Numerous animal studies have demonstrated that commonly used general anesthetics may result in cognitive impairment in the immature brain. The prevailing theory is that general anesthetics could induce developmental neurotoxicity via enhanced apoptosis. In addition, inhibited proliferation induced by anesthetics has also been reported. So far, whether autophagy, a well-conserved cellular process that is critical for cell fate, also participates in anesthesia-induced neurotoxicity remains elusive. Here, we first examined autophagy-related changes after sevoflurane exposure and the effect of autophagy on apoptosis and proliferation, and we also explored the underlying mechanisms of autophagy activation. Pregnant rats were exposed to 2 or 3.5% sevoflurane for 2 h on gestational day 14 (G14); then, markers of autophagy and expression of autophagy pathway components were measured in fetal brains 2, 12, 24, and 48 h after anesthesia. Changes in neural stem cell (NSC) apoptosis, neurogenesis, neuron quantity and learning and memory function were examined after administration of an autophagy or PTEN inhibitor. The expression of microtubule-associated protein 1 light chain 3 (LC3)-II, Beclin-1 and phosphatase and tensin homolog on chromosome 10 (PTEN) were increased in the 3.5% sevoflurane group, while Sequestosome 1 (P62/SQSTM1), phospho-protein kinase B/protein kinase B (p-Akt/Akt) and mammalian target of rapamycin (mTOR) were decreased. 3-methyladenine (3-MA), an inhibitor of autophagy, or dipotassium bisperoxo-(5-hydroxypyridine-2-carboxyl)-oxovanadate (V) (bpV), a PTEN inhibitor, significantly attenuated the activation of autophagy, reversed the decreased expression of B-cell lymphoma-2 (Bcl-2) and reduced the number of terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) positive cells, ameliorated the decline of Nestin expression, Ki67 positive cell rate, neuron quantity and cross platform times, and shortened the prolonged escape latency. Our results demonstrated that 2 h 3.5% sevoflurane exposure at G14 induced excessive autophagy in the fetal brain via the PTEN/Akt/mTOR pathway. Autophagy inhibition reversed anesthesia-induced NSC apoptosis, proliferation decline and memory deficits. Advanced surgical technologies have made it possible to conduct more fetal intervention operations. And most of them are carried out in the second trimester, which is regarded as a relatively safe period compared with earlier or later gestation. However, the fetal brain is still sensitive to changes in the external environment during this period.Mammalian neural stem cells (NSCs) have attracted much attention as they play a significant role in brain development . Owing tA large body of animal research has demonstrated that use of common general anesthetics may result in cognitive impairment in immature brains . Most stA agonists, we hypothesized that sevoflurane exposure may also increase autophagy level in fetal brain. In addition, emerging evidence indicates that, not only is autophagy related to cell proliferation, there are also complex interactions between autophagy and apoptosis  rat model since the mid-pregnancy is considered safe for fetal intervention . We hypoAll animal experiments were conducted following the National Institute of Health Guideline for the Care and Use of Laboratory Animals. This study was approved by the Institutional Animal Care and Use Committee of Shengjing Hospital, China Medical University (No. 2015PS211K).Two-month-old female Sprague-Dawley rats (220–250 g) and male SD rats (250–280 g) used in the study were procured from Research and Development Center of Shengjing Hospital. The rats were housed at a constant temperature of 22 ± 1°C with free access to water and food. They were maintained under a 12 h light/12 h dark cycle (lights-on from 7 AM to 7 PM). Each male rat was put in the cage together with 3–4 female rats. The next morning, male rats were removed, and vaginal smear of female rats were conducted. If sperm was observed, the female rat was considered pregnant and the day was recorded as gastation day 0 (G0) with respect to the pregnant rats and embryonic day 0 (E0) with respect to the fetus.Rats were put into a acrylic chamber for anesthesia with two holes on the opposite sides of the chamber, each connected to a duct. One duct was connected to a sevoflurane vaporizer transporting mixed gas including oxygen, nitrogen and sevoflurane; the other duct was used for transporting the gas samples to the monitor. The temperature in the chamber was maintained via heating pool. In the control (CON) group, rats were placed in a chamber which was ventilated with 30% oxygen at a flow rate of 2 L/min for 2 h. 2% sevoflurane (2%SEV) group and 3.5% sevoflurane (3.5%SEV) group received 2 or 3.5% sevoflurane for 2 h with 30% oxygen. The rats breathed spontaneously during the gas exposure. When the anesthesia was terminated, the rats were transferred into the normal cages, and the fetuses in the uterus were obtained through cesarean section 2, 12, 24, and 48 h after gas exposure or natural birth, respectively.Rats in 3.5% sevoflurane plus 3-Methyladenine group (3.5%SEV + 3-MA) were intraperitoneally injected with 3-MA (40 mg/kg), and rats in 3.5% sevoflurane plus bpV group (3.5%SEV+bpV) were intraperitoneally injected with bpV (0.2 mg/kg). Drug administration mentioned above were all initially conducted 1 h before anesthesia. Other groups that were compared with the two groups above were treated with the same volume of vehicle solution.3) from the fetal brain was completed in the 2% glutaraldehyde and the samples were post-fixed in 1% osmium tetraoxide for 2 h. Then the tissues were infiltrated in a mixture of acetone and resin before undergoing dehydration with graded ethanol. The tissue samples were then embedded in resin. Sections were obtained and followed by staining of 2% uranyl acetate and lead citrate. Autophagic specific structures in the NSCs were observed.G14 rats anesthetized with pentobarbital 2 h after 3.5% sevoflurane exposure were transcardially given 200mL normal saline followed by 200 mL 4% paraformaldehyde. The immersion fixation of tissue samples . The primary antibodies were: LC3B Anitibody , Beclin-1 Antibody , P62/SQSTM1 Antibody , PTEN Antibody , Akt Antibody , Phospho-Akt Antibody , mTOR Antibody , Nestin Antibody , Bcl-2 Antibody . Number of rats per group in Western blot experiment was 5 (n = 5).The whole brains of fetuses were removed immediately when the female rats were under cesarean section, and stored at -80°C before use. The samples were homogenzied and ultrasonicated in radio-immunoprecipitation assay buffer  containing phenylmethylsulfonyl fluoride solution . The homogenate was centrifuged at 1200 g for 30 min, and the supernatant was collected then measured by BCA Protein Assay Kit  in order for equal concentration. Forty micrograms of protein per lane was separated by electrophoresis using 10 or 12.5% SDS-polyacrylamide gel and then electrotransferred to polyvinylidene fluoride members . The membranes were blocked in 5% non-fat milk or BSA and incubated with primary antibodies overnight at 4°C. The next day, the membranes were incubated with the second antigen-antibodies at room temperature (RT) for 2 h. Proteins were detected and photographed by GE Amersham Imager 600 using SuperSignal2) at 400× magnification. Average values of three determinations were converted to the ratio of the CON group values and used to calculate the relative neuron quantities. Number of rats per group in immunohistochemistry experiment was 5 (n = 5).Whole brains were removed from fetuses. Fetal brains were first immersed in 4% PFA at 4°C for 24 h, then embedded in paraffin after dehydration in graded ethanol. Transverse plane sections  were deparaffinized and heated in citrate buffer at 121°C for 7 min. PBS containing 10% fetal bovine serum and 3% hydrogen peroxide was added to each section to reduce background staining. Next, sections were incubated with the Nestin Antibody  or NeuN Antibody  at 4°C for 12 h in a humidified chamber. After three 5 min rinses in PBS, the sections were incubated with peroxidase-conjugated secondary antibody and DAB was applied for chromogenic staining. The nuclei were stained with hematoxylin. Finally the sections were photographed with Nikon C1 microscope by an investigator who was blinded to the experimental intervention. Each brain section of Nestin staining was photographed at least 2 random fields of view and OD value was quantified by NIS-Elements AR Analysis 4.50.00 software. We sliced each brain tissue serially and selected 3 comparable brain slices per rat for NeuN staining. NeuN-positive cells were counted in a random reticle (approximately 0.01 mmn = 5).The brain sections were deparaffinized and heated in citrate buffer for antigen retrieval. PBS containing 10% fetal bovine serum was added to each section to reduce background staining for 30 min at room temperature. Sections were incubated with two kinds of primary antibodies simultaneously at 4°C overnight in a humidified chamber for immunofluorescence double staining, and then they were incubated with secondary antibodies for 2 h at RT. When performing the immunofluorescence staining for Ki67, PBS with 0.3% Triton X-100 was used to rinse the sections instead of PBS. The antibodies used were: LC3B Antibody , Nestin Antibody  and Ki67 Antibody . The nuclei were stained with DAPI. Sections were photographed with Nikon C1 microscope by an investigator who was blinded to the experimental interventions. Each Ki67 staining section with 1–2 random fields of view was photographed, and the numbers of Ki67 positive cells and total cells were counted at 400× magnification by NIS-Elements AR Analysis 4.50.00 software, then we calculated the ratio of the number of Ki67 positive cells to the total number of cells. Number of rats per group in immunofluorescence staining experiment was 5 (n = 5).The brain sections were deparaffinized and heated in citrate buffer for antigen retrieval. PBS containing 10% fetal bovine serum was added to each section at room temperature for 30 min to reduce background staining. The sections were incubated with the Nestin Antibody  at 4°C overnight in a humidified chamber and then incubated with secondary antibody and terminal deoxynucleotidyl transferase (TdT) and dUTP  simultaneously at 37°C for 1 h. Nuclei were stained with DAPI. Each brain section with 1 random field of view was photographed with Nikon C1 microscope by an investigator who was blinded to the experimental interventions. The number of TUNEL/Nestin-positive cells was counted at 400× magnification by NIS-Elements AR Analysis 4.50.00 software. Number of rats per group in TUNEL experiment was 5  were put into the water to search for the hidden platform from four quadrants facing the pool wall. The duration spent on finding the platform (escape latency) was within 90 s. When the rats did not find the platform within 90 s, they were guided toward the platform and the escape latency was recorded as 90 s. After each trail, rats were made to stand on the platform for 20 s. During spatial probe test, rats were permitted to swim for 90 s freely after the platform was taken away from the maze. The entire process of MWM experiment was recorded by a camera located above the pool and analyzed using image analysis software .To test the spatial learning and memory ability of the rat offspring, Morris water maze (MWM) experiments were conducted 28–33 days after birth. The MWM