Background: Auditory processing disorders (APD) may be one of the problems experienced by children with listening difficulties (LiD). The combination of auditory behavioural and electrophysiological tests could help to provide a better understanding of the abilities/disabilities of children with LiD. The current study aimed to quantify the auditory processing abilities and function in children with LiD. Methods: Twenty children, ten with LiD  and ten typically developing (TD)  participated in this study. All children were evaluated with auditory processing tests as well as with attention and phonemic synthesis tasks. Electrophysiological measures were also conducted with click and speech auditory brainstem responses (ABR). Results: Children with LiD performed significantly worse than TD children for most behavioural tasks, indicating shortcomings in functional auditory processing. Moreover, the click-ABR wave I amplitude was smaller, and the speech-ABR waves D and E latencies were longer for the LiD children compared to the results of TD children. No significant difference was found when evaluating neural correlates between groups. Conclusions: Combining behavioural testing with click-ABR and speech-ABR can highlight functional and neurophysiological deficiencies in children with learning and listening issues, especially at the brainstem level. Listening difficulties (LiD) can have a negative impact on academic learning as 60% of school time for children in the primary school level is devoted to listening, according to Imhof 2008) . As sugg008 . As Many APD definitions have been proposed ,5,6,7,8,Accordingly, electrophysiological measurements could be utilized to reduce the contribution of attention, motivation and other non-auditory factors when evaluating auditory abilities in children. Since these measures do not rely on the listener’s participation, they can provide a better insight into understanding auditory system function . The cliWhile click-like stimuli were utilized in the early investigations of ABR to optimize the transient auditory brainstem responses, they are not a good representation of relevant behavioural sounds heard in the real world, such as speech and music, non-speech vocal, and environmental sounds . AdditioPrevious studies using FFR reported significantly longer latencies of some transient and/or sustained elements in children with LiD relative to the controls ,35,36,37In summary, there is little literature on the auditory capacities and function of children with LiD in order to determine whether their difficulties might be caused primarily by APD. Moreover, none of the above studies on FFR investigated the neural consistency of recorded electrophysiological responses in children with LiD. The aim of the present study was to explore the auditory abilities and function of children with LiD and learning difficulties. Two sub-objectives were targeted: (1) identify whether their LiD was specifically related to APD and (2) examine whether the click-evoked ABR and FFR components could be sensitive to differences between responses of children with LiD and those of TD children.The present study was approved by the Office of Research Ethics and Integrity at the University of Ottawa . The study was explained to the participants and legal guardians. Legal guardians were given a consent form to read, and all gave written consent for their child to participate in the current study. Children also provided their assent before the data collection. p = 0.481). The control group participants resided in the same province as the participants of the experimental group. They had no known otologic, congenital, neurological, developmental or metabolic disorders. None of the participants had a diagnosis of intellectual disability. Participants in both groups had normal peripheral auditory function: otoscopy was unremarkable, tympanometry values were between 0.5 and 2.5 mL for ear canal volume; middle ear pressure was between −150 to 50 daPa; tympanic membrane compliance was between 0.3 to 2 mmho, and conventional pure tone audiometry revealed hearing thresholds equal to or below 20 dB HL for all frequencies tested (250 to 8000 Hz). All participants spoke English fluently.This cross-sectional comparative study was conducted at the University of Ottawa. Eleven experimental group participants  were recruited at a clinic that tests for APD. They were referred to the clinic because of their difficulties in school  auditory processing evaluation and (2) electrophysiological evaluation. The auditory processing tests and the electrophysiological recordings were administered to the participants at the APD clinic or at the University of Ottawa. All the evaluations occurred in a soundproof booth, respecting standard noise floor levels  .Participants underwent a battery of behavioural auditory processing tests. All children performed at least one test of each of the following auditory ability categories: binaural integration, binaural separation, figure-ground separation, sequential organization with variations in frequency, temporal resolution, and auditory closure. Two complementary tests were included in this battery, one assessing phonemic synthesis ability and one evaluating sustained auditory attention. During the testing, multiple breaks were given as needed by the children. The Dichotic Digit test (DD)  or the SBinaural separation ability was evaluated with the Competing Sentences Test (CST) . The parThe Bamford–Kowal–Bench Speech in Noise test (BKBSIN) ,44 was uAuditory closure ability was evaluated with the Low Pass Filtered Speech test (LPFST) ,46, wherAuditory sequential organization was evaluated with the Pitch Pattern Sequence Test (PPST) , where pThe Random Gap Detection Test (RGDT)  assessedTwo complimentary evaluations were conducted: (1) The Phonemic Synthesis (PS) test , in whic® : (1) a click of 100 μsec broad-spectrum square wave and (2) a five-formant 40 msec /da/, with an initial noise burst and a formant transition between the vowel and the consonant. They were presented at 80 dB SPL in the right ear only as there is evidence of a speech-specific right ear advantage [Throughout the electrophysiological testing, participants were seated comfortably watching a muted movie with subtitles in a dimly lit room. They were instructed to remain relaxed and to refrain from moving or speaking. Both click- and speech-ABR took approximately 40 min, and breaks were given as needed. Two stimuli were generated by BioMARKdvantage  through dvantage . ElectriResults for the electrophysiological measures were computed in four ways: (1) transient responses for click-ABR and speech-ABR, (2) sustained responses, (3) neural response consistency, and (4) the stimulus-to-response consistency for speech-ABRs.Peak selection was completed through the BioMARK software . For click-ABR, three peaks—I, III, and V—were identified and marked manually. For speech-ABR, seven peaks were identified and marked manually: two onsets—V and A, a consonant-vowel transition C, three steady states—D, E, F—and an offset—O. Two evaluators identified all peaks on each recording separately, and the inter-judge concordance was 96% for the click-ABR and 98% for the speech-ABR. If both evaluators could not agree on a peak, a third evaluator would break the tie. Following the peak selection, electrophysiological data were extracted with the MATLAB  powered To calculate the sustained responses of the speech-ABR, data were computed with the Brainstem Toolbox Version 2013 . The FFRr-scores with Fisher’s transformation for all statistical computations [Inter-response consistency reflects the fidelity of the response morphology when compared with itself ,55,56. Iutations .Statistical analysis was completed with IBM SPSS Statistics software (Version 27) . To explore differences between groups for the behavioural and electrophysiological tests, the non-parametric Mann–Whitney U test was selected conservatively as the number of participants in the groups was limited and varied between groups and tests.The results of the statistical tests comparing the values between the two groups are presented below for the auditory processing and supplementary tests. As part of this retrospective study, some children in the experimental group had missing data in their files. Children in the control group were prospectively tested and missing data for a few children was due to time constraints see . p = 0.019, r = −0.56, left ear: U = 3.00, z = −3.13, p = 0.001, r = −0.76), the Staggered Spondaic Word test , the Competing Sentences test, in the left ear only , the Random Gap Detection test , the Pitch Pattern Sequence test , the BKB Speech in Noise test in the binaural condition  and the Low Pass Filtered Speech test . The effect size for significant results was found to be of a moderate to large effect. The Mann−Whitney test results indicated that the children in the experimental group had significantly lower performance than children in the control group in eight of the nine behavioural tests : the Dicp = 0.009, r = −0.60) and for three of the four conditions of the Auditory Continuous Performance test: Impulsivity , Inattention  and Total . The vigilance component of the test was not significantly different between groups   and wave E   and , respectively  . The eff= −0.50) . For the = 0.45) . The effectively .Regarding the statistical test results for the root mean square, F0, F1, and HF indicated no significant differences between groups for any amplitude measures despite the fact that the overall amplitude of the component was lower in the experimental group than the control group .p = 0.089, r = −0.39) .The Mann−Whitney test results revealed no significant difference between the two groups for neural response consistency, but the odd/even condition approached significance, where the experimental group had lower neural response consistency than the control group  . For the= −0.39) , there wOne objective of the current study was to investigate if LiD in children having academic difficulties could be solely attributed to APD. All children in the experimental group fulfilled the APD diagnostic criteria of ASHA (2005) . They haAnother objective of the study was to explore if click-evoked ABR and FFR were sensitive enough tools to identify children with LiD, and if there was any difference in the neural consistency of FFR responses between children with LiD and TD school-aged children. In click-ABR assessments, there were no significant differences in wave I, III, and V latency; however, the amplitude of wave I was significantly smaller in children with LiD than in the control group. The reduction in wave I amplitude was previously reported in children with APD and language disorders ,66, and Regarding the speech-ABR test results, the latencies of waves V, D, E, and O were longer in children with LiD compared to TD children. The difference was, however, significant only for waves D and E. These findings were in accordance with studies reporting prolonged latencies of waves evoked in response to transient and/or sustained elements of speech stimuli in children with APD, LD, dyslexia, and ADHD ,69,70,71Another finding of this study was the observation of greater variability/inconsistency of neural responses to speech sounds across trials in children with LiD compared to the controls, despite the fact that there was no significant difference in neural consistency between the two groups. Poor response consistency indicates differences in FFR morphology between trials . This inChildren with LiD and children with typical development were assessed with auditory processing tests, attention and phonemic synthesis screening tests, and click- and speech-ABR measures. The findings revealed poor auditory processing, language, and attentional skills in children in the experimental group, which suggested that their LiD was not related exclusively to APD. The prolonged latency of waves D and E could indicate slower processing of speech stimuli at the brainstem level and might be an indication of an auditory processing deficit. As such, FFR could be used as a complementary objective tool in assessing auditory processing information at the brainstem level in children with LiD and learning problems. However, the low sample size made it difficult to generalize the results of the present study to the population of children with LiD. Further studies with greater sample sizes are needed to determine probable relationships between the results of behavioural tests and specific manifestations of FFR. In the present study, correlation analyses could not be performed due to the limited number of participants. Moreover, studies could also explore whether FFR recordings in adverse listening situations, such as in background noise, can shed further light on neural inconsistencies in processing speech stimuli at the subcortical level in children with LiD. The outcomes of these studies may reveal that the use of FFR measurements in noise and behavioral auditory tests is a suitable combination to identify APD in school-aged children with LiD.


