To investigate the etiological characteristics of plastic bronchitis (PB) caused by pulmonary infections in children and to identify any differences in the clinical features of PB cases caused by different pathogens.We collected data on children diagnosed with PB and admitted to the Respiratory Department at Soochow University Children’s Hospital between July 2021 and March 2023 utilizing electronic bronchoscopy. We analyzed clinical characteristics and the species of pathogens causing the illness in these children.Mycoplasma pneumoniae (MP) had the highest detection rate at 53.3%, followed by the Boca virus at 26.7%. MP-induced PB typically occurs in older children with an average age of 7.46 ± 2.36 years, with the main symptoms including high fever (85.7%) and local hyporespiration (42.9%). In contrast, Boca virus-induced PB tends to occur in younger children, with the main symptoms of moderate fever (54.5%), and wheezing (54.5%). The MP group exhibited a higher incidence of both internal and external pulmonary complications, including pleural effusion (42.9%), elevated aspartate aminotransferase (52.4%), lactic dehydrogenase (76.2%), and D-D dimer (90.5%). Conversely, the Boca virus group primarily showed pulmonary imaging of atelectasis (81.8%), with no pleural effusion. The average number of bronchoscopic interventions in the MP group was 2.24 ± 0.62, which was significantly higher than that required in the Boca virus group (1.55 ± 0.52). During the second bronchoscopy, 57.1% of children in the MP group still had visible mucus plugs, while none were observed in the Boca virus group.A total of 45 children were enrolled. The main clinical symptoms observed were cough (100%), fever (80%), shortness of breath (28.9%), and wheezing (20.0%). Pathogens were identified in 38 (84.4%) patients. MP and Boca virus are the primary pathogens responsible for PB among children. The clinical manifestations of PB typically vary significantly based on the pathogen causing the condition. Plastic bronchitis (PB) is a rare, partial or complete airway obstructive disease characterized by the formation of arborising, thick, tenacious casts of the tracheobronchial tree . It occuElectronic bronchoscopy and bronchoalveolar lavage (BAL) are indispensable techniques for investigating and treating pediatric patients with PB and both are carried out as routine procedures in many health centers –10. In tA retrospective study was conducted on 45 pediatric patients who were diagnosed with PB through electronic bronchoscopy and admitted to the Respiratory Department of Children’s Hospital of Soochow University from July 2021 to March 2023. Patients with congenital heart disease and sickle-cell disease were excluded from the study.Upon admission, demographic and clinical information was collected from all patients. Within four hours of admission, venous blood samples were taken for blood routine, liver and kidney function, coagulation routine, and pre-transfusion examination. Chest radiography was performed within 24 h prior to admission or within the next 24 h. All patients underwent electronic bronchoscopy within 72 h after admission and BAL samples were collected. In some cases, electronic bronchoscopy was reviewed 3–4 days after initial examination.MP).Before the electronic bronchoscopy procedure, parents were notified of the potential surgical risks and provided informed consent. Pediatric patients were required to fast from both solids and liquids for at least 6–8 h prior to the procedure. Premedication in the form of intramuscular atropine sulfate, at a dose of 0.01–0.02 mg/kg, was given to the children. The procedure was performed under the care of experienced anesthesiologists. An electronic bronchoscopy  was wedged into each lobe. Foreign body forceps or brush were used to clean and remove the plastic sputum suppository in order to restore unobstructed airway passage Normal saline (0.9%) was used for local irrigation before and after cleaning up the plastic phlegm suppository. Local irrigations were 1ml/kg each time, and first 3 lavages were collected by a sterile sputum-collecting pipe . The collected BAL sample was used for cell counts, viral analysis, and microbiological analysis . At least 500 cells were examined for each specimen. The ratios of various cell types in total cell counts were reported.Mycoplasma Pneumoniae (MP). Bacteria were tested by inoculating BAL samples onto blood plates and examining them after incubation for 18–20 h. Bacterial growth > 103 colony-forming units/ml was considered significant. MP, Chlamydia pneumonia and viruses, including respiratory syncytial virus, adenovirus, influenza virus , parainfluenza virus, Boca virus, human rhinal virus, human coronavirus, human metapneumovirus were investigated by polymerase chain reaction using a 13 Respiratory Pathogen Multiplex Detection Kits   according to the manufacturer’s instructions.BAL samples from plastic bronchitis were tested for 13 types of viruses and bacteria, as well as P value < 0.05 was considered statistically significant.Measurement data were presented as the mean ± standard deviation (SD). Counting data is expressed as a percentage. Comparison between groups were performed by t test, or Fisher’s exact probability method. A Between July 2021 and March 2023, our department diagnosed 45 cases of PB. The oldest patient was 12 years and 8 months old, while the youngest was 1 year and 3 months old. The average age of the patients was (5.63 ± 2.94) years old, with 28 (62.2%) being male and 17 (37.8%) being female.All patients undergoing electronic bronchoscopy showed blockage of the lumen by mucus sputum plugs. The plastic sputum plugs obstructing the bronchus were successfully removed after cleaning with foreign body forceps and brush. The plastic phlegm products were found to be located in the left bronchus or its branches in 22 cases (48.9%), in the right bronchus or its branches in 20 cases (44.4%), and in both in 3 cases (6.7%).The BAL sample analysis revealed neutrophilic inflammation in PB. The mean percentage of neutrophils was (67.00 ± 22.58)%. Eosinophil levels were elevated in only two patients, with 6% present in one case and 7% in the other.All 45 cases (100%) presented with cough symptoms. Additionally, 36 cases (80%) had a fever, with 1 case experiencing low fever (37.3℃-38.0℃), 9 cases with moderate fever (38.1℃-39.0℃), and 26 cases with high fever (39.1℃-41.0℃). Wheezing was observed in 9 cases (20.0%), shortness of breath in 13 cases (28.9%), and the three depressions sign in 9 cases (20.0%).Pulmonary signs: 14 cases (31.1%) had reduced breath sounds on auscultation in a localized area. 15 cases (33.3%) had wheezing sounds. 10 cases (22.2%) had wet rales. And in 10 cases (22.2%) no positive pulmonary signs were identified.All children exhibited a large flake shadow on their lung imaging. Out of them, 21 patients (46.7%) displayed atelectasis, 12 patients (26.7%) had pleural effusion, 1 patient (2.2%) exhibited pneumothorax, and 1 patient (2.2%) had bronchial expansion as a complication.MP, 12 (26.6%) tested positive for Boca virus, 2 (4.4%) tested positive for Influenza A, and 1 (2.2%) tested positive for human rhinovirus. Of these cases, 2 were concurrently diagnosed with both MP and Streptococcus pneumoniae infections, 1 with MP and Boca virus infections, and 1 with Influenza A and Streptococcus pneumoniae infections.All patients underwent testing to determine the etiology of the BAL sample, with a specific pathogen identified in 38 cases (84.4%), while the remaining 7 cases yielded negative results. Among the children with a positive pathogen identification, 24 (53.3%) tested positive for MP or Boca virus alone. Data specifically related to these groups can be found in Table Boca virus group was significantly lower than that in the MP group. This difference was found to be statistically significant (P < 0.001). There was no significant difference in sex ratio between the two groups (P = 0.712). The average length of hospital stay for the MP group (11.62 ± 3.12 days) was slightly longer than that for the Boca virus group (10.18 ± 3.49 days), but the difference was not statistically significant (P = 0.244).For comparative analysis, we included only children infected with either MP group exhibited fever, primarily high fever, accounting for 85.7%, whereas most of the children in the Boca virus group experienced moderate fever (54.5%). There was a statistically significant difference in the peak temperature between the two groups (P = 0.010). Wheezing was observed in 54.5% of children in the Boca virus group, but none in the MP group, and there was a statistically significant difference between the two groups (P = 0.001). Tachypnea was present in 45.5% of children in the Boca virus group, marginally higher than in the MP group (19.0%), although the difference was not statistically significant (P = 0.213). Similarly, 45.5% of children in the Boca virus group exhibited three depressions sign, which was significantly higher compared to 4.8% in the MP group (P = 0.011).All the children in the Boca virus group, whereas none in the MP group exhibited wheezing sounds. This difference was statistically significant (P < 0.001). Conversely, 42.9% of the children in the MP group displayed local respiratory hypotonia, which was slightly higher compared to the Boca virus group, though the difference was not significant (P = 0.248).During pulmonary auscultation, palpable wheezing sounds were present in 72.7% of children in the Boca virus group (81.8%) compared to those in the MP group (38.1%). Conversely, children in the MP group had a higher likelihood of developing pleural effusion (42.9%), while none exhibited pleural effusion in the Boca virus group. These differences were statistically significant .The prevalence of atelectasis was higher in children in the P > 0.05). The percentage of neutrophils and lymphocytes in the MP group was considerably higher than that in the Boca virus group . The proportion of phagocytic cells in the Boca virus group was significantly greater than that in the MP group (P = 0.001).There was no statistical discrepancy between the two groups in terms of the formation site of plastic phlegm suppository (MP group (P = 0.009), while the percentage and absolute value of lymphocytes were significantly higher in the Boca virus group . Furthermore, the elevated percentage of eosinophils in children with Boca virus was higher than that in the MP group (P = 0.009). Additionally, the MP group was more prone to experiencing other systemic complications. The proportions of increased AST and D-D dimer were significantly higher in this group compared to the Boca virus group . No elevated aminotransferase was identified in the Boca virus group.There were no significant discrepancies in the total number of white blood cells, absolute value of neutrophils, and platelet count between the two groups. The percentage of neutrophils was significantly elevated in the MP group was 2.24 ± 0.62, which was greater than the Boca virus group (1.55 ± 0.52), and the difference was statistically significant (P = 0.004). During the second bronchoscopy, 57.1% of the children in the MP group still had visible mucus plugs, whereas none of the children in the Boca virus group had visible mucus plugs (P = 0.002). These findings suggest that performing a second tracheoscopy intervention may not be necessary in the Boca virus group.The average number of bronchoscopic interventions needed in the MP [influenza virus A [adenovirus [Boca virus [MP and around 1/4 were associated with Boca virus, indicating that these two are the primary pathogens responsible for infection-related PB. Other causative agents include influenza virus A and rhinovirus.PB is characterized by the presence of thickened bronchial casts that can be coughed up or detected during bronchoscopy or surgical procedures. The etiology of PB is multifaceted, with infectious pathogens being an important contributing factor. Several pathogens have been reported in the literature as potential causes of PB, including MP , influen virus A , adenovienovirus , 12 and ca virus . Our stuPlastic bronchitis, due to the formation of sputum in the bronchial cavity, causing severe blockage of the airway, causing clinical symptoms such as shortness of breath and breathing difficulties, which can be life-threatening in severe cases , 15. TheMP and boca virus can cause PB, there are noticeable differences in their clinical manifestations. Currently, much research has been conducted on PB caused by MP [MP pneumonia [MP was primarily characterized by high fever. Previous studies have shown that MP can also cause wheezing in children [MP wheezed, but the local respiratory sound was low. It may be related to the complete blockage of the lumen by the plastic-shaped sputum plug formed by MP infection. Studies showed that MUC5AC and MUC5B played an important role in the formation of mucin plugs [MP [MP infection clogs the lumen more firmly, leading to low local breathing sounds rather than wheezing. Of course, we still need more cases to observe in the later stage. There were also more internal and external pulmonary complications in MP cases, including pleural effusion, elevated aminotransferase, elevated LDH and D-D dimer levels. The BAL fluid showed significant neutrophilic inflammation.Although both ed by MP , 16, 17.children  However,in plugs . The elein plugs . Our prelugs [MP . This maBoca virus-induced PB, with most literature limited to case reports [Boca virus infected identical twins, leading to the development of PB in both children. This suggests that even a simple Boca virus infection can trigger PB in otherwise healthy children [Boca virus is a common cause of PB, with a higher incidence in young children, with an average age of 2.86 years. Clinical symptoms typically include moderate fever and wheezing, with nearly all children presenting wheezing sounds in their lungs, with half experiencing difficulty breathing. Pulmonary imaging studies mainly show atelectasis, with no accompanying pleural effusion. As opposed to MP, there are fewer extrapulmonary complications in children infected with Boca virus, and no elevations in transaminase levels are typically observed.At present, there is a relative shortage of studies focused on  reports . One stuchildren . In our MP required significantly more bronchoscopic interventions on average than those caused by Boca virus. More than half of the children with MP infection still had mucus plugs present during the second bronchoscopic intervention, indicating that a single intervention was often insufficient, whereas those with Boca virus-induced PB typically required only one intervention.The treatment for PB typically involves targeted therapies to address the underlying pulmonary condition, as well as interventions to facilitate the removal of mucus plugs. Bronchoscopy is both a diagnostic tool and a primary method of treatment for PB . In thisMP and Boca virus are the primary pathogens associated with PB in children. There are significant differences in the clinical manifestations of PB caused by different pathogens.To summarize, infection is a significant contributor to PB in children, presenting as symptoms such as cough, fever, wheezing, and shortness of breath.


