Mass screening and treatment (MSaT) aims at reducing the spread of malaria in communities by identifying and treating infected persons regardless of the symptoms. This study was conducted to identify and treat asymptomatic cases using MSaT approaches in the community.Three rounds of MSaT using cluster combination approaches were carried out during September 2018 to December 2019 to identify and treat asymptomatic malaria cases in the community. All individuals who were present in the household were screened using RDT irrespective of malaria related symptoms. Simultaneously thick and thin blood smear and blood spot were collected for further analysis using microscopy and diagnostic PCR done in a subset of the samples.Logistic regression analysis revealed that asymptomatic malaria cases significantly less among the older age groups compared with < 5 years children , lowest in cluster 4 ; during third round of MSaT survey  and significantly higher in moderate to high endemic areas .Over the three rounds of MSaT, the number of asymptomatic cases were significantly less in the older age groups, and during third round. Similarly, the asymptomatic cases were significantly less in the low endemic area with API < 1 (cluster four). Therefore, the malaria elimination programme may consider the MSaT strategy to identify asymptomatic cases that would be otherwise missed by routine fever based surveillance. This MSaT strategy would help accomplish the malaria elimination goal in an expedited manner. India has committed to the global malaria elimination goal of 2030, which translates to achieving zero indigenous cases by 2027 . Over thThe Mass Screening and Treatment (MSaT) aims to detect and treat all malarial parasite infections, including asymptomatic parasite carriers within the community. The World Health Organization recommends MSaT as an intervention strategy targeting asymptomatic infections to reduce the prevalence . In anotIn the present study, three rounds of MSaT were conducted using RDTs, microscopy, and PCR to identify asymptomatic malaria-infected individuals. The MSaT was accompanied by vector control interventions such as LLIN and IRS, along with active fever surveillance and case management. These three round of MSaT were done during the pre-transmission, transmission, and post-transmission period for identifying and treating the asymptomatic cases in the community of district Mandla, Madhya Pradesh, India.This study was part of the Malaria Elimination Demonstration Project, which was a first-of-its-kind public–private-partnership between the Indian Council of Medical Research (ICMR) through the National Institute for Research in Tribal Health (NIRTH) Jabalpur, Government of Madhya Pradesh (GoMP), and the Foundation for Disease Elimination and Control of India .2, inhabited by ethnic tribal communities, mainly ‘Gonds’ and ‘Baiga’.The study was conducted in district Mandla, located between coordinates of 22°02′ and 23°22′ N latitudes and 80°18′ and 81°50′ E longitudes in the east-central region of the state of Madhya Pradesh (MP), India. The total geographical area of the district was 8771 kmA cross sectional MSaT was conducted during the malaria transmission season in September 2018, pre-transmission season in June 2019 and post-transmission season in December 2019 to assess the asymptomatic cases of malaria in the community. A multistage sampling method was adapted. In September 2018, for the first round of MSaT, a cluster sampling method was used in which all the villages of the district Mandla were divided into four clusters based on their Annual Parasite Incidence  during five preceding years (2013–2017) and accessibility to the study areas.The clusters were categorized as: (1) Hard-to-reach or inaccessible villages; (2) More than or equal to API 5; (3) API between 1 to 4.99; and (4) API between 0 and 0.99. The second round of the MSaT survey was conducted in June 2019 to validate the findings from the first round of MSaT and the entire study area was further stratified into moderate to high endemic (> 1 API) and low endemic (0–1 API) villages. Further, a third round of the MSaT survey was carried out in December 2019 in 50 houses surrounding the cryptic cases  diagnoseSample size was determined using the formula of simple random sampling for a finite population as given below:The sample size for the different clusters and malaria endemic areas was determined based on the lowest asymptomatic malaria prevalence reported by Wangchuk et al.  in BhutaDoor-to-door visits were done by trained Village Malaria Workers (VMWs) to screen the community for malaria parasites. The VMWs screened each consenting individual in the study area using bivalent malaria RDTs  per field, a blood slide was considered negative. All the samples having discordant results were re-checked by an independent WHO certified microscopist for quality assurance of light microscopy.Plasmodium species was determined using species specific nested PCR by targeting 18Sr RNA gene. To set up the primary PCR, 5 µL of genomic DNA as template was taken to amplify the 18S rRNA gene for the Plasmodium genus using forward and reverse primer [Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale. The PCR product was analysed on 1.2% Agarose gel electrophoresis.Molecular analysis was done in about one-third of the randomly selected samples. Genomic DNA was isolated from the dried blood spots using the Chelex method [Chelex-100 Sodium form (50–100 Mesh) Himedia Laboratories . The pree primer . The priThe demographic  variables, clinical symptoms related to malaria, results of RDT, microscopy, and PCR were entered in Microsoft Excel 2007 worksheet, and the numerically coded data was exported in R 4.1.2 for Windows  for statistical analysis. Logistic regression analysis was used to examine the association of independent variable(s).A total of 88142 individuals, irrespective of malaria-related symptoms, were tested using RDTs during door-to-door visits. Out of which, 34694 (39.36%), 18550 (21.05%) and 34898 (39.59%) were screened in the first, second and third rounds of the MSaT surveys, respectively. In clusters one to four; 4437 (5.03%), 17616 (19.99%), 9709 (11.02%) and 56380 (63.96%) individuals were screened, respectively. About three-fourths of the individuals were adults with a mean age of 31.86 ± 20.07 years, and about 51% were female . Out of which, 0.20% (69/34694), 0.15% (28/18550), and 0.10% (35/34898) cases were recorded during the first, second and third rounds of MSaT, respectively, with a statistically significant declining trend (p = 0.003).The cluster-wise (one to four) prevalence of malaria was 1.19% (53/4437), 0.28% (50/17616), 0.29% (28/9709), and 0.002% (1/56380), respectively, which was statistically significant (p < 0.0001). Amongst the residents of low malaria endemic areas, the prevalence was 0.002% (1/56380). Whereas the prevalence in moderate-to-high malaria endemic areas was 0.41% (131/31762), which was significantly higher (p < 0.0001). The PCR based diagnosis was done in a sample of 24357, which was randomly selected from the total 88142 MSaT samples. Out of these 24357 cases tested by PCR, 23681 were asymptomatic, and the rest 676 were symptomatic. Amongst the samples tested by PCR, the malaria positive cases by RDT were 0.12% (30/24357), microscopy were 0.14% (35/ 24357), and PCR were 1.06% (258/24357).The prevalence of asymptomatic malaria infections in the first, second, and third rounds was 0.53% (179/34013), 0.57% 103/18202) and 0.06% (20/33797), respectively. Further, analysis revealed that the asymptomatic malaria prevalence significantly declined during the third round as compared to the first round . Similar declining trend was recorded in all the clusters (p < 0.0001). Neither asymptomatic nor symptomatic malaria cases was reported from clusters three (low endemic) and four (moderate endemic) during the second and third rounds of the MSaT survey  than first round 2.64% (18/681) of MSaT survey (p = 0.002), while it increased in the third round  but not statistically significant. A similar trend was reported in clusters one and two (hard to reach and high endemic areas). None of the symptomatic malaria cases was reported in cluster three and four during second and third round of MSaT survey , 0.48% (21/4348) and 0.89% (12/1347) in cluster one; 0.14% (24/1726), 0.10% (18/1726) and 1.33% (151/11348) in cluster two; 0.16% (15/9400), 0.08% (8/9400) and 2.72% (64/2349) in cluster three; and 0% (0/54995), 0.002% (1/54995) and 0.06% (5/8637) in cluster four, respectively. The analysis revealed that a significantly higher prevalence of asymptomatic malaria was reported using PCR diagnosis in all the clusters (p < 0.0001).This prevalence varied significantly over the MSaT rounds through all the clusters and diagnostic methods. Among all the diagnostic methods, the asymptomatic malaria prevalence significantly declined in the third MSaT round as compared to the first MSaT round (p < 0.001) except in cluster one, where the asymptomatic malaria prevalence was found to be significantly higher in the third round than the first round (p < 0.001). However, a higher prevalence of symptomatic malaria was found during the third round of MSaT as compared to the first round in all the diagnosis methods, and it was significant in cases diagnosed using RDT and PCR (p < 0.001) Table .Table 3CUnivariate logistic regression analysis revealed that asymptomatic malaria was significantly lower among the age groups 5–15 years , 15–25 years , 25–50 years  and above 50 years  in reference to the < 5 years children. Further analysis showed asymptomatic malaria was significantly lower in cluster four , during the third round of MSaT survey  and significantly higher in the moderate to high endemic areas  than their reference categories. However, the asymptomatic malaria cases did not significantly differ between age groups and gender (p > 0.05). Further, symptomatic malaria cases were significantly lower in the clusters two  and three  as compared to the cluster one.Symptomatic malaria cases did not differ significantly through different age groups, gender, endemicity and rounds of the MSaT surveys (p > 0.05). The total malaria prevalence in MSaT significantly decreased among the older age groups than < 5 years children (p < 0.05); in round three ; in cluster two  and cluster four  than their reference categories, while it was 104 times likely to be higher in moderate-to-high endemic areas than low endemic areas , 0.15% (second round), and 0.10% (third round). This observation was similar to a study done in Zambia that revealed 17% reduction in the incidence of clinical malaria after three rounds of MSaT . The groThe present study revealed a significantly higher prevalence of asymptomatic infections in moderate to high endemic areas (0.41%) as compared to the low endemic areas (0.0%). Compared to this, an opposite result was found in western Kenya, but the sample size was much lower .Durgama Anchalare Malaria Nirakarana (DAMaN)’ focussing on these hard-to-reach areas was conducted in Odisha and reported a significant reduction in malaria cases using MSaT, LLINs, and community mobilisation [The hard-to-reach areas remain a bottleneck of malaria elimination due to inadequate health care services/ service providers. The cluster wise analysis of the present study reported a prevalence of 1.19% (hard to reach area), 0.28% (API: > 5), 0.29% (API: 1–4.99), and 0.002% (API: 0–1) with a declining trend over different rounds (p < 0.0001). A study known as the ‘lisation .In another MSaT study conducted in Malawi in school children, a decrease in malaria transmission was reported , which iThe present study has revealed that the asymptomatic malaria infections were significantly reduced in the third round 0.06% of MSaT as compared to the first round (0.53%), which suggest that MSaT should be considered as a strategy along with other case management and vector control tools for effectively halting malaria transmission in the community.Another important observation of this study is the finding that PCR detects more than three times higher cases as compared to RDT / microscopy. The asymptomatic cases diagnosed by PCR were higher in all clusters than the conventional methods and were highest in moderate to high endemic areas. In a similar study from Zanzibar, it was found that MSaT detected more than 10 times higher cases using species-specific PCR (2.5%) as compared to RDT (0.2%) . This fiP. falciparum using PCR tools [The usefulness of a sensitive point-of-care PCR-based diagnostic tool for MSaT was highlighted by Von Seidlein L , which iCR tools , and repThe evidence generated by this Malaria Elimination Demonstration Project , 32–41 iThis study has demonstrated the significance of MSaT as an effective surveillance tool to detect and treat asymptomatic cases of malaria. Given the limitations of RDT and microscopy, it is recommended that national programmes and the WHO should implement a policy that recommends the use of nucleic acid-based detection tools, such as PCR, for detecting asymptomatic reservoirs of malaria during elimination phase.


