This study aimed to evaluate the functional impact of a shared intervention model by the mobile physiotherapy and rehabilitation team (MPRT) and primary care case management nurses (PCCMNs) on chronic patients. This was a prospective, observational study involving 1086 patients  in the province of Almeria, which was conducted between 2004 and 2018. Most of the registered diseases included cerebrovascular and neurological diseases (56.7%), osteoarticular diseases (45.3%), diabetes mellitus (25.7%), cardiovascular diseases (25.5%), and chronic respiratory diseases. The study included a home care intervention by the MPRT and PCCMNs and included the following main outcome measures: age, sex, main caregiver, disabling process (ICD-9), type and number of inclusion categories for chronic disease, initial and final Barthel index (BI), treatment or intervention on the patient (techniques), objectives, and number of sessions. The main techniques used were kinesiotherapy (44.6%) and caregiver training (23%), along with technical aid. An equation predicting the patients’ final BI, according to the initial BI, was constructed using multiple linear regression modelling. A marked improvement in functional capacity was found after an average of 10 physiotherapy sessions. A lower patient age was correlated with a higher functional capacity, both initial and final BI, as well as a greater number of sessions.  Currently, one of the main goals of health service providers is to enable patient-centered care in their own environment. This is achieved by ensuring cooperative and coordinated services among different care providers, including the patient’s family and social and community resources . In Andalusia (Spain), mobile physiotherapy and rehabilitation teams (MPRTs) were established in primary care (PC) in 2002 and included in the regional Family Support Plan with the aim of improving patient and caregiver access to these services in the home environment , althougChronic diseases are long-term, slow-developing conditions with serious adverse side effects that affect one’s quality of life and have important economic repercussions, causing a high degree of disability and dependence . They reChronic diseases are one of the foremost challenges to healthcare, owing to their high prevalence and morbidity. Although, owing to medical progress, the aging population experiences an increased survival rate, many chronic patients turn to emergency services, develop comorbidities and complications, and require complex treatments and hospitalization . In SpaiThe increased demand for home care (HC) for chronic patients may be explained by the fact that most elderly people and dependents prefer to be treated at home . PredictFor these reasons, strategic guidelines should be established to achieve a fully developed integral HC system for chronic patients that guarantees the best results in terms of overall health and quality of life, delivery of improved services, cost-effective interventions, greater cohesion of health care teams, a sustainable healthcare system, and high patient satisfaction levels .According to a previous study ,15,16, wWe performed a prospective observational study to analyze the frequency, determinants, and factors related to different diseases in PC chronic patients in the province of Almeria. The clinical history of 4762 patients, registered between 2004 and 2018, were analyzed, and 1086 chronic patients were included who met the following criteria: patients referred by PCCMNs to the MPRT who had received care according to the service action protocol , suitabiPatients were excluded if they refused to receive physiotherapy, or if it was contraindicated. The study variables were as follows: age, sex, main caregiver identity, disabling process (ICD-9), type, and number of inclusion categories for chronic disease , and thePrior to commencing the study, permission was sought from the pertinent ethics and sciences committee of the province of Almeria (CEIC-AL 39/2012). Data were obtained in strict adherence to current privacy and data protection laws, and guidelines from the International Committee of Medical Journal Editors. All patients provided written informed consent before treatment, in accordance with the Helsinki Declaration.t-tests for related samples were performed to calculate the changes between scores for the initial and final BI in each clinical category. t-tests for independent samples were used to calculate differences in functionality (BI) through sex. We performed an analysis of variance (ANOVA) test for the initial and final BI values, and the patients’ age groups . If all criteria were met , multiple comparisons were made using the Bonferroni test; when homogeneity was not fulfilled, a Dunnett’s T3 test was performed.Descriptive and bivariate analyses were carried out using SPSS version 23 ; analyses sought possible associations between dependent and independent variables. p2.A multivariate analysis (MANCOVA) was conducted to assess the differences between clinical categories, with sex and age (independent variables) and initial BI as covariates; this analysis was complemented by the calculation of effect size by ηAdditionally, relationships between quantitative variables  were assessed through correlation measurements (Pearson linear), and the multiple regression was obtained as determined by the predictive model in relation to the BI values of the patients.The average age of the 1086 patients was 80.42 years (SD = 12.3), of which 63.7% were women. The reasons for referral to the unit were as follows: multimorbidity, advanced age (85.2%), and architectural barriers in the home (14.8%).The main caregiver in the home was identified primarily as the daughter (30.1%), followed by the family (17.9%); wife (12.5%); others (7.3%); son (6.5%); husband (5.1%); children (4.9%); and, finally, grandchildren (0.8%). Home assistants collaborated in 11.7% of cases, while 3.3% of elderly people had no caregiver.The most common disabling processes were as follows: consequences of immobility (25.7%); motor impairment (19.2%), cerebrovascular disease (12.3%), Alzheimer’s disease (10.8%), fractured hip (5.3%), chronic obstructive pulmonary disease , and Parkinson’s disease (3.2%); the remainder represented less than 2% of the total.The main objectives of treatment were to improve functioning (27.5%), caregiver training (14.9%), walk training (15.1%), and pain relief (2.5%), and to increase respiratory capacity (3.9%). The different rehabilitation techniques applied were grouped under five categories as follows: functional exercises (44.6%), functional exercises and electrotherapy (5.8%), caregiver training (23%), functional exercises and caregiver training (17.3%), and technical aids (9.3%). The average number of physiotherapy sessions was 9.8 (SD = 10.2).t = −20.47; confidence interval, CI 95%: −12.32 to −10.16; and p < 0.001). The difference between the average initial BI (n = 326) had one inclusion category, 28.2% had two categories (n = 292), 21.3% (n = 220) had three, 8.8% (n = 91) had four, 3.4% (n = 35) had five, and 0.3% (n = 3) had six. The most prevalent category for referral was E , stroke, and neurological and cognitive impairment, followed by H  chronic osteoarticular disease; F, 25.7% (n = 265), peripheral artery disease (PAD), and diabetes mellitus with retinopathy; A, 25.5% (n = 263), heart failure; C, 17,6% (n = 181), chronic respiratory disease; G, 14.3% (n = 147), chronic anemia and solid or hematologic neoplasm; B, 13.3% (n = 137), autoimmune disease and renal disease; and D, 7.9% (n = 81), inflammatory bowel disease (IBD) and chronic liver disease.Analysis of the inclusion criteria for the patients into single or multiple chronic pathologies revealed that 31.5% of the sample .The quantitative variables  were analyzed to determine the existence of any sex-related differences . No statp < 0.001). These age groups also fulfilled the applicability hypothesis; in the case of the initial BI, all the conditions required to carry out an ANOVA test were fulfilled, indicating a statistically significant difference between groups ; analysis of post hoc Bonferroni data  showed differences between the 60–69 years (p = 0.001) were also found between the 70–79  relative to the initial and final BIs, statistically significant differences were found in some groups (p = 0.000). The post hoc Bonferroni test  revealed differences between the 70–79 years group (p = 0.004) between the 80–89 years group  = 14,540, Wilks’ Lambda = 0.881; η2 = 0.119). There were statistically significant differences due to sex  = 6505, Wilks’ Lambda = 0.943; η2 = 0.057] and age  = 1568, Wilks´ Lambda = 0.957; η2 = 0.014). There were no significant differences due to sex × age  = 1057, Wilks’ Lambda = 0.971; η2 = 0.010). Considering the effect size, these differences showed a low influence.A MANCOVA inferential analysis concluded that there were statistically significant differences between the initial BI indices by clinical category . An inferential analysis on the saturated model parameters concluded that only the initial BI  = 57.024 and semipartial correlation = 0.839) and the number of sessions  = 14.555 and semipartial correlation = 0.214) were significant; however, age was not significant  = 1.841, and semipartial correlation = 0.027). The relationship between the three predictive variables and the final BI is strong because the correlation coefficient was 0.892 (saturated linear model); hence, 80% of the final BI variance was explained by the combination of predictive variables. As confirmed by the ANOVA, the regression was significant , optimization of the remaining physical and psychic faculties that are not affected, and recovery of those affected, maintaining functional independence and ambulation as long as possible . In lineThe case management nurse´s activities were carried out on behalf of patients and their specific needs. These included procedures, assessments, providing physical care, and counselling; promoting innovative patient care and facilitating the optimal progression of patients through the healthcare system. They assessed patient/family response to therapy and modified the plan of care, performing interdisciplinary coordination in planning in order to optimize the patient’s health.The rehabilitation techniques applied with the aim of achieving these objectives were, principally, functional exercises (approximately 50%), caregiver training (a third), functional exercises combined with caregiver training (17.3%), and functional exercises combined with electrotherapy . These results are in line with those obtained in previous studies , althougThere was a difference of approximately 10 points between the initial and final BI, once interventions by PCCMNs and the MPRT were initiated. In a study by García Morillo et al. , 16% of In our study, a higher age correlated with a lower initial and final BI, and a lower number of treatment sessions; these results agree with those of a similar study on patients with motor impairment by Vega-Ramírez et al . HoweverRecently, the Cochrane Library reviewed the effectiveness of patient-centric interventions and health services designed to improve the condition of patients with multiple pathologies (those with two or more chronic diseases) in PC and the community . The revThe advent of new roles, such as the case management nurse, improvements in the care, attention to certain processes, and changes in the location of certain services (such as mobile physiotherapy and rehabilitation) are key to services that are adapted to the needs of the chronic patient . The solSeveral studies confirm the role of PCCMNs in facilitating the sustainability of services and the effectiveness of results obtained in chronic and complex-chronic patients . The maiThe effectiveness of PCCMNs in Andalusia is the subject of continued study, with the aim of making more improvements. In 2016, López-Liria et al  concludeDescriptive studies such as these help create predictive models that allow for the stratification of the population in accordance with their healthcare needs, which enables health services to be proactive, arranging specific intervention according to their concrete needs .This study has some limitations. The patients were not randomly chosen but arbitrarily or intentionally, which may affect the external validity. The selection was conditioned by the availability of resources in the geographic area of the mobile team’s operations. However, the exhaustive data collection and the way these were collected provided information on the needs of these patients and the resources available to them. Similarly, we also obtained information from the professionals involved, their shared interdisciplinary knowledge, and consensus on the best strategies, in order to offer improved quality care for this particularly vulnerable population. This information has enabled this service to be quantitatively and qualitatively described, including its benefits and the needs detected, so that approximate estimates can be made of the socio-health resources required, both in the service offered and in the patients’ homes, as well as to propose plans for improvement. With similar studies that measure the cost effectiveness of these treatments, we could save on economic resources dedicated to polymedication or dependence care , offering greater functional independence to people, and improving the quality of life of their families and caregivers. It is necessary to integrate these services in order to provide more effective and efficient care to patients with complex health problems, beyond individual approaches with disparate organizational alternatives, and pending to be conclusively evaluated.This study examines the care of chronic patients. Patient-centered care in their environment presents a current and future socio-health challenge, including an increasingly prioritized healthcare trend. The home intervention model shared between the PCCMN and MPRT in PC allows for an improvement of functional capacity in these chronic patients, with an average of 10 physiotherapy sessions. A lower patient age correlates with higher initial and final functional capacity, as well as a greater number of sessions.The findings of this study have enabled a quantitative and qualitative description of the integrated services provided to a chronic HC patient. The results also reveal the benefits and limitations of the combined services, which are undeniably useful to estimate the socio-health resources needed in the service offered and in the patients’ homes, as well as propose future plans for improvement. The introductions of new roles, such as the PCCMN, or changes in the location of certain services, such as the MPRT, are two examples of the adaptation of healthcare services to the needs of chronic patients.It is necessary to improve the delivery of HC for chronic patients seeking superior cost-effective health interventions, quality of life, and satisfaction, as well as that of their caregivers and the care team, thereby contributing to the sustainability of the healthcare system.


