The subjects who consumed alcoholic beverages never or almost never had an upward change of 27.04%, and the time of daily exercise was reduced by 10.16 ± 48.68 min (p < 0.001). After quarantine, cigarettes per day increased by 1.14 ± 3.62 (p < 0.001) and the awakenings during night time increased by 0.37 ± 1.93 (p < 0.001). Our results suggest that the quarantine brought about significant changes in smoking, sleeping habits, physical activity, dietary habits, and the consumption of alcoholic beverages, some of which continue after its termination. Due to the evolution of COVID-19,restrictive measures were implemented. The quarantine resulted in significant changes in the social, economic, and psychological status of the population; however, its long-term effects have not yet been elucidated, especially in young adults. In the present study, people aged 18–25 years were studied, in relation to their sleeping, smoking, eating, and drinking habits and their physical activity before, during, and after the implementation of quarantine. We included 540 respondents . During quarantine, from 23 March to 4 May 2020, we observed an increase in sleep hours by 1.17 ± 1.98 h ( In December 2019 a new coronavirus strain . The firDuring the quarantine, people were faced with many and important changes in their lives and in their daily routines, with significant social , economiThe lifestyle changes  that occurred during quarantine may have a long-term impact on health; restrictive measures may carry a long-term increase in the risk of cardiovascular disease . In the In the present study, we aimed to assess the long-term effects of restrictive measures on an underrepresented population in the literature . To this end, young people, aged 18–25 years, were studied regarding smoking, sleeping habits and behaviors, consumption of meals, drinks, and cigarettes, and physical activity-exercise, in order to investigate the changes that may occur as a result of quarantine enforcement measures.This is a retrospective cohort observational study that used data collected using an online survey that included all regions of Greece and was conducted from 2 September to 27 November 2020. The study concerned young adults, aged 18–25 years, residents of Greece. Participants were asked to answer a questionnaire that was structured using the Google Form website The full questionnaire has been provided as The questionnaire was distributed through social media. This method was effective in terms of geographical distribution taking into account the restrictive measures due to the pandemic, mainly due to the systematic involvement of young people with the Internet ,28. The questionnaire included a total of 62 questions divided into 5 different sections. The first section (6 questions) included demographics, the second section (17 questions) addressed smoking habits, the third section was about sleep–wake habits, the fourth section (6 questions) assessed diet and alcohol consumption habits, and the fifth section concerned physical activity and exercise (9 questions). The questions in A pilot test of the first draft of the questionnaire was performed with a limited number of participants in order to assess ability to complete the questionnaire. We estimated that the time required for the completion of the questionnaire was about 10–20 min. We modified the questionnaire according to the results of the pilot testing. We did not include the responses of the draft questionnaire in the final analysis.2), (2) normal weight (18.5–24.9 kgr/m2), (3) overweight (25–29.9 kgr/m2), (4) obese (≥30 kgr/m2). Smokers were classified according to their smoking habit depending on the number of cigarettes per day . Additionally, subjects were classified according to the hours of sleep  and the time of sleep arrival . All variables were tested for regularity according to the criteria of the Kolmogorov–Smirnov test. Bivariate correlations were performed with the use of the Pearson coefficient or Spearman coefficient, according to the distribution of the variables. In the cases of normality, the t-test was used for the statistical comparison between groups and the paired-samples t-test or ANOVA for variables, which were measured in the same individuals PRE, DURING, and POST the mobility restrictions. In the cases of non-normality, we used the Mann–Whitney U, Kruskal Wallis, Wilcoxon z, and the Friedman test, respectively. The correlations between nominal variables were calculated by the chi-square test (x2) and Cramer’s V [2) was used to estimate the percentage of effect explained by the model. The level of statistical significance was set at p < 0.05. The statistical analysis was performed with the statistical program SPSS, version 20 . Data are presented as mean and standard deviation (mean ± SD), frequencies (N), and percentages (%). Body Mass Index (BMI) categories was classified as follows: (1) underweight . When we compared the smoking habits before the lockdown with the smoking habits during the lockdown there was no significant change in the total of subjects. However, when comparing the PRE to the POST phase there was an increase of cigarette consumption by 1.14 ± 3.62 (p < 0.001) in the total of subjects. DURING quarantine, the percentage of young men and women who smoked 5–10 cigarettes per day decreased, as opposed to the percentage of those who smoked <5 and >10 cigarettes per day which increased. The percentage of non-smokers remained relatively stable   and the PRE vs. POST phases . Moreover, there was a significant increase in the number of cigarettes per day of 1.54 ± 3.84 cigarettes/day  and of 1.5 ± 3.22 cigarettes/day  in women and in normal weight subjects, respectively. When we stratified participants by gender, we observed that the number of cigarettes per day showed a significant difference between the two sexes in all three phases of quarantine  . Additiop < 0.001, Wilcoxon z) in the whole group. POST-lockdown sleeping hours increased by 0.27 ± 1.52 h . DURING quarantine, the percentage of those who slept >9 h increased by 23.07% while after lockdown it increased by 5.1%  and in women by 1.25 ± 2.07 h . when taking BMI into account, we demonstrated that POST-lockdown sleeping hours increased by 0.84 ± 2.02 h  in underweight subjects, and increased by 0.26 ± 1.39 h  in normal weight participants. We stratified the sample according to gender and observed that in men, sleeping hours increased by 1.04 ± 1.82 h  and in the PRE vs. POST phase . The percentage of the study population with sleep arrival >15 min, increased by 14.43% DURING quarantine and by 6.47% POST-quarantine . A significant change was found in the total group, in the comparison of PRE vs. DURING phase  and in the PRE vs. POST phase . Sleep awakenings between men and women demonstrated a significant difference DURING the quarantine  and POST-quarantine . Regarding restless sleep, difficulty in getting up, morning headaches, and morning irritability, in all phases of the pandemic, there was a significant difference between men’s and women’s percentages , DURING , POST , Cramer’s V)  . p < 0.001, Wilcoxon z). The percentage of those who consumed ≥5 meals increased by 23.1% DURING the quarantine compared to the PRE-lockdown phase, and by 2.4% POST-lockdown compared to the PRE-lockdown phase . We observed a significant difference in the total population in the PRE vs. DURING phase , DURING , POST , Cramer’s V)  . p < 0.001, Wilcoxon z) in terms of sex. The mean exercise days per week were 3.11 ± 1.61 days in the PRE-lockdown phase. No difference was observed between the phases of the pandemic . A signic = 0.714, p < 0.001, Cramer’s V). On the other hand, no significant difference was found between men and women in terms of the type of physical activity-exercise DURING quarantine  and the years of physical activity-exercise ,  (Mean exercise days did not differ significantly according to sex . Howevern women) . p = 0.027, Mann–Whitney U), DURING , and POST phase . There was a significant difference in daily exercise time in minutes between men and women in the PRE  and the years of smoking history . The number of cigarettes per day DURING quarantine was significantly associated with BMI , years of smoking history , pack-years  and the number of cigarettes consumed PRE-lockdown . The number of cigarettes per day POST-lockdown were significantly correlated with years of smoking history , pack-years , the number of cigarettes per day PRE-lockdown  and the number of cigarettes consumed per day DURING lockdown . The hours of sleep DURING lockdown were significantly correlated with the hours of sleep PRE-lockdown  and the number of meals per day PRE-lockdown . The hours of sleep POST-lockdown were significantly correlated with age , the hours of sleep PRE-lockdown , and the hours of sleep DURING lockdown . The time to sleep arrival PRE-lockdown was significantly correlated with sleep awakenings PRE-lockdown . The time to sleep arrival DURING quarantine was significantly correlated with the number of cigarettes per day PRE-lockdown  and the number of sleep awakenings DURING lockdown . The time to sleep arrival POST-lockdown was significantly correlated with age , time to sleep arrival PRE-lockdown , time to sleep arrival DURING lockdown , sleep awakenings DURING lockdown , sleep awakenings POST-lockdown , and the number of meals per day DURING lockdown . Sleep awakenings PRE-lockdown were significantly correlated with time to sleep arrival PRE-lockdown. Sleep awakenings DURING lockdown were significantly correlated with time to sleep arrival PRE-quarantine , time to sleep arrival DURING lockdown , and sleep awakenings PRE-lockdown . Sleep awakenings POST-lockdown were significantly correlated with hours of sleep DURING lockdown , time to sleep arrival PRE-lockdown , time to sleep arrival DURING lockdown , time to sleep arrival POST-lockdown , sleep awakenings PRE-lockdown , and sleep awakenings DURING lockdown . The number of meals per day PRE-lockdown were significantly correlated with age  and pack-years . The number of meals per day DURING lockdown were significantly correlated with BMI , pack-years , and the number of meals per day PRE-lockdown . The number of meals per day POST-lockdown were significantly correlated with pack-years , number of meals per day PRE-lockdown , and the number of meals per day DURING lockdown .We assessed for significant correlations between the numerical variables examined in the questionnaire. We observed that the number of cigarettes per day PRE lockdown were significantly correlated with BMI  and the number of cigarettes during the lockdown  were independently predictive of the number of cigarettes per day POST-lockdown (r2 = 0.806). The following variables were predictive of sleep hours POST lockdown: age , sleep hours before the quarantine , and sleep hours during the restrictive measures  (r2 = 0.236). For time of sleep arrival POST-lockdown, time of sleep arrival before the lockdown , time of sleep arrival during the lockdown , sleep awakenings during the lockdown , and sleep awakenings after the restrictive measures  were independently predictive (r2 = 0.523). The following variables were predictive for sleep awakenings POST-lockdown (r2 = 0.382): time of sleep arrival DURING the lockdown , time of sleep arrival POST-lockdown , sleep awakenings PRE-lockdown , and sleep awakenings POST-lockdown . For the number of daily meals POST-lockdown, we observed that only the number of