Saccharomyces cerevisiae fermentation product (NTK) in response to a mastitis challenge. Eighteen mid-lactation multiparous Holstein cows (n = 9/group) were fed the control diet (CON) or CON supplemented with 19 g/d NTK for 45 d  and then infected in the right rear quarter with 2500 CFU of Streptococcus uberis . After 36-h, mammary gland and liver biopsies were collected and antibiotic treatment started until the end of P2 . Cows were then followed until day 75 . Milk yield (MY) and dry matter intake (DMI) were recorded daily. Milk samples for somatic cell score were collected, and rectal and udder temperature, heart and respiration rate were recorded during the challenge period (P2) together with blood samples for metabolite and immune function analyses. Data were analyzed by phase using the PROC MIXED procedure in SAS. Biopsies were used for transcriptomic analysis via RNA-sequencing, followed by pathway analysis.We aimed to characterize the protective effects and the molecular mechanisms of action of a CATHL4, NOS2), epithelial tissue protection (e.g. IL17C), and anti-inflammatory activity . Pathway analysis indicated upregulation of tumor necrosis factor α, heat shock protein response, and p21 related pathways in the response to mastitis in NTK cows. Other pathways for detoxification and cytoprotection functions along with the tight junction pathway were also upregulated in NTK-fed cows.DMI and MY were not affected by diet in P1, but an interaction with time was recorded in P2 indicating a better recovery from the challenge in NTK compared with CON. NTK reduced rectal temperature, somatic cell score, and temperature of the infected quarter during the challenge. Transcriptome data supported these findings, as NTK supplementation upregulated mammary genes related to immune cell antibacterial function (e.g., Overall, results highlighted molecular networks involved in the protective effect of NTK prophylactic supplementation on udder health during a subclinical mastitic event.The online version contains supplementary material available at 10.1186/s40104-021-00560-8. Escherichia coli, Streptococcus uberis, and Staphylococcus aureus [Bovine mastitis, an inflammation of the mammary gland, is one of the most-common and economically-important diseases in the dairy industry. Primarily caused by bacterial intramammary infection (IMI), predominantly s aureus , clinicas aureus . Despites aureus –4. With s aureus , 6, and s aureus , 7, 8, tSaccharomyces cerevisiae (SCFP) were reported to increase leukocyte function both in vitro and in vivo [Traditionally, treatment of clinical cases of mastitis and control of mastitis in dairy herds utilize antimicrobial agents as a blanket approach of antibiotic. However, given societal concerns, dairy production systems have a renewed focus to reduce the reliance on nonselective antibiotic use and develop new and sustainable strategies to manage and control infectious disease . Nutriti in vivo , 10, and in vivo –14. When in vivo . The SCF in vivo , 17.Strep. uberis, and to address its molecular mechanism of action. Strep. uberis, a Gram-positive and coagulase-negative bacteria, was chosen as one of the major pathogen that causes environmental mastitis, leading to both clinical and subclinical cases, some of which could turn chronic and eventually cow-associated [In the current study, our objective was to investigate the efficacy of SCFP  during a case of subclinical mastitis, modeled via an IMI challenge involving sociated .Animal systemic response and the effect of SCFP supplementation on clinical parameters and a comprehensive blood biomarker panel were characterized. Furthermore, the local response and physiological mechanisms of mammary gland tissue were assessed via RNA-sequencing combined with bioinformatics analysis. Our hypothesis was that SCFP supplementation (bioactive mixture of compounds) would reduce the severity of mastitis by activating local leukocyte responses and epithelial tissue defenses.All procedures involving animals received approval from the Institutional Care and Use Committee at the University of Illinois, Urbana (protocol no. 17166).n = 9), and a supplemented group receiving 19 g/d of a Saccharomyces cerevisiae fermentation product  manually top-dressed over the basal diet, as per manufacturer recommendations. Groups were balanced for DIM, lactation number, intake, milk production, body weight, body condition score, and somatic cell count (Suppl. Table Eighteen multiparous Holstein cows past peak lactation (> 60 d postpartum) from the University of Illinois dairy herd were used for this study. To be eligible, cows must have exhibited composite milk somatic cell count < 200,000 cells/mL in two consecutive Dairy Herd Improvement Association (DHIA) samplings, and cows must not have been treated for clinical mastitis or any other diseases during early lactation. Furthermore, the current lactation must have not been the result of a twin pregnancy. Eligible cows were housed in individual tie-stalls, had free access to water, and were milked three times daily at 04:00, 12:00, and 19:30 h. Diet was provided ad libitum as a TMR once daily at 08:00 h. Ration was formulated to meet NRC (2001) requirements  phase 1, from the beginning of the experimental period until day 44, (ii) phase 2, from day 45 to 53, which represents the mastitis challenge period, and (iii) phase 3, from day 54 to 75, to observe the recovery from the challenge.Strep. uberis  measured by spectrophotometry . The bacterial solution was then serially diluted to a final concentration of 2500 CFU/mL in sterile PBS. Part of the inoculum was plated overnight at 37 °C on TSA to confirm the concentration. Resulting counts were between 2000 and 2500 CFU/mL.At day 45 of the experimental period, the first day of phase 2, all cows from both groups were subjected to a mastitis challenge via intramammary injection of 2500 colony forming units (CFU) of esville, ). Upon dStrep. uberis challenge. Before inoculation, the right rear quarter was cleaned with 70% isopropyl alcohol pads  and infused with 1 mL of bacterial inoculum with a sterile disposable syringe fitted with a plastic disposable teat cannula . Immediately following inoculation, the inoculum was massaged upward in the quarter, and both rear teats were immersed in a teat disinfectant containing 1% iodine. Due to the labor intense sampling post inoculation, cows were inoculated in batches of 2 or 4, balanced per treatment. As indicated by visual and manual inspection and SCS data prior to inoculation, all inoculated quarters were healthy at the time of inoculation. After 36 h from the challenge, following the afternoon milking of the following day, both rear quarters were biopsied and treated with a daily infusion of 125 mg of ceftiofur HCl  for 7 consecutive days, terminating the challenge. Day 53 of the experimental period represents the end of phase 2, as the first day without antibiotic treatment. Animals were then followed (phase 3) to monitor their recovery from the mastitis event up to day 75 of the experimental period, which represent a full month  from the inoculation intramammary of Strep. uberis. At the end of phase 2, two animals (one per each treatment group) developed severe mastitis in non-challenged quarters that sustained a reduction of DMI and near total cessation of lactation due to cross contamination of the infection to other quarters, and were removed from the study, reducing the number of animals per group to 8 in phase 3.After the morning milking, cows from both treatments  were moved back to their stall for the Dry matter intake and milk production were recorded daily. Composite milk samples were prepared in proportion to milk yield at each milking, preserved , and analyzed for contents of fat, protein, lactose, solid-not-fat (SNF), milk urea nitrogen (MUN), and SCC by mid-infrared procedures  in a commercial laboratory . Due to the consequences of the biopsy procedure on the challenged quarter (bloody milk and clots), daily production data from day 47 through 49 of the experiment were dropped from the final data set, and composite milk collection was not conducted during phase 2. On day 46 of the experimental period, production data from the evening was dropped as well as the first data after the surgical procedure. Body weight (BW) and body condition score (BCS) were recorded weekly. Three individual scorers were used at each time point and their scores were averaged to avoid bias.Strep. uberis. Furthermore, samples were taken also at 3, 7, and 9 d after inoculation, which corresponded to 72, 168, and 216 h from the challenge. These samples were collected after the morning milking before feed distribution into vacutainer tubes containing lithium heparin  and placed on ice. Plasma was obtained by centrifugation at 2000×g for 15 min at 4 °C and aliquots stored at − 80 °C until further analysis. Concentration of non-esterified fatty acids (NEFA), β-hydroxybutyrate (BHB), glucose, cholesterol, urea, minerals , ceruloplasmin, albumin, aspartate aminotransferase (AST), γ-glutamyl transferase (GGT), bilirubin, alkaline phosphatase, haptoglobin, creatinine, paraoxonase (PON), total reactive oxygen metabolites (tROM), myeloperoxidase (MPO), ferric reducing ability of plasma (FRAP), interleukin-1β (IL1β), interleukin-6 (IL6), and nitric oxides  were analyzed as previously described, using a combination of ELISA, colorimetric, and UV methodologies [Blood was sampled from the coccygeal vein at 0 (right after inoculation), 12, and 36 (right before biopsy) h from inoculation with Samples collected at 0, 12, and 36 h from bacterial inoculation, including a baseline sample 48 h prior to inoculation, were also analyzed for innate immune cells response, measuring monocyte and neutrophil phagocytosis capacity and oxidative burst activity via a flow cytometry-based assay as previously described .Strep. uberis infusion. At the same time points udder temperature for each rear quarter was measured using an infrared camera . Individual rear quarter milk samples were collected at − 1, 6, 13, 22, and 30 h approximately relative to Strep. uberis infusion  for SCC analysis. SCC were converted to the linear somatic cell score (SCS), calculated as log2 + 3 where SCC is in units of cells/mL. For udder quarter data (temperature and SCS) a delta was calculated between the right rear  and left rear  of each cow to better account for the individual response to the intramammary challenge .During phase 2, several health and clinical parameters were collected to assess the animal response to the intramammary challenge. Rectal body temperature, and heart and respiration rates were measured at − 24, 0, 6, 12, 18, 24, 30, and 36 h relative to Liver and mammary gland rear quarter  tissues were harvested approximately 36 h post intramammary challenge and on day 73 of the experimental period  from all animals enrolled via percutaneous biopsy. The procedure was conducted under mild general anesthesia with xylazine and local anesthesia with lidocaine HCl. Due to the systemic nature of the liver as an organ, its tissue was sampled first. Liver biopsies were harvested using the same procedures as described previously . For mamStrep. uberis inoculum) was used for mammary gland RNA extraction and subsequent analysis. Tissue was weighted (~ 0.05 g), immediately placed in QIAzol Lysis Reagent  (1 mL) and homogenized using a Mini-Beadbeater-24  with two 30 s cycles, and 1 min incubation on ice in between the cycles. Samples were then centrifuged for 10 min at 12,000×g and 4 °C, and the supernatant was transferred to a separate tube and mixed with chloroform (0.2 mL). After centrifugation for 15 min at 12,000×g and 4 °C, the aqueous phase was transferred to a new tube, mixed with 100% ethanol (0.75 mL), and total RNA was cleaned using miRNeasy mini kit columns  following manufacturer’s protocols. During purification, genomic DNA was removed using the RNase-Free DNase Set . Quantity was determined using a Qubit Fluorometer , while integrity was assessed via a Fragment Analyzer™ . All samples had an RQN  score greater than 7.0. RNA samples were stored at − 80 °C until analysis.Only the rear right quarter  were used for analysis. After quality control, samples were handed to the DNA Service Lab of the University of Illinois Roy J. Carver Biotechnology Center for library preparation and sequencing. RNA-seq libraries were constructed with the TruSeq Stranded mRNA Sample Prep kit . The final