The characterization of subcellular protein localization provides a basis for furtherunderstanding cellular behaviors. A delineation of subcellular localization of proteins oncytosolic membrane-bound organelles in human liver cancer cell lines (hLCCLs) has yet tobe performed. To obtain its proteome-wide view, we isolated and enriched six cytosolicmembrane-bound organelles in one of the hLCCLs (SK_HEP1) and quantified their proteinsusing mass spectrometry. The vigorous selection of marker proteins and amachine-learning-based algorithm were implemented to localize proteins at cluster andneighborhood levels. We validated the performance of the proposed method by comparing thepredicted subcellular protein localization with publicly available resources. The profilesenabled investigating the correlation of protein domains with their subcellularlocalization and colocalization of protein complex members. A subcellular proteomedatabase for SK_HEP1, including (i) the subcellular protein localization and (ii) thesubcellular locations of protein complex members and their interactions, was constructed.Our research provides resources for further research on hLCCLs proteomics.Database URL: http://www.igenetics.org.cn/project/PSL-LCCL/ The presence of subcellular compartments within eukaryotic cells provides specializedlocation and physical and chemical environment for protein expression, playing an essentialrole in cellular homeostasis . Furtheret al.  based protein identification . It has Human liver cancer is the sixth incidence and the third leading cause of cancer-relatedmortality worldwide . The Canhttp://www.igenetics.org.cn/project/PSL-LCCL/).Our previous study identified a component of the retromer complex—VPS35—exerting itsoncogenic role on LIHC through FGFR3 recycling .To furtThe framework for the present study included isolation and enrichment of six membrane-boundorganelles, MS and data analysis .The parental SK_HEP1 was obtained from the Chinese Academy of Sciences and was confirmed free of mycoplasma . Inaddition, short tandem repeat profiling confirmed cell authentication . We have previously established a VPS35-knockout (KO)model and demonstrated the oncogenic role of VPS35 in the development of liver cancer. Both pag for 10 min.The supernatants were collected, and the protein concentration was measured using Bradfordprotein assay. Extracted proteins were reduced in 10 mM dithiothreitol at 56℃ for 60 minand then alkylated in 45 mM iodoacetamide at room temperature for 30 min in darkness. Thesample was diluted four times by adding 25 mM ammonium bicarbonate buffer, then underwenttrypsin digestion (enzyme-to-substrate ratio of 1:50 at 37℃ for 16 h) followed bydesalting through C18 cartridges  and vacuum-dried bySpeed Vac. The extracted proteins for each organelle were subjected to Western Blotanalysis using organelle-specific marker proteins for confirmation , respectively. These organelles werederived from the cultured cells in simultaneous batches in technical triplicate. Theorganelle was lysed in lysis buffer supplemented with protease inhibitors for 20 min on ice. Samples were then sonicated for2 min (3 s on and 3 s off) on ice and centrifuged at 14 000 irmation .A 120-min gradient elution separated peptides at a flow rate of 0.300 μL/min with theEASY-nLC 1000 system, directly interfaced with the Thermo Orbitrap Fusion massspectrometer. The analytical column was purchased from Thermofisher . Mobile phase A consisted of 0.1% formic acid, and mobilephase B consisted of 100% acetonitrile and 0.1% formic acid. The Orbitrap Fusion massspectrometer was operated in the data-dependent acquisition mode using Xcalibur3.0software, and there is a single full-scan mass spectrum in the Orbitrap  followed by 3-s data-dependent MS/MS scans in an Ion Routing Multipoleat 38% normalized collision energy (HCD). MS was conducted in Beijing Qinglian Biotech.The MS/MS spectra from each LC-MS/MS run were searched against protein sequences fromUniProt using Maxqant .The search criteria included that a complete tryptic specificity was required, two missedcleavage was allowed and carbamidomethylation (C) was set as the fixed modifications. Theoxidation (M) was set as the variable modification, and precursor ion mass tolerances wereset at 15 ppm for all MS acquired in an orbitrap mass analyzer. The fragment ion masstolerance was set at 20 mmu for all MS2 spectra acquired. The peptide false discovery rate(FDR) was calculated using Target Decoy PSM Validator provided by maxquant. When searchedagainst the reverse decoy database, FDR was determined based on PSMs. Peptides onlyassigned to a given protein group were considered unique. The FDR was set to 0.01 forprotein identifications.Marker proteins specifically localized in organelles are required to be highly replicableand have robust fractionation profiles under different conditions . Therefofiltered . The remn = 1481) using mClust (v5.4.6), which assigned proteins to differentclusters with probability based on the expectation-maximization algorithm on a mixture ofGaussians model  implemented in the ‘rtsne’ (v0.15) in R . Two hypns model . A totalns model . Theideb is the protein number of the target organelle in the cluster,n is the total number of proteins in the cluster, B isthe total protein number of the target organelle and N is the totalnumber of proteins in all clusters. Significance levels were estimated using thehypergeometric test (‘Phyper’ in R) and were corrected for multiple testing(Benjamini–Hochberg)  . We assister 18).Second,ster 18). The anaster 18).We followed the definition of neighborhoods as ‘secretory’ (Clusters 1–3), ‘mitochondria’(Clusters 4–8), ‘nuclear’ (Clusters 9–11) and ‘cytosol’ (Clusters 12–17) according to theknown relationship of subcellular compartments .n = 984) and testing (n = 497) sets. Ten-foldcross-validation was used to avoid over-fitting on training data. We searched the best-fitparameters of the cost (i.e. misclassification rate) from We used the support vector machine (SVM) with a Gaussian radial basis function kernel toclassify the identified proteins by inputting the relative quantification of markerproteins. To build a classifier, we randomly split the marker proteins into the training. Proteins classified into a single cluster weredisplayed as corresponding clusters, and proteins without cluster classifications weredesignated as neighborhood classifications. The network was visualized in Cytoscape(v.4.0.1).The localization network was constructed based on proteins with a single neighborhoodclassification . We obtained the proteins withsequence similarity >40% and then extracted the domains for proteins in each cluster inthe Pfam database. The domains enriched in each neighborhood and the number of occurrencesof the domains are obtained, and domains that appear to be less than three times in eachneighborhood were filtered.We first obtained proteins with signal peptides, transit peptides and transmembranedomains from UniProt. Then, we searched the Pfam database (Pfam-A.hmm.gz) for annotatingother protein domains using ‘hmmscan’ in HMMER (v3.3.2) . Signifi of 0.05 . Next, wA list of human core complexes was obtained from the Comprehensive Resource of MammalianProtein Complexes (CORUM) database, which collected experimentally verified mammalianprotein complexes . For eachttp://www.igenetics.org.cn/project/PSL-LCCL/. The database provided accessto both the raw fractionation data and the prediction for subcellular proteinlocalization.For visualization and access to the subcellular protein localization, we created thePSL-LCCL portal developed by the shiny framework for R, which is available at n = 4097) in triplicate showed the robustness of the proposed method and testing (n = 497) sets, balanced tocover the 18 clusters. Of the identified proteins in triplicate(n = 4464), we successfully classified 2510 (56%) proteins into clusters(n = 2510) with those having single localization in the UniProt(n = 7147) or GO (n = 6664) and noted that 44% ofproteins (n = 1105) were consistent as single-localization proteins were classified intoneighborhoods, i.e. approximately 1300 proteins were rescued at the neighborhood level,significantly greater than the coverage of 56% at the cluster level. A localized networkdemonstrated an overview of the classification by mapping proteins into specific clustersor neighborhoods, as well as their relationship (n = 3803) with proteins having single localization inUniProt or GO, and 45% (n = 1710) proteins were consistent were classified as the cluster level. We followed the definition of ‘neighborhoods’  to classtionship . The neitionship . The clationship . We compnsistent . Organelnsistent , e.g. thn = 3803) against single-localization proteins annotated inthe public database. The protein localization of the public database was first assigned tothe five defined neighborhoods. Of the 1686 proteins overlapped with UniProt and GO, anagreement was increased from 65.9% to 70.6% for proteins with the same localizationannotated in both UniProt and GO (n = 4647) (n = 243), 64% with ‘Supported’ (n = 319),41% with ‘Approved’ (n = 144) and 27% with ‘Uncertain’ (n = 487) were present in the mitochondrial database . The ovecertain’ . The ovecertain’ . In the database  . Of notet} 0.05) .We also clustered proteins in each neighborhood using CD-HIT. For protein clusters withsequence similarity >40%, 27 domains appeared to more than three times in secretory, 2in mitochondria, 10 in nucleus, 22 in cytosol and 2 in ribosome . We showWe next investigated the domains and their localization of the members in the retromercomplex, which comprises a VPS26-VPS29-VPS35 heterotrimer implicated in cargo recognitionand various combinations of sorting nexin (SNX) proteins, contributing to membranerecruitment and formation of recycling tubules . The SNXentified . In addiProtein complexes composed of multiple proteins play critical roles in various biologicalprocesses . Using tent}0.8) . Howeverent}0.8) , e.g.fient}0.8) . Incompent}0.8) , and prohttp://www.igenetics.org.cn/project/PSL-LCCL/).We created a database of PSL-LCCL, including two layers of information. At the proteinlevel, it included the classification probabilities of proteins in the 18 clusters and 5neighborhoods. For demonstration purpose, we created a simple cell map to visualize thelocalization of a given protein by keyword query. Users can also view the