MET proto-oncogene that has a significant role in cancer cell progression. Several drugs targeting MET are under development for the treatment of different cancers, including non-small-cell lung cancer (NSCLC). However, until now, relatively few of these drugs have shown sufficient clinical activity and obtained regulatory approval. One of the reasons for this could be the lack of effective biomarkers to select the right patients for treatment. In a number of clinical trials, different biomarkers have been studied, but so far, MET exon 14 skipping mutation is the only one that has shown sufficient predictive properties. Another interesting biomarker is MET amplification detected by fluorescence in situ hybridization (FISH), which has shown promising results in the treatment of patients with NSCLC. Future clinical research will show whether MET amplification by FISH is an effective predictive biomarker for MET-targeted therapy.MET is a receptor tyrosine kinase encoded by the MET exon 14 skipping. An alternative predictive biomarker for MET therapy is MET amplification, which has been identified as a resistance mechanism in patients with EGFR-mutated NSCLC. Results obtained from different clinical trials seem to indicate that the MET/CEP7 ratio detected by FISH possesses the best predictive properties, likely because this method excludes MET amplification caused by polysomy. In this article, the concept of CDx assays will be discussed, with a focus on the currently FDA-approved MET targeted therapies for the treatment of NSCLC.Dysregulation of the MET tyrosine kinase receptor is a known oncogenic driver, and multiple genetic alterations can lead to a clinically relevant oncogenesis. Currently, a number of drugs targeting MET are under development as potential therapeutics for different cancer indications, including non-small cell lung cancer (NSCLC). However, relatively few of these drugs have shown sufficient clinical activity and obtained regulatory approval. One of the reasons for this could be the lack of effective predictive biomarkers to select the right patient populations for treatment. So far, capmatinib is the only MET-targeted drug approved with a companion diagnostic (CDx) assay, which is indicated for the treatment of metastatic NSCLC in patients having a mutation resulting in  By the end of 2021, this number was close to 50 [For more than 20 years, companion diagnostics (CDx) and predictive biomarkers have had a significant impact on the development of a number of targeted hematological and oncological drugs as well as their subsequent use in the clinic. The first drug to have a CDx linked to its use was the monoclonal antibody trastuzumab , which was approved for the treatment of HER2-positive metastatic breast cancer together with the immunohistochemical assay (IHC) HercepTestse to 50 . For mosMET was originally discovered as the transforming gene in a chemically transformed cell line derived from human osteosarcoma [MET is a proto-oncogene on chromosome 7q31, and it encodes a transmembrane receptor with intrinsic tyrosine kinase activity. The receptor tyrosine kinase is also called c-Met or hepatocyte growth factor receptor (HGFR) after its ligand, hepatocyte growth factor (HGF) [MET gene is special, as different genomic states such as amplification, mutation, and rearrangement can lead to a clinically relevant oncogenesis [MET exon 14 skipping (METex14) [osarcoma . Since tor (HGF) ,5,6. Dysogenesis . One of METex14) ,10. In tClinical Cancer Research [The successful development of trastuzumab for HER2-positive metastatic breast cancer opened the way for the drug-diagnostic co-development model, in which a predictive biomarker test is developed along with the drug. The use of a biomarker-guided clinical enrichment strategy often leads to an increased power of the individual clinical trial and a higher likelihood of a positive outcome . A few yResearch . In thisResearch . One of Research ,12. In 2014, the FDA issued the first guideline on In Vitro Companion Diagnostic Devices in which they officially defined a CDx assay . This deSeveral other countries worldwide, inducing the European Union (EU), have introduced similar definitions of a CDx assay as the FDA, and have tightened documentation requirements and regulatory approval procedures. In 2017, the European Parliament passed new regulations on In Vitro Diagnostic Medical Devices (IVDR) that will have a great impact on the development and use of CDx assays in European countries, as CE-IVD marking based on a self-declaration will no longer be possible . The newMET dysregulations can serve as primary drivers in promoting tumor growth, invasion, angiogenesis, and metastasis. NSCLC MET dysregulations in the form of MET amplification (METamp) have also been shown to act as secondary drivers that can mediate resistance to targeted therapy for other oncogenes such as epidermal growth factor receptor (EGFR) mutations [MET dysregulations have been found in 5% to 26% of NSCLC patients following treatment with an EGFR tyrosine kinase inhibitor (TKI) [ALK-rearranged NSCLC more than 10 years ago [Within different cancers, utations ,8,16. MEor (TKI) . The firears ago . SubsequCrizotinib is a small molecule inhibitor of receptor tyrosine kinases including ALK, MET, and ROS1, and belongs to the class Ia MET inhibitors ,18. DiffALK-rearrangement, which led to a regulatory approval of the drug by the FDA in 2011 [ROS1 rearrangement [ALK- and ROS1-rearrangements [ALK or ROS1 rearrangement, but when it comes to patients with MET dysregulations, the documentation is less convincing, and a regulatory approval for this indication has not yet been granted. The first indication in which crizotinib demonstrated important clinical activities was in metastatic NSCLC patients with  in 2011 ,21,22. Iangement ,23. For ngements . Today, METamp or an METex14 mutation, with objective response rates (ORR) in the range of 12% to 32% depending on the type of MET dysregulation [METex14 mutation was detected by next-generation sequencing (NGS) and METamp by fluorescence in situ hybridization (FISH). Different cut-off values were applied with regard to the detection of METamp by FISH. In one of the trials, the cut-off value was based on a MET/CEP7 ratio > 2.2, and in another trial, it was based on a MET gene copy number (GCN) ≥ 6. The results of the different reported MET trials with crizotinib are shown in A few clinical trials have shown varying activity of crizotinib in NSCLC patients with gulation ,25,26. IMETex14 mutation or METamp [METamp, as well as MET-mediated phosphorylation of the different downstream signaling proteins, which results in the impaired proliferation and survival of the MET-dependent tumor cells [Capmatinib is a small molecule kinase inhibitor that belongs to the MET-class Ib inhibitors . In murir METamp ,9. CapmaMET GCN ≥ 5 or MET/CEP7 ratio ≥ 2.0 by FISH. In addition, the METex14 mutation was detected in a small subset of patient samples using NGS. Overall, for all enrolled patients, an ORR of 20% was observed. For the subgroup of patients with a MET GCN ≥ 6 (n = 15), an ORR of 47% was shown. For the group of patients with MET overexpression as IHC3+ (n = 37), the ORR was 27%. METex14 mutation was detected in four patients, and all responded to treatment with capmatinib. For more results from the phase I trial, please see MET GCN ≥ 6 or an METex14 mutation. When it comes to MET overexpression by IHC, this method was not considered a reliable predictive biomarker for the efficacy of capmatinib [In a phase I trial, a possible biomarker enrichment strategy for capmatinib was investigated. Here, 55 pretreated metastatic NSCLC patients with MET dysregulation were treated with capmatinib as monotherapy . SeveralMETamp by FISH or MET overexpression by IHC and were treated with capmatinib plus gefitinib . For the phase Ib part, the patient selection criteria were either MET GCN ≥ 5 and/or a MET/CEP7 ratio ≥ 2.0 or MET overexpression as IHC2+ or IHC3+ in ≥ 50% of the tumor cells. For the phase II part, the selection criteria were initially defined as MET GCN ≥ 5 or MET IHC2+ or IHC3+ overexpression in ≥ 50% of tumor cells. However, in a protocol amendment, these criteria were revised to MET IHC2+ or IHC3+ plus MET GCN ≥ 5; these were subsequently changed once more to MET IHC3+ or MET GCN ≥ 4. Across the phase Ib/II trial and the different MET or MET selection criteria, the observed ORR was 27%. In a post-hoc subgroup analysis, an ORR of 47% was shown for phase II patients with MET GCN > 6 (n = 36). For patients with MET IHC3+ (n = 78), the ORR was 32%, and for the MET IHC2+ group (n = 16), the ORR was 19%. For more results from the phase Ib/II trial, please see MET FISH using a cut-off value of MET GCN > 6 was more accurate compared to MET IHC in predicting the response in NSCLC patients receiving a combined treatment of capmatinib and gefitinib [Another phase Ib/II trial also showed clinical activity of capmatinib in MET-dysregulated NSCLC patients with acquired EFGR-TKI resistance . In thisefitinib . METex14 mutation or METamp [MET status and previous treatment. METex14 mutation was initially determined by a qualitative real-time reverse-transcription PCR (RT-PCR) assay. Detection of METamp, as GCN, was initially determined by FISH. Subsequently, the METex14 mutation and MET GCN were retrospectively retested based on the baseline tissue samples from the trial using the NGS FoundationOne CDx assay (Foundation Medicine). A total of 364 patients were assigned to different study cohorts. For the group with the METex14 mutation, an ORR of 68% was obtained in the treatment-naïve patients (n = 28) compared to the previously treated patients (n = 69) with an ORR of 41%. In the patients with METamp and a GCN ≥ 10, the ORR was 40% in the treatment-naïve (n = 15) and 29% in those