Closteroviridae, designated as grapevine leafroll-associated viruses (GLRaVs). However, the specific etiological role of any of these GLRaVs in GLRD has not been demonstrated. Even though GLRaV-3 is considered the chief GLRD agent, little is known about the molecular, cellular, and pathological properties of this virus. Such a knowledge gap is due to multiple factors, including the unavailability of biologically active virus cDNA clones and the lack of reliable experimental systems for launching grapevine infection using such clones. In this work, we tested four methods for inoculating tissue-cultured grapevine plantlets with cDNA clones of GLRaV-3: (i) vacuum agro-infiltration; (ii) agro-pricking; (iii) agro-drenching; and (iv) agro-injection. We showed that vacuum agro-infiltration was the most effective of these methods. Furthermore, we examined the impacts of different experimental conditions on the survival and infectivity rate of grapevines after infiltration. To verify the infectivity rate for different treatments, we used RT-PCR, RT-qPCR, and Western blotting. We found that humidity plays a critical role in the survival of plantlets after agro-infiltration and that the use of RNA silencing suppressor and dormancy treatment both had strong effects on the infection rates. To our knowledge, the experimental protocol reported herein is the most effective system for launching the infection of grapevine using cDNA clones of grapevine viruses featuring up to a 70% infection rate. This system has strong potential to facilitate grapevine virology research including the fulfillment of Koch’s postulates for GLRD and other major virus diseases as well as identifying the molecular, cellular, and pathological properties of GLRaVs and, potentially, other important grapevine viruses.Grapevine leafroll disease (GLRD) is the most globally prevalent and destructive disease complex responsible for significant reductions in grape yield and quality as well as wine production. GLRD is associated with several positive-strand RNA viruses of the family  Vitis spp.), the largest number of viruses detected in a particular plant species [To date, 86 distinct viruses belonging to 17 families and 34 genera have been demonstrated to infect grapevine , and the fanleaf degeneration caused by GFLV, is the oldest known and one of the most widespread viral diseases of grapevine [Betafexiviridae. This group includes Grapevine rupestris stem pitting-associated virus (GRSPaV), Grapevine virus A (GVA), and Grapevine virus B (GVB), among other related viruses [Grapevine fleck virus (GFKV) belongs to the family Tymoviridae, which is associated with the fleck disease complex [Closteroviridae, which is associated with the grapevine leafroll disease (GLRD) complex [Grapevine red blotch virus’ (GRBV) and grapevine leaf mottling and deformation caused by Grapevine Pinot gris virus (GPGV) have been identified around the world [Currently, most of the documented grapevine viruses are classified into four major groups, which are associated with four disease complexes. One group of viruses belongs to the family rapevine . The sec viruses ,7. The t complex ,7. Final complex ,8. In rehe world ,11,12,13Closteroviridae family, Grapevine leafroll-associated virus 3 (GLRaV-3) from the Ampelovirus genus is regarded as the most economically destructive of the GLRaVs due to its global distribution, high prevalence, and disease severity [Among all these diseases, GLRD is inarguably the most important viral disease in grapevines economically, comparable to those caused by fungal pathogens. The GLRD complex occurs in all major grape-growing regions worldwide, causing significant losses to the grape and wine industry. Grapevine infections with GLRD result in major reductions in yield and fruit quality and shorten the productive lifespan of vineyards. Although GLRD is associated with six species of grapevine-leafroll-associated viruses (GLRaVs) belonging to three genera of the severity ,15,16.To investigate the molecular or cellular aspects of RNA virus infection or virus gene functions, it is essential to generate experimentally amenable, biologically active cDNA clones of the virus genomes. These cDNA clones can initiate virus infection either upon mechanical inoculation of the susceptible plants or upon agro-infiltration ,18. The Agrobacterium tumefaciens to deliver target DNA into the nuclei of plant cells. Agro-infection can be achieved using four available inoculation methods, depending on the virus–host combination. In the first method, an agrobacterium suspension is directly rubbed on the leaf surface or injected into the leaves using a needleless syringe. This method is suitable for the viruses of herbaceous hosts [Solanaceae species via the drenching of soil with agrobacterium suspensions [Grapevine virus A (GVA) and Grapevine rupestris stem-pitting-associated virus (GRSPaV) [Grapevine red blotch virus (GRBV). Based on their report, this method worked well as it resulted in a good infection rate. The fourth, more efficient method is vacuum-mediated agro-infiltration developed for woody plant viruses such as Grapevine leafroll-associated virus-2(GLRaV-2), Apple chlorotic leaf spot virus (ACLSV), GRBV, and Grapevine Pinot gris virus (GPGV) [Unlike many herbaceous plants that can be readily infected via mechanical inoculation with cloned virus cDNA, grapevine and other woody perennials are recalcitrant toward mechanical inoculation. An alternative to mechanical inoculation is agro-infection, which relies on the natural ability of us hosts ,18,27,28pensions . Agro-dr(GRSPaV) ,22. Howe(GRSPaV)  in the as (GPGV) ,22,26,30Despite substantial progress in developing methods for the delivery of cDNA clones, further improvements of these methods remain crucial for empowering the research and practical applications of the viruses of grapevine and other woody plants. Accordingly, the main objective of this study was to optimize an experimental system for launching grapevine infections with virus cDNA clones in general and GLRaV-3 in particular. We first compared the performance of the four mentioned inoculation methods in initiating grapevine infection with GLRaV-3 cDNA clones and found that vacuum-mediated agro-infiltration is the most effective of these. We further tested a series of experimental conditions impacting the survival of the infiltrated plantlets and infectivity rate and developed the optimized protocol for the entire process of vacuum agro-infiltration and the subsequent plant recovery. This protocol has a strong potential for broad applications in woody plant virology, including the fulfillment of Koch’s postulates, virus–host interactions including pathogenesis, as well as virus and plant functional genomics using virus vectors for gene expression and virus-induced RNA interference.2/s. These tubes were periodically checked for contamination. After approximately 2 weeks, once the new shoots started growing, a GS1 liquid medium containing 0.2 g/L of cefotaxime was added to the glass tubes  containing the binary vector pCB301 carrying a full-length GLRaV-3 cDNA clone (which we designate as pLR3 from here on) or its GFP-tagged variant (which we designate as pLR3-GFP), which were recently constructed in our lab (data not published), and vLR2-p24 (GLRaV-2 RNA silencing suppressor) (RSS) was used [600) of 1.0. Then, the agrobacterial cultures were centrifuged for 10 min at 5000 rpm, and the resultant pellets were re-suspended and washed in an infiltration buffer (10 mM of MES (pH 5.85), 10 mM of MgCl2, and 150 μM of acetosyringone). A second round of centrifugation was carried out, and the pellets were re-suspended in an appropriate amount of the infiltration buffer. The final OD600 of the agrobacterial suspension was adjusted to 2.0 for infectious viral clones or to 0.5 for vLR2-p24, followed by induction at room temperature for two hours [Agro-infiltration was conducted using was used . Brieflywo hours . Four different techniques including vacuum-based agro-infiltration, agro-pricking, agro-drenching, and agro-injection were tested and compared in this study. In all methods, 8–11-week-old, healthy-looking, tissue-cultured grapevine plantlets were used. To prepare them for agro-inoculation, the plantlets were trimmed with a pair of scissors to remove most of the leaves and side shoots, especially near the bottom of the plantlets. In addition, some of the roots were trimmed to one-third of their length. In the vacuum infiltration and pricking methods, trimmed plantlets were poked on their stems and roots with a 31-gauge needle a–c. The 600 of 2.0 for the viral clone and OD600 of 0.5 for the RSS p24. To examine the infectivity of our pLR3, we followed the protocol of Kurth et al.  with min2/s  to find out which cultivar is more conducive to the vacuum infiltration procedure, as judged by both the survival and infection rate. In this experiment, the pLR3-containing agrobacterium at an OD600 of 2.0 and p24 at anOD600 of 0.5 was infiltrated into the Syrah and Cabernet franc plantlets following the standard procedure, as described previously. After infiltration and potting the plantlets in 2.5-inch round plastic nursery pots, these small pots were placed in 3-gallon plastic pots, which were covered