proteins ofinterest in the organelles. When multiple proteins are inquired, the classification forall proteins will be displayed simultaneously, enabling a convenient way to compare thelocalization of various proteins. Examples of well-known compartment markers were shown. Atthe We isolated and enriched six cytosolic membrane-bound organelles individually andimplemented a machine-learning-based algorithm. We predicted the subcellular proteinlocalization for the identified proteins in the SK_HEP1 cell line. We successfullyclassified 2510 (56%) and 3803 (85%) out of 4464 identified proteins into 18 clusters and 5neighborhoods, respectively. The prediction accuracy for marker proteins and the comparisonof the subcellular protein localization with the well-known public resources confirmed thereliability of our results. Furthermore, the classification enabled investigating theassociation between subcellular localization with protein domains and complexes. Forresource convenience, a user-friendly subcellular proteome database of ‘PSL-LCCL’ forSK_HEP1 was provided.Recently, MS and machine-learning algorithms have been used to study subcellular proteinlocalization , 8, 35. The annotation for each cluster (i.e. the assignment of organelle based on the publicresources) may not be unique . OrganelPrevious studies have shown that protein domains have implications in the localization ofproteins, e.g. signal peptide  and traninvitro could improve the reliability of the localization, especially for proteinswith inconsistent annotations. However, it may be beyond the scope of the present study.Finally, protein relocation is essential for cell signal transmission and rapid adaptationto environmental changes. A dynamic organelle map can be used to identify globaltranslocation proteins for our further study.The present study has several limitations. First, proteins in ‘unexpected’ organelles wereidentified under the proposed method. An impure subcellular fractionation for sixmembrane-bound cytosolic organelles may include proteins in unseparated cytosolic or nucleiccomponents. A consistent rate of 89% cytosolic proteins classified in the present study andSubCellBarCode was obtained. However, the lack of separation for cytosolic and nucleiccomponents resulted in a relatively lower protein coverage. Separating more organelles couldimprove the classification accuracy and the coverage depth. Second, although our method candetermine the single dominant localization for proteins, proteins with multilocalizationremain undetermined. A previous study showed that multilocalization of proteins wasuncommon, and ocalized, these pIn conclusion, our study characterized subcellular protein localization of proteinsidentified in six membrane-bound cytosolic organelles for the SK_HEP1 cell line. A proteincomplex interaction map integrating member localization and protein–protein interactions wasprovided. Our resources have implications for further research on the proteomics of livercancers.baab087_SuppClick here for additional data file.


To compare the prognostic value of tumor-infiltrating lymphocytes (TILs) and CD3 + cells and CD20 + cells between schistosomal colorectal cancer (SCRC) and non-schistosomal CRC (NSCRC).Although schistosomiasis has been basically eliminated, it has not been completely extinction in China, and occasional outbreaks occur in Europe recently. The role of immune cells in the immune microenvironment of SCRC and NSCRC is remaining obscure, and the inflammation-based prognostic systems of SCRC has rarely been reported.HE-stained sections of 349 colorectal cancer (CRC) tumors, which were completely resected, were evaluated for density of TILs. Meanwhile, we evaluated CD3 + T lymphocytes and CD20 + B lymphocytes by immunochemistry. The relationship of these infiltrating immune cells with clinicopathological features, including schistosomiasis, and clinical outcomes was evaluated, and the prognostic roles of TILs in SCRC and NSCRC were explored.P < 0.0001), there were no significant differences between NSCRC and SCRC patients in clinicopathological features (P > 0.05). Beside, the positive expression pattern of sTILs, iTILs, CD3, and CD20 between NSCRC and SCRC patients was also similar (P > 0.05). In the whole cohort, sTILs and CD3 were defined as independent prognostic factors . CD3 was an independent prognostic factor both in the NSCRC and SCRC set . Higher sTILs, CD3, and CD20 were correlated with less aggressive tumor characteristics in the whole cohort and in subgroups.Except for age (Although CD3 was an independent prognostic factor for both NSCRC and SCRC set, there were no significant differences between SCRC and NSCRC patients in sTILs, CD3, CD20, and in other clinicopathological features.The online version contains supplementary material available at 10.1186/s12957-023-02911-3. Schistosoma japonicum infection is considered as a risk factor for colorectal cancer. Qingpu District of Shanghai is used to be one of the 10 areas with serious schistosomiasis epidemic in China [Oncomelania snails. Thus, there is still a risk of a rebound in the incidence in some areas [Schistosomiasis is an infectious disease that affects more than 230 million people worldwide, according to conservative estimate . In Asiame areas . In addiGrowing evidences have arisen in recent decades that inflammation is the cause of many malignant tumors , 6. As tThe immune system is known to act against tumors, and it has been postulated that TILs reflect a tumor-related immune response –16. TILsA total of 349 CRC patients were enrolled in this retrospective study after applying the following inclusion/exclusion criteria. All patients had received curative resection without preoperative chemotherapy at Qingpu Branch of Zhongshan Hospital affiliated to Fudan University, from January 2008 to August 2016. And all of operations followed the principle: adequate resection margins, en bloc high ligation of the inferior mesenteric artery (IMA), and lymphadenectomy. All circumferential margins were cleared. The number of positive lymph nodes and total number of retrieved lymph nodes were recorded. The inpatient medical records and pathological reports were reviewed, and the patients were followed up by telephone. OS is defined as the interval from the surgical operation date to the last follow-up or death caused by CRC. Inclusion criteria included the following: (i) patients with CRC as primary focus, (ii) none of these patients had received any prior anti-tumor therapy, and (iii) patients were diagnosed as adenocarcinoma by pathology after resection of CRC. Exclusion criteria included the following: (i) Tis tumors; (ii) patients who lacked complete information; (iii) patients with synchronous malignancy, such as liver cancer, lung cancer, and ovarian cancer, were excluded; and (iv) patients with survival time less than 1 month. Two expert pathologists reviewed HE-stained slides to determine the diagnosis and to restage the tumors according to the eighth edition of American Joint Committee on Cancer (AJCC).This study was approved by the medical ethics committee of Fudan University, in accordance with the Helsinki Declaration of 1975. Prior written informed consent was obtained from all patients.Schistosoma eggs in the intestinal tissues.Schistosome ova were observed in all of original HE-stained formalin-fixed paraffin-embedded (FFPE) sections , which were examined at × 10 and × 40 magnification fields using a conventional light microscope by two pathologists who were blinded to clinic data. The diagnosis of schistosomiasis was done by finding schistosome eggs in anywhere in the HE-stained slides. Patients with schistosomal CRC were identified by the presence of Tumor budding was defined as the presence of dedifferentiated single cells or small clusters of up to 5 cells at the invasive front of CRC . To asseEvaluation of TILs was performed as previously described . TILs weThree- to 5-μm-thick CRC tissues were consecutively cut and subsequently dewaxed and rehydrated through graded alcohols. Slides were immunohistochemically stained in Roche Ventana BenchMark XT automated slide stainer  according to the manufacturer’s instructions. Monoclonal and polyclonal antihuman antibodies were used for identification of CD3 + T cells , and CD20 + TILs . Quantitative evaluation of CD3 + and CD20 + TILs was performed in 10 most representative high-power fields (magnification × 40) per tissue section using a Leica DM2000 microscope . The results were carried out blindly to the clinical data.2, and the average value of the four fields is utilized.Evaluation of T-cell marker density was carried out blinded to clinicopathologic information. Individual cores were examined by two observers and annotated to ensure that only normal colonic epithelium or viable tumor tissue was included in the area of analysis. No attempt was made to evaluate the various tumor compartments separately . The whole section slides were observed under low magnification × 20. Then each tissue is divided into four quadrants, and the percentage of CD3 and CD20-positive cells in the four quadrants is calculated in the unit of the number of positive cells/mmP ≤ 0.05 in the univariate analysis were included in a multivariate analysis. Finally, multivariate Cox regression analysis was performed to identify predictive factors for OS. Clinically relevant variables that may have impacted outcomes are as follows: age, gender, TNM stage, lymph node metastasis, and histological type. P < 0.05 was considered to indicate a statistically significant difference.Data were analyzed using SPSS  and GraphPad 5.0. Fisher’s exact and chi-square tests were utilized to perform correlation between clinicopathological characteristics and TILs and schistosomiasis. The main reasons are as follows: Firstly, there is more than 300 cases enrolled in the cohort, and the sample size is large enough. Secondly, the theoretical frequency T in each grid is greater than 5. Thirdly, some indexes are continuous variables, and some are noncontinuous variables, which are converted into categorical variables. K-M curves with log-rank tests were used to determine the prognostic significance for OS. Every variable was analyzed using univariate analysis to identify all potentially important predictors, and then, variables with Schistosoma , there were no significant differences between SCRC and NSCRC patients in