The societal costs were calculated for healthcare and non-healthcare costs in the first two years after stroke. The QoL was measured using EQ-5D-3L. The differences between (sub)groups over time were investigated using a non-parametric bootstrapping method. (3) Results: A total of 344 post-stroke patients were included. The total two-year societal costs of a post-stroke were EUR 47,502 (standard deviation (SD = EUR 2628)). The healthcare costs decreased by two thirds in the second year −EUR 14,277 . In the second year, over 50% of the total societal costs were connected to non-healthcare costs . Sensitivity analyses confirmed the importance of including non-healthcare costs for long-term follow-up. The subgroup analyses showed that patients who did not return home after discharge, and those with moderate to severe stroke symptoms, incurred significantly more costs compared to patients who went directly home and those who reported fewer symptoms. QoL was stable over time except for the stroke patients over 75 years of age, where a significant and clinically meaningful decrease in QoL over time was observed. (4) Conclusions: The non-healthcare costs have a substantial impact on the first- and second-year total societal costs post-stroke. Therefore, to obtain a complete picture of all the relevant costs related to a stroke, a societal perspective with a follow-up of at least two years is highly recommended. Additionally, more research is needed to investigate the decline in QoL found in stroke patients above the age of 75 years. Based on the Global Burden of Diseases Study data, the lifetime risk of stroke from the age of 25 years onward is approximately 25% among both men and women , while sThey also estimated an annual total healthcare cost of USD 30.8 billion in 2016 and 2017 as a result of strokes . CurrentBuilding on previous work from the Restore4Stroke study , the oneThe current study involves a bottom-up Burden of Disease (BoD) study from a societal perspective with a two-year follow-up. More specifically, the aims of the current study were: (1) to estimate the total societal costs two years post stroke; (2) to compare both the healthcare and the non-healthcare costs measured in the first and second-years post-stroke; (3) to measure the impact of strokes on generic health-related QoL at one and two years post-stroke; and (4) to identify relevant stroke subgroups. The current study was embedded in a prospective, multi-center, observational cohort study entitled ”Restore4Stroke”, which aims to gain insight into care received post-stroke, and the economic consequences of psychosocial care in the first two years post-stroke. The design of this BoD study , the shoStroke survivors were recruited from stroke units in six general hospitals throughout the Netherlands: St. Antonius Hospital (Nieuwegein); Diakonessenhuis (Utrecht); Canisius Wilhelmina Hospital (Nijmegen); TweeSteden Hospital (Tilburg); St. Elisabeth Hospital (Tilburg); Catharina Hospital (Eindhoven). Patients were eligible for inclusion if they had a clinically confirmed stroke (either first or recurrent stroke) within the last seven days and were at least 18 years of age. The exclusion criteria were if the patient (1) had any other condition that may be expected to interfere with the study outcomes , (2) were clinically judged unable to sufficiently understand and complete Dutch questionnaires, (3) were rated physically dependent as defined by a Barthel Index (BI)  score ofPatients received information on the study and were asked to give informed consent by the nurse practitioner or the trial nurse during the hospital stay in the first-week post-stroke. After informed consent was obtained, the patients’ characteristics were collected. The generic health-related QoL questionnaires (EuroQoL-5D-3L)  and the A bottom-up costing approach from a societal perspective using a specifically designed 11-item cost questionnaire was performed . The queThe valuation of the costs was conducted using the updated Dutch Manual for Cost Analysis in Health Care Research . An overThe descriptive system of the three-level EuroQoL (EQ-5D-3L) is a widely used and recommended preference-based measure of health . The fivWhen two or more complete assessments were missing from the cost and/or quality of life questionnaires, patients were excluded from the analyses. Diseased patients were always included in the analysis. The EQ-5D-3L scores for diseased patients were set to the maximum ranking for all dimensions  and the costs were defined as zero for all measurements after their reported date of death.Cost questionnaires were filled in at two months (retrospective for two months), six months (retrospective for four months), and at twelve and twenty-four months (retrospective for six months). In the base case analysis, to calculate the total resource use of the second year , the 24-month measurement was doubled (twice the reported 6 months of resource use).Multiple imputations were applied to replace missing cost and QoL data using the following predictors: gender; marital status; age; treatment location; severity of stroke  rating) ,33. The Four sensitivity analyses were performed to test for differences between follow-up measurements or follow-up periods. In the first sensitivity analysis, the subject of investigation was the extrapolation of the costs from a 12 to 18-month period. In the base case analyses, to estimate the total costs for the second year after stroke, the total costs at 24 months (retrospective measurement for 6 months) were multiplied by two. In the sensitivity analysis, the costs at 12 months (retrospective measurement for 6 months) and the costs at 24 months were added to estimate the costs of the same period. In the second sensitivity analysis, the societal perspective  was compared to the healthcare perspective . In the two other sensitivity analyses, the base case Dutch tariffs  for the Both generic health-related quality of life and the total societal costs were investigated in seven subgroups. These groups were gender , age (65−/65+), education level (low/high), stroke type (infarction/hemorrhage), recurrent stroke (yes/no), home discharge (yes/no), and severity of stroke (NHISS 0–4/>5). n = 344) of the initial Restore4Stroke Cohort (n = 395) was included in the current study. A total of 35 patients (8.9%) dropped out and another 16 patients (4.4%) were excluded as they had two or more assessments of costs and/or QoL measurements missing. More details on the missing data are reported in A total of 78%  or to a nursing home .The average total societal cost for patients two years post-stroke was EUR 47,502 (SD = EUR 2628). The mean healthcare cost was estimated to be EUR 27,159 (SD = EUR 1611) and the mean non-healthcare costs were on average EUR 20,330 (SD = EUR 1603) per patient. Over 80%  of the healthcare costs were related to outpatient rehabilitation and inpatient hospital and rehabilitation stay. The other 20% of the healthcare costs were related to the remaining healthcare cost categories, such as costs of medication, general practitioner, and specialist visits. 60% of the non-healthcare costs in the two years after a stroke are related to paid home care, informal care, and the cost of productivity losses. The other 40% of costs (EUR 8284) are due to the patients’ inability to perform unpaid labor. For further details on resource use and costs in the 24 months post-stroke, see A significant difference was reported in societal costs between the first year and the second year after a stroke  see . The totThe scores with respect to the five dimensions of the EQ-5D-3L were comparable at 12 months post-stroke compared to 24 months post-stroke. This also accounts for the utilities estimated using the Dutch tariffs for either group. When the patients were divided into subgroups for “gender”  and “severity of stroke” , no significant differences in utilities were reported in time. Only patients over 75 years of age had a significant decrease and clinically meaningful lower utility score at 24 months compared to 12 months post-stroke . For details, see The sensitivity analysis shows that the cost extrapolation method in the second year after stroke did not change the base case results. In another sensitivity analysis, it was found that societal costs were significantly higher compared to healthcare costs .When the Dutch utilities were compared with UK tariffs at one year, no significant difference was reported . At 24 months, however, significantly different utilities were observed . However, this difference could not be defined as clinically meaningful. For details, see n = 97) compared to home discharge (n = 49) incurred twice as many costs as patients who went home (n = 247) or were transferred to rehabilitation clinics (n = 48) in the second year  and “discharge at home” (Yes/No), the 24 months utility measurements, and for the subgroup “stroke type” (Infarction/Hemorrhage). However, only the subgroup “stroke type” could also be defined as clinically meaningful. In other words, in the hemorrhage subgroup, the utilities were lower compared to the infarction group. After two years, the societal costs were significantly higher when the patients were transferred to rehabilitation clinics or geriatric rehabilitation centers . A decline in societal costs in the second year was observed . However, the non-healthcare costs, such as (in)formal care costs and the cost of not being able to perform unpaid labor, have a substantial impact on the total costs in the second year after a stroke. The sensitivity analyses confirmed the importance of including non-healthcare costs for the long-term follow-up. The subgroup analyses showed that the patients who were transferred to rehabilitation clinics or geriatric centers, and those with moderate to severe stroke symptoms, incurred significantly more costs compared to patients who went directly home and reported fewer stroke symptoms. The QoL was stable over time except for stroke patients over 75 years of age, where a significant and clinically meaningful decrease in QoL over time was observed.In general, studies investigating the economic burden of strokes often do not document non-healthcare costs , despite their significant contribution to the total costs ,14. In aThe costs of a stroke have previously been associated with stroke severity in the first-year post-stroke ,44 and wBesides the costs, the QoL was also investigated. For stroke survivors, a QoL utility score of 0.68 to 0.73 was reported in previous research ,47, whicThe strengths of the current study are: (1) the data were obtained through a prospective multi-center observational cohort study entitled ‘’Restore4Stroke’’, of which the design ,17 was pThis study has the following limitations: (1) the use of retrospective, patient-reported resource use might have led to recall bias ; (2) onlCurrently, there is a lack of recent data regarding long-term follow-up (>18 months) of BoD studies, using a societal perspective, a prospective design, and a bottom-up costing approach for stroke survivors. Detailed information on the BoD methodology used, the population included, and the treatment given is needed to compare different study findings; it is clear that guidance is highly recommended on how to interpret and report the results of different BoD studies .The data of the BoD studies are also essential for developing reliable model-based economic evaluations, which are needed to estimate the cost-effectiveness of stroke treatments with a lifelong follow-up. These models can be used by policymakers to make better-informed decisions by taking into account both the costs and effects of stroke treatments.In addition, these studies provide policymakers with better insights into national health reforms in society. The current Dutch policy aims to stimulate the informal care of (stroke) patients, rather than reimbursing paid home care to decrease healthcare spending. This study shows that the hours spent by informal caregivers of stroke patients in the first two years are relatively high. Besides other factors such as the patient’s health-related quality of life and the severity of the stroke, the number of hours informal care received are highly associated with the risk of burnout for caregivers .Finally, more research is needed to investigate the impact of older age (>75 years) on generic QoL ratings in post-stroke patients. For instance, does this group require special monitoring and help to improve their QoL after stroke?The average total societal cost for patients two years post-stroke was almost EUR 50,000. The total healthcare cost decreased in the second year compared to the first-year post-stroke due to a decrease in the costs of inpatient care and “rehabilitation treatments” in the second year. However, the healthcare costs in the second year post-stroke could still be considered high. On the other hand, non-healthcare costs have a substantial impact on the total societal costs at 24 months. Home care, informal care, the inability to perform unpaid labor, and productivity losses are all important cost categories to consider for the long-term cost of illness studies. The severity of disease and discharge destination have an impact on total societal costs. The stroke patient scores included in the Restore4Stroke cohort reported a lower QoL than the age group norms but was stable over the first two years post-stroke. Patients over 75 years of age show a clinically meaningful decline in QoL in the first two years post-stroke, which requires further attention.