with plastic sheets and kept in a growth chamber, and one week later, the plastic covers were removed instantly or gradually  on the infection rates, several such suppressors derived from different viruses including p24 of GLRaV-2, p19 of To study the influence of dormancy on the infectivity rate, viral titer, and symptom development, grapevine plants that were inoculated via agro-infiltration 8–10 months earlier were subjected to either one or two cycles of dormancy. As negative controls, grapevines derived from the same agro-infiltration procedure were kept under normal growth conditions. Each dormancy treatment comprised 50 grapevines, 25 of which tested positive, whereas the remaining 25 plants tested negative for GLRaV-3 based on nested RT-PCR. The dormancy treatment was repeated once for all experiments. Before inducing dormancy, each plant was trimmed, such that only three to four leaves were left. In the single dormancy treatment, 50 grapevine plantlets were subjected to an incremental drop in temperature over a 3-week period. In week one, the temperature was reduced from 22 to 16 °C; in week two, the temperature was further dropped to 10 °C; and from the third week onward, the plants were kept at 4 °C for 2 months. At the end of the cold treatment cycle, the temperature was returned to 22 °C using the same increment but in reverse order. The double dormancy treatment simply comprised two cycles of the single dormancy treatment that were interrupted by a 2-month break period between them. In other words, at the completion of the first dormancy cycle, plants were returned to 22 °C for 2 months to recover and grow followed by a second cycle of dormancy.Different methods were used to detect the virus after infiltration. The best and most sensitive method, especially in the early months post-infiltration, was based on the detection of virus RNA, such as nested RT-PCR and RT-qPCR. Other assays such as sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, which are based on the expression of the virus capsid protein, were also helpful for testing the infectivity success but were slightly less sensitive. A schematic of these methods is shown in To test for the systemic infection of the virus resulting from agro-infiltration, new and non-infiltrated leaves of grapevines were collected at two months post-infiltration (mpi). Subsequently, the veins and petioles were obtained from the leaf samples and ground to fine powders in liquid nitrogen using a mortar and pestle and stored at −80 °C. Total RNA was extracted from 50 mg of the ground tissue samples following the protocol described by Xiao et al. . The qua2), 0.2 mM of dNTPs, 0.2 µM of each primer (F1_16289 and R_17240), and 1.0 unit of Taq DNA polymerase  to amplify a 952 bp DNA fragment. The PCR conditions included an initial denaturation step at 94 °C for 5 min, then 35 cycles at 94 °C for 30 s, 53 °C for 30 s, and 72 °C for 1 min, followed by a final extension at 72 °C for 7 min. In the second round of PCR, everything remained the same as in the first round of PCR except the following: (1) 2 µL of the amplification product from the first round of PCR was used as a template instead of cDNA; (2) an internal forward primer (F2_16896) and the same reverse primer were used to amplify a 345 bp DNA fragment; and (3) PCR was conducted for 20 cycles instead of 35 cycles. The primers used for these assays are shown in Conventional RT-PCR was conducted by using virus-specific primers following Shabanian et al. , with a Western blot was performed following the protocol of Shabanian et al.  with somFour inoculation methods were tested and compared to assess their efficacy in launching the infection of grapevine plantlets using agrobacteria containing pLR3  to be confidently called positive. However, after a single dormancy cycle, the Cq value was reduced to 25–30 at 12 mpi, whereas the Cq values of the non-dormancy treatment group collected at the same time point were between 30 and 35. Taken together, these data demonstrate that dormancy had a considerable positive impact on the viral titer a. FurtheNicotiana benthamiana plants [Cuscuta spp.) have shown different levels of success in transferring some viruses to woody plants [Cauliflower mosaic virus. Since then, agrobacterium-based inoculation has been widely used in the plant virology community. In fact, this approach has become the method of choice when it comes to studies on viruses that infect grapevines and other woody perennials.The state of research on viruses of woody perennial plants including grapevine lags far behind the research on those that infect herbaceous plants due to the compound effect of several factors. First, grapevines are commonly infected with a mixture of distinct viruses and strains, complicating the elucidation of the etiological contribution of individual viruses in a disease. Second, many pathogenic viruses of woody plants do not infect herbaceous plants, which are incomparably more amenable to investigating all aspects of viral infections. In this respect, GLRaV-2 seems to be a rare exception in being readily inoculated into a plants . Third, y plants ,38. Thery plants  to launcGrapevine Pinot gris virus cDNA clones [Drenching was the first agrobacterium-based inoculation method that was developed to launch grapevine infection using micro-propagated plantlets with an infectious DNA clone of GVA . This meA clones ,26. UnfoA clones .Geminiviridae responsible for an emerging grapevine red blotch disease [V. vinifera cultivars and four rootstocks was tested. It was concluded that(1) both vacuum agro-infiltration and agro-pricking worked equally well; (2) 4–6-week-old plantlets were superior to 8–12-week-old plantlets; (3) none of the three silencing suppressors  were necessary for initiating GRBV infection; and (4) the vacuum duration and level were specific to the grapevine cultivars tested [In 2012, a breakthrough was achieved through the development of a vacuum-based agro-infiltration method to launch the infection of micro-propagated grapevine plantlets with a GFP-tagged cDNA clone of GLRaV-2 . The aut disease . In the In this study, we used a systematic approach to test a set of factors that may influence the survival and infection rate of grapevine plantlets. First, four inoculation methods for the grapevine plantlets with a cDNA clone of GLRaV-3 (unpublished data) were applied. Among these, the vacuum agro-infiltration method was the most effective, followed by pricking, whereas soil drenching and direct infiltration with a needleless syringe failed to produce infection. We demonstrated that plantlets of 8–11 weeks of age gave the best performance both in terms of survival and infectivity. We compared five different silencing suppressors in agro-infiltration and demonstrated that four of them were highly effective as each substantially increased the infection rate by 40–50%. In line with previous research ,24, mainIt was also reconfirmed that the grapevine cultivar and the age of plantlets have direct impacts on survival and infection rates. The 8–11-week-old plantlets showed the best outcomes in survival (65–82%) and infectivity (44–63%) rates, while the older and younger plantlets resulted in either reduced infectivity or survival rates similar to the findings of Yepes et al. . Also coWe also found that the application of virus RNA-silencing suppressors was an important factor in the virus infection rates and viral titer, in agreement with the results of Kurth et al.  but contAs mentioned above, the GLRaV-3 titer and infection rates exhibited a sharp variation before and after cold treatment. According to the RT-qPCR and nested-RT-PCR analyses of the samples collected prior to and after dormancy treatment, the cold treatment increased the titer and infection rates by16% and 19% in Syrah and Cabernet franc, respectively, similar to the findings of Yepes et al.  with an To our knowledge, this is the most comprehensive report on the optimization of agro-inoculation methods for launching grapevine infections with a viral cDNA clone. In particular, we established that the best material for inoculation is 2–4-month-old micro-propagated Syrah or Cabernet franc plantlets. We also found that co-infiltration of the plantlets with a strong suppressor of RNA silencing, such as HC-Pro, p19, or p24, significantly increases the infection rate. Upon agro-infiltration, plantlets must be protected from loss of humidity during the first 3 weeks. Finally, we showed that a single cycle of dormancy treatment of lignified plantlets at 4 °C for 2 months significantly increases the viral titer.This work is certain to facilitate overall progress in the field of grapevine virology by providing an optimized protocol for grapevine agro-infection. This protocol constitutes a critical tool for investigations into the etiological roles of distinct viruses in grapevine disease complexes often caused by several viruses, as well as advanced studies on the molecular and cellular aspects of virus–host interactions in grapevines.Furthermore, the methodical approaches developed in this work have clear potential applications for studying challenging but important pathogenic viruses of other woody crop plants such as citrus, stone fruits, and pome trees.


