Social media has changed the way we live and communicate, as well as offering unprecedented opportunities to improve many aspects of our lives, including health promotion and disease prevention. However, there is also a darker side to social media that is not always as evident as its possible benefits. In fact, social media has also opened the door to new social and health risks that are linked to health misinformation.This study aimed to study the role of social media bots during the COVID-19 outbreak.The Twitter streaming API was used to collect tweets regarding COVID-19 during the early stages of the outbreak. The Botometer tool was then used to obtain the likelihood of whether each account is a bot or not. Bot classification and topic-modeling techniques were used to interpret the Twitter conversation. Finally, the sentiment associated with the tweets was compared depending on the source of the tweet.Regarding the conversation topics, there were notable differences between the different accounts. The content of nonbot accounts was associated with the evolution of the pandemic, support, and advice. On the other hand, in the case of self-declared bots, the content consisted mainly of news, such as the existence of diagnostic tests, the evolution of the pandemic, and scientific findings. Finally, in the case of bots, the content was mostly political. Above all, there was a general overriding tone of criticism and disagreement. In relation to the sentiment analysis, the main differences were associated with the tone of the conversation. In the case of self-declared bots, this tended to be neutral, whereas the conversation of normal users scored positively. In contrast, bots tended to score negatively.By classifying the accounts according to their likelihood of being bots and performing topic modeling, we were able to segment the Twitter conversation regarding COVID-19. Bot accounts tended to criticize the measures imposed to curb the pandemic, express disagreement with politicians, or question the veracity of the information shared on social media. Social media has radically changed the way we live and communicate. These new communication platforms offer unprecedented opportunities to improve many aspects of our lives, including public health ,2. They Nevertheless, social media has also opened the door to new social and health risks ,5. PolicTherefore, the role of social media during the COVID-19 pandemic has been critical. Although these new platforms have been useful to keep the public informed during the most critical moments of the pandemic, the responses by health authorities to combat the outbreak have been followed by a massive “infodemic,” recently defined as “an overabundance of information—some accurate and some not—that makes it hard for people to find trustworthy sources and reliable guidance when they need it” . InformaRecently, misinformation dynamics have increased their complexity due to the emergence of so-called “social bots” . The role of social bots in the spread of misinformation on social media platforms has been widely recognized during political campaigns and election periods  and in rThe activity of social bots has dramatically increased in the context of the COVID-19 infodemic  due to tcovid_19, covid19, covid, and coronavirus. These hashtags were used during this period to capture the conversation during the first wave of COVID-19. To simplify the subsequent analysis, only tweets written in the English language were selected. The resulting data sample contained approximately 14 million tweets from about 285,000 different Twitter accounts.Data collection started on March 16 and ended on June 15, 2020, using the Twitter streaming API with the following hashtags: Botometer  [Botometer is a publicly available service that leverages more than 1000 features to evaluate the extent to which a Twitter account exhibits similarity to the known characteristics of social bots. As in other studies [We used project)  to obtai studies ,30, 0.8  studies . In our Botometer also gives specific scores for 6 different bot types: echo chamber, fake follower, financial, self-declared, spammer, and other. Given the differing nature of social bots, it was considered necessary to draw a distinction between self-declared bots and other types of bots. Self-declared bots are extracted from Botwiki [In addition to the overall likelihood of being a bot,  Botwiki ,32.Finally, together with the bot classification, we also applied topic-modeling techniques. This unsupervised classification approach allows the classification of texts, using techniques such as clustering to find groups of texts with similar content. In this case, we used latent dirichlet allocation (LDA), a popular topic-modeling technique which considers each document as a random mixture of various topics and each topic as a mixture of words .To correctly interpret the results, we considered the distribution of the topics within the corpus, the keywords of each of the topics, and the intertopic distance . Based oIn addition, we plotted an intertopic distance map  to visuaFor each of the groups, we used sentiment analysis to examine the tone or sentiment associated with the content. Sentiment analysis is an area of knowledge in the field of natural language processing, text analysis, and computational linguistics used to identify and extract subjective information from resources. In the case of text mining, sentiment analysis involves automatically mass-classifying documents based on the positive or negative connotation of the language in the document .For the sentiment extraction, we used Valence Aware Dictionary and Sentiment Reasoner (VADER), a rule-based tool specifically attuned to sentiments expressed on social media platforms . VADER uAnother feature of this method is the output value. Most sentiment analyses classify texts as positive, negative, and neutral; for example, texts with a predominance of words, expressions, or ways of writing perceived as positive are classified as positive. However, the method used here returns a sentiment score between –1 and 1, allowing a higher level of comparison between the different types of accounts.F2,284,814=1056; P<.001). As also noted in Broniatowski et al [ki et al , the mosNot all groups contributed to the same extent. Likewise, the contribution of the participants in the global discussion was highly unequal. The Gini index was used to measure this inequality. This index is a measure of the distribution, with a higher Gini index indicating greater inequality. In the case of self-declared bots, the most active accounts spread official data . Second, several of these accounts were digital magazines or independent news agencies. The descriptions of these accounts mentioned that they created messages to provide periodic reports on the situation and communicate the global evolution of COVID-19 or substantial changes in the evolution of the pandemic. Many of these accounts indicated that their purpose was informative. Given this situation, these profiles were separated from those classified as regular bots in the analysis.The descriptions of the accounts classified as bots were very different from each other. Many accounts identified themselves with technology companies. Others identified themselves as activists, either political, environmental, or even military. These accounts tweeted about the pandemic, the political measures taken, or complaints about the situation resulting from inaction.After classifying the accounts, the topics were extracted using LDA. To select the correct number of topics, we relied on the coefficient of variation, which measures the coherence between the topics inferred by a model. In other words, the coefficient indicates which combination of topics is the most coherent. Higher values indicate that the topics are semantically interpretable. Topic coherence measures score a single topic by measuring the degree of semantic similarity between high-scoring words in the topic. This concept brings together several measures to assess the coherence between the topics. To choose the number of topics, the LDA model was reapplied with different outputs, and those with the highest coefficient of variation were selected . In totaIn the intertopic distance map below , each buP(w), by its distinctiveness, a measure of how informative the specific term is to determine the generative topic. Saliency is therefore a measure of the degree to which the word appears a small number of times or not at all in other topics [We also plotted the most common terms in a bar chart . The terr topics .We then carried out the qualitative phase, with both authors agreeing in 89% (16/18) of the cases. stay, home, and family. Other tweets shared this kind of information but for specific regions. For example, Topic 9 was focused on regions in Africa, and Topic 13 was focused on the lockdown in India.In These 2 topics have the most substantial differences between self-declared bots and the rest of the accounts. Topic 9 accumulated the highest percentage  of self-declared bot accounts, compared to bots  and nonbots . Likewise, the percentage of self-declared bots in Topic 13 is 5.7% (495/8690), whereas the percentage for bots is 3.4% (293/8616) and 3.8%  for nonbots.case, death, report, and total. In the following topics, the model groups’ contents were related to specific measures to curb the pandemic. Topic 3 mentioned the lack of testing. Some topics reminded people to stay at home (Topic 4), of the importance of wearing a face mask (Topic 11), or of washing one’s hands (Topic 12).Topic 2 contained information about the evolution of the pandemic. This topic was focused on the second wave and information on the number of deaths. The most common keywords were president, Trump, China, virus, year, and world. This topic had the biggest difference between the percentage of bots and the rest of the accounts. In Topic 16, most of the tweets mentioned the lack of honesty of the US President. There were also complaints about the need to share true information and disregard rumors (Topic 15). In these last 2 topics, the percentage of bot accounts was slightly higher than the rest of the accounts.Other messages were related to US politics or President Trump. Most of the tweets in Topic 17 were about decisions by the US Congress. Topic 18 mentioned certain national political scandals. Topic 8 was focused on criticizing President Trump’s policies. These tweets cast President Trump as a liar and irresponsible. Some of the most common keywords were F2,284,814=5216; P<.001). The mean value of the VADER score for each group was 0.0109 (SD 0.414) for nonbots, 0.00784 (SD 0.383) for self-declared bots, and –0.0155 (SD 0.427) for bots. An ANOVA test was used to check for statistically significant differences in the mean values of the groups . Although the percentage of undeclared bots on Twitter is relatively low compared to the large number of human users, it has been established that bots are generally linked to web-based conversations characterized by controversy and polarization. In this sense, the role of these automatic agents is far from negligible, considering the role they play in the amplification of ideas and opinions that generate conflict in our societies ,43.The classification adopted has allowed the comparison of the different topics arising in the conversations of 3 different profiles of Twitter users during the initial months of the pandemic. Furthermore, to the best of our knowledge, this study has several advantages compared to other works that analyze sentiment in a general manner and regardless of the information source and type -46. FirsOn the other hand, the sentiment analysis also gives an idea of the strategy of undeclared bots or automated accounts in the context of the first months of the COVID-19 pandemic. Our study shows that social bots were used to criticize and harass political opponents rather than to provide useful information on health measures and self-protection behavior in a context where quality information was sorely needed in the face of widespread misinformation . In-lineDespite these findings, additional evidence is needed to determine the social and health impacts of the misuse of social bots during the early months of the pandemic. Likewise, it is necessary to determine to what extent these agents have hindered the prevention and control of the health crisis by the different governments. In any case, this is a new working hypothesis that remains open and should be analyzed in detail in future studies.This study is subject to several limitations. First, the data collected from Twitter is limited by the technical characteristics of the Twitter streaming API. Although the streaming API is more accurate than the REST API, it never returns the total number of tweets about the conversation . MoreoveOn the other hand, this study also has several strengths. First, it takes into account the credibility of the information source. This aspect is rarely addressed in studies of social media platforms . Second,By classifying the accounts according to the likelihood of being bots and applying topic modeling, we were able to segment the Twitter conversations regarding the COVID-19 pandemic. Nonbot accounts, for example, tended to share information or give advice on how to deal with the pandemic. The accounts declared as bots mostly shared information and statistics on the pandemic. Finally, accounts not declared as bots tended to criticize the measures imposed to curb the pandemic, express disagreement with politicians, or question the veracity of the information shared on social media platforms. We also used sentiment analysis to compare the tone of the conversations in these different groups. Self-declared bots had conversations with a neutral tone. The tone of messages written by nonbot accounts tended to be more positive than the former. On the contrary, the tone of undeclared bots was always more negative than the tone of self-declared bots. Therefore, it is necessary to work on the identification and monitoring of these agents in times of infodemics.


