The role of prophylactic drainage (PD) in gastrectomy for gastric cancer (GC) is not well-established. The purpose of this study is to compare the perioperative outcomes between the PD and non-drainage (ND) in GC patients undergoing gastrectomy.A systematic review of electronic databases including PubMed, Embase, Web of Science, the Cochrane Library, and China National Knowledge Infrastructure was performed up to December 2022. All eligible randomized controlled trials (RCTs) and observational studies were included and meta-analyzed separately. The registration number of this protocol is PROSPERO CRD42022371102.P = 0.04; I2 = 0%), earlier time to soft diet  and shorter length of hospital stay . While other outcomes including anastomotic leakage, duodenal stump leakage, pancreatic leakage, intra-abdominal abscess, surgical-site infection, pulmonary infection, need for additional drainage, reoperation rate, readmission rate, and mortality were not significantly different between the two groups. Meta-analyses on observational studies showed good agreement with the pooled results from RCTs, with higher statistical power.Overall, 7 RCTs (783 patients) and 14 observational studies (4359 patients) were ultimately included. Data from RCTs indicated that patients in the ND group had a lower total complications rate (OR = 0.68; 95%CI:0.47–0.98; The present meta-analysis suggests that routine use of PD may not be necessary and even harmful in GC patients following gastrectomy. However, well-designed RCTs with risk-stratified randomization are still needed to validate the results of our study.The online version contains supplementary material available at 10.1186/s12957-023-03054-1. Gastric cancer (GC) is one of the most common causes of cancer-related deaths worldwide , 2. DespProphylactic drainage (PD) has long been routinely performed in abdominal surgery for the purpose of preventing and managing potential postoperative abdominal complications , 6. HoweHowever, evidence for avoiding routine PD after surgery in patients with gastric cancer is sparse. In 2020, a meta-analysis based on 10 studies concluded that PD avoidance may favor a reduction in morbidities and a trend towards a decrease in length of stay [Our meta-analysis was performed in line with the requirements from PRISMA  guidelines  and asseRelevant studies from electronic datasets including PubMed, Embase, Web of Science, the Cochrane Central Register of Controlled Trials, and China National Knowledge Infrastructure were systematically examined up to December 15, 2022. The following key words (limited to title or abstract) were combined with Boolean operators AND or OR, to comprehensively capture potential articles: “drainage,” “drain,” “gastric cancer,” “gastric carcinoma,” “stomach cancer,” and “stomach neoplasm”. The search strategy was applied to suit each database, and the complete search strategy was reported in the supplementary file: Table SP: Patients were pathologically diagnosed with GC and underwent gastrectomy; I: non-drainage; C: prophylactic drainage; O: perioperative outcomes; S: Comparative studies including RCTs, cohort and case-controlled studies.The inclusion criteria were determined according to the PICOS approach as follows. The exclusion criteria were studies (1) reported as case reports, reviews, letters, and abstracts and (2) with overlapping data.Two independent reviewers (HY-P and LH-C) conducted the data extraction and cross-checked all the results, and any discrepancies were resolved by a third reviewer (H S). The following data were extracted from each study: first author, publication year, study interval, country, study design and sample size, age, sex, neoadjuvant therapy, surgical approach, gastrectomy extent, combined organ resection, surgical margin, lymphadenectomy extent, TNM stage, time of drainage removal, and a series of perioperative outcomes.The Cochrane Risk-of-Bias 2.0 (RoB 2.0)  tool wasPerioperative outcomes that occur during hospitalization or within 30 days after surgery were assessed in this study, including total complications, anastomotic leakage, duodenal stump leakage, pancreatic leakage, intra-abdominal abscess, surgical-site infection, pulmonary infection, need for additional drainage, time to first soft diet, length of hospital stay, reoperation, readmission, mortality and drain-related complications.2 statistic. In the present study, all meta-analyses were performed assuming the random-effects model, which accounts for variance across included studies. Subgroup group analysis and meta-regression analysis were performed to investigate the sources of heterogeneity. Publication bias was tested using Begg’s funnel plot when there were at least 10 studies included. A two-tailed P value < 0.05 was considered statistically significant. All of these statistical analyses were performed by Review Manager Software, version 5.3 , and Stata, version 12.0 .The odds ratios (ORs) and mean differences (MDs) with their 95% confidence intervals (CIs) were used as the effect sizes for dichotomous variables and continuous variables, respectively. For studies that reported median with range or inter-quartile range, data were converted into mean with standard deviation (SD) using the method reported by McGrath et al. . HeterogA flow chart of the selection process was shown in Fig. P = 0.04; I2 = 0%); observational data showed concordant result, however, without significant difference . In addition, we compared the incidence of specific complications between the ND and PD groups. As shown in Table All seven RCTs including 783 patients and 11 observational studies including 2146 patients contributed data for total complications . The pooled result from RCTs suggested that there was no evidence that ND increased the risk of early death , which was in line with the results of observational studies . While the results of the heterogeneity test demonstrated a high heterogeneity among the observational studies.A total of 4 RCTs involving 438 patients and 6 observational studies involving 698 patients reported on time to first soft diet . The observational data were consistent, although the statistical difference threshold was not reached . The results of the heterogeneity test demonstrated a moderate and high heterogeneity among the RCTs and observational studies, respectively.Data regarding postoperative hospital stay were available from 6 RCTs and 12 observational studies, including 716 and 2303 patients, respectively Table . In the P = 0.95; I2 = 0%) and observational studies  demonstrated a similar rate of additional drainage between the ND and PD patients. However, the results of the heterogeneity test showed a high heterogeneity among the observational studies.As shown in Table P = 0.41; I2 = 25%; Table P = 0.11; I2 = 0%; Table Two RCTs comprising 168 patients reported on reoperation. Pooled data from RCTs showed no significant difference in reoperation rate between the ND and PD groups  tended to be lower in the ND group than in the PD group.Subgroup analyses stratified by the sample size (≥ 100 vs. < 100) and academic institution (Yes vs. No) were performed to explore the potential discrepant treatment effect of different subgroups. Moreover, the efficacy of PD was also explored in GC patients who underwent laparoscopic surgery or a total gastrectomy. As shown in Fig. SP values > 0.05).For pooled outcomes with significant heterogeneity , meta-regression analyses based on the following covariates were also performed to investigate the sources of heterogeneity: study design (RCT vs. non-RCT), sample size (≥ 100 vs. < 100), academic institution (Yes vs. No), surgical approach (laparoscopic gastrectomy or not) and surgical procedure . As shown in Table SAs shown in Fig. P values were greater than 0.05, indicating that these pooled outcomes had a low risk of publication bias.The Begg’s funnel plot was used to assess the potential publication bias of the pooled outcomes including at least 10 studies. As shown in Table Currently, the routine placement of abdominal drainage tubes after gastrectomy is still widely used worldwide for the early diagnosis and management of critical abdominal complications such as post-operative bleeding, anastomotic leakage, and intra-abdominal infections . SuccessTo our knowledge, this is the largest meta-analysis (21 studies including 5142 patients) to evaluate the role of PD in perioperative outcomes of GC surgery. In this study, we found that the routine use of PD after surgery did not reduce the incidence of abdominal complications such as anastomotic leakage and pancreatic leakage. In contrast, the overall complication rate was significantly higher in the PD group. In addition, the length of hospital stay and the time to soft diet were much longer in the PD group than in the ND group. Moreover, PD did not also show any benefit in reducing readmission, reoperation, or mortality in GC surgery.Several previously published meta-analyses , 38–40 hP = 0.11) and pancreatic leakage (P = 0.07) was slightly lower in the ND group, although there was no strong evidence at the pooled analyses that routine ND has an effect on reducing these adverse outcomes. With advances in surgical techniques and laparoscopic equipment, laparoscopic surgery has been shown to be less likely to result in serious postoperative complications in experienced centers, due to its minimally invasive nature [In recent years, laparoscopic surgery has been widely performed in GC, but the role of PD in laparoscopic gastrectomy is still unclear. Therefore, we performed a subgroup analysis for laparoscopic resections. Based on the results from 567 patients who underwent laparoscopic gastrectomy, our finding of the benefit of ND in these patients remained unchanged. Besides, we found that in this subgroup, the incidence of anastomotic leakage  and hospital nature (academic institution vs. non-academic institution) on the perioperative outcomes of PD in GC patients. As shown in Fig. SNevertheless, our findings are based on literature, some uncertainties exist in the evidence included in this meta-analysis. The lack of stratified information in the original literature prevented us from analyzing the applicability of ND in certain specific subgroups, such as patient demographics , surgical parameters  and oncological variables (neoadjuvant therapy and TNM stage). Therefore, the current evidence does not mean that abdominal drainage should be discontinued in all patients after GC surgery. What we can conclude is the avoidance of routine drainage of a prophylactic nature. Drainage is strongly recommended in some cases, such as abdominal contamination due to perforation and obvious iatrogenic organ injury , 47. In Recently, the first nomogram for predicting the risk of postoperative percutaneous drain placement has been constructed . This prThe present study has some limitations that should be acknowledged. First, although 7 RCTs were included in our study, the quality of these RCTs was not high and did not also perform stratified analyses in specific populations, which had a certain impact on the reliability of the results of this study. Therefore, more well-designed RCTs with large sample sizes are expected to provide more credible evidence on this issue. Second, several included studies , 26, 33 The present meta-analysis suggests that the routine use of PD after GC surgery is not beneficial, and even harmful with increased morbidities, and prolonged time to soft diet and hospital stay. However, based on the abovementioned limitations and low level of evidence of the comparisons, more multicenter RCTs with risk-stratified randomization are needed to confirm these questions.Additional file 1:Table S1. Detailed search strategies of each database. Table S2. Outcomes of the meta-regression analyses. Figure S1. Forest plots of perioperative outcomes including: A. anastomotic leakage; B. Duodenal stump leakage; C. Pancreatic leakage; D. Intra-abdominal abscess; E. Surgical-site infection; F. Pulmonary infection; G. Mortality; H. Need for additional drainage; I. Readmission; J. Reoperation. Figure S2. Subgroup analyses of perioperative outcomes based on sample size (≥100 vs. <100). A. total complications; B. anastomotic leakage; C. Duodenal stump leakage; D. Pancreatic leakage; E. Intra-abdominal abscess; F. Surgical-site infection; G. Pulmonary infection; H. Mortality; I. Time to first soft diet; J. Length of hospital stay; K. Need for additional drainage; L. Readmission; M. Reoperation. Figure S3. Subgroup analyses of perioperative outcomes based on academic institution (Yes vs. No). A. total complications; B. anastomotic leakage; C. Duodenal stump leakage; D. Pancreatic leakage; E. Intra-abdominal abscess; F. Surgical-site infection; G. Pulmonary infection; H. Mortality; I. Time to first soft diet; J. Length of hospital stay; K. Need for additional drainage; L. Readmission; M. Reoperation. Figure S4. Subgroup analyses of perioperative outcomes in GC patients who underwent laparoscopic surgery. A. total complications; B. anastomotic leakage; C. Duodenal stump leakage; D. Pancreatic leakage; E. Intra-abdominal abscess; F. Surgical-site infection; G. Pulmonary infection; H. Time to first soft diet; I. Length of hospital stay; J. Reoperation. Figure S5. Subgroup analyses of perioperative outcomes in GC patients who underwent total gastrectomy. A. total complications; B. anastomotic leakage; C. Duodenal stump leakage; D. Pancreatic leakage; E. Intra-abdominal abscess; F. Surgical-site infection; G. Pulmonary infection; H. Mortality; I. Time to first soft diet; J. Length of hospital stay; K. Need for additional drainage; L. Readmission; M. Reoperation. Figure S6. Begg’s funnel plot of perioperative outcomes including: A. total complications; B. anastomotic leakage; C. Intra-abdominal abscess; D. Surgical-site infection; E. Pulmonary infection; F. Time to first soft diet; G. Length of hospital stay. All P values >0.05.


Candida auris is an emerging pathogen that constitutes a serious global health threat. It is difficult to identify without specific approaches, and it can be misidentified with standard laboratory methods, what may lead to inappropriate management.C. auris isolation from blood cultures and wound swabs of a young male following meningococcal septicaemia, in February 2019. The patient had been previously hospitalized in the United Arab Emirates. The isolate was rapidly identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry and therefore clinicians were promptly informed on the alert pathogen isolation. The targeted antifungal treatment was successful and infection control measures seemed effective. ITS-based identification and subsequent whole genome sequencing showed that the C. auris isolate belongs to South Asian lineage (clade I).We report, probably the first in Poland, C. auris is able to cause outbreaks in healthcare settings. Therefore, it is important to quickly identify C. auris isolates in hospital settings so that healthcare facilities can take proper precautions to limit its spread. Candida auris has been widely known as an emerging multi-drug resistant fungal pathogen. It might be resistant to all available classes of antifungals [Candida species, C. auris spreads easily in health-care facilities causing sporadic cases and outbreaks [ifungals –4. This ifungals . In contutbreaks –5.C. auris from a patient in Poland, the successful targeted antifungal treatment of an invasive infection, and the effective infection control procedures.Here we report the isolation of An eighteen years-old male was admitted in February 2019 to the Dr Antoni Jurasz University Hospital No. 1 in Bydgoszcz, Poland, to the Department of Transplantation and General Surgery, for a treatment of severe complications of a meningococcal septicaemia. The patient was transferred directly from the district hospital in Greater Poland, after two days of hospitalization and following three weeks of hospitalization in the United Arab Emirates (UAE), due to a meningococcal septicaemia.At the admission, two blood samples and seven swabs from necrotic chronic wounds were collected. The aerobic blood culture became positive after two days of incubation . Microscopic examination revealed the presence of budding, non-pseudohyphae-forming, cylindrical shaped yeast cells Fig. . An empiC. auris isolation.Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) using the MALDI Biotyper system  provided a confident species-level identification of the isolate (score 2.16). The clinicians were immediately informed on C. auris colonization of necrotic chronic wounds on upper and lower limbs, and subsequently—stumps, was documented. A total of 20 C. auris isolates were cultured  according to the manufacturer's instructions. The following minimum inhibitory concentrations (MICs) values were observed for the first obtained isolate: fluconazole > 256 mg/L, amphotericin B 1 mg/L, anidulafungin 0.047 mg/L, caspofungin 0.25 mg/L, micafungin 0.064 mg/L. The results of the susceptibility tests performed on subsequently obtained ® and Octenisept®  were used topically. Altogether, four positive blood cultures were obtained, all in aerobic conditions. Simultaneously incubated blood cultures in an anaerobic conditions were negative. The first negative blood culture in the follow-up investigation was noticed after seven days of treatment with micafungin. The treatment was continued for two weeks in total. The susceptibility of the isolates to micafungin was monitored, and they remained susceptible to the drug. However, a MIC value increase from 0.047 to 0.125 mg/L was observed.Therapy with micafungin 100 mg/day was initiated. OctenilinC. auris from blood, a persistent colonization of necrotic chronic wounds of upper and lower limbs with C. auris was documented.In contrast to the eradication of C. auris. Any additional changes for environmental cleaning methods were not applied, since regularly used disinfectants in our hospital have sufficient antifungal activity—Incidin Plus (active ingredient (AI): glucoprotamin), Incidin Pro  dodecylamine, benzalkoniumchloride), Incidin Liquid Spray , Sani Cloth Active (AI: didecyldimethylammonium chloride), and Incides N  .According to the local procedures, contact precautions were implemented from the admission day, but the ICU and Surgical Department staff were additionally warned about the epidemic potential of C. auris isolate (no. 5049–2019) and type C. auris DSMZ 21,092 strain with an application of ExtractMe DNA Yeast Kit , performed according to manufacturer’s instruction. The DNA samples concentration quality was checked spectrophotometrically and the isolation protocol efficiency was confirmed additionally electrophoretically in 1.5% agarose in TBE buffer . DNA quality control was performed by measuring the absorbance at 260/280 nm, the template concentration was determined using Qubit fluorimeter . DNA integrity was analyzed by 0.8% agarose gel electrophoresis.DNA was isolated from the 48 h culture of the clinical C. auris DSMZ 21,092 strain DNA sequence of the ITS locus was identical to the reference sequence of clade II. Clinical C. auris isolate was recognized as clade I or III, therefore whole genome sequencing (WGS) was performed to define the clade.The ribosomal DNA ITS-region was amplified using primers ITS1 and ITS4 as previously described . An amplhttp://www.bioinformatics.babraham.ac.uk/projects/fastqc/) [Paired-end sequencing library was constructed using the NEB Ultra II FS Preparation Kit  according to the manufacturer’s instructions. Library was sequenced using an Illumina NextSeq platform  with 2 × 75 paired-end reads. Sequence quality metrics were assessed using FASTQC (fastqc/) .https://github.com/CDCgov/mycosnp-nf) [Filtered Illumina reads were assembled into contigs using SPAdes v.3.13.1 . Phylogeosnp-nf)  with defC. auris from National Center for Biotechnology Information (NCBI) BioProject PRJNA328792 [Candida-auris-PL1 isolate have been deposited in NCBI Genome under accession numbers: BioProject: PRJNA863457, SRA: SRS1478113, BioSample: SAMN30096895.Illumina sequencing yielded 131 million reads and 9.88 Gb of sequence data. Genome assembly generated 930 contigs with N50 41 kb and total assembly size was 12,257,419 bp. Clade identification strategy was done as described previously . In brieC. auris isolate (Candida-auris-PL1) reveals that it belongs to clade I. The reference strain for the phylogeny analysis was C. auris B8441, which belongs to clade I (NCBI Genbank ID: PEKT00000000.2). WGS was performed and the obtained data were analysed at the DNA Sequencing and Oligonucleotide Synthesis Laboratory, Institute