The aim of this study was to assess the prognostic value of ABCDE-SE in a prospective, large scale, multicentre, international, effectiveness study. Stress echocardiography (SE) was recently upgraded to the ABCDE protocol: step A, regional wall motion abnormalities; step B, B lines; step C, left ventricular contractile reserve; step D, Doppler-based coronary flow velocity reserve in left anterior descending coronary artery; and step E, electrocardiogram-based heart rate reserve.n = 952, with semi-supine bike, n = 887, or treadmill, n = 65 with adenosine for step D) or pharmacological stress . SE response ranged from score 0  to score 5 . All-cause death was the only endpoint. Rate of abnormal results was 16% for A, 30% for B, 36% for C, 28% for D, and 37% for E steps. During a median follow-up of 21 months (interquartile range: 13–36), 73 deaths occurred. Global X 2 was 49.5 considering clinical variables, 50.7 after step A only (P = NS (not significant)) and 80.6 after B–E steps (P < 0.001 vs. step A). Annual mortality rate ranged from 0.4% person-year for score 0 up to 2.7% person-year for score 5.From July 2016 to November 2020, we enrolled 3574 all-comers  with known or suspected chronic coronary syndromes referred from 13 certified laboratories. All patients underwent clinically indicated ABCDE-SE. The employed stress modality was exercise (ABCDE-SE allows an effective prediction of survival in patients with chronic coronary syndromes.  This approach has a recognized clinical value but it seems unfit to describe the emerging complexity and heterogeneity of the individual patient vulnerability. Therefore, SE was recently upgraded to the ABCDE protocol:The study hypothesis was that each pathophysiological variable may contribute independently and incrementally to the prognostic vulnerability in the individual patient and, therefore, the outcome would progressively worsen with the increasing number of abnormal steps during cardiac functional stress. To test this hypothesis, ABCDE-SE was performed in patients with chronic coronary syndromes, referred for clinically driven SE in accredited laboratoriesn = 281), step C (n = 18), or step D (n = 286). The main reasons were that the step was performed prior to specific training and certification for steps B and C, or the test was attempted but data were not obtained because of technical failure in step D. An additional 54 patients were lost to follow-up. The final study population included 3574 patients all studied with the full ABCDE protocol with outcome information.In this prospective study, we initially screened 4213 patients recruited from July 2016 to November 2020 by 13 certified laboratories of 5 countries . Of these initial 4213, 639 did not complete the full protocol with missing information on step B (The inclusion criteria were: (i) age >18 years; (ii) referral for known or suspected CAD; (iii) no severe primary valvular or congenital heart disease, or presence of prognosis-limiting comorbidities, such as advanced cancer, reducing life expectancy to <1 year; (iv) echocardiography of acceptable quality at rest and during stress; (v) willingness to give their written informed consent allowing scientific utilization of observational data, respectful of privacy rights; and (vi) recruitment in centres with systematic follow-up program.n = 1400, 39%); inconclusive exercise ECG or unable to exercise ; and prognosis assessment or re-assessment in stable CAD, with or without global left ventricular dysfunction .All patients underwent SE testing as part of a clinically driven evaluation and according to the referring physician’s indications. Indications were in recommendation Class I or IIa according to the European Society of CardiologyWritten informed consent was obtained from all patients before testing. The study protocol was reviewed and approved by the institutional ethics committees as a part of the SE 2020 study . The study was funded partly by the Italian National Research Council (Ageing project) and with travel grants of the Italian Society of Echocardiography and Cardiovascular Imaging with dedicated sessions during national meetings. No support from industry was received.We used commercially available ultrasound machines. All patients underwent comprehensive transthoracic echocardiography at restCriteria for terminating the test were severe chest pain, diagnostic ST-segment shift, excessive blood pressure increase , limiting dyspnoea, maximal predicted heart rate, and significant arrhythmias. Echocardiographic imaging was performed from parasternal long- and short-axis views, and apical four- and two-chamber views, using conventional two-dimensional echocardiography. Contrast use was recommended in cases of suboptimal acoustic window, when ≥2 segments were not optimally visualized for 50% or more of endocardial length, and its use ranged from 0% to 40% in recruiting centres, depending on local lab policy and reimbursement issues. Anti-anginal drugs were usually not suspended before testing.Step A included assessment of wall motion abnormalities and was performed in all patients. Wall motion score index (WMSI) was calculated in each patient at baseline and peak stress, in a 4-point score ranging from 1  to 4 (dyskinetic) in a 17-segment model of the left ventricle.Step B of protocol included the assessment of B lines with lung ultrasound and the four-site simplified scan, from mid-axillary to mid-clavicular lines on the third intercostal space, each site scored from 0  to 10 .Step C of protocol included the force-based assessment of LVCR as the stress/rest ratio of force, calculated as systolic blood pressure/end-systolic volume.Coronary flow velocity reserve (step D) was assessed during the standard SE examination using intermittent imaging of wall motion and LAD.Heart rate reserve (step E) was calculated as the peak/rest heart rate from 12-lead ECG.Figure 1).All steps were performed by the same sonographer/cardiologist with the same transducer for cardiac, lung, and coronary scan—although occasionally a different high-frequency transducer was used for coronary flow. All steps were acquired at rest and peak stress. If needed, steps were repeated after 5 min in the recovery phase .A detailed visual description of the scanning procedure is also available in a 9-min movie from the consortium (YouTube. ABCDE Stress Echo 2030: How I Make It. More easily done than said. Available at: All positivity criteria were determined a priori.The A criterion was considered positive in the presence of stress-induced RWMA (WMSI stress > rest), when at least two adjacent segments of the same vascular territory of the left ventricle showed an increment of at least one point of segmental score during SE.The B criterion was considered positive in the presence of stress or rest B lines ≥2 units.The C criterion was considered positive in the presence of force-based LVCR ≤2.0 for exercise or dobutamine and ≤1.1 for vasodilators.The D criterion was considered positive in the presence of CFVR ≤2.0.The E criterion was considered positive in the presence of HRR <1.80 for exercise or dobutamine or <1.22 for dipyridamole or adenosine.As required by SE 2020 protocol, all readers had passed the quality control for each of the four imaging parameters upstream to starting patient recruitment.Stress echocardiography response was summarized with a score ranging from 0 to 5 as follows: score 0  or scores 1–5, according to the number of abnormal steps .The results for each test were entered in the data bank at the time of testing by each recruiting centre and sent monthly to the core lab with the electronic case report form with clinical data. After checking for internal consistency by trained technical staff, and double-checking with the centre for data verification on possibly inconsistent input, the data were added to the data bank and locked.n = 1421) or non-invasive multidetector coronary angiography (n = 20) showing no CAD was available in 1441 patients. Coronary angiography was indicated by the referring physician based on symptoms, individual clinical characteristics, and non-invasive imaging results. Obstructive significant CAD was defined by a quantitatively assessed coronary diameter reduction ≥50% in the view showing the most severe stenosis. Images were read by experienced invasive cardiologists unaware of the SE results.Invasive coronary angiography  depending on variable distribution. For continuous variables, differences among groups were tested with one-way analysis of variance followed by Bonferroni P-value <0.10 at univariate analysis were considered for inclusion in the multivariate Cox proportional hazards model. Collinearity was verified for all the models. We used the variance inflation factors to check the presence of collinearity and, for the multivariable model containing the positivity components, the overall mean variance inflation factor was 1.16. Moreover, values for all the variables were below 1.4, thus suggesting no evidence of collinearity in the model.Survival was estimated using the Kaplan–Meier method and survival curves were compared by means of the log-rank test. Univariate Cox proportional hazards models were used to identify candidate predictors for selected endpoints. All variables with X 2 value to evaluate improvement of goodness-of-fit as well as continuous net reclassification index and integrated discrimination index to assess its incremental value. Statistical significance was set at P-value <0.05. All analyses were performed using STATA .Non-proportionality of hazard was assessed using the Schoenfeld test. The incremental value of ABCDE-SE was evaluated comparing multivariable models with and without individual steps using global Table 1. The employed stress was exercise , or pharmacological (n = 2622) with vasodilator , or dobutamine (n = 471). Patients with more advanced ABCDE score (4 or 5), when compared to patients with lower scores, were more often hypertensives and studied under anti-ischaemic therapy with beta-blockers and nitrates (Table 1).The main clinical characteristics of the 3574 study patients are described in Table 2. Figures 3 show examples of ABCDE negativity and ABCDE positivity, respectively. Rate of abnormal results was 16% for A, 30% for B, 36% for C, 28% for D, and 37% for E steps.The main SE findings are described in The positivity rate in various centres differed significantly, possibly due to different referral patterns and also because of the limited sample in some centres that determined a high degree of variability. In details, positivity rate ranged from 0% to 56% for step A, from 0% to 60% for step B, from 16% to 100% for step C, from 13% to 100% for step D, and from 8% to 86% for step E Table S1.All five parameters were normal (score 0) in 1114 patients (31%), all five were abnormal (score 5) in 172 patients (5%). The abnormal score was 1 in 1064 (30%), 2 in 610 (17%), 3 in 349 (10%), and 4 in 265 (7%) patients.Figure 4).In the 1441 patients with coronary angiography available, 848 patients (59%) showed no CAD, 373 patients (26%) showed one-vessel CAD, and 220 patients (15%) showed multivessel CAD. ABCDE score increased progressively with increasing extent of CAD , and step E , but not step A  and step C  (Table 3). In the multivariable analysis, ABCDE-SE was an independent predictor of mortality with scores 3 , 4 , and 5 . Annual mortality rate ranged from 0.4% person-year for score 0 up to 2.7% person-year for score 5. Survival was worse in patients with ABCDE score 5 and best in patients with ABCDE score 0 .During a median follow-up of 21 months (interquartile range: 13–36), 73 deaths occurred. In the univariable analysis, predictors of all-cause mortality were step B [hazard ratio (HR) 2.621, 95% confidence interval (CI) 1.654–4.152, X 2 of clinical model for the prediction of death increased from 49.5 considering clinical variables positive at univariate analysis to 50.7 after step A only (P = NS), and 80.6 after BCDE steps (P < 0.001 vs. step A). The increase over clinical evaluation was not significant after step A  and step C (after considering step B) and significant for steps B, D, and E .In the incremental analysis, global P = 0.048). Integrated discrimination index was 0.51% .Adding ABCDE-SE, risk reclassification was significantly improved with net reclassification index: 23.4%  for valvular or ischaemic heart disease to step G (for gradients) in hypertrophic cardiomyopathy up to step R (for right ventricular function) in valvular and some forms of congenital heart disease.ABCDE-SE allows assessment of multiple different vulnerabilities of the patient above and beyond epicardial artery stenosis. Vulnerabilities include pulmonary congestion, reduced contractile reserve, coronary microvascular dysfunction, and cardiac autonomic unbalance. This spectrum of phenotypes identifies the individual risk more accurately than inducible ischaemia and RWMA. Each step is important in modulating outcome and is a potential selective therapeutic target, paving the way to personalized assessment of risk and tailored therapy approaches.European Heart Journal online.The study was partially funded by CNR-MIUR  Ageing subproject .Conflict of interest: none declared.The data presented in this study are available on request from the principal investigator (QC). The data are not publicly available due to privacy.ehab493_Supplementary_DataClick here for additional data file.


