Brodsky classification, MMS, modified Friedman’s staging system and retropalatal lateral-to-lateral (L–L) collapse of Muller’s test were significantly correlated with VOTE count (p < 0.05). The concordance between VOTE under DISE and Brodsky classification or modified Friedman’s staging system was moderate. In contrast, the concordance between VOTE under DISE and MMS or Muller’s test was slight. The study revealed that Brodsky classification and Friedman staging had a significant relationship with DISE on the velum and oropharynx, but the level of tongue base is uncertain between DISE and MMS. Correlation of awake evaluation of tongue base is still not correlated to the DISE findings. Pre-treatment evaluation of DISE is still warranted.The Velum, Oropharynx, Tongue base and Epiglottis (VOTE) classification on drug-induced sleep endoscopy (DISE) is used widely for obstructive sleep apnea (OSA) syndrome, though research into comparative physical examinations with VOTE on DISE is still limited. The aim of this study was to evaluate the relationship between the findings of physical examinations and DISE in patients with OSA. Fifty-five patients with OSA were enrolled in this retrospective study. All of the patients received clinical explorations including a Brodsky classification, a modified Mallampati score (MMS), a modified Friedman’s staging system, and a Muller’s test. Drug-induced sleep endoscopy was further evaluated in the operating room. There were significant relationships between Brodsky classification, modified Friedman’s staging system, Muller’s test and oropharynx collapse during DISE ( Obstructive sleep apnea (OSA) syndrome is characterized by a repetitive upper airway collapse during sleep, narrowing the pathway for airflow and causing periods with hypopnea and apnea with decreased blood oxygen levels . PolysomThe level of obstruction is essential for determining correct surgical management of patients with OSA. Conventionally, airway evaluation has relied on physical examination with Brodsky classification, modified Mallampati score (MMS), and modified Friedman’s staging system. Brodsky classification is used for palatine tonsil size evaluation . MMS is This is a retrospective study. The charts of fifty five patients with OSA were enrolled for review from January 2016 to July 2018 in China Medical University Hospital. This study for waiver of informed consent was approved by the local ethics committee of China Medical University Hospital(No. CMUH103-REC1-078). The inclusion criteria for this study were: (1) adult (age older than 18), (2) apnea-hypopnea index (AHI) > 5 per hour by PSG, and (3) having the records of physical examinations, such as Brodsky classification, MMS, modified Friedman’s staging system and Muller’s test. The exclusion criteria were: (1) previous sleep surgery history, (2) contraindication for anaesthesia. Overnight PSG was performed at a sleep center, which equates to a whole night of PSG, the number of channels included electrocardiogram, electrooculography, electromyography, O2 oximeter, flowmeter, and nasal–oral thermocouple and nasal pressure cannula. The AHI was clarified as events per hour, and evaluated by type I PSG, together with the definitions of apneas and hypopneas under the American Academy of Sleep Medicine (AASM) scoring manual v2.5 edited by an “accepted” scoring rule. The incomplete awake diagnostic tests were excluded as well. The medical records of the patients were reviewed. Basic data, including age and body mass index (BMI), were recorded. Physical examinations including Brodsky classification, MMS, modified Friedman’s staging system and Muller’s test were performed by a senior sleep surgeon with a qualified sleep fellow. The DISE was evaluated via VOTE under total intravenous anesthesia (TIVA) in target control infusion (TCI) mode used with propofol as the sedating agent, administered by a senior sleep surgeon together with a senior anesthesiologist in the operation room. Each of the clinical awakening clinical physical examinations was surveyed and correlations with DISE was assessed by VOTE.Brodsky classification was used for palatine tonsil size evaluation. The grading is classified as follows: grade 0 = tonsils limited to the bilateral tonsillar fossa; grade 1+ = tonsils occupy less than 25% of the space between the anterior pillars in the oropharynx; grade 2+ = tonsils occupy 25% to 50% of the space between the anterior pillars; grade 3+ = tonsils occupy 50% to 75% of the space between the anterior pillars; grade 4+ = tonsils occupy 75% to 100% of the space between the anterior pillars .MMS was used for grading in accordance with tongue size and oral cavity, and higher MMS refers to a larger tongue size. Patients were classified as follows: class I = complete visualization of uvula, tonsils, and palatal arches; class IIa = complete visualization of the uvula while the tonsils and arches are partly hidden; class IIb = visualization of the soft palate and base of the uvula but not tonsils or pillars; class III = visualization of some of the soft palate but not tonsils, pillars, distal soft palate, or base of the uvula; and class IV = only the hard palate is visible .Modified Friedman’s staging system is used to predict the success of uvulopalatopharyngoplasty. The system is based on tonsil size, tongue positions, and BMI . The staThe patient was evaluated while sitting in an awake condition, and retropalatal and retrolingual obstruction was assessed. All the patients underwent Muller’s test with topical nasal anesthesia and/or vasoconstrictor. An Olympus 3.2 mm fibro-optic scope passed through the nose to survey velum, oropharynx, and tongue base; epiglottis was used for evaluation. Every patient received level dimensional change by inhaling with a blocked nose to cause negative pressure and the collapse percentage of the airway in the velum, oropharynx, and tongue base areas; the epiglottic area was assessed and graded under Wang’s grading system . The colDISE was performed for every patient in the supine position in the operating room, under pulse-oximetry and cardiac rhythm monitoring. One percent lidocaine with topical phenylephrine mixture spray was applied to nares, before an Olympus 3.2 mm fibro-optic scope was passed through the nose to survey velum, oropharynx, tongue base, and epiglottis. During DISE, IV propofol 100 mcg/kg/min was infused by TIVA under TCI-inducing sedation and was maintained by 0.1 to 0.3 mg/kg intermittently for DISE. The sleep depth was evaluated by bispectral index monitoring, in which the level below 70 was considered suitable for DISE records. The VOTE classification was a structure-based evaluation  encompasStatistical analyses were performed using SPSS 19 software package . Data were expressed by descriptive statistics, and the t-test was used to compare clinical characteristics differences between “not severe” and “severe” OSA patients. The categorical variable of proportion testing was analyzed by chi-square and Fisher’s test. The statistical relationship between different levels of physical examinations  and DISE (grade 0 or 1 + 2) was analyzed by chi-square test. Spearman’s correlation coefficient was used to analyze the relationships between the physical examinations and related VOTE classification. Cohen’s kappa coefficient (κ) was used to measure inter-rater reliability of different levels of physical examinations and related VOTE classification. Kappa values < 0 were taken as indicating no agreement, 0~0.20 as slight agreement, 0.21~0.40 as fair agreement, 0.41~0.60 as moderate agreement, 0.61~0.80 as substantial agreement, and 0.81~1 as almost-perfect agreement. P-value < 0.05 was considered a statistically significant difference.2; and AHI = 44.6 ± 22.0/hr). Twenty eight of 55 (50.9%) OSA patients had BMI ≧ 30, and 36 of them (65.5%) had AHI ≧ 30/hr. Comparing the clinical characteristics and physical examinations between the patients with severe OSA (AHI > 30/hr) and not-severe OSA (AHI ≤ 30/hr), there were no significant differences in age, Brodsky classification grade, MMS, modified Friedman’s staging system, Muller’s test and collapse level under DISE (p > 0.05) except BMI . Tongue assessment of VOTE during DISE also had a significantly positive correlation with modified Friedman’s staging system . Conversely, no correlation was noted between MMS and tongue assessment of VOTE (p > 0.05). VOTE level, retrolingual A–P of Muller’s test and MMS had significant positive correlations . The VOTE count, Brodsky classification, MMS, modified Friedman’s staging system, and retropalatal L–L of Muller’s test were significantly correlated (p < 0.05). The analysis results for correlation coefficients analysis of physical examinations and VOTE during DISE are shown in A kappa statistic was used to further evaluate the different levels of four physical examinations and related VOTE under DISE in p < 0.05, p > 0.05).Because the Brodsky classification and oropharynx level in DISE had a moderate concordance in p < 0.05). In grades 1 and 2 of oropharynx scoring during DISE, grade 0 of Muller’s test were noted in 6 of 18 patients (33.33%). A higher Muller’s test (grades 1 and 2) was accompanied by oropharynx collapse in 24 of 37 patients (64.87%). There was a significant relationship between Muller’s test and oropharynx scoring during DISE (p < 0.05).When OSA patients were in stage I of modified Friedman’s staging, oropharynx scoring during DISE was noted in 40% of patients (6/17 patients). A modified Friedman’s staging as stages II, III, and IV was accompanied by oropharynx scoring during DISE in 70% of patients (28/40 patients). Modified Friedman’s staging had a significant relationship with oropharynx collapse during DISE (For the OSA patients, a major goal of non-CPAP treatment is the identification of the correct collapse level of the upper airway. We used to evaluate patients with a clinical examination and a grading system, but these methods may show limited value because they are performed while patients are awake. DISE was first described in 1991, providing a similar condition to natural sleep and dynamic assessments . In the On oropharynx assessment, Fernandez-Julian et al. showed acceptable concordance . M. BlumFernandez-Julian et al. showed good correlation between DISE and Friedman staging regarding the suggestion of operation . Stage IOur study aimed to compare the findings of clinical examination with DISE. We found that not all clinical findings showed a significant relationship between DISE findings. A higher score in MMS does not always indicate that obstruction is at the level of the tongue base. Personalizing the level of the obstruction site is essential for correct surgical management in patients with OSA. Certal V.F. et al. showed that surgical treatment changed after DISE in 50.24% cases . In concThe physical examination is considered to have correlation with DISE because both are used to evaluate the upper airway. However, in our study, the highest correlation coefficient was 0.69, between Brodsky classification and oropharynx, which represented a moderate correlation, while the others showed a weak-to-low correlation. Muller’s test was also used to evaluate the correlation with DISE. We found that the site of collapse determined at retropalatal level with L–L direction had a weak correlation with VOTE count and the site of collapse determined at the retrolingual level with an A–P direction had a weak correlation with VOTE level. Therefore, in clinical practice, we should not just perform a physical examination but should also arrange DISE. In a North American DISE-oriented sleep surgery outcome study, the conclusion was offered that surgical outcomes were not correlated with velum and epiglottis-related obstruction or the degree of epiglottis-related obstruction, but were instead associated with tonsil size and BMI. They still consider DISE to be an important tool for evaluating collapse at tongue base and oropharyngeal lateral walls and an important diagnostic tool toward making proper sleep surgery plans for each OSA patient . There are several limitations in our study. First, the case number of this study was limited. Second, we use propofol under bispectral index monitoring for DISE. Generally, propofol has a quicker onset, a shorter half-life, and can demonstrate larger degrees of obstruction, which may reflect more accurately what happens during rapid-eye-movement sleep . HoweverThe study revealed that physical examinations, i.e., Brodsky classification and Friedman staging, have a significant relationship with DISE, but the level of tongue base is uncertain between DISE and MMS. DISE can be a reliable method for evaluating upper airway dynamics during sleep. DISE, with all its limitations and the relevance of its findings so far, goes beyond the objective of simply identifying collapse with precision, but rather helps in understanding the collapse of the identified sites.


