Still, there are large gaps in our knowledge and large parts of modern enuresis management guidelines are (still) not based on firm evidence. In this review I will question the following commonly made assumptions regarding enuresis evaluation and treatment:In this review I will argue that much of what we do with these children is based more on experience and well-meant but poorly supported assumptions than on evidence. Some advice and therapies are probably ineffective whereas for other treatments we lack reliable predictors of treatment response. More research is obviously needed, but awaiting new results enuresis management could be substantially simplified. Enuresis used to be viewed as a purely psychiatric disorder. Until the 1980s the evaluation of bedwetting children was focused on behavior, early trauma and other psychological factors, and therapy—if any therapy was advocated—was usually psychotherapy in various forms.But since the seminal work in the late 80s by the Aarhus group we know more . EnuresiThe recommended strategy for managing these children has changed accordingly, as reflected by international guidelines –10. Now,Nowadays, children with enuresis are expected to be taken seriously and the wait-and-see attitude is no longer accepted, at least for children aged six years or more. Neither is psychotherapy advocated as a primary (or indeed secondary) therapy. Instead the recommendation is often given that the LUT function of these children be “normalised” by the institution of regular drinking  and voidThe new prevailing strategy for the management of children with enuresis is surely a great step forward compared with the views of several decades ago, but there are still problems. Much of what we now do is (still) based not on firm evidence but on experience and assumptions. These assumptions are not by any means unreasonable, just not properly tested.The aim of this review is to scrutinize some of the central assumptions underlying modern enuresis management. By doing this I do not mean neither to polemize or criticize the experts nor distance myself from my contribution to the current guideline documents but, hopefully, to underline fields needing more research and to suggest ways that—pending that research—enuresis management may be simplified, at least outside the university setting.According to the International Children's Continence Society (ICCS) nocturnal enuresis can, and should, be subdivided into monosymptomatic nocturnal enuresis (MNE) and nonmonosymptomatic nocturnal enuresis (NMNE) on the basis of whether daytime symptoms of LUT dysfunction are also present or not , 14. TheThe MNE/NMNE definitions were an update of the previous terminology document that stated that MNE, defined only as enuresis without daytime incontinence, involved “urodynamically normal voidings” whereas NMNE did not . When weStill, we did make assumptions regarding the underlying pathogenesis and the expected response to therapy. The argument went something like this: (1) we know that detrusor overactivity is one crucial pathogenic factor behind enuresis, (2) we assume that symptoms such as daytime incontinence and urgency, and findings such as a high daytime micturition frequency, indicate underlying detrusor overactivity , 17, (3)We have usually assumed that children with NMNE constitute the minority, but this has been questioned –23, 29. Urgency is a particularly problematic symptom. It is assumed to indicate underlying detrusor overactivity , a phenoLooking for studies addressing whether children with MNE and NMNE require different therapies the result is meager. In very many studies, perhaps the majority, only children with presumed MNE have been included. And even though many of those studies have not had a verified “high” or “low” (daytime) micturition frequency among the exclusion criteria  the result is that we know very little about which therapy works and does not work in children with NMNE. Studies expressly including children with both MNE and NMNE while clearly characterizing them, and giving them the same therapy while looking for differences in therapy response between the groups, are very few indeed . The resPerhaps the only remaining argument for giving different treatment across the MNE/NMNE divide is that if there is nocturnal polyuria there is no need to assume concomittant detrusor overactivity and desmopressin could be tested straight away. But even this assumption has not been properly tested, due to a lack of studies of desmopressin treatment in children with properly defined NMNE . And howIn the latest enuresis guideline document from the ICCS these uncertainties have been acknowledged by joining the previously separate NMNE and MNE documents , 7 into In future guidelines, I suggest that much less emphasis is put on the MNE-NMNE subdivision.via weighing of diapers or sheet covers  the parents told the child to void, (b) the child believed that it was supposed to void, (c) it was socially convenient to go to the toilet, (d) the bladder was full, or (e) uninhibited detrusor contractions? It's really only the last reason for voiding which is interesting for us, the others are just obscuring the picture.Sadly, the evidence for a link between voiding chart data such as voided volumes or voiding frequency and cystometric findings is very tenuous , 32. AndThe value of the voiding chart as a predictor of enuresis therapy response is also meager. There are data suggesting that normal voided volumes are more common among desmopressin responders –38, but therapy, regardless of whether it is useful as a diagnostic tool or not. By documenting the micturitions in a chart it is probably easier to adhere to the voiding schedule according to the instructions given  predicts a likelihood that desmopressin will work . HoweverA problem here is that nocturnal urine production measurements, to be reasonably reliable as predictors of desmopressin response, need to be performed several times. Once or twice is not enough , 47. AndIt could also be questioned—given the less than perfect correlation between nocturnal urine production and desmopressin response—whether the absence of nocturnal polyuria in a child who does not respond to the enuresis alarm means that we should let anticholinergics or tricyclics be the next step before testing desmopressin. I think not.Although old psychodynamic explanations regarding enuresis pathogenesis are clearly obsolete it has been convincingly shown that children with enuresis—especially if they also have daytime incontinence and/or fecal incontinence—are more prone to behavioral problems or neuropsychiatric disorders than their nonenuretic peers , 48, 49.The central motivation for this recommendation is neither that we consider the psychiatric issues to be causative nor that treatment of them will by itself make the children dry. Instead it rests on the assumption that concomittant problems such as ADHD will negatively influence treatment response.This assumption does not seem unreasonable regarding the enuresis alarm or urotherapy—treatments that demand much active cooperation from the child. But there is no reason to believe that response to pharmacological treatment is affected .And although it seems fair to suspect that successful alarm treatment is difficult to achieve in a child who scores positive on a screening tool for, say, ADHD, this has not been put to the test in prospective studies. Intriguingly, in an American study comparing 95 enuretic children with ADHD and 95 children without ADHD no differences were found regarding alarm treatment results . Maybe tIt should be kept in mind that in many countries and settings child psychiatry and psychology are scarce resources. Can the healthcare system take care of all the new referrals, should this recommendation be followed? Thus, given the current state of evidence, I would suggest that if screening tools are used and indicate behavioral/psychiatric issues, then the child should not be automatically referred unless there are also substantial problems with social interaction apart from the wetting. Or perhaps wait until one serious alarm attempt has been tried and failed.This recommendation is also based on assumptions regarding detrusor overactivity, which is the main cause behind daytime incontinence . First, But we have no firm evidence for the truth of either the first or the second of these arguments. In fact, there are earlier studies indicating that it may be the other way around, i.e., that enuresis alarm treatment may work regardless of concomittant daytime incontinence , that itThe third argument, about the impact of daytime incontinence being greater for the child, may certainly be true but that's not for us to decide.Thus, pending new evidence, the best strategy is probably to let the families decide about which problem to address first. Or treat both conditions simultaneously. As the evidence now stands we have no grounds for delaying the enuresis therapy just because the child also wets during daytime.vice versa  may boost the child's self esteem, it should be noted that it is time-consuming for both the family and the healthcare provider.Based on these considerations it is fair to say that daytime urotherapy has no place in the initial treatment of children with enuresis.The purpose of this review has not been to criticize the experts behind the existing guidelines. I have myself been very active in the creation of the relevant ICCS documents a contribution which I do not regret. These documents have been based on the available evidence, and, whenever evidence has been unavailable, the collective clinical experiences and reasonable assumptions of the experts. My aim here has been to highlight the many existing uncertainties and the areas in which recent evidence contradict our previous assumptions. My views and recommendations are summarized in •The role (if any) of voiding charts in the evaluation of enuretic children•Desmopressin and alarm response in enuretic children with concomittant daytime symptoms•The effect of treatment for neuropsychiatric disorders on response to first-line enuresis therapy•Studies on the need for, or benefit of, treatment of non-bothersome constipation in children with enuresisIt should be obvious that there are many areas in which new research is sorely needed, and these include such basic questions as what to focus on during the primary evaluation and how to choose first-line therapy for vast numbers of children. Most of the research needed is not hi-tech or expensive, but the impact for the many affected children will be considerable. Here are some suggested fields that deserve further study:•Put less emphasis on the differentiation of enuresis into monosymptomatic and nonmonosymptomatic varieties•If voiding charts are used, make sure that families who don’t manage to complete them are not lost•Don't let concomittant daytime incontinence be a contraindication to enuresis treatment•Stop using (daytime) urotherapy as a treatment of enuresisBased on the available evidence today, I suggest that the following changes are made to the recommended enuresis management:•At the first visit, focus on warning signals that indicate serious underlying conditions •No need for voiding charts or measurement of nocturnal urine production•Do not let concomittant daytime incontinence delay enuresis therapy•Treat constipation only if it bothers the child or if there is also daytime incontinence•Start directly with alarm or desmopressin treatment according to family preferences•Seek the help of a psychiatrist/psychologist if the child has substantial problems with social interaction or school, but do not let this delay enuresis therapySituations with strained resources, for the families and/or the healthcare system, deserve special mention. Here, the perfect can be the enemy of the good. We cannot expect that all families of enuretic children who seek healthcare assistance for the first time are able to adhere to time-consuming or labor-intensive evaluation methods or therapies. Likewise, we cannot expect primary care healthcare professionals without expertise regarding the pediatric LUT to be able to conduct state-of-the art enuresis management the way we experts would do it. In this setting—awaiting new research findings—I suggest the following cornerstones of a simplified, “bare-bones” enuresis management strategy for primary care:This way, an immense benefit could be gained for millions of children while we keep doing research in order to make future management strategies more evidence-based.


