Malaria in the Amazon is often perceived as an exclusively rural disease, but transmission has been increasingly documented within and near urban centers. Here we explore patterns and causes of urban-to-rural mobility, which places travelers at risk of malaria in Mâncio Lima, the main malaria hotspot in northwestern Brazil. We also analyze rural-to-urban mobility caused by malaria treatment seeking, which poses an additional risk of infection to urban residents. We show that the rural localities most frequently visited by urban residents–typically farming settlements in the vicinity of the town–are those with the most intense malaria transmission and also the most frequent source localities of imported malaria cases diagnosed in the town. The most mobile urban residents are typically poor males 16 to 60-years old from multi-sited households who lack a formal job. Highly mobile residents represent a priority target for more intensive and effective malaria control interventions, that cannot be readily delivered to the entire community, in this and similar urbanized endemic settings across the Amazon. Although the overall burden of malaria in Latin America and the Caribbean has decreased dramatically over the past two decades, transmission persists in 21 countries in the region, where 120 million people are estimated to be currently exposed to some risk of infection [Malaria in the Amazon has traditionally been perceived as a disease affecting poor rural communities, with most reported infections acquired in remote riverine villages ,4, frontThe Amazon Basin of Brazil is facing an accelerated urban growth–estimated at 2.4% per year between 2000 and 2010  and charMalaria transmission rates in Brazil are nowadays greatest in the upper Juruá Valley, next to the border with Peru . With <0Here we combine epidemiological surveillance data and travel histories to explore human mobility patterns in Mâncio Lima, the main urban malaria hotspot of Brazil. We found that the localities that are most frequently visited by urban residents are typically those with the most intense malaria transmission. These localities also contribute the vast majority of imported malaria infections diagnosed in the urban area. Importantly, source communities with more intense transmission, which are likely to drive most urban malaria risk, are not hard-to-reach riverine villages; instead, they are situated in the vicinity of the town, where effective public health interventions are easier to implement.2 in the upper Juruá Valley region of Acre State, westernmost Brazil  for a municipality in Brazil, estimated at 422.8 in 2018 . With a A population census performed by our field team between November 2015 and April 2016 enumerated 9,124 permanent residents in the urban area of Mâncio Lima, with ages ranging between <1 month and 105 years  and distributed into 2,329 households . Our stuTo measure the spatial mobility of urban residents, individual reports of overnight trips within the past 6 months were collected during two consecutive cross-sectional surveys carried out 6 months apart. Reports included the total duration of each trip and its destination, allowing us to calculate the total number of days each individual spent in each destination between September 2018 and August 2019. Only trips for which the destination, resolved at the locality level (as defined below), was situated in the upper Juruá Valley region were considered . Original anonymized data are available in We retrieved all malaria case notifications from the upper Juruá Valley region  that were entered into the electronic malaria notification system of the Ministry of Health of Brazil between January 2016 and December 2018. Because malaria is a notifiable disease in Brazil and diagnostic testing and treatment are not available outside the network of government-run health care facilities, the database comprises the vast majority of laboratory-confirmed malaria episodes countrywide .imported infections [The upper Juruá Valley has been divided, for operational malaria control purposes, into smaller geographic units, or “localities”, with shared epidemiological and ecological characteristics . The cenfections ). An inffections .vulnerability [From these data retrieved from the electronic malaria notification system we estimated the average API between 2016 and 2018 for every locality in the region and quantified rural-to-urban mobility caused by malaria-treatment seeking during this period, which determines the frequency of influx of infected individuals into the town . The nu2 tests and correlations were investigated using the Pearson′s correlation test.Field-collected data, entered using tablets programed with REDCap , were clMultivariate regression models with either dichotomic or continuous outcome variables were run to identify correlates of urban-to-rural mobility. The dichotomic outcomes analyzed with logistic regression models were: (1) overnight trip outside the town within the past 12 months (no/yes) and (2) overnight trip to high-risk localities–i.e., those with an API greater than that of the town of Mâncio Lima –within the past 12 months (no/yes). Given the nested structure of the data , we used the “meqrlogit” STATA command to build mixed-effects logistic regression models that included the grouping variable “household” as a random factor. These models performed better than their Poisson counterparts, as judged by Akaike’s and Bayesian information criteria; therefore, only logistic regression results are presented here. The continuous outcome variables analyzed with negative binomial models were: (3) total number of overnights outside the town within the past 12 months and (4) total number of overnights in high-risk localities. Models were built with the “menbreg” STATA command, including the grouping variable “household” as random factor. For each outcome we ran two separate regression models: (a) one for the entire population and (b) one for economically active males (i.e. aged 16–60 years old), who tend to be more mobile due to occupational activities and behavior. For each analysis we present both adjusted and unadjusted coefficients.Bolsa Família conditional cash transfer program [Variable selection for the final models followed the hierarchical approach based on conceptual frameworks as suggested by Victora and colleagues ; hierarc program   of the Ministry of Health of Brazil (CAAE number 64767416.6.0000.5467). Written informed consent and assent were obtained from all study participants.Complete mobility information was available from 1,903 individuals, distributed into 504 households, who participated in both cross-sectional surveys. This total corresponds to 94.4% of individuals interviewed in May-June 2019 and 89.3% of those interviewed in September-October 2019. Sociodemographic characteristics of the study population are shown in P. vivax and P. falciparum are given in The average API for the town of Mâncio Lima during the same period was 442 cases per 1,000 population per year, which is surprisingly high for an urban setting. For comparison, the three other urban centers in the upper Juruá Valley region–namely, Cruzeiro do Sul , Rodrigues Alves , and Guajará , whose locations are indicated in P. falciparum infections, which mostly cluster in the vicinity of the town, being much less frequent in more remote sites  of the study participants reported at least one overnight trip between September 2018 and August 2019, with a total of 34,150 overnights outside the town, corresponding to approximately 5% of their nights during the study period. Nearly half (47.0%) of the study participants who reported overnights outside the town were away for ≥1 month during the study period (one or more trips); 24.4% of them were away for ≥3 months.The three most frequent destinations are two small rural settlements . We note, however, that the three most visited localities  are outliers in the regression analysis, being disproportionately more visited than expected from their distance from the town of Mâncio Lima , suggesting that study participants tend to spend more overnights in proximate localities, with the exceptions noted above.The number of overnights in each locality is not significantly correlated with its distance from the town of Mâncio Lima . The riverine village of Timbauba, along the Moa river (54 km northwest of the town in Euclidian distance), is the second most frequent site for a second residence and also the second most visited locality . Likewise, the farming settlement of Tonico (16 km west of the town by road), the most visited locality  and the highest API in the region, is among the top-five locations where study participants have a second residence.Not surprisingly, members of multi-sited households display increased mobility. Indeed, 55.4% of study participants whose families have a second residence reported at least one overnight trip in the previous 12 months, compared with only 30.7% of those with no second residence  male gender, (b) age between 16 and 60 years, (c) informal employment, (d) regular fishing, and (e) second residence outside the urban area . We noteP. vivax infections (82.4%) and 3,500 P. falciparum infections (17.6%)–were diagnosed, treated, and notified in the town of Mâncio Lima between January 2016 and December 2018. Of them, 5,389 (33.0%) P. vivax infections and 1,545 (44.1%) P. falciparum infections were classified as imported during routine case investigation  had one of the 65 localities shown in P. vivax and 69.3% of P. falciparum infections imported into the town of Mâncio Lima. Permanent malaria diagnosis outposts are not available in these localities. Importantly, the number of imported cases correlates negatively with the distance between the putative source locality and the town .We next investigated the geographic origin of imported malaria infections notified in Mâncio Lima. The vast majority of them . Larvaler human ,53 and cer human .Third, imported infections diagnosed in urban communities across the Amazon do not necessarily originate in hard-to-reach traditional settlements. Instead, our results indicate that they are mostly acquired in farming settlements in the vicinity of the city or town, where farmers usually sell their crops and purchase goods. These findings imply that more intensive control interventions and better infrastructure for laboratory diagnosis and prompt treatment in nearby rural localities might drastically reduce the number of cases imported into the urban area. Moreover, as malaria transmission has been clearly linked to deforestation , environThis study has some limitations. One is the possible recall bias in travel histories obtained at approximately six-month intervals, given the long time elapsed between the event and the interview. Alternatives to travel histories include the use of mobile phone data, which have been increasingly used to track human movements over time . To thisDespite these potential study limitations, we show that surveillance data combined with additional sociodemographic and mobility information from population-based surveys provides valuable information that can be explored for evidence-based planning and deployment of interventions aimed to reduce urban malaria risk across the Amazon.S1 Checklist(DOC)Click here for additional data file.S1 File(PDF)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.S1 Fig(TIFF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 FigGeoreferenced localities are represented by circles with size proportional to their population size and filled with tones from light yellow to dark brown that are proportional to malaria transmission intensity, using the APIs as a proxy (higher APIs in darker tones).(TIF)Click here for additional data file.S4 FigGeoreferenced localities are represented by circles with size proportional to their population size and filled with tones from light yellow to dark brown that are proportional to malaria transmission intensity, using the APIs as a proxy (higher APIs in darker tones).(TIF)Click here for additional data file.S5 Fig(TIFF)Click here for additional data file.


