Gallstone disease (GSD) is thought to be associated with the risk of coronary heart disease (CHD) or acute myocardial infarction (AMI), which may be due to abnormal cholesterol metabolism. We used multiple Mendelian randomization (MR) methods based on publicly available genome-wide association study data to assess whether this association is genetically causal and to search for loci driving causality. Pooled data for GSD were obtained from FinnGen Biobank and Biobank Japan, while CHD and AMI were obtained as pooled data from the CARDIoGRAMplusC4D consortium. In this MR study, we found a significant negative causal effect of genetic susceptibility to GSD on AMI in the Finnish population, but no causal effect was found on CHD. This causal effect was not confounded by reverse causality and the same findings were obtained in the Japanese population. Furthermore, the negative causal effect of GSD on AMI risk may be driven by the rs4245791-regulated ABCG5/8 protein. In conclusion, the results of this MR study support a negative causal effect of GSD on AMI and suggest that rs4245791 is the causal driver locus of this effect, which provides new ideas and evidence for the prevention and etiologic study of AMI in patients with GSD. Most patients with GSD are initially asymptomatic; as the disease progresses, GSD can lead to biliary pancreatitis, acute cholecystitis and acute cholangitis and may even be fatal3. Three types of gallstones are typically recognized, cholesterol stones, pigment stones and mixed stones, which are present in 90% of patients with GSD4. The etiology of GSD is diverse, and the formation of cholesterol gallstones is mainly due to the following causes: supersaturation of cholesterol in the bile of the gallbladder (excessive cholesterol secretion by the liver); impaired gallbladder motility; increased absorption of cholesterol from the small intestine to the liver, the intestinal microbiota and genetic factors6. These factors can accelerate production or growth of cholesterol crystals and eventually lead to stone formation.Gallstone disease (GSD) is one of the most common diseases of the digestive system, with high costs. Approximately 10–20% of adults worldwide have gallstones; as a result, more patients with GSD are hospitalized than people with any other digestive disease8. Acute myocardial infarction (AMI) is essentially a type of CHD that results from irreversible necrosis of cardiac myocytes due to acute ischemia. Each year, approximately 1.7 million Americans are diagnosed with myocardial infarction, and more than 8 million are hospitalized due to signs and symptoms of AMI9. These findings undoubtedly emphasize the need to clarify the possible etiology of CHD and AMI. Altered cholesterol metabolism leads to atherosclerosis, and some studies have shown that residual cholesterol is an independent predictor of CHD or AMI11. Because cholesterol is a major component of most gallstones, there may be a link between GSD and CHD through cholesterol metabolism13. Indeed, there is evidence of a positive association between GSD and risk of CHD or AMI. However, because the mechanism of the association remains unclear and the number of available studies is small, debate regarding whether such an association exists continues14. Additionally, if GSD has a direct effect on CHD or AMI risk, the potential causal relationship and direction of the two diseases is uncertain owing to possible bias caused by confounding factors in previous experiments13.Coronary artery disease (CHD) is the leading cause of death globally, and its prevalence has increased in recent decades. CHD also imposes a heavy economic burden of $160 billion annually worldwide15. Compared to RCT, MR has the advantages of time savings, low cost, analysis of a large amount of data and no ethical restrictions16. MR is a novel epidemiological approach that uses genetic variation in genome-wide association studies (GWASs) as an instrumental variable (IV) to proxy exposure factors and to infer causal relationships between exposures and outcomes. As genetic variation arises randomly during meiosis, MR can minimize the bias of results due to reverse causality and confounding factors without interference of reverse factors.To better address this issue, the aim of this study was to further explore the causal effect of GSD on CHD or AMI risk using multiple Mendelian randomization (MR) methods. Although the randomized controlled trial (RCT) design is the gold standard for determining causality, RCT studies have not been widely implemented in the clinical setting due to resource-intensive and severe ethical constraints. MR studies, which can address the shortcomings of traditional epidemiological studies in determining etiology, have become one of the best epidemiological tools to compensate for RCT studies; they have been widely applied in recent years to assess causality and are considered a "natural" RCT studyhttps://gwas.mrcieu.ac.uk/). The GWAS contains thousands of sequences of human genetic variants and the effect sizes of corresponding genetic variants, which can be used to identify risk factors for disease etiology in the population. In addition, all aggregated GWAS statistics used in this study are publicly available, ethical approval was obtained in the original study, and written informed consent was provided by all participants. The analysis design strictly followed the STROBE-MR statement18, the complete STROBE-MR checklist can be found in the Supplementary Table In our study, MR analysis was used to explore a potential causal effect between a patient's genetic susceptibility to the exposure factor GSD and CHD or AMI risk outcomes. This analysis was performed on the basis of summary-level data from a GWAS of European, Finnish, and Japanese populations, for which relevant data are available from the IEU OpenGWAS project (https://www.finngen.fi/en/access_results (r8.finngen.fi))19. FinnGen is a large public–private partnership that aims to collect and analyze genomic and health data for 500,000 Finnish participants, and now FinnGen is regarded as a world-class database of advances in medicine and therapeutics. The GWAS dataset for GSD in the external validation was obtained from Biobank Japan (https://biobankjp.org/en/), with a total of 487,553 samples of East Asian ancestry 20. Diagnosis of GSD was according to International Classification of Diseases .We obtained pooled data for the GSD GWAS from FinnGen Biobank with 32,894 cases and 301,383 controls of European ancestry . As mentioned in published guidelines, diagnostic criteria for AMI include the following: (1) persistent symptoms of chest pain; (2) persistent progression of ischemic signs on the electrocardiogram; and (3) increased levels of infarct-related biomarkers. Summary-level data on risk factors for AMI were obtained from the IEU OpenGWAS database. We selected several recognized risk factors for AMI, including low-density lipoprotein cholesterol (LDL-C), smoking, and hypertension22.Summarized data on CHD GWAS were contributed by the CARDIoGRAMplusC4D and UK Biobank CardioMetabolic Consortium CHD working group, which used the UK Biobank Resource (application number 9922). The study involved 184,305 participants  and evaluated 9.4 million variants. AMI data, also from CARDIoGRAMplusC4D, contain 43,676 AMI cases and 128,199 control cases. Source GWAS data for CARDIoGRAMplusC4D are based on a meta-analysis of a predominantly European GWAS16  IVs must be significantly correlated with exposure factors and not directly correlated with outcomes, (2) IVs must be independent of common confounders, and (3) IVs can only influence outcomes through exposure IVs must–6) and removed those associated with CHD or AMI (p < 5 × 10–5) from the corresponding GWAS database as IVs. Then, ensuring that all IVs satisfied the independence assumption, we used linkage disequilibrium to exclude ineligible IVs . The threshold for r2 was determined by 1000 Genomes Project for populations of European ancestry. During the analysis, we removed all palindromic single-nucleotide polymorphisms (SNPs), duplicate SNPs and SNPs with missing information. To ensure that IVs are not associated with the remaining confounders of exposure and outcomes, we used the PhenoScanner website (http://www.phenoscanner.medschl.cam.ac.uk/) to find and remove SNPs corresponding to factors known to be associated with GSD, CHD or AMI . In addition, we used the F statistic to infer the presence of weak (F-statistic < 10) IVs: F = R2*(N-2)/1-R223, where R2 represents the variance of the IV corresponding to the exposure factor and N is the sample size (R2 = 2*MAF*(1-MAF)*β2). After calculation, the F values of all IVs were much higher than 10, which correlates strongly with exposure and guarantees the validity of SNPs24.First, we extracted variants significantly associated with GSD , Mendelian randomization-Egger (MR‒Egger), weighted median, and maximum likelihood27. Funnel plots allow researchers to judge horizontal pleiotropy by visual effects, and the symmetry of the plots indicates a low probability of the presence of pleiotropy28. The degree of heterogeneity was assessed by calculating the Cochran-Q statistic. Furthermore, as a correction treatment for the heterogeneity of IVs, we used MR Pleiotropy RESidual Sum and Outlier (MR-PRESSO) to identify and correct for