Liangshan Yi Autonomous Prefecture is one of the areas that most severely affected by human immunodeficiency virus (HIV) in China, and virological failure on antiretroviral therapy (ART) is serious in this area. Analyses of prevalence and determinants of ART failure, the genetic diversity and drug resistance among people living with HIV (PLWH) helps improve HIV treatment efficiency and prevent HIV transmission.A total of 5157 PLWH were recruited from 2016 to 2017. The venous blood samples were subjected to RT-PCR, followed by sequencing of the HIV-1 pol gene, targeting the protease and reverse transcriptase fragments. HIV-1 diversity was analyzed using the DNAStar software and drug resistance mutations were analyzed using the Stanford University HIV Drug Resistance Database.A total of 2156 (41.81%) PLWH showed virological failure on ART. Males (ORm = 1.25), heterosexual behaviors and drug injection (ORm = 1.44) and mother to child transmission routes (ORm = 1.58), the clinical stage of AIDS (ORm = 1.35), having used illicit drugs and shared the needles , having ever replaced ART regimen (ORm = 1.48) increased the risk of virological failure among PLWH, while higher education lever (ORm = 0.77) and ≥ 12 months on ART  was associated with lower likelihood of virological failure. The data revealed that CRF07_BC  were the most common strains, and the drug-resistant rate was 32.10% among PLWH with virological failure in this area. The high frequencies of drug resistance were found in EFV and NVP of NNRTIs, ABC, FTC and 3TC of NRTIs, and TPV/r in PIs. The most common mutations in NNRTIs, NRTIs and PIs were K103N/KN (64.69%), M184V/MV/I (36.29%) and Q58E/QE (4.93%), respectively.We concluded that surveillance of virological failure, HIV-1 subtypes, and drug resistance to understand HIV-1 epidemiology and guide modification of ART guidelines, and target prevention and control strategies should be formatted to reduce the virological failure and drug resistance to promote viral suppression and prevent HIV-1 transmission. Human immunodeficiency virus (HIV) infections in humans affect more than 37 million individuals worldwide, and approximately 0.85 million people living with HIV (PLWH) live in China. The number of PLWH has increased by 14% from 2016 to 2017 [Antiretroviral therapy (ART) is highly effective in suppressing HIV, stopping the progression of HIV disease and reducing the risk of HIV transmissions , 6. The HIV is characterized by its great genetic variability and responsibility for developing drug-resistant mutations with high viral replication rates. This not only increases the risk of virological failure on antiretroviral treatment, but also results in the spread of drug-resistant strains, which brings a huge challenge to controlling the HIV epidemic and preventing the HIV spread. The genotype of HIV-1 is related to the route of transmission, and there are differences in epidemic scale and distribution characteristics , 17. TheParticipants were recruited through a multi-stage cluster sampling strategy. The county with the most severe HIV epidemic was selected from all 17 counties in Liangshan Prefecture. According to the basic information system for Acquired Immune Deficiency Syndrome (AIDS) prevention and treatment, all PLWH in this county were included in our study. We have tried to identify as many PLWH as possible through all three possible channels: (1) Provider-Initiated HIV Testing & Counseling (PITC) service for HIV, (2) Voluntary Counseling and Testing (VCT) service through fixed VCT sites and non-government organizations (NGOs), (3) HIV sentinel surveillance (HSS) for unmarried youths, pregnant women, community population, and drug users. The inclusion criteria for PLWH were: (1) receiving confirmatory HIV diagnosis, (2) being permanent residents or had stayed in Liangshan area for more than 5 years, (3) receiving ART in the local township health center for more than 6 months. The exclusion criteria for participants were having have major psychiatric illness  would be collected and inputted into system.Each participant was asked to provide 5 mL of venous blood for detecting the viral load of HIV-1. The plasma samples were isolated from each participant, and preserved in a − 80 °C freezer until analysis. HIV viral load was measured in the Sichuan Center for Disease Control and Prevention. Virological failure in ART was defined as HIV RNA level ≥ 1000 copies/ml , 21 aftePol region and the first 300 codons of the reverse transcriptase gene was amplified by using reverse transcription polymerase chain reaction (RT-PCR). The amplified products were purified in and sequenced at Beijing Genomics Research Center Ltd., in China.Total viral nucleic acid was extracted from 200-μl plasma using an automatic extraction machine . The full-length protease gene in The HIV-1 pol sequences obtained in the study, together with reference sequences of different subtypes and CRFs, were edited and aligned using ChromasProl.33, and the sequence alignments were manually performed by BIOEDIT Sequence Alignment Editor software . The detailed amplification, sequencing and drug resistance analyses were detailed described previously , 23.2) test. Using the presence of virological failure in ART as the dependent variable, univariate odds ratios (ORu) and 95% confidence interval (CI) for socio-demographic information, possible route of HIV transmission, HIV-related behaviors and disease-related characteristics were estimated. A summary model was obtained by fitting a multiple regression model that considered all variables that were significant in the respective univariate analysis as candidates. Multivariate odds ratios (ORm) and respective 95%CI were obtained. Was used for data analysis, with p < 0.05 taken as statistically significant.Data were analyzed using SPSS version 21.0 for Windows . Categorical variables were expressed as frequencies and percentages and compared using the chi-squared  PLWH showed virological failure on ART from 2016 to 2017. Among PLWH with virological failure, majority of them were male, peasant, married, illiteracy, Yi minority, having HIV transmission route of drug injection, the first CD4+ counts of 200 ~ 500 counts/ul, having never used illicit drugs and shared the needle, having no casual sexual partners and history of sexually transmitted diseases except for HIV . In multivariate logistic regression model, we found that males, heterosexual behaviors and drug injection and mother to child transmission routes, stage of AIDS, having used illicit drugs and shared the needles, having ever replaced ART regimen significantly increased the risk of virological failure among PLWH (p < 0.05), while higher education level and time on ART ≥12 months were associated with lower likelihood of virological failure (p < 0.05)  samples were successfully detected. The phylogenetic analyses based on the pol regions showed that CRF07_BC  were the most common strains in this area, and followed by CRF08_BC  and C  subtypes were the most common strains in Sichuan province . The prevalence of drug resistance was high in nonnucleoside reverse transcriptase inhibitors (NNRTIs) in all treatment regimens, then followed by nucleoside reverse transcriptase inhibitors (NRTIs) and protease inhibitors (PIs)  was much higher than that to NRTIs  and PIs  among PLWH with virological failure. The most common mutations in NNRTIs were K103N/KN (64.69%), V179D/E (23.47%) and Y181C/YC/I (14.00%), they were M184V/MV/I (36.29%), T215F/FS/TNSY (7.50%) and K219Q (5.92%) in NRTIs, and they were Q58E/QE (4.93%), L10F/LFI (0.39%) and M46L (0.39%) in PIs  among PLWH was high in this area. A high prevalence of subtype CRF07_BC (95.62%) was observed among PLWH with virological failure on ART, which was different from other areas of China , 25. ThuOur study found that male PLWH and illiteracy were associated with a higher likelihood of incomplete viral suppression of virological failure on ART. Compared to females, males are more likely to discontinue from ART, which results in virological failure , 27. ParWe also found that patients who had been on ART ≤12 months, and had changed ART regimens were more likely to experience virologic failure. Time on ART ≤12 months was associated with a high risk of virological failure than time on ART ≥12 months. This result was similar to a finding from a study in Zimbabwe, which showed that compare to time on ART ≥4 years, patients whose time on ART ≤4 years had more risk of treatment failure. Because the latter may have taken the wrong medicine or forgotten it due to lack of experience , 31. TheOur studies found that the subtype CRF07_BC 95.62%) was most prevalent and was the predominant subtype, followed by CRF08_BC. CRF07_BC, which was one of the most prevalent CRFs in China –39 and w% was mosAccording to our findings, drug-resistant frequency to NNRTIs was much higher than that to NRTIs and PIs in this area among PLWH with virologic failure in ART. A similar pattern was also found in other places in China and low- and middle-income countries –46, but There were several limitations in this study. First, this study was a cross-sectional survey, time-based sequence and cause-effect relationships among these variables cannot be established. Second, some HIV related behaviors were self-reporting, which may lead to recall bias. Third, some important factors of treatment failure in ART, such as ART adherence, should be considered as potential factors in our study, but such information did not collect during the follow-up. Fourth, the participants enrolled in the study were not newly diagnosed with HIV-1 infection, and some patients may have pretreatment drug resistance. Therefore, we could not identify the time of virologic failure and the resistance to ART before and after genotyping in the first virologic failure.The findings confirmed that the prevalence of incomplete viral suppression of ART virologic failure in ART among PLWH was high in this area. On the one hand, we should develop more suitable health education measures for medication compliance for this kind of population; on the other, the early identification of the drug resistance of PLWH with ART virologic failure are of great significance. Timely replacement of new regimens for PLWH with drug resistance could prevent additional drug resistance mutation or multiple drug resistance. Last but not the least, continuum in care and retention in care are important factors in preventing virological failure , and enh


Routine Health Information Systems (RHIS) of low-income countries function below the globally expected standard, characterised by the production and use of poor-quality data, or the non-use of good quality data for informed decision making.This has negatively influenced the health service delivery and uptake. This study focuses on identifying the factors associated with the performance of RHIS of the health facilities (HF) in Yaoundé, so as to guide targeted RHIS strengthening.p-value of less than 0.05 was considered statistically significant.A HF-based cross-sectional study in the 6 health districts (HDs) of Yaoundé was conducted. HFs were chosen using stratified sampling with probability proportional to size per HD. Data were collected, entered into Microsoft Excel 2013 and analysed with IBM- SPSS version 25. Consistency of the questionnaire was measured using Cronbach’s alpha coefficient. Pearson’s chi-square (and Fisher exact where relevant) tests were used to establish relationships between qualitative variables. Associations were further quantified using unadjusted Odd ratio (OR) for univariable analysis and adjusted odds ratio (aOR) for multivariable analysis with 95% confidence interval (CI). A p < 0.001), proving that the questionnaire was reliable in measuring RHIS performances. At univariable level, the following factors were positively associated with good performances: supportive supervision ; p = 0.02), receiving feedback from hierarchy ; p = 0.05), having received training on health information ; p = 0.003), and presence of a performance evaluation plan , p = 0.007). At multivariable level, the only significantly associated factor was having received training on health information , p = 0.04).Of 111 selected HFs; 16 (14.4%) were public and 95 (85.6%) private. Respondents aged 24–60 years with an average of 38.3 ± 9.3 years; 58 (52.3%) males and 53(47.7%) females. Cronbach’s alpha was 0.96 (95%CI: 0.95–0.98, Training of