daily meals PRE-lockdown  and the number of daily meals DURING the lockdown  were independently predictive (r2 = 0.462). For exercise days per week POST-lockdown, we demonstrated that time of sleep arrival DURING lockdown , exercise days per week PRE-lockdown , and exercise days per week DURING lockdown  were independently predictive (r2 = 0.241).We performed multiple linear regression analysis to assess for predictors of sleep quality and lifestyle parameters POST the restrictive measures. In the multiple linear regression model, the number of cigarettes before the quarantine  that occurred in young adults, aged 18–25 years, due to the COVID-19 pandemic, PRE, DURING, and POST-quarantine. Importantly, we investigated changes in the aforementioned parameters POST-lockdown since most studies focus on behavioral modifications before and during restrictive measures. During the quarantine, we observed no significant change in cigarette consumption per day in the total of the subjects, except for the obese subjects who demonstrated an increase. During the lockdown, the percentage of those who smoked 5–10 cigarettes decreased, and the percentage of those who smoked <5 and >10 cigarettes increased. A similar study in Italy in subjects aged 12–86 years showed a decrease in cigarette consumption for the PRE and DURING phases , althougRegarding smoking during the lockdown, a study in young people at a university in Saudi Arabia showed an increase in smoking , while iThe restrictive measures and the long stays in the house led to a parallel increase in sleep hours in the entire population of the study in question. DURING the quarantine, the percentage of those who slept <7 h and 7–9 h decreased, while a spectacular increase (23.07%) was recorded in those who slept >9 h. Interestingly, the increase was maintained, to a lesser degree, even after the end of the lockdown. In the POST-quarantine phase there was a significant increase in sleeping hours in the total of the researched population, with the underweight and normal weight subjects showing the greater increase. Moreover, an increase in those sleeping >9 h was also observed in Italy  and in tThe young people in the researched population, and to an even greater extent, the women, needed more time to fall asleep DURING the quarantine, a need that remained even after the lockdown. A similar increase in sleep time was observed in Italy , as wellRestless sleep, morning headaches, morning irritability, and low morning alertness were also parameters that increased significantly DURING the quarantine and remained high even in the POST-quarantine phase. DURING the quarantine, young people consumed more meals, with women ahead. An increase in quarantine meals was also observed in the United Kingdom , Poland Those who consumed alcoholic beverages every day increased their percentage DURING the quarantine (2.97 vs. 4.44%), while those who never or almost never consumed them doubled their consumption (21.48 vs. 48.52%). An increase in alcohol consumption was recorded in students at a Saudi Arabian university , in GermAlthough exercise days did not differ between the phases of the pandemic, we observed that daily exercise time was reduced DURING the quarantine with women exercising less time than men. Although some studies provide data that support an increase in physical activity during the pandemic , most puOur results have some important clinical and public health implications. We observed an increase in cigarette consumption and a reduction in physical activity that persisted after the expiration of restrictive measures. The aforementioned findings highlight the need for public health system policies that will support healthy lifestyles in young adults in order to restore or to improve the negative impact of the lockdown measures. In the same context, others have advised on the development of psychological intervention programs that aim to limit the adverse effects of COVID-19 pandemic on children’s psychological well-being . The present study, besides its strengths, has several limitations. The retrospective design of the present investigation is a limitation since it may be associated with recall bias. We studied subjects aged 18–25 years since they have been underrepresented in previous studies. However, we acknowledge that our results cannot be extrapolated to other age groups. Additionally, the questionnaire was distributed through online social media. This method could not ensure compliance with the parameterization but met the requirements of massive participation of young adults of the Greek population. It was effective in terms of geographical distribution taking into account the restrictive measures due to the pandemic, mainly due to the systematic involvement of young people with the Internet ,28. AnotThe restrictive measures implied during the COVID-19 pandemic display significant long-term adverse effects on sleep quality as well as on lifestyle parameters, such as smoking habits, alcohol consumption, and physical activity. With the exception of the smoking habits and the consumption of alcoholic beverages that differ greatly between different countries and age groups, we observe that the other parameters were affected in a similar way around the world during and/or because of quarantine. We observed that young people in general increased their meal consumption, sleeping hours, their sleep onset time, while their daily exercise time decreased significantly. Our results support the need for the development of public health policies that will support healthy lifestyles in this population during the recovery from the COVID-19 pandemic. It is worth noting that in the present study, some additional parameters related to the quality of sleep were exclusively investigated, with significant results. It was observed that sleep awakenings, restless sleep, as well as morning irritability and headache showed a significant increase during the quarantine, parameters that were maintained even after the end of the lockdown measures in the study population. Further studies should be designed to elucidate whether these changes persist over longer time spans.


To compare the difference between magnetic resonance imaging (MRI) and computed tomography (CT) in delineating the target area of lung cancer with atelectasis.A retrospective analysis was performed on 15 patients with lung cancer accompanied by atelectasis. All positioning images were transferred to Eclipse treatment planning systems (TPSs). Six MRI sequences  were registered with positioning CT. Five radiation oncologists delineated the tumor boundary to obtain the gross tumor volume (GTV). Conformity index (CI) and dice coefficient (DC) were used to measure differences among observers.The differences in delineation mean volumes, CI, and DC among CT and MRIs were significant. Multiple comparisons were made between MRI sequences and CT. Among them, DWI, T2WI, and T1WI+C 10 minutes sequences were statistically significant with CT in mean volumes, DC, and CI. The mean volume of DWI, T2WI, and T1WI+C 10 minutes sequence in the target area is significantly smaller than that on the CT sequence, but the consistency is higher than that of CT sequences.The recognition of atelectasis by MRI was better than that by CT, which could reduce interobserver variability of primary tumor delineation in lung cancer with atelectasis. Among them, DWI, T2WI, T1WI+C 10 minutes may be a better choice to improve the GTV delineation of lung cancer patients with atelectasis. For both men and women, lung cancer is the most common diagnosis of cancer and the leading cause of cancer deaths . RadiothHowever, there is no in-depth study to explore the effect of different MRI sequences on the interobserver difference of target delineation in lung cancer with atelectasis. The purpose of this study is to explore the differences between different MRI-CT fusion sequences and single CT, so as to select the optimal MRI sequence for GTV target delineation of lung cancer with atelectasis.We retrospectively included 15 lung cancer patients who underwent positioning CT and MRI scans between May 2019 and June 2021. Due to the small number of lung cancer patients who underwent MRI localization, only 15 patients who met the criteria for enrollment were included during the study period. The 15 patients recruited in the study were pathologically diagnosed with lung cancer and imaging diagnosed with atelectasis. The diagnosis of atelectasis was decided by a senior doctor in the imaging department. CT- and MRI-simulated localization images were scanned in the same supine position within a week. Among them, six MRI sequences  were all scanned completely during MRI location scanning. Patients with surgically removed or metastatic lung tumors, unclear tumor display, contraindications to MRI, or intolerance were excluded. All the 15 patients in the group were diagnosed with central lung cancer. Among them, one patient had not received treatment, 11 patients had received chemotherapy, one patient had received chemotherapy and immunotherapy, one patient had received chemotherapy and anti-angiogenesis therapy, and one patient had received chemotherapy, immunity, and anti-angiogenesis therapy. The detailed data of the patients are listed in All patients underwent CT simulation  before radiotherapy. All of them were fixed with a vacuum negative pressure bag in supine position. Respiratory gating was used during the scan. The CT scan area covered at least the chest. The interslice thickness of the CT scan was 3 mm. At the end of the scan, the enhanced scan was performed by injecting 90 ml iohexol (350 mg/ml).2.The supine position of MRI was the same as the simulated position of CT. A 3.0-T, 70-cm Bore MR scanner  was used for MRI simulation positioning in our hospital. A 20-ml high-pressure injector was used to inject gadoterate meglumine (0.2 mmol/kg) at 2 ml/s followed by 20 ml saline to obtain the enhanced image. Meglumine gadolinate is a paramagnetic contrast agent. A low-dose injection only shortens the T1WI relaxation time and does not affect T2WI imaging. The MRI scanning sequence includes T1WI (WATER : BH AX LAVA-Flex), T1WI+C (WATER : BH AX LAVA-Flex-C), T1WI+C Delay (WATER : BH AX LAVA-Flex-C delay), T1WI+C 10 minutes (WATER : BH AX LAVA-Flex-C 10min), T2WI (RTr Ax T2 fs Propeller), and DWI, in which T1WI+C 10 minutes was specially scanned by our organization. T1WI+C, T1WI+C Delay, and T1WI+C 10 minutes sequences were scanned 15 s, 2 min, and 10 min after injection of a contrast medium, respectively. Because of the large endothelial space and immaturity of the vascular endothelium in the tumor, the contrast medium enters and exits faster than the normal tissue. During the early delayed imaging scan, the contrast medium enters the tumor tissue, and the normal lung tissue begins to strengthen, but the enhancement is not complete. At this time, the boundary between tumor and normal tissue is not clear. When scanning on the 10-min image, the normal lung tissue was completely enhanced, while the tumor tissue showed fast-in and fast-out realization. At this time, all the contrast media had been leaked, and the low signal of the tumor tissue was more obvious compared with that of the fibrous tissue and normal tissue, and the tumor boundary was clearer. We wonder whether T1WI+C 10 minutes sequences can also be used to distinguish lung cancer from atelectasis. This sequence has been proved to be