Food quality and safety are important to protect consumers from foodborne illnesses. Currently, laboratory scale analysis, which takes several days to complete, is the main way to ensure the absence of pathogenic microorganisms in a wide range of food products. However, new methods such as PCR, ELISA, or even accelerated plate culture tests have been proposed for the rapid detection of pathogens. Lab-on-chip (LOC) devices and microfluidics are miniaturized devices that can enable faster, easier, and at the point of interest analysis. Nowadays, methods such as PCR are often coupled with microfluidics, providing new LOC devices that can replace or complement the standard methods by offering highly sensitive, fast, and on-site analysis. This review’s objective is to present an overview of recent advances in LOCs used for the identification of the most prevalent foodborne and waterborne pathogens that put consumer health at risk. In particular, the paper is organized as follows: first, we discuss the main fabrication methods of microfluidics as well as the most popular materials used, and then we present recent literature examples for LOCs used for the detection of pathogenic bacteria found in water and other food samples. In the final section, we summarize our findings and also provide our point of view on the challenges and opportunities in the field. Lab-on-a-chip devices (LOCs) are miniaturized devices in which biological, chemical, or biochemical analyses that are normally conducted in a laboratory are performed inside a single device . Such deSimilarly to the μTAS definition, LOCs are devices in which all necessary steps during a sample analysis can be completed in a single device. Basic functions such as sample preparation, mixing, filtration, heating, separation, and detection can be integrated into a LOC operation . It is tIn terms of materials used in LOC fabrication, silicon and glass were the first materials used since the first introduction of μTAS in 1990 .Salmonella, Campylobacter, E. coli O157:H7, and Listeria monocytogenes, are mainly responsible for food and water deterioration and are causing most of the foodborne illnesses.As stated above, LOC devices have been used in various fields, such as chemistry, drug production, medicine, proteomics, and food safety, and their usage has only been expanding during the past few years ,18,19,20Thus, the application of LOC devices in food safety-related applications is blooming since LOCs can provide rapid, accurate, and on-site analysis of food samples. In this review paper, we provide an overview of the main fabrication techniques and the applications of microfluidic devices, as well as a complete literature review of efforts about polymeric and paper-based LOCs used for detection of food-borne and water-borne pathogens. In the final section, we summarize the most important advances in the field together with their advantages and disadvantages, and we present our opinion on the remaining tasks required in order to accelerate the industry’s uptake of this new type of devices.Since the introduction of the μTAS concept in the 1990s, scientists have gradually shifted their interest in polymers due to the demand for cheap and disposable devices. Polymer materials typically used in microfluidic applications can be divided into three categories according to their physical properties: (a) thermosets, (b) thermoplastics, and (c) elastomers .Thermosets are polymers that are irreversibly hardened and come from soft, solid, or viscous liquid prepolymers. Curing results in chemical reactions that create extensive cross-linking between polymer chains to produce an infusible, insoluble, and thermally stable polymer network . ExampleThermoplastics, on the other hand, do not form irreversible chemical bonds during the curing process because their polymer chains are not cross-linked, so they can move inside the bulk at elevated temperatures, which is the reason why they are deformable . They arElastomers consist of cross-linked polymer chains that can be deformed under pressure but come back to their initial shape when the force stops. The most common elastomer is PDMS (polydimethylsiloxane) which belongs to the group of silicon elastomers and can be deformed under force or air pressure . PDMS isPaper is another material commonly used in microfluidics and LOCs; it is produced from a dilute aqueous suspension of cellulose fibers that are drained through a sieve, then pressed and dried to yield a sheet formed by a network of randomly interwoven fibers . Its uniThanks to their properties, polymers can be easily used as base materials for several parts, objects, and components in many industry sectors. The main production method for plastic objects is molding. In microfluidics, molding polymers on the microscale is one of the most common techniques for the realization of microfluidics and LOCs. In the following sections, we provide examples of the different molding techniques used in microfluidics.Hot embossing is a technique that uses the very simple concept of transferring patterns of micro- and nanoscale features from a mold to a target substrate and has been extensively used for the fabrication of various microfluidic devices. In the past two decades, this technique has successfully broken through the barrier of laboratory-scale production and become an industrial-scale production technique . OverallSeveral examples of microfluidics or LOCs fabricated using hot embossing exist in the literature, such as the fabrication of a microfluidic platform for ELISA on beads targeting the detection of two important human proteins (IL6 & PDGF-2). The device included superhydrophobic inlet and outlet valves as well as an incubation chamber, and it was created by hot embossing the Si master onto a PMMA substrate . SimilarE. coli K12 cells. The negatively charged nanofiber mats managed to capture 72% of the E. coli cells while reducing nonspecific analyte retention within the channel. The microfluidic channels  were embossed into pieces of PMMA using a Hot Press at 130 °C and 10,000 lb  of force for 5 min. Furthermore, UVO-assisted thermal bonding was used to produce microfluidic devices. In another example, a deep (28  μm) cyclo-olefin polymer (COP) microcolumn with high aspect ratio (3:1) features was fabricated using a two-step process: (a) hot embossing of a-Si master and (b) pressure-assisted thermal bonding. The column was used to separate substances used in the food industry such as sodium benzoate, methylparaben, and propylparaben. To reduce processing time, techniques such as ultrasonic hot embossing have also been developed ..L. monocE. coli O157:H7, Salmonella Typhimurium, Vibrio parahaemolyticus, and Listeria monocytogenes on a centrifugal lab on a disc. The lab on a disc was made from PMMA and incorporated many operations, including DNA amplification, extraction, purification, and detection. The results of the DNA amplification were evaluated by a colorimetric test based on the change of color from purple to light blue. The lab on a disc achieved a LOD of 10 cells for every pathogenic bacterium, in a duration of fewer than 65 min [L. monocytogenes cells. In particular, a gold concentric-3-electrode was fabricated, consisting of a working, a reference, and a counter electrode. The biosensor and the microchip were fabricated using the 3D printing technique. The LAMP amplification and the specially designed primers could detect 12 serotypes of L. monocytogenes. As an electrochemical transducer, the methylene blue redox-active molecule was utilized to specifically distinguish the positive from the negative tests. The attained LOD was 1.25 pg DNA per reaction. The electrochemical biosensor was tested in three food samples: dairy milk, fresh cheese, and smoked salmon [The loop-mediated amplification (LAMP) technique has also been used for the detection of n 65 min . The nexd salmon .Salmonella Typhimurium, S. aureus, and Listeria monocytogenes at a high sample flow rate. To do so, they used a microdevice based on magnetophoresis that consists of a magnet and a tube surrounding it. The innovation is that the tube can be replaced with tubes of varying lengths, allowing processing at high flow rates. The microdevice’s tube was manufactured from polyethylene, while the microchannels were created using PDMS and soft lithography. The proposed device was evaluated on two food samples, milk and homogenized cabbage, with the separation and concentration efficiency exceeding 92% and the flow rate above 40%, demonstrating that this device has a better throughput than other devices. Although it is important to note that high flow rates reduce the device’s performance because they create an unsteady flow. To minimize the related vortex, both the tube’s diameter and the microchannel’s shape could be modified [The majority of developed microdevices have low throughputs, which results in longer processing times for a few milliliters of samples to detect the target bacterium. To address this fact, a study processed, separated, and concentrated samples of modified .Staphylococcus aureus is a gram-positive, spherical bacterium that inhabits the skin and nasal cavity of humans. Its presence in the human body may not cause symptoms; however, food can be contaminated by human carriers who handle it. The contamination of food with staphylococcus promotes its growth and the production of toxins, which affect the food’s organoleptic qualities, causing external spoilage and a foul odor. Staphylococcus is susceptible to heat treatment, whereas the produced toxins are not [S. aureus as well as the assurance of food quality and consumer safety. This section will discuss advances using biosensors and LOCs. are not ,200. NumStaphylococcus aureus cells, has been proposed. In this case, NCs act as natural enzymes, and reactions with catalysts cause a change in color. Here, TMB and H2O2 were the catalysts used. The observation of blue color is evidence of the existence of bacteria and is observed by the naked eye but also measured from the change in absorbance at 652 nm. The bacteria quantification is conducted through the paper-based microchip, which is fabricated by Whatman’s No.1 filter paper after CO2 laser treatment, forming a Y-shaped micro-device, with a 600 μm Y-shaped microchannel. The color intensity depends on the concentration of bacterial cells (102–108 cfu/mL) and detection limit of 80 cfu/mL was achieved within 5 min [S. aureus, methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA), in clinical samples from China and the United States. As far as MRSA is concerned, the detection of this type of S. aureus was conducted by the recognition of the mecA gene. The samples were processed with the PCR method and multilocus sequence typing (MLST). The exploited materials were SU-8 photoresist, forming the channel’s mold, PDMS, and glass. The proposed method appeared to be sensitive and could identify the sample’s origin. The biosensor detected 17.400 SERS spectra for a duration of 3.5 h [A paper microchip in combination with a biosensor that is based on bimetallic nanoclusters Au/Pt, with peroxidase properties, aiming to identify whole in 5 min . Anotherof 3.5 h .E. coli O157:H7 and S. aureus has also been proposed. The immobilization of the target cells is conducted via the self-assembling (3-glycidoxypropyl) trimethoxysilane (GPMS) silane. The impedance change was then recorded for frequencies between 1 and 100 KHz. The