Aeromonas bacteria can cause an infection characterized by septicemia and is one of the most common pathogens in tropical fish. This disease is responsible for high morbidity and mortality rates, causing considerable losses in aquaculture. Thus, the understanding of its pathophysiology is crucial to develop control strategies of this bacterial infection in farmed fish. This study aimed to characterize early pathological aspects of acute sepsis in pacu (Piaractus mesopotamicus) experimentally infected with Aeromonas hydrophila. A total of 160 juvenile pacus were inoculated intraperitoneally with A. hydrophila (1.78 x 109 CFU/mL) and at 0 (control), 1, 3, 6, and 9 hours post-inoculation (hpi), animals were anesthetized and samples were collected for microbiological, light microscopy and transmission electron microscopy (TEM) analyzes. The results showed the occurrence of hemodynamic alterations, such as hemorrhage and congestion, which were observed mainly after 6 and 9 hpi. It was possible to re-isolate Aeromonas at all sampling times except in control group. However, just after 9 hpi it was possible to find the bacteria in all fish and tissues. Light microscopy analyses revealed a degenerative process, necrosis and vascular damage mainly at 6 and 9 hpi. According to the ultrastructural examination, areas of cellular death were identified in all examined tissues, especially at 6 and 9 hpi. However, the most severe, related to necrosis, were observed after 6 and 9 hpi. The findings suggested that this bacterium spreads in the first hpi through the fish organs, mainly affecting spleen, liver and kidney, causing irreversible lesions at the molecular level. In find death ,9.A. hydrophila, are consequences of endotoxins, triggering sepsis or endotoxemia; complex syndromes that are defined by the presence of a systemic inflammatory response (SIRS) [A. hydrophila in fish [Aeromonas infection, wich were petechiaes and suffusions, 9 hours post-inoculation of A. hydrophila in pacus, showing the importance of understanding aeromonosis.The deleterious effects caused by Gram-negative bacteria, such as e (SIRS) ,11. The e (SIRS) . Interna in fish . SimilarA. hydrophila.Based on the aforementioned, this study aimed to characterize by morphological, ultrastructural and microbiological studies, the early stages of the septic process in different tissues of pacus experimentally infected with P. mesopotamicus) (250 ± 99.3 g and 15 ± 2 cm), originating from the same spawning of fish rared at Fish Repoduction Laboratory of Aquaculture Center of Unesp (Caunesp), were randomly distributed in five fiber tanks of 250 L (n = 10 fish/tank), supplied with chlorine-free running water from spring, at a flow of one liter per minute and constant aeration. Fish were acclimated for two weeks before the experiment. During this period, fish were fed to apparent satiety twice a day. Tanks were siphoned once a week and water quality was monitored daily using multiparameters probes . Water quality parameters were kept as follows: dissolved oxygen = 5.1 ± 0.6 mg.L-1; temperature = 29.47 ± 1.58°C; pH = 7.66 ± 0.36 and electrical conductivity = 117.96 ± 6.12 μScm-1. Parameters remaining within the adequate ranges for this species [A total of 50 healthy pacus . DNA concentration was 1690.9 ng/μL and absorbance ratio 260/280 and 260/230, varying between 2.02 and 2.04. Subsequently obtaining the DNA, ribosomal gene 16S rRNA was amplified according to Sarcar et al. [http://www.ncbi.nlm.nih.gov), which presented 99% similarly with A. hydrophila (Accession number: CP007518-2). The challenge strain was grown on tryptic soy agar (TSA)  and incubated at 28°C for 24 h. Bacterial suspensions were prepared by transferring a single colony of A. hydrophila to Falcon tubes with 50 mL of tryptic soy broth (TSB)  and reincubated at 28°C in a bacteriological incubator . Twenty-four hours after incubation bacterial suspensions were centrifuged  at 1792 g/10 min/4°C. Bacterial mass was resuspended in sterile 0.65% NaCl solution. Bacterial concentration was estimated by spectrophotometry and confirmed by determining the colony-forming units (CFU) of the original culture , reaching a concentration of 1.78 x 109 CFU/mL.n = 10). Control group fish were injected by intraperitonal route with 0.5 mL of sterile 0.65% NaCl solution, while fish from the other groups were inoculated by the same route with 0.5 mL of A. hydrophila solution (1.78 x 109 CFU/mL) [-1) [After acclimation period, 50 pacus were randomly distributed into five groups for biological samples obtainment, according to time in hours post-inoculation (hpi): control group or 0 hpi; group 1 hpi; group 3 hpi; group 6 hpi and group 9 hpi ( CFU/mL) . SpecimemL) [-1) .After 0, 1, 3, 6 and 9 hpi, fish were anesthetized for blood sampling collection by puncture of the caudal vein using with sterile needles and syringes. Blood was immediately used for microbiological examination. After blood collection, anesthetized fish were euthanized by pithing and submitted to necropsy. Fragments of the spleen, heart, brain, liver and anterior kidney were aseptically sampled after the procedures of antisepsis and necropsy using sterile surgical instruments , aiming to perform microbiological, histopathological and ultrastructural examinations. Additionally, intestine, gills and pancreas were sampled for the histopathological exam.Animals were evaluated continually throughout the experiment, to verify possible macroscopic alterations and/or clinical behavior.-1, and incubated for 24 h at 28°C, in aerophilic atmosphere [−4. By means of the “Spread-plate” method, 50 μL of each dilution  and the fresh blood were spread on TSA containing 10 mgL-1 of sodium ampicillin. Plates were incubated for 24 h, at 28°C. Then, colony counting was made in an electronic counter. Random samples of the bacterial colonies were confirmed as A. hydrophila by Polymerase Chain Reaction (PCR).In all sampling times, an aseptically removed fragment of each sampled organ and fresh blood were immediately streaked with a platinum loop in TSA plates, supplemented with sodium ampicillin to a final concentration of 10 mgLmosphere . A seconTissue samples were fixed in 3.5% neutral buffered formalin for 24 h, and then preserved in alcohol 70%. Then, samples were placed in plastic cassettes and processed by gradual dehydration in 70–100% alcohol, clearing in xylene, and embedding in paraffin wax. Five-micron thick sections were cut using a microtome , and then stained with hematoxylin and eosin (H&E) . Slides Tissue fragments of spleen, heart, brain, liver and head kidney were fixed in Karnovsky’s solution  for 24 h at 4 ºC, bathed three times in phosphate buffer 0.1 M, pH 7.0, post-fixed in 1% osmium tetroxide in sodium cacodylate buffer  for 1 h, and then processed for TEM . The secData from microbiological analyzes were tested for normality  and then submitted to a variance analysis, and Dunn test was applied in order to verify differences within the medians (p < 0.05).No behavioral alterations were observed in any of the groups. Macroscopic lesions observed during necropsies were evident only at 6 and 9 hpi, with the last being characterized by a greater severity of lesions. Petechial cutaneous hemorrhages were evident, especially close to the inoculation area , and in A. hydrophila in different organs and blood after the pre-established experimental times. The data has shown that only at 9 hpi 100% of animals inoculated with the bacteria obtained positive results for A. hydrophila re-isolation in all analyzed tissues  compared with the control group (0 hpi), which did not present bacterial growth, in all examined organs and blood . MoreoveA. hydrophila from diseased pacus was capable of inducing aeromonosis in healthy fish, which presented characteristic lesions of the disease and presented a positive re-isolation of this bacterial strain in the afflicted tissues of these animals. The strain was confirmed by PCR.Macroscopic alterations and microbiological exams showed that the isolated strain of In general, lesions were observed at 6 and 9 hpi in all evaluated organs, being more frequent and intense in the spleen, liver and kidney. The 1 and 3 hpi groups presented no lesions in most of the organs or rare and discrete histopathological alterations, which were characterized by discrete congestion and cellular edema in some organs . Control group was used as a parameter to compare the lesions to the other groups, since it did not show significant histopathological alterations in all organs.In the heart, bacterial colonies were observed adhered to the pericardium, as well as the presence of leukocyte infiltrate surrounding these areas ; a discrThe pancreatic tissue was found in the mesentery of pyloric sacks, in a capsular region of the spleen and liver. We observed extensive areas of cellular death with disorganization of the tissue’s architecture, with the main alterations being the congestion of large vessels , hemorrhIn the kidney, larger quantities of melanomacrophages were found , as wellSinusoidal capillaries and large vessels congestion was verified in the liver , as wellInfected fish presented congestion of large vessels of gill filaments and extensive areas of epithelial cells detachment at the base of interlamellar spaces . Other fExtensive areas of cellular death were identified in all examined tissues, especially at 6 and 9 hpi, in which the findings were more severe, in comparison to groups 1 hpi, 3 hpi and control. The most common ultrastructural alterations observed at the present study were karyolysis or chromatolysis ; condensAeromonas are pathogens that have several virulence factors, thus the infection is complex and multifactorial [This study showed the kinetics of histopathological lesions in the acute stage of experimental septicemic aeromonosis in pacus, revealing that the appearance of macro and microscopic lesions initiates at 6 hours post-inoculation (hpi), with more severe results at 9 hpi. The development and severity of lesions are related to bacteria pathogenicity, seen that actorial –23.A. salmonicida infection in zebrafish, the signs related to swimming disturbances occurred only 12 hours after the experimental infection [Altered behavioral manifestations were not shown by fish throughout the clinical observation period up to and including 9 hpi, which may be related to the absence of severe injuries in its nervous system; neurological disturbances such as erratic swimming are one of the main behavioral alterations in fish affected by aeromonosis ,25. Anotnfection .Aeromonas spp. infection in cyprinids, demonstrated external macroscopic findings, such as skin darkening and hemorrhage in the body surface, fins, gills and eyes [Concerning macroscopic examinations, inoculated fish presented petechiae hemorrhage