previously treated (n = 69). For the patient cohorts previously treated with MET GCN < 10, the activity of capmatinib seemed to be limited, with ORR in the range of 7 to 12% [METex14 mutation in the GENOMETRY mono-1 trial, capmatinib obtained FDA approval [METex14 mutation in NSCLC patients who may benefit from treatment with capmatinib [In a prospective, open-labeled, multiple-cohort phase 2 trial (GENOMETRY mono-1), capmatinib was further investigated in metastatic NSCLC patients with an r METamp . The patents n =  comparednts n = 6 with an pmatinib . Tepotinib  is a small molecule-class Ib inhibitor that targets MET by inhibiting HGF-dependent and -independent MET phosphorylation as well as the MET-dependent downstream signaling pathways . In vitrMETamp as GCN ≥ 5 or MET/CEP7 ratio ≥ 2.0 by FISH. In the phase 1b part, four of the seven patients with MET IHC3+ responded, which was similar for four of the six patients with METamp. In the phase 2 part of the trial, 13 of the 19 MET IHC3+ patients responded (ORR 68%), which was similar to the METamp patients, with 8 of 12 patients responding (ORR 67%). For the chemotherapy group, the ORR was 33% for MET IHC3+ and 43% for METamp patients [METamp patients. In a phase Ib/II trial, the clinical activity of tepotinib was demonstrated in metastatic NSCLC patients with MET dysregulation who had developed a resistance to EGFR-TKI . A totalpatients . Based oMETex14 mutation [METex14 mutation was detected from a tissue biopsy or from plasma as circulating tumor DNA (ctDNA). For the tumor tissue biopsies, the METex14 mutation was assessed using the NGS Oncomine Focus Assay , and for the liquid biopsy, the ctDNA was analyzed using another NGS assay, the Guardant360 . Testing by both biopsy methods was not a requirement for inclusion in the trial. A total of 152 patients were enrolled in the trial, and in the combined biopsy group (n = 99), the ORR was 46%. For the 66 patients with liquid biopsies, the ORR was 48%, and it was 50% for the 60 patients with tissue biopsies. In 2021, based on clinical data from the VISION trial, tepotinib obtained FDA approval for the treatment of patients with metastatic NSCLC harboring the METex14 mutation [METex14 mutations. The full prescribing information for tepotinib emphasizes that an FDA-approved test for the detection of METex14 mutations in NSCLC for selecting patients for treatment is not available. Furthermore, it is stated that testing for the presence of METex14 mutations in plasma specimens is recommended only in patients in whom a tumor biopsy cannot be obtained. If a METex14 mutation is not detected in a plasma specimen, the possibility of a tumor biopsy should be reconsidered [METex14 mutation), because some tumors do not shed a sufficient amount of DNA into the circulation to be detected by this method [In another phase II trial (VISION), tepotinib was investigated in patients with metastatic NSCLC who harbored a mutation . Furthermutation . Somewhansidered . Informansidered . The reas method  Amivantamab  is a bispecific antibody targeting the EGFR and MET . In vitrEGFR exon 20 insertion mutation, METex14 mutation, and METamp. So far, only the results from the EGFR exon 20 insertion mutation cohort (n = 81) have been reported, and here, amivantamab given as monotherapy showed an ORR of 40% [EGFR exon 20 insertion mutation, whose disease has progressed on or after platinum-based chemotherapy [EGFR exon 20 insertions [METex14 mutation and METamp, and to see if they can support an expansion of the current indication for amivantamab. In a multicohort open-labeled phase I trial (CHRYSALIS) in patients with metastatic NSCLC, amivantamab was investigated in different molecular-defined subgroups, including patients with R of 40% . The clisertions . HoweverTaking into consideration the relatively large number of MET inhibitors that have been or are under development for different indications, it is disappointing to see that only a small proportion have obtained regulatory approval and subsequently reached the clinic. Besides being investigated for treatment of NSCLC, MET inhibitors are under clinical development for different indications, such as gastric and gastroesophageal cancer, hepatocellular carcinoma, and renal cell carcinoma ,8,35. ThMET/CEP7 ratios by FISH, GCN by NGS, and METex14 mutation by NGS. The use of MET overexpression, either MET IHC2+ or IHC3+, has given inconsistent results, and the data in One of the reasons for the relatively low success rate of MET-targeted therapy in NSCLC might be the lack of predictive biomarkers with sufficient accuracy. For the FDA-approved drugs, METamp in MET GCN > 6 or a MET/CEP7 ratio ≥ 2.0. For a MET/CEP7 ratio > 2.0, the ORR following treatment with MET-targeted therapy ranges from 33% to 67%, whereas MET GCN > 6 predicts ORR outcomes in the range of 16% to 67%. MET gene copy number gains can occur through both polysomy and amplification, but it seems that true amplification is more likely to lead to oncogenic addiction, which might explain the possibly better predictive properties of MET/CEP7 over MET GCN [METamp in patients with NSCLC, but results from several comparative studies with FISH have shown poor reliability in detecting the various levels of METamp [METamp identified by FISH remains the optimal biomarker to identify suitable candidates for MET-TKI therapy [MET gene copy number could be the result of polysomy rather than a true METamp [The data for f METamp ,39,40. BMETamp seems to play a central role [METamp has been established as a mechanism of acquired resistance, and evidence is accumulating that this could also occur in NSCLC with targeted therapies related to ALK-, RET-, and ROS1-rearrangements. For patients with METamp who have developed resistance to EGFR inhibitors, the combination with a MET inhibitor seems to overcome the resistance [METamp in patients with NSCLC, or whether the recommendation should be to use a FISH assay going forward.For NSCLC patients with identified oncogenic drivers, the use of targeted therapy has significantly improved treatment outcome, with high response rates and improved progression-free survival. However, resistance to this type of therapy will be developed sooner or later, and here, ral role ,8,16. Fosistance ,29. In tMETex14 mutation detected by NGS also shows variability regarding the ORR observed. However, if data originate from larger populations, it seems to show a higher degree of consistency with ORR in the range of 32% to 64% [In % to 64% ,28,30. IMETex14 mutation was performed based on samples and clinical data from the GENOMETRY mono-1 trial [METex14 mutation RT-PCR assay. To establish concordance between the two assays, both cohorts of treatment-naïve and previously treated patients were analyzed. Based on the assay results, positive percent agreement (PPA), negative percent agreement (NPA), and overall agreement (OPA) were calculated. For the previously treated group of patients, PPA was 96.8%, NPA was 100%, and OPA was 99.1%. For the group of treatment-naïve patients, PPA, NPA, and OPA were all 100% and, thereby, a complete concordance between the two assays was achieved and likewise with respect to the clinical outcome [So far, the FoundationOne CDx is the only assay linked to a MET inhibitor that has obtained FDA approval ,31. This-1 trial . Here, aMET/CEP7 ≥ 2.0 as a cut-off was used [MET-targeted therapy has also been investigated outside NSCLC, and here, it is more or less the same type of predictive biomarkers that have been used for patient selection. In a phase II trial, the experimental small molecule MET inhibitor AMG 337 was investigated in patients with gastric/gastroesophageal junction/esophageal adenocarcinoma, and a FISH assay with was used ,42. Furtwas used ,44. DespMETex14 mutation. Likewise, in different clinical trials, the METex14 mutation has also shown predictive properties for drugs such as tepotinib and crizotinib in patients with metastatic NSCLC. Another candidate biomarker is MET amplification, which plays an important role in the development of achieved resistance to EGFR inhibitors. Results obtained from different clinical trials indicate that determination of the MET/CEP7 ratio by FISH possesses the best predictive property, likely because this approach excludes MET amplification caused by polysomy. However, further clinical research will have to show whether MET/CEP7 by FISH is an effective predictive biomarker for MET-targeted therapy. Despite intensive research and development efforts, relatively few MET inhibitors have shown sufficient clinical activity. One of the reasons for this could be the lack of effective predictive biomarkers to select the right patient population for treatment. So far, capmatinib is the only MET inhibitor that has been approved with a CDx assay. In 2020, capmatinib obtained FDA approval for the treatment of patients with metastatic NSCLC whose tumors harbor a