libraries were quantitated with a Qubit Fluorometer  and the average library fragment length was determined on a Fragment Analyzer™ . The libraries were diluted to 10 nM and further quantitated by qPCR on a CFX Connect Real-Time qPCR system  for accurate pooling of the barcoded libraries and maximization of number of clusters in the flowcell. Libraries were pooled by tissue and loaded on 4 lanes (2 per tissue) of an 8-lane flowcell for cluster formation and sequenced on an Illumina HiSeq 4000. The libraries were sequenced from one end of the cDNA fragments for a total of 100 bp. The fastq read files were generated and demultiplexed with the bcl2fastq v2.20 Conversion Software . The quality of the resulting fastq files was evaluated with the FastQC software, which generates reports with the quality scores, base composition, k-mer, GC and N contents, sequence duplication levels and overrepresented sequences. On average, 20–25 million reads per samples were obtained , and conducted by phase. All data were subjected to repeated measures ANOVA using PROC MIXED. Dietary treatment (TRT), time, and their interaction (TRT×time) were used as fixed effect. Time was omitted as a factor when only one data point was collected  for some measurements. Cow was the random effect. For analysis in phase 1, data collected during the adaptation phase, prior to treatment imposition, were used as a covariate. The Kenward-Roger statement was used to compute the degrees of freedom. Covariance structure for the repeated measurement were tested and the most appropriate  was chosen for each analysis. For intake, milk production and composition, BW, and BCS Autoregressive 1 was chosen. Spatial power was implemented for clinical data , blood analysis, and immune function data, while compound symmetry was selected for SCS data around the challenge. Normality of the residuals was checked via PROC UNIVARIATE. All data were normally distributed. Statistical significance was determined at Bos taurus UMD 3.1.1 reference genome using default settings of STAR 2.6.0 [Data were analyzed separately by tissue . Alignments and counts were performed on the Carl R. Woese Institute for Genomic Biology Biocluster of the University of Illinois High-Performance Biological Computing. Single-end reads were first filtered using Trimmomatic 0.33  with a mAR 2.6.0 . UniquelAR 2.6.0  in the S2-transformed, edgeR was used to conduct differential expression analyses. The applied statistical model included dietary treatment, time, and their interaction as fixed effect. Differentially expressed genes (DEG) across time points were determined with a combination of fold-change (> 1.5 or < − 1.5) and raw P-value (< 0.05) thresholds to balance for reproducibility, sensitivity, and specificity or results [Further data analysis was conducted using R. 3.5.1 . Reads uniquely assigned to a gene were used for subsequent analysis. Genes were filtered if 3 samples did not have > 1 count per million mapped reads. A TMM  normalization was applied to all samples using edgeR . After d results , 28 as d results –31. For The dynamic impact approach (DIA) was used for Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of DEG. The detailed methodology of DIA is described elsewhere . DiffereP(TRT) = 0.07), no main effect of treatment was detected (P(TRT) > 0.10). NTK cows tended to have slightly higher BCS (Table P(Time) = 0.14), time effects were detected for all parameters (P(Time) < 0.10). Milk yield, DMI (kg and %BW), fat and solids percentage, and SCS had a decreasing trend over time, while BW, BCS, and protein and lactose percentage tended to increase  = 0.02), but no statistical differences between treatment groups were detected at any time point.During phase 1, except for BCS  > 0.10). The imposition of the challenge caused an initial depression of daily milk yield and DMI, with lowest values reached at day 46 and 47, respectively, followed by recovery of both parameters towards the end of the phase (P(Time) < 0.05). (Table P(TRT×Time) = 0.08) and per milking (P(TRT×Time) = 0.01) milk yield in phase 2. For daily milking no difference between groups was detected within each time point, while NTK cows had higher (P < 0.05) milk production in morning and night milkings on day 51 and 52, respectively, while there was a tendency (P < 0.10) for higher milk yield for NTK in the day 52 morning milking and day 53 evening milkings  > 0.10), while a time effect was observed for daily milk yield, percentage of MUN, and SCS (P(Time) < 0.05), with all decreasing over time  = 0.08), as NTK supplemented cows had greater (P < 0.05) milk lactose percentage at 10 weeks.  ≤ 0.05) except for delta udder temperature (P(Time) = 0.08), and monocyte oxidative burst (P(Time) = 0.26, Table A time effect was observed for all parameters (P(TRT) = 0.02 and P(TRT×Time) = 0.05). At the udder, NTK supplemented cows tended to have a lower delta temperature (P(TRT) = 0.06), and overall lower (P(TRT) = 0.05) delta SCS (Table P(TRT) = 0.05) overall circulating phagocytosis capacity of monocytes (Table P(TRT×Time) < 0.05) revealed how feeding NTK lowered both monocyte and neutrophil phagocytosis capacity at 36 h post inoculation  < 0.05) with circadian concentration fluctuation in the first 36 h, and up or downwards trends post biopsy (Table P(TRT×Time) = 0.05, Suppl. Fig. All metabolic parameters changed over time (sy Table . A trendP(TRT) > 0.10) was observed for inflammation biomarkers and acute phase proteins. Concentrations of MPO, haptoglobin, albumin, and ceruloplasmin were affected by time (P(Time) < 0.0001), with a downward trend for albumin and an upward trend for others  = 0.06), as its concentration tended to increase 36 h post inoculation. An interaction of dietary supplementation and time was observed for IL-1β (P(TRT×Time) = 0.05)  > 0.10). Except for NO3 concentration (P(Time) = 0.15), there was a Time effect for other parameters in this category (P(Time) < 0.05). ROM concentration was higher at 168 and 216 h post inoculation, while NOx, NO2, FRAP, and PON concentrations were at their lowest at the same time points  < 0.0001), with downward trends over time  > 0.10) for greater AST in NTK supplemented cows  = 0.10), concentrations of minerals were affected by time (P(Time) < 0.01). Magnesium and zinc concentrations were lowest at 36 and 72 h post inoculation, then increased to pre-challenge levels  = 0.01), and an interaction with time was identified for sodium concentration (P(TRT×Time) = 0.05). NTK cows displayed greater overall levels of calcium, and higher levels of sodium at 0 and 36 h post inoculation  collected 36 h post inoculation and on day 73 of the experimental period were used for transcriptomic analysis. However, the current manuscript focuses specifically on the treatment comparison (NTK vs. CON) at 36 h post inoculation.Liver and mammary gland samples from the right rear quarter . The lack of systemic changes could be due to the short duration of the challenge (36 h), while the systemic increase or decrease observed in these biomarkers beyond 36 h post inoculation were likely imputable to the surgical procedure (biopsy) of both mammary gland rear quarters. Despite the lack of changes in the first phase, when animals were kept under observation without the addition of external insults, effects of NTK supplementation were observed once the IMI challenge with Strep. uberis was imposed. The overall lower rectal temperature and SCS, together with lower corrected (infected minus control quarter) udder temperature and SCS underscored the effectiveness of NTK in reducing the severity of the IMI. In fact, by 30 h post inoculation, the infected quarter of NTK-supplemented cows had a SCS of 3.84, which corresponded to a SCC of 179,415 cells/mL, lower than the accepted threshold for a subclinical case [The occurrence of clinical signs  indicated effectiveness of the pathogen in creating a subclinical mastitic event, an outcome frequently observed in the field during ris IMIs . This chhallenge  h, whileStrep. uberis IMI challenge as a mastitis model was established, and NTK supplementation counteracted its effects, the transcriptomic and bioinformatic analysis allowed a deeper understanding of the molecular mechanisms (at the tissue level) associated with the observed responses. A graphical model was built to summarize the information gathered via RNA-seq  in NTK supplemented animals might suggest a greater chemotaxis and influx of immune cells to the mammary gland [IL17C in NTK supplemented cows supports the ability of SCFP to stimulate epithelial tissue production of antibacterial peptides and proinflammatory molecules enhancing host defense [Once the validity of seq Fig. . As an iry gland , 38. Lowry gland , 40, sug defense .TNF, CATHL4, and NOS2. TNF is the master-regulator of the inflammatory response, which orchestrates a powerful antimicrobial response by a variety of mechanisms [CATHL4 (cathelicidin-4 or indolicidin) is a tridecapeptide amide found in bovine neutrophil cytoplasmic granules with potent bactericidal function, capable of virtual sterilization of a suspension of S. aureus and E. coli [CATHL4 was upregulated more than 20-fold in these animals.When evaluating effect of increased host bactericidal capacity, the transcriptomic analysis revealed upregulation of chanisms , while C E. coli . Its higNOS2 is responsible for the synthesis of nitric oxide (NO), a potent cytotoxic molecule produced by neutrophils with the ability to further modulate their functionality  [CATHL4 expression in the parenchymal tissue of lactating cows was observed to be reduced during mastitic events [CATHL4 and NOS2 expression could indicate a stronger antimicrobial activity of the infiltrating immune cells in NTK-supplemented cows.The protein encoded by r traps) . Synthesc events . Howeverc events , 47. As c events , we beliSTS in control cows [Candida albicans, they displayed a greater antifungal activity [Strep. uberis IMI challenge. These two hypotheses match the observed clinical data, but need to be further tested in vitro and in vivo experiments.Bioinformatics analysis may have also detected two additional molecular signatures of a lower, or milder, mastitic event in the NTK-supplemented cows. The negative flux of ‘arachidonic acid metabolism’ suggested greater activation in control cows compared with NTK. This response was due to a combination of genes responsible for the synthesis of HETE (5- and 15-hydroxyeicosatetraenoic acids). Compared with healthy cows, a study involving dairy cows affected by coliform mastitis reported an elevation of these compounds in milk. Furthermore, the negative flux of ‘steroid hormone biosynthesis’ induced by greater expression of rol cows  suggesteactivity . Thus, tMT3 expression might have allowed the tissue to respond to the oxidative challenge imposed by the production of ROS from phagocytes [The inflammatory response is a beneficial process with the goal of containing and eradicating threats to the host organism. However, both dysregulation of its magnitude or duration can lead to detrimental effects such as damage to the host tissue and its functionality, and multiple chronic pathologies . Consideagocytes , 52. Actagocytes .HSPA6 and 1A , DNAJB13 and DNAJB1 , and HSPH1 (2.9 folds). Additionally, NTK also upregulated genes indirectly involved [BAG3 and ZFAND2A, 2.5 and 2.6 folds, respectively).Besides these mechanisms in response to specific stimuli, supplementation of NTK upregulated two more systems, the first of which was the heat shock protein (HSP) response, responsible for broader cytoprotection. HSPs, or stress proteins, are highly-conserved and present in all cells of all organisms . They arinvolved , 58 withCDKN1A , which impacted and drove the positive flux of ‘p53 signaling pathway’ and ‘Cellular senescence’ pathways, suggested a resolution of the inflammation via immune cell apoptosis [ATF3 (2.9 folds) and IER3 (2.2 folds) further supported the idea of a resolving inflammatory response. ATF3 is a transcription factor that controls inflammation by suppressing its signaling cascade to drive the tissue towards re-establishing homeostasis. In contrast, IER3 is a regulatory gene activated in response to stressors which define subsets of regulatory networks in the elimination of pathogens, and in the restoration of epithelial barrier functions, while exerting effects as regulator of apoptosis [The second system activated by NTK supplementation was the p21 cascade, responsible