Physical activity (PA) counseling by health professionals has promising results in behavior change. However, few studies have evaluated its prevalence in Primary Health Care in Latin American countries. This study aimed to describe the prevalence and analyze the associated factors of PA counseling in adults in Primary Health Care in Brazil. This is a cross-sectional study with a representative sample of 779 adults (70% women). Counseling was identified among those who reported having received PA counseling during a health professional consultation in the last 12 months. Sociodemographic factors, health conditions, and leisure-time PA were analyzed with Poisson regression. The prevalence of counseling was 43% , higher in people aged ≥40 years , obese , take prescription medication , and walk for leisure . People with more education were less likely to receive PA counseling . In conclusion, 4 out of 10 users reported receiving PA counseling and this was associated with sociodemographic factors, health conditions, and walking for leisure. These results can guide PA promotion in Primary Health Care. In most countries, the current health scenario is a consequence of an increase in chronic diseases due to the adoption of unhealthy lifestyles, emphasizing physical inactivity . DespiteThus, integrated programs of PA promotion at the population level have been implemented in many countries globally, which presuppose the interrelationship between different community sectors, such as school, active commuting, built environment, and health professionals ,5,6. In In this context, all health professionals should counsel or guide their patients about PA ,11, and PA counseling by health professionals has had promising results in behavior change and is recommended as part of integrated community interventions ,12,16,17Patient PA counseling is underutilized , and fewIt is essential to expand this knowledge, based on robust studies with a representative sample from BHUs in order to identify the individuals who are more exposed to PA counseling. This approach and its results are essential so that health professionals can implement and direct specific actions to promote PA towards disadvantaged population groups and those facing barriers to starting PA, whether due to psychosocial issues or lack of information, based on each city’s context or region. Thus, this study aimed to describe the prevalence and to analyze the associated factors of PA counseling in adults in Primary Health Care centers in Brazil.This study was reported according to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines. In 2019, a quantitative, observational, and cross-sectional study was conducted in a representative sample of adults in the BHUs of an urban area of São José dos Pinhais . The general project aimed to evaluate PA or sedentary behavior counseling, LTPA, and some of their determinants .BHUs are public clinics strategically distributed within a city, with free access for Primary Health Care provided by physicians, nurses, pharmacists, physiotherapists, nutritionists, psychologists, and community health agents . In some2 (21% urban), there is an estimated population of 329,058 inhabitants (medium size), a population density of 280 inhabitants km2, and a high Human Development Index (0.758). Some indicators among São José dos Pinhais, Curitiba, and Brazil are similar , a distance of 18 km from downtown to downtown. It has an area of 946 km similar  34]..2 (21% un = 15) were intentionally selected, as these are accessible to 90% of the city’s population. The study was approved by the National Commission for Ethics in Research (CONEP) of the National Health Council, with the Certificate of Presentation of Ethical Appreciation (CAAE) under protocol number 95985118.0.0000.0020, and by the Research Ethics Committee of the Pontifical Catholic University of Parana under the number 2.882.260. Participants were consulted, informed about voluntariness, and agreed to participate in the research by signing an informed consent form, according to the recommendations of CONEP.The city has 413 health establishments, among which are 27 BHUs (56% in the urban area). Only BHUs in the urban area . For sample representation, we considered the prevalence of PA counseling received in BHU (30%—based on a literature review available during the project planning and pilot study) and a 95% confidence level. A sampling error of four percentage points and a design effect of 1.5 were also regarded [n = 920) to reduce the estimation errors for multivariate analyses in future studies. The sample size was proportionally calculated by the number of visits to each BHU and varied between 31 to 92 users.The number of participants was estimated based on the average number of visits made in each BHU between January and February 2019 . A posteriori statistical analyses showed that, based on the predictor variables, our sample had an average power of 90% (β: 20%) and a confidence level of 95% (α: 5%) to detect prevalence ratio (PR), identified as significant in our study. This analysis was conducted using software G*Power .The participants were systematically selected based on their position in the waiting room at the BHU, counting between the first and fifth, from left to right of the entrance door . The thin = 9).Only adults (≥18 years) were eligible and invited. Among them, we excluded people who lived outside the urban area, and were using the BHU for the first time, had some physical limitation for PA practice , or had some cognitive or phonation limitation that prevented comprehension of the questionnaire  . The data was collected between April and October 2019.PA counseling was evaluated based on dichotomous response to the question: “During the last year (12 months), any time you were at the BHU, did you receive PA counseling during the consultation by a health professional ?” . This measure has been used in similar studies and adapted to the local context ,28,29,30Based on the literature review, predictive variables included sociodemographic factors , health conditions , and LTPA ,29,30,36The sex was observed , and the participants grouped into three age groups . The marital status was evaluated into three categories  and grouped into two . Skin color was self-reported into five categories  and categorized as white and non-white (other categories). Education was evaluated into five levels , and then regrouped into three categories: elementary school or less, junior high school, high school or more. All variables were measured according to the method proposed by the Brazilian Institute of Geography and Statistics and adopted by the Brazilian Health Surveillance System .Socioeconomic status (SES) was evaluated by a standardized questionnaire  which co2, overweight: 25–29.9 kg/m2, obese: ≥30.0 kg/m2) [The BMI was calculated using self-reported weight and height data and classified into three categories  . Smoking0 kg/m2) .The PA in a usual week was measured with the leisure module of the long version of the International Physical Activity Questionnaire (IPAQ) ,41,42. Pp < 0.001). Fourth, data entry was conducted by the field coordinator using the EpiData software . Fifth, data was cleaned using exploratory analysis, with SPSS software , to identify possible typing errors in data entry for each variable, to detect outliers, and to verify all variables’ distributions. Finally, each variable outlier was personally checked in the questionnaire and manually corrected in the database.Quality control of data was assured through six steps. First, all interviewers  received a 20 h theoretical and practical training on technical procedures for interviewing  based on an instruction manual prepared by the core project team. The interviewers strictly followed all these procedures; they were blinded to the investigation’s objectives and hypotheses and supervised by a field coordinator. Second, a pilot study was conducted on a random sample of 81 participants from three BHUs to test the data collection procedures and the comprehension of the questions translated from other studies and adapted to the local context. Third, all pilot study participants were re-interviewed at an interval of 7 to 10 days to analyze the temporal stability of the main variables of the study. Temporal stability (reproducibility) of PA counseling was analyzed with percentage agreement and Cohen’s kappa test, which showed 88% agreement and good Kappa value . Initially, variance inflation factor (VIF) tests were performed and rejected the hypothesis of multicollinearity (1/VIF ≥ 0.71). After bivariate analysis, all variables at the same or higher level, which presented a  3: LTPA . All anan = 134), and losses 2% (n = 22), which resulted in 779 participants interviewed. Most participants were women (69.8%), aged between 18–39 years (45.2%), married (64.0%), identified as having white skin color (73.0%), with education level of high school or higher (50.4%), and low economic level (71.2%)  .2), 14.9% smokers, 35.9% hypertensive, 15.7% diabetics, 15.9% dyslipidemic, and 6.5% had received a medical diagnosis of coronary artery disease. Just over half of the participants reported zero chronic diseases (54.8%) and consumed one or more prescribed medications (51.3%)  . Walking (51.3%) .p < 0.05). Walking  and total LTPA  showed a positive association with a higher prevalence of PA counseling.In the bivariate analysis , the soc2  and those who take continuous use medication  were more likely to receive PA counseling. Finally, leisure walking was positively associated with PA counseling received by health professionals .After adjusting for possible confounding parameters , the higOur study aimed to explore the possible factors associated with PA counseling received by adults in BHUs of Primary Health Care in a medium-sized city in southern Brazil. With low risk of bias, the method used allowed us to represent the adult users of BHUs in an urban area, besides exploring the main individual predictors of counseling, analyzing the association’s strength and direction. Also, the variables were measured by standardized procedures and instruments, and these are our study’s strengths. Counseling was higher among middle-aged adults and the elderly, those with less education, married, obese, those who consume medications for chronic diseases, and those who walk in leisure-time. Studies have shown a positive effect of counseling received and PA levels ,16,17. TOur results showed that the participants had worse health conditions when compared to adults in the capital Curitiba, a city next to São José dos Pinhais  . In partAbout 4 out of 10 users reported receiving PA counseling (43%). A