helpful in the delineation of breast cancer lumpectomy cavity . The speWe used respiratory gating in CT and MRI positioning scans to reduce motion artifacts. The analog positioning images of CT and MRI were uploaded to Eclipse treatment planning systems (TPSs). According to the anatomy of the sternum, rib, vertebral body, pulmonary artery, and tumor, each MRI sequence  was automatically registered with CT through the planning system, and the fusion stability was increased by manual correction.Five radiation oncologists come from our hospital with 17, 16, 14, 7, and 6 years of working experience, respectively. The five observers, all of whom have rich clinical experience and are good at target delineation radiotherapy, were chosen in order to reduce the intra-observer variability. This paper mainly discusses the differences between observations. The five radiation oncologists sketched the tumor contours on CT and CT-MRI fusion images, respectively. The boundary between tumor and atelectasis was determined by referring to the CT structure and coronal and sagittal MRI images when drawing CT-MRI fusion images. The target areas of CT and MRI-CT images were sketched on the same day. We stipulate that CT images are drawn first. Then, CT-MRI image fusion was performed. The fusion image was drawn in the order of T1WI, T1WI+C, T1WI+C Delay, T1WI+C 10 minutes, T2WI, and DWI sequences. According to previous reports, observers were trained in advance to outline only the primary tumor and not the metastatic lymph nodes –14. If tP< 0.05 was used to determine statistical significance. The Shapiro–Wilk test was used to test the normality of the distribution of tumor volume, CI, and DC, respectively (P< 0.01), 0.97213 (P = 0.02592), and 0.96955 (P = 0.01611), respectively, suggesting that the distribution of tumor volume, DC, and CI is not normal, as shown in P-value <0.0083 was considered to be statistically significant.All analyses were conducted using R software (version 4.0.2) and SPSS software (Version 25.0). ectively . The W vP<0.01).The median and quartile spacing of the volumes of CT and MRI sequences as well as the results of statistical analysis are shown in The six MRI sequences were compared with CT sequences in pairs. The results showed that there were statistical differences in the T1WI+C 10 minutes, T2WI, and DWI sequences compared with the CT sequences . As seen in Every descriptive statistic of CI and DC is also found in In this study, we compared the differences of target volume, CI, and DC in locating MRI and CT in patients with lung cancer with atelectasis. DWI, T2WI, and T1WI+C 10 minutes sequences were statistically significant with CT in volume, CI, and DC. We can conclude that contours of the observers on CT overestimate the GTV, while MRI, especially the DWI, T2WI, and T1WI+C 10 minutes sequences, can better delineate the target area of lung cancer with atelectasis.GTV definition in lung cancer is one of the cornerstones in quality assurance of radiotherapy , 18. HowMany related studies have shown that MRI has a certain advantage in distinguishing atelectasis from tumor tissue because of its good soft tissue recognition ability –21. AmonThe study by Zhang et al.  comparedSix MRI sequences and not only DWI were studied in this study. Moreover, the effects of T1WI enhancement sequences on each time image were studied from pulmonary vascular perfusion imaging. At present, there is no literature to study whether MRI sequence injection contrast medium 10 minutes scan images can help distinguish tumor and atelectasis. In addition, few articles use CI and DC values to further quantify the differences between interobservers. This is the innovation point of this article.Studies by Weiss et al.  suggest The overall trend shows that the T1WI+C 10 minutes, T2WI, and DWI sequences are obviously superior to those of CT in target delineation, which may help improve the consistency of target delineation in patients with atelectasis of lung cancer and reduce the target volume and normal tissue volume and be better used in clinical practice. In recent years, MRI has been increasingly used in treatment planning in radiotherapy. With the advent of the era of precision radiotherapy, the definition of the tumor target volume is particularly important for conformal intensity-modulated radiotherapy. Compared with CT, MRI can provide better soft-tissue features, coupled with its multi-plane ability and enhanced imaging function; these advantages for target volume description outweigh its lack of electronic density information and potential image distortion. MRI radiation-less imaging, high time resolution, fast sequence, and functional imaging highlight the potential of MRI to improve accuracy. MRI-based delineation provides a better target description for radiotherapy treatment planning (RTP). Furthermore, MRI not only is suitable for initial radiotherapy of tumors but also may be used for retreatment, because it can distinguish between changes caused by cancer recurrence and changes caused by fibrosis after treatment. It can also better depict risk organs (OAR) to avoid doses in RTP –32. At p3 (20–369 cm3) for atelectasis and 40 cm3 (-7 to 131 cm3) for primary tumor. It is very important to detect the changes in atelectasis volume during treatment. In practical clinical work, lung cancer patients with atelectasis will be scanned by CBCT imaging every time they are treated with radiotherapy to closely observe the changes of atelectasis, modify the target area in time, and reduce the dose of normal tissue. At present, there is no related research on predicting the geometric changes of lung tissue caused by radiotherapy for atelectasis of lung cancer, which may be a direction of future research.In the course of radiotherapy, the regression or progression of the tumor can lead to the regression or expansion of atelectasis. Atelectasis changes in 10% to 30% of patients with non-small cell lung cancer during treatment . TennysoThis study does contain some limitations. Firstly, due to the strict inclusion criteria, the sample size is significantly reduced. Our team is now expanding the sample size for follow-up automatic sketching-related research. Secondly, target delineation lacks the gold standard of pathology, which may need to be further confirmed by prospective studies. Thirdly, the imaging findings of atelectasis and obstructive pneumonia are different in different periods. In the process of delineating, the inflammation period cannot be determined, which undoubtedly increases the difficulty of delineating the target area, which may increase the difference between the observers. Fourthly, the limitation of rigid registration itself could result in inevitable errors, which increases the contingency of the results. Fifthly, PET-CT could have been another modality studied.In addition, there is inevitably some recall bias. Finally, heterogeneity of the study group may affect the results.In summary, compared with CT, MRI may help to distinguish tumor from atelectasis. In particular, compared with other MRI sequences, T1WI+C 10 minutes is a new discovery. The results of T2WI and DWI are consistent with those of other studies, and they have good identification ability. The formulation of the MRI and CT dual-localization radiotherapy plan may be helpful to reduce the target volume and normal-tissue dose in patients with lung cancer with atelectasis. This study may provide a new direction for MRI-guided radiotherapy. In addition, the prediction of geometric changes of the target area before treatment in patients with lung cancer atelectasis is also worthy of further study.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by ethics committee of Shandong Cancer Hospital. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.HZ is responsible for the research design, planning implementation, statistical analysis, and drafting of the manuscript. CF, MF, LL, YC, HS, QZ, DH, and CL participated in the study design and data acquisition. MF and LL carried out the literature search and data aggregation. BL provided the theoretical proof and academic advice. WH is responsible for the topic selection, overall research guidance, and revision of 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 (No. 81773232), Academic Promotion Program of Shandong First Medical University  (No. 2020RC002), and Project of Young Taishan Scholars (No. Tsqn201909187).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.


Sixty (18.8%) participants showed anxiety symptoms and 14 (4.4%) had moderate or severe anxiety. The average score of SCL-90 was 140.88 ± 44.77, and 96 (30.0%) participants showed SCL-90 scores ≥160. Compared with the norm, significant differences were shown in eight of the nine SCL-90 factor scores, i.e., somatization, obsessive-compulsive, depression, anxiety, hostility, phobic anxiety, paranoid ideation, and psychoticism. The average score of ESHAC was 19.92 ± 4.54, and 114 (35.6%) participants did not reach the qualifying standard. Significant correlations were observed between the SAS score, SCL-90 total and factor scores, and ESHAC scores. The Logistic regression analysis showed that being born at very high altitude was an independent influencing factor  after controlling for other factors. Conclusion: Permanently living at very high altitude can influence the psychological and social fitness of healthy adults.Background: Environmental factors of high altitude, especially hypobaric hypoxia, may directly and persistently affect human physical and mental health. Our study was designed to assess the psychological and social fitness in healthy adults permanently living at very high altitude, i.e., an average elevation of 3650 m. Methods: In our observational study, 320 participants were included, among which 218 (68.1%) had resided in such a setting for more than 20 years. Participants underwent 138 assessments, including the Self-Rating Anxiety Scale (SAS), Symptom Check List 90 (SCL-90) and the Evaluation Scale of Human Adaptation Capability (ESHAC). SAS (20 items) and SCL-90 (90 items) were used to assess psychological fitness, and the ESHAC (28 items) was used to assess social fitness. Pearson analysis was used to assess correlations and Logistic regression analysis was performed to determine independent influencing factors. Results: The highest SAS score was 80 and the mean score was 43.26 ± 8.88, which was higher than the norm in China ( High altitudes (HA), defined as 2500 m or more above sea level, are characterized by lower atmospheric oxygen pressure, increased ultraviolet radiation exposure, cold climate and extremely low absolute humidity ,2. TheseHealth has been redefined by the World Health Organization as a state of complete physical, mental and social well-being, and not merely the absence of disease or infirmity . This dePsychological and social fitness are two important components of health. They can be assessed using a number of evaluation scales, for example, Symptom Check List 90 (SCL-90), Self-Rating Anxiety Scale (SAS), Self-Rating Depression Scale (SDS), Social Adaptation Self-evaluation Scale (SASS) and Vineland Adaptive Behavior Scales (VABS) ,11,12. CThirteen cultural stations run by neighborhood committees and the Public Cultural Service Center affiliated with the Chengguan District Culture and Tourism Bureau provide public cultural services for the residents living in all the communities in Lhasa City, the capital of the Tibet