Females, individuals with normal body mass index, history of COVID-19 infection, and influenza-unvaccinated individuals were significantly associated with declining the booster dose. Higher fear levels were observed in females, rural citizens, and chronic and immunosuppressed patients. Our results suggest that vaccine hesitancy and fear in several highlighted groups continue to be challenges for healthcare providers, necessitating public health intervention, prioritizing the need for targeted awareness campaigns, and facilitating the spread of evidence-based scientific communication.COVID-19 vaccines are crucial to control the pandemic and avoid COVID-19 severe infections. The rapid evolution of COVID-19 variants such as B.1.1.529 is alarming, especially with the gradual decrease in serum antibody levels in vaccinated individuals. Middle Eastern countries were less likely to accept the initial doses of vaccines. This study was directed to determine COVID-19 vaccine booster acceptance and its associated factors in the general population in the MENA region to attain public herd immunity. We conducted an online survey in five countries  in November and December 2021. The questionnaire included self-reported information about the vaccine type, side effects, fear level, and several demographic factors. Kruskal–Wallis ANOVA was used to associate the fear level with the type of COVID-19 vaccine. Logistic regression was performed to confirm the results and reported as odds ratios (ORs) and 95% confidence intervals. The final analysis included 3041 fully vaccinated participants. Overall, 60.2% of the respondents reported willingness to receive the COVID-19 booster dose, while 20.4% were hesitant. Safety uncertainties and opinions that the booster dose is not necessary were the primary reasons for refusing the booster dose. The willingness to receive the booster dose was in a triangular relationship with the side effects of first and second doses and the fear ( The coronavirus disease 2019 (COVID-19) outbreak has prompted remarkable research responses in various fields ,4,5. TheSix COVID-19 vaccines were approved in the Middle East and North Africa (MENA) region: mRNA-1273 , given as two doses with at least 28 days between doses ; Ad26.COThe clinical studies supported by legitimate reports and observations showed great effectiveness toward SARS-CoV-2 infection ,13. HoweDuring the follow-up process of 6 months, participants in the BNT162b2 vaccine clinical study revealed that effectiveness towards infection declined by around 6% every 8 weeks . While vThe Arab world’s vaccine acceptance level is lower than that worldwide . A studyAccording to Bartsch et al., achieving public herd immunity requires vaccinating 70% of the population, and that in turn relies on the willingness of the public to accept it .Thus, assessing willingness to receive a COVID-19 booster dose is vital. To the best of our knowledge, no such reports or studies have been conducted in the MENA region. Hence, this study aimed to assess the attitudes of fully vaccinated adults in the MENA region toward receiving a COVID-19 booster dose and identify the related hesitation variables that contributed to their decisions.An online survey was conducted in the MENA region in November and December 2021. After translating from English to Arabic, we used a validated survey  originally designed by Rzymski et al.  after obThe main survey questions included: (Ⅰ) Are you fully vaccinated against COVID-19? (Ⅱ) Which COVID-19 did you receive? (Ⅲ) Please rate the severity of the side effects after receiving the first dose of the COVID-19 vaccine. (Ⅳ) Please rate the severity of the side effects that occurred after receiving the second dose of the COVID-19 vaccine. (Ⅴ) Are you willing to receive a booster dose of the COVID-19 vaccine if it is available? (Ⅵ) Please assess the level of fear associated with receiving the possible additional dose of the COVID-19 vaccine. (Ⅶ) Have you been infected with COVID-19? Moreover, fear and side effects were reported on a 10-point Likert scale (1 is the lowest and 10 is the highest).Participants had to be Arabic speakers, have received the first and second dose of COVID-19 vaccination (or only one dose in case of Ad26.COV2.S), be living in any MENA region country , and be at least 18 years old. The online survey was available in Arabic and was designed and hosted on Microsoft Forms. Participation was voluntary, and before filling out the survey, each participant had to confirm their consent to participate. We distributed the survey using social media and mailing lists.U test. Chi-squared test with Bonferroni correction was used to identify significant predictors for fear and willingness to receive the booster dose. Kruskal–Wallis ANOVA was used to associate the fear level with the type of COVID-19 vaccine. Moreover, univariate and multifactorial logistic regression were performed to confirm the results and reported as odds ratios (ORs) and 95% confidence intervals (95% CI). p-value < 0.05 was considered statistically significant for all the results.Data were analyzed using TIBCO Statistica  and PQStat Software (version 1.8.2). OpenEpi was used to calculate the sample size according to the proportion. The following parameters were used: the population size was 100,000,000, and 5% was the confidence limit. The hypothesized frequency was set to 50%, and the design effect was 1. Therefore, for a 95% confidence level, the estimated minimum sample size was 384 per country. Missing data were treated by listwise and pairwise deletions. Categorical data were reported as frequency/percentage, and continuous data as median . Normality was calculated using Shapiro–Wilk tests. Differences between side effects and fear among those willing to receive the booster dose were calculated by Mann–Whitney A total of 4056 responses were collected, and only 3041 (75.0%) met the study criteria for further assessments (see n = 1520); (iii) were settled in urban areas; (iv) had received a bachelor’s degree ; (v) were not infected by SARS-CoV-2; and (vi) had never been administered the influenza vaccine. Approximately 11.9% of respondents (n = 361) had at least one chronic disease, with asthma the most common , and there were  immunosuppressed patients. The majority of participants were vaccinated with BNT162b2 , followed by AZD1222  and BBIBP-CorV . The frequency of the administered vaccines is shown in A majority of the participants (i) were below 50 years old; (ii) had higher BMI  of respondents were willing to receive the COVID-19 vaccine booster dose, while 20.4% (n = 619) were hesitant. The principal reasons to refuse the vaccine were safety uncertainties , belief in the non-necessity of the booster dose , and side effects associated with previous COVID-19 vaccine doses .Overall, 60.2% (n = 133), followed by those vaccinated with Ad26.COV2.S  and BBIBP-CorV , while among those vaccinated with BNT162b2, 29.5% (n = 267) stated no desire to be vaccinated again.Individuals earlier vaccinated with AZD1222 were more willing to accept the booster dose , individuals with obesity (p < 0.02), those who regularly or irregularly receive influenza vaccination (p < 0.001), and who have not been infected by SARS-CoV-2 (p < 0.0001). Saudi citizens were more likely not willing to receive the booster dose compared with citizens in other countries in the region , n = 144; respectively; p = 0.01, z = 2.59).More serious side effects after the first and second dose were significantly higher among participants unwilling to receive the booster dose than those willing to receive it —includinn = 263) with a fear level above 5. No significant differences were found between the type of COVID-19 vaccine and the fear of booster dose (p = 0.15). Participants aged <50 years, females, rural residents, immunosuppressed patients, and Saudi citizens reported higher fear levels . In general, 9.5% (n = 218) said they had no specific interest in the type of vaccine, while 17.8% (n = 410) said they could not decide at the time of the study. Nevertheless, the majority of individuals that fulfilled their first BNT162b2 and Ad26.COV2.S vaccination regimens wanted to get a booster dose with the same type of vaccine, if possible . In the case of mRNA-1273, 54.1% (n = 47) of the individuals questioned expressed interest in getting it as a possible booster dose. In contrast, the survey question about the AZD1222 type showed that only 46.6% (n = 342) of those questioned expressed interest in receiving it as a booster dose. Overall frequencies of top-selected booster doses appear in The participants willing to receive the COVID-19 booster did not prefer the same type of vaccine as previously. The individual responses regarding a specific COVID-19 vaccination they preferred as a future booster dose are shown in Several factors increase the willingness to accept a booster dose, such as being fully vaccinated with Ad26.COV2.S, AZD1222 or BBIBP-CorV. Those who received the mRNA-1273 vaccine showed less likelihood of accepting the booster dose. Regardless of the received COVID-19 vaccine, the side effects of previous doses and fear were associated with declining the booster dose. Participants infected with SARS-CoV-2 after the first dose were not likely to accept the booster dose. Males, Sudanese, Egyptians, or those with an influenza vaccination history were willing to accept the booster dose. The binary and multifactorial backward stepwise logistics of the significant predictors of accepting a COVID-19 vaccine booster dose are shown in The majority of the participants (60.2%) were willing to receive a COVID-19 booster dose, according to our findings. Fortunately, it seems that vaccine hesitancy is declining as more information on the vaccine’s safety and effectiveness becomes available. However, there are still certain factors affecting individuals’ decisions whether to receive the COVID-19 booster dose, including fear, side effects, several demographic factors, and previous vaccination with influenza vaccine.n = 2925) of the population of some Middle Eastern countries agreed to be vaccinated against COVID-19, while 32.6% were hesitant and 42.5% objected [It was reported that the COVID-19 vaccine acceptance rate in Middle Eastern countries was relatively low. Abu-Farha et al. reported that only 24.9%  showed that age variations were significant in Brazil, Ecuador, Mexico, and South Africa, where older people were more inclined to follow their employer’s vaccination recommendations. However, younger people were much more likely to do so in France and the USA. Results were similar when compared to respondents from Italy, Poland, and Russia, in which younger people were more likely to express their intention to accept an employer’s vaccine recommendation [p = 0.003, respectively). This can be explained as the elderly are considered a high-risk population for serious COVID-19 infection, suggesting that a booster dose might be advantageous in providing them with further infection protection [In many countries, younger generations are much more vaccine-hesitant, which impedes immunization progress ,36,37,38endation . Our stuotection .p <0.000001, respectively).Furthermore, vaccination reluctance has been higher among younger women in various nations ,42,43. In = 162). Several publications show that relying on social media can be dangerous, as they create an environment for spreading misleading information that may affect public mental health [Moreover, Thomas and Darling reported that the educational level more strongly influences people’s willingness to obtain the vaccine. People of Asian heritage may be the sole exception, since they showed a high willingness to be vaccinated regardless of their educational level. In addition, people with lower levels of education were less trusting of the vaccines . Moreovel health ,46,47,48Regarding vaccine hesitancy and its association with the area of residence , the results from a survey of 1676 adults living in the United States, including Alaska and Hawaii (including interviews from 298 Hispanic adults and 390 non-Hispanic Black adults), showed that those who live in rural areas are still more likely to be vaccine-hesitant than those who live in suburban and urban areas . MoreoveSARS-CoV-2 infection status has also influenced people’s decisions. After at least one vaccination dose, infected participants had an almost 50% lower probability of accepting a booster dose. At the same time, those who were regularly or irregularly vaccinated with the influenza vaccine had higher odds  of accepting a booster dose. These findings imply that previous vaccination experience dramatically influenced people’s decisions to receive a booster dose.The immunosuppressed and chronic patients were willing to receive the vaccine but reported a higher fear level than normal individuals. However, immunosuppressed patients with a fear level above 5 had higher odds of rejecting the vaccine, as shown in A tendency to favor American-based vaccines was observed in a survey released in Saudi Arabia, Lebanon, Jordan, and Iraq, since 45.2% of the participants favored BNT162b2, whereas 30% were unfamiliar with different types of COVID-19 vaccines . Almost Finally, it is worth mentioning that this study exhibits some strengths and potential limitations. First, regarding time constraints, the timing of the study was essential to achieve proper interventions since people in the MENA region had started receiving the booster dose. Second, we implemented strict inclusion and exclusion criteria to obtain accurate results. Third, the sample size covered five different MENA region countries to provide a general overview of this geographic location. Concerning the limitations, self-reporting of the fear scale might not be accurate and may reflect misreporting. Additionally, we asked participants to rate the side effects on a Likert scale; however, we did not know what these side effects were —a topic that can be further addressed in future studies. Some groups were underrepresented, such as people with different educational backgrounds (primary and secondary). Additionally, the anonymity of the survey may have attracted individuals interested in manipulating the data; even though we removed all responses below 1 min in length to prevent any usage of Macros (survey filling bots), this point still needed to be declared for transparency. Even after extending the collection time, the response rate was low, allowing only a 5% error margin, which could have been improved if we had a higher response rate. Moreover, the national coordinators distributed the survey using each country’s most commonly used social media channels; hence, the design effect should have been considered to calculate the sample size per country. Further research can benefit from mailing lists or by promoting their surveys on popular local websites. Finally, the responses represented personal opinions at a given point in time that did not fully reflect the respondents’ future decisions. As we mentioned, several factors could affect that, including the prospective status of the COVID-19 pandemic and the spread of the infodemic.The main reasons to refuse the booster dose were uncertainties over their safety, belief that the booster dose is unnecessary, and side effects associated with previous COVID-19 vaccine doses. Since the introduction of booster doses has been firmly evidence-driven, efforts to improve public awareness should target the population groups in greatest need through effective scientific communication to overcome vaccine hesitancy and fear.