Antimicrobial drug resistance is one of the top ten threats to global health according to the World Health Organization. Urinary tract infections (UTIs) are among the most common bacterial infections and main reason for antibiotic prescription. The incidence of UTIs appears to be high among people living with HIV. We sought to determine the most common UTI pathogens among HIV infected patients and evaluate their susceptibility towards antibiotics.We performed a cross-sectional study among HIV-infected patients aged ≥ 18 years presenting at an HIV care specialized clinic with symptoms suggestive of a urethritis. Urine cultures were subjected to antibiotic susceptibility testing according to Clinical Laboratory Standards Institute. The data was analyzed using STATA, we performed Pearson’s Chi-square and Fisher’s exact tests to compare differences between proportions.Escherichia coli was the most commonly isolated uropathogen (72%), followed by Klebsiella pneumoniae (9%). E. coli was completely resistant to cotrimoxazole and ampicillin; resistance to ciprofloxacin and ceftriaxone was 44% and 35% respectively; 9% to gentamicin; no resistance detected to nitrofurantoin and imipenem.Out of the 200 patients, 123 (62%) were female. The median age was 41.9 years (IQR 34.7–49.3). Only 32 (16%) urine cultures showed bacterial growth. Our findings are congruent with the Uganda national clinical guidelines which recommends nitrofurantoin as the first line antibiotic for uncomplicated UTI. Significant ciprofloxacin and ceftriaxone resistance was detected. In the era of emerging antibiotic resistance, understanding the local susceptibilities among sub-populations such as HIV infected patients is crucial. Further investigation is needed to address reasons for the low bacterial growth rate observed in the urine cultures.The online version contains supplementary material available at 10.1186/s12879-021-06865-3. Escherichia coli, Klebsiella pneumoniae, various other Enterobacteriaceae, as well as Staphylococcus saprophyticus [Urinary tract infections (UTIs) are one of the most common bacterial infections globally, with an estimated annual incidence of more than 150 million cases worldwide , 3. Theyphyticus . In recephyticus . World Hphyticus . Furtherphyticus .Sub-Saharan Africa (SSA), a region with a high burden of infectious diseases, is especially vulnerable to antimicrobial resistance . In Soutpneumocystis jirovecii pneumonia (PCP) [Generally, the incidence of UTIs appears to be high among people living with HIV, especially in those with lower CD4 lymphocyte count . HIV-infia (PCP) . Cotrimoia (PCP) . In Ugania (PCP) , 19.This study was conducted through the Infectious Diseases Institute (IDI), Makerere University, Uganda and University of Zurich (UZH), Switzerland research collaboration. The study describes findings of a UTI surveillance project implemented in an out-patient HIV clinic of the IDI of the Makerere University College of Health Sciences in Kampala, Uganda. We sought to determine the most common UTI pathogens in the HIV-positive population and evaluate their susceptibility towards commonly prescribed antibiotic agents.The study was conducted between September 2017 and December 2018 in an out-patient HIV clinic at IDI in Kampala, Uganda. It is an urban center of excellence for HIV care and treatment, with over 8000 patients.We performed a cross-sectional study among HIV-infected patients aged ≥ 18 years presenting with symptoms suggestive of urethritis. This was defined as showing one or more of the following symptoms; dysuria, urgency, frequency and fever. The patient data was collected as part of routine surveillance at the out-patient HIV-Clinic for a period of 16 months.Patients were instructed on how to provide an adequate midstream urine sample (MSU) by a nurse with the help of a leaflet with visual step-by-step instructions of the procedure. The MSU was then taken to the laboratory at the Department of Medical Microbiology, Makerere University School of Biomedical Sciences within 2 h or within 24 h if refrigerated at 4 °C.4 colony forming units (CFU)/ml, or > 105 CFU/ml if two types of pathogens were present, were classified as showing significant growth and used for further identification and sensitivity testing using Müller-Hinton Agar. Plates with no visible colonies were reported as no bacterial growth. Growth results ≥ 102 CFU/ml, but below the threshold for significant growth were reported as insignificant growth. Cultures with more than two types of organisms were reported as mixed growth, suggesting contamination. Drug susceptibility testing (DST) was performed for the following antimicrobial agents, as per the laboratory SOP: ciprofloxacin, cotrimoxazole, nitrofurantoin, ampicillin, ceftazidime, cefuroxime, ceftriaxone, chloramphenicol, gentamicin, imipenem, and nalidixic acid. Sensitivity was evaluated with the Kirby-Bauer disc diffusion method. More details on microbiology are provided in the Additional file The MSU was subjected to urine dipstick and urine cultures were subjected to antibiotic susceptibility testing according to Clinical Laboratory Standards Institute (CLSI). Sheep Blood and Cystine Lactose Electrolyte Deficient (CLED) agar were used for urinary culture. Cultures were incubated at 35–37 °C and checked for growth after 18–24 h. Agar plates with pure growth > 10In case the cultured uropathogen showed resistance towards the empirically prescribed antibiotic regimen, patients were contacted and asked to report to the clinic. Therapy was then adjusted by the assigned follow-up physician according to the resistance test results.Demographic and clinical characteristics, UTI related information including urine test results was collected using Epidata®. UTI-symptoms and further information about the patients’ medical history were obtained using a standard evaluation form that was filled out by the patients with the support of a nurse.The data was analyzed using STATA version 14.2 . Statistics for proportions, means with standard deviations (SD) and medians with interquartile ranges (IQR) were acquired to describe the data. We performed Pearson’s Chi-square and Fisher’s exact tests to compare differences between proportions.A total of 200 HIV infected patients presenting with symptoms suggestive of UTI were recruited and included in the study. Of these, 123 (61.5%) were female. The median age was 41.9 years (IQR 34.7–49.3); 40.1 years (IQR 33.8–48.7) for females and 43.9 years (IQR 36.7–50.9) for males. The majority of patients presented with dysuria . Nearly all the patients  reported to be taking cotrimoxazole or dapsone prophylaxis, which was recommended by the Uganda HIV treatment guidelines at the time of the study. The median duration on cotrimoxazole or dapsone prophylaxis in months was 110 (IQR 64–142). 83 (41.2%) patients reported using other antibiotics within the past month, of these 59 (29.5%) were taking antibiotics at the time of recruitment. Almost all the patients, 195 (97.5%) were on antiretroviral treatment (ART) and the median duration on ART in months was 103 (IQR 60–144). Majority of them, 195 (97.5%) had had a viral load test within the past 12 months; of these 175 (87.5%) were undetectable (< 75 copies/ml). Comprehensive details on the baseline characteristics of patients recruited in the study are presented in Table Escherichia coli was the most commonly isolated uropathogen (71.9%), followed by Klebsiella pneumoniae (9.4%) and Morganella morganii (6.3%). Other isolates included Acinetobacter baumannii, Enterobacter spp, Citrobacter freundii, Providencia spp., Enterococcus spp. and Group B Salmonella.Out of 200 cultures, only 32 (16.0%) showed bacterial growth. 36 (18.0%) cultures showed mixed bacterial growth, and 132 (66.0%) cultures had no bacterial growth. Escherichia coli was completely resistance to ampicillin and cotrimoxazole. Resistance to ciprofloxacin and ceftriaxone was 44.4 and 35.0% respectively; to gentamicin was 9.1%, no resistance was observed to nitrofurantoin and imipenem. Klebsiella pneumoniae was cultured in three cases, all were resistant to cotrimoxazole and ampicillin. One out of two isolates was resistant to ciprofloxacin; one out of three to ceftriaxone, gentamicin and nitrofurantoin; none was resistant to imipenem. Table Table Escherichia coli (71.8%). This finding is congruent with other studies conducted in Uganda, Sub-Saharan African countries and other parts of the world. In Uganda, two studies found Escherichia coli to be the responsible pathogen in 57.5% and 50% of culturally confirmed UTI respectively [Escherichia coli in 52.7% of urine cultures [Escherichia coli is estimated to be responsible for 60–70% of UTIs [This surveillance study describes urinary tract pathogens and their resistance patterns among Ugandan patients living with HIV who presented with symptoms suggestive of a UTI. The most commonly isolated pathogen in our study was ectively , 20. A scultures . In Euro of UTIs , 23. HowE. coli, the most isolated uropathogen was completely resistant to ampicillin and cotrimoxazole, as well as significantly resistant to ciprofloxacin and ceftriaxone. Susceptibility was high to nitrofurantoin, imipenem and gentamicin. In Uganda, high resistance in uropathogens to cotrimoxazole, amoxicillin, and nalidixic acid and considerable resistance towards ciprofloxacin has been observed [observed . This reobserved . Anotherobserved . High reobserved , 17, 24.observed . This faobserved , 25. A sobserved . The samobserved . Howeverobserved , 18, 20.observed . Since gOut of 200 patients, only 32 (16%) cultures had significant bacterial growth. Other studies in Sub-Saharan African countries showed similarly low rates of culture growth , 20, 26.The limitations of this study are we included only HIV-infected patients presenting with symptoms suggestive of urethritis. Furthermore, we did not have recent CD4 count results of the patients because CD4 is no longer routinely measured and replaced by viral load count for monitoring purposes of HIV patients. Lastly, we did not collect data on previous hospitalizations of the patients.E. Coli to be completely resistant to cotrimoxazole and ampicillin. Furthermore, significant resistance to ciprofloxacin and ceftriaxone was observed. Susceptibility was high to gentamicin, nitrofurantoin and imipenem. It is noteworthy that majority of urine cultures showed no bacterial growth in symptomatic patients. This is a concern and further studies are needed to address potential reasons behind the observed low growth rate. Finally, in the era of emerging antibiotic resistance, understanding the epidemiology and the local susceptibilities among sub-populations such as HIV infected individuals is crucial. This study is aligned with the WHO global action plan on antimicrobial resistance and provides further information that will contribute to the database on antimicrobial resistance in UTI in Uganda.Our study found the most isolated pathogen, Additional file 1: Appendix S1. SOP Microbiology.


