Bacillus licheniformis DSM 17236 and Saccharomyces cerevisiae cell wall extract on dextran sulfate sodium (DSS)-induced colitis in Sprague Dawley rats.Uncertain effects of probiotics and/or prebiotics have been reported in experimental and clinical colitis. This study aims to examine the effects of a synbiotic combination comprising Acute colitis was induced in rats by oral administration of DSS 3.5% for 7 days. Fifty rats were divided equally into five groups; one control group and the other groups were induced with colitis and treated with or without the tested synbiotic, mixed with diet, for 28 days and sulfasalazine (100 mg/kg) via intragastric tube once daily for 14 days.p < 0.05) amplification of WBCs, myeloperoxidase (MPO), malondialdehyde (MDA), nuclear factor kappa B (NF-kB) expression and proinflammatory cytokines tumor necrosis factor alpha (TNFα), interferon gamma (INFγ), and interleukin-1 beta (IL-1β) while depressed the antioxidant enzymes glutathione peroxidase (GPx), catalase (CAT), and superoxide dismutase (SOD) when compared with the DSS and control groups. The DSS intoxicated and Synbiotic+DSS groups showed desquamations of the covering epithelium, noticeable diffuse leukocytic infiltrations, sever catarrhal enteritis, ischemic colitis with diffuse coagulative necrosis of the entire colonic mucosa. Contrarily, sulfasalazine proved to be effective in the reduction of the tested inflammatory markers and the pathological degenerative changes of the DSS ulcerative colitis.Symptomatically, the synbiotic administration raised the disease activity index (DAI) to comparable scores of the DSS group, specially from the 2nd to 7th days post DSS intoxication. It also induced a significant (The examined synbiotic did not ameliorate but aggravated the DSS-induced colitis, so it should be subjected to intensive experimental and clinical testing before their use in animals and human. Inflammatory bowel disease (IBD) involves a group of multifactorial disorders including two major forms; Crohn’s disease (CD) and ulcerative colitis (UC), that affect the integrity of the intestinal mucosa , 2. AlthCytokines specially TNFα, INFγ and interleukin 1-beta (IL-1β) are key components of IBD pathogenesis by mediating the communication between innate and adaptive immune cells and stimulate the recruitment of inflammatory cells to the intestinal mucosa –7. DSS dRecent approaches dedicated that oxidative stress (OS) including reactive oxygen and nitrogen species  play a deleterious role in the pathogenesis of IBD . Gut comThe current medications of IBD involving immunosuppressants and anti-inflammatory drugs as sulfasalazine, corticosteroids and anti-TNFα  antibody are used to depress the aberrant immune responses and inflammatory cascades . HoweverBacillus subtilis, Bacillus clausii, Bacillus cereus, Bacillus coagulans and Bacillus licheniformis. Being a spore former, it has the advantages of heat stability and can be stored at room temperature, in addition to its ability to survive the acidity of the stomach [Bacillus licheniformis were found to be safe in the in-vivo toxicity studies, non-toxinogenic, and sensitive to the antibiotics listed in european food safety authority (EFSA), therefore it may be considered as non-pathogenic and safe for human consumption [Probiotics and prebiotics have been used more or less successfully in IBD patients and their protective and therapeutic effects mainly depend on the strains used –20, wher stomach , 23. Accsumption .Bacillus licheniformis ameliorated diarrhea in growing pigs [E. coli [Veterinary studies showed that ing pigs , stimula[E. coli , increas[E. coli , enhance[E. coli , improve[E. coli , and dim[E. coli .Prebiotics are nondigestible dietary substances that have the ability to stimulate growth of native and probiotic bacteria . PotentiBacillus licheniformis on the chemically induced colitis. Therefore, the present study was conducted to investigate the ability of the synbiotic mixture of Bacillus licheniformis DSM 17236 and an extracted prebiotic from cell walls of the baker yeast, Saccharomyces cerevisiae, to modify DSS-induced experimental colitis in Spargue Dawley rats in comparison to the sulfasalazine drug. The assessment included the detection of DAI and the colonic tissue levels of MPO, oxidative stress parameters, NF-kB expression. Furthermore, the leukocytic count and serum inflammatory cytokines  level were assayed. Histopathological examination of intestinal colitis was also performed.As far as our research team knows, a scarcity of studies is available in scientific literature regarding the effect of P < 0.05) of WBCs count , desquamations of the covering epithelium and degenerations of the intestinal gland lining epithelium , and prominent vascular dilatation with congestion in the inter glandular areas and hemosiderosis . Meanwhile the negative control group showing normal and intact colonic wall that is mainly consisted of normal tunica mucosa having folds of tubular glands or crypts lined by columnar epithelium rich with goblet cells, submucosa of highly vascularized connective tissue, then well-defined muscularis and serosa .The intoxicated rats with DSS showed severe degenerations of the covering epithelium with noticeable diffuse leukocytic infiltration extending in between the mucosa and submucosa Fig. b, c, desium Fig. d, e, andsis Fig. f. Meanwhosa Fig. a.Fig. 3Ip < 0.05) of intestinal tissue levels of SOD, CAT, and GPx , while it augmented the production of colonic MDA by 8.9 folds when compared with the control group, respectively  in the serum levels of TNFα (116.91 ± 3.74), INFγ (148.03 ± 6.37), and IL-1β (1931.55 ± 44.91) in comparison to the control group; TNFα (65.12 ± 1.322), INFγ (51.57 ± 51.57), and Il-1β (1309.65 ± 28.02), respectively as shown in Fig. The DSS clearly amplified the colonic tissue MPO by 288% more than the untreated rats  than the control group (4.16- and 3.2-fold changes). The oxidative stress parameters: CAT, SOD, GPx, and MDA, in the colonic tissue of DSS + Synbiotic treated rats were noticeably deteriorated , severe catarrhal enteritis represented in diffuse inflammatory cells infiltration in the mucosa, congestion in the inter glandular area with goblet cells hyperplasia with excessive mucous secretions filling the intestinal glands , hemorrhage , and moderate hemosiderosis . In addition, ischemic colitis with diffuse coagulative necrosis of the entire intestinal mucosa characterized by maintaining tissue architecture but with completely loss of cellular details was also clarified of some examined sections .On the histological level the synbiotic treatment produced intense degenerations and desquamations of the covering epithelium with loss of the cell’s details Fig. a, severends Fig. b, c, hemage Fig. d, and mosis Fig. e. In addons Fig. f.Fig. 6CP < 0.05) by 36% for TNFα, 50% for INFγ, and 26% for IL-1β from the DSS group , mild to moderate inflammatory cells infiltrations in the mucosa , hyperplastic changes of the intestinal gland and its lining goblet cells with excessive mucous secretions, and minute hemorrhage .Sulfasalazine treatment ameliorated the pathological damage in the colon induced with DSS and demonstrated mild degeneration and desquamation of the covering epithelium Fig. a, b, milosa Fig. c, hyperpage Fig. d.Fig. 7C.Furthermore, the sulfasalazine reduced the inflammatory condition caused by the tested synbiotic and DSS in (DSS + Syn + Sulfa) group. This group presented moderate DAI Fig. , proinflFig. d, e, f.FBacillus licheniformis DSM 17236 and an extracted prebiotic derived from cell walls of the baker yeast Saccharomyces cerevisiae (It distinguishes itself by high amounts of Mannan-Oligosaccharides (MOS) and ß-Glucans), in comparison to sulfasalazine regarding their role on the severity of DSS-induced colitis in Sprague Dawley rats.Probiotics and/or prebiotics have been evaluated for the management of some of gastrointestinal disorders including UC , 38, pouIt was found that the synbiotic combination was not able to reduce the symptomatic, heamatological, biochemical and histopathological features of DSS colitis. Interestingly, synbiotic treatment actually increased most of the tested inflammatory indicators of DSS-colitis.In our study, DSS intoxication for 7 days developed the symptoms of the inflammatory conditions in colon, markedly increased the DAI with the 2nd day, and elevated the WBCs count in blood. These findings are similar to what was mentioned by other authors who had applied the same way of induction , 41, 42.DSS model for induction of ulcerative colitis was originally reported by Okayasu et al. . DSS-indThe main histological feature of colitis is the leukocytic infiltration of the colonic tissue specially with the polymorph nuclear cells and monocytes , 46. NeuThe imbalance between the increased amounts of toxic reactive oxygen and nitrogen species (ROS and RNS) and the capacity of the tissue antioxidant defense mechanisms; CAT, SOD, and GPx, leads to oxidative injury and increases mucosal injury in colitic patients , 50. TheThe free radicals and the cytokine like property of MPOinduces nuclear factor NF-κB which is a transcription factor that controls the expression of genes of the proinflammatory cytokine; TNF-α, IFN-γ and IL-1β . IncreasDespite the DSS-colitis is caused through the toxic damage of the intestinal and colonic epithelial cells, the inflammatory reactions ensue after the proinflammatory cytokines  production by tissue macrophages that seems to play an essential role in the disease process , 57. In TNF-α exerts its pro-inflammatory impacts via the raised production of IL-1β and IL-6, as well as initiation of cytotoxic and apoptotic response –60. TNF-On the histological level, the synbiotic treatment induced severe damage