LSD is an important transboundary disease affecting the cattle industry worldwide. The objectives of this study were to determine trends and significant change points, and to forecast the number of LSD outbreak reports in Africa, Europe, and Asia. LSD outbreak report data (January 2005 to January 2022) from the World Organization for Animal Health were analyzed. We determined statistically significant change points in the data using binary segmentation, and forecast the number of LSD reports using auto-regressive moving average (ARIMA) and neural network auto-regressive (NNAR) models. Four significant change points were identified for each continent. The year between the third and fourth change points (2016–2019) in the African data was the period with the highest mean of number of LSD reports. All change points of LSD outbreaks in Europe corresponded with massive outbreaks during 2015–2017. Asia had the highest number of LSD reports in 2019 after the third detected change point in 2018. For the next three years (2022–2024), both ARIMA and NNAR forecast a rise in the number of LSD reports in Africa and a steady number in Europe. However, ARIMA predicts a stable number of outbreaks in Asia, whereas NNAR predicts an increase in 2023–2024. This study provides information that contributes to a better understanding of the epidemiology of LSD. Capripoxvirus genus of the poxviridae family [Lumpy skin disease (LSD) is an emerging transboundary viral disease that is caused by the lumpy skin disease virus (LSDV), which belongs to the e family . Cattle e family , but some family . Arthrope family ,5,6,7. Te family ,9. It cae family . The Wore family .In 1929, the first outbreak of LSD occurred in Zambia, and in the next decade, the virus extended to sub-Saharan Africa . LSD wasChange point analysis and trend analysis are statistical methods that are generally utilized to determine and monitor the behavior of time series data . The terSeveral research publications have provided critical information on the global status and regional or country situation of LSD outbreaks. For example, the spread of LSD from Africa to Europe, the Middle East, and Asia ; the epiDisease forecasting utilizing well-accepted prediction methods is critical for developing strategic plans to monitor and prevent disease outbreaks. Predictions of COVID-19, which appeared in hundreds of publications, are a prime example of the widespread application of forecasting methodology ,36. ForeSystematically, LSD outbreak reports from various regions around the globe have been published continuously by the WOAH. For a better understanding of LSD epidemiology, the trends, change points of disease trends, and forecasts of LSD outbreaks are worth investigating. Thus, the aims of this study were: (i) to determine the trends and change points in the time series data, and (ii) to forecast the number of LSD reports based on data from Africa, Europe, and Asia.https://wahis.woah.org, accessed on 14 August 2020), were imported and analyzed. Based on the WOAH report file, the numbers of LSD reports are shown as biannual data. For instance, 2020 has two semesters, with the first semester covering total LSD reports from January to June 2020, and the second semester covering July to December 2020.In this study, data on the number of LSD reports in Africa, Europe, and Asia from January 2005 to January 2022, publicly available on the official WOAH website . The segments ,39.Given m segments of the time series data, change point detection based on this technique is achieved by minimizing the function :(1)∑i=1mThe ARIMA and NNAR models were utilized to predict the number of LSD reports over the next 3 years (2022–2024) for each continent. The ARIMA technique is based on the principle that future values of a time series are generated from a linear function of past observations and white noise terms . The ARIp, d, q), where ARIMA has three parameters, which can be written as ARIMA , with The NNAR model uses lagged values of the time series data as inputs to a neural network. For non-seasonal data, it has the notation NNAR  data differencing until the data become stationary, (ii) examining ACF and PACF for the differenced data and selecting potential candidate models, and (iii) comparing the selected models using the Akaike information criterion (AIC) [nnar function, an automatic algorithm in the forecast package, provides a procedure to determine the best-fitting NNAR model as output [The forecasting of LSD outbreak reports was carried out using R statistical software and the “dplyr”, “xts”, “tsbox”, “TSstudio”, and “forecast” packages. The on (AIC) ,44. Techs output .Additionally, the African data were split into two datasets: one covering the years 2005–2015 (training set) and another covering the years 2016–2020 . The training set was used to build an ARIMA and NNAR model, both of which were utilized to generate forecast values. Further, the forecasted values were compared to the actual ones in the validation set. In addition, error metrics, including mean absolute percentage error (MAPE), mean absolute scale error (MASE), and root mean square error (RMSE), were calculated using functions from the “Metrics” package in order to measure the predictive abilities of the ARIMA and NNAR models ,45. Overall, Africa had 29,966, Asia had 8837, and Europe had 2471 outbreak reports during the study period. Africa had an undulating trend during 2005–2019, and by the end of 2020, outbreaks had dropped sharply and remained consistently low, whereas Europe had a peak in 2016, a sharp decline in 2017, and then became stable, and Asia had three peaks throughout the period .n = 18,072), with the highest number occurring in 2014 (n = 1915). Ethiopia, ranked second, has been reporting outbreaks for several years.Regarding the top five African nations reporting the most LSD outbreaks , Zimbabwn = 524), observed in 2016. North Macedonia, Albania, Montenegro, Russia, and Greece were the top five European nations to report LSD outbreaks that year  during the whole study period, with the maximum in 2019. From 2013 to 2019, Turkey reported notably high numbers of LSD epidemics in 2014 and 2015. Iran had its highest number in 2019. During the period from 2021 to January 2022, Thailand had the highest number of LSD reports.In Asia , Oman haThe time-series data of the number of LSD reports have four change points for each continent. Technically, once the change points have been identified, the segments that correspond to them are represented. For example, the second segment is found between the first and second change points . In thisIt was observed that the fourth segment of the African data  had the For Europe, all four change points were detected during 2015–2017 . The firFor Asia, four change points and five segments corresponding to them were identified . The firp, d, q) and NNAR  notations obtained from the analysis are shown in The forecasting of LSD outbreaks in Africa, Europe, and Asia by ARIMA and NNAR is shown in Change points in LSD outbreak time series data provide information on times when significant changes occurred in the data, which is essential information for epidemiology, particularly in the temporal dimension. Forecasts of the number of LSD reports based on well-accepted forecast methods offer useful baseline data that can assist authorities with planning disease surveillance and prevention efforts.After the first outbreak in Zambia in 1929, the disease became prevalent in several regions of Africa . ZimbabwOur findings further show that the fourth segment  had the In Europe, four change points and only two segments were identified. These change points correspond to the situation of the disease in 2015–2016. The first change point was found in early 2015, when the first LSD outbreak occurred in Greece . The secIn Asia, Turkey reported numerous LSD outbreaks from 2013 to 2016 . The incIn this study, we applied ARIMA and NNAR models to forecast the numbers of LSD outbreak reports. Overall, the number of outbreaks in Africa is expected to be higher than that reported in 2020–2021, whereas the number of outbreaks in Europe is projected to remain consistent. Forecasts of LSD outbreaks in Asia show an increasing trend in 2023–2024 based on the NNAR model, whereas ARIMA predicts a larger number of outbreaks than what occurred in January 2022. Notably, the results demonstrate that the prediction capabilities of both ARIMA and NNAR models tested with African data are not highly accurate, which may be influenced by the limited number of observations employed for model training, A follow-up study with more observations would allow for the development of more accurate forecast model models. Moreover, our results revealed that the prediction abilities of the ARIMA and NNAR were approximately comparable. This could be due to the fact that the data set contains both linear and non-linear patterns, and, therefore, the strengths of one model may not provide an advantage over another ,45.Our forecasts offer authorities useful information that can be incorporated into strategies to