Trypanosomacruzi were found in an endomyocardial biopsy in the third month aftertransplantation.A 26-year-old man with cardiopathy due to congenital Chagas’ disease was submitted toheart transplantation for heart failure; amastigote forms of The patient had been diagnosed with Chagas’ disease due to transplacental transmissionand was followed at Hospital das Clínicas of Faculdade de Medicina ofUniversidade de São Paulo (FMUSP) until the age of 2 years, when he wasdischarged from the follow-up. He was diagnosed with cardiopathy due to Chagas’ diseaseat age 20.He sought emergency medical care on July 23, 2016, at age 25, for dyspnea on moderateexertion and paroxysmal nocturnal dyspnea and edema for three weeks.The physical examination disclosed blood pressure of 118/98 mmHg, heart rate of 87 bpm,respiratory rate of 28 breaths/min, oxygen saturation of 99% and pulmonary auscultationshowed decreased vesicular murmur at the bases. Cardiovascular examination disclosedincreased jugular venous pressure, thin pulses, cardiac stroke deviated 2 cm beyond thenipple line, 2 digital pulps, arrhythmic heart sounds, normal heart sounds with thepresence of a third heart sound and mitral systolic murmur. Abdominal examinationrevealed painful hepatomegaly, with the liver palpated 6 cm from the right costal borderand ++ /4 edema in the lower limbs.The electrocardiogram showed sinus rhythm, heart rate of 107 bpm, PR interval of 187 ms,QRS duration of 146 ms, left atrial overload, right bundle-branch block andanterosuperior left bundle-branch block and probable left ventricular overload .The posteroanterior chest x-ray disclosed veil-like opacification of the bothhemithoraces, compatible with pleural effusion, increased pulmonary hila with signs ofpulmonary congestion and cephalization of the pulmonary vasculature network and ++++ / 4global cardiomegaly .The patient was re-hospitalized on July 29, after two episodes of syncope that occurredon the day before hospitalization.The physical examination disclosed a patient in regular overall status, hydrated,eupneic, acyanotic, conscious and oriented. Blood pressure was 90x60 mmHg, heart ratewas 88 bpm, oxygen saturation was 97%; pulmonary auscultation was normal; cardiacauscultation disclosed an irregular heart rhythm, with no heart murmurs or accessorysounds; the abdominal examination showed no visceromegaly, no lower-limb edema, and nocalf stiffness.Laboratory tests  showed: red blood cells: 5200000/mm³, hemoglobin15.6g/dL, hematocrit of 47%, leukocytes 14,570/mm³ (74% of neutrophils), platelets157,000/mm³, potassium 3.8 mEq/L, sodium 137 mEq/L, BNP 1,128 pg/mL, ALT 54 U/L, AST 42U/L, gamma-GT 99 U/L. Urinalysis was normal.The chest x-ray  disclosed clear pulmonary fields and cardiomegaly at theexpense of the right ventricle  andB.2, normal value < 34 mL/m2); moderate enlargement of theleft ventricle and right atrium; slight enlargement of the right ventricle. Systolicfunction was decreased due to diffuse hypokinesia. The right ventricle showed mildhypokinesia. There was also mild to moderate mitral regurgitation, as well as mild tomoderate tricuspid regurgitation.The transthoracic echocardiogram  showed: left atrium, 52 mm; rightventricle, 43x32 mm; septum, 8 mm; posterior wall, 8 mm; left ventricle 66x60 mm; leftventricular ejection fraction, 20%; pulmonary artery systolic pressure, 39 mmHg. Therewas a marked increase in left atrium  showed marked right ventricular dilatation with anejection fraction of 17%, left ventricle with diffuse hypokinesia, with late mesocardialenhancement in the basal segment of the septum and transmural enhancement in the basal,middle and apical segments of the anterior, lateral and inferior walls, and less than50% in the basal inferosseptal segment. The ejection fraction of this ventricle was also17% .In the electrophysiological study  there was no onset of atrial orventricular arrhythmias after the extra stimuli.Right catheterization  showed mean right atrial pressure of 11 mmHg,right ventricular end-systolic and diastolic pressures of 45/29 mmHg, and pulmonarycapillary pressure of 30 mmHg. The pulmonary vascular resistance was 3.6 Wood Units and the cardiac index was 2.1L / min / m². After the use of10 µg/kg/min of dobutamine, pulmonary vascular resistance decreased to 1.6 WoodU.The patient was placed on a transplant waiting list with a priority status, as vasoactivedrug weaning was not achieved due to hypotension. Orthotopic heart transplantation wasperformed with no complications on December 6, 2016. The donor was positive forcytomegalovirus.The anatomopathological analysis of the para-aortic lymph nodes revealed reactivelymphadenitis with no granulomas.The post-transplantation electrocardiogram  showed low voltage in thefrontal plane and end-conduction disorder .The biopsy on December 16, 2016 showed focal mild-degree fiber aggression; focal mildhistiocytic proliferation and mild focal lymphocytic infiltrate; there was moderatediffuse edema. Compatible with acute grade 1R cellular rejection (low grade mildrejection). The search for antibody-mediated rejection by immunohistochemical reactionto complement C4d fraction was negative.The serology was negative for cytomegalovirus; however, molecular biology screening forthe parasite was positive since the end of December and viral load reached 47417 U/mL inFebruary 2017, when the patient received ganciclovir for 21 days.The echocardiogram performed in February 2017 was normal, except for an increase in theleft atrium, whereas the one performed in March showed all measures within the normalrange.Trypanosoma cruzi antigens.Benznidazole was then prescribed.In a biopsy carried out in March 2017, moderate and focal fiber aggression, moderatediffuse proliferation, moderate focal lymphocytic infiltrate and mild diffuse edema wereobserved. Amastigote nests were observed inside the myocytes, with protozoalmyocarditis. This biopsy was suggestive of Chagas' disease reactivation, with moderatemononuclear myocarditis. Kinetoplasts were observed in the parasites and theimmunohistochemistry was positive for Trypanosoma cruzi.A biopsy carried out in September 2017 disclosed acute grade 2R cellular rejection. Both histological and immunohistologicalanalysis were negative for The serology for Chagas' disease was negative in November 2017.At an outpatient consultation on May 3 2019, the patient was asymptomatic and thephysical examination was normal.1 This multifaceteddisease is caused by the protozoan Trypanosoma cruzi, which can betransmitted in different ways. Vector transmission through hematophagous insects isthe most classic one, although it has declined in importance in recent years withmeasures to control the vector population.2 Transfusion transmission, as well as vector transmission,have shown a drastic reduction in the last decades, and no cases have been reportedin Brazil for years.3 In contrast,there was an increase in reports of oral transmission. This form of contagion waslittle known but gained importance, with several descriptions of micro-epidemicevents in the country, especially related to the consumption of açai berry.The development of the acute phase of the disease is more common in the oraltransmission.4 In thecontext of controlling the main forms of contagion, vertical transmission has becomerelevant.Chagas' disease was first described by Brazilian physician and scientist CarlosChagas in 1909.5 Most of these cases are found in Latin America, and Brazilis the country with the majority of infected individuals . The decrease in transmission was accompanied by a reduction inmortality, estimated in 2007 to be 2.78 deaths/year for every 10,000inhabitants.6 Anotherchange in the disease epidemiology in recent years has been the increase in cases innon-endemic regions, such as the USA and Europe, which has contributed to theincreased attention given by the international scientific community to Chagas’disease.5The World Health Organization (WHO) estimates there are approximately 8 millionindividuals infected with Chagas disease worldwide, with an annual mortality of10,000 people due to disease complications.7 It is known that the inflammatory response triggeredby the parasite plays a crucial role in this pathophysiology.8 This hypothesis is supported by lowtissue parasitism and low parasitemia in the chronic phases of the disease. Morerecent studies have identified an autoimmune response triggered by thecross-reaction between parasite antigens and host proteins, such astroponin.9 Diagnosis isattained by serological tests in the vast majority of cases, with the directinvestigation of the parasitic agent being reserved for acute phases orreactivations, situations in which the parasitemia may be higher.7The pathophysiology of Chagas' disease is multifactorial, depending on severalcharacteristics of both the host and the parasite.10 demonstrated that the meanprevalence of infected pregnant women is 1.7%, with a mean percentage of congenitaltransmission of 1.7%. Extrapolating these data to the population based on the 2010census, Brazil would have approximately 34,629 infected pregnant women, with anincidence of 587 children born with congenital Chagas per year.10 Due to these numbers, the WHOrecommended in 2018 increased attention to cases of congenital Chagas throughmaternal-fetal transmission, not only in Brazil, but in all countries with endemicdisease.Congenital Chagas’ disease is a separate group. It occurs when there is verticaltransmission, that is, during pregnancy. In Brazil, Martins-Melo et al.11 demonstrated that children of pregnant women whoreceived treatment with antitrypanosomal drugs during their lives have a much lowerchance of developing congenital Chagas’ disease than children of women who have notbeen treated.11 Thus, in additionto indications of etiological treatment included in the I Latin American Guidelinefor Chagas' disease in 2011, it is recommended that women of childbearing age alsoreceive antitrypanosomal drugs. In Brazil, the available drug is benznidazole, whichshould be used at a dose of 5mg/kg/day divided into 2 or 3 doses a day for 60days.7 It is worth notingthat benznidazole use is contraindicated during pregnancy, due to the risk ofteratogenicity found in animal studies.12In a study carried out in Argentina in 2014, Fabbro et al.13 Thediagnosis, in addition to the clinical picture, is based on the direct screening forthe parasite up to 6 months of age and on the serological tests after 9 months, dueto the presence of circulating antibodies from the mother. All cases should betreated with antitrypanosomal drugs as soon as the diagnosis is confirmed.14 The earlier treatment isimplemented, the lower the incidence of side effects and the higher the cure rate,which is 100% when implemented in the first year of life.12The clinical picture of congenital Chagas disease is extremely variable andnon-specific, being similar to several other infections seen in the neonatal period,such as toxoplasmosis, rubella, HIV and syphilis. The main symptoms are pretermbirth, intrauterine growth restriction, neuropsychomotor development deficit, lowApgar score, respiratory distress syndrome, jaundice, and hepatosplenomegaly. Thesesymptoms may appear days or even weeks after birth. Mortality is approximately 5%and is usually related to more severe manifestations, such as meningitis andmyocarditis.14 Amongpatients with Chagas cardiopathy submitted to heart transplantation, the incidencevaries between 21 and 45% depending on the studied series.15 Review studies show that mortality is low whenappropriate treatment is implemented and, therefore, diagnosis is a crucial part ofa favorable outcome.16Chagas' disease reactivation can affect any individual with the chronic forms of thedisease, especially when they are submitted to immunosuppression. In this context,the most important conditions are HIV coinfection and/or organtransplantation.Trypanosoma cruzi amastigotes may bepresent in the Chagas’ disease reactivation biopsy, observed by hematoxylin-eosinstaining or by immunohistochemistry analysis.15The diagnosis of reactivation in the transplanted patient is based on the clinicalpicture and the routine screening for the parasite in endomyocardial