The average follow-up was 6.5 years. The age- and sex-adjusted decline in eGFR was greater in patients with NAFLD  compared to those without it . In multivariable adjusted models, the average difference in annual percent change in decline in eGFR comparing patients with NAFLD to those without NAFLD was −1.06% . The decline in eGFR associated with NAFLD was greater in patients with higher NAFLD fibrosis score, in those with proteinuria or with low eGFR at baseline ( <45 ml/min/1.73 m2), and in those who were smokers and hypertensive. Therefore, NAFLD is independently associated with CKD progression.This study aimed to investigate the association of nonalcoholic fatty liver disease (NAFLD) and its severity with the decline in kidney function in patients with chronic kidney disease (CKD). We conducted a cohort study of 1,525 CKD patients who underwent repeated health check-up examinations from January 2003 through December 2013. NAFLD was diagnosed by ultrasonography and its severity was assessed by the NAFLD fibrosis score. At baseline, the prevalence of NAFLD was 40.9%, and the mean estimated glomerular filtration rate (eGFR) was 59.1 ml/min/1.73 m The global prevalence of CKD was estimated at 10.4–13.4%3. End-stage renal disease (ESRD), the last stage of CKD requiring renal replacement therapy, is rapidly increasing worldwide and expected to further increase in the next decade4. There is a substantial interest in identifying novel and potentially reversible causes of progression from early stages of CKD to ESRD.Chronic kidney disease (CKD) is associated with high morbidity and mortality, and is rapidly becoming a major burden to the global health care system6. The global prevalence of NAFLD was estimated at 25.2%7. Furthermore, the consequences of NAFLD extend beyond the liver8. NAFLD is associated with increased risk of type 2 diabetes and cardiovascular disease11, and with increased prevalence and incidence of CKD14. No study, however, has evaluated the role of NAFLD on the decline in kidney function among patients who already have reduced kidney function (CKD stage 3 or more). NAFLD is frequently accompanied by multiple traditional risk factors for CKD progression including obesity and diabetes, but evidence supporting the independent role of NAFLD on CKD progression is still lacking.Nonalcoholic fatty liver disease (NAFLD), characterized by excessive hepatic fat accumulation without significant alcohol intake, use of medications causing fatty liver, or other traditional causes of fatty liver, is the most common cause of chronic liver disease worldwide, especially in developed countriesWe thus investigated the association of NAFLD and its severity with the longitudinal decline in kidney function in a cohort of CKD patients, adjusting for traditional risk factors for CKD.2 . While eGFR at baseline was higher in patients with NAFLD compared to those without NAFLD at baseline , patients with NAFLD had higher proportion of proteinuria than those without NAFLD .The mean  age of study patients was 60.8 11.3) years, and the prevalence of NAFLD at baseline was 40.9% . During follow-up, the age- and sex-adjusted decline in eGFR was greater in patients with NAFLD  compared to those without it .After adjustment for age, sex, and year of visit, the average difference in annual percent change of eGFR between patients with NAFLD and those without NAFLD was −1.09% 5. Future studies will also need to evaluate whether NAFLD resolution can reduce the risk of CKD progression independently of traditional risk factors40.Screening for NAFLD in CKD patients is not routinely performed and there is no specific guideline for screening NAFLD in CKD patients2. Our findings suggest that NAFLD may contribute to CKD progression and may help identify patients with a higher risk of rapid progression. CKD patients may benefit from screening and management of NAFLD, which warrants prospective validation.In summary, NAFLD was associated with declining kidney function in CKD patients independently of established risk factors, and the association was stronger in patients with advanced NAFLD, and in those with proteinuria or with eGFR lower than 45 ml/min/1.73 m2 or proteinuria ≥2 + on urinalysis), and underwent an abdominal ultrasound (US) at baseline, and had at least 1 additional follow up for serum creatinine . We then excluded patients who had any of the following conditions at baseline: history of cancer (n = 34), history of liver cirrhosis, positive hepatitis B surface antigen, or hepatitis C virus antibodies (n = 70), alcohol intake ≥ 30 g/day in men or ≥20 g/day in women (n = 85), or had received a kidney transplant or started dialysis within 1 year after the baseline examination (n = 13). We also excluded patients who had missing information on alcohol intake (n = 113), NFS (n = 3), or less than 6 months follow up (n = 6). Since study patients could have more than one exclusion criteria, the final sample size was 1,525 . The Institutional Review Board of the Samsung Medical Center approved this study and waived the requirement for informed consent as we used only de-identified data routinely collected during health screening visits. All methods were performed in accordance with the relevant guidelines and regulations.We conducted a retrospective cohort analysis of men and women 18 years of age or older who underwent a comprehensive health screening examination at the Samsung Medical Center Health Promotion Center in Seoul, South Korea, from January 2003 to December 2013 . Height, weight, and sitting blood pressure were measured by trained nurses. BMI was calculated as weight in kilograms divided by height in meters squared.Serum creatinine was measured with the kinetic alkaline picrate method (Jaffe method) using automated chemistry analyzers Hitachi 7600  from 2003 to 2009 and Modular DP  from 2009 to 2013. Serum glucose was measured by the hexokinase/glucose-6-phosphate dehydrogenase method. Hemoglobin A1c (HbA1c) was measured by high performance liquid chromatography. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), and gamma-glutamyltransferase (GGT) were measured following the International Federation of Clinical Chemistry method. Urine protein was measured semi-quantitatively by urine dipstick using urine chemistry analyzers, Cliniteck Atlas  or Urisys 2400 (Roche). The Department of Laboratory Medicine and Genetics at Samsung Medical Center has participated in several proficiency testing programs operated by the Korean Association of Quality Assurance for Clinical Laboratory, the Asian Network of Clinical Laboratory Standardization and Harmonization, and the College of American Pathologists.42. Since we had already excluded patients with excessive alcohol use (≥30 g/day for men and ≥20 g/day for women) as well as other identifiable causes of fatty liver at baseline as described in the exclusion criteria, fatty liver was considered NAFLD.Abdominal US imaging was performed using LogiQ E9 , iU22 xMatrix  or ACUSON Sequoia 512  equipments by experienced radiologists unaware of the study aims. Images were captured in a standard fashion with the patient in the supine position with the right arm raised above the head. An US diagnosis of fatty liver was made based on standard criteria, including parenchymal brightness, liver-to-kidney contrast, deep beam attenuation and bright vessel walls2) + 1.13 × impaired fasting glucose/diabetes  + 0.99 × AST/ALT ratio − 0.013 × platelet count (×109/l) − 0.66 × albumin (g/dl)43. Based on NFS, patients with NAFLD were classified as high-intermediate (NFS ≥ −1.455) and low probability (NFS < −1.455) of advanced fibrosis.Among individuals with NAFLD, we used the NFS to assess severity of fibrosis. The NFS was calculated as −1.675 + 0.037 × age (years) + 0.094 × BMI −0.329× (0.993)age; for women with serum creatinine >0.7, eGFR was calculated as 144 × (serum creatinine/0.7)−1.209 × (0.993)age; for men with serum creatinine ≤ 0.9, eGFR was calculated as 141 × (serum creatinine/0.9)−0.411 × (0.993)age; and for men with serum creatinine > 0.9, eGFR was calculated as 141 × (serum creatinine/0.9)−1.209 × (0.993)age. Significant proteinuria was defined as protein ≥2+ on urinalysis. CKD was defined as eGFR < 60 ml/min/1.73 m2 or protein ≥2+ on urinalysis at the baseline examination. The degree of CKD progression was evaluated as the average annual percent change in eGFR from baseline eGFR. For the severity of CKD, patients were further divided into two groups using a cut-off value of eGFR ≥45 ml/min/1.73 m2 vs. <45 ml/min/1.73 m2 (dividing G3a and G3b) at baseline.EGFR was calculated from serum creatinine using the CKD epidemiology collaboration (CKD-EPI) equation45. To account for correlations in eGFR from repeated measurements over time in the same participant, the main analyses consisted of two-level linear mixed models for longitudinal data. We modeled linear trajectories in eGFR with time at the first level, and variations in eGFR trajectories across participants at the second level. We used loge-transformed (eGFR) as the outcome and we estimated the average difference in annual percent change in eGFR  comparing patients with NAFLD to those without NAFLD at baseline. The 95% CIs were obtained from linear mixed models with different intersecting linear trends during follow-up time, interactions between linear time and baseline NAFLD status, and random variations in linear time among participants.We compared serial changes in eGFR among CKD patients with or without NAFLD at baseline using linear mixed models for longitudinal data with random intercepts and random slopesWe used three models with increasing degrees of adjustment to account for potential confounding factors at baseline. Model 1 adjusted for age, sex, and year of visit. Model 2 further adjusted for smoking , alcohol consumption (none and moderate), and BMI. In addition, to evaluate potential mediation of the association between NAFLD and eGFR changes by metabolic factors, model 3 further adjusted for hypertension, diabetes, hyperlipidemia, systolic blood pressure, HbA1c, low-density lipoprotein (LDL) cholesterol, and triglycerides.2 only, proteinuria only, and both. In addition, we performed stratified analyses to evaluate if the association of NAFLD with CKD progression differed in pre-specified subgroups defined by age (<60 vs. ≥ 60 years), sex, smoking (never or former vs. current), alcohol drinking (none vs. moderate), obese (defined as BMI ≥ 25 kg/m2), hypertension , diabetes , hyperlipidemia (defined as high-density lipoprotein (HDL)-cholesterol < 40 mg/dl in men or < 50 mg/dl in women, triglycerides ≥ 150 mg/dl, or the use of lipid-lowering medication), or baseline eGFR (<45 vs. ≥ 45 ml/min/1.73 m2). All reported P values were two-sided and the significance level was set at 0.05. Statistical analyses were performed using Stata version 14 .We conducted sensitivity analyses depending on CKD criteria at baseline: eGFR < 60 ml/min/1.73 mSupplementary Table


