P = 0.016]. Men with stroke were also found to be associated with a significant decline in International Index of Erectile Function −5 (IIEF-5) score as compared with the healthy controls . The prevalence of ED in post-stroke patients among 14 studies ranged from 32.1 to 77.8%, which was dramatically higher than that of the general population. The result of the GRADE-pro revealed that the quality of the evidence in this study was moderate. The present study has confirmed the high prevalence of ED in men with stroke. ED in stroke patients is a result of both neurological and psychological factors. Rehabilitative interventions rather than phosphodiesterase-5 (PDE-5) inhibitors are recommended to improve the erectile function for those survivors with ED.Men with erectile dysfunction (ED) are considered to be at risk from stroke events. Conversely, post-stroke patients are also at high risk of ED, whereas a quantitative result from all the relevant studies has not been previously addressed. Therefore, we have performed a comprehensive review and meta-analysis on this issue. This study was registered on PROSPERO (ID No. CRD42021226618). Twenty studies with a total of 3,382 stroke events were included, of which six studies were included for quantitative analysis, and the remaining 14 studies were calculated for the ratio of ED. Synthetic results from four eligible studies providing the ED cases showed that stroke patients were associated with a significantly higher risk of ED than the general population [pooled relative risk (RR) = 3.32, 95% confidence interval (CI): 1.25–8.82,  Stroke is a debilitating cerebrovascular event characterized by impaired blood flow to the brain causing rapid neuron death due to hemorrhage or extremely serious oxygen and energy deprivation. According to the common causes, stroke can be classified into four main types, including ischemic, hemorrhagic, transient ischemic attack, and cryptogenic . IschemiStroke also serves as the third most common cause of disability globally . Disabilvice versa. Several meta-analyses (P < 0.001).Stroke leads to ED and analyses –23 suggeanalyses  showed tanalyses  demonstrCurrently, however, how risky ED is in post-stroke patients compared with the general population is still unknown. In the present study, we aim to summarize all the evidence on the association between stroke and ED and further calculate a quantitative result by a meta-analysis to facilitate clinical understanding of this issue.As shown in Four common databases, including MEDLINE (PubMed), EMBASE (OVID), Cochrane Library, and PsycINFO, were systematically searched to filter the eligible studies prior to December 1, 2020. We only included those studies reporting with English language and investigating with human participants. The search strategy used for detecting the potential studies in PubMed databases was: (((((((((((((((((((((((((((((“Stroke”[Mesh]) OR (Strokes)) OR (Cerebrovascular Accident)) OR (Cerebrovascular Accidents)) OR (CVA (Cerebrovascular Accident))) OR (CVAs (Cerebrovascular Accident))) OR (Cerebrovascular Apoplexy)) OR ) OR ) OR (Brain Vascular Accident)) OR (Brain Vascular Accidents)) OR ) OR (Cerebrovascular Stroke)) OR (Cerebrovascular Strokes)) OR ) OR ) OR (Apoplexy)) OR ) OR ) OR ) OR ) OR ) OR (Acute Stroke)) OR (Acute Strokes)) OR ) OR ) OR (Acute Cerebrovascular Accident)) OR (Acute Cerebrovascular Accidents)) OR ) AND ((((((“Erectile Dysfunction”[Mesh]) OR sexual function) OR sexual dysfunction) OR “Sexual Dysfunctions, Psychological”[Mesh]) OR “Sexual Dysfunction, Physiological”[Mesh]) OR Impotence). Furthermore, we attempted to identify additional studies by manual inspection of reference lists in the relevant articles.Both of the definitions of stroke and ED were based on the World Health Organization (WHO) and the International Classification of Diseases (ICDs) codes. The diagnosis of stroke was commonly confirmed by computerized tomography (CT) and/or magnetic resonance imaging (MRI). We accepted all types of stroke, including ischemic, hemorrhagic, transient ischemic attack, and cryptogenic stroke. ED is diagnosed with any of the validated tools as reported in the eligible studies, such as the International Index of Erectile Function −5 (IIEF-5), ICD, enquired by the clinicians, questionnaires, and prescription for ED.©, the confirmation of ED (O), and any study designs (S). In addition, those studies representing with the RR, hazard ratio (HR), or odds ratio (OR) with 95% CI or sufficient data to calculate the effect sizes were included. Besides, in this study, we further included those relevant studies without providing the information of the control group due to most of the related studies investigating the relationship between stroke and ED only focused on the crude prevalence of ED in stroke patients but lack of the ED prevalence in a control group with similar comorbidities in clinical practice. Since this is a comprehensive review, including all relevant studies could better facilitate clinical understanding of the prevalence of ED in post-stroke patients among different countries and samples. The exclusion criteria in this study were: (1) review articles, editorial letters, comments for published studies, and case reports; (2) duplicated data or previous publications for the same samples and the scientific questions; and (3) animal or in vitro experiments. The process of study selection was performed by two authors independently. Any ambiguities were resolved through a third author or the corresponding author.Any studies reporting the prevalence of ED in post-stroke patients with or without a control group were considered eligible. The inclusion criteria for this systematic review and meta-analysis were followed with the standard of Patient, Intervention, Comparison, Outcome, and Study design (PICOS). The question guiding for this study was: is there a positive association between stroke and ED? The components for the PICOS evidence were: adult men with ED or impotence (P), a history of stroke (I), compared with the normal healthy subjectsThe following information was extracted according to a conventional data collection form, including the first authors' names of the included studies, publication year, country, study design, age of the participants, ED cases (ratio), assessment and type/classification of stroke, assessment of ED, and scores of IIEF-5 with mean and standard deviation.The methodological quality of the included cross-sectional studies was dependent on the gained scores from the cross-sectional study quality methodology checklist: 0–3 , 4–7 , and 8–11 . While the methodological quality of cohort and case–control studies was followed by the Newcastle–Ottawa Scale (NOS), low quality, moderate quality, and high quality have corresponded to scores of 0–3, 4–6, and 7–9, respectively. To further rank the overall quality of the evidence from this meta-analysis, the evaluation of the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach was employed.The risk of bias assessments for the included studies was conducted by using the software Review Manager 5.3 .P < 0.05. I2 statistic and Cochrane Q statistic were used for the heterogeneity test. Of which, I2 >50% was considered as substantial heterogeneity, and the P-value of the Q test <0.10 was supposed to be statistically significant. A random-effect model rather than a fixed-effects model was applied in this study due to a high likelihood of between-study variance for differences in both studies designed and demographic characteristics. Moreover, sensitivity analyses were conducted to find the potential origin of the between-study heterogeneity. Finally, the funnel plot, Begg's rank correlation test, and Egger's regression asymmetry test were used to identifying the publication bias.All analyses were completed using STATA version 13.0 software for Windows . In this study, the overall RR with its 95% CI synthesized by all the eligible studies was used to quantitatively estimate the strength of the association between stroke and ED. In addition, the standard mean differences (SMD) and the corresponding 95% CI were applied to calculate the combined effects for the continuous variables . Statistical significance was assumed at a two-tailed The search flowchart for screening the relevant studies is shown in As aforementioned, we further included those studies that only provided the ED cases in the stroke sample but without a healthy control group because we want to show the crude ED prevalence among stroke patients for different countries or races. After exhaustive searching, we have identified 14 eligible studies , 30–40 tAs shown in To rank the quality of the evidence from this meta-analysis, the GRADE approach was applied . ResultsThe results of the risk of bias table and graph are illustrated in P = 0.016) by using a random-effects model. However, statistical heterogeneity was detected during this analysis .As displayed in P = 0.002; heterogeneity: I2 = 95.8%, P < 0.001). Of note, the study by Sikiru et al.  and the substantial heterogeneity were eliminated after omitting the study developed by Chung et al. . On the other hand, sensitivity analysis also indicated that there was no significant association between stroke and ED after omitting the studies by Parazzini et al.  or Tibaek et al. . These results revealed that there were some variations in the combined RR and heterogeneity after omitting one of the included studies. The main reason for this inconsistency was that only four studies were included and the sample size was distinctly different among each study.To evaluate the influence of an independent study on the overall RR and heterogeneity derived from four studies , 27, 29 g et al.  .As shown in Sexual health is an integral part of the quality of life. Various extragenitourinary diseases have been confirmed to contribute to the impairment of sexual life, e.g., diabetes mellitus, cardiovascular disease, dyslipidemia, and some digestive diseases. In 1981, Bray et al.  were theP = 0.016). Ascertained to the GRADE evaluation, the average rates of ED events were 256/1,030 (24.9%) in subjects with stroke and 1,562/10,159 (15.4%) in the healthy controls without stroke. Furthermore, the quality of evidence of this analysis was judged to be moderate. In keeping with the RR calculated from the dichotomous variables, the combined SMD from three eligible studies have suggested that men with stroke have 1.8 scores less than the healthy control subjects and this gap has achieved statistical significance . Based on the 14 different clinical trials providing the ED cases in post-stroke patients but lacking a control group, the results showed that the crude prevalence of ED in all these studies (32.1–77.8%) was obviously higher than that of the general population in Europe (19%) or the United States (22%). The methodological quality of the six eligible studies for meta-analysis was moderate to high. Besides, no significant publication bias was identified among these studies. Based on the combined RR, SMD, and crude prevalence of ED in this study, all these results were inclined to confirm that stroke might serve as a risk factor for ED development. Stroke affects erectile functioning and vice versa. Several studies have revealed that men with ED were at high risk of stroke  and psychosocial causes . Stroke-induced ED was mainly dependent upon impairment of brain areas controlling penile erection or governing sexual behavior. Erections can be modulated by the medial preoptic area, paraventricular nucleus, and anterior hypothalamic regions in the brain. Erectile function is maintained by the autonomic nervous system that controls the interaction of the corpora cavernosa smooth fibers and endothelial cells. The neural pathways for erection include the central nervous system (CNS)-driven sexual behavior, the CNS control of the autonomic system, and the activity of the peripheral nerve . The neuBrain damage for stroke has long been recognized as the dominating cause of ED development. However, in addition to CNS impairment, co-morbidity and psychological elements might also play roles in post-stroke erectile functions. Mobility restrictions  and spasticity after stroke might cause coital position to be cumbersome  or poor ED is highly prevalent in men after stroke, and it is an integral part of the quality of life for the survivors. A systematic evaluation and targeted physical examination should be performed when the post-stroke man's presenting concern is ED. More importantly, post-stroke patients with physical and psychological difficulties need to be taken into account in rehabilitation programs, including sexual counseling and pharmacological and rehabilitative interventions. It was suggested that the knowledge possessed by post-stroke patients and their spouses on sexual function was directly associated with sexual frequency and satisfaction . PsychosBased on the published data of the different clinical characteristics of the post-stroke patients with ED and the non-ED stroke patients, in the risk stratification of patients before the stroke event, individuals with psychosocial disorders , medication use , comorbidities , and co-existing medical conditions  were more vulnerable to suffer from ED than those without these risk factors. In addition to these clinical characteristics, the endothelial dysfunction markers, endothelial progenitor cells (EPCs), and endothelial microparticles are considered to serve as the serum biomarkers for both ED  and strovia a comprehensive review and meta-analysis. However, some inherent limitations of the study should be noted during the interpretation in clinical practice. First, 20 clinical trials were included in this study, but only six studies provided both the study and control groups that could be used for pooling RR or SMD via a meta-analysis. The strength of this study is that all the studies reporting the ED prevalence in stroke samples are included and further analyzed. Referring to the present study, we can not only figure out how risky ED is in post-stroke patients as compared with the general population but also know the crude prevalence of ED in stroke men among different studies. Second, a statistical heterogeneity (I2 = 95.1%) existed in this meta-analysis. A diverse study design; sample size; age of the participants; countries, types, and locations of the stroke; stroke duration and severity; measurements of ED; and comorbidities could be partly responsible for such heterogeneity.To our knowledge, this is the first study to demonstrate a significant association between stroke and ED Results from the pooled RR, SMD, and crude prevalence of ED in stroke patients demonstrated that there is a significant positive association between stroke and ED. Neurological profiles, physical factors, and psychological elements are the important determinants of post-stroke ED. Rehabilitative interventions, psychological therapies, and sexual counseling rather than PDE-5 inhibitors are recommended to improve sexual life or erectile functioning for stroke patients with ED.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.SZ and LF: conceptualization. WW, PW, CD, and MS: methodology. BX, ZX, and YH: investigation. SZ, PW, and CD: writing—original draft. SZ: writing—review and editing. LF and MS: supervision. All authors contributed to the article and approved the submitted version.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.


In fact, the risk of dying from CVD is significant when compared to the risk of developing end-stage renal disease (ESRD). Moreover, patients with severe CKD are often excluded from randomized controlled trials, making evidence-based therapy of comorbidities like CVD complicated. Thus, the goal of this study was to use an integrated bioinformatics approach to not only uncover Differentially Expressed Genes (DEGs), their associated functions, and pathways but also give a glimpse of how these two conditions are related at the molecular level. We started with GEO2R/R program  to get DEGs by comparing gene expression microarray data from CVD and CKD. Thereafter, the online STRING version 11.1 program was used to look for any correlations between all these common and/or overlapping DEGs, and the results were visualized using Cytoscape (version 3.8.0). Further, we used MCODE, a cytoscape plugin, and identified a total of 15 modules/clusters of the primary network. Interestingly, 10 of these modules contained our genes of interest (key genes). Out of these 10 modules that consist of 19 key genes (11 downregulated and 8 up-regulated), Module 1  and module 5  had the highest number of these genes. Then we used ClueGO to add a layer of GO terms with pathways to get a functionally ordered network. Finally, to identify the most influential nodes, we employed a novel technique called Integrated Value of Influence (IVI) by combining the network's most critical topological attributes. This method suggests that the nodes with many connections  and high spreading potential (the spreader nodes are intended to have the most impact on the information flow in the network) are the most influential or essential nodes in a network. Thus, based on IVI values, hubness score, and spreading score, top 20 nodes were extracted, in which RPS27A non-seed gene and RPS2, a seed gene, came out to be the important node in the network. The risk of getting cardiovascular disease (CVD) in patients with chronic kidney disease (CKD) is more than without CKD as discussed by Jankowski et al. . ChronicNetwork theory is a useful tool for deciphering the topological features and dynamics of complex systems and their functional modules. Many extant networks can be classified as scale-free, small world, random, or hierarchical networks. The hierarchical form of the network includes modules and sparsely dispersed hubs to manage the network which is of particular interest to biologists . Moreovewww.ncbi.nlm.nih.gov/geo/;  NCBI . These ov/geo/; . The detcs tools .http://www.ncbi.nlm.nih.gov/geo/geo2r/) was used. GEO2R is a web-based analytical tool with a built-in R program and GEO query for Linear Models for Microarray Data (Limma). The default settings were utilized to prepare the datasets  correction method was utilized to adjust the significant value of p obtained by the original hypothesis test during differential expression analysis. Finally, for DEGs screening, log Fold change was employed as the key index. Log fold change |0.5–2| was employed as a DEG screening condition in this study. The criteria  were selected in order to expand the total number of DEGs between healthy control and diseases samples. Since very few genes were differentially expressed with respect to this threshold, therefore it made the threshold more stringent and would lead to nearly no or very few DEGs. If the fold change is altered, the resulting DEGs will be changed as well, and vice versa. There is no standard value for fold change when it comes to selecting DEGs  and also provides gene-centric views of the network data . The ProThe MCODE method (A cytoscape plugin) was used to detect the modules/clusters/communities (Strongly interconnected regions) in the native network. In a PPI network, protein complexes and pathways are prevalent groupings (clusters) . To sepaGene