatients care and CNY 853.2 (USD 135.2) for outpatient care. The treatment and surgery fees accounted for the largest part of direct medical costs (52.0%) for IHD patients. The direct medical costs of IHD patients covered by the UEBMI schemes   were higher than those of patients covered by the URBMI schemes  . The direct medical costs and OOP expenses for all patients increased from 2008 to 2009, and then decreased during the period of 2009–2012. The time trends of direct medical costs between the UEBMI and URBMI patients were different during the period of 2008–2012. This study also discovered that the type of medical insurance, gender, age, hospital levels, ICU admission, PCI operation, LOS, and comorbidities (hypertension and diabetes mellitus) were significantly associated with total direct medical costs and OOP expenses of IHD. The UEBMI enrollees had higher direct medical costs but had lower OOP expenses than the URBMI enrollees. This was the first study to estimate the direct medical costs and OOP expenses of IHD for the urban population from all levels of hospitals using an entire city's claims datasets, including both inpatient and outpatient costs. Different from previous China-based studies, this research also compared the medical expenses and OOP expenses of IHD patients between two different urban health insurance schemes and examined the time trend of direct medical costs and OOP expenses in China.Compared with studies in other countries, we found that there were plenty of disparities in costs estimation results for IHD patients. In this study, the direct medical costs of IHD were CNY 27,136.4 , much lower than the results in the US  , Korea , CamerooComparing with prior studies conducted in China, the average direct medical costs of IHD in this study  were higher than that of Le et al.'s study   and WangHowever, the direct medical costs in this study were lower than that of Ding et al.'s study  . A possiIn terms of costs composition, treatment and surgery fees occupied the largest proportion of direct medical costs for IHD patients (52.0%) in this study. This may be due to substantially higher cost of invasive therapeutic procedures . This reThe spending on medical consumables such as heart stent implantation was also a critical component for treatment and surgery fees. Evidence in previous China-based studies , 51 illuThe medication costs took up the second largest part of direct medical costs for IHD, including Western drug fees 24.3%) and traditional Chinese drug fees (4.8%). The proportion of total medication costs (29.1%) was similar to the figure in Poland (30%) , but low.3% and tDifferent from previous China-based studies, discrepancies in direct medical costs and OOP expenses of patients with IHD across two types of basic health insurance schemes were observed in this study for the first time. The direct medical costs of IHD patients enrolled in UEBMI schemes  (USD 4395.9) were higher than those of patients covered by the URBMI schemes  (USD 3335.9). But the proportion of OOP expenses out of direct medical costs for the UEBMI patients (28.7%) was approximately half of the OOP percentage for the URBMI patients (52.0%). The regression findings also suggested that UEBMI enrollees had significantly higher direct medical costs with IHD including both inpatient and outpatient care, but had lower OOP expenses than URBMI enrollees.These findings were in line with previous studies estimating discrepancies in expenditures of other diseases under different types of social health insurance –60. UEBMThis study also found that the time trends of direct medical costs between the UEBMI and URBMI patients were different during the period of 2008–2012 for the first time. The direct medical costs of URBMI patients kept increasing during the period of 2008–2012. However, the direct medical costs of UEBMI enrollees had decreased since 2009. This was probably associated with a new policy on improving outpatient benefit packages only for UEBMI enrollees initiated in 2009 , which mIn this study, we demonstrated that those aged 80 and above had significantly lower direct medical costs and lower OOP expenses than those aged under 60. Previous studies found that older patients with IHD incurred higher medical costs than younger patients , 29. HowIn consistent with previous studies , 28, 29,Patients treated in higher levels of hospitals incurred significantly higher direct medical costs in our study, consistent with previous research in China , 37, 48.In accordance with other literature , 29, lonThere were some limitations in this research. First, we only included urban patients with IHD insured by two urban basic medical insurance schemes, and excluded rural residents, which might cause selection bias. Second, the study only estimated the direct medical costs of IHD and did not evaluate the indirect costs such as informal care and loss of productivity. Third, the dataset was a little bit old due to administrative restrictions on data availability, which may not reveal the IHD costs and differences at present. Future studies could consider using a more recent claims dataset, as well as including people covered by all types of insurance, and examining the indirect costs for a more comprehensive evaluation of IHD costs. Fourth, individual factors and disease factors, which were more critical to determine the economic burden, were not introduced in this study because such data were not available in the claims dataset. Finally, the specificity of IHD, such as case characteristics and treatment methods, was not reflected in our analysis due to the unavailability of such data in the claims dataset. Further studies could consider adding case characteristics and treatment methods to examine the determinants of IHD costs.The direct medical costs and OOP expenses of patients with IHD for Chinese urban population were high and different across two basic health insurance schemes. Types of health insurance, gender, age, hospital levels, LOS, ICU admission, PCI operation and comorbidities were significantly associated with the direct medical costs and OOP expenses of IHD. Therefore, policymakers should aim to reduce disparities across different health insurance schemes in terms of benefit packages and narrow the economic burden gap among IHD patients covered by different insurance schemes.The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.The studies involving human participants were reviewed and approved by the Institutional Review Board of the School of Public Health, Sun Yat-sen University, China . Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.Conceptualization, methodology, resources, supervision, project administration, and funding acquisition: HZ. Software and formal analysis: PX. Validation: HZ and DZ. Investigation: PX, XL, and FG. Data curation and writing—original draft preparation: HZ and PX. Writing—review and editing: HZ, DZ, and XL. All authors have read and agreed to the submitted version of the manuscript.


Although several studies compare the clinical outcomes and costs of laparoscopic and robotic proctectomy, most of them reflect the outcomes of the utilisation of older generation robotic platforms. The aim of this study is to compare the financial and clinical outcomes of robotic and laparoscopic proctectomy within a public healthcare system, utilising a multi-quadrant platform.Consecutive patients undergoing laparoscopic and robotic proctectomy between January 2017 and June 2020 in a public quaternary centre were included. Demographic characteristics, baseline clinical, tumour and operative variables, perioperative, histopathological outcomes and costs were compared between the laparoscopic and robotic groups. Simple linear regression and generalised linear model analyses with gamma distribution and log-link function were used to determine the impact of the surgical approach on overall costs.During the study period, 113 patients underwent minimally invasive proctectomy. Of these, 81 (71.7%) underwent a robotic proctectomy. A robotic approach was associated with a lower conversion rate  at the expense of longer operating times . Regarding financial outcomes, robotic surgery was associated with increased theatre costs  and overall costs . Hospitalisation costs were similar between both approaches. An ASA ≥ 3, non-metastatic disease, low rectal cancer, neoadjuvant therapy, non-restorative resection, extended resection, and a robotic approach were identified as drivers of overall costs in the univariate analysis. However, after performing a multivariate analysis, a robotic approach was not identified as an independent driver of overall costs during the inpatient episode (P = 0.1).Robotic proctectomy was associated with increased theatre costs but not with increased overall inpatient costs within a public healthcare setting. Conversion was less common for robotic proctectomy at the expense of increased operating time. Larger studies will be needed to confirm these findings and examine the cost-effectiveness of robotic proctectomy to further justify its penetration in the public healthcare system.The online version contains supplementary material available at 10.1007/s00384-023-04446-1. The worldwide adoption of minimally invasive colorectal surgery over the last decades has been remarkable but it is still limited , 2. SpecRobotic surgery offers advantages over conventional laparoscopic surgery, including the ability to rotate and articulate instruments, a surgeon-controlled camera with three-dimensional vision, and improved ergonomics. However, one disadvantage of robotic surgery is the lack of tactile feedback. Despite this limitation, several randomized controlled trials have demonstrated that robotic proctectomy is not only feasible and safe, but also associated with lower conversion rates compared to laparoscopic proctectomy, albeit with longer operating times , 12. NevThe costs of robotic colorectal surgery are perceived to be substantially increased compared to conventional laparoscopic surgery –15. AlthA retrospective review of a prospectively maintained rectal cancer databases in a public quaternary centre  was carried out. Consecutive patients undergoing minimally invasive (laparoscopic and robotic) proctectomy between January 2017 and June 2020 were included. Patients who had resections due to lesions higher than 15 cm from the anal verge were excluded. Demographic characteristics, baseline clinical, tumour and operative variables, perioperative, histopathological