device was fabricated by PDMS and attached to aluminum foil. The PDMS layer was treated with oxygen plasma to bond with the aluminum. Sealing of the PDMS to the aluminum was achieved with adhesive epoxy. The immunosensor achieved a LOD of 102 cfu/mL in just 2 h [S. aureus, and the procedure can be conducted in approximately 10 min. The advantage of this study is that no process of the DNA is necessary. S. aureus was detected at concentrations between 5.2 × 104, and 5.4 × 105 cfu/mL in spiked samples [A microfluidic immunosensor with a nanoporous membrane and specific antibodies to detect pathogen bacteria cells of just 2 h . An apta samples .S. aureus, a self-sufficient lab-on-a-foil system was constructed; the device utilizes recombinase polymerase amplification (RPA) for DNA processing. PDMS was used for the fabrication of this device, implementing techniques such as micro-milling and hot embossing. This lab-on-chip device was constructed to be capable of processing up to 30 samples due to its cartridges. It also achieved the detection of S. aureus in less than 10 copies, while the duration of this procedure was less than 20 min [For the detection of the antibiotic resistance mecA gene of n 20 min .Staphylococcus in order to detect different types of Staphylococcus cells and identify the Methicillin resistance type. More specifically, this micro-device could detect a variety of Staphylococcus types, such as S. aureus, S. epidermidis, S. haemolyticus, and S. hominis. The material in this case was polycarbonate (PC), which was processed using injection molding. The microchip consisted of 10 microchambers, which were connected to the microchannels. The reported LOD was 20 cfu/reaction for S. aureus, S. epidermidis, S. hominis, and methicillin-resistant S. aureus (MRSA) and 200 cfu/reaction for S. haemolyticus in just 70 min [Loop-mediated amplification (LAMP) was employed and integrated into a microchip for the amplification of the mecA and femA genes of t 70 min .S. aureus. The microchip was mounted on top of an aluminum block, with a plate heater underneath.  to conduct PCR on the peanut’s DNA and the hsp gene of erneath. . In thisrodevice .E. coli and S. aureus cells has been presented. As far as E. coli is concerned, the platform was able to detect E. coli at concentrations between 105–3.2 × 107 cfu/mL in a solution containing phosphate-buffered saline (PBS) and peritoneal dialysis fluid (PD), whereas S. aureus was contained in PBS. The microchip was made of PMMA and consisted of a single microchannel, a sample injection inlet, and an outlet. The microchip was also gold-coated and modified with specific antigens to capture E. coli and S. aureus cells. Detection and quantification were performed by bright field and fluorescence imaging [A microdevice incorporating a plasmon resonance platform, as shown in  imaging ,208.S. aureus via labeling and the use of immune microspheres with 50–90 nm diameter for surface modification. After a 4-min procedure (reaction), S. aureus was detected at a LOD of 1.5 × 101 cfu/ μL. Practically, the procedure of detecting the target cells is based on the binding of the target cells with specific antibodies, that bind respectively with antigens. Then the antigens bind with the anti-S. aureus antibody (FITC). For the creation of the mold spin-coating SU8-3025 photoresist was used and processed by silane treatment. After 5 min, PDMS is poured and baked at 80 °C for 10 min. For 2 μL/min flow rate, the fluorescence intensity reaches its maximum functionality, while a 5 μL/min flow rate and a 4 min duration are the optimal conditions for the optimal injection and reaction. At this flow rate, the target bacteria can be seen from the microsphere’s surface [A self-made microfluidic chip was fabricated for the detection of  surface .Campylobacter is a pathogenic, gram-negative, non-spore-forming bacterium responsible for many outbreaks of foodborne illness. Morphologically, it is mainly found in an S-robbed shape and has a length of 0.5–5 μm. Campylobacter is sensitive to atmospheric oxygen and can be killed by heating. Nevertheless, it shows the ability to persist in contaminated food, one of the reasons being the development of biofilms, which are strong structures with high resilience. Campylobacter is present in animals, through which it is transferred to humans or animal-derived products. The products that most often contain campylobacter are raw meat, with the most basic form being chicken, and raw/unpasteurized milk. However, campylobacter can contaminate water or even ice [even ice ,211,212.Campylobacter spp. and AST. 100% specificity was achieved in detecting multiple Campylobacter species in artificially contaminated milk and poultry meat. On-chip AST determined Campylobacter’s susceptibilities to multiple clinically significant antibiotics with a high degree of concordance (91–100%). The lab-on-a-chip device completed on-chip identification and AST in 24 h, whereas traditional methods require days. The developed method decreased analysis time, streamlined food sample preparation and chip operation, and utilized up to 1000 times fewer reagents than standard reference methods, making it competitive for rapid screening and surveillance studies in the food industry. This microfluidic device could be used for food safety management and clinical diagnostics in areas with limited resources.In the context of combating the antibiotic resistance crisis, Ma et al.  developeCampylobacter jejuni. The method simultaneously completed 22 genetic analyses in an automated format, with a detection limit ranging from 7.9 × 10−3 to 9.54 × 10−1 pg of genomic DNA of pure bacteria per reaction, and the LAMP reaction was completed in just 35 min. In the analysis of coastal water samples, the clinical sensitivity and specificity were 93.1% and 98.0%, respectively. The established dual-sample on-chip LAMP assay provides a dependable and efficient multiple-pathogen analysis of waterborne bacterial pathogens and is appropriate for on-site detection and routine monitoring of waterborne bacteria in aquatic environments. A droplet PCR method for the identification of pathogen DNA biomarkers utilizing polystyrene micro-beads with fluorescent color coding has also been proposed. More specifically, using a commercial bead set, the authors were able to encode several singleplex droplet PCRs. It is highlighted that this technique provides more scalability than the limited quantity provided by fluorescent detection probes like TaqMan probes. The approach was verified for three distinct bead sets coupled with target-specific capture oligonucleotides to detect the hybridization of three pathogens infecting poultry: avian influenza, infectious laryngotracheitis virus, and Campylobacter jejuni. The target DNA was amplified using fluorescently labeled primers and monodisperse picolitre droplets. The hybridization technique recognized the target DNA of all three species with high specificity from samples containing an average of one DNA template molecule per droplet. It is also worth mentioning that, with the scalability enabled by the color-coded detection beads, the droplet PCR assay detection panel could simultaneously detect many targets [Building on the capabilities of the dual-sample-on-a-chip LAMP assay, J. Jin et al.  created  targets .Listeria monocytogenes, Salmonella typhimurium, EHEC, S. aureus, Citrobacter freundii, and Campylobacter jejuni. This was validated for 10 pg, 1 pg, and 0.1 pg per pathogen DNA quantities. In quantitative mode, one DNA sample per cartridge can be quantitatively analyzed for the presence of two pathogens using standard curves that have been pre-made and created on a disk. 50 pg and 500 pg samples of L. monocytogenes genomic DNA were measured to contain 83 ± 17 pg and 540 ± 116 pg DNA, respectively, while 50 pg and 500 pg samples of S. Typhimurium DNA contained 48 ± 4 pg and 643 ± 211 pg DNA, respectively. It is shown that in both operating modes, microfluidic liquid routing was accomplished by spinning the cartridge on a low-cost centrifugal test apparatus [Using a microfluidic device for the continuous separation of bacteria from food samples, a recent study proposes a novel method for pathogen detection that builds on previous research. A microfluidic “LabDisk” capable of parallel, PCR detection of up to six distinct food-borne pathogens. It is composed of a microfluidic network for integrating positive controls, no-template controls (NTCs), and standards (STDs) into a centrifugal microfluidic PCR cartridge. In qualitative mode, each cartridge may test two DNA samples for the presence of six food pathogens, including PCs and NTCs, which are pparatus .E. coli O157:H7, Campylobacter jejuni, and adenovirus to ensure the safety of water resources. In this study, a poly (methylmethacrylate) (PMMA) microfluidic biosensor was developed and integrated with an array of organic blend heterojunction photodiodes (OPDs) for chemiluminescent detection of the target pathogens. The PMMA chip allowed for the capture of pathogens, which were then detected by poly/fullerene OPDs with a sensitivity greater than 0.20 A/W at 425 nm. The limits of chemiluminescent detection for E. coli, C. jejuni, and adenovirus were determined to be 5 × 105 cells/mL, 1 × 105 cells/mL, and 1 × 108 mg/mL, respectively. In less than 35 min, the integrated biosensor enabled the simultaneous analysis of all three analytes. As illustrated by the recovery tests, the study also demonstrated the capability of the integrated biosensor to detect bacteria in actual water samples. The findings indicate that the PMMA-OPD biosensor can be used to detect waterborne pathogens rapidly and sensitively in environmental samples.Lastly, Pires & Dong  detectedIn E. coli, Salmonella, and Listeria are the most common bacteria discovered in food matrices, these are the microbes that have received the most attention in the scientific community. Regarding the fabrication techniques to produce microchips, thermoplastics are the most common materials used in microfluidics since there is a demand for cheap and disposable microfluidic devices. Microfluidics and more complex LOC devices are usually made of thermoplastics, because of the existing mass-scale production methods existing (injection molding) and their unique properties , which enable coupling with established detection methods. Such approaches can provide LOC devices with low detection limit capable for real life applications.Microfluidic chips and LOCs are miniaturized devices that can exploit phenomena such as the capillary flow of fluids in conjunction with technologies like PCR and can offer attractive solutions for the on-site detection of several pollutants. They can also serve as an important tool for quality control in food and water, as well as in the medical industry, where they are primarily applied. From what was presented in the previous sections, it is clear that a substantial amount of research has been undertaken in relation to food quality and pathogenic microorganisms. Since There is also great interest in the paper-based chips due to their low cost and user friendliness. Paper-based devices are compact and lightweight, meaning they are ideal for use in point-of-care applications and also an eco-friendly option since paper is a biodegradable and environmentally friendly material. However, the sensitivity of μPADs is usually limited; they cannot meet the regulatory requirements in food