The increasing prevalence of Chronic Obstructive Pulmonary Disease (COPD) has imposed a considerable economic burden. However, there remains a paucity of relevant evidence regarding the hospitalization costs of COPD cases. Therefore, in this study, we aimed to assess the hospitalization costs among COPD cases and investigate the factors that contribute to their costs in Henan Province, China.We enrolled a total of 1697 cases who were discharged with a diagnosis of COPD from January 1, 2020 to December 31, 2020, into the study. Demographic and clinical characteristics of the cases were obtained from the hospital information system (HIS) of two large tertiary hospitals in Henan Province, China. The factors associated with hospitalization costs were examined using a multiple linear regression model.Total hospitalization costs of 1697 COPD cases were $5,419,011, and the median was $1952 (IQR:2031). Out-of-pocket fees accounted for 43.95% of the total hospitalization costs, and the median was $938 (IQR:956). Multiple linear regression analysis revealed that hospitalization costs were higher among older cases, cases with more comorbidities, and cases with longer length of stay. Furthermore, hospitalization costs were higher in cases who paid through private expenses compared to those covered by Urban Employee Basic Medical Insurance. Additionally, we found that cases admitted through an outpatient clinic had higher hospitalization costs than those admitted through the emergency department.Hospitalization costs of COPD cases are substantial. Strategies to reduce hospitalization costs, such as shortening LOS, optimizing payment plans, and preventing or managing complications, should be implemented to alleviate the economic burden associated with COPD hospitalizations. Chronic Obstructive Pulmonary Disease (COPD), the most common chronic respiratory disease, is a major global public health concern , 2. AccoCOPD is associated with many comorbidities, including pulmonary artery disease, diabetes, cardiovascular disease, and malnutrition , 11. COPUnderstanding the hospitalization costs and associated factors of COPD cases is vital in estimating the direct costs of COPD and providing suggestions for reducing the economic burden on cases. This issue has been extensively researched globally –20, but Due to the prevalence and preventable nature of COPD, accurate estimates of its hospitalization costs are crucial to identify cost-effective interventions for COPD management. Hence, we aimed to provide an up-to-date estimation of COPD hospitalization costs and investigate the associated factors of COPD using the 2020 data from two large public tertiary hospitals in Henan Province, China.The study was carried out in the First Affiliated Hospital of Zhengzhou University and Henan Provincial People’s Hospital, two of the largest tertiary general hospitals in Henan Province, China. Data for cases from January 1, 2020 to December 31, 2020 were extracted from the Hospital Information System (HIS), with diagnosis of COPD coded according to the International Statistical Classification of Diseases and Related Health Problems 10th Revision (ICD-10). To ensure the accuracy of ICD-10 coding, diagnoses were determined by checking the details in medical records. Criteria for inclusion in this study were as follow: (1) the main discharge diagnosis ICD-10 coding were J40 to J44; (2) age ≥ 18 years. The exclusion criterion was the presence of primary data deficits. For cases undergoing two or more hospitalizations, each hospitalization was studied as a separate case, as our research focused on COPD cases.Medical record information of COPD hospitalized cases was exported from the HIS to Microsoft Excel, which included the following data: (1) demographic characteristics . (2) hospitalization costs, encompassing total fees, out-of-pocket fees, service fees , diagnostic fees , treatment fees , medical fees  and other fees .U test (with 2 subgroups) or Kruskal-Wallis test (with multiple subgroups) was carried out to test differences. Prior to conducting a multiple linear regression model, the hospitalization costs were log transformed and all continuous independent variables were z-standardized. The independent variables were included in the multiple linear regression, if they were statistical significant in the univariate analysis (p < 0.2) [p < 0.05 was considered statistically significant in multiple linear regression.For categorical variables, we described them using the percentages (%). As hospitalization costs were negatively skewed distribution, we used the median and interquartile range (IQR) to describe the hospitalization costs of COPD cases. Mann-Whitney p < 0.2) . No collp < 0.2) . A probaAll fees were converted to US dollars using the average exchange rate of 6.7986 RMB to USD published by the China Foreign Exchange Trade Center for the year 2020.As shown in Table Table p = 0.919), and number of hospital admissions (p = 0.249) were not associated with hospitalization costs. Age group (p < 0.001), comorbidities (p < 0.001), type of medical insurance (p = 0.185), LOS (p < 0.001), and mode of admission (p = 0.021) were significantly associated with the hospitalization costs (p < 0.2)  Table .β = 0.090, p = 0.005), 60–69 , 70–79 , and over 80 years old  had significantly higher costs. Compared with cases with fewer than three comorbidities, those with 3–4 , 5–6 , or more than 6 comorbidities  had significantly higher costs. Cases with LOS 6–8 , 9–12 , or more than 12 days  had significantly higher costs than those under six days. Cases with Urban Employee Basic Medical Insurance (UEBMI) had lower costs  than those with private medical expenses. Cases admitted through the outpatient clinics had higher costs than those admitted through emergency department .Multiple linear regression analysis showed that age group, number of comorbidities, type of medical insurance, length of stay (LOS), and mode of admission are associated with hospitalization costs Table . SpecifiIn this cross-sectional study, we assessed COPD hospitalization costs and their components in Henan province, China, and examined the factors associated with COPD hospitalization costs. Our findings revealed that hospitalization costs increased with older age, more comorbidities, private expenses, longer LOS, and admission through outpatient clinic. As expected, health insurance significantly reduced out-of-pocket costs, with an average reimbursement rate of 56.05% for COPD cases. Our research can provide a reference for comparison with other countries and other regions in China.The median hospitalization cost for COPD case in this study was $1,952, which is consistent with the findings of a previous study conducted in Guangdong, China . One posWe further found that the hospitalization costs of COPD cases in this study were different from other countries. According to previous studies –26, the In regards to the breakdown of hospitalization costs, this study found that medication fees for COPD cases accounted for 37.90%, with diagnostic fees following at 24.53%. Service fees and treatment fees accounted for only 14.32% and 9.47%, respectively. These results are consistent with previous findings in China and Turkey , 28. GivThe study found that older adults accounted for the majority of COPD cases and had notably higher hospitalization costs. Previous studies have reported that a significant portion of COPD-related morbidity, severity, and mortality occurs among the elderly population , 31. ThiWe observed higher hospitalization costs in COPD cases with multiple comorbidities. In this study, comorbidities were present in 98.2% of COPD cases, echoing findings from other studies that reported comorbidities in most COPD cases –37. HoweUnlike other research referred to other diseases , 44, thiConsistent with other studies , 19, ourThere are some limitations to this study. First, only hospitalization costs were analyzed, and outpatient and indirect costs were not included. Second, the data were collected from two hospitals in Henan Province, China and may not be representative of other regions or hospital types. Third, the data was limited to the period between January 1, 2020 and December 31, 2020, a relatively short time span; thus, a continuous trend comparison could not be made. Fourth, for cases undergoing two or more hospitalizations, each hospitalization was studied as a separate case, which may have led to an underestimation. Finally, data on demographic and clinical characteristics were limited , futureIn summary, the cost of each hospitalization for COPD cases is substantial. Older age, more comorbidities, private expense, longer LOS, and admission through outpatient clinic are strongly associated with higher hospitalization costs in COPD cases, highlighting the need for targeted prevention and control measures in this population. Our study has important implications for estimating direct costs and providing suggestions for reducing the economic burden of this disease.


In this study, we detected a circular replication-associated protein (Rep)-encoding single-stranded (CRESS) DNA virus [named Po-Circo-like (PCL) virus] in intestinal tissue and fecal samples of pigs. PCL virus contains a single-stranded DNA genome, and ORF1 encodes the Rep and not the typical capsid protein encoded in PCV. The Rep protein may be responsible for viral genome replication. In addition, PCL virus may be one of the pathogens causing diarrhea symptoms in pigs. We identified four strains of PCL virus in two different pig farms with severe diarrhea outbreaks in Hunan Province, China. The strains in this study share 85.7–99.7% nucleic acid identity and 84.7–100% amino acid identity with Rep of the reference strains. A multiple sequence alignment of these PCL viruses and Bo-Circo-like CH showed a identity of 93.2% for the Rep protein, and the nucleotide identity was 86.7–89.3%. Moreover, Bo-Circo-like CH and HN75, HN39-01, HN39-02 had similar stem-loop sequences. In conclusion, the present study is the first detailed report of the PCL virus in Hunan provinces, which is a potential new virus in pigs that might be involved in cross-species transmission. Further investigation is needed to determine the pathogenesis of this virus and its epidemiologic impact. In receCircoviridae is divided into two genera of Circovirus and Cyclovirus, and can infect a variety of animals  and repeatedly freeze-thawed three times. The viral DNA/RNA is then extracted using RaPure Viral RNA/DNA Kit . Polymerase chain reaction (PCR) assays for the detection of several common pig viruses w-ere used including porcine epidemic diarrhea virus (PEDV), Transmissible gastroenteritis virus of swine (TGEV), Swine acute diarrhea syndrome coronavirus (SADS-CoV) and porcine deltacoronavirus (PDCoV). Five primer pairs were designed based on the reference sequence of the PCL virus 21 and 22 strain determined in the United States and the PCL virus detected in China. The primers are described in 2.3.PCR products of PCL viruses positive samples were amplified and purified. We cloned it into the pMD 19-T vector (TaKaRa Bio Inc.) and then transformed it into DH5α competent cells. The positive clones were screened by PCR and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.2.4.snapgene.com). Subsequently, all gene sequences arrangements were further aligned with MegAlign (Lasergene) using the Clustal W alignment method. A phylogenetic tree was built using the maximum likelihood method with 1,000 bootstrap replicates in MEGA 7.0 software. The reference strains of this study are shown in The complete gene sequences of PCL viruses obtained in this study have been uploaded to Genbank with the accession numbers . BLAST analysis3.3.1.Since mixed infection of pathogens is common in clinically infected pigs, we used polymerase chain reaction (PCR) to detect PEDV, TGEV, SADS-CoV, PDCoV, PCL virus. The primers are described in 3.2.snapgene.com) was used to assemble the genome sequences together and draw the complete gene map. The NCBI Open Reading Frame (ORF) FinderSubsequently, the full genomes of four strains were sequenced, and their gene characteristics were further analyzed . First, The DNASTAR package  was used for sequence alignment and identity analysis. The strains in this study share 85.7–99.7% nucleic acid identity and 84.7–100% amino acid identity with Rep of the reference strains . A multi4.A novel CRESS DNA virus was first detected in pig intestinal tissue in the United States . The virThe original contributions presented in the study are included in the article/The animal study was reviewed and approved by South China Agricultural University Experimental Animal Welfare Ethics Committee.JM and YS: conceived and designed the experiments. CJ, MZ, and XL: performed the experiments. CJ, MZ, and YW: sample collection. CJ and MZ: analyzed the data and contributed to the writing. All authors contributed to the article and approved the submitted version.This work was financially supported by the Guangdong Major Project of Basic and Applied Basic Research (no. 2020B0301030007) and Natural Science Foundation of Guangdong Province (nos. 2022A1515012473 and 2020A1515010295).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.


