The use of pyronaridine-artesunate (PA) has been associated with scarce transaminitis in patients. This analysis aimed to evaluate the hepatic safety profile of repeated treatment with PA versus artemether–lumefantrine (AL) in patients with consecutive uncomplicated malaria episodes in Bobo-Dioulasso, Burkina Faso.This study analysed data from a clinical trial conducted from 2012 to 2015, in which participants with uncomplicated malaria were assigned to either PA or AL arms and followed up to 42 days. Subsequent malaria episodes within a 2-years follow up period were also treated with the same ACT initially allocated. Transaminases (AST/ALT), alkaline phosphatase (ALP), total and direct bilirubin were measured at days 0 (baseline), 3, 7, 28 and on some unscheduled days if required. The proportions of non-clinical hepatic adverse events (AEs) following first and repeated treatments with PA and AL were compared within study arms. The association of these AEs with retreatment in each arm was also determined using a logistic regression model.A total of 1379 malaria episodes were included in the intention to treat analysis with 60% of all cases occurring in the AL arm. Overall, 179 non-clinical hepatic AEs were recorded in the AL arm versus 145 in the PA arm. Elevated ALT was noted in 3.05% of treated malaria episodes, elevated AST 3.34%, elevated ALP 1.81%, and elevated total and direct bilirubin in 7.90% and 7.40% respectively. Retreated participants were less likely to experience elevated ALT and AST than first episode treated participants in both arms. One case of Hy’s law condition was recorded in a first treated participant of the PA arm. Participants from the retreatment group were 76% and 84% less likely to have elevated ALT and AST, respectively, in the AL arm and 68% less likely to present elevated ALT in the PA arm. In contrast, they were almost 2 times more likely to experience elevated total bilirubin in both arms.Pyronaridine-artesunate and artemether–lumefantrine showed similar hepatic safety when used repeatedly in participants with uncomplicated malaria. Pyronaridine-artesunate represents therefore a suitable alternative to the current first line anti-malarial drugs in use in endemic areas.Trial registration Pan African Clinical Trials Registry. PACTR201105000286876 Artemisinin-based combination therapy (ACT) represents the mainstay of recommended treatment for uncomplicated malaria since the early 2000s. According to the World Health Organization (WHO), no alternative to artemisinin derivatives is expected for several years . CurrentIn sub-Saharan Africa, where malaria is endemic, several episodes may occur within a year requiring repeated treatment either with the same drug or with different drugs. However, longitudinal data on safety evaluation on patients exposed to repeated treatment with the same artemisinin-based combination are lacking. In addition, following a period of ultimate efficacity of ACT against uncomplicated malaria, parasites with very prolonged elimination time were detected following the use of ACT ; since 2This report is a secondary analysis of data from the WANECAM 1 study site of Bobo-Dioulasso, in Burkina Faso. The protocol and standard procedures of this trial were published elsewhere –20. BrieFrom August 22, 2012 to November 8, 2015 patients of either gender, diagnosed with uncomplicated malaria were enrolled in two primary health facilities, Sakaby and Colsama in the health district of Do . Globally, participants were allocated to either PA or AL arms according to a computer-generated randomization list. Subsequent uncomplicated malaria episodes were treated with the same artemisinin-based combination as at enrolment during the follow-up period.This secondary analysis used the available dataset on the site of Bobo-Dioulasso. According to the study protocol, a total size of 224 participants was needed in the PA arm versus 296 participants in the AL arm to meet the country/site individual size requirement. This sample size was calculated to give sufficient power for an independent country/site data analysis. The full sample size calculation process is described elsewhere .During each malaria episode, PA and AL were administered orally over three days . PA  was given either as tablets (180 mg of pyronaridine and 60 mg of artesunate) or as granule sachets (60 mg of pyronaridine and 20 mg of artesunate) once daily. AL  was given twice daily as non-dispersible tablets (20 mg of artemether and 120 mg of lumefantrine). All doses were bodyweight-based and directly supervised at the study clinics from day 0 to day2 as described elsewhere  and the retreatment group , p = 0.40, as well as in the PA arm between 1.78%, 95% CI  in the first treatment group versus 2.08%, 95% CI  in the retreatment group, p = 1.0.In direct comparison, proportions of elevated direct bilirubin, elevated total bilirubin and elevated ALP among retreated participants in both study arms were similar. There was no evidence of any significant difference between proportions of elevated direct bilirubin in retreated participants with AL (9.56%) versus PA (8.92%) arms, p = 0.810. The proportions of elevated direct bilirubin in retreated participants in the AL arm (8.80%) was close to that of the PA arm (8.63%), p = 1.00. Similarly, the proportion of elevated ALP after retreatment with AL and that of those retreated with PA was all alike with 2.10% in the PA arm versus 2.08% in the PA arm, p = 1.00.In Fig. Table In the PA arm, participants in the retreatment group were 67% less likely to experience elevated ALT compared to those of the first treatment group, COR = 0.33, 95% CI . Retreatment with PA was not significantly associated with neither elevated AST, elevated ALP nor elevated total and direct bilirubin.After adjusting for age and gender retreatment with AL was significantly associated with elevated ALT, elevated AST, and elevated total bilirubin. Furthermore, in the AL arm, participants in the retreatment group were 76% and 84% less likely to have elevated ALT  and elevated AST , respectively, than those of the first treatment group. Each unit increase in participants’ age was associated with 17% reduced chance to present with elevated AST, aOR = 0.83, 95% CI .In addition, participants in the retreatment group were 2 times more likely to experience elevated total bilirubin, aOR = 2.48, 95% CI 1.31; 4.69). Each unit increase in participants’ age was associated with 5% more chance to present with elevated total bilirubin [aOR = 1.05 95% CI ] as shown in Table 1; 4.69. After adjustment, retreatment with PA was significantly associated with elevated ALT and total bilirubin. In fact, participants who were retreated with PA were 68% less likely to experience elevated ALT compared to participants of the first treatment group, aOR = 0.32, 95% CI 0.14; 0.74). Repeatedly treated participants of this arm were also 2 times more likely to present with elevated total bilirubin compare to those from the first treatment group, aOR = 2.09, 95% CI . In addition, each unit increase in participants’ age was associated with 12% more chance to present with elevated total bilirubin, aOR was 1.12, 95% CI . However, there was no evidence of significant association of elevated AST with retreatment with PA, aOR = 0.59, 95% CI  as shown in Table ; 0.74. RThe efficacy of pyronaridine-artesunate has been largely studied and proven elsewhere , 20, 23.This study shows that the risk of hepatic adverse events occurrence following repeated treatments was similar across study arms. This result argues for the wide and repeated use of pyronaridine-artesunate in any routine health care systems in malaria endemic areas. In fact, artemether–lumefantrine has been intensively studied in clinical trial settings and has shown a satisfactory safety profile. Artemether–lumefantrine has been adopted for several years as a first-line therapy against uncomplicated malaria in many endemic sub-Saharan countries –29. By sThe results of this study have also shown the particular strength of the association between repeated treatments with pyronaridine-artesunate and artemether–lumefantrine and elevated total bilirubin. Hyperbilirubinaemia following anti-malarial treatment in general, and especially artemisinin-based combinations, is common. In such a context, hyperbilirubinaemia is attributed to the haemolytic process of parasitized red blood cells. This haemolytic process can secondarily be worsened by the ACT-induced haemolysis leading to an unconjugated or indirect hyperbilirubinaemia , 32. HalThis study has also shown that repeated treatments with pyronaridine-artesunate and with artemether–lumefantrine did not increase the specific risks of liver injury (with elevated ALT/AST) and of Hy’s law condition. By contrast, repeated treatments with pyronaridine-artesunate and with artemether–lumefantrine trend to reduce these risks with significant lower proportions than those noted after first treatments. This finding could be explained first by the clinical trial setting where participants have been encouraged to visit the health facilities earlier as soon as they feel unwell whatever the symptoms or signs were. In addition, due to the weekly parasitological follow-up procedure until day 42, many late treatment failures have been diagnosed before the onset of clinical symptoms and signs. According to the study protocol, late parasitological or clinical failures identified by day 28 were considered and treated as new episodes. This raises the hypothesis that early diagnosis and treatment of subsequent malaria episodes protected participants from massive hepatocytes and erythrocytes infection and destruction. In contrast, at enrolment participants were similar to those diagnosed in routine settings where delay in healthcare-seeking is common , 36. ThiThis study has two main limitations. First, the impact of concomitant medications on the liver has not been investigated aside of that of pyronaridine-artesunate and artemether–lumefantrine. In fact, a concomitant drug could independently induced hepatic AEs or interact with study drugs (pyronaridine-artesunate and artemether–lumefantrine) and, therefore, could improve or worsen their impact on the liver –39. CompSecondarily, during this study, artemether–lumefantrine was not administered with fatty food to optimize the intestinal absorption of lumefantrine. There was also no daily dietary assessment as recommended in previous studies on artemether–lumefantrine specific efficacy and safety , 42, 43.Pyronaridine-artesunate has a potential as an alternative to artemether–lumefantrine and can help to address the shortage of available, effective and well-tolerated artemisinin-based combinations and to cope with the emergence and spread of anti-malarial drugs resistance. Focusing on the hepatic safety of pyronaridine-artesunate, this analysis of the WANECAM 1 data from the site of Bobo-Dioulasso confirmed that there is no specific increased risk of liver injury with repeated use of pyronaridine-artesunate in participants with uncomplicated malaria. Taking account of its already proven efficacy, the use of pyronaridine-artesunate is, therefore, suitable in malaria endemic countries where patients may experience several episodes mostly during high transmission seasons. In such areas, National Malaria Control Programmes (NMCP) should consider pyronaridine-artesunate as a potential alternative to their actual first-line anti-malarial drugs. However, pharmacovigilance of ACT should be a reality in malaria endemic countries; since artemisinin-based combinations are the most frequently used drugs in these areas, NMCP should undertake effective actions to monitor adopted (first- and second-line) artemisinin-based combinations safety by setting up and effectively running specialized pharmacovigilance centres.