biopsies,because often there are no symptoms, or the symptoms are non-specific. Symptoms maybe cardiac, such as congestive and low-output symptoms in cases of myocarditis orchanges in the electrocardiogram, such as cardiac rhythm disturbances or new blocks.The most frequent extracardiac symptoms are fever and skin lesions. Thus, a highdegree of suspicion is necessary for diagnosis to be attained. When theendomyocardial biopsy is altered, the main differential diagnosis is acute cellularrejection, since there is lymphocyte/monocyte infiltration in both cases. Thedifference is that nests of 15When the reactivation diagnosis is made, the recommended treatment is benznidazoleuse, at the previously mentioned doses. It should be emphasized that this treatmentdoes not result in the cure of the chronic infection, and the patient is subject torecurrent reactivations. As a consequence of this risk, it is recommended that theimmunosuppression in these patients be as little as possible, aiming at an adequatebalance between the risk of reactivation and rejection.17 Thus, despitethe drastic decrease in the disease transmission in the country, Chagas' cardiopathycontinues to be very frequent in Brazil, both due to the number of patients with thechronic forms of the disease and to other forms of transmission that were notpreviously very relevant, such as vertical transmission. The care of pregnant womenand the treatment of those infected at reproductive age should be improved. Theactive screening for infection in the children of infected mothers is essential,aiming at establishing the earliest possible antitrypanosomal treatment in infectedchildren, thus achieving the cure and reducing the potential number of patients withthe chronic forms of the disease. (Dr. Henrique Trombini Pinesi)Azathioprine use should be preferred instead of mycophenolate in the Chagasicpopulation, since it is associated with a lower reactivation rate, without worseningof other outcomes.Diagnostic hypothesis: Chagas disease reactivation in transplantedheart. (Dr. Henrique Trombini Pinesi)15 For this purpose, peripheral bloodsamples should be collected for direct detection of the parasite in the buffy coat,which increases the likelihood of its finding, and for parasite screening bymolecular biology. This method seems to be more sensitive, detecting the increase ofthe parasitic load before the onset of clinical and/or histopathologicalmanifestations. It can be qualitative or quantitative. Monthly or quarterlymonitoring is recommended in the first year after cardiac transplantation, when thelevel of immunosuppression is higher and after the treatment of rejection episodes.(Prof. Dr. Tânia Mara Varejão Strabelli)The laboratory monitoring of Chagas' disease reactivation after cardiactransplantation is recommended. Due to the difficulty of attaining a clinicaldiagnosis of Chagas’ disease reactivation, with the exception of skin lesions, thelaboratory monitoring of transplanted patients is recommended.The explanted heart weighed 332g. It had a globose shape and the external surface wascovered by smooth serosa, with small white and prominent nodules being noticed,focally, in the trajectory of the coronary vessels. The opening showed dilatation ofall chambers , predomiThe post-transplantation endomyocardial biopsy carried out in March 2017 showed goodtissue representativeness, with a moderate inflammatory process and severalcardiomyocyte aggression foci .Trypanosoma cruzi parasites, which were positive inpseudocysts containing amastigotes ((Dr. Vera DemarchiAiello)As this was a patient with Chagas’ disease, an immunohistochemical reaction wascarried out for stigotes .New secstigotes . This case shows a young patient with chronic Chagasic cardiopathy who had clinicalmanifestations around the age of 20 years, after a diagnosis of congenital Chagas'disease. There was no clear history of childhood disease treatment. The explantedheart showed a typical picture of Chagas’ chronic heart disease.18 However, as this is the case of apatient with Chagas' disease as the primary cardiopathy, one must carry out a moredetailed investigation of parasites, since the histological picture of acute 2Rcellular rejection is identical to that of disease reactivation. The investigationwas then carried out by immunohistochemistry and then in more detailed sections ofthe biopsy block, we concluded it was a reactivation of Chagas' disease in thetransplanted heart, which allowed the appropriate treatment to be implemented.Regarding the post-transplant endomyocardial biopsy findings, the presence ofinflammation with more than one focus of cardiomyocyte aggression is, at first,compatible with the diagnosis of acute grade 2R cellular rejection.19 also showed that thefirst episode of reactivation occurred at a median of 6.6 monthspost-transplantation. Therefore, the routine evaluation of endomyocardial biopsiesin Chagasic patients with heart transplantation should include, whenever there is anR2 or higher grade acute cellular rejection, a very detailed evaluation of thehistological sections for possible detection of parasites. (Dr. Vera DemarchiAiello)It is known that the rate of reactivation depends on the implementedimmunosuppressive treatment, as previously described. The work of Vidal etal.


Risk stratification and management of acute myocardial infarction patients continue to be challenging despite considerable efforts made in the last decades by many clinicians and researchers. The aim of this study was to investigate the metabolomic fingerprint of acute myocardial infarction using nuclear magnetic resonance spectroscopy on patient serum samples and to evaluate the possible role of metabolomics in the prognostic stratification of acute myocardial infarction patients.1H-nuclear magnetic resonance spectroscopy and the spectra were used to characterize the metabolic fingerprint of patients. Multivariate statistics were used to create a prognostic model for the prediction of death within 2 years after the cardiovascular event.In total, 978 acute myocardial infarction patients were enrolled in this study; of these, 146 died and 832 survived during 2 years of follow-up after the acute myocardial infarction. Serum samples were analyzed via high-resolution p = 2.16×10−16). Moreover, in the multivariate regression only age, dyslipidemia, previous cerebrovascular disease, Killip class, and random forest score remained statistically significant, demonstrating their independence from the other variables.In the training set, metabolomics showed significant differential clustering of the two outcomes cohorts. A prognostic risk model predicted death with 76.9% sensitivity, 79.5% specificity, and 78.2% accuracy, and an area under the receiver operating characteristics curve of 0.859. These results were reproduced in the validation set, obtaining 72.6% sensitivity, 72.6% specificity, and 72.6% accuracy. Cox models were used to compare the known prognostic factors  with the metabolomic random forest risk score. In the univariate analysis, many prognostic factors were statistically associated with the outcomes; among them, the random forest score calculated from the nuclear magnetic resonance data showed a statistically relevant hazard ratio of 6.45  contains supplementary material, which is available to authorized users. Among cardiovascular diseases (CVDs), acute coronary syndrome (ACS) represents the most common cause of emergency hospital admission and it is associated with the highest mortality and morbidity , 2. The Several studies have evaluated the potential clinical usefulness of new biomarkers able to identify patients who had a poor outcome. In particular, high levels of inflammatory markers such as C-reactive protein and interleukin-8 had long-term prognostic utility in patients with ACS that undergone coronary revascularization , 6. HoweA number of studies have evidenced that a global approach, such as genomics, proteomics, or metabolomics, may represent a valid strategy for improving current knowledge about pathophysiological mechanisms and for identifying ACS patients at high risk of secondary atherothrombotic events or premature death.3 [2 different small molecules present in a biological sample [Metabolomics is the accepted name for the -omic science that deals with the characterization of the metabolome, in turn defined as the whole set of metabolites in a certain biological system, such as a cell, tissue, organ, or entire organism . The two3 ), with a3 , and NMR3  and intr3 . NMR givl sample , and hasRecently, the metabolomic approach has been applied to identify a risk profile in heart failure patients –14, atriRisk stratification should identify individuals at high risk who require more intensive therapy, or, conversely, help avoid drug overuse and associated side effects in patients with a favorable prognosis. In this framework, the aim of the present study was to evaluate the impact of the metabolomic fingerprint on the occurrence of cardiovascular death in acute myocardial infarction (AMI) patients after percutaneous coronary intervention.This study was part of a collaborative project between the Department of Medical and Surgical Critical Care of the University of Florence and the Magnetic Resonance Centre (CERM) of the University of Florence.t test as the test statistic, and fixing an alpha level of 0.05 for a significant comparison, it was found that having 146 patients who died and 832 patients who survived was enough to detect small to medium effects (Cohen’s d ~0.25) with a statistical power of 80%; furthermore, the training and validation sets separately showed adequate statistical power . Blood samples were collected 24–48 h after percutaneous coronary intervention (PCI) and overnight fasting. All information about inclusion criteria and treatment of the patients are detailed in Additional file The study population comprised 978 out of 1496 patients admitted to the coronary units of the six hospitals  of the Florence health district between April 2008 and April 2009, and enrolled in the frame of the Florence Acute Myocardial Infarction-2 (AMI-Florence 2) registry . In the All subjects gave written informed consent. The study (number 11/2008) complies with the Declaration of Helsinki and was approved by the ethics committees of the local health unit, the University of Florence, and Careggi Hospital (19 March 2008).1H-NMR spectra, namely nuclear Overhauser effect spectroscopy (NOESY), Carr–Purcell–Meiboom–Gill (CPMG), and Diffusion-edited spectra, were acquired, allowing the selective detection of different molecular weight metabolites. A detailed description of the sample preparation and experiments is presented in the Additional file Samples were prepared following the standard protocols detailed by Bernini et al. . AccordiEach one-dimensional spectrum in the range 0.2–10.00 ppm was segmented into 0.02 ppm chemical shift bins and the corresponding spectral areas were integrated using AMIX software . The region between 4.5 and 5.0 ppm containing the residual water signal was removed and the dimension of the system was reduced to 466 bins. The total spectral area was calculated on the remaining bins and total area normalization was carried out on the data prior to pattern recognition.t test was used for comparison between groups and the chi-square test for comparison between categorical variables.Data analyses were performed using the open source software R. For the demographic and baseline characteristics, the For the multivariate data analyses of the NMR data, the group of 978 patients was randomly split into two independent cohorts : a trainThe initial analysis was restricted to the training set and the first step was to establish if serum metabolomic profiles could distinguish between patients who survived and died within 2 years after the cardiovascular event. For this purpose, a random forest (RF) classifier  was builThe next step was to test the hypothesis that a metabolomic signature similar to that of one of the patients who died would be predictive of death within 2 years after the cardiovascular event. Using receiver operating characteristics (ROC) analysis (“colAUC” function of the R