The performance of the KCHA zeolite membrane in extracting potassium from seawater and sea bittern at room temperature was studied in detail. The XRD results show that the prepared KCHA zeolite was a KCHA membrane. The EDS test indicated that the potassium content of the KCHA zeolite membrane reached a value of 18.33 wt.%. The morphology of the KCHA zeolite grown on the surface of the alumina sphere was similar to a sphere, and it had good symmetry. The potassium ion-exchange capacities of the KCHA zeolite membrane reached 32 mg/g in seawater and 77 mg/g in sea bittern at room temperature. Ion exchange between the ammonium ions and potassium ions in the KCHA zeolite membrane could be completed in a short time at room temperature. The KCHA zeolite membrane was proven to have good reusability in seawater and sea bittern. The selective ion-exchange mechanism of the KCHA zeolite membrane was controlled by a specific K Potassium is an element essential to maintain the growth of plants. Among the many plant nutrients, potassium plays an especially critical role in all living organisms . About nZeolites are regarded as important ion-exchange materials for potassium extraction and are widely used in selective separation because of their high ion-exchange capacity and specific surface area ,11,12,13+ was studied in detail at room temperature. In this work, a KCHA zeolite membrane was prepared and characterized using X-ray diffraction (XRD), energy dispersive X-ray spectroscopy (EDS), and scanning electron microscopy (SEM). Thus, the performance of the KCHA zeolite membrane for extracting potassium from seawater and sea bittern were studied for the first time.At present, zeolites usually need to be granulated in practical applications, which causes a reduction in purity and in the potassium extraction performance of zeolite. In addition, the regeneration of zeolite needs a high temperature and a long time, resulting in the high cost of potassium extraction. In order to solve the first problem, in this paper, coating technology was adopted to maintain the high exchange performance of zeolite, which is more feasible. In order to solve the second problem, KCHA zeolite membrane coated alumina microspheres with rapid adsorption and desorption for K2), and ammonium chloride (NH4Cl), were purchased from Shanghai Sinopharm Chemical Reagent Co., Ltd. Soluble silicate  was purchased from Qingdao, China. A porous alumina sphere  was purchased from Zhengzhou, China. Seawater and sea bittern were prepared manually , sodium aluminate  was modified with ammonium ions to obtain an ammonium-ion-loaded KCHA zeolite membrane labeled as KCHA-2. The KCHA zeolite membrane labeled as KCHA-3 was recovered after KCHA-2 was added to 500 mL of artificial seawater to absorb potassium for 10 min at room temperature. KCHA-3 was modified again with ammonium ions to obtain an ammonium-ion-loaded KCHA zeolite membrane labeled as KCHA-4. When KCHA-2 was added to 25 mL of artificial sea bittern to absorb potassium for 10 min at room temperature, a KCHA zeolite membrane labeled as KCHA-5 was obtained. KCHA-5 was modified again with ammonium ions to obtain an ammonium-ion-loaded KCHA zeolite membrane labeled as KCHA-6. A flow diagram of the potassium extraction and removal process is shown in α radiation. The surface morphology of the KCHA zeolite membrane was examined using SEM , and elemental analysis was performed using EDS .The structural feature of KCHA zeolite was evaluated using XRD  with λ = 1.5418 Å Cu KThe XRD pattern of the KCHA zeolite powder scrapped from the surface of the prepared KCHA zeolite membrane is exhibited in Micrographs of the KCHA zeolite membrane were obtained using SEM. The surface and cross-section SEM photographs are shown in The elemental components of the synthetic zeolite membranes were determined using EDS, and the results are shown in The weight %s of the elements  in KCHA-1, KCHA-2, KCHA-3, KCHA-4, KCHA-5, and KCHA-6 during the potassium extraction and removal processes as shown in The content of potassium in KCHA-2 was very low, indicating that potassium was mostly replaced by ammonium ions. After KCHA-3 and KCHA-5 were modified again with ammonium ions, nitrogen comprised a large proportion of KCHA-4 and KCHA-6. The main ions adsorbed from seawater were potassium and sodium, with very low amounts of magnesium and calcium found in KCHA-3. The main ions adsorbed from sea bittern were also potassium and sodium for KCHA-5. Because the concentration ratio of potassium and sodium in sea bittern is higher than that in seawater, the amount of potassium adsorbed from sea bittern by the ammonium-ion-loaded KCHA zeolite membrane was significantly higher than that of sodium. In sea bittern, the difficulty of separating potassium from sodium was reduced. The potassium ion-exchange capacities of the KCHA zeolite membrane were 32 mg/g in seawater and 77 mg/g in sea bittern. The potassium ion-exchange capacity in sea bittern was more than twice that in seawater. It was basically consistent with the potassium ion-exchange capacity of merlinoite previously reported by us, but it was much higher than that of P zeolite reported by Cao . The conQK (mg/g) is the potassium ion-exchange capacity of the KCHA zeolite membrane, m (g) is the mass of the KCHA zeolite membrane, m1 (mg) is the mass of KCHA-3 (KCHA-5), ω1 is the weight % of potassium in KCHA-3 (KCHA-5), m2 (mg) is the mass of KCHA-4 (KCHA-6), and ω2 is the weight % of potassium in KCHA-4 (KCHA-6).The potassium ion-exchange capacities of the KCHA zeolite membrane were calculated using the formula given below:QK of the KCHA zeolite membrane is shown in QK of the KCHA zeolite membrane did not change noticeably. It was found that the KCHA zeolite membrane possessed great reusability, and it can be sustainably used to extract potassium from seawater and sea bittern.The KCHA zeolite membrane was first modified with ammonium ions and then added to seawater or sea bittern to extract potassium. This was repeated five times. Five + ions, which played a major part in preparing the KCHA zeolite membrane. Potassium ions were first introduced into the KCHA zeolite membrane as target ions. As a result, the ammonium-ion-loaded KCHA zeolite membrane prepared from the modification of the KCHA zeolite membrane had a particular ability to remember K+ ions. The diameter of sodium ions is similar to that of potassium ions, causing sodium ions to be competitive ions with respect to potassium ions when replacing ammonium ions of the ammonium-ion-loaded KCHA zeolite membrane. Sodium ions are major disruptive ions for extracting potassium from seawater and sea bittern [The selective ion-exchange mechanism of the KCHA zeolite membrane is proposed as follows: Potassium hydroxide was used for the preparation of the KCHA zeolite membrane. Potassium hydroxide not only provided alkalinity but also extra K bittern ,26.QK of the KCHA zeolite membrane reached 32 mg/g in seawater and 77 mg/g in sea bittern. The potassium ion-exchange capacity in sea bittern was more than twice that in seawater. The concentration ratio of potassium to sodium in sea bittern was obviously higher than that in seawater, so it was relatively easy to separate potassium and sodium from sea bittern. Potassium ions in the KCHA zeolite membrane were exchanged with ammonium ions in a short time at room temperature. The KCHA zeolite membrane presented fine reusability, and it can be sustainably used to extract potassium from seawater and sea bittern. The KCHA zeolite membrane had the ability of K+ ion memory, allowing for the selective identification of K+ ions. The alumina-sphere-supported KCHA zeolite membrane has the characteristics of rapid potassium extraction and removal at room temperature, and it is expected to be widely used and popularized in the future.The potassium extraction performance of KCHA zeolite membranes was studied for the first time. A KCHA zeolite membrane with a high potassium content of 18.33 wt.% was successfully prepared. The


Antibiotics are frequently prescribed during viral respiratory infection episodes in primary care. There is limited information about antibiotic prescription during the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic in primary care and its association with risk factors for an adverse course.To compare the proportion of antibiotic prescriptions between patients with COVID-19 and influenza or influenza-like symptoms, and to assess the association between antibiotic prescriptions and risk factors for an adverse course of COVID-19.An observational cohort study using pseudonymised and coded routine healthcare data extracted from 85 primary care practices in the Netherlands.Adult patients with influenza and influenza-like symptoms were included from the 2017 influenza season to the 2020 season. Adult patients with suspected or confirmed COVID-19 were included from the first (15 February 2020–1 August 2020) and second (1 August 2020–1 January 2021) SARS-CoV-2 waves. Proportions of antibiotic prescriptions were calculated for influenza and COVID-19 patients. Odds ratios (ORs) were used to compare the associations of antibiotic prescriptions in COVID-19 patients with risk factors, hospital admission, intensive care unit (ICU) admission, and mortality.The proportion of antibiotic prescriptions during the first SARS-CoV-2 wave was lower than during the 2020 influenza season (9.6% versus 20.7%), difference 11.1% . During the second SARS-CoV-2 wave, antibiotic prescriptions were associated with being aged ≥70 years , the number of comorbidities , and admission to hospital  or ICU .Antibiotic prescription was less common during the SARS-CoV-2 pandemic than during influenza seasons, and was associated with an adverse course and its risk factors. The findings suggest a relatively targeted prescription policy of antibiotics in primary care during COVID-19. Antibiotics are frequently prescribed during viral respiratory infection episodes in primary care to treat a presumed bacterial superinfection. This may also have occurred during the SAR-CoV-2 pandemic to treat COVID-19. To date, there is limited information on patterns of antibiotic prescription during the SARS-CoV-2 pandemic in primary care. This study shows antibiotics were less frequently prescribed in primary care during the SARS-CoV-2 pandemic compared with preceding influenza seasons. This likely points to more appropriate prescription of antibiotics when guided by dedicated diagnostic tests. Antibiotic prescription was associated with a more severe course of COVID-19, as would be expected. This suggests that GPs are able to estimate the risk of an adverse course. Antibiotics are often prescribed by GPs to reduce morbidity and mortality owing to these bacterial superinfections, particularly in the presence of certain risk factors. Influenza is a recognised major seasonal cause of viral RTIs and a trigger comparable with SARS-CoV-2 with regard to the risk of bacterial superinfections.The new SARS-CoV-2, like all viral respiratory tract infections (RTIs), carries a risk of bacterial superinfection. Another downside is the occurrence of potential side effects of antibiotics. Prudent antibiotic prescription is therefore still indicated and should be sustained in the current pandemic circumstances to reduce the risk of inappropriate