monitor and prevent future LSD outbreaks. Of note, the forecasts are generated from past observations; thus, they do not account for any future situation or implementation. If interventions such as more effective control measures are adopted, it is likely that fewer outbreak reports will be received than anticipated. In this aspect, we suggest using the forecast numbers as basic information or benchmarks, with the goal of keeping the number of outbreaks below these figures.The current study has several limitations. We were unable to determine the seasonality characteristics of the number of LSD outbreak reports due to the biannual format of the available data. Accordingly, it would be advantageous for future research if the data from WOAH were made public in a monthly format. Moreover, it important to note that forecast results should be interpreted with caution. Because forecasts are based on previous observations and patterns, some interventions and changes in disease drives in the future, which may change the patterns, will have an impact on the actual disease occurrences, and, therefore, our forecast may be over- or underestimated. Furthermore, there may be underreporting of LSD outbreaks in some countries during certain periods, so the reports used in this study may not represent the actual situation. Moreover, forecasting was limited to two methods. Thus, follow-up studies to investigate other methods of forecasting the number of LSD outbreak reports are warranted.It is notable that LSDV isolates from outbreaks in some countries are genetically related ,62,63; tIn this work, we used a statistical approach to identify major changes in the data underlying LSD outbreak reports. Additionally, we utilized time series models to forecast the number of LSD outbreak reports in Africa, Europe, and Asia during 2022–2024. Although LSD outbreak reports in Africa appear to be decreasing since 2020, it is expected that the number of reports will increase slightly. The number of LSD outbreak reports in Europe is projected to continue the previous 5-year steady trend. Additionally, the forecast predicts an increase in the number of outbreak reports in Asia. These findings indicate that LSD remains a substantial threat to the cattle industry in various countries; thus, efforts should be made to monitor its spread within and between regions. Additionally, because LSD is regarded as a significant transboundary disease, strict disease prevention and control in every country are critical. Furthermore, coordination among nations to control and eradicate the disease is essential.


Pseudomonas aeruginosa (Pa) (108 CFU ml–1) was inoculated to wheat plants with and without foliar-applied MLEs at two different concentrations (MLE 1 = 1:15 v/v and MLE 2 = 1:30 v/v) twice at 25 and 35 days after seed sowing (50 ml per plant) after the establishment of drought stress. Results revealed that Pa + MLE 2 significantly increased fresh weight (FW), dry weight (DW), lengths of roots and shoot and photosynthetic contents of wheat. A significant enhancement in total soluble sugars, total soluble proteins, calcium, potassium, phosphate, and nitrate contents validated the efficacious effect of Pa + MLE 2 over control-treated plants. Significant decrease in sodium, proline, glycine betaine, electrolyte leakage, malondialdehyde, hydrogen peroxide, superoxide dismutase (SOD), and peroxide (POD) concentrations in wheat cultivated under drought stress conditions also represents the imperative role of Pa + MLE 2 over control. In conclusion, Pa + MLE 2 can alleviate nutritional stress and drought effects in wheat. More research in this field is required to proclaim Pa + MLE 2 as the most effective amendment against drought stress in distinct agroecological zones, different soil types, and contrasting wheat cultivars worldwide.Less nutrient availability and drought stress are some serious concerns of agriculture. Both biotic and abiotic stress factors have the potential to limit crop productivity. However, several organic extracts obtained from moringa leaves may induce immunity in plants under nutritional and drought stress for increasing their survival. Additionally, some rhizobacterial strains have the ability to enhance root growth for better nutrient and water uptake in stress conditions. To cover the knowledge gap on the interactive effects of beneficial rhizobacteria and moringa leaf extracts (MLEs), this study was conducted. The aim of this experimental study was to investigate the effectiveness of sole and combined use of rhizobacteria and MLEs against nutritional and drought stress in wheat. Nitrogen-fixing bacteria  Crop production is always under pressure to increase and sustain the food demands, considering the estimated increase in global population that might increase from the current 7.7 billion to approximately 9.6 billion in the year 2050 . MoreoveWater constitutes approximately 80–90% of the total biomass of herbaceous plants and is crucial in almost all plant physiological processes, a principal means of nutrients and metabolite transport . Water sThe induction of drought stress tolerance has been described by several physiological and biochemical changes . In spitSeveral microorganisms, mainly bacteria, colonize the plant root zone . The benMoringa oleifera Lam belongs to the Moringaceae family and native to the subcontinent (Moringa leaf extract (MLE), as a plant biostimulant in the foliar application, enhances the growth of plants that are grown even under abiotic stress conditions . Moringaontinent . Moringaontinent . These montinent . Sprayinontinent . This vaontinent .Triticum aestivum L.) is one of the most important cereal crops worldwide, considering its production and human consumption. Wheat supplements almost one-third of the total global population. Harvesting more yields to meet the future food demands of the increasing population is a major agricultural concern at all times. Different environmental factors contribute to crop yields  were studied solely on wheat grown at high temperatures. This study covers the knowledge gap in the combined use of NFB and MLE under normal and heat stress situations. In earlier studies, treating plants with rhizobacteria and the effects of foliar sprayed MLE were assessed solely on wheat grown under drought stress conditions by considering their ameliorative implications on various physiological and biochemical attributes but their combined implications are still not confirmed.Therefore, this study aimed to investigate the best treatment combination for alleviation of heat stress in wheat. Anaj 17 genotype was selected because it is one of the latest genotypes developed in the past 5 years in Pakistan and it performed well under abiotic stress conditions so we wanted to check its response to the application of MLE and rhizobacteria. It is hypothesized that the combined use of MLE and NFB might be a better approach than the sole application to improve wheat growth attributes under heat stress. This study was conducted to analyze the ameliorative and best co-application of applied amendments in alleviating the drought stress effects on wheat plants. It is hypothesized that MLE and rhizobacterial combined application might be an effective approach to improve wheat attributes than their sole applications in drought effects. In earlier studies, treating plants with rhizobacteria and the effects of foliar sprayed MLE were assessed solely on wheat grown under drought stress conditions by considering their ameliorative implications on various physiological and biochemical attributes but their combined implications are still not confirmed. Therefore, this study is conducted to analyze the ameliorative and best co-application of applied amendments in alleviating the drought stress effects on wheat plants. It is hypothesized that MLE and rhizobacterial combined application might be an effective approach to improve wheat attributes than their sole applications in drought effects.The wheat crop was sown at the experimental site of the Government College University Faisalabad . A greenhouse pot experiment was carried out from November to January. The experimental layout was a randomized complete block design (RCBD) that was replicated three times.