package “caTools”) and Harrell’s c index (“cindex” function on the R package “dynpred”), the performances of the RF risk scores were compared with the actual outcome. To delineate high risk of death, a cut-off for the RF risk score was calculated in the training set that optimized accuracy, sensitivity, and specificity, and the performance of the model was subsequently tested in the validation set.The performances of the NOESY RF score were evaluated by calculating a Cox proportional hazards regression model  using thP-value < 0.05 was deemed significant. Effect size using Cliff’s delta [The spectral regions related to 23 metabolites, present in concentrations above the detection limit >1 μM) in all samples (up to 30–40 different metabolites could be quantified in each sample ), were a μM in al’s delta  was calcThe statistical approach described above was also used to build sex-specific statistical models based on NOESY spectra.http://www.ebi.ac.uk/metabolights).NMR and clinical data are freely available in the Open-Access Database Repository MetaboLights from October 2017 with the accession number MTBLS395  were classified using the RF classifier. The profiles showed significant differential clustering, with good separation of the two groups using each type of NMR spectra: NOESY Fig. a, CPMG, A threshold of ≥ 0.454 for the NOESY RF score was set to optimize accuracy, sensitivity, and specificity; this optimized threshold yielded 76.9%  76.5–77.3%) sensitivity, 79.5% (95% CI 78.4–80.6%) specificity, and 78.2% (95% CI 77.6–78.7%) accuracy.p-value of 3.45×10−09 calculated using the likelihood-ratio test, and a concordance coefficient of 0.73.The goodness of fit of the NOESY RF model was calculated using a Cox proportional hazards regression model: NOESY RF showed a hazard ratio of 7.4 (95% CI 3.51–15.6) with a Moreover, the NOESY RF model was robust with respect to different strategies of model validation; indeed, a mean AUC of 0.805 (95% CI 0.795–0.814) was obtained with 100 cycles of classical Monte Carlo cross-validation with data split 80 to 20% . Furthermore, our approach was robust with respect to the origin of samples: only slight differences in the AUC were obtained when using samples from the Careggi University Hospital as the training set to build the RF model and samples from all the other hospitals as the validation set (and vice versa)  was evaluated using an unsupervised analysis. Spectra of the validation samples were classified as either ‘dead’ or ‘survivor’ using the optimized NOESY RF risk score model derived from the training set. Comparison between metabolomic classification and actual outcome demonstrated high correlation with an AUC of 0.801 .The AUC calculated on the RF score was assessed for significance against the null hypothesis of no prediction accuracy in the data by means of 10,000 randomized class-permutations tests: the estimated AUC obtained after randomization was 0.570 (95% CI 0.569–0.571), demonstrating the significance of our result  with a The known prognostic factors age, sex, previous CABG, previous PCI, heart failure, atrial fibrillation, cerebrovascular disease, diabetes, creatinine concentration, Killip class, and ACS classification were compared with the NOESY RF risk score, calculated on the entire dataset, in univariate and multivariate Cox regression analyses. The results are displayed in Table In the univariate analysis, many prognostic factors were statistically associated with the outcomes, among them the NOESY RF score showed a Cox hazard ratio of 6.45. In the multivariate analysis, only age, dyslipidemia, hypertension, previous cerebrovascular disease, Killip class, and NOESY RF score remained statistically significant, demonstrating their independence with respect to the other variables. The smoker’s paradox, already described by other studies , was alsp-value < 0.01 after Bonferroni correction) with respect to the two separate scores in both the training and validation sets. A comparison of these two scores with the NOESY RF score is included in the Cox regression analyses in Table The performance of the NOESY RF score in predicting 2-year outcomes was also compared with the performance of the GRACE hospital discharge risk score  for thosUsing ROC analyses, the performance of the NOESY RF and GRACE scores was tested against some well-known parameters that are commonly measured in the clinical practice . Training and validation sets were considered separately and only data from patients for whom all the clinical parameters were available were included (80.5% of the entire cohort). As shown in Fig. The performance of our approach was also tested by dividing patients in two distinct subgroups of patients according to AMI severity  and non-STEMI). The results demonstrated that our approach is marginally affected by these two subcategories  higher levels of 3-hydroxybutyrate, proline, creatinine, acetate, acetone, formate, and mannose, and significantly lower levels of valine and histidine  were included in the analysis. The following 23 metabolites were unambiguously assigned and quantified in the spectra: acetate, acetone, alanine, citrate, creatine, creatinine, formate, glucose, glutamate, glutamine, glycine, histidine, isobutyrate, isoleucine, lactate, leucine, mannose, methionine, phenylalanine, proline, tyrosine, valine, and 3-hydroxybutyrate. We observed that patients who died were characterized by significantly , and the analysis replication in a validation set, some limitations should also be mentioned. First, sample collections were done exclusively in the acute phase of the disease, impairing the acquisition of data correlated to the biochemical mechanisms of the transition to the quiescent phase. Owing to the importance of this aspect, further efforts in this direction are required. Second, even though the data were replicated in a validation set, a totally independent cohort for validation is lacking. However, before attempting to replicate these findings in very large multicenter studies, common standard operating procedures are required for sample collection and storage, otherwise samples collected in different centers will not be comparable. Our group is strongly committed to this, and have contributed to the development of the optimal pre-analytical procedures for metabolomics . FinallyLifestyle and the medication administered to a patient influence the molecular signatures in plasma and serum samples, and the relative metabolite concentrations reflect tissue lesions and organ dysfunctions. In this framework, previous studies have underlined the usefulness of metabolomics of serum and plasma in determining the individual’s disease risk, prognosis, and therapeutic options in different clinical settings , 50, 51.In a prospective study of three population-based cohorts from Finland that were free of CVD at baseline , a metabThe usefulness of the metabolomic approach was also demonstrated in a general cohort of patients at risk for cardiovascular events undergoing cardiac catheterization ; at baseOur study, performed in a large sample population of AMI patients followed for 2 years, provided new data about the role of high 3-hydroxybutyrate circulating levels on post-AMI mortality. A previous study that evaluated the ketone bodies in the urine of five ACS patients demonstrated that ketone bodies, and particularly 3-hydroxybutyrate, were altered during the acute event . FurtherFurthermore, it is worth of mentioning that formate has already been proposed as a possible biomarker of ACS .As expected, patients with worst prognosis showed higher level of serum creatinine, a well-known marker of renal insufficiency. It has already been demonstrated that elevated serum levels of creatinine on admission are associated with impaired myocardial flow and poor prognosis for 1-year mortality , 62.Previous studies evidenced an interaction between sex and adverse cardiovascular events in CAD patients –65, withBuilding sex-specific models enabled us to improve the outcome prediction in the male cohort, but not in the female one. Thus, the male metabolic fingerprint seems to show a higher association with cardiovascular mortality than the female one. However, the female cohort was smaller than the male cohort and this may have affected the predictive capability of the model; therefore, larger cohorts of patients are needed to build robust sex-specific models.Our data support the usefulness of the metabolomic approach for identifying a more precise risk profile in AMI patients. Metabolomic analysis by NMR enables fast, approachable, and reproducible characterization of the AMI metabolic fingerprint associated with a poor prognosis, improving the cardiovascular risk assessment beyond that achieved by established risk factors, and identifying those patients who need to undergo a very early and aggressive treatment. Thus, metabolomics could represent a valid addition to the already established risk stratification tools. Further, as demonstrated by the combination of the NOESY RF scores with the Grace score, metabolomics, in combination with classical tests, is able to significantly improve risk classification over the two separate scores.Additional file 1:Supplementary material. Expanded methods and results. Table S1. Demographic and Clinical Characteristics divided according to gender. Table S2. Univariate Metabolites Analyses. Table S3. Results for the gender-specific models. Figure S1. Flow chart explaining sample exclusion reasons from the NMR metabolomic analysis. Figure S2. Metabolite signal deconvolution using our in-house-developed algorithm for quantification. Figure S3. Metabolomic profiles for one randomly selected patient from each group of outcomes. Figure S4. Discrimination between patients from different centres using PCA. Figure S5. Receiver Operating Characteristic Curves and the corresponding Area for the two models. Figure S6. Discrimination between NSTEMI and STEMI using RF. Figure S7. Metabolite concentrations. Figure S8. Gender-specific models and predictions using NOESY1D spectra. (DOCX 3630 kb)


The present clinical study aims to describe protocol to evaluate the effects of vitamin D3 supplementation on the cardiovascular risk factors in a population of rotating shift workers.A randomized, double-blind, placebo-controlled, parallel group clinical trial testing 2 oral dosages of cholecalciferol  for 12 months.P < .05 will be considered significant. The analysis of primary and secondary outcomes will use an intention-to-treat population and a per-protocol population. The primary and secondary outcomes will be compared separately between each treatment group and placebo, using binary logistic regression or regressão de Poisson for proportions (for binary outcomes) and using linear regression for differences in means (for continuous endpoints), with 95% confidence intervals.The primary outcome for evaluation is an 18% reduction in hypertriglyceridemia (≥150 mg/dL) between pre and postintervention measurements. Baseline characteristics of the study population will be summarized separately within each randomized group, and will use tests for continuous and categorical variables. For all tests, a Rotating shift workers, adults aged between 18 and 60 years, with hypovitaminosis D and alterations in at least 1 of the following parameters: fasting glucose, high-density lipoprotein cholesterol, triglycerides, low-density lipoprotein cholesterol, blood pressure, and waist circumference.This clinical trial aims to contribute to the gap in knowledge about the potential, dose, and time of vitamin D supplementation to generate beneficial effects on triglycerides in a population at increased risk for hypertriglyceridemia and vitamin D deficiency. There are still controversies about the reference values of vitamin D [25(OH)D]; the Brazilian Society of Endocrinology and Metabolism and the Endocrine Society define sufficient levels as values ≥30 ng/mL, and the Institute of Medicine defines sufficient levels as values ≥20 ng/mL. The literature data on vitamin D status in occupational groups are still scarce. However, when analyzing vitamin D status according to occupational activity, it is observed that shift workers and night-shift workers have lower levels of vitamin D and higher prevalence of deficiency and insufficiency when compared to day workers.,6Hypovitaminosis D has affected populations in different age groups around the world.