This pilot study did not demonstrate an association between sarcopenia and SIRS or bacterial translocation. These results should be confirmed in future larger studies, encompassing a greater number of chronic inflammation events and quantifying levels of bacterial DNA.Advanced liver disease is associated with a persistent inflammatory state, derived from abnormal bacterial translocation from the gut, which may contribute to the development of sarcopenia in cirrhosis. We aim to document the association of chronic inflammation and bacterial translocation with the presence of sarcopenia in cirrhosis. We prospectively followed cirrhotic patients aged 18–70 years with medically refractory ascites at a single tertiary care center in Toronto, Canada. The baseline data included patient demographic variables, the presence of bacterial DNA in serum/ascitic fluid, systemic inflammatory response syndrome (SIRS) status, and nutritional assessment. Thirty-one patients were enrolled, 18 (58.1%) were sarcopenic, 9 (29%) had bacterial DNA in serum and ascites fluid. The mean MELD score was 11.5 ± 4.0 (6–23). Sarcopenic and non-sarcopenic patients did not differ significantly in their baseline MELD scores, caloric intake, resting energy expenditure, the incidence of bacterial translocation, or SIRS. While sarcopenia was not linked to increased hospital admissions or death, it was strongly associated with increased episodes of acute kidney injury (3 vs. 0,  Sarcopenia, or loss of muscle mass, is a common complication of advanced liver disease, occurring in 40–68% of cirrhotic patients ,2. The pIt is hypothesized that the persistent inflammatory state observed in advanced liver disease derives from an abnormal bacterial translocation from the gut . When viThe paucity of data concerning molecular mechanisms of sarcopenia in liver disease, as well as the demonstrated key contribution of sarcopenia to liver disease outcomes, point to an urgent need to research the associated etiological mechanisms. In this pilot study, we aim to document the association of suspected risk factors, including inflammation and bacterial translocation, to the presence of sarcopenia, with the goal of identifying relevant research targets for future investigation.This study was approved by the Research Ethics Board of the University Health Network, Toronto, Canada (REB #10-0325). Written informed consent was obtained from all patients.3), positive ascitic fluid and/or blood cultures, existing renal impairment (creatinine ≥133 umol/L), chronic obstructive pulmonary disease, active malignancy, and individuals who were currently on antibiotics or had been treated with antibiotics within the last two weeks.Participants were prospectively recruited at Toronto General Hospital , a single tertiary care center, between September 2010 and March 2015. Eligible participants were aged 18–70 years, diagnosed with decompensated cirrhosis based on liver biopsy or clinical, radiologic, and biochemical findings, and had medically refractory ascites [Patient data collected included age, gender, and variables related to the etiology and severity of liver disease and liver disease outcomes. The severity of liver disease for each participant was determined by calculating both their Child-Pugh class  and MELDAfter obtaining informed consent, participants underwent a therapeutic paracentesis under sterile technique, and ascitic fluid (AF) was obtained for cell count, culture (a set of anaerobic and aerobic culture bottles inoculated with 10 mL each of AF), and subsequent analysis for the presence of bacterial DNA (bactDNA), as described below. Peripheral blood cultures were also obtained at the time of paracentesis, as well as blood samples for routine hematologic, biochemical, and coagulation studies.The ideal location of bactDNA isolation for predicting liver disease outcomes has not been well defined and varies in the literature. Therefore, we assessed bactDNA presence in both blood and ascitic fluid and assessed outcomes related to the DNA findings in either of these sources or in a combination of the two.DNA isolation, DNA extraction and PCR amplification of the complete 16S ribosomal RNA gene were performed in all serum and AF samples using the previously described methodology . SpecimeDNA amplification and PCR reactions for the complete amplification of the 16S ribosomal RNA gene were carried out. Eubacterial primers of a conserved region of the 16SrRNA gene used were: 5′-AGA GTT TGA TCA TGG CTC AG-3′ as forward (located at positions 8–27) and 59-GGT TAC CTT GTT ACG ACT T-39 as reverse (positions 1509–1491). Approximately 10–100 ng of template was added into a reaction mix containing 20 mM Tris HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 200 mM of each deoxynucleoside triphosphate, 0.4 mM of each primer, and 2.5 U Taq DNA Polymerase  to complete a final volume of 50 µL.To avoid false-positive results, positive and negative controls were performed in duplicate in each assay. DNA from Escherichia coli was added as a positive control, and sterile water and PCR mixtures (without template) were used as negative controls. PCR was carried out in a Mastercycler personal (Eppendorf) through the cycles as follows: an initial cycle of 95 °C for four minutes was followed by 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 90 s, with a final extension period at 72 °C for 10 min to complete the cycling sequence. The Total PCR reaction volume was filtered through QIAquick Spin Columns  to remove primers and nucleotides. Purified products were visualized on 1.5% agarose gels stained with ethidium bromide. A band of approximately 1500 base pairs (bp) was obtained, corresponding to the specific amplification of the prokaryotic 16S ribosomal RNA gene.2 < 32 mmHg, (4) White blood cell count >12.0 × 109/L or <4.0 × 109/L [Participants were evaluated for systemic inflammatory response syndrome (SIRS) criteria, and had a positive diagnosis if they fulfilled two or more of the following four criteria: (1) Temperature >38 °C or < 36 °C, (2) Heart rate ≥90 beats per minute, (3) Respiratory rate ≥20 breaths per minute or pCO × 109/L .Assessment of the participants’ overall nutritional status was performed using the subjective nutritional assessment (SGA), a scoring system based on clinical and physical exam findings . The SGAThe presence of hypermetabolism, as defined by an elevated corrected resting energy expenditure (REE % predicted), was measured via indirect calorimetry (described in the next section).2) (kg/m2). A three-day dietary recall was used to assess individuals’ oral intake [In all subjects, body height was measured to the nearest 0.5 cm and body weight was measured to the nearest 0.1 kg after therapeutic paracentesis, but we did not correct for any pedal edema. Body mass index (BMI) was calculated as weight (kg) divided by height (ml intake .Anthropometric measurements were performed by a single investigator (CT) who measured the mid-arm circumference (MAC) and triceps skinfold thickness (TST) to the nearest 1.0 mm at a point midway between the acromion and medial epicondyle of the humerus using a Lang SF caliper . The MAC was used to calculate the mid-arm muscle circumference (MAMC) using the formula MAC − (TST × 0.314) . Patient2 and expired VCO2). Once the patient attained steady-state, i.e., a period of ten minutes during which the variation in VO2 and VCO2 measurements was ≤10% and variation in respiratory quotient (RQ) ≤5%, the metabolic cart measured the gas exchange over another ten-minute period. The RQ, measuring fuel consumption, and mREE were calculated automatically using the manufacturer’s equations  × 1440) [In order to measure resting energy expenditure (mREE), indirect calorimetry was performed on all subjects using the Vmax Encore 29 machine . After an overnight fast, each subject was placed in a supine position and a transparent canopy was placed over the head in order to measure gas exchange (inspired VO × 1440) . HypermeIn order to assess how representative our population is of other liver disease patient populations, we assessed liver disease outcomes for all patients based on sarcopenia status. Since little is known about the role of bacterial translocation in liver disease, we also assessed outcome variables based on bacterial translocation status. Patients were prospectively followed with routine hematologic and biochemical bloodwork and outpatient follow-up every four months for hospitalization, development of complications, or transjugular intrahepatic shunt (TIPS) insertion, until death or liver transplant, or until they were lost to follow-up. All complications, including the development of SBP, acute kidney injury (AKI), hepatic encephalopathy (HE) or variceal bleeds, as well as hospitalizations were documented, including the reason for admission, in-hospital treatment, and discharge diagnosis. Episodes of AKI were defined as either an absolute increase in serum creatinine of ≥26.4 µmol/L or a 50% increase from baseline . Standar® .Demographic, risk factor and outcome variable distributions were calculated using descriptive statistics. Continuous variables are expressed as means and categorical variables as proportions. Pearson Chi-square tests were performed to assess associations between risk factor variables and sarcopenia. P-values of <0.05 were considered statistically significant. Statistical analyses were performed using JMPn = 21, 65.6%) with a mean age of 56 ± 9 years. The most common cause of cirrhosis was alcohol  followed by hepatitis B or C . Twenty-four subjects were classified as Child-Pugh B  and seven were classified as Child-Pugh class C . Two patients (6.3%) had a prior history of hepatic encephalopathy, and four (12.5%) had a history of variceal bleed. Twelve patients (37.5%) were on non-selective beta-blockers. The mean MELD score was 11.5 ± 4.0 (6–23). The mean period of follow-up was 12.7 ± 17.5 months.A total of 68 patients were screened . Thirty-p = 0.059), BIA-measured fat mass , triceps skinfold thickness , midarm muscle area  and midarm fat area . Conversely, patients in the two groups did not differ in total caloric intake or diet composition, as well as measured or corrected (% predicted) resting energy expenditure .Sarcopenia was present in 18 (58.1%) subjects. There was no significant difference in the baseline demographic characteristics of patients who were not sarcopenic vs. those who were . As predenditure . Both sap = 0.860) (p = 0.083) .In our pilot population, liver disease etiology was not found to significantly contribute to sarcopenia status (= 0.860) . Neither= 0.083) . SIRS wap = 0.211 for serum bactDNA, p = 0.739 for ascites, and p = 0.857 for both serum and ascites) (p = 0.399), but patients who were bactDNA +ve demonstrated lower protein consumption as compared to patients who were bactDNA −ve (0.042). Indirect calorimetry did not demonstrate a significant difference between groups in measured resting energy expenditure or fuel consumption using RQ. MELD and Child-Pugh scores were not dependent on bacterial translocation status , although the presence of bactDNA was associated with lower serum creatinine levels  and albumin levels . Despite this, sarcopenia was not more prevalent in patients who were bactDNA positive. SIRS occurred exclusively in patients with bacterial translocation to both ascites and serum.BactDNA was detected in only serum in six patients (19.4%), in only ascites in nine patients (29%), while another nine patients had detectable bactDNA in both AF and serum (29%). Bacterial translocation was not significantly different between sarcopenic and non-sarcopenic patients, irrespective of location of bactDNA detected (ascites) . Baselinp = 0.981 and p = 0.132, respectively) (p = 0.429), all due to complications from liver disease.Seven patients in the sarcopenia group underwent transjugular intrahepatic shunt (TIPS) insertion (38.9%) for treatment of refractory ascites, and two patients received liver transplant (11.1%), compared to six (46.2%) and zero patients in the non-sarcopenic group (ctively) . Patientp = 0.05). Time to the event  based on sarcopenia status is presented in Sarcopenia was not found to correlate with hospital admissions, or episodes of SBP, HE, and infections , but wasp < 0.005). However, during follow-up, this did not translate into significantly increased hospital admissions or episodes of infection (p = 0.435). No patients required hospitalization after their outpatient paracentesis. There were no significant differences in episodes of AKI, HE or SBP, based on bacterial translocation status.The presence of bactDNA in blood and ascitic fluid was associated with a significantly higher prevalence of SIRS at baseline as compared to patients who were bactDNA negative (nfection . During p = 0.019), while serum bactDNA was associated with an increased number of episodes of HE (p = 0.059).A significantly higher number of patients with AF bactDNA received TIPS (p = 0.545). Two of the patients died within 3 months after study enrolment, and two within 6 months, all from complications of their end-stage liver disease. Presence of bactDNA in serum tended towards a significant association with occurrence of death. Time to the event, including transplant, death or loss to follow-up based on bacterial translocation status is presented in During follow-up, one death (11.1%) occurred in the group of patients with bactDNA present in both blood and AF, compared to 3 deaths (13.6%) in other patients . No patients had a MELD score higher than 24. Despite this, we found that sarcopenia was still present in the majority of our study population (58.1%). Similar to previously published studies, we found no association with sarcopenia and Child-Pugh score [This is the first study to examine the potential associations between sarcopenia, bacterial translocation, and markers of inflammation in patients with decompensated cirrhosis. We had a unique study population of cirrhotic patients with advanced portal hypertension, but low MELD scores. The mean MELD score was only 11.5, and the majority of subjects had a MELD score of less than 15  and no history of SBP, both excellent prognostic factors reflective of the patients’ early decompensated state. Despite this, sarcopenia was extremely prevalent, reflecting the fact that sarcopenia is not dependent on the severity of liver dysfunction. Of note, while there was no significant difference in serum creatinine between sarcopenic and non-sarcopenic patients, serum creatinine level is influenced by muscle mass, and may have overestimated the patients’ true glomerular filtration rate in the sarcopenic cohort. Therefore, the sarcopenic patients may have had more significant renal dysfunction than estimated by the serum creatinine, leading to increased episodes of AKI.Research into the pathophysiological mechanisms underlying sarcopenia in cirrhosis has shown that sarcopenia develops as a result of dysregulation of muscle protein synthesis and protein breakdown . Human dWhile the exact molecular mechanisms by which inflammation regulates muscle mass are still under investigation, it has been suggested that such proinflammatory mediators upregulate protein breakdown through the activation of FOXO3a and the ubiquitin-proteasome system , and dowZapater et al. hypothesized that bacterial DNA in AF and serum leads to systemic inflammation through activation of the innate immune system, release of nitric oxide and other soluble inflammatory cytokines, and subsequent increase in liver damage . This isThe low occurrence of SIRS may also account for our excellent clinical outcomes and relatively lower-than-expected mortality rate. Despite 29 hospitalizations that were required by 11 subjects (34.4%), renal function remained well-preserved, and there were only three episodes of acute kidney injury. Only three patients developed episodes of infection, and another three patients developed episodes of hepatic encephalopathy. In addition, there were only four deaths over the follow-up period (12.9%), which is a significantly better prognosis than has been previously reported in the literature . We did This is the first paper to examine the relationship between sarcopenia and markers of bacterial translocation and inflammation. In our patient population, we did not find an association between these factors and the development of sarcopenia, although these results should be confirmed in a larger clinical setting. While sarcopenia has been shown to be an independent predictor of complications of cirrhosis , our pat


Neoadjuvant chemotherapy (NAC) is increasingly used to treat locally advanced breast cancer (LABC). Improved response to NAC correlates with better survival outcomes. The dual purpose of this study is to report recurrence and survival outcomes for LABC patients treated with NAC, surgery and adjuvant radiotherapy and to correlate these outcomes with tumour response after NAC using multiple response assessment methods.All LABC patients treated for curative intent with NAC, surgery, and adjuvant radiotherapy at our institute between January 2009 and December 2014 were included for analysis. NAC was mostly anthracycline and taxane-based; radiotherapy consisted of 50 Gy to the breast/chest wall and regional lymph nodes. Response to NAC was categorized using synoptic pathology reports, modified-RECIST and Chevallier scores. Survival curves were generated by the Kaplan-Meier method and compared using the log-rank test.n = 53) and Stage III (n = 50). Rates of locoregional control (LRC), recurrence-free survival (RFS), and overall survival (OS) were 99, 98, and 100% at 1 year and 89, 69 and 77% at 5 years, respectively. Responses to NAC did not correlate with LRC (p > 0.05) but did correlate with RFS and OS (p < 0.05), except that the Chevallier score did not predict RFS (p = 0.06). Using bivariate Cox modeling tumour size before (p = 0.003) and after (p < 0.001) NAC, stage group (p = 0.05), and response assessed by synoptic pathology (p = 0.05), modified-RECIST (p = 0.001), and Chevallier score (p = 0.015) all predicted for RFS. No factors predicted for LRC.The cohort included 103 patients nearly equally divided between Stage II (Pathologic response by all tested methods correlated with improved survival but were not associated with decreased LRC. Neoadjuvant chemotherapy (NAC) is increasingly used to treat patients with locally advanced breast cancer (LABC) –3. Such The NSABP B-18 and B-27 trials delivered anthracycline-based NAC (and included taxanes in B-27). Patients who underwent breast conserving surgery (BCS) after NAC received whole breast radiation therapy (RT) alone, while mastectomy patients did not receive RT. These trials demonstrated locoregional recurrence rates of 12.3% for mastectomy patients and 10.3% for BCS patients . SubsequPatients who achieve a pathologic complete response (pCR) to NAC have improved survival compared to patients who do not achieve pCR , 15, 16.In this study, response to NAC was measured using three methods. The first used data from synoptic pathology reports prepared by our institutional pathologists. The second, Chevallier score , provideThis study had two main goals. First, we investigated recurrence and survival outcomes for a cohort of LABC patients treated with NAC, surgery, and RT. Patients treated with all three modalities were selected as this represented the majority of patients treated at our centre and we wanted to ensure that results were reported from as homogeneous a cohort as possible. Second, we assessed tumour response after NAC using synoptic pathology reports, Chevallier score and a modified RECIST criteria-based (MR) response score and correlated these responses with recurrence and survival outcomes in our cohort.Institutional review board approval was obtained prior to data collection and analyses for this retrospective study. Data was collected via electronic medical records for all female breast cancer patients treated with NAC, surgery and radiotherapy with curative intent at our center between January 2009 and December 2014 for whom complete clinical data was available. Clinical tumour (T) staging used physical exam or pre-chemotherapy imaging . Pre-chemotherapy clinical nodal (N) assessment consisted of physical exam and imaging. Pre-chemotherapy lymph node involvement was assessed via core or fine needle aspiration biopsy, if available, but was not included in the clinical stage grouping. Stage grouping used the American Joint Committee on Cancer (AJCC) 7th edition staging guidelines.Prior to beginning NAC, all patients had biopsy-confirmed breast cancer that was either larger than 5 cm, invading into the skin/chest wall, or involving the axillary lymph nodes. Patients received NAC followed by mastectomy or BCT including surgical excision of the axillary lymph nodes. Adjuvant RT delivered 45–50 Gy in 25 fractions to the chest wall , axillary and supraclavicular lymph node regions. The RT was delivered via monoisocentric three or four field technique consisting of opposed tangent fields to the breast/chestwall and lymph node coverage with either opposed anterior-posterior fields or anterior field alone. Radiotherapy to the internal mammary lymph node chain and boost RT to the tumour cavity were left to the discretion of the treating radiation oncologist; generally boost RT was offered to patients treated with BCT who were younger than age 50 or had high risk features such as high grade disease or close/positive margins . All patTumours were grouped into molecular subtypes based on hormone and HER2 receptor status, according to the St. Gallen consensus . SubgrouResponse to NAC for all patients was categorized using three methods. The first (hereafter referred to as “standard pathology”) reviewed synoptic pathology reports available for each patient produced using the American Joint Committee on Cancer (AJCC) 8th edition criteria for staging and synoptic reporting . At the The second method was the Chevallier score, based on combined macroscopic and microscopic assessment: Grade 1 denotes complete disappearance of macroscopic or microscopic tumour, grade 2 has carcinoma in situ but no remaining invasive tumour or lymph node involvement, grade 3 indicates invasive disease present with stromal alteration and grade 4 indicates little or no modification of tumoural appearance .The third method was the MR system, which categorizes response based on tumor size using RECIST criteria. Size was assessed pre-chemotherapy by imaging or physical exam; the proportion of measurements by different modalities is shown in Table Three survival parameters were calculated: locoregional control (LRC) was defined as survival without LRR defined as recurrence in the breast, chest wall, or axillary, supraclavicular, or internal mammary lymph nodes; recurrence-free survival (RFS) was defined as survival without LRR or distant metastasis; and OS was defined as survival without death. Documentation of recurrences was based on available medical records, including dictated clinical notes and radiological studies. Time of recurrence was defined as the date when definitive recurrence or disease progression was first noted in a clinical or radiology report. Death was determined via clinical notes, hospital records and obituary search. The start date of NAC was used as a surrogate for the date of diagnosis; survival times and time to recurrence were calculated from this date.All patients in the cohort had their response categorized using all three methods. Each method was then assessed to determine if they were predictive of LRC, RFS, and OS. Survival curves were generated by the Kaplan-Meier method and survival differences between groups were compared via the log-rank test. Cox proportional hazard regression modeling was used to analyze factors contributing to survival rates. The association between MP and Chevallier scores  was examined using Spearman correlation. To assess the correlation between standard pathology response  and MP and Chevallier scores, a regression analysis was carried out. Analysis was performed using SAS Version 9.4 .Our cohort consisted of 103 patients and the median follow up was 45.6 months. Median age was 46.7 years old. An additional three patients were identified who otherwise met our inclusion criteria but were not included in the analysis due to lack of clinical follow up after completion of therapy. Pre-treatment patient and disease characteristics are shown in Table There were 23 patients (21.3%) who experienced disease recurrence. One patient had synchronous regional (lymph node) and distant recurrence. Seven patients (6.8%) had LRR only; five of these (4.8%) were in the ipsilateral chest wall or intact breast, the other two (1.9%) had recurrences in the axillary lymph nodes. Of 15 patients (14.6%) whose first episode of recurrence was distant (without LRR), six had recurrence in the lungs, nine in the bones, six in the liver, and four in the brain. Median time to recurrence was 22.5 months. During the analysis period, 14 deaths (13.6% of patients) were recorded.For the entire cohort, LRC rates were 99% at 1 year, 94% at 2 years, and 89% at 5-years. Rates of RFS rates were 98% at 1 year, 85% at 2 years, and 69% at 5-years. Rates of OS were 100% at 1 year, 95% at 2 years, and 77% at 5-years. Kaplan-Meier curves with these results are shown in Fig. p = 0.51). A significant RFS difference (p = 0.04) was seen, with 5-year RFS for stage II patients of 83 and 58% for stage III patients. 