clinicopathological features (Table P > 0.05). Unexpectedly, there were also no significant differences between the two groups in sTILs, iTILs, CD3, and CD20 (Table P > 0.05).Patients were divided into two groups: NSCRC and SCRC set based on the status of schistosomal infection. The comparison of clinicopathological features and infiltrating immune cells between NSCRC and SCRC patients was performed. Except for age (Table P < 0.001), less deeper pathological T stage (P < 0.001), well/moderately differentiated tumors (P = 0.034), and less number of tumor budding (P < 0.001), which is an important additional prognostic factor for patients with CRC [P = 0.016), CD3 (P < 0.001), and CD20 (P = 0.019). As shown in Supplementary Table P = 0.011), less number of tumor budding (P = 0.013), earlier TNM stage (P < 0.001), and less deeper pathological T stage (P = 0.010). CD3 was also positively correlated with CD20 (P = 0.001). There was no association between CD20 and clinicopathological features.The correlation between sTILs and CD3 and CD20 and clinicopathological features was shown in Supplementary Table with CRC , 28. BesP = 0.031) and CD3 (P = 0.003) as independent prognostic factors as shown in Table P = 0.001), TNM stage (P < 0.0001), differentiation (P = 0.039), lymph vascular invasion (P = 0.012), and tumor deposit (P < 0.0001).Representative infiltrating immune cells staining is shown in Fig. schistosomiasis, respectively. The optimum cutoff value of sTILs, iTILs, CD3, and CD20 in NSCRC and SCRC was also determined by X-tile program. In the NSCRC group, gender, sTILs, CD3, CD20, pathological T stage, lymph node metastasis, TNM stage, tumor budding, tumor differentiation, lymph vascular invasion, tumor budding, and tumor deposit were significant prognostic factors for OS (P < 0.05) as shown in Table P ≤ 0.05 in the univariate analysis were included in a multivariate analysis, CD3 (P = 0.026), sTILs (P = 0.034), gender (P = 0.021), tumor budding (P = 0.050), tumor differentiation (P = 0.021), lymph vascular invasion (P = 0.006), and tumor deposit (P < 0.001) were independent prognostic factors in the NSCRC group. However, merely TNM stage (P < 0.0001) and CD3 (P = 0.045) were independent predictors in SCRC group  months, respectively. During the follow-up, there were 42.6% (134 out of 314) patients who died.P = 0.0007). Meanwhile, patients with CD3 rich were associated with favorable OS compared with that of with CD3 poor . Besides, patients with CD20 rich were also associated with favorable OS compared with those of CD20 poor .Patients with sTILs rich gained significant survival benefit compared with those with sTILs poor . In the NSCRC group, higher sTILs and CD3 and CD20 were also significantly associated with favorable OS .Considering that schistosomiasis was a risk factor in the univariate analysis as shown in Table Schistosomiasis is a common disease worldwide. In endemic areas, schistosomal infestation has been implicated in the etiology of several human malignancies including the bladder, liver, and CRC , 31. Ye Specimens from patients with schistosomiasis showed associated inflammatory changes, pseudopolyps, and transitional mucosal changes of schistosomal granulomatous disease progressing to mucosal atypia and to carcinoma were reminiscent of colorectal carcinoma in patients with ulcerative colitis . It was In present study, we evaluated iTILs and sTILs, respectively. However, we found that iTILs are typically present in lower numbers and detected in fewer cases; they are more heterogeneous and are uneasy to observe on H&E-stained slides . The perGiven the functional heterogeneity of intratumoral lymphocytes, and negative immune regulators are present as part of a normal feedback loop reacting to an active and ongoing antitumor immune response, a focused evaluation of individual subsets may have limited value. Besides, the degree of lymphocytic infiltration assessed by simple evaluation of hematoxylin and eosin (H&E)-stained tumor sections has been shown to have predictive and prognostic value despite a lack of detailed information on the immune subpopulations of the infiltrate , 40–42.CD20 + B cells form a major proportion of tumor-infiltrating lymphocytes in a variety of cancers –45. TherThe activation of T cells requires the T-cell receptor complex, which includes the co-receptor CD3 . Our finSeveral limitations associated with the present study warrant mentioning. Firstly, the diagnosis of schistosomiasis was done by finding schistosome eggs in HE-stained slides, lacking of other methods to prove schistosome infection. This may lead to the missing of the number of schistosomiasis-positive cases. In order to reduce this limitation, schistosome ova were observed in all original HE-stained slides , which were obtained from original individual paraffin blocks and used for routine diagnosis. Besides, the medical records of the selected cases were checked carefully to further screen schistosome-positive reports. Secondly, the working group states that evaluating sTIL as a continuous variable will allow for more accurate statistical analysis , but in In summary, the results of this study identified CD3 + cell, but not TILs and CD20 + cell as an independent prognostic indicator for schistosomal and non-schistosomal CRC patients. And the clinicopathological features and positive expression pattern of these infiltrating immune cells between NSCRC and SCRC patients were similar. However, further work to confirm the prognostic role of CD3 in a larger cohort or in multi-organization is needed in further study.Additional file 1:Supplementary Fig. 1. The relationship between schistosomial infection and age distribution. The association was evaluated by using Kruskal-Wallis statistic. Supplementary Fig. 2. Kaplan-Meier curves of Overall Survival (OS) revealing prognostic significance of sTILs in CRC patients with different age group. Supplementary Table. 1. The association between clinicopathological characteristics and schistosomiasis and infiltrating immune cells.


Mucosal healing (MH) has become the treatment goal of patients with Crohn’s disease (CD). This study aims to develop a noninvasive and reliable clinical tool for individual evaluation of mucosal healing in patients with Crohn’s disease.A multicenter retrospective cohort was established. Clinical and serological variables were collected. Separate risk factors were incorporated into a binary logistic regression model. A primary model and a simple model were established, respectively. The model performance was evaluated with C-index, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy. Internal validation was performed in patients with small intestinal lesions.p = 0.004). There was no statistical significance between these two models in sensitivity , specificity , PPV , NPV , and accuracy . The primary model had good calibration and high levels of explained variation and discrimination in validation cohort.A total of 348 consecutive patients diagnosed with CD who underwent endoscopic examination and review after treatment from January 2010 to June 2021 were composed in the derivation cohort, and 112 patients with small intestinal lesions were included in the validation cohort. The following variables were independently associated with the MH and were subsequently included into the primary prediction model: PLR (platelet to lymphocyte ratio), CAR , ESR (erythrocyte sedimentation rate), HBI (Harvey-Bradshaw Index) score and infliximab treatment. The simple model only included factors of PLR, CAR and ESR. The primary model performed better than the simple one in C-index (87.5% vs. 83.0%, This model can be used to predict MH in post-treatment patients with CD. It can also be used as an indication of endoscopic surveillance to evaluate mucosal healing in patients with CD after treatment.The online version contains supplementary material available at 10.1186/s12876-022-02304-y. Crohn’s disease (CD) is a life-long and progressive inflammatory disease with symptoms evolving in a remitting and relapsing manner . Most paMucosal healing (MH) is confirmed lead to a lower rate of relapse, hospitalization and surgical resection . In receIt is reported that clinical characteristics such as mild clinical manifestation and early introduction of biologicals associated with MH in patients with CD –11. In aIn this study, we aimed to address the predictive role of serum inflammatory index and clinical features in patients with CD who diagnosed and treated in two tertiary hospitals in China. We analyzed the pre-treatment and post-treatment data individually and explored their relationship with MH after treatment including Corticosteroids, Immunomodulators or Infliximab in patients with CD. Subsequently, we used hematological data with or without clinical features to construct assessment models for MH prediction. Model with superior performance is recommended for clinical use.This was a retrospective, multi-center observational cohort study of consecutive patients with CD from Inflammatory Bowel Disease Center of The First Affiliated Hospital of Nanjing Medical University and the Second Affiliated Hospital of Soochow University, China, between 2010 and 2021. Diagnoses of CD were determined according to standard clinical, laboratory, radiological, endoscopic, and histopathologic findings . Data reInclusion criteria were listed as follows: 1) Patients underwent at least twice endoscopic procedures and serological examination both pre-treatment and post-treatment during the study period. 