IgG4 related disease (IgG4-RD) is a rare, immune-mediated inflammatory disease that varies widely in its presentation because it can affect nearly any organ. We present a case of a 73-year-old male who presented with an ill-defined mass of the parotid gland, found to be IgG4-RD, after several months of work up and tissue sampling. Most cases of salivary gland involvement in IgG4-RD present as bilateral swelling of the submandibular glands. We present this case as a unique manifestation of salivary gland disease in IgG4-RD as a persistent, non-discrete unilateral mass in the parotid gland. It is critical that clinicians who regularly treat salivary gland pathologies are familiar with this rare disease and its potential manifestations in the oral cavity. IgG4-related disease (IgG4-RD) is a rare, immune-mediated inflammatory disease that can affect nearly every organ. It is characterized by lesions of tumor-like swelling, lymphoplasmacytic infiltrate rich in IgG4-staining plasma cells, storiform fibrosis, and elevated serum IgG4 levels . The disClinicians that regularly treat salivary gland pathologies must be well-familiarized with IgG4-RD for early diagnosis and treatment. We present one case of IgG4-RD presenting as a persistent, fluctuating, non-discrete, unilateral mass in the parotid gland.A 73-year-old male presents to the otology clinic with right-sided otalgia, with pain around the pre- and post-auricular area. Initially, the physical exam did not exhibit a palpable mass, and the patient was closely followed. When the patient returned two months later, the physical exam showed an ill-defined 4-cm hypermobile mass over the right parotid that was tender to palpation. He did not have any focal neurological deficits. MRI showed an infiltrative, ill-defined, enhancing mass in the right parotid gland that involved both the superficial and deep lobes with ipsilateral lymphadenopathy in levels 2A and 2B Figure . Fine neAt the time of the incisional biopsy, the patient had an infected tooth root and implant. The patient’s symptoms were attributed to the odontogenic infection. He was treated with oral antibiotics and underwent tooth extraction and replacement of the implant. The patient also had a basic autoimmune workup, which incidentally found the patient to have a latent tuberculosis infection and was placed on appropriate treatment.The patient returned to the clinic several months later with no change in mass size. Given the persistence of symptoms, he underwent a right parotidectomy with facial nerve monitoring. With the results of the FNA and the appearance intraoperatively, the mass was presumed to be lymphoma and was transected at the level of the nerve leaving the deep lobe to minimize the risk of facial nerve paralysis.The final pathology showed focal suppurative granulomatous sialadenitis, periductal fibrosis, reactive lymphoid hyperplasia, and a marked increase in IgG4-staining plasma cells, indicative of IgG4-RD.The patient was referred to rheumatology, and a CT chest/abdomen/pelvis was reassuring for isolated disease to the right parotid gland. He was started on a steroid taper and did well with the resolution of the parotid gland swelling. He healed from surgery with transient weakness of the upper branch of the facial nerve.IgG4-RD was only recently recognized as a systemic disease in 2003 [The disease typically affects middle-aged to elderly males, unlike typical autoimmune conditions. Patients usually present with single-organ involvement but, upon further work-up, are found to have involvement of other organs .40% of cases of systemic IgG4-RD have major salivary gland involvement. The submandibular glands were the most commonly involved (94%), while the parotid glands (29%) and sublingual glands are less commonly involved . The mosMiculicz’s disease and Küttner’s tumor are salivary gland diseases that fall on the IgG4-RD spectrum, both of which previously had been considered a subcategory of Sjogren's Syndrome (SS). Miculicz’s disease is characterized by bilateral swelling of the lacrimal, parotid, and submandibular glands with histologic characteristics of mononuclear cell infiltration, while Küttner’s tumor is defined by unilateral or bilateral swelling of the submandibular glands with histologic characteristics of fibrosis mononuclear cell infiltration ,9.The workup for IgG4-RD can be challenging as it presents with relatively non-specific findings. A thorough history and physical exam, along with lab testing, imaging, and histopathological examination, are critical.Laboratory testing with elevated serum IgG4 was previously a hallmark of the disease. However, recently, it has been determined that this is not necessary for diagnosis because elevated IgG4 levels are non-specific for IgG4-RD, and low levels are not sufficient to rule out the disease. For example, other diseases like autoimmune conditions, lymphoma, and ANCA-associated vasculitis are also associated with elevated IgG4 levels. Therefore, elevated serum IgG4 should raise suspicion for IgG4-RD but does not confirm the diagnosis .While there are no definitive diagnostic requirements for IgG4-RD, proposed diagnostic criteria include diffuse/localized swelling or mass lesions, elevated serum IgG4 levels, and histopathologic findings of lymphoplasmacytic infiltration and fibrosis with IgG4-staining plasma cells . Needle There are a variety of imaging techniques that can be used to investigate salivary gland pathologies. Ultrasonography remains the most used. Salivary gland involvement in IgG4-RD and SS can have similar ultrasonographic features but, recently, color Doppler has been shown to identify specific features of IgG4-RD in salivary glands, such as increased Doppler signaling ratios. PET is also being investigated as a diagnostic tool for IgG4-RD. While it cannot distinguish between inflammatory and malignant lesions, it can be useful in determining whether multi-organ involvement is present .The treatment is widely variable for IgG4-RD. Watchful waiting is often recommended for indolent courses of IgG4-related lymphadenopathy, especially when asymptomatic. Glucocorticoids remain the mainstay of treatment without well-defined guidelines surrounding dosing and duration. Glucocorticoids are commonly effective in initial treatment, but disease flares are common . While tWith regard to other treatment options, there is a lack of evidence surrounding steroid-sparing treatment options. The most commonly used non-steroid treatment was azathioprine, followed by mycophenolate mofetil and methotrexate. However, several patients have experienced relapses after the use of these treatments. Biologic agents, such as rituximab, have shown promise in treating IgG4-RD and are being investigated as treatment options after a relapse. However, there is still a lack of randomized controlled trials supporting this evidence .IgG4-RD is a rare and diagnostically challenging disease for clinicians. It can affect nearly every organ of the body and commonly presents with multi-organ involvement. When there is salivary gland involvement, presentation typically includes bilateral swelling, most commonly in the submandibular glands. However, this case highlights a unique presentation of IgG4-RD as an isolated, unilateral mass in the parotid gland. Our case is especially unique because of its non-discrete, fluctuating characteristics. It is critical that clinicians that encounter salivary gland pathologies, even when unilateral, are aware of the potential presentations of IgG4-RD in the head and neck region for early diagnosis and treatment.


IgG4-related disease (IgG4-RD) is a systemic autoimmune disorder that has been defined in various organs. The disease is characterized by typical clinicopathological features including a dense lymphoplasmacytic infiltrate rich in IgG4 positive plasma cells, storiform fibrosis, obliterative phlebitis, and often an elevated serum IgG4 level. Esophageal IgG4-RD is rare, and its presentation as a solid mass is even more rare. Only 15 previous cases of IgG4-related esophageal disease have been described. We herein present a case of giant IgG4-related pseudotumor of the esophagus resected with endoscopic submucosal dissection (ESD) and a review of the literature. The patient was a 67-year-old man who was admitted to our hospital for assessment of progressive dysphagia. Upper gastrointestinal endoscopy revealed a 9 cm mass in the cervical esophagus. In the previous two hospitals, the patient’s mass could not be diagnosed despite repeated biopsies. Because of concerns regarding malignancy, endoscopic submucosal dissection was performed. Histopathological examination showed dense lymphoplasmacytic infiltration with predominant IgG4-positive plasma cells on a sclerotic background. The patient was diagnosed with IgG4-RD. During the follow-up, no residual mass was detected but the patient was diagnosed with lung adenocarcinoma. We present a unique case of giant IgG4-related pseudotumor of the esophagus. Resection with ESD of such a big mass of IgG4-RD in the esophageal region has never been reported before in the literature. IgG4-related disease (IgG4-RD), originally described in the pancreas by Sarles et al. in 1961 . It has A 67-year-old Caucasian man presented with progressive dysphagia to solids over the last 2 years. His past medical history was uneventful. He had no other autoimmune diseases. He was an ex-smoker with a smoking history of 43 pack-years. The patient had previously visited 2 different hospitals, where he underwent upper gastrointestinal endoscopy, neck computed tomography (CT), and FDG-positron emission tomography (PET) because of the suspicion of malignancy. Upper gastrointestinal endoscopy revealed a mass-forming lesion prolapsing into the lumen of the cervical esophagus. The neck CT demonstrated a suspicion of gastrointestinal stromal tumor (GIST) and no concomitant lymphadenopathy. FDG-PET showed that the SUVmax of the mass was 10.0. Because of the suspicion of esophageal cancer, multiple biopsies were taken from the mass. However, the biopsy specimens were evaluated as squamous epithelial hyperplasia and inflammatory granulation tissue histologically which did not explain the tumoral nature of the lesion.The patient presented to the gastroenterology department of our hospital. The physical examination was unremarkable. All laboratory data were within normal limits. Upper gastrointestinal endoscopy revealed an approximately 9 cm polypoid tumor partially obstructing the esophageal lumen, located about 15 cm from the incisors . BecauseHe underwent a successful ESD. After marking the lesion, a mixture of saline, indigocarmin and hyaluronic acid was locally injected into the submucosal space to elevate the lesion. The borders were marked circumferentially using a needle knife tip  and careful dissection of the lateral borders and submucosal space was performed. En bloc removal of the lesion was performed successfully and it was sent to the pathology department for histological evaluation. Cauterization for bleeding was accomplished and enteral feeding was started in order to allow the esophageal mucosa to heal.Gross examination of the resected specimen revealed a polypoid tumor that measured 9x7x5 cm with mucosal surface erosion and a tan-white, solid, partially rubbery cut surface . HistoloWith the help of histopathological findings raising concern for IgG4-RD, the serum IgG4 level was checked afterwards. Ten days after the ESD procedure, the patient’s serum IgG4 level was found to be elevated at 179 mg/dL . Based on comprehensive diagnostic criteria for IgG4-RD , the patThe patient did not receive any medication including corticosteroid therapy after the ESD procedure. He underwent neck and thorax CT two months after the ESD procedure. Neck CT showed no residual mass but unexpectedly the thorax CT showed a solid mass with an irregular spiculated contour of 24 mm size in the perihilar area of the lower lobe in the left lung. FDG-PET showed that SUVmax of the mass was 14.68 and there were concomitant hypermetabolic lymph nodes in the mediastinum. Due to the suspicion of lung cancer, bronchoscopy was performed. However, the mass could not be sampled despite repeated bronchoscopies. Bone scintigraphy showed a lesion suspicious in terms of metastasis in the left iliac crest. Biopsy taken from the lesion was evaluated as adenocarcinoma favoring the lung as primary origin. The patient received chemotherapy but unfortunately, 21 months after the ESD procedure, he passed away with unknown cause, and no autopsy was performed.IgG4-RD is an immune-mediated fibroinflammatory condition that has been described in a variety of organs . AccordiIn IgG4-related pancreatitis, especially in resection specimens, it has been reported that >30 IgG4+ plasma cells per HPF have acceptable specificity, and >50 IgG4+ plasma cells per HPF is highly specific. However, it is emphasized that the appropriate cut-off point may vary from organ to organ .In 2019, American College of Rheumatology/European League Against Rheumatism developed the classification criteria for IgG4-RD as validated in a wide cohort of patients . The claEsophageal involvement of IgG4-RD (IgG4-RDE) was uncommonly reported in the literature. To our knowledge only 15 cases ,14–18 haIn previous reports, the size of the IgG4-RDE mass in cases with one ranged from 1.5 cm to 3.9 cm ,8. The cIn 2017, a series of IgG4-RDE cases that consisted of 8 subjects was compared with chronic esophagitis, not otherwise specified, by Obiorah et al. Storiform fibrosis and high-density lymphoplasmacytic infiltrate were present in all cases with IgG4-RDE, while obliterative phlebitis was found in only 3 of 8 cases . It was Steroid therapy is an effective and primary choice for the treatment of IgG4-RD . In our IgG4-RD is considered an autoimmune disorder, and malignancy is associated with the dysregulated immune system . Some coIn conclusion, IgG4-RDE can present with a mass and may lead to a misdiagnosis of malignancy, both clinically and radiologically. Diagnosis of IgG4-RDE requires a careful sampling of the lesion in order to perform a correct histopathological examination. However, it may be difficult to diagnose IgG4-RDE with small endoscopic biopsy specimens in some patients. In cases of suspicion, the biopsy/resection specimens should be stained with IgG4 and IgG immunohistochemically and the serum IgG4 level of the patient should be checked. In patients with an esophageal mass, consideration of IgG4-RDE in the differential diagnosis by the clinicians and pathologists will ensure the correct diagnosis and prevent unnecessary overtreatment.None of the authors have any conflicts of interest to declare.