in the body, fins and operculum surfaces, gills congestion and ocular hemorrhage. Similarly, studies of and eyes ,28.Carassius carassius) infected by A. sobria.The findings regarding the necropsy performed using fish evidenced hemorrhages in serous and organs, a discrete accumulation of serosanguinolent liquid in the cavity, congestion and a slight increase of the organs and superficial vessels volume, corroborating the results described in the literature for others species of fish affected by aeromonosis . SimilarMacroscopic hemorrhagic alterations possibly occurred due to the acute inflammatory response to the infection. Leukocytes release several vasoactive substances that cause vasodilation and increased vascular permeability, leading to hemorrhages –32.A. hydrophila through the tissues supposedly occurred as a function of the motile capacity of these bacteria, which moves with the aid of flagella.Microbiological examination revealed that, even in low percentages, bacteria were detected at the first hour post-inoculation (1 hpi), reaching 100% of positive isolations at 9 hpi in all organs and blood of infected fish. This rapid dissemination of Aeromonas spp. in the initial colonization of tissues relates to the presence of polar flagella, which allows the organism to move in liquid environments, and lateral flagella, aiding in viscous surfaces, giving a greater invasive capacity for these bacteria [Tenacibaculum maritimum infection, a motile microorganism that does not have flagella and moves by sliding, Faílde et al. [Psetta maxima L.) only six hours after experimental inoculation. By comparing these data with our findings, it can be inferred that the presence of flagella may have conferred a greater precocity in the initial colonization of pacus tissues, seen that A. hydrophila was already found in the first hour after infection.One of the most important virulence factors of bacteria . In stude et al.  detectedA. hydrophila in pacus, thus facilitating the diagnosis of this pathology, even in early cases through the preventive diagnosis of outbreaks. In studies of acute experimental streptococcosis, Abdullah et al. [Streptococcus agalactiae in red tilapia (Oreochromis spp.), determining the encephalon as the target-organ of this microorganism in this fish.The upward curve of bacterial quantification in organs and blood, emphasizing the greatest values for spleen, liver and kidney in all experimental times. This fact may indicate that these organs are most susceptible to infection by h et al.  isolatedA. salmonicida subspecies salmonicida, two avirulent and one considered virulent, in organs of experimentally infected preached fish (Psetta maxima L.). The results indicated that the virulent strain was isolated in internal organs, especially liver and kidney, in the first 12 hpi, which remained present in all experimental times until death, after seven days. Conversely, avirulent strains were rarely isolated from internal organs, being eliminated four days after the challenge.Bacterial virulence factors are related to the capacity of invasion, replication and evasion of the host immune system, causing lesions during pathogenesis . Farto eA. hydrophila, when directly inoculated in the coelom of pacus, causes lesions in multiple organs. These lesions were observed at 6 hpi and were more severe at 9 hpi. As Aeromonas spp. possesses the capacity of adhering to tissue cells, the illness severity depends on the types of virulence factors involved and the immune status of the host, which leads to several degrees of injury on tissues [Histopathological findings showed that  tissues –39.Histopathological lesions occurred likely due to the liberation of bacterial toxins and other virulence factors inherent to aeromonosis. This bacterium produces endotoxins and exotoxins, such as hemolysin and aerolysin, which cause the rupture of cellular membranes ,41, enteA. salmonicida subsp. salmonicida.The present results showed that, after the bacteria inoculation in the coelom, probably, there was colonization of serosa or capsular compartment of the organs in this cavity . Following that colonization, bacteria crossed the epithelial barriers and leaded to the infection and, consequently, inducing damage to the cells inside these organs. Concomitantly, some bacteria crossed mesothelium of peritoneum vessels and reached the blood circulation and colonized even distant organs . Farto et al.  observedLactococcus garvieae in zebrafish. Chong et al. [Erysipelothrix rhusiopathiae, moderate brain and hepatic vascular congestion. Carraschi et al. [A. hydrophila, and Avci et al. [Lactococcus garvieae.No available reports of histopathological findings in the acute phase of aeromonosis were found until this time, however studies on advanced stages of septicemic diseases caused by pathogenic bacteria in fish showed similar histopathological alterations to the ones found in this study. Aguado-Urda et al.  observedg et al.  found, ii et al.  reportedi et al.  found thAeromonas spp., such as flagella, fimbria, membrane proteins and lipopolysaccharides (LPS), which allows the adherence of bacteria to the fish tissues [Bacteria that causes sepsis are frequently observed in injured areas or associated to inflammatory processes in teleosts previously challenged or naturally infected ,44,47, c tissues .Clarias macrocephalus x Clarias gariepinus) infected with Edwardsiella ictaluri presented lymphocyte infiltrate in the renal glomeruli [Puntius sarana) challenged with A. hydrophila presented increased number of melanomacrophages and congestion of central vessels in the liver [Similar to our histopathological findings in kidney and liver, other studies of bacterial septicemia in fish reported that hybrid catfish  infected with Vibrio vulnificus manifested the presence of the bacteria in a close association with necrotic cells, which presented alterations in the cell membrane and organelle damage [In this study, the observed lesions in transmission electron microscopy reinforce the histopathological observations, cellular death was verified, and organelle degeneration and a strict association between lesions and the presence of bacteria in infected organs. Likewise, eels (e damage .Streptococcus iniae inside macrophage phagosomes of gilthead seabream (Sparus aurata) and red porgy (Pagrus pagrus), the authors demonstrated by means of ultrastructural analyzes, that these cells may be important sources of bacteria dissemination in the nervous system, since bacteria may cross the blood-brain barrier without any resistance [In a study concerning the resistance of sistance .in situ hybridization, that both gills and intestines are target organs of the acute infection by herpesvirus in carps (Cyprinus carpio) after challenged by immersion. According to these authors, these organs were relevant as a gateway in the initial stage of the illness, reinforcing the importance of the determination of target organs in the understanding of infectious diseases.The organs that presented more severe microscopic lesions were liver, spleen and kidney. These organs also presented the greatest bacterial quantifications, indicating that these are the target organs of the acute stage of septicemic aeromonosis in pacu. Monaghan et al.  determinA. hydrophila in the pathophysiology of the acute stage of septicemic aeromonosis in pacu organs. Spleen, liver and kidneys were the most affected organs and are suggested as the target organs of the infection, which may assist the diagnosis of acute cases of this pathology in pacus. These findings help to improve the knowledge about the infection process model and may orientate and assist the diagnosis of acute cases of this pathology in pacus.The findings showed that this bacterium spreads in the first hpi through the pacus organs. Morphological and microbiological findings showed necrosis, degenerative processes, vascular alterations and an association between bacteria and lesions, especially at 6 and 9 hpi, demonstrating the action of the virulence factors of


Heatwave studies typically estimate heat-related mortality and morbidity risks at the city level; few have addressed the heterogeneous risks by socioeconomic status (SES) and location within a city. This study aimed to examine the impacts of heatwaves on mortality outcomes in Memphis, Tennessee, a Mid-South metropolitan area top-ranked in morbidity and poverty rates, and to investigate the effects of SES and urbanicity. Mortality data were retrieved from the death records in 2008–2017, and temperature data from the Applied Climate Information System. Heatwave days were defined based on four temperature metrics. Heatwave effects on daily total-cause, cardiovascular, and respiratory mortality were evaluated using Poisson regression, accounting for temporal trends, sociodemographic factors, urbanicity, and air pollution. We found higher cardiovascular mortality risk (cumulative RR (relative risk) = 1.25, 95% CI : 1.01–1.55) in heatwave days defined as those with maximum daily temperature >95th percentile for more than two consecutive days. The effects of heatwaves on mortality did not differ by SES, race, or urbanicity. The findings of this study provided evidence to support future heatwave planning and studies of heatwave and health impacts at a coarser geographic resolution. The overall rising temperature in the 21st century resulting from climate change is likely to be the most serious global health threat of the century. Heatwaves are natural hazards that pose significant risks to human health ,4,5,6. HDespite the well-documented association between heatwaves and mortality, fewer studies have examined how heat-related mortality risks differ by socioeconomic status (SES) and location within a city or county. Social disadvantages, such as low SES, minority, blighted communities, and social isolation, are likely to increase an individual’s vulnerability; however, previous findings were inconsistent regarding their interactions with heat-related health risks. Several studies demonstrated that heatwaves disproportionally affected mortality among black ,17,18,19Memphis, Tennessee (TN), is a Mid-South metropolitan area with high morbidity, poverty, and racial segregation rates, serving as a good testbed for investigating heatwave impacts and the sociodemographic modifiers. The city of Memphis is the regional center and seat of Shelby County, TN. Memphis has a high percentage of African Americans (67%) and a poverty rate of 30% among African Americans . The leaA few national and regional heatwave studies have briefly reported mortality risks during extreme heat events in Memphis ,8,9. HowWe focused on the warm season, i.e., May through September, in Shelby County for the latest 10-year period from 2008 to 2017. Elevated mortality risks