Antimicrobial resistance (AMR) is one of the biggest threats to global public health. Selection of resistant bacteria is driven by inappropriate use of antibiotics, amongst other factors. COVID-19 may have exacerbated AMR due to unnecessary antibiotic prescribing. Country-level knowledge is needed to understand options for action.To review the current situation with respect to AMR in Vietnam and initiatives addressing it. Identifying areas where more information is required will provide a call to action to minimize any further rises in AMR within Vietnam and improve patient outcomes.Streptococcus pneumoniae and Haemophilus influenzae, were identified. National and international antibiotic prescribing guidelines for CA-RTIs  commonly used locally were also reviewed, plus local antibiotic availability. Insights from clinicians in Vietnam were sought to contextualize this information.National initiatives to address AMR in Vietnam, antibiotic use and prescribing, and availability of susceptibility data, in particular for the key community-acquired respiratory tract infection (CA-RTI) pathogens In Vietnam there have been some initiatives addressing AMR; Vietnam was the first country in the Western Pacific Region to develop a national action plan to combat AMR, which according to the WHO is being implemented. Vietnam also has one of the highest rates of AMR in Asia due, in part, to the overuse of antimicrobial drugs, both in the animal health sector and in humans in both hospitals and the community. In addition, despite a 2005 law requiring antibiotic prescription, there is unrestricted access to over-the-counter antibiotics. Several global surveillance studies provide antibiotic susceptibility data for CA-RTI pathogens in Vietnam including Survey of Antibiotic Resistance (SOAR) and SENTRY . For management of the common CA-RTIs in Vietnam there are several country-specific local antibiotic prescribing guidelines and in addition, there is a range of international guidelines referred to, but these may have been created based on pathogen resistance patterns that might be very different to those in Vietnam. Expert clinician opinion confirms the high resistance rates among common respiratory pathogens. A more standardized inclusive approach in developing local guidelines, using up-to-date surveillance data of isolates from community-acquired infections in Vietnam, could make management guideline use more locally relevant for clinicians. This would pave the way for a higher level of appropriate antibiotic prescribing and improved adherence. This would, in turn, potentially limit AMR development and improve clinical outcomes for patients. Even if new medicines are developed, without behaviour change, antibiotic resistance will remain a major threat’.3 The first paper in this Supplement included details about the multiple factors which can drive a rise in AMR along with the global initiatives that are in place to address this threat.2 Each country and/or region must also play their part through local initiatives.Antimicrobial resistance (AMR) is one of the biggest threats to public health throughout the worldStreptococcus pneumoniae and Haemophilus influenzae. National and international antibiotic prescribing guidelines for CA-RTIs, specifically community-acquired pneumonia (CAP), acute otitis media (AOM) and acute bacterial rhinosinusitis (ABRS), commonly used by healthcare professionals in Vietnam were also reviewed, along with how these link to local antibiotic availability. Insights from a clinician in Vietnam were sought to contextualize this information. In addition, we aimed to identify areas where more information is required and present a call to action to improve clinical outcome for patients and to minimize further rises in AMR within Vietnam.In order to identify how AMR can be addressed in Vietnam in the future, it is necessary to review what is happening now. In this paper, we present the current situation in Vietnam, determined by using published information  to ascertain any national initiatives to address AMR, antibiotic use and prescribing, and availability of susceptibility data, in particular for the key community-acquired respiratory tract infection (CA-RTI) pathogens 4 many countries began to develop their own National Action Plan (NAP). For the future, enhanced regulation, patient/public education, provision of information on local resistance patterns, establishing antimicrobial stewardship programmes and developing national and regional consensus programmes have been proposed to help combat AMR.5 Vietnam was the first country in the WHO Western Pacific Region to develop a NAP to combat AMR. It was produced by the Ministry of Health in 2013 and entitled the ‘National Action Plan on Combating Drug Resistance in the Period 2013–2020’.7 The plan focused principally on human health and a further plan was developed to combat drug resistance in livestock and aquaculture. Antibiotic use in food production is perceived as a key driver of AMR in humans. As addressing AMR requires a coordinated approach, relating to not only human use, but also to the use of antibiotics in animals and agriculture, there is potential to strengthen multi-sectorial collaboration between the animal and human health sectors and create cross-disciplinary awareness and participation, to provide a One Health Approach to strengthen the animal health sector and reduce the spread of AMR.8 According to the report by the WHO for 2020–21, Vietnam’s NAP is being implemented.9Following the formulation by the World Health Assembly in 2015 of a Global Action Plan (GAP) for AMR10Vietnam is currently classified as a lower middle-income country with a population of 94.6 million and GDP per capita of US $2171. In 2016, health expenditure accounted for 5.7% of GDP. Antimicrobials accounted for one-third of the total spend on medications in public hospitals in Vietnam and currently there are many different forms and formulations of antimicrobials available on the Vietnamese market.11 That study emphasized the need for more surveillance of antibiotic resistance.11 In rural Vietnam, a study of resistance of S. pneumoniae in preschool children reported that multidrug resistance increased from 31% to 80% over a 15 year period (1999–2014).12Vietnam has one of the highest rates of AMR in Asia. In an AMR surveillance study using a network of 16 hospitals in Vietnam between 2012 and 2013, the proportion of AMR was high among all pathogens isolated from clinical specimens and in most, resistance had increased since an analysis in 2009.11 A study of antibacterial consumption in 76 countries between 2000 and 2015 found that Vietnam ranked 11th in antibacterial consumption with 32.0 DDD per 1000 inhabitants per day; this is a higher rate of prescribing than that seen in most EU countries.13 Another study extracted antimicrobial usage data for key animal species and humans from different published sources. Total antimicrobial use in Vietnam equated to 261.7 mg/kg and 247.3 mg/kg of human and animal biomass, respectively, compared with 122.0 mg/kg and 151.5 mg/kg in the EU.14 A review of the consumption of antibiotics in rural Vietnam, in relation to the WHO Access, Watch, Reserve classification showed a high consumption of antibiotics from the Watch group, particularly in children. Watch group antibiotics were provided frequently from private pharmacies showing the requirement for putting into practice pharmacy-specific interventions.15Several factors have contributed to the increase in AMR. In Vietnam, there is substantial overuse of antimicrobial drugs, both in the animal health sector and in humans in both hospitals and the community and this is an important driver for the emergence and spread of AMR.5 Consumers of antibiotics in Vietnam are influenced by what constitutes a high-quality medicine and give less emphasis to the importance of a correct diagnosis. They regard antibiotics as a ‘trusted remedy’ but have a limited understanding of antibiotic resistance. Social media also influences consumption and choice of antibiotics.16Despite a 2005 law requiring antibiotic prescription, there is unrestricted access to over-the-counter (OTC) antibiotics in Vietnam, with 38% of caregivers still accessing antibiotics without any formal medical assessment. Limited knowledge amongst members of the public is also an issue, with many people buying antibiotics to treat the common cold.5 Unnecessary hospitalization has been reported in children with non-severe pneumonia and there is a reluctance to step down from intravenous to oral antibiotics.17A large proportion of inpatients are reported as receiving inappropriate antibiotic treatment in Vietnamese hospitals.5Several physician-related factors also contribute to the overuse of antibiotics, including patient pressure, lack of access to microbiology services and limited knowledge of local resistance patterns.S. pneumoniae and H. influenzae are the two most common causative pathogens, both in children and adults. This has been confirmed by various investigations in Vietnam including a recent study which made use of combined culture and real-time PCR results to investigate the pathogens causing CAP in Vietnamese outpatients. Microbial pathogens were detected in 94% of cases, of which 18.7% involved single pathogens, including S. pneumoniae  and H. influenzae . Multiple pathogens were detected in 76.3% of cases and most of these were S. pneumoniae and H. influenzae and/or combined with other pathogens; S. pneumoniae and other pathogens; and H. influenzae and other pathogens.18 For this reason antibiotic susceptibility of these two pathogens is the focus of surveillance studies in the community.Pneumonia is the most common infection in the community and S. pneumoniae and H. influenzae, the most commonly isolated respiratory bacterial pathogens in the community.19 Key features of SOAR are that it focuses on only these pathogens, and identification and susceptibility testing are performed in an independent centralized laboratory using standardized methodology (CLSI) allowing for comparisons to be made between countries/regions and for the identification of trends over time. SOAR data is analysed based on three different