Implantation of an implantable cardioverter defibrillator (ICD) is standard care for primary prevention of sudden cardiac death. However, ICD-related complications are increasing as the population of ICD recipients grows.ICD-related complications in a national DO-IT Registry cohort of 1442 primary prevention ICD patients were assessed in terms of additional use of hospital care resources and costs.During a median follow-up of 28.7 months (IQR 25.2–33.7) one or more complications occurred in 13.5% of patients. A complication resulted in a surgical intervention in 53% of cases and required on average 3.65 additional hospital days. The additional hospital costs were €6,876 per complication or €8,110 per patient, to which clinical re-interventions and additional hospital days contributed most. Per category of complications, infections required most hospital utilisation and were most expensive at an average of €22,892. The mean costs were €5,800 for lead-related complications, €2,291 for pocket-related complications and €5,619 for complications due to other causes. We estimate that the total yearly incidence-based costs in the Netherlands for hospital management of ICD-related complications following ICD implantation for primary prevention are €2.7 million.Complications following ICD implantation are related to a substantial additional need for hospital resources. When performing cost-effectiveness analyses of ICD implantation, including the costs associated with complications, one should be aware that real-world complication rates may deviate from trial data. Considering the economic implications, strategies to reduce the incidence of complications are encouraged.The online version of this article (10.1007/s12471-022-01733-4) contains supplementary material, which is available to authorized users. In a large multicentre cohort of real-world primary prevention implantable cardioverter defibrillator (ICD) patients, we showed that device-related complications occur in a significant proportion of patients and are associated with substantial clinical consequences and hospital costs.Mean hospital costs for different types of complications, per complication and per patient with one or more complications are reported as well as a national estimate of yearly costs associated with complications of ICD implantation for primary prevention.The cost data can be used for health economic modelling (research practice) of new cardiac implantable electronic devices for heart rhythm disorders and facilitate ranking of targets for prevention of ICD-related complications.Implantable cardioverter defibrillator (ICD) therapy has been proven effective for primary prevention of sudden cardiac death due to ventricular tachycardia or fibrillation , 2. In aThe increase in ICD implantations and related increase in device-related complications have economic implications and may pose a challenge to the healthcare budget. Although several studies have described ICD-related complications and their associations with device type and patient clinical characteristics, little information is available on the impact of ICD-related complications on healthcare resource utilisation and the associated costs. Furthermore, these implications for healthcare should be acknowledged and it should be investigated whether the different ICD-related complications show differences in type, intensity or cost of care. Furthermore, the need for improved risk stratification of primary ICD indications has been emphasised and if better patient selection is possible, then these complication costs can be prevented too .To better understand the economic impact of ICD-related complications and their associated management, this paper addresses the hospital provider costs associated with complications that occurred within 2 years after device implantation for primary prevention as observed in a national clinical practice registry.Patients who received their first ICD for primary prevention of sudden cardiac death between September 2014 and June 2016 were prospectively enrolled in the DO-IT (Dutch Outcome in Implantable cardioverter defibrillator Therapy) Registry . This ICAfter obtaining the patients’ informed consent, baseline data were collected on demographics, medical history, diagnostics, left ventricular ejection fraction and implant-related data. Data regarding mortality, (in)appropriate ICD therapy and ICD-related complications were extracted from medical records during regular protocol-based follow-up. All registry data were extensively monitored. More details of the registry and baseline characteristics of the patients included have been published elsewhere , followeThe primary outcome measure for the current analysis was the hospital provider costs for any ICD-related complication. Complications were defined as any undesirable clinical occurrence related to the ICD implantation and function. These complications were further categorised as related to the lead, the pocket, an infection or other causes. Any patient having one of these complications at any time during the observation period was included in the analysis. The secondary outcome measure was the hospital provider costs per type of complication.Device interrogation records, electrophysical procedure reports, the hospitals’ patient administration and patient medical records were used to determine the patients’ clinical course directly related to a complication. The following information was gathered: details of the complication, re-interventions, length of additional hospital stay, extra consultations, and extra diagnostic or laboratory procedures.Unit costs were obtained from the latest complete unit cost sheet from one of the participating major hospitals  and fromBaseline characteristics Tab.  are presp-value < 0.05 was considered statistically significant. Statistical analyses were performed with SPSS version 24.0 .A two-sided Inclusion and follow-up data were collected for 1,442 patients. During a median follow-up of 28.7 months  230 complications occurred in 195 patients (13.5%). Baseline characteristics of this cohort are listed in Tab. The mean and total number of surgical interventions, hospital admissions, outpatient consultations and laboratory or diagnostic procedures for each complication type and each complication category are shown in Table S2 . An ICD-related complication required surgical intervention in 53%, with an average additional use of hospital resources per complication of 0.61 surgical interventions, 3.65 hospitalisation days, 1.6 outpatient consultation visits and 5 diagnostic or laboratory procedures. Most surgical re-interventions occurred in patients experiencing a lead-related complication. However, on average, infections resulted in the most surgical interventions per patient. In terms of hospital resources used, (systemic) infections had the most impact, followed by complications related to the lead.Tab. p = 0.66). The mean costs per ICD-related complication in patients receiving a single-chamber ICD, dual-chamber ICD, CRT‑D and subcutaneous ICD were €6,825, €10,162, €8,267 and €6,425, respectively. Higher costs were observed in patients implanted with a dual-chamber or a CRT‑D device compared to those receiving a single-lead or subcutaneous ICD, but the difference was not significant. The costs resulting from an ICD-related complication for patients with ischaemic or non-ischaemic aetiology were similar .Complication costs were non-significantly higher in women than in men  interventions, outpatient consultations and additional hospitalisations. With one in seven patients experiencing an ICD-related complication, the complication rate in the DO-IT Registry is high compared to that in the landmark trials , 2. HoweTo estimate the national hospital cost impact, we performed an extrapolation of the economic implications due to ICD-related complications based on our own registry data. Because official data on the yearly number of primary prevention device implementations in particular are not available, a conservatively calculated yearly incidence of 2,500 patients was applied. Based on the reported average costs per complication type, extrapolation to other countries with purchasing power parities  is possiThis study has several limitations. First, these results may only be pertinent to the situation in the Netherlands, as we studied the hospital costs incurred as a result of all ICD-related complications in a large Dutch primary prevention cohort. With incidences and hospital care utilisation potentially being different in cohorts elsewhere, our results need further confirmation. In addition, the presented costs associated with ICD-related complications are likely an underestimation of the costs to society, since costs for out-of-hospital healthcare, out-of-pocket expenses and costs resulting from productivity loss could not be included. Second, our data were not sufficient to adequately report on the costs related to inappropriate shocks. However, with regard to the total costs associated with adverse consequences of defibrillator devices and the subsequent total costs of ICD implantation, the costs incurred due to inappropriate shocks are also relevant and should be addressed in future studies. Nevertheless, our findings indicate (data not shown) that the mean hospital costs per inappropriate shock were €884 , including the inappropriate shocks not resulting in hospital care. Third, the time horizon of the analysis was limited to approximately 2 years after device implantation; therefore, device replacements in the longer term and their subsequent complications and associated costs were not captured. Fourth, with data stemming from a registry there is a possibility of under-reporting in terms of complications and subsequent hospital care utilisation. In addition, 21 patients were lost to follow-up at some point in time and as a consequence complications may have been missed. No follow-up data were available for one patient, while seven patients emigrated during follow-up. A complication would be expected in approximately 3 patients (13.5% of 21 patients); however, during an average follow-up of 16.18 months we observed complications in 5 patients. Therefore, we think it is unlikely complications were missed and that this does not bias our results. Moreover, patient data were extensively monitored, and the relatively high complication rate suggests that under-reporting, if any, would have been minimal. The most dominant related in-hospital costs were accounted for. Rare use of related hospital resources (e.g. blood transfusion) was not included and co-medication for co-morbidities present was not recorded. Lastly, unit costs for clinical or surgical interventions and diagnostic and laboratory measures were partly based on the latest unit costing data of one of the participating major hospitals and may vary from one institution to another. Hence, the results should just be considered as strongly indicative.In conclusion, data from this nationwide registry showed that additional hospital utilisation following ICD-related complications is substantial and treatment of complications may be expensive. This study demonstrates that complication-related costs can be an important component in the overall cost-effectiveness of device therapy. Our findings suggest that strategies to reduce the incidence of complications, reducing the costs of managing complications and sharpened ICD indications may result in significant reductions in hospital care utilisation and expenditures. Additionally, this financial assessment provides more accurate information on the cost implications of health outcomes and this information is of importance for reimbursement or hospital healthcare management.The Electronic Supplementary Material provides a list of all DO-IT investigators and supplemental tables to give readers additional information about their work