To investigate the association between underweight and early childhood caries (ECC) among children aged one to three years in rural Cambodia.Z scores were below two and three standard deviations were considered as moderately-underweight and severely-underweight. Children whose mid-upper arm circumstance (MUAC) was below 12.5 cm, were regarded as having malnutrition. ECC was recorded following the WHO guidelines. Associated factors were obtained through interviews with caregivers.A total of 200 Cambodian children aged one to three years at several villages in Kampong Cham province participated in this study. The children whose P < 0.05). The prevalence of ECC was higher prevalence of those who with severely-underweight, although significant differences were not observed (P = 0.054). Logistic regressions showed that those with low birth weight  and malnutrition  were likely to be moderately-underweight and severely-underweight, whereas those who with ECC had more moderately-underweight, although it was not significant . Those with low birth weight  and ECC  were likely to be severely-underweight.33.5% and 7.0% of the children were identified as being moderately-underweight and severely-underweight, respectively. The prevalence of ECC was 61.5%. ECC was significantly associated with children being moderately-underweight (The findings of this study suggest that low birth weight, malnutrition and ECC were associated factors of underweight in this population. Children’s physical growth is rapid during the first three year after birth . InternaUnderweight (low weight-for-age) is a main indicator for malnutrition in children  and moreEarly childhood caries (ECC) is defined as the presence of one or more decayed (non-cavitated or cavitated lesions), missing (due to caries), or filled tooth surfaces in any primary tooth in a child 71 months of age or younger . A previThe associations between body weight and oral health status of children have been reported in several studies in Asian countries. Several of these showed that children with severe dental caries were likely to be underweight , 17. In This cross-sectional study was conducted in Steung Trang district, Kampong Cham province from May 2018 to January 2019, Cambodia. Initially, we recruited children aged one to three who had registered births with several villages whose families attending the Khpob Ta Nguon Health Center in Khpob Ta Nguon. Before commencing the study, we obtained the permission from the local health administrative district in Cambodia and the chiefs in each village. After explaining the purpose and the protocol of the study, we obtained written informed consent from the caregivers. Children who declined participation in the study and those who had less than eight erupted teeth were excluded. Data from 200 children  were analyzed. The sample size was calculated with the following formula.N = Z2α/2 P(1 − P)/d2N = required minimum sample sizeα/2 = confidence level at 95% (1.96)ZP = estimated prevalence of an indicator (proportion of underweight among 3-year-old children in Cambodia at 25%) [ at 25%) d = maximum allowable error at 6%N = (1.96)2(0.25)(1 − 0.25)/(0.06)2 = 200.08≒ 200Z score curves (weight-for-age) following the WHO Child Growth Standards [Z scores, below two (−2SD) and below three standard deviations (−3SD) were considered as moderately-underweight and severely-underweight, respectively [All parts of the survey were conducted in the Khpob Ta Nguon Health Center. Exact ages and birth weights of the children were obtained by referring to the birth registration records and their maternal health handbooks, respectively. Low birth weight was categorized as below 2500 g . The curtandards . In the ectively . To asseectively .Before the oral examinations commenced, intra-examiner calibration was carried out on 20 subjects by one trained dentist. An obtained kappa score of 0.87 indicated a high level of agreement. The oral examinations were conducted under natural light with the examiner and mother sitting “knee-to-knee” and the child in the supine position on their laps. A visual examination of the teeth was conducted following the WHO Oral Health Surveys Basic Methods .The questionnaire interviews were conducted by local trained interviewers and included questions on socio-demographic background, and child-rearing practices, including dietary and oral hygiene practices related to ECC.p < 0.05 was set as indicating statistical significance.Data was entered into an Excel spread sheet and transferred for statistical analysis using the statistical software package . The Shapiro–Wilk Test was performed to verify normal distribution of the data. The Chi-square test was used to investigate associations between variables and weight status. Logistic regression analyses were conducted by inputting variables with <0.1 at the baseline and adjusting for sex, age and the number of erupted primary teeth to determine the predictors of underweight among children. A This study was conducted according to the guidelines of the Declaration in Helsinki and was approval by the Ethical Committee of Niigata University (2017-0187).P < 0.05). No significant differences were found between males and females. In this study, the prevalence of children with low birth weight was 15.2%. These children were significantly more likely to be moderately- (P < 0.05) and severely-underweight (P < 0.01) compared with the other children. 5.1% of children whose mid upper arm circumstance was <12.5 cm showed malnutrition. These children were significantly more likely to be moderately- and severely-underweight (P < 0.05).Table P < 0.05). Among child-rearing practices, no significant differences were observed between underweight status, and daytime and night-time breast-feeding after 18 months, bottle-feeding after 18 months, the introduction of tooth-brushing after 18 months and sugary food intake. However, more than once a weekly sugary beverage intake by children was significantly associated with moderately-underweight (P < 0.05). The children with high frequency of sugary beverage consumption had higher moderately-underweight status than those who did not by 20% (41.2–22.2).In this study population, 61.5% of children had ECC and all teeth with ECC were untreated. The children with ECC had a higher chance of being moderately-underweight than those children without ECC by 16.4% (39.8–23.4) , whereas those with ECC were more likely to be moderately-underweight, although this was not significant since the confidence interval included 1    after 18 months . WHO recIn the present study, children with a frequent sugary drink intake had a higher prevalence of underweight. Sugary drinks are cheaper to buy than cows’ milk and infant formula, and are particularly harmful to the teeth. To foster child growth and development, healthy weaning practices are essential. These weaning practices can vary widely from country, largely influenced by sociocultural factors. In Cambodia, age-appropriate weaning which meets WHO recommendations should be promoted, taking into account the existing dietary culture and child-rearing environments.Previous studies have shown that the oral health of Cambodian preschool children is poor , 13, 38.This study has several limitations. First of all, only children who attended for health check-ups were included in the study. These caregivers may have had a higher level of health concern for their child compared to other mothers, resulting in selection bias. Second, the study was conducted on a small population size in one rural part of Cambodia, and since it was cross-sectional, causal relationships between ECC and underweight could not be established. Further longitudinal studies should be conducted to investigate the role of ECC in underweight.In conclusion, the prevalence of underweight and ECC were high in this group of one to three years old children, and both increased with age. The findings of this study suggest that low birthweight, malnutrition based on MUAC scores and ECC were predictors of underweight in this population. Comprehensive health and nutrition programs targeting children during the weaning period should be conducted in order to address underweight and ECC in Cambodia.


Pseudomonas. Finally, a techno-economic analysis highlighted potential economic advantages to air chilling compared to water chilling in facility locations where water costs are a more significant factor than energy costs.The United States’ large-scale poultry meat industry is energy and water intensive, and opportunities may exist to improve sustainability during the broiler chilling process. By USDA regulation, after harvest the internal temperature of the chicken must be reduced to 40°F or less within 16 h to inhibit bacterial growth that would otherwise compromise the safety of the product. This step is accomplished most commonly by water immersion chilling in the United States, while air chilling methods dominate other global markets. A comprehensive understanding of the differences between these chilling methods is lacking. Therefore, we assessed the meat quality, shelf-life, microbial ecology, and techno-economic impacts of chilling methods on chicken broilers in a university meat laboratory setting. We discovered that air chilling methods resulted in superior chicken odor and shelf-life, especially prior to 14 days of dark storage. Moreover, we demonstrated that air chilling resulted in a more diverse microbiome that we hypothesize may delay the dominance of the spoilage organism IMPORTANCE As the poultry industry works to become more sustainable and to reduce the volume of food waste, it is critical to consider points in the processing system that can be altered to make the process more efficient. In this study, we demonstrate that the method used during chilling (air versus water chilling) influences the final product microbial community, quality, and physiochemistry. Notably, the use of air chilling appears to delay the bloom of Pseudomonas spp. that are the primary spoilers in packaged meat products. By using air chilling to reduce carcass temperatures instead of water chilling, producers may extend the time until spoilage of the products and, depending on the cost of water in the area, may have economic and sustainability advantages. As a next step, a similar experiment should be done in an industrial setting to confirm these results generated in a small-scale university lab facility. Currently, the United States is the largest producer and second-largest exporter of poultry meat worldwide . The pouTemperature control during production and processing is a critical point in ensuring the safety and quality of poultry products. Thus, it is common for broiler production systems to reduce the internal temperature of chicken meat from 40°C to 4°C within 1 to 2 h following harvest. This step, though critical to maintaining the safety of the product, is time-consuming and requires significant investments in energy and water, depending on the chilling method utilized , 6. Duri7–The rapid reduction in carcass temperature during poultry processing provides a unique thermodynamic challenge that requires significant energy inputs. Although AC results in a carcass with a slightly reduced yield (due to evaporative water loss), it has been estimated to require almost 50 times less gross energy than water chilling systems, when entire energy expenditures are considered , 12. In To address meat quality, shelf-life, microbial, and techno-economic impacts of chilling methods, we conducted an experiment at the University of California, Davis (UC Davis) Meat Science Laboratory, in which chickens were chilled via either AC or WC. Novel to this experiment was the assessment of the impacts each chilling system had on the microbiome of poultry products and how that may relate to the quality of products from each system. This experiment yielded results that enhance the current knowledge regarding not only the quality of broiler meat produced using either chilling method but also the important techno-economics, which may guide industry investment or utilization in either system.In this experiment, 256 chicken carcasses were subjected to either air chilling (AC) or water immersion chilling (WC), then fabricated into either bone-in or boneless breasts before being placed under dark storage for either 7 or 14 days see . At eachP < 0.05; see P > 0.05; data not presented).The chilling method significantly impacted the carcass weight changes