ontology words are a standardized set of terms separated into three categories: Molecular function, Biological Processes, and Protein Class . Thus, DFor gene annotation and gene list enrichment analysis, Metascapes leverages a variety of databases and technologies. Metascape uses lists of gene identifiers to extract rich annotations, find statistically enriched pathways, and build PPIN networks. Pathway or process enrichment analysis uses the standard accumulative hypergeometric statistical test to identify ontology words with significant input genes given a gene list . We giveThe IVI is a new method for detecting influential nodes. Hubness and spreading values combine to form the IVI algorithm. IVI captures all the network's topological dimensions. In domains ranging from transportation to biological systems, identifying critical individuals within a network is a persistent challenge. Identifying the most powerful nodes with the ability to have the most influence on the PPIN network , degree 1, X2, X3, X4, X5, and X6) and 5 GSE series of Y .The GSE series of CVD denoted by X and the GSE series of CKD was denoted by Y. There are 6 GSE series of X  with (3) and (2) with (4), as follows:p ≤ 0.05 and log fold change |0.5–2.0| were considered statistically significant and differentially expressed. The total upregulated genes in all 11 GSE series of CVD and CKD was Xu∩Yu = {1295}, whereas total downregulated genes are Xd∩Yd = {1478}. Total up and downregulated genes in CKD and CVD, separately, are 437 and 528, and 858 and 950 while the total upregulated genes in all 11 GSE series of CVD and CKD are 1,295, and downregulated genes are 1478 (The intersection of (5) and (6) gives the Overlapped genes. Genes showing a value of are 1478 . After care 1478 .C), betweenness centrality (BC), and Neighborhood Connectivity (NK) centrality metrics.Based on mRNA profiling and protein networks, to generate subnetwork biomarkers (interconnected genes whose aggregate expression levels are predictive of disease state), networks provide a rich source of biomarkers for illness classification. To construct a CVD and CKD interaction network and infer gene-disease correlations using network features , we starA network representation and analysis is a powerful tool for studying the complicated behavior of biological systems' physiology and pathology . Throughp < 0.05  inside each cluster, and then connects all term pairs with Kappa similarity >0.3 , organism (human), visuals styles (group), evidence of experiments, all experimental , and value of p < 0.05 . ClueGO p < 0.05 . WikiPatity >0.3 . Cytoscaemblance . To mainemblance .Ec represents number of edges in a cluster, Nc for number of nodes, and the gamma constant γ = 0.8. The HE calculation for a network within the modules considers contributions from the organization of nodes and edges in a competitive manner, and this energy is used in organizing or re-organizing the network at various levels. This approach can also magnify the significant changes in the network organization when it goes down to various levels of organization, which capture the importance of hubs in the network and at the modular level. Therefore, HE formalism proves to be a useful technique for considering variations in the network organization  is a method for identifying the most influential nodes in a network that considers all topological aspects. The IVI formula combines the most important local , semi-local , and global  centrality measures in such a way that their effects are synergized and their biases are eliminated . The degtherwise . Betweentherwise . Local Htherwise . In additherwise .P-value for the GSE43484 and GSE67492. The current study is more like the meta-analysis of gene expression datasets using common microarray platforms which is quite widespread in many species. Apart from all the technicalities, the sharing of similar functions associated with transcripts of similar expression has persistently been used to annotate functions for humans in bioinformatics. Thus, using multiple datasets within functional premises governed by genes expressing in cell/tissue for a phenotype can be considered to involve similar pathways. This study retrieved 2,773 DEGs from 11 GSE Series (6 CVD and 5 CKD) series with thousands of genes. The 2,873 genes were overlapped in both cases during the process of distinguishing between the significant genes. Finally, based on our filtering criterion, we got to 43 key (or seed) genes in both conditions (24 down and 19 up-regulated genes) represented by Venn diagram in The risk of CVD is increased with stages of CKD by promoting myocardial hypertrophy, coronary (CAD), atherosclerosis, and fluid overload. As the two most important organs of the body, the heart and kidneys work in close relation to each other. So, to unroll this methodical relationship at the molecular level, the present study was intervened. In this study, we have used integrated network-based approach and IVI methods to extracts the information , hub genes, functions, and pathways. One of the limitations of this kind of study is that we have different cell and tissue types. Using p-adjustment is not the right choice under such circumstances as we did get no significant genes at 0.5 adjusted Our retrieved key genes might be of significance in understanding the pathological degree of CRS in a better way. The network was constructed from 43 seed genes  using String (a web tools) with an interaction score of 0.9, the network was then imported to Cytoscape (3.8.1) for further downstream analysis. Out of 43, only 19 seed genes (8 up and 11 downregulated genes) were retained by the native network.p-value, and gene symbol, along with their GO-TERM are represented in the table.To find the involvement of the DEG(s) at different levels in the constructed network, the native or primary network was further broken down to 15 subnetworks or modules using MCODE  and one upregulated (RPS24) gene, module 5 contains one downregulated gene (MYC) and two  upregulated genes, module 6 contains two genes, one (MAP2K3) downregulated and one (TNFSF10) up-regulated gene. Modules 7 and 8 consist of one downregulated (PER3) and one (IRAK3) up-regulated gene. Module 9 contains two genes, one BCL6 (upregulated) and one HSPB1 (downregulated). Module 10 contains one downregulated gene (ENO1). Module 11 contains the gene ATP1A1 (downregulated), and module 13 and 15 contain two genes each, namely, LYN, FCGR3A (downregulated) and NPR3, NPPB (up-regulated) as shown in Based on GO term enrichment, the highest represented a group of 8 genes  perform negative regulation of intracellular signal transduction (GO:1902532) while the least suggested function is negative regulation of apoptotic signaling pathway (GO:2001234) see . Thus, gFurthermore, we used various other available tools for identifying the most influential nodes with IVI like based on its centrality measurements , 10. TheThese results offer DEGs that may act as therapeutic targets for CVD and CKD in the future. This study reveals many aspects of CVD and CKD, such as gene-gene interaction, gene enrichment analysis, and pathways of hub genes. The hub genes may be the biomarker of the two conditions (CVD and CKD) need to be validated further but there are limitations such as few genes may be up-regulated and downregulated in the separate entity (means either in CVD or CKD). Maybe those genes are the key genes in CVD or CKD further research will reveal this gap. The study mainly focuses only on overlapped genes, despite all up and down genes. The non-seed genes are the most influential nodes in the network, not related to CVD and CKD. Because of the PPIN networks from the String database construct, the gene-gene interaction network using the source selected (human) and not disease specific genes interact with the seed genes. So, the study focuses only on the seed genes or related genes with the disease, but the results show the IVI -score, hubness score, and Spreading score, etc., of the non-seed genes .Our computational approach offers a comprehensive study, revealing the biomarkers of CVD and CKD using network approach and IVI methods , signifying the importance of nodes in the network, which help in Discover and understanding the various aspects of this disease. CKD Patients exhibit a pronounced risk for CVD events, utmost 50% of patients with CKD (stage 4 to 5) have CVD.in-silico approaches focus to produce that potential evidence based on past knowledge, thus making this integrated process fast and efficient. On the other hand, there is great efforts are ongoing with the aim of reducing CVD residual risk by developing reliable prognostic and predictive biomarkers. Apart from many challenges, finding seed genes is one of the first challenges and we have proposed a very simple and efficient way to do that using set theory. The resulting 43 seed genes (that defines the molecular relationship between CVD and CKD) can then be used to construct the GGiN (gene-gene interaction network) to uncover the possible biological and functional meaning. On the other hand, we have used IVI method to calculate the influence of the nodes in the network, which further minimizes the gene list to a more realistic one (that can be tested in-vivo or vitro). Our study finds 19 genes out of many for being more prominent in the CRS (published data), whereas many other genes show a good expressions level. Although, RPS27A, a non-seed gene, was found to be the most influential node in the network followed by RPS2 and MYC. Based on these findings, new validation experiments can be constructed to further prove these as markers or good drug targets. Thus, giving us an opportunity to reduce the risks of CKD and CVD.Evidence-based approach has always been the center of clinical studies, while The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/MA and RI conceptualized the work. MA and AAla did data curation. MA, RA, SH, and AAls prepared the figures. MA, RA, ST, AAla, SA, and RI wrote the manuscript. HB, SA, MA, RI, and RB revised the manuscript. All authors contributed to the article and approved the submitted version.MA, ST, and RA were financially supported by the Indian Council of Medical Research, Government of India under Senior Research Fellow. FTS No. ISRM 11/(04)/2019. AAla financially was supported by the Department of Health and Research, (DHR) Ministry of Health and Family Welfare, Government of India under young scientist F.No. 12014/06/2019-HR.