Experiments were duplicated for each frequency to ensure outcome reproducibility, and examinations were blinded for quality assurance. All rats survived without significant clinical signs until the end of experiments. Some changes in body weight between the MF‐exposed and control groups were observed over the course of exposure, although the directions of the changes were inconsistent and not statistically significant after subchronic exposure. There were significant differences between MF‐exposed and control groups in some organ weights and parameters in hematology and clinical chemistry, but these were minor in magnitude and not repeated in duplicate experiments. Histopathological findings reflecting toxicity were sporadic. Frequencies of other findings were similar to historic data in this rat strain, and findings had no specific relationship to changes in organ weight or parameters of hematology and clinical chemistry in each animal. The changes observed throughout this study were considered biologically isolated and were attributable to chance associations rather than to MF exposure. The results, in particular the histopathological evidence, indicate an absence of toxicity in IF MF‐exposed rats and do not support the hypothesis that IF MF exposure produces significant toxicity. Copyright © 2015. The Authors. Journal of Applied Toxicology Published by John Wiley & Sons Ltd.Despite increasing use of intermediate frequency (IF) magnetic fields (MFs) in occupational and domestic settings, scientific evidence necessary for health risk assessments of IF MF is insufficient. Male and female Crl:CD(SD) rats (12 per sex per group) were exposed to 20 kHz, 0.20 mT or 60 kHz, 0.10 mT(rms) sinusoidal MFs for 22 h day −1 for 14 days (acute) or 13 weeks (subchronic). MF‐exposed rats did not exhibit significant and reproducible changes in body and organ weights, hematology, clinical chemistry and histopathology. Our results indicate that IF MF exposure does not carry a significant health risk to mammals.Despite increasing use of intermediate frequency (IF) magnetic fields (MFs) in occupational and domestic settings, scientific evidence is insufficient for IF MF health risk assessments. Rats were exposed to 20 kHz or 60 kHz sinusoidal MFs for 22 h day EHC 238 stated that MFs < 100 kHz have stimulating effects on excitable cells present in tissues such as muscles and nerves. Based on the criteria, the International Commission on Non‐Ionizing Radiation Protection (ICNIRP) set exposure guidelines for MF of < 100 kHz ICNIRP, , while e. EHC 238et al.  remarkedet al. conducted acute and subchronic toxicity studies on very intense and graduated IF MF exposures. They exposed male and female B6C3F1 mice to 0.08, 0.28 or 1.0 mT sinusoidal MFs at 10 kHz for 14 or 90 days, and failed to demonstrate any biological changes related to toxicity produced by IF MF exposure. The modeled 10 kHz sinusoidal MF is employed in inductively coupled power transmission (or wireless/contactless power transfer) systems that are used to transfer electric power efficiently through large gaps between pairs of coils  or subchronic (13 weeks) exposure to IF MFs produced toxicological effects in the rat. These exposure periods were chosen to be comparable to a previous toxicity study of 10 kHz MF  group.This study comprised 10 independent (six acute and four subchronic) experiments and each employed the same experimental protocol. Two of the six acute experiments were conducted as sham–sham studies, where animals were placed in each facility but not exposed to MF, to address concerns regarding the equivalency of the two facilities. For each exposure period (14 days or 13 weeks), two identical experiments (experiments 1 and 2) were conducted using 20 kHz or 60 kHz MF exposure, respectively, to confirm the reproducibility of the outcomes. Irreproducible biological responses to MFs have become an issue in this research field due to inter‐ and intralaboratory variability, and this was of particular concern in our studies due to the limited number of exposure doses in each frequency.The institutional Animal Experiment Committee of the Environmental Science Research Laboratory, Central Research Institute of Electric Power Industry, approved all of the animal experimental procedures used in this study in accordance with the Committee's guidelines under the Japanese Law Concerning the Protection and Management of Animals. Following the Organization for Economic Co‐operation and Development OECD,  and InteFive‐week‐old SPF/VAF Crl:CD(SD) rats were purchased from Charles River Laboratories Japan Inc. . The rats were quarantined for 1 week in the barrier system at the magnetic field exposure facility, where background MFs were extremely low (< 0.001 μT(rms) at 20 kHz and 60 kHz, and 0.03 μT(rms) at 50 Hz). Room air was HEPA‐filtered, and room temperature and humidity were maintained at 23 ± 2 °C and 50 ± 20%, respectively. The light cycle was 12 h/12 h (on from 07.00 to 19.00 h). The rats were housed in polycarbonate cages  and allowed access to food pellets  and water  ad libitum. Commercial wood chips  were used as bedding material. Cage positions within and among the racks were changed twice per week to minimize the influence of subtle environmental differences, such as illumination and airflow. On the last day of the quarantine period, the male and female rats were weighed and randomly assigned to either the MF‐exposed (12 per sex) or control (12 per sex) groups, stratified by body weight.et al., The MF exposure facility has been explicitly described in previous papers  for 14 days or 13 weeks. The electric current in the exposure coils, which is linearly related to MF production, was monitored continuously and recorded every 10 min by a data logger. Animal care and clinical observations were conducted daily during the 2 h when animals were not exposed to MFs. This exposure regime was maintained throughout the course of the study. Rats were individually housed and specially made polycarbonate cage lids, glass water nozzles and glass pellet containers were used instead of the standard metal versions to avoid induced current during MF exposure.Rats were exposed to 20 kHz, 0.20 mT(rms) MFs, to 60 kHz, 0.10 mT(rms) MFs, or to the facility with no MF (sham‐exposed) for 22 h dayAnimals were observed daily for morbidity and clinical signs of toxicity throughout the exposure periods, and abnormal signs were recorded. Body weights were measured at the initiation and termination of exposure periods, on the first, third and seventh days of acute exposure, or once a week during subchronic exposure. Food and water consumption were not measured because the flat cage lids did not accommodate the containers required for food and water consumption measurements. The animals were fasted overnight before necropsy and blood collection.On the termination of exposure, all animals were anesthetized by isoflurane inhalation. Blood samples were drawn from the abdominal aorta and placed into tubes containing the anticoagulant, EDTA‐2K. An automated analyzer  was used to measure hematological parameters, including erythrocyte count, hemoglobin concentration, hematocrit percentage, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration, platelet count, reticulocyte count (RET) and leukocyte count. In subchronic toxicity studies, differential leukocyte counts and morphological evaluations of blood cells were performed under the microscope on blood smears stained with Wright–Giemsa.Aliquots of blood samples for clinical chemistry examination were placed in tubes containing heparin sodium and centrifuged, and the plasma was separated. Clinical chemistry variables were measured using an automated chemistry analyzer . Parameters included blood glucose (BGLU), total protein (TP), albumin, albumin/globulin ratio, total cholesterol (TCHO), triglycerides, total bilirubin, creatinine (CRE), blood urea nitrogen, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALKP), calcium and inorganic phosphoric acid (IPHS). Plasma sodium, potassium and chloride were measured by an electrolyte analyzer .Complete gross postmortem observations were performed on all animals, and abnormalities were recorded. Absolute organ weights were measured, and organ weights relative to the body weight at necropsy were calculated for the brain, heart, lung with bronchus, thymus, liver, kidneys, adrenal glands, spleen and either the testes or the ovaries and uterus.The following organs and tissues were obtained from all animals: the heart, aorta, spleen, thymus, bone marrow (sternum and right thigh bone), submandibular lymph node, mesenteric lymph node, trachea, lung with bronchus, salivary gland , parotid gland (left), tongue, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, liver, pancreas, kidneys (left and right), urinary bladder, testes (left and right), epididymides (left and right), seminal vesicle, prostate gland, ovaries (left and right), uterus, vagina, brain, spinal cord, sciatic nerve (right), eyes (left and right), pituitary, thyroid with parathyroid gland, adrenal glands (left and right), skeletal muscle (right thigh), skin (back) and mammary gland. These tissue samples were fixed with 10% neutral buffered formalin solution, embedded in paraffin, sectioned and stained with hematoxylin and eosin for microscopic examination. All gross lesions, as defined by the study pathologist, were also included in the examination. All examinations and measurements after exposure, as well as statistical analyses, were conducted by an independent GLP‐licensed laboratory that was blind to the treatment conditions.t‐test (two‐tailed test) was used for statistical comparison of the MF‐exposed and control group data, with a significance threshold of P < 0.05. The data analyzed included body weight, organ and tissue weight, ratio of organ or tissue to body weight, and parameters from hematological and clinical chemistry examinations. Fisher's exact test was applied to evaluate group differences in mortality, clinical findings, gross pathological findings and histopathological findings, with differences between groups considered significant at P < 0.05 (one‐tailed test). Statistical analyses were performed using SAS  and EXSUS  software.Data were presented as the group means ± SD. Welch's In the acute exposure experiments, including the sham–sham experiments, no mortality or abnormal clinical findings were observed in MF‐exposed or control groups of either sex after exposure to 20 kHz or 60 kHz MFs. The body weights of the rats in the MF‐exposed and control groups were similar over the 14 days of experimentation.In the subchronic exposure experiments, one MF‐exposed female rat in experiment 1 of the 60 kHz MF exposure study exhibited a muzzle nodule after 7 weeks of exposure; and this rat grew normally and continuously gained weight until the termination of exposure. Other than this incidence, no mortality or abnormality was recorded in any animal. The body weights of male and female rats are shown in Table In sham–sham experiments, the male rats in the 60 kHz MF facility had MCV and MCH significantly lower than those in the 20 kHz MF facility. However, the differences were relatively small  and not reproduced in replicate experiments or in female rats. No other hematological variables showed significant differences between the two sham–sham groups. These results indicated that environmental differences between the exposure facilities did not affect hematology in the rats.2 µl−1), and this observation was not reproduced in replicate experiments or in male rats. Other hematological parameters assayed did not show significant changes between groups in any acute exposure experiment.In acute exposure experiments, the 60 kHz MF‐exposed female rats had white blood cells significantly lower than that of the control rats. However, the means showed large SDs and a relatively small difference , most were not common to both sexes, and none were reproduced in replicate experiments. These isolated changes were observed in 17 parameters, but no other significant group differences were observed for clinical chemistry parameters. Overall results did not indicate that rats bred in the two facilities differed in clinical chemistry.