–9 Shift work leads to deregulation of the circadian cycle, which promotes metabolic changes such as deregulation of cortisol, leptin, ghrelin, and melatonin, which contribute to the development of these risk factors.,10 In a previous study by our research group with rotating shift workers, Batista et al showed that workers with hypovitaminosis D (<30 ng/mL) had a 5.9 times greater chance of having altered low-density lipoprotein (LDL) cholesterol (≥160 mg/dL) and a 2.3 times greater chance of having altered triglycerides (≥150 mg/dL) in relation to workers with sufficient levels of vitamin D.Shift workers have a higher prevalence of risk factors for cardiovascular diseases, such as increased glucose, increased blood pressure, altered lipid profiles, and obesity when compared to day-shift workers.–16 It is not yet clear whether there is a cause–effect relationship. There are few randomized clinical trials and with inconsistent results to provide an evidence on the benefit of vitamin D in reducing the risk of extraskeletal diseases, and most of those studies have been done in postmenopausal women,18 (above 50 years), with normal sleep–wake rhythms. Thus, there are no data in the literature on the effects of vitamin D replacement in adult men (<60 years) who work in shifts.Cross-sectional studies of several age groups suggest that hypovitaminosis D is associated with cardiovascular disease and risk factors, isolated or clustered, such as metabolic syndrome, waist circumference, body mass index (BMI), fasting glucose, blood pressure, total cholesterol, high-density lipoprotein (HDL) cholesterol, LDL cholesterol, and triglycerides.Considering that there is still no consensus on the dose and time required for vitamin D replacement to generate beneficial effects in the cardiovascular profile, this clinical study aims to describe protocol to evaluate the effects of vitamin D3 supplementation at doses of 14,000 IU and 28,000 IU weekly for 12 months on the cardiovascular risk factors in a population of rotating shift workers. The hypothesis of the present study is that there will be an 18% reduction in hypertriglyceridemia (≥150 mg/dL) between pre and postintervention measurements in adult males with work activity that breaks the usual circadian cycle.22.1A randomized, double-blind, placebo-controlled, parallel group clinical trial testing 2 oral dosages of cholecalciferol  per week for 12 months with a 1:1:1 allocation ratio. This study will demonstrate the efficacy of supplementation with both doses of vitamin D in reducing hypertriglyceridemia.2.2The study participants consisted of rotating shift workers from 5 units of a Brazilian iron ore mining company; all were adults aged between 18 and 60 years. The company's rotating shift schedule is 6 hours of work, followed by 12 hours of rest. All participants work 4 shifts on the following schedule: 7 PM to 1 AM, 1 PM to 7 PM, 7 AM to 1 PM, and 1 AM to 7 AM. After completing the 4-shift weekly cycle, they have a day off. The interviews and blood sample collection will occur in the workplaces.The inclusion criteria are as follows:25(OH)D levels <30 ng/mL (75 nmol/L)With at least 1 of these parameters:; HDL cholesterol <50 mg/dL (1.29 mmol/L) for women and <40 mg/dL (1.04 mmol/L) for men;Fasting glucose ≥100 mg/dL (5.55 mmol/L);Triglycerides ≥150 mg/dL (3.88 mmol/L);LDL cholesterol ≥130 mg/dL (3.36 mmol/L);Blood pressure systolic ≥130 mm Hg or diastolic ≥85 mm HgWaist circumference ≥80 cm for women and ≥90 cm for men.Subjects are excluded with evidence as following: malabsorption syndrome, kidney, liver, or thyroid diseases; specific drug use ; or altered levels of serum creatinine, parathyroid hormone (PTH), serum calcium, or albumin. Among women, current breast feeding or pregnancy are also considered exclusion criteria.After each visit (every 4 months), the follow-up will be interrupted if subjects evidence 25(OH)D >50 ng/mL (or 159 nmol/L) associated with altered serum calcium levels (>11 mg/dL) and with confirmation of a second test with altered serum calcium (>11 mg/dL) and PTH (<10 pg/mL) levels or evidence of vitamin D intoxication symptoms ; recent diagnosis of malabsorption syndrome, kidney, liver, or thyroid diseases; specific drug use ; or participant request. In cases of interrupted due to intoxication, the code will be broken to clarify the diagnosis, only by the code keeper.2.3This trial use 2 interventions groups and a placebo group. One of the intervention groups will receive 14,000 IU of cholecalciferol oral supplementation per week, and the other intervention group will receive 28,000 IU of cholecalciferol oral supplementation per week. The active compound is cholecalciferol (vitamin D3) from a pill containing 7000 IU per unit (SanyD) already existing in the Brazilian market .For the double-blind masking of the groups, the vitamins were distributed as capsules; the encapsulation process was carried out by Amphora specialized handling pharmacy. Inside each capsule, the pills were intact to maintain the phycochemical integrity of the active compound. The placebo group's capsules contained only the inert formulation with the same color, smell, and weight of the other capsules distributed to the intervention groups.Participants were individually instructed, by the responsible team, to take 1 capsule per week, preferably close to the time of eating large meals (lunch/dinner) or foods containing fat. The delivery of the capsules was carried out every 2 months with support from the company's medical team to accommodate the varied to work shifts and workplaces. Each participant has received a bottle, identified by the name of the participant, containing capsules for 10 weeks at each follow-up visit, with the last bottle containing capsules for 12 weeks, completing 52 weeks of intervention.Compliance is ascertained by interview at follow-up visits and by phone, or participants’ message to the responsible team. To improve adherence, the responsible teams send a message every week for participants to remember to take the pill, also send messages with information about healthy lifestyle, and deliver the lipids tests results to participants after follow-up visits every 4 months.2.4The primary outcome to be evaluated is an 18% reduction in hypertriglyceridemia (≥150 mg/dL) between the pre and postintervention measurements. The secondary outcomes to be evaluated are a 36% reduction in hypertriglyceridemia (≥150 mg/dL) between the pre and postintervention measurements with 28,000 IU of vitamin D; an 18% reduction in high LDL cholesterol (≥130 mg/dL) between the pre and postintervention measurements; an 18% reduction in waist circumference  between the pre and postintervention measurements; and a reduction in low cognitive impairment (by cognition tests) between the pre and postintervention measurements.2.5 and Endocrine Society. The second is double of maintenance therapy. It was decided to use this low dose to verify if the maintenance therapy is able to reduce hypertriglyceridemia, and double dose in order to increase the probability of generating a significant difference in the vitamin D status between the treatment groups and placebo. The clinical trial lasts for 12 months due to the seasonality of the incidence of ultraviolet radiation. In addition, vitamin D has endogenous synthesis via exposure to the sun and can be stored in adipose tissue, factors that make the placebo group necessary.The 25(OH)D used in this trial already exists in the market, which has safety and quality. It was decided to 2 doses of vitamin D: 14,000 IU and 28,000 IU per week. The first one corresponds to maintenance therapy (1500 to 2000 IU per day) for replacement adult population at risk for hypovitaminosis D, in accordance with the Brazilian Society of Endocrinology and Metabolism,3 The safe value of serum vitamin D levels suggested to avoid hypercalcemia is less than 150 ng/mL.The risk of intoxication should be considered, but this condition is rare and caused by the intake of extremely high doses of vitamin D for long periods. The clinical symptoms of intoxication are characterized by hypercalcemia, hyperphosphatemia, suppression of PTH levels which can lead to nephrocalcinosis, and soft tissues calcification, especially blood vessels, in addition to fatigue, weight loss, anorexia, and impairment of renal function. and by others authors, who did not observe serious adverse events. In a randomized trial, vitamin D supplementation with 6000 IU daily for 3 months, after 3000 IU daily for more 3 months and 2200 IU daily for more 3 months, was not associated with any adverse events or hypercalcemia during all 9 months of the study. In the vitamin D, diet and activity study, 2000 IU of cholecalciferol daily were supplemented for 12 months, and 4 women reported light-headedness, severe headaches, nausea, rash/hives, weakness/numbness, and constipation.Safety evidence for 25(OH)D supplementation has been reported by the Endocrine SocietySymptoms of vitamin D intoxication include dizziness, headache, nausea, rash, urticaria, weakness, numbness and constipation, hypercalcemia, hypercalciuria, and associated kidney stones. To minimize the risk of vitamin D supplementation, individuals with a diagnosis of renal, hepatic, or thyroid disease were excluded. As a safety precaution, participants with 25(OH)D >50 ng/mL (or 159 nmol/L) associated with altered serum calcium levels (>11 mg/dL) and confirmation of a second test with altered serum calcium (>11 mg/dL) and PTH (<10 pg/mL) levels or evidence of vitamin D intoxication symptoms  are excluded from trial. Additionally, measures will be taken to record adverse events or reactions: by the follow-up questionnaire every 4 months or in spontaneously by the participant by telephone/message or email to the responsible team.2.6; the percentage of hypertriglyceridemia in the group exposed to vitamin D replacement (22%); and the likelihood ratio equal to 2.3, considering 80% power and a 95% confidence level. As a result, this study requires 106 participants in each group; after adding 10% for losses, a total of 350 participants at least were enrolled. To estimate the sample size, OpenEpi software  version 3.01, a web-based epidemiologic and statistical calculator for public health, was used.The sample was calculated with the following assumptions: the percentage of hypertriglyceridemia in the group not exposed to vitamin D replacement (40%)2.7All rotating shift workers from 5 units of an iron ore mining company are invited by study researcher to participate in screening for a general health assessment. The screening visit is used to identify eligible participants. After identifying the potential participant, the study investigator invites the individual to participate in the clinical trial. The invitation is made for individual or group face-to-face interviews, to clarify any doubts, to sign the consent form and to delivery to the participant a copy of the consent form.2.8Randomization will be used to ensure a balance between the 2 intervention groups and placebo group for deficient and insufficient vitamin D frequencies. The groups were also balanced by the following cardiovascular risk factors: fasting glucose, HDL cholesterol, triglycerides, LDL cholesterol, blood pressure, and waist circumference Table . similar to propensity score matching. The steps used in the allocation process of the patients in the groups of this clinical trial are presented below, considering a sample of p variables with a total of m categories:The allocation of the individuals were performed by an algorithm developed in R software version 3.5.1  language that guarantees the similarity of the groups in relation to the total sample (eligible) considering a final sample (selected) weighted by the parameters and categories considered ;Second step: The algorithm receives the proportions of the categories of each variable and creates an ordered vector containing the proportions of