health staff in the RHIS favors RHIS good performance. Hence, emphasis should be laid on training and empowering staff, frequent and regular RHIS supervision, and frequent and regular feedback, for an efficient RHIS strengthening in Yaoundé. The health information system (HIS) makes up one of the pillars of the health system (HS) whose responsibility is the generation of data to facilitate the functioning of the other HS components: service delivery, health workforce, access to essential medicines, financing, and leadership . A good Since data generation is done at the level of the health facility, emphasis should be placed on the staff at this level through implicating them in the elaboration of RHI tools, planning, implementation, and evaluation of RHIS interventions. In Benin, exhaustibility of reporting increased from 16 to 89%, and the proportion of reports with sufficient data quality increased from 18.8 to 45.8% after implicating staff in the conception and elaboration of data collection tools [State-related factors that would affect the functioning of an RHIS include governance, planning, availability of material, financial and human resources, supportive supervision, information dissemination, and promotion of a culture of information use . The ideThe Cameroon health system is divided into 3 pyramidal levels. These include: the central , the intermediate , and the peripheral  levels. Three sub-sections exist in the system: the public, private (private non-profit-making and private profit-making), and the traditional sub-sections , 16. AllThe health sector is further segmented into 5 components . Vertical components are: health promotion, disease prevention, and disease management. Horizontal components include: health system strengthening, and governance . Thus, sFollowing the standardization of data collection by the Cameroon Ministry of Public Health (MOPH) through the implementation of DHIS 2 , there iBy 2027, ensure the development of health research and the availability of quality health information system for evidence-based decision-making at all levels of the health pyramid” [Cameroon Health Sector Strategy (HSS) for the health information system (HIS) states: “pyramid” . HSS aimpyramid” . To redupyramid”  who deteBiyem-assi, Cité Verte, Djoungolo, Efoulan, Nkolbisson and Nkolndongo.We carried out a facility-based cross-sectional study for a period of 5 months extending from 1st May 2019 to 30th September 2019 in the 6 health districts (HDs) in Yaoundé. These HDs include: The study variables were the socio-professional characteristics of participants, HF-related, and health system (HS)-related characteristics of participating HFs, and Health Facility and Community Information System Standards. Socio-professional characteristics included age, sex, professional qualification, years of experience, and function.The independent variables were the HF-related, and health system (HS)-related characteristics: Status of the HF (public/private), Presence of health information unit ), Stable person in charge of statistics/data management (Yes/No), Available functional computer for data management (Yes/No), Stable internet (Yes/No), Availability of call credit (Yes/No), RHIS supervision received (Yes/No), Receiving feedback from hierarchy (Yes/No), Receiving training on HI (Yes/No), and Presence of a performance evaluation plan . A PEP is a written document that describes the process of carrying out the monitoring and evaluation of the RHIS performance, detailing the “What”, the “How”, and the “Why It Matters” for the RHIS evaluation, as well as exploiting evaluation results for RHIS performance improvement and decision making .Management and Governance Data and Decision Support Needs Data Collection and Processing )Data Analysis, Dissemination, and Use The dependent variable was the score of the Health Facility and Community Information System Standards. This variable was defined and classified into domains and subdomains by WHO and MEASURE Evaluation  as folloi)Proportion of scores for the various subdomains: we summed the score for each item (question) in the subdomain, multiplied by 100 and divided by the maximum score for that subdomain.ii)Proportion of scores for the domains: we summed up the obtained scores of the subdomains in the given domain, multiplied by 100 and divided by the maximum score for that domain.iii)Global score for all the domains, we summed up all the obtained scores of all the domains, multiplied by 100 and divided by the maximum global score. The obtained global score was then grouped into good score (scoring 60% and above) and poor score (less than 60%).The score of all the domains was calculated as follows :i)ProporA minimum sample size (n) of 106 HF that were visited was obtained using the formula: 2×P1-Pd2 , where Z2×P1-Pd2 , d is th2×P1-Pd2 , and nonThe recruitment criteria for HFs included functional public and private HFs of the operational level whose consent for participation was obtained. Respondents (one per HF) were either the head, person in charge of statistics/data management, or any other responsible staff, capable to provide the needed responses.0 (no answer/not applicable); 1 ; 2 (needs a lot of strengthening); 3 (needs some strengthening); and 4 .Interviewers were recruited and trained to understand the objectives and the methodology of the study. Data were collected using the WHO/MEASURE Evaluation pre-established Rapid Assessment questionnaire  that wasunacceptable if α < 0.7, (ii) acceptable if 0.7 ≤ α < 0.8, (iii) good if 0.8 ≤ α < 0.9, (vi) excellent if α ≥ 0.9. Pearson’s Chi-square test (Fisher exact test where relevant) was used to establish relationships between qualitative variables. Associations were further quantified using unadjusted Odds ratio (OR) for univariable analysis and adjusted Odds ratio (aOR) for multiple logistic regression analysis with 95% confidence interval (CI). The Hosmer-Lemeshow (HL test)‘s goodness of fit test was used to assess the adequacy of the multiple logistic regression. A p-value of less than 0.05 was considered statistically significant.Data were entered into Microsoft Excel 2013, cleaned and then exported for analyses using IBM-SPSS version 25 . Frequen0 00786/CRERSHC/2019 from the Regional Research Ethics Committee for Human Health of the Centre and the authorization N0 00756−/AP/MINSANTE/SG/DRSPC/CRERSH from the Regional Delegate of Public Health for the Centre Region. Study procedures were described to participants, during which they were briefly and clearly informed of their voluntary participation in the study; and that refusal to participate would have no negative consequences. Their informed consents were then obtained prior to the interview.The study received ethical approval CE NOf 111 selected HFs (out of 799); 16 (14.4%) were public and 95 (85.6%) were private. Respondents aged 24–60 years with an average of 38.3 ± 9.3 years; 58 (52.3%) males and 53(47.7%) females.HIU, stable internet and PEP were available only in 27.9, 39.6 and 27.9% of HFs respectively. The other characteristics: having received training on HI (63.1%), feedback received from hierarchy (74%), RHIS supervision (60.4%), and presence of functional computer (55.9%) were present in more than 50% but less than 75% of the HFs. However, stable persons in charge of statistics/data management were available in 78.4% of HFs.As presented in Table p < 0.001), proving that the questionnaire was reliable in measuring RHIS performances. Table Collection and Management of Individual Client Data) and 0.88 (Management); and for domains between 0.90  and 0.93 (Management and Governance).The consistency of the items of the RHIS tool was assessed using Cronbach’s alpha. Overall Cronbach’s alpha was 0.96 . HFs with a HIU performed slightly poorer (22.6%) compared to HFs without a HIU (28.8%), with non-significant difference (p = 0.50). Facilities with stable persons in charge of statistics (27.7%), functional computer (31.7%), stable internet (35.7%), and call credit (30.3%) performed better compared to those that did not possess the resources or benefitted from activities . Facilities that benefitted from RHIS supervision (34.8%), received feedback from hierarchy (32.1%), facilities trained on HI (36.8%) and those with a PEP (45.2%) also presented better and statistically significant performances with respect to HFs that did not. Thus, the following factors were positively associated with good performances after univariable analysis: RHIS supervision ; p = 0.02), receiving feedback from hierarchy ; p = 0.05), having received training on health information ; p = 0.003), and presence of a performance evaluation plan , p = 0.007). At multivariable level, the only significantly associated factor was having received training on health information , p = 0.04). The Hosmer-Lemeshow’s goodness of fit test showed that the multiple logistic model adequately fitted the data .Table This study assessed the factors associated with the performance of the RHIS as a whole component of the health system. It was evidenced that HFs that have benefited from HI training were more likely to perform better than their counterparts. Training on HI enables staff to acquire skills in basic computer use, planning, data analysis and management, data use, and interpretation. This also includes training on DHIS 2 which is the software used by the Ministry of Public Health for health data management. In a cross-sectional descriptive study carried out in Kenya , 80.8% op < 0.001). We noticed that 32.1% of HFs who have received feedback from superiors presented a good performance compared to 11.5% among HFs that did not receive feedback. It however points out the importance of regular and frequent feedback mechanisms to enable immediate discussion and resolution of problems for RHIS strengthening [Sixty percent of HFs reported having been supervised, even though most of these facilities (34.8%) presented better performance compared to their counterparts. This calls to question the quality and frequency of the supervision visits. Supervision aims to empower and support staff in carrying out their daily tasks . This shgthening .Functional computers for data management was present in about half (55.9%) of the health facilities. Of these facilities, 31.7% had a good performance against 21.3% among facilities without one. This finding was similar to that obtained by Shiferaw et al.  in EthioThe presence of a functional computer alone in an HF or department will not guarantee effective and efficient HI management. This is complemented by the services of a stable and computer literate person in charge of statistics. The difficulties experienced by most HFs are that persons in charge of data management are unstable, untrained in HI management and computer skills, or occupy other posts . Apart fDHIS 2 was implemented with the hope of meeting the health sector strategic objective of attaining 80% completeness in monthly activities reporting by 2027 . With thSome limitations of the study include the fact that it didn’t focus enough on other behavioural determinants such as the knowledge, skills, attitudes, values, and motivation of the people who collect, analyse, and use health data. In addition, our cross-sectional study design prevented the findings from showing causal relationships.This study revealed that training of health staff in the RHIS is a determinant of the good performance of the RHIS system in Yaoundé. In this light, emphasis should be laid on training and empowering staff. Likewise, frequent and regular RHIS supervision, and frequent and regular feedback should also be implemented for an efficient RHIS strengthening in Yaoundé. Health authorities should carry the greater responsibility in the strengthening process through the creation of an enabling environment. This involves advocating and providing sufficient resources, monitoring, and evaluation of the implementation of RHIS policies by both public and private HFs for better management of data for evidence-based decisions making. However, the findings of this study also pave the way for further research in the domain of RHIS. Post-implementation evaluation studies will be better indicated to measure the change in the performance of the RHIS after the implementing these interventions. Secondly, cost-effective studies will be indicated to measure the cost effectiveness of these interventions especially in a low-income country setting.