2) Corticosteroids had been discontinued for more than 12 weeks. Exclusion criteria: (1) Acute or chronic infections during the inspection; (2) Previous medical history of hematologic or rheumatic autoimmune disease; (3) Acute or chronic renal failure, heart diseases, cirrhosis or cancer; (4) A previous history of taking aspirin or warfarin; (5) Missing complete blood count, ALB, ESR or CRP data. (6) Any other conditions that affect the blood routine results or inflammatory markers. (7) Patients treated with novel biologicals including adalimumab (ADA), ustekinumab (UST) or vedolizumab (VDZ). In the present study, serological indicators were used to construct mucosal healing model of CD patients. So, we ruled out the effects of drugs on blood cells including corticosteroids, aspirin and warfarin. The clinical, endoscopic features and laboratory data of the study population are summarized in Tables  Patients CorticosEthical approval for the study was approved by Clinical Research Ethics Committee of The First Affiliated Hospital of Nanjing Medical University (ref: 2021-SR-235), in compliance with the Declaration of Helsinki. All patients in the study gave their informed consent for reviewing their clinical data.Baseline blood values had been collected at the time of CD diagnosis when patients were admitted to hospital before administration of any treatment. Post-treatment hematology was completed within one week of the patient's endoscopic review. Venous blood specimens were drawn into sterile standard tubes containing ethylene diamine tetraacetic acid (EDTA) as an anticoagulant and evaluated within 1 h after venipuncture using a Beckman Coulter UniCel DxH800 hematology analyzer. The Beckman Coulter UniCel® DxH 800 was used for analyzing ESR and routine blood markers including White Blood Cell (WBC), neutrophils (NE), monocytes (MO), lymphocytes (LY), Eosinophils (EO), Basophilic (BA), Hemoglobin (HGB), platelet (PLT) and hematocrit (HCT). The Beckman Coulter AU5800 Clinical Chemistry Analyzer was used for assessing ALB and CRP. Inflammatory markers of NLR, Monocyte-to-Lymphocyte Ratio (MLR), PLR, CRP-ALB Ratio (CAR) and Platelet-ALB Ratio (PAR) were calculated subsequently.Patients underwent at least twice endoscopic examination during the study, before treatment and approximately one year after treatment (10–14 months), respectively. Mucosal status was assessed by double-balloon enteroscopy or colonoscopy according to the lesion site. Endoscopic procedures were performed with the standard protocol and the static endoscopic images were reassessed retrospectively by two experienced gastroenterologists. MH was defined as a mucosal activity of gastrointestinal tract as remission or mild inflammatory activity, without ulcer , 17. Dist test (2-tailed) was applied for data with normal distribution while Mann–Whitney U test were performed in data with abnormal distribution. Chi-square tests or Fisher's exact test were used to compare the nonparametric categorical data between groups.The statistical analyses were performed by using SPSS 26.0 software . Normality test were applied by Shapiro–Wilk test. Data with normal distribution are presented as mean with Standard deviation (SD), and data with non-normal distribution are presented as median with Interquartile Range (Q). The p Value less than 0.05 was considered statistically significant.Univariate and multivariant analyses were applied in SPSS. R software (version 3.3.2) was used to build the nomogram and evaluation of model performance (“rms” package). Parameters inclusive of the interaction terms and of clinical significance were included in a full multivariate model subsequently. The model performance was evaluated with C-index, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy. To simplify the logistic regression results and create a practical tool, the coefficients derived from the multivariate analysis were used as weights to elaborate a nomogram, which facilitates the practical application of the model for evaluating probability of MH expected for a given patient. Internal validation was performed in patients with small intestinal lesions. p < 0.001). Thus, Mann–Whitney U test was performed and identified that levels of HBI (p = 0.006) and age at diagnosis (p = 0.003) were significantly associate with MH, while the disease course was not associated with MH (p = 0.893). In addition, chi-square analyze showed that patients without lumen stenosis (p = 0.005) and treatment with infliximab (p < 0.001) are associated with MH. . Thus Mann–Whitney U test was performed and identified that the levels of Eosinophils (p = 0.021), MLR (p = 0.020), PLR (p = 0.015) and CAR (p = 0.044) were significantly associate with MH (Table p = 0.037). However, the ROC curve analysis showed that AUC of PLR were only 0.58  and specificity was only 0.313, lacking of clinical application significance.Pre-treatment laboratory blood parameters of patients with CD were summarized in Table s p = 0.01, MLR (pp < 0.001). Mann–Whitney U test identified that the levels of NE (p < 0.001), MO (p = 0.001), HGB (p < 0.001), HCT (p = 0.003), PLT (p < 0.001), CRP (p < 0.001), ESR (p < 0.001), NLR (p < 0.001), MLR (p < 0.001), PLR (p < 0.001), CAR (p < 0.001), PAR (p < 0.001) were significantly associate with MH  only contains serum biomarkers including PLR, CAR and ESR. The primary model (model-2) was consisted of the following variables: PLR, CAR, ESR, HBI score and treatment with infliximab. Variables included in the simple and primary models are showed in Table Diagnostic value was compared between the two models. The golden standard is whether MH has been achieved under endoscopy. Table p < 0.001)  . Primary model was superior to simple model in C-index . There was no statistical significance between primary model and simple model in sensitivity , specificity , PPV , NPV , and accuracy   Fig. . Primary01) Fig. . Accordip < 0.001)  Serum biomarkers such as PLR, CAR and ESR after approximately 1 year treatment was independently associated with MH. (2) Patients with lower HBI score and the use of infliximab are more likely to achieve MH. Based on this, we developed prediction model with the above significant variables to evaluate MH in patients with CD.The routine blood test is the most fundamental and accessible examination, which has long been proposed as an essential assistant tool for disease assessment . In our Some clinical characteristics can also predict MH in patients with CD. It is reported that early introduction of tumor necrosis factor (TNF) antagonists, particularly in combination with immunosuppressives associate with MH in patients with CD.  In our sA simple model and a primary model were established in the study. The simple model is simpler, easier to operate clinically, and with favorable accuracy. However, taking into account of the C-index and the calibration plots, primary model showed better discrimination ability. Reliable nomogram based on aforementioned factors was constructed and showed excellent evaluation abilities for MH among patients with CD. Parameters in the monogram are easy to obtain, which increases the clinical practicality. This nomogram can predict MH probability in patients with CD after one year of treatment and provide reference for doctors to perform endoscopic review. If the prediction results indicate low probability of MH, doctors could temporarily eliminate endoscopy and adjust treatment regimen, avoiding repeated and unnecessary invasive endoscopy.Fecal calprotectin (FC) has been widely clarified for the correlation with endoscopically proven CD activity –30. HoweOur study has some limitations. Firstly, although the patients we included from two tertiary hospitals in Eastern China, the results may not represent the general population of patients with CD. Secondly, this is a retrospective study and novel biologics such as Vedolizumab and Ustekinumab were not included as evaluation item in the model. The present model still could be improved by follow-up study and improved to broaden the scope in evaluation of CD.In summary, this study provides comprehensive insights into serum inflammatory index and clinical information to evaluate MH after treatment in patients with CD. We conducted a nomogram, providing a portable decision tool for early MH screening and clinical decision of endoscopic review time. More prospective studies in the future are warrant to perform.Additional file 1. Excel 1. Patients’ data before treatment.Additional file 2. Excel 2. Patients’ data after treatment.Additional file 3. Excel 3. General information of the patients.Additional file 4. Table S1. Baseline demographic and clinical characteristics of patients


Graft-derived cell-free DNA (GcfDNA) is a promising non-invasive biomarker for detecting allograft injury. In this study, we aimed to evaluate the efficacy of programmed monitoring of GcfDNA for identifying BK polyomavirus-associated nephropathy (BKPyVAN) in kidney transplant recipients. We recruited 158 kidney transplant recipients between November 2020 and December 2021. Plasma GcfDNA was collected on the tenth day, first month, third month, and sixth month for programmed monitoring and one day before biopsy. ΔGcfDNA (cp/mL) was obtained by subtracting the baseline GcfDNA (cp/mL) from GcfDNA (cp/mL) of the latest programmed monitoring before biopsy. The receiver operating characteristic curve showed the diagnostic performance of GcfDNA (cp/mL) at biopsy time and an optimal area under the curve (AUC) of 0.68 in distinguishing pathologically proven BKPyVAN from pathologically unconfirmed BKPyVAN. In contrast, ΔGcfDNA (cp/mL) had a sensitivity and specificity of 80% and 84.6%, respectively, and an AUC of 0.83. When distinguishing clinically diagnosed BKPyVAN from clinical excluded BKPyVAN, the AUC of GcfDNA (cp/mL) was 0.59 at biopsy time, and ΔGcfDNA (cp/mL) had a sensitivity and specificity of 81.0% and 76.5%, respectively, and an AUC of 0.81. Plasma ΔGcfDNA (cp/mL) was not significantly different between TCMR [0.15  cp/mL] and pathologically proven BKPyVAN[0.34  cp/mL]. In conclusion, we recommend programmed monitoring of plasma GcfDNA levels after a kidney transplant. Based on our findings from the programmed monitoring, we have developed a novel algorithm that shows promising results in identifying and predicting BKPyVAN. BK polyomavirus (BKPyV) infection frequently causes BK polyomavirus-associated nephropathy (BKPyVAN) in up to 10% of kidney transplant recipients (KTRs) . The priGraft-derived cell-free DNA (GcfDNA) originates from allografts and is considered a potential noninvasive marker for evaluating graft injury, especially in distinguishing antibody-mediated rejection (ABMR) –10. As wThus far, the role of plasma GcfDNA in the diagnosis of BKPyVAN has not been confirmed. We performed this study to investigate the value of plasma GcfDNA level in programmed monitoring and during BKPyV infection. The diagnostic capabilities of plasma GcfDNA in BKPyVAN were further analyzed and used to develop algorithms to detect BKPyVAN.10 copies/mL. Those with urine BKPyV DNA load > 7 log10 copies/mL would undergo further kidney biopsy to confirm the diagnosis of BKPyVAN (biopsy cohort). Written informed consent was obtained from all the patients. Kidney allografts from