Numerous mechanisms have shown that long noncoding RNAs (lncRNAs) promote the development of colorectal cancer (CRC), but the role of lnc-LRRTM4 in the progression of CRC remains unclear. In this article, we found that lnc-LRRTM4 was highly expressed in CRC tissues and cell lines and that lnc-LRRTM4 could promote the proliferation and metastasis of CRC cells. These consequences were achieved by lnc-LRRTM4 directly binding to the promoter of LRRTM4 to induce its transcription. Moreover, lnc-LRRTM4 enhanced the growth of CRC cells in vivo by promoting cell cycle progression and reducing apoptosis. Taken together, our results revealed that lnc-LRRTM4 promotes the proliferation and metastasis of CRC cells, suggesting that it may be a potential diagnostic and therapeutic target for CRC.The online version contains supplementary material available at 10.1186/s12935-023-02986-8. Colorectal cancer (CRC), a rapidly growing and easily metastatic solid tumor, has become the third leading cause of morbidity and mortality among all kinds of tumors . MeanwhiThe process of epithelial-mesenchymal transformation (EMT) involves the transdifferentiation of epithelial cells into motile mesenchymal cells , it is iLong noncoding RNAs (lncRNAs) are a class of noncoding RNAs that are longer than 200 bp and generally do not have the ability to encode proteins. In a variety of tumor-related studies, it has been confirmed that lncRNAs participate in the tumorigenesis and development of cancers by regulating EMT progression , for exaIn this study, we identified that lnc-LRRTM4 is highly expressed in CRC tissues and cell lines and that high expression of lnc-LRRTM4 predicts poor overall survival (OS) and disease-free survival (DFS) rates in CRC. Further studies confirmed that lnc-LRRTM4 promotes proliferation, metastasis and EMT by binding to the promoter of LRRTM4 to enhance its transcription. We also verified that lnc-LRRTM4 accelerates the growth of CRC cells in vivo. Collectively, these results revealed that lnc-LRRTM4 plays a carcinogenic role in CRC.This study was approved by the ethics committee of The Second Affiliated Hospital of Baotou Medical College, and informed consent of patients was obtained for surgical specimens. 50 pairs of CRC and para-tumor tissues were collected from 2019.09.01 to 2021.09.01 at the Department of General Surgery, The Second Affiliated Hospital of Baotou Medical College.2.The human normal colonic epithelial cell line FHC and CRC cell lines HCT116, SW480, DLD1, LoVo, HCT8, SW620 and HT29 were purchased from National Collection of Authenticated Cell Cultures . HCT116 and HT29 cells were cultured in McCoy’s 5 A medium  with 10% FBS , 1% penicillin and streptomycin  while FHC, SW480, DLD1, LoVo and HCT8 cells were cultured in RPMI 1640 medium and SW620 cells were cultured in L15 medium. All cells were cultured at 37℃ in a humidified incubator with 5% COThe CRC cells were transfected with siRNA and plasmid  using Lipofectamine 2000 transfection reagent  according to the manufacturer’s protocol. Briefly, cells were transfected with siRNA at about 30% alignment and with plasmid at about 80% alignment using serum-free medium and incubated for 6 h, then cells were washed with PBS and replaced with fresh complete medium, cells were collected for further experiments 48 h after transfection. The sequences of siRNA are as follows:si-lnc-LRRTM4#1:5’-GGAACUACAGAAGGACCAATT-3’.si-lnc-LRRTM4#2:5’-GCUCCUGUCCUUUCUUGAUTT-3’.si-LRRTM4#1:5’-GAGGGTCACAAGGCTTATCAT-3’.si-LRRTM4#2:5’-GCAAGCTCATCGTTCCTGTTA-3’.si-Ctrl:5’-UUCUCCGAACGUGUCACGUTT-3’.Total RNA was extracted using RNA isolater Total RNA Extraction Reagent after transfection, then were reversed using HiScript Q RT SuperMix for qPCR and detected using AceQ qPCR SYBR Green Master Mix  according to the manufacturers’ protocol, the expression was detected using LightCycler480-II fluorescence quantitative PCR instrument  and GAPDH was used as reference. The sequences of primers are as follows:lnc-LRRTM4-Forward:5’-CTGGGCTCAAGCAATCT-3’.lnc-LRRTM4-Reverse:5’-CAGGACCTAGTTCAAAGTG-3’.LRRTM4-Forward:5’-GGCCTTAACCAGCTTATATGGC-3’.LRRTM4-Reverse:5’-TTGGGAACTGGGTGAAATGTTT-3’.GAPDH-Forward:5’-CTGGGCTACACTGAGCACC-3’.GAPDH-Reverse:5’-AAGTGGTCGTTGAGGGCAATG-3’.LRRTM4 Promoter-Forward:5’- TCCTTGGCATAAACCT-3’.LRRTM4 Promoter-Reverse:5’- ACGGGAACAAGTGAGC-3’.Total protein was extracted using RIPA lysis buffer  and the concentration was detected using BCA Protein Assay Kit , then protein was separated in 10% SDS-PAGE and transferred to PVDF membrane , after incubation with primary and secondary antibodies, every band was exposed in Chemiluminescene Imager  using Ultra-sensitive ECL Chemiluminescene Kit . The antibodies used are as follows: anti-LRRTM4 , anti-caspase3 , anti-caspase9 , anti-Cyclin D1 , anti-Cyclin E2 , anti-E-cadherin , anti-N-cadherin , anti-SNAL1 , anti-GAPDH .3 cells were plated into the 96-well plate, then the cells were incubated with 100 µl serum-free medium and 10 µl CCK-8 solution  at 37℃ for 2 h and detected the optical density (OD) at 450 nm.Cells after transfection were collected and counted, 4 × 105 cells in serum-free medium were placed above the dish with or without MatriGel for 24 and 48 h, the dishes were fixed with 4% paraformaldehyde and stained with Crystal Violet solution , the images were acquired using the Inverted microscope .MatriGel  was mixed with serum-free medium at 1:9 proportion and placed above Insert cell culture dish  the night before and placed in a 37℃ incubator, then 1 × 10Cells after transfection were collected using trypsin without EDTA  and fixed with 75% alcohol overnight, then cells were incubated with RNase and PI for 30 min at RT and detected by Flow Cytometry  according to the manufacturer’s protocol of Cell Cycle Detection Kit (KeyGEN BioTECH).Cells were collected in the same way as cell cycle detection assay, then cells were incubated with Annexin V and PI solution for 10 min at RT and detected by Flow Cytometry  according to the manufacturer’s protocol of Apoptosis Detection Kit (KeyGEN BioTECH).FISH was assessed with a Fluorescent In Situ Hybridization Kit  according to the manufacturer’s protocol, specific probes against lnc-LRRTM4 was also synthesized by RiboBio. Cells were fixed with 4% paraformaldehyde  and permeabilized with 0.5% Triton X-100/PBS, then the cells were prehybridized at 37 °C and hybridized with lnc-LRRTM4 fluorescent probes at 37 °C overnight. On day 2, the cells were stained with DAPI and observed with a confocal microscope .The experiment was conducted according to the manufacturer’s protocol of ChIRP Kit . Cells were cross-linked with 1% paraformaldehyde and lysed by sonication, then biotin-labeled probe and magnetic beads were incubated with the lysis solution, at last the DNA was extracted from magnetic beads and detected by qPCR.Luciferase report assay was conducted according to the manufacturer’s protocol of Dual Luciferase Reporter Gene Assay Kit . Cells transfected with pGL3-basic plasmid containing LRRTM4 promoter  and pRL-TK control plasmid with or without lnc-LRRTM4 siRNA were collected for lysis, then the firefly luciferase detection reagent and renilla luciferase detection reagent was added into the solution and measured by Multifunctional microplate reader  separately. The sequence of LRRTM4 promoter used in pGL3-basic plasmid was as follow: 5’- ccttggcataaaccttttaaattattggagagaaaaaaatagtgattctgtgcctttaatttccctttctgagcaagggaaggctgcacaccactgacaggctcacttgttcccgt-3’.Tumors of xenografts were fixed in formaldehyde solution and embed into paraffin blocks, then tumors sections were performed and dewaxed and rehydrated using xylene and echelon alcohol, antigen of sections were repaired using citrate solution, blocked with goat serum and combined with antibodies. Finally, the sections were stained with DAB chromogenic agent and hematoxylin solution and images were acquired using the inverted microscope  according to the manufacturer’s protocol of IHC Kit .7 cells with lnc-LRRTM4 knockdown or control cells were injected into the flank region of mice and the tumor was measured every 5 days. At the 21 days after injection, the mice were sacrificed and the tumors were harvested. All animal experiments complied with the policies of the Animal Care Committee of The Second Affiliated Hospital of Baotou Medical College. The sequences of shRNA are as follows:Cells were generated by infection of sh-lnc-LRRTM4 lentivirus  with the help of Polybrene, then cells were selected with puromycin  for 2 weeks. Six-week-old male BALB/c nude mice  were obtained and randomly divided into 2 groups. 1 × 10sh-lnc-LRRTM4:5’-CACCGGGAACUACAGAAGGACCAATTTTCGAAAAAATTGATCAATGCCGAGGA-3’.sh-Ctrl:5’-CACCGTTCTCCGAACGTGTCACGTTTCGAAAAACGTGACACGTTCGGAGAA-3’.http://www.ncbi.nlm.nih.gov/geo/) and analyzed using R (https://www.r-project.org) to determine the differentially expressed lncRNAs, and then filter was carried out with logFC≤-2 or logFC ≥ 2 and p < 0.05 as the criterion, while the heatmap was formed with differentially expressed lncRNAs filtered with logFC≤-4 or logFC ≥ 4 so that the most significantly differentially expressed lncRNAs could be labeled in the picture. Similarly, we also used the data downloaded from TCGA database  to draw the survival curve with R.The data of GSE156732 was downloaded from GEO database .All values were expressed as mean ± standard deviation (SD). The significance of the difference was determined by student’s t-test or one-way ANOVA and Kaplan-Meier analysis was used for survival analysis. https://www.lncipedia.org) and named it lnc-LRRTM4 (data not shown). We assessed the expression of lnc-LRRTM4 in TCGA database and 50 pairs of CRC and para-tumor tissues, confirming that lnc-LRRTM4 was highly expressed in CRC tissues compared with para-tumor tissues  and found that LRRTM4 was the nearest gene of lnc-LRRTM4  , 27–29. d in cis . Currentd in cis . One of immunity , and P53igenesis . Since tigenesis , PD-L1-ligenesis . LRRTM4 igenesis , 37. Sevigenesis , 39, butTaken together, our results suggest that lnc-LRRTM4 could promote the proliferation, metastasis and EMT of CRC. Mechanistically, lnc-LRRTM4 binds to the promoter of LRRTM4 to enhance the transcription of LRRTM4 and thus promotes CRC progression. Our findings of this study could provide a reference for further understanding the tumorigenesis and development of CRC and formulating diagnostic and therapeutic strategies.Below is the link to the electronic supplementary material.Supplementary Material 1


