In Canada, approximately 12,000 people annually are hospitalized with influenza. While vaccination is the most effective method for reducing the burden of seasonal influenza, the propagation of vaccine virus strains in eggs can result in egg adaption, resulting in reduced antigenic similarity to circulating strains and thus lower vaccine effectiveness (VE). Cell-based propagation methods avoid these alterations and therefore may be more effective than egg-propagation vaccines. We evaluated three different scenarios: (1) egg-based quadrivalent influenza vaccine (QIVe) for individuals <65 years and adjuvanted trivalent influenza vaccine (aTIV) for ≥65 years; (2) QIVe (<65 years) and high-dose QIV ; and (3) cell-based derived QIV  and aTIV (≥65 years) compared with a baseline scenario of QIVe for all age groups. Modelling was performed using a dynamic age-structured SEIR model, which assessed each strain individually using data from the 2012–2019 seasons. Probabilistic sensitivity analysis assessed the robustness of the results with respect to variation in absolute VE, relative VE, number of egg-adapted seasons, and economic parameters. QIVe + aTIV was cost-saving compared with the baseline scenario , and QIVe + HD − QIV was not cost-effective in the majority of simulations, reflecting the high acquisition cost of HD − QIV. Overall, while the incremental benefits may vary by influenza season, QIVc + aTIV resulted in the greatest reductions in cases, hospitalizations, and mortality, and was cost-effective  in all simulations. Influenza is one of the leading causes of morbidity and mortality in Canada, associated with approximately 12,200 hospitalizations and 3500 deaths annually ,5. In CaIn recent seasons, the effectiveness of influenza vaccines has decreased against the A/H3N2 strain, particularly in seasons where there have been antigenic differences between the circulating and vaccine strains ,10,11. W® Quadrivalent, Seqirus Inc, Holly Springs, CA, USA) was first introduced in Canada in 2019 for persons aged 9 years and older and has demonstrated non-inferiority to cell-based TIVs in paediatric and adult clinical trials, with a favourable safety profile )Data on circulating influenza strains from 2012 to 2019 were obtained from Flunet, using specific data for Canada . As no sThe economic model was based on that described in Fisman et al. , using tThree scenarios were compared to a standard baseline scenario of QIVe for all age groups, assuming six unmatched influenza seasons and an rVE for A/H3N2 of 15.6%, as estimated from the pooled retrospective studies analysis for the 2017–2018 seasons. Scenario 1 evaluated the use of QIVe for all individuals aged 6 months to 64 years and adjuvanted TIV (aTIV) for adults ≥65 years. Scenario 2 replaced aTIV with high-dose QIVe (HD-QIV) for adults ≥65 years. Scenario 3 evaluated QIVc for 6-month-olds to 64-year-olds and aTIV for adults ≥65 years. Individuals <65 years of age were assumed to receive the same vaccine, irrespective of whether they were considered low or high risk for influenza. Coverage rates by age group are shown in Stochastic probability sensitivity analysis was performed on vaccine effectiveness, season profile , economic inputs, and the number of egg-adapted seasons. For the simulation, 500 sets of parameters were randomly drawn from a normal distribution, as described previously by Fisman et al. . A ±10% All three scenarios assessed against the baseline scenario resulted in reduced cases, hospitalizations, and deaths from influenza . The greScenario 1 (QIVe+aTIV) was cost-saving compared with the base case scenario, with total direct savings of approximately CAD 20 million . ScenariAssessment of the ICER across different rVE estimates showed that scenario 1 was cost-saving with all three rVE estimates, whereas scenario 2 was above the CAD 50,000 threshold in all three cases . ScenariThese findings were confirmed by sensitivity analysis, which showed that scenario 1 remained cost-saving and that the upper bound of the 95% CI remained under the cost-effectiveness threshold for scenario 3 in all situations assessed . ScenariWhile high levels of antigenic similarity have been observed worldwide for the past 15 seasons (both in the Northern Hemisphere and in the Southern Hemisphere) between vaccine and circulating A/H1N1 strains, egg adaptation of the A/H3N2 vaccine strain is of particular concern for seasonal influenza vaccination . AdaptioPoor VE against A/H3N2, together with the challenges of limited immune response in older adults, has led many countries to routinely recommend enhanced vaccines  for seasonal use in older adults. In Canada, enhanced vaccines are among the vaccines recommended to be offered to adults aged ≥65 years, although standard-dose and unadjuvanted vaccines are also recommended . aQIV isEgg adaption appears to have an important impact on the effectiveness of vaccines against A/H3N2. Analysis in recent years has shown multiple influenza seasons where the circulating A/H3N2 strain was antigenically different to the vaccine virus in QIVe vaccines, whereas this difference was less likely to occur with QIVc vaccines: across the 2012–2016 seasons, 20% of egg-based and 100% of cell-based A/H3N2 vaccine viruses were antigenically similar to circulating strains . OverallSeveral studies over the past three Northern Hemisphere seasons have demonstrated an increased effectiveness of QIVc compared with standard-dose egg-based vaccines, although absolute VE estimates vary by season, in line with the unpredictability of circulating A/H3N2 strains. Part of this observed variability is due to differences in study designs, with retrospective cohort studies showing significantly increased effectiveness of QIVc over QIVe, whereas only trends with large confidence intervals were observed with test negative design studies ,28,43,44While administration of QIVe to all ages may have a limited budget impact, the economic model confirmed that a combination of QIVc and aTIV would be cost-effective in all of the simulations assessed due to lower acquisition vaccine costs and the incremental efficacy in preventing medical visits, hospitalizations, and deaths in the most at-risk populations. This estimate is robust, as we used data collected over several seasons, which allowed us to capture the heterogeneity of influenza epidemiology from one season to another. This approach increased the external validity of the results, as it captured the main uncertainty of the variables that impact the results, i.e., the number of egg-adapted seasons and varying VE. However, there were a number of limitations to our analysis. Although we considered VE by strain, we did not vary VE by age group, and there are currently no data available on QIVc in infants and children <4 years of age. Additionally, the data used are based on the US influenza seasons from 2012–2019 and therefore may not be broadly generalizable across countries with different influenza dynamics or population structures. Thirdly, we did not evaluate the impact of aQIV in older adults, as it is not currently available in Canada. The use of aQIV compared with aTIV would potentially provide increased protection against influenza in this high-risk population and therefore may increase cost-effectiveness estimates when it becomes available in future seasons. Finally, our base-case scenario assumed six egg-adapted seasons out of the eight analyzed. While sensitivity analysis still indicated the cost-effectiveness of the QIVc+aTIV scenario with three egg-adapted seasons and with varying VE, these results may have been different with fewer egg-adapted seasons. In the unlikely scenario of only one egg-adapted season in the eight seasons evaluated, QIVc+aTIV would no longer have been cost-effective. In summary, this analysis has shown that vaccination of 6-month- to 64-year-olds with a cell-based QIV together with aTIV for ≥65-year-olds is cost-effective across varying assumptions of rVE and numbers of egg-adapted influenza seasons. Overall, this vaccine combination resulted in the greatest reductions in cases, hospitalizations, and deaths due to influenza compared with the other scenarios evaluated. While the incremental advantages of QIVc+aTIV will vary between individual influenza seasons, sensitivity analysis reveals that this vaccine combination would be favourable in nearly all scenarios.


