The generation of these predictive equations represents a step towards updating the ME:DE default conversion factor value of 0.82 adopted from the National Research Council to meet the ME requirements of beef cattle in Korea. The new recommended predictive equation enables the adjustment of the nutrient requirements, thus enhancing animal productivity and maximising the economic return for beef farmers.The available energy in feedstuff represents the largest proportion of the total cost for intensive beef production. Therefore, the energy content of feeds must be known before diet formulation. The determination of digestible energy (DE) and metabolizable energy (ME) values by animal experiments is both time-consuming and costly. Predictive equations to estimate the ME from DE can be useful for feed ingredient evaluations and diet formulations. A range of regression equations were developed in the present study, taking into consideration the gender and body weights of the animals, as well as the feed nutrients, to predict the relationship between the DE and ME. An evaluation of these equations suggested predicting the ME value based on ME = 0.9215 × DE − 0.1434 (R2) = 0.946, root mean square error (RMSE) = 0.107, p < 0.001 for intercept and slope). Mixed-model regression analyses to adjust for the effects of the experiment from which the data were obtained similarly showed a strong linear relationship between the DE and ME : ME = 0.9215 × DE − 0.1434 . The DE was strongly related to the ME for both genders: ME = 0.8621 × DE + 0.0808  and ME = 0.7785 × DE + 0.1546  for male and female Hanwoo cattle, respectively. By BW, the simple linear regression similarly showed a strong relationship between the DE and ME for Hanwoo above and below 350 kg BW: ME = 0.9833 × DE − 0.2760  and ME = 0.72975 × DE + 0.38744 , respectively. A multiple regression using the DE and dietary factors as independent variables did not improve the accuracy of the ME prediction .This study was performed to update and generate prediction equations for converting digestible energy (DE) to metabolizable energy (ME) for Korean Hanwoo beef cattle, taking into consideration the gender  and body weights (BW above and below 350 kg) of the animals. The data consisted of 141 measurements from respiratory chambers with a wide range of diets and energy intake levels. A simple linear regression of the overall unadjusted data suggested a strong relationship between the DE and ME : ME = 0.8722 × DE + 0.0016 (coefficient of determination (R Energy is a vital component for biological reactions and an important nutrient to meet the requirements for the maintenance, growth and reproduction of beef cattle. The energy requirements depend mainly on age, gender, body weight (BW), animal genotype, physiological state and environment . To meetn = 87 means) from different cattle breeds. Scarce calorimetric studies have been conducted in Korea to evaluate the efficiency of converting the DE to ME of the Korean native beef cattle (Hanwoo) [Hanwoo is a cattle breed native to the Republic of Korea and is one of the most economically important domestic animals in the country. The Hanwoo beef industry has experienced a considerable change between 2005 and 2017, with the total number of cattle increasing from 1.8 to 2.6 million head and an improvement of Hanwoo cattle performance in terms of the quantity and quality. The average carcass weight of Hanwoo steers increased from 343 to 437 kg and the marbling score improved from 3.6 to 5.6 within the same period, andthis trend affected the basal metabolism of the animals. Hence, Hanwoo beef cattle tended to have higher metabolic rates and require more energy for maintenance and production. Currently, farmers rely more on the use of high energy-based concentrate diets imported mainly from international markets . Vermore(Hanwoo) . Kim et (Hanwoo)  estimateTherefore, we hypothesised that Hanwoo beef cattle have been fed more digestible diets with different ME:DE conversion factors compared to the NRC conversion factor. Therefore, there is an urgent need to update this conversion factor according to the Korean context (diet and genotype). Hence, the aim of this study was to analyse the relationship between the DE and ME of typical diets for Korean Hanwoo beef cattle, taking into consideration the gender  and BW (above and below 350 kg) of the animals.The data used for the present work were from published research papers and reports. Therefore, the requirement for animal care and use committee approval was waived, because no animals were used in this study.n = 36) [n = 54) [n = 20) [n = 31) [Statistical evaluation of the relationship between DE and ME was performed using published data. The data included individual treatments from three different studies published in Korean Journals and national reports from 1983 to 2013. Data were assembled in a dataset of 141 individual animal measurements from 35 respiration calorimetry experiments with a wide range of diets and energy intake levels. In addition, data were generated from experimental studies carried out in the National Institute of Animal Science (NIAS), Republic of Korea (average data are presented in n = 36) ,11,12, f[n = 31) ,14 and hThe mixed model methods as described by Littell et al.  were usep-value of 0.05 to remain in the model. The correlation coefficients between the chemical components  and energy contents  of the dataset treatments were analysed using the PROC CORR procedure.Similar to Galyean et al. , citatio2) and root mean square error (RMSE) were determined for all the models. The R2 is an indicator that defines the best-fit equations, and the RMSE is an indicator of the model accuracy [The coefficient of determination : intercept  and slope ), and the linear regression equation was: The results of the linear regression analyses of the relationship between ME as a dependent variable and DE as an independent variable using the whole data (unadjusted and adjusted for random coefficients) are graphically represented in 2 value (R2 = 0.9999 and RMSE = 0.00403), in which the ME and DE were expressed in Mcal/kg of the DM. The 95% CI for the slope and intercept were  and , respectively. The adjusted linear regression equation was: The scatterplot of the adjusted citations for random coefficients presented in The ME:DE was about 0.87 at 2.80 Mcal/kg of the DE (approximate mean of the whole dataset).2 values of 0.9600 and 0.9718 for male (n = 90) and female (n = 51) beef cattle, respectively. The adjusted citation showed increases in the precision of the prediction equations for both male and female cattle, with R2 of 0.9929 and 0.9999, respectively. The steers showed a higher ME:DE (0.89) compared to female cattle (0.83). By BW, the unadjusted data showed a strong positive correlation between the DE and ME, where the R2 of the generated equations were 0.9907 and 0.9139 for animals with BW < 350 kg and >350 kg, respectively. The adjusted data improved the R2 of the ME prediction equations to 0.9998 and 0.9999, respectively. In addition, the results showed a difference in the ME:DE between animals with BW below and above 350 kg . The unadjusted and adjusted linear regression equations expressing the DE and ME relations for animals by gender and BW are shown in The unadjusted DE and ME relationship between the gender  and BW (<350 kg and >350 kg) are plotted in p < 0.001) with the NDF and ADF dietary contents. In addition, the ME showed strong positive correlations with the DE, EE and CP dietary contents. Specific dietary nutrients that modify the ruminal fermentation may affect the relationship between the DE and ME. The results of the residual analyses (n = 141) of the predicted ME using a simple linear regression with EE, NDF and ADF are presented in p < 0.001) of the EE (r = 0.67), NDF (r = –0.87) and ADF (r = –0.79) with the ME prediction errors, suggesting that the ME prediction will probably be biased by these dietary factors, and their inclusion could improve the precision of the predictive equations.The Pearson correlation coefficients (r) of the various chemical compositions  to the GE, DE and ME contents of the dataset treatments are presented in p < 0.001) for predicting the ME. However, the EE concentration in the diet as an independent factor was not significant (p > 0.05)  . Therefo2 = 0.9621); the intercept and slope coefficients in the model were significant at p < 0.001 , DE , CP , NDF  and ADF ).The DE and ME were expressed in Mcal/kg of the DM, and the CP, NDF and ADF were expressed as percentages of the DM (R2 = 0.9995) than the NRC equation . In addition, Equation (2) was more accurate and had a lower RSME (0.0059) than