This study aims to investigate patterns of antibiotic prescribing and to determine patient-specific factors associated with the choice of antibiotics by the World Health Organization's Access-Watch-Reserve (WHO AWaRe) class for acute respiratory infections (ARIs) in rural primary care settings in northern Vietnam.We retrospectively reviewed health records for outpatients who were registered with the Vietnamese Health Insurance Scheme, visited one of 112 commune health centres in 6 rural districts of Nam Dinh province, Vietnam during 2019, and were diagnosed with ARIs. Patient-level prescription data were collected from the electronic patient databases. We used descriptive statistics to investigate patterns of antibiotic prescribing, with the primary outcomes including total antibiotic prescriptions and prescriptions by WHO AWaRe group. We identified patient-specific factors associated with watch-group antibiotic prescribing through multivariable logistic regression analysis.Among 193,010 outpatient visits for ARIs observed in this study, 187,144 (97.0%) resulted in an antibiotic prescription, of which 172,976 (92.5%) were access-antibiotics, 10,765 (5.6%) were watch-antibiotics, 3366 (1.8%) were not-recommended antibiotics. No patients were treated with reserve-antibiotics. The proportion of watch-antibiotic prescription was highest amongst children under 5-years old . In multivariable logistic regression, children, district, ARI-type, comobid chronic respiratory illness, and follow-up visit were associated with higher likelihood of prescribing watch-group antibiotics.The alarmingly high proportion of antibiotic prescriptions for ARIs in primary care, and the frequent use of watch-antibiotics for children, heighten concerns around antibiotic overuse at the community level. Antimicrobial stewardship interventions and policy attention are needed in primary care settings to tackle the growing threat of antibiotic resistance.This work was supported through Australian government and UK aid from the UK government funding to FIND (Foundation for Innovative New Diagnostics) grant number FO17-0015, in addition to a Wellcome Trust grant (213920/Z/18/Z), and an Oxford University Clinical Research Unit internal grant from the Wellcome Trust Africa Asia Programme core grant in Vietnam (106680/Z/14/Z). We searched PubMed and Google Scholar for articles published from 1st January 2000 to 30th May 2021 using key concept terms such as “antibiotic” , "primary care" ("community"), "prescribing" , "respiratory tract infection" ("respiratory infection") and "low middle income". We only considered the articles published in English.We found that the previous studies on similar topics conducted in low-middle income countries mainly collected data from surveys or a sample of traditional handwritten prescriptions, which often contained a small sample size and lacked detailed and reliable clinical information on diagnoses and drug names, and possibly resulted in recall bias and documentation errors. Only a few studies have explored the factors associated with restricted antibiotic prescription, most of which used qualitative approaches that might limit representativeness, and they did not identify the most important determinants. Because the World Health Organization Access, Watch and Reserve antibiotic classification (WHO AWaRe) was published in 2017, the number of studies in low-middle income countries that have so far used it for data analysis remains limited.This is the first study in Vietnam to use routinely collected electronic health record data to characterize antibiotic prescribing for acute respiratory infections (ARIs) in primary care, and to determine which factors are associated with restricted antibiotic prescription by the World Health Organization Access, Watch and Reserve classification (WHO AWaRe). Electronic health record data are reliable because they are regularly checked and used as evidence of prescribers’ clinical practice at CHC for routine audits by local health authorities and the national reimbursement agency. Including nearly 200,000 outpatient visits from 112 commune health centres in Northern Vietnam during 2019, this study is the largest study in Vietnam on this topic to date making the findings robust. The findings of this study, including the patterns of antibiotic prescribing, and determinants of watch-antibiotic prescribing are critical to inform future policy-making on antibiotic prescribing and antibiotic stewardship in Vietnam, and as evidence to explain the alarming rise of antimicrobial resistance (AMR) in the country.Among 193,010 outpatient visits for ARIs observed in this study, 187,144 (97.0%) resulted in an antibiotic prescription, , and no patients were treated with reserve antibiotics. Antibiotic prescription proportion was higher than 97% in the most commonly seen ARIs (acute pharyngitis [97.2%] and acute bronchitis [97.3%]) and higher than 90% in ten of eleven types of ARI observed. Influenza, with the lowest antibiotic prescription proportion, still reached 77.3%. To our knowledge, it is the highest all-age proportion of antibiotic prescribing for common infections which has been observed in Vietnamese primary care settings. This proportion is much higher than those reported from High Income Countries’ health systems as well as other AMR hotspots. Of concern, our study also found that the proportion of watch-antibiotic prescribing in children was higher than that in adults, especially for those under 5-year old , reflected by both descriptive data and regression analyses. This pattern is in line with what has been observed in other primary care settings in Vietnam, such as community pharmacies and outpatient clinics at urban hospitals, underscoring that children should be an important target for antibiotic stewardship interventions. To address these situations, we believe that strengthening primary healthcare prescribers’ capacity through interventions such as education, training in communication skills, developing appropriate guidelines, and providing them with point-of-care diagnostic tests to guide antibiotic prescribing is crucial.Alt-text: Unlabelled box,Acute respiratory infections (ARIs) are the most frequent infections presenting to primary care in low- and middle- income countries (LMICs).,Streptococcus pneumoniae with reduced susceptibility to penicillin increased from 8% to 75%.S.pneumoniae and Haemophilus influenza was only 3% for macrolides and 5% for 1st and 2nd generation cephalosporins, consequently ranking the antibiotic susceptibility in Vietnam among the lowest in Asia.,As the world is exiting the fast and intense pandemic of COVID-19, a slower and more insidious pandemic of AMR is gradually worsening.,The current health system in Vietnam is a mixed public-private system, in which the public system plays a critical role in preventive and curative care for the population nationwide, and almost 90% of the population have national health insurance.There are concerns that over-prescription of antibiotics for ARI symptoms in CHCs in Vietnam has contributed to the emergence and spread of AMR. A large survey in 217 CHCs in Hai Phong Province from 1994 to 1996 showed that over 51% of patients visiting CHCs were for ARIs and 65% were prescribed antibiotics.These previous studies in Vietnam mainly collected data from surveys or a sample of traditional handwritten prescriptions, which often lacked detailed and reliable clinical information on diagnoses and drug names, and possibly resulted in recall bias and documentation errors. Since the release of the Vietnamese National Action Plan on AMR in 2013, large-scale studies updating evidence on the context of antibiotic treatment in primary care settings in Vietnam has been limited. Therefore, we conducted this study to provide updated data on antibiotic prescribing by general practitioners in rural primary healthcare centres in northern Vietnam and to identify patient-specific factors associated with choice of antibiotics.2). It includes 1 city and 9 rural districts with 194 rural communes. The monthly average income per capita of residents in 2021 in Nam Dinh province (4.615 million VND) was a bit higher than that in the whole country (4.30 million VND).We conducted a retrospective review of claims data from Electronic Health Records (EHRs) for outpatients at 112 CHCs in six rural districts  in Nam Dinh province, northern Vietnam. Profiles of these districts are provided in Supplementary Table 1. Nam Dinh is 90 km southeast of Hanoi, the capital city. The province covers an area of 1,669 km2 and supports a population of approximately 1.781 million  to guide antibiotic prescribing for ARIs, included 48 CHCs in three districts .st January and 31st December 2019. Afterwards, these data were gathered and transferred to study coordinators (HTN and TTV). The coordinators checked for missing or potentially invalid data (e.g.. data on a visit was available but data on treatment was missing) and gave feedback to the study collaborators. The study collaborators discussed with CHC prescribers and re-checked the CHC's electronic database, or compared electronic data and data from the CHC's health record notebook to validate these cases and provided adjustments if needed. Finally, raw data were sent to our data manager (DTVV) to integrate and convert into a cleaned, validated and ready-to-use format. The data manager also removed patients’ and prescribers’ names to ensure confidentiality before analysis.From the claims databases of 112 selected CHCs in this study, our study collaborators who were staff at district health centres extracted electronic health record (EHR) data (raw data) for all patients registered with the Vietnamese Health Insurance Scheme and who had at least one health visit at any of these centres between 1The EHRs include health insurance code, demographic data , clinical diagnosis, clinical symptoms, the international classification of diseases - 10th revision (ICD-10) code assigned for the main diagnosis, medications prescribed, and the location of the CHC.,,We obtained EHRs of all outpatients who visited for ARIs, defined by an ICD-10 code relating to ARIs , or a clinical diagnosis with ARI. We chose these codes and diagnoses by reviewing previously published research on a similar topic.In this study, we defined an antibiotic prescription for ARI as related to a visit if at least one systemic antibiotic was prescribed. For the ARI visits with multiple diagnoses relating to infectious diseases, a study pharmacist (NVN) reviewed the relevant record to determine if antibiotic prescription targeted the ARI or the other diagnoses. An infectious disease specialist (NTDT) in the study team participated in the assessment process if the