SARS-CoV-2 seroprevalence was reported as substantially increased in medical personnel and decreased in smokers after the first wave in spring 2020, including in our population-based Tirschenreuth Study (TiKoCo). However, it is unclear whether these associations were limited to the early pandemic and whether the decrease in smokers was due to reduced infection or antibody response. We evaluated the association of occupation and smoking with period-specific seropositivity: for the first wave until July 2020 , the low infection period in summer , and the second/third wave . We measured binding antibodies directed to SARS-CoV-2 nucleoprotein (N), viral spike protein (S), and neutralizing antibodies at BL, FU1, and FU2. Previous infection, vaccination, smoking, and occupation were assessed by questionnaires. The 4181 participants (3513/3374 at FU1/FU2) included 6.5% medical personnel and 20.4% current smokers. At all three timepoints, new seropositivity was higher in medical personnel with ORs = 1.99 (95%-CI = 1.36–2.93), 1.41 (0.29–6.80), and 3.17 (1.92–5.24) at BL, FU1, and FU2, respectively, and nearly halved among current smokers with ORs = 0.47 (95%-CI = 0.33–0.66), 0.40 (0.09–1.81), and 0.56 (0.33–0.94). Current smokers compared to never-smokers had similar antibody levels after infection or vaccination and reduced odds of a positive SARS-CoV-2 result among tested. Our data suggest that decreased seroprevalence among smokers results from fewer infections rather than reduced antibody response. The persistently higher infection risk of medical staff across infection waves, despite improved means of protection over time, underscores the burden for health care personnel. SARS-CoV-2 antibodies measured in serum of population-based studies’ participants provide insights into the proportion of individuals who have experienced infection. SARS-CoV-2 infection-mediated seroprevalence has been reported to be increased for medical personnel and decreased for smokers in numerous studies ,7,8,9,10Increased seropositivity among medical personnel during the first infection wave in Europe is readily explained by higher infection risk due to occupational exposure. The early pandemic increased infection risk for medical personnel could have been due to limited knowledge regarding transmission routes or limited availability of protective gear. Some early studies suggested that health care workers supplied with appropriate protective gear were not at increased risk for SARS-CoV-2 infection . HoweverThe reasons for the association between smoking and decreased seropositivity are more elusive. In principle, such results could point towards a reduced risk of infection among smokers ,16. HoweHere, we set out to investigate whether the association between working in a medical occupation or smoking with seropositivity was persistent across infection waves. We also aimed to evaluate a possible link between smoking and the development of SARS-CoV-2 specific antibodies after infection or vaccination. For this, we conducted longitudinal analyses in our cohort study from the Tirschenreuth population, the hardest-hit county in Germany during the first SARS-CoV-2 wave. We analyzed the association of smoking and medical occupation status with seropositivity, registered infections, and antibody response after infection or vaccination for three observation periods: pre-pandemic to July 2020 (first wave), July 2020 to November 2020 (few infections), and November 2020 to April 2021 (second/third wave).The study was designed as a cohort study of the population aged at least 14 years living in the Tirschenreuth county, Germany, as described previously . BrieflyLocal health authorities registered 1109 SARS-CoV-2 infections in the county until 4 July 2020, 513 additional infections until 18 November 2020, and 3100 additional infections until 21 April 2021  and fromUsing the pre-sent, written questionnaire in German language, participants provided information on smoking and occupational status. Specifically, participants were asked at BL, FU1, and FU2 whether they were currently smoking cigarettes, have previously smoked, or have never smoked. Current smokers were asked as to how many cigarettes they were smoking, and ex-smokers about their age when stopping. If participants reported to be current or ex-smokers previously and later on to be never-smokers, they were classified as ex-smokers for the later time point. The smoking status in the association analysis reflected the status at blood draw. Participants were asked at BL whether they were employed in February 2020 and, if yes, in which occupation . The options were: (i) medical occupation in medical care, care home, or midwife, (ii) teacher at school or pre-school, (iv) service or cook in food service, (v) cashier or shop assistant at groceries, (vi) hairdresser, (vii) other service with customer contact, or (viii) any other. In our analysis on the association of occupation and N-seropositivity as proxy for previous infection, we were particularly interested in three occupation groups, namely medical staff, grocery workers, and teachers, as for those groups, an increased exposure to the SARS-CoV-2 virus in the work setting is well imaginable. Grocery workers were one of the few occupational groups who were working during the first SARS-CoV-2 wave despite lockdown measures and strong restrictions on work with personal customer contact. Teachers can be suspected to be at high exposure during the 2nd/3rd wave and medical staff potentially over the whole period of the pandemic. From this motivation and with the intention to avoid small subgroups, we classified the occupational status of the study participants into four distinct groups: the three mentioned as well as a fourth group of “others” including all other types of self-reported occupations as well as unemployed individuals. Note that all analyses regarding occupation are restricted to individuals of working age (20–69 years).Via the questionnaire at BL, as reported previously ,21, partBy questionnaire, participants were asked whether they had undergone a SARS-CoV-2 test by trained staff  and whether this test was positive: ever (BL), since 17 July 2020 (FU1), or since 27 November 2020 (FU2). Self-reported positive tests were validated by health authorities. At FU2, participants were also asked whether they were vaccinated against SARS-CoV-2 and, if yes, how often, which vaccine, and when. Individuals were classified as fully vaccinated, if they had two vaccinations by Comirnaty , SpikeVax , or Vaxzevria  ≥ 14 days before their respective blood draw.Sample processing and antibody measurements were reported previously ,21,24. BWhereas S-specific binding and neutralizing antibodies offer insights into possible protection , neitherThus, for the main analysis, seropositivity from infection was judged by N-antibody levels (cutoff = 1.0 Arbitrary Units (AU)/mL): participants N-seronegative at BL and N-seropositive at FU1 were classified as newly seropositive at FU1, and participants N-seronegative at FU1 and N-seropositive at FU2 as newly seropositive at FU2. Antibody response after PCR-confirmed infection or after full vaccination was based on N-, S- (S-cutoff = 0.8 Binding Antibody Units (BAU)/mL), or neutralizing antibodies (cutoff Inhibitory Dose 50 (ID50) = 20, as performed in ). For thAt each of the three observation periods, we analyzed the association of smoking or occupational status with (new) N-seropositivity. Participants were included in these analyses when they had an N-antibody measurement available at the respective time point  and, for FU1 and FU2, when they had N-antibodies measured and negative at the previous time point. For the smoking association, we restricted this to participants with information on smoking status at the respective time point. For the occupation association, we focused on participants at working age (20–69 years old) and analyzed medical staff, preschool/school teachers, and grocery employees vs. any other . We applied logistic regression (R package mgcv ) using tFor the smoking association, we conducted additional analyses: (i) we evaluated antibody response after infection  by smoking status: we analyzed seropositivity and median levels of N-, S-, and neutralizing antibodies at FU2 by smoking status. For the S- and neutralizing antibodies, we restricted this analysis to unvaccinated individuals to exclude distortions due to vaccine-induced antibodies. (ii) We evaluated fully vaccinated participants at FU2 analyzing S- and neutralizing antibody levels measured at FU2 by smoking status. (iii) We evaluated the association of antibody levels with age and time since vaccination using a generalized additive model . We compared this with an extended model adding smoking status and its interaction with age and time since vaccination and tested the null hypothesis of “no association of smoking with expected antibody levels” (approximate F-test). Finally, we evaluated, for each of the three observation periods, the proportion of participants with a health authority-validated positive test among those tested by smoking status and medical occupation and the proportion of tested among all participants.All statistical analyses were performed in the R environment for statistical computing, version 4.1.0 . These analyses included 4181 participants of the longitudinal Tirschenreuth Study with valid measurements of N-antibodies at BL . At BL, participants were aged 14 to 102 years, 51.6% women, 20.4% current cigarette smokers, and 8.0% of the 3303 participants aged 20 to 69 years worked in medical occupation . ResponsSARS-CoV-2 vaccination roll-out in Germany started in late December 2020, yielding the first fully vaccinated individuals at the end of January. During the early phase of the vaccination program, individuals at increased risk of severe disease and individuals at increased risk of exposure were prioritized. As a consequence, we observed increased fractions of vaccinated individuals at FU2 in the higher age groups , we evaluated the association of smoking and occupation status with new N-seropositivity. New N-seropositivity was about half among current smokers compared to never-smokers for all three observation periods. p < 0.001), 0.40 , and 0.56 , respectively. Note that the estimate at FU1 shows large uncertainty, as the overall number of new seropositive cases was rather low, but the point estimate is in line with results from BL and FU2. The smoking association with N-seropositivity was not observed for ex-smokers compared to never-smokers . The current smoking association was underscored by a dose–response association when analyzing the number of daily currently smoked cigarettes at BL and FU2 , 1.41 , and 3.17 , respectively  seropositivity at BL and at FU2 (compared to FU1) remained when adjusted for each other and for age, sex, education, household size, physical activity, alcohol consumption, and BMI (The smoking association with (new) seropositivity at BL and at FU2 (compared to FU1) was consistent across subgroups  to define “infected” individuals independent of antibody-based serostatus and analyzed levels of N-, S-, and neutralizing antibodies at FU2 . We found similar median antibody levels by smoking status . The prop = 0.274 and p = 0.266 for S- and neutralizing antibodies, respectively). Since time since full vaccination was relatively short  and the dose was standardized, this is an ideal model for antibody response. Second, we sought further support of the hypothesis that smokers’ antibody responses resembled those of non-smokers by evaluating response after vaccination as a general model for antibody response. We restricted this to participants fully vaccinated without previous infection . We found similar levels of S- and neutralizing antibodies at FU2 by smoking status A,C, alsoOverall, we found no evidence that current smokers developed fewer quantitative antibodies after infection or vaccination or that smokers were more likely seronegative than ex-/never-smokers based on three different serological tests.As a third piece of evidence, we analyzed the proportion of participants who had tested positive for SARS-CoV-2 infection among those who reported a test for the respective observation period by smoking or medical occupation status. Interpreting these proportions is challenged by the potentially varying testing intensity by smoking or medical occupation status and over time. We thus began by evaluating the fraction of tested participants for the three observation periods : among all participants, the fractions tested were 12.1%, 27.2%, and 45.0%, respectively; this documented the increased testing intensity over time. These testing fractions were similar for current smokers, but medical staff were tested markedly more often A. p ≥ 0.05). Together with the similar testing intensity by smoking status, the lower proportion of positive tests among current smokers supported the idea of fewer infections among current smokers.Next, we evaluated the proportion of positive tests  among tested participants by observation period: this proportion was substantially lower among current smokers than among never-smokers for each of the three observation periods B: age-/sp = 0.81, 0.69, 0.12, The observed increased testing intensity among medical occupations (see above) can distort the comparison of the proportion of tested positives among medical staff compared to other participants. In addition, the higher seropositivity for medical personnel is realistically attributable to more infections and requires no further support. Still, we compared the proportion of positive test reports among those tested between medical staff vs. “other”: we found little difference , occupational role , extent of patient contact, number of working hours, etc. Due to the population-based sample of our participants, the proportions of the specific medical subspecialties among participants roughly reflect the proportions in the medical staff population, and our effect estimates can be considered an average across these subspecialties. A detailed work history and meticulous quantification of patient contact would be warranted in future studies in order to quantify the occupational infection risk more specifically and to identify those at highest risk.The finding of decreased seropositivity among smokers requires more detailed consideration of potential bias and possible reasons. The observed association of smoking with decreased seropositivity was strong, stable across timepoints and subgroups, persistent when adjusted for potential confounding factors, and underscored by a dose–response association. The observed association with odds ratios around 0.5 were similarly observed in previously published seroprevalence studies of different designs ,10,11,12A possible bias can derive from differential participation with respect to smoking and the outcome of interest. We did observe reduced follow-up participation among current smokers compared to never-smokers, but both at a relatively high level with 77% vs. 87% participation at FU1 and 71% vs. 84% at FU2. Our outcome was new seropositivity among those never seropositive until the start of the respective observation period . When coLower seropositivity among smokers can derive from lower infection probability or from lower antibody response. In order to fully explain the strong association, antibody response for smokers would need to be substantially decreased, basically nulled for ~50% of smokers. In our data, smokers and never-smokers showed similar levels of antibody response after infection in a variety of antibody tests. For this analysis, we defined “infection” by having had a previous positive PCR test validated by health authorities, yielding 237 participants with validated infection independent of serostatus. This supports the hypothesis that the decreased seropositivity among smokers observed here and by others was not explained by differential antibody response after infection, but rather due to fewer infections. We also evaluated antibody levels after vaccination by smoking status in a further 287 participants under the notion that vaccination-induced antibody generation is a role model for infection-induced generation where the “exposure” dose is standardized and time since “exposure” is known. A recent meta-analysis  concludeIn contrast to the lack of any support for differential antibody response by smoking status, our data indicated lower infection probability for smokers when evaluating individuals who reported previous SARS-CoV-2 testing: comparing current with never-smokers, the odds of a positive PCR test among individuals having been tested was similarly reduced as the odds of seropositivity, and this was consistent across the three observation periods. Together with the fact that the probability of having been tested did not vary much by smoking status, the magnitude of the observed smoking association with both seropositivity and PCR-detected infection was in line with a ~50% decreased infection risk for smokers. Together, our data suggest that lower seroprevalence among smokers is due to lower infection risk. The reason for this is still elusive. This might be behavioral or biological. Decreased risk of infection among smokers can derive from a smoking-specific behavioral pattern, e.g., leaving indoor gatherings to smoke outside. However, there is no evidence that the association is modulated by socio-demographic or health-related factors: we found similar associations of smoking and seropositivity across age groups, between men and women, with high or low education, individuals with chronic disease or without, and among individuals in medical occupation or not. Our results imply that any behavioral pattern of smokers leading to reduced infection risk was stable across such subgroups and across infection waves, rendering this explanation less plausible. MUC4 and MUC16 were supported by genetic variants that increased mucin expression in cilial lung tissue that might be protective for infection [MUC4−/− mouse models suggest a protective role of mucin 4 during coronavirus pathogenesis [If the reason for the smoking association is biological, it might be smoking-related differential gene expression . Transcrnfection . Cigaretnfection , mucin 5nfection  or MUC16nfection . Early aogenesis ,36. It iMUC4 overexpression has been associated with various types of cancer [If substantiated, smoking will certainly never be a preventive option against infection due to the severe well-known effects on cancer and cardiac risk. Particularly f cancer ,38, whicThe strength of our study is the population-based longitudinal approach covering individuals aged 14 to 102 and the infection occurrence over more than 1 year. Further strengths are detailed participant information on socio-demographic and lifestyle factors as well as several types of antibody measurements. When using antibody status as an indicator of previous viral infection, it is important to consider the potential existence of vaccine-induced antibodies. In the case of SARS-CoV-2, this is particularly important as the early vaccination campaign had to deal with initially limited vaccine availability by prioritizing individuals at increased risk of severe disease and individuals at increased risk of exposure, such as medical staff. In line with this, we observed a larger proportion of medical staff fully vaccinated until April 2021 than other individuals at working age. Using N-specific seropositivity as a marker for a previous infection and restricting the analysis of S- or neutralization antibodies to unvaccinated individuals enabled the focus on infection-related antibodies for our association analyses without vaccination-related distortions.Limitations of our study are typical to cohort studies, including non-perfect response at BL and FUs. However, our response was rather high with 64% at BL and >80% at FUs. The time between BL and FU1 assessments captured a period of low incidence with overall few new infections during summer 2020, yielding low power for detecting statistically significant associations with new seropositivity at this time point of our study. It is nevertheless interesting and noteworthy that we observed consistent, albeit not statistically significant associations at FU1. Another limitation is that infection occurrence was predominantly assessed based on serostatus with limited knowledge of the infection date or viral dose, as for other seroprevalence studies. Our analyses of individuals who had undergone community-based PCR testing allowed for insights independent of serological tests. A strength of seroprevalence studies is that they capture symptomatic as well as asymptomatic infections and are independent of the community testing strategy and its changes over time. The presented results allow for a comparison of risk factors for the winter 2020/21 infection wave with the first infection wave in spring 2020. Our data do not stretch into the current Omicron-induced infections. However, given the multiple infections on top of a mostly vaccinated population at the current time point, these previous pandemic results enable an isolated view of mainly single, first-time infections and might serve as a role model for SARS-CoV-2 infections in principle. Further results from studies with longer follow-up into the current pandemic situation are warranted, but will be substantially complicated by the mix of infections.Altogether, our results are in line with the notion that smoking-related pathways—behavioral or biological—overlap with infection-protective pathways that eventually might inspire behavioral measures and also the development of novel therapeutics. Our results also highlight a continued high infection risk for medical personnel well into winter 2021 despite full protection gear in place.