microbiology, and this is one of the main reasons that most efforts have not yet been commercialized.For the detection of pathogenic microbes present in food and/or water samples, a wide range of detection techniques are available. In this review paper, we presented recent studies, each of which utilized or combined different detection techniques for the identification of various bacteria. In conclusion, the detection methods that are implemented into LOCs can be categorized into four main categories, Biosensors, Nucleic acid-based assays, immunoassays, and spectroscopic techniques. Biosensors are usually coupled with microfluidic devices, and they can be used to detect changes, such as magnetic, voltammetry, impedance, color, or resistance. In addition to biosensors, there are techniques based on nucleic acids that are used to detect pathogenic microorganisms. These methods rely on the detection of pathogen-specific DNA or RNA sequences. qPCR, for example, is a commonly used method for the identification of bacteria and is based on the use of fluorescent dyes or probes to measure the quantity of DNA or RNA amplified during the PCR reaction. These methods are currently very popular because they offer a very low limit of detection. Immunoassays employ specific antibodies capable of binding to the pathogenic microorganisms of interest, and the detection is completed using a label. Immunoassays are really fast and easy to perform, but again, in most cases, they exhibit limited sensitivity and can be mainly used as screening, rapid tests in food microbiology. Finally, the spectroscopic techniques such as Raman spectroscopy and surface-enhanced Raman spectroscopy (SERS) can be incorporated into LOC devices to create highly accurate, portable, and miniaturized analytical instruments for various medical, environmental, and chemical applications. Overall, all these methods have been successfully implemented in LOCs for research purposes, usually under ideal conditions, and few of them have passed to the next level of commercial exploitation. Therefore, there is a need for research and development on existing methods rather than proposing new ones, utilizing them on real samples and in industrially relevant settings, in order to accelerate the industry’s uptake of already developed concepts. In this direction, the technological advancements that have significantly simplified the detection protocols  and reduced the number of pretreatment steps are expected to provide more “success stories” of microfluidic based and LOC approaches being commercialized during the next few years.Other state-of-the-art applications of microfluidics and LOCs in food science also include, but are not limited to, microfluidics to test the antibacterial action of emulsions or antibacterial agents, microfluidics to synthesize antibacterial composites and particles, as well as microfluidics used in nanoencapsulation of functional substances.As far as the industry is concerned, the market for microfluidic chips is projected to reach $7.1 billion by 2025, indicating strong development potential. The fast expansion of the point-of-care testing industry and the rising need for downsizing and automation of laboratory procedures are driving market growth. Geographically, the microfluidic chip market is dominated by North America and Europe, with the United States being the largest contributor. Asia-Pacific, led by China and Japan, is also rising as a significant area. These areas have a substantial market presence due to factors such as technological breakthroughs, favorable government regulations, and the existence of key industry players. Infrastructure, strong investment in research and development (R&D), the presence of key industrial players, and favorable government assistance are all characteristics of a prosperous economy.The United States has emerged as the industry leader for microfluidic chips, holding a large market share. This is the result of several variables, including a well-established healthcare infrastructure, substantial investment in research and development (R&D), the presence of large industry players, and supportive government policies. The U.S. healthcare system is robust, with considerable demand for innovative medical technology and products. This, in turn, increases the need for microfluidic chips, which are indispensable for medical diagnosis and treatment. The United States offers a favorable climate for R&D, with considerable government and commercial investment. This investment has resulted in innovation in the microfluidic chip market as companies strive to produce new and innovative products.While it has many benefits, it is crucial to highlight that LOC devices appear to have some drawbacks; despite being a very large market, LOC devices are not yet mass-produced, resulting in higher prices. There is a link between the quantity of created devices and their price. Moreover, the competent authorities  have not yet allowed their extensive usage in quality control/safety. Another significant disadvantage is that in most cases up to now, LOC devices have been predominantly evaluated under optimal conditions, creating doubt regarding the precision and dependability of the results under sub-optimal situations. Therefore, more examination and testing are necessary to guarantee their full functionality.


This study investigates the impact of various sources of social support on the mental health of unaccompanied children under residential education in China. Unaccompanied children refer to those whose parents are still alive but unable to raise them due to various reasons. The study utilized self-reported questionnaires administered at two time waves, with the first wave (T1) evaluating family support, teacher support, and peer support, and the second wave (T2) evaluating depression, subjective well-being, and resilience. A total of 202 participants completed both surveys. To examine the predictive effect of different sources of social support on the mental health of these children, the study used the structural equation model with depression and subjective well-being as indicators. The results show that neither family support nor teacher support (T1) had a significant effect on the mental health (T2) of the children. However, peer support (T1) had a significant positive predictive effect on mental health (T2), indicating the unique role of peer support in promoting the mental health of unaccompanied children. The study also explored the mediating role of resilience between social support and well-being, revealing that though the direct effect of teacher support (T1) on mental health (T2) was not significant, the indirect mediating effect of resilience on the relationship between teacher support and mental health was significant. Both the direct and indirect effect of family support (T1) on mental health (T2) were not significant. These findings highlight the importance of creating a positive peer environment for unaccompanied children to promote their mental health. This study has important practical implications for the development of effective intervention programs aimed at improving the mental health of this population. In China, unaccompanied children refer to a specific population of children whose parents are still alive but unable to care for them due to various reasons. Unlike the Western world, where unaccompanied minors are often immigrants fleeing from war, poverty, or exploitation, or searching for family members ,2, unaccAs a result of these hardships, the mental health of unaccompanied children in China has been significantly challenged. Both Chinese research on left-behind children ,8 and inSocial support plays a vital role in children’s mental health. It encompasses both tangible and emotional assistance that individuals receive through interactions with others in their social networks ,18. SuchPrevious research has compared the impact of social support from various systems, such as relatives, family members, teachers, and peers on the mental health of children in distress, such as orphans ,22,23 anThis study examines the role of peer support for unaccompanied children living in two non-profit boarding schools in south-central China. Residential education refers to a type of educational program where students live on campus or in a residential facility while attending school. It has been found to be an effective mode for rural children to establish deep friendships in school ,25. FurtThus, this study assumes that peer support is of great and unique value in the social support system of unaccompanied children, especially those in residential education settings.Firstly, peer support is the most stable and accessible form of social support for children under residential education. According to ecological systems theory , the micSecondly, peer support meets the needs of school-age unaccompanied children in different development phases. To begin with, Erikson’s eight-stage theory of personality development  states tThirdly, peer support is an important medium for the transmission of values in residential education. The theory of group socialization  highlighFinally, peer support is closely related to children’s mental health. On the one hand, a higher level of peer support can directly improve children’s daily mental health ,31 and mBased on the reasons mentioned above, we expect that peer support will be a stronger predictor of mental health outcomes in unaccompanied children compared to other forms of support.One potential mechanism through which social support enhances mental health for children facing adversity is by fostering resilience. According to the integrated resilience model , the intH1: Among three sources of social support, peer support would be the strongest predictor of mental health of unaccompanied children under residential education.H2: Unaccompanied children would perceive more social support, which predicted higher levels of resilience that could improve their mental health outcomes.The data for this study were collected from two public welfare schools located in Hunan province, China. In both schools, Children are mostly enrolled in the third grade. During the semester, they study and stay in school, and maintain contact with their current caregivers through the school’s telephone. During holidays, students return to their homes. A total of 244 children and their caregivers provided informed consent and completed questionnaires in two waves. After quality checking, we finally collected 202 valid questionnaires. The testing materials and procedures were approved by the Human Experiment Ethics Committee of East China Normal University . The sample consisted of 108 boys and 94 girls with an average age of 11 years (range: 8–15).This study was conducted with two rounds of data collection performed separately. In both rounds, participants who agreed to participate in the study were concentrated in their classrooms. Researchers included 5 graduate students and a doctoral student in the psychology major, and all of them had been trained in the testing process before participating in the study. After distributing the questionnaires to each participant, for third and fourth grade participants, researchers read each item of the questionnaire twice to make sure children could understand. Participants from other grades read and answered on their own. After participants finished answering, they raised their hands and