breakpoints: CLSI, EUCAST dose-specific, and PK/PD breakpoints.Several global surveillance studies provide antibiotic susceptibility data in Vietnam. The Survey of Antibiotic Resistance (SOAR) is a multinational antibiotic surveillance study, ongoing in an expanding range of countries since 2002. The study aims to collect and make available in published, peer-reviewed papers, antibiotic susceptibility data, specifically for 21 EUCAST breakpoints are dose-specific and use the EMA-approved doses that are included in the Summary of Product Characteristics of an antibiotic. This means that by application of breakpoints for higher doses, the effect of using a raised dose on the clinical efficacy of a particular antibiotic can be predicted. Currently, most clinical microbiology laboratories in Vietnam use CLSI breakpoints, however the international application of the EUCAST breakpoints is expanding,22 so it is possible that dose-specific breakpoints could at some time also be applied in Vietnam. The EUCAST dose-specific breakpoints can also be used retrospectively to calculate the susceptibility of previously collected isolates to show the susceptibility levels that would have been achieved at higher doses.Clinical breakpoints are cut-off MIC values used to classify microorganisms into the clinical categories susceptible (S), intermediate (I) and resistant (R) to assist in the prediction of the clinical success or failure of a specific antibiotic. Two international organizations define breakpoint values: CLSI and EUCAST. Due to variation in criteria for their definition, there are some differences between CLSI and EUCAST in the clinical breakpoint values for certain bacteria for some antibiotics and this can impact susceptibility interpretation of clinical isolates.S. pneumoniae, the most commonly isolated respiratory bacterial pathogen24 for infections such as CAP, AOM and ABRS has over time become less susceptible to amoxicillin/clavulanic acid in some countries25 since the MIC of some isolates has increased. When treating infections, it is important to be able to eradicate bacterial pathogens with raised MICs to optimize the clinical outcome while at the same time minimizing the risk of selecting variants with even higher MICs. This is possible because β-lactams, unlike many other antibiotics, have time-dependent killing properties. Their efficacy depends on the amount of time an adequate drug concentration is present at the site of action. Although increasing the concentration at the infection site above a particular concentration will not have any effect on the efficacy, the use of higher doses and/or more frequent dosing allows for successful eradication of infections caused by pathogens with higher MICs because the time the antibiotic is above the MIC is increased.26Use of the EUCAST dose-specific breakpoints shows the effect of increasing the antibiotic dose on the susceptibility of a pathogen, providing additional information so the prescriber can decide if a higher dose would be of benefit. For example, S. pneumoniae isolates (n  = 161) were susceptible to amoxicillin/clavulanic acid but susceptibility fell to 4.4%, 2.5% and 1.9%, respectively, for the macrolides azithromycin, clarithromycin and erythromycin. Susceptibility to cephalosporins  was also generally low, except for ceftriaxone for which susceptibility was 62.1%. For the fluoroquinolones levofloxacin and moxifloxacin, S. pneumoniae susceptibility was 90.1% and 93.2%, respectively  was 60.3%, to cefixime and cefpodoxime, both 5.6%, to ceftriaxone, 62.1% and to azithromycin, 2.5%.19The most recent SOAR results for Vietnam, from pathogens isolated from samples collected at two sites in 2016 to 2018 from outpatients with confirmed CA-RTIs, show that when applying the CLSI breakpoints, 59.6% of S. pneumoniae isolates in Vietnam for 2009–11 with the results for isolates collected in the period 2016–18 and shows that S. pneumoniae isolates had become less susceptible to penicillin, amoxicillin/clavulanic acid, cefaclor and cefuroxime during that time period.27S. pneumoniae susceptibility to azithromycin was consistently low across both time periods.27By using SOAR data from different time periods, susceptibility trends over time can be identified. Figure H. influenzae isolates  from Vietnam, a high susceptibility of 97.4% to amoxicillin/clavulanic acid was seen in 2009–11, using CLSI criteria  to cefixime and cefpodoxime was 70.8% and 60.4%, respectively.19For all a Figure  and thisS. pneumoniae isolates collected in Vietnam in 2018 but the number of isolates was very low (n  = 9).28The SENTRY Antimicrobial Surveillance Program was initiated in January 1997 and was designed to monitor the predominant pathogens and AMR for nosocomial and community-acquired infections globally. Data are available for susceptibility of 29 to provide the supporting data required to ensure that countries can design cost-effective, evidence-based AMR response strategies. During the first 4 years, 91 countries or territories enrolled in GLASS and data for over two million patients from 66 countries are included.30 Pathogens currently included in GLASS-AMR are: Acinetobacter spp., Escherichia coli, Klebsiella pneumoniae, Neisseria gonorrhoeae, Salmonella spp., Shigella spp., Staphylococcus aureus, and S. pneumoniae and a new and important component is the inclusion of antimicrobial consumption (AMC) surveillance at the national level.31 GLASS data is analysed based on CLSI and EUCAST breakpoints. However, Vietnam does not currently participate in this initiative.In 2015, the WHO launched the Global Antimicrobial Resistance and Use Surveillance System (GLASS). GLASS is the first global system to collect national AMR data for selected bacterial pathogens that cause common infections. The aim is to monitor the prevalence of AMR among major pathogens in clinical settings32 was set up to generate information which could be used to inform a new schedule for infant immunization in developing countries and support the efficient use of pneumococcal conjugate vaccines. This trial involved 1200 infants from two areas in Ho Chi Minh City, Vietnam and involved taking nasopharyngeal swabs for analysis of pneumococcal carriage and susceptibility testing of the isolates s Figure . A recenFor management of the common RTIs, CAP, AOM and ABRS in Vietnam there are several country-specific local antibiotic prescribing guidelines which may not always be adhered to by clinicians. In addition, there are a range of international guidelines referred to, but these may have been created based on pathogen resistance patterns that might be very different to those in Vietnam. Examples of some of the published management guidelines in use in Vietnam are included in Table Most guidelines suggest a first-line antibiotic or antibiotics along with alternative(s) and then a second-line antibiotic or antibiotics, also with an alternative(s). The first-line antibiotic is the recommended first choice that should be prescribed by the clinician following diagnosis of the infection, supported by the criteria defined by the organization or committee; alternative(s) may be provided for use in particular circumstances. For example, if the first-line antibiotic is a β-lactam then alternative suggestions may be for use in the case of allergy. The second-line antibiotic is for use if the first-line one does not achieve the anticipated outcome, and again alternative(s) may be included for use under specific circumstances.41 and the Infectious Diseases Society of America (IDSA)50For the management of CAP in adults and paediatrics, international guidelines referred to by clinicians in Vietnam include those from the British Thoracic Society (BTS)50 however, one of the differences between the 2019 and 2007 American Thoracic Society/Infectious Diseases Society of America Community-acquired Pneumonia Guidelines is that in the 2019 guideline, macrolide monotherapy is not strongly recommended for outpatients, but rather conditionally depending on local pneumococcal resistance levels, and for standard empirical therapy for severe CAP there is stronger evidence in favour of β-lactam/macrolide combinations than β-lactam/fluoroquinolone combinations.50 Similar guidance is also given by Insitut national d’excellence en santé et en services sociaux (INESSS) 2017 CAP in adults.49Both UK’s NICE guidance and Vietnam’s ‘Guidance to using antibiotic in children’ recommend amoxicillin/clavulanic acid (suspension) as alternative empirical therapy or as first-choice antibiotic in children with CAP. A first-line recommendation by the IDSA 2019 for treating adults with comorbidities and CAP is amoxicillin/clavulanic acid 875 mg/125 mg twice daily or 500 mg/125 mg given three times daily (with either a macrolide or doxycycline)43 and INESSS 2016: AOM in children 3 months of age or older.47 For the management of ABRS in adults and paediatrics the international guidelines referred to in Vietnam include those from the IDSA42 and INESSS 2016: Acute Rhinosinusitis in Adults,43 INESSS 2016: Acute Rhinosinusitis in Children46 and those from NICE 2017.48For the management of AOM, the international guidelines referred to in Vietnam include those from the American Academy of Pediatrics (AAP)A range of national guidelines are referred to by physicians in Vietnam, examples of which are listed in Table 55 Substandard poor-quality or falsified antibiotics promote AMR and the spread of drug-resistant infections.56 Since poor-quality antibiotics are unlikely to contain the full dose needed to eliminate all of the infecting pathogens this would encourage resistance to develop and allow resistant strains to survive and be transmitted.57Access to antibiotics may be an issue for patients in low- and middle-income countries (LMICs) due to cost and insufficient government expenditure or support in this area. Drug supply chains may also contribute to the problem. Limited access to the most appropriate antibiotic to treat a specific infection may result in raised mortality from treatable bacterial infections, and the use of suboptimal amounts of antibiotic facilitates resistance development and allows resistant strains to persist.58 Empirical treatment with antibiotics