The greater part of this illness is associated with beef meat, which is used in many traditional dishes across the world. Beneath the low microbiological quality of beef lies (pathogenic) bacterial contamination during primary production as well as inadequate hygiene operations along the farm-to-fork chain. Therefore, this study seeks to understand the microbiological quality pathway of minced beef processed for fast-food restaurants over three years using an artificial neural network (ANN) system. This simultaneous approach provided adequate precision for the prediction of a microbiological profile of minced beef meat as one of the easily spoiled products with a short shelf life. For the first time, an ANN model was developed to predict the microbiological profile of beef minced meat in fast-food restaurants according to meat and storage temperatures, butcher identification, and working shift. Predictive challenges were identified and included standardized microbiological analyses that are recommended for freshly processed meat. The obtained predictive models (an overall  The presence of microbes in food is a major issue in the food industry. This problem arises as a global issue because it poses a constant threat to health. For example, foodborne illnesses related to food-producing animals affect millions of people every year and cause approximately 5000 deaths . In ordeMeat is an easily spoiled product with a short shelf life . Some veEscherichia coli infection [Salmonella spp. and E. coli may be present in the gastrointestinal tract of cattle [E. coli represents an illness-causing agent, and much effort has been invested in risk assessment of the public health impact of this bacterium, especially of E. coli O157:H7 in ground beef [E. coli, and it has been supposed that such organisms in the faeces of cattle are spread to meat during processing steps [E. coli, Staphylococcus aureus is also one of the impacts of meat processing. Namely, Mohamed [S. aureus can be expected as meat contamination, because this bacterium represents normal flora in humans and its presence in foods indicates inadequate butcher handling. As one of the bacteria that can grow and produce toxins under different environmental and nutritional conditions, S. aureus can cause food poisoning by enterotoxins, while meat and meat products represent frequently incriminated foods in staphylococcal food poisoning [Salmonella spp. and Listeria monocytogenes are recognized for many types of food products and can be transferred to contaminated raw or undercooked red meats [Pseudomonas and Clostridium representatives, as well as Enterobacteriaceae, Shewanella putrefaciens, and Brochothrix thermosphacta [The most potent phases for microbial spreading in meat processing are the mincing and mixing phases ,11. The nfection ,13,14,15f cattle , which cf cattle . As one und beef . Chilledng steps . Due to  Mohamed  reportedoisoning . As two ed meats ,22. In ted meats ,11,23,24ed meats . Other, osphacta .Monitoring of the microbiological profile is very difficult during each step in the supply chain, although there is adequate standardized microbiological analysis. Obtaining results requires a lot of time, while the meat is usually transported immediately after processing to the retail phase. Due to this fact, many meat industry producers are open to the implementation of intelligent systems that can predict the microbiological quality of the meat and meat products based on the experimental data collected over time. Using predictive microbiologically techniques, meat producers can decrease the cost and time spent on the microbiological analysis and also have a better picture of microbial contamination, growth, mortality, and survival of microorganisms . In receTherefore, the formation of a system for long-time monitoring of the microbiological quality of beef meat based on the predictive capabilities of the ANN models was tested, using standardized microbiological analysis, as well as meat temperature, storage temperature, butcher working shift, and sampling weekday . Sampling was conducted for three consecutive years (2019–2021) for the same meat production after the mincing and mixing process. The obtained experimental data were served for mathematical modelling and determination of the relative influence of independent variables on the microbiological profile of beef minced meat.The minced meat samples were obtained from a local butcher specializing in beef, located in Novi Pazar, Republic of Serbia. All processed meat batches were transported at 4 °C to the fast-food restaurant in Novi Sad, Republic of Serbia. Samples represent ground meat without bones that is minced and contains less than 1% salt. Approximately one hundred grams of minced meat was obtained from each batch immediately after transport and placed in a sterilized sampling box. The sample collection was conducted for three consecutive years  for each batch transported to the restaurant after the phase of mincing and mixing.Escherichia coli [Guide to Microbiological Criteria for Food [Salmonella spp. or L. monocytogenes, where the absence of the bacterium is required. All analyses were performed in five repetitions.The microbiological profile of minced meat samples was determined following standard methods presented in hia coli —microbiofor Food  and prevAccording to the Rulebook, the obtained results can be classified as satisfactory , acceptable , or unsatisfactory . The m- and M-value represent defined limit values, minimum and maximum for each analysis separately, and depend on the type of food. Briefly, to determine compliance with food safety or hygiene criteria for one batch of food, it is mandatory to take an adequate number of samples in a few repetitions . If the results of the microbiological testing are less than or equal to the limit , the tested sample can be evaluated as satisfactory. In the case when the maximum allowed results are between m- and M-value and all other results are less than or equal to the m-value, the sample can be defined as acceptable. On the other hand, when one or more results exceed the M-value, a sample is unsatisfactory. If one or more tested microorganisms are deemed acceptable and if the results for other tested microorganisms are consistent, then the sample is considered acceptable. If one or more results for a particular parameter are assessed as non-compliant, then the sample is unsatisfactory .E. coli, Salmonella spp., S. aureus, and LAB) and to classify objects into groups in the factor space.As one of the potential solutions for long-time monitoring of microbiological criteria in food production processes, advanced mathematical tools can be included in the interpretation of the obtained results. In view of practical application, this type of mathematical tool can potentially decrease some crucial time-consuming and economic parameters of microbiological profiling of food samples, such as the number of samples, frequency of analyses, etc. Therefore, this study used a mathematical modelling approach to evaluate the obtained results of microbiological testing. Descriptive statistics were used for the collected data, while mathematical modelling was performed using STATISTICA 10.0 software . The independent variables used for ANN modelling were: meat temperature, storage temperature, butcher identification , and sampling weekday —equally with weekday for primary production and distribution, while the output variables were the microbiological profile of beef minced meat. Butchers (meatmen) are permanent employees in the local butcher located in Novi Pazar, Republic of Serbia. In order to classify them, each butcher is marked with A, B, C, or D instead of their names, which is in accordance with their shifts. The selected four independent variables were chosen due to their reproducibility and traceability in any other butcher without special equipment or investments. In this way, processing and sampling parameters as well as a human influence were included in this study. In the whole process, butchers A, B, C, and D were the same persons working in continuous shifts at the butcher. The storage temperature was determined by an inside/outside thermometer , while meat temperature was monitored by a penetration laboratory thermometer . Principal component analysis (PCA) was selected as a tool to discover the possible correlations among measured parameters  presents the ANN by using matrix notation.Y is the matrix of the output variables, f1 and f2 are transfer functions in the hidden and output layers, respectively, and X is the matrix of input variables. The W1 and W2 are determined during the ANN learning cycle using minimization of the error between the network and experimental outputs, according to the sum of squares (SOS) and the BFGS algorithm used to speed up and stabilize convergence. The coefficients of determination were used to check the performance of the gained ANN model [The ANN model was built as a three-layer perceptron model, having input, hidden, and output layers. The data were normalized prior to the ANN modelling, in order to facilitate the improvement of the ANN model’s conduct . The expNN model ,40. r2), reduced chi-squared (χ2), mean bias error (MBE), root mean square error (RMSE), and mean percentage error (MPE) using Equations (2)–(5) [A numerical verification of the obtained models in the previous step was tested using the coefficient of determination ( (2)–(5) ,41. A stexp,ix represents the experimental values, and pre,ix represents the predicted values obtained by calculating the model for these measurements. N and n are the number of observations and constants, respectively.In Equations (2)–(5), Yoon’s interpretation method was used to determine the relative influence of independent variables on the microbiological profile of beef minced meat . This meGuide to Microbiological Criteria for Minced Meat Samples [Minced meat represents a good medium for microbiological growth, which can primarily be spotted on the meat’s surface but is also distributed into the meat product during the mincing and mixing process . Therefo Samples . All anaConsidering that a specific limitation for a number of lactic acid bacteria does not exist, the summarizing distribution of lactic acid bacteria determination was individually presented in E. coli, lactic acid bacteria (LAB), and S. aureus. From this statistical analysis, Salmonella spp. and L. monocytogenes are excluded, because they were not influential in any of the tested samples. For a better understanding of the obtained results, each gained result is classified using different colours based on levels represented in the Rulebook and the Guide to Microbiological Criteria for Food. The green colour represents a satisfactory level of the obtained results, while yellow and red signify an acceptable and unsatisfactory level of tested samples, respectively.Additionally, The PCA of the presented data  explaineE. coli, LAB, and S. aureus prediction was equal to 10 (network MLP 9-10-4) to obtain high values of r2  and low values of SOS , as well as the elements of matrix W2 and vector B2 (bias) for the hidden layer, used for calculation in Equation (1).2), root mean square error, (RMSE), mean bias error (MBE), coefficient of determination (r2), skewness (Skew), kurtosis (Kurt), mean of the residuals (Mean), standard deviation of the residuals (SD), and variance of the residuals (Var.). Except for the quality of the model fit, the residual analysis of the developed predictive model was presented in the same table. The presented four-parameter sigmoidal mathematical model appears to be simple, robust, and accurate. Mathematical models had an insignificant lack of fit tests, which means that all the models represented the data satisfactorily. A high coefficient of determination (2)r is indicative that the variation was accounted for and that the data fitted adequately to the proposed model. The adequacy of the model can be summarized by comparing the microbiological parameters as follows (in descending order): S. aureus, E. coli, lactic acid bacteria, and mesophilic and aerobic bacteria  was used. The obtained results are presented in According to the following Rulebook  and the Salmonella spp. and Listeria monocytogenes (food safety criteria) and mesophilic and aerobic bacteria (MAB), Escherichia coli, and Staphylococcus aureus (criteria of hygiene). Additionally, lactic acid bacteria (LAB) as one of the specific spoilage groups of microorganisms for meat are tested [In order to test both food safety criteria as well as criteria of hygiene, this study involves the determination of the microbiological profile of freshly processed minced meat based on e tested . In thise tested ,11,23,24e tested , who repE. coli analysis with an acceptable level according to the Guideline. The minced meat contaminated with E. coli can be a source of several illnesses, including haemolytic uremic syndrome, haemolysis, thrombocytopenia, renal failure, etc. [E. coli is detected in meat products, meat production needs to revise current hygiene practices and provide a safer product. L. monocytogenes were not noticed in any of the 216 samples, while Salmonella spp. was present in only 6.9% of samples. The non-appearance of Salmonella spp. is especially important considering the widespread presence of Salmonella in minced meat, salmonellosis outbreak, and the frequency of its consumption via many traditional products [L. monocytogenes, especially by the three serotypes 1/2a, 1/2b, and 4b [L. monocytogenes in the samples after the production process does not provide security for recontamination during final packaging or later handling [S. aureus contamination, the total percent of samples at on satisfactory level was 94%, which is a greater value than previously reported (62%) by Shawish and Al-Humam [Out of the total number of meat samples, more than 95% of samples were satisfactory in view of re, etc. . When E., and 4b . On the handling . In the Al-Humam  as well Al-Humam .E. coli and S. aureus [Additionally, . aureus .S. aureus (49.8%) and E. coli (49.8%) exhibited negative scores according to the second principal component (PC2). E. coli (51.2%) showed a positive influence on the third principal component, while S. aureus (43.8%) showed a negative influence toward the third principal coordinate (PC3)  .r2 value, for ANN models. The ANN model anticipated experimental aerobic and mesophilic bacteria, E. coli, LAB, and S. aureus rather well for a broad range of the parameters, as seen in r2 is illustrative that the data fitted satisfactorily to the developed model.Many empirical models are used for understanding microbiological profiles of meat and meat products, but a step forward in advanced mathematical and statistical modelling is still not widely applied. Only a few studies included using predictive tools for an explanation of microbiological experimental data, such as partial least squares regression analysis , growth E. coli in beef minced meat, with a relative importance of 19.08%, while the influence of Butcher B showed the most negative impact, with −26.62% negative importance. Butcher D was the most important factor for predicting the amount of S. aureus in beef minced meat, with a relative importance of 19.60%, while the influence of Butcher A showed the most negative impact, with −15.67% negative importance. Due to the obtained results of the relatively high influence of butcher shift on the microbiological quality of meat, it can be suggested that certain workers maintain much more hygiene in the working space than others, doing the work in accordance with good hygienic practice and in the shortest possible time. Accordingly, it would be recommended that a specific aspect of corrective action be taken in relation to these results. Although storage and meat temperature do not have a high relative influence on the number of MAB, these two variables have a great influence on other tested microorganisms. Consequently, it can be seen in E. coli, and S. aureus. According to In the meat industry, all production, storage, and transport processes need to be under cold conditions, in a temperature range between 2 and 7 °C . FrequenE. coli, Salmonella spp., and S. aureus appeared randomly during the examination period. The butcher shift and temperature were the most important factors for predicting the microbiological profile of beef minced meat, with a high relative importance of tested microbiological parameters. Furthermore, the developed artificial neural network provided a good prediction of the microbiological profile of beef minced meat with an overall r2 of 0.867 during the training cycle. It can be concluded that maintenance of good hygiene practices in a meat production and distribution process can prevent increasing the number and occurrence of some microorganisms, but potential critical points are already in the primary production process. In terms of practicality, ANN models offer a simpler alternative to traditional models for the prediction of microorganisms but require a greater amount of parameters and data. Furthermore, using statistical analysis of the gained microbiological results in the everyday routine of meat processing can facilitate the establishment of microbiological quality control system and minimize economic losses. In future work, it is recommended to carry out the prediction model of other indicators of the physicochemical and microbiological quality of meat for other meat industries based on the proposed methodology. In addition, it is recommended to extend the prediction model to the supply chain, taking into account other types of storage and exploitation categories. For example, all parameters in maintaining hygiene during production and the whole transport chain can be based on a better understanding.A parallel mathematical investigation of feasible analysis and the construction of a mathematical model displayed suitable accuracy for the anticipation of the quality of minced beef meat, which is one of the most consumable types of meat, but also a food product with short shelf life due to propensities for microbial growth. According to the obtained results, all tested samples had a relatively high number of aerobic and mesophilic bacteria, while


P = 0.001), have a history of migrant worker , have commercial sexual behaviour , married , have a history of HIV antibody testing , HIV-related knowledge  were associated with new HIV infection among older men. The present study revealed some potential risky/protective factors altogether. The results highlighted the direction of HIV/AIDS prevention and control among older men, and it is a social issue that requires the joint participation of the whole society.Human immunodeficiency virus (HIV) has been widely prevalent among older men (aged ≥50 years old) in Sichuan Province. The study aimed to discover associated factors with the new HIV infection in older men, and provide a scientific basis for the prevention and control of acquired immunodeficiency syndrome (AIDS) in this group. A cross-sectional survey study of newly reported HIV/AIDS and general male residents aged 50 years and older was conducted between April and June 2019, with a resample of respondents to identify cases and controls, followed by a case–control study. Logistic regression was applied to analyse the association between the selected factors and new HIV infection among older men. At last, 242 cases and 968 controls were included. The results of multiple logistic regression suggested that many factors including living alone/concentrated (OR 1.56, 95% CI 1.20–2.04,  Since the emergence of human immunodeficiency virus (HIV), the causative pathogen of acquired immunodeficiency syndrome (AIDS), at the beginning of the 1980s , with thSichuan Province is located in southwestern China and has the largest number of HIV-infected people in China, with more than 160 000 HIV/AIDS cases at the end of 2019 . SimilarHowever, in Sichuan, the factors associated with new HIV infection among older adults are not clear, and related studies are insufficient. To better help policymakers identify and address their specific needs for HIV prevention among older adults, it is particularly urgent to carry out studies on the related factors of new HIV infection in older adults. In addition, between 2008 and 2019, 73.0% of newly reported HIV infections among older adults in Sichuan Province were males , and theDuring April and June 2019, we conducted a cross-sectional survey aimed at the newly reported HIV/AIDS cases between January 2018 and June 2019 and elderly male residents aged 50 years and older in Sichuan Province, China.In the cross-sectional survey, HIV/AIDS cases were newly diagnosed and reported in the National Case Reporting System (NCRS) from January 2018 to June 2019; ordinary elderly male residents, were randomly selected in the HIV/AIDS cases investigation areas, who have lived for more than half a year. We excluded those (both cases and ordinary residents) who were incapacitated, severely mentally impaired, and refused to take part in the survey. In addition, we also tested ordinary residents for HIV antibody.The survey areas were determined by a step-by-step sampling method. According to the number of HIV/AIDS cases and the proportion of older men in all cities (prefectures) of Sichuan Province, Chengdu, Zigong, Yibin, Meishan and Guangyuan were finally determined to carry out the investigation. Then three counties or urban districts were selected in each city by the same method. Finally, according to the number of HIV/AIDS cases, geographical location and traffic conditions in each township and community of the 15 counties (districts), two townships/communities were selected in each county (district). Fifteen counties (districts) for HIV/AIDS cases investigation, 30 townships/communities for general older men investigation, a survey of 100 older men was conducted at each township/community by convenience sampling, and a total of 3000 older men were interviewed.The limiting antigen avidity enzyme immunoassay (LAg-Avidity EIA) was developed from the HIV-1 subtypes B, E and D IgG-capture enzyme immunoassay by the US Centers for Diseases Control and Prevention . In 2020An interview-administered questionnaire was applied to obtain the required data. A pre-investigation was conducted before the formal investigation, and the questionnaire was evaluated and revised according to the results. The interview was completed by public health personnel who have been trained uniformly by the research group. The investigators came from