associated with heatwaves are most likely to occur during these months in the U.S. . The tothttp://www.rcc-acis.org/). Previous studies used various definitions of heatwaves to quantify the health impacts of heatwaves since population acclimatization to heat is diverse across different regions [Meteorological data and heatwave definitions. Daily temperature measurements were used to investigate the association between heatwaves and mortality, as earlier studies generally reported the heatwave effect on health was acute and lasted only for three to four days ,26. Temp regions . This st regions , as listMortality data. The daily mortality data of 77,662 death records in 2008–2017 were based on Shelby County resident death certificate data from the Tennessee Department of Health and analyzed in collaboration with the Shelby County Health Department epidemiology team. Heatwave events and mortality are more likely associated among elderly populations, and thus we limited our analyses to the elderly population aged 65 and above. As a result, a total of 17,935 deaths were included, with 7948 (44%) being aged 65–79 and 9987 (56%) aged 80 and above. Daily counts of deaths were computed for each of the four underlying causes of deaths considered in this study.Covariates of analysis. The covariates and potential confounding and interaction variables included the year, day of the week (DOW), age (65–79 and 80+), sex, race , urbanicity of the residence, poverty level, and air pollution. We classified the residence as urban or rural following the U.S. Census Bureau’s definition of the urban area . We clasNumerous studies have reported elevated mortality resulting from exposure to air pollution ,33,34, at) is the expected mortality on day t assumed to follow an over-dispersed Poisson distribution [t = 1 if day t was a heatwave day by a heatwave definition, and 0 otherwise; lag1 = 1 if it was a heatwave day 1 day before, and 0 otherwise; lag2 = 1 if it was a heatwave day 2 days before, and 0 otherwise; lag3 = 1 if it was a heatwave day 3 days before, and 0 otherwise; Year = categorical variable for year; DOWt = categorical variable for day of the week. Model (1a) estimated the county-level RRs, which are adjusted for the overall year trend and short-term DOW effects. Model (1a) fitted with a canonical log link was equivalent to a log-linear model.The dependent variables for all the analyses were daily death counts, which might be stratified by individual demographic factors  and neighborhood characteristics (see below for model-specific dependent variables). The association between daily mortality and heatwave events was examined using Poisson regression models, as deaths are rare events. We started with the following model to estimate the relative risks (RRs) of mortality on heatwave days and 1, 2, and 3 days after the heatwave days .ribution . HWt = 1In addition, we also estimated the cumulative risks of mortality of HW days using a distributed lag nonlinear model (DLNM) for Model (1) ,39,40,41In the DLNM model, HW statuses of both current and 1–3 lag days were in the same model, represented by a bi-dimensional cross-basis transformation of both current and lag terms, fitted with a natural cubic spline function to model the possible non-linear association between HW and daily mortality. The DLNM means the current day mortality is predicted by both current and 1–3 lag days, which is equivalent to the idea that the effect of HW on mortality will be distributed over the current day and three follow-up days (forward interpretation in time). The cumulative effect will be roughly the sum of effects from the heatwave day and three subsequent days. Details of DLNM methodologies can be found in references ,42.Models (1a and 1b) were run separately for HW days, 3 lag days, and cumulative heatwave and lag days, 4 heatwave definitions, and 3 categories of mortality . RRs in these different scenarios were generated from a total of 60 models.We then added an interaction item in Model (1) to evaluate whether heatwave effects differed by sex, age group, poverty level, urbanicity, or race:t was the daily death counts for either males or females separately. If there was no death for males  on a specific day, zero was filled for that day.Model (2) was then run for 4 heatwave definitions and 3 categories of deaths, respectively, yielding a total of 60 models. In this interaction model, we included only the heatwave status of the current day. Separate lag effect interaction models were also explored but not reported in this paper due to non-significant results. The dependent variable, daily death counts, were cross-tabbed by modifiers. For example, for assessing the interaction between HW and sex, YWe also constructed the following complete model to evaluate the heatwave effects on mortality, accounting for each of the covariates and potential confounders:Model (3) was repeated for the three types of deaths; for the four HW definitions; and for heatwave days, lag1, lag2, and lag3 days. In this model, daily death counts were cross-tabbed by age group, poverty, urbanicity, race, and sex. Zeros were filled for cells with no death, resulting in a sparse dataset. Model (3) did not include relative humidity (RH) as a covariate because it was a function of temperature, and a recent global study reported that RH did not influence heat-related mortality . We conswww.r-project.org). No multiple comparison adjustment was made, as most of the comparisons are non-significant at the 0.05 level.The address geocoding was completed in ArcGIS . All the statistical analyses were performed in SAS , except that the DLNM models were performed using the package “dlnm”  in R . Only three events, all in 2010, occurred on a day that was classified as a heatwave day based on all four definitions. The only event that occurred on a day not considered a heatwave day based on any of the four heatwave definitions was also checked for possible lag effects. Even when a lag1, 2, or 3 was taken into account, none of the lag days included any heatwave classified days based on any of four definitions. Overall, direct heat-related deaths were relatively rare in Memphis during the study period.The total number of all-cause deaths of people aged 65 years and above was 17,935 in Memphis over the 10 years. The distribution of deaths across age groups, poverty levels, genders, and races was generally balanced, except that numbers of other races were small . The majThe mortality risk varied by different heatwave definitions. It was notable that the HW4 definition captured many more deaths 2350,  than theFor all-cause mortality, there were significant risks on lag3 day based on HW2  and lag2 day and lag3 day based on HW3 . Based on HW3, cardiovascular mortality increased by 20% on lag2 day  and by 20% on lag3 day , with an overall increase of 25%  during heatwave days. HW days appeared to have no effects on respiratory mortality .The results summarized in Moreover, Model (3) revealed the effects of the time, SES, and urbanicity. For the two temporal variables, the year had significant effects on mortality, indicating the mortality rate changed over the years, but DOW did not show significant effects on mortality. Poverty had significant effects on all-cause mortality , but notWe found that there was no interaction between heatwave and sociodemographic variables in predicting daily mortality. As summarized in Excess mortality during heatwave events did not appear to be a significant public health issue in Memphis. The 1980 single heatwave event resulted in 83 direct heat-related deaths due to heat exhaustion, heatstroke, or systemic hyperthermia . In contLocal heat-health early warning systems should be based on identified local heatwave definitions that are optimal for protecting public health ,44. In tp = 0.007 for lag2 and 0.018 for lag3), as shown in We did not find significant impacts of heatwaves on the overall mortality in Memphis during the study period. An earlier U.S. national study on heat-related mortality risks reported lower risks in the South than those in the Northeast or Midwest and no mortality increase during heatwaves in some southern cities, such as Charlotte, NC, which has a similar warm season climate pattern to Memphis . Our finMoreover, our individual-level analysis did not find heterogeneity in heat-mortality risks among populations in different races, SES, or residential locations across the county. Notably, income and neighborhood urbanicity levels did not appear to be an effect modifier that exacerbates vulnerability to heatwaves in Shelby County. Studies have documented considerable variation in SES, racial distributions, land use types, and health outcomes by census tract in Memphis ,48,49, ePrevious studies reported positive associations between short-term changes in ozone and mortality ,50 and iWe acknowledge several limitations of this study. First, the temperature data were obtained from only one single weather station, which might not account for the within-city variations in temperature, especially the differences between urban and rural areas. The urban center may have higher surface temperatures due to the “urban heat island” effect. Further analysis may use high-resolution temperature data to capture the variation in heat exposure. Second, the mortality rate calculations were based on 2010, assuming a stable population size over the entire study period. Nevertheless, this approach was legitimate, given the stable population size in Shelby County. Third, for heatwave definitions 1, 2, and 3, we had small numbers of heatwave days over the study period. The small daily mortality numbers might lead to reduced statistical power when exploring various interactions between heatwave and SES. Fourth, due to the unavailability of individual income data, poverty at the census tract level was used to represent an individual’s income level, which may not capture the variability among individuals. Fifth, we have small death counts for some disease-specific deaths  of cardiovascular mortality based on the HW3 definition, i.e., maximum daily temperature >95th percentile for more than two consecutive days. We did not find significant interaction effects between heatwave effects and sociodemographic parameters  or air pollution. Our findings suggested that the cardiovascular mortality risk due to heatwave events remained nontrivial in the South region of the U.S., despite the high availability of air conditioning and population acclimatization, contributing to the relatively lower vulnerability to heat-related mortality. Future research is needed to extend the analysis to a longer period, validate the generalizability of our findings in other large urban areas, and improve heat exposure assessment at the community and individual levels.