the NRC equation (RSME = 0.0079).This study highlighted the importance of updating the NRC equation used for converting the DE to ME in the Hanwoo beef industry. The data analysed represented a broad range in terms of animal age ; BW and diet type (forage and concentrate feeds) of Hanwoo beef cattle. This study showed that the ME:DE is higher than the fixed NRC conversion factor (0.82). This equation seems to not be adequate for Hanwoo beef cattle, which led to overfeeding animals to meet their energy requirements. Our DE and ME values were similar to those reported by Galyean and Tedeschi  for seveWe found a strong relationship between the DE and ME for gender and BW. This relationship was similar to that of Galyean et al. , who revThe Agriculture and Food Research Council (AFRC)  suggestsWe observed differences in energy utilisation according to gender. This difference may reflect differences in the energy costs of the maintenance and production of beef cattle of different physiological status and productivity. Our ME:DE were 0.89 in males and 0.83 in females. The steers were more efficient in converting DE to ME compared to female cattle because of the specific feeding program for steers where the animals received high metabolic diets, in particular, to produce high fat depositions in the intramuscular tissues. Steers were fed diets with a forage-to-concentrate ratio ranging from 40:60 in the growing phase (7–13 months of age) to 30:70 in the early fattening phase (14–21 months of age) and 10:90 in the late fattening phase (22–30 months of age) ,21. Furtp < 0.01) as the forage-to-concentrate ratio decreased, 0.86 to 0.92.Moreover, the BW is another key driver determining the relationship between the DE and ME in the feeding management of Hanwoo cattle. A BW of 350 kg at around 13 months of age is the typical switching point from the growing to the fattening phase for Hanwoo beef cattle in Korea , and thep < 0.001), initial analyses using stepwise regression indicated that only the CP, NDF and ADF concentrations in the diet were significantly independent variables, along with the DE (p < 0.001), for predicting the dietary ME (Equation (9)). This study did not consider the data points of the starch content, as some trials did not report the starch content in the diet. Moreover, the results showed that the inclusion of dietary factors did not improve the accuracy of the prediction of the ME (R2 = 0.9621) compared to the estimation of the ME using a single-variable (DE) regression approach (R2 = 0.9999).Galyean et al.  reported2 > 0.9000). However, Equation (2) gave a higher prediction precision in terms of the R2 (0.9995) compared to the NRC (2000) equation (R2 = 0.9461). Therefore, Equation (2) has higher predictive power than the previously published ME:DE [A comparative slaughter studydetermined the net energy requirements for the growth of Hanwoo steers from 6 to 30 months of age . Eight sed ME:DE , the fixThis study highlighted the importance of updating the relationship between the DE and ME recommended by the NRC using Hanwoo beef cattle data. Our results strongly suggest that the use of a constant value of 0.82 to convert the dietary concentration of DE into ME for beef cattle diets should be replaced with a new model that considers the national specificity of animal performances and the nature of the diet energy contents. Therefore, it is recommended to adopt the newly generated equation (ME = 0.9215 × DE − 0.1434) for Hanwoo beef cattle instead of the NRC ME:DE constant conversion factor of 0.82. Our results will be useful for future studies that aim to update the energy requirements for the maintenance and growth of beef cattle in Korea. Future research is warranted to re-evaluate the ME:DE conversion factor of cattle in other regions and continents according to the specificity of the breed and feed quality in order to understand the underlying mechanisms that affect the conversion of DE to ME.


Compared to warfarin, all reduced-dose NOACs showed significantly lower risk of S/SE , 0.63 (0.52–0.75) for apixaban; 0.51 (0.42–0.61) for dabigatran; and 0.67 (0.57–0.79) for rivaroxaban) and MB (0.54 (0.45–0.65) for apixaban; 0.58 (0.49–0.69) for dabigatran; 0.73 (0.63–0.85) for rivaroxaban). In the real-world practice among Asians with NVAF, all reduced-dose NOACs were associated with a significantly lower risk of S/SE and MB compared to those of warfarin.Reduced-dose nonvitamin K antagonist oral anticoagulants (NOACs) are commonly prescribed to Asian patients with nonvalvular atrial fibrillation (NVAF). We aimed to compare the risk of stroke/systemic embolism (S/SE) and major bleeding (MB) between patients treated with reduced-dose NOACs and those treated with warfarin, using the claims database in Korea. Patients with NVAF newly initiated on oral anticoagulants  between 1 July 2015 and 30 November 2016 were included. Among all patients with NVAF treated with OACs, 5249, 6033, 7602, and 8648 patients were treated with reduced-dose apixaban, dabigatran, rivaroxaban, and warfarin, respectively. Patients treated with reduced-dose NOACs were older and had higher CHA Oral anticoagulants (OACs) have taken a crucial role in the treatment strategy of atrial fibrillation (AF) to prevent stroke ,2,3. AltNonvitamin K OACs (NOACs), now recommended as the choice of treatment, are widely used based on evidence from recent large randomized controlled trials (RCTs), which have demonstrated their noninferiority and superior efficacy/tolerability compared with those of warfarin ,13,14,15Therefore, this study aimed to compare the risk of stroke/systemic embolism (S/SE) and major bleeding (MB) in Korean patients treated with reduced-dose NOACs versus warfarin under real clinical practice.The Korean Health Insurance Review and Assessment (HIRA) Service claims data from 1 January 2007 to 30 November 2016 were used in this study. The HIRA database includes data covering the entire population under the universal health insurance system in Korea [2DS2-VASc score of 2 or higher, all included patients are considered indicated for anticoagulation therapy.The study included OAC-naïve patients who had one or more prescriptions for apixaban, dabigatran, or rivaroxaban during the intake period of 1 July 2015–30 November 2016. We defined the index date as the first OAC prescription date. The inclusion criteria were patients above 18 years of age on the index date and a minimum of two outpatient visits or one hospitalization with AF diagnosis  before or on the index date. The latter inclusion criterion performed well in a previous validation study with a positive predictive value of 94.1% . As the Patients with the following conditions were excluded: hip or knee replacement surgery within six weeks prior to or on the index date; valvular AF, prosthetic heart valves, venous thromboembolism, thyrotoxicosis, hypertrophic cardiomyopathy, end-stage chronic kidney disease, kidney transplant, dialysis, pericarditis, elective defibrillation, radiofrequency ablation, or left atrial appendage occlusion during the 12-month baseline period; NOAC or warfarin use within 1 year prior to the index date; more than one OAC prescription on the index date; both standard and reduced dose on the index date; both standard and other dose on the index date; or both reduced and other dose on the index date.Standard dose was defined as the general recommended dose for patients with AF as specified in the package insert . Reduced dose was defined as the recommended dose for patients who have renal dysfunction and/or low body weight or are elderly as specified in the package insert . Other dose was defined as a dose lower than the reduced dose on label or higher than the standard dose .The follow-up period was from the index date to the date of switching from index OAC treatment to another OAC, treatment discontinuation, death, or 30 November 2016, whichever came first. Discontinuation was defined as the absence of prescription for any OACs within 30 days after the last day of supply of the last filled prescription. Medication switch was defined as the presence of a prescription filled for nonindex OAC treatment within 30 days after the last day of supply of the last filled prescription.S/SE was the effectiveness outcome and included ischemic stroke, hemorrhagic stroke, and systemic embolism. Diagnosis codes with hospitalization and brain computed tomography (CT) or magnetic resonance imaging (MRI) records were used to identify stroke ,23, wher2DS2-VASc score, HAS-BLED score, and individual risk factors of CHA2DS2-VASc and HAS-BLED scores. The details on CHA2DS2-VASc and HAS-BLED scores, baseline medication use, and CCI are