Background: Given the prevalence of the omicron variant and decreased immunity provided by vaccines, it is imperative to enhance resistance to COVID-19 in the old population. We planned to explore the hesitancy rate toward the booster dose of the COVID-19 vaccine and the association between risk perception and the abovementioned rate among people aged 60 and older. Methods: This national cross-sectional study was conducted in mainland China from 25 May to 8 June 2022, targeting people who were 60 years old or above. Four dimensions were extracted from the Health Belief Model (HBM) to assess participants’ perceived risk levels, including perceived susceptibility, perceived severity, perceived barriers, and perceived benefit. An independent Chi-square test was used to compare the vaccine hesitancy rates among different groups stratified by characteristics. Univariable and multivariable logistic regression models were performed to explore the associations between risk perception and hesitancy rate. Results: Of 3321 participants, 17.2% (95% CI: 15.9–18.5%) were hesitant about booster shots of COVID-19 vaccines. Believing that they were ineligible for vaccination due to certain illnesses (38.3%), concern about vaccine safety (32.0%), believing the booster shots were unnecessary (33.1%), and their limitation on movements (28.0%) were the main reasons for vaccine hesitation. Adjusted by all the selected covariates, people with low perception level of susceptibility  and benefit  were less likely to receiving the booster dose, and the same results were found in people with higher perceived barriers . Our estimates were stable in all four models. Conclusions: In total, 17.2% of the people aged 60 years and older in China were hesitant about booster dose of COVID-19 vaccines, and it was closely associated with a lower level of perceived susceptibility and benefit, as well as a higher level of perceived barriers. Concerns about contraindications, vaccine safety, and limited movements were the main reasons for vaccine hesitancy. Targeted public health measure is a priority to improve the understanding of the elderly on their own susceptibility and vulnerability and clear the obstacles to vaccination. As one of the worst plagues in nearly a century, coronavirus disease 2019 (COVID-19) has caused an incalculable disease and economic burden across the globe . UndoubtVaccine hesitancy, one of the ten major threats to global health in 2019, refers to the reluctance or refusal of people to be vaccinated when available, especially among the older population . AccordiThe health belief model (HBM) is widely used to assess public attitudes toward vaccines and predict their behavior toward vaccination, revealing significant perception factors such as perceived susceptibility, perceived severity, perceived barriers, and perceived benefit ,19,20. RUp to now, no study has been performed to specifically investigate the hesitancy toward the booster dose of the COVID-19 vaccine and the association between risk perceptions among the Chinese elderly (≥60 years old). Accelerating global coverage of vaccination in the old population, especially the booster dose, is indeed a priority for achieving herd immunity against COVID-19 . To bettThis national cross-sectional survey was conducted based on an online platform called Wen Juan Xing . Owing clear personal information  of nearly 3 million registered members in China, it could accurately deliver questionnaires to the representative respondents we expect. The target participants of this study were senior citizens aged 60 or above in China. For users who have trouble answering questions using electronic devices (without cognitive impairment), they can enlist the help of those around them, and questions could be relayed to the old people by those young adults living with them. Each question was followed by a reminder that the purpose of the questionnaire was to find out the true thoughts and situations of people aged 60 and above.p = 0.13) among old people, calculating the sample size with α as 0.05 and the confidence interval width as 0.1p (0.013). In total, 2647 valid questionnaires were expected when using the exact (Clopper–Pearson) method for calculation by the PASS software 15.0 . Anonymous questionnaires were randomly allocated among 31 provinces from 25 May to 8 June 2022. The minimum number of valid questionnaires for each province was allocated according to the proportion of the older adults aged 60 or above reported in the Seventh National Census [According to previous studies that examined the willingness of Chinese adults to receive booster shots, 10.56% (95% CI: 6.99–14.14%) were unwilling to receive booster vaccination among those aged 60 and older . Thus, wl Census . A totalp < 0.001). Then the expressions of some items were modified according to the feedback of the respondents, making them easier to understand. The reliability of this questionnaire was confirmed by Cronbach’s alpha coefficient by different dimensions.This structured questionnaire was divided into 4 sections in total, which consisted of the following parts: (1) sociodemographic characteristics and health status, (2) knowledge of COVID-19 and COVID-19 vaccines, (3) four dimensions of risk perception based on health belief model (HBM), and (4) attitude toward the booster dose of COVID-19. All items in our questionnaire were stated by a panel of experts, including one public health expert and two epidemiologists specializing in infectious diseases. We conducted a pilot study involving 40 old people before it was officially released to test the questionnaire’s validity  is a conceptual framework based on motivation theory, cognitive theory, and expectancy-value theory, which has been widely used to assess public attitudes toward vaccines and predict their behavior toward vaccination ,20,21. FTo assess their attitude toward the booster dose of the COVID-19 vaccine, all eligible participants were required to answer the question, “Are you willing to receive the booster dose of COVID-19 vaccine if available?” The hesitancy rate was defined as the proportion of participants who answered “No” or “Not sure”, and if the participants had any concerns about the booster dose, the specific reasons for the reluctance were further asked at the same time.Frequencies and percentages were used to describe all categorical variables, consisting of sociodemographic characteristics, health status, knowledge about COVID-19 and the COVID-19 vaccine, and four dimensions of risk perception. An independent Chi-square test was used to compare the vaccine hesitancy rates among different groups stratified by the abovementioned characters. Logistic regression analyses were performed to explore the associations between risk perception and hesitancy rate toward the booster dose among old people (≥60 years old), and we finally constructed four models to examine the robustness of the estimations. Model A is a univariable logistic regression model. Region, age group, sex, marital status, education, and monthly household income were adjusted in model B. All covariates were included along with the other three dimensions of risk perceptions in model C. Additionally, only covariates with significant differences and the other three dimensions of risk perception were contained in model D. Crude odds ratios (cORs) and adjusted odds ratios (aORs) with 95% CIs were calculated to explain the effect size in different risk perception groups.p-value less than 0.05 was regarded as statistically significant.All statistical analyses were conducted by SPSS 26.0  in this study, and a two-sided A total of 3331 people aged 60 years and older in China were recruited to fill in this questionnaire, of which 3321 participants were eligible for the final analysis . Among tp < 0.05). People with lower perceptions of susceptibility (21.6%) and benefit (45.2%) were less likely to receive a booster dose of vaccination. Perceived barriers had a negative impact on vaccination willingness in older adults. However, no significant difference in vaccination hesitancy was found among groups with different degrees of perceived severity. A total of 571 old people were reluctant about the booster dose of COVID-19 vaccines. Believing that they were ineligible for vaccination due to certain illnesses (38.3%), concern about vaccine safety (32.0%), believing the booster shots were unnecessary (33.1%), and their limitation on movements (28.0%) were the main reasons for vaccine hesitation  were hesitant about the booster shots of COVID-19 vaccines . People sitation .As shown in p for interaction > 0.05). People who perceived low susceptibility in western China  or having lower income  were less likely to accept a booster dose of COVID-19 vaccine  was negatively correlated with hesitation, while perceived barriers  were positively correlated [According to our research, vaccination hesitancy toward the booster dose in the older population was closely associated with low perceived susceptibility, low perceived benefit, and high perceived barriers, but not with perceived severity. As people’s subjective judgment of the characteristics and severity of a particular risk, Risk perception will eventually affect people’s behavior ,21. Somerrelated . The assrrelated ,37,38,39rrelated . Chen etrrelated . At diffrrelated ,42,43,44Unlike young people, older people are more worried and concerned about booster shots. In this survey, 38.3% of the elderly worried that they were not eligible for booster shots for their existing diseases. Evidently, advanced age and comorbidities such as hypertension, diabetes, cardiovascular disease, and chronic respiratory disease were risk factors for SARS-CoV-2 infection and poor prognosis ,7. PeoplAs with other online cross-sectional surveys, our study also has some limitations. First, selection bias may exist. Considering the accessibility of online surveys for the elderly, questionnaires were answered only by Internet users. Moreover, some elderly people who had difficulty answering questions using electronic devices may seek help from young people around them and answer questions orally through young people’s dictation. This may also increase the selection bias of this study. Although we have set up tips at the end of each question and repeatedly emphasized that the subjective questions were supposed to be answered by the elderly orally, we cannot completely rule out proxy answers. However, under the current situation, it is urgent to understand the vaccination willingness toward the booster shots and promote vaccination coverage among the elderly. In addition, people’s acceptance of the booster dose of the COVID-19 vaccine was measured only by self-report, and we were unable to develop a standard scale to assess their willingness. Third, this survey was only conducted in China and based on a specific theoretical model, so results need to be interpreted carefully when extrapolated to other countries or compared with other models. Since this was an online survey, our depth was limited to some extent. Our results were only a crude supplement to the current research gap, and we hope that large-scale offline surveys with more participants will be implemented as soon as feasible. Moreover, this is only a cross-sectional study, which cannot fully verify the relationship between risk perception and vaccination hesitancy from a causal perspective.Of 3321 participants, 17.2% were hesitant about the booster dose of the COVID-19 vaccine, and it was closely associated with a lower level of perceived susceptibility and benefit, as well as a higher level of perceived barriers. Concerns about contraindications, vaccine safety, and limited movements were the main reasons for vaccine hesitancy. Under this circumstance, it is urgent to improve the understanding of the elderly on their own susceptibility and vulnerability, reasonably judge whether they could be vaccinated, and clear the obstacles to vaccination. Therefore, our results have important theoretical and practical implications for the subsequent promotion of the coverage of booster doses in the elderly population.