Surgery in patients with non-functioning pituitary macroadenomas (NFMA) is effective in ameliorating visual function. The urgency for decompression, and preferred timing of surgery related to the preoperative severity of dysfunction is unknown.Systematic review for evidence to provide clinical guidance for timing of surgical decompression of the optic chiasm, and a cohort study of 30 NFMA patients, in whom mean deviation (MD), and severity of visual dysfunction was assessed.Systematic review 44 studies were included with a total of 4789 patients. Postoperatively, visual field defects improved in 87.0% of patients, stabilized in 12.8% and worsened in 1.0%. Specific protocols regarding timing of surgery were not reported. Only seven studies (16.7%) reported on either the duration of visual symptoms, or diagnostic, or treatment delay.Cohort study 30 NFMA patients , had a median MD of − 5.3 decibel (IQR − 3.1 to − 10.1). MD was strongly correlated with clinical severity , and were used for severity of defects cut-off values: (1) normal >  − 2 dB, (2) mild − 2 dB to − 4 dB, (3) moderate − 4 to − 8 dB, (4) severe − 8 to − 17 dB, (5) very severe <  − 17 dB.Surgical decompression is highly effective in improving visual function. Uniform, quantitative grading of visual dysfunction was lacking. MD is a promising quantitative outcome measure. We provide recommendations for the evaluation of timing of surgery, considering severity of visual impairment, which will need further validation based on expert clinical practice.The online version of this article (10.1007/s11102-020-01044-0) contains supplementary material, which is available to authorized users. Non-functioning macroadenomas (NFMA) are the most prevalent pituitary macroadenomas (25%), and are often diagnosed due to pituitary insufficiency (80%) or visual impairment (85%) –3. MostlFollowing compression of the optic chiasm, the sequential order of development of visual field defects (VFD) is typical: first in the temporal upper quadrant(s), followed by the temporal lower quadrant(s). Subsequently, defects in the nasal upper and lower quadrants appear, often accompanied by deterioration of visual acuity (VA) and, if no intervention is provided, this can ultimately result in blindness. As the central aspect of the optic chiasm is consistently exposed to higher pressures than the temporal aspects, the intersecting fibers are affected more severely than non-intersecting fibers, leading to the typical bi-temporal VFDs . By contExperts agree that in cases of diminished VA, and severe VFDs immediate surgical decompression is required. Presentation with, or progression of, milder VFDs, and mild deterioration of VA are ‘semi-urgent’, and ‘urgent’ indications for surgical intervention, aiming at restoration of visual function whilst preserving  pituitary function . Tumor gMoreover, VFDs in pituitary disease have typically been described as a qualitative or semi-quantitative measure, somehow subject to individual interpretation. In other ophthalmological diseases, such as glaucoma, the mainstay of reporting severity of disease has become the mean deviation (MD). As provided by automated perimetry, MD is a numerical value in decibel (dB) representing deviation of the VF from age-matched controls, and could therefore be a quantitative measure of severity of VFDs . Note, wThe clinical relevance of prevention of visual morbidity as a benchmark for quality of care in pituitary adenoma patients is highly acknowledged . The aimFirst, we performed a systematic review on the topic of timing of surgical intervention in NFMA patients, adhering to PRISMA guidelines .A literature search strategy was developed using medical subject headings (MeSH) and text words related to surgical treatment of NFMA, visual parameters and function tests and timing variables (see Supplementary File). The following databases were searched: PubMed, EMBASE, Web of Science and the Cochrane Central Register of Controlled Trials.Randomized controlled trials (RCTs), controlled clinical trials (CCTs), prospective and retrospective comparative cohort studies and case–control studies were included for initial screening. Studies were eligible for inclusion if: (1) the article was published in English; (2) the reported cohort consisted of patients with NFMA; (3) the reported study cohort consisted of at least 10 patients; (4) patients were treated surgically with transsphenoidal microscopic, or endoscopic techniques, or transcranial techniques; (5) visual parameters were assessed prior to and after surgery. Studies on patients with various types of pituitary adenomas were excluded if the published results were not stratified by pituitary tumor type. Reviews, letters to the editor and expert opinions were excluded. Two independent reviewers (ICMP and MJTV) assessed eligibility and disagreements were resolved by discussion and consensus.An assessment was formulated by our research group to assess the quality of the included studies. Fourteen items were defined, including timing of surgery and visual function test, pre- and post-operative magnetic resonance imaging (MRI) and visual function characteristics and follow-up time (Supplementary Table 1). Individual scores ranged from 0 to 34 points. The median of the quality score percentages was used as a cut-off point for low- and high-quality papers. Studies were not in- or excluded based on the quality assessment.The following data were extracted: sample size, gender distribution, age, adenoma characteristics, timing of surgery, surgical technique, surgical outcome, visual function and the timing of visual diagnostics. Factors described in two studies or more were considered eligible for inclusion in the systematic review. Unfortunately, the reporting of the outcome of similar variables varied greatly amongst studies. Therefore, all parameters described in this review were (re)defined by the authors. Following data extraction, lead authors were contacted regarding remaining questions, when applicable.To validate the use of MD instead of a subjective semi-quantitative description of severity of VFD , a cohort of patients with an NFMA operated in our center followed in our Value Based Health Care (VHBC) care path with a dedicated clinical and patient-reported outcome set (2016–2018) was selected . PatientFor all patients, age, adenoma characteristics and the outcome of pre- and postoperative visual function tests were recorded. Treatment delay was calculated using the date of diagnosis of VFD and the date of surgery. All included patients were studied using perimetry performed with an automated central 30–2 Humphrey field analyzer (HFA)  in the presence of an ophthalmic technician. From the perimetry assessment, MD and Visual Flied Index (VFI) were recorded for each eye individually.From our multidisciplinary team (MDT), eight treating physicians were selected: two neuro-ophthalmologists, one neuro-ophthalmologist in training, one neuro-endocrinologist, one endocrinologist-in-training, and three pituitary neurosurgeons. All physicians are specialized in pituitary disease and set the indication and time frame for surgical decompression in the clinical care setting.Using a standardized survey, we assessed the physician’s view on the pattern and severity of VFD based on HFA assessment blinded for MD and VFI. Since all physicians were very proficient in Dutch, the questionnaire was in Dutch. The questionnaire started with one general question regarding their assessment of the HFA assessment . Next, the individual eyes were scored for pattern and severity of VFD. Rating of patterns was scored as follows (1) no defects, (2) defects due to glasses etc., (3) quadrantanopia (4) temporal hemianopia, (5) superior hemianopia (6) inferior hemianopia, (7) nasal hemianopia and, (8) other. Rating of the severity was scored as follows: low (0 points), mild (1 point), moderate (2 points), severe (3 points), very severe (4 points). Based on the assessment of the two eyes of one individual, recommendation of the indication and timing of surgical decompression .For the severity, average severity scores of all eight assessing physicians  were calculated. Additionally, average severity scores were calculated for the ophthalmologists only. Following the averaging of the scores, patients were divided into severity categories. Scores from 0 to 0.5 were considered low, 0.5 to 1.5 mild, 1.5 to 2.5 moderate, 2.5–3.5 severe and 3.5 and higher very severe. These obtained scores were compared to and associated with MD.All data was collected using Microsoft Excel  and graphs were made using GraphPad Prism 7 . For the systematic review, individual study data are depicted as median (interquartile range (IQR)) or n (%) unless otherwise specified. No comparative statistical analyses were performed because of the heterogeneity of the outcome data. For the cohort study, all data were tested for normal distribution and reported as mean ± SD or median (IQR), when applicable. Intra-class correlations (ICC) were calculated using Reliability Analysis. Spearman’s correlations were performed for severity scores and MD.The search conducted in December 2019 yielded 3322 articles, of which 3068 articles were excluded based on title and abstract . Ultimately, 44 articles were included , 15–57. All 44 articles were assessed using the Quality Assessment Tool (QAT), of which the results are shown in Supplementary Table 2. Twenty-two studies (50.0%) were classified as high-quality studies (> 39.7%). Of the QAT, three items were most important for the quality of this review: reported timing of surgery, pre-operative visual function tests, and postoperative visual function tests. Timing of surgery scores were low, as solely three studies reported this factor (sub-scores ranging from 1 to 3).As depicted in Supplementary Fig. 2a, preoperative VFDs were reported in 69.0%  of 2305 patients (48.1% of included population) in 27 studies (61.4%) , 53, 55.Preoperative impairment of VA was reported in 51.0%  of 1354 patients (28.3% of included population) in 12 studies (27.3%), as depicted in Supplementary Fig. 2d , 53, 55.With the exception of Visual Impairment Score (VIS) and Visual Evoked Potentials (VEPs), no other visual function parameters were assessed in the included articles. Nakao et al. reported a preoperative VIS of 73.1 ± 18.4, which improved to 41.7 ± 18.8 postoperatively . WatanabNone of the 44 included articles provided details of protocols or policies regarding timing of surgery in relation to severity of visual symptoms. As depicted in Table . stated that duration of visual symptoms significantly affected visual outcome based on the recovery rates in the following patient groups: CR in 90% patients and PR in 10% of patients with symptom duration < 6 months, CR in 54.5% of patients and PR in 45.5% of patients with symptom duration 6–12 months and, CR in 5% of patients and PR in 95% of patients with symptom duration > 12 months [When assessing the effects of time-dependent parameters, and surgical outcome, three studies stated that earlier surgical intervention improved postoperative results. Jahangiri et al. reported that patients with complete recovery (CR) had a median symptom duration of 3.5 months, whereas the median symptom duration was 12 months in patients with partial recovery (PR) . Nakao e2 months . Treatme. and Mortini et al. described improved outcomes with increased surgical experience [. reported that age at diagnosis was not predictive for recovery rates, although age correlated with increased symptom duration [Decompression of the chiasm on intraoperative imaging, surgical experience, age, and tumor size were reported as significant risk or protective factors in 7 of 44 articles (15.9%). Berkmann et al. reported that a decompressed optic chiasm on intra-operative MR imaging was correlated with postoperative VF and VA improvement . Magro eperience , 44. Morperience , 43. By duration . Additioduration .Of the 44 included studies, 7 specifically reported on patients with deterioration of visual function. Ferreli et al. stated that the surgical procedure was efficacious and safe with only one patient experiencing deterioration of visual acuity postoperatively . In a seOf the 30 studied patients, 15 were female (50%) and the mean age was 58.5 ± 14.8 years. Two patients had a giant adenoma (maximum diameter > 4 cm), and the remaining 28 patients had a macroadenoma (maximum diameter 1-4 cm). All 60 eyes were assessed, of which individual outcomes of the visual function tests are shown in Supplementary Table 3. Median MD was − 5.3 decibel (dB) (IQR − 3.1 to − 10.1) and median VFI was 91.5 (IQR 67.3–96.0).Reliability for pattern determination as assessed by ophthalmological physicians was low, with an ICC of 0.37 (95% CI 0.27–0.49). By contrast, for the severity scores as assessed by all physicians, the reliability was high, with an ICC of 0.81 (95% CI 0.74–0.87). Therefore, severity scores were averaged for all physicians, which were highly correlated with the MD of the 60 eyes . Following the averaging of the severity scores, patients were divided into the aforementioned severity categories. Severity grading was recorded as low in 7 eyes, mild in 20 eyes, moderate in 13 eyes, severe in 14 eyes, and very severe in 6 eyes. MD for the separate severity categories were assessed, and scores until − 2 dB were considered normal. MD for mild defects ranged from − 2 to − 4 dB, MD for moderate defects ranged from − 4 to − 8 dB, and MD for severe defects ranged from − 8 to − 17 dB. MD below − 17 dB were considered very severe.Of the 30 patients, four patients with radiological compression of the chiasm with still normal visual function were operated for other reasons. In the remaining 26 patients, the operation indication was worsening of VFD or VA. Therefore, treatment delay was calculated for these 26 patients, resulting in a median delay of 13 days (IQR 3–35). Patients were divided into the patient categories proposed below, as is shown in Fig. Decompression of the optic chiasm is without doubt very effective in improving visual function in the clear majority of patients. Improvement in VFDs and VA is to be expected in 87.0% , 50, 55,A clear limitation of the available literature includes the heterogeneity of the studied population. Patients present with VFDs and impaired VA prior to surgery, in 20–100% , 53, 55,Since clear grading systems to quantify the severity of VFD were unavailable, we aimed to provide an easy and objective alternative for reporting on the severity of VFD. For this purpose, we assessed which MD reflects expert-physician-judged severity of VFDs. MD is well known in glaucoma care and research, and recent reports have started to use this promising objective measure in sellar, and suprasellar tumors , 59. IntThe evidence obtained from the systematic review that a longer duration of symptoms is adversely related to postoperative visual outcome is weak. Duration of symptoms consists of two components: first, time between the start of symptoms and diagnosis (i.e. the diagnostic delay), and second, timing of surgery, defined as the time between diagnosis and surgical intervention (i.e. the treatment delay). Unfortunately, patients might not notice mild loss of peripheral vision, and therefore estimated diagnostic delay is unreliable. Longer symptom duration was related to decreased recovery rates , 45, andTreatment delay, in contrast to diagnostic delay, is a modifiable factor, but has been scarcely studied. Treatment delay was reported in only three studies, with extremely variable individual timeframes ranging from 1 day to 48 months following NFMA diagnosis, refraining from mentioning outcome and severity of visual dysfunction at presentation , 18, 35.Ultimately, all patients with an NFMA and concomitant compression of the optic chiasm are candidates for surgical removal of the adenoma. However, consequences of surgical delay will depend on multiple individual factors. In scarcity situations, identification and prioritization of patients at risk for adverse outcomes when treatment is delayed is pivotal. Consequently, uniforms definitions of visual (dys)function and treatment delay are needed. The following clinical recommendations address this clinical unmet need in a stepwise approach, according to the Plan-Do-Check-Act cycle: Plan: (1) Establishment of a patient classification based on severity or progression of visual dysfunction using a uniform clinician- and patient-reported outcome set. (2) Proposal of preferred and undesirable time frames for timing of surgery for each group for clinical benchmarking. Do: (3) Prospective data collection with proposed uniform outcome set and timeframes. Check: (4) Evaluation of data. Act: (5) Refinement of patient classification, outcome set, time frames based on evaluation of prospectively collected data, consensus discussion, and further development by a network of PTCOEs, and implementation in official guidelines.In the current management of pituitary adenomas, an optimal ophthalmological examination is paramount to ensure optimal timing of treatment . Two maiBased on these different categories, preferred, standard, and undesirable time frames for surgical intervention are proposed  , 65. TheAs an example, we provided data from clinical practice for clinical benchmarking in our patient cohort. Both preferred and undesirable treatment delay was seen in every patient group. Strikingly, preferred performance was best met in urgent and acute cases (Group 5), whereas standard and undesirable performance was common in severe and moderate cases (Group 3 and 4), reflecting a mismatch between desired and achieved delay in this group. In this small series, no significant adverse outcome was recorded. However, this patient stratification system needs to be validated in larger patient cohorts.The success of effective surgical decompression of the optic system in NFMA patients is very high as improvement of VFDs and VA is observed in the clear majority of patients. A practical conclusion from this systematic review is that in case of deterioration or no improvement of visual function after surgery, pituitary imaging should exclude hemorrhage. Providing uniform measures of severity of visual dysfunction from literature remains unattainable. Moreover, evidence regarding the optimal timing of surgical intervention for the individual patient is not available. However, patients with shorter treatment delay had higher postoperative recovery rates. We propose clinical recommendations to support future clinical benchmarking for the timing of surgical intervention in various categories of patients, based on objective measures of severity of VFDs and VA impairment. The proposed timelines need to be evaluated and adapted in future research projects, using international clinical networks of pituitary reference centers, such as PTCOE and Endo-ERN.Supplementary file1 (DOCX 25 kb)Supplementary file2 (PDF 94 kb)Supplementary file3 (PDF 156 kb)Supplementary file4 (DOCX 33 kb)Below is the link to the electronic supplementary material.


We identified multiple QTLs for each trait and revealed that additive effects from various loci drive the inheritance of Cd distribution, while epistatic effects between various loci contribute to differences in Cd tolerance. One pleiotropic locus, qCddis8, was found to affect the Cd distribution from both roots to shoots and from leaf sheaths to leaf blades. The results expand our understanding of the diversity of genetic control over Cd distribution and Cd tolerance in rice. The findings provide information on potential QTLs for genetic improvement of Cd distribution in rice varieties.Rice, a staple crop for nearly half the planet’s population, tends to absorb and accumulate excessive cadmium (Cd) when grown in Cd-contaminated fields. Low levels of Cd can degrade the quality of rice grains, while high levels can inhibit the growth of rice plants. There is genotypic diversity in Cd distribution and Cd tolerance in different rice varieties, but their underlying genetic mechanisms are far from elucidated, which hinders genetic improvements. In this study, a joint study of phenotypic investigation with quantitative trait loci (QTLs) analyses of genetic patterns of Cd distribution and Cd tolerance was performed using a biparent population derived from  Cadmium (Cd), a heavy metal element that is nonessential and toxic to most organisms, is ubiquitously distributed in soil as a consequence of anthropogenic activities, where it can bring serious damage to crop production . ExposurIn vitro, Cd is first absorbed in rice roots then released into the xylem. After xylem-to-phloem loading and detoxification processes, including sequestering in vacuoles and binding to cell wall compounds, absorbed Cd is distributed through vascular bundles (VBs) in nodes, which are interconnected and linked with roots, stems, sheathes, leaves, and panicles  for analysis of Cd distribution  or 25 μM Cd  for analysis of Cd tolerance . Additionally, two biological replicates were simultaneously cultured without Cd (pH 5.4) as controls in Cd tolerance experiments. Cd exposure treatments lasted 1 week, 1/2 KB solution was renewed every 2 days, and the pH was maintained at pH 5.4 using 1 M KOH solution.To investigate the differences in Cd distribution and Cd tolerance, physiological assays were carried out on RILs using hydroponic system with 1/2 Kimura B solution (KB) made from a 20× KB nutrient stock solution prepared as described previously . After p1, were screened for available markers, displayed as distinct PCR product polymorphisms between “93-11” and “IRAT129.” All STS markers were developed and labeled in a previous study  method with minor modifications . A totalus study . Meanwhietic map . The SSRetic map . A totaletic map . The graetic map . In geneetic map .4 conditions, 1-month-old seedlings were soaked in a solution of 5 mM CaCl2 for 15 min to remove adhered Cd, then rinsed three times with distilled water. To investigate the Cd distribution patterns, seedlings were partitioned into root, basal internode , leaf sheath (Ls), and leaf blade (Lb) samples. All tissue samples, including roots, nodes, and leaves  were then dried in an oven at 80°C to constant weight, and ∼0.3000 g of each sample was digested by concentrated nitric acid (100%) in a microwave digestion system . The Cd level of each digested solution was determined by inductively coupled plasma optical emission spectrometry (ICP-OES) using an Agilent 700 Series instrument . Blank and quality controls  were included in parallel to confirm the accuracy of Cd determinations.All tested individuals, including the two parental lines and the 147 RIL lines, were harvested after culturing in the presence of Cd in hydroponic assays. After culturing under 2.5 μM CdSO4 hydroponic assays were harvested. To investigate the phenotypic variation in Cd tolerance, the lengths of shoots and roots were measured after removing adhered Cd. Shoot length was measured from the basal internode to the tip of the longest leaf, root length was measured from the basal internode to the tip of the longest root, and five to eight individuals from each line were surveyed. Growth conditions for each seedling were defined as the growing ratio calculated as the growth ratio dividing the length of shoots to that of the roots. The relative growing ratios between control (0 μM CdSO4) and Cd exposure (2.5 μM CdSO4) groups were estimated to assess Cd tolerance in both parental lines and the lines from the RIL population.For Cd tolerance, plants simultaneously cultured in both 0 and 25 μM CdSO4 (Cd-Tol) was calculated to describe Cd tolerance differences in the RILs.In this study, we used normalization of phenotypic variation to enhance genetic factors to further investigate the variations in Cd distribution and Cd tolerance. Four indicators were developed. For Cd distribution, the Cd concentration ratio between shoots (leaves and nodes) and roots (as S/R), the Cd concentration ratio between developing tissues (leaf sheaths and leaf blades) to nodes (as Lf/Nd), and the Cd concentration ratio between leaf blades and leaf sheaths (as Lb/Ls) were calculated. S/R was used to describe Cd translocation from roots to shoots, Lf/Nd was used to describe Cd transportation from nodes to developing tissues, and Lb/Ls was used to describe Cd distribution between developing tissues. For