Journal by P. Lazure et al. on the barriers in the implementation and functioning of antimicrobial stewardship programmes (AMS),1 and we would like to congratulate the authors for this interesting article and make some complementary contributions to this important topic from our experience in a university hospital in a Latin American country.We have read the recent article published in the et al. and that may play an individual-level significant role during antibiotic prescription in the clinical practice: It is the fear of patient deterioration or complications.In the article, it was indicated that the lack of knowledge and skills pertaining to best practices in the diagnosis of infections and antibiotic prescription is one of the existing barriers to the suitable functioning of AMS, concluding on the need to work on the education of health professionals involved in antibiotic prescribing, especially in countries where AMS may not be fully implemented. And, although we agree, we believe it is important to keep in mind an additional aspect that is not mentioned in the study by Lazure 2–4 However, we identified that some antibiotic prescriptions were not concordant with the guidelines, due to improper antibiotic, dose, interval or duration. During feedback sessions by the infectious diseases staff, a survey was conducted that asked about the reason for these prescriptions.At the Hospital Universitario Nacional de Colombia, a referral centre of high complexity in Bogotá , as part of the continual training activities for health professionals within the framework of the AMS, we developed a virtual course and a mobile application with the main recommendations of diagnosis and antibiotic prescription of the most prevalent infections.The survey was carried out during four consecutive months, from 1 June to 30 September 2022, and feedback was provided to healthcare professionals who prescribed an antibiotic that was not concordant with guidelines. The feedback was provided by telephone and reference to the clinical guidelines was also stated.n = 50) corresponded to prescriptions in patients undergoing surgical procedures, 16% (n = 11) in the setting of skin and soft tissue infections, and the remaining 9% of inappropriate prescriptions were identified in the management of community-acquired pneumonia (n = 4) and intra-abdominal infections (n = 2). Most of the cases with inappropriate prescriptions were found in general hospitalization , and the antibiotics used corresponded to: cefazolin or cefepime , combination therapy , and ampicillin/sulbactam or piperacillin/tazobactam . Ninety-three percent of the prescribers were male (n = 62), with an age range of 31 to 50 years (n = 38), and the main reason reported for inappropriate prescribing was fear of patient deterioration or complications , followed by rush or lack of time .In total, 485 antibiotic prescriptions were analysed, 67 cases of non-adherence to the recommendations were identified, 75% (5–7 in which fear and time pressure have been identified as common barriers for appropriate antimicrobial prescribing. It is worth mentioning the limited number of studies with this approach carried out in Latin American countries, another reason to congratulate the authors of the article published in your journal,1 which includes data from Mexico.These findings correlate with the results of studies conducted in other countries around the world,However, most of the strategies for the implementation and strengthening of the AMS still fail to address these issues. Therefore, we consider it necessary to highlight to readers, who can be stakeholders in the healthcare system from different perspectives, the importance of including these aspects of beliefs and attitudes when designing educational strategies for healthcare professionals involved in the prescription of antibiotics.


The power density and specific capacity of the Zn‐air battery with FeCoNC/SL as air cathode are as high as 224.8 mW cm−2 and 803 mAh g−1, respectively. Morphologically, FeCoNC/SL endows more reactive sites and accelerates the process of oxygen reaction. Density functional theory reveals the active site of the heteronuclear diatomic, and the formation of FeCoN5C configuration can effectively tune the d‐band center and electronic structure. The redistribution of electrons provides conditions for fast electron exchange, and the change of the center of the d‐band avoids the strong adsorption of intermediate species to simultaneously take into account both ORR and OER and thus achieve high‐performance Zn‐air batteries.Combining morphological control engineering and diatomic coupling strategies, heteronuclear Fe—Co bimetals are efficiently intercalated into nitrogen‐doped carbon materials with star‐like to simultaneously accelerate oxygen reduction reaction (ORR) and oxygen evolution reaction (OER). The half‐wave potential and kinetic current density of the ORR driven by FeCoNC/SL surpass the commercial Pt/C catalyst. The overpotential of OER is as low as 316 mV ( Morphology‐controlled engineering combined with diatomic coupling strategy to induce heteronuclear Fe—Co bimetallic with differentiated electron distribution accelerates oxygen reaction for efficient operation of zinc‐air battery energy storage devices. In this context, the development of a new generation of electric energy storage and conversion equipment is of great significance to the storage of clean energy and the application of different scenarios. Although lithium‐ion batteries have achieved great success in commercialization, they still have to face problems such as low energy density (less than 350 W h kgZn−1) and potential safety hazards. Different from lithium, sodium, potassium, magnesium, and aluminum‐air batteries, zinc‐air batteries have the advantages of high safety, low cost, higher energy density (about 1086 W h kgZn−1) and discharge voltage (about 1.65 V), so it has become an ideal energy storage and conversion equipment. At present, the performance of zinc‐air batteries (ZAB) is still limited by the slow oxygen reduction reaction (ORR) and oxygen evolution reaction (OER), so the development of efficient ORR and OER catalysts is the priority.Nearly 64.5% of the world's electricity supply comes from fossil fuel, which situation should be changed today when nonrenewable resources are continuously depleted and environmental problems are prominent.1, 2 However, the problems of Pt/C catalysts also affect its widespread use, such as high cost (scarce resources), poor electrochemical stability, and easy CO poisoning. In this context, researchers have to consider other potential ORR catalysts to achieve a balance between performance and cost. Fortunately, it has been inspired by biological porphyrin catalysts that single‐atom materials with M‐N‐C configuration are conducive to achieving moderate O2 adsorption (formation of M—O bonds), such as heme‐like Fe‐N‐C materials. Many ORR catalysts with M‐N‐C configuration have been developed and effective ORR has been achieved, including Fe‐N‐C, Co‐N‐C, Zn‐N‐C, Cu‐N‐C, Se‐N‐C, etc., these achievements have shaken the status of Pt/C catalysts to a certain extent. Even so, there is still a long way to go to design non‐noble metal single‐atom materials with ORR activities surpassing those of Pt/C catalyst. On the other hand, although the catalysts with OER activity are not Pt‐based materials, the catalysts exhibiting excellent activity are Ru‐ and Ir‐based materials, such as RuO2 and IrO2. The use of these noble metal compounds (or complexes) will also increase the cost burden, so it is also necessary to find ways to reduce costs while ensuring catalytic activity. Currently, many studies are focusing on the development of non‐noble metal‐based materials with OER activity, mainly Ni, Co, Fe, Mn and their oxides, carbides, or hydroxides. It has been reported that the OER activity of some non‐noble metal‐based catalysts has surpassed that of RuO2 or IrO2, such as Co/N‐CNT, Fe/Fe3C/MC, Co‐CeO2/C, and CoFe2O4. Non‐noble metal‐based catalysts have shown attractive potential in catalyzing OER, but they also face urgent problems to be solved. Among them, stability is the biggest obstacle restricting non‐noble metals and their oxides, carbides, and hydroxides as OER catalysts, whose physical and chemical properties will be changed in the redox reaction and thus lose OER activity. In this case, it is necessary to explore reasonable ways to improve the electrochemical stability of non‐noble metal catalysts, and the formation of a single‐atom state in a coordinated form may be the best way. For example, after the formation of FeN4, the central Fe atom will combine with N in the form of coordination bonds, so it can resist the destruction of oxidation or reduction potential. However, the OER activity of single‐atom catalysts seems to be at a low value, and the performance is far behind Ru‐ and Ir‐based materials, and some Co‐, Fe‐, and Ni‐containing oxides and hydroxides. Therefore, it is necessary to further regulate the state of the single‐atom central metal to stimulate its OER activity.As we all know, so far Pt/C materials are still the best catalyst for driving ORR and have been commercialized.7 Howe Among them, the formation of a bimetallic atom structure is more conducive to the splitting of O2, because the difference in electronegativity between different metals is conducive to the transfer of electrons. Jiang et al. reported that Co2@C2N has a higher adsorption energy for O2 than Co@C2N, the former being able to elongate the O—O of O2 to 1.47 Å, while the latter was only able to elongate to 1.33 Å. Therefore, Co2@C2N has better ORR activity than Co@C2N, which is attributed to the shift of the d‐band center after diatom formation. In the report of Sun et al., the spin polarization of Fe—Co diatoms can promote the parallel spin alignment of O2 molecules, thus promoting the activation of O2 and the formation of O—O bonds in OER. Another report also showed that the formation of Fe—Co diatoms can enhance the ORR and OER activities, in which the activity of Co is higher than that of Fe, because the presence of Fe can change the electronic structure of Co, thereby improving the bonding behavior of *O intermediates. These phenomena suggest that the introduction of another metal atom in the single‐atom site is expected to obtain bifunctional catalysts with ORR and OER activities. In addition, the morphology of the material will also affect the distribution (or exposure) of the active sites of the catalyst to a certain extent, so it is also one of the concerns. For example, zeolite imidazole frame (ZIF) material with large specific surface area is an excellent porous carrier.The performance of catalysts in terms of ORR and OER can be improved to a certain extent by adjusting the electronic structure of the metal site in the single atom or controlling the