Statistically significant differences were found among quantified volumes for healthy residual parenchyma, GGO, and consolidation  between dead patients and discharged patients. CT was not performed on cadavers; the death was an outcome, which was retrospectively included to differentiate findings of patients who survived vs. patients who died during hospitalization. Among discharged patients, complete disease resolutions on CT scans were observed in 62/129 patients with lung disease involvement ≤5%; lung disease involvement from 5% to 15% was found in 40/129 patients, while 27/129 patients had lung disease involvement between 16 and 30%. Moreover, 8–21 days  was an “advanced period” with the most severe lung disease involvement. After the extent of involvement started to decrease—particularly after 21 days—the absorption was more obvious. Conclusions: a complete disease resolution on chest CT scans was observed in 48.1% of discharged patients using a computer-aided tool to quantify the GGO and consolidation volumes; after 16 days of hospital admission, the abnormalities identified by chest CT began to improve; in particular, the absorption was more obvious after 21 days.Purpose: the purpose of this study was to assess the evolution of computed tomography (CT) findings and lung residue in patients with COVID-19 pneumonia, via quantified evaluation of the disease, using a computer aided tool. Materials and methods: we retrospectively evaluated 341 CT examinations of 140 patients (68 years of median age) infected with COVID-19 ), who were hospitalized, and who received clinical and CT examinations. All CTs were evaluated by two expert radiologists, in consensus, at the same reading session, using a computer-aided tool for quantification of the pulmonary disease. The parameters obtained using the computer tool included the healthy residual parenchyma, ground glass opacity, consolidation, and total lung volume. Results: statistically significant differences ( The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has already assumed pandemic proportions, affecting over 100 countries in few weeks [Currently, the “gold standard” for diagnosis of COVID-19 infection is a real-time reverse transcriptase polymerase chain reaction (RT-PCR) amplification of the viral DNA. However, radiological imaging is of great significance in the surveillance of COVID-19 infection ,4,5. RecHowever, the consolidation, or GGO with consolidation, increased, and reticular was observed in the later stages (scan > 1 week after symptom onset), this represents the conversion of findings from GGO to consolidation, and an increase in the reticulation pattern in affected lung parenchyma. CT features had rapid sever changes, from focal unilateral pulmonary parenchyma to diffuse bilateral GGO, or GGO with consolidation, within 1–3 weeks ,7. AlthoCT investigation in patients with suspected COVID-19 pneumonia involves the use of high-resolution techniques. Artificial intelligence (AI) software for quantification of pneumonia lesions has been employed to integrate CT diagnosis ,16. CompTo the best of our knowledge, no study in the literature reports on the temporal changes of CT findings, using an automatic tool to quantify the abnormality in lung parenchyma, due to COVID-19 pneumonia, in a large dataset of patients.We investigate the use of a computer-aided tool in order to quantify the abnormalities visible on chest CT images in patients with COVID-19 pneumonia.The aim of this study was to assess the evolution of CT findings and lung residue in patients with COVID-19 pneumonia, performing quantitative analysis of the disease with the commercially available system.This retrospective study included patients enrolled by “Hospital of Colli ” in Naples. In relation to the ongoing epidemic emergency, the institutional local review boards gave up written informed consent for this retrospective study that evaluated anonymized data and involved no potential risk to patients. The population included 140 patients  subjected to the nucleic acid amplification test of the respiratory tract or blood specimens, using a reverse transcription real-time fluorescence polymerase chain reaction test, for suspicion of COVID-19, between 2 March 2020 and 5 May 2020. The virus investigation for etiological diagnosis was executed by the current gold standard test. All patients with a positive RT-PCR test at hospital admission and with respiratory distress were hospitalized and followed-up. The clinical evolution of the disease was subdivided in stable, improved, and worsened. The parameters considered took into account the fever  and the breathing with SpO2 value in ambient air, and the ratio PaO2/FiO2 . The following laboratory parameters were assessed: white blood cells , PCR, VES, procalcitonin (PCT), D-dimer. The worsened picture was evaluated, considering organ dysfunction with the delta sequential organ failure assessment score (SOFA), in ranges from 0 to 24, and included points related to six organ systems: respiratory (hypoxemia), coagulatory (thrombocytopenia), liver (hyperbilirubinemia), cardiovascular system (hypotension), neurologic (low-level consciousness), and renal (oliguria or elevated creatinine).Chest CT scans were performed at the time of hospital admission and during the hospital stay, with a 64-slice scanner  dedicated to COVID-19 patients. CT examinations were performed with the patient in the supine position using a standard dose protocol, without contrast intravenous injection. The scanning range was from the apex to the base of the lungs. The tube voltage and the current tube were 120 kV and 100–200 mA, respectively. All images were obtained with a standard dose scanning protocol, reconstructed at 1.0 mm slice thickness, with 1 mm increment, 512 × 512 mm. Images were reconstructed with a sharp reconstruction kernel for parenchyma (FC13 on Toshiba). The lung window setting was at a window level of −600 Hounsfield units (HU) and window width of 1600 HU.DICOM data were transferred into a PACS workstation and CT images were evaluated by two expert radiologists, in consensus, at the same reading session, using the clinically available computer tool Thoracic VCAR software . The software provides automatic segmentation of the lungs and automatic segmentation and tracking of the airway tree. It provides the classification of voxels based on Hounsfield units and a color-coded display of the thresholds within a segmented region. Thoracic VCAR provided automatic segmentation of the lungs, and was performed using adaptive density based morphology. The lungs were extracted by using an optimal thresholding to identify low-density fields in the scans, region growing  and void filling. The three-dimensional hole filling was used to fill the lung cavities created by the elimination of normal blood vessels during the thresholding process, while airways were automatically segmented and exempted by iterative application of increasingly restrictive constraints, to a thresholding and 3D region growing process. The software complies with the regulatory requirements of Council Directive 93/42/EEC concerning medical devices (CE 0459) and FDA regulations. Lung parenchyma was divided by Hounsfield unit (HU) intervals from−1024 to less than −977 HU, representing emphysematous changes ,26; valuContinuous data were expressed in terms of median value and range.p value < 0.05 was considered significant for all tests.The Mann–Whitney test and Kruskal–Wallis test were used to assess statistically significant differences among groups. All analyses were performed using Statistics Toolbox of MATLAB R2007a .A total of 341 CT examinations, including baseline and follow-up CTs, were analyzed. Thoracic VCAR software was unable to perform the quantification in 16/341 (4.7%) cases, both automatically and manually; therefore, the findings of 325 CTs were reported in the results. Among 140 enrolled patients, 11 patients died, while 129 patients were discharged after a median hospitalization period of 14 days .p value > 0.23 at Mann–Whitney test).No statistically significant difference was found in the quantified volume distribution in the right and left lungs  among quantified volumes of healthy residual parenchyma, GGO, consolidation and total pulmonary volume, considering different clinical conditions  (see ved) see .p value ≤ 0.05 at Kruskal–Wallis test), based on patients outcomes between dead patients and discharged patients, for quantified volumes of healthy residual parenchyma , of GGO , and of consolidation   . GGO andIn Several previous studies ,14,15,16Zhou et al.  investigPan et al.  assessedWang at al  reportedHowever, to the best of our knowledge, there is no study in the literature reporting on the temporal changes of CT findings, using an automatic tool to quantify the abnormality in lung parenchyma (due to COVID-19 pneumonia).According to the recent literature, we reported that GGO is the most representative sign of COVID-19 disease on chest CT, and that statistically significant differences were found among quantified volumes of healthy residual parenchyma, GGO, consolidation and total pulmonary volume, considering different clinical conditions . Statistically significant differences were also found, based on patient outcomes between dead patients and discharged patients, for quantified volumes of healthy residual parenchyma , of GGO , and of consolidation . We reported that, among discharged patients, a complete disease resolution on CT scans was observed in 62/129 patients with lung disease involvement ≤5; lung disease involvement ranging from 5% to 15% was found in 40/129 patients, while 27/129 (20.9%) patients had lung disease involvement, between 16 and 30%. The discharged patients at the last follow-up had a percentage change of lung disease involvement of 12.5% while the dead patients of 57.1%.Moreover, we demonstrated that GGO and consolidation at the last follow-up were almost completely absorbed, and that 8–21 days of hospital admission was the advanced period with the most severe lung involvement. After 16 days of hospital admission, the abnormalities identified by chest CTs started to improve and, in particular, after 21 days, the absorption was more obvious.In this study, we reported that no statistically significant difference was found in the quantified volume distribution in the right and left lung—in contrast to what was reported by Li et al.  and NagrWe believe that analysis of CT findings, using a computer tool based on different thresholding Hounsfield unit settings, could identify pulmonary abnormalities and lung recruitment, and we believe that knowledge of the percentage of potentially recruitable lung evolution may be important to establish the therapeutic efficacy in COVID-19 disease.There are still some limitations in this study. First, the time for CT re-examination of each patient is not standardized. Second, the retrospective and monocentric nature of the study. Third, the absence of laboratory findings to correlate with the CT results.In conclusion, we reported that CT findings, using a computer automatic tool based on different thresholding Hounsfield Unit settings, could identify pulmonary abnormalities and lung recruitment. Moreover, we demonstrated that discharged patients had lung disease involvement of 12.5%, while for dead patients it was 57.1%; a complete disease resolution on chest CT scans was observed in 48.1% of patients using a computer aided tool to quantify the GGO and consolidation volumes. Moreover, 8–21 days of hospital admission is the advanced stage, with peak levels of abnormalities on CTs; after 16 days, the abnormalities started to improve. Therefore, CT has proven to be a useful tool in following the evolution of the disease, by clarifying the progression/regression timing of the disease.