was performed in a circular pool with black walls . The pool was filled with 30-cm-deep water at 20°C. An escape platform (diameter: 12 cm) was located 1.5 cm below the surface of water and at the center of the target quadrant. Probe trial sessions began at 8: 00 am for 5 days and were conducted four times (once per quadrant) daily with 30 min rest time. Rats  followed by Bonferroni post test for the four groups’ comparisons. The spatial probe test data were analyzed using Kruskal–Wallis with Dunn’s Multiple comparison test. All the data were analyzed with GraphPad Prism 5.0 software or SPSS 20.0 software. Differences were considered to be statistically significant if P < 0.05. Data were presented as mean ± SD.The data were checked by Bartlett’s test for equal variances and Shapiro–Wilk test for normality. Parametric data of three groups were compared by one-way analysis of variance (ANOVA) followed by the Student–Newman–Keuls Figure 1). LC3 has two forms: when autophagy is initiated, LC3II is formed by conjugating LC3I to phosphatidylethanolamine. Since the expression of LC3I is quite abundant in brain tissue . To further verify these results, fetal brains were harvested 2 h after the 3.5% sevoflurane exposure and examined under the transmission electron microscope (TEM), which is considered as the gold standard for detecting the autophagic process. TEM revealed the presence of double-membrane vacuolar structures containing cytoplasmic content in the cytoplasm of NSCs, representing the formation of autophagosomes and autolysosomes . To further verify autophagy induction in NSCs, we performed double immunostaining of LC3B and Nestin, which is a maker of NSCs. In the brain sections from the 3.5%SEV group, LC3B co-localized with Nestin presenting punctate pattern, whereas brain sections from both the CON group and 2%SEV group showed diffused green light labeling. Taken together, these results indicated that sevoflurane activated autophagy in NSCs after exposure to 3.5% sevoflurane; and there were no differences observed between the 2%SEV group and CON group .In our previous study, we found that 3.5% sevoflurane anesthesia resulted in acute neurotoxicity with learning and memory impairment in rat offsprings; whereas no differences were observed between rats in the 2% sevoflurane group and the control group. To further study the autophagic activity in fetal brains, we examined the expression of the key autophagy-related proteins including LC3BII, Beclin-1 and P62/SQSTM1 2, 12, 24, and 48 h after the exposure . In accordance with the above findings, there was no difference between the 2%SEV and CON group.The results we demonstrated above was 3.5% sevoflurane upregulated the level of autophagy. However, the mechanism by which this occurred is unclear. To further investigate the mechanism of autophagy activation induced by 3.5% sevoflurane, we examined the PTEN/Akt/mTOR activity with Western blotting 2, 12, 24, and 48 h after gas exposure. 3.5% sevoflurane had significantly upregulated PTEN and downregualted p-Akt/Akt and mTOR in the fetal brains, compared to the CON and 2%SEV group , and LC3BII and Beclin-1 levels were reduced in the 3.5%SEV + bpV group than the 3.5%SEV group . Besides the role of regulating autophagy, we also observed a decline in the number of TUNEL positive cells and an increase in both Ki67 positive cell rate and Nestin expression in the 3.5%SEV + bpV group compared with the 3.5%SEV group , which indicated that autophagy might regulate apoptosis and proliferation when the fetal brains were exposed to 3.5% sevoflurane.In order to prove that autophagy was mediated by PTEN/Akt/mTOR pathway, the PTEN inhibitor, bpV, was intraperitoneally injected into G14 rats 1 h before gas treatment. Then the brains were harvested 24 h after anesthesia. The results showed p-Akt and mTOR levels were increased . Then we assessed the extend of apoptosis via with detecting TUNEL and Western blotting of the anti-apoptosis protein, Bcl-2. Exposure to 3.5% sevoflurane for 2 h induced NSC apoptosis in the immature brains with decreased Bcl-2 expression compared to the CON group . The Bcl-2 expression in the 3.5% sevoflurane plus 3-MA group was increased compared to the 3.5% sevoflurane group . TUNEL results also validated the anti-apoptotic effects of 3-MA on the developing brains exposed to 3.5% sevoflurane. More TUNEL-Nestin double positive cells were observed in the 3.5%SEV group than CON group , and fewer positive cells were observed in the 3.5%SEV+3-MA group than 3.5%SEV group at all of the four time points . Collectively, the results indicated that the hyperactive autophagy could lead to NSC apoptosis, and inhibiting autophagy could ameliorate NSC apoptosis.To reveal the effects of autophagy on NSC apoptosis, we injected 3-MA intraperitoneally into G14 rats 1 h prior to sevoflurane treatment. Then fetal brains were harvested at the four time points mentioned above. We evaluated the expression of LC3BII and Beclin-1 to verify the effects of 3-MA on the inhibition of autophagy. Both LC3BII and Beclin-1 levels were reduced as compared with the 3.5%SEV group . The Nestin expression in the 3.5% sevoflurane plus 3-MA group was increased compared to the 3.5% sevoflurane group , which was consistent with the results of Nestin immunohisochemistry . Then we investigated the proliferation of NSCs with immunofluorescence staining. Neurogenesis in the fetal brain was evaluated by the ratio between Ki67 positive cells and total cells. We observed that Ki67 positive rate was decreased in the 3.5% SEV group compared to the CON group , and the 3.5%SEV + 3-MA group presented a higher positive rate compared to the 3.5%SEV group . The fundings above suggested that hyperactive autophagy could contribute to the descending proliferation of NSCs.To examine whether autophagy was involved in the inhibited NSC proliferation, 3-MA was applied as described above in the G14 rats. Then we assessed the proliferation of NSCs by detecting Nestin expression with Western blotting and immunohistochemistry, and Ki67 expression with immunofluorescence staining. Exposure to 3.5% sevoflurane for 2 h disturbed NSC proliferation in the immature brains with a declined Nestin expression compared to CON group . Consistent with previous findings, offsprings of the 3.5%SEV group took longer time to reach the platform as compared with the offsprings of the CON group from the third day of training. The application of 3-MA or bpV could significantly shorten the escape latency after the exposure of sevoflurane; there was no significant difference among else offsprings of the 3.5%SEV + 3-MA group, 3.5%SEV + bpV group and CON group . During the spatial probe test, offspring from the 3.5%SEV group crossed the platform fewer times than offsprings from the CON group , 3.5%SEV + 3-MA group  and 3.5%SEV + bpV group , and there was no difference in 3.5%SEV + 3-MA group, 3.5%SEV + bpV group and CON group. After the spatial probe test, we assessed the quantity of neurons in CA1 hippocampal region by NeuN immunohistochemistry. The rats exposed to 3.5% sevoflurane for 2 h at E14 showed a declined relative neuron quantity compared with the CON group , the relative numbers of NeuN-positive cells in the 3.5%SEV + 3-MA and 3.5%SEV + bpV group were increased compared to the 3.5% sevoflurane group , and there was no difference compared with the CON group.We also explored the role of autophagy in learning and memory deficiencies by investigating the effect of 3-MA and bpV on neuron quantity in the hippocampal CA1 area and MWM performance. Pregnant rats delivered pups on G21 day. Learning and cognition of the offsprings were tested with the MWM on P28 to P33. There was no motor impairment among groups. We detected significant difference between groups during the learning phase using two-way ANOVA analysis (group: in utero interventions in recent years. Midtrimester is a proper stage to perform these procedures as embryogenesis is then complete (The extensive and growing use of anesthetics in pregnant women and young children raises significant concern regarding to their safety on developing brains . Accordicomplete . Neverthcomplete . In the complete . It has complete .in utero interventions are usually performed with relative high-dose general anesthetics to prevent preterm labor and for a longer duration (Given the FDA warning, anesthesia duration and frequency are considered key factors in anesthesia-induced neurotoxicity; however, the dose of the anesthetics may also play an important role . Compareduration . Since tduration , we specBesides apoptosis and neurogenesis, autophagy also participates in anesthesia-induced neurotoxicity. Autophagy is indispensable for normal brain development. In NSCs from E13.5-15.5 mice, the expression of autophagy related genes increase gradually . InterfeTo our knowledge, this study is the first to provide the evidence of NSC autophagy in the fetus exposed to sevoflurane, and our results indicated that autophagy was regulated via the PTEN/Akt/mTOR pathway. And the effect could be alleviated by PTEN inhibitor bpV. The PI3K/Akt/mTOR signaling pathway has been reported to be an important negative regulator of autophagy . PTEN caThe relationship between sevoflurane anesthesia-induced apoptosis and autophagy in the NSCs remains poorly understood. Both apoptosis and autophagy participate in development of the central nervous system. During brain development, apoptosis is the main mechanism to prevent excessive cell survival . Our resIt has been proved that neuroapoptosis is not the only reason to be responsible for developmental anesthesia related neurotoxicity . After eApart from abnormal neurogenesis and neuroapoptosis, other factors like autophagy might also lead to adverse neurocognitive sequelae. However, there was rare evidence on the relationship between autophagy and sevoflurane induced cognitive dysfunction. Contrary to the our results that inhibition of autophagy alleviated cognitive dysfunction, blocking autophagy by chloroquine exacerbated sevoflurane induced neuronal apoptosis and memory impairment . This diOur study does have a few limitations. First, we did not observe dynamic changes of autophagy over time. Instead, we compared the changes in autophagy levels in different treatment groups; also, in terms of clinical applications, general anesthesia usually does not involve only one single general anesthetic agent. Combined use of general anesthetics may perplex the neurotoxicity outcome. Further studies on the effect of combined use of anesthetic agents are necessary to elucidate the issue.In summary, our research demonstrated that autophagy was upregulated through the PTEN/Akt/mTOR pathway when the E14 rats were exposed to 3.5% sevoflurane for 2 h, and that the activated autophagy led to increased apoptosis, declined NSCs proliferation, decreased relative neuron quantity, and impaired neurocognitive function. Understanding the role of NSC autophagy induced by 3.5% sevoflurane may provide novel strategies to alleviate the neurodevelopment impairment in the future.XL and PZ designed the experiments. XL, ZW, and PZ contributed to the planning of the work. XL performed all the experiments with the help of ZW, YZ, YX, and GH. ZW, YZ, YX, and GH participated in the data collection. XL, YZ, YX, and GH analyzed and interpreted the results. XL wrote the manuscript with the help of YX and GH. PZ supervised the project and revised the article.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.