the county-level Center for Diseases Control and Prevention (CDC) and primary medical and health institutions  in the investigation areas.Except for the questions on HIV infection, the questionnaire for HIV/AIDS cases was the same as general residents, including socio-demographic characteristics , daily activities, living status, history of migrant worker (being away from home for six months or more), HIV-related knowledge and attitudes, and income. Commercial sexual behaviour and HIV antibody testing (excluding the testing to be diagnosed) in the past six months were also inquired in the questionnaire. The awareness of HIV-related knowledge was judged by the eight questions in the ‘AIDS Monitoring and Evaluation Framework of China ’, and those who correctly answered six or more questions were considered to be aware of HIV-related knowledge .The interview was completed face-to-face by investigators in a private environment. The questionnaire did not contain any personal information, such as the name, ID number, current address or telephone number, to fully protect the privacy of the respondents.Cases and controls were identified by resampling the investigated HIV/AIDS cases and general older men. Cases were selected from older adult HIV/AIDS cases newly reported to the NCRS and determined to be newly infected by LAg-Avidity EIA, and 242 cases were selected finally. Controls were selected among the 3000 general older men (HIV-positive ones were excluded) by age (±1 year) in a ratio of 1:4, and the number of control was 968.P < 0.05 was considered statistically significant. All analyses were conducted using SPSS 23.0 .The Chi-square test was applied to measure the unadjusted associations between the new HIV infection of older men and categorical variables. The Cochran-Armitage test was used to identify the potential change in the trend of the proportion of commercial sexual behaviour and HIV-related knowledge among ordinal categorical variables. The adjusted associations for the study variables and new HIV infection of older men were measured by Multiple logistic regression. The backward variable selection strategy was employed to include variables in the regression model. Odds ratio (OR) and 95% confidence interval (CI) were calculated. P = 0.016), 158 cases (65.3%) and 583 controls (60.2%) were illiterate or had elementary education (P = 0.003), most cases (62.0%) and controls (79.1%) were married with spouse (P < 0.001), 163 cases (67.4%) and 581 controls (60.0%) on HIV-related knowledge (P = 0.036), 172 cases (71.4%) and 75 controls (7.8%) had commercial sexual behaviours in the past six months (P < 0.001). Trend analysis showed that with the increase in age, the proportion of HIV-related knowledge awareness in both cases  and controls  showed a downward trend, while the changing trend of the proportion of commercial sexual behaviours was not statistically significant .There were 242 cases and 968 age-matched controls in the analysis. Overall, most cases (72.2%) and controls (63.9%) came from rural area .P = 0.001), have a history of migrant work , have commercial sexual behaviour  were significantly associated with a higher risk of new HIV infection among older men. Furthermore, married , have a neutral/opposition attitude to finding FSW , have a history of HIV antibody testing , HIV-related knowledge , and have two or more ways to acquire HIV-related knowledge  were found to be inversely associated with the new HIV infection in older men.The results of the current study suggested that several variables including, HIV-related knowledge and the number of ways to acquire HIV-related knowledge, marriage status, living status, history of migrant worker, attitude to finding FSW, commercial sexual behaviour, and HIV antibody testing may affect the risk of new HIV infection among older men.This study revealed that HIV-related knowledge was inversely associated with new HIV infection in older men. Published studies showed that HIV-related knowledge and the awareness of the risk of HIV infection among older adults were generally low . They beHeterosexual transmission was a dominant way of HIV transmission among older men, and many studies have indicated the paramount transmission mode was commercial heterosexual contact , 31, 32.After controlling for other variables, a significant association was observed between living alone/concentrated and the risk of new HIV infection. As the harbour of the soul, the family is the place of psychological sustenance. They lack the support, help, care and love from their families and relatives. Older men tend to have emptiness, monotonous life, lack of spiritual life, coupled with the influence of unhealthy behaviour outside, leading to high-risk sexual behaviour, particularly in under-developed western regions of China , 40. OldThe present study also showed that the history of migrant worker was a significant risk factor for new HIV infection among older men. These people are far away from home, their spouses are not around, and they are prone to commercial sex under the dual pressure of life and work. Studies have shown that population mobility promotes the spread of sexually transmitted diseases and other infectious diseases, including AIDS, and the infection rate of this population is high . Male-doIn our study, the history of HIV antibody testing (in the past six months) was associated with a lower risk of new HIV infection. Sichuan has carried out an expanded screening strategy for many years. Whether they were active or passive participants in testing, it was an opportunity to receive AIDS publicity and education, which deepened their awareness of AIDS. An important purpose of AIDS publicity and education is to raise awareness of AIDS prevention. Voluntary counselling and testing (VCT) is a concrete manifestation of self-protection awareness in AIDS prevention. Currently, the detection of HIV/AIDS cases in older men in Sichuan Province is mainly passively diagnosed in general hospitals, and the proportion of VCT is relatively low . It is sThere are, nonetheless, some limitations that should be acknowledged. This study was conducted through a retrospective cross-sectional survey, and recall bias was unavoidable. The investigators of the cases and controls are different, the former is the staff of the county-level CDC, and the latter is the staff of the primary medical and health institutions. Despite the unified training, the ability of different investigators also has a certain impact on the investigation. The content of this survey about personal sexual behaviour is sensitive, and it is difficult to guarantee that the results obtained are real. Cases and controls were age-matched, and the effect of age on new HIV infection has not been studied. And controls were selected by convenience sampling methods that can lead to a sampling bias. In addition, no information on injection drug use was collected in the study, but the influence on the results of this study may be small, because the anti-drug work has always been under high pressure state in China, the proportion of traditional injection drug use is getting smaller and smaller, and the survey points were not serious drug epidemic area.This innovative study used new HIV-infected individuals as cases to study the influencing factors of HIV infection among older men. We revealed that the factors affecting their infection include personal behaviours, attitudes and external factors, which can guide HIV prevention and control professionals and policymakers. For older men with the above factors, a precise AIDS prevention and control strategy could be formulated, including AIDS publicity, education and testing, to detect the infected people as soon as possible, and prevent new infections. Moreover, AIDS prevention and control is a social issue that requires the participation of the whole society, to enrich the lives of older adults in their later years, and advocate healthy lifestyles and safe sexual behaviours.


Aeromonas, which is an increasing threat to environmental and human health. To date, no epidemiological cut-off values (COWT) for Aeromonas spp. have been established by the Clinical and Laboratory Standards Institute nor the European Commission on Antimicrobial Susceptibility Testing. In this study, commercially prepared minimum inhibitory concentration (MIC) test 96-well plates (dry-form plates) were used to determine the MIC of eight antimicrobial agents against 556 Aeromonas strains. The obtained MIC distributions were simulated and analyzed by NRI and ECOFFinder to obtain tentative COWT values for Aeromonas spp. The COWT values of eight kinds of representative antimicrobial agents including trimethoprim–sulfamethoxazole, erythromycin, doxycycline, neomycin, colistin, florfenicol, enrofloxacin, and ceftazidime for Aeromonas spp. were established and were 0.25, 64/32, 4/2, 8, 4, 1, 0.062/0.125, and 0.5 μg/mL, respectively. Results showed that Aeromonas spp. had a very high proportion of non-wild-type strains to enrofloxacin, florfenicol, and doxycycline, which are the most widely used antimicrobials in aquaculture. The COWT values for Aeromonas spp. obtained in this study can contribute to the final establishment of COWT for Aeromonas spp. internationally.The abuse of antibiotics in aquaculture has led to the increasing rate of antibiotic resistance of aquatic bacteria including  Antimicrobial resistance (AMR) has become a growing threat to global public health . The conAeromonas is a Gram-negative facultative anaerobic bacillus widely distributed in aquatic ecosystems, which can infect not only fish and other aquatic animals but also humans and livestock. It is an opportunistic pathogen of human–fish–animal symbiosis. Aeromonas not only causes a wide range of diseases in humans such as bacteremia/septicemia, gastroenteritis, wound infection, and infection immunodeficiency. Some diseases caused in fish also seriously restrict the healthy development of the aquatic animal breeding industry [Aeromonas can acquire antibiotic resistance mechanisms and has the potential for horizontal gene transfer, so it may be a good candidate for tracking the spread of antibiotic resistance in the water [Aeromonas has been accepted as an indicator organism for monitoring water quality and sewage pollution, and it is suitable for evaluating the prevalence, development, and spread of AMR in the aquatic environment [The aquatic environment may provide an ideal environment for the acquisition and spread of antibiotic resistance because it is closely related to human activities , such asindustry . Aeromonhe water . At presironment .WT)/epidemiological cut-off values (ECOFF) and clinical breakpoints (CBPs). The main purpose of the establishment of COWT/ECOFF is to epidemiologically monitor AMR phenotypes, investigate whether bacteria are sensitive or resistant to antimicrobial drugs and distinguish bacteria into wild type (WT) and non-wild type (NWT), and clinical effects are not considered. CBPs are mainly used to distinguish between good prognosis and treatment failure bacteria, which is obtained by comparing the clinical treatment effect of different MIC pathogens [To determine whether bacteria are susceptible to antimicrobial drugs, a drug resistance criterion needs to be established, that is, a breakpoint, a