Autonomous Region. Chengguan District has 473,586 inhabitants. During the period between July 2021 and June 2022, 341 people were contacted to partake in our observational study. A total of 320 participants who participated in cultural activities actively and followed the inclusion criteria were included in the final analysis. The inclusion criteria were as follows: (1) older than 20 years and less than 59 years of age; (2) absence of severe diseases of major organs, including chronic heart failure, chronic respiratory failure, end stage liver disease, uremia and advanced malignant tumors, as determined by interviews conducted by the researchers; (3) no affective disorders, according to their medical history and medication history; (4) permanent residence in Lhasa City for at least three years, with an average elevation of 3650 m; and (5) sufficient educational level to correctly understand the meanings of three assessment scales. The participants filled in the questionnaires on paper independently and only one time. Completion of the questionnaire took less than one hour. Our research strictly followed the Declaration of Helsinki, and the written informed consent was obtained from all the participants.Both the SAS, which was developed by Zung in 1971, and the Symptom Check List 90 (SCL-90) assessments were used to assess the psychological fitness of our participants in this study, considering that anxiety was the most commonly observed disorder ,14. TwenThe SCL-90 questionnaire, which was proposed by Derogatis, was used to further assess psychological fitness based on the SAS assessment. A total of 90 items are included in the SCL-90 assessment. A scale of 1–5 is used for each item, and the maximum score is 450 points. Evaluative standard: An SCL-90 score ≥160 is indicates symptoms of psychopathology and psychological distress. The 10 factors of SCL-90 are somatization (12 items), obsessive-compulsive (10 items), interpersonal sensitivity (9 items), depression (13 items), anxiety (10 items), hostility (6 items), phobic anxiety (7 items), paranoid ideation (6 items), psychoticism (10 items) and other (7 items). For each SCL-90 factor, the average score is calculated and the total factor score is 5 points. The results of SCL-90 in our study were compared with the norm in China.The social fitness of our participants was assessed using the ESHAC, which was developed by Hong Ren and issued at the 9th National Sports Science Conference in China in 2011. Twenty-eight items are included in the ESHAC, which contains three subscales: adaptation to the natural environment (9 items), adaptation to the social environment (10 items) and adaptation to physiological change and resistance to diseases (9 items). Each item is rated on a ten-point scale (from 1 to 10); the maximal scores of the three subscales are 90, 100 and 90 points, respectively. To simplify the analysis, the final scores of the three subscales are the total scores divided by the number of items, and the final total score is 10 points for each subscale. The ESHAC score is the sum of the scores of the three subscales; therefore, the highest possible score is 30 points. An ESHAC score ≥18 is considered to be the qualifying standard.SPSS for Windows, version 21.0  was used for statistical analyses. The mean ± standard deviation and Independent-Samples T-test were used for continuous variables. The numbers (proportions) and Pearson Chi-Square test were used for categorical variables. The Single-Sample T-test was used to compare the SAS and SCL-90 scores of our participants with Chinese norms. Subgroup analyses were performed and the 320 participants were subgrouped according to gender, age, ethnicity, birthplace, marital status and duration of residence in Lhasa City. Pearson analysis was used to assess the correlation between the SAS standard score, SCL-90 total score, SCL-90 factor scores, ESHAC total score and the scores in the three subscales of the ESHAC. Logistic regression analysis was performed to determine the independent influencing factors for psychological and social fitness. The dependent variable was performance in the psychological and social fitness assessments, and six independent variables were analyzed. Participants who met one of the following criteria were considered to have poor psychological and social fitness: (1) SAS score ≥ 50; (2) SCL-90 score ≥ 160; and (3) ESHAC score < 18. The results are expressed as odds ratios (ORs) or adjusted odds ratios (AORs) and their 95% confidence intervals (CIs).Three hundred and twenty healthy participants were included in our study; their demographic characteristics are shown in p < 0.001), which indicated that the SAS score of our population was higher than the norm. For the incidence and severity of anxiety, 60 (18.8%) participants were assessed to have anxiety symptoms, including 46 (14.4%) with mild anxiety, 10 (3.1%) with moderate anxiety and 4 (1.3%) with severe anxiety. A comparative analysis of 60 participants with anxiety and 260 participants without anxiety is shown in Anxiety was the most common disorder among mental illnesses ,14. The p < 0.001), obsessive-compulsive , depression , anxiety , hostility , phobic anxiety , paranoid ideation  and psychoticism . Compared with the norm in China, higher scores were observed in our participants.Based on the SAS assessment, SCL-90 was used to further assess psychological fitness. The results are shown in p = 0.012), obsessive-compulsive  and depression . Participants aged 20–29 years showed higher scores in phobic anxiety  than those aged 30–59 years, and the Tibetan group had higher scores in phobic anxiety  than those with non-Tibetan nationalities. No significant differences were found in the other comparisons.A comparison of the total score of SCL-90 and the factor scores between males and females, between participants aged 20–29 and 30–59 years, and between Tibetan and non-Tibetan are shown in The ESHAC assessment may be used to assess the social fitness. It contains three subscales: adaptation to the natural environment, adaptation to the social environment and adaptation to the physiological environment . Among op = 0.003), birthplace  and duration of residence in Lhasa . For the three subscales, compared with Tibetans, the non-Tibetans had higher scores in the adaptation to natural environment (p < 0.001) and adaptation to physiological environment (p = 0.006) categories; however, no statistical significance was observed in the adaptation to social environment (p = 0.248) category. Similar results were shown for birthplace and duration of residence.Subgroup analyses were performed, and the results are shown in p < 0.001), whereas negative correlations were found between the SAS score and the ESHAC total score , and between the SCL-90 total score and the ESHAC score . Moreover, significant correlations were shown between the SAS score, nine SCL-90 factor scores and the scores in three subscales of the ESHAC.Pearson analysis was used to assess the correlation between psychological and social fitness. As shown in p = 0.004) and born at very high altitude  cohorts. Multivariable analysis showed that born at very high altitude was an independent influencing factor  after controlling for other factors.Logistic regression analysis was performed to determine the independent influencing factors of psychological and social fitness. Based on the results of SAS, SCL-90 and ESHAC, the 320 participants were divided into two groups, comprising 162 (50.6%) participants who had at least one positive result among the three assessments and the remaining 158 (49.4%) participants who had no positive result and served as the control group. The dependent variable was the psychological and social fitness assessments, and six independent variables were analyzed, namely, gender, age, ethnicity, birthplace, marital status and duration of residence . UnivariLhasa City is situated at the Qinghai–Tibet Plateau, which as the highest plateau on Earth, has long been known as the roof of the world. Permanently living at very high altitude may affect human physical and mental health . Our stuMental health is one of the important dimensions of overall health. Indeed, as stated by the World Health Organization, “there is no health without mental health” . Mental SAS and SCL-90 questionnaires were used to assess the psychological fitness of our participants. Considering that anxiety is the most common disorder among mental diseases ,14, the The SCL-90 assessment in our study showed that the total score was 140.88 ± 44.77; the scores in eight of the nine factors were higher than the norms. Moreover, statistical significances were found in some SCL-90 factor scores between males and females, between participants aged 20–29 and 30–59 years and between Tibetans and non-Tibetans. Our results were compared with those obtained from other researchers . One stuThe social fitness of our participants was assessed by the ESHAC, which was developed by a Chinese scholar. We believed that this questionnaire is particularly suited to Chinese populations. Firstly, the positive results in the SCL-90 and ESHAC were compared. Ninety-six (30.0%) participants showed SCL-90 score ≥ 160, and 114 (35.6%) did not reach the qualifying standard. The two rates were very close, and no significant difference was found. Secondly, the correlation between the ESHAC, SAS and SCL-90 scores was determined by Pearson analysis. Significant correlations were shown between the ESHAC total score, scores in three subscales of ESHAC, the SAS score and nine SCL-90 factor scores. In addition, subgroup analyses were performed and significant differences in ethnicity, birthplace and duration of residence were observed. Further analysis showed that the differences were mainly due to the adaptation to the natural and physiological environment, as opposed to the social environment.Influencing factors of poor psychological and social fitness were determined by a Logistic regression analysis. The results showed that being born at very high altitude is an independent influencing factor , after controlling for other factors. One study from China included 644 older adults and found that cognitive impairment was more common in people living at high altitude . AnotherFor marital status, 130 (40.6%) participants lived without a spouse or partner; this proportion seemed to be higher than that of the general population. The main reason for this may have been that more young people who actively participated in cultural activities were included in our study; the mean age of our cohort was 30.5 ± 8.2 years. This study was intended to promote the safety of participants living at very high altitude, although our approach may have led to a selection bias. For the differences between male and female participants, no statistical significances were found in the SAS score, SCL-90 total score or ESHAC scores. However, for the SCL-90 factor scores, males had lower scores in somatization, obsessive-compulsive and depression than female participants.Our study showed that permanently living at very high altitude may influence the psychological and social fitness of otherwise healthy adults. Psychological and social fitness are the two important components of health, and they also need the same attention as physical fitness. In future, some effective strategies should be implemented to improve the psychological and social fitness in people residing at high altitude.