Soy sauce oil, a byproduct of whole soybean processing by the soy sauce industry, was evaluated as a source of linoleic acid for dairy cows for the purpose of manipulating the composition of milk.Eight dairy Holstein cows fitted with rumen cannulas were used for ruminal administration of soy sauce oil for a 28-day period using a 4×4 Latin square study design with 4 doses .Although dry matter intake and milk yield were not affected by soy sauce oil administration, ruminal concentrations of total volatile fatty acids and acetate were decreased, specifically at 600 g/d administration. While milk fat percentage was decreased with administration of soy sauce oil, proportions of linoleic, vaccenic and conjugated linoleic acids in the rumen, blood and milk were increased with increasing soy sauce oil dose.These results suggest that soy sauce oil feeding could be useful for improving milk functionality without adverse effects on animal production performance when fed at less than 400 g/d. The fatty acid composition of soy sauce oil is similar to that of soybean oil and mainly consists of linoleic acid . TherefoTherefore, to clarify the suitable range of soy sauce oil feeding, we carried out an experiment in which different doses of soy sauce oil were ruminally administered to dairy cows. The application of soy sauce oil to dairy feed is discussed in the context of the effects of soy sauce oil administration on DMI, ruminal fermentation and milk production.The research protocols regarding animal care followed the Guidelines for Animal Experimentation of the local independent administrative agency of the Hokkaido Research Organization.Eight primiparous Holstein cows (574±29 kg body weight with 116±22 days in milk) were used in the present study. Cows were housed in tie stalls with rubber mats bedded with sawdust. The animals had been previously fitted with rumen cannulas . The experiment was carried out using a 4×4 Latin square design with a 28-day treatment period, in which the first 21 days were for diet adaptation and the last 7 days were for sampling and data collection. Cows were randomly assigned to one of four treatment groups: 0, 200, 400, or 600 g/d soy source oil administration. Soy sauce oil was administered into the rumen via the cannula twice daily (0830 and 1730 h) in two equal doses.ad libitum, which was equivalent to 110% of the expected intake. The amounts of diet offered and refused were recorded daily. Fresh water was available at all times. Cows were milked twice daily (0900 and 1900 h) and milk production was recorded at each milking. Total digestible nutrient (TDN) requirement was calculated based on body weight and milk production of each cow [Ingredients and chemical composition of diet used in the present study are shown in each cow  and TDN Cows were weighed at the end of each treatment period. Milk was sampled on 4 consecutive days during the collection period and pooled to obtain representative samples. An aliquot of the milk sample was placed into plastic vials and stored at −30°C for long chain fatty acid analysis.Rumen and blood samples were obtained on day 28 via the ruminal cannula and jugular vein catheter at −0.5, 0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, and 9 h after the morning administration of soy sauce oil . The blood catheter was inserted 12 h before the first sampling and removed just after the last sampling of each sampling day. Rumen samples were taken from the dorsal and ventral sac by hand, mixed, and then strained through 4 layers of surgical gauze to obtain representative filtrates in a plastic vial. The filtrates were used for pH measurement using a HM-50G electrode , and then stored at −30°C for further analysis. Blood samples were centrifuged to obtain serum and stored at −30°C until analysis.Feed samples including grass silage, corn and soybean meal were collected in each period and dried in a 60°C forced air oven for 48 h for DM determination. The dried feed samples were ground and sieved through a 1-mm screen using a cutting mill and used for chemical analysis.Feed samples were analyzed for DM, crude protein (CP), ether extract (EE) and neutral detergent fiber using standard procedures . Fat, prFrozen rumen, milk and blood serum samples were thawed at 4°C and assessed using the following chemical analyses. Ammonia nitrogen was analyzed using the phenol hypochlorite method . VFA wasFatty acid analysis was performed after conversion of fatty acids into fatty acid methyl esters (FAME), with different protocols used for milk and other samples (rumen and blood serum) as follows. Milk fatty acids were analyzed according to the method of Aii et al  with minData were analyzed using JMP  as a replicated 4×4 Latin square design. When the fixed effect was significant, the difference among means was determined with Tukey’s multiple comparison test. Linear and quadratic effects were also evaluated through administration levels. A level of p<0.05 was defined as significant.Ruminal fermentation parameters, including pH, ammonia N, and VFA, as affected by administration of soy sauce oil are shown in trans-11 vaccenic acid (vaccenic acid) and CLA in cows administered soy sauce oil at different levels are shown in Ruminal concentrations of linoleic acid, Concentrations of linoleic acid, vaccenic acid and CLA in blood serum of cows administered soy sauce oil are shown in New feed materials such as soy sauce oil are expected to have insignificant effects on feed intake, as DMI is an important factor in the production performance of dairy cows. In general, the amount and fatty acid profile of oil, especially the fatty acid composition of digesta reaching the small intestine, are known as determinants of feed intake . In the Although the decrease of total VFA concentration at 2 h after feeding of soy sauce oil was previously reported Shibata et al , no suchThe biggest impact of the present study results is the dose-dependent increase in concentration of functional CLA in the rumen, blood and milk with soy sauce oil administration  to 4. ThWhen calculated from the CLA proportion of total fatty acids of milk  and milkRuminal supplementation of soy sauce oil can shift fatty acid profiles of rumen content, blood serum and milk toward higher proportions of linoleic acid, vaccenic acid and CLA, depending on the administration level. Daily administration of up to 400 g/cow is proposed to be suitable for this purpose without detrimental effects on VFA and milk fat production.


Procalcitonin (PCT) has received much attention as a serum marker for bacterial infection. Elevated serum PCT is occasionally seen in severe trauma, heatstroke, and neoplastic diseases, including lung cancer with neuroendocrine component.In the present study, we evaluated PCT expression in the specimen of pulmonary neuroendocrine tumors, comparing large cell neuroendocrine carcinoma (LCNEC), carcinoid, and small cell lung carcinoma (SCLC). Pathological specimens of 10 LCNEC, 4 carcinoid, and 7 SCLC cases were evaluated with immunochemical staining of PCT. Clinical characteristics and serum levels of PCT and C-reactive protein were also evaluated. We observed positive PCT expression in 5 (50%) LCNEC and 2 (50%) carcinoid specimens that were surgically resected. Whereas serum PCT levels were not elevated in patients with PCT-positive carcinoid, two out of three LCNEC patients with high PCT expression in the tumor had elevated serum PCT levels that reflected disease progression. In patients with SCLC, PCT was not detected in the tumor or serum. This is the first immunohistochemical study of the PCT expression in the lung tumor specimens. We concluded that, in patients with LCNEC, high serum PCT levels may be indicative of disease activity and serve as a biomarker. Procalcitonin (PCT) is a peptide consisting of 116 amino acids and is synthesized primarily in the C-cells of the thyroid gland as a calcitonin precursor protein . In 40.6PCT is secreted from neuroendocrine cells present in the lungs, adrenal glands, liver, kidneys, adipose tissue, and muscle during sepsis, suggesting increased production of PCT may be associated with NETs. Although elevated levels of serum PCT are occasionally seen in patients with pulmonary NETs , PCT expWe retrospectively evaluated data from 10 consecutive patients with surgically resected LCNEC and 4 with carcinoid who were diagnosed at Hirosaki University Hospital between 2013 and 2019. Pathological diagnosis of LCNEC was based upon neuroendocrine morphology and immunostaining of the neuroendocrine markers, such as chromogranin A, synaptophysin and neural cell adhesion molecule (NCAM), according to the 2015 WHO classification of lung tumors [Immunohistochemical staining was performed using anti-PCT monoclonal antibody obtained from CUSABIO . Formalin-fixed and paraffin-embedded tissues were sectioned at a thickness of 4 µm and stained on positively charged glass slides stored at 4 °C within 3 days after sectioning. We used Bond-III Leica automated slide stainer, and signals were detected using the Bond polymer refine detection kit . After peroxide blocking, staining was developed with diaminobenzidine.The proportion of tumor cells found to express PCT (proportion score) was assessed according to the following scale: 0 0–10%); 1 (11 to 25%); 2 (26 to 50%); 3 (51 to 70%); and 4 (71 to 100%) of tumor cells. The intensity of staining (intensity score) was evaluated according to the following scale: 0 (no staining); 1 (weak staining); and 2 (strong staining) in 10% of cancer cells  following the manufacturer's instructions. The lower detection limit was 0.02 ng/mL. The cut-off value was 0.5 ng/mL, which is widely used for a diagnosis of bacteremia.The median age was 67 years  in patients with LCNEC, 58 years  in those with carcinoid, and 69 years  in SCLC patient. There were 6 female patients . Most of the patients with LCNEC (90%) and SCLC (86%) had smoking history. The Brinkman indices of the patients with LCNEC and those with SCLC were 653 ± 148 and 832 ± 283, respectively.Immunostaining for PCT was performed on 21 specimens, including 15 resected tumors and 6 transbronchial biopsy specimens (Table PCT levels were measured using the serum samples collected at the time of diagnosis Table . Serum PIn two LCNEC patients with elevated serum PCT levels at the time of diagnosis, we evaluated the serial changes and found that serum PCT levels changed in association with the disease progression or treatment. In Case 7, which was a stage IV disease with adrenal and brain metastases in a male patient, the disease progression could not be controlled after a total of four courses of chemotherapy. The serum PCT level was as high as 170.4 ng/mL at the time of diagnosis and gradually increased thereafter. Case 9 was also a stage IV disease with multiple brain and bone metastases and pleural dissemination in a male patient. His PCT level in serum was 16.72 ng/mL at diagnosis and around 20 ng/mL while the disease was controlled by 4 cycles of chemotherapy (Fig. PCT has been drawing attention as a serum marker of serious bacterial infection such as sepsis. In the present study, we observed positive PCT expression in a half of the specimens of surgically resected LCNECs and carcinoids. Whereas serum PCT levels were not elevated in patients with PCT-positive carcinoid, two out of three LCNEC patients with high PCT expression in the tumor had elevated serum PCT levels that reflected disease course. In patients with SCLC, PCT was not detected in the tumor or serum. To the best of our knowledge, this is the first immunohistochemical study of the PCT expression in the lung tumor specimens.There have been some reports of elevated serum PCT in patients with primary lung cancers , 6, 7. PIn this study, we observed positive PCT expression in a half of the specimens of LCNEC and carcinoid, whereas none of the SCLC specimens showed PCT expression. Because all the SCLC specimens showed positive immunostaining of synaptophysin and NCAM, we considered that positive immunostaining of PCT in tissue specimens may be useful to exclude SCLC from other pulmonary NETs.PCT is secreted from neuroendocrine cells in various organs, suggesting PCT production in NETs. In fact, immunohistochemical diagnosis of PCT-secreting NETs has been made in cases of medullary thyroid carcinoma  and pancPCT has a variety of biological effects. For example, PCT promotes the induction of nitric oxide synthase from sites of inflammation resulting in the onset of vasodilatation . It alsoIn this study, our immunohistochemical analysis revealed positive PCT expression in a half of the specimens of surgically resected LCNECs and carcinoids. Some of the LCNEC patients with high PCT expression in the tumor showed elevated PCT levels in serum, which was associated with disease progression. In patients with LCNEC, high serum PCT levels may be indicative of disease activity and serve as a biomarker.This study has some limitations. First, the sample size was relatively small, because LCNEC is a rare type of lung cancer and its diagnosis usually needs a surgical specimen. Second, most specimens of SCLC were obtained with transbronchial biopsy, which might affect the immunohistochemical evaluation.


