Pyoderma gangrenosum (PG) is a neutrophilic dermatosis that presents as painful, rapidly growing skin ulcers with undermined edges and a violaceous, ragged border at the periphery and is non-responsive to conventional treatments. The average onset age is in the fourth decade, with a female preponderance. Genital PG is uncommon and may present singly or coexist with common sexually transmitted genital ulcerative diseases, which causes delays in the diagnosis and treatment of genital PG, thereby adding to the morbidity. Here, we highlight a case of non-healing genital ulcers that did not respond to conventional antibiotic treatment and aggravated each month with menstruation. In this case, menstruation acted as a trigger factor for the development of a herpes genital infection. The latter acts as a pathergy for the monthly aggravation of genital PG. The patient responded to treatment with anti-viral medications and immunosuppressive medications. Pyoderma gangrenosum (PG) is a non-infectious neutrophilic dermatosis that presents as painful, rapidly growing skin ulcers with undermined edges and a violaceous, ragged border at the periphery . The avePG uncommonly presents in the genital areas and may present singly or in coexistence with common sexually transmitted genital ulcerative diseases. It can mimic various other causes of non-healing ulcers, like sexually transmitted diseases , infectious diseases like tubercular ulcers, vascular causes , and others ,3. Such Hence, we present the case of non-healing genital ulcers, which did not respond to conventional antibiotic treatment and aggravated each month with menstruation. In this case, menstruation acted as a trigger factor for the development of a herpes genital infection. The latter acts as a pathergy for the monthly aggravation of genital PG. The patient responded to treatment with anti-viral medications and immunosuppressive medications.A 33-year-old married woman, with no history of high-risk sexual behavior and one sexually active partner (her husband), presented with complaints of multiple painful raw areas over her genitals for six weeks. She gave a history of multiple yellowish-raised lesions on the groin that formed raw areas within three to four days and were associated with severe pain.On examination, three well-defined shallow ulcers with violaceous margins and granulation tissue at the base were present, one on the mons pubis and two on the vulva Figure .Histopathological examination showed a dense perivascular neutrophilic infiltrate Figure .Hematological and biochemical investigations were within normal limits, and pus cultures showed no growth. Viral markers were non-reactive. On the basis of Delphi criteria, a diagnosis of genital ulcerative PG was made .The patient's initial treatment was intra-lesional triamcinolone acetate, administered once every three weeks and three times in total. While on treatment, she complained of an increase in burning and pain that aggravated with menstruation for two months. She was then started on oral cyclosporine 100 mg twice daily and topical tacrolimus 0.1% ointment twice daily. But even after this, the monthly aggravation continued, even after the initial response to the treatment.The patient was then diagnosed with genital herpes triggering before the onset of menses. This was treated initially with episodic treatment with oral acyclovir 400 mg thrice a day for five days. This was done for two months. After observing a favorable response to this treatment, the patient was shifted to a suppressive dose of oral acyclovir 400mg twice a day, along with continued oral cyclosporine and topical tacrolimus. The patient responded to this treatment Figure . The herPyoderma gangrenosum is a chronic inflammatory skin condition in which a painful papule progresses to form an ulcer with undermined margins . PG causThe diagnosis in our case was based on the Delphi criteria . This crPG is commonly associated with inflammatory bowel disease, rheumatoid arthritis, and hematologic malignancies . An assoCorticosteroids and/or cyclosporine are frequently used as the first step in the treatment of PG to reduce inflammation, followed by the addition of slower-acting immunosuppressive medications with better adverse event profiles, such as biologics . In our Genital PG is uncommon and sometimes misdiagnosed. Therefore, it should be taken into account when treating refractory genital ulcers . ClinicaIn each patient with PG with a hematological malignancy or compromised immunity, a thorough investigation for a viral agent should be conducted, particularly if the lesions are on the face or genital area . Our casThe association between PG and HSV has been infrequently reported in the literature . The firThe monthly aggravation of the genital PG ulcers with menstruation, even while on treatment, acted as a challenge. On close examination, grouped vesicles and ulcerations were noted on the labia majora, which led us to think about a genital herpes infection triggering the PG. Subsequently, IgG for HSV-2 was also reported to be elevated. We postulate that menstruation acted as a stressor for the development of recurrent genital herpes infection, which acted as a pathergy phenomenon for the monthly aggravation of the genital PG.Recurrent herpes virus infection can act as a trigger factor for the pathergy phenomenon that leads to the aggravation of pyoderma gangrenosum (PG). Timely identification of herpes simplex and an early start of treatment can prevent the development of pathergy.


Handwashing is an important intervention which can reduce indirect disease transmission, however soap and water for handwashing purposes is not available in some low-resource regions. When handwashing with soap and water is not possible, individuals may use alternatives such as the Supertowel . Testing of viral inactivation as a result of antimicrobial treatment on the Supertowel, however, has been limited. The goal of this study is to provide information about the performance of the Supertowel’s antimicrobial treatment against viruses, which will help inform the use of the towels as handwashing alternatives.We seeded the Supertowel and a regular microfiber towel with two bacteriophages (enveloped Phi6 and non-enveloped MS2) and monitored viral inactivation over time. Additionally, we assessed if temperature, humidity, whether the towel was initially wet or dry, or virus type had an effect on viral decay rate constants. Virus concentrations were measured repeatedly over 24 h.P = 0.06 and P = 0.22, respectively). We found that the variables of temperature, whether towels were initially wet versus dry, and virus type were significantly different from 0, suggesting that these variables explained variance in the decay rate constant . Higher temperatures, dry towels, and enveloped viruses all resulted in increases in the decay rate constant.We found that neither towel type (whether the towel was a Supertowel or a regular microfiber towel) nor humidity were significant variables in our model of decay rate constants (Viruses seeded onto a Supertowel decay similar to viruses seeded onto a regular towel indicating that the virucidal potential of the Supertowel is minimal. Indirect transmission of viruses occurs when viruses are released into the environment from an infected host, a susceptible individual comes into contact with the virus, and the susceptible individual becomes infected. Indirect transmission can occur through exposure to virus-laden air, food, water, or fomites . Hands cHandwashing with soap and water is recommended by the World Health Organization (WHO) and Center for Disease Control and Prevention (CDC) , howeverThe goal of the present study is to investigate the inactivation of viruses Phi6 (a surrogate enveloped virus) and MS2 (a surrogate non-enveloped virus) seeded onto a Supertowel and a regular microfiber towel; inactivation was assessed at multiple time points over 24 h at a number of realistic environmental conditions. In particular, we carried out the experiments at three different temperatures , two humidities (33% and 75%), and wet and dry towels. The results of study will inform the use of the Supertowel and regular towels as handwashing alternatives.Pseudomonas syringae . Full descriptions of the culture media for the virus and host is described in Anderson and Boehm  for 48 h. Phi6 stock was created by mixing the soft agar of lysed plaque assay plate with phosphate buffered saline . The virus-agar-PBS mixture was centrifuged and the supernatant was filtered through a 0.2 µm pore-size membrane, concentrated using an Amicon® Ultra-15 centrifugal filter unit , and stored at −80 °C in a 15% glycerol solution. Phi6 stock concentration was approximately 1011 plaque forming units (PFU)/mL. MS2 (DMS No. 13767) was propagated in its host Escherichia coli  based on Anderson and Boehm and EPA Method 1602 ( E. coli stock and incubated (without shaking) at 37 °C until the logarithmic growth phase. MS2 virus stock was created by mixing the soft agar of lysed plaque assay plate with PBS. The mixture was centrifuged, the supernatant was filtered through a 0.2 µL pore-size membrane, and the filtrate was stored at −80 °C in a 15% glycerol solution without ultrafiltration, because the concentration without ultrafiltration was adequately high. MS2 concentration of the stock was approximately 1010 − 1011 PFU/mL.Phi6 (NBRC 105899) was propagated in its host nd Boehm . Brieflyhod 1602 . BrieflyA total of 24 experimental treatments were investigated (3 temperatures × 2 humidities × 2 towel types × wet/dry). Towel coupons were destructively sampled at fixed times up to 24 h and virus was recovered from the towel and enumerated. Each experimental treatment was repeated three times, creating three trials  that serve as biological replicates. A full list of temperatures, towel conditions, humidity, and time points is in 2 coupons using sterile scissors. Dry towels were used as purchased. A subset of the coupons was wet using 500 µL of autoclaved deionized (DI) water at the start of experiments (towels were not re-wet throughout the experiment). Thereafter, 100 µL of virus suspension (50 µL of approximately 1010 PFU/mL Phi6 stock and 50 µL of approximately 109 PFU/mL MS2 stock) was applied to the coupons. The towel was stored in an airtight tupperware containers (Rubbermaid) at the specified humidity (high (75% relative humidity (RH)) or low (33% RH)) and temperature  for up to 24 h. RH was maintained using saturated salt solutions of MgCl2 for 33% RH and NaCl for 75% RH within the containers  and new, unused regular towels  were used in the experiments. The only difference between the Supertowels and the regular towels was that the Supertowel had an antimicrobial coating per the manufacturer. Towels were cut into 2 cmntainers . TemperaVirus was recovered from towels at each time point by submerging the entire towel coupon in 4 mL of tryptic soy broth (TSB) and vortexing for 20 s. The coupon was then carefully removed from the TSB and inserted into a 5 mL syringe and excess liquid was pressed out of the coupon and into the TSB see . This al11 PFU/mL.Negative controls were included with each experiment and consisted of a Supertowel coupon, stored in environmental conditions for 24 h, and put through the viral recovery process before sample plating. Positive controls were included with each experiment and consisted of plating diluted −80° C viral stock, which we expected to have a concentration of ∼10P. syringae host and 100 µL of diluted sample were added to soft nutrient agar (0.3% agar), then poured onto hard nutrient agar plates (2.3% agar). Phi6 plates were incubated upright overnight at 30 °C, then PFUs were counted. To enumerate