specific inhibition zone diameter, or a minimum inhibitory concentration (MIC) value. If there is no drug resistance criterion, drug resistance cannot be measured. The purpose of establishing the drug resistance criterion is to evaluate the status of bacterial drug resistance, guide clinical medication, and assist clinical treatment . At presathogens .WT values is to conduct an antimicrobial susceptibility test (AST) and then perform statistical fitting analysis on the AST results. CLSI and EUCAST have published AST methods including micro-/macro-broth dilution method, agar dilution method, and disk diffusion method. The micro-broth dilution method is currently the most commonly used method for aquatic bacteria. It quantitatively obtains the MICs of antimicrobials on bacteria by setting a two-fold serial dilution of the drug concentration gradient [The main method to formulate COgradient ,13,14,15gradient .Aeromonas. CLSI [A. salmonicida to oxytetracycline and oxolinic acid and the epidemiological cut-off values to seven drugs including gentamicin, erythromycin, florfenicol, ormetoprim–sulfadimethoxine, trimethoprim–sulfamethoxazole, oxytetracycline, and oxolinic acid. It also provides the epidemiological cut-off values for A. hydrophila to six drugs including gentamicin, erythromycin, florfenicol, oxytetracycline, enrofloxacin, and oxolinic acid. The only MIC or zone diameter breakpoints established by EUCAST [Aeromonas spp. to six drugs including cefepime, ceftazidime, aztreonam, ciprofloxacin, levofloxacin, and trimethoprim–sulfamethoxazole. Most of these antimicrobials are not commonly used in aquaculture, so the reference values of these CBPs or COWT values are limited. Literature [WT values for A. bestiarum, A. salmonicida, A. sobria, and A. eucrenophila and Aeromonas spp. against 15 antimicrobials such as florfenicol, colistin, and enrofloxacin. However, these results have not been verified by other studies.At present, there are very few reference standards and studies for CBPs for as. CLSI  only proy EUCAST  are the terature  reportedAeromonas, the CBPs used in these studies mainly referred to ones of CLSI, which are unsuitable for the clinical treatment of fish diseases nor suitable for the analysis of the prevalence of AMR in aquaculture. And whether the COWT values established in the above literature [China is the most important country in the world for aquaculture, with high aquaculture production and complex use of antimicrobial drugs . Althougterature  are equaWT criteria of Aeromonas spp. to investigate and analyze the current AMR status of Aeromonas spp. in typical aquaculture ponds, a reservoir, and a natural river in China.Therefore, this study aimed to establish COAeromonas selection medium). The water sample or sediment sample diluted with 0.86% sterile normal saline was pipetted onto the sterile ampicillin MacConkey agar and spread evenly with a triangular glass rod, and all plates were incubated at 28 °C for 12–24 h. The pink single colonies were picked and cultivated overnight in a 1 mL BHI broth medium for bacterial identification.From November 2020 to October 2021, sick and healthy fish, water, or sediment samples from fish ponds in Jingzhou City, Hubei Province; sick and healthy fish samples from fish ponds in Wuhan City, Hubei Province, and Xiashan reservoir in Weifang City, Shandong Province; and healthy fish samples from Liuchong River in Bijie City, Guizhou Province, were collected and transported to the laboratory through ice boxes to perform bacterial isolation. The clinical symptoms of the sick fish were swirling on the surface of the water body, multiple congestions on the body surface, body ulceration; yellowing of the body, increased mucus, swelling of the abdomen with ascites; split head, khaki liver; ulcerative erythema on the body surface; anal swelling and gently press on the abdomen that had yellow mucus or blood coming out of the anus, with a shriveled gut and congested gut walls. And fish without any of the above symptoms was judged as healthy fish. The intestinal, kidney, liver, spleen, brain, or blood of the sampled fish were streaked onto sterile ampicillin MacConkey agar  (accessed on 8 February 2022). The obtained Aeromonas bacterial suspensions were then stored in a −80 °C refrigerator with 20% glycerol.The above bacterial suspension was used as the DNA template. The bacterial universal primer pair 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) was used for 16S rRNA gene PCR amplification. Amplification conditions were as follows: 94 °C for 5 min at pretreatment; 30 cycles of 94 °C for 30 s, 55 °C for 30 s, 72 °C for 90 s, and final extension of 72 °C for 7 min. The PCR products were sent to Wuhan Aikangjian Biotechnology Co., Ltd. (Wuhan China) for sequencing. Sequence homology analysis  was performed using the Nucleotide BLAST in NCBI  was used in this study. The assay plate contained lyophilized powder of the drug, and a two-fold dilution method was used to prepare the drug concentration gradient. The assay plates were manufactured in full compliance with the relevant CLSI protocols ,21, and 5 CFU/mL. About 100 μL of this bacterial solution was added to each well of the MIC assay plate, and positive and negative controls were set up simultaneously. The plates were incubated at 28 °C for 24–28 h, and then MIC values were recorded. The quality control strain used was ATCC 25922, and the results were considered valid only if the MIC of the quality control strain was within the specified quality control range.The MIC was determined according to the instructions of the MIC assay plate. The specific method is as follows: pick fresh single colonies suspended in cation-adjusted Mueller–Hinton broth, adjust the concentration of the bacterial solution to 0.5 McFarland concentration, and then dilute at a ratio of 1:200 so that the inoculum concentration of the bacterial solution is about 5 × 10WT values were determined using the free and automated EXCEL spreadsheet programs ECOFFinder (available online: https://clsi.org/meetings/microbiology/ecoffinder/) and NRI (available online: http://www.bioscand.se/nri/) (both were accessed on 11 February 2022). These two methods were proposed by Turnidge et al. [WT values of bacteria. The NRI program directly provides COWT values, whereas the ECOFFinder program selects the MICs with a confidence interval of 95.0% or 97.5% as the final COWT values. ECV and EOCFF are used for epidemiological cut-off values set by two international institutions, CLSI and EUCAST, respectively. To avoid confusion, the epidemiological cut-off values are abbreviated as COWT in this study.COe et al.  and Krone et al. , respect50 is the MIC required to inhibit 50% bacterial growth, and MIC90 is the MIC required to inhibit 90% bacterial growth. The strains with MIC values greater than the breakpoint value of the 95% or 97.5% confidence interval are all NWT strains. The strains with MIC values less than or equal to the breakpoint value of the 95% or 97.5% confidence interval are WT strains. The resistance rate is equal to the ratio of the number of NWT strains to the total number of strains tested in the present study.MICAeromonas were assayed for their MIC, and the proportion of strains in four sampling areas was as follows: 369 strains in Jingzhou City, Hubei Province; 30 strains in Wuhan City, Hubei Province; 16 strains in Weifang City, Shandong Province; and 141 strains in Bijie City, Guizhou Province. The 556 Aeromonas strains were derived from 183 fish, 14 water samples, and 5 sediment samples. The specific source and quantity distribution of the strains are shown in A. veronii , A. sobria , A. hydrophila , and A. allosaccharophila  were very abundant, accounting for 88.3% of the total number of Aeromonas.A total of 556 strains of Aeromonas strains to eight drugs are shown in n = 212), florfenicol , enrofloxacin , and ceftazidime . Drugs with MIC higher than the highest concentration measured were trimethoprim–sulfamethoxazole, erythromycin, neomycin, colistin, florfenicol, and ceftazidime. Only one drug, doxycycline, had MICs that were always within the drug dilution range.A total of four batches of MIC assay plates were used in this study. Quality control tests were performed for each batch using the standard strain ATCC 25922, and the results were all within the quality control range except for neomycin and erythromycin, for which no quality control range was available. The MIC distributions of 556 Aeromonas spp. are shown in Aeromonas spp. obtained by the Kronvall and Turnidge methods were almost identical. The MIC distribution of enrofloxacin against Aeromonas spp. was very wide and multimodal, with two peaks, one large and one small, on the right side. The MICs for doxycycline and florfenicol were widely distributed and showed no obvious bimodal distribution. The MIC distributions for the remaining five drugs were narrow and unimodal. The COWT values can be preliminarily observed with the naked eye, which is the upper limit of the 95% or 97.5% confidence interval of the abscissa of the fitting curve. Erythromycin had the largest COWT value, followed by neomycin and colistin, doxycycline, florfenicol, ceftazidime, trimethoprim–sulfamethoxazole, and enrofloxacin.The Kronvall and Turnidge method nonlinear regression fitting distributions of eight drugs against WT, MIC50, and MIC90 (μg/mL) and NWT (%) of 556 Aeromonas strains against eight drugs are shown in WT of Aeromonas spp. against each drug obtained by the NRI or ECOFFinder table processing program was consistent with that observed with the naked eye. The COWT values calculated by the Kronvall and Turnidge methods were the same for the five drugs, including trimethoprim–sulfamethoxazole, neomycin, colistin, florfenicol, and ceftazidime. The COWT values calculated by the two methods for the other three drugs had only a difference of one step of drug dilution concentration. The similarity rate of COWT obtained by the two methods could reach 62.5%. Using the COWT established in this study to calculate the number (percentage) of NWT strains among all strains collected in the present study, NWT (%), it could be concluded that enrofloxacin had the highest proportion of NWT strains of Aeromonas, exceeding 50%, followed by florfenicol. The two drugs with lower proportions were ceftazidime and neomycin. The difference between the MIC50 and MIC90 of trimethoprim–sulfamethoxazole was the largest, and its MIC90 exceeds the dilution range of the drug. The difference between the MIC50 and MIC90 of neomycin and ceftazidime was the smallest, with only two drug dilution gradients. The drug with the largest MIC50 and MIC90 was erythromycin, followed by enrofloxacin and doxycycline, and ceftazidime.The COWT, MIC50, and MIC90 (μg/mL) of the four Aeromonas spp. to the eight drugs are shown in WT of A. veronii was the closest to that of Aeromonas spp., followed by A. sobria. The COWT of the four Aeromonas spp. differed for the eight drugs. A. veronii obtained the highest COWT similarity rate by Kronvall and Turnidge methods (up to 87.5%), and A. allosaccharophila obtained the lowest COWT similarity rate by the two methods. The largest difference between MIC50 and MIC90 was trimethoprim–sulfamethoxazole of A. veronii, and the smallest difference was trimethoprim–sulfamethoxazole