The coronavirus disease 2019 (COVID-19) vaccines are considered to be an effective way to prevent the spread of the infection. Our previous study has shown that about 75% of healthcare workers (HCWs) in China were willing to receive the vaccine when it became available. Here, we examined the acceptance of a third booster dose among Chinese people and identified the influencing factors.A cross-sectional online survey was conducted and the snowball sampling method was utilized. An online questionnaire was provided to all the participants in the form of a quick response (QR) code. The questionnaire included general demographic information, views on vaccines, the General Health Questionnaire-12 (GHQ-12), and the Depression, Anxiety, and Stress Scale-21 (DASS-21). The univariate analysis was done between all the variables and our dependent variable. Then, we used the multivariate logistic regression model to examine the influencing factors of the third booster dose acceptance.n = 960) declared that they would accept the booster dose. Knowing more about the vaccine and recognizing the efficacy of vaccines were significantly associated with greater acceptance of the booster dose. People willing to take the booster dose had better psychological health. A belief that the booster dose could prevent severe infection caused by COVID-19 and enhance the effectiveness of the first two doses were the main contributing factors to vaccine acceptance. Vaccine hesitancy was mainly due to a low perceived risk of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and rapid mutation of SARS-CoV-2.We collected 1,062 complete answers. Of these, 90.39% (This study revealed that Chinese people were very receptive to the third booster dose, which is an inspiring result. More positive attitudes regarding COVID-19 vaccination were supported by its efficacy and few side effects. The ongoing pandemic of coronavirus disease 2019 (COVID-19) has caused extensive damage worldwide. Despite established supportive therapies and the development of new antiviral drugs, vaccination is considered an effective method to prevent infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), particularly for at-risk populations. In China, the COVID-19 vaccines started to be used in priority groups on 15 December 2020. The Chinese government accelerated free vaccination for all the Chinese citizens starting in late March 2021 , regardless of region, occupation, or status, were eligible to participate in our study.The study protocol was approved (2022-S-27) by the Ethics Committee of Third People's Hospital of Chengdu . Informed consent was obtained before study enrollment. This was a cross-sectional online survey using a social media platform (WeChat™)-based survey program “Questionnaire Star” between 1 December and 31 December 2021. The online questionnaire was provided to all the participants in the form of a quick response (QR) code Before initiating the study, we first consulted psychologists working at the Third People's Hospital of Chengdu and the psychologists recommended two commonly used scales, as described below, suitable for measuring psychological status. The questionnaire gathered information on: (i) general demographics ; (ii) whether or not to accept the third booster dose; and (iii) the reasons for being willing or not willing to be vaccinated. Moreover, this questionnaire included the General Health Questionnaire-12 (GHQ-12) and the Depression, Anxiety, and Stress Scale-21 (DASS-21) to investigate the psychological health of respondents.Participants were asked if they would accept the third booster dose of the COVID-19 vaccine. They rated the item regarding their attitudes toward a booster dose on a four-point scale . Those answering 1 or 2 were identified as the vaccine-agree group. Those who answered 3 or 4 were identified as the vaccine-disagree group. Then, we listed common or possible causes of people's willingness or unwillingness to be vaccinated. People who agreed to take the vaccine could voluntarily select factors promoting their willingness to be vaccinated. Those who did not agree to take the vaccine could voluntarily choose the factors causing their resistance. The percentage of people who chose different factors was calculated.With the help of a consultant psychologist, we chose the scores for the GHQ-12 and the DASS-21 to measure participants' mental health. The GHQ-12 is widely used to identify common psychiatric conditions , 16. TheOnly complete questionnaires could be collected and incomplete data could not be submitted through the Questionnaire Star. The Questionnaire Star automatically collected data. We could convert all the data into text format and numeric form and export them to spreadsheets.n), and percentage. We used the univariate analysis with the t-test or the chi-squared test to compare the two groups (agree and disagree) to identify the factors associated with vaccine hesitancy. Then, the multivariate logistic regression model was employed to examine and identify the factors associated with the acceptance of the COVID-19 vaccine. Statistical analyses were performed using SPSS version 23.0 . p ≤ 0.05 was considered statistically significant.Descriptive analyses were conducted on all the study variables, which were reported as the mean, SD, number  declared that they would accept a booster dose. Of those who were willing to accept the third booster dose, 884 responders (83.2%) were strongly willing and 76 responders (7.1%) were willing but waiting to review more data. Of the 1,062 participants, only 102 (9.6%) participants were not willing to accept the third booster dose. Of them, 71 (6.7%) participants did not plan to receive the COVID-19 vaccine and 31 (2.9%) participants were strongly against taking the booster dose. We found that 95.6%  of respondents had completed the two-dose regimen and 97.3% (935) of respondents who had completed the two doses were willing to receive the third dose .2 = 5.577, p = 0.018), had a better understanding of how the vaccine works , had nearly completed a two-dose regimen, were more likely to think that the vaccine works , and had better mental health based on the GHQ-12 . However, the two groups did not differ significantly in terms of sex, age, occupation, educational level, marital status, contact with a person with COVID-19, living in a high-risk area, or income.The univariate analysis was used to decide significant differences between the two groups . People who had completed the two-dose regimen were more likely to accept the third dose compared with those who had not completed the two-dose regimen . People who believed that the vaccine was effective had 7.949 times greater odds of accepting the third dose compared with those who believed that the vaccine was not effective . People with the lower GHQ-12 score were more likely to accept the third dose  compared to those with the higher GHQ-12.To determine the factors associated with the willingness to be vaccinated, we used the multivariate logistic regression models. Results are given in Of 960 respondents who were willing to receive the third dose, 927 (96.6%) respondents chose the reasons for accepting the third dose. Of 102 respondents who were not willing to receive the third dose, 88 (86.3%) respondents chose the reasons for rejecting the third dose. The top two reasons for accepting the third dose were that the booster dose can prevent severe infections (69.6%) and enhance the effect of the first two doses 63.2%; . The topDespite newly approved antiviral drugs, the role of vaccination remains crucial. However, the efficacy of vaccination diminishes over time. Cohn and their colleagues have demonstrated that the efficacy of the COVID-19 vaccine against infection declined from 87 to 48% from February to October 2021 . ProtectMost surveys on COVID-19 have focused on the safety and immunogenicity of the third dose. Only a few studies have assessed the acceptance of the third booster dose. Suman and their colleagues have found that nearly two-thirds of respondents were concerned that vaccination may be ineffective against new strains of SARS-CoV-2 and that booster doses may be required. However, acceptance by vaccine-hesitant respondents of a hypothetical booster dose was only 14.3% . AnotherIn our study, 90.39% of participants declared that they would accept the booster dose. This acceptance rate was higher than that of the first two doses in our previous study (76.63%)  among HCOur study indicated that people with a high perceived risk of being infected had twice the odds of vaccine acceptance compared with those without a perceived risk of being infected. This concern is not surprising given that vaccines remain the main protection method against COVID-19. This result is similar to the findings noted by Harapan and collaborators and Rajamoorthy and coworkers , 34. AnoOur study indicated that learning more about the COVID-19 vaccine might have contributed to people being more willing to take the vaccine compared with those who had known less about the vaccine. This finding is consistent with data from our previous study, suggesting that greater education efforts toward vaccination against COVID-19 should be considered to increase public understanding of vaccines. People who believed that the COVID-19 vaccine was effective had 7.949 times greater odds of accepting the third dose compared with those who believed that the COVID-19 vaccine was not effective. Harapan and colleagues have reported similar findings . We founOur study had two major strengths. First, it was the first study to evaluate the acceptance of the third booster dose among the general population in China. Second, we evaluated mental health to investigate if it influenced vaccine hesitancy.Our study had two main limitations. First, we employed an electronic questionnaire to collect data (instead of a face-to-face interview), which resulted in uncontrolled conditions during questionnaire completion. Second, as we used a snowball sampling method, some individuals in this sample have still not been found and some individuals are probably omitted by a provider, leading to a biased sample. Third, the sample size was small, limiting the generalizability of our findings.In China, about 1.2 billion people have completed the two-dose regimen. The safety and efficacy of the COVID-19 vaccines have been recognized. People's acceptance of the booster dose has also been improved. Our study findings make us optimistic about the COVID-19 vaccination. We believe that the COVID-19 vaccination campaign will progress smoothly and will eventually provide herd immunity against COVID-19.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by the Ethics Committee of Third People's Hospital of Chengdu.YS, TX, PW, and XZ conceived and designed the questionnaire. JZ, DC, and YH recruited participants. YS, TX, and HD analyzed the data. YS wrote and revised the manuscript. All authors have approved the final version of the manuscript and agreed with submission to your esteemed journal.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.