during the chilling process. On average, carcasses chilled using the WC system gained 5% of the prechilled weight, whereas carcasses chilled using the AC system lost 1.6% of the prechilled weight  or water chilling (WC). On average, the WC carcasses gained 5% of their prechilling weight, while AC carcasses lost 1.6% of their prechill weight. Download Copyright © 2021 Belk et al.2021Belk et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P < 0.05) psychrotrophic bacteria (1.05 log CFU/g), organisms capable of growing at low temperatures, prior to fabrication (cutting of the chicken carcasses into breasts) than the AC chicken (2.12 log CFU/g). However, no difference in mesophilic bacteria, organisms that grow best at moderate temperatures, was observed between the two treatments for prefabrication samples . Petrifilms were then incubated at 7°C and 35°C to obtain counts of psychrotrophic and mesophilic aerobic organisms, respectively. The WC chicken had fewer ( samples . On day  samples . WC and  samples .10.1128/mSystems.00912-20.2TABLE S2P < 0.05). D00, day 00. Download Table S2, TIF file, 1.5 MB.Least square means of mesophilic bacterial counts (log CFU/g) for bone-in and boneless chicken breast cooled by either air chilling or water chilling pre- and postfabrication, pre- and postchilling, during storage, and during and after display. Least square mean values within a column with different superscripts (a to e) differ  and b* (yellowness) values were greater (P < 0.05) for AC breasts than WC breasts throughout the display period, indicative of more desirable red and yellow tones within the muscle of AC breasts (P > 0.05). Additionally, trained sensory panelists detected no differences in flavor or texture attributes between the chilling methods (P > 0.05) . These results demonstrated that the International Commission on Illumination (CIE)  breasts . Similar breasts . Chillin > 0.05) .10.1128/mSystems.00912-20.3TABLE S3P > 0.05). Download Table S3, TIF file, 0.8 MB.Trained panelists were asked to evaluate chicken breasts for texture and flavor attributes on a 100-point scale. Within a column, values with the same letter were not significantly different , though there were differences when comparing sampling groups across these days. The greatest differences between treatments were seen on the samples collected after 14 days of dark storage with a 3-day retail display. Among samples from this time point, the boneless WC breasts had a higher degree of lipid oxidation compared to the boneless AC breasts (P < 0.05).As expected, lipid oxidation increased as the time after chilling progressed . Lipid oP > 0.05). The same pattern was detected in moisture, as moisture and dry matter are inversely related. Measurements of ash and crude protein revealed a few differences between treatment groups, but these did not reveal significant patterns (P < 0.05); carcass samples and bone-in breasts had higher crude fat content within almost all chilling methods and sampling days . Following this trend, the relative abundance of fatty acids was not grossly different between chilling methods on any sampling day. However, among fatty acids with less than 10% relative abundance, linoleate methyl ester (C18 and C18:9c12c) were more abundant in WC than AC breasts after 7 days of dark storage with 3 days of retail display . Download Table S4, TIF file, 0.8 MB.Analysis of nutrient composition  values. Data are organized first by chilling method, including hot carcass (HC), air chilled (AC) and water chilled (WC) samples. Within a day, data are then organized by sampling day, which includes not stored (day 0), 7 or 14 days of dark storage, and 3 days of retail display after removal from dark storage (day 10 and day 17). Within a day, data are organized by fabrication method, including unfabricated carcass, bone-in breasts, and boneless breasts. Values with the same letter within a column are not significantly different  and water chilled (WC) chicken meat. (A) All fatty acids present in the samples. (B) The “rare” fatty acids present at <10% relative abundance. Download Copyright © 2021 Belk et al.2021Belk et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the https://www.earthmicrobiome.org/protocols-and-standards/16s/). Sequencing a total of 286 samples and controls resulted in a total of 3,837,564 demultiplexed reads. After denoising, quality filtering, read joining, and chimera removal via the DADA2 pipeline, 3,262,269 sequence reads  were assigned to 774 amplicon sequence variants (ASVs). Subsequently, we filtered out ASVs that were taxonomically identified as representing mitochondria and chloroplasts, resulting in 3,261,944 sequence reads and 752 ASVs. The commercial positive-control sample  resulted in the expected community of five microbes with no unexpected taxa, indicating no major contamination  and the standard, expected composition as described by the company. Download Copyright © 2021 Belk et al.2021Belk et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P < 0.05) in AC. After fabrication, or the cutting of the chicken carcasses into breasts, the alpha diversity increased, though not to the original levels. The high microbial diversity on chicken carcasses before product storage was associated with microbial communities dominated by organisms in the family Enterobacteriaceae. These communities also included bacteria at lower relative abundance from families Clostridiaceae, Bacillaceae, and Pseudomonadaceae  bacterial diversity of chicken products was reduced during chilling and processing . The hotnadaceae . There wP < 0.05) than the diversity of AC bone-in samples and AC boneless samples. After the 7-day time point, the mean diversities were similar . During these poststorage and postdisplay sampling points, Pseudomonas bacteria became the dominant group. For the chicken that was stored for 7 days, followed by 3 days of retail display, communities from WC chicken became dominated by Pseudomonas before AC chicken. When the beta diversity was calculated and visualized using robust Aitchison principal component analysis, samples separate initially by sampling day—all products that were stored, regardless of storage or display time, separated from samples that were not stored  come to dominate the microbial community during storage and display , At day 10 (7 days with 3-day retail display), there was also one ; at day 14 there were three , and at day 17 (14 days with 3-day retail display) there were two (ASV7 and ASV20).In order to focus on particular ASVs, we performed an analysis of the composition of microbiomes (ANCOM) analysis at each sampling point  comparing the two chilling methods (pooling fabrication method). Prior to storage, none of the ASVs that were differentially abundant were ASVs assigned to Economic viability is a critical aspect of technology adoption. The levelized chilling cost (dollars/metric ton) for the baseline AC and WC models are shown in 10.1128/mSystems.00912-20.10FIG S52), processing rate (metric ton/hour), chilling energy (kilowatt hour/metric ton), worker capacity (metric ton/hour), water use (liter/metric ton), water price plus water treatment cost (dollarsliter), and electricity cost (dollars/kilowatt hour). The resulting levelized costs for AC (upper panel) and WC (lower panel) systems demonstrate which aspects of each system have the most influence over the total levelized cost of chilling. For AC systems, capital costs and labor costs have the most impact on the total levelized chilling cost, while water and labor costs have the most impact on WC systems. Download FIG S5, TIF file, 1.8 MB.The sensitivity analyses varied the following model values by ±50%: capital cost (dollars/tonne or metric ton), chilling floor space  with advantages in areas of limited water and low-cost electricity.This study demonstrates that the method of chilling poultry carcasses not only influences the shelf-life and quality of chicken breasts but also has important implications for energy and water usage. Overall, in this study, chicken breasts from carcasses chilled using air chilling (AC) methods had superior odor and shelf-life . Moreover, the microbial communities associated with AC products maintained diversity postchilling, and therefore may be more favorable by slowing the growth of spoilage organisms such as Throughout dark storage and retail display, quality and shelf-life attributes were impacted by the chilling method. While all product was spoiled after 14 days of dark storage, notable quality and physical differences were observed among chicken treated with different chilling methods after 7 days of dark storage and 3 days of retail display. In this time period (7 days of dark storage to retail display), chicken chilled using AC demonstrated more desirable quality attributes, including more yellow tones and a lower abundance of spoilage-associated fatty acids, including linoleate methyl esters. These fatty acids have been associated with odor, color, and shelf-life challenges in previous studies 1416. DiffeWe did not detect a significant impact on texture, flavor attributes, or nutritive composition by chicken chilling method. Previous studies have shown mixed impacts of chilling method on flavor and sensory attributes. For example, Hale et al.  suggesteEnterobacteriaceae, Clostridiaceae, and Bacillaceae, in agreement with findings by Handley et al. , ASV1 (no difference), and ASV11 (no difference) were found in a clade containing P. lurida, P. poae, P. trivialis, P. palleronia, P. tolaasii, P. costantinii, P. extremorientalis, and P. simiae. ASV20 (higher abundance in WC at days 14 and 17) and ASV14 (no difference) were found in a clade with P. veronii. While none of these latter Pseudomonas species has been directly shown to be involved in food spoilage, we hypothesize based on these data that at least some of them likely play a role in chicken spoilage.While spoilage , they arand meat –34 and w. putida , 34, werAlthough our research suggests that AC may have shelf-life and quality advantages over WC, techno-economics are more nuanced. The WC systems can use up to 10 times more water than the AC system, but the electrical costs of the AC system are 2 times that of the WC system. Our baseline models do not support the claims by two other studies that AC systems require almost 50 times less gross energy than WC systems, when entire energy expenditures are considered , 12. RatThis experiment was designed to be a laboratory-based, pilot-sized representation of the larger process of chicken chilling, fabrication, storage, and retail display. We made efforts to represent industry conditions as accurately as possible in a small laboratory setting; however, there were a few conditions we were unable to replicate. We were unable to reproduce an antimicrobial application, either in the WC chill water or sprayed on the AC carcasses, which could modify some of the microbiological results. Therefore, our experiment represents a worse-case scenario. Moreover, all chicken carcasses used in this experiment were obtained from the same production lot in order to start all chilling processes at the same time. This may have led to a more homogeneous outcome than a more randomized selection. Future experiments should confirm the current findings using real industry conditions.The overarching goal of this study was to combine multidisciplinary approaches to determine the impact of chilling method on the overall system efficiency and sustainability of chicken production. We were able to conclude that AC methods had an advantage in quality, spoilage, and consumer appeal prior to 14 days of dark storage, that AC appeared to result in a more favorable, diverse microbial community, and that AC requires less gross energy and, depending on the local price of water, may be the more economically favorable system.n = 256) were obtained from a commercial processing facility in California and transported to the USDA-inspected Meat Science Laboratory at the University of California, Davis (UCD)  within 2 h of harvest. Carcasses were transported in sterile 150-quart coolers  at a mean temperature of 30.25°C. Upon arrival at UCD, carcasses were divided into sampling groups following the scheme in n = 110 birds/chilling method). Following chilling (described below), 10 carcasses from each AC and WC treatment group were sampled, and the remaining were evenly assigned to fabrication pathways (n = 50 birds/fabrication) yielding either bone-in or boneless chicken