morphology of the single‐atom material.22 AmoE1/2) of the ORR with FeCoNC/SL participation reached 0.876 V, and the E1/2 of the commercial 20 wt% Pt/C, CoNC/SL, and FeNC/SL were only 0.866, 0.825, and 0.806 V versus RHE under the same conditions. At 0.4 V versus RHE, the kinetic current density (Jk) of FeCoNC/SL reaches 4.21 mA cm−2, which is much higher than the 3.29 mA cm−2 of commercial 20 wt% Pt/C. More beneficially, the power density of the Zn‐air battery assembled with FeCoNC/SL material as the air cathode is as high as 224.8 mW cm−2, and the specific capacity is closer to the theoretical value, reaching 803 mAh gZn−1. For ORR and Zn‐air batteries, FeCoNC/SL also exhibits fascinating electrochemical long‐lasting stability. These outstanding properties are all due to the high active site exposure ratio given by the bimetallic synergistic effect and the morphology engineering control carrier.Against the above background, here we try to find highly active bifunctional catalysts for ORR and OER through morphology regulation and construction of diatomic sites. Specifically, under the induction of surfactants, heteronuclear Fe and Co bimetal sites were successfully introduced into the starfish like ZIF framework (named FeCoNC/SL) to achieve efficient catalysis of ORR and the construction of alkaline Zn‐air cells with close to theoretical energy density. The half‐wave potential   is higher than that of Zn2+ (common in four coordination), therefore, Fe3+ and Co2+ can easily replace the Zn2+ ion that coordinated with 2‐methylimidazole. When the pyrolysis temperature is higher than 906°C, the Zn in ZIF‐8 will evaporate and leave the ZIF‐8 matrix, leaving only the dodecahedron nitrogen–carbon (CN) framework, but Fe and Co will not leave the NC framework. Based on these principles, a CN material with a star‐like morphology doped with Fe and Co bimetal was successfully prepared. After pyrolysis treatment at 950 °C, the material still retains the star‐like morphology before pyrolysis, and the material shows a tendency of inward shrinkage only due to the departure of metal Zn and the increase of surface energy. In the scanning electron microscopy (SEM) and aberration corrected scanning transmission electron microscopy (AC‐TEM) images , the signals of Fe and Co in FeCoNC/SL in element mapping are strong and evenly distributed . Similarly, the EXAFS of Co foil showed strong signals of Co—Co bonds, where the signals at 2.15 and 3.9 Å correspond to the first and second shells of the Co—Co bond , so it is easy to form Fe—N and Co—N bonds in situ after pyrolysis. The 2D color patch image obtained by EXAFS after wavelet transformation has carried out a visual transformation of EXAFS  spectrum, FeCoNC/SL, FeNC/SL, and CoNC/SL show consistent signal characteristics Figure 2a. Exceps Figure .29]29Figud Figure . The EXAS Figure . Fe foilS Figure . The higS Figure . In contS Figure , consist and the EXAFS and q space signal simulated by the FeN4C and CoN4C structure also conforms to the results of the experimental collection to a certain extent  saturated with O2, an electrochemical reduction peak appeared in the cyclic voltammetry (CV) curves obtained by using CoNC/SL, FeNC/SL, and FeCoNC/SL as catalysts, respectively  curve shows a similar trend to that of CV. The half‐wave potential (E1/2) and limiting current density (JL) of the LSV curve obtained when FeCoNC/SL was used as the RDE electrode are both higher than the commercial platinum‐carbon catalyst (20 wt% Pt/C), reaching 0.876 V and ≈5.8 mA cm−2 (the RDE rotation speed is 1600 rpm)  as a comprehensive performance parameter is the best choice for evaluating ORR performance. When the potential is 0.4 V versus RHE, the Jk of FeCoNC/SL exceeds 20 wt% Pt/C catalyst (3.29 mA cm−2) and is much higher than CoNC/SL and FeNC/SL, reaching 4.21 mA cm−2 −1 through the Koutecky–Levich equation, the slopes of the straight lines obtained by the fitting are all close to 4  Figure . In contR Figure . Kinetic2 Figure . To clard Figure . After t4 Figure . Therefoe Figure , which ie Figure . As we ae Figure . When us2.4Figure 2, the OER activity is much higher than that of CoNC/SL and FeNC/SL, and the overpotential is only 316 mV (at 10 mA cm−2). This phenomenon indicates that the co‐doping of Fe and Co has higher OER activity than single metal doping. In addition, FeCoNC/SL can obtain a lower Tafel slope when used as OER catalyst  is given. In other words, the oxidation thermodynamics of Pt within the OER window is irreversible. In contrast, Fe and Co in FeCoNC/SL existing in a high‐valence state can withstand the erosion of the high oxidation potential of OER. For example, after running 5000 cycles of cyclic voltammetry, the collected LSV curve is similar to the initial state, and the potential difference when the current density reaches 50, 100, and 200 mA cm−2 is only 0, 4, and 9 mV  is also important. This is because in practical applications, a reversible operation of a certain reaction is required to realize the reinitialization of the device, such as in the field of metal‐air batteries. Here, the activity characteristics of FeCoNC/SL as OER catalyst were evaluated in detail. In the LSV curve Figure 4a, the ct Figure , which r1 Figure . AlthougV Figure , which v2.5Figure m KOH solution containing 2 m Zn(AcO)2. When the FeCoNC/SL input is 10 mg and the geometric area of the air cathode is about 1 cm2, the open‐circuit voltage of the zinc‐air battery reaches 1.438 V  values, so the electron transfer rates of the three are similar. The difference is that the diffusion coefficient of FeCoNC/SL is higher than that of FeNC/SL and CoNC/SL, which can promote the adsorption of O2 on the catalyst surface. In addition, the CV curves (in 0.1 m KOH solution)   capacitance values of FeCoNC/SL, FeNC/SL, and CoNC/SL are 7.65, 0.925, and 1.42 mF cm−2, respectively. The larger the Cdl value, the more electrochemically active area. As the bimetallic bimetal can adjust the electronic structure of each other and affect the electronic atmosphere of the nearby NC, the effective electrochemically active area in FeCoNC/SL is larger than that in FeNC/SL and CoNC/SL.Compared with single‐metal‐doped FeNC/SL and CoNC/SL, the ORR activity and OER activity of FeCoNC/SL containing bimetal have been greatly improved, so the synergistic mechanism here needs to be further explored. In Nyquist plots  of ORR, which is increased by about 33.8% for jL when the morphology is transformed from dodecahedron‐like to star‐like. The difference in morphology also significantly affects the activity of OER. The OER overpotential is 316 mV (at 10 mA cm−2) when FeCoNC/SL was used as catalyst, while it reaches 533 mV when FeCoNC/DL was used as catalyst  of Fe and Co is displayed in Figure  In FeCoN5C  was further used to reveal the role of Fe—Co diatomic pairs in ORR and OER in FeCoNC/SL. Based on the HAADF images, XPS signal, EXAFS and their fitting results, a model (after optimization) for DFT calculation was constructed as shown in Figure mediates.31 FeCC Figure , althoug2O in the ORR process on FeCoN5C is more likely to occur than FeN4C and CoN4C. Specifically, when the applied voltage is 0 V, the Gibbs free energy drop of the PDS in the ORR on FeCoN5C is more negative, which is a spontaneous process. When the voltage is 1.23 V, the PDS of ORR on FeCoN5C, CoN4C, and FeN4C all need to overcome a positive Gibbs free energy change, but FeCoN5C requires the smallest energy barrier. Unlike ORR, the PDS of OER in the reverse reaction is from OOH* to O2. Regardless of whether the applied voltage is 0 or 1.23 V, the PDS of the OER on FeCoN5C needs to overcome the smallest Gibbs free energy barrier, which is favorable for OER to occur. Through DFT analysis, the phenomenon produced in the experiment and the advantages of FeCoN5C structure are well revealed. O2 activation is a key step toward ORR. We note that at U = 0 V and U = 1.23 V, the formation of the heteronuclear FeCoN5C biatomic pair configuration is more favorable for PDS of ORR than the formation of FeFeN4C or CoCoN4C. Another noteworthy result is that the change of Fe species from FeN4C form to FeFeN5C or Co species from CoN4C form to CoCoN5C is overall favorable for ORR, suggesting that the formation of diatomic centers can promote ORR. As shown in the Figure 2 activation is easier to be conducted on the surface of FeCoN5C catalyst. Meanwhile, adsorption energy (Eads) of *O2 on the Co sites of FeCoN5C, Fe sites of FeCoN5C, Co sites of CoN4C, and Fe sites of FeN4C are −3.58, −3.51, −3.35, and −3.22 eV, respectively, displaying high concentration O2 covered on the surface of FeCoN5C. In addition, the catalytic ORR efficiencies of FeCoN5C, CoN4C, and FeN4C showed a trend of FeCoN5C > FeN4C > CoN4C, and the theoretical overpotential (ηtheo) were 0.41, 0.52, and 0.67 eV  of Co is about 23.4% higher than that of Fe. As DFT calculations only focus on single metal atoms, CoNC/SL contains more metal sites than FeNC/SL and thus exhibits better performance.3−2, respectively. The overpotential of OER driven by FeCoNC/SL is as low as 316 mV (at 10 mA cm−2), and the mass activity is at least 3.2 and 9.4 times that of mononuclear CoNC/SL and FeNC/SL, respectively. When FeCoN5C/SL was used as the air cathode of Zn‐air battery, the power density, specific capacity and open circuit voltage are as high as 224.8 mW cm−2, 803 mAh g−1, and 1.438 V, respectively. Whether in ORR, OER, or Zn‐air batteries, FeCoN5C/SL has long‐lasting stability, which is beneficial for practical applications. DFT calculation shows that the formation of heteronuclear Fe—Co diatomic pairs can effectively regulate the electronic structure and improve the electron integration ability, which is also the main factor for FeCoNC/SL to have high ORR and OER bifunctional activities and achieve efficient operation of effective Zn‐Air batteries. In addition, converting the nearly spherical structure into a star‐shaped structure with a higher specific surface area allows more active sites to be exposed, which is the key to increasing the limiting current density.Using ZIF‐8 as the precursor, morphology control engineering and coordination atom exchange strategies were used to effectively embed heteronuclear Fe—Co bimetals on nitrogen‐doped carbon materials. The FeCoNC/SL obtained after annealing treatment shows excellent dual‐functional activity of oxygen reduction reaction and oxygen evolution reaction. In alkaline medium, the half‐wave potential and kinetic current density of the ORR driven by FeCoNC/SL surpassed the commercial Pt/C (20 wt%) catalyst, reaching 0.876 V and 4.21 mA cmThe authors declare no conflict of interest.Supporting InformationClick here for additional data file.