antibiotic prescriptions to avoid unnecessary harm.There is limited information on the extent of antibiotic prescriptions in COVID-19 patients in primary care and the associations of these prescriptions with outcomes of interest. The main disadvantage of the use of antibiotics is the development of antimicrobial resistance (AMR).Antibiotic prescriptions were compared during recent influenza seasons with those of the first and second SARS-CoV-2 waves in The Netherlands. In addition, associations between antibiotic prescriptions and hospital admissions, ICU admissions, mortality, and various known risk factors were calculated.For this observational study, pseudonymised, coded routine healthcare data were used from patients enlisted between 2016 and 2020 with one of the 85 general practices participating in the Extramural LUMC Academic Network (ELAN) medical registry, operating out of the Leiden and The Hague area. GPs involved in this network provide complete and actively updated longitudinal data on their patients via their electronic medical records (EMRs). An informed opt-out procedure for the use of these pseudonymised data is in place.Patients aged ≥18 years with influenza, upper RTIs, or flu-like symptoms were identified in the ELAN registry by searching the dossiers for the International Classification of Primary Care first edition (ICPC-1) codes . PatientBox 1An influenza season is defined as more than 51 patients per 100 000 inhabitants with influenza-like illness or symptoms visiting their GP.For season 2019–2020, the threshold was 58 patients per 100 000 inhabitants per week.2017: 28 November 2016 up to including 6 March 2017.2018: 11 December 2017 up to including 9 April 2018.2019: 10 December 2018 up to including 11 March 2019.2020: 27 January 2020 up to including 15 March 2020. The first wave lasted from 15 February 2020–1 August 2020. The second wave lasted from 1 August 2020–1 January 2021. The SARS-CoV-2 index lineage was dominant in The Netherlands during both waves.The following two definitions for diagnosis of a COVID-19 infection were accepted: (1) COVID-19 confirmed with a positive polymerase chain reaction (PCR) test and an appropriate ICPC code in the EMR ; and (2)Box 2 CoughingColdSore throatShortness of breath while resting or during light exertionLoss of taste or smellFeverSudden fatigueDiarrhoeaHeadacheConjunctivitisMuscle and joint painsThe Anatomical Therapeutic Chemical Classification System code J01 was used to identify and extract data on oral antibiotic prescriptions from the ELAN registry. Prescriptions were linked with patients with influenza and patients with COVID-19 through the pseudonymised patient numbers following a check that the date of the antibiotic prescription corresponded with the registration date of the ICPC-1 code. If the date of the antibiotic prescription and the registration date did not correspond, the antibiotic prescription was not included.An adverse course of COVID-19 was defined in the study as a hospital admission, ICU admission, or mortality. Data on this adverse course were extracted from the EMR in the ELAN registry through examination of the free text in the EMR of each patient with COVID-19. Included risk factors were as follows: age, sex, obesity, smoking, heart disease, diabetes mellitus, severe chronic respiratory disease, HIV infection, severe renal disease, severe liver disease, and Down's syndrome. The definitions are listed in Risk factors tested for association with a severe course of COVID-19 were based on the definition by the Dutch National Institute for Public Health and the Environment  and outcomes of recent literature reviews on risk factors for an adverse course of COVID-19.The outcome measures were as follows: (a) number of antibiotic prescriptions and (b) proportion of patient contacts resulting in antibiotic prescriptions during influenza seasons 2017–2020 and during the two waves of the SARS-CoV-2 pandemic (2020); (c) the number of hospital admissions; (d) ICU admissions; and (e) deaths among patients with COVID-19.t-tests. Association testing between risk factors and outcome measures was performed using multivariate logistic regression with age, sex, obesity, and smoking added to the model as covariates with the additional risk factors, heart disease, diabetes mellitus, severe chronic respiratory disease, HIV infection, severe renal disease, severe liver disease, and Down's syndrome, merged into a composite comorbidity variable. For calculation of this composite variable, the presence of each risk factor or disease was counted as one and added together as a count variable. The multivariate logistic regression model tested the associations between these risk factors and outcome measures (a and b) antibiotic prescriptions, (c) hospital admissions, (d) ICU admissions, and (e) mortality.For comparison of extent of antibiotic prescription between SARS-CoV-2 waves and influenza seasons, the number of antibiotic prescriptions and proportion of patient contacts resulting in antibiotic prescriptions were compared via unpaired Multiple imputation was used to address missing data for risk factors smoking and obesity. The imputation model included all covariates and outcomes (details of multiple imputation model in supplement 1). SPSS statistics (version 25) was used for statistical analysis.In total, 1702 patients were diagnosed by their GP with suspected or confirmed COVID-19 in the first wave of 2020 with 6904 patients diagnosed in the second wave . The totSimilar effect estimates were found with multivariate logistic regression using original or pooled imputed data. Therefore, results from multivariate logistic regression with pooled imputed data are presented. During the second wave, an antibiotic prescription was positively associated with an age of ≥70 years , the number of comorbidities  , a hospiIn this study, the frequencies of antibiotic prescription during SARS-CoV-2 episodes were compared with those of preceding influenza episodes. Antibiotic prescriptions were found to be less frequently used in primary care during SARS-CoV-2 waves than during influenza seasons 2017 up to and including 2020. Antibiotic prescriptions during the second SARS-CoV-2 wave were associated with older age, the number of comorbidities, and also with hospital or ICU admission later. This association was not observed during the first wave. However, these studies include different symptoms and diseases, which makes them difficult to compare directly. The prescription of antibiotics was less common during the SARS-CoV-2 pandemic in The Netherlands compared with the rates recorded for RTIs pre-SARS-CoV-2.In the study population, antibiotics were prescribed for 20–30% of patients with influenza-like illness or influenza. This may, according to the guidelines, be interpreted as inappropriate prescription. Other Dutch studies likewise show excessive antibiotic prescription during viral RTI episodes by GPs.et al during the SARS-CoV-2 pandemic (7.1%).The proportion of antibiotic prescriptions per contact for COVID-19 during the first wave (9.6%) was comparable with antibiotic prescribing in the management of RTI symptoms in Dutch primary care reported in a study of van der Velden  The relatively higher frequency of antibiotic prescriptions during the first wave may partly be owing to registration bias, as not all COVID-19 patients during the first wave were registered. Another reason for the less frequent prescription of antibiotics during the second wave may be the increasing knowledge on disease course and risk factors for severe deterioration of COVID-19. Further, there were fewer non-COVID RTIs during the SARS-CoV-2 pandemic. The high probability of a SARS-CoV-2 infection combined with accessible PCR testing aids the GP with diagnostic accuracy and likely decreases antibiotic prescription.In the present study, the total sum of antibiotic prescriptions during SARS-CoV-2 did not differ much between the first and second waves. This, in spite of the burden of the SARS-CoV-2 pandemic being higher during the second compared with the first wave, reflected by the higher number of hospital admissions for COVID-19 patients in The Netherlands.A strength of the study is the comparison of antibiotic prescriptions during influenza seasons with those during the SARS-Cov-2 pandemic. Influenza was already a major seasonal cause of viral RTIs and antibiotic prescriptions, and now SARS-CoV-2, at least initially, may have the same effect on GPs' prescribing behaviour in primary health care. Influenza patients and patients with COVID-19 present with similar symptoms. Therefore, the initial assessment does not differ between the two diseases. However, the study revealed increasing differences in antibiotic prescriptions, which may reflect increasing experience among physicians in judging disease severity, or better estimates of potential adverse disease course development. Until 1 June 2020, there was a lack of PCR-testing capacity in The Netherlands. As a consequence, only patients with COVID-19 symptoms assessed at an emergency department were tested. Until June 1 2020, GPs mainly based a COVID-19 diagnosis on the medical history, patient characteristic, and reported and observed symptoms. Patients were advised to contact their GP if they experienced severe symptoms. This led to under-registration of COVID-19 patients in the first wave, leading to a higher proportion of patients with a severe course of COVID-19 being registered. From June 2020 onwards, all patients with symptoms could be tested for SARS-CoV-2 by the municipal health services and test results were quickly passed on to GPs. But patients could have to wait up to 3 days before a PCR test was performed and the results were passed on. Meanwhile, they may have contacted their GPs, leading to a registration of suspected COVID-19.The results of the study may be hindered by registration bias as not all COVID-19 patients were registered (correctly) before 1 June 2020. The gold standard for diagnosing COVID-19 patients is a positive PCR test from a nasal and throat swab. As such, the analysis of the second wave was addressed in the primary discussion.At the start of the SARS-CoV-2 pandemic in The Netherlands, patients with (suspicion of) COVID-19 were not uniformly registered in the EMR with the same ICPC code. A separate ICPC code, R83.03 SARS-CoV-2, was introduced in November 2020, and slowly implemented. Most patients were registered according to their 'influenza-like' symptoms. For this reason, patients aged ≥18 years with the ICPC codes listed in It was found antibiotic prescriptions were given less often during SARS-CoV-2 waves compared with influenza seasons. This may be owing to proper testing of patients for COVID-19, along with a coinciding lower prevalence of influenza and other respiratory viruses, leading to less diagnostic uncertainty about potentially missing a bacterial infection. This may have led to more confidence in the diagnostic accuracy among physicians and hence to communicating a diagnosis to a patient with more certainty.As a result, antibiotics to prevent or treat a possible bacterial superinfection were largely restricted to those assessed to be at risk of developing or having a more adverse course of COVID-19. Since COVID-19 testing might be the most probable explanation of increased appropriateness in antibiotic prescriptions over time, rapid point-of-care tests for influenza and other viral RTIs may further reduce diagnostic uncertainty and result in fewer antibiotic prescriptions during viral RTI episodes. A Dutch study in primary care has already suggested that point-of-care testing for patients with RTIs may decrease antibiotic prescriptions.In conclusion, this study confirmed that a high proportion of patients with influenza in the past four seasons were treated with antibiotics by their GP. In contrast, the rate of antibiotic prescription in primary care during the first two waves of the SARS-CoV-2 pandemic in The Netherlands was lower than the influenza seasons studied. Patients with COVID-19 who were prescribed an antibiotic were more likely to have risk factors and more often experienced an adverse course of COVID-19, as is shown by an increased number of hospital or ICU admissions among those prescribed antibiotics. These observations suggest a relatively targeted antibiotic prescription policy during COVID-19, but also clearly suggest that inappropriate antibiotic prescription would potentially decrease further with diagnostic testing for other specific viral infections.