Cd tolerance, the relative growth ratio between 0 and 25 μM CdSOP value for the stepwise regression-based likelihood ratio test was set at 0.05 for additive QTLs and 0.01 for epistatic QTLs. To avoid false-positive detection and ensure the reliability of QTL analysis, chromosomal regions containing marker loci with a logarithm of odds (LODs) score ≥2.50 were considered significant additive QTLs, and to declare a significant epistatic QTL, the threshold for the LOD score was set at 5.0. Other statistical analyses, such as linear regression and Student’s t-tests, were performed mainly with Microsoft Excel 2016 (Microsoft Corporation) and SPSS 18.0 (SPSS Inc. PASW Statistics for Windows).QTL identification was performed using the average performance of each trait from two biological replicates under an inclusive composite interval mapping (ICIM) approach with the QTL Icimapping 4.0 program, including additive QTLs and epistatic QTLs . ICIM wajaponica rice subspecies accumulated low levels of grain-Cd, while “93-11” from indica rice subspecies accumulated high levels of Cd in grains . By contrast, the value for Cd in the nodes of “93-11” was 22.4%, significantly higher than in “IRAT129” . Cd in leaf-sheath tissues of “93-11” and “IRAT129,” respectively, accounted for 17.7 and 16.4% of total Cd in plants, which was not significantly different (P = 0.401). These results indicate that more Cd remained in the roots and leaf blades in “IRAT129” than in “93-11.”The proportions of Cd in tissues relative to total Cd in plants were also estimated to explore Cd distribution differences between the two parental varieties . Cd distP = 4.98E-5; P = 0.003), while Lf/Nd was significantly higher in “IRAT129” than in “93-11” . The average Cd-Tol value for “IRAT129” was 4.04 under Cd stress compared with 4.40 for controls, indicating no significant difference (P = 0.106). Reduction curves were subsequently plotted  ranged from −0.021 to 0.175 (P > 0.05). These results suggest that the genetic basis for determining the Cd distribution between different tissues differs. Meanwhile, no significant correlation was detected between Cd-Tol and any of the Cd distribution indicators  was identified as pleiotropic. This QTL could explain 9.01% of the S/R variation in the RILs with a negative effect of −0.041, and this locus could also explain 7.20% of the Lb/Ls variation in the RILs with a negative effect of −0.019. According to the phenotypic variation, the qCddis8 allele from “IRAT129” could significantly decrease Cd transport from roots to shoots  and could explain 8.62% of Lb/Ls variations in the RILs with a negative effect of -0.021. According to the phenotypic variation after discounting other loci, the qCddis7.2 allele from “IRAT129” could distribute more Cd to leaf sheaths than to leaf blades  was identified, accounting for 5.43% of phenotypic variation with a positive effect of 0.068 (LOD = 2.69).To explore the genetic controls driving Cd distribution and Cd tolerance, QTL mapping approaches were performed for additive QTLs and epistatic QTLs using average values for four Cd distribution indicators and one Cd tolerance indicator from the RILs. The additive QTL mapping approach revealed a total of 19 additive QTLs explaining the variation in Cd distribution and one additive QTL for Cd tolerance . AdditivA total of 24 epistatic loci were identified for explaining the variation in Cd tolerance, and one epistatic locus was identified for explaining the variation in S/R for Cd distribution . EpistatP < 0.05; r) between the number of additive loci and the phenotypes were 0.26 for S/R and 0.23 in Lb/Ls . Therefore, the results revealed that the additive effects from these loci determined the variation in Cd distribution in the RIL population.In the present study, QTL analyses revealed that additive effects from loci, rather than interactions between loci, dominated the variation in Cd distribution in the RILs . Thus, iTogether, the results of QTL analysis revealed quite different genetic patterns between Cd distribution and Cd tolerance. Additive effects from loci played crucial roles in determining variation in Cd distribution, while interaction effects between different loci contributed to variation in Cd tolerance.indica and japonica rice.Extensive studies on Cd distribution and Cd tolerance in rice in recent decades has revealed details of the mechanisms underpinning these physiological processes . As a re4 treatment  responsible for Cd distribution were identified as additive QTLs , and mosAt present, geneticists and plant breeders are trying to develop rice varieties that accumulate low levels of Cd and exhibit better Cd tolerance to ease the risk of excessive Cd consumption and yield losses due to Cd toxicity . The finjaponica gene pools, such as QTLs on chromosome 7 (Another important aspect of the present study was exploring loci to systematically reduce Cd transport in rice . Althougmosome 7 , few allmosome 7 . Few allmosome 7 . After ejaponica rice variety “IRAT129” and indica rice variety “93-11.” The results showed that the genetic patterns controlling Cd distribution and Cd tolerance are quite different. Additive effects from different loci contribute to the inheritance of Cd distribution. By contrast, epistatic effects from loci are the main factors determining Cd tolerance variation. Meanwhile, one pleiotropic locus, qCddis8, was found to simultaneously affect Cd distribution from both roots to shoots and from leaf sheaths to leaf blades.In this present study, we performed a joint study of phenotypic investigations with QTL analysis to identify the genetic controls governing Cd distribution and Cd tolerance in a biparent population derived from The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.LS and W-BT: methodology and supervision. Y-BC and J-RW: validation. Y-BC and Y-CC: formal analysis. LS and Y-CC: investigation. Y-BC and SL: data curation. Y-XZ and H-bD: writing—original draft preparation. LS: writing—review and editing. LS, W-BT, Y-XZ, and H-bD: visualization. All authors have read and agreed to the published version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Caryodendron orinocense Karst.) oil is poorly investigated and explored. Thus, this study focuses on the physicochemical characterization , fatty acid composition as determined by gas chromatograph mass spectrometry (GC/MS), total phenolic content (TPC), and antioxidant activity  of cacay oil, coconut oil and a coconut/cacay oil blend, also known as cacay butter. The antibacterial activity of cacay oil was additionally evaluated. Our study demonstrated that cacay oil presents a high amount of polyunsaturated fatty acid (PUFA) (58.3%) with an emphasis on linoleic acid and a lower acidity value (2.67 ± 0.01 cg I2/g) than butter and coconut oil, indicating a low concentration of free fatty acids. In contrast, cacay butter and coconut oil presented higher saturated fatty acid percentages  and higher saponification values . The samples showed low moisture and relative density between 912 and 916 kg/m3. The hydrophilic fraction of cacay oil was highlighted in the quantification of TPC (326.27 ± 6.79 mg GAE/kg) and antioxidant capacity in vitro by DPPH radical scavenging assay (156.57 ± 2.25 μmol TE/g). Cacay oil inhibited the growth of Bacillus cereus (44.99 ± 7.68%), Enterococcus faecalis (27.76 ± 0.00%), and Staphylococcus aureus (11.81 ± 3.75%). At long last, this is the first study reporting the physicochemical characterization and bioactive properties of cacay butter. Coconut oil and cacay butter showed great oxidative stability potential due to higher contents of saturated fatty acids. Moreover, cacay oil presents as an alternative source of raw materials for cosmetic and biotechnology industries due to its high concentration of PUFA and for being a rich source of phenolic compounds.The Amazon region is rich in genetic resources such as oilseeds which have potentially important local commercial exploitation. Despite its high concentration of bioactive compounds, cacay ( Interest in non-conventional sources of oils and fats has increased due to increased demand for industrial use . The AmaCaryodendron orinocense Karst.) is a Euphorbiaceae plant which grows along the Andes base adjacent to the Amazonian lowland [Cacay  using either mechanical or thermal processing [Virgin coconut oil is an edible oil obtained from extracting a matured kernel from the coconut  oil and butter and coconut (Cocos nucifera L.) oil were kindly provided by Plantus LTDA . The cacay butter used in this study is a commercial product obtained by mixing 70% coconut oil and 30% cacay oil. This proportion is used to obtain a semi-solid consistency which is better-accepted by consumers (unpublished data). Samples were stored at 4°C in plastic containers. The present study was conducted under authorization from the National System for Management of Genetic Heritage and Associated Traditional Knowledge (SisGen) no. A679EA3.Cacay  and the results were expressed as kg/m3 at 20°C.Moisture and volatile matter (method Ca 2d-25), saponification (method Cd 3–25), iodine (method Cd 1d-92), and acidity (method Cd 3d-63) were determined in the cacay oil, cacay butter and coconut oil using the AOCS standard . DensityFatty acids were initially obtained using the conventional methylation procedure previously described by Hartman and Lago . The chrThe TPC in the HF was determined according to Singleton and Rossi , with soDPPH  radical scavenging assay in HF, LF, and total unfractionated samples (TS) was evaluated using the method by Brand-Williams et al. , with moABTS ) radical scavenging assay in HF, LF and total unfractionated samples (TS) was evaluated using the method by Rufino et al. , with moEscherichia coli ATCC 25912, Pseudomonas aeruginosa ATCC 27853, and Salmonella paratyphi ATCC 14028) and three Gram-positive .Antibacterial activity in the sample with higher values of polyunsaturated fatty acids was performed according to the methodology described by the Clinical and Laboratory Standards Institute (CLSI) , with moThe microorganism suspension in Mueller Hinton (MH) broth  was added to different sample concentrations (2.25–144.00 mg/mL) dissolved in 1% (v/v) tween 80 in a 96-well microplate. The microplates were then incubated at 35°C under shaking at 200 rpm . The optical density at 595 nm was determined at times 0 and 24 h.Wells containing MH broth, microorganisms, and saline solution 0.9% with 1% tween 80 were considered as positive growth control . Vancomycin (0.4 mg/mL) was added to the Gram-positive and gentamicin (0.3 mg/mL) to the Gram-negative bacteria for the microbial sensibility profile control.The inhibition percentage of bacterial growth was calculated according to pc represents the absorbance variation in the positive control , and Δabssample the absorbance variation in the sample.Where ΔAbsone-way analysis of variance (ANOVA) with Tukey HSD post hoc test, considering a 95% confidence level (p ≤ 0.05) as a significant result.Results were expressed as arithmetic mean ± standard deviation. The Statistica software  program was used for The results showed that saturated fatty acids are dominant for coconut oil (78.4%) and cacay butter 69.1%), especially lauric acid . Correia9.1%, espCacay oil, coconut oil, and cacay butter presented similar percentages of oleic acid among the detected monounsaturated acids. Lower results were reported by Correia et al.  and PéreLinoleic acid ω-6) was the most abundant fatty acid in cacay oil regarding polyunsaturated fatty acids (PUFA). Radice et al. [ was the Thus, the high presence of polyunsaturated fatty acids in cacay oil demonstrates its potential as a raw material for food, pharmaceutical, and cosmetic products since polyunsaturated fatty acids may have bioactive properties of industrial interest .3. The saponification value found for coconut oil was similar to that obtained by Mansor et al. [The density of the samples evaluated in the present study agrees with Abollé et al. , since vr et al.  by the cr et al. .2/g). This fact can be associated with the high PUFA content. On the other hand, a low iodine value was found for cacay butter due to the high saturated fatty acid concentration