Primary adrenal insufficiency (PAI) is a rare but severe and potentially life-threatening condition. No previous studies have characterized Portuguese patients with PAI.To characterize the clinical presentation, diagnostic workup, treatment and follow‐up of Portuguese patients with confirmed PAI.This multicentre retrospective study examined PAI patients in 12 Portuguese hospitals.We investigated 278 patients with PAI , with a mean age of 33.6 ± 19.3 years at diagnosis. The most frequent presenting clinical features were asthenia (60.1%), mucocutaneous hyperpigmentation (55.0%) and weight loss (43.2%); 29.1% of the patients presented with adrenal crisis. Diagnosis was established by high plasma ACTH and low serum cortisol in most patients (43.9%). The most common aetiology of PAI was autoimmune adrenalitis (61.0%). There were 38 idiopathic cases. Autoimmune comorbidities were found in 70% of the patients, the most frequent being autoimmune thyroiditis (60.7%) and type 1 diabetes mellitus (17.3%). Seventy-nine percent were treated with hydrocortisone (mean dose 26.3 ± 8.3 mg/day) mostly in three (57.5%) or two (37.4%) daily doses. The remaining patients were treated with prednisolone (10.1%), dexamethasone (6.2%) and methylprednisolone (0.7%); 66.2% were also on fludrocortisone (median dose of 100 µg/day). Since diagnosis, 33.5% of patients were hospitalized for disease decompensation. In the last appointment, 17.2% of patients had complaints (7.6% asthenia and 6.5% depression) and 9.7% had electrolyte disturbances.This is the first multicentre Portuguese study regarding PAI. The results emphasize the need for standardization in diagnostic tests and etiological investigation and provide a framework for improving treatment. Addison disease (AD) or primary adrenal insufficiency (PAI) is a life-threatening disease that results from bilateral destruction or dysfunction of the adrenal cortex . PAI is Tuberculosis has historically been the predominant cause of AD , 11, 12,AIRE) gene and is characterized by the presence of two of the three main components: AD, chronic mucocutaneous candidiasis and hypoparathyroidism. The more common APS-2 is characterized by two or more concurrent autoimmune endocrinopathies, such as AD, autoimmune hypo- or hyperthyroidism and type 1 diabetes mellitus (DM) . APS-1 is a very rare monogenic disorder caused by mutations in the autoimmune regulator (tus (DM) . Isolatetus (DM) .To date, no data have been available about the epidemiology, clinical features and management of Portuguese patients with PAI. The main objective of this study is to characterize the clinical presentation, diagnostic workup, treatment and follow‐up of such patients. Other objectives of this study are to explore the treatment modalities in this group and identify the most prevalent concomitant diseases.This observational, multicentre, retrospective study investigated patients older than 18 years with a diagnosis of PAI followed by endocrinology in 12 national hospitals . Hospitals in the Adrenal Tumours Study Group (GET-SR) of the Portuguese Society of Endocrinology were invited to participate in the study. Patient selection and data collection occurred between September and November 2016. All patient clinical data were anonymized and analysed by an independent reviewer. Consent has been obtained from each patient after full explanation of the purpose and nature of all procedures used. Ethical approval was granted by the Ethics Committee of Centro Hospitalar de São João.The diagnosis of PAI was verified by review of medical records by 15 endocrinologists and endocrinology residents from the 12 Portuguese centres. AD was diagnosed based on one of the following criteria: (1) a plasma adrenocorticotropic hormone (ACTH) concentration exceeding 66 pmol/L in combination with a low serum cortisol (<5 µg/dL); (2) an abnormal standard dose ACTH stimulation test (peak plasma cortisol <18 µg/dL); (3) characteristic clinical signs and symptoms such as hyperpigmentation, salt craving, typical electrolyte disturbances and chronic treatment with glucocorticoids and fludrocortisone in patients who were diagnosed decades ago. The diagnosis of autoimmune AD was based on positive autoantibodies toward 21-hydroxylase or associated autoimmune diseases.Cases with no known cause were classified as idiopathic. Patients were excluded if their adrenal insufficiency was deemed to be secondary to adrenalectomy, pituitary surgery or prolonged use of glucocorticoids. Patients who died since diagnosis were also excluded.We studied patient demographic and clinical data, particularly regarding diagnosis, aetiology, concomitant autoimmune disorders, family history and replacement regimens, such as the type, daily dosage and dose frequency of glucocorticoids, as well as the use of mineralocorticoids. We assessed the occurrence of hospitalization due to adrenal crisis through the review of all hospital medical records since the diagnosis. Adrenal crisis (AC) was defined as an acute impairment of general health requiring hospital admission and administration of intravenous saline and glucocorticoids in patients with AD. Furthermore, symptoms and electrolyte disturbances at the last appointment were evaluated.s.d.) or medians (range) for variables that did not conform to a normal distribution. The crude incidence rates of hospitalization due to AC were estimated as the number of hospitalization cases divided by the total follow-up time and were reported as number of cases per 100 patient-years. For independent samples, two-way comparisons for proportions were performed using a chi-square test (χ²) for categorical variables and a t-test or Mann–Whitney U test for continuous variables. Statistical significance was taken as two-tailed at the level of 0.05. All phases of preparation for the study were in line with the ethical and deontological principles regarding data collection and statistical analysis.A comparative analysis was performed to investigate the type of glucocorticoid replacement and aetiology, mean daily hydrocortisone equivalent dose, mean daily fludrocortisone dose, occurrence of AC and the presence of symptoms or electrolyte disturbances. Data were initially collected in an Excel 2011 database, and statistical analysis was performed with SPSS Statistics, V.22. Data are presented as proportions, means , mucocutaneous hyperpigmentation (55.0%), weight loss (43.2%), hypotension (42.8%) and hypoglycaemia (8.6%). Hyponatraemia was documented in 36.3% of cases, while hyperkalaemia occurred in 25.9%. AC was diagnosed in 29.1% of the patients. The median lag time between onset of symptoms and confirmed diagnosis was 3 (0–36) months.The diagnoses were established by high ACTH and low serum cortisol in 122 (43.9%) patients; by an abnormal standard dose ACTH stimulation test (peak plasma cortisol <18 µg/dL) in 39 (14.0%) patients and by characteristic clinical symptoms/signs and chronic treatment with glucocorticoids and fludrocortisone in 48 (17.3%) patients who were diagnosed decades ago. These data were missing for 69 (24.8%) patients.Data regarding etiological investigation were available in 246 patients. The most common causes were autoimmune adrenalitis (61.0%), genetic-related AD (14.2%) and infectious adrenalitis (7.3%). Thirty-eight cases (15.4%) were considered idiopathic. We also documented three cases secondary to bilateral adrenal haemorrhage and two cases of adrenal metastatic infiltration. P < 0.001).Among 150 patients with autoimmune AD, 65.3% were female and the mean age at diagnosis was 34.9 ± 15.9 years. The mean age of diagnosis was higher for females, 37.9 ± 15.3 years than for males, 29.2 ± 15.5 years (Ninety-three percent of the patients had autoantibodies directed against 21-hydroxylase. Computed tomography (CT) scans of the adrenal glands were performed in 50 patients, among which adrenal morphology was described as normal in 37 cases, while 13 patients presented reduced size or volume.Isolated autoimmune AD occurred in 30% of the patients, whereas 70% were diagnosed with one or more associated autoimmune endocrinopathies. The most common of these were autoimmune thyroiditis (60.7%), type 1 diabetes mellitus (17.3%) and pernicious anaemia (6.7%), with 103 (68.7%) of the patients meeting the criteria for APS-2. We also found two patients with associated chronic mucocutaneous candidiasis and meeting the criteria for APS-1. Other recorded comorbidities are presented in Congenital adrenal hyperplasia (CAH) resulting from 21-hydroxylase deficiency occurred in 27 patients , with a median age at onset of 3  years. The diagnosis of CAH was based on the molecular analysis of CYP21A2 gene. Five patients had X-linked adrenoleukodystrophy (ALD), with a median age at diagnosis of 25  years. Three patients had X-linked congenital adrenal hypoplasia with a mutation of DAX1, who were all diagnosed within the first year of life.Out of the 18 cases  of infectious adrenalitis, 17 resulted from tuberculosis and one resulted from histoplasmosis. The median age at diagnosis was 50  years. Ten patients had records of radiological studies, of which seven showed an increase in adrenal volume and three had adrenal glands with normal morphology.In the 38 cases considered idiopathic, the median age at diagnosis was 41  years. Only two of these patients had records of anti-adrenal antibodies (negative), and 18 patients had radiological imaging of the adrenal glands .P < 0.05) and dexamethasone . Replacement with dexamethasone was significantly more frequent among patients with CAH . There were no differences between the other types of glucocorticoid replacement and aetiology.All patients received glucocorticoid replacement therapy, 79.1%t were treated with hydrocortisone, 10.1% with prednisolone, 6.1% with dexamethasone and 0.7% methylprednisolone (missing data = 11). The mean daily dose for patients with hydrocortisone treatment was 26.3 ± 8.3 mg/day (missing data = 1), which was divided into one (3.5%), two (37.4%), three (57.5%) or four (0.5%) daily doses (missing data = 3). Other glucocorticoid replacement doses were recalculated as the hydrocortisone equivalent dose . PatientP = 0.021) or dexamethasone , but there were no differences regarding daily fludrocortisone dose (P = 0.46). The mean hydrocortisone equivalent dose was not significantly different in patients with or without mineralocorticoid replacement .Mineralocorticoid replacement was utilized by 66.5% of the patients, and the median fludrocortisone dose was 100  µg/day. The two patients under methylprednisolone replacement did not receive mineralocorticoid. The prevalence of fludrocortisone replacement was higher in patients with prednisolone (88.9%) than in patients with hydrocortisone . There were no differences between dose of glucocorticoid, mineralocorticoid replacement or