Treatments with etanercept , or saline (control group) were carried out on days 0, 6, 12, and 18 of gestation. On the morning of the 21st day of pregnancy, rats were euthanized in a CO2 chamber and submitted to laparotomy to remove the fetuses, placentas, ovaries, and maternal organs. There were no differences between groups in the following parameters: water and food consumption; placental efficiency; reproductive parameters, including number of corpora lutea and implants, reabsorption, and pre- and post-implantation losses. However, etanercept treatment increased liver weight, reduced fetal and placental weight, decreased the placental junction zone, reduced the percentage of normal fetuses, and increased visceral or skeletal fetal abnormalities. Therefore, etanercept resulted in damages more related to fetus and placenta. However, more studies with different doses are required to better predict possible injuries elicited using etanercept during pregnancy.Etanercept is a tumor necrosis factor alpha (TNF-α) inhibitor chronically used to treat autoimmune diseases. However, the use of etanercept during pregnancy still needs to be further investigated. The aim of this study is to evaluate the etanercept treatment during pregnancy, analyzing maternal reproductive performance, fetal outcomes, and placental repercussions. Wistar rats (200–250 g) were mated and randomly distributed into two experimental groups: control and etanercept ( During pregnancy, the maternal environment undergoes many physiological adaptations to enable the presence of the concept in the body, ensuring the immunotolerance to the developing fetus . To achiAmong them, tumor necrosis factor alpha (TNF-α), a Th1 cytokine, is one of the earliest and most potent mediators of the inflammatory response . In factTumor necrosis factor alpha overproduction mediates a large spectrum of diseases, including some autoimmune conditions. On this regard, TNF-α inhibitors have been successfully used to control several autoimmune diseases including ankylosing spondylitis, rheumatoid arthritis, polyarticular juvenile idiopathic arthritis, psoriasis as well as psoriatic arthritis . Most ofEtanercept is a soluble recombinant TNF-Fc complex, formed by extracellular fragments of TNF-α receptors attached to a human IgG Fc fraction , which btrans-placental permeability of etanercept.However, recently, it was demonstrated that some TNF-α inhibitors may display a cumulative outcome, since spontaneous abortion, preterm labor and low birth weight rates were increased when they are used during the entire pregnancy, as was the case for infliximab . With thEven this negative evidence, women with autoimmune disease may benefits from anti-TNF therapies. Unfortunately, due to the limited available data, the recommendations differ on the continuity or discontinuation of anti-TNF treatment during pregnancy, and discontinuation may carry a risk of relapse of the maternal disease . EtanercTo answer this question, we formulated the hypothesis that the use of etanercept during the gestational process impacts maternal reproductive performance, fetal and placental changes in Wistar rats. In the present study, we investigated Wistar rats submitted to etanercept treatment during pregnancy, to assess maternal reproductive performance and possible fetal and placental alterations.The animals used in the research were kept in the animal facilities of the Federal University of Mato Grosso, in 12-h light/dark cycles, under controlled temperature (23 ± 1°C) and free access to food (standard chow) and water. All procedures were performed in accordance with the guidelines provided by the Brazilian College of Animal Experiments, approved by the Animal Ethics Committee (CEUA #23108.038471/2019-14) of the Federal University of Mato Grosso.Wistar rats in reproductive age, weighing between 200 and 250 g, were used. For mating, housed in a proportion of four females to one male Wistar rats. Vaginal swabs were collected daily, and pregnancy was confirmed by the presence of sperm concurrent with the occurrence of the female estrous cycle, and this was designated the day 0 of gestation.n = 10) and etanercept  groups. The treated group received a dose of etanercept (0.8 mg/kg) subcutaneously on days 0, 6, 12, and 18 of gestation  × 100, where the final weight corresponds to rat weighted on 21st days of pregnancy, minus litter weight. Fetuses and their respective placentas were also immediately removed and individually weighed.On the morning of the 21st day of pregnancy, the rats were killed in a COThe points of implantation, reabsorption (embryonic death) and numbers of living and dead fetuses were observed and counted. The ovaries were removed for observation and counting of corpora lutea. The pre-implantation loss percentage (embryo loss rate in the period prior to implantation) was calculated as follows: (Number of corpus luteum-Number of implantation/Number of corpus luteum) × 100. The percentage of post-implantation loss (death of embryos after implantation) was calculated as follows: (Number of implantations-Number of living fetuses/Number of implantations) × 100 . The plaPups from the etanercept-treated dams whose birth weights were lower (<10th percentile) or greater (>90th percentile) than the mean birth weight of the control pups were classified as small for gestational age (SGA) or large for gestational age (LGA), respectively, and reported as percentage. The mean birth weight of the control pups was 5.05 ± 0.49 g. Therefore, pups from mothers treated with etanercept with birth weights smaller than 4.40 g (<10th percentile) were considered SGA and weighting more than 5.51 g (>90th percentile), as LGA .After weighing the fetuses, the analysis of external anomalies was performed. Newborns were examined externally, with detailed analysis of eyes, mouth, ear implantation, cranial conformation, fore and hind limbs, anal perforation, and tail . After eThus, half of the newborns of each litter were conditioned in alcohol (70%), and later were eviscerated, diaphanized with potassium hydroxide and stained with alizarin. For the analysis of skeletal anomalies, the method of circC, followed by the battery for inclusion of placentas in paraffin, left at room temperature for 24 h for paraffin solidification. Sections (4 μm) of the placenta were serially performed from the central region, with the aid of a microtome (microtome Thermo Fisher Scientific HM355S). Then, the cuts were stretched in a 50°C floating bath and were collected from the bath on glass slides previously treated with 0.1% poly-L-lysine (Sigma), for better adherence of the cuts.Placentas were removed, dived in the central region, and immediately immersed in Methacarn  for 3 h, at a temperature of 4°C, under constant agitation. Then, the placentas were dehydrated and were clarified in xilol. Subsequently, tissue infiltration was performed in two paraffin baths, inside a heating oven adjusted to 60The slides containing the sections were submitted to the deparaffinization process. Subsequently, they were gradually hydrated. For morphological analysis, staining with hematoxylin-eosin was performed. The slides were then dehydrated in a 95% alcohol bath and in two absolute alcohol baths, for 10 min each, and cleared in two xylene baths, for 5 min each. Finally, the slides were assembled using PERMOUNT (Fisher-SP15-500) as a means of adhesion between the slide and cover slip.2 using the IMAGE—PRO PLUS software .Images were observed through a microscope (Nikon Eclipse–E200) with a 4× magnification objective. Placental images were captured using a digital camera (Option ISH-500). The entire area of the junctional zone and the labyrinth was measured in mmn” represents the number of animals used in the experiment. Statistical analysis was performed . The data was submitted to the Shapiro-Wilk normality test, to ensure its normal distribution. Student’s t-test was used to make parametric comparisons and Mann Whitney test was used for non-parametric comparisons. For percentage analysis, Fisher’s exact test was used in. Values of p < 0.05 were considered statistically significant.Data are presented as mean ± SD and “As expected, weight gain was observed over the weeks of gestation in both groups. The relative weight (g) of the heart, spleen, and kidneys of rats did not differ between the experimental groups. However, the liver relative weight in the etanercept group was higher than in the control group .There was no difference between pre- and post-implantation losses between the experimental groups .2 vs. 10.05 ± 1.28 mm2, respectively; p = 0.03). In the labyrinth, there was no difference between the control and etanercept-treated group   .The main finding of this work is that fetus from Wistar rats displayed augmented visceral and skeletal anomalies upon Etanercept-treatment. This finding raises one important concern regarding the risk/benefit profile of anti-TNF therapy during pregnancy, since this drug is widely used by women with autoimmune disease, during reproductive age.Previous work reported that etanercept treatment improved maternal blood pressure, pregnancy outcome and uterine artery function in an experimental model of hypertension . BesidesTumor necrosis factor alpha regulates the gestational process since the early stages of development, and progressively increases with the course of the pregnancy, playing an important role in the inflammatory mechanisms that regulate implantation . When evAs pregnancy progresses, the placenta turns to the major source of TNF-α production and plays an essential role in fetal growth and support . EtanercIn the present study, etanercept treatment resulted in decreased placental weight, conversely to the higher maternal liver relative weight. Etanercept is known to cross the placental barrier, being detected in the serum of children born from patients treated with etanercept during pregnancy . HoweverPlacental function may be also related to morphometric evaluation, since morphological and functional placental adaptations can affect the nutrient transport capacity and consequently, placental efficiency . EtanercRegarding fetal anomalies, the etanercept-treatment had a lower percentage of normal fetuses, which comprise individuals without any skeletal or visceral anomaly. Skeletal alterations most frequently found in newborns, such as incomplete ossification and abnormally shaped sternebra, are considered merely observed variations and have no teratogenic effect . FurtherThe use of TNF-α inhibitors may be useful during pregnancy, specially to conditions, were exacerbated TNF-α elicits obstetric complications. For example, the use of etanercept during pregnancy in stroke prone spontaneously hypertensive rats (SHRSP) resulted in important gestational gains such as reduced maternal blood pressure, partial reversion of endothelial dysfunction and improvement of gestational parameters including, reversing growth restriction, and restoring placental morphology . This istrans-placental permeability of etanercept. Considering the importance of TNF-α for the gestational process, it is important to further address how the trans-placental transport of etanercept could impact pregnancy.Compared to other pharmacological therapies used to reduce TNF-α actions, including monoclonal antibodies, only a small portion of etanercept cross the placenta . Even inThe most important limitations of the current study are that a unique dose was evaluated, the etanercept-treatment before and being continued during pregnancy was not performed; and the detection of maternal and fetal blood levels of etanercept were not addressed. It is possible that treatments being performed with doses higher than the one used here would result in greater malformations or toxicity. Also, the cumulative effect of treatment since before the pregnancy period would refer to a treatment model closer to that used therapeutically, unraveling possible implications on parameters related to implantation and pregnancy success. Finally, it would be important to measure whether etanercept reaches fetal circulation in this experimental model.Therefore, taken together, these findings demonstrate that etanercept favored morphological alteration in the placenta, along with growth restriction and augmented rate of fetal malformation in Wistar rats. However, it remains a challenge to treat chronic conditions in women in reproductive age, raising the concept of safety both to mother and fetus. Studies in this area should be encouraged to further attest the use of these biological compounds during pregnancy.