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer MA declared a shared affiliation, with six of the authors MA, ST, RA, MS, AAla, and RI to the handling editor at the time of the review.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Eimeria spp. cause the disease coccidiosis, which results in chronic wasting of livestock and can lead to the death of the animal. The disease, common worldwide, has caused huge economic losses to the cattle industry in particular. This is the first systematic review and meta-analysis of the prevalence of bovine Eimeria in China. Our search of five databases including PubMed, ScienceDirect, China National Knowledge Infrastructure (CNKI), Chongqing VIP, and Wan Fang for articles published up to February 29, 2020 on the prevalence of Eimeria in cattle in mainland China yielded 46 articles, in which the prevalence of cattle ranged from 4.6% to 87.5%. The rate of bovine Eimeria infection has been decreasing year by year, from 57.9% before 2000 to 25.0% after 2015, but it is still high. We also analyzed the region, sampling years, detection methods, feeding model, seasons, and species of bovine Eimeria. We recommend that prevention strategies should focus on strengthening detection of Eimeria in calves in the intensive farming model. Eimeria, one of the most common parasitoses in cattle throughout the world  OR Eimeriidas OR Eimeriidae)”. In the same way, we search for “Cattle”[Mesh] and “China”[Mesh] in turn, and the retrieval formulas obtained are: ; “China”[Mesh]) OR People’s Republic of China) OR Mainland China) OR Manchuria) OR Sinkiang) OR Inner Mongolia). The Boolean operator “AND” is used to connect the three search queries. The final search formula is “(“Eimeriida”[Mesh]) OR Eimeriidas) OR Eimeriidae)” AND  AND (“China”[Mesh] OR People’s Republic of China OR Mainland China OR Manchuria OR Sinkiang OR Inner Mongolia)”. In the VIP Chinese Journal Databases, the search strategy set was “title or keywords: coccidiosis AND cattle”. In CNKI and Wanfang Data, the search strategy was “theme: coccidiosis AND cattle”. In the three Chinese databases, all the retrieval processes included fuzzy searches and synonym expansion. We neither contacted the authors of original studies for additional information nor identified related unpublished data. Endnote X9 was used to edit the articles retrieved.Eimeria in cattle; (2) the study provided the total number of cattle tested and prevalence; (3) each sample was from one type of cattle (not a mixed sample); (4) the study sample size was greater than 30; and (5) the study design was cross-sectional. Articles that did not meet these criteria were removed.After removing duplicates, we selected each article based on the title and abstract. Then we applied the following inclusion criteria: (1) the purpose of the study was to check the prevalence of Eimeria species, species of cattle, detection methods, feeding model, total number of cattle, and number of eimeriosis seropositive cattle. The data collection form is presented in Table 1. The quality of eligible publications was estimated based on criteria derived from the Grading of Recommendations Assessment, Development and Evaluation method [Four authors  extracted data with standardized data collection forms to identify eligible studies . Any disn method . In shorEimeria based on numerous studies was calculated by meta-analysis. A high chance of heterogeneity in the included studies was presupposed; by using R software for analysis, the results were linear distributions with W-values greater than 0.9. Thus, a random-effects model was employed to calculate and prepare forest plots using Stata 12 software  [I2 and Cochran’s Q  statistics were used to assess the variations. The potential sources of heterogeneity were further investigated by subgroup analysis and meta-regression analysis. The investigated factors comprised the geographical region, the sampled year , the age of cattle , the species of Eimeria, season (comparison of spring and summer with autumn and winter), feeding model (comparison of extensive with intensive), the cattle species and detection method . We performed a sensitivity analysis for the included studies to verify the stability of the results. Publication bias of the studies included in this meta-analysis was statistically examined with Egger’s test and trim and fill analysis using Stata software (version 12.0). The meta-analysis was performed according to the PRISMA guidelines [The pooled prevalence of bovine TX, USA) . Heterogidelines .In this study, we searched 1330 articles in five databases, and 46 articles were selected for inclusion in our systematic review and meta-analysis . We evalEimeria. The funnel plot did not visually indicate whether there was publication bias, so we conducted Egger’s test and trim and fill analysis to further evaluate the presence of publication bias. Egger’s test result showed P = 0.011 (Table S1), and the result of the trim and fill analysis demonstrated that the publication bias disappeared after the addition of two related studies, implying that the studies we included had publication bias or small-study effect bias .The extent of publication bias in the selected studies was measured and demonstrated by a forest map and funnel plot  and 3. TFig. S3), in which one study at a time was removed and the others analyzed to estimate whether the results could have been aﬀected markedly by a single study. The results showed that after excluding a certain study, the results of the reorganization analysis are basically consistent with the previous results, so we believe that the results of our meta-analysis are relatively stable and reliable.A sensitivity analysis was also conducted , in North America in cattle (91.7%), and buffalo (81.5%) in three regions of Italy [Bovine eimeriosis is a parasitic gastrointestinal disease caused by industry . To dater forage . In calvprotozoa . Becauseexico 60.%, in Norof Italy . By contof Italy , 57.Eimeria species oocysts can survive within −30 °C, and the survival time at −5 to 8 °C is much longer than −30 °C [Eimeria. Secondly, because a dry climate is not suitable for the survival of Eimeria oocysts, infection with bovine Eimeria is more serious in the humid southerly regions than in the slightly dry north [p > 0.05), probably because the number of articles in each region we included varies greatly. It is worth noting that we were not able to assess the prevalence of bovine Eimeria in the northeast because no studies on bovine Eimeria in the northeast were included . Importantly, the high prevalence in Hunan Province might be because there was only one article in the province and the total number of samples was only 32. Therefore, in order to more accurately reflect the true prevalence of Eimeria in cattle in China, it is recommended to further expand the scope of investigation of Eimeria in cattle, increase the sample size of the investigation, and reduce small-study effects bias.In the regions we studied, the prevalence in northern China was much lower than those in southern China. This may be caused by the following reasons: first of all, n −30 °C  The climn −30 °C , so a sury north . In addiEimeria. The fecal flotation test is the most commonly used technique in clinical laboratory, medical and veterinary medicine for separation of oocysts and eggs. Compared with noncentrifugal flotation methods, centrifugal flotation methods are obviously faster and more efficient [Eimeria species by polymerase chain reaction (PCR) targeting the species-specific ITS-1 region. Considering sensitivity and reliability, PCR appears to be better than conventional oocyst stool examination and can identify important species of bovine Eimeria [Eimeria. In addition, it can also be distinguished according to the size, shape and color of the oocysts [Eimeria species that can be distinguished by PCR technology, highly pathogenic Eimeria can be detected, which provides an effective basis for the prevention and control of Eimeria. When the experimental conditions permit, we believe that the PCR method should be selected to detect bovine Eimeria.In meta-analysis of rates, the detection method is usually a source of heterogeneity. All the studies we included used traditional fecal testing methods to detect fficient . The sat Eimeria . Compare Eimeria . Traditi oocysts , but the oocysts . The detEimeria in China showed a significant downward trend (p < 0.05). At the beginning of the reform and opening up, the stock of cattle increased rapidly while little attention was paid to the prevention and control of Eimeria emmetaria, so the parasite spread quickly. This is consistent with our research results. In our study, the prevalence of bovine Eimeria showed a downward trend year by year, which may have the following reasons: first, since 2000, the Chinese