In recent years, Ghana has been recognised as a leading player in addressing antimicrobial resistance (AMR) in Africa. However, based on our literature review, we could not ascertain whether the core elements of the national action plan (NAP) were implemented in practice. In this paper, we present a qualitative analysis of the development of AMR-related policies in Ghana, including the NAP. We conducted 13 semi-structured expert interviews to obtain at a more thorough understanding of the implementation process for the AMR NAP and to highlight its accomplishments and shortcomings. The results show that AMR policies, as embodied in the NAP, have led to an extended network of cooperation between stakeholders in many political fields. Broadly, limited allocation of financial resources from the government and from international cooperation have been deplored. Furthermore, the opportunity for using the NAP in mainstreaming the response to the threat of AMR has not been seized. To the general public, this remained hidden behind a number of other relevant health topics such as infection prevention, veterinary services and pharmaceutical regulation. As a One Health (OH) challenge, developing countries could integrate AMR NAPs into other health and environmental programmes to improve its implementation in practice. A recent systematic analysis in the Lancet  providesRegulations and programmes relevant for fighting AMR in Ghana date as far back to 1998 when Ghana adopted the Rational Use of Medicines Programme (RUM), which complemented the “Essential Drugs List and National Formulary with Therapeutic Guidelines” , which was separately published later as the Standard Treatment Guidelines and Essential Medicines List. Since 2000, the Ghana National Drugs Programme (GNDP), a pharmaceutical policy unit of the Ministry of Health (MoH), has controlled these activities . In 2013RUM included the formation of Drugs and Therapeutic Committees (DTCs) in the majority of the teaching, regional and district hospitals. Their tasks focussed on the monitoring of antibiotic use in their facilities. The RUM programme contributed to a decrease in antibiotic prescription at the level of Outpatient Departments (OPDs) from 56.3% in 1999 to 41.4% in 2012. Nevertheless, between 2002 and 2004, a first nationwide surveillance of antibiotic resistance in Ghana observed a high percentage of resistance for tetracycline, cotrimoxazole and ampicillin, which are frequently used antibiotics in Ghana .The discourse on AMR, which led to the launch of the Ghanaian NAP on 11t April 2018, was initiated by a Danish research cooperation project on “Antibiotic Drug use, Monitoring and Evaluation of Resistance in Ghana—A research capacity building project (ADMER)”, which operated from 2009 through 2015 in partnership with the University of Ghana Medical School (UGMS) in Accra and the Kwame Nkrumah University of Science and Technology (KNUST) in Kumasi  meeting every quarter. … we know what is happening in the human health sector, we know what is happening in the environment. This is what we are doing now as a committee at the AMR level”(R13)Other respondents also stressed the integrative impact of OH: “…the NAP is developed in one health. So that is one of the major successes….the WHO Ghana Office, the FAO and then OIE… [are] working with the platform” .While in the human health sector there is no direct legal conflict between policies to control AMR and economic interests (besides issues of finance), the situation is different in the field of animal farming, where “rearing animals for food and jobs”, using “European style” raising of animals has been supported by development politics . “We are also working with the OIE and all the others, FAO, we also trained poultry farmers in Dormaa Ahenkro” ; this is a model field school to stop the “inappropriate use of antibiotics” in poultry production .In principle, the legal paths for procurement of medicines and the control of the access to antimicrobials are strictly controlled by several institutions , MoH pharmacy) and various laws and regulations focussing on the control of quality of medicines and preventing the entry and sale of fake medicines  Medical Products”). Respondents, however, pointed out that there was no effective enforcement of these regulations. “…the pharmaceutical- the health professional regulatory act, talks about what we’re supposed to do when a medicine is dispensed in Ghana. Such implementations are not ongoing. There’s no national intervention to ensure that …the recommendation from the law is being implemented. So, then we still have, I’ll say, lack of control… over the dispensing of drugs in the country”.(R2)The lack of control coincides with income opportunities for groups who illegally procure antibiotics either by effectively using the loopholes of the local pharmaceutical markets, or by various forms of smuggling.“There may be promoters of resistance in the society by their activities. They have no knowledge about the antimicrobial that they sell around. All they want is money. So, any sickness mentioned, they’ll give you the antibiotics. Whether it will kill the bacteria, whether the sickness is caused by a bacterial infection, they don’t care”.(R2)“[Smugglers] pass through the unapproved routes, the borders. Yes. So, they always smuggle antibiotics into the country. Whether they are safe or they are efficacious or not, is another issue” (R2). “It’s a whole cartel that is working, a kind of a global menace. …These drugs come through illegal routes … Let’s stick to antibiotics. Antibiotics will need to be stored appropriately… to retain its efficacy. So, I go out I buy some boxes of amoxicillin injection or a few parts of …meropenem or, aminoglycosides…Because they’re not bulky, so if I have a suitcase, I can buy let’s say 10, 20 right, then I come and supply it…. I can buy it at a cheaper cost than the ones that have been imported by those who have registered the products to use. So, we cannot be sure of the supply chain integrity of such products”.(R1)One of the main issues constitutes the control of counterfeit medicines. Bright Simons, a renowned Ghanaian innovator, has developed a widely used system to use mobile phone-based technology to check for counterfeit medicines, mPedigree. However, they were not successful in rolling it out in Ghana: “we’ve been trying for… more than a decade. …Finally, it became quite clear that we will not be able to make any headway in Ghana. So, we left Ghana and we spent time in Nigeria and Kenya and other places. And yes, in those countries, particularly in Nigeria which is the most advanced, the solution and others like it is used for controlling authenticity.” He further stressed: “… I see the traceability and supply chain solution as the base on which you can build other higher value adding solutions some of which include antimicrobial resistance, surveillance and monitoring systems”.(R12)Other respondents confirmed this, but added that the MoH is now using another medicine traceability strategy, which is currently being developed . Nevertheless, the situation described above seems to prevail. Concerning core capacities , progress and shortcomings to the implementation of the NAP alike have been stressed by the experts, drawing attention to the system for governance of fighting AMR. While the “Ghana AMR Policy Process Flow”  gives an“Well, health systems in general in Ghana, is not at its peak... if you take one aspect, governance alone is good on paper but practically it’s not so strong”.(R9)enforcement is weak. It is a system-wide problem. It is not just about the regulatory agencies. I keep saying that it is more of access and equity …” (referring to difficult access to pharmacies in remote regions).“When you look at the NAP or the policy, there is a governance structure … and then how relevant it is towards the NAP implementation”.(R1)“But then we have had these challenges, where even at a point in time, you know, equipment that are needed in the field to work and generate the needed data has not still gone down to the field, in spite of the fact that capacity of the practitioners has been developed . There are also fears that even once [human] capacity is developed, and they get some lucrative job, they may leave so there may be the need for retraining, once we’re able to get things in place. But I agree that there has to be, … somebody who is looking at; the … AMR Secretariat is being actually tasked, … but I don’t know if they have the powers to work with [international organisations]”.(R1)A detail was added by R2: “There is a desk in the Ministry that is responsible for that…at the PPME directorate.” In fact, this directorate comprises a Resource Mobilization Unit with the following tasks: “The Unit develops and reviews the financial strategies or options for policies/programmes and projects of the Ministry. It leads in the sourcing, monitoring and coordination of external funding for healthcare delivery, to include sourcing external funding for the procurement of health commodities, services and works”. There is, however, no further link to enter the PPME directorate .Many respondents referred to the central role of the tripartite organisations  in coordination and in supporting ongoing activities important to combat AMR, that is antimicrobial stewardship (AMS): “We cannot implement AMR activities without international cooperation; most of the funding comes from WHO and FAO. They supported in developing the National Action Plan. Coordination at the national level is spearheaded by WHO. So, if WHO is going to undertake some activity, they consult FAO, OIE and now UNEP. MoH hosts the AMR secretariat and they organize the platform meetings”.(R10)“I’ll say that the Tripartite in general actively supported the country to come out with the AMR policy and then the NAP and of course, we are still supporting them to implement some aspects of it… We basically provide technical support and some funding to ensure activities are implemented. …Collaborations with FAO and OIE are excellent, I know, there are AMS activities ongoing by various agencies . What I can say too is that there is a project coming... the Multi-Partner Trust Fund Project which seeks to pool these agencies together in One Health. Out of that there are crosscutting activities which are supposed to be implemented across the three agencies and that project would hopefully begin somewhere April if all things are put in perspective..