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the Brazilian College of Animal Experiments, the Animal Ethics Committee (CEUA #23108.038471/2019-14) of the Federal University of Mato Grosso.GA, RP, and TS conducted the data collection. GV and GA conducted the fetal analysis. RP and FG conducted the placental morphometry. RL supervised the statistical evaluation. FG, VL, and GA conducted the hypothesis and experimental design. All authors listed have made a substantial, direct, and intellectual contribution to the text, and data discussion.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Type 2 diabetes mellitus (T2DM) is a complex disorder comprehensively influenced by genetic and environmental risk, and research increasingly has indicated the role of microbial dysbiosis in T2DM pathogenesis. However, studies comparing the microbiome characteristics between T2DM and healthy controls have reported inconsistent results. To further identify and describe the characteristics of the intestinal flora of T2DM patients, we performed a systematic review and meta-analysis of stool microbial profiles to discern and describe microbial dysbiosis in T2DM and to explore heterogeneity among 7 studies . Using a random effects model and a fixed effects model, we observed significant differences in beta diversity, but not alpha diversity, between individuals with T2DM and controls. We identified various operational taxonomic unit (OTUs) and bacterial genera with significant odds ratios for T2DM. The T2DM signatures derived from a single study by stepwise feature selection could be applied in other studies. By training on multiple studies, we improved the detection accuracy and disease specificity for T2DM. We also discuss the relationship between T2DM-enriched or T2DM-depleted genera and probiotics and provide new ideas for diabetes prevention and improvement. Tcf7l2, Kcnq1) and environmental lifestyle factors   , 9. TherRoseburia has been shown to causally improve glucose tolerance  and increased the levels of erythrocyte superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity and total antioxidant capacity. Similarly, the blood glucose, insulin, homeostasis model assessment for insulin resistance (HOMA-IR) index and inflammation were significantly reduced by probiotic intervention in a randomized double-blind placebo-controlled study of 61 Saudi T2DM patients  guidelines , a systeThe V4 or V3-V4 region of the 16S rRNA gene was the most frequently sequenced fragment with the Illumina (MiSeq or HiSeq) or Ion Torrent platform (PGM or S5) among the included studies  with -fa4 permutations in vegan , Shannon index, and evenness (J) were calculated based on OTU tables of each study. Significance tests between T2DM patients and healthy controls were conducted by the Wilcoxon test method. Differences in community structure across samples (β diversity) were visualized by principal coordinates analysis (PCoA) plots based on Bray-Curtis distance. Significance tests were determined using permutational multivariate analysis of variance (PERMANOVA) with 10in vegan . Meta-anin vegan . GeneralRandom forest  models were trained for individual studies, and datasets combined all studies together at the OTU and genus levels to test whether a mixture of featured taxa can predict T2DM. We evaluated their performance using leave-one-out (LOO) cross-validation and scored the predictive power in a receiver operating characteristic (ROC) analysis. Meanwhile, to refine microbiome signatures for diabetic detection, we developed a two-step procedure modeling workflows with rigorous external validation to avoid overfitting and overoptimistic reports of model accuracy. In the first step, we ranked the common OTUs and genera by their relative abundances. Next, as a precaution against overoptimistic evaluation, stepwise feature selection was employed to select predictive microbial features and eliminate uninformative features based on 10-fold cross-validation (the depict in p<0.001) when combining all samples from the 7 individual studies together. However, samples were distinctly clustered primarily by individual studies in PCoA  from 7 studies were retained for downstream analyses , none of the ORs of alpha diversity metrics were significantly higher than 1.0 for T2DM in either the RE model or FE model with low heterogeneity  for OTUs and median AUC = 0.59, ranging in  for genera], compared to the single study’s own test set   >0.1, Clostridium_sensu_stricto_1, Blautia, and Dorea, were positively correlated with Bifidobacterium breve and Bifidobacterium adolescnts and were negatively correlated with L. acidophils. Meanwhile, Clostridium_sensu_stricto_1, Blautia, and Lactobacillus were correlated negatively with Bifidobacterium bifidum. Also, Lactobacillus that was enriched in T2DM patients was positively correlated with Lactobacillus delbruecki, B. breve, and Lactobacillus salivarius. By observing correlations with probiotics, supplementation with specific probiotics may effectively regulate the gut microbiota and improve T2DM, implying that balancing the intestinal microecology could provide a new prevention and treatment method for T2DM patients. Indeed, studies have shown that there is a significant association between certain genera and diabetes.In the context of diabetes mellitus, experimental and clinical studies have demonstrated that different species of bacteria reduce oxidative stress, showing antidiabetic effects . Thus, tThis study systematically evaluated the differences in intestinal flora between T2DM patients and healthy controls based on 1143 samples from China , 31, Colp<0.001). This heterogeneity in 7 studies of cases and controls may be due to the methodological, clinical, or study heterogeneity  of the included studies.Our sequence-based analysis portrayed non-significant differences in OTU richness, evenness and Shannon diversity index between T2DM cases and controls. This is consistent with the results of most included studies, among which only two studies reported significant differences in Chao1 in patients with T2DM compared to controls , 29 and Lactobacillus and T2DM  genera and probiotics shows that Clostridium_sensu_stricto_1 and Blautia were positively correlated with B. breve, and that Lactobacillus enriched in T2DM patients was correlated negatively with B. bifidum. Previously, Clostridium_sensu_stricto_1 was reported to be negatively correlated with insulin ] and 0.84 [ranging in ], respectively, in six out of seven data sets compared to a single study . This may be attributed to the fact that the samples studied by a single center are not universal. Meanwhile, our RF analysis identified several OTUs related to  insulin , C-pepti insulin . Lactobamoglobin . L. reut reuteri  used as There were limitations in this study. First, the study methodology in the included studies varied. These studies were performed with different sequencing platforms and sequencing regions, and differences in research methods have certain effects on the intestinal microbiota. Second, the included reports had relatively small sample sizes, with three of the seven studies recruiting more than 50 participants with T2DM. Some authors were reluctant to share data. Third, the studies included in our analysis used 16S rRNA sequencing to analyze the changes in bacterial groups, which underestimated the complexity of the gut microbiota. Despite these limitations, we systematically searched all raw sequencing data and meta-data and analyzed them in a suitable and uniform manner to minimize heterogeneity, which is important to detect alterations in the gut microbiota in patients with diabetes.In summary, our study analyzed diverse fecal 16S rRNA gene sequencing datasets in a uniform manner and revealed shifts in fecal bacterial diversity and taxa in T2DM. By selecting bacterial features and building an RF model, we raise the possibility of a fecal bacterial mode of monitoring gut health and a complementary approach for risk assessment of T2DM. Furthermore, by analyzing the interaction between T2DM-associated genera and probiotics, we provide evidence for the therapeutic potential of probiotics applied in T2DM to restore and maintain a healthy gut microbiota state.The original contributions presented in the study are included in the article/BZ, CX, KH, and MC conceived of the study. JH, ZC, AL, LY, QZ, QC, CY, ZW, DZ, and FC were responsible for collecting published data sets, YQ, MC, JH, and BZ were involved with the meta-analysis, YQ, MC, CX, ZC, and KH completed the statistical analysis, and YQ, MC, JH, CX, KH, and BZ wrote the paper. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Foundation of China , Postdoctoral Research Foundation of China , and National Health Commission of China NHC Key Laboratory of Health Economics and Policy Research (NHC-HEPR2019003).AL was employed by company Xiamen Treatgut Biotechnology Co., Ltd.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The production of safe and healthy foodstuffs is considered as one of the most important challenges in the food industry, and achieving this important goal is impossible without using various processes and preservatives. However, recently, there has been a growing concern about the use of chemical preservatives and attention has been focused on minimal process and/or free of chemical preservatives in food products. Therefore, researchers and food manufacturers have been induced to utilize natural-based preservatives such as antimicrobial enzymes in their production. Lactoperoxidase, as an example of antimicrobial enzymes, is the second most abundant natural enzyme in the milk and due to its wide range of antibacterial activities, it could be potentially applied as a natural preservative in various food products. On the other hand, due to the diffusion of lactoperoxidase into the whole food matrix and its interaction and/or neutralization with food components, the direct use of lactoperoxidase in food can sometimes be restricted. In this regard, lactoperoxidase can be used as a part of packaging material, especially edible and coating, to keep its antimicrobial properties to extend food shelf-life and food safety maintenance. Therefore, this study aims to review various antimicrobial enzymes and introduce lactoperoxidase as a natural antimicrobial enzyme, its antimicrobial properties, and its functionality in combination with an edible film to extend the shelf-life of food products. Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus, and Escherichia coli through the consumption of most foods, including dairy products, meat products , fresh fruit and vegetable produces, and sea foods as well (Food safety (the opposite of food risk) is defined as the absence of chemical, microbial, or physical hazards in consuming foods . Microbi as well . They ar as well –9. To th as well –13.Traditionally, several methods, including cooling, freezing, pasteurization, canning, modified atmosphere packaging (MAP), and the utilization of chemical preservatives in different fresh food products, are ascertained, which mean that the shelf-life of foods is increased and the growth of microorganisms causing human health risks is prevented . HoweverIn recent years, consumers have a tendency to use natural bio-preservatives  due to new concerns about the safety of synthetic preservatives –19. TodaRecently, the development of packaging (plastic or edible films and coatings) for the entrapment of antimicrobial compounds (as a kind of active packaging) could be a promising alternative approach to extend food shelf-life and food safety maintenance without affecting food's sensory characteristics . In addiEdible films and coatings can be produced from diverse natural constituents, such as polysaccharides , proteins , and lipids (waxes) alone or in combination, taken from renewable agricultural sources or food processing wastes , 29–31. 2O2), and  and hypothiocyanous acid (HOSCN), with the aid of H2O2 , and  thiocyan of H2O2 , 7. An ahe cells , 27, 41.he cells . Furtherhe cells .The incorporation of the LPOS, generally recognized as a safe (GRAS) compound, into different kinds of edible coatings and films to control the food spoilage originated from pathogenic microorganisms has been investigated in recent years , 28, 42.There are two main purposes associated with food preservation including decreasing the prevalence of foodborne diseases resulting from food pathogens and stopping or postponing microbial spoilage. The latter causes the food unpleasant but not essentially unsafe for consumers –46. ConcEnzymes are highly specific and efficient in minor concentrations and are working in mild temperature and pH conditions. Furthermore, they react quickly, making them a cost-effective approach in food processing. Enzymes have been experientially consumed since very old ages. They are mainly extracted from natural sources  and may be simply inactivated following the occurrence of an appropriate transformation. So, they are considered to be natural and safe food components that are more desirable than chemical compounds, as food-processing agents, by producers and consumers. Regarding the enormous number of enzymes successfully employed in the food industry , it is reasonable to respect the potential of enzymes in the food preservation sector . AntimicThe most promising antimicrobial enzymes are shown in 2O2-producing enzymes (oxidases) and (2) H2O2-responsive enzymes (peroxidases). Oxidases, like glucose oxidase, generate H2O2 in the absence of catalase or peroxidases, which could be lethal due to the toxicity of generated H2O2 and the consumption of oxygen necessary for aerobic microorganisms  are efficient as antimicrobial compounds against different kinds of food spoilage microorganisms and food borne pathogens , and LPO, present in several plants, animals, and humans. They use H2O2 as a substrate to oxidize halides  or pseudohalide (like thiocyanate) to more strong antimicrobial agents (like OSCN− in the case of LPOS)  in the microorganisms cells . This grrganisms . H2O2 kiathogens , 55. H2Oof LPOS) , 56. It ocyanate . Table 1Despite several advantages of antimicrobial enzymes, the main disadvantage is sensitivity at different conditions like temperatures, salt, and pH, which could result in enzyme denaturation and inhibiting their activity . Therefo2O2, and thiocyanate (SCN−) and it is only active when all of these three compounds exist together , and cyanosulfurous acid (HO SCN) with an antimicrobial activity against bacteria, fungi, and viruses , Htogether , 59, 60.tabolism , 62. In  viruses , 64.2O2 to oxidize isocyanate and halides to stronger antimicrobial agents against invasive microorganisms , a heme-containing chain glycoprotein and as a member of the mammalian peroxidase family, is a natural enzyme secreted by mammalian glands and found in tears, saliva, and milk , 69. It Peroxidases secreted in the lacrimal, salivary, and mammary glands are chemically and immunologically similar . LPO wasLactoperoxidase enzyme, a glycoprotein with 8–10% carbohydrate, consists of a single peptide chain containing 612 amino acids with a molecular weight of ~78 kDa , 76, 77.2O2 causes an irreversible inactivation of LPO  to its ground state using H2O2 occurs. Following this reaction, propagation reactions take place. These reactions involve converting the ground state of LPO into the so-called compound I state by a reaction with H2O2. At low concentrations of SCN− (<3 μM) and halide, compound I reacts with existing one-electron donors such as proteins and peptides, to create compound II that is consistently reduced to the ground state at a low rate. Compound II may react with excess H2O2 (>5 mM) and form compound III resulting in ferrylperoxidase adduct and an irreversible inactivation of LPO. Compound I created from the reaction of peroxidase with H2O2 participates in the oxidation of SCN− and halides 2], cyanogen thiocyanate (NC-SCN), cyanosulfurous acid (HO2SCN), and cyanosulfuric acid (HO3SCN) may be formed , light, pH, glycerol, and ammonium sulfate  and were inoculated either onto agar before placing the film disc or onto the top of the film disc. In a similar study, a whey protein isolate film containing 29 mg/g LPOS prevented 4.2 CFU/cm2 of L. monocytogenes inoculated on agar media. The application of LPOS-whey protein isolate coating (with 40 mg LPOS/g coating) in cold-smoked salmon decreased to near 3 and 1 log CFU/g of L. monocytogenes and total aerobic microorganisms, respectively. Also, whey protein coating containing LPOS inhibited L. monocytogenes in smoked salmon at 4 and 10°C for 35 and 14 days, respectively  or E. coli O157:H7 (5.6 log CFU/g), coated with whey protein containing LPOS (5 and 3% w/w) and stored at 4 and 10°C for 42 days. The results indicated an inhibitory effect against both bacteria at both temperatures during storage and the inhibition was more remarkable when the coating was formed on the surface of the turkey before inoculation than when the coating was formed on the inoculated surface.In another study by Min et al. , the LPOE. coli (NRRL B-3008), Listeria innocua (NRRL B-33314), and Pseudomonas fluorescens (NRRLB-253) in the presence of different levels of H2O2  and KSCN . It was indicated that the level of H2O2 and KSCN affected an antimicrobial activity of the LPOS. All bacteria were inhibited by using the LPOS along with 0.4 or 0.8 mM H2O2 and 4 mM KSCN during a 6-h incubation. Also, by applying 0.8 mM H2O2 and 4 mM KSCN, the LPOS showed an inhibitory effect against L. innocua and P. fluorescens for a 24-h incubation period. The highest and lowest resistant bacteria to the LPOS were E. coli and P. fluorescens, respectively.Yener et al.  evaluateXanthomonas campestris pv. Mangifera indica, Colletotrichum gloeosporioides , and Lasiodiplodia theobromae ngr 05A were investigated. The results showed that chitosan at the levels of 1 and 1.5% containing LPOS with or without iodine completely inhibited X. campestris pv. M. indica. It was stated that, compared to Gram-negative bacteria, Gram-positive bacteria had a lower resistance to LPOS without iodine. In the case of molds, 1 and 1.5% chitosan incorporated by LPOS with or without iodine inhibited C. gloeosporioides C64 and L. theobromae, whereas C. gloeosporioides C4612 was susceptible to iodine and C62 was resistant  on esistant .Shewanella putrefaciens, P. fluorescens, and psychrotrophic and mesophilic bacteria during a 16-day storage. Regarding the production of total volatile basic nitrogen (TVBN) and thiobarbituric acid values, lower levels were detected in LPOS-coated chitosan  was added to chitosan coating, which was applied for the shelf-life extension of the rainbow trout. The results indicated an inhibitory activity against chitosan . In a sichitosan  investigCissé et al.  studied S. putrefaciens and P. fluorescens and the total viable count as well as psychrotrophic bacteria remained below 7 log CFU/g at the end of storage. The highest and the lowest TVBN values were detected in whey protein coating and coating + LPOS + MAP, respectively. The TVBN level in samples coated with whey protein, whey protein + LPOS, and whey protein + MAP was higher than the acceptable limit of 30–35 mg N/100 g. It was noted that the use of MAP in combination with the LPOS reduced fat oxidation and consequently thiobarbituric acid reactive substances due to the decreased oxygen level. Regarding pH, samples coated with whey protein + LPOS and whey protein + LPOS showed a lower pH increase due to lower bacterial growth. Also, a sensory analysis was indicative of higher acceptability of samples coated with whey protein + LPOS + MAP during a 16-day storage. It was highlighted that in MAP packaging, replacing the oxygen with nitrogen decreased the growth of aerobic bacteria during the logarithmic phase, and the lag phase was prolonged by carbon dioxide. Similarly, Shokri and Ehsani  and the LPOS (5% v/v) individually and in combination against L. monocytogenes and E. coli O157:H7 in trout filets during a 16-day storage. The lowest bacterial count was observed in samples coated with alginate solution containing 0.5% and 1% essential oil, and the LPOS was indicative of a synergistic effect of these two antimicrobial agents.Sharifi et al.  studied 2O and the reduction of H+ concentration. Alginate-LPOS coating decreased TVBN formation in chicken filets. Chicken breast filets coated with alginate-LPOS at levels of 4% and 6% had lower Enterobacteriaceae, Pseudomonas aeruginosa, and aerobic mesophilic bacteria. Also, coated samples with 6% LPOS had the highest overall acceptability. The antimicrobial activity of coating containing LPOS was attributed to the interaction of OSCN− and HOSCN with SH groups of proteins in the cell membrane and the release of cell components. Also, it has been reported that an uptake of glucose, purines, and pyrimidines and the production of proteins, DNA, and RNA were prevented.Yousefi et al.  investigEnterobacteriaceae, and P. aeruginosa in chicken thigh meat. It was observed that increasing the level of LPOS enhanced an antibacterial activity and in this regard, coating with 8% v/v LPOS showed the highest activity. It was mentioned that the LPOS in the coating required 2 days for a complete activation against Enterobacteriaceae. Also, the highest inhibitory effect of the LPOS against aerobic mesophilic bacteria occurred after 4 days.In a study by Molayi et al. , whey pr, Pseudomonas spp., P. fluorescens, and S. purefaciens