systematic review synthesized Primary Health Care results in several countries and showed an average prevalence of 37% (±14%) . HoweverIn our study, middle-aged and elderly adults received more counseling compared to young adults. Many studies have explored these variables, showing consistent results of higher prevalence of PA counseling according to age ,46,47,48The married status was positively associated with receiving counseling. A few studies have explored this variable and found contradictory associations ,24,25. ORegarding education, people who completed high school or more were less likely to receive counseling. These results are not well understood in the literature, as studies have shown that higher education levels were positively associated with counseling ,36,47. AThe consumption of a high number of medications showed a positive association with counseling. This result is similar to other studies found in the literature . In someThe positive association between counseling and walking can be explained by the recurrent counseling offered in Primary Health Care , especiaContrary to what was expected, some predictors  had no association with counseling received. However, other studies have pointed to a positive association between some of these factors and counseling ,49. SuchOur results must be interpreted considering some limitations. The sample was restricted to adult BHU users from an urban area of a medium-sized city in southern Brazil. This city has a broad rural area (79% of the territory), comprising 12 BHUs. Thus, our findings cannot be extrapolated to other contexts . There were no physical education professionals in the health teams or PA programs of the selected BHUs. These may, in part, have altered the prevalence of counseling received, as well as associations between counseling and PA ,54. FinaThe prevalence of PA counseling was 43%, and the populational groups more exposed to counseling were middle-aged and elderly adults, married, those with less education, obese, those who consume medications and walk in leisure-time.Primary Health Care actions in Brazilian BHUs should be expanded, with health professionals’ training towards promoting PA, emphasizing epidemiology, public health, and PA in addition to developing counseling methods in routine service, with valid and useful protocols to evaluate actions’ effectiveness . Health


Leprosy still represents a negleted public health problem in Brazil. Early and adequate treatment of leprosy, carried out in a primary health network is essential to reduce morbidity and sequelae.To analyze the therapeutic management of leprosy patients referred from primary healthy services to a specialized service.An analytical retrospective study using medical records and the Notifiable Diseases Information System. Patients diagnosed with leprosy, referred to a specialized outpatient clinic, between 2016 and 2017, in Goiás state, were included. The treatment carried out in the primary health services was compared to the Ministry of Health guidelines.2 = 11.43 and p < 0.001). Regarding the leprosy reactions and sequelae management, 56.9% and 19.5% were considered inadequate, respectively. There was no difference between the percentage of adequate or inadequate management when considering the Goiás health macroregions .Two-hundred twenty-five leprosy patients were included, of whom 33.3% were referred by leprosy reactions, 27.1% by sequelae, and 10.2% by suspected recurrence or reinfection. Reviewing the therapeutic management, 123 (54.7%) were considered inadequate, 92 (40.9%) adequate, and 10 (4.4%) inconclusive. Of the 200 multibacillary patients, 39.5% had adequate management. In contrast, 12 (85.1%) out of 14 paucibacillary patients had adequate management (χUse of recorded data, with incomplete medical records and lack of patient follow-up.The study demonstrated the equivocal multibacillaryleprosy management conducted in healthy primary care, with an emphasis on leprosy reactions and sequelae. Training and monitoring the medical staff in the primary healthy services could reduce the morbidity and sequelae of leprosy. Mycobacterium leprae (M. leprae), bacillus discovered in 1873 by Amauer Hansen.Leprosy, also known as Hansen's disease, is a chronic infectious-contagious disease known to humankind since ancient times. It is characterized by predominant involvement of skin and peripheral nerves, caused by the In 1991, the World Health Organization (WHO) approved a resolution to “eliminate leprosy as a public health problem” by the year 2000, with the elimination being defined as the reduction of prevalence to less than 1 case per 10,000 inhabitants.In 2018, three countries reported more than 10,000 new cases of leprosy, including India , Brazil , and Indonesia .Sistema Único de Saúde), patients notified with leprosy must be forwarded to the primary basic health unit closest to their homes, so that they have easier access to health care and a reduced chance of irregular or discontinuation of their treatment.With the administrative and organizational decentralization of the Brazilian Unified Health System  and the difficulty in analyzing the health care service routines.This is an analytical retrospective study whose recorded data compiled from patients` medical charts of the Leprosy Outpatient Clinic at the University Hospital from January 1, 2016 to December 30, 2017 were evaluated.Patients seen at the first consultation for diagnostic investigation of leprosy or for the management of complications, sequelae, or leprosy reactions, who had a referral letter from a primary public health service, without restriction of age, sex, or municipality of origin were included. Patients with a history of previous leprosy treatment at the Dermatology Outpatient Clinic of University Hospital, patients whose medical records did not include the treatments received for leprosy, their sequelae and leprosy reactions, or those whose data could not be retrieved from the leprosy notification/investigation forms of the Notifiable Diseases Information System (SINAN) were excluded.The data were obtained from the patients’ medical records, letters of referral to the outpatient clinic, and the leprosy notification/investigation forms of SINAN (ICD-10 A30), made available by the Health Secretariat of the state of Goiás. As for the data collection procedure, initially, the Medical Archive Service at University Hospital provided a list with the nominal identification and medical record number of all patients treated at the leprosy outpatient clinic during the study period. The main researcher (PPS) reviewed the medical records and referral letters, and identified the cases that fulfilled the inclusion criteria.The data were compiled in the Microsoft Excel® spreadsheet. The analyzed variables were: identification, sex, age, city of origin, initial diagnosis, the reason for referral, treatments performed at the service of origin, degree of physical disability, management in the specialized clinic, and final diagnosis.The municipalities of origin were grouped inside the five health macroregions of the state of Goiás, namely: Northeast, Midwest, Mid-North, Southeast, and Southwest.The patients who were treated according to the Ministry of Health protocols had their management considered as adequate, as follows:Paucibacillary cases - Multidrug Therapy (MDT) with rifampicin and dapsone for 6 months.Multibacillary cases - MDT with rifampicin, dapsone, and clofazimine for 12 months.Type 1 leprosy reaction - Corticosteroids (first-line drug) and preventive therapeutic regimens for strongyloidiasis, diabetes, hypertension, and osteoporosis; alternative drugs: azathioprine, cyclosporine, when corticosteroids are contraindicated or cases are unresponsive to them.Type 2 leprosy reaction - Thalidomide at varying doses, depending on the reaction severity, which may be associated with corticosteroids in cases of necrotic erythema nodosum leprosum, nerve trunk involvement (neuritis), orchiepididymitis, iritis or iridocyclitis, reactional hands, and feet; women of childbearing age and/or pregnant women – corticosteroids and/or clofazimine (or pentoxifylline). In childbearing age women, thalidomide can be prescribed if provided using two contraceptive methods, with one being a barrier method.Patients treated differently from what is recommended were classified as receiving inappropriate treatment and those whose data were not possible to be recovered were classified as inconclusive.The Operational Classification was used for the classification of cases: Paucibacillary (PB) and Multibacillary (MB), taking into account the bacilloscopy.Regarding the criteria for the diagnosis of leprosy reaction, the ones recommended by the Ministry of Health were considered, in which type 1 reaction is characterized by an exacerbation of previous lesions or the appearance of new lesions, making them edematous; also, ulcerations may occur, while nerve trunks can increase in volume and become painful. In the type 2 reaction, the most common clinical manifestation is erythema nodosum leprosum, characterized by the onset of erythematous and painful nodules and nerve thickening. Other clinical presentations are necrotic erythema nodosum leprosum and erythema multiforme.M. leprae is not cultivated in vitro.As for recurrence, leprosy cases that were regularly treated with official standardized and correctly indicated regimens, who were discharged after cure, and who once again showed new clinical signs and symptoms of active infectious disease and who did not respond to the anti-reactional treatment were considered as cases of recurring disease.Statistical analysis was performed using the Statistical Package for Social Sciences software v.21 . Continuous variables were described considering the minimum and maximum values and the mean and standard deviation. Categorical variables were shown as absolute and relative values (percentages). The chi-square test was performed to compare categorical variables. Variable data potentially associated with inadequate management were evaluated. The significance level was established at p < 0.05.The research project was approved by the Research Ethics Committee of the University Hospital (CAAE: 06122919.4.0000.5078).During the study period, 403 patients were treated at the Leprosy Outpatient Clinic, of whom 290 were attended by the first-time. However, 16 were excluded from the study because the evaluated data were not available. Of the 274 included patients, 163 were males (59.5%). The patients’ age ranged from 2 to 84 years, with a mean of 48.9 and a Standard Deviation (SD) of 15.9 years. Seven patients under the age of 15 years were