breasts (n = 20 breasts/fabrication pathway). Immediately following fabrication, 10 chicken breasts from each group were sampled, and the remaining breasts were placed on expanded polystyrene trays and overwrapped with polyvinyl chloride film . Overwrapped trays were placed in rigid cardboard boxes (n = 8 trays/box) and stored at 4°C (3 to 6°C) for either 7 or 14 days, a time frame that reflects industry standards. At each storage interval (7 or 14 days), individual packages of chicken breasts (bone-in and boneless) were removed from dark storage. Ten breasts from each group were sampled immediately after removal from storage, and the remaining packaged breasts were placed in a retail display case  maintained at 4°C (3 to 6°C) for 3 days.The experiment was conducted using a 2 × 2 × 2 factorial design to evaluate three factors: chilling method (air chilling [AC] versus water immersion chilling [WC]), fabrication method (bone-in versus boneless), and dark storage period (7 days versus 14 days). Eviscerated, hot chicken carcasses (n = 256) were removed from the processing line, placed in sterile plastic bags (n = 30 to 32 carcasses/bag), and bags were placed in sealed sterile coolers for transportation to the Meat Science Laboratory at University of California-Davis (UC-Davis) . Additionally, temperature recorders  were placed in the coolers to monitor temperature during transportation.A commercial chicken processing facility in California was utilized to procure hot chicken carcasses for this research. Live birds were subjected to standard poultry harvest protocols as implemented by the commercial processing facility. Carcasses used for this experiment were obtained following defeathering, evisceration, and application of an initial postharvest antimicrobial carcass spray. Prior to chilling, the carcasses , while the remaining carcasses were randomly and evenly assigned to one of two chilling methods  as shown in n = 50/fabrication method) for the generation of bone-in and boneless chicken breasts. Carcasses were fabricated, meaning cut from carcasses into individual parts, by trained personnel in the UC Davis Meat Science Laboratory using sterile instruments, and WC and AC carcasses were fabricated separately. Bone-in chicken breasts contained the ribs and part of the spine, while boneless breasts had these bones further removed. Immediately following fabrication, 10 chicken breasts from each group were sampled, and the remaining breasts were placed on expanded polystyrene trays and overwrapped with polyvinyl chloride film . Overwrapped trays were placed in rigid cardboard boxes (n = 4 trays/box) and stored at 4°C (3 to 6°C) for either 7 or 14 days.Immediately following chilling, carcasses within each chilling method (AC and WC) were randomly and evenly assigned to one of two fabrication methods (L* (lightness), a* (redness), and b* (yellowness) values were taken using an illuminant A/10° observer for each breast. Measurements were taken through the packaging material at three separate locations on the chicken breast and were averaged prior to analyses. Packages were rotated in the display case every 12 h to ensure equitable temperature and light exposure.At each storage interval (7 or 14 days), individual packages of chicken breasts (bone-in and boneless) were removed from dark storage. Ten breasts from each group were sampled immediately after removal from storage, and the remaining packaged breasts were placed in a retail display case  maintained at 4°C (3°C to 6°C). Packages remained in the retail display case for 3 days. Instrumental meat color, measured via evaluating the lean color of the boneless samples and skin color of the bone-in samples, was taken using a portable spectrophotometer  that was standardized before each use. A total of three readings of the International Commission on Illumination (CIE) At each sampling point, the microbial communities of the chicken products were collected using a rinsate method. At prefabrication time points, the entire chicken carcass was placed in a sterile collection bag  with 200 ml of phosphate-buffered saline (PBS)  and agitated for 60 s to dislodge surface bacteria. After this, the carcass was removed from the rinsate and saved for physicochemical analysis. At the postfabrication time points, each chicken breast was divided in half. One half was placed in a sterile collection bag  with 50 ml of PBS and agitated for 60 s. The second half was reserved for physicochemical analysis. At all time points, the rinsate was collected from the sampling bag into 50-ml falcon tubes  for analysis. A 10-ml aliquot of each sample was separated to be used for aerobic bacterial population analysis, and the remainder was frozen to –80°C and transported to Colorado State University   for microbial ecology analysis.g for 15 min in a swing bucket rotor . The supernatant was poured off, and a portion of the pellet equivalent to approximately 600 μl was used for analysis. DNA was extracted from the pellet following standard protocols utilizing the Qiagen PowerSoil DNA 96-well extraction kit  following the manufacturer’s protocol for low biomass samples, which included the additional step of allowing the EB solution to be heated to 65°C before adding to the DNA plate wells for 5 min before eluting. DNA was eluted in two steps. Initially, 60 μl was eluted and considered our more concentrated DNA extraction. Next, an additional 80 μl of DNA was eluted. Each 96-well plate included eight mock extractions (no sample added) and one positive control (ZymoBIOMICS D6300). The 16S rRNA gene (V4 region) was amplified using primers 515F and 806R universal primers with the forward primer barcoded to allow for multiplexing during sequencing, following the Earth Microbiome Project protocols (https://www.earthmicrobiome.org/protocols-and-standards/16s/). The forward primer 515F included the unique sample barcode following Parada et al.  (https://github.com/smirarab/sepp/issues/40]). SEPP 4.3.10 was then run with the following parameters (-P = 33 -A 10) to optimize the alignment breakdown using the ASV file, the RAxML tree, the RAxML info file, and the reference alignment as input.All ct (RDP) , along wct (RDP)  using thAs described by Martin et al. , aerobicNumerous biochemical and physicochemical changes that affect shelf-life occur in postharvest meat products . Thus, aFatty acid composition was obtained using gas chromatography following methods described by Engle et al. and Kang and Wang , 50. FirLipid oxidation was measured using the thiobarbituric acid-reactive substance (TBARs) assay as described by Yin et al. . BrieflyNutrient composition analysis  was conducted to determine the dry matter, moisture, ash, crude fat, and crude protein composition within each sample. Dry matter and moisture were measured using the AOAC oven drying method, 950.46 and 934.01 . Two graEight untrained participants were asked to evaluate the acceptability of three sensory attributes  during retail display using a three-point sensory scale described by Lytou et al. . In addiIn addition to evaluation of chicken breast color, odor, and texture, trained taste panelists were asked to evaluate various palatability attributes  of bone-in and boneless chicken breasts representing both chilling methods following 7 days of dark storage. Panelists consisted of graduate students from the CSU Center for Meat Safety and Quality and were trained to recognize the aforementioned attributes using methods and references described by Solo . Samplesnth plant economic assumptions from literature and assumed a 10% internal rate of return (IRR), 20-year facility life, 8% loan interest rate on a 10-year loan with 40% equity, and the 2019 U.S. corporate tax rate of 21% , water use , and chilling energy costs . Due to the complexity and high variability in WC and AC system designs and operational characteristics, sensitivity analyses were used to evaluate how system parameters can impact the levelized cost associated with chilling poultry carcasses. The sensitivity analysis varied the following model values by ±50%: capital cost (dollars/metric ton), chilling floor space (square meters), processing rate (metric ton/hour), chilling energy (kilowatt hour/metric ton), worker capacity (metric ton/hour), water use (liter/metric ton), water price plus water treatment cost (dollars/liter), and electricity cost (dollars/kilowatt hour). The end result is a direct comparison of the two technologies in terms of costs with sensitivity used to highlight high impact variables.All baseline values were taken from literature or acquired through communication with poultry chilling equipment manufacturers and poultry processing facilities. In particular, the system layout and energy consumptions reported in the literature were found to be antiquated, and thus, most of these data were acquired through communications with industry. The mutual baseline inputs were plant operation , poultry processing rate (16.5 metric ton/hour), water treatment Human participation in taste and food quality evaluation was reviewed by the Institutional Review Board at Colorado State University with IRB ID 364-18H and declared to be exempt from the requirements of the human subject protection regulations.PRJEB41700, and in QIITA, study 12193. Analysis details can be accessed at https://github.com/Metcalf-Lab/Air-versus-water-chilling-of-chicken.16S rRNA gene sequencing data are available in the EBI-ENA database, accession number


The findings show that families either led by a Black/Latino father or mother have a significant impact on the sexual health-seeking behavior of BLMSM and their perception of HIV and PrEP.This study employs the ecodevelopmental theory to examine the influence of mother and father bonding, family engagement in healthcare, and family support on PrEP stigma among BLMSM. We used a cross-sectional sample from wave five of the Healthy Young Men (HYM) study, with a survey sample of 399 participants aged 16–24 years. We conducted two-path analyses to test multiple hypotheses: (1) mother/father bonding is associated with an increase in family engagement in healthcare; (2) family engagement in healthcare is associated with family social support; and (3) family social support is associated with PrEP stigma. Family social support was negatively correlated with PrEP stigma (r = −0.15;  Although pre-exposure prophylaxis (PrEP) proves efficacious in reducing HIV transmission, not all populations benefit from the advancement equally ,2. BlackSocial and structural factors have impacted PrEP uptake among BLMSM. These factors are structural health care barriers, such as lack of access, insurance status, and discrimination/racism. Social factors emanating from homophobia, HIV stigma, and additional personal elements within the cultural/community context have also contributed to low PrEP use ,9,10,11.Parental bonding is a subjective experience of affection from a parent towards a child. The core tenet of the bond is the perceived feeling of “love” expressed in parental behavior ,16,17. TTo address the gaps in knowledge surrounding this area, this study used the ecodevelopmental theory to examine how mother and father bonding, family engagement in healthcare, and family support influence PrEP stigma among BLMSM and fills a critical gap in the literature on the same. We hypothesized the following:Hypothesis 1 (H1).Mother and Father Bonding will predict Family Sexual Health.Hypothesis 2 (H2).Family Sexual Health will predict Family Social Support.Hypothesis 3 (H3).Family Social Support will predict PrEP Stigma.The ecodevelopmental theory guided this study to investigate how the family context  influences PrEP stigma among BLMSM. Based on Bronfenbrenner’s social-ecological model, the theory frames the social ecology of an individual in the context of four interrelated systems: microsystem, mesosystem, exosystem, and macrosystem ,21. To dIn understanding how stigma influences HIV prevention behaviors such as PrEP, it is critical to recognize the intersecting identities of BLMSM and the potential operation of these identities in the family context. Intersectionality can complement and enhance the ecodevelopment theory, as intersecting stigmas may be critical drivers of PrEP-related stigma among BLMSM . While bn = 498) of MSM of color. The HYM research aims to prevent and reduce HIV acquisition among MSM of color by investigating barriers and protective factors that contribute to their engagement in care. Young men who