This study aimed to evaluate the efficacy of the 1-minute Sit to Stand test (1MSTS) to determine physical activity capacities for patients with chronic disease.2max). We considered two groups of patients based on the results of the functional evaluation of exercise performance: (1) No limitation in exercise performance  and (2) limited exercise performance . All patients also received an 1MSTS on the same day.For this cross-sectional study, we studied fifty patients with chronic disease and no beta-blocker treatment. They systematically performed a cardiopulmonary exercise test to determine maximal oxygen uptake (V'O2max measured during the exercise test and 1MSTS repetitions displayed a positive slope [r = 0.401 (95% CI 0.114–625)].We found 22 (44.0%) patients with normal exercise capacity and 28 (56.0%) patients with limited exercise performance. In this sample, mean 1MSTS repetitions were 27.1 (7.1) and 25.2 (8.7), respectively. There were no significant differences between the two groups for repetition and Borg Scale end test. The correlation between V'O2max for patients with chronic disease. 1MSTS did not permit the precise determination of physical activity capacities in this sample.This study demonstrated a moderate relationship between 1MSTS and V'O We classified the patient's referral department categories behind the APA program into four groups: Obesity, Rheumatology and Others. A maximum cardiopulmonary exercise test was carried out on all included patients.2max) ≥80% of the reference value] and (2) limitation of exercise performance capacities defined by a reduced V'O2max . We used Wasserman equation for reference values . For each patient, a progressively increasing work rate protocol was designed  so that volitional exhaustion is reached following 8–12 min of exercise. Respiratory and metabolic variables  were measured breath-by-breath through a mask connected to a pneumotachograph and O2 and CO2 analyzers . Criteria for the achievement of V'O2max were HR >90% of the maximal reference value HR (210–0.65 × age), respiratory exchange ratio [(RER) = V'CO2/V'O2] > 1.1 or V'O2 plateau . We used Pearson's correlation to analyze the relationship between the 1MSTS test and V'O2max. For interpreting Pearson's correlation we used this scale: 0.00–0.10 Negligible correlation, 0.10–0.39 Weak correlation, 0.40–0.69 Moderate correlation, 0.70–0.89 Strong correlation, 0.90–1.00 Very strong correlation  or with the median and interquartile range (IQR). The chi-square test or Fisher's exact test with, if necessary, the exact calculation of Fisher, was used for the ordinal or nominal data analysis. We used the student All data used were obtained from the medical records. No supplementary examination was necessary for patients to meet the inclusion criteria. This study is registered with the Information Technology and Freedoms Commission for the University Hospital of Nancy (file number: 2021PI191) and on Clinicaltrials.gov (NCT05146544) and was designed in accordance with the general ethical principles outlined in the Declaration of Helsinki. The protocol of this study was approved by the Information Technology and Freedoms Commission. All patients gave their consent for the use of their medical data during the period they received medical care at the University Hospital. The Ethics Committee waived the requirement of written informed consent for participation.2. We found 28 (56.0%) patients with limited exercise capacities and 22 (44.0%) patients with normal exercise performance. There were no significant differences between the two groups for demographic and clinical characteristics, except for gender (p = 0.028) (p = 0.021 (p = 0.056 for gender).Fifty patients were included in the study, 41 (82%) of whom were women. Mean age was 44.1 (±10.9) years with a mean BMI of 36.5 (±9.1) kg/m= 0.028) . But mulp = 0.386). There were no significant differences between the two groups for HR and Borg Scale after the test .In this sample, for normal and limited exercise patients, the 1MSTS mean repetitions were 27.1 (7.1) and 25.2 (8.7), respectively, with no significant difference (2max measured during the exercise test and 1MSTS repetition by exercise performance level showed a positive slope [r = 0.401 (95% CI 0.114–625)] (The correlation between V'O14–625)] .2max in patients suffering from chronic disease following APA. Indeed, it was not possible to ascertain exercise aerobic performance capacity with the 1MSTS. The 1MSTS test was developed in 2002 to assess the aerobic exercise capacities of patients with chronic disease  unlike our result which based on absolute repetition number  . Indeed, the influence of methods and mental state influence performance. On the other hand, in our population, VO2max is a faithful reflection of exercise capacities.In the case of elite athletes, the V'Or = 0.8) in healthy subjects aged 55–70, with no chronic disease, while the association is weaker in patients presenting a Chronic Obstructive Pulmonary Disease (r = 0.50). It is probably the reason for the moderate correlation observed in our sample of patients with capacity limitations.The first publication referring to the Sit to Stand test aimed only at assessing the capacity of the lower limb extensor muscles .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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A survey of California (CA) dairies was performed in spring 2018 to characterize antimicrobial stewardship practices, antimicrobial drug (AMD) use, and health management of adult cows on CA dairies since the implementation of the Veterinary Feed Directive (VFD) and the CA Senate Bill 27 (SB 27). Effective January 1, 2017, the U.S. Food and Drug Administration (FDA) implemented regulatory changes requiring veterinary oversight for therapeutic uses of medically-important antimicrobial drugs (MIADs) administered in feed (VFD) and water (veterinary prescription). Similarly, effective January 1, 2018, the CA legislature enacted California Food and Agricultural Code (FAC) 14400–14408, formerly known as Senate Bill 27 (SB 27) requiring veterinary prescriptions for all other dosage forms of MIADs.The questionnaire consisted of 43 questions partitioned into three sections to assess herd information, management practices, and AMD use and perspectives. The questionnaire was mailed to 1,282 grade A licensed dairies in CA and 149 responses (11.6%) were collected from 19 counties across the three defined regions of CA: Northern CA (NCA), Northern San Joaquin Valley (NSJV), and Greater Southern CA (GSCA).Most dairies reported treating all dry cows with intramammary AMD and/or teat sealant at the end of a lactation (87.2%). In 92.3% of dairies, producers relied on the veterinarian for information about AMD used to treat cows. Treatment duration for cows treated with AMD was based on the drug manufacturer’s label and veterinarian’s instructions in most dairies (98.6%). Most respondents to the survey confirmed having a valid veterinarian-client-patient-relationship (VCPR) for their dairies (91.7%), participated in animal welfare audit programs (81.8%) and dairy quality assurance programs (52.9%). Approximately 98.6% respondents were aware that all uses of MIADs in livestock required a veterinary feed directive (VFD) or prescription and are no longer sold over-the-counter (OTC) in CA since January 1, 2018. Multiple factor analysis (MFA) was performed and identified seven components composed of 21 variables (questions) that explained 99.7% of the total variance in the data. Hierarchical cluster analysis on the principal coordinates of the MFA based on conventional dairy survey responses identified two clusters characterized as large conventional dairies  and mid-sized conventional dairies (median herd size: 715 cows) mostly in GSCA and NSJV. The organic dairies grouped into a single cluster of median herd size of 325 cows mostly in NCA.The survey results contribute to the knowledge of AMD use and antimicrobial stewardship practices on CA dairies since the implementation of the SB 27 and VFD laws and provide useful information for future evaluation of resistance-related risk in adult cows. California (CA) is the leading dairy producing state in the United States, with over 1.7 million dairy cows producing 18.5 percent of the nation’s milk supply on 1,331 dairies . MaintaiThe development of AMR is a complex multifactorial process driven by numerous factors . Exposurst 2017  in June 2015 announced stricter requirements for administering medically important antimicrobial drugs (MIADs) to animals through feed and water. The law, known as the Veterinary Feed Directive (VFD), mandates supervision by a licensed veterinarian for any use of MIADs in animal feed as of January 1st 2017 . Califorst 2017 . The FAC.A list of all 1,282 licensed, grade A dairies in CA in 2017 provided by CDFA served as a sampling frame. A grade A dairy produces milk used in any products intended for consumption in fluid form . A confidential identification number was randomly assigned to all dairies on the list. Each dairy was uniquely identified by this number during analysis to maintain confidentiality. Dairies were mailed a survey packet containing a copy of the questionnaire, a postage-paid addressed business reply envelope, and an information cover letter. The cover letter introduced the dairy producers to the survey and provided an explanation that the term antimicrobial included antibiotics, drugs that are naturally produced by other microorganisms and that can kill or inhibit the growth of other microorganisms, and also synthetic chemicals such as sulfonamides. Producers were instructed that for the purpose of this survey questions will refer to all AMDs as antibiotics regardless of their origin. The estimated time for producers to complete the survey was 30 minutes. To improve questionnaire response rate, each dairy was mailed a reminder card two weeks after the initial questionnaire package. A second and third copy of the questionnaire, each followed by a reminder card, were similarly mailed a month and six months later, respectively, to dairies that had not previously responded. All mailings were completed between April and September 2018.This section focused on gathering baseline information about the responding dairy. Questions included: the respondent’s role on the dairy, the county where the dairy was located, the breeds of milking cows, the number of milking cows (herd size), the annual rolling herd average for milk production (RHA), the previous month’s average bulk tank somatic cell count in the herd (BTSCC), and if the dairy was a USDA certified organic milk producer.The questions in this section addressed the farms’ dry-off protocols, types of AMD used at dry-off, disease prevention, and vaccinations used in lactating cows. Additional questions highlighted sources of information on AMD used to treat sick cows, who made the decisions on what AMD are stocked or used to treat sick cows, and whether producers had written or computerized animal health protocols or used a drug inventory log on their dairies. Other questions were considered indicators for AMD usage on the dairy, such as how and which AMD treatment information is tracked, if the dairy tracked AMD withdrawal intervals for treated cows, the farm’s disease diagnosis and management practices, and if the dairy had a working relationship with a veterinarian or veterinary practice.The final section posed questions relating to the respondent’s participation in animal welfare audit programs and/or dairy quality assurance programs, the respondent’s familiarity with the FDA’s “medically important antimicrobial drugs (MIADs)” term, and awareness that all MIADs required a prescription and were no longer sold OTC in CA after January 1, 2018. Additional questions addressed differences that may have occurred in the periods before and after January 1, 2018 in: the use of OTC and prescription AMD on the dairy, changes made regarding previously available OTC AMD, and changes in the dairy’s AMD drug costs. Similarly, questions