researchers would collect the questionnaires one by one.The first round of data collection, referred to as T1, was conducted at the end of the first semester, and the second round, referred to as T2, was conducted 5 months later at the end of the second semester. At T1, data were collected from 244 unaccompanied children in grades 3 to 8 from the two schools on teacher support, peer support, and family support. At T2, data were collected from the same 244 individuals on resilience, self-reported depression, and subjective well-being. After that, we excluded participants who could not understand the meaning of the questions or who failed to effectively report their scores on the questionnaires above. Finally, 202 valid questionnaires were collected .2/df = 1.37, RMSEA = 0.043, CFI = 0.952, TLI = 0.946.Questionnaire on social support status of left-behind children. The scale was compiled by Zhao  to asses“Delighted/Terrible” (D/T) Scale. The subjective well-being of participants is measured using the single-item face scale developed by Andrews and Withey . The sca2/df = 1.28, RMSEA = 0.038, CFI = 0.911.Children’s Depression Inventory (CDI). The questionnaire, compiled by Kovacs , assesse2/df = 1.86, RMSEA = 0.065, CFI = 0.941.The Connor-Davidson Resilience Scale(CD-RISC-10). The scale, extracted by Campbell-Sills and Stein  from 25 For this study, the data were processed and analyzed using SPSS 23.0 and AMOS 23.0 statistical software. The mean and standard deviation of social support, subjective well-being, self-reported depression, and resilience levels of unaccompanied children were first investigated. The effects of demographic variables, such as gender, age, grade, number of siblings, and parental marital status, on the subjective well-being, self-reported depression, and resilience of the children were then analyzed using one-way ANOVA and Pearson correlation analysis. Additionally, the correlation between family, teacher, and peer support at T1 and the subjective well-being, depression level, and resilience level of children at T2 was investigated.2/df value; the root mean square error of approximation (RMSEA); and the comparative fit index (CFI). To confirm a good model fit, the χ2/df should be less than 3.0, the CFI value should be larger than 0.90, and the RMSEA values should be smaller than 0.08 [To examine the mediating effect of resilience between social support and children’s mental health, structural equation modeling (SEM) was conducted as a powerful multivariate analytical tool . Family han 0.08 .To test the significance of the indirect effects in the mediation model, we conducted bias-corrected bootstrap tests with a 95% confidence interval (CI). The mediation analysis was evaluated using 2000 bootstrap samples to calculate the 95% bias-corrected and accelerated bootstrap CIs, a method that is considered more statistically robust than traditional approaches . A signip > 0.05). Furthermore, children’s teacher support was negatively correlated with depression levels at T2 , and was positively correlated with their resilience at T2 , indicating that the mental health and resilience of unaccompanied children under residential education are partially influenced by their teachers’ support.The results reveal that teacher support at T1 was not significantly associated with children’s subjective well-being at T2 (p < 0.05) and resilience  at T2, though weakly (r < 0.3). Additionally, family support at T1 was negatively correlated with children’s depression levels at T2 . These results suggest that the mental health and resilience of unaccompanied children under residential education are weakly correlated to their family support.Family support of unaccompanied children at T1 was positively correlated with children’s subjective well-being  and resilience  at T2. Additionally, peer support was negatively correlated with children’s depression levels at T2 . These findings indicate that the mental health and resilience levels of unaccompanied children in residential education are positively influenced by their peer support.Furthermore, peer support at T1 was positively correlated with children’s subjective well-being  = 338.64, CFI (0.941) is greater than the critical value of 0.9, and RMSEA (0.044) is less than the critical value of 0.08. The standard factor loading of all the observed variables are ranged from 0.493 to 0.885 (ps < 0.001), which means the observed variables adequately represented their respective latent variables in the model.The measurement model in this study includes four latent variables: family support, peer support, teacher support, and mental health. The measurement model was tested and found to fit well according to several indicators, χ2/df = 1.502, CFI = 0.941, TLI = 0.933, RMSEA = 0.044.To test the predictive effect of peer, family, and teacher support on the mental health of unaccompanied children, a model inspection was conducted using social support data at T1 and mental health and resilience data collected at T2 . The resp = 0.007) and direct effect  between peer support and mental health of unaccompanied children under residential education. Moreover, there was a significant indirect effect between teacher support and children’s mental health . There was neither direct effect nor indirect effect between family support and mental health outcomes.The bias-corrected bootstrap test results regarding the mediation effects are shown in These findings support the hypothesis of this study, suggesting that peer support, compared to family and teacher support, has the strongest predictive effect on the mental health of unaccompanied children. Additionally, resilience plays a mediating role in the relationship between children’s teacher support and their mental health. The present study aimed to investigate the effects of teacher, family, and peer support on the mental health of unaccompanied children under residential education, as well as the mediating role of resilience. The results indicate that among the three types of social support, only peer support at T1 had a significant positive predictive effect on mental health at T2. The findings also suggest that resilience plays a mediating role in the relationship between teacher support and unaccompanied children’s mental health. These results highlight the unique role of peer support in the social support system for unaccompanied children and have implications for evidence-based social policy and social work intervention aimed at promoting the mental health of unaccompanied children in China. Overall, this study contributes to the growing body of literature on the social support system and mental health outcomes of unaccompanied children in China, and sheds light on effective interventions to improve their mental health.Consistent with previous research ,22,45, tOf the three sources of social support, only peer support was significantly correlated with the subjective well-being, resilience, and depression level of unaccompanied children simultaneously. Specifically, when family, teachers, and peer support were included in the same model, only peer support had a significant predictive effect on the mental health outcomes of unaccompanied children. Therefore, it is reasonable to conclude that, among the three sources of social support that unaccompanied children have direct contact with, peer support is the most important for their mental health.This conclusion differs from some previous studies examining children in distress, such as orphans ,22,23, wIn the future, interventions to promote the mental health of unaccompanied children could consider the construction of a peer support system in order to provide more effective support. These findings can also inform evidence-based social policy and social work interventions to improve the mental health outcomes of unaccompanied children in China.Consistent with previous research on other vulnerable children ,23,46, tKohut’s theory  offered It is important to note that the study reveals that resilience did not mediate the impact of peer support on children’s mental health, suggesting that the influence of peer support on mental health involves a more complex mechanism.This study has provided insights into the implications of different sources of social support for unaccompanied children, particularly those under residential education. By comparing the effects of different sources of social support on the mental health of these children, we have been able to refine the scope of social support and discuss the mechanism of resilience in this context.The results of this study have important practical implications for the construction of social support systems for unaccompanied children. In particular, the construction of a peer support system should be given priority. However, the current rescue mode still mainly relies on external resources, such as good school supplies and teachers, while the resources within the school, such as the peer support system, may be overlooked. Based on the present findings, managers and teachers of schools for unaccompanied children should invest more in discovering peer resources within the school. For instance, the current support that involves older children taking care of younger children in the dormitory is a creative attempt. Still, additional group activities in school can be organized to strengthen the peer support systems. These practical initiatives can lead to the improvement of the mental health and resilience of unaccompanied children under residential education.Several limitations should be taken into account when interpreting the findings of this study. Firstly, the small sample size limits the generalizability of the findings, as the specific environment in which unaccompanied children in China reside makes it difficult to collect larger samples. Future research with larger sample sizes could yield more comprehensive results. Secondly, as the sample size is small, in order to ensure the statistical reliability of the results, this study did not consider the impact of differences between the two schools. However, the two schools are close in distance, have the same educational philosophy and modes, and belong to the same foundation. The difference between the two schools may have little impact on results. Future research can include consideration of the school affiliation’s bias. Thirdly, due to the self-reported method used in this study, younger children (below grade 3) who lack sufficient reading and writing abilities were not included in the sample. Moreover, this study is limited to examining the effect of social support on the mental health of unaccompanied children under residential education. Future research could investigate whether social support from different sources has varying effects on the mental health of unaccompanied children in different environments.Social support, including support from family members, teachers, and peers, is correlated with the mental health and resilience of unaccompanied children under residential education. Among them, peer support was found to be the most important form of social support in promoting the mental health of unaccompanied children, as it was the only source of social support that directly predicted mental health of unaccompanied children after 5 months. Future interventions aimed at promoting the mental health of unaccompanied children should focus on building strong peer support systems for these children.