of the covering epithelium, coagulative necrosis, and catarrhal enteritis.Bacillus licheniformis Zhengchangsheng® succeeded to attenuate the severity of colitis induced by DSS in mice where it reduced the weight loss, colonic mucosal damage and increased the DAI [Bifidobacterium [Lactobacillus strains [Bifidobacterium infantis, Lactobacillus acidophilus, Enterococcus faecalis, and aerobic Bacillus cereus [Streptococcus salivarius subsp. thermophiles [These results were in contrary to Li Y et al. who reported that  the DAI . Moreoveacterium , or Lact strains , or mixts cereus , or VSL#mophiles –74, havemophiles . Inulin mophiles , 76.On the other hand, because probiotics have influences on both the innate and acquired immune systems, including effects on cytokine secretion and dendritic cell function , 78, exaBacillus subtilis Gallipro DSM 17299 and MOS depressed the growth rate and elevated serum C-reactive protein (CRP), acute phase protein, that is enhanced in response to stimulation by both IL-1 and IL-6, such increase reflects intestinal inflammatory process [Bacillus licheniformis to diet amplified mRNA expression of NF-κB in the jejunum of broilers and in turn activation of their signaling cascade leading to initiation of cellular responses of innate immune cells and production of proinflammatory cytokines [Bacillus licheniformis showed increased production of the proinflammatory cytokine, IL-8, significantly more than the control group, while Bacillus subtilis increased IL-6 secretion [In accordance with our work, feeding chicks with  process , 81. Addytokines . Porcineecretion . This inecretion .Lactobacillus crispatus M206119 suspension induced a higher severity of colitis in the form of elevated DAI involving diarrhea, bloody stools, greater weight loss, higher histopathological damage and leukocytic infiltration when compared to the control group [Lactobacillus salivarius subsp. salivarius 433,118 (UCC118) did not have a beneficial effect on DSS colitis or on pathological or physiological parameters [L. acidophilus NCFM, L. planetarium NCIMB8826 and L. rhamnosus GG also did not attenuate but deteriorate some colitis symptoms [In the same line, Zhou et al. demonstrated that animals treated with ol group . Moreoverameters . Other ssymptoms –89.Lactobacillus reuteri in healthy adults and increased fecal calprotectin [In a double-blind placebo-controlled trial, Mangalat et al. observed an eminent proinflammatory triggering following the administration of rotectin . Moreoverotectin , 92.Saccharomyces cerevisiae antibodies (ASCAs) was discovered in the serum of CD patients [Saccharomyces cerevisiae exhibited intestinal epithelial damage, more crypt loss, and inflammatory cells infiltration [There are numerous clinical and experimental studies that yeast might induce intestinal inflammation. Anti–patients , 94 whicpatients . In the ltration .L. fermentum BR11, elevated the rigorousness of some indicators of DSS-colitis [Saccharomyces cerevisiae and contains about 55% β-glucans) aggravated the progression of DSS-induced colitis and amplified colonic levels of pro-inflammatory cytokines [Interestingly, FOS treatment, alone and in synbiotic combination with -colitis . β-gluca-colitis . Similarytokines .The interactions of different oligosaccharides  with different carbohydrate receptors located on either epithelial or immune cells can trigger the immune reactions in the gastrointestinal tract including the inflammation , 99. We Sulfasalazine, the union of 5-aminosalicylic acid  and sulfapyridine moiety, is an analgesic and non-steroidal anti-inflammatory drug (NSAID) from the salicylates group, which has been extensively used for the management of moderate ulcerative colitis , 101. ItHowever, there is a lack of availability to address some key points in the current study regarding exploring the colitis associated and the effect of the tested symbiotic on T-cell clonotypes through analyzing the colonic T cell receptor repertoire of the diseased, synbiotic treated, and control animals. The potential implication of immunophenotyping of T cell subsets associated with colitis needs further investigations in the future.Bacillus licheniformis DSM 17236 and an extracted prebiotic derived from cell walls of the baker yeast Saccharomyces cerevisiae does not reduce but increased the severity of experimentally-induced colitis in Spargue dawley rats. Future studies could investigate the effects of this synbiotic on the inflammatory reactions in the gut, which may affect the extent of colonic injury. The application of probiotics and/or prebiotics as a potential adjunctive treatment for IBD remains controversial and further experimental studies are indicated.We can conclude that synbiotic mixture of Sulfasalazine® was purchased from Rosemont Pharmaceuticals Co., UK, and dextran sulfate sodium (DSS) from MP Biomedicals Co., UK. Commercial, double sandwich, enzyme-linked immunosorbent assays (ELISA) kits for the accurate quantification of Interferon γ (IFN-γ) with Cat. No. E0105Hu, Interleukin 1β(IL-1β) with Cat. No. E0094Hu and Tumor necrosis factor α (TNF-α) with Cat. No. E0082Hu in serum. These ELISA kits were obtained from Bioassay Technology Laboratory Co., China. MDA diagnostic kit was purchased from Biodiagnostics Co. , while the MPO kit was bought from Jiancheng biotech Co (China). All other chemicals were purchased from standard commercial suppliers and were of analytical grade.Bacillus licheniformis DSM 17236  is purchased from . This probiotic strain has been isolated from soil and is a non-genetically modified organism (GMO). Bacillus licheniformis are Gram positive organisms, Spore formers and facultative aerobe but at presence of nitrate/nitrite it can grow anaerobic. The product was mixed at a final concentration of 1.6 × 109 CFU/kg of the basal diet [sal diet .) is an extracted prebiotic derived from cell walls of the baker yeast Saccharomyces cerevisiae. It distinguishes itself by high amounts of Mannan-Oligosaccharides (MOS) and ß-Glucans. It is mixed at 0.1% (1 g/kg) of the basic diet [TechnoMos® , and Approval number (ZU-IACUC/2/F/31/2022) and all methods are reported in accordance with ARRIVE guidelines.Fifty adult male Sprague Dawley rats (200–250 g) were used. The age of rats was about 8 months. They were obtained from the laboratory animal unit, Faculty of Veterinary Medicine, Misurata University. They were clinically healthy, kept under hygienic condition and housed in plastic cages with hard wood shavings as bedding and at a controlled temperature (21–24 °C) with a relative humidity of 50–60% and a 12-hr light-dark cycle. They were fed a balanced diet and had free access to clean water. All animals were accommodated for laboratory conditions for 2 weeks before being experimented. Animals were managed according to the Guidelines for the Care and Use of Laboratory Animals of the Institutional Animal Care and Use Committee of the Zagazig University, Egypt  and fed on the basal diet.DSS + Syn + Sulfa group: animals with chemically induced ulcerative colitis, treated with intragastric sulfasalazine (100 mg/kg) and fed with the synbiotic combination that was mixed with the normal basal diet.As described previously  ulceratiDetection of serum TNFα, INFγ, and IL-1-β using double antibodies sandwich ELISAs were adopted for estimation of the three cytokines according the manufacturer’s guidelines.A sample volume of a whole blood specimen is aspirated into the analyzer. M- 52DIFF LYSE applies to BC-5000 Auto Hematology Analyzer manufactured by Mindray to dissolve the WBCs for their counting according to the manufacturer’s instructions.MPO was measured in the homogenate supernatant of the intestinal tissue as a marker of neutrophile accumulation in colonic mucosa as preciously described .Lipid Peroxides (MDA) was estimated in intestinal tissue homogenate according to instructions described in the kits and the method of .Total RNA was extracted from 20 mg of colon tissues using the RNeasy kit of Qiagen (Cat. No.74104). The high purity of the extracted RNA samples were confirmed using a NanoDrop spectrophotometer technologies . The gene-specific primer sets used in the present study were as follows: -kB primers, F:5ˋ-AAT TGC CCC GGC AT -3ˋ, R: 5ˋ- TCC CGT AACCGC GTA -3ˋ (Accession No. XM_342346.4) and β-actin primers, F:5ˋ- TCA CTA TCGGCAATGTGC GG -3ˋ, R: 5ˋ- GCT CAG GAG GAG CAA TGA TG -3ˋ (Accession No.NM_007393). cDNA was prepared by the reverse transcription reaction . The real-time PCR reaction was performed in a total volume of 20 μl of the following reaction mixture: 1 μl of each primer (20 pmol), 6 μl template DNA, 10 μl SYPR® Green (1X) and 2 μl water nuclease-free. The samples were transferred to each well of a PCR plate. Rotor-Gene Q2 plex  was used to carry out the amplification reaction. The relative quantitation detection method used for calculating the transcripts of the replicates and β-actin was used as a house keeping gene for normalization .Specimens of colon were collected and immediately fixed in 10% neutral buffered formalin for 48 hrs and then processed histologically. These specimens were dehydrated in ascending grades of ethanol, cleared in xylene and embedded in paraffin wax forming paraffin blocks. 4–5 μm thick sections were obtained and stained with Harris Hematoxylin and Eosin (H&E) . The micP values of less than 0.05 were considered to indicate statistical significance.All data were tested for normality by the Shapiro-Wilk test and homogeneity of variance by Levene’s mean test before being analyzed with a one-way ANOVA followed by a Tukey’s multiple-comparison test using SPSS/21 software. The figures were generated using GraphPad Prism 7.0. The data were expressed as mean ± standard error of mean (SEM) for ten animals in each group.