Some HCWs broadly accepted the value of vaccination, and believed that boosters were necessary to effectively combat emergent new virus strains, which contrasted with peers who believed that boosters offered little defence against virus mutations. Fear prohibited some HCWs from getting the booster, with some having experienced adverse side effects from their initial vaccination, whilst others were concerned about future complications. Waning booster uptake rates could be arrested through invigorated communication strategies, while effective evidence-based training can potentially create positive normative vaccination practices amongst HCWs.This study aimed examin the factors associated with the uptake and non-acceptance of COVID-19 vaccine booster doses among healthcare workers (HCWs) in South Africa. We used a mixed-methods design with data from a web-based self-administered survey followed by semi-structured in-depth interviews (IDIs) with selected participants. Of the 6235 HCWs included in our analysis who had fully vaccinated, 3470 (56%) had taken their booster dose with a further 17% intending to get the booster. HCWs aged 35 to 49 years (OR = 1.30 [95% CI: 1.15–1.46]), and those aged 50 years or older (OR = 2.66 [95% CI: 2.32–3.05]) were more likely to get the booster dose. Females were less likely to have received the booster dose (OR = 0.88 [95% CI: 0.79–0.98]) with doctors more likely (OR = 1.58 [95% CI: 1.35–1.84]) than Nurses to have received the booster dose. HCWs in direct contact with patients (OR = 1.17 [95% CI: 1.00–1.38]) and who had previously received a flu vaccine (OR = 1.99 [95% CI: 1.56–2.55]) were more likely to have received the booster dose. Four themes emerged from the qualitative data analysis:  Healthcare Workers (HCWs) remain susceptible to SARS-CoV-2 infection, making this a priority group for vaccination and access to booster options, as outlined in the WHO’s Strategy to Achieve Global COVID-19 Vaccination by mid-2022 .Research among HCWs in SA revealed high initial COVID-19 vaccine uptakes rates . These dStudies of the acceptance and uptake of COVID-19 vaccine booster doses have focused on HCWs in high-income settings, with little evidence from the African continent and South Africa specifically. This study, therefore, aimed to gain a better understanding of the factors associated with vaccine booster uptake among HCWs in SA. Survey data were augmented by qualitative data revealing HCW perspectives on COVID-19 vaccine booster doses, with the summative evidence providing not only guidance for COVID-19 vaccine booster uptake interventions, but also other routine immunisation programmes.The study was granted ethical clearance by the University of KwaZulu-Natal Biomedical Research Ethics Committee (BREC/3970/2022) in compliance with all regulations and policies regarding ethical conduct of research. Written consent was obtained at the beginning of the online survey by providing formal paragraph-wise information about the study, with the participant requiring to click on a button for providing consent on the same online survey platform before moving on to filling the rest of the online survey questionnaire.This study used a mixed-methods design with data from a web-based self-administered survey followed by in-depth interviews (IDIs) with selected participants. Mixed methods research allows for combining elements of qualitative and quantitative research approaches for the broad purposes of providing depth of understanding and corroboration on a particular research topic , 13. Datrandbetween formula in Microsoft Excel for IDIs and scheduled for a virtual interview. In total we interviewed 30 HCWs, 10 vaccinated and 20 unvaccinated.The Foundation for Professional Development’s (FPD) database, which comprised contact details of 88 000 HCWs at the commencement of the study, was used to recruit HCWs for this study. Participants were offered compensation in the form of an entry into a draw for one of ten ZAR500 (~US$33) cash vouchers. The draw was not linked to participants’ survey responses. Participants interested in participating in IDIs were asked to provide their contact details following completion of the self-administered survey. A total of 7 763 HCWs participated in the survey. For this paper, we excluded all participants that were either unvaccinated or did not disclose their vaccination status. Thus, final analysis was undertaken on 6 325 vaccinated participants. As part of the survey, participants had the option to indicate their willingness to be contacted for a follow-up interview (IDI). Participants who had provided their contact details were organised into two groups (vaccinated and unvaccinated), then randomly selected using the The survey captured socio-demographic information, COVID-19 history, chronic conditions, and questions on vaccination behaviour. Survey questions were derived from a review of studies evaluating HCW hesitancy towards COVID-19 vaccines, described elsewhere .Two outcome measures were created based on the question; “Are you planning to get a booster vaccination against COVID-19?” with respondents indicating either “I have already received the booster dose”, “yes, as soon as possible”, “yes, but in a few months up to a year”, “yes, but in a year or more”, “I am unsure”, “no, but I might consider it in the future”, and “no, never”. The first outcome measure was coded as a binary variable where: 1 = “I have already received the booster dose” (Received Booster), and 0 = any other response to the question (No Booster). The second outcome measure was coded as a binary variable where: 1 = “I have already received the booster dose” or “yes, as soon as possible” or “yes, but in a few months up to a year” or “yes, but in a year or more” (Received and Intend), and 0 = “I am unsure” or “no, but I might consider it in the future” or “no, never” (Hesitant and Against).For the IDIs, two interview guides were developed, one for vaccinated HCWs and another for unvaccinated HCWs, with open-ended questions and probes regarding; (1) HCWs’ vaccination behaviour (including vaccine booster doses); (2) HCWs’ experience with administering vaccines; (3) HCWs’ perspectives of the vaccine programme; (4) how HCWs gather and appraise information sources; and (5) HCWs’ perspectives on educational resources that can be used to support them. Interviews were conducted by authors PBN and GG, who are experienced in conducting qualitative interviews using interview guides. IDIs lasted approximately 30 minutes and were recorded and transcribed.Personal information from study participants is available to the study principal investigator (PI) and co PI’s, this data was transmitted via a password-protected secure web-based file sharing dashboard service. Study personnel also transferred all data to password-protected computers.For the quantitative data, we ran a series of univariate logistic regression models to determine; how significantly a number of measures influenced a participant’s likelihood of having received a booster compared to those who hadn’t, and how significantly the same measures influenced a participant’s likelihood of having received or intended to receive a booster dose, compared to those who were hesitant or against receiving a booster dose. Finally, we conducted descriptive analysis (frequencies and proportions) of the underlying reasons for hesitancy towards vaccination boosters, stratified by gender.Qualitative data were analysed thematically using an inductive approach as prescribed by Braun and Clarke  six-stepIn this study, the point of integration was at the data analysis phase where the qualitative data provided in-depth understanding of the quantitative findings, revealing trends on HCWs vaccination attitudes and behaviours. The quantitative and qualitative data were kept analytically distinct where a statistical technique was used to analyse the survey data while thematic analysis was used to analyse interview data, as explained by Tariq and Woodman . FindingThere were 7 763 participants in the study. Of these, 6 235 participants were vaccinated, with 3 470 (56%) having received at least one booster dose. See Doctors were more likely to have received the booster dose than nurses (OR = 1.58 [95% CI: 1.35–1.84]) while all other HCWs  were less likely to have received the booster dose when compared to nurses (OR = 0.81 [95% CI: 0.72–0.92]). HCWs who were in direct contact with patients were more likely to have received the booster dose than those not in direct contact with patients (OR = 1.17 [95% CI: 1.00–1.38]). HCWs who had worked for 10 or more years were more likely to have received the booster dose (OR = 1.79 [95% CI: 1.52–2.11]) than their less experienced colleagues.In In the following tables, we conducted univariate logistic regression analysis with our second outcome variable and our demographic measures , and sevThe data further revealed that 15.84% of females had not received their booster dose because they had experienced adverse effects after initial vaccination, compared to 8.24% of males who listed this as a reason.(1) Vaccination as a routine practice among HCWs; (2) Emergence of new COVID-19 variants necessitating vaccine boosters; (3) Fear of potential side-effects from COVID-19 vaccine boosters; and (4) Limited value of COVID-19 vaccine boosters. Overall, participants held positive views towards COVID-19 vaccine boosters, with most highlighting the importance of boosters in maintaining population immunity. However, some HCWs remained both hesitant and against getting the booster, questioning its value, while raising safety concerns, which largely emanated from personal and observed experiences of adverse events following initial vaccination or after receiving a booster dose. These are discussed more fully below.We identified four themes regarding HCWs’ uptake and experiences of COVID-19 vaccine boosters: Participants recognized the value of regular vaccination, often citing that the influenza vaccine was part of their regular health routine. These participants were easily able to accept the importance of regular COVID-19 vaccine boosters, both to provide ongoing protection against COVID-19 disease and severe illness, and for protecting against potential virus variants or mutations.I actually said to my family, this is going to end up being like a flu vaccine. It’s something that we’re going to have to get annually, like we all get our flu vaccines annually. So for me it was never a question of maybe getting only one or two vaccines, and then it’s going to stop. This is a virus, and it changes, and it mutates, and we have to try and keep up with it.Another participant went on to explain:Personally, I think it is important. I think that again I’m a believer, so I have taken flu vaccine annually, and so if I believe that you know I don’t take a flu vaccine once and expect it to cover me for the rest of my life […] I think boosters are important in terms of coverage, to be fully vaccinated.HCWs continue to feel