Background: Kappa free light chains (KFLC) are a promising new biomarker to detect neuroinflammation. Still, the impact of pre-analytical effects on KFLC concentrations was not investigated. Methods: KFLC concentrations were measured in serum and cerebrospinal fluid (CSF) of patients with a newly diagnosed multiple sclerosis (MS) or clinically isolated syndrome (CIS) before (n = 42) or after therapy with high-dose methylprednisolone (n = 65). In prospective experiments, KFLC concentrations were analyzed in the same patients in serum before and after treatment with high-dose methylprednisolone (n = 16), plasma exchange (n = 12), immunoadsorption (n = 10), or intravenous immunoglobulins (n = 10). In addition, the influence of storage time, sample method, and contamination of CSF with blood were investigated. Results: Patients diagnosed with MS/CIS and treated with methylprednisolone showed significantly lower KFLC concentrations in serum as untreated patients. Repeated longitudinal investigations revealed that serum KFLC concentrations continuously decreased after each application of methylprednisolone. In contrast, other immune therapies and further pre-analytical conditions did not influence KFLC concentrations. Conclusion: Our results show prominent effects of steroids on KFLC concentrations. In contrast, various other pre-analytical conditions did not influence KFLC concentrations, indicating the stability of this biomarker. An immunoglobulin synthesis within the central nervous system is frequently observed in a broad spectrum of autoimmune and infectious neurological diseases ,2. When Medical records were screened for patients who presented with symptoms suggestive for a first demyelinating episode at the Department of Neurology of the Hannover Medical School between 2010 and 2015. Patients were included when they were either newly diagnosed with MS according to the 2017 McDonald criteria or converted to MS during follow-up. Some of these patients were previously investigated with a focus on different other aspects ,17,18. AIn the prospective part of the study, serum samples were taken from patients who were either treated with intravenous methylprednisolone, immunoadsorption, plasma exchange, or intravenous immunoglobulins at the Department of Neurology of the Hannover Medical School in the time from 2018 to 2019. For the analysis of pre-analytic variables, CSF and corresponding serum samples originated from patients that underwent routine lumbar puncture between 2018 and 2019. All prospectively investigated patients gave their informed consent for inclusion before they participated in the study. Demographic data of these patients are depicted in In the group of patients treated with intravenous methylprednisolone, all 16 patients received 1000 mg methylprednisolone per day for at least 3 days, while 9 of these patients were treated for an additional 2 days. Serum samples were taken before treatment as well as after 24 h and 48 h from all 16 patients. In addition, blood was sampled after 96 h from 9 patients who were treated five days with steroids.Plasma exchange therapy was administered for at least 3 cycles with 1 cycle each day for all 12 patients, while 7 of these patients received 2 additional cycles. Serum samples were taken before treatment, after 24 h, 48 h, and 72 h from all patients and additionally from 7 patients after 96 h and 120 h after the start of plasma exchange.All 10 patients who were treated with immunoadsorption received 5 cycles of therapy in total with 1 cycle each day. Serum samples were taken before and after 24 h, 48 h, 72 h, 96 h, and 120 h of treatment.Serum samples were taken from 10 patients who were treated with intravenous immunoglobulins. Samples were taken before the treatment with immunoglobulins was started and within 12 h after the last infusion of the cycle with cumulative dose of 60–160 g immunoglobulins.This part of the study aimed to investigate whether contamination with 5000 erythrocytes per mL CSF or 20,000 erythrocytes per mL CSF might cause an influence on KFLC concentrations (n = 17). Further, CSF and serum samples were stored for 14 days at 4 °C and room temperature and subsequently compared with baseline values (n = 16). EDTA and serum samples of the same patients were compared to investigate the influence of different sample methods on KFLC concentrations (n = 33).For the retrospectively collected data, all paired CSF and serum samples were analyzed according to routine diagnostic procedures in the Neurochemistry Laboratory of the Department of Neurology. CSF cells were counted manually using a Fuchs–Rosenthal counting chamber. Total protein in CSF was determined by Bradford dye-binding procedure in centrifuged samples . ConcentGraphPad Prism version 5.02 was used for statistical analysis. The level of statistical significance was set to 5%. Data were described by medians, standard deviation, and interquartile ranges. The D’Agostino and Pearson omnibus normality test was used to asses for normal distribution of values. Mann–Whitney U-Test was used for independent values. Kruskal–Wallis test and Friedman test with Dunn’s Multiple Comparison post hoc test were used for group comparison and Wilcoxon signed-rank test and paired t-test for comparison of two groups with repeated measurements.p < 0.0001) and 3000 mg (p = 0.0009) methylprednisolone. In contrast, the mean serum concentrations of the immunoglobulins IgG, IgA, and IgM and of albumin did not differ significantly between untreated patients and patients who received methylprednisolone.The results of retrospectively analyzed serum and CSF are shown in In CSF, the mean KFLC concentration was slightly lower in patients after 3000 mg, 4000 mg, and 5000 mg methylprednisolone but this did not reach significant differences. In addition, no differences were found between mean CSF concentrations of the immunoglobulins IgG, IgA, and IgM and of albumin between untreated patients and patients who received methylprednisolone.Calculating the KFLC index revealed slightly higher mean values for patients who were treated with methylprednisolone than for untreated patients but did not reach significant differences. The KFLC index was above the threshold of 5.9 in 60/65 patients who were treated with methylprednisolone and in 36/42 patients who were untreated. No significant difference between the quotients of IgG, IgA, and IgM was found between treated and untreated patients .To analyze the effects of high-dose steroids on serum KFLC concentrations, serum samples were collected before therapy and after 24 h and 48 h in each patient (n = 16) treated three days with 1000 mg methylprednisolone per day. Furthermore, serum was sampled after 96 h from 9/16 patients who were treated five days with steroids. Our results show that serum KFLC concentrations continuously decreased after the increasing dosage applications of methylprednisolone. Significant differences were found between baseline KFLC values (18.7 ± 9.1 mg/L) and KFLC values measured after 48 h (10.9 ± 4.4 mg/L) and 96 h (7.6 ± 2.6 mg/L). The mean percentage decrease of the KFLC concentration in serum after 24 h was 28% (± 9%), after 48 h 40% (± 12%), and after 96 h 49% (± 10%) of the initial concentration, independently whether a high or a low concentration was measured before treatment. While the mean serum concentrations of the immunoglobulins IgG, IgA, and IgM and albumin decreased slightly after each application of methylprednisolone, the differences reached significant levels for albumin after 96 h only as depicted in Serum samples were collected before therapy and after 24 h, 48 h, and 72 h in each patient (n = 5) treated three times with one plasma exchange therapy per day in order to assess the impact of plasma exchange on serum KFLC concentrations. Furthermore, serum was sampled after 96 h and 120 h from 7/12 patients who were treated with five cycles of therapy. Our results show no significant effects of plasma exchange therapy on serum KFCL concentrations. In contrast, the concentrations of the immunoglobulins IgG, IgA, and IgM decreased after each cycle of plasmapheresis and reached significant levels . High coThe influence of immunoadsorption on serum KFLC concentrations was investigated in serum samples that were collected before therapy and after 24 h, 48 h, 72 h, 96 h, and 120 h in each patient (n = 10) treated five times with one immunoadsorption therapy per day. The effects of immunoadsorption on serum KFLC and immunoglobulin concentrations were comparable to the effects of plasmapheresis. Serum KFLC concentration decreased slightly after each cycle of immunoadsorption but did not reach significant differences. The concentrations of the immunoglobulins IgG, IgA, and IgM decreased after each cycle of plasmaphereses and reached significant differences . ConcentTo analyze the effects of intravenous immunoglobulins (IVIG) on serum KFLC values, serum samples were collected before and after two to five days of a daily infusion therapy of 30 g to 40 g of intravenous immunoglobulin in 10 patients. Our results show no significant effects of intravenous immunoglobulins on KFLC concentrations; however, a tendency to higher KFLC concentrations in serum after the infusion was observed . Since iDifferent pre-analytical conditions were investigated as a possible influence on serum and CSF KFLC concentrations. Measurements of serum and EDTA samples from the same patients did not differ in KFLC concentrations . StorageThe main finding of this study is that intravenous steroids significantly influence the concentrations of KFLC in serum. KFLC concentrations continuously decreased as a consequence of increasing dosage applications of methylprednisolone. The amount of KFLC decreased to 58% after 48 h and further to 41% after 96 h as compared to values before therapy with steroids. The previously unknown influence of steroids on the concentration of KFLC is an important issue since high-dose methylprednisolone is the treatment of choice in various neuro-immunological disorders especially in events suggesting MS ,24.The presence of oligoclonal bands in CSF, which indicate an intrathecal immunoglobulin synthesis, is a hallmark biomarker of MS and has been demonstrated to be highly prevalent ,25,26. CIn clinical practice, therapy with methylprednisolone often starts in patients who present with symptoms indicating MS such as optic neuritis even before the complete diagnostic work-up including lumbar puncture could be completed. In our retrospective cohort of patients with first clinical relapse indicating a demyelinating disease, methylprednisolone was administered in 61% (65/107) of patients before lumbar puncture