outcomes and costs were compared between the laparoscopic and robotic groups. Subsequently, univariate, and multivariate analyses utilising demographic, baseline clinical and surgical variables were performed to identify the drivers of costs during the inpatient episode and examine the impact of the surgical approach on the overall costs.All cancer cases were discussed in a multidisciplinary team meeting. The surgical approach was decided on a case-by-case basis with input from a multidisciplinary team discussion and the robotic platform availability.th Edition for Cancer Staging) [The clinical and histopathological staging was recorded according to the TNM classification (AJCC 8Staging) . The preen-bloc removal of an adjacent pelvic organ due to invasion or circumferential margin threatening by the primary rectal tumour [Extended resections were defined as per the Beyond TME Collaborative as any procedure that required an l tumour . Pelvic Complications, readmissions, and mortality up to 90 days after surgery were considered and recorded. Complications were classified according to the Clavien-Dindo classification .The histopathological evaluation considered an R0 resection as a resection margin of > 1 mm. R1 resection was the presence of microscopic residual disease 1 mm or less from the resection margin, whereas R2 resection was the presence of macroscopic residual disease.Individual patients’ costs were retrieved via the Business Intelligence and Analytics Department of the hospital. These included real costs from the inpatient episode, thus no estimated values were used. All costs were recorded as Australian Dollars (A$) and were obtained from the databases in November 2021. Costs were classified in theatre, hospitalisation, and overall costs. Theatre costs included consumables, medications, nursing staff, theatre utilisation and recovery expenses. Hospitalisation costs included ICU and ward expenses, medical and nursing staff, allied health staff, medication, transfusions and laboratory and imaging testing costs. Overall costs were the combination of total theatre and hospitalisation costs.In our institution, a robotic colorectal surgery program was started in 2010 and taTME started in 2015. More than 200 robotic cases and more than 100 taTME procedures were performed by 4 and 2 surgeons, respectively, before the period of this study. All surgeons were formally trained in colorectal surgery and beyond the learning curve for laparoscopic TME. All surgeons were robotically credentialed by the industry . All patients had mechanical bowel preparation. General anaesthetic was given, and prophylactic antibiotics were administered at induction. An indwelling urinary catheter was inserted, and the patient was placed in the Lloyd-Davies position. All patients had sequential compression devices and low molecular weight heparin was given on induction, and throughout the hospital stay. The splenic flexure was mobilised in all restorative cases and selectively in non-restorative cases. For cancer patients, a high ligation of the inferior mesenteric vessels was performed. The approach was selected by the treating surgeon based on patient and team factors, plus the availability of the robotic platform. Laparoscopic TME was undertaken utilising a five-trocar technique. The dissection followed the TME plane which was continued down to the level of the pelvic floor and then, the rectal transection was carried out using laparoscopic linear staplers in restorative cases. All robotic TME cases were performed with the Da Vinci Xi Surgical System . Four robotic ports and an assistant port were used in all cases. Procedures were performed in a totally robotic fashion. After the pelvic dissection, rectal transection was carried out utilising robotic linear staplers if a restorative case was performed. When a taTME approach was added, the procedure was carried out as a synchronous two-team operation. The transabdominal component of taTME was either robotic or laparoscopic according to the surgeons’ preference and platform availability. Details of our setup and steps have been previously published by the authors . AnastomAll patients followed an ERAS protocol perioperatively.Continuous variables were described as mean and SD. Categorical variables were described as frequencies and percentages. Chi-square or Fisher’s exact tests were utilised to compare categorical variables. Students’ t-test was used to compare continuous variables. Given the skewed distributions of economic outcomes, a GLM with gamma distribution and log-link function was used to identify the drivers of overall costs in this study. Those variables with a P value < 0.1 in the simple linear regression analysis were considered significant and were entered into the GLM. Additionally, a GLM in a backwards fashion was conducted by manually removing variables starting from the one with the highest P-value until only keeping those significantly related to overall costs. All statistical analyses were performed using the IBM SPSS Statistics for Windows, Version 27.0 .A local Institutional Review Board ethics approval was obtained (QA/80014/PMCC-2021-283105).One hundred and thirteen patients underwent MIS proctectomy during the study period. Of these, 81 (71.7%) underwent a robotic proctectomy. In terms of operation variables, a taTME component was more often utilised in the laparoscopic approach . Two patients had a re-do proctectomy in the robotic group. The rest of the patients had a primary rectal resection. The complete list of demographics, clinical and tumour baseline variables and operative variables are summarised in Table Regarding perioperative outcomes, a robotic approach was associated with a lower conversion rate  and longer operating times . Causes for conversion in the robotic group were a proximal IMA lesion (n = 1), obesity (n = 1), and intraoperative bleeding (n =1). In the laparoscopic group the reasons for conversion included a difficult pelvic dissection (n = 4), inadequate bowel perfusion (n = 1), an anterior tumour perforation (n = 1) and an iliac artery lesion which was controlled with only 50 ml of bleeding (n = 1). Complications, reoperations, length of stay, readmissions, and surrogate oncologic markers were similar between groups  and overall costs . Hospitalisation costs were similar between both approaches . However, after performing a GLM including these factors, a robotic approach was not identified as an independent driver of overall costs during the inpatient episode Table . When adAn ASA ≥ 3, non-metastatic disease, and extended resections were significantly associated with increased overall costs in the multivariate analysis. Moreover, similar results were obtained after running the GLM in a backwards fashion and when adjusting for postoperative complications.The current study shows that a robotic approach is associated with a decreased conversion rate at the expense of a longer operating time compared to laparoscopic proctectomy. Moreover, despite theatre costs being higher for robotic proctectomy, overall inpatient costs seem to be similar to those of laparoscopic proctectomy when adjusted for confounders.Although robotic surgery is generally perceived as being more expensive than a conventional laparoscopic proctectomy, it could be said that the specialised literature is at least conflicting about the cost-benefit and cost-effectiveness outcome of robotic proctectomy. Several studies in the literature compare clinical outcomes and costs of laparoscopic and robotic proctectomy –19. MostOnly a few studies analyse the cost-effectiveness of robotic colorectal surgery. Simianu et al.  comparedAs opposed to the previously mentioned studies, Quijano et al.  in 2020 Explanations to this dissonance may be related to the different platforms utilised and institutional and individual surgeons’ learning curves. Newer platforms allow for an easier delivery of totally robotic procedures as opposed to hybrid approaches which require the concomitant use of laparoscopic and robotic equipment. In this line, recent studies show that the evolution of robotic platforms is associated with an increasing case volume and reduced operating times and costs within this field –27. It mThis study has several limitations to be addressed. As a retrospective cohort study, selection biases cannot be excluded, and the small number of patients make it subject to statistical type II errors. The fact of having a high rate of an added taTME component may also bias our results. However, our data shows that the addition of a taTME component to either a laparoscopic or a robotic approach was not associated with higher overall costs . The lower costs associated with taTME in the robotic group may be due to a statistical error secondary to the limited sample size (11 of 81 cases). Furthermore, indirect costs incurred due to losses such as lost wages, productivity, and costs resulting from the need for home care and others, were not measured in this case. Also, despite theatre costs are considered, the capital expenses of running and maintaining a robotic and laparoscopic surgery program are not evaluated in this study. Moreover, this work does not include the necessary variables to deliver a cost-effectiveness analysis which would be the ideal template to compare both approaches. The ideal setting to give an answer to this matter would be to perform a randomised controlled trial with costing and quality of life outcomes. Nevertheless, to our best knowledge, this is the first study comparing the costs of minimally invasive proctectomy within a public Australian healthcare system and provides an insight to potential areas of cost reduction to maximise the cost-benefit ratio of the approach.Robotic colorectal surgery is still in the early adopters’ phase where institutions are negotiating their learning curves. Regardless, limited trials show that robotic proctectomy is associated with an improved bowel recovery and lower conversion rates, at the expense of increased operating time . AdditioRobotic proctectomy is associated with increased theatre costs but not with increased overall inpatient costs within a public healthcare setting. Conversion was less common for robotic proctectomy at the expense of increased operating time. Larger studies will be needed to confirm these findings and examine the cost-effectiveness of robotic proctectomy.Supplementary file1 (DOCX 14 KB)Below is the link to the electronic supplementary material.