outliers in IVW linear regression and conducted sensitivity analysis to assess the reliability of the results29. The results of MR-PRESSO Raw can also provide complementary analysis for causality in addition to the four traditional MR analysis methods. We detected possible outliers among the selected SNPs in a multiple outlier test, and after removing the outliers using "MR-PRESSO outlier test". Furthermore, we tested whether the GSD risk estimates for CHD or AMI remained essentially the same for each SNP removed, which is called the "leave-one-out" analysis. Finally, on the basis of the traditional two-sample MR analysis, we also performed reverse MR analysis, external validation MR analysis and two-step MR analysis to make the results more comprehensive and reliable.Although MR is a powerful technique for causal inference, several issues, including horizontal pleiotropy, epigenetic effects, and disequilibrium, may interfere with the three main assumptions of IV30.After MR analyses identified negative causality of GSD on AMI risk, we used co-localization analyses to explore loci mediating causality. Co-localization analysis is a method for estimating the posterior probability of assessing whether the same locus of genetic variation leads to a causal linkage between two phenotype or simply different causal variants that are in linkage disequilibrium with each other. The PPH4 in the posterior probability corresponds to the hypothesis that phenotype 1 (GSD) and phenotype 2 (AMI) are significantly associated with SNP loci in the genomic region and are driven by the same causally variable locus. In this study, we hypothesized that co-localization significance exists when PP.H4 > 0.80, and the greater the PP.H4 the greater the probability that the negative causality of GSD on the risk of AMI is driven by a common variant locus. LocusCompare plots were used to demonstrate the results of the co-localization analysis28 and the "MR-PRESSO" package. The coloc R package was used to perform co-localization analysis between GSD and AMI risk31 (R version 4.2.2). As two rounds of analysis were performed on the dataset, statistical significance was set at P < 0.05/2 = 0.025 for this study based on Bonferroni correction.All data analyses for MR were performed in the R environment with the "TwoSampleMR" packageAll data used in this study were obtained from publicly available databases; further ethical approval was not required.We assessed whether there was a causal effect of GSD on AMI risk using four MR methods, which ultimately included 64 SNPs after removing confounders and outliers  and the smallest sum of p-values for the correlation between the two phenotypes  is a mitogenic factor that promotes tissue repair and cell proliferation, and there is evidence that IGF-1 serum levels may be associated with susceptibility to gallstones40. In addition, IGF-1 deficiency is an important factor in development of atherosclerosis, and IGF-1 levels may be negatively associated with cholesterol deposition and plaque formation41. However, at present, this association is not sufficient to prove that IGF-1 is a causal influence of GSD on risk of CHD or AMI42. Similarly, metabolites of the gut microbiota and inflammation have been suggested to link GSD to CHD or AMI risk, but whether they are causal influences remains to be demonstrated by intrinsic mechanisms45. In our study, in order to investigate whether the causal relationship between GSD and AMI is caused by traditional cardiovascular risk factors, we performed two-step MR analysis with LDL-C, smoke, and hypertension as mediators. However, the results suggest that the causal relationship is not independently mediated by the above factors, and the specific causal drivers may be otherwise.The positive correlation exhibited by GSD with CHD or AMI risk in most studies may be due to a large number of common risk factors. For example, obesity, diabetes mellitus and hypercholesterolemia, which can lead to crystallization of supersaturated cholesterol in bile to form gallstones, increase cholesterol deposition in the lining and accelerate the AS process, respectively, do not show an intrinsic causal influence46. The D19H polymorphism in ABCG8 is by far the most prominent genetic determinant of GSD, GWAS data also showed that mutations in some loci of ABCG5/8 were strongly associated with GSD47. The link between GSD and cardiovascular disease is thought to arise through ABCG5/8, a transporter protein that promotes the secretion of cholesterol and phytosterols into the bile48. In animal studies, researchers found that high expression of ABCG5/8 attenuated hypercholesterolemia in Ldlr−/− mice, which in turn significantly ameliorated atherosclerotic lesions in the aorta49. In humans, gain-of-function due to variants in the ABCG5/8 gene has been shown to increase the amount of cholesterol discharged from hepatocytes to the gallbladder, thereby directly increasing the lithogenicity of bile and indirectly decreasing LDL-C concentrations and the risk of AMI50. The above studies provide strong evidence to support our conclusion that the negative causality of GSD on AMI risk is mediated by ABCG5/8 gain of function, which makes the results more reliable and valuable. miRNA-223, on the other hand, has been found by researchers to directly target the inhibition of ABCG5/8 to reduce the occurrence of GSD, but whether it will inversely lead to an increased risk of AMI is not conclusive51. In addition, animal studies have shown that deletion of the ABCG5/8 gene does not eliminate hepatic cholesterol secretion to the gallbladder and that other ABCG5/8 nondependent pathways involved in hepatic cholesterol secretion exist53. This implies that enhanced ABCG5/8 expression in GSD patients reduces serum cholesterol levels, but only to a limited extent. In general, AMI patients have higher cholesterol levels than CHD patients, and some cholesterol-lowering effect of ABCG5/8 may be sufficient to significantly reduce risk of AMI, an acute CHD type disease, but not sufficient to cause a significant change in overall CHD risk. This explains the significant negative causal effect of GSD on AMI risk found in our study, though this negative effect was present but not significant for CHD risk. In conclusion, ABCG5/8 can still be regarded as a potential drug target for lipid-lowering therapy, and its gain-of-function regulatory mechanisms and ameliorative effects on AMI risk will continue to be deeply investigated in future studies.A number of potential mechanisms may provide an explanation for our conclusions. In the results of co-localization analysis we found that rs4245791 is a key locus mediating the causal relationship between GSD and AMI, and it has been shown that rs4245791 is strongly correlated with ABCG8The strengths of our study are as follows. First, our study is the first MR study to analyze the causal effect of GSD on CHD or AMI. We included GWAS datasets from multiple populations in our study and also performed external validation as well as reverse MR analysis. This makes our findings more comprehensive and reliable and can be applied to a wider range of populations. Second, MR‒Egger regression results showed no cross-sectional polymorphisms in the IVs; after removing possible heterozygous loci, both the leave-one-out method and the funnel plot showed our conclusions to be robust and plausible. Finally, co-localization analysis was used by us to explore the specific sites mediating causality, which allowed for more in-depth studies. The results provide support for the causal relationship observed by MR analysis at the mechanistic level and provide precise targets for researchers to modulate this relationship.However, there are also limitations that cannot be ignored. Initially, the study identified a key role for rs4245791 in reducing the risk of AMI in GSD patients, but we did not further explore its potential as a therapeutic target nor did animal experiments validate the conclusions. In addition, although the database used in this study included two major populations of Finnish and East Asian ancestry, this is still not representative of the rest of the global population, and the applicability of the findings to ethnic groups other than those of Finnish and Japanese origin needs to be further explored. Ultimately, sex and age factors may also influence the causal relationship between exposure and outcome. Further analysis of sex-age stratified cohorts could be conducted, as data allow, to explore the impact.Overall, the results of this MR study found a protective causal effect of GSD on AMI, suggesting that patients with GSD may have a lower risk of AMI but no benefit on the risk of CHD. Co-localization analyses further identified a key role for rs4245791 and its regulated ABCG5/8 proteins in driving the causal effect of GSD on AMI, providing strong evidence to support the conclusions. We believe that the present study makes a valuable contribution to the field by providing a novel perspective and deeper explanation of the correlation between GSD and AMI. More prospective studies as well as basic experiments are needed in future studies to continue to explore the mechanism of association between GSD and AMI and the possible clinical benefits of targeting ABCG5/8.Supplementary Figures.Supplementary Table S1.Supplementary Tables.