seen, with three as suspected leprosy cases, one with type 2 leprosy reaction, one patient for post-treatment reassessment because a residual lesion persisted, another for a medical report, and an 11-year-old patient was referred due to suspected recurrence (inconclusive due to loss of follow-up). The three suspected cases were not ultimately confirmed as leprosy, receiving the diagnoses of atopic dermatitis and pityriasis alba.Regarding the Health Macro-regions, 31 patients (11.3%) were referred from the Northeast region, 52 (19%) from the Mid-North region, 112 (40.9%) from the Midwest region, 56 (20.4 %) from the Mid-Southeast region, and 23 (8.4%) from the Southwest region.The main reason for referring patients was type 2 leprosy reaction, in 52 (19%) patients, followed by suspected leprosy diagnosis in 49 (17.9%) patients. Of 49 patients who were referred as suspected cases, 16 had their diagnosis confirmed, with a prompt notification, and 12 were lost to follow-up. Twenty-one patients had other diagnoses: macular amyloidosis, Dupuytren's disease, pityriasis alba, systemic sclerosis, drug reaction, lichen simplex chronicus, pityriasis versicolor, pellagra, pre-neoplasms/neoplasms of the skin .Table 1FIn summary 225 patients had a confirmed diagnosis of leprosy. Of these, 210 (93.3%) patients were classified as multibacillary, 14 (6.2%) as paucibacillary and one was not classified. Thirty-two patients had neural sequelae, such as loss of thermal, tactile, painful sensibility, in addition to permanent paresthesia (degree 1 of disability) and 29 physical sequelae (degree 2 of disability), such as plantar ulcers, claw hand deformity, foot drop, bone resorption, totaling 27.1%. Twenty (8.9%) patients were referred for different reasons, for instance, lack of thalidomide in the public network or for the treatment of several dermatoses such as tinea corporis, pellagra, stasis ulcer, pityriasis versicolor, and many of these patients had already been discharged after leprosy cure. Eight (3.5%) patients required an evaluation due to reaction to polychemotherapy drugs.Twenty-two (9.8%) patients were referred due to type 1 leprosy reaction. Two patients had type 1 and type 2 reactions. Seven (2.6%) patients were referred due to a positive bacilloscopy test, even after the end of treatment.For the classification of patients regarding the adequacy of treatment, those referred as a suspected case and, therefore, who had not undergone any treatment, were excluded, as well as those referred for biopsy whose diagnosis had not yet been attained. For treatment management reviewing, 225 patients had a confirmed leprosy diagnosis; 92 (40.9%) and 123 (54.7%) of whom had adequate and inadequate treatment, respectively, and 10 (4.4%) patients were classified as inconclusive.2 = 11.43 and p < 0.001). One patient was not classified on the notification form.Of the 200 MB patients 79 (39.5%) had adequate treatment management. In contrast, 12 (85.1%) out of 14 PB pattients had adequate management . Inconclusive cases were excluded from this analysis.2 = 0.81; p = 0.37). Inconclusive cases and cases with concomitant type 1 and 2 reactions were excluded from statistical analysis.When analyzing the treatment management for leprosy reactions, out of 22 patients having type 1 leprosy reaction, 86.3% were treated inadequately, while of those 52 patients having type 2 leprosy reaction, this percentage was 78.9%  , with 19.5% .Table 4CAccording to the Health Macroregions of the state of Goiás, the percentages of inadequate management related to leprosy reactions were similar in all of them .Figure 2Of the 206 patients whose degrees of disability were obtained from the SINAN, 116 (42.3%) had grade 0 disability, 55 (20.1%) had grade 1, and 26 (9.5%) grade 2. Nine (3.3%) patients were not assessed regarding the disability degree at the diagnosis. When comparing the degrees of disability attributted in the primary healthy care and in the specialized outpatient clinic, 13 patients who were initially classified as grade 0, were considered as grade 2. On the other hand, three patients who received a grade 2 classification in the primary care, were classified as grade 0 at the leprosy outpatient clinic of the University Hospital.The present study allowed the identification of equivocal therapeutic management of leprosy patients referred from the primary health network, to a dermatology referral service in the state of Goiás, Brazil. In approximately 60% of the cases, there was some divergence of therapeutic management in disagreement with the Ministry of Health guidelines. Leprosy reactions and physical and neural sequelae were the main reasons for referral to a specialized outpatient clinic. A predominance of type 2 leprosy reactions was observed, with approximately 80% of cases having been conducted diversely from the Ministry of Health guidelines. This information draws attention to the risk of disease progression to physical disabilities , social stigma, and worsening quality of life.Reactions type 1 and 2 of leprosy comprise very frequent complications, being responsible for the suffering and permanent disabilities of patients.The subdivision of the state of Goiás into health macroregions showed that the inadequacy of the performed treatments was similar in the different regions. It is noteworthy that the Midwest region encompasses cities such as Goiânia and its surroundings, that is, places where access to information and diagnostic methods would be more accessible. Therefore, the need for training programs aimed at professionals working in the primary health care network regarding the correct diagnosis and management of these patients is necessary.Among the cases referred to the specialized service as suspected leprosy, approximately 30% were confirmed, suggesting that the primary care physician has difficulty in identifying these cases. Another fact that draws attention concerns diagnostic errors, in which cases notified as leprosy were excluded at the specialty clinic or even cases of leprosy reactions being treated as recurrence cases. It is known that in multibacillar patients, the bacilloscopy shows the presence of viable bacilli in the initial stages of treatment, while the proportion of granular bacilli increases with the continuity of treatment.One patient was referred due to pregnancy while receiving MDT for MB leprosy. Treatment in these cases consists of the standard protocol with the use of multibacillary polychemotherapy and, if applicable, systemic corticosteroids to control the reactions. Although there is a recommendation to restrict the intake of these drugs in the first trimester of pregnancy, the benefits of treatment outweigh the risk. Thalidomide is contraindicated, as it is a teratogenic drug, and can be used by women of childbearing age only if the woman is using two contraceptive methods, with one being a barrier type.Examples of inadequate management, according to the Ministry of Health guidelines, constitute, for example, patients using prednisone for a very long time, without weaning, and without prophylaxis for strongyloidiasis or osteopenia/osteoporosis. Some of these patients had cushingoid facies, drug-related acne, dyslipidemia, hypertension, diabetes mellitus, striae, and cataracts.Leprosy learning during undergraduate medical school is mostly theoretical, with few cases seen by the students in medical practice. Therefore, primary care professionals often feel insecure in the management of leprosy patients, which can lead to diagnostic delays and iatrogenesis.28The present study has limitations, including those inherent to retrospective studies using recorded data. Among these, we can mention the incompleted data in the referral forms and medical records and the lack of patient follow-up, difficulting the clinical outcome assessment. Also, the study did not consider the year of diagnosis and the length of treatment prior to referral to the specialized service as variables that could be potentially associated with inadequate management.Despite the limitations, the study showed a high percentage of leprosy patients referred to a specialized outpatient clinic whom had been managed in disagreement with the guidelines of the Ministry of Health, in all health macroregions of the state.Overall, in-person consultation at the tertiary level adds obstacles to the access, such as difficulties for intra and inter-municipal displacements, costs and delays in scheduling medical appointments. The training and supervision of primary care teams are essential to reduce problems related to the management of leprosy patients. In this context, the application of health information technologies  makes it possible to expand the training of professionals and increase the resolution of leprosy treatment in primary care.Different remote assistance and monitoring strategies could contribute to having leprosy patients evaluated by dermatologists, in referral centers, when necessary, using a more agile and interactive approach, with a consequent reduction in morbidity and sequelae.30Leprosy reactions and physical/neural sequelae represented about 60% of the cases referred from the health primary care network to a tertiary referral center for leprosy, in Goiás. In a significant number of cases, the adoption of therapeutic management approaches was in disagreement with the guidelines recommended by the Ministry of Health. The study indicates the need to reinforce health education strategies for primary care professionals, as well as streamline the assessment of patients in tertiary centers, not necessarily through in-person appointments. The primary purpose of comprehensive care for the patient with leprosy is not only the reduction of the disease burden and its sequelae but also the correct diagnosis, to avoid unnecessary treatments and disease progression, which lead to physical disabilities.The authors also suggest that prospective studies should be carried out to better assess the therapeutic management of leprosy patients treated at the primary basic care network, as well as the creation of specialized rehabilitation services in different regions of the country, aiming to prevent morbidity and sequelae.None declared.Pétra Pereira de Sousa: Conception and planning of the study; data collection and analysis; literature review; writing of the manuscript.Ana Lúcia Maroccolo de Sousa: Conception and planning of the study; participation in the drafting of the manuscript; approval of the final version.Marília Dalva Turchi: Planning of the study; statistical analysis and data interpretation; participation in the drafting of the manuscript; critical review of the manuscript.None declared.