Environmental changes and global warming may promote the emergence of unknown viruses, whose spread is favored by the trade in plant products. Viruses represent a major threat to viticulture and the wine industry. Their management is challenging and mostly relies on prophylactic measures that are intended to prevent the introduction of viruses into vineyards. Besides the use of virus-free planting material, the employment of agrochemicals is a major strategy to prevent the spread of insect vectors in vineyards. According to the goal of the European Green Deal, a 50% decrease in the use of agrochemicals is expected before 2030. Thus, the development of alternative strategies that allow the sustainable control of viral diseases in vineyards is strongly needed. Here, we present a set of innovative biotechnological tools that have been developed to induce virus resistance in plants. From transgenesis to the still-debated genome editing technologies and RNAi-based strategies, this review discusses numerous illustrative studies that highlight the effectiveness of these promising tools for the management of viral infections in grapevine. Finally, the development of viral vectors from grapevine viruses is described, revealing their positive and unconventional roles, from targets to tools, in emerging biotechnologies. Vitis vinifera) is one of the world’s most significant fruit crops, standing out among cultivated plant species for its paramount socio-economic relevance. According to 2020 statistics from the Food and Agriculture Organization (FAO) of the United Nations, grapevines are cultivated on almost 6.9 million hectares, producing 78 million tons of fruit. More than 70% of the crops are earmarked for wine production, while limited fractions are consumed as fresh (table grapes) or dried fruit (raisins), processed to grape juice, or distilled to brandy.Grapevine  Panel on Plant Health (PLH) established a comprehensive list of viruses and viroids of different plant species, including ifera L. . About 8ifera L. ,4. Degenifera L. . The degifera L. . Both clifera L. ,8. Rugosifera L. .Agricultural practices, mixed infections, spillover from reservoir host species, and impacts of a changing environment and global warming pose continuous challenges for the management of epidemics that result from the emergence of novel plant viruses . In the Grapevines infected by viruses cannot heal, since the plants are not able to defeat the virus(es). The outcome of the never-ending process of grapevine–virus co-evolution is extremely difficult to predict. A latent infection that has never caused severe symptoms or drastic physiological alterations may scale up quickly to a serious disease following environmental changes and/or in the presence of mixed infections . TherefoSeveral factors may affect the outcome of sanitization procedures, significantly decreasing their efficacy. Thermotherapy is highly recommended against viruses located in parenchymatic tissues, while heat treatments are less effective for phloem-restricted viruses . SimilarBesides such prophylactic measures, agronomic practices, including rogueing of infected grapevines and replacement of entire diseased vineyards with healthy planting material  and the According to the recently published European Green Deal plan, a 50% reduction in the use of agrochemical products is expected before 2030 . Thus, tTraditional plant breeding has been widely employed to improve disease resistance and tolerance to abiotic stresses in elite cultivars of grapevine. This approach relies on the exploitation of the genetic variability among individuals of the same species by combining the desired traits into new and improved varieties. V. vinifera is the most commonly used Vitis species in the grape and wine industry, favored for its superior aroma and flavor characteristics. Interestingly, V. vinifera has little or no genetic resistance against pathogens, rendering it highly susceptible to numerous diseases [Vitis species have been identified, impeding the employment of traditional plant breeding to confer virus resistance in grapevine [omic” technologies [Xiphinema index [rgflv1. The most important feature of the resistance provided by rgflv1 is its monogenic determinism, which makes it stable through transmission to offspring and easy to use in breeding programs, despite being recessive. The discovery of rgflv1 may pave the way for the first effective and environmentally friendly solution to control grapevine fanleaf disease through the development of new GFLV-resistant grapevine rootstocks, which was hitherto an unthinkable prospect [rgflv1 against GFLV F13 strain may be effective against other GFLV strains.The Eurasian grapevine species diseases . To daterapevine ,35. Neverapevine ,39,40,41nologies  have sigma index . This reprospect , althougSimilarly, comparative genomics has revealed a myriad of viral sequences that are integrated, or ‘endogenized’, in the nuclear genomes of plants ,45,46. ICaulimoviridae family) was identified through an in silico approach in annotated genomes of two Pinot noir plants [The presence of numerous potential ORFs whose deduced protein sequences exhibited high identity to the protein sequences of distinct pararetroviruses  prior to the occurrence of viral infection represents an efficient strategy to protect plants from destructive viruses ,50. RNA Historically, the early activation of antiviral RNA silencing before the occurrence of viral infection was achieved by the cross-protection strategy, which entailed plant pre-immunization with mild or avirulent viral strains that impeded the onset of severe viral infections due to the initiation of superinfection exclusion mechanisms ,54. DespAntiviral defense may also be activated transgenically through the host-induced gene silencing (HIGS) approach. This mechanism relies on the insertion of a viral transgene into the grapevine genome which readily activates RNA silencing . Le GallGrapevine fanleaf disease threatens vineyards worldwide and it has always drawn a great deal of attention, which is likely due to the devasting impact and the challenging management of the disease in vineyards ,62. In tTechnical and biological difficulties, the long time required, and strong rejection by consumers, as well as restriction by international legislation, have strongly hampered the wide exploitation of transgenic approaches in grapevine. Spray-induced gene silencing (SIGS) may be considered the “non-transgenic alternative” to the HIGS strategy . This meThe triggering of RNA silencing via exogenous delivery of RNA molecules represents a more complex phenomenon, mostly due to the requirement for local and systemic spread of RNA molecules in plant tissues ,72,73. AElucidating the molecular mechanisms of SIGS and overcoming technical limits are key objectives for the successful future implementation of this novel biotechnological approach in agriculture. Since the early 2000s, new breeding technologies (NBTs) have evolved rapidly, providing a suite of innovative biotechnology-based methods designed to improve plant traits more rapidly and precisely than traditional plant breeding approaches ,77. Among NBTs, genome editing strategies are sparking increasing interest in agriculture, representing valuable tools for crop improvement and enhancement of disease resistance. These technologies entail the use of sequence-specific nucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR/Cas9 system, which can be programmed to target virtually any DNA sequence of interest in the genome . CompareThe CRISPR system was first discovered in prokaryotes as a component of their adaptive immunity, conferring resistance to invading pathogens, such as bacteriophages and viruses . scaffold” sequence, which directly binds the Cas9 nuclease, and a 20 nt sequence named “spacer”, which plays a crucial role in Cas9 recruitment [The use of CRISPR/Cas9 as an RNA-programmable DNA editing platform requires single-guide RNA (sgRNA), consisting of a “ruitment . Essentiruitment ). Once truitment ,82, or vruitment . In recent years, the use of CRISPR/Cas9 to gain virus-resistant grapevine cultivars has raised significant interest. In this regard, Jiao and colleagues exploited this technology to target GLRaV 3, attempting to improve grapevine resistance to viral infection . The autInstead of targeting viral RNA (virus-mediated resistance), CRISPR/Cas technology may also be employed to manipulate the plant genome (plant-mediated resistance) by editing gene sequences that are putatively involved in viral infections . Among tNevertheless, CRISPR/Cas9 technology also shows several limitations that should be considered in order to establish the most effective antiviral strategies. The occurrence of off targets represents the main drawback of the CRISPR/Cas system because they may lead to the knockout of essential host factors, resulting in the impairment of plant development, or, in the most severe cases, be lethal to the plant . Agrobacterium-mediated transformation, and they may be further reduced by the use of Cas variants with higher specificity [Despite the frequency of off-target mutations in grapevine due to the CRISPR/Cas system, they are likely insignificant compared to variations caused by tissue culturing and/or cificity  or by emcificity ,94. Moreover, to avoid the evolution of viruses with mutations allowing escape from CRISPR/Cas cleavage, an accurate selection of reliable sgRNA sites is strongly recommended ,96. In tThanks to their small-sized genomes and their autonomous replication, plant viruses are ideal for engineering as vectors for the expression of heterologous proteins, avoiding stable transformation and the production of genetically modified (GM) plants ,98. SeveN. benthamiana plants [Agrobacterium tumefaciens-mediated expression between the cauliflower mosaic virus (CaMV) 35S promoter and ribozyme sequence immediately upstream of the nopaline synthase terminator sequence [Infectious cDNA clones of GLRaV-2 have been exploited in a reverse genetic study to investigate the functional role of papain-like L1 and L2 proteases in the viral replicative cycle, assessing their eventual involvement in the systemic spread of the virus in a plants . The entsequence . The ressequence . N. benthamiana. Elsewhere, the GLRaV 2 replicons did not show tropism, since the expression of reporter genes was exclusively detected in proximity to the agroinfiltrated leaf areas [In RNA virus-based vectors, the insertion of sg promoters belonging to closely related species has been shown to be preferable to promoter duplication because of the rapid loss of identical sequences due to homologous recombination . In addiaf areas . N. benthamiana led to the adaptation of the virus to the new host plant, selecting a viral variant of GLRaV-2 that was unable to infect grapevine [This first generation of full-length cDNA clones of GLRaV 2, however, was not able to systemically infect grapevine and therefore could not be exploited as a viral vector in this host . The mairapevine . V. vinifera plants. Expression of GFP was monitored in phloem cells of roots and leaves, as well as in berries. In contrast to other viral vectors based on tobamoviruses and potyviruses, vLR2-GFP revealed considerable genetic stability, persisting in grapevine and maintaining the exogenous insert for more than a year after agroinfiltration [A new GLRaV 2 vector was generated from RNA collected from Pinot noir plants . This noltration . Furtherltration . This second generation of GLRaV 2 vectors was successfully used to promote VIGS. Instead of (or immediately after) the GFP expression cassette, the introduction of cDNA sequences encoding the phytoene desaturase (PDS) or the subunit I of magnesium-protoporphyrin chelatase (ChlI) promoted suppression of the cognate gene in grapevine through the activation of gene silencing mechanisms. The simultaneous expression of GFP and the adjacent coding sequence targeting the grapevine’s endogenous genes allowed observation of the VIGS response in relation to viral tropism in the plant. After infection, chlorophyll bleaching was observed first in the veins where GLRaV 2 replicated, then, following transitivity, in phloem tissues far from the viral replication site. VIGS persisted for months after viral inoculation, with symptoms appearing cyclically and depending on the growth stage of the plant, tissue differentiation, and viral spread . Moreover, since GLRaV 2 has no natural vectors and can only be transmitted by grafting, any potential biosafety risk(s) related to the release and fate of GLRaV 2-derived vector(s) in the environment is limited. Vitivirus, can also be used as a viral vector to express exogenous genes or for VIGS experiments [N. benthamiana to express reporter genes  or proteins of unrelated viruses [GVA, belonging to the genus eriments . The GVA viruses . As a pr viruses . N. benthamiana and in grapevine plants. The viral cDNA was inserted into a binary vector under the transcriptional control of two CaMV-35S promoters and a single CaMV-35S terminator. To assess the effectiveness