Foot deformity is a risk factor for diabetic foot ulcer. This study was aimed to investigate the relationship between hallux valgus (HV) and diabetic foot through the radiographic measurement.The patients with diabetic foot hospitalizing in the Department of Endocrinology, the First Affiliated Hospital of Chongqing Medical University from September 2016 to June 2020 were selected. Then the foot plain X-ray radiographs were completed, and the size of HV angle (HVA) was measured. Their clinical data were collected, and the ulcer recurrence rate, amputation rate and mortality rate of the patients were followed up.A total of 370 patients were included. According to HVA, patients were divided into non-HV group (HVA<15°), and mild (15°≤HVA ≤ 20°), moderate (20°<HVA ≤ 40°) and severe (HVA>40°) HV groups. The age, height, BMI, smoking history and glycosylated hemoglobin level among the non-HVA, mild, moderate, and severe HV group (P<0.05), while smoking history, HbA1c, eGFR and autonomic neuropathy were significantly lower in HV group than those in non-HV group (P<0.05). The ulcer area in patients with moderate HV was larger than that in non-HV patients, and the severity of infection in patients with severe HV was significantly higher than that the other three groups (P<0.05).The occurrence of HV is not only related to age and BMI, but also to the creatinine and eGFR level, autonomic neuropathy, lower limb arteriosclerosis occlusion, coronary heart disease and hypertension. Therefore, more attention should be paid to renal function screening, neuropathy screening and evaluation of lower extremity vascular lesions in patients with diabetes, especially those with moderate or higher HV. The occurrence of HV was related to demographic factors including age and BMI.The occurrence of HV was related to the creatinine and eGFR level.HV was related to autonomic neuropathy, lower limb arteriosclerosis occlusion, coronary heart disease and hypertension.The severity of HV seems to be related to ulcer area and degree of infection.Diabetes prevalence is increasing rapidly due to economic development and aging of population . CurrentDiabetic neuropathy (DPN) is the most common chronic complication of diabetes and the most common predictor of foot ulcers . Among tStudies have shown that digitized radiologic images are commonly used to assess the severity objectively in patients with HV deformity –24. The The patients with diabetic foot who were hospitalized in the Department of Endocrinology, the First Affiliated Hospital of XXX University from September 2016 to June 2020 were selected, and the patients who met the inclusion criteria were selected as the research subjects.The inclusion criteria of diabetic patients were: 1) who met the diagnostic criteria for type 2 diabetes  diagnosed with ischemic diabetic foot, neurological diabetic foot or mixed diabetic foot according to the Guidelines for the Prevention and Treatment of Diabetic Foot in China (2019 Edition) (II) and clinical examination; 3) with complete foot weight-bearing X-rays after admission.The exclusion criteria were: 1) Patients with foot deformity, requiring dialysis treatment, diagnosed tumors, severe Malnourished patients, long-term bedridden due to cerebrovascular accident, or diagnosed with other neurological diseases were excluded.This study was in line with the ethical principles of World Medical Association Declaration of Helsinki, and was approved by the ethics committee of the First Affiliated Hospital of XXX University, and the guardians signed the informed consent.©2005-2014 MedCalc software) based on the foot plain X-ray radiographs of included patients After meeting the inclusion criteria, the plain foot X-ray films of diabetic foot patients were collected. The hallux valgus angle (HVA) was measured using Digimizer software  classification system  and the The SPSS26.0 statistical software was used for statistical analysis, and K-S single-sample test was used to test whether data conformed to the normal distribution. Normally distributed data was expressed as mean ± standard deviation (X ± S), and analyzed by t test; non-normally distributed data was expressed as median (quartile), and analyzed by the rank sum test. Categorical and rank variables were expressed as percentages or frequencies, and comparisons between groups were performed using the chi-square test or Fisher’s exact test. Spearman correlation analysis was used to evaluate the relationship between HVA and related positive variables. Univariate logistic regression analysis was performed on all potential variables and the occurrence of HV. Then, all positive predictors (P<0.1) were analyzed by a multivariate logistic regression model. A P value < 0.05 was considered statistically significant. Graphs were drawn using GraphPad Prism 9.3.1.A total of 370 patients with diabetic foot were included and the HVA was measured. The basic data of the total population was shown in The incidence of hypertension in the mild HV group was significantly higher than that in the non-HV group, and the incidence of coronary heart disease in the mild HV and moderate group was higher than that in the non-HV group (P<0.05). The eGFR level of the mild and severe HV groups were significantly lower than that of the non-HV group.The proportion of Charcot’s foot of the severe HV group was significantly higher than the other three groups. The comparison of PEDIS classification among the four groups showed that the ulcer area in patients with moderate HV was greater than that in patients without HV Table 2;A total of 370 patients were followed up by telephone, of which 282 patients or their family members answered relevant follow-up questions, and 88 patients were lost to follow-up due to wrong phone calls, empty numbers, shutdown, shutdown, and refusal to answer. The median follow-up time of the 282 patients was 3.92  years, of which the median follow-up time of non-HV patients (N=110) was 3.96  years, and the median follow-up time of HV patients (N=127) was 3.96  years. The follow-up time was 3.92  years, and there was no statistical difference between the two groups . Of the 282 patients, 92 patients had ulcer recurrence (32.6%), 21 patients were amputated again due to diabetic foot/toe (7.4%), and 82 patients died due to diabetic foot, diabetes-related complications, or cardiovascular and cerebrovascular events (29.1%). However, there was no significant difference in recurrence, re-amputation/toe and mortality between the non-HV group and the HV group (P>0.05).Spearman correlation analysis showed that the angle of HV was positively correlated with age , BMI , and arteriosclerosis obliterans , hypertension  and coronary heart disease , while it was negatively correlated with smoking history , HbA1c , autonomic neuropathy , and eGER .After univariate and multivariate logistic regression analysis, autonomic neuropathy was found to be a protective factor for the development of HV Table 3.HV as one of the common foot deformities  has a moAmong the 370 patients, there was no difference between the occurrence of HV and diabetic nephropathy, but the patients with HV had higher creatinine and lower eGFR, suggesting that the renal function of HV patients was worse than that of non-HV patients. The incidence of lower limb arteriosclerosis obliterans, hypertension and coronary heart disease in patients with HV was significantly higher than that in patients without HV. Diabetes can cause peripheral arterial disease, endothelial dysfunction and autonomic nervous system dysfunction, etc., resulting in blood flow regulation disorders , which iHV has been identified as a risk factor for foot ulcers . By gradMoreover, the increased plantar pressure and load in diabetic patients with HV may be a result of their larger ulcer size, while the increased ulcer size also increases the risk of infection. Studies suggest that HV is related to the degree of infection , but curAt present, there only a few articles have reported that neuropathy is related to the occurrence of foot deformity , and neuHowever, there are some disadvantages of this study that must be considered: this study was a cross-sectional study; the included patients were all diagnosed with diabetic foot, and there was no diabetes without diabetic foot as control; the objects were all from the same hospital with severe condition, therefore there could be selection bias.In conclusion, the occurrence of HV in diabetic foot was not only related to age and BMI, but also related to the occurrence of creatinine level, HbA1c level, eGFR level, autonomic neuropathy, lower limb arteriosclerosis occlusion, coronary heart disease and hypertension. Moreover, HV seems to be related to ulcer area and severity of infection, and autonomic neuropathy was found to be a protective factor for the development of HV. Therefore, patients with diabetes mellitus, especially those with moderate or higher HV, should pay more attention to their renal function, neuropathy screening and assessment of lower extremity vascular lesions, such as ABI, TBI, lower extremity vascular ultrasound, and even CTA of lower extremity arteries if necessary to assess the vascular lesions more visually. At the same time, in addition to strengthening the screening for complications, it is also important to assess the ulcer area and the degree of infection. When the infection is suspected to have invaded the bone, enhanced MRI or probe-to-bone test should be performed to assess whether it is combined with osteomyelitis.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.The studies involving human participants were reviewed and approved by First Affiliated Hospital of Chongqing Medical University. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.Conception and design of the research: XL and CZ. Acquisition of data: QH and ZD. Analysis and interpretation of data: XN, QZ and QC. Drafting the manuscript: XL and CZ. Revision of manuscript for important intellectual content: QZ and QC. All authors contributed to the article and approved the submitted version.


Animals are frequently used to determine whether new compounds have the potential to be used as drugs against the parasitic disease leishmaniasis. There is, however, no consensus on the best animal model, and many different ones are used. This review explored the many different models and identified their strengths and weaknesses. The work showed that many papers on animal models for leishmaniasis lack a thorough description of the experimental procedures used, are deficient in appropriate ethical review , and measures to reduce, refine or replace animal studies are hardly addressed. Mice and hamsters are most frequently used, but there are many differences in how the animal experiment is set up. We propose a more generalized animal model, in particular to ensure that different experiments can be better compared. Furthermore, we make a plea to report all animal experiments in the same way, and all experiments must be scrutinized by appropriate ethical review committees.L. amazonensis or L. major were most frequently used to establish experimental infections (single inoculum). Animal welfare was poorly addressed in all included studies, as the definition of a human end-point or consideration of the 3Rs  was hardly addressed. Most animals were euthanized at the termination of the experiment. The majority of the studies had an unknown or high risk of bias. Animal experiments for drug development for leishmaniasis mainly poorly designed and of low quality, lack appropriate ethical review, and are deficient in essential information needed to replicate and interpret the study. Importantly, aspects of animal welfare are hardly considered. This underpins the need to better consider and record the details of the study design and animal welfare.Many different animal models are in use for drug development for leishmaniasis, but a universal model does not exist. There is a plethora of models, and this review assesses their design, quality, and limitations, including the attention paid to animal welfare in the study design and execution. A systematic review was performed following the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines of available literature after the year 2000 describing animal models for leishmaniasis. The risk of bias was determined using the SYstematic Review Centre for Laboratory animal Experimentation (SYRCLE) risk of bias assessment tool. A total of 10,980 records were initially identified after searching the databases PubMed, EMBASE, LILACS, and SciELO. Based on the application of predetermined exclusion and inclusion criteria, a total of 203 papers describing 216 animal experiments were available for full analysis. Major reasons for exclusion were a lack of essential study information