Pyoderma Gangrenosum (PG) is a rare neutrophilic dermatosis with multiple different clinical presentations and associated comorbidities. PG has historically been a challenging disorder to diagnose, leading to the development of new diagnostic criteria rather than the traditional approach of a diagnosis of exclusion. The pathophysiology is thought to involve both innate and adaptive immune system dysregulation, neutrophilic abnormalities, environmental, and genetic factors. As of today, no gold standard therapy exists for the treatment of PG, and the literature is restricted to mainly case reports, case series, and 2 small randomized clinical trials. Topical, systemic, and biologic therapy, as well as adequate analgesia and proper wound care all play a role in the management of PG. Recent studies have identified additional cytokines and signalling cascades thought to be involved in the pathogenesis of PG, ultimately leading to the development of new targeted therapies. This review will focus on recent advances in the pathophysiology, clinical presentation and associated comorbidities, diagnosis, and management of PG. However, given the lack of gold standard for diagnosis, the exact prevalence has yet to be elucidated since PG is commonly under- and over-diagnosed. PG is classified as a neutrophilic dermatosis because of a predominant neutrophilic inflammatory infiltrate in the lesions3. The pathophysiology of PG is not completely understood and is thought to be multifactorial, involving both the innate and adaptive immune system and having a genetic influence5.Pyoderma gangrenosum (PG) is a rare auto-inflammatory ulcerative dermatosis with an overall incidence of 5.8 per 100,000 individuals and an increased mortality rate when compared with the general population4.There is no widely accepted “gold standard” treatment in the management of PG. Proper analgesia, wound care, and compression therapy are all important tenets in the management of PG. Both topical and systemic therapy can be used, and choice of therapy is dependent on numerous factors, including the number of lesions, size, location, comorbid systemic disease, side effects of the medications, and patient preferencesThis review will summarize new advances in the pathophysiology, diagnosis, and management of PG in order to assist physicians in better understanding and managing PG and ultimately improving patient care.4.The pathophysiology of PG is poorly understood and is thought to involve adaptive and innate immune system dysregulation, neutrophilic abnormalities , abnormal phagocytosis, and genetics6 and multiple novel therapeutic targets have been explored. These are described in more detail below , rheumatoid arthritis (RA), seronegative arthritis, hematologic disorders, and malignancies such as acute myeloid leukemia (AML)7. Interleukin-6 (IL-6), a pro-inflammatory cytokine that plays a role in activation and accumulation of neutrophils, has been found to be elevated in PG lesions8. Interestingly, some studies have shown PG lesions to be related to defects in adhesion and function of neutrophils, suggesting a multifactorial pathogenesis7.The abundance of neutrophils in the histopathology of PG has been reported in typical untreated lesions. Histologic analysis of PG lesions demonstrates dermal edema, neutrophilic abscesses, and suppurative inflammation in the dermis that can reach the underlying subcutaneous fat4. For example, PAPA  syndrome presents as sterile arthritis in childhood, severe cystic acne, pathergy, and recurrent ulcerations. The underlying mutation in PAPA syndrome leads to uncontrolled production of IL-1, thus leading to auto-inflammation9.Genetics play a role in the pathogenesis of PG, best exemplified by the PG-associated genetic syndromes. The specific mutations that give rise to PG-associated genetic syndromes are all associated with a pro-inflammatory state. Such syndromes and their associated gene mutations are listed in6, and vitamin B12. There are cases of PG associated with this mutation in the literature, improved with vitamin B treatment9.Methylene tetrahydrofolate reductase (MTHFR) is an enzyme that assists in the conversion of homocysteine to methionine. Mutations lead to increased levels of homocysteine, resulting in a pro-inflammatory state. Enzymatic co-factors include folic acid, vitamin B12. Other case reports describe the development of PG in family members after abdominal surgery and trauma13. In addition, there are familial cases of PG-associated genetic syndromes in the literature14. These case reports support the role of genetics in the pathogenesis of PG.There are some familial cases of PG in the literature. Three case reports in the pediatric population describe familial PG in both the setting and absence of systemic disease16. In comparisons of lesional skin of individuals with classic ulcerative PG and PG, acne, and suppurative hidradenitis (PASH) syndrome, both showed overexpression of IL-1β, tumor necrosis factor alpha (TNFα), IL-17, endothelial- and leukocyte-selectin, and chemokines IL-8, CXCL16, and RANTES18. Matrix metalloproteinases, a group of enzymes involved in tissue damage, were overexpressed as well7. Biopsies of pre-lesional PG  showed CD3+ infiltrates and increased inflammatory cytokines, whereas PG lesions showed significant overexpression of IL-1α, IL-1β, IL-6, IL-8, and IL-36α20.Current research is continuing to identify new—and confirm already-known—cytokines and signaling cascades involved in the pathogenesis of PG. The innate immune system signaling pathways, pattern recognition receptor (PRR) pathways (which are associated with autoimmune diseases such as IBD and RA), and Janus kinase (JAK) 1–3 and signal transducer and activator of transcription (STAT) pathways were upregulated in lesional skin compared with non-lesional skin in patients with PG7. The ratio between T regulatory cells and T helper 17 (Th-17) cells was found to be reduced in PG lesions but not in sweet syndrome (which is associated with a milder skin inflammation). T regulatory cells are involved in preventing auto-inflammation and therefore an impairment in the level of these cells may allow the uncontrolled activation of other pro-inflammatory cells and cytokines, such as Th-17 and IL-1721. In addition, T-cell clonal expansions have been seen in patients with PG early in the disease course6. Histologically, the “cigarette paper” appearance of PG scars was not consistent with a thinning of the dermis, but rather significantly fewer T helper cells and fibroblasts20.The adaptive immune system is also thought to play a role in PG given that medications interfering with T-cell function and promoting apoptosis improve symptoms5. Pathergy has been suggested to be caused by increased activity of polymorphonuclear cells because of the presence of neutrophils in pathergic lesions of Behçet disease7. However, further research is needed to confirm this theory in PG.The relationship between external triggers  and genetic factors is poorly understoodet al.20 (2017) hypothesized that abnormal cytokine expression and an adaptive immune response targeted at pilosebaceous units are responsible for the development of PG. In contrast with psoriasis and venous stasis, ulcerations in PG never occurred at sites of previous ulcers; biopsies of healed and non-lesional skin resulted in ulceration (pathergy) only in the latter. Wanget al. hypothesize that this is due to the loss of an autoantigen target, specifically follicular adnexal structures. To further this theory, PG ulcerations  do not occur in areas lacking follicular adnexal structures, such as the nipple-areolar complex, palmar surface of the hand, and plantar surface of the foot20. Again, future studies are required to test this hypothesis.Wang6. Given the association between PG and IBD, RA, and hematologic conditions, other extra-cutaneous manifestations may also be present. This includes ocular (scleritis and ulceration), pulmonary (aseptic nodules), splenic, and musculoskeletal (sterile polyarthritis and neutrophilic myositis) involvement4.There are multiple subtypes of PG, including ulcerative, bullous, pustular, vegetative, and peristomal and post-surgical PG, the latter two being the most commonly pathergic variants. Ulcerative (classic) PG is the most common subtype and usually is seen on the legs. Subtypes can vary in aggression and are associated with different systemic diseases . The maj24. PG was often diagnosed as a wound infection at first, leading to debridement and ultimately worsening the lesion because of pathergy. Earlier diagnosis and recognition may help prevent morbidity and lessen health-care costs22.The average time between surgery and the first symptoms of postoperative PG was 11 days. More invasive procedures and patients with chronic disease have a higher risk of developing this subtype. In one retrospective analysis, about 15% of individuals had post-surgical recurrence or exacerbation of existing lesionset al., who found that the majority of individuals with PG in the literature were middle-aged white women with an average age at presentation of 51.6 years25. Ulcers were most common on the lower extremities but may also be seen surrounding stomas, in individuals with IBD , on the trunk, upper extremities, head or neck, or other locations26. Pathergy was seen in about one third of individuals, was more common over the age of 65, and may be underestimated (if no trauma occurred)27.In 2018, a large retrospective review was completed by Ashchyan25. In individuals with IBD and concomitant PG, IBD is diagnosed at a younger age, and they are more likely to have a family history of UC, be of black African origin, have a stoma, and be on immunosuppressive medications. The mean time between diagnosis of IBD and PG was about 6 years, and PG occasionally preceded the diagnosis of IBD28.Two thirds of individuals have an associated comorbidity and IBD is most common (occurring in 0.5% of patients with IBD), especially in individuals under 6527. Hematologic disorders are more common in individuals over 65 years of age25. Three phenotypes of arthritis are seen in association with PG: RA, bowel-associated arthropathy, and progressive erosive seronegative arthritis. PG (bullous PG in particular) can be the initial presentation of leukemia (most commonly AML) and is a poor prognostic sign9. One study found that the mortality rate in patients with leukemia and PG was higher than in patients with non-leukemia hematologic malignancies, but further studies are needed to investigate this relationship29. The paraneoplastic phenomenon can also occur with atypical presentations of PG  and is seen more frequently in patients with underlying hematologic disorders6.Other associated comorbidities include arthritis, malignancy (acute and chronic myeloid leukemia [AML and CML] and non-Hodgkin lymphoma), hematologic disorders , arthritis, hidradenitis suppurativa, and depression30.Necrotizing neutrophilic dermatosis (NND)—specifically, necrotizing PG (NPG)—is a more severe form of PG seen in critically ill patients with a previous diagnosis of PG. NPG is most commonly associated with hematologic disorders and malignant neoplasms. These patients often mimic necrotizing fasciitis, sepsis, or septic shock given their level of systemic inflammation. Common features of NND include erythematous and necrotizing violaceous margins, ulcerations, pathergy, elevated inflammatory markers, and neutrophil invasion. Tissue biopsy and cultures are critical in distinguishing NPG from necrotizing fasciitis, as NPG does not respond to antibiotics but rather immunosuppressive therapy 31. PG therefore has a high misdiagnosis rate: in a study by Weeniget al., PG was initially diagnosed and treated in 67% of patients before an alternative diagnosis was