of this VIGS approach, a sequence homologous to partial regions of PDS genes from either N. benthamiana or grapevine was introduced in place of the reporter gene cassette [N. benthamiana plants agroinfiltrated with a GVA-derived vector carrying the PDS construct, chlorophyll photo-bleaching was limited to the leaf veins [In subsequent work, the vector was further engineered to perform VIGS experiments in cassette . In N. baf veins . Surprisaf veins , symptomaf veins .Flexiviridae family, has also been engineered to express enhanced-GFP (eGFP) in both N. benthamiana and grapevine [N. benthamiana in proximity to the agroinfiltrated area, in grapevine the GRSPaV viral vector harboring the eGFP construct was able to spread systemically, since fluorescence signals were readily reported in distal tissues as well. However, confocal microscopy observations showed eGFP fluorescence only in restricted clusters of a few cells. This aspect has been putatively correlated either with a low titer of virus-derived vector or with loss of the eGFP expression cassette. Despite its potential, the ability of the GRSPaV viral vector to induce host gene silencing has still not been analyzed [The foveavirus grapevine rupestris stem pitting-associated virus (GRSPaV), which, like GVA, belongs to the rapevine . The GRSanalyzed .Limonium sinuatum) and nipple fruit (Solanum mammosum) [N. benthamiana and several grapevine varieties, including Syrah, Brachetto, Nebbiolo, Sultana, and Corvina, which were found to be susceptible to systemic infection by the synthetic virus [N. benthamiana plants, the GFP sequence was added to the construct, which was named pGMG [N. benthamiana following knockout of the p19 suppressor [N. benthamiana plants. The typical chlorophyll bleaching phenotype was clearly detected in systemic leaves of infiltrated plants, demonstrating the potential of the GALV-derived pGMG19m vector to be employed in a VIGS approach [V. vinifera has yet to be assessed.Additionally, the tombusvirus grapevine Algerian latent virus (GALV) has been employed as a viral vector. GALV was initially detected in grapevine  and lateammosum) ,109. GALammosum) . Infectiic virus . Afterwaic virus . In ordemed pGMG . The necmed pGMG . A weak ppressor . This noapproach . DespiteGeminiviridae family represents the largest family of DNA viruses, consisting of numerous viral species characterized by circular ssDNA genomes [DNA viruses can also be employed as vectors for the delivery of genetic information into host cells and/or for gene editing technology . The Gem genomes . Geminiv genomes ,115. In  genomes ). When u genomes . Besides genomes . Agrobacterium as a carrier [Although some geminirus-derived vectors, such as tomato yellow leaf curl virus (TYLCV) and bean yellow dwarf virus (BeYDV), have already been successfully employed in grapevine studies involving functional genetic  and geno carrier . Environmental change, global warming, and trade in propagation material are only a few of the factors that are favoring the onset and spread of novel viruses ,120,121,Given the impending drastic reduction in the use of chemical pesticides , implementation of the innovative biotechnological tools that have been developed so far represents the only promising strategy for the sustainable control of disease in vineyards.Vitis germplasm, severe inbreeding depression, long lifecycle, and lack of knowledge of resistance genes or the genetic architectures of the relevant traits have strongly limited the use of traditional plant breeding in controlling grapevine viruses, thus promoting the use of transgenic strategies [The scarcity of sources of viral resistance in the rategies .In grapevine, transgenic approaches are highly laborious due to its recalcitrance to both transformation and in vitro regeneration. Moreover, these techniques are expensive and time-consuming, requiring several years, to produce new varieties. Besides these technical and biological limitations, the constitutive expression of viral transgenes with the potential to induce virus resistance was also found to drastically interfere with plant development, causing serious morphological and physiological anomalies in transgenic grapevine . These aThe powerful new breeding technologies (NBTs) are expected to make substantial contributions to tackling the future challenges faced by the grapevine and wine industry. Among them, CRISPR/Cas9-mediated genome editing represents a promising technology for the development of virus-resistant varieties. However, three main drawbacks are impeding the implementation of genome editing: controversial regulation, high costs, and social and market acceptance. The regulatory aspects have a major influence on the others, since the lack of a clear recognition of NBT-derived varieties as either genetically modified (GM) or non-GM organisms may encourage their acceptance, promoting investments in both research and commercialization. In fact, unlike classical transgenic approaches, NBTs include a range of new strategies, such as genome editing technologies, such that the resulting varieties may not be considered genetically modified organisms (GMOs), depending on how the NBTs are applied. As an alternative to NBTs, plant immunization via the exogenous application of RNA molecules that are capable of activating plant defenses prior to the establishment of pathogenic infections  provides a novel, non-transgenic means for the sustainable control of disease in grapevine, which may replace the use of agrochemicals in vineyards.


Many different enteric viruses can cause acute gastroenteritis in humans worldwide. While a single virus can indeed cause disease, multiple-virus infections are commonly reported. However, data regarding a comparison between single- and multiple-virus infections upon clinical manifestations of acute gastroenteritis are relatively limited. In this study, a total of 2383 fecal specimens were collected from children with acute gastroenteritis during June 2014–July 2017 in a pediatric clinic in Japan and tested for 11 viruses by multiplex RT-PCR. At least 1 virus was found in 1706 (71.6%) specimens and norovirus GII was the most frequent agent, followed by rotavirus A and other viruses. Multiple-virus infections were identified in 565 cases (33.1%). While major clinical symptoms were found to be significantly different in some single- vs. multiple-virus infections, the disease severity was statistically non-significant. Our study highlights the burden of multiple-virus infections for acute gastroenteritis and the clinical features of patients with multiple-virus infections. Acute gastroenteritis (AGE) is a major cause of morbidity and mortality, particularly in children and the elderly. While mortality rates declined quite rapidly in many countries, AGE is still responsible for an estimated 533,768 deaths in children under 5 years old . AGE is A total of 2383 fecal specimens were collected from 1245 children with AGE during July 2014–June 2017 and tested for enteric viruses. It was found that 1706 were positive for 1 or more viruses. NoV GII, the most common virus, was detected in 1229 fecal specimens, followed by RVA and EV in 514 and 241 fecal specimens, respectively . Other vp = 0.008), abdominal pain (p = 0.036), and diarrhea (p = 0.001) were observed between NoV GII and RVA; however, there was no significant difference in vomiting (p = 0.1) between NoV GII and RVA. It is interesting that the significant differences in vomiting were seen between NoV GII and other viruses, including EV (p = 0.0038), PeV-A (p < 0.001), and HAdV (p = 0.015). Similar to NoV GII vs. RVA (p = 0.001), significant differences in diarrhea were also seen between NoV GII vs. PeV-A (p = 0.0001) and NoV GII vs. HAdV (p = 0.0009), except NoV GII vs. EV (p = 0.73). Pairwise comparisons of the major symptoms’ characteristics were also performed for common single-virus infections and multiple-virus infections, as shown in p < 0.0001) and duration (p = 0.0024) of fever were significant in only EV vs. NoV GII. There were significant differences in both the frequency and duration of vomiting in four groups, including EV vs. NoV GII, EV/NoV GII vs. NoV GII, PeV-A/NoV GII vs. PeV-A, and PeV-A vs. NoV GII. In addition, both the frequency and duration of diarrhea were significantly different in three groups, including RVA/NoV GII vs. NoV GII, RVA vs. NoV GII, and PeV-A vs. NoV GII. Disease severity was assessed using a clinical Vesikari score, and its median score was four or five for these common single-virus infections and multiple-virus infections. Disease severity was considered to be statistically non-significant  were performed for some single-virus infections and multiple-virus infections, which were the most commonly detected, as shown in nificant .®  performed an epidemiological study in outpatient children with AGE in China and reported that RVA (43.3%) was the most prevalent virus, followed by NoV GII (28.9%) . In anot15 perforlent G1P  live-att3, G4, P  live-att3, G4, P . Taken tp = 0.047 and p = 0.0088, respectively); fever and vomiting were not in this combination , as shown in p = 0.0004 and p = 0.017, respectively), other symptoms (fever and vomiting) were statistically non-significant  test was used for the comparison of single-vs. multiple-virus infections with major symptoms such as fever (>38 °C), abdominal pain, vomiting, or diarrhea. The Mann–Whitney U test was used for a comparison of single- vs. multiple-virus infections with other clinical features such as the maximal degree and duration of fever or frequency per day and the duration of vomiting or diarrhea. Data were analyzed using the JMP statistical software program . Differences with p < 0.05 were considered statistically significant. This study was performed with the approval of the Ethical Committee of Nihon University School of Medicine (No. 29-9-2) in accordance with the guidelines of the Declaration of Helsinki . Informed consent was obtained from the parents of the children enrolled in this study.Fecal specimens were collected from all children with diarrhea who visited Hikita Pediatric Clinic in Kiryu city, Gunma prefecture, Japan, at any time from June 2014 to July 2017. Diarrhea was defined as ≥3 loose or watery feces per 24 h. The study population included 1245 children  with a median age of 2.6 years. At the time of clinic visits, these children did not have other disease conditions and no medications were being taken. Fecal specimens were stored at −30 °C until use. The viral genome was extracted by using a QIAamp Viral RNA mini kit . Reverse transcription (RT) was performed with superscript reverse transcriptase III  with a random primer . The specimens were analyzed using multiplex RT-PCR assays with three different primer sets. The first primer set could detect RVA, RVB, RVC, and HAdV, the second set NoV GI, NoV GII, HAstV, and SaV, and the third set AiV, PeV-A, and EV . The dat


Background: Previous research demonstrated that utilization management (UM) such as prior authorization (PA) or non-formulary (NF) restrictions may reduce pharmacy costs when designed and applied appropriately to certain drug classes. However, such access barriers may also have unintended consequences. Few studies systemically analyzed the impact of major UM strategies to extended-release (ER) opioids on different types of health plans.Objective: This study evaluated, from payer perspective, the impact of formulary restrictions  for branded oxycodone HCl extended release (OER) on market share, and healthcare resource utilization/costs in ER opioids patients for multiple types of health plans in the United States.Methods: This retrospective, longitudinal case-control study analyzed prescription and outpatient medical claims data (2012 to 2015) for adult ER opioid patients from US plans  that instituted OER PA, NF, or ST. Patients from each restricted plan (cases) were matched to patients in an unrestricted plan (controls) on key patient characteristics. ER opioid market share and healthcare resource utilization/costs for both cases and controls were evaluated for the 6-month period before and after the formulary restriction dates. A difference-in-differences (DiD) approach was utilized to evaluate change in the total per patient per month (PPPM) healthcare utilization and costs.Results: The study comprised 1622 , 2020 , 34 703 , and 4372  cases and equivalent number of controls. OER market share decreased after the formulary restrictions, with the national Medicare NF plan showing the greatest decrease (9.2%). DiD analyses indicated that PPPM office visit change in the PA and NF plans were non-significant , but significant in the ST plan . For most plans, no significant total monthly cost change was observed; PPPM costs decreased by $48.74 and $59.87 in ST and regional PA plans and increased by $37.90 in national NF plans .Conclusions: This study observed that despite reducing the market share of OER, OER formulary restrictions had negligible impact on overall ER opioid utilization, and did not result in substantial pharmacy/medical cost savings.