The efficacy of standard operating procedures (SOPs) for the decontamination of ambulances against SARS-CoV-2 has been debated. In Italy, the differential use of ambulances was implemented by regional health authorities, with selected vehicles being used exclusively for transporting COVID-19 patients. We investigated the presence of SARS-CoV-2 on high-touch surfaces in ambulances to assess contamination dynamics and the effectiveness of decontamination SOPs. Four high-touch surfaces were sampled before and after decontamination . The gloves of the EMS crew chief were also sampled. RNA extraction was performed with a commercial kit, followed by RT-qPCR molecular detection of SARS-CoV-2. A total of 11 transports were considered. Seven transports had at least one positive sample; all were related to a COVID-19 patient. Three of the negative transports had dealt with COVID-19 case, and one had dealt with a COVID-19-negative patient. One door handle and one oxygen knob were positive at T0, with negative T1 swabs. The monitors were positive in 5 transports at T0, yet they were never positive at T1. Three stretcher handles tested positive at T0, and two of them also at T1, possibly having bypassed decontamination during personnel dismounting. Gloves were contaminated in five transports, in which 1 to 3 additional samples  resulted as positive. Overall, the efficacy of decontamination SOPs was confirmed under the unprecedented conditions of the COVID-19 emergency. However, the importance of correct hand-hygiene and glove-disposal should be further emphasized through the dedicated training of EMS personnel. Shortly after the onset of the ongoing coronavirus pandemic, concerns were raised about the efficacy of standard operating procedures (SOP) for ambulance decontamination operated by emergency medical services (EMS) crew, following the transport of both COVID-19 patients and persons under investigation (PUI). Given the degree of evidence-based uncertainty and the unprecedented burden on healthcare systems, a possible decline in the effectiveness of decontamination SOPs was hypothesized.Prepandemic literature on the bacterial contamination of ambulances dates back to the early 2000s . LateIn addition, considering that EMS crews were at the forefront of the fight against COVID-19, tiredness, fatigue, and a possible lack of time and resources were recognized as additional risk factors. Moreover, insufficient compliance on behalf of EMS crews with decontamination SOPs had been reported even before the pandemic . The stuCriticisms concerning the lack of adequate training programs for ambulance personnel were also emphasized . Indeed,In the pandemic scenario, the prompt implementation of prevention and control measures against SARS-CoV-2 resulted in being fundamental. Possible transmission mechanisms for SARS-CoV-2 include the inhalation of contaminated carriers ; the deposition of such carriers onto mucous membranes; and contact of mucous membranes with contaminated hands or environmental surfaces and objects . ShiftinTo the authors’ knowledge, only a few works are available in the literature that discuss the possible viral transmission pathways through contaminated surfaces in ambulances ,14,15. TThe present investigation was carried out in the Veneto Region  during the first stages of the COVID-19 pandemic (10 March 2020–30 April 2020). Two EMS bases were involved in the study: the Italian Red Cross Committee of Selvazzano Dentro (province of Padova) and the Emergency Department of Bassano del Grappa (province of Vicenza). Convenience sampling of COVID-19 EMS transports was performed by adopting a set of real-time decisional processes from information provided by the EMS Operations Center. One heart-failure transport was also considered, serving as an experimental blank. Transported patients were not directly involved in the study, and their sensitive personal data were not accessible, nor needed, by the investigators. Results of SARS-CoV-2 molecular testing performed upon hospital admission were also recovered for each patient for experimental completeness.For each EMS base, one crew that expressed a willingness to participate in an internal quality improvement initiative attended a 1-hour training on COVID-19 surface sampling techniques, held by the Laboratory of Hygiene and Applied Microbiology (LIMA) of the University of Padova. The training included a theoretical webinar, a dedicated surface-swabbing video tutorial and a conclusive practical session. The experimental details and the precise scope of the study were divulged to neither the EMS trainees nor the decontamination technicians, so as to adopt blinded-experiment precautions and to reduce the risk of experimental bias arising from the awareness and knowledge of the field investigators.® swabs  were used for sampling. The swabbed surface area was 25 × 25 cm for flat items  and consisted of the whole outer surface for non-flat objects . After sampling, the swabs were refrigerated at 4 °C and processed within 24 h.As a reference the WHO’s environmental surface sampling protocol for SARS-CoV-2 was adopted . Four hiTM XenoTM IPC, Thermo Fisher Scientific, Waltham, MA, USA) was added to each sample prior to extraction, so as to assess the extraction process efficiency and the presence of PCR inhibitors. The abovementioned kit also contained a dedicated, real-time PCR assay. Molecular detection of SARS-CoV-2 RNA was performed with a previously published RT-qPCR assay, targeting gene N with a limit of detection (LOD) of 2.5 genome copies/µL [Swabs were thoroughly vortexed for 15 s. Viral nucleic acid extraction was then performed on 140 µL of the vortexed sample with a commercial kit , following the manufacturer’s instructions, and yielding 60 µL of RNA extract. An internal RNA positive control  had at least one positive surface swab: all were related to a confirmed COVID-19 case. Among the 5 negative transports, 1 dealt with the heart-failure patient and 4 with confirmed COVID-19 cases.Among the 6 positive transports, when considering the pre/post decontamination variable, the monitor resulted as the most frequently contaminated surface at T0 (83.3% of transports) although it always tested negative at T1. Three stretcher handles tested positive at T0 (50.0%), and two of them maintained positivity at T1. One sliding-door handle and 1 oxygen knob were positive at T0 (16.7% each), while all of their respective T1 swabs tested negative.The EMS crew chiefs’ gloves resulted as contaminated in four transports (66.7%). Whenever gloves scored positive, 1–3 additional surfaces  also resulted contaminated by SARS-CoV-2.The Essential Assistance Levels (LEAs) are the services that the Italian National Health Service provides to Italian citizens, free of charge or upon participation, using public funds collected through taxation. The LEAs also describe the territorial EMS network, which in Italy is mainly entrusted to the public 118 Health Emergency System. Currently, the unique European Emergency Number, 112, in which various types of distress calls can converge, is active in only about half of the country’s administrative regions.Specific organizational choices and the programming of EMS services fall within each region’s competence to meet local peculiarities. Operating models for EMS services can thus slightly differ from region to region. Some entrust the EMS management to a specific regional agency. Elsewhere, the coordination and interlacing of distinct health agencies have been established. Finally, some Italian regions have adopted a centralized management model, which appears to correlate with a greater degree of autonomy and management efficiency.The present study was conducted in the Veneto region , where the organization of EMS services is coordinated by seven Operations Centers (OCs), each located in a distinct province. Within each province, there are multiple Territorial Stations from which emergency vehicles depart in response to the received distress calls. OCs coordinate the whole sequence of activities, ensuring the transport of patients to the nearest and most appropriate hospital. OCs also assess the degree of complexity of the interventions to dispatch the most appropriate resources, using shared procedures and protocols. The degree of complexity of the intervention is defined by the dispatch system. This specific OC function encompasses the different phases of the rescue, starting from the reception of the distress call, up to the on-site arrival of the basic or advanced life-support crews.Since the beginning of the COVID-19 pandemic, the Veneto regional health system has promoted a differentiated use of ambulances. A set number of emergency vehicles was exclusively engaged for either COVID-19-confirmed cases or PUIs. Nevertheless, should the effectiveness of decontamination be inadequate, non-infected patients and EMS crews could have been exposed to SARS-CoV-2 during the transport.The present study aimed to assess the effectiveness of the national SOPs for the decontamination of ambulances, adopting a pre-/poststudy design, in which selected environmental surfaces were sampled both before and after selected EMS transports. Proper ambulance decontamination is known to drastically reduce the microbial contamination of surfaces touched by patients, especially those with a high viral load. Past studies have also revealed the risk of the microbial contamination of high-touch surfaces in the ambulance setting by the EMS crew, resulting in potential healthcare-associated infections (HAIs) . On the In the present study a non-COVID-19 EMS transport was considered  for analytical reference; no positive environmental sample was detected at either T0 or T1. The ten remaining transports concerned COVID-19 confirmed cases, except Transport 2, since investigators could not retrieve the result of the SARS-CoV-2 testing. Four out of the ten COVID-19 transports  were characterized by negative environmental samples, both at T0 and T1, whereas 6 ambulances had at least 1 positive environmental swab, supporting the SARS-CoV-2 