for the resolution of inflammation. Once inflammation is established and has accomplished its goal , it must be resolved to restore tissue homeostasis and prevent long-term damage. Together with an increased activation of the ‘apoptosis’ pathway in NTK-fed cows already at 36 h post-inoculation, the upregulation of poptosis . In factpoptosis . Its defpoptosis , 61.Strep. uberis invasion. Furthermore, the higher calcium concentration in peripheral blood suggested a lower presence of pyrogen in NTK-supplemented cows. Calcium concentration, in fact, was shown to drop when LPS was infused via the jugular vein in cattle [S. uberis is gram-positive) are quickly catabolized and not easy to detect in the circulation. An intact epithelium should lead to better recovery from the production drop induced by the mastitic event. Due to the limited number of animals, as previously discussed, our analysis was underpowered to effectively detect performance differences. However, towards the end of phase 2, few daily differences or tendencies were detected for greater milk production in NTK-supplemented cows. In phase 3, NTK-supplemented cows maintained numerically, but not statistically due to low number of cows and high standard errors, greater milk production compared with controls, returning to production values comparable to those pre-inoculation. Furthermore, despite the lack of statistical power and significance, a difference of 110 kg of cumulative milk production (or average of 5.3 kg/d/head) in phase 3 was observed for NTK animals.These data taken together suggest a greater cytoprotective activity in the udder of NTK-supplemented cows. No histological analysis was undertaken in the current experiment. However, by using other data as proxy, we can speculate that NTK supplementation helped maintain integrity of the mammary gland epithelial tissue, preventing damage induced by the invading pathogen. In NTK cows, the bioinformatics analysis highlighted upregulation of the ‘tight junction’ pathway responsible for the synthesis and modulation of the cell-to-cell connection, maintaining an intact barrier as a first line of defense from n cattle , 63, as n cattle . Lastly,Strep. uberis inoculation. However, some of the transcriptomic changes may be indicative of a priming effect induced by NTK supplementation. For instance, the HSPs system was in fact not only activated in the mammary gland, but upregulation of its components was observed in the liver as well , with as high as 26-fold change in expression, and six common genes  were upregulated in both mammary gland and liver , we do not place much emphasis in the discussion of the limited transcriptional changes  observed at the hepatic level . Molecular data generated highlight key features of the mechanisms of action of this product. Supplemented animals displayed a greater pathogen-killing capacity of such cells, and the activation of cellular mechanisms to enhance mammary gland cytoprotection against side effects of inflammation, while maintaining tissue integrity and health. Furthermore, similar effects were highlighted in the liver , which might explain the broad ameliorating effect of NTK supplementation against other environmental, immunological, and physiological stresses reported in the literature.Supplementation with a commercially-available Additional file 1. Provides detailed about diet composition (Suppl. Table 1), animal data prior to experiment (Suppl. Table 2), and complete results about the RNA-sequencing analysis, including sequencing and alignment performance (Suppl. Table 3), and full list of differentially expressed genes with relative fold-changes . Furthermore, it includes graphical representation of results reported only in tabular format in the main body: DMI as %BW , BW and BCS , milk composition , blood biomarkers of metabolic status, inflammation and APP, oxidative status, liver enzymes, and minerals . Included are also the full results of liver DIA pathway analysis .


Observational studies indicate that periodontal disease may increase the risk of colorectal, lung, and pancreatic cancers. Using a 2-sample Mendelian randomization (MR) analysis, we assessed whether a genetic predisposition index for periodontal disease was associated with colorectal, lung, or pancreatic cancer risks.Our primary instrument included single nucleotide polymorphisms with strong genome-wide association study evidence for associations with chronic, aggressive, and/or severe periodontal disease . We used summary-level genetic data for colorectal cancer , lung cancer , and pancreatic cancer . Four MR approaches were employed for this analysis: random-effects inverse‐variance weighted , Mendelian Randomization-Pleiotropy RESidual Sum and Outlier, simple median, and weighted median. We conducted secondary analyses to determine if associations varied by cancer subtype , sex , or smoking history (lung and pancreatic cancer). All statistical tests were 2-sided.P = .03), 3% increased risk of colon cancer (P = .02), 4% increased risk of proximal colon cancer (P = .01), and 3% increased risk of colorectal cancer among females (P = .04); however, it was not statistically significantly associated with the risk of lung cancer or pancreatic cancer, overall or within most subgroups.The genetic predisposition index for chronic or aggressive periodontitis was statistically significantly associated with a 3% increased risk of colorectal cancer (per unit increase in genetic index of periodontal disease; Genetic predisposition to periodontitis may be associated with colorectal cancer risk. Further research should determine whether increased periodontitis prevention and increased cancer surveillance of patients with periodontitis is warranted. Colorectal cancer, lung cancer , and pancreatic cancer together account for >2.9 million deaths per year globally . ColorecTwo-sample MR is an approach that uses summary association estimates (often from genome-wide association studies [GWAS]) to develop a genetic instrument index for the exposure and then applies the index to assess the association with the outcome in a different sample of the same underlying source population ,10. The Previous 2-sample MR studies used genetic instruments for periodontal disease  and otheP < 5 × 10−8 in a pooled analysis of multiple cohorts (focusing on populations of European descent to match the population demographics of our outcome data). We considered the evidence strong for SNPs that were positively associated with chronic periodontitis in 1 cohort (P < 5 × 10−6), nominally positively associated (P < .05) with severe chronic periodontitis in an independent replication cohort, and positively associated (P < 5 × 10−6) with chronic periodontitis in a meta-analysis of over 5000 European American individuals. Our secondary instrument included the 8 SNPs in the primary instrument and 6 additional SNPs  with moderate evidence for an association with periodontitis   ,22. We aWe used summary-level genetic data for colorectal cancer  from 125 478 participants  in the Genetics and Epidemiology of Colorectal Cancer Consortium , Colon Cancer Family Registry (CCFR), and Colorectal Transdisciplinary study (CORECT) ; lung caBecause we used only deidentified data, we did not need institutional review board approval for our analysis.2I statistic  (MRPRESSO (version 1.0). We constructed plots showing the genetic associations of the SNPs with periodontitis . Default parameters were used for each analysis. All statistical tests were 2-sided. Our cut point for statistical significance was .05.In our primary analysis, we estimated the association between each genetic predisposition index for having chronic or aggressive periodontal disease and colorectal, lung, and pancreatic cancer risks using the random-effects inverse-variance weighted (IVW) method. We also considered 3 other MR estimation methods , simple median, and weighted median) with different assumptions to evaluate the robustness of the IVW findings. A description of the strengths and weaknesses of each MR approach is provided in the iations) ,47. We aiotropy) . All staP = .03) but not with the risk of lung  or pancreatic cancers  and limited evidence of horizontal pleiotropy for the secondary analyses . Separate sensitivity analyses removing each of rs1537415 , rs3826782 , rs12461706 , rs1537415 and rs12461706 , and rs9984417  did not substantively change any of these results , a 4% increased risk of proximal colon cancer (P = .01), and a 3% increased risk of colorectal cancer among females , distal cancer , or colorectal cancer in men , with the MR-PRESSO method, a 1-unit increase in the genetic predisposition index for chronic or aggressive periodontitis was associated with a 2% increased risk of distal colorectal cancer (P = .03) and a 2% increased risk of colorectal cancer in men (P = .01). In secondary analyses including the 6 additional SNPs with moderate evidence for an association with periodontitis, none of the associations assessed with any of the MR methods remained statistically significant  and sex .We also investigated associations between genetic predisposition to having chronic or aggressive periodontitis and risk of lung cancer stratified by histologic type , smoker status (current or not current), and the combination of histologic type and smoker status . We did P = .02) and MR-PRESSO methods , colon cancer, and proximal colon cancer. Conversely, our 2-sample MR results were not consistent with the hypothesis that genetically predicted periodontal disease is linked to lung cancer or pancreatic cancer risk.Our observation of an MR association between genetic predisposition to periodontitis and increased colorectal cancer risk is supported by several observational studies ,49,50, tFusobacteria is among the quantitatively dominant microorganisms in dental plaque   that migrate to extra oral sites ,57. For l plaque ; it intel plaque . Notablyl cases) . Furtherl cases) . These ol cases) , supportIn contrast to meta-analyses of observational studies suggesting that periodontal disease is associated with the risk of lung and pancreatic cancer ,69,70, oAs with all MR studies, 1 limitation of our analysis is the potential for violations of the MR assumptions. For example, we could not directly test for the presence (or impact of) directional pleiotropy using MR-Egger due to the small number of genetic variants in our instrument. However, the overall consistency of our primary analyses using the IVW method and secondary analyses using different MR methods   suggestsStrengths of our study include the large number of cancer cases included in each analysis, our MR approach that limited the potential for confounding or reverse causation, and our systematic approach to SNP selection for inclusion in our genetic instrument. Previous 2-sample MR analyses that used genetic instruments for periodontal disease (examining noncancer outcomes) included SNPs identified from single GWAS articles without clear justification for using those specific articles and SNPs ,17. AnotOur 2-sample MR analysis utilizing a systematically developed genetic instrument suggests that a genetic predisposition to having chronic or aggressive periodontal disease may be associated with colorectal cancer risk. Additionally, our results suggest confounding is unlikely to fully explain previous observational studies’ claims for an association between periodontal disease and colorectal cancer. Our results were not consistent with the hypothesis that a genetic predisposition to having periodontal disease is associated with lung or pancreatic cancer risk; however, we cannot entirely rule out the possibility that periodontal disease is associated with either of these cancers. Taken together, our results suggest that increased attention to preventative oral health measures and increased cancer surveillance of patients with periodontitis may be warranted. Future research is needed to further elucidate biological pathways underlying the associations between periodontitis and cancer risk.This work was supported the AACR-Johnson & Johnson Lung Cancer Innovation Science grant number 18–90-52-MICH. LC was supported by the National Institute of Child Health & Human Development at the National Institutes of Health (grant number K12HD092535). KKT was supported by Cancer Research UK (grant number C18281/A29019). CA was a Research Scholar supported by Cancer Prevention Research Institute of Texas grant RR170048. Funding for the cancer consortia that provided genetic data for this analysis is listed in the Role of the funder: None of the funders had any role in this analysis or interpretation of the data; the writing of the manuscript; or the decision to submit this manuscript for publication.Disclosures: No authors have any conflicts of interest to disclose.Author contributions: Conceptualization: EAP, DSM. Data curation: MR, DSM, consortia . Validation: KKT, EB. Formal analysis: MR. Supervision: DSM. Writing—Original Draft: LC. Writing—Review and Editing: all authors.Acknowledgements: The authors would like to thank the study participants and staff of the Seattle Colon Cancer Family Registry and the Hormones and Colon Cancer study (CORE Studies). They would also like to thank Kimon Divaris for helpful guidance on selecting periodontitis-associated SNPs and genes.https://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/analysis.cgi?study_id=phs001273.v3.p2&phv=282571&phd=7215&pha=4930&pht=6171&phvf=&phdf=&phaf=&phtf=&dssp=1&consent=&temp=1; https://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs000206.v3.p2; https://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs000648.v1.p1; https://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs001078.v1.p1; and https://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs001499.v1.p1. Data unavailable in dbGaP can be requested from the respective consortia.Data underlying this article are available through dbGAP through the Oncoarray Consortium—Lung Cancer Studies dbGaP Study Accession: phs001273.v3.p2 at pkab037_Supplementary_DataClick here for additional data file.