MS2, 200 µL of log-phase E. coli and 300 µL of diluted sample were added to soft tryptic soy agar (0.7% agar), then poured onto hard tryptic soy agar plates (1.5% agar). MS2 plates were incubated upside-down overnight at 37 °C, then PFUs were counted.Viral concentration was evaluated using double-layer plaque assays to quantify infective viruses using 60 mm diameter Petri dishes. To enumerate Phi6, 100 µL of 6 for MS2 and 1:107 for Phi6. Although 500 PFU is a large number of plaques to count, plaques formed by these viruses with the hosts are small, and 500 PFU was deemed countable by the technicians. Plates with more than 500 PFUs were classified as TNTC (too numerous to count). In some instances, all the dilutions we plated were TNTC and in these cases, the plaque assay was repeated using the archived sample which was held at 4 °C using greater dilutions. Repeated plaque assays were completed within 24 h of sample collection. Three to four dilutions were plated for each sample.The TSB samples were diluted prior to plating according to preliminary experiments which provided information on how to dilute the virus-containing TSB at each time point so as to obtain a countable number (between 0 and 500) of PFUs. Dilutions ranged from undiluted to 1:10t-test to determine if the median viral recovery differed between Supertowel and regular towels.Figures and statistical tests were performed with R . A Wilcok, we used the first order inactivation model described in N0 is the average initial viral concentration (PFU/mL) of replicates recovered from the towel sample at t = 0, Nt is the average viral concentration (PFU/mL) of replicates recovered from the towel at each time point, t is time (hour), and k is the first-order rate constant (h−1). Using the left-hand-side of  t as our independent variable, we were able to determine the slope of the model (-k) using a linear regression fit function in R.To calculate k: temperature, humidity, towel type, wet versus dry towels, and virus type. The latter four variables were categorical and temperature was continuous. The relationship between the first-order rate constant k and temperature is typically described through the Arrhenius A is the pre-exponential factor, Ea is the activation energy (J/mol), R is the universal gas constant (J/(molK)), and T is the absolute temperature (K). We investigated the following independent variables which may affect the dependent variable The Arrhenius equation in the form of a linear equation is shown in A) is replaced with a, and −Ea/R is replaced with βT. To evaluate the effect of the categorical variables, we assigned a dummy variable to the variable. These dummy variables were used in addition to the Arrhenius equation to develop k), our independent variable, are linear, that observations are independent, and that independent variables are not highly correlated with one another. The linear regression also assumes that model residuals are normally distributed and that the variance of error is similar across independent variables. Confirmation of these assumptions is detailed in the SI.Multiple linear regression assumes that the relationships between the dependent variables and ln(f2) of 0.30. Cohen’s f2 is a unitless value, calculated with model R2 values  . Using an alpha of 0.05 for specificity, a power of 0.80, and our 5 predictors, our total sample size of 48 data points was able to measure an effect size (Cohen’s ulation) . Cohen’se effect . Therefok, determined using a threshold of four times the mean Cook’s Distance, were removed from the data set of k values.In instances where there were zero plaques in all plates of the undiluted samples, the sample was recorded as a non-detect and 0.5  was substituted as the undiluted sample PFU. Outliers of 11 PFU/mL for Phi6 and MS2 stock) indicating that the assays performed as expected with no contamination. In total, we measured Phi6 and MS2 each in 324 samples. Samples were below the limit of detection and substituted with 0.5 PFU 40 times out of 324 data points for Phi6 and 0 times for MS2. Median virus recovery using the recovery procedure was 0.69% for Supertowels (n = 72) and 0.72% for regular towels (n = 72) .All negative controls for the experiments had zero plaques and all stock concentrations results were as expected . Of the 38 k values that were significantly different from 0, 37 were positive indicating decreases in concentration with time while 1 was negative indicating an increase over time. Across all tested conditions where k was significantly different from 0, k varied from −0.04 to 0.27h−1 for MS2 and 0.16 to 3.19 h−1 for Phi6  . Towel type was not a significant variable in the model nor was RH . The unitless measure of effect size, Cohen’s f2, of temperature, wet versus dry towels, and virus type were 3.00 (large effect), 0.33 (medium effect), and 9.46 (large effect), respectively. The results of the analysis indicate that k values tended to be larger at higher temperatures, on dry towels, and for Phi6 compared to MS2.We found that coefficients for temperature, for the dummy variable describing whether towels were initially wet versus dry, and for the dummy variable describing virus type were significantly different from 0, suggesting that these variables explained variance in ln(k) of an enveloped and non-enveloped bacteriophage seeded on Supertowels were not different from those seeded onto a regular towel without the antimicrobial coating. This suggests that the antimicrobial coating on the Supertowel does not have enhanced antiviral activity relative to a regular towel. A previous study  after one hour was 0.81 log, suggesting that the Supertowel would not be an effective antimicrobial surface according to US EPA standards.An effective antimicrobial surface, according to the United States Environmental Protection Agency’s (US EPA’s) interim guidelines for effective antimicrobial surface coatings, should result in a minimum 3 log reduction after 1–2 h . SupertoEscherichia coli seeded onto volunteer hands. Therefore, while the results from this study indicate that the Supertowel antimicrobial surface may not be effective against viruses, it could still be effective against bacteria and fungi.When the Supertowel and a regular towel were actually used in practice for handwashing, k also increased. This is consistent with previous studies which found that temperature and viral decay rate constants are positively associated for viruses on surfaces (f2 effect size of the virus-type variable was 9.46 (unitless), which is considered a large effect size, and was the largest effect size of the variables we measured in this study. Previous studies have also found that enveloped virus inactivation is greater than non-enveloped virus inactivation on surfaces  and monitor viral inactivation over time, which will help inform the use of the towels as handwashing alternatives. Results suggest that the Supertowel provides inconsistent inactivation of viruses, as differences in viral decay rate constants were not explained by towel type. These viral results differ from tests with bacteria, which suggested the Supertowel was an effective antimicrobial tool . Results10.7717/peerj.15202/supp-1Supplemental Information 1Click here for additional data file.


Infectious NoV could be recovered and quantified in the HIEs from lettuce, frozen raspberries, or frozen strawberries seeded with high titers of either of these strains. While many experimental challenges still remain to be overcome, the results of this study represent an important step toward the detection of infectious norovirus from representative produce items.Norovirus (NoV) is the leading cause of viral foodborne gastroenteritis globally. Currently, the gold standard for detecting NoV in clinical, food, and environmental samples is via molecular-based methods, primarily RT-PCR. Nevertheless, there is a great need for confirmatory assays that can determine the infectivity of viral particles recovered from contaminated matrices. The use of the human intestinal enteroids system (HIEs) has allowed for the expansion of norovirus replication, although it still suffers from limitations of strain preferences and the requirement of high titer stocks for infection. In this study, we wanted to explore the feasibility of using the HIEs to support the replication of NoV that had been recovered from representative food matrices that have been associated with foodborne illness. We first confirmed that HIEs can support the replication of several strains of NoV as measured by RT-qPCR. We subsequently chose two of those strains that reproducibly replicated, GII.4 and GII.6, to evaluate in a TCID Norovirus (NoV) is the leading cause of gastroenteritis worldwide, causing over 100,000 hospitalizations, 465,000 emergency department visits, and 2.3 million outpatient visits in the US alone per year . One sucHistorically, human norovirus has proven difficult to culture ,14. Unti50 assay. Finally, we used the replication-competent strains GII.4[P16] and GII.6[P7] in the enteroids culture system to measure infectious norovirus recovered from a variety of fresh and frozen artificially spiked produce items toward establishing the utility of this culture system for the detection of infectious virus particles recovered from foods. In this study, we used the human intestinal enteroids system to screen a variety of norovirus genotypes for replication and identify those that demonstrated reproducibly positive virus replication. We selected two different norovirus genotypes (GII.4[P16] and GII.6[P7])  for the Human jejunal enteroids (J2) were generously provided by Dr. Mary Estes . The enteroids were passaged, and monolayers were prepared on 96-well plates for infection experiments, as reported previously . Three-dg for 5 min at 4 °C. Some stool samples were further filtered onto pre-wet 0.2 µm syringe filters  and aliquoted. A minimum of 50 µL was extracted for RNA, and RT-qPCR was used to determine virus copy number/µL. For screening and infection experiments, the number of viral genome copies added per well for cell infection is summarized in Norovirus-positive stools or vomit were obtained either from collaborators  or from sporadic cases. An amount of 100 µL stool or vomit was mixed with 900 µL PBS, vortexed, sonicated briefly, and then clarified by centrifugation at 25,000× 50) genomic copies and tissue culture infectious dose 50% (TCID50) for infectivity of these two strains was determined by applying the Reed–Muench method as previously reported and described for human norovirus replication in HIEs [50 and was used to determine 95% confidence intervals for the respective genotypes.For determination of the infectious virus titer of the GII.4P16] and GII.6[P7] fecal filtrates, monolayers of J2 HIEs in 96-well plates were used, with 8 wells for each virus dilution, ) on separate plates. Each assay was performed in triplicate for each virus strain. Viruses were serially diluted at a 1:3 ratio beginning at 1:10 or 1:30 of a 10% fecal suspension, and 6 dilutions were used per virus. For the 0 hpi wells, following a 1.5 h adsorption, plates were washed three times with CMGF media, overlaid with 50 µL CMGF media and stored at −20 °C until RNA extraction of the entire well. For the 5 dpi plate, which was adsorbed and washed in parallel with the 0 h plate, the wells were overlaid with 150 µL differentiation medium containing 500 µM GCDCA, which was replenished with an additional 75 µL medium on day 2 post-infection. Plates that were 5 dpi were frozen and thawed three times, after which 80 µL was removed from each well for RNA extraction to determine total virus RNA copies per well. Individual 5 dpi wells were scored as replication positive if there was a ≥5-fold increase in viral genome copies over the average of genome copies in 0 h wells at the same dilution. Viral genome copy numbers were determined by RT-qPCR analysis as described in  and GII. in HIEs , yielded7 genome copies per virus). After 1 h incubation at room temperature, food was stored overnight at 4 °C. The following day, the virus was eluted and concentrated with ultracentrifugation, as described in BAM chapter 26. The concentrated pellet was resuspended in 600 µL of enteroids infection media , sonicated for 5 min, and spun at 13,800 rpm for 5 min at 4 °C in a microcentrifuge to clarify the virus particles from accumulated solids. In some experiments, a portion of the recovered virus was further diluted as indicated using CMGF with GCDCA and Pen/Strep. The virus extracts were stored at −80 °C prior to use in culture or RNA isolation but typically were used within 1–2 weeks after recovery. Virus extraction was completed as described in US FDA BAM chapter 26  without RNA extraction of infected cells and viral inputs (including the virus recovered from berries and lettuce) was performed using Trizol™  to lyse the samples, followed by the use of the Direct-zol RNA extraction kit, following the manufacturer’s instructions . RNA recovered from berries was purified using the PCR Inhibitor Removal Kit (Zymo Research), according to the manufacturer’s instructions. 