Chlamydia trachomatis is one of the most common sexually infections that cause infertility, and its genital infection induces tubal adhesion and hydrosalpinx. Intravaginal Chlamydia muridarum infection in mice can induce hydrosalpinx in the upper genital tract and it has been used for studying C. trachomatis pathogenicity. DBA2/J strain mice were known to be resistant to the chlamydial induction of hydrosalpinx. In this study, we took advantage of this feature of DBA2/J mice to evaluate the role of antibiotic induced dysbiosis in chlamydial pathogenicity. Antibiotics (vancomycin and gentamicin) were orally administrated to induce dysbiosis in the gut of DBA2/J mice. The mice with or without antibiotic treatment were evaluated for gut and genital dysbiosis and then intravaginally challenged by C. muridarum. Chlamydial burden was tested and genital pathologies were evaluated. We found that oral antibiotics significantly enhanced chlamydial induction of genital hydrosalpinx. And the antibiotic treatment induced severe dysbiosis in the GI tract, including significantly reduced fecal DNA and increased ratios of firmicutes over bacteroidetes. The oral antibiotic did not alter chlamydial infection or microbiota in the mouse genital tracts. Our study showed that the oral antibiotics-enhanced hydrosalpinx correlated with dysbiosis in gut, providing the evidence for associating gut microbiome with chlamydial genital pathogenicity. Chlamydia trachomatis can cause long-lasting genital fibrosis and may result in tubal infertility  was used in this study. The Chlamydia muridarum organisms were grown in HeLa cells and purified as elementary bodies as previously described  were purchased from Vital River, Beijing. Mice were bred and maintained under specific pathogen free (SPF) conditions in the institutional animal facility of the Institute of Pasteur of Shanghai Chinese Academy of Sciences. Mice were treated with or without a cocktail of vancomycin  and gentamicin  C. muridarum organisms the same as the swabs and as previously described  was gift from Jingyue Ma in Tianjin Medical University General Hospital. After primary antibody incubation, the secondary antibody  was added and the nuclei was visualized by DNA dye Hoechst 33258 . The doubly labeled samples were used for counting for chlamydial inclusions under a fluorescence microscope  with a CCD camera (Hamamatsu).The primary antibody: A rabbit antibody , hydrosalpinx detectable only under microscope (1 score), hydrosalpinx visible with naked eyes but the size was smaller than the ovary on the same side (2 score), equal to the ovary on the same side (3 score) or larger than the ovary on the same side (4 score). Scores from both sides of the oviduct from the same mouse were added up to represent the score for a given mouse. The individual mouse scores were calculated into means ± standard errors.Fecal samples were collected from female DBA2/J mice both before and after the oral ABX treatment. The fecal samples were frozen in liquid nitrogen and stored at -80°C until DNA extraction. The fecal genomic DNA was extracted using the QIAamp DNA Stool Mini kit  according to the manufacturers’ instructions. The purity and quantity of DNA samples were determined by absorbance ratios at 260/280. A real time/quantitative PCR  was used to quantitate bacterial 16S rRNA genes in each sample. The following PCR condition was used: 95°C x 5 min for denaturation, then 95°C x 5 s, 60°C x 30s to 90s for 45 cycles as described previously . The priThe number of live organisms expressed as IFUs, Log10 IFUs, 16S rRNA gene copies. The chlamydial genome copies and hydrosalpinx scores were compared between groups using Wilcoxon rank sum test. The incidences of hydrosalpinx between groups were evaluated by Fisher’s Exact Probability Test.Correlations of chlamydial pathogenicity in the upper genital tract with chlamydial colonization in different tissues were analyzed by calculating Spearman Rank-Order Correlation Coefficients.C. muridarum  and the correlation co-efficient between the hydrosalpinx scores and vaginal F/B ratios or rectal IFUs vaginal IFUs were −0.0416 (p = 0.89), 0.0048 (p = 0.9763), and 0.1094 (p = 0.5665) respectively . As a result, we identified a significant association of gut dysbiosis with chlamydial pathogenicity in the upper genital tract.The correlation co-efficient between the hydrosalpinx scores and gut dysbiosis was 0.51  to QT and (32000138) to TZ.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.The reviewer ZZ declared a shared parent affiliation with the authors LW, XS to the handling editor at the time of review.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. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The objective of this research was to encapsulate probiotics by alginate hydrogel beads based on an in situ cultivation method and investigate the influences on the cell loading capacity, surface and internal structure of hydrogel beads and in vitro gastrointestinal digestion property of cells. Hydrogel beads were prepared by extrusion and cultured in MRS broth to allow probiotics to grow inside. Up to 10.34 ± 0.02 Log CFU/g of viable cell concentration was obtained after 24 h of in situ cultivation, which broke through the bottleneck of low viable cell counts in the traditional extrusion method. Morphology and rheological analyses showed that the structure of the eventually formed probiotic hydrogel beads can be loosed by the existence of hydrogen bond interaction with water molecules and the internal growth of probiotic microcolonies, while it can be tightened by the acids metabolized by the probiotic bacteria during cultivation. In vitro gastrointestinal digestion analysis showed that great improvement with only 1.09 Log CFU/g of loss in viable cells was found after the entire 6 h of digestion. In conclusion, the current study demonstrated that probiotic microcapsules fabricated by in situ cultivation method have the advantages of both high loading capacity of encapsulated viable cells and good protection during gastrointestinal digestion. Probiotics have multiple health benefits for humans and are used not only as health supplements but also for the prevention and treatment of diseases such as diarrhea, colon cancer, obesity, diabetes, and inflammation . HoweverLactobacillus paracasei BGP-1) microcapsules and found that the formulation produced with alginate–shellac and coconut fat can reduce the porosity of microcapsules and was the most effective in improving probiotic survival in simulated gastrointestinal fluids, in which 7.5 Log CFU/g of probiotics survived at the end of gastrointestinal digestion. Khan et al. [Bifidobacterium adolescentum was significantly improved over the pH-challenge test with end-point cell reduction values of 2.0–2.6 Log CFU/g of capsule compared to that of 4.5 Log CFU/g in free cells. Sandoval et al. [Lactobacillus casei using the extrusion method for application in yogurt and found that the binary calcium–alginate–pectinate beads matrices provided good protection against bile salts.Extrusion is one of the most commonly used technologies to prepare probiotic microcapsules, where the microcapsules are formed by adding the probiotic to the wall solution and dropping it into the curing solution as droplets (using a nozzle) to form tiny particles. The extrusion method exhibits numerous advantages, such as simplicity, low cost, mild process conditions, uniform particle size, and high cell viability. For instance, Marluci et al.  used co-n et al.  developezoogloea ramigera (KCTC 2582) cells into hydrogel beads consisting of sodium alginate and found that the specific uptake capacity of cadmium was 1.45 mg/g of the crosslinked capsule biosorbent, which can maintain the mechanical strength and adsorption/desorption capacity even after 30 cycles of repeated uses. Heumann et al. [Lacticaseibacillus paracasei (ATCC334) into hydrogel beads consisting of low methoxyl pectin and found that the cells encased in these pectinate beads exhibited increased resistance to acidic stress (pH 1.5), osmotic stress (4.5 M NaCl), and freeze-drying processes. Kim et al. [Gluconacetobacter xylinus into alginate beads and with 36 h of cultivation; the entire bead surface was covered with cellulose fibers (~30 nm), which was metabolized by the cells. After immobilizing lipase into this kind alginate/bacterial cellulose beads, the enzyme activity and specific activity were 2.6- and 3.8-fold higher than that of lipase immobilized in cellulose beads.Recently, probiotic hydrogel beads fabricated by inoculating cells into blank hydrogel beads and then in situ co-cultured has caught the attention of researchers and has been applied in the area of reusable heavy metal ions biosorbents, biofilm, and bacterial cellulose nanocomposite beads. For instance, Park et al.  inoculatn et al.  inoculata KCTC 252 cells iAlthough in situ cultivation technology has shown its superiority regarding protection of bioactive core materials to the outside adverse environments, few studies have been performed as to whether the application of this technology could improve the gastrointestinal target delivery property of probiotic bacteria, which gives us a chance to further move on. Thus, the aim of this study was to encapsulate probiotic bacteria through in situ cultivation technology and analyze its effects on the loading capacity, surface and internal structure, viscoelasticity, as well as the gastrointestinal properties of probiotic hydrogel beads and the corresponding mechanisms.Lactobacillus plantarum 550 was purchased from Sichuan Gaofuji Biological Technology Co., Ltd. . Sodium alginate is pharmaceutical grade, purchased from Aladdin Biochemical Technology Co., Ltd. , calcium chloride ; pepsin ; pancreatic protease ; porcine bile salt .2 powders were added to the solution and stirred until completely dissolved. All instruments and reagents were sterilized using an autoclave at 121 °C for 15 min prior to sample preparation.Dissolve the appropriate amount of sodium alginate powder in water and stir with a cantilever stirrer for about 4 h to allow complete hydration. Dried anhydrous CaClL. plantarum 550 were harvested by centrifugation at 4000 rpm for 10 min. Then, L. plantarum 550 cells were dispersed (6 LogCFU/g) in the alginate solution until a uniform suspension was obtained. The cell suspension was dispersed dropwise using a syringe auto-pump into sterile CaCl2 (1 %wt) as a hardening solution at the rate of 1 mL/min in order to produce small