living or deceased organ donors who met the ethical guidelines for kidney donation were used in this study. No kidney transplant recipients received donor organs from executed prisoners or other institutionalized individuals. This study adhered to the tenets of the Declaration of Helsinki and was approved by the Ethics Committee of the Sichuan Provincial People’s Hospital (No. 2020405).We enrolled primary KTRs at the organ transplant center of Sichuan Provincial People’s Hospital . The inclusion criteria were as follows: (a) adult male or female aged > 18 years and (b) voluntary participation with informed consent. The exclusion criteria were as follows: (a) multi-organ transplant, (b) repeated kidney transplant, (c) severe pneumonia, (d) refusal to participate in the study, and (e) loss to follow-up. Urine BKPyV infection in our study included patients with urine BKPyV load > 3 log10 copies/mL during the postoperative follow-up. Additional blood samples for GcfDNA were collected 1 d before kidney biopsy (biopsy time). Within 10 h of blood collection, plasma was separated by centrifugation for 10 min at 2000 × g, after which the supernatant was centrifuged for 20 min at 4000 × g. Then, the plasma was stored at −80°C until circulating cell-free DNA (cfDNA) was extracted. cfDNA was extracted using the QIAamp Circulating Nucleic Acid Kit , according to the manufacturer’s instructions.For each recipient, 10 mL peripheral blood samples were collected regularly on the tenth day (D10), first month (M1), third month (M3), and sixth month (M6) after transplantation. A kidney biopsy was required for KTRs who were diagnosed with a urine BKPyV DNA load > 7 log®” by S&KM Biotechnology Co., Ltd. GcfDNA fractions (%) were calculated using the following formula:The fractional abundance (%) and genomic copies per mL plasma (cp/mL) of the GcfDNA were measured using a technical testing service called “YiLeShu-Graft sentinalAbsolute quantification of haploid GcDNA genomic copies per mL plasma (cp/mL) is calculated by multiplying the total concentration of cfDNA (cp/mL) in a sample by the GcfDNA fraction (%). The concentration of the total cfDNA is determined by a digital droplet PCR. The assay was performed using the Bio-Rad QX200 Droplet Digital System , and the data were processed using QuantaSoft™ version 1.7.4 software .The pathological diagnosis of BKPyVAN is according to the 2019 American Society of Transplantation guidelines in Table 1 The detection method of IHC staining is as follows: 2–3 µm paraffin sections, dewaxed in water, were thermally repaired with EDTA antigen repair solution (pH 8.0) for 3–5 min. An appropriate amount of endogenous peroxidase blocker was added and incubated at room temperature for 10 min, after which an appropriate amount of primary antibody SV40 was added and incubated overnight at 4°C. Next, an appropriate amount of secondary antibody was added and incubated at 37°C for 30 min; DAB chromogenic solution was added to the sections and incubated for 5 min at room temperature; they were then counterstained with hematoxylin and sealed.3 copies/mL, and the upper limit is 5 × 108 copies/mL.We used the BK virus nucleic acid detection kit and real-time fluorescent PCR probe (TaqMan) technology to detect BK virus DNA quantitatively. For the plasma sample, 35 mL of the patient’s venous blood was drawn, and the detection was completed within 4 h at room temperature. For urine samples, we obtained 10–20 mL of interrupted morning urine and completed the detection within 24 h at room temperature. The minimum detection limit of the kit is 2 × 10Considering the fact that the pathological diagnosis of BKPyVAN was far from perfect, patients with negative pathological results who responded to CNI dose reduction could be diagnosed with clinical BKPyVAN if drug-induced renal injury and other possible causes were ruled out.t-test. Skewed distributed variables were presented as medians (interquartile range [IQR]) and analyzed using the Mann–Whitney U test. Categorical variables were presented as percentages and were tested using the χ2 test. The Kolmogorov–Smirnov normality test was used to assess for normality. Pearson’s test was used to identify correlations among variables. Logistic regression modeling was used for binary covariates. Discrimination was quantified using the area under the curve (AUC) of the receiver operating characteristic (ROC) curve. The least absolute shrinkage and selection operator (LASSO) regression was used to predict BKPyVAN after kidney transplantation using various clinical variables and early GcfDNA. The predictions were then compared with the actual diagnosis of BKPyVAN to assess the effectiveness of the predictions. These were used to develop algorithms to predict BKPyVAN using GcfDNA. Statistical analyses were performed using GraphPad Prism 8.0 and R version 4.0.3. All statistical tests adopted a 95% confidence interval (95% CI) and a two-tailed value of P< 0.05, indicating statistical significance.Continuous variables with normal distributions were presented as means with standard deviation (SD) and evaluated using an independent A flow diagram depicting the criteria used for patient inclusion is shown in According to the biopsy results, 17 were possible BKPyVAN, 9 were presumptive BKPyVAN, and 14 were proven BKPyVAN. No patient was diagnosed with probable BKPyVAN during the study period. Based on clinical judgment, in addition to 14 proven BKPyVAN, 2 out of 17 possible BKPyVAN and 2 out of 9 presumptive BKPyVAN were classified as clinically diagnosed BKPyVAN (n = 18). Serum creatinine levels before kidney transplant in the proven BKPyVAN group [85.8  µmol/L] were comparable to those in the presumptive BKPyVAN group  but greater than those in the possible BKPyVAN group . The warm ischemia time (WIT) in the proven BKPyVAN group [11  min] was similar to that in the presumptive BKPyVAN group [8  min (P = 0.58)] but significantly higher than that in the possible BKPyVAN group [2  min (P = 0.006)]. The cold ischemia time (CIT) in the possible BKPyVAN group [230  min] was comparable to that in the presumptive BKPyVAN group [360  min (P = 0.33)] but was significantly lower than that in the proven BKPyVAN group [790  min ] Table 3.At biopsy time, the median GcfDNA (cp/mL) in the proven BKPyVAN group [0.90 (0.63–1.06) cp/mL] was comparable to that in the presumptive BKPyVAN group [0.80 (0.70–1.05) cp/mL] but was significantly higher than that in the possible BKPyVAN group [0.52 (0.42–0.78) cp/mL (P = 0.037] (The ROC curve showed the diagnostic performance of both GcfDNA (cp/mL)  and GcfDNA (%)  at biopsy time in distinguishing pathologically proven BKPyVAN from pathologically unconfirmed BKPyVAN. Moreover, the sensitivity and specificity of GcfDNA (cp/mL) were 92.9% and 46.2%, respectively, at a cutoff value of 0.57 cp/mL  before biopsy time and baseline of GcfDNA (cp/mL) and found that 76.92% (30/39) of programmed monitoring values before biopsy were increased; plasma GcfDNA of programmed monitoring time and biopsy time of possible BKPyVAN, presumptive BKPyVAN, and proven BKPyVAN recipients are summarized in Two cases were excluded from the analysis: one patient was diagnosed with proven BKPyVAN on day 5 (baseline GcfDNA not available), and another patient was diagnosed with presumptive BKPyVAN on day 339 . The ROC curve showed that the diagnostic performance of ΔGcfDNA (cp/mL) had a sensitivity and specificity of 80% and 84.6%, respectively, when distinguishing pathologically proven BKPyVAN from pathologically unconfirmed BKPyVAN (AUC = 0.83)  could help distinguish them. Eight patients were diagnosed as biopsy-proven TCMR, We found programmed monitoring values before biopsy were also increased in the TCMR group, but plasma ΔGcfDNA (cp/mL) was not significantly different between TCMR [0.15  cp/mL] and pathologically proven BKPyVAN[0.34  cp/mL] Figure 5In our previous studies, we found that early GcfDNA can predict eGFR values 90 days post-transplant . FurtherWe further performed multivariate logistic regression to investigate whether GcfDNA alone had a predictive value. We include recipient age, donor age, serum creatinine level before transplant, WIT, CIT, HLA-MM, and creatinine level at D10 and GcfDNA at D10 in the model. And the multivariate logistic regression shows that GcfDNA at D10  and HLA-MM  are independent predictors  and GcfDNA (%) , 7, 21. Our results were consistent with those reported by Chen , that plConsidering patients’ programmed monitoring of GcfDNA on D10, M1, M3, and M6, we proposed a new algorithm for ΔGcfDNA (cp/mL), which is obtained by subtracting the baseline GcfDNA (cp/mL) from the measured GcfDNA(cp/mL) of the latest programmed monitoring time before biopsy time. The results showed that the diagnostic performance of ΔGcfDNA (cp/mL) has a better discriminatory ability for the diagnosis of BKPyVAN than GcfDNA (cp/mL) at the biopsy time, regardless of the diagnostic criteria of pathologically proven BKPyVAN or clinically diagnosed BKPyVAN. This result further suggests that BKPyVAN injury occurs before clinical manifestations and that programmed monitoring of plasma GcfDNA (cp/mL) may help identify kidney transplant injury earlier, thereby triggering earlier kidney biopsy and therapeutic intervention.Shen et al.  found thEarly prediction of BKPyVAN can greatly improve its treatment outcomes. To our knowledge, this is the first study that combined clinical features with GcfDNA (cp/mL) to develop an algorithmic model for predicting BKPyVAN. Previous kidney transplant prediction models have been developed based on multiple sets of clinical data and algorithms, which could inform clinicians regarding graft rejection, graft acceptance, and patient survival . In the The main limitation of this study is that the number of patients recruited from a single hospital with a concurrent diagnosis of BKPyVAN was low, which requires the continued expansion of the cohort. Patients with multiorgan transplants, repeated kidney transplants, and severe pneumonia were not included in this study; therefore, these findings need to be extrapolated to these patients with caution.In conclusion, this study found that plasma GcfDNA (cp/mL) and GcfDNA (%) in BKPyV-infected recipients did not differ significantly from all transplant patients in programmed monitoring. However, they showed significant elevation in proven BKPyVAN. Moreover, GcfDNA (cp/mL) has a low diagnostic efficacy for BKPyVAN at biopsy time. Our novel algorithms based on programmed monitoring, ΔGcfDNA (cp/mL), and the LASSO regression model show promising results in identifying and predicting BKPyVAN.