present (69.1%).The iodine value measures the degree of unsaturated fatty acids that can absorb halogens . A higheThe acidity value of cacay oil was very similar to that found by Radice et al.   was higher than in the coconut oil (292.06 ± 10.04 mg GAE/kg) and cacay butter (300.45 ± 4.62 mg GAE/kg) . Moreover, cacay oil has higher TPC than other oils reported in the literature. Seneviratne et al.  obtainedThe antioxidant contribution of the fractions and unfractionated samples was evaluated, as shown in Overall, cacay oil showed a promising antioxidant capacity for the evaluated methods when compared to coconut oil and cacay butter. Radice et al.  showed pHF showed significantly higher results (p<0.05) than the lipophilic fraction among the analyzed fractions for oil and cacay butter. This may be attributed to the presence of a larger TPC present in HF, since these compounds are capable of donating hydrogen to free radicals and inhibiting the propagation chain of reactions promoted by oxidative stress .In contrast, no significant difference was observed between antioxidant activities in the HF and LF (p = 0.072) of coconut oil. Such results may be justified by the complex composition present in vegetable oils and the synergistic effect with other phytochemical compounds not evaluated in the present study , 37.Cnidoscolus quercifolius) oil and its fractions. This suggests that the compounds present in the samples can lead to an antagonistic effect when mixed, thereby decreasing their antioxidant activity [Unlike that found by Espín et al. , the sumactivity .in vitro of cacay oil was evaluated due to the higher concentration of polyunsaturated fatty acids (58.3%), since the antibacterial activity can be attributed to the presence of these compounds [Natural oils have high biological potential due to the broad diversity of bioactive components . Thus, tompounds , 40. FigB. cereus, E. faecalis, and S. aureus strains at all tested concentrations. However, there was a decrease in the inhibition of bacterial growth in the concentrations of 144.00 and 2.25 mg/mL for E. faecalis. It is possible that, like some antimicrobial agents, the cacay oil had no exerted antibacterial action in a concentration-dependent manner. Physicochemical factors, such as solubility and diffusion, may account for this [Cacay oil showed active biological properties to inhibit the bacterial growth for Gram-positive for this .E. coli, P. aeruginosa, and S. paratyphi strains. This was probably due to structural differences in the outer membrane of bacteria. The thick layer of the lipopolysaccharide outer membrane of Gram-negative bacteria may have shown to be more resistant to a hydrophobic substance (cacay oil) compared with the Gram-positive bacteria, which possess a single peptidoglycan layer structure [On the other hand, no inhibition was observed for Gram-negative tructure . Accorditructure , the antTo the best of our knowledge, this is the first report regarding the antibacterial effects of cacay oil. Thus, given this potential of cacay oil, the results of the present study may support further research aimed at applying oil in topical formulations for skincare and repair. In addition, the use of cacay oil can be a useful strategy for obtaining products with a longer shelf life, as well as safer products due to their ability to slow or prevent the growth of contaminating bacteria.Oils and butter evaluated in the present study had distinct composition profiles and physicochemical characterization. Coconut oil and cacay butter showed high potential to oxidative stability due to high contents of saturated fatty acids such as lauric, myristic, and palmitic acids, which influenced the iodine value. Regarding cacay oil, its high concentration of polyunsaturated fatty acids (PUFA) such as linoleic acid and being a rich source of phenolic compounds presents it as an alternative source of raw materials for the cosmetic and biotechnology industries, which can be used as health promoting products. Satisfactory results in antioxidant and antibacterial activity reinforce its commercial exploitation. Furthermore, it is expected that additional refining of the oils will improve the product characteristics and stability.S1 Dataset(XLSX)Click here for additional data file.


Numerous studies have revealed that acupuncture can increase the somatic pain threshold. Electro-acupuncture (EA) can help pain-relieving with minimal physiologic disturbance. Various painful disorders, as well as pain following various surgeries, like cesarean section, gastrostomy, and enterectomy were managed properly with acupuncture. Therefore we studied the postoperative analgesic effect of EA in patients undergoing abdominal hysterectomy.A randomized, prospective clinical trial study was carried out on 56 women undergoing hysterectomy under spinal anesthesia. Patients were allocated randomly to receive either spinal anesthesia and electric ear acupuncture (EEA group) or spinal anesthesia alone (control group). EEA was done by fine needles to anatomically defined 4 points of the ear: Shen Men Point, thalamus Point 26, Analgesia Point 3, and Uterus Point 58, and connected to EA therapeutic apparatus. After finishing surgery, the fine needles were substituted by permanent press needles to be removed after 24 hours. The primary outcome was the postoperative 24 h morphine consumption by patient-controlled analgesia, while secondary outcomes included Post-operative pain scores and postoperative 1st request of analgesia.p-value =0.000). The postoperative pain scores were significantly reduced in the EEA group in comparison to the control group, with delayed 1st request of postoperative analgesia.Total morphine consumption in the first 24 postoperative hours was obviously reduced in the EEA group versus the control group  database .The trial was registered before enrolment of the first patient at the Pan African Clinical Trial Registry ( Acupuncture is considered as a traditional therapeutic technique with a long history of 4000 years. Electro-acupuncture (EA) is a new type of hand acupuncture that can deliver effective analgesia with a minute physiological disturbance , 2. It wGenerally, acupuncture can boost physical and emotional health through the release of certain neurochemicals from the nervous system, as well as affecting cerebral areas responsible for reducing pain and anxiety , 8. Acuphttp://www.randomizer.org). The allocation sequence was concealed in opaque numbered envelopes. The outcome assessor was another anesthesiologist who was blind to the study group. Inclusion criteria included patients aged between 21-60 years old and have an American society of anaesthesiologist (ASA) physical status I & II. Patients who have contraindications for spinal anesthesia as , allergy to morphine, and/or bupivacaine, morbid obesity (body mass index (BMI) > 35 Kg/m2), psychological disease, narcotic abuse, prolonged use of sedatives or opioid drug use, contraindications for ear acupuncture (infection at the acupuncture site) and patients refused to participate in the study were excluded. The primary objective was to assess the influence of auricular acupuncture regarding the postoperative analgesic requirement in patients scheduled for abdominal hysterectomy, while secondary objectives were intraoperative hemodynamic parameters, Post-operative pain scores, and Postoperative 1st request of analgesia.The current study is a single-blinded randomized controlled clinical trial. After Obtaining approval by the Hospital Ethics Committee, and Written informed patient consent with an explanation regarding the purpose, methods, effects, and complications of techniques, 56 patients were randomly allocated into two equal groups using simple random tables generated from a computer software program (Group I (EEA group): 28 females received electric ear acupuncture after spinal anesthesia.Group II (control group): 28 females received spinal anesthesia without ear acupuncture.Explanation of the whole procedure including the expected benefits and complications was done. All Patients were instructed preoperatively for the use of the PCA apparatus.The patient’s age, ASA status, and BMI were recorded. Medical history was reviewed to rule out excluded patients from the study. General examination, vital signs: heart rate (HR), blood pressure (BP), temperature, chest, heart, abdominal examination, and airway evaluation were done. Investigations done were complete blood count, random blood sugar, bleeding profile as prothrombin time, International Randomized Ratio, and partial thromboplastin time.The patient was reassured and the procedure was explained to her. Airway devices and anesthesia machines, ventilators, flowmeters, and equipment were checked promptly. Routine Monitoring equipment (Datex-Ohmeda®) was attached to the patient including 3 leads electrocardiogram (ECG), non-invasive BP, and pulse oximetry. An 18- gauge intravenous cannula was inserted into the patient’s forearm and 500 mills of intravenous (I.V.) fluid (ringer acetate) were administrated. No premedications were given. All patients in both groups were given spinal anesthesia by 25 G needle with hyperbaric bupivacaine 0.5% (15-20 mg) according to the patient’s height, in the L4- L5 intervertebral space. During spinal injection, no supplementary opioids were given; the level of anesthesia was assessed by pin-prick every 5 min in the first 20 min till it reached T4-T6 which would be considered sufficient to start the surgery.Acupuncture was done based on our institutional experience. EEA was done by fine needles (10 mm x 30guage) to the anatomically defined 4 points of ear acupuncture which are called; Shen Men Point (Divine Gate Point) or point 55 which is a sedative analgesic anti-inflammatory point, thalamus Point 26 which is an analgesic point in acute and chronic severe pain, Analgesia Point 3, and Uterus Point 58 , 8.Then four pre-sterilized single-use acupuncture needles were inserted in the predetermined acupoints and connected to clamps of electrode cords that were connected to the electro-acupuncture therapeutic apparatus  and oxygen saturation were recorded and analyzed every 5 min in the first 20 min, then every 20 min for the rest of the surgery. At the end of the operation, the duration of surgery and spinal level were recorded for both groups, and Diclofenac potassium 75 mg was administered to all patients by intramuscular injection. Postoperative BP and HR were recorded immediately after transfer to the post-anesthesia care unit (PACU), then after 1, 3,6,12, and 24 hours. Post-operative pain assessment was done by visual analog scale .when the reported visual analog scale (VAS) was 3 or more, a loading dose of morphine 0.05 mg/kg was administered via slow I.V. route. Then, a PCA was administered. The PCA pump was loaded with 1 mg / ml of morphine and set to deliver on-demand doses of 2 ml with 10 min lockout intervals and 4 h limit of 20 mg. No background infusion was allowed. The pain score was reassessed at the end of the 1st postoperative 24 hours. The time to the 1st postoperative request of analgesia was recorded using the PCA delivery system. The total amount of morphine consumption over the 1st 24 hours postoperatively was recorded. Side effects associated with morphine consumption as nausea, vomiting, and over sedation were recorded. Nausea and vomiting were assessed using a 5 point scale from zero to four, where 0 is no nausea or vomiting, 1 = mild nausea, 2 = severe nausea, 3 = vomiting once, 4 = vomiting more than one. These assessments were performed in the PACU immediately after discharge and after 1, 3, 6, 12, and 24 h postoperatively. Side effects associated with auricular acupuncture-like bleeding, excessive pain, fainting, broken needles, and local infection were recorded.. Normality was assessed using the Kolmogorov–Smirnov test and visual inspection of histograms. For demographic and baseline comparison, the standardized difference was calculated using a web-based effect-size calculator [P-value < 0.05 was considered significant. Presentation of the statistical outcomes in the form of tables and graphs was performed using the “Microsoft Office Excel® 2010” program.The statistical analysis was performed using IBM SPSS Statistics® 22 for Windows 10 operating systemlculator . 