provided in Propensity score matching (PSM) using propensity scores was conducted to balance baseline demographics and clinical characteristics. Propensity score is the probability of receiving treatment, which is based on the observed characteristics . We usedFor analysis, the following three comparisons were performed: (1) reduced-dose apixaban versus warfarin, (2) reduced-dose dabigatran versus warfarin, and (3) reduced-dose rivaroxaban versus warfarin. All statistical analyses were performed using SAS 9.4 .2DS2-VASc and HAS-BLED scores compared to those of patients treated with warfarin , 87% of patients were prescribed on-label NOAC doses; the rates of underdosing and overdosing were 9.4% and 3.4%, respectively [In a US prospective registry for patients with AF receiving NOACs , this study’s results provide additional information on the benefit of NOACs over warfarin in Asian patients requiring anticoagulation for stroke prevention.Although this study could not differentiate between the appropriate and inappropriate use of reduced-dose NOACs, the study findings revealed the clinically significant data on reduced-dose NOACs that may provide benefits in real-world practice compared to warfarin therapy. However, caution is warranted in its interpretation and use of reduced-dose NOACs should be strictly based on prescribing labels. This finding suggests that NOACs, regardless of dosage, may provide better clinical outcomes in patients with AF for whom warfarin is indicated. Poor INR control and low treatment satisfaction of VKA users among Korean patients with AF might be one of the potential underlying reasons for the observed differences in outcomes between warfarin and reduced-dose NOACs in this study . In anotThis study has some inherent limitations associated with the retrospective analysis of claims data. First, the HIRA database does not include data on some patient characteristics such as body weight or laboratory data, including INR and those related to renal and liver function. Therefore, assessing whether patients were managed with appropriate doses of anticoagulants was challenging. In addition, the adequacy of anticoagulation in patients in the warfarin group could not be determined due to lack of information on INR, which may affect the clinical interpretation of the study results, as Asian AF patients tend to be kept at a lower INR range compared to that of non-Asians. Second, the claims database that we utilized in this study cannot discern the specific type of AF . Third, despite the careful adjustment of confounders by PSM, thereby allowing a close balance between the groups, the presence of unmeasured confounding factors cannot be ruled out. Finally, the study follow-up period was relatively short as reimbursement of NOACs as first-line therapy was initiated on 1 July 2015. Therefore, further studies are necessary to explore the long-term effectiveness and safety of NOACs. However, the Cox proportional hazards model revealed significant differences in all comparisons; therefore, the relatively short follow-up period might not have had a significant impact on the main findings of this study.In a large observational study of reduced-dose NOACs, NOACs were associated with lower thromboembolic and bleeding risk compared to those of warfarin. Nevertheless, the use of NOACs should adhere to dose-reduction criteria indicated in the therapeutic label. This study provides further evidence for the use of NOACs in Asian patients with NVAF.


The titer of the anti-SARS-CoV-2 antibodies produced after vaccination shows a relevant decay over time, as demonstrated in several studies. However, less is known on the possible factors affecting the entity of this decay. The aim of this study is to analyze a group of individual factors which are possibly associated with anti-SARS-CoV-2 antibody titer decay six months after the second vaccine dose. We report here the results of a follow-up serological analysis and a questionnaire-based evaluation of a sample of workers from an Italian nursing home, vaccinated with two doses of BNT162b2 vaccine in early 2021. The baseline data were collected one month after the vaccine, while in the present analysis we report the data collected six months later. Our data show a relevant decay of the neutralizing antibody titer, even if for all the workers a largely positive response was detected. Moreover, our results demonstrate a possible association between younger age and the absence of previous COVID-19 infection, and a higher decay rate of the anti-SARS-CoV-2 antibodies titer. The decrease in the protection against SARS-CoV-2 new infections and re-infections after a few months from the second vaccine dose has highlighted the need for a third booster dose. The administration of this further anti-COVID-19 vaccine dose began in European and North American Countries in the second half of the year 2021 ,4. CurreAccording to these premises, the objective of this work is to evaluate a series of individual factors which are possibly associated with a significant decrease of the anti-RBD IgG antibody titer in a sample of healthcare workers (HCW) six months after the second dose of the COVID-19 BNT162b2 vaccine.We performed a new evaluation of a previously described group of workers employed in a nursing home in northern Italy, who received two doses of the anti-SARS-CoV-2 BNT162b2 vaccine in early 2021 . The nat-being an employee of the nursing home at work during the period January–August 2021;-being vaccinated with two doses of the Pfizer/BioNTech BNT162b2 anti-SARS-CoV-2 mRna vaccine between 12 January and 17 February of 2021 ;-having participated to our first baseline investigation, with an available anti-SARS-CoV-2 IgG antibody titer determined via a serological test performed in our lab.The inclusion criteria of this follow-up study were:No other exclusion criteria were applied.We did not only consider healthcare operators for our analysis, but also included other workers employed in the nursing home such as administrative personnel, cleaning service and kitchen staff and maintenance workers. Healthcare workers (HCW) were categorized as follows: nurses, nurses’ assistants, physicians and other HCW . All the personnel voluntarily agreed to participating in our study, and signed a written informed consent. The study was approved by our local institutional review board.As reported in our prior publication , four weDuring the baseline investigation we collected a self-reported questionnaire  Supplem and a set test, and one-way analysis of variance (ANOVA) with the Bonferroni test were applied whenever necessary. Mann–Whitney test was used to compare the percentage of anti-SARS-CoV-2 neutralizing IgG titer decline between two subgroups and Kruskal–Wallis test for the comparison of three or more subgroups. Multivariate linear regression analysis was performed to determine whether the participants’ characteristics  influenced anti-SARS-CoV-2 neutralizing IgG titer decline six months after the vaccination. Data collected were analyzed using STATA Software . Regarding statistical significance, a p value < 0.05 was considered.Logarithmic transformation was used to normalize anti-SARS-CoV-2 neutralizing IgG and the results are expressed as median and ranges. The chi-square test, paired n = 28), 20.6% by other HCWs (n = 13), 14.3% by kitchen and cleaning personnel (n = 9) and 20.6% by technical and administrative personnel (n = 13).A total of 63 out of 74 employees of the nursing home (85.1%) investigated in our first study agreed to participate in the follow-up and gave a further blood sample for the determination of the anti-SARS-CoV-2 IgG titer. Workers were for the 79.4% females, with a median age of 54 years old (range: 25–73). Regarding job type, 44.5% of the sample was composed of nurses, nurses’ assistants and physicians (p < 0.001), also considering the subjects grouped according to the different variables investigated  , while after six months the titer was 876 (156–6034). For all the subjects, a decrease of the titer was observed and the difference between the first and the second determination was highly significant (iseases) . In Figuiseases) .In Overall, the decline of the antibody titer reached 83%, slightly higher in males compared to females (84.5 vs. 82%), even if the difference is not statistically significant .p = 0.04, p = 0.001).We observed a significant difference evaluating the decay of the titer with respect to age-classes: in the younger group, the highest decrease of the titer was observed , while in the older group the decrease of 78% is lower compared to the other age-classes (p = 0.085) (p = 0.017), as well as for those with