Diaphorina citri Kuwayama) that fed solely on mandarin (Citrus ×aurantium L.) plants grafted with buds infected with ‘Candidatus Liberibacter asiaticus’ (CLas), a phloem-limited bacterium associated with the severe Asian variant of huanglongbing (HLB), the most destructive disease of citrus. Brassica yellows virus (BrYV), the causative agent of yellowing or leafroll symptoms in brassicaceous plants, and its associated RNA (named as BrYVaRNA) were detected in ACP. In addition, the porcine reproductive and respiratory syndrome virus (PRRSV), which affects pigs and is economically important to pig production, was also found in ACP. These viruses were not detected in insects feeding on plants grafted with CLas-free buds. Changes in the concentrations of insect-specific viruses within the psyllid were caused by coinfection with CLas.Detection of new viruses or new virus hosts is essential for the protection of economically important agroecosystems and human health. Increasingly, metatranscriptomic data are being used to facilitate this process. Such data were obtained from adult Asian citrus psyllids (ACP)  trees from a commercial orchard and grafted them onto CLas-free mandarin plants under laboratory conditions. Through metatranscriptome sequencing, we first identified the Asian citrus psyllids feeding on plants grafted with CLas-infected buds carried the plant pathogen, brassica yellows virus and its associated RNA, and the swine pathogen, porcine reproductive and respiratory syndrome virus. These discoveries indicate that both viruses can be transmitted by grafting and acquired by ACP from CLas+ mandarin seedlings. Plants are infected with a diverse range of microbes that interact with their host and with each other. The majority of these microbes are not causal agents of disease , and manBrassica, Raphanus, and Sinapis (Polerovirus (Solemoviridae); it has since been reported in Korea (–Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) (Brassica yellows virus (BrYV) is a recently discovered virus that mainly infects cruciferous vegetables in which it causes mottling, yellowing, or leaf roll symptoms , 11. It  Sinapis . BrYV wa Sinapis  and is tin Korea  and Austin Korea . Moleculin Korea , 14, 15,in Korea –19; one rodidae) . A recenrodidae) .ca. 15 kb in length (–Aedes vexans (Meigen) (Diptera: Culicidae) and houseflies (Musca domestica L. [Diptera: Muscidae]) from infected pigs and in which it can be retained for a short period; the virus did not replicate within the insects  (Hemiptera: Sternorrhyncha: Psyllidae) is an economically important pest of citrus. It is the major vector of the pathogenic bacterium ‘Candidatus Liberibacter asiaticus’ (CLas), which is the putative causal agent of the most severe form of huanglongbing (HLB), the most serious disease of citrus (Citrus tristeza virus (CTV: Closteroviridae) (Diaphorina citri-associated C virus (DcACV: family not assigned), Diaphorina citri bunyavirus (DcBV: Bunyaviridae), Diaphorina citri cimodo-like virus (DcCLV: Reoviridae), Diaphorina citri densovirus (DcDNV: Parvoviridae), Diaphorina citri flavi-like virus (DcFLV: Flaviviridae), Diaphorina citri picorna-like virus (DcPLV: Picorna-like virus), and Diaphorina citri reovirus (DcRV: Rheoviridae) (–f citrus . Besidesviridae)  and seveviridae) –40.CLas+ and CLas– plants, to determine if the virome composition differs due to coinfection with CLas. Two viruses, the brassica yellows virus and the porcine reproductive and respiratory syndrome virus, are the first reports in ACP.Traditional viral screening often uses PCR and qPCR , 41, whiCLas+ ACP and passed into intestinal epithelial cells by incorporation of secretory granules .A total of 38,676,951 and 34,843,908 clean reads were obtained from the fication  and a BLCLas+ (2 contigs) and CLas– (3 contigs) samples of the Guangdong population. These sequences ranged from 326 to 2,339 nucleotides (nt) in length and showed >98% similarity to an isolate from a Taiwan population. Second, a BLAST search showed that contigs of 9,951 and 9,920 nt amplified from CLas+ and CLas– samples both showed 96.0% similarity to DcPLV found in an ACP population in Brazil. Lastly, a total of 111 contigs from CLas+ samples and 10 contigs from CLas– samples were identified as DcRV and showed >99% similarity to the DcRV-FL isolate recorded in Florida.First, DcDNV sequences were identified in CLas+ and CLas– ACP samples, respectively, as CTV sequences. The contigs ranged from 260 to 3,997 nt in length and had a close relation to the CTV-FN08 isolate from Guangdong, China.The Kaiju classification and BLAST searches confirmed that 5 and 15 contigs from CLas+ sample was named as BrYV-DC; this virus was not detected in the CLas– samples. It has the typical genomic organization of the BrYV genome, including 6 ORFs  (To compare the new BrYV isolate obtained from ACP to previously characterized BrYV isolates (Table S2), a phylogenetic analysis using maximum likelihood (ML) was performed. The analysis showed most of the BrYV isolates being in the same monophyletic clade. BrYV-DC clustered with BrYV-CC and BrYV-CR, which were both associated with the plant host icaceae) .CLas+ sample and shares 95.1% nt identity with a partial sequence of the Turnip yellows virus (TuYV)-associated RNA  and 94.9% nt identity with the complete sequence of beet western yellows virus (BWYV)-associated RNA (BWYVaRNA: KF533709). It has a typical genomic organization of Polerovirus-associated RNAs, encoding overlapping ORF1a and ORF1b , ranging from 301 to 1,664 nt in length, were identified as PRRSV type 2 (Table S1). To infer the phylogenetic position of PRRSV-DC, we obtained 1,198 PRRSV type 2 and one PRRSV type 1 isolates from NCBI (Table S4). Based on the sequences of ORF5, phylogenetic analysis showed that PRRSV-DC belongs to the North American type (type 2) and grouped with isolates rV63, rV68, YN-2011, BJ4, GS2002, GS2003, and GS2004    including orchard species and roadside weeds, need (s) to be determined. Some Polerovirus-associated RNAs cause severe of symptoms when in combination with a helper virus, e.g., BWYV ST9-associated RNA and Carrot red leaf virus-associated RNA  Heynh. , which facilitates DNA glycosylase activity to decrease viral DNA methylation thereby promoting viral virulence   and strasaceae]) . In thisated RNA . In addiirulence . This isCLas+ plants, and be acquired by ACP. The successful colonization of BrYV in mandarin in orchards is most likely related to transmission by other insect vectors—particularly phloem-feeding insects other than ACP—commonly found on citrus. There is also a possibility that organisms in the soil rhizosphere, phyllosphere fungi or microbial biofilms could be involved in transmission. For example, COVID-19 can be transmitted through biofilms, aerosols and wastewater (–Aphis (Toxoptera) aurantii Boyer de Fonscolombe, Aphis (Toxoptera) citricidus , Aphis gossypii Glover, Aphis spiraecola Patch, and Myzus persicae (Sulzer) (Sternorrhyncha: Aphidae), associated with plant pathogens of citrus in China (Aphis gossypii is known to feed on Brassica rapa L. (syn. B. campestris L.) and from which it can transmit turnip mosaic virus and cucumber mosaic virus 2  cascades and the subsequent induction of defense responses   , and thebacteria . In the bacteria . In addibacteria . The combacteria . Furtherbacteria , 76. Duebacteria , 78. In stewater . Hence, iversity .CLas, weakness of trees and poor immune balance of plants caused by CLas may lead to susceptibility to viruses. For example, sec-delivered effectors (SDE15) secreted by CLas can inhibit multiple members of papain-like cysteine proteases (PLCPs), which are extracellular broad-spectrum defense proteins  in east-central Guangdong province, China, and buds from them were cut and grafted onto CLas–free mandarin plants. Buds from the CLas-free plants were also budded onto CLas–free mandarin plants to act as controls. All the grafted citrus plants were kept in a small, insect-proof net house. After 6 months, each plant was assayed by qPCR for the presence of CLas with the primer pair, HLBas/HLBr, targeting the 16S rRNA gene (CLas+) ‘Shatangju’ plants with CT values <25, and the uninfected (CLas-) control with CT values >36 were chosen for further experimentation.RNA gene . The infMurraya paniculata (L.) Jack (Aurantieae) at South China Agricultural University, Guangzhou, Guangdong, in 2013, was used in the present study. The strain had been maintained on M. paniculata in controlled environment chambers under a 14 h: 10 h light: dark cycle at 28 ± 1°C and 60% relative humidity for more than 25 generations.A laboratory strain of ACP collected from CLas+ and uninfected (CLas–) ‘Shatangju’ plants for 14 days under a 14 h: 10 h light: dark cycle at 28 ± 1°C and 50–60% relative humidity. After this period, the heads of individual adult psyllids were dissected for DNA extraction using a Tiangen DNA extraction kit  following which the CLas status of each individual was determined by qPCR as above.Third to fourth instar psyllid nymphs were selected and reared on the CLas-infected ACP adult was dissected then fixed in 2.5% glutaraldehyde in 0.1 M phosphate-buffered saline  at 4°C overnight. The tissue was then rinsed in 0.1 M PBS, secondarily fixed with 2% osmium tetroxide in 0.1 M PBS at room temperature for 4 h then embedded in Spurr’s resin. Sections (100 nm) of abdomen tissue were cut with a diamond knife, stained with 2% uranyl acetate and alkaline lead citrate. Sections were then examined using a transmission electron microscope  and photographed using a digital camera .The abdomen of a CLas+ and two aliquots 20 CLas– ACP were each pooled to constitute a sample to give two samples of each psyllid type and the thoraxes and abdomens ground in liquid nitrogen. RNA was extracted from each sample using TRIzol. Three biological replicates were made from every sample to give 12 extracts in total and used for RNA-seq. The resulting Illumina reads were trimmed using Trimmomatic v0.33 . Viral sequences were aligned using MAFFT v7.471 with the l-INS-I strategy (The reads and assembled contigs were preliminarily classified using the Kaiju virus database . Then, tstrategy  and maxistrategy  to inferhttps://github.com/ljmhaha/data.of.ACP.All the RNA samples are deposited in −80°C refrigerator in the College of Natural Resource and Environment of China Agricultural University. And all the raw data and clean data of metatranscriptome sequencing are deposited in the private account of corresponding author of data storage center of College of Advanced Agricultural Science of Zhejiang A&F University. The newly generated viral sequences were deposited in