Patients with cardiovascular disease are vulnerable to severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) infection. Although SARS-CoV2 vaccination may be effective, its impact on surgical patients is not well studied. We investigated the effects of cardiovascular surgery, especially under cardiopulmonary bypass (CPB), on the antibody titres after SARS-CoV2 vaccination.A prospective observational study was designed for patients undergoing surgery between July and November 2021. The immunoglobulin G against the receptor-binding domain was measured and antibody preserved rate (APR) was calculated from perioperative titres comparison.n = 58, 92%) and mRNA-1273 (Moderna) . While immunoglobulin G against the receptor-binding domain titres did not significantly decrease after surgery in Group None, they decreased significantly in Group CPB from 21.80  to 11.95  U/ml (P < 0.001) a day after surgery, 11.40  U/ml (P < 0.001) 14 days after surgery and 7.60  U/ml (P < 0.001) a month after surgery. The APRs a day after the surgery were significantly lower in Group CPB  than in Group None  (P < 0.001).Enrolled 63 study patients were divided into 39 undergoing surgery with CPB (Group CPB) and 24 without CPB (Group None). Preoperative vaccines were BNT162b2 (Pfizer/BioNTech)  in December of 2019, a fear and a threat impacted humanity socially and economically. Since the first outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) in December of 2019, a fear and a threat impacted humanity socially and economically. The SARS-CoV2 is a type of coronavirus which accounts for series of symptoms including fever, myalgia, headache, dyspnoea, cough, sore throat, smell or taste abnormalities, and severe acute respiratory syndrome (SARS) which World Health Organization officially designated as Coronavirus disease 2019 (COVID-19) in February 2020 . In a fiAlthough pharmaceutical treatments are being developed around the globe, vaccination plays a key role in the prevention of COVID-19. World Health Organization recommends the vaccination for people older than 18 years old, especially for those with vulnerable medical conditions . As of 2To determine the efficacy of the SARS-CoV2 vaccine, a serological antibody correlation with vaccine-induced immune response has been reported previously . While tThis study was approved by Fujita Health University Ethics Committee (HM 21-164). It was also conducted with the informed consent, according to the ethical guidelines for clinical study published by the Ministry of Health and the Declaration of Helsinki.This prospective observational study in the patients who had already been vaccinated and underwent surgery at Fujita Health University between July 2021 and November 2021.In this study, patients were divided into 2 groups. The first group included the patients undergone surgery without CPB (Group None), as opposed to the group including the patients undergone surgery with CPB (Group CPB).2 and above 60 mmHg, respectively, with norepinephrine. Inotropic support with epinephrine was used during CPB weaning.In all patients of Group CPB and some patients in Group None, anaesthesia was induced with intravenous midazolam, fentanyl and vecuronium bromide, followed by endotracheal intubation. Anaesthesia was subsequently maintained with sevoflurane, propofol, remifentanil and vecuronium bromide. CPB was performed using a heparin-coated circuit and a membrane oxygenator. In Group CPB, oxygen concentration and ventilation frequency were adjusted to maintain normoxemia (>90 mmHg) and normocapnia (35–45 mmHg). Cardiac arrest was induced by blood-cardioplegia. Pump flow and mean arterial pressure were maintained at 2.5 l/min/mIn some patients of Group None, the surgery was performed with local anaesthesia and sedation with propofol and dexmedetomidine without endotracheal intubation. In both groups, patients were transferred to the intensive care unit (ICU) after the surgery, where they were extubated following institutional criteria.The patient characteristics were collected and analysed from electronic health records, including age at surgery, sex, smoking history and comorbidity . The surgical procedures performed (type of surgery), a total operation duration, a CPB duration, a cardiac arrest duration, a selective cerebral perfusion duration, total amount of bleeding, an amount of blood transfusion administered, a duration of tracheal intubation, time in ICU and length of hospital stay were recorded.The SARS-CoV2 vaccination dates and the types of vaccine were interviewed in each patient and were also verified with the vaccination certificates issued by each healthcare institute. Patients with 2 doses of vaccine are enrolled in the study, but those with third booster vaccination are not included. We also checked the history of possible SARS-CoV2 exposure in each patient. The check-ups for SARS-COV2 infection or COVID-19 disease were performed before admission, about 1 week, 2 weeks and 1 month after discharge.Blood samples for antibody titre measurement were taken at 4 correspondent times, including prior to the surgery, a day after the surgery (7–16 hours after surgery), 2 weeks after surgery and a month after surgery, as visualized in The IgG against the receptor-binding domain (RBD-IgG) was measured as an antibody titre from each blood samples. A chemiluminescent enzyme immunoassay using an Accuraseed COVID-19 antibody reagent with the Accuraseed automated immunoassay system from FUJIFILM Wako Pure Chemical Corporation was used for the measurement. The details of the method and performance such as limit of quantification, precision, and accuracy were published previously .t-test or a nonparametric test, the Mann–Whitney U-test. A correlation between 2 variables were analysed using Spearman’s rank correlation coefficient. All statistical analyses were performed with statistical software ‘EZR’ (Easy R) available on website (http://www.jichi.ac.jp/saitama-sct/SaitamaHP.files/statmed.html)  U/ml (P < 0.001) and 7.60  U/ml (P < 0.001), a day after surgery, 14 days after surgery and a month after surgery, respectively. In contrast, RBD-IgG titres in Group None did not significantly decrease from 36.85  U/ml before surgery to 28.00  U/ml (P = 0.15) a day after surgery and 17.50  U/ml (P = 0.23) 14 days after surgery. However, the RBD-IgG titres in Group None decreased significantly to 22.50  U/ml (P = 0.03) a month after surgery, although those were significantly higher than those in Group CPB (P = 0.002). As also shown in Table P = 0.835).As shown in Table 40 7.85, 2.65 U/mlP < 0.001). Also, the APRs a month after the surgery were significantly lower in Group CPB  than in Group None  (P = 0.05). However, the APRs 14 days after the surgery were similar between the 2 groups (P = 0.77): Group None: 0.65  and Group CPB: 0.52 .We labelled antibody preserved rate (APR), calculated from the ratio between the antibody titres before and after the surgery, for the perioperative titres’ comparison. As shown in Fig. r = −0.003, P = 0.99 and Group CPB: r = −0.122, P = 0.48) and 14 days after  the surgery were observed, as shown in Fig. r = 0.488, P < 0.01) but not in Group None , as shown in No significant correlations between the vaccination to surgery days and the APRs in both groups a day after . At 2 weeks after surgery, the APRs were 0.48  (BNT162b2) and 0.43  (mRNA-1273) (P = 0.85). At a month after the surgery, they were 0.51  (BNT162b2) and 0.34  (mRNA-1273), respectively (P = 0.43).There were no relationships between the types of vaccine and the APRs, as shown in Table To our best knowledge, this is the first report investigating the effects of cardiovascular surgery under CPB on the IgG antibody titres after SARS-CoV2 vaccination. The main findings of our study were as follows: (i) There was significant reduction, up to half of the preoperative values, of SARS-CoV2 antibody titres immediately after the surgery in Group CPB. (ii) After a month, antibody titres reduced significantly in both groups. (iii) Although an interval from vaccination to surgery was irrelevant to APR in Group None, positive correlation was observed between the interval and APR at 1 month after the surgery in Group CPB.Patients queueing cardiovascular surgery are vulnerable to SARS-Cov2 infection not only because of their cardiovascular disease, but along with their background and comorbidities related to their lifestyles. They are recommended for vaccination and priority is high among them , 12.Aside from the novel SARS-CoV2 mRNA vaccine, pre-existed live nor inactive vaccine’s information regarding perioperative vaccination is limited. Even annual influenza vaccination after open heart surgery varies among institutions, lacking consensus , 14.et al. [Haemophiles influenzae vaccination between before and after the congenital cardiac surgery; however, they had reduced significantly among Bordetella and hepatitis B vaccination. With that in mind, significant reduction of SARS-CoV2 antibody titres in Group CPB but not in Group None in our result is very informative. Although the effect of CPB on the antibody titres was not significantly apparent 14 days after the surgery, there was greater reduction of APR in Group CPB than in Group None after a day and a month, as shown in The inflammatory and immune system are known to get altered after any surgery, due to general anaesthesia and its invasive procedure , 16. Furet al.  reports It is also informative that the antibody titres a month after surgery reduced significantly in both groups. This might reflect the natural wane off of an antibody titre reported in the literature , 20, 21 A period from vaccination to surgery seemed irrelevant as of APRs in Group None, suggesting that there was no alteration in vaccine acquired immunity after surgery without CPB. However, since a positive correlation between the interval from vaccination to surgery and the APR at a month after the surgery was observed in Group CPB, a careful scheduling of a vaccination may be proposed for surgery under CPB. The lower antibody titres and lower APRs in Group CPB compared to that in Group None a day after and a month after surgery, as shown in There are number of limitations in this research. First, a small number of study sample size influenced the statistical result, making it difficult to analyse the underlying conditions. Also, conduction of single institution research could have added sampling bias. The second limitation is different subject characteristics between 2 comparison groups. Smoking history and vaccination to surgery interval were statistically implicated different in background characteristics. Factors of surgical intervention also weighed more on Group CPB, such as more operation time, more tracheal intubation period, more ICU stay, and more hospital stay, shown in Table A significant reduction of antibody acquired by SARS-CoV2 vaccination was perceived immediately after the surgery with CPB. A positive correlation between the interval from vaccination to surgery and the APR at a month after the surgery was observed in Group CPB, although there was similar tendency of reduction in APR in the patients undergoing with and without CPB. Based upon our informative results, careful considerations of vaccination schedule might be required for the patients undergoing surgery with CPB.The analysis obtained in this research could support the argument of additional vaccination among perioperative patients. Further study is demanded to provide better preventive medication for perioperative patients. A shared decision-making on additional dose of SARS-CoV2 vaccination among perioperative patients is desired.ICVTS online.ivac123_Supplementary_DataClick here for additional data file.