Tranexamic acid (TXA) has shown significant reductions in blood loss and transfusion rates in total knee arthroplasty (TKA). However, the optimal administration route continues to be debated. The aim of this trial was to compare the effectiveness of intravenous (IV) versus peri-articular injection (PAI) application of tranexamic acid in patients undergoing total knee arthroplasty.We conducted a randomized controlled, double-blinded study. A total of 93 patients undergoing primary unilateral TKA were randomly distributed between 2 groups: the IV group  and the PAI group . The amount of total and hidden blood loss (HBL), drainage, transfusion rate, hemoglobin and hematocrit drift, and complications were recorded.P < 0.001) and HBL more than IV use of TXA (P < 0.001). No patients in either group received a transfusion. No symptomatic deep venous thrombosis or other severe complications occurred.Peri-articular injection of TXA reduced total blood loss (Peri-articular injection of TXA significantly reduced total blood loss and hidden blood loss to a greater degree than IV injection in total knee arthroplasty without reduction of drainage volume.ChiCTR-INR-16010270. Date of registration: December 27, 2016.Chinese Clinical Trial Registry,  Total knee arthroplasty (TKA) can result in an estimated 800 to 1200 ml of blood loss, which has traditionally resulted in a transfusion rate as high as 53% . The manTXA is a competitive inhibitor of plasminogen activation that interferes with fibrinolysis improving hemostasis and helping prevent additional blood loss after tourniquet deflation during TKA . This prHowever, the ideal method of topical or local techniques varies widely from the simple soaking of the wound prior to joint closure to intra-articular injections after deep fascial closure. However, the limited exposure time of TXA with local tissues raises concerns about both simple soaking and intra-articular injections, especially in patients undergoing significant soft tissue releases. Peri-articular injections (PAI) of TXA eliminate the concern of limited local effect and have been shown to significantly improve blood loss and transfusion rates when compared to intra-articular injections .Peri-articular injections aimed at multimodal analgesia are widely used in TKA and have proven to be more effective in providing analgesia compared to traditional methods , 13. To Given the above, we evaluated the benefits and complications of an analgesic peri-articular injection with the addition of TXA to a standard IV TXA injection.This double-blinded randomized control trial was conducted from January 2017 to July 2018, and the study protocol was approved by our institutional review board and is registered in the Chinese Clinical Trial Registry (ChiCTR-INR-16010270). Written informed consent and research authorization for participation in this study were obtained from each patient before surgery.3; (6) a history of a pro-thrombotic condition; (7) pregnancy; (8) breastfeeding; (9) donated preoperative autologous blood; (10) an age of < 18 years or > 80 years; and/or (11) a preoperative hemoglobin level of < 10 g/dl.Patients presenting during the study time frame undergoing elective unilateral, primary TKA for end-stage osteoarthritis or rheumatoid arthritis were screened for inclusion into the study. Patients were then excluded for any of the following reasons: (1) an allergy to TXA; (2) preoperative hepatic or renal dysfunction; (3) serious cardiac or respiratory disease, including coronary artery stent placement; (4) congenital or acquired coagulopathy, as evidenced by an international normalized ratio (INR) of > 1.4 or a partial thromboplastin time (PTT) of > 1.4 times normal; (5) thrombocytopenia, as identified by a preoperative platelet count of < 150,000/mmPatients were then randomized to 1 of 2 groups: IV administration of TXA or PAI TXA, in accordance with the random number table. The randomization schedule was generated after the initiation of study enrollment  with ropivacaine, 200 mg/20 ml ; morphine, 10 mg/1 ml (domestic company); flurbiprofen axetil injection, 50 mg/5 ml ; adrenaline, 0.25 mg (1:1000); betamethasone, 7 mg/1 ml ; and 34 ml of normal saline solution was prepared in three 20-ml syringes. Ten minutes before skin incision, patients received 1000 mg of IV TXA  IV administration.In the PAI group, the 60-ml MCPI was the same as that in the IV TXA group, except that TXA 1000 mg/10 ml  was added. Before incision, patients received 110 ml of saline IV administration as a placebo.All patients underwent TKA under tourniquet control by a single surgeon . A medial parapatellar approach was utilized, and either a G-II PS  or Scorpio  was inserted. No patella was resurfaced during the TKA procedure. The peri-articular injections were performed with the following technique: the first 20 ml of the cocktail was injected into the posterior aspect of the capsule and structures of the knee joint immediately before implanting the prosthesis. After implantation, the remaining 40 ml of the mixture was injected into the extensor mechanism, synovium, anterior capsule, pes anserinus, retinaculum, periosteum, iliotibial band, and collateral ligaments. A drain was placed within the lateral gutter in all patients prior to deep fascial closure.Postoperatively, a vacuum wound drainage dressing was applied in every patient and a standardized rehabilitation protocol was implemented starting on day 1. In both groups, the drains were removed at 48 h, and the volume of fluid at 24 and 48 h was measured. Chemical and mechanical deep vein thrombosis (DVT) prophylaxis was instituted in all patients and consisted of thromboembolic deterrent stockings along with the daily subcutaneous injection of low molecular weight heparin (0.1 ml/10 kg once a day) until discharge. The criteria for the transfusion of blood products included a hemoglobin level of < 8 g/dl or a hemoglobin level of < 10 g/dl in a patient with symptomatic anemia, or a patient deemed at high risk because of notable underlying cardiac comorbidities. After hospital discharge, patients were seen at 2 weeks, 4 weeks, and 12 weeks postoperatively. They were then seen twice a year or more frequently if complications had occurred.The primary outcomes measured were the total blood loss (TBL), hidden blood loss (HBL), hemoglobin (HGB) and hematocrit (HCT) drift, and need for a blood transfusion. Hidden blood loss was calculated using the method described by Sehat et al. . HGB andAdditionally, secondary outcomes included postoperative duplex ultrasounds at post-op days 3 and 14 to detect DVT. D-dimer levels were measured at 24 h postoperatively. Complications were measured up to 3 months postoperatively and included local soft tissue complications, skin necrosis, peroneal nerve palsies, superficial and deep surgical site infections, symptomatic VTEs, cerebrovascular accident, and myocardial infarction.The study design of this trial was an equivalence test, and the sample size was calculated based on the measured postoperative blood loss. We assumed an alpha error of 0.05 and applied an allocation ratio of 1. A sample size of 37 participants, which allowed for a dropout rate of 10% (4 participants), was calculated to provide 80% power in detecting a difference of 150 ml or reducing postoperative blood loss by 30% in favor of the PAI TXA (from 20 patients of our earlier pilot study); we considered these parameters to be clinically relevant [t tests. For categorical variables, data was summarized as the frequency and proportion. Proportions were compared between the groups using Fisher’s exact test on univariate analysis.The Statistical Package for Social Sciences  was used. For continuous variables, the mean and range were compared between the groups using Student A total of 114 unilateral primary total knee arthroplasties were screened for eligibility to be included in the trial. Ninety-eight patients met inclusion criteria and were randomized into the study groups, with each group consisting of 49 patients. Of the 98 patients, five were lost to follow-up, leaving 47 patients in the IV TXA group and 46 patients in the PAI TXA group for statistical analysis. A participant flowchart is provided in Fig. 2 . There were no significant differences between the 2 groups for baseline variables including age, sex, medical history, American Society of Anaesthesiologists (ASA) score, BMI, and preoperative laboratory values (P > 0.05) , and the mean body mass index (BMI) was 27 kg/mP = 0.00; HBL 419.8 ± 239.9 ml vs 651.7 ± 243.7 ml, P = 0.00)  . The D-dimer values 24 h after operation were not significantly different (P > 0.05)  of the patients sustained asymptomatic DVT compared with 2 (4.3%) of the patients in the PAI TXA cohort, as confirmed by postoperative ultrasound  are widely used in peri-operative pain protocols , 13. ThiThe optimal dosage of TXA in peri-articular injections has yet to be determined. Mao et al. found 2000 mg of TXA to be effective in reducing postoperative blood loss during TKA . While PGood et al.  reportedIn this study, there were no symptomatic VTE in either group, and the results of asymptomatic DVT confirmed by ultrasound and D-dimer measurements revealed no significant difference between the two groups. Although there was no control group to determine the effects TXA may have had on VTE development, the latest meta-analysis supports the safety of TXA in joint arthroplasty .We do acknowledge the limitations of our study. First, we did not assess functional factors such as VAS score, knee range of motion, knee swelling, and walking ability. Second, we did not measure serum levels of PAI TXA vs IV TXA, thus limiting our interpretation of its systemic effects. Third, this study excluded patients who would be deemed at the most risk if they were to receive IV TXA. To compare the safety of each modality, we would require a large number of patients and may not be practical to investigate in a prospective, randomized study. Fourth, the timing was also different with IV being given immediately preoperatively and PAI being injected in the later stages of the surgery, which may have made it difficult to identify the local effect of PAI TXA in reducing blood loss. In addition, our study did not involve the determination of the optimal dosage of TXA, which also is a shortage and needs further studies to compare whether different doses of TXA can produce different results.This randomized controlled trial has demonstrated encouraging results regarding the novel use of TXA as part of a combined peri-articular injection in TKA with a reduction in blood loss versus the more commonly used IV administration of TXA. The use of locally injected TXA is an effective and simple method of reducing blood loss and should be considered as an alternative to IV or topical application in primary TKA.