inquired about differences before and after January 1, 2018, in usage of alternatives to AMD , any changes in management to prevent a disease outbreak or disease spread, and changes noted in animal health on the dairy. The final questions in this section considered how important survey respondents ranked certain AMD use stewardship practices on the dairy, and their level of agreement on statements relating to AMR.Data on the counties where the dairies were located were categorized into three regions —NorthernProportions and their standard error (SE) were computed for categorical and ordinal variables. Mean and SE were computed for continuous variables. Confidence intervals for proportions were calculated using the normal distribution approximation method . DescripMultiple Factor Analysis (MFA), is a dimensionality reduction approach used for the analysis of several groups of quantitative and/or categorical variables . The MFAA total of 149 (11.6%) survey responses from 19 of the 31 dairy producing counties in CA were received through the three mailings . FollowiOverall, 87.2% of the respondents blanket treated cows with intramammary (IMM) AMD and/or teat sealant at dry-off . ApproxiClostridium, pinkeye, and footrot, respectively.The survey showed that 57.7% of respondents provided separate fresh pens, other than the hospital pens, for recently calved cows; 93.9% of the responding dairies’ management included harvesting colostrum from fresh cows to feed newborn calves . ApproxiThe majority of respondents (92.3%) indicated they relied on a veterinarian or a veterinarian in addition to other sources for information about AMD used to treat their cows . Other sApproximately one third (38.5%) of dairies included a veterinarian in their decision on which AMD is used to treat sick cows, otherwise, decisions were made by the herd manager (68.4% ± 3.8%), dairy owner (54.3% ± 4.0%), treatment crew (16.1% ± 3.0%), and/or milker (0.7% ± 0.6%), (Note: percentages do not add up to 100% due to multiple selection). For producers who did not include a veterinarian in the decision on which AMD is used to treat sick cows, analysis showed that the majority (91.7% ± 2.9%) had a VCPR for their dairies, while the others had a veterinarian observe or discuss the health of their cows (7.1% ± 2.7%) and served as a source of information about AMD used to treat their cows (1.1% ± 1.1%).Three quarters (75.0% ± 3.5%) of the respondents indicated having written/computerized animal health protocols . In addiApproximately 85.4% of the respondents trained their treatment crew members or milkers on treatment protocols for sick cows. A veterinarian was involved in training the treatment crew members or milkers on treatment protocols for sick cows for approximately half (52.7%) of the respondents. Health protocols were reviewed or revised once to twice a year for 60.7% of dairies, when a new product was added for 22.5% of dairies, and every few years for 16.6% of study dairies. Three quarters (75.0%) of the study dairies included veterinarians when reviewing their animal health protocols; the remaining quarter of respondents involved the dairy owner (73.0% ± 4.3%), herd manager (43.2% ± 4.8%), and nutritionist (6.7% ± 2.4%) in protocol review.Approximately half (50.3%) of the surveyed dairies did not keep a drug inventory log for their dairies . For theSelection of a second AMD to treat a sick animal if the first was not successful was based on veterinarian input for approximately two thirds (63.2% ± 4.3%) of the respondents . The remA majority, four-fifths, (80.3% ± 3.3%) of the surveyed dairies confirmed tracking/recording milk, meat, or milk and meat withdrawal intervals along with any of: date, dose, and route of AMD treatment . The majThe statewide mastitis treatment incidence estimated using the survey responses was 2.9 ± 0.3 mastitis cases/100 milking cow months . The repThe statewide metritis treatment incidence estimated using the survey responses was 1.9 ± 0.2 metritis cases/100 milking cow months . The repThe statewide lameness treatment incidence estimated using the survey responses was 1.5 ± 0.2 lameness cases/100 milking cow months . The repThe statewide pneumonia treatment incidence estimated using the survey responses was 0.3 ± 0.1 pneumonia cases/100 milking cow months . The repThe statewide postoperative care treatment incidence estimated using the survey responses was 0.04 ± 0.01 postoperative care cases/100 milking cow months . The rep® Program. Personnel on half (52.9% ± 4.2%) of surveyed dairies participated in a dairy quality assurance program in the previous year. Approximately 68.1% ± 3.9% of the survey dairies were familiar with the FDA “MIADs” term, i.e. they recognized that MIADs are further classified as important, highly important or critically important drugs, and are available for livestock only via prescription or VFD pursuant to a VCPR with a licensed veterinarian. The majority (98.6% ± 9.8%) of surveyed dairies were aware that since January 1, 2018, all uses of MIADs in livestock, including injectable AMD such as Penicillin Injectable, Liquamycin® LA 200 (oxytetracycline), and Tylan® Injection (tylosin), and boluses, such as Supra Sulfa® III or Sustain III (sulfamethazine), required a prescription and were no longer sold OTC in CA.Approximately four-fifths (81.8% ± 3.1%) of surveyed dairies participated in animal welfare audit programs . The aniThe majority (85.2% ± 2.9%) of survey respondents confirmed the use of OTC and/or prescription AMD on their dairies prior to January 1, 2018. Approximately half (56.0% ± 4.1%) of the studied dairies had not made any changes since January 1, 2018 compared to 2017 with regard to injectable and/or IMM AMD that were previously available OTC. Approximately 43.9% ± 4.1% made changes which included: treating fewer animals with AMD (69.3% ± 5.8%); discontinued one or more AMD (43.5% ± 6.2%); use same AMD but the dosage and duration decreased (19.3% ± 5.0%) or increased (3.2% ± 2.2%); added one or more AMD (1.6% ± 1.5%). No respondents indicated treating more animals with AMD after January 1, 2018 compared to 2017. A quarter (26.5% ± 3.6%) of the study dairies confirmed usage or increased use of alternatives to AMD (vitamins (66.6% ± 8.2%), minerals (60.6% ± 8.5%), herbal remedies (36.3% ± 8.3%), and vaccines (36.3% ± 8.3%)) since January 2018. Approximately 40.8% ± 4.1% confirmed making changes in management to prevent disease outbreaks or spread since January 2018; specifically, these changes included: improvements in vaccination programs to prevent disease (83.0% ± 5.1%); quarantine purchased/returning animals from offsite locations  (20.7% ± 5.5%); improved biosecurity  (11.3% ± 4.3%); and pre-purchase testing of animals before adding to the herd (5.6% ± 3.1%). More than a quarter (28.6% ± 3.7%) of surveyed dairies confirmed decreased AMD costs on their dairies since January 2018 compared to 2017 and earlier. Additionally, 24.4% ± 3.5% and 46.8% ± 4.1% of the dairies confirmed an increase, and no change, respectively, in costs of AMD on their dairies since January 2018 compared to 2017 and earlier. Furthermore, a third (31.6% ± 3.9%) of the surveyed dairies confirmed better animal health on their dairies since January 2018 compared to 2017 and earlier. Only 7.3% ± 2.2% confirmed worse animal health, while 61.0% ± 4.1% confirmed no change in animal health since January 2018 compared to 2017 and earlier.The percentage of respondents that blanket treated cows with IMM AMD and/or teat sealant at dry-off were comparable across herd sizes; however, respondents whose average number of milking cows were 3,500 or more exclusively blanket treated cows at dry-off . ConversThe first two principal component dimensions of the MFA explained approximately 14.02% of variability in the survey responses, 9.59% for the first dimension and 4.43% for the second. Seven components with correlation coefficient ≥ 0.4 were identified and retained . On the The profiles of the conventional dairy clusters are described in Most of the cluster C1 dairies (95.1%) implemented blanket treatment of dry cows with IMM AMD and teat sealant, compared to dairies in cluster C2 (80.0%). Notably, dairies in cluster C1 used multiple AMD classes  for dry cow treatment, dairies in cluster C2 used mainly cephalosporins. Approximately two thirds of the dairies in cluster C1 tracked both AMD treatments given to cows (69.7%) and withdrawal periods for AMD treated cows (66.4%) using a computer, in addition to other methods. The reverse was true for dairies in cluster C2, approximately two fifths (60.0%) do not track AMD treatments and two thirds (77.8%) do not track AMD withdrawal periods using a computer.For mastitis treatment, dairies in cluster C1 relied mostly on clinical signs and laboratory testing, and tended to treat cows while culture results, if any, were pending. Dairies in cluster C2 relied mostly on clinical signs. Most C1 dairies (98.4%) treat mastitis with AMD, compared to 75.0% in C2. For metritis treatment, dairies in cluster C2 totally relied on oral/injectable AMD, while cluster C1 relied on both oral/injectable AMD and intrauterine treatment. Similarly, dairies in cluster C2 managed lameness in adult cattle using mostly hoof treatment and less of oral/injectable AMD compared to dairies in C1.Any use of antibiotics may result in infections more difficult to treat in the future”, whereas, fewer dairies in cluster C1 (11.6%) agreed with same statement.Most dairies in cluster C1 (97.5%) strongly agreed that administration of appropriate AMD, dose, route and duration are important AMD stewardship practices compared to less in C2 (80.0%). However, responding to questions relating to AMD resistance, more dairies in cluster C2 (40.0%) strongly agreed with the statement “All the survey’s organic dairies formed a single cluster characterized by multiple breeds with Holstein and Jersey making up 37.5% and 12.5%, respectively and the remaining majority being other breeds. The dairies in the cluster were predominantly in NCA (81.2%) with three respondent in GSCA (18.8%), had a median herd size of 325 milking cows, and a median annual rolling herd average of 8,235 kg per cow. Two organic dairy (12.5%) reported use of blanket treatment of dry-cows depending on the season with an external teat sealant, one organic dairy (6.3%) reported use of blanket treatment of dry-cows with organic intramammary infusion . One organic dairy (6.3%) reported use of selective dry-cow treatment with an organic intramammary infusion (Phyto-Mast), and one other dairy reported use of selective dry-cow treatment with external teat sealant depending on season and mastitis history. Approximately two thirds (60.0%) of the organic dairies reported use of abnormal milk and sample testing at a milk quality laboratory as basis for mastitis treatment decisions, the remaining relied on abnormal milk findings only. Of the organic dairy respondents, 18.8% indicated treatment of mastitis with AMD, while 50% reported treatment of metritis with intrauterine bolus, and 100% reported treatment of lameness with bolus or injection of AMD and hoof treatment. Few organic dairies in the cluster used computers to track AMD treatments (41.7%) and withdrawal periods (37.5%). Three fifths (60.0%) of the survey’s organic dairies indicated use of OTC AMD, or OTC and prescription AMD prior to January 1st 2018. The remaining organic dairy responses (40.0%) indicated no OTC AMD, prescription but no OTC AMD, neither OTC nor prescription, or no response.Most of the organic respondents (76.9%) identified the following AMD stewardship practices as very important: administration of the appropriate dose, route and duration of AMD; good record keeping of treatments and treatment dates; and observing withdrawal periods and drug residue avoidance. However, on a statement that any use of AMD may result in infections more difficult to treat in future, 38.5% of the survey’s organic dairies agreed or strongly agreed, 38.5% disagreed or strongly disagreed, while 23.0% were neutral.The purpose of the