aetiology and the incidence of hospitalizations.Since the diagnosis, 33.5% of patients had been hospitalized due to AC. The overall incidence of AC was 4.36 (95% CI 3.98–4.74) per 100 patient-years. The incidence rate of hospital admission was higher in the group receiving hydrocortisone  than in the group receiving prednisolone  per 100 patient-years  with most common being asthenia (7.6%) and depression (6.5%), while 9.7% presented electrolyte disturbances (missing data = 17). No association was found between complaints and the type of glucocorticoid replacement, mineralocorticoid therapy, the presence of symptoms or electrolyte disturbances.We reviewed 278 adult patients with AD followed by endocrinology in 12 Portuguese hospitals. To date, this is the only Portuguese cohort of patients with PAI. Several patients may be followed in other centres or by other specialties, so we were not able to estimate the prevalence of this disease in Portugal. However, considering the dimension and the good distribution of patients across the country, we assume that this cohort is representative of the Portuguese population with AD.P = 0.017).Several recent studies have suggested an increasing incidence of the disease in the last decades , 8, 15. In our cohort, only 14% of patients had records of a corticotropin test. Current guidelines by the Endocrine Society suggest that only if a corticotropin test is not feasible should the combination of low plasma cortisol and ACTH >2 times the upper reference limit be used as a preliminary test for suggesting adrenal insufficiency . HoweverThe most common aetiology was autoimmune AD, and 68% of this subset of patients had other autoimmune endocrinopathy. The most frequent concomitant diseases were hypothyroidism, DM, hyperthyroidism and pernicious anaemia, which is in concordance with studies from Norway , Italy , the UniWe found 35 patients with genetic forms of adrenal insufficiency, and CAH was the most frequent. CAH is the most common cause of adrenal insufficiency in infancy . ConsideThe frequency of idiopathic cases was somewhat higher than that reported in other studies , 21. OnlCurrent treatment recommendations suggest using short-acting glucocorticoids, hydrocortisone 15–25 mg) or cortisone acetate (20–35 mg) in two or three divided oral doses per day to avoid overtreatment and enable a diurnal physiological pattern . Our stu5–25 mg oMineralocorticoid replacement therapy with fludrocortisone is recommended in all patients with PAI . The minThe incidence rate of hospitalizations due to AC found in this population was lower than that reported in Dutch (5.2/100 person-year) and German (6.3/100 patient-years) patients , 24. HowOn the last appointment, 17% of our patients had complaints, and 10% had electrolyte disturbances. Unfortunately, it was not possible to evaluate in this study the morbidity and mortality associated with AD. There is growing evidence that current replacement regimens are associated with a significant reduction in health status and working ability , 29. MorOne of the strengths of this study is the inclusion of patients from most tertiary centres in the northern, central and southern regions of Portugal. It is also the first national cohort of patients with AD, and it was possible to characterize the clinical presentation, diagnosis, treatment and follow-up of the disease.The main limitations of this study stem from its retrospective observational nature in which data were collected from medical records by several investigators. We cannot draw any conclusions about the real incidence of the disease in our country, as this cohort just includes adult patients followed on endocrinology department. Furthermore, as aforementioned, the exclusion of deaths probably leads to an underestimation of the disease and incidence of AC.In conclusion, autoimmune AD is the most frequent aetiology in this population, but there is still a high prevalence of cases considered idiopathic. This study emphasizes the need for standardization in diagnostic tests, etiological investigation and the investigation of associated autoimmune comorbidities. It is also important to advocate the treatment of these patients according to current recommendations and to minimize symptoms associated with over- or under-substitution of glucocorticoids and under replacement of mineralocorticoid.The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.


A hand-foot-mouth disease outbreak occurred in 2014 around Guangdong. The purpose of this study was investigating the status and susceptibility of infectious neutralizing antibodies to enterovirus 71 among children so as to provide scientific evidence for the population immunity level of hand-foot-mouth disease and prepare for enterovirus 71 vaccination implementation. Serum specimens were collected from children in communities from January 2014 to March 2015 in Guangzhou. A total of 197 serum samples from children 1–5 years old were collected for this cross-sectional study via non-probabilistic sampling from the database of Chinese National Science and Technique Major Project. Neutralization activity was measured via micro neutralization test in vitro. The positive rate of enterovirus 71 neutralizing antibodies was 59.4%, whereas the geometric mean titre was 1:12.7. A statistically significant difference in true positive rates was found between different age groups but not between different genders. Being the most susceptible population of hand–foot–mouth disease, children under 3 years of age are more likely to be infected with enterovirus 71, and the immunity of children increases with increasing age. Further cohort studies should be conducted, and measures for prevention and vaccination should be taken. Enterovirus 71  is a major causative agent of HFMD. Jin . Our study was conducted as a cross-sectional study via non-probabilistic sampling. We calculated the sample size as 119 with an estimated neutralizing antibody positive rate (abbreviation as “p”) of 59%, significance level of 0.05, and permissible error of 0.15 “p”. Considering the small sample number for each age group, we collected 197 samples between 2014 and 2015. All serum specimens and demographic characteristics of children in Guangzhou were collected from the blood sample database set up by Chinese National Science and Technique Major Project (2012ZX10004912). Child participants were divided into four groups: under 2 years old, between 2 and 3 years old, between 3 and 4 years old, and between 4 and 5 years old. As positive control, the EV71 positive serum was collected from child patients identified by Yuebei Hospital in Shaoguan City. Human rhabdomyoma (RD) cells were provided by Guangdong Centre for Diseases Control (CDC) and used for virus sub-cultivation and antibody neutralization test. EV71 (virus strain number 2014XN37281) was collected from HFMD cases, isolated, cultured, and sequencing identified by Guangzhou CDC.−1 to 10−10 and then cultured in 96-well culture plates (8 × 12 wells). Eight wells per vertical row in each culture plate were for one dilution level of virus liquid inoculated (50 µL was added to each well). Each culture plate included one cultivating liquid group and one cell suspension group as controls. To prevent the infection from being trailed from non-neutralized virus wells to the cell culture suspension, all tips were changed for each new row. A 50% Tissue Culture Infective Dose (TCID50) was generated to measure the EV71 antibody titre, and mathematically calculated using the Reed–Muench method [Virus titration was conducted via endpoint dilution assay. This assay was selected because of its higher sensitivity and cost-effectiveness compared with plaque assay ,18. Viruh method . Observa−1 to 2−9 and cultured in 96-well culture plates within two wells for one dilution level (50 µL per well). Then, diluted 100 TCID50 virus liquid (50 µL per well) was added. To prevent cross infection, all tips were changed for each new row. After homogeneous vibration, these culture plates were placed in a carbon dioxide incubator for 2 h neutralization. RD cell liquid was added (100 µL per well), and the plates were placed back into the carbon dioxide incubator at 35 °C. RD cells were used for virus subcultivation. Occurrence of CPE in RD cell culture indicates the presence of virus activities. When EV71 neutralizing antibody is present in RD cell culture, the infectivity of EV71 is reduced, leading to the absence of CPE in the cell culture. Therefore, the absence of CPE proved the positive result of EV71 neutralizing antibodies. CPE of these 96-well culture plates was observed under an inverted microscope daily for 3 days. The serum control group, virus control group, and blank control group were added simultaneously.EV71 neutralizing antibody titre was determined by the absence of cytopathic effects (CPE) in the micro neutralization test ,21. SeruStandard prediction was performed after the micro neutralization test. The multiplicative inverse of the highest dilution level under CPE absence was the determined point of neutralizing antibody titre. The antibody titre value determined the positive or negative results of EV71 neutralizing antibodies. If the neutralizing antibody titre values were less than or equal to 1:4, 1:4 was recorded as negative. The value was classified as positive if the neutralizing antibody titre values were greater than or equal to 1:8. All neutralizing antibody titre values that were greater than 1:32 were recorded as ≥1:32. Calculations of the geometric mean titre (GMT) of EV71 neutralizing antibodies and its 95% confidence interval (95% CI) were generated.Statistical analysis of data was performed by SPSS 19.0 . Chi-square test was used to compare the positive rates of EV71 neutralizing antibodies within different ages, genders, and years. Analysis of variance (ANOVA) was used to compare the logarithm values of neutralizing antibodies GMT within different ages, genders, and years. Cochran-Armitage Trend Test was performed to verify the trend of positive rates along with age. The significance level was set to 5% (α = 0.05). Pairwise comparison of the neutralizing antibody GMT between two age groups was processed in Fisher’s least significant difference (LSD) with significant level correction.In total, 197 children aged 1–5 years participated in this study. The participants comprised 112 males and 85 females . Groups of children aged <2, 2–3, 3–4, and 4–5 years included 17, 60, 60, and 60 samples, respectively.RD cells can support the replications of EV71; therefore, CPE occurred in the RD cell culture as shown in z = 3.361, p < 0.001). Statistically significant differences in positive rates were observed among the four groups . The total GMT value of EV71 neutralizing antibodies was 1:12.7 . Furthermore, the GMT values were 1:8.0 , 