Sichuan province is one of the highest AIDS epidemic provinces in China, with a large number of floating population. The annual number of cases of HIV/AIDS reported in Sichuan has been the highest province in China for several successive years. There is a lack of widespread and representative data on the distribution of HIV genotypes in Sichuan. We aim to investigate the characteristics of HIV-1 molecular epidemiology and transmitted drug-resistance in newly diagnosed HIV-infected patients in Sichuan, China.pol) gene sequences. We also analysed the association of demographic and virological factors with transmitted drug-resistance (TDR) and transmission clusters.Archived plasma samples (n = 1524) from HIV-1 newly-diagnosed individuals in April 2019 were selected by cross-sectional investigation from all 21 cities in Sichuan province. Phylogenetic relationship, transmission cluster, and genotypic drug resistance analyses were performed using HIV-1 polymerase (pol gene sequences were obtained from 1297 cases. HIV-1 epidemic strains in Sichuan province: the majority of genotypes were circulating recombinant form (CRF) 07_BC , CRF01_AE , CRF08_BC , CRF85_BC , subtype B , the other genotypes only accounted for 4.93%, and unique recombinant forms (URFs)  were observed in the study, and the difference of age, ethnicity, education, occupation, region and transmission pathway of different genotypes were statistically significant. According to WHO HIVDR surveillance threshold, the level of TDR has reached a medium level, with 72 of 1297 (5.55%) cases carrying drug-resistance mutation sites, TDR mutation frequency to nonnucleoside reverse transcriptase inhibitors  was much higher than nucleoside reverse transcriptase inhibitors  and protease inhibitors , and CRF08_BC was a risk factor for TDR . The most common drug resistance HIV-1 mutation pattern for NNRTI was V106  and E138 , and for PI was M46 . A total of 205 (15.8%) pol sequences were involved in the genetic transmission network clusters, CRF01_AE , subtype B , drug resistance  and different levels of education (P < 0.05) were significantly associated to be in clusters.Partial The distribution of HIV-1 genotypes in Sichuan is more diverse and complex, and the Men who have sex with men (MSM) is underrated, arguing for behavior scaling up intervention in this specific population besides the elderly people with heterosexual transmission risk groups. The risk of TDR mutation frequency increased in newly diagnosed patients highlights the significance of genotypic drug resistance monitoring and molecular surveillance of pretreatment HIV-1 drug resistance. The regimen composed of TDF, 3TC and EFV was still currently the preferred solution used free first-line therapy. The prevalence of human immunodeficiency virus type 1 (HIV-1) remains a major public health burden in China. Sichuan province is in the interior of Southwest China, adjacent to Yunnan and Guizhou in the South and Tibet in the West. The epidemic of HIV/AIDS has kept rising in Sichuan province since reported the first case of imported AIDS in 1991, the number of newly reported cases per year has increased by about 20% since 2006, by 2019, Sichuan province reported more than 170 thousand cases of HIV/AIDS, with the number of surviving cases ranking first in the country [https://www.hiv.lanl.gov/content/sequence/HIV/CRFs/CRFs.html) within HIV-1 group M. With the genetic variation and cross recombination of genes between different genotypes, numerous new circulating recombinant forms (CRFs)/unique recombinant forms (URFs) are emerging, and the global epidemic situations of HIV has changed significantly. HIV enormous genetic variability and rapid evolution have led to its epidemic and therapeutic challenges. Worldwide, the proportion of subtype C was the highest, accounting for 48%, followed by subtype A (12%), subtype B (11%) and CRF02_AG (8%), CRF01_AE (5%), G subtype (5%) and D subtype (2%) [Among the four phylogeny groups that constitute HIV-1 , M group is the virulence factor of AIDS pandemic . There wype (2%) . A natioCRF01_AE %, G subtype (2%) . With thype (2%) . AdditioSince Chinese government officially launched the National Free Antiretroviral Treatment Program (NFATP) in 2003, the number of patients enrolled in this program has increased rapidly year by year, with the increasing coverage of antiviral therapy; the occurrence of drug resistance is also increasing. The emergence of HIV drug resistance (HIVDR) threatens the global scale-up of antiretroviral treatment (ART) for treating HIV infection, which could increase the risk of ART failure. It is important to conduct HIVDR surveys to estimate the transmitted drug-resistance (TDR) rate of ART-naive people living with HIV (PLWH) to provide baseline information for effective ART programs, delaying HIVDR occurrence and developing a rational public health strategy to control HIV prevalence. However, HIVDR testing for ART-naive PLWH is not routinely performed in China as a developing country with limited conditions. The rates of TDR vary throughout the world. In China, the overall prevalence of TDR among ART-naïve individuals in 2004 and 2005 was 3.8% , and theSichuan province has a population of approximately 85 million people, and is a developing area, Sichuan province is the most severely HIV affected area in China . In a prpol sequence  information was successfully exported from 1297 PLWH in this study.A total of 1524 plasma samples were obtained from individuals newly diagnosed as HIV-1 infected patients in April 2019 were selected by cross-sectional investigation from all 21 cities in Sichuan province. The epidemiological data of demographic characteristics  were acquired from China Information System for Disease Control and Prevention. After eliminating duplicate samples, About 5 ml of venous blood was extracted. The EDTA-K2 anticoagulated plasma samples were isolated from each participant and preserved in a – 80 °C freezer before sending the cold chain to the Sichuan CDC.pol. The Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was used to amplify the full-length protease gene in the pol region and the first 300 codons of the reverse transcriptase gene. The PCR products were dealt with electrophoresis with 1% agarose gel, and the amplified positive products were purified and sequenced by Beijing Genomics Research Center Ltd. The detailed amplification and sequencing performed as previously described [The viral nucleic acid was obtained from 200 μl plasma of PLWH by extraction machines . Sequences were generated from the HIV-1 escribed .pol sequences using the GTR + G + I nucleotide substitution model [https://www.hiv.lanl.gov/content/index) was used to review the results. Presented phylogenetic tree by using FigTree v1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/). If the sequence whose genotype cannot be confirmed by phylogenetic tree and HIV Databases BLAST were considered as URFs. For example, the sequence between the CRF_01AE and B subtype reference strains in the phylogenetic tree, and the similar sequences in HIV Databases BLAST were 01B genotype, we judged this URF as URF_01B.The obtained sequence was spliced by using the analysis software Sequencher 5.1. The BioEdit Sequence Alignment Editor was used to edit and correct the sequence. MEGA 7.0 software was used to determine the genotype. All assembled sequences were aligned together with the reference sequences  using Clustal W program in MEGA 7.0, and then further checked manually in Bioedit . FastTreon model , using ton model , 18. Thepol sequences do not diverge more than 1% during the first 3 years of infection [The gene distance between the clusters was calculated using the software hyphy 2.2.4 in TN93 model, and the relationship between the two sequences was determined by the minimum gene distance method. The transmission network was constructed with a threshold of pairwise genetic distance of 0.5%, which is more appropriate for identifying rapidly growing clusters, and was based on study findings showing that within monoinfected individuals nfection . The molnfection .https://hivdb.stanford.edu/) was used to screen the pol sequences for surveillance drug resistance mutations (DRMs), it was evaluated relevant resistance for nucleoside reverse transcriptase inhibitors (NRTIs), nonnucleoside reverse transcriptase inhibitors (NNRTIs), and protease inhibitors (PIs) [Drug-resistance were analyzed based on genotypic interpretations defined by the Surveillance Drug Resistance Mutation list recommended by the World Health Organization. The Stanford University HIV DRUG RESISTANCE DATABASE (rs (PIs) , 22. If 2 test. Univariate logistic regression model was used to test the independent variables (i.e. sociodemographic) and transmission drug resistance individuals , multivariate logistic regression model was used to further test the significance of all micro significant (P < 0.1), variables in univariate logistic regression model. Potential demographic differences between patients in cluster and those out of cluster were evaluated by uni-multivariate logistic regression analysis. P value < 0.05 was considered statistically significant.Data were entered in a spreadsheet (Micorsoft Office Excel 2013) and analyzed using SPSS 25 software . The potential demographic characteristics  were expressed in percentage, differences between the variables group for genotypes were evaluated using the χpol sequences from 1524 infected patients with HIV-1, 15 genotypes were identified . The majority groups of CRF85_BC  and CRF105_0108  with age ≥ 50 years old were found in the heterosexual transmission; 89.39% of ethnic Yi infected with CRF07_BC; 49.75% of ethnic Han was infected with CRF07_BC, which was found their infection rate was higher than that of other age groups in the 25 year old  and 25–40 years old  age group. Among the transmission route of IDUs, mother to child transmission (MCT), and MSM, CRF07_BC was the dominant strain and the composition ratio of was 100%, 77.78% and 54.43%. Furthermore, 13/13 CRF55_01B, 5/5 CRF79_0107, 2/3 CRF59_01B was detected in men.Demographic information is summarized in Table Of the 1297 subjects, 72 had drug-resistant mutations, among which 12 had high drug resistance, 21 had moderate drug resistance and 55 had low drug resistance, potential drug resistance were 66 cases. 50 cases  were found to have non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance mutation, the mutation probability of E138 , K103  and V106  was significantly higher than that of other sites. The mutation probability of drug resistance site in CRF07_BC, CRF08_BC, CRF01_AE was higher than that in other genotype, which were resistant to all NNRTIs in varying degrees, and the high resistance was mostly caused by K103N mutation. Four cases  had NRTI resistance mutation, the mutation rate was relatively low, but d4T, ddI and TDF caused by K65KR/R mutation were highly resistant. 22 subjects  had protease inhibitor (PI) resistance mutation, M46  and Q58  had the highest mutation probability, most of them were low-grade resistance. Additionally, the most frequent mutation was V106, which was observed in 23.6% (17/72) of patients, HIV-1 strains with this mutation exhibited the degree of potential low-level resistance, except for V106VIM resulted in NVF and EFV were highly resistant and dot was moderately resistant. Followed by E138 , V179  and K103 , all of them were NNRTI related mutations. Moreover, the most resistant drugs were RPV , NVP , DOR . The distribution of drug resistance and mutation sites was shown in Fig. The drug resistance level is shown in Fig. P < 0.05), while other variables had no significant effect on drug resistance (P > 0.05).Taking drug resistance as an independent variable, 8 variables including gender, age at the time of HIV diagnosis, nationality, education level, marital status, occupation, route of infection and genotype as dependent variables, logistic stepwise regression model were used to analyze the risk factors of TDR Table . The resP < 0.05), subtype B , drug resistance and different levels of education(P < 0.05) were significantly associated to be in clusters , the cluster sizes ranged between 2 and 24, there were 72 (94.7%) clusters with size < 5, and 4 5.3%) clusters with size ≥ 5. With regard to the characteristics of individuals involved in clusters in comparison with those out of clusters, CRF01_AE  according to WHO HIVDR surveillance threshold . The resAccording to our study, TDR mutation frequency to NNRTIs was much higher than NRTIs and PIs, due to NNRTI had a low resistance barrier, and it was more prone to drug resistance . BecauseHowever, our study has limitations. First, a potential sampling bias, on the one hand, in our study only one cross-sectional study was conducted, and the results could not be used to observe the dynamics of the local HIV epidemic, on the other hand, we could analyze only the samples that had been diagnosed, but those that had been infected but not diagnosed could not be included in the analysis. Second, our analysis of TDR concentrated only on NRTI, NNRTI and PI, not containing integrase inhibitors, which are increasingly infected people began using this drug.In summary, our study provides a comprehensive molecular epidemiologic dataset to understand the diversity and distribution of HIV genotypes in newly diagnosed HIV-infected patients, and supplements in transmitted drug-resistance in Sichuan province. The distribution of HIV-1 genotypes in Sichuan is more diverse and complex than 2014, with CRF07_BC and CRF01_AE still as the predominant genotypes. The MSM is underrated, arguing for behavior scaling up intervention in this specific population besides the elderly people with heterosexual transmission risk groups. The risk of TDR mutation frequency increased in newly diagnosed patients highlights the significance of genotypic drug resistance monitoring and molecular surveillance of pretreatment HIV-1 drug resistance, the regimen composed of TDF, 3TC and EFV was still currently the preferred solution used free first-line therapy.