current study was to explore the antimicrobial stewardship practices, AMD use and health management practices in adult cows on CA dairies following the passage of California SB 27 and FDA’s VFD rules on the judicious use of MIADs in food producing animals. While the current survey achieved an 11.6% response rate, the responses represented 19 (61%) of the dairy producing counties in CA, responsible for over 94.9% of total CA milk production. Producers who participated in our survey were distributed across the three milk sheds in CA, specifically 14% in NCA, 39% in NSJV, and 47% in GSCA. The survey respondent’s distribution was similar to the distribution of dairy operations across CA published by the CDFA, California Dairy Statistics 2017  which reAdditional survey findings categorized CA dairy producers into three clusters characterized by demographic parameters, dry-off protocol, disease management, and AMD stewardship practices. Our findings identified important management practices, such as dry cow treatment practices, cow health protocols and treatment practices, where extension and outreach efforts should focus on improving antimicrobial stewardship. One of the goals of such extension education is to introduce and reinforce preventive measures that may reduce the use of AMDs on dairies, including vaccines, supplements, and preventive measures, such as internal teat sealants at dry-off and hoof trimming . Other g®, Tomorrow®) class of AMD in dry cow treatment, 33.6% used AMD containing penicillins , while 14.4% used penicillin-aminoglycoside combinations (Quartermaster®). Dry cow treatment statistics based on the NAHMS 2014 dairy study showed comparable estimate for the use of cephalosporins (63.3%) on Western US dairy operations , while 34.3% used Spectramast® . The contrast was seen with use of AMD containing penicillins on dry-off treatment which was reportedly greater in our study 33.6% compared to the 26.8%  in NAHMS 2014 Dairy study; and with Quartermaster® which was less in our study (14.4% compared to 24.0% in NAHMS 2014 Dairy study)  treated all end-of-lactation cows presenting for dry-off with IMM AMD and/or teat sealants. About half (49.6%) of the respondents used IMM AMD, 44.1% used both IMM AMD and teat sealant, while 6.3% used only teat sealant at dry-off. The USDA-National Animal Health Monitoring System 2014 Dairy Study, , showed y study) . Severaly study) . The pray period . Howevery period . Recent y period . Some coy period . Selectiy period . HoweverMost dairy producers in the survey vaccinated lactating cows to prevent mastitis due to coliform bacteria (85.6%), respiratory disease (87.0%), and abortion and infertility (73.0%). Furthermore, 95.3% of the study dairies administered at least one type of vaccine to lactating cows for disease prevention. This percentage is higher than the 73.8% of dairy operations that administered any vaccine to cows for disease prevention reported by Overall, 92.3% of the study dairy producers relied on a veterinarian only or veterinarian and other sources for information regarding AMD to use for treatment of adult cows. Half (49.3%) of the dairies included a veterinarian in their decision on which oral and injectable AMD are purchased and stocked, while 37.9% included a veterinarian in their decision on which AMD is used to treat sick cows. These proportions are consistent with the In our study, 75.0% of dairies confirmed having written/computerized animal health protocols for cows, mainly developed with veterinarian inputs for both treatment and preventative therapeutic uses. In a 2007 study aimed at determining South Carolina (SC) dairy farmers’ knowledge on the importance of prudent antibiotic use on dairy cows, Drug inventory and treatment records help dairy producers track drug use and treated cows. Approximately half of the surveyed dairies reported not keeping a drug inventory log on their dairies, information which otherwise may help the producers monitor drug use on the dairy over time, track availability and stocking of drugs to avoid shortages or waste due to their expiration, and prevent accidental administration of expired drugs. A survey of dairies in Pennsylvania found only 50% of the dairies kept written or computerized records of antibiotic treatment that could be verified . Other iAt least 36.8% of our survey respondents relied on bacterial culture and antibiotic sensitivity results in choosing a second antibiotic drug to treat a sick animal if the first was not successful. In addition to consulting with a veterinarian, laboratory testing may be a better approach after a treatment failure to confirm the bacterial etiology or its antimicrobial sensitivity if the diagnosis was correct.Most (80.3%) of the dairies in our survey confirmed tracking/recording milk, meat, or milk and meat withdrawal intervals. According to NAHMS, more than 90.0% of U.S. dairy operations administered drugs that required a milk withdrawal period or a milk and meat withdrawal period . The NatAccording to the survey, the number one reason for AMD use on CA dairies was for mastitis treatment, followed by metritis, lameness, pneumonia, and postoperative care; similar to the report by According to NAHMS 2014 dairy study , 58.8%, The basic elements of a VCPR are that the veterinarian agreed to assume the responsibility for the animal’s health and the producer agreed to comply with the veterinarian’s instructions; the veterinarian has sufficient knowledge of the animals to make a general diagnosis and assumes responsibility for follow-up care, including adverse reactions and/or treatment failures; and the veterinarian maintains all animal records (16 CCR 2032.1). Most dairies (92%) in our survey confirmed having a valid VCPR for their dairies. Further analysis of the survey questions revealed that the 8.0% (12/143) of producers who said they do not have a VCPR for their dairies may have either misunderstood the question, were not familiar with the term, or mistakenly selected the incorrect answer, as their responses to other questions relating to cow health on their farms showed direct involvement of a veterinarian in cow health care decisions. Specifically, of the 12 producers who indicated not having a VCPR, 10 included veterinarians as one of the sources they relied on for information about AMD used to treat cows. The remaining two dairies had either submitted non-routine samples for infectious disease diagnosis or had used other on-farm diagnostic techniques to guide treatment decisions with AMD for cows; both diagnostic activities were most likely associated with a veterinarian. Hence, continued education of dairy producers is necessary to update their understanding of the requirements of the current law and what a valid VCPR entails.Animal welfare audit programs are designed to improve welfare standards in livestock operations by ensuring animals are humanely raised. Dairy quality assurance programs, on the other hand, are designed to improve product quality through assessments and monitoring, thereby promoting consumer health, environmental health, and dairy cattle health and welfare. Most dairies in our survey participated in welfare (97.5%) and quality assurance programs (52.9%) in the previous year, highlighting the dedication of CA producers to maintaining high standards of animal welfare and product quality. Based on the NAHMS 2014 dairy study, approximately 45.9% of all dairy operations nationwide participated in a quality assurance program, with higher participation by operations in Western US (66.8%) compared to in the Eastern US (43.9%) . The majThe MFA identified seven components that accounted for 99.7% of the variation in the data. The seven components identified the important questions that could be considered for a risk assessment tool for AMD use and AMR on CA dairies in future studies, similar to the approaches adopted for bovine respiratory disease  and JohnCluster C1 consisted of large-scale conventional dairies  situated mainly in the GSCA and NSJV region. Dairies in this cluster were characterized by their AMD usage in dry cow management  at the end of each lactation, and in the management of disease conditions such as mastitis, metritis, and lameness in adult cows. The cluster can be linked with good knowledge of AMD stewardship practices as evident by the high frequency of AMD treatment and withdrawal tracking, most often using a computer, and overall agreement to statements regarding drug use stewardship practices and AMR in their dairy operations.Cluster C2 consisted of mid-sized conventional dairies  located in NSJV and GSCA regions. Producers in this cluster could be linked with some knowledge of AMD use stewardship practices as characterized by their high level of agreement (100%) to statements regarding AMD use stewardship practices. They tend to show more agreement regarding AMR issues raised in the study compared to dairies in clusters C1. Producers in this cluster could benefit from outreach and education relating to use of computer in record keeping, AMD use stewardship practices and their impact on AMR in dairy operations.The organic cluster consisted of small-sized dairies mainly located in NCA and reported little or no usage of AMD in their dairy operations. Organic dairies do not use AMD amongst other prohibited chemicals  unless sick animals require antibiotics. Emergency AMD treatments to maintain the welfare of the sick animals are allowed on organic dairies; however, such animals have to be segregated from the organic herd and sold to a non-organic market in addition to strict documentation and reporting requirements . The surThe current study data were collected based on voluntary participation, in accordance with the SB 27 from CA licensed grade A dairy producers. The mean dairy herd size in our survey was 1,550 cows per herd, which was greater than a 2017 estimate of average herd size of 1,304 cows per herd ; which mThe current study provided a description of the current AMD use, treatments, and AMD stewardship practices that may influence AMR on CA dairies. The majority of the current practices reported by respondent CA dairy producers are in line with the provisions of the SB 27 and VFD regulations. The purpose of such regulations is to protect human and animal health through the judicious use of MIADs in food-producing animals, which has the potential to slow or prevent the development of bacterial resistance to AMDs. Support for producers in understanding and enhancing their VCPRs is likely to enhance judicious use of antimicrobial drugs. Producers and veterinarians may want to consider implementation of selective dry cow therapy, increased use of alternatives to AMDs, and adoption of electronic record-keeping as means to further implement antimicrobial stewardship.10.7717/peerj.11515/supp-1Supplemental Information 1a BVD = Bovine viral disease; b SRP = siderophore receptor and porinClick here for additional data file.10.7717/peerj.11515/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.11515/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.11515/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.11515/supp-5Supplemental Information 5a DA = displaced abomasum; b C-Section = Cesarean sectionClick here for additional data file.10.7717/peerj.11515/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.11515/supp-7Supplemental Information 7Click here for additional data file.