2 ≥ 0.98) over the concentration range of 1.7 × 105 to 17 genomic copies/5 µL RNA per reaction with slopes indicating reaction efficiencies from 91 to 98%; corresponding Ct values ranged from 22.57 to 39.03, respectively. Deviation from linearity (Ct vs. genome copy number per reaction) and decreased detection  frequency of <100%  occurred at concentrations of <17 copies/5 µL RNA per reaction. In order to (i) provide a minimum value for “quantification” on the nonlinear portion of the curve, (ii) acknowledge that an “undetermined” signal may represent either an absence of an RNA target or the presence of a target at a concentration that may give <100% detection frequency, and (iii) limit overestimation of fold increase for samples containing low copy numbers, we assigned a value of 8 genome copies/5 µL RNA per assay to “undetermined” signals from 0 hpi where 3 to 5 dpi yielded a Ct value. The 8 genome copies/5 µL RNA thus convert to 128 genome copies in an 80 µL volume of extract. Quantitative reverse-transcription PCR (RT-qPCR) on 5 µL RNA (input and cell) extracts was performed as described previously . A full-Analysis of variance (ANOVA) and linear regression analyses were used to evaluate standard curves and assay performance using GraphPad Prism (version 9.5.1).5 genome copies added per well at a 1:200 dilution of the 10% filtrate). The virus from the adult’s sample did replicate, although at lower levels of efficiency than for those typically seen with some of the GII.4 strains (range of 11–a 200-fold increase from t0). During the screening process, we observed that one strain of GII.4[P16] occasionally caused a cytopathic effect  were averaged to obtain the data for the GII.4[P16] (50 of the GII.4[P16] was determined as 8.1 × 104 genome copies (at 1 TCID50); e.g., for a given set of wells, 8.1 × 104 gc per well are required to successfully infect (a five-fold increase over t0) half of the wells. A 570 TCID50 /1 µL of 10% stool suspension was determined for GII.4[P16. The ID50 for GII.6[P7] was 8.8 × 104 genome copies (at 1 TCID50) and 81 TCID50 /1 µL of 10% suspension. Our first goal was to determine the IDI.4[P16] a and GIII.4[P16] b strains7 genome copies per sample) on three separate occasions, generating enough viral samples to perform infections on four batches of cells  of the recovered virus. Cytotoxicity was never observed in control wells containing samples derived from unspiked produce, and a positive control (derived from stool resuspension) was included in each experiment. After extraction from raspberries, only 3 out of 8 virus inputs for GII.4[P16] and 1 of 6 for GII.6[P7] could be detected by RT-qPCR prior to their addition to cells, even after the use of a PCR inhibitor removal kit on all the sample inputs, followed by a 1:10 dilution of the RNA , neither of these replicated, although input titers were not unusually low. A recent publication from Hagbom et al.  strain (referred to as GII.4 (011617) in ) replica strain  using a slightly more statistically robust method than previously published  infection in the supernatant and cellular fractions from 1 to 5 dpi. Similar to what has been reported  infectio. We deci3 range. Standardized, validated methods that are currently used for virus recovery and detection in foods ,44 are b50/TCID50 values for this particular genotype. For the experiments conducted with lettuce, we were able to calculate recoveries that ranged from 0.3 to 12% for GII.4[P16] and 0.6–5% for GII.6[P7], similarly variable to other studies with HIEs [It has been suggested that different NoV genotypes may perform differently in terms of environmental persistence, inactivation patterns, and binding to putative receptors such as histo-blood group antigens, fucose, etc. ,52. In aith HIEs  or studiith HIEs ,54. Over2 to 103 genome copies per gr [Our study had several limitations. We used a high-titered virus as the inoculum for the raspberry, strawberry, and lettuce seeding studies as a proof-of-concept study and an inoculum level that may be closer to what has been often found in naturally contaminated foods (typically closer to 10s per gr ,48. Inde


Alteromonas sp. Ni1-LEM supernatant as a cleaning agent for ROMs in a desalination seawater plant in northern Chile (Aguas Antofagasta S.A.), which is responsible for supplying drinking water to the city of Antofagasta. ROMs treated with Altermonas sp. Ni1-LEM supernatant exhibited statistically significant results (p < 0.05) in terms of seawater permeability (Pi), permeability recovery (PR), and the conductivity of permeated water compared with control biofouling ROMs and those treated with the chemical cleaning protocol applied by the Aguas Antofagasta S.A. desalination plant.Biofouling refers to the undesirable growth of microorganisms on water-submerged surfaces. Microfouling, the initial state of biofouling, is characterized by aggregates of microbial cells enclosed in a matrix of extracellular polymeric substances (EPSs). In seawater desalination plants, filtration systems, such as reverse-osmosis membranes (ROMs), are affected by microfouling, which decreases their efficiency in obtaining permeate water. The existing chemical and physical treatments are expensive and ineffective; therefore, controlling microfouling on ROMs is a considerable challenge. Thus, new approaches are necessary to improve the current ROM cleaning treatments. This study demonstrates the application of  Biofouling is the undesirable growth of microorganisms on water-submerged equipment, infrastructure, or surfaces ,2. The iBiofouling is one of the major problems affecting seawater desalination plants. Biofouling negatively affects the water quality of filtration system products, such as reverse-osmosis membranes (ROMs), through biofilm-enhanced concentration polarization (BECP) and enhanced solute passage, resulting in an increased number of pretreatment steps . Maddah Since the existing chemical and physical treatments are expensive and ineffective, controlling microfouling on ROMs is a considerable challenge. It damages ROM structures, thus producing mechanical interference and a decrease in the permeability of seawater ,9. AccorBiological approaches have been proposed to solve this problem, such as the use of molecules with quorum-quenching activity, isolated enzymes, and the use of bacterial supernatants with mixes of enzymatic activities ,14,15. QAlteromonas sp. Ni1-LEM [Alteromonas sp. Ni1-LEM disrupt the matrix of the extracellular polymeric substances (EPSs), thus modifying the microbial communities observed in ROMs on a laboratory scale [Alteromonas sp. Ni1-LEM supernatant’s cleaning effect on ROMs exposed to the pressure, temperature, and flux conditions that are normally used in reverse-osmosis plants. In the present study, we evaluated the efficacy of the supernatant obtained from Alteromonas sp. Ni1-LEM as a cleaning treatment for ROMs using the same conditions applied by Aguas Antofagasta S.A., a company dedicated to supplying drinking water through reverse-osmosis technology in the city of Antofagasta, Chile. The results show that the application of the supernatant obtained from Alteromonas sp. Ni1-LEM in the ROMs allows the considerable recovery of permeate production capacity, restoring the topography of the membranes under the pressure and flow conditions used in the seawater desalination industry.Thus, if the use of these new technologies is effective, they could represent an eco-friendly product that decreases the operating costs of the cleaning procedures associated with ROMs maintenance . In this Ni1-LEM , which p Ni1-LEM ,25,26,27Alteromonas sp. Ni1-LEM [2HPO4 6 g/L, KH2PO4 3 g/L, NH4Cl 1 g/L, NaCl 21 g/L, 0.1 M MgSO4·7H2O 10 mL, 0.01 M CaCl2·2H2O 10 mL, vitamin B1 (1%) 0.2 mL, and glucose (20%) 20 mL) until the stationary phase was reached. Afterward, the culture was centrifuged at 8500 rpm at 4 °C for 15 min. The supernatant was filtered in a tangential flow filtration system  with a 0.2 µm pore size  and stored at 4 °C until further use. For ROM cleaning treatments, the total protein concentration was determined using the Pierce BCA protein assay kit . Ni1-LEM  was growAlteromonas sp. Ni1-LEM supernatant as a cleaning agent for ROMs, 42 cm2 sections were obtained from 3.5-year-old ROMs  with homogeneous microfouling and kept in sterilized seawater at 4 °C until use. The brand-new ROM  was used as a clean control.A ROM model SWC6-LD Hydranautics system with 3.5 years of continuous operation was used in all experiments. The SWC6-LD is a seawater reverse-osmosis membrane manufactured by Hydranautics made of composite polyamide polymer and a low-fouling spiral-wound configuration with dimensions of 40 × 8 inches, and a performance of 12,000 gallons/day, 1200 PSI max pressure, <0.1 PPM of max chlorine concentration, and 45 °C of max operating temperature. ROMs were selected on the basis of the first membranes located in the RO system . The selThe cleaning protocol applied by Aguas Antofagasta S.A. was used with certain modifications (varying cleaning solutions), and it was escalated to the Sterlitech CF042 system. This consisted of the passage of different cleaning solutions for a total estimated time of 10 h at 58 psi (4 bar), with recirculation and rest cycles using the following steps:First Rinse Step: Permeate water was recirculated for 15 min at 58 psi to eliminate the remaining salinity from ROM operations. Next, a solution of 0.1% ethylenediaminetetraacetic acid (EDTA), pH 12, at 35 °C was recirculated for 5 min to eliminate salt precipitates.w/v, EDTA 0.6% w/v, pH 12) at 35 °C, and 4 cycles consisting of 30 min of recirculation at 58 psi and 45 min of incubation were applied. After every cycle, the alkaline wash solution was replaced with a fresh solution with the same properties.Wash Step 1: The membranes were washed with an alkaline solution  at room temperature. In total, 3 cycles consisting of 30 min of recirculation at 58 psi and 45 min of incubation were applied. After every cycle, the acid wash solution was replaced with a fresh solution with the same properties.Wash Step 1: The membranes were washed with an acid solution  were applied and are listed in To evaluate the effects of the −1, pressure: 825 psi, intake water temperature: 19–21 °C, water flux rate: 2 L/min).ROMs previously cleaned with