5-year OS rates were 86% for stage II patients and 72% for stage III patients, although this difference was not statistically significant (p = 0.46). Survival curves by stage are shown in Fig. p = 0.11), RFS (p = 0.45), or OS (p = 0.60).The cohort was nearly equally divided between patients with stage II (51%) and stage III (49%) disease. No significant difference in LRR was seen between these two groups (p = 0.48), but significant differences were observed for RFS (p = 0.015) and OS (p = 0.015). No patients achieving a pCR died within 5-year of treatment completion.Kaplan-Meier curves for recurrence and survival outcomes with patients divided by standard pathology response category are shown in Fig. p = 0.45), but significant differences were observed for RFS (p = 0.003) and OS (p = 0.014), as shown in Fig. For patients divided by MR score, local recurrence rates were not significantly different p = 0.5, but sip = 0.96) was seen between patients grouped by Chevallier score, as shown in Fig. p = 0.06). Nevertheless, significant differences in OS were observed (p = 0.005), with no deaths reported at 5 years for patients with Chevallier score of 1 or 2.No difference in LRR rates  and MR score (p < 0.001). The Spearman correlation between MR and Chevallier showed a strong negative association . Regression analysis showed bivariate correlation between MR and standard pathology was 0.874 and between Chevallier and standard pathology was 0.775.Standard pathology response was significantly associated with both Chevallier , pre-treatment stage group, and pathological response assessed by all three methods  significantly predicted for RFS. Analysis of OS is not reported due to the small number of deaths in our cohort. The results of this analysis are shown in Table All 103 patients in our cohort were evaluated for primary tumour response to NAC using three methods: standard pathology, MR score, and Chevallier score. Our results suggest that breast cancer treated with NAC followed by surgery and RT is associated with low rates of LRR (approximately 10% at 5 years) and relatively high OS (greater than 75% at 5 years). Rates of RFS and OS were significantly improved with increasing response to NAC, but LRR was not affected. Distant recurrence alone (15 patients) was twice as common as LRR alone (7 patients), similar to previously reported results of RT after NAC and surgery . MoleculRates of 5-year RFS for pCR, MR score 5, and Chevallier score 1 were 92%, 93%, and 86% respectively, while 5-year OS was 100% for all three groups. The similarity in outcomes suggests that all are valid methods for predicting outcomes after NAC, especially for those patients who achieve pCR. Patients grouped by MR score of 3 or 4  or 1 or 2 (“non-responder”) tended to have worse outcomes than similar categories using standard pathology.In our cohort, the three response methods employed detected similar rates of pCR: 25.2% of patients achieved pCR by standard pathology, 30.1% achieved MR score of 5 and 21.4% had Chevallier score of 1. These rates are similar to the 26.1% pCR rate in NSABP B-27. Over 95% of patients received anthracycline and taxane-based chemotherapy, similar to that given to NSABP B-27 patients [p = 0.06) significant predictive ability was measured for RFS. Strong correlations between categorizations using the different methods were also demonstrated.All three treatment response methods showed similar predictive abilities for clinical outcomes. Using bivariate Cox modeling, all three methods were predictive of RFS, but none were predictive of LRR. Using Kaplan-Meier methodology, when patients were stratified by standard response assessment and MP, response to NAC did not predict for LRR but did predict both RFS and OS. Chevallier score did not predict LRR and did predict for OS; a borderline  all serve as adequate predictors of response and ultimate clinical outcomes. The results agree with those of Romero et al. who reported that RCBI and RECIST-based assessments both predicted for RFS in a cohort of 151 patients . This coRecurrence and survival results in our cohort were similar to those reported in the initial publication by Ogston et al. that validated the Miller-Payne pathology scoring system . The iniDifferent pathologic subtypes of breast cancer may behave differently in response to NAC. Von Minckwitz et al. studied responses to NAC by different pathologic subtypes, in a cohort of over 6000 patients. They found that HER2-positive and triple-negative cancers achieved the highest pCR rates with Luminal A patients experiencing the lowest. If pCR was defined to allow residual in situ disease, pCR rates were 8.9, 51, and 35.8% for Luminal A, HER2-positive, and triple-negative, respectively . Our resLee et al. studied over 500 patients and assessed response by both “relative” methods (i.e. those that compare size or cellularity of post-NAC samples with pre-NAC data) such as MR score and “absolute” methods (i.e. those that use only the post-NAC sample) such as RCBI. They concluded that for triple negative cancer, all response assessment methods can predict for disease-free survival. However, for hormone-receptor positive, HER2-receptor negative disease, only absolute methods had prognostic value . Our cohAll three response assessment methods we studied showed the highest rates of RFS and OS among patients who achieved pCR. The NSABP B-18 and B-27 studies compared outcomes after randomizing patients with operable breast cancer between NAC and post-operative chemotherapy. Patients in the NSABP B-18 trial received AC chemotherapy (doxorubicin and cyclophosphamide), while the B-27 study used AC plus docetaxel. Hazard ratios for recurrence and death for patients not achieving pCR compared with those who did not were 0.47 and 0.32, respectively, after 15-year follow-up in the B-18 trial and 0.49 and 0.36, respectively, after 8 year follow-up in B-27. Neither study demonstrated statistically significant differences between the NAC and adjuvant chemotherapy groups for either disease-free survival or OS .Our results support the hypothesis that pCR  is predictive of RFS and OS, even when RT is given after surgery (which was not given in the B-18 and B-27 studies). However, based on all three response methods assessed in this study, pCR does not appear to be predictive of LRR when RT is given. In the combined B-18 and B-27 cohort, predictors of LRR on multivariable analysis were: age at randomization, tumor size before NAC, clinical nodal status before NAC, and pCR after NAC. In our cohort, these same factors predicted for RFS . However, we did not identify any factors that significantly predicted for LRR in our cohort. Our data support the NSABP findings that these factors are significant prognostic markers, but the RT that our patients received seems to have reduced these factors’ influence on LRR rates .p = 0.0001), and RT significantly improved cause-specific survival for patients with stage IIIB or IV disease, clinical T4 tumours, and four or more positive lymph nodes. McGuire et al. [p = 0.04) and RT also improved OS (p = 0.0017). No recurrence benefit was seen for patients with stage I and II breast cancer who achieved pCR however.The role of adjuvant RT after NAC and surgery has not been evaluated in randomized trials but is generally recommended for patients with cT3-T4 and/or N2-N3 disease , 35. It e et al.  studied Our cohort had a similar stage distribution  to these other retrospective reports. Our cohort's LRR rate (approximately 10% recurrence at 5 years) agrees closely with the results reported by Huang and McGuire for patients who received adjuvant RT. In the McGuire study, 5-year OS among patients who received RT was over 80%. Patients in our cohort who achieved a pCR had 5-year OS of 100% compared with 71% for partial responders and 65% for non-responders. The low rates of LRR seen in our cohort (particularly among patients who achieved pCR) mirrors the low rate of LRR in previous retrospective analyses. These retrospective cohorts together appear to support the routine use of adjuvant RT after NAC for clinical stage III disease, even after pCR. The question of RT after pCR is the subject of ongoing trials, including NSABP B-51 which is randomizing patients with T1–3, N1 disease between nodal RT and no nodal RT.Buccholz et al.  reportedIn the NSABP B-27 trial, LRR at 10 years for patients who received AC-T chemotherapy was 9.5%. In the combined NSABP B-18 and B-27 dataset, the 10 year LRR rate was 11.1% . Local recurrences accounted for 71% of 10-year LRR in patients treated with mastectomy and for 79% of 10-year LRR in patients treated with lumpectomy plus breast XRT . Rates oTo our knowledge, this study represents the largest North American cohort to date with response to NAC assessed using multiple pathologic scoring systems, which represents a major strength. We observed strong correlations between all three response assessment methods and similar clinical outcomes between pathological pCR, MR-5 and Chevallier-1 . These results suggest that all three methods are valid predictors of clinical outcomes when assessing completeness of response. Our study also presents a cohort of relatively homogeneous patients who all received NAC followed by surgery and RT.Limitations include the study’s retrospective nature, which depends on the completeness and accuracy of the information in clinical notes. Ki67 levels were generally not available, limiting our ability to distinguish between Luminal A and Luminal B (HER2-negative) cancers; however, this likely did not alter our conclusions.Three patients who were otherwise eligible were excluded from the analysis due to lack of follow up. It is possible that these patients were lost to follow up due to poor outcomes (e.g. morbidity or death), which could bias our results. However, the number of such patients was small compared to the studied cohort size.We did not have a sufficient number of deaths to study predictors of OS. Longer follow up may allow for more recurrences and deaths to be observed and late recurrence may be more likely in patients with certain outcomes . We also could not combine pathologic response assessment with molecular subtypes as was done by Lee et al. . A largeLocal recurrence rates after NAC, surgery, and RT are low and the dominant risk for patients is distant recurrence disease. Pathologic complete response, as measured by different pathologic and radiologic methods, is associated with improved survival for these patients but may not be associated with decreased local recurrence. Our data supports the use of adjuvant RT after NAC, but this question remains the subject of ongoing prospective trials.