Candida auris clades/subclades by means of Raman spectroscopy and imaging. Representative Raman spectra from different Candida auris samples were systematically deconvoluted by means of a customized machine-learning algorithm linked to a Raman database in order to decode structural differences at the molecular scale. Raman analyses of metabolites revealed clear differences in cell walls and membrane structure among clades/subclades. Such differences are key in maintaining the integrity and physical strength of the cell walls in the dynamic response to external stress and drugs. It was found that Candida cells use the glucan structure of the extracellular matrix, the degree of α-chitin crystallinity, and the concentration of hydrogen bonds between its antiparallel chains to tailor cell walls’ flexibility. Besides being an effective ploy in survivorship by providing stiff shields in the α–1,3–glucan polymorph, the α–1,3–glycosidic linkages are also water-insoluble, thus forming a rigid and hydrophobic scaffold surrounded by a matrix of pliable and hydrated β–glucans. Raman analysis revealed a variety of strategies by different clades to balance stiffness, hydrophobicity, and impermeability in their cell walls. The selected strategies lead to differences in resistance toward specific environmental stresses of cationic/osmotic, oxidative, and nitrosative origins. A statistical validation based on principal component analysis was found only partially capable of distinguishing among Raman spectra of clades and subclades. Raman barcoding based on an algorithm converting spectrally deconvoluted Raman sub-bands into barcodes allowed for circumventing any speciation deficiency. Empowered by barcoding bioinformatics, Raman analyses, which are fast and require no sample preparation, allow on-site speciation and real-time selection of appropriate treatments. This study targets on-site/real-time taxonomic identification and metabolic profiling of seven different  Candida auris (C. auris) has emerged as a multidrug-resistant human fungal pathogen causing invasive nosocomial infections with high morbidity and mortality [C. auris clinical isolates were reported to exhibit a diversified resistance to multiple drug classes including azoles and polyenes [C. auris at both clade and subclade levels. In the past decade, ortality ,2. Its cortality , exhibitortality ,5. Exceppolyenes ,7. The hpolyenes ,9,10 hasC. auris to the existing antifungal remedies resides in the lack of a real-time, sensitive, and reliable diagnostic tool to unequivocally identify it at the species, clade, and sub-clade levels. This has been the main cause of nosocomial outbreaks, which has made this pathogen a superbug. Only several years after its first isolation in 2009 [C. auris clinical isolates became available, and the simultaneous emergence of different clades from various geographical regions could be revealed. C. auris is indeed hardly distinguishable from other, more common species of Candida by simply examining its phenotypic traits . Accordingly, the last decade has seen a rapid evolution of diagnostic methods for its identification. Precise C. auris speciation requires resorting to molecular methods, which include DNA sequencing [C. auris from its close relatives, and further implementations are ongoing to distinguish among subclades and isolates from different geographical areas.The root of the developed resistance of  in 2009 , the whoquencing , proteomquencing , and metquencing . These mC. auris at the clade/subclade level requires whole-genome DNA sequencing and multilocus sequence typing. These analyses greatly impact on costs and turnaround time for collection of the results. Accordingly, efforts have been made to refine genome-mapping criteria to save time and cut down costs. Advanced studies have recently succeeded in locating unique DNA sequence junctions that map clade-specific regions, a procedure greatly reducing the time necessary to locate the four major clades of C. auris [C. auris strains from all four phylogenetic clades. Such advanced databases allow overcoming clade identification challenges with high identification scores [C. auris clades through selectively collecting differences in the structure of their isolated mannans. Despite these improvements and newly obtained insights, the unique-DNA sequence-junction mapping still impacts labor costs, and MALDI-TOF proteomics and NMR metabolomics require elaborate sample preparation, sophisticate databases, and the availability of expensive equipment. For this body of reasons, a suitable procedure for on-site/real-time profiling of C. auris species/clades/subclades is nowadays unavailable to clinical practitioners.In principle, a full identification of C. auris . Regardin scores . Bruno en scores  have sucCandida species with completely sequenced genomes increases, extensive research is being conducted in the search for the specific genes responsible for their virulence and drug resistance. Recent studies have revealed the presence of unique chemical fingerprints and metabolite profiles enabling not only Candida speciation but also the identification of functional links between metabolic and virulence traits of different species [Candida species. Confocal Raman spectroscopy (and related imaging) is a relatively new tool in metabolomics, which allows extracting and spatially visualizing biological information from living cultures of cells, yeasts, and bacteria with high spectral and spatial resolutions [Candida species in nearly real time. Moreover, modern Raman instruments are relatively inexpensive as compared to MALDI-TOF or NMR devices and can be equipped with optical hardware that allows for the collection of maps of millions of spectra in minutes. Once empowered with deep learning approaches and multivariate statistical methods, the Raman spectra can be decomposed into a series of images representing chemical signatures of specific molecules [As the number of  species ,18. Owinolutions ,20. The olecules . HyperspCandida species/clades [C. auris clades and subclades, while also locating links between their virulence, stress resistance, and metabolite structure. The presented Raman methodology opens a new path toward Raman metabolomics of C. auris, which not only enables the correct selection and patient customization of drug dosing, but also uniquely provides hints for developing new strategies in counteracting candidiasis/candidemia pathophysiology.In our previous Raman studies of s/clades ,23,24, wC. auris clades and their related subclades were provided by Teikyo University for a total of seven different cell samples, as follows: LSEM3690 , LSEM3682 and LSEM643T , LSEM3672 and LSEM3673 , and LSEM3662 and LSEM3663 . All clades were cultured in brain–heart infusion (BHI) broth  at 36 °C for 24 h under atmospheric pressure on a glass-bottom plate . Portions of the clade samples were then mounted in distilled water on a microscope slide  and observed with a laser microscope  at a magnification of 400~1000×. The remainder of the samples of each colony was characterized by Raman spectroscopy, and the spectra were analyzed and compared.C. albicans and other yeasts [A library of reference Raman spectra was built by using 40 selected elementary compounds, including polysaccharides , mono– and disaccharides , lipids , polyols –Arabitol and L–(−)–Arabitol), and other key molecules such as adenine, ergosterol, and glycine. The elementary compounds were selected and their spectra deconvoluted in order to feed a computer program designed to pick up a series of Gaussian–Lorentzian sub-bands from a library  to calibrate the spectrometer at each measurement. The Raman-scattered light was monitored by means of a single monochromator interfaced with an air-cooled charge-coupled device (CCD) detector . The acquisition time for a single spectrum was typically 10 s. Twenty spectra were collected at different locations over a total area of ~2 mm2 for each Candida sample and averaged in order to obtain a representative spectrum for each clade/subtype. Raman spectra were collected in situ using a spectrometer specially designed for measurements on biological samples . A 20× objective lens was used throughout the experiments, and the optical circuit was set in confocal mode. High efficiency and high spectral resolution could be concurrently achieved by employing a holographic notch filter. The incoming light was a 532 nm solid-state laser source operating at 10 mW. A spectral resolution of better than 1 cm−1  with an accuracy in a laser spot spatial location of 100 nm. Raman hyperspectral images were generated using commercially available software . Raman images were generated using intensity ratios from normalized spectra. In order to minimize errors related to spectral resolution and possible shifts in band position, we used the average intensity of the pixels at the band nominal location ± 3 pixels, rather than single pixel intensity. Selected areas of the samples were scanned with a lateral displacement step of 500 nm for the laser focal point.Raman images were collected using a dedicated Raman device  operated in microscopic measurement mode (100× lens) with confocal imaging capability in two dimensions. As a peculiarity, this Raman microscope is capable of achieving ultra-fast simultaneous image acquisition of up to 400 spectra in order to be compatible with cells’ lives. The excitation source was a 532 nm solid-state laser, and the spectral resolution was ~2 cm−1). Detailed descriptions of spectral deconvolution criteria have been given in previous works [Candida species. Following the pre-selection of elementary molecules from the library, the algorithm located the best fit to the experimental spectra by preserving relative intensities, spectral positions, and full-width at half-maximum values from each elementary compound . Mathematical constraints on band positions, relative intensity, and bandwidths allow for a univocal deconvolution of the experimental spectra. When specific sub-bands appeared, which the solver could not properly fit according to the pre-selected library compounds, a new compound was selected from the library to match the unknown band and a new run of adjustments was performed for determining the intensity contributions of all elementary compounds.Experimental spectra were preliminarily treated with a polynomial baseline subtraction and then deconvoluted into a series of Gaussian–Lorentzian sub-bands. The baseline subtraction procedure was performed using options available in commercial software  by following the same criteria for all spectra collected on different clades. The automatic software applied a polynomial-fitting criterion for baseline subtraction. All spectra were analyzed for their relative intensity after normalization to the glucose ring signal  and enabled the display of statistical information with a set of “summary indices”, referred to as principal components PC1 and PC2.Statistical analyses and visualization of the large Raman data sets obtained during high-resolution imaging were performed according to principal component analysis (PCA). The number of spectra collected in Raman imaging was in the order of 10A barcode was built from deconvoluted Raman spectra in order to enable efficient electronic recordkeeping and to increase data accessibility and structural characteristics of different species through apps and user-friendly software. Sequences of deconvoluted Gaussian–Lorentzian sub-bands were converted into barcodes by means of an algorithm assigning to each band a line with a thickness equal to 1/50 of the sub-band full width at half maximum and a distance from the successive line proportional to the sub-band area.t-test [p-values < 0.05 and <0.01 were considered as statistically significant and labeled with one and two asterisks, respectively.The statistical relevance of the experimental data was analyzed by computing mean values and standard deviations. Their statistical validity was evaluated by applying the unpaired Student’s t-test . For eacC. auris clades and related subclades . Cell size and aspect ratio, which were directly measured (and averaged) on the respective laser micrographs, showed some statistically relevant differences among the studied clades/subclades . Cells of LSEM3673 (Clade III) were the largest in size (4.5 ± 0.2 μm in average) and the most elongated ones , whereas those of LSEM3690 (Clade I) were the smallest in size (2.9 ± 0.25 μm) and almost roundish in shape . Cells belonging to all remaining clades/subclades investigated presented intermediate average sizes within narrow intervals . The above morphological observations are in line with microscopy observations presented by other authors on C. auris clades [C. auris clades/subclades cultured under exactly the same conditions could be found in the published literature. Despite the aforementioned morphological differences, it