Chronic obstructive pulmonary disease (COPD) affects approximately 15 million people in the United States and accounts for approximately $36 billion in economic burden, primarily due to medical costs. To address the increasing clinical and economic burden, the Global Initiative for Chronic Obstructive Lung Disease emphasizes the use of therapies that help prevent COPD exacerbations, including inhaled corticosteroid/long-acting beta2-agonist (ICS/LABA).To evaluate health care costs and utilization among COPD patients newly initiating ICS/LABA combination therapy with budesonide/formoterol (BFC) or fluticasone/salmeterol (FSC) in a managed care system.COPD patients aged 40 years and older who initiated BFC (160/4.5 μg) or FSC (250/50 μg) treatment between March 1, 2009, and March 31, 2012, were identified using claims data from major U.S. health plans. BFC and FSC patients were propensity score matched (1:1) on age, sex, prior asthma diagnosis, prior COPD-related health care utilization, and respiratory medication use. COPD-related, pneumonia-related, and all-cause costs and utilization were analyzed during the 12-month follow-up period. Post-index costs were assessed with generalized linear models (GLMs) with gamma distribution. Health care utilization data were analyzed via logistic regression (any event vs. none) and GLMs with negative binomial distribution (number of visits) and were adjusted for the analogous pre-index variable as well as pre-index characteristics that remained imbalanced after matching.P = 0.003), reflecting lower inpatient , pharmacy , and outpatient/office  costs, but higher emergency department  costs. Pneumonia-related health care costs were also lower on average for BFC patients . Similarly, initiating BFC was associated with lower all-use health care costs versus initiating FSC . No differences in health care utilization were found between the 2 groups.After matching, each cohort had 3,697 patients balanced on age (mean 64 years), sex , asthma and other comorbid conditions, prior COPD-related health care utilization, and respiratory medication use. During the 12-month follow-up, COPD-related costs averaged $316 less for BFC versus FSC patients ($4,326 vs. $4,846; In this study, although no difference was observed in rates of health care utilization, COPD patients initiating BFC treatment incurred lower average COPD-related, pneumonia-related, and all-cause costs versus FSC initiators, which was driven by cumulative differences in inpatient, outpatient, and pharmacy costs. What is already known about this subjectInhaled corticosteroid/long-acting beta2-agonist (ICS/LABA) therapies are common first-line treatment options for the treatment of chronic obstructive pulmonary disease (COPD) in patients with a history of exacerbations.Prior research on the health care cost differences between the ICS/LABA therapies budesonide/formoterol (BFC) and fluticasone/salmeterol (FSC) in the United States is extremely limited.What this study addsThis study adds to previous work analyzing differences in healthcare costs for patients initiating the 2 therapies by providing alonger follow-up period and the use of multivariable models toadjust for pre-index differences.This study demonstrated that COPD patients initiating BFC therapy had lower COPD-related, pneumonia-related, and all-cause costs than patients initiating FSC treatment.This study found no significant differences in health care utilization between patients initiating BFC therapy versus those initiating FSC therapy.5 The disease is characterized by inflammation and declining lung function and is linked to increasing rates of morbidity and mortality. The severe effects of COPD are manifest in several aspects of patients’ lives and activities: 700,000 inpatient hospitalizations, 10.3 million outpatient visits, and 1.5 million emergency department (ED) visits in 2010,6 along with considerable absenteeism, in the United States.7 COPD results in substantial economic burden ($36 billion), estimated at $32.1 billion for medical costs and $3.9 billion for worker absenteeism—about 16.4 million lost work days—in the United States in 2010. By 2020, annual COPD-related medical costs are projected to increase to $49 billion.6Chronic obstructive pulmonary disease (COPD) is an increasingly burdensome respiratory illness that affects about 6.5% of the adult population (approximately 15 million people) in the United States.8 The use of controller medications is supported by the GOLD 2015 treatment guidelines, which endorsed inhaled corticosteroid/long-acting beta2-agonist (ICS/LABA) combination therapies as a first-line maintenance therapy for patients with a history of COPD exacerbation.9 At the time of this study, 2 ICS/LABA combination agents, budesonide/formoterol 10 and fluticasone/salmeterol 11 were indicated for maintenance treatment of airflow obstruction in COPD patients in the United States, and are included in this study. A third ICS/LABA, fluticasone/vilanterol, is now available for maintenance treatment of COPD12; however, this therapy was approved in May 2013, after our study period ended in March 2013, and thus was not included.In the face of such serious economic and clinical challenges, the Global Initiative for Chronic Obstructive Lung Disease (GOLD) emphasized the use of therapies that help to prevent COPD exacerbations as a central element of its disease management recommendations.14; however, information on costs associated with these ICS/LABA combination treatments is limited. To our knowledge, only 1 U.S. study evaluated medical service costs for patients receiving BFC and FSC treatments.4 The authors found no difference in mean total costs between the COPD patient groups receiving BFC and FSC agents.Previous studies have shown that patients initiating BFC have fewer exacerbations and pneumonia events compared with FSC patients4 might have contributed to the lack of observed difference in mean costs between BFC and FSC cohorts, this study used a longer follow-up time of 12 months. Furthermore, in recognition of pneumonia as an important safety endpoint and cost driver among patients receiving ICS/LABA combination therapy, this study also evaluated mean pneumonia-related costs for the 2 cohorts.16 The cost outcomes analyzed in this study were a secondary endpoint in a study primarily designed to compare the rates of COPD exacerbations in the year following initiation of the study medications .17To begin addressing this important evidence gap, we evaluated health care costs and utilization among COPD patients naïve to ICS/LABA fixed-dose combination medications who initiated BFC or FSC therapies in a large, real-world, managed care setting. To reduce the likelihood that a shorter follow-up time, as evidenced by a prior study,17 This analysis used the intent-to-treat approach and attributed all post-index treatment outcomes to the index medications of interest. The treatment index date was defined as the first date of treatment initiation corresponding to the first prescription fill of either BFC or FSC within the study intake period. All patients initiating BFC and FSC therapy between March 1, 2009, and March 31, 2012, were identified in HIRE. The HIRE contains enrollees in 14 major regional health plans and at the time of this study contained longitudinal claims data for more than 31 million enrollees from all U.S. census regions. This study was conducted in accordance with the Agency for Healthcare Research and Quality’s guidelines for conducting comparative effectiveness research.18 The management of all data and study materials conformed with all the relevant Health Insurance Portability and Accountability Act of 1996 (HIPAA) rules. A limited dataset, which excluded patient-identifying information, was used for all analyses, as defined by the HIPAA Privacy Rule. A national, independent institutional review board approved this study.In this retrospective, claims-based study, patients identified in the HealthCore Integrated Research Environment (HIRE) were followed for 12 months after initiating treatment with ICS/LABA combination agents BFC (160/4.5 μg) or FSC (250/50 μg). The primary objective of this study was to examine differences in COPD exacerbation rates between the 2 groups, the results of which have been published elsewhere.International Classification of Diseases, Ninth Revision, Clinical Modification [ICD-9-CM] diagnosis code 491.xx, 492.xx, or 496.xx), and/or at least 1 ED visit with a COPD diagnosis (either primary or secondary), and/or at least 2 other medical claims with a COPD diagnosis (either primary or secondary) during the pre-index period were required for inclusion in the study. Patients diagnosed with cancer, those who received ≥ 180 days of oral corticosteroids (OCS) in the 12-month pre-index period, and those who initiated both study medications on the same date were ineligible for the study.Only patients who were naïve to ICS/LABA during the 12 months prior to initiating BFC (160/4.5 μg) or FSC (250/50 μg) were included in the study. The pharmacy claim date of treatment initiation was considered the index date. To be included, patients were required to be aged at least 40 years on the index date and have continuous active health plan enrollment, with both medical and pharmacy benefits, encompassing at least 12 months before (pre-index period) and 12 months after (post-index period) the index date. Furthermore, at least 1 inpatient visit with a primary diagnosis for COPD . The total cost of care, including actual costs paid by the patient and those paid by the health plan, were included in all cost calculations. The sum of all costs was considered the total health care cost, and the sum of all non-pharmacy costs was considered medical costs. COPD-related costs include the costs of events that were linked to a COPD diagnosis code as a primary diagnosis for inpatient stays and any diagnosis for other services. A COPD-related pharmacy cost was defined as the cost of any of the following medications and medication classes, and includes the cost of the index drug: ICS/LABA, ICS, LABA, long-acting muscarinic antagonist (LAMA), short-acting beta2-agonist (SABA), short-acting muscarinic antagonist (SAMA), SABA/SAMA combination, OCS, theophylline, roflumilast, and antibiotics. Pneumonia-related costs did not require a primary pneumonia diagnosis and included visits with any pneumonia diagnosis (480.xx-486.xx), including viral, bacterial, and other/unspecified pneumonia. Pneumonia-related costs did not include any pharmacy costs, because of the inability to determine whether prescribed antibiotics  were specific to pneumonia or for the treatment of another infection. All-cause health care costs include the costs of all health care encounters regardless of reason or diagnosis. For all cost data, the cost of procedures were included in their applicable place of service; for example, if a pulmonary function test was performed during a COPD-related office visit, the cost of that test would be included in the COPD-related office visit costs.19The presence of a visit (any vs. none) and the number of visits by place of service  were captured. As with cost calculations, health care utilization was considered COPD related if the medical claim contained an applicable COPD diagnosis code; a primary diagnosis was required for inpatient stays, and a claim in any position was adequate for other service locations. Pneumonia-related utilizations included visits with a diagnosis of viral, bacterial, or other/unspecified pneumonia in any position. The diagnostic criteria using the claims database was validated against abstracted medical records with 80% positive predictive value.Comorbid conditions were based on the presence of at least 1 medical claim with an ICD-9-CM diagnosis code for the condition of interest. Adherence to