Vitamin-D deficiency is a universal health problem caused primarily by inadequate exposure to sunlight. This study aimed to assess the vitamin D status and investigate the factors affecting vitamin D distribution among Saudi males.A cross-sectional study was conducted at the King Saud University Medical City from December 2015 to August 2016. Saudi males attending the outpatient primary health care clinics were invited to participate in the study. Data were collected on the current and past health status along with biochemical investigations for total 25-hydroxyvitamin D (25OHD), blood sugar, and cholesterol.Majority of the participants (76.1%) had vitamin D deficiency. Blood sugar level, age, and cholesterol level were the most significant factors associated with vitamin D status. The highest percentage of deficiency was observed in the youngest age group (30-40 years). With increasing age, the percentage of deficiency decreased significantly. Those with normal blood sugar and cholesterol level had higher serum vitamin D levels compared to those with diabetes and hypercholesterolemia.Vitamin D deficiency is still endemic in Saudi Arabia, particularly among younger males and those with diabetes and hypercholesterolemia. Vitamin D screening, supplementations, and vitamin D-fortified foods should be provided especially for these groups.The online version of this article (10.1186/s12889-019-6527-5) contains supplementary material, which is available to authorized users. Vitamin D deficiency is a universal health problem caused primarily by inadequate exposure to sunlight. Vitamin D is an important vitamin with powerful effects on several systems of the body . Unlike A severe vitamin D deficiency could lead to variety of presentations like fatigue, body aches, and myopathy up to association with increased risk of prostate cancer, dementia, Schizophrenia and cardiovascular disease , 5. SaudThe current study was carried out to assess the status of vitamin D levels among Saudi males and investigate the significant correlations that influence the vitamin D levels in this cohort.This cross-sectional study was conducted at the King Saud University Medical City from December 2015 to August 2016. All Saudi males attending the outpatient primary health care clinics during the morning shift were invited to participate in the study. Written informed consent was obtained from all the participants before commencement of the study. A short questionnaire was administered to collect data on the current and past health status, history of smoking, prostate diseases, vitamin D supplementation, and sunlight exposure  using the Roche diagnostics test.Biochemical investigations included analyzing blood samples for total 25-hydroxyvitamin D (25OHD), sugar level, and lipid profile. Blood samples (4 ml) were collected from all participants in the morning and serum samples were isolated. The sera were centrifuged at 2000 G for 10 mins and stored at -202; normal, BMI 18. 5-24.9 kg/m2; overweight, BMI 25. 5-29.9 kg/m2; and obese, BMI ≥30 kg/m2.Age was categorized into 4 groups: 30-40 years, 41–50 years, 51–60 years, and above 60 years. According to World Health Organization (WHO)  classifiAccording to the laboratory of King Saud University Medical City reference values, vitamin D deficiency was defined based on serum levels of 25OHD: low, 25OHD < 75 nmol/L; low normal to high normal, 25OHD between 75 and 250 nmol/L; and toxic, 25OHD > 250 nmol/L.According to the National Health and Nutrition Examination Survey (NHANES), diabetes mellitus (DM) patients were identified as those participants with measured Hemoglobin A1c (HbA1c) levels ≥6.5% or those taking medication for diabetes. HbA1c levels were categorized as: normal (< 5.7%) and pre-diabetic (5. 7-6.4%). Participants were identified as having hypercholesterolemia if the cholesterol level was ≥6.2 mmol/L.It is worth mentioning here that all Saudi populations are related to the same ethnic group, there is no difference in skin color, and all males wear similar traditional clothing.The exclusion criteria were participants with chronic conditions that affect vitamin D status such as malabsorption, chronic liver disease, renal impairment or nephrotic syndrome; those who were on medications that can affect vitamin Df metabolisms such as anticonvulsants or corticosteroids; and those with a family history of hypocalcemia or vitamin D disorders.P values < 0.05 using a confidence interval (CI) of 95% were considered significant.The data were expressed as mean and standard deviation. One way analysis of variance (ANOVA) and t-test were used to test the significance of the difference in vitamin D levels between more than two groups and two groups respectively. Factors affecting vitamin D were determined using linear regression analysis. The total number of participants who met the inclusion criteria of the study was 1702. All of them were residents of Riyadh or the neighboring cities where they had plenty of sunlight exposure. The age ranged from 30-95 years and the mean age was 54.25 ± 13.1 years.Figure Exposure to sunlight varies between respondents; while 79% mentioned that they are exposed to sunlight daily, those who are exposed to sunlight, 3-4 times weekly and 1-2 time weekly represented 11.3 and 3.2% of our sample respectively, Those who are rarely exposed to sun comprised 6.5% of the participants. Only, more than one third (37.1%) of the participants mentioned that they spend more than one hour outside home exposing to sun, while the remaining percentage (62.9%) mentioned that their exposure to sunlight is barely less than one hour.p = 0.08).After excluding participants with toxic levels of vitamin D > 250 nmol/L), Table 50 nmol/Lp > 0.1) (Table p = 0.01). Similar results were seen in males with normal serum cholesterol levels vs. those with hypercholesterolemia, though not statistically significant  , though not statistically significant (1) Table . The meaIn the current study, 76.1% of the participants were found to be vitamin D deficient, which was higher than the figures reported in other studies. Age, blood sugar levels, and cholesterol were significant factors associated with vitamin D status in Saudi men.Despite the higher serum 25OHD cut-off (75 nmol/L) used in the current study, the prevalence of vitamin D deficiency among this cohort of Saudi males was higher in comparison to other studies conducted in Saudi Arabia. Most of these studies have used a cut-off ranging from 25 nmol/L to 50 nmol/L, and the prevalence of vitamin D deficiency among Saudi Males ranged from 17.7 to 87.8% –13. UsinAge was one of the significant correlates of serum vitamin D levels in our study cohort. The vitamin D levels were lowest in the younger age group, which significantly increased until they reached the highest level in the older age group. These results are in agreement with earlier studies , 12, 15 With respect to the blood sugar levels, the mean vitamin D serum levels were higher amongst non-diabetic individuals in our study, which is in contrast to other studies that show higher levels of 25OHD in subjects with DM compared to non-diabetic individuals –23. ThisData on the association between cholesterol and vitamin D shows varied results. Population studies indicate that people with lower levels of vitamin D are more likely to have higher cholesterol levels. In 2012, a study showed that vitamin D supplements had no cholesterol lowering effects at least in the short term, and then too only low-density lipoproteins levels may increase. On the other hand, a study in 2014 found that taking calcium and vitamin D supplements together enhances cholesterol levels in postmenopausal overweight or obese women . The stuOur results indicated no significant association of BMI with vitamin D levels. Baradaran et al. reported results similar to our study , in contThough our study has some interesting findings, it has certain limitations. First, it was a cross-sectional, hospital-based study and therefore, we could not assess causality. In addition, this might have exposed the study to some sources of bias resulting from the manner in which study subjects were recruited or due to differences arising due to the participants’ cultural background, age, and socio-economic status. However, participants in the current study referred to the King Saud University Medical City were not limited to the capital city of Riyadh as all neighboring areas and governments centers refer to this tertiary hospital. This makes the selection of cases devoid of bias and could be considered representative of the Saudi population. Second, the sample size was relatively small and the dietary intake was not assessed. The study did not take into consideration the difference in sunlight exposure arising due to seasonal variation though, given the availability of sunshine nearly throughout the year, the seasonality might not be a significant factor in our case. However, the study addressed the significantly high deficiency of vitamin D amongst participants living in a country where they are exposed to sunny environment and high temperature throughout the year.Vitamin D deficiency is still endemic in Saudi Arabia, despite plenty of sun in the area. Increased awareness about the importance of vitamin D, particularly among younger age group, in addition to the integration of vitamin D testing in the primary health care centers, vitamin supplementations and vitamin D-fortified foods are warranted, especially for those with DM and hypercholesterolemia.Additional file 1:Vitamin D distribution questionnaire. (PDF 235 kb)