beads. After a gelation time of 10 min in CaCl2, the beads containing L. plantarum 550 were filtered through a 200-mesh gauze filter and washed with bacteria-free water 2–3 times. Finally, the surface of the hydrogel beads was blotted with bacteria-free paper to obtain the sample of hydrogel beads containing about 6 LogCFU/g L. plantarum 550 called before incubation, containing a low concentration of the bacteria group (BLP).Bacterial cells of L. plantarum 550 (BC) and the control group (CLP) that contained a high concentration of L. plantarum 550 (about 9 LogCFU/g) were prepared in the same way.In order to describe and compare the changes in the apparent morphology, internal structure, and resistance to external damage of the hydrogel bead samples during the incubation process, the hydrogel bead samples without L. plantarum 550 and the sample named BC without bacteria were put into the appropriate amount of MRS broth and incubated in a constant temperature incubator at 37 °C for a period of time.The sample named BLP containing approximately 6 LogCFU/g L. plantarum 550 and the after-incubation non-bacteria group (AC) samples obtained from the cultivation of BC samples without bacteria, respectively. The composition and preparation of each hydrogel bead sample are shown in After 24 h of incubation, the samples were filtered through a 200-mesh gauze filter and washed with bacteria-free water 2–3 times, and finally the surface of the hydrogel beads was blotted with bacteria-free paper to collect the after-incubation bacteria-containing group (ALP) obtained from the cultivation of BLP containing about 6 LogCFU/g L. plantarum 550 encapsulated in the hydrogel beads, and the plates were spread to count the viable bacteria.The pH of the external MRS broth was measured at different times during the incubation process, while a certain quantity of hydrogel beads containing the bacteria was taken in a previously prepared PBS solution and dispersed in a high-speed disperser at 4000 rpm for about 10 min to fully release the Ten hydrogel beads were randomly selected from each group and their diameters were measured using a micrometer. The mean diameter ± standard deviation was calculated, as well as the percentage change between corresponding groups before and after cultivation.Each group of hydrogel beads was placed entirely on a slide, and a microscope was used to observe the whole and partial position of the hydrogel beads; magnification was amplified at 50–400×. The observation was repeated 3 or more times.The hydrogel beads were first pre-frozen in a −80 °C refrigerator for 4 h, followed by freeze-drying in a vertical freeze-dryer  at 0.025 mbar vacuum and −55 °C for 72 h. The freeze-dried beads were split in half, then the surface of the complete freeze-dried beads and the cross-section of the freeze-dried beads were sprayed with gold. Finally, the surface and cross-section microstructure of the beads were observed by field emission scanning electron microscope . The observation was repeated 3 or more times.The method of Dai et al.  was usedη′ is called the dynamic viscosity and is the energy dissipation part of the complex viscosity, which is related to the loss modulus and indicates the contribution of viscosity. η′ is calculated by the following Equation (1):η″ is called the imaginary viscosity and is a measure of elasticity and energy storage, which is related to the dynamic modulus and indicates the contribution of elasticity. η″ is calculated by the following Equation (2):The utilization of dynamic viscosity and imaginary viscosity can effectively characterize the viscoelastic properties of polymer systems.Firstly, the preparation of simulated gastric solution and simulated intestinal solution was carried out. The gastric solution consisted of 0.32 g of pepsin and 0.2 g of NaCl in 100 g of ultrapure water, mixed well, and then the pH of the solution was adjusted to 2.0 with 1 M HCl. The intestinal solution included 0.1 g of trypsin and 0.08 g of pig bile salt in 100 g of 0.2 M PBS solution, mixed well. Then, the simulated intestinal solution and simulated gastric solution were incubated in 37 °C water for 20 min and then passed through a filter membrane with a pore size of 0.22 μm, respectively, for sterilization.L. plantarum 550 from the hydrogel beads, and the solution in each of the 4 tubes was diluted in a gradient and counted using plate coating. Immediately after 120 min of simulated gastric solution incubation, 10 mL of simulated intestinal solution was added to each of the other 4 centrifuge tubes, which were also shaken and mixed continuously at 180 rpm in a 37 °C shaker, and the solutions in these 4 centrifuge tubes were diluted in a gradient at 1, 2, 3, and 4 h of incubation time, respectively; additionally, the viable bacteria were counted using a plate.Then, 1 g of hydrogel beads or 1 mL of probiotic suspension with different concentrations of ALP and CLP were added to 8 centrifuge tubes containing 9 mL of simulated gastric solution, respectively, and mixed with constant vibration at 180 rpm in a shaker at 37 °C. At 30, 60, 90, and 120 min of incubation time, 4 centrifuge tubes were taken and 10 mL of 0.2 M PBS solution was added immediately, and the solution in each tube was decapsulated at 4000 rpm for 10 min in a high-speed disperser to release p < 0.05. Origin 2018(Version 9.0) was applied to form figures.All measurements were carried out in triplicate. The data were expressed as mean ± standard deviation and subjected to analysis of variance (ANOVA). Statistical analysis was performed with SPSS Statistics Software 24.0. Differences were considered significant when L. plantarum 550 was optimal after encapsulation and subsequent culturing in the MRS medium, and the three-dimensional hydrogel network of beads did not inhibit bacterial growth and metabolism, which could be attributed to the porous structure inside the calcium alginate hydrogel beads and the channels that existed on the surface [ surface . On one  surface . On the L. roxellanae. The author observed that the viable counts in the hydrogel beads before and after lyophilization were around 107–108 Log CFU/mL and 106–107 Log CFU/mL, respectively; Mirmazloum et al. [L. acidophilus and Ganoderma lucidum  extracts within calcium alginate beads and determined the viable counts inside of approximately 7 Log CFU/mL. Exhilaratingly, in our study, viable bacterial counts up to 10.34 ± 0.02 Log CFU/g of hydrogel beads were attained and were around 1000 times higher than those obtained with the traditional method, which broke through the bottleneck of low viable cell counts in traditional alginate beads.In the conventional method, the maximum viable bacterial count in sodium alginate microspheres was approximately 7 Log CFU/mL. For instance, Ding et al.  incorporm et al.  co-encap2+ to form more stable “egg box” type structures, leading to better sphericity of the hydrogel beads. Moreover, an increase in sodium alginate concentration also increased the viscosity of the extrusion solution leading to the occurrence of severe entanglement between the alginate molecular chains and finally resulting in the formation of hydrogel beads with larger particle sizes [le sizes . The parle sizes . As repole sizes , throughThe increasing proportions of particle size between samples with the same formula before and after incubation were shown in The optical microscopy images of different hydrogel beads were shown in The surface morphology of lyophilized probiotic microcapsules was further observed using SEM a. The paSmall-amplitude oscillation strain and frequency scans of hydrogel beads were performed to compare the viscoelastic variations before and after incubation a,b. OverComparing AC to BC and ALP to BLP samples that have the same alginate concentration, the cultured beads exhibited lower G′ than that in the uncultured samples, which can be attributed to the absorption of water and corresponding swelling. The hydrogen bonding interaction mediated by water has two effects: firstly, it weakens the internal hydrogen bonds of carbohydrates; secondly, it reduces the ability of alginate binding to calcium ions, resulting in the loosening of the hydrogel network structure and ultimately leading to a decrease in G′ ,16.L. plantarum filled the inner space of the hydrogel beads, forming microcolonies that can enhance G′. Secondly, bacterial growth was accompanied by the metabolism of acids. The charge density of hydrogel beads increased with the decrease in pH, thereby decreasing chain flexibility, shortening the distance between molecular chains. Eventually, the number of interactions and entanglements between alginate molecules would increase, leading to the rising in G′. Similar results were also reported by Bastos et al. [Comparing AC to ALP samples with the same alginate concentrations, the cultured beads exhibited higher G′ than that in the uncultured samples. There were mainly two reasons for this variation: firstly, the proliferation of s et al. , who stuThe utilization of dynamic viscosity and imaginary viscosity can effectively characterize the viscoelastic properties of polymer systems. For all the samples, with the increasing in frequency, both dynamic viscosity and imaginary viscosity decreased, indicating shear-thinning behavior . The sheWhen comparing the difference between imaginary viscosities of AC and ALP groups, we found that the absolute slope values of η″ curves in the ALP group increased to different degrees compared to those in the AC group, which originated from the electrostatic interactions between the polysaccharide chains . During The gastrointestinal digestion properties of hydrogel beads fabricated using in situ co-culturing (ALP) and traditional method (CLP) were shown in The current study demonstrated that probiotic microcapsules fabricated by an in situ cultivation method could not only increase the loading capacity of viable cells after encapsulation, but also greatly improve the survival of cells during gastrointestinal digestion. Up to 10.34 ± 0.02 Log CFU/g of viable cell concentration was achieved after 24 h of cultivation, and only a loss of 1.09 Log CFU/g of viable bacteria was observed after digestion in the gastrointestinal tract, resulting in an increase of approximately 2.44 Log CFU/g after the end of digestive process. The method developed in this research holds potential for application to a broad range of probiotics, and further investigation and optimization of this method may lead to commercial products.