the categories of each variable of the complete original sample k groups of equal size n/k, where n is the sample size. If the result obtained from n/k is not an integer algorithm, the size of each group will be rounded to the next integer. Thus, the number of elements in the groups is defined;Third step: It randomly divides the initial sample into k groups, the proportions of the categories of each variable and each of the groups are calculated according to 1st and 2nd steps and the proportions vector Pk1 = ;Fourth step: After dividing the k subsamples and the original proportions of the total sample, is defined as D0. That is: Fifth step: Sum of the Euclidean distances between the vectors of proportions of the This will be the metric used in the simulation.Sixth step: New subsamples are generated according to 4th step and a new metric is calculated for the new subsamples according to 6th step, namely: D is maintained as the most probable sample to the original and will be the basis of the next simulation. The sample with the highest value of D will be discarded.Seventh step: The subsample metrics are compared. The sample whose subgroup division presents the lowest value of D reaches a predetermined stopping criterion or until a certain maximum number of simulations occurs. The sample with the lowest D value will be the one with the highest similarity in terms of the proportion of the categorical variables between the groups when compared to the original proportions.Eighth step: Repeat 6th–7th steps until the metric 3Double-blind masking was maintained for the participants and the team members in direct contact with the participants. Only 1 researcher in the Laboratory of Cardiometabolism, School of Medicine of the Federal University of Ouro Preto, who is not involved in the data collection, has the knowledge of this information. This researcher was responsible for allocating the participants to the intervention or placebo groups and for receiving the packaged and encapsulated supplements.3.1The screening was performed in a population of 686 rotating shift workers; all individuals were invited to a general health assessment. All data were collected at the company's clinic through face-to-face interviews with a structured questionnaire assessment and a blood draw. The team responsible was composed of nurses, biochemists, nutritionists, and students, who were trained to collect the data before the study.Figure The blood samples were collected after 10 hours of fasting by nurse and pharmacist of responsible team. Every follow-up visit the blood samples are analyzed immediately on fresh samples, and aliquots are stored frozen at −80°C. The procedures and biochemical analyses were performed by pharmacist of responsible team at the Laboratory of Pilot and Clinical Analyses (LAPAC/UFOP) and Laboratory of Epidemiology of Parasitic Diseases of the Federal University of Ouro Preto School of Medicine Table . with their feet placed on the 2 lower electrodes while holding the 2 upper electrodes with their arms extended away from the trunk, and without moving or talking during measurement. Four hours of fasting, an empty bladder, minimal dress, absence of heavy metal, and no strenuous exercise was also required before measurement, according to the Tanita manual . The following data were collected: body fat percentage , visceral body fat, skeletal muscle mass , total body water, total bone tissue, and basal metabolic rate. For the measuring process, the participant were standing barefoot in the platform center, without wearing or holding any heavy objects, looking straight ahead,al Table .3.2P < .05 will be considered significant.Baseline characteristics and follow-up measurements of the study population will be summarized separately within each group. The baseline characteristics are vitamin D, fasting glucose, HDL cholesterol, triglycerides, LDL cholesterol, blood pressure systolic, waist circumference, cognition, sex, age, race, calcium, PTH, albumin, creatinine, total cholesterol, sun exposure, alcohol, physical activity, and food consumption. The follow-up measurements will be demonstrated as continuous variables, which are vitamin D, HDL cholesterol, triglycerides, LDL cholesterol, waist circumference, and total cholesterol. For continuous variables, the means and standard deviations or medians, 25th and 75th percentiles will be presented. The ANOVA or the Kruskal–Wallis test will be use for continuous variables after evaluating normality and the equality of variances using the Kolmogorov–Smirnov and Levene tests, respectively. For categorical variables, the number and percentage of participants within each category will be presented. The Pearson chi-squared test or Fisher exact test will be used. For each variable , the percentage of missing values will be reported. For all tests, a  for proportions (for binary outcomes), and using linear regression for differences in means (for continuous endpoints), with 95% confidence interval (CI). An analysis will be performed to check whether adjusting for age, duration of shift work, physical activity level, alcohol consumption, lower cholesterol drugs, and foods consumption  has any impact on the estimated treatment effects; if it has no impact, then they will not be included in the model. Statistical analyses will be calculated using PASW  version 17.0 and R software (R Development Core Team) version 3.5.1.The analysis of primary and secondary outcomes will use an intention-to-treat (ITT) population and a per-protocol (PP) population. ITT population includes all participants for whom outcome data are available, in the group to which they were randomized, regardless of the treatment actually received. PP population excludes individuals who did not comply with the protocol. The primary and secondary outcomes, which are hypertriglyceridemia, high LDL cholesterol, waist circumference, and low cognitive impairment, will be compared separately between each treatment group and placebo, using binary logistic regression or regressão de Poisson, if the outcome is rare, 5% or less, and the relative risk (RR) is less than about 23.3This trial will be monitored by a data monitoring committee (DMC), Trial Steering Committee (TSC). That includes biostatistics, epidemiologist, cardiologist, occupational physician, nutritionist, and internal member of the company. The TSC performs the following indicators of the work execution, through reports and seminars. The DMC will perform the monitoring during and at the end of the study for endpoints settings and code opening. Interim analysis is performed to assess the sample losses after each follow-up visit.3.4A unique numeric identifier is assigned to each trial participant. All data are stored on the drive and in the cloud, and the links to this information are available only to the study coordination team. Consent forms and questionnaire data are stored in paper on Laboratory of Cardiometabolism of Medicine School of the Federal University of Ouro Preto. The questionnaire data are single-entered by the study team and the range checked twice for data values, to assure the quality of data entry.3.5This clinical trial was approved by the Research Ethics Committee of the Federal University of Ouro Preto (CEP 1.381.376) on December 17, 2015; and registered as “Fatigue Management Project” in the Brazilian Registry of Clinical Trials (RBR-6CS352). The responsible institution for this clinical trial is the Medicine School of Federal University of Ouro Preto. All participants sign consent form. If further other analyses will be required, prior authorization from the participants will be solicited.If there is presence of side effects or proportion of individuals with hypervitaminosis higher than expected, there will be a change in protocol with communication to investigators, research ethics committee/institutional review board, trial participants, trial registries, journals, and regulators. This study does not have financial compensation, but to those who suffer damages resulting from the study participation, it is assured the referral to the company's health service.The results of the trial will be reported to participants, health professionals and the public through seminars open to participants, technical report, and scientific articles.4 Shift workers correspond to 14.8% of the workforce, with 2.5% rotating shift workers. In the mining area, the proportion of workers in rotating shifts is higher, reaching 31.9%.This is the first clinical trial evaluating 12 months of vitamin D supplementation in rotating shift workers. Shift work schedules are those that occur irregularly or uncommonly in comparison to usual daytime work (7/8 AM–5/6 PM), and can be fixed or rotating, continuous or discontinuous, and include or not include a night shift. However, this work schedule contributes to the desynchronization of the circadian cycle. The circadian cycles are biological rhythms that are synchronized with external stimuli in order to adapt the organism to the environment in which it lives, thus allowing corporal homeostasis. Among the environmental factors, luminosity is considered the main external stimulus that promotes the synchronization of endogenous rhythms. Nighttime activities contribute to the misalignment of the circadian cycle, which can lead to adverse health consequences. Studies have reported that shift workers are at an increased risk for obesity, diabetes, metabolic syndrome, and cardiovascular diseases, as well as for increased glucose, triglycerides, total cholesterol, blood pressure, waist circumference and BMI, and for decreased HDL cholesterol –10,50.The shift work schedule allows activities 24 hours a day and aims to meet the needs of services, industries, and leisure. in a systematic review with 71 articles and a total sample of 53,345 workers, found, in shift workers, approximately 80% hypovitaminosis D (≤20 ng/mL), a higher risk of developing vitamin D deficiency  and a higher risk of developing vitamin D insufficiency  when compared to other occupational groups. This study suggests that these workers are more vulnerable to hypovitaminosis D. In a previous study conducted by our research group, with 391 rotating shift workers, we observed a high prevalence (73%) of hypovitaminosis D (<30 ng/mL).In addition, Sowah et al,Therefore, the present study aims to contribute knowledge about the role of the dose and time of vitamin D supplementation in generating beneficial effects on triglycerides in a population at increased risk for hypertriglyceridemia and hypovitaminosis D.4.1This trial is not concluded. This trial began with 362 participants enrolled in November 2016.Authors thank Medicine School and Nutrition School of Federal University of Ouro Preto for the transportation of interviews. Authors also thank Laboratory of Pilot and Clinical Analyses and Laboratory of Epidemiology of Parasitic Diseases of Medicine School of the Federal University of Ouro Preto for the equipment of procedures and biochemical analysis.Conceptualization: Fernando Luiz Pereira Oliveira, George Luiz Lins Machado-Coelho, Fausto Aloísio Pedrosa Pimenta, Silvia Nascimento Freitas, Raimundo Marques Nascimento Neto.Formal analysis: Virginia Capistrano Fajardo, Fernando Luiz Pereira Oliveira.Funding acquisition: Raimundo Marques Nascimento Neto.Methodology: Virginia Capistrano Fajardo, Fernando Luiz Pereira Oliveira, George Luiz Lins Machado-Coelho, Fausto Aloísio Pedrosa Pimenta, Silvia Nascimento Freitas, Antônio Luiz Pinho Ribeiro, Maria Marta Sarquis Soares, Marcio Weissheimer Lauria, Rosielle da Costa Farias, Raimundo Marques Nascimento Neto.Project administration: Fernando Luiz Pereira Oliveira, George Luiz Lins Machado-Coelho.Supervision: Virginia Capistrano Fajardo, Fausto Aloísio Pedrosa Pimenta, Silvia Nascimento Freitas, Raimundo Marques Nascimento Neto.Writing – original draft: Virginia Capistrano Fajardo.Writing – review & editing: Fernando Luiz Pereira Oliveira, George Luiz Lins Machado-Coelho, Fausto Aloísio Pedrosa Pimenta, Silvia Nascimento Freitas, Antônio Luiz Pinho Ribeiro, Maria Marta Sarquis Soares, Marcio Weissheimer Lauria, Ive Bahia França, Raimundo Marques Nascimento Neto.