Previous clinical studies have suggested that trimethylamine-N-oxide (TMAO) could contribute to the development of atherosclerosis cardiovascular disease. However, the synthetic analysis in coronary heart disease (CHD) was not yet performed. We aimed to clarify the relationship between elevated plasma concentrations of TMAO and the incidence of major adverse cardiovascular events (MACE) in CHD patients.Meta-analysis and dose-response analysis of hazard ratio data from prospective observational studies reporting on the association between TMAO plasma concentrations and the incidence of MACE in patients with CHD were conducted.P = 0.000). For follow-up ≥ 1 year, it was associated with 62% higher risk of MACE in patients with longer-term than shorter-term . The dose-response analysis revealed a ‘J’ shaped association between TMAO concentration and the incidence of MACE (P = 0.033), with the concentration above 5.1 μmol/L being associated with HR of > 1.Of the 2369 published articles identified in the search, seven papers, with data from nine cohort studies , were included in the meta-analysis. Combined data showed that elevated plasma TMAO concentrations could increase 58% higher risk of MACE in patients with CHD (hazard ratios [HR]: 1.58; 95% confidence interval [CI] = 1.35–1.84, Elevated levels of TMAO are associated with an increased incidence of MACE in patients with CHD. TMAO concentration of 5.1 μmol/L may be a cut-off value for prognosis. Coronary heart disease (CHD) has become the leading cause of death throughout the world in recent years , 2. HencClinical epidemiology demonstrates a positive correlation between higher plasma TMAO concentrations and an increased incidence of MACE –10. ReceClinicalTrials.gov, Chinese VIP Information [VIP], China National Knowledge Infrastructure [CNKI], SinoMed and Wanfang Databases) up to 7 November 2019 for prospective, observational clinical studies, reporting on the association between TMAO and MACE in patients with CHD. We used wide search terms  or chronic CHD . Publications without detailed data were excluded. In each publication, relative risk estimates  for increments of one standard deviation, or at least two TMAO categories, must have been available.Three or more TMAO categories were required  for the dose-response analysis. For this analysis, a quantitative measure of TMAO and the number of each group must also have been available.When multiple publications were published from the same source study, we used the publication with larger sample size or more relative data.The following data from each study were extracted: the first author’s name, publication year, country of study conduct, sample size, patient characteristics , follow-up period, TMAO plasma concentrations, collection times and quantification, TMAO exposure level, relative risk estimates and 95% CIs for at least two categories of TMAO.In the dose-response analysis, the proportions of patients who experienced MACE, HRs, and 95% CIs for all categories of TMAO, were necessary. These data were not explicitly presented in every publication, so we sent emails to the authors to request this information and waited 2 months for the reply. After this time, we only included those publications where we could calculate HRs and 95% CIs using published methods .Data were extracted by one author (M.E.Y), and were checked for accuracy by another author (P.D.L). Study quality was assessed using the Newcastle-Ottawa scale (NOS) , containP < 0.05 from a two tailed analysis to be statistically significant. Subgroup analyses for patients with ACS [Stata/SE 14.0 software was used to perform all statistical analyses. Summary HR and 95% CI were calculated for the risk of MACE associated with high concentrations of TMAO in comparison with low concentrations , 7, 9–11with ACS , 8–11 anwith ACS , one to P < 0.05 is calculated, a dose-response relationship is considered for curve nonlinearity. All categories of TMAO, HR level of TMAO, and number of patients per study and per group, were included in the model , I2 = 25–50% (moderate), I2 = 50–75% , I2 = 75–100% (extreme). Because the heterogeneity was substantial (50% < I2 < 60%), a random-effects model was used for the meta-analysis. For sensitivity analysis, a fixed-effects model was conducted to test whether the heterogeneity or pooled estimates could represent a reverse association. Given that fewer than ten studies were included in our analysis, the potential publication bias could not be quantified by using the Begg’s and Egger’s test or the funnel plot.Heterogeneity was assessed with Chi-squared, Q-tests, and Iatistics . In Q-teThe search process and study selection , and 1.37  group was substantial and the chronic CHD group (I2 = 34.4%) moderate.The pooled HR estimates for MACE in patients with ACS , 8–11 ann = 3, P < 0.001) and 1.96  in studies with 1–3 years [2 = 0.0%). The in-hospital observation of one study [P = 0.001) for MACE.When stratified by the duration of the follow-up period, the pooled HR estimates for MACE were 1.34 . Heterogeneity and pooled HR were not significantly different between patients with ACS and chronic CHD (P = 0.058).There was significant difference in pooled HR among groups with different durations of follow-up, where heterogeneity was also significantly different  [The pooled HR for MACE in all studies when comparing the highest plasma concentrations of TMAO with the lowest was 1.58  . High TMThe results of sensitivity analysis were summarized in Additional file The dose-response analysis used a total of twelve HR estimates, from three studies, with four concentrations of TMAO in each , it may be that TMAO has a stronger prognostic value over the long term vs the short term. This may be partially caused by the accumulation effect of longer-term choline intake with more TMAO generating. The HR estimate (HR 6.01) was significantly highest for the in-hospital observation [This meta-analysis evaluated the accumulating evidence for an association between higher TMAO plasma concentration and the incidence of MACE  in CHD patients. From the 2369 literature records identified, data from nine relevant cohort studies were included in the meta-analysis. We found that higher levels of plasma TMAO significantly increase the risk of MACE in patients with CHD, both in those with ACS and chronic CHD , and over a short and long follow-up duration . When the follow-up duration ≥ 1 year, we found significant heterogeneity of HR between groups with a longer and shorter term , which increased almost linearly with increasing concentrations. This suggests that low TMAO concentrations (< 3.9 μmol/L) may even predict a lower incidence of MACE in CHD patients. TMAO was only associated with a HR > 1 when the concentration rose more than 5.1 μmol/L for populations with CHD. This suggests that 5.1 μmol/L may be a cut-off value. The result was in accordance with the median plasma TMAO concentrations of patients with MACE in two involved trials, the Cleveland Cohort study [Our dose-response analysis found that high plasma concentrations of TMAO (> 5.1 μmol/L) were associated with highly increased risk of MACE. A couple of experiments study showed that TMAO has the potential to accelerate the pathological progress by promoting atherogenesis, thrombosis, and vascular Inflammation , 27. Cel−/− mice has also shown that TMAO can slow aortic lesion formation [Furthermore, our analysis showed that TMAO may not be associated with a pathogenic process when the plasma concentration was under 3.9 μmol/L in patients with CHD. The nature of these processes is not yet clear. As a natural osmolyte, TMAO has a protein-stabilizing effect , 34 and ormation , suggestAlthough the pathway for TMAO generation has been well established , the mecAlthough meta-analysis of TMAO and the risk of MACE has been performed previously , 26, ourThe nine included studies were prospective and had an average NOS score of 7.56; failing to get stars mainly because they lacked demonstration of the outcome of interest, adequate control factors, or a statement of follow-up adequacy. There is potential for publication bias considering that no subgroups contain data from more than ten studies. Although the HRs identified from two studies  were unaA limitation of the meta-analysis was that we found nine relevant studies to include, with only three contributing to the dose-response analysis. This limits our ability to summarize ‘real-world’ relative risk with precision. Furthermore, there were several differences in the methodology of these studies. TMAO concentrations can be influenced by several patient characteristics, such as diet , renal function, liver function, excretory function of gut , 40, or Further studies should investigate the method to regulate the generation of TMAO, such as modulating intestinal flora, inhibiting the activity of FMOs . In addiThis meta-analysis revealed a significant association between higher plasma TMAO concentrations and the long term risk of MACE in CHD patients, including those with ACS and chronic CHD. Our dose-response analysis found a non-linear relationship between TMAO concentration and the HR for MACE, with a concentration above 5.1 μmol/L being associated with a HR of > 1. Our findings are based on limited study data but clearly suggest that TMAO concentration has prognostic value for patients with CHD. More prospective researches are needed to evaluate this relationship and the mechanisms that drive it.Additional file 1. Literature searches.Additional file 2. MOOSE Checklist.Additional file 3. Data used in the ‘TMAO concentration-risk of MACE’ analysis.Additional file 4. Dose-response analysis by Stata/SE 14.0 (commands).Additional file 5. Summary of 57 disqualified papers by evaluating full texts.Additional file 6. Summarized results on the relative risk of MACE of elevated TMAO in patients with CHD.