were HIV negative (n = 448) and positive (n = 50) were eligible to participate in the study. Inclusion criteria were: (1) between the ages of 16 to 24 years; (2) assigned as male at birth; (3) self-identified as gay, bisexual, or uncertain about their sexual orientation; (4) reported a sexual experience with a man within the last 12 months; (5) self-identified as of African American/Black, Hispanic/Latino, or multiracial ethnicity; and (6) resided in Los Angeles city or county with no plans on moving for at least six months.This secondary analysis used wave five data from the Healthy Young Men’s (HYM) cohort study, a longitudinal study conducted with a sample  to 5 (strongly agree), with a higher score indicating lower stigma (α = 0.84). Sample items provided here were in response to the prompt, stating, “Please decide how much you agree with the following statements: (1) “I think people should take PrEP.” (2) “Having sex with someone on PrEP is risky”, and (3) “People have different opinions about PrEP.”PrEP attitudes were measured by a 3-item, 5-point Likert scale with values ranging from 1 (strongly disagree) to 5 (strongly agree), with higher scores reflecting positive attitudes towards PrEP (α = 0.83). A sample item stated, “I think people should take PrEP” .Family social support was measured using a 4-item Likert scale (ranging from 1 (strongly disagree) to 4 (agree)), asking the respondents to agree or disagree with the following: (1) “My family tries to help”, (2) “I can talk about my problems with my family”, (3) “My family is willing to help me make decisions”, and (4) “I get the emotional help and support I need from family”. Family Social Support (FSS) scores were created by averages across item responses (α = 0.91). Mother and father bonding were two separate measures created using a 2-item scale , asking about participants’ receiving love and support from their mother and father. Sample items included (1) “Would you say this woman/man was loving?” and (2) “Would you say this woman/man was very supportive?”. The scores were achieved by averaging across item responses, and the Cronbach’s alpha for fathers was α = 0.76 and mothers α = 0.80.Family engagement in healthcare was measured by two items, consisting of 5-point Likert-type questions, with values ranging from 1 (strongly disagree) to 5 (strongly agree) and a higher score indicating more family engagement in healthcare (α = 0.70). A sample item includes “When I was growing up, my parent(s) or guardian(s) made sure I had regular check-ups with my doctor”.n = 498). p-value greater than 0.05. A mean score of the scale items was generated for participants with non-missing data for survey scales. All analyses were conducted using STATA 17.All analyses were conducted on observations inclusive of non-missing data for the outcome of PrEP stigma. Overall, approximately 50% of BLMSM experienced some form of PrEP stigma. Generally, 72% harbored positive attitudes towards PrEP, and 71% reported having family support. Moreover, only 63% of the sample reported their family being engaged in healthcare. Most of the sample reported having strong bonds with their mothers (89%), and 66% reported strong bonds with their fathers.p < 0.001). Family social support was negatively correlated with PrEP stigma . Family engagement in healthcare was negatively associated with PrEP stigma  and positively associated with PrEP attitudes  and family social support . History of mother bonding was positively associated with family social support  and family engagement healthcare . History of father bonding was positively correlated with family social support  and mother bonding .p = 0.67; CFI = 0.99; TLI = 0.99; RMSEA = 0.01; AIC = 45; and BIC = 46). n = 399). Results indicated that a history of mother bonding was statistically significant and directly linked to family engagement in healthcare . Family engagement in healthcare was significant and directly linked to family social support . Family social support was significant and negatively associated with PrEP stigma . Mother bonding was significant and indirectly associated with family social support . Lastly, mother bonding was significant and indirectly linked with PrEP stigma  , < 0.001) .p = 0.99; CFI = 0.99; TLI = 0.99; RMSEA = 0.01; AIC = 46; and BIC = 47). n = 245). Family engagement in healthcare was significant and directly associated with family social support . Family social support was significant and negatively linked with PrEP stigma . Father bonding was significant and indirectly associated family social support . In addition, father bonding was significant and indirectly associated with PrEP stigma  ,  < 0.05) .The purpose of this study was to use the ecodevelopment theory to examine the impact of family-level factors on PrEP stigma among BLMSM. Despite the low PrEP use among BLMSM and the significant public health implications, few studies have examined the contextual factors associated with mother and father bonding, family engagement in healthcare, and family social support on PrEP stigma ,15,16,17Studies examining the effects of the ecodevelopmental theory in the family context on BLMSM health, HIV, and stigma are limited . EcodeveOur study results indicated that mother bonding both positively predicted family engagement in healthcare but not for father bonding , which iThe path analyses indicated that family engagement in healthcare was associated with family social support. Past research has shown that family engagement and support in care are essential for optimal health outcomes . AdditioOur study findings indicated that family social support in both path analyses (mother and father) was independently associated with a reduction in PrEP stigma. This is an important finding because PrEP stigma has been linked to lower PrEP interest, intentions, comfort discussing PrEP with a provider, and uptake ,11,28. PSurprisingly, our results indicated that mother and father bonding was indirectly associated with reducing PrEP stigma. This is critical as PrEP stigma minimizes the likelihood of engagement in PrEP ,11. HoweAlthough our study findings contribute to the literature on family social support and PrEP stigma, there are several limitations. The analysis was conducted using secondary data, so we were dependent on the pre-existing measures to determine variables related to family context. Additionally, the landscape for the availability and administration of PrEP  is ever changing. We are unsure how the availability of PrEP may or may not influence its uptake in the future. The data analyzed were cross sectional, and we cannot predict changes in family dynamics, including divorce or single parenthood, from the data. Future studies should examine the longitudinal impact of family bonding and how it changes over time in different household settings. We plan to augment our research findings by examining the differences between household types based on socioeconomic factors and demographics such as single parenthood.The purpose of our ecodevelopmental study was to examine family-level factors on PrEP stigma among BLMSM. Based on our findings, there is an urgent need to optimize HIV outcomes by including the family unit in Black and Latino HIV prevention. Utilizing the family unit and providing comprehensive resources to increase HIV prevention awareness may reduce stigma around PrEP and encourage use.Attention to family dynamics and leveraging the strengths of BLMSM may help reduce PrEP stigma and negative attitudes towards PrEP. Much bio-behavioral research focuses on risk factors associated with PrEP ; however


GH3 genes in apple  and provided evidence that MdGH3-2 and MdGH3-12 play important roles during AM symbiosis. The expression of both MdGH3-2 and MdGH3-12 was upregulated during mycorrhization, and the silencing of MdGH3-2/12 had a negative impact on AM colonization. MdGH3-2/12 silencing resulted in the downregulation of five genes involved in strigolactone synthesis, and there was a corresponding change in root strigolactone content. Furthermore, we observed lower root dry weights in RNAi lines under AM inoculation conditions. Mycorrhizal transgenic plants showed greater sensitivity to drought stress than WT, as indicated by their higher relative electrolytic leakage and lower relative water contents, osmotic adjustment ability, ROS scavenging ability, photosynthetic capacity, chlorophyll fluorescence values, and abscisic acid contents. Taken together, these data demonstrate that MdGH3-2/12 plays an important role in AM symbiosis and drought stress tolerance in apple.Drought leads to reductions in plant growth and crop yields. Arbuscular mycorrhizal fungi (AMF), which form symbioses with the roots of the most important crop species, alleviate drought stress in plants. In the present work, we identified 14  Arbuscular mycorrhizal (AM) symbiosis is one of the most complex and beneficial interrelationships and is formed between the roots of nearly 80% of terrestrial plants and fungi from the phylum Glomeromycota4. This symbiosis evolved more than 450 million years ago, making it one of the most ancient symbioses, and it provides a significant selective advantage to both symbiotic partners5. After a molecular dialog between the two partners, the AM fungus penetrates the host roots and forms branched structures in cortical cells in which nutrient exchange occurs6. As obligate symbionts, AM fungi (AMF) complete their life cycle using the carbon sources provided by their host plants. In exchange, fungi not only improve the availability of mineral nutrients  for the partner but also enhance its resistance to various environmental stresses8.To cope with a variety of environmental challenges, plants have evolved many strategies, including interactions with other microorganisms10. Over the past decade, great progress has been made in understanding how AM symbiosis is established13. The process of symbiosis formation is relatively consistent between different plant and AMF species and can be divided into several stages. First, before coming into contact with the AMF, the plant roots secrete strigolactone (SL) “branching factors”. These branching factors stimulate the spores of AM fungi germination and branching, which continue to approach the roots14. At the same time, AMF also secrete signaling substances called “mycorrhizal factors”, which include sulfated and nonsulfated lipochitooligosaccharides16. After the plant roots recognize these substances, the host plants activate common symbiotic signaling pathways17. When the fungal mycelium comes into contact with the plant root epidermal cells, it differentiates into an attachment structure called a hyphopodium, and the root epidermal cells also undergo cytoskeletal changes. The fungal hyphae penetrate the epidermal cells and continue to elongate toward the cortical cells, continuously growing through the root tissue18. When the mycelium extends to the endothelial layer of cells and penetrates the cell wall, it continues to branch in the cells, forming a highly branched, tree-like structure that becomes an arbuscule. Arbuscules are the main site of the exchange of mineral nutrients between the host cell and the fungi13.The establishment of AM symbiosis is a complex process that requires molecular communication between the two partner organisms22. Several studies have reported that different hormones are involved in AM formation24, and auxin and its derivatives are thought to play a particularly important role. Hanlon and Coenen25 showed that the roots of two auxin-related mutants were poorly colonized. Similarly, a low-auxin bsh mutant of pea (Pisum sativum) exhibited a reduction in mycorrhizal colonization and strigolactone exudation26. Guillotin et al.9 showed that Sl-IAA27 may control SL biosynthesis via the regulation of NSP1. Although all plant hormones can perform their biological functions independently, the regulation of a plant’s development throughout its lifespan requires the synergistic action of multiple hormones24.Plant hormones are multifunctional molecules that act as central regulators of all plant developmental processes, including the development of the plant-AMF interaction28. AM symbiosis can improve the performance of plants under various environmental stresses, such as drought, and can alter plant water relations under conditions of both water sufficiency and water stress31. The regulation of plant drought resistance by AMF is a very complex process involving many metabolites and