contamination of the vehicle’s cabin. The rational use and removal of personal protective equipment (PPE) for coronavirus disease, both for healthcare workers and for transported patients, are eligible as the main factors preventing the environmental contamination of the ambulance setting  and MTAIAt T0, Transport 1 had three positive samples , plus the gloves of the crew chief. However, no positive swabs were found at T1, suggesting the correct accomplishment of the decontamination SOPs. Transport 2 and Transport 4 both shared a similar pattern: the gloves, monitor, and stretcher were positive at T0, and the positivity of the latter was also maintained at T1, possibly due to the stretcher being dismounted from the ambulance and thus bypassing the correct decontamination SOPs. The remaining three EMS transports had at least one positive sample at T0, but none at T1: Transport 5 had positive gloves and monitor; Transports 7 and 9 showed positivity for the stretcher and monitor, respectively.Switching to a sampling-point perspective, it emerged that SARS-CoV-2-positive gloves always resulted in the contamination of the ambulance cabin. Overall, this finding reprises the critical role of correct hand hygiene and PPE use. Some pre-pandemic studies have already discussed how EMS personnel could mediate the transmission of microbial pathogens if hand hygiene is not performed correctly . In addiOnly one sliding-door handle resulted as contaminated, marking this sampling point as at low contamination risk. Consistently, the opening and closing of the vehicle’s sliding door is usually performed by the driver, who has no direct interaction with the transported patient. Similarly, the oxygen knob seemed to be a low-risk sampling site, with only one positive swab at T0. However, since we could not retrieve any information on oxygen usage during each transport, correlating to the severity of the transported patient, the actual contamination risk for the knob remains uncertain.The multiparameter monitors (positivity at T0 in 5 transports) and the stretcher handrail (positivity at T0 in 3 transports) emerged as the surfaces with the highest probability of contamination, especially if the gloves of the crew chief were infected, too. However, the monitor decontamination always resulted successfully, whereas two out of three EMS transports also maintained positivity for the stretcher at T1. On the one hand, the flat and smooth monitor surface can be easily decontaminated. On the other hand, the haste of EMS operations during patient dismount, together with the juxtaposed actions of multiple healthcare workers, probably interfered with the correct stretcher decontamination.Thus far, the present study has the following two limitations: First, it would have been preferable to conduct field investigations on a larger number of EMS transports and, especially, to recruit more than two EMS bases/crews, so as to lessen the effect of possible operator bias. By enlarging the sampling activities, we could also strengthen our inferences on possible faults of decontamination SOPs over a wider territory; second, the severity of each patient’s health condition during the EMS transport was not available, and we could not evaluate its influence on surface contamination nor on the accuracy of the decontamination procedures .Research perspectives, strongly supported by the authors, should therefore include further field research to increase pragmatic knowledge on the proper meeting of hygienic quality requirements for the particular indoor setting of ambulances and EMS vehicles. In particular, the definition of specific methods to perform the assessment of microbiological air quality in the ambulance cabin is needed.Since the early stages of the COVID-19 pandemic, effective decontamination SOPs have played a crucial role in preventing SARS-CoV-2 MTAIs on ambulances. Moreover, the proven persistence of SARS-CoV-2 on surfaces in hospital settings has highlighted the importance of hygiene fundamentals to control the virus’s environmental transmission. The present study suggested how theoretical efficiency  of decontamination SOPs can be considered safe. Nevertheless, their effectiveness  seemed to be easily flawed by the cursory behaviors of the EMS crews, such as imperfect hand hygiene and hasty stretcher management.Improving compliance with correct SOPs appears to be the most efficient and achievable approach. The dedicated and continuous training of EMS personnel and the consolidation of critical decontamination steps should be supported and promoted by local EMS bases under the guidance of the regional health authorities. Moreover, additional environmental evaluations  and scheduled monitoring programs should be encouraged, as complimentary research investigation to further elucidate possible discrepancies between the theoretical efficacy and the pragmatic effectiveness of decontamination SOPs.


Background: Since the SARS-CoV-2 pandemic, lateral flow assays (LFA) detecting specific antibodies have entered the market in abundance. Despite being CE-IVD-labeled, the antigenic compounds of the assays are often unknown, the performance characteristics provided by the manufacturer are often incomplete, and the samples used to obtain the data are not detailed. Objective: To perform a comparative evaluation of nine lateral flow assays to detect IgG responses against SARS-CoV-2. For the evaluation, a carefully designed serum panel containing post-infection samples and post-vaccination (both mRNA vaccine and inactivated virus vaccine) samples was used. Results: The sensitivity of the assays overall ranged from 9 to 90.3% and the specificity ranged from 94.2 to 100%. Spike protein-containing assays performed generally better than the assays with only nucleocapsid protein. The sensitivity of some assays was higher on post-infection samples, while other assays had a higher sensitivity to post-vaccination samples. Conclusion: A comparative approach in the verification of LFAs with an adequately designed serum panel enabled the identification of the antigens used in the assays. Sensitivities differed between post-infection and post-vaccination samples, depending on the assays used. This demonstrates that the verification of assays must be performed with samples representative of the intended use of the assay. A novel coronavirus first identified in Wuhan, China, in December 2019 has led to a pandemic, eliciting unprecedented global measures .Testing strategies, in addition to control and treatment strategies, are crucial in fighting the pandemic. The early detection of the virus in both the symptomatic and the asymptomatic population is critical to accurately identify and isolate cases in an attempt to limit the spread of the virus. Methods based on molecular principles or antigen testing are mostly used for this purpose . At the To date, there is still no consensus on the use of serology, and the proposed protection thresholds vary according to the study. Nevertheless, it is important to deepen the knowledge of all analytical tools used in order to define diagnostic strategies adapted to any epidemiological, logistical, and economic scenario.Before the appearance of antigen tests, lateral flow tests (LFAs) allowed serological tests to be performed at point of care (POC) quickly and inexpensively. However, they must meet certain quality criteria—e.g., having a specificity >98% and a sensitivity >95%  to accelIn the literature, several studies can be found concerning SARS-CoV-2 LFAs ,7,8,9. TIn this study, we determined the performance of nine commercially available SARS-CoV-2 lateral flow assays. We selected four tests that were available in Europe and previously evaluated by one of the authors (Dr. Giuseppe Togni) for his activity in the company Unilabs SA (Switzerland) and randomly selected five from a large panel of available tests. All tests were analyzed using a sample panel consisting of 219 samples characterized as positive (reactive with anti-spike and anti-nucleocapsid IgG ELISA tests for infected patients and reactive with anti-spike IgG ELISA test for vaccinated patients) and 69 samples characterized as negative (non-reactive with ELISA tests). This allowed for a direct comparison of the different characteristics of the tests. Taken together, our study highlights the need to standardize the comparative approach and shows that the verification of these assays in the context of their intended use is needed.Cohorts: Samples (taken between 5 May 2020 and 12 July 2021) from five patient cohorts were used for this study. The first panel was from patients who were followed after a primary COVID-19 infection (35 patients). A second panel was from health personnel who wished to participate in a serological control study (32 positive and 69 negative samples defined following the criteria described below). The third panel included people who took part in a seroprevalence study in the Italian Graubünden (36 patients). The other two panels were from vaccinated patients, which included one panel of patients vaccinated with mRNA vaccines (Comirnaty COVID-19 from Pfizer (22 patients) and Spikevax from Moderna (30 patients)), and a panel of patients vaccinated with inactivated virus vaccine ). Characteristics of the samples are given in The choice of samples was made to compose a panel with potentially positive samples from both infected and vaccinated people, supplemented with negative samples. All the samples were first analyzed with two immuno-assay tests . The criterion for determining whether a patient had been infected (positive sample) was as follows: reactive with anti-spike and anti-nucleocapsid IgG ELISA tests. Samples from vaccinated patients were analyzed to verify the presence of anti-spike antibodies. By these criteria, 8 samples from the infected groups were excluded and 16 samples from the vaccinated groups were excluded from analysis, resulting in 195 positive samples. All patients consented to the study.Immuno-assay procedure: Lateral flow assays (LFAs): The following nine commercial lateral flow assays (LFAs) for the detection of SARS-CoV-2 specific IgM and IgG were analyzed in this study  and a weakly positive test (slightly visible test band). According to the instructions of the suppliers, both results are considered positive in our analysis.ELISA and CMIAassays: Samples were tested to detect anti-spike IgG , anti-nucleocapsid IgG . The analyses were performed following the manufacturer’s instructions (the reaction volumes were 10 μL for the ELISA test and 25 μL for the CLIA test). The reported sensitivity is 96% (ELISA) and 94% (CMIA) for samples from patients with severe infection collected >14 days post-onset, and the reported specificity is 99.8% (ELISA) and 100% (CMIA) [Statistical analysis: A power calculation  based on expected sensitivity  was used to determine the minimum sample size required to assess the sensitivity of several serological tests for COVID-19, using expected binomial exact 95% confidence limits, resulting in an estimated number of positive cases of 45. All the test results were analyzed using R [ using R ,15,16,17Reliable LFAs must meet both specificity and sensitivity criteria. Specificity was determined with 69 control samples, all of which were determined to have negative anti-spike IgG by