study pharmacist needed further consultation.We presented data on patterns of antibiotic prescribing according to the 2021 Access-Watch-Reserve (AWaRe) antibiotic classification of the World Health Organization (WHO). According to this classification, 258 common antibiotics were classified into 4 groups: access, watch, reserve and not recommended. Access-group antibiotics are the first or second choice antibiotics that offer the best therapeutic value while minimizing the potential for resistance. Watch-group antibiotics are the first and second choice antibiotics for a specific, limited number of infective syndromes because these agents are more prone to development of antibiotic resistance and thus prioritized as targets of stewardship programs and monitoring. Finally, reserve-group antibiotics should be considered antibiotics of last resort, which should be tailored to highly selected patients (life-threatening infections due to multi-drug resistant bacteria) when all alternatives have failed or are not suitable. The classification also lists those antibiotics whose use is not recommended by WHO, namely fixed-dose combinations of multiple broad-spectrum antibiotics that lack evidence-based indications for use or recommendations in high-quality international guidelines. For visits with a combination of antibiotics, our classification was based on the antibiotic with the highest level of restriction .Descriptive statistics were used to investigate the patterns of antibiotic prescribing for ARIs. The primary descriptive analysis includes the antibiotic prescription proportions for outpatient visits for ARIs, defined by the number of visits with antibiotic prescriptions divided by the total number of visits in selected CHCs during the studied duration. These proportions were measured for total antibiotics and for each AWaRe antibiotic category. We also calculated the prescription proportions of total antibiotics and of AWaRe-specific groups for each ARI, and for each sub-population stratified by age or by selected districts.,,,,We analysed determinants of prescribing restricted antibiotics (either watch- or reserve-antibiotics). These agents have higher toxicity and/or resistance potential compared with access antibiotics and are not recommended as first choices for common infections such as community-acquired ARIs. Since reserve antibiotics are not available at primary care level in Vietnam, and were not found in our study population, our analysis only reviewed the determinants associated with prescribing watch-antibiotics. We performed a mixed effects logistic regression model to estimate odd ratios (ORs) and 95% CIs for the outcome of being prescribed at least one watch-group antibiotic for ARI. The variables included in the model were identified through a literature review of previously published studies with similar topics.The protocol of the ICAT trial was approved by the Oxford University Tropical Research Ethics Committee  and the ethical committee of the National Hospital for Tropical Diseases in Vietnam (Reference: 07/HDDD-NDTW/2019). The protocol of the Co-Act trial  was approved by the Oxford University Tropical Research Ethics Committee  and the ethical committee of the National Institute of Hygiene and Epidemiology (Reference: HDDD-30/2019). Permission to carry out this study was obtained from local authorities.Among 409,139 outpatient visits at 112 CHCs in 6 districts in rural Nam Dinh in 2019, we identified 193,010 visits for ARIs  were prescribed at least one systemic antibiotic. Antibiotic prescription proportion was higher than 97% in the most commonly seen ARIs (acute pharyngitis [97.2%] and acute bronchitis [97.3%]) and higher than 90% in ten of eleven types of ARI observed. Even in patients diagnosed with influenza, by definition of viral aetiology, the proportion of patients prescribed an antibiotic reached 77.3%. Between WHO-AWaRe antibiotic groups, access-group antibiotics were the most frequently prescribed . Watch-group and not-recommended antibiotics were less frequently prescribed, at 5.9% and 1.8%, respectively. No reserve-group antibiotics were prescribed. Prescriptions of watch-group antibiotics was highest in pneumonia and acute tonsillitis, at 11.3% and 10.0 %, respectively, and prescription of not-recommended antibiotics was highest in acute nasopharyngitis, at 7.7% . Proportions of not-recommended antibiotic prescribing for all age groups were below 5% . PatientIn all studied districts, antibiotic prescription proportions were higher than 90%. Watch-group antibiotic prescription proportions were lower than 10% in all districts except for Hai Hau (10.6%). Y Yen was the only district where not-recommended antibiotics were prescribed .Figure 3We found that younger age, district, type of ARI, presence of comorbid chronic respiratory illness, and follow-up visits were associated with prescribing watch-antibiotics. Compared with adults aged 18–49-years, children aged under 5-years , and those aged 5–17-years  had more risk to be prescribed watch-antibiotics, while older adults aged 50–64-years  and the elderly aged 65-and-above-years  were less likely to be prescribed watch-antibiotics. Between districts, Nghia Hung, Xuan Truong and Hai Hau had higher watch-antibiotic prescribing compared with Truc Ninh. Most of the less common ARIs had higher watch-antibiotic prescription compared with acute pharyngitis (the most commonly seen ARI), with the exception of acute nasopharyngitis (lower prescribing) and unspecified lower respiratory tract infection. Watch-antibiotics were more likely prescribed in follow-up visits rather than first visits .  Patients,,,In this study, 97% of the total visits for ARIs received antibiotic prescriptions. To our knowledge, it is the highest all-age proportion of antibiotic prescribing for common infections which has been observed in Vietnamese primary care settings. This proportion eclipses those reported in high income countries such as in the US (41%), the UK 7.5–30.9% in) and Japan (52.7%), as well as other AMR hotspots in India (45.1%), China (52.9%) and South America 35–40%)..9% in an–40%..9% The prescription proportion of access-antibiotics was much higher than watch- antibiotics , which is in line with the WHO recommendations on prioritizing access-antibiotics as first-line treatment for community-acquired infections. This highlights the success of tightening prescribing policies in improving appropriate use of antibiotics.,Of concern, our study found that primary care doctors in rural Vietnam use more watch-antibiotics for children than for adults, especially for those under 5-year old, reflected by both descriptive data and regression analyses. Watch-antibiotics prescribed for children in our study population included broad-spectrum antibiotics such as second generation cephalosporins and macrolides, which were previously shown not to improve clinical outcomes in children with ARI compared with narrow-spectrum antibiotics, while increasing the risks of drug-related adverse events.We also observed different patterns of antibiotic prescribing by district. In this study, Hai Hau district, the one with the highest population and number of health facilities, also had the highest proportion of watch-antibiotic prescribing.Vietnam was one of the first countries to develop a National Action Plan to combat AMR in the World Health Organization (WHO) Western Pacific Region. This plan addresses raising awareness of AMR within the community, safeguarding access to antimicrobials and encouraging the safe use of drugs as among key ways to prevent the spread of AMR. However, prescribers’ habits and diagnostic uncertainty contribute to antibiotic misuse.To our knowledge, this is the first study in Vietnam to use routinely collected EHR data to characterize antibiotic prescribing for ARIs in primary care, and to determine which factors are associated with restricted antibiotic prescription. Including outpatients from 112 CHCs, this study is the largest study on this topic to date  making the findings robust. This also minimizes impacts of recall and responder bias because data was immediately collected at the time of patient visits and documented in CHCs’ electronic database. Electronic health record data are reliable because they are regularly checked and used as evidence of prescribers’ clinical practice at CHC for routine audits by local health authorities and the national reimbursement agency.In conclusion, our study finds an alarmingly high proportion of antibiotic prescription for ARIs in public primary care settings in Vietnam. These findings corroborate earlier reports regarding inappropriate antibiotic prescribing in rural Vietnam, and other LMICs. There is an urgent need for multi-faceted interventions to tackle both over-prescribing and choice of antibiotics. These could include training in antimicrobial stewardship and communication skills, and point-of-care diagnostic tests to guide antibiotic treatment decisions.NVN, NTTD, NTDT, RCG, SD, MV, HRvD, YL and SOL were responsible for conceptualization and study design. HTN and TTV coordinated study activities across the sites with supervision from NTTD and SOL, and local support and oversight by TNP, NTDT, TQP, VTK, TTBL and THC. DTVV and TST accessed, cleaned, managed and verified the data. NVN led the data analysis with supervision from DTVV and SOL. LNN supported the data analysis and visualization. NVN drafted the manuscript with supervision from SOL. All authors contributed to the final version of the manuscript and approved the submission. The corresponding author had final responsibility to submit for publication.Data may be available according to data sharing policy of the relevant partners contributing to this study upon request to the corresponding author.The authors declared no conflicts of interest.