1:9.1 , 1:13.9 , and 1:18.4  in the four groups (As shown in r groups . More nep < 0.05). However, statistically significant differences in GMT only were found between the groups of children aged <2 and 4–5 years (p = 0.002), as well as between the groups of children aged 2–3 and 4–5 years (p = 0.000077).Statistically significant differences in the logarithm values of GMT were observed among the four groups . The GMT values of EV71 neutralizing antibodies were 1:13.1  for boys and 1:12.1 .The positive rates of EV71 neutralizing antibodies were 59.8% for boys and 58.8% for girls. Among the boys’ titre values, 40.2% (45 samples out of 112) were less than 1:4, 8.0% (9/112) were 1:16, and 51.8% (58/112) were equal or greater than 1:32. Among the girls’ titre values, 41.2% (35/85) were less than 1:4, 4.2% (4/85) were 1:8, 7.1% (6/85) were 1:16, and 47.1% (40/85) were equal or greater than 1:32. No statistically significant difference in positive rates of EV71 neutralizing antibodies were observed between different genders  antibodies was 45.64% in 2012 among children below 5 years of age . AccordiFocusing on the details for each group, we found the lowest positive rate of EV71 neutralizing antibodies in children under 2 years old 35.3%), and the GMT of this group was 1:8.0. Similar results were obtained by Li  in Guang5.3%, andp = 0.18). Consistent with the results of these studies, the transmission of EV71 can be inferred to be nonselective toward gender in the Guangzhou population. By contrast, no significant differences in positive rate or GMT were found among children in Guangzhou between 2014 and 2015. This outcome differed from the findings of Li [The positive rates of EV71 neutralizing antibodies for boys and girls were 59.8% and 58.8%, showing no statistical significance between genders. In a German study on 696 individuals, Rabenau  found thgs of Li  in 2013 5) can be found in children between 6 and 11 months old. Therefore, further seroepidemiological study on EV71 should be continued, including sample collection from under children below 1 year of age. As an important factor of the detection prevalence validation of EV71 neutralizing antibodies, the precise duration of EV71 neutralizing antibodies should be investigated. Given that the susceptible population involves children under 3 years old, specific medical treatments and vaccination to EV71 for children should be under the national and regional implementations.The limitation of our study is the different number of samples for each age group. The sample size of children under 2 years of age was markedly smaller than those of the three other groups. Chang  indicateChildren under 3 years of age are highly susceptible to HFMD. During 2014 and 2015, the positive rate of EV71 neutralizing antibodies among 197 serum specimens from children aged 1–5 was 59.4%, whereas the GMT was 1:12.7. Statistically significant differences in positive rates were observed between different age groups but not between different genders. Given the HFMD transmission routes and the characteristics of susceptible population, prevention and control measures should be taken. Further epidemiological studies and vaccination to EV71 should be continued under the national and regional protocols of implementation.


Magnetic resonance imaging (MRI) offers a rapid and powerful tool for the detection of lymph node metastasis (LNM) and in the current manuscript is assessed as a critical tool in the preoperative protocol for liver resection for treatment of CCA. However, the accuracy of MRI to detect LNM from CCA had yet to be comprehensively evaluated.Treatment planning especially liver resection in cholangiocarcinoma (CCA) depends on the extension of tumor and lymph node metastasis which is included as a key criterion for operabilityThe accuracy of MRI to detect LNM was assessed in a cohort of individuals with CCA from the Cholangiocarcinoma Screening and Care Program (CASCAP), a screening program designed to reduce CCA in Northeastern Thailand by community-based ultrasound (US) for CCA. CCA-positive individuals are referred to one of the nine tertiary centers in the study to undergo a preoperative protocol that included enhanced imaging by MRI. Additionally, these individuals also underwent lymph node biopsies for histological confirmation of LNM (the “gold standard”) to determine the accuracy of the MRI results.MRI accurately detected the presence or absence of LNM in only 29 out of the 51 CCA cases , resulting in a sensitivity of 57.1% (95% CI 34.0–78.2) and specificity of 56.7% (95% CI 37.4–74.5), with positive and negative predictive values of 48.0% (95% CI 27.8–68.7) and 65.4% (95% CI 44.3–82.8), respectively. The positive likelihood ratio was 1.32 (95% CI 0.76–2.29), and the negative likelihood ratio was 0.76 (95% CI 0.42–1.36).MRI showed limited sensitivity and a poor positive predictive value for the diagnosis of LNM for CCA, which is of particular concern in this resource-limited setting, where simpler detection methods could be utilized that are more cost-effective in this region of Thailand. Therefore, the inclusion of MRI, a costly imaging method, should be reconsidered as part of protocol for treatment planning of CCA, given the number of false positives, especially as it is critical in determining the operability for CCA subjects.The online version of this article (10.1186/s12957-017-1246-9) contains supplementary material, which is available to authorized users. Opisthorchis viverrini (Ov), one of the few carcinogenic pathogens [Cholangiocarcinoma (CCA) is a primary hepatic malignancy that arises along the intrahepatic and extrahepatic bile ducts. It is the second most common liver cancer globally  and the athogens . When deathogens , 5, withathogens .As the objective of CASCAP is to increase survival from CCA by aggressively screening for this cancer by mobile US units in Ov-endemic areas, a critical component of this endeavor was to establish a preoperative evaluation protocol for liver resection for CCA, which would include factors such as (1) tumor infiltration beyond the second order bile duct branches, (2) tumor invasion of major vessels such as main hepatic artery or portal vein, and (3) the extent of lymph node metastasis (LNM) –15. Due www.cascap.in.th) and nine tertiary care hospitals in this same region. The CASCAP study is comprised of two cohorts. The first cohort is the “screening cohort,” which includes individuals who were risk stratified using criteria consisting of (1) their residence in Northeastern Thailand, (2) age of 40 years or older (inclusive), (3) previous reported infection with the carcinogenic liver fluke Ov, (4) previously reported treatment for Ov infection with praziquantel, and (5) self-reported consumption of raw or undercooked freshwater fish, the second intermediate host of Ov. Individuals considered at risk for Ov-induced CCA underwent liver US as detailed in Khuntikeo et al. [This study was based on data from CASCAP, which is a combined community and hospital-based cohort study conducted in Ov-endemic areas in Northeastern Thailand  or computerized tomography (CT) scanning at one of nine tertiary care centers. The MRI used a 1.5–3.0 T system. Two different MRCP techniques were applied: a single-shot rapid acquisition with relaxation enhancement (RARE) and a multislice half-Fourier acquisition single-shot turbo spin echo (HASTE). The slabs of the single-shot RARE sequence were obtained on various planes  to allow for the optimal visualization of the bile ducts. The number of thick-slab acquisitions ranged from seven to nine per patient. Next, multislice HASTE images were obtained in the coronal and oblique planes. Each examination was performed during a single breath-hold.The imaging parameters for the single-shot RARE sequence were as follows: repetition time of ∞, effective echo time of 1200 ms, echo train length of 240, flip angle of 150°, slab thickness of 70 to 90 mm, field of view of 300 to 340 mm, matrix of 240 × 256, and an acquisition time of 2.32 s. The imaging parameters for the multislice HASTE sequence were as follows: repetition time of ∞, effective echo time of 95 ms, echo train length of 128; flip angle of 150°, section thickness of 4 mm without a gap and 13 to 15 slices (range of coverage 52–60 mm), field of view of 300 to 340 mm, matrix of 240 × 256, and an acquisition time of 18 to 20 s. Fat saturation was used to reduce a strong fat signal during image acquisition. The total acquisition time for all of the imaging steps in the MR imaging were less than 15 min.The determination of LNM by MRI was made by a consensus between two board-certified radiologists using the following morphological criteria: nodal size greater than 10 mm in short axis diameter, central necrosis, and inhomogeneous enhancement following intravenous contrast medium injection –24. In eIn the case when a surgical treatment occurred, the operation types and plans were discussed with the doctors before the procedure. The time interval between MRI scan and operation was about 1 month. Lymph node dissections were performed in case of the presence of an enlarged or abnormal consistency of the lymph nodes. These dissected lymph nodes were sent for pathological examination. The diagnosis of pathological LNM was done by two board-certified pathologists. The procedures included each pathologist performing a gross and microscopic examination of the tissue specimen separately with the pathological diagnosis and lymph node metastasis made by consensus. In case of differences in the diagnosis between the two pathologists, immunohistochemistry strains were used to determine a consensus between both pathologists.The descriptive statistics of patients and the type of tumor were presented as mean and standard deviations, such as age. Categorical variables were presented as numbers and percentages. A comparison between LNM diagnosis using an MRI scan and pathological diagnosis included the following parameters: sensitivity, specificity, positive predictive values (PPV), negative predictive values (NPV), false positive rates, and false negative rates. They were calculated along with their 95% confidence intervals (95% CI). These diagnostic parameters were presented as either the overall CCA or CCA stratified into intrahepatic, perihilar, and distal type based on the American Joint Committee on Cancer (AJCC) criteria. All statistical analyses were conducted using Stata13 . Attached data sets 1-3 were used for all analysis , the Declaration of Helsinki, and the national laws and regulations about clinical studies. CASCAP was approved by Khon Kaen University Ethics Committee (HE551404) for Human Research and received written informed consent from all patient participants.A total of 433 individuals suspected of CCA by US were sent for MRI scanning, with a