–1), pH (water) 7.3; organic matter contents 1.38%; available N 0.032 ppm, available P 5.93 ppm, and available K 32.3 ppm.The clay loam soil (8–12 inches in depth) collected from the experimental site of the Government College University Faisalabad was air-dried and sieved through a 2-mm sieve. The collected soils were sterilized through solarization, by covering the soil with a thin layer of plastic sheet. The heat from the sun builds up the temperature of the soil to kill most of the bacteria, weeds, and pests . Some soA well-adapted wheat genotype Anaj 17, which performs well under abiotic stress conditions, was selected. Seeds were disinfected using 95% ethanol and washed using 70% sodium hypochlorite solution followed by rinsing with distilled water three times.Pseudomonas aeruginosa (Pa) (strain) [inoculation with Pa], (iii) MLE 1 [foliar sprayed MLE at 1:15], (iv) MLE 2 [foliar sprayed MLE at 1:30], (v) Pa + MLE 1 [inoculation with Pa bacteria + foliar sprayed MLE at 1:15], and (vi) Pa + MLE 2 [inoculation with Pa bacteria + foliar sprayed MLE at 1:30].The treatments (two sets of pots) were as follows: (i) control  2O), and diammonium phosphate  [24]. Initially, ten seeds were sown in each pot containing 12 kg clay loamy soil (25 cm diameter × 30 cm height), and after complete emergence, they were thinned to six plants per pot.At the start of the experiment, the soil was fertilized with a basal dose of N–P–K fertilizer (0.51–0.45–0.38 N–P–K g) using urea (46% N), sulfate of potash (50% K–7 were spread on Luria-Bertani (LB) agar plates at 37°C temperature and inoculated overnight. Bacterial growth was determined by measuring optical density at 600 nm using a spectrophotometer  was shifted to canopies for the imposition of drought stress. Well-watered conditions were maintained at 70 FC (70% field capacity), while drought stress was maintained at 45 FC (45% field capacity). The plants that remained were kept at different water regimes until harvesting.An aerobic PGPR strain of free-living soil nitrogen-fixing bacteria; Pa (Pa strain) isolated from the rhizosphere of wheat roots growing in local field areas by serial dilution method was used in this study. The serial dilutions up to 10otometer . Before –1 FW, carotenoids = 1.58 mg g–1 FW, total phenolics = 1.68 μmol g–1 FW, mg g–1 FW, nitrogen = 14.13 mg g–1 DW, phosphorous = 2.98 mg g–1 DW, potassium = 11.97 mg g–1 DW, and calcium = 16.8 mg g–1 DW. The extracts were freshly prepared before their application. Distilled water was sprayed on control plants in both applications. MLE was sprayed to a pot-grown wheat twice after 25 and 35 days of sowing date (50 ml per plant) after the establishment of drought stress. Tween-20 surfactant was used in a foliar spray (0.1% v/v).Fresh and disease-free moringa leaves were collected and rinsed with water. Notably, a 100-g leaf sample was extracted in 1 L distilled water (1:10 w/v) for 15 min . Later, Harvest was done after 45 days of planting. Root and shoot fresh weights (FWs) and lengths were measured immediately after harvest at the experimental site. Fresh samples were stored at -30°C in a biomedical refrigerator for fresh analysis. Three samples per treatment were oven-dried (65°C) for 3 days to determine their dry weights (DWs) and ionic content analysis using the acid digestion method.The chlorophyll contents of wheat leaves were determined as described by Stomatal conductance (gs) of fully developed leaves (three plants per treatment) was measured by putting them in a portable infrared gas analyzer chamber . The measurements were made 6 days after the first MLE foliar spray.Fully expanded leaves from each replicate were taken, wrapped in aluminum foils, immersed in liquid nitrogen, and transferred into plastic zipper bags. These samples were stored at –80°C for further analysis. Following, biochemical analysis was performed using a spectrophotometer .To determine osmolytes as sugars and non-enzymatic antioxidants, 50 mg of dried leaves were homogenized in 10 ml of 80% ethanol and filtered followed by the re-extraction in 10 ml ethanol, and a 20 ml of the final volume was maintained. This obtained solution was used to evaluate flavonoids , solubleFor the determination of P contents in wheat molybdate/ascorbic acid, the blue technique was used, and nitrate contents were assessed by post-hoc test, which was performed to measure specific differences between treatments using the Duncan’s Multiple Range Test (DMRT) in a completely randomized block design. The significant diffidences between treatment means were determined using analysis of variance and mean separation at a 5% significance level (p ≤ 0.05). In addition, the Pearson correlation of different wheat attributes under drought stress and well-watered conditions was performed. Logarithmic data transformation to obtain near-normal distribution was implemented before analysis, where required.Statistical analysis of data was performed using a All applied treatments had a significant positive effect on root and shoot FWs, DWs, and lengths under control and drought stress conditions . In thisAll photosynthetic pigments such as chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids were significantly affected when subjected to drought stress conditions (45 FC) as compared with well-irrigated conditions (70 FC) . All appFlavonoids, phenolics, total soluble sugars, total soluble proteins, and stomatal conductance of wheat plants were significantly affected in drought stress (45 FC) compared with well-watered plants (70 FC) . Both so2O2) increased significantly in plants subjected to 45 FC water conditions. Furthermore, antioxidant enzyme [superoxide dismutase (SOD) and peroxide (POD)] activities were increased in drought stress.Ionic nutrient contents increased significantly in plants that were irrigated at 70 FC compared with plants irrigated with water to reach only 45 FC . However2O2, and SOD and POD contents significantly. Also, they increased calcium, potassium, phosphate, and nitrate concentrations in plant tissues. It is worth mentioning in this study that sole and combined applications of either MLE or Pa were better in decreasing the oxidative stress indicators and increasing nutrient contents of wheat, and this signifies the success of these ameliorating treatments for drought stress. Pa + MLE 1 and Pa + MLE 2 recorded the least oxidative stress indicators and enzymatic antioxidant activities during more nutrient assimilation in plants.All additives had significant positive improvements in ameliorating drought effects in wheat. These amendments were more detectable when the dose of MLE foliar application was increased. Pa and MLE decreased sodium, proline, GB, EL, MDA, HP. aeruginosa inoculated plants, MLE 1 = foliar applied MLE at 1:15, and MLE 2 = foliar applied MLE at 1:30] .All values are the means of three replicates ± SD. Different labels represent significant different alphabets using the least significant difference (LSD) test. [70 FC = well-irrigated conditions, 45 FC = drought stress conditions; C = control, Pa (strain) = A significant positive correlation exists between plant morphological attributes and photosynthetic pigments . This miThis study evaluates the effect of MLE (MLE 1 = 1:20 v/v and MLE 2 = 1:30 v/v) and PGPR strain (Pa) on drought stress tolerance ability of wheat (Anaj 17 genotype) under two irrigation regimes (45 FC and 70 FC). The use of organic or biofertilizers as a global initiative instead of costly chemical fertilizers is addressed by several researchers . Plant-dBacillus sp. and Klebsiella sp. and concluded that inoculated plants have more DW than non-inoculated ones. In this sense, + available to the plants. Moreover, according to the study by The highest dry matter contents in both root and shoot were obtained with the combined application of Pa + MLE 2 mixture that agrees with previous findings describing that soil beneficial bacteria can promote plant growth in abiotic stress conditions. Pseudomonas sp. and Azospirillum sp.).Photosynthetic pigment contents are considered a valuable physiological indicator for evaluating the damage caused by stress intensity. The wheat genotype in this study had more chlorophyll contents when supplemented with Pa and MLE with the highest value recorded in their combined treatment at both water regimes. This alteration is directly related to plant macronutrient contents such as nitrogen. The reactive oxygen species (ROS) produced during abiotic stress cause the reduction of chlorophyll contents by damaging photosynthetic machinery . SimilarP. aeruginosa and MLE applied separately and in combination increased levels of sodium, calcium, potassium, phosphate, and nitrate in wheat submitted to drought. This might be due to the nutrient solubilization by added microorganisms due to the production of ACC deaminase  and foliar sprayed MLE either in sole or combined treatments can alleviate nutrient and drought stress effects. Nevertheless, the combined application of Pa and MLE 2 (Pa + MLE 2) can efficiently improve wheat growth attributes, photosynthetic pigment contents, and nutrient uptake under drought stress. The combined supplementation of Pa and MLE had more significant positive effects compared with their sole applications. Pa + MLE 2 was also efficient in decreasing oxidative stress indicators and enzymatic antioxidants of wheat under drought stress. There is a need for more investigations at field levels by considering the effect of other environmental constraints to effectively validate these findings.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.IL, SA-D, LA-A, RH, KAM, NM, SA, MA, AA, PP, KM, and TG: researching and writing. AA, SA, and LA-A: writing. 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. The reviewer FZ declared a shared affiliation with one of the author, RH, to the handling editor at the time of the review.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.