2-taile24 patients per group were needed to detect 5.39 mg difference between the means of total 24 hours postoperative morphine consumption in patients undergoing cesarean section between electro-acupuncture and control groups, at a standard deviation of 5.18 , with 95Fifty-six patients were randomly assigned for two equal groups EEA group and the control group Fig. Fig. 2FlThe two groups were comparable in age, BMI, ASA status, history of chronic illness, duration of surgery, and level of the block at end of surgery. The duration of surgery was about 80 min in both groups. Table  no statip-value =0.000). Total post-operative 24 hours morphine consumption was markedly reduced in favor of the EEA group (6.214± 2.1319 mg) versus (15.714 ± 3.3428 mg) in the control group with statistically significant difference . postoperative VAS score after 24 hours was lower in the favor of the EEA group in comparison to the control group (mean ± SD: 2.536 ± 1.1380VS 5.036 ± 1.3467) in both groups respectively with statistically significant difference .  in both groups respectively with statistically significant difference  between the two groups regarding the incidence of nausea at 1 h, 3 h, 6 h, and 12 h in the favor of the EEA group. .  can influence pain threshold in the abdominal area; they advocated that LFEA can be helpful for analgesia following abdominal surgery .The effect of LFEA and TENS on the deep pain threshold at the skin surface and deeper layers has been examined by a few studies , 18. KitSeveral mechanisms for acupuncture pain have been reported as the release of spinal natural opioids like dynorphin, endorphin, and encephalin, which are potent pain suppressors, increase in local blood flow that encourages the process of healing, gate control theory of pain, the endogenous release of adrenocorticotrophic hormones, myofibrillary entanglement relaxation, and creating a balance in the mesolimbic neural pain pathway .In 2016, De Freitas et al., have reported in an animal study that acupuncture has a pre-emptive analgesic effect . Sim et In the current study, we decided to use preoperative EEA along with continuous postoperative acupoint stimulation by press needles to achieve the maximum analgesic effect. Our study demonstrated that the total postoperative PCA morphine consumption in the first 24 hours was reduced in the EEA group versus the control group; also the time of the first request for supplemental analgesia was delayed in the EEA group when compared with the control group.Similar to our findings: In a prospective, randomized study done by WU Hung-Chien et al.  and condIn our study, we had a delayed first morphine request and less total morphine dose which may be due to our usage of diclofenac potassium by intramuscular injection, the pre-emptive effect of acupuncture, and also acupuncture needles were replaced in the EEA group by press needles.In our study, the VAS pain scores were lower in the EEA group versus the control group. Similarly, WU Hung-Chien, et al.  found thSimilarly, Iacobone,m et al. 2014 , found lChen, C. C et al., 2015 , have inIn our study, there was a statistical difference between both groups as regards the incidence of nausea at some time points in the favor of the EEA group; also we found a significant difference between both study groups concerning the incidence of vomiting after 6 h in the favor of acupuncture group. This may be explained by a reduced total amount of 24 hours of morphine consumption in the acupuncture group in comparison to the control group.Usichenk et al. 2004  found thP < 0.05), with markedly improved passive knee motion by the third postoperative day in patients treated with acupressure (P < 0.05) [Chang et al. 2012  have con < 0.05) .Lastly, we have some limitations. The analgesic effect found in the current study can be the result of either preoperative EEA, continuous post-operative acupoint stimulation by press needles, or combined effect of both techniques. Comparison between EEA and pressure acupuncture in the same acupoints is recommended in future studies. The other limitation in the current study was the use of a single postoperative non-opioid analgesic. In future studies, at least two non-opioid analgesics should be used to determine the real influence of acupuncture on postoperative analgesia requirements.Auricular acupuncture is a useful analgesic adjuvant as it reduced postoperative analgesic requirements following abdominal hysterectomy. It provided more patient satisfaction regarding postoperative pain control without any hazard.


Escherichia coli, the predominant fecal indicator bacteria isolated from a major urban river of northern India Yamuna. The genetic environment of blaCTX–M–15 was also investigated. Our results indicated that 57.5% of the E. coli strains were resistant to at least two antibiotic classes and 20% strains were multidrug resistant, i.e., resistant to three or more antibiotic classes. The multiple antibiotic resistance index of about one-third of the E. coli strains was quite high (>0.2), reflecting high contamination of river Yamuna with antibiotics. With regard to plasmid-mediated AMR genes, blaTEM–1 was present in 95% of the strains, followed by qnrS1 and armA (17% each), blaCTX–M–15 (15%), strA-strB (12%), and tetA (7%). Contrary to the earlier reports where blaCTX–M–15 was mostly associated with pathogenic phylogroup B2, our study revealed that the CTX-M-15 type extended-spectrum β-lactamases (ESBLs) were present in the commensal phylogroups A and B1, also. The genetic organization of blaCTX–M–15 was similar to that reported for E. coli, isolated from other parts of the world; and ISEcp1 was present upstream of blaCTX–M–15. The integrons of classes 2 and 3 were absent, but class 1 integron gene intI1 was present in 75% of the isolates, denoting its high prevalence in E. coli of river Yamuna. These evidences indicate that due to high prevalence of plasmid-mediated AMR genes and intI1, commensal E. coli can become vehicles for widespread dissemination of AMR in the environment. Thus, regular surveillance and management of urban rivers is necessary to curtail the spread of AMR and associated health risks.Globally, urban water bodies have emerged as an environmental reservoir of antimicrobial resistance (AMR) genes because resistant bacteria residing here might easily disseminate these traits to other waterborne pathogens. In the present study, we have investigated the AMR phenotypes, prevalent plasmid-mediated AMR genes, and integrons in commensal strains of  Escherichia coli. Besides its natural habitat, E. coli is also found in secondary habitats like aquatic and terrestrial reservoirs   are PCR-amplified , plasmids, and transposons  , multilomplified . Based omplified . Severalnsposons . Integronsposons  because E. coli populations and an ideal ecological niche for studying strains with diverse phenotypes, genotypes, and AMR. In an earlier study published from our laboratory, we had described the β-lactam susceptibilities and β-lactamase genes in 61 E. coli strains of all phylogroups  isolated from river Yamuna  of E. coli from river Yamuna, India.River Yamuna is a major river of northern India, which is associated with several anthropogenic activities of the population residing in the National Capital Region of India. It gets contaminated with effluents originating from hospital and municipal wastewaters; discharge from livestock, poultry, and agriculture production plants; industries; etc. The high levels of pollutants are expected to provide a positive selection pressure for increasing AMR in bacterial population residing there . Thus, ir Yamuna . Though Escherichia coli was performed using a published method (E. coli colonies were selected and maintained as pure cultures on Luria–Bertani (LB) agar slants at 4°C. Of these, 126 isolates were presumptively identified as E. coli using API 20E strips . API 20E is a standardized kit of biochemical tests used to identify members of the family Enterobacteriaceae and other non-fastidious Gram-negative rods.Two hundred water samples were collected from different sites along the entire stretch of the river Yamuna, which flows through the National Capital Region of India in sterile screw-capped bottles, transported to the laboratory on ice, and processed within 6 h of the sample collection. A schematic figure showing the details of the sampling sites has been published earlier . Enrichmd method . BrieflyE. coli strains using the boilinglysis method , 200 μM of the four dNTPs, 1 U of Taq DNA polymerase , 10 pmol of forward and reverse primers, and 1 ng of genomic DNA in a final volume of 25 μl. The PCR conditions and methods for purification of PCR amplicons and sequencing have been described earlier  and sequenced at a commercial facility using Sanger sequencing . The nucleotide sequence homology was analyzed using the nucleotide BLAST (BLASTn) algorithm available at the National Center for Biotechnology Information (NCBI).DNA was extracted from the s method . The gen earlier . BrieflyThe phylogenetic profiles of all the isolates were determined by triplex PCR ; and 40 E. coli strains for various classes of antibiotics like β-lactams, aminoglycosides, quinolones, and tetracycline were determined by Kirby–Bauer disk diffusion method. The antibiotic disks  that were used in this study were (charge in μg/disk) as follows: ampicillin (10 μg), piperacillin (100 μg), amoxicillin–clavulanic acid (20/10 μg), cefazolin (30 μg), cefuroxime (30 μg), cefotaxime (30 μg), cefepime (30 μg), streptomycin (10 μg), kanamycin (30 μg), tobramycin (10 μg), netilmicin (30 μg), amikacin (30 μg), nalidixic acid (30 μg), ciprofloxacin (5 μg), ofloxacin (5 μg), and tetracycline (30 μg). The results of antimicrobial susceptibility testing were interpreted following the guidelines of Clinical and Laboratory Standards Institute (E. coli strain exhibited resistance was divided by 16 because susceptibility of each strain was tested for 16 antibiotics. The E. coli strains were tested for production of extended-spectrum β-lactamases (ESBLs) using a phenotypic confirmatory test recommended by the CLSI ; (ii) aac(6’)-Ib-cr acetyltransferase (aac), which modifies fluoroquinolones like ciprofloxacin and enrofloxacin; and (iii) active efflux pump (qepA).The presence of genes encoding plasmid-mediated quinolone resistance (PMQR) was determined by PCR amplification of genes encoding for (i) proteins that protect DNA from quinolone binding  of a representative strain (KP20) was submitted to GenBank (NCBI) with the accession number KF040057. The partial CDS of blaTEM–1 was also identical to each other; hence, the sequence of one representative strain (IP1N) was submitted to GenBank (NCBI) with the accession number KF055435. The partial CDS of qnrS was also identical; hence, the CDS of a representative strain (KP20) was submitted to NCBI GenBank under accession number KF055436. The partial CDS of tetA gene of all the three strains  was submitted under the accession numbers KJ409940–KJ409942.Gene sequencing revealed that the Escherichia coli. The results of the triplex PCR and Clermont classification indicated that 50% (n = 20) of the strains belonged to phylogroup A  while 50% (n = 20) to phylogroup B1 . Earlier studies have reported that E. coli strains of all phylogroups were present in river Yamuna  were r Yamuna . Phylogr strains . Several strains  but thatn = 38) of the commensal E. coli strains wereresistant to ampicillin, while 32% (n = 13) of the strains were resistant to piperacillin. Among the cephalosporins, 42.5% (n = 17) strains were resistant to cefazolin , 17.5% (n = 7) to cefuroxime , 22.5% (n = 9) to cefotaxime , and 15% (n = 6) to cefepime . The fact that commensal waterborne E. coli were less resistant to new-generation cephalosporins than ampicillin is normal because ampicillin was a widely prescribed broad-spectrum penicillin, and over time, bacteria might have developed resistance to this antibiotic. Earlier studies have also reported that ampicillin resistance was highly prevalent in commensal strains of E. coli isolated from India