There was no significant association between ESRD vs. pre-dialysis CKD and PSQI global scores and no significant co-relation between eGFR and global PSQI score  in pre-dialysis CKD patients. Poor sleep quality is common in patients with CKD including hemodialysis patients in a developing country, which is independent of kidney function in non-dialysis patients. There is no difference in quality of sleep between pre-dialysis CKD and ESRD patients.Few studies have compared quality of sleep between pre-dialysis chronic kidney disease  patients and end-stage renal disease patients on dialysis (ESRD) and have found inconsistent results. Objective of this study is to compare quality of sleep between patients with pre-dialysis CKD and ESRD in a developing country. This study was conducted in an out-patient department and hemodialysis unit of a tertiary care facility. Patients included had either pre-dialysis CKD or ESRD. Assessment of quality of sleep was done using Pittsburgh sleep quality index (PSQI). A total of 152 patients were included in the study. Out of these patients, 79 (52%) had ESRD and 73 (48%) had pre-dialysis CKD. Median PSQI score was 6 (IQR 3–8.8). Poor sleep quality (PSQI ≥5) was present in 100 (65.8%) patients. Only hemoglobin ( Chronic kidney disease (CKD) especially end-stage renal disease (ESRD) is associated with reduced sleep quality, sleep disordered breathing, excessive day time sleepiness and periodic leg movement .Several studies have addressed prevalence of poor sleep quality in patients on hemodialysis and its correlates. Reported prevalence of poor sleep quality has been found to be 34–78% in hemodialysis patients in majority of the studies . StudiesIn a developing country like Pakistan, patients are often diagnosed with CKD at a later stage . Due to 2 but excluding patients on renal replacement therapy [This study was conducted in an out-patient department and hemodialysis unit of a tertiary care facility. Study was approved by institutional review board. Informed consent was obtained from all patients. Patients included in the study were above 18 years of age. Sample size was estimated to be 150 based on estimated prevalence of poor sleep quality as 50% with 95% confidence and 8% precision. Patients included had either chronic kidney disease, not on hemodialysis  or patients who had end-stage renal disease on hemodialysis (ESRD). Pre-dialysis CKD was defined as estimated glomerular filtration rate (eGFR) of less than <60 mL/min/1.73m therapy . eGFR wa therapy .We obtained patients’ history and laboratory data. We collected information on following variables: age, sex, height, weight, address, educational level, socio-economic, marital, employment, smoking status, duration of renal disease, hypertension, diabetes mellitus, cardiovascular disease, hepatitis C, blood hemoglobin, serum calcium, phosphorous, albumin and serum creatinine.Cardiovascular disease was defined as known prior history of coronary artery disease, cerebrovascular disease or peripheral vascular disease based on history and review of prior medical records. Patient was considered to be in lower income class if daily income was less than twice the upper limit of accepted poverty line, i.e., $2 per household person . HyperteAssessment of quality of sleep was done using Pittsburgh sleep quality index (PSQI). The Pittsburgh Sleep Quality Index (PSQI) is a self-reported questionnaire that consists of 19 individual items, creating 7 components that produce once global score ranging from 0 to 21. A lower score indicates healthier sleep quality. A score of 5 or greater is indicative of “poor sleep quality”. PSQI has been found to be valid and reliable in the assessment of self-reported sleep problems compared with actigraphic measures .The level of anxiety and depression was assessed using hospital anxiety and depression scale (HADS) . This sct-test for parametric data and Mann–Whitney U-test for non-parametric data. Spearman correlation was used to evaluate association between PSQI global score and eGFR in patients with pre-dialysis CKD. Since there were significant differences in several variables between pre-dialysis CKD and ESRD groups, we conducted a multiple linear regression analysis to study unadjusted and adjusted association between various predictor variables and PSQI global score in all patients. We included pre-dialysis CKD versus ESRD as a predictor variable in multiple linear regression analysis. Final model included demographic variables, pre-dialysis CKD versus ESRD and variables with an unadjusted p value of .10 or less. For all tests, p values of <.05 were considered statistically significant.All statistical analyses were performed using SPSS 20.0 . Continuous parametric variables were reported as means ± standard deviation; non-parametric continuous variables were reported as median with 25–75 inter quartile range; and categorical variables were expressed as percentages. Normality was assessed by using QQ plots. Categorical variables were compared using the chi-square test, and continuous variables were compared using 2 (IQR 7.8–24.5). Of these patients, 15.8% had stage III, 43.9% had stage IV, and 40.4% had stage V CKD. There was no significant co-relation between eGFR and global PSQI score  in pre-dialysis CKD patients.A total of 152 patients were included in the study. Mean age of all patients was 47.3 ± 18.3 years, 93 (61.2%) were males, and 59 (38.8%) were females. Out of these patients, 79 (52%) had ESRD and 73 (48%) had pre-dialysis CKD. Median duration of renal disease was 16 months (IQR 8–36 months). In patients with pre-dialysis CKD, median eGFR was 17.6 mL/min/1.73 mHypertension, diabetes mellitus, cardiovascular disease and hepatitis C were present in 81.6%, 46.7%, 19.7% and 28.3% of all patients, respectively. Mean hemoglobin was 9.8 ± 1.6 g/dl and median serum albumin was 3.9 g/dl (IQR 3.4-4.3 g/dl). Median PSQI score was 6 (IQR 3–8.8). Poor sleep quality (PSQI ≥5) was present in 100 (65.8%) patients.A comparison of characteristics of patients with pre-dialysis CKD and ESRD is shown in In our study, we found that poor sleep quality was present in two-third of patients with CKD including patients on hemodialysis. There was no significant difference between quality of sleep between non-dialysis CKD patients and patients on hemodialysis after adjustment of other variables.Our study results are comparable with other studies. Frequency of poor sleep quality was found in 57–78.5% in various studies involving hemodialysis patients . SimilarIn this study, we found no difference in sleep quality between patients with chronic kidney disease not on hemodialysis and hemodialysis patients even after adjusting for other variables. In addition, we found no significant co-relation between eGFR and quality of sleep in patients with CKD not on hemodialysis. This finding can be explained by the fact that our patients had advanced CKD with 40.8% of patients had stage V CKD. This is consistent with late presentation of patients with CKD in our health care set up . Few stuIn our study, we found that history of cardiovascular disease, high-depression score and lower hemoglobin were associated with poor sleep quality in a multivariate analysis. Association between lower hemoglobin and poor sleep quality was found in other studies ,31. DeprPoor sleep quality may have several implications. Poor sleep quality has been found to be associated with increased mortality  in hemodOur study has several limitations. This is a single center study with sizable but still limited sample size. We have assessed substantial but not all possible variables which could be associated with poor sleep quality. In addition, we do not have a longitudinal follow up of patients to examine impact of poor sleep quality on patient outcomes.In summary, poor sleep quality is common in patients with CKD including hemodialysis patients in a developing country, which is independent of kidney function in pre-dialysis patients. There is no difference in quality of sleep between patients on hemodialysis and pre-dialysis CKD patients even after adjustment of confounding variables. Clinicians taking care of these patients should routinely assess and address sleep quality in these patients including those not yet on dialysis. Further studies are needed to evaluate impact of sleep quality on risk of hospitalization, end-stage renal disease and mortality in our patient population.


We sought to study the association between sedation status, medications , and clinical outcomes in a resource-limited setting.A longitudinal study of critically ill participants on mechanical ventilation.Five intensive care units (ICUs) in four public hospitals in Lima, Peru.One thousand six hundred fifty-seven critically ill participants were assessed daily for sedation status during 28 days and vital status by day 90.p < 0.001) and a significant decrease in ventilator , ICU , and hospital  free days. Agitation was also associated with higher mortality . The most commonly used sedatives were opioids and benzodiazepines (9259 and 8453 patient days respectively), and the latter were associated with a 41% higher mortality in participants with a higher cumulative dose . The overall cumulative dose of benzodiazepines and opioids was high, 774.5 mg and 16.8 g, respectively, by day 7 and by day 28; these doses approximately doubled. Haloperidol was only used in 3% of ICU days; however, the use of it was associated with a 70% lower mortality .After excluding data of participants without a Richmond Agitation Sedation Scale score and without sedation, we followed 1338 (81%) participants longitudinally for 18,645 ICU days. Deep sedation was present in 98% of participants at some point of the study and in 12,942 ICU days. Deep sedation was associated with higher mortality (interquartile odds ratio (OR) = 5.42, 4.23–6.95; Deep sedation, agitation, and cumulative dose of benzodiazepines were all independently associated with higher 90-day mortality. Additionally, deep sedation was associated with less ventilator-, ICU-, and hospital-free days. In contrast, haloperidol was associated with lower mortality in our study.The online version of this article (10.1186/s13054-019-2394-9) contains supplementary material, which is available to authorized users. Sedation and analgesia are essential components in the care of mechanically ventilated patients in the intensive care unit (ICU) to provide comfort, improve patient-ventilator synchrony, and reduce anxiety and agitation . HoweverDespite these recognized benefits, sedation practices remain variable, with a tendency towards over-sedation and a lack of routine delirium assessment , 15. LesA detailed description of the protocol and participating ICUs was provided elsewhere . Adults Eligibility criteria included age ≥ 18 years, at least 24 h of invasive mechanical ventilation at one of the intensive care units participating in the study and enrollment into the study within 48 h of mechanical ventilation onset. Data collection and quality control methods were reported elsewhere . At enroWe collected daily cumulative dose of sedatives, neuromuscular blockers, analgesics, and antipsychotic medications. Opioid doses were converted to fentanyl equivalents, and benzodiazepines were converted to midazolam equivalents for comparison  as 0 if a participant died ≤ 28 days or if mechanically ventilated for > 28 days, or as the number of days between successful weaning from mechanical ventilation and day 28 after study enrollment. Similarly, we defined ICU-free days (IFDS) as 0 if a participant died ≤ 28 days or stayed in the ICU > 28 days, or as the number of days between ICU discharge and day 28 after study enrollment. Hospital-free days (HFDS) were defined as 0 if a participant died ≤ 60 days or was hospitalized > 60 days, or as the number of days between hospital discharge and day 60 after study enrollment.th and 25th percentiles of percent days with deep sedation or agitation. We then constructed a second multivariable logistic regression model to evaluate the association between 90-day mortality and cumulative opioids and benzodiazepines, and use of antipsychotics adjusted for the same variables as above. To express effect size, we used the interquartile odds ratio, i.e., the ratios of odds between the 75th and 25th percentiles of cumulative use of opioids and benzodiazepines by 28 days. We also used multivariable linear regression models to examine the above-described relationships with ventilator-free days, ICU-free days, and hospital-free days. We used R (www.r-project.org) for statistical analysis [The main objective of this analysis was to determine the relationship between sedation status and 90-day mortality. Specifically, we evaluated sedation depth based on the RASS score, cumulative use of benzodiazepines and opioids, and use of antipsychotics in single-variable and multivariable regressions. We first constructed a multivariable logistic regression model to evaluate the association between 90-day mortality and percent of days with deep sedation or agitation, adjusted for age, sex, APACHE III, and indicator variables for ICU site. To express effect size, we used the interquartile odds ratio, i.e., the ratios of odds between the 75analysis .n = 294) and those without a RASS, Ramsay, or Glasgow Coma Scale score (n = 25). This gave us a final sample of 1338 participants (81%) followed for 18,645 ICU days. From the 1338 patients in our cohort, 869 (65%) had RASS measured at least once during their stay. From the remaining patients, 357 (27%) had at least one Ramsay measurement during their ICU stay and 112 (8%) only had been evaluated using the Glasgow Coma Scale. There were differences in age, sex, event-free days, mortality, and disease severity between participants in the final sample and those excluded . Secondary outcomes and their relationship with sedation status were assessed  was associated with a 41% higher mortality. Additionally, the use of antipsychotics was associated with lower mortality by nearly 70%.We evaluated the cumulative dose of benzodiazepines and opioids, adjusting for variables associated with mortality