Contact-based education, offering meaningful contact with individuals living in recovery with mental illness, reduces stigma. This study evaluated the effectiveness of the National Alliance on Mental Illness Provider Education Program (NAMI PEP) when implemented as a curricular requirement across two cohorts of third-year osteopathic medical students, comparing traditional, passive learning and active, online delivery formats.Participants were two cohorts of third-year medical students  who completed questionnaires measuring affect, beliefs, and behaviors toward patients with mental illness at pre-program, 1-week follow-up, and 6-month follow-up. For Cohort 1, the existing community-based NAMI PEP was implemented. For Cohort 2, the program was adapted to an online, active learning format tailored to medical students, and an additional 3-month follow-up assessment was added to better identify intermediate-term effects.The NAMI PEP was associated with longitudinal improvements in target outcomes, with enhanced effects with the adapted curriculum in Cohort 2. At 6-month follow-up, students reported less stigma, fewer stereotyping negative attitudes, and lower anxiety treating patients with mental illness. They also reported increased confidence integrating psychiatry into routine care and increased competence in principles of collaborative mental health treatment.This study demonstrates the longitudinal effectiveness of the NAMI PEP across two cohorts of medical students, with strengthened effects observed when the program is tailored to contemporary medical education. Consistent with mental health stigma prevalent in the general public, physicians report negative affect (emotions), unfavorable attitudes, and low perceived self-efficacy related to working with patients with mental illness (MI) . PhysiciContact-based educational programs, consisting of focused social interactions with individuals with MI, represent promising adjunctive training opportunities for changing negative affect, attitudes, and behavioral intentions related to working with patients with MI . OptimalWithin and beyond the field of psychiatry, existing research evaluating direct contact has several noted limitations . Issues The NAMI PEP is a standardized, 15-hour contact-based program aiming to increase health-care professionals’ delivery of patient-centered and collaborative care for patients with MI. It entails small-group instruction by a team of three NAMI-trained facilitators representing a person living well in mental health recovery, a family member of a person with MI, and a health-care provider with personal or familial experience with MI. It promotes greater understanding of the impact of MI on individuals and their families, with facilitators sharing personal, familial, and clinical experiences with MI and recovery. It also includes didactic presentations, small-group discussions, clinical scenarios, and experiential exercises.Although originally designed for practicing health-care professionals, the NAMI PEP is a promising program for medical students. It incorporates each of the above-referenced optimal conditions for contact-based education and offers inherent implementation and scale potential as a standardized program with existing delivery infrastructure through NAMI affiliates in communities across the USA. In a non-randomized, controlled trial in a self-selected medical student sample, the NAMI PEP was associated with improved affect, beliefs, and behaviors towards patients with MI at one-week and 3-month follow-up . While tThe present study evaluated the longitudinal effectiveness of the NAMI PEP across two cohorts of third-year medical students. In both cohorts, the NAMI PEP was a required course, with participation in this research study of course outcomes being optional. In the first cohort 2019), we implemented the standard NAMI PEP. In the second cohort (2020), a team of medical educators, national NAMI staff, and NAMI PEP facilitators tailored the delivery of the NAMI PEP to medical students’ specific educational needs and to reflect current pedagogical approaches in academic medicine. Specifically, we updated instructional methods to increase student engagement in learning and to integrate content with other educational experiences. Alterations included a flipped classroom model where didactic content was presented in advance via asynchronous e-modules and class time was used for active application of the material; increased amounts of pair share and breakout group discussions; increased use of case examples; integration of content with clerkship experiences; and embedded opportunities for students to document specific lessons to apply in future patient care. Additionally, we recruited psychiatry residents to serve in the health-care provider facilitator roles to enhance students’ perceived credibility and practicability of the material and provide physician modeling. Finally, we delivered the program virtually through synchronous videoconferencing  instead of in person, utilizing recommended best practices for engaging students in this platform [19, we imOverall, the third-year student samples and the content of the NAMI PEP curriculum were consistent across both cohorts, with Cohort 2 receiving the adapted delivery methods described above. Thus, the present study aimed to: 1) establish baseline pragmatic longitudinal effectiveness of the standard NAMI PEP when delivered as a required course to an entire cohort of medical students , and 2) evaluate the effects of adapting instructional methods to align the program with contemporary medical school education . We hypothesized that the adapted curriculum would demonstrate enhanced outcomes across all measures. Our aims address identified gaps in the contact-based education literature, including an extended 6-month follow-up period and utilization of an existing community-based program with potential for national dissemination .During both Cohort 1 (standard) and Cohort 2 (adapted) programs, all third-year students at our osteopathic college of medicine  were required to attend the NAMI PEP. In both cohorts, this required course occurred at the end of the third year, following pre-clinical education in psychiatry and all core clerkships including a four-week psychiatry clerkship. The course was taught simultaneously to groups of 32–37 students, each led by a separate team of NAMI facilitators, to allow for enhanced discussion. We randomly assigned students to these group sections and further randomly subdivided students into smaller breakout discussion groups for certain activities.The NAMI PEP was a curricular requirement at the institution; however, participation in research evaluating program outcomes was optional. Directly preceding the start of the NAMI PEP in each cohort, we invited all enrolled students to participate in the optional research and provided time to complete a pre-program survey before beginning the course. In Cohort 1 (standard), we made an in-person verbal research invitation to students. In Cohort 2 (adapted), the flipped classroom approach necessitated an updated recruitment format, as initial exposure to the NAMI PEP now came during required pre-course asynchronous modules. To ensure completion of pre-program surveys prior to exposure to course content in these modules, students viewed a recorded research invitation prior to accessing the modules. If students agreed to participate in the study, we only provided access to the pre-program survey prior to accessing these modules. In Cohort 1, we assessed participants at pre-program, 1-week follow-up, and 6-month follow-up. Cohort 2 included these same assessment time points, with the addition of a 3-month follow-up assessment to better identify intermediate-term effects. The present study focused on changes from pre-program to 6-month follow-up. We compensated participants $5 for completion of the initial survey and $10 for each subsequent survey. All study procedures were approved by the Des Moines University IRB (IRB-2019-30). All research participation was confidential and voluntary. Names of research participants were not disclosed to course evaluators or linked to responses on surveys.affect, we used Day’s Mental Illness Stigma Scale – Anxiety subscale (DMISS) [beliefs, we used: 1) the Opening Minds Scale for Health Care Providers (OMS-HC) [behavioral intentions, we used: 1) the Attitudes and Confidence in the Integration of Psychiatry Scale (ACIP) [Surveys assessed target domains of affect, beliefs, and behavioral intentions regarding mental illness. Measures were selected based on validity, brevity, and applicability to these constructs of interest. We utilized the following validated measures at all study time points across both cohorts; internal reliability (Cronbach’s α) was calculated for each scale across time points in each cohort. In the domain of  (DMISS) , a 7-ite(OMS-HC) ,25, a 7-(OMS-HC) , a 9-ite(OMS-HC) . In the e (ACIP) , a 6-itee (ACIP) , a 4-itee (ACIP) . InternaWe completed analyses using R software, version 4.1.1. We treated all but the time factor and categorical demographic covariates as measurements on continuous scales. We performed summary descriptive statistical calculations and simple univariate analyses  on the outcome measures and demographic covariates. Changes in outcome measures over time were of primary interest, with the pre-program time point used as a baseline reference point for improvements. The data bear the characteristics of repeated measurements on the same participants, since more than one measurement was taken on each participant over time. Thus, it is plausible to assume that measurements on the same individual participants are correlated. Ignoring the covariance between such measurements may result in erroneous statistical inference and avoiding it by data transformation or related techniques may result in inefficient statistical inference.multcomp, to compute the adjusted p-values for the multiple comparisons of means between the baseline and later time points. The adjusted p-value cutoff of <0.05 was used to determine significance for all statistical tests. We calculated effect size (ES) using Cohen’s d, defining effect sizes ≥0.20 as small effects, effect sizes ≥0.50 as medium effects, and effect sizes ≥0.80 as large effects.The statistical technique of linear mixed modeling for repeated measures allows the covariance structure to be integrated into the modeling while accounting for the fixed time effects of the NAMI PEP as well as the randomness of participants. We built a linear mixed-effect model for repeated measures to model the time effects of the NAMI PEP while controlling for the additive effects of an optimal set of demographic and personality covariates, which were determined based on the Akaike information criterion (AIC) by searching all modeling options of the covariates. Upon determining the model’s predictor variables, we selected and evaluated several candidate covariance structures according to the study design , out of which we used the AIC criteria to select one final covariance structure. From the chosen final model, we assessed the impact of the NAMI PEP on the respective measures across time while accounting for covariates. We used the single-step method of the R software package, χ2(2) = 10.69, p = .005) and personality traits of agreeableness (t(293) = 2.59, p = .01) and emotional stability (t(290) = 2.94, p = .004) between