(R9)The role of CSOs in international cooperation remains unclear; in some cases, they might be included just as a fig leaf, as in the case of “Health for Future Generations” in the Fleming Fund Grant: “We don’t have any role, we were part of the development; our organization contributed, we supported with a letter, everything but when the grant came, we are not involved so we don’t know what exactly they are doing…Our social media team also support creating […] a lot of information but we don’t have any project that is funded by anybody. …For now… ReAct Africa is doing a lot of awareness work”. (R7)As these quotes show, also among experts who have been involved in international cooperation, there was considerable insecurity about a central management of cooperation projects within the Ghanaian government. Two other new cooperation projects were mentioned in the interviews, which are also referred to in the literature: “SORT IT  and WHO) supports countries to build sustainable capacity to conduct and publish operational research and use the evidence for informed decision making to improve public health. The goal is to make countries “data rich, information rich and action rich’”   which is being rolled out nationally, we are hoping that we will be able to obtain consumption data moving forward. This roll out is ongoing…, we are planning a meeting where we will ask each region and even each district to nominate a focal AMR person, who will be tasked to make sure the consumption data is collected and reported nationally to feed into the [..] systems and eventually into the WHO GLASS”.(R6)According to the Ghanaian Ministry of Health “expected that by November 2020, all health centres and “functional” CHPS [Community Based Health Planning and Services] would have been successfully signed onto the system fully” ,48. InteRespondents agreed that AMR had not been integrated into university teaching as a specific module, but that it played a significant role in many courses, basically in infectious disease management. It was revealed that many students showed interest in the topic with some Master’s and PhD dissertations being written in this field. However, the perceptions of MoH impact on university programmes differed among respondents.[Modules of AMR and AMS?] “No. Currently there are not. … But you know, universities are independent, so we are suggesting but universities have to take time to buy it into their own system…I would not say that there’s no strong interest on AMR in the universities. I suspect that because the NAP has come and has to be integrated into the curricula of the universities, it is now something that they are trying to put in place, because they already had their own programme before and I think that curriculum is not changed every year in the universities”.(R13)The respondents also referred to a number of theses and publications carried out at the universities. Furthermore, it should be pointed out that AMR and AMS is included in teaching on infectious disease management:“…in pharmacy, we have actually incorporated antimicrobial stewardship and the role of the pharmacists in AMR containment, at different levels, graduate levels, undergraduate levels and all of that is actually part of the curricula for infectious disease management. But I think we should go beyond that, we should be able to see that in the curricula of Veterinary Medicine, we should see that in the curricula of School of Medicine and Dentistry, we should see that in the curricula of Laboratory Science, you know, all of that so I think we need to move on”.[Interest among students?]” Yes, huge, you know, … very, very, very huge. A lot of them even take up mini projects, alright, on that …. So, if what is happening in pharmacy can also be replicated, you know, medical school, nursing, at least in their final years or a year to the final year, if they are actually taught about some of these issues, I think that would really be great. But I don’t know which institution or organisation will lead that kind of advocacy”.(R1)[Oversight by the secretariat] “You know the NAP recommended, that we should have AMR issues included in the curriculum of the teaching of the universities… So, we know that one university, KNUST, started and they’ve done some inclusion already. But the others, we’re yet to find out from them whether they’ve included issues of AMR in their curriculum…. From time to time, the secretariat goes through some of the training institutions, to create awareness…”.(R2)In addition, it should be mentioned that WHO and FAO are carrying out a number of training programs on specific issues, such as the SORT IT mentioned above, or training for professionals in agriculture and fisheries, a “Private Sector Engagement Workshop on Antimicrobial Use and Resistance” and programmes for journalists .While academic institutions have provided an important contribution to awareness building on the professional level, AMR Goal 1 rightly goes beyond that to increase awareness in the general population. A respondent from the Environmental Protection Agency replied: “It will interest you to know that I was not even aware of this AMR thing, until I joined the whole AMR discussion. I think in 2018 there was the WAAW week, they held a programme at the Ministry of Health, that was the first time I participated in it and I understood the whole AMR discussion. So, you see it means that a lot of people are not aware of this whole AMR issue. And I think the awareness creation has been limiting for a long time. Apart from the WAAW week I don’t see any rigorous awareness on this whole AMR thing”. (R10)“At least every November onwards [WAAW] something should happen and people are aware. … But I can tell you a lot of plans are put on paper but our basic challenge has been funding because people are prepared to work…. People don’t put money into awareness issues because the deliverables are not tangible. I’ll say that gradually the awareness is catching up”.(R9)“I think the problem with the media is …to do more of the training. I think they have to understand the AMR issues. … even when you have spoken to them about it, when they are reporting, sometimes you realise that the substance in what you shared with them is not what actually they are reporting… When we are doing World Antimicrobial Awareness Week we are going to see more of the media, … we see people going there, buying airtime, or maybe as part of their corporate social responsibility, giving them the platform to talk about AMR issues. … And I think that, you know, we should also work very hard to make the media guys very strong advocates, once they understand the issues, and they make noise about it, and I think that’s one sure way of ensuring massively a real change”.(R1)[Around 2015] “… the awareness level was measured to be so low, even among the health workers, let alone the general public. The Kintampo Health Research Centre for instance, did the KAPP  have projects actively working, yes. …So, if you ask me about awareness, I think its limited, I will say maybe 0.5, 0.3% of people are aware, maybe listening to TV and radio. You know, when we are celebrating AMR week… I don’t think people are aware because when you talk to people … they become surprised, so we are not doing much when it comes to awareness creation.… Civil society can do more than what they are doing because that is our field; because working with community members, communicating in their language is our work. So, I think we need to just really appreciate, there is a gap that we have, big gap that we must address…you know we work a lot in the communities. When we are meeting mothers for our maternal health, we talk about it, but receiving money purposely for this, no.… Communication [with the NPAR] was very active when we set up the platform. Now the communication, I don’t see it very active but periodically you will see some information on the platform”.(R7)The statements from our respondents confirm the assumption based on internet research, that AMR is not presented as a priority topic in the Ghanaian mass media and in the general public. The impact of the WAAW remains limited, as long as AMR remains hidden behind other health topics such as malaria, and recently, COVID-19. Among the experts interviewed, there was a high level of self-consciousness concerning the role of Ghana in the international fight against AMR: “Ghana plays a leading role in the fight against AMR in the sub-region and also globally” (R4). “But …I think countries like Kenya, South Africa, okay, and perhaps Ethiopia are overtaking Ghana, right when it comes to implementation… So, we need to step up”.(R1)“The Call to Action Conference that was funded by Wellcome and the other partners, was really really great. I mean, it brought in a lot more stakeholders, you know, experts who are working in that area, to share ideas…. Usually at the conferences of the International Pharmaceutical Federation, students who have done [work] with the AMR space have had the opportunity to showcase their work”.(R1)There were, however, more voices insisting that Ghana should not repose on the international acknowledgement received in the past. There were concerns that the country was losing ground.“So far, research and conferences are more geared towards trying to achieve targets for the development of researchers. You are under pressure to publish or perish. So, regarding policy implementation and informing policy that is on our mind, we try to do that, but we focus more on publish or perish. So much more has to be done regarding impact on policy”.(R5)“… if you look at GLASS reporting… Ghana has not reported anything yet… I think we have worked very hard to be putting the systems and infrastructure in place and probably when we take off we would have smooth systems running. So, we contribute, attending international conferences for most of the international engagement; on the African continent and beyond. For me, I mostly have the opportunity to be sharing data… but that is not enough as far as I am concerned. We must hit the ground and get the evidence. The evidence at the moment is not there, so it is like we are not doing anything”.(R11)The international role of Ghana has been based on close cooperation with multilateral organisations: “I think without them we would have just been sitting without doing anything. They have been very pressing and pushy. These abilities have gotten us this far. We alone would have been standing aloof”.(R5)Considering the assessment by most respondents that in spite of the important preparatory work on the NAP, cooperation between institutions facilitated through the NPAR and the significant output of biomedical research, Ghana has “not made as much progress as some other countries” (R1), one should expect a growing interest in evaluating the progress and the obstacles during the implementation of the NAP. There has been a mid-term rapid review of the implementation of the NAP, which was not seen as a profound accomplishment by the respondents who referred to it: [Enforcement of existing regulations?] “… during the mid-term review, the AMR secretariat did a rapid review on the implementation of the NAP, and one of the main activities is enforcement of regulation on antimicrobial use in Ghana. No work has started on that arm”.(R2)“In fact, I happen to be part of a team that did an evaluation of the NAP. We did it early last year. What we did was that we looked at the various actions and evaluated the progress made. All focal persons from the various institutions were on board. So, you ask yourself, this one have we done it, this one have we not done, why? Then we raise the questions; if we have done it, to what level, what is left? It was teamwork, I will say that the focal points of the various institutions involved did their own evaluation. The thinking was that an external person will be contracted to do the evaluation again, but you know all these things require financial resources to undertake so I don’t think that has happened”.