Hypera postica , is an economically important pest of alfalfa throughout the western United States. Coccinellids and nabids are among the most important natural enemies of this species, contributing to the biological control of H. postica in alfalfa fields. The spatio‐temporal dynamics of H. postica and these two predator groups were investigated using 81 (= 9 × 9 grid) sample points in each of five alfalfa fields in north‐central Montana. The data were analyzed using variogram and spatial analysis by distance indices (SADIE).Understanding the spatio‐temporal dynamics of prey and predator distributions can provide valuable insights into pest management strategies and conservation of natural enemies in agro‐ecosystems. The alfalfa weevil, H. postica in 17 of 19 sampling times for larvae, and three of 12 sampling times for adults. Using SADIE, statistically significant aggregation distribution was evident in four of 19 sampling times for larvae, and five of 12 sampling times for adults of H. postica. Combined variogram and SADIE showed strong evidence of spatial aggregation of H. postica larval population (~95%) while a moderate level of aggregation in the adult population (~67%) of the sampling times analyzed. The average aggregation distances based on the range value of the variogram were 22.3 m and 14.7 m for larvae and adults, respectively. Based on variogram results, populations of natural enemies, coccinellids and Nabis spp. were found spatially aggregated in 57.9% and 5.6% of the sampling times, respectively. SADIE further supported the variogram results as coccinellid populations (52.6% of sampling times) were highly aggregated in contrast with the Nabis spp. populations (5.6% of sampling times) in alfalfa fields. There was no evidence of significant spatial synchrony between H. postica and its predators, coccinellids and Nabis spp.Variogram analysis revealed the spatial dependence (aggregation) of H. postica and its two natural enemies (coccinellids and nabids) in irrigated alfalfa fields. The possible implications of these findings for integrated pest management (IPM) of alfalfa weevil populations are discussed.Our study was able to determine the spatial and temporal distribution of  Hypera postica and the population of its two natural enemies (coccinellids and nabids) in irrigated alfalfa fields.This study determined the spatial and temporal distribution of  It is believed to be native to Asia, Europe, and North Africa, but has spread to most of the alfalfa‐growing regions of the world.Hypera postica has been the most significant pest of alfalfa in the United States since its introduction more than 60 years ago.The alfalfa weevil, H. postica overwinters as an adult. The female becomes active when temperature increases (~ 9 °C threshold) in the spring and lay clusters of 5 to 20 eggs inside stems.H. postica feeding is greatest on the first alfalfa cutting.In cooler parts of the United States, including Montana, Bathyplectes curculionis (Thomson), B. anurus (Thomson) (Hymenoptera: Braconidae), Microctonus aethiopoides (Loan) (Hymenoptera: Ichneumonidae), and Oomyzus incertus (Ratzenberg) (Hymenoptera: Eulophidae) are considered effective natural enemies of alfalfa weevil.H. postica outbreaks.As a perennial and nitrogen‐rich plant, alfalfa offers a favorable habitat for many beneficial arthropods, including pollinators and natural enemies.Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae), spotted alfalfa aphid, Therioaphis maculata (Buckton) (Hemiptera: Aphididae), and corn rootworm, Diabrotica virgifera (LeConte) (Coleoptera: Chrysomelidae) were determined in tomato, alfalfa and cornfields, respectively, using a geostatistical analysis.Oulema melanopus (L.) (Coleoptera: Chrysomelidae), in wheatVitacea polistiformis (Harris) (Lepidoptera: Sesiidae), in grape vineyards.Understanding the spatial distribution of a pest and its natural enemies is useful for developing an effective pest monitoring and management program.The spatial distribution of natural enemies helps to understand the pest–natural enemy relationship in the field.H. postica and these natural enemies. Therefore, this study aimed to determine the spatio‐temporal distribution of H. postica, and two natural enemy groups – coccinellids, and nabids using two geospatial methods, SADIE and variogram.The spatial distribution and association of pests and their natural enemies can be characterized using several methods, including variogram and SADIE. Both methods have their strengths and weaknesses, and the combination of the two methods is recommended in ecological studies.22.1o 35.192 W112o 21.169; and Field B: N 48o 30.206 W112 o14.350), Ledger  and Valier (Field E: N 48o 35.192 W112o 21.169), in Pondera County, Montana, USA. All fields were in north‐central Montana. Alfalfa plants were grown multiple years on the same piece of land after broadcast seeding with 1–2 harvesting (i.e. cuttings) per year; therefore, plant stand age in the fields used in this study ranged from two to five years. Field sizes  were 40, 16, 30, 12 and 68 ha, respectively. Irrigation was applied one to two times before and after alfalfa cutting using wheel‐line or center‐pivot systems. None of the fields received insecticides during the growing period of the sampling year nor in the two previous years (2014–2015). Two cuttings were made, which is the typical harvest practice for irrigated alfalfa in Montana, where the active growing season for alfalfa is between May and August. The average seasonal temperature (May–August) for Conrad, Ledger and Valier fields ranged 10–18, 9–18 and 9–17 °C, respectively.Five irrigated commercial alfalfa fields were selected from three locations: Conrad (Field A: N 482.2H. postica (larvae and adults), lady beetle larvae and adults (Coccinellidae), and damsel bugs nymphs and adults (Nabidae) were conducted in a portion (i.e. sampling area ~0.2 ha) of the five fields in 2016. The sampling area contained 81 sampling points distributed at 5 m intervals in a square grid (i.e. nine sampling points across X‐coordinates and nine sampling points across Y‐coordinates) marked with 1‐m tall red‐painted wooden sticks. Sampling was conducted using a standard 180°‐sweep net (diameter 38 cm) sampling (ipm.ucanr.edu/PMG/r1300511.html), taking 20 sweeps at each sampling point . The collected insect samples were placed in plastic Ziploc® bags and taken to the laboratory on the day of collection, where they were either processed immediately or frozen at −20 °C for later identification and counting. Samples were collected on four dates: two before and two after the first alfalfa cutting. After the second sampling (before first cutting), wooden sticks were removed from study sites and replaced with plastic ear tags. Before the start of the third sampling, plastic ear tags were removed, and wooden sticks were relocated to the study sites. The placement of plastic ear tags allowed us to find the same spots that were sampled before the first alfalfa cutting. Sampling was performed roughly every 10‐days: first sampling from June 1–3 [i.e. Julian week (JW) 23]; second sampling from June 14–16 (i.e. JW 25); third sampling from July 27–29 (i.e. JW 31); and fourth sampling from August 5–7 (i.e. JW 32). Sampling was conducted 3–4 times for each field with a total of 19 samplings across five fields, and these sampling instances were hereafter referred to as sampling times.Samplings of 2.3H. postica and its two natural enemies in alfalfa fields. SADIE accounts for the underlying patchiness of insect counts from spatially referenced locations, whereas the variogram assumes a gradual change in abundance for local insect populations, thus both methods can produce significantly different results and conclusions.H. postica larva, H. postica adult, coccinellids, and Nabis spp. respectively, were used for geospatial analyses, namely variogram and SADIE.Two different geospatial techniques, that is the variogram and SADIE, were used to characterize the spatial distribution of 2.4γ) is calculated as follows:γ^h is the estimated semivariance for the entity of interest (z) at all points (xi) separated by lag distance (h), and N(h) is the number of pairs of samples separated by lag distance h.Variogram plots depict spatial dependence by calculating the autocorrelation among sample points, making them a geostatistical method for determining the spatial distribution pattern of arthropods.X and Y values) of individual sample points as the independent variables.P < 0.05) indicated the presence of the trend in the dataset, and the standard residuals of those datasets were used to develop variograms. Out of 19 sampling weeks, standard residuals were used in six, four, four, and ten datasets of H. postica adult, H. postica larvae, Nabis spp., and coccinellids respectively to develop variograms. Variograms were developed using geostatistical software, GS+ (version 9.0.11).Insect counts that did not meet the assumption of normality were transformed using C0/C0 + C) describe the extent of aggregation,Three parameters  of the variogram model determine the shape of the plots. The maximum distance over which the spatial dependence persists is called the range,2.5D), which represents the minimum total distance that individual samples would need to move in order to obtain the same number (i.e. mean) for individual sample points. The aggregation is expressed in the form of clustering areas with either greater (i.e. patches) or smaller counts (i.e. gaps) compared to the mean. The magnitude of D can be calculated by a randomization test in which permutations of all observed counts from sample