which have broad-spectrum coverage of the bacterial causes of CAP, such as amoxicillin/clavulanic acid, is used for outpatient treatment.58 Amoxicillin/clavulanic acid treatment recommendations for CAP include 500 mg/125 mg three times daily,59 amoxicillin/clavulanic acid 875 mg/125 mg twice daily in mild/moderate infections or a dosage of 1000 mg/125 mg given twice daily.59 In severe infections (including those of the lower respiratory tract) 1000 mg/125 mg is recommended three times daily in adults.59 Amoxicillin/clavulanic acid given three times daily is currently unavailable in Vietnam but is undergoing regulatory approval.In Vietnam, some currently available formulations of amoxicillin or amoxicillin/clavulanic acid, cephalosporins, macrolides, and fluoroquinolones are mentioned as first- or second-line recommendations by the RTI management guidelines and endorsed by both national and international guidelines for the treatment of a wide range of infections. Using amoxicillin/clavulanic acid as an example, this antibiotic is indicated for upper and lower RTIs, genito-urinary tract infections, skin and soft tissue infections in adults and children.57 The GSMS aims to work with WHO member states to improve the quality of reporting of substandard and falsified medical products, and, importantly, to ensure the data collected are analysed and used to influence policy, procedure, and processes to protect public health, at the national, regional and the global level. Use of substandard or falsified antibiotics not only compromises clinical outcome but also risks increased AMR. The most recent summary (2013–17) reported substandard and falsified medicines in 46 member states (including Vietnam) and antibiotics represent 16.9% of all products reported, second only to malaria drugs (19.6%).57The quality of medicines, specifically antibiotics, is an important consideration for countries worldwide. The WHO launched a Global Surveillance and Monitoring System (GSMS) for substandard and falsified products.S. pneumoniae and H. influenzae, are decreasing. For S. pneumoniae, increasing resistance is seen to all antibiotics, including penicillins, cephalosporins, fluoroquinolones and macrolides. In efforts to address the problem of rising AMR worldwide, one of the strategic objectives outlined by the WHO was to strengthen the knowledge and evidence base through surveillance. Early studies aimed to implement AMR surveillance with national and international efforts in Vietnam. These showed the importance of local susceptibility data, providing solid evidence on the high AMR pattern in Vietnam. In the most recent study (Viet Nam Resistance: VINARES 2016–17), which was a hospital antibiotic resistance surveillance study covering a network of hospitals, results showed that 58% (663/1136) of S. pneumoniae had reduced susceptibility to penicillin and 88% (804/911) of H. influenzae were resistant to ampicillin;60 local surveillance on CA-RTI also revealed similar alarming messages. In the SOAR studies, as described in this paper, Vietnam had the lowest susceptibilities of S. pneumoniae and H. influenzae to most of the antibiotics tested among the four Asian countries reported. It is noticeable that only 1.2% of S. pneumoniae isolates were susceptible to intravenous penicillin, 59.6% were susceptible to amoxicillin whereas only 11.2% of H. influenzae isolates showed susceptibility to ampicillin.18 These pathogens are commonly involved in other community-acquired infections such as AOM and ABRS, but minimal data are available so far for these infections specifically. Therefore, it is even more challenging to have optimal antibiotic management in Vietnam when there is limited information available regarding the AMR profiles.Vietnam is a hotspot of AMR in Asia, where the burden of resistant infections is disproportionate. In particular, the susceptibilities of the two most common respiratory pathogens, responsible for many of the incidences of CAP in both adults and children, 61 Irrational antibiotic use along with guideline non-compliance might be explained by the lack of local microbiology data-driven guidance, patient-expectation oriented prescribing behaviour, lack of consultation time with each patient and promotion of broad-spectrum antibiotic use by pharmaceutical companies.62 Therefore, antimicrobial stewardship teams in Vietnam should elaborate the guidelines with local microbiological data, as well as integrate and disseminate the guidelines to doctors.36The Vietnamese Ministry of Health has issued a general antibiotic treatment guideline and also specific guidelines for CA-RTI, AOM, and ABRS for both adults and children. However, guidelines on infectious diseases at the national level have had limited impact, with low rates of adherence. In a multicentre study on CAP, widespread use of intravenous third-generation cephalosporins was observed (29.3% as monotherapy and 40.4% as combination therapy) regardless of infection severity.13 Stratifying antibiotics by the WHO Access framework, Vietnam has a high rate of consumption of Watch-group antibiotics, particularly in children. A 2018 study in pharmacies showed that 54.8% of pharmacy encounters, 53.0% of days of treatment and 53.6% of DDD in children were Watch-group antibiotics. Vietnam had the largest increase in Watch antibiotic consumption (10.6 DDDs per 1000 inhabitants per day) our of 75 countries from 2000 to 2015.13Vietnam is an emerging pharmaceutical market with antimicrobial consumption ranked 11th out of 76 countries.63 A study involving children <5 years of age in rural Vietnam showed that although most caregivers knew that antibiotics were not required for mild ARI without fever, such as the common cold, 71.0% of children were actually prescribed them and most (82%) of the unnecessary antibiotics had been recommended by a healthcare professional.63 Inappropriate antibiotic dispensing was considered the main reason for the increase in the AMR rate of S. pneumoniae in the community. A trend towards prescribing of broader spectrum antibiotics was common, with cephalosporins gradually replacing penicillins from 1991 to 2007.12The Vietnamese Pharmaceutical Law (2005) made antibiotics prescription-only drugs. Despite this, antibiotic dispensing without prescription is still a major problem in Vietnam. A common reason for buying antibiotics is mild acute respiratory infection (ARI) without fever.65 Surveillance data are therefore critical to inform infection treatment guidelines, monitor trends, and to assess interventions, although most surveillance programmes are passive and pathogen-based, which will inevitably lead to bias. Addition of clinical and patient outcome data would provide considerable added value to pathogen-based surveillance, as would continuously monitoring local antibiotic use and bacterial resistance. In addition, in terms of guidelines, clinicians need to move from expert-based guidelines to evidence-based ones and institutions also need to follow strict infection control.Pharmacies in Vietnam are rarely penalized for antibiotic dispensing without prescription. The accessibility to antibiotics is only controlled by pre-authorization policy as recommended in the national antimicrobial stewardship guideline. More-restrictive measures are needed to control, monitor, and optimize antibiotic use, both in the community and in hospital.In an era of rising AMR throughout the world, this paper aims to define areas where action is required to address AMR by analysing and understanding the current situation within a country or region. Information is presented for Vietnam concerning antibiotic use and prescribing, approach to AMR, availability of local susceptibility data, use of international and/or local management guidelines and how these link to antibiotic availability. To our knowledge this is the first time this information has been reviewed and presented in detail by country.Antibiotic use in Vietnam is extremely high and continues to rise. OTC sale of antibiotics plays an important role in increasing the rate of AMR. Education and enforcement concerning restricting OTC use of antibiotics will be needed for pharmacists and the same topics along with the harm of overuse and misuse of antibiotics would form the basis of a country-wide education campaign for the public, which would need to be repeated to reinforce these messages.Antibiotic susceptibility is low for most antibiotic classes in Vietnam; the fluoroquinolone antibiotics have so far retained higher levels of activity, although guidelines and regulatory bodies urge caution, restricting their use to limited situations due to serious safety concerns.There are some international and local surveillance studies for CA-RTI pathogens in Vietnam but development of a coordinated national antibiotic surveillance system for Vietnam in which the data are collected by accredited laboratories under international standards, followed by data sharing would provide a centralized accurate picture of antibiotic susceptibility throughout the country. In addition, according to the expert clinician comments, whilst surveillance studies include clinical isolates from CAP patients, these pathogens are commonly involved in other community-acquired infections such as AOM and ABRS, but minimal data are available so far for these specific infections.2 This would pave the way for improved adherence and a higher level of appropriate antibiotic prescribing in CA-RTIs which could, in turn, potentially limit AMR development and improve clinical outcomes for patients.Whilst a range of international and Vietnamese guidelines is utilized by clinicians in Vietnam, a more standardized inclusive approach could be helpful to develop local country-specific guidelines avoiding any reliance on the international guidelines which have been developed in countries or regions with markedly different susceptibility patterns to those that are very specific to Vietnam. These guidelines would be based on the up-to-date surveillance data of isolates from community-acquired infections which would make them more locally relevant for clinicians, reiterating the Consensus Principles as described in the introductory paper to this Supplement.