cohorts. We controlled for these differences by inclusion of these variables in the final linear mixed models. All other baseline individual characteristics were consistent across cohorts.Of the 418 students enrolled in the required course across the two cohorts, a total of N = 325 chose to participate in the present research evaluation (78%), suggesting favorable generalizability. Specifically, initial research enrollment rates were 88% in Cohort 1 (186/211 eligible students enrolled) and 67% in Cohort 2 (139/207 enrolled). Retention rates at 6-month follow-up were 65% in Cohort 1 (120/186) and 78% in Cohort 2 (109/139). Frequencies, means, and standard deviations for select demographic variables are presented in affect, students reported reduced anxiety concerning interacting with someone with a mental illness. In the domain of beliefs, students demonstrated reductions in stereotyping negative attitudes towards patients with MI and, in Cohort 2 only, reductions in stigma. In the domain of behavioral intentions, students demonstrated increased confidence integrating psychiatric practice into routine medical care and improvements on a vignette-based measure of behavior demonstrating competent caring for mental health in a general medical setting. Furthermore, in Cohort 2 there were improved behavioral intentions regarding managing mental health during routine medical care that were not observed in Cohort 1.Across cohorts, longitudinal improvements in each of the three target domains of affect, beliefs, and behavioral intentions toward individuals with MI were consistently identified at the 6-month follow-up. In addition, effect sizes with the adapted curriculum were larger and observed across all six outcome measures . The adapted NAMI PEP had the largest impact on the domains of behavioral intentions and affect regarding MI, with large Cohort 2 effect sizes observed for increased confidence integrating psychiatry into routine care and reduced anxiety interacting with someone with MI see . The larThis study evaluated longitudinal outcomes of the NAMI PEP when implemented as a curricular requirement across two cohorts of third-year medical students, comparing standard implementation of the existing community program (Cohort 1) with an adapted program tailored to medical students (Cohort 2). The NAMI PEP resulted in either reductions in unwanted affective and attitudinal outcomes, or improvement in desired behavioral outcomes, with enhanced impact following its adaptation for medical students (Cohort 2). Six months after the NAMI PEP, students were less anxious about interacting with patients with mental illness, had less stigma and stereotyping negative attitudes about mental illness, reported more confidence integrating psychiatric practice into routine medical care, and demonstrated increased competence in principles of collaborative mental health treatment in a general practice setting. As longitudinal results were derived from two separate cohorts with their own respective baseline reference points, the consistent findings of direct improvements in target outcomes increase confidence in the effectiveness of the intervention and provide support for the adaptations made for medical students.This study expands existing knowledge of contact-based psychiatry education in meaningful ways. First, it demonstrates the pragmatic longitudinal effectiveness of the NAMI PEP when integrated as a medical school curricular requirement. In showing that the NAMI PEP offers benefits for an array of students, not only those who voluntarily seek it as an elective educational opportunity, the results suggest potential broad workforce impact for future physicians across a range of medical specialties. This has important implications for provision of quality medical care for individuals with comorbid psychiatric illness. Further, the study demonstrates the feasibility of adapting an existing community-based program for contemporary medical education, with updates including a flipped classroom model, enhanced student participation and small group discussion, integration with other educational experiences, physician modeling, and virtual delivery. Together, these findings demonstrate the external validity and generalizability of the results under realistic medical education conditions.By directly comparing the same contact-based psychiatry education program implemented in-person versus virtually using the same multidimensional outcome measures, this study also extends existing evidence that virtual interactions are a meaningful means of contact-based psychiatry education. Live virtual contact in lieu of in-person contact increases dissemination potential of such programs and may be more feasible for institutions with a distributed clerkship model, students who are geographically isolated from institutions during clerkships, or implementation with specific stigmatized patient populations. Additionally, videoconferencing features may increase the diversity of interaction opportunities to better engage certain learners  and provide some with enhanced psychological safety  ,21.Further, the demonstration of magnified and sustained improvements 6 months after the program suggests longer-term benefits of the NAMI PEP than previously identified . This hiThe present study is not without limitations. We primarily relied on student self-report, which may be subject to demand characteristics. Although a practical challenge throughout contact-based education research , assessmOur multiyear comparison across two cohorts is also limited by differential research enrollment, retention, and participant characteristics. Cohort 2 had a lower rate of research enrollment, yet improved participant retention as compared to Cohort 1. Ultimately, this resulted in comparable sample sizes at the primary 6-month follow-up across both cohorts. The use of a pre-recorded recruitment message in Cohort 2 (due to changes necessitated by the flipped classroom approach) may have impacted enrollment. Despite a strong overall enrollment rate across the two cohorts, it is possible that students who enrolled in the research differed from those who did not. Further, more students endorsed lived experience of mental illness in Cohort 2 than Cohort 1. Given past research suggesting that lived experience of mental illness is associated with reduced mental illness stigma , our anaFuture research should examine the generalizability of findings to other medical institutions. Although this represents the first known integration of the NAMI PEP into a medical school curriculum, the standardized nature of the curriculum and existing national infrastructure of local NAMI affiliates lends itself naturally to extension to other institutions in the USA. Still, the 15-hour length of the course may limit its implementation at many institutions. Although we chose to hold the course in condensed form across 2 days, there is flexibility to the delivery model. For example, the NAMI PEP can be subdivided into multiple smaller sessions, such as for offering multiple 2–3-hour sessions throughout a psychiatry clerkship. Moreover, future directions of our research program include empirical evaluation of a condensed, 4-hour NAMI PEP seminar offering an abbreviated version of content from this 15-hour program. The condensed nature of this program may increase its implementation potential.Examination of ways to further streamline the NAMI PEP implementation to optimize dissemination and scalability in medical schools, without detracting from its benefits, is ongoing. Future directions also include expanding the program and its evaluation beyond medical students. The NAMI PEP educational benefits may generalize to other health professional students, increasing the potential scope of contact-based psychiatry education to a broader cross-section of the future health-care workforce while promoting collaborative interprofessional care of patients with mental illness.Individuals living with mental illness face barriers to receiving competent medical care, contributing to health disparities such as increased morbidity and mortality. In addition to systemic issues such as limited access to specialty psychiatry services or integrated primary and behavioral health care, there is a critical need to increase all medical providers’ competencies and reduce stigma when working with this patient population . These f


Interestingly, the release of testosterone in response to human chorionic gonadotropin  was enhanced by eightfold in the control group, but elevenfold in the fructose-fed group. LH receptor expression in rat Leydig cells was also increased. Moreover, LH secretion from the anterior pituitary was altered in the fructose diet-fed group. These results suggest that fructose diet-fed rats have lower plasma testosterone levels, which can lead to a higher sensitivity of hCG in Leydig cells.Plasma testosterone levels have been found to decrease in older insulin-resistant male patients. Both lower total testosterone levels and a higher incidence of metabolic syndrome have also been reported. The aim of this study was to investigate the effects of high-fructose diet-induced diabetes on both the testosterone release by Leydig cells and the activity of the hypothalamus–pituitary–gonadal (HPG) axis in male rats. Male rats were fed with either standard chow (control group) or a high-fructose diet (fructose-fed group) for 21 weeks. Hyperglycemia, hyperinsulinemia, and hypertension were observed in the fructose-fed group. Moreover, plasma testosterone and LH levels decreased in the fructose-fed group compared to the control group. Sperm motility was also reduced by 15% in the fructose-fed rats. In contrast, the basal release of testosterone from rat Leydig cells was not altered by fructose feeding. Moreover, in vitro studies showed that the testosterone release, in response to different stimulants, including forskolin (an adenylyl cyclase activator, 10 Diabetes mellitus (DM), a metabolic disorder characterized by chronic hyperglycemia, derives from either a lack of or resistance to insulin . DM and The hypothalamic–pituitary–gonadal (HPG) axis plays a critical role in the development and regulation of the reproductive system. The gonadotropin-releasing hormone (GnRH), a hormone secreted from the hypothalamus, stimulates luteinizing hormone (LH) production from the anterior pituitary, thereby stimulating androgen synthesis and secretion from Leydig cells. Androgen is required for the spermatogenesis and maturation of secondary sexual characteristics. Male infertility affects approximately 7% of all men, and is mainly characterized by azoospermia or HPG axis alterations .Diabetes is linked to the impairment of male reproductive function. There is a bidirectional relationship between lower testosterone levels and insulin resistance status ,5. MoreoLow androgen caused by diabetes may be related to the occurrence of other diseases. For example, hypogonadal males with diabetes have a higher plaque score, coronary artery disease, and cardiovascular mortality risk ,14, whicPrevious studies have shown an inverse association between diabetes and male fertility. Moreover, this effect is related to the disturbance of the HPG axis. However, the mechanisms of the effects of diabetes on the HPG axis are still obscure. The purpose of the present study was to evaluate the effects of a high-fructose diet on the HPG axis in rats with