points are performed.Ia, and probability, Pa. Values, Ia > 1, Ia = 1, and Ia < 1, indicate the aggregation, random, and uniform distribution patterns. The associated probability (i.e. Pa < 0.025) determines the statistical significance of the resultant distribution pattern.SADIE is a geospatial technique that can be used to determine the spatial distribution pattern of arthropod pests and plant diseases using spatially referenced count data.2.6X0) was used to determine any spatial synchrony between H. postica and two natural enemies, Coccinellid spp., and Nabis spp. This information may better explain the ecological roles of different factors in spatial distribution and sampling. Significant positive association  indicates the presence of either a gap or a patch for both variables  in that particular week of sampling while significant negative association  indicates association of a patch of one variable with a gap of the other variable or vice versa.The spatial association between two datasets was conducted using N_AShell (version 1.0), a part of the SADIE.33.1H. postica larvae was collected across all five fields. The overall distribution of larvae at each field was presented in violin plot  were found in all five fields. The total numbers of coccinellids and nabids collected were 2356 and 988, respectively across five fields. The overall distribution of predators at each field was presented in violin plot  (> 97% of the collected samples), followed by the two‐spotted lady beetle  (Coleoptera: Coccinellidae) across five fields. Coccinellid population density fluctuated depending on sampling time and field location. At JW 23, the mean coccinellid population density was 1.6 times higher in Fields A, B, C and E contrasted to Field D  in similar proportions in all locations. Among the five field locations, Field C generally had a lower population density throughout the sampling times, except the minimal level of increase at JWs 31 and 32. The mean level varied from 0.19 to 0.83 per 20 sweeps. In Fields A and D, nabid density remained relatively unchanged for earlier sampling dates (i.e. JWs 23 and 25) until JW 31, but increased two‐fold by the last sampling time, JW 32. In Field E, nabid. density increased sharply after the first alfalfa cutting (i.e. JWs 31 and 32) with mean densities of 1.50 and 2.00 adults per 20 sweeps at JWs 31 and 32, respectively , Field B  and Field D   Table . In othen = 10) followed by spherical (n = 5), Gaussian (n = 2) and linear (n = 2). The nugget‐to‐sill ratios which measure the degree of aggregation of H. postica larvae were < 0.25 (i.e. strong aggregation), 0.25–0.75 (i.e. moderate aggregation), and > 0.75 (i.e. weak aggregation) in 14, three, and zero of 17 sampling times, respectively  followed by spherical (n = 2) and exponential (n = 1). All three sample times showed a strong spatially aggregated distribution of adults, as indicated by < 0.25 nugget‐to‐sill ratio  in four of 19 sampling times. Regarding adult distribution, five of 12 sampling times showed a statistically significant aggregation pattern (P < 0.025)  compared to the exponential (n = 5), spherical (n = 4), or Gaussian (n = 2). Conversely, 17 of 18 nabid data fitted the linear (n = 17) model, while one dataset (i.e. JW 31 of Field C) fitted to the exponential model. The nugget‐to‐sill ratio was < 0.25, indicating strong aggregation in 11 and two of the sampling times for coccinellids and nabids, respectively . The range values of the variograms of coccinellids were between 13.1 m and 25.6 m, with a mean of 16.6 m, while only one sampling date showed aggregation in 3.4.2P < 0.025) in 10 of 19 sampling times, with at least one significant sampling date occurring in each field  and H. quinquesignata (Kirby) (Coleoptera: Coccinellidae). Unlike coccinellids, the aggregation pattern of Nabis spp. was found at a low level, 5.6% of the total datasets used for both variogram and SADIE across five fields. These results are in line with previous reports of none to moderate aggregation of Nabis spp. nymphal populations in soybean fields using non‐geospatial methods.Nabis spp. could be due to their prey‐searching behavior, with continuous movements of individuals in the field, much more than coccinellids.Natural enemies can play an important role in balancing the prey density. In our study, out of the total sampling times evaluated, the spatial aggregation pattern of coccinellids was found in 57.9% and 52.6% based on variogram and SADIE, respectively. Using mean–variance methods, Evans and TrentH. postica and Nabis spp., but also between H. postica and coccinellids in alfalfa fields. Several factors such as the harvest timing,versus alternative prey), and the presence of other natural enemies can influence probability of association between coccinellids and its prey.et al.Acyrthosiphon pisum Harr.) (Hemiptera: Aphididae) was high, particularly during the second (JW 25) and third (JW 31) sampling periods . It is known that coccinellids prefer aphids over their alternative prey that includes H. postica, and this could have resulted in a weak association between H. postica and coccinellids in these alfalfa fields. Asynchronous distribution between prey and natural enemies can be useful in certain site‐specific pest management situations such as applying insecticide when populations of prey, but not natural enemies, aggregate in the field. Under this scenario, properly timed insecticide applications would be less disruptive to natural enemy populations.Hassell and MayThe range value of the variogram models, which indicate aggregated distribution patterns , can be used to develop reliable sampling methods for insect pest monitoring and population assessment. Sweep net, stem‐count, and shake‐bucket are the most common sampling methods that alfalfa growers practice for scouting and monitoring of alfalfa weevil, particularly for the larval population. It is recommended to sample randomly from several locations in a field to determine the economic threshold level.H. postica samples are spatially dependent on average distances of 22.3 m for larvae and 14.7 m for adults. The integration of range value information  into sampling methods may help to improve current alfalfa weevil larvae scouting and monitoring programs. The range value of the variogram can be used to develop sampling plans for two purposes. If the intention is to develop H. postica abundance hot‐spot maps and conduct site‐specific pest management as it has been used for some agricultural pests,H. postica management in alfalfa fields.Our study is the first to demonstrate that Nabis spp. within the field, the comprehensive sampling plan developed for H. postica and coccinellids should also work for Nabis spp. as minimum sampling distance does not apply to the randomly‐distributed population. A total of 81 sample points in each field were used to obtain an empirical minimum sampling distance using variogram analyses. With this new sampling distance guidelines, a high level of sampling intensity is not required for routine pest sampling. The sample size can be reduced by 50% to make the sampling plan more cost‐effective under field conditions. Based on the spatial distribution information generated in our study, we recommend a systematic sampling scheme, including the use of a minimum of 23 m sampling distance  and a minimum of 40 sample points in a grid when conducting alfalfa weevil and predator sampling when using either sweep net or damaged stem counts. This sampling plan should provide a reasonable estimation of alfalfa weevil larvae and its natural enemies populations in the alfalfa field. This allows the alfalfa producers to accurately estimate the weevil population as well as predator(s) density within the field accurately. This approach helps to reduce the unnecessary use of pesticides by properly determining the need and timing for insecticide applications. These are important aspects of integrated pest management (IPM) in order to reduce environmental pollution, promote biological control, minimize the risks of pesticide resistance, and overall reduce the pesticide‐related expense.In our study, coccinellid predators showed a spatial dependency within the distance of 16.6 m for most of the sampling times (~58%). The minimum sampling distance for coccinellids to obtain independent samples is 17 m, and this information can be integrated to develop a comprehensive sampling plan. Since the majority of the sampling time (> 94%) datasets indicated a random distribution of H. postica and the population of its two natural enemies (coccinellids and nabids) in irrigated alfalfa fields and develop a comprehensive sampling plan for their population assessment. Although this study was conducted in Montana, the study results and sampling recommendations should apply to other irrigated alfalfa producing regions in the western United States that include Pacific Northwest, California, Arizona, New Mexico, and others. Future studies can validate this sampling scheme for major alfalfa‐growing areas in the western United States and examine the spatial distribution of additional natural enemies, such as parasitoids, to improve pest management programs for alfalfa weevil.In conclusion, our study was able to characterize the spatial and temporal distribution of The authors declare no conflict of interest. Permission to collect insect samples was granted by local private landowners of Pondera County of Montana: Jeremy Curry, Tony Verstrate, John Balkenbush and Zane Drishinski. The research activities reported here did not involve, pose a risk to, or harm any endangered or protected species.