Total knee arthroplasty are among the most frequently conducted surgeries, due to an aging society. Since hospital costs are subsequently rising, adequate preparation of patients and reimbursement becomes more and more important. Recent literature revealed anemia as a risk factor for enhanced length of stay (LOS) and complications. This study analyzed whether preoperative hemoglobin (Hb) and postoperative Hb were associated with total hospital costs and general ward costs.The study comprised 367 patients from a single high-volume hospital in Germany. Hospital costs were calculated with standardized cost accounting methods. Generalized linear models were applied to account for confounders, such as age, comorbidities, body mass index, insurance status, health-related quality of life, implant types, incision-suture-time and tranexamic acid.Preoperative anemic women had 426 Euros higher general ward costs (p < 0.01), due to increased LOS. For men, 1 g/dl less Hb loss between the preoperative value and the value before discharge reduced total costs by 292 Euros (p < 0.001) and 161 Euros fewer general ward costs (p < 0.001). Total hospital costs were reduced by 144 Euros with 1 g/dl higher Hb on day 2 postoperatively for women (p < 0.01).Preoperative anemia was associated with increased general ward costs for women and Hb loss with decreasing total hospital costs for men and women. Cost containment, especially reduced utilization of the general ward, may be feasible with the correction of anemia for women. Postoperative Hb values may be a factor for adjustments of reimbursement systems.Retrospective cohort study, III.The online version contains supplementary material available at 10.1007/s00402-023-04929-4. End-stage osteoarthritis (OA) is a global burden with a high prevalence, due to an aging society, with total knee arthroplasty (TKA) as a common surgery to improve pain and function . The proDue to the challenging correction of anemia preoperatively, the correction of iron deficiency anemia is also done postoperatively, but the improvements were not immediately significant during the hospital stay . AdditioSince Hb values are important screening parameters, preoperatively and postoperatively, this study investigated their association to hospital costs, in order to bridge the gap between medical care and financial management. The preoperative Hb values were examined, to reveal whether anemia is associated with not only negative effects, such as complications and increased LOS for the patient, but also whether its economic impact may provide a link to cost containment. Associations of preoperative anemia and costs might be an argument for standardized correction of anemia to reduce costs for the healthcare system with simultaneous positive effects for the patients. Although, there may be patient groups where a correction is not feasible, as mentioned implicitly in the discussion about preoperative anemia in the bundled-payment system, which revealed the research gap of postoperative Hb values and their use for risk adjustments into reimbursement systems. In this study the hospital costs were calculated for the reimbursement system of diagnoses related groups (DRG) in Germany. These hospital costs included inpatient costs and were sub-divided into general ward costs, which allowed a detailed analyses of cost components, especially with regard to LOS. An association of these hospital costs with postoperative Hb values may be the basis for risk adjustments in reimbursement systems. To the best of our knowledge this is the first paper to investigate preoperative anemia and its association with hospital costs for TKA as well as the course of postoperative Hb values and their association with hospital costs. In the last decade, transfusion rates were reduced sharply, and surgical techniques were improved, due to TRX. Hence, only patients without transfusions were analyzed to explore whether preoperative and postoperative Hb values were associated with hospital costs. The following research (RQ) questions were developed:RQ1: Was preoperative anemia associated with increased total hospital costs and general ward costs for TKA?RQ2: Which postoperative Hb values were associated with total hospital costs and general ward costs for TKA?®). The study is a part of the research project Munich Network Health Care Research—MobilE-Net. Patients with primary TKA, due to OA, were retrospectively included. All patients had one TKA, no simultaneous bilateral TKA. This study was approved by the ethics committee of Ludwig-Maximilians-Universität München (reference number: 18–274). All patients had a surgery between 5 August 2019 and 30 November 2020 and signed the consent to participate. The cross-sectional data was split into men and women, because Hb values differ between genders.The observational study comprised 367 patients from a single hospital in Germany, which was certified as an arthroplasty center of maximum medical care . Tourniquets or drains were not used.Thromboprophylaxis was performed in all cases with low-molecular-weight heparin from the evening of the day of surgery for 11–14 days postoperatively according to the applicable guideline. In cases with an increased thromboembolic risk, semi-therapeutic doses were administered from the 3rd postoperative day. In exceptional cases , new oral anticoagulants (NOACs) were also used.About 10% of patients were on antiplatelet medication . In patients taking warfarin or other oral anticoagulants prior to surgery, the medication was paused preoperatively and bridged with a low molecular weight heparin if necessary.TRX was used according to an in-house standard in all cases in which there was no contraindication to its administration, regardless of the presence of preoperative anemia, except patients had a thrombosis, deep vein thrombosis or pulmonary embolism. Additionally, TRX was not given to patients with an apoplex, cardiac infarction or stent implantation within 12 months before surgery.Hospital costs were collected for each patient from the financial department of the hospital and were calculated according to the standard of the Institute for the Hospital Remuneration System in Germany . Total hPreoperative anemia was included as a binary variable with one indicating anemia and zero otherwise. The definition of anemia follows the suggestions of the World Health Organization (WHO) , for menConfounders were selected thoroughly, because the cross-sectional, observational data were not randomized. The statistical analyses were based on a regression model, because with univariate statistical tests confounding is not possible. Hence, the study evaluated several variables as confounders, with reasons.General health before surgery was measured with the EQ VAS , part of the EQ-5D-5L questionnaire . This coSince the study was conducted during the COVID-19 pandemic, the lockdown status was included . The locFrom the 367 patients, 1 patient with 0 Euros total costs, 1 patient with 0 Euros implant costs, 1 patient with 0 days of LOS and 1 patient with a BMI > 200 were excluded, because all these values seemed to be not meaningful. Outliers were detected using boxplots and excluded above and below 1.5 times the 75% quantiles (22 patients). 341 patients remained for the analyses.2 from McFadden was used to evaluate the fit of the models, with a R2 between 0.2 and 0.4 indicating an excellent fit [® and the statistical analyses with R [Generalized linear models (GLM) with gamma distribution and log-link function were applied to account for confounders, due to the positive values of hospital costs. Coefficients below 1 indicate a negative association with the respective costs and coefficients above 1 indicate a positive association. The coefficients were interpreted in percentages of the mean. Anemia and each of the 4 Hb values were included in the models with the same confounders for men and women, referring to total- and ward costs respectively, resulting in 20 models. Model selection was done with the Akaike information criterion (AIC)  and the lent fit . The dats with R , 40–42.Several sensitivity analyses were performed . The maiThe descriptive results show that 147 men and 194 women received TKA with 21 preoperative anemic men and 21 preoperative anemic women. Total hospital costs were 8119 Euros for men with preoperative anemia and 7859 Euros for men without preoperative anemia. For women, the total hospital costs were 8204 Euros with preoperative anemia and 8015 Euros without preoperative anemia. Total hospital costs for these patients were about 260 Euros higher for men and 189 Euros higher for women with preoperative anemia and total hospital costs were generally higher for women compared to men. Normal ward costs were also increased for patients with preoperative anemia . The mean Hb values comprised 11.9 g/dl for men with preoperative anemia and 11.4 g/dl for preoperative anemic women. For patients without preoperative anemia the mean Hb were 14.9 g/dl for men and 13.7 g/dl for women. The course of Hb was similar for both genders, with − 1.8 g/dl for men with preoperative anemia and − 1.6 g/dl for women (without preoperative anemia − 2.8 g/dl for men and − 2.6 g/dl for women). The LOS was increased by 1 day for anemic women, but only by a third of a day for men. TRX was applied by roughly 78% of all patients with very low costs, because the drug costs per patient amounted in approximately 75 Euros. The implant costs were a major cost component with approximately 2000 Euros per patient .For men, the Hb loss between the preoperative Hb and the day before discharge was highly significantly associated with general ward costs and total costs. The general ward costs were reduced by 5.7% (approximately 161 Euros) with 1 g/dl less loss in Hb. The reductions for total costs were 3.7% (approximately 292 Euros) for 1 g/dl less loss in Hb. For women, the Hb on day 2 postoperatively was decisive, but only slightly significant for total costs (p < 0.05) and moderately significant for general ward costs (p < 0.01). General ward costs decreased by 2.6% (approximately 79 Euros) and total costs decreased by 1.8% (approximately 144 Euros) with 1 g/dl increased Hb.  was analyzed. There were only 21 women and 21 men with preoperative anemia. This is a relatively small number, hence there might be a bias, due to other reasons than anemia. Patients with blood transfusion were excluded, because the transfusion rate was approximately 1%, which was regarded to be too low for an analysis without a bias. Although the lockdown status for the COVID-19 pandemic was included as a dummy variable, a bias due to the pandemic cannot be excluded. TRX was generally applied for all patients with a consent on the medical information, despite not being approved for this indication. There are few contraindications where TRX was not given, including thrombosis, deep vein thrombosis and pulmonary embolism. Additionally, it was not given whether a patient had an apoplex, cardiac infarction or stent implantation within the last 12 months before surgery. This might cause a bias because patients were not randomly selected with and without TRX. All patients were treated with similar standards and therefore regarded to be comparable with respect to surgical approach and peri- and postoperative care. Nevertheless, there may be biases, due to different surgeons . Moreover, the model selection was explorative, hence further confirmation is required. Costs were calculated according to the InEK standard, not distinguishing between direct and indirect costs. General ward costs and total hospital costs included indirect and direct costs. Whether solely the direct or indirect costs would be evaluated, the results might change.Preoperative anemia was a risk factor for high costs for women and general ward costs, but not for men. Total hospital costs decreased for specific Hb values, especially the difference between discharge and preoperative Hb for men, and day 2 postoperatively for women. Hence, preparation of patients and further development of reimbursement systems should consider both, preoperative anemia, especially for women, and postoperative Hb values. Monitoring Hb values appeared to be a goal congruence between medicine and financial management, because all significant results revealed cost reductions with improved Hb.Supplementary file1 (PDF 35 KB)Below is the link to the electronic supplementary material.