metabolic syndrome.Five-week-old male Sprague–Dawley rats were purchased from BioLasco Co., Ltd. and adapted for one week by housing in a temperature-controlled room (22 ± 1 °C) with 14 h of artificial illumination daily (600–2000 h) and ad libitum access to water and food. Thereafter, rats were randomly divided into two groups to be fed on either a 65% high-fructose diet or a standard chow diet for 21 weeks ,19. Hype3H]-testosterone from Amersham Pharmacia Biotech . All the other chemicals were purchased from Sigma–Aldrich Chemical Co. . The doses of drugs are being as the final molar concentrations used in the flask.We obtained [5 cells/mL) and viability (over 95%) were determined by using a hemocytometer and the trypan blue method. Approximately 87% of Leydig cells were found after 3β-hydroxysteroid dehydrogenase (3β-HSD) staining.Animals were sacrificed, and the testes were decapsulated. Leydig cells were isolated and digested by collagenase and Percoll separation, as our previous study described ,21. In b5 cells/tube) were incubated in a 1% (w/v) BSA-M199 medium for 60 min at 34 °C under 95% O2–5% CO2. Different stimulators including human chorionic gonadotropin , 8-Br-cAMP , and forskolin  were used to evaluate the alteration of LH and cAMP/PKA signaling pathway in rat Leydig cells after fructose feeding. Furthermore, different precursors, including 25-hydroxycholesterol , pregnenolone , progesterone , and androstenedione  were used to evaluate the activity of steroidogenesis enzymes.Rat Leydig cells (1 × 10We used a radioimmunoassay (RIA)  4.1%, inter-assay 4.7%) to determine plasma and media testosterone levels ,21.The sperm vitality was evaluated according to our previous study . The rat6 cells/tube) were evaluated by Western blotting according to previous described methods [w/v) nonfat dry milk in TBST buffer for 2 h in RT. Thereafter, membranes were incubated with specific primary antibodies overnight at 4 °C. After being washed three times, membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit antibodies for 2 h in RT. Protein levels were detected by chemiluminescence.The protein expressions of LHR  and β-actin  in rat Leydig cells  and used the Shapiro–Wilk test to test the normality. In the normal distribution condition, differences among all groups were assessed by one-way analysis of variance (ANOVA), followed by Duncan’s multiple-range test, which was used for comparison among different treatment groups. A Student’s unpaired p = 0.017). Moreover, the percentage of dead sperm was significantly increased in the fructose-fed group as compared to the control group .Furthermore, the testosterone secretion capacity of primary rat Leydig cells was evaluated . InteresTo further evaluate whether the fructose-rich diet can affect LH sensitivity in response to hCG in vitro, rat Leydig cells were purified and then treated with different concentrations of hCG  . As our Furthermore, we further evaluated the LH secretion ability of the anterior pituitary isolated from the two groups. Higher basal LH secretion was found in the fructose diet group. In contrast, there was a lower LH secretion ability in response to GnRH (10 nM) administration compared to the control group . These rIn order to study the activity of key steroidogenic enzymes in the two groups, testosterone levels were evaluated after incubating rat Leydig cells with different precursors . The tesThe protein expression of LH receptors on rat Leydig cells was examined and evaluated by Western blot analysis. The results indicate that LH receptor protein expression was increased in the fructose-fed group as compared to the control group . These eThe consumption of fructose-rich foods results in the development of metabolic syndrome features and therefore diabetes . TestostPrevious studies have demonstrated that metabolic syndrome reduces the total plasma testosterone levels, which in turn impairs sperm production and motility after 12 weeks of a high-fat diet . MoreoveElevated fructose consumption is considered as one of the riskiest lifestyles as part of the Western-diet-related diabetes model . Energy Male reproductive activities such as sexual behavior, spermatogenesis, and fertility are regulated largely by the hormones of the HPG axis. The synthesis of testosterone, the hormone that plays a vital role in regulating sexual behavior and increasing muscle mass, is mainly regulated by the activation of LH receptors, thereby acting on the cAMP/PKA pathway, and thus elevating the expression and activity of steroidogenic enzymes . The incThe present study had some limitations. First, we did not measure plasma GnRH levels in the hypothalamic–hypophyseal portal system, which connects the hypothalamus to the anterior pituitary and regulates hormone secretion from the pituitary. Secondly, we did not determine the protein expression of steroidogenic acute regulatory (StAR) protein and cholesterol side-chain cleavage (P450scc), which are the two proteins of the rate-limiting step in steroidogenesis. Therefore, despite the fact that high-fructose feeding affects LH secretion and promotes increased sensitivity of Leydig cells, leading to increased testosterone synthesis, we cannot confirm the notion that hypothalamic GnRH secretion is indeed altered. Additional studies are required to determine the GnRH levels in testis tissues of rats fed with a high-fructose diet.In summary, the present results demonstrate that the consumption of a high-fructose diet induces metabolic syndrome, thereby abolishing the physiological regulatory function of the HPG axis. Steroidogenesis and sperm motility were reduced in fructose-fed rats. These effects might be due to the inability of the hypothalamus and anterior pituitary gland to appropriately respond to a decline in plasma testosterone levels .In this study, we demonstrated that a high-fructose diet decreased plasma testosterone levels and altered the steroidogenesis, which may cause spermatogenesis dysfunction and increase the sensitivity of LH, increasing the risk of male infertility.


Stagnating COVID-19 vaccination rates and vaccine hesitancy remain a threat to public health. Improved strategies for real-time tracking and estimation of population-level behavior regarding vaccinations are needed. The aim of this study was to evaluate whether online search trends for COIVD-19 and influenza mirror vaccination rates. State-level weekly fraction of online searches for top vaccination-related search terms and CDC vaccination data were obtained from June 1, 2020, to May 31, 2021. Next, trends in online search and vaccination data for COVID-19 and influenza were analyzed for visual and quantitative correlation patterns using Spearman’s rank correlation analysis. Online searches in the US for COVID-19 vaccinations increased 2.71-fold (95% CI: 1.98-3.45) in the 4 weeks after the FDA emergency authorization compared to the precedent 4 weeks. In March-April 2021, US online searches reached a plateau that was followed by a decline of 83.3% (95% CI: 31.2%-135.3%) until May 31, 2021. The timing of peaks in online searches varied across US states. Online searches were strongly correlated with vaccination rates , preceding actual reported vaccination rates in 44 of 51 states. Online search trends preceded vaccination trends by a median of 3.0 weeks (95% CI: 2.0-4.0 weeks) across all states. For influenza vaccination searches, seasonal peaks in September-October between 2016-2020 were noted. Influenza search trends highly correlated with the timing of actual vaccinations for the 2019-2020  and 2020-2021 season . Search trends and real-world vaccination rates are highly correlated. Temporal alignment and correlation levels were higher for influenza vaccinations; however, only online searches for COVID-19 vaccination preceded vaccination trends. These findings indicate that US online search data can potentially guide public health efforts, including policy changes and identifying geographical areas to expand vaccination campaigns. The ongoing coronavirus disease 2019 (COVID-19) pandemic has challenged health care systems around the world since late 2019 –5. As ofOver the past decade, influenza vaccination campaigns have been shown to be an important public health measure . During Search data from Google, the most common search engine, can be extracted from Google Trends (GT) together with information on regional and temporal specifics of online searches. GT represents an effective tool that can mirror public health trends and might predict patient behavior in response to health-related topics –25. The This study compares trends in online search interest for influenza and COVID-19 vaccinations and correlates these trends with actual vaccine administration rates across the United States. The overall aim is to  investigData were extracted from Google Trends as query fractions per 10 million search queries for influenza and COVID-19/SARS-CoV-2 vaccination-related terms in the US using Google Trends for Health Application Programming Interface. Weekly search data were retrieved for a 12-month period between June 1, 2020, and May 31, 2021, to explore short-term trends. This timeframe was selected to cover a period of 6 months before and after the first COVID-19 vaccine approval. Monthly search queries were extracted for the analysis of long-term trends between June 1, 2016, and May 31, 2021.The respective terms were selected using the “top related queries” field in the Google Trends application after removing unrelated/unspecific terms  as well as duplicates. Search trends for COVID-19 vaccinations were analyzed as the average of the top 10 terms related to “vaccine covid 19”  databases providing publicly available information on the administered vaccine doses in the US for both influenza  and COVID-19 (CDC COVID Tracker) . AdminisTo compare search trends, Microsoft Excel, version 16.52, was used to compute time-averaged searches and weekly COVID-19 vaccinations. The rcompanion package in R software, version 4.1.1 , was applied to calculate the non-parametric Spearman’s rank correlation between weekly search rates and administered vaccine doses. Comparisons between correlation coefficients were performed using Fisher’s z-transformation and Student’s t-test. Visualization of trends in vaccinations and online searches were performed using the ggplot2 package in R. We also forecasted search interest for COVID-19 vaccination-related terms pre- and post-vaccine approval using an autoregressive integrated moving average (ARIMA) starting on November 1, 2020 accounting for the first positive trial results for a COVID-19 vaccine and the FDA authorization request. December 12, 2020, was set as cut-off as this was representative for the first COVID-19 vaccine authorization in the US on December 11, 2020. ARIMA calculation was performed using the gtrendR package in R software, version 4.1.1.Trends in online searches, as represented by the query fraction per 10 million searches, for the top 10 terms related to COVID-19 and influenza vaccinations as well as weekly COVID-19 case numbers between June 2020 and May 2021 are shown in ). During March 2021, online search trends reached a plateau level with highest search volumes  and COVID-19 vaccine side effects (‘covid vaccine side effects’) exceeded the expected online searches after the first approval of a COVID-19 vaccine on December 11, 2021 2. BetweThe following states showed COVID-19 vaccination rates above