metabolic pathways33. AMF symbiosis has been shown to improve plant drought adaptability by up- and downregulating many physiological and biochemical processes. Specifically, AMF can directly promote the absorption and transport of plant nutrients and water, increase plant osmotic regulation, improve plant gas exchange capacity and water use efficiency, and increase plant antioxidant capacity34.In agricultural ecosystems, drought is one of the most serious environmental factors that limit plant growth and crop productivityMdGH3-2 (MD02G1180300) and MdGH3-12 (MD15G1290900), was shown to be significantly induced by AM fungi. To further understand their potential roles in the mycorrhizal process, an RNA interference (RNAi) approach was used to repress their expression. We hypothesized that their repression would not only affect mycorrhization but would also affect drought resistance in apple under drought conditions. Physiological parameters such as photosynthetic parameters, antioxidant system activity, osmotic adjustment, and ABA levels under AM inoculation were assessed. Overall, the data show that MdGH3-2/12 are involved in AM symbiosis and drought stress tolerance in apple.In this study, the expression of two apple early auxin-responsive genes, MdGH3 family genes were identified. However, we found that there was one gene (MD09G1091000) with 280 amino acids, and the predicted conserved domain indicated that it was incomplete. Therefore, this gene was not analyzed further. The basic information for the MdGH3 genes, including gene names, gene IDs, chromosome locations, protein lengths, theoretical isoelectric points (pIs), and molecular weights (MWs), is listed in Table In total, fifteen ene MD09G091000 wiArabidopsis , and rice (Oryza sativa L.) GH3s proteins, we constructed a phylogenetic tree  plants  similar  apple trees and the fruits, leaves, and roots of ‘Winter Red’, a cultivar of crabapple , grown at the horticultural experimental station of Northwest A&F University  were sampled. The mature leaves of ‘Qinguan’ apple were used for gene cloning. A line with high regeneration capacity isolated from ‘Royal Gala’ (M. domestica) named GL-3 was used for genetic transformation46. After rooting on MS agar medium, the plants were transferred to small plastic pots (8 × 8 cm) that contained a substrate/perlite/vermiculite mixture  that had been sterilized by autoclaving at 121 °C for 2 h. After one month, the mycorrhization assay plants were moved to larger plastic pots (9 × 10 cm) that contained the sterilized mixture described above. Ten grams of fungal inoculum was added, and noninoculated control plants received the same amount of sterilized mycorrhizal inoculum. The AM fungus was R. irregularis. Two months after transplantation, AMF seedlings and non-AMF seedlings were exposed to well-watered or drought conditions, with 40 pots in each treatment. Water was withheld from the drought-stressed seedlings for 9 days.The leaves, branches, bark, flower buds, and mature flowers of ‘Royal Gala’  was used to locate the fluorescent proteins in the nucleus. Instantaneous transformation and fluorescence signal observation were performed as described by Liu et al.51.The coding regions without stop codons of or Fig. S. The AtC52.After 8 weeks of the NM treatment, the roots were placed in envelopes in a 105 °C oven for 1 h. The oven temperature was then adjusted to 80 °C, and the roots were fully dried. The supernatant obtained by centrifugation was diluted with methanol to final concentrations of 10 and 1 mg/L. Germination bioassays were performed according to Ma et al.53.Frozen root tissue (0.1 g) was ground into a 1 mL extraction solution containing methanol:isopropanol, 20:80 (v/v), with 1% glacial acetic acid. After standing at 4 °C for 12 h, the mixture was centrifuged at 12000 g for 5 min at 4 °C. Afterward, the supernatants were combined and passed through a 0.22-μm PTFE filter. The concentrations of ABA were determined as described by Guo et al.v/Fm was measured using a MINI-PAM-II fluorometer .Gas exchange parameters, including the net photosynthetic rate (Pn) and transpiration rate (Tr), were monitored using a portable photosynthesis system . For each treatment, the 4th–6th leaves from the stem base were selected for measurement. F44 and Dionisio-Sese and Tobita54.The RWC and REL were determined and calculated according to the method described by Sun et al.2O2, superoxide radical (O2-), and proline and the activities of superoxide dismutase (SOD) and peroxidase (POD) were detected via Suzhou Comin Biotechnology test kits .The levels of H55. Malate dehydrogenase (MDH) transcription was used to normalize the levels of different genes. Table Total RNA was extracted from apple plants via a Wolact plant RNA isolation Kit . Apple genomic DNA was isolated with a Wolact Plant Genomic DNA Purification Kit . qRT-PCR analysis was carried out as previously described by Huo et al.Arabidopsis were downloaded from the NCBI database (https://www.ncbi.nlm.nih.gov) according to Xu et al.56. All AtGH3 proteins were used as queries in a BLASTP search against the apple genome database . Candidate sequences were confirmed using the Conserved Domain Database (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) and SMART Database (http://smart.emblheidelberg.de/) and compared with Yuan et al.57. The OsGH3 protein sequences were downloaded from the NCBI database (https://www.ncbi.nlm.nih.gov) according to Jain et al.58. The phylogenetic trees were estimated with the MEGA5 program using the neighbor-joining method. Gene structures were constructed using TBtools (version 1.046). Sequence alignment of MdGH3-2 and MdGH3-12 was performed with DNAMAN (version 6.0.3.99).The protein sequences of 19 GH3 genes from SPSS 16.0 was used for the statistical analysis. The data were subjected to independent-samples t-tests and one-way ANOVA and expressed as means ± SDs.Supplementary materials


Case Presentation. A 13-year-old boy with ASD presented with bilateral leg pain after an unwitnessed fall. Laboratory investigations revealed severe hypocalcemia  and severe vitamin D deficiency (25(OH)D < 4 ng/mL). Lower extremity X-rays revealed bilateral distal femoral metaphyseal fractures.  Femur fractures in adolescents are rare. Severe vitamin D deficiency has important implications for bone health. We describe the case of a 13-year-old boy with autism spectrum disorder (ASD) who presented with low-impact bilateral femur fractures in the setting of severe vitamin D deficiency.  This is the youngest known case of bilateral femoral fractures in the setting of severe 25(OH)D deficiency. Children with ASD are especially at risk for 25(OH)D deficiency as many have inadequate nutritional intake. As such, primary care providers may provide a pivotal role in the routine laboratory screening of 25(OH)D in this population. Vitamin D (25(OH)D) deficiency has important implications for bone health. Decreased exposure to sunlight, a major source of vitamin D, has become an important factor in the development of vitamin D deficiency, especially as children spend much time indoors. Individuals with darker skin tones have also been found to require longer sun exposure in order to synthesize cholecalciferol and are at greater risk for deficiency as well , 2. Few A 13-year-old boy presented to the pediatric emergency department with bilateral leg pain after an unwitnessed fall onto the street from the sidewalk. There was no history of major trauma, syncope, or seizure. He had severe autism spectrum disorder (ASD) with poor verbal skills. On examination, he was crying and had tenderness over the bilateral knees and right ankle. The range of motion of the hip and knee was limited by pain. There were no bony deformities. Lower extremity X-rays revealed acute comminuted and impacted fractures of the bilateral distal femoral metaphyses with anterolateral apical angulation of the distal fracture fragments Figures . Diffuseth percentile . His weight was at the 52nd percentile and his body mass index (BMI) at the 87th percentile, meeting the criteria for overweight. His physical exam, apart from his limited lower extremity movement, was remarkable for a positive Chvostek sign, early Tanner stage 3 development with 8 cc testes descended bilaterally, and vertical fingernail ridges. Skin examination was negative for bruising, acanthosis, and striae. The differential diagnosis included vitamin D deficiency, malabsorptive disorder , osteogenesis imperfecta, prolonged steroid use, renal failure, hypothyroidism, and juvenile osteoporosis.On physical examination, his height was at the 4Initial laboratory investigations revealed a serum calcium of 4.9 mg/dL with serum albumin of 4.4 g/dL. An ionized calcium of 0.61 mmol/L confirmed hypocalcemia. Parathyroid hormone (PTH) was elevated at 280.7 pg/mL and there was evidence of severe deficiency in both 25(OH)D (<4 ng/mL) and 1,25(OH)2D (17.8 pg/mL). Nutritional labs revealed a low vitamin A, vitamin B12, vitamin B6, and zinc as described in The patient did not have a history of apparent numbness, weakness, muscle cramps, or bone pain. In the prior few months, however, he was noted to go down steps with two feet, one step at a time, and this had been a change in behavior. He was raised in the Northeast United States in New York and was of Hispanic ethnicity. His mother described him as a picky eater. On further dietary recall, he was noted to eat potato chips, French fries, corn, crackers, pizza without cheese, and chicken nuggets. He mainly drank ginger ale and apple juice. His lifestyle was sedentary with most time spent indoors or riding on the train. There was no personal or family history of metabolic bone disease. He had no history of prolonged steroid use.Given his hypocalcemia, treatment was initiated with intravenous calcium gluconate 2 grams every 2-3 hours with a goal total calcium of greater than 8 mg/dL so that he would be stable for orthopedic surgery. After 48 hours of intravenous calcium treatment, he underwent bilateral antegrade nailing of the bilateral femurs. He was started on vitamin D3 50,000 international units once weekly. Postoperatively, enteral calcium carbonate of 100 mg/kg/day divided every 6 hours was ordered. However, due to his refusal to take enteral calcium, a nasogastric tube was placed and his dose was increased to 120 mg/kg/day divided every 8 hours. Total calcium levels continued to improve and enteral calcium was slowly weaned to 60 mg/kg/day divided twice daily. By the day of discharge, his calcium level had improved to 9.9 mg/dL and 25(OH)D concentration improved to 13 ng/mL. He was discharged to a rehabilitation center for further feeding therapy and physical therapy.Vitamin D is a key regulator in calcium and phosphate homeostasis, aiding in normal bone mineralization. 25(OH)D deficiency therefore has important implications for overall bone health. As 25(OH)D deficiency has increased in prevalence in children and adolescents worldwide , 5, seveOsteopenia is one of the earliest radiographic signs of 25(OH)D deficiency. However, as the deficiency persists, signs of rickets can be noted, such as metaphyseal widening and fraying . MoreoveFew cases have described bilateral femur fractures in children with moderate to severe 25(OH)D deficiency. There have been reports of older adolescents with severe vitamin D deficiency who presented in the setting of hypocalcemic seizure. Schnadower et al.  describeInterestingly, our patient developed femoral fractures, which are rare as compared to the more common distal forearm fractures seen in adolescence . AlthougA history of ASD likely contributed to our patient's degree and presentation of 25(OH)D deficiency. Children with ASD are at increased risk for nutritional deficiencies, including vitamin D, given their food selectivity, often severely restricting their diet to up to a few foods . FactorsAs children with ASD have varying degrees of verbal skills , it can To our knowledge, this is the youngest reported case of bilateral femur fractures in the setting of severe 25(OH)D deficiency. When an adolescent presents with low-impact femur fractures, 25(OH)D deficiency should be considered. Clinicians should recognize that children with ASD are at higher risk for vitamin and mineral deficiencies and require close nutritional evaluation for adequate calcium and vitamin D intake. Primary care providers may therefore provide a pivotal role in the routine laboratory screening of 25(OH)D in this population.