The direction the evolution of virulence takes in connection with any pathogen is a long-standing question. Formerly, it was theorized that pathogens should always evolve to be less virulent. As observations were not in line with this theoretical outcome, new theories emerged, chief among them the transmission–virulence trade-off hypotheses, which predicts an intermediate level of virulence as the endpoint of evolution. At the moment, we are very much interested in the future evolution of COVID-19’s virulence. Here, we show that the disease does not fulfill all the assumptions of the hypothesis. In the case of COVID-19, a higher viral load does not mean a higher risk of death; immunity is not long-lasting; other hosts can act as reservoirs for the virus; and death as a consequence of viral infection does not shorten the infectious period. Consequently, we cannot predict the short- or long-term evolution of the virulence of COVID-19. During the waves of the COVID-19 pandemic, people often wonder about the future of the virus and its effect on us. One such effect in the spotlight these days is the evolution of virulence, the hereditary change in the relative ability of an infectious agent to cause disease. It is worth mentioning, as reviews on the subject often do, that there are different notions of virulence. Here, we use the definition of decreasing the fitness of the host mostly via increased mortality but also by decreased fecundity , and δ is the excess death rate caused by the infection. The basic reproduction number, the number of secondary infections an infected individual produces on average in a susceptible population, is:Around the beginning of the twentieth century, a view of virulence evolution emerged, which held sway for most of that century Méthot : pathogeβ), and decreasing the recovery (γ) or death rate (δ) would increase the basic reproduction number and thus fitness. The transmission rate (β) can increase but not indefinitely: Both the speed of infection cannot be instantaneous, and the number of susceptible hosts within reach cannot be unlimited . Recovery rate (γ) can also decrease, meaning it takes longer and longer to eliminate the pathogen. In the end, a persistent productive infection (γ = 0) is possible. Pathogen-caused excess death rate (δ) could also decrease and can theoretically reach zero. This evolutionary outcome is avirulence. The outcome heavily rests on the assumption of independence of the parameters.If the above three parameters are independent of each other, then increasing the transmission rate  and recovery rate (γ) are functions of virulence, measured here as excess death rate (δ). Thus, Eq. . Theory suggests that in such case the evolutionarily stable level of virulence is higher  and shortens the infectious period. There seems to be ample evidence for the pathogen’s replication being detrimental to the host  do not show any symptoms of Simian immunodeficiency virus (SIVsmm) infection  nor 100% lethality are optimal, and evolution would lead away from them . As the initial lethality was close to 100%, myxomatosis in rabbits is bound to evolve to lower virulence (or stay the same). Myxomatosis is caused by the myxoma virus belonging to the Poxviridae family of viruses. It causes a mild disease in its original host, the Brazilian cottontail (Sylvilagus brasiliensis). But in the European rabbit, the myxoma virus caused a mostly fatal disease, which is why it was used to control the invasive rabbit population in Australia. The virus is carried from one rabbit to another by an arthropod vector. The virus just hijacks the arthropod, and it does not replicate in it. While vector-borne pathogens are generally out of scope for the transmission–virulence trade-off hypothesis, as myxoma virus does not replicate in the vector, the hypothesis was still applied to it. It is worth mentioning that while on average, it seems that the lethality of the Myxoma virus has lowered in the European rabbits, very virulent strains are still circulating . HIVs are retroviruses which switched hosts from other primates to humans in the early twentieth century . Because of agricultural importance, a vaccine was developed for an otherwise mild disease that rarely if ever was lethal. The vaccine successfully kept symptoms at bay, but virus replication and transmission were not inhibited. Selection on limiting virulence was lifted as vaccinated birds do not suffer from the heightened virulence because of the vaccine  plays no role in the epidemic . The reIt is transmitted directly from living individuals as stated above.Coinfection by different strains of SARS-CoV-2 is not widespread. There is a possibility of coinfection with different strains of SARS-COV-2 . But as reinfection is possible and former infections protect from severe disease, then, on a population level, COVID-19 will become a mild, seasonal infectious disease (like influenza).Antia and coworkers  of 2–2.5  and a basic reproduction number , an endpoint of evolution (in an otherwise constant environment), it needs to be locally evolutionary stable and should be reachable from its genetic vicinity  of SARS-CoV2 will decrease, increase, or stay the same.The maturity of a field in natural science can be seen in the number of predictive mathematical formulas employed. It is high in physics, moderate in chemistry, and very few in biology. Biology is still in its juvenile stage. New data are generated faster than we can make sense of it. Still, data are not always generated on what we need but what is available and easy to do. Pathogen management should have a sound theoretical basis (Dieckmann et al.