was performed. Since the CSF concentration of KFLC was similar between untreated patients and patients under infusion of methylprednisolone and our findings indicate a significant reduction of serum KFLC concentrations, the ratio of CSF and serum KFLC (Q KFLC) might be falsely elevated. In methods to evaluate the origin of KFLC in CSF such as KFLC index or diagrams, falsely elevated Q KFLC might falsify the results and, therefore, pretend an intrathecal inflammation ,27. ThisInterestingly, we did not find significant effects of plasma exchange or immunoadsorption on serum concentrations of KFLC. Therapeutic apheresis is commonly used as second-line therapy in various neuroimmunological diseases including MS and autoimmune encephalitis after insufficient clinical response to methylprednisolone. The effects of apheresis on serum immunoglobulin concentrations have been described before and are in line with our observations of a significant decrease .Since CSF samples were understandably not obtained in patients who underwent therapeutic apheresis, the effect on intrathecal synthesis can only be speculated. Spurious quantitative intrathecal immunoglobulin syntheses have been described after plasma exchange or immunoadsorption in one previous study . The misOn the other hand, the missing decrease of serum KFLC using immunoadsorption is expectable as during an immunoadsorption therapy the high affinity of staphylococcal-protein-A-columns to the Fc portion of IgG is used to selectively remove IgG antibodies, aggregated IgG, and IgG-containing immune complexes . It, theN-methyl-D-aspartate (NMDA) receptor encephalitis [Intravenous immunoglobulins are commonly used in a wide spectrum of autoimmune diseases including immune-mediated neuropathies and anti-phalitis . Intravephalitis . It is sphalitis . Althougphalitis .Serum-free light chains concentrations are supposed to have a short half-life of 2–6 h in vivo, which is in contrast to immunoglobulins having a half-time of 21 days . HoweverOne limitation of this study is the retrospective approach when analyzing CSF and serum samples of patients who presented for the first time with symptoms suggestive for a MS relapse. The limited number of patients who were retrospectively and prospectively analyzed is caused by the strict inclusion criteria, which also hazards a selection bias. Therefore, the results should be confirmed in further studies with larger cohorts of patients.In conclusion, our results show prominent effects of steroid therapy on KFLC concentrations, which should be considered while interpreting results. In contrast, various other pre-analytical conditions including therapeutic apheresis did not influence KFLC concentrations indicating stability of this biomarker.


As a main component of the stratum corneum, ceramides can construct protective lamellae to provide an epidermal barrier against dehydration or external microorganisms. However, as ceramide molecules can easily form the isolated crystalline phase through self-assembly due to the amphipathic nature of bioactive lipids, the effective incorporation of ceramides into liquid media is the remaining issue for controlled release. Here, we report an unprecedented effective strategy to fabricate a completely amorphous and highly sustainable hierarchical ceramide polymer microcapsule for promising epidermal barrier by using the interpenetrating and cooperative self-construction of conical amphiphiles with a different critical packing parameter. The self-constructed amorphous architecture of ceramides in polymer microcapsule is achieved by the facile doping of conical amphiphiles and subsequent in situ polymerization of shell polymer in the core-shell geometry. It is experimentally revealed that an irregular cooperative packing structure formed by adaptive hydrophobic–hydrophilic interactions of cylindrical ceramides and conical amphiphiles in the confined microcapsule geometry enables a completely amorphous morphology of ceramides to be realized during the spontaneous encapsulation process. Furthermore, this elegant approach affords a highly dispersible and uniform hierarchical amorphous ceramide microcapsule with a greatly enhanced long-term stability compared to conventional crystalline ceramides. Human and animal skin plays an important role in the prevention of dehydration and temperature control, as well as in protection against physical, chemical, and biological attacks. Most of these functions are fulfilled by the skin lipids of stratum corneum, located in the outermost layers of the epidermis ,2,3. AmoThe importance of ceramides is well known, but their stable and uniform incorporation into the existing medical or cosmetic liquid media remains difficult. Although the amphipathic properties of ceramides as bioactive lipids play a key role in forming an epidermal barrier with a highly robust lamellar structure, recrystallization or significant agglomeration of ceramides frequently occur in liquid media due to the formation of the isolated crystalline phase through self-assembly. As a consequence, the poor solubility and dispersibility in liquid media make it difficult to introduce the efficacy of ceramides for potential epidermal barrier ,10.Microcapsules, which are microsized molecular carriers for wrapping functional materials in the core region, are widely applied in the food, pharmaceutical, printing, and cosmetic industries ,13,14,15Herein, we present an unprecedented yet simple, effective, scalable strategy for the fabrication of completely amorphous and highly sustainable hierarchical ceramide microcapsules for potential epidermal barrier based on the interpenetrating and cooperative self-construction of conical amphiphiles with a different critical packing parameter in the confined geometry of polymeric microcapsule . The inhMn = 124,000–186,000) were purchased from Sigma Aldrich . The MMA monomer was purified to remove the polymerization inhibitors. Ethylene glycol dimethacrylate (EGDMA) was purchased from Acros . 2,2-Azobisisobutyronitrile (AIBN) was purchased from Junsei Chemicals  and used after purification. Methylene chloride (MC) and benzene used as dispersion solvents were purchased from Daejung Chemicals & Metals . Ceramide (Y-30) and sodium dilauramidoglutamide lysine  were obtained from Cosmax .Methyl methacrylate (MMA), sodium nitrite, and poly  water. PVA acts as a dispersion stabilizer to maintain spherical particles when synthesizing amorphous ceramide microcapsules via in situ suspension polymerization. 50 mg of ceramide and 50 mg of DLGL were dissolved in 2.5 mL of MC and 2.5 mL of benzene, respectively, and then the combined MC and benzene solution were stirred at 1000 rpm and 35 °C for 30 min. Then, 1.4 g of MMA, 0.002 g of EGDMA, and 0.02 g of AIBN were added to the combined solution, followed by stirring at 1000 rpm and 35 °C for 30 min. Next, this mixture was added to the PVA aqueous solution and stirred at 1000 rpm and 25 °C for 5 min. After stirring, 0.0025 g of sodium nitrite was added, and subsequently, the solution was polymerized in a two-necked flask equipped with a reflux condenser and nitrogen injector. Polymerization was carried out by stirring at 200 rpm and 80 °C for 9 h under a nitrogen atmosphere. Finally, the solution was purified with DI water and ethanol using a centrifuge at 10,000 rpm for 30 min at room temperature. The final product was dried in a vacuum oven at room temperature for 24 h. To investigate the effect of DLGL amphiphile on the self-constructed hyperstructure of ceramides in the core of polymer microcapsule, PMMA microcapsule containing only ceramide molecules was prepared according to the above synthetic procedure.−1 under nitrogen atmosphere. Thermogravimetric analysis  was performed from 30 to 700 °C under nitrogen flow.The structure and size of hierarchical ceramide microcapsules were analyzed by Fourier-transform infrared  spectrometer equipped with an attenuated total reflection (ATR) accessory, field emission scanning electron microscope , and energy dispersive X-ray spectroscopy . The dispersion properties and morphology of the ceramide microcapsules were examined using an optical microscope  and polarized optical microscope . The morphological structure and crystallinity of the fabricated microcapsules were investigated by X-ray diffraction  and differential scanning calorimetry . The DSC thermograms were obtained at a heating rate of 10 °C minao) at the hydrophobic–hydrophilic interface, the length (lc) of the hydrophobic alkyl chain, and the volume (v) of molecule  derived from the head group area (molecule  19]. Th. Thao) aTo examine the effect of molecular geometry of amphiphile on the inner self-assembled structure of the polymer microcapsule, two types of PMMA microcapsules were prepared, as shown in −1, respectively. In addition, the broad peak at 3290 cm−1 was assigned to the N-H stretching vibration of amide group. The CDP and CP microcapsules synthesized through the in situ polymerization of MMA monomer exhibited characteristic bands of amide group stemming from inner core materials similar to those of the pristine ceramide and DLGL. Moreover, a C=O stretching vibration of acrylate group was observed at 1725 cm−1, and the absorbance of C=C double bond at 1630 cm−1 was greatly reduced by MMA polymerization. These results confirm that hierarchical ceramide microcapsules containing the interpenetrated ceramide and DLGL as core materials were successfully prepared using facile doping of DLGL amphiphile and subsequent in situ polymerization of PMMA in the core-shell geometry. In comparison between CDP and CP microcapsules, the increased absorbance at 1550 cm−1 was observed for CDP microcapsule due to the stronger N-H bending vibration of DLGL dopant, indicating the efficient encapsulation of both ceramide and DLGL amphiphiles.A structural analysis of the CDP and CP microcapsules was carried out by Fourier-transform infrared spectroscopy c. The prTo investigate the existence and structural stability of inner core materials in the hierarchical PMMA microcapsules, thermogravimetric analysis was conducted on CDP and CP microcapsules with different core materials. While the pristine ceramide maintained the initial weight even at 360 °C due to the highly ordered crystalline structure, an abrupt weight loss at 105 °C was observed for pure DLGL molecules with a spherical self-assembled structure d. In theEnergy dispersive X-ray spectroscopy was carried out to confirm the elemental composition and distribution of the hierarchical amorphous ceramide microcapsule, as shown in In order to examine the morphological characteristics of hierarchical ceramide microcapsules dispersed in