in shrimp samples. Coating of shrimp with whey protein and different levels of LPOS decreased TVBN formation, which remained below the unacceptable limit even after a 16-day storage. LPOS-coated samples (5 and 7.5%) showed the highest scores in different sensory parameters  to whey protein coating and its effect on the microbial, chemical, and sensory properties of shrimp was investigated and stored at the refrigerated temperature for 16 days. It was revealed that the LPOS at the level of 7.5% significantly decreased mesophilic aerobic bacteria, psychrotrophic bacteriarameters .Bunium persicum essential oil (0.5% v/v) on the microbial and chemical characteristics of Gouda cheese during a 90-day storage. It was indicated that bacteria number was constant in all essential oil and essential oil-LPOS-coated cheese samples during a 90-day storage. However, LABs and Enterobacter spp. were less susceptible to the antibacterial effects of LPOS. In addition, the occurrence of lipid oxidation decreased in cheese samples coated with whey protein-essential oil and whey protein-essential oil-LPOS. It was suggested to utilize LPOS with essential oil to achieve an antibacterial activity for both Gram-negative and Gram-positive bacteria. In a similar study, the LPOS (5% v/v) and B. persicum essential oil (0.5 or 1% w/v) were incorporated into alginate coating and its effect on L. monocytogenes in chicken breast filets was evaluated during a 20-day storage at 4°C. The lowest count of L. monocytogenes was obtained in the coated samples containing LPOS + 1% essential oil , or both on microbial and chemical characteristics of rainbow trout filets has been investigated during a 16-day storage. The results revealed that the combination of essential oil (1%) and LPOS (5%) was efficient in reducing mesophilic viable counts, psychrotrophic bacteria, S. putrefaciens, and P. fluorescens as well as the control of pH increase and the reduction of total volatile nitrogen. Also, the use of the LOPS individually was more effective than Z. multiflora essential oil alone , il alone .Pseudomonas spp. and Shewanella spp., TBARS, pH, and the sensory properties of the samples were determined during refrigeration. It was observed that chitosan films containing LPOS had the highest inhibitory activity against all bacterial growth. The underlying mechanisms included synergistic interactions between the components, the prevention of biochemical pathways and enzymes, and an interaction with cell wall components and pore formation in the cell wall. Also, chitosan films with LOPS reduced TBARS and showed the highest acceptability.In a study by Ehsani et al. , differePhomopsis sp. RP257 and Pestalotiopsis sp. isolated from mango. It was indicated that in vitro condition, Pestalotiopsis sp. was inhibited by chitosan alone at the levels of 1 and 1.5% and incorporation of the LPOS and LPOSI to 0.5% chitosan resulted in an increase of inhibition from 26 to 93%. In the case of Phomopsis sp. RP257, chitosan films with the LPOS and LPOSI were effective, especially at 1 and 1.5% chitosan levels. Similarly, it was stated that chitosan films completely prevented Pestalotiopsis sp. in mango at 1 and 1.5% while Phomopsis sp. RP257 inhibited 50% at the mentioned levels. Also, LPOS addition improved an antifungal activity.Mohamed et al.  preparedvia these intermediate oxidizing products leading to a change in cellular functions, such as membrane integrity, passage systems, and metabolic enzymes and subsequent death of the cells. This system has bacteriostatic and bactericidal activities against Gram-positive and Gram-negative foodborne microorganisms, respectively. Additionally, it shows antifungal and antiviral performances. However, a direct incorporation of LPOS in food matrices could lead to the reduction of its activity as a consequence of an interaction with food components and its utilization in edible films and coatings. Conducted researches in respect of films and coatings containing LPOS showed an antimicrobial activity and improved food product shelf-life and in some cases, reduced chemical spoilage in food products without a negative impact on sensory properties. Nevertheless, the types of films and coating with LPOS were limited to alginate, chitosan, gelatin, and whey protein and further researches are required to examine the antimicrobial effects of the different types of edible films and coatings incorporating LPOS. Additionally, an antimicrobial effect of the LPOS in combination with other natural antimicrobial agents and hurdle technology can be a subject of future researches.Recently, general trend has emerged that consumers are concerned about the safety of chemical preservatives utilized in food. Hence, extensive and integrated efforts have been carried out to discover natural-based preservatives that are able to prevent microbial growth in food and improve the safety and shelf-life of food products. This study revealed that lactoperoxidase as a natural antimicrobial enzyme in the LPOS could be potentially applied as a replacement for chemical preservatives to improve the quality and shelf-life of food products. An antimicrobial activity of LPO is supposed to arise from the oxidation of SH groups present in microbial proteins and enzymes This study was designed by NK. The manuscript was written by MY, NK, AN, MS, and AM. MY and NK critically revised the manuscript. All authors listed have approved the manuscript for publication, contributed to this article, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Focal epithelial hyperplasia (FEH) or Heck’s disease is a rare, benign, oral condition that is associated with infection by human papillomavirus type 13, 32 or both. The whiteish to mucosal-colored, soft, papular or nodular elevated lesions in the oral cavity are normally asymptomatic but can grow to a size or at a location where treatment is needed. The diagnosis is often based on clinical presentation and histopathology, and the HPV genotype can be determined using PCR utilizing specific primers or DNA sequencing. While FEH was reported to often affect several members of the same family and exist primarily among indigenous populations around the world, the number of reported cases within the European region is increasing. This contemporary review summarizes the main findings in relation to HPV genotypes, impact of superinfection exclusion and vaccination, transmission, diagnosis, geographical and ethnical distribution, comorbidities and treatment of FEH with an emphasis on including the most recent case reports within the field. Furthermore, we describe for the first time a FEH lesion infected with the low-risk HPV90. Focal epithelial hyperplasia (FEH), also known as Heck’s disease or multifocal epithelial hyperplasia (MEH) is a rare, benign oral condition that is induced by infection with human papillomavirus type 13 (HPV13), 32 (HPV32) or both ,5,6 Figa, which HPV13 and HPV32 are low-risk, non-oncogenic and the most prevalent HPV types found in FEH ,19. HoweAlthough multiple FEH cases with high-risk HPV coinfections were reported, to this end, no case of malignant transformation in FEH has been described. Furthermore, an in vitro study confirmed that HPV32 did not have tumor-transforming properties . This isWhen multiple genotypes are at play, superinfection exclusion may limit or block secondary coinfections. Superinfection exclusion is an event in which a single cell infected by a specific type of virus is unable to become infected with a secondary virus of the same type. Hence, coinfection with more than one HPV genotype can be rare . Althougn = 53, age 4–73 years, mean age 13.2 years), answered that they had experienced sharing toothbrushes and kitchen utensils, such as cups, cutlery and dishes, and only had access to poor sanitary conditions [The household transmission of FEH is believed to be through saliva, as free HPV13 viruses were detected in the saliva of infected individuals . In a sunditions .A diagnosis of FEH is often based solely on clinical presentation and histopathology, while the HPV involvement is rarely assessed ,13,17,29In some instances, a pan-anti-HPV antibody is used for immunohistochemistry (IHC) on biopsy material. However, this gives no information regarding the specific genotype involved ,11. The FEH was primarily reported in the indigenous populations around the world, but the number of reported cases has been increasing in the European region. From 1966–2005, Sethi and colleagues reported nine published cases. In 2001–2019, this number had risen to 20 published cases ,17, whiln = 2147) [n = 460, 46% of the inhabitants) among the indigenous Greenlanders from Nanortalik in southwest Greenland [n = 343) [In Greenland, the Danish medical doctor Helms described symptoms associated with FEH among the indigenous population back in 1894 , and les = 2147) ; in contreenland  and 32.3In many cases, FEH was described to affect several close relatives ,10,18,20n = 29) had FEH, 26% of which were infected with HPV13 and 31% with HPV32 [There are several reports describing FEH as a comorbidity with other diseases. In particular, immunosuppressed patients seem to have a higher incidence compared to the general population. Diseases with a lower level of naïve CD4+ T-cells, such as HIV and intestinal lymphangiectasis, were described in relation to FEH ,16,32. Ith HPV32  and leukth HPV32 , lung trth HPV32 , chronicth HPV32  and rheuth HPV32 .Preclinical research into FEH is complicated by the fact that the HPV life cycle is heavily dependent on host cell differentiation. Therefore, native HPV is only produced in vivo, while in vitro studies utilizing, e.g., immortalized cell lines rely on synthetic virus particles that can bypass the need for cell differentiation . To thisWhen it comes to clinical manifestations of FEH, in some instances, the characteristic nodules spontaneously disappear, leaving no residual functional or esthetic defects ,6. The dThat the implementation of these vaccines has been vastly successful was shown by e.g., a Swedish study investigating the HPV vaccination effects, who found that the oral HPV prevalence decreased from ~10% in unvaccinated youth from 2009–2011 to <2% after 2013, where more than 70% of the individuals were vaccinated . The preIn cases where the lesions grow to a size or at a location where treatment is warranted ,16, treaFEH is primarily associated with HPV13 and HPV32 infection, and coinfection with high-risk HPV16 and HPV18 was reported. HPV16 and HPV18 are covered by current HPV vaccines. However, the impact of the vaccines on infection with HPV13 and 32, and thus the overall prevalence of FEH, is currently unknown, but vaccinees might experience some cross-protection to related genotypes. Presently, the vaccines do not include HPV13 and HPV32.For the first time, we present a histological picture of FEH with proven HPV90 involvement. The diagnosis of FEH is often based solely on clinical examination and histopathology, risking a differential diagnosis. HPV assessment is therefore recommended. Despite being described primarily among the indigenous populations around the world, a recent report has found the number of reported cases in the European region to be increasing. Hence, more research in the field is of utmost importance to establish the exact pattern of the spread of this disease.