no previous COVID-19 infection (p = 0.016) (p = 0.024), while for previous COVID-19 the coefficient was −2.57 .No significant differences in the decrease of neutralizing antibodies’ titer were observed according to BMI and smoking habit, and, as expected, according to job type . A lower= 0.085) . A signi= 0.016) . Using mConsidering the number of concomitant diseases reported, no differences in the reduction of the titer were identified when comparing workers with no diseases vs. personnel with 1 or ≥2 pathologies .Our results are a further confirmation of the relevant decline of the anti-SARS-CoV-2 IgG titer six months after the second dose of anti-COVID-19 vaccination ,4,5,6. NThe second objective of this work was to identify possible factors associated with different decay rates of anti-SARS-CoV-2 IgG titer six months after the vaccines. It should be underlined that, considering our data, the most relevant decline was observed in those subjects who had a higher antibody titer at the baseline, confirming recent data . This isWith regards to the role of previous SARS-CoV-2 infection, it is well documented that HCW who had a SARS-CoV-2 infection plus a vaccination reach higher anti-SARS-CoV-2 antibody titer one month after the vaccination when compared to subjects with no infection history, and our results are in agreement with these findings ,24,28. MConsidering recognized risk factors for COVIID-19 as high BMI, smoking status and the number of concomitant diseases , we did Among the main limitations of our study there is the small sample size and the observational nature of the research, analyzing self-reported data collected with a survey. Among all the variables analyzed, the self-reporting may have been particularly critical for the variables related to the medical anamnesis of the subjects, including the data on anti-flu vaccination and those on concomitant diseases. This data would be better captured with more objective methods, based on the direct collection of clinical data by health professionals. Regarding the diseases, we tried to overcome this limitation by performing a blind and rigorous ex-post coding of the answers to the open questions given by the respondents. Moreover, we tested various approaches, categorizing the diseases in different ways, considering the specific types of pathologies, finally keeping the most reliable variable we used in the multivariate analysis . Regarding the anti-flu vaccine, in this case the possibility of further cleaning and testing the self-reported data in different ways was not reliable, as we had only one yes/no question based on the survey administered. For this reason, the finding related to anti-influenza vaccine must be interpreted with extra caution, and has to be confirmed with an additional analysis of larger samples, possibly including more objective data on the performance of the vaccine. Our study may have also technical limitations, referred in particular to the determination of the anti-SARS-CoV-2 antibody titers and to the assay performed, including the possible presence of different immunoglobulins’ isotopes and their relations with the total level of circulating antibodies. Moreover, other limitations of our study are intrinsically related to its nature and to the source population, which is selected from a small nursing home located in northern Italy, where the spread of SARS-CoV-2 was particularly relevant compared to other areas of Italia and the world during the first few months of the pandemic. Furthermore, it should be taken into consideration that this is a professionally exposed population, with a significantly higher exposure to SARS-CoV-2 compared to the general public: this peculiarity, together with the already mentioned issue of the small sample size, makes the extrapolation of our findings to a wider public not possible. Finally, the limitations intrinsically related to the variability over time of the virus, and in particular of SARS-CoV-2, should also be mentioned here: the changes of the virus, considering the quite long duration of our study, may have influenced the antibodies’ titer of our population, with different individual responses based on the virus variant and viral load exposure.Despite these limitations, our study has also important strengths, as almost all the subjects investigated one month after the vaccination agreed to participate again six months later, filling in the same questionnaire administered the first time and being evaluated for the anti-SARS-CoV-2 with the same test applied for the first serological determination. Moreover, all the subjects had been vaccinated with two doses of the same anti-COVID-19 vaccine, indicating that the possible differences in the decline of the antibodies’ titers, identified across sub-groups, cannot be related to different vaccines used or different number of doses administered.Our study in a sample of Italian nursing home workers shows a relevant decay of anti-SARS-CoV-2 neutralizing antibodies six months after the second BNT162b2 vaccine dose. A largely positive antibody response was detected in the totality of the sample. We found that the absence of previous COVID-19 infection and a younger age were significantly associated with a higher decay of the antibody titer.


Objectives: This is a longitudinal prospective study which was designed to assess the trend of anti-SARS-CoV-2 antibodies targeting the Spike (anti-S) and Nucleocapside protein (anti-N) viral antigens over a 9-month period after the administration of an anti-SARS-CoV-2 vaccine in a big COVID-19 hospital located in Northern Italy. Participants: 7411 vaccinated workers were included in a linear mixed-effect model analysis performed to model the anti-S decay over the 9 months following the vaccination, during serological screening performed approximately 2, 4, and 9 months following the first jab administration. Serological tests performed in the 9 months preceding vaccine administration were retrospectively analysed to identify the burden of infections occurring before vaccination. Results: The serological assays were used for monitoring the antibody titres during the observational period. Vaccination significantly reduced the rate of infection and elicited a specific humoral response, which lasted during the whole observational period (9 months). A decay was observed in all considered subgroups. At 35 weeks, workers with no history of pre-vaccine infection showed a significantly lower anti-S titre (−2522 U/mL on average (−2589.7 to −2445.7)); younger workers showed significantly higher anti-S titres (140.2 U/mL on average (82.4 to 201.3)). Only seven immunocompromised workers did not show significant levels of anti-S antibodies; three of them, all females, showed a specific T-cell response. Conclusions: Comparing the 9-month periods before and after the first vaccine dose, a significant reduction in infection rate was observed (1708 cases vs. 156). Pre-vaccine infection, especially if contracted during the first pandemic wave, greatly enhanced the response to vaccination, which was significantly affected also by age both in extent and duration (inversely related). A gender effect on the T-cell immune response was observed in a small group of workers who did not produce antibodies after vaccine administration. Vaccination against SARS-CoV-2 started in Italy, as well as other European countries, on 27 December 2020 (European Vaccine Day), soon after the EMA released a conditional marketing authorization for the BNT162b mRNA vaccine , hence mASST Spedali Civili is a public, major European COVID-19 centre in Lombardy, Northern Italy, an area strongly hit by the pandemic since its early beginning, which accounted for around 500,000 cases and over 25,000 deaths in 2020 . Before The main objective of the present study was to evaluate the 9-month trend of anti-SARS-CoV-2-S (anti-S) antibody titres in vaccinated workers, also considering age, gender, and pre-vaccine SARS-CoV-2 infections, as well as to evaluate the effectiveness of the vaccination campaign over time. The trend of anti-N antibody titres was also monitored, and anti-N serological conversions in vaccinated workers were used to identify new SARS-CoV-2 infections. Finally, we evaluated the anti-SARS-CoV-2 T-cell response induced by vaccination in a very small group of vaccinated workers who did not develop detectable amounts of anti-S antibodies after two vaccine doses.0) and then in March–April 2021 (T1), May–June 2021 (T2), and August–October 2021 (T3) for determination of both anti-S and anti-N antibody titres. All samples collected within 24 h from the vaccine 1st jab were included in the T0 group; T1, T2, and T3 groups