the respective treatments were placed in two cells of a CF042D crossflow RO system , and their performance was assessed for 15 h of continuous operation. To perform these experiments, pre-osmosis water from the Aguas Antofagasta operation plant was used , and the operation conditions were similar to those applied by Aguas Antofagasta S.A. in their routine applications (water flux speed: 37.49 cm × segPi), the permeate was obtained in a glass container positioned on a digital scale , and the difference in the weight was monitored every hour for a total time of 15 h. Equation (1) was applied according to Nagaraj et al.’s work (2017) [Pi is water permeability (L·m−2·h−1·bar−1), V is the volume in liters of the permeate obtained under constant pressure, P is the operating pressure (bar), t is time (h), and A is active ROM area (m2).To calculate seawater permeability (k (2017) :(1)Pi=VAPi values obtained during the experiments were used to estimate the permeability ratio according to Equation (2).Pi is the permeability after the respective cleaning treatments and Pcontrol is the control related to a clean membrane (ROM-CT).The average −1) and pH were measured every hour in the pre-osmosis and permeate water during all experiments.In addition, to observe membrane integrity and permeate water quality, conductivity  coupled to a model DU970N-BV (EMCCD) CCD camera, and pictures from a 100 µmAlteromonas sp. Ni1 LEM supernatant as a cleaning treatment was studied via one-way analysis of variance (ANOVA) using STATGRAPHICS Centurion XV Professional . The means were compared using multiple comparison tests of least significant differences .The values were expressed as mean ± standard error (ES) using Microsoft Excel . The effect of Pi) over 15 h from ROMs treated with the different cleaning treatments (p < 0.05). The other treatments, in order of decreasing seawater permeability, were as follows: ROM-SN, ROM-QT, ROM-SN-QTac, ROM-QTAl-SN, ROM-M9, and fouled membranes without cleaning treatments (ROM-BF). In addition, the ROM-M9 treatment, corresponding to the bacterial culture medium containing Alteromonas sp. Ni1-LEM, was used. This treatment was carried out without the antifouling bioactive substance and exerted no antifouling effect; no significant differences were found (p < 0.05) compared with the ROM-BF condition. In contrast, when water permeability was compared between different ROM cleaning treatments, the results showed that during the 15 h of the experiment, the highest permeability was obtained in the ROM-SN treatment, a condition that corresponded to a cleaning procedure using only the bacterial supernatant  measurements conducted over 15 h for ROMs treated with different cleaning treatments are shown in p < 0.05; p > 0.05).As observed in relation to the (Pi) values, when PR was compared between different ROM cleaning treatments, the PR results obtained after 15 h of experiments showed that the highest recovery was achieved by ROMs treated with the bacterial supernatant ROM-SN as a cleaning treatment (−1) obtained after 15 h of the ROMs experiment and those previously cleaned with different treatments are shown in Al-SN treatment, followed, in decreasing order, with significant differences (p < 0.05), by ROM-BF, ROM-QT, ROM-SN-QTac, ROM-M9, ROM-SN, and finally ROM-CT. This tendency was observed in each hour sampled throughout the experiment. No significant differences were observed in pH values (p > 0.05) between ROM cleaning treatments. However, a decrease in pH was observed in the permeated water in all treatments compared with the pH observed in feed water (pre-osmosis), with an average value of 6.93 ± 0.04.The results of permeate water conductivity (µS × cmAlteromonas sp. Ni1-LEM supernatant (ROM-SN), either as a single treatment (Alteromonas sp. Ni1-LEM supernatant effects were maintained under operating conditions (high pressure and continuous flow).To observe how cleaning treatments modified the physical aspects of ROMs, the ROM surface was analyzed through atomic force microscopy (AFM). The results showed that ROM-CT (brand new membranes) samples had a rough texture, highlighting the presence of small regular peaks on their surface A. Howevereatment E or mixereatment F. This rAlteromonas sp. Ni-LEM strain as a cleaning agent under real-life operating conditions according to the protocols used by a desalination plant in charge of supplying drinking water to the city of Antofagasta, Chile (Aguas Antofagasta S.A.).Researchers have long searched for novel economical and eco-friendly methodologies for cleaning ROMs, and diverse approaches have been evaluated in an attempt to achieve this goal. Several authors have proposed modifications in ROMs structures to improve their chemical and physical properties , as wellThe results showed that the Ni1-LEM supernatant exhibited considerable potential as a cleaning agent under real-life conditions. The data showed that the Ni1-LEM supernatant resulted in the best outcomes in relation to water permeability, the recovery of permeability (PR), and conductivity among all analyzed treatments, and it reached the closest values to the control conditions (ROM-CT).Alteromonas sp. Ni1-LEM supernatant also resulted in improved permeate quality according to the conductivity values. Lower permeated water conductivity implies a higher retention of salts by ROMs [−1), resulting in permeate water with fewer salts compared with the other treatments. However, the permeated water conductivity from the ROM-CT treatment was used as a reference; thus, treatments with conductivity values near to those of ROM-CT showed better cleaning performance. In this context, the treatment using the bacterial supernatant (ROM-SN), which exhibited lower permeated water conductivity compared with other cleaning treatments, led to better performance when comparing the conditions.The use of the  by ROMs ,31,32, mAlteromonas sp. Ni1-LEM supernatant is a temperature-sensitive solution [Alteromonas sp. supernatant on a laboratory scale (without pressure and water flux) were obtained at room temperature [Alteromonas sp. Ni1-LEM supernatant only interacted with biofouling components, such as the formed EPS, and could thus be successfully used against a broad spectrum of microalgal biofilms without negatively affecting the ROM structures [Traditional chemical treatments, primarily those that use alkali solutions to increase cleaning efficiency, require an operating temperature from 35 °C to 38 °C, which is reflected in the increased costs associated with cleaning treatments due to energy use (Aguas Antofagasta SA). In contrast, although the solution , this diperature , a condiperature ,33,34. Tructures ,27.Altermonas sp. Ni1-LEM supernatant was used as a cleaning treatment, a recovery in terms of the topography was observed (similar to the control samples: ROM-CT). This recovery was not observed when chemical treatments were used as cleaning methods. The topography observed in the ROM-SN treatment was also correlated with the results obtained regarding the comparison of permeate volumes, because the ROM-SN treatment (treatment with the Altermonas sp. Ni1-LEM supernatant) had the highest performance compared with the other chemical treatments. These data support the idea that the Alteromonas sp. Ni1-LEM supernatant can be applied in operating conditions as an eco-friendly alternative for ROM cleaning in the desalination industry.Atomic force microscopy (AFM) provides several tools with which to evaluate the physical and micromechanical properties of samples. This is achieved through the interaction of a cantilever probe with the surface to be studied . Thus, AAlteromonas sp. Ni1-LEM to obtain and evaluate the production of a supernatant with antifouling effects. The data showed that this massive culture had a supernatant production yield with a concentration of 614 µg mL−1 of protein. As mentioned above, the application of the supernatant as an ROM-cleaning treatment at a concentration of 200 µg mL−1 of protein as a working solution indicated that the massive culture generated a 3× concentrate of the working solution.One of the biggest bottlenecks preventing the industrial application of these new eco-friendly approaches, allowing them to compete with more traditional treatments, is the scaling of these processes for higher-volume production . In this−1) has an approximate cost between USD 0.14 to 0.19, which would allow generating a business model for its large-scale production.Additionally, preliminary economic studies showed that the production cost of 1 L of working solution of supernatant (200 µg mLAlteromonas sp. Ni-LEM supernatant is a promising substance with a high potential for industrial application because it can be applied under the conditions of pressure, water flow, and temperature normally used in seawater desalination plants. Future work will focus on the application of a semi-industrial-scale Ni1-LEM supernatant based on technological transference projects.The results show that the


B646L gene, yet 10 discrete fragments accounting for over 56% of their genomes are derived from genotype II virus. Animal studies with one of the recombinant viruses indicate high lethality and transmissibility in pigs, and deletion of the virulence-related genes MGF_505/360 and EP402R derived from virulent genotype II virus highly attenuates its virulence. The live attenuated vaccine derived from genotype II ASFV is not protective against challenge of the recombinant virus. These naturally occurring recombinants of genotype I and II ASFVs have the potential to pose a challenge to the global pig industry.African swine fever virus (ASFV) poses a great threat to the global pig industry and food security. Currently, 24 ASFV genotypes have been reported but it is unclear whether recombination of different genotype viruses occurs in nature. In this study, we detect three recombinants of genotype I and II ASFVs in pigs in China. These recombinants are genetically similar and classified as genotype I according to their  Potential recombination of African swine fever virus genotypes is not well understood. Here, Zhao et al. report naturally occurring ASFVs carrying mosaic genomes of genotype I and II and show that the recombinant virus is lethal in pigs and evades the immunity induced by genotype II live vaccine. ASF viruses (ASFVs) belong to the genus Asfivirus and Asfarviridae family2. The ASFV B646L gene encodes the viral capsid protein P72 that plays an essential role in virion assembly and virus attachment to the host cell3. ASFVs are divided into 24 genotypes based on the C-terminal sequence of their B646L gene with 86.2–99.5% nucleotide identity4. All 24 genotypes of ASFVs have been detected in sub-Saharan Africa, and only two of these genotypes have spread outside of Africa5. In 1957, genotype I ASFV was introduced into Europe and caused ASF outbreaks in many European countries6. In 2007, a highly lethal genotype II ASFV (Georgia07) appeared in Georgia, and quickly spread to neighboring countries (www.woah.org). In 2018, Georgia07-like ASFV spread to China and other Asian countries7, and has since been responsible for the loss of over seven million swine in Eurasian countries (www.woah.org).African swine fever (ASF) is a devastating infectious disease in swine that is a severe threat to the global pig industry  and were genetically similar to the NH/P68 strain reported in Portugal in the 1960s13. Both genotype I and genotype II ASFVs have been found in pigs in China, so we asked: is it possible that these viruses recombine in nature? And, if so, what challenges will be posed by the recombinant strains?Pigs are not vaccinated against ASF; therefore, due to the lack of an effective control