Salmo macrostigma) that colonized the Biferno and Volturno rivers of the Molise region . The partial 16S rRNA gene sequences of these strains were determined for the species-level taxonomic placement. The phylogenetic analysis revealed that the isolated LABs belonged to seven genera . The study of the enzymatic activities showed that these LABs could contribute to the breakdown of polysaccharides, proteins, and lipids. In future studies, a greater understanding of how the LABs act against pathogens and trigger the fish immune response may provide practical means to engineer the indigenous fish microbiome and enhance disease control and fish health.Knowledge of the composition of the gut microbiota in freshwater fish living in their natural habitat has taxonomic and ecological importance. Few reports have been produced on the composition of the gut microbiota and on the presence of LAB in the intestines of freshwater fish that inhabit river environments. In this study, we investigated the LAB community that was present in the gastrointestinal tract (GIT) of Mediterranean trout ( Lactobacillus, Lactococcus, Leuconostoc, Enterococcus, Streptococcus, Carnobacterium, Pediococcus, and Weissella genera have adapted to grow under very different environmental conditions and are important representatives of the gut microbiota of both freshwater and marine fish [The intestinal tracts of fishes contain a complex and dynamic community of microorganisms ,2. Amongine fish . In receine fish ,9,10. LAine fish ,12,13,14Salmo macrostigma is an endemic freshwater trout species of the Mediterranean area  that inhabits river environments. At present no similar studies are available on this freshwater fish species.Knowledge of the composition of the gut microbiota in freshwater fish living in their natural habitat has taxonomic and ecological importance. In particular, the study of the gut microbiome in threatened wildlife species has enormous potential to improve conservation efforts and to gain insights into the host–microbe coevolution. The interaction of animals with their respective symbiotic microorganisms can provide important tools for the management of various issues related to the protection of endangered animal species . Salmo mean area . This saA total of 18 adult specimens of Mediterranean trout that accidentally died in the fish traps used in the scientific activities of the LIFE Nat. Sal. Mo Project (LIFE17 NAT/IT/000547) was collected. The fish were taken from 7 different study sites on the Biferno (4 sites) and Volturno (3 sites) rivers  between November and January during the daily inspection of the fish traps and immediately transported in portable refrigerators (2–8 °C) to the microbiology laboratory. The exterior of the fish was wiped clean with 70% ethanol, the abdomen opened at the ventral midline, and the whole intestine was aseptically removed from the abdominal cavity and separated into the proximal  and midgut (MG) sections .Once the specimens were longitudinally opened, the MG sections were rinsed using 3% NaCl to remove non-adherent  bacteria and digesta. Later, the PC and MG matrices were homogenized together in sterile physiological water (0.9% NaCl) and serial decimal dilutions were obtained. The bacterial colonies were isolated by plating serial decimal dilutions on MRS and M17 media  that were supplemented with cycloheximide (40 mg/L). Plates were incubated at 30 °C under anaerobic conditions using an anaerobic system . After 48–72 h, approximately 5% of the colonies were isolated and purified from culture plates by streaking them on the respective culture media. The bacterial colonies were randomly selected according to morphological differences (colony size and shape).Prior to genotypic identification, presumptive LABs isolates were examined for their Gram reactions and catalase activity. The Gram reactions were performed by dissolving a loop full of freshly grown colony material in a drop of 3% KOH  on a microscope slide. Like the Gram stain reaction, the KOH test is based on differences in the chemistry of the bacterial cell wall. In the presence of potassium hydroxide, Gram-negative cell walls are broken down. KOH easily dissolves the thin layer of peptidoglycan of the cell walls of Gram-negative bacteria. On the other hand, Gram-positive bacteria are not affected by KOH because they have a thicker peptidoglycan layer in the cell wall. The isolates, which did not give a viscid product, were selected since LAB are known as Gram-positive cells .2O2; Sigma-Aldrich) solution (5%) to a small quantity of colony mass on a glass slide. The catalase test differentiates bacteria that produce a catalase enzyme that can degrade H2O2 in water and free oxygen. The production of oxygen bubbles can be observed.The catalase activity was determined by adding a drop of hydrogen peroxide  according to the manufacturer’s instructions.Genomic DNA from pure cultures of putative LABs was extracted using a Bacterial Genomic DNA Isolation Kit  according to the manufacturer’s instructions. The 16S rRNA gene fragments were amplified using the 27F and 1492R primer pair .w/v) agarose gel in a 1× Tris-Borate-EDTA (TBE) buffer.The polymerase chain reaction (PCR) mixture contained 10 μL 2× PCR master mix (Norgen Biotek), 0.5 μL of each primer (2.5 μM), 7 μL Milli-Q water, and 2 μL template DNA. In the negative control for the PCR reactions, Milli-Q water was used instead of DNA. The PCR reactions were performed with a Mastercycler Nexus PCR thermal cycler . PCR amplifications were achieved using the following program: pre-denaturation at 95 °C for 10 min, then 30 cycles of denaturation at 95 °C for 30 s, annealing for 1 min, and extension at 72 °C for 1.5 min. The last cycle was followed by a 7 min extension at 72 °C . PCR proThe bands were visualized under a UV transilluminator  and the sizes were estimated by comparison against 1 kb DNA ladder (Norgen Biotek).PCR products were purified using a QIAquick PCR purification kit  and sent to a commercial facility for sequencing . The 16S rRNA sequences were examined using the Basic Local Alignment Search Tool (BLAST)  program http://www.ncbi.nlm.nih.gov/BLAST/ (accessed on 26 June 2021). The sequence of the closest related type strain was compared with the respective sequence of our collected strains.In total, sixty-one Gram-positive and catalase-negative bacterial strains were presumptively considered LABs. The partial 16S rRNA gene sequences of these strains were determined for taxonomic placement. The BLASTN algorithm was applied to the GenBank database to identify sequences (Sequence matches that showed high identity scores (98% and above) were considered acceptable for taxonomic placement at the species level .According to the 16S rRNA gene sequences, all isolated strains and their related type strains were used to construct a phylogenetic tree  using thCarnobacterium, Enterococcus, Lactobacillus, Lactiplantibacillus, Vagococcus, Lactococcus, and Weissella). The majority of the LAB isolates were identified as Carnobacterium maltoaromaticum (28 strains), Lactiplantibacillus plantarum (14 strains), Enterococcus faecalis (7 strains), Lactococcus lactis (6 strains), and Weisella paramesenteroides (3 strains). A minority of LABs were identified as Lactobacillus acidophilus and Vagococcus fluvialis (1 strain each). It was not possible to determine whether the L8 strain belonged to the species Lp. plantarum or Lp. pensosus. Our results confirmed that the definition of the phylogenetic distances and sometimes even the differentiation using 16S RNA sequencing was not feasible for Lp. plantarum and Lp. pentosus because of the high similarity. In fact, these two species belong to the same phylogenetic group (Lp. plantarum group) [C. maltoaromaticum was widely found in 12 trout specimens (percentage frequency 66.67%), Lp. plantarum was detected in 7 trout specimens (44.44%), while Lc. lactis and E. faecalis were detected in 27.78% of the trout specimens. The phylogenetic analysis revealed that the 61 strains belonged to 7 genera (m group) ,30. In FLp. plantarum strains, which instead exhibited the following enzymatic activities: alkaline phosphatase, β-galactosidase, α-glucosidase, β-glucosidase, and N-acetyl-β-glucosaminidase. All the strains belonging to C. maltaromaticum species showed acid phosphatase, cystine arylamidase, leucine arylamidase, valine arylamidase, naphthol-AS-BI-phosphohydrolase, and N-acetyl-β-glucosaminidase activities. Meanwhile, α-mannosidase, α-fucosidase, α-chymotrypsin, and lipase activities were not detected in C. maltaromaticum species. Lipase and α-chymotrypsin activities were detected only in Lc. lactis strains.The enzymatic activities assayed using the API-ZYM system are presented in C. maltaromaticum, Lp.plantarum, and Lc. Lactis species, there was variability in some enzymatic activities; the positive strains percentage is highlighted in In the group of bacteria ascribed to the C. maltaromaticum and Lp. plantarum. There are no similar studies available on this freshwater fish for comparison. Diet and environmental temperature are among the factors that have the greatest impact on the gut bacterial communities of fishes [Lp. plantarum and the absence of C. maltaromaticum. Instead, other studies showed that C. maltaromaticum are important inhabitants of the gastrointestinal tract of freshwater and marine fishes [Our results highlight that in the LAB community of the gut microbiota of the Mediterranean trout, there is a predominance of f fishes ,10,31,32f fishes . The resf fishes  in a stue fishes ,36,37,38e fishes .C. maltaromaticum were reported [Lp. plantarum is a versatile and ubiquitous microorganism that is capable of colonizing several ecological niches, including the gastrointestinal tract of mammals, insects, and fishes [S. macrostigma. Other studies have shown the presence of Lp. plantarum in the intestinal tract of other freshwater fish [Lc. lactis species was isolated amongst the LAB in the Mediterranean trout intestine, which is in agreement with other studies carried out on salmonids [Lc. lactis strains are highly adaptable to different environments, including animal sources, dairy products, and silages [Lc. lactis is a LAB found in milk and milk derivatives, it may also be present in the disposal effluents of dairy factories that are released into the environment [Lc. lactis strains derived from fish intestines and Lc. lactis strains derived from milk derivatives were demonstrated [Lc. lactis were selected and proposed as probiotics in the fish diet [Although a fish probiotic culture was used ,41, severeported ,43,44. Hreported . Lp. plad fishes ,47. Our ter fish  and someter fish ,49,50. Ialmonids ,52. It h silages . Some reironment . Neverthironment ,56,57. Fnstrated ,59. Receish diet ,60.Enterococcus genus includes species that predominantly reside in the gastrointestinal tract of humans and animals; nonetheless, they are widely distributed in the environment [E. faecalis in the intestinal tract of S. macrostigma, confirming its frequent presence in the gut microbiota of fishes [E. faecalis are resilient and versatile species that are able to survive under harsh conditions [The ironment ,62. The f fishes ,63. The nditions . Some stnditions ,65.V. fluvialis, W. pseudomesenteroides, and L. acidophilus species can be isolated from fish guts [Our results confirmed that, more or less frequently, some LABs belonging to the ish guts ,51,66,67Bacteria present in the aquatic environment and introduced with food may influence the composition of the gut microbiota in fish .C. maltoaromaticum and Lp. plantarum in the aquatic environment [LABs are ubiquitous microorganisms and several studies found the presence of species such as ironment ,70 and iironment ,71. The Our study was conducted using a culture-dependent method; further studies using culture-independent methods will be applied in the future ) for the assessment of biodiversity in gut microbiota communities of wild Mediterranean trout. However, the technique we adopted allowed us to isolate the most numerically representative LABs from the intestinal tract of this salmonid and to submit them to a first characterization based on the in vitro evaluation of their enzymatic profile.The environmental temperature and the host trophic levels  influenced the composition and metabolic capacity of the gut microbiota of wild freshwater fishes ,72,73,74C. maltoaromaticum strains displayed aminopeptidase activity  as proteolytic indicators and the alkaline and acid phosphatase activities involved in lipidic metabolism [The LABs are producers of extracellular enzymes that are involved in the breakdown of cellulose, starch, proteins, and lipids ,78,79,80tabolism ,82.C. maltoaromaticum and Lp. plantarum strains showed N-acetyl-β-glucosaminidase activity. LABs producing this enzyme could facilitate the breakdown of the exoskeleton of many invertebrates, including insects, which are the prey of the Mediterranean trout in its natural habitat [All the  habitat . The exo habitat .Lc. lactis strains. These data confirm the potential of this bacterial species in the breakdown of proteins and lipids [C. maltaromaticum, Lp. plantarum, and Lc. lactis, there was variability in some enzymatic activities. However, the limited number of isolated LAB and the techniques used did not allow us to carry out a meaningful analysis on the intraspecific variability (phenotypic and genotypic). In the future, when our collection of bacteria is numerically more substantial, further studies on these important aspects will be carried out.The lipase and α-chymotrypsin activities were detected only in d lipids . In the The characterization of microbial populations in the intestinal microenvironment of fish and understanding the physiological interactions between the indigenous microbiota and the host might have important implications.However, the enzymatic properties of all the isolate strains shown in vitro did not axiomatically result in a positive contribution