follow-up confirmation of 255 cases of CCA (58.9%). There were 133 (52.2%) individuals with intrahepatic CCA, 112 (43.9%) with perihilar CCA, and 10 (3.92%) with distal CCA. In the CCA group confirmed by MRI scans, 248 (97.3%) subjects also had radiographic lymph node findings, with 125 (50.4%) showing LNM. Of these 255 MRI-confirmed CCA subjects, 130 received surgical treatments with pathologically confirmed CCA. The 51 underwent lymph node dissection, with only 21 (41.2%) having confirmed LNM based on the conventional pathological diagnosis , with 33 out of 51 (64.7%) being males. Of these individuals, 35.3% had intrahepatic, 60.8% perihilar, and 3.9% distal CCA , resulting in a sensitivity of 57.1% 95% CI 34.0–78.2) and a specificity of 56.7% (95% CI 37.4–74.5), with a PPV and a NPV of 48.0% (95% CI 27.8–68.7) and 65.4% (95% CI 44.3–82.8), respectively. The positive likelihood ratio was 1.32 (95% CI 0.76–2.29), and the negative likelihood ratio was 0.76 (95% CI 0.42–1.36)  and a specificity of 88.9% (95% CI 51.8–99.7), with a PPV and a NPV of 80.0% (95% CI 28.4–99.5) and 66.7% (95% CI 34.9–90.1), respectively. The positive likelihood ratio was 4.50 (95% CI 0.63–32.4), and the negative likelihood ratio was 0.56 (95% CI 0.27–1.17)  extension of the cancer to the hepatic artery or portal vein and (2) extension of lymph node metastasis (LNM) –15. CCA In the current study, MRIs were observed to have limited utility for preoperative evaluation for CCA compared to the “gold standard” of pathological confirmation of LNM. An especially concerning finding was the high false positive rate of MRIs for CCA LNM, which would exclude individuals otherwise eligible for surgery. We quantified the sensitivity of MRI scans in determining LNM for CCA at 57.1%, which was far lower than other preoperative resectability criteria such as the bile duct and vascular involvement , 20, 37.Liver resection has long been shown to increase survival from CCA, though it is highly dependent on LNM. Herein, we utilized the unique early screening program and a large sample size (CASCAP) in Northeastern Thailand to determine the quality of data for MRI scanning to determine LNM in the preoperative evaluation for CCA. We found that MRI scanning for LNM had limited sensitivity, with a low positive predictive value, and a high number of false positives, which would define eligible patients as inoperable. The evaluation of the nodal metastasis based on this radiological imaging modality has significant clinical implications for the surgical treatment of CCA. As such, we strongly recommend a “reconsideration” of the inclusion of MRI results for LNM in the preoperative evaluation for CCA, especially when it is used as the single or principle criterion for whether or not an individual will have curative liver resection for CCA.MRI has limited sensitivity and positive predictive value for the diagnosis of LNM in CCA. Therefore, surgeons should be aware of the number of false positives in determining the inoperability of CCA subjects.Additional file 1:Dataset 1. (CSV 16 kb)Additional file 2:Dataset 2. (CSV 22 kb)Additional file 3:Dataset 3. (CSV 52 kb)


From swimming pools, bathers may acquire many potential pathogens or may be affected by the physicochemical characteristics of water used during bathing. Hence, this study aimed at assessing the physicochemical and microbiological quality of public swimming pools located at different hotels and recreation center in Addis Ababa, Ethiopia.E. coli were determined. Data was analyzed using SPSS Version 20.A cross sectional study was carried out from February to May, 2016. Nine hotels and one recreation center which recognized to have public swimming services were included. A total of 60 swimming pool water samples from 10 swimming pools were collected at deeper, shallow and intake point twice on a weekly basis using a 250 ml sterile bottle containing sodium thiosulphate. PH, residual chlorine and temperature of samples were recorded at the time of collection. Sample containing bottles were transported in ice box to microbiological laboratory and analyzed on the same day. Standard cultural and biochemical methods were used for isolation and characterization of the main microbial groups. Total viable count, total coliform count, fecal coliform count and oC respectively. Of all analyzed water samples, 58.4% (n=35/60) of them had PH range of 7.2-7.8, 58.3% (n=35/60) of samples had temperature in the range of 21oC-32oC and 25% (n=15/60) of water samples had residual chlorine in the range of 2-3mg/l. 73.3% (n=44/60) of the samples had a total viable count below 200 MPN/ml and 70% (n-42/60) of the samples had Total Coliform Count values less than 2 MPN/100 ml. Moreover, 66.7% (n=40/60) of the samples had fecal coliform counts falling below 1 MPN /100 ml. E. coli was absent in 70% (n=42/60) of the samples while it was present in 30% (n=18/60) of the samples.Average PH and temperature of swimming pool water samples were 7.1 and 29PH, residual chlorine and temperature value of majority of the swimming pools’ water samples were within the acceptable limit. Regarding microbial quality, most swimming pools’ water samples complied to the WHO standard. Swimming pools that did not comply to the standard both in physicochemical levels and microbial quality need improvement due to their significant health implication. The use of public swimming pools for recreational activities, rehabilitative treatment or sport is growing worldwide , 2. Swimetc.), transport on the body or growth within the filter bed [e.g., sewage, storm water, and agricultural runoff) [Salmonella typhi, Salmonella paratyphi, Shigella dysenteriae, Vibrio cholera, Pseudomonas aeruginosa, Mycobacterium spp., Staphylococcus aureus, Legionellae, Cryptosporidium parvum, Giardia, Microsporidia, Dermatophytes and Keratinophilic fungi and Molluscum contagiosum, hepatitis A and E [Recreational waters may be contaminated by direct excretion by bathers  , 6. Most A and E , 7, 8. T A and E , 6.Escherichia coli presence is a strong indication of the presence of enteric pathogenic bacteria such as Salmonella typhi, Salmonella paratyphi, Shigella dysenteriae, Vibrio cholerae and parasites in the pool [If there is no control over health standards for swimming pools, they can be a serious source of microbial contamination since wide range of people with different levels of economic, social and health status use swimming pools . To monithe pool , 8. Thesthe pool . Hence tA cross sectional study was carried out from February to May, 2016 in Addis Ababa, the capital city of Ethiopia with a population of 3,384,569 according to the 2007 population census with annual growth rate of 3.8%. Addis Ababa culture and tourism bureau gave recognition for nine hotels and one recreation center to have swimming services for the public where only two of these swimming pools were indoors while the rest were outdoors. In this study, all these ten swimming pools which were recognized by Addis Ababa culture and tourism bureau were included.A total of 60 water samples were collected from ten swimming pools . Swimming water samples were collected at three points  from each swimming pool. A 250 ml sterile bottle with sodium thiosulphate was immersed to an elbow depth with its opening facing the water. Its opening was then inverted so that water could get in to it. After the bottle was full, it was withdrawn from the water. PH, residual chlorine and temperature of samples obtained from each point were measured at the time of collection. Sample containing bottles were transported in ice box to microbiological laboratory within two hours of collection. All the samples were analyzed on the day of collection.Total Viable Count: after mixing bottles containing swimming pool water sample by gentle inversion, 1ml of sample was poured in to a sterile culture plate. Then, 20 ml of plate count agar (PCA) was poured on to the plate. The sample and the PCA were mixed and were made to stay at room temperature until the mixture solidified. All plates were incubated for 48 hours at 37 degrees Celsius and total viable counts were then counted by using digital colony counter (24).Total Coliform Count: Three sets of test tubes  containing Durham’s tubes within them were arranged. Double strength MacConkey broth was poured in to the 50 ml and the 10 ml test tubes (50ml in to the 50 ml and 10 ml in to the 10 ml) while 1 ml of single strength MacConkey broth was added in to each of the 5ml test tubes. Pool water was then added to the three sets of test tubes.The contents of each test tube were then mixed. Every test tube was observed for the presence of air bubbles in the Durham’s tubes and air bubbles were removed from test tubes which had them. The lids of each test tube were then loosened and all the tubes were incubated at 37 degrees Celsius. After 48 hours, each test tube was examined for gas production and for change in color. Test tubes which became yellow and produced gas were considered positive for total coliform and those with no color change and gas production were considered negative for total oliform. Finally, the total coliform count was obtained from the Most Probable Number (MPN) .Fecal coliform: Each positive test tube from each set of test tubes was sub-cultured in to test tubes containing Escherichia coli broth with Durham`s tube. Contents were mixed and air bubbles were removed from the test tubes. Lids of each test tube were loosened and the test tubes were incubated at 44.4 degrees Celsius for 24 hours, and then all the test tubes were examined for turbidity and gas production. Those with turbidity and gas production were taken as positive for fecal coliforms and the fecal coliform count was determined from the Most Probable Number (MPN).Escherichia coli: Samples from positive test tubes for fecal coliform were inoculated into nutrient broth and incubated at 44.4 degrees Celsius for 24 hours. After 24 hours incubation, a drop of Kovacs reagent was added and Formation of red ring was used as a proof for indole positivity and hence for the presence of E. coli .Escherichia coli ATCC 25922 standard control strains.Standard Operating Procedures (SOP) were strictly followed verifying that media meet expiration date and quality control parameters and manufacturer`s instructions regarding media preparation were followed meticulously. Visual inspections of cracks in media or plastic Petri-dishes, unequal fill, evidence of freezing, bubbles, and contamination were carried out. Quality control was performed to check the quality of medium and each new lot was quality controlled before use by testing Data were entered, cleaned and analyzed by SPSS version 20. Descriptive statistics  were calculated and a Chi square test was used to check association between the variables. Results were then presented in