Ae. albopictus (Zhejiang/Aedes-1/2017) were determined. Phylogenetic analyses were performed using the neighbor-joining method with the Tajima-Nei model. Phylogenetically, DVs of all four serotypes with multiple genotypes  were present in the indigenous and imported cases in Zhejiang during the same period. Most of the isolates probably originated from South-East Asia and Western Pacific countries. The imported cases, high density of mosquito vector, and missed diagnosis might contribute to the 2017 outbreak in Zhejiang.Dengue, a mosquito-borne disease caused by the dengue virus (DV), has been recognized as a global public health threat. In 2017, an unexpected dengue outbreak occurred in Zhejiang, China. To clarify and characterize the causative agent of this outbreak, data on dengue fever cases were collected from the China Information System for Disease Control and Prevention in Zhejiang province for subsequent epidemiological analysis. A total of 1,229 cases were reported, including 1,149 indigenous and 80 imported cases. Most indigenous cases  were in Hangzhou. The epidemic peak occurred in late August and early September, and the incidence rate of elderly people  was relatively high. Imported cases were reported all year round, and most were from South-East Asia and Western Pacific regions. Young people and men accounted for a large fraction of the cases. Acute phase serums of patients were collected for virus isolation. And 35 isolates  were obtained after inoculation and culture in mosquito C6/36 cells. The E genes of the 35 new DV isolates and the complete genome of a DV-2 isolate (Zhejiang/HZ33/2017), and the E gene of a DV-2 isolate from  Flavivirus, family Flaviviridae. Its genome is approximately 10,700 bp, comprising a single open reading frame that encodes three structural proteins  and seven non-structural proteins  . The vast distribution of mosquito vectors, which is fueled by frequent human migration, and a lack of adequate listed vaccines and effective drugs have resulted in a dramatic increase in dengue virus (DV) infections, which have undergone 30-fold growth over the past 50 years  but no deaths were reported in Zhejiang, China. The DV cases in Zhejiang Province were mainly DV serotype 2, with a small fraction of DV serotype 1, 3, and 4. To analyze the molecular evolution of the virus, a phylogenetic tree can be constructed by using specific regions of the DV genome, such as the full-length sequence, E, or E/NS1  at the China Information System for Disease Control and Prevention in Zhejiang province. In China, DF is classified as a class-B notifiable infectious disease. Cases of DF must be reported to NNIDRIS within 24 h after diagnosis. Dengue cases were diagnosed according to the criteria for DF from the Chinese Ministry of Health. Information on each dengue case included demographic characteristics, laboratory confirmation, onset data, diagnosis data, and whether the case was imported or indigenous. An imported dengue case was defined as an infected person whose whereabouts could be traced to an origin in a dengue-endemic area outside Zhejiang Province within 15 days of the onset data; otherwise, cases were defined as indigenous. Characteristics of dengue cases were described by age, sex, occupation composition, and temporal and spatial distribution. Incidence was calculated as the number of cases divided by the population size during the study period. Temporal and spatial analyses of indigenous and imported cases were performed in Zhejiang Province, and spatiotemporal analysis of indigenous cases was performed in 13 districts of Hangzhou. Statistical analyses were performed by using ArcGIS 10.3  and R statistical software, version 3.2.2 (R Development Core Team 2015).Ae. albopictus infestation levels. Vector surveillance continued until BI was less than 5 according to dengue guidelines for diagnosis, treatment, prevention and control of the World Health Organization.The mosquitoes were collected in sweeping nets at various communities located in the main urban area of Hangzhou during the 2017 dengue outbreak. The pooled mosquitoes were then stored at −80°C until virus detection. DV nucleotide detection, serotype identification and E gene amplification used the methods that mentioned below. Breteau index  was used to record A total of 90 serum samples of patients were obtained from the municipal Centers for Disease Control and Prevention (CDC) of Zhejiang. DV nucleotide detection and serotype identification were performed according to diagnostic criteria for DF (WS 216–2008) of the Ministry of Health of China.Ae. albopictus mosquito C6/36 cells, which were a gift from the National Institute for Viral Disease Control and Prevention. The C6/36 cell line was cultured in minimum essential medium (MEM)  supplemented with 2% fetal bovine serum  at 28°C in 5% CO2. When complete cytopathic effects (CPE) were observed, culture supernatant was collected and stored at −80°C until use.Patients' positive acute phase serums (collected within 1–5 days of illness onset) were inoculated in Viral RNA was extracted with a RNeasy Mini Kit  according to the manufacturer's instructions. Complete genome sequencing of the DV-2 isolate from an indigenous case in Hangzhou (Zhejiang/HZ33/2017) was performed as previously described . Bootstrap values ≥70%, calculated from 1,000 replicates, are shown at the tree branches. DV genotype was analyzed by including related reference sequences with known genotypes in the phylogenetic tree.The sequence was spliced in SEQMAN from the LaserGene package . The nucleotide sequences were aligned using the Clustal W multiple sequence alignment program. DV reference sequences were downloaded from the GenBank database. The phylogenetic analysis based on full genome and E gene sequences was carried out by using the neighbor-joining method with the Tajima-Nei model in MEGA version 6.06 , and the incidence rate was 2.00 and 2.11 per 100,000, respectively. The median age was 51 (IQR: 35–63). The incidence rate of elderly people was relatively high, and retirees accounted for nearly one third of all cases . The median age was 35 (IQR: 27.5–50.5), and the incidences in the age groups of 20–39 years old were relatively high. Farmer, commercial service people, industrial worker and cadres accounted for most cases . With the process of mosquito eradication, the BIs decreased rapidly and were all less than 5 in 37th week .A total of 136 mosquitoes were collected in a community in Shangcheng district of Hangzhou, of which 52 were identified based on morphology as Ae. albopictus mosquitoes homogenates to amplify the whole genome and E gene. The complete genome sequences of DV-2 isolate Zhejiang/HZ33/2017 and the E gene of 35 additional isolates were obtained and submitted to the NCBI GenBank database (GenBank accession numbers: MH010595-MH010630).Eighty-one of the 90 collected serum samples were DV RNA positive, including 55 indigenous cases and 26 imported cases. Serotype identification results indicated the presence of all four serotypes DVs in Zhejiang Province in 2017: 68 DV-2 cases (50 indigenous and 18 imported), 10 DV-1 cases (5 indigenous and 5 imported), 2 DV-3 imported cases, and one DV-4 imported case. Among 43 randomly-selected acute phase serums that were positive for DV RNA, 35 DV isolates were successfully obtained after inoculation and culture in C6/36 cells. Routine RT-PCR was performed with virus culture and the supernatant of the pooled Phylogenetic analysis was performed based on the sequences obtained, including reference sequences from the NCBI GenBank database. This analysis indicated that DVs of all four serotypes were present in indigenous and/or imported cases in Zhejiang Province, 2017 Table .The complete genome sequences of isolate Zhejiang/HZ33/2017 and sequences from representative isolates of different countries were chosen to construct a phylogenetic tree, and sequences of the DV-1 strain Hawaii, DV-3 strain H87, and DV-4 strain H241 were used as outgroups. The phylogenetic reconstruction classified all the DV-2 isolates into five genotypes, and Zhejiang/HZ33/2017 was identified as the Cosmopolitan genotype and was closely related to viruses from Malaysia and Singapore Figure . The resThe phylogenetic tree based on E gene categorized all DV-1 isolates into five genotypes and revealed that DV-1 from Zhejiang Province had a different genotype Figure . Two paiDV-3 and DV-4 isolated from DF cases in Zhejiang in 2017 were all imported. Phylogenetic analysis indicated that the DV-3 isolate Zhejiang/17-16/2017 belonged to genotype III, clustering closely with virus from Africa (2013) Figure .DF was clearly reported in the coastal provinces of China in the early twentieth century; and the first DF outbreak was identified in Guangdong province in 1978. Since the 1990s, dengue epidemics have spread gradually from Guangdong, Hainan, and Guangxi provinces in the southern coastal regions to the relatively northern and western regions including Fujian, Zhejiang, and Yunnan provinces.  were also analyzed based on their E gene sequences, respectively. Four DV-1 isolates from indigenous cases in Taizhou were located in two clusters belonging to genotypes I and V Figure . Case 17This was a large outbreak with 1,229 cases, including 1,153 cases  in Hangzhou, the provincial capital of Zhejiang. DV-2 was the dominant serotype epidemic in Zhejiang; however, all four serotypes emerged during the same period. Although Zhejiang does not belong to the endemic area of dengue, there are vector mosquitoes and environmental conditions supporting DV transmission in some regions of Zhejiang. There is always a risk of local outbreak caused by imported cases, such as the DV-1 and DV-3 outbreaks in 2004 and 2009, respectively. There are imported dengue cases in Hangzhou every year that do not cause a large scale endemic transmission every time. Hence, why was there a dengue outbreak in 2017? We inferred some probable reasons that may have led to this unexpected large outbreak. First, for Hangzhou residents, some South-East Asia and Western Pacific countries, including Thailand, Myanmar, Sri Lanka, Vietnam, Malaysia, are popular tourist destinations. Second, Hangzhou is a beautiful and economically advanced city in China that experiences high population mobility every year, as notably occurred after the Group 20 Summit in 2016. Third, long periods of high temperature and abundant precipitation in the summer and fall of 2017 in Hangzhou probably led to more frequent mosquito activity and facilitated mosquito breeding (Yang et al., In our study, it was found that DVs of all four serotypes have been identified in Zhejiang Province. During 2017, the DV-2 Cosmopolitan genotype and DV-1 genotypes I and V were verified in indigenous cases. Multiple isolates of each serotype were present, and this may have increased patients' risk of dengue hemorrhagic fever and dengue shock syndrome, and threatened patients' lives Morens, . FortunaAe. albopictus in urban areas increase the risk of indigenous dengue transmission and indigenous DF epidemics in Zhejiang Province. Because no effective vaccines and drugs are available for dengue, prevention and control measures should be strengthened. Determining the origin of the 2017 isolates from Zhejiang Province might provide information that could lead to more effective control measures. Efforts should be made in improving the capabilities of clinicians to diagnose this disease, improving and disseminating proper preventive measures in high-risk populations, surveillance of imported cases, strengthening vector control, and prevention and control of local epidemics.Our results indicated that imported DF patients from South-East Asia and Western Pacific countries were probably the primary cause of the DF epidemics in Zhejiang Province. Widely distributed of YZ designed and supervised the experiments. HY, JY, WY, JP, and XL carried out the experiments. HY, ZD, JY, JP, JS, HW, and CW analyzed the data. HY and ZD wrote the paper. JY, HY, JH, and HM performed sample collections. ZY, HM, and JL provided helpful suggestions about the study. All authors read and approved the final 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.