Background: Antibiotics are prescribed for children both in hospital and community settings, particularly at preschool age. Italy shows a high rate of inappropriate antibiotic prescriptions which may represent a serious problem in the hospital scenario. Thus, the aim of this study was to investigate appropriateness of antibiotic prescribing in the context of different paediatric subspecialties in a hospital setting.Methods: Antibiotics prescribing was retrospectively analysed in paediatric patients (0–18 years) admitted in the emergency paediatrics, general paediatrics, paediatric nephrology and rheumatology units between January and December 2019. Patients were stratified by age in neonates, infants, toddlers, children and adolescents. Assessments were conducted by trained local assessors and appropriateness was classified as appropriate, inappropriate and not assessable.Results: Empirical antibiotics were mainly prescribed following a diagnosis of respiratory, gastrointestinal and/or urinary infection. A total of 825 antibiotic prescriptions were recorded in the three subspecialties; 462 antibiotic prescriptions (56%) out of 825 were assessed as inappropriate and 55 prescriptions (6.7%) were not assessable. Inappropriateness considerably varied within subspecialties: the risk of inappropriate antibiotic prescribing was higher in emergency paediatrics and general paediatric than in children, according to age. Ceftriaxone and clarithromycin were the most inappropriate prescribed antibiotics in the emergency paediatrics whereas amoxicillin/clavulanic acid represented the most inappropriate antibiotic prescribed in general paediatrics.Conclusion: The present data may be useful in order to reduce inappropriate antibiotic prescribing in the paediatric setting; antibiotic stewardship and clinical improvement programs in hospital paediatric care are strongly recommended. Antibiotics are frequently prescribed for children both in hospital and community settings , particuClostridium difficile infection  were detected among non-European paediatric patients  . The Antnfection  as well nfection . For thenfection . HoweverIn a retrospective observational study, antibiotics prescribing was analysed in paediatric patients (0–18 years) admitted to the University Hospital “G. Martino” of Messina,  for the period between January and December 2019. Antibiotics were classified according to the Anatomical Therapeutic Chemical Classification (ATC) and in particular the prescription of drugs in 2nd level ATC = J01 was evaluated. Emergency paediatrics, general paediatrics, paediatric nephrology and rheumatology were involved in this study and its protocol was approved by the Ethics Committee of the A.O.U. “G. Martino” of Messina  according to the legal requirements concerning observational studies; patient’s consent to participate was not requested for this kind of study. Data were anonymously recorded by clinical doctors and pharmacists during hospitalization, including patient demographic features, indication for antibiotics, route of antibiotic administration and type of antibiotics as norm of clinical practice. All paediatric patients were stratified by age group: neonates (<28 days), infants (28 days to <1 year), toddlers (1year to <3 years), children (3 years to <12 years), adolescents (12 years to <18 years). Data about consumption and cost of antibiotics prescriptions were extracted from the pharmacy administrative database; the used drugs by ATC code, the consumption in unit dose and the cost in euro (€) were extracted for each unit.The appropriateness of antibiotic prescribing was investigated by consulting the patient’s clinical records of the emergency paediatrics, paediatrics, paediatrics nephrology and rheumatology wards. Patients with at least one antibiotic administration were selected. All clinical information  and socio-demographic (age and sex) features of each patient were collected. The appropriateness of antibiotic prescribing was assessed by the World Health Organization (WHO) AWaRe classification guidelines and by the regional guidelines for appropriate antibiotic prescribing in children . AssessmU test for independent sample and two—tailed Pearson chi—squared test was performed to compare continuous variables and categorical variables. Odds ratios (ORs) with 95% CIs were estimated for each covariate of interest in univariate (Crude OR) and multivariate (adjusted OR) regression models. A p-value < 0.05 was assessed as statistically significant. Statistical analysis was carried out using the SPSS version 23.0 (IBM Corp. SPSS Statistics).The results were expressed as absolute and relative frequencies of the categorical variables, with 95% confidence intervals (CIs) and as medians and interquartile range (Q1—Q3), respectively. The Mann—Whitney N = 325) and mostly were male (57.8%) and children (37.4%).Between January and December 2019, 626 patients were hospitalized in the units of emergency paediatrics, general paediatrics, paediatric nephrology and rheumatology . PatientDuring the hospital stay, 825 prescriptions of antibiotics were recorded ; more spn = 246; 53.24%) followed by general paediatrics  and paediatric nephrology and rheumatology   out of 825 were assessed as inappropriate and 55 (6.7%) were not assessable . Inappro 12.34%) . Figure  12.34%) . A regre 12.34%) .In our study, drug utilization and expenditure were also evaluated in the paediatric units throughout the observation period. The 18.8% of the 26.617 consumed drugs in all paediatric units was antibiotics and more than half of the antibiotic utilization (54.9%) was observed in the emergency paediatric unit . MoreoveThis retrospective study provides an analysis of appropriateness of antibiotic prescribing in hospitalized paediatric patients allocated in different subspecialties in agreement with their medical needs. As far as we know, no previous study has focused attention on the antibiotic prescribing related to the different paediatric subspecialties. This issue was investigated taking advantage of the availability of a real word scenario: a hospital characterized by the presence of several paediatric units. Our results showed the occurrence of a high rate of antibiotic inappropriate prescription; more than half of the prescribed antibiotics was inappropriate. Therefore, the co-presence of several paediatric subspecialties units does not allow a better management of antibiotic prescribing, even if they are empirically prescribed based on the clinical diagnosis. Furthermore, we tried to investigate the “clinical scenario” more “at risk” of antibiotic inappropriateness. As expected, the emergency paediatrics unit showed the higher rate of inappropriate antibiotic prescribing. This is not surprisingly if the “characteristics of emergency” of this clinical setting are taking into account. Indeed, previous data have suggested that antibiotics represent the most commonly drugs prescribed with inappropriateness in the general emergency department either in adults and children, in turn causing unwanted adverse events and contributing to the development of therapeutic failure and antimicrobial resistance . HoweverThe present study has some limitations. Firstly, this is a single-centre study; secondly, enrolled patients were only hospitalized, and our survey does not capture data on community or primary care, which represent the bulk of antibiotic prescribing. Moreover, antibiogram was not considered during the assessment of the suitability of antibiotics. Also, we did not analyse precise diagnoses in accordance with International Statistical Classification of Diseases, Injuries and Causes of Death (ICD) but only groups of diagnosis.Our study suggests the need to implement measures of a close monitoring of antibiotic prescriptions in the paediatric population. Furthermore, our results on antibiotic prescribing in the different paediatric units can be useful for future interventions directed towards reducing inappropriate antibiotic use. More specifically, we have clearly shown that emergency paediatrics unit deserves a close monitoring and additional attention. We suggest that antibiotic stewardship and clinical improvement programs in hospital paediatric care should be strongly recommended. Finally, our study confirms the need to create professional’s positions of clinical pharmacologists with the aim to cooperate and synergize with paediatric clinicians to reduce the exposure of hospitalized patients to an inappropriate antibiotic treatment.


For common infections, previous studies suggest more seasoned prescribers more commonly use inappropriately long antibiotic durations however, there is a paucity of data regarding patient and provider factors associated with antibiotic prescription duration for pediatric skin and soft tissue (SSTI).Retrospective analysis of patients age 0-21 years prescribed enteral antibiotics for cellulitis, abscess, impetigo, or other SSTI in one of 13 Nationwide Children’s Hospital’s primary care clinics in 2020. Encounters for paronychia, facial abscess, and immunocompromised patients were excluded. Based on national and local SSTI guidelines, antibiotic duration was considered inappropriately long if >5 days for cellulitis or drained abscess or >7 days for undrained abscess, impetigo, or other SSTI. Patient and prescriber factors were collected (Table 1) then univariate and multivariable analyses performed to determine which factors were associated with long antibiotic duration. Statistical analyses accounted for within prescriber correlation.From 1/1/2020 to 12/31/2020, we identified 1248 encounters for SSTI. After exclusions, 577 encounters with enteral prescriptions written by 89 prescribers were analyzed. Patient age ranged from 11 days to 20 years, 314 (54%) were female, and 497 (86%) had public insurance. The most common diagnosis was impetigo in 165 (29%). Similar rates of long duration were seen across patients of all ages and insurance categories. No difference in prescriber degree type was associated with antibiotic duration. Mean years since completion of medical training for prescribers of long vs guideline concordant prescriptions was 13 vs 10 in univariate analysis (p=.001). In the multivariable analysis, only diagnosis was associated with increased odds of long duration (Table 2). Compared to impetigo, the adjusted odds ratio for inappropriately long duration was 9.36 for cellulitis and 2.03 for abscess.Patient and Prescriber Characteristics for SSTI Antibiotic PrescriptionsIQR, interquartile range, MD, Doctor of Medicine, DO, Doctor of Osteopathic Medicine, NP, Nurse Practitioner†Mann Whitney ‡Fisher’s exact *Chi-square ¶N=556 due to missing data for 21 encountersMultivariable Analysis of Patient and Provider Factors and Inappropriately Long Antibiotic Duration for SSTIMultivariable Analysis of Patient and Provider Factors and adjusted odds ratios for inappropriately long antibiotic duration. *Unknown insurance type for 24 encounters †Missing data for 21 encountersIn this study of antibiotic duration for SSTI, diagnosis of cellulitis was most strongly associated with inappropriately long duration- providing a focus for future stewardship efforts. Contrary to prior studies, no other patient or provider factor was associated with long duration in our pediatric primary care network.Jack Stevens, PhD, Colgate Palmolive : Stocks/Bonds|Procter and Gamble : Stocks/Bonds.