government has successively promulgated policies such as the “Animal Husbandry Law” and “Animal Epidemic Prevention Law” to strengthen support for the development of animal husbandry [Eimeria. Commonly used preventive drugs included monensin, amprolium, diclazuril and toltrazuril, and therapeutic drugs included furacilin [Eimeria on the cattle industry. Third, since 2012, the Chinese government has begun to pay more and more attention to environmental pollution caused by animal husbandry: for example, the 12th Five-Year Plan of National Livestock and Poultry Pollution Control issued by the Ministry of Environmental Protection (MEP) and the Ministry of Agriculture (MOA) to strengthen environmental governance [Eimeria prevalence. Therefore, formulating corresponding prevention and control policies may play a positive role in reducing bovine Eimeria infection. The above measures have reduced the risk of bovine eimeriosis infection.We found that in the past 32 years, the prevalence of bovine usbandry . Second,uracilin , 65. Thivernance . In 2015Eimeria infection, which is consistent with the results of our study. Our research found that although the point estimate of the intensive farming model is lower than that of extensive, the difference is not significant, and the prevalence of Eimeria in cattle under the two farming models is greater than 35%. Intensive farming is denser, and a large amount of manure may not be processed in time, leading to widespread epidemics of infectious diseases [Eimeria infection in intensive farming [Eimeria in intensive farming showed a downward trend. We suggest further implementation of high-level intensive farming to increase the utilization of cattle industry resources, while reducing the spread of diseases such as Eimeria.There are two main breeding methods in China’s cattle industry: intensive farming model and free-range farming . Intensidiseases , 84, whi farming . Further farming . The “Im farming . The “AcEimeria prevalence in cattle of all age groups varies greatly, mainly affecting calves. The prevalence of calves over 6 months old is higher than that of calves 1–6 months old, which may be related to good care and colostrum feeding, especially colostrum can improve the immunity of calves. In addition, 18-month-old cattle are also unlikely to be infected with bovine Eimeria due to their highly immunity [Eimeria in calves is characterized by abdominal pain, watery to hemorrhagic diarrhea, fever, and dehydration [Eimeria in calves.According to reports, immunity , 37, 57 immunity . The cliEimeria. There are more than 11 common species of Eimeria in water buffalo. Among them, E. bareillyi is a unique species in buffalo [Eimeria in buffalo may be expected. In our research, the Eimeria prevalence of dairy cows is the lowest, which may be due to the high requirements for food safety and the rapid promotion of mechanization, and intelligence and information technology in dairy farming facilities [Eimeria infection. In addition, in China, yak and yellow cattle are generally regarded as labor cattle, and they have wider home ranges, so they have more opportunities for contact with infectious Eimeria oocysts.Many breeds of cattle can be infected with  buffalo , a host  buffalo , so highcilities . The daiEimeria oocysts in winter results in fewer spores and a lower shedding rate [Eimeria infection.Although season is not the main factor affecting eimeriosis infection in cattle, prevalence vareies with season . A studying rate ; therefoEimeria species have arisen in mixed infections [Eimeria are pathogenic. Eimeria zuernii and E. bovis are considered the most pathogenic [E. zuernii or E. bovis may lead to severe diarrhea including blood, intestinal tissue, and fibrin [E. bareillyi had the highest prevalence, which may be due to the fact that the total number of samples taken in the study of E. bareillyi is only 50 cattle, and the total number of samples is too small, which leads to biased research results. Meanwhile, there are relatively few studies on E. kwangsiensis, E. mandela, E. illinoisensis and E. stiedai-like. The prevalence derived from our analysis should be generalized with caution because several included articles did not use random sampling.Up to eight different fections , though thogenic . Infectid fibrin . Lasprild fibrin , which iEimeria in the selected databases by searching using several different MeSH terms; however, these searches may not have found all the relevant studies. Secondly, some of the factors included in some studies and the sample size are too small, so there may be a small-sample size bias, resulting in unstable outcomes. Finally, the research we included may not be particularly accurate because some of the samples in the included articles were not randomly sampled.The advantages of this research are the wide range of the research, the long time span of the research, the large sample size, and the thorough examination of potential risk factors. There were, however, several limitations to our analysis. Firstly, we identified studies related to bovine Eimeria occurs in most regions of China, and the prevalence has been declining year by year. The age of cattle is one of the main reasons that affects the prevalence of Eimeria. To reduce the risk of bovine Eimeria infection, farmers should pay special attention to calves, control the density in intensive breeding, keep the breeding environment clean, and strengthen precautions recommended in policy documents. At the same time, it is necessary to develop more convenient, faster, and more accurate methods to detect Eimeria.This systematic review and meta-analysis showed that bovine https://www.parasite-journal.org/10.1051/parasite/2021055/olmSupplementary material is available at Figure S1. Egger's publication bias plot.Figure S2. Publication bias of studies by Trim ad Fill analysis.Figure S3. Sensitive analysis.Table S1. Egger’s for publication bias.Table S2. Trimming estimator.Table S3. Filled meta-analysis.Table S4. PRISMA checklist item.Table S5. Included studies and quality scores.This work was funded by the Science and Technology Development Program of Jilin Province (20190304004YY).The authors declare that they have no competing interests.


Furthermore, the nanosized functionalization of the AuNP surface brought about the formation of self-assembled monolayers through the employment of polymer coatings as capping agents covalently bonded to the nanoparticles. The most common chemical–physical characterization techniques to determine the size, shape and surface coverage of AuNPs are described underlining the structure–activity correlation in the frame of their applications in the biomedical and biotechnology sectors.Relevant properties of gold nanoparticles, such as stability and biocompatibility, together with their peculiar optical and electronic behavior, make them excellent candidates for medical and biological applications. This review describes the different approaches to the synthesis, surface modification, and characterization of gold nanoparticles (AuNPs) related to increasing their stability and available features useful for employment as drug delivery systems or in hyperthermia and photothermal therapy. The synthetic methods reported span from the well-known Turkevich synthesis, reduction with NaBH Nowadays, nanotechnology and nanochemistry are very often combined in order to develop nanostructured materials and, also, determine to what extent the manipulation of matter on an atomic, molecular, and supramolecular level may affect the desired nanomaterials properties . The atoThis review collects the synthesis and chemical–physical characterization methods of AuNPs with interesting shapes that are requested in common applications . Their u-Sensors: AuNPs can be used for protein detection in Raman spectroscopy utilized as support for the implementation of the analysis of vibrational energies of chemical bonds .-Probes: used for biological imaging application. AuNPs can produce an array of colors employed in dark-field microscopy .-Diagnostics: AuNPs are able to detect biomarkers as a valid tool in the diagnosis of cancers, infectious agents, and heart diseases .-Treatment Agent Transport: AuNP surfaces can be functionalized with hundreds of biomolecules, which are delivered to target cells .