To clarify the frequency and outcome of patients with systemic lupus erythematosus (SLE) who achieved the clinical state as serologically active clinically quiescent (SACQ) and to identify factors associated with the flare of disease.Clinical data of patients diagnosed as SLE and followed in Peking University First Hospital from 2009 to 2015 were retrospectively reviewed. Six hundred eighty-two patients who were followed up for more than 6 months were analyzed. SACQ was defined as an at least a 6-month period with persistent serologic activity and without clinical activity and daily dose of prednisone or equivalent were less than 7.5 mg. Serologically quiescent clinically quiescent (SQCQ) patients served as control groups. Data including demographics, initial symptoms, duration to SACQ, treatments before and after SACQ, and characteristics of the patients suffered from flare were analyzed.p < 0.001). Of 170 SACQ patients, 32.9% experienced flare that was significantly higher than 15.5% of SQCQ patients (29/187). Corticosteroids  was an independent risk factor for flare, while antimalarials  and immunosuppressants  were protective factors in SACQ patients; however, only antimalarials was protective factors in SQCQ patients .Among the 682 patients, 170 patients were SACQ (24.9%) and 187 patients were SQCQ. SQCQ patients (38.61 ± 15.08 years old) were older at baseline than SACQ patients (38.61 ± 15.08 years vs. 32.09 ± 14.35 years, About one third of SLE patients with SACQ experience flare, significantly more frequent than that of patients with SQCQ. Thus, approach to prevent flare in SACQ patient is required. Maintenance therapy of hydroxychloroquine and immunosuppressant agents may be protective and beneficial treatment strategy in these patients.The online version contains supplementary material available at 10.1186/s13075-021-02641-5. Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by multisystem involvements and recurrent flares after the induction of remission. Anti-double-stranded DNA (anti-dsDNA) antibodies are considered as specific markers for SLE present in 60–80% of the patients –3. Anti-In recent years, the therapeutic goal for SLE has evolved from a symptom-based to a target-based approach. With the strategy of treat-to-target (T2T) being proposed in 2014, it is a consensus that flare prevention should be a therapeutic goal and that it is a realistic target . And it In 1979, Gladman et al.  reportedSACQ patients with SLE appears to account for 6–12% of all patients with SLE, but there is disagreement about whether such patients are indeed clinically stable –19. In tThis is a retrospective observational study. All consecutive patients recruited in this study were identified from a large, single-center cohort of patients with SLE attending the Rheumatology and Clinical Immunology Department of Peking University First Hospital from January 2007 to December 2015. The patients were followed up for more than 6 months with visits no more than 6 months apart. All patients fulfilled either the 1997 American College of Rheumatology (ACR) Modified Classification Criteria for SLE .SACQ was defined as an at least a 6-month period with persistent serologic activity and without clinical activity. Each of these patients had a SLE Disease Activity Index 2000 (SLEDAI-2 K) of 2 or 4, from positive anti-dsDNA antibody and/or hypocomplementemia only, at each clinic visit. Serologically quiescent clinically quiescent (SQCQ) patients had at least a 6-month period without serologic or clinical activity (SLEDAI-2 K score = 0). Both SACQ and SQCQ patients could be taking a daily dose of prednisone or equivalent 7.5 mg or less and immunosuppressants with stable or decreasing dosage and/or antimalarials were allowed. The SACQ period was calculated from the first SACQ visit to either the date of known flare or serologic inactivity or to the most recent known SACQ clinic visit. Some patients had more than one SACQ period during follow-up, only the first one was analyzed. Other patients were defined as serologically active clinically active (SACA) in case (1) SLEDAI-2 K score presented from both clinical domain and anti-dsDNA or hypocomplementemia, (2) absent of SLEDAI-2 K score from clinical domain or serological domain less than 6 months or (3) applied more than 7.5 mg prednisone or equivalent per day. Disease flare in clinically quiescent (either SACQ or SQCQ) patients was defined as (1) any increase in the SLEDAI-2 K score accounted by neither hypocomplementemia nor anti-dsDNA, (2) an increase in the prednisone dosage to more than 7.5 mg per day, or (3) judged as flare by physician at a visit during follow-up.The demographics, laboratory results , and treatments including corticosteroids, antimalarials, and immunosuppressants were recorded. And corticosteroid doses were converted to milligrams (mg) of prednisone equivalent. All immunosuppressive medicines applied more than 3 months were recorded, including azathioprine 50–100 mg per day, mycophenolate mofetil 0.75–2.0 g per day, methotrexate 7.5–15 mg per week, leflunomide 10–20 mg per day, cyclophosphamide 0.4–0.6 g every 2 weeks and cyclosporin A 100–200 mg per day. SLE-related organ involvements at initial diagnosis, such as lupus nephritis (LN), neuropsychiatric SLE (NPSLE), hematological involvement(thrombocytopenia and hemolytic anemia), pulmonary hypertension, myocardial involvement, interstitial lung disease (ILD), alveolar hemorrhage, smooth muscle involvement, polyserositis and co-morbidities including antiphospholipid antibody syndrome (APS), thrombotic thrombocytopenic purpura (TTP), or thrombotic microangiopathy (TMA), and Sjogren’s syndrome (SS), were collected from the medical records of all recruited patients. The time of the onset of diseases, time to achieve SACQ/SQCQ, duration of SACQ/SQCQ time, and duration of serological activity or inactivity before SACQ/SQCQ were calculated. And for the flare group, the time to first flare after SACQ period and the onset of SLE, the clinical manifestations and SLEDAI-2K score when relapsed, and their prognosis were also collected.t test with unequal variances, whereas abnormally distributed continuous variables were compared using Mann-Whitney U tests. Comparison of categorical data was performed using chi-square tests.Continuous variables were presented as mean ± standard deviation (SD) or median (interquartile range) for abnormally distributed continuous variables. Categorical variables were described as numbers (percentages or proportions). Normally distributed continuous variables were compared using the Student Flare rate over time was estimated using Kaplan-Meier survival analysis, and comparisons of differences in outcome between groups were analyzed using the log-rank test. Logistic regression models, with odds ratios (ORs) and 95% confidence intervals (CIs), were used to determine the variables associated with flare. Age at diagnosis, genders, positive anti-dsDNA antibodies, hypocomplementemia, duration of serological activity or inactivity before SACQ/SQCQ, and therapy including immunosuppressants, corticosteroids, and antimalarials were included in the multivariable Logistic regression model. Besides, as the definitions of disease flare are same in both SACQ and SQCQ groups, we also put the two groups together and tried to identify factors associated with recurrence in these patients.p values are 2-sided and only associations with a p value less than 0.05 were considered statistically significant. All statistical analyses were performed using SPSS 20.0.All reported A total of 924 SLE patients visited our center between January 2007 and December 2015, and 682 of them were enrolled. We identified 170 patients (24.9%) who fulfilled the SACQ criteria as defined in the “Methods,” and 187 (27.4%) were SQCQ patients. The number of patients who did not achieve clinical remission was 325 (47.7%) . The SQCQ patients reached the steady state faster and were followed up longer until flare or the last visit than SACQ . In the comparison of SACQ and SQCQ, there was no difference in the dose of antimalarials and whether to use immunosuppressants at the start of SACQ or SQCQ (Table The median time from the time of being diagnosed to SACQ occurred was 22.55 (12.70–50.36) months. The patient demographics are summarized in Table p < 0.001). Patients with the state of SQCQ were more likely to maintain relapse-free survival than those with the state of SACQ . The time from they reached the SACQ or SQCQ to recurrence occurred showed a trend of longer time in the SQCQ group than that in the SACQ group, although it was not statistically significant . There was no difference in the treatments of the flare patients between the group of SACQ and SQCQ  had sustained SACQ state until last visits. There was at least 1 flare in 32.9% (56/170) SACQ patients and 15.5% (29/187) SQCQ patients (p = 0.002) and SQCQ (p = 0.001) patients. And the use of immunosuppressants when relapsed or at the end of follow-up followed the same trend in both SACQ (p = 0.017) and SQCQ (p = 0.041) patients. But the dose of corticosteroids is different between the flare and non-flare group only in SACQ patients (p = 0.011). There were no consistent clinical or serologic characteristics that distinguished the recurrent patients from the rest of the SACQ/SQCQ patients  was an independent risk factor for flare, while antimalarials  and immunosuppressants  were protective factors (Table p = 0.033) as protective factors (Table Logistic regression models showed differences in the therapy when the patients relapsed (flare group) or at the end of followed-up (non-flare group) between groups. In the SACQ group, dose of corticosteroids .A similar conclusion was reached when the two groups (SACQ and SQCQ patients) were analyzed together Table . The theSLE is a chronic disease characterized by varying degrees of severity with multiple episodes of relapse and remission, and the main purpose of treatments includes induction and maintenance of remission. SACQ is a specific subgroup of lupus patients with clinically stable but serological activity. In our cohort, the average age of SACQ was 32.1 years old and was younger than those of SQCQ. Whether these SACQ patients are truly stable is still controversial. Since the first report in 1979 , there hPrevious studies described the natural history of SACQ patients, with varying rates of disease flare from 59 to 81%, given the different definitions of SACQ , 22. TheCurrently, no factors are reliable to predict the course of SLE to maintain remission or to occur relapse. Previous studies have discussed the relationship between serological results and flare, with incongruent results. Steiman AJ et al.  investigTreatment is the focus of our analysis. The target of treatment of SLE should include the prevention of flares . HoweverIn a prospective, randomized, double-blind, placebo-controlled trial , the finThere were some limitations of the study, which include its limited number and retrospective nature. Additionally, the conclusions of our study based on Chinese patients may not be generalized to other ethnic populations.In conclusion, the clinician needs to pay attention to SLE patients with SACQ since about one third of them experience flare. Our study showed that more intensive treatment strategies, including antimalarials and immunosuppressants, are beneficial to prevent flare in SACQ patients. These results must be validated in other independent cohorts and studies with larger sample size must be undertaken to determine the optimal therapeutic strategy for SACQ SLE patients.Additional file 1.