Cytopenias, abnormal liver tests, and coagulation disorders were significantly more frequent in patients with MAS; lactate dehydrogenase and serum ferritin levels were significantly higher. An optimized threshold of 3500 μg/L for serum ferritin yielded a sensitivity (Se) of 85% and a negative predictive value (NPV) of 97% for identifying patients with/without MAS. Survival analysis showed that a high ferritin level at the time of SD diagnosis was predictive of MAS development (p < 0.001). Specific factors, including neurological symptoms, cytopenias, elevated LDH, and coagulopathy, may contribute to the early detection of MAS. Extreme hyperferritinemia at the onset of SD is a prognostic factor for the development of MAS.Distinguishing between macrophage activation syndrome (MAS) and a simple flare of Still’s disease (SD) may be challenging. We sought to clarify the clinical features and outcome of MAS in SD and to explore predictive factors of MAS development. Demographic and clinical data, treatments, and outcomes were recorded in a cohort of 206 SD patients. SD patients with and without MAS were compared. To explore predictive factors for the development of MAS, patients were compared at the time of SD diagnosis. Twenty (9.7%) patients experienced MAS, which was inaugural in 12 cases. Patients with MAS were more likely to have hepatomegaly (OR, 3.71; 95% CI, 1.14–11.2;  Still’s disease (SD) is a rare, non-monogenic autoinflammatory disorder characterized by high fever, evanescent rash, polyarthritis, and elevated WBC count ,2. By deSD has a good prognosis, with an estimated specific mortality rate of 1–3% ,13,14,15In addition, MAS and SD flares share many clinical and biological signs and symptoms, making the identification of MAS difficult ,26,27,28In this multicenter study, we sought to determine the specific characteristics that distinguish MAS from an SD flare and to explore the predictive factors of the secondary occurrence of MAS.All patients followed for SJIA or AOSD in nine regional clinical centers, between January 2001 and March 2021, were included in the observational cohort (AURAL STILL study). Patients were excluded if data at the time of diagnosis were missing. For each patient, data were collected from medical records and biological software using an anonymous and standardized electronic Case Report Form . Demographic data, medical history, clinical, biological, and imaging data at the time of diagnosis were collected. If available, histopathological and other related test results were collected. Medical management, treatments, side effects, and outcome were recorded chronologically. Antinuclear antibodies (ANA) were considered positive if their titer was ≥160. HScore and HLH-2004 score were retrospectively calculated . All datThe Yamaguchi or Fautrel criteria for AOSD ,43 and Ip-value of ≤0.05 was considered significant. A receiver operating characteristic (ROC) validation analysis was carried out by obtaining the area under the ROC curve to evaluate biological data and score in MAS prediction. The best cutoff was determined to be the one with the highest Youden’s index. Kaplan–Meyer curve analyses representing the probability of survival without MAS over time were compared using the maximally selected rank statistics from the maxstat and the survminer packages. All analyses were performed using the statistical software RStudio, v1 3.1093 .SD patients with and without MAS were compared, respectively, at the time of MAS onset and at the time of SD diagnosis to look for factors that could distinguish MAS from a simple SD flare . This analysis was also dichotomized between SJIA  and AOSD (onset after 16) to look for differences in more homogeneous groups. In a subsequent analysis, to look for predictive factors of secondary MAS, we excluded patients who had an inaugural MAS from the analysis, and compared the initial data of patients with late-onset MAS to the remaining patients without MAS. Descriptive analyses are presented as medians  for non-normally distributed continuous variables and as frequencies and percentages for categorical variables. For the percentage calculation of each variable, the number of missing values was excluded from the denominator . Nonparametric statistical methods were used to compare the study continuous variables by Mann–Whitney U-test, and the chi-square test or Fisher’s exact test as appropriate for categorical variables. A ClinicalTrials.gov (accessed on 29 November 2021) under the ID #NCT05055882. All patients were sent written information about the study. In accordance with French legislation on non-interventional retrospective studies, no written informed consent was required for inclusion.Ethical approval has been obtained from the institutional review board and the French CNIL (#19_348). This study was registered on A total of 206 patients with SD were included, of whom 86 (42%) were male. The median age at the time of SD diagnosis was 27  years. A total of 78 (38%) patients had a diagnosis of SJIA. ILAR criteria were met in 43% of cases and PReS criteria in 53% of cases. Of the 128 patients with AOSD, 60% met the Yamaguchi criteria and 65% met the Fautrel criteria. Winter was the season with the highest frequency of SD onset (33%), while spring was the season with the lowest frequency of onset (19%). The median time from symptom onset to diagnosis of SD was 39  days. Epidemiological data, as well as clinical and laboratory findings, are shown in n = 77/171) of cases. The rash was typical  in 13% (n = 18/135) of cases and was concomitant with fever peaks in 47% (n = 63/135) of cases. In total, 21% (n = 38/189) of patients had heart involvement, mostly pericarditis , complicated by tamponade in five cases. Autoimmunity was present in 28% (n = 45/163), with antinuclear antibodies and rheumatoid factor being positive in 20% (n = 32/160) and 10% (n = 11/114) of cases, respectively. The disease course was mainly systemic, with 36% and 35% of monocyclic and polycyclic patterns, respectively . Steroids were the first-line treatment used in 85% (n = 171/202) of patients, then IL-1-blockers . Patients who did not receive corticosteroids often received a combination of other treatments: 11 patients with monocyclic form , 11 with polycyclic form , 8 patients with chronic form . At the end of the follow-up, 38% of patients (n = 72/192) were considered cured, while 2.7% (n = 5/185) of patients had died.Fever and joint involvement were the most common symptoms. Arthritis was present in 45% (ectively . More thp < 0.001). The only clinical features that were significantly different between SD patients with and without MAS were a higher frequency of hepatomegaly  and neurological symptoms  in patients with MAS (We then compared SD patients with and without MAS at the time of MAS occurrence to look for factors that discriminate MAS from an SD flare. MAS complicated SD in 20 patients (9.7%). Twelve patients had inaugural MAS , while the remaining eight had late-onset MAS . MAS complicated 11 AOSD and 9 SJIA, with no preponderant seasonality. Compared to SD patients without MAS, those who developed MAS were more frequently immunocompromised  and platelet counts (p < 0.001). They had higher ferritin levels , p < 0.001) but there was no difference in ferritin glycosylated fraction. Patients with MAS were more likely to have abnormal liver function tests, and their lactate dehydrogenase (LDH) levels were significantly higher (p < 0.001), as well as the median triglyceride levels (p = 0.001). They were more likely to have coagulopathy and hypofibrinogenemia . Patients with MAS had no autoimmunity (p = 0.004). As expected, hemophagocytosis was more frequent in patients with MAS . Overall, the HLH-2004 and the HScore were significantly higher in patients with MAS (p < 0.001).While no bacterial triggers were identified in the MAS group, a viral replication was observed in seven cases (35%) with a majority of EBV replication , and a polycyclic course was more frequently observed in this group (55%). Finally, none of the patients from the MAS group had died at the end of the study period.There was no difference in steroid use, but interleukin blockers were more frequently used in patients with MAS . However, this difference was not significant and the small size of the cohort precludes formal conclusions. Patients with SJIA + MAS were more frequently immunocompromised than other SJIA patients (p < 0.001). They had less joint involvement (p = 0.037) and a significantly lower prothrombin time . A greater proportion of SJIA + MAS patients received anti-IL-1 therapy (p = 0.033), intravenous immunoglobulins , and immunosuppressive drugs (p = 0.003), mostly ciclosporin. In 89% of cases, MAS occurrence was associated with a polycyclic course of SJIA. For AOSD patients, we found no additional differences in clinical and biological features. Yet, a greater proportion of AOSD + MAS received IVIg (p = 0.007) or anti-IL-6 therapy (p = 0.006).Although a continuum between AOSD and SJIA is admitted, we analyzed each condition separately to gain potential in-group homogeneity . There wTo better define the parameters that distinguish MAS from SD flares, we calculated the areas under the curves (AUC), the best thresholds, and associated sensitivities (Se) and specificities (Sp) for the most significant ones .The Youden’s index reached a maximum with a threshold of 3500 μg/L for serum ferritin, yielding a Se of 85%, a Sp of 62%, and a negative predictive value (NPV) of 97% for identifying patients with/without MAS. Similarly, an LDH level of 459 U/L yielded a Se of 90%, a Sp of 72%, and an NPV of 98%, and a fibrinogenemia of 4.39 g/L yielded an NPV of 94%. The best threshold for the HScore was 135, yielding a Se of 90%, a Sp of 85%, and an NPV of 99%. In our series, this cutoff value had a higher performance than the previously reported value of 169 .p < 0.001, The median time to MAS onset was 30  days. The survival analysis assessed the risk of MAS development by comparing SD patients according to their baseline ferritin levels. This analysis showed that patients with a ferritin >10,448 μg/L at the time of SD diagnosis had a significantly higher risk of developing MAS during the course of the disease (p = 0.012) and a lower median PT (p = 0.004), none of the characteristics present at the time of SD diagnosis were predictive of secondary MAS development. The proportion of patients with initial lung or heart involvement was greater in the group with late-onset MAS, but this difference was not statistically significant.Finally, to explore the predictive factors of late-onset MAS, we