We investigated the incremental benefit of PET over the mDC in NVE.International guidance recognizes the shortcomings of the modified Duke Criteria (mDC) in diagnosing infective endocarditis (IE) when transoesophageal echocardiography (TOE) is equivocal. Dual-center retrospective study (2010-2018) of patients undergoing myocardial suppression PET for NVE and PVE. Cases were classified by mDC pre- and post-PET, and evaluated against discharge diagnosis. Receiver Operating Characteristic (ROC) analysis and net reclassification index (NRI) assessed diagnostic performance. Valve standardized uptake value (SUV) was recorded.69/88 PET studies were evaluated across 668 patients. At discharge, 20/32 had confirmed NVE, 22/37 PVE, and 19/69 patients required surgery. PET accurately re-classified patients from possible, to definite or rejected , with significant incremental benefit in both NVE (AUC 0.883 vs 0.750) and PVE (0.877 vs 0.633). Sensitivity and specificity were 75% and 92% in NVE; 87% and 86% in PVE. Duration of antibiotics and C-reactive Protein level did not impact performance. No diagnostic SUV cut-off was identified.PET improves diagnostic certainty when combined with mDC in NVE and PVE.The online version contains supplementary material available at 10.1007/s12350-021-02689-5. Infective endocarditis (IE) is classically viewed as a rare diagnosis, however its incidence has risen since the turn of the century, associated with a surge in cardiovascular intervention in an aging population.218F-Fluorodeoxyglucose positron emission tomography with computed tomography  to aid in the diagnosis of prosthetic valve endocarditis (PVE) has been increasingly reported with high sensitivity and specificity,12The use of Barts Heart Centre (BHC) was formed in May 2015 following a merger of the cardiology departments of Barts Health NHS Trust and University College London Hospitals (UCLH) NHS Trust. This merger made BHC the single cardiac surgery referral center for North Central and East London, and resulted in a significant increase in the number of IE cases seen. In line with the European Society of Cardiology (ESC) IE guidelines, this prompted formalization of the UCLH model to form an Endocarditis Team.Under terms of an overarching audit, a dual center retrospective review identified all patients undergoing PET for IE from January 2010 to December 2018. Patients imaged early in the post-operative period following valve surgery for IE (< 3 months), those with CIED-only IE, and studies with failure of myocardial suppression were excluded  in consensus. The definitive discharge diagnosis was recorded by surgical specimen in those who underwent operative intervention, or by Endocarditis Team consensus in those medically managed (excluding the PET findings).ad hoc bedside discussion.The Endocarditis Team review all cases of IE referred to our Institution on a weekly basis. Prior to the formation of the BHC team, the core members led in the clinical care of patients with IE at UCLH via 18F-FDG (4.5 MBq/kg) at a mean time of 64 minutes (SD 13 minutes) (mean activity 157 ± 39 MBq), we performed combined imaging with an MI DR PET-CT scanner . An unenhanced, ungated CT was performed  from vertex to thigh for attenuation correction. A subsequent PET was performed at a bed overlap of 49% and a time per bed position of 100s. The reconstruction method was VUE Point FX, with 2 iterations, 12 subsets and a 5 mm Gaussian filter. All studies were standardized for display and reading with an SUV window threshold of 0-10All patients underwent myocardial suppression technique to suppress metabolic activity in the myocardium. This was achieved using a > 24-hour high fat, carbohydrate-restricted diet, a > 12-hour fast and intravenous injection of unfractionated heparin (50 IU/kg) 60 minutes prior to assessment with PET.PET images were read in a blinded fashion by two independent investigators (CP & LM) with joint reading to resolve discrepancies in consensus, using attenuation and non-attenuation corrected images (the latter in particular for PVE). Myocardial suppression was graded as good, fair, poor or non-diagnostic,Studies were assessed for avidity of the culprit valve. The pattern and distribution of avidity was categorized as focal, heterogenous, homogenous or none . An overall verdict (yes/no) was given on a case-by-case basis as to whether the PET was suspicious for IE or not, with a study considered positive if uptake was either ‘focal’ or ‘heterogenous’. Note was made if PET suggested an alternative diagnosis.An elliptical region of interest (ROI) was placed over the valve, mediastinal blood pool and liver for semi-quantitative assessment of avidity using absolute mean and maximum standardized uptake values (SUV) allowing target-to-background analysis. In addition to avidity at the level of the cardiac valves, note was made of the presence of extracardiac uptake .Studies were analyzed using the freely available Horos (version 3.3.5).χ2 for categorical data. Diagnostic performance was evaluated using Receiver Operating Characteristic (ROC) analysis and net reclassification index (NRI). Respective sensitivities, specificities, positive and negative predictive values for PET in both native and prosthetic valve disease were calculated using the discharge diagnoses, categorized as either confirmed or rejected IE as described above.GraphPad Prism (version 7.0) and SPSS (version 25) were used for statistical analyses. Descriptive statistics were calculated for continuous variables, and PET was undertaken in 88/668 patients, with 69 studies 10.3%) eligible for inclusion; 59/404 (14.6%) following the formalization of the Endocarditis Team in October 2015. The cohort featured 48 male patients, with an overall mean age of 61 years (range 21–89 years). Thirty-two (46%) were native valve patients and 37 cases had prosthetic valves, of which 20 were tissue and 17 mechanical prostheses. All patients, except one NVE patient, underwent assessment with TOE . Further baseline characteristics are described in Table 0.3% eligStaphylococcus aureus was isolated in 20/69 (29.0%) patients; 18/69 (26.1%) were peripheral blood culture-negative (BCNIE)  had confirmed NVE and 22/37 (59%) confirmed PVE, giving a total prevalence for IE in our cohort of 61%. Nineteen (28%) patients required surgical intervention, 9 (28%) NVE and 10 (27%) PVE, with the remaining 50/69 cases managed medically, as per European Society of Cardiology (ESC) and British Society for Antimicrobial Chemotherapy (BSAC) guidance.18P > 0.10). Mean CRP at the time of PET was 38.6 (SD ± 29.8) mg/L in 20/32 NVE cases and 39.3 (SD ±4 5.4) mg/L in 29/37 PVE cases. Prolonged duration of antibiotic therapy was associated with a downward trend in CRP at time of PET . There was no difference in PET performance with CRP < 40 and ≥ 40 in NVE, PVE or overall (P > 0.10).12The median duration of IE-targeted antibiotic therapy pre-PET was 20.6 (IQR 9.5-25.0) days in 30/32 NVE cases and 17.0 (IQR 11.5-33.0) days in 33/37 PVE cases. Categorical analysis of median duration of antibiotics showed no significant impact on PET performance in NVE, PVE or overall , there were 9 episodes of further IE in 8 patients, with 1 treatment failure in a medically managed patient unfit for surgery. Median IE free duration was 379 days in these individuals (range 28-1095 days). Of these episodes, 5 required surgical intervention during the index admission and four were managed medically. Independent review of mDC post-PET and discharge diagnosis made by the Endocarditis Team showed PET to have correctly confirmed or refuted IE in all cases.P < 0.001) and PVE  compared to discharge diagnosis  were excluded from analysis due to complete failure of myocardial suppression, rendering the scans non-diagnostic Figure . All rems Figure . PET senmax or SUVmean, nor when these parameters were normalized to hepatic and mediastinal blood pool uptake  cases of NVE and 18 49%) in PVE Table . Diffuse9% in PVEThe climbing incidence of IE, and clear benefits of the Endocarditis Team mandate a guideline-driven approach to the diagnosis of IE.1217IE remains a diagnostic challenge, especially in the absence of positive major criterion of the mDC. In our cohort, all patients had ongoing clinical suspicion of IE following equivocal TOE in 68/69 cases , and 26% with likely BCNIE Table , as wellOur data add further weight to the growing body of evidence advocating the sensitivity and specificity of PET in PVE, and corroborate our ability to utilize PET appropriately in IE. However, we have now been able to additionally demonstrate comparable sensitivity and specificity in NVE, at 75% and 92%, respectively, as with one similar series to date.8Given the lack of typical TOE findings of significant valvular insufficiency and/or presence of vegetations in this cohort, it is unsurprising that fifty patients (72%) lacked a surgical indication and were managed medically. This is despite 99% of patients having at least one TOE, a high rate compared to 81%-88% of patients in other studies. Identifying the factors responsible for the high performance of PET in the current cohort is critical. Factors thought responsible for the low sensitivity of PET in NVE are well summarized in the literature, from both technical limitations of the modality as well as the pathophysiology of NVE.Achieving adequate myocardial suppression is imperative to the successful use of PET in IE, with ~ 85% of suppression graded as good or fair in our cohort, and only 3/72 (4%) studies found to be non-diagnostic and therefore excluded from analysis. Even when myocardial suppression was poor, meaningful valvular assessment was still possible in this cohort based on visual analysis. This is in comparison to failure of myocardial suppression in 5%-32% of the cohort in similar studies of NVE and PVE.2318F-FDG.23In our practice, PET is used where the diagnosis remains unclear despite 99% utilization