or appropriate ethical review and approval. Mice  and hamsters  were the most frequently used animals, mostly commercially sourced, in the included studies. All studies lacked a formal sample size analysis. The promastigote stages of  Leishmania species [Phlebotominae), and there are about 20 Leishmania species that can infect humans [Leishmaniasis is a protozoan parasitic disease caused by  species ,2. The pt humans ,2. Leisht humans ,2,3. Next humans , includit humans ,6. Leisht humans ,4. The treatment of leishmaniasis is difficult and painful, and many drugs have significant toxic side effects ,2,3. ManLeishmania species used for experimental infection, the dose and route of the inoculation of the challenge strains, responses to the disease, and the clinical presentation of symptoms [A key step in the development of new drugs for leishmaniasis remains the pre-clinical assessment of the safety and efficacy of new components in animal models . There asymptoms ,14. ThisThis review aims to assess the design, quality, and limitations of various animal models for leishmaniasis their use in drug discovery and evaluation. It further focuses on the attention that is being paid to animal welfare in study design and execution.https://pubmed.ncbi.nlm.nih.gov (accessed on 29 March 2020), Embase https://www.embase.com (accessed on 29 March 2020), the Latin American and the Caribbean Health Sciences Literature database (LILACS); http://lilacs.bvsalud.org (accessed on 29 March 2020), and the Scientific Electronic Library Online (SciELO); https://www.scielo.br (accessed on 29 March 2020) databases.This systematic review followed the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines . A systeMus spp.), rabbits (Oryctolagus spp.) or hamsters (Cricetinae spp.) [Many animal models for leishmaniasis are mice (ae spp.) ,14. For ae spp.)  and PubMae spp.)  were appThe databases were last searched on 29 March 2020. The complete search syntaxes are presented in Full papers presenting original data of which the full text could be retrieved were used for review. Excluded were publications of which the full text was not retrievable, editorials, conference abstracts, conference proceedings, case reports, and (systematic) reviews.https://endnote.com accessed on 22 March 2023), and replicates were removed using this software package followed by the further removal of replicates by using Rayyan web-based  software [The retrieved records were imported into EndNote .A second screening was performed on full-text articles. Discrepant results were resolved by discussion until a unanimous decision was reached.Leishmania, in vitro studies, non-mammalian studies or papers that addressed infections other than leishmaniasis were excluded.We included papers for review that reported in vivo animal experiments on the safety or efficacy of compounds for the treatment of leishmaniasis. Papers addressing vaccine development, studies on immune responses to Leishmania species, stage, and number of parasites used for inoculation (promastigotes or amastigotes), route and frequency of inoculation, duration of inoculation; whether next to an experimental treatment a competitor treatment, placebo treatment, and/or no treatment was applied; duration to read-out, primary organs targeted for read-out and experimental method used for read-out ; physiological parameters reported ; and statistical analysis performed . In addition, information on animal welfare ; consideration of a human end-point [For each eligible publication, the following study variables were extracted: whether there was an ethical review  of the study; species, breed and gender of animal used for experimentation; weight and/or age of the experimental animal; number of animals used in total and per group; source ; housing ; pre-treatment(s) given; use of pregnant animals; information on the experimental challenge: i.e., nd-point , considend-point , and whaLeishmania infection.A manuscript was immediately excluded from full analysis if an ethical review  of the study protocol was not performed or reported. Furthermore, a study was not further analyzed if one of the following primary parameters was missing : specieThe quality of the animal experiments described in papers included for full analysis was assessed with the SYRCLE´s risk of bias (RoB) assessment tool . The RoBhttps://www.crd.york.ac.uk/prospero/ accessed on 12 May 2023) under registration number CRD42019092382.This systematic review is registered in the international prospective register of systematic reviews  or a review.Finally, 539 articles, describing 634 animal experiments, were available for further analysis. From this set of articles, 216 papers were excluded because appropriate ethical review or approval was not reported. Furthermore, another 120 articles were excluded because essential study information was not reported. This finally resulted in 203 articles that remained for data extraction and full analysis. A full list of the references of the papers included in this systematic review is presented as Leishmania infection, were not given to the animals in the analyzed experiments.None of the included studies used pregnant animals. Pre-treatments, for example, steroids to modulate the immune response or to make the animals more susceptible to Mus musculus), and in particular the breed BALB/c , were by far the most frequently used experimental animals in the analyzed animal studies  of the studies used male mice, 2.2% (4/179) used both female and male mice in the experiment, and 16.2% (29/179) did not report the gender of the used mice. The mean age of the mice was 6.8 weeks (95% CI: 6.5–7.1), and the mean weight was 22.7 g (95% CI: 21.3–24.1).Female mice were used in 69.3% (124/179) of the animal studies with Mesocricetus auratus) was used in 37 analyzed studies , but these were not included in the final analysis because of a lack of essential study information.Rats and dogs were reported in very few initially included studies —see The mean number of hamsters per study group was 7.4 animals —see n = 196; 90.7%) did report on the statistical method that was applied. The most frequently used statistical analysis was ANOVA, followed by Student’s t-test or Tukey test.In total, 20 studies (9.3%) did not report the application of a formal statistical analysis of the data generated by the animal experiment. All other studies . In contrast, (axenic) amastigote stages were used in 34 experiments (15.7%).L. major . L. donoLeishmania infection. None of the animals was repeatedly infected with the parasites. The mean number of parasites used to establish an experimental infection in mice was 1.25 × 107 ± 2.31 × 107. In general, the inoculum in hamsters was higher: 2.36 × 107 ± 3.26 × 107. A sub-cutaneous inoculation was the most frequently used method of administration  to establish the experimental infection  or blood parameters including cytokines, antibodies, and inflammatory markers (33.8%).Leishmania infection-associated lesions. Most animals were sacrificed at the end of the experiment  of the studies; 60.2% (130/216) of these studies used animals obtained from a commercial source, and 9.3% (20/216) used animals reared in-house or from another non-commercial source. However, sourcing was not reported in 30.6% (66/216) of the studies .n = 181; 83.8%). Housing in groups was reported for 10 studies (4.6%), and individual housing was reported in 25 studies (11.6%). Furthermore, 91 studies (42.1%) provided information on access to food and water, lighting conditions, humidity or housing under spf conditions.Housing conditions were reported for 35 (16.2%) of the analyzed studies, but not for the majority  did not report on randomization at all. Moreover, none of the studies (100%) reported whether the allocation was adequately concealed (high risk of selection bias). In contrast, with the exception of three studies, in most studies, the study groups were similar at baseline (low risk of selection bias). All analyzed studies had a high risk of performance bias as none reported on the random housing of the animals during the experiment and whether caregivers and/or investigators were blinded from knowledge on which intervention each animal received during the experiment. Further, in terms of detection bias, the risk was determined to be high as most studies failed to report whether the animals were selected at random for outcome assessment, and it was not reported whether the outcome assessor was blinded. For all studies, there was an unknown risk of attrition bias. None of the studies reported on incomplete outcome data or reported on having encountered these, but all presented complete data sets. Finally, it was determined that almost all studies (with the exception of two) were free of selective outcome reporting (low risk of bias) and free of other problems that could result in a high risk of bias. This was most likely due to the fact that the inclusion and exclusion criteria for this systematic review were stringent.This literature review presents a systematic analysis of the animal models that are in use to determine the efficacy of  drugs for leishmaniasis. Although there is a wealth of publications in this field, a critical review signals that many of these studies are poorly designed, lack essential information that is needed to reproduce experimental data, and have an unknown or high risk of bias. This is despite the availability of guidelines, i.e., the ARRIVE guidelines  that were originally developed in 2010 to improve the reporting of animal research  and updaLeishmania species), duration of experiments, and read-outs. This makes it impossible to extract a complete generalized protocol or model to study  drug efficacy in animals from the analyzed studies. Nevertheless, having a standardized model available would greatly improve the comparability of different studies on the efficacy of anti-leishmanial  drugs and would contribute to the reduction in the number of animals used [A multiplicity of experimental animal protocols is presented with a huge variation in the choice of animals (mainly mice and hamsters), all with different ages, weights and genders, inocula  as compared to, for example, Swiss mice that are highly resistant to n humans .Leishmania species will strongly depend on what clinical type of leishmaniasis will be studied, but we propose that for visceral leishmaniasis (VL) drug studies, in the first instance, L. donovani should be used, compared with L. major for Old World cutaneous leishmaniasis (CL) and L. amazonensis for New World CL. Preferably, WHO reference strains of Leishmania will be used to further optimize the standardization of the model. Other species can be studied when sufficient efficacy has been established. We propose an initial infective dose of 1.25 × 107 promastigotes and a duration of 21 days of infection to establish disease. The group size could be six to seven animals per experimental group, although a formal sample size analysis has not been done. The experiment should include at least one group that is treated with an established drug to treat leishmaniasis of interest and a placebo group. Most likely, a non-treated (as this is covered by the placebo) and a non-infected group  are not needed. Several different read-out systems are used to determine the efficacy of the drug under investigation. Many of them are in-house developed and/or prone to subjective interpretation, such as microscopy. To circumvent this and to establish an objective quantifiable read-out system, we propose the use of standardized quantitative PCR methods for VL and CL [Leishmania parasite burden over multiple time points in the same animal, the use of in vivo imaging using luciferase/fluorescent parasites could be used as a more advanced method [Leishmania strains and extensive laboratory infrastructure. A multi-center validation of the proposed animal model, including read-out systems, should be performed.The selection of infecting L and CL ,30,31. Td method ,33. HoweThe study protocol must always be ethically reviewed by a competent committee and must include sufficient information on the expected efficacy of the drug under experimental investigation that justifies the initiation of an in vivo animal experiment. Finally, aspects of animal welfare  should always be addressed in the protocol and reported in the scientific publication ,25,26,27Animal experiments for the drug development for leishmaniasis are in mainly poorly designed and of low quality, lack appropriate ethical review, and are deficient in essential information needed to replicate and interpret the studies. Importantly, aspects of animal welfare are hardly considered. This underpins the need to better consider and record the details of the study design and animal welfare.