of Biochemistry and Biophysics of the Polish Academy of Sciences (IBB PAS).A genomic relationship of tested C. auris was firstly identified in 2009, when isolated from the external ear canal of an inpatient in a Japanese hospital [C. auris has emerged as healthcare-associated alert pathogen. C. auris isolations, sporadic cases and outbreaks, have been reported in around 40 countries, on six continents: Asia , Africa (2014), Europe (2015), South America (2016), North America (2016), and Australia (2018) [hospital . Next, wC. auris isolates discovered four major clades: South Asian (clade I), East Asian (clade II), African (clade III), South American (clade IV), and Iranian (clade V), which means simultaneous emergence of multidrug-resistant C. auris on different continents [C. auris isolates in health care facilities within several United States.WGS analysis of clinical ntinents , 14, 15.ntinents  revealedC. auris strains firstly appeared in Europe in 2015 [ in 2015 , 17–20.C. auris from Poland. Applied molecular methods confirmed that the isolate belongs to clade I, also known as South Asian. It is presumed that C. auris infection and colonization was ongoing when the patient was admitted to our hospital, but the Candida sp. was not isolated or identified to the species-level in the United Arab Emirates (UAE). A sporadic case of C. auris isolation in the UAE has been previously reported [Candida species. At present, mass spectrometry  and DNA sequencing of the ITS and/or D1/D2 ribosomal DNA regions reliably identify C. auris [C. auris accurately in a large number of countries.To our knowledge this case is the first report of reported . Widely C. auris . Still tFortunately, we were able to identify the pathogen rapidly and reliably, and inform the clinician promptly. Hence, contact precautions were introduced immediately to prevent this pathogen transmission.C. auris is a lack of standards for disinfection procedures effective against C. auris. The protocols applied for infection control in our healthcare facility seem effective as until time of publication, any other C. auris isolation ensued.The actual problem of C. auris strains [Likewise the majority of  strains , 3, our C. auris tends to persists in patients, especially on skin of groins and axillae, in ear canals, and in nostrils [nostrils . We haveCandida spp. identification to the species level is of great importance. There is a high need of a reliable and rapid identification of C. auris in all medical microbiology laboratories using new technologies approved for microbiological diagnostic. This is the basic tool for an effective prevention of infection and control measures.Our case proved that careful and precise C. auris into European hospitals and the role of a reliable and rapid C. auris identification in prevention of further transmission and healthcare-associated outbreaks. Difficulties in an identification may delay early pathogen detection, increasing the risk for C. auris transmission. A cooperation of microbiologists, clinicians, nurses and epidemiologists is particularly important in this case. There is a constant need to evolve standards for infection prevention and control measures effective against C. auris.In conclusion, our report confirms the ongoing introduction of


Neisseria meningitidis and Neisseria gonorrhoeae. This raises a question as to the origin of the ChoP used for macromolecule biosynthesis in these species. In the current study, we used in silico analyses to identify the potential pathways involved in ChoP biosynthesis in genomes of the 26 bacterial species reported to express a ChoP-modified biomolecule. We used the four known ChoP biosynthetic pathways and a ChoP transferase as search terms to probe for their presence in these genomes. We found that the Lic-1 pathway is primarily associated with organisms producing ChoP-modified carbohydrates, such as lipooligosaccharide. Pilin phosphorylcholine transferase A (PptA) homologs were detected in all bacteria that express ChoP-modified proteins. Additionally, ChoP biosynthesis pathways, such as phospholipid N-methyltransferase (PmtA), phosphatidylcholine synthase (Pcs), or the acylation-dependent phosphatidylcholine biosynthesis pathway, which generate phosphatidylcholine, were also identified in species that produce ChoP-modified proteins. Thus, a major finding of this study is the association of a particular ChoP biosynthetic pathway with a cognate, target ChoP-modified surface factor; i.e., protein versus carbohydrate. This survey failed to identify a known biosynthetic pathway for some species that express ChoP, indicating that a novel ChoP biosynthetic pathway(s) may remain to be identified.Many bacterial surface proteins and carbohydrates are modified with phosphorylcholine (ChoP), which contributes to host mimicry and can also promote colonization and survival in the host. However, the ChoP biosynthetic pathways that are used in bacterial species that express ChoP have not been systematically studied. For example, the well-studied Lic-1 pathway is absent in some ChoP-expressing bacteria, such as IMPORTANCE The modification of bacterial surface virulence factors with phosphorylcholine (ChoP) plays an important role in bacterial virulence and pathogenesis. However, the ChoP biosynthetic pathways in bacteria have not been fully understood. In this study, we used in silico analysis to identify potential ChoP biosynthetic pathways in bacteria that express ChoP-modified biomolecules and found the association between a specific ChoP biosynthesis pathway and the cognate target ChoP-modified surface factor. Streptococcus pneumoniae, the pneumococcus [Haemophilus influenzae (Neisseria spp. [Neisseria meningitidis and Neisseria gonorrhoeae (Aggregatibacter actinomycetemcomitans (Acinetobacter baumannii (H. influenzae (NTHi) (H. influenzae colonization of the human nasopharynx (Phosphorylcholine (ChoP) is a small zwitterionic molecule and can be found in Gram-positive bacteria  and Grafluenzae  and commria spp. ). ChoP morrhoeae , Flp fimcomitans , and poraumannii . It has aumannii  and nonte (NTHi)  adhesione (NTHi) , 13. Furopharynx  and matuopharynx . The benopharynx .licABCD operon (–H. influenzae utilizes glycerophosphodiester phosphodiesterase (GlpQ) to acquire choline from the respiratory tract epithelial cells  to phosphatidylethanolamine , phosphse (PmtA ), and acnolamine , startinnolamine . WhereasLike the Lic-1 pathway, exogenous choline is required in the Pcs pathway. Choline transporters such as ChoXWV take up the choline from the environment , and PcsPseudomonas aeruginosa. This posttranslational modification of a lysine to add additional methyl groups on elongation factor protein, EF-Tu, results in a structural mimic of ChoP that reacts with the ChoP-specific monoclonal antibody TEPC15. Collectively, this new information will enable a correlation between the class of ChoP biosynthetic pathway and the target biomolecules that are modified by ChoP and will facilitate a more complete understanding of the factors controlling the expression of this important virulence factor.This study aimed to identify the ChoP biosynthetic pathway that is used by each of the 26 species of bacteria that have been reported to express ChoP. This was achieved by bioinformatic analysis of ChoP synthesis-related genes (as presented above ) presentH. influenzae as the query in a Basic Local Alignment Search Tool for protein sequences (BLASTp) search against the NCBI nonredundant (nr) protein database. H. influenzae and the summary of Lic-1 pathways protein found in other bacteria. The presence of Lic-1 pathway enzymes corresponds well with the bacterial species that are reported to express carbohydrate-linked ChoP, including representatives of Haemophilus haemolyticus H. influeolyticus , Histophus somni , S. pneueumoniae , 33, Strs oralis , Streptous mitis , Pasteurultocida , commensllinarum , Erysipeopathiae , and Mycrmentans –41 have irabilis  and Morgmorganii , both ofN. meningitidis, N. gonorrhoeae  modification at Ser68 and Ser156 (QXZ29786) protein sequence from the fully annotated sequenced N. meningitidis C311 genome . The two PptA-like ORFs are not solely in A. actinomycetemcomitans D7S (ORF1_AFI87984 and ORF2_AFI87430); 90% of strains have two PptA-like proteins, such as ORF1_TYB21792 and ORF2_TYB20790 from strain HK_907. In A. baumannii, six PptA-like ORFs were detected  with PptA, whereas the identities among Ab_ORF4, Ab_ORF5, and Ab_ORF6 were low (26%).We observed that all organisms with a ChoP-modified protein, including homologs . Previouacterium . Consistdetected , with soN. meningitidis PE transferases, LptA and Lpt3  of LPS with PE. PptA clustered with EptB but did not appear to be closely related to it, implying that they have distinct functional properties. However, the ORF2 of A. actinomycetemcomitans and A. baumannii shared a node with EptB, and Aggr.a_ORF2 clustered together with EptB on the same branch. This indicates that these PptA-like ORFs may not act as ChoP transferases but rather may transfer PE. In contrast, PptA ORF1 of A. actinomycetemcomitans (Aggr.a_ORF1) clustered with N. meningitidisandN. gonorrhoeae PptA on the same node, suggesting that PptA functions for Aggr.a_ORF1 are probable. As shown in A. baumannii, only ORF1 and ORF2 exhibit relatively close proximity to PptA, while the remaining ORFs are grouped with other PE transferases, represented by EptA. Additionally, it is noteworthy that A. baumannii ATCC 17978, which contains ChoP-modified porin D (QNT80540) is annotated as a PE transferase, and deletion of this gene could result in the loss of lipid A modification  and phosphatidylcholine synthase (Pcs). Recently, a further, acylation-dependent PC biosynthesis pathway was proposed as a novel PC biosynthesis pathway (There remains a gap in our knowledge as to what the ChoP donor used by PptA might be and the pathway(s) required for ChoP donor biosynthesis prior to pilin posttranslational modification. In this regard, phosphatidylcholine (PC) could be a potential ChoP donor. The headgroups of phospholipids, such as phosphatidylglycerol and phosphatidylethanolamine, are used for the biosynthesis of phosphoglycerol and phosphoethanolamine , 59. Cipoprotein . PptA isngitidis , 54. It  pathway .Rhodobacter sphaeroides (NCBI:protein