Euroimmun ELISA or negative anti-nucleocapsid IgG by Abbot immunoassay. Six of the nine assays had a specificity >98% .The sensitivity of the LFAs was determined with 195 positive samples and ranged from 9.0 to 90.3%. Three assays had sensitivities above 80%, but none fulfilled the criterium of >95%. Discriminating the groups in post-infection and post-vaccination showed that sensitivities ranged from 4.6–93.6% . Two assVaccination responses were divided by response after mRNA vaccine versus inactivated virus vaccine, since after mRNA vaccine only antibodies to the spike protein will be generated . The LFAThe highest sensitivities were found for spike-protein-containing assays with post mRNA vaccine samples. The confidence interval for the sensitivity of two assays  included 95%, and for two more assays, it was almost as large .LFAs responses were plotted against the anti-spike IgG levels . If any Displaying the data per vaccine, it becomes evident that, except for the Abnova test, all LFAs showed a clear response correlation with the anti-spike IgG response after vaccination using a virus inactivated vaccine (Sinovac). As expected, IgG generated with the mRNA vaccines  gave positive responses in spike-protein-containing LFAs and negative responses in nucleocapsid-containing LFAs .These similarities and differences are more obvious when observing the correlation matrices. With these correlation matrices, it appears that Ring Biotech, Abnova, and Dynamiker all use nucleocapsid targets exclusively, and the others target at least the spike protein A. The seSupporting these findings, linear model results are presented in the Government guidelines should always be consulted when selecting appropriate LFAs. In addition, independent assessment with predefined conditions applied to a wide selection of kits allows a comprehensive insight into the performance of these kits. In this study, except for the first cohort, for which we had confirmatory PCR, we based our positive/negative selection of all samples with best-in-class quantitative serological assays in a clinically certified environment. Since these assays are known to have very high specificity ,19, the Our assessment succeeded in the classification of LFAs kits in performance and guided our choice for the most performant kit in terms of sensitivity and specificity, independent of the LFAs kit manufacturer’s documentation. There is not a lot of literature about the minimum performance criteria of serological SARS-CoV-2 assays. Van Walle et al. , in theiAlthough all LFAs were able to detect both IgG and IgM, for this study we only included the IgG responses for higher assay robustness. All positive samples had been shown to have positive IgG responses against spike protein (post-vaccine samples) and both spike and nucleocapsid (post-infection) in the immuno-assays. In addition, most samples were taken >20 days after infection or vaccination, so an analysis of the IgM response would not be of additional value.Based on the results obtained with the carefully composed study cohort containing post-infection samples, as well as samples from either mRNA-vaccinated or inactivated-virus-vaccinated people, we could cluster the LFAs with regard to the responses seen. Both post-infection and samples from subjects vaccinated with the inactivated virus are expected to contain anti-nucleocapsid antibodies in addition to the anti-spike antibodies, while sera will contain only anti-spike antibodies after mRNA vaccination. For six assays, we had a claim from the manufacturer about the antigenic target. Two out of six were not correct, in such that apart from the claim that the antigenic target was nucleocapsid protein, spike protein had to be present as well, since we found a high correlation with the semi-quantitative LFA response and the anti-spike IgG response and the heat map clustering of these assays with the assays containing spike protein exclusively. Two of the three assays of which we did not receive any information about the antigenic target contained spike protein according to our heatmap analysis. The third assay with an unknown antigenic target performed very poorly, with sensitivities of less than 20% in all groups. The fact that this assay met the specificity criterium probably reflects the low reactivity of the test system and should not be considered as a pass.In general, the sensitivities of the spike-antigen-containing LFAs were highest when analyzing post-mRNA vaccine samples, reflecting the significantly higher anti-spike IgG responses in these samples compared to the other groups. It cannot be excluded that subjects in the post-mRNA vaccine group may have had a SARS-CoV-2 infection in the past, either symptomatic or asymptomatic. We demonstrated anti-nucleocapsid antibodies in five samples besides the anti-spike antibodies, which must have been induced by infection. However, these samples did not have higher anti-spike IgG titers compared to samples in this group that did not have anti-nucleocapsid antibodies, so it is unlikely that a past infection influenced the results.We did not find any effect of gender, date between sampling and infection/vaccination, or age. However, the different groups were not evenly balanced: we had twice as many females as males, infected people were all under the age of 60, and only the mRNA-vaccinated group contained people over 60.The difficult time of the pandemic induced the emergence in the market of a multitude of available tests with variable specifications ,20, someDespite the technical limitations that we have highlighted in our study, we believe that there may be possible applications for these devices. Studies suggest that monitoring antibody concentrations can be used to optimize vaccination strategies and monitoring of individual patients  by estimating antibody levels . The pro


In Europe, half of people living with HIV (PLWH) present late to care, with associated higher morbidity and mortality. This study aims to assess short- and long-term costs of HIV-care based on time of presentation and identify other factors contributing to higher costs in the first and fifth year after antiretroviral therapy (ART) initiation.We included ATHENA cohort data which prospectively includes 98% of PLWH in the Netherlands. PLWH who initiated ART in 2013 were included and followed over five years. PLWH were divided in three categories based on CD4 cell-count at time of ART initiation: timely presentation (CD4>350cells/μL), late presentation (CD4 200-350cells/μL or >350cells/μL with AIDS-defining illness) and very late presentation (CD4<200cells/μL). The total HIV-care cost was calculated distinguishing ART medication and non-ART medication costs .From 1,296 PLWH, 273 (21%) presented late and 179 (14%) very late. Nearly half of those who entered HIV-care in a very late stage were of non-Dutch origin, with 21% originating from sub-Saharan Africa. The mean cost per patient in the first year was €12,902 , of which about two-thirds due to ART ). ART costs in the first and fifth year were comparable regardless of time of presentation. During the first year on treatment, non-ART medication costs were substantially higher among those with late presentation ) and very late presentation ), compared with timely presentation ). Higher non-ART costs were attributable to hospitalization and co-medication. The total non-ART costs incurred across five years on treatment were 56% and 246% higher for late and very late presentation respectively as compared to timely presentation.Very late presentation is associated with substantial costs, with non-ART costs nearly seven times higher than for those presenting timely. Hospitalization and co-medication costs are likely to continue to drive higher costs for individuals with late presentation into the future. Programs that identify individuals earlier will therefore likely provide significant short- and long-term health cost savings. Late presentation, or accessing care with a CD4 cell-count <350 cells/μL or with an AIDS-defining illness, has several consequences on an individual clinical, public health, and health systems level . On the In 2018, 5.6 per 100,000 people were newly infected with HIV in the European region . DespiteThe comprehensive cost of late presentation has not been assessed with a longer duration of follow-up since the World Health Organization recommended to initiate ART at CD4 cell-count <500μL . We therIn the Netherlands, HIV-care is provided by designated treatment centres As an integral part of HIV-care, the HIV Monitoring Foundation is responsible for prospectively collecting demographic data and relevant HIV and treatment data, as well as data on viral hepatitis coinfection, from PLWH in the Netherlands and receiving care in one of these 27 treatment centres .This data collection is known as the ATHENA cohort which was initiated in 1998 and captures data from >98% of all patients with a diagnosed HIV-infection who are in care in the Netherlands . Th. Th20]. Costs are reported in 2018 Euro and presented as cost per patient per year (using means and standard deviations).At initiation, the cohort was approved by the institutional review board of all participating centers. People entering HIV care receive written material about participation in the ATHENA cohort and are being informed by their treating physician of the purpose of collection of data, after which they can consent verbally or elect to opt-out. Data are pseudonymized before being provided to investigators and may be used for scientific purposes. A designated quality management coordinator safeguards compliance with the European General Data Protection Regulation .In total 1529 PLWH initiated ART between 1 January 2013 and 31 December 2013. After exclusion of PLWH who enrolled the SHM database > 6 months after start of ART and adolescents (<18 years of age), a full year of follow-up was available for 1316 of PLWH . Of PLWHThe majority of PLWH included in our analysis were male, comparable to the overall PLWH population in the Netherlands that year . The majHIV is mainly transmitted among MSM . IndividDuring the first year after ART initiation PLWH with very late presentation used the most healthcare resources . The strAfter the first year of ART initiation the total HIV-care cost per individual is €12,902 , of which €8,250  was for ART medication and €4,652  for all non-ART medication costs . There iSimilar as to timely presentation non-ART costs were mostly attributed to the use of co-medications. However, co-medication costs were twice the amount ) for presentation late and ten times the amount ) for presenting very late. Additionally, hospitalization costs are more common among individuals with late presentation ) and very late presentation ), while negligible for those who presented timely ) .Over the years, the total annual HIV-care cost per individuals remained similar for PLWH with a timely presentation  . AlthougOur results showed that apart from higher morbidity, mortality, and prolonged possibility of transmission, late presentation- and more specifically very late presentation- resulted in considerably higher short-term HIV-care costs. The higher cost per patient that initiated timeously compared to those with a late presentation persisted through to the end of our follow-up period at five