the liquid medium, optical microscopic measurements were conducted under parallel or crossed polarizers. θ = 4.59°, 6.93°, 11.54°, 19.87°, 20.47°, and 21.07°, which are attributed to the lamellar crystalline structure of self-assembled cylindrical ceramide molecules [θ = 4.59° and 6.93° in the small-angle region and only a broad halo centered at 2θ = 15° corresponding to the disordered hydrophobic alkyl chains in the wide-angle region, indicating that the ceramide amphiphiles surrounded by PMMA shell cannot construct the highly ordered crystalline phase with a long-range order due to the confinement effect of the microcapsule. The encapsulation of crystalline ceramides inside the PMMA microcapsule hindered their crystallization and therefore afforded a less-ordered supramolecular structure. The similar XRD results have been reported on the capsule systems including crystalline curcumin materials [To investigate the structural difference in the self-organized phases of amphiphilic molecules, X-ray diffraction (XRD) experiments were performed on the pristine ceramide, CP microcapsule, and CDP microcapsule a. In theolecules . On the aterials ,29. In t−1) compared to that of pure ceramide (94.0 J g−1), suggesting a lower crystallinity in the internal area of the microcapsule. It is supposed that the low crystallinity of CP microcapsule is ascribed to the confinement of ceramide molecules inside the PMMA microcapsule, and this result is in accord with the POM and XRD observations. In CDP microcapsule, no endothermic peak for pure ceramide was observed in the temperature range of the crystalline phase, which is indicative of a completely disordered amorphous phase. As a consequence, it is confirmed that the proposed approach based on the polymeric microencapsulation of cylindrical ceramide with conical DLGL dopant can afford a completely amorphous structure of conventional ceramide material.Thermal phase transitions and crystallinity of pure ceramide and hierarchical ceramide microcapsules were studied by the differential scanning calorimetry. To examine the effect of polymeric microencapsulation on the long-term physical stability of ceramides, which is an essential attribute of controlled release system for epidermal barrier application, dispersibility test was performed in mineral oil at 25 °C for seven days after mechanical agitation for 1 h. The particle size analysis supported the long-term stability of hierarchical amorphous ceramide microcapsules, as shown in In summary, a facile yet effective, scalable strategy to fabricate completely amorphous and highly sustainable hierarchical ceramide microcapsules for a potential epidermal barrier has been newly developed using the polymeric encapsulation of the interpenetrating conical amphiphiles with a different critical packing parameter. Even though the amphipathic cylindrical ceramide as a bioactive lipid plays a crucial role in constructing an epidermal barrier with a robust bilayer architecture, there is no precedent for the fabrication of a completely amorphous ceramide structure for efficient penetration into the epidermis. The elegant hybrid concept using polymeric microencapsulation and critical packing parameter is applied to the first successful attempt to prepare the self-assembled amorphous architecture of ceramides in the confined polymer microcapsule. To attain a completely amorphous morphology of ceramides, sodium dilauramidoglutamide lysine as a conical amphiphilic dopant with a different critical packing parameter was incorporated into the core of PMMA microcapsule containing ceramides. The specific hydrophobic–hydrophilic interactions between cylindrical ceramides and conical amphiphiles induced an irregular packing structure in the restricted core region of the polymeric microcapsule, and consequently, endowed the hierarchical ceramide microcapsule with a completely amorphous structure after polymeric microencapsulation. This unprecedented approach rendered it possible to achieve the long-term physical stability of amorphous ceramide microcapsule with an excellent dispersibility in the liquid medium compared to common crystalline ceramides. Furthermore, the self-constructed amorphous ceramide microcapsule exhibited excellent thermal durability up to 175 °C in high-temperature conditions. Notably, the proposed hierarchical amorphous ceramide microcapsule could be simply prepared in large quantities at low cost through simple suspension polymerization without the need of complex synthetic procedures. It is believed that the proposed approach opens a new way to prepare functional amorphous bioactive materials with potential applications, such as epidermal barrier, controlled-release microsystem, drug delivery vehicle, and biosensor.


Complications due to prematurity are a threat to child survival and full developmental potential particularly in low-income settings.The aim of the study was to determine the neurodevelopmental outcomes among preterm infants and identify any modifiable factors associated with neurodevelopmental disability (NDD)We recruited 454 babies  in a cohort study at birth from Iganga hospital between May and July 2018. We followed up the babies at an average age of 7 months (adjusted for prematurity) and assessed 211 preterm and 187 term infants for neurodevelopmental outcomes using the Malawi Developmental Assessment tool. Mothers were interviewed on care practices for the infants. Data were analyzed using STATA version 14.The study revealed a high incidence of NDD of 20.4% (43/211) among preterm infants compared to 7.5% (14/187) among the term babies, p < 0.001, of the same age. The most affected domain was fine motor (11.8%), followed by language (9.0%). At multivariate analysis, malnutrition and Kangaroo Mother Care (KMC) at home after discharge were the key factors that were significantly associated with NDD among preterm babies. The prevalence of malnutrition among preterm infants was 20% and this significantly increased the odds of developing NDD, OR = 2.92 (95% CI: 1.27–6.71). KMC practice at home reduced the odds of developing NDD, OR = 0.46, (95% CI: 0.21–1.00). Re-admission of preterm infants after discharge (a sign of severe illness) increased the odds of developing NDD but this was not statistically significant, OR = 2.33 (95% CI: 0.91–5.94).Our study has shown that preterm infants are at a high risk of developing NDD, especially those with malnutrition. Health system readiness should be improved to provide follow-up care with emphasis on improving nutrition and continuity of KMC at home. Complications due to prematurity and low birth weight are the leading cause of neonatal deaths worldwide, and in 2018 they contributed about 34% of the 2.5 million newborn deaths ,7. SurviInstitutional deliveries have increased over time in LICs, and in Uganda over 70% of mothers deliver from health facilities . EvidencThe few studies conducted in sub-Saharan Africa have focused on long-term outcomes of preterm babies, usually starting in the post-infancy period –18. A stThe aim of the current study was to determine the neurodevelopmental outcomes of preterm infants as compared to term normal birth weight babies, and identify any modifiable factors associated with NDD among preterm infants at 6–8 months of age in eastern Uganda in order to inform interventions that can appropriately address these challenges.The study utilized a prospective cohort design where preterm babies (exposed) and term/normal birth weight babies (unexposed) delivered in Iganga district hospital in Busoga region in eastern Uganda were followed up until 6 months (corrected age for the preterms) to establish their neurodevelopmental outcomes. Busoga region is the third largest region in Uganda with a population of about four million people, where over 80% reside in rural areas and depend on subsistence farming. The area is served by five general hospitals, including Iganga hospital and one regional referral hospital, with varying levels of capacity of neonatal care, with no access to neonatal intensive care or cerebral imaging.Iganga hospital is a 100-bed capacity hospital although by the time of the study it was admitting about 200 patients. The hospital served another five adjacent districts without hospitals. The annual deliveries in Iganga hospital were 7,113 in 2018, 10% of whom were preterm and low birth weight babies admitted to the neonatal care unit (NCU). The NCU was run by three midwives, student nurses, and a pediatrician who was based on the pediatric ward and visited the unit occasionally. The unit had incubators, oxygen cylinders and concentrators, a phototherapy machine, an improvised continuous positive airway pressure (CPAP) machine and other supplies essential for neonatal care. The hospital was one of the study sites for the Preterm Birth Initiative study (2016 to 2019) which provided some of the equipment and essential supplies at the start of the intervention ,24. HoweThe study population included babies born in Iganga hospital, who were preterm (with gestation age <37 weeks and Birth weight <2.5 kg) or term/normal birth weight babies, whose mothers were residents within a 20 km radius area of that hospital. The recruitment occurred at discharge for 3 months from May to July 2018. Neurodevelopmental assessment was carried out from November 2018 to May 2019 when the babies were 6–8 months of age (corrected for the preterm babies). We excluded all newborn babies who died before discharge and those with any congenital malformations such as spina bifida. Babies who died after recruitment were documented and reported but were excluded from the analysis for neurodevelopmental outcomes.A sample size of 454 participants (242 preterm-exposed and 212 term babies-non-exposed) achieving a power of 80% was computed using a sample size formula with the continuity correction (CC) factor by Fleiss , where wWe recruited preterm babies with gestation age (GA) <37 weeks and a birth weight of less than 2.5 kg consecutively until the sample size of 242 babies was attained. We assessed GA using the New Ballard score sheet  within 2Three midwives (one per duty shift) employed by the hospital were trained by the first author for 2 days to identify and register the eligible preterm and term infants in the same hospital. After obtaining informed consent, a questionnaire was administered to capture mothers’ socio-demographic characteristics, the antenatal care practices including use of anti-anemia medicines (Ferrous/Folate tablets), and any maternal morbidities like malaria in pregnancy, high blood pressure, diabetes, and history of admission during pregnancy. The physical location description to aid follow-up