accession no. ABA79897) (Sinorhizobium meliloti (AAG10237)  with undefined ChoP-modified structures. As shown in R. sphaeroides PmtA homologs with 30% and 32% identities in the N. meningitidis C311 strain that have ChoP posttranslational modification on pilin (Rhodobacter PmtA ORFs from N. gonorrhoeae shared 32% identities with R. sphaeroides PmtA. Certain A. baumannii stains contain both R. sphaeroides and S. meliloti PmtA ORFs with high sequence identities (30% to 34%)  with R. sphaeroides PmtA (The representative PmtA protein sequences from BA79897)  and SinoAG10237)  were useon pilin . Rhodoba to 34%) . Howeverdes PmtA .Bradyrhizobium japonicum contains four Pmt proteins , and only the R. sphaeroides PmtA-like PmtA and PmtX1 can produce PC via subsequent methylation  has not been confirmed. The N. meningitidis homolog of R. sphaeroides ORF1 (NMB1270) has been studied previously  with S. meliloti Pcs  to lysophosphatidylcholine (lyso-PC) via two acylation reactions on GPC (X. campestris acyltransferases (Xc_0188 and Xc_0238) that perform the second acylation from lyso-PC to PC have been identified  (AAY47325) (WP_000899054) (WP_000004616) , X, XE. col0899054) , and Lpl0004616)  as the qg method .S. pneumoniae and A. actinomycetemcomitans, both of which clustered with Aas of E. coli on the same main clade with a bootstrap value of 100 .Similar to ynthesis . As showPseudomonas aeruginosa . Results were then filtered according to the identity threshold of 20%. The best-hit matches for each organism were retained for downstream analysis.The accession numbers of the ChoP enzyme sequences used as search terms are listed in Amino acid sequences retrieved by BLASTp search in the nr protein database were first aligned with MUSCLE (Codons) available in MEGA software (version X)  using th


C. difficile is the primary cause for nosocomial infective diarrhoea. For a successful infection, C. difficile must navigate between resident gut bacteria and the harsh host environment. The perturbation of the intestinal microbiota by broad-spectrum antibiotics alters the composition and the geography of the gut microbiota, deterring colonization resistance, and enabling C. difficile to colonize. This review will discuss how C. difficile interacts with and exploits the microbiota and the host epithelium to infect and persist. We provide an overview of C. difficile virulence factors and their interactions with the gut to aid adhesion, cause epithelial damage and mediate persistence. Finally, we document the host responses to C. difficile, describing the immune cells and host pathways that are associated and triggered during C. difficile infection. Clostridioides difficile, a Gram-positive, spore-forming, obligate anaerobic bacterium is the aetiological cause for C. difficile infection (CDI). This bacterium is ubiquitous in nature, readily isolated from soil, water, animals and importantly, nosocomial environments [ronments . Accordironments . Elderlyronments . Clinicaronments . The cosronments .C. difficile colonization is usually prevented by the native gut microbiota in healthy individuals and recent studies have revealed a number of mechanisms underlying microbiota-mediated resistance, which have been extensively reviewed elsewhere [C. difficile with the host epithelium and the immune system play a crucial role in the pathogenesis and the outcome of CDI. This review will primarily focus on the pathogen and host factors that control infection at the gut interface.Colonization of the gut by a pathogen is a highly complex process. lsewhere . Along wC. difficile forms highly resistant spores, which can survive under unfavourable environmental conditions. These spores traverse the gastrointestinal (GI) tract through ingestion, resisting physical defence mechanisms like stomach acid and mucus . Spore germination is induced by bile acids, cholate or taurocholate, but inhibited by primary bile acid CDCA. The generation of secondary bile acids like deoxycholic acid (DCA), which is transformed from conjugated primary bile acids produced by the host, are strong inhibitors of spore germination and vegetative cell growth [Clostridium scindens, which results in DCA formation from cholate was associated with protection from CDI [C. difficile was previously thought to be a bystander, it was recently reported to deconjugate taurine-conjugated bile acids to produce cholate [C. scindens was shown to trigger and promote C. difficile biofilm formation [C. difficile interactions with the microbiota are highly complex, warranting further studies to probe the inter-species interactions occurring at the gut interface.Finally, the role of bile acid (BA) in the lifecycle of escribed . Intestil growth . The 7α-from CDI . Althoug cholate . Additioormation . Thus, C. difficile produces a wide range of virulence-associated factors, including many determinants that mediate bacterial attachment to the mucosal surface, that are considered key for colonization  and TcdB (269.6 kDa), belong to a family of large glucosylating toxins. They are considered to be the major virulence determinants of this pathogen and have been reviewed extensively [tcdA) and toxin B (tcdB) are found on the 19.6 kb pathogenicity locus (PaLoc) nestled between three regulatory open reading frames; tcdR, tcdE and tcdC. TcdR (previously known as TcdD and TexR), is a positive regulator that acts as an alternative sigma factor and mediates the recognition of the RNA polymerase core to the toxin promoters [tcdR and/or tcdR-containing RNA polymerase [ensively . Their eensively . The genromoters . TcdC islymerase , althouglymerase . TcdE, alymerase .Clostridium sordellii [et al., as sublethal concentrations of toxin A were able to alter cell polarity of Caco-2 colonocytes, causing an increase in adhesion and penetration of the host mucosal barrier [Both toxins belong in the family of large clostridial toxins (LCTs) alongside TscL of dellii . The quadellii  and are dellii . Endosomdellii  and the dellii . Toxin Adellii . The toxdellii . An indi barrier .C. difficile binary toxin, also known as C. difficile transferase (CDT) is present in approximately 4–45 % of strains isolated, but more common in hypervirulent strains such as ribotype 027 [Cdt locus, CdtLoc, with a CDT regulator, cdtR upstream. Interestingly, strains can exhibit the complete CdtLoc, a 4.2 kb truncated version or be ‘absent’ with a 68 bp region [The type 027 . The twop region . Unlike p region . The CDTp region . Each cop region .C. difficile receptor [Cell intoxication occurs with the proteolytic cleavage of the CDTb 98.8 kDa) activation domain by serine type proteases to form a mature 75 kDa protein . The tox.8 kDa acreceptor  found wireceptor . Thus, CC. difficile, various ribotypes are differently flagellated; ribotype 012 strain CD630 is peritrichously  flagellated, whilst the ‘hypervirulent’ ribotype 027 R20291 possesses a single monotrichous flagellum [Flagella are commonly responsible for bacterial motility, evasion of host defences and colonization of apical surfaces. Within lagellum .C. difficile flagellum has three distinctive components, the membrane bound basal body, hook and a helicoidal filament encoded on three operons termed F1, F2 and F3 [As seen in other bacteria, 2 and F3 . The F3 2 and F3 . F2 is t2 and F3 . Transcr2 and F3 . The fla2 and F3 . SigD is2 and F3 .C. difficile, the role of the flagella has been associated with colonization and virulence in the hamster and murine infection models [sigD, fliC and fliD) in overall bacterial virulence [C. difficile infection, although its precise role, which appears to be strain-dependent, remains elusive.In n models , althougn models . Furthern models . Mutatioirulence . TherefoC. difficile were first described by Borriello et al., over 3 decades ago as fimbriae, describing multipolar protrusions of approximately 4–9 nm diameter and 6 µm long, although no correlation between binding efficiencies of strains with/without these fimbriae were demonstrated [C. difficile genome encodes the type IV pili (T4P) system, consisting of nine different pilin genes, assembly and scaffold proteins [pilA1 and pilB1, which encode for the major pilin and pilus assembly ATPase, respectively [Pili structures in nstrated . The Cproteins . The mosectively . Bacteriectively .C. difficile strain 630 adhering to crypt cells of hamsters [pilA1 or pilB1 in CD630 exhibited a reduction in adherence to Caco-2, HT-29 epithelial cells and MDCK cells. The adherence defects were more pronounced in pilB1 mutants, indicating that the assembly of minor pilins play a functional role in adhesion and persistence [pilA1 mutants in vitro using confocal microscopy [pilA1 was upregulated threefold in R20291 suggesting a T4P-mediated biofilm formation in this strain. Pilus-assisted motility was also higher in R20291 across agar concentrations compared to CD630 [Pilus-like filaments were demonstrated using immunogold labelling with PilA antisera in hamsters . Mutantssistence . Deficiecroscopy . Furtherto CD630 . Interesto CD630  have beeto CD630 .C. difficile has an surface layer (S-layer) that is linked to the peptidoglycan-containing layer and accounts for approximately 15 % of the total protein within this bacterium. C. difficile is unique as this bacterium is coated by a mostly heterodimeric, proteinaceous paracrystalline array [C. difficile S-layer shows a considerable degree of variation between strains and thirteen S-layer cassette types have been identified [ne array , which ientified .slpA. SlpA has three sub-domains: N-terminal secretion signal, LMW SLP and HMW SLP. The secretion signal translocates the peptide across the cell membrane by an accessory Sec system [slpA called cell-wall protein 84 (cwp84) [C. difficile S-layer assembly, based on high-resolution crystal structure and electron microscopy was recently reported, which surprisingly revealed a tightly packed array with narrow pores, possibly allowing the passage of small metabolites. While more flexible and permeable points in this compact structure are indicated, it remains to be understood how large proteins are secreted out through this layer [Strains are divided into two groups based on varying sizes of the glycoprotein: group I have 32 kDa LMW SLP and 42–48 kDa HMW SLP, while group II has 38 kDa LMW SLP and 42 kDa HMW SLP . The