-Photodynamic Therapy: AuNPs generate heat when they are irradiated by 700–800-nm wavelengths of light. The heat of these nanoparticles produced when they are inside cancer cells is then exploited to induce death .In particular, AuNPs are employed for medical purposes as: Most of the aforementioned products are still restricted to the research and development stages, with human tests, delivery systems, and toxicological assessments that have yet to be analyzed and developed.3+ and Au+ to metallic gold. Nowadays, many reduction agents are used for the synthesis of AuNPs, such as citric acid and citrate, borohydrides, polyols, sulfites, etc. /[citrate] molar ratio, pH, and temperature, may influence the size and stability of the nanoparticles. The colloidal gold nanoparticle formation process is summarized in A classical method of AuNP synthesis, introduced by Turkevich in 1951 , involveIn the first step, metal ions are rapidly reduced, forming metallic clusters of 1 to 2 nm in size. In the second step, the reduction persists, and the newly formed particles undergo coalescence processes by which two or more clusters join each other during contact to create a single, more significant metallic cluster, thus leading to a decrease in the number of particles. Where the average particle size reaches 2.5 nm in diameter, the number of particles remains unchanged, but the particles keep growing in size. Afterwards, the AuNPs begin growing as a result of the incorporation of the gold atoms reduced in the solution. In the third step, when the average radius of nanoparticles reaches nearly 4 to 5 nm, the growth rate drastically increases, and 70–80% of the gold ions, dissolved in the solution, is rapidly reduced . After t4 and sodium citrate in water under reflux [4 in an ethanol solution added into a boiling solution of tri-sodium citrate under vigorous stirring conditions; in this case, the AuNP size was 4 nm [Seitz et al. prepared a single 40-nm gold nanoparticle by mixing HAuClr reflux . Yang etwas 4 nm .Gold nanoparticles were synthesized by Huang et al. , Akiyama4 was heated up to 100 °C for 15 min and, finally, added with sodium citrate. During the inverse method, altering the order of the additions of the reagents, the experiment was repeated using the same amounts of reagents but mixing sodium citrate with HAuCl4 before the heating step. All the reaction solutions were kept at the boiling point for 5 min before cooling them down to room temperature. The experimental results showed that, while AuNPs synthesized by the direct method had an average diameter of about 18 nm, the indirect method yielded AuNPs with a size of nearly 9 nm.Ojea-Jiménez et al. , investi4). Some of these summary procedures are illustrated below. These syntheses were carried out by varying the Au: citrate molar ratios and/or by altering the reaction conditions. The methods are reported in detail in In order to perform AuNPs synthesis in milder conditions, in the absence of heating, the Turkevich method was modified with the addition of sodium borohydride  by altering the concentrations of the citrate and reducing agent .The Brust–Schiffrin method is one of the most well-known procedures to synthesize spherical AuNPs soluble in organic solvents. The gold surface of nanoparticles presents a high affinity to thiol ligands; this interaction prevents AuNPs from growing and makes possible the formation of small nanoparticles with an average size lower than 10 nm .4 was mixed with a toluene solution of tetraoctylammonium bromide (TOAB). The two-phase mixture was vigorously stirred to induce the transfer of tetrachloroaurate into the organic phase. Then, dodecanethiol and sodium borohydride were added to the mixture and vigorously stirred for 3 h. After that, the two phases were separated, and the organic one was evaporated in a rotary evaporator to reduce the volume. Subsequently, with the aim of removing excess thiol, ethanol was added, and the mixture was kept for 4 h at 18 °C. Once the dark brown precipitate was collected by filtering it off and washing it with ethanol, the resulting product was dissolved in toluene and precipitated one more time with ethanol. The AuNPs yielded by this method presented an average size of 2.5 nm [The “Brust–Schiffrin” method is described as follows: an aqueous solution of HAuClf 2.5 nm . Some sy4 solution layer. Consequent to shaking the mixture, AuCl4− ions were transferred from the aqueous to the organic layer. The gold solution was branched into two vials containing distinct organic compounds: CTAC (cetyltrimethylammonium chloride) and CTAB (cetyltrimethylammonium bromide). Both mixtures were well-stirred, and then, NaBH4 was added to each solution. At this point, the reaction mixtures were vigorously shaken. During this stage, a change in the color was observed; firstly, the yellow tone due to the presence of AuCl4− vanished, and the solution became colorless; afterwards, it turned into a wine-red color after additional stirring. The final particle size was 10 nm [4 solution was mixed with a toluene TOAB solution. Then, an aqueous MPA solution was mixed, and then, an aqueous NaBH4 solution was promptly added to the mixture, which was stirred overnight. The aqueous phase, containing MPA-protected AuNPs with 2 nm of diameter, was collected. The MPA carboxylic groups were deprotonated into carboxylate groups by sodium hydroxide produced by reducing NaBH4, which allows MPA-protected AuNPs to disperse in water [4 aqueous solution with NaBH4 as a reducing agent and DMAP ) as a stabilizer. In a typical synthesis, an aqueous HAuCl4 solution was combined with a toluene TOAB solution acting as a phase transfer reagent. This results in the rapid migration of the AuCl4− ions to the organic layer within a few seconds. Then, a freshly prepared aqueous solution of NaBH4 was added to the mixture and vigorously stirred. Consequent to the addition of the reducing agent, a change in the organic phase color was observed—that is, from light yellow to wine red, within a few minutes. After the separation of two phases, the organic phase was washed with sulfuric acid, sodium hydroxide, and water and consequently dried over anhydrous sodium sulfate. Afterwards, the precipitation of nanoparticles was inducted by adding DMAP to the organic phase, and the excess of the unreacted DMAP was removed by washing the residue three times with toluene. Finally, the precipitate was suspended again in water to collect only DMAP-protected AuNPs with a 20-nm average size [Praharaj et al. prepared AuNPs as follows: TOAB was added in toluene and was introduced above an aqueous HAuClas 10 nm . Kuroda in water . Ghosh eage size . 4 solution with a toluene TOAB solution. A color change was observed; the yellow aqueous phase became colorless, while the organic one turned orange. Subsequently, a freshly prepared aqueous NaBH4 solution was slowly added into the reaction mixture over a period of 30 min, and the mixture was vigorously stirred for 30 min more. The organic layer was then separated and washed with H2SO4 and nanopure water. Finally, the organic phase was dehydrated using MgSO4 and filtered over a filter paper, yielding 52-nm AuNPs [4 solution was added to the toluene TOAB solution and stirred. Chlorobenzenemethanethiol was dissolved in toluene, and NaBH4 was added in the water phase. The mixture solution was agitated for 3 h; the toluene phase was separated, and its volume was reduced by evaporation. The AuNPs produced were precipitated into methanol, filtered, and then washed again with methanol. The AuNPs collected had a final size ranging between 3 and 4 nm [Wang et al. proposed their AuNP synthesis, mixing an aqueous HAuClnm AuNPs . Kim et and 4 nm . Other s4 solution was added to an aqueous trisodium citrate solution. Then, NaBH4, the freshly prepared solution, was incorporated under constant stirring conditions. The reaction mixture turned pink rapidly after the addition of NaBH4, suggesting the formation of AuNPs. In this case, citrate acted only as a capping agent, since it is not able to reduce gold salt at room temperature. For the growth solution, cetyltrimethylammonium bromide (CTAB) was added to an aqueous HAuCl4 solution. After heating the mixture until a clear orange color change was observed, it was cooled at room temperature and employed as a stock growth solution. Then, four 50-mL conical flasks were coded as A, B, C, and D. In set A, the prepared growth solution was mixed with an ascorbic acid solution, and after the addition of the seed solution, the mixture was stirred for 10 min. A change in the color into wine red was observed, and finally, the AuNPs yielded were spherical, with 5.5-nm sizes. Likewise, in set B, both the growth solution and ascorbic acid solution were put together, and the seed solution was added under vigorously stirring conditions for 10 min. The solution’s color changed into a deep red, and the collected AuNPs were spherical, with an 8-nm diameter. In set C, the growth solution was mixed with an ascorbic acid solution, and solution B was added under vigorously stirring conditions for 10 min. The AuNPs prepared following this procedure were approximately spherical, with a diameter of 17 nm, and the final color of the mixture was reddish-brown. The latter was used as a seed in set D, where the growth solution was mixed with a solution of set C and an ascorbic acid solution. The collected AuNPs were both spherical, with 37-nm diameters and rods, with a major axis of 200 nm and a minor axis of 17 nm, and the resulting color of the solution was brown. Overall, the prepared solutions—namely A, B, C, and D—were stable for nearly two months thanks to the presence of CTAB as a stabilizer agent [4, CTAB, acetone, and cyclohexane, which was consequently mixed with AgNO3 and ascorbic acid. Then, the growth solution was divided into four parts containing 5 mL each and, subsequently, treated with the seeding-growth technique, as previously described. The mean size of the AuNPs was 10 nm [In the proposed AuNP seeded-growth synthesis, a reducing agent is employed to produce in a first reaction step: Au (0) nanoseeds from a gold precursor. In a second reaction step, these nanoproducts are induced to grow into gold nanorods, using the compound as the cationic surfactant, which selectively adsorbs specific crystal facets showing high-surface energies. Usually, the nanorod’s growth is allowed by using small concentrations of additional ions  as surface passivation components in a structure-directing role . Jana eter agent . Sahoo eas 10 nm . sn-glycero-3-phosphocholine (PL) for the preparation of the growth solution. The seed solution was prepared with an aqueous HAuCl4 and trisodium citrate solution. He et al. reported a synthesis using 1,2-bis-Ascorbic acid is an eco-friendly molecule well-known for its antioxidant properties in biochemical reactions. It is biodegradable and biocompatible, it has a low toxicity, and it is highly soluble in an aqueous medium. Different papers, reported in the