Adherence to recommended age-specific immunisation schedules is critical in ensuring vaccine effectiveness against vaccine preventable diseases (VPDs). There is limited data on immunisation timeliness in sub-Saharan Africa. Therefore, this study assessed the timeliness of age-specific routine childhood immunisation within the Western Cape Province of South Africa. Participant records (N = 709) from a prospective health-facility based study conducted in Cape Town, SA in 2012–2016 were analysed. The outcome measure was receiving age-specific immunisations ≥4 weeks of that recommended for age as per the South African Expanded Programme on Immunisation (EPI-SA) schedule. Proportions, medians, inter-quartile ranges (IQR) and regression were used to obtain the prevalence, time-at-risk, and risk factors for delayed immunisation. A total of 652 /709 (91.9%) participants were eligible. Immunisation coverage declined with age from 94.9% (95% CI 92.9–96.4) at birth to 72.0% (95% CI 65.7–77.6) at 18 months. The highest delay in the uptake of vaccine doses was observed among the 3 rd dose of the DTP vaccine [163 (34.6% (95% CI 30.3–39.1)], while the lowest was seen among BCG [40 (6.5% (95% CI 4.7–8.8)]. The longest median time-at-risk of VPDs was among the 2 nd dose of the measles vaccine [12.9 (IQR 6.7–38.6) weeks] and the lowest was OPV birth dose [IQR 6.3 (5.3–9.1) weeks]. Low and upper-middle socio-economic quartiles were associated with delayed uptake of vaccine doses. Delayed vaccination increases the time of susceptibility to VPDs during infancy and childhood. There is a need to develop strategies aimed at mitigating factors associated with delay in uptake of routine childhood vaccines in the Western Cape. Mitigation strategies should provide vaccine education and mobile reminder systems. Education about timely vaccine uptake will aid in the provision of informed council from healthcare providers to caregivers. Multiple reminder systems could cater for low network coverage areas and caregivers with busy schedules. The World Health Organisation (WHO) first recommended the administration of routine childhood vaccines through the Expanded Programme on Immunisation (EPI) in 1974. Four decades on, this initiative has proven to be the most cost-effective public health strategy globally, reducing childhood morbidity, disability and death associated with vaccine preventable diseases (VPDs) In South Africa, routine childhood immunisation is reported to avert an estimated 2.5 million deaths annually Outbreaks of VPDs such as measles and rubella in Gauteng, Kwa-Zulu Natal and the Western Cape provinces have been reported as recent as 2017 Immunisation coverage is defined as the proportion of people who receive vaccines at a certain age, regardless of the timing of administration Immunisation coverage is related to the receipt or non-receipt of immunisations. While immunisation timeliness addresses the analytical questions such as whether immunisations were received on time, early, or delayed. Delays in immunisations are associated with inadequate developments of immune protection, which predisposes to the acquisition of VPDs To mitigate the potential detrimental impact of delayed uptake of routine childhood vaccines, catch up immunisations are necessary to protect populations who missed or delayed immunisations. These strategies not only give individuals another chance of protection from VPDs, but population-based catch-up strategies can also reach areas of political conflict This study sought to assess the timeliness of age-specific routine immunisation in children presenting with respiratory tract infections in the City of Cape Town, Western Cape Province, South Africa. Primarily, the study set to describe the proportion of children with delayed timeliness of age-specific routine immunisation and to describe the degree of this delay. As a secondary outcome, the study intended to investigate for risk factors that lead to children not receiving age-specific immunisations on time.This study retrospectively analysed data collected during a parent prospective study that was conducted between 2012 and 2016. The parent study investigated the incidence and risk factors for pertussis in South African children less than 13 years of age. The children presented to four health facilities  with mild to severe respiratory tract infections We estimated that a minimum of 500 children’s medical records of the original database would meet the inclusion criteria. This would give a sample size sufficient to give precisions within 5% of all possible estimates of the primary outcome between 20% and 50%, which we deemed to be acceptable.The study included all participants from the original database for whom immunisation information as noted in the handheld immunisation record, the Road to Health Card (RTHC) was available. The information had to include the date each dose of the recommended vaccines was received. In addition, the participants’ Case Report Forms (CRFs) had to contain sufficient data to assess for potential risk factors.Variables such as immunisation history, socio- demographic and economic data  were retrieved from the research database. Socio-economic status was categorized into inter-quartile ranges (IQRs) based on a validated weighted composite score that included asset ownership, employment, and education Eligibility at each time point was described as participants who were old enough to receive vaccines as recommended. Up-to-date was described as receiving all the recommended vaccines at the recommended age time points. On-time uptake of vaccines was described as vaccine doses received less than 5 days before and not longer than 28 days after the recommended age as per the EPI-SA schedule The primary outcome of interest was the receipt of a vaccine dose later than 28 days of that recommended for age as per the EPI-SA schedule (birth doses are no exception). To evaluate timeliness, proportions of participants receiving and not receiving age-specific immunisations, at the recommended time were described for each vaccine. Timeliness in immunisation was calculated as the difference in the expected date and the actual date of receipt of the age-specific immunisation. Duration of delay was described for those who did not receive timely immunisations. The Bacillus Calmette–Guérin (BCG) birth dose was used as a reference to compare the median duration in delays with age. BCG is expected to have the lowest delay in uptake, and therefore when used as a reference, presents an opportunity to depict trends (increase/decrease) for delays with age.Data were analysed using R version 3.5.1 (2018–07-02) and R studio version 1.3.1073 Factors associated with delayed immunisation for each vaccine was evaluated using logistic regression using the binary outcome of Yes/No (Delayed/On time). All covariates in univariable analyses with a p-value of 0.05 were included in multivariable analyses.The study was approved by the University of Cape Town Faculty of Health Sciences Human Research Ethics Committee [HREC 027/2020].A total of 652/709 (92%) participants were eligible for analysis . The medMost (360 [55.2%]) of the participants were male. Of the total 605 participants who were eligible to attend creche, 457 (75.5%) did not attend. Of the available 642 records in which participants divulged their racial background, 389 (60.6%) identified as Black, 247 (38.5%) as Coloured (mixed ancestry) and 6 (0.9%) as other races. Overall, 639 (98%) of caregivers were mothers, with a median age of 28 [IQR 24–34] years. A total of 622 (95.4%) caregivers reported having attained a basic level of education, with grade 11 [IQR 10–12] being the median highest grade reached .Table 1PThe findings on vaccine coverage and timeliness of immunisation within the study population is reported for each of the age time-points indicated in the EPI-SA .Table 2IVaccination with BCG vaccine was received by 619 (94.9%) of the 652 participants. Of the 619 participants who received BCG, 40 (6.5%) delayed uptake. The oral polio vaccine (OPV) birth dose was received by 616 (94.5%) of the study participants. Of the 616 participants who received OPV, 43 (7.0%) had delayed uptake A and B.Fst dose of hepatitis B vaccine was received by 579/634 (91.3%), and the 1st dose of PCV was received by 546/634 (86.1%)  was received by 599/634 (94.5%); the1 (86.1%) . Amongst (86.1%) C-G.Immunisation coverage of the 2nd dose of DTP/IPV/HiB and hepatitis B vaccines was 537/594 (90.4%) and 533/595 (89.6%), respectively . DelayedThe 3rd dose of the DTP/IPV/HiB vaccine was received by 471/556 (84.7%) participants. Of those, 163 (34.6%) delayed vaccine uptake, while 286 (60.7%) had timely uptake. The 3rd dose of hepatitis B vaccine was received by 471/556 (84.7%), and of those, 105 (22.3%) delayed uptake, while 345 (73.2%) of participants had timely uptake. Immunisation coverage for the 2nd dose of PCV was 421/556 (75.7%). Amongst those 117 (27.8%) delayed uptake, while 262 (62.2%) had timely uptake of doses, as recommended. Regarding the vaccine doses administered at 14 weeks, the 3rd dose of hepatitis had the lowest proportion of participants with delayed vaccine uptake, while the 3rd dose of DTP/IPV/HiB vaccine had the highest proportion of participants who delayed vaccine uptake. When the timeliness of uptake of the vaccines administered at the 14-weeks timepoint was compared to their 10-week doses, there was an 8.7% increase for the 2nd dose of DTP/IPV/HiB vaccine, 7.3% increase for the 3rd hepatitis B vaccine dose, 9.3% increase for the 2nd PCV dose and 7.9% increase for the 2nd Rotavirus vaccine dose in delaying vaccine uptake. The 2nd dose of PCV had the highest (42 [10%]) early vaccine uptake in the 14-week age timepoint A-D.Fig. The 1st dose of measles vaccine was received by 314/370 (84.9%). Amongst those 70 (22.3%) delayed vaccine uptake, while 223 (71%) had timely uptake of the vaccine. The 3rd dose of PCV was received by 242/370 (65.4%) and of those, 55 (22.7%) of participants delayed uptake, while 175 (72.3%) had timely 3rd dose PCV vaccine uptake. While coverage of the 3rd dose of PCV decreased by 10.3% between the 2nd and 3rd doses, delayed uptake reduced by 5.1% from the 14 weeks timepoint. The 1st dose of the measles vaccine had 21 (6.7%) and the 3rd dose of PCV had 12 (5%) of participants who had early uptake of vaccines E and F.The 2nd dose of measles and the 4th dose of DTP/IPV/HiB vaccines were both received by 167/232 (72.0%) of participants. Delayed uptake among the measles and the DTP/IPV/HiB doses was observed in 49 (29.3%) and 47 (28.1%) of participants, respectively. Both 2nd dose of measles and the 4th dose of the DTP/IPV/HiB vaccines had 100 (59.9%) of participants who received timely uptake of vaccines. A reduction of 12.9% for the measles dose from the 9-month age timepoint and a 12.7% reduction for the DTP/IPV/Hib dose from the 14-week age timepoint in immunisation coverage was observed for both vaccines recommended for administration at 18 months, but the delay in uptake of the 4th dose of DTP/IPV/HiB reduced by 6.5% compared to the 3rd dose of DTP/IPV/HiB, while the delay in uptake of the 2nd dose of measles vaccine increased by 7% from 1st dose. Of all the recommended age timepoints, the 18-months age timepoint had the highest early uptake of vaccines. Early uptake was observed in 18 (12%) participants for the measles vaccine and 20 (10%) participants for the DTP/IPV/HiB vaccine G and H.The degree of delayed age-specific routine immunisation was described using medians and inter-quartile ranges (IQR).The median delay in uptake of the BCG vaccine was 6.6 [IQR 5.4 – 9.1] weeks, while the OPV birth dose had a median delay in uptake of 6.3 [IQR 5.3 – 9.1] weeks. The median delay in uptake of the OPV birth vaccine dose was not significantly different to BCG (p = 0.97).The median duration of delay in uptake of DTP/IPV/HiB vaccine doses increased with increasing age. The median duration of delay in uptake of the 1st, 2nd, and 3rd doses of the DTP/IPV/HiB vaccine administered at 6, 10 and 14 weeks was 7.0 [IQRs 4.7 – 11.2], 7.6 [IQRs 5.0 – 12.4] and 7.9 [IQRs 5.3 – 17.1] weeks, respectively .The median duration of delay in uptake of the hepatitis B vaccine increased from 7.6 [IQR 5.9 – 13.8] weeks for the 1st dose to 8.9 [IQR 5.29 – 14.3] weeks for the 2nd dose. The median duration of delay of the 3rd dose of hepatitis B vaccine decreased to 7.9 [IQR 5.1 – 13.3] weeks. The median differences for all hepatitis B vaccine doses compared to BCG vaccine were not significant .Fig. 4ChThe median duration of delay amongst the 1st and 2nd doses of the PCV vaccines given at 6 and 10 weeks declined with increasing age. The median duration of