HMWc system . Post-tr (cwp84) . LMW SLP (cwp84) . Binding (cwp84) . An atomis layer .C. difficile have been isolated without a S-layer, accompanied with a reported reduction in spore production and survival rate, toxin release and increased susceptibility to lysozymes [The S-layer is thought to have a role in adhesion to cell surfaces and colonization. Utilizing purified recombinant SLPs, SLP was demonstrated to bind via the HMW SLP to collagen I, thrombospondin and vitronectin. High levels of avidity were also observed between LMW SLP to epithelial cell Caco-2 but paradoxically to Hep-2 cells . Specifiysozymes .C. difficile has the relevant gene clusters, there is a lack of evidence for glycosylation [Furthermore, most bacterial S-layers are glycosylated, however, although sylation . There asylation , which msylation . The esssylation .C. difficile, which are part of the S-layer. SlpA and 11 slpA paralogs can be found in the slpA locus and 17 more within the genome [To date, 28 other cell-wall proteins (Cwp) have been identified in e genome . These pe genome . The strcwp84 mutant generated uncleaved SlpA peptides, which would translocate onto the cell surface; these mutants grew slowly and had a propensity for aggregation [Cwp84 mediates the cleavage of SlpA. As isogenic regation . Howeverregation . Additioregation .C. difficile 630 demonstrated decreased adherence to Caco-2 cells and increased toxin A release [cwp22 mutants on the other hand showed reduced toxin production, autolysis, cell growth and adherence to HCT-8 cells. Furthermore, a reduction in virulence and bacterial fitness was observed in a mouse infection model [Cwp66 was one of the first proteins to be associated with cell adhesion, conferring an increased adhesive ability to Vero cells, which was reversed upon treatment with anti-cwp66 C-terminus antibodies . CwpV is release . R20291 on model . Cwp19 ion model . Thus, Cin vivo transcriptomics study of C. difficile 630 murine infection. CbpA displayed an increased expression rate early in infection and was shown to interact with collagen and adhere to extracellular matrix producing cells [et al. demonstrated a potential redundancy in proteins binding to collagen, which may explain its lack of impact on bacterial colonization in vivo. CD2831, another collagen binding protein was also reported to bind to collagen-rich tissues such as human fibroblasts [In vivo infection in monoxenic mouse, fbp68 mutants had a reduced capacity to colonize the caecum, while in dixenic mice the colonization rates were similar, albeit slightly slower [Other surface proteins have also been associated with cell adhesion and virulence. Collagen binding protein A  was identified through an ng cells . Tulli eroblasts . The surroblasts . Howevery slower .A lipoprotein CD0873 was discovered to be involved in adhesion to Caco-2 cells, with a mutant lacking this protein displaying a reduction in attachment and antibodies against CD0873 inhibiting adherence . Using ain vitro and fibronectin produced by human fibroblasts, indicating potential roles of extracellular metalloproteases in infection [Finally, there are other secreted proteins that modulate bacterial adherence. A secreted zinc metalloprotease, proline-proline endopeptidase (PPEP-1), was reported to efficiently cleave CD2831 and CD3246 to mediate better adherence. Furthermore, PPEP-1 is able to cleave fibrinogen nfection .C. difficile surface proteins have been studied, further understanding of their role during infection is necessary, as these may have excellent potential as vaccine candidates that could help prevent and disrupt the process of C. difficile colonization. It is important to note that some of the putative multifunctional adhesins discussed above were characterized only using in vitro assays. The presence of multiple surface proteins in C. difficile makes it difficult to study specific functions during infection, however, this could suggest redundancy or specializations specific to the host environment. Additionally, challenges associated with genetic manipulation of C. difficile have limited targeted structure-function studies of surface proteins.Although functions of C. difficile and other anaerobes from clinical samples were investigated, and were found to be able to produce hyaluronidases, chondroitin sulfatases, gelatinases, collagenases, proteases and heparinases [C. difficile may utilize hyaluronidase to degrade hyaluronate found as a major component of ground substance, a viscous substance secreted by connective tissues, which acts as a natural physical barrier to prevent bacterial penetration. The breakdown of this barrier may allow an easier course of infection for C. difficile. Furthermore, the substrates produced are dissacharrides, which are thought to provide another nutritional source for the bacterium [Clostridia can express and secrete hydrolytic enzymes to increase their range of nutritional sources . Enzymatarinases . C. diacterium . A similacterium .C. difficile can persist in different environments including the human gut through two main mechanisms, formation of spores and biofilms.C. difficile necessitates the need for stress-resistant spores to maintain its lifecycle outside of host environments. Initiation of sporulation is a common path utilized by Bacillus and Clostridioides spp. when exposed to environmental stresses such as nutrient limitations, quorum sensing, oxygen, desiccation and ultraviolet light [The anaerobic nature of et light .C. difficile strain [Bacillus, Spo0A acts as the master regulator for sporulation [spo0A coordinates expression and activation of sporulation sigma factors: σE, σF, σG, and σK [F and σG are active in the forespore, while σE and σK in the mother cell. Briefly, early sigma factors σF and σE are activated during the polar septum formation and mother cell engulfment, respectively. σF proteolytically activates late sporulation σG, but unlike B. subtilis, lacks proteolytic activation of σK by the products of σG. σK becomes active upon synthesis to contribute to the remainder of the sporulation pathway of cortex synthesis, exosporium assembly and release. The sequential order activation is not as completely clear in C. difficile and in general sporulation pathways in C. difficile are distinct to Bacillus [Sporulation frequencies vary between strains and in different laboratory conditions, with CD630 being the most studied  strain . Like Barulation . Transcr, and σK  and a ne, and σK . The spocillus .F [sinRR’ and tcd operon [While Spo0A also regulates biofilm formation, flagella production, expression of cell-wall proteins  and indiF , while Cd operon .C. difficile spores provides potential continual sources of infection, in both the nosocomial and community environments [spo0A mutants of strains 630 and R20291 were able to cause acute disease but unable to persist or transmit infection in a murine infection model [The ubiquitous nature of ronments . The abion model . In a dion model . Notablyon model , though on model . Spores on model , induce on model , adhere on model  A recenton model .C. difficile biofilms may contribute to prolonged and repeated episodes of infection [Biofilm formation is believed to be involved with the progression of colonization, maintaining persistence and resisting environmental stresses, with a positive correlation of the duration of hospital occupancy and biofilm-associated infections with other species . Therefonfection .C. difficile can form either mono- or poly-species biofilms with other gut bacteria [in vitro and in vivo [C. difficile biofilm can harbour dormant spores with reduced germination efficiency [bacteria  both in  in vivo , influen in vivo . Externa in vivo , which i in vivo . The den in vivo . Additioficiency .In vivo C. difficile is primarily associated with the caecum and colon [In vivo studies in murine models suggests that formation of biofilms occurs on the outer layer of the mucosal lining of these epithelial cells and in conjunction with other Bacteriodota and Bacillota [C. difficile were shown to cause recurrent infection in a human gut model [nd colon . In vivoillota . Furtherut model . While tut model .C. difficile infection was also shown to be impacted by stem-cell damage induced by toxin B [Most work at the epithelial interface has been performed with the toxins A, B and CDT. Multiple cellular receptors have been reported to interact with toxin A and toxin B. In a recent CRISPR-Cas screen, sulfated glycosaminoglycans and low-density lipoprotein receptor were identified to impact toxin A endocytosis . Toxin B toxin B .C. difficile hsmRA was shown to capture and utilize haem, thus reducing the inflammatory response it induces, while another haem system HatRM system detoxifies haem through efflux [zupT mutant, suggesting metal homeostasis is key for infection [C. difficile growth by limiting Zn availability, was shown to be an important factor in protection against CDI [A few other bacterial factors have been described to utilize host pathways. Heme transporter system encoded by h efflux . Furtherh efflux . Competinfection . Furtherinst CDI .et al. reported decreased MUC2 production in CDI patients, with no changes in MUC1 secretion [C. difficile to stool mucus from patients with CDI. C. difficile was further shown to chemotax towards MUC2 and utilize mucin-derived oligosaccharides. However, as C. difficile lacks the glycosyl hydrolases required to degrade mucins, it is unable to utilize MUC2 directly, and breakdown of mucins to monosaccharides by other mucin degrading gut commensals was necessary [C. difficile colonization in mice [Finally, the mucus is a key host component that gut pathogens need to interact with. Engevik ecretion . They alecessary . Further in mice .The innate immune response plays a key role in CDI, which is primarily been attributed to increased neutrophilic infiltration at the gut mucosa. Antibody responses against toxins have been well studied although recent work has highlighted epithelial immunity mechanisms in CDI. It is evident now that a plethora of complex interactions between both arms of immunity, the bacterium and the microbiota impact the disease severity and clinical manifestation.The classical symptom of pseudomembranous colitis in severe CDI is associated with epithelial tissue damage and heavy