Plasmodium vivax malaria in South Asian countries, the genetic diversity of circulating parasite populations is not well described. Determinants of antimalarial drug susceptibility for P. vivax in the region have not been characterised. Our genomic analysis of global P. vivax (n = 558) establishes South Asian isolates (n = 92) as a distinct subpopulation, which shares ancestry with some East African and South East Asian parasites. Signals of positive selection are linked to drug resistance-associated loci including pvkelch10, pvmrp1, pvdhfr and pvdhps, and two loci linked to P. vivax invasion of reticulocytes, pvrbp1a and pvrbp1b. Significant identity-by-descent was found in extended chromosome regions common to P. vivax from India and Ethiopia, including the pvdbp gene associated with Duffy blood group binding. Our investigation provides new understanding of global P. vivax population structure and genomic diversity, and genetic evidence of recent directional selection in this important human pathogen.Despite the high burden of  Plasmodium vivax strains in South Asia isn’t well described. Here, the authors sequence P. vivax from returning UK travelers and establish South Asian isolates as subpopulation distinct from East African and South East Asian isolates.The genetic diversity of  Plasmodium vivax is now the leading cause of human malaria outside of sub-Saharan Africa1, where intensive disease-control interventions prioritising the control of P. falciparum, have had less impact on reducing the transmission of P. vivax2. Despite an estimated overall reduction of 61.0% in the burden of vivax malaria worldwide since 2000, there were 6.4 million cases reported in 20191. Some countries have seen recent worrying plateauing in rates of case decline, or even increases , partly associated with regions affected by political and economic instability, making the support of effective malaria control strategies difficult2. Further, drug resistance is potentially hindering malaria elimination. Resistance of P. vivax to chloroquine was first reported in 2003 in Papua New Guinea, and is now present in many P. vivax endemic countries3. As chloroquine susceptibility falls in P. vivax from these regions, the rates of treatment failure and relapse are increasing1. P. vivax has also developed resistance to the anti-folate drug combination sulfadoxine-pyrimethamine (SP), and possibly other antimalarial drugs such as mefloquine, used for P. falciparum where the two species are sympatric. As a result, artemisinin-based combination therapy (ACT) is now recommended for treating vivax malaria4. Other control measures which are traditionally effective against other Plasmodium parasites  have proven to be less effective against P. vivax5 due to its ability to form dormant liver-stage parasites6. These hypnozoites cause delayed blood-stage relapses, but can be cleared from the liver with primaquine or tafenoquine treatments, although both regimens can cause severe haemolysis in individuals with G6PD deficiency7.P. vivax malaria cases globally, but there is little understanding of the parasite ancestry, genomic diversity and underlying factors governing transmission and drug resistance. Similarly, Eastern Africa accounts for a further 8% of the global cases1, but only in southern Ethiopia has genomic diversity been studied8. This contrasts with sequencing studies in South East Asia and South America, where genome-wide analyses have provided evidence of clustering of P. vivax in geographically distinct subpopulations, and for selective pressure on genetic regions encompassing some P. falciparum orthologous drug-resistance loci, including pvdhps and pvdhfr genes linked to SP11. Applying such population genetic approaches to identify determinants of drug resistance is important, because the understanding of biological susceptibility mechanisms including frontline chloroquine is poor; in part, hampered by the inability to long-term culture P. vivax parasites in the laboratory. Further, studies of in vivo drug efficacy are complicated by the difficulty of determining whether parasite reappearance comes from a new infection, recrudescence after treatment, or from relapse due to hypnozoite activation.Countries in South Asia, including India, Afghanistan and Pakistan, account for nearly two-thirds of the total estimated P. vivax populations approaching elimination, which typically undergo a transition from high recombination rates and widespread intermixing, to clonal expansions with reduced genetic diversity12. Tracking this evolution of transmission patterns and inferring the geographical origin of isolates is aided by newly designed molecular barcodes13. Complementing these barcodes with South Asian and East African markers in putative drug-resistance genes will further assist infection control. Given the expansive P. vivax populations in the South Asia region are largely absent from population-based genomic studies, we have sequenced >118 P. vivax isolates in UK travellers with unambiguous travel histories returning from Bangladesh, India, Pakistan and Afghanistan. In general, travellers returning from malaria-endemic countries tend to have monoinfections, and provide an opportunity to investigate infections and drug efficacy in a potentially high-risk group14. The inclusion of these new South Asian parasite whole-genome sequences, together with new data generated from travellers to three East African countries  and previously published data, enables the largest study of P. vivax genomics to date (n = 558). By introducing data from little-studied geographic sources, our analysis provides expanded insights into the global ancestry, genomic diversity and population structure of P. vivax. We sought evidence that the substantial P. vivax populations of South Asia bear specific signals of selection in their genomes, and may harbour geographically distinct variants of drug susceptibility-associated loci and genes encoding red cell invasion proteins considered vaccine candidates. This knowledge can potentially inform the control of a disease with a high burden in this heavily populated region.Whole-genome sequencing analysis has provided insights into P. vivax. The final dataset comprised 558 isolates from 25 different countries , East Africa , South America , South East Asia , and Southern SEA . A total of 118  P. vivax isolates from travellers entering the UK with unambiguous histories were sequenced de novo, covering five countries that have not previously undergone whole-genome sequencing.A total of 388,933 high-quality SNPs were identified in the non-hypervariable regions in the genome of WS metric >95%), was more common among SEA non-traveller isolates , than among non-traveller isolates from South America  or returning traveller isolates from both South Asia  and East Africa   S. This obA SNP-based neighbour-joining tree revealed that South Asian and East African isolates occupy distinct positions as independent subpopulations within a global setting Fig. . An admipvmsp1 gene located on chromosome 7 (0.064) , a cluster of genes including PHIST proteins  and the pvdbp gene and its neighbouring regions . Three Afghanistan-specific signals were observed, one on chromosome 1 (710–740 kbp) spanning six genes, and two on chromosome 11, including 630–660 kbp, spanning 7 genes , and 1050–1070 kbp spanning 6 genes . One signal specific to Pakistan corresponded to a region on chromosome 8 (280– 300 kbp) spanning five genes, including pvgbp2 and pvtmem147. These regions could be candidates for inclusion in molecular barcodes for P. vivax.Analysis of identity-by-descent (IBD) at the country level was performed in order to understand the chromosome-level structure in the subpopulations. The isolates obtained from returning travellers from India (median: 0.024), Pakistan (median: 0.013) and Afghanistan (median: 0.013) presented lower fractions of pairwise IBD overall than other isolates sampled in more geographically limited regions, such as Mexico (median: 0.23) or Malaysia (median: 0.33); but similar to other subpopulations in SEA, such as Thailand (median: 0.026) or Cambodia (median: 0.017) (SFST) analysis. The FST distributions between putative drug resistance and other loci were different between SEA and South Asian or East African populations (Wilcoxon P < 0.012) (SFST > 0.8) varied in number, with South Asia (n = 5) and SEA (n = 6) having much less than the other regions  (Table pvdmt1 (S310R and V217I) and pvmrp1 ; for East Africa, South Asia and South America there was high differentiation in members of the CRMP gene family , implicated in mosquito transmission. Mutations in genes associated with mosquito stages of the parasite life cycle differentiated South Asian (pvs28 and pvs47) and South American  populations, likely linked to differences in species of mosquito vectors, where pvs47 has a role in parasite-mosquito interactions and its haplotypes present a marked geographical population structure15. Other loci with mutations with high FST in South Asia include the PVP01_0514700 gene (FST = 0.95) which has an unknown function, and the protein disulfide isomerase PDI8 (FST = 0.83). Similarly, East African-specific mutations occurred in the pvclamp gene linked to parasite motility, and PVP01_1444400 (max. FST = 0.91) with unknown function; whereas, there was a cluster of mutations on chromosome 11 surrounding PVP01_1129700 (max. FST = 0.99), which was South American-specific.The SNPs underlying differences between subpopulations were identified using a fixation index  0.012) S. The num2 S. The Plasmodium drug susceptibility loci in SEA, we identified non-synonymous (NS) mutations in P. vivax genes of interest and assessed their frequency across all regions: pvcrt (4 NS), pvdhfr (17), pvdhps (21), pvkelch12 , pvmdr1 (27), pvmrp1 (41) and pvpm4  , one in Mexico (E207Q), and one in Pakistan . Similarly, for pvkelch12, two mutations outside the propeller domain were observed in South Asia . No mutations in the pvcrt or pvkelch12 genes were identified in East Africa. Finally, in the pvpm4 gene, orthologous to piperaquine resistance-associated pfpm4 in P. falciparum, only one mutation (I165V) was present across all regions including both South Asia (7.7%) and East Africa (24.5%), and two other mutations were present only in Papua New Guinea.With strong population-differentiating effects implicated in s: pvcrt  NS, pvdhpvdhfr (PVP01_0526600) and pvdhps (PVP01_1429500) genes in the anti-folate pathway are thought to contribute to SP drug resistance, although there are very few published studies that associate genotypes of these loci with anti-folate susceptibility phenotypes18. We observed several pvdhfr mutations  in SEA or Southern SEA, but these were absent from South Asia and East Africa  at low frequencies (range: 3.6–10.7%). For pvdhps, previously described mutations A553G and A383G were observed in high frequency in SEA (67.6% and 96.3%) and Southern SEA (54.1% and 68.9%), with lower frequencies in South Asia (11.0% and 13.3%) and East Africa (0% and 32.0%). Six mutations were unique to the South Asian region  all present in low frequency across any of the three main countries (<13%). A further two mutations (L534I and E142G) were observed only in