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethics Committee of the Sichuan Provincial People’s Hospital (No.2020405). The patients/participants provided their written informed consent to participate in this study.JW and YH conceived the study. RS and HY collected the data. JW and QR analyzed the data. JW wrote the manuscript. YH, HY, and QR reviewed and revised the manuscript. All authors contributed to the article and approved the submitted version.The study was supported by the “key R&D projects of Department of Science and Technology Sichuan Provincial” (no. 2022YFS0093) and “Youth Fund of Sichuan Provincial People’s Hospital” (no. 2022QN26).The authors thank Sichuan Provincial People’s Hospital, University of Electronic Science and Technology of China, for their support of this work and the reviewers for allowing us to make improvements to the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The health-related quality of life of patients awaiting a total knee arthroplasty (TKA) deteriorates with increasing time to surgery and identification of those with the worst quality of life may help to prioritize patients. The aims were to identify and validate independent variable(s) associated with a health state worse than death (WTD) in patients awaiting a TKA and whether these variables influenced patients-reported outcome measures.n = 3076) was used to validate the variable that was most predictive of a state WTD and to assess the influence on patient-reported outcomes.A retrospective cohort of 5857 patients undergoing a primary TKA was identified from an established arthroplasty database. Patient demographics, body mass index (BMI), index of multiple deprivation, Oxford Knee Score (OKS), EuroQoL five dimension (EQ-5D) 3 level, and visual analogue scale (EQ-VAS) were collected preoperatively and one year postoperatively. An EQ-5D utility of less than zero was defined as WTD. A randomly selected subset of patients (P = 0.050), worse preoperative OKS (P < 0.001), or EQ-VAS (P < 0.001) were independently associated with a health state WTD. The OKS was the most reliable predictor  of a health state WTD. A threshold value of 16 or less, 80% sensitive and specific, was validated and confirmed to have a negative predictive value of 97.5%. Patients with an OKS of 16 or less had a significantly greater improvement in their OKS  and EQ-5D score . When adjusting for confounding factors, a health status WTD was not associated with worse postoperative OKS , EQ-5D  or patient satisfaction .There were 771 (13.2%) patients with a health state WTD. Increasing social deprivation (A threshold score 16 or less in OKS was a reliable predictor of a health status WTD and was associated with a greater improvement in knee-specific and health-related quality of life following TKA. The coronavirus disease 2019 (COVID-19) pandemic has resulted in an unprecedented number of patients on waiting lists for elective surgery, due to the disruption to healthcare services . The numThe Federation of Surgical Specialty Association has defined surgical priority according to four levels . PrioritThe primary aim of this study was to identify independent variables associated with a health state WTD in patients awaiting a TKA. The secondary aims were to (1) identify and (2) validate the most reliable variable associated with a health state WTD, and (3) to assess whether this influenced the patient-reported outcomes following TKA.2. Socioeconomic deprivation was measured using Index of Multiple Deprivation (IMD) deciles (1 being the most deprived and 10 being the least deprived) [Patients for this study were identified retrospectively from a prospectively compiled arthroplasty database held at the study centre. During a 9-year period (June 2007 to November 2016), 5857 patients undergoing primary TKA at the study centre were asked to complete a preoperative patient questionnaire. There were 2204 male patients and 3653 female patients with a mean age of 71.1  years. The mean body mass index (BMI) was 30.6 (SD 5.5) kg/meprived) .The Oxford knee score (OKS)  and Eurowww.random.org) to identify the group according to the power calculation [Patients were randomly selected from the cohort to validate the identified threshold value in the OKS that was predictive of a health state WTD. Patients were assigned a study number and a random number generator was used  were used to assess the relationships between scalar variables. Multivariate Logistic regression analysis was used to identify independent predictors of health state WTD. Multivariate linear regression analyses (MVLRA) were used to assess independent association of a health state WTD with patient-reported outcomes . Variables were assessed for multicollinearity and the variance inflation factor was below 2 for all included factors. Receiver operating characteristic (ROC) curve analysis was used to identify thresholds in the OKS, EQ-VAS and IMD that identified a health state WTD. The area under the ROC curve (AUC) ranges from 0.5, indicating a test with no accuracy in distinguishing whether a patient was satisfied or if their expectation was fulfilled, to 1.0 where the test was perfectly accurate identifying all patients with a health state WTD. The threshold was defined as the point (OKS) at which the sensitivity and specificity were maximal in predicting a health state WTD. A P-value of < 0.05 was defined as statistically significant.Statistical analysis was performed using Statistical Package for Social Sciences version 17.0 . The data assessed demonstrated a normal distribution and parametric tests were used to assess continuous variables for significant differences between groups. A Student’s vs. 1933).A power calculation for the validation cohort was performed using an effect size of 0.2, two-tailed analyses, and alpha of 0.05 (with Bonferroni correction for multiple testing), an allocation of 1:3 (due to lower proportion of patients having an OKS of less than 16) and a power of 95% would require a minimum of 2578 patients , younger age (P < 0.001), increasing BMI category (P < 0.001), increasing social deprivation (P < 0.001), and worse preoperative OKS (P < 0.001), EQ-5D (P < 0.001) or EQ-VAS (P < 0.001) were associated with a state WTD (Table P = 0.050), worse preoperative OKS (P < 0.001), or EQ-VAS (P < 0.001) were independently associated with a health state WTD (Table P < 0.001) correlations with preoperative EQ-5D and the OKS , EQ-VAS  and indices of multiple deprivation , with the greatest correlation being observed with the OKS  patients with a preoperative HRQoL state WTD. Unadjusted analysis demonstrated that female gender , and 957 (31.1%) had a preoperative OKS of 16 or less. The threshold value of 16 or less in the OKS was 85.7% specific and 76.3% sensitive in predicting a health state WTD and had a negative predictive value of 97.5%. Patients with a preoperative OKS of 16 or less had a significantly greater improvement in their OKS and EQ-5D score postoperatively when compared to those who had a higher score which was beyond the MCID , EQ-5D  or patient satisfaction . No patient had a health state WTD postoperatively.In the validation cohort, there were 370 12.0%) patients with a preoperative HRQoL state WTD, which was not significantly different from the overall cohort . The OKS was a reliable predictor of a health status WTD and a score of 16 or less offered 80% sensitivity and specificity, which was validated and shown to have a negative predictive value of 97.5%. Patients with a preoperative OKS of 16 or less had a greater improvement in their OKS and EQ-5D scores, and although they had worse postoperative scores and a lower level of satisfaction with the outcome of their operation, when adjusting for confounding factors these outcomes were equal to those with an OKS of more than 16 points.Rosser and Kind  were amoet al. (2019) [et al. (2021) [et al. (2019) [et al. (2019) [vs. 80%) and specificity (64% vs. 80%) than the current study. Furthermore, the current study validated this threshold using a smaller cohort of patients and demonstrated a similar sensitivity and specificity of 85.7% and 76.3% respectively, suggesting that the OKS is a reliable predictor of a health state WTD.The percentage of patients in a health state WTD observed in the current study (13%) is supported by Scott . (2019) , who des. (2021)  demonstr. (2019)  who also. (2019)  also fouet al. (2018) [et al. (2020) [To use a patient-reported outcome measure such as the OKS to prioritize patients for TKA may not be an ideal tool and may be liable to patients “gaming” the system. The proposed threshold value would enable those with the worst quality of life to be identified and who would benefit the most from TKA. An OKS of 32 or more has been shown to be a reliable predictor of patients not requiring a TKA . However. 2018)  have dem. (2018) . Price e. (2020)  more rec  have de. (2020) , 28. Rat. (2020) . Using t. (2020) .Prioritizing patients on waiting lists has been recognized by several authors either by assigning surgical priority according to indications or using tools to help quantify prioritization , 11–13. et al. (2021) [et al. (2021) [A recent study by Kulkarni . (2021)  highligh. (2021)  highligh. (2021) . If thisThere are several limitations to the current study. All outcomes pre- and postoperatively were prior to the COVID-19 pandemic and may not be fully reflective of patients currently awaiting surgery. The study did not assess the influence of patient comorbidities or American Society of Anesthesiologists grade on health-related quality of life, which may have influenced their quality of life pre- and/or postoperatively. A further limitation was that this study only assessed one year postoperative outcome scores which may not be reflected with longer term follow-up. However, this does seem to be the optimal time-point to assess patient-reported outcomes . FinallyA threshold score 16 or less in OKS was a reliable predictor of a health status WTD and was associated with a greater improvement in knee-specific indicators and HRQoL following TKA. The OKS could be used as a tool to prioritize patients awaiting a TKA, identifying those with the worst quality of life.