in digestive processes; this aspect deserves further investigation.In contrast to endothermic animals, the exact role of gut microbiota in fish nutrition is difficult to conclude as a consequence of the complex and variable ecology of the GI tract of fish .Moreover, among the microbial population in the fish gut, beneficial enzyme-producing bacteria continuously compete with pathogens through competitive exclusion; thus, this topic should be addressed in studies conducted in vitro and in vivo.In general, the microbial population of the gut represents a very important and diversified enzymatic potential, and the enzymatic mass present in the digestive tract might interfere in a considerable way with a major part of the metabolism of the host animal ,72.Furthermore, the role of enzyme-producing fish gut bacteria as probiotics in the enhancement of food digestibility and their effect on gut enzyme activity was evaluated through several investigations ,88.Some studies have shown that the formation of the microbiota of the digestive tract from the larval stage to adult fish is formed gradually ,90,91,92The enzyme-producing microorganisms isolated from fish digestive tracts can be beneficially used as a probiotic, especially in the larval stages . The maiIn this context, the search for beneficial extracellular enzyme-producing gut bacteria to be used as probiotics for the fish may be of interest. Most studies on enzyme-producing gut bacteria isolated from fish were conducted on different fish species, while few studies have been carried out on salmonids .Therefore, we believe that the topic enzyme-producing LABs isolated from fish deserves further investigations, especially in relation to chitinase activity, as chitin is one of the most renewable biopolymers on earth and might be useful as a constitutive material in formulated fish feed in the future. Even though dietary chitin modulates the intestinal microbiota and influences disease resistance, susceptibility, and innate immune parameters, these topics are not fully understood; as a consequence, further studies are needed .S. macrostigma is a cold stenothermal fish species, which, in the natural environment, has a diet that is almost exclusively carnivorous. Our results suggest that these factors are important in shaping the gut microbial community of these fish. The microbial communities that constitute fish microbiomes are essential for the host’s health. Therefore, a better understanding of the natural bacterial communities of healthy individuals and how they interact with the host and other environmental factors is of crucial importance [S. macrostigma but also for every fish species suffering a demographic decline [The environmental temperature and the host diet create strong selective pressure in the gut, which shapes the structure of the gut microbial community ,33,73,96portance . Captiveportance ,54,98. Tportance . This in decline .C. maltoaromaticum and Lp. plantarum species.The results obtained in this study show that the dominant LABs that are associated with the intestinal tract of the Mediterranean trout specimens examined belong to Our study is certainly not exhaustive and further genotypic and phenotypic investigations are underway on the gut microbiota of Mediterranean trout. These additional studies are needed to produce a greater understanding of the interactions between environment–host–microbiota and how specific microbes, such as LABs, can be used as resources to improve the health and wellbeing of this fish ,82.


This review aimed to compare the clinical features and CT imaging features between patients with pulmonary tuberculosis (PTB) and lung cancer and patients with PTB alone. That would help to analyse the differences between the two and consequently providing a theoretical basis for the clinical diagnosis and treatment for the patients. Relevant case-control studies focusing on the clinical and CT imaging characteristics between PTB with lung cancer and PTB alone were systematically searched from five electronic databases. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated for comparison. As of 2021-07-06, a total of 1735 articles were retrieved. But only 15 articles were finally included for meta-analysis. The results showed a higher proportion of irritable cough, haemorrhagic pleural effusion and lower proportion of night sweating in PTB patients with lung cancer than in PTB patients, and the differences were statistically significant . In addition, there are many differences in the imaging characteristics of the two types of patients. In conclusion, this review summarises the similarities and differences in clinical symptoms and imaging features between patients with PTB and lung cancer and patients with PTB alone, suggesting that we should be alert to the occurrence of lung cancer in patients with obsolete PTB relapse. Mycobacterium tuberculosis. It often invades multiple organs throughout the body, with pulmonary tuberculosis (PTB) as the most common one [Tuberculosis (TB) is a chronic infectious disease caused by mmon one . The maimmon one . In recemmon one . Primarymmon one . PTB, itmmon one . And themmon one , 4. TherBecause the early aetiology of lung cancer is unknown and both lung cancer and PTB are respiratory and consumption diseases, there are similarities in the clinical and imaging features between the two , 6. ThesThe systematic review followed the methodology outlined in Cochrane Handbook for Systematic Reviews of Interventions Version 6.0 . And thiCase-control studies related to the clinical and CT imaging characteristics between PTB with lung cancer and PTB alone were systematically search from PubMed, Embase, Cochrane, WangFang Data and China National Knowledge Infrastructure up to 6 July 2021, to provide a comprehensive comparison of clinical and imaging features between the two. The search items were as follows: ‘lung cancer’, ‘lung cancer with pulmonary tuberculosis’, ‘pulmonary tuberculosis’, ‘clinical features’ and ‘CT images’. In addition, the references of the preliminarily included articles in the above systematic search were also searched to prevent the omission of any related articles. Based on the literature retrieval, a comprehensive comparison and report on the similarities and differences of clinical and imaging features between the two types of patients had been achieved.Two researchers independently assessed the titles and abstracts of the articles obtained from the initial search according to the inclusion and exclusion criteria. In case of disagreement during this process, the third researcher would be consulted, who would finally determine whether to include the controversial article or exclude based on the opinion of the former two. Inclusion criteria were: (1) retrospective case-control study; (2) the subjects were PTB patients, and were divided into the study (PTB with lung cancer) and control  groups; (3) analysis indexes included clinical symptoms and imaging features of the two groups. Exclusion criteria were: (1) duplicated publication of the same trial; (2) the full text was not available, or the data were incomplete and were not available through reasonable channels; (3) with major deficiencies in study design  or major biases in the reporting of results. Studies that met any of the criteria were excluded.Two researchers independently extracted the following information provided by each included study into a table: title, first author, journal of publication, year of publication, number of included study subjects, grouping, age of study subjects, inclusion criteria and exclusion criteria, diagnostic criteria for PTB and lung cancer, clinical characteristics and imaging characteristics of patients, and study design-related indicators . After data extraction, a third researcher checked the consistency of the data extracted by the former two researchers.The quality of the included studies was assessed by two researchers independently using the Newcastle-Ottawa Scale (NOS) for observational case-control studies . The evaQ test and I2 statistics. I2 values of 0–39%, 40–59% and 60–90% indicated low, moderate and high heterogeneity among studies, respectively [P < 0.05 suggested a significant difference except that P < 0.10 in the results of Egger's test was considered statistically significant.Results were merged across studies with STATA version 15.1  , 11. Stuectively . In caseectively , 13. P <n = 193; review or in vitro/animal studies or letter or editorial or conference paper, n = 161; not related to comparison between patients with PTB accompanying lung cancer and patients with PTB alone, n = 903; not related to clinical features or radiologic characteristic, n = 94). Subsequently, by reading full text, 23 articles without valid data were excluded. Finally, 15 studies were included in the meta-analysis (In total, 1733 articles were obtained by systematic retrieval in five database, and two articles by manual retrieval from the references of the initially included articles. After removal of 346 duplicate articles, the titles and abstracts of the resulting article were screened and 1351 articles that did not meet the inclusion criteria were then excluded (not related to PTB, analysis , includiIn terms of quality assessment, all the 15 studies were considered to be of medium or high quality, with scores of 5–9 . In addiThe meta-analysis results showed no significant difference in most clinical symptoms of the patients in the two groups (persistent chest pain: OR 2.07, 95% CI 1.00–4.27; chest distress: OR 1.62, 95% CI 0.92–2.82; haemoptysis: OR 0.92, 95% CI 0.44–1.91; fever: OR 0.78, 95% CI 0.47–1.31; emaciation: OR 1.69, 95% CI 0.93–3.06; shortness of breath: OR 1.22, 95% CI 0.74–2.03; cervical lymphadenectasis: OR 1.02, 95% CI 0.71–1.46, However, the meta-analysis results also summarised some clinical characteristics to distinguish the two types of patients. Specifically, by using random-effects model, irritable cough was found to be of a significantly higher frequency in patients with PTB and lung cancer in comparison with that in patients with PTB alone (OR 2.43, 95% CI 1.43–4.11; Imaging characteristics between the two types of patients were also summarised and compared to obtain the similarities and differences. No significant difference was identified in calcified shadow (OR 1.93, 95% CI 0.51–7.25; We used Egger's test to analyse the publication bias of each indicator. The test result found publication bias in lobulation sign. Duval and Tweedie's trim and fill test proved the stability and guiding significance of the effect size of each index .Table 3In quantitative analysis, the high heterogeneity obtained by meta-analysis and the existing publication bias have no serious impact on the comparison result of clinical and imaging characteristics in the two groups of patients. That means that this meta-analysis truly reflects the possible situation in the process of clinical examination and treatment for those two types of patients. First of all, for high heterogeneity and publication bias in lobulation sign, the significance of its effect sizes is questionable. However, the meta-analysis of this indicator showed good consistency, that is, patients with PTB with lung cancer have a higher proportion of lobulation sign than patients with PTB alone , Fig. 4bThe imaging findings summary confirmed that CT imaging is effective in distinguishing patients with PTB and lung cancer from patients with PTB alone. There are significant differences in the imaging features between the two groups. Specifically, compared with the other group, patients with PTB and lung cancer showed a higher proportion of lobulation sign, mass, nodular shadow, satellite lesion, small vacuole sign, vacuole sign, spicule sign and pleural indentation, but a lower proportion of cord-like shadow and cavitary lesions. And good consistency of each index was observed , Fig. 4.+ T cells, will lead to the weakening of anti-tumour immune function and the increase of cancer cell escape, thus promoting tumour formation [PTB is one of the most common risk factors of lung cancer. Compared with the general population, PTB patients have a 50% increased risk of lung cancer. The presence of potential TB is significantly related to the increased risk of lung cancer. Especially in patients with more than 20 years of history of PTB, the risk of developing lung cancer is more than 2.5 times higher than that of the general population , 30. Theormation . In addiormation .In summary, when PTB patients, especially those with a history of more than 20 years, suddenly experience symptoms such as irritable cough and haemorrhagic pleural effusion after regular anti-PTB treatment, attention should be paid to whether they have lung cancer in addition to PTB recurrence. At the same time, it is necessary to combine the imaging features and laboratory results to accurately diagnose and treat the patients, aiming to improve the survival rate and prognosis of those with PTB and lung cancer.This review still has some limitations. First, only retrospective case-control studies are included in this review, which can't confirm the direct correlation of clinical symptoms and imaging features with PTB complicated with lung cancer, but only play a suggestive role. Second, the meta-analysis results of some indexes have high heterogeneity, so their guiding significance for clinical diagnosis and treatment is difficult to clarify.In conclusion, the meta-analysis summarises the similarities and differences in clinical symptoms and imaging characteristics between patients with PTB and lung cancer and patients with PTB alone, suggesting that we should be alert to the occurrence of lung cancer in patients with old PTB recurrence. Specifically, we should pay close attention to the clinical symptoms and follow-up the imaging features of the patients, thus timely identifying whether the patients are accompanied by lung cancer and consequently prompt treatment for improving patients' prognosis.