Actb, Pgk1, Sdha, Gapdh, Rnu6b) as a function of hypoxia exposure and hypothermic treatment in the neonatal rat cerebral cortex–in order to identify RGs that are stably expressed under these experimental conditions–using several statistical approaches that have been proposed to validate RGs. In doing so, we first analyzed RG ranking stability proposed by several widely used statistical methods and related tools, i.e. the Coefficient of Variation (CV) analysis, GeNorm, NormFinder, BestKeeper, and the ΔCt method. Using the Geometric mean rank, Pgk1 was identified as the most stable gene. Subsequently, we compared RG expression patterns between the various experimental groups. We found that these statistical methods, next to producing different rankings per se, all ranked RGs displaying significant differences in expression levels between groups as the most stable RG. As a consequence, when assessing the impact of RG selection on target gene expression quantification, substantial differences in target gene expression profiles were observed. Altogether, by assessing mRNA expression profiles within the neonatal rat brain cortex in hypoxia and hypothermia as a showcase, this study underlines the importance of further validating RGs for each individual experimental paradigm, considering the limitations of the statistical methods used for this aim.Real-time reverse transcription PCR (qPCR) normalized to an internal reference gene (RG), is a frequently used method for quantifying gene expression changes in neuroscience. Although RG expression is assumed to be constant independent of physiological or experimental conditions, several studies have shown that commonly used RGs are not expressed stably. The use of unstable RGs has a profound effect on the conclusions drawn from studies on gene expression, and almost universally results in spurious estimation of target gene expression. Approaches aimed at selecting and validating RGs often make use of different statistical methods, which may lead to conflicting results. Based on published RG validation studies involving hypoxia the present study evaluates the expression of 5 candidate RGs ( In qPCR analysis, reference genes (RGs) with stable expression levels are essential internal controls for relative quantification of mRNA expression. RGs normalize variations of candidate gene expression under different conditions , 2. The RG selection should be performed using the same samples that will be compared when looking at genes of interest. For this purpose, several statistical methods have been proposed, i.e. GeNorm , qBase , BestKeeTo combine these stability rankings, two main approaches have been proposed, i) a weighted rank –20 and iIn the present study, we applied this same approach in the evaluation of the stability of five candidate RGs in a murine model of perinatal asphyxia and therapeutic hypothermia. Perinatal asphyxia is a clinical condition defined as oxygen deprivation that occurs around the time of birth and may be caused by perinatal events such as placental abruption, cord prolapse, or tight nuchal cord, limiting the supply of oxygenated blood to the fetus . Recentlin vivo and in vitro studies on hypoxia, making use of qPCR, have been reported [Here, we used a murine model of perinatal hypoxic-ischemic encephalopathy that causes well-described physiological and behavioral impairments and recapitulate several key features of human perinatal hypoxic-ischemic injury –31, to areported –38, indiRest), a gene that has been shown to be upregulated by hypoxic-ischemic injury in the peri-infarct cortex of adult rats following transient focal ischemia induced by middle cerebral artery occlusion (MCAO) [Bad), a gene that has been shown to be up-regulated by hypoxia in the MCAO rat model, was assessed [We selected five candidate RGs based on published RG validation studies involving hypoxia . Subsequn (MCAO) . Moreoveassessed . This stSprague–Dawley albino rats with genetic quality and sanitary certification from the animal facility of our Institution were used following the international rules and guidelines of the Federation of European Laboratory Animal Science Associations (FELASA). Animals were kept under standard laboratory conditions, with light/dark cycles of 12/12 h. Standard rat chow and water were given ad libitum. During housing, animals were monitored twice daily for health status. No adverse events were observed. All the procedures concerning the animal manipulation and treatment were performed according to the Guide of Animal laboratory Care (revisited in 1996) by the National Institute of Health Guide for the Care and Use of Laboratory Animals (Publications No. 80–23). The animal model described below has been approved by the Ethical Committee of CICUAL: “Comité Institucional para el Uso y Cuidado de Animales de Laboratorio” (Resolution N° 2079/07), Facultad de Medicina, Universidad de Buenos Aires, Argentina. All sections of this report adhere to the ARRIVE Guidelines for reporting animal research . A complRattus norvegicus) obtained from the Animal Facility of the Facultad de Medicina, Universidad de Buenos Aires, Argentina were housed in an individual delivery-cage, maintained at controlled temperature (22±1°C), humidity (50–60%), with a fixed 12-h light/dark cycle (light on 7:00 AM to 7:00 PM), and standard rat chow and water present ad libitum.Fifteen pregnant albino Sprague-Dawley rats  following the manufacturer’s instructions. The residual DNA was removed by the TURBO DNA free kit . Yield and purity of RNA were determined by the NanoDrop ND-1000 spectrophotometer . RNA samples with an absorbance ratio OD 260/280 between 1.9–2.2 and OD 260/230 greater than 2.0 were used for further analysis. RNA integrity was assessed using agarose gel electrophoresis. One microgram of RNA from each sample was reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer’s instructions. cDNA was stored at −20°C for future use. For qPCR analysis, each cDNA sample was diluted 20 times with nuclease-free water.Real-time PCRs were conducted using the LightCycler 480 Multiwell Plate 96  containing 1μM of each primer. For each reaction, the 20 μl mixture contained 1 μl of diluted cDNA, 5 pmol each of the forward and reverse primers, and 10 μl 2 × SensiMix SYBR No-ROX Kit . The amplification program was as follows: 95°C for 30 sec, 40 cycles at 95°C for 15 sec, and 60°C for 15 sec, and 72°C for 15 sec. After amplification, a thermal denaturing cycle was conducted to derive the dissociation curve of the PCR product to verify amplification specificity. Reactions for each sample were carried out in triplicate. qPCR efficiencies in the exponential phase were calculated for each primer pair using standard curves . The mean threshold cycle (Ct) values for each serial dilution were plotted against the logarithm of the cDNA dilution factor and calculated according to the equation E = 10(−1/slope) × 100, where the slope is the gradient of the linear regression line.Actb, Pgk1, Gapdh, Sdha, Rnu6b. These genes represent commonly used endogenous control genes chosen from the relevant literature and have been previously validated in rat, mouse and human brain tissues exposed to hypoxia. The selected RGs belong to different molecular pathways to minimize the risk of co-regulation between genes. The primers were designed from nucleotide sequences identified using NCBI BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Rnu6b TaqMan MicroRNA Assay (Rnu6b) was commercially available . All other primers were ordered from Thermo Fisher Scientific with their certificates of analysis. The primer characteristics of nominated RGs are listed in Based on their common usage as endogenous control genes in previous studies , five caRest;—F, AACTCACACAGGAGAACGCC—R, GAGGTTTAGGCCCGTTGTGA.Bad;—F, GCCCTAGGCTTGAGGAAGTC—R, CAAACTCTGGGATCTGGAACA.To assess the stability of candidate RGs, five statistical methods, each with unique characteristics, were used: GeNorm, BestKeeper, NormFinder, Coefficient of Variation analysis, and the comparative ΔCt method. Ct values were converted to non-normalized relative quantities according to the formula: 2−ΔCt. CV analysis, GeNorm and NormFinder calculations are based on these converted quantities, whereas BestKeeper and the ΔCt method make use of raw Cq values. To obtain a integrated ranking, we used the workflows as described in more detail previously , 21.Rest and Bad. The relative expression profiles of Rest and Bad were determined and normalized with all tested RGs. Relative fold changes in gene expression were calculated using the DDCt and Pfaffl methods. Data was expressed as mean ± standard error of the mean (SEM) from six independent samples/group with triple qPCR reactions. One-way analysis of variance (ANOVA) test was applied to analyze significant differences between conditions for each house-keeping gene. Differences were reported as statistically significant when p<0.05. GraphPad Prism 6  was used for statistical procedures and graph plotting.The impact of RG selection on gene expression quantification was assessed via examining the expression of Pilot assays were performed to optimize cDNA and primer quantities. A total of 0.9 mg of RNA that was previously treated with DNase was used for the reverse transcription reaction in a total volume of 40ml. One microliter of the resulting cDNA was used for the qPCR reaction. Each gene amplification was analyzed, and a melting curve analysis was performed, showing a single peak indicating the temperature of dissociation. Efficiencies are shown in Gapdh as the most stable RG, and Actb as the least stable RG. This method however does not consider potential expression differences between different conditions; hence, a CV analysis alone cannot determine the best set of RGs.We calculated the raw expression profiles of RGs as changes of Ct values across groups and ranked the gene stability according to the CV. The CV estimates the variation of a gene across all samples, therefore, a lower CV value indicates higher stability . This anSdha, Rnu6b, Pgk1, Actb) showed significant variation in mRNA levels when comparing different conditions , the expression stability of candidate RGs was analyzed using four well known statistical methods .Rnu6b, which presented the highest M-value (M = 1.923), all of the other candidate RGs presented M-values lower than 1.5, which is considered to be the cut-off for suitability [Pgk1 and Actb. This is in contrast to the CV analysis, that showed those genes as the least stable ones (higher CV), and to the expression profiles that showed inter-group differences.First, a GeNorm analysis was performed on all five candidate genes. GeNorm calculates a stability value (M) based on pairwise variation of every two genes. In our analysis, except for tability . Based oActb, Sdha and Pgk1 were the most stable RGs, presented stability values lower than 0.3. Gapdh (SV = 1.736) and Rnu6b (SV = 3.17) were the least stable.NormFinder calculates the stability score (S) based on the inter- and intra-group variation. However, it has been reported that including genes with high overall variation can affect the stability ranking of all genes with this method . Actb, SPgk1 (r = 0.825), while Rnu6b was considered the least stable gene (r = 0.106). The ranking obtained from this analysis was the same as the one obtained with GeNorm.BestKeeper uses the cycle threshold (Ct) values to calculate a standard deviation (SD), coefficient of variance (CV), and Pearson correlation coefficient (r) for each gene. Lower SD and CV values indicate more stable gene expression, and genes that exhibit a SD in Ct values above 1.0 should be eliminated and regarded as unreliable controls. Then, the remaining RG are ranked according to r values, with a higher r value indicating more stable gene expression. None of the genes analyzed were excluded for having SD above 1. The most stable RG was Pgk1 (Av. SD = 1.41) and Sdha (Av.SD = 1.45), and the least stable Rnu6b (Av.SD = 3.23). The overall ranking depicted in Pgk1.Using the Δ-Ct method, the ranking was similar to previous rankings. The most stable RGs were Rest and Bad. These genes have shown to be influenced by hypoxia and hypothermia. Five gene expression normalizing strategies were used to select the least and most stable RGs, and the best combination of two genes, Actb/Pgk1  and the primer efficiency method Click here for additional data file.