compared SD patient characteristics at the time of SD onset, but excluding patients with inaugural MAS . Except MAS is a severe condition that can lead to multiple organ dysfunction syndrome, especially if not recognized early. The overlap between the symptoms of MAS and SD flares makes the identification of MAS challenging. In this large multicenter study, we combined data from adults and children with SD to maximize the chance of identifying discriminating factors. This combination was possible because previous studies have shown that these conditions share the same pathophysiology, clinico-biological features, disease course, and outcome ,4,5,47. In our series, overt MAS occurred in 9.7% of cases, a frequency similar to those described previously ,48. HepaNeurological involvement has been reported in 10–73% of cases in reactive hemophagocytic lymphohistiocytosis (reHLH). Disorders such as encephalopathy, meningitis, epilepsy, or posterior reversible encephalopathy syndrome have been described ,58,59. IWith regard to biological parameters, our results also confirm the need for close monitoring of leukocyte and platelet counts and fibrinogen levels. Indeed, while these biomarkers are usually increased during SD flare , it is classic to suspect MAS when their levels are paradoxically normal or decreased. As expected, hemophagocytosis on bone marrow aspirates was more frequent in patients with MAS. However, it was also observed in 5% of SD patients without MAS. Such a finding supports a common pathophysiological spectrum encompassing MAS and SD ,61. IndeTo facilitate the identification of MAS, we sought to determine the optimal thresholds for the most specific biomarkers. We found that, although this is a usual finding during SD flares, elevated ferritin levels were significantly higher during MAS . A ferriConversely, we found significant differences in the HScore , which wLDH has been repeatedly reported as a useful biomarker for the diagnosis of MAS, notably in SJIA ,69, but Like some previous studies, we showed that MAS is associated with the polycyclic course of SD and with a higher proportion of relapsing patients ,70. ThesStudies addressing the risk of late-onset MAS are scarce  and mostAt odds with previous publications, we did not observe a significantly reduced survival rate in patients undergoing MAS ,41, as aAlthough it has one of the largest sample sizes in the literature, our study has limitations. First, it has a retrospective design; there is a significant amount of missing data and the long recruitment period and changes in practices make the results more heterogeneous. Despite a large sample of SD patients, the number of cases with MAS remains limited, which may have prevented the identification of some prognostic factors. In the same way, statistical analysis should be interpreted with caution due to the small number of MAS patients. Finally, due to the absence of death in the MAS group, we were not able to perform a survival analysis.MAS is a frequent complication and a challenging diagnosis in SD, mainly due to overlapping signs and symptoms. However, some specific factors, including cytopenias, elevated LDH, and coagulopathy, may help in its early detection. In contrast to the ferritin glycosylated fraction, the previously published HScore appears to be useful for the detection of MAS complicating SD. Its diagnostic performance in this particular context may be optimized. Extreme hyperferritinemia at the onset of SD is prognostic for the subsequent occurrence of MAS. However, larger studies are needed to clarify its exact value.


Brochothrix thermosphacta was highly abundant at the beginning of the shelf-life period, but was later overtaken by Leuconostoc carnosum and Lactococcus piscium. For cooked chicken products, Latilactobacillus sakei was most abundant throughout the entire period. Additionally, 13 cooked ham and 16 cooked chicken samples were analyzed using metabarcoding. Findings obtained with this method were generally in accordance with the results from the culture-dependent approach, yet they additionally demonstrated the presence of Photobacterium at the beginning of the shelf-life period in both product types. The results indicated that combining culture-dependent methods with metabarcoding can give complementary insights into the evolution of microorganisms in perishable foods.Although refrigeration and modified-atmosphere packaging (MAP) allow for an extended shelf life of cooked charcuterie products, they are still susceptible to bacterial spoilage. To obtain better insights into factors that govern product deterioration, ample information is needed on the associated microbiota. In this study, sliced MAP cooked ham and cooked chicken samples were subjected to culture-dependent and culture-independent microbial analysis. In total, 683 bacterial isolates were obtained and identified from 60 samples collected throughout the storage period. For both charcuterie types, lactic acid bacteria (LAB) constituted the most abundant microbial group. In cooked ham,  According to estimates by the United Nations Environment programme, approximately 931 tons of food waste were generated in 2019, which suggests that 17% of global food production may be wasted [Spoilage of meat is usually of microbial origin, which is also the case for the specific category of cooked meat products. The latter are rich in proteins, fats and some (added) carbohydrates which can be broken down and metabolized by specific spoilage organisms (SSOs), resulting in off-flavors, off-odors, discoloration, gas formation, changes in texture and slime-formation, making the product unsuitable for consumption ,5,6,7. ABrochothrix thermosphacta is generally favored under anaerobic MAP conditions with N2 and CO2. Due to competitive effects, the growth of acid-sensitive Gram-negative bacteria is largely inhibited [Refrigeration and modified-atmosphere packaging (MAP) allows for an extension of the shelf-life period of cooked meat products with minimal changes in the physical and chemical properties of the product ,13. Grownhibited ,15,16,17nhibited .The end of the shelf-life period is defined by a maximum acceptable level of either SSOs or sensory alterations in the product . TherefoIn the present study, the microbiota of two types of MAP sliced cooked charcuterie , produced at different production sites but sliced in the same facility, were investigated during storage using conventional culture-dependent microbiological analysis in combination with 16S rRNA gene metabarcoding during the shelf-life period. The metabarcoding approach was included to generate a culture-independent and cost-effective approach to obtain a complementary overview of the microbial composition of the samples on different taxonomic levels, although underrepresented communities may be overlooked ,19,20. TTwo types of food products were sampled in this study: (1) cooked ham and (2) cooked chicken products. Both products were pressed into meat logs and underwent a cooking process wherein cooked ham reached a core temperature of approximately 69 °C, whereas cooked chicken reached at least 72 °C. The samples were collected within the premises of the food business operator (FBO), at the moment of slicing and packaging. Samples were taken aseptically of the log of each product type, right before slicing occurred, as well as of the sliced products derived thereof. The logs had a shelf-life period of three weeks each, and were sliced by the FBO between one and three weeks of storage at −1.5 °C, depending on the market demand. To reflect this variability, each batch of each product type was sampled twice. This was done when the log was sliced one week after its production and when another log of the same batch was sliced two weeks later, at the end of the log’s shelf-life period. A schematic overview of the sample collection procedure is presented in 2 and 30% CO2. Each sliced MAP product was analyzed on the day of slicing (D0) as well as after 28 d of storage (D28), corresponding with the end of the shelf-life period of the sliced product. The 28-d storage period consisted of 19 d at 7 °C and an additional 9 d at 8 °C, per usual practice of storage studies by the FBO.All sliced products derived from the various logs were packaged under MAP conditions of 70% NFive batches were analyzed per product type, resulting in 30 analyzed samples per product type or 60 analyzed samples in total. Samples were stored and transported under cooled conditions to the laboratory for further analysis (with a transport duration of maximum 30 min).2 each; A4 format) was performed using sponge sticks , moistened with 10 mL of maximum recovery diluent .An additional environmental sampling was conducted eight months after the original samplings. Environment samples were taken of food contact surfaces from two slicers during production; one slicer was used to slice cooked ham and another one to slice cooked chicken. Surface sampling  was used for the enumeration of TAC, applying incubation at 21 °C during 5 d. Reinforced clostridial agar  was incubated at 21 °C during 5 d for the enumeration of TANC. For the enumeration of presumptive LAB, de Man, Rogosa and Sharpe agar  and M17 agar (Oxoid) were both incubated at 21 °C during 5 d. This was done to obtain an enhanced overview of the LAB present, as MRS medium contains acetate, whereas M17 does not. Acetate inhibits growth of Carnobacterium spp., which is known to occur in meat products [B. cereus, of which the presence was further confirmed using blood agar base (Oxoid) with addition of 7% of defibrinated sheep blood . Blood plates were incubated at 30 °C during 24 h. Streptomycin thallous acetate actidione agar  was incubated at 21 °C for 48 h for the enumeration of B. thermosphacta. The confirmation of B. thermosphacta was performed by both a catalase and oxidase test . Oxytetracycline-glucose-yeast extract agar  was incubated at 25 °C for 5 days for the enumeration of yeast and moulds. For PCA, OGYE, STAA, Slanetz and Bartley, MRS, M17, and VRBG agar, dilutions were used to prepare pour plates, which were incubated aerobically. For MRS, M17 and VRBG, an overlay of 10 mL was added. For RCA and TSC, dilutions were used to prepare pour plates, which were incubated anaerobically using AnaeroGen 3.5 L (Oxoid) in an airtight jar. For MYP agar, the spread-plating technique was used followed by aerobic incubation. The lower limit for enumeration for all media was 1 log colony forming unit (CFU)/g.Ten grams of each food product sample were subsampled and analyzed. After preparing appropriate dilutions in MRD, the following