include all samples collected more than 14 days, more than 90 days, and more than 180 days from the vaccine 1st jab, respectively , involved 382 workers at T0). Further serological blood samples were taken on average after 2 (T1), 4 (T2), and 8 (T3) months. A total of 7411 individuals performed at least a test during the observational period and were enrolled in the study. A full description of the entire cohort, which includes administrative staff, assistant personnel, nurses, physicians, other HCW and “external” workers , is presented in the 0 and in the 9 months before (in spring 2020 and autumn 2020), as they had shown higher sensitivity in detecting previous SARS-CoV-2 infections among our personnel [0) to further minimize the risk of misclassification. New cases of SARS-CoV-2 infection in vaccinated workers were identified by anti-N sero-conversion.We planned a prospective longitudinal cohort study involving the WF (mostly HCW) of the ASST Spedali Civili di Brescia, one of the largest tertiary university hospitals in Italy, with over 1500 beds and an estimated workforce of 9436 individuals, including those who are not directly employed but who are continuously working in the hospital. Every worker receiving the 1st dose of the anti-SARS-CoV-2 vaccine (N = 8648 people) had the chance to join the study and undergo a baseline serological screening ; written informed consent was obtained from all participants. The study was reported according to the Strengthening the Reporting of Observational Study in Epidemiology (STROBE) Statement.None.® SARS CoV2 S1/S2 IgG assay , whereas, during autumn 2020, electrochemiluminescence immunoassay (ECLIA) Elecsys® Anti-SARS-CoV-2, which detects immunoglobulins (IgG/A/M) anti-N , was used. The response to the vaccine (from T1 onwards) was assessed using the ECLIA Elecsys® Anti-SARS-CoV-2 S for anti-S (IgG/A/M) detection .During spring 2020, serum samples were tested using the chemiluminescent immunoassay Liaison® SARS CoV2 S1/S2 IgG is a CLIA assay for the in vitro quantitative detection of IgG anti-S (anti-S1 and anti-S2) in serum and plasma. Recombinant S1 and S2 antigens bound to magnetic beads and the mouse monoclonal antibody anti-human IgG were used to detect and quantitate IgG in human samples. The results are expressed as U/mL, and specimens are considered negative if <12 U/mL, equivocal between 12 and 15 U/mL, and positive if ≥15 U/mL.Liaison® Anti-SARS-CoV-2 is an ECLIA immunoassay for the in vitro qualitative detection of antibodies (IgG/A/M) against SARS-CoV-2 in human serum and plasma. The assay uses a recombinant protein representing the nucleocapsid (N) antigen in a double-antigen sandwich assay format. The results are expressed as the cut-off index, the cut-off being 1.Elecsys® Anti-SARS-CoV-2 is an immunoassay for the in vitro quantitative determination of antibodies (IgG/A/M) to the SARS-CoV-2 Spike (S) protein receptor binding domain (RBD) in human serum and plasma. The assay uses a recombinant protein representing the RBD of the S antigen in a double-antigen sandwich assay format. The results are expressed as U/mL, the cut-off was 0.8 U/mL, and the upper limit of detection was 250 U/mL. Since the antibody titres elicited in immunized individuals were very high, we tested all serum samples at a dilution of 1:20, in accordance with Roche, so the upper limit of detection raised to 5000 U/mL and the dynamic range could be extended.Elecsys®, Venlo, The Netherlands), an interferon-gamma release assay (IGRA) consisting of two antigen tubes that use a combination of proprietary antigen peptides specific to SARS-CoV-2 to stimulate lymphocytes involved in cell-mediated immunity in heparinized whole blood samples. The QuantiFERON SARS CoV-2 Ag1 tube contains peptides targeting CD4+ epitopes from the receptor binding domain (RBD) of the spike protein with overlap to span the entire RBD, whereas the Ag2 tube contains immunodominant CD8+ epitopes selected from the entire spike protein and CD4+ epitopes from the RBD. Plasma from stimulated samples can be used for the detection of IFN-γ using an enzyme-linked immunosorbent assay (ELISA)-based platform. Specimens were processed according to the manufacturer’s guidelines. Following ELISA, quantitative results (IFN-γ concentration in U/mL) were recorded and used for analysis. According to the manufacturer’s instructions, a positive response was defined as a value at least 0.20 U/mL greater than the background U/mL value from the QuantiFERON SARS-CoV-2 Nil tube; the Nil tube value was subtracted to mitigate against background IFN-γ in the sample that was not a result of SARS-CoV-2-specific T-cell stimulation. Median (min–max) Nil subtracted IFN-γ responses were plotted, and the median was chosen to illustrate the central measurement of the dataset in which biological variation could skew results. Minimum and maximum values were provided to inform the range of responses in addition to the central (median) value.The T-cell response against SARS-CoV-2 was evaluated via the QuantiFERON SARS-CoV-2 assay  and R (version 4.1.0).Normality of distributions was assessed using the Kolmogorov–Smirnov test. Categorical variables are presented as frequencies or percentages and compared by the chi-squared test or Fisher’s exact test, as more relevant. Continuous variables were summarised by the means ± standard deviations (SD) when normally distributed or as medians, interquartile range (IQR) when a skewed distribution was observed, and a bootstrapped two-way ANOVA for repeated measures was applied to test differences among groups over time. Multivariate logistic regression was used to identify groups at higher risk of infection. A multivariable linear mixed effect model was performed to estimate the anti-S decay over time adjusted for pre-vaccine SARS-CoV-2 infection, sex, and age to address potential sources of bias and to test group differences through the introduction of interaction terms. Covariates were included based on the hypothesis that they could have an influence on the anti-S trajectories. A restricted cubic spline with 3 knots, in which outer quantiles were set at the 1st and 9th decile, was applied to allow for a non-linear relationship between time and anti-S levels. A total of 500 bootstrap iterations were used to account for the non-normal outcome distribution. The choice of the mixed-effect models also allowed us to deal with missing data at T®, Pfizer®, New York, NY, USA/BioNTech®, Mainz, Germany; >99.1%), and 6862 (79.3%) were included into the T0 group of the SeroCoVax-BS Project  were vaccinated against SARS-CoV-2, most of them with the BNT162b2 mRNA vaccine , 493 (29%) males and 1215 (71%) females. Pooling together the serological tests performed in 2020 and those performed at T0 vs. 23.1 U/mL at T1, 20.1 U/mL at T2 and 14.4 U/mL at T3) together with a significant increase with age. Higher antibody titres were measured in the older age groups at the different sampling times  identified by RPS.A significant decrease of anti-N antibody levels over time was observed , p = 0.037 for 50–59 years and over 60 years age groups, respectively, compared to the reference age group 20–29).During the entire observational period, sero-reversion of anti-N antibody titres was observed in 85 individuals, about 6% of all workers who tested positive for anti-N serological assays. Such event was not affected by gender and was inversely associated with age groups (OR 0.36 (95%CI 0.19–0.69), 1, anti-S assays demonstrated a 99.9% effectiveness of the vaccine in terms of serological conversion . During the whole observational period, two serological reversions were detected at T3 in two over-50 aged males. p < 0.001). In contrast to anti-N antibodies, an inverse relationship of anti-S antibodies with age groups was observed at T1, T2, and T3 (p < 0.001).At T0–T1), we observed an average decrease of −42.3 U/mL per week ; at the average time of the second interval , there was a reduction of −37.4 U/mL per week  while at the average time of the third interval  the decrease per week is almost null .The bootstrapped simple linear mixed-effect regression model showed an overall non-linear antibody decay over time that stabilizes at around 1477.4 U/mL  after 39 weeks since the first jab A. The deWhen introducing an interaction term between time and pre-vaccine infection , we observed significant differences at any time between both groups  as well When comparing the trends between genders in the whole sample, we only observed a significant difference during the first weeks , always on the sample. In this case, we could observe statistically significant differences at any time, where younger subjects had higher