Results showed that sanitary status of each bull was not associated with semen quality. It was found that the quality of the semen from the second collection was better due to the interruption of sexual rest. The evidence thus revealed that a bull infected with BLV generated good-quality contaminated semen and, therefore, that it is essential to detect contaminated seminal samples to prevent the spread of BLV. A multivariate analysis showed the presence of four sperm subpopulations in Brahman bulls that differ significantly in their kinematic patterns and with respect to sanitary status (P < 0.05), indicating that infection-free and seronegative bulls present the best kinematic parameters, which improved discrimination of sperm quality according to sanitary status. Overall, the analyses indicate that the seropositive-infected bulls with BLV and BHV-1 should be excluded from beef cattle farms.Enzootic bovine leukosis virus (BLV) and bovine herpesvirus 1 (BHV-1) are very important infectious agents for the livestock industry worldwide. The present study aimed to explore the association between natural exposure to BLV and BHV-1 with sperm quality analyzed by Computer-Assisted Semen Analysis (CASA) systems. Ten sexually mature Brahman bulls, with sanitary status BLV Fertility potential of a bull is observed mainly through the analysis of sperm quality2. In extensive systems of beef cattle breeding, fertility evaluations revealed that only 20% of bulls are suitable for mating2. Consequently, the low availability of bulls with acceptable fertility is a problem and predicting it is complex3, even more when the number of infertile or sub-fertile bulls is related to infectious diseases during their productive life. These infections can cause temporal or permanent infertility problems.Reproductive efficiency has an important impact on the profitability of the livestock industry. Reproduction is a very complex process which depends on the fertility qualities of the cow and the bull, with a significant percentage of reproductive failure attributable to bull subfertilityTritrichomonas, Neospora and Campylobacter. Other important endemic agents are enzootic bovine leukosis virus (BLV), Leptospira, and Brucella abortus6. A single exposure to BHV-1 can have a high economic impact and commercial importance for artificial insemination (AI) centers due to impaired sperm quality or transmission risk to healthy bulls7. The same applies to BLV10. Both viruses, BLV and BHV-1, are frequently found in multiple infections in endemic regions12.Among the most common infectious agents associated with reproductive failure are bovine viral diarrhea virus (BVDV), bovine herpesvirus 1 (BHV-1), 15. It is known that the association of BLV and BHV-1 have a negative effect on semen quality9, but their multiple effect on bull sperm function has not been elucidated. BHV-1 has been reported to cause infertility or reduced reproductive efficiency, although other authors found no significant association between simple exposure to the virus and seminal variables such as concentration, volume, mass and progressive motility2. Another study determined in bulls infected with BHV-1 an increase in sperm morphological abnormalities, decreased motility and swimming capacity, membrane integrity, and sperm concentration compared to non-infected bulls7, However, in both studies seminal parameters were assessed by subjective analyses and sperm quality has been just examined using optical microscopy observations.Depending on the animals' physiological conditions and environmental factors, simultaneous viral infections show interactions that can lead both to increased or reduced adverse effects on health status18. Therefore, the aim of this study was to explore the association between exposure to BLV and/or BHV-1 with semen quality determined by CASA technology.Currently, a commercial computer-assisted semen analysis system (CASA) allows to determine with precision and objectivity parameters such as sperm swimming, kinematics, morphometrics sperm variables and sperm concentration to evaluate semen quality and to be able to predict bull fertility+/BHV-1+), six bulls were seropositive and latently infected with BHV-1 (BLV−/BHV-1+), and two bulls were negative for the presence of antibodies and antigen associated with the BLV and BHV-1 (BLV−/BHV-1−). All bulls maintained the sanitary status throughout the investigation. Furthermore, only one seminal sample from bull 498 was positive to BLV at 0 day but it was negative at 30 days . The BLV detected in blood and seminal samples of bull were characterized as genotype 5 of BLV . No blood and semen samples were positive for the presence of BHV-1.In this study, blood samples showed that two bulls were seropositive and infected with BLV and BHV-1  and kinematics variables did not show a change in trend between evaluations (0 and 30 days) or between the sanitary status of the bulls that would allow differentiation or increase the discriminant potential on the seminal quality initially characterized based on the percentage of normal morphology and viability  of the bulls showed at least one of the seminal parameters below the expected limit of detection for a sample with satisfactory seminal quality. At 30 days, only 40% (4/10) of the bulls showed doubtful seminal quality and the proportion of bulls with satisfactory seminal quality increased  of bulls had sperm with proximal droplets, which ranged from 0.47 to 9.34%; 90% (9/10) of bulls had sperm with loose heads, which ranged from 0.47 to 18.93%; 90% (9/10) had sperm with tightly coiled tail, which ranged from 0.41 to 7.14% and, finally, 80% (8/10) had distal droplets, which ranged from 0.41 to 1.90%. There were also two rare anomalies in bulls, namely double heads and bent midpiece Table .Table 3SThe PFA rendered two principal components (PCs) for sperm kinematic variables, explaining 84.51% of the total variance  were identified; they showed clear differences in most kinematic parameters Table . SP1 had−/BHV-1+ (SS2) had the highest percentage of spermatozoa (54.3%), followed by BLV−/BHV-1+(SS1) with 23.1%, and finally by BLV−/BHV-1− (SS3) with 22.7% of total spermatozoa analyzed. There was a higher percentage of SP4 in SS1 y SS2 , but in SS3 was SP3 (7.6%)  with VCL and VAP exhibiting the highest differences between the sub-populations Table . There w21 but contrast with other studies which found that the percentage of abnormal spermatozoa was not a good predictor of fertility level22.The results of this study showed that the percentage of normal sperm morphology and sperm viability were the variables with the greatest discriminating potential for samples with satisfactory, doubtful, and unsatisfactory seminal quality. These results agree with those reported earlier25. However, there were three abnormal sperm morphology defects  that overcame 6% of individual thresholds for each abnormality26. The concept of sperm with compensable or uncompensable traits allows to differentiate thresholds as the case may be27. For example, compensable traits such as loose heads preclude affected sperm from fertilizing the ovum and the threshold in this case can be 30%26. For uncompensable traits the authors suggested that threshold of such abnormalities should not surpass 20%28. At 30 days, all the bulls showed a percentage of abnormal spermatozoa lower than 30%, a value associated with satisfactory quality for the seminal sample29. The prevalence of morphological abnormalities provides very valuable information to assess reproductive health associated with spermatogenesis26. When considering the prevalence of each sperm abnormality in each bull, the presence of proximal droplets was the most prevalent sperm abnormality, followed by loose heads, tightly coiled tails and distal droplets, which are within the 3 major types of sperm defects described for beef bulls30. The most serious morphological abnormalities were proximal droplets and loose heads. The high percentage of proximal droplets was 8.5% (Bull 446) but counts of 10–15% have been associated with decreased fertility31. Also, the highest percentage of detached heads was 18.9; although the bull can still be considered “fertile” with 30–40% of this defect, the ejaculate was considered infertile due to low viability (4%) (Bull 545). It has been described that the main causes of spermatozoa with detached heads are testicular hypoplasia, testicular degeneration and senescence of spermatozoa due to sexual inactivity. In this study the seminal samples of bulls were evaluated after the first ejaculation30. Our results were below threshold sperm values for compensable or uncompensable traits and provide evidence for the association between the sanitary status of seropositive-latently infected bull with BHV-1 and sperm morphology in Brahman bulls.Our results indicate that bulls presented a