East Africa, with L534I exclusively in Uganda and E142G present across all four constituent countries. Based on in silico protein modelling, eight mutations in the PVDHFR  and four in the PVDHPS  are within a distance of 10 Å of the drug-binding site and could be expected to change the affinity of the drugs and proteins  with the exception of South America (11.9%). The Y976F mutation was at high frequency in Southern SEA (88.1%) and also present in East Africa (54.0%) and SEA (32.3%), and at lower frequency in South Asia (2.2%) and South America (10.1%). Two mutations  were found at higher frequency in South Asia and East Africa (>35%) than in SEA and Southern SEA (< 1%), with S698G fixed in South America (100%). The N1657S mutation is found only in Malaysia. Eight mutations were observed in South Asia only, but only three  were common (>7%)  coincided with the high population differentiation FST results  revealed the proximity of pvmrp1 mutations  to established drug-resistance phenotype causing mutations in P. falciparum in the vicinity of the 2nd ABC transporter domain  at a country and region level, for populations with at least ten isolates. We found regions containing the pvmrp1 in SEA, the antigenic and potential vaccine targets pvmsp4/5 genes in SEA, South America and Papua New Guinea; as well as further regions upstream of the pvdhfr gene, a region surrounding the MSP7-like cluster of genes on chromosome 12 in South America, and a cluster of PHIST exported genes on chromosome 14 in Papua New Guinea  , which included clusters surrounding pvmrp1, pvcrmp2, antigenic targets , uncharacterised genes on chromosome 6 (PVP01_0620400 to _0621000), reticulocyte-binding and invasion loci (pvrbp1a/b), the pvdhfs gene that encodes the bifunctional enzyme dihydrofolate synthase/folylpolyglutamate synthase (upstream of DHFR in the folate biosynthesis pathway), and uncharacterised exported proteins on chromosome 14 (PVP01_1427400 to _1427800)  (SPVP01_1126100 to _1126500) and 14 (PVP01_1469900 to _1470700) which included tryptophan-rich protein-coding genes . Similarly, for the isolates from Afghanistan, the most significant SNPs were found in a cluster of exported proteins on chromosome 8 (P < 1 × 10−10), as well as a region surrounding the pvkelch06 (P = 1 × 10−6), being orthologous to the artemisinin susceptibility-related pfkelch10 gene21.For the South Asian subpopulations, ten chromosomal regions had multiple SNP hits  , as well as a cluster including pvmsp4/5, and multiple genes on chromosomes 5 (PVP01_0525800 to _0526100), 11 (PVP01_1132900 to _1133600) and 14 (PVP01_1401000 to _1401900). These results were supported by individual South Asian country comparisons with Thailand, including finding regions encompassing pvdhfr and pvdhps genes , and  pvmrp1 (India P < 1 × 10−15). A comparison between South Asia and East Africa highlighted pvdbp, involved in red blood cell invasion, and pvpk4 which is a drug target for inhibiting PK4 kinase activity with potency across species of malaria parasites. P. vivax isolates from Ethiopia displayed evidence of selection on regions of chromosomes 2 (including pvmrp1) and 10 (spanning pvmsp8), but with differential signals across South Asian population comparisons. Between country comparisons within South Asia (Supplementary Data Rsb identified 71 regions (P < 1 × 10−5), and reinforced differences with isolates in India, including genetic regions encompassing pvdbp, pvmrp1, pvmrp2, sporozoite invasion-associated protein 2 (pvsiap2) and antigen UB05 . Only 17 differences occurred between Afghanistan and Pakistan, including pvcrmp2 and a high density of exported proteins on chromosomes 6 and 8.We identified evidence of geographically distinct signals of selection using the P. vivax or directional selection (SPVP01_0525100), antigenic targets  and conserved proteins of unknown function . From above, the pathogenesis-related pvmsp5 locus also exhibited evidence of differential selection between populations, more broadly, reflecting observed complex haplotype structures of different strains circulating in different populations8.To study allele frequency distributions for individual genes across populations, our analysis focused on the 4826 loci that had at least five SNPs. Tajima’s D values were predominantly negative , being most negative in Thailand (median −1.545), consistent with a historical population expansion of lection S Figure. lection S includedpvmrp1, pvdhfr and pvdhps) with population-specific mutations within India, Afghanistan/Pakistan and Thailand, with signatures of directional selection. There was a modest correlation between selection (Rsb) and population differentiation (FST) statistics in regional comparisons for coding SNPs , where high values for both metrics included mutations in pvdhps, pvdbp, p28/47 mosquito-related and the CRMP family of genes  are unlikely to have had a major impact on our findings. However, we recognise that the exclusion of polyclonal infections disproportionately from endemic populations may have led to some underestimation of genetic diversity among these isolates compared to those from travellers. The lower multiplicity of infection in travellers  cannot be taken as an indication of lower transmission intensity in the source country. Further, the returning traveller samples were almost exclusively derived from passive sampling. Several studies have suggested that parasites derived from passively detected symptomatic infections have similar genotypes to actively detected asymptomatic infections22. These findings suggest that the development of clinical symptoms may be more attributable to host factors such as immunity and red blood cell polymorphisms, than to intrinsic parasite factors resulting in a higher multiplication rate. There are likely to be differences in demographic , and health characteristics  in returning travellers compared to resident populations, which might increase either susceptibility to malaria infection or the risk of severe disease.Despite South Asian countries contributing collectively to more than half of the estimated 12. This subpopulation encompasses India, Afghanistan, Pakistan, Bangladesh and Sri Lanka, although isolates do share some features with parasite genomes from East Africa and SEA. This distinct characteristic is consistent with allopatric isolation by distance. Furthermore, at an intra-regional level, a degree of genetic separation was observed for isolates from India compared to Afghanistan and Pakistan, which are indistinguishable from each other, and an indication of parasite migration and gene flow between these two countries. In East Africa, despite the small sample size, a clear separation was observed for isolates from travellers returning from Uganda compared to other East African countries. Furthermore, isolates from Eritrea and Sudan seemed to cluster together and were clearly separated from Ethiopian isolates in the principal component analysis, although ADMIXTURE analysis did not reveal a separate ancestral population. This observation is consistent with recent reports of genetic diversity of P. vivax in Sudan using microsatellites23, but further studies of P. vivax in this region are warranted, given the recent history of cross-border refugee movements in the 20th and 21st centuries. P. vivax isolates from travellers to Guyana and The Philippines clustered near the South American and South East Asian populations as expected. The consistency of results at the geographic level combined with the observation that newly sequenced Ethiopian isolates clustered together with previously described Ethiopian sequences8 indicates that using returning travellers can be a robust approach to study difficult-to-access parasite populations. However, the typical lack of resolution of geographic information below national or, at best, provincial level, mean results should be backed up by future studies using systematically collected population samples from endemic communities in South Asia and East Africa.Population genetics analysis of the sequenced South Asian isolates provides evidence of an ancestral parasite subpopulation distinct from those in Africa, SEA and South America25. Analysis of the overall proportion of the genome that presented evidence of IBD revealed that isolates obtained from returning travellers displayed greater genetic diversity than in some highly related sampling populations, in particular Mexico and Malaysia. This result suggests that perhaps infections from returning travellers may be more diverse because they are not geographically defined by a given catchment area, whereas studies of endemic malaria tend to be geographically centred, and thus represent more coherent population samples. However, an alternative explanation might be that the parasite populations in Mexico and Malaysia are less diverse and highly related compared to all populations, because of lower transmission, leading to smaller effective population size. Our observed levels of IBD among traveller isolates from each country of interest were comparable to those of other populations such as Thailand or Cambodia, further supporting this alternative explanation. The genomic regions identified with a high proportion of IBD in different subpopulations included the liver-specific protein 2 (LISP2) on chromosome 3, a region on chromosome 10 including the Myosin B protein, and the pvmsp1 gene region located on chromosome 7. A remarkable similarity was found between the regions of high IBD in India and Ethiopia, including a strong signal surrounding the pvdbp gene, the main invasion receptor that interacts with the human Duffy antigen to invade red blood cells. P vivax infections have now also been reported in Duffy-negative individuals, and pvdbp gene alterations, such as duplications have been suggested as a mechanism contributing to this adaptation27.Identity-by-descent (IBD) analysis was applied to further understand the structure and selection within parasite populationspvmrp1, pvdhfr and pvdhps genes11 in South Asia and other previously studied populations , as well as in the pvmsp4/5 regions on chromosome 4 for Ethiopia. Importantly, the analysis also revealed new regions which had not been previously reported as presenting with positive selection, including those surrounding the pvrbp1a and pvrbp1b genes and the folate pathway gene pvdhfs in South Asia. Furthermore, a region surrounding the pvkelch10 gene was identified in Afghanistan as being under selection pressure, where this locus is an orthologue to the pfkelch13 gene driving resistance to artemisinin in P. falciparum28; although no record of artemisinin resistance has been described in P. vivax currently, ACT regimens are now commonly used to treat P. vivax infections in many countries, reflecting guidance from the WHO29. Phenotypic information from in vivo ACT susceptibility studies linked to further evaluation of genetic variants at these loci is now needed to understand any selection acting upon the kelch propeller domain proteins of P. vivax.Population genetic analysis revealed genomic regions undergoing recent positive selection, including some encompassing loci linked to drug resistance such as the pfmrp1 gene, encoding an ABC transporter transmembrane