and from other parts of the globe like Vietnam, China, Sudan, and Thailand (n = 7) of the commensal E. coli strains tested positive, while none of the strain tested positive for AmpC production (n = 4) of the ESBL producers were resistant to ciprofloxacin. Since, penicillins and cephalosporins are the most frequently used antibiotics in India, it is normal that all the seven ESBL-producing E. coli strains were resistant to many antibiotics of these classes. The fact that some ESBL producers were also resistant to ciprofloxacin and streptomycin/kanamycin suggests that besides β-lactam antibiotics, resistance to other antibiotic classes also exhibited co-selection.Antibiotic susceptibility testing revealed that 95% (Thailand . With reoduction . All then = 14) strains were resistant to older quinolones like nalidixic acid, while 20% (n = 8) and 15% (n = 6) strains were resistant to newer quinolones like ciprofloxacin and ofloxacin, respectively. In this regard, our results are similar to those of other studies that reported lower ciprofloxacin and ofloxacin resistance in waterborne E. coli isolated from other parts of the world (n = 5) strains resistant to older aminoglycosides like streptomycin and less than 7% strains resistant to new aminoglycosides like kanamycin, tobramycin, netilmicin, and amikacin. An earlier study also reported that a low level of aminoglycoside resistance was present in E. coli strains isolated from aquatic environments of Kuala Lumpur, Malaysia (n = 11) of the strains exhibited resistance. The frequent use/misuse of ampicillin, streptomycin, and tetracycline due to frequent prescription, availability, and affordability might be a probable reason for higher bacterial resistance to these antibiotics (n = 23) E. coli strains were resistant to at least two antibiotic classes, and 20% (n = 8) of the strains were multidrug resistant (MDR), i.e., resistant to three or more antibiotic classes. MDR E. coli were defined as bacteria resistant to antibiotics belonging to three or more antimicrobial classes (E. coli strains were quite high (>0.2). E. coli strains observed in this study is not surprising because river Yamuna is highly contaminated with effluents originating from hospital and municipal wastewaters; discharge from livestock, poultry, and agriculture production plants; etc.With regard to quinolone resistance, 35%  followed by blaCTX–M–15, which was present in 15% (n = 7) of the E. coli strains. The plasmid-encoded AmpC enzymes (CMY types) were not found in any strain. The presence of blaTEM–1 correlated well with ampicillin resistance in all the strains. Similarly, all the blaCTX–M–15-positive strains showed phenotypic production of ESBLs. Earlier studies had reported that blaTEM–1 was widely present in E. coli strains isolated from water bodies of India andother countries like Spain, Australia, France, China, and Poland  and D  and D . HoweverstrA-strB genes are the most widely prevalent streptomycin resistance genes in E. coli worldwide and encode for phosphotransferases (strA-strB genes were present in only 12% (n = 5) of the waterborne E. coli. Of these, four strains exhibited phenotypic resistance to streptomycin, while one strain (ISF), despite harboring strA-strB, was phenotypically susceptible for streptomycin. The 16S rRNA methylases methylate certain amino acid residues of the 16S RNA, resulting in resistance to amikacin, tobramycin, gentamicin, and netilmicin (armA was found to be present in 17.5% (n = 7) of the strains. Earlier studies also reported that a low level of aminoglycoside resistance was present in E. coli strains isolated from aquatic environments in Kuala Lumpur, Malaysia  tested, only qnrS was detected in the E. coli strains isolated from river Yamuna. The qnrS1 was detected as the predominant PMQR gene in about 17% of the aquatic strains (n = 7). Earlier studies have also reported that the qnrS type gene was the most frequently detected PMQR gene in E. coli isolated from environmental E. coli worldwide  of the strains. Earlier studies have also indicated that tetracycline efflux-related genes like tetA, tetB, and tetC were more prevalent than ribosomal protection-related genes (like tetM and tetW) in waterborne E. coli  harbored the blaCTX–M–15 gene. The upstream region of the blaCTX–M–15 was analyzed in these seven strains by PCR amplification and sequencing. Gene sequencing revealed that the ISEcp1 was present in the upstream region of blaCTX–M–15 and orf477 was present in the downstream region of all the seven strains  and −10 (TACAAT) regions were present within 3′ terminus end of ISEcp1 and 48 bp away from the blaCTX–M–15 start codon. The same organization was previously reported from E. coli strains isolated from different countries of the world  of the isolates  of the intI1-positive E. coli strains. Three different types of gene cassette arrays of class 1 were present in the downstream region of intI1 whose size ranged from 1.7 to 2.8 kb (dhfr12 and dfrA1), aminoglycoside (aad) resistance gene family , and chloramphenicol (CHL) resistance gene family (catB3) were present (intI1 in Indian aquatic isolates was quite high (75%) and alarmingly more than that reported for E. coli isolated from global aquatic environments , blaCTX–M–15 (15%), strA-strB (12%), and tetA (7%). Though most of the earlier studies have reported that blaCTX–M–15 in waterborne E. coli was mostly present in pathogenic phylogroup B2, our study revealed that CTX-M-15 type ESBLs were present in the commensal phylogroups A and B1, also. The genetic organization of blaCTX–M–15 was similar to that reported for E. coli globally, and ISEcp1 was present in the upstream region of blaCTX–M–15. Though integrons of classes 2 and 3 were absent, class 1 integron gene intI was detected in 75% of the isolates, which indicates its high prevalence in the E. coli isolates of the river Yamuna than that reported, globally. The presence of MDR phenotypes, plasmid-mediated AMR genes, and class 1 integron gene intI1 in E. coli is a serious public health risk, because these commensal strains can become potent vehicles for widespread dissemination of AMR determinants to pathogenic E. coli and other waterborne pathogens. Thus, our study suggests an urgent need for regular surveillance and management of natural water bodies to curtail the spread of antibiotic resistance in microorganisms.Our results indicated a high prevalence of drug resistance in The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material.NSS, NS, MK, and JV analyzed the data. NSS and NS wrote the manuscript. JV conceptualized and supervised. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To investigate the potential value of volumetric iodine quantification using preoperative dual-energy computed tomography (DECT) for predicting microvascular invasion (MVI) of hepatocellular carcinoma (HCC).O1, tumor plus outer layer 1; VOIO2, tumor plus outer layers 1 and 2; VOII1, tumor minus inner layer 1; VOII2, tumor minus inner layers 1 and 2). In addition, qualitative CT features, including peritumoral enhancement and tumor margin, were assessed. Qualitative and quantitative CT features were compared between HCC patients with and without MVI. Diagnostic performance of DECT parameters of layers and VOIs was assessed using receiver operating characteristic curve analysis.This retrospective study included patients with single HCC treated through surgical resection who underwent preoperative DECT. Quantitative DECT features, including normalized iodine concentration (NIC) to the aorta and mixed-energy CT attenuation value in the arterial phase, were three-dimensionally measured for peritumoral and intratumoral regions: (i) layer-by-layer analysis for peritumoral layers  and intratumoral layers (inner layers 1 and 2) with 2-mm layer thickness and (ii) volume of interest (VOI)-based analysis with different volume coverage  and without MVI (n = 22) were included. HCCs with MVI showed significantly higher NICs of outer layer 1, outer layer 2, VOIO1, and VOIO2 than those without MVI . Among the NICs of layers and VOIs, the highest area under the curve was obtained in outer layer 1 (0.747). Qualitative features, including peritumoral enhancement and tumor margin, and the mean CT attenuation of each layer and each VOI were not significantly different between HCCs with and without MVI (both P >  0.05).A total of 36 patients  with MVI (Volumetric iodine quantification of peritumoral and intratumoral regions in arterial phase using DECT may help predict the MVI of HCC. Hepatocellular carcinoma (HCC) is the most common primary liver cancer and is the second leading cause of cancer-related deaths in the Asia-Pacific region . Liver rPrevious studies reported that several imaging features of the tumor border on preoperative imaging were associated with MVI of HCC. These features include non-smooth tumor margins, peritumoral enhancement in the arterial phase, peritumoral hypointensity in the hepatobiliary phase, and radiogenomic algorithm based on the association between imaging features  –10. ThisTherefore, the purpose of this study was to identify the potential diagnostic value of iodine quantification in the peritumoral region of HCC by volumetric tumor segmentation with dual-energy computed tomography (DECT) for identifying MVI.The institutional review board of our institution approved this retrospective study and waived the requirement for informed consent.We retrospectively searched our radiology database system between April 2017 and December 2019 to identify eligible patients according to the following criteria: (a) underwent four-phase liver CT at dual-source dual-energy CT ; (b) underwent hepatic resection within 4 weeks after CT; and (c) surgically confirmed single, treatment-naïve HCC. The exclusion criteria were as follows: (a) preoperative local treatment for the index tumor; (b) < 1 cm, which may increase registration error; and (c) definite macrovascular invasion.All surgical specimens were examined by experienced pathologists. The histologic parameters included size, number, Edmondson-Steiner grade, and MVI status of the resected tumor. MVI was defined as the presence of tumor cells in the portal vein, hepatic vein, or a large capsular vessel of the surrounding hepatic tissue lined by the endothelium that was visible only on microscopy. Additionally, the histological grade of fibrosis in the background liver parenchyma was reported on the basis of the staging system for chronic hepatitis by the Korean Study Group for the Pathology of Digestive Diseases  .Liver CT consisted of precontrast, arterial, portal venous, and equilibrium phases. Precontrast, portal venous, and equilibrium phase images were obtained using 90-kVp tube energy before, 70 s, and 180 s after contrast media administration  with weight-based dosing (1.6 mL/kg).The arterial phase was scanned using dual-energy 17 s after the attenuation of the abdominal aorta reached 80 Hounsfield unit (HU) at 100 kVp, using the care bolus technique of the vendor. For arterial phase dual-energy scanning, a tube potential pair of 80/150 kV with a tin filter was used. The quality reference effective mAs was set to 250 mAs for the 80-kV tube and 125 mAs for the 150-kV tube. Detector configuration, gantry rotation time, and pitch were 192 × 0.6 mm, 0.5 s, and 0.6, respectively. Images were reconstructed with semi-smooth quantitative body kernels in all phases.The 80- and 150-kV images of the arterial phase were imported into the prototype software . The software automatically creates three image sets: the arterial phase of the blended images with a mixed ratio of 0.6 (60% 80 kV and 40% tin-filtered 150 kV), virtual noncontrast, and material density iodine images. Image analysis was performed in mixed-volume images. Two fellowship-trained body radiologists  identified the HCC in the mixed-volume images and independently drew a region of interest (ROI) across the maximum dimension of the tumor in any plane   . In addition, five VOIs were generated with different volume coverage  to minimize intersubject variations. The software estimates the NIC by dividing vital iodine concentration of the ROI by the iodine concentration in the aorta. Additionally, the