Heavy menstrual bleeding (HMB) or abnormal uterine bleeding (AUB) is a common cause of gynecological complaints in perimenopausal women. The treatment chosen by most of the women having HMB/AUB in the perimenopausal age group when medical therapy fails is hysterectomy, which unfortunately has significant issues related to safety and long-term quality of life apart from being a burden on the health care system and cost to patients. Minimal access surgical techniques like hysteroscopic targeted therapies are available which are conservative and spare the uterus and major surgery and its complications and sequelae. Hysteroscopic management of HMB is a minimally invasive and targeted approach in diagnosing and treating the pathological lesions causing the symptoms and not radical like hysterectomy which has more chances of occurrence of adverse events both intra and postoperatively. In terms of health-related quality of life, women suffering from HMB who underwent a hysteroscopic conservative approach had better scores when compared to those with hysterectomy.The present review aims to review the evidence generated to compare the two surgical modalities, hysteroscopic targeted therapy and hysterectomy, in terms of their effectiveness, safety, and effect on the quality of life of these perimenopausal women having HMB/AUB. One of the most important symptoms with which women seek consultation in gynecology is abnormal uterine bleeding (AUB) or heavy menstrual bleeding (HMB). The prevalence of AUB/HMB is reported between 11-13% in all age groups of women increasing to as high as 25 to 30% in those aged 36-40 years  . It is sIn a study done by Selvanathan et al., a significant number of women who underwent hysterectomy had late and delayed adverse events at the end of one year when compared to hysteroscopy suggesting hysteroscopic surgical modality is found safer even when women are followed up for one year. They further state women who underwent hysterectomy had a higher occurrence of pelvic pain, bladder symptoms, sexual discomfort, and menopausal symptoms and they lasted for a longer duration of time when compared to hysteroscopic therapy .Quality of Life (QoL)Matteson et al. analyzed six studies that evaluated QoL and concluded that both the modalities had improvement. They further suggested that there is a need to study the long-term effects on QoL to compare the two surgical modalities . SimilarIn a study by Acharya et al., in both groups, viz. hysteroscopic therapy and hysterectomy group, the mean scores in all domains increased progressively at one week, six months, and one-year follow-up, but the hysteroscopy group had significantly better scores and improved rapidly and earlier when compared to those of hysterectomy group at all set of postoperative follow-up visits and resumed work early .Abbott et al. studied quality of life as the primary outcome in terms of treatment results of endometrial ablation and concluded quality of life of women suffering from HMB improved with hysteroscopic ablation and it is a successful method to treat these women . Table 1Heavy menstrual bleeding is a common cause of gynecologic consultation with a 24% incidence of overall gynecologic consultations. Hysteroscopic targeted therapy is effective in controlling menstrual bleeding in cases of AUB/HMB. It is also effective in relieving burdensome menstrual symptoms as a chief complaint in AUB/HMB. Hysteroscopic therapeutic procedures establish eumenorrhea or hypomenorrhea and amenorrhea in most of the patients at short-term and long-term follow-up which is a desirable effect with the benefit of conserving the uterus and avoiding hysterectomy. Those few cases who have persistent menstrual symptoms have a fair chance of responding to medical therapy as the primary cause is removed by hysteroscopy. Hence hysteroscopic therapeutic procedures are comparable in terms of effectiveness in relieving primary complaints of menstrual abnormality for women suffering from AUB/HMB and having the potential benefit of avoiding major and radical surgical procedures of hysterectomy.Hysteroscopic therapeutic procedures are safer having reduced relative risk of intraoperative, early, late, and delayed postoperative complications as a surgical modality for the management of AUB/HMB when compared to hysterectomy. Hysteroscopic surgeries also have the benefit of less postoperative pain, shorter operation time, and hospital stay thereby having the benefit of early postoperative recovery and avoiding loss of working hours when compared to hysterectomy. Women who undergo hysterectomy, have significantly more risk of pelvic pain, bladder dysfunction, sexual dysfunction, and menopausal symptoms which last for a longer duration of time when compared to hysteroscopic therapy. Patients suffering from AUB/HMB have poor health-related quality of life (QoL). Both hysteroscopic therapy and hysterectomy as therapeutic modalities for HMB improve all domains of quality of life. The improvement in QoL in all domains is rapid and earlier in hysteroscopic therapy than that observed in the hysterectomy group.


N4BP1 and WASHP, along with 331 new genes. Furthermore, we conducted a comprehensive analysis to determine the critical impact pathways of virus integration by combining our findings with the results of three major global studies on HBV integration. Meanwhile, we found common characteristics of virus integration hotspots among different ethnic groups. To specify the direct impact of virus integration on genomic instability, we explained the causes of inversion and the frequent occurrence of translocation due to HBV integration. This study detected a series of hotspot integration genes and specified common characteristics of critical hotspot integration genes. These hotspot genes are universal across different ethnic groups, providing an effective target for better research on the pathogenic mechanism. We also demonstrated more comprehensive key pathways affected by HBV integration and elucidated the mechanism for inversion and frequent translocation events due to virus integration. Apart from the great significance of the rule of HBV integration, the current study also provides valuable insights into the mechanism of virus integration.Hepatitis B virus (HBV) integration is closely associated with the onset and progression of tumors. This study utilized the DNA of 27 liver cancer samples for high-throughput Viral Integration Detection (HIVID), with the overarching goal of detecting HBV integration. KEGG pathway analysis of breakpoints was performed using the ClusterProfiler software. The breakpoints were annotated using the latest ANNOVAR software. We identified 775 integration sites and detected two new hotspot genes for virus integration,  TERT (n = 18), MLL4 (n = 9), and CCNE1 (n = 4)1. Later, new high-throughput Viral Integration Detection (HIVID) methods significantly promoted the research on virus integration2. For example, Zhao et al. identified a series of HBV integration hotspots in liver cancer, such as TERT (101), KMT2B (31), CCNE1 (7), and CCNA2 (8), by utilizing the HIVID technology to detect 426 liver cancer samples from China3. In addition, they ascertained that virus integration is directly associated with clinical prognosis. Péneau et al. found that the gene hotspots of virus integration were TERT (n = 48), CCNE1 (n = 4), and KMT2B (n = 3) by carrying out virus integration detection on 177 liver cancer samples from France4. Over the years, the research on HBV integration has gradually increased. HBV integration is known to cause cancer in at least three aspects: (1) HBV integration leads to abnormal gene expression at, before, and behind the virus integration site; (2) Viral protein is produced after the virus is integrated within an appropriate site; (3) Virus integration disturbs normal chromosome structure and chromatin accessibility, leading to the instability of many forms of genomes6. Thus, it is evident that virus integration induces hepatocellular diseases by disturbing the normal transcription and translation of hepatocytes.Hepatitis B virus (HBV) integration has been frequently detected in liver cancer. With the advancement of high-throughput sequencing technology, significant progress has been made in understanding virus integration. First, Sung et al. found that there were HBV integration hotspots in cancer tissues by sequencing the whole genome of 88 tissue samples of liver cancer patients from Southeast Asia, and the hotspot genes were Although a series of new hotspots of virus integration have been identified from the research on HBV integration, the data on HBV integration sites obtained by high-throughput technology is still minimal. Therefore, new HBV integration sites, especially new integration hotspots, need further exploration. Moreover, the key pathways affected by HBV integration and genomic instability due to virus integration remain unclear.N4BP1 and WASHP, and 331 new genes. Furthermore, we combined our results with three significant global studies on HBV integration to determine the pathways and functions related to virus integration sites. Results revealed that the essential hotspot genes of virus integration have common characteristics across ethnic groups. The study also found that the viral genome is integrated into the forward and reverse directions and frequently leads to events such as inversion in the genome. Consequently, we clarified the possible mechanism for the occurrence of inversion and the reason for frequent translocation events caused by HBV integration. Overall, this study detected new integration genes, hotspot genes, and more comprehensive critical pathways associated with the HBV integration sites. Our findings provide valuable insights into the mechanisms underlying inversion and translocation events due to virus integration. This study not only plays a positive role in the clinical application of virus integration but also effectively promotes elucidating the mechanism behind virus