the index medication was measured by the proportion of days covered (PDC). The PDC was calculated as the ratio of the sum of days supply of all prescription fills during the 12 month follow-up period divided by 365 days.21 Although adequate clinical information was not available, propensity score matching, using proxies for clinical patient attributes, enabled the formation of comparable cohorts. Random forest methods22 were used to estimate the propensity scores as the probability of receiving BFC therapy, and patients were matched with the greedy nearest neighbor (1:1) matching technique without replacement.21 The outcome variable in the model was dichotomous, indicating whether a patient received BFC (1) or FSC (0) therapy. Treatment cohorts were considered well balanced when the P value for the difference between groups was > 0.05 for each of the prespecified variables listed below. The matching scheme specified a priori the balancing of age, sex, prior asthma diagnosis, COPD-related inpatient hospitalizations and ED visits, OCS fills, antibiotic fills, SABA or SABA/SAMA fills, LABA fills, and LAMA fills.Patients in the BFC and FSC cohorts were propensity score matched to reduce selection bias and create more comparable groups.P values are reported. All costs were adjusted for calendar year and are reported in 2012 dollars. To evaluate utilization outcomes, a GLM with a negative binomial distribution and log link was used for the number of visits and other count data, and logistic models were used for dichotomous outcomes. Adjusted mean differences (AMDs) in costs were calculated as the difference in predicted mean cost values between groups after adjusting for covariates in the statistical model.A generalized linear model (GLM) using the gamma distribution and a log-link function was used to model health care costs. Model adjusted mean differences, 95% confidence intervals, and P < 0.05 was considered statistically significant for all outcomes, effect estimates and confidence intervals were primarily used in the interpretation of results. No adjustment was made for multiple testing. The propensity scores used for matching were obtained using R,23 and all other analyses were performed using SAS version 9.2 .Covariates identified in the 12-month pre-index period that remained imbalanced after matching  were incA total of 3,788 BFC and 6,439 FSC initiators met all inclusion/exclusion criteria prior to matching. Following propensity score matching, a total of 3,697 patients from each cohort were included. On average, patients were aged 64 years in each treatment group, and the groups had a similar proportion of females (52% BFC and 54% FSC). In addition, there were similar rates of previously diagnosed pneumonia , asthma , and hypertension , among other conditions, during the 12-month pre-index period. Proxies for COPD severity were also well balanced, including the mean number of prior COPD-related inpatient visits , COPD-related ED visits , and antibiotic fills (2.7 in each group). The use of prior respiratory medications at baseline was also similar for the 2 cohorts, as reflected by proportions of patients with at least 1 fill of OCS , ICS , SABA , and SABA/SAMA combination (23% for both). During the 12-month follow-up period, adherence to the index medication was similar in each group, as evidenced by a PDC of 33% in the BFC cohort and 34% for FSC patients.Overall, the rates of COPD-related and all-cause utilization were similar for patients in the 2 cohorts during the 12-month follow-up period, as shown in Pneumonia-related encounters were also similar for patients in the 2 cohorts . InpatieSimilar proportions of patients in the BFC and FSC cohorts, respectively, had at least 1 all-cause inpatient hospitalization (30.6% and 31.9%), ICU stay (6% and 6.5%), ED visit (27.4% and 27.1%), and outpatient/office visit (99.5% and 99.2%). When examining those that had at least 1 event, the average number of visits was similar between the 2 cohorts, including the number of inpatient hospitalizations (1.9 vs. 2.0), ICU stays (1.2 vs. 1.3), ED visits (1.6 vs. 1.7), and outpatient/office visits (33.1 vs. 34.9). Although not statistically significant, those initiating BFC had an average length of inpatient hospitalization stays nearly 2 days fewer than those who initiated FSC (9.3 days vs. 11.2 days).P = 0.003, respectively). The lower mean costs in the BFC relative to the FSC cohort were driven by lower COPD-related inpatient costs , outpatient/office visit costs , and pharmacy costs , despite higher COPD-related ED visit costs , as shown in Although similar rates of health care utilization were observed between the 2 cohorts, BFC patients incurred $316 less in COPD-related costs than FSC patients during the first year after treatment initiation , which was mainly driven by lower pneumonia-related inpatient costs .Pneumonia-related costs followed a pattern similar to that of COPD-related costs. Patients in the BFC group incurred significantly lower pneumonia-related medical costs compared with patients in the FSC cohort . The difference was driven mainly by lower all-cause inpatient costs , and lower outpatient/office visit costs , among BFC versus FSC patients, respectively.In all, those initiating BFC incurred lower all-cause health care costs versus FSC patients during the first year after treatment initiation  and had longer inpatients lengths of stay on average (mean days: 11.2 vs. 9.3), both of which are associated with high costs; thus, small nonstatistical differences in their utilization may be responsible for statistically significant differences in overall health care costs.4 The authors reported that pharmacy costs were $43 greater for FSC relative to BFC patients during the limited follow-up period (compared with $121 over a 12-month follow-up in this study), which were attributed to the greater mean medication possession ratio among patients receiving FSC treatment.4 However, in the current study, adherence to the index medication, as measured by the PDC, was similar between the 2 groups. The Roberts et al. study was similar to our study in a number of key respects: patients were health plan enrollees, and sample size and age and sex distributions were generally similar. However, the difference in follow-up time, 6 months versus 1 year, was a salient difference.4 Although evidence from prior studies is not currently available, it is not implausible that as COPD patients continue to receive controller medications, the rate at which they require other services, especially costly inpatient hospitalization, could change and impact their overall costs. Although no differences in the number of hospitalizations and other services were detected, small, nonstatistically significant differences, including the length of inpatient hospitalization and prevalence of ICU stays, were seen between the 2 groups. Further evidence, including additional real-world effectiveness studies or randomized trials, is needed to clarify potential additional differences between these 2 agents.In contrast to these findings, a study by Roberts et al. (2011) reported no significant differences in COPD-related costs for medical services during the 3- to 6-month follow-up period.26A possible difference in costs may exist if different health plans offer different levels of coverage for the 2 medications being compared. However, in this study it is believed that both drugs, which are of the same class, were covered similarly, and the most recent health plan formularies show no difference in coverage between the 2 medications being studied.22 A subset of pneumonia diagnoses in this study was validated via medical chart abstraction and review, which found a positive predictive value of 80%.19A key strength of this study was the use of a robust design that incorporated propensity score matching to create cohorts with solid bases for comparison over the 12-month follow-up period. Furthermore, the random forest method utilized in the propensity score algorithm has been shown to be more effective in balancing treatment cohorts with less bias than traditional logistic regression techniques.The other important observation in the study of costs is that COPD-related costs are just a portion of total health care costs, although in this study they accounted for a significant fraction, with more than 20% of all-cause costs related to COPD. Additionally, because the presence of COPD is associated with a number of comorbid conditions, better COPD control may plausibly decrease total health care spending directly and indirectly. Further study is needed to determine whether a strategy of improved ICS/LBA compliance might lead to lower total health care costs while marginally increasing pharmacy costs.28 They may be generalizable only to commercially insured populations in the United States. Patients were not required to take the index medication during follow-up. As a result, they filled index medication an average of roughly 4 times during follow-up, which resulted in an average of just 33%-34% of post-index days covered with supply of the index medication. Nonetheless, the results of this intent-to-treat analysis were evaluated as the outcomes from the initiation of the study medications without prior ICS/LABA use.The results of this study must be viewed against important limitations, including those associated with a reliance on secondary data.8There was a lack of detailed clinical information, including smoking status, pulmonary function assessment, and patient reported outcomes, to adjust for COPD disease severity and activity or to confirm COPD diagnoses, because such information is absent from the primary data source, administrative claims. However, the study matched on prior COPD exacerbations, which is the best predictor of future exacerbations.29 Thus, the results of this study might not be generalized to BFC or FSC users with COPD overall.Pharmacy medications administered during inpatient hospitalizations were not included because that information was not available in these data. It was not possible to determine the primary reason for outpatient and ED visits via claims data; as a result, even when a COPD diagnosis code was present, the visit might have been for routine follow-up or non-COPD-related reasons.No difference was found in rates of health care utilization between the 2 groups; however, this study demonstrated an association between COPD patients initiating BFC therapy and lower COPD-related costs relative to patients initiating FSC treatment. This result represented the cumulative effect of lower mean costs for inpatient hospitalizations, pharmacy, and outpatient/office visits counterbalancing slightly higher average ED visit costs. BFC patients also had lower all-cause costs, which were driven by lower hospitalization and outpatient/office visit costs incurred by BFC versus FSC patients, and a similar trend was observed for pneumonia-related costs. This is the first study comparing resource utilization and costs of BFC and FSC administered to ICS/LABA-naïve patients for a full year following treatment initiation. Additional studies are needed to evaluate other key aspects of the economic outcomes of BFC and FSC use among COPD patients.