susceptible to infections, with older HCWs and those with chronic diseases feeling particularly vulnerable to COVID-19.I’ve been boosted yes…Because it’s a virus so that’s what this virus job is to infiltrate and cause chaos, so you need to be one step ahead, just like the flu, so you need to get boosted, especially my age, I’m over 50.Participants recognised the limited effect vaccines had on the new emerging variants, relying therefore on boosters to provide protection against virus mutations.I’ve had the booster. I think it is important because of the variants […] it’s changing.Participants felt that the booster dose improved individual’s immune response.I did get a booster. I think you know, it’s probably quite important just to help your immune system to create memory cells adequately, and to make sure that you know, you’re actually able to bolster an adequate response.Some HCWs experienced adverse side-effects following their initial vaccination. This, coupled with observing colleagues and others suffering from side-effects after receiving the vaccine or booster shots, dissuaded them from returning for their booster doses.There were many stories about the vaccine and one ended up not getting the booster […] I was concerned that maybe one would have side effects and getting a booster and I thought, maybe I also would have side effects as well.Another HCW added:Most of my colleagues have not yet gone for the booster but I think most of them had…a few of them still have that fear of, like I mentioned experiencing severe side effects of the vaccine […]I think, with the booster dose, I delayed it due to the side effects that I had with the second dose of the vaccine.In addition to the side-effects, participants raised concerned around the safety of booster doses for those that were pregnant.The few colleagues that I spoke to, they said to me that they felt so bad after the second vaccination, they’re not going for the boosters. And I also have a colleague now that’s pregnant and she’s not sure what the effects might be on the foetus.Some participants questioned the value of additional booster doses, suggesting that the initial vaccine provided adequate protection against infection and severe illness.I had COVID, I think, last year December, and it was not bad, so I don’t think the booster is going to make any difference, because the symptoms were not bad, so I’m not doing it.Some participants did not feel particularly vulnerable to COVID-19 infection, suggesting that their risk profile did not warrant getting booster doses.I’ve not gone on to receive the booster dosages. I personally, for my own personal risk perspective, I didn’t think it was that important. I don’t have particularly high-risk factors for severe COVID-19, and I didn’t feel that it was necessary for me personally.Even among unvaccinated participants, getting the initial vaccine and the booster dose was often expressed as unnecessary.I would opt for my natural response… you know development of antibodies etcetera, and see what will happen. Actually, my option was natural response versus COVID-19 vaccines and boosters.In our study, 56% of HCWs had received their initial COVID-19 booster vaccination while a further 17% reported their intention to get the booster dose within 12 months of completing the survey. Of those that had received their first booster dose, 23% had already received their second at the time of the study. These percentages are higher than those observed among the general South African public, where only approximately 24% had received a vaccine booster by March 2023 . These pPreviously published data on primary COVID-19 vaccination among the same study sample highlighted significant disparities in vaccine uptake by age, gender, race, job roles and presence or absence of a chronic health condition . This stThere were, however, some notable incongruities with respect to findings from initial vaccine uptake behaviour , with feTwo further dominant reasons for not getting the booster doses–for both males and females–centred around the belief that the initial vaccination provided adequate protection and that subsequent boosters were unnecessary or ineffective. The IDIs revealed contrasting perspectives, with some believing that boosters were necessary in combating the new virus strains whereas others questioned the efficacy of available vaccine boosters. HCWs operating in other contexts also reported that boosters would be required to counter new emerging strains of the virus .This study also found disparities in COVID-19 booster vaccination uptake among HCWs who were patient facing, which can be viewed as a proxy for increased perceived risk and fear of transmitting to patients, both factors correlating with increased uptake of boosters in another study . HCWs whVaccination booster rates, along with the intention to get the booster dose, remain high among HCWs in South Africa. However, not all HCWs who had vaccinated were committed to receiving boosters. Enthusiasm for further boosters appears to be waning as indicated by the low percentage 23%) who had received their second booster, even though most would have been eligible. The slowing uptake of boosters is evident not only among the South African general population  but amon% who hadOur study adds to the limited available data regarding COVID-19 booster vaccination uptake among HCWs, especially data available from Africa. Findings are expected to guide future vaccine campaigns and public health strategies to build vaccine confidence among HCWs and the general population.The study is limited by using an unrestricted self-administered survey that was dependent on the online reachability of HCWs on selected databases. Limitations in study design may have introduced selection bias and may have limited generalizability amongst HCWs in South Africa.This is the first study examining the uptake of vaccine booster doses among South African HCWs, offering valuable insights into drivers of hesitancy, revealing the perspectives of HCWs, all of which will hopefully influence the design of future vaccination programmes. This study shows that key demographics influence uptake of boosters, as was the case with primary vaccine doses. Age, race, gender, job roles and chronic health conditions were all factors contributing to the uptake of vaccine booster doses. Booster rates in our sample of HCWs were high, with the majority having had or intending to get their booster doses. The data revealed that females were concerned about the potential side effects and future complications which may arise from booster doses. Hesitancy was also fuelled by those who questioned the value of boosters relative to the protection afforded to them from their primary vaccination, or whether these boosters were effective against the emerging virus strains. Efforts are required to ensure that vaccination against disease becomes normative practice amongst HCWs, with this study showing participants who had historically taken the influenza vaccine more willing to get the COVID-19 booster doses. These normative practices can only be inculcated amongst HCWs through effective evidence-based training and communication strategies.S1 Text(DOCX)Click here for additional data file.


This cross-sectional survey explored the attitudes and the reasons, as well their associated factors, for receiving the second booster dose of the COVID-19 vaccine among a sample of all old adults and of people with chronic medical conditions attending two randomly selected immunization centers in Naples . A total of 438 questionnaires were collected. The majority were male (55.1%) and the median age was 71 years. A higher perception of the vaccine’s utility, measured with a 10-point Likert type scale, has been observed among males, individuals with a higher perception that COVID-19 is a severe illness, with a higher self-awareness of being at risk of infection, and with a higher trust in the information received. The most reported reasons for receiving the second booster dose included protection of themselves and of their family members from getting COVID-19, fear of acquiring the disease, and having a physician’s recommendation. Younger participants, married/cohabitant, and with a higher perception that COVID-19 is a severe illness were more likely to have indicated protecting themselves and their family members as reason for receiving the booster dose. Respondents with a chronic medical condition, with a higher perception that COVID-19 is a severe illness, with a lower trust in the information received, and informed by physicians were more likely to have received the vaccine because they perceived of being at risk of getting a severe form of the SARS-CoV-2 infection. Physicians should play a pivotal role in stressing the importance of the second booster dose and in helping individuals to make decisions. The coronavirus disease 2019 (COVID-19) continues to determine global public health concern with the emergence of the Omicron variant as the dominant strain around the world. As of 7 March 2023,there were more than 755 million confirmed COVID-19 cases and almost 7 million deaths in the world . Since 1Understanding the attitudes regarding the second booster dose and the reasons for receiving this dose are crucial for developing specific interventions to increase vaccine uptake and to generate support for health policy makers in the prevention activities. However, no literature is available on this topic. Therefore, to address this gap, this present cross-sectional survey was designed to characterize the attitudes regarding the second booster dose of the COVID-19 vaccine and the reasons for receiving it among a large sample of vaccinated adults and people with chronic medical conditions in Southern Italy, as well as to identify the associated factors.This survey was conducted as part of a larger project aimed at investigating perceptions and behaviors towards the COVID-19 vaccination among different groups of people in Southern Italy ,14,15,16The minimum target sample size of 427participants was determined assuming an expected proportion of 50% of respondents reporting the protection of themselves and their family members from getting COVID-19 as reason for having received the second booster dose of the COVID-19 vaccine, with a margin of error of 5%, considering a confidence interval of 95%, and allowing for an expected response rate of 90%.Before enrollment, well-trained research investigators, with professional skills in recruiting respondents and knowledge on the topic, approached each potential participant or parent/guardian for those younger than 18 years of age who had been registered for the administration of the second booster dose of the COVID-19 vaccine in the waiting rooms of the centers. The research investigators illustrated for each participant the study objectives and procedures, that the survey was answered on a voluntary basis, that no subject specific identifiers were recorded, that information was