Both MRI and VGS had a good concordance with the pathology report . However, MRI had a false-negative rate (38.1%) that was greater than VGS (0%) in cases of cervical tumor size <2 cm. We found a good concordance between histology and VGS in the stromal infiltration assessment, with 89% sensitivity (95% CI 0.44–0.83) and 89% specificity (95% CI 0.52–0.86). VGS is a simple, inexpensive, widely available, and fast execution method that can complement ultrasound in particular cases and show a good correlation with MRI in the assessment of tumor dimensions, with a better performance in detecting small tumors (<2 cm).This study aims to analyze the sensitivity of vaginosonography (VGS) and magnetic resonance imaging (MRI) in the preoperative local evaluation of early-stage cervical cancers and to assess their accuracy in the detection of tumors, size of the lesions and stromal invasion by comparing them with the final histopathology report. This single-center study included 56 consecutive patients with cervical cancer who underwent VGS and MRI from November 2012 to January 2021. VGS significantly overestimated the lesion size by 2.7 mm ( Cervical cancer is one of the leading causes of morbidity and mortality in women, representing the second most common cancer in developing countries and the fourth worldwide .Although a pelvic examination continues to be the first approach to detecting cervical cancer, the 2018 FIGO classification has updated the cancer staging, highlighting the central role of imaging. In fact, imaging techniques allow a better definition of the tumor size, parametrial invasion, extension to the pelvic wall or adjacent organs, and lymph node involvement [Many diagnostic methods are used in clinical practice; their use depends on availability, cost, and the expertise of clinicians and radiologists. Although magnetic resonance imaging (MRI) is considered the main technique to assess tumor size and parametrial extension ,4,5,6, tVaginosonography (VGS) is a technique first described by Dessole et al. in 2003  that comOur study aims to analyze the sensitivity of VGS and MRI in the preoperative local evaluation of early-stage cervical cancers and to assess their accuracy in the detection of tumor presence, size of the lesion, and stromal invasion by comparing them with the final histopathology report. We enrolled 81 consecutive women diagnosed with cervical cancer by clinical examination and referred to the European Institute of Oncology from November 2012 to January 2021. All patients underwent diagnostic evaluation, including a cervical biopsy or a pathological review of the original slides if the initial diagnosis was made at a different institution. All tumors were staged according to the FIGO (2018) criteria using vaginal and rectal examinations, standard chest and abdominal computed tomography (CT), TVUS or TRUS, and MRI. Twenty-five patients were excluded either because they received non-surgical treatments . Data frThe results of MRI and VGS were compared with the pathology report. If the MRI scan was performed outside the European Institute of Oncology, an internal gynecological oncology radiologist reviewed the images.All patients underwent both TVUS and VGS. The scans were performed by two experienced ultrasound examiners with 25 and 15 years of experience in ultrasound for gynecologic oncology (D.F. and A.M.V.U.), respectively. The examiners were blinded to the MRI results.All women were examined in the lithotomy position with an empty bladder using high-end ultrasound equipment, with the frequency of the vaginal probes varying between 5.0 and 9.0 MHz. Ultrasonographic images were acquired according to a standardized time gray-scale examination technique.The VGS procedure consists of four steps: (1) a 5.3 Fr sonohysterography catheter is inserted into the vagina by an assistant; then, (2) the probe is inserted by the operator; (3) the assistant closes the vaginal channel by narrowing the major labia; (4) approximately 60 cc of room temperature saline solution is instilled while the ultrasound is performed. The following parameters were evaluated by VGS and TVUS: tumor presence, maximum diameter of the tumor, parametrial and fornix infiltration, vaginal involvement, stromal invasion, anterior and posterior septum involvement, and blood flow of the lesion. The blood flow was assessed by a color Doppler score based on the intensity of the color signal with the following value ranges: (1) no flow, (2) minimal flow, (3) moderate flow, and (4) high vascular flow.A dedicated pathologist, highly experienced in gynecologic cancers, assessed the specimens and described the size and extent of the tumor, histology type, grading, stromal invasion, minimum free thickness, and maximum deep infiltration.Approval was obtained from the ethics committee of the European Institute of Oncology (UID 2731), and all patients signed an informed consent form.Patients’ characteristics were summarized either by count and percent or mean and standard deviation (SD) for categorical and continuous variables, respectively. Methods agreement was estimated using Cohen’s kappa coefficient and Lin’s correlation. Lesion size (maximum diameter) differences between VGS and MRI with respect to histology were summarized by mean, SD, and 95% confidence intervals (95% CI) and compared using Bland–Altman and Passing–Bablok regression methods. All method comparisons considered histology as the gold standard. Cross-tabulation of the categorical variables was tested for significance using Fisher’s exact test; the mean comparison was made using the unpaired t-test. All tests were two-tailed and considered significant at the 5% level. All analyses were done using SAS 9.4  and STATA .The demographic and clinicopathological characteristics of the 56 patients included in the study and their previous treatments are reported in p < 0.001).Forty-two (75%) lesions were described as highly vascularized (color score 4—CS 4), 12 (21.4%) as moderately vascularized (CS 3), and 2 (3.6%) as minimally vascularized. We did not find any correlation between vascularization and histotype, but we found a statistically significant correlation between vascularization and the largest dimension of the lesion, with a mean of 26.9 mm in CS 4 and 12.4 mm in CS 3 . Squamous tumors were predominantly hypoechoic , while adenocarcinomas were mainly hyperechoic  , a specificity of 89% (95% CI 0.52–0.86), and a Cohen’s kappa of 0.78. When the stromal invasion was >2/3, the histological exam identified five of six (83.3%) cases with positive lymph nodes (6), while VGS predicted four cases (66.6%).Although we did not compare the fornix infiltration to the gold standard because of the early stage of the tumors, we observed eight false-positive cases of fornix infiltration with MRI and two with VGS, with a specificity of 85.7% and 96.4% and a negative predictive value (NPV) of 88.9% and 100%, respectively.VGS significantly overestimated the lesion size by 2.7 mm compared to the gold standard. On the other hand, MRI underestimated it by 1.9 mm . Figure When comparing the size of the lesions, both MRI and VGS had a good concordance with the pathology report , showingThe role of ultrasound in the pre-treatment evaluation of cervical cancer is well established ,16,17,18To the best of our knowledge, this is the first study that investigates the accuracy of VGS in the assessment of early-stage cervical cancer. VGS is an inexpensive and well-tolerated ultrasound technique that can increase the image quality of cervical tumors. The acoustic windows created by saline solution results in a distance of the probe to the lesion, the distension of vaginal walls, and the exclusion of hindering factors, including vaginal collapse, bleeding, and/or mucus secretions.Compared to histology (used as the gold standard), VGS and MRI assess the dimension of the lesion with similar accuracy. However, we found that VGS tended to overestimate the dimensions, whereas MRI underestimated them. Previous studies have reported that ultrasounds overestimate tumor dimensions ,17. DespThe value of Lin’s concordance in the assessment of the lesion dimensions was better between VGS and histology than MRI and histology. This is because MRI did not detect some small tumors, recorded as 0 mm in our study. Indeed, when the tumor was <2 cm, MRI was not able to identify 38.1% of the tumors. Therefore, the real detection rate of MRI was 47% (10 out of 21) versus 76.1% of VGS (16 out of 21). Hence, VGS is more sensitive than MRI in identifying small lesions. Prior studies did not find that TVUS was better than MRI in the detection of lesions <2 cm ,20. OnlyAs our study focuses on early-stage cervical cancers, we have limited data on fornix infiltration. Nevertheless, we found that VGS was more sensitive than MRI in excluding fornix infiltration, with an NPV of 100% and 88.9%, respectively. Recently, similar results have been reported by comparing the accuracy of histology to TVUS, MRI, and clinical examination under anesthesia . TVUS caWe confirmed that different histotypes have different echogenicities, as demonstrated in a previous report . EpsteinAs observed in previous studies ,9,11,22,Stromal infiltration is another interesting issue. Some studies reported that comparing ultrasound and MRI results with the final histology report led to high false-positive rates ,17,18, aIt is a well-known matter that the incidence of lymph node metastasis in cervical cancer depends on the stage: 2% in IA2, 14–36% in IB, 38–51% in IIA, and 47% in IIB (14). Stromal infiltration is one of the independent factors identified for the risk of lymph node metastases in patients with cervical cancers, along with age, tumor size, lymph vascular space invasion, histological grade, and type ,24. In oVGS is a simple, inexpensive, widely available, and fast execution method that can complement ultrasound in particular cases. To our knowledge, this is the first study that has evaluated the diagnostic accuracy of VGS and MRI in cancer and compared it with the final histology. Our results show a good correlation between VGS and MRI in the assessment of tumor dimensions, highlighting the better performance of VGS in detecting small tumors (<2 cm) and in predicting the absence of fornix infiltration as it shows higher sensitivity.Moreover, the acoustic windows created between the ultrasound probe and the tumor can improve the study of the echogenicity of squamous tumors and adenocarcinomas by emphasizing the hyperechogenicity of the latter. There was a very good concordance between stromal invasion predicted by VGS and histology, which is an independent factor for the risk of lymph node involvement.However, VGS has some limitations: despite its widespread availability, it is an operator-dependent technique, and this can limit its application to centers with highly qualified examiners.Future studies are needed to assess the use of VGS in patients with advanced tumor stages, especially the ability to correctly identify patients with vaginal spread and septal infiltration.