Erythropoiesis-stimulating agents (ESAs) are commonly used to treat anemia due to chronic kidney disease (CKD). In addition to drug acquisition costs, the administration of ESAs can include direct and indirect costs due to the needle-based route of administration . However, a comprehensive assessment of the costs associated with the administration of ESAs is lacking.To estimate the excess costs associated with the needle-based administration of ESAs for the treatment of anemia due to non-dialysis-dependent (NDD) CKD in the United States in 2019 from a societal perspective.Excess costs associated with ESA administration were estimated as the sum of annual costs that could be avoided with the introduction of an oral treatment with comparable safety and efficacy to ESAs. Cost components included direct health care costs, transportation costs, and work productivity loss costs from the perspective of both patients and caregivers (as applicable). Costs were estimated based on scientific publications, governmental agencies, and the results of a recent survey of US patients and caregivers of patients with anemia and CKD. The setting of the administration , frequency of administration, and insurance type were considered.At the societal level, annual excess costs associated with ESA administration were estimated at $2.5 billion in the United States in 2019, based on an estimated 462,005 patients with anemia and NDD-CKD treated with ESAs. Overall, 94.4% ($2.4 billion) of these costs were incurred from in-clinic ESA administration. When stratifying costs by insurance type, Medicare-insured patients accounted for 79.4% ($2.0 billion) of total annual excess costs. The largest contributor to total annual excess costs was direct health care costs , followed by patient work productivity loss costs , caregiver work productivity loss costs , and transportation costs . Total annual excess costs of in-clinic administration ranged from $2,572 per patient receiving monthly administration to $20,948 per patient receiving thrice-weekly administration, while the total annual excess costs of at-home administration ranged from $1,123 per patient receiving monthly administration to $2,109 per patient receiving thrice-weekly administration. At the ESA administration level , total excess costs were estimated at $128 per in-clinic ESA administration and $7 per at-home ESA administration, excluding monitoring costs.The needle-based administration of ESAs in patients with NDD-CKD is associated with a substantial economic burden. The introduction of an oral treatment has the potential to result in important cost savings from a societal perspective. What is already known about this subjectNeedle-based erythropoiesis-stimulating agents (ESAs) are commonly used to treat anemia resulting from chronic kidney disease (CKD).Despite the clinical utility of ESAs, their use is associated with a considerable economic and societal burden resulting from the logistics of therapy administration for both patients and caregivers, especially for non-dialysis-dependent (NDD) patients who do not have prescheduled regular visits for dialysis during which ESAs can be administered.The magnitude of the administration costs that could potentially be avoided with the introduction of a new oral treatment for anemia due to CKD with comparable safety and efficacy to ESAs has not been assessed.What this study addsThis study estimated the direct and indirect excess costs associated with the needle-based administration of ESAs for the treatment of anemia due to NDD-CKD at $2.5 billion in the United States in 2019, or $5,399 per treated patient.More than 90% of this excess cost was due to in-clinic ESA administration, suggesting that resources associated with ESA administration visits account for the vast majority of costs that could potentially be avoided with the introduction of novel oral medications.Direct health care costs and patients’ work productivity loss costs, particularly those resulting from in-clinic ESA administration, were the 2 main drivers of total excess costs, respectively, accounting for 54.9% and 33.9% of total excess costs.3 The prevalence of CKD increases with age,4 such that the prevalence among the Medicare-insured population is estimated to be approximately 22% compared with approximate prevalence of 4% among Medicaid-insured, 1% among commercially insured, and < 1% among uninsured populations.5 Further, both the prevalence and economic burden of the disease are on the rise,6 with total Medicare spending for CKD  exceeding $120 billion in 2017, up by 5.3% relative to 2016.7Chronic kidney disease (CKD) is characterized by persistent abnormalities in kidney structure, function, or both, with implications for the health of the affected individual.9 Anemia is a common complication of CKD whose prevalence markedly increases with disease progression; a study by Stauffer et al reported that anemia prevalence increases from 8.4% among patients with stage 1 CKD to 53.4% among those with stage 5 CKD.10 Anemia due to CKD is characterized by several symptoms  that adversely affect patients’ health-related quality of life (HRQOL).11Further, the economic burden of CKD, including health care resource use and costs, significantly increases with CKD progression, largely due to the emergence of complications.13 Treatments currently recommended for anemia due to CKD include iron administration, erythropoiesis-stimulating agents (ESAs), and red blood cell transfusion.14 ESAs, which can be administered intravenously or subcutaneously, are commonly used for the treatment of anemia due to CKD but may introduce a substantial burden.14 In addition to drug acquisition costs, the time spent by health care professionals on ESA administration significantly contributes to the total treatment costs.16 These direct health care costs are further compounded by indirect work productivity loss costs due to patients’ time spent receiving therapy. Furthermore, caregivers’ time spent accompanying patients to therapy contributes to the societal burden of ESA treatment.17 The costs associated with ESA administration may be particularly significant in the non-dialysis-dependent (NDD) population since these patients do not have prescheduled regular visits for dialysis during which ESAs can be administered.18The symptoms associated with anemia and their resulting adverse impact on HRQOL can be alleviated with appropriate treatments of anemia.20 Hypoxia-inducible factor prolyl hydroxylase inhibitors (HIF-PHIs) are a novel class of oral treatments that have demonstrated promising results in treating anemia due to CKD in recent trials.22 However, as prior studies have not provided a comprehensive assessment of costs incurred by patients, caregivers, and payers for the needle-based administration of ESAs,24 the potential savings that might result from the introduction of oral HIF-PHIs remain largely unknown. Consistent with the approach advocated by several health technology assessment bodies, assessing costs from a societal perspective is important to reflect the perspective of decision makers, who must take into account the broader impact of health care policies on society in order to appropriately allocate resources.The direct and indirect costs associated with the needle-based administration of ESAs may be substantially reduced with the introduction of new oral treatments for anemia due to CKD.The goal of this study was to estimate the excess costs associated with the needle-based administration of ESAs for the treatment of anemia due to NDD-CKD in the United States in 2019 from a societal perspective. This study focused on patients with NDD-CKD, since it is challenging to accurately distinguish costs specific to the administration of ESAs from those associated with dialysis in the dialysis-dependent (DD) population .25Total excess costs associated with the administration of ESAs in patients with anemia due to NDD-CKD were estimated as the sum of costs expected to be avoided with an oral treatment instead of a needle-based treatment, assuming a comparable safety and efficacy profile. Inputs used to estimate the total excess costs in the United States  were obtained from the literature, including scientific publications and governmental agencies, as well as the results of a survey conducted in 2020 among US patients and caregivers of patients with anemia due to CKD (hereafter referred to as the “CKD survey”).Total excess costs comprised direct health care costs, transportation costs, and work productivity loss costs. Direct health care costs were reported from a payer’s perspective and included costs associated with ESA administration. Transportation costs and work productivity loss costs included costs incurred by patients and caregivers separately. All costs were expressed in 2019 US dollars, consistent with the most recent complete year  with the available Consumer Price Index (CPI) at the time the analyses were conducted. Direct health care costs were inflated to 2019 US dollars using the CPI for All Urban Consumers (CPI-U): Medical Care. Transportation costs and work productivity loss costs were inflated to 2019 US dollars using the CPI-U: All Items and CPI-U: Hourly Compensation, respectively.25Total excess costs were reported at the societal level annually, at the patient level annually , and at the ESA administration level . At the societal level, a prevalence-based approach was used to estimate annual costs of ESA administration, where costs per patient were multiplied by the number of prevalent ESA users in the US population. Annual per-patient costs of ESA administration were calculated by multiplying the total cost per administration by the annual number of administrations separately for each available frequency of administration .Total excess costs were reported overall and then stratified by insurance type  and administration setting .27 The distribution of the US population by payer type was estimated by classifying individuals into mutually exclusive payer types using the following hierarchy: commercial , Medicare , Medicaid, VA, and uninsured.28Data on the US adult population in 2019 were obtained from the US Census Bureau.29 Due to the limited availability of prevalence estimates for certain payer types, the prevalence of diagnosed NDD-CKD among VA-insured patients was assumed to be equal to that of commercially insured patients.4 Similarly, the prevalence of ESA use among Medicaid-insured, VA-insured, and uninsured patients was assumed to be equal to that of commercially insured patients.29The number of adults with NDD-CKD receiving ESAs for the treatment of anemia in 2019 was estimated using publicly available sources that derived payer-specific estimates of the prevalence of diagnosed NDD-CKD and ESA use among patients with NDD-CKD in the United States from claims data.25 To account for the fact that some population parameters were estimated prior to 2019, these parameters were adjusted for population growth based on data from the US Census Bureau.27The overall number of patients with NDD-CKD receiving ESAs in the United States was calculated as the sum of patients across payer types. Due to the lack of publicly available US-specific estimates, the number of patients receiving in-clinic and at-home ESA administration, as well as the frequency of in-clinic and at-home ESA administration, were estimated based on results from the CKD survey.Direct health care costs included costs of ESA administration and monitoring costs. These costs were reported from a payer’s perspective and were assumed to be the same across all payer types due to limited data on payer-specific estimates available in the literature.19 Only patients receiving in-clinic ESAs were assumed to incur these costs. It was assumed that at-home ESA administration was conducted by patients or caregivers directly, and therefore, was not associated with direct health care costs.Costs of ESA administration included fees such as examination fees and facility fees per ESA administration but excluded drug acquisition costs .19 Monitoring costs were assumed to be the same across administration settings . Annual monitoring costs per patient were calculated by multiplying the cost per monitoring event by the expected annual number of monitoring events related to ESA administration. The frequency of monitoring events was estimated based on the US Food and Drug Administration recommendation to monitor patients on a monthly basis during the maintenance phase .31Monitoring costs included fees associated with the assessment of blood hemoglobin levels through laboratory tests and nurse consultations.33 which was then multiplied by the average cost per mile.34 Only patients receiving in-clinic ESAs were assumed to incur these costs.Transportation costs included costs incurred for transportation to and from health care facilities to receive in-clinic ESA treatment. Per-patient costs included both patient and caregiver costs and accounted for the proportion of patients who were accompanied by a caregiver during ESA administration visits. It was assumed that no additional transportation costs were incurred for monitoring . The average transportation cost per administration was calculated by multiplying the average total travel time per visit, as reported by persons seeking ambulatory care in the United States (patients and caregivers separately), by the average driving speed to obtain the average distance traveled,Work productivity loss costs were estimated based on a human capital approach, which assumed productivity was valued at an individual’s market earnings. Per-patient costs included both patient and caregiver costs, accounting for the proportion of patients who were accompanied to visits by a caregiver.32 For in-clinic ESA recipients, the