of high-quality TOE (with lower rates in other series), including in patients with NVE. This is particularly important as this group of patients typically lack valve findings that mandate early surgery, hence diagnostic equipoise. Our approach is to therefore repeat echocardiography prior to PET, in order to easily identify those in whom valve dysfunction may have developed. However, this is reflected in the long median duration of antibiotics  prior to PET and downward trend in CRP  when compared to other studies.We would suggest the use of PET in IE requires involvement of an Endocarditis Team with high volume throughput. This will both optimize case selection and improve technical reading of the study. This is particularly important to correctly distinguish valve from myocardial tracer uptake, and recognize patterns of uptake typical and atypical for IE, especially following previous cardiac surgery Figure . Our anaThe results of this study suggest that the real-world application of PET to patients with IE has meaningful benefit. Nonetheless, a formal prospective multicenter diagnostic accuracy study with hard endpoints is warranted, and we would argue should include patients with both NVE and PVE.Advances in IE have been hindered by low incidence and a lack of randomized trials as a result.The incidence of IE and the limitations of the mDC may cause bias when relying on expert consensus to confirm or refute IE in medically managed patients, and is a significant issue in all IE studies without surgical specimens. However, this is where the guidance currently supports the use of PET, where the diagnosis is not clear, and therefore surgical intervention is not necessarily mandated. Blinded scoring of the mDC and imaging analysis by the research team reduce the limitations of expert consensus in our study, and is supported by net reclassification following PET, highlighting the benefit of PET overall and for reclassification of individual patients. This impact is further mitigated by follow-up data that suggest the correct diagnosis was made, especially given a low incidence of recurrent episodes of IE over 212 patient-years.Despite these limitations, the incremental benefit of PET in both NVE and PVE described herein suggests meaningful benefit. However, we would only advocate the routine use of PET where the diagnosis is equivocal after high-quality TOE and surgery is not mandated for another indication.In this retrospective analysis, we highlight the incremental benefit of PET for the diagnosis of IE in both native and prosthetic disease. PET performs well irrespective of inflammatory markers or duration of IE-focussed antibiotic treatment. We advocate the use of PET by expert Endocarditis Teams where both NVE and PVE is suspected, but TOE remains equivocal.The literature consistently identify poor sensitivity of PET/CT for NVE. We highlight that in a high-volume center, PET can be used to contribute to the diagnosis of both NVE and PVE in a meaningful manner. PET provides meaningful information at valve level in PVE and NVE, to help confirm and refute the diagnosis (NRI), outperforms mDC alone (AUC) and has higher than reported sensitivity in NVE. We further explore duration of antibiotics, CRP at time of PET and time to PET to explain why our findings differ to the rest of the literature.Supplementary file1 (PPTX 3408 kb)Supplementary file2 (PPTX 3221 kb)Below is the link to the electronic supplementary material.


N = 290) were collected from farms (N = 63), collection centers (N = 5), shops/kiosks (N = 37), milk bars (N = 17), roadside vendors (N = 14), restaurants (N = 3), milk vending machines (N = 2), mobile traders (N = 2) and a supermarket (N = 1). Mean values of colony-forming units for TBC and TCC were referenced to East African Standards (EAS). Logistic regression analysis assessed differences in milk acceptability based on EAS. The raw milk from farms and collection centers was relatively within acceptable EAS limits in terms of TBC (3.5 × 105 and 1.4 × 106 respectively) but TCC in the milk from farms was 3 times higher than EAS limits (1.5 × 105). Compared to farms, the odds ratio of milk acceptability based on TBC was lower on milk bars (0.02), restaurants (0.02), roadside vendors (0.03), shops/kiosks (0.07), and supermarkets (0.17). For TCC, the odds that milk samples from collection centers, milk bars, restaurants, roadside vendors, and shops/kiosks were acceptable was less than the odds of samples collected from farms . Comparison of raw milk across the nodes showed that the odds of milk samples from restaurants, roadside vendors, and shops/kiosks being acceptable were less than the odds of samples collected the farm for TBC . For TCC, the odds of raw milk from collection centers, restaurants, roadside vendors, milk bars, and shops/kiosks being acceptable were lower than the odds of acceptability for the farm samples . Practices with possible influence on milk bacterial quality included muddy cowsheds, unconventional animal feed sources, re-use of spoilt raw milk, milk adulteration, acceptance of low-quality milk for processing, and lack of cold chain. Therefore, milk contamination occurs at various points, and the designing of interventions should focus on every node.Food networks present varying food safety concerns because of the complexity of interactions, production, and handling practices. We investigated total bacteria counts (TBCs) and total coliform counts (TCCs) in various nodes of a Nairobi dairy value chain and identified practices that influence food safety. A value chain analysis framework facilitated qualitative data collection through 23 key informant interviews and 20 focus group discussions. Content thematic analysis identified food safety challenges. Cow milk products ( The global dairy industry, comprised of ~265 million cows, has continued to grow over the past decade, with milk production increasing from 590 million tons in 2009 to 683 million tons in 2018 . For exaIncreased demand for milk consumption coupled with its predicted low supply will put pressure to existing value chains, and this will trigger the evolution of more milk supply chains, which will complicate already complex food systems. Food systems present some of the most complicated networks, especially in urban areas where production and distribution are through simple to complex value chains [–8; HuestAccording to the national livestock production report of 2012, Nairobi, one of the fastest-growing urban cities in Africa  producedBased on the annual per capita consumption and projected growth rate of the city, approximately at 4% , it meanAchieving food safety in such complex food systems is a challenge, particularly because milk is produced primarily by small-scale farmers, and marketing channels are dominated by informal systems –20. The There are many factors that may contribute to unsafe milk , and chaMycobacterium species , Kenya. The overall objective of the Urban Zoo project was to understand mechanisms leading to the introduction and transmission of pathogens to urban populations through livestock commodity value chains in 33 sub-locations of Nairobi. Most of the qualitative data used in this study were collected during the mapping of the dairy value chain  implemenTo comprehensively map the bacteriological (TBC and TCC) landscape of milk in Nairobi, milk sampling was conducted on nodes that were identified during the initial mapping by Kiambi et al. . These iThe selection of participants for focus group discussions (FGDs) was facilitated by government animal health assistants (AHAs) in the sub-county of focus. This, however, was in contrast to the FGD with KDB licensing officers, which were organized by the KDB head office and the Kibera FGDs, which were facilitated by a community mobilizer . The guidance on selection of participants was given to the AHAs/community mobilizer such that for each group there was adequate gender representation, wider geographical coverage of the people , and participant's vastness in understanding of the dairy systems in the area. Each group of participants was selected based on their specific type of business/enterprise as described by the stakeholder analysis. The various FGDs that were conducted included dairy cow farmers from Kibera, an urban informal settlement), farmers in Kikuyu and Dagoretti (peri-urban areas) dairy cooperatives, traders not associated with DTA, retailers, LPOs, PHOs, KDB officers in charge of licensing and the city council of Nairobi. Key informant interviews were conducted with managers of a feed manufacturing company, officials of DTA, various managers of large processing companies , KDB, and representatives from the Directorate of Veterinary services and Department of Livestock Production.Twenty FGDs with 105 people and 23 key informant interviews (KIIs) with 35 people were conducted. From each of the FGDs, a checklist with open-ended questions was used for data collection. Qualitative data were collected on: (i) production practices including housing of the livestock, sourcing of livestock feeds, management of animal health, management of milk obtained from cows undergoing treatment, (ii) milk handling practices in bulking centers including assurance of milk safety/quality, management of milk that has been rejected for poor quality or spoilage, transportation of raw milk from farms, and processing practices that could influence food safety, (iii) practices at a retail level including sourcing of milk, transportation, value addition, management of spoilt milk, and (iv) waste management and food safety management practices. In addition, all the participants were asked to describe challenges that they or other people working in the milk system experienced that would lead to compromise in food safety. The information gathered through FGDs was triangulated during KIIs. When discrepancies were detected, additional interviews were conducted