Progranulin (PGRN), endothelial cell-specific molecule-1, clusterin (CLU), and human epididymis protein 4 (HE-4) are novel proteins reported to have diagnostic and prognostic potential in lung cancer. Here, we aimed to identify the markers with high sensitivity and specificity in distinguishing malignant pleural fluids from other pleural fluids.This prospective, descriptive study was conducted at a medical faculty hospital between 2016 and 2019. The study population consisted of 90 patients <18 years of age with pleural effusion (PE). Levels of pleural fluids of PGRN, endothelial cell-specific molecule-1, CLU, and HE-4 were measured with enzyme-linked immunosorbent assay kits under the manufacturer’s manual.P < .05). Cutoff values were achieved by receiver operating characteristics analysis for CLU, HE-4, and PGRN to distinguish between malignant and benign groups. For diagnosis of MPE, the sensitivity and specificity values were found as 0.66 and 0.67 for a cutoff value of CLU of 18.29 mg/L (P = .00), as 0.76 and 0.76 for a cutoff value of HE-4 of 9.33 mg/L (P = .00), and as 0.66 and 0.67 for a cutoff value of PGRN of 105.91 mg/L (P = .001).Of 90 patients, 54 were men, and 36 were women (mean age 65 ± 16 years). Of pleural fluids investigated, 23 (25%) and 67 (74%) were transudates and exudates, respectively. Of exudates, while 27 (40%) and 19 (28%) were parapneumonic PE and tuberculous PE, respectively, 20 (29%) were malignant pleural effusion (MPE). Levels of all biomarkers in exudate fluids were found significantly higher than those of transudate fluids. CLU, HE-4, and PGRN levels in MPE were also found significantly higher than benign fluids (HE-4 having high sensitivity and specificity can be a potential diagnostic marker in distinguishing between malignant and benign effusions, and these findings can constitute a basis for future research. Common causes of exudative pleural effusion (EPE) are malignancy, parapneumonic effusion, and tuberculosis (TB). In various studies, malignant pleural effusions (MPE) are shown as one of the most common causes of exudate fluids (42%–72%). MPE can be due to the complication of any malignancy; however, MPE incidence ranges between 7% and 23% in patients with lung cancer. Malignant effusions in lung cancer not only impairs the quality of life of patients but indicate a poor prognosis, as well. Although PEs are malignant in most lung cancer patients, benign effusions may also develop during the diagnosis or the clinical course of the condition. In a study by Porcel et al, benign causes of PEs in those with lung cancer were shown to consist of up to 20% of all effusions. Therefore, the diagnosis of malignant effusion in lung cancer is of vital importance in terms of the early initiation of therapy and management of symptoms. Although critical, accurate diagnosis of MPE still remains a difficult procedure. The cytological examination is the traditional and standard method for its diagnosis; however, its sensitivity varies between 30% and 60%. Additionally, the efficacy of sequential blind pleural biopsies has been asserted to be limited, and so blind biopsies of MPEs are diagnostic in only 20% of patients with negative cytology of malignant effusion. Even though it is the “gold standard” in the diagnosis of malignancy, the thoracoscopic pleural biopsy is more invasive and costly. For these reasons, medical science now keeps seeking an answer to the question of “Can PEs be evaluated with less invasive and inexpensive methods?”The first step in the differential diagnosis of pleural effusion (PE) is to determine whether the effusion is transudative or exudative. Light criteria are used to distinguish between transudates and exudates.,9 Vascular endothelial growth factor-A (VEGF-A) is a critical cytokine provoking the extravasation and formation of MPEs. Even so, VEGF is not the only vasoactive cytokine involved in MPE. Other molecules provoking the formation of MPEs are pro-inflammatory cytokines, such as tumor necrosis factor (TNF), chemokine (C-C motif) ligand 2 (CCL2), and osteopontin (OPN) Osteopontin (OPN), also known as bone sialoprotein I (BSP-1 or BNSP).MPE has traditionally been associated with the obstruction of parietal pleural stomata or the occlusion of lymph vessels in mediastinal lymph nodes. However, such features as the lymphatic block, inflammation, increased angiogenesis, and vascular leakage are deprived of referring fully to MPE. and in the later period, ESM-1 expression was found out to be associated with lung, kidney, and breast cancers.–15Endothelial cell-specific molecule-1 (ESM-1) is a 50 kDa soluble proteoglycon composed of a mature polypeptide of 165 amino acids and associated with the endothelial cells expressed by the renal and pulmonary endothelium. ESM-1 was originally cloned from a human endothelial complementary DNA by Lassalle et al,Progranulin (PGRN) is a glycoprotein with 593 amino acids, the mRNA of which is expressed both in vitro and in vivo by many epithelial cells. PGRN levels have been identified as a new parameter determining the rate of epithelial proliferation leading to tumor growth in some cell lines. This enables PGRN to play a critical role in epithelial proliferative homeostasis and may lead to important consequences in the development and growth of carcinomas. The abnormal expression of CLU protein is reported to be associated with cardiovascular diseases, inflammatory diseases, and tumorigenesis.,19 In most of the clinical studies, it was shown that CLU is an indicator of poor prognosis associated with tumor recurrence and metastases, including lung cancer, and that secretory clusterin (sCLU) is associated with increased survival and proliferation of non-small cell lung cancer (NSCLC), thus leading to poor prognosis.Clusterin (CLU) exists widely in various tissues and organs and is involved in several biological processes, including fluid transport, cell apoptosis, and cell adhesion, etc. In a meta-analysis including a total of 1412 patients from eight studies, it was reported that higher HE-4 expression was associated with poorer overall survival in Asian lung cancer patients although no significant relationship was found in Caucasian lung cancer patients. Another meta-analysis including a total of 16 studies (1756 lung cancer and 1446 controls) also revealed that HE-4 had a moderate diagnostic accuracy rate for lung cancer; the analysis of HE-4 should be interpreted in parallel with the clinical findings and results of other traditional tests; more studies are needed to meticulously evaluate the diagnostic accuracy of HE-4 for lung cancer.The protein of human epididymis protein 4 (HE-4) is encoded by a gene located on chromosome 20q12-13.1 and is known as a precursor of the human epididymis protein. HE-4 is frequently overexpressed in ovarian cancers; however, other expressions are also found in lung, endometrial, breast adenocarcinomas, and mesotheliomas, and to a lesser degree, in the gastrointestinal, kidney, and transitional cell carcinomas. In our study, it was investigated whether ESM-1, PGRN, CLU, and HE-4 can be utilized in PE to differentiate between malignant patients and nonmalignant diseases causing PEs.For MPEs, a standard biomarker has yet to be identified. Ideal biomarkers should be cost-effective and easily measurable, sensitive, and specific to the disease investigated, and contribute to decision-making for the diagnosis.Ninety patients with PE admitted to the department of chest diseases of a university hospital between 2016 and 2019 were prospectively enrolled in the study. On admission, pleural fluids were drawn from each patient through the thoracentesis and centrifuged at 3000 rpm for 10 minutes. The supernatants were frozen and stored at −80° C until analysis. In order to investigate PGRN, ESM-1, CLU, HE-4 concentrations in Pes, the levels of concentrations were measured for PGRN with the human progranulin immunoassay kit , for ESM-1 with the enzyme-linked immunosorbent assay (ELISA) kits , for CLU , and HE-4 . The levels were evaluated using the immunoassay kits under the manufacturers’ instructions. The biochemists assessing all samples were blinded to perform the assays, and all levels were determined in a duplicated form. The present study was conducted after written informed consent was obtained from all participants. The study protocol was approved by the ethics committee of the institution.The determination of PE etiology for each patient was performed, based on the clinical presentation, diagnostic test results, and response to the treatment. Based on Light’s criteria for exudate fluids, while levels of pleural fluid/serum protein and pleural fluid/serum lactate dehydrogenase (LDH) were defined as >0.5 and >0.6 respectively, level of pleural fluid LDH was defined as >2/3 of the upper limit of the reference range for serum LDH. If malignant cells were observed on cytological examination or in the pleural biopsy specimen, MPE was diagnosed. As consistent with the design of the study, only malignant effusions associated with lung cancer were recorded. Parapneumonic effusion (PPE) was defined as the patient’s novel fever, purulent sputum, and the presence of pulmonary infection with pneumonic infiltration on chest radiography, responding to antibiotic treatment. However, tuberculous pleural effusion (TPE) was diagnosed through the pathological confirmation by pleural biopsy or the positivity of biopsy material or TB culture of pleural fluid in those with lymphocyte predominance and high level of adenosine deaminase (>40 IU/L) in the pleural fluid.U test was used for nonparametric variables. The receiver operating characteristics (ROC) curves were analyzed to determine the optimum cutoff values and to compare the diagnostic accuracy of the markers. The cutoff value for each marker was determined based on the best diagnostic efficiency obtaining the balance between the sensitivity and specificity. If a P value was below 0.05, the difference was considered statistically significant. The statistical analysis was performed using the Statistical Package for the Social Sciences software version 18.0 for Windows .The data detected in the study are presented at median and interquartile intervals. To compare the differences between the groups, the Kruskal–Wallis or the Mann–Whitney P > .05).A total of 90 patients, including 21 malign PE in primary lung carcinomas group and 69 in the benign PE group were enrolled in the study. Among lung cancer patients, 11 adenocarcinomas, eight squamous cell carcinomas, and two small cell carcinomas were detected. There were 19 TPE, 27 PPE, and 23 transudate PE in the group with benign PE. Of 90 study participants, 54 (69%) were male, and the age level was detected not to be significantly different between malignant and benign PE groups (P = .001). The clinical features of each group are summarized in Table However, the age level of the patients with TPE was found to be significantly lower, compared with the other groups P = .00. The cliP < .05). Even so, there was a borderline significance for CLU (P = .05) (Table P < .05). Average concentrations of CLU, ESM-1, HE-4, and PGRN in patients with MPE, TPE, PPE, and transudate are shown in Table The pleural fluid levels of ESM-1, HE-4, and PGRN were significantly higher in the exudative PE group, compared with the transudate group (5) Table . AdditioP < .001)   Fig. .P < .001) . However, no significant difference was found between MPE and PPE in terms of PGRN (P = .747).Mean fluid levels of PGRN were found as 317.06, 133.97, 380.33, and 65.37 ng/mL for MPE, TPE, PPE, and transudate groups, respectively Table . The levP = .001, χ2 0015.487); however, ESM-1 was not significantly different from that of the patients with TPE and PPE  . Considering the cutoff value for HE-4 as 9.33 mg/L, the sensitivity and specificity rates were found to be 0.76 and 0.76, respectively; AUC was 0.874  (P = .00). Given the cutoff value as 105.91 mg/L for PGRN, the sensitivity and specificity rates were calculated as 0.66 and 0.67 respectively, and AUC was 0.733  (P = .001).Given the cutoff value as 18.29 mg/L for CLU in the diagnosis of MPE, the sensitivity and specificity were calculated as 0.66 and 0.67, respectively. The area under the curve (AUC) was also found as 0.776   (P < .001). In previous studies, the