was also captured. Records were checked for perinatal factors surrounding the birth of the baby including duration of labor, labor complications , and the mode of delivery. The essential newborn care within the health facility was also documented. This included birth weight, head circumference measurement, APGAR score of the baby, and KMC for the preterm babies.Mothers were reached on phone at 3-months post discharge to ascertain whether the place of residence of the mother-baby pair had not changed. We employed five nurses/midwives as research assistants, supervised by the first author, for follow-up activities at 6 months of infants’ age (corrected age for preterm infants). We used the physical location description obtained from the mother either at the time of recruitment, or at 3 months (if location changed) and the telephone contacts given to locate the residence of the mother/baby pair. In some situations, babies could not be located at 6 months but at a later date due to challenges in identifying the physical location, or where mothers had travelled for various reasons, and gave appointments of home visit upon return. In other situations, the mother together with the infant had shifted from the original location to faraway places outside the catchment/study area. Such infants were considered as lost to follow-up. Similarly, those whose phone contacts were off on five attempts, and the physical locations given could not be traced, were registered as lost to follow-up.The research assistants were trained for 5 days to conduct physical and neurodevelopmental assessment of infants at 6 months (or more) after birth (corrected age for preterms). We used the Malawi Developmental Assessment Tool (MDAT) to assess the neurodevelopmental outcomes . The MDANeurodevelopmental assessment including Gross and Fine motor, Language and Social behavioral development were determined through use of the MDAT tool at 6–8 months’ chronological age for term infants, and corrected (adjusted) months of age for the preterm infants. Each child was scored either Pass or Fail for each domain per given age. The child was scored as fail in a given domain, if he/she failed more than two parameters of the expected at that age . Data frDescriptive statistics were used, stratified by term/preterm status of the infants, to determine the incidence of neurodevelopmental delay/disability. Chi-square tests were applied to categorical variables to assess any differences in maternal and infant characteristics as well as neurodevelopmental outcomes, between the preterm and term babies. In addition, odds ratios and confidence intervals were used to determine any factors associated with NDD among preterm babies.The Z-test was used to determine the infants’ physical development in terms of weight gain for age. Those whose Z-scores were less than −2 were considered to be under weight, while between −2 and −3 were said to be having moderate malnutrition, and those whose Z-scores were <-3 were categorized as severely malnourished. The physical development data were analyzed using the two – sample t-test to assess whether there is a difference in rate of physical development in terms of the mean weight, and mean head circumference between the term and preterm babies.A multivariable logistic regression was used to model the relationship between neurodevelopmental outcome among infants born preterm and the following independent factors: readmission of the infant after discharge following birth, the nutrition status , KMC at home, mother’s parity, and father’s occupation as a proxy for socioeconomic status. We examined the model goodness of fit using the Hosmer Lemeshows goodness of fit test, the model was considered a good fit if the p-value was less than 0.05. Non-significant factors or predictors were dropped except for those known predictors from literature. We used the log-likelihood ratio test to compare two models that is the full model and the reduced model and the AIC/BIC criteria to select a parsimonious model. We tested for presence of multicollinearity using variance inflation factor (VIF) analyses. Multicollinearity was present if VIF was >10 or tolerance (1/VIF) was <0.1 for any independent factors in the model. A parsimonious model was presented along with model fit statistics.Of the 454 infants recruited 398 (87.7%) were reached for neurodevelopmental assessment. Fifteen (15) infants (3.3%), all preterms, died between recruitment at discharge from the health facility and at 7 months of follow up .Figure 1The results show that there were significant variations in maternal characteristics among mothers with preterm babies compared to those with term babies . SpecifiThe study findings revealed no differences in gender between the preterm and term infants . The meaThe mean age at follow up was 7.2 months for term infants and 6.6 months corrected age for preterm babies. There were no differences in the percentage of preterm and term babies who developed complications after discharge, and those who sought care at the health facility. However, more preterm babies (22.4%) were admitted between discharge and the follow-up period compared to 12.7% term babies, p = 0.083 .The study results revealed significant differences in neurodevelopmental disabilities between term and preterm infants, with higher incidence of NDD among preterm babies (at 6.6 months of corrected age): 20.4% compared to 7.5% among term babies (p < 0.001). The disabilities among preterm infants were mostly in the Fine motor domain (11.8%), followed by Language development (9.0%), while among the term infants the disabilities were predominantly in the Social development domain (4.8%). There were no significant statistical differences in disabilities in the Social domain across the two groups of infants .Table 3.Similarly, there were significantly more infants with malnutrition as demonstrated by Z-score of less than −2 of weight for age among the preterm infants (18.6%) as compared to the term infants (2.1%), p = 0.001. Severe malnutrition was seen in 5.7% among preterm infants while there were no cases of severe malnutrition identified among the term babies.At bivariate analysis, malnutrition was found to be significantly associated with NDD, with 32.5% of cases with malnutrition having NDD , OR = 2.The results at multivariate analysis revealed a good fit for the model of the relationship of five independent factors and NDD among preterm infants, Hosmer-Lemeshow’s goodness of fit test, LR-chi of 15.71, p = 0.028 . MalnutrThis is one of the first studies in this setting to determine the neurodevelopmental outcomes and modifiable factors among preterm infants. The findings revealed a high incidence of NDD of 20.4%, and severe disability of 3.3% associated with malnutrition among preterm infants at 6.6 months of corrected age, as compared to 7.5%, and 0.5%, respectively (p = 0.001), among term normal birth weight babies. The most affected developmental domain among preterm infants was Fine motor, followed by the Language domain. There was no statistically significant difference in social behavioral development among preterms and term babies. This may be explained by the similarities in parental engagement and stimulation of these infants irrespective of GA at birth. These findings are important as they suggest that mortality, NDD and malnutrition are higher and occur early in preterm infants after hospital discharge in our setting.Our findings are interestingly similar in the domains affected to those of Do et al. (2019) in Vietnam  where prAlthough Gladstone et al. (2011) did not find any significant neurodevelopmental differences among term and preterm babies at 12 months, significant differences were reported at 18 months of corrected age . Others We found that malnutrition was the main factor associated with NDD among preterm babies. About 20% of preterm infants had malnutrition, of whom a third had NDD. Malnutrition due to poor feeding, infections, and/or poor gut development increases the vulnerability of preterm babies to developing NDD ,35. ChilAlthough almost all preterm infants had KMC at the health facility, only 77% continued KMC practice at home for 4.5 hours a day on average. KMC is effective in reducing morbidity, and improved growth and neurodevelopment –44. A CoPolicy and program implications of this work include: as we improve preterm survival, we should be aware of NDD with its social, health system, and economic implications. Systematic follow-up of these infants for nutrition, growth and neurodevelopment monitoring until at least catch-up growth is recommended , to acceThe study involved only one district-level hospital, hence limiting generalizability of the findings to the preterm babies in the whole country. However, this particular hospital is a typical district hospital in the country though may be considered to have had a higher-level quality of care compared to others in the country at that time as it had concerted capacity building efforts for newborn care under the Preterm Birth Initiative, meaning that findings elsewhere might be worse. In addition, we used the Ballard score for GA estimation due to lack of first trimester/early ultrasound scan (USS) during pregnancy for most of the mothers which may limit the accuracy of gestation age dating. In this setting, there were no mechanisms of diagnosing intraventricular hemorrhage, one of the commonest causes of disabilities among preterm babies, and no preventive treatment modalities for this complication. Despite these limitations, the findings are still useful since malnutrition can be prevented in this setting. Although the MDAT tool used has some areas of personal care, it is currently the only developmental assessment tool we know of which was developed and validated for rural African setting. In this study, we had an attrition rate of 12.3%. In estimating the sample size for our study, we took care of loss to follow-up by adjusting the sample size by more than 20% for the preterm babies. Therefore, the attrition rate of 12.3% would have very minimal or no effect on our results.Preterm infants in the study setting are at a high risk of developing neurodevelopmental disabilities. Under-nourished infants and those whose mothers did not consistently practice KMC at home were more likely to have NDD. Health system readiness should, therefore, be improved to provide systematic follow-up care, inclusive of growth and neurodevelopmental assessments, to prevent and address malnutrition. Increased nutrition support, and continuity with KMC at home after discharge from health facility can be helpful in curbing NDD among preterm infants in low resource settings.