Sunitinib and pazopanib are among the most prescribed targeted therapies for the systemic management of advanced renal cell carcinoma (RCC), but published cost comparisons between the 2 agents are few and limited by methodological and population differences. Also, sunitinib is administered on a 4-week on/2-week off cycle, and pazopanib is taken continuously. Thus, appropriate use and cost comparisons between the 2 drugs require methodological approaches to account for these differences. One way to accomplish this is to substitute expected for observed days supply. Recognizing the effects of nonrepresentative days supply values is important for assessing real-world treatment patterns and costs.To (a) characterize demographic and clinical characteristics among patients with RCC newly initiating sunitinib or pazopanib, using a large administrative claims dataset; (b) characterize treatment patterns, persistence, and costs for each treatment group; and (c) assess the effect on treatment patterns and costs for sunitinib by substituting 42 days for prescriptions with 28- or 30-day supplies to account for sunitinib’s 4-week on/2-week off dosing schedule.P < 0.05 for all comparisons except for interactions with significance set at P < 0.10. The effects of substituting 42 days supply for sunitinib prescription records with 28 or 30 days supply were determined.This was a retrospective cohort study using health care claims data from the Truven MarketScan Research Databases, which include enrollment information and medical and pharmacy claims. Baseline patient demographic and clinical characteristics and treatment patterns  were compared. Health care costs were calculated as mean daily index medication costs and as total, medical, and medication  costs over the 12 months post-index period. Inclusion criteria were continuous health plan enrollment between 6 months pre-index and 12 months post-index; no RCC medications 6 months pre-index; ≥ 2 RCC diagnoses within ±180 days of index; and age ≥ 20 years. For demographic and clinical characteristics, treatment patterns, and costs, means (± standard deviations) for continuous data and relative frequencies for categorical data were reported. Chi-square tests or Student t-tests were used to evaluate differences other than costs. A generalized linear model with gamma distribution and log link was used for evaluating costs, controlling for patient demographic and pre-index clinical characteristics, persistence days, and index medication. All statistical tests were 2-tailed with significance set at P = 0.0409), with fewer patients taking sunitinib continuing treatment than patients taking pazopanib. However, with substitution, treatment patterns differed significantly , but with more sunitinib patients than pazopanib patients continuing treatment. Without substitution, unadjusted daily mean index medication costs were significantly different for sunitinib ($216) versus pazopanib . Substitution of sunitinib days supply eliminated the significant differences in daily index medication costs between treatment groups. The 1-year RCC-related and all-cause medication, medical, and total unadjusted costs were not significantly different between treatment groups, and substitution had no effect on these costs. After adjustment for possible confounding factors, these cost results were similar to those found with unadjusted analyses.In total, 609  sunitinib patients and 183  pazopanib patients were included. Demographic and clinical characteristics were similar for each treatment cohort. The persistence periods and number of prescriptions filled were also similar. Without substitution, significant differences were observed between treatment groups in patterns of index medication use (overall In this study, patients with RCC who were initiating sunitinib and pazopanib had similar demographic and clinical characteristics and drug persistence patterns. The effect of substituting days supply values was demonstrated as an approach to considering differences in dosing cycles. Substitution significantly reduced sunitinib mean daily index medication costs and eliminated or reversed the direction of significant differences in costs between drugs during the persistence period. No significant differences were observed in unadjusted or adjusted 1-year costs. What is already known about this subjectSunitinib and pazopanib are commonly used as first-line treatment for advanced renal cell carcinoma (RCC).Persistence with self-administered oral oncology medications is vital to the success of treatment.Assessment of persistence (duration of therapy) and related costs of sunitinib requires consideration of the 4-week on/2-week off dosing schedule.What this study addsThe majority of prescription records contains days supply values that do not accurately reflect the recommended dosing schedule for sunitinib.Substitution of days supply to account for the recommended sunitinib dosing schedule eliminated the difference between groups in the proportion of patients continuing index treatment and in the mean daily index medication cost that resulted from using observed days supply.Total all-cause and RCC-related costs did not differ between index drug treatment groups over 1 year of follow-up.1 Approximately 62,700 new cases and 14,240 deaths due to kidney cancer are estimated to occur in 2016.1 A substantial proportion (15%-40%) of cases are diagnosed in late stages or will develop distant metastases,4 and estimates of 5-year relative survival are only 8%-12% for late-stage metastatic cases.5 Sunitinib and pazopanib are among the most prescribed targeted treatments for RCC,7 and pazopanib was found to be noninferior to sunitinib in a randomized clinical trial.8 However, compliance with therapy is critical to treatment success, and safety, adverse events, quality of life, and costs are factors that may affect compliance with treatment in real-world practice. Recent studies employing health care claims data reported similar persistence and compliance patterns between these agents.10Renal cell carcinoma (RCC) accounts for 2%-3% of all malignancies diagnosed in adults and approximately 85% of kidney cancers.11 treatment costs are known to affect patient adherence to self-administered oncologic medication and are of increasing importance to U.S. payers and oncology professional organizations.12 In choosing between efficacious drugs, costs may influence treatment preference for payers and patients. Yet, cost comparisons between the commonly used RCC first-line drugs sunitinib and pazopanib in real-world populations are few, and methodological differences limit interpretation across studies. Hansen et al. (2015) suggested that costs were lower for pazopanib-treated patients than for patients treated with sunitinib; however, the cohorts were not compared within the context of real-world treatment, but rather in the context of patients participating in a clinical trial, which does not provide an accurate profile of costs incurred in routine clinical practice.13 Racsa et al. (2015) presented differences between patients newly treated with sunitinib and pazopanib, suggesting higher annual costs for sunitinib, yet differences did not persist with adjusted models and were only statistically significant for nonadherent patients, perhaps because of the inclusion of patients previously treated for RCC.10In addition to adverse events,A preliminary analysis of sunitinib prescription records revealed that the majority (75%) of records had 28 or 30 days supplies indicated, although 42 days is the accurate supply for 1 prescription, given the recommended dosing schedule of 4 weeks on therapy followed by 2 weeks off. Objectives of the current study were to (a) characterize demographic and clinical characteristics among treatment-naïve patients with RCC newly initiating sunitinib or pazopanib from a large administrative claims dataset; (b) characterize treatment patterns, persistence, and costs associated with each treatment group; and (c) assess the effect on treatment patterns and costs for sunitinib by substituting 42 days for prescriptions that have 28 or 30 days supplies to account for sunitinib’s dosing schedule. Also examined were differences in costs by patient age (< 65 vs. ≥ 65 years) to serve as a possible proxy for commercial versus Medicare-insured status.This study used data from the Truven Health MarketScan Research Database, which provides enrollment data, paid medical claims for inpatient and outpatient utilization, and pharmacy claims for over 170 million U.S. patients. Truven Health databases comprise the health care characteristics of covered populations and providers, including employer-sponsored commercial plans as well as Medicare-supplemental employer-sponsored plans (both Medicare- and employer-paid portions). This study used HIPAA-compliant, de-identified data, as specified in US 45 CFR 46 and was thus exempt from review and approval by an institutional review board.International Classifications of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) diagnosis codes (189.0) for RCC and at least 1 prescription fill for sunitinib or pazopanib ; and prescription records with nonpositive days supply.Pre-index (baseline) demographic characteristics included age, gender, geographic region of residence  and continuous health plan enrollment (days) pre-index and post-index. Pre-index (baseline) clinical characteristics included the following:14Deyo Charlson Comorbidity Index (CCI) score and number and percentage (n %) of patients with each CCI comorbidity in 6 months pre-index period (Days from the earliest RCC diagnosis to the index date (back to January 2008).Six-month pre-index RCC-related (records with ICD-9-CM 189.0) and all-cause inpatient admissions, number and percentage of patients with an admission and per-patient mean (± standard deviation [SD]) number of admissions.Metastases (ICD-9-CM codes 196.xx-199.xx): Percentage of patients during 6 months pre-index and January 1, 2008-September 30, 2013.The persistence period consisted of the number of days from the start of the index medication to the end date of the last prescription’s days supply or the day before the start of a non-index RCC medication prescription within the allowed gap 30 days; . The folContinued: Patients did not switch or discontinue index medication .Early Switch: Patients switched to a nonindex medication while in possession of index medication (before run-out of last prescription’s days supply). There was at least 1 > 30-day gap for index medication during follow-up and at least 1 nonindex RCC medication within the 30-day gap.Delayed Switch: Patients switched to a nonindex medication after completion of the last index medication prescription’s days supply. There was at least 1 > 30-day gap for index medication and at least 1 nonindex RCC medication > 30 days after the last index prescription’s days supply before reinitiation of index medication or end of follow-up.Interrupted: Patients’ treatments were halted but reinitiated with index medication. That is, there was reinitiation of the index medication after the last index prescription’s days supply plus at least 30 days, with no nonindex RCC medications before reinitiation.Discontinued: Patients ceased treatment with index medication, with no switch to nonindex RCC medications from last index prescription through follow-up and no reinitiation of index medication from last index prescription’s days supply plus at least 30 days through the end of follow-up.The RCC medications included sunitinib, pazopanib, temsirolimus, axitinib, everolimus, interferon alfa-2B recombinant, bevacizumab, sorafenib, aldesleukin, and interferon alfa-2a .Costs were categorized into the following 3 groups:Index medication costs: Mean daily index medication cost during the persistence period was determined using persistence days as the denominator.1-year RCC-related costs: Payer-paid costs were determined over the 12-month follow-up for RCC medical (diagnosis-related), RCC medication, and total RCC-related  costs. RCC-related medical costs were based on inpatient and outpatient visits with an ICD-9-CM code of 189.0. Costs related to RCC medication included administrations and prescriptions for sunitinib, pazopanib, aldesleukin, axitinib, bevacizumab, everolimus, interferon α-2a, sorafenib, interferon α-2B recombinant, and temsirolimus.1-year all-cause costs: All-cause medical (inpatient + outpatient), all-cause