appears extremely difficult to speciate C. auris clades by means of mere morphological analyses. An important detail was that some specific subclades, such as LSEM3682 (Clade II) and LSEM3673 (Clade III), appeared to be abundantly embedded into an extracellular matrix (ECM), whereas others, i.e., both Clade IV subclades, preferred to aggregate into small clusters of cells without sharing any ECM. The observed ECM corresponded to the general description given by Flemming and Wingender [Candida species: a polymeric gel-like hydrated three-dimensional structure in which the cells become partially immobilized. The ECM plays a number of different roles in the overall physiological behavior of Candida yeast cells. These include providing a defense against phagocytosis and obstructing drug diffusion. The ECM structure is both species- and environment-dependent, but, as a general characteristic, its major components are polysaccharides (glucans and mannans), monosaccharides (including) hexosamines, with additional fractions of proteins (and their glycosylated counterparts), phosphorus, and uronic acid [s clades ,31,32. Hingender  for Candnic acid ,35. The C. auris are shown in C. auris clades upon performing detailed spectroscopic analyses. A description of the main spectral differences is offered in the following.Averaged and deconvoluted Raman spectra for the seven investigated clades/subclades of Biofilm-related signals—One striking difference that immediately appeared in comparing spectra from different clades over the whole recorded wavenumber interval  [−1 region exactly corresponded to samples for which the presence of ECM could be observed  was the most intense band in the recorded spectra of two of the three ECM-embedded subclades  and LSEM3673 (Clade III)), the spectrum of the third subclade (LSEM3682 (Clade II)) presented its strongest signal at around 895 cm−1, namely, in a spectral zone dominated by signals from β-glucans. This difference points at peculiar chemical fingerprints for the ECM of different clades and will be discussed later in the context of glucans’ signals.interval  was a man modes) . RemarkaMarkers of cell energetic state—An important marker of cell physiology can be identified in the band at ~750 cm−1 , and can be taken as an intrinsic measure of the energetic state of the Candida cells. The two subclades of Clade IV, which presented the strongest relative intensities for the 750 cm−1 signal  of the momentary metabolic/stress state of Candida cells. C. auris clades/subclades.cm−1 cf. . This Rachrome c . Cytochrysiology ,40. Varignal cf. f,g, shouGlucans—The Raman spectra of glucans present clear fingerprints that allow for their recognition from their specific secondary structures [−1 and ~941 cm−1, respectively. These bands have a common origin (C–O–C glycosidic linkage vibrations) but differ by ~50 cm−1 in wavenumber as a consequence of their different bond environment [−1 signals, GRα/β = I941/I890, is thus linked to the volumetric fraction of α– to β–glucans. The value of the ratio, GRα/β, differed among C. auris clades/subclades (GRα/β = 1.14) was found in LSEM643T (Clade II) and LSEM3673 (Clade III), and the lowest one was consistently found in both Clade IV subclades (GRα/β = 0.14~0.15). A striking finding was that the other LSEM3672 subclade of Clade III showed a quite low value (GRα/β = 0.15), proving that C. auris metabolomics is not only clade- but also strongly subclade-dependent. On the other hand, the GRα/β values measured in LSEM3690 (Clade) I and LSEM3682 (Clade II) were both moderately high  is crucial in the structural and dynamical performance of both EMC and cell walls. Gα3/α6R ratios for the seven C. auris samples under investigation. The highest Gα3/α6R ratios were found in the subclades with the highest Gα/βR values, namely, LSEM643T (Clade II), LSEM3673 (Clade III), and LSEM3682 (Clade II) . These are the three samples embedded in abundant ECM . The mismatch between the two Clade III subclades, already found for the Gα/βR ratio, is thus also confirmed for the Gα3/α6R ratio, with values of 0.38 and 0.93 detected in LSEM3672 and LSEM3673, respectively. This confirms that the high Gα3/α6R value of the latter is related to the presence of ECM (similar to the two subclades of Clade II). In addition to its high level of insolubility in water, α–1,3–glucan is also a very rigid molecule. Therefore, LSEM 3682 (Clade II), LSEM643T (Clade II) and LSEM3673 (Clade III) are capable of building a very rigid and water-insoluble ECM structure, whereas the two Clade IV subclades lack ECM and share a relatively flexible glucan structure in their cell walls. This point will be examined in more detail in the forthcoming An additional spectroscopic parameter related to the glucan structure can be located in the ratio between Raman signals from α–1,3–glucans and α–1,6–glucans. The aforementioned Raman signal peculiar to the α–1,3–glucan structure , LSEM643T (Clade II) and LSEM3673 (Clade III)) displayed high Mα/Rβ ratios . However, the highest in fraction of α anomer of D–mannose was the LSEM3690 (Clade I) (Mα/Rβ = 0.68), which lacks ECM (cf. Mα/Rβ ratio was recorded in LSEM3672 (Clade III) (0.28). The remaining samples showed intermediate values, again showing clear heterogeneity between subclades of the same clade  skeletal stretching in all polysaccharides. Regarding the aforementioned chitin-related triplet, it enables a distinction between C–O–C bonds located within and between rings. The stretching of C–O–C bonds within rings produces the Raman signal at 1054 cm−1, whereas C–O–C stretching between rings is seen at 1107 cm−1. Accordingly, the band intensity ratio, E/RR = I1107/I1054, can be defined as the “esterification ratio” and taken as representative of the ether-to-ring C–O–C bond fraction in chitin. According to this definition, the E/RR ratio reflects whether the chitin structure incorporates long crystalline chains. Chitin structures with higher E/RR ratios contain a higher fraction of ether C–O–C bonds, and thus they consist of longer chains and are expected to bestow higher crystallinity (as demonstrated later). C. auris investigated in comparison with their respective low-frequency band at 645 cm−1. The computed E/RR values are given in the inset. LSEM3662 (Clade IV) appears to possess the chitin structure with the highest E/RR ratio (1.64), followed by LSEM3673 (Clade III) (1.48) and LSEM3672 (Clade III) (1.24). The remaining subclades investigated all experienced lower E/RR values, ranging between 1.12 and 1.19.charides . The maicharides . This stChitin sub-structures—Another peculiarity of the chitin structure, among other polysaccharides, is the presence of amide groups that partly replace OH groups as structural sub-units in the polymeric chain. Amides I, II, and III Raman signals, which appear in the wavenumber intervals 1600~1700 cm−1, 1400~1500 cm−1, and at around 1300 cm−1, respectively, are peculiar to the N-acetyl group of chitin  and intramolecular bonds between N-acetyl and hydroxyl groups or between two N-acetyl groups (C=O…HO(C6) or C=O…HN) belonging to the same molecule, respectively [C. auris clades/subclades according to the degree of acetylation of their chitin structure. The relative intensity ratio, i/iR = I1644/I1660, namely, the Raman ratio of intermolecular-to-intramolecular hydrogen bonds, could be taken as a parameter measuring the degree of acetylation of α-chitin: the higher the ratio, the more acetylated the structure. As discussed later, a highly acetylated structure of chitin serves to reduce membrane permeability against external molecules, including drugs [f chitin d. Accordectively ,52. A spng drugs .−1. These signals can be assigned to C=O stretching and associated with the presence of different chitin secondary structures or defects in small crystalline chitin fibrils, respectively [i/iR ratios , and LSEM3682 (Clade II) and LSEM3690 (Clade I) showed the lowest ones . The degree of acetylation, namely, the fraction of acetyl groups present in the α-chitin structure, is linked to the crystalline structure of chitin, because a high degree of acetylation stabilizes the highly stable α-chitin crystalline structure through a network of hydrogen bonds that the amine acetyl groups form with NH groups [Additional signals might also be found in the Amide I region as shoulder bands at 1675~1685 and >1688 cmectively . The twoH groups ,54,55. C−1 that could unequivocally represent the β–chitin allomorph  contributed by membrane sterols [−1 (within rings) and 1666 cm−1 (in acyl chains). However, only the former band can be clearly detected (as a shoulder) in the C. auris spectra, whereas the latter strongly overlaps with Amide I signals and is not univocally resolvable. Further, the relatively intense and isolate band at ~713 cm−1 is contributed by ergosterol (cumulative of C–H bending and ring deformation) [C. auris clades/subclades. An important finding was that the doublet at 597/620 cm−1 showed a wide range of variability with respect to its relative intensity among the investigated samples. In pure ergosterol, the low-frequency band is more intense than the high-frequency one, and vice versa in ergostane [−1 appeared in the spectra (cf. red asterisk in −1 stretching mode of –C=C– bonds in the second sterol ring when the 597 cm−1 band displayed intensity higher than the 620 cm−1 one. These three spectroscopic characteristics suggest the presence of a fraction of ergostane-type steroid structures (besides ergosterol) in all studied clades/subclades , LSEM3673 (Clade III), LSEM643T (Clade II), and LSEM3682 (Clade II) in decreasing order . sterols . Fingerprmation) , but it rgostane . Concurrades cf. e. AccordC. auris clades/subclades revealed significant differences and located a series of spectroscopic parameters  representing vibrational fingerprints of polysaccharides and lipids. The computed Raman biochemical parameters are summarized in C. auris samples investigated. The plots vividly reflect a variety in the fractions of primary metabolites, which are linked to key structural characteristics of C. auris clades/subclades and reveal a breadth in their functional diversity.In summary, the above analysis of deconvoluted Raman spectra collected on C. auris cell cultures, Raman imaging performed with sub-micrometric spatial resolution made a large number of spectra (in the order of 106) available, which served to typify the clades/subclades with respect to the five Raman biochemical parameters described in the previous section with high statistical validity. The Raman-imaging approach is complementary to the obtainment of representative Raman spectra for each subclade upon averaging over 20 spectra collected with a low-magnification optical probe (20×), including multiple cells (10~20) in each acquired spectrum . Upon entering the average Gα3/α6R value computed for each image in the plot of α3 . The monotonic character of the Gα3/Rα6–Fα3 relationship preserves the order described in the previous section , LSEM3673 (Clade III), and LSEM3682 (Clade II); cf. α3 values >60%. However, LSEM3690 (Clade I) also showed a Fα3 value as high as 59.2%, despite not showing ECM. On the other hand, the lowest amount of α–1,3–glucans, recorded in LSEM3672 (Clade III), was computed as 42.1%. The two Clade IV subclades displayed intermediate Fα3 values at around 50%.Mα/βR = I960/I974 + I960, to the α–D–mannopyranose fraction, FM. The linear plot is based on data reported in Ref. [Mα/βR ratio recorded on the investigated C. auris clades/subclades (cf. labels). Average Mα/βR values were computed for each image and introduced in M . The linear calibration plot unveils a trend similar to that shown for α–1,3–glucans, with the three ECM-embedded subclades (LSEM643T (Clade II), LSEM3682 (Clade II), and LSEM3673 (Clade III)) showing relatively high FM fractions . However, the highest fraction was recorded for the ECM-free LSEM3690 (Clade I) (68.6%), thus suggesting that α–D–mannopyranose molecules in this subclade predominantly belong to cell walls. Of note was also that, as compared with the fraction Fα3 of α–1,3–glucans, the α–D–mannopyranose fraction, FM, fluctuated over a wider  interval among different C. auris samples. The minimum FM values were displayed for LSEM3672 (Clade III) (28.3%), thus confirming the possibility of wide fluctuations between samples belonging to the same clade as further proof of intra-clade functional diversity. in Ref. . Figure E/RR = I1107/I1054 intensity ratio from the data in Ref. [−1) is plotted in (b) as a function of crystallinity values, DC (determined by wide-angle X-ray diffraction measurements). As seen, the E/RR ratio sharply increased with increasing chitin crystallinity. E/RR ratio collected on different C. auris samples (cf. labels). The average E/RR values computed for each image were then inserted into the plot of C . Except for LSEM3662 (Clade IV) and LSEM3673 (Clade III) , all other C. auris samples displayed relatively low crystallinity values . Since the length of a chitin dimer is 1.032 nm [C. auris samples with DC ~20% or less likely only incorporate a small subgroup of crystalline α-chitin allomorph to form rigid microfibrils, whereas the remainders might take a different allomorphic structure or lie in a disordered state. This point will be detailed in In  in Ref.  (after n1.032 nm  and the 1.032 nm . Assumin…HN) and intramolecular (C=O…HO(C6) or C=O…HN hydrogen bonds) and the structure of a partly deacetylated α-chitin including primary amine groups to replace acetyl amines. Zhang et al. [A [i/iR = I1644/I1660, referred to as the Raman ratio of intermolecular-to-intramolecular hydrogen bonds as a function of DA is given in C. auris samples. i/iR ratio for different C. auris samples (cf. labels), from which average values were computed and matched with the calibration plot in A values , with the highest and lowest values being recorded for LSEM3662 (Clade IV) (94.7%) and LSEM3682 (Clade II) (79.3%), respectively. Similar to other metabolite-related parameters, DA also showed differences between subclades of the same clade , the ErgR = I597/I620 ratio was plotted as a function of the ergostane fraction, FEs, (ErgR ratio are given for different C. auris samples (cf. labels in inset). Average values were computed for each map and matched with the calibration plot in Es values. In line with previous reports on the sterol composition of other Candida species [Es values could also be found in clades/subclades belonging to the C. auris species. LSEM3672 (Clade III) and LSEM3690 (Clade I) were the richest and poorest samples in ergostane (FEs = 86.0% and 36.7%), respectively. These differences in sterol composition can greatly impact the drug resistance  of different clades/subclades, as discussed in a later section.on, FEs, c accordi species ,66,67,68α3, FM, DC, DA, and FEs, respectively, as determined from Raman experiments on the C. auris samples investigated in this study. Standard deviations are indicated in the inset together with statistical evaluations performed on Raman maps collected at different locations . The potential impact of such wide variations in cell metabolites on virulence and drug resistance will be discussed in the forthcoming C. auris clades/subclades investigated are shown in 2 in size were selected (cf. red squares in each image), in which 200~400 Raman spectra were collected and averaged to obtain a representative spectrum for each selected location. The obtained 10 (average) spectra per each sample were then subjected to PCA analyses. −1. This plot shows that PCA analysis is capable of separating all C. auris clades/subclades except for the LSEM643T (Clade II) and LSEM3673 (Clade III). The present application of the PCA statistical method reduces the dimensionality of the Raman data matrix to two orthogonal variables and provides evidence for the usefulness and limitations of a standard chemometric analysis in identifying C. auris clades/subclades upon discriminating among their Raman spectra.Images of the seven C. auris sample. C. auris clades/subclades. Once translated into line patterns, such spectral differences became clearly distinguishable by the naked eye for all subclades, including LSEM643T (Clade II) and LSEM3673 (Clade III)  stress-driven cell expansion while preventing cell breakage [Candida cells maintain the delicate balance between rigidity and compliance of their walls. The Raman metabolomics data shown in C. auris subclades producing ECM, as well as fundamental differences in the chemical structure of cell walls among different C. auris subclades. Raman analyses also suggest that both balance and redundancy exist in the strategies employed by different subclades to assemble their cell walls.As common to all  species ,72, C. acf. also , and higman host . CandidaC. auris subclades in terms of polysaccharides structural characteristics. With reference to the four quantitative parameters located as characteristic of cell wall structure  showed the lowest values with respect to both parameters. The low degree of α-chitin crystallinity and the scarce presence of hydrogen bonds between its antiparallel chains confer high cell walls’ flexibility to the Clade II sample. Clearly, the LSEM3682 (Clade II) roots its surviving strategy in building copious ECM with a substantial fraction of stiff and insoluble α-glucan structure, which makes its Fα3 value the second-highest among C. auris samples  and LSEM3673 (Clade III), both also embedded in ECM; cf. C, and degree of acetylation, DA), LSEM 3673 (Clade III) possesses relatively high values of both chitin parameters, thus displaying strategic redundancy in concurrently producing ECM and strengthening the chitin structure of its cell walls.An instructive exercise in discussing different survivorship strategies and structural differences could reside in comparing LSEM3682 (Clade II) and LSEM3662 (Clade IV) with respect to both their propensity to produce ECM and composition of their cell walls. These two samples might be considered the most contrastive among Candida cells in survivorship. Moreover, the α–1,3–glycosidic linkages are water-insoluble, as compared with water-soluble α–1,6–glycosidic linkages [ECM provides the yeast cells with a stiff shield, since the α–1,3–glucan polymorph is by far the most rigid molecule among polysaccharide structures  . Tailorilinkages ,44,45. Nlinkages . Therefolinkages .C. auris clades, Kwon et al. [C. auris exposed to Amphotericin B [C. auris clades.Looking at the published literature on structural characteristics of n et al.  studied n et al. . Microscn et al. . In a prericin B , a lack ericin B  in orderC. auris clades/subclades also displayed different structures and complexities of glucan mannans  cell walls, reveal the bi-functional strategy of this subclade: supporting cell walls through the formation of a strongly hydrophobic scaffold while increasing virulence by disabling host-cell detection.The cell walls of different Upon extending the comparison of the two somewhat antithetical structural characteristics/strategies of LSEM3682 (Clade II) and LSEM3662 (Clade IV) to the other clades investigated, one finds that the LSEM3690 (Clade I) lacks ECM but contains the highest fraction of α–D–mannopyranose, while also exploiting a relatively high fraction of core α–1,3–glucans. On the other hand, only LSEM3672 (Clade III) includes quite low contents in both core α–1,3–glucans and peripheral α–D–mannopyranose. In other words, though lacking ECM, this latter subclade is neither “buying” the immune-recognition blocking strategy of the LSEM3682 (Clade II), nor packing its α–1,3–glucans in the inner core of the cell walls to develop a strongly hydrophobic scaffold as in the cases of both Clade II subclades. Accordingly, the origin of high virulence/drug resistance of LSEM3672 (Clade III) should reside in a different metabolite strategy.Candida metabolites in As discussed above, on the basis of the Raman analysis of C. auris subclade incorporating the highest ergostane fraction. Ergostane-type steroids are chemical compounds characteristic of a variety of fungi, with a wide spectrum of biological activity also including immunological functions. Raman spectroscopy does not allow for judging the location of the detected ergostane molecules, namely, whether they are buried in the plasma membrane or cling to the outer dynamic cell-wall domain as glycosylated sterols. In either case, however, a key-role could be foreseen for the ergostane moiety. Modifications of the sterol composition of the plasma membrane could be interpreted as a strategy to stabilize the structure of the plasma membrane in order to obstruct the action of pore formation exerted by Amphotericin B [C. auris clades/subclades.As seen in ericin B . On the ericin B  reportedCandida genes are continuously updated, molecular techniques such as polymerase chain reactions are systematically applied to document the mechanisms of drug resistance arising from genetic mutations [C. auris clades/subclades, Raman metabolomics has the potential to improve this situation by providing real-time speciation and key information about the structural variety of individual clades/subclades. Structural differences can be used as biomarkers to speciate the clades and to interpret the strategies followed by their specific isolates. Additional advantages given by Raman imaging are the possibility to monitor, in situ and in time lapse, drugs’ effectiveness, and to predict therapeutic outcomes. Raman speciation could boost modern methods of companion diagnostics [modus operandi in precision medicine [Candida infections could provide a new path for safely and effectively selecting drugs, giving healthcare professionals a robust platform to reduce patients’ risks and greatly lowering the chances of dangerous genetic mutations of the Candida pathogen. Time-lapse Raman imaging of selected Candida metabolite levels could provide the “reasons” for genomic mutations and enable monitoring the effects of drug compounds on specific metabolic pathways. Complementary to genomics, Raman-based pharmacodynamic approaches may provide insightful snapshots of cell metabolism and allow for visualizing the pathways and mechanisms involved in cell responses to different treatments. Raman metabolomics of Candida clades and subclades could also help to reproducibly assess observed interpatient variability by interpreting Candida metabolome diversities related to the physiological variables of different patients.Though databases of utations . Despitegnostics , and in medicine ,87. The Candida albicans causing fungemia in a large number of patients from the same hospital. These researchers found that up to 25% of patients were infected by endemic C. albicans genotypes , thus demonstrating that the isolates might have originated from common sources, i.e., contact with healthcare workers, the use of catheters and other biomedical devices, parenteral nutrition, or other sources in the hospital environment. Despite the importance of these findings, the authors also pointed out the main limitation of their study as the failure to precisely determine the source(s) of infection and the route(s) of transmission in the hospital. The reason for such a failure should be tracked down to the extremely cumbersome amount of genomic analyses required to characterize isolates from the overall hospital environment, from health care workers, and from other anatomical sites of patients affected by candidemia. The availability of an agile, precise, and relatively inexpensive tool, such as Raman spectroscopic profiling of Candida isolates, could simplify and empower tracking procedures, thus greatly advancing the epidemiologic management of invasive candidiasis.Escribano et al.  studied Candida infections. The initial delivery of an appropriate antifungal drug should take place within the first 12 h after the first positive test of blood culture in order to maximize the cure outcome [Candida infections represents just one specific aspect of a global trend aimed at accurately identifying diseases at their prodromal stage in order to avoid the acute stage and elongate the span of healthy living. Disease prevention and early diagnostics appear nowadays as the two main goals of global healthcare, with patients increasingly focused on self-care and quality of life and governments pinpointing reduced healthcare costs. In our modern times, several new tools have become available to make these goals possible; these include improved analytical techniques, algorithms of machine learning, and artificial intelligence approaches. The combination of these innovative tools could in the near future enable the collection and handling of a large volume of high-quality data in nearly real time, thus opening the way for new routes for high-efficiency healthcare management. As discussed in the previous section, the prompt administration of the appropriate antifungal drug(s) is key in the successful treatment of patients affected by  outcome . HoweverC. auris by applying a “Raman barcode” approach built on machine-learning-construed spectral deconvolutions. Though the barcode technology is not new in modern healthcare, the method proposed here presents substantial differences with those presently in use. Barcodes are currently employed from manufacturing to dosing, in tracking batches of medicine, and in making distribution processes more efficient. In clinical settings, using barcodes has been shown to greatly increase accuracy in medication management [Candida species. Therefore, the Raman barcode, which requires no or minimal sample preparation, might surpass the DNA barcode in swiftness and flexibility regarding on-site diagnostics.In the present work, we have explored one possible high-efficiency route by demonstrating the possibility of speciating all