expression of HE-4 has also been shown in lung cancer cell lines.,26In our study, we investigated the pleural fluid levels of ESM-1, PGRN, CLU, and HE-4, considered being a useful diagnostic approach in distinguishing between pleural fluids, especially malignant fluids from exudate fluids. We revealed that all of the biomarkers studied in the study had higher levels in the exudative fluid than those in transudative pleural fluid. However, grouping PEs as transudates and exudates does not provide a disease-specific diagnosis. In previous studies, various researchers, including Light,P = .001). With the use of an optimal threshold of 652.2 pmol/L, the HE-4 level distinguished malignant lung cancer from benign lesions with a sensitivity of 78.3% and specificity of 75.0%. Additionally, patients with lung adenocarcinoma demonstrated significantly higher levels of HE-4 than those with squamous cell carcinoma and small cell carcinoma. In another study conducted in 88 patients with different types of PE by Elsammak et al, both serum HE-4 levels and HE-4 levels of PE were detected to be significantly higher in the patients with lung and extrapulmonary malignant effusions than those with transudative or nonmalignant exudative effusion. In the study, a cutoff value of 1.675 pmol/L was revealed in pleural fluid with a diagnostic sensitivity of 85.3% and a specificity of 90.7%, predicting malignant PEs.In light of the literature, there are so few studies investigating the diagnostic value of HE-4 in malignant PE among those with lung cancer. In a study performed by Lv et al, HE-4 protein in PE of 60 patients with lung cancer was compared with 56 patients with benign lung disease. In the study by Lv et al, as consistent with our findings, the HE-4 level was significantly higher among lung cancer patients than that of those with benign lung disease (P < .0001); the area under the ROC curve was 0.84 for HE-4 . In the same study, HE-4 levels were observed to increase significantly in all different histological subgroups of lung cancer, and also among those with the smallest tumors (P = .002). In another study, when compared with small-cell, squamous, and large-cell lung cancers HE-4 tissue expression was shown to be the highest in adenocarcinomas of the lung.–33 In our study, while no significant difference was found between the types of malignancies, most of the malignant fluids were detected in the patients with adenocarcinomas, and the number of small cell carcinomas was seen to be very low. For this reason, we may have found no significant difference between HE-4 levels of the fluids. Finally, in the study by Galgano et al, it was demonstrated that of 47 patients with mesothelioma, 28 had HE-4 expression somewhat.In the study where the serum samples obtained from 100 patients with lung cancer, 57 patients with benign lung disease, and 274 healthy controls by Choi et al, HE-4 levels were found to be significantly higher in the patients with lung cancer than those with benign lung disease and healthy controls  from MPE could not be obtained in our study.In the study by Scherpereel et al, a significant increase was observed in the serum levels of ESM-1 in patients diagnosed with lung cancer. To the best of our knowledge, there is no previous study evaluating the differential role of PGRN in pleural fluids. In our study, PGRN levels of parapneumonic, malignant, and tuberculosis PEs were higher than the transudate level. PGRN level was also found to be significantly higher in patients with lung cancer, compared to TB effusions; even so, no significant difference was found in distinguishing between MPE and PPE. Among the exudates, the highest level of PGRN was observed in PPE.PGRN is a multifunctional glycoprotein playing a role in the formation, development, inflammation, and repair of tumors. Immunohistochemistry staining results also revealed that CLU is highly expressed in vascular cells  in lung cancer tissues, indicating that CLU may be involved in tumor angiogenesis. In the meta-analysis of 26 immunohistochemical studies, where the prognostic role of CLU was investigated in multiple malignant neoplasms, CLU has been reported to be a potential biomarker for both recurrence-free survival and disease-free survival. Evidence also suggests that CLU is overexpressed in patients with metastatic tumors and experimental metastasis models. Specifically, CLU has been shown to play a role in the antiapoptotic capacities, the enhancement of treatment resistance, and the induction of epithelial-mesenchymal transition, all of which are associated with cancer metastasis. In a study, it has been reported that inhibition of CLU increases the cytotoxic effects of chemotherapeutic agents and improves the survival of stage-III and IV cancer patients. In our study, CLU was detected to be higher levels in MPEs, compared to transudate and nonmalignant fluids; however, the specificity and sensitivity rates were found to be lower in distinguishing between malignant and nonmalignant fluid.CLU is a secretory glycoprotein and has been stated to be upregulated in various human cancers, including breast, colon, prostate, and lung cancers.The level of HE-4 was found to be higher in patients with primary lung carcinomas, compared with transudative fluids and other exudative fluids. HE-4 could be a potential marker in detecting tumors in the pleural fluids of primary lung carcinomas. However, we consider that to benefit as a diagnostic tumor marker in the clinical setting, HE-4 should be investigated further in tissue expression and at serum levels in benign and malignant lung diseases, as well as the substrates of different lung cancers.The present study was performed only in a single province, and the number of study population was limited. For this reason, more comprehensive studies with larger populations are needed to shed light on the entity.Investigation: Fatma Hümeyra Yerlikaya, Sebnem Yosunkaya, Soner Demirbas, Ummgülsüm Can.Methodology: Ummgülsüm Can.Resources: Soner Demirbas.Supervision: Soner Demirbas.Writing – original draft: Soner Demirbas.Writing – review & editing: Celalettin Korkmaz.


Avibacterium paragallinarum is the aetiological agent of infectious coryza in poultry. Vaccines are the best means of control, helping reduce clinical signs and colonization of this bacterium. Most vaccines are based on international reference strains, or, lately, regional strains, but, generally, without any information regarding their virulence. The characterization of the pathogenicity of 24 Av. paragallinarum strains of the three Page serogroups, including four variant strains of serogroup B, all isolated from infectious coryza outbreaks in Peru, was performed. After experimental inoculation into the infraorbital sinuses, information regarding their capacity to induce infectious coryza typical clinical signs, spreading, and colonization was recorded. Furthermore, after intraperitoneal inoculation, septicaemia and death were registered. Differences among strains in these parameters were observed, even within strains from the same serogroup. Finally, the four most pathogenic strains, one from each serogroup, were chosen to formulate an experimental vaccine that was tested successfully against homologous challenges in reducing clinical signs and colonization in vaccinated birds compared to unvaccinated ones. This is the first time that Av. paragallinarum strains from Peru were studied thoroughly for their virulence in a search for improving vaccine formulation.Worldwide,  Avibacterium paragallinarum, is an acute respiratory disease that affects poultry worldwide [Av. paragallinarum, and it is mainly reported in layers with a reduction of up to 40% in egg production, but it can get complicated when other pathogens are associated, increasing these losses up to 85% with considerable mortality rates [Av. paragallinarum [Infectious coryza, caused by orldwide . Page clorldwide . Later, orldwide ,4. Neverorldwide ,6,7,8. Iorldwide . There ity rates ,8,9,10. ty rates . On the ty rates ,13,14,15ty rates ,16,17 anllinarum .Av. paragallinarum has been isolated even from inside eyeballs in acute cases. Columbia agar base plates plus 7% equine hemolyzed blood [The distribution of the three Page serogroups differs from country to country. Nevertheless, not all the countries report the distribution of the different serogroups. The three Page serogroups were reported in Argentina , Brazil ed blood  may be ued blood . The moled blood .Av. paragallinarum infections, spreading, and reduce economic losses, good management practices and strict biosecurity measures should be applied, such as in the case of other diseases. The use of some antibiotics may reduce clinical signs, but the birds remain carriers for life, and, even more, Av. paragallinarum is likely to develop resistance [Av. paragallinarum. Hence, a good vaccination program is very efficient, as inactivated vaccines are very protective when they are well prepared [To protect the chickens against sistance ,42,43. Asistance ,45 but tprepared . Generalprepared ,47,48, aprepared ,50. Furtprepared .Av. paragallinarum strains is variable and was evidenced using field and reference strains [The pathogenicity of  strains ,53. Thes strains ,49. Simi strains ,54. NeveAv. paragallinarum strains in inactivated vaccines might be improved after being selected not only for their serotype but also according to their pathogenicity. For that purpose, the classification of local Peruvian stains of Av. paragallinarum was performed to study their capacity to cause clinical signs, horizontal infection as well as cause septicaemia and death in experimentally inoculated chickens. Afterwards, the most pathogenic strains of serogroups A, B, Bvar, and C were included in an experimental bacterin, and its efficacy was tested in vivo against homologous experimental infection with the same four serogroups.Therefore, it is suggested that the inclusion of Av. paragallinarum, isolated from poultry farms in Peru, were used , H8 (serogroup B), and H32 (serogroup C), as it was not possible to determine their serogroup using the international reference antiserums and were regarded as variants of serogroup B (Bvar) [Furthermore, these strains were serotyped and classified into the three Page serogroups using inhibition of the haemagglutination assays  using raB (Bvar) ,44,50. SAv. paragallinarum strain was thawed from the frozen vial and was streaked onto Columbia Blood agar base  + 7% equine hemolyzed blood (CLBA). These plates were incubated for 48 h at 37 °C in a microaerophilic atmosphere using the candle jar method. Afterwards, a few isolated colonies were used to seed a Brain Heart Infusion (BHI) broth  supplemented with 25 µg/mL of reduced NAD . This tube was incubated as above and before inoculation, bacterial enumeration was performed using triplicate CLBA plates [Before each trial, the corresponding avianized A plates .in vivo by inoculation of 0.2 mL of the correspondent inoculum into the infraorbital sinus of two 25-days-old SPF chickens. Two days after the inoculation, upon the manifestation of clinical signs, the birds were euthanized, each strain was re-isolated, and the growth was aliquoted and kept frozen (−80 °C) until usage.To increase the virulence of the strains  and stanSalmonella-free status was confirmed by bacteriological analysis of the meconium of all chicks. These samples were pooled together into 100 mL Tetrathionate Broth (TB) (Merck 1.05285), followed by subcultures onto XLD  + 0.46% Tergitol 4 (Sigma 100H0494) (XLDT4) agar plates. Salmonella-like colonies were further checked by biochemical tests to discard the presence of Salmonella sp.One-day-old Hy-Line layers chicks were used . ad libitum from the outside through plastic pipes into the feeders. Animals received Salmonella-free balanced food based on a vegetable protein diet, free from meat or fishmeal, without adding any antibiotic or coccidiostat drug. The absence of Salmonella spp. in feed was confirmed by bacteriological cultures of all batches using Lactose broth followed by a subculture into TB and XLDT4 agar plates as previously described [All chicks were reared until the beginning of each trial under strict biosecurity measures. During the first week of life, all birds were individually identified with numbered wing tags. The distribution