As opioid-related fatalities continue to rise, the need for novel opioid use disorder (OUD) treatments could not be more urgent. Two separate hypothalamic neuropeptide systems have shown promise in preclinical OUD models. The oxytocin system, originating in the paraventricular nucleus (PVN), may protect against OUD severity. By contrast, the orexin system, originating in the lateral hypothalamus (LH), may exacerbate OUD severity. Thus, activating the oxytocin system or inhibiting the orexin system are potential therapeutic strategies. The specific role of these systems with regard to specific OUD outcomes, however, is not fully understood. Here, we probed the therapeutic efficacy of pharmacological interventions targeting the orexin or oxytocin system on two distinct metrics of OUD severity in rats—heroin choice  and cued reward seeking. Using a preclinical model that generates approximately equal choice between heroin and food reward, we examined the impact of exogenously administered oxytocin, an oxytocin receptor antagonist , and a dual orexin receptor antagonist (DORA-12) on opioid choice. Whereas these agents did not alter heroin choice when rewards (heroin and food) were available, oxytocin and DORA-12 each significantly reduced heroin seeking in the presence of competing reward cues when no rewards were available. In addition, the number of LH orexin neurons and PVN oxytocin neurons correlated with specific behavioral economic variables indicative of heroin versus food motivation. These data identify a novel bidirectional role of the oxytocin and orexin systems in the ability of opioid-related cues to bias reward seeking. Drug choice and the ability of drug cues to motivate drug seeking are conceptually dissociable processes that contribute to the pervasive opioid taking and seeking that characterize opioid use disorder (OUD). Over the past several decades, preclinical models of relapse have been the focus of efforts to advance OUD therapies. In contrast, preclinical models of opioid choice have only recently begun to receive significant attention. The vast majority of these preclinical choice models have revealed that rodents will primarily choose natural rewards  over drug rewards under conditions of mutually exclusive choice, equivalent price , and equal delay . Thus, sOxytocin is a neuropeptide produced by a subset of neurons located in the paraventricular nucleus of the hypothalamus (PVN). Aside from its known role in parturition and maternal behaviors , oxytociTM) reduces several relapse-related and self-reported craving indices in patients with cocaine or opioid use disorder [Orexins A and B are neuropeptides produced in the lateral hypothalamus (LH) that regulate motivated drug seeking. Orexin-expressing neurons are activated by stimuli associated with morphine, cocaine, and alcohol –35, and disorder , 52. Basdisorder , althougDespite the growing popularity of choice models in preclinical addiction research, to date no study has investigated the contributions of hypothalamic neuropeptide systems under choice conditions. To address this gap in knowledge, we tested whether pharmacological treatment with either oxytocin or a DORA could alter choice behavior, under conditions when rewards are available or unavailable . We also applied principles from the field of behavioral economics  to invesn = 32) and female (n = 32) Wistar rats . Animals were single-housed in a temperature and humidity-controlled environment (lights on 8am-8pm) with free access to standard laboratory chow (Envigo 2020X) and water. Procedures followed the guidelines outlined in the Guide for the Care and Use of Laboratory Animals [All animal procedures followed guidelines approved by the University of Colorado Anschutz Medical Campus Institutional Animal Care and Use Committee. Subjects were age-matched P55–60) male  was dissolved in sterile water and administered at doses of 1 or 3 mg/kg  30 min prior to testing. This pretreatment interval and dose range were based on prior literature showing an effective reduction in drug seeking with minimal side effects , 56, 57.Rats were surgically implanted with an intravenous jugular catheter as previously described . After rC indicates consumption  and constant k (set to 8.85) specifies the range of consumption values [α , Q0 , and Pmax  [We used a between-session behavioral economics paradigm wherein the price  for rewards increased over daily sessions . Food ann values . An elasgorithm) , 59, 61.Because EV is independent of reinforcer magnitude, it is useful for comparing different reinforcers directly , 62. TheChoice procedures were implemented after self-administration training. During choice sessions, rats were given 14 trials to choose food or heroin (FR3). Once the FR requirement was reached on one lever, the respective reward and cue was delivered, then levers retracted for a 10 min time-out between trials. The first 2 choice sessions began with two forced-choice trials for each reinforcer  during which only the food or heroin lever was extended in alternating order, followed by 10 free-choice trials with both lever options. Thereafter, the forced choice trials were dropped and only free-choice sessions  were conducted. Following at least 6 stable choice sessions , rats began repeated choice testing, with each test separated by two choice sessions. Heroin choice was expressed as the percentage of heroin choices over total choice trials. Importantly, there were no omissions; all rats completed all 14 trials on each test. Choice sessions (including choice tests) were the only behavioral sessions where both food and heroin rewards were available simultaneously, with both food and heroin levers extended at the same time. Rats were assigned to the oxytocin or orexin study in a balanced manner based on the percentage of heroin choice and sex. Figure After completing choice testing, rats were returned to FR3 self-administration procedures identical to those during training. During the first 30 min of each session, the food lever was available, followed 10 min of levers retracted, and then the heroin lever was available for 2 h. After at least three reacquisition sessions, rats underwent a single cued reward seeking test. During the test, both food and heroin levers were simultaneously available for 1 h. Pressing on each lever resulted in cue delivery (food-tone or heroin-light) on an FR3 schedule , but no rewards. Note that repeated testing is not feasible under these conditions, as responding extinguishes rapidly; hence, a single dose of each agent was selected for testing. Each rat received either vehicle (water or 5% DMSO + 40% captisol in water), 1 mg/kg oxytocin, or 30 mg/kg DORA-12, 30 min prior to the cue test. The 1 mg/kg dose of oxytocin has been commonly used to reduce cued reward seeking in other studies , 18. The® 594  secondary antibody. Sections were mounted onto slides and coverslipped with ProLong Diamond Antifade mounting medium with DAPI (Invitrogen Cat# P36971). Images were acquired with a slide scanner microscope  and imported into Imaris software . Oxytocin neurons were counted semi-automatically using the Imaris spot detection function. The number of oxytocin cells in the PVN was counted in both hemispheres and averaged across two sections per animal. For orexin counts, images were acquired using a Zeiss Axio Zoom V16 microscope. Tiled photographs were compiled at ×20 magnification using Zen 2 imaging software (Carl Zeiss Microscopy). As in our previous studies [The number of orexin neurons and oxytocin neurons were counted in the brains of male rats from our previous study , which u studies , 64, the studies , 64 by dt test where appropriate. Cued reward seeking test data were analyzed using a 2-way RM-ANOVA with lever (heroin or food) as within-subject factor and treatment as between-subject factor, followed by Sidak post hoc tests. Linear relationships between variables were analyzed using Pearson’s Correlation coefficient.All statistical analyses were performed in Prism . Differences in behavioral economics variables were assessed using 2-way repeated measures (RM) analyses of variance (ANOVA) with reward (food and heroin) as the within-subjects factor and sex as the between-subject factor. Choice data and lever presses for food or heroin during choice testing were analyzed using a one-way RM-ANOVA with treatment as within-subject factor or a 2-tailed paired F = 15.39, p = 0.0006; paired t test: t(25) = 3.826, p = 0.0008) and Pmax  = 20.77, p = 0.0001; paired t test: t(25) = 4.582, p = 0.0001) were significantly higher for heroin than for food, and heroin α was lower than food α  = 12.30, p = 0.0018; paired t test: t(25) = 3.14, p = 0.0043). These results indicate that rats had a more inelastic demand curve for heroin, suggesting that under these experimental conditions, rats treat heroin as a necessity and food as a luxury  = 22.63, p < 0.0001; paired t test: t(25) = 4.8436, p < 0.0001), indicating that rats respond significantly more for food than for heroin under “free” access conditions, consistent with our prior work [Representative demand curves for food and heroin self-administration over increasing price points from a single animal are shown in Fig. F = 7.18, p = 0.0131; α food versus α heroin: p = 0.0004). However, no significant differences between sexes were observed in any behavioral economics variables  = 2.21, p = 0.0366).Heroin preferring rats were defined as those showing >65% heroin choice, and food preferring rats were defined by <35% heroin choice, leaving those choosing 35–65% as equal preferers. Seven of the ten heroin preferring rats in this study were female; equal preferring rats were roughly equivalent in sex , whereas pellet preferring rats were predominantly male . Thus, in this study, ~38% of the population was heroin preferring, ~35% equal preferring, and ~27% pellet preferring, yielding a population average of 56 ± 6% heroin choice  or vehicle was administered 30 min prior to the choice test session in rats assigned to the oxytocin study. A within-subjects design was used, with at least two drug-free choice sessions in between tests. No treatment effects of oxytocin were observed on opioid choice Fig. , left, hIn rats assigned to the orexin study, the dual orexin receptor antagonist DORA-12  or vehicle was administered 30 min prior to the choice test session, again using a within-subjects design, with drug-free choice sessions separating each test. There were no effects of DORA-12 on opioid choice Fig. , left, hF = 49.93, p < 0.0001), but there were no a priori differences between treatment groups.After choice testing was complete, all rats were returned to food and heroin self-administration (on FR3) for at least 3 sessions before the cued reward seeking test. During this final test, rats were able to respond on both heroin and food levers, which delivered the heroin- or food-related cues (on FR3) but did not deliver the rewards. Thus, this was a cue