(R10)The AMR Secretariat, with the support of WHO, conducted a mid-term rapid review of the implementation as recommended by the NAP, but with limited methodological effort as described by R10). Results were published by the WHO Ghana Office , but notFew respondents reflected on the fact that the NAP expires in 2021 and that there is the need for a new plan for the following years: “…because the plan that we have currently is expiring and we need a new plan. The problem is, you know, they will be doing some monitoring and evaluation, it will be highly technocratic, they will hire some consultation, they’ll do the evaluation but you’ll be very surprised to learn that real consultation in Ghana is often conducted in a manner that, to me, suggest it is perfunctory; they are just checking the box. And I think a lot of that problem persist. I don’t blame the technocrats at specific ministries or bureaucracies alone. I think it is just a general culture that we have in the country that makes accountability just difficult”.(R12)“I can say our implementation is quite slow from where I sit. If you look at it critically people are prepared to work but everything is about money and if government should put in some money, because I can say the government has taken some of the NAP into the NDPC Plan. And you know, the president also co-chairs the SDGs, …, I remember … he asked that some of the indicators be pushed into SDGs and the National Development... forty-year plan” , of which only an Outline is available online .Hardly any links to a future policy on AMR can be found online. On the webpage of the NDPC  there is an entry on Social development/ health and health services with no further links, but in “Medium-term Plans–Sectors” there is no entry pertaining to more recent developments, other than “Health Sector Medium-Term Development Plan 2014–2017”. There is a Medium Term Expenditure Framework (MTEF) for 2018–2021 from the Ministry of Health without any mention of AMR measures .In a recent text by the MoH, “National Health Policy” (revised on January 2020) , “The PoConsidering the work on other internationally demanded national action plans, such as the National Action Plan for Health Security (NAPHS) launched in 2020 , and worGhana’s NAP on AMR has been guided by WHO standards . Though many aspects of it have proven to be challenging to fully implement in the short term, it has constituted an important basis for a continued discourse among stakeholders within a broad institutional framework. To a large extent, this framework is supported by the OH concept, linking the whole stakeholder network in the field of AMR and creating a broad awareness among professional stakeholders about strategies to control AMR.Antimicrobial stewardship has been acknowledged as key to combatting AMR. In Ghana, the regulations, policy documents and guidelines have been well established. However, our findings show that due to the poor coordination, reporting and publication among the various stakeholders, the progress made on the implementation of the NAP is fragmented. Moreover, based on the international accolade received during the development and launch of the Ghana NAP, the fact that Ghana had not yet submitted any report to the WHO Global Antimicrobial Resistance Surveillance System (GLASS) leaves much to be desired relative to other African countries such as Tanzania which also launched their NAP in the same year as Ghana . FightinThe results from the interviews and literature on recent developments point to the following key findings, which demonstrate that both accomplishments and shortcomings regarding the Ghana NAP are ambiguous. They point to insufficient financial resources, stakeholder coordination, infrastructure and human capacity, awareness creation and the impact of a lack of communication on AMR policies.Practical outcomes of the implementation process for the NAP have been limited due to (1) the lack effective enforcement of regulations, (2) limited implementation of larger projects because of a lack of financial resources provided by the Ghanaian government and (3) insufficient supplementation of efforts by international cooperation. These challenges have been highlighted in Ghana and in other developing countries ,18,60,61For many countries in sub-Saharan Africa, there is a lack of adequate infrastructure and capacity for robust AMR surveillance systems . The impThere is also a need to continuously improve the qualification of medical and other personnel involved in activities related to the NAP. To promote the effective implantation of the national strategies on AMR in Thailand for instance, technical capacities among implementing agencies were highlighted as essential to translate policies into practice . HoweverConcerning the critical analysis of the lack of explicit reference to AMR in many health-related documents, we found a certain caveat. Attention to AMR is indeed implicitly included in a number of other health-related programmes. This, however, reduces the visibility of AMR policies to the general public and also in international communication. It also reduces the transparency concerning the implementation of the NAP. Similar to other developing countries ,18,63,64Awareness has considerably grown in professional groups beyond the original field of human health, which now particularly includes the agricultural sector, animal health and the environment. However, awareness remains weak in the rest of the population . Our finThe Ghanaian experience as well as the implementation problems of NAPs on AMR in other low- and middle-income countries point to the need for further research on their implementability regarding the time and the space dimensions. The formulation of global action plans by international institutions should consider the national preconditions for implementations, which differ significantly in the global context, and related to that, the specific time frames which are required to implement different elements of an action plan to fight AMR. Sequencing the implementation implies a step-by-step approach based on periodic strong evaluations of what has been achieved on specific goals, and it demands a longer-term perspective than just one five-year plan.The lack of financial resources and a lack of full (and more explicit) integration of AMR policies into the Ghanaian health system and in Ghanaian society in general can be seen as the most important factors preventing a comprehensive implementation of all NAP dimensions. Improving diagnostics, institutional healthcare and better-organised access to medicines in marginal regions also would reduce the incentives to use informal markets for accessing (frequently sub-standard) antibiotics without prescriptions. The effective functioning of the National Health Insurance System (NHIS)  could improve the health-seeking behaviour of the general population and translate into better enforcement of existing regulations. This implies the task of mainstreaming the societal embedding of fighting AMR, but is also a general problem of the level of national per capita income and budgetary allocation to health interventions.


Steroidal-compound-loaded chitosan (ST-CH) nanofibers were fabricated using the electrospinning technique in the presence of polyvinylpyrrolidone (CH/PVP). The electrospun nanofibers were characterized by scanning electron microscopy (SEM). The swelling degree of the electrospun nanofibers and their steroidal compound release performance were studied as well. Furthermore, their antibacterial activity against gram-positive (Staphylococcus aurous) and gram-negative bacteria (Escherichia coli) was evaluated. The experimental data revealed that identical and bead-free nanofiber mats loaded with 10 Wt. % of synthesized steroidal derivatives had been obtained. The FTIR spectrum proved that no change occurred in the chitosan structure during the electrospinning process. The synthesized nanofiber mats showed a high swelling degree and a burst release of steroidal compounds after 2 h doping in phosphate buffer saline. In addition, the electrospun nanofibers containing 3β-chloro-N-amido-5α-cholestano-aziridine and those containing 3β-acetoxy-N-amido-5α-cholestano-aziridine were the most active, with activity indices of 91 and 104% in the case of S. aureus and 52% and 61% in the case of E. coli, respectively. The release mechanism by CH/PVP of the drug samples was studied based on the charge density and diffusion controlled factors. The oxazoline derivatives release mechanism from CH/PVP was evaluated by applying the suppositions of the Ritger-Peppas kinetic model and by estimating the transport exponent; the latter revealed the involvement of the solvent diffusion and chain relaxation processes. Tailored steroidal loaded-chitosan (ST-CH) nanofibers are expected to be feasible and efficient drug delivery systems.A novel drug delivery system based on chitosan nanofibers containing some steroidal derivatives was developed using an electrospinning process. Oxazolines and aziridines from the cholestane series of steroidal epoxides were successfully synthesized and characterized by elemental analysis, Fourier transforms infrared spectroscopy (FTIR), proton nuclear magnetic resonance ( Chitosan is a naturally occurring polymer of deacetylated chitin, which is the second most plentiful polysaccharide, occurring in crab, shrimp shells, fungal mycelia, and some mushrooms (zygote fungi). Furthermore, chitosan is the only pseudo-natural cationic with plenty of primary amines. It exhibits remarkable biocompatibility, biodegradability, adsorption activity, and antimicrobial ability . Chitosa2 group which exists at the C-2 position of the unhydroglucose unit. Through this mechanism, the polysaccharide is transformed into a polyelectrolyte. The solubility of chitosan in acetic acid can provide an ionically conductive solution due to the presence of protons from the acid  was measured in a mixture of 0.1 M acetic acid and 0.2 M sodium chloride at 25 °C. Polyvinyl pyrrolidone (PVP)  was supplied by Sigma Aldrich. All samples were used without further purification.The synthesis processes are presented in the following schematic diagram .At room temperature, 75 mL of freshly purified SOCl₂ (thionyl chloride) was added slowly to 100 g of cholesterol. An energetic reaction ensured the creation of gaseous products. The mixture was heated at 323–333 K in a water bath for 60 min and then decanted by stirring onto crushed ice. The obtained compound was filtered under vacuum, and then washed many times with iced water and air-dried. Recrystallizations of the raw product from acetone yielded 5.50 g of the desired compound (m.p. 95–96 °C).A mixture of pyridine , 50.0 g of cholesterol, and 50 mL of acetic anhydride were heated in a water bath for 120 min. The resulting brown solution was stirred into a crushed ice–water mixture. A light brown solid was produced, which was then air-dried. The crude product recrystallized from acetone, yielding 45.0 g of the compound .II) in 100 mL chloroform was pickled with per benzoic acid solution  and left for 20 h at a temperature of −8 °C. Subsequently, the blend was washed consecutively with an ice-cold 5% solution of NaHCO3, 5% solution of