integration.This study used 27 tumor samples for HIVID, which resulted in identifying 775 integration sites and detecting two new hotspot genes for virus integration, TERT gene and six in KMT2B. We found that the hotspot genes of virus integration were TERT (4), KMT2B (6), and POTEA (6)  Figs. . There w6) Figs. . After cPathway analysis of all integrated genes from the four studies revealed that the significant pathways were the Rap1 signaling pathway, Axon guidance, Calcium signaling pathway, Cholinergic synapse, Growth hormone synthesis, secretion and action, and GABAergic synapse Fig. . In addiAmong the 775 integration sites, 388 viruses were connected with the human genome in a forward direction and 387 in a reverse direction , but also found that the sample integration frequencies of the TERT gene were 23.7% (101/426), 20.5% (18/88), 27.1% (48/177), and 14.8% (4/27), respectively, after comparing with previous studies. In addition, we found that the integration frequencies of KMT2B were 7.3% (31/426), 10.2% (9/88), 1.7% (3/177), and 22.2% (6/27), respectively4. This indicated significant differences among different studies, and the integration frequency of the KMT2B gene in Asia was much higher than that in Europe. Moreover, a comparison between the Asian and European populations revealed that there were a series of common hotspot genes , indicating that there were some common characteristics of virus integration among the different ethnic groups. This suggests that the common virus integration events have certain regularity in enhancing tumor evolution, but the hypothesis requires further study. In addition, we found that N4BP1 enables mRNA binding activity, ribonuclease activity, and ubiquitin-binding activity. It is also involved in the cellular response to UV and negative regulation of viral genome replication7. It acts as a restriction factor against some viruses, such as HIV-1: restricts HIV-1 replication by binding to HIV-1 mRNAs and mediating their degradation via its ribonuclease activity8. WASH8P (WAS Protein Family Homolog 8) is a pseudogene. Although little research has been done on this gene, it was recently been linked to rectal cancer9. Considering the limited knowledge of N4BP1 and WASH8P genes and virus integration information, this study could not elaborate on how virus integration affects the two genes.This study detected the breakpoints of cancer tissues and adjacent tissues in 27 HCC patients. It was found that the intersection of the breakpoints between cancer and adjacent tissues was 3  or reverse connection  during HBV integration. We identified 775 points and found that the same direction connection accounted for 388/775. In contrast, the reverse connection accounted for 387/775, indicating that insertion of virus integration happens in various directions. The proportion of reverse connection cases shows that virus integration leads to the frequent reverse connection of genomes. The reverse connection at both endpoints after virus integration indicates similar inversion events. However, although this phenomenon has been observed in many studies on HBV integration modelsism Fig.  and 7 ofion Fig. . The aboTERT and KMT2B. We found that these hotspot genes are universal across different ethnic groups, providing an effective target for better research on the pathogenic mechanism and treatment of liver cancer through virus integration. Virus integration is widely believed to cause changes in genome structure, genomic instability, and abnormal expression of tumor suppressor genes, oncogenes, and viral genes. This study established that virus integration could directly cause inversion and translocation at the genomic structural level and found that virus integration can be closely associated with nerve axons and crucial synapses. Although the mechanism of virus integration affecting nerve axons and synaptic pathways requires further study, the close relationship with the nervous system has been gradually uncovered. Li et al. also observed that genes shared by HBV and HPV integration are enriched in nerve axons and essential synapses. These phenomena indicate that virus integration is closely linked with nervous system abnormalities during the carcinogenic process11. This study confirmed the commonness of virus integration hotspots among different ethnic groups and discovered new hotspot genes, describing the close relationship between virus integration and the nervous system. Moreover, the study explained the mechanism of inversion and translocation due to HBV integration. Overall, the research achievements will be significant for the clinical application of HBV integration and for studying carcinogenic mechanisms and drug targets.Conclusion: This study identified a series of new virus integration genes and expanded the latest knowledge of virus integration hotspots in the field of HBV integration. There is an extensive background of random integration and evident hotspot genes, such as Twenty-seven HBV-positive hepatocellular carcinoma (HCC) samples were obtained from Jining Medical University. Supplementary Table All the procedures performed in this study involving human participants followed the ethical standards of the institutional research committee based on the 1964 Helsinki Declaration and the later amendments or comparable ethical standards. The study was approved by the Ethics Review Committee of Jining Medical University. Written informed consent was obtained from each participant.Genomic DNA was extracted from all samples using the Tiangen kit following the manufacturer’s instructions. The capture probes were designed from the DNA sequences of eight genotypes of HBV and synthesized by MyGenostics. The extracted DNA (1 ug) from each sample was sheared into fragments of 150–200 bp length using Covaris M220 . The fragments were then purified, end blunted, ‘A’ tailed, and adapter ligated to obtain a DNA library. Next, the hybridization process was carried according to MyGenostics GenCap Target Enrichment Protocol , followed by PE150 DNA sequencing .2. First, low-quality, duplicate reads and adapter-contaminated reads are filtered out to obtain the clean data. Then, clean reads were mapped on to human  and HBV genomes by Burrows-Wheeler Aligner (BWA). Paired-end reads could be perfectly mapped to the human or HBV reference genome were removed. The remaining reads were used to reconstruct fragment based on the overlap of paired-end reads. Subsequently, the paired-end assembled reads were mapped onto human and HBV genomes using BWA18. The junction position (Breakpoints) of the human and HBV sequences was detected out in the paired-end assembled reads. Finally, the breakpoints of HBV integration were obtained. The breakpoints with total support read ≥ 5 were retained.HBV integration sites were detected using the HIVID method19, an intelligent bioinformatic tool for statistical and network analysis. The significance threshold for altered biological processes/pathways was set at a corrected hypergeometric P-value of 0.0521. The protein-protein interaction (PPI) networks were constructed based on the data from the STRING (https://cn.string-db.org/) database. The breakpoints were annotated using the latest ANNOVAR in hg19 coordinates22. The integrated viral genome has been considered a strong cis-activator of the flanking genes, and the cis-acting enhancers influence their target genes over long distances. Thus, genes near the breakpoints in the intergenic region were included in determining the affected gene in HBV-integrated samples24.KEGG pathway enrichment analysis of the integration breakpoints was performed using the Clusterprofiler softwaresupplementary information


Monkeypox virus after a recent trip to Brazil, where he engaged in condomless sexual intercourse with multiple male partners.A healthy young man first diagnosed with mpox in May 2022 presented again in November 2022 with anal proctitis and a positive polymerase chain reaction on a rectal swab for   Monkeypox virus (MPXV), was considered a sporadic zoonotic disease limited to West and Central African countries until the current worldwide outbreak was declared in May 2022. MPXV belongs to the Orthopoxvirus genus, as do Vaccinia virus, Cowpox virus, and Variola virus, all of which are infectious to humans. MPXV infection generates humoral and cellular immunity that is expected to provide long-term protection against reinfection [Variola virus, the agent of smallpox disease [Vaccinia virus provided almost complete protection from infection with Variola virus thanks to cross-immunity [Monkeypox virus reinfection in an otherwise healthy adult.Mpox, caused by nfection . These aimmunity . MPXV reimmunity , so far Orthopoxvirus polymerase chain reaction (PCR) [Chlamydia trachomatis infection. Within 2 weeks, skin lesions spontaneously resolved without complications.A 34-year-old man in good general health presented in May 2022 with 4 umbilicated lesions on the penis. He was on preexposure prophylaxis for human immunodeficiency virus (HIV) and reported having had condomless sex with men, including receptive anal intercourse. He did not report previous smallpox infection or vaccination. On physical examination, no lymphadenopathy was found, and oropharyngeal and anal inspection was normal. The patient did not report fever or any other symptoms. Skin lesions and pharyngeal swabs tested positive for MPXV by real-time on (PCR)  with cycOn 1 December 2022, the patient reported persistent perianal pain without bleeding nor discharge. Symptoms had started 2 weeks before, on his return from Brazil in November 2022, where he had engaged in condomless anal intercourse with multiple male partners. No other complaints or symptoms were reported. He related a last receptive anal intercourse 2 weeks before the presentation. The patient did not recall any signs or symptoms of mpox among his partners, nor was he aware of an MPXV infection among them. Clinical examination found no skin lesion in the perianal region or other body parts. Apart