Exposure to benzene may lead to the appearance of a series of signs, symptoms, and complications, which are characterized by benzene poisoning, which is an occupational disease. This study evaluated the presence of signs and symptoms related to occupational exposure and whether occupational exposure to BTX is associated with the development of hematological changes. (2) Material and Methods: This cross-sectional epidemiological study included 542 participants, in which 324 were gas station workers (GSWs) and 218 were office workers (OWs) with no occupational exposure to benzene. To characterize the type of exposure (exposed and not exposed), trans,trans-Muconic acid (tt-MA), Hippuric acid (HA), and Methylhippuric acid (MHA) were used as exposure biomarkers. The tt-MA analysis revealed that the GSWs had 0.29 mg/g of urinary creatinine and the OWs had 0.13 mg/g of urinary creatinine. For HA, the GSWs presented 0.49 g/g of creatinine while the OWs presented 0.07. MHA analysis revealed that the GSWs had 1.57 g/g creatinine and the OWs had 0.01 g/g creatinine. Occupation habits and clinical symptoms were collected by questionnaire and blood samples were analyzed for hematological parameters. The persistence of hematological changes was evaluated with three serial blood collections every 15 days followed by laboratory hematological analysis. A descriptive analysis by the Chi-square test method was performed to evaluate the association between occupational exposure to fuels and the occurrence of changes in hematological parameters. (3) Results: In the GSWs, the most described signs and symptoms were somnolence (45.1%), headache (38.3%), dizziness (27.5%), tingling (25.4%), and involuntary movement (25%). Twenty GSWs that presented hematological alterations performed serial collections fifteen days apart. In addition, these workers presented total leukocyte counts above the upper limit and lymphocyte counts close to the lower limit. Leukocytosis and lymphopenia are hematological alterations present in chronic benzene poisoning. (4) Conclusions: The results found an initial change in different hematological parameters routinely used in clinics to evaluate health conditions. These findings reveal the importance of valuing clinical changes, even in the absence of disease, during the health monitoring of gas station workers and other groups that share the same space. Gasoline produced from oil refining is widely marketed at gas stations throughout Brazil . BenzeneIn addition to benzene, gasoline contains other volatile organic compounds (VOCs), including toluene and xylene isomers, represented by the term BTX. Workers involved in the production, transport, and commercialization of fuels are exposed to various levels of BTX ,3,4,5,6 In Brazil, in addition to the filling station attendants at gas stations, workers in convenience stores or even snack bars also often occupy the same space, increasing the number of workers exposed to fuel. Considering the context of gas stations in Brazil, occupational exposure may occur through direct contact  with gasoline, but also by breathing  , dependiAcute or chronic exposure to benzene may lead to a series of signs, symptoms, and complications which are characteristic of benzene poisoning , known iGiven the adverse health problems caused by benzene, this study aimed to evaluate the presence of signs and symptoms related to occupational exposure and whether occupational exposure to fuels is associated with the development of hematological changes in gas station workers in the city of Rio de Janeiro, Brazil.This is a cross-sectional epidemiological study. Workers from gas stations in the city of Rio de Janeiro, Brazil, were recruited and interviewed after signing the informed consent form for the study. All gas station workers (GSWs) were invited to participate: both gas station attendants and managers—who incur inhalation and dermal exposure to the fuels and supply vehicles, receive and/or collect tank truck samples, and perform tank measurements underground of the station; and convenience store workers—who incur exposure to the fuels by inhalation .To characterize the hematological changes from BTX, the exposed workers who presented hematological alterations performed two more collections within 15-day intervals . Workers with no occupational exposure to fuels were also selected to form the comparison group who were not occupationally exposed—office workers (OWs)—in the same region as the gas station.Thirty-eight gas stations were visited, but after refusals and withdrawal of participation, 21 gas stations were included, 9 of which were located in the Central region of Rio de Janeiro and 12 in the South Zone. These regions were chosen because gas stations are concentrated in the same geographical region, thus, environmental and social issues were expected to be more homogenous. In addition, short distances between sites facilitated interviews and sample collection. Out of these, 324 GSWs participated in the study and 20 workers were included in the SCWs, regardless of their function.For the OW group, 218 workers enrolled from the National Cancer Institute (INCA) and the Federal University of the State of Rio de Janeiro (UNIRIO), with no occupational exposure to benzene. In all cases, workers of both sexes participated in this study, who were over 18 years old and had at least 6 months of employment or contract.This work was submitted and approved by the Human Research Ethics Committee of INCA (CEP/INCA) under No. 121/09. All workers invited to participate in the study signed an informed consent form prior to any intervention. The results obtained were delivered to individual workers, and if any changes in health conditions were perceived, the workers were referred for medical follow-up.The study participants answered a standardized questionnaire applied through an interview with a trained professional. Information regarding socioeconomic aspects and chemical agent exposure during the workday were collected. A clinical questionnaire on current and prior history of complaints, signs, and symptoms was applied. Occupational habits were also evaluated.The following variables were evaluated: sex; age; smoking; alcohol consumption; processed food consumption; working time; headache; weight loss; drowsiness; dizziness; tremor; loss of muscular strength; weakness; tingling; involuntary movements; petechial; bruising; epistaxis, and night sweats. The participants answered questions about common practices of the GSW: (1) clothes wet with fuel; (2) siphoning fuel with a hose; (3) trusting the automatic nozzle; (4) sniffing the fuel tank cap; (5) bringing their face closer to the fuel tank; (6) using a cloth.n = 304), serial collection workers (SCWs) (n = 20), and office workers (OWs) (n = 218).The peripheral blood was collected by a professional through venipuncture of the forearm in a vacuum collection tube (EDTA anticoagulant) and kept refrigerated at approximately 8 °C in a Styrofoam box. The samples were then sent to the Laboratory of Clinical Pathology of the INCA for hematological analyses. This analysis considered gas station workers (GSWs) (n = 255), SCWs (n = 20), and OWs (n = 100) were considered because some participants had creatinine urinary levels higher than the limit value.To confirm workers’ exposure to BTX, urine samples were collected at the end of the working day from all 8 h shifts . Samples were collected once for each worker and the samples were analyzed separately. After collection, the urine samples were placed in a Styrofoam box containing recyclable ice, kept cold, and transported to the laboratory. Samples from the OWs  were collected at the end of the working day and manipulated in a similar way as samples from the exposed group (GSWs). For these analyses, GSWs . The total cell count parameter was selected, in which quantification of white blood cells, differential count of leukocytes  as well as erythrocytes, hemoglobin, hematocrit, and platelets were performed for all study participants.® chromatography column (250 × 4.6 mm) . The standard was obtained from Sigma-Aldrich® , and results were normalized using urinary creatinine values. Samples were cleaned by solid phase extraction (SPE) in Applied Separations® cartridges , N+ Quaternary Amino (SAX), 500 mg, 3 mL. The SPE conditioning was as follows: 3 mL of methanol, 3 mL of ultrapure water. One milliliter of urine was added, followed by a prewashing of 3 mL 1% acetic acid. Finally, elution occurred in 10% acetic acid (pH 2.7). Subsequently, an aliquot of 20 μL was injected into the HPLC by manual injection and the chromatographic conditions described by Geraldino et al. [The urinary levels of trans, trans-muconic acid (tt-MA-metabolite of benzene) were evaluated by high efficiency liquid chromatography, a method initially proposed by Ducos et al. , modifieo et al. .®  [® , and results were normalized using the urinary creatinine levels. One milliliter of sample was added to 1 mL of methanol and then centrifuged for 7 min at 3000 rpm, with subsequent manual injection of 20 µL into the HPLC-UV.The urinary levels of hippuric acid (HA-metabolite of toluene) and Methyl Hippuric Acid (MHA-metabolite of xylene) were evaluated by high-performance liquid chromatography (HPLC) using an ultraviolet detector (HPLC-UV) equipped with a Lichrosorb RP18 chromatography column (244  ×  4 mm) with Merck 5 mm particlesGermany) . The staThe validation parameters followed the recommendations by ANVISA . PrecisiFor the construction of the calibration curve, a linear range of tt-MA ranging from 0.01 to 100 mg/L was used. The RSD was less than 3%. The limit of detection and quantification were, respectively, 0.00075 and 0.0013 mg/L. The linearity coefficient was r = 0.996, within the acceptable range. The cartridge recovery study proved adequate (98–102%). The matrix effect and stability study were adequate.The RSD estimate was less than 2% and a linear range of HA concentration from 0.01 to 10 g/L was observed. Seven points were used to construct the calibration curve, whose linearity coefficient had an acceptable performance (r = 0.998). The limit of detection and limit of quantification were, respectively, 0.0075 and 0.001 mg/L.The linearity coefficient remained within the acceptable limit range (r = 0.999). The RSD was less than 2% and the linear range of MHA concentrations was 0.001 to 10 g/L. Seven points were used in the construction of the calibration curve. The limits of detection and the limit of quantification were, respectively, 0.0005 and 0.005 mg/L.A descriptive analysis of the characteristics of the study population was carried out using a frequency distribution of the categorical variables, comparing the two groups of workers (GSWs and SCWs) with each other and to the OWs by the Chi-square test method. For the continuous variables, the measures of central trend and dispersion were calculated. The normality of the continuous variables was evaluated using the Kolmogorov–Smirnov test. As the variables presented a non-normal distribution, they were analyzed with the nonparametric tests of comparison of the Mann–Whitney test. For the categorical variables, the Chi-square or Fisher test was employed. To confirm the relationship between variables, linear regression analysis was used. Statistical analyses were performed using the program Statistical Package for Social Sciences  for Windows, version 17.0.This study involved the participation of 324 GSWs and 218 OWs. Analysis of biomarkers of exposure to BTX was performed on urine samples from 258 GSWs, 17 SCWs, and 100 OWs because some workers had high limit values of creatinine and were excluded. The