medication, and all-cause total  costs were determined over the 12 months post-index. Any health care cost, regardless of diagnosis or type of medication, was included.Demographic and pre-index clinical characteristics, treatment patterns, and costs between treatment groups were compared using means [± SD] for continuous variables and relative frequencies  for categorical data.The current starting dose schedule recommendation for sunitinib is 50 mg orally every day for 4 weeks followed by 2 weeks off therapy . The days supply value in the prescription record is integral to calculations of persistence, yet the majority of records in a preliminary analysis had 28 or 30 days supply, which does not account for the 2 weeks off therapy. Thus, we evaluated the effect of substituting 42 days for sunitinib prescription records with 28 or 30 days supply on calculations of mean days supply, treatment patterns including persistence, and costs. To serve as a possible proxy for commercial versus Medicare-insured status, differences in costs were evaluated for patients aged < 65 years versus patients aged ≥ 65 years.P < 0.05, except for treatment group by age interactions where significance was set at P < 0.10.Chi-square tests were used to test for differences in categorical variables, and Student t-tests were used for continuous variables other than costs. A multivariable regression model was used to examine differences in costs between sunitinib and pazopanib. Since health care costs are typically non-normally distributed, a generalized linear model with a gamma distribution and log link was used for evaluating costs (dependent variable), controlling for patient demographics and pre-index characteristics, persistence days, and index medication (as predictor variables). All statistical tests were 2-tailed with significance set at Following application of inclusion/exclusion criteria, 609 patients initiating treatment with sunitinib and 183 patients initiating treatment with pazopanib were identified as the study sample . Of thesP = 0.002). Of note, the length of time between the earliest pre-index RCC diagnosis  and the index date was also shorter for sunitinib (373 days) than pazopanib .No significant differences in mean age, gender, geographic region, pre-index inpatient admissions, or metastatic diagnosis were observed between sunitinib or pazopanib treatment groups . Mean  daily costs were similar between comparators: $178.49 [51.89] for sunitinib versus 176.65 [48.93] for pazopanib (P = 0.669). The adjusted 1-year RCC-related and all-cause costs were not significantly different between index medication treatment groups, and substitution had no effect on these costs.After adjusting for possible confounding factors , we foun13 Using health care claims, we compared patient characteristics and medication use patterns between treatment-naïve RCC patients initiating sunitinib and pazopanib as first-line therapy. This study contributes unique health care cost information and demonstrates the effect of using substitution of days supply to address the difference in dosing schedules of these 2 drugs. There are few studies comparing first-line RCC targeted treatment groups, but we highlight key similarities and differences with this study.Accurate understanding of treatment persistence and costs in a real-world setting is an important contributor to decision making regarding the first-line treatment of RCC. Existing literature describing cost differences between sunitinib and pazopanib include a study applying costs to a clinical trial population and a real-world study of pazopanib and sunitinib in first or later lines of therapy for RCC.15 Similar to our results, the results of the only other real-world study comparing sunitinib and pazopanib treatment patterns using CCI scores found a similar extent of comorbidity between treatment groups.9 In the current study, a significantly longer time from RCC diagnosis to index date and longer pre-index enrollment period for pazopanib patients than sunitinib patients was observed, although reasons for these differences are not clear. It is possible that the baseline health of patients or physician experience influenced the choice of index treatment. Because pazopanib was approved for advanced RCC late in 2009, physicians may have been more inclined to prescribe sunitinib as first-line treatment in the early years of the study period. This might also explain the larger proportion of sunitinib patients in the study overall.Mean age, the predominance of males, and geographic distribution of patients were similar between treatment groups and comparable to national statistics and previous studies of various targeted RCC treatments using health care claims.16 A few retrospective studies have assessed persistence to tyrosine kinase inhibitors in RCC, but comparisons are hampered by dissimilar measures and methodology. Important factors that must be considered when comparing treatment patterns and costs over time are similarities or differences in dosing schedules. The problem of drug exposure misclassification with inaccurate pharmacist-recorded days supply has been described, and correction of days supply by substitution has been used in disease states where noncontinuous daily drug dosing occurs.19 Packaging of sunitinib as 28 count and U.S. health plan dispensing requirements likely contribute to pharmacist entry of 28 count or 30 count in the days supply field of the prescription record.Increasing use of oral oncology agents provides convenience advantages over clinic-delivered treatments, but it has been reported that persistence to self-administered drugs may be problematic.10 Despite different methods and patient selection criteria, both studies yielded similar conclusions as the current study, that is, no significant differences in persistence between treatment groups. Taken together, all studies further emphasize that consideration of dosing schedule is needed when comparing sunitinib to other first-line RCC treatments.For the current study, the need to accommodate for non-continuous daily dosing through correction of days supply by substitution was informed through preliminary analysis. This analysis revealed that most sunitinib prescriptions had days supply values of 28 or 30, both less than the dosing cycle of 42 days, but prescription date intervals were consistent with a dosing cycle of 42 days. The effect of substitution was evident through changes in persistence results and consequent conclusions regarding differences between treatment groups that involve this measure. In their studies comparing treatment patterns between sunitinib and pazopanib, DaCosta Byfield et al. (2015) adjusted for sunitinib’s dosing schedule using different methods, and Racsa et al. used substitution methods similar to the current study.The final objective of this study was to compare mean daily index medication costs and all-cause and RCC-related health care costs during the 12-month follow-up period. It was hypothesized that substitution would affect cost calculations because days supply affects calculations of the persistence period and, as a result, costs calculated over the persistence period. Without substitution, sunitinib had a significantly higher average daily medication cost, but substitution eliminated the difference. No significant differences were observed between treatment groups stratified by the age 65 threshold in adjusted daily costs, RCC-related costs, or all-cause 12-month costs. Before adjustment for demographic and clinical characteristics, all-cause medical costs were significantly higher in the sunitinib group in patients aged ≥ 65 years but were not different after adjustment.7The authors reported an average across all drugs based on data from 2004-2010, but the results are comparable to the 1-year post-index RCC medication costs for sunitinib and pazopanib observed in the current study. Racsa et al. reported higher total health care costs than in the current study (with no significant differences between treatment groups) and also found that adjusted pharmacy costs for sunitinib and pazopanib patients were not significantly different, including index and non-index RCC medications.10 However, the magnitude of cost results are not comparable to the current study because of the much shorter treatment duration in the Racsa et al. study and because that study’s sample had more Medicare patients, whereas the current study had more commercial patients.10 Our examination by age group was intended to serve as a possible proxy for commercial versus Medicare insured status. Gross covered payments for both treatment groups were higher in patients aged < 65 years compared with those patients aged ≥ 65 years, but no significant differences were observed between treatment groups for either age group.Few studies have examined health care costs associated with RCC treatment. Geynisman et al. (2015) reported similar first-year, first-line oral drug costs for patients aged < 65 years.13 Hansen et al. used a clinical trial population and applied wholesale acquisition drug costs to treatment and assigned price weights from insurance data to select adverse events rather than study patients from routine clinical practice and the actual costs associated with their care.13 The authors’ conclusion that overall costs were greater for patients treated with sunitinib was based solely on differences observed in drug costs, and it is important to note that the reference dates for drug costs used were from different time points: preceding a price increase for pazopanib and following a price increase for sunitinib. Racsa et al. observed no difference in overall annual costs between the 2 treatment groups but did observe significantly higher annual costs in nonadherent sunitinib patients compared with nonadherent pazopanib patients.10 This difference may be explained by unreported factors and/or patient selection, since patients previously treated for RCC were included, and there was a significantly higher rate of previous treatment in the pazopanib group.10 Comparisons of cost in the published literature are few, and understanding the methodologies employed, results observed, and conclusions made is critical. Accurate data are vital in cost studies, as well as patient, physician, and payer considerations regarding treatment.Any economic comparison must have accurate cost data as its foundation, and so far, real-world studies using actual costs are lacking. To date, only 2 studies have directly compared health care costs between patients receiving sunitinib and pazopanib.Possible limitations to this study should be considered in the interpretation of the findings, in part because health care claims data were not created specifically for research purposes. Claims may contain errors in coding or may be incomplete. The presence of prescription fill claims cannot be considered to represent proper self-administration or adherence or include any drugs obtained outside of the pharmacies filing the claims, such as physician samples, or patients’ participation in a clinical trial. Nevertheless, there is no reason to believe these limitations would be present to differing degrees in patients taking sunitinib versus pazopanib. This study also did not assess survival or adverse events, which might influence persistence results. Also, this study’s results are not necessarily generalizable to patients who are not enrolled in similar health plans as those in this study, since the characteristics and gross covered payments may differ among different insured samples.This analysis of persistence and actual costs represents the experience of real-world clinical practice of first-line treatment of RCC with sunitinib and pazopanib. Accounting for sunitinib’s unique dosing cycle for persistence and cost calculations is critical when comparing first-line RCC targeted treatments. The substitution of sunitinib days supply corrected for the values not reflecting actual patient experience. Calculations affected by this measure include daily index medication costs. There were no significant differences in persistence and similar RCC-related and all-cause costs over the 12-month period following the initiation of sunitinib or pazopanib as first-line therapy for RCC. The results of this study support selection of initial systemic treatment for RCC based on patients’ individual clinical factors and physician experience in using these agents.