the US average: NY 344%, TX 299%, FL 228%, and CA 577% above the US mean between the first CDC-recorded vaccinations January 10 and May 31, 2021 Figure 2For terms related to COVID-19 vaccinations, peaks in online searches were reached in March-April 2021 with FL on April 04, TX on March 07, and CA on March 28, while searches in NY peaked earlier on February 07, 2021. Similarly, and consistent with overall US trends, online search trends for influenza vaccinations reached a maximum in late September – early October 2020 with peaks for NY, FL, and TX on October 4, 2020, and for CA on September 27, 2020 –E.Weekly online searches and vaccination rates per 1 million persons for COVID-19 are shown in Correlation analyses revealed a clear correlation for both COVID-19 and influenza in terms of online searches and vaccination rates 2. For COnline search trends for influenza vaccinations and actual administrated vaccine doses throughout the US between 2019 and 2021 are depicted in The results of the present study show a strong correlation between online search behavior for COVID-19 and influenza vaccinations and administered vaccination doses for the US. This association was strongest for influenza vaccinations. For COVID-19 vaccinations, but not for influenza vaccinations, trends in online searches clearly preceded trends in vaccine uptake, which was consistent throughout most US states.To our knowledge, this is the first study to evaluate the potential role of online search behavior for COVID-19 and influenza vaccinations to predict vaccination rates. The observed peaks in online searches for influenza vaccinations during September-December, which clearly aligned with vaccination rates, are in accordance with the CDC recommendations for the timing of the influenza vaccination campaigns , 30.For COVID-19 vaccinations, online searches showed increases well before vaccination rates increased. One explanation for this offset between online searches and actual vaccination trends might be the potential need for information prior to an individual’s decision in favor of the vaccination. Secondly, the allocation and distribution processes, largely because of the initially limited vaccine supplies, might have contributed to the observed offset with maximum vaccination rates in April 2021. This was coincident with the removal of vaccine prioritizations making all adults eligible for vaccination across the US states . As suchIn the light of stagnating vaccination rates and continuing COVID-19 vaccine hesitancy, simultaneously decreasing online search interest and vaccination rates after April 2021 might be relevant to the development and implementation of future directions in vaccination campaigns , 32. COVSeasonal trends in influenza vaccination rates are largely a result of the recommended vaccination schedule according to CDC and guideline recommendations, which particularly indicate the months of September and October as optimal , 34. OnlThe analyzed search terms were selected at the authors’ discretion based on the top related search terms as provided by GT to ensure overall representativity by considering multiple terms for the present analyses. During this selection process, duplicates and unrelated terms were removed to ensure relevance and specificity. There are some relevant limitations to this study: First, the use of Google searches is likely to overrepresent a younger, health-oriented population that is more likely to routinely access online information. As such, for example individuals aged ≥ 65 years might be underrepresented by online searches because of a less frequent internet usage despite generally higher vaccination rates in this age group. Second, GT data do not provide demographic or geographic information on the individual users. Therefore, subgroup analysis by demographic characteristics such as age, sex, income level could not be done. Third, the present data did not identify the purpose of vaccination-related searches. Therefore, the present search data might include online searches performed for other less related purposes, such as research or educational purposes. Ultimately, GT exclusively reports data from the Google search engine, which represents the most frequently used online search tool.We observed a strong correlation of online search volumes with vaccination rates for both COVID-19 and influenza, while for COVID-19 vaccinations, online searches precede trends in vaccinations by about 3.0 weeks (95% CI: 2.0-4.0 weeks). Thus, our findings provide support to the use of online search behavior for tracking both COVID-19 and influenza vaccination behavior and may be able to mirror COVID-19 vaccination rates. In addition, these data might complement directly gathered population-based data and provide additional guidance for decision-makers to optimize vaccination communication and vaccine distribution in vaccination campaigns. Overall, the observed correlations between online searches and actual vaccination rates for COVID-19 add further details in the predictive capability of GT for near-real-time public health analyses.The original contributions presented in the study are included in the article/PB, LH and OD participated in the conception and drafted the manuscript. AR, EB, NO, AS, SM, JA and MB revised subsequent drafts critically for important intellectual content. All authors contributed to the article and approved the submitted version.Author OD received support from National Institutes of Health grant T32 HL007227.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Somatostatin receptor ligands (SRLs) represent a true milestone in the medical therapy for acromegaly. The first-generation SRLs (FG-SRLs), octreotide and lanreotide, have demonstrated good efficacy in disease control and tumor shrinkage, and are still considered first-line medical therapies. The development of long-acting release (LAR) formulations has certainly improved the therapeutic tolerability of these drugs, although many patients still experience therapy-related burden. As such, new formulations have recently been developed to improve adherence and therapeutic efficacy and more solutions are on the way. In the case of FG-SRL-resistant disease, pasireotide, the only second generation SRL currently available, demonstrated superiority in disease control and tumor shrinkage compared to FG-SRLs. However, its use in clinical practice is still limited due to concern for impairment in glucose homeostasis. In this review, we discuss the news about the present and future role of SRLs in acromegaly, exploring the therapeutical frontiers of this drug class. Moreover, we provide practical guidance on the use of pasireotide, based on the data in the literature and our clinical experience. Acromegaly is a rare disorder characterized by excessive circulating growth hormone (GH) levels, which in more than 95% of cases is caused by a GH-secreting pituitary adenoma. Due to the wide range of comorbidities associated to GH excess , acromegaly may significantly impair quality of life (QoL) and increase risk of death ,2.Universally, the first-line therapy for acromegaly is the surgical removal of the pituitary adenoma via a trans-sphenoidal approach. In specialized centers, surgery results are effective in 80–90% of microadenomas and 50–75% of macroadenomas, with minor efficacy in the case of very large or invasive adenomas ,4. When The goal of medical therapy is to achieve optimal disease control from both a clinical and biochemical perspective, normalizing insulin-like growth factor 1 (IGF-1) levels within an age-specific reference range and limiting the development of disease-related complications. In addition to biochemical control, treatment aims to prevent tumor growth or, ideally, induce tumor shrinkage. Medical therapy includes somatostatin receptor ligands (SRLs), pegvisomant, and dopamine agonists, although the role of the latter is currently considered suitable for patients with minor hormonal hypersecretion.The release of FG-SRLs, octreotide and lanreotide, dates back to 1980s . Both drIn patients unresponsive to FG-SRLs, the second line of therapy includes pegvisomant (PEGV), a GH receptor antagonist, or pasireotide (PAS), the only second-generation SRLs currently available . PEGV isPAS is a multi-receptor somatostatin ligand approved for acromegaly by both FDA and EMA in 2014, which compared to FG-SRLs expresses a significantly higher affinity for SSTR5 and a lower affinity for SSTR2 . In bothIn recent years, more in-depth studies on pharmacokinetic and mechanisms of response to therapy have shed new light on the role of SRLs in the treatment of acromegaly. New formulations have been recently released and highly selective drugs are currently under development. As such, the purpose of this review is to summarize the novelties in the world of SRLs, exploring from new perspectives the present and future role these drugs play in the clinical management of acromegaly.The most widely used formulations of FG-SRLs are octreotide in long-acting-release formulation (octreotide LAR), administered via intramuscular injection at a standard dose of 10–30 mg every 4 weeks, and lanreotide Autogel, administered via deep subcutaneous injection at a standard dose of 60–120 mg every 4 weeks.Despite an overall improved QoL thanks to disease control, patients treated with FG-SRLs still experience reduced QoL because of therapy-related burden . A recenAn oral formulation of octreotide was approved by Food and Drug Administration (FDA) in June 2020 for acromegalic patients already responsive to injective SRLs. This new formulation was tested in two sponsored randomized controlled trials (CH-ACM-01 and CHIASMA OPTIMAL) including one-hundred-and-fifty-five patients and fifty-six patients, respectively, who were already controlled in FG-SRLs therapy ,17. TherThe main limitation of the study was the inclusion in the treatment phase only of patients already responsive to oral octreotide, despite demonstrating the efficacy and safety of the oral formulation. Moreover, despite the presence of substantially overlapping side effects between the two formulations, the satisfaction in patients undergoing oral treatment proved to be significantly superior to injective formulation, as measured with AcroTSQ . Oral ocAn octreotide subcutaneous (SC) depot formulation name CAM2029), is also under development. This formulation uses FluidCrystal technology, allowing both monthly administration and use of thin needles. In a phase 2 trial performed on patients with either acromegaly or functioning neuroendocrine tumors, the SC depot formulation demonstrated to increase octreotide plasma levels more than the IM formulation, with good biochemical control of disease and safety profile [29, is alIn addition, a prolonged release formulation (PRF) of lanreotide is under development, with the purpose of increasing the interval of therapy administration from 4 to 12 weeks. In a phase 2 clinical trial, lanreotide PRF was administered at multiple doses of 180 mg, 270 mg, and 360 mg. The primary endpoint of the study was to find the maximum tolerated dose (MTD), demonstrating good tolerability. Moreover, GH and IGF-1 levels remained substantially stable throughout the study . TherefoPAS is currently considered in acromegaly therapeutic scenario as a second-line therapy. While for Cushing’s disease the drug is only approved in a short acting formulation, acromegalic patients can benefit