confidential, that they had the option to withdraw their participation at any stage without justification, and that by answering the questionnaire they gave the consent to take part in the survey. Informed written consent was obtained from each parent/guardian for the participants younger than 18 years of age. The research investigators asked each participant to complete the questionnaire and to return it immediately once filled. Individuals who had difficulties writing had the option to be interviewed face-to-face by the research investigators. No compensation or incentive was given to the individuals completing the questionnaire.The Ethics Committee of the Teaching Hospital of the University of Campania “Luigi Vanvitelli” approved the study protocol and the questionnaire.The questionnaire was designed based on those used in similar previously published surveys carried out by some of us among a variety of populations ,9,10,11.The questionnaire, with instructions that aided self-administration by participants, consisted of three sections . In the p-value less than or equal to 0.25 were selected for inclusion into the multivariable analysis. Multivariable linear and logistic regression models with the stepwise variable selection procedure with a threshold of p = 0.2 to retain and of p = 0.4 to exclude the variables were used to explore the association between several variables and the following outcomes of interest: perception of the utility of the second booster dose of the COVID-19 vaccine (Model 1); protection of themselves and of their family members from getting COVID-19 as reason for having received the second booster dose of the COVID-19 vaccine (Model 2); and perception of being at risk of getting a severe form of SARS-CoV-2 infection as reason for having received the second booster dose of the COVID-19 vaccine (Model 3). The following independent variables have been included in all models: sex , age (continuous), marital status , at least one chronic medical condition , baccalaureate/graduate degree , having been infected by SARS-CoV-2 , at least one family member/colleague/friend who has been infected by SARS-CoV-2 , level of perception that COVID-19 is a severe illness (continuous), level of trust in the sources of information used about the second booster dose of the COVID-19 vaccine (continuous), self-awareness of being at risk of getting the SARS-CoV-2 infection (continuous), physicians as source of information about the second booster dose of the COVID-19 vaccine , and need of additional information about the second booster dose of the COVID-19 vaccine . The variable level of the perception of the utility of the second booster dose of the COVID-19 vaccine (continuous) was included in Models 2 and 3.The following descriptive statistics were computed to summarize the answers of the respondents: frequencies, proportions, means, median, and interquartile range. The chi-square test or Student’s t-test were used to test the association between each variable and the continuous or dichotomous outcome. Variables associated in the bivariate analysis with a p-value equal to or less than 0.05. STATA software version 15.1 was used for performing the statistical analysis.Odds ratios (OR) with a 95% confidence interval (CI) and standardized regression coefficients (ß) for potential determinants associated with the study outcomes were estimated respectively in the logistic and in the linear regression models. Statistical significance was considered with a two-sided Of the 452 subjects approached, 438 agreed to participate for an overall response rate of 96.9%. Survey participants were asked to rate their level of agreement, using a 10-point Likert type scale, with statements regarding attitudes towards the COVID-19 infection and the second booster dose of the COVID-19 vaccine. Regarding the perception that COVID-19 was a serious illness, most respondents (42%) reported the value of 10 with an overall mean value of 8.4. Regarding the self-awareness of being at risk of getting the SARS-CoV-2 infection, the overall mean score was 7.4, and 35.9% of respondents had the higher value of 10. The perception towards the usefulness and utility of the second booster dose was very high with an overall mean value of 8.4 and 8.2, respectively. The final multivariable linear and logistic regression models displayed in Most of the sample (91.6%) had learned about the second booster dose of the COVID-19 vaccine. More than half of the respondents indicated mass media (57.1%) as the main source of information. Additional sources were physicians (36.9%) and institutional organizations (24.9%). Only a small number (11.2%) stated that they needed additional information on the second booster dose of the COVID-19 vaccine.The current findings are important as, to the best of our knowledge, this was the first and largest survey that evaluated vaccinated adults and people with chronic medical conditions about their attitudes regarding the second booster dose of the COVID-19 vaccine and the reasons for receiving it, and quantified the relative contribution of several factors.The present survey sheds light on the main reasons why adults and people with chronic medical conditions in Southern Italy received the second booster dose of the COVID-19 vaccine. Protection of themselves and of their family members from getting COVID-19, fear of acquiring the disease, having the vaccine recommended by a physician, and the perception of being at risk of getting a severe form of infection were the primary reasons cited by the survey participants. A possible explanation for indicating the protection as the main reason is the extensive and persistent diffusion of SARS-CoV-2 infection that may have contributed to the awareness among the sample with a consequent high sense of urgency regarding this vaccination. Earlier studies in different geographic areas showed comparable results ,18,19,20A substantial number of survey participants (91.6%) stated that they had learned about the second booster dose. The most important source of information was the mass media and it is interesting to observe that the physicians ranked second. The heavy use of mass media is an issue of concern. Indeed, even though during the pandemic mass media routinely reported the epidemiological data, some misinformation has been generated especially on the vaccines. It is important to note that physicians as a source of information have a positive significant impact. Indeed, individuals who had received information from a physician were more likely to receive the second booster because they perceive to be at risk of having a severe form of COVID-19 than those who did not use this source. This result highlights that physicians, as all healthcare providers, are a reputable source for disseminating health information and for health promotion and prevention of diseases. They play a pivotal role with a direct impact in helping individuals to acquire and to understand COVID-19-related information and, therefore, in influencing individuals’ decision-making regarding healthcare issues including COVID-19 vaccination. This finding is aligned with the mounting evidence showing that advice or recommendation by physicians is very effective for increasing the level of knowledge, awareness, and vaccination uptake in different groups ,26,27,28In addition to the association with the sources of information, the multivariable linear and logistic regression analysis revealed several other significant independent predictors of the three outcomes of interest. It is important to note that among all the socio-demographic and general respondent’s characteristics, age, sex, relationship status, and health status have been identified as important determinants. Males were more likely to have a higher perception of the utility of the second booster dose, whereas young and married/cohabitant respondents were more likely to have received the second booster dose for protection of themselves and of their family members from getting COVID-19 than individuals older and unmarried/not cohabiting. The association with sex may be explained by the number of deaths in Italy, which was higher among males despite the lower numbers of cases compared to females. Regarding the age, younger respondents may be more exposed to SARS-CoV-2 and more worried about the risk of acquiring and transmitting it and, therefore, decide to receive the second booster dose for preventing this infection. Individuals with at least one chronic medical condition were more likely to have received the second booster dose because they perceive to be at more risk of getting a severe form of SARS-CoV-2 infection than healthy individuals. This association may be explained by the fact that they put more emphasis and interest in health issues and thus they are more interested in this vaccination. The associations with socio-demographic and general respondent’s characteristics observed in this survey are consistent with the literature ,31,32,33The present survey has some potential methodological limitations that should be considered when interpreting the findings. First, as in all cross-sectional study design, there is no evidence of a temporal relationship between independent variables and outcomes of interest. Second, the survey has been conducted in immunization centers in a single geographic area, thus the findings should be interpreted carefully as they may not be generalized to the overall populations of adults and people with chronic medical conditions in Italy. Third, respondents using self-reporting questionnaires may have introduced response and social desirability bias. However, these types of bias, common disadvantages of this data collection method, have been reduced by using a completely anonymous questionnaire. Although this survey has these limitations, there are several advantages of this type of data collection that should be emphasized, such as the low cost, the rapidity, the easier data collection, and the high response rate. Moreover, this is the first survey about the reasons for receiving the second booster dose of the COVID-19 vaccine in adults and in people with chronic medical conditions in Southern Italy and the results have potential intervention implications for the health policy makers.In sum, this survey provides useful information about the second booster dose of the COVID-19 vaccine for adults and for people with chronic medical conditions in Southern Italy and underlines the need for implementation and policymaking in order to expedite the achievement of an optimal coverage rate. Lack of recommendations for receiving the second booster dose from physicians is of great concern, since they should play, as with all public health professionals, a pivotal role in stressing the importance of this vaccination and in advising and helping individuals to make decisions, while self-protection and protection of their family members from infection were the main reasons for this sample to receive the booster dose.