−1) and TCHO (63.4 ± 13.9 vs. 50.8 ± 5.9 mg dl−1) levels were increased in 20 kHz MF‐exposed male rats in comparison with control rats, and ALKP (590 ± 97 vs. 691 ± 130 IU l−1) was decreased. In addition, AST (62.3 ± 3.6 vs. 68.8 ± 9.7 IU l−1) was decreased in male rats exposed to 60 kHz MF. However, these changes in clinical chemistry parameters were not reproduced in replicate experiments or in female rats. No other significant differences between the study groups were observed.In acute exposure experiments, TP  than those included in the present study, Nagase et al.  reportedIn sham–sham experiments, a kidney cyst was observed in a male rat held in the 20 kHz MF facility. In the animals in the 60 kHz MF facility, kidney hypoplasia and dilatation of the uterus were observed in a female rat, and brown foci were found in the liver of a different female rat.In 20 kHz MF acute exposure experiments, dilatation of the renal pelvis and a uterine cyst were found in a female MF‐exposed rat, and a control female had a uterine cyst. In 60 kHz MF exposure experiments, nodules in the spleen and two kidney cysts were recorded in three male rats in the MF‐exposed groups. One control male rat had liver congestion and another had a kidney cyst. One control male rat exhibited a localized loss of fur. Overall, the frequencies of gross pathological findings in sham–sham and acute exposure experiments were low across animals in the control and MF‐exposed groups.Pathological changes in the subchronic exposure experiments were also very low in frequency, and the changes showed no association with the MF exposure, specific organs or the sex of the animal. These changes were brown foci in the brain, a unilateral kidney cyst, caudate lobe torsion and abnormal lobulation in the liver, muzzle nodules and a pituitary cyst in the 60 kHz MF‐exposed rats. No change was observed in the 20 kHz MF‐exposed rats. Control rats exhibited diverticulum in the ileum and swelling of the medial iliac lymph node. Observations of brown foci in the brain, nodules in the muzzle and swelling of the medial iliac lymph node were toxicologically noteworthy but no statistically significant group differences in the frequencies of any of the findings were evident.In sham–sham experiments, there were two statistically significant differences in relative organ to body weights between the study groups. Male rats in the 60 kHz MF facility had a lower relative lung weight than male rats in the 20 kHz MF facility (0.376 ± 0.023 vs. 0.399 ± 0.031%), and female rats in the 60 kHz MF facility had a higher relative spleen weight than female rats in the 20 kHz MF facility (0.226 ± 0.028 vs. 0.204 ± 0.016%). No other significant differences were observed. These results indicated that the breeding environments of the exposure facilities did not influence relative organ weights.In the acute exposure experiments, four statistically significant differences were found. The relative heart and adrenal gland weights in male rats in the 20 kHz MF‐exposed group were higher than in the control group . The relative weights of the epididymides and ovaries in rats exposed to 20 kHz MF were lower than the control rats . No other significant differences were observed.Table The histopathological findings are shown in Table Toxicologically relevant findings in MF‐exposed rats included astrocytoma in the brain, moderate congestion and severe necrosis in the caudate lobe in the liver, and squamous cell papilloma in the skin. In control rats, moderate cellular infiltrations in the pancreas and increased numbers of lymphocytes in the sinus of the medial iliac lymph nodes were found. However, these were isolated events, which did not occur uniformly within treatment groups.We investigated the toxicity of IF MF exposure in rats because there are insufficient biological studies to allow accurate health risk assessment of such exposure, which occurs in the proximity of widely used commercial and domestic appliances. For 14 days or 13 weeks, groups of 12 male or female rats were exposed to either 20 kHz, 0.2 mT(rms) or 60 kHz, 0.1 mT(rms) sinusoidal MFs, or sham‐exposed, and a series of pathological examinations was performed in a treatment‐blind manner. Experiments were performed in duplicate in each frequency and exposure period.et al., In sham–sham experiments, except for some observations that showed no consistency in replicate experiments, the body and organ weights and hematological and clinical chemistry parameters were similar in the animals in the 20 kHz and 60 kHz MF‐facilities. This result supported the equivalency of the animal breeding environments in these facilities. Furthermore, the validity of this study was supported by the comparison that sham–sham and control data were similar and within historical data ranges reported for Crl:CD(SD) rats  exposure of juvenile rats to a very intense (3.8 mT(rms), sinusoidal), 21 kHz MF for 14 days did not influence hematological, serological or immunological parameters. While the exposure time on each day was less than one‐twentieth of that used in the present study, the MF to which the animals were exposed was almost 20 times stronger and their results were in accordance with the hematological and clinical chemistry observations in our acute toxicity studies. Their study was designed to expose the entire rat body to an intense and uniform MF. Consequently, the coils and the designated exposure space were rather small relative to the rat body size. The rats were individually restrained in a plastic chamber, which limited the daily exposure time. The present study aimed to identify the possible health hazards associated with IF MFs and the target organs affected. Therefore, we also exposed the whole body of the rats to uniform MFs, but the animals were able to move freely in a cage while being exposed continuously for 22 h day−1 for up to 13 weeks, to maximize the opportunity for hazard identification.Recently, Ushiyama et al.  reportedet al., et al.  or 60 kHz, 0.1 mT(rms) sinusoidal MFs showed no toxicity in rats. No MF frequency‐specific response was observed in any experiment, and these results do not support the hypothesis that IF MF exposure is toxic to mammals. As stated in the introduction, negative results were reported following acute and subchronic toxicity studies of 10 kHz MFs at up to 1.0 mT(rms) in mice (Robertson , et al.  and Lee  (et al. , 2010 al (et al.  reportedThe authors did not report any conflict of interest.


Organic–inorganic lead halide perovskites are promising optoelectronic materials resulting from their significant light absorption properties and unique long carrier dynamics, such as a long carrier lifetime, carrier diffusion length, and high carrier mobility. These advantageous properties have allowed for the utilization of lead halide perovskite materials in solar cells, LEDs, photodetectors, lasers, etc. To further explore their potential, intrinsic properties should be thoroughly investigated. Single crystals with few defects are the best candidates to disclose a variety of interesting and important properties of these materials, ultimately, showing the increased importance of single‐crystalline perovskite research. In this review, recent progress on the crystallization, investigation, and primary device applications of single‐crystalline perovskites are summarized and analyzed. Further improvements in device design and preparation are also discussed. Since then, materials with the formula ABX3 and similar crystal structures as CaTiO3 are defined as perovskites. Depending on the ionic or elemental radii of the A, B, and X elements, the crystal structure could change from a high‐symmetry cubic phase to a tetragonal phase or to a low‐symmetry orthorhombic phase  and CH7N2+ (FA), having similar sized inorganic ions, can also form perovskite materials, called organic–inorganic hybrid perovskites. The organic group size will determine whether the crystal structure is 3D or 2D. In this review, unless otherwise specified, perovskite represents an inorganic–organic hybrid perovskite. These materials have been investigated since the 1970s,3NH3PbI3 and CH3NH3PbBr3 as absorbers in sensitized solar cells brought them into the scientific spotlight.Organic groups, such as CHJ–V) curve hysteresis,Furthermore, additional optoelectronic properties have been discovered, including long balanced hole–electron diffusion length,Over the last 2 years, an increasing number of researchers have begun studying single‐crystalline perovskites, including crystal preparations,2In this section, the crystal growth strategies and basic principles for single‐crystalline perovskites and single‐crystalline thin films are summarized. Furthermore, the optoelectronic properties and corresponding applications of perovskite crystals are included and discussed, which includes properties in optics, carrier dynamics and applications in solar cells, photodetectors, and lasers.2.1N,N‐dimethylformamide (DMF)/γ‐butyrolactone (GBL)/dimethyl sulfoxide (DMSO)‐based solutions, and an antisolvent method.Methods used to grow single crystals are classified by growth environment and include solid growth, liquid growth, and vapor growth. To date, all reported perovskite crystals have been prepared with the liquid growth method.2.1.1Figure2+ and CH3NH3+ from ≈100 °C to room temperature, allows for MAPbX3  crystals to be obtained. Except for MAPbI3, the temperature should be controlled above a temperature of 40 °C,4PbI6·2H2O will be formed. Without careful control of the crystal growth process, it is hard to obtain large and regular‐shaped crystals. Recently, this method has been modified and used to grow larger crystals with a regular shape.The perovskite solubility in a HX‐based solvent will decrease with a lower solution temperature Figure2a.61 F3NH2 and HI blending solution with MAI, the Tao group also produced large crystals with sizes exceeding 10 mm 3, MASnI3, and FAPbI3, have also been successfully prepared by this method.The Huang group applied a top‐seeded solution growth (TSSG) to prepare large single crystals Figure d–f.21 Fi2.1.23  in different solvents.3 and MAPbI3 crystals called the antisolvent vapor‐assistant crystallization (AVC) method  were dissolved in DMF to form the precursor solution. The solution and dichloromethane (DCM) were then sealed in a closed vessel and stored. The DCM will slowly evaporate, and its vapor will diffuse into the precursor solution because DCM mixes well with DMF. The diffusion of DCM in DMF will consume some DMF solvent. Since PbBr2 and MABr cannot dissolve in DCM, the consumption of DMF by DCM will increase the concentration of MABr and PbBr2 in the solution. This will result in the formation of MAPbBr3 crystals. Recently, the Huang group found that it was difficult to get high‐quality MAPbBr3 with a 1:1 mole ratio of MABr and PbBr2 because of their different solubility's in DMF. The PbBr2 and MABr molar ratio of 0.8 can produce high‐quality crystals with higher carrier mobility and lifetime.In 2014, the Cheng and Seok groups found antisolvent‐assisted crystalline methods, which are called the fast crystallization deposition2.1.33 in GBL drops almost twofold as the temperature of the solution increased from 60 to 100 °C.3 in the solution could be controlled by adjusting the temperature. During the heating of the saturated precursor solution, perovskite crystals will precipitate at the bottom of the solution since the precursor concentration is larger than what the solubility allows. This method quickly produced high‐quality perovskite crystals, with a 5 mm‐sized crystal obtained in 3 h.3, DMF works best for MAPbBr3 and GBL works best for MAPbI3.3, it was difficult to obtain MAPbI3 crystals. Furthermore, by repeating and carefully controlling the ITC process for several times, 70 mm‐sized perovskite single crystals could be obtained ,3NH3CaI3,3NH3SnI3,3)3SnBr3,3)3SnCl3,2AgBiBr6,2PdBr6)3, which possesses higher thermal stability, could also been fabricated using the Bridgman method, with the highest furnace temperature approximately 1470 °C. The obtained CsPbBr3 shows unprecedent carrier mobility as high as 2290 cm2 V−1 s and a trap density as low as 1.9 × 109 cm−3, demonstrating its high electronic quality and crystal quality.Except organic–inorganic lead halide perovskites, inorganic lead halide perovskites ,11 to ≈109 cm−3 )*hν)2 represents the product of the absorption coefficient and photon energy. Much longer light absorption regions could absorb a larger number of photons and may produce higher