strategy, ASFV is widespread and continues to evolve in many countriesHere, we isolated three recombinants of genotype I and II ASFVs in fields in China. The recombinants were fully characterized for their genomes, and their virulence and immune escape capability were tested in pigs. The results revealed the fast evolution of ASFVs through genomic recombination between different genotypes, which poses a new challenge to disease control efforts and vaccine development.B646L gene sequence , Pig/Henan/123014/2022 (HeN/123014/22), and Pig/Inner Mongolia/DQDM/2022 (IM/DQDM/22), respectively. We identified these strains as genotype I according to their B646L and EP402R genes from the field samples as well as purified virus stocks. The PCR amplification for the B646L and EP402R genes were cloned into T-Vector pMD19, and 20 positive clones for each reaction were randomly picked for sequencing by using the Sanger sequencing method. The results indicate that the B646L gene and EP402R gene of these newly emerging viruses are from genotype I virus and genotype II virus, respectively.We then performed next-generation sequencing for the OQ504954 for HeN/123014/22, OQ504955 for IM/DQDM/22, and OQ504956 for JS/LG/21) by using primers as described previously in ref. 16, and adjacent PCR amplification segments share an overlap of 100–200 base pairs (bps) to cover the sequences generated by the primers. We then performed a phylogenic analysis of the three viruses and 56 reference viruses of the eight different genotypes that are available in GenBank. The lengths of the genomes of JS/LG/21, HeN/123014/22, and IM/DQDM/22 are 185,431, 185,395, and 185,342 bps, respectively, and each of them has 172 open reading frames (ORFs). The three emerging viruses share 99.97–99.99% homology with each other at the nucleotide level, and form a unique branch in the phylogenic tree, which clusters between the genotype I and II viruses  and genotype I virus SD/DY-I/21 (GenBank: MZ945537), which share 97.38% nucleotide identity16. Compared with that of SD/DY-I/21, the genome of HLJ/18 has 21,062 bp insertions, 4237 bp deletions, and 3840 bp mutations. Both viruses have genotype-specific single nucleotide polymorphisms (SNPs) in the coding and noncoding regions of their genomes , which is from genotype I virus, whereas the EP402R gene encoding CD2v is in fragment 6 (F6), which is from genotype II virus  (we refer to these six genes as the “MGF_505/360 genes” in this report)19. To investigate whether the genes from the lethal genotype II virus contribute to the pathogenicity and transmissibility of the recombinant ASFV, groups of six 7-week-old specific-pathogen-free (SPF) pigs were inoculated with 103 HAD50 or 106 HAD50 of JS/LG/21. Two uninoculated pigs were cohoused with each group of inoculated pigs to evaluate virus transmissibility. Pigs were monitored daily for clinical signs. Oral and rectal swabs and blood were collected from all of the pigs every other day starting on day 3 post-inoculation (p.i.) to detect viral shedding and viremia by qPCR.The ten discrete fragments from highly virulent genotype II virus in the novel recombinant viruses bear 106 ORFs  of the Chinese Academy of Agricultural Sciences (CAAS) as approved by the Ministry of Agriculture and Rural Affairs. This study was performed in accordance with the Guide for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of the People’s Republic of China. The protocols were approved by the Committee on the Ethics of Animal Experiments of the HVRI of CAAS .15. Blood or homogenized tissue samples, including lung, spleen, and lymph nodes, were used to inoculate PAMs. On day 5 post-inoculation (p.i.), the cell supernatants were examined in a qPCR targeting the B646L gene of ASFVs. ASFV-positive cell supernatants were collected and virus purification was performed in PAMs by three rounds of limiting dilution. The ASFV isolate was titrated in PAMs by using an immunofluorescence assay or hemadsorption assay, and determined to be free of contamination with bacteria, porcine circovirus type 2, pseudorabies virus, porcine respiratory and reproductive syndrome virus, or classical swine fever virus. The recombinant virus with deletion of EP402R, MGF_505-1R, MGF_505-2R, MGF_505-3R, MGF_360-12L, MGF_360-13L, and MGF_360-14L genes was generated by using homologous recombination as previously described10. All virus stock was aliquoted and stored at −80 °C.Primary porcine alveolar macrophages (PAMs) and peripheral blood mononuclear cells (PBMCs) were prepared from the bronchoalveolar lavage and EDTA-treated blood of 30-day-old specific-pathogen-free (SPF) pigs as previously described15. Briefly, tenfold serially diluted virus solutions were inoculated into PBMCs with 0.1% porcine red blood cells in 96-well plates, respectively. HAD was observed daily for 7 days after inoculation under a microscope. The 50% HAD dose (HAD50) for each virus was measured using the Reed and Muench method40.The HAD activity of each ASFV was assessed as previously described in ref. 41.ASFV genomic DNA in swabs, tissue homogenates, serum, EDTA-treated whole peripheral blood samples, and cell supernatants was extracted using QIAamp® DNA Mini Kits  and detected using WOAH-recommended qPCR as described previously in refs. 12. To construct the phylogenetic tree, all ASFV genomes were aligned using E-INS-i of the program MAFFT v742 and the ambiguously aligned regions were excluded using Gblocks-0.9143. Phylogenetic analysis was inferred using maximum likelihood (ML), and ModelFinder was used to select the best-fit model according to the Bayesian information criterion (BIC)44. ML analysis was inferred using IQ-Tree45 and the best model was GTR + F + R3. Bootstrap branch support values (MLBS) were obtained with 1000 rapid bootstrap inferences and subsequently sought in a thorough ML search of the dataset. The diagram of the ASFV genome was created using the BLAST Ring Image Generator (BRIG) with the whole genome sequence of JS/LG/21. The BLAST option of BRIG was ncbi-blast-2.13.0+. Different recombination regions and the open reading frames (ORFs) were analyzed in comparison with those of genotype II virus HLJ/18 and genotype I virus SD/DY-I/21, respectively. Biological software, including Snapgene® 4.1.8 and MEGA X (https://www.megasoftware.net), were used for the alignment of multiple sequences. Primer sequences for PCR amplification and sequencing used in this study are shown in Supplementary Dataset ASFV genome segments were amplified by PCR using specific primers and a high-fidelity reaction system and sequenced using the Sanger DNA sequencing method as described previously in refs. 6 HAD50 or 103 HAD50 of JS/LG/21. Two additional naïve SPF pigs were then cohoused with the infected pigs in each group from the first day of infection to evaluate the transmission of JS/LG/21. All pigs were monitored daily for survival and clinical signs. Oral swabs, rectal swabs, and EDTA-treated blood were collected for virus DNA quantification using qPCR at the indicated times p.i. or post-contact (p.ct.). Organs and lymph nodes were collected from dead or euthanized pigs and examined for viral DNA using qPCR as described previously41.To evaluate the virulence of ASFV in pigs, groups of 7-week-old SPF Landrace pigs, regardless of sex, from the Laboratory Animal Center of HVRI were intramuscularly (i.m.) inoculated with 106 TCID50 of JS/LG/21-7GD. All pigs were monitored daily for rectal temperature, survival, and clinical signs for 28 days p.i. Pig sera were collected at the indicated timepoints for ASFV-specific antibody detection using a blocking ELISA kit (in-house)46.To examine the virulence of JS/LG/21-7GD in pigs, ten 7-week-old SPF Landrace pigs, regardless of sex, were i.m. inoculated with 10i.m. inoculated with 106 TCID50 of HLJ/18-7GD, and were then i.m. challenged with 103 HAD50 of JS/LG/21 or HLJ/18 on day 28 post-vaccination (p.v.). Groups of four unvaccinated pigs were parallelly challenged as controls. All pigs were daily monitored for rectal temperature, survival, and clinical signs for 28 days post-challenge (p.ch.). Organ tissues, including heart, liver, spleen, lung, kidney, tonsil, and lymph nodes, were collected from the dead pigs or surviving pigs euthanized at the end of the observation period to detect virus loading using qPCR.To evaluate the protective efficacy of genotype II live attenuated vaccine HLJ/18-7GD against the new emerging ASFV isolates, groups of 7-week-old SPF Landrace pigs regardless of sex were Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Reporting Summary


Bos indicus). Fourteen Nelore (Bos indicus) bulls were castrated and assessed pre- and post-surgically. Parameters evaluated were agonistic  and affiliative (head-play) behavior, plasma testosterone concentrations, average daily weight gain (ADG), and a score for severity of post-surgical infection. Exploratory statistics included social network analysis (SNA), hierarchy rank delta (Δ), and principal component analysis (PCA). Furthermore, statistical inferences included the Wilcoxon test, multiple logistic regression models, and Spearman's correlation.Confinement of cattle imposes spatial restrictions and predisposes to aversive social encounters that can lead to contusions, wounds, pain, stress, fright, and reduced productivity. Although endogenous testosterone concentrations are linked to agonistic dominance behaviors in males, it is unknown whether decreased blood testosterone concentrations after castration alter social hierarchy rank in Nelore bulls. Therefore, in this study, we investigated the impact of the surgical would inflammation post-orchiectomy on social dynamics in a group of Nelore bulls (The social dynamic of Nelore bulls was modified after castration based on the findings of the SNA and the PCA. The moderate correlation between the postoperative inflammation level with the Δ, and the significant effect of this level in the logistic model post-castration were partially attributed to effects of pain on social relations.Our findings suggest the severity of post-surgical inflammation, which has an association with pain intensity, was closely associated with changes in the social hierarchy.The online version contains supplementary material available at 10.1186/s12917-023-03638-9. In the past few decades, there is increasing worldwide concern regarding production animal welfare . WelfareConfinement imposes spatial restrictions, resulting in competitive encounters and violation of individual spaces; consequently, there is intensification of agonistic social behaviors  –10, as wBos taurus males recovered physical activity and sexual behavior [Exacerbated displays of agonistic behavior in males are apparently modulated by endogenous testosterone . In non-behavior . Similarbehavior .Castration of bulls has been associated with easier and safer animal handling and reductions in lesions and aggressive behavior –29. Howevs post-castration moments.Regarding social network analysis (SNA), numbers of connections and reciprocity of the agonistic and affiliative interactions in the social networks Table , as wellOn the first dimension of the PCA, the loading value of testosterone concentrations (0.57) indicated it had limited association with other variables and only limited contributions in the dimension Table S. The SNAComparisons between moments indicated that the group of cattle had increased tolerance in receiving agonistic behaviors (ReceptorsAG). Furthermore, the interactive capacity between the whole group for affiliative behaviors (ClosenessAF), body weight and ADG, also a decreased total number of affiliative interactions (Degree ALLAF), tolerance to receive (Degree INAF) affiliative behaviors, testosterone, and