from a LAR formulation to be administered at the standard dose of 20–60 mg every 4 weeks. PAS-LAR is suggested for treatment of patients poorly responsive to FG-SRL, especially if there is concern for tumor growth . Indeed,In our one-center experience (unpublished data), nineteen acromegalic patients  resistant to FG-SRLs at high doses and/or intolerant to pegvisomant were switched to PAS-LAR. Eleven had persistent disease after neurosurgery and two had also undergone radiosurgery (12 and 24 months before starting PAS-LAR). Six complained of acromegalic headache (symptomatic score was 3/3 in 5 and 2/3 in the last). On FG-SRLs, IGF-1 and GH were  193% upper limit normal age-matched range (ULNR) (120–303) and 5.2 ng/mL (0.6–25), respectively. PAS-LAR was injected every 28 days, starting with 40 mg for 3 months, up-titrated to 60 mg if IGF-1 pathologically persisted, or down-titrated to 20 mg if IGF-1 was <50% ULNR. GH and IGF-1 were assessed at 28, 84, and 168 days after starting protocol. Treatment was withdrawn if IGF-1 remained pathologic after 3 months on 60 mg q 28 days. PAS-LAR normalized IGF-1 in 10/19 patients after the first injection and was withdrawn in five unresponsive patients at 6 months. After 12 months, IGF-1 was 74% ULNR  and GH 1.2 ng/mL (0.2–3.9). At the last follow-up  IGF-1 was 74% ULNR  and GH 0.7 ng/mL (0.1–2.5). Headache almost disappeared in all patients (in 5/6 after the first injection) and reappeared with pathologic IGF-1 levels after PAS-LAR withdrawal in one irradiated patient. Tumor shrinkage  was observed in 6/7 evaluated patients without previous irradiation at 6–36 months after the start of PAS-LAR. In two patients, PAS-LAR was withdrawn at 36 and 60 months due to poor compliance in the first, and QTc lengthening in the second, who had started amiodarone treatment.Recent studies also suggest that the positive effect of PAS in acromegaly may go beyond simple control of disease. A longitudinal retrospective study performed by Chiloiro et al. on patients with resistant acromegaly showed that treatment with PAS reduced the incidence of vertebral fractures (VF), also independently from IGF-1 levels . This fiPAS may also represent the best therapeutic option for rare types of acromegaly associated with large pituitary tumors, such as X-linked acrogigantism (X-LAG) or aryl hydrocarbon receptor-interacting protein (AIP) mutation positive acromegaly, which are often resistant to FG-SRLs . In a caThe use of PAS is still limited in clinical practice by concerns about the development of alterations in glucose metabolism. As such, we considered useful to include in this review a section regarding clinical management of PAS-induced hyperglycemia.The pathophysiology underpinning PAS-induced hyperglycemia depends on its higher affinity for SSTR5 than SSTR2. In fact, SSTR5 is highly expressed in pancreatic beta cells, responsible for insulin production, and in entero-endocrine cells is responsible for incretin release , while SSTR2 expression prevails in alpha pancreatic cells, mainly responsible for glucagon production. As such, PAS affinity for SSTR5 induces a marked inhibition on both insulin and GLP-1 secretion, with a minor effect on glucagon, whose secretion is instead inhibited by FG-SRLs . A studyNevertheless, PAS-induced diabetes mellitus generally involves only a minority of patients. A post hoc analysis performed on the population recruited in the PAOLA study reported that starting an antidiabetic treatment (OAD) was necessary in only 25% of patients, as another 25% was already under diabetic treatment at baseline, and 50% required no specific therapy. Real-life studies show worsening on glucose metabolism or necessity for intensification of OAD in almost 60% of patients, although adding an OAD was required in only one-third of patients and a therapeutically different form metformin was added only in 20% of patients . In our As such, PAS certainly exerts a worsening effect on glucose metabolism. In few cases, glucose homeostasis derangement can be significant, increasing the risk of DKA and requiring prompt insulin therapy, but in most cases it unlikely progresses to overt diabetes or requires substantial modification of OAD. Several studies have investigated the possible factors contributing to this progression, and the most relevant was the presence of an already altered glucose metabolism at baseline; other possible contributors include increasing age, dyslipidemia and hypertension . Thus, iIt has been suggested that in the first three months of treatment, home fasting glucose measurement should be performed once or twice a week in case of normal glucose tolerance at baseline, and daily in case of impaired fasting glucose (IFG) or impaired glucose tolerance (IGT). If no glucose metabolism alteration develops during the first 3 months, home glucose monitoring can be discontinued and only a three month assessment of HbA1C is suggested ,32. In pThe choice of these two classes of oral antidiabetic therapy relies on the mechanism of PAS-induced hyperglycemia, as the use of a DPP4-I or a GLP1RA can counterbalance the inhibition on endogenous GLP1 release induced by PAS. The positive effect of incretin drugs on PAS-induced hyperglycemia was confirmed in a recent phase 4 trial, including 249 patients with either acromegaly (190) or Cushing’s disease (59) . PatientNoticeably, in most of patients of the study from Sanson et al. , no modiAside from alterations in glucose metabolism, PAS has demonstrated both in clinical trials and in real-life settings mostly minor adverse drug reactions (AE). In the PAOLA extension study, cholelithiasis occurred in almost 30% of patients, although not requiring significant intervention except for one case reported of bile duct stone and cholecystitis. In the ACCESS study, evaluating safety of PAS-LAR 40 mg in forty-four acromegalic patients, gastrointestinal symptoms were reported as the most common non-glucose-related AEs: diarrhea in 38.6% of patients, nausea in 27.3%, and abdominal pain in 18.2% . FDA alsCombining FG-SRLs with PEGV is a therapeutic strategy increasingly used in real-world setting. At the moment, expert consensus recommends combination therapy in patients with resistance to monotherapy and concern for tumor growth and impaired glucose metabolism .However, a recent study on French ACROSTUDY population showed that combination therapy was prescribed in almost half of patients treated with PEGV . InteresRegarding the addition of PEGV to SRLs, the main reason was the raise of IGF-1 levels in the presence of an aggressive disease or tumor with extrasellar invasion. SRLs and PEGV combination therapy has actually proved to achieve a better biochemical control of disease than SRLs alone . Bonert While adding PEGV to SRLs appears to be a logical therapeutical escalation, it is more difficult to justify the addition of SRLs to PEGV, whose main reasons may be uncontrolled disease in PEGV monotherapy, concerning tumor growth and the reduction of frequency of PEGV injections. In the French ACROSTUDY, authors report that 53.3% of patients with uncontrolled disease achieved IGF-1 normalization after addition of SRLs and that PEGV frequency of administration significantly reduced (16% vs. 44.3% of patients receiving less than seven injections per week) . InteresRegarding glucose metabolism, two recent meta-analyses showed an overall neutral effect of combination therapy on fasting glucose and HbA1c with a significant reduction of fasting plasmatic insulin ,43. AssoIn selected cases, a novel therapeutic perspective is represented by the combination therapy of PEGV with PAS-LAR . PotentiThe effect of combination therapy PAS + PEGV was analyzed in the PAPE study, involving sixty acromegalic patients previously controlled with FG-SRLs + medium to high doses of PEGV (medium dose at baseline 134 mg/week). After 12 weeks of follow up, PEGV doses were arbitrarily halved (medium dose 61 mg/week), and patients were either randomized to start PAS-LAR 60 mg together with low dose PEGV if IGF-1 levels had risen above the range after halving PEGV, or to PAS-LAR 60 mg monotherapy if IGF-1 levels were within the reference range. After 24 weeks from baseline, patients in PAS-LAR monotherapy group maintained optimal control of disease in 93% of cases, while in the combination therapy group, PEGV could be further reduced to a medium dose 48 mg/week and even discontinued in 68% of patients. Overall, 12 weeks of PAS-LAR therapy led to a 66% reduction of PEGV dose . In addiIn a 2019 longitudinal study, Chiloiro et al. described the efficacy of PEGV + PAS combination therapy in six patients with active acromegaly despite previous treatment with PAS, PEGV or FG-SRLs + PEGV. The patients of this study had large invasive pituitary adenomas with a mean Ki67 of 3.5% and very high GH levels at baseline. All of them reached biochemical control of disease within the first month of PEGV + PAS combination therapy and one patient experienced a significant reduction in tumor size. Despite the small number of patients and unclear dosage of therapies, this study suggests that PAS + PEGV combination therapy might represent the ultimate pharmacological strategy in patients with very aggressive disease and unresponsive to any other treatment .After almost 10 years since the release of the last SRL, research is continuing to explore this drug category and new selective receptor ligands are currently under development.Paltusotine (former CRN00808) is a highly potent, orally administered, SST2 agonist with a >4000-fold selectivity for SST2 over other somatostatin receptor subtypes. In a phase 1 trial, pharmacokinetic analysis proved an estimated half-life of 22–34 h, supporting a once-daily dose. The drug should be administered in a fasting state as a high-fat, high-calorie meal showed to markedly lower plasma concentrations, although a new spray-dried dispersion (SDD) tablet formulation has proved less sensitivity to food administration than the original capsule formulation . PaltusoSomatropim, also known as DG3173 or COR-005, is another SRL showing high affinity to SST2, SST4, and SST5. Somatropin has demonstrated in studies on primary cultures of GH-secreting adenomas to suppress GH release in more tumors than octreotide  and to reduce GH secretion in 38% (6/16) of tumors that were unresponsive to octreotide . A phaseIn addition, two more selective SST2 agonists are currently in development: ONO-5788 and ONO ST-468. The former demonstrated in studies in vivo on rats to significantly reduce basal and GHRH-stimulated GH ; phase 1An overview of emerging treatment strategies for acromegaly is given in After almost forty years since their release, SRLs still represent a milestone for medical therapy for acromegaly. The development of new formulations has opened up new possibilities, with a view to an increasingly personalized therapy based on the patient’s needs. Moreover, the development of more potent drugs such as PAS has improved the therapeutic outcome and adherence for resistant acromegalic patients, allowing an optimal control in most patients with a single monthly injection. In addition, the possibility of combining these drugs with other therapies makes them particularly suitable for a wide range of applications even in difficult clinical scenarios. In conclusion, the development of SRLs drugs is still progressing, with promising new solutions that may be introduced in future clinical practice.