words and depicted in tables and diagrams.An ethical clearance was obtained from departmental research and review committee of School of Medical Laboratory Science, Addis Ababa University. Purpose of the research was then clearly explained by the principal investigator to hotels with swimming pools in Addis Ababa. Persons in charge of the hotels were also informed that information fetched from the swimming pools would be kept confidential. Finally informed consent was obtained from the respective hotels and data collection was started after then.1). The study has shown that swimming pool water samples had temperature values ranging from 20oC-37oC with an average of 29oC. More than half i.e. 58.3% of (n=35/60) the swimming pool water samples were found to have temperatures ranging from 21oC-32oC.However, 11.7% (n= 7 /60) of the swimming pool water samples had a temperature of 20oC. .The average PH value of the swimming pool water samples was 7.1 with a range of 5-10. Among the tested swimming pool water samples, 58.4% (n=35/60) had PH values within WHO recommended limit of 7.2-7.8 while 41.6% (n=25/60) of the water samples had pH values out of the recommended limits where 20% (n=12/60) and 21.7% (n=13/60) were below 7.2 and above 7.8, respectively. The residual chlorine of swimming pools water samples was between 0.0 and 4.0 mg/L where 73.3% (n=44/60) of swimming pool water samples had residual chlorine values in the range of 0.0-1.9mg/L while only 1.7% (n=1/60) of the swimming pool water sample was found to have residual chlorine values of 4mg/L. Majority 73.3% (n=44/60) of the swimming pool water samples had residual chlorine values below 2.0 which is the WHO minimum recommended limit whereas 25% (n=15/60) had values within the WHO recommended range (2-3mg/L) (Table 4 cfu/ml where 73.3% (n=44/60) of swimming pool water samples had a total viable count below 2x102 CFU/ml while 26.7% (n=16/60) of the swimming pool water samples had a total viable count above 2x102 CFU/ml. Regarding total coliform counts (TCC) done on the 60 water samples, 66.7% (n=40/60) of the swimming pool water samples had TCC values of 0 MPN /100ml. Swimming water samples which met the WHO standard with a TCC value of less than 2 MPN /100ml were 70.0% (n=42 /60) while 30% (n=18/60) did not show TCC values above 2 MPN /100ml.Swimming pool water samples had a total viable count values in the range of 0 cfu/ml-7.6x102 MPN /100ml and 66.7% (n=40/60) swimming pool water samples had fecal coliform counts falling below 1 MPN /100ml while 33.3% (n=20/60) had counts above 1 MPN /100ml. 8.3% (n=5/60) of the swimming pool water samples were found to have a fecal coliform count of 1.8x102 MPN /100ml and 5% (n=3/60) swimming pool water samples had fecal coliform counts of 2.5x101 MPN /100ml, 2x101 MPN /100ml and 1.8x101 MPN /100ml while the rest had counts below the aforementioned values. The swimming pool water samples were also checked for the presence or absence of E. coli. Accordingly, E. coli was present in 30% (n=18/60) of swimming pool water samples while it was absent in 70% (n=42/60) of the pool water samples.The fecal coliform counts ranged from 0 MPN /100ml -1.8x10E. coli nor fecal coliform showed compliance while 31.7% (n=19/60) of samples which had either E. coli or fecal coliform did not comply to the WHO standard. (Table 2).Overall, the compliance of swimming pool water samples was evaluated against the WHO standard and 68.3% (n=41/60) of the swimming pool water samples which had neither Average PH value of 7.1 of swimming pool water samples with a range from 5-10 was comparable to the findings of a study from Iran . Our stuThe residual chlorine of swimming pools water was in the range of 0 to 4mg/l with an average value of 1.16 mg/L which was in accordance with a finding from Iran . This vaoC which was in close agreement with the result of another study [oC-32oC for swimming pool water, 58.3% (n=35/60) of swimming pool water samples were found to have temperatures range falling within the limit. This implies that there was still a problem in monitoring the temperature of swimming pool waters.With regard to temperature, the study indicated that swimming pool water samples had an average temperature of 29er study . Compare2 cfu/ml met the standard while 26.7% with a count above 2x102 cfu/ml did not meet the set standard. The finding of 26.7% of the swimming pool water samples that did not meet the standard was relatively higher as compared with another study [In our study, 73.3% swimming pool water samples with a count of 2x10er study . The dis2 MPN /100ml with a study conducted in Iran [1 cfu/100 ml which was not comparable with the result of this study [With regard to total coliform count, data from this study showed that a comparable total coliform counts ranged from 0 MPN /100ml to 1.8x10is study . When ev2 MPN /100ml while 5% had values of 2.5x101 MPN /100ml, 2x101 MPN /100ml and 1.8x101 MPN /100ml.Our study indicated that 33.3% of the swimming pool water samples were positive for fecal coliforms which was a much higher finding than a study from Greece which had 3% fecal coliform counts out of 200 samples . AnotherE. coli, 30%√(n=18/60) of the samples were positive for E. coli while from 70% (n=42/60) water samples, E. coli were not detected thus meeting the WHO standard. From a study [E. coli which was a lower finding than our study. The non conformance to the standard might be due to differences in the sanitation practice, bather attitude, sample size and study period. According to WHO, for water to be potable, E. coli/fecal coliforms should not be detected. In this respect, 68.3% (n=41/60) of the samples had no E. coli thus were potable while 31.7% (n=19/60) had E. coli and/or E. coli indicating non-potability.Regarding the presence or absence of  a study , 16.6% oE. coli nor fecal coliform. However, swimming pool water samples which had either E. coli or fecal coliform that did not comply to the WHO standard indicated the need for swimming pools’ water quality improvement.Most swimming pools’ water samples showed PH, residual chlorine and temperature value within the acceptable limit recommended by WHO. Good monitoring of residual chlorine, temperature and PH of swimming pools is a must considering their significant impact on the microbial safety of swimming pools which has a health implication on the public who use these pools. Overall, 68.3% (n=41/60) swimming pools water samples complied to the microbial standard which had neither


We compared effects of shorter moderate-intensity exercise time (<10 min bouts) on cardiometabolic parameters with the current recommendations among elderly adults.S and FS, respectively), andL and FL, respectively). Short-duration bouts consisted three 5–10 min moderate-intensity jogging sessions daily, and long-duration bouts consisted 30–60 min sessions 3–5 days weekly. Cumulative exercise times were equivalent. Physical activity (PA) was measured by log and activity monitors. Fasting venous blood at baseline and 8 weekly intervals was used for blood chemistry.Fifty-three sedentary individuals aged ≥50 years were divided into exercise groupsS and FS with total cholesterol (TC) of >5.2 mmol/L and >5.3 mmol/L decreased from 22.2% to 14.8% and from 30.9% to 11.5%, respectively. For ML, this decreased from 25.9% to 3.7%, while FL had 0% change. In MS and ML, TC/high-density lipoproteins (HDLs) of >5.0 mmol/L dropped from 22.2% to 7.4% and from 22.2% to 15.4%, respectively. In FS and FL, TC/HDL of >4.5 mmol/L declined from 19.2% to 7.7% and from 19.2% to 3.8%, respectively. MS and ML with fasting blood glucose of ≥5.5 mmol/L declined from 40.7% to 11.1% and from 33.3% to 3.7%, respectively. Similarly, it declined from 46.2% to 0% and 42.3% to 11.5% for FS and FL, respectively. There were no differences in the changes between regimes throughout the study.After 24 weeks, MBouts lasting <10 min per session are as good as those lasting;≥30 min in improving cardiometabolic profiles of sedentary adults aged ≥50 years. Majority of sedentary Kenyans aged above 50 years have metabolic health risks.Moderate-intensity exercise offered in <10 min bouts has similar effects to longer bouts in modifying cardio-metabolic markers in sedentary adults.Regardless of the sex, accumulated short bouts of moderate-intensity exercise that last less than 10 min have beneficial health effects.The current recommendation for weekly duration of moderate-intensity PA for health benefit in adults is a minimum of 150 min, traditionally achieved in 3–5 sessions of 30–60 min per week, or accumulated through bouts that last more than 10 min.In the 1990s, suggestions were made that short exercise bouts were as effective as single continuous sessions of similar intensities provided that the cumulative weekly exercise time is equal.The cardiometabolic health benefits received from exercise for the elderly is clear, with a strong linkage to improved cardiorespiratory fitness (CRF),Healthy sedentary males (n=27) and females (n=26) aged ≥50 years were recruited from Eldoret, Kenya, following a local print advertisement. Sedentariness was defined as exercise/activity amounting to <600 metabolic equivalent minutes, using the WHO Global Physical Activity Questionnaire (GPAQ). A physical examination to rule out existing health problems was performed on all volunteers on their verbal declaration they were in good health. Individuals with cardiorespiratory disease or other physical ailments/injuries were excluded. Also excluded were participants on β-blockers and participants with diabetes on therapy. Signed informed consent was obtained from all participants.S),S),L) and female long-duration bouts of exercise (FL). In the short-duration bouts of exercise groups, the participants engaged in three sessions of 5–10 min of moderate-intensity jogging daily. In the long-duration bouts of exercise groups, participants engaged in 30–60 min jogging sessions for 3–5 days each week. Data from the participants were collected and analysed at the recruitment stage (September 2016) and then at 8-week intervals for a period of 24 weeks after the start of the exercise intervention to culminate in May 2017. Participants kept an exercise log, and objective verification of adherence to the exercise regime was done weekly using Polar Wearlink ActITrainer Accelerometers , and activity monitors were fastened onto participants on select days. The data, metabolic equivalent minutes achieved from the activity monitors, were analysed weekly to ascertain that participants in the different exercise regimes had comparable cumulative exercise time. Since this was only available for select days and used to primarily verify adherence, exercise intensity was monitored using WHO GPAQ. For clinical analysis, 4 mL of venous blood was collected from an average of two