BI post castration Table .Table 2MP = 0.0073) with level of inflammation. However, there was no difference for post-castration average daily weight gain (ADG) between bulls with Δ negative or Δ positive ranking .In the post-castration evaluation, four bulls had a mild inflammation of their surgical wound , two had a moderate inflammation (Bulls 10 and 13), and one had severe inflammation (Bull 5)  and feeding a maintenance diet. In 37 Friesian bulls, there was an effect of social ranking on ADG [Interestingly, the process of establishing a social hierarchy can reduce cattle welfare, including potential lesions, pain, stress, and fear , in addig on ADG , whereasg on ADG .p > 0.05) in the modeling of the Δ of the pre-castration ranking indicated the absence of an evident influence of testosterone in modulation of social hierarchy, as observed in rats [We hypothesized that after surgical castration testosterone reductions affect social hierarchy rank in Nelore bulls. For non-human primates –20, rats in rats –34. Furt in rats –38. The Pain can be a stress-inducing factor capable of modulating social interactions in laboratory animals . For exaIn the present study, modifications in the social ranking of bulls post-castration, the moderate correlation between the surgical wound inflammation post-orchiectomy level with the Δ of the ranking, and the significant effect of this level in the logistic model post-castration were partially attributed to effects of pain on social relations. We inferred that pain originating from surgical wound inflammation post-orchiectomy modified hierarchy. Furthermore, another perspective is that the treatment of such surgical wounds post castration could have increased stress, potentializing the indisposition towards socialization and impairing its social position, since most bulls in this study with any degree of inflammation downgraded their position in social rank. Perhaps maintenance of the social hierarchy is dynamic and sensitive to the perceptible opportunities for each bull, allowing upgrading in social ranking in the face of stressful factors impacting the group. This can be understood as an adaptive value for species' survival, based on the Darwinian theory. In summary, there was a modification in social hierarchy in a medium-term after the would inflammatory episode; however, the short-term response needs to be evaluated to improve understanding of the influence of pain on social hierarchy of the bulls. This is important, since several events may have occurred between the pre- and post-castration behavioral evaluations, which were not documented in our study, and which should be investigated in the future. Our results represent the first step in the investigation about surgical castration and social dynamics of bulls.n = 14), they displayed social interactions sufficiently capable of differentiating pre- and post-castration social hierarchy. Plausibly, a survey with more bulls, such as in commercial feedlots, would result in an even further increase in social interactions. Lastly, to castrate only three bulls every 15 d could have impacted our study's findings; however, the logistic model of the Δ of the ranking post-castration did not indicate any significant effects related to the sequence in which the bulls were castrated. We emphasize that this was an opportunistic study, and the experimental design employed here was limited by the main experiment with the same animals [The present study was not without limitations. Despite only a limited number of animals used . A fortunate opportunity of another study from our team  taking pFourteen Nelore bulls  were used in the study, all derived from the same commercial herd. After arrival at our institution, the bulls were individually restrained in a cattle handling chute, body weight recorded and a 10-mL blood sample collected from coccygeal vessels into heparinized tubes  that were centrifuged  and plasma removed and frozen (-20 ºC) pending a testosterone assay.The bulls were housed together in one outdoor pen  with 1.25 m of feeder area and ad libitum water provided through an automatic auto-fill water tank . For acclimatization, for 15 d after arrival, there was minimal human contact, only providing feed and cleaning the feeder and water tank. Twice daily, the bulls were fed a maintenance diet comprised of corn silage (63.18%), cornmeal (20.58%), cottonseed meal (10.77%), sugarcane bagasse (4.11%), urea (0.69%), limestone (0.5%) and mineral salt (0.17%).After 15-d acclimatization, bulls were weighed a second time, average daily weight gain (ADG) calculated, and the data related to the pre-castration period. Throughout the subsequent 5-d interval, behavioral assessments were performed (described below); on the first day, the animals underwent general anesthesia, were exposed to testicular heat stress (3 h at ≤ 40 ºC) and castrated immediately thereafter, as described . Three bProphylactic antimicrobial therapy was administered before castration . Furthermore, a nonsteroidal anti-inflammatory and analgesic treatment was given . Post-surgical inflammations were treated symptomatically. The wound was cleaned with water and antiseptic solution  followed by application of insect repellent  and an agent to promote cicatrisation . Furthermore, the animals were treated with 12,000 IU/kg of benzylpenicillin benzathine, benzylpenicillin procaine, benzylpenicillin potassium IM, and 5 mg/kg disulfate dihydrostreptomycin disulfate base and streptomycin base IM . Furthermore, they were weighed twice (3 and 20 d) after the last castration, and post-surgical ADG was calculated. Lastly, at 21 d post castration, a blood sample was collected (as described above) and used to determine post-castration testosterone concentration.After the end of the experimental period, all animals were incorporated into the faculty herd.After the 15-d acclimatization period, the social hierarchy pre-castration was determined at ~ 07:30 am, based on the record of the frequencies of affiliative and agonistic interactions through the application of the ethogram described in Table The degree of surgical wound inflammation post-orchiectomy was evaluated by the same evaluator (HDGM) using a four-level score. Scoring was based on clinical evaluation of regional edema and swelling and the presence of haemorrhage or myiasis, and graded as follows: 0, 1, 2 and 3, representing no, mild, moderate, and severe inflammation, respectively.Plasma testosterone concentrations were measured with a commercial ELISA kit , with a 3.9 pg/mL sensitivity. Pre-castration samples were diluted 1:24 with the ELISA Buffer included in the kit. Duplicates were performed for the first and last samples measured; the intra-assay CV was 7.4%. Although no dilution was performed, the samples did not reach the minimum limit of identification. Therefore, the minimum detectable level indicated by the company (3.9 pg/mL) was used.All statistical analyses were performed by the same data scientist (PHET), using the software R with integrated developing system RStudio (Version 4.0.2 (2020–06-22), RStudio, Inc.). Various packages and functions were used in the format 'function{package}' according to the programming language in R. An α level of 5% was used for all tests. For the analyses below, bodyweight considered was the last bodyweight of either the pre- or post-castration intervals.The ADG was calculated using the following equation:ADG = average daily gain; Wf = body weight at the end of the experiment; Wi = body weight at the beginning of the experiment; d = average interval (d) between the two weights.A priori, the inter-relation between bulls was analyzed with exploratory statistical techniques. The frequency of agonistic behaviors was the sum of the frequency of mounts, fights, and headbutts. Four social network analyses (SNA) were performed ('as_data_frame{igraph}'), one for each behavioral category (agonistic and affiliative) in pre- and post-castration moments. The following parameters were extracted: asymmetry; diameter; connections; reciprocity; betweenness, closeness, degree , hub score (authors), and authorities score (receptors).Furthermore, based on data extracted from the SNA, the balance index (BI) was calculated based on a previous report in dairy bulls , using the following equation:INaffiliative: frequency that the animal was the receptor of affiliative behavior; INagonistic: frequency that the animal was the receptor of agonistic behavior OUTaffiliative: frequency that the animal was the author of affiliative behavior; OUTagonistic: frequency that the animal was the author of agonistic behavior; F1: correction factor of 0,005; F2: correction factor of 0,001. A small value was used as a correction factor to avoid a zero quotient in the absence of behavior display (adapted from ).As indicated in previous studies that demonstrated the importance of agonistic and affiliative behaviors in bovine social hierarchy –52, the Parameters from the SNA, in association with weight, ADG, testosterone, BI, and ranking, were subjected to a principal component analysis . First, on the biplot of the PCA ('fviz_pca{factoextra}'), confidence ellipses were prepared based on the distribution of the density of the bulls in each moment. Then, through a qualitative judgment, the distance between each ellipsis and their distribution in the four quadrants of the biplot were analyzed, and the distance between ellipses and their relationship with the origin (convergence between the x and y axes of ellipses).A posteriori, to confirm and interpret inferences of the results, confirmatory statistical analyses were performed. Data distributions were analyzed through histograms ('hist{graphics}'), quantile–quantile plots ('qqnorm{stats}') and boxplots ('ggboxplot{ggpubr}'); graphically, all variables were defined as non-normal. Therefore, variables were compared over time (pre- vs post- castration) with paired two-tailed Wilcoxon tests ('wilcox.test{stats}').The delta (Δ) of the ranking was extracted by subtracting the classification attributed post- minus pre-castration. Thus, individuals with ascension in the social hierarchy ranking post castration were considered Δ positive, whereas those that downgraded were Δ negative.To further investigate which factors related to the castration influenced the downgrading of individuals in the ranking, two logistic multilinear regression was done for each moment ('glm{stats}'). For the pre-castration moment, the possibility of the ranking be considered as Δ positive or negative (dichotomic) was used as the dependent variable, whereas the weight, ADG, and testosterone concentration were used as independent variables. For the post-castration moment, the same parameter was used as the dependent variable, whereas the weight, ADG, surgery order, and inflammation level were used as independent variables.If the independent variable (inflammation level) had a significant effect, a Spearman correlation was performed ('rcorr{Hmisc}') with the independent variable – possibility of ranking of Δ being positive or negative. Lastly, for ADG after the castration, bulls with social hierarchy rank with negative Δ were compared to positive Δ using a non-paired, two-tailed Wilcoxon test.Additional file 1:Table S1. Individual values for variables used in social net analyses for agonistic behavior (A), affiliative behavior (B) and hierarchy ranking, balance index (BI), testosterone, average daily weight gain (ADG), and scrotal circumference (C) before and after castration of Nelore Bulls. Table S2. Charge value, eigenvalue and variance of the principal component analyzes before and after castration of the Nelore bulls. In bold are charge values > 0.60 or < - 0.60.