time required for ESA administration included all time spent from the moment the patient left his or her residence to the moment he or she returned .32 For at-home ESA recipients, the time required for ESA administration included all time spent preparing and administering ESA treatment at home.25Patients’ work productivity loss costs were estimated using the average time required for ESA administration and patients’ mean hourly wage.32Caregivers’ work productivity loss costs were estimated similarly, using the average time required for ESA administration from the caregivers’ perspective, caregivers’ mean hourly wage, and the proportion of patients with a caregiver assisting during administration.Two sensitivity analyses were conducted assuming (1) monitoring requirements were equivalent for needle-based and oral formulations  and (2) the prevalence of ESA use among uninsured individuals was 0% .Annual excess costs associated with ESA administration in the United States in 2019 were estimated at $2.5 billion, or $5,399 per patient. In-clinic ESA administration accounted for 94.4% ($2.4 billion) of total annual excess costs, and at-home ESA administration accounted for the remaining 5.6% .Total annual excess costs of in-clinic ESA administration ranged from $2,572 per patient receiving monthly administration to $20,948 per patient receiving thrice-weekly administration, while total annual excess costs of at-home ESA administration ranged from $1,123 for monthly administration to $2,109 for thrice-weekly administration . At the 35 Medicare-insured (22.0%),35 Medicaid-insured (3.5%),35 and uninsured patients (0.1%).5The number of adults with diagnosed NDD-CKD in the United States in 2019 was estimated to be 8,916,742 based on an overall prevalence of 3.6% . This pr29Among patients with NDD-CKD, it was estimated that 462,005 received ESAs for the treatment of anemia, based on an overall prevalence of ESA use of 5.2% in the NDD-CKD population . This prBased on the results of the CKD survey, the numbers of in-clinic and at-home ESA recipients were estimated to be 367,893 and 94,112, respectively . Among iAnnual excess direct health care costs associated with ESA administration  were estimated at $1.4 billion, or $2,965 per patient. Annual direct health care costs associated with in-clinic ESA administration ranged from $1,731 per patient receiving monthly administration to $10,018 per patient receiving thrice-weekly administration. Annual per-patient direct health care costs associated with at-home ESA administration were $1,041, regardless of dosing frequency.Supplementary Table 1).19 Annual excess costs of in-clinic ESA administration ranged from $691 per patient receiving monthly administration to $8,978 per patient receiving thrice-weekly administration. Direct costs of at-home ESA administration  were assumed to be $0 (Supplementary Table 1).Direct costs per in-clinic ESA administration  were estimated at $58 (Supplementary Table 1), corresponding to an annual cost of $1,041, assuming monthly monitoring visits. Costs per visit included those associated with hemoglobin tests ($3), the cost of a blood draw ($3), and the cost of a nurse consultation ($80).19Direct health care costs associated with each monitoring visit were estimated at $87  ranged from $63 for monthly administration to $822 for thrice-weekly administration. At-home administration was assumed to incur $0 in transportation costs.Supplementary Table 1). From the patient perspective, each in-clinic administration visit incurred $4 of excess transportation costs. This cost was estimated based on an average self-reported total travel time of 33 minutes for an in-clinic appointment,32 an average driving speed of 37.1 mph,33 and an average cost per mile of 20 cents.34 From the caregiver perspective, each in-clinic administration visit incurred an average excess transportation cost of $1. This cost was estimated based on an average self-reported total travel time of 48 minutes for an in-clinic appointment,32 an average driving speed of 37.1 mph,33 an average cost per mile of 20 cents,34 and an assumption that 20% of patients were accompanied to visits by a caregiver.32At the ESA administration level, total transportation costs (including both patient and caregiver costs) associated with in-clinic ESA administration were estimated at $5  ranged from $777 per patient receiving monthly administration to $10,107 per patient receiving thrice-weekly administration; for at-home administration, these costs ranged from $82 per patient receiving monthly administration to $1,069 per patient receiving thrice-weekly administration.Supplementary Table 1). This cost was calculated using an average hourly wage of $28 (based on the characteristics of an average patient) and an average time of 115 minutes to seek care.32 At-home ESA administration was estimated to incur excess work productivity loss costs of $5 per administration (Supplementary Table 1) based on an average hourly wage of $28 and an average duration of ESA administration of 10 minutes.32For patients, excess work productivity loss costs were estimated at $53 per in-clinic ESA administration (Supplementary Table 1). This cost was calculated using an average hourly wage of $26 (based on the characteristics of an average caregiver), an average of 139 minutes spent accompanying patients to receive care, with the assumption that 20% of patients are accompanied by a caregiver.32 Each at-home ESA administration was estimated to incur excess work productivity loss costs of $2 per administration. This cost was calculated using an average hourly wage of $26 and an average duration of ESA administration of 10 minutes,32 with the assumption that 53% of patients are assisted by a caregiver for at-home administration.25For caregivers, excess work productivity loss costs were estimated at $12 per in-clinic ESA administration .Assuming monitoring requirements were equivalent for needle-based and oral formulations , total annual excess costs associated with ESA administration in the United States in 2019 were estimated at $2.0 billion, or $4,358 per patient, which represents a $0.5 billion (19%) reduction compared with the base-case scenario , the total estimated number of adults receiving ESAs in the United States was 461,254, which represents a reduction of 751 (0.2%) patients receiving ESAs. Total annual excess costs were comparable to the base-case scenario ($2.5 billion).The findings of this study suggest that the needle-based administration of ESAs is associated with a considerable economic burden, which was estimated at $2.5 billion in the United States in 2019. More than 90% of this excess cost was due to in-clinic ESA administration, suggesting that resources associated with ESA administration visits account for the vast majority of costs that could be avoided with the introduction of novel oral medications. In addition, direct health care costs and patients’ work productivity loss costs, particularly those resulting from in-clinic ESA administration, were the 2 main drivers of total excess costs, respectively accounting for 54.9% and 33.9% of total excess costs. Further, in both administration settings, Medicare-insured patients incurred the majority of costs due to the high prevalence of NDD-CKD and ESA use in this population, which resulted in nearly 80% of total excess costs incurred by Medicare-insured patients.39To the best of our knowledge, this study is the first to provide a societal perspective on ESA administration costs that could potentially be avoided with the introduction of a new oral treatment for anemia due to CKD with comparable safety and efficacy to ESAs. Most prior studies that assessed the economic burden associated with ESAs and their administration did not focus on patients with NDD-CKD, who may incur the greatest excess costs associated with ESA administration.42 Further, most of these studies exclusively assessed the burden associated with ESAs from the patient perspective or only considered direct health care costs,24 which does not allow for a comprehensive assessment of excess costs associated with the needle-based administration of ESAs.In addition, studies that included patients with NDD-CKD were conducted more than a decade ago and thus may not provide reliable contemporary estimates due to changes in ESA reimbursement policies and prescribing labels over the past decade.43 an estimate that included costs incurred by patients, payers, and third parties. The present estimate of $2,965 per year in direct health care costs incurred exclusively by payers due to the needle-based route of administration of ESAs and associated monitoring requirements may account for an important proportion of total health care costs among ESA-treated patients with NDD-CKD.The results of the current study suggest that the needle-based administration of ESAs may account for a notable proportion of the economic burden of ESA-treated patients. A 2007 study by Maddux et al reported that ESA recipients with NDD-CKD incurred total direct health care costs of $3,876 per month ,31 Furthermore, while the current study assessed the economic impact of time spent receiving therapy, the intrinsic value of an individual’s time spent receiving therapy also warrants consideration. Clinic visits, transportation time, and work loss may result in diminished quality of life and add to the already high burden of anemia due to CKD. Therefore, an oral treatment merits consideration as a convenient alternative to ESAs for the treatment of anemia due to CKD.The needle-based route of ESA administration may also have an impact on patient and caregiver HRQOL, which was not measured as part of this study. For example, patients’ fear of needles and potential injection-site reactions  may impact HRQOL and further compound the overall burden of ESA administration.25 Therefore, the use of an oral treatment with a safety and efficacy profile comparable to that of ESAs has the potential to improve patients’ satisfaction with treatment in addition to avoiding costs associated with ESA administration.Despite the cost and burden associated with ESA administration, current treatment options for anemia in patients with CKD requiring ESAs remain limited to needle-based medications. In a recent survey, nearly 50% of patients and caregivers of patients with anemia and NDD-CKD expressed a preference for an oral anemia treatment.44 Although comparative data are currently limited, it is thought that this novel mechanism will enable a safer and more effective correction of anemia compared with ESAs.45 Once additional clinical data are available, further studies will be warranted to account for potential differences in the safety and efficacy of HIF-PHIs compared with ESAs and the potential cost implications of these differences.HIF-PHIs have been shown to improve anemia by stimulating endogenous erythropoietin production and restoring iron metabolism in ongoing clinical trials.The present study is subject to some limitations. For certain payer types, the prevalence of NDD-CKD and ESA use was assumed to be equal to that of commercially insured patients due to a lack of published payer-specific estimates. This assumption may have underestimated the societal cost associated with ESA administration, as estimates for the commercially insured population were the lowest estimates among available payer types.32Data on the setting  and frequency of ESA administration were derived from a recent survey in which the sample may not be representative of the general US population. Moreover, there was a scarcity of information on certain cost components  specific to the ESA-treated NDD-CKD population. Hence, values derived from the general population receiving ambulatory care were used for these components. However, values in the general population were very similar to CKD-specific estimates found in the CKD survey. For example, 20% of adults in the US general population were reportedly accompanied to ambulatory visits by a caregiver compared with 23.7% of patients accompanied to ESA administration visits as reported in the CKD survey.48Because of the scarcity of publicly available estimates of comparable direct health care costs for all payer types, direct health care costs of Medicare-insured patients were applied to individuals with other types of insurance coverage; this extrapolation may have underestimated health care costs, especially among commercially insured individuals who are known to incur higher costs than Medicare beneficiaries.Although this analysis adopted a societal perspective, which encompassed costs beyond direct health care costs, this study was limited to specific costs for which estimates were available in the literature. Accordingly, total excess costs may be underestimated due to additional factors related to the needle-based administration of ESAs  or potential consequences of increased caregiver burden , which were not captured in this study due to a lack of cost estimates in the literature.Finally, this study focused on the NDD-CKD population, and costs may not be generalizable to the DD-CKD population.The findings of this study suggest that needle-based administration of ESAs in patients with NDD-CKD may be associated with a substantial economic burden. If all ESA-treated patients with NDD-CKD and anemia were to receive an oral treatment with comparable safety and efficacy as when receiving ESAs, potential savings associated with oral administration would amount to approximately $2.5 billion from a societal perspective. As new data become available, further studies will be warranted to assess excess costs associated with ESA administration, taking into account potential differences between new oral treatments and ESAs in terms of safety and efficacy.