from a slightly palpable painless right inguinal lymphadenopathy, the medical examination was normal. Four weeks after symptom onset, a proctologic examination revealed a small anal fissure but no typical mpox lesion.C. trachomatis (non–lymphogranuloma venereum). The latter was treated with a 7-day course of doxycycline. HIV serology and viremia were negative, and syphilis was serology negative for acute infection. On 13 December 2022, rectal and pharyngeal swabs were collected again and turned out negative. Orthopoxvirus PCR was also negative in the blood and urine on 13 December 2022. Total MPXV antibodies were tested  on sera collected prior to the first episode in May 2022 and after the second in December 2022: they came back negative and positive, respectively. Unfortunately, no serum had been collected between the 2 episodes.Sexually transmitted infectious diseases were screened using standard diagnostic tests. The anal swab done on 1 December 2022 came back positive for MPXV with a Ct of 27 and for Orthopoxvirus PCR returned positive on an anal swab 14 days after the last receptive anal intercourse and 6 months after a previously confirmed MPXV infection. It is not possible to determine if this second mpox episode contributed to the proctitis in the context of concomitant C. trachomatis infection and the presence of an anal fissure. Specific mpox mucosal lesions might have been missed as the proctologic examination was carried out 4 weeks after symptom onset. It is therefore impossible to distinguish between symptomatic and asymptomatic reinfection. Though viral culture could not be performed to confirm active infection with certainty, a trusted physician’s detailed anamnesis undertaken during the second episode makes anal contamination following condomless anal intercourse with an MPVX-positive partner in the days before sampling unlikely. The disease course was mild, and the patient recovered completely. No immunosuppressive condition were found and HIV could be excluded.We report a case of probable MPXV reinfection. The patient's This case illustrates that sterilizing immunity may be limited despite prior systemic infection. Studies suggest that historical smallpox infection or vaccination produces cross-immunity against MPXV , 6. The Vaccinia virus [Reinfection in our patient most likely occurred in Brazil, which in November 2022 had one of the highest reported daily new cases of mpox. Because of the nature of MPXV, a double-stranded DNA virus that has shown few variations since the outbreak's beginning, it seems unlikely that reinfection occurred because of a mutation-driven immune escape of a Brazilian variant. The patient's first isolate was sequenced confirming MPXV B.1., but a technical issue and lack of sufficient specimen precluded obtaining the second sequence (Oxford Nanopore MinION), so strain comparison could not be made. With only 2 Brazilian MPXV sequences reported on Nextstrain, the hypothesis of a mutation-driven immune escape cannot be excluded entirely. Yet, the large cross-immunity between different orthopoxviruses and the large number of shared epitopes driving the immune response  make theia virus  but alsoia virus . Yet, thIn this case, the high rate of mucosal lesions associated with anal intercourse leading to high viral load exposure might have outweighed the host's local immune response and led to local and transient replication of MPXV. Asymptomatic shedding of MPXV in MPXV-naive individuals has previously been described . In MPXVFurthermore, persistent MPXV-positive PCR results after infection have been reported in the literature and are suggestive for prolonged MPXV shedding . Our patWhile the next few months will be decisive in determining whether this case is an exception or the first of a beginning series, it is crucial to raise awareness among clinicians that MPXV may not develop effective protection in time and that if suspicion criteria are met, patients should be tested again for MPXV. With the growing evidence for asymptomatic and/or prolonged shedding of MPXV, screening among naive, recovered, and vaccinated individuals also needs to be addressed. Meanwhile, public health authorities should be informed and prepare for the consequences that increasing MPXV reinfections would imply for the management of the current mpox epidemic. Further tools may be useful, such as the development of specific MPXV vaccines.


Surveillance of SARS-CoV-2 in wastewater offers a near real-time tool to track circulation of SARS-CoV-2 at a local scale. However, individual measurements of SARS-CoV-2 in sewage are noisy, inherently variable and can be left-censored.We aimed to infer latent virus loads in a comprehensive sewage surveillance programme that includes all sewage treatment plants (STPs) in the Netherlands and covers 99.6% of the Dutch population.We applied a multilevel Bayesian penalised spline model to estimate time- and STP-specific virus loads based on water flow-adjusted SARS-CoV-2 qRT-PCR data for one to four sewage samples per week for each of the more than 300 STPs.12 virus particles per 100,000 persons per day in the epidemic trough in August 2020 to almost 1015 per 100,000 in many STPs in January 2022. The timing of epidemics at the local level was slightly shifted between STPs and municipalities, which resulted in less pronounced peaks and troughs at the national level.The model captured the epidemic upsurges and downturns in the Netherlands, despite substantial day-to-day variation in the measurements. Estimated STP virus loads varied by more than two orders of magnitude, from ca 10Although substantial day-to-day variation is observed in virus load measurements, wastewater-based surveillance of SARS-CoV-2 that is performed at high sampling frequency can track long-term progression of an epidemic at a local scale in near real time. What did you want to address in this study?The primary aim of this study was to estimate SARS-CoV-2 virus loads in sewage in the Netherlands, using data from a comprehensive national surveillance programme. The secondary aim was to compare estimated virus loads in sewage with hospital admission data in order to use the sewage signal as a main indicator of morbidity.What have we learnt from this study?Virus loads in sewage water can vary by more than two orders of magnitude. The dynamics of the COVID-19 epidemic in wastewater were largely synchronised between municipalities. Estimated virus loads in sewage closely followed the daily number of hospitalisations and case notifications.What are the implications of your findings for public health?Surveillance of SARS-CoV-2 in wastewater can track in near real-time how the COVID-19 epidemic develops within a defined local area, even in the presence of substantial background signals and day-to-day variation.The COVID-19 pandemic has posed one of the most severe threats to public health in recent history. Severity and progression of the disease are variable, depending on host risk factors and pathogen variants . TransmiIn contrast with earlier applications of wastewater-based surveillance such as the identification of emerging enteric viruses , monitorHere we report data and statistical analyses from a national wastewater-based surveillance programme in the Netherlands. Since 7 September 2020, all sewage treatment plants (STPs) in the Netherlands have been providing between one and four samples per week to the Dutch National Institute for Public Health and the Environment (RIVM). Since almost every Dutch household is connected to a sewer, coverage of the programme is close to 100%. Samples are subjected to a standardised extraction protocol and analysed by real-time RT-PCR within 2–4 days after sampling. As measurements of SARS-CoV-2 RNA in sewage are inherently variable and can be left-censored (i.e. not able to detect RNA at low virus loads), our aim was to integrate the available data to provide estimates of the latent true SARS-CoV-2 virus loads over time for all STPs. We analysed data from 1 August 2020  up to and including 8 February 2022 (when coverage exceeded 99%). Hence, the time series data cover the winter epidemic of 2020/21, the 2021 summer surge after many restrictions had been lifted, and the 2021/22 winter epidemic.In the Netherlands, sewage of 99.6% of the population is treated at 317 STPs, of which four closed between start and end of the period of analysis . CoveragAt the central laboratory, RNA extraction was performed on each sample using the Viral NA Large Volume kit on the MagNA Pure 96 instrument . Detection of SARS-CoV-2 RNA was based on an assay described by the Centers for Disease Control and Prevention (CDC) [10-transformed virus concentration data, normalised by 24 h sewage water flow rates . The analyses employed a Bayesian multilevel spline model with random effects at the level of the STPs [10-transformed virus load at time t and STP i, i(t),c was given byThe basis for the analyses were logthe STPs . In the c(t) represents the national trend and the i(t)d represent the STP-specific deviations. The central goal was to infer the unknown latent virus loads i(t)c in the presence of measurement error and left-censoring. The national trend c(t) was modelled with a penalised spline (p-spline) with cubic b-spline basis functions and 15 equidistant knots (yielding 17 regression coefficients). The results presented here were obtained with a first-order random walk prior for the regression coefficients [N prior distribution for the first regression coefficient and an inverse gamma prior distribution for the variance parameter [10-transformed virus load at the first time point was normally distributed a priori, with a mean of 12 per 100,000 persons per day and approximate 95% prior range of 10–14 per 100,000 persons per day. This provided good coverage for virus loads at the first time points (August 2020). In a similar manner, STP-specific deviations from the trend were modelled with cubic b-splines with 15 equidistant knots (yielding 17 coefficients per STP), and with