Here, we announce the complete coding sequence of two strains of feline panleukopenia virus (FPLV) that were obtained from deceased domestic cats in animal shelters in Tennessee. The provided sequence data will contribute to a deeper comprehension of the genetic characteristics and evolutionary patterns of FPLV in the USA. Parvovirus genus, is a non-enveloped, single-stranded DNA virus that infects a wide range of animals beyond domestic cats, including raccoons, tigers, lions, and minks, making it a significant concern for both domestic and wild feline populations (–Feline panleukopenia virus (FPLV), belonging to the ulations –5. It calations –, 7. FPLVlations –.GAATGATAGGCGGTTTGTGTGT-3′) and reverse primer (5′-CCTACGCGGTCTGGTTGATT-3′) were designed to amplify the coding sequence of FPVL using Geneious Prime  cells  , following the manufacturer’s instructions. The amplicons were purified using GenCleanTurbo kit https://www.ncbi.nlm.nih.gov/orffinder/) was used to predict each ORF using default parameters.PCR amplicon libraries were prepared using the Nextera DNA Flex kit  following the manufacturer’s instructions. Two FPLV strains were sequenced using Illumina MiniSeq (150 bp × 2)  resulting in 621,221 and 604,347 high-quality reads for each VI2882 and VI2534 strains, respectively. The raw reads for each sequence were trimmed using BBDuk followed by mapping to reference by Geneious Prime version 2023.0.1. Default parameters were used for all software unless otherwise specified . The NatVP2 gene of VI 2882 and VI 2534 FPVL strains, along with feline parvovirus strains from various continents, was retrieved from the National Center for Biotechnology Information virus  contents for VI2882 and VI2534 were 36.2% and 35.9%, respectively. The full-length on virus . The seqon virus , 13. Phy20180621 . The str


SABA prescriptions (number of canisters per year) were defined as high (≥3) or low (1–2). Association of SABA prescriptions with HCRU was assessed by negative binominal model adjusted for covariates. The UK unit costs from the NHS were applied to estimate total healthcare costs (2020). Medication costs were based on the annual average number of canisters per year per patient.Overall, 186 061 patients with SABA prescriptions were included, of whom 51% were prescribed high SABA. Total annual average costs (HCRU and medication) were 52% higher in the high SABA group versus the low SABA group (£2 256 091 per 1000 patients/year versus £1 480 640 per 1000 patients/year). Medication costs accounted for the majority of asthma-related costs. Across both groups, most HCRU costs were for non–exacerbation-related primary care or hospital outpatient visits. The annual average HCRU cost difference for high SABA versus low SABA was the greatest for hospitalisations  and exacerbation-related primary care visits . Asthma-related HCRU extrapolated to the broader UK asthma population was £108.5 million per year higher with high SABA versus low SABA.High SABA versus low SABA prescriptions are associated with higher asthma-related HCRU costs.


Oropharyngeal squamous cell carcinoma (OPSCC) can be treated with definitive (chemo)radiotherapy ((C)RT) or primary surgical treatment (PST) with or without postoperative oncologic treatment. The prognosis of OPSCC does not essentially depend on the treatment modality, which allows to consider secondary decision-making aspects such as treatment costs when recommending an individual treatment modality. We attempted to analyze the costs associated with definitive (C)RT and PST in the treatment of OPSCC in Finland.We included 73 patients diagnosed with OPSCC at the Helsinki University Hospital (HUS)  in 2019 and 2020. Treatment costs were defined as the costs incurred in the specialized medical care during the first 12 months after the diagnosis was established.Definitive RT and definitive CRT were on a 1-year horizon associated with median costs of approximately 10 700€ and 13 300€, respectively; while, the median costs of PST equaled about 40 600€. The costs of definitive (C)RT mostly consisted of the costs of (chemo)radiotherapy sessions; while, the operating room costs and the costs of intensive care and stay on a ward drove the costs of PST.PST is associated with 2–3 times higher median costs than definitive (C)RT in Finland. The finding differs from the results previously reported in North America, which is related, e.g., to differences in the treatment practices as well as in the regulation of the health care systems. Head and neck cancers (HNCs) form the seventh most common cancer type globally . The incThe treatment of OPSCC can be based on either definitive (chemo)radiotherapy ((C)RT) or primary surgical treatment (PST) with or without postoperative (C)RT. The choice of treatment modality depends, e.g., on the T and N classes of the disease. A small and local carcinoma can be treated with a single modality; whereas, the use of a combined approach is applied along with higher T and N classes . The proPreviously, the treatment costs of OPSCC have been analyzed in some North American studies. Tam et al.  analyzedThe afore-mentioned results cannot be directly applied to other countries because of differences in healthcare systems and treatment practices. For instance, the use of TORS in the treatment of OPSCC is still unestablished in Finland. We, therefore, compared the costs of surgery and (C)RT in the management of OPSCC in Finland.n = 11) and the patients for whom data on the treatment costs were not available for a sufficiently long time period (n = 37), the final series consisted of 73 patients   in 2019 and 2020. After excluding the patients treated with a non-curative intent  was in the financial data divided between outpatient costs (40€) and outpatient procedure costs (139€). The treatment of OPSCC typically consisted of 30–35 radiotherapy sessions, indicating that the total costs of a radiotherapy period were approximately 5 400–6 300€.The operating room costs were generally not essential for patients receiving definitive RT and equaled zero for a median patient. For some patients, however, swallowing difficulties caused by radiotherapy established the need for percutaneous endoscopic gastrostomy. In addition, one patient in the sample underwent surgical resection of the primary tumor due to insufficient response to radiotherapy. Since operations and related inpatient periods were expensive compared to radiotherapy sessions, such operations performed on individual patients led the average total costs of definitive RT to be considerably higher than the median total costs of definitive RT.The costs of clinical physiology formed the third-largest cost category when examining median costs. The costs of PET CT scan, typically performed a few months after the last radiotherapy session, accounted for most of these costs. Imaging costs, e.g., ultrasound and MRI of the neck, formed less than 10% of the total costs of definitive RT.The median and average costs of definitive CRT are shown in Table The operating room and inpatient costs were mostly low for patients treated with definitive CRT. Most of the patients receiving definitive CRT did not undergo any operations as indicated by the median operating room costs of zero, although procedures such as dissection of the lymph nodes of the neck and percutaneous endoscopic gastrostomy were performed to some of the patients. The relatively long periods in the bed ward of some individual patients treated with definitive RT led the average inpatient costs to be lower for definitive CRT patients than for definitive RT patients.The costs of clinical physiology—essentially, the cost of the PET CT scan performed a few months after the treatment—was an important cost category in the group that received definitive CRT as well. The laboratory costs were higher for patients treated with definitive CRT than for those treated with definitive RT, reflecting the fact that use of cisplatin, the most commonly used chemotherapy drug for OPSCC patients in Finland , requireTable www.fshno.fi 2020). We compared the costs of surgical and oncological treatment and found the PST approach being 2–3 times more expensive than definitive oncologic treatment. Since PST and definitive RT are considered alternative treatment options especially for OPSCC patients with a small and local disease [Definitive oncologic treatment has during the past decades become more common than primary surgical treatment (PST) both in the U.S.  and in F disease , it is nThe present findings are inconsistent with previously published North American studies , 9, 14, Compared to previous studies, the partly different treatment practices of OPSCC also contribute to the opposite results. In the studies by Tam et al. , de AlmeIn this study, the patient groups treated with alternative methods are not stage matched, meaning that this study rather attempts to describe the costs related to the treatment modalities, given the fact that different patients are treated differently, than to estimate how the costs related to modalities would compare to each other if the patient groups were comparable as in the study by Tam et al. . The facThe relative affordability of definitive RT and definitive CRT found in this study forms only one aspect in the decision-making in OPSCC treatment planning. While the choice of treatment modality must be primarily based on expectations on its effectiveness as well as on the ability of the patient to tolerate the treatment and potential adverse effects, the impacts on later quality of life must be taken into consideration. In England, for example, the National Institute for Health and Care Excellence has defined society’s willingness to pay per one quality-adjusted life year to be about £20 000–£30 000. Therefore, it could make economic sense to prefer more expensive treatment modalities if they were associated with significantly higher future quality of life. Unfortunately, the data applied in this study do not allow analysis of the impacts of the treatments on the prognosis and future quality of life of the patients.There are many potential ways to further expand the analysis on the treatment costs of OPSCC. This study follows the approach adopted by Tam et al.  by consiWhile the treatment of OPSCC can be based on either definitive (chemo)radiotherapy or primary surgical treatment, the prognosis of OPSCC is not significantly impacted by the chosen treatment modality. This enables to consider secondary factors, including treatment costs, when deciding on the treatment modality. This study shows that PST—with median total costs of 40 600€—is in Finland significantly more expensive than definitive RT and definitive CRT, the median total costs of which are 10 700€ and 13 300€, respectively. The present results differ from those previously reported in North American studies, which can be explained, e.g., by differences in the regulation of the health care systems as well as differences in the treatment practices.