Psidium guajava L leaves and phenytoin as a standard agent on the induced oral mucosal wound.The aim of this study was to evaluate the biological effects of hydroalcoholic extract of th and 10th, and 10 rats from each group were sacrificed on the 14th day. Interleukin-6 and total antioxidant capacity were determined in the serum. The tissues were evaluated for pathological and stereological assessments. Phytochemical analyses were performed on the hydroalcoholic extract of Psidium guajava L to determine the antioxidant potency.Hundred seventy Sprague Dawley rats were grouped in 5 clusters randomly. Oral mucosal wounds were induced in all rats except for the control group. Phenytoin and guajava leaf extract were used as a mouthwash. Twelve rats from the 5 groups were euthanized on day 7Psidium guajava L. Decreasing IL-6 and increasing TAC were seen in the guajava hydroalcoholic extract and phenytoin groups. The difference of IL-6 between the wound treated guajava group and the wounded group was significant. The wound treated guajava group and wound treated phenytoin group on the 14th day increased the number of fibroblast cells and volume density of sub-mucosae effectively to the same thickness to be considered as a healed sub-mucosae layer. The volume density of the epithelium changes showed statistically significant different responses based on gender.Total phenolic content test and DPPH analysis demonstrated the high potential of antioxidant capacity of Psidium guajava L leaves might exert theraputic effects on oral mucositis.In conclusion, hydroalcoholic extract of The online version contains supplementary material available at 10.1186/s12906-022-03655-5. Oral mucositis (OM) is a frequent inflammatory complication of cancer chemo- or radiotherapy that is characterized by pain, erythema, and formation of ulcers. OM patients have difficulty in speaking, swallowing, and alimentation, which contribute to weakness in daily functions and wellbeing . Current15H12N2O2) is an insoluble weak acid, usually administered as salt to improve solubility [Phenytoin C1H12N2O2 ilubility . The comlubility . The efflubility .Topical steroids and/or local anesthetics are not recommended on the inflammated oral wounds as they suppress the immune functions, or might mask other severe symptoms. Attempts are now focusing on developing novel substances with minimal side effects with profound therapeutic effects , 9.Psidium guajava L., popularly known as guajava, is growing wild in the torrid zone and subtropics [Psidium guajava L is cultivated in Taiwan, South American countries, Brazil, Colombia, Mexico, Venezuela, and southeast areas of Iran [Recently, research on phytochemicals and their effects on public health has been intensively increased. In particular, research has focused on finding powerful natural antioxidants with wound healing properties. btropics . Psidium of Iran .P. guajava indicated the immense potential of this plant in the treatment of diarrhea, wounds, acne, dental plaque, malaria, allergies, coughs, diabetes, and cardiovascular disorder [P. guajava Linn leaves and Curcuma longa Linn rhizomes were used to treat mouth ulcers. The results showed that the mentioned extract had significant antimicrobial activities which are important to develop anti-ulcer agents [Candida albicans to 24 mm compared to positive control with 27 mm [C. albicans [Guajava flowers and green leaves contain high amounts of α-pinene, β-pinene limonene, tannins, flavonoids, flavonoids, rutin, saponins as well as quercetin, luteolin, guajavolide, and kaempferol . The phadisorder . Recentlr agents . The antth 27 mm . Also, oalbicans . On the albicans , 18 and albicans . Also, talbicans –22.Common causes of mouth ulcers are nutritional deficiencies , poor oral hygiene, and infections. On the other hand, microbial biofilms, overexpression of inflammatory cytokines, and high levels of reactive oxygen/nitrogen species  activate the inflammatory phase, inhibiting progression to the proliferation and reepithelialization stages –25. It wThe aim of the present study was to evaluate the therapeutic effect of guava as a source of natural antioxidant and bioactive compounds on the wound healing process on the induced oral mucosal wound in rats. Also, the total phenolic compound (TPC) and antioxidant potential of the extract were measured. Phenytoin was used as a reference compound for wound healing activity.Ethical approval was confirmed by the Animal Care Committee of Shiraz University of Medical Sciences (IR.SUMS.REC 0.1395. S928). The authors followed up all institutional and international guidelines for animal care and use during this study under the supervision of the ethical committee of Shiraz University of Medical Sciences. The Animal Research Reporting in Vivo Experiments guidelines (ARRIVE) and international legislation, as well as AVMA Guidelines for the Euthanasia of Animals, were also followed up.Group 1: control group (no induced wound)Group 2: wound + no interventionGroup 3: wound + normal saline (W + N.S), 0.9% mouthwash, every day.Group 4: wound + phenytoin suspension (W + P), 30 mg /5 ml mouthwash, every day.Group 5: wound + hydroalcoholic extract of the guajava leaves (W + G), 10% mouthwash, every day.The experiments were carried out on 170 Sprague Dawley rats with equal utility in both sexes . Animals were obtained from the Laboratory Animal Center of Shiraz University of Medical Sciences. They were fed with a standard diet and water ad libitum . In the 2 increases around 100% in the induction chamber.The rats were anesthetized with intramuscular injection of ketamine hydrochloride 10% (100 mg/kg) and xylazine 2% (10 mg/kg). Excising the soft tissue of oral buccal mucosa was done with a punch  of 5 mm diameter in all rats, except for group 1.  in large quantities to reduce the variety of components in the plant. A voucher specimen was deposited in the herbarium of the Faculty of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran; then, the plants were identified (Voucher No. 771). The leaves were air-dried at ambient temperature. The hydro-ethanolic extract was prepared by adding 100 g of dried leaves to 500 ml hydro-ethanol solution (70% ethanol) at room temperature, through fractionated percolation, and then the final mixture was filtered. The obtained extract after filtration was concentrated in vacuum using a rotary flash evaporator . There wth and 10th and 10 rats from each group on day 14th. Serum was obtained via cardiocentesis for assessments of IL-6 and TAC on determined days. The sample tissues were sectioned into pieces (4 µm and 25 µm in thickness) and stained with hematoxylin and eosin (H&E) for histopathological and stereological evaluations.Suspension of phenytoin  and normal saline 0.9%, (Tehran-Daru Co. Iran) were used for mouth washing. Mouth washing was done for each rat by using 5 ml of the specified solution for 2 min over the wound surface twice daily. 12 rats  from every 5 groups were euthanized ethically on days 750. The experiment was repeated 3 times and the IC50 value was calculated by best-fit equations using the Curve Expert statistical program [Assay for the scavenging of stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) was done according to Brand-Williams et al.’s (1995) method with slight modifications , 34. Que program –37.The TPC of Guajava extract was determined using the Foline–Ciocalteu procedure, as described previously with minor modifications , 39. TheAn Ebiosience kit  was obtained and the serum level of inflammatory factor interleukin-6 (IL-6) was determined by enzyme-linked immunosorbent assay (ELISA). Besides, the total antioxidant capacity (TAC) in the serum was evaluated by colorimetric method using a commercial kit  based on a previous study .Stereological evaluation of buccal mucosal tissue of the wounded area was performed according to previously reported procedures . BrieflyThe numerical density of the fibroblasts was also measured using the “optical disector” method Fig. C.Score 0: normal epithelium and connective tissue without vasodilatation; absence of discreet cellular infiltration; absence of hemorrhagic areas, ulcerations or abscesses.Score 1: discreet vascular ingurgitation, re-epithelialization, discreet inflammatory infiltration with mononuclear prevalence; absence of hemorrhagic areas, edema, ulcerations or abscesses.Score 2: moderate vascular ingurgitation, areas of hydropic epithelial degeneration, inflammatory infiltration with neutrophil prevalence, presence of hemorrhagic areas, edema and eventual ulcerations, absence of abscesses.Score 3: severe vascular ingurgitation and dilatation, inflammatory infiltration with neutrophil prevalence, presence of hemorrhagic areas, edema, and extensive ulcerations and abscesses.The histological evaluation was based on the degree of tissue healing using a 4-point scoring system ranging from grade 0–3 as Lima’s method :Score 0:P < 0.05.All the statistical analyses were performed using the SPSS statistical software (v. 25). For all the above methods, the results were expressed as mean ± standard error. Statistical analysis was done using parametric (multiple comparison test) or non-parametric statistics based on the normality test. Then, the data were analyzed using the Mann–Whitney U test. Besides, differences were considered significant at 50 ± SD). The DPPH radical scavenging activity expressed as IC50 was 172 ± 3.592 ng/ml. It seems that Guajava extract was found effective to prevent cell damage against ROS. Quercetin as positive control demonstrated IC50 of 9.1 ± 0.42 µM.The stable free radical DPPH was used to estimate the activity of Guajava extract as a source of antioxidants to decrease and inactivate the initial DPPH radical concentration to 50% in 30 min ,  . Also, the IL6 concentration of female rats on the 10th day in the W + N.S group showed a significant decrease compared to the 7th day (p = 0.037).  . At the end of the study, higher levels of TAC in the guajava and phenytoin treatment groups were seen and this increase was more for the guajava group in female rats.No statistically significant changes were seen in the TAC outcomes between each specific intervention group during the study period in male rats Fig. . There wth and 14th days wound score evaluations revealed that guajava and phenytoin treatment decreased the wound mean scores significantly  compared to the wound and W + N.S groups. Overall, it can be understood that the administration of guajava in females . Comparisons of the guajava-treated group and phenytoin treatment groups revealed no significant differences between both groups in the repaired epithelial thicknesses.As depicted in Fig. ρ value < 0.001) through increase in the thicknesses of the lamina propria significantly even more than this thickness in the no wound rats (control).Similar effects were observed on the lamina propria thickness compared to the epithelial thickness Fig. . The lamData in Fig. ρ value = 0.008). Interestingly, the administration of guajava extract increased the number of fibroblasts during the treatment. No significant difference was observed between the guajava-treated rats and the phenytoin-treated rats. Also, the original tissue section depicted fewer fibroblasts cells in the wound and W + N.S rats. Treatment of the wound rats with the phenytoin and extract ameliorated the loss of fibroblast cells. The arrow indicates the fibroblast cell , and enzymes [Psidium guajava [Psidium guajava were catechin (846 mgr), gallic acid (681 mgr), and resveratrol (71 mgr) [diarrhea . The mai enzymes , 48. Que enzymes . Also, g enzymes , 50, as  guajava . Another(71 mgr) . The ant(71 mgr) . Our resAn increase in the oxidative enzyme activity and/or reduction in the activity of the antioxidative enzymes cause oxidative stress and this situation leads to cell damage, non-wound healing in the pathogenesis of chronic, premature aging and even neoplastic transformation. Therefore, ROS balance is essential for the normal repair process . Under tPsidium guajava using the excision wound model and observed more than 90% wound healing after 14 days post-surgery [These antioxidant properties of the guajava leaf are associated with its phenolic compounds such as gallic acid, ellagic acid, ascorbic acid, quercetin, and resveratrol . Many st-surgery .TAC was used to measure the total antioxidant capacity of the plasma and cell lysates . At the th day, the decreasing trend of IL-6 in male rats (more androgens) which received phenytoin was more than female rats. Our interventions improved the volume density of the epithelium and made the healing process shorter. Although wounded male rats in the phenytoin and guajava treatment groups showed a significantly higher volume density of the epithelium compared to female rats in their same group. Mariotti et al. (2013) evaluated the endocrinology of sex steroid hormones and cell dynamics in the periodontium and concluded specific populations of the fibroblasts and epithelial cells would be modulated by sex steroid hormones [Epidemiologic studies have previously reported controversial results about the impact of sex on wound repair. One critical mediator of wound healing is the androgens hormone, which accelerates repair in both human and animal models . In an ihormones . The reshormones . ReductiRegarding natural and homeopathic agents, the application of guajava leaves hydroalcoholic extract as a mouthwash, patch, syrup, or topical product with anti-inflammatory and anti-oxidant properties on oral wounds and ulcers may result in fast epithelialization from the biochemical and histopathological points of view which contribute to tissue healing.Additional file 1: Figure S1. Graphical illustration of IL-6 changes in Female and Male rats during the intervention.