of A. sobria. The MIC50 and MIC90 of eight drugs against A. sobria were smaller. For these four Aeromonas spp., the MIC50 of erythromycin was relatively high, whereas those of trimethoprim–sulfamethoxazole, ceftazidime, and enrofloxacin were relatively low.The COWT K and COWT T of Aeromonas spp. against eight drugs, the resistance rate was calculated according to the MIC distributions of Aeromonas spp. against eight drugs in each region. The results are shown in Aeromonas spp. in culture ponds in Jingzhou City, Hubei Province, had high resistance rates to enrofloxacin, florfenicol, and trimethoprim–sulfamethoxazole. Aeromonas spp. in culture ponds in Wuhan City, Hubei Province, had high resistance rates to enrofloxacin, florfenicol, erythromycin, and doxycycline, but zero resistance to ceftazidime. Aeromonas spp. in the two ponds had the highest resistance rate to enrofloxacin (up to 82.7%), followed by florfenicol. The resistance rates of Aeromonas spp. in Xiashan Reservoir of Shandong Province to five drugs including trimethoprim–sulfamethoxazole, erythromycin, neomycin, florfenicol, and ceftazidime were 0, but the resistance rates of Aeromonas spp. to enrofloxacin and colistin were high. Aeromonas spp. in Liuchong River, Guizhou Province, had a high resistance rate to colistin, but their resistance rates to the other seven drugs were all low.Using the above obtained COAeromonas as indicator bacteria for antimicrobial susceptibility in aquatic environments [Aeromonas spp. are ubiquitous and pathogenic to many aquatic animals and can be isolated in freshwater systems at any time of the year [A. hydrophila, A. veronii, A. sobria, and many other Aeromonas species are the most common pathogenic bacteria in aquaculture in China and have a negative impact on the Chinese aquaculture industry [Aeromonas spp. as the first aquatic bacterial species to establish the resistance threshold. This is the first study of this type conducted using Chinese isolates.The establishment of resistance breakpoints is influenced by different laboratories in terms of assay protocols, reagents, and human factors, and the results of the established breakpoints may vary greatly. Therefore, only the breakpoints established following the standard experimental protocols, such as that of CLSI, can be widely accepted and recognized. Many researchers have used ronments ,30,31. Athe year . A. hydrindustry . TherefoAeromonas were isolated from fish collected from a natural non-aquaculture river and aquaculture ponds in China. The bacterial strains from these two sources are defined as environmental or pathogen strains. The protocols reported in the literature [WT values for Aeromonas. The results obtained in the present study can be used for comparison with those reported in the literature [WT values are influenced by the geographical origin of the bacterial strains. Additionally, it can provide breakpoints for determining the epidemiological characteristics of Aeromonas AMR to antimicrobials of greatest concern to the Chinese aquaculture industry. Commercially prepared MIC test 96-well plates containing dehydrated antimicrobial agents (dry-form plates) were used in this study, this standardized method made the experimental results more accurate and reproducible. During the actual operation with NRI and ECOFFinder, the MICs of the 95% and 97.5% confidence intervals of ECOFFinder were the same, and most of the COWT values obtained by selecting the 95% or 97.5% confidence intervals of ECOFFinder were the same or similar to the COWT values obtained by NRI.In this study, terature  were useterature  to verifWT obtained in this study was compared with the results reported in CLSI [WT T values of A. hydrophila to erythromycin and florfenicol were consistent with the corresponding results of CLSI, and other COWT T values or COWT K values were similar to the corresponding result of CLSI. The maximum difference did not exceed two drug dilution gradients. The COWT obtained in this study was compared with the results reported by Baron et al. [WT T values and COWT K values of Aeromonas spp. to trimethoprim–sulfamethoxazole and ceftazidime were exactly the same with the corresponding results reported by Baron et al. The COWT T values of Aeromonas spp. against erythromycin and enrofloxacin and A. sobria to trimethoprim–sulfamethoxazole and the COWT K values of A. sobria to ceftazidime were the same with the corresponding results reported by Baron et al. The rest of the results were similar to the corresponding results reported by Baron et al., except for the COWT T of A. sobria to enrofloxacin. The maximum difference did not exceed two drug dilution gradients. In conclusion, the COWT T values obtained in this study were closer to the corresponding results reported by CLSI and Baron et al. In addition, the results obtained in this study are the same or similar to those reported by CLSI and Baron et al., which indicates that the experimental methods referenced in this study are reliable and credible. On the other hand, it also shows that if a standard set of experimental methods for establishing breakpoints is used to establish COWT values, the COWT values obtained are generally the same or similar and are not greatly affected by regions.The CO in CLSI . The resn et al. . The COWWT can be observed with the naked eye, that is, the upper limit of the MIC distribution of the WT strain. If the MIC distribution of the strain exhibits insignificant bimodal distribution or continuous multimodal characteristics, the fitting analysis of the MIC distributions can be performed with ECOFFinder or NRI to obtain more accurate COWT values [Aeromonas spp. had a wide range and a multimodal distribution, indicating the existence of a higher proportion of enrofloxacin-resistant strains. Aeromonas spp. indeed had the highest resistance rate to enrofloxacin, as high as 61.5%/53.8%. The MICs of doxycycline and florfenicol against Aeromonas spp. were widely distributed and showed an insignificant bimodal distribution, indicating that a certain proportion of strains were resistant to doxycycline and florfenicol. The resistance rate of Aeromonas spp. to doxycycline and florfenicol was higher . This study found that the resistance rates of Aeromonas spp. collected from Xiashan reservoir and Liuchong river were low for most drugs compared with those of Aeromonas strains collected from aquaculture ponds, which may be related to the fact that these two natural water bodies have almost no drugs present. However, the resistance rates of these two water bodies to colistin or enrofloxacin were high. This was probably due to the surrounding industrial and agricultural production wastewater and domestic pollution. Aeromonas spp. in culture ponds had higher resistance rates to enrofloxacin, florfenicol, doxycycline, and trimethoprim–sulfamethoxazole. More importantly, enrofloxacin, doxycycline, and florfenicol are commonly used antibacterial drugs in aquaculture [Aeromonas to these three kinds of drugs, and these antibacterial drugs should be rationally used in aquaculture practice to avoid the outbreak of drug-resistant strains caused by the abuse of antibiotics.In general, the MIC distribution of the drug against the strain presents a unimodal or bimodal distribution. For a typical bimodal distribution diagram, the right peak represents the MIC distribution of NWT strains, and the left peak represents the MIC distribution of WT strains. If the MIC distribution of a certain drug to the strain has an obvious bimodal distribution, COT values . In thisaculture ,27. Ther50 or MIC90 of Aeromonas for the same drug in different studies was mostly different or quite different. This may be related to different human factors such as drug use, artificial breeding, and water pollution in different regions. However, the MIC50 and MIC90 of Aeromonas spp. and A. sobria to ceftazidime in this study was the same as the corresponding results of Baron et al. [Aeromonas spp. to ceftazidime were low, at 4.1% and 9.4%, respectively. This may be related to the fact that ceftazidime was less used in these two regions, and most of the existing Aeromonas were naturally sensitive to ceftazidime. The MIC50 and MIC90 of eight drugs against Aeromonas spp. obtained in this study can provide a theoretical reference for the usage and dosage of drugs in aquaculture.Compared with previous studies ,33,34,35n et al. , and theWT values of bacteria of a species, whereas at least 500 bacteria need to be collected to establish the COWT values of a genus. However, the CLSI [WT values. The number of strains collected in this study met the above requirements.The CLSI  documentthe CLSI  documentthe CLSI  suggesteWT values of Aeromonas for a variety of antimicrobial agents obtained in this study still need to be compared and integrated with data obtained by other laboratories to obtain internationally accepted COWT values for Aeromonas [The COeromonas , which wAeromonas. The tentative epidemiological cut-off values  of these eight drugs against Aeromonas spp. were established by ECOFFinder and NRI methods, which were 0.25, 64/32, 4/2, 8, 4, 1, 0.062/0.125, and 0.5 μg/mL, respectively. Using these breakpoints as the standard, the proportion of NWT strains of Aeromonas spp. in different water bodies in China was analyzed. The results showed that Aeromonas spp. had a higher proportion of NWT strains against enrofloxacin, doxycycline, and florfenicol. Therefore, it is necessary to focus on the resistance changes of Aeromonas to these three drugs in the aquaculture environment and to conduct early monitoring of the resistance of Aeromonas to avoid the spread of drug-resistant strains.In this study, the optimized micro-broth dilution method was used for the first determination of the MIC of eight drugs including trimethoprim–sulfamethoxazole, erythromycin, doxycycline, neomycin, colistin, florfenicol, enrofloxacin, and ceftazidime against aquatic