photocurrents using the same material. Except for the extended absorption region, mix‐halide perovskites have also been successfully prepared, which show tunable light absorption and photoemission across the entire UV–vis–NIR light region.3, MAPbBr3, MAPbI3 are located at 402, 537, and 784 nm, respectively, which are smaller than the absorption onsets positions. The difference, which may have arose from the direct and indirect band gap profile of perovskite,Compared to thin films, single crystals show extended light absorption (redshift).2.3Semiconductor materials have very special optical and electronic properties. They are the base of sensors, detectors, CPUs, etc. Hybrid perovskite possess great optoelectronic properties, as summarized in last section, and shows great potential in solar cells, lasers, photodetectors, etc. The typically applications of these materials are summarized in the following sections.2.3.1FigureAs a semiconductor material with a large absorption coefficient and long carrier diffusion length, solar cells should be the best model device to show their advantages, which are similar to a perovskite single crystal. However, there are obstacles in direct usage of a large single crystal. The Huang group built a solar cell device, including a semitransparent Au/perovskite/Ga device Figure6a,b,21Jsc, Voc, and FF decreased dramatically because the charge carrier cannot be collected efficiently. Recently, using space confine strategy, the Kuang group successfully obtained a microscale MAPbBr3 single‐crystalline film with similar results as the Bark group.3 because they have similar electronic and carrier dynamic properties. However, to date, it is difficult to prepare thin single‐crystalline MAPbI3 films at the 5 µm scale.The Bakr group developed a CTAC strategy Figure d to prepTo overcome the drawbacks of this high intrinsic resistance, the Huang group designed and prepared a lateral structured single‐crystalline perovskite solar cell Figure e,f.128 I2.3.2Photodetectors have the potential to transform background electromagnetic waves into electronic signal and provide useful and important data for automatic control, information transportation, medical diagnosis, etc. Depending on how the generated carriers are utilized, current photodetectors can be classified as a photoconductive or photovoltaic. Next, the basic mechanism of single‐crystalline photodetector devices will be introduced.Photoconductive Detectors: In a photoconductive photodetector, symmetrical electrodes , a variation of resistance will result in current changes. Turning the light on and off will make the carrier concentration of the device vary dramatically, which will result in a large difference in device resistance (R) and corresponding current (I)  Figure b. This p9 cm−3). This is a major reason for the observed large resistivity . After being excited by photons, the carrier concentration will increase dramatically, which will alter the resistivity of the single‐crystalline perovskite. This results in a large difference in device current, which could allow single crystals to be used in photoconductive detectors. Equation R is the responsivity, Iph and Idr are the current of device under light illumination and dark, Pin is the power of the incident photons, s is the effective area of device, and nph and ndr are the carrier concentration in light and dark, respectively. Under the same photocarrier generation rate with a lower intrinsic carrier concentration, larger responsivity values are seen for the photodetector. From this point of view, the single‐crystalline perovskites are great candidates for photodetector applications due to their extremely low intrinsic carrier concentration. Using the simple lateral electrode configuration described in Figure 3‐based device achieved a responsivity as high as 953 A W−1 and an EQE as large as 2.22 × 105% under a 1 V bias voltage and 2.12 nW cm−2 532 nm light illumination.−2 light illumination. In addition to high responsivity and EQE, the simple‐structured photodetector also shows a very fast response time, rise time, and decay time, with values as small as 74 and 58 µs, respectively; other groups have reported similar results  Te single‐crystal X‐ray detector and 4 times higher than that of the currently used α‐Se X‐ray detector. The lowest detectable dose was as low as 0.5 µC Gyair s−1, which is lower than required by regular medical diagnostics. This new X‐ray detector could reduce the radiation dose during medical and security checks, with a very fast response speed of approximately 730 µs.The Huang group first developed Au/mix‐halide perovskites/Ga structured p–i–n type detectors,s Figure g,h.106 A2.3.32, a polycrystalline MAPbI3 film still shows a strong amplified spontaneous emission, demonstrating its high optical gain.−2.Q factor. This may be a result of the trap state generated by the grain boundaries, which will broaden the photoluminescence.Hybrid perovskites are high‐gain materials for lasing because of their high absorption coefficient, high photoluminescence quantum yield, slow Auger recombination rate, long carrier diffusion length, low defect density, etc. Sandwiched between spiro‐OMeTAD and TiOQ factor. Using a MAPbI3 nanoplate, they can achieve 780 nm laser emission with a threshold of 37 µJ cm−2 and Q factor of ≈650 3 nanowire lasers with an even smaller threshold of ≈1 µJ cm2 and a Q factor exceeding 2000 , the successive improvements of perovskite film quality, such as film coverage, grain size, and surface passivation, offer more incremental PCE improvements. The basic properties, including the charger carrier lifetime, light absorption region, and carrier diffusion length, single‐crystalline perovskites have shown great advantages in laser and photodetector applications and have the potential to be better candidates for solar cell applications. However, there are few reports on highly efficient single‐crystalline perovskite solar cells. To further obtain high efficiency single‐crystalline solar cells, three problems should be considered: the large light absorption coefficient, the intrinsic low conductivity, and the device structure. The large light absorption coefficient determines that the position of the photon‐generated carriers is close to the top surface of the light incident side. To successfully extracted the carriers, (1) a thin single‐crystalline perovskite film  should be prepared; (2) thick perovskite films should be doped to make the carrier concentration high enough, which can reduce the ohmic loss during the carrier collection in a thick single‐crystalline perovskite film; (3) the interface should be manipulated to achieve increased electronic contact, as well as less defects; and (4) in addition to material optimization, new device structures, such as lateral design, should be applied for special morphologies and electrical properties. These great optoelectronic properties also endow perovskite materials with potential application in high‐speed photon detection because of their high carrier mobility and long carrier lifetime, highly sensitive X‐ray or γ‐ray detection because of Pb, as well as large mobility and lifetime product, efficient lasers, etc. Further developments in these applications should be focused on: (1) device integration to achieve a highly efficient X‐ray detection system to alleviate bio‐damage during safety and medical examination and (2) integrating perovskite crystals into a laser cavity to achieve a low threshold for an energy efficient laser device with whole spectrum coverage.The authors declare no conflict of interest.


Osteoporosis is a potential metabolic complication of diabetes mellitus (DM). Therefore, patients with DM should have adequate osteoporosis knowledge and beliefs in order to get engaged in osteoporosis preventive behaviors. The objective of this study was to assess osteoporosis knowledge and beliefs among diabetic patients.This was a cross sectional study carried out at Al-Makhfiah governmental primary healthcare unit in Nablus, Palestine from September 2016 to December 2016. The tools used to assess knowledge and beliefs were Osteoporosis Health Belief Scale (OHBS) and the Osteoporosis Knowledge Test (OKT) respectively.p = 0.037), exercise (p = 0.043), and OKT score (p = 0.021) than males. Regarding OHBS, female participants had significantly higher belief score of susceptibility (p <  0.01) and seriousness (p <  0.01) of osteoporosis compared to males.Three hundred diabetic patients were interviewed regarding their knowledge and belief about osteoporosis. The study sample included 192 (64.0%) females. Mean ± standard deviation (SD) of the participants was 58.5 ± 9.3 years. Regarding co-morbidities, 229 (76.3%) had at least one co-morbidity other than DM. The majority of participants incorrectly answered 19 out of 32 questions of OKT scale. The mean OKT score was 13.5 ± 4.2 indicating poor osteoporosis – related knowledge. Females had significantly higher nutrition  contains supplementary material, which is available to authorized users. Diabetes mellitus (DM) is a chronic medical illness that requires careful and continuous adherence to and implementation of daily treatment regimens, dietary restrictions, and change in life style that can be demanding and difficult to implement by some diabetic patients . AccordiLong term microvascular and macrovscular complications of DM are well known and include nephropathy, retinopathy, neuropathy, acute coronary syndrome, and stroke . AnotherThe exact pathogenesis of diabetes-induced risk of osteoporosis is still controversial. For patients with type I DM, the absolute deficiency of insulin and the potential presence of autoimmune diseases can lead to bone fragility and poor bone health –20. In tWorldwide literature in osteoporosis knowledge focused mainly on knowledge and belief in females of different age categories –28. OsteThis was a cross sectional study. Participants in this study were diabetic patients attending Al-Makhfiah primary healthcare unit in Nablus, north of Palestine. The study was carried out for four months, from September to December 2016. Al-Makhfiah clinic offers primary healthcare services and medications for individuals with chronic diseases who have governmental medical insurance. The number of diabetic individuals who attend Al-Makhfiah clinic on regular basis is less than 700. Recruited participants for this study were given full information regarding the purpose of the study and were asked to give informed consent before the interview. Participants were asked to complete a questionnaire through a face-to-face interview. The participants were interviewed by two co-authors who are senior medical students (G.I and Y.A). Participants in this study were limited to diabetic patients regardless of type. Participants who reported being diagnosed with osteoporosis or had previous bone fracture or currently taking calcium/vitamin D were excluded.To estimate the sample size, a margin of error less of than 5% and a confidence level of 95% were entered in Raosoft calculator . The totTools used to collect the required data were the “Osteoporosis Health Belief Scale” (OHBS) and the “Osteoporosis Knowledge Test (OKT)”. Both were obtained from the developer with permission to use in Palestine . The OKTThe OKT is a 32-multiple choice instrument and has two subscales, a nutrition, and an exercise - related subscale. The two subscales had 14 items in common. In scoring the OKT, correct answers were coded as 1 while incorrect answers were coded as 0. Total score for OKT scale ranges from 0 to 32. The range for nutrition subscale was from 0 to 26 while that for exercise subscale ranges from 0 to 20. For each interviewed patient, scores of the OKT and the two subscales were analyzed as continuous variables and no categorization of scores was used. Examples of questions in the nutrition and exercise subscales include: “best source of calcium?: apple, cheese, cucumber”, “best way to reduce osteoporosis: swim, walk, stretching?”