literature, have described its remarkable reduction properties.4 was mixed with ascorbic acid and CTAB. Following the color change of the solution, the formation of a precipitate occurred due to the difficulty of complexation and/or solubility of the CTAB. It has been shown that, by reacting solutions of ascorbic acid with solutions of HAuCl4 and CTAB at different molar concentrations, AuNPs of irregular shapes with dimensions from 24 to 42 nm were obtained [The synthesis proposed by Khan et al. can be summarized as follows: HAuClobtained . Polyhedobtained . Boca etobtained . Recently, AuNP green syntheses have been widely documented in the literature; they consist of alternative eco-friendly and biocompatible procedures performed by using plant extracts. Various chemical compositions and amounts of reducing agents may be found in organic extracts, altering the resulting product. Different geometrical shapes and sizes may be obtained, affecting the function and, thus, the final application. Different amino acids, proteins, enzymes, amines, aldehydes, ketones, carboxylic acids, phenols, flavonoids, and alkaloids can provide electrons to induce the reduction of cationic gold into AuNPs. The properties of the resulting products depend on the concentrations of the plant extract, metal salt, pH value of the reaction mixture, and temperature . CommonlAvena sativa biomass. Firstly, a sample of oat ground stems was washed with HCl and then rinsed with deionized water; the pH value was adjusted by using diluted HCl and NaOH solutions. In a separate beaker, a KAuCl4 solution was prepared and moved to three test tubes containing the oat biomass. While smaller AuNPs were collected at pH values of 3 and 4 (5–20 nm), larger AuNPs were obtained at a lower pH value—namely, 2 (25–85 nm).Armendariz et al.  describe4 solution at room temperature and constantly stirred for 1 h to complete the reduction process. The formation of AuNPs was determined by observing the color change of the solution to dark red, which was then centrifuged, and the surfactant was discarded. Subsequently, the collected AuNPs were washed several times with deionized water to remove the excess biological materials, uncoordinated, and, finally, dried at 50 °C in an oven. The AuNPs had diameters that ranged between 6 and 26 nm.Hamelian et al.  produced4 solution was synthesized by the oxidation of a required amount of metallic gold foil in aqua regia and subsequent cycles of evaporation/the addition of concentrated HCl. The HAuCl4 solution was combined with IMC solutions, kept at 25 °C, and permanently stirred for at least 7 days. The yielded AuNPs were ultra-small, with an average diameter of 2.3 nm. Nazirov et al.  suggesteHibiscus sabdariffa L. extract as a reduction agent for AuNP synthesis. The fresh H. sabdariffa L. was washed several times with distilled water to clear away its impurities and dried for 2 days at 65 °C. Once the product was dry, it was powdered and dispersed into distilled water for 30 min at 60 °C. The mixture was then separated by filtration, leaving a pale orange color residual extract with a pH of 3.1. This extract was finally used in 9-nm AuNP synthesis [Taib employed a ynthesis . 4 solution and stirred at 70 °C until the AuNPs were formed when the color of the solution turned from pale yellow to red [AuNPs with an average size of 10–15 nm were synthesized using biopolymer chitosan as a reducing agent. A newly prepared solution of chitosan dissolved into an acetic acid solution was mixed with a HAuClw to red .Guggulutiktham Kashayam (GK), an ayurvedic herbal medicine, as a reduction agent for metallic AuNP synthesis with a size range between 15 and 50 nm. GK was added to an aqueous HAuCl4 solution at room temperature, and, in this condition, the synthesis took nearly 1 h for a complete reduction. By contrast, the synthesis of the AuNPs carried out at 100 °C was found to be faster, taking just 1 min [Suvith et al. used st 1 min .4 boiling solution was added to different volumes of the aforementioned coconut oil and acetone one. It was then possible to conclude that the smaller the volume of reducing agent solution added, the larger the AuNP size [Meena Kumari et al. employed commercial edible coconut oil of high purity as a reducing agent. A solution including coconut oil and acetone was prepared. An aqueous HAuCluNP size .Anacardium occidentale (AO) for AuNP synthesis: AO fresh leaf was hydrodistilled using a Clevenger apparatus, producing oil, which was solubilized acetone, to get the reducing agent solution for the synthesis of AuNPs. An aqueous solution of HAuCl4 was added to the oil, both at 100 °C and at room temperature. The AuNPs prepared at room temperature were hexagonal in shape, and the average length was 36 nm. The AuNPs prepared in 100 °C were both triangular and hexagonal in shape, with their sizes ranging from 15 to 37 nm [Sheny et al. used to 37 nm .w/w) of honey and 42% (w/v) HAuCl4 was prepared, mixed, and vigorously stirred [Spherical AuNPs of 15 nm in size were prepared by Philip et al. using natural honey as the reducing agent. An aqueous solution containing 28% ( stirred .Volvariella volvacea (VV) mushroom as a reducing agent to obtain AuNPs from 20 to 150 nm in size; VV was boiled in water and then filtered with the aim of collecting the VV extract. Different volumes of mushroom extract were added to the same aliquot of HAuCl4 solution at two different temperatures—namely, 40 °C and 80 °C [Philip et al. used nd 80 °C . Coffea arabica L.) pulp extract as a reducing agent. HAuCl4 at concentrations of 3, 4, and 5% was added to the extract coffee pulp solution and kept at 85 °C for 20, 40, or 60 min, respectively. In this work, three factors were also considered as the precursor concentration, reducing agent concentration, and reaction time, and their combined effects on the maximum intensity, particle size, and particle density were analyzed [Recently, AuNPs with an average particle size of 5–22 nm were obtained by Bonilla-Nepomuceno et al. using an aqueous coffee  ligands, attached on the AuNP surface, support their stability, (ii) linkers, bonded on AuNP surfaces, allow additional functionalization reactions, and (iii) functional ligands and biomolecules, directly immobilized on AuNP surfaces, allow further functionalization or bioconjugation, providing a more comprehensive application range . The excThe secondary modification technique is based on the “place exchange” of a thiol ligand to incorporate various surface functionalities on AuNPs. A literature survey showed several works proposing the functionalization of AuNP surfaces. The thiol exchange ligands chosen can vary to a great extent, including organic/inorganic dyes, smart polymers , biomoleAuNP incubation with specific ligands is a simple method for the rapid immobilization of functional molecules. Functionalization by the physical sorption of ligands or biomolecules on AuNP surfaces is essentially driven by electrostatic and hydrophobic interactions. Thus, the control of the pH value of the incubation solution is essential, since it modifies the AuNP charge state and the degree of immobilization of the functional molecules . In factSurface modification based on a dative bond among metallic atoms of the AuNP surface and the ligands, including available sulfur, oxygen, or nitrogen atoms, by giving their unshared electron pairs is one of the most-used procedures for ligand or biomolecule functionalization on AuNPs . Sulfur,Cys-linked proteins have been immobilized following the procedure described by Jeong et al. [Bifunctional linkers with spacers like PEG are commonly used for AuNP surface modification because of the ability of PEG to sustain colloidal stability and decrease the nonspecific binding of proteins from the environment. Furthermore, its terminal reactive functional group can be used for the coupling of peptides or proteins . The dirg et al. . Contrarg et al. ,130,131.The employment of polymer coatings as spacers for ligand immobilization is a popular approach for AuNP surface modifications. The aggregation of AuNPs driven by Van der Waals interactions is prevented thanks to the steric hindrance caused by the long-chain structure of neutral polymers yielding more stable AuNPs . Ionic p4− to aldehyde groups. In the second step, the aldehyde groups were derived with a bifunctional thiolated PEG linker containing a terminal hydrazide group. Then, the protein conjugate was trapped on AuNPs through the Au–S bond, producing a highly stable AuNPs@antibody conjugate, which was oriented and had a higher binding capacity of antigen compared to that of the antibody, leading to a random orientation. The typical covalent immobilization of ligands on AuNPs is carried out through a two-step approach. In the first step, the AuNP surface is preactivated with reactive functional molecules, such as carboxylic acid or amine groups, often introduced via SAM immobilization of bifunctional linkers containing a reactive thiol for inducing the formation of an Au–S bond on one end and the other reactive functional group on the other end . SimilarFullerene has been commonly used for the synthesis of composite materials due to its mechanical, spectral, and structural properties. In many research works, different thiolated fullerenes with alkyl chains have been employed to stabilize AuNPs and tested for electrochemical or photoelectric applications . Carbon Kd = 10−15 mol/L, facilitates the essentially irreversible immobilization of biotinylated ligands on streptavidin–AuNP conjugates. This method allows biotinylated nucleic acids [Biomolecule and ligand immobilization through affinity binding is a