anti-S antibody titres  as well When we stratified by gender and pre-vaccine infection groups, we observed significantly higher anti-S antibody titres in females compared to males among subjects that were not infected during the first and last weeks of observation but not in the central part of the follow-up . Both thWe then analysed our sample stratifying by age and pre-vaccine infection groups. Among not infected subjects, those below 50 years old showed higher anti-S antibody titres compared to older subjects . Both cuWe finally compared the subjects with no pre-vaccine infection, those who got an infection during the first wave and those who got a pre-vaccine infection later in time. The last two groups had similar anti-S antibody titres during the first weeks but those who got the infection during the first wave showed higher anti-S antibody titres  and a leOverall, seven vaccinated workers did not develop a significant amount of anti-S antibodies. All such workers were in treatment with immunosuppressive drugs due to their clinical conditions, which are summarized in 0 confirmed that our hospital, with a prevalence rate of infected workers of 19.8%, was severely hit by the first (March–April 2020) and second (October–November 2020) pandemic waves. Such a high infection rate can possibly explain the in-mass adhesion (>90%) of workers to the vaccination campaign, well before the enaction of the Italian Law n. 76/2021 on 28 May 2021, which made SARS-CoV-2 vaccination mandatory for HCW. Females showed a significantly lower risk of infection, whereas a higher risk was found both for nurses  and “other HCW” , as well as in the youngest age group . Vaccination, which mostly occurred with the BNT162b2 vaccine , succeeded in reducing the rate of infection among our workforce despite the local increase of cases observed during the first trimester of 2021 [To the best of our knowledge, this is the first longitudinal study investigating the trends of anti-N and -S antibody titres in such a large sample for such a long time. Since the beginning of the SARS-CoV-2 pandemic, six in-mass serological screenings were performed on the WF of the ASST Spedali Civili of Brescia Hospital, the last four in the context of the SeroCoVax-BS prospective study. The 2020 serological screenings and those at T of 2021 . A compa of 2021 .1–T2 period plus a further 22% during the T2–T3 interval). In general, the production of anti-SARS-CoV-2 antibodies was strongly influenced by age, showing a complementary pattern: while higher anti-S antibody titres were observed in younger workers, higher anti-N antibody levels were found in the older age groups. The hypothesis that a dysregulated antibody response could be related to the severity of the disease observed in different age groups needs to be further investigated [Despite the persistence of a positive serological response, our data documented a progressive decay of anti-S antibodies of approximately 46% in 9 months . This phenomenon may lead to misdiagnosis of previous infections. While a positive test for anti-N could be considered a reliable marker of previous SARS-CoV-2 infection, a negative test should not rule it out, especially in younger individuals, who are more prone to lose this type of antibodies over time. This finding again agrees with what was observed in the REINASSANCE study [We also observed a reduction in anti-N antibody titres and 85 cases of sero-reversion in individuals who tested positive at TCE study . Only a CE study . RegardlCE study ,21. FurtCE study ,23,24.0). Misclassification due to possible anti-N sero-reversions was addressed considering the results of serological tests performed during 2020, at the end of the first and second pandemic waves, which were cumulated with the baseline. Preferring serological test results to RPS increased the sensibility and specificity [Based on the sero-conversion of anti-N antibodies, we could detect 156 new infections during the 9-month follow-up, mostly occurring in the first two months after the first vaccine jab . Such a figure and the constant pauci-symptomatic clinical course of all such cases allowed us to estimate a protection from SARS-CoV-2 infection above 95% and a 100% protection from hospitalization during the first nine months following the first jab administration. A possible limitation of the study is that no anti-S assay was performed at the baseline (Tcificity  for iden


In recent years, evolutionary algorithms based on swarm intelligence have drawn much attention from researchers. This kind of artificial intelligent algorithms can be utilized for various applications, including the ones of big data information processing in nowadays modern world with heterogeneous sensor and IoT systems. Differential evolution (DE) algorithm is one of the important algorithms in the field of optimization because of its powerful and simple characteristics. The DE has excellent development performance and can approach global optimal solution quickly. At the same time, it is also easy to get into local optimal, so it could converge prematurely. In the view of these shortcomings, this article focuses on the improvement of the algorithm of DE and proposes an adaptive dimension differential evolution (ADDE) algorithm that can adapt to dimension updating properly and balance the search and the development better. In addition, this article uses the elitism to improve the location update strategy to improve the efficiency and accuracy of the search. In order to verify the performance of the new ADDE, this study carried out experiments with other famous algorithms on the CEC2014 test suite. The comparison results show that the ADDE is more competitive. Optimization exists in various fields of engineering and applications , and theTo solve the problem of numerical optimization, Price and Storn firstly proposed differential evolution algorithm (DE) in 1995. Since the 21st century, more and more advanced DE variants have been proposed to be applied in different fields . It has The rest of this article is arranged as follows: in the second section, we briefly review the development of DE, the ABC algorithm, and their famous variants; The third section introduces the principle and structure of the proposed algorithm in detail. In “Experiment and Result”, experiments are carried out in the CEC2014 test suite , and theIn this section, two important branches of the stochastic optimization algorithm, the famous ABC and the powerful DE, are reviewed as well as their variants.In the ABC algorithm, the global optimal solution is found through three different stages. In the first stage, the leader bee looks for a better honey source in the neighborhood of the honey source (feasible solution); in the second stage, the following bee selects one of the leading bees to follow and collect honey according to the information of the quality of the honey source found by the leader bee, so as to find a better honey source in the neighborhood of the honey source; in the third stage, when the leader bee cannot find a better honey source for many times, he will become a reconnaissance bee, The new honey source location was randomly searched within the search range. The standard functions of the location update process of the original ABC algorithm are shown as follows.λ is a random number and in the range of , when the function value of the new honey source is better, the new honey source is used to replace the original honey source, otherwise the original honey source is retained. The original ABC algorithm updates the position by randomly approaching or away from other bees, which greatly enhances its search ability, but also weakens the exploitability and is not easy to converge to the global optimal solution. In the development of the ABC algorithm, many excellent ABC variants have been proposed . For exaDE is a powerful evolutionary algorithm, the core idea of which is survival of the fittest. The evolution process of DE imitates the operation of mutation, crossover, and selection in genetics. It is a robust global optimization algorithm. The operation mechanism of DE consists of four steps . First, In the past 20 years, scholars have done a lot of research on DE, and many DE variants , 2013b hAs we all know, search and development are equally important for the performance of swarm intelligence algorithms. DE is well known for its powerful development performance, which largely benefits from its multidimensional search strategy. In the search process, it can quickly approach the global optimal solution, but there will be premature convergence. This is caused by the lack of search performance in the late iteration, and