Antimicrobial resistance (AMR) is one of the biggest threats to global public health. Selection of resistant bacteria is driven by inappropriate use of antibiotics, amongst other factors. COVID-19 may have exacerbated AMR due to unnecessary antibiotic prescribing. Country-level knowledge is needed to understand options for action.To review the current situation with respect to AMR in Pakistan and initiatives addressing it. Identifying areas where more information is required will provide a call to action to minimize any further rises in AMR and improve patient outcomes.Streptococcus pneumoniae and Haemophilus influenzae) were identified. National and international antibiotic prescribing guidelines for specific CA-RTIs  commonly used locally were also reviewed, plus local antibiotic availability. Insights from a local clinician and clinical microbiologist were sought to contextualize this information.National AMR initiatives, antibiotic use and prescribing in Pakistan, and availability of susceptibility data, in particular for the key community-acquired respiratory tract infection (CA-RTI) pathogens (Pakistan is active in developing initiatives to address AMR such as compiling a National Action Plan. However, antibiotic consumption is high and although there is legislation in place prohibiting over-the-counter purchase of antibiotics, this is still possible. Healthcare professionals use local and international antibiotic prescribing guidelines for CA-RTIs when managing patients. As highlighted by the clinical microbiologist’s expert comments, surveillance of AMR in locally prevalent microorganisms is lacking. A more standardized inclusive approach in developing local guidelines, using up-to-date local surveillance data of isolates from community-acquired infections, could make management guideline use more locally relevant for clinicians. This would pave the way for a higher level of appropriate antibiotic prescribing and improved adherence. This would, in turn, potentially limit AMR development and improve clinical outcomes for patients. Even if new medicines are developed, without behaviour change, antibiotic resistance will remain a major threat’.3 The first paper in this Supplement included details about the multiple factors which can drive a rise in AMR, along with the global initiatives that are in place to address this threat.2 Each country and/or region is expected to play their part through local initiatives.Antimicrobial resistance (AMR) is one of the biggest threats to public health throughout the world,Streptococcus pneumoniae and Haemophilus influenzae. National and international antibiotic prescribing guidelines for CA-RTIs  commonly used by healthcare professionals in Pakistan were also reviewed, along with how these link to local antibiotic availability. Insights from a clinician and a clinical microbiologist were sought to contextualize this information. In addition, we aimed to identify areas where more information is required and present a call to action to improve clinical outcomes for patients and to minimize further rise in AMR within Pakistan.In order to identify how AMR can be addressed in Pakistan in the future, it is necessary to review what is happening now. In this paper, we present the current situation in Pakistan, determined by using published information  to ascertain any national initiatives to address AMR, antibiotic use and prescribing, and availability of susceptibility data, in particular for the key community-acquired respiratory tract infection (CA-RTI) pathogens 4 many countries began to develop their own National Action Plan (NAP). The government of Pakistan formed an intra-sectoral committee on AMR in recognition of the severity of the problem and the AMR NAP for Pakistan contained the vision that no Pakistani citizen should suffer as a result of AMR in the coming years. The mission statement encompasses having a functional, coordinated, collaborative and sustainable AMR containment system using the ‘One Health’ approach which is aligned with the WHO GAP on AMR. The strategic priorities of the Pakistan NAP5 include developing and implementing a national awareness-raising and behavioural-changing strategy on AMR; establishing an integrated national AMR surveillance system; improving prevention and control of all infections; updating and enforcing regulations for human and veterinary antimicrobial use; phasing out use of antimicrobials as growth promoters; integrating AMR in all public health research agendas including vaccine research; and estimating the health and economic burden of AMR for decision making.5 The Pakistan chapter of the Alliance for the Prudent Use of Antibiotics  states that AMR problems in Pakistan stem from lack of: access to appropriate antimicrobial therapy; regulation in human use; regulation in agricultural use, surveillance of antibiotic use and resistance; updated antibiotic use and treatment guidelines; continuing medical education on antibiotic use for prescribers; and microbiological laboratory capacity/laboratory training/diagnostic tools. They also note that AMR complicates the treatment of infectious diseases such as malaria, tuberculosis, typhoid, acute respiratory tract infections (RTIs), and diarrhoeal diseases.6Following the formulation by the World Health Assembly in 2015 of a Global Action Plan (GAP) for AMR,7 In common with the rest of the world, there has been a reported upsurge in infections due to resistant strains in Pakistan. For example, a 2019 WHO report ranked Pakistan among the top five countries with the highest number of neonatal deaths caused by resistant bacteria.8It has been well recognized that the threat of AMR in developing countries, such as Pakistan, where a proportion of the population has limited access to education and healthcare, is significant.9 When the consumption of cephalosporins and fluoroquinolones was analysed for the 5 year period 2014 to 2018, a significant increase (65%) in consumption of cephalosporins included in the WHO ‘Watch’ group of antibiotics was noted, while the population treated with ‘Reserve’ cephalosporins doubled. Amongst the fluoroquinolones, there was a major increase in consumption of ciprofloxacin.7Pakistan is the third-highest antibiotic-consuming country among low- and middle-income countries (LMICs). Between 2000 and 2015, the number of defined daily doses (DDDs) antibiotics increased by 65%, to a level of consumption of around 20 DDDs per 1000 inhabitants per day in 2015. This placed Pakistan in the mid-table position of 34th in terms of antibiotic consumption, in a study of 71 countries.10 The Drug Act of 1967 in Pakistan stated that antibiotics are not over-the-counter (OTC) medications, and the Act made their availability without a prescription illegal. Despite this, throughout Pakistan it is possible to obtain antibiotics without a legitimate prescription, generally following a simple request without any clarification or explanation.11 It has been suggested that in some situations, such as when there is limited access to a healthcare professional or in resource-limited settings, OTC purchase of antibiotics may be needed, but self-medication risks antibiotic misuse and can result in increasing resistance.13 A 2011 global study estimated that 9% of antibiotic sales in Pakistan took place without a prescription.13Pakistan is a developing country where the supply of antibiotics is regulated in principle by well-established legislation and antibiotics should only be dispensed with a valid prescription written by a medical practitioner.14Many individual surveillance studies have investigated the local prevalence of AMR in Pakistan, but unfortunately, overall comprehensive studies that would provide a national picture are lacking.Streptococcus pneumoniae and Haemophilus influenzae, the most commonly isolated respiratory pathogens in the community.15 Key features of the SOAR study are that it focuses only on these pathogens, and that identification and susceptibility testing are performed in an independent centralized laboratory using standardized methodology (CLSI) allowing for comparisons to be made between countries or regions and for the identification of trends over time. SOAR data is analysed based on three different breakpoints: CLSI, EUCAST dose-specific and PK/PD breakpoints.The Survey of Antibiotic Resistance (SOAR), a multinational antibiotic surveillance study, has been run in a range of countries including Pakistan since 2002. The study aims to collect and make available in published, peer-reviewed papers, antibiotic susceptibility data, specifically for 16 Two international organizations define breakpoint values, namely CLSI and EUCAST. Due to variation in criteria for their definition, there are some differences between CLSI and EUCAST in the clinical breakpoint values for certain bacteria for some antibiotics and this can impact susceptibility interpretation of clinical isolates.17 EUCAST breakpoints are dose-specific and use the EMA-approved doses that are included in the Summary of Product Characteristics of an antibiotic. This means that by application of breakpoints for higher doses, the effect of using a raised dose on the clinical efficacy of a particular antibiotic can be predicted. Currently, clinical microbiology laboratories in Pakistan use CLSI breakpoints, however the international application of the EUCAST breakpoints is expanding18 so it is possible that dose-specific breakpoints could also be applied in Pakistan in future. The EUCAST dose-specific breakpoints can also be used retrospectively to calculate the susceptibility of previously collected isolates to show the susceptibility levels that would have been achieved at higher doses.Clinical breakpoints are cut-off MIC values used to classify microorganisms into the clinical categories susceptible (S), intermediate (I) and resistant (R) in order to assist in the prediction of the clinical success or failure of a specific antibiotic.S. pneumoniae, the most isolated respiratory pathogen20 for clinical indications such as CAP, AOM and ABRS, has over time become less susceptible to amoxicillin/clavulanic acid in some countries21 since the MICs of some isolates have increased. When treating infections, it is important to be able to eradicate bacterial pathogens with raised MICs to optimize clinical outcome while at the same time minimizing the risk of selecting variants with even higher MICs. This is possible because β-lactams, unlike many other antibiotics, have time-dependent killing properties. Their efficacy depends on the amount of time the drug concentration is present at the site of action. Although increasing the concentration at the infection site over a particular concentration will not have any effect on the efficacy, the use of higher doses and/or more-frequent dosing allows for successful eradication of pathogens with higher MICs because the time above the MIC is increased.22Use of the EUCAST dose-specific breakpoints shows the effect of increasing the antibiotic dose on the susceptibility of a pathogen, providing additional