Body image (BI) is an important issue for cancer patients, as patients with BI concerns are susceptible to depression, anxiety, difficulty coping, and poor quality of life (QoL). While this concern has been documented in patients with other malignancies, no data exists of this QoL issue in patients with primary brain tumors (PBT).A cross-sectional survey of 100 PBT patients was conducted on an IRB approved prospective protocol using structured questionnaires. Participants completed the body image scale (BIS), Appearance Scheme Inventory Revised (ASI-R), MD Anderson Symptom Inventory Brain Tumor (MDASI-BT), and Patient-Reported Outcomes Measurement Information System (PROMIS) Depression, Anxiety, and Psychosocial Impact Positive measures.r = 0.53, 95% CI 0.37 to 0.65). The mean PROMIS Depression score was 48.4 (SD = 8.9), PROMIS Anxiety score was 49.4 (SD = 9.9), and PROMIS Psychosocial Illness Impact Positive score was 48.9 (SD = 9.7). BIS was significantly correlated with age, and trended with BMI and sex. The PROMIS Psychosocial Illness Impact Positive and PROMIS Anxiety scores were the most strongly related to BIS.The prevalence of clinically significant body image dissatisfaction (BIS ≥ 10) was 28% (95% CI 19–37%), median BIS score was 5 (range 0–27). The median ASI-R composite score was 2.9 (range 1.5–4.7). BIS was significantly correlated with the ASI-R  contains supplementary material, which is available to authorized users. Body image is a multidimensional concept that includes one’s physical appearance and the cognitive, emotional, and relational elements that influence an individual’s sense of identity . It inclBody image has been found to be an important concern for cancer patients. It has been noted that the physical signs of disease or its treatment can be a constant reminder of the reality of cancer, leading to adaptation difficulties or the emergence of emotional frailty . Body imWhile body image concerns and their psychosocial outcomes have been documented in patients with many cancer pathologies, currently no data exists of this quality of life (QoL) issue in patients with primary brain tumors (PBT) . PatientThe goal of this study was to address the prevalence of body image concerns in PBT patients using validated questionnaires previously used in other cancer populations, and explore any contributing psychological, disease, and treatment related factors. This information is of importance as the prevalence of people in the US living with PBT was estimated to be 688,000 in 2010 and projecting that 86,970 will be diagnosed in 2019 , 16.Patients enrolled on an Institutional Review Board approved protocol between December 2017 and April 2018 were screened and approached if eligible. This was a cross-sectional design at a single time point. and eligibility requirements were adult (≥ 18 years old) patients with histologically confirmed PBT, with intracranial only disease, who were proficient in English. Informed consent was obtained from all participants. Patient medical data was collected prospectively from patient charts such as age, gender, Karnofsky Performance Score (KPS), height, weight, cancer diagnosis, and previous treatment history.The Body Image Scale (BIS) was used to assess body image dissatisfaction. The questionnaire allows cancer patients to self-rate change in body image after diagnosis or treatment . It is aInvestment in body image and appearance was assessed by the Appearance Schemas Inventory–Revised, a 20-item scale with scores calculated based on the mean of responses for a Composite score . It consSymptom burden was assessed using the MD Anderson Symptom Inventory-Brain Tumor Module (MDASI-BT). The 28-item MDASI-BT assesses symptom severity and symptom interference . The MDAEmotional state was assessed with three Patient Reported Outcomes Measurement Information System (PROMIS®) measures. The PROMIS Short Form v1.0—Depression 8a assesses mood, views of self, and social cognition . The PROAdditionally, an open response, Body Image Feedback Form  was provided to the participants. It allowed participants to respond to open-ended questions about their body image and cosmetic concerns, and the effects of the tumor and its treatment on their lives.p < 0.05. Qualitative analysis was conducted using MAXQDA 2018  for text coding of response data on the Body Image Feedback form to identify recurring themes in body image concerns.IBM SPSS Version 25 was used for the statistical analysis . DescripOne hundred patients were enrolled and completed the BIS and ASI-R. Patients’ age ranged from 23 to 74 years old (median 48 years), and 56% of patients were male. The most common diagnosis was glioblastoma (32%), and 30% had low grade (WHO I–II) malignancies. The median time from diagnosis was 5 years (range 0–22), and 85% of the participants had a KPS ≥ 80. 66% of patients were overweight or obese, and all participants had undergone brain tumor surgery. Patient characteristics are presented in Table r = − 0.24, 95% CI − 0.44, − 0.05, p = 0.015), with younger patients having increased body image dissatisfaction. The BIS demonstrated very good reliability in the PBT population with a Cronbach’s alpha of 0.91 with all ten items contributing to its reliability.The BIS score was used to evaluate body image dissatisfaction. The median BIS score was 5.0 range 0–27) with a mean of 7.0 (SD = 6.7). Clinically significant body image dissatisfaction (BIS score ≥ 10) was present in 28% (95% CI 19–37%) with 14% of patients reporting no body image dissatisfaction (BIS = 0). The relationship between BIS, demographic, and clinical factors were assessed , the ASI-R Self-Evaluative score , but not with the ASI-R Motivational score . As body image dissatisfaction increased so did the negativity of the patient’s belief about their appearance. Cronbach’s alpha for the ASI-R composite, and self-evaluative and motivational subscales were 0.87, 0.85, and 0.81 respectively.The ASI-R evaluated body image investment. The ASI-R composite scores ranged from 1.5 to 4.7 (mean = median = 2.9 (SD = 0.6)). Subscale scores for Self-Evaluative salience ranged from 1.2 to 4.5 (mean = 2.6 (SD = 0.7), median = 2.5); and Motivational salience ranged from 1.9 to 5.0 (mean = 3.3 (SD = 0.7), median = 3.4). The BIS score was significantly correlated with the ASI-R Composite score  characterized patients body image challenges. Twenty-six patients (36%) contributed to the most prevalent theme relating to lifestyle factors , daily routine impacted , changes in relationships , and loss of independence . One patient reported, “It has affected my daily routine or just doing the things in life like walking, kneeling, steps, hills, things we take for granted ”. Patients described common short and long- term symptoms , vision problems , pain , fatigue  impacting their quality of life. Additionally, treatment and surgery impacted changes in appearance , as patients mentioned visible symptoms , indentation on the head , and dry skin . One patient reported, “Whenever I meet someone, I feel like they will judge me for my scar on my head and I do feel especially unattractive because of the scar in my head ”. Only 3 out of the 23 (13%) patients affirmed making a lifestyle adjustment to accept unavoidable changes to their appearance. A negative outlook , difficulty dealing with changes  endorsed by nine patients (13%) described their concerns with long-term physical changes, however a positive change in perspective , acceptance of changes , exercise , hope/importance of physical health/enjoyment of life (1/13%) provided new meaning to patients’ outlook. On the open-ended questionnaires, fifteen patients (21%) reported no body image concerns.This protocol investigates a previously unstudied QoL concern for patients with PBT . Body imThe current data supports the importance of body image concerns in the PBT population as well, with 28% of patients having clinically significant body image dissatisfaction, and only 14% of patients noting none on the BIS measure. Age and BMI showed small mean differences in BIS scores, with younger patients or patients with a larger BMI reporting more body image dissatisfaction. No difference was seen in the body image outcomes in terms of sex, tumor grade, performance status, or previous therapy.The prevalence and amount of body image dissatisfaction among PBT patients presents at a similar level to that seen in other solid tumor populations. Harrington et al.  reportedIt was hypothesized that parallels could be drawn between the disease related dysfunction and treatment effects in the head and neck cancer and PBT populations . HoweverSimilarly, a sample of patients with oral cavity cancers were found to have a mean BIS score of 2.51 (SD = 3.14) . CorrelaIn the general head and neck population study, 33% of patients endorsed behavioral changes including reassurance seeking, increased grooming or checking behaviors, or avoidance of grooming due to heightened concern with appearance which is more in keeping with the results of the ASI-R in PBT patients . HoweverAn analysis of body image dissatisfaction among patients with advanced cancers of the breast, GI, GU, head and neck, gynecologic, hematologic and respiratory system demonstrated a 58% rate of clinically significant body image dissatisfaction (BIS ≥ 10), which significantly correlated with increased physical and psychological distress and depression . This reWith increasing longevity and survival in a variety of brain tumors, the potential impact of body image will become a more significant patient survivorship concern. The importance of this survivorship issue in other tumor sites is well recognized even amongst patients with poor prognosis , 6, 29. Establishing a model of body image dissatisfaction among PBT patients is of clinical importance and may allow for intervention in this patient population, as has been done in other tumor types , 30. TheThe BIS and ASI-R demonstrated good internal consistency and reliability in the PBT population and are useful measures for future analysis. A BIS score of ≥ 10 has been suggested as a clinical cutoff for body image dissatisfaction. It has been used in a variety of cancer pathologies, and patients with advance disease, supporting its use in PBTs, however, the optimum cutoff for the PBT population remains an important area of future research , 17, 18.This study explored the prevalence of body image concerns in the previously unstudied PBT population. In determining the factors associated with body image concerns limitations of this study include patients numbers, single institution, and cross sectional design. Future research may focus on change in body image dissatisfaction over time to determine opportune time points for intervention, optimal cutoff values of body image scales, and primary CNS tumors of the spinal cord –34.Despite the potential significant impact of tumor and treatment effects on body image and subsequently self-esteem, sexuality, and interpersonal relationships in the PBT population, very little was known. The present study demonstrated that 28% of PBT patients are affected by clinically significant body image dissatisfaction. Exploratory analysis of related factors included BMI, age, anxiety, and patient outlook on the impact of their illness. Based on the knowledge of the significant QoL impact of body image in other tumor types, this data supports future research into the characteristics and interventions for this QoL issue among PBT patients.Supplementary file1 (DOCX 15 kb)Below is the link to the electronic supplementary material.