inflammation from neutrophil infiltration . The infet al., showed hypervirulent strains expressing the binary toxin (CDT) was able to suppress host eosinophil response through activation of the Toll-like Receptor 2 (TLR2)-dependent pathway; subsequent adoptive transfer of TLR2-deficient eosinophils was able to restore survival rates of TLR2-/- mice [C. difficile can also elicit the activation of other TLRs. Flagellin is recognized by TLR-5 on epithelial cells, which induces the MAPK and NFκB cell signalling cascade, resulting in an inflammatory response and mucosal injury [The role of eosinophils remains unclear in CDI, however Cowardin -/- mice . C. dil injury . The secl injury . Mice inl injury . Additiol injury .in vitro in THP-1 and J774A.1 macrophages [C. difficile persistence; spores were seen to survive in Raw 264.7 macrophages for up to 72 h and subsequently induce macrophage death [Phagocytic activity in CDI has been understudied, despite the abundance of macrophages in the lamina propria . Toxin erophages . Interesge death .et al., demonstrated that NFil3-/- mice, which lack NK cells and ILC1s [C. difficile spores VPI 10463 [-/- (B- and T-lymphocytes deficient) and Rag-γc-/- mice (ILC and B- and T-lymphocytes deficient) were assessed using a similar infection model, and Rag-γc-/- mice developed more severe CDI compared to the WT and Rag1-/- mice [+/CD127+ ILCs into the Rag-γc-/- mice, resulted in recovery from infection. The study also distinguished the role of IFN-γ (ILC1s) and IL-22 (from ILC3s) in more severe cases of CDI, as Rag1 IL-22-/- mice with IFN-γ neutralized, showed a higher mortality rate [et al., suggesting IL-22 is able to regulate host glycosylation, promoting the growth of Phascolarctobacterium spp. and additional nutritional competition for succinate [Recent studies have focused on another group of innate cells, the innate lymphoid cells (ILCs), which are further sub-categorized into ILC1s, ILC2s and ILC3s based on their differential developmental transcription factors, cytokine expression and effector functions . Geiger nd ILC1s , were moPI 10463 . Rag1-/--/- mice . Furtherity rate . The rolity rate . The assuccinate . Further+ T cells have been attributed to the prevention of recurrent infections [Large numbers of T cells and B memory cells can be found in the lamina propria in CDI . The intfections , althougfections .C. difficile spores [C. difficile, vaccines based on toxins A and B have not yet been successful at phase-III clinicals trials indicating that other bacterial components may be needed for inducing effective protection.Colonic biopsies of CDI patients and immunohistological assays revealed a reduction in B lymphocytes and mucosal macrophages, with larger decreases in recurrent episodes . The cir spores . Sequenc spores . Despite spores . While tC. difficile with the host and gut microbiota are highly intricate. A minor change in one dimension can alter C. difficile responses and the severity of the disease. Although significant advances have been made within the last two decades, there is a need for multifaceted assays to truly understand three-way interactions between these systems. However, such studies are challenging due the variability within systems, such as strain-dependent differences, diverse microbiota compositions and the lack of good model systems. Triangulating and understanding these dynamic processes will hopefully lead to new and better therapeutics for C. difficile infection.The interactions of the anaerobic pathogen


Changes in blood glucose concentrations are underpinned by blood glucose kinetics (endogenous and exogenous glucose appearance rates and glucose disappearance rates). Exercise potently alters blood glucose kinetics and can thereby be used as a tool to control blood glucose concentration. However, most studies of exercise-induced changes in glucose kinetics are conducted in a fasted state, and therefore less is known about the effects of exercise on glucose kinetics when exercise is conducted in a postprandial state. Emerging evidence suggests that food intake prior to exercise can increase postprandial blood glucose flux compared with when meals are consumed after exercise, whereby both glucose appearance rates and disappearance rates are increased. The mechanisms underlying the mediating effect of exercise conducted in the fed versus the fasted state are yet to be fully elucidated. Current evidence demonstrates that exercise in the postprandial state increased glucose appearance rates due to both increased exogenous and endogenous appearance and may be due to changes in splanchnic blood flow, intestinal permeability, and/or hepatic glucose extraction. On the other hand, increased glucose disappearance rates after exercise in the fed state have been shown to be associated with increased intramuscular AMPK signaling via a mismatch between carbohydrate utilization and delivery. Due to differences in blood glucose kinetics and other physiological differences, studies conducted in the fasted state cannot be immediately translated to the fed state. Therefore, conducting studies in the fed state could improve the external validity of data pertaining to glucose kinetics and intramuscular signaling in response to nutrition and exercise. The maintenance of blood glucose concentrations within a physiological range is of primary importance to human health. If blood glucose concentrations are too low (hypoglycemia), then individuals can become comatose; if too high for prolonged periods (hyperglycemia), then micro- and macrovascular damage can occur, increasing morbidity and mortality. Therefore, understanding the physiological mechanisms which underpin blood glucose concentrations can provide evidence to inform rational approaches to promoting and/or maintaining human health.a) and disappearance (glucose Rd) govern blood glucose concentrations over time. Glucose Ra represents the summation of fluxes from endogenous glucose production (EGP) and exogenous glucose entering the circulation from the intestine, whereas Rd represents glucose exiting the circulation and entering tissues. Although many tissues contribute to Rd—and the relative proportions will depend on the circumstances (discussed later)—the liver is the principal source of EGP in most physiological states  secrete, two incretins, glucose-dependent insulinotropic peptide (GIP) and glucagon-like peptide-1 (GLP-1)  , 14 that (GLP-1) . Moreovenditions . Therefoe uptake .a, but also via effects on EGP and glucose Rd due to changes in insulinemia , combined with increased early-phase insulin secretion, reduced hepatic glucose production, and enhanced skeletal muscle glucose disposal  and markedly decreases hepatic glucose production (d (d/glucose concentration) is also higher during exercise following carbohydrate ingestion, relatively higher plasma insulin , oral glucose tolerance should be logically improved following exercise. Nevertheless, there is evidence that one bout of endurance exercise tends to reduce , possibly due to intestinal cell damage  prior to exercise  results in greater splanchnic blood flow during exercise, maintaining greater availability of exogenous glucose to appear in the circulation  before exercise led to a further increase in both glucose Ra and Rd . Plasma culation . The rolculation . In addiculation . Future a following preexercise feeding, more evidence is available on the potential mechanisms contributing to glucose Rd. In the same study demonstrating increased postexercise glucose Rd with preexercise feeding, intramuscular activation of Adenosine 5′-monophosphate-activated protein kinase (AMPK) was also increased, as indicated by increased phosphorylation of AMPKThr172 and Acetyl-CoA carboxylase (ACC)Ser79 Ser79 . AMPK caactivity .It might be expected that a high-carbohydrate, preexercise meal would result in a higher muscle glycogen concentration and therefore decrease, not increase, postexercise AMPK activity. Following ingestion of a carbohydrate-rich meal, however, muscle glycogen concentrations can first decrease, before starting to show a net increase by 2–3 h later postmeal . The mosa (exogenous or/and endogenous) or decreased glucose Rd. In addition, further research is needed to clarify adaptations in glucose flux during exercise after a fed or fasted training program. Postprandial insulin sensitivity could be even deteriorated in an intensity-dependent manner immediately after exercise due to hormonal changes [i.e., catecholamines  conducted in a fasted state can also increase the postprandial insulin sensitivity and glucose disappearance rates, but this can be offset by increases in endogenous and exogenous glucose appearance rates. When exercise is conducted in a fed state, postprandial glucose kinetics after exercise can show a further increase in flux compared with exercise in a fasted state. Increased glucose appearance rates could be due to changes in splanchnic blood flow, intestinal integrity, and/or splanchnic glucose extraction, meanwhile, increased glucose disappearance rates could be explained by increased activation of AMPK, which can stimulate GLUT4 translocation and thus potentiate postexercise skeletal muscle glucose uptake. Therefore, current evidence suggests that food intake before exercise alters blood glucose kinetics after exercise, and evidence from exercise in the fasted state cannot be immediately translated to exercise in the fed state.This work was funded by British Heart Foundation PG/19/43/34432 (to J.T.G.).J.T.G. has received research funding from Biotechnology and Biological Sciences Research Council (BBSRC), Medical Research Council (MRC), British Heart Foundation, Clasado Biosciences, Lucozade Ribena Suntory, ARLA Foods Ingredients and Cosun Nutrition Center. J.T.G. is a scientific advisory board member to ZOE and 6d Sports Nutrition and has completed paid consultancy for The Dairy Council, PepsiCo, Violicom Medical, Tour Racing Ltd., and SVGC.A.M.-C. and J.T.G. conceived and designed research; A.M.-C. and J.T.G. performed experiments; A.M.-C. and J.T.G. analyzed data; A.M.-C. and J.T.G. interpreted results of experiments; A.M.-C. and J.T.G. prepared figures; A.M.-C. and J.T.G. drafted manuscript; A.M.-C. and J.T.G. edited and revised manuscript; A.M.-C. and J.T.G. approved final version of manuscript.