spermiogram, which contained < 30% abnormal sperm, as a threshold value described in other studies30. In this sense, the highest values for normal morphology, progressive motility and viability are positively associated with a higher non return rate than the average fertility in the group of bulls, but lower values of the seminal parameters expected as satisfactory for an ejaculate can be due to variability and do not indicate that the bull is subfertile. However, they do suggest that the bull with lower values should not be used for natural mating or as a semen donor in AI programs at the time32. Consequently, in this study high variability in sperm viability was observed between bulls with values that ranged from 4 to 93.8%. Particularly, in the group of bulls positive for BHV-1, it was observed that 50% (3/6) did not exceed 50% of viability, in accordance with what was reported by El-Mohamady et al.33 although it indicated that BHV-5, a virus similar to BHV-1, does not induce functional and morphological damage of infected spermatozoa. In this study the type of morphological abnormalities was very diverse and the percentage of abnormalities decreased in the second sample  compared to the first evaluation , which suggests an effect of the sampling period rather than the effect of the virus on semen quality, contrary to what was reported by El-Mohamady et al.7 because it was probably due to sperm turnover, in accordance to that indicated by the ABBA (2015). Recently, it has been reported in Brahman bulls that sexual rest decreases sperm quality34. More research is necessary on the issue of dead sperm in inactive/rested bulls and exploring the possibility that sperm quality is better in the second sample.In general, it is accepted that bulls with values for progressive motility greater than 50% and normal morphology greater than 70% should be above the average fertility calculated for the sire group35. The differences observed in values of semen parameters for each bull, considering the sanitary status and the evaluations (0 and 30 days), show considerable variability and no clear associations were observed2. Furthermore, some authors suggested that BHV-1 does not affect semen quality36. Our findings indicate that although there were differences between sanitary status of Brahman bulls in kinematics variables, this did not appear to have a biological relationship with velocity, linearity, or undulation of sperm cells.In reference to the CASA variables, it seems that they do not contribute to differentiate seminal quality with a discriminant effect37. The analysis of sperm subpopulations by sanitary status provides evidence that the pattern of high velocity and progressive motility is associated with BLV−/BHV-1+ and BLV−/BHV-1−, this suggestes that seropositive-infected bulls with BLV and BHV-1 should be discarded as sires.Based on CASA-Mot variables, our results identified four sperm subpopulations in Brahman bulls. The differences between sperm subpopulations (P < 0.05) suggest that sperm kinematics patterns with very active but non-progressive movement or rapid and progressive sperm movement may be indicative of early capacitation and hyperactivated-like motile spermatozoa. Other sperm subpopulations contained poorly motile sperm, and others presented non-progressive sperm and low velocity but greater progressive kinematics traits which highlights the heterogeneous nature of sperm ejaculates of fresh semen in Brahman bullsThe evaluation of seminal quality is very complex. Apparently, the variables that best discriminate between the quality of the ejaculates are normal morphology and sperm viability. The CASA-Mot variables do not reveal a clear trend with regards to the quality of semen samples. Taken together, the sanitary status of bulls did not show a clear change in the trend of the variables that define semen quality. However, the results indicated that the bulls infected with BLV or BHV-1 produce semen with variable quality, and that in most of the bulls the semen was free of the presence of the virus, but the use of bulls infected with BLV can generate semen contaminated in an intermittent way. The sperm subpopulation analysis indicated that seropositive-infected bulls with BLV and BHV-1 should be excluded from beef cattle farms.https://arriveguidelines.org/; accessed on 8 November 2021).This study was performed following ethical principles and with the approval of the Committee of Centro de Investigación y Desarrollo de la Agricultura Sostenible para el Trópico Húmedo at the Costa Rica Institute of Technology (CIDASTH-ITCR) according to section 01/2019, article 1.0, DAGSC-074-2019. The care of the animals during the research complied with the animal welfare guidelines of Costa Rica Institute of Technology. Bulls were grazed and sampling procedures were reviewed and approved by the Production Committee of the Agricultural Production Program of the School of Agronomy at the Costa Rica Institute of Technology (PPASC-106-2021). A statement about plant ethics is not relevant because no experimentation is done on plants. The study was carried out in compliance with ARRIVE guidelines  were employed in this study. The animals came from a commercial farm located in San Carlos, Costa Rica. The bulls had passed a standard breeding soundness evaluation and had produced sperm with acceptable post-thaw characteristics (progressive motile sperm > 60%) and fertility (non-return rates > 65%).+/BHV-1+, (n = 2); BLV−/BHV-1+, (n = 6) and BLV−/BHV-1−, (n = 2)]. The age of the animals ranged from 25 to 84 months, body weight ranged from 400 to 850 kg, mean scrotal circumference was 37.6 ± 2.9 cm, and mean scrotal temperature was 32.1 ± 0.5 °C. Bulls included in the study were negative for the presence of antibodies and antigens associated with circulating bovine viral diarrhea virus. On the farm, the animals were not vaccinated against infectious bovine rhinotracheitis.The sanitary status of bulls against BLV and BHV-1 was previously established by enzyme-linked immunosorbent assay (ELISA) [BLVIschaemum indicum) and Mombaza (Panicum maximum). Animals were fed with grass without restrictions of access to forage consumption and they were supplied with salt, minerals and water ad libitum. The sanitary management of the bulls were an external dewormer , water-soluble vitamins  and minerals (Matsuda breeding Top Line). General inspection of the bulls was made, focusing on anatomical aspects such as conformation of legs and feet, body condition, coloration of mucous membranes and particularities of each animal. The condition of the external reproductive organs, such as the testicles, scrotum, foreskin, and penis were also examined.During the experimental period, the animals were handled in an area of 18 hectares, a rotational system of seven apartments was established, with six days of occupation and 42 days of rest, with Ratana grass  using a commercial egg yolk-free diluent . Prior to semen dilution, a 1 mL aliquot of raw semen was placed in 1.5 mL sterile microtubes. Semen samples were assessed for gross motility, by placing 20 µL of fresh raw semen on a prewarmed slide at 37 °C, and for concentration, employing a bovine photometer AccuRead®  at 530 nm wavelength. Diluted semen samples were placed on a heating plate at 37 °C and then transported in a polyurethane cooler at 25 °C. Subsequently, the raw semen samples were frozen at − 20 °C until nucleic acid extraction.Semen was collected by electroejaculation using the Pulsator V® equipment  as described by Montoya-Monsalve et al., and Víquez et al.38 and at least 200 spermatozoa were counted to determine the percentage of abnormalities22. All samples were coded in such a way that the technician who performed the analysis did not know the number of the bull, the number of the ejaculate, or which ejaculate belonged to a particular bull.Viability, percentage of sperm abnormalities and sperm morphology were assessed by preparing a smear of the semen sample using 10 µL of diluted semen stained with eosin-nigrosin as described by Kumar et al.g 5 min, an aliquot of 1.5 mL was taken and stored in 1.5 mL microtubes at − 20 °C until assessment.After semen extraction, blood samples with and without anticoagulant were taken by coccygeal vein puncture using the Vacutainer system, and placed in a cooler at 4 °C. An aliquot of 1.5 mL of blood with anticoagulant was deposited in 1.5 mL microtubes and frozen at − 20 °C until the extraction of nucleic acids. Serum was separated by centrifugation at 3500×39. After thorough mixing of the diluted semen samples, 2.7 µL of diluted semen were placed in the counting chamber tracks by drop displacement according to Valverde et al.40. Analyses were conducted with the CASA-Mot system ISAS®v1  fitted with a video-camera , a frame rate of 50 frames per second (fps) and a final resolution of 768 × 576 pixels