made32.Historically, the diagnosis of PG has been challenging given its numerous presentations, clinical similarities with other dermatoses (and other neutrophilic dermatoses), and various associated systemic diseaseset al.25 have proposed an age-focused initial evaluation for PG: they use a different approach depending on whether the patient is over or under 65 years old. In their study, diagnosis was based on the criteria proposed by Suet al.32 (described in the paragraph and table below),which are centered on PG being a diagnosis of exclusion33. For patients under 65, the history and physical exam should evaluate for the presence of IBD and the threshold for referral to gastroenterology is lower. For patients over 65, physicians should evaluate for hematologic disorders and malignancy with a possible work-up, including a blood smear, monoclonal gammopathy evaluation, and a lower threshold for referral to hematology for bone marrow studies. They suggest that, regardless of age, all patients receive a skin biopsy with tissue culture, age-appropriate malignancy screening, and complete blood count with differential. Depending on the history and physical, an inflammatory arthritis evaluation, autoimmune work-up, and vasculitis screen may be conducted25.Given the differences in presentation depending on age, Ashchyanet al.34 (2004). The diagnostic criteria, which are based on PG’s being a diagnosis of exclusion, are listed in34.Numerous articles in the literature used the proposed diagnostic criteria for classic ulcerative PG by Su31. Consensus was not reached regarding the inclusion of hematologic disease as a minor criterion (unlike IBD and inflammatory arthritis). Given the prevalence of hematologic disease in PG, future iterations should consider its addition as a minor criterion. In addition, the use of biopsy as a major criterion may not reflect actual clinical practice33. Interestingly, one study found that only 12% of individuals had histological evidence of a neutrophilic infiltrate and that less than 10% had a biopsy consistent with PG27.Given the difficulty in diagnosing PG and excluding other diagnoses, 12 physicians collaborated to create a diagnostic criterion for classic ulcerative PG. The Delphi Consensus of International Experts diagnostic criteria are composed of one major and eight minor criteria . In ordeet al.35 developed the PARACELSUS score as a separate diagnostic tool for PG. This score was developed on the basis of a review of the literature. The criteria are listed in35. All three criteria are compared inAround the same time as the development of the Delphi criteria above, Jockenhöfer4.PG is a challenging condition to manage, and treatment focuses on reducing systemic inflammation. There is a lack of large randomized controlled trials (RCTs) in the literature and therefore the majority of treatment decisions are based on expert opinion, case reports and case series, and small cohort studies. Treatment decisions are personalized to reflect the location, number, and size of lesion(s), extra-cutaneous involvement, underlying systemic disease, side effect profiles, cost, and patient preference2)37. One review found that topical tacrolimus 0.3% promoted lesion resolution in mild and localized PG37. Intralesional steroids, applied to the active border of the lesion surrounding the ulcerated area, can also be used in small and localized PG4. However, there is little information on which steroid class to use, the frequency of application, the dosage of tacrolimus to use, and the best dressing to apply afterwards36. Other topical therapies include sodium cromoglycate, nicotine, dapsone, and 5-aminosalicylic acid (5-ASA)4.The best evidence in the literature regarding topical therapy is for corticosteroids and tacrolimus. A case series conducted in 2011 found topical therapy to be used most often in peristomal PG, smaller lesions, and localized PG , and ulcer size was an important predictive factor in lesion resolution. A prospective cohort study found that less than 50% of individuals had healed (no longer required dressings) with topical therapy alone in a 6-month period and that one third of individuals required systemic therapy6.Monotherapy can be used in patients with mild PG. Examples of typical monotherapy agents include steroids, tacrolimus, topical sodium cromoglycate, nicotine, 5-ASA, intralesional triamcinolone, and intralesional cyclosporine. Current research is targeted at the development of new biologic agents that target different inflammatory cytokines and signaling pathways39. An RCT comparing oral cyclosporin (4 mg/kg per day) with prednisolone (0.75 mg/kg per day) found no difference between these medications in lesion-healing speed (in a 6-week period), treatment response, resolution of wounds, pain, quality of life, treatment failure, and recurrence. Overall, about half of all PG ulcers had healed by 6 months. Therefore, treatment is guided by the side effect profiles of these two medications 39.The most commonly used first-line treatment in the management of PG is systemic steroids4. The addition of topical or systemic antibiotics or anti-neutrophilic agents (dapsone and colchicine) has traditionally been based on the provider’s preference. The benefits of using anti-neutrophilic agents are for both their anti-inflammatory effects and prophylaxis againstPneumocystis jiroveci5.Other systemic therapies  have been used in the literature, but more data are needed to evaluate their efficacy in treating PG27. There are few studies comparing different combination therapies in the literature. In two observational studies,100% of in-patients achieved either partial or complete healing when given combination therapy with systemic steroids and another immunomodulator 41. Currently, the use of well-studied combinations of immunomodulators  is also recommended in PG6.Combination therapy is often used in the treatment of PG4. Steroids appear to be less efficacious in treating PG with comorbid IBD when compared with biologics42. However, the use of biologics is not without potential harm. Rare adverse effects of biologics include lymphoma, congestive heart failure, multiple sclerosis, peripheral neuropathy, and anti-DNA antibody formation. Reactivation of tuberculosis has been seen in the use of anti-TNFα therapy6. Patients should be made aware of the risks of therapy before beginning treatment.Multiple different biologics have been proposed for the treatment of PG. Agents targeting TNFα are the best studied, given their ability to treat coexisting IBD (other than etanercept)Tumor necrosis factor antagonists. No TNFα antagonist has been proven to be more efficacious than others in the treatment of PG. Their use has been associated with a decrease in C-reactive protein (CRP), IL-1, IL-6, and immune cell adhesion markers8.4. Given this RCT and its rapid onset of effect, infliximab is often preferred in a clinical setting. Thirty patients were randomly assigned to receive either an infusion of infliximab (5 mg/kg) or placebo at week 0 and were reassessed 2 weeks later. If there was no improvement by week 2, everyone was offered open-labelled infliximab at the same dose; 46% of individuals showed clinical improvement with infliximab (compared with 6% with placebo) by 2 weeks, and 69% had improved by week 6 (21% complete resolution). Individuals with lesions of less than 12 weeks’ duration had a higher improvement/remission rate than those with a longer duration43.Infliximab, the only biologic to have an associated RCT, functions by restoring the ability of T regulatory cells to inhibit aberrant cytokine production8. However, the sample size was small and evidence is limited4.Adalimumab is a humanized IgG1 monoclonal antibody with activity against TNFα. The literature surrounding adalimumab is composed of case reports and small case series; in some of these, it was added to or replaced current therapy because of treatment failure. The majority of the literature showed either complete resolution or partial improvement8. However, etanercept is less efficacious than other TNF antagonists in the treatment of coexisting IBD6.Etanercept functions as a decoy receptor for TNFα and has activity against TNFβ. Data are limited to case reports and small case series, the majority of which showed clinical improvement or complete resolution44.Golimumab, a newer TNFα inhibitor, led to complete ulcer resolution in 24 weeks in a patient who had failed infliximab and adalimumab. Another novel TNFα inhibitor is certolizumab pegol. Future studies are needed to further evaluate the use of Golimumab and Certrolizumab Pegol PGIL-12 and IL-23 antagonists. Ustekinumab blocks the common p40 subunit of IL-12 and IL-23. These two cytokines are important in neutrophil recruitment through their interaction with Th1 and Th17 cells, respectively. Case reports in the literature demonstrate either partial or complete resolution of PG lesions with ustekinumab; however, more studies are needed to confirm efficacy8.8.Tildrakizumab and guselkumab are IL-23 antagonists without simultaneous IL-12 antagonism. Future research is needed to assess their efficacyIL-1 antagonists. As mentioned above, some PG-associated genetic syndromes are associated with a mutation in thePSTPIP1 gene, leading to increased IL-1 production. IL-1 inhibitors, therefore, have the potential of blocking the downstream effects of this mutation, but the evidence is still limited8.8. In comparison with other biologics, anakinra may be less effective in its management of PG4.Anakinra is a competitive inhibitor of IL-1 (both subtypes) with a short half-life (4 to 6 hours). Although the majority of case reports demonstrated partial or complete resolution of ulcers, large daily doses were needed8. Five patients with PG (without systemic disease) who had all failed steroids were given canakinumab. Four of the five individuals had clinical improvement in 16 weeks, and three individuals had complete resolution of their lesions in this time period. However, one patient in this study had new-onset rapidly progressive genital ulcers, likely representing PG at a different location19.Canakinumab is a monoclonal antibody targeted against IL-1β with a longer half-life (about 1 month)8.Gevokizumab, another monoclonal antibody targeting IL-1β, showed promise in the treatment of PG; however, the rights to this drug were sold in 2016IL-6 antagonists. Tocilizumab has been successful in treating PG in a patient with RA and interstitial lung disease (ILD), as ILD is a contraindication to TNFα inhibitors5.JAK/STAT inhibitors. Tofacitinib is an oral JAK 1 and 3 inhibitor that is currently approved for use in RA and ulcerative colitis. Three patients with treatment-resistant PG and both comorbid Crohn’s disease and inflammatory arthritis were given tofacitinib, leading to complete resolution in two patients and symptom improvement in the third by 12 weeks45.46.Ruxolitinib, a JAK-2 inhibitor, was used in a case report of a 64-year-old female with polycythemia vera and bilateral lower-leg PG who had failed multiple immunosuppressive regimens, intravenous immunoglobulin (IVIG), and anakinra. Dramatic healing of the PG lesions was seen within 10 weeks, and complete healing of her lesions was seen after 4 yearsIntravenous immunoglobulin therapy. IVIG has been used as an adjunctive strategy for treatment-refractive PG, most commonly as a combination therapy with systemic steroids. One study found that patients with solitary PG lesions are more responsive to IVIG than those with two or more lesions whereas factors such as ulcer location had no meaningful significance47. The majority of patients showed clinical improvement, and 53% of these individuals had complete resolution. The most common side effects were headache and nausea. However, patients in