.The OHBS consists of 42 items divided into seven subscales: susceptibility (items 1–6), seriousness (items 7–12), benefits of exercise (items 13–18), benefits of calcium intake (items 19–24), barriers of exercise (items 25–30), barriers of calcium intake (items 31–36), and health motivation (items 37–42). The internal reliability testing for the OHBS yielded a Cronbach’s alpha value of 0.720. The internal reliability of the seven OHBS subscales were 0.83, 0.71, 0.73, 0.72, 0.68, 0.69, and 0.66 for susceptibility, seriousness, benefits of exercise, benefits of calcium intake, barriers of exercise, barriers of calcium intake and health motivation subscales respectively. The OHBS is scored by awarding 5 for responses of “Strongly Agree” to 1 for “Strongly Disagree” for each item. Since there are 6 items in each subscale, the possible score for each subscale ranges from 6 to 30 with higher scores indicating higher belief in the tested subscale. The total score for the whole OHBS scale ranges from 42 to 210. Total score for each OHBS subscale was analyzed separately and as a continuous variable with no categorization of scores. The Arabic version of the OHBS subscales is shown in the additional file.Examples of questions in the OHBS subscales include: “chances of getting osteoporosis high”; “when think about osteoporosis, get depressed”; “regular exercise helps build strong bones”; “taking enough calcium cuts chance of broken bones”; “have no place where can exercise”; “to eat more calcium foods have to give up other I like”; “look for new information related to health”.To maintain the rights of participants in the study an Institutional Review Board (IRB) application was submitted to An-Najah National University. The IRB approved the study and asked for verbal consent from the participants and not a written consent because the study does not involve any invasive procedure. Furthermore, the Palestinian Ministry of Health approved the study and gave permission to authors to conduct the study after obtaining verbal approval from the participants. Before the collection of data, the participants were handed an informed consent that included information about all aspects of the study. Developers of the questionnaire gave permission to use the tool for the study.Descriptive statistics including mean ± standard deviation (SD) or frequency (percentage) were presented for each variable, stratified by gender. Scores for all subscales were tested for normality using Kolmogrov - Smirnov test and were found to be not normally distributed. In Bivariate analysis, Mann-Whitney U test was used for ordinal variables while correlation between total knowledge scores and various belief subscales were carried out using Spearman correlation.Three hundred diabetic patients were recruited; 192 (64%) were females. The mean ± SD age of the participants was 58.5 ± 9.3 years. The majority of participants were living in urban areas . The majority also reported being currently married . For the level of education, the majority  did not have a college degree. Regarding occupation, the majority  of participants were unemployed. Participants had mean ± SD duration of DM of 11.3 ± 8.0 years. More than half of the participants  were insulin users and two thirds of the participants were hypertensive. Regarding co-morbidities, 229 (76.3%) of studied patients have at least one co-morbidity other than DM and 91 patients (30.3%) had retinopathy.p <  0.01). Furthermore, females had significantly shorter duration of diabetes than males . Significant differences between males and females were also shown in place of residence (p = 0.022), marital status (p <  0.01), level of education (p <  0.01), employment status (p <  0.01), use of insulin (p = 0.012), and presence of hypertension (p <  0.01).Comparison of the demographic and clinical characteristics between female and male participants Table  showed tp = 0.021), higher exercise knowledge score (p = 0.043), and higher nutrition knowledge score (p = 0.037)  and question 10   and perceived seriousness  of osteoporosis when compared to male participants. No significant difference was found between males and females in other belief subscales (Table When belief scores were examined by gender, female participants showed significantly higher scores in perceived susceptibility (17.4 ± 4.1 for females versus 15.8 ± 3.8 for males; This study was carried out to assess osteoporosis knowledge and beliefs in diabetic patients in a governmental diabetic primary health care center in Nablus, Palestine. The levels of osteoporosis knowledge and perception were poor necessitating preventive educational programs to increase awareness about risks of osteoporosis. In particular, these preventive measures could target young diabetic females and those who are unemployed since they have poor perception toward osteoporosis.Our results regarding poor knowledge and perception were expected given that the majority of participants were not well educated. Studies from non-Arab Middle Eastern countries such as Turkey and Iran highlighted that educational level of females can determine the extent of awareness of osteoporosis , 46. OurIn our study, both males and females did not believe that they were susceptible to osteoporosis. Furthermore, participants did not have a high perception of seriousness of osteoporosis. The relatively poor perception toward susceptibility and seriousness of osteoporosis could be due, low awareness and/or poor health promotion system in Palestine. In Palestinian ministry of health had launched several awareness workshops and campaigns to fight several diseases such as breast cancer, smoking, childhood vaccination. However, osteoporosis awareness campaigns and workshops and research are absent, not only in Palestine but in Arab countries as well . Our finTo the best of our knowledge, no previous studies about knowledge or belief about osteoporosis among diabetic patients had been published from Arab countries, and therefore, comparison of our results with published studies in other Arab communities was not possible. However, several studies have been published about knowledge and belief about osteoporosis among different groups of women or particular types of patients , 48–53. Our study has limitations and points of weaknesses. The cross-sectional design and convenience sampling technique used to recruit the participants could have created some bias. Future studies need to include larger sample of DM patients with various educational background and from different regions in Palestine. Furthermore, future studies need to shed light on daily practices of patients with regard to nutrition such as protein and calcium intake to link belief with daily practices.Overall, diabetic patients had poor osteoporosis knowledge, moderate perception of susceptibility and seriousness of osteoporosis despite that they have relative high score in health motivation subscale. Implementation of awareness and educational programs among diabetic patients might increase preventive practices and measures toward osteoporosis. Such preventive practices need to focus on calcium rich nutrition and regular exercise.


Aim. The aim of the current study is to determine the practice patterns of physiotherapists for patients undergoing thoracic surgeries in India. Materials and Methodology. A cross-sectional survey was conducted across India in which 600 questionnaires were sent in emails to physiotherapists. The questionnaire addressed assessment and treatment techniques of thoracic surgery. Results. A total of 234 completed questionnaires were returned with a response rate of 39%, with the majority of responses received from Telangana, Karnataka, and Andhra Pradesh. More than 90% of the responders practiced physical examination, chest expansion, chest X-ray, ABG analysis, pulmonary function test, and SpO2 (oxygen saturation) as the assessment measures in both the pre- and the postoperative phase. Breathing exercises, incentive spirometry, thoracic expansion exercises, coughing and huffing, positioning, and modified postural drainage are found to be commonly used physiotherapy interventions, both pre- and postoperatively, with a response rate of more than 90%. A response rate of more than 84.6% indicated that patients are made to dangle their lower limbs over the edge of the bed on the 1st postoperative day. Mobilization, such as walking up to a chair, sit to stand exercises, and perambulation within the patient's room, was started on the 2nd postoperative day, as stated by more than 65% of the physiotherapists. Staircase climbing was started on the 5th postoperative day. The most commonly used functional evaluation prior to discharge was 6-minute walk test. This was, in fact, practiced by 77.4% of the physiotherapists in their clinical settings. Conclusion. The most predominantly employed assessment measures included were physical examination, chest expansion, ABG analysis, pulmonary function test, chest X-ray, SpO2 (oxygen saturation), peripheral muscle strength, and cardiopulmonary exercise. The physiotherapy interventions most commonly used were breathing exercises, thoracic expansion exercises, incentive spirometry, and coughing and huffing techniques, in both the pre- and the postoperative phase. Thoracotomy is the most frequently used open procedure in thoracic surgery and is performed primarily for lung resections . FollowiThe changes in lung function occurring may be both procedure- and patient-related and occur intra- and postoperatively. Many of these changes are expected following surgery and are transient and self-limiting. These changes include characteristics reduction in lung volume which is primarily restrictive in nature, reduction in functional residual capacity, slowing of mucociliary clearance, and abnormalities in gaseous exchange .This leads to postoperative pulmonary complications [PPC] like atelectasis, pulmonary collapse, consolidation, pleural effusion, persistent air leaks, and pneumonia. Other frequently observed postoperative complications are postthoracotomy pain syndrome and ipsilateral reduction in upper extremity range of motion [ROM] and strength –5.Physiotherapy is widely considered to be important in limiting the development of postoperative pulmonary complications, which are associated with significant clinical and economic impact, and in the prevention and treatment of shoulder dysfunction, which has been reported extensively following thoracotomy .The physiotherapy management of patients after major surgery forms the basis of much debate among physiotherapists worldwide . A growiThere is scarcity of retrievable literature regarding pre- and postoperative assessment and treatment for patients undergoing thoracic surgeries in India. The aim of the study was to analyse current practice patterns among physiotherapists in pre- and postoperative assessment and treatment for patients undergoing thoracic surgeries in India.A written approval was obtained from the Institutional Ethics Committee and Scientific Committee of KMC, Mangalore. Upon approval by the committee, a list of hospitals where thoracic surgeries are done was obtained from the NABH  and MCI  websites, and a survey was conducted across India in which around 600 questionnaires were sent via email to physiotherapists who were involved in the management of patients undergoing thoracic surgery.This email included a hyperlink which directed the participants to a webpage with the Informed Consent Form and those consenting to participate could access the questionnaire.A period of 2 weeks was given to the physiotherapists to fill in the questionnaire in an attempt to ensure good response rate. If no response was obtained within the stipulated period, telephonic or email reminders were sent and the response