microbiological enumerations were performed: total psychrotolerant and psychrophilic aerobic (TAC) and anaerobic microbial counts (TANC), psychrotolerant and psychrophilic lactic acid bacteria (LAB), products . For theAfter making appropriate dilutions in MRD, the surface swab samples were plated on PCA for the enumeration of TAC, RCA for the enumeration of TANC and MRS agar for the enumeration of presumptive LAB, following the same incubation period and temperature as mentioned above.w) was measured for each food sample using the AQUALAB 4TE water activity meter . The pH was measured using a pHenomal pH 2100 L in combination with a SF113 electrode .Water activity , total anaerobic count (RCA) and LAB (MRS agar) were used. On the agar media representing the highest decimal dilution showing bacterial growth, five to ten colonies were randomly picked from each of the three types of media, conforming to a median level of 31% of colonies available. Pure cultures were inoculated in brain heart infusion  for cultures originating from PCA and RCA or in MRS broth (Oxoid) for cultures originating from MRS agar, with 15% glycerol , incubated for 2 d at 21 °C and kept at −80 °C. From PCA, RCA and MRS agar, a total of 259, 227 and 197 isolates were collected, respectively.5, PCR fingerprinting was carried out [5 fingerprint clusters, respective totals of 147, 144 and 104 isolates were selected for identification. They were selected as representative isolates of visually defined clusters grouping isolates with the same band patterns. For clusters containing up to three isolates, one representative isolate was selected. A minimum of two isolates was selected for identification of clusters containing four or more isolates. A 1127 bp-region of the 16S rRNA gene was amplified for identification of the selected isolates, as previously described [From each isolate, except for those that could not be cultivated after storage at −80 °C (128 out of 811 isolates), DNA was collected and stored at −20 °C. DNA was extracted according to Strandén et al. . On thesried out . The obtescribed . PCR proescribed .From the slicers, 148 isolates were identified, including 58 isolates from PCA plates, 53 from RCA plates and 37 from MRS agar.g for 3 min to remove potentially inhibiting food matrix material. The resulting supernatant was again centrifuged at 10,000 RCF for 10 min to pellet the bacterial cells. The resulting pellet was frozen using liquid N2 and stored at −80 °C prior to DNA extraction. DNA extraction was performed using the DNeasy mericon Food Kit  following the manufacturer’s instructions. The DNA concentration was measured using the Quantus Fluorometer  in combination with the NanoPhotometer N60 . Amplicon sequencing of the V3–V4 region of the 16S rRNA gene was performed as described by the Illumina protocol and with primers of Klindworth et al. [Ten grams of each sample were suspended into a 1:1 dilution using MRD. This suspension was gently kneaded to allow bacterial cells to detach from the matrix’s surface. Then, the suspension was centrifuged at 200× h et al.  on an IlThe amplicon sequencing dataset was demultiplexed by the sequence provider, and barcodes were clipped off. The processing pipeline was entirely performed in RStudio 1.3.1093. Reads were imported into R and the ShortRead package was used to remove primer sequences . Then, qp-value < 0.05).Analysis of variance  and Tukey’s test were performed using Version 1.3.1093  to verify whether enumerations were significantly different  for cooked ham and cooked chicken were obtained . Tukey’sBacillus cereus, Brochothrix thermosphacta and Enterococcus spp. were rarely above the lower limit of enumeration . Leuconostoc carnosum, although not found in the unsliced log samples, was also relatively well-represented after slicing . At the end of the shelf-life period, species diversity was lower, and L. carnosum was clearly highly abundant . Also always present at the end of the shelf-life period, albeit to a lesser extent, was Lactococcus piscium. The latter species represented 12, 7 and 4% of the PCA, RCA and MRS agar isolates, respectively. In contrast, B. thermosphacta could no longer be recovered.For the cooked ham samples, a total of 425 colonies was identified. In the unsliced log samples, Latilactobacillus sakei was highly abundant throughout the shelf-life period, although this was less clearly demonstrated on PCA  than on RCA  and MRS agar . Carnobacteria also showed a clear presence throughout the shelf-life period, although a successive pattern of different species was found. Carnobacterium inhibens was only found on the unsliced logs  and at day 0 . Carnobacterium maltaromaticum was first identified at day 0, representing 25% of the RCA isolates, and was still present at the end of the shelf-life period . Carnobacterium divergens, on the other hand, was mostly dominant at the end of the shelf-life period, representing 23, 12 and 24% of the PCA, RCA and MRS isolates, respectively. Prior to that, it was only found at day 0 on MRS agar (11% of the isolates).For the cooked chicken samples, a total of 258 isolates was identified at species level. L. carnosum in cooked ham samples and Lb. sakei in cooked chicken samples).The changes in biodiversity indexes between isolates obtained from the unsliced log as well as the samples at day 0 and 28 are summarized in Serratia proteamaculans was identified but represented only 3% of the isolates obtained from RCA.Gram-negative bacteria were rarely present at the end of the shelf-life period. None of the isolates from cooked ham obtained for the samples at day 28 showed any presence of Gram-negative bacteria. For cooked chicken, Enterococcus gilvus and Streptococcus parauberis. Coincidentally, E. gilvus was largely found in the cooked ham log before slicing, while merely two colonies were identified as S. parauberis in the cooked ham samples, both right after slicing. Brochothrix thermosphacta was only identified once in the environmental samples, while this organism was highly present in the product log and right after slicing. Additionally, surface samplings indicated the presence of C. divergens, C. maltaromaticum, L. carnosum, Psychrobacter maritimus, Psychrobacter faecalis and Vagococcus spp., all of which were not or barely found in the unsliced log samples but could be identified during the later stage of the cooked ham storage.Identities of surface sample isolates taken from the cooked ham slicer and the cooked chicken slicer were also determined  and two of the six analyzed cooked chicken samples (22% and 83%).The metabarcoding analysis was performed on all samples of each product type (30 per type). Based on rarefaction curves, 13 cooked ham samples  and 15 cooked chicken samples  were retained for further analysis. The number of reads obtained per sample varied from 9051 to 341,236 reads, with an average of 81,489 reads per sample. Shannon–Wiener diversity and Chao1 richness indices are represented in Photobacterium was consistently found throughout the shelf-life period at a relative abundance of >5%, namely Pseudomonas. While the recovered taxa from the unsliced log samples showed clear similarities, the recovered taxa at day 0 of the two samples seemed more sample-specific with Aeribacillus, Enterococcus and Thermicanus being the most abundant taxa in one of the two analyzed samples and Acinetobacter, Pseudomonas, Photobacterium and Vibrio in the other. At the end of the shelf-life period, the genera Leuconostoc, Serratia and Lactococcus were most often recovered . Other more often recovered genera at D28 were Carnobacterium and Vibrio, each found in two samples.In cooked ham, one genus other than Photobacterium, that were consistently recovered from the cooked chicken samples throughout the shelf-life period were reported as Lactobacillus (mainly found at day 28), and, similar to the cooked ham samples, Pseudomonas (mainly found at day 0). The taxa recovered and their abundance seemed sample-specific in cooked chicken, although similarities within batches could be found. Samples obtained from the unsliced logs in batch 3 and day-0 samples from batch 1 and 3 showed very similar recovered taxa and relative abundances within batches, while the other samples displayed different profiles. At the end of the shelf-life period, Lactobacillus was most often reported , followed by Carnobacterium (which was recovered in two samples at an abundance varying between 10–52%). Brochothrix was found in only one sample, but at the very high abundance of 96%.The genera, besides Acinetobacter, Photobacterium, Pseudomonas, Serratia and Vibrio were recovered in cooked ham samples at day 28, while cooked chicken samples from the same time point were characterized by the presence of Acinetobacter, Photobacterium, Pseudomonas and Psychrobacter.Contrary to culture-dependent results, Gram-negative bacteria were more often recovered through metabarcoding analysis at the end of the shelf-life period. Latilactobacillus sakei, for instance, is known to efficiently reduce the development of Enterobacterales in cooked ham [Lb. sakei was indeed typically dominant throughout the shelf-life period, in agreement with previous findings for MAP cooked poultry products [Lb. sakei was only minimally present in cooked ham, whereas Enterobacterales were detectable in more than half of the investigated samples at the average level of 4 log CFU/g at the end of the shelf-life period. On the other hand, cooked ham samples were typified by the development of high levels of L. carnosum populations towards the end of the shelf-life period, in line with the fact that this is a commonly encountered species in such products [Leuconostoc spp. can be the cause of buttery aroma’s, slime formation, blowing of package and green discoloration [L. carnosum strains are known to display bioprotective potential, suppressing the growth of the background microbiota and limiting metabolite activity of B. thermosphacta in cooked ham environments [Lc. piscium was present, which has been shown to effectively reduce B. thermosphacta in cooked shrimp stored at 8 °C [B. thermosphacta counts at the end of the shelf-life period of cooked ham were lower than for cooked chicken, where the presence of L. carnosum was minimal and Lc. piscium was not found.The microbiota of the two types of MAP cooked charcuterie products that were investigated in the present study were both dominated by LAB, whereas the counts of Gram-negative bacteria were relatively low. This is a common pattern for such products, when oxygen availability is low due to packaging ,16,17,29oked ham , being