the one-dimensional search strategy can make up for this deficiency. ABC algorithm is famous for its powerful searching ability. It uses a one-dimensional search strategy to update the location. Inspired by ABC, a new DE algorithm is proposed in this study, which update by the adaptive selection of single or multi-dimensional search strategy. The arrangement of this chapter is as follows: In the first part, the improved location update equation is given. In the second part, the adaptive selection scheme for the multi-dimensional update or single dimensional update is described. Finally, the pseudo-code of the proposed ADDE algorithm is given in the third section.Generally speaking, elitism is an effective technique to enhance the performance of the algorithm in the optimization process: using elitist individuals  to guide other individuals to complete the evolution, will more quickly approach the global optimal solution. However, when only one elite is used, for example, only the global optimal position of the current iteration is used for guidance, it is easy to fall into the local optimal solution and converge prematurely.In the second stage of the ABC algorithm, it is easier for observation bees to follow the better bees to collect honey. This means that the better the individual is, the easier it is to be selected for one-dimensional location updating. This is undoubtedly beneficial to the whole optimization process. The difference is that the poor individuals in the population usually mean that they are far away from the global optimal solution, and multidimensional updating can speed up the whole optimization process. Based on this, this article proposes an adaptive selection scheme based on ranking. Its selection mechanism is as follows:Among them, As shown above, the higher the current individual ranking, the easier it is to choose a single-dimensional update strategy, while the lower the ranking, the easier it is to choose a multi-dimensional update strategy. In particular, when using a multi-dimensional strategy, there is still a need for crossover, and when using a single-dimensional strategy, there is no need for crossover. In the adaptive dimension selection scheme, according to the ranking of multi-dimensional or single dimensional update strategy selection, is a kind of effective play to their expertise technology, to achieve the balance of search and development.In our experiments, the CEC2014 benchmark set is used to evaluate the performance of the proposed algorithm. In the test benchmark suite, all benchmark functions can be divided into four categories: single-mode function  fitness value will get more position update opportunities. These two improvements are reflected in the location update strategy and adaptive selection scheme of the ADDE algorithm. Therefore, we compare our algorithm with some famous ABC variants and advanced DE variants to verify the effectiveness of our algorithm. ABC variants include: GABC, MABC, IABC, QABC, and DE variants include: DE/rand/1, RBDE, DEGL, SADE, QUATRE, S-QUATRE, QUATRE-DEG. The parameters of all algorithms are in line with the original recommended values, and the detailed parameters are listed in maxFES, is set to 10e4*D, where D is the dimension. We then compared the average value of fitness error ∆f and std (standard deviation) of 51 runs of each algorithm. In order to evaluate the significance of the two algorithms, a paired nonparametric statistical procedure of Wilcoxon’s signed rank test, was performed on experimental data  benchmarks, and some benchmarks still got worse. In addition, both ADDE and RBDE improved better than DE/RAND/1 and RBDE on high-dimensional problems, both achieving 28 better and a similar result on the 50D basis. Secondly, compared with the improved results of SADE, which is also an adaptive scheme, ADDE has the best effect on the basis of 30D, achieving 26 better results and one similar results. It only becomes worse on F6, F7 and F27. Finally, the improvement of ADDE over advanced QUATRE-DEG deteriorates as the dimension increase, becoming best at 10D, obtaining 26 better benchmarks, and 24 and 23 better benchmarks at 30D and 50D, respectively. It can be analyzed that compared with the more advanced DE variants, the effect on the high-dimensional benchmark is slightly decreased. This is because ADDE algorithm has enhanced certain search ability (from ABC), so the development energy will be relatively weaker, and some high-dimensional problems need to be developed more. Overall, however, we still got the best performance compared to these DE variants.We select the representatives of four benchmark functions in the CEC2014 test suite and analyze their convergence rate, including basic single-mode function FUNC1, FUNC3, basic multi-mode function FUNC6, FUNC9, FUNC15, FUNC16, mixed function FUNC17–FUNC22, combined function FUNC27–FUNC30. We evaluate the algorithm complexity of the novel ADDE on 30D, and the algorithm complexity of the CEC2014 is given in DE is a simple and powerful algorithm, which has attracted more and more attention in recent years. We know that the outstanding development ability of DE is largely due to its multidimensional search strategy, which enables it to quickly approach the global optimal solution. But for a better solution, the one-dimensional search strategy can ensure a greater update success rate. Especially in the late stage of the optimization iteration, the one-dimensional search strategy can slow down the particles falling into the local optimal solution. Based on this, this article proposes an adaptive updating scheme, which uses multi-dimensional and one-dimensional updating strategies to achieve a better balance between search and development. The multi-dimensional updating strategy comes from the improvement of the standard DE algorithm, while the one-dimensional updating strategy comes from the improvement of the standard ABC algorithm. The main idea to improve them is to increase elitism which is used to improve the overall optimization speed. To verify the performance of our ADDE algorithm, we compared it with other famous ABC variants and DE variants on the CEC2014 dataset. The results show that the new algorithm is competitive. In the future, we will further use ADDE to solve practical engineering problems .10.7717/peerj-cs.1007/supp-1Supplemental Information 1Click here for additional data file.


Althaea officinalis L., radix), which we can find in European pharmacies, are widely used among the European population. However, recently, voices have been heard in the public about alleged PTE occurrence. In this article, the levels of Pb and Cd impurities were measured in samples of THMPs with marshmallow root available in Polish pharmacies (n = 10). Our proposed toxicological approach was based on two important issues. The first was PTE levels (Pb: 1.60–6.80 μg/L and Cd: 0.80–3.81 μg/L presented as raw results) in comparison with the permissible limit set by FAO/WHO for medicinal herbs  and also ICH Q3D guidelines . The second was the estimation of exposure of investigated PTE in a single dose and daily dose for each THMP. It should be noted that the content of analyzed heavy metals in a single dose of analyzed products was very low, and hence is not a threat for patients. The obtained daily intake of heavy metals impurities in comparison with PDE values confirms the safety of all analyzed phytopharmaceuticals . It can be summarized that each of the analyzed THMP with marshmallow root available in Poland are safe for the patients. Based on literature review, this article is the first study about heavy metals impurities level in final THMPs with A. officinalis L., radix  available in European pharmacies.The level of potentially toxic elements (PTE) in phytopharmaceuticals can be a potential threat to human health through the food chain. The traditional herbal medicinal products (THMPs) with the marshmallow root ( Althaea officinalis L., radix . This herbal plant has been used since ancient times in different European countries [Althaea officinalis L. [Sirupus althaeae is prepared by maceration of 5 parts of roots with 1 part of ethanol (760 g/L) and 40 parts of purified water for 3 h without stirring. In macerate obtained 64 parts of sucrose and 0.1 part of benzoic acid are solved”. This kind of pharmaceutical product is widely applied for irritation of the pharyngeal and oral mucosa and associated dry cough [A. officinalis L. [Lead and cadmium are potentially toxic elements (PTE), but usually scientific studies about impurities of these heavy metals in pharmaceutical products are treated very marginally. It seems that nowadays lead and cadmium