information so the prescriber can decide if a higher dose would be of benefit. For example, S. pneumoniae in Pakistan over four different time periods from 2002 until 2017. These data suggest that whilst the susceptibility of S. pneumoniae isolates remains high to amoxicillin, amoxicillin/clavulanic acid, cefuroxime, cefpodoxime, ceftriaxone and moxifloxacin, for penicillin and cefaclor, the susceptibility decreased with time so that in the 2015–17 study, the levels of susceptibility were 23.4%, 28.7%, respectively. Susceptibility for the macrolide antibiotics also decreased so that by 2015–17 the level was 33% 23Figure H. influenzae isolates collected in Pakistan in 2007–17 remained highly susceptible to most of the antibiotics tested  and 2017 (n = 8). ATLAS data is analysed based on CLSI and EUCAST breakpoints.The Antimicrobial Testing Leadership and Surveillance (ATLAS) database is a global AMR surveillance programme which is fully accessible and covers susceptibilities of a range of bacterial and fungal pathogens to a bank of antimicrobials with reference to the different breakpoints.25 to provide the supporting data required to ensure that countries can design cost-effective, evidence-based AMR response strategies. During the first four years, 91 countries/territories enrolled in GLASS and data for over two million patients from 66 countries are included.26 Pathogens currently included in GLASS-AMR are: Acinetobacter spp., Escherichia coli, Klebsiella pneumoniae, Neisseria gonorrhoeae, Salmonella spp., Shigella spp., Staphylococcus aureus, and S. pneumoniae. GLASS data is analysed based on CLSI and EUCAST breakpoints. A new and important component is the inclusion of antimicrobial consumption (AMC) surveillance at the national level.27In 2015, WHO launched the Global Antimicrobial Resistance and Use Surveillance System (GLASS). GLASS is a global system that collects national AMR data for selected bacterial pathogens that cause common infections. The aim is to monitor the prevalence of AMR among major pathogens in clinical settings27Pakistan started AMR surveillance with enrolment in GLASS in 2017. In 2019, the National Surveillance System was established, the primary goal of which is to generate evidence on the burden of AMR amongst priority pathogens. According to the May 2021 report, 40 surveillance sites are participating in the national surveillance system, these sites include 19 hospitals, one outpatient facility and 20 in/outpatient facilities.For management of the common RTIs, CAP, AOM and ABRS in Pakistan, clinicians use several country-specific local antibiotic prescribing guidelines plus a range of international guidelines as shown in Table 32, the 2011 and 2019 IDSA guidelines35 and additionally those from the IDSA/American Thoracic Society published in 2007.29 For example, a first-line antibiotic treatment recommendation for CAP management from the IDSA 2019 guideline for treating adults with no comorbidities or risk factors for MRSA or Pseudomonas aeruginosa is amoxicillin or doxycycline or a macrolide  but if the patient has comorbidities, the recommendation is combination therapy with amoxicillin/clavulanic acid or a cephalosporin plus a macrolide or doxycycline, or monotherapy with a respiratory fluoroquinolone. The recommended dosage for adults with comorbidities is amoxicillin/clavulanic acid, 500 mg/125 mg given three times daily, 875 mg/125 mg or 2000 mg/125 mg both given twice daily in combination with a macrolide or doxycycline.35 The BTS also suggests amoxicillin/clavulanic acid, cefaclor, and macrolides as an alternative to first-line amoxicillin in children with low-severity pneumonia.32 For AOM management, the international guidelines referred to in Pakistan include those from the American Academy of Pediatrics (AAP)34 and for the management of ABRS in adults and paediatrics, the international guidelines referred to in Pakistan are from the IDSA.33 For ABRS in children, the IDSA recommends as initial empirical treatment first-line, amoxicillin/clavulanic acid 45 mg/kg/day twice daily; and in adults first-line amoxicillin/clavulanic acid 500 mg/125 mg three times daily or 875 mg/125 mg twice daily.33For CAP management in adults and paediatrics, the international guidelines referred to by clinicians in Pakistan include the 2009 and 2011 British Thoracic Society (BTS) guidelines28 are commonly referred to. For AOM, the MMIDSP guidelines recommend as the preferred antibiotic, in adults amoxicillin/clavulanic acid 1 g given twice daily and in children amoxicillin/clavulanic acid 20–45 mg/kg/day every 8–12 h (the dose can be up to 80–90 mg/kg). For ABRS, the preferred antibiotic treatment in adults is amoxicillin 500 mg given three times daily or amoxicillin/clavulanic acid 625 mg given three times daily.Considering local Pakistan guidelines for the management of CAP, AOM and ABRS, the Medical Microbiology Infectious Disease Society of Pakistan’s (MMIDSP) Guidelines for Antimicrobial Use37Access to antibiotics may be a challenge for patients in LMICs due to cost and insufficient government expenditure or support in this area. Unreliable drug supply may also contribute to the problem. Limited access to the most-appropriate antibiotic to treat a specific infection may result in raised mortality from treatable bacterial infections and, the use of suboptimal amounts of antibiotic facilitates resistance development and allows resistant strains to persist.38 and the spread of drug-resistant infections. Since poor-quality antibiotics are unlikely to contain the full dose needed to eliminate all of the infecting pathogens, use of these will encourage resistance to develop and allow resistant strains to survive and be transmitted.39Substandard poor-quality or falsified antibiotics promote AMR39 GSMS aims to work with WHO member states to improve the quality of reporting of substandard and falsified medical products, and, importantly, to ensure the data collected are analysed and used to influence policy, procedure, and processes to protect public health, at the national, regional and the global level. Use of substandard or falsified antibiotics not only compromises clinical outcome but also risks increased AMR. The most recent GSMS summary (2013–17) reported substandard and falsified medicines in 46 member states (including Pakistan). Antibiotics represent 16.9% of all products reported, second only to malaria drugs (19.6%).The quality of medicines, specifically antibiotics, is an important consideration for countries worldwide. WHO launched a Global Surveillance and Monitoring System (GSMS) for substandard and falsified products.35 The same recommendation is made in the IDSA 2012 guidelines as an initial empirical treatment for rhinosinusitis in adults and additionally by the MMIDSP’s Guidelines for Antimicrobial Use28 for rhinosinusitis in adults.In Pakistan, several formulations of amoxicillin/clavulanic acid currently available are mentioned as first- or second-line recommendations by the RTI management guidelines. Examples include amoxicillin/clavulanic acid 500 mg/125 mg three times daily (with either a macrolide or doxycycline) which is recommended in the IDSA 2019 guidelines for the treatment of CAP in adult outpatients with comorbidities.33In terms of management of infection in children, amoxicillin/clavulanic acid 45 mg/kg/day twice daily is recommended by IDSA 2012 as an initial empirical therapy for rhinosinusitis.In Pakistan, public health amenities such as clean drinking water, proper sanitation, and infection control practices are mostly lacking which not only gives rise to an increase in the incidence of communicable diseases but also increased antibiotic consumption and a resulting rise in resistance. Most classes of antibiotics are sold OTC, which often leads to misuse and overuse. Sale of counterfeit drugs and use of broad-spectrum antibiotics without the appropriate indication are also very common. In addition, Pakistan lacks good quality diagnostic facilities and credible data that would help ascertain the scale of prevailing AMR. Lack of some, or all, of the following factors leads to increasing AMR: access to appropriate antimicrobial therapy; regulation of antibiotic use in humans, agriculture and livestock; surveillance of antibiotic use and resistance; updated antibiotic use and treatment guidelines; continuing medical education on antibiotic use for prescribers; microbiological laboratory capacity and training; and diagnostic tools. Increasing AMR complicates the management of treatable infectious diseases such as malaria, tuberculosis, acute RTIs, and diarrhoeal diseases.Salmonella typhi. Emergence of microbes developing resistance against last resort antibiotics is a major health challenge. Positive gains could be made if practices were modified with the prudent use of antibiotics including using narrow-spectrum antibiotics and recommending the right antibiotic at the right dose, at the right time and for the right duration. Monitoring antibiotic misuse in livestock and agriculture is also important.The prevailing conditions in Pakistan have already led to the emergence of carbapenem-resistant organisms along with the ceftriaxone-resistant 40 highlighting the main burdens caused by malaria, tuberculosis, RTIs (upper and lower), and diarrhoeal diseases.It is difficult to ascertain the burden of infectious diseases in a country as large as Pakistan, especially with a lack of local surveillance studies, however a 5 year audit (published in 2018) conducted at a tertiary care hospital in Karachi, Pakistan gives a snapshot of the commonly occurring infectious diseasesSurveillance of AMR of locally prevalent microorganisms is also lacking in Pakistan and would be required to guide an infection prevention and control programme, which ultimately would help in the reduction and spread of multiresistant organisms. Surveillance studies that monitor antibiotic consumption can further guide improvements in antibiotic use and ease of availability, provided they are used judiciously. In 2015, WHO launched GLASS, the first global collaborative effort to standardize AMR surveillance. However, it has been challenging for NIH Pakistan to recruit centres to participate, and more focused, localized as well as disease-specific surveillance studies are also required.There is also a need for a unified and nationally endorsed set of treatment guidelines for communicable diseases, such as upper and lower RTIs along with gastrointestinal infections. The MMIDSP management guidelines for adults as well as children were released in 2019, but dissemination and acceptance still needs full realization. Although international treatment guidelines exist and are already in use in various centres around Pakistan, they are not tailor-made for local settings, especially with the variable prevalent resistance patterns. Moreover, many antibiotics are not available in Pakistan, which makes adherence to the international guidelines more difficult.The main daily challenges faced within the clinical microbiology laboratory during AST