this study were older and more likely to have comorbidities than other patients in the literature48. The anti-inflammatory activity of IVIG is likely the reason for lesion resolution49.Phosphodiesterase 4 inhibitors. Phosphodiesterase 4 (PDE4) is an enzyme produced by immune cells, and inhibition helps modulate different pro-inflammatory signaling cascades. Apremilast, an oral PDE4 inhibitor, inhibits multiple cytokines involved in these signaling cascades8. In the literature, there is one case report of resistant vegetative PG with underlying IgA MGUS. Complete healing of one lesion and partial healing of another were seen when apremilast was used concomitantly with oral prednisone50. Further studies are needed to better characterize this treatment.4.Treatment of PG is multifaceted and is not limited to pharmacotherapy. Other aspects of treatment include lifestyle modification , the avoidance of triggers and trauma , wound care, analgesia, prevention of superimposed infection, and compression therapy (to minimize edema)5. However, about 30% of individuals have undergone debridement by a wound care specialist because of diagnostic uncertainty26. Other wound management modalities include negative pressure wound therapy and hyperbaric oxygen, both of which show promise5.Gentle cleaning of the wound, proper use of topical antimicrobial agents  if indicated, a moist wound environment, and control of edema are essential in the management of PG. Multiple dressings have been used in the literature for the treatment of PG. Dressing choice depends on the ulcer’s characteristics . Sharp debridement should be avoided in PG given the high rate of pathergy but may be needed depending on the amount of non-viable tissue51.Compression therapy is used as an adjunct to immunosuppression in the management of PG as it is paramount in reducing any associated edema and promoting wound healing. One report commented on the importance of multimodal therapy and specifically compression therapy, given the lack of wound healing likely due to prednisone-induced edema5. Opioids may be associated with decreased healing in venous ulcers (in addition to their other adverse effects) and therefore alternative strategies for analgesia should be considered first52.Pain control is an important tenet in the management of PG and often involves the use of non-steroidal anti-inflammatory drugs and opioids. Neuropathic medications can also be used if nerve damage is present27.Given the association between PG and underlying systemic disease, referrals to appropriate subspecialties  are required. In addition, mental health support is warranted given the association between PG and major depressive disorderPG is a rare neutrophilic dermatosis with a complex pathophysiology and difficult diagnosis. It is important to note that owing to the rarity of this condition, there are very few RCTs. The majority of other publications are cohort studies, case reports, and case studies, many of which have a small sample size. However, current research has been promising and continues to provide new potential targets for therapy. Recently, multiple diagnostic criteria have been proposed to improve the accuracy of diagnosis. Although the pathophysiology of PG is still incompletely understood, the discovery of new inflammatory cytokines and signal cascades has led to the development of novel biologic therapy. Long-term data on the use of immunosuppressive medications, biologics, and small-molecule therapy are lacking. Moreover, it is difficult to assess long-term efficacy, adverse events, and remission rates as outcome measures for PG are not widely available. Additional studies are needed to better characterize these medications and subsequently to compare available treatments.Written informed consent was obtained from the patient in


P = .038) in the study group as compared with the control group. The rates of parathyroid gland preservation in situ were significantly improved in the left superior (P = .001), left inferior (P = .002), and right inferior glands (P = .005). Furthermore, the incidence of transient hypoparathyroidism decreased significantly (P = .028). Our study indicated that normal parathyroid glands were identified by ultrasonography, and the anatomical localization improved the rate of parathyroid gland preservation in situ and decreased the incidence of transient hypoparathyroidism.Postoperative hypoparathyroidism due to dysfunction of the parathyroid gland is the most common complication after thyroidectomy. Our objective was to introduce the method of anatomical localization of normal parathyroid glands before thyroidectomy through ultrasonography and to evaluate its efficiency. The study group included 52 patients subjected to anatomical localization of the parathyroid gland prethyroidectomy through ultrasonography. The control group included 52 sex- and age-matched patients without parathyroid gland localization. The proportion of parathyroid glands preserved in situ and postoperative hypoparathyroidism rates in the 2 groups were compared. The rates of normal parathyroid glands identified according to ultrasonography for left superior, left inferior, right superior, and right inferior glands were 78.8%, 90.4%, 57.7%, and 82.7%, respectively. The rate of parathyroid gland excised inadvertently was significantly decreased ( The most common reasons for hypoparathyroidism include mechanical damage, devascularization, or inadvertent removal of the parathyroid gland.,7 A recent study by Lorente-Poch et al demonstrated that the more numbers of parathyroid glands preserved in situ, the less incidence of transient and permanent hypocalcemia. The study suggested that the parathyroid glands are identified and preserved in situ, and autotransplantation should be limited to those accidentally devascularized glands that cannot be preserved. To date, various studies have focused on the early detection of hypocalcemia or replacement with calcium supplementation. However, an exhaustive literature search retrieved few formal data regarding methods for in situ preservation of parathyroid gland, thereby reducing the incidence of hypoparathyroidism.Postoperative hypoparathyroidism is the most common complication of thyroidectomy and leads to prolonged hospitalization and medical care.The present study hypothesized that identifying the anatomical position of the normal parathyroid gland and the correlation between the thyroid lobe, common carotid artery (innominate artery), trachea, esophagus, and thyroid vein before the operation could facilitate the detection, identification, and the in situ preservation of parathyroid gland during thyroidectomy, thereby decreasing the incidence of hypoparathyroidism. Thus, this study aimed to introduce the method of anatomical localization of normal parathyroid glands before thyroidectomy through ultrasonography and to evaluate its effects on hypoparathyroidism in patients, who underwent thyroidectomy and central neck dissection (CND).22.1Patients with thyroid carcinoma or nodular goiter, who underwent thyroidectomy or CND between May 1, 2017 and July 31, 2018, were reviewed in a retrospective study. The exclusion criteria were as follows: previous thyroidectomy or parathyroidectomy, preoperative hypocalcaemia, surgical extent less than thyroid lobectomy, and osteoporosis requiring treatment with calcium or vitamin D.The identification of the anatomical position of normal parathyroid glands by ultrasonography before thyroidectomy was first introduced by Jinduo Shou in May 2017, and the patients who were subjected to this procedure were enrolled in the present study. The cohort consisted of 52 patients (47 women and 5 men) with mean age 46.8 years . This study group was compared to a control group of sex- and age-matched patients who did not undergo ultrasonography of the parathyroid gland between May 1, 2017 and July 31, 2018 .The present study protocol was approved by the Ethics Committee of Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University.2.2We used EPIQ 5 iU22  or LOGIC E8  machines equipped with L523 (5–15 MHz) linear transducer for ultrasonography examination of normal parathyroid glands before thyroidectomy. The conventional gray-scale and power-Doppler ultrasounds were used to search and identify the parathyroid gland during ultrasonography. All examinations were performed by the same investigator with 10 years of experience in thyroid ultrasound. The patients were placed in a supine position with the neck slightly extended by a roll beneath the shoulders.The thyroid lobe was selected as the reference, and the longitudinal positions of the parathyroid gland were divided into cranial to the upper thyroid lobe, dorsal to the upper third thyroid lobe, dorsal to the middle third thyroid lobe, dorsal to the lower third thyroid lobe, and caudal to the lower thyroid lobe Fig. . Next, t2.3All the procedures involved were conventional open surgeries and performed by 1 senior surgeon or 2 surgical fellows under his direct supervision. In the study group, the surgeon established the anatomical position of the parathyroid glands and the correlations between the thyroid lobe, common carotid artery (innominate artery), trachea, esophagus, and thyroid vein as provided by ultrasonography preoperatively. In the control group, the surgeon lacked any information about the position of the parathyroid glands before surgery. Bilateral CND involved the removal of prelaryngeal, pretracheal, and both the right and left paratracheal nodal basins, whereas the unilateral CND involved the removal of the prelaryngeal, pretracheal, and one paratracheal nodal basin. During thyroidectomy and CND, the parathyroid gland was searched and preserved. After thyroidectomy and CND, the parathyroid gland was identified, and the blood supply was determined by the fine-needle pricking test. A gland with oozing blood after the test was considered to be vascularized, whereas otherwise devascularized. Moreover, any devascularized parathyroid gland was removed and autotransplanted into the sternocleidomastoid muscle.Thyroidectomy was performed using a standard technique of meticulous capsular dissection. The recurrent laryngeal nerves and all parathyroid glands were routinely identified and preserved under direct vision. The CND was conducted strictly according to the American Thyroid Association (ATA) guidelines.2.4The data were collected on parameters including sex, age, Hashimoto's thyroiditis, body mass index (BMI), voice change, maximum tumor size, extrathyroidal extension, multifocality, number of harvested and metastatic lymph nodes, number of parathyroid glands identified during thyroidectomy, and number of parathyroid glands preserved in situ, autotransplanted, and inadvertently excised. The number of parathyroid glands excised inadvertently was examined based on the paraffin-embedded specimens of thyroid lobe and the fibrofatty tissue of the central compartment.2.5,10 Permanent hypoparathyroidism was defined as a subnormal serum iPTH level, calcium level <2 mmol/L, or requirement of calcium and/or vitamin D supplement to treat hypocalcaemia-related symptoms for >6 months. The calcium supplements containing 750 mg calcium carbonate plus 60 units vitamin D3 were administered 2 times daily to treat the severe symptomatic hypocalcaemia.Transient hypoparathyroidism was defined as a subnormal level of serum intact parathyroid hormone (iPTH) , serum calcium level <2 mmol/L (8 mg/dL), or requirement of calcium supplement to treat the clinical symptoms of hypocalcaemia, such as distal digital paraesthesia or tetany during the hospital stay.2.6t test or Mann–Whitney U test for continuous variables and Pearson χ2 test for categorical variables. P<.05 was considered statistically significant. Data were analyzed