We estimated healthcare resource utilization (HRU) and costs in patients with generalized myasthenia gravis (gMG) in Taiwan.This retrospective population-based, matched cohort study used the National Health Insurance Research Database to identify prevalent patients with gMG (cases) in 2019. In total, 2537 cases were matched (1:4) by age, sex, and urbanization level to 10148 randomly selected patients without gMG (comparators). A generalized linear regression model predicted the frequency of HRU and costs among service users. Costs attributable to gMG were obtained by subtracting all-cause HRU costs incurred by comparators from cases.p < 0.0001 for all). The mean (standard deviation) difference in all-cause healthcare costs between cases and comparators was NT$ 94997 (76431) [US$ 3133 (2521)], and was significantly higher for all categories . Among employed persons, 13.18%/7.59% of cases/comparators changed employment status during the study (p < 0.0001).The mean age of all patients was 54.99 years and 55.97% were female. Compared with comparators, cases had significantly higher rates of hypertension (33.03%/24.26%), diabetes mellitus (18.92%/11.37%), malignancies (16.00%/4.08%), cardiovascular disease (11.35%/8.12%), thyroid-related conditions (5.99%/1.16%), respiratory illness/disorders (4.38%/1.22%), and neurotic disorders (4.65%/2.6%). Amongst users of healthcare resources, cases had a mean 10 additional outpatient visits, 0.62 inpatient stays, and 0.49 emergency room visits in 2019 compared with comparators (gMG presents a substantial burden on HRU and healthcare costs in Taiwan. A high attrition rate from full-time employment suggests additional societal costs. Improved treatments are needed to alleviate the burden of disease on individuals, healthcare systems, and economies. PatientgMG requires life-long treatment with individualized combinations of anti-cholinesterase inhibitors, thymectomy when indicated, immunosuppression using corticosteroids and steroid-sparing agents such as azathioprine, mycophenolate mofetil, cyclosporine, cyclophosphamide, or methotrexate . PatientA systematic review of the literature published until 2020 found that information about the costs associated with the treatment of MG was sparse, and that health resource utilization (HRU) and associated costs varied markedly between and within countries . Key driWe previously reported the epidemiology of gMG in Taiwan using the population-based Taiwan National Healthcare Insurance Research Database (NHIRD). Between 2009 until 2019, the prevalence of patients with gMG in Taiwan increased significantly, from 6.83 per 100000 population to 11.18 per 100000 population (submitted), with an increasing proportion of older adults with gMG. We observed that the epidemiology of gMG in Taiwan is evolving rapidly, pointing to a growing burden of disease with associated increases in HRU and healthcare costs. In parallel, new treatments for gMG are becoming available, and information about HRU and healthcare costs will be needed to inform health technology assessments. In this study, we estimated HRU and costs associated with gMG by conducting a population-based study using the NHIRD. The costs associated with gMG were assessed by comparing outcomes in patients with and without gMG.2.2.1.Taiwan’s National Health Insurance (NHI) program provides compulsory health insurance to 99.9% of the Taiwanese population of approximately 24 million  in the NHIRD in 2019. Patients with gMG were identified as having an inpatient claim with MG as the main or sub-diagnosis under any discipline ; and a prescription for azathioprine or at least one treatment period of steroids at a minimum dose of 20 mg prednisolone  daily for 28 days. Patients with gMG who died during 2019 were excluded from the analysis.A comparator cohort of patients without gMG was identified by randomly selecting individuals with no claim for a diagnosis of MG during 2019 in the database. Four comparators were matched to each case by age, sex, and residential urbanization level . Costs w2.3.The Charlson comorbidity index (CCI) was used for evaluating comorbidities using ICD-10 codes.All-cause HRU and costs were captured for cases and comparators during 2019. Costs associated with outpatient, inpatient, and emergency department (ED) services were evaluated. Costs were further broken down by medication and non-medication costs. Non-medication costs included all fees for physician consultations, inpatient episodes, diagnostic and laboratory tests, procedures, and surgery. An estimate of the HRU and costs attributable to gMG was obtained by subtracting the all-cause HRU and costs incurred by patients with gMG from all-cause HRU and costs incurred by their matched comparators.Costs are presented in New Taiwan dollars (NT$). In early 2023, 100 NT$ converts to around 3.0 Euros and 3.3 USD.2.4.t-tests were applied to assess potential differences between cases and comparators for categorical and continuous variables, respectively.Continuous variables were described using means, standard deviations (SD), medians, and interquartile ranges (IQR). Categorical variables were described using frequencies and percentages. chi-square and Raw mean all-cause HRU and costs in 2019 were reported for patients with gMG and comparators. To account for the potential over-representation of zeros and right-skewed distribution of HRU and cost data, a two-part model was performed: a logistic regression to predict the probability of service use and a generalized linear model to predict the frequency of HRU and costs among those with positive frequency of service. Covariates entered into the models to predict HRU and costs included age, sex, urbanization index, and comorbidities.Cases/comparators who had a change in employment status during 2019, either becoming unemployed and dependent on their children/relatives, or who moved into a lower income employment insurance category, were identified. Cases/comparators who turned 65 during 2019 (the age of retirement from the NHI) were excluded from this analysis.All statistical analyses were performed using SAS Version 9.4 .3.There were 2537 prevalent patients (cases) with gMG in the NHIRD in 2019 matched to 10148 comparators . The mea3.1.The majority of cases and comparators (99.92% and 94.79%) used outpatient services in 2019. Inpatient and ED episodes were recorded for 44.42% and 37.33% of cases, respectively, and 10.68% and 17.75% of comparators . Among up < 0.0001 for all comparisons)  .3.2.The total mean all-cause cost of healthcare in cases who used healthcare resources in 2019 was NT$ 135219.23 (SD 224174.16) , versus NT$ 40904.12 (SD 106625.26) (US$ 1347.82 SD 3513.38) in comparators . Costs op < 0.0001) . The mean cost of inpatient stays in 2019 was NT$ 54001 (SD 29982)  more for patients with gMG than comparators. Outpatient visits cost NT$ 21422 (SD 29055)  more, and drug treatments cost NT$ 16887 (SD 20363)  more for cases than comparators.In the regression analysis, the mean difference in healthcare costs between cases and comparators in 2019 was NT$ 94997 (SD 76431)  ( 0.0001) . The dif3.3.p < 0.0001)  became either unemployed (dependents) or transferred to a lower income level insurance category, compared with 266 out of 3506 (7.59%) comparators ( 0.0001) . Women w4.We used the NHIRD to identify HRU and costs associated with gMG in 2019. Over the 12 months study period, HRU and associated healthcare costs were significantly higher in cases than a cohort of matched comparators without gMG. While this difference was mainly driven by inpatient costs, outpatient episodes and drug treatments were also substantially higher in cases than comparators, reflecting the chronicity of the disease, the need for life-long treatment, and the costs associated with management of acute episodes such as myasthenic crisis.We found that over a 12 months period, 13% of employed patients with gMG changed their employment status, of whom 6% became unemployed and classified as dependents in the NHI. This high rate of attrition from the workforce suggests a substantial burden of disease in some patients that negatively impacts their ability to work. Women were impacted more than men, and became unemployed at twice the rate of men.Few studies have assessed the costs associated with MG in Asia . In one A study in China reported that, in patients hospitalized for MG, the median length of hospital stay was 8 days (IQR 4–15) at a cost of approximately USD$ 1037 (IQR 493–2925). For patients admitted to hospital for myasthenic crisis, the median length of hospital stay was 14–15 days, at a cost of approximately USD$ 3521 .The costs associated with the inpatient management of MG in the United States increased by 13-fold between 2003 and 2013, attributed in part to a new requirement for IVIg to be administered as an inpatient, but mainly to changes in physician decision-making toward admission . In anotThe available evidence suggests that the costs associated with the management of MG vary widely according to physician thresholds for hospitalization and accepted (or insured) medical practices such as administration of IVIg at home, as an outpatient, or as an inpatient . The extThe strengths of our study are the use of the population-based NHIRD that allows comprehensive data capture, including employment status, across the entire population, and the use of recent data that provide a contemporary picture of the healthcare burden associated with gMG in Taiwan. Potential limitations are the absence of a specific ICD code for gMG, which we accounted for by using a combination of hospitalization and treatment criteria. The absence of clinical information in the claims database prevented the identification of patient subgroups which would have allowed a more nuanced understanding of the HRU associated with gMG. Patients with milder forms of gMG not requiring hospitalization in 2010 were not captured in our analysis, suggesting that our results may underestimate the true cost of gMG in Taiwan. We were unable to assess indirect healthcare costs but used change in employment status as an indicator of reduced quality of life.In conclusion, our study shows that gMG presents a substantial burden on HRU and healthcare costs in Taiwan that exceeds that in individuals without gMG. A high attrition rate from full-time employment suggests additional societal and economic costs that my go unaccounted for in health technology assessments. Improved treatment for MG is needed to alleviate the burden of disease on individuals, healthcare systems, and economies.http://dep.mohw.gov.tw/DOS/np-2497-113.html).Publicly available datasets were analyzed in this study. This data can be found here: the data underlying this study are from the NHIRD which has been transferred to the HWDC. The Taiwan government prohibits release of the NHI claims dataset to the public domain. Interested researchers can obtain the data through formal application to the HWDC, Department of Statistics, Ministry of Health and Welfare, Taiwan (Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.S-PS: software, formal analysis, data curation, visualisation, and writing—review and editing. KH: project administration and writing—review and editing. YL: conceptualisation, methodology, supervision, and writing—review and editing. C-CY: writing—review and editing. C-HT: study conceptualisation, methodology, data curation, formal analysis, and writing—review and editing. All authors contributed to the article and approved the submitted version.This work was supported by Janssen Asia Pacific, a division of Johnson & Johnson Pte Ltd.KH is an employee of Janssen Asia Pacific and YL is an employee of Epidemiology, Office of the Chief Medical Officer, Johnson & Johnson. KH and YL hold stock in Johnson & Johnson.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.