reporting to a physician include explaining why results of certain antibiotics were withheld and, importantly, when the organism is pan-resistant and there are no alternative antibiotics to offer. Additional challenges include explaining why it is better to aim for a narrow- rather than a broad-spectrum antibiotic. Possible solutions to bridging communication between clinical microbiologists and physicians could be to discuss results by telephone or on the clinical ward rounds and to have orientation sessions for physicians concerning microbiology and infectious diseases. In addition, online forums could be useful for posting questions and responses.Antibiotic prescribing practices are inconsistent in Pakistan. The reasons for this are multiple and include the quality of undergraduate medical education, a lack of opportunities for postgraduate training, the unregulated and fragmented healthcare system, poor diagnostics, the scarceness of quality microbiology laboratories, (hence non-availability or underutilization of culture and AST) unrestricted OTC antibiotic availability, extensive veterinary use and unregulated waste disposal into natural bodies of water. All of these factors contribute to the relentless upward trajectory of AMR and poor patient outcomes.41 The website gives extremely useful information on laboratory diagnostics and susceptibility of major bacterial pathogens in Pakistan and is a useful resource for guiding empirical and definitive therapy for various conditions.Antibiotic susceptibility data are available from a few, select institutions in Pakistan, on an annual or biannual basis, on a website created for this purpose .28 The Society, through its various academic and awareness activities, has been actively involved in imparting basic knowledge about antibiotic choice and use in different clinical situations. Prominent MMIDSP members are leading infection, prevention and control (IPC) and AMR activities in Pakistan and the Society also aims to engage pharmacists to promote antibiotic stewardship activities.Guidelines on the use of antibiotics released by MMIDSP are in the process of revision.Clinical guidelines are generally useful. However, in the context of Pakistan, one should be cognizant of the local susceptibility data before ‘adopting’ the guidelines in totality. The logical process would be that of ‘adaptation’ of guidelines based on local disease spectrum and susceptibility patterns.To optimize use of antibiotics in Pakistan, with the objectives of improving patient outcomes, reducing cost and preventing rising AMR, the approach has to be multipronged. In addition to improving training of doctors , microbiology laboratory systems have to be strengthened. There has to be a sustained move towards establishing aetiology early in the course of infection, rather than consistently relying on prolonged empirical use of broad-spectrum antibiotics. Susceptibility data should be made easily available to general practitioners along with instructions on how to interpret and use the data appropriately for the benefit of our patients, both in terms of cost and outcomes.In an era of rising AMR throughout the world, this paper aims to define areas where action is required to address AMR by analysing and understanding the current situation within Pakistan. Information is presented for Pakistan concerning antibiotic use and prescribing, approach to AMR, availability of local susceptibility data, use of international and/or local management guidelines, and how these link to antibiotic availability. To our knowledge this is the first time this information has been reviewed and presented in detail by country.Antibiotic use in Pakistan is extremely high and although legislation exists prohibiting the OTC purchase of antibiotics, this continues to be a major problem. In terms of surveillance, there are a small number of studies from individual units or hospitals which have provided information on local AMR. Several global surveillance studies also include data for Pakistan (SOAR and ATLAS). The WHO GLASS study will also provide useful data in future. The surveillance data that do exist show a trend towards decreasing susceptibility amongst the common respiratory pathogens for some antibiotic classes such as the macrolides and oral penicillin, but amoxicillin/clavulanic acid and fluoroquinolone have so far retained their activity, although guidelines and regulatory bodies urge caution with the fluoroquinolones, restricting their use to limited situations due to serious safety concerns.For management of the common RTIs in Pakistan, clinicians make use of several country-specific local antibiotic prescribing guidelines plus a wide range of international antibiotic prescribing guidelines. For the management of CA-RTIs, amoxicillin/clavulanic acid is a commonly recommended antibiotic amongst both international and local prescribing guidelines, and its choice is confirmed by currently available susceptibility data.2 This would pave the way for improved adherence and a higher level of appropriate antibiotic prescribing in CA-RTIs, which could, in turn, potentially limit AMR development and improve clinical outcomes for patients.The clinical microbiologist and clinician view in Pakistan is in alignment in the wish for local up-to-date susceptibility data to support empirical antibiotic prescribing, since national data for a country as large as Pakistan is unlikely to be representative of the local situation. An increase in local surveillance studies would lead to the development of regularly updated management guidelines which, if applied along with updated guidelines, for example those relating to the management of COVID-19, would improve clinical outcomes and minimize further rises in AMR. A more standardized inclusive approach is needed to develop local Pakistan-specific guidelines. These guidelines would be based on up-to-date surveillance data of isolates from community-acquired infections which would make them more locally relevant for clinicians, reiterating the Consensus Principles as described in the introductory paper to this Supplement.


Establishment of an in vitro osteoclast induction model under nuclear factor-Cathepsin K, osteoclast associated receptor (OSCAR), and TRAP, and the effect of Met on Mitogen-activated protein kinases (MAPK) signaling pathway was detected by Western blot.  RANKL induced the differentiation of mouse bone marrow macrophages (BMMs) into osteoclasts in vitro, and Met was added at different concentrations for intervention during the induction process. After 5 d of culture and fixation, the number of osteoclasts was counted by tartrate-resistant acid phosphatase (TRAP) staining and F-actin staining, and the function of osteoclasts was examined with hydroxyapatite-coated plates. Real-time fluorescence quantitative PCR was performed to detect the expression of Cathepsin K, OSCAR, and TRAP. The Western blotting study demonstrated that Met inhibited the MAPK signaling pathway by decreasing the phosphorylation of extracellular regulated protein kinase (ERK), which plays important roles in osteoclast formation.  Met significantly reduced osteoclast formation, F-actin ring formation, bone resorption, and the expression of relevant genes  Metformin hydrochloride inhibited the differentiation of osteoclasts, decreased the bone resorption area, and suppressed phosphorylation of ERK in vitro. Bone reconstruction in the human body includes two mutually balanced processes of bone formation and bone resorption, which are regulated by osteoblasts and osteoclasts, respectively . ExcessiCurrently, it has been found that all types of diabetes increase the risk of osteoporosis . The useBMMs were isolated as previously described by our work . Brieflyµg/mL streptomycin , and 50 ng/mL of macrophage colony-stimulating factor . When the cells were grown to 90% fusion, the adnexal cells were gently scraped off with a cell scraper and these were considered to be BMMs.Centrifuge the rinse solution to collect the cells and then incubate in Dulbecco's Modified Eagle Medium  containing 10% fetal bovine serum , 100 IU/mL penicillin, 100 3 cells/well) were seeded in a 96-well plate and incubated with M-CSF (50 ng/mL) and RANKL . Subsequently, cells were treated with the indicated doses of Met  for 72 h. Then, the culture solution in each well was discarded and DMEM culture solution (without FBS) containing 10 μg CCK-8 assay solution was added and incubated for 2 h. The absorbance at 450 nm was then measured by a microplate reader , charted, and statistically analyzed.To evaluate the Met-dose used in this research without toxic effects on BMMs, a cell viability assay (cell counting kit-8 (CCK-8); Jiangsu KeyGen Biotech Co., Ltd., Nanjing, China) was performed. BMMs (5 × 10μM), and the culture medium was changed every 2 d for a total of 5 d.BMMs were induced to become mature osteoclasts by RANKL in 96-well plates with 8000 cells per well containing 2 mL of complete medium, 50 ng/mL M-CSF, and 100 ng/mL RANKL with different concentrations of Met .F-actin: After 5 d, the cells were fixed for 10–15 min. The fixative was discarded, and the cell was rinsed with PBS and permeabilized with 0.1% (v/v) Triton X-100 for 5 min. Subsequently add 1 : 100 dilution of Alexa-Fluor 647 phalloidin  and incubate for 1 h at room temperature. Then, BMMs were rinsed three times with PBS, and the nuclei were counterstained with DAPI. Photograph and observe the stained cells under a confocal microscope .4 cells per well containing 2 mL of complete medium, 50 ng/mL M-CSF, and 100 ng/mL RANKL were added at different concentrations of Met (200 and 400 μM). After 5 d of incubation, the cells were washed off the plates with 10% bleach solution, and then, the bone resorption traps were photographed with a microscope and the bone resorption area was calculated by Image J .BMMs were induced to become osteoclasts by RANKL in a 6-well plate, and the cells were obtained by digestion and seeded on a bone resorption plate covered with hydroxy-limestone . 