Background: Recent evidence has shown that the updated COVID-19 bivalent booster is effective in preventing COVID-19 compared with no previous vaccination and prior monovalent vaccination. Despite its effectiveness, uptake has been poor, and a minority of eligible recipients have received the booster. Understanding healthcare worker (HCW) attitudes for and against voluntary uptake of the bivalent booster dose against COVID-19 can help guide communication strategy to maximize uptake. In this survey study, we investigated attitudes toward updated and/or bivalent booster uptake in a behavioral health hospital shortly after a COVID-19 outbreak. Methods: A survey tool was developed and sent to all HCWs at the Yale New Haven Psychiatric Hospital in December 2022. The survey queried demographic data, job category, history of COVID-19, prior COVID-19 vaccinations, perception of COVID-19 exposure, and updated and/or bivalent booster doses. The survey was administered several weeks after a COVID-19 outbreak on multiple inpatient behavioral health units. Receipt of the COVID-19 primary vaccination series and the first booster dose were mandated for HCWs; however, receipt of the bivalent booster was voluntary. Results: The survey was sent to 664 HCWs with primary assignments in behavioral health settings. In total, 182 (27.4%) provided complete responses to the survey and are included in these data. Moreover, 91 HCWs (50.0%) reported previously having COVID-19 at least once. Overall, 100 HCWs (55.0%) received the bivalent booster. The most identified reasons for receiving the bivalent booster were wanting to protect family and friends (n = 113), importance of staying healthy (n = 112), and protecting colleagues and patients (n = 103). The most identified reasons for not wanting to receive the bivalent booster dose were not thinking it provides additional protection (n = 33), “too many” shots already received (n = 31), and concern about side effects (n = 30). Discussion: Bivalent booster dose uptake in HCWs on behavioral health units shortly after a COVID-19 outbreak was greater than the general population. HCWs reported varying reasons for and against receipt of the bivalent booster dose, with the most common being protection of family and friends and perceptions of no additional protection, respectively. A limitation of this study was voluntary response bias, in which results are biased toward individuals more likely to receive a bivalent booster vaccine. It is unclear whether reasons for declining the vaccine are representative of HCWs who did not complete the survey. Assessing attitudes for the bivalent booster dose can assist in guiding communication and outreach strategies to increase vaccine uptake by HCWs.Disclosures: None


This study aimed to investigate factors influencing the uptake of first and second COVID-19 booster vaccines among adults in Belgium, particularly age, sex, region of residence and laboratory confirmed COVID-19 infection history.A binomial regression model was used with having received the first or second booster as outcome and age, sex, region of residence and infection history as fixed variables. Among adults, there was generally a higher uptake to receive the first booster among older age groups compared to younger ones. Females, individuals residing in Flanders and those with no previous COVID-19 infection were more likely to receive the first booster. For the second booster, the same age trend was seen as for the first booster. Males, individuals residing in Flanders and those who tested positive for COVID-19 once after first booster were more likely to receive the second booster. Individuals with multiple positive COVID-19 tests before and after primary course or first booster were less likely to receive the subsequent booster dose compared to COVID-naïve individuals. This information could be used to guide future vaccination campaigns during a pandemic and can provide valuable insights into booster uptake patterns.The online version contains supplementary material available at 10.1186/s13104-023-06608-4. In the second half of 2021, the World Health Organization (WHO) recommended administering booster vaccines against COVID-19 to adults who had already completed their primary course of COVID-19 vaccination (PC). Several studies have aimed to identify socio-demographic, socio-economic and perception-related factors associated with booster vaccine intention and hesitancy. Findings indicated that older age was positively associated with the intention to receive both first and second booster doses  while thAlthough these studies provided insight into booster acceptance among different populations, they did not delve into actual booster uptake nor its influencing factors. Retrospective cohort studies utilizing registry-linked data could provide more comprehensive insights into the determinants of booster uptake. We identified only few articles that described registry-based studies examining the association between socio-demographic and economic factors, and COVID-19 (booster) vaccination. These studies showed a higher vaccine uptake among older individuals, females and those without prior COVID-19 infection while lower uptake was observed among socially deprived individuals and those with a migrant background –16. EvenIn Belgium, the first booster campaign, for which the entire adult population was invited , took plSeveral COVID-19 waves preceded and coincided with the Belgian booster campaigns, resulting in breakthrough infections after PC. These individuals may harbor scepticism about vaccine effectiveness compared to those infected before vaccination or uninfected individuals , 6. ThisTwo databases were used in this analysis: (1) the COVID-19 national vaccination registry (VaccinNet +) which contains demographic information  and COVID-19 vaccination details , and (2) the COVID-19 Health Data test database which contains individual COVID-19 test data (PCR and antigen test results and testing dates). These databases were linked using the unique Belgian national registry number. Data were pseudonymised before conducting the analyses.® (Pfizer/BioNtech), Spikevax® (Moderna), Vaxzevria® (AstraZeneca-Oxford) or 1 dose of Jcovden® (Johnson & Johnson)—were included in the analyses focusing on first booster uptake. Persons with incomplete PC (only one dose over two) or a heterologous PC  were excluded from the analyses. For analyses regarding the second booster uptake, those who received one booster dose after a PC and were aged 50 years or older were included (as only persons over 50 were actively invited for a second booster during the Belgian vaccination campaign). Only individuals who received a booster with Comirnaty® (Pfizer/BioNtech) or Spikevax® (Moderna) were included in the analyses. First boosters administered until March 1st 2022 and second boosters until January 31st were considered. We only included persons alive on the 31st of January 2023.All adults (≥ 18 years) who had PC in Belgium—2 doses of ComirnatyInfection history of individuals was based on their laboratory-confirmed COVID-19 infection status, utilizing PCR and antigenic COVID-19 tests. This was considered relative to the last preceding COVID-19 vaccination. As shown in Table We fitted a logistic regression model, assuming a quasi-binomial data distribution (number of boosted over number of people who received previous vaccination), with having received a first or a second booster as outcome (number of successes) and with age, sex, region of residence and infection history as categorical fixed effects. Additionally, a sensitivity analysis was performed based on stratification by age, by fitting logistic regression models with the same fixed effects apart from age. The model coefficients were used to calculate the adjusted odds ratio (aOR). All statistical calculations were performed in R (version 4.1.3).The study population comprised 7 857 113 individuals, of whom 51.42% were female. The mean age was 50.6 ± 18.8 years. Among those who received the PC, 82.55% received the first booster and 46.57% received the second booster. Among the first booster recipients, 84.00% were COVID-naïve, 8.00% had at least one prior infection before PC and 7.68% had one infection after PC. Less than 1% of the study population had multiple infections before and after, or only after PC. Among second booster recipients, 74.16% were COVID-naïve, 11.99% had at least one infection before the first booster and 12.73% had one infection after the first booster. Less than 2% had multiple infections before and after or only after the first booster , older age groups showed higher likelihood of receiving the first booster, with the 75–84 age group being the most likely . Men had a slightly lower likelihood to receive the first booster compared to women. Residents of Flanders had the highest odds of receiving a first booster compared to Wallonia and Brussels.Compared to COVID-naïve individuals, those with one infection after first booster were slightly more likely to receive the second booster . However, individuals with other infection history patterns were consistently less likely to receive one, compared to COVID-naïve individuals .Compared to the youngest age group (50–54 years), higher age groups were more likely to receive the second booster, with the over 85 age group having the highest likelihood . Men had a slightly higher likelihood to get the second booster compared to women. Individuals residing in Flanders had the highest likelihood of receiving the second booster compared to Wallonia and Brussels , but also to changes in testing strategies and non-pharmaceutical interventions during booster campaigns. During the first booster campaign, Belgium was still in a “crisis mode” with various restrictions and a more elaborate testing strategy. However, as 2022 progressed, the testing capacity and test prescriptions decreased, resulting in a decrease in registered PCR tests. This led to an underestimation of individuals’ infection history relative to the first booster uptake, which causes a vast overestimation of COVID-naïve individuals. The COVID-naïve group was used as reference for infection history in both uptake models, considering its heterogenous nature in the second booster uptake model, it will influence the model outcome directly. Individuals with all other infection history patterns were consistently less likely to have received the second booster, following the trends observed in the first booster uptake. Nevertheless, given the aOR, infection history had a more sizable effect on vaccine uptake for the first booster campaign compared to the second. Bennett et al. and Della Polla et al. reported on the possible effect of previous COVID-19 infection on first and second booster intention respectively, based on self-reported surveys with limited study samples, but the results were not significant . A studyWe observed an increasing likelihood of first and second COVID-19 booster uptake with increasing age, consistent with studies on booster acceptance , 11, 12.Older age, residing in Flanders (compared to the other Belgian regions) were positively associated with both first and second booster uptake. Having had multiple confirmed COVID-19 infections before or after preceding vaccination, were negatively associated with the first and second booster uptake. These findings highlight the potential influence of being previously infected with COVID-19 on individuals’ booster vaccine uptake. This information could give a better estimation of expected booster vaccine uptake in future booster vaccine campaigns.We acknowledge three main limitations. Firstly, the infection history data is limited due to the evolving testing strategy. The incomplete testing of symptomatic individuals and the absence of antigenic self-test results, particularly during the period in between the first and second booster (start of Omicron dominance), resulted in an overestimation of COVID-naïve individuals. However, we believe that this limitation has minimal impact on the overall direction of our estimates given the repeated negative effect of having received multiple infections on booster uptake. Secondly, we cannot asses the influence of individuals perception regarding booster vaccines. Several studies have shown the inherent link between perception, regarding politics, religion, vaccine safety and effectiveness, risk of (severe) disease and peer behaviour, and booster hesitancy , 6, 10. Additional file 1:Figure S1. Comparison of drivers booster uptake in general models and age group-stratified models. A Odds ratio for the general model for first booster uptake. B Odds ratio for the general model for second booster uptake. C Odds ratio for the age group-stratified model for first booster uptake. CI is not shown for ‘>1 infect. after’ due to out of bounds. D Odds ratio for the age group-stratified model for second booster uptake