Visual fatigue and discomfort are very common complaints for video display operators (VDTs). The aim of our study was to study work-related visual symptoms in relation to refractive disorders and psychosocial factors in 3054 public employees by way of follow-ups for 10 years with periodic medical examinations with eye evaluation in the period 2000–2009. Factors related to visual fatigue were evaluated in the follow-up using generalized equation estimation. Visual fatigue was very common in VDT operators (64.03%). During the follow-up, no relationship between visual fatigue and age, sex, seniority of work, visual acuity and refractory disorders was found. Visual fatigue was significantly associated with anxiety perception in a dose-related matter (odds ratio (OR) 7.40, confidence interval (CI) 95% 1.77–31.3), psychosocial factors , use of lenses  and time of VDT usage . This study confirmed that visual fatigue is common in VDT users and is related to anxiety perception, time of VDT usage, use of lenses and stress. No relationship was found between visual fatigue and refractory disorders or visual acuity. Workers that spend many hours on video display terminals (VDTs) can report visual fatigue and discomfort related to their work environment as well as vision defects, psychosocial stress and anxiety perception ,2,3,4. MEye symptoms are very common in VDT users, and the American Optometric Association has started to refer to computer vision syndrome (CVS) as the combination of eye and vision problems associated with the use of computers. This derives from insufficient visual capabilities to perform the computer task comfortably. CVS is characterized by itching, redness, burning, eye tearing, headache, double vision, eye strain and blurred vision . The preVisual fatigue is a major complaint in subjects with CVS and is characterized by weakness of the eyes, usually accompanied by headache and dimming of vision—symptoms that are transitory and recede with rest ,11,12,13The subjective asthenopic symptoms, however, can also be related to environmental factors, such as lighting, screen resolution and work arrangements . The surVisual fatigue prevalence ranges between 19.6%  and 72.1In addition to ergonomic and environmental factors ,22,23, sVisual fatigue and stress are common in VDT users ,26,27 anWe evaluated 3054 computer operators working with VDTs for more than 20 h per week in different public offices in Trieste  that underwent periodical health surveillance in the Clinical Unit of Occupational Medicine from 2000 to 2009. Two thousand two hundred and eight workers completed the follow-up with 3 or 4 medical examinations according to age: workers underwent a periodic medical evaluation with eye examination, as required by Italian law (every 5 years for workers <50 years-old and every 2 years for those ≥50 years-old). Lenses in use were defined using a lensmeter . Myopia was defined when >−0.25 dioptres (D). Hyperopia and presbyopia were defined when >0.25D and astigmatism was defined when >0.25D. Visual acuity using lenses was evaluated for far, near and intermediate distances (57 cm) using the Vision test .During the medical examinations, workers filled in a standardized questionnaire, divided into three sections. (1) The first part comprised 59 questions concerning personal data, years of use of a VDT, hours of use per day, computer programs used, screen size and type, work breaks, interruptions during VDT work, work environment in terms of lighting, glare on the screen, distance between the computer and operator, working desk, environmental noise and temperature; (2) the second part analyzed workers’ life and health, marital status, schooling, sport, smoking habit, coffee consumption, eye illnesses, use of lenses, eye examinations, and joint diseases; (3) the third part investigated the frequency of eye-symptoms related to VDT use: fatigue, redness, burning, tearing, headache, photophobia, far and near blurred vision, myodesopsia, double vision, and eye drop application—defined as never, sometimes, and often in relation to VDTs or not related to VDTs; (4) anxiety perception was defined as never, sometimes, and often in relation to VDTs or not related to VDTs ; (5) psychosocial factors were evaluated using a simplified questionnaire that investigated discomfort (1—yes or 0—no) during work at VDTs regarding the following aspects: overcrowding, relationship with users, relationship with colleagues, work condition, responsibility, repetitiveness, excessive concentration, excessive attention, low possibility of correcting errors, frequent interruption during work, computer slowness, excessive variability of work tasks, low control of work tasks, slow cadence of work tasks, and fast cadence of work tasks. Psychosocial factors were summarized as the sum of all answers. All subjects signed an informed consent, and data were analyzed anonymously. Periodical medical surveillance is compulsory in Italy under the law (81/2008) and, according to Italian rules, the Local Ethical Committee was informed about the study.p < 0.05.Data were collected using the program Excel for Windows, and statistical analyses were performed using the STATA program . Continuous data were compared using the t-Student test and t-pair test for repeated measures; categorical data were compared with the chi-square test and with Mc Nemar test for repeated measures. Relationships between visual fatigue, occupational data and other symptoms were evaluated with Pearson correlation and univariate logistic regression. Significant findings were studied with multivariate logistic regression. The relationship between visual fatigue and potentially associated variables during follow-up was evaluated using generalized estimating equations (GEE) that permit the study of the same subject over time. Statistical significance was settled at p < 0.005. A similar trend was found for work seniority starting at 7.4 ± 6.7 years in the first control and reaching 14.5 ± 7.4 in the last follow-up (p < 0.001). The time of VDT use was around 5 h per day in the period considered. Refractive defects in workers over time are shown in the same table. Myopia was quite stable in terms of years; astigmatism ranged from 37.14% to 42.97%. Presbyopia increased according to the age of people (from 36.82% in the first examination to 52.26% in the last one (p < 0.001). Hyperopia grew from 7.5% in the first examination to 9.94% at the intermediate follow-up and to 11.59% at the last control (p < 0.001).The characteristics of the workers involved in the study are reported in The use of lenses was very common during the follow-up, with more than 80% of people using lenses.p < 0.001), ocular redness , and near blurred vision . Headache and anxiety were reported in similar figures during the follow-up (12% to 14% and 3.5% to 4.2%), respectively.The most common eye symptom was visual fatigue, followed by ocular redness, burning and tearing. In general, eye symptoms increased during follow up, reaching statistical significance for visual fatigue  and OR 4.97 (CI 95% 1.48–6.84) for “sometimes” or “often”, respectively. VDT working hours, use of lenses, hyperopia and psychosocial factors were significantly related to visual fatigue.To analyze factors related to visual fatigue in the follow-up, we used the generalized estimating equation (GEE) as shown in Age was inversely associated with visual fatigue, and no relationship was found with seniority of work and gender. Anxiety perception was strongly associated with visual fatigue in a dose-related matter with OR 2.69 (CI 95% 1.57–4.61) for “sometimes” feeling anxious during VDT use and OR 7.10 (CI 95% 1.77–31.03) for “often” feeling anxious during VDT use. A significant relationship was demonstrated also with hours of work with VDTs , use of lenses , environmental factors  and psychosocial factors .Many epidemiological studies on VDT users have reported the presence of computer vision syndrome , and ourRegarding refractive disorders, myopia prevalence did not increase in follow-up, while presbyopia and hyperopia were clearly age-related and astigmatism increased in small amounts during follow-up. We found visual defects to be higher than those in the literature  because Visual fatigue was also related to environmental and ergonomic factors such as monitor characteristics and workplace illumination, in accordance with the literature ,43,44. PPsychosocial factors were associated with visual fatigue. They are, in addiction to ergonomic factors, the basis of the occurrence of somatic disorders (such as headache and insomnia), generalized tension and stress ,46,47. ASome factors were more related to visual fatigue than others. Work in crowded places, having problems with colleagues, performing tasks that require too much attention and concentration, and having little control over decisions about work, intensity of work have relevant impacts on the occurrence of visual symptoms. Other important factors are job position and the repetitiveness of working duties.Another important issue that must be considered in people using VDTs is motion sickness, which has been studied mainly in video game users  or in peOur study is one of the largest and longest available in the literature, to our best knowledge, and demonstrated that visual fatigue did not increase with refractive disorders or with visual acuity with lenses, or in relation to work seniority and age, confirming that symptoms are not harmful for the eye. Visual symptoms are mainly caused by anxiety perception and psychosocial factors: this finding suggests the need to consider this and to suggest interventions to increase wellness in workplaces, but also the need to emphasize the safe characteristic of work with VDT, avoiding the overestimation of symptoms without signs of diseases. Moreover, visual symptoms in general are mild. To confirm this, the majority of workers reported “visual fatigue in relation to VDTs sometimes”, while “visual fatigue in relation to VDTs often” was reported by a lower percentage of workers. Irritant symptoms such as “red eye” and “burning eye” were reported by less than 7% of subjects, meaning that important symptoms were rare, and acute and disabling symptoms were never reported . MoreoveOur study, in a long follow-up, failed to find an increase of refractory disorders in VDT users. Visual fatigue was common and symptoms were related to many factors, but not to refractive disorders and visual acuity with lenses. Anxiety perception was of paramount importance compared to environmental factors, use of lenses, time of VDT use and psychosocial factors. Visual fatigue did not increase in relation to age, and seniority of work and gender did not play any role in the occurrence of symptoms. In our 10-year follow-up, refractive disorders increased mainly in relation to age as presbyopia and hyperopia, while no increase in the prevalence of myopia was found, confirming that VDT use is not harmful for vision.This study emphasizes the need to promote the wellness of workers, focusing on interventions to reduce anxiety, increasing good relationships between workers, and promoting a good psychosocial environment together with a good control of environmental variables and the correction of refractive disorders, which seems to be not relevant compared to other factors. Effort can be put into the good organization of the workplace, the environmental control of lights and a better evaluation of psychological factors.