software automatically performs segmentation of the entire liver parenchyma to evaluate lesion-to-normal parenchyma ratios of mean attenuation by dividing the mean attenuation of the ROI into those of the normal liver parenchyma.One radiologist  blinded to the histologic findings performed the measurement. Additional measurements were performed in separate session by same radiologist (T.M.K.) at least 2 weeks apart and in one session by another radiologist  to evaluate intraobserver and interobserver reliability, respectively. In addition, the aforementioned quantitative analysis was performed in the same manner but with different layer thicknesses (4 mm) to evaluate the effect of different peritumoral or intratumoral areas.Two fellowship-trained body radiologists , who were blinded to the clinical and pathological data, independently assessed the images to evaluate the following qualitative features for MVI: (a) presence of peritumoral enhancement, which was defined as the existence of a detectable enhancing portion adjacent to the tumor border in the arterial phase, later becoming isoattenuation in the equilibrium phase , 17, andOur primary end point was to evaluate the association between the NIC of peritumoral regions on DECT and MVI in HCC. Secondary end points were as follows: (a) to compare the diagnostic performance of peritumoral NIC with qualitative analysis by a human observer for identifying MVI, (b) to compare the diagnostic performance of peritumoral NIC with peritumoral mean attenuation for identifying MVI, and (c) to investigate the effect of different peritumoral areas (2 mm vs. 4 mm) on the diagnostic performance of MVI.P < 0.10 in univariate analysis were applied to binary logistic regression analysis. Continuous variables were converted to categorical variables with optimal cutoff values to perform multivariate analysis. Among the NIC of multiple layers and VOIs, only one layer or VOI that showed the highest AUC was included in the multivariate analysis to avoid multicollinearity of parameters.To compare parameters between the HCCs with and without MVI, continuous data were assessed using the Mann-Whitney test, while categorical variables were analyzed using the chi-square test. Additionally, to investigate the association between subjective peritumoral enhancement and DECT parameters of peritumoral layers, the mean attenuation and layer-to-normal parenchyma ratio of mean attenuation of peritumoral layers were compared between the groups with absent and present peritumoral enhancement. A receiver operating characteristic (ROC) curve analysis was performed using quantitative parameters of layers and VOIs and qualitative parameters to determine the diagnostic performance for the prediction of MVI. Areas under the ROC curves (AUCs) with 95% confidence intervals (CIs) were calculated for the significant CT parameters, and a comparison analysis of AUCs was performed. Variables with P < 0.05 were considered statistically significant.Intraobserver and interobserver agreements were evaluated using intraclass coefficient class (ICC) and Cohen κ-statistics. The agreements of DECT parameters in volumetric HCCs between manual and semiautomatic segmentation, and agreements of maximal diameter of HCCs obtained in preoperative CT by both segmentation techniques and histopathology were also evaluated using ICC. The strength of agreement via ICC and κ values < 0.4, 0.4–0.6, 0.6–0.8, and >  0.8 were categorized poor, moderate, good, and excellent agreement, respectively. Statistical analyses were performed using a statistical software package . Differences of n = 7), small size (< 1 cm) of the HCC (n = 2), and macrovascular invasion (n = 2). Finally, 36 patients  with MVI (n = 14) and without MVI (n = 22) were included. The median tumor size was 2.7 cm . The surgical procedures included segmentectomy (n = 23), right anterior sectionectomy (n = 1), left lateral sectionectomy (n = 3), right hemihepatectomy (n = 2), left hemihepatectomy (n = 3), and total hepatectomy for transplantation (n = 4). The mean time between dual-energy CT and surgery was 16 days . The detailed information of the study group is summarized in Table A total of 47 patients with HCC underwent preoperative liver CT within 4 weeks of hepatic resection. Among them, 11 patients were excluded because of preoperative transarterial chemoembolization . The group with MVI showed significantly higher serum alpha-fetoprotein (AFP) levels  compared with those without MVI .Between the two groups with and without MVI, no significant difference was observed in age, sex, histologic grade of hepatic fibrosis, underlying liver disease, and Edmondson-Steiner grade of HCCs .The NICs of outer layer 1, outer layer 2, VOIP > 0.05).For entire volumetric HCCs, quantitative parameters including total volume, maximal diameter, mean HU in mixed-energy images, lesion-to-normal parenchyma ratio of mean attenuation value, total iodine concentration, and NIC showed no difference between HCCs with and without MVI (P > 0.05).The AUCs of NICs of layers and VOIs ranged from 0.67 to 0.75 Table , Fig. 3.P = 0.10, 0.13). The AUCs of peritumoral enhancement and non-smooth tumor margin were 0.64 (95% CI 0.45–0.83) and 0.63 (95% CI 0.44–0.82), respectively. The AUCs of the NICs and qualitative image features showed no significant differences (P > 0.05).Peritumoral enhancement of HCCs was present in 36.4% (8/22) of HCCs without MVI and in 64.3% (9/14) of HCCs with MVI. The MVI absent group showed non-smooth tumor margins in 45.5% (10/22), while the MVI present group showed 71.4% (10/14). There were no significant differences between the groups with and without MVI in peritumoral enhancement and non-smooth tumor margins (P = 0.05), larger maximal diameter (P = 0.04), and higher NIC of outer layer 1 (P = 0.02) than the MVI absent group. In multivariate analysis, NIC of outer layer 1 was the only independent factor for predicting MVI, with an odds ratio of 7.14  level . However, the mean HU and NIC showed no significant difference between the two groups .The mean attenuations of all layers and VOIs showed no significant difference between the two groups . There were no significant differences between areas in a pairwise comparison of ROC curves, including the values obtained in 2-mm layer thickness .In the 4-mm layer thickness, the NICs of outer layer 1, VOIICC = 0.97–1.0, Supplementary Table 4, Additional file ICC = 0.94) and semiautomatic  segmentation methods.The agreements of DECT parameters of volumetric HCCs between two segmentation techniques were excellent . In qualitative parameters, interobserver and intraobserver agreements were good to excellent and excellent, respectively  also showed different NIC values between the MVI present and absent groups with comparable AUCs to those of peritumoral regions only. This might imply that MVI status could be predicted by analyzing NICs of both intratumoral and peritumoral regions as well as peritumoral regions only. We also found that the NIC of the peritumoral region up to 2 mm from the tumor margin was an independent predictive factor for MVI in multivariate analysis. Yang et al. indicated that the NICs of HCCs with MVI were significantly higher than those of HCCs without MVI [Our study demonstrated that the NIC of the peritumoral zones and intratumoral zones of HCCs measured by DECT was useful for the prediction of MVI. In more detail, outer layer 1 and outer layer 2, which corresponded to the peritumoral layers, showed significantly higher NIC in the MVI present group than in the MVI absent group. Interestingly, the combination of HCCs and peritumoral regions  than peritumoral NICs (0.67–0.75), there was no statistical difference. It might be contributed to the small study population. However, in contrast to peritumoral NICs, there were no significant factors for the prediction of MVI in peritumoral attenuation coefficients. A previous study revealed that dual-energy CT demonstrated a linear relationship with low relative error (less than 10%) between measured and actual iodine concentrations in an in vitro experiment . Based oA recent study demonstrated radiomic analysis of contrast-enhanced CT for predicting MVI . They inThe main issue of our study was how to correctly draw the tumor boundary. The initial process for tumor segmentation is the most important step because it affects all of the peritumoral layers and intratumoral layers in the following process. We used a dedicated software prototype (eXamine: “DE Tumor Segmentation”) that allows for a refined volumetric semiautomatic segmentation of the entire tumor and a respective evaluation of spectral data. This application is designed to evaluate different parameters of a tumor scanned with Siemens DECT technology using an iodine contrast agent. It also provides semiautomatic segmentation of the tumor peel datasets. Once the user selects the number of peels in the prototype, the user obtains the respective number of peels to the outside and inside of the original tumor automatically and a respective analysis of the different layers from a spectral perspective. We additionally performed manual segmentation of HCCs to evaluate the performance of semiautomatic segmentation, and found high agreement between the DECT parameters of HCCs in both segmentation techniques (ICC 0.97–1.0). One remarkable observation in our study is that we found strong correlations between the macroscopic diameter of the surgical tumors and the maximal diameter of HCCs obtained by semiautomatic segmentation (ICC 0.91), as well as manual segmentation (ICC 0.94). These results suggest, although we had to edit the tumor margins manually in a few HCCs with ill-defined margins, semiautomatic segmentation technique used in the current study can perform tumor segmentation accurately and efficiently. It minimized the hand-related artifacts and showed excellent intra- and interobserver agreements. The manual drawing in each slice was also accurate and simple but takes a lot of time and effort. Our study is in concordance with previous studies that demonstrated good correlation between manual and semiautomatic segmentation, while latter technique can reduce the time for measurement and show excellent accuracy and repeatability –30. ThisThis study has some limitations. First, this retrospective study was performed in a single center with a small number of patients. Additionally, only surgically confirmed HCCs were included in this study. A potential selection bias might have occurred. However, because MVI can only be confirmed by pathology, strict inclusion criteria are inevitable. Second, tumors less than 1 cm were excluded from our study because of the potential measurement error. Although it was not intended, all HCCs of the patients were less than 5 cm in maximal diameter. Therefore, our results cannot be generalized to small (< 1 cm) or large (> 5 cm) HCCs. Third, various degree of liver fibrosis in our study population may affect the enhancement characteristics of peritumoral region and results of our study. A larger study with subgroup analysis according to the fibrosis degree would be helpful to decrease this bias. Fourth, the software used in our study was only available in dual-source dual-energy CT. Other dual-energy techniques, such as rapid kVp switching or dual-layer spectral CT, were not used in our study. Further studies using other dual-energy CT vendors are warranted.In conclusion, the volumetric iodine quantification of peritumoral regions in arterial phase may be useful for predicting MVI in preoperative dual-energy CT.Additional file 1: Table S1. Relationship between peritumoral enhancement and quantitative parameters of peritumoral layers.Additional file 2: Table S2. The Hounsfield units of peritumoral and intratumoral regions between MVI absent and MVI present groups.Additional file 3: Table S3. The normalized iodine concentration (NICs) of peritumoral and intratumoral regions in 4 mm layer thickness between MVI absent and MVI present groups.Additional file 4: Table S4.. Comparison of semiautomatic segmentation and manual segmentation.Additional file 5: Table S5. Intraobserver and interobserver agreements of quantitative and qualitative parameters.