Na2S2O3·5H2O, and finally, with water. Solvent evaporation provided an oil that was chromate-graphed over a silica gel column. The substance was then rinsed with petroleum ether-ether (10:1) to produce a solid product that was recrystallized from acetone as needles, yielding give 8.0 g epoxide (III) .First, 10 g of 3β-Acetoxycholest-5ene (I) in 100 mL chloroform was dried with a per benzoic acid solution  and left for 20 h at a temperature of −8.0 °C. Subsequently, the mixture was washed sequentially with an ice-cold 5% solution of NaHCO3, 5% solution of Na2S2O3·5H2O, and finally, with water. Solvent evaporation yielded compound (IV) as an oil that was crystallized from acetone as 8.10 g of needles .First, 11.0 g of 3β-Chloro cholest-5ene (IV)  dissolved in 50 mL DMF, was treated with 1.0 g of acetamide in the presence of AlCl3 anhydrous as a catalyst. Subsequently, the mixture was analyzed by thin-layer chromatography (TLC); the solvent and the residue (1.62) were column chromatographed over 35.0 g of silica gel.First, 2.0 g of compound (II)  and 1.0 g of acetamide were dissolved in 50 mL of DMF. The mixture was heated under reflux in the presence of anhydrous AlCl3 as a catalyst. TLC was used to monitor the progress of the reaction. Upon completion of the reaction, the solvent was evaporated under condensed pressure. The produced compound was removed with ether. The ethereal layer was washed many times with water and dried over anhydrous Na2SO4. Removal of the solvent yielded oil (1.65 g) (VI), which was chromatographed over silica gel (40.0 g).First, 2.0 g of compound .First, 3β-chloro-5,6α-epoxy-5α-cholestane was dissolved in DMF and treated with semi-carbazide in the presence of anhydrous AlCl3 anhydrous as a catalyst and refluxed for 8 h. The reaction progress was followed with TLC. Upon completion, the reaction blend was dried over anhydrous Na2SO4. Following the evaporation of the solvent, the remaining compound (1.30 g) was column chromatographed over 30.0 g of silica gel.A mix of 1.5 g semi-carbazide and 1.5 g 3β-acetoxy-5,6α-epoxy-5α-cholestane were dissolved in 25 mL DMF in the presence of AlClw/v) and 15% PVP (w/v) in glacial acetic acid 90% (v/v). The prepared solutions of CH/PVP were then mixed with 10wt % in petroleum ether separately; 3β-Chloro-5α-Cholestano-2′-methyl-2-oxazoline (V), 3β-acetoxy-5α-Cholestano-2′-methyl-2-oxazoline (VI), 3β-chloro-N-amido-5α-cholestano-aziridine (VII) and 3β-acetoxy–N-amido-5α-cholestano aziridine (VIII) were named as samples A, B, C, and D, respectively. All prepared solutions were stirred with a homogenizing ultrasonic stirrer for 30 min. Electrospinning of the prepared solutions was performed according to the method reported in [Chitosan (CH) solution was prepared by dissolving (5wt %) of chitosan in 90% glacial acetic acid. Solutions with constant mass ratios (1:3) gm% of CH and PVP, respectively, were prepared by dissolving 5% chitosan (orted in  using a −1. The 1HNMR spectra were recorded using a CDCl3 NMR Bruker (400 MHz) spectrometer  with Tetramethylsilane as the interior standard; the results are reported in ppm (o). Mass spectra were measured using a GC–MS  with an AOC 20i auto-injector. The TLC plate was coated with silica gel and sprayed with 20% perchloric acid solution. Next, 20.0 g of silica gel was utilized for 1.0 g of the material in column-chromatography. Petroleum ether was used (b.p. 40–60 °C). Finally, anhydrous Na2SO4 was utilized as the dehydrating agent.The melting points of the aforementioned compounds were determined using a melting point device. FTIR spectra were attained with a Cary 830 FTIR  spectrophotometer; FTIR values are reported in cmThe electrospun nanofibers were characterized using scanning electron microscopy .sW indicates the weight of the swelled mat and W0 indicates the weight of the mat in dry form after 24 h impregnation in phosphate-buffered saline.The swelling degree of the electrospun mats was determined by a gravimetric method. The electrospun mats were impregnated with phosphate-buffered saline (pH = 7.4) at 25 °C for 24 h. The immersed nanofiber samples were taken out, the extra surface water on the samples was removed with filter paper, and the swollen nanofibers were weighed. The ratio of swelling was calculated using the following Equation (1): 2 squares and the extracted compound was estimated as a function of the weight mat. The drug loading into CH-PVP nanofibres was 12 mmole/100 gm CH-PVP from each sample, i.e., A, B, C, and D. The specimens were placed in individual vessels containing 50 mL buffer saline phosphate (pH = 7.40), and the vessels were incubated at 310 K. Release experiments were performed by dispersing 200 mg of the ST-CH nanofibers in 50 mL of the buffer solution. After a certain time, 5 mL of the buffer was replaced with the same volume of fresh buffer to maintain a constant volume. A mixture of 3 mL methanol and 2 mL distilled water was prepared and added to each buffer to determine the absorbance by UV-Visible spectroscopy at 300 nm. A standard calibration curve was applied to measure the concentration of the free steroidal oxazoline and aziridine derivatives. The percentage of the released steroidal oxazoline and aziridine derivatives was then calculated based on the initial weight of these compounds incorporated in the electrospun nanofiber mats. The results were plotted over time, up to 60 h.UV-Visible  absorbance was considered as an indicator of the release of steroidal oxazoline and aziridine derivatives from electrospun mats. The dehydrated, electrospun, extract-loaded-nanofibrous mats were first divided into 12 × 12 cmStaphylococcus aureus gram-positive and Escherichia coli gram-negative bacteria using an agar well diffusion assay, as described in [The antibacterial activity of the electrospun CH/PVP nanofibers with and without steroidal oxazoline and aziridine derivatives was evaluated against ribed in ,28. Brie29H48NOCl. As shown in m/z at 461 (M+), followed by important fragmentations at m/z 404 and m/z 57, as shown in The desired m.p. of 110 °C was confirmed for CVI). The compound was obtained as a semi-solid and analyzed as C31H51NO3. The mass spectrum of the compound revealed a molecular ion peak at m/z 484/485 (M+), as well as important fragmentations at m/z 368, m/z 57, m/z 59. Fragmentations of lower masses as shown in VII), i.e., C28H47N2OCl. The synthesis process is described in m/z 484/486 (M+), followed by some significant fragment peaks at m/z 368/369, m/z 57 (NH2CO–N), m/z 59 (M+-CH3COO). Lower fragmentation mass peaks are shown in Electrospinning and the characterization of CH, as well as CH/PVP without and with synthesized 10% 3β-cloro-5α-cholestano-2′-methyl-2-oxazoline, 3β-acetoxy-5α cholestano-2′-methyl-2-oxazoline, 3β-chloro-N-amido-5α-cholestano-aziridine, and 3β-acetoxy- N-amido-5α-cholestano-aziridine, were investigated. A horizontal design setup for the electrospinning apparatus was used to obtain nanofibers from aqueous solutions. To examine the ability of the chitosan-based nanofibers to absorb water, the swelling performance of the nanofibers was studied. The swelling ratio (%) correlates to the water absorption ability. The swelling ratio (%) of the electrospun nanofibers is detailed in Drug release from nanofibers should take place at the target location. The spatial control of drug carriage can be ensured by employing electrospun nanofibers at the targeted location via invasive or non-invasive means. Recently, the chronological control of drug release from electrospun nanofibers has been measured according to drug dissolution rates, drug dispersal rates, fiber diameters (lengths of diffusion barrier), drug physical-desorption rates, and/or polymer degradation/erosion rates ,32,33,34Drug release (A–D) was approximately constant between 10 and 30 h. The accumulated drug amount decreased with an increase in release time. The amount of released drug from the nanofiber sample showed the following decreasing order: A > B > C > D.2 groups in the chitosan-based matrix. Intercalated anions exist in the functional groups of the obtained drugs. However, the release amount showed the order A > B > C > D in the first 2 h. This conceivably resulted from the fact that samples C (~26 mmol/L) and D (~24 mmol/L) had greater charge density than samples A (~30 mmol/L) and B (~28 mmol/L), as shown through in bulk characterizations above. This higher charge density brought about stronger interactions with the Chitosan-based matrix. This can have the effect of increasing the energy barrier to release, further slowing the release rate [A further explanation for this behavior can be provided in terms of the charge density as well as the phenomenon of diffusion-controlled release. Electrostatic interactions among the incorporated drugs (samples A–D) within the electrospun CH/PVP nanofibers enhanced the charge density and the physical electrostatic binding between the protonated –NHase rate .Moreover, the diffusion-controlled release factor can also affect the behavior of the illustrated drugs. This performance can perhaps be attributed to the diffusion path length of the drug molecules within the release milieu . A direck (rate constant) and exponent n will be given by the linear relation of log  within 30 h. The equation is as follows :(3)MtM∞=2 value of 0.986. This value of n was between 0.5 and 1, considering the release mechanism as irregular . Next, it was declared that drug release by the solvent diffusion and relaxation of the polymeric chains occurred consecutively. This was due to the dominant effect of the diffusion process with the influence of the relaxation process [After plotting this relationship, the intercept and slope values were calculated. Then, transport exponent n was estimated to be ~0.52 with an R process .Staphylococcus aureus (gram-positive) and Escherichia coli (gram-negative). The respective diameters of the inhibition zone (in mm) of the nanofibers are listed in Next, we directed our attention to the antibacterial properties of the newly synthesized oxazoline and aziridine steroidal derivatives-loaded CH/PVP electrospun nanofibers. The antimicrobial properties were assessed against two bacterial strains, namely S. aureus compared to E. coli. This was probably due to the differential selective permeability of the bacteria cell wall. Escherichia coli (gram-negative) bacteria have two cytoplasmic membranes which hamper drug diffusion through the lipid membrane into the cytoplasm. On the other hand, Staphylococcus aureus (gram-positive) lacks an outer lipid membrane and has only a thin cytoplasmic membrane, which facilitates drug penetration  against S. aureus. These results indicate that the steroidal derivative had been released from CH/PVP electrospun nanofiber and was effective against S. aureus. Indeed, these results encorage further investigations employing a more expansive arsenal of bacterial strains.The antibacterial activity of the electrospun nanofibers containing the synthesized steroidal aziridines was greater than that of the nanofibers containing steroidal oxazolines. The antibacterial activity of electrospun nanofibers containing 3β-cloro-5α-cholestano-2′-methyl-2-oxazoline and 3β-chloro-1H NMR, and mass spectra analysis. An electrospun chitosan