was awaited for another 2 weeks beyond which nonresponders were excluded from the study.Responses were numerically coded to allow for descriptive summaries and frequency analysis of the data using SPSS version 16. Frequency variables regarding assessment and treatment for thoracic surgery patients were merged in order to create three responses: “always or frequently,” “sometimes,” and “rarely or never.”A total of 600 questionnaires were mailed to physiotherapists all over India, of which 234 completed questionnaires were returned. Data are reported as the number of respondents for each individual question, which varied throughout the survey. The physiotherapy profile is displayed in n = 218  [93.1%]), chest expansion (n = 217  [92.7%]), ABG analysis (n = 183  [78.2%]), pulmonary function test (n = 175  [74.7%]), chest X-ray (n = 215  [93.5%]), oxygen saturation (n = 211  [90.1%]), peripheral muscle strength (n = 175  [74.7%]), cardiopulmonary exercise test (n = 159  [67.9%]), 6 min and 12 min walk test (n = 138  [60.0%]), Barthel index (n = 157  [70.4%]), and functional independence measure (n = 164  [72.3%]) of the responders.The frequencies with which different assessment measures were used preoperatively are given in n = 217  [93.9%]), huffing and coughing (n = 204  [88.7%]), active cycle of breathing exercises (n = 203  [86.7%]), incentive spirometry (n = 216  [92.3%]), and thoracic mobility exercises (n = 213  [91.0%]) of the responders (The preoperative physiotherapy interventions included deep breathing exercises (sponders .n = 223  [95.2%]), chest expansion (n = 221  [94.4%]), ABG analysis (n = 198  [84.6%]), pulmonary function test (n = 187  [79.9%]), chest X-ray (n = 211  [90.1%]), and oxygen saturation (n = 216  [88.4%]).The frequencies with which different assessment measures were used postoperatively are given in n = 221  [94.4%]), relaxation techniques (n = 215  [91.8%]), thoracic expansion exercises (n = 193  [82.4%]), incentive spirometry (n = 198  [84.6%]), coughing and huffing techniques (n = 177  [75.6%]), positioning (n = 138  [58.9%]), modified postural drainage (n = 206  [88%]), upper extremities range of motion exercises (n = 172  [73.5%]), lower extremities range of motion exercises (n = 201  [85.8%]), and dangle (n = 198  [84.6%]).The frequencies of various physiotherapy treatment techniques used on postoperative day 1 are given in n = 228  [97.4%]), relaxation techniques (n = 217  [92.7%]), thoracic expansion exercises (n = 217  [92.7%]), incentive spirometry (n = 217  [92.7%]), coughing and huffing techniques (n = 192  [82%]), percussion and vibrations (n = 156  [66.6%]), positioning (n = 214  [91.4%]), modified postural drainage (n = 179  [76.4%]), upper extremities range of motion exercises (n = 216  [92.3%]), lower extremities range of motion exercises (n = 211  [90.1%]), dangle (n = 174  [74.3%]), up to chair (n = 156  [66.6%]), sit to stand (n = 125  [53.4%]), walking in the room (n = 168  [71.7%]), and walking in the corridor (n = 86  [39.8%]).The frequencies with which different treatment techniques were used on postoperative day 2 are given in n = 228  [97.4%]), relaxation techniques (n = 216  [92.3%]), incentive spirometry (n = 226  [96.5%]), modified postural drainage (n = 197  [84.1%]), upper extremities range of motion exercises (n = 222  [94.8%]), lower extremities range of motion exercises (n = 216  [92.3%]), dangle (n = 197  [84.1%]), up to chair (n = 194  [82.9%]), sit to stand (n = 177  [75.6%]), and staircase climbing (n = 86 [36.7%]) of the responders.The frequencies with which different treatment techniques were used on postoperative day 3 are given in n = 221) of the physiotherapists responded with data on postoperative day 2. The patients were made to walk 25 to 50 feet by 66.2% (n = 155), 60 to 70 feet by 24.7% (n = 58), and 80 to 90 feet by 3.5% (n = 8); and the patients were made to walk twice daily by 61.1% (n = 143), once a day by 1.3% (n = 3), thrice daily by 7.3% (n = 17), and four times a day by 0.9% (n = 2). A total of 99.1% (n = 232) of the physiotherapists responded with data on postoperative day 3. The patients were made to walk 110 to 130 feet by 67.5% (n = 158), 140 to 160 feet by 24.8% (n = 58), 170 to 190 feet by 5.1% (n = 12), and >200 feet by 1.7% (n = 4) of the responding physiotherapists.Data pertaining to mobilization of patients on postoperative days is given in n = 113); on the 4th postoperative day by 29.1% (n = 68); on the 6th postoperative day by 9.8% (n = 23); and on the 7th postoperative day by 5.6% (n = 13) of the responders. The “functional evaluation prior to discharge” included a 6-minute walk test employed by 77.4% (n = 181); a 2-minute walk test employed by 16.7% (n = 39); a 12-minute walk test employed by 2.6% (n = 6); and a shuttle walk test employed by 0.9% (n = 2) of the responders. Discharge teaching was provided by the responders in one of four ways: by an individual [50.9% (n = 119)], in written form [27.4% (n = 64)], using an audiovisual method [13.2% (n = 31)], and by a group [8.1% (n = 19)].Staircase climbing was started on the 5th postoperative day by 48.3% , and peripheral muscle strength. Other impairment measures like cardiopulmonary exercise test, 2 min walk test, 6 min walk test, and 12 min walk test were used by fewer responders in their clinical practice. There was limited use by the responders of functional measures like the Barthel index and functional independence measure.The assessment primarily focused on physical examination, chest expansion, ABG analysis, pulmonary function test, chest X-ray, SpOThe most commonly used treatment techniques included deep breathing exercises, relaxation techniques, incentive spirometry, thoracic mobility exercises, modified postural drainage, and range of motion exercises of the upper and the lower limbs. A smaller number of responders also practice percussion, vibrations, and coughing and huffing techniques.The patients were made to dangle the lower limbs over the edge of the bed on postoperative day 1, along with common treatment techniques. Mobilization techniques like walking up to a chair, sit to stand exercises, and mobilization in the room were started on postoperative day 2. The patients were made to start with staircase climbing on the 4th postoperative day. The most regularly used functional evaluation test prior to discharge was the 6-minute walk test for thoracic surgery patients.The novel findings of the study were as follows:2 (oxygen saturation), peripheral muscle strength, and cardiopulmonary exercise testing.In our survey, the assessment of patients focused primarily on certain impairment measures such as physical examination, chest expansion, ABG analysis, pulmonary function test, chest X-ray, SpOIn the preoperative phase, physical examination should address the presence of dyspnoea, exercise tolerance, cough, and expectoration. Examination should also focus on respiratory rate, pattern of breathing, and wheezing. Patients may show either normal or altered breathing pattern on physical examination. Postoperatively, that is, after thoracic surgery, patients usually present with monotonous shallow breathing without spontaneous deep breaths, increase in respiratory rate, decreased tidal volume, and significant change in minute ventilation. Wheeze, rales, or prolonged breath sounds will be revealed on auscultation . The curChest expansion is reduced in thoracic surgery patients, particularly on the side operated on. Clinically, the decreased chest movement may lead to hypoventilation and complications like atelectasis or lung collapse . After p2 retention. In a patient with sudden onset of alveolar hypoventilation causing a rise in PCO2, there would be a sudden fall in pH leading to uncompensated respiratory acidosis [Postoperatively, ABG analysis typically shows respiratory acidosis in the initial stages, while respiratory alkalosis develops in later stages . During acidosis , 12. 80.The chest radiographs of thoracic surgery patients show disease limited to one lobe or less . Chest XPulmonary function test is used to determine the degree to which the preexisting obstructive and restrictive components of pulmonary function may compromise the ability to ventilate adequately and to maintain clear lungs after thoracic surgery. Pulmonary function abnormalities in thoracic surgery include a decrease in forced expiratory volume, increased airway resistance, a decreased inspiratory capacity, and a decrease in maximum voluntary ventilation (MVV) . The sur2) is used to measure and record oxygen saturation. In thoracic surgery patients, there is a significant fall in PaO2, as well as abnormalities of gaseous exchange, disturbances in coordination of ventilation, and perfusion of lungs [Pulse oximetry .Deep breathing exercises are taught to the patients in the preoperative period. These can reduce the effects of an altered breathing pattern after thoracotomy and can help to obtain full expansion of the chest wall during spontaneous breathing. This is essential to help restore lung function and to prevent subsequent chest deformity. A large inflating volume and transpulmonary pressure gradient is needed to be maintained for several seconds in order to achieve lung reexpansion . In thisIncentive spirometry is used to encourage sustained maximal inspiration, promote reinflation of lung tissue, and prevent or resolve atelectasis. Incentive spirometry involves deep breathing through a device with visual feedback, thought to maximize accuracy of breathing technique and motivation . The resCoughing and huffing techniques are used in clearing secretions from large airways. The rapid, forceful expulsion of air from the lungs helps remove secretions from the large airways. In addition to mobilizing and expelling secretions, the high pressures generated during a cough may be an important factor in reexpanding lung tissue . The curActive cycle breathing exercises (ACBTs) are used in improving pulmonary function and airway clearance. The ACBTs are combinations of breathing control, thoracic expansion control, and forced expiratory techniques (FET). Breathing control has been referred to as diaphragmatic breathing, or as gentle breathing using the lower chest. Thoracic expansion exercises are simply active inspirations with larger than normal breaths, followed by relaxed expiration. This larger lung volume increases airflow through peripheral airways and collateral ventilation channels, which increases the gas volume available to mobilize secretions during expiration . AccordiThe results of our survey showed that predominantly the same treatment techniques were employed postoperatively as employed preoperatively.Breathing exercises are used to improve the efficiency of ventilation and gas exchange, increasing the excursion of the diaphragm and easing the mobilization of secretions . BreathiIncentive spirometry has the same effect as shown in preoperative management. It is used primarily to prevent alveolar collapse and atelectasis in postoperative patients . In the Thoracic expansion exercises were given to improve the mobility of chest wall, trunk, and shoulder girdles. Exercises that combine stretching of these muscles with deep breathing improve ventilation on that side of the chest. These exercises are also used to reinforce or emphasize the depth of inspiration or controlled expiration . AccordiCoughing techniques enhance the clearance of retained bronchial secretions, thus preventing atelectasis and infection. During a normal cough, airflow velocity varies in airways, creating high linear velocities, increased turbulence, and high shearing forces within the airway. These forces shear secretions from the airway walls, propelling them towards the larger airways and trachea which causes expelling of secretions . CoughinPercussion is used to augment mobilization of secretions by mechanically dislodging viscous or adherent mucus from airways. Vibrations