of the chickens into the experimental groups, as well as the selection of the isolator, were random and coded in a manner that the operators or investigators were blind to treatments. Each isolator was used to house no more than 10 chickens. The rearing facility consisted of a closed room with a ventilation system and automatically controlled temperature and lighting systems. Before entering the building, all personnel had to take a shower and used disposable gloves, hats, masks, clean clothes, and boots. Each isolator has glassed front doors and a wire net floor to avoid contact with faeces. Water was administered by nipples and feed was given escribed .Four sets of trials were performed: the first two sets of trials were done to investigate the pathogenicity of all strains regarding their capacity to cause clinical signs after inoculation  and their capacity to horizontally infect non-inoculated chickens . The capacity to colonize the inoculated infraorbital sinuses was also studied in both trials by bacteriology cultures.The third set of trials was performed with the most pathogenic strains that were selected from Trials 1 and 2 to learn the capability to cause septicaemia after intraperitoneal inoculation and its lethal capacity. Finally, after performing a qualitative analysis of the results from the three trials, the most pathogenic strains, one for each serogroup, were selected for the elaboration of an experimental bacterin that was tested for efficacy against homologous challenges with the same strains. A description of the experimental design is available in In all trials, similar procedures were used. The chickens were distributed into two isolators for each experimental group. The inoculations were done by injection of 0.2 mL of the correspondent inoculum into the left infraorbital sinus of each chicken. Clinical-signs scores of 0–4 were registered individually for each chicken. In Trials 1 and 4, on day 5 post-inoculation, all chickens were euthanized, heads were separated from the body and a sterile cotton swab, previously moistened in sterile BHI, was introduced separately into each of both infraorbital sinuses and was immediately streaked onto CLBA plates that were incubated as before. Differently, in Trial 2, inoculated chickens were housed with non-inoculated birds in the same isolator. In this set of trials, the registration of clinical signs scores to all birds was performed on days 2, 5, and 7 post-inoculation. Euthanasia of birds was done on day 7 post-inoculation, followed by bacteriological culturing as above. On the other hand, Trial 3 was performed by intraperitoneal infections, and mortality was registered until day 3 post-inoculation before all birds were euthanized. Livers from dead and euthanized birds were directly cultured on CLBA plates that were followed as before.Av. paragallinarum infections at all times to maintain the validity of the results.The number of strains that were tested at the same time was determined according to the total number of isolators in the experimental unit. During all four trials, in each experiment, two experimental groups of 10 chickens each were added; in the first group, the chicks were inoculated with sterile BHI, while in the other, 10 birds remained as the negative control. These 20 birds were housed at the same facilities under the same conditions and had to be negative for clinical signs and stay free from Trial 1 was performed using 20 chickens of 20 days of age for each experimental group, housing 10 birds in each isolator. Birds were inoculated by depositing 0.2 mL of the correspondent inoculum into the infraorbital sinus. The clinical signs score was registered on days 2 and 5 post-infection and bacteriological examinations were performed on euthanized birds on that day.Trial 2 was performed by inoculating four chicks, as in Trial 1, and housing them together with another six chicks in close contact within the same isolator. Clinical signs score was registered on days 2, 5, and 7 post-infection and bacteriological examination was performed on euthanized birds on that day.According to the pathogenicity and horizontal infection results from Trials 1 and 2, twelve strains were chosen: three from serogroup A, two from serogroup B, four from serogroup Bvar, and three from serogroup C. These strains were used in Trial 3 where 20 seven-days-old birds for each strain were inoculated intraperitoneally with 0.5 mL of the correspondent inoculum and mortality was observed during 72 h post-infection. Bacteriological examination of the livers of the dead birds as well as of euthanized surviving chickens was performed.8 for each strain. Calcium Phosphate was added as an adjuvant at a final concentration of 0.7%.The most pathogenic strains from Trials 1, 2, and 3 were selected. For each strain, a few colonies from CLBA plates of these strains were used to seed BHI supplemented with reduced NAD, which was incubated for 48 h at 37 °C. Afterwards, thimerosal was added (0.01%) and left at 2–8 °C for inactivation for 14 days. Inactivation was confirmed by bacteriological culturing. The final bacterial concentration was 5 × 10Av. paragallinarum was re-isolated from one or two infra-orbital sinuses.Vaccination of four experimental groups was done at weeks 5 and 8 of life. The vaccine was administered by subcutaneous injection of 0.5 mL of the vaccine at the back of the neck. Each experimental group consisted of 12 chickens and a similar number of unvaccinated chickens was used as the negative control for each of the four selected most pathogenic strains. As above-mentioned, another group of 10 birds was added as non-vaccinated and non-inoculated chickens for internal control. The infection trials were performed three weeks after the second vaccination dose comparing clinical signs scores and bacteriological re-isolation rates between vaccinated and unvaccinated chickens. Clinical signs scores were registered on days 2 and 5 post-infection and bacteriological examinations were performed on euthanized birds on that day. In this trial, a bird was considered diseased if it scored at least 2 points on one of both days of observations of clinical signs or if In all trials, clinical signs of each facial side were registered according to the following criteria: 0, no clinical signs; 1, light conjunctivitis; 2, facial inflammation, infraorbital sinusitis, and/or conjunctivitis and/or tears; 3, oedema and facial inflammation, infraorbital sinusitis, abundant nasal and eye secretion, and partially closed eye; and 4, severe oedema and facial inflammation, abundant nasal and eye secretion, and complete closure of the eye. Scores of 0 and 1 were considered negative, while scores of 2, 3, or 4 were considered positive ,50.Av. paragallinarum from the liver gave an additional point. The total number of points from Trial 3 together with the previous ones was used to select the most pathogenic strains to formulate the experimental bacterin and as challenge strains for Trial 4.In trials 1 and 2, each bird was given a score between 0 and 4 points for each infraorbital sinus, with a theoretical maximum of 8 points per bird on each observation day. Additionally, bacteriological examination for each sinus for each infraorbital sinus was also considered as an additional theoretical maximum of 2 points. This way, in Trial 1, each bird could sum up to 18 points while in Trial 2, each bird could sum up to 26 points . The total number of points from Trials 1 and 2 was used to select the more pathogenic strains of each serogroup to be further studied. This way, 12 strains were chosen for Trial 3, where a dead bird was given 1 point for each strain and the re-isolation of Each animal was considered as the experimental unit, with the only exception of the horizontal infection trial  where the six non-infected chickens were considered as the experimental unit.Avibacterium paragallinarum strains [In Trials 1, 2, and 3, the number of chickens was based on previous trials that described the pathogenicity of  strains ,57,58. I strains .Av. paragallinarum.Nevertheless, the results of Trials 1 and 2 were evaluated without any statistical interpretation based on qualitative information about the presentation of clinical signs . Within the experimental groups in these two trials, there were no statistical differences regarding the re-isolation rates of In Trial 3, the chi-square test of independence was performed for the results obtained comparing the number of dead birds or the number of positive livers within strains of the same serogroup with a level of significance of 95%.In Trial 4, the Fisher Exact test was used to compare the number of birds with clinical signs, the number of positive infraorbital sinuses, and the number of diseased birds comparing vaccinated birds in comparison with non-vaccinated ones, for each serogroup with a level of significance of 95%.Handling of birds was performed according to the Committee for the Update of the Guide for the Care and Use of Laboratory Animals  and euthWorldwide, infectious coryza affects poultry and it is a cause of important economic losses in layers as well as in broilers. Nevertheless, the employment of good biosecurity measures together with good vaccination programs should be sufficient to protect against the disease when the bacterin is adequately prepared . Most ofAv. paragallinarum strains to be included in the vaccines should contain some facts regarding their pathogenicity and capability to cause clinical signs, spread to non-infected birds, and cause septicaemia and death of experimentally inoculated birds. This information may be added to the ability of the strains to colonize the inoculated infraorbital sinus as well as the not-inoculated one.A good vaccine should be composed of local/regional strains and should include at least one strain from each serogroup . AdditioAv. paragallinarum.All hens remained healthy and free from any external infection until challenged. In every inoculation trial, two experimental groups of 10 chickens each were added; in the first group, the chicks were inoculated with sterile BHI, while in the other, 10 birds remained as the negative control. The trial was considered valid only if none of these birds demonstrated clinical signs and stayed free from infection by Av. paragallinarum strains was studied. These strains were collected from clinical cases in Peru during the last 20 years. As the pathogenicity of Av. paragallinarum might be altered upon long conservation and an undefined number of passages in artificial culture media, all strains were passaged in live birds, re-isolated, and kept with a standardized number of subcultures [In the present work, the pathogenicity of 24 Peruvian cultures ,14. ThisAv. paragallinarum was re-isolated from all inoculated infraorbital sinuses and almost from all the non-inoculated ones and varied between 36 and 40.Later, inoculation studies were performed comparing their capacity to cause clinical signs  and to spread to non-infected birds . The use of clinical signs score for pathogenicity studies was used by other authors, although criteria may vary, it is a good way to describe the course of infection and its severity ,65,66,67p < 0.05).Strains Q10 and Q13 from serogroup B were not lethal as well as strain Q24 of serogroup C, with only 2, 3, and 4 positive livers, respectively. Mortality rates varied among the other nine strains: strains Q2 and Q17 resulted in 7 and 6 dead birds, respectively, and were the two strains with more dead birds than all the others. Within serogroup Bvar, these 6 dead birds (Q17) were significantly more than the only 1 dead bird for each of the strains Q15 and Q16  than strain Q15 with only one.On the other side, the re-isolation of the challenge strains was significantly higher in strains Q2, Q7 (both from serogroup A), Q12, and Q18 (serogroup C) with 18, 18, 18, and 12 positive livers, respectively, in comparison with the other strains of the same serogroup. Within serogroup Bvar, strains Q17, Q16, and Q14 had, respectively, 11, 7, and 6 positive livers, significantly more  and test the efficacy in a complete vaccination program in layers during production under controlled conditions or in field trials [The results of the present trials suggest an alternative methodology for the selection of d trials . If fiel