extinction test, similar to other forms of cued reinstatement , 65, conF = 16.15, p = 0.0006) compared to the food lever, consistent with the higher motivation to seek heroin when both cues were present. Treatment with either oxytocin or orexin agents produced a significant reduction in responding  = 5.662, p = 0.0108; lever × drug interaction, F = 3.646, p = 0.0437), and post-hoc analyses indicated that this reduction in responding was specific to the heroin lever  with no significant effects on the food lever. Furthermore, responding for the heroin cue was greater than responding for the food cue only in the Veh group (p = 0.0003). These results indicate that activating the oxytocin system with exogenous oxytocin, or inhibiting the orexin system with DORA-12, is capable of normalizing rates of reward seeking in the presence of heroin cues to those of food.During the cued reward seeking test, rats responded at higher rates on the heroin lever for the heroin cue  and Pmax , and negatively correlated with heroin α . Interestingly, no food variables correlated with orexin cell counts (data not shown). Moreover, the number of orexin neurons in the DMH/PF did not correlate with any behavioral economics variables for heroin or food  and food α . No other variables correlated with oxytocin cell counts (data not shown). This suggests a possible generalized involvement of the oxytocin system in motivation for both drug and natural rewards.We then examined correlations between these counts and the behavioral economics variables from each individual rat’s demand curves for heroin and food, as well as reward seeking during the cue test. Figure α, allowing us to directly compare the elasticity of demand between these two different rewards. Pmax, or the maximum price the animal is willing to pay (in lever presses) to defend desired consumption (Q0), was also higher for heroin than food. Moreover, heroin α correlated with both the number of orexin and oxytocin neurons, indicating that these hypothalamic systems underlie motivation for heroin.Altogether these results provide evidence that two hypothalamic neuropeptides, oxytocin and orexin, bidirectionally regulate the ability of opioid cues to bias choice in acute (~24 h) withdrawal. We confirmed previous findings in a similar choice model that rats are willing to work more for heroin than natural reward (food) . Indeed,α was higher than heroin α, again pointing to a higher motivational state for heroin than food, contrary to other reports [A few preclinical studies have investigated the relationship of behavioral economics variables and choice between drugs and non-drug alternative rewards. Early work suggested that rodents value sweet rewards  as much or more than drug rewards , 69. In  reports , 72, 73.The hypothalamus, especially its oxytocin and orexin neuropeptide systems, has been strongly implicated in addiction-related behaviors , 74, 75.Both clinical and preclinical studies support the therapeutic potential of oxytocin-based medications for SUDs, including OUD . A recenInterestingly, neither oxytocin nor DORA altered choice behavior when heroin and food were present. Consistent with these negative behavioral findings, neither oxytocin nor orexin cell numbers correlated with choice behavior. However, both compounds were effective at reducing opioid seeking in the presence of heroin cues (food cues simultaneously available). Thus, activating the oxytocin system or inhibiting the orexin system reduces reward seeking triggered by heroin cues to levels similar to those of food cues. A couple of key differences between the test conditions may account for these apparent disparate results. First, during the choice tests, response rates are low and rewards are available and being consumed. This type of behavior may be less amenable to disruption than behavior under conditions like the cue test, where competing heroin and food cues are available, but not their respective rewards, and response rates are high. Furthermore, the particular choice conditions used here do not capture all elements of a free economy, as heroin was only available during operant sessions –90. ThisDespite the considerations noted above, the specificity of the effects of DORA-12 and oxytocin on cued heroin seeking suggest that these therapies may be particularly effective at preventing relapse triggered by drug-associated cues. Though we did not explore the neural circuitry underlying these therapeutic effects here, the fact that DORA-12 and oxytocin affected the same behavioral outcomes, but in opposite directions, raises the possibility that their actions are mediated via similar brain regions and mechanisms. A potential common site of action is the basolateral amygdala; orexin inputs to the basolateral amygdala, particularly involving the Ox1R, have been implicated in cued reinstatement of drug seeking , and eviPmax. Notably, the maximum amount of effort exerted to maintain drug consumption at Pmax, has received considerable attention as a reliable predictor of SUD severity in humans, particular for alcohol. In humans with recent binge drinking episodes, the demand curve for alcohol becomes more inelastic [Although neither oxytocin nor orexin cell counts correlated with heroin seeking during the cued reward seeking test, correlations with α were observed. The variable α has been shown repeatedly to correlate with drug-seeking for multiple drugs of abuse  and is anelastic , and evenelastic , again snelastic . CollectIn contrast with the negative correlation observed between orexin cell numbers and heroin α, oxytocin cell numbers positively correlated with both heroin α and food α, highlighting the bidirectional functional role of these systems in motivation and suggesting a more general involvement of the oxytocin system in motivation for both drug and non-drug rewards. For instance, acute administration of oxytocin decreases sucrose taking during self-administration as well as seeking triggered by sucrose cues . While tFinally, both oxytocin and orexin play an important role in arousal and sleep, and soporific effects of exogenous oxytocin and DORAs have been reported under some circumstances. However, it is unlikely that the behavioral effects of oxytocin and DORA-12 on opioid-seeking behavior observed here are the result of impairment in general activity, as (1) these compounds had no effect on choice behavior, a task that required sustained operant responding across the session; and (2) in the case of DORA-12, several studies have reported no effect of similar compounds on animals’ locomotor activity profile, low-effort drug intake, or performance on cognitive tasks that require a high level of task engagement and responding –49. NeveIn sum, these results point towards the hypothalamic oxytocin and orexin systems as two potential therapeutic targets capable of bidirectionally regulating reward seeking in a world of competing drug and non-drug cues. Since relapse rarely occurs in an isolated setting where only drug cues are available, these results have implications for relapse that occurs under conditions where alternatives are available. Future studies should thoroughly examine the potential to exploit the oxytocin and orexin systems to achieve remission in SUDs, by biasing choice towards natural rewards and reducing craving triggered by drug-associated cues.Supplemental material


Post-traumatic stress disorder (PTSD) and opioid use disorder (OUD) are comorbid in clinical populations. However, both pre-clinical and clinical studies of these co-occurring disorders have disproportionately represented male subjects, limiting the applicability of these findings. Our previous work has identified chronic escalating heroin administration and withdrawal can produce enhanced fear learning. This behavior is associated with an increase in dorsal hippocampal (DH) interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and glial fibrillary acidic protein (GFAP) immunoreactivity. Further, we have shown that these increases in IL-1β and TNF-α are mechanistically necessary for the development of enhanced fear learning. Although these are exciting findings, this paradigm has only been studied in males. The current studies aim to examine sex differences in the behavioral and neuroimmune effects of chronic heroin withdrawal and future enhanced fear learning. In turn, we determined that chronic escalating heroin administration can produce withdrawal in female rats comparable to male rats. Subsequently, we examined the consequence of heroin withdrawal on future enhanced fear learning and IL-1β, TNF-α, and GFAP immunoreactivity. Strikingly, we identified sex differences in these neuroimmune measures, as chronic heroin administration and withdrawal does not produce enhanced fear learning or immunoreactivity changes in females. Moreover, we determined whether heroin withdrawal produces short-term and long-term anxiety behaviors in both female and males. Collectively, these novel experiments are the first to test whether heroin withdrawal can sensitize future fear learning, produce neurobiological changes, and cause short-term and long-term anxiety behaviors in female rats. Post-traumatic stress disorder (PTSD) and opioid use disorder (OUD) are highly comorbid in clinical populations, as nearly 35% of individuals with an opioid use disorder have comorbid PTSD   paradigm ; however, 7 days following withdrawal, only males exhibited these symptoms. Lastly, experiment 4 tested whether heroin withdrawal affects IL-1β, TNF-α, and GFAP immunoreactivity in females. Collectively, these novel experiments are the first to test whether heroin withdrawal can sensitize future fear learning, produce neurobiological changes, and cause short-term and long-term anxiety behaviors in female rats.Adult female and male Sprague–Dawley rats  were individually housed under a reversed 12-h light/dark cycle. Rats were given ad libitum access to food and water and were regularly handled throughout experimentation. All procedures were conducted with approval from the University of North Carolina at Chapel Hill Institutional Animal Care and Use Committee.Heroin  Drug Supply Program, Bethesda, MD, USA) was dissolved in sterile 0.9% saline to produce 1.0, 2.5, 5.0, 7.5, or 10.0 mg/mL solutions and stored at 4 °C until time of injection.Animals were randomly assigned to drug  treatment and heroin withdrawal timepoint  and underwent chronic escalating heroin administration as described previously , two opposing open arms (30 cm × 5 cm), and two opposing closed arms (30 cm × 5 cm) with a 15-cm height . Each animal was placed in the central part of the maze facing an open arm at the beginning of the session. The maze was cleaned with 75% ethanol and dried between each use. Exploratory behavior on the maze was filmed with a GoPro HERO10 camera, and later measured by an observer blind to experimental condition. Open/closed arm entries, open/closed arm time, and stretch