are used in conjunction with percussion to help move secretions to larger airways. The air cushion between the cupped hand and the chest wall generates the typical hollow sound, transmits the energy to the underlying lung, and helps mobilize secretions. The atelectatic areas of the lung can be reinflated and total lung/thorax compliance can be increased following percussion and vibration , 17. In Positioning is advised for promoting relaxation, reducing postoperative pain, and causing lung expansion as also for draining secretions. In pneumonectomy, the lateral position is advised, as perfusion of the dependent lung is increased due to gravitational changes though ventilation is more difficult. The prone position is advantageous when a large quantity of bronchial secretions is present in the segmentectomy . In thisModified postural drainage  is used for airway clearance by mobilizing secretions from different lung segments to the central airways by placing the patient in various positions so that gravity assists in draining secretions. Postural drainage attempts to use gravity to move secretions from peripheral airways to the larger bronchi, from which they are more easily expectorated . A totalAn upper limb exercise programme is commonly used to prevent or minimize shoulder dysfunction and to maintain joint range of motion after thoracotomy. Active shoulder exercises like flexion, abduction, external rotation and elevation, shoulder shrugging, and progressive shoulder and thoracic cage exercises are commonly used after thoracic surgery. Shoulder strengthening exercises for flexion, extension, abduction, and external rotations are usually performed after thoracic surgery . Upper lLower limb exercises like hip and knee flexion and extension exercises and active leg, foot, and ankle exercises are taught to prevent circulatory problems like deep vein thrombosis and pulmonary embolism . Thus, rMobilization in the upright position coordinated with breathing control and supported coughing maneuvers is encouraged in order to reduce atelectasis and impaired mucociliary transport associated with surgery. Early mobilization elicits cardiopulmonary responses, resulting in enhancement of oxygen transport. These beneficial effects are enhanced by improved chest wall motion, improved gut mobility, and reduced intra-abdominal pressure. Also, a study stated that early walking has a good psychological effect on stable and beneficial breathing potentially leading to recovery of pulmonary function after surgery . ExtremiRoom mobilization was started on the 2nd postoperative day by 71.1% of the responders and corridor mobilization was started on the same day by 39.8% of the responders. This response rate is lower than the response obtained in a similar study conducted in Australia and New Zealand (71.7%) [One of the limitations of the study was that the majority of the responders were from Telangana, Karnataka, and Andhra Pradesh and this is not truly representative of India. Another possibility is that participants responded to the survey questionnaire with what they considered to be the best or most appropriate answer to each question. Therefore, their responses may not reflect the actual practice patterns of physiotherapists in India. And one more limitation is that we did not consider the minimally invasive thoracic surgery.2 monitoring. Preoperative treatment techniques practiced are deep breathing exercises, huffing and coughing, active cycle of breathing exercises, incentive spirometry, and thoracic mobility exercises.This survey provides an overview of current physiotherapy practices across India in thoracic surgeries. Commonly used assessment measures in the preoperative phase are physical examination, chest expansion, ABG analysis, pulmonary function test, chest X-ray, and SpO2. The postoperative treatment techniques regularly used are breathing exercises, relaxation techniques, thoracic expansion exercises, incentive spirometry, coughing and huffing, and modified postural drainage. Mobilization which includes dangle, room and corridor mobilization, and the 6-minute walk test is commonly used as a functional evaluation test.The most commonly used postoperative assessment measures are physical examination, chest expansion, ABG analysis, chest X-ray, pulmonary function test, and SpO


Hypertension is a major health problem worldwide. It can lead to cardiovascular disease and also leads to functional disturbances including hematological parameters. The abnormalities of haematological parameters may enhance an end-organ damage. Therefore, the aim of this study was to assess some hematological parameters of hypertensive individuals in comparison with normotensive individuals at University of Gondar hospital, northwest Ethiopia.t-test and the Mann Whitney u-test were used to find out significant difference and Pearson’s and Spearman’s correlation were used for correlation test. P values less than 0.05 was considered the level of significance.A cross sectional comparative study was conducted from October to November 2015 on a total of 126 hypertensive and 126 normotensive individuals at University of Gondar Hospital. All participants after taking informed consent were interviewed for detailed history and 3 ml of blood was collected for hematological test analysis. Independent From a total of 252 study subjects, about 67.5% were females. The mean age of study subjects was 50.3 ± 11 years for hypertensive individuals and 49.8 ± 11.6 years for normotensive individuals with range of 18–65 years. In the present study, the median (IQR) value of WBC, RBC, Hgb, HCT, MCV and the mean value of MCHC, RDW, MPV and PDW were significantly higher in hypertensive group compared to apparently healthy normotensive groups. Additionally, WBC, RBC, Hgb, HCT and PLT showed statistically significant positive correlations with blood pressure indices. Platelet count and MCH did not show statistically significant difference between the two groups.Hypertension has impact on hematological parameters. In this study, the mean and median values of haematological parameters in hypertensive individuals were significantly different compared to apparently healthy normotensive individuals. Hence, hematological parameters can be used to monitor the prognosis of the disease and manage hypertensive related complications, and it is important to assess hematological parameters for hypertensive individuals which may help to prevent complications associated hematological disorders.The online version of this article (doi: 10.1186/s12878-017-0093-9) contains supplementary material, which is available to authorized users. Hypertension (HTN) is also called high blood pressure. It is a condition in which systemic arterial pressure is elevated above the threshold value . It is eHTN can be categorized in to two; primary and secondary hypertensions. Primary HTN, which consists of about 95% cases, can occur without any obvious underlying causes while secondary HTN is developed due to secondary to diseases such as kidney disease, endocrine disorders and narrowing of the aorta or kidney arteries .HTN is a major health problem worldwide that affects 20–30% of the adult population . It is rHTN may lead to severe end-organ damage, coronary heart disease and stroke which constitute the leading cause of mortality , 12. It On the other hand, HTN has an impact on hematological parameter such as hematocrit (HCT), hemoglobin (Hgb), red blood cell (RBC) count, white blood cell (WBC) count and platelet (PLT) count. Impaired hematological parameters may strongly indicate hypertensive end-organ damage, specifically kidney failure , 16, 17.Generally, there are contradictory results regarding hematological parameters of hypertensive patients in different countries. Moreover, there is lack of information regarding to hematological parameters in hypertensive patients in Ethiopia. Therefore, this study was aimed at assessing hematological parameters in hypertensive patients in comparison with apparently healthy individuals and correlating hematological parameters with blood pressure indices  at university of Gondar hospital, Northwest Ethiopia.A comparative cross-sectional study was conducted from October to November 2015 at university of Gondar hospital chronic illness clinic. The hospital is located in Gondar town which is 740 km from the capital of Ethiopia, Addis Ababa, in the Northwest of Ethiopia. Gondar University Hospital is the only referral hospital in North West Ethiopia serving a population of about 5 million. This hospital gives different types of service for the population including treatment of chronic diseases. A total of 252  study subjects were included conveniently in this study. The study subjects with hypertensive and healthy controls were age and sex matched. Study subjects with history of infectious diseases, alcohol consumers, smokers, taking antibiotic, treatment for anemia, patients with systemic diseases and with secondary hypertension were excluded from this study with face to face interview and review of medical records  was analyzed by using Sysmex KX-21 automated hematology analyzer. White blood cells (WBC), red blood cell parameters  and platelet parameters  were analyzed and collected for each study participants by using laboratory result registration form  was assessed by Pearson’s and Spearman’s correlation. In any condition, P value < 0.05 was considered as statistically significant.Data were cleaned, edited and checked for completeness and entered in to SPSS version 20 statistical software for analysis. The normality of data distribution was checked by statistical tools of Kolmogorov-Smirnov (K-S). The results were expressed as mean ± standard deviation and median with interquartile range. Comparison of parameters between hypertensive subjects and apparently healthy normotensive controls was done with independent A total of 252 study subjects grouped into two groups  were involved in this study. From them 67.5%  were female study subjects which makes 1:2 ratio of male to female. The mean age of study subjects was 50.3 ± 11 years for hypertensive individuals and 49.8 ± 11.6 years for apparently healthy individuals with range of 18–65 years. About 200 (79.4%) of the total study subjects and 84 (66.7%) of the cases group were urban dwellers. The mean and standard deviation value of the BMI, MAP, SBP and DBP were 24.0 ± 2.3, 109.4 ± 5.4 mmHg, 143.2 ± 8.0 mmHg, and 92.4 ± 4.6 mmHg, respectively. The average duration of illness was 3.47 ± 2.8 years (Table P < 0.05). Although the median (IQR) value of PLT count is relatively higher in hypertensive groups, there is no statistically significant difference between these groups (P = 0.262) (Table Hypertensive groups had significantly higher median (IQR) value of WBC, RBC, Hgb, HCT, MCV and mean (SD) value of MCHC, RDW, MPV and PDW were significantly higher in hypertensive patient than apparently healthy normotensive individuals .In the present study correlation of the various haematological parameters and the blood pressure indices among hypertensive individuals was determined. As Table 3/μl (3.7 × 103/μl) when compared to apparently healthy normotensive controls 5.2 × 103/μl (2.2 × 103/μl). This finding is in agreement with other similar studies by Babu KR et al. [A total of 252 study subjects  were involved in this study to compare some hematological parameters among hypertensive and normotensive individuals. The mean age of hypertensive and control individuals were 50.3 ± 11 and 49.8 ± 11.6 years, respectively. Based on the result, hypertensive groups had significantly higher median (IQR) WBC value 6.9 × 103/μl 3. × 103/μlR et al. . Also, tThere is a causal relationship between vascular function and different hematological disorders , 20. MosThe relationship between WBC and hypertension may be explained by an increased concentration of stem cell factor (SCF) in serum . During 