blend with polyvinyl pyrrolidone nanofibers loaded with synthesized steroidal derivatives of oxazolines and aziridines was fabricated by an electrospinning process. The surface morphology and diameter of electrospun nanofibers were characterized with SEM. The obtained data revealed that nanofibers without beads had been formed with regular flat shapes with smooth surfaces, with an average diameter size in the range of 140–150 nm. Furthermore, there was no effect on the surface smoothness or flatness of the loaded electrospun nanofibers. The diameter of loaded electrospun nanofibers increased slightly, and there was no difference in the diameters of the loaded nanofibers. The diameters of obtained loaded electrospun nanofibers ranged from 167 to 170 nm. Furthermore, the swelling ratio of the CH/PVP mats did not decrease significantly following the incorporation of different synthesized steroidal epoxides. All samples showed a high swelling ratio, i.e., between 800–900%, which indicated a high water absorption capability. The release of drugs increased within the first 2 h, which was a consequence of the high charge density and the diffusion-controlled release mechanism. Moreover, the release rate was affected by the concentration of the drug inside the electrospun nanofibers. The drug release mechanism of derivative B over CH/PVP revealed both the diffusion and polymeric relaxation processes in light of the assessed value of exponent n as the transport factor using the Ritger-Peppas kinetic model, with a dominant effect of solvent diffusion. Steroidal oxazolines and aziridines were synthesized from steroidal epoxides with acetamide, benzamide, and semicarbazide. The structures of these compounds were established by elemental analysis, FTIR, E. coli, but had slight activity toward S. aureus. Both bacteria were sensitive to steroidal oxazoline and aziridine-loaded chitosan/PVP electrospun nanofibers.Finally, the electrospun CH/PVP did not display antibacterial activity against


The objective of this study was to determine the rates of hypogonadism and prescription of testosterone replacement therapy (TRT) in men with Klinefelter syndrome (KS). We hypothesized that men with KS are under-treated for testosterone deficiency with TRT due to a combination of factors, including a poor understanding of hypogonadism in this population and neurocognitive issues leading to delay in seeking of treatment for hypogonadism.We queried TriNetX, a large multicenter electronic health record database, to identify all men with a diagnosis of KS (ICD-10-CM Q98.4). Prevalence of testosterone deficiency was determined as defined by testosterone level < 300 ng/dL. The primary outcome of the study was prescription of any of the following forms of TRT on the day of diagnosis or later.There were in total 5437 men with diagnosis of KS. A total of 1581 men with KS received laboratory measurement of testosterone level, 1113 (70.4%) of whom were hypogonadal. Mean testosterone level in this group was 354 ng/dL [50–658]. Of the 1113 men found to be hypogonadal, only 657 (59.0%) men were given prescription for TRT.This is the first study to evaluate TRT prescribing habits in men with KS. In this large retrospective study, TRT was underprescribed in men with KS. Further studies are needed to corroborate these findings and to evaluate barriers to receiving care in this population. In this study, we aimed to investigate the prevalence of hypogonadism in men with KS, and to describe the rates of TRT prescription among men with KS. We hypothesized that patients with diagnosed KS would be undertreated with TRT.We queried TriNetX, a large multicenter electronic health record database, to identify men with a diagnosis of KS (ICD-10-CM Q98.4). Exclusion criteria were then applied to create the initial cohort, which included men with documented use of testosterone before diagnosis of KS or men with diagnosis of KS >20 years ago due to incomplete information available in the electronic health record. Data on comorbidities, including mental retardation (F70-F79), type 2 diabetes mellitus (E11), obesity (E66), hyperlipidemia (E78.5), hypertension (I10), osteoporosis (M81), pulmonary embolism (I26), and other vascular thrombosis (I74), were collected for all patients in addition to demographic characteristics. We then identified a laboratory cohort by identifying men in the initial cohort who had received a testosterone laboratory measurement on the day of diagnosis of KS or later. A brief flowchart detailing the creation of each cohort is shown in 10 Rates of TRT prescription among hypogonadal men with KS were assessed separately. IRB waiver was obtained from the University of Miami.The primary outcome of the study was prescription of any of the following forms of TRT on the day of diagnosis or later using associated medication codes: testosterone, testosterone 17-phenylpropionate, testosterone enanthate, testosterone cypionate, and testosterone undecanoate. In addition, we investigated the prevalence of hypogonadism in men with diagnosis of KS and documented testosterone laboratory measurement, as defined by testosterone level <300 ng/dL.There were in total 6071 men in the initial cohort with diagnosis of KS in the TriNetX database. After excluding patients with a diagnosis of KS >20 years ago or use of any form of TRT before diagnosis with KS, 5347 patients remained. The majority of men were White (71%), followed by men of unknown race (17%) and African American (9%). Mean age at diagnosis was 33 years [13–53]. Descriptive analysis of comorbidities revealed 350 (6.5%) patients with a diagnosis of intellectual disability, 761 (14.2%) with type 2 diabetes mellitus, 995 (18.6%) with obesity, 919 (17.2%) with hypertension, 246 (4.6%) with osteoporosis, 757 (14.2%) with hyperlipidemia, 153 (2.9%) with history of pulmonary embolism, and 60 (1.1%) with a history of vascular thrombosis.In total, of these 5347 patients, 1484 (27.8%) were prescribed any form of TRT . A total11 in those who are hypogonadal and FDA approval for management of hypogonadism in this population,12 men with KS would be undertreated with TRT.Hypogonadism poses a significant burden on the quality of life of men. Despite the recommendation to provide TRT to hypogonadal men with KS, there is a paucity of data describing real-world prescription of TRT in these men. We aimed to determine treatment rates with TRT in men diagnosed with KS, in addition to the prevalence of hypogonadism in this patient population using a large multi-institutional electronic health record database. We hypothesized that despite recent recommendations for lifelong treatmentT < 300 ng/dL) by testosterone laboratory assessment received testosterone replacement. Thus, there is significant room for improvement in properly treating men with KS and hypogonadism. To our knowledge, there are no previous studies in the literature that characterize the rates of prescription of TRT for patients with KS. By understanding treatment rates in men with KS, we can begin to explore the social, mental, and clinical considerations in managing these patients.Our study of a large global federated research network highlights that just 27.8% of men with diagnosis of KS subsequently received any form of TRT, and only 26% of men were evaluated further with laboratory measurement of serum testosterone level. We found that only 59.0% of men who were hypogonadal .To our knowledge, this study is the first to describe the rates of TRT prescription in men with KS, and is one of few studies in the literature to objectively describe the prevalence of hypogonadism in patients with KS in a large patient population. Additional strengths of our findings include the large sample size and the ability to establish the chronology of diagnosis and further evaluation and management. However, our findings are not without limitations. First, the retrospective nature of our study may introduce incomplete or inaccurate capture of data. It is also possible that men with a diagnosis of KS followed up for clinical workup and subsequent TRT prescription at a health care organization that does not report data to the TriNetX database, potentially leading to underestimation of the true rate of treatment with TRT in this cohort.Although we assume that physicians only provided TRT to men who were found to be hypogonadal, this database does not allow us to draw the conclusion that all men treated with TRT were hypogonadal. In addition, there are no data available on if patients experienced symptoms associated with hypogonadism in our cohort. It is possible that men who were asymptomatic were less likely to receive treatment with TRT even if they were hypogonadal by laboratory assessment. Furthermore, this study lacks data on clinical follow-up, so we are unable to assess whether or not these men were undertreated due to the fact that they did not follow up with a specialist to discuss their diagnosis or laboratory results and receive TRT.In this retrospective epidemiological study, we found that men with a diagnosis of KS are undertreated with TRT despite the recommendation for lifelong testosterone replacement after puberty in this population. In a large cohort of 5347 patients with KS, only 27.8% subsequently received TRT. Furthermore, a majority of hypogonadal KS patients were not treated with TRT. Future studies are needed to corroborate our findings. We are especially interested in further study into other problems that men with KS face such as sexual dysfunction. In particular, research exploring the psychological, social, and socioeconomic considerations in management of patients with KS could shed light on important underlying mechanisms for underdiagnosis and undertreatment in this population.