Table . Benzodith vs 25th percentile of days in deep sedation) was associated with a fivefold greater odds of mortality and a 4- to 7-point reduction in ventilator-free, ICU-free, and hospital-free days. Agitation status had a 40-fold higher mortality. The interquartile cumulative difference in benzodiazepine usage was associated with a 41% higher odds of 90-day mortality. Additionally, we reported that the usage of antipsychotics was associated with lower 90-day mortality. We identified that most of our critically ill participants undergoing mechanical ventilation were deeply sedated throughout their ICU stay. The most common used sedatives were opioids and benzodiazepines.We found that deep sedation, agitation, and benzodiazepines were independently associated with worse clinical outcomes. Specifically, a greater percentage of days spent in deep sedation . This way, the adverse outcomes related to these preventable measures can be avoided.Additional file 1:Table S1. Dose Equivalency of Benzodiazepines Relative to Midazolam and Opioids relative to Fentanyl. Table S2. Ramsay Sedation and Glasgow Coma Scales conversion to the Richmond Agitation-Sedation Scale. Table S3. Sedation Status corresponding to Richmond Agitation-Sedation score. Table S4. Sedation Status and Use of Sedatives, Antipsychotics and Neuromuscular Blockers. Table S5. Sedation Status and Secondary Outcomes. Figure S1. Cumulative Incidence Plots Evaluating Sedation Status and Vital Status during ICU Stay stratified by ARDS Status at Enrollment. We plot the cumulative incidence of death (broken red line), unassisted breathing (broken blue line), and sedation status (shaded areas) among those who are receiving assisting breathing, stratified by whether participants have ARDS on admission (panel A) or no ARDS (panel B). The shaded areas were proportional to the percentage of participants who were deeply (dark blue), moderately (blue) or adequately sedated (light blue), and agitated (pink). Based on the Richmond Agitation Sedation Scale score or its conversion Figure S2. Cumulative Incidence Plots Evaluating Sedation Status and Vital Status during ICU Stay Stratified by APACHE III Score at Enrollment. We plot the cumulative incidence of death (broken red line), unassisted breathing (broken blue line), and sedation status (shaded areas) among those who are receiving assisting breathing, stratified by APACHE III score . Shaded areas and categorization of sedation same as in Figure S1. Figure S3. Cumulative Incidence Plots Evaluating Sedation Status and Vital Status during ICU Stay Stratified by Admission Type. We plot the cumulative incidence of death (broken red line), unassisted breathing (blue line), and sedation status (shaded areas) among those who are receiving assisting breathing, stratified by whether admission was medical (panel A) or surgical (panel B). Shaded areas and categorization of sedation same as in Figure S1. (PDF 1953 kb)


Based on a 1H NMR-based metabolomics analysis, CLP-induced septic mice had increased levels of acetate, pyruvate, and lactate in serum and decreased levels of alanine, aspartate, glutamate, and fumarate in lungs. However, these metabolic disorders, mainly involving energy and amino acid metabolism, can be recovered by MaR1 treatment. Therefore, our results suggest that the protective effects of MaR1 on sepsis could be related to the recovery of metabolic dysfunction and the alleviation of inflammation and mitochondrial damage.Maresin1 (MaR1), a new anti-inflammatory and proresolving lipid mediator, has been proven to exert organ-protective effects in septic animal models. However, the potential mechanisms are still not fully elucidated. In this study, we sought to explore the impact of MaR1 on metabolic dysfunction in cecal ligation and puncture- (CLP-) induced septic mice. We found that MaR1 significantly increased the overall survival rate and attenuated lung and liver injuries in septic mice. In addition, MaR1 markedly reduced the levels of proinflammatory cytokines (TNF- Sepsis is a life-threatening clinical syndrome characterized by multiple organ dysfunctions due to uncontrolled host inflammatory responses towards infection or injury . Sepsis Maresin1 (MaR1), derived from docosahexaenoic acid (DHA) by macrophages via 12-lipoxygenase , possessMetabolomics is an “omics” technique that attempts to analyze all low-molecular-weight metabolites in biological samples and explore metabolic mechanisms underlying disease development or drug treatment . Metabolα and IL-6 ELISA kits were from R&D Systems . Blood biochemistry assay kits (ALT/AST) were from Jiancheng Bioengineering Institute .Maresin1  was from Cayman Chemical Company . The chemical structure of MaR1 is shown in Eight-week-old male C57B6/L mice weighing 20–25 g  were housed four per cage and maintained in a specific pathogen-free room with controlled temperature (22–24°C) and humidity (60–65%) under a 12 h light/dark cycle. The mice were given standard laboratory chow and water ad libitum. Principles of laboratory animal care were followed, and all procedures were conducted according to the guidelines established by the National Institutes of Health , and every effort was made to minimize suffering. This study was approved by the Animal Care and Use Committee of Wenzhou Medical University.Mice were anesthetized with 2% sodium pentobarbital  and randomly assigned to three groups: sham group, CLP group, and CLP + MaR1 group . Sepsis was induced surgically by CLP as described previously . Midlinen = 12/per group). The survival rate of mice was monitored for 8 days after CLP surgery and recorded every day.The mice which had undergone CLP surgery were randomly administered MaR1 (100 ng) or saline in the sham group , and aspartate aminotransferase (AST) were, respectively, determined using ELISA kits according to the manufacturer's protocols.The lung and liver samples were obtained immediately after exsanguination (24 h after CLP) then embedded in paraffin and stained with haematoxylin and eosin (H&E) for light microscope analysis. Organ injury scores were performed by two independent investigators blinded to the research assignment following the applicable criteria . Whole bFor electron microscopy experiments, the lung tissues were harvested 24 h after CLP and the effect of MaR1 was assessed by transmission electron microscopy analysis. Mice were euthanized and then perfused with cold PBS, followed by 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4), and processed for transmission electron microscopy (TEM), as described previously . Images μL D2O containing sodium trimethylsilyl propionate-d4  and then transferred to a 5 mm NMR tube for NMR analysis. In addition, 200 μL of serum sample was thawed and diluted with 250 μL of phosphate buffer and 50 μL of D2O. The diluted serum was mixed and centrifuged at 12,000 g at 4°C for 15 min. After that, 500 μL of supernatant was transferred into a NMR tube for analysis.Lung and liver samples were collected, frozen in liquid nitrogen rapidly, and stored at −80°C until use. The extraction was performed as previously described . Prior t1H NMR spectra were measured by using a Bruker Avance III 600 spectrometer equipped with a triple resonance probe  at 128 K. A standard single-pulse sequence (ZGPR) with water signal presaturation was employed to acquire NMR spectra of tissue extracts. For serum sample, the Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence with a fixed receiver-gain value was performed in order to minimize broad NMR peaks from proteins and lipids. The main acquisition parameters were set as follows: spectral width = 12,000 Hz, data points = 256 K, relaxation delay = 4 s, and acquisition time = 2.66 s per scan.The 1H NMR spectra were corrected manually for phase and baseline using TopSpin 3.0 software . The spectra of serum samples were referenced to the methyl peak of lactate at 1.33 ppm, while the spectra of tissue samples were referenced to the TSP peak at 0 ppm. The “icoshift” procedure was used to align NMR spectra in MATLAB software  [The MA, USA) . The speP value <0.05. Heat map and metabolic pathway analyses were conducted by using MetaboAnalyst 3.0 [P values from the pathway enrichment analysis and pathway impact values from the pathway topology analysis, and the metabolic pathway with high values of these two parameters, namely, located in the top-right region, was identified as the most important pathway.In this study, all mice were randomly assigned to experimental procedures including housing and feeding, animal grouping, CLP surgery, and MaR1 treatment. Principal component analysis (PCA) was used to obtain an overview of the metabolic pattern of animal models using the SIMCA software . Prior to PCA, NMR data were Pareto-scaled for improving data comparability. All data were analyzed by one-way analysis of variance (ANOVA) followed by Fisher's LSD test using SPSS software . Survival of the two subgroups was estimated by Kaplan–Meier survival curves and compared by the log-rank test. In this study, a statistically significant difference was considered when lyst 3.0 . The resP < 0.05). Pathologic examination revealed that mice in the sham group displayed normal tissue histology , while their levels were notably downregulated after MaR1 treatment , aspartate (P < 0.01), glutamate (P < 0.001), and fumarate (P > 0.05) were decreased in lung tissue of the CLP group; however, their levels were marginally increased after MaR1 treatment.In lung tissue, we identified alanine, aspartate, and glutamate metabolism as a key metabolic pathway Figures . Compareα and IL-6 in plasma, which may influence the patients' outcome in critical illness [Maresin1 (MaR1) is a new family of special proresolving mediators (SPMs) that are derived from endogenous DHA. SPMs have been shown to exert potent protective effects in humans' biology and health , 22. Our illness . Korner  illness . Moreove illness , 25.Although MaR1 has demonstrated a potential protective effect on sepsis, its clinical application is still in its infancy . Explori2 and H2O via the tricarboxylic acid (TCA) cycle or transformed to lactate by anaerobic glycolysis. In the present study, our results reveal that MaR1 can effectively recover abnormal increases in serum pyruvate and lactate levels in CLP-induced septic mice, suggesting a recovery of pyruvate metabolism after MaR1 treatment. Of note, lactate is an established indicator in tissue hypoxia and mitochondrial dysfunction in critical illness [Drosophila melanogaster model of surviving sepsis. Taken together, the recovery of pyruvate metabolism induced by MaR1 treatment could be contributed to its potential therapeutic effect in CLP-induced septic mice.Pyruvate metabolism has been closely associated with human disease by maintaining energy metabolism homeostasis . Pyruvat illness , 28. Rec illness , 30. Our illness . Therefo illness  detected illness  reportedMetabolomics results also reveal that MaR1 treatment recovered abnormal metabolism of alanine, aspartate, and glutamate in CLP-induced septic mice. Glutamate plays a vital role in the biosynthesis of proteins in living beings. The reduction in glutamate level in plasma has been linked to poor outcomes in septic shock patients . In the α-ketoglutarate and form pyruvate and glutamate. However, Izquierdo-Garcia et al. [Alanine has a close association with multiple metabolic pathways such as glycolysis, gluconeogenesis, and the TCA cycle. In this study, the alanine level in lung tissue was significantly reduced in CLP-induced septic mice as compared with normal control mice. This finding may be due to a significant increase in ALT level in CLP-induced septic mice, since ALT can catalyse the transfer of an amino group from alanine to a et al.  found anAspartate is synthesized from an intermediate of the TCA cycle, oxaloacetate, by transamination. Our results show that MaR1 treatment mildly recovered the CLP-induced reduction in lung aspartate level. The same trend was also obtained in the level of lung fumarate, which is an important intermediate of the TCA cycle. Therefore, MaR1 could maintain energy metabolism homeostasis in the lung of CLP-induced septic mice. Consistent with this finding, we also found that MaR1 ameliorated the CLP-induced ultrastructural damage of mitochondria in lung tissue by transmission electron microscopy analysis.Oxidative stress has been implicated in the pathogenesis of sepsis , 37. TauThe present study suggested that MaR1 treatment improved the survival rate of CLP-induced septic mice in multiple ways, including the recovery of lung and liver injuries, the reduction of inflammatory cytokines, and the attenuation of mitochondrial damage. Furthermore, using a metabolomics analysis, we also found that MaR1 could alleviate metabolic disorders in CLP-induced septic mice, mainly involving pyruvate metabolism, alanine, aspartate, and glutamate metabolism, as well as lung taurine level. Yet, it is worth noting that MaR1 significantly regulated the metabolic changes in serum and lung but not liver in septic mice. The potential explanation underlying this phenomenon could be due to the time effect of MaR1 treatment on sepsis. Therefore, the impacts of MaR1 on hepatic metabolic response might occur for an extended period. Our findings reveal a novel metabolic mechanism for the anti-inflammatory and proresolving actions of MaR1 in sepsis. MaR1 may provide new opportunities to design “metabolic targeted” therapies with high accuracy in treating sepsis. However, several limitations or future works should be considered: on the one hand, based on the present preliminary study, cause-effect associations among MaR1, metabolism, and sepsis cannot be proved. Therefore, cellular experiments or other animal models are recommended to discover more evidence on the therapeutic mechanisms of MaR1 in sepsis. On the other hand, metabolomics needs to be coupled with other omics techniques  for a better understanding of the effect of MaR1 on metabolic regulation and sepsis treatment. We believe that a deeper insight into the MaR1 therapeutic mechanisms will promote its clinical development and application.