analysis of urinary tt-MA revealed that the GSWs and SCWs had exposure to benzene at work (0.28 mg/g and 0.23 mg/g of urinary creatinine) when compared with the OWs (0.13 mg/g of urinary creatinine). Similar results were found for toluene and xylene that presented a higher mean of HA and MHA in the GSWs and SCWs than in the OWs, as can be seen in p-value 0.965; smoker, p-value 0.466), alcohol consumption , and processed food consumption . For HA, the adjusted model was not affected by smoking , alcohol consumption , and industrialized food consumption . For MHA, the adjusted model was not affected by smoking , alcohol consumption , and industrialized food consumption . Therefore, no influences of confounding variables were found in the concentration of the analyzed biomarkers.Exposure biomarkers can be influenced by confounding variables. For this reason, a linear regression analysis was performed considering smoking, alcohol, and processed food consumption as confounding factors. For tt-MA, the adjusted model was not affected by smoking , convenience store employee (25.3%), and manager/employer in charge (15.1%). Their most commonly reported length of work was up to ten years for 81.1% of GSWs and 70% of SCWs . Some coThe most described signs and symptoms of the GSWs, including convenience store employees, were drowsiness (45.1%), headache (38.3%), dizziness (27.5%), tingling (25.4%), involuntary movement (25%), and weakness (21.9%) as can be seen in The hematological results showed that GSWs had significantly higher levels of erythrocyte, hemoglobin, hematocrit, and lymphocyte, and lower levels of neutrophil, than OWs . The obsTo analyze benzene poisoning, the important parameters to monitor in the hemogram include leukocyte and lymphocyte counts. Those analyses were performed three times with 15-day intervals. Of the 20 volunteers who presented hematological alterations, 5 remained with the same alterations with an interval of 45 days between collections. These exposed workers presented leukocytosis in the three collections and the number of lymphocytes was very close to the lower limit .3), while the ACGIH established the value of 0.5 ppm (1.6 mg/m3) for the TLV-TWA, which corresponds to the occupational exposure limit value, which takes into account a weighted average over an 8 h working day and a 40 h working week [3) for chemical and petrochemical sectors, such as gas stations.This study indicated that GSWs with different jobs in the same gas station environment, such as the filling station attendants, manager/employer, convenience store workers/employees, cleaners, and the administrative sector, are exposed to a high concentration range of BTX vapors in the workplace. There is concern worldwide to reduce, as much as possible, the exposure of these workers to benzene. The National Institute for Occupational Safety and Health (NIOSH)  establising week . In Brazing week , which d3, a variation between 0.04 mg/m3 and 0.43 mg/m3 was observed. Thus, 33% of the stations had an environmental concentration of benzene greater than 1.0 ppm [Cruz et al.  determin 1.0 ppm .In this study, the environmental monitoring of exposure to BTX was not performed, but exposure biomarkers present in the urine of workers were analyzed. According to Brazilian legislation, urinary tt-MA is indicated as a biomarker of benzene exposure. The correlation between tt-MA and benzene is detected at environmental levels of the solvent below 1.0 ppm; this fact allows its use as a biomarker of exposure in gas station workers, since the maximum concentration allowed in these environments is 1.0 ppm. Scherer et al. (1998) observed a linear correlation between urinary excretion levels of tt-MA and occupational exposure to benzene in workers exposed to concentrations above 0.1 ppm . As a reSome habits may interfere with urinary tt-MA, HA, and MHA levels such as smoking, consumption of processed foods containing sorbic acid , and alcoholic beverages . For thiSome occupational habits that were identified during the interview could increase exposure to benzene present in gasoline through its contact with skin and mucous membranes. During analysis of the frequency of the main occupational habits or procedures that could enhance exposure, 29.3% of workers reported that they had worked in uniforms wet with fuel. This often occurs after a fuel spill or improper handling of fuel pumps. Being in contact with a piece of clothing soaked with fuel increases contamination through dermal absorption and consequently increases exposure ,22. ReseIn Brazil, filling vehicles can only be conducted with the automatic filling spout. They have a lock that, among other functions, prevents spillage/leakage of fuel during fueling. It was observed that 53% of the workers trust the automatic filling nozzle of the fuel pump, but the remaining 47% do not trust it. When fuel spills occur during refueling, filling station attendants usually use a cloth to clean the vehicle. Many of them keep this cloth close to their body, on their shoulders or waist so that it can be used promptly in case a spill occurs during another filling . It is kBrazilian legislation set 2033 as a deadline for gas stations to install vapor recovery systems at their pumps to reduce workers’ exposure to gasoline components that evaporate during filling . MoreoveOther occupational habits that increase contact with fuels were also reported by the filling station attendants. The habit of siphoning fuel with the mouth occurs when the attendant fills the vehicle with the wrong fuel and the worker uses this mechanism to remove the wrong fuel from the tank. It is known that siphoning through the oral mucosa is increased and, therefore, the filling station attendants who reported performing this activity may increase their exposure .Gasoline has a sweet smell characteristic of benzene , and thiThe main occupation reported in the exposed groups was filling station attendant. This result was similar to that reported by Amaral et al. , who obsAccording to Fundacentro , benzene poisoning is characterized by the presence of signs, symptoms, and complications after acute or chronic exposure to benzene, and chronic exposure can promote diverse signs and symptoms, with the possibility of preferential complications in the hematopoietic system . The OWsThe main signs and symptoms of the GSWs were analyzed, and the literature indicates that most effects related to acute benzene intoxication are mucosal irritation; central nervous system effects, such as drowsiness, dizziness, excitation, headache; tremors; loss of consciousness; and death ,8,23. InSince there is no established effect biomarker for benzene in Brazil, the present article evaluated the levels of exposure of workers to benzene through tt-MA, the presence of signs and symptoms related to occupational exposure, and whether occupational exposure to fuels with BTX is associated with the development of hematological changes in gas station workers.Major hematological changes in benzene poisoning include persistent leukocytosis; mean corpuscular enlargement (macrocytosis); eosinophilia; decrease in the absolute number of lymphocytes (lymphopenia or lymphocytopenia); neutrophilic changes: basophilic stippling, hyposegmentation of neutrophils (pseudo-Pelger); presence of macroplatelets; and leukopenia with association of other cytopenias (thrombocytopenia) . In thisThe hematological analysis revealed that of the 324 GSWs whose blood was collected, 304 maintained their results within the reference limits and, for this reason, did not participate in the serial collection. The median values presented by the GSWs were similar to those presented by the OWs; nevertheless, a statistical difference occurred between the groups for erythrocytes, hemoglobin, hematocrit, neutrophils, and lymphocytes.Data from the literature revealed several types of changes in the hematopoietic system in workers with benzene exposure, either with an increase or decrease in the parameters evaluated. In contrast with our study, many studies about gas station workers showed a decrease in the number of erythrocytes and hemoglobin among those exposed ,32,33,34Of the 20 workers who performed the serial collection, 5 workers remained with an increased number of leukocytes and a low number of lymphocytes, close to the lower limit of the reference value. A previous study observed that some GSWs, regardless of their occupation, were more likely to present DNA damage and alterations in cells of the immune system than workers belonging to the comparison group without occupational exposure to BTX .According to the Health Surveillance Standard for workers exposed to benzene, they must be workers who present hematological alterations in serial blood counts, without other clinical findings that justify them, among others: persistent leukocytosis and lymphopenia. In situations where the alterations persist in this minimum period of 45 days, the suspected case for benzene poisoning is considered . ChronicThere is little investment in safety measures and training of gas station workers in Brazil. In addition, other factors, such as high temperatures throughout most of the year, and tampering with gasoline with higher concentrations of benzene and other solvents than permitted by law, increase the exposure level and the risk of disease. This should be aggregated with information about the presence of signs and symptoms related to occupational exposure and whether occupational exposure to fuels with BTX is associated with the development of hematological changes in gas station workers in the city of Rio de Janeiro, Brazil, in order to contribute to health surveillance strategies of benzene-exposed workers in the country.The observed results reveal initial changes in parameters routinely used to evaluate the health conditions of the population, when different groups are compared. This can help to reinforce adoption of practical measures in worker health surveillance, such as recognizing clinical changes even in the normal laboratory results, considering the care and monitoring of the health of GSWs, as well as other groups of workers who share the same spaces as the service stations.This study showed that occupational exposure of gas station workers by inhalation and/or dermal route increased the frequency of signs and symptoms related to benzene intoxication such as somnolence, headache, dizziness, tingling, involuntary movements, and weakness. Hematologic changes such as leukocytosis and lymphopenia were also present in the serial collection group. Although concentrations of benzene and other solvents in gas stations are considered low, continuous exposure may lead to chronic effects for workers who are in contact with these compounds during their workday, causing the greater risk of developing hematological damage by these workers and possibly for nearby residents. The results of this article may contribute to technical and scientific support to raise discussions around the theme, and contribute to the establishment of stricter standards for the reduction/elimination of human exposure to carcinogens, such as benzene, so that effective surveillance and control measures in work environments can be taken routinely and consistently. We hope that these results can contribute to the prevention and surveillance of cancer and other work-related diseases, since the monitoring of workers exposed to agents with high human toxicity are pillars of the surveillance of populations exposed to carcinogens. Therefore, our findings corroborate the importance of periodically monitoring the health of gas station workers, including convenience store employees.