2.4 × 104 cells per well, containing 2 mL complete medium, 50 ng/mL M-CSF, and 100 ng/mL RANKL with different concentrations of Met (200 and 400 μM). The solution was changed every other day and cultured for 5 d. We used TRIzol reagent  to extract total ribonucleic acid (RNA) from the cells, which was followed by RNA reverse transcription using the Revert Aid First Strand cDNA Synthesis kit (Thermo Fisher) according to the manufacturer's instructions and amplification using a suitable primer as follows: Cathepsin K (forward 5′-GGGAGAAAAACCTGAAG-3′ and reverse: 5′-ATTCTGGGGACTCAGAGAGC-3′); OSCAR (forward: 5′- CTGCTGGTAACGGATCAGCTCCCCAGA -3′ and reverse 5′-CCAAGGAGCCAGAACCTTCGAAACT-3′); TRAP (forward 5′-TGTGGCCATCTTTATGCT-3′ and reverse 5′-GTCATTTCTTTGGGGCTT-3′); and GAPDH (forward: 5′-CCTCGTCCCGTAGACAAAATG-3′ and reverse 5′-TGAGGTCAATGAAGGGGTCGT-3′). GAPDH was used as an internal reference to determine the messenger RNA (mRNA) levels.Osteoclast-related genes were detected by real-time fluorescence quantitative PCR. BMMs were inoculated in 6-well plates with 2.4 × 104 cells per well, containing 2 mL of complete medium, 50 ng/mL M-CSF, and 100 ng/mL RANKL, and different concentrations of metformin hydrochloride (200 and 400 μM) were added. The medium was changed every other day and discarded after 5 d of incubation. Cells were lysed by shaking in radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with protease inhibitor . The bicinchoninic acid (BCA) assay (Jiangsu KeyGen Biotech) measured the protein concentration. Cell extract (30 μg) was separated using 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes; 5% skim milk dissolved in Tris-buffered saline (TBS)-Tween  was used to block the membranes for 1 h. The membrane was incubated overnight at 4°C with primary antibodies .BMMs were inoculated in 6-well plates with 2.4 × 10β-Actin was used as the internal reference.On the next day, the membrane was washed three times with PBST and incubated with HRP-antirabbit secondary antibody  for 1 h. The bands were visualized by a BIO-RAD imaging system . t-test. All statistical analyses were performed using the SPSS 22.0 , and ∗p < 0.05 was considered significant.Measured data were analyzed using analysis of variance (ANOVA). The difference between the two groups was analyzed using Student's μM, according to the results (BMMs were cultured to see how Met affected osteoclastogenesis. We used a CCK-8 test to determine Met's cytotoxicity before confirming the safe dose. Met did not affect BMMs viability at concentrations less than 400  results .μM), and TRAP and F-actin tests were performed to see how Met affected osteoclastogenesis. Met reduced RANKL-stimulated osteoclastogenesis in a dose-dependent manner, as evidenced by the considerably smaller area of TRAP-positive osteoclasts and F-actin ring in Met-treated groups compared to Sham-treated groups  were used to interfere with osteoclast differentiation, and then, the expression of osteoclast-related genes  was examined. The results showed that Met inhibited osteoclast-specific genes  at 200 μM and 400 μM. This inhibition was concentration-dependent  to interfere with osteoclast differentiation, and then, the effect of Met on the MAPK signaling pathway was examined. According to the findings by collecting the cell proteins of each group after 5 d of Met treatment, Met had a limited effect on the activation of the MAPK subfamilies JNK and p38, according to the findings. The degree of phosphorylated ERK in the Met-treated group was much lower, and the impact was concentration-dependent, indicating that Met may block ERK phosphorylation (Met was used at different concentrations (200 and 400 rylation .With the aging of the world population, osteoporosis has become an important disease affecting the quality of life, health, and safety of life of the global population. In diabetic patients, osteoporosis was first reported in 1948 and was named diabetoporosis in 2015 [Met is currently the first-line drug for the treatment of diabetes mellitus, with high efficiency and low side effects. Current literature reports that Met has anti-inflammatory, antitumor, hypolipidemic, and cardioprotective effects in addition to hypoglycemic effects . It is wCathepsin K, OSCAR, and TRAP. The mRNA levels of these genes were elevated after stimulation with RANKL for 5 days, whereas simultaneous treatment with Met inhibited this increase in the Cathepsin K, OSCAR, and TRAP.Bone tissue is in a dynamic balance between osteoblasts leading to bone formation and osteoclasts leading to bone resorption. OPG, RANKL, and RANK are the three main cytokines that couple the differentiation and activation of osteoblasts, and osteoclasts play a very important regulatory role in the process of bone metabolism . OsteoclTRAP, CTSK, and OSCAR in a dose-dependent way. These impressive findings imply that Met suppresses phosphorylation of the ERK signaling pathway during osteoclastogenesis while having a limited effect on p38 and JNK signaling pathway activation.We next investigate the underlying mechanisms of Met in osteoclast formation by Western blotting. Met inhibited the RANKL-induced ERK signaling pathway but had a very limited effect on p38 or the JNK signaling pathway, according to our data. MAPKs are a group of serine-threonine protein kinases that regulate inflammatory and innate immune responses . The MAPDespite these intriguing results, our research has certain limitations. First, bone metabolism is a delicate balance between osteoclastic bone loss and osteoblastic bone production . Bone prIn conclusion, the present study demonstrated that Met suppressed the ERK signaling pathway, which decreased osteoclastogenesis and the expression of osteoclastogenesis-related genes. This study suggests that Met might be an effective therapy for osteoporosis .


A 67-year-old man was admitted to our cardiology ward for a diagnostic work-up.Panels A and B) and multidetector computed tomography coronary angiography  revealed an aneurysm of the proximal segment of the left anterior descending artery.Coronary angiography  is an infrequent finding and an aetiologic diagnosis is crucial for the treatment selection.A comprehensive diagnostic work-up revealed elevated IgG4 levels  that, according to medical history and histological findings, confirmed the diagnosis of IgG4-related disease (IgG4-RD).Panels E and F), highlighting the importance to consider conservative approaches for this condition.The patient was treated with corticosteroids and anticoagulants. One-year follow-up CTCA showed unchanged findings (2 The history of pancreatitis, sialadenitis, dacryoadenitis and lymphadenopathy in a patient with CAA should alert cardiologists to suspect IgG4-RD.IgG4-RD often presents with pancreatitis, dacryoadenitis and sialadenitis but clinical manifestations are recognized in every organ system, including coronary arteries. Glucocorticoids are the first-line treatment of IgG4-RD, potentially resulting in disease remission.Guarantor: The scientific guarantor of this publication is Antonio Iaconelli as corresponding author.Statistics and biometry: No complex statistical methods were necessary for this paper.Informed consent: Written informed consent was obtained from the patient involved in this study.Methodology: Case.Funding: None declared.


The World Health Organization expressed concern that antimicrobial resistance would increase during the COVID-19 pandemic due to the excessive use of antibiotics. This study aimed to explore if antibiotic prescribing patterns in general practices located in the North Denmark Region changed during the COVID-19 pandemic. The study was conducted as a registry-based study. Data was collected for every antibiotic prescription issued in general practices located in the North Denmark Region during the first year of the pandemic (1 February 2020 to 31 January 2021) and the year prior to the pandemic (1 February 2019 to 31 January 2020). Data were compared regarding antibiotic agents and the type of consultation linked to each antibiotic prescription. Results showed that antibiotic prescriptions decreased by 18.5% during the first pandemic year. The use of macrolides and lincosamides, along with combinations of penicillins and beta-lactamase -sensitive penicillins, was reduced the most. Face-to-face consultations related to an antibiotic prescription decreased by 28.5%, while the use of video consultations increased markedly. In Denmark, COVID-19 restrictions have contributed to both a lower consumption of antibiotics and a change in prescription patterns in general practice. Probably some of the COVID-19 -preventing initiatives could be of importance moving forward in the fight against antimicrobial resistance. The discovery of antibiotics in 1928 became a revolution in modern medicine . Any antFor more than a decade, antibiotic use in the Danish primary healthcare sector has steadily decreased from 17.05 DID (2011) to 12.83 DID (2020) ,7. In adThe reduction in overall antibiotic use contradicts WHO’s anticipations that AMR would rise during the pandemic due to inappropriate use of antibiotics . The redThis study aimed to describe if the antibiotic prescribing patterns in general practices located in the North Denmark Region changed during the COVID-19 pandemic compared to one year prior.p-value < 0.0001). This finding corresponds to an overall decrease of 18.5% .The distribution among the various types of antibiotics remained the same during both observation periods despite an overall reduction in the number of antibiotic prescriptions issued during the two periods , while a minor increase in the use of nitrofurantoin (J01XE) was observed. The large reduction in the use of macrolides and lincosamides can perhaps be explained by a change in the Danish venereology guidelines in 2019 for treating pandemic ,19. The Combinations of penicillins (J01CR) and beta-lactamase sensitive penicillins (J01CE) also decreased considerably during the pandemic, which must be directly linked to the COVID-19 initiatives introduced to minimise acute respiratory tract infections, where both antibiotic agents are often prescribed in Danish general practice. A finding of great potential moving forward.The consultation types related to antibiotic prescribing changed significantly during the pandemic. Face-to-face consultations decreased—yet remained the most frequent consultation type related to an antibiotic prescription. The use of video consultations increased markedly during the pandemic.The findings of this study are in line with studies from other countries, including studies from the Netherlands ,16,17,18This study demonstrates an 18.5% reduction in the use of antibiotics in general practices in the North Denmark Region during the COVID-19 pandemic. Interestingly, the Danish national surveillance system DANMAP reports a reduction of only 6.8% in the overall antibiotic consumption in the Danish primary healthcare sector in 2020 compared to 2019 . This diHaemophilus influenza and Streptococcus pneumoniae, where a reduction in invasive infections was reported dismissing the matter in question [The reduced use of antibiotics in general practice during the COVID-19 pandemic can be explained by both the restrictions introduced in society and on a personal level, especially including social distancing to prevent infective transmission. Other initiatives, such as the use of hand sanitisers and face masks, may also have contributed to diminishing infections, although it is questionable if the effect of face masks is sufficient in preventing virus transmission ,25. The question .A marked change in the type of consultation linked to the prescribing of antibiotics was seen. The most obvious explanation is that most patients with symptoms and signs of acute infection were booked for a video consultation instead of a face-to-face consultation. Furthermore, telephone consultations linked to an antibiotic prescription increased during the pandemic, consistent with the total number of telephone consultations increasing significantly in the entire primary healthcare sector . In addiA 