delay in the 1st dose of PCV was 8.0 [IQR 5.3 – 12.9] weeks, while the median duration of delay for the 2nd dose of PCV was 7.7 [IQR 5.3 – 19.9] weeks . HoweverThe median duration of delay for the 9 months measles vaccine dose was 8.6 [IQR 5.5 – 25.3] compared to 12.9 [IQRs 6.7 – 38.8] weeks at 18 months; p = 0.07. .The median duration of delay for the 3rd dose of PCV at 9 months further declined from the 14 weeks age timepoint from 7.7 [IQR 5.3 – 19.9] to 7.3 [IQR 5.0 – 23.4] weeks . There wThe 4th dose of the DTP/IPV/HiB vaccine had a median duration of delay that increased to 10.9 [IQR 8.0 – 28.7] weeks from that of the 3rd dose of the DTP/IPV/HiB vaccine which was 7.9 [IQR 5.3 – 17.1] weeks. .To determine which demographic and socio-economic factors are associated with delayed immunisations, logistic regressions analyses included sex, creche (preschool) attendance, caregiver age, education, and socio-economic status (SES) as covariates. Where data on these variables were limited or not available, these were excluded from the analysis . CaregivNone of the factors assessed were found to be statistically associated with the delay in uptake of the BCG and OPV vaccine doses .Table 3LOn average, those whose caregivers were adults had lower odds of delaying vaccine uptake, with 0.33 (CI 0.13 – 0.94) times the odds of delaying the 1st dose of rotavirus vaccine compared to those whose parents were adolescents .An adjusted model for the 1st dose of the measles vaccine suggested a harmful association, where those of upper-middle SES compared to those of high SES had 4.39 (CI 1.37 – 19.71) times the odds of delaying the 1st dose of the measles vaccine. Low SES was a risk factor in delaying the 3rd dose of the PCV vaccine, with 5.09 (CI 1.15 – 28.17) times the odds compared to those of high SES .Attending creche was protective in delaying the 4th dose of DTP/IPV/HiB, but this protective effect was attenuated in the adjusted model. Creche attendance was protective for the 2nd dose of measles, compared to those who did not attend creche .This study found that proportions of children with delay in timely uptake of immunisations increases with age. Similarly, the median duration of delay increases with age. These trends are coupled with a decline in immunisation coverage and an increased probability of delaying uptake of subsequent doses when previous doses have been delayed. The highest vaccine coverage rates were seen amongst the birth doses, while the lowest was amongst the 9- and 18-months vaccine doses. Delay in receiving doses was independently associated with creche attendance, having an adult caregiver, and being in low and upper-middle socioeconomic quartiles.Declining immunisation coverage with increasing age is not unique to this study. Prior studies have reported low immunisation coverage with vaccines given at 9- and 18- months and high immunisation coverage for the birth doses The observed trends of increasing delay [Highest: DTP-3 34.6% (CI 30.3–39.1) and lowest BCG: 6.5% (CI 4.7–8.8)] in the uptake of immunisation and increased time at risk of VPD was not surprising. Several studies have found similar findings where significant increases in delayed uptake of vaccine doses with age were seen, especially for vaccines given after the first year of life meta-analysis found that administering the primary measles vaccine dose before 9 months of age can elicit a good immune response in high-risk settings, and that a subsequent dose further increases vaccine effectiveness, rather than attenuate the immune response In this study, vaccine doses given at 9- and 18 months had the highest proportion of early uptake of vaccines. Due to the known high proportions of delay, early administration of measles has been observed in health care facilities as means to alleviate the delay in uptake of vaccines in some populations When we explored risk factors associated with the delay in uptake of scheduled vaccines, creche attendance was a common protective factor at the 18-months age timepoint. It is not surprising to see a protective effect from creche attendance, as the Department of Basic Education in South Africa requires parents or caregivers to provide up-to-date immunisation records as part of applications for admission to public or independent schools. Similarly, the Western Cape Education Department’s regulations requires up-to-date RTHCs before a child can be registered in any public school (independent schools in the province have their own admission rules) Education about timely vaccine uptake will aid in the provision of informed council from healthcare providers to – not only adult caregivers - but adolescent caregivers as well, with the aim to reduce delayed uptake of vaccine amongst those raised by adolescent caregivers. The health system and the EPI-SA should couple these interventions with effective mobile health strategies . These reminder systems will particularly serve to remind those caregivers who delay uptake of vaccines because of busy work schedules. A recent South African multi-centre study reported the use of mobile reminders to effectively increase uptake of recommend maternal and childhood immunisations Taken together, these findings suggest sub-optimal immunisation coverage and lack of immunisation timeliness in this population. The considerable increase in the delay in uptake of vaccines and the increased time at risk of VPDs is concerning as this may increase the pool of infants and children susceptible to VPDs. These findings emphasize the need of interventions targeting immunisation timeliness at infancy and early childhood.This study has several strengths which are worth noting. Firstly, our study provides an update on the coverage and timeliness of routine childhood vaccines in the Western Cape province. Secondly, quantifying delay has the potential to aid in better understanding potential outbreaks of VPDs in the Western Cape province, thus informing about which age-groups should be of interest to immunisation program managers and policy makers. Limitations of this study include the use of RTHCs in verifying vaccination history which could be misleading in instances where the accuracy and quality of the data was compromised. This limitation emphasise the need for electronic immunisation records This study underscores the distinction between immunisation coverage and timeliness as separate entities, which are both critical in understanding the effectiveness of routine childhood immunisation programmes in preventing the high burden of VPDs. Our findings of declining immunisation coverage, increasing delay and increased time-at-risk of VPDs with increasing age calls for immediate attention as the results have the potential to undermine the EPI-SA. Catch-up immunisation campaigns seem to be effective in minimising missed immunisation opportunities in the Western Cape province. However, more effort is required from the health system including immunisation service providers in firstly ensuring timely uptake to ensure optimal protection from VPDs and secondly, to ensure that catch-up immunisation services are readily available and accessible to caregivers. Effective surveillance and monitoring of immunisation programmes at all levels is required to achieve effective immunisation service delivery and uptake. In the current COVID-19 context, it will be important to understand how disruptions to immunisation services have further impacted timely uptake of routine childhood vaccines and potentially derailed existing VPD control efforts.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Childhood obesity is an emerging public health problem globally. Although previously a problem of high-income countries, overweight and obesity is on the rise in low- and middle-income countries. This paper explores the factors associated with childhood obesity and overweight in Uganda using data from the Uganda Demographic and Health Survey (UDHS) of 2016.We used Uganda Demographic and Health Survey (UDHS) 2016 data of 4338 children less than 5 years. Multistage stratified sampling was used to select study participants and data were collected using validated questionnaires. Overweight and obesity were combined as the primary outcome. Children whose BMI z score was over two were considered as overweight while those with a BMI z score greater than three were considered as obese. We used multivariable logistic regression to determine factors associated with obesity and overweight among children under 5 years of age in Uganda.The prevalence of overweight and obesity was 5.0% (217/4338) (95% CI: 4.3–5.6), with overweight at 3.9% (168/4338: 95% CI: 3.2–4.3) and obesity at 1.1% (49/4338: 95% CI: 0.8–1.5). Mother’s nutritional status, sex of the child, and child’s age were associated with childhood obesity and overweight. Boys were more likely to be overweight or obese  compared to girls. Children who were younger (36 months and below) and those with mothers who were overweight or obese were more likely to have obesity or overweight compared to those aged 49–59 months and those with underweight mothers respectively. Children from the western region were more likely to be overweight or obese compared to those that were from the North.The present study showed male sex, older age of the children, nutritional status of the mothers and region of residence were associated with obesity and overweight among children under 5 years of age. Sub-Sahara Africa is facing an epidemiological transition towards non-communicable diseases (NCDs) such as cardiovascular illnesses and diabetes, with about one third of current disability-adjusted life years (DALYs) attributable to NCDs . The keyChildhood obesity is an emerging public health burden. Globally over 38 million children under the age of 5 years are affected . ChildreObesity arises from a complex interaction between behavioral, metabolic, environmental, and socioeconomic determinants ranging from changing food systems and reduced physical activity to indiscriminate marketing that promotes obesogenic foods , 11–13. Tackling childhood obesity is important because it is associated with unfavorable health and socioeconomic consequences. Excessive weight gain is linked to premature mortality and adverse short term and long-term effects on mental health. Furthermore, overweight and obese children have higher risk of early chronic diseases onset, such as diabetes, dyslipidemias, cardiovascular diseases and some cancers , 10, 20.A plethora of studies from Africa have primarily focused on the study of undernutrition and not overweight or obesity among children. The few studies on obesity have mostly come from Nigeria and South Africa, and almost none from other countries in Sub-Sahara Africa . In UganUDHS 2016 was a nationally representative cross-sectional study conducted using validated questionnaires. UDHS is a periodical survey that is carried out every 5 years as part of the MEASURE DHS global survey and collects Information on demographic, health and nutrition indicators. The survey was conducted between June 2016 and December 2016 using stratified two-stage cluster sampling design that resulted in the random selection of a representative sample of 20,880 households , 23. Thehttps://dhsprogram.com/.Our secondary analysis excluded children whose BMI z-score were missing or was recorded as “Flagged cases”. Flagged cases were defined as more than 5 SD above or below the standard population median (Z-scores) based on the WHO Child Growth Standards . In the The BMI z-scores based on WHO 2006 reference population were used to assess obesity and overweight . ChildreIndependent variables were categorized into children, parents’ and household characteristics that were chosen basing on previous studies –27 and a2, normal between 18.50 kg/m2 and 24.99 kg/m2 and overweight or obesity between 25.0 kg/m2 and above 30.0 kg/m2) [Maternal nutritional status (underweight defined as body mass index (BMI) less than (<) 18.50 kg/m0 kg/m2) , 28, motWealth index is a measure of relative household economic status and was