aoked ham ,32. In cproducts ,14, whilproducts ,34. Hereloration . Beyond ronments . Additio at 8 °C . Be thatCarnobacterium spp. were present in both products, usually throughout the shelf-life period. However, they were never able to manifest themselves to the same degree as was the case for Lb. sakei in cooked chicken or L. carnosum in cooked ham. Although carnobacteria, specifically C. divergens and C. maltaromaticum, are often described as meat spoilers when low-oxygen packaging is applied [Leuconostoc spp. and Lactobacillus spp. (or genera derived from Lactobacillus such as Latilactobacillus) and, therefore, less likely to prevail at temperatures below 12 °C [Carnobacterium inhibens was identified in the unsliced log samples. This species is mostly recognized in relation to Atlantic salmon intestines, while carnobacteria in meat products usually belong to C. divergens and C. maltaromaticum [ applied ,38, theyow 12 °C . An idioomaticum ,40. The Enterococci were only found in the samples derived from cooked ham, specifically at the end of the shelf-life period and in some product log samples. Enterococci can pose spoilage problems in cooked meats due to their ability to survive heating at 60 °C for 30 min ,42. PersLeuconostoc and Lactococcus as the most abundant genera at the end of the shelf-life period, they also revealed some differences and added additional information. For instance, it was shown that most of the genera present, other than Leuconostoc, were more sample dependent than product dependent. In addition, the metabarcoding results only detected Enterococcus once with a significant relative abundance, while they yielded a high abundance of Vibrio, mainly found in the logs and at day 0, but also in some of the cooked ham samples from day 28. Raimondi et al. [Vibrio, which they further identified as V. rumoiensis, as a major component in cooked hams, mainly at the start of the shelf-life period. Vibrio spp. are normally considered marine bacteria, adapted to salty environments and commonly present on seafood products originating from warm water climates [While the metabarcoding results were generally confirmative of the above-mentioned data obtained by the culture-dependent methods, for instance by identifying i et al.  also recclimates . Howeverclimates .Photobacterium was often present in the unsliced logs and at day 0 for both the cooked ham and cooked chicken samples. Photobacteria have been found in meat products before when using culture-independent approaches [Photobacterium that could be relevant for spoilage and have found overlap with the metabolisms of the common meat spoilers Brochothrix and Carnobacterium. Even though Photobacterium was often recovered at the beginning of the shelf-life period, it nonetheless appeared to be outcompeted towards the end.The most revealing finding from the metabarcoding analysis, however, was that proaches ,48,49,50proaches . Numbersproaches . Therefoproaches . Höll etproaches  have prePhotobacterium risk being overlooked. Such microorganisms that are hard or impossible to cultivate with standard procedures can thus be recovered using the metabarcoding approach, resulting in a more complete overview of the product’s microbiota involved. A disadvantage, however, is that a reliable identification can only go down to genus level, and no distinction can be made between viable and non-viable bacteria. Another limit in this study, which affects both used methods, was the low abundance of microbiota present on the logs right before slicing. As enumerations in these types of samples are generally low, less information on the identity of the microbiota that are present can be amassed and certain bacteria can be overlooked when using the culture-dependent method. This is also an issue with the metabarcoding approach, as a high yield of pure DNA is desired after extraction to obtain reliable results, resulting in a large number of samples to be discarded after rarefaction curve analysis.Both the culture-dependent and culture-independent techniques used in this study obviously come with their own set of advantages and disadvantages. One limit of the use of the former methodology is that certain viable organisms will not be cultivable under the conditions used, so that microorganisms such as


Although there is a general assumption that a phenylalanine (Phe)-restricted diet promotes overweight in patients with phenylketonuria (PKU), it is unclear if this presumption is supported by scientific evidence. This systematic review aimed to determine if patients with PKU are at a higher risk of overweight compared to healthy individuals. A literature search was carried out on PubMed, Cochrane Library, and Embase databases. Risk of bias of individual studies was assessed using the Quality Assessment Tool for Observational Cohort and Cross-Sectional Studies, and the quality of the evidence for each outcome was assessed using the NutriGrade scoring system. From 829 articles identified, 15 were included in the systematic review and 12 in the meta-analysis. Body mass index (BMI) was similar between patients with PKU and healthy controls, providing no evidence to support the idea that a Phe-restricted diet is a risk factor for the development of overweight. However, a subgroup of patients with classical PKU had a significantly higher BMI than healthy controls. Given the increasing prevalence of overweight in the general population, patients with PKU require lifelong follow-up, receiving personalised nutritional counselling, with methodical nutritional status monitoring from a multidisciplinary team in inherited metabolic disorders. In phenylketonuria (PKU), the prevalence and patient susceptibility to overweight and obesity has been widely discussed. Several retrospective studies have reported a higher body mass index (BMI) and a higher prevalence of overweight in patients with PKU compared to the normal population ,2,3,4, eThe World Health Organisation (WHO) defines overweight and obesity as abnormal or excessive fat accumulation. This has numerous negative health consequences including cardiovascular diseases, non-insulin-dependent diabetes mellitus, musculoskeletal disorders, pulmonary diseases, and cancer ,13,14.PKU is a rare autosomal recessive inborn error of phenylalanine (Phe) metabolism, and if untreated, can cause severe and irreversible neurological damage . The maiAdequate dietary energy is essential to maintain blood Phe stability, particularly in patients with classical PKU, by promoting anabolism and counteracting catabolism, which increases blood Phe levels . Energy Due to concerns over increasing obesity in PKU, industry has reformulated many of their PS, adding less CHO to their products . FurtherThis lack of consensus highlights the need to assess the quality of evidence that reports the prevalence of overweight and obesity in PKU. This systematic review aims to (1) determine if patients with PKU are at a higher risk of overweight compared to healthy individuals, and to (2) understand the association between early exposure to Phe restriction and overweight in patients with PKU.This systematic review with meta-analysis was developed according to preferred reporting items for systematic reviews and meta-analyses (PRISMA) statement  and the Inclusion and exclusion criteria were defined according to the PECO  strategy. Inclusion criteria: (1) patients with PKU (Population) on a Phe-restricted diet (Exposure) and followed up at a PKU centre; (2) studies included healthy controls (Comparator); (3) reported anthropometric measures or prevalence of overweight (Outcome); (4) published as a full paper; and (5) included only randomised controlled trials (RCTs), non-randomised controlled trials (non-RCTs), or observational  studies.Non-human studies, review articles, systematic reviews, meta-analysis, letters, conference abstracts, case reports, case series, position papers, and authors’ replies were excluded. Only studies published in English were included.A literature search was carried out on PubMed, The Cochrane Library, and Embase databases on the 16 January 2020. Both medical subject headings (MeSH or Emtree) and text words related to overweight, obesity, and PKU were used. The PubMed search strategy was converted to search in other databases as described in detail in the All articles identified in the search were included in the screening process and duplicates excluded. Two independent reviewers (A.M. and J.C.R.) screened the titles and abstracts of the articles for relevance, and full-text articles were reviewed when title and abstract did not provide enough information. Once potentially relevant studies were identified, full-text articles were then assessed for eligibility according to previously established criteria. The reference lists of the included articles were screened to ensure that no relevant studies were missed.Data items were extracted by two authors (C.R. and A.P.) using a standard data extraction form. For each study, first author, year of publication, country of origin, study design, sample characteristics, methods, and outcomes were extracted. In cases where information was missing or incomplete, the correspondence authors were contacted requesting further information.Risk of bias of individual studies was assessed by two independent reviewers (C.R. and A.P.) using the National Institutes of Health (NIH) Quality Assessment Tool for Observational Cohort and Cross-Sectional Studies . The folStandardised mean difference (SMD) was used as an effect measure for the continuous variable ‘BMI’. Odds ratio (OR) was used as an effect measure for the dichotomous variable ‘prevalence of overweight’. The SMD and OR were converted to a common metric and then combined across studies. A sensitivity analysis was performed to compare the meta-analysis results with and without the converted study . Effect 2 tests were used to assess heterogeneity. According to the Cochrane guidelines [2 values were interpreted as follows: 0% to 40% might not be important; 30% to 60% may represent moderate heterogeneity; 50% to 90% may represent substantial heterogeneity; 75% to 100% represent considerable heterogeneity.The Cochran’s Q (significance level of 0.1) and Iidelines , the I2 Mean BMI from Evans et al.  was calcp-value < 0.05 was considered as statistically significant. Statistical analysis was performed using Review Manager (RevMan), version 5.4, The Cochrane Collaboration, 2020.Pooled estimates were computed and weighted using generic inverse-variance and random-effect modelling. A Funnel plots were used to assess evidence of publication bias. Quality