using SPSS version 16.0 .Continuous data are presented as mean (SD) or median (range). The baseline patient characteristics, the incidence of hypoparathyroidism, the proportion of parathyroid glands identified, and the in situ preservations were compared between the study and control groups using the Student 33.1A total of 104 patients were eligible for the present study. The male:female ratio was 11:93 (1: 8.5), and the mean age was 46.8 years. The study group consisted of 52 patients, including 47 patients with thyroid carcinoma (46 cases of papillary thyroid carcinoma [PTC] and 1 case medullary thyroid carcinoma) and 5 patients with nodular goiter, and the control group consisted of 52 patients, including 48 patients with PTC and 4 patients with nodular goiter. Parameters such as age, sex, the prevalence of Hashimoto's thyroiditis, BMI, tumor size, rates of multifocal malignant disease and extrathyroidal extension, and the number of harvested and metastatic lymph nodes did not differ significantly between the study and control groups  on the left side and in 30 patients (57.7%) on the right side, respectively. In addition, the inferior parathyroid gland was identified in 47 patients (90.4%) on the left side and in 43 patients (82.7%) on the right side, respectively (Table 3.3P = .004) (Table P = .038)  Table . MoreoveP = .001; inferior parathyroid gland on the left side: 54.1% vs 16.7%, P = .002; inferior parathyroid gland on the right side: 61.3% vs 28.2%, P = .005) . However, permanent hypoparathyroidism was not observed in either of the groups.In the study group, 6/52 patients (11.5%) presented transient hypoparathyroidism as compared to 15/52 patients (28.8%) in the control group  undergoes dissection 2 times. It is first exposed and preserved by a meticulous capsular dissection during thyroid lobectomy, followed by identification and preservation in situ, whereas the central neck fibrofatty tissue is removed along with the lymph nodes. Failure to identify the parathyroid gland during thyroid lobectomy makes the in situ preservation in CND difficult owing to the variable positions of the glands in the adult neck The ultrasonography in the present study displayed the normal parathyroid glands and the positional correlations between the thyroid lobe, common carotid artery (innominate artery), trachea, and esophagus. The rates of normal parathyroid glands displayed on ultrasonography for left superior, left inferior, right superior, and right inferior glands were 78.8%, 90.4%, 57.7%, and 82.7%%, respectively.Herein, we established the method of anatomical localization of normal parathyroid glands through ultrasonography. The findings showed that anatomical localization of parathyroid gland before thyroidectomy increased the rate of identification and preservation in situ during thyroidectomy, and decreased the incidence of parathyroid gland excised inadvertently as well as the prevalence of transient hypoparathyroidism.Furthermore, the ultrasonography examination of the parathyroid gland before thyroidectomy revealed the longitudinal position of the parathyroid gland in reference to the thyroid gland lobe, the transverse position of the gland in reference to the common carotid artery and the trachea, and the located layer of the gland in reference to the thyroid lobe, esophagus, and common carotid artery. Strikingly, the insight into the three-dimensional position of the parathyroid glands would assist the surgeons in detecting and identifying the glands during thyroid surgery. classified the parathyroid gland into type A (close contact) and type B (nonclose contact). Type A included A1 (planar attachment), A2 (embedded attachment), and A3 (intrathyroid), whereas type B comprised B1 (around the thyroid), B2 (intrathymus), and B3 (blood supply from thymus or mediastinum). The ultrasonography examination of the parathyroid gland revealed the type of the gland before surgery. In this study, we found that the proportion of type A according to ultrasonography was 85.4% for the left superior gland, 59.6% for the left inferior gland, 83.3% for the right superior gland, and 69.8% for the right inferior gland, respectively  to preserve the parathyroid gland closely attached to the thyroid gland. In the case of the parathyroid gland of type B, surgeons should focus on CND in order to preserve the gland in situ.–15 Paek et al reported that the parathyroid gland excised inadvertently was a major risk factor for both postoperative transient and permanent hypoparathyroidism. The study demonstrated that the frequency of such removal of the parathyroid gland was 29%. Recently, Applewhite et al found that patients with incidental parathyroidectomy exhibited postoperative biochemical and symptomatic hypocalcemia than the controls, and the number of parathyroid glands identified intraoperatively was inversely correlated with the number of parathyroid glands in the specimen. In the present study, we observed that the anatomical localization of normal parathyroid glands before operation significantly increased the rate of parathyroid glands identified intraoperatively, thereby decreasing the rate of parathyroid gland excised inadvertently .Despite careful dissection and familiarity with anatomy, the parathyroid tissue is frequently removed from the neck inadvertently during thyroid operations. Reportedly, the incidental parathyroidectomy occurs in 5.2% to 21.6% of the thyroid operations. So, it is hard to confirm whether the low incidence of hypoparathyroidism was related to the application of microsurgical technique and loupes or just own to the author's excellent surgical experience.In 2016, Dorazi et al reported the incidences of transient and permanent hypoparathyroidism were 10.7% (84 of 782 patients) and 0.38% (3 of 782 patients), respectively, with the use of loupes magnification and microsurgical technique in thyroid surgery. Dorazi et al suggested the use of loupes magnification and microsurgical technique as a common tool to visualize and preserve the parathyroid glands in thyroid surgery. However, Dorazi's study did not have a control group of patients undergoing standard thyroid surgery.m)-sestaMIBI is used as radiotracer for intraoperative localization of parathyroid adenomas and the injection is usually performed 2 to 3 hours before surgery because Tc-99m-sestaMIBI is washed out more quickly from the normal thyroid and parathyroid tissue than from the parathyroid adenomas and hyperplastic glands.,19 Recently, Pasta et al further developed this technology through inhibit the interference of uptake of the thyroid gland by means of the administration of 10% Lugol's solution, which is useful both to the certain identification and intraoperative localization of the parathyroid adenoma by gamma probe. However, up to now, Tc-99m-sestaMIBI is only used for the localization of parathyroid adenomas, whereas normal parathyroid glands still canot be detected by Tc-99m-sestaMIBI scintigraphy.As is known, technetium  have been used to localize the parathyroid glands intraoperatively, IV injection is essential that might lead to symptoms such as neurotoxicity, photobleaching, and pain at the infusion site.Nevertheless, the present study had some limitations, such as the limited number of enrolled patients, the retrospective design of the study, and the nonconsecutive collection of the patients. In addition, the control group in this study was only age- and sex-matched but not randomized. Although the baseline characteristics and TNM stage did not exhibit significant differences between the study and control groups, selective biases may be observed. Thus, the usefulness of the anatomical localization technique of normal parathyroid glands before thyroidectomy to prevent hypoparathyroidism needs further substantiation by randomized control trials.In conclusion, we developed a method to identify and anatomically localize the normal parathyroid glands before thyroidectomy using ultrasonography examination. This technique of anatomical localization preoperatively can aid the surgeons in preserving the parathyroid glands in situ during thyroidectomy and CND, thereby reducing the transient hypoparathyroidism postthyroidectomy.Conceptualization: Jin-Duo Shou, Jian-Biao Wang.Data curation: Jin-Duo Shou, Shui-Ming He, Xian-Feng Jiang, Liu-Hong Shi.Formal analysis: Shui-Ming He, Jian-Biao Wang.Funding acquisition: Jian-Biao Wang.Investigation: Jin-Duo Shou, Shui-Ming He, Xian-Feng Jiang, Liu-Hong Shi, Lei Xie, Jian-Biao Wang.Methodology: Jin-Duo Shou, Shui-Ming He, Xian-Feng Jiang, Liu-Hong Shi, Lei Xie, Jian-Biao Wang.Supervision: Jian-Biao Wang.Writing – original draft: Jin-Duo Shou, Shui-Ming He, Lei Xie, Jian-Biao Wang.Writing – review and editing: Jian-Biao Wang.


Eimeria tenella (EtMIC2) was cloned, characterized, and its protective efficacy as a DNA vaccine investigated. The EtMIC2 gene, which codes for a 35.07 kDa protein in E. tenella sporulated oocysts, was cloned and recombinant EtMIC2 protein (rEtMIC2) was produced in an Escherichia coli expression system. Immunostaining with an anti-rEtMIC2 antibody showed that the EtMIC2 protein mainly localized in the anterior region and membrane of sporozoites, in the cytoplasm of first- and second-generation merozoites, and was strongly expressed during first-stage schizogony. In addition, incubation with specific antibodies against EtMIC2 was found to efficiently reduce the ability of E. tenella sporozoites to invade host cells. Furthermore, animal-challenge experiments demonstrated that immunization with pcDNA3.1(+)-EtMIC2 significantly increased average body weight gain, while decreasing the mean lesion score and oocyst output in chickens. Taken together, these results suggest that EtMIC2 plays an important role in parasite cell invasion and may be a viable candidate for the development of new vaccines against E. tenella infection in chickens.Microneme proteins play an important role in the adherence of apicomplexan parasites to host cells during the invasion process. In this study, the microneme 2 protein from the protozoan parasite  Eimeria spp., leads to heavy economic losses in the poultry industry worldwide , instead of the oocyst production, together with weight gains and cecal lesion scores, were used to evaluate the protective efficacy of this DNA vaccine. While these are small-scale experiments, the consistency of the trial and level of efficacy with an approximate reduction of ~75.30% in oocyst shedding following vaccination, being higher than what has been previously seen in many studies with other antigens, indicates that EtMIC2 should be considered as an ideal candidate antigen in the development of a new vaccine against E. tenella in chickens.Rotational treatment with anticoccidial drugs and a commercial live vaccine is currently the best way to control infection in chicken flocks. Due to the high expense of scaling-up the production of the live parasite vaccine, there have been a number of recent efforts to develop subunit and recombinant coccidiosis vaccines using both DNA and protein-based antigens , 21, 30. studies , 45, 46.cidiosis , 43, 44.Williams , becauseE. tenella infection in chickensIn summary, EtMIC2 was found to be located in the anterior region and membrane of sporozoites, was strongly expressed during first schizogony, and was mainly located in the cytoplasm of first- and second-generation merozoites. Additionally, rEtMIC2-specific antibody was shown to inhibit parasite invasion. The recombinant plasmid pcDNA3.1(+)-EtMIC2 induced partial protective immunity in immunized chickens. Overall, these results suggest that EtMIC2 may play an important role in parasite cell invasion and may be an ideal candidate for the development of new vaccines against The authors declare that there are no competing interests.