6/μl (0.88 × 106/μl) when compared to apparently healthy controls 4.88 × 106/μl (0.58 × 106/μl). This is supported by Babu KR et al. [Similarly, the current study showed significantly higher median (IQR) RBC value 5 × 10R et al. , Reis RSR et al.  and BrusR et al. . Also, RR et al. . Since iR et al. .In the present study, Hgb value was significantly increased in the hypertensive group compared to normotensive groups. This findings is in agreement with supported studies done by Babu KR et al.  and Al-MThe association between HTN and Hgb level may be explained by Hgb and arginase enzyme effects on nitric oxide (NO) bioavailability , 34. DurIn our study, the median (IQR) value of HCT significantly increased in hypertensive individuals compare to normotensive individuals. These findings are also familiar to Babu KR et al. . In bivaIn our study, RDW increased significantly in hypertensive groups compared to normotensive individuals. Most studies suggest that higher RDW, which is a measure of the variability in the circulating erythrocytes’ size, may be resulted from ineffective erythropoiesis due to chronic inflammation during hypertension , 40.In this study MCV and MCHC were increased significantly in hypertensive groups but there were no significant differences in MCH. But other studies in these parameters showed contradicted ideas. For example a study conducted by Babu KR et al.  showed sIn the present study, the median (IQR) value of PLT count, mean value of MPV and PDW were increased in hypertensive groups than controls. Even though statistically not significant, median value of PLT count was slightly higher in hypertensive groups. The possible explanation for this could be related to consumption of platelets. During hypertension, there is endothelial dysfunction and this leads to platelet activation and clot formation. Then platelets will be consumed and there number does not increase as expected , 41, 42.In our study, PLT count positively correlated with blood pressure indices. The possible mechanisms might be related to vascular complication in hypertensive groups. High blood pressure causes endothelial damage via shear stress, which results in an increase in platelet activation . When plSince antihypertensive therapy reduces blood pressure and improve endothelial function, their effect didn’t assess in this study. Additionally, cell free hemoglobin analysis was not considered. Therefore, further cohort study is required.In the present study, the median (IQR) value of WBC, RBC, Hgb, HCT, MCV and the mean value of MCHC, RDW, MPV and PDW were significantly higher in the hypertensive group compared to apparently healthy controls. But platelet count and MCH showed no statistically significant difference between hypertensive and normotensive groups. In this study there was statistically significant positive correlations of WBC, RBC, Hgb, HCT and PLT with blood pressure indices  among hypertensive individuals. Impaired hematological parameters may strongly indicate hypertensive end-organ damage. Hematological complication such as RBCs reduction in deformability and an increase in the size causes hemolysis, high Hgb and low levels are associated with cardiovascular risk and activation of platelets is risk factor for thrombotic diseases observed during hypertension. Therefore, it is important to assess changes in hematological parameters for hypertensive patients which may help to prevent such complications associated hematological disorders.Additional file 1:Questionnaire. Data collection questionnaire designed for the comparative cross-sectional study of some hematological parameters of hypertensive and normotensive individuals at the university of Gondar hospital, Northwest Ethiopia. (DOCX 15 kb)Additional file 2:Laboratory result registration form. Laboratory result registration form designed for the registration of hematological parameter results of hypertensive patients and normotensive controls at university of Gondar Hospital, Northwest Ethiopia. (DOCX 13 kb)


Use of molecular beacons avoids this problem because molecular beacons produce fluorescence signals only when binding to target DNA, thus acting as a direct indicator of amplification products. Our analyses determined the optimal conditions for molecular beacons as an evaluation tool in LAMP: beacon length of 25–45 bp, beacon concentration of 0.6–1 pmol/μL, and reaction temperature of 60–65 °C. In conclusion, we validated a novel molecular beacon loop-mediated isothermal amplification method (MB-LAMP), realizing the direct detection of LAMP product.This study established a constant-temperature fluorescence quantitative detection method, combining loop-mediated isothermal amplification (LAMP) with molecular beacons. The advantages of LAMP are its convenience and efficiency, as it does not require a thermocycler and results are easily visualized by the naked eye. However, a major disadvantage of current LAMP techniques is the use of indirect evaluation methods (e.g., electrophoresis, SYBR Green I dye, precipitation, hydroxynaphthol blue dye, the turbidimetric method, calcein/Mn This method uses a DNA polymerase with strand displacement activity and yields large quantities of DNA in less than an hour under constant temperature (thus obviating the need for expensive thermocyclers). Moreover, a correspondingly large amount of white magnesium pyrophosphate precipitate is synthesized as a by-product, enabling direct visual confirmation of amplification success through monitoring of mixture turbidity. These characteristics provide results in a fast, efficient, and cost-effective method that theoretically should amplify target sequences with higher specificity than PCR. Since its introduction, LAMP has seen wide interdisciplinary application, for example in detecting microorganisms  suggests higher specificity and sensitivity than standard PCR, which uses only two primers, the increase in primers can also cause nonspecific amplification2+ 2+ is consumed in the reaction solution, they fail to assess whether the LAMP reaction has successfully amplified the target sequences. TurbidityThe first technique, SYBR Green I stainingIn summary, the indirect nature of existing detection methods signifies a major shortcoming of LAMP technology. In this study, we therefore developed and validated a method to detect LAMP products directly. Both TaqMan and molecular beacons were under consideration for direct validation of amplification, but we chose to use molecular beacon probes because, in contrast to TaqMan, they are not dependent on Taq exonuclease activity, which many DNA polymerases do not possess. Based on insights from fluorescence quantitative PCR technology, we named our technique the molecular beacon loop-mediated isothermal amplification (MB-LAMP). Molecular beacons (MBs) are fluorescent nucleic acid probes with a hairpin structure, developed in 1996−4 mM and 65 °C were used in the probe selection experiments.To monitor LAMP reactions successfully, molecular beacons must hybridize to their targets at the isothermal temperature suitable for the reaction, which can be achieved through selection of the appropriate length of the probe and arm sequences. In PCR, the length of the probe sequence is usually 15–30 nucleotides and the length of the arm sequence is 5–7 nucleotides. In our experiment, we aimed to explore the appropriate MB length under isothermic conditions. We examined lengths of 3–23 nucleotides in our tests as the head and neck sequences and found that both overly short and overly long MBs were unusable as a validation tool for LAMP. We selected the extreme 3 bp and 23 bp lengths and the common 13 bp length for head and neck sequences for evaluation; the nucleic acid sequences are listed at −4 mM. To determine optimal MB concentrations for MB-LAMP, we tested five concentrations in our assay: 0.8 × 10−4 mM, 1.6 × 10−4 mM, 2.4 × 10−4 mM, 3.2 × 10−4 mM, and 4.0 × 10−4 mM . After several experiments, we found that fluorescence intensity increased significantly with increases in probe concentration until a probe concentration of 3.2 × 10−4 mM, and no real difference was visible between the two highest MB concentrations  is approximately 3.2 × 10trations . We thusThe temperature range of LAMP is approximately 60–69 °C, with an optimum temperature of approximately 65 °C. The temperature strongly influences MB fluorescence intensityompW gene was significantly amplified at Ct = 28, or 14 min. The other standard strains . To test the sensitivity of the technique, we serially diluted the plasmid from 10 ng/μL to 1 × 10ary LAMP , in whicThe convenience of LAMP has increased its popularity in fields requiring rapid gene detection, but the indirect detection methods commonly employed with this technique lead to difficulty in identifying nonspecific amplification. Although all amplification methods are subject to possible contamination that can lead to false positives, nonspecific amplification is a particular problem with LAMP because of the greater sensitivity associated with an increased number of primers. To provide convincing data, satisfactory LAMP results have to be obtained by screening and designing primers or increasing the reaction temperature, as we did in these experiments. In this study, we developed and validated the use of MBs in conjunction with LAMP. Because MBs can directly detect and bind to amplification products, our detection method performs better than traditional LAMP in terms of specificity.In addition to avoiding false positives caused by nonspecific amplification, the short reaction time and low equipment costs associated with isothermal amplification suggest that MB-LAMP may have a similar impact to the introduction of quantitative PCR in the future. Numerous inexpensive and small isothermic fluorescence detectors are widely available . Attempts to pair fluorescent probes with isothermal amplification methods have been previously successful, such as the use of fluorescence energy transfer probes with strand displacement amplification (SDA), molecular beacons with nucleic acid sequence-based amplification (NASBA), and padlock probes with rolling circle amplification (RCA)1718The promise of MB-LAMP as a widespread diagnostic tool was recently demonstrated by its successful use to test for the Zika virus in our laboratory . The MB-15Genomic DNA of several bacterial strains  was extrompW gene from Vibrio cholerae (GenBank: X51948.1). A 222-bp synthesized target plasmid of the ompW gene (−8 ng/μL in ddH2O for use as templates.We used MB-LAMP to detect a part of the 1031-bp mpW gene  was quanhttp:/primerexplorer.jp/lamp). Two additional loop primers  were designed to accelerate amplification. The fluorescent nucleic-acid probes in MB-LAMP (LBP and LFP) were directly modified from LB and LF  were designed in Primer Explorer V4  was added. The LAMP was performed at the traditional temperature of 65 °C, and the optimum conditions for MB-LAMP were determined in the experiments described in this manuscript. We used the concentration of 3.2 × 10−4 mM and temperature of 63 °C once they were established as the optimal conditions.The reaction mixture  for LAMP and MB-LAMP were 25 μL per reaction tube , containing the following reagents : 20 mM Tris-HCl (pH = 8.8), 10 mM KCl, 10 mM (NH4)Turbidity in the LAMP assay was monitored using a Loop Amp real-time turbidimeter , and the OD was recorded at 650 nm in 6-s intervals. Fluorescence of MB-LAMP was monitored with a CFX Connect Real-Time PCR Detection System  and amplifications were performed under 120 cycles of constant temperature for 30 s. The fluorescence value was read in 30-s (one cycle) intervals.How to cite this article: Liu, W. et al. Establishment of an accurate and fast detection method using molecular beacons in loop-mediated isothermal amplification assay. Sci. Rep.7, 40125; doi: 10.1038/srep40125 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.