Patients with knee osteoarthritis (OA) who are obese have problems performing land-based exercises. The reduced joint stress associated with aquatic exercise may benefit these patients. This study aimed to develop an underwater treadmill (UTM) machine that is affordable and suitable for use in developing countries, and to evaluate its efficacy in decreasing pain and increasing functional improvement.2 were recruited. The UTM exercise protocol was 30 min/session, 3 days/week, for 4 weeks. The main outcomes were NRS pain score, 6-min walk distance (6MWD), quadriceps strength (QS) and body weight. Those outcomes were evaluated at baseline and at week 4.Clinical testing of the UTM machine was performed in an outpatient setting at Siriraj Hospital during January–June 2017. Patients with knee OA, aged 50–85 years, numerical rating scale (NRS) ≥5/10, and body mass index (BMI) ≥25 kg/m 2. Most patients (65.0%) developed bilateral knee OA, used pain medication (56.7%), and engaged in regular knee exercise (73.3%). Of the 30 enrolled patients, 6 withdrew. All of the remaining 24 patients attended all 12 sessions. The mean difference between baseline and the end of the study was − 2.3  for NRS pain; 34.9 m  for 6MWD; and, 1.8 kg  for QS. Concerning adverse events, 4 patients (15.4%) developed muscle pain, 2 patients (7.7%) had joint pain, and 1 patient (3.9%) withdrew due to severe knee pain. Two-thirds of patients described themselves as being ‘very satisfied’ with UTM exercise, and approximately 90% of participants rated their symptoms as ‘improved’ or ‘much improved’.The UTM was constructed with safety, ergonomically designed and user-friendly control panel with push button for emergency stopping. Thirty patients were included for clinical testing. The mean age was 62.8 years, and almost all were female. The mean BMI was 28.9 kg/m 4-week exercise with UTM can significantly improve NRS pain, 6MWD, and QS. UTM could be an alternative treatment for patients with knee OA who are obese due to small size, durability, and ecofriendly design as an exercise modality. Knee osteoarthritis (OA) is the most common degenerative disease of the musculoskeletal system worldwide. Overweight and obese older adults are the most likely subpopulation to develop knee OA. The prevalence of knee OA among older adults living in the community in Thailand was reported to be 34.5–45.6% . Knee OAMany studies have investigated the effect of aquatic exercise. Roper, et al. studied the effect of aquatic treadmill exercise in 14 patients with knee OA. That study had a cross-over design that compared between aquatic and land treadmill exercise for 1 week (3 sessions). They found that aquatic treadmill exercise improved angular velocity and decreased pain compared to land treadmill exercise . Mackay-In 1996, Mangione, et al. suggested using a land treadmill with body weight support in older adults with OA, but this type of exercise/therapy could not relieve pain . AquaticThe aim of this project was to design, develop, and clinically test a UTM that is both affordable and suitable for use in developing countries. UTM development-related information is presented in the supplementary file. The UTM was developed and assembled at the Department of Mechanical Engineering, Faculty of Engineering, Kasetsart University, Bangkok, Thailand, and it was installed at the Department of Rehabilitation Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand. Clinical testing of the UTM machine was performed in an outpatient setting during January to June 2017. This study was conducted in accordance with the ethical principles of the Declaration of Helsinki. This study was approved by the Institutional Review Board of Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand (COA no. Si 185/2013). All recruited participants provided written informed consent.2) [Participants were recruited via publicly displayed printed posters that introduced and described the study. Participants with knee OA who satisfied all of the inclusion criteria and none of the exclusion criteria were recruited. The inclusion criteria were patients with knee OA, aged 50–85 years, numerical rating scale (NRS) ≥5 (range: 0–10), and body mass index of Asian obese population (BMI ≥25 kg/m2) . PatientThe primary outcome was NRS pain score, and the secondary outcomes included 6-min walking distance (6MWD), body weight, and quadriceps strength. NRS pain score ranges from 0 to 10, with a higher score representing a higher level of pain intensity . 6MWD isThe sample size for this study was calculated using NRS pain score from a study by Dias, et al. , with 90t-test was used to analyze the mean differences between baseline and end of the study for primary and secondary outcomes with normally distributed data. Per-protocol analysis was used to analyze the outcomes. A p-value of less than 0.05 was considered statistically significant for all tests. SPSS Statistics  was used to perform all statistical analyses.Baseline demographic, anthropometric, and clinical characteristics of study participants are presented as mean ± standard deviation  or median and range  for continuous data, and as number and percentage for categorical data. Paired The design and construction details of the UTM machine are presented in the supplementary file, and the UTM specification and design characteristics are shown in Fig. 2. Two-thirds of patients had an education level below bachelor’s degree. More than half of patients (65.0%) developed bilateral knee OA, used pain medication (56.7%), and engaged in regular knee exercise (73.3%). Some used knee support (40.0%), and a few used gait aids (13.3%). Of the 30 enrolled patients, 6 (20%) withdrew from the study due to inconvenience (n = 4), lost contact (n = 1), and severe knee pain (n = 1)  for NRS pain; 34.9 m  for 6MWD; and, 1.8 kg  for quadriceps strength.Mean NRS pain score, body weight, 6MWD, and quadriceps strength at week 0 and week 4, and the mean difference between pre- and post-treatment using per-protocol analysis are shown in Table Concerning adverse events (AE) that occurred after recruitment (including patients lost due to AE), 4 patients (15.4%) developed muscle pain, 2 (7.7%) had joint pain, and 1 3.9%) was lost to follow-up due to severe knee pain. No study participants reported falling, joint swelling, or fatigue  of our findings, we found that UTM therapy could reduce the mean NRS pain score (mean difference: 2.3), which was a greater reduction than that reported by Farrar, et al. (mean difference: 2.0) . HoweverRegarding complaints of musculoskeletal pain, 15.4% of patients reported muscle pain, and 7.7% of patients described having joint pain. These adverse events may be due to inadequate warm-up, and/or to excessive muscle stress. Participants have to exercise against water resistance during continuous walking, and one-fourth of our patients did not exercise regularly before entering the study. Therefore, therapists should emphasize the importance of warm-up before commencing UTM exercise.The aim of this study was to assess the applicability and efficacy of the UTM, which was developed by our team. A sample size of 30 patients was calculated based on previous study . Since oThis study has some mentionable limitations. First, the non-randomized design of our study renders it vulnerable to selection bias. In addition, the grade of evidence was considered low in this study due to its pre-post design. However, this study design was adopted to facilitate the collection of preliminary data prior to testing using a randomized controlled design. Second, due to reasons that included transportation availability, travel distance, disability-related challenges, and frequency of hospital visits per week, 20% of our study population withdrew from this study. This substantial reduction in sample size may have introduced selection bias, and may have given our study insufficient statistical power to identify all significant differences and associations. Third, most participants in this study were female, and this may limit the generalizability of our findings to males. This difference between males and females in our study may be due to a higher reported prevalence of knee OA in females than in males (39.8% vs. 22.6% respectively) . Fourth,Aquatread”. It is a low-cost, small footprint, ecofriendly therapeutic/exercise modality that can provide aquatic exercise to older obese patients with knee OA via continuous treadmill walking. Since its installation over 2 years ago, Aquatread is still functioning fully to its design and performance specifications. With regard to user satisfaction, most participants described being satisfied to very satisfied, and their global improvement rating increased from improved to much improved after beginning use of the Aquatread. Taken together, these findings may support the benefit of underwater treadmill for treatment of patients with knee OA who are obese. As recommended by the study of Bartels, et al., aquatic exercise could be used during the initial phase of exercise program [The author-designed UTM profiled in this study was given the name “ program .Aquatread therapy had no significant effect on body weight. Although adverse events were reported by 7 patients (1 of those withdrew from the study due to severe knee pain), two-thirds of patients described themselves as being ‘very satisfied’ with Aquatread, and approximately 90% of participants rated their symptoms as ‘improved’ or ‘much improved’. After 2 years of use, Aquatread continues to function reliably and deliver according to its designed performance specifications.The UTM profiled in this report was designed, fabricated, and clinically tested by the authors of this study. Of the 4 measured parameters, NRS pain, 6MWD, and quadriceps strength were all significantly improved at the end of the study. However,