The purpose of this study was to evaluate the incidence of vitreomacular adhesion (VMA) release after anti-VEGF therapy for the treatment of diabetic macular edema (DME) and to evaluate further changes in outcome.This was a retrospective study that enrolled 66 eyes of 66 patients with DME who presented with VMA diagnosed by spectral-domain optical coherence tomography (OCT) at baseline. VMA was classified as focal (attachment: ≤ 1500 μm) or broad (attachment: > 1500 μm). All patients received at least three monthly intravitreal injections of an anti-VEGF agent. Follow-up visits were performed 1 month after each injection to evaluate the incidence of VMA release.The mean patient age was 61.4 years (range: 29 to 78 years), and 72.7 % were male. The mean best-corrected visual acuity was 0.62 logMAR, and the mean central retinal thickness (CRT) was 473 μm at baseline. The mean length of follow-up was 18.5 months, and the mean number of injections was 5.8. The intravitreal drugs used were aflibercept (40.9 %), ranibizumab (37.9 %) and bevacizumab (21.2 %). Forty-seven eyes had broad VMA, and 19 had focal VMA. Twenty-two eyes (33.3 %) developed VMA release following a mean of 5.7 injections (range: 3–13). Sixteen eyes (72.7 %) with focal VMA and 6 eyes (27.3 %) with broad VMA at baseline developed VMA release. Twenty-one eyes that developed VMA release showed an improvement in CRT following VMA release .VMA release occurs in approximately 1/3 of patients with DME following anti-VEGF therapy. Most of them show a short-term decrease in CRT. Diabetic macular edema (DME) is the most common cause of visual impairment in patients with diabetes mellitus, occurring in approximately 7–8 % of the diabetic population . The patMany studies have evaluated the influence of vitreomacular adhesion (VMA) on the response to anti-VEGF agents in macular diseases. A recent retrospective study did not identify an association between vitreoretinal interface status and treatment outcomes with anti-VEGF agents for macular edema secondary to retinal vein occlusions . In caseSince the configuration of the vitreomacular interface in macular diseases seems to have an effect on visual outcomes, it would be important to know the incidence of VMA release following intravitreal injections of anti-VEGF agents, which could be an independent factor on its own. A recent study showed that only 5.6 % of neovascular AMD patients developed posterior vitreous detachment (PVD) following intravitreal anti-VEGF therapy . It is nThis was a retrospective study designed to evaluate the incidence of PVD after intravitreal injections of currently used anti-VEGF drugs for DME. All subjects were informed about the nature of the study and signed a written informed consent in accordance with the tenets of The Declaration of Helsinki.The Ethics Committee of the Federal University of Minas Gerais in Belo Horizonte, Brazil, approved the study.From March 2011 to June 2019, all patients newly diagnosed with DME at the Institute of Vision were enrolled in this study. At baseline, all patients underwent a complete ophthalmological examination, including slit lamp biomicroscopy, color fundus photography, fluorescein angiography and optical coherence tomography (OCT). Inclusion criteria were (a) presence of intraretinal cysts and/or subretinal fluid detected by SD-OCT located at least 750 μm from the central fovea or central retinal thickness ≥ 300 μm; (b) presence of VMA; (c) visual acuity ≥ 20/800 at baseline; (d) follow-up of at least 3 months; and (e) random enrollment of only one eye into this study. If both eyes were eligible, the study eye was randomly selected for entry.The exclusion criteria were (a) eyes with other conditions known to affect the vitreomacular interface, such as retinal vascular disease, age-related macular degeneration, uveitis and pathologic myopia; (b) ocular conditions that may affect visual acuity; (c) vitreomacular traction; (d) eyes previously submitted to pars plana vitrectomy (PPV) and (e) eyes previously treated with laser, anti-VEGF or corticosteroids for the last three months.The evaluation of the vitreomacular interface was based on the flowchart shown in Fig. pro re nata regimen. Furthermore, after the first injection, there was the possibility of switching of the intravitreal drug in some cases. Retreatment criteria were (a) persistence of or increase in intra- or subretinal fluid and (b) worsening of at least one line of VA. A single retinal specialist (M.B.N.) performed all intravitreal injections.Intravitreal anti-VEGF injections were performed using bevacizumab , ranibizumab  or aflibercept . All intravitreal anti-VEGF injections were performed in the operating room with aseptic techniques, including the prophylactic use of topical iodopovidone 5 %. All patients were subjected to a treatment protocol that included a loading dose with three intravitreal injections of anti-VEGF agents at one-month intervals. After the third dose, they followed a Best-corrected visual acuity (BCVA) and CRT were measured before and 1 month after each intravitreal injection. BCVA was determined using a Snellen chart and then converted to logarithm of the minimal angle of resolution (logMAR) values. CRT was measured based on central 1 mm subfield thickness. The automated segmentation of retinal boundaries was used and, in case of any segmentation error, corrected manually.A total of 280 eyes of 196 patients were diagnosed with DME during the study period. Of these eyes, 128 eyes (45.7 %) had VMA, and a total of 66 eyes of 66 patients met the inclusion criteria and were evaluated in this study. The mean age was 61.4 years (range: 29 to 78 years), and 47 out of 66 patients (72.7 %) were male. The mean baseline BCVA was 0.62 logMAR, and the mean baseline CRT was 473 μm. Fifty-three eyes (80.3 %) were phakic, and 13 (19.7 %) were pseudophakic. Forty-three eyes (65.1 %) were treated with focal laser treatment before or during the course of the study. The mean follow-up period was 18.5 months (range: 3 to 42 months). The mean number of intravitreal injections was 5.8 (range: 3 to 21 injections). The intravitreal drugs used in the study were aflibercept (40.9 %), ranibizumab (37.9 %) and bevacizumab (21.2 %). Of 66 eyes with DME and VMA, 47 (71.2 %) had broad VMA, and 19 (28.8 %) had focal VMA at baseline.A total of 22 eyes (33.3 %) presented VMA release following a mean of 5.7 intravitreal anti-VEGF injections (range: 3–13), while 44 eyes (66.7 %) maintained persistent VMA. VMA release developed after the intravitreal injection of aflibercept in 11 eyes, ranibizumab in 6 eyes and bevacizumab in 5 eyes. CRT decreased at the following visit in 21 eyes that developed VMA release after intravitreal injection   that presented VMA release were phakic, and 5 (22.7 %) were pseudophakic. Among 22 eyes that developed VMA release, 16 eyes (72.7 %) had focal VMA at baseline, with diameters of vitreous attachment ranging from 365 to 1120 μm (mean: 618 μm); 6 eyes (27.3 %) were classified as having broad VMA at baseline. The area of attachment in all eyes classified as having broad VMA at baseline showed a progressive reduction prior to the development of VMA release, so that all eyes presented focal VMA by the time this release occurred.The role of the vitreomacular interface in the pathogenesis and outcome of DME has been previously studied but is not completely understood. Previous studies indicate that DME may be exacerbated due to persistent vitreomacular traction, a thickened and taut posterior hyaloid that may or may not be adherent to the internal limiting membrane, macular traction due to tractional proliferative membranes, or loculation of cytokines in the premacular vitreous pocket .There are two landmark papers dealing with DME and the vitreous in clinical terms. Hikichi et al. showed that if PVD occurs in the presence of DME, it leads to resolution of edema and improvement in visual acuity. In their report, spontaneous resolution of DME was seen in 55 % of eyes in which PVD developed compared with only 25 % in those with persistent VMA . GaucherPPV has been proposed for the treatment of DME in select cases. After PPV, VEGF and other proinflammatory cytokines would be expected to diffuse away from the macula more easily. Therefore, PPV can produce structural and functional improvements in some eyes with DME. However, a recent meta-analysis showed that the visual gains are not significantly better than those obtained with laser or observation .More recently, several studies evaluated the effects of VMA on anti-VEGF treatment for neovascular AMD, suggesting that VMA may be associated with a worse VA outcome or the need for more intravitreal injections –10. It wIn the present study, 45.7 % of the eyes presented VMA, a higher percentage than those in other recent studies of DME that reported a prevalence ranging from 20.9 to 37.5 % , 14–18. We found that among 66 eyes with VMA, a total of 22 eyes (33.3 %) developed VMA release following intravitreal anti-VEGF injections, while 44 eyes (66.7 %) maintained persistent VMA. Since all eyes developed VMA release within a relatively short period after intravitreal injection, this indicates that rather than being coincidental, the release in these cases might have been induced by the intravitreal injection. However, it is not clear whether the VMA release occurred based on a pharmacologic or a mechanical effect of the injection. In fact, many intravitreal procedures, including gas injections, can lead to VMA release. For a better analysis of this subject, it would be ideal to have a control group receiving sham injections, which, obviously, would be unethical. The incidence of VMA release after treatment was higher in our study than in previous investigations. A recent study based on data from the READ-3 (Ranibizumab for Edema of the Macula in Diabetes: Protocol 3 with High Doses) clinical trial retrospectively evaluated the role of VMA in visual and anatomic outcomes in patients with DME treated with 6 monthly injections of ranibizumab. They noted that at month 6, among the 26 VMA(+) eyes, 7 eyes (26.9 %) developed PVD . AnotherPrevious studies showed that the majority of patients with either diabetic retinopathy or neovascular AMD presented with a broad area of vitreomacular attachment when they were selected for anti-VEGF therapy. Regarding diabetic patients, a previous study showed that among 26 eyes with DME and VMA at baseline, 5 eyes (19.2 %) had focal VMA, and 21 eyes (80.8 %) had broad VMA . RegardiIn our study, of 28 eyes that developed VMA release, 16 eyes (72.7 %) had broad VMA, and 6 eyes (27.3 %) had focal VMA at baseline. However, the area of attachment in all eyes classified as having broad VMA showed a progressive reduction prior to the development of its release, so that all eyes presented focal VMA by the time VMA release occurred. In a previous study, 4 of the 5 eyes (80.0 %) that had focal VMA at baseline developed PVD, whereas 3 of the 21 eyes (14.3 %) that had broad VMA at baseline developed PVD (p = 0.01) . In anotIn our study, CRT decreased in almost all eyes that developed VMA release after intravitreal injection, suggesting that this release may favor short-term anatomical outcomes. Even though this could have a benefit in a short-term period, if left untreated, macular edema may recur, which highlights the complex and multifactorial pathophysiology of this disease. We believe that a short-term evaluation immediately after VMA release, as performed in this study, could offer a more reliable analysis of the influence of this factor on the therapeutic response. In terms of functional improvement, BCVA improved in only 1/3 of the eyes that presented VMA release. It is known that other prognostic factors could influence the functional outcome, including lower hemoglobin A1c levels, younger age and absence of prior panretinal photocoagulation . AlthougConsidering that, in our study, anti-VEGF agents could be switched after the first injection, it was not possible to know the effect of each drug alone. For this purpose, it would be ideal to analyze subgroups of patients, each one treated with only one type of anti-VEGF agent. Furthermore, the data in this study do not allow us to know whether VMA release after intravitreal anti-VEGF injection would be influenced by the stage of diabetic retinopathy. Studies involving a larger number of patients and a longer follow-up would be necessary for such an assessment. Future investigations are necessary to better establish the possible functional and anatomical long-term benefit of VMA release in eyes with DME treated with anti-VEGF agents.In conclusion, we showed that approximately one-third of eyes with DME treated with anti-VEGF agents presented VMA release during the study period. VMA release leads to short-term anatomical improvement.