protein, is a potential multidrug-resistance candidate in P. falciparum with mutations associated with reduced susceptibility to SP (R1466K)30, chloroquine (F1390I)32, mefloquine (T1007M)31 and pyronaridine (H785N)33. Previous population genomic analyses of P. vivax populations in SEA and Oceania found evidence that the pvmrp1 gene is under strong selection11, and our study provides evidence for this in South Asian isolates. Further, we found that pvmrp1 contributes to IBD structure in other populations such as in Ethiopia. Previously unreported mutations were observed in South Asian populations, where pvmrp1 exhibited a high frequency of two non-synonymous mutations (D1393Y and G1419A) compared to other regional subpopulations, supporting the strong positive selection signal. Furthermore, a comparison of the pvmrp1 and pfmrp1 sequence similarity revealed that non-synonymous mutations found in pvmrp1 specific to the South Asia region, overlap with pfmrp1 locations residing between the ABC transmembrane and the second ABC transporter domains. Pfmrp1 mutations have been shown to confer resistance experimentally in P. falciparum32. In the absence of a long-term P. vivax in vitro culture system for validation, experimental strategies such as orthologue replacement transgenesis in P. knowlesi, in which P. vivax alleles of interest replace endogenous loci, can allow in vitro assessment of drug susceptibility for these variants19.The P. vivax population structure and genomic diversity in three high burden South Asian countries, and further advanced our understanding of the genomic composition of P. vivax around the globe10. Despite some limitations of sampling from returning travellers, including reduced levels of the multiplicity of infection and therefore disconnection from transmission intensity levels in the origin populations, this approach has delivered valuable genome-wide data from otherwise uncharted P. vivax parasite populations. The evidence presented relating to positive selection evidence in the South Asian region for drug-resistance candidates such as pvmrp1 is founded on previously established findings in other endemic regions12. The direct investigation of the phenotypes of gene variants in P. vivax19, and identification of potential genetic drivers of drug resistance using recent advancements in laboratory strategies such as orthologue replacement, is now possible.Overall, this study has provided analyses of P. vivax isolates were included in the study, including previously publicly available data13 (n = 598) and newly sequenced (n = 132) isolates from imported UK cases with blood samples sent to the Public Health England Malaria Reference Laboratory. After quality control, the final dataset included 558 isolates from 25 countries spanning all the P. vivax endemic regions , Colombia (34), Guyana (3), Peru (58), Mexico (20)), East Africa , Ethiopia (29), Madagascar (1), Sudan (5), Uganda (3)), South Asia , Bangladesh (1), India (36), Sri Lanka (1), Pakistan (32)), South East Asia , China (1), Laos (2), Myanmar (9), Thailand (128), Vietnam (14)) and the Western Pacific and southern South East Asia , Papua New Guinea (25), Indonesia (9), The Philippines (1)). These included both publicly available data  and newly sequenced samples . Sequencing of the newly generated isolates (n = 132) used DNA extracted from blood samples sourced from returning travellers to the UK from endemic areas between 2017 and 2018 and was performed on an Illumina HiSeq 4000 platform. The 132 samples were sourced from travellers to Afghanistan (n = 26), Bangladesh (n = 1), Eritrea (n = 11), Ethiopia (n = 6), Guyana (n = 3), India (n = 38), Pakistan (n = 35), The Philippines (n = 1), Sudan (n = 7) and Uganda (n = 4). For the isolates from India, country regional level data were available for 32 isolates, with 10 being from Mumbai, 8 from Gujarat, 7 from Goa and 7 from Punjab. The UK National Research Ethics Service (Ref: 18/LO/0738) and LSHTM Research Ethics Committee (Ref: 14710) provided approval for the project “Drug susceptibility and genetic diversity of imported malaria parasites from UK travellers”. Informed consent was obtained from all UK traveller study participants.A total of 730 trimmomatic (version 0.39) and the parameters: LEADING:3 TRAILING:3 SLIDINGWINDOW:4:20 MINLEN:36. Reads were then mapped to the P. vivax P01  reference genome34 using bwa-mem (version 0.7.12). The samtools35 (version 1.9) functions fixmate and markdup were applied to the resulting BAM files. The GATK’s BaseRecalibrator and ApplyBQSR functions were applied using a combined VCF file with SNPs identified in previous datasets as being high quality and presenting a minimum allele frequency (MAF) > 5%11. A set of 730 improved BAM files was obtained. Alignment metrics were calculated using the flagstats option within the samtools software.The Illumina sequencing reads were first filtered using 36. GATK was run with default parameters and using the option -ERC GVCF. The GVCFs were validated using the GATK ValidateVariants function, then imported into a GenomicDB using the GATK function GenomicsDBImport, and a combined VCF was created using the GATK GenotypeGVCFs function. A total of 1,384,569 SNPs were identified. Variants in subtelomeric regions and internal vir genes regions were excluded. Subtelomeric regions in the 14 chromosomal sequences were identified by mapping previously published regions from P vivax Salvador I  into the PvP01 reference  and followed recommended best practice37 with the following options: -no-downstream -no-upstream. A summary of the analysis pipeline applied is presented in SA total of 172 isolates with >40% of SNPs missing genotype data, as a result of poor sequencing coverage, were excluded from downstream analysis. The excluded isolates consisted of 158 (91.9%) publicly available  and 14 (8.1%) newly sequenced traveller samples . Source countries with excluded isolates had other samples that passed quality control, ensuring no loss in global representation. SNPs were then processed to replace the genotype call in the VCF for a mixed call whenever the secondary MAF was at least 20% in a given SNP on each individual sample. A subset of only biallelic SNPs was also obtained from this matrix. The final matrix consisted of 558 isolates (118 from travellers) and 388,933 high-quality SNPs. Variants were annotated using snpEff (version 4.1)FWS score38, as well as at an individual isolate level using estMOI(version 2.0)39. Only SNPs in coding regions which were biallelic were used for the calculation of FWS. FWS was calculated using the moimix (version 0.0.2.9001) package (https://github.com/bahlolab/moimix).Multiplicity of infection (MOI) was assessed by grouping isolate data at a country level and calculating the ape package (version 5.4-1)40. A total of 135,954 high-quality SNPs with MAF > 1% were used to assess the population structure within the ADMIXTURE (version 1.3.0) package, where the most likely number of subpopulations (K) was obtained using cross-validation error 41. In order to assess the SNPs driving the allele frequency differences between populations, we identified high fixation index FST42 values using the PopGenome library (version 2.7.5) in R. Isolate populations were compared at both a regional and country level.A principal component analysis (PCA) was performed on the high-quality SNPs and isolates by calculating the pairwise genetic distance using the Manhattan method. A neighbour-joining tree was obtained to represent these similarities, and was generated using the rehh package (version 3.2.1) in R43. Isolates with FWS < 95% were excluded from this analysis in order to use only monoclonal infections. EstMOI estimated that MOI was 1 for the majority of evaluable included isolates, with less than thirty displaying an MOI of 2, and for these, dominant clones had a relative abundance of at least 90% in every case. Both the integrated haplotype score (iHS)44 for within-population selection and Rsb for identification of selection between two populations45 were calculated. Calls obtained from the P. cynomolgi data (Accession number: DRR000476) were used as the ancestral states of the SNPs in our dataset12. Critical regions were identified using a 10 kbp sliding window including at least three SNPs with a P value <1 × 10−4 for iHS and a P value <1 × 10−5 for Rsb. Tajima’s D was calculated at a country level using the package pegas (version 0.14) in R for populations with >10 isolates and for genes with >5 SNPs. A negative Tajima’s D value may indicate population size expansion and/or purifying selection. Whilst a positive value may indicate a decrease in population size and/or balancing selection. Identity-by-descent (IBD) screening was also performed at a country level using the package hmmIBD (version 2.0.4)24 for populations with >10 isolates and excluding isolates with FWS > 95%. Default parameters, including recombination rate, were used and based on P. falciparum estimates24. The proportion of pairwise comparisons for isolates presenting evidence of IBD was plotted by genome location using a sliding window of 10 kbp.Isolate populations at both country and regional level with >10 isolates were screened for positive selection using the P. vivax orthologues of P. falciparum genes are based on high levels of identity and similarity in the encoded amino acid sequences46. The protein sequences for pvmrp1 and pfmrp1 genes were downloaded from PlasmoDB (https://plasmodb.org) with gene ID PF3D7_0112200 for pfmrp1 and PVP01_0203000 for pvmrp1, and then aligned using the mafft protein aligner . Protein domains predictions were also accessed through the PlasmoDB website.https://www.rcsb.org/) . The structures were visualised and annotated using UCSC chimera. Distance from the ligand was calculated using the mCSM-lig webserver (http://biosig.unimelb.edu.au/mcsm_lig).Protein structures for DHFR and DHPS were downloaded from the Protein Data Bank (PDB) (Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Supplementary Data 8Supplementary Data 9Supplementary Data 10Supplementary Data 11Supplementary Data 12Reporting Summary