It is found that defects will occur when the process parameters are not controlled properly and the main defects of composite materials include inadequate resin impregnation, weak interlaminar binding force, fiber displacement warping, hole and brittle fracture. Moreover, there are significant differences in the infiltration microstructure, bending properties, and fracture morphology of the composite materials with different defects. When the defects of weak interlaminar binding force and brittle fracture occur, bending properties of composite materials are relatively low, and they are 220 MPa and 245 MPa, respectively, which reach 34.9% and 38.9% of the bending strength of composite material whose defects are effectively controlled. When the process parameters are reasonable and the defects of the composite materials are effectively eliminated, the bending strength can reach 630 MPa. This will lay a foundation for the preparation of 2D-T700/E44 composite materials with ideal microstructures and properties by ICM. Fiber-reinforced composites have a series of excellent properties, such as low density, high specific strength, and specific stiffness, low wear resistance, low thermal expansion coefficient, good fatigue resistance, good dimensional stability, etc. They have wide application prospects in aerospace, weapon equipment, communications electronics and transportation ,2,3,4. CAt present, the preparation methods of carbon fiber-reinforced resin matrix composites mainly include hand paste molding, winding molding, extrusion molding, resin transfer molding and molding process ,8,9,10. In this paper, an improved experimental system of improved compression molding process (ICM) is proposed on the basis of traditional molding method. Firstly, the precut and impregnated fiber material are put into the high temperature curing box to preliminarily realize the flow and impregnation of the resin. Then, after the initial curing of the resin, it is transferred to the hot pressing mold to complete the final curing of the composite material. This will prevent the resin from sticking to the mold so that it is easy to take out the parts, and fiber segregation and displacement defects do not occur easily, which are beneficial to the preparation of 2D-T700/E44 composite with high properties. However, when this process is used to prepare composite materials, it involves parameters such as solution ratio, curing temperature, pressure and time, etc. When the process parameters are not controlled properly, it is easy to produce defects, which will affect the successful preparation of composite materials with ideal microstructure and performance. Some scholars have studied the fabrication defects of composites ,12,13, bIn this paper, 2D-T700/E44 composite materials were prepared by ICM. Then combined with microstructure observation analysis and bending strength test, the defects such as insufficient impregnation of solidified mixed solution, weak interlaminar binding force, fiber displacement warping, pore hole and brittle fracture of composite materials during the preparation of composite materials were thoroughly analyzed. Finally, the main causes of defects and the control measures to avoid defects were obtained, which would lay a foundation for the successful preparation of ideal composite materials.The reinforcement used in the study is 12K unidirectional T700 carbon fiber cloth produced by Toray in Japan, and the matrix used is E44 epoxy resin produced by Xi’an resin factory in China. The curing agent used is 593 curing agent of Sanmu Group Co. Ltd. In the experiment, T700 carbon fiber non-woven fabric was cut into rectangular thin sections, and its length and width were 100 mm and 50 mm, respectively. The cutting carbon fiber cloth with the two fiber directions was shown in 2D-T700/E44 composite materials were prepared by ICM. The steps of using this process to prepare 2D-T700/E44 composite materials are as follows. (1) E-44 epoxy resin and 593 curing agent were mixed into a curing mixed solution according to the mass ratio of 5:1. (2) The curing mixed solution was spread on both sides of the carbon fiber cloth sheets, and then sheets were laminated and compacted. The rotation angle between adjacent fiber laminations is 90°, and lamination thickness of the composites prepared is 2~2.5 mm. (3) Carbon fiber laminations were placed at room temperature under the condition of 6 h to achieve natural curing process, and then they were transferred to the constant temperature drying box to achieve pressure-free heating curing process, whose curing temperature and time were 115 °C and 110 min, respectively. (4) The carbon fiber lamination was taken out from the constant temperature drying box and put into a preheated hot pressing mold to achieve pressure heating curing process. The curing temperature and pressure were 50 °C and 10 MPa respectively, and pressure holding time was 25 min. (5) The heating and pressure were stopped. After the mold was restored to room temperature, 2D-T700/E44 composite material was obtained by taking out from the mold. The process of preparing 2D-T700/E44 composite materials by ICM was shown in The impregnation microstructure and bending fracture morphology of 2D-T700/E44 composite material were observed by JEOL JSM-6390A scanning electron microscope (SEM). The bending strength of 2D-T700/E44 composite was tested by DNS100 electronic universal testing machine of Changchun institute of mechanical science and the test method followed Test Method for Flexural Properties of Fiber-reinforced Plastics (GB/T1449-2005). The bending sample size is 50 mm × 15 mm × 2 mm, and the loading upper head radius R is 5 mm. To obtain reasonable bending strength value of each composite, three bending strength specimens are tested, and representative bending strength of the composite is chosen in this study. The test loading speed is 10 mm/min, and the span is 40 mm. The experimental schematic diagram is shown in The improved compression molding process (ICM) for the preparation of 2D-T700/E44 composites involves the processes of preparation of the curing mixed solution, coating on the carbon fiber cloth of the curing mixed solution, natural curing, pressure-free heating curing, and pressure heating curing. It also involves process parameters such as curing mixed ratio, natural curing time, pressure-free heating curing temperature and time, pressure heating curing pressure, and pressure heating curing temperature and time. All these process parameters can affect the preparation effect of 2D-T700/E44 composite materials, and defects such as insufficient resin impregnation, weak interlaminar binding force, fiber displacement warping, hole and brittle fracture of composite materials will occur, when they are not properly controlled. Therefore, it is necessary to conduct in-depth study on the causes of these defects and the control methods of reducing the defects to prepare 2D-T700/E44 composite materials with ideal microstructures and properties.When the curing temperature and pressure are not appropriate, the insufficient resin impregnation occurs easily . Figure When the process parameters such as the ratio of the curing mixture solution or the curing temperature and pressure are inappropriate, the bonding force between the layers will be weak, so the properties of the materials will be reduced due to the lamination under loads. As shown in the microscopic picture of fiber stratification b, it canWhen 2D-T700/E44 composite materials were prepared by ICM, if the curing temperature and time were not reasonable, fiber displacement or warping would occur, and the carbon fiber in the same layer was not distributed in a plane . Its preWhen 2D-T700/E44 composite materials were prepared by ICM, hole defects would appear in the composites if the pressure, temperature and time of pressure heating curing were not feasible. The hole is mainly formed by gas accumulation which is not eliminated in time, which leads to irregular shape and size under load. The discharge of gas is affected by the fluidity of the curing mixed solution, and fluidity is influenced by its temperature. If the temperature of curing mixed solution is too low, its fluidity is poor, and the gas discharge rate is slow, so bubbles occur easily. If the temperature is too high, the mixture has been solidified before gas discharges, and this will result in hole. The formation of holes indicates that the composite is not compacted enough, and the porosity is large. The size of porosity is directly related to the bending properties of composites. The higher the porosity is, the worse of the bending strength of materials is . The benWhen the curing temperature is high and the curing time was short, the curing mixed solution is rapidly solidified, and brittle composite material will be formed. When the material is loaded, brittle fracture is likely to occur, resulting in low bending property . Figure When the process parameters of ICM are appropriate, the defects of 2D-T700/E44 composite can be effectively avoided. The process parameters in From the microscopic fracture in According to the above analysis, when the parameters of 2D-T700/E44 composite materials prepared by ICM are not reasonable, the defects such as insufficient resin impregnation, weak interlaminar adhesion, fiber displacement and warping, holes and brittle fracture of the composite materials are easy to occur, which will affect the impregnation microstructure, bending properties and fracture morphology of the composite materials. Meanwhile, these defects will affect the preparation of ideal composite materials ,19. BendBending strength values of the composites were 315 MPa and 335 MPa  under th(1)2D-T700/E44 composite materials can be prepared by using ICM. When the process parameters of curing mixed ratio, curing temperature, pressure, and time are not controlled properly, the preparation defects such as insufficient impregnation, weak interlaminar binding force, fiber displacement and warping, holes, and brittle fracture of the composite material occur easily.(2)The effects of different defect forms on the bending properties of CFRP composites are obviously different. Bending strength values of composites under the defects such as weak interlaminar binding force, brittle fracture of composite material and fiber displacement, and warping are only 34.9%, 41.3%, and 38.9%ofthe bending strength of 2D-T700/E44 composite without defects. Bending strength values of the composites under the defects such as insufficient resin impregnation and hole only reach 50% and 53.2% of the bending strength of 2D-T700/E44 composite without defects, respectively.(3)When the process parameters are reasonable and the defects of composite materials are effectively eliminated, the bending strength can reach 630MPa. If defects occur, the bending property of the composite material will be greatly reduced. Too large or too small parameter values are unfavorable to the preparation of composite materials with ideal microstructure and properties. The defects of composite materials need to be reduced and avoided through process improvement and parameter control.