Sleep disturbance and related improvement in quality of life as a result of arthroscopic repair in rotator cuff tear (RCT) patients can be considered as an important parameter. The aim of our study is to evaluate the rotator cuff by ultrasonography (USG) in the first postoperative year and to examine whether there is a difference between sleep disturbance and quality of life between cases of rupture and healthy patients. In addition, we aim to compare the preoperative and at least the first postoperative year’s sleep disturbances and quality of life among patients who underwent arthroscopic RCT repair and to examine the effects of factors affecting this situation.We retrospectively reviewed the records of patients who were operated on for RCT. In this process, 257 patients were examined and 76 patients who met the inclusion criteria were included in the study. The Pittsburgh sleep quality index (PSQI), Constant–Murley shoulder score (CSS), and Oxford shoulder score (OSS) were used to evaluate the results preoperatively and at the last control visit of each patient. In the USG performed in the postoperative first year, the rotor cuff was evaluated in terms of rerupture.It was observed that 14 (18.4%) patients’ rotor cuffs were reruptured and those of 62 (81.6%) patients were intact. The preoperative PSQI, CSS, and OSS values of the patients were calculated as an average of 10.79 ± 3.58, 35.61 ± 8.88, and 17.61 ± 4.20 and the mean postoperative values were calculated as 5.45 ± 1.68, 81.55 ± 5.27, and 38.05 ± 3.06, respectively. The postoperative PSQI value was statistically significantly lower in patients with tears of <1 cm (PSQI: 4.29 ± 0.73) than in those with tears of 1–3 cm (PSQI: 5.50 ± 2.17) and 3–5 cm (PSQI: 5.88 ± 1.25) (P < 0.001). The mean CSS and OSS values were significantly higher in postoperative measurements for all tear types. According to the size of the tear, postoperative CSS and OSS values were statistically significantly lower in patients with tears of 3–5 cm (CSS: 78.59 ± 4.50 and OSS: 36.18 ± 2.47) than those with <1 cm (CSS: 85.43 ± 2.14 and OSS: 40.57 ± 1.55) and 1–3 cm (CSS: 83.21 ± 5.35 and OSS: 39.07 ± 2.94) tears (P < 0.001).In the USG performed in the postoperative first year, it was determined that the patients with healthy rotator cuffs recovered better than those with rerupture. Rotator cuff tear (RCT) is the second most common musculoskeletal pathology and one of the most common shoulder ailments for which patients need treatment . RCT causes pain, weakness, stiffness, and limitation of movement in the shoulder [3]. Sleep disturbance, especially caused by night pain, is an important source of complaints in patients, but the exact degree of pain that prevents sleep is unknown . This problem may cause negative emotional, behavioral, and cognitive effects in the patient and increase the prevalence of anxiety and depression . According to the American Academy of Sleep Medicine and the Sleep Research Society, regularly sleeping fewer than 7 h per night can cause health problems such as obesity, diabetes mellitus (DM), hypertension (HT), heart disease, stroke and depression . These problems alone are sufficient to create the need for treatment in a patient [9]. Adequate sleep is important for the recovery and satisfaction of the patient, and sleep disturbance and related improvement in quality of life as a result of arthroscopic repair in RCT patients can be considered as an important parameter.Arthroscopic RCT repair can generally improve shoulder pain, joint functions, psychological status, physical function, and general health status, but it may not provide sleep recovery in every patient due to patient-specific pathologies such as psychiatric disorders [9–11]. It has been shown that sleep disorders occur in between 15% and 35% of the general population . There may be different factors such as functional limitations, challenging positions, and shoulder stiffness that play important roles in reducing sleep quality [9]. In addition, it was concluded that preoperative and postoperative use of narcotic pain medications increased wakefulness and inhibited REM sleep, resulting in sleep disturbance .There is no consensus on improving sleep disturbance and quality of life after arthroscopic RCT repair. To date, it has been shown that there are relationships between sleep quality and factors such as female gender, HT, DM, body mass index (BMI), depression, low back pain, corticosteroid injections, and osteoarthritis in patients with RCT, while no statistically significant relationship was found between RCT features and sleep quality . When the literature is reviewed, there is no study conducted to investigate the effect of rotator cuff (RC) rerupture on sleep and quality of life in the postoperative period [12].The aim of our study is to evaluate RCs by ultrasonography (USG) in the first postoperative year and to examine whether there is a difference between sleep disturbance and quality of life between cases of rupture and healthy patients. In addition, we aim to compare the preoperative and at least the first postoperative year’s sleep disturbances and quality of life among patients who underwent arthroscopic RCT repair and to examine the effects of factors affecting this situation. Our hypothesis in this study was as follows: With the USG performed in the postoperative first year, the improvement in sleep and quality of life will be significantly higher in patients with healthy RCs compared to those with ruptures.We retrospectively reviewed the records of patients who were operated on for RCT by the same surgeon in a single center between 2015 and 2019. In this process, 257 patients treated surgically for RCT were examined. The study’s inclusion criteria were as follows: Patients over 18 years of age who underwent arthroscopic repair due to RCT; atraumatic rerupture without implant failure and without revision surgery according to the Cofield classification [15]; and <1 cm , 1–3 cm (medium), or 3–5 cm (large) full-thickness RCT in patients who underwent arthroscopic repair with at least 1 year of follow-up. Exclusion criteria were as follows: Patients who had undergone previous shoulder surgery; massive (>5 cm) RCT; simultaneous adhesive capsulitis; advanced glenohumeral arthritis; history of psychiatric illness; treatment of history for a sleep disorder; use of narcotic analgesics or tenodesis for biceps pathology; and the unwillingness to be included in the study. As a result, 76 patients who met the criteria were included in the study.The study protocol was approved by the Ethics Committee of Ankara City Hospital (reference number: E1-20-1070).In this study, the Pittsburgh sleep quality index (PSQI) [16], Constant–Murley shoulder score (CSS) [17], and Oxford shoulder score (OSS) [18] were used to evaluate the results preoperatively and at the last control visit of each patient. Demographic data of the patients such as BMI [19] and nicotine and alcohol use were collected along with age, sex, affected side, chronic diseases , height, and weight. In the USG performed because it is easier and less costly in the postoperative first year, the RC was evaluated in terms of rerupture.All patients were operated on in the sunbed position under hypotensive (systolic blood pressure: 80–100 mmHg) general anesthesia, keeping the joint pump pressure around 50 mmHg. Postoperative intravenous analgesic was administered to patients in whom an interscalene block could not be performed. According to the Cofield classification, the definition of the tear, repair technique, materials used, additional interventions such as subacromial decompression, distal clavicle excision, biceps intervention, and intraoperative complications were recorded after surgery. For tear sizes, AP lengths were measured by arthroscopic imaging using a calibrated probe. Considering the tension of the RC, a single row or transosseous-equivalent double row was repaired.Oral analgesics and nonsteroidal antiinflammatory drugs (NSAIDs) were prescribed at the time of discharge. A similar rehabilitation protocol was applied for all patients postoperatively and a shoulder/arm sling was used for immobilization for 4 weeks. Pendular exercise was started on the postoperative first day. On the 15th day, the sutures were removed and four-way isometric exercises and passive exercises of up to 90° were started. In addition to isometric exercises, active exercises of 90° and passive exercises of 90–120° were added in the first month’s control visits. Active full-range motion exercises were started after the eighth week. From the third month, strengthening exercises against resistance were started. After the fourth month, it was possible to return to daily activities without any restrictions.Patient outcome scores were collected and scored by one research coordinator and were recorded preoperatively and postoperatively.The PSQI is designed to measure sleep quality [16]. It has been generally used in evaluating sleep quality in clinical practice. Patients complete a 19-question survey, from which 7 component scores are obtained: subjective sleep quality, sleep latency, sleep duration, habitual sleep efficiency, sleep disorder, sleep medication use, and daytime functional disorder. This questionnaire yields scores from 0 to 21 points. A higher score indicates worse sleep dysfunction and a total score of >5 is indicative of poor sleep quality.The CSS is a comprehensive and comparable assessment of shoulder function [17]. This patient and clinician completed survey contains four subscales: pain (15 points), activities of daily living (20 points), strength (25 points), and range of motion including forward elevation, external rotation, abduction, and internal rotation of the shoulder (40 points). A higher score represents higher quality of function.The OSS aims to provide a self-assessment of pain and function of the shoulder and is commonly used in shoulder operations other than stabilization [18]. It comprises 12 items: 4 about pain  and 8 about daily functions. Each item in the original scale is scored according to a 5-point Likert-type scale, where 1 = no pain/easy to do, 2 = mild pain/little difficulty, 3 = moderate pain/moderate difficulty, 4 = severe pain/extreme difficulty, and 5 = unbearable/impossible to do. In the revision study and the online form, the item scoring is from 0 (worst) to 4 (best).Data analyses were performed using SPSS 22.0 for Windows . Whether the distribution of continuous variables was normal or not was determined by the