The effects of psychological interventions on symptoms and psychology of functional dyspepsia (FD) remain unclear. We aimed to comprehensively evaluate the effects of psychological interventions on symptoms and psychology of FD.We searched the PubMed, Cochrane Library, and Embase electronic databases for randomized controlled trials (RCTs) evaluating the role of psychological interventions in FD patients published before July 2021. Standardized mean differences (SMDs), risk ratios (RRs) and 95% confidence intervals (CIs) were calculated by a random effects model. Subgroup analyses and sensitivity analyses were also performed.p = 0.01], relieve gastrointestinal symptoms scores at follow up , relieve gastrointestinal symptoms scores at end of treatment , decrease anxiety  and depression levels . The results of the subgroup analysis showed that psychotherapy was more likely to symptom improvement, relieve gastrointestinal symptoms scores and decreased depression levels compared to the control.Fourteen RCTs with a total of 1,434 FD patients were included. Compared with the control group, psychological interventions were significantly more likely to symptom improvement [RR = 1.74, 95% CI , Psychological interventions may be effective in alleviating the symptoms and psychology of FD, but the effect appears to be limited to psychotherapy with fewer trials for other psychological interventions. More data from high-quality RCTs are needed to confirm their use in the treatment of FD. Functional dyspepsia (FD) is a chronic gastrointestinal disease originating from the gastroduodenum without structural diseases .The patients were 18 years or older.The experimental group received psychological interventions . The control group received supportive therapy or no psychological intervention.The outcomes included at least one measure of symptom relief, gastrointestinal symptoms scores, quality of life, psychological symptom, anxiety, or depression.The study design was a randomized controlled trial (RCT).The patient had any type of organic gastrointestinal disease.Heartburn and acid regurgitation were the main symptoms.Data extraction was performed independently by two researchers. For each study, we collected the following data: name of the first author, article publication years, country of study population, study intervention, number of patients, age, symptom improvement, gastrointestinal symptom scores, quality of life, psychological symptom scores, anxiety, depression, follow-up time, and information needed for quality assessment. The gastrointestinal symptom scores were based on patients assessed symptoms. In the process of data extraction, any inconsistencies were resolved by consulting the third researcher.All the studies we included were RCTs, and the quality assessment was conducted by GRADEPro software. The quality of evidence was assessed by considering the following factors: risk of bias, inconsistency, indirectness, imprecision, publication bias, large effect, plausible confounding and dose-response gradient. The quality was rated as high, medium, low, or extremely low. Certainty of evidence was assessed for each outcome and risk of bias was assessed for each study. The risk of bias was assessed using the Cochrane Risk Bias Tool (ROB version 1.0). The key factors included the following: selection bias, performance bias, detection bias, attrition bias, reporting bias, and other biases. The quality assessment was conducted independently by two researchers, and any inconsistencies were resolved by consulting the third researcher.p < 0.1 and I2 > 50% to represent substantial heterogeneity. The meta-analysis was performed using a random effects model (DerSimonian–Laird method). Data were extracted from the intention-to-treat analysis of the original article. If the outcomes in the original article were shown as the median and interquartile range, the mean and SD values were calculated according to the method of Wan et al. (2 = (SD pretreatment)2 + (SD posttreatment)2 − (2R * SD pretreatment* SD posttreatment), and R was calculated from a included study reported in considerable detail  and 95% confidence intervals (CIs) were used to evaluate continuous variables. The interpretation for this effect size: 0.2 represents a small effect, 0.5 a moderate effect, and 0.8 a large effect. For dichotomous variables, risk ratios (RRs) and 95% CIs were evaluated. The heterogeneity analysis was performed by Q statistic test, and we considered n et al. . In studA total of 844 studies were found through the literature search, including 279 studies from PubMed, 307 from the Cochrane Library and 258 from the Embase database. After the elimination of 186 duplicate studies, the titles and abstracts of the remaining 658 studies were screened, and 633 studies were eliminated. In addition, 11 of the remaining studies did not meet the inclusion criteria: nine of them were review articles, 2 had incomplete data. Therefore, a total of 14 RCTs were included in our meta-analysis .The study characteristics are shown in There is a certain degree of heterogeneity in our meta-analysis. The clinical heterogeneity was evidenced by different numbers of patients, a wide range of study locations, and different psychological interventions, and the length of follow-up was also different among the included studies (2 weeks to 14 months). The methodological heterogeneity was evidenced by different methods of measurement, although the studies we included were all RCTs. We used SMDs and 95% CIs to evaluate continuous variables because the same outcome may have been evaluated differently. We also performed a subgroup analysis to find the source of heterogeneity. We conducted a subgroup analysis of gastrointestinal symptoms scores, anxiety levels, and depression levels. After subgroup analysis, the heterogeneity between the psychotherapy group and other groups was not significantly reduced, indicating that the type of psychological intervention was not a source of heterogeneity. And considering the existence of heterogeneity, a random effects model was used.p = 0.01] . In the subgroup analysis, compared with the control group, psychotherapy was more likely to symptom improvement , and heterogeneity was significantly reduced   .p < 0.0001] . The results of the subgroup analysis of psychological intervention types showed that there was a significant effect of psychotherapy on reducing gastrointestinal symptoms scores compared to the control , and there was significant heterogeneity between studies   .p < 0.001] .A total of four RCTs  . In the subgroup analysis, compared with the control group, psychotherapy was more likely to decrease anxiety levels, but the difference was not statistically significant . Significant heterogeneity was observed in the subgroup analysis .Six studies  . Heterogp < 0.0001] .A total of seven RCTs Fan,  with 933 0.0001] . The resp = 0.001, I2 = 81%). If Calvert's . And there was a significant change in the heterogeneity of improving the quality of life . If Hjelland's . Sensitivity analysis was also conducted on other outcomes that were reported in ≥3 included studies, and the heterogeneity was not significantly changed after removing studies one by one.By removing studies one by one to evaluate the impact of each study, we found that there was a significant change in the heterogeneity of symptom improvement .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


This study was performed to evaluate the effects of exercise on positive and negative symptoms and depression in patients with schizophrenia through a systematic review and meta-analysis focusing on randomized controlled trials (RCTs). PubMed, Embase, CINAHL, MEDLINE, Cochrane Library, PsycINFO, and Web of Science were searched from their inception to 31 October 2022. We also conducted a manual search using Google Scholar. This meta-analysis was conducted according to the PRISMA guidelines. The methodological quality of the studies was assessed using the Cochrane risk-of-bias tool for randomized trials. To identify the cause of heterogeneity, subgroup analysis, meta-ANOVA, and meta-regression analyses were performed as moderator analyses. Fifteen studies were included. The meta-analysis (random-effects model) for overall exercise showed a medium significant effect  on negative symptoms, a small significant effect  on positive symptoms, and a nonsignificant effect  on depression. Our findings demonstrate that exercise can relieve the negative and positive symptoms of schizophrenia. However, the quality of some included studies was low, limiting our results for clear recommendations. Approximately 1% of the global population suffers from schizophrenia. Schizophrenia is a persistent mental health problem worldwide, and long-term care and management are crucial throughout life . PatientPositive symptoms can cause violent behavior and influence patients’ social interactions and daily functioning . These fPatients with schizophrenia frequently have comorbid mental illness, such as depression . The preTherefore, health providers have tried to treat and care for the positive and negative symptoms of schizophrenia, in addition to other psychological problems such as depression. In clinical practice, pharmacological approaches, including antipsychotics, are the first-line treatments, and they have been used to treat clinical symptoms in patients with schizophrenia. However, a study on the effectiveness of antipsychotics for treating schizophrenia reported symptom reduction of only approximately 60% following antipsychotic treatment . PositivAlthough the main treatment for schizophrenia is antipsychotic medication, other modalities such as cognitive therapy and exercise have been proposed as adjuvant treatments . NonpharSeveral studies have shown that physical activity can significantly improve positive and negative symptoms and other psychological symptoms in this population . In contEvidence indicates that physical activity can affect symptoms of schizophrenia. Low levels of brain-derived neurotropic factor (BDNF) are related to schizophrenia symptoms. There may be a remarkable association between psychotic symptoms and BDNF levels in the cognitive functioning areas . In addiThe hippocampus is a brain region associated with depression, and physical activity may contribute to a decrease in depressive symptoms by inducing an increase in BDNF levels in the hippocampus . FurtherPrevious systematic reviews and meta-analyses have investigated the effects of physical activity on the positive and negative symptoms in patients with schizophrenia. However, some of these reviews included both randomized controlled trials (RCTs) and non-RCT studies , and somThis study aimed to determine the effect of physical activity on positive symptoms and negative symptoms, alongside depression, one of the most common mental health issues in people with schizophrenia, through a systematic review and meta-analysis of controlled trials. We also explored the impact of various intervention characteristics that affect the outcomes of physical activity. The findings of this study can be used as basic data for developing interventions to manage symptoms and psychological problems in people with schizophrenia.The eligibility criteria for this review were as follows: (1) participants: patients aged 18 years or older who met the diagnostic criteria for schizophrenia or schizoaffective disorder; (2) interventions: any type of exercise ; (3) intervention in the control group: usual care, no intervention, and stretching; (4) outcomes: positive symptoms, negative symptoms, and depression; (5) study design: RCTs only. We excluded studies that combined other types of interventions with exercise, studies that evaluated exercise without a control group, studies that included participants with schizophrenia and other diagnoses, and studies with missing data essential for the meta-analysis. We included all studies published in English between 2011 and 31 October 2022. This review was performed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. The review protocol was registered in the International Prospective Register of Systematic Reviews (PROSPERO) (CRD42022318841).We performed a literature search in the following international databases in the period of 18 September 2022 to 31 October 2022: PubMed, Embase, Cumulative Index of Nursing and Allied Health Literature (CINAHL), MEDLINE, Cochrane Central Register of Controlled Trials, PsycINFO, and Web of Science. Additionally, to identify literature that was not identified through the search strategy, we conducted a manual search on Google Scholar.A search strategy was formulated using Medical Subject