Antimicrobial resistance (AMR) is a global security threat that accounts for about 700 000 deaths annually. Studies have shown that antimicrobial resistance could result in a 2% to 3.5% reduction in global Gross Domestic Product by 2050 and a loss of between 60 and 100 trillion US dollars, worth of economic output resulting in significant and widespread human suffering. Low- and middle-income countries (LMICs) will be worse hit by an unchecked rise of AMR. For example, it is predicted that AMR could kill about 4.1 million people in Africa by 2050 if it is not curbed. Similarly rising rates of AMR will lead to increased treatment costs and an inability to attain universal health coverage, in LMICs with fragile health systems. Sadly, AMR is driven by the inappropriate use of antimicrobials, especially antibiotics. Inappropriate antibiotic use is a pertinent problem in LMICs where regulatory frame works are weak. Inappropriate antibiotic use in LMICs is a multifaceted problem that cuts across clinical and veterinary medicine and agriculture. Therefore, efforts geared at curbing inappropriate antibiotic use in LMICs must identify the factors that drive this problem (i.e. inappropriate antibiotic use) in these countries. A clear knowledge of these factors will guide effective policy and decision making to curb inappropriate antibiotic use and ultimately AMR. The focus of this review is to discuss the factors that drive inappropriate antibiotic use in LMICs. In this regard antibiotic use is deemed inappropriate if any of the following scenarios have occurred:It is important to clearly define the concept of inappropriate antibiotic use to have a precise understanding of the factors that drive inappropriate antibiotic use in LMICs. The term inappropriate antibiotic use implies that antibiotics are used in an inappropriate manner or injudiciously.Inappropriate indication: this implies that antibiotics are prescribed or consumed when not indicated.17 It also includes prescribing antibiotics for prophylaxis even when available guidelines clearly obviate the need for prophylaxis.17Inappropriate choice or selection: examples of wrong antibiotic choices include choosing antibiotics that are not recommended in guidelines and using antibiotics that do not cover the microbial spectrum or that lack activity at the target organ or system.18 The concept of inappropriate choice may also be contextual and may include the administration of antibiotics to patients in the light of clear contraindications, risks of adverse events or other patient specific issues that make the choice of a particular antibiotic inappropriate.19Inappropriate quality: this refers to the consumption of sub-standard, falsified or expired antibiotics. The use of sub-standard antibiotics results in sub-optimal inhibitory concentrations of the active pharmaceutical ingredient, treatment failures and the emergence of antimicrobial resistance.21Inappropriate dosing: administering sub-optimal doses of antibiotics  are examples of inappropriate antibiotic use.17Inappropriate dispensing: guidelines for appropriate dispensing emphasize that prescribed antibiotics should be dispensed by qualified and licensed dispensers.23 At the point of dispensing, patients should be provided appropriate information regarding the antibiotic, e.g. dosage, possible adverse effects, the need to adhere to the prescription etc.23 When antibiotics are not appropriately dispensed then patients are put at the risk of inappropriate antibiotic use and associated adverse outcomes.23Lack of adherence of the patient to antibiotic prescriptions: Some patients may not adhere strictly to antibiotic prescriptions due to inadequate information at the point of dispensing or financial constraints (which may hinder some patients from completing doses of unaffordable or expensive antibiotics).25Several factors drive inappropriate antibiotic use in LMICs. Some factors are peculiar to LMICs while other factors are common to all countries . This review draws attention to those factors that are peculiar to LMICs. Factors that are common to all countries irrespective of their income categorizations are discussed later in this review. The factors that drive inappropriate antibiotic use in LMICs include:26 The political will of an incumbent government is a strong factor that influences decision making, budgetary allocations, public private partnerships and the inflow of technical expertise and/or funds into a nation.27 Political will is a key element required to curb inappropriate antibiotic use in LMICs as it fosters the development and implementation of National Action Plans (NAPs) on AMR.28 The dearth of political will in LMICs results in poor or absent regulatory frameworks to curb inappropriate antibiotic use and AMR.28 The major factor impeding the development and implementation of NAPs in LMICs is the dearth of political will.28The term ‘political will’ is defined as ‘the commitment of political leaders and bureaucrats to undertake actions to achieve a set of objectives and to sustain the costs of those actions over time’.In addition to poor political will, some other factors that impede the implementation of NAPs on AMR include:1. Insufficient funds: Developing and implementing NAPs involves a wide range of cost intensive, multidisciplinary activities.28 Many LMICs, however, lack the financial resources required to implement NAPs.28 For example, in 2017, Zimbabwe’s 5-year NAP on AMR was estimated to cost about US$44.6 million.29 This amount of money is clearly expensive for many LMICs.282. A lack of multi-sectoral coordination: The implementation of NAPs is also hindered by poor multi-sectoral coordination in LMICs.30 The problem of inappropriate antibiotic use involves diverse stakeholders, and traverses ecological and geographical boundaries.30 Since many LMICs lack policies and frameworks required to support multi-sectoral coordination, efforts to curb inappropriate antibiotic use and AMR usually occur in a fragmented, uncoordinated and non-inclusive manner.30 For example, in some LMICs the Ministry of Finance  is not involved in discussions regarding the development or implementation of NAPs.283. A paucity of data and weak surveillance structures: Data collection in many LMICs is manual and usually consists of incomplete data sets, which fail to synergize laboratory data (e.g. antibiotic susceptibility patterns) with clinical data.31 This results in poor surveillance of local antibiotic consumption and resistance rates.31 In the absence of data and effective surveillance structures, such information is not readily available to guide antibiotic therapy at institutional levels, nor policy formulation at national levels.314. A dearth of technical capacity: many LMICs lack personnel with adequate training in antimicrobial stewardship and other skills required for effective networking, communication and governance of initiatives to curb inappropriate antibiotic use and AMR.3032 Lack of access to quality laboratory services in LMICs may be regarded from two perspectives: inadequate access and inequitable access.33–36A major challenge in LMICs is the lack of access to quality laboratory services, which results in diagnostic uncertainty, poor clinical decision making, inappropriate antibiotic use and the emergence of AMR.Inadequate access: this refers to an insufficient quantity and quality of laboratory services in many LMICs.34 A study showed that only 380 laboratories in Sub-Saharan Africa were accredited to international standards and that 37 of the 49 countries surveyed had no laboratories accredited to international quality standards.35 In many LMICs, laboratories are challenged by deficiencies in the quality of services they render.36 Examples of these deficiencies include lack of qualified manpower, inadequate infrastructure, insufficient (or non-existent) laboratory supplies, deficits in supply chain management, poor equipment maintenance, inadequate quality management systems and a dearth of regulation by government.36 In addition, pathogen detection and antibiotic susceptibility testing involve the use of obsolete phenotypic methods that are often slow, unstandardized and prone to errors, and therefore result in inaccurate clinical decision making.37Inequitable access: this refers to the uneven distribution of quality laboratory services in LMICs.35 The previously mentioned study showed that 91% of internationally accredited laboratories in Africa were in South Africa.35 Also, many rural communities in LMICs lack laboratory services because services in LMICs are usually located in urban areas.39 In the absence of guidance, by diagnostic laboratory services, doctors practising in rural areas usually resort to presumptive management of patients, blind prescription of antibiotics or referral of very sick cases.39 This results in significant morbidity and mortality among these patients.3941; subservience to hierarchy (e.g. a senior physician instructs a junior physician not to use the laboratory)41; poor communication between physicians and the hospital’s laboratory41; a lack of awareness of the problem of inappropriate antibiotic use and the role of the clinical microbiology laboratory in curbing AMR.41 Other reasons include long turn-around times or delays in sending out results to clinicians (these delays subsequently render such results irrelevant to patient management) 41; lack of confidence in the accuracy of results; absence of clinical microbiologists41 and the patients not able to afford laboratory tests.41 When physicians fail to use clinical microbiology laboratory services, they are usually left with the option of blind prescription of broad-spectrum antibiotics.41 Subsequently, when treatment failure occurs, physicians resort to repeated  antibiotic switches that result in patients bearing the adverse clinical and economic consequences of inappropriate antibiotic use.41Another driver of inappropriate antibiotic use in LMICs, is the poor use of laboratory services by clinicians due to the following reasons: the wrong assumption that clinical diagnosis is sufficient and that laboratories are not necessary to manage patients with infections42–45:Poor IPC protocols in the community and healthcare setting may be viewed from three perspectives1.Lack of access to WASH  facilities in communities43 According to a 2021 report, approximately 48 million Nigerians still defecate in the open, whereas only 8% of the population practices safe hand washing.46 The same report also showed that 23% of Nigerians lack access to basic water supply services and only 10% of the population had access to basic water, sanitation and hygiene services combined.46 Unfortunately, available data show that diarrhoeal diseases disproportionately affect places with inadequate access to WASH facilities, as well as low-income or marginalized populations.47 For example, in 2016, diarrhoea was the eighth leading cause of death, responsible for more than 1.6 million deaths, globally.48 More than a quarter (26.93%) of these diarrhoeal deaths occurred among children younger than 5 years of age, and about 90% of these diarrhoeal deaths occurred in south Asia and sub-Saharan Africa.48 High diarrhoeal burdens also result in increased consumption of antibiotics.43 For example about 494 million cases of diarrhoea are treated with antibiotics each year in Brazil, Indonesia, India and Nigeria alone.43 Such high antibiotic consumption rates are drivers of AMR in these countries and other LMICs in which antibiotics are used inappropriately.43In many LMICs, lack of access to WASH facilities results in a vicious cycle of high burdens of communicable diseases, increased antibiotic consumption and the emergence of AMR.2.Poor vaccine coverage and uptake in communities49 It is estimated that vaccination yields a return on investment valued at about US$44 for every dollar invested.50 By preventing the occurrence or spread of bacterial infections, vaccination reduces antibiotic consumption and the emergence of AMR.51 However, one in five African children and one in four children in South America do not receive all required childhood immunizations resulting in the occurrence