calculated by DHS from information on household asset ownership using Principal Component Analysis  , type ofAge of the child in months , sex of the child  and stunting status (categorized as stunted and not stunted) defined as height-for-age Z-score is below minus two standard deviations (− 2 SD) from the median of the reference population .p-values. Variables included in our multivariable model were determined a priori during literature review [We used the SPSS analytic software version 25.0 Complex Samples package for this analysis. Weighted data was used to account for the unequal probability sampling in different strata. Frequency distributions were used to describe the background characteristics of the children. Pearson’s chi-squared tests were used to investigate the significant differences between childhood obesity and overweight and the explanatory variables. Bivariable logistic regression was also conducted and we present crude odds ratio (COR), 95% confidence interval (CI) and e review . All varOf the 4338 children, 5.0% 217/4338) (95% CI: 4.3–5.6) were overweight or obese (overweight 3.9% (95% CI: 3.2–4.3) and obesity 1.1% (95% CI: 0.8–1.5). Mean age of the children and that of their mothers was 28.34 months (SD 17.20) and 28.80 years (SD 6.83) respectively. Boys were slightly more than girls. The highest number of children were those less than 1 year. Majority of the children resided in rural areas and belonged to households with five or more members. The mean weight, height, number of household members and BMI z-score were 11.5 kg (SD 3.57), 83.5 cm (SD 14.0), 6 members (SD 2.7) and 0.20 (SD 1.19) respectively. More detailed characteristics of study participants are shown in Table 7/4338  (95% CI: 3.7–5.2) with overweight at 3.7% (95% CI: 2.9–4.2) and obesity 0.9% (95% CI: 0.6–1.3). Only sex and age of the child were the significant factors. The association between sex of the child with being overweight or obese was stronger compared to the primary analysis with boys (AOR: 2.10 (95% CI: 1.32–3.34) being twice more likely to be overweight or obese compared to the girls. Younger children less than 12 months (AOR: 2.31 (95% CI: 1.03–5.18), 13–24 months (AOR: 4.15 (95% CI: 1.87–9.19) and 25–36 months (AOR: 3.37 (95% CI: 1.35–8.40) were more likely to be overweight or obese compared to the older children aged 49–59 months. After excluding children below 24 months, the prevalence of overweight or obesity was 4.0% (99/2487) (95% CI: 3.1–4.7) with overweight at 3.5% (95% CI: 2.7–4.1) and obesity 0.5% (95% CI: 0.3–0.9). Sex and age of the child were still the only factors significantly associated with overweight or obesity. Children less than 36 months  were more likely to be overweight or obese compared to their older counterparts (49–59 months). Boys (AOR: 2.30 (95% CI: 1.38–3.84) were more likely to be overweight or obese compared to girls.This study provides evidence on the factors associated with childhood obesity and overweight in Uganda from the UDHS conducted in 2016. We found that maternal overweight and obesity, age and sex of the child as well as region of residence were associated with the child’s overweight or obesity status. For instance, children of mother’s who were overweight were almost three (2.7) times more likely to be overweight or obese compared to those whose mothers were underweight.The strong association between maternal obesity and childhood overweight/obesity has also been demonstrated elsewhere. Two previous studies showed similar findings with this study where the risk of childhood obesity was increased in cases of maternal overweight/obesity both before and during pregnancy, which also predisposed them to obesity in later life , 31. ThiThis study demonstrated that boys had a statistically significant higher risk of being overweight or obese compared to girls, which is consistent with the findings of Beatrice J et al.  and DuboChildren aged 36 months and below were more likely to be overweight or obese compared to those aged between 49 and 59 months. A study conducted in Cameroon found a similar association  where chRegional differences in the odds of being overweight or obese were imprecise in this study. However, some pertinent regional differences have been documented that may play a role in the observed differences. The western region of Uganda, for example, receives more rainfall annually, has higher crop yields hence better food security, and has the second highest GDP per capita , 37. TheWe used a nationally representative sample with data weighted to account for the unequal probability sampling in different strata, so our results are generalizable to all Ugandan children below the age of 5 years. Secondly, our study was adequately powered to study all these associations because of the large sample size from the UDHS.The lack of other measures of overweight and obesity, such as skin fold thickness measurements, measure of physical activity, analysis of nutritional characteristics such as dietary habits was major limitation. This data/information would have allowed us to assess the development of overweight and obesity. The cross-sectional design is limited by lack of temporality hence causality inferences could not be made. BMI adjusted for-age for children under 2-years is a contested measure because in this age group, BMI is based on Length other than height. BMI for age  in infancy has un unanswered questions however recent studies have found a high level of agreement between BMI for age and length for age and found BMI for age to be more appropriate .This study established maternal nutritional status (BMI), region of residence, sex and age a child as significant determinants of childhood overweight/ obesity. Failure to observe any significant differences between wealth quintiles implies that strategies to improve child nutrition should be applied across all socioeconomic groups. Preventive interventions will also need to consider regional differences in outcomes and context. Efforts should be made to promote healthy weight in mothers across the spectrum of motherhood , as a healthy mother will likely have a healthy child.Further studies including nutritional characteristics are needed to understand the association with child age and sex and will help in refining preventive strategies against childhood overweight and obesity in Uganda.


Nevertheless, data on spatial and temporal heterogeneity of tumor-infiltrating lymphocytes in advanced UC are lacking. The major aims of this study were to explore spatial heterogeneity for lymphocyte infiltration and to investigate how the immune landscape changes during the disease course. We performed multiplex immunohistochemistry to assess the density of intratumoral and stromal CD3+, CD8+, FoxP3+ and CD20+ immune cells in longitudinally collected samples of 49 UC patients. Within these samples, spatial heterogeneity for lymphocyte infiltration was observed. Regions the size of a 0.6 tissue microarray core (0.28 mm2) provided a representative sample in 60.6 to 71.6% of cases, depending on the cell type of interest. Regions of 3.30 mm2, the median tumor surface area in our biopsies, were representative in 58.8 to 73.8% of cases. Immune cell densities did not significantly differ between untreated primary tumors and metachronous distant metastases. Interestingly, CD3+, CD8+ and FoxP3+ T cell densities decreased during chemotherapy in two small cohorts of patients treated with neoadjuvant or palliative platinum-based chemotherapy. In conclusion, spatial heterogeneity in advanced UC challenges the use of immune cell infiltration in biopsies as biomarker for response prediction. Our data also suggests a decrease in tumor-infiltrating T cells during platinum-based chemotherapy.Checkpoint inhibitors targeting PD-(L)1 induce objective responses in 20% of patients with metastatic urothelial cancer (UC). CD8 Whereas cisplatin-based chemotherapy remains the standard first-line treatment for patients with metastatic urothelial cancer (UC), immune checkpoint inhibitors have become available as an additional treatment option for these patients in the last few years. Checkpoint inhibitors targeting programmed cell death protein-1 (PD-1) or its ligand (PD-L1) are currently available for those that progressed on first-line platinum-based chemotherapy and those that are cisplatin-ineligible and have a PD-L1 positive tumor. Although anti-PD-(L)1 is able to induce durable responses in some patients, objective responses and disease control are achieved in only 21.1% and 38.5% of patients with metastatic UC, respectively  compared to watchful waiting in patients who achieved a response or stable disease with first-line chemotherapy (21.4 vs 14.3 months) ] ] . Neverth is used .+ T cell infiltration and response to checkpoint inhibitors and the interest in the use of checkpoint inhibitors in other disease settings, knowledge on the evolvement of the immune infiltrate during the disease course is desired. Several studies have investigated the relationship between T cell infiltration and disease stage in localized UC. These studies did not find a strong association with tumor stage and/or grade (Considering the association between CD8or grade . Little +), cytolytic (CD8+) and regulatory (FoxP3+) T cells and B cells (CD20+) in longitudinally collected samples of 49 UC patients using multiplex immunohistochemistry (mIHC). The major aims of this study were to explore spatial heterogeneity for lymphocyte infiltration, both between different tissue sites and within individual tumor masses, and to investigate how the immune landscape changes during the disease course.In this exploratory study, we evaluated the density of total ureterectomy. When this was unavailable, we used tissue obtained during transurethral resection (TURT) instead. Whenever a patient had received neoadjuvant chemotherapy (NAC) before radical cystectomy, we included both tissue obtained before (TURT) and after NAC (cystectomy). In the rare cases that a patient developed two primary tumors (in different parts of the urinary tract), tissue of both primary tumors was used. Further, if the patient had local lymph node metastases at the time of surgery, we also included tissue of a synchronous lymph node metastasis. To assess lymphocyte infiltration in distant metastases, archival tumor tissue obtained in the metastatic setting was used. If a patient had undergone multiple biopsies in the metastatic setting, all samples were used for mIHC.This study was approved by the local Radboudumc medical ethical committee (file number 2017-3934). All patients provided written informed consent to scientific use of leftover tissue, unless deceased.Formalin-fixed paraffin-embedded tissue blocks were cut into 4 µm thick tissue sections. Consecutive tissue sections were used for hematoxylin and eosin (H&E), 7-color mIHC, GATA3 and cytokeratin 5/6 (KRT5/6) stainings.The 7-color mIHC stainings were performed using primary antibodies against CD3 , CD8 , FoxP3 , CD20 , CD45RO  and pan cytokeratin . In this panel, pan cytokeratin served as tumor marker. For details on antibody order and fluorochrome pairing, we refer to The fully automated BOND-RX IHC stainer (Leica Biosystems) was used to perform the 7-color mIHC stainings. First, slides were deparaffinized using Bond Dewax Solution . Before application of the primary antibody, antigen retrieval was performed in Bond Epitope Retrieval 2 solution  at 95°C for 20 minutes. After this, slides were incubated in Opal antibody diluent  for 10 minutes to reduce nonspecific background staining. Next, the primary antibody was applied for 1 hour at room temperature. Slides were then washed three times with Bond Wash Solution , before applying Opal Polymer anti-mouse/anti-rabbit HRP secondary antibodies . After this, slides were washed again three times, Opal TSA substrate was applied in a 1:50 dilution  or a 1:200 dilution  for 10 minutes and the slides were washed again . This process was repeated for all six markers. Finally, DAPI nuclear counterstain, diluted in TBST, was applied