To investigate plaque inhibition of 0.1% octenidine mouthwash (OCT) vs. placebo over 5 days in the absence of mechanical plaque control.p < 0.05.For this randomized, placebo-controlled, double-blind, parallel group, multi-center phase 3 study, 201 healthy adults were recruited. After baseline recording of plaque index (PI) and gingival index (GI), collection of salivary samples, and dental prophylaxis, subjects were randomly assigned to OCT or placebo mouthwash in a 3:1 ratio. Rinsing was performed twice daily for 30 s. Colony forming units in saliva were determined before and after the first rinse. At day 5, PI, GI, and tooth discoloration index (DI) were assessed. Non-parametric van Elteren tests were applied with a significance level of p < 0.0001). OCT reduced GI  and salivary bacterial counts . Tooth discoloration was slightly higher under OCT . Mild tongue staining and dysgeusia occurred.Treatment with OCT inhibited plaque formation more than treatment with placebo (PI: 0.36 vs. 1.29; OCT 0.1% mouthwash inhibits plaque formation over 5 days. It therefore can be recommended when regular oral hygiene is temporarily compromised.When individual plaque control is compromised, rinsing with octenidine mouthwash is recommended to maintain healthy oral conditions while side effects are limited.The online version contains supplementary material available at 10.1007/s00784-021-03781-3. Antiseptic mouthwashes are commonly used in oral home care. They are recommended particularly in situations where the performance of efficacious mechanical plaque control is temporarily or permanently impaired, like in individuals in need of care, after oral surgical interventions, during orthodontic therapy with fixed appliances or as adjunct for gingivitis or periodontitis therapy –4. Next Presently, chlorhexidine (CHX), a bisbiguanide antiseptic, comprehensively meeting all those requirements, is the most widely used antimicrobial agent in mouthwashes. Its clinical efficacy has been verified by a multitude of studies , 10.More recently, octenidine dihydrochloride, a bispyridinamine, came into the focus of interest as another particularly suitable agent to be used in oral antiseptics due to properties rivaling those of CHX , 12. It It was the aim of this clinical trial to evaluate the inhibitory effect of a 0.1% OCT mouthwash on plaque re-growth over 5 days in the absence of mechanical plaque control in healthy adult subjects, using a modified plaque re-growth model .No significant difference between both experimental groups regarding plaque re-growth assessed by plaque index (PI)  5 days aclinicaltrials.gov  and the European Clinical Trials Database . All participants were informed about the study and signed the informed consent declaration. The studies were registered at clinicaltrials.gov: NCT03322124, NCT03378401.The investigation was designed as a prospective, randomized, placebo-controlled, double-blind, parallel group, multi-center phase 3 study, divided into two studies OML-III-A and OML-III-B. OML-III-A took place at the study centers of Dresden, Frankfurt, and Leipzig from January to December 2018. OML-III-B was performed at the study centers in Wuerzburg, Cologne, and Vienna from January to September 2018. The study was conducted in accordance with the principles of Good Clinical Practice (GCP) and the Declaration of Helsinki. It was approved by the ethics committees of the participating centers  and was registered at The results presented in the following are based on the joint data set of the OML-III-A and OML-III-B studies, which followed identical study protocols and were performed concomitantly.Participants  were recruited among systemically healthy dental patients visiting the study centers in Dresden, Frankfurt, Cologne, Vienna, and Wuerzburg or among healthy volunteers from the resident population being asked for study participation by local advertisement.Inclusion criteria were as follows: age ≥ 18 years; total mean gingival index GI ≤ 1.5 ; a minimExclusion criteria were as follows: severe systemic diseases; necessity of antibiotic endocarditis prophylaxis; untreated caries with cavitation; presence of a GI score 3 at any tooth ; manifesThe experimental mouthwashes were manufactured by NextPharma GmbH . They contained glycerol, sodium gluconate, citric acid, disodium phosphate dihydrate, macrogolglycerol hydroxystearate 40 EO, mint cool flavor PHL-167319, sucralose, and 0.1% octenidine (OCT mouthwash only) or phenoxyethanol 0.5% (placebo mouthwash only).Study participants were instructed to rinse their mouth with 10 ml of the assigned mouthwash twice daily for 30 s each over a period of 5 days, resulting in a total of 10 applications. For dosing, a measuring cup was provided with the study drug package.Primary study outcome was the amount of plaque re-growth assessed by plaque index (PI)  5 days aSecondary study outcomes were the reduction of salivary bacterial counts after a single application of the experimental mouthwashes for 30 s at baseline and changes in mean GI scores  and DI sFor the assessment of salivary bacterial counts, the study participants were asked to rinse their mouth with 5 ml of sterile water for 30 s and to spit the originating saliva-water volume into a sterile screw-cap test tube. The samples were subsequently serially diluted, inoculated on Columbia blood agar plates + 5% sheep blood  and incubated at 37 °C for 48 h. The number of detected colony forming units (CFU) was used to calculate the number of CFU/ml for the respective dilution.Gingival health was assessed by recording the gingival index  at Vsc, The presence and extent of tooth discoloration were assessed at V1 after PMPR and after 5 days of rinsing (V2) at the vestibular sites of the anterior teeth according to the criteria of the discoloration index .Furthermore, the study participants were instructed to document their daily consumption of staining beverages like coffee, tea, red wine, and juices, the use of chewing gum and menthol-containing lozenges, as well as tobacco smoking by a self-reported questionnaire.At baseline and at the end of the study (day 5), the study participants were interviewed for the occurrence of related or unrelated adverse events.Application compliance was verified by instructing the study participants to return all empty, partially used, or unused containers of the experimental mouthwashes at the end of the study (V2). The volume of applied mouthwashes was calculated as difference in the weight of the assigned supply of mouthwash containers before handing out and after returning them to the study center.All clinical examiners of the participating study centers had been trained and calibrated prior to the onset of the study following established guidelines .Investigators and other study personnel were blinded to the assignment of the study participants to the treatment groups throughout the study. Both experimental mouthwashes (OCT/placebo) were provided in bottles with identical packaging and labelling and had an identical appearance, color, and taste to ensure blinding of participants. The handing out to the study subjects in a 3:1 ratio (OCT vs. placebo) was performed by a study nurse not involved in the recording of the study data using a computer-generated randomization list. Randomization was stratified for GI baseline score (mean GI ≤ 1.0 vs. mean GI > 1.0).At the screening visit, eligibility criteria were checked, and demographics, medical history, concomitant medication, and smoking habits were documented. PI  and GI  were recAt visit 1, eligibility criteria were re-checked, GI baseline scores were recorded, and a saliva sample was collected  revealed a number of 75 and 25 study subjects (OCT/placebo respectively) to be sufficient to detect a difference in mean PI of − 0.9 between the groups with a given power of p < 0.025). To assess the potential impact of co-variables, an additional analysis of co-variance (ANCOVA) with independent variables for treatment group, gingival status at V1 (factor for stratified randomization), total mean PI at screening, and study center was performed.Due to lack of normal distribution of data, the primary study outcome mean PI at day 5 of the trial was analyzed using van Elteren test  or the placebo (n = 49) group. Two hundred of them completed the study with a full data set. One participant of the OCT group did not use the assigned mouthwash. Because there were no statistically significant differences between the intention to treat and the per protocol analyses regarding any of the assessed variables, all results depicted in the following are based on the intention to treat analysis.Recruitment and drop-outs are depicted in the CONSORT  flow diaThe demographic data of the study participants are displayed in Table p < 0.0001, van Elteren test) than the median PI score of the placebo group . Among all confounding variables tested, ANCOVA only revealed a center effect (p < 0.0001). The null hypothesis of no difference between both experimental groups at V2 therefore had to be rejected.The results of the plaque re-growth analysis are depicted in Table p < 0.0001; van Elteren test; Table A single rinse with the OCT mouthwash reduced salivary bacterial counts significantly stronger than the placebo mouthwash. The observed median decrease in salivary bacterial counts was 2.73 lgCFU/ml for the OCT group vs. 0.24 lgCFU/ml for the placebo group .The variation of recorded median GI scores within the observation period is displayed in Table p < 0.0011, van Elteren test, Table The median change in the DI scores from V1 to V2 was significantly more pronounced in the OCT group when compared to the placebo group , but fewer subjects in this group smoked (25.8% vs. 32.7%) and consumed menthol dragées (13.9% vs. 20.4%). Other eating habits were similar between the two groups.n = 59) or moderate (n = 4) TEAEs. In the OCT group, 53 out of 151 participants (35.1%) reported 71 TEAEs. In the placebo group, 10 out of 49 subjects (20.4%) reported the occurrence of 12 TEAEs. The most frequently reported TEAEs in the OCT group were dysgeusia (n = 32), tongue discoloration (n = 9), and headache (n = 4). In the placebo group, oral discomfort (n = 3), dysgeusia (n = 2), and headache (n = 2) occurred most often. The remaining events were reported by < 5% of individuals in any treatment group. Out of 83 TEAEs, 63 TEAEs were considered being treatment-related , 56 of them were reported by OCT-group members and 7 by participants of the placebo group.There were no severe treatment-emergent adverse events (TEAE). Overall, 63 subjects (31.5%) experienced mild  supporting the conclusion that the regular use of the 0.1% OCT mouthwash has the same antibacterial efficacy as routine mechanical oral hygiene measures. Similar observations were made in other studies using mouthrinses containing 0.1% octenidine , 37–40 o supportiIn comparison to the golden standard CHX, bacterial reduction and plaque inhibition occurred to a similar extent –44. So fWhile the inclusion criteria allowed to include study subjects with mild to moderate chronic gingivitis up to a mean GI score of 1.5, the overall baseline mean GI score of the study population was GI 0.40. Only 13 recruited study participants (6%) had a mean GI score > 1.0 and were suffering from moderate gingivitis. Nevertheless, the use of the OCT mouthwash resulted in a statistically significant inhibition of gingival inflammation, although the observed median reduction of GI = 0.04 may not be considered clinically relevant.p > 0.05).As reported in many other studies evaluating antiseptic mouthrinses before, tooth and tongue discoloration was the most frequently reported adverse event followed by dysgeusia , 37. TheAs the recording of PI and GI scores was restricted to the Ramfjord teeth, the true extent of plaque re-growth and accompanied gingival inflammation within the dentition might have been underestimated. However, this systematic bias would have affected both groups equally. The general validity of study data gained by the evaluation of the Ramfjord teeth has been verified in various other studies , 46, 47.Repeated rinsing with a 0.1% octenidine mouthwash is an efficacious measure for the temporary inhibition of plaque re-growth and the maintenance of gingival health in the absence of personal mechanical plaque control during 5 days.ESM 1(DOC 218 kb)