as described by Valverde et al41. The camera was attached to a microscope UB203 (UOP/Proiser R + D) with a 1X eyepiece and a 10X negative-phase contrast objective (AN 0.25), and an integrated heated stage maintained at 37 ± 0.5 °C.Reusable counting chambers with cover slide for the analysis of motility and kinematics variables  were used after pre-warming them to 37 °C−1), corresponding to the straight line from the beginning to the end of the track; curvilinear velocity , measured over the actual point-to-point track followed by the cell; average path velocity , the average velocity over the smoothed cell path; amplitude of lateral head displacement , defined as the maximum of the measured width of the head oscillation as the sperm swims; beat-cross frequency , defined as the frequency with which the actual track crosses the smoothed track in either direction; motility (%), the percentage of total motile cells and progressive motility (%), corresponding to spermatozoa swimming rapidly forward in a straight line . Three progression ratios, expressed as percentages, were calculated from the velocity measurements described above: linearity of forward progression (LIN = VSL/VCL·100), straightness (STR = VSL/VAP·100), and wobble (WOB = VAP/VCL·100)17.CASA analyses were performed recording seven microscope fields with a total of at least 600 cells per sample. The CASA-Mot variables assessed in this study included: straight-line velocity  according to the manufacturer's instructions, respectively.42.Genomic DNA extraction was performed from 300 µL of blood with EDTA and from semen diluted 1:5 (vol:vol) using Wizard® Genomic DNA Purification Kit  according to the manufacturer’s instructions for DNA extraction from samples of frozen blood. The DNA obtained was stored at -75 °C until use. BLV proviral DNA was identified by semi-nested PCR. In the first round, a set of primer targeting on gene encoding gp51 (envelope protein) were used, the forward primer was Env1: 5′-ATGCCYAARRAACGACGRYCCCG-3′ (4826–4848), and the reverse primer was Env3: 5′-GAGGGCGAGRCCGGGTCCAGAGC-3′ (5550–5528), while the second round was performed with the forward primer Env2: 5′-CAAGGGCGRCGCCRRTTYGGAGC-3′ (5108–5124) and the reverse primer Env3 described above. The final volume of each PCR reaction was 20 µL and the mix consisted of 10 µL of Phusion U Multiplex PCR Master Mix #F-562S ; 1.2 μL (0.3 μM/μL) of each primer pair Env1(+)/Env3(−) in the first round and Env2(+)/Env3(−) in the second round, 6.6 μL of RNase-free water  and 1 μL of proviral DNA in the first reaction or 1 μL of the initial PCR mixture of the first reaction after amplification in the second round to complete the volume for nested PCR. The amplification protocol was the same in both PCRs, as follows: initial denaturation at 98 °C for 3 min, 35 cycles of denaturation at 98 °C for 20 s, annealing at 70 °C for 30 s, and extension at 72 °C for 15 s, followed by a final extension at 72 °C during 5 min and maintenance at 4 °C thereafter. DNA from strain 280918-4418-CR isolated in Costa Rica was used as positive control (GenBank accession number: MN830810), while Rnase free water  was used as negative control. The expected products for BLV were 725 and 443 bp when the Env1(+)/Env3(−) primer pair was used in the first round or when the Env2(+)/Env3(−) primer pair was used in the second round, respectively, according to Khudhair et alBHV-1 was identified using a set of primers that were based on region of the gE gen of BHV-1. The forward primer was BHV1-gE3-883F: 5′-CAATAACAGCGTAGACCTGGTC-3′ , and the reverse primer was BHV1-gE3-989R: 5′-GCTGTAGTCCCAAGCTTCCAC-3′  . The final volume of each PCR reaction was 20 µL and consisted of 10 µL of Phusion U Multiplex PCR Master Mix #F562S , 1 µL of each primer IBR-IgE-F(+)/IBR-IgE(−) (0.25 µM/µL), 7 µL of Rnase-free H2O  and 1 µL of DNA. The amplification protocol consisted of an initial denaturation cycle at 98 °C for 2 min; 35 cycles of denaturation at 98 °C for 20 s, annealing at 63 °C for 30 s and amplification at 72 °C for 15 s, followed by a final amplification at 72 °C during 5 min and holding at 4 °C thereafter. DNA from the Colorado strain contained in the commercial vaccine EXPRESS® FP 10-HS  was used as a positive control. The expected product was 106 bp. All reactions were carried out in the 2720 thermocycler . The products amplified by PCR were stained with GelRed Nucleic Acid Stain 10000X in Water  and separated on a 2% agarose gel using the Fotodyne Model N°1-1430 electrophoresis equipment at 80 V during 40 min. Once separated they were visualized in the imager and ultraviolet illuminator .S = total progressive motility > 50%, normal morphology > 70% and viability > 50%), doubtful  and unsatisfactory . The response variables were semen volume, total and progressive motility, swimming patterns (kinematics of spermatozoa), sperm concentration, normal and abnormal sperm (%), and viability (%). A normal distribution with an identity link function was assumed for all response variables. The quality of the seminal samples was categorized by analyzing the percentage of three variables , as follows: satisfactory seminal quality  was performed on these data to derive a small number of linear combinations that still retained as much information as possible from the original variables. Prior communalities for this analysis were estimated from the maximum absolute correlation coefficient between each variable and any other. The number of principal factors (PF) to be extracted was determined from the Kaiser criterion, namely by selecting only those with an eigenvalue > 1. The KMO (Kaiser–Meyer–Olkin) statistic was also obtained45. From this analysis, an optimal number of clusters was determined based on criteria such as the Cubic Clustering Criterion (CCC), Pseudo-T, Pseudo-F, and partial R2. Second, the optimal number of clusters obtained in the previous analysis was used as the target number of clusters in a non-hierarchical K-means model cluster analysis46.Further, an analysis was conducted to classify the ejaculates into a reduced number of clusters, based on scores obtained from factor analysis. This was accomplished in two-step phases, combining hierarchical and non-hierarchical clustering procedures with the sperm-derived indices obtained after the PFA. First, factor scores for all ejaculates were clustered hierarchically using the Ward Minimum Variance method47. This analysis allowed the identification of sperm subpopulation and the detection of outliers. Analysis of variance (ANOVA) was further applied to evaluate statistical differences between clusters for all morphometric variables. Furthermore, an interaction effect between sanitary status and sperm subpopulation was considered. The threshold for significance was defined as P < 0.05. Multiple comparisons between cluster means were performed by the Tukey adjustment. Results were presented as mean ± standard error of the mean. All data were analyzed using the IBM SPSS statistical program, version 23.0 for Windows .This classifies the spermatozoa of the data set into a small number of cluster (subpopulations) according to their head dimensions or kinematics variables, as has been described previously by Barquero et al.The study was conducted following ethical principles and the approval of the Committee of Centro de Investigación y Desarrollo de la Agricultura Sostenible para el Trópico Húmedo at the Costa Rica Institute of Technology (CIDASTH-ITCR) according to Section 01/2019, article 1.0, DAGSC-074-2019, and Production Committee of the Agricultural Production Program of the School of Agronomy at the Costa Rica Institute of Technology (PPASC-106-2021).


We report a sequencing protocol and 121-kb poxvirus sequence from a clinical sample from a horse in Finland with dermatitis. Based on phylogenetic analyses, the virus is a novel parapoxvirus associated with a recent epidemic; previous data suggest zoonotic potential. Increased awareness of this virus and specific diagnostic protocols are needed. Parapoxvirus encompasses the following recognized species: Orf virus, bovine papular stomatitis virus, pseudocowpoxvirus, red deerpox virus, and grey sealpox virus (GSEPV) (Parapoxviruses (PPVs) usually cause contagious skin infections in ruminants and occasionally infect other species such as humans (A severe infection caused by a parapox-like virus (F14.1158H) was first verified from a horse euthanized in Finland in 2013 . PartialTo better characterize the virus, we analyzed DNA extracted directly from a skin lesion of the 2013 equine case . The firWe conducted phylogenetic analysis for the following poxvirus core genes . ConsistMost known PPVs are zoonotic, and any novel virus detected in animals should be treated with concern . In termAdditional information for partial genome characterization of novel parapoxvirus in a horse, Finland.