of vaccine-preventable diseases and deaths.52 In Africa, more than 30 million children under five still suffer from vaccine-preventable diseases and of these number of children, over half a million die annually, representing approximately 58% of global deaths due to vaccine-preventable diseases.53 Also, poor vaccine uptake in LMICs results in increased consumption of antibiotics, increased inappropriate antibiotic use and the eventual emergence of AMR.54Vaccination has a positive impact on population health, productivity and education and adds value by reducing disease burden on individuals, families and communities.44 The COVID 19 pandemic has further worsened this picture.55 Before the beginning of the COVID 19 pandemic, in 2019 about 19 million children under the age of 1 year failed to receive basic vaccines through routine childhood immunization programmes.55 However, by 2021 this figure had risen to about 25 million children under the age of 1 year. In addition, 18 million children who have never received any routine childhood vaccine doses (i.e. zero-dose children) did not receive any vaccines in 2021.55 This is the highest number of poor vaccine uptake among zero-dose children since 2005.55 Sadly, almost all zero-dose children live in LMICs, primarily in Africa and South-East Asia.56Factors responsible for low vaccine coverage include distrust of parents and caregivers in vaccination, considerable distance between the home and the vaccination centre, language barriers and inadequate infrastructures to maintain the cold chain and adequate supply of vaccines.3.Poor adherence to IPC protocols in healthcare facilities45 Many healthcare facilities in LMICs lack adequate WASH facilities.45 This results in a vicious circle characterized by low quality of care, the occurrence of healthcare-associated infections (HCAIs), increased antibiotic consumption and significant morbidity and mortality.45Poor adherence to IPC protocols is also a major challenge in healthcare settings in LMICs.58 When healthcare workers fail to take required vaccinations they are at considerable risk of developing vaccine-preventable diseases  and transmitting these diseases to patients.58Also, a dearth of vaccination programmes for health workers and vaccination hesitancy among health workers exacerbates the problems posed by defective IPC protocols and HCAIs.59 Dispensing remains a vital element of the rational use of antibiotics and efforts to promote appropriate antibiotic use must incorporate good dispensing practices.23 All resources used in patient care before dispensing may be wasted if the designated patient does not receive the proper antibiotic in an effective form, in the proper packaging, at the proper dosage and advice .22 For proper dispensing to occur, the dispenser(s) must be well trained and have a basic knowledge about the antibiotics being dispensed. Such knowledge includes the correct dosage of the antibiotic, side effects, possible interactions with other medicines etc.22 In addition, the dispenser must have the right attitude and skills required to communicate effectively with patients.22 However, there is a dearth of trained pharmacists in many LMICs. This results in drug dispensing being done by poorly trained staff who lack the required knowledge, attitude and skills of proper drug dispensing.60 The lack of training therefore compromises their ability to contribute to curbing inappropriate antibiotic use and AMR in health facilities.60Dispensing involves ‘the review of a prescription and the preparation, packaging, labelling, record keeping and transfer of the prescribed medicine including counselling to a patient, their agent, or another person who is responsible for the administration of the medicine to that patient’.61 Illegal drug vendors contribute significantly to inappropriate antibiotic use and the emergence of AMR in LMICs.61 Globally, over 50% of antimicrobials are purchased without prescriptions in the community.61 These antimicrobials are sold by illegal drug vendors in markets, stalls and commuter vehicles in many LMICs.61Closely related to the problem of inadequately trained drug dispensers is the problem of illegal drug vendors in the community.62 It is defined as ‘having drugs continuously available and affordable at public or private health facilities or drug outlets that are within one hour’s walk of the population’.63Access to effective medicines is a fundamental human right and a key component of the SDGs.22 Several factors are responsible for a lack of access to effective antibiotics in LMICs. These factors include:A major challenge in LMICs is a lack of access, to effective antibiotics.Poor regulatory frameworks that result in the manufacture, importation and distribution of sub-standard or falsified medicines.64 About 17% of these sub-standard or falsified medicines are antibiotics.64 Poor regulatory frameworks also result in individuals with no pharmaceutical training being involved in the importation and distribution of antibiotics in LMICs.22 These individuals lack basic knowledge of standard procurement and distribution practices of antibiotics (and other medicines) and therefore contribute to the problem of inappropriate antibiotic use by their distribution of sub-standard antibiotics in LMICs.22Poverty and inadequate funding of the health sector deny patients from accessing quality healthcare and effective antibiotics in LMICs.21 Inadequate budgetary allocations result in infrastructural deficits and chronic shortages of antibiotics and other medicines that (if available) are usually sold to patients at subsidized rates in public health facilities.21 However, when these antibiotics are not available, patients are made to bear high medical costs when they buy antibiotics (at higher prices) from private pharmacies or drugstores.21 In many cases, patients cannot afford to buy these antibiotics.21 Subsequently, their failure to use antibiotics compromises their quality of care.The diminishing antibiotic pipeline and waning research and development efforts in new antibiotics reduces the global portfolio of available and effective antibiotics.8 Even after the discovery of a new antibiotic, regulatory hurdles, weak health systems and unreliable supply chains can delay market entry of new antibiotics into LMICs.2113The factors that follow are common to all countries irrespective of income levels . However, they are more pronounced in LMICs due to weak regulatory structures and a dearth of political will to curb inappropriate antibiotic use.These factors include:21 The attitudes, beliefs and socio-cultural values of consumers influence the consumption of antibioitcs.65 Even when antibiotics are deemed unnecessary, individuals, relatives or care givers are known to demand for antibiotics.66 This is a common occurrence in all countries irrespective of income level.15 However, certain socio-cultural differences that are peculiar to some LMICs may accentuate this problem. For example, in LMICs with strong patrilineal, traditional or religious cultures, the decision to use antibiotics or seek healthcare is largely dependent on the husband , the traditional ruler or religious leader.67 In addition, strong socio-cultural preferences for a particular route of administration may also result in inappropriate dosing.21 For example, it is not uncommon for outpatients in some LMICs to insist on intravenous or intramuscular antibiotics even when oral alternatives may suffice.21Consumers or end users of antibiotics include individuals, families and communities.69 Examples include:Several factors may influence physicians to prescribe antibiotics inappropriately.69The physicians’ personal attributes: These include clinical experience, exposure to continuous medical education and speciality.68–70The physician’s attitude to the problems of inappropriate antibiotic use and antibiotic resistance  and engaging policy entrepreneurs77 .A major recommendation is to build political will to curb inappropriate antibiotic use and AMR in LMICs. Building political will can be a complex issue and individuals or groups seeking any health reform must have an understanding of their nation’s political peculiarities, the political process and the dynamics involved in getting issues on the agenda of government.28 These NAPs must incorporate the One Health approach of curbing AMR in humans, agriculture and the environment.80 Funding and technical expertise to drive the implementation of NAPs may be sought from donor organizations and global institutions that are committed to curbing inappropriate antibiotic use and AMR.28 LMIC governments should also develop and implement National Strategic Laboratory Plans that would holistically address deficits and gaps in laboratory capacity and better position laboratories to contribute to curbing inappropriate antibiotic use and AMR.81Also, LMIC governments must be committed to developing and implementing NAPs on AMR.83 and LMIC governments must be committed to improving vaccine uptake and coverage through increased funding for infrastructure and surveillance of vaccine-preventable diseases.84 Establishing IPC teams in healthcare facilities in LMICs will curb the occurrence of HCAIs and reduce antibiotic consumption.85 Digital technology also offers opportunities to enhance IPC in healthcare facilities in LMICs.86 For example, mobile apps and wearable devices  could be used to improve hand hygiene practices by giving health workers, visual or audible reminders to perform hand hygiene.86Budgetary allocations and development aid for WASH facilities should be significantly increased87 However, effective AMS programmes must be integrated with other components required for health systems strengthening i.e. IPC, WASH facilities, adequate diagnostic microbiology services and efficient governance frameworks.88Antimicrobial Stewardship (AMS) Programmes are instrumental in curbing inappropriate antibiotic use and AMR and should be implemented in healthcare settings in LMICs.89Digital tools such as mobile apps are low-hanging fruit that could be easily deployed by AMS programmes to guide appropriate prescribing of antibiotics in healthcare settings in LMICs.21 Some LMICs have efficient supply chain management systems for drugs required in the management of HIV, tuberculosis and malaria.90 Such supply chain management systems could be expanded to improve access to antibiotics. LMICs with National Health Insurance Schemes could deploy funds from these schemes to procure essential medicines (including antibiotics) and provide them at subsidized rates to patients.21Efforts should be made to increase access to antibiotics in LMICs to reduce the infectious disease burden in these countries.91 Topics such as appropriate antibiotic use and AMR should be included in the curricula of medical and agricultural schools in LMICs to sensitize students to the problems of inappropriate antibiotic use and AMR.91Increased public enlightenment and education of health workers on appropriate antibiotic use and the danger posed by AMR is also recommended.92–95 The huge financial, technical and infrastructural gaps in many LMICs make it difficult for LMIC governments to effectively curb inappropriate antibiotic use and AMR.95 However, the private sector can provide critical bridge financing, technical expertise and infrastructure.93 LMIC governments should engage the private sector through public private partnerships and create a favourable investment climate (e.g. removing import barriers that raise the costs of acquiring diagnostic microbiology equipment) for private sector participation in efforts to curb inappropriate antibiotic use and AMR.95The private sector has an immense role to play in the fight against inappropriate antibiotic use and AMR.Rising rates of antimicrobial resistance (AMR) and waning research and development regarding new and effective antibiotics have made it imperative to curb the inappropriate use of antibiotics in LMICs. It is however, necessary to identify the factors which drive inappropriate antibiotic use in LMICs. A proper knowledge of these factors will guide policy formulation and other concerted efforts to curb this problem.