with other experts working in or conducting research on the dairy value chain.In addition to data from the FGDs and KIIs, epidemiological data were collected from nodes of the value chain where milk samples were collected. In each node, a pretested questionnaire was administered to collect data on: (i) type of node, (ii) amount of milk handled per day, (iii) type of milk and milk products handled, (iv) main sources of milk, (v) methods of milk preservation, and (vi) costs related to buying and selling of milk. Documentation of the interview processes was aided by video and voice recording (following consenting of the participants) and taking notes.http://www.wikihow.com/Create-a-Random-Sample-in-Excel) to facilitate random selection of one sub-location from a peri-urban area and another from an informal settlement to represent milk chains in the two different settings. The Uthiru and Korogocho areas (2 (The selection of sampling sites for this study involved a process whereby the 33 sub-locations (where the Urban Zoo project was working) were entered in a Microsoft Excel worksheet  in the sub-location was aided by the area administrative officer (chief). These segments included various production units (farms), collection centers, distributors, and milk retailers. The nearest dairy farm from the chief's office was the first to be enrolled in the study upon obtaining consent. Subsequently, the next nearest farm in the same village was identified, and the procedure was repeated up to a maximum of four farms within one single area in a village. This was considered cluster one. Then, the research team moved about 200–300 m in the Uthiru area and about every 50 m in the Korogocho area (because of much higher household density) from the first cluster to another within the same village, and the process was repeated. This was done throughout the village until the teams got to the start point before proceeding to the next village where the same procedure was repeated. For retailers, up to four milk vendors identified between and within the clusters and between the villages were enrolled. These included shops/kiosks, restaurants, milk bars, roadside vendors, supermarkets, automated milk machines, milk collection centers, and mobile traders (hawkers).A biological sampling of cow milk was conducted in farms, milk bars, shops/kiosks, supermarkets, restaurants, roadside vendors, milk vending machines, and milk collection centers. Different milk types that were sampled included: raw, pasteurized liquid milk, ultra heat treated, fermented, and yogurt. In farms, milk samples were collected only in morning hours when the temperature was cool. A farmer was requested to obtain about 50 ml of milk directly into a sterile barcoded falcon tube. However, if the farmer was unable to milk for whatever reason, they were requested to provide whatever remained from the last milking. To obtain about 50 ml of milk from the other nodes (retail and bulking centers), participants were requested to transfer the sample directly into the sterile barcoded falcon tubes. However, if the milk was in packets or sealed bottles, the entire content was purchased. All the milk samples were immediately placed in a cool box that was packed with ice packs and transported to the laboratory within 2–4 h of collection.−1-10−4) were prepared in a sterile phosphate-buffered diluent (0.0425 g of potassium dihydrogen phosphate per L of distilled water), pH 7.2. Enumeration of TBC was conducted using sterile standard plate count agar (SPCA);  that was prepared according to the manufacturer's instructions. One milliliter of an undiluted milk sample and each of the four serial dilutions were aseptically pipetted into a separate sterile pre-labeled disposable 90-mm diameter Petri dish on which freshly prepared agar was poured. The mixture (sample plus media) was gently but thoroughly mixed by whirling to ensure even distribution of the sample into the culture medium. The content was left to solidify at room temperature, and the plates were incubated at 32°C for 48 h. This was followed by an assessment of plates that had countable colony-forming units (CFUs). Plates that had between 25 and 250 CFUs per plate were selected for enumeration.For the determination of TBC, samples were prepared according to the protocol described by Christen et al. . For eac®) guided by the manufacturer's instructions. Culture and isolation were carried out as described elsewhere . Data entry was complemented with data collected in notebooks. The first step was to collate data in pre-formatted word documents. This allowed for systematic organization of the emerging food safety themes. The second stage of analysis entailed thorough reading of the templates and organization of the data in distinct sections based on the emerging food safety themes, which were categorized as challenges. These included a category on what practice(s) was of food safety concern, who said it (during the interview), where the practice(s) was mentioned to occur, and why the practice was said to occur. To comprehensively explore factors that may impact food quality and safety, the qualitative analysis contextualized the main practices that were mentioned in milk production, bulking centers, processing, transportation, and retailing.Data cleaning, coding, and analysis were conducted in Stata 16 . DescripA logistic regression analysis was performed to detect differences in milk TBC and TCC between various nodes and milk types. Two binary outcome variables were used as an indicator of whether a sample was acceptable or not based on levels according to the EAS standards  for TBC Model coefficients are reported at odds ratios (OR) where coefficients above 1 indicate increase in odds and coefficients below 1 indicate decrease in odds. Because of the clustered nature of the data , the variance-covariance matrix corresponding to the parameter estimates was specified using a clustered sandwich estimator, i.e., vce (cluster) command in Stata. This estimator allowed us to account for intragroup correlation for the estimation of standard errors. The model specification was performed in Stata 16.1 .Ethical approval for this study was obtained from the ILRI Institutional Research Ethics Committee (project reference: ILRIIREC2014–04/1). ILRI IREC is accredited by the National Commission for Science, Technology, and Innovation (NACOSTI) in Kenya. Ethical approval was also obtained from the Royal Veterinary College ethics committee (project reference: URN 2013 0084H).N = 82) were women. The age of participants ranged from 18 to 86 years, with a mean age of 41.69 and mode of 45 years. Most of the respondents (≈85%) reported to own an enterprise, while the rest were either employees (≈12%) or relatives (≈3%). For those who kept cows (farmers), the majority of people (≈84%) reared between 2 and 3 milking cows, followed by those keeping 6–9 cows (≈13%). Only a small proportion of farmers (≈3%) kept 10–13 milking cows. In terms of volumes of milk handled per day, the majority of people (≈59%) reported to handle between 0.5 and 20 L of milk, followed by 21–100 L (≈34%), while only a small proportion (≈7%) handled more than 100 L per day.One hundred and forty four people were interviewed during milk sampling. Of these, 56.9% (N = 63), milk collection centers (N = 5), kiosks (N = 37), milk bars (N = 17), roadside vendors (N = 14), restaurants (N = 3), mobile traders (N = 2), milk vending machines (N = 2), and a supermarket (N = 1). The different types of samples collected included raw milk (N = 203), homemade fermented milk (N = 12), pasteurized milk (N = 35), ultra heat-treated milk (N = 13), processed yogurt (N = 13), and processed fermented milk (N = 11).Two hundred and ninety (290) cow milk samples were collected from various nodes represented by the respondents. These included farms (About 44% of the milk from which these samples were obtained was described to have come from within Nairobi County. The rest (≈50%) was described to be sourced from Kiambu County (peri-urban area neighboring Nairobi), about 3.4% from further rural areas, and a small proportion of milk (≈2.6%) was of unknown origin. Delivery of milk from various sources was reported to be mainly (≈88%) direct (own cows or own transport). The rest reported to have milk delivered by traders (≈9%), dairy cooperatives (≈3%), and a small proportion (≈0.5%) by processors (processed products). Information regarding recent use of antibiotics in cows from which samples were obtained was not known by 50% of the respondents, while the rest of the participants said that antibiotics had not been used in cows for about 2 weeks prior to the sampling.5 cfu/ml, at grade II of EAS (>2 × 105–1 × 106), while that from collection centers was within the limits, at grade III of EAS (1 × 106–2 × 106) at 1.4 × 106. This shows that the mean values for the milk from farms were better in terms of TBC than that from collection centers. Furthermore, the bacterial quality of milk deteriorated at the retail level . The liquid milk from all nodes except for pasteurized and UHT had higher mean values than the raw milk from farms which had a mean of 3.5 × 105 cfu/ml. For example, cfu/ml mean value for the raw milk from collection centers was four times higher than the EAS limits recommended for raw milk from farms, 11.4 times for restaurants, 12.3 times for milk bars, 22.6 times for roadside vendors, and 9.4 times for milk collected from shops/kiosks. Mean values for processed (pasteurized and UHT) products were within the EAS limits. When comparing the mean values of milk samples that had values with unacceptable EAS standards, the milk from roadside vendors was worst in terms of TBC, followed by milk bars, restaurants, and shops/kiosks in that order.3–5 × 104), the TCC in milk from farms was three times higher, indicating unacceptable contamination of milk, which should not be sold to consumers. Quality of