In Krabbe disease (KD), mutations in β-galactosylceramidase (GALC), a lysosomal enzyme responsible for the catabolism of galactolipids, leads to the accumulation of its substrates galactocerebroside and psychosine. This neurologic condition is characterized by a severe and progressive demyelination together with neuron-autonomous defects and degeneration. Twitcher mice mimic the infantile form of KD, which is the most common form of the human disease. The Twitcher CNS and PNS present demyelination, axonal loss and neuronal defects including decreased levels of acetylated tubulin, decreased microtubule stability and impaired axonal transport.We tested whether inhibiting the α-tubulin deacetylase HDAC6 with a specific inhibitor, ACY-738, was able to counteract the early neuropathology and neuronal defects of Twitcher mice.Our data show that delivery of ACY-738 corrects the low levels of acetylated tubulin in the Twitcher nervous system. Furthermore, it reverts the loss myelinated axons in the sciatic nerve and in the optic nerve when administered from birth to postnatal day 9, suggesting that the drug holds neuroprotective properties. The extended delivery of ACY-738 to Twitcher mice delayed axonal degeneration in the CNS and ameliorated the general presentation of the disease. ACY-738 was effective in rescuing neuronal defects of Twitcher neurons, stabilizing microtubule dynamics and increasing the axonal transport of mitochondria.Overall, our results support that ACY-738 has a neuroprotective effect in KD and should be considered as an add-on therapy combined with strategies targeting metabolic correction. As a consequence of GALC deficiency, the metabolism of several galactosphingolipids is impaired and GALC substrates - galactosylceramide and galactosylsphingosine , accumulate , or globoid cell leukodystrophy, is a lysosomal storage disorder caused by mutations in the cumulate . Whereascumulate  and leadcumulate . Consequcumulate . Among tcumulate . The Twinal GALC , mimics nal GALC . Similarnal GALC .Hematopoietic stem cell transplantation (HSCT) emerged as one of the most valuable treatments for KD, delaying disease progression and extending life time of KD patients if transplantation is performed before the onset of symptoms . In spitWith respect to neuroprotection, it is important to note that in addition to demyelination, neuronal-autonomous defects are also central players in KD progression . In fact50–1,7 nM) with a high selectivity of 60-1,500-fold over class I HDACs. Although this compound has a rapid elimination from plasma (T1/2–0.2 h), upon systemic administration, it leads to a ratio brain-plasma exposure of 1.22:1, demonstrating a higher capacity to cross the blood–brain barrier than other HDAC6 inhibitors such as tubastatin A  and a specific histone deacetylase, HDAC6, that has a cytoplasmic location and thereby does not interfere with histone acetylation . Studiesstatin A . As stabstatin A . Currentnditions , and in gression , whereasgression . In the lifespan . ACY-738lifespan . In viewGiven the impairment caused by the defect in tubulin acetylation in Twitcher nerves, we tested whether ACY-738 delivery could counteract the early neuropathology of this KD model. Here, we show that the early systemic delivery of ACY-738 robustly increases tubulin acetylation, stabilizes microtubule dynamics, increases axonal transport, and rescues the early loss of myelinated axons in the optic and sciatic nerves of Twitcher mice. Our results thus support that ACY-738 has neuroprotective effect in KD and should be considered as an add-on therapy in KD.2.2.1.Galc allele possesses the mutation, as described  mice and wild-type (WT) littermates were obtained from heterozygous breeding pairs (Jackson Laboratory). Genotyping of Twitcher mice was based on the use of a PCR mismatched primer that creates a restriction site for EcoRV if the escribed . Mice we2.2.Animals were monitored twice per week until P25 and then daily until humane endpoints were reached. Parameters including weight loss, natural/provoked behavior, and activity level were scored. Natural behavior was assessed by observation of the animals in their home cage; animals with normal behaviour, i.e., moving, grooming and feeding, and alert received the highest score 3, animals with minor changes to these parameters were scored as 2, animals with severely decreased mobility but that were still alert received a score of 1, whereas less motile and not alert animals scored 0. The animals were then touched to provoke a response, which was scored with 3 if the animal reacted normally , 2 if minor changes to normal behavior were found, 1 if moderate changes to the escaping and freezing responses were seen or 0 is the escaping and freezing responses were absent. Activity level was evaluated by observation of the animals in their home cage during 5 min and recording the time spent ambulating and exploring; animals active for more than 4 min received the highest score (3), and animals with less than 1 min of activity scored (0). Twitching frequency was accessed also twice a week using a similar scoring system, with animals with rare twitching scoring 4 and animals with severe tremors soring 1.2.3.n = 10 for each condition) or maintained until humane endpoints were reached . WT mice were also injected subcutaneously daily with ACY-738 (n = 8–10) or vehicle (n = 8–10) to serve as controls. Either at P9 or when animals reached humane endpoints, brains, sciatic nerves, optic nerves and dorsal root ganglia (DRG) were collected and processed for morphometric analysis, western blot analysis or primary neuron cultures, as described below. During the delivery period, no signs of toxicity of the drug were detected.ACY-738  was prepared in 0.5% hydroxypropyl methylcellulose (HPMC) diluted in PBS at a final concentration of 0.5 mg/mL. Starting at P0, Twitcher mice were injected subcutaneously every day (3 mg/kg) with either ACY-738 (in 0.5% HPMC) or vehicle (0.5% HPMC) and euthanized at P9 (2.4.n = 10) or control Twitcher mice (n = 8–9) were homogenized in lysis buffer (PBS containing 0.3% Triton X-100), 1 mM sodium orthovanadate and protease inhibitors . Total protein of each sample was determined using the Bio-Rad DC kit  and protein lysates were run on 10% SDS-PAGE (5 μg/lane) and transferred to supported nitrocellulose membranes . Membranes were incubated overnight at 4°C with mouse anti-acetylated tubulin , rat anti-tyrosinated tubulin  and mouse anti-α-tubulin . Given the difficulty to perform stripping of anti-tubulin antibodies from the membranes, two separated gels were loaded and transferred simultaneously to probe for either acetylated- or α-tubulin. Secondary antibodies were HRP-conjugated anti-mouse IgG  or the IRDye® 800CW Goat anti-Mouse IgG . Proteins were detected using Luminata Forte  and quantification was performed by densitometry using QuantityOne software (Bio-Rad). Alternatively, the Odyssey CLx (LI-COR) imaging system was used for detection of membrane total protein  and dye-conjugated labelled secondary antibodies .Sciatic nerves, optic nerves and brains from ACY-738-treated Twitcher mice (2.5.n = 8 ACY-738-treated and n = 8 control Twitcher mice), the right sciatic and optic nerves were used for neuropathological analyses. All tissues were isolated and fixed by immersion in 4% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 5 days and processed for electronic microscopy as previously described  until humane endpoints  supplemented with 10% fetal bovine serum  and 1% penicillin/streptomycin . DRG were digested with collagenase IV-S  for 1 h and 30 min. Neurons were isolated centrifuging the cell suspension into a 15% bovine serum albumin  cushion, for 10 min at 1000 rpm. Cells were resuspended in DMEM:F12 supplemented with 1% P/S, 2× B27 , 2 mM L-glutamine  and 50 ng/mL nerve growth factor .2.7.For the analysis of microtubule dynamics in the growth cone, DRG neurons from Twitcher and WT mice from animals euthanized humane endpoints were isolated as described above and nucleofected with a plasmid encoding pEGFP-eEB3 (Addgene #190164). After transfection, cells were left in suspension for 24 h. At DIV1, cells were plated at a density of 10,000 cells/well in glass-bottom 8-well μ-dishes. At DIV5, 2 h before imaging, half of the medium was replaced with complete media without phenol red and supplemented with 100 nM ACY-738 in 0.1% DMSO or 0.1% DMSO alone. Transfected cells were visualized using the 100x objective of a spinning disk confocal system Andor Revolution XD with an iXonEM+ DU-897 camera and IQ 1.10.1 software (ANDOR Technology). Time-lapse recordings  were acquired in three planes separated by 0.2 μm each at 37°C and 5% CO2. For the quantification of EB3 comet growth speed, kymographs were performed using the Fiji KymoResliceWide plugin . Starting and end positions of the traces were defined using the Fiji Cell Counter plugin.2.8.For live imaging of axonal transport, DRG neurons from ACY-738-treated and control mice were isolated as previously described from animals euthanized at humane endpoints. Dissociated DRG neurons  were cultured in DMEM:F12 supplemented with NGF, Pen/Strep, B27 and L-glutamine for 16 h in 8-well μ-slides ibiTreat (Ibidi) coated with PLL and laminin. DRG neurons isolated from ACY-738-treated WT and Twitcher mice were maintained in culture in the presence of 100 nM ACY-738 in 0.1% DMSO. Control WT and Twitcher DRG neurons were maintained in 0.1% DMSO. For the analysis of axonal transport of mitochondria, DRG neurons were incubated with Mitotracker  in DMEM:F12 for 45 min at 37°C. Axonal transport was visualized in a confocal SP5II Leica microscope. Photomicrographs were taken for 2 min with 1 s frame intervals with 5 Z-stacks per frame. For each condition, the movement of at least 50 organelles was tracked. Analysis of axonal transport (velocity and flux) was done using the Fiji software.2.9.t-test, one-way or two-way ANOVA followed by multiple comparison tests or Welch ANOVA test if unequal variances were observed between the groups. Sample sizes are indicated in figure legends and significance was defined as p value* < 0.05, p** < 0.01, p*** < 0.001, p**** < 0.0001, ns: not significant.Data is shown as mean and standard error of the mean (SEM). The statistical analysis for all experiments was performed with GraphPad Prism 9. Unless elsewhere stated, the following statistical tests were used as indicated in figure legends: two-tailed unpaired 3.3.1.To test the possible neuroprotective effect of ACY-738 in KD, the drug was delivered subcutaneously daily to Twitcher mice. Since Twitcher mice present axonal loss that can be detected readily at P9, prior to the onset of demyelination , we expl3.2.Twitcher neurons exhibit decreased acetylated tubulin levels, decreased microtubule cytoskeleton stability , and impAs axonal transport efficiency is highly influenced by microtubule organization and dynamics , we hypo3.3.Twitcher mice develop tremors at around 18–20 days of age and muscle weakness and weight loss at P20-25 and usually reach the humane endpoints established in our facility (and recommended by FELASA) at 30–35 days of age. To determine the possible effect of ACY-738 treatment in disease progression, animals were monitored daily for their functional performance. ACY-738-treatment resulted in a substantial improvement of natural  and prov3.4.Given the functional improvement of Twitcher mice treated with ACY-738, we further investigated its potential to correct the early neuropathology of this model. In the optic nerve, as previously reported , P9 TwitSimilar to the optic nerve, P9 Twitcher sciatic nerves displayed a reduced density of both myelinated and unmyelinated axons in comparison with WT sciatic nerves –H, as pr3.5.The results obtained from the subcutaneous delivery of ACY-738 from P0-P9 warranted the analysis of treated Twitcher mice for extended time periods, i.e., until humane endpoints were reached. Detailed analysis of control and ACY-738-treated Twitcher mice showed that the number of small caliber myelinated axons was increased by ACY-738 treatment . Specifi4.Galc, in long-term surviving AAV-treated Twitcher mice, multiple demyelinating areas are still detected in the brain, suggesting a possible late onset reduction of AAV efficacy , University of Porto, Porto, Portugal.MS and JN-R coordinated research. MJ, MS, and JN-R designed the study. SB, PB, MJ, MS, and JN-R analysed experiments. SB, MM, MP, AM, and JN-R performed the experiments. SB, MS, and JN-R wrote the manuscript. All authors contributed to the article and approved the submitted version.The Nerve Regeneration group was supported by a Sponsored Research Agreement with Acetylon Pharmaceuticals Inc. that provided ACY-738. The work was also supported by Portuguese funds through FCT - Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the project EJPRD/0002/2019. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication.OG and MJ were employed by Acetylon Pharmaceuticals Inc.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.