P. ovale spp. infections are endemic across multiple African countries and are caused by two distinct non-recombining species, P. ovale curtisi (Poc) and P. ovale wallikeri (Pow). These species are thought to differ in clinical symptomatology and latency, but existing diagnostic assays have limited ability to detect and distinguish them. In this study, we developed a new duplex assay for the detection and differentiation of Poc and Pow that can be used to improve our understanding of these parasites.Poc and Pow genomes and used for assay development and validation. We evaluated the analytical sensitivity and specificity of the best-performing assay using a panel of samples from Tanzania and the Democratic Republic of the Congo (DRC), then validated its performance using 55 P. ovale spp. samples and 40 non-ovale Plasmodium samples from the DRC. Poc and Pow prevalence among symptomatic individuals sampled across three provinces of the DRC were estimated.Repetitive sequence motifs were identified in available Poc and Pow targets had 9 and 8 copies within the reference genomes, respectively. Our duplex assay had 100% specificity and 95% confidence lower limits of detection of 4.2 and 41.2 parasite genome equivalents/μl for Poc and Pow, respectively. Species was determined in 80% of all P. ovale spp.-positive field samples and 100% of those with >10 parasites/μl. Most P. ovale spp. field samples from the DRC were found to be Poc infections.The best-performing Poc and Pow and used them to develop a new duplex real-time PCR assay that performed well when applied to diverse field samples. Though low-density Pow infections are not reliably detected, the assay is highly specific and can be used for high-throughput studies of P. ovale spp. epidemiology among symptomatic cases in malaria-endemic countries like the DRC.We identified promising multi-copy targets for molecular detection and differentiation of  P. falciparum transmission is declining. Plasmodium ovale curtisi (Poc) and wallikeri (Pow) are neglected parasites that can cause relapsing malaria and are thought to differ in clinical symptomatology and latency. However, existing diagnostic assays have limited ability to detect and distinguish Poc and Pow and are not well-suited for high-throughput use, hindering our understanding of P. ovale spp. epidemiology. Mining recently available Poc and Pow reference genomes, we identify new multi-copy targets for molecular detection and develop a novel duplex qualitative real-time PCR assay capable of species differentiation. The assay is highly specific and requires short turn-around time. While sensitivity can be improved for low-density Pow infections, this new assay can be used for high-throughput studies of symptomatic P. ovale spp. infections in malaria-endemic countries. We apply this tool to samples collected during a large study conducted in the DRC and investigate P. ovale spp. epidemiology across health centers in three provinces.Non-falciparum malaria is gaining attention, especially in settings where  Plasmodium falciparum, the parasite species responsible for most deaths, increasing evidence confirms co-circulation of other neglected Plasmodium species that cause human malaria [Plasmodium ovale spp. in multiple African countries [P. ovale spp. may prove to be an obstacle to malaria elimination efforts [P. ovale comprises two distinct non-recombining species, P. ovale curtisi (Poc) and P. ovale wallikeri (Pow) [Poc and Pow have potential differences in clinical symptomatology and latency [Malaria remains a major global health concern despite decades of sustained investment in elimination efforts. Though most malaria programs prioritize  malaria . Recent ountries , 3, wher efforts –6. P. ovri (Pow) . Poc and latency , but exi latency .Poc and Pow is not currently possible using conventional malaria diagnostic assays relying on microscopy. Microscopic examination of blood smears remains the gold standard for malaria diagnosis, but differentiation of parasite species and examination of low parasite density or mixed-species infections is challenging and requires significant expertise [Poc and Pow infections often occur as mixed infections at low density, and are morphologically indistinguishable on blood slides [P. falciparum and Plasmodium vivax [Detection and differentiation of xpertise . Poc andd slides , 12. Furum vivax , 14. ThuPlasmodium detection than microscopic examination or RDTs, but most existing assays target the 18S rRNA gene of both P. ovale spp. leading to potential cross-reactivity and a lack of complete species specificity for Poc and Pow. A duplex real-time PCR assay for Poc and Pow detection was published in 2011, however, results of the melt-curve analysis can be hard to interpret [Poc and Pow [P. ovale spp. have not distinguished Poc and Pow. However, recently released Poc and Pow genomes (PocGH01 and PowCR01) provide opportunities for improved molecular assay development [P. ovale spp. malaria, we mined publicly available Poc and Pow genomes to identify novel multi-copy targets and developed a new duplex qualitative real-time PCR assay for detection and differentiation of Poc and Pow infection.Molecular methods are more sensitive and specific for nterpret . A nestenterpret , and whi and Pow , 17, 18.elopment . To imprPoc (PocGH01) and Pow (PowCR01) reference genomes obtained from the NIH National Center for Biotechnology Information (NCBI) database, we identified sequence motifs of 100 base-pairs (bp) in length with ≥6 copies using Jellyfish (version 2.2.10) [blastn to investigate their specificity. Sequences aligned to other Plasmodium parasites were excluded. We then re-aligned the remaining targets to the Poc and Pow genomes separately using blastn to investigate their copy numbers in each genome. Candidate diagnostic assay targets for Poc and Pow were selected based on species-specificity and copy numbers. Primer and probe sets were designed manually using Oligo Calc [Using the publicly available  2.2.10)  Fig 1)..Poc  samples from Tanzania and the Democratic Republic of the Congo (DRC). Laboratory controls included two P. falciparum, one P. malariae, two P. vivax, and one P. knowlesi samples from an external quality assurance program [P. ovale parasite densities were estimated using a semi-quantitative real-time PCR assay targeting the 18S rRNA gene of both P. ovale spp. as previously described [Poc versus Pow species was determined using two singleplex real-time PCR assays as previously described [A panel of 15 well-characterized  program . DNA was program  and the escribed . Poc verescribed .Poc and Pow versus this panel of samples were selected. A duplex qualitative real-time PCR assay was developed based on the selected primer sets and the corresponding probes. Assay optimization was then performed using synthetic plasmids containing targets for Poc and Pow detection . We tested a range of annealing temperatures and of primer and probe concentrations to identify optimal reaction conditions. All optimization analyses were performed in duplicate.Primer sets with the best specificity for Poc and Pow plasmid DNA comprising 104 and 161 total replicates, respectively (S2 Table). Analytical specificity of the present assay was assessed using the same panel of 15 well-characterized Poc and Pow field samples and six non-ovale Plasmodium laboratory controls described above in duplicate. Samples with Ct values less than 45 in both reactions were considered positive.We further characterized the best performing assay to determine its analytical sensitivity and specificity. Analytical sensitivity estimates were determined using probit analysis  with serP. ovale spp. samples and 40 non-ovale Plasmodium samples  [Plasmodium species and parasite densities were identified using real-time PCR assays for P. ovale, P. falciparum and P. malariae as previously described, with samples positive in duplicate selected for use during validation of the present assay, and the previously published singleplex real-time PCR assay for both P. ovale spp. used as the gold standard for clinical sensitivity and specificity calculations [The assay’s clinical sensitivity and specificity were assessed using 95 dried blood spot samples selected from a large sample set from a previous study conducted in the DRC , includiections) . DNA wasulations , 27. SamP. ovale spp. infection and the distribution of Poc vs Pow infections using samples from a study of symptomatic malaria across three provinces in the DRC. Among a randomly selected group of 1,000 symptomatic individuals, 64 samples were previously found be positive for P. ovale spp. by real-time PCR testing [2 test or Fisher’s exact test was used for comparison with categorical variables that might be associated with P. ovale spp.-infections based on results of previously published studies [P. falciparum co-infection, bed net use, education level, recurrent malaria infection (any prior Plasmodium species infections within 6 months), and rural residence. Though no significant association between pregnancy and malaria was reported in previous studies, pregnancy was included in the present analysis.We investigated the epidemiology of  testing , 25. The studies –33, inclP. ovale infections. Because DNA degradation is possible during long-term storage, we repeated the previously published singleplex P. ovale spp. real-time PCR assay on the 64 samples previously identified as P. ovale spp.-positive. Among these, 44 tested positive in duplicate and were selected for species determination using the present duplex assay. To determine the proportions of Poc and Pow within these P. ovale spp.