Influenza B virus (IBV) is highly contagious, spreads rapidly, and causes seasonal epidemic respiratory disease in the human population, especially in immunocompromised people and young children. Clinical manifestations in this high-risk population are often more severe than in immunocompetent hosts and sometimes atypical. Therefore, rapid, and accurate detection of IBV is important.An amplified luminescent proximity homogeneous assay linked immunosorbent assay (AlphaLISA) was developed for detection of IBV by optimizing the ratio of IBV antibody-labeled receptor beads, streptavidin-conjugated donor beads and biotinylated IBV antibody, as well as the optimal temperature and time conditions for incubation. Assay sensitivity, specificity and reproducibility were evaluated. A total of 228 throat swab samples and inactivated influenza B virus were tested by AlphaLISA and lateral flow colloidal gold-based immunoassay (LFIA).AlphaLISA produced the best results for detection of inactivated influenza B virus when IBV antibody-labeled acceptor beads were 50 μg/ mL, streptavidin-conjugated donor beads were 40 μg/mL, and biotinylated IBV antibody was 0.5 μg/mL at 37°C for 15–10 min. Under these conditions, AlphaLISA had a limit of detection of 0.24 ng/mL for the detection of influenza B nucleoprotein, did not cross react with other common respiratory viruses, and showed good reproducibility with inter-assay coefficient of variation (CV) and intra-assay CV < 5%. The results of 228 clinical throat swab samples showed good agreement between AlphaLISA and LFIA (Kappa = 0.982), and AlphaLISA showed better sensitivity than LFIA for detecting inactivated influenza B virus.AlphaLISA showed higher sensitivity and throughput in the detection of IBV and can be used for IBV diagnosis and epidemic control. IAV infects a variety of hosts, including domestic and wild birds, humans and marine mammals and the antigen is readily mutated, predisposing to cause a pandemic. For IBV, it is primarily isolated from humans and in a rare event from a seal and is less antigenically mutated, which limits the risk of pandemic outbreaks , 2. In aEarly and appropriate antiviral therapy requires rapid, sensitive and specific diagnosis of IBV. Currently, several methods are commonly used for the detection of IBV, including viral culture, molecular diagnosis and immunological methods. Both viral culture and molecular diagnostic methods are considered the gold standard for diagnosing viral infections, but viral culture is time consuming and laborious, so it is not suitable for rapid screening of IBV. In molecular diagnostic methods, the development has been relatively mature, and methods have been developed to achieve rapid diagnosis of IBV. On the basis of PCR, the whole experimental time is significantly shortened. For example, CRISPR technology takes only 50–60 min  and the Immunological methods are mainly based on serological antibody detection and rapid antigen detection. Serological detection includes hemagglutination inhibition test  and viruAmplified luminescent proximity homogeneous assay linked immunosorbent assay (AlphaLISA) has always been an attractive method in the field of immunoassay due to its speed, simple operation and easy automation. AlphaLISA is a novel immunoassay technology, reducing hands-on and total assay time by separating bound and unbound assay components without multiple washes compared to previous enzyme linked immunosorbent assay (ELISA) and chemiluminescent immunoassays (CLIA). The method works by intermolecular interaction, that is, antigen–antibody specific binding makes the donor beads close to the acceptor beads (distance less than 200 nm). The donor beads are excited by a wavelength of 680 nm, and the photosensitive material in the donor beads converts oxygen in the surrounding environment to singlet oxygen, which diffuses to the acceptor beads. Following cascade reactions, the acceptor beads emit light at 615 nm . AlphaLI2.2.1.A total of 228 throat swab samples were collected from The Fifth Medical Center of Chinese PLA General Hospital from February 14 to July 11, 2019. Eu acceptor beads, streptavidin donor beads, and 1/2 AREAPLATE-96-well plates were purchased from PerkinElmer (United States). A pair of monoclonal antibodies (MAbs), 10-I55E and 10-I55B were purchased from Fitzgerald (United States). The inactivated influenza B virus was obtained from our laboratory and inactivated using β-propiolactone. The influenza B (B/Florida/4/2006) nucleoprotein (NP) with His tag was purchased from Sino Biological . The influenza B antigen and the influenza A antigens  were purchased from Sinovac . The antigens included respiratory syncytial virus (RSV), parainfluenza virus (PIV), and adenovirus (ADV) were purchased from Microbix . Real time quantitative PCR (RT-qPCR) kits were purchased from BGI GBI . TRNzol total RNA extraction kit was purchased from Tiangen . LFIA detection kits were purchased from Abon Biopharm . EZ-Link Sulfo-NHS-LC-Biotinylation kits and real-time PCR instrument were purchased from Thermo (United States). SPECTRAMAX i3 multiplate reader was purchased from Molecular Devices (United States). The study protocol has got approval of the ethics committee of the hospital (ky-2019-1-4). All methods were performed in accordance with the relevant guidelines and regulations.2.2.3CN (400 mM), 0.625 μL Tween-20 (10%), 50 μg IBV antibody and HEPES  were added in a total volume of 100 μL. 5 μL carboxymethoxyamine (65 mg/mL) was added after 24 h (h) rotating incubation at 37°C to block unreacted sites. After blocking for 1 h at 37°C, the beads were pelleted by centrifugation at 16,000 g for 15 min and the supernatant discarded. Beads were washed twice with Tris–HCl , followed by resuspension in 100 μL PBS  and stored at 4°C. Biotinylation of IBV antibody was performed according to the instructions of the biotin labeling kit.The 500 μg receptor beads were washed by resuspension in PH7.4 phosphate buffered saline (PBS), the supernatant discarded, and 5 μL NaBH2.3.The principle of AlphaLISA detection for IBV was based on a sandwich assay . A two-sOptimal concentrations of acceptor beads, donor beads, and biotinylated antibody were screened with the same incubation temperature (37°C) and time (15–10 min) conditions. The effects of: acceptor beads concentrations , biotinylated antibody dilutions  and donor beads at different concentrations  on the S/N ratio of 200-fold dilution of inactivated influenza B virus were compared. After optimization of beads and biotinylated antibody concentrations, the S/N ratios of 100-, 200-, 400-, and 800-fold dilutions of inactivated influenza B virus were tested at different incubation temperatures (37°C or 25°C) and different incubation time .The influenza B NP was serially diluted 2-fold  and then detected with AlphaLISA. Each concentration was repeated three times and the standard curve of influenza B antigen detection was modeled. The specificity of the AlphaLISA assay was evaluated and compared to other common respiratory viruses including IAV , RSV, PIV and ADV. The influenza B antigen and seven other respiratory viral antigens were diluted to 100 μg/mL, 10 μg/mL and 1 μg/mL in triplicate wells for each antigen dilution to compare the corresponding S/N ratio. 100- and 1,000-fold dilutions of inactivated influenza B virus was used to verify the repeatability of AlphaLISA. Three independent experiments in triplicate wells for each virus dilution were performed to validate inter-assay coefficient of variation (CV) of AlphaLISA; In one experiment, 12 replicate wells were tested for each virus dilution to verify the intra-assay CV.2.4.The detection of LFIA was performed according to the kit instructions. 228 throat swab samples were tested in the hospital’s department of laboratory medicine. The detection of IBV was performed in our laboratory. Inactivated influenza B virus was diluted 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, and 1:6400 using the lysate in the kit, and three replicates were made for each virus dilution gradient. The reagent strips were immersed in diluted virus diluent and the results were interpreted after 15 min incubation at room temperature.2.5.Fourteen throat swab samples were tested by RT-qPCR. RNA was extracted from samples using Trizol extraction. According to the RT-qPCR kit instructions, 19.7 μL of nucleic acid reaction solution, 0.3 μL of reverse transcriptase and 10 μL of extracted RNA were added to the reaction tube. The reaction tube was put into the fluorescence PCR detection instrument, and the program of fluorescence quantitative PCR instrument was set up according to the requirements: 50°C for 30 min 1 cycle; 95°C 15 min 1 cycle; 95°C 15 s, 58°C 45 s 40 cycle. The experimental process was carried out in accordance with the kit instructions and the instrument operation requirements.2.6.t-test was used for comparison between groups and the weighted kappa coefficient was used for concordance analysis. Diagnostic performance was evaluated using receiver operating characteristic (ROC) analysis, and the area under the curve (AUC) was calculated for comparison. When the Youden index reached the maximum value, the optimal cut-off value was determined. p < 0.05 was considered statistically significant.Graphpad Prism 8.0, SPSS 26.0 and Sigmaplot 14.0 software were used for statistical analysis of all data. The student’s 3.3.1.In order to avoid high background signal value caused by high concentration of reactant or weak signal value caused by low concentration of reactant, we tested the dilution concentrations of acceptor beads, biotinylated IBV antibody and donor beads. We found that the S/N ratio was maximum when acceptor beads were 50 μg/mL, biotinylated antibody was 0.5 μg/mL, and the donor beads were 40 μg/mL –C.In addition, for obtaining higher S/N ratio, we screened optimized incubation temperature and time for AlphaLISA. The temperature and time affect not only the binding of antibodies and analytes, but also the generation and diffusion of singlet oxygen . The res3.2.R2 = 0.9986), with a limit of detection (LOD) of 0.24 ng/mL.A standard curve was calculated with AlphaLISA signal (S/N ratio) as function of the influenza B NP . The resSince the clinical manifestations of IBV infection are nonspecifically different from those of other respiratory virus infections, and the therapeutic strategies corresponding to different viruses vary, the specificity of the method was validated. It showed that the S/N ratios for antigens of other seven respiratory viruses were all below the cut-off value, except for the influenza B antigen  (p < 0.001), and the asymptotic 95% confidence interval was 0.956–1.007. Two samples showed negative results using LFIA but positive results using AlphaLISA. Given that RT-qPCR is the gold standard for the detection of IBV and offers high sensitivity and good specificity, we performed RT-qPCR validation on these two samples, both of which showed positive by RT-qPCR, validating the AlphaLISA results. To further validate the accuracy of AlphaLISA, 12 additional throat swab samples with the S/N ratios close to cut-off value were tested by RT-qPCR, which again were concordant with a positive RT-qPCR test  of positive and negative samples were 13.91 ± 0.16 and 0.77 ± 0.09, respectively. The results showed that there was a significant statistical difference in S/N ratio between positive and negative samples ( 0.0001) . The ROC 0.0001) . AccordiPCR test .To further compare the sensitivity of the AlphaLISA method with LFIA, inactivated influenza B virus was detected at different dilution gradients. We found that AlphaLISA could detect diluted inactivated influenza B virus at a dilution of 1:25,600, whereas LFIA could only detect inactivated influenza B virus in 1:3,200 dilution . AlphaLI4.via endocytosis playing a critical role during viral infection , which enables virus attachment to host cells and uptake nfection . IBV HA nfection . Furthernfection . During nfection . TherefoAlphaLISA is a homogeneous light-stimulated chemiluminescence sandwich immunoassay. At present, this assay is useful for detection of viral and food toxins as well as human molecular proteins. It showed good specificity and sensitivity in the detection of SARS-CoV-2 and African swine fever virus , 20.In tThe ideal approach for pathogen detection should be rapid, sensitive, specific, affordable, sometimes instrument-free and suitable for Point-of-care-testing (POCT) . Current5.In summary, AlphaLISA had good sensitivity, specificity and repeatability in the detection of IBV and had the advantages of easy operation and rapid reaction.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by The Fifth Medical Center of Chinese PLA General Hospital. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.HZ, SZ, XS, HJ, ZZ, SC, XW, YW, YJ, XL, LT, PL, QL, and YL participated in the study concept and design. HZ, PL, and QL performed the data analysis and wrote the draft. SZ performed the sample collection. All authors contributed to the article and approved the submitted version.This work was funded by the National Science and Technology Major Projects (2018ZX10711001003 and 2018ZX10713002002004).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.