manually for 5 minutes  and slides were embedded in Fluormount-G .DAB stainings for GATA3  and KRT5/6  were performed using the semi-automated Lab Vision Autostainer (Thermo Scientific). Antigen retrieval was performed with Target Retrieval Solution, High pH  at 97°C for 10 minutes. After application of the primary antibody, visualization was achieved with EnVision Flex, High pH .The H&E, GATA3 and KRT5/6 stainings were visually assessed by a dedicated genitourinary pathologist. H&E staining served to confirm the presence of tumor tissue and to evaluate the histological subtype. In the case of discrepancies with the original pathology report regarding histological subtype, all available tissue sections of the particular specimen were reevaluated. The GATA3 and KRT5/6 stainings were performed in order to classify tumors into basal and luminal subtypes –14. Alth® 3 Automated Quantitative Pathology Imaging System, with software version 3.0.4. Single stainings were used to set the exposure times. Slides were first scanned at 4x magnification. Using the PerkinElmer Phenochart software (version 1.0.9), tumor regions plus one surrounding region of stroma (669 × 500 µm) were selected for imaging at 20X magnification  was used for spectral unmixing, removal of autofluorescence signal and tissue segmentation. For tissue segmentation, an algorithm was trained based on the expression of pan cytokeratin, DAPI and autofluorescence to discriminate between tumor, stroma and background , cytotoxic T cells (CD8+), regulatory T cells (FoxP3+) and B cells (CD20+). Unfortunately, we were not able to accurately distinguish CD45RO+ from CD45RO- cells. Therefore, CD45RO+ was excluded from analysis.In this study, we differentiated between intratumoral immune cells and immune cells located in the surrounding stroma. If the tumor or stroma region was smaller than 0.10 mm2/field) from stained whole-slides for assessment (2 (similar to a 0.6 mm diameter core on a TMA). These regions were predominantly segmented as tumor tissue by inForm tissue segmentation, but small stromal bands  , 19–22 osessment –26. To s2). As many samples were too small to select four of these larger tumor regions, these regions could only be selected in a minority of samples (n=20).In addition, to evaluate whether biopsies are representative for immune cell infiltration in a tumor, we randomly selected four tumor regions the size of the median tumor surface area in our biopsies , the intratumoral and stromal cell densities per tissue site were visualized in boxplots. A Kruskal-Wallis test was performed to test whether there were significant differences  between tissue sites. If significant, a post-hoc Dunn’s test with Bonferroni correction was performed for pairwise comparisons.First, we evaluated whether lymphocyte infiltration differed depending on the tissue of origin. To assess this, we selected all tissue sites for which we had at least 5 samples available. For each cell subset  quartile. This procedure was repeated for the larger tumor regions (3.30 mm2).Next, we assessed heterogeneity within samples. For this, four tumor regions of 0.28 mmData from paired tumor samples were used to study longitudinal changes in lymphocyte density within individual patients. First, immune cell infiltration in untreated primary tumors was compared with immune cell infiltration in metachronous metastases. If there had been two primary tumors or if multiple biopsies from the metastatic setting were available, the cell densities of these samples were averaged. Differences were analyzed using a Wilcoxon signed-rank test. Again, a p-value below 0.05 was considered statistically significant. Subsequently, samples obtained before and after chemotherapy were compared in two cohorts of patients . Because of the small sample size, no statical tests were performed. The median change and range were used to describe changes during chemotherapy.All statistical analyses were performed in R version 4.0.2.Immune cell infiltration was assessed in 111 longitudinally collected samples of 49 patients with metastatic UC. The study population consisted of both patients with upper (n=11) and lower urinary tract disease (n=35). In three cases the origin of the metastases was unclear, because the patient presented with metastatic disease without a detectable primary tumor (n=1) or because the patient had a history of both invasive UC of the bladder and upper urinary tract (n=2). All patients had a tumor of urothelial origin. Sixteen patients (32.7%) had a component of divergent differentiation. This mainly concerned squamous (n=8) or sarcomatoid differentiation (n=2). Further patient characteristics are shown in 2 (range 0.14 – 150.40) and 10.1 mm2 (range 0.0083 – 117.47), respectively. In one sample, the stromal cell density could not be assessed due to a stromal surface of less than 0.10 mm2. The intratumoral and stromal surface area in biopsies was smaller compared to samples that were obtained through surgery ureterectomy). The median surface area of the intratumoral and stromal regions in biopsies was 3.30 mm2 (range 0.14 – 14.93) and 1.51 mm2 (range 0.0083 – 20.78), respectively.Across all 111 samples, the median surface area of the intratumoral and stromal compartment was 7.23 mm+, CD8+, FoxP3+ and CD20+ cells in the tumor compartment were 376.6 cells/mm2 (range 28.1 – 3497.6), 140.0 cells/mm2 (range 1.1 – 2511.9), 71.0 cells/mm2 (range 2.5 – 539.8) and 8.3 cells/mm2 (range 0 – 1494.8), respectively. Median cell counts in stroma were 1255.4 cells/mm2 (99.5 – 8959.5), 394.0 cells/mm2 (range 19.3 – 2539.9), 251.7 cells/mm2 (range 29.2 – 1760.9) and 124.9 cells/mm2 (range 0 – 3784.0). CD20+ cells were frequently clustered in tertiary lymphoid structures (TLS), which can be recognized as CD20+ B cell follicles, adjacent to a CD3+ T cell zone , 55 were only positive for GATA3 , and 15 were only positive for KRT5/6 . Ten samples were negative for both markers (9.5%).+ and FoxP3+ T cells were significantly higher in lymph node metastases compared to bone metastases  or tumors located in the urinary tract . A significant difference between lymph node and bone metastases was also observed for intratumoral CD8+ T cells (p=0.035). Finally, the intratumoral CD20+ cell density significantly differed between lymph node metastases and lesions in all other locations  as well as between urinary tract and soft tissue lesions (p=0.015). Nevertheless, the median cell densities in the latter two locations were both very low (8.4 versus 0.7 cells/mm2). Comparable results were obtained for stromal immune cell densities , lymph node (n=29), soft tissue (n=10), liver (n=7) and bone (n=5). The cell densities per tissue site are shown in GATA3 and KRT5/6 expression also differed between the five tissue sites. Whereas 51-52% of urinary tract and lymph node were classified as luminal, this was the case for 30% of soft tissue, 71.4% of liver and 100% of bone lesions. Nevertheless, when we selected only the luminal samples, we still observed large differences between tissue sites, with lymph node metastases still containing the most dense and bone metastases the least dense infiltrates (data not shown). The basal subgroup was too small to compare tissue sites.TLSs, which can only be assessed in non-lymphoid tissues, were observed in most urinary tract samples (43/50), but were infrequent in samples from other tissue sites . This difference may be explained by the larger area of stroma in urinary tract samples, as TLSs were mostly located in the stromal compartment.+ and CD8+ cell densities did not clearly differ between urinary tract and lymph node samples. However, intratumoral FoxP3+ and CD20+ cell densities were higher in the lymph node metastases in four and five out of six patients, respectively. As expected stromal immune cell densities were also higher in the lymph nodes  and +, FoxP3+ and CD20+ cells). The median difference in cell count/mm2 between the region with the highest and the region with the lowest cell density was 515.40 for CD3+ cells (range 10.75 - 5884.79), 221.57 for CD8+ cells (range 0 – 2957.72), 136.16 for FoxP3+ cells (range 0 – 155.88) and 16.07 for CD20+ cells (range 0 – 2264.49).Next, we assessed heterogeneity within tumor samples. On visual inspection, we observed substantial heterogeneity within samples . The median difference in cell count/mm2 between the region with the highest and the region with the lowest cell density was low compared to the 0.28 mm2 regions: 188.97 for CD3+ cells (range 38.46 – 659.73), 79.76 for CD8+ cells (range 17.87 – 460.34), 48.49 for FoxP3+ cells (range 8.48 – 343.99) and 5.45 for CD20+ cells (range 0 – 160.31). Nevertheless, the correct classification rate for CD3+ and CD8+ cells was only slightly higher  and the correct classification rate for FoxP3+ and CD20+ cells was poorer   and -59.58 cells/mm2 (-423.09 to 616.90), respectively. FoxP3+ cell density also decreased in seven patients, but these changes were small . No clear trend was observed for the CD20+ cell density . The changes observed in stromal immune cell density were comparable to the changes in the tumor compartment. There was no evident change in the FoxP3+/CD8+ T cell ratio  and after NAC (cystectomy) (+ and CD8+ cell density decreased. Median change in CD3+ and CD8+ cell density was -415.58 cells/mm2 (range -1003.08 to -89.07) and -134.74 cells/mm2 (range -319.55 to -41.45), respectively. Intratumoral FoxP3+ and CD20+ cell density decreased in four out of five patients. Median changes in FoxP3+ and CD20+ cell density were -51.71 cell/mm2 (range -216.24 to 182.12) and -21.30 cells/mm2 (-177.18 to 11.75). Stromal CD3+, CD8+, FoxP3+ and CD20+ cell densities decreased in three, three, four and two out of five patients, respectively. Again, no clear trend in the ratio between FoxP3+ and CD8+ T cells was observed  or tumors located in the urinary tract . Although the difference in intratumoral CD3+ T cell density between urinary tract and lymph node lesions could not be confirmed in a small cohort of patients with simultaneously resected urinary tract and lymph node samples (n=6), we did observe higher intratumoral FoxP3+ and CD20+ cell densities in lymph node metastases of these patients. In line with our findings, studies in other cancer types have also reported differences in immune cell infiltration between tissue sites  selected. By statistical comparison of interquartile range (IQR) distributions, the minimum number of regions for an accurate estimate of CD8+ cell infiltration was determined. Here, the bootstrapping estimate for 10 regions functioned as reference value. A limitation of this method is that, from a mathematical point of view, the IQR is expected to get smaller when the number of selected regions approaches the reference value. Therefore, we decided to use another method to analyze heterogeneity. We classified the samples into four quartiles based on the mean cell densities of the four regions and determined the percentage of regions that was classified into the same quartile.Another important finding of this study is that there is considerable spatial heterogeneity within tumors. Tumor regions of 0.28 mmor types . That st+ T cell infiltration in UC has been defined so far. Secondly, we classified samples into quartiles based on the mean cell density of the four regions instead of the whole slide cell density. We felt that this was more appropriate because there was large variation in whole slide tumor surface area. Moreover, although our selected tumor regions were largely classified as tumor tissue by inForm segmentation, the selected regions often included small stromal bands (areas without pan cytokeratin expression). While it is commonly accepted to consider these small areas part of the tumor compartment (2) regions in small subset of samples, most of which were derived from the primary tumor. It is unclear whether heterogeneity in primary muscle-invasive tumors is comparable with heterogeneity in distant metastases.Although our analysis contributes to a better understanding regarding the significance of the observed heterogeneity, some remarks should be made with respect to the used method. Firstly, the decision to classify samples into quartiles instead of tertiles or quintiles was somewhat arbitrary. However, no generally accepted cutoff value for CD8partment , 21, 26,Based on the observed heterogeneity, we conclude that median-sized biopsies do not provide a representative sample for the evaluation of immune cell infiltration. Although biopsies might reveal an association between immune cell infiltration and response to checkpoint inhibitors on a group level, they are likely not appropriate to predict response or prognosis in individual patients. There is a substantial risk that the sample is not representative and that the patient is thus classified in the wrong risk group.Another aim of this study was to evaluate longitudinal changes in lymphocyte density within individual patients. In this study, the density of tumor-infiltrating immune cells did not clearly decrease or increase during progression to metastatic disease. Nevertheless, we should acknowledge that our cohort is heterogeneous, not only in terms of biopsy site but also in terms of the treatments given between primary tumor and metastasis sampling.