The displacements observed in the 1H-NMR presented the complexion between LAs and HP-βCD. The diffusion coefficients of free BTB and RVC were 7.70 × 10−10 m2 s−1 and 4.07 × 10−10 m2 s−1, and in the complex with HP-βCD were 1.90 × 10−10 m2 s−1 and 3.64 × 10−10 m2 s−1, respectively, which indicate a strong interaction between the BTB molecule and HP-βCD (98.3% molar fraction and Ka = 72.279 L/mol). With STD-NMR, the encapsulation of the BTB/HP-βCD and RVC/HP-βCD in SL vesicles was proven. Beyond the saturation transfer to the LAs, there is the magnetization transfer to the hydrogens of HP-βCD. BTB and RVC have already been studied in normal liposome systems; however, little is known of their behavior in SL.The drugs delivery system in the treatment of diseases has advantages such as reduced toxicity, increased availability of the drug, etc. Therefore, studies of the supramolecular interactions between local anesthetics (LAs) butamben (BTB) or ropivacaine (RVC) complexed with 2-hydroxypropyl-β-cyclodextrin (HP-βCD) and carried in Stealth liposomal (SL) are performed.  The mixture was left on a stirrer table for 24 h and then filtered in polycarbonate membrane .For the preparation of the calibration curve, BTB samples were prepared in phosphate pH buffer solution 7.4, varying the concentration from 0.014 to 0.066 mM. In order to obtain the RVC curve form, samples of RVC were prepared in phosphate-buffered solution pH 7.4 with concentration ranging from 0.1 to 0.54 mM. After that, the samples were taken to UV-vis.For the determination of the maximum solubility of BTB in sodium phosphate buffer pH 7.4, the methodology available in the Brazilian Pharmacopoeia 5th Edition  was used. The preparation of the solutions consisted of the addition of excess drug (BTB)  to constant volumes of solvent (3 mL), aiming at obtaining a saturated solution. According to the procedure systematized by Pharmacopoeia, the total content of solute in UV-vis solutions was determined.S0 is the aqueous solubility of LA in the absence of HP-βCD.The complex formed between BTB and HP-βCD was prepared in pH 7.4 buffer solution (sodium phosphate buffer 0.05 mM), varying the concentrations of HP-βCD 0.001 to 0.5 mM, with an excess BTB (150 mM). The solutions were left on an agitator table for 24 h. The samples were filtered in polycarbonate membrane  and taken for UV-vis analysis. For the RVC and HP-βCD complex, the variation in HP-βCD concentration was 0.001 to 0.4 mM with excess RVC (70 mM). The mixture was stirred at room temperature for 24 h to achieve balance . The samLiposomes were prepared using the lipid film method . StealthThe stealth liposomes were prepared from the compounds of egg phosphatidylcholine (EPC), cholesterol , and DSPE-PEG-2000 (Avast lipids) at a molar ratio of 54:41:5. The components were solubilized in chloroform and left at room temperature to obtain a lipid film . Then, tThe efficiency of LA encapsulated in liposomes was determined using the ultrafiltration/ultracentrifugation technique  . A samplNMR experiments were carried out at the Laboratory of Nuclear Magnetic Resonance of the State University of Londrina—UEL. The equipment used was the Bruker 400 MHz for hydrogen frequency , carried out with “software” Bruker standards under typical conditions. The experiments were conducted at 25 °C, applying as reference the peak of the residual deuterium (4.70 ppm), which was used as field lock and adjusted the homogeneity of the magnetic field.2s−1 × 10−10) in another dimension. The mean time of acquisition of the experiment was 25 min. The complex diffusion coefficient is determined based on the exchange between a free and complex state of the ligand. The complexed molar fraction (fx) and association constant (Ka) can be calculated using Equations (9) and (10), respectively [DOSY design: For all experiments, 16 different gradient amplitudes were used with diffusion time optimization of 0.06 s. The DOSY Toolbox  data proectively .(9)fx=.1H chemical shift, which the aromatic rings of the drugs interacted with the HP-βCD cavity. It was observed in the normalized 1H-STD that the BTB/HP-βCD complex showed variations in intensity of all 1H signals, with aromatics hydrogens showing the highest intensity values. HP-βCD 1H signals in the region between 3 and 4 ppm were also observed. This result can confirm the encapsulation of the binary complex in the liposomal vesicle. For the RVC anesthetic, lower values of encapsulation were observed both in the binary complex and in the liposomes (DOSY). The complex RVC/HP-βCD showed an alternative of topology whit the aromatic hydrogens and the piperidine ring in hydrophobic cavity of HP-βCD. For the 1H-STD of RVC/HP-βCD complex, the RVC hydrogens with higher saturation transfer intensity were those of the aromatic ring, and there are HP-βCD 1H signals in the region between 3 and 4 ppm too. In general, these results are promising, indicating that 1H NMR techniques for mapping the bonds intermolecular of complexes may be utilized. This opens the door to further studies, aiming at better systems for drug protection and release, since the field of study in supramolecular chemistry grows exponentially.The molecular interactions of local anesthetics BTB and RVC with HP-βCD were identified by the DOSY technique and also by the


Antimicrobial resistance (AMR) is rising at an alarming rate and complicating the management of infectious diseases including lower respiratory tract infections (LRTI). Metagenomic next-generation sequencing (mNGS) is a recently established method for culture-independent LRTI diagnosis, but its utility for predicting AMR has remained unclear. We aimed to assess the performance of mNGS for AMR prediction in bacterial LRTI and demonstrate proof of concept for epidemiological AMR surveillance and rapid AMR gene detection using Cas9 enrichment and nanopore sequencing.n = 27 patients, n = 32 pathogens). Secondary outcomes included the association between hospital exposure and AMR gene burden in the respiratory microbiome (n = 88 patients), and AMR gene detection using Cas9 targeted enrichment and nanopore sequencing (n = 10 patients).We studied 88 patients with acute respiratory failure between 07/2013 and 9/2018, enrolled through a previous observational study of LRTI. Inclusion criteria were age ≥ 18, need for mechanical ventilation, and respiratory specimen collection within 72 h of intubation. Exclusion criteria were decline of study participation, unclear LRTI status, or no matched RNA and DNA mNGS data from a respiratory specimen. Patients with LRTI were identified by clinical adjudication. mNGS was performed on lower respiratory tract specimens. The primary outcome was mNGS performance for predicting phenotypic antimicrobial susceptibility and was assessed in patients with LRTI from culture-confirmed bacterial pathogens with clinical antimicrobial susceptibility testing (p = 0.00030), or those without LRTI (p = 0.0024). We found that Cas9 targeted sequencing could enrich for low abundance AMR genes by > 2500-fold and enabled their rapid detection using a nanopore platform.Compared to clinical antimicrobial susceptibility testing, the performance of respiratory mNGS for predicting AMR varied by pathogen, antimicrobial, and nucleic acid type sequenced. For gram-positive bacteria, a combination of RNA + DNA mNGS achieved a sensitivity of 70%  47–87%) and specificity of 95% (CI 85–99%). For gram-negative bacteria, sensitivity was 100% (CI 87–100%) and specificity 64% (CI 48–78%). Patients with hospital-onset LRTI had a greater AMR gene burden in their respiratory microbiome versus those with community-onset LRTI (mNGS has utility for the detection and surveillance of resistant bacterial LRTI pathogens.The online version contains supplementary material available at 10.1186/s13073-022-01072-4. Antimicrobial resistance (AMR) presents a clear threat to human health and is responsible for increasing rates of treatment failure in patients with lower respiratory tract infections (LRTI), the leading cause of infectious disease-related mortality . ImplemeMetagenomic next-generation sequencing (mNGS) holds promise for overcoming the limitations of traditional respiratory diagnostics by affording culture-independent detection of pathogens and simultaneous profiling of host gene expression signatures of infection . In prinThis is in part due to the low abundance of pathogen AMR genes in respiratory and other clinical body fluids, which challenges their detection using conventional mNGS methods . Recent Here, we address these gaps by studying a cohort of critically ill patients to assess the potential of both DNA and RNA mNGS to predict LRTI bacterial pathogen AMR, facilitate epidemiological AMR surveillance, and rapidly detect clinically relevant resistance genes using CRISPR/Cas9 targeted enrichment coupled with real-time nanopore sequencing.We studied 70 mechanically ventilated patients with LRTI and 18 with non-infectious respiratory illnesses   was made using ontology in ARG-ANNOT ) were also identified by FLASH-nanopore sequencing (median sequencing depth of 1.19 × 106 reads [IQR 1.02 × 106–1.46 × 106])  eight days later while undergoing treatment with aztreonam, an antibiotic lacking MRSA coverage. mNGS analysis of a TA specimen obtained 7 days before VAP onset revealed the mecA gene, providing an early indication of the subsequent AMR VAP pathogen.Prior work has demonstrated the utility of mNGS in cases of culture-negative LRTI, which represent more than half of all pneumonia cases , 8, 10. Public health surveillance is essential for pandemic preparedness, understanding trends in AMR, and preventing outbreaks of resistant organisms. Our findings demonstrate the feasibility of mNGS for epidemiological surveillance of AMR and highlight an association between hospital exposure and AMR gene burden in the lower respiratory tract microbiome. Importantly, our analysis included culture-negative LRTI cases and adds to prior literature demonstrating a similar association in cases of culture-confirmed bacterial pneumonia .Nanopore sequencing has proven useful for microbial detection from respiratory samples with high pathogen abundance , 10, 31;Rapid detection of AMR is essential in critically ill patients with severe bacterial infections given that time to appropriate antimicrobials correlates with mortality . FLASH aStrengths of this study include that it is the largest to assess the performance of mNGS AMR prediction in LRTI and that it systematically assessed performance for multiple classes of antimicrobials against a clinical reference standard. Further, we provide the first culture-independent assessment of healthcare exposure and resistance gene burden in the respiratory tract, and a novel demonstration of Cas9 targeted enrichment coupled with rapid nanopore sequencing. Our study also has several limitations, including sample size, spectrum of antimicrobial classes assessed, spectrum of pathogens assessed, and the need for independent validation of findings. Future work in a larger, prospective cohort with a greater diversity of bacterial pathogens and resistance mechanisms can address these limitations. In addition, a randomized clinical trial will be needed to assess the potential impact of mNGS on time to appropriate antimicrobial treatment, antimicrobial stewardship, and LRTI outcomes. Lastly, genotype to phenotype prediction remains imperfect, even for cultured isolates , 35. As In summary, we characterize the utility of mNGS for predicting AMR in bacterial LRTI and demonstrate proof of concept for both epidemiological AMR surveillance and rapid resistance gene detection using Cas9 and nanopore sequencing.Additional file 1: Table S1. Clinical and demographic features of cohort.Additional file 2: Table S2. mNGS detection of respiratory pathogens compared to a reference standard of clinical microbiological testing.Additional file 3: Dataset 1. AMR gene database utilized for analyses.Additional file 4: Dataset 2. AMR genes and guide RNAs for FLASH targeted mNGS. AMR genes and guide RNAs for FLASH targeted mNGS. A) AMR genes targeted by FLASH. B) AMR alleles and guide RNA targets. Legend: Bla = beta lactam; MLS = macrolide, Tmp/Sul = trimethoprim/sulfamethoxazole.Additional file 5: Table S3. Time to return of clinical antimicrobial susceptibility testing results for culture-confirmed bacterial pathogens.Additional file 6: Table S4. AMR genes detected by mNGS.Additional file 7: Table S5. Performance of RNA-seq, DNA-seq and combined RNA-seq + DNA-seq for AMR prediction compared to clinical antimicrobial susceptibility testing.Additional file 8: Table S6. Pathogen AMR genes detected by FLASH-mNGS.Additional file 9: Table S7. AMR genes detected by FLASH coupled with Nanopore mNGS.