Headings (MeSH) and Embase Subject Headings database (Emtree) terms, synonyms, free text, and truncations. The search terms were the following: (schizophrenia or schizoaffective disorder), (exercise), and , as well as derivatives of these terms .Two reviewers independently searched the databases and performed the eligibility assessments. The titles and abstracts of the studies were screened according to the eligibility criteria. The full text of the studies was reviewed, and eligible studies were selected. We then extracted the data independently using a standard data extraction form, which included information on the authors, publication year, country, study design, sample size, and intervention . Discrepancies during inclusion of studies in the final review were resolved by discussion between the two reviewers or, if unresolved, by a third reviewer.This study assessed the risk of bias using the revised Cochrane risk-of-bias tool  for randomized trials (ROB 2) . Two rev2 and I2 statistics. An I2 value greater than 50% and a p-value less than 0.1 indicated substantial heterogeneity according to the criteria of the Cochrane Handbook for Systematic Reviews of Intervention. In addition, the interpretation of the I2 value depends on many factors, but a rough guide for the same in a meta-analysis of randomized trials is as follows: 0–40%, might not be important; 30–60%, may represent moderate heterogeneity; 50–90%, may represent substantial heterogeneity; 75–100%, considerable heterogeneity [The meta-analyses were performed using the R software package Meta version 4.0.3 . We used a random-effects model assuming that the true effect samples may differ across studies. The effect size was estimated on the basis of the standardized mean difference  with a 95% confidence interval (CI). The effect size was represented according to Cohen’s classification  . We analogeneity . To inveA total of 1109 studies were identified in the database and manual search. After removing 816 duplicate articles, the titles and abstracts of 286 studies were screened, and 246 studies were excluded. Next, 40 studies were sought for retrieval, and six studies were excluded as they were not retrieved. After excluding 23 studies that did not meet the inclusion criteria, 11 studies were reviewed. Meanwhile, we identified seven additional studies from websites and through a manual search of citation; after excluding three of them, four were included in the review. Finally, we selected 15 studies in the systematic review  1,17,18,38,39,40n = 884) and control groups (n = 758). On analyzing the negative and positive symptoms, the withdrawal rate was found to be 16.67% in the exercise group and 11.68% in the control group; according to the depression analysis, it was 11.87% in the exercise group and 12.65% in the control group.Fifteen RCTs were included in this study. The characteristics of the included studies are presented in In the intervention group, the types of exercises included aerobic exercise in 10 studies, neuromotor exercise in four studies, and multicomponent exercise in three studies according to the criteria presented by the American College of Sports Medicine (ACSM) . ExercisThe outcomes of negative and positive symptoms in 15 studies were measured using the Positive and Negative Syndrome Scale (PANSS). The outcome of depression was measured using the Chinese version of the PANSS, Montgomery and Åsberg Depression Scale (MADRS), and Calgary Depression Scale for Schizophrenia (CDSS).The findings of the risk-of-bias (RoB) assessment are presented in We conducted a basic meta-analysis for each outcome: negative symptoms, positive symptoms, and depression.As shown in I2 = 45.0%, p = 0.02). Therefore, we conducted a subgroup analysis and meta-analysis of variance to investigate the potential causes of heterogeneity. First, a subgroup analysis based on the effect of negative symptoms by exercise type revealed a mean effect (Hedges’ ĝ) of −0.68 (95% CI: −1.19 to −0.16) for multicomponent exercise, −0.53 (95% CI: −0.82 to −0.23) for aerobic exercise, and −0.41 (95% CI: −0.84 to 0.03) for neuromotor exercise. However, the type of neuromotor exercise did not significantly improve the negative symptoms in patients with schizophrenia. The meta-analysis of variance showed no significant differences across the types of exercises .According to the criteria of the Cochrane Handbook for Systematic Reviews of Intervention , 45% of p = 0.701). In addition, meta-regression indicated that the duration of exercise did not account for the heterogeneity of the mean effect (0%). Lastly, we performed a subgroup analysis based on exercise session. In the subgroup analysis based on sessions of exercise, the mean effects (Hedges’ ĝ) were −1.03 (95% CI: −1.39 to −0.68) for 13–24 times, −0.46 (95% CI: −1.39 to −0.68) for 25–48 times, −0.44 (95% CI: −0.75 to −0.13) for 49 times or more, and −0.10 (95% CI: −1.35 to 0.14) for 12 times or less. Twelve or fewer sessions did not significantly improve negative symptoms. In addition, a meta-analysis of variance showed significant differences between the exercise sessions . Moreover, the meta-regression indicated that the exercise session significantly explained 100% of the variation in the mean effects .Furthermore, we performed subgroup analyses based on exercise duration. In the subgroup analysis based on duration of exercise, the mean effects (Hedges’ ĝ) were −0.60 (95% CI: −0.88 to −0.31) for exercise duration of 8 weeks, −0.55 (95% CI: −0.98 to −0.13) for 20 weeks or more, and −0.33 (95% CI: −0.52 to −0.15) for 12 weeks. The meta-analysis of variance showed no significant differences in the duration of exercise . First, to investigate the cause of heterogeneity, we conducted a subgroup analysis by exercise type, which showed that the mean effect (Hedges’ ĝ) was −0.61 (95% CI: −1.08 to −0.14) for multicomponent exercise. Only the types of multicomponent exercise significantly improved positive symptoms among patients with schizophrenia. In addition, the meta-analysis of variance showed no significant differences between the types of exercise .Seventeen RCTs reported on the effects of exercise on positive symptoms . The exep = 0.051).In addition, we performed subgroup analyses based on exercise duration. In the subgroup analysis based on duration of exercise, the mean effects (Hedges’ ĝ) were −0.79 (95% CI: −1.28 to −0.30) for exercise duration of 20 weeks or more, −0.15 (95% CI: −0.39 to 0.08) for 12 weeks, and −0.11 (95% CI: −0.43 to 0.20) for 8 weeks. Only an exercise duration of 20 weeks or more significantly improved positive symptoms among patients with schizophrenia. However, the meta-analysis of variance showed no significant differences in the duration of exercise  indicated statistical significance and, thus, a potential publication bias. To confirm the effect of this publication bias on our study outcomes, we used the trim-and-fill method. After correcting the asymmetrical funnel plot to be symmetric, including six studies that were assumed to be omitted  was not statistically significant, indicating a significant relationship between the effect size and standard error. The results confirmed a statistically significant improvement in positive symptoms among patients with schizophrenia. Regarding depression, we did not examine for publication bias because the power of the test was low due to the small number of studies.For positive symptoms, the funnel plot of the standard error and effect sizes seemed symmetrical C. HoweveA sensitivity analysis was performed to investigate whether within-study bias influenced the mean effect of exercise on negative symptoms A. After Schizophrenia is a chronic illness often associated with an incomplete recovery of clinical symptoms. Some symptoms of schizophrenia are difficult to resolve with antipsychotics alone . ClinicaThis study was a systematic review and meta-analysis conducted to confirm the effect of exercise intervention on positive symptoms, negative symptoms, and depression in the schizophrenia population. Our review included 15 studies designed only as RCTs, and all 15 studies were included in the meta-analysis. Although previous meta-analyses were conducted to evaluate the effect of exercise in patients with schizophrenia, some included both controlled and uncontrolled studies  or selecNegative symptoms in schizophrenia are defined as the absence or diminution of normal behaviors and functions and are more difficult to treat than positive symptoms using antipsychotic medications ,44. SomeNext, in this study, the effect of exercise intervention on the positive symptoms of schizophrenia was found to have a small effect size . The systematic review by Dauwan et al. , which iLastly, in this study, the effect of exercise on depression in schizophrenia was not significant , whereas other studies showed a significant effect (SMD = 0.71) . Since tOur analyzed studies had at least 30 min/day of exercise time and ranged from one to seven times per week. Most exercise interventions followed the recommendations of ACSM . HoweverThe findings of this review indicate that exercise is effective in improving the negative and positive symptoms of schizophrenia, which is associated with difficulty in cognition and social functioning. This can be explained by the biopsychosocial model, according to which physical activity affects an individual’s biological, social, and psychological factors ,24.Eight of our selected studies were performed in Asia. The cultural background and attitudes of participants in the exercise intervention could not be excluded, and this should be considered when interpreting our results.Our study had some limitations. Firstly, the percentage of heterogeneity ranged from moderate to high. Although meta-analysis and subgroup analyses were attempted, they did not account for this heterogeneity. This could be explained by the high variety of the different exercise protocols. Secondly, some of the included studies were concerning for their methodological quality of randomized controlled trials. Thirdly, not all studies were included in our analysis because of language limitations. Lastly, it is difficult to be sure whether the changes in symptoms and depression scores were the result of exercise interventions only because all confounding factors related to experimental studies were not excluded.This study aimed to evaluate the effects of exercise on positive symptoms, negative symptoms, and depression in patients with schizophrenia through a systematic review and meta-analysis focusing on RCTs, including recently updated studies. Our findings suggest that exercise interventions, including aerobic, multicomponent, or neuromotor exercises, can help improve clinical negative and positive symptoms. In particular, multicomponent exercise intervention combined with aerobic and resistance exercises had a moderate effect size in improving the positive and negative symptoms of schizophrenia. In addition, an exercise duration of 20 weeks or more had a moderate effect size in relieving the positive and negative symptoms. We recommend future reviews focusing on the level of clinical symptoms in patients with schizophrenia. It is proposed to compare the effect size between physical exercises and mind-body exercises on the symptoms of schizophrenia.