+, CD8+ and FoxP3+ T cells during chemotherapy, both in the neoadjuvant and palliative setting. Preclinical evidence has suggested that cisplatin and gemcitabine increase the infiltration and cytotoxic activity of CD8+ T cells   or results from disease progression .Although most patients in our cohort eventually received checkpoint inhibitors, a minority of patients underwent a biopsy right before initiation of treatment. This, in combination with the small sample size and the different biopsy locations, prevented us from studying associations between lymphocyte infiltration and response to checkpoint inhibitors.+, CD8+ and FoxP3+ cell densities decrease during treatment with platinum-based chemotherapy.In conclusion, this exploratory study provides a first insight into spatial and temporal heterogeneity in advanced UC. Our data demonstrate that spatial heterogeneity in UC samples distorts immune cell quantification in median-sized biopsies, challenging the use of immune cell infiltration as prognostic or predictive biomarker. In addition, our results indicate that CD3The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Radboudumc Medical Ethical Committee. All patients provided written informed consent to the scientific use of leftover tissue, unless deceased.SW and NM participated in the conceptualization. SW, MG, LW, SS, and IV participated in the methodology. SW participated in the formal analysis. SW and MS participated in the investigation. SW, MG, LW, SS, IV, and NM provided resources. SW participated in the data curation. SW participated in the writing—original draft preparation. All authors participated in the writing—review and editing. SW participated in the visualization. NM participated in the supervision. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Background: Our aim was to compare the bowel function and oncologic outcomes following these two treatment modalities.Materials and methods: This was a single-center study with 67 patients included between 2009 and 2018. A total of 32 patients underwent total mesorectal excision (TME) group and 35 transanal local excisions (LE) ± chemoradiation. We performed a case-matched analysis: we matched the patients by age, cancer stage, and comorbidities. Duration of operation, postoperative complications, length of hospital stay, and long-term functional and oncological outcomes were compared. We calculated oncological outcomes using Kaplan–Meier Cox diagrams. In addition, we used a low anterior resection syndrome (LARS) score for the bowel function assessment.Results: Mean operation time in the LE group was 58.8 ± 45 min compared with the TME group that was 121.1 ± 42 min (p = 0.032). Complications were seen in 5.7% in LE group and 15.62% in TME group (p = 0.043). ~85.2% of the patients had no LARS in LE group compared with 54.5% in TME group (p = 0.018). Minor LARS was 7.4% in LE group compared with 31.8% in TME group (p = 0.018); major LARS was 7.4 and 13.7%, respectively (p = 0.474). Hospital stay was 2.77 days in LE group compared with 9.21 days in TME group (p = 0.036). The overall survival was 68.78 months in LE group compared with 74.81 months in TME group (p = 0.964).Conclusion: Our results of a small sample size showed that local excision ± chemoradiation is a rather safe method for early rectal cancer compared with gold standard treatment. In addition, better bowel function is preserved with less postoperative complications and shorter hospital stays. Colorectal cancer is common cancer worldwide with rectal cancer accounting for approximately 30% of all colorectal malignancies . Due to Recently, thanks to cancer screening programs, the proportion of rectal cancers diagnosed at an early stage are increasing in Western countries, which gives the capability of reducing the size of the operation and minimizing negative effects on low anterior resection with TME . Now, miWe aimed to compare the long-term bowel function and oncologic outcomes following these two treatment modalities.The National Cancer Institute Review Board has approved the study . All the patients signed the written informed consent.Data from the consecutively recruited patients who were treated at the National Cancer Institute between 2009 and 2018 were investigated. Patients who had T1-T2 rectal cancer with no lymph node or distant metastases (staging was done by using CT scan of the chest and abdomen and MRI of the pelvis) and with final pathology were included. We excluded patients with more than pT2 cancers and patients with positive lymph nodes . During the study period, more than 1,600 rectal cancer surgeries were performed see in . All theClavien–Dindo classification (We used a low anterior resection syndrome (LARS) score for bowel function assessment for at least 2 years following the procedure , 11. Comfication .If the final pathology report following local excision was T2 cancer (10 patients) or T1 cancer with poor prognostic factors (seven patients) , the patient was offered completion of TME or adjuvant chemoradiotherapy, if the patient was unfit or unwilling for the surgery. Patients received 50.4–54.0 Gy of radiation to the pelvis concomitant to 5-fluorouracil-based chemotherapy for 5 weeks (1.8–2 Gy per day). Seventeen patients underwent chemoradiotherapy. We have excluded nine patients who had poor prognostic factors and underwent completion of TME .We have also performed a subgroup analysis and compared the survival and bowel function in three groups: LE only, LE + chemoradiotherapy, and TME group.Kaplan–Meier Cox diagrams were calculated for oncologic outcomes.We performed statistical analysis using SPSS Statistics 23.0 . The *Power 3.1.9.4 sample size calculator and the free version was available from https://stats.idre.ucla.edu/other/gpower/ . The value of alpha—the probability of a false positive was set at 5% and, hence, the familiar p < 0.05. Power is 1-beta, so in percentage terms, these were expressed as 80%. The effect size was set at 0.15 (the expected difference of patients having major LARS between the two groups of 15%). For 1:1 randomization, it showed that 44 patients (22 in each arm) would provide 80% power for a two-sample proportion test. There are likely to be patients lost to follow-up, so the target recruitment was set at 50.The sample size was calculated by using Gp = 0.032). Two patients (5.7%) in the LE group had complications: one patient was treated conservatively, one had grade IIIB complication—fistula, which required additional surgical intervention and five patients in TME group (15.62%) (p = 0.043) had grade II-IIIA complications. The length of hospital stay in LE group was 2.77 days and 9.21 days in the TME group  underwent TME.p = 0.018). Minor LARS was 7.4% in LE group compared to 31.8% in TME group (p = 0.018); major LARS was 7.4 and 13.7%, respectively (p = 0.474) (p = 0.964) (No LARS was found in 85.2% of the patients in LE group compared to 54.5% of the patients in the TME group (= 0.474) . There w= 0.964) . Local rp = 0.045). Accordingly, major LARS was present in three (13.6%) patients, one (7.7%) patient, and one (7.1%) patient (p = 0.7330). Moreover, we found no survival difference between the three groups (p = 0.236)  patients who underwent TME, in 12 (92.3%) patients with LE ± chemoradiation, and in 11 (78.6%) patients with LE only (= 0.236) .In both groups, 13 patients (seven patients in the LE group and six patients in the TME group) had poor prognostic factors. However, as the numbers are very small, no further analysis was performed.We found that LE with or without chemoradiation can provide good oncological and functional outcomes compared with radical surgery (TME). LE remains an evolving area in the management of rectal cancer, requiring comprehensive screening and selection of patients. Nevertheless, the right choice of treatment can significantly improve quality of life of the patient without compromising survival. However, LE for high-risk T1 or T2 rectal carcinomas is a relative contraindication because it is associated with a high risk of local or distant recurrence compared to radical surgery , 14.Borstlap et al. found the overall local recurrence after LE following adjuvant chemoradiotherapy that was 5% for pT1 rectal cancer and 14.3% for pT2 rectal cancer. Distant recurrence for pT1/pT2 rectal cancer was 8.2%  for T1 cancer and 78.2% (65.5–90.9%) for T2 cancer. In addition, they stated that the LE and chemoradiotherapy were not recommended to be used in advanced disease (high-risk T2/T3 cancer). Interestingly, they found no local recurrence in the patients with T3 cancer. It is possible because these patients were typically very friable and died even before the development of recurrence with a 5-year overall survival rate of just 20 , developed fecal incontinence (LE−9.9% vs. TME−24.8%), pain (LE−3.6% vs. TME−15.3%), and impotence (LE−33.3% vs. TME−62.3%)  . SimilarE−62.3%) , 28. In nor LARS . In thisRecently, the issue of treatment of early rectal cancer brought even more attention. Two systematic reviews and meta-analyses have been just published , 31. In Our study is limited by the small sample size and retrospective approach. However, previous studies had very similar numbers of included patients. Moreover, in the LE group, there might have been more fragile older patients with the inability to survive the radical surgery. In addition, the follow-up of our last patients included is only 3 years—this weakens our statement on equal survival rates. As only one patient within the surveillance period had local recurrence, counting disease-free survival or local recurrence-free survival becomes irrelevant. Finally, the lack of endoanal ultrasound for preoperative examination is another limitation. The strength of our study is the assessment of bowel function in two selected groups by using a validated questionnaire.According to our small group, LE ± chemoradiation probably gives comparable results to TME in survival rates. On the contrary, it has better bowel function, causes fewer postoperative complications, and helps to shorten the length of hospital stay. However, patients with T2 cancer should be warned of the high risk of recurrence.The data are available upon reasonable request from the corresponding author.The studies involving human participants were reviewed and approved by National Cancer Institute review board. The patients/participants provided their written informed consent to participate in this study.AD, JP, and LP contributed to the study design and edition, patient enrollment, follow-up, data collection, final statistical analysis, text creation, and final text supervision. VP contributed with statistical analysis, text creation, and final text supervision. All authors listed have made a substantial, direct, intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.