attend postures were measured.Estrous cycle was assessed during the experiment at the following time points: 24-h withdrawal time point, B-shock, and test days 1, 2, 7, and 14. During assessment, the animals were restrained and the animal’s tail was elevated to visualize the vagina. The vaginal cells were flushed by gently introducing approximately 100 μl of saline using a sterile transfer pipette. The pipette was placed at the entrance of the vaginal canal, and saline was slowly released into vagina and then drawn back into the tip about 4 to 5 times in the same pipette. The fluid was then introduced on a glass slide, air-dried, and stained using a Wright Giemsa Stain, modified (Sigma-Aldrich) method. Vaginal cell distribution was examined using a Leica DM 6000B microscope  and estrous phase  was determined according to methods described by Cora and colleagues  ketamine hydrochloride (100 mg/mL) mixed with xylazine (100 mg/mL), and transcardially perfused with ice-cold phosphate buffer  followed by 4% paraformaldehyde in 0.1 M PB. Brains were extracted and post-fixed in 4% paraformaldehyde for 6 h, and used 30% sucrose for cryoprotection with 0.1% sodium azide at 4 °C. Once the brains were saturated with sucrose, brains were cut into 40-μm coronal sections on a cryostat .Fluorescent immunohistochemistry (IHC) was used to examine alterations in dorsal hippocampus (DH) TNF-α, IL-1β, and GFAP in the dentate gyrus (DG) at the 24-h heroin withdrawal time point. The IHC protocol used here has been described previously  was used to capture color images. The microscopy protocol used here has been described previously  × 7 (Day) repeated measures ANOVA. Weight change and withdrawal composite score were analyzed using an unpaired, two-tailed Student’s t test. Weight change sex comparison was analyzed using a 2 (Drug Treatment) × 2 (Sex) ANOVA. In experiment 2, a two-way  ANOVA was used to analyze baseline freezing data. A 2 (Drug Treatment) × 2 (Sex) × 4 (Test Day) ANOVA was used to analyze freezing behavior across test days between sexes. Significant interactions between main effects in ANOVAs of experiment 2 were examined using Tukey’s post hoc comparisons. In experiment 3, a 2 × 2 ANOVA analysis was used to analyze elevated plus maze behavior. Unpaired, two-tailed Student’s t tests were used to analyze the differences between saline-treated and heroin-treated animals within groups in the EPM as well as the stretch attend posture behaviors. In experiment 4, IL-1β, TNF-α, and GFAP immunoreactivity and cell counts were analyzed using an unpaired, two-tailed Student’s t test.In experiment 1, withdrawal behaviors were analyzed using a 2 analysis revealed no difference between the heroin withdrawal and saline controls for rearing behavior (χ2n = 23). Importantly χ2 analysis revealed significant increases in withdrawal behaviors between the heroin withdrawal and saline controls for wet dog shakes (χ2n = 23), teeth chattering (χ2n = 23), diarrhea (χ2n = 23), and weight loss (χ2n = 23)  = 28.824, p < 0.001) , indicating no pre-existing weight differences. Additionally, both heroin and saline animals gained weight relatively similarly. Student’s t test revealed a significant difference between heroin and saline animals on day 11 (t(20) = 2.618, p = 0.016), revealing heroin animals lost weight following the 24-h withdrawal in comparison to female animals. Student’s t test comparing weight change between day 10 and day 11  revealed that heroin-treated animals lost significantly more weight than saline-treated animals (t(21) = 7.212, p < 0.001)  = 102.418, p < 0.001), but no main effect of sex  Fig. . A 2 × 701) Fig. . Student01) Fig. . A 2 × 2sex Fig. , indicatt test comparing composite withdrawal scores between heroin-treated animals and saline-treated animals revealed that heroin-treated animals had a significantly higher composite withdrawal score than saline-treated animals (t(21) = − 2.922, p = 0.008)  Fig. . These r08) Fig.  for maleF = 1.425, p = 0.242), indicating that there was no heroin-produced generalized fear to the novel context. There was a main effect of sex  = 49.355, p < 0.001), indicating that male animals had a lower baseline freezing to the novel context than females. To avoid stress to female rodents induced by male pheromones, the sexes were run at different times, so slight differences in baseline freezing can be expected. However, all animals had below the 20% freezing threshold for baseline. A 2 (sex) × 2 (drug) × 4 (test day) repeated measures ANOVA was used to analyze test days and revealed a significant main effect of heroin treatment  = 10.380, p = 0.003) and a significant main effect of sex  = 7.932, p = 0.008). These main effects of heroin treatment and sex were on contextual freezing. There was also a significant effect of test day  = 45.512, p < 0.001), indicating that conditioned freezing behavior diminished over time. Importantly, there was a significant heroin treatment by sex interaction  = 12.333, p = 0.001). Tukey’s post hoc comparisons revealed heroin-treated males exhibited significantly higher freezing behaviors to saline-treated males and all female animals on test days 1 and 2 (p < 0.05)  ANOVA was used to analyze baseline freezing. There was no main effect of heroin treatment on baseline contextual freezing (05) Fig. . These rn = 8–12) when split across stage did not yield a large enough sample size to probe effects of estrous on heroin withdrawal behaviors and enhanced fear learning. Therefore, in experiments 1 and 2, samples in diestrus and proestrus were characterized as “increasing/high estradiol,” while those in estrus and metestrus were charactered as “low estradiol” , indicating that estrous cycle played no role in the female heroin withdrawal behaviors. There was also a significant effect of drug  = 16.047, p < 0.001), indicating that heroin-withdrawn animals still lost weight , indicating that there is no effect of estrous cycle on baseline freezing. Additionally, 2 (drug) × 2 (estrous cycle) × 4 (test day) repeated measures ANOVA revealed no significant main effects of heroin treatment (p > 0.05) or estrous cycle (p > 0.05), indicating that there is no effect of estrous cycle on contextual freezing. There was a significant effect of test day  = 5.624, p = 0.034), indicating that conditioned freezing behavior diminished over time  = 28.775, p < 0.001) and closed arms  = 21.636, p < 0.001. There was no main effect of sex in the open arms or closed arms, but a significant main effect of sex in the center  = 6.370, p = 0.017). Post hoc tests revealed, 24 h following heroin administration, female heroin-treated animals spent less time in the open arms (t(18) = 4.310, p < 0.001), more time in the closed arms (t(18) = − 4.329, p < 0.001), and an equal time in the center (t(18) = − 1.238, p = 0.232) compared to their saline-treated counterparts  = 3.213, p = 0.006), more time in the closed arms (t(15) = − 2.631, p = 0.019), and an equal time in the center (t(15) = − 0.027, p = 0.979) compared to their saline-treated counterparts  = 9.061, p = 0.005), closed arms  = 6.841, p = 0.014), and center  = 5.035, p = 0.033). There was a main effect of sex in the closed arms  = 4.384, p = 0.045) and center  = 18.486, p < 0.001). Post hoc tests revealed 7 days following heroin administration, the time spent in open arms (t(13) = 0.841, p = 0.416), closed arms (t(13) = − 0.976, p = 0.347), and center (t(13) = − 0.107, p = 0.917) was not significantly different in female heroin-treated animals and saline-treated animals  = 4.880, p < 0.001), less time in the closed arms (t(15) = − 3.504, p = 0.003), and more time in the center (t(15) = − 3.309, p = 0.005) compared to their saline-treated counterparts  = − 12.059, p < 0.001) and males (t(13) = − 12.953, p < 0.001) exhibited significantly higher behavior counts than their saline-treated counterparts. Seven days following heroin administration, heroin-treated females and saline-treated females had no significant difference in behaviors (t(12) = 0.178, p = 0.862); however, heroin treated males exhibited significantly higher behavior counts than their saline-treated counterparts (t(11) = − 9.797, p < 0.000)  Fig. . This fut(19) = − 1.232, p = 0.233) and cell counts (t(19) = − 0.021, p = 0.983) were not significantly enhanced by heroin withdrawal in females  = 0.692, p = 0.497) and cell counts (t(20) = 0.147, p = 0.885) were not significantly enhanced by heroin withdrawal in females  = − 0.065, p = 0.949) and cell counts (t(20) = 1.785, p = 0.089) were not significantly enhanced by heroin withdrawal in females  or low estradiol (estrus and metestrus) stages. We demonstrate in the current studies that females in both the high and low estradiol phases exhibit heroin withdrawal behaviors as well as freezing.Although we did not see any evidence of enhanced cytokine production in females following heroin withdrawal, or an effect of estrous cycle on heroin withdrawal behaviors or enhanced fear learning, other immune measures may be modified by female sex hormones. The current study was optimized to maximally detect changes in heroin withdrawal-induced cytokines especially in the dorsal hippocampus, as this is a direct comparison to our previous work in males  and found that heroin withdrawal produces short-term anxiety in both males and females (observed at the 24-h time point), but surprisingly, only produces long-term anxiety in males (assessed at the 7-day time point). We predict that the two anxiety behaviors we observe following withdrawal  are mediated by separate, distinct biological mechanisms and neural circuitry than fear learning behaviors. In turn, alterations in hippocampal cytokine levels may not be related to the enhanced generalized anxiety response, and may instead be mediated by other mechanisms, such as the reward system. For example, the central nucleus of the amygdala is well-established in increasing reward thresholds, anxiety-like responses, and extracellular levels of corticotropin-releasing factor during opioid withdrawal (Back et al. In summary, our exciting findings demonstrate that heroin administration and withdrawal produce withdrawal behaviors in females that are similar to those of males. However, chronic escalating heroin administration does not produce enhanced fear learning in female rats or increased immunoreactivity of IL-1β, TNF-α, and GFAP in the dentate gyrus of the dorsal hippocampus in female rats as previously seen in males. Additionally, heroin withdrawal produces short-term anxiety in both males and females, but only produces long-term anxiety in males. Collectively, these data provide important new evidence of sex differences following heroin withdrawal and evidence that females may have a differential fear-learning and anxiety response to stress. The current study demonstrates the need to further study sex differences in both drug and trauma responses to uncover the neurobiological mechanisms underlying sex differences in symptoms.