milk from roadside vendors was 13 times poorer than the EAS limits, 8.2 times poorer for milk from milk bars, 3.2 times poorer for milk from shops/kiosks, and 1.6 times poorer for milk from restaurants. Hence, the worst raw milk in terms of TCC when compared to EAS was from roadside vendors, milk bars, shops/kiosks, farms, and restaurants, in that order.For analysis based on the type of nodes, the reference node type was farm so coefficients represent differences between a farm and a node type. For TBC, the results show that milk samples from milk bars and restaurants were less acceptable than samples collected from farms (odds 0.02). Similarly, the odds of samples from roadside vendors, shops/kiosks, and supermarkets  being acceptable were less than the odds of samples collected from farms . For TCCAn analysis comparing the TBC in raw milk from various nodes of the value chain showed that the odds that milk samples from restaurants and roadside vendors were acceptable were less than the odds that samples collected from farms were acceptable (0.03). Similarly, the odds that samples from milk bars and shops/kiosks were acceptable were lower than the odds of milk samples from farms . For TCC, the results showed that the odds that milk was acceptable from various nodes were all lower than the odds of acceptability for farm samples. That is, 0.12 for samples from collection centers, 0.12 for restaurants, 0.02 for roadside vendors, 0.06 for milk bars, and 0.05 for samples from shops/kiosks .For the analysis based on type of milk, the reference was raw milk . There wThere were several practices that were mentioned during key informant interviews and FGDs that could possibly influence food safety . In prodsince not every farmer will have bad milk or will have used antibiotics at the farm, the good milk will neutralize the bad milk, and overall all the milk will be fairly good. So we don't reject all that need to rejected except when it is grossly curdled or dirty. Our competitors who don't care about quality, especially the informal traders will be waiting for it, and they will sell it, since milk market is ever ready.” Milk cooling and basic screening tests were said to be lacking in most collection centers, with screening relying on organoleptic tests. On disposal of milk that had been rejected in bulking centers, dairy cooperatives and large processors reported that such milk were sent back to suppliers, and they reported that some of it was returned back to the food chain.In bulking centers, the FGD and KII with dairy cooperatives and some large processors reported that they sometimes, especially during milk scarcity, accepted milk that should be rejected. They argued that rejection of milk in such periods of scarcity and in the midst of the liberalized dairy sector would set their competitors at an undue advantage of selling what they rejected. One of the managers in the bulking centers argued that bad milk would be neutralized (unacceptable contents would be diluted) by good milk. He said, “In retail, milk adulteration by addition of water was reported to be a frequent occurrence aiming at increasing the volume of milk. This was reported to occur mainly during dry seasons when milk production was low. According to traders and retailers, the practice was mentioned to occur at the farm level, in traders (those selling milk to retailers), retailers, including roadside vendors, milk bars, restaurants, and in shops/kiosks. Another food safety challenge mentioned in retail was the lack of cooling facilities during transportation and at sale points. Raw milk that got spoilt either in transit or at sale points was said to be sold either as fresh liquid milk at a price than lower than that of raw milk, or was converted into yogurt or fermented milk. Fermented milk is basically prepared by letting the raw milk that had curdled to stay for a few more days in a container to ferment more, while the preparation of yogurt entailed the addition of flavors and color to the raw fermented curdled milk. Finally, there was a glaring gap in regulation and enforcement of food safety practices in the value chain, as several businesses (traders and retailers) were reported and observed to be operating without necessary government permits and licenses.By 2050, the demand for milk consumption will triple in Sub-Sharan Africa , with coThis study utilized the previously developed framework of Nairobi's dairy value chain  to invesThis study found that TBC levels in production nodes (farms and collections centers) were generally good and within the acceptable limits of the EAS for TBC, but that TCC limits were higher (than EAS) in farms. This agrees with several other studies that have demonstrated good bacterial quality of milk at farm levels , 66, 67.We noted that the bacterial quality of milk deteriorated at retail level. For example, the logistic regression analysis for TBC showed that the odds that milk samples from retailers were acceptable were less than the odds of samples collected from farm as follows: 0.02 (milk bars), 0.02 (restaurants), 0.07 (shops/kiosks), 0.03 (roadside vendors), and 0.11 (supermarkets). For TCC, the model results indicated that the odds of milk samples from milk collection centers were acceptable were less than the odds of samples collected from farms (0.18), and that the odds of milk from milk bars and restaurants were less than the odds of farm samples . Similarly, the odds of samples from roadside vendors and shops/kiosks being acceptable were less than those of samples collected from farms . Our results agree with findings from other studies that show such bacterial deterioration of milk as it flows from farms through the retailing system , 42, 74.accidentally curdled,” and it was reported to be converted into fermented milk  or yogurt (addition of flavors and colors to the fermented milk). This probably explains the high TBC and TCC in homemade fermented milk and homemade yogurt. On the regression analysis, our results indicated that the odds that the homemade (yogurt and fermented) and processed (yogurt and fermented) types of milk were acceptable for TBC was 97 and 90% lower, respectively, than raw milk, while for TCC, the homemade (yogurt and fermented) had lower odds (94%) than raw milk. While most Kenyans normally boil milk before consumption , processed fermented milk (2.7 × 101), and processed yogurt (5.6 × 104). Some practices elucidated during FGDs and KIIs with the dairy cooperatives and large processing companies that may contribute to high bacterial load in processed milk included lack of cold chain in collection centers and acceptance of milk that should be rejected so as not to benefit competitors who care less about quality. Unfair competition among value chain actors has been identified as one major factor that hinders achieving optimal food safety in the Nairobi dairy value chain  were acceptable were less than the odds of samples collected from farm being acceptable for TBC and TCC. Likewise, the odds that raw milk samples from retail  were acceptable was less than the odds of raw samples collected from farm being acceptable for TBC and TCC. With raw milk as the reference, the analysis by milk type showed few differences in the odds of TBC and TCC acceptability across milk types.Several practices with possible influence on food safety were mentioned. In production, these were related to keeping cows in very muddy cowsheds, obtaining animal feeds from dumpsites and market leftovers, obtaining feeds by the roadside, treatment of cows by unqualified personnel coupled with compromise on withdrawal periods following treatment, resale of milk that has been rejected from dairy cooperatives or allowing it to ferment further (and taken as fermented milk), and addition of water to increase the volume of milk. In bulking centers, the practices were related to accepting milk that should be rejected, lack of cold chain and basic screening tests, and lack of procedures for the management of milk that has been rejected. In retail, there was milk adulteration by addition of water and other chemicals, lack of cooling facilities during transportation and at sale points, sale to consumers of raw milk that got spoilt, and conversion into yogurt or fermented milk.The analytical methodology presented in this study demonstrates a practical approach for strategic policy decisions. To achieve milk quality and safety, the authors suggest the implementation of more robust training of people involved in the milk system. However, this needs to be guided by a critical analysis of prevailing challenges in every segment of the value chain. Every node of the value chain should be considered prior to designing and implementing any intervention, as further underscored in Kiambi et al. , FAO 2626, and Ahttps://doi.org/10.17638/datacat.liverpool.ac.uk/1639.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: Data repository of the University of Liverpool available at SK, EF, PA, JR, and EK designed the study and data collection tools. SK and JM collected data. SK, JO, PA, and EF drafted the manuscript. SK, GA, and NG developed the culture and isolation standard procedures, facilitated culture and isolation and interpreted TBD and TCC results. All authors read, commented on, and approved the final manuscript for publication.This study was supported by the United Kingdom Medical Research Council, Biotechnology and Biological Science Research Council (United Kingdom), the Economic and Social Research Council (United Kingdom), the Natural Environment Research Council (United Kingdom), through the Environmental & Social Ecology of Human Infectious Diseases Initiative (ESEI), grant reference: G1100783/1. This study also received support from the CGIAR Research Program on Agriculture for Nutrition and Health (A4NH) led by the International Food Policy Research Institute (IFPRI).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.