Antimicrobial resistance is of critical concern for both human and veterinary medicine worldwide. Many bacterial infections are currently very difficult to treat due to the presence of several mechanisms involved in bacterial resistance to marketed antimicrobials. Preserving the effectiveness of currently available antimicrobials and reducing the burden of infections caused by resistant bacteria is a priority for competent authorities operating in both sectors. It has been perceived that the livestock sector is a primary contributor to the spread of bacterial resistance in both humans and the environment. This review examined the recent scientific literature on this topic in order to explore the relationship between antimicrobials use in farm animals and the selection of bacterial resistance in this sector and its subsequent dissemination to humans. Recent data indicated that the global biomass-adjusted amount of antimicrobials consumed by farm animals was slightly higher than the amounts used in humans, while the reel contribution of farm animals in the spread of AMR to humans is probably very low compared to initial estimations. However, this review highlights the importance of the close collaboration between veterinary and human medicine, as part of the ‘One Health’ approach, to preserve the longevity of antimicrobials.Antimicrobial resistance (AMR) represents a global threat to both human and animal health and has received increasing attention over the years from different stakeholders. Certain AMR bacteria circulate between humans, animals, and the environment, while AMR genes can be found in all ecosystems. The aim of the present review was to provide an overview of antimicrobial use in food-producing animals and to document the current status of the role of farm animals in the spread of AMR to humans. The available body of scientific evidence supported the notion that restricted use of antimicrobials in farm animals was effective in reducing AMR in livestock and, in some cases, in humans. However, most recent studies have reported that livestock have little contribution to the acquisition of AMR bacteria and/or AMR genes by humans. Overall, strategies applied on farms that target the reduction of all antimicrobials are recommended, as these are apparently associated with notable reduction in AMR . The interconnection between human and animal health as well as the environment requires the acceleration of the implementation of the ‘One Health’ approach to effectively fight AMR while preserving the effectiveness of antimicrobials. The introduction of antimicrobials for clinical use is certainly the greatest medical breakthrough of the 20th century, being responsible for the treatment of serious bacterial diseases, as well as for major advances in surgery  . HoweverAntimicrobials have been used, since the 1950s, therapeutically, prophylactically, and even for growth promotion in farm animals to maintain animal health and increase overall productivity ,9. The mE. coli and the mobile colistin resistance (mcr)-1-positive E. coli in animals, humans, and the environment [Several studies have reported that the application of different strategies aimed at reducing the use of antimicrobials in veterinary medicine  have positively contributed to a decrease in the prevalence of colistin-resistant ironment ,14. In tironment ,16. Thisironment . Howeverironment ,19. Neveironment . The One Health approach has been recently defined by the Food and Agriculture Organization of the United Nations (FAO), the World Organization for Animal Health , the World Health Organization (WHO), and the United Nations Environment Programme (UNEP) (tripartite and UNEP) as “an integrated, unifying approach that aims to sustainably balance and optimize the health of people, animals, and ecosystems” . It is nIn farm animals, antimicrobials are used therapeutically  for prophylaxis , for metaphylaxis  ,30, and Drug classification is an important tool for addressing AMR against medically important antimicrobials (MIAs). Restricting the use of these antimicrobials in both animals and humans may prolong their usefulness in the two sectors . The OIEThe global trends in AMU in farm animals has been extensively discussed elsewhere ,40,41,42Monitoring and surveillance of AMU in animals is generally not harmonized among the different countries. As a consequence, the results are not always directly comparable . Indeed,The estimated predictions on AMU for 2030 or 2050  should bE. coli following the use of colistin in pigs, whether used at therapeutic or at higher doses [E. coli strains of dairy herd origin [Several studies explored the selective effect of the level of AMU and the selection for AMR bacteria in farm animals. However, their results were not conclusive in most cases . Indeed,er doses ,62,63. Td origin ,64. E. coli from healthy broilers [E. coli in food-producing animals , but also for other antimicrobials such as fluoroquinolones, aminopenicillin, polymyxin, and tetracycline and different bacterial species (including Campylobacter jejuni) [E. coli isolates had significantly decreased in the sampled dairy farms  [mcr-1 gene, in pigs and poultry, significantly decreased [vanA, mecA, blaCTX-M, mcr-1, aadA2, tet(E), tet(P), vat(E), sul2, dfrA5 and dfrA13) and humans (vanA), while no effects were detected for β-lactamases other than blaCTX-M and the remaining tet genes [On the other hand, several other studies could establish a link between AMU and AMR in farm animals ,65,66. I jejuni) . Two yeasamples) . In Chinecreased ,69. Alsoecreased . Howeverecreased ,72,73. Aecreased . Also, iecreased . Howeverecreased ,75. Alsoet genes .As can be seen from the information above, the causal link between the use of antimicrobials in animal production and the selection of resistant bacteria and their genetic determinants is not universal. This indicates a complex causality involving the interaction of several other factors that determine the magnitude of selection pressure, the spread, and the persistence of AMR bacteria and genes within the animal intestinal microbiota and in the farms environment . SeveralThe link between AMR in humans and AMR in animals, and thus ultimately the use of antimicrobials in livestock and pets, is still puzzling the scientific community. Only a few studies have focused on this problem with a ‘One Health’ approach. When studying the transfer of resistance, a first distinction should be made between zoonotic bacteria and other bacteria. Since zoonotic bacteria are present in animals, and frequently without any harm, AMR is selected in animals before it infects humans. The odds that this bacterium goes back to the animal from an infected human is very unlikely. It is thus clear that resistance in zoonotic bacteria is mainly selected in animals and that there is a direct transfer of these bacteria with their AMR genes from animals to humans. The indirect transfer, however, is more complex. Hereby, AMR genes from bacteria of animals transfer to bacteria from humans. It is problematic that AMR genes are present in both animal and human bacteria as well as in bacteria in the environment, and that the contribution of resistance to any of them is thereby very hard to evaluate (as there is no difference between AMR genes). Thus, a direct source attribution of AMR genes is not possible. Campylobacter clinical isolates was observed in several countries  [C. jejuni from broilers and fluoroquinolone resistance of C. jejuni from humans [Several studies reported that after fluoroquinolones were introduced for farm animals use in 1995, a significant increase in the prevalence of resistance toward this antimicrobial in animal and human , Spain) ,81. The m humans . This coStaphylococcus aureus (LA-MRSA). Multiple sources of evidence support the role of swine as reservoirs of LA-MRSA for humans [Salmonella Typhimurium DT104 and its resistance genes were widely maintained in cattle and humans separately, with limited transmission in both direction [Campylobacter and Salmonella (which are foodborne) and LA-MRSA (which is rather transmitted through direct contact). This causes only a temporary colonisation, rather than contamination, and as such gives different results.However, there are differences in the methods of transmission of zoonotic bacteria that should be taken into account. This is exemplified by studies carried out on livestock-associated methicillin-resistant r humans ,86. Thisr humans . This str humans . Humans r humans . Howeverr humans . Moreoveirection . This diThe first studies investigating this transfer used in vivo models and were limited in their set up. One such study examined the transfer of vancomycin-resistant enterococci (VRE) , whereinOther studies examined the potential transfer of ESBLs from animal to human bacteria. The first ex vivo studies conducted have shown that the genes were readily transferred even without selective pressure . HoweverE. coli and plasmid-mediated AmpC producing E. coli might possibly be attributable to foodstuffs of animal origin [E. coli between broiler flocks, broiler farmers, and the open community, the colonization of the open community could primarily be attributed to the open community itself (62%), followed by vegetable consumption (29.5%) and contact with farmers (8.5%) [E. coli, involve community-associated infection of genitourinary or gastrointestinal origins, while non-human reservoirs made little contribution to invasive human disease in this country [E. coli (ESCR-EC) infections originate from farm animals and that poultry appears to be the more likely source than other farm animals [E. coli isolates (including ESBL-producing isolates) from cross sectional surveys of livestock, retail meat, and human patients in East England were genetically distinct, suggesting that E. coli causing serious human invasive disease had not directly originated from livestock [E. coli isolates from humans or human-polluted environments  with isolates from livestock or animal-polluted environments  on Reunion Island [E. coli by humans [A study conducted in the Netherlands demonstrated that 19% of community-acquired ESBL-producing l origin . Meanwhil origin . In the s (8.5%) . Likewis country . In a sy animals . However animals . Whole givestock . Similarn Island  confirmey humans . This sty humans  or, if ty humans ,101. ThuE. coli from animals  [E. coli strains and the subsequent transfer in persons with direct contact [Nevertheless there are other studies showing a closer link between ESBL positive d chain) . On the d chain) . Other s contact ,105. IndDiscrepancies between studies related to the existence or not of a link between farm animals and humans regarding the transmission of AMR could be explained by several factors. The use of a variety of protocols for the determination of the susceptibility of bacteria to antimicrobials and for the detection of ARGs are probably of minor influence in the comparison of the results between studies . The varMuch research has been carried out regarding the association between AMU in livestock and AMR in humans. The effect of interventions that restrict AMU in food-producing animals on AMR in humans is still under investigation ,76. NeveE. faecium (GRE) in broilers, from 72.7% in 1995 to 5.8% in 2000 was measured [vanA gene are resistant to vancomycin and teicoplanin [After the ban of avoparcin as a growth promotor in 1995 in Denmark a decrease in the occurrence of glycopeptide-resistant measured ,74. The measured . Moreovemeasured . It is ncoplanin . Howevercoplanin ,110,111.E. coli and the prevalence of mcr-1 gene was observed in chicken and pigs as well as in humans in comparison with data collected before the entry into force of this policy [E. coli and Salmonella enterica serovar Heidelberg in both retail chicken and humans isolates [E. coli isolates from humans [After the ban of colistin use as a growth promoter in farm animals in China, in April 2017, a significant reduction in colistin-resistant s policy . Similarisolates . Interesm humans . E. coli strains from livestock [A systematic review, commissioned by the WHO, demonstrated that the reducing of AMU in livestock has been associated with a decrease in the prevalence of AMR bacteria in animals by about 15% . This reivestock . WhetherAntimicrobial resistance compromises the possibility of effectively treating bacterial diseases in humans and animals and represents a global challenge to agriculture and public health. The extensive use of antimicrobials in animals and humans is being recognized as a major driver for the emergence and spread of AMR worldwide. The high occurrence of resistant bacteria and their resistance determinants in the intestinal microbiota of farm animals is a major concern for animal and human health (as well as for the environment). The amount of antimicrobial drugs (biomass-adjusted (mg/PCU)) consumed by farm animals, based on the available global data, was only slightly higher than the antimicrobial drugs used for humans. Banning the global use of growth promotors will likely bring the use of antimicrobials in livestock lower than in humans. Care should be taken with the interpretation of these data, as the use of mg is not very precise as some antibiotics are more potent than others. The contribution of farm animals to the spread of AMR to humans continues to be debated within the scientific community, with a lot of uncertainties and contradicting results having emerged. Therefore, there is definitely a need for more research in this field. Nevertheless, it can be concluded that AMR in zoonotic bacteria is of animal origin and that these resistant bacteria can cause infections in humans and thus pose a threat to human health as possible resistant bacterial infections. The contribution of commensal resistant bacteria is still a matter of debate, although we can conclude that the impact is probably much smaller than first anticipated. Very specific interventions that restrict the use of only a single antimicrobial or antimicrobial class in farm animals was associated with a limited effectiveness in reducing AMR in both sectors. It is believed that part of this can be attributed to co-resistance selection. Overall reduction of AMU may have much larger effects. This has been shown in animal bacteria, although the impact on human bacteria is still a matter of debate. Nevertheless, combating AMR  will have major benefits, and there is a need for accelerating the implementation of the One Health approach which needs strong multisectoral collaboration and partnerships between all stakeholders including physicians, veterinarians, environmental professionals, researchers, pharmaceutical companies, hospitals, policymakers, and patients for reducing AMU and AMR. Finally, while global public health efforts continue to focus on the management of the COVID-19 pandemic, this public health priority should not overshadow the efforts required to contain the current “not so silent” AMR pandemic.