Kolmogorov–Smirnov test. Levene’s test was used for the evaluation of homogeneity of variances. Unless otherwise specified, continuous data were described as mean ± SD for normal distributions and as mean ± SD and median (interquartile range) for skewed distributions. Categorical data were described as number of cases (%). In the statistical analysis, differences between nonnormally distributed variables in two independent groups were compared with the Mann–Whitney U test while the differences between nonnormally distributed variables among more than two independent groups were analyzed by the Kruskal–Wallis test. When the P-values from Kruskal–Wallis test statistics were statistically significant, posthoc Conover–Iman tests/multiple comparison tests were used to determine which group differed from the others. Statistically significant differences for nonnormally distributed variables between two dependent groups were compared with the Wilcoxon test. Categorical variables were compared using Pearson’s chi-square test or Fisher’s exact test. Degrees of relation between variables were evaluated with Spearman’s correlation analysis. Multivariate linear regression (backward method) was first used with risk factors thought to be related to postoperative PSQI, OSS, and CSS values. R2 values were analyzed to determine how much independent variables explained dependent variables. The model adaptation of estimates was also analyzed with the ANOVA test model adaptation test. ROC curve analysis was used to determine the cutoff points. Values of P < 0.05 were accepted as significant in all statistical analysis.The average age of our patients was 60.39 ± 9.04 years, with 28 men and 48 women. The demographic characteristics of the patients are presented in the Table 1. It was observed that 14 (18.4%) patients’ RCs was reruptured and those of 62 (81.6%) patients were intact. Those with rerupture had statistically significantly higher postoperative PSQI scores and lower CSS and OSS scores compared to those without rerupture (P < 0.001) (Table 2).The postoperative PSQI value among all patients was thus statistically significantly lower than the preoperative value (P < 0.001). The postoperative CSS value among all patients was statistically significantly increased compared to the preoperative value (P < 0.001). The postoperative OSS value increased statistically significantly among all patients compared to the preoperative value (P < 0.001) (Table 3).According to the RCT Cofield classification, 25 patients had ruptures of <1 cm , 23 patients of 1–3 cm (medium), and 13 patients of 3–5 cm (large). Sleep disturbances had improved significantly in the postoperative measurements for all tear types. According to the size of the tears, the postoperative PSQI value was statistically significantly lower in patients with tears of <1 cm than those with tears of 1–3 cm and 3–5 cm (P < 0.001). The mean CSS and OSS values were significantly higher in the postoperative measurements of all tear types. According to the size of the tear, postoperative CSS and OSS values were statistically significantly lower in patients with tears of 3–5 cm than those with tears of <1 cm and 1–3 cm (P < 0.001) (Table 4).It has been observed that alcohol use affects postoperative PSQI results. Specifically, alcohol use increased the postoperative PSQI score in this study (P = 0.015). None of the patients used narcotics. The variables of age, sex, follow-up time, surgery side, BMI, trauma history, DM, HT, HL, smoking, and surgical technique did not independently affect sleep disorder . No complication that might require treatment developed in any of our patients during the postoperative period.When the literature is reviewed, there is no study conducted to investigate the effect of rerupture and robustness of the RC on the quality of sleep and life in the postoperative period of patients who have undergone arthroscopic repair due to RCT [12]. When patients with rupture and those without were compared in our study, it was found that those with rupture had lower sleep quality.Although many studies have been reported on the relations between shoulder pathologies such as subacromial impingement, glenohumeral arthrosis, and adhesive capsulitis and sleep disturbance, only a few studies have been reported on the effects of RCT on sleep disturbance . Cho et al. reported sleep disturbance (PSQI: 8.1) caused by chronic pain in RCT patients [20]. Mulligan et al. reported that 93% of patients with RCT had night pain and 71% had sleep disturbance (PSQI: >5), and the cause of the sleep disturbance was related to night pain [5]. Gumina et al. claimed that RCT is only one of the causes of sleep disturbance in middle and old ages. In addition, they reported that night pain was the main cause of sleep disorders in RCT patients [21]. Khazzam et al. stated that 94% of patients with RCT complained of night pain (PSQI: 9.48), and 89% of patients with RC tendinitis (impingement) had night pain (PSQI: 8.66). They stated that shoulder pain symptoms may not be clearly related to the severity of RC disorders [7]. Our present findings for patients with RCT are compatible with those presented in the literature, with an average PSQI value of 10.79 ± 3.58. This result shows a correlation between sleep disturbance and RCT.Recovery from sleep disturbance after arthroscopic RCT repair was first described in the literature by Austin et al. [9], who stated that sleep disturbance (PSQI: >5) decreased from 89% preoperatively to 38% in the sixth postoperative month and that recovery continued for 6 months. Cho et al. reported that sleep disturbance improved in the 12-month follow-up of 47 patients who underwent RCT repair, but for 42% of the patients, problems still continued (PSQI: >5) [20]. Serbest et al. found a dramatic improvement in sleep disturbance in a 6-month follow-up period (PSQI: 15 to 6) for 31 patients who underwent RCT repair. Although they found a great improvement in sleep disturbance, 58% of patients still complained of sleep disturbance (PSQI: >5) 6 months after surgery [22]. Hornef et al. reported that 89% of RCT patients with preoperative sleep disorders (PSQI: 11.7) recovered postoperatively, while41% (PSQI: 5.5) recovere after at least 24 months of follow-up [23]. In our study, it was observed that the mean PSQI score decreased from 10.79 ± 3.58 to 5.45 ± 1.68. The mean follow-up time was 31.44 ± 4.67 months, which was longer than in previous studies. In this case, it was observed that there was a great improvement in sleep disturbance after at least 1 year of follow-up after arthroscopic RCT repair. We can thus say that sleep disturbance decreases after arthroscopic RCT repair.According to Austin et al., RCT dimensions did not correlate with sleep disorders [9]. Supporting this study, there are many studies showing that tear size does not affect sleep disorder . Gumina et al. reported that patients with small tears had lower sleep quality than those with larger tears. However, in this study, sleep disturbance was reported only in the preoperative period, and sleep improvement was not reported in the postoperative period [21]. According to the study of Cicekli, large tears caused lower sleep quality than other tears [24]. In our study, tears of 3–5 cm (large) caused lower sleep quality than other tears.Austin et al. demonstrated correlations among the simple shoulder test (SST), visual analog scale for pain (VAS), and PSQI. An increase in SST was accompanied by decreases in VAS and PSQI [9]. Horneff et al. reported that the correlation between SST, VAS, and PSQI scores continued for up to 24 months [23]. Serbest et al. found a correlation between shoulder scores and sleep disturbance [22]. In our study, the improvements in shoulder scores and sleep quality were associated. Good results in PSQI scores together with good results in CSS and OSS scores have been demonstrated after at least 1 year of follow-up.Khazzam et al. showed that factors causing sleep disturbance in cases of RCT are female gender, depression, low back pain, DM, cervical involvement, and high BMI, but they did not examine the factors affecting the results of RCT repair [7]. Austin et al. stated that the only independent demographic or surgical factor affecting postoperative sleep was the use of narcotic pain medication [9]. Serbest et al. reported that there was no correlation between demographic variables and chronic disease and sleep disturbance in patients who underwent RCT repair [22]. Additionally, less improvement in sleep quality after RCT repair was reported in patients with concurrent DM and HT [24]. In our study, demographic data of the patients such as age, sex, follow-up time, surgery side, BMI, trauma history, DM, HT, HL, smoking, and surgical technique were analyzed, and it was found that there was no effect on sleep disorders after RCT repair.The most important feature of our study is the evaluation of RCs with USG in the first postoperative year among patients who underwent arthroscopic repair due to RCT. We consider the exclusion of patients who underwent previous shoulder surgery or had massive (>5 cm) RCT, simultaneous adhesive capsulitis, advanced glenohumeral arthritis, psychiatric disease history, history of treatment for sleep disorder, use of narcotic analgesics, and tenodesis for biceps pathology as an advantage of this study. The limitations of this study are that it was retrospective and there was not a control group or isolated RCT patient group. It is necessary to study patients with the same types of tears belonging to the same sex and similar age groups together with a control group comprising individuals who do not have additional shoulder pathologies or chronic diseases with RCT.In this study, in the USG performed in the first postoperative year, it was determined that the patients with healthy rotator cuffs recovered better than those with ruptures. According to this, we can say that in the follow-up of patients who underwent arthroscopic repair due to RCT, patients with intact RC have more significant improvement in sleep disturbance and quality of life compared to patients with ruptures. Due to the limitations of this study, it is difficult to say that the sleep disorders of patients who underwent RCT arthroscopic repair recovered specifically due to that repair. However, we can say that sleep disturbance and quality of life improved as a result of the arthroscopic repair performed for RCT and its accompanying pathologies and the rehabilitation protocol applied during the follow-up process.