-positive samples, inverse probability weighting (IPW) was used to account for differences between selected samples and the original 64 P. ovale spp.-positive samples. Selection weights were calculated by multiple logistic regression analysis with the following covariates: gender, age, area, and P. ovale spp. parasitemia.We calculated crude odds ratios (cORs) and their 95% confidence intervals (CI) to evaluate associations between each of these eight factors and ggplot2 (version 3.4.1) and forestplot (version 3.1.1) packages. Mapping was done via pixelmap [Statistical analysis was performed using R software  in RStudio (version 2022.02.2). Figures were generated using the pixelmap .Existing samples from previous studies were chosen based on convenience. DRC samples were collected as part of a 2017 study investigating malaria diagnostic test performance in three provinces, Kinshasa, Bas-Uele, and Sud-Kivu. TanzaniPoc and Pow reference genomes, respectively. Targets with low GC content, highly repetitive short sequences, or aligned to other Plasmodium parasite genomes were excluded. A total of three potential assay targets with ⩾8 copies in each of the Poc and Pow genomes were selected. Focusing on these potential targets, we designed five and three primer and probe sets for Poc and Pow, respectively (S1 Table). After testing all primer and probe sets using a panel of 15 well-characterized Poc and Pow field samples and six laboratory non-ovale Plasmodium controls, we selected two primer and probe sets with the best specificity for Poc and Pow, respectively, for additional laboratory testing (Poc target had nine copies within putative liver stage antigen 3 (lsa3) gene on chromosome 4 , while the Pow target had eight copies in a non-coding region on chromosome 14 . Short distances (< 50 bp) were noted between the repetitive Poc target motifs and between Pow target motifs.A total of 2,585 and 3,978 sequences of 100 bp in length with ⩾ 6 repeats were found in the  testing . The selPoc and Pow primers and probes, we optimized a duplex, qualitative real-time PCR assay for the detection and differentiation for Poc and Pow. The final, optimized duplex assay was performed in a small final volume of 10μl, including 7μl of reaction master-mix containing 2x FastStart Universal Probe Master (Rox) , primers and probes ; and 3μl of DNA template. Optimal thermocycling conditions were two min at 50°C, 10 min at 95°C, followed by 45 cycles of 15 s at 95°C and 60 s at 58°C, allowing for detection of parasite DNA in less than two hours.Combining these Poc and Pow were 4.2 and 41.2 parasite genome equivalents/μl, respectively  had parasite densities <10 parasites/μl. The assay’s overall sensitivity was 80%, successfully determining P. ovale species in 44 of the P. ovale spp.-positive field samples , 2.8% (10/353), and 2.2% (7/319), respectively. Among these 1,000 participants, urban residence  and bed net use  had significant protective associations with P. ovale spp. infection; while recurrent malaria infection (within 6 months)  and coinfection with P. falciparum  was associated with P. ovale spp. infection  samples using the present assay: 28 (75.7%), 6 (16.2%), and 3 (8.1%) were Poc infections, Pow infections, and Poc-Pow mixed infections, respectively. Inverse probability weighting analysis indicated that 89.8% of all P. ovale spp. infections in our cohort included Poc species, alone or as a Poc-Pow mixed infection, and 15.2% included Pow species, alone or as a Poc-Pow mixed infection.Among the subsample of 44 Poc and Pow to develop a highly specific duplex real-time PCR assay that can be used to improve our understanding of their epidemiology in malaria-endemic countries. Recent studies have revealed a previously unappreciated burden of P. ovale spp. in Africa [Poc and Pow are distinct non-recombining species, the few existing assays capable of distinguishing them are not well-suited to large studies, requiring separate assays for each species and higher volumes of DNA, with potential for cross-reactivity at higher parasite densities. Because current assays require multiple steps, long turnaround time, or lack complete species specificity [Poc and Pow, and their prevalence and clinical features remain understudied [We mined recently published genomes of n Africa , 36, 37.cificity –18, mostrstudied –40. Our lsa3 gene on Poc chromosome 4 and a non-coding region on Pow chromosome 14, respectively. Studies of P. falciparum lsa3 indicate that it is a mutable non-essential gene expressed during the pre-erythrocytic stages of infection that encodes an antigen with tetrapeptide repeats of unclear function [P. falciparum lsa3 was shown to be largely conserved across isolates collected from geographically diverse sites [Pow repetitive motif we targeted has unclear function, with no obvious orthologues identified in publicly available databases. Though non-essential genes are more likely to be lost over time based on in vitro P. falciparum studies, genes with putative roles in antigenic variation such as lsa3 might be important in human infection [Poc and Pow targets to develop an assay with several advantageous performance characteristics.Our assay targets are distinct from those used in prior assays and take advantage of 100 bp repetitive motifs in the putative function , 42. P. nfection –44. We lPoc and Pow 18S rRNA genes [Poc and Pow targets enabled development of a highly specific assay. These targets’ copy numbers are higher than those reported for 18S rRNA genes in Plasmodium genomes [Poc and Pow detection, respectively [Poc targets and between Pow targets decrease the PCR efficiency, offsetting improved sensitivity that might otherwise be achieved from their increased copy number. Validation using field samples from the DRC confirmed robust species differentiation when the assay was applied to P. ovale spp. samples with >10 parasites/μL and 100% specificity across all parasite densities. Though its ability to identify Pow in particular was limited at lower parasite densities, the simultaneous amplification of Poc and Pow DNA in a single reaction tube allows our assay to have shorter turnaround time and require less materials compared to published singleplex assays [P. ovale infection.Compared to published real-time PCR assays that usually target NA genes , 17, 18, genomes , 46. Howx assays . The dupP. ovale infections in symptomatic individuals from the published study was 6.4% [P. ovale infections were found in nationally representative, asymptomatic adults (0.5%) and children (4.7%) in DRC [P. ovale spp. infections in symptomatic individuals had significant association with rural residence and co-infection with P. falciparum, which is consistent with previous studies [P. ovale malaria in our study, indicating that it is an effective strategy to reduce P. ovale-related malaria burden. People who reported malaria infection within six months before enrollment showed higher odds of P. ovale-related malaria infection; this finding could be attributed to re-infection, inadequate treatment of previous malaria infections, or P. ovale relapse [P. ovale spp. vary across different countries [Poc and Pow across three provinces in the DRC, with Poc more prevalent in symptomatic individuals. A previous, nationally representative study in asymptomatic and symptomatic school-age children in DRC also showed high prevalence of Poc [The prevalence of was 6.4% , while l) in DRC . P. oval studies , 37, 47. studies , 49, bed relapse . Existinountries . Using tPow detection, limits its utility in low-density or asymptomatic infections. Thus, the prevalence of Poc and particularly Pow in our study are likely underestimated. This limitation could be overcome in the future by combining a pan-P. ovale spp. 18S rRNA assay  and deductive identification of Pow mono-infection . Second, our Poc and Pow prevalence estimates are derived from people presenting with malaria symptoms to health facilities in three DRC provinces. The DRC is a large and diverse country. Our results provide further insight into the epidemiology of Poc and Pow in three large regions but do not provide precise, nationally representative estimates, nor information about asymptomatic infections. Third, crude associations were analyzed in this study to explore possible factors related to P. ovale; no causal relationships were assessed. Fourth, the assay targets two non-essential genomic regions at risk of deletion or disruption if future treatment choices are tied to diagnosis, as has been proposed for P. falciparum and observed for Chlamydia trachomatis non-essential diagnostic targets [P. falciparum and do not routinely offer radical cure to clear P. ovale spp. hypnozoites.Several limitations of our approach should be highlighted. First, the assay’s relatively low sensitivity at lower parasite densities, particularly for l et al. ) with ou targets , 53. HowPoc and Pow. Though our assay’s sensitivity at lower parasite densities could be further improved, its streamlined work-flow reduces complexity and is well suited for high-throughput use in field studies. As some countries progress toward malaria elimination, improved assays for Poc and Pow like that presented here will become more important and open the way to improved understanding of P. ovale spp. epidemiology, clinical impact, to inform eradication strategies.In conclusion, we developed and validated a novel, highly specific duplex real-time PCR assay capable of detection and differentiation of