impurities are not a popular problem in scientific literature; however, herbal plants in traditional herbal medicinal products (THMPs) can especially be a potential source of these elements impurities . One of ountries . The THMountries . The marnalis L. . The pronalis L. : “Sirupuethanol 70 g/L andry cough . Even thnalis L. , not in It should be mentioned that exposure to Pb and Cd may cause serious health effects—especially ,11,12 neA. officinalis L. is required. For this purpose, the best strategy is based on the ICH Q3D  guideline about acceptable limits of EI [Hence, comprehensive health assessment of Pb and Cd impurities in THMPs with ts of EI . In thists of EI . Althaea officinalis L., radix, available in Polish pharmacies. Our studies included all available THMPs in the market with marshmallow root extracts in Poland (n = 10). The justification for the choice of this study was the lack of appropriate investigations about Pb and Cd impurities in such pharmaceutical products. Additionally, there are common opinions among the Polish population about the alleged PTE impurities in THMPs available in Poland, without any scientific background.Hence, our work aimed at a comprehensive assessment of Pb and Cd impurities (based on the ICH Q3D guideline mentioned earlier) in THMPs with 3)2 in 0.5 mol/L HNO3 and 1000 mg/L Pb CertiPUR®  and cadmium (Cd standard solution traceable to SRM from NIST–Cd(NO3)2 in 0.5 mol/L HNO3, 1 g/L Cd CertiPUR®, catalog product: 1.19777.0500) were prepared by dilution of certified standard solutions, 1 mg/L MERC of corresponding metal ions. The purchased certified reference material  was material prepared from lichen. Additionally, the second certified reference material  was purchased from the Institute of Nuclear Chemistry and Technology—Department of Analytical Chemistry .For the experimental work, demineralized water was applied during sample processing. Nitric acid (65%) was of spectroscopic grade . Standard solution for Pb was standard solution traceable to SRM from NIST—Pb. All samples were denoted by letters (A–J). It should be noted that only a few independent manufacturers produce these kinds of pharmaceutical products in Poland. The investigated products were over-the-counter medicines collected from pharmacies in Kraków, Niepołomice, Bochnia, Wieliczka, and Rzeszów in 2021 (April–May). All investigated THMPs were syrups and contained A. officinalis L. (root). The preparation in all cases was the same. The detailed information about each product (based on manufacturer information) is described in more detail in THMPs containing Althaea officinalis L., radix was homogenized. From each sample, 0.5 mL was measured , poured into Teflon vessels, and digested (1 h) with 5.0 mL of concentrated nitric acid . After this operation, all samples were cooled to a room temperature (25 °C). The final volume was 20 mL. Five replications were performed for all samples to increase the precision of the results. The time–temperature program in the graphite furnace atomic absorption spectrometer for Pb and Cd determination is presented in The acid digestion of samples was applied using a microwave oven MDS 2000 . Each THMP with The analytical calibration function includes levels 0.0; 1.0; 2.0; 5.0; 10.0 µg/L for Pb, and 0.0; 0.5; 1.0; 2.0 µg/L for Cd. The values of correlation coefficients (R) were 0.998 for Pb and 0.998 for Cd, which indicated that the analyses were both precise and accurate.The obtained values for recoveries were 98.2% and 97.5% for Pb and Cd, respectively.The calculated limits of detection (LODs) were 0.46 µg/L for Pb and 0.16 µg/L for Cd. The calculated limits of quantification (LOQs) were 0.96 µg/L for Pb and 0.35 µg/L for Cd.The CRM was prepared from lichen  and corn flour, INCT-CF-3. Until the analysis, samples of CRM were dried at 70 °C for 24 h. After drying, the samples were transferred for microwave digestion . In the first step, 5 mL of nitric acid (concentration 65%) was added to 300 mg of the sample in Teflon reaction vessels and left to predigest for 24 h. In the next step, digestion was carried out, and after cooling the vessels, the extracts were transferred to Sarstedt vessels and completed with demineralized water to a total volume of 15 mL. Samples prepared were analyzed using a Perkin-Elmer 5100 ZL atomic absorption spectrometer with a graphite furnace. The declared value of Pb was 40.9 ± 1.4 mg/kg and the obtained value was 38.05 ± 0.23 mg/kg for BCR-482; IRMM. The declared value of Pb was 0.052 ± 0.009 mg/kg and the obtained value was 0.050 ± 0.009 mg/kg for corn flour, INCT-CF-3. The analysis of the mentioned certified reference materials was helpful for assessing the traceability of the results. The applied methodology was similar to our previous studies using the same apparatus ,19,20,21Obtained results were calculated using the scientific statistical software Excel 2010 (Microsoft Office) and Origin 2021 Pro.The strategy for our toxicological assessment of investigated PTE impurities is shown schematically in The first step was the preparation of raw results impurities for each THMP. The results were compared with the permissible limit set by FAO/WHO for medicinal herbs in different countries. The next step was the assessment of exposure of lead impurities after the application of THMP. For this purpose, the first step was the estimation of Pb levels in a single administration (ng/single dose) based on posology. The last step was to estimate the daily dose of PTE (ng/day) and compare with permitted daily exposure (PDE) based on the ICH Q3D guideline.The results of all analyzed samples of THMP samples (A–H) are presented in The comprehensive toxicological assessment of investigated PTE impurities also requires estimation of exposure after oral application. Hence, based on the posology for each product . Azizov et al. [A. officinalis L., radix collected in Syrdarinsk District of Uzbekistan. As was mentioned in the introduction, lead may be taken up by plant roots [4 µg/kg [The general characteristics indicate that lead impurities were present at a relatively low level (mean = 3.73 μg/L) in all analyzed samples (1.62–6.74 μg/L). The lowest level of Pb was in sample F (1.62 ± 0.13 μg/L), and the highest level was observed in sample G 6.74 ± 0.24 μg/L). It should be noted that there are limited studies about Pb level in v et al.  and Esmav et al.  describent roots . Additiont roots . In ment μg/L. ItA. officinalis L., radix . Azizov et al. [A. officinalis L., radix collected in Syrdarinsk District of Uzbekistan. This element is readily taken up by plants, and an important problem can be that the toxicity level is in the range of 0.5–1 mg/kg dry plant material, whereas crop plants tolerate at least ten-fold of that concentration in tissue [Similar results were observed for cadmium; the Cd impurities were present at a very low level mean = 1.88 μg/L) in all investigated samples (0.85–3.81μg/L). The highest Cd level was observed in sample E (3.81 ± 0.13 μg/L), and the lowest level was in samples D (0.85 ± 0.09 μg/L) and H (0.85 ± 0.07 μg/L). As in the case of Pb, there are limited studies about Cd levels in v et al.  and Esmav et al.  describen tissue . The abon tissue ). Additi.88 μg/L −6/day) are variable. Based on the Integrated Exposure Uptake Biokinetic (IEUBK) Model [−6/day—sample G). Additionally, the estimated daily exposure levels for Cd are variable (21.25–228.6 mg 10−6/day). The main point for toxicological assessment of Cd by oral route is renal toxicity [The obtained results  of dailyK) Model , PDE valK) Model . Hence, toxicity ; hence tAlthaea officinalis L., radix. Additionally, all results were below the permissible limit set by FAO/WHO for medicinal herbs and plants in different countries . The contents of these elements in a single dose were also at a very low level; hence, there is no potential hazard for people. Additionally, the estimated daily intake of Pb and Cd impurities compared to the PDE value confirm all samples safety. Hence, it can be summarized that all samples meet the standards of the ICH Q3D guideline due to the PTE impurities.Our studies are extremely important for regulatory purposes (regulatory toxicology), especially for the pharmaceutical industry. Conducted studies show that lead (1.62–6.74 μg/L) and cadmium (0.80–3.81 μg/L) impurities as raw results were at a very low level in all analyzed THMPs with n = 10) but this could be a limit for global interpretation .The broader toxicological assessment considering other heavy metals (Hg and As) in THMPs available in European pharmacies will be valuable in future studies. It should be noted that we analyzed all available products in Poland (