years post ART initiation. Although the cost of ART medication comprises the greatest proportion of the total annual HIV-care costs, non-ART costs drive the increasing cost of late presentation. The short-term non-ART costs were nearly twice as high for late presentation and nearly seven-times higher for very late presentation as compared with those who presented timely in HIV-care. Over a five-year period, the total HIV-care cost for late presentation were 56% higher for late presentation and 246% higher for very late presentation as compared to timely presentation.Cost components driving the higher short-term non-ART cost among PLWH presenting late in care not surprisingly mainly included hospitalization and co-medication costs. Given the likelihood of such individuals suffering from conditions requiring frequent and prolonged hospitalization and the use of multiple co-medications. Unfortunately, after five years this increase in costs is still present mainly in the form of increased co-medication costs. This suggests that even though the clinical condition of PLWH presenting later in care improved, over time their health state remains worse than those presenting timely. Similar findings were found by Krentz et al. who found higher continuing medical costs for PLWH presenting later in care related to non-ART drugs .We identified several patient characteristics associated with late presentation such as heterosexual transmission, elderly, and origin in line with other studies , 15, 21.Another strategy which may help in earlier identification of HIV disease, would be through HIV indicator condition related testing , 25. HIVMigrants, from low- and middle-income countries, are a subgroup which are disproportionately affected by HIV in high-income countries and frequently present late for care High levels of HIV-related stigma, unawareness, and limited access to care result in insufficient engagement with HIV prevention and treatment services. Therefore migrants are placed at risk of poor HIV related clinical outcomes . In ordeFree and easy to access self-tests could also lower the barrier for stigma among several risk groups . In addiApart from the individual clinical benefits of timely presentation into HIV-care, our findings stress the importance of earlier identification of HIV-infection from a cost perspective in both the short and long run. Moreover, investing in earlier identification will also results in a decrease of transmission. Importantly, given that an individual presenting late incurs €8,463 and very late incurs €26,404 of additional costs over the first five years on treatment compared to those presenting timeously, these financial resources could be redirected to novel testing and HIV screening programmes. If a new HIV-infection could be identified for that cost, then those testing or screening programmes would quickly become cost-saving, particularly since the cost savings that we calculate here do not include the cost of onward transmission.A major strength of our study is that the population studied can be considered to be highly representative of the PLWH and in care in the Netherlands, as more than 98 percent of those in care consent to their data being collected as part of the ATHENA cohort . As 91% Our study was the first study analyzing the long-term follow-up after ART treatment is recommended for patients with CD4 cell-counts <500 μL . The guiOne of the limitations of this study is that we did not have access to all costs of HIV-care as appointments or procedures performed by a general practitioner are not included. This could result in a slight under-estimation of costs for timely presentation, since they possibly use more care provided by general practitioners. Care provided by a general practitioner is, however, less expensive than hospital care. Moreover, our resource utilization showed that PLWH with timely presentation only have slightly less outpatient visits. Therefore, it would be unlikely that visits to the general practitioner office would make a substantial difference. Secondly, we did not include cost to the patient. If considering costs from the patient perspective, those presenting very late, due to the greater number of inpatient days and outpatient visits incurred, are likely to have incurred more personal costs than those presenting on time. Including costs from the patient perspective would only have further strengthened our conclusions. Lastly, we excluded PLWH who died during the study period as we calculated a full year and consecutive full four years of HIV health care utilization. The number of PLWH who died that presented very late in care was much higher compared to individuals with timely and late presentation even after five years. Although these PLWH utilized less health care costs, due to short hospital admission stay and often not receiving ARTs, they have the worst health care outcome, death. This further strengthens the need for programs focused on early identification of new HIV infections.In conclusion, late presentation can result in substantial additional non-ART-medication costs over one- and five-year time periods. Several patient characteristics as country of origin, transmission route, and age are predictors of presenting late in care highlighting populations where aggressive testing strategies should be implemented, and awareness should be increased, to maximize both individual benefit and reduce costs. Investment in these strategies could improve clinical outcomes, prevent onward HIV transmission, and avoid these long-term excess patient costs.S1 TableCosts are presented per unit in €.(DOCX)Click here for additional data file.S2 TableThe cost of co-medications per day in €. Costs are calculated based on the average of the lowest and highest price of the available medication for a 70kg adult and 2018 list price.(DOCX)Click here for additional data file.S3 TableThe cost of ART per day in €. Costs are calculated based on the average of the lowest and highest price of the available medication and 2018 list price.(DOCX)Click here for additional data file.


Despite entering an endemic phase, SARS-CoV-2 remains a significant burden to public health across the global community. Wastewater sampling has consistently proven utility to understanding SARS-CoV-2 prevalence trends and genetic variation as it represents a less biased assessment of the corresponding communities. Here, we report that ongoing monitoring of SARS-CoV-2 genetic variation in samples obtained from the wastewatersheds of the city of Louisville in Jefferson county Kentucky has revealed the periodic reemergence of the Delta strain in the presence of the presumed dominant Omicron strain. Unlike previous SARS-CoV-2 waves/emergence events, the Delta reemergence events were geographically restricted in the community and failed to spread into other areas as determined by wastewater analyses. Moreover, the reemergence of the Delta strain did not correlate with vaccination rates as communities with lower relative vaccination have been, to date, not affected. Importantly, Delta reemergence events correlate with increased public health burdens, as indicated by increased daily case rates and mortality relative to non-Delta wastewatershed communities. While the underlying reasons for the reemergence of the Delta variant remain unclear, these data reaffirm the ongoing importance of wastewater genomic analyses towards understanding SARS-CoV-2 as it enters the endemic phase. Throughout the COVID-19 Pandemic, many developed and developing communities have relied on surveillance programs to monitor the prevalence/burden of SARS-CoV-2 infection and to define the genetic composition of the circulating SARS-CoV-2 strains ,4. TheseAs the pandemic has begun to enter the endemic phase in many global communities, clinical and public health efforts have largely focused on the Omicron variant. This focus is understandable since the Omicron variant and its associated sublineages have become the dominant strain of SARS-CoV-2 . The expHere, we report the findings of ongoing SARS-CoV-2 wastewater monitoring efforts of a robust partnership between the University of Louisville’s Envirome Institute, the Rockefeller Foundation’s Pandemic Prevention Institute, and the city of Louisville KY. Importantly, these efforts have led to the detection of a series of reemergence events in the city of Louisville KY after the dominant emergence of the Omicron variant, Altogether, these observations indicate that it is important to continue the observation of SARS-CoV-2 community infection by wastewater surveillance and underscore the im-portance of wastewater monitoring to pandemic readiness and response.g for 30 min at 4 °C. The supernatant was discarded, and the pellet was resuspended in 1.1 mL of TRIzol  and transferred to a sterile microcentrifuge tube. After vortexing to ensure complete resuspension, the sample was incubated for 5 min at room temperature and then clarified via centrifugation at 12,000× g for 5 min at 4 °C. The sample was then divided into two new microcentrifuge tubes, with one half of the sample processed further, and the other half archived at −80 °C for later assessment.Aggregate samples from the five major water quality treatment centers servicing the city of Louisville in Jefferson County Kentucky were collected using a composite sampler, with sample volumes collected over a 24-h period with a rate of sampling was 30 mL per 15-min period. The composite samples were then homogenized and aliquoted prior to further assessment. Samples were maintained on ice and processed within 12 h of collection. Each 45 mL sample was initially passed through a 70-micron filter  and PEG-8000  and NaCl were added to a final concentration of 12.5 and 210 mM, respectively. The samples were thoroughly mixed and refrigerated overnight prior to being subjected to centrifugation at 15,000× A total of 500 uL of TRIzol was added to the sample being processed, and the sample was processed using a Direct-zol 96 MagBead RNA kit  according to the manufacturer’s instructions. The RNA sample was eluted into 50 uL of molecular grade water, and RNA cleanup was achieved using the Zymo RNA Clean & Concentrator-5 kit  according to the manufacturer’s instructions. The final sample yield was 62 uL of molecular grade water, and the purity/concentration of the extracted RNA was determined using a NanoDrop 1000. The sample was then divided into aliquots for two purposes- the quantification of viral load via qRT-PCR, and the development of cDNA libraries for next-generation sequencing. From the above samples, cDNA was synthesized using the Superscript IV First-Strand Synthesis System  using random hexamer priming. The RT reactions were assembled as according to the manufacturer’s instructions, with the final RT reaction being 40 uL in volume, 7.5 uL of which was the purified RNA sample from above. The reverse transcription reaction cycling conditions were slightly modified, as recommended by the Swift Biosciences SNAP low input protocol . Briefly, sequential incubations of 23 °C for 10 min, 50 °C for 30 min, and 80 °C for 10 min were used. Libraries were prepared using the Swift Biosciences SNAP low input protocol for SARS-CoV-2 to generate whole genome amplicons. Briefly, 10 uL of cDNA was combined with 20 uL of reaction mix