Pre-diabetes is an emerging public health challenge in sub-Saharan Africa.To estimate prevalence of pre-diabetes and assess its associated factors among adolescents. The risk factors were divided into individual, interpersonal and community factors, adapting socio-ecological model.This study utilised a cross-sectional descriptive survey. The target population was secondary school adolescents of Osogbo Local Government. Questionnaire was used to interview 405 participants through multi-stage sampling. Prediabetes was measure through fasting blood glucose.Findings revealed prevalence rate of 9.4%. Individual factors identified to be significant include age, religion and family history. Further analysis showed adolescents with normal BMI and high BP are likely to develop pre-diabetes when compared to those with underweight and normal BP respectively. Among interpersonal factors, parents' dietary habit was significant. Also, adolescents with employed parents were likely to develop pre-diabetes compared to those with unemployed parents. Lastly, availability of healthy food in school was the only statistically significant community factor. Hence, the more availability of food, fruits and vegetables in schools, the less likelihood of developing prediabetes.These findings affirmed that prediabetes is becoming common problem among Nigerian adolescents. There is therefore need for stakeholders to face this challenge before it becomes endemic. More African people will die from non-communicable diseases (NCDs) than infectious diseases by 2030; with 27% increase in deaths expected over next ten yearsPrediabetes is defined as plasma glucose levels that are elevated above the normal range, but below the threshold for diabetes. World Health Organisation (WHO) defines prediabetes as Fasting Plasma Glucose, FPG level of 110 – 125 mg/dL and/or Oral Glucose Tolerance Test, OGTT of 140 – 200 mg/dl; while International Society for Pediatric Adolescent Diabetes (ISPAD) defines it as FPG of 100 – 125 mg/dl or OGTT of 140 – 200 mg/dl Globally, an increase in epidemiological trends of prediabetes' prevalence among adolescents has been reported - as in the general population – regardless of the raceThough the prevalence of prediabetes among children is reaching alarming rates across the globe, the actual global prevalence remains unknownDespite prediabetes being an emerging public health challenge in sub-Saharan Africa, Nigeria inclusive, there is little research focus on adolescentsSocial Ecological Model, the theoretical framework for the study, was into three-level to suit the study. The model was used to consider the complex interplay between individual, relationship and community factors. An individual's various traits and identities make up the first level – interpersonal factors. It identifies biological and personal history factors that increase the likelihood of becoming pre-diabetic. Some of these factors are age, gender, financial resources, socio-economic status, race/ethnicity, religious identity, physical health, knowledge, attitudes, behaviour, self-concept, skills, developmental history, genetis, health literacy and personal preferences are some of the many attributes noted at this intervalThis study utilised a cross-sectional descriptive survey to conduct the study in Osogbo Local Government Area (LGA) – the major LGA in Osogbo metropolis. The target population for this study was secondary school adolescents of the LGA, while study population was the adolescents of the four selected schools. The minimum sample size calculated for this study was 310 participants, using Cochran formula – with adjustment of 20% for non-response and 10% for those the researchers were unable to contactInclusion criteria were secondary school adolescents between the ages of 10 and 19 yearsApproval letter and ethical approval certificate were obtained from Osun State Ministries of Education and Health respectively, after which permission was sought from respective school principals. An information sheet/consent form was sent to parents of all adolescents attending the selected schools. Only adolescents whose parents signed the consent form were included in the study. Parents were asked to ensure their children did not take breakfast on the day of the study until they have been tested. For the adolescents, purpose and procedure of the study, as well as the importance of not eating until the test is done were explained to thm on the day prior data collection. After this, adolescents who met the inclusion criteria and gave verbal assent were interviewed. Confidentiality of all data gathered was maintained. To avoid contamination, universal safety precautions were strictly adhered to when carrying out the finger prick test. For ethical reasons, awareness on prediabetes was created among parents, teachers and students. Results of the tests were communicated to the adolescents and their parents by sending Short Message Service (SMS) to their respective parents; and all students with abnormal glucose and Blood pressure (BP) levels were referred to their family physician.th percentile), normal weight (5th to < 85th percentile), overweight (85th to < 95th percentile) and obesity (≥ 95th percentile). BP was classified with the aid of 2017 American Academy of Pediatrics Guidelines for Childhood Hypertension. The BP percentiles based on age, gender and height was then determined and classifiedParticipants' B/P was first checked, to avoid being stressed, with the aid of Accosson mercury sphygmomanometer - while sitting and well comfortable on chair. After this, the FBG was measured by finger prick test with the aid of Accu-chek active glucometer while still on sit. Weight and height were then be measured while standing on the Generic Height and Weight with no shoes. Body Mass Index (BMI) was categorized according to BMI percentile charts for age and sex by WHO using BMI-for-Age growth charts into underweight  were middle adolescents, while 49.4% (n=200) were late adolescents. Also, 74.6% (n=302) were females, while 25.4% (n=103) were males; 44.9% (n=182) were Christians and 55.1% (n=223) were Muslims; 74.3% (n=301) were from public schools while 25.7% (n=104) were from private schools.2, mean FBG 88.4 + 9.2mg/dl, mean Systolic BP 102.5 + 12.3 mmHg while the mean Diastolic BP was 66.6 + 11.5mmHg. Furthermore, it was deduced that 20.2% (n=82) of participants were underweight, 73.8% (n=299) had normal BMI, 3.5% (n=14) overweight while 2.5% (n=10) were obese; 88.4% (n=358) had normal BP, 7.9% (n=32) had elevated BP, 3.0% (n=12) had stage I hypertension while 0.7% (n = 3) had stage II hypertension. From The mean weight of participants was 46.3 + 10.4kg, median height 1.57m, mean BMI 18.7 + 3.1kg/mFor the first hypothesis, which predicts no statistically significant relationship between the individual factors  and presence of pre-diabetes, 90.6% of cases were correctly predicted by the model. Among the individual factors tested, only age, religion and family history were statistically significant see . HoweverFor the second hypothesis, which predicts no statistically significant relationship between the interpersonal factors  and presence of pre-diabetes, 91.4% of cases were correctly predicted by the model. Among the interpersonal factors tested, only parents' dietary habit was statistically significant See . HoweverFor the third hypothesis, which predicts no statistically significant relationship between the community factors  and presence of pre-diabetes, 90.6% of cases were correctly predicted by the model. Among the community factors tested, only availability of healthy food in school was statistically significant see . FurtherConsidering the age distribution of participants with elevated parameters, more overweight participants were middle adolescents, while majority of the obese were late adolescents. This means that as they grow older, overweight adolescents are likely to become obese unless they modify their lifestyleThis study reported the prevalence rate of prediabetes among the participants to be 9.4%. This implies prediabetes is becoming common in our communities. Urgent steps are therefore needed to be taken to curtail its menace. Such steps include raising awareness of the disease and regular screening exercise among this population - as none of the participants had a previous knowledge of having the disease. This finding is in line with that of previous studies conducted in within and outside NigeriaIn addition, among the individual factors, age, religion and family history were statistically significant in developing prediabetes. That is, participants who are in middle adolescence, Muslim and had family history of DM are at more risk of developing prediabetes than their counterparts in late adolescence, Christian and had family history respectively. Meanwhile, findings from several studies have identified age as significant risk factor for development of pre-diabetesHowever, gender, type of school attended, physical activity, dietary habit and knowledge on diabetes mellitus were not statistically significant. As for gender, the finding was corroborated by that conducted in Cote d'Ivoire, which reported no significant difference in glycemic status of participants in terms of genderFurther findings revealed that those with normal BMI are likely to develop prediabetes while obese/overweight adolescents are not, when compared to those that are underweight. This finding is not in line with many previous studies, which reported overweight/obesity as a risk factor associated with developing prediabetes and DMAmong the interpersonal factors tested, only parents' dietary habit was statistically significant. That is, the healthier the dietary habit of the parents, the less likely their adolescents developing prediabetes. This finding is in line with that of previous studiesLastly, among the community factors, only availability of healthy food in the school was statistically significant. That is, the more availability of food, fruits and vegetables and less availability of soft drink, snacks and biscuits in school, the less likelihood of the adolescents developing prediabetes. This implies schools are important stakeholder when considering healthy dietary habits in adolescents. This finding was in accordance with the report that schools play a critical role in improving the dietary behaviour of adolescents, by creating environments that are supportive of healthy eating and implementing policies and practicesPre-diabetes is becoming a common problem among Nigerian adolescents, like their counterparts worldwide. There are many challenges associated with this; as this is an emerging health issue for this population. There is therefore need for stakeholders at all levels, to face this challenge before it becomes endemic problem in our country.