further method for the synthesis of chemically stable protein@AuNP conjugates. Common high-affinity binding couples consisting of protein A/G–immunoglobulin, streptavidin–biotin, and antigen–antibody molecular recognition systems have been employed for this purpose. In a series of applications, the bio-affinity of biotin and streptavidin has been widely used for the immobilization of ligands to the AuNPs surface. The low dissociation constant between biotin and streptavidin, which is in the order of ic acids , antibodic acids . A speciic acids .AuNPs can be characterized by a great number of optical and physical methods. The specific optical and physical characteristics of AuNPs are basically caused by the different sizes, morphology, shapes, and colloidal stability of the AuNPs. Therefore, exhaustive characterization with various techniques is of great importance for the quality control of the produced AuNPs. To prevent ambiguous interpretations, a sequence of multiple techniques is generally suggested for suitable and complementary characterizations .D (ln ε = 3.32·ln D + 10.81) [Ultraviolet–visible spectroscopy is one of the most common techniques for the characterization of the localized surface plasmon resonance (LSPR) of AuNPs . LSPR in+ 10.81) . In a re+ 10.81) . Once th+ 10.81) . Since b+ 10.81) . Further+ 10.81) .6, where D is the diameter of the particles (I ~ D6). This relationship is valid for small systems whose diameters are less than 1/10th of the laser wavelength (λ). AuNP diameter distributions appear to be different when the measurements are based on the intensity, volume, and number distributions. Moreover, DLS measures the hydrodynamic diameter instead of the dry state diameter like TEM. The DLS technique is useful when it is necessary to analyze the state of particles in a solution, and what is more is that the shell thickness around AuNPs could be determined by comparing the hydrodynamic diameters of AuNPs before and after functionalization [Dynamic light scattering (DLS) is an appropriate and nondestructive technique to obtain the hydrodynamic size distribution of AuNPs and to determine their aggregation state . Having lization .Transmission electron microscopy (TEM) is a fundamental technique used in nanoscience, because it furnishes high-resolution images of AuNPs at the nanometer level. TEM images consent to determine the sizes and shapes of AuNPs in the dry state. Reliable size measurements and the clear structural morphology of AuNPs can be observed . NeverthThe physicochemical characterization of nanomaterials needs a higher number of analytical techniques to understand the morphology and composition of the nanoparticles. In fact, their surface compositions may change due to exposure to biological fluids or other environmental factors. When the nanoparticles are modified on the surface, the total and uniform coverage of the nanoparticle needs to be guaranteed; it is therefore necessary to accurately characterize the surfaces and functional coatings on the nanoparticles . TGA is X-ray photoelectron spectroscopy (XPS) is a highly specific analytical technique for the characterization of the chemical composition of surfaces of a wide variety of materials. It is a fundamental tool for analyzing the surface atomic layers and surface chemical structures of AuNPs within several nanometers of the surfaces . Some ofThe characterization of the AuNP surface coating is an essential tool for the quality control and regulatory approval of the AuNPs for specific applications in the analysis and biomedical sectors. It is also important for determining the catalytic efficiencies of bionanocatalysts. Quantitative methods developed for the characterization of AuNP shell thickness, ligand orientation, and the number of active parts on AuNPs are briefly summarized in the following sections .N-glycosylated peptides. The surface hydrazide densities were quantified with a UV detection technique. For this reason, the hydrazide groups linked to AuNPs were protected with an excess of 4-nitrobenzaldehyde (4-NBA); the solution was then centrifuged, and the NBA-coupled AuNPs were removed from the solution. The supernatant was analyzed by UV spectroscopy at 265 nm to quantify the concentration of the unreacted amount of free NBA. The immobilized hydrazide groups were determined by back calculation. Xia et al. described four different methods for the quantification of the surface coverage of the spacer (HS-PEG-NH2) on AuNP conjugates: two indirect methods named (i) the fluorescamine-based assay and (ii) ninhydrin-based assay and two direct methods: (iii) the FITC-labeling assay and (iv) Cu2+-labeling assay [2 molecule in the supernatant. These two indirect methods may be imprecise if the reaction is carried out with a large excess of ligands [Indirect methods are extensively employed for determining the ligand densities on AuNP surfaces by quantification of the free ligand in the reaction mixture, such as the supernatant after the functionalization reaction of the AuNP surface . Then, tng assay . Fluores ligands .2 surface shell using FITC or DOTA containing Cu2+ to label the terminal –NH2 groups of the surface ligand. The marked ligands were detected by the ICP-MS technique or fluorescence after dissolving the AuNPs conjugated in aqua regia. In another example, the thiol ligand density of AuNPs conjugated with different bifunctional thiolated ligands was quantified by ICP-MS measurement through the simultaneous detection and quantification of AuNPs and the thiol group [Direct quantification methods furnish more precise and accurate results compared to indirect methods. Usually, both the AuNPs and the ligand density concentration are in relatively low ranges. Thus, for accurate quantification, highly sensitive detection assays are required. To develop a sensitive technique, a different mark is used before the quantification analysis. Xia et al. directly detected an “active” –NHol group . The expWhen the AuNP size is gradually reduced, below 10 nm, important differences in the physical and chemical properties can be observed. The most critical result of the reduction in the particle size is the development of the surface/volume ratio. The modifications involve: (i) a lowering in the melting temperature and (ii) a decrease of the interatomic distance . SurfaceS.aureus) and Gram-negative (E. coli) bacteria, with a minimum inhibitory concentration starting from 30 nmol mL−1 [3O4 magnetic nanoparticles loaded with AuNP and streptomycin. The latter exhibited good antibacterial activity against Gram-negative E. coli and Gram-positive S. aureus bacteria, indicating a good synergy between Fe3O4–AuNP and antibiotics [C. ternatea leaves. In particular, the inhibition of the rate of formation of Pseudomonas aeruginosa by 94.4% was observed when the AuNP concentration reached 100 μg/mL [E. coli, S. Typhimuriumm, S. aureus, and L.monocytogenese, showing how the antibacterial activity is favored at the minimal concentrations of 0.05 μg/mL, 0.2 μg/mL, 0.008 μg/mL, and 0.0002 μg/mL, respectively [Due to the increase in the clinical resistance to bacteria, antibiotics have started to lose their bacteriostatic efficacy. Indeed, the hydrophilic nature of antibiotics causes the passage of proteins through channels and pores of the bacteria membrane, resulting in a shorter stay inside the cells and a weakening of their bactericidal effect . The commol mL−1 . Khandelmol mL−1  and gentmol mL−1 . The expibiotics . The maiibiotics . Gold naibiotics . In geneibiotics , and higibiotics , while Aibiotics . As regaibiotics . Vanaraj00 μg/mL . Lanh anectively . Surface plasmon resonance (SPR) supports different standard devices for measuring material adsorption onto metal nanoparticles. SPR is defined as the resonant oscillation of conduction electrons excited by incident light. In AuNPs, the electron oscillation is dissimilar from the SPR. If AuNP sizes are around 20 nm—that is, smaller than the wavelength of the electromagnetic wave—the AuNP electron cloud is detected by the electric field. Thus, the entire group of electrons is polarized, inducing the formation of a dipole moment in the nanoparticle’s surface, and, as a consequence, this oscillating polarization generates an electric field opposite to that of excitation, resulting in a restoring force. This oscillation damping takes place following two different pathways: heat production and light scattering. LSPR is a bulk effect occurring in a nanometric volume of metals. The principal characteristics of LSPR can be summarized as follows: (i) the plasmon resonance is displayed in the visible or near-infrared spectral ranges for AuNPs that display light scattering, with a cross-section wider than conventional dye . (ii) Th®, Abraxis BioScience Inc., Los Angeles, CA, USA), a 130-nm albumin particle, has been authorized by the US Food And Drug Administration (US FDA) for metastatic breast cancer [Specific optical properties of AuNPs make them powerful nanometric thermal sources, thanks to an intrinsic energy exchange brought to light–heat conversion, generally called the thermo-optical response of nanomaterials. This response is mainly controlled by the behavior of electrons. In stationary conditions, a temperature variation provokes a modification of the material optical index and properties. The thermo-optical response of AuNPs is greatly influenced by the surface electromagnetic field improvement at the SPR . AuNPs ct cancer . Doxorubt cancer . Methodst cancer  without The combination of nanoscience and biotechnology has spawned nanobiotechnology; this research area offers a huge opportunity to advance in medical and health