to proceed with the multiplex PCR according to the manufacturer’s instructions. To increase yield, one additional amplification cycle was performed. The resulting PCR amplicons were size selected using SPRIselect beads  at a 1:1 ratio. Purified amplicons were resuspended in 17.4 uL of RNAse-free TE buffer. Individual samples were indexed via PCR using the SNAP Unique Dual Indexing Primers . Again, to increase yield, two additional amplification cycles were performed. The resulting crude libraries were further cleaned using a 0.65 × PEG NaCl purification. The purified libraries were then eluted in 22 uL of TE buffer and stored at −20 °C until needed. Libraries were normalized using Swift Biosciences Normalase 2 nM final pooling protocol, as per the manufacturer’s instructions.Library pools were spike with PhiX were denatured and diluted as per Illumina’s directions. Libraries with a 1% PhiX spike were sequenced using a NextSeq 500/550 Mid Output Kit v2.5 300 cycle kit , with a read length of 2 × 149 bp to provide a target of 1 to 5 million reads per library. The resulting sequence reads were analyzed via a custom bioinformatics pipeline as previously described . Briefly, low quality bases were trimmed using Trimmomatic v0.38, and were then aligned to the NC_045512.2 reference genome using bwa mem v 0.7.17-r1188 [https://COVID-19-in-jefferson-county-ky-lojic.hub.arcgis.com/ (accessed on 16 September 2022)). All public health data used in this study may be found as part of the Publicly available public health data was obtained via the accession of databases maintained by the Department of Public Health and Wellness ; the first week of June 2022 (week 23 of 2022); and the third week of August 2022 (week 33 of 2022), in samples obtained from a single aggregate wastewater quality treatment center. Specifically, as shown in As mentioned earlier, the monitoring of SARS-CoV-2 prevalence and genetic variation in the wastewater of Louisville KY has been instrumental towards a complete understanding of the pandemic in the community ,7. A lonAs the vaccines currently available at the time were known to be effective in limiting the health impacts of the Delta variant, we first examined whether vaccine uptake in the communities of Louisville, KY correlated with the reemergence events of the Delta variant. As shown in To assess whether the reemergence of the Delta strain negatively impacted community health the testing and clinical incidence of SARS-CoV-2 infections with respect to geographic location and time was examined. As shown in To better quantify and understand the impact of the Delta reemergence events on human health we examined the rate of SARS-CoV-2 associated deaths for the time periods in which the Delta variant accounted for >20% of wastewater prevalence. As SARS-CoV-2 deaths typically occur several weeks after the initial diagnosis, a three-week rolling average was applied to determine potential adverse outcomes associated with Delta reemergence events . As showAt this stage of the SARS-CoV-2 pandemic there have been renewed discussions as to the importance of continuing pathogen surveillance efforts using aggregate or sewershed-specific wastewater monitoring. As demonstrated by the data presented here, the SARS-CoV-2 pandemic continues to be a dynamic situation in regard to the circulation of variants of concern/interest, despite the ongoing dominance of the Omicron lineage. Nonetheless, the cost–benefit comparisons for many communities play a significant role in public health decisions, as finite resources must be used to instill the greatest value to the communities which they serve. Previously we have reported that focusing efforts on sampling aggregate wastewater sources, such as wastewater quality treatment centers, provides a community level assessment of SARS-CoV-2 burden and genetic composition in an unbiased manner . Here, wOur SARS-CoV-2 wastewater monitoring group has selected next-generation sequencing over the use of RT-qPCR methodologies to monitor VOI/C prevalence. While similar results may be obtained using RT-qPCR, the next-generation sequencing approach has several notable advantages. First, next-generation sequencing approaches have inherent flexibilities that RT-qPCR cannot provide, most importantly is the capacity to perform unbiased analyses without a need for prior target identification, probe generation, probe validation, and the determination of detection efficiency for each qPCR primer/probe set. As alluded to above, next-generation sequencing enables the detection of known and unknown polymorphism signatures without relying on the specific detection of individual polymorphisms through a targeted means. Thus, wastewater sequencing is superior to RT-qPCR as it is a readily deployable platform that requires no forethought as to target detection. Secondly, next-generation sequencing based approaches build historical catalogs of SARS-CoV-2 wastewater variation data which can be reexamined at later dates to determine if, and when, specific polymorphisms were present. Detection approaches based on RT-qPCR platforms would require reassessment of the individual cDNAs (including the regeneration of new cDNA if supplies were insufficient) to provide such information. Moreover, communities relying on RT-qPCR would need to decide whether continuing with specific VOI/C primer/probe sets in the absence of known infections in the community was an effective use of resources. As the Omicron variant is highly dominant one could envision a scenario where RT-qPCR schemes to detect Delta were discontinued, which would preclude the detection of the events reported here in this manuscript. Third, multiplexing allows for the costs of next-generation sequencing to be defrayed over many samples, driving the per run costs down to the point where per reaction costs are competitive with those associated with RT-qPCR in the long term. Finally, next-generation sequencing enables the identification of novel variants in real time, whereas RT-qPCR detection paradigms do not allow for the unbiased detection of novel variants at any stage of the process. Altogether, it is for these reasons that we posit that the costs and benefits of next-generation sequencing are superior to those of RT-qPCR based approaches. A considerable benefit of the RT-qPCR method is that many communities may lack access to the necessary equipment and expertise for next-generation sequencing, and investments in RT-qPCR technologies require smaller capital investments. Nonetheless, many partners in global public health are actively seeking to remove the financial barriers to these efforts for smaller communities so that the benefits of next-generation sequencing are accessible. As demonstrated by the data above, notable reemergence events involving the Delta strain of SARS-CoV-2 were observed in Louisville, KY after the introduction of Omicron variants into the community. However, it is important to note that efforts to sequence individual clinical specimens in the state of Kentucky have been limited in scope, and thus a complementary set of clinical isolate sequences from the period and location in question does not exist. Hence, we are not able to directly confirm the presence of clinical Delta infections during these reemergence events. While signatures of the Delta variant were readily detected in the community, the overall impacts of Delta reemergence were muted relative to historical expectations deriving from the first Delta wave. While public health data from the afflicted wastewatershed did reflect increases in clinical incidence and mortality during the reemergence events, the overall magnitudes of negative health outcomes were disproportionate suggesting that infections associated with Delta reemergence events are milder than those of the initial Delta wave . The cauSurprisingly, these reemergence events have not been uniformly observed across the Louisville metropolitan area, despite a clear history of interregional spread of SARS-CoV-2 variants in the Louisville communities. This observation is puzzling for several reasons, one of which is that areas with lower relative vaccination rates were not affected by the reemergence of the Delta variant. The precise reason behind this phenomenon is unknown, as the CCWQTC and DRGWQTC (which has the lowest relative vaccination rate of Louisville KY) are neighboring regions that have largely similar demographics, median household incomes, and population densities. Specifically, the CCWQTC serves a community of roughly 56,000 individuals with an estimated number of individual households of 21,500, as per the most current US Census data. These demographic values are in line with those of FFWQTC and HCWQTC with these wastewatershed communities being slightly smaller in comparison to CCWQTC as they serve populations of approximately 32,500 and 31,200, respectively. In contrast, the MFWQTC and DRGWQTC serve much larger and comparatively more diverse populations, with populations of approximately 350,000 and 296,000, respectively. Due to population size, it remains possible that a minority of cases may have a disproportionate effect within smaller wastewatersheds; however, as wastewater sequencing revealed the emergence of the Omicron variant within the MFWQTC community we have confidence that a failure to detect reemergence of the Delta variant in other wastewatersheds was not strictly a function of population size. Regardless, despite the underlying causes being unknown, reemergence of Delta events in the background of Omicron are clearly possible, and capable of negatively impacting community health ,20. CrypWhether reemergence events become more commonplace as vaccine and infection-associated immunity wanes with respect to time remains to be seen; however, the likelihood of such is high.The data presented above indicates that despite the circulation of dominant SARS-CoV-2 strains, prior VOI/Cs may reemerge within a population with demonstrable negative consequences to individual health. As the SARS-CoV-2 standards of care are largely determined on a regional basis, reemergence events may constitute lost opportunities for clinical interventions if the standard lacks consideration of real time variant analysis of wastewater or pathogen sequence analyses in the individual patient. For instance, the shift towards Omicron dominance changed the standard of care to utilize therapeutic monoclonal antibodies with known specific impacts to the Omicron variant . Thus, iWhile the burden posed by SARS-CoV-2 remains ongoing and significant, contemporary outbreaks of Monkeypox and Poliovirus have renewed interest in wastewater pathogen surveillance ,25,26. AAs the COVID-19 pandemic is now globally reaching an endemic status, it is important to continue efforts to characterize infection rates and pathogen genetic variation within communities by wastewater surveillance efforts. Continued monitoring has led to a comprehensive view of pandemic dynamics at a community level and has enabled the deployment of targeted public health interventions to increase public wellbeing. Additionally, a fine resolution of infection over a community level has allowed us to draw conclusions as to how vaccination status may allow or lead to the reemergence and continuation of variants of concern in a community.