Accipiter gentilis 2 years earlier in the same area, belonging to the WNV-L1 Western-Mediterranean sub-cluster. These results underline that WNV-L1, after reintroduction in 2020, has probably silently circulated during a 2-year eclipse, with no positive sample revealed by both serological and biomolecular examinations in horses, birds, and mosquitoes. The climate changes that have occurred in the last decades are evolving the epidemiology of WNV, with introductions or re-introductions of the virus in areas that were previously considered low risk. Thereby, the virus may easily amplify and establish itself to reappear with sporadic evident cases in susceptible hosts after several months or even years.West Nile virus (WNV) is the most widespread arbovirus worldwide, responsible for severe neurological symptoms in humans as well as in horses and birds. The main reservoir and amplifier of the virus are birds, and migratory birds seem to have a key role in the introduction and spread of WNV during their migratory routes. WNV lineage 1 (L1) has been missing in Italy for almost 10 years, only to reappear in 2020 in two dead raptor birds in southern Italy. The present study reports the first equine outbreak in the Campania region. A 7-year-old horse died because of worsening neurological signs and underwent necropsy and biomolecular analyses. WNV-L1 was detected by real-time RT-PCR in the heart, brain, gut, liver, and spleen. Next Generation Sequence and phylogenetic analysis revealed that the strain responsible for the outbreak showed a nucleotide identity of over 98% with the strain found in  This virus is included in the Japanese encephalitis virus serocomplex, along with the Usutu virus, the Murray Valley encephalitis virus, the Stratford virus, the Alfui virus, the Kunjin virus, and the Saint Louis encephalitis virus (West Nile virus (WNV) is a zoonotic enveloped single-stranded RNA agent that belongs to the is virus . Althougis virus , it was is virus . Indeed,is virus , 5. In iis virus , 5.WNV can infect a plethora of animals, such as birds, horses, sheep, cats, rodents, reptiles, Indian elephants, Indian rhinoceros, ring-tailed lemurs, red pandas, snow leopards, and babirusas , 3, 6–9.Horses, instead, as well as humans, are dead-end hosts. In this species, the infection is usually asymptomatic, while in 20% of cases, clinical and neurological signs may develop, which can range from fever and mild ataxia to total recumbence, leading to a mortality rate of 30–40% in unvaccinated animals .Currently, strains of WNV are phylogenetically divided into eight distinct lineages, of which 1 (L1) and 2 (L2) are responsible for severe disease in humans and horses . NeverthRecently, WNV has emerged in numerous geographic areas, even becoming endemic in several countries. Major epidemics have been reported globally , 17. In Falco tinnunculus) and a goshawk (Accipiter gentilis), were found WNV-L1 positive in the Campania region  aims to monitor WNV circulation on resident birds of targeted species and on dead wild birds, as well as on mosquitoes, equids, and humans. According to this plan, Italian regions are risk-based classified, identifying three different classes: high-risk areas where the virus is currently circulating or has circulated during the previous 5 years, thus outbreaks are frequently observed; low-risk areas where the virus has sporadically circulated in the past or the virus has never been detected, but eco-climatic conditions may be favourable to virus circulation; and minimum-risk areas where the virus has never been detected and eco-climatic conditions are considered non-favourable to virus circulation.Before the detection of WNV-L1 in the kestrel and goshawk, the Campania region was considered a low-risk area since the circulation of the virus had never been revealed by either passive or active surveillance. Nevertheless, after these cases, no further positive samples were observed for 2 years. Hereby, we describe the first clinical case of a horse infected by WNV-L1 in the Campania region and its phylogenetic relationship with the WNV-L1 strain previously found in a wild bird in the same area.22.1At the end of September 2022, an unexpected death of a 7-year-old male horse with neurological signs occurred. The owner referred the animal in the morning began to show worsening neurological symptoms, characterised by unsheathing of the penis and an inability to urinate. Within 2 h, these symptoms were followed by flaccid paresis of the rear limbs, an inability to stand, and proprioceptive deficits that progressed to recumbency. The horse died in the night. The official veterinary health authorities performed the necropsy in the field. At the opening of the body cavities, no gross lesions were observed except for an intestinal volvulus. After the outbreak was confirmed, an epidemiological investigation was conducted in order to analyse the possible source of infection.2.23 of samples from the inner parts of each organ were taken. Official veterinary authorities also collected serum and whole blood samples from 12 horses and 3 chickens from the same stable. Horse blood was collected from the jugular vein, while chicken blood was collected from the alar vein using sterile evacuated tubes with Serum Clot Activator  and EDTA, following the dictates of the relevant guidelines and regulations on animal welfare.The horse carcass underwent a post-mortem examination in the field. Heart, head, gut, spleen, and liver were entirely collected by the official veterinary services and brought to the Istituto Zooprofilattico Sperimentale del Mezzogiorno (IZSM) at refrigerated temperature. Next, in the necropsy room, the head was dissected by craniotomy, and brain samples were collected by veterinarians from the Unit of Forensic Veterinary Medicine. Furthermore, after preliminary cauterisation of their surfaces, approximately 2 cm2.3g for 5 min. Aliquots of 200 μL of supernatant were collected, and VetMAX Xeno Internal Positive Control RNA  was added. Nucleic acid extraction and purification were carried out using QIAsymphony DSP Virus/Pathogen Mini Kit (Qiagen) on the QIAsymphony automated system (Qiagen) following the manufacturer’s instructions, eluted in 60 μL, and stored at −80°C until use. The eluates underwent a one-step RT-PCR assay for the simultaneous detection of West Nile virus lineages 1 and 2 according to the protocol already described by Del Almo et al. (In the Biosafety Level 3 (BLS-3) laboratory, 2 mg of each tissue sample was suspended in 1.8 mL of sterile phosphate buffer saline (PBS) in 2 ml tubes. Samples were homogenised with a 4.8-mm-diameter stainless steel bead to allow mechanical lysis using a TissueLyser  for 5 min at 30 Hz and subsequently centrifuged at 1,650 × o et al.  and the o et al. . In brieNext, the RNA detected in the brain sample was fully sequenced by next-generation sequencing (NGS) technology. Briefly, RNA concentration was quantified using the Qubit RNA HS Assay Kit  and diluted to reach 100 ng final concentration. Diluted RNA was then used for library preparation, which was carried out with Illumina RNA Prep with Enrichment (L) Tagmentation , according to the manufacturer’s instructions. Finally, the enriched library quality check was performed using Tape Station 4,150  and quantified using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific). A final concentration of 10 pM was obtained and loaded onto the MiSeq platform using the v2 Reagent Cartridge, 300 cycles, and 150 bp paired-end reads. FASTQ files were generated with BaseSpace Sequence Hub (Illumina) and assembled using Geneious R9.13. The final assembly contained a total of 10,977 bp. The WNV strain sequence is available on GenBank2.4Accipiter gentilis (n = 2), Blyth’s reed warbler (n = 1), Culex pipiens (n = 2), Homo sapiens (n = 4), horse brain (n = 3), house sparrow (n = 1), Jay (n = 1), and magpie (n = 2), as well as sequences isolated from different countries, such as France (n = 1), Israel (n = 1), Italy (n = 10), Morocco (n = 1), Senegal (n = 1), Spain (n = 1), and Turkey (n = 1). All sequences belonged to lineage 1. The tree was rooted by using the strain ON032498.1, belonging to lineage 2 as an outgroup.A phylogenetic analysis was performed on the WNV L1 strain detected in the horse and on the other 18 complete coding DNA sequences downloaded from Genbank . SequencNucleotide sequences were aligned using the ClustalW algorithm implemented in the Molecular Evolutionary Genetics Analysis (MEGA11) software and edited manually. The maximum likelihood approach was used to infer phylogeny, and Bayesian Inference (BI) was performed using a general time reversible model with Gamma Distributed with Invariant Sites (G + I) and a gamma category count of 4. The robustness of branching patterns was tested by 500 bootstrap replications using the Hasegawa-Kishino-Yano model.2.5g for 10 min . In line with the PNA 2020–2025, horse and chicken serum samples were tested for the presence of WNV IgM and IgG antibodies using ID Screen West Nile IgM Capture  and ID Screen West Nile Competition Multi-species (IDvet), respectively. ELISA-positive sera were then conferred to the National Reference Laboratory for exotic diseases of animals (CESME),  to confirm the ELISA positivity using a virus neutralisation (VN) assay.Horse and chicken samples were transported to the IZSM laboratories under refrigerated conditions. Whole blood samples underwent real-time RT-PCR as reported above, while sera were obtained by centrifugation at 1,300 × For this study, ethical review and approval were waived because sample collection was conducted during official routine activities in accordance with national and regional regulations and internal policy.2.6A map was produced to highlight the area where the tested-positive WNV-L1 goshawk and the dead horse of the present study were found in 2020 and 2022, respectively. Data were obtained by the Epidemiologic and Biostatistics Regional Observatory (ORSA) using qgis version 3.22 open source software33.1It was revealed that in the stable, there were a total of 12 horses and three chickens. No animal movements to and from the farm were reported in the previous 10 months. One of the 12 horses present on the farm, which originated from the Piedmont region of Northern Italy, was vaccinated against WNV. No signs of fever, apathy, or other symptoms were reported in the horses during the vector