Carassius gibelio). Two isonitrogenous and isolipidic diets, containing 25%  and 45% (HCD) corn starch, were formulated to feed gibel carp (14.82 ± 0.04 g) for 8 weeks. The experimental fish were sampled at 2nd, 4th, 6th, and 8th week. In HCD group, the hyperlipidemia and significant hepatic lipid deposition (oil red O area and triglyceride content) was found at 4th, 6th, and 8th week, while the significant hyperglycemia was found at 2nd, 4th, and 8th week, compared to NCD group (P < 0.05). HCD induced hepatic lipid deposition via increased hepatic lipogenesis  but not decreased hepatic lipolysis (hsl and cpt1a). When compared with NCD group, HCD significantly elevated the hepatic sterol regulatory element binding proteins 1 (SREBP1) signals (positive hepatocytes and fluorescence intensity) at 4th, 6th, and 8th week (P < 0.05). The hepatic SREBP1 signals increased from 2nd to 6th week, but decreased at 8th week due to substantiated insulin resistance  in HCD group. Importantly, the hepatic carbohydrate response element binding protein (ChREBP) signals  were all significantly elevated by HCD-induced glucose-6-phosphate (G6P) accumulation at 2nd, 4th, 6th, and 8th week (P < 0.05). Compared to 2nd and 4th week, the hepatic ChREBP signals and G6P contents was significantly increased by HCD at 6th and 8th week (P < 0.05). The HCD-induced G6P accumulation was caused by the significantly increased expression of hepatic gck, pklr, and glut2 (P < 0.05) but not 6pfk at 4th, 6th, and 8th week, compared to NCD group. These results suggested that the HCD-induced hepatic lipid deposition was mainly promoted by SREBP1 in earlier stage and by ChREBP in later stage for gibel carp. This study revealed the sequential regulation pathways of the conversion from feed carbohydrate to body lipid in fish.The present study investigated the sequential regulation signals of high-carbohydrate diet (HCD)-induced hepatic lipid deposition in gibel carp ( Carassius gibelio) [Carbohydrates are widely applied in aquatic feeds as energy-supplying nutrients for their excellent price-performance ratio , 2. Howegibelio) . In recegibelio) , 11 and gibelio) –14. HoweCtenopharyngodon idella) [Salmo salar L.) [In general, the lipogenesis process is mainly regulated by insulin signaling pathway . After c idella) , and Atlalar L.) . Notablyalar L.) , 20. Accalar L.) , 22. ChRalar L.) , 24. Unlalar L.)  and frucalar L.) . ChREBP alar L.) . Severalalar L.) , 28. AltC. gibelio) are widely cultured in China with an annual production of 2.78–million tons in 2021 [th and 6th) and by G6P-ChREBP in later stage (6th and 8th) during the 8 week experiment. Thus, we clarified a sequential regulation manner of the conversion from feed carbohydrate to body lipid in fish. These findings present sequential and precise targets for ameliorating the HCD-induced hepatic steatosis in cultured fish.Gibel carp ( in 2021 . As an o in 2021 . In the Two isonitrogenous (29.91% crude protein) and isolipidic (6.72% crude lipid) experimental diets were formulated, which contained 25% corn starch  and 45% corn starch (HCD), respectively. The formulation and approximate chemical compositions of the experimental diets are shown in C. gibelio var. CAS V) were obtained from the Institute of Hydrobiology, Chinese Academy of Sciences . Four weeks prior to the feeding trial, gibel carp were acclimated in an indoor rearing system with fiber glass cylinder and fed to satiation twice a day at 8:30 and 16:30 with a commercial feed . After a 24 hr fasting, the fish of healthy appearance and uniform size  were randomly distributed into six indoor fiber glass tanks (water volume: 120 L) with 20 fish density for each tank. Triplicate tanks were assigned for each treatment. During the trial, fish were fed twice a day at 8:30 and 16:30 for 8 weeks. The water temperature was recorded daily and maintained at 27–29°C. Ammonia nitrogen (Ammonia-N), dissolved oxygen (DO), and pH were monitored every 3 days. The values showed that the concentration of Ammonia-N was below 0.3 mg/kg, the DO was 6.0–7.5 mg/L, and the pH was 6.8–7.3. The photoperiod was 12 hr light (8:00–20:00): 12 hr dark.The experimental gibel carp  at postprandial 4 hr. The body length and weight of the anesthetized fish were measured, then two of them were sampled blood from the caudal vein by heparinized syringe. The blood was centrifuged at 3,500 g for 10 min to obtain the plasma and stored at −80°C for further analysis. Immediately, the liver tissues were removed on ice. A small part of each liver tissues was fixed by 4% paraformaldehyde for histological staining and observation, while the rest was stored at −80°C for further analysis. The specific growth rate (SGR) and condition factor (CF) were calculated as follows:Sample collections were conducted at 2Total RNA from the liver tissues was extracted by TRIzol Reagent  according to the product instructions. The quality and concentration of extracted total RNA were evaluated according to our previous methods . The totPlasma glucose levels were measured by Glucose Assay Kit with O-toluidine . Hepatic and plasma triglycerides (TG) contents were measured using a Triglyceride Assay Kit . Plasma insulin level was measured using a commercial ELISA kit . Hepatic G6P content were measured using commercial Glucose-6-phosphate Assay Kit . All measurements were performed following the manufacturers' instructions.μm with a cryostat . For oil red O (ORO) staining, the liver cryosections were stained with ORO to visualize the lipid droplets. Images were collected by Leica automatic digital slide scanner  and quantified with ImageJ . Immunofluorescence on the liver cryosections was performed according to a previous study [Ser473 , and anti-CHREBP , followed by Alexa Fluor 568  or Alexa Fluor 488  conjugated secondary antibody for visualization. Nuclei were stained by Hoechst 33342 . Images were collected with Leica laser-scanning confocal microscope , analyzed by Imaris Viewer , and quantified by ImageJ .The liver tissues initially fixed with 4% paraformaldehyde plus 20% sucrose overnight and sectioned to 8 us study . PrimaryP Values < 0.05 were considered significant. All statistical analyses were carried out and graphed with GraphPad Prism 8 .All results are expressed as means ± SEMs (standard error of the mean). Normality was tested by 1-sample Kolmogorov–Smirnov test. Homogeneity of variance was examined by the Levene test. A two-tailed independent t test was used to evaluate the significant differences of measured parameters between two groups (data between NCD and HCD at each week). A one-way ANOVA with Duncan's multiple-range test was used to evaluate significant differences of measured parameters among several groups (data among four weeks of each treatment). Differences with th and 8th week in HCD group (P < 0.05), while there was no significant difference between HCD and NCD groups at 2nd or 4th week (nd to 8th week (th week (P < 0.05)   . However < 0.05) .nd, 4th, and 8th week (P < 0.05) (th and 6th week displayed significantly decreased (P < 0.05), compared to that of 2nd week     (nd to 8th week (The plasma glucose levels of HCD group were significantly higher than those of NCD group at 2 < 0.05) . Within 2nd week . The pla < 0.05) . Within  < 0.05) . The ORO Figures . The hep Figures . Within  < 0.05) . Besides8th week .acc), fatty acid synthase (fasn), and ATP-citrate lyase (acly) were all significantly increased in HCD group at 2nd, 4th, 6th, and 8th week, compared to those in NCD group (P < 0.05) (hsl) and carnitine palmitoyl transferase 1a (cpt1a) were significantly increased in HCD group at 6th and 8th week, while acyl-CoA oxidase 3 (aco3) significantly increased in HCD group only at 8th week, compared to those in NCD group (P < 0.05)  . However < 0.05) .th, 6th, and 8th week, compared to those in NCD group (P < 0.05) ; however, they were both significantly decreased at 8th week, compared to those at 6th week (P < 0.05)   , while there was no significant difference among 4th, 6th, and 8th week (The immunostaining of hepatic SREBP1 showed that the percentage of SREBP1 positive hepatocytes and the fluorescence intensity of SREBP1 were significantly increased in HCD group at 4 < 0.05) . Within  < 0.05) . Besides < 0.05) . The pla < 0.05) . Within 8th week .irs) genes showed that the expression levels of hepatic irs1 and irs2 were significantly increased in HCD group at 2nd, 4th, and 6th week, but only irs2 was significantly increased at 8th week, compared to those in NCD group (P < 0.05)   (P < 0.05)  ; however, there was a further increase in the percentage of ChREBP positive hepatocytes at 8th week, compared to those at 6th week (P < 0.05)    (th and 8th week displayed significantly increased compared to those of 2nd and 4th week (P < 0.05), respectively  . Within  < 0.05) .Within N < 0.05) . Besides < 0.05) . Moreove < 0.05) . Within ectively .gck), pyruvate kinase L/R (pklr), and glucose transporter 2 (glut2) were all significantly increased in HCD group at 4th, 6th, and 8th week, while 6-phosphofructokinase (6pfk) only significantly increased at 2nd week, compared to those in NCD group (P < 0.05) (g6pase) and phosphoenolpyruvate carboxykinase (pepck) were significantly decreased in HCD group at 2nd week, while significantly increased in HCD group at 6th and 8th week, compared to those in NCD group (P < 0.05)  . However < 0.05) .C. gibelio). The results demonstrated that HCD promoted hepatic lipid deposition and lipogenesis mainly via insulin-SREBP1 in earlier stage (4th and 6th) and via G6P-ChREBP in later stage (6th and 8th) for gibel carp during the 8-week feeding experiment.With the rapid development of artificial feeds, carbohydrates have become a basic dietary composition for aquatic animals. Fish have been thought to be inefficient in utilizing dietary carbohydrate, though carbohydrate is the most abundant energy-supplying nutrient in nature . Interesth week via supraphysiological lipogenesis in gibel carp. Excessive carbohydrate intake leads to metabolic disorders, that is particularly true for fish [th to 8th week, while the serve fatty liver started from the 6th week under HCD treatment. The HCD-induced lipid accumulation in fish has been observed [th and 8th week. The studies in largemouth bass  and zebrafish (Danio rerio) also indicated that the HCD-induced lipid accumulation was accompanied with activated lipolysis [Pelteobagrus fulvidraco) and barramundi  showed that dietary glucose and gelatinized wheat starch did not alter the lipolysis [HCD induced lipid overdeposition from 6for fish , 34. Incfor fish . In the observed , 36; howobserved –39. In tipolysis , 40. Howipolysis , which ith week to 8th week, when we compared the dynamic changes of expression and activation level of SREBP1 within HCD group. This phenomenon has not been reported by previous studies. Persistent HCD usually leads to insulin resistance in mammalians [ser473 level became insensitive to HCD, while the hepatic gluconeogenesis, plasma insulin, and plasma glucose levels were still higher in HCD group at 8th week. These results indicated the insulin resistance was established in gibel carp after an 8-week HCD challenge. The similar results were also observed in rainbow trout (Oncorhynchus mykiss) [Long-term HCD abated SREBP1 signal via insulin resistance in gibel carp. Insulin-SREBP1 is a key pathway in the motivation of lipogenesis in mammalians . Plenty mmalians , 44, whimmalians . Interes mykiss) . Therefoth week, the hepatic lipogenesis and lipid deposition did not show any mitigating trend in HCD group. In mammalians, the elevated lipogenesis and nonalcoholic fatty liver disease also developed in the setting of insulin resistance [nd to 8th week in gibel carp. The expression and activation levels of ChREBP were even further increased at 6th and 8th week in HCD group. The increased gene expression levels of chrebp were also observed in yellow catfish [Acipenser schrenckii) [gck were increased more than 2.28 fold to those in NCD group from 4th to 8th week, while the expression levels of 6pfk showed no change between HCD and NCD groups. Thus, there was a fine-tuning in the glycolysis of gibel carp in HCD group. Since G6P is generated from glycolysis [Although the SREBP1 signal was decreased by the insulin resistance at 8sistance , 48. Thisistance , as it ssistance . ChREBP sistance , 27. A psistance . Interes catfish , and amurenckii)  with carrenckii) . The intycolysis , produceth and 6th) while by G6P-ChREBP in later stage (6th and 8th) for gibel carp during an 8-week feeding experiment (In conclusion, the present study demonstrated that HCD induced dynamic hepatic lipid deposition via increasing lipogenesis, that was mainly promoted by insulin-SREBP1 in earlier stage (4periment . Therefo