assessment of the evidence for each outcome was performed by two independent authors (C.R. and A.P.) using the NutriGrade scoring system . The metn = 41)  . Two stuA summary of the main characteristics of included studies is given in The methods used to assess dietary intake varied between the included studies and are given in Patients with PKU were compared to 593 healthy controls, 455 of which were included in the meta-analysis. Healthy controls were from both genders, and the age range varied from 1 month to 50 years. The majority were matched for age and gender, and some studies included family relatives, friends, or healthy individuals with similar characteristics in the PKU group.Most studies examined the association between a Phe-restricted diet and BMI ,36,37,38From 15 studies included in the qualitative synthesis, 12 did not find significant differences in BMI and overweight prevalence between patients with PKU on a Phe-restricted diet, compared with healthy controls ,39,40,41On the basis of the NutriGrade assessment , we founUsing the NIH Quality Assessment Tool for Observational Cohort and Cross-Sectional Studies, we found that 4 studies were assessed as fair with moderate risk of bias ,30,31,34Visual inspection of the funnel plot did not indicate substantial asymmetry .p = 0.14; I2 = 27%, p = 0.18; In the 12 studies included in the meta-analysis, there were no differences for BMI of patients with PKU compared with healthy controls . Studies assessed as poor with high risk of bias [p = 0.02; I2 = 1%, p = 0.42).A subgroup analysis was conducted according to the risk of bias for each study . Studies of bias ,36,37,38p = 0.32; I2 = 35%, p = 0.15). Moreover, the subgroup of studies including both early and late diagnosed patients found no differences between patients with PKU and healthy controls . There were no statistical differences between the two subgroups (p = 0.73).Three studies included late diagnosed patients in their samples ,37,38, ap = 0.15), and a higher heterogeneity in the subgroup of studies that included all age groups (I2 = 61%). The subgroup that included adults only had one study [The studies included in the meta-analysis covered a wide patient age. We performed a subgroup analysis  comparinne study  that ideFour studies included patients prescribed BH4 in their patient cohort ,25,30,37p = 0.01; I2 = 0%, p = 0.97). Studies that included only patients on a Phe-restricted diet [p = 0.74; I2 = 35%, p = 0.15).Studies that included some patients with PKU treated with diet and BH4 ,25,30,37ted diet ,35,36,38Four studies in the meta-analysis included only patients with classical PKU ,18,26,32Several authors of the included studies provided individual participant data, including disease severity ,34,35,36p = 0.02; I2 = 31%, p = 0.17).We found that patients with classical PKU had a significantly higher BMI than healthy controls .To reject the hypothesis that this result was due to the removal of the three studies, whose individual participant data is unknown, we performed the first meta-analysis  without Only six studies provided adequate information to establish a comparison on sex, which limits the subsequent interpretation of its effect on overweight. However, when comparing females with PKU and healthy females, all studies found a trend towards a higher BMI in females with PKU .2 = 58%, p = 0.05); thus, we were unable to present accurate data on metabolic control.We tried to explore the association between metabolic control and BMI. However, only five studies provided information on metabolic control, and the comparison between patients with poor metabolic control and healthy controls  had subsThe methods used to assess body fat percentage across studies were different. This led to a heterogeneous overall result, rendering it unfeasible to present and compare body fat results .To the best of our knowledge, this is the first systematic review with meta-analysis evaluating the association between a Phe-restricted diet and overweight and obesity in patients with PKU. We pooled data from 12 observational studies for the meta-analysis and found no differences between patients with PKU and healthy controls for BMI. The pooled data included diverse patient phenotypes with variable Phe-restriction, with dissimilar contributions from the PS and SLPFs to total protein and energy intake ,42,43. OHowever, patients with classical PKU had a significantly higher BMI than healthy controls. This observation resulted from nine studies, including only patients with classical PKU and studies whose authors provided additional individual participant data, although these results should be considered with caution. One plausible explanation is that more calories may be given to patients with classical PKU in order to prevent catabolism that causes higher blood Phe levels. This may lead to the development of overweight.Among the studies included in qualitative synthesis, 4 studies had a moderate risk of bias and 11 had a high risk of bias using the NIH Quality Assessment Tool. The subgroup of studies with moderate risk of bias did not find a higher BMI in patients with PKU. In contrast, studies assessed as poor due to their methodological flaws found a significantly higher BMI in patients with PKU compared to healthy controls. Therefore, this work highlights the fragility of the evidence supporting the idea that a Phe-restricted diet promotes overweight and indicates the need for controlled studies with improved methodology and comprehensive data collection.Three of the seven most common flaws observed in the studies were limited description of the study population using demographics (who), location (where), and time period (when) (question 2 of the NIH tool) ,39,40,41Eleven studies were cross-sectional ,38,39,40For the different levels of exposure assessment (question 8 of the NIH tool), from the 10 studies that included patients with different phenotypes, the use of BH4 with a relaxed Phe-restriction or patients who were late diagnosed with PKU, only five studies considered these factors ,32,34,35In addition, between 20 and 50% of patients with PKU are responsive to the synthetic form of the cofactor (BH4), meaning that a less restricted diet is followed. Evidence suggests that 51% of patients on BH4 therapy completely stop PS intake . In our Of the 12 studies included in the meta-analysis, 4 did not assess patients’ dietary intake ,35,37,38We also tried to determine if there was an association between patients’ BMI and metabolic control (which may reflect patients’ exposure to the Phe-restricted diet). However, most of the studies did not report patients’ BMI, nor its comparison with metabolic control. In the literature, some studies have found a positive correlation between mean Phe levels and BMI ,36,48, aMost of the included studies did not adjust for key prognostic variables, such as physical activity, family history, socioeconomic status, parents’ weight, and epigenetics, among other determinant factors that may be associated with overweight.Finally, none of the included studies considered the regular follow-up of patients by a nutritionist. Nutritionists play a crucial role in monitoring the patient’s weight while ensuring they meet their complex dietary needs . ConsequSeveral limitations in this systematic review should be acknowledged. First, our systematic review included observational studies only. Observational evidence usually provides lower strength evidence than RCTs, due to confounding variables. Nevertheless, RCTs addressing our question have not been conducted, which is unsurprising, given that PKU is a rare disease and the exposure to an unrestricted Phe-diet is clinical and ethically unacceptable. In addition, there was large heterogeneity in the design of observational studies and in the reporting of results.The diversity of the study populations also contributes to the heterogeneity of the results. For instance, some studies included patients with different disease severities, with variable degrees of Phe-restriction, being diagnosed early and later on, patients on BH4 treatment, and patients with poor metabolic control. Additionally, patients had a wide age range.The Phe-restricted diet was not always well defined: not all studies reported patients’ dietary intake, and some studies did not assess it.In relation to the comparator, we did not define any inclusion criteria for healthy controls. Most of them were matched for age and gender only, and the number of controls included in our work was less than the number of patients with PKU.Regarding the outcome, one study  only preFinally, most of the included studies had a high risk of bias according to the NIH tool. On the basis of the NutriGrade assessment, we found that the quality of the meta-analysis comparing all patients with PKU to controls was ‘low’, and the quality of the meta-analysis comparing patients with classical PKU to controls was ‘very low’.In order to strengthen the conclusions of our systematic review with meta-analysis, we used the best methodology, namely, (1) following the PRISMA guidelines and registering on the PROSPERO database—studies that do appear to be of higher quality ,52; (2) As the study of risk factors is based on comparisons between exposed and unexposed individuals , only stFinally, our systematic review provides a clear overview of the available evidence on the topic overweight and PKU and will be useful in guideline development. It also identifies the main flaws and pitfalls that should be avoided when designing novel studies to address this question in the future.We found no differences between patients with PKU and healthy controls in BMI. Thus, there is no evidence to support the concept of Phe-restricted diet as a risk factor for the development of overweight. However, a subgroup of patients with classical PKU had a significantly higher BMI than healthy controls. In addition, studies assessed as poor with high risk of bias and studies that included both diet-treated and BH4-treated patients found a significantly higher BMI in patients with PKU compared to healthy controls.Given the increasing prevalence of overweight in the general population, patients with PKU should remain in long-term follow-up, receiving personalised nutritional advice with systematic nutritional status monitoring by a multidisciplinary team in inherited metabolic disorders. This is essential to prevent overweight, obesity, and its related comorbidities.Future studies with improved methodology are needed to properly address this question and to help in guiding the clinical practice of health professionals.