season. The landscape where the stable was located was characterised by wetlands that were marshlands before reclamation in the 1930s, with irrigation channels and four artificial lakes in the immediate vicinity. During spring and summer, there was an unusual abundance of mosquitoes. Periodically, the owner had performed adulticide treatments using piretroids, but no action was taken against larvae. Moreover, within 6–7 km, there is a bird-watching area widely populated by resident and migratory birds; indeed, it was a frequent observation area for wild birds, in particular ducks, corvids, hawks, and magpies. Nevertheless, no dead bird was reported in the area, except for a WNV-L1-positive goshawk found in 2020 at 17 km distance . Further3.2Culex perfucus in Italy (Veneto Region) in 2021 and 2022. The second cluster contains five WNV-L1 strains isolated in Italy between 2008 and 2009  from humans, magpies, and jays. The third cluster is the largest and encloses two sub-clusters with a common ancestor. One includes strains isolated  from Russia, Turkey, Israel, Italy, Morocco, and France. The other includes the strain sequenced in the present study, together with the strains MW627239 isolated in the Campania region (2020), the ON813215 strain isolated in Senegal (2012), and the JF719069 strain isolated in Spain (2010). They share a sequence identity of > 98%.Biomolecular tests done at IZSAM and IZSM showed that all the collected organs had WNV lineage 1. The phylogenetic analysis showed that the consensus sequence obtained by NGS  was similar to those of the 17 WNV L1 retrieved from GenBank . The valThree horses and one chicken tested positive for WNV IgM and IgG, respectively. No WNV RNA was detected using real-time RT-PCR. By VN assay, the three positive horse sera showed 1:20, 1:40, and 1:320 WNV titres, while the chicken serum was 1:20.4During the past 30 years, several outbreaks of West Nile virus have been observed in Europe, both in humans and animals, with two important peaks in 2018 and 2022 , 26. IndThis study evidenced a close genetic correlation between the WNV isolated from the horse and the MW627239 goshawk strain previously isolated in the Campania region in 2020 , but alsFlavivirus introduction  can be found at: Ethical approval was not required for the studies involving animals in accordance with the local legislation and institutional requirements because the Istituto Zooprofilattico Sperimentale del Mezzogiorno is the official laboratory designed by the Italian Ministry of Health. According to the national regulations and internal policies, ethical approval was deemed unnecessary. Written informed consent was obtained from the owners for the participation of their animals in this study.CM: Investigation, Writing – original draft. LCa: Investigation, Writing – original draft. FP: Methodology, Writing – original draft. MV: Methodology, Writing – original draft. LCo: Formal analysis, Methodology, Writing – original draft. RP: Data curation, Formal analysis, Writing – original draft. SC: Data curation, Formal analysis, Software, Writing – original draft. MA: Methodology, Writing – original draft. MG: Supervision, Writing – review & editing. FM: Supervision, Writing – review & editing. GS: Supervision, Writing – review & editing. FD’O: Conceptualization, Writing – review & editing. RP: Funding acquisition, Supervision, Writing – review & editing. GF: Conceptualization, Funding acquisition, Resources, Supervision, Writing – review & editing.


Viruses provided a fascinating snapshot of plant and fungus virus research being undertaken in Australasia during the final year of the official COVID-19 pandemic. Eight papers published from April 2022 to May 2023 were presented in this Special Issue, all originating from Australia and New Zealand.The Special Issue ‘State-of-the-Art Plant Virus Research in Australasia’ in Nicotiana benthamiana. This variable species has an uneven distribution across Northern Australia , but previous sleuthing had indicated the accession used by virologists was unusual in several aspects and originated from a single source at a gold-mining site called The Granites, a remote group of low granite hills located hundreds of kilometres north-west of the nearest large town of Alice Springs. The authors obtained a herbarium specimen of N. benthamiana, collected by Dr John Cleland on a trip he undertook to The Granites in 1936 to study a group of Warlpiri Aboriginal people living there, who still maintained a largely traditional hunter–gatherer lifestyle. The Warlpiri used N. benthamiana as a source of the addictive alkaloid nicotine, yet maintained taboos against young women using it as such. Nowadays, we all know of the damage to the unborn foetus that tobacco use causes, and it seems that back then, the Warlpiri were well aware of the danger too. The 86-year-old herbarium specimen held the same mutation in its RdRp1 gene seen in the laboratory accession of N. benthamiana today, confirming it to be a ‘natural’ mutation. A paper trail of historical records, scientific publications, and new scientific data led from the N. benthamiana population at The Granites in 1936 to Professor Thomas H. Goodspeed at UC Berkeley in 1939, and from there to the Division of Tobacco Investigations in Maryland by 1945, and onwards to its ubiquitous distribution in virology labs today.Obliquely associated with plant virus research, the first paper in the series  providedThe second paper in this series  concerneAnthocercis is a genus of solanaceous shrubs and small trees indigenous to Australia. Yellow tailflower mild mottle virus (YTMMV) is a tobamovirus described from several Anthocercis species. The authors examined its response to the presence of the introduced weed Solanum nigrum, which sometimes grows in physical contact with infected Anthocercis plants. Would YTMMV spillover to S. nigrum? Populations of S. nigrum growing adjacent to and distant from infected Anthocercis plants were examined and found to be infected with YTMMV. The existence of S. nigrum populations growing in suburban locations far from Anthocercis populations suggested seed-borne transmission and spread of YTMMV facilitated by fruit-eating birds. Seed transmission of YTMMV in S. nigrum was then proven in the laboratory. The threat of infection of YTMMV to solanaceous crops such as potato is discussed in light of the export of such crops providing potential opportunities for international travel and spread by YTMMV.The third paper  also desPoleroviruses of peas and other crops and surrounding weeds in Tasmania were the main focus of the fourth paper in the series , althougPaper five in the series  was abouThe authors of paper six  examinedPaper seven  reportedClosterovirus known to infect grapevine. Since 2001, 11,257 vine samples were received, of which 313 tested positive for GLRaV-2, an overall incidence of 2.7%. This incidence rate remained steady over 20 years because this virus does not have a natural vector and spreads mainly through grafts from infected vines. GLRaV-2 isolates exist as six phylogenetic groups based on coat protein and p24 nucleotide sequences, which to some extent reflect their pathogenic properties. The groups are designated PN, 93/955, H4, BD, RG and PT20. Members of PN, which are more prevalent, as well as the 93/955 group, are associated with both leafroll symptoms and graft incompatibility. The BD group appears to be asymptomatic and is rarely associated with leafroll symptoms or with graft incompatibility. The RG group is associated with the graft incompatibility symptoms which only develop following grafting, but it is symptomless in ungrafted vines. Although three different phylogroups of GLRaV-2 were detected in the same vine, no evidence of recombination was observed among them. GLRaV-2 is a debilitating virus, especially in phylloxera-infested regions where the grafting practice is mandatory. The authors propose that in such areas the use of rootstock genotypes sensitive to the virus should be avoided.The final paper  in the sIn summary, there is investment in a broad range of plant virus-related topics in Australasia. From a biosecurity viewpoint, the region has the advantage of a continuous oceanic border around the island continent of Australia and the islands of New Zealand, which forms a barrier to the natural spread of viruses. However, as the international trade in fresh produce and propagules increases, and the climate continues to change, opportunities for viruses (and other pathogens) to jump over oceanic barriers and expand their ranges to Australasia and its crops and flora will probably increase. Similarly, we have a responsibility to keep the viruses that evolved in the region from escaping and establishing in new continents.The indigenous viromes of Australasian floras have largely been ignored to date. This is understandable given the limited sources of funding for research in ‘non-commercial’ plants and fungi. However, we urge that a more thorough knowledge of Australasian viromes be obtained in years to come, not only to forestall future spillover events but also simply to pursue knowledge for the sake of knowledge, a noble scientific endeavour. For the undergraduate student considering undertaking a higher degree in a biological discipline, there are almost infinite choices of virus-based research projects to consider. Additionally, if one decides to study ancient symbiotic interactions between indigenous viruses and their hosts, there is a very good chance the researcher will have the opportunity to describe and understand one or more new-to-science species. We have only scratched the surface of the unique Australasian virome; the adage ‘more research is needed’ is especially true in the flora of this region.This Special Issue provided a snapshot of some of the plant and fungal virological research being conducted in Australasia at the start of the third decade of the twenty-first century. Although much has been discovered, the field remains in its infancy. We are confident that future generations of virologists in the decades and centuries to come will build on what we have started, will endeavour to understand in greater detail the complex interactions between host and virus, between virus and virus, the evolutionary forces that act upon viruses when they jump hosts, as well as building better tools to detect and defeat the viruses that cause us harm.