In Tanzania, data on antibiotic use at the patient level is scarce, and intervention measures to optimize antibiotic use and reduce antimicrobial resistance are rarely performed.To describe antibiotic use at Muhimbili National Hospital.This was a point prevalence survey on antibiotic use conducted at Muhimbili National Hospital in August-September 2022. The World Health Organization point prevalence survey data collection tool was used to collect patients’ information from the files. All patients admitted to the wards on the day of the survey were included.Overall, 47% (185/397) of admitted patients were on at least one antibiotic during the survey. All antibiotics prescribed were for empirical treatment and guideline compliance was low, at 45%. Of 185 patients who received antibiotics, the most common indication was community acquired infection (55%) and 36% had no documentation of the reasons for prescribing antibiotics. Almost 75% of the antibiotics were administered parenterally, with only 2% switching to oral route. Microbiological tests were performed in only 9 (5%) patients out of 185 and results were available for only one patient. Of all participants, 52% received two or more antibiotic in combination, with the combination ceftriaxone-metronidazole being most frequently prescribed, followed by the combination of ampicillin, cloxacillin, and gentamicin. For individual antibiotics, ceftriaxone was the most frequently prescribed antibiotic accounting for 28% (79/283), followed by metronidazole (24%) and amoxicillin-clavulanic acid (11%).The findings of a high prevalence of antibiotic use, inadequate use of bacterial culture, and frequent empiric antibiotic treatment suggests the need for strengthening diagnostic and antimicrobial stewardship programs. Furthermore, this study has identified areas for quality improvement, including education programs focusing on prescription practice. Antimicrobial resistance (AMR) in resource-limited countries is on the rise, with increasing number of infections caused by antimicrobial-resistant bacteria. The magnitude of AMR, its implications, and risk factors for its development are often not documented. Irrational use of antibiotics has been documented as driving the emergence of AMR. Despite this existing evidence, the use of antibiotics in resource-limited countries is poorly tracked, further fueling the emergency of AMR.S. aureus and extended-spectrum beta-lactamase-producing bacteria causing surgical site infections and bloodstream infections.In Tanzania, studies have demonstrated a high burden of both community- and healthcare-acquired infections caused by antibiotic-resistant bacteria , 2. Desp , 4, withThe overall prevalence of antibiotic use in health care facilities in Tanzania has been reported to vary from 30 to 62%, depending on the level of the facilities –11. TherMisuse and overuse of antimicrobial agents are well known to drive the emergence of antimicrobial resistance through selective pressure. Monitoring antibiotic use by patients and antibiotic prescription behavior by health care workers is essential for planning containment measures to prevent the spread of AMR and optimize antibiotic use. Point prevalence surveys (PPS) on antibiotic use have been successful worldwide in optimizing antibiotic use in hospitals and planning interventions to reduce AMR –16. It iThis study was conducted at Muhimbili National Hospital (MNH) for the duration of three consecutive weeks, starting from August 29, 2022, to September 18, 2022. MNH is located in Dar-es-Salaam, the economic hub of Tanzania. MNH has a 1500-bed capacity and is the largest tertiary and main consultant referral hospital in the country, attending approximately 2000 outpatients per day. MNH regularly receives referrals from other hospitals and has highly specialized staff, technical equipment, specialized imaging units, and a well-equipped clinical laboratory. It also serves as a teaching hospital for Muhimbili University of Health and Allied Sciences.The hospital wards include pediatric wards , neonatal wards , adult wards , and mixed wards .This study adopted the WHO methodology for a PPS on antibiotic use in hospitals . The stuData were collected using a modified Excel WHO PPS data collection tool. The tools consisted of wards, patients, indications, and antibiotic information. All information was collected from the patient’s files or other medical records, including the hospital information management system. No patient interviews and no clinician or health facility staff interviews were performed.p-value of < 0.05 was considered statistically significant.The collected data was analyzed using Stata version 16 . Categorical variables were presented as frequencies, percentages, and proportions. The chi-square test was used to compare the association between two categorical variables. A A total of 397 patients were surveyed between August 29, 2022, to September 18, 2022.Participants aged 16–65 were most common , followed by children 1 month-15 years , neonates  and the elderly aged > 65 . 56% were females. Surgical , adult medical  and Obstetrics/gynecology  wards accounted for the highest number of patients. One-fourths and 72% (300/397) of patients, had urethral catheter and peripheral vascular catheterization, respectively. Table Overall, 185/397 (47%) of admitted patients received one or two antibiotics during the survey. There were no differences in antibiotic use by sex, age groups, intubation or type of wards where patients were admitted. Patients with urethral catheter 64%, 62/97) were more frequently on antibiotic treatment compared to those who were not catheterized , p-value < 0.001  had community acquired infection as indication, 67 (36%) were given antibiotics without documentation of the reasons for prescribing antibiotics, and for 11 (6%) patients the indication was hospital associated infection. The most common clinical diagnoses for patients who received antibiotics were respiratory tract infections , gastrointestinal tract infections  and genital or urinary tract infections . However, a disproportionate number of patients received antibiotics without documentation of the clinical diagnosis . For the majority of the patients antibiotics were dosed twice  or thrice daily . Across all patients, almost 75% of antibiotics were administered parenterally, with only 2% switching to oral route. Of all, 43% received a combination of two antibiotics and 9% received more than two antibiotics. Of participants receiving two or more antibiotics, the combination of ceftriaxone and metronidazole was the most common , followed by ampicillin, cloxacillin, and gentamicin .Culture sample were performed in only 9 (5%) patients out of 185 and results were available for only one patient Table .A total of 283 antibiotic prescriptions were made during the survey. The most prescribed antibiotic during the survey period was third generation cephalosporins (ceftriaxone) accounting for 28% (79/283), followed by metronidazole 24% (67/283), amoxicillin-clavulanic acid 11% (30/283) and ciprofloxacin 7% (19/283). Figure Tanzania is currently implementing its national action plan 2023–2028 to combat antimicrobial resistance, and the use of PPS on antimicrobial use to improve antibiotic prescription is one of the core activities in strategic objective number three . We condIn this survey, antibiotic prescriptions were entirely empiric without evidence from microbiological test results, which could have illuminated the causative bacteria and effective antibiotics. Bacterial culture in this survey was only requested for 5% of the participants. While this is in line with other studies from the region , it is dWe observed that ceftriaxone, which belongs to the WHO “watch” category, was the most commonly prescribed antibiotic, followed by metronidazole and amoxicillin-clavulanic acid, both belonging to the WHO “access” group of antibiotics. However, ampicillin-cloxacillin and ceftriaxone-sulbactam, which the WHO does not recommend for use in clinical practice, were prescribed to some patients. Prior studies in Tanzania showed similar patterns of antibiotic prescriptions, where ceftriaxone, metronidazole, and amoxicillin-clavulanic acid were frequently prescribed antibiotics , 9. FurtIn this survey, we found that parental routes of antibiotic administration were common, and that only 2% of those switched to oral routes of administration during the whole course of hospital admission. These findings indicate that in ward rounds, antibiotic prescriptions are not discussed in detail, and decisions about the escalation or de-escalation of antibiotics are certainly not often considered. Obviously, the lack of microbiological results makes it even more difficult to target and de-escalate antibiotic treatment. Similar findings have been reported in other studies from the country and other developing countries , 22, 29.We observed that community-acquired infections were the most common indication for antibiotic administration, and one-third of patients received antibiotics without documentation of the reasons for prescribing antibiotics. In addition, compliance to the treatment guideline was low, which reflects that antimicrobial stewardship at this setting is rarely implemented. Despite community-acquired infections being a common indication, neither of these patients were confirmed to have bacterial infections as bacterial cultures were not performed. The consequence of this empiric treatment in a setting without a hospital cumulative antibiogram is the emergence and spread of antimicrobial resistance. Other studies from the region have documented the same findings, were community acquired infections are a common indication and limited documentation exists for the reason for antibiotic prescription , 19.This study observed that 52% of patients received two (43%) or more 9%) antibiotics in combinations; ceftriaxone combined with metronidazole, followed by the combination of ampicillin, cloxacillin, and gentamicin were the most common. The combinations of ceftriaxone and metronidazole was likely targeting suspected polymicrobial infections (aerobic and anaerobic infections). The combination of ampicillin, cloxacillin and gentamicin may have been chosen to widen the coverage compared to individual drugs alone, reflecting that the clinicians treat blindly without any microbiological evidence to guide therapy. Often, the use of combination antibiotics in empirical therapy is considered to widen the coverage of susceptibility over individual antibiotics alone. This means the second antibiotic offers increased coverage compared to the first antibiotic. The practice of combining metronidazole with other antibiotics without considering the presence or absence of risk factors for anaerobic infections has been common in sub-Saharan Africa % antibio, 23, 30.A weakness of this study is that it is a single-center report, and the findings may not reflect practice in other settings. However, a strength of the study is that by assessing a main national and teaching hospital in Tanzania, the study is well situated to highlight gaps in knowledge and practices on addressing AMR and provide opportunities for improvement in combating AMR. Another strength of the study is that it addresses a topic that has been under-researched across the continent. Despite being a single-center study, our findings are similar to those studies from sub-Saharan Africa, , 29, 30,In conclusion, the findings of a high prevalence of antibiotic use, underutilization of bacterial culture, and total reliance on empiric antibiotic treatment suggest the need for strengthening diagnostic and antimicrobial stewardship programs. The management should provide the committee with resources, including human resources like microbiologists and infectious diseases specialists who should be dedicated to performing bedside consultations for antimicrobial use, PPS, and prospective audits on antimicrobial use and providing feedback. Furthermore, this study has identified areas for quality improvement, including education programs for healthcare workers on antibiotic prescription behaviors and practice change, as well as training of healthcare workers on the importance of performing culture and susceptibility.