zero-mean normal prior distributions ). Hence, plant-specific deviations were a priori expected to be no more than ca 100-fold (i.e. 2 standard deviations) lower or higher than the trend. Of note, this choice covered almost all (> 99.9%) individual measurements for all STPs over time. where ficients , using aarameter . In thisN prior distributions for the regression coefficient, and evaluated competing models using the leave-one-out information criterion [10-transformed virus load measurements itY at plant i and time t were normally distributed, it~N(ci(t), σ)Y, and we used an uninformative (improper uniform) prior distribution for σ>0.We also explored a suite of alternatives, e.g. estimating the variance of the deviations, or replacing the p-spline for the national virus load with a b-spline using riterion . Here we0c (load at which detection occurs with a probability of 0.5) and k (steepness of the detection curve) were estimated. Hence, denoting the probability of detection byThe probability of virus detection in a sample is determined by the detection limit of the qRT-PCR, the daily flow rates of water through the sewerage, and possibly also by the composition of the sewage. Hence, there is no fixed predefined cut-off for RNA detection that can be applied to all STPs at all time points, and we included in the analyses a two-parameter logistic function that determined the probability of detection. The parameters 1-p(detection|i(t))c to the likelihood. In this manner, samples in which no RNA is detected tended to pull the estimated load i(t)c to lower values. The parameter 0c was given a weakly informative N prior distribution (a priori of 50% probability of detection ranges from log virus load of ca 11–13), and k was provided with an uninformative (improper) uniform prior distribution (k>2). The event of a detection was Bernoulli-distributed, D~Bernoulli(p(detection|i(t)))c. Putting it together, the log-likelihood contributions were given bya non-detect result added a contribution Df and Yf denote the log Bernoulli and log normal probability densities, respectively.where 10-transformed virus load at time t in municipality i, i(t)b, was given byTo tie the results at the STP level to other organisational levels  we applied posterior weighting of the estimated virus loads (i.e. the exponentiated estimated log loads), where weighting was proportional to the numbers of persons contributing to the various organisational units. Demographic data were obtained from Statistics the Netherlands using the census of 1 January 2020 , with thj, and i,jIwere the number of inhabitants in municipality i serviced by STP j. Log-transformed virus loads at other organizational levels  were calculated similarly. Of note, the analyses reported here included all but the three overseas extraordinary municipalities .where we summed over all STPs https://github.com/rivm-syso/SARS-CoV-2_sewage.We analysed sewage data of more than 47,000 measurements spanning the period from 1 August 2020  up to and including 8 February 2022. For comparison of the results with other epidemic indicators, we also made use of the daily incidence of hospital admissions  and the daily number of positive tests, both stratified by municipality. All analyses were performed with Stan (version 2.21.0) and R (version 4.1.3), using RStan (version 2.21.2) as interface to Stan . We ran 12.5). It is noteworthy that STPs often process sewage from multiple municipalities, and it is not uncommon that a municipality is served by multiple STPs. Hence, STPs do not follow the hierarchical organisational structure of the Dutch administrative divisions, which makes averaging from STP-level analyses to municipalities, provinces and safety regions not straightforward.The Dutch sewage surveillance programme for SARS-CoV-2 was initiated in the first months of 2020. Since its inception, the number of included STPs has increased over time such that 80 STPs provided samples in August 2020, and all 313–317 STPs provided samples from September 2020 onwards. An overview of the location and size of STPs in the Netherlands is provided in https://github.com/rivm-syso/SARS-CoV-2_sewage. In general, the model described the data well, and indicators suggested that the estimation procedure yielded satisfying results. Specifically, the scale reduction factor was close to 1, there were no divergent transitions, components of the posterior distribution were located well within the support of the prior distributions yielding strong posterior contraction, there were no systematic deviations of residuals from posterior medians. Further, in Supplementary Figure S1 we show that approximately 95% of observations were within the 95% posterior prediction intervals.12–1013 virus particles per 100,000 inhabitants per day. At these virus loads, a sizeable fraction of samples can yield a non-detection, especially at the lower end of this range. This is illustrated in 11, close to 50% at a virus load of 1012, and almost 1 at a virus load of 1013. Estimated parameters of the detection function were 0 c= 12.1 : 12.1–12.2) for the posterior median of the logistic midpoint, and k = 4.2 (95% CrI: 4.0–4.4) for the posterior median of the logistic growth rate.Estimated virus loads were generally low in August and September 2020 and were estimated between 1014 particles per 100,000 per day in many STPs. Thereafter, virus loads started to decline gradually in all STPs. From May 2021 onwards, when the vaccination coverage was strongly increasing, virus loads started to decrease at a faster pace until the early summer. Subsequently, virus loads increased and reached a peak in early August 2021 by the temporary lifting of restrictions for vaccinated persons. Finally, from October 2021, virus loads started increasing again, first to just under 1014 particles per 100,000 per day in most STPs in December 2021, and subsequently to well over 1014 and even up to 1015 particles per 100,000 per day in February 2022.Between August 2020 and November 2020, virus loads increased by more than one order of magnitude to a peak value close to ca 10σ=0.353 (95% CrI: 0.351–0.355), such that individual measurements could be 102σ ≈ 5-fold lower or higher than the estimated virus load. This, however, is still substantially smaller than the 100- to 1,000-fold increases and decreases in virus loads from epidemic troughs to peaks and vice versa.Overall, we observed that trends in virus loads corresponded to main epidemiological events. This was true even though we estimate that measurement variation was substantial. In fact, the posterior median of the standard deviation of the observation model was Supplementary Figure S2 the estimated virus loads in the nine largest municipalities with the daily number of positive tests, showing a similar correspondence between virus loads and case notifications as we observed between virus loads and hospitalisations.The estimated virus loads in the nine largest municipalities in the Netherlands are presented in 12 particles per 100,000 per day in many STPs in August 2020 but almost 1013 at the national level, and almost 1015 in the February 2022 peak but only just over 1014 at the national level. The fact that local epidemics were not fully synchronised is the reason that the national trend was less pronounced than those in the individual STPs . In the analyses, the standard deviation is estimated at 0.353, such that the estimated latent virus load can be approximately fivefold lower or higher than the individual measurements.More than 99% of the Dutch population is connected to the sewerage and thus to an STP. This makes it possible to perform high resolution and near real-time surveillance of pathogens that are shed in faeces, urine and other excreta and secreta ,16. HoweOur analyses using multilevel splines provide a convenient research tool to analyse sewage data, as it enables a natural borrowing of information from the national virus load and samples taken around the same time. This has not hitherto been included in other smoothing approaches of sewage data . In thisAlthough our analyses provide an adequate fit to the data, we do not claim that the results are optimal for all SPTs at all time points. Rather, they provide a parameter-sparse description of the data, linking the 317 STP time series through the national virus load. This seemed to work well in general, but it should be noted that the model has difficulty following very sudden changes in virus load data. A prominent example were the sudden and strong drops in virus loads around October 2021. This is only partially resolved by adding more knots to the splines, at the cost of greatly increasing computation times (data not shown). Future extensions could also focus on adding STP-specific measurement noise or perhaps even directly modelling the PCR data and water flow rates jointly.Our statistical modelling has provided a description of trends in virus loads at a local level by weighing all available wastewater measurements. In contrast, other modelling studies have focused on relating national or subnational virus loads to case notification data or hospitalisations using mechanistic modelling, with the aim to estimate the incidence and prevalence of the number of infections over time (-25 and rOur analyses provide steps to integrate noisy individual virus load measurements in sewage into more smooth estimates of the underlying latent virus load. This is valuable in itself, but we believe that the future value of the analyses will be mostly in combining the sewage data with other epidemic indicators such as hospitalisations or positive tests ,27. In aWastewater-based surveillance of SARS-CoV-2 can track long-term epidemic progression at a local scale in near real time, even in the presence of substantial noise and day-to-day variation. This can support public health authorities by early notification of local increases in virus circulation in a semi-endemic situation with decreasing numbers of case notifications and hospital admissions.