participants daily following an overnight 12 hours’ fast, usually between 06:00 and 08:00. Two drops of fleshly drawn blood were immediately used to measure blood glucose using Freestyle Optium Glucometer . For lipid profiles, centrifugation of blood was done within an hour of collection following which the serum was frozen and kept at −12°C, awaiting to run accumulated weekly samples together. This analysis into total cholesterols (TCs), high-density lipoproteins (HDLs), low-density lipoproteins (LDLs) and triglycerides (TGs) was done using Cobas Integra 400 Plus  at the Academic Model Providing Access to Healthcare Reference Laboratories, a certified clinical laboratory run by Indiana University/Moi University partnership.A biodemographics questionnaire on exercise history was completed for each participant following which they were randomly allocated into four groupsData analysis was done at univariate, bivariate and multivariate levels based on sex of participant and their exercise regime. Paired t-tests were used for intragender differences of metabolic markers following the different exercise regimes. The outcome variables were further analysed at the multivariate level by performing multiple analysis of variance for the dependent variables of lipid profiles and blood glucose and, further, linear regressions controlling for sexgroup performed. For comparison across the four phases of the experiment, repeated measures analysis of variance was conducted for equality of means of these blood chemistry results. Analysis was done using STATA V.13; p value was set at ≤0.05.S (n=14) and FS (n=13) group adhered to their 24-week exercise regime, compared with 61.5% of the ML (n=14) and 76.9% of FL (n=13). The weekly cumulative exercise minutes was similar for MS and ML  and also for FS and FL . Specific baseline characteristics for participants from the four groups are presented in The mean age of the males (n=27) was 55.5±3.0 years and 53.9±3.0 years for the females (n=26). The proportion of participants who had completed tertiary education was 88.4% among the male and 70.4% among the female. Ninety-one per cent of participants were in white-collar jobs. All (100%) male and female participants in the MS group), 33.3% (ML group), 46.2% (FS group) and 42.3% (FL group). In 22.2% (MS), 25.9% (ML), 30.8% (FS) and 11.5% (FL), baseline TC was above the cut-off of >5.2 mmol/L for males and >5.3 mmol/L for females. HDLs below 0.9 mmol/L was found in 18.5% (MS), 11.1% (ML), 7.7% (FS) and 15.4% (FL). TC/HDL ratio of >5.0 mmol/L and>4.5 mmol/L, the cut-offs for males and females, respectively, was found in 44.4% of the males (22.2% each in MS (mean=6.58±1.21) and ML (mean=6.63±1.29)) and 38.5% of the females (19.2% each in FS (mean=5.38±0.92) and FL (mean=5.42±0.59)). LDL/HDL ratio >3.5 was found in 14.8% MS group (mean=4.07±0.53) and in 18.5% ML group (mean=4.79±1). LDL/HDL ratio >3.0 was found in 23.1% FS group (mean=3.53±0.27) and 11.1% FL group (mean=3.32±0.43).Mean fasting blood glucose (FBG) of all participants at the start of the study was 5.92±1.4 mmol/L  and 6.23±0.74 mmol/L . The percentage of males and females with FBG >5.5 mmol/L was 40.7% , and in FS group, those with the ratio >4.5 decreased from 19.2% to 7.7% (mean decreased from 5.38±0.92 to 5.14±0.03). In the ML group, the decrease was from 22.2% to 15.4% (mean change from 6.63±1.29 to 5.66±0.5), and in FL group, the decrease was from 19.2% to 3.8% (mean change from 5.42±0.59 to 5.18±0). Percentage of MS group with LDL/HDL >3.5 mmol/L was halved to 7.4% with mean LDL/HDL decreasing to 3.61±0.14. In the ML group, the decrease was from 18.5% to 11.1% with mean LDL/HDL decreasing to 4.65±0.67 mmol/L. In FS group, participants with LDL/HDL ratio >3.0 decreased from 23.1% to 15.4% (mean 3.49±0.55) and to 3.8% from 11.1% (mean 3.49±0) in the FL group. Mean changes for the lipid ratios are provided in At the end of the 24-week period, male participants with FBG of ≥5.5 mmol/L in both the M regimes . All MS In summary, except for TC in females where the short bouts appeared superior to the long bouts, there was no difference in actual value change between the two bouts for the various cardiometabolic parameters over the 24-week period as shown in Lipid profiling is crucial in CRF assessment among healthy individuals and those with metabolic syndrome. At the start of the study, nearly half of the males and females had TC above their reference cut-offs . A third of the males and females had unfavourable levels of both LDL and HDL. Additionally, one-fifth and one-tenth of males and females had high TG levels. For TC/HDL associated with poor CRF and cardiovascular disease,S group with unfavourable TC at the start reached values associated with better CRF. Furthermore, mean TC in these males decreased as did the mean for those who still did not reach the recommended ranges. The finding was similar among ML group, suggesting the two exercise regimes produced comparable affect. This ML group also had a reduced percentage that remained with values above recommended cut-off and a decline in mean values for the whole group and, importantly, those who did not attain TC values <5.2 mmol/L as well. The two exercise regimes produced similar changes in the mean TC at the end of the study period. In females, except for the decrease in mean TC being higher in FS group, which suggests short-bout exercise could have better outcome compared with the currently recommended exercise regime, no other differences were observed in TC. Also, two-thirds of females with high baseline TC in FS group reached the recommended range at the end of the study; none from the FL group did. The two exercise regimes, however, had comparable decrease in the overall TC means, even among participants who were unable to achieve the reference cut-off. When baseline and end-point values were compared, mean TC change was similar in the two regimes. Thus, based on TC alone, no demonstrable difference could be shown between the exercise regimes for either sex. No study that we are aware of has described this exercise effect in individuals of comparable age and setting and followed for similar time-length as our study.After 24 weeks of prescribed moderate-intensity exercise, both males and females showed improved metabolic profiles. Nearly half of the ML group, those with HDL <0.9 mmol/L, also decreased from 15.4% to 3.8%, and there was no change in the ML group. What our study adds and that could be a beneficial interventional approach is that the short sessions appeared more beneficial in improving HDL levels when compared with the traditional regimes. For LDL and TG, the change was marginal. Males and females on long-bout exercise regime had slight rise in both LDL and TG, but there was slight improvement in the short-bout exercise participants.Males and females in both exercise regimes had an increase in HDL. This finding is consistent with other studies that show exercise affects HDL.S and FS group showed reduced TC/HDL . In ML, it was a quarter, and in FL, it was four-fifths. Previously, it has been suggested that intermittent exercise regimes may actually be more beneficial than current exercise regimes in regulation of attributes such as blood pressure and maximal oxygen consumption.et al,et al,For lipid ratios, two-thirds of MS group with baseline values of >3.5 showed lower ratios. Slightly less than half of ML regime participants also showed LDL/HDL ≤3.5. There was no difference between the groups in overall mean change of LDL/HDL for the duration of the study. The exercises regimes also had comparable mean values in those not reaching the recommended LDL/HDL ranges. Fifty per cent of FS group that started with high values of LDL/HDL reached the recommended threshold, compared with about two-thirds in the FL group. Thus, shorter exercise regime was more favourable in LDL/HDL regulation in males, although females still attained satisfactory results since drop in LDL/HDL for traditional group was not statistically superior to that in experimental group. Based on TC/HDL and LDL/HDL, it is apparent the two exercise regimes had similar reduction of cardiovascular disease risk. This could also address the recent finding, which our baseline data also support, that majority of Kenyans aged above 50 years are at cardiovascular disease risk based on combination of various lipid ratios.LDL/HDL had lower change in rate throughout the study period. However, the overall trend was similar in both sexes from the two exercise regimes. Half of MS group, participants with FBG >5.5 mmol/L decreased from 40.7% to 11.1%, while in the ML from 33.3% to 3.7. Absolute FBG mean change, however, was similar between the groups, and participants retaining prediabetic-to-diabetic values were less in MS and FS than in the ML and FL groups. In the FS group, all participants with prediabetic-to-diabetic baseline FBG levels (46.2%) achieved normal levels after the 24 weeks. In the FL, this was from 42.3% to 11.5%. The mean change in absolute FBG values between baseline and end-point were similar for FS and FL groups. Regression analysis controlling for gender showed no significant difference between the short-duration and long-duration bouts of exercise in FBG mean change. The view that longer exercise sessions among the males and the shorter regime for the females were superior based on percentage change  therefore failed to hold further. This, coupled with the observation of no difference in mean change for FBG after 24 weeks for both sexes from the two different exercise regimes, supports our hypothesis that these exercise regimes have similar effects. Thus, shorter/intermittent exercise sessions lasting <10 min each regulate elevated FBG to the same extent as the longer traditional sessions lasting >30 min per session, as long as the cumulative exercise times are similar.FBG levels decreased for all groups. In MThe design of the protocol may have caused some limitations. Our sample was recruited through print advertisement, which self-selection may have favoured those able to read, compromising inference to the general elderly population. Furthermore, the design could not allow for blinding, depended on subjective health assessment at recruitment with only a physical examination the only form of objective verification, and did not consider lifestyle behaviours such as smoking history and diet, which could have affected health status of participants. These potential limitations may have confounded our results and affected generalisation.An exercise regime of accumulated short bouts lasting <10 min shows improvement of cardiometabolic measurements among sedentary adults aged ≥50 years comparable with the currently advocated longer sessions that last >30 min each, as long as the intensity and cumulative exercise times are similar.