four clades (and related subclades) of pathogenic nagement ,91. The nagement ,93. DNA Candida cells, it gives indications on profiling and reveals metabolism-related parameters, which could be used in evaluating and predicting environmental stress adaptations and drug resistance. Raman barcodes, which unambiguously locate C. auris clades and subclades (cf. As shown in this study, when Raman barcoding is applied to ades cf. , could bC. auris clades and related subclades for a total of seven different samples. First, representative Raman spectra were computed from averaging over 20 spectra collected at different locations on each sample with high spectral resolution and an optical probe including multiple cells in each acquired spectrum. Average spectra were deconvoluted according to a specifically tailored machine-learning algorithm linked to a previously built database of Raman spectra from elementary molecules. Then, several millions of spatially resolved spectra were taken with sub-micrometric spatial resolution in order to map the cell cultures. Maps were then converted into hyperspectral images of selected parameters for the purpose of metabolomic profiling. These two complementary Raman procedures gave consistent results, showing excellent reproducibility and thus the possibility of establishing a satisfactory level of standardization. The Raman analyses unveiled important differences in the way different clades/subclades are structured, with important implications for cellular morphogenesis, virulence, and drug resistance. This study demonstrates the importance of metabolomics as a parallel and complementary approach to genomics in profiling C. auris clades and subclades. Raman identification of Candida species down to the subclade level can be obtained in a span of minutes, thus reducing the time necessary for optimizing antifungal treatment decisions to a minimum.A dual Raman approach has been applied to analyze the metabolites of four Candida infections down to the sub-clade level. The Raman approach could operate hand-in-hand with other global health trends, such as enabling companion diagnostics, point-of-care testing, and remote healthcare. Personalized medicine carried out through on-site real-time Raman analyses, decentralization of healthcare exploiting relatively inexpensive Raman devices, and acquisition of cloud-based big data through applying Raman barcodes could represent a new path toward the medicine of the future.In conclusion, Raman metabolomics empowered by machine-learning algorithms and barcode technology, as presented in this paper, could become a powerful tool in efficiently profiling and tracking


Obesity and cardiovascular disease are major global public health problems. Maternal obesity has been linked to multiple adverse health consequences for both mother and baby. Obesity during pregnancy may adversely alter the intrauterine environment, which has been hypothesised to predispose the offspring to poorer cardiovascular health throughout life. In this paper, we systematically review current literature examining the links between maternal obesity and offspring cardiovascular health.This study is registered with PROSPERO (CRD42021278567) and was conducted in accordance with the PRISMA guidelines. A comprehensive systematic literature search was conducted, including two electronic databases , cross-referencing, author searching, and grey literature searches. We selected studies exploring the relationship between maternal obesity and offspring cardiovascular health, using pre-defined eligibility criteria. Studies were critically appraised using the ROBINS-I tool.From 1,214 results, 27 articles met the eligibility criteria. Multiple cardiovascular outcomes were considered, including congenital heart disease, cardiometabolic parameters, and cardiovascular diseases in neonates, children, and adults. In these studies, maternal obesity was consistently associated with congenital heart disease, several adverse cardiometabolic parameters throughout life including higher body mass index and insulin levels, and greater risk of cardiovascular disease in adulthood. Hypothesized underlying mechanisms are complex and multifactorial comprising genetic, environmental, and socioeconomic components, which can be difficult to quantify. Heterogeneity in study designs, highly selected study samples, and high risk of bias in some studies limit conclusions regarding causality.We identified consistent evidence of links between maternal obesity and poorer offspring cardiovascular health throughout the lifecourse, extending from the neonatal period into adulthood. Although underlying mechanisms are unclear, our findings support consideration of targeted maternal obesity prevention for promotion of offspring cardiovascular health. This all-encompassing systematic review provides critical appraisal of the latest evidence, defines gaps and biases of existing literature, and may inform potential new public health strategies for cardiovascular disease prevention.https://www.crd.york.ac.uk/prospero], identifier PROSPERO (CRD42021278567).[ The World Health Organisation (WHO) has characterised the rising prevalence of obesity as an epidemic. As a risk factor for a multitude of diseases, obesity poses a major global public health challenge .The prevalence of obesity in women of reproductive age is increasing alongside that of the general population . Currentin-utero is a critical determinant of long-term offspring health   protocol. Prior to conducting the review, the study was registered on the International Prospective Register of Systematic Reviews  . MethodsWe set a broad remit for the review with pre-defined study inclusion criteria Table 1.The searches were carried out by two independent investigators  using the Ovid Medline (1946-October 2020) and EMBASE electronic databases. In Ovid Medline, we used the advanced search option to select relevant MeSH  terms. To capture broad results, we used the explode function, included all subheadings, and included a keyword search for each term using the multi-purpose (mp) function. Search terms were combined using Boolean operators. The results were limited to English language articles and human studies Table 2.The studies were assessed for their risk of bias using the ROBINS-I (Risk of Bias in Non-randomised Studies – of Interventions) tool . This toThe search generated 1,213 results, including 9 duplicates. One study was identified from unpublished literature  searching. 1,159 papers were excluded following the title and abstract screen. This left 46 articles, which were read in full. Of these, 27 studies met the eligibility criteria and are included in the review Figure 1The 27 included studies were published between 2011 and 2020 in 11 different countries. All were observational studies. All used Maternal pre-pregnancy body mass index (MppBMI) or maternal body mass index (BMI) during pregnancy as the main exposure of interest. Six studies used gestational weight gain (GWG) as an additional exposure. One study also used skinfold thickness and fat percentage by bioimpedance. The studies measured cardiovascular health outcomes in offspring at various ages: 10 papers studied neonates, 11 studied children (0-18years), and eight studied adults (17-86 years). A diverse range of outcomes were considered, including CHD, cardiometabolic parameters  measurement, serum lipids, and serum insulin levels), and incident/prevalent CVD.We organised the identified studies according to the age range studied and the outcome measured: neonatal CHD (n=7), neonatal cardiometabolic parameters (n=1), childhood cardiometabolic parameters (n=11), adult cardiometabolic parameters (n=5) and adult CVD (n=3). The characteristics, outcomes, and exposures of studies are summarised in The studies were evaluated for their risk of bias using the ROBINS-I tool. 16 studies were appraised to be of moderate risk of bias, while 5 were of severe risk and 5 of critical risk. One study did not have enough information available to come to a bias assessment. No studies were at a low risk of bias. The results of the bias assessment are displayed in 2) compared to mothers with BMI<30kg/m2. Models were adjusted for study site, age, race, insurance status, and maternal smoking. A further analysis on a subset of 5,131 pairs was also adjusted for maternal glucose tolerance. The sample included 1,388 (1%) cases of CHD. The authors report a dose-dependent increase in odds of any CHD across increasing BMI categories. Compared to normal weight mothers, mothers with morbid obesity (BMI ≥40kg/m2) were at highest risk of having a child with CHD [OR 1.36 (1.03-1.78)], followed by mothers with obesity [OR 1.25 (1.08-1.45)], and mothers who were overweight [OR 1.18 (1.03-1.34)]. Compared to mothers without obesity, those with BMI ≥30kg/m2 had significantly greater odds of having an infant with conotruncal defects [OR 1.33 (1.03–1.72)], atrial septal defects [OR 1.22 (1.04–1.43)], or ventricular septal defects [OR 1.38 (1.06–1.79)]. In the subset of 5,131 pairs adjusted for maternal glucose tolerance, these results remained consistent.Several studies examined the link between maternal obesity and neonatal CHD. Brite et al.  conducteMadsen et al.  report aA small case control study by Alvarado-Terrones et al. , investiComplementary to these findings, Tang et al.  report aSeveral studies have failed to reproduce the observed associations between maternal BMI and infant CHD risk. Ghaderian et al.  present Dolk et al.  also previa Children’s Hospital of Philadelphia (1992-2010) and the Paediatric Cardiac Genomic Consortium (2010-2012). This study reported no significant association , suggese et al. . HoweverLemas et al.  recruiteA total of 12 studies investigated the association between maternal BMI and cardiometabolic parameters during childhood Table 5.Nine studies report positive associations between MppBMI and offspring childhood anthropometrics , 40, 41.Filler et al.  conducte2 increase in MppBMI was associated with 0.92kg higher total fat, 0.27 BMI z-score and 0.39kg trunk fat in the offspring . To assess this, Perng et al.  and Tan Tan et al.  also useAdiposity has been strongly linked to insulin resistance. Therefore, it was important that studies adjusted for current offspring anthropometrics: in the four studies that adjusted for this, the association was partially attenuated , 38, 40.Four studies investigated the role of MppBMI on offspring lipid levels, although results were mixed. Gaillard et al.  and Tan Two studies used echocardiography to measure offspring cardiovascular structure and function in childhood , 36. In There is a paucity of evidence in this area, which limits the conclusions that can be drawn. The results available suggest that higher MppBMI is associated with adverse cardiovascular phenotypes in childhood, but that this may be wholly related to childhood BMI. Further studies are required before definitive conclusions can be drawn in this field.Two studies assessed the association between MppBMI on the offspring vasculature , 32. Toe2 and waist circumference (WC) increased by 3.5 cm, and similarly, greater GWG was associated with higher offspring BMI in adulthood . This was described by Barker following the observation that death rates distributions from coronary heart disease mirrored infant mortality and low birth-weight rates 70 years earlier  and also from the CAP-AI programme, London’s first AI enabling programme focused on stimulating growth in the capital’s AI Sector. CAP-AI is led by Capital Enterprise in partnership with Barts Health NHS Trust and Digital Catapult and is funded by the European Regional Development Fund and Barts Charity. SEP has also received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 825903 (euCanSHare project). CM and SN were supported by the Oxford NIHR Biomedical Research Centre (IS-BRC-1215-20008). SN was additionally supported by the Oxford British Heart Foundation Centre of Research Excellence. NCH acknowledges support from the UK Medical Research Council (MRC #405050259 and #MC_UU_12011/1), NIHR Southampton Biomedical Research Centre, University of Southampton, and University Hospital Southampton. AJL is funded by a British Heart Foundation Intermediate Research Fellowship (FS/18/3/33292). The funders provided support in the form of salaries for authors as detailed above but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.ZR-E recognizes the National Institute for Health Research (NIHR) Integrated Academic Training programme which supports her Academic Clinical Lectureship post and was also supported by British Heart Foundation Clinical Research Training Fellowship No. FS/17/81/33318. SEP acknowledges support from the ‘SmartHeart’ EPSRC programme grant (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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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