7% increase in the total number of consultations in Danish general practice in 2020 compared to 2019 has been reported [This study included information exclusively from general practices in the North Denmark Region issuing one or more antibiotic prescriptions within the study period. When interpreting the results of this study, some limitations must be kept in mind. The study measured antibiotic consumption by the number of prescriptions issued by a GP. This method does not accommodate the amount or dose of antibiotic used, which is an important element when considering the increasing problems with resistant bacteria. The use of Defined Daily Doses per 1000 inhabitants per day (DID) could have been implemented to accommodate this limitation. However, an assumption can be made that the same guidelines for treating infections in Danish general practice have been followed during the two periods, and, thereby, prescriptions for the same infectious diseases are alike. Due to the data source (the North Denmark Region prescription database), it is not possible to obtain information about whether patients redeemed their prescriptions at the pharmacies. Nor whether the patients were compliant in administering the antibiotics as prescribed by the GP. Finally, information about consultation type was not available for all antibiotic prescriptions resulting in a rather large proportion of missing data for this outcome.COVID-19 restrictions have contributed to lower consumption of antibiotic agents by reducing viral and bacterial transmission in society. Antibiotic consumption can potentially remain at this low level if restrictions and behaviours are continued beyond the pandemic. However, several restrictions are hard to implement permanently, such as social distancing without a reduction in quality of life. Contrariwise, other restrictions such as frequent hand sanitising, applying face masks when having an acute respiratory tract infection, and staying or working from home when sick could be implementable in the future in order to prevent viral and/or bacterial transmission. Furthermore, initiatives in general practices could be implemented beyond the pandemic, such as, for example, the use of video consultations for patients with symptoms of acute infection and thereby continue to minimise bacterial and viral transmission to both medical professionals and other patients. By January 2022, video consultations will be established as a permanent solution and option for consultations in Danish general practice ,30.Lastly, it would be very relevant to also explore how medical specialists other than GPs in the Danish primary healthcare sector have prescribed antibiotics during the COVID-19 pandemic. Not only to estimate the exact numbers for the different settings but also to clarify the exact contribution of Danish general practice in the observed overall reduction of antibiotic use in the Danish primary healthcare sector.The study was conducted as a register-based study, including antibiotic prescriptions issued by GPs in the North Denmark Region between 1 February 2019 to 31 January 2021. Data was extracted from the North Denmark Region prescription database and pseudonymised before being handed over. Antibiotic agents were classified according to the Anatomical Therapeutic Chemical (ATC) system, and both J01  and P01AB01 (metronidazole) were included. Information about each prescription , patient , and date of any type of consultation in general practice  was obtained. The consultation type related to each of the antibiotic prescriptions was found in relation to the antibiotic prescription date by noting any consultation performed within three days (+/−) of the issue. Since prescriptions were noted for patients based on their Central Person Registration number (CPR-number), antibiotics for foreigners were not included in the study. Information about whether the prescription was redeemed at a pharmacy was not available.To describe the prescribing patterns, data were divided into two comparable periods; a pre-pandemic period running from 1 February 2019 to 31 January 2020 and a pandemic period accounting for 1 February 2020 to 31 January 2021 .The number of prescriptions was calculated on a monthly basis, and the difference was noted as percentages. Data were normally distributed, and a paired t-test was conducted to decide if the variation was statistically significant for the two periods. Changes in the distribution of prescriptions within the various groups of antibiotics were determined by using a paired t-test. Missing data were handled by pairwise deletion, as it was not possible to identify a consultation type in relation to all antibiotic prescriptions. Data were analysed using the program IBM SPSS Software version 27.0 .In conclusion, this study found that the COVID-19 pandemic has had profound impact on antibiotic prescribing patterns in general practices located in the North Denmark Region. This finding indicates that COVID-19 prevention initiatives can probably be of importance moving forward in the fight against increasing problems with antimicrobial resistance. No evidence of an increase in observed invasive infections during the COVID-19 pandemic has been reported, implicating a safe reduction in antibiotic prescribing.


Gender disparities exist in academia and are disproportionately affecting females. We conducted a cross‐sectional study to analyze gender disparities in multiple myeloma (MM) publications. A total of 679 publications with 8898 authorships were analyzed. The mean number of authors for females vs. males, per publication, was 4.4 and 8.7, respectively. Females constituted a third of the total authors. Female first authors, corresponding authors, and last/senior authors were 34%, 21%, and 18%, respectively. Note that, 17% of authors of clinical trial publications were females. Gender disparities in MM publications exist and are more obvious in the last/corresponding authorship. Efforts should be made to identify factors that contribute to these disparities and work to resolve them. TherefoPrevious studies have reported significant gender disparities and underrepresentation of women in medical peer‐reviewed publications which may result in job dissatisfaction and burnout , 3. Wome2Data on MM publications were collected through Medline/PubMed database search and filtered to only include articles from specific journals in the studied period. The following search items were used: “multiple myeloma”, “myeloma”, and “plasma cell dyscrasia”. Top journals in hematology/oncology, determined by an impact factor greater than 10, were included. Impact factor data was based on 2016 InCites Journal Citation Reports, an annual publication by Clarivate Analytics. We included all journals with an impact factor ≥10 with at least 10 MM‐related publications in the five years studied period. The selected journals include: the New England Journal of Medicine, Journal of Clinical Oncology, The Lancet, The Lancet Hematology, The Lancet Oncology, Blood, Nature Communications, Journal of Clinical Investigation, Nature Reviews of Clinical Oncology, Leukemia, and JAMA Oncology.3n = 28) and specific publications types  due to low research significance (n = 22). Of the remaining 700 publications, the following variables were collected: year of publication, field , type of article , number of authors, number of male authors, number of female authors, gender(s) of first author(s), gender(s) of last author(s), gender(s) of corresponding author(s), country of the first author, and country of the last author.Our search yielded 750 publications which we further refined by excluding publications unrelated to multiple myeloma (n = 21) were excluded from our analysis. Per inclusion criteria, a total of 679 publications with 8898 authorships were included in our analysis. [Figure To determine the gender of the authors, we used Genderize, a validated database used in prior research to assess gender disparities in medical research publications , 10. GenThe average number of authors per publication was 13 . Females constituted around one third of total authors across all publications in the studied period. Females as first authors, corresponding authors and last/senior authors were 34%, 21%, and 18%, respectively. Figure Original investigations and clinical trial publications constituted 61% of all publications in the studied period. Only 17% of authors of clinical trial publications were females. The proportion of females in original investigations publications was higher at 35%. The lower proportion of female authors was similar in basic vs. clinical research . Data related to publication by country also revealed comparable percentages .The proportion of female authorship was similar with no change in 2020 when compared to 2016 (both at 33%). There was a slight improvement in females as first authors (32%–37% from 2016–2020), last authors (16%–21% from 2016–2020), and corresponding authors (17%–22% from 2016–2020).4Our findings showed gender disparity in MM publications. Gender disparities were previously reported in other fields of medicine including in the hematology/oncology field , 13. FemOur analysis has some limitations. We only included publications of MM and we did not study the frequency of authors with multiple publications. Furthermore, the use of Genderize, an external database, to classify gender poses a risk of gender misclassification. Despite these limitations, our study provides a detailed analysis of all MM publications in medical journals focusing on the highest impact factor which has previously not been done.In conclusion, gender disparities exist in MM peer‐review publications both in the US and other countries especially in the last/corresponding authorship. There is a slight improvement in the proportion of female authors, however, this is far from optimal given the growing proportion of female physicians in the medical field. Such disparities may contribute to increased burnout encountered by female physicians and may result in loss of interest in academic positions. Efforts should be made to identify factors that contribute to these disparities at all levels and implement strategies to support females and eliminate gender disparities in academia.The authors received no specific funding for this work.Aala Dweik, Hadeel Dweik, Meera Mohan, and Samer Al Hadidi report no relevant conflict of interest. Hira Mian receives advisory fees from Janssen, GSK, Celgene/BMS, Takeda, Sanofi, and research funding from Janssen. Hira Mian is the recipient of an early career research award from Hamilton Health Sciences.Samer Al Hadidi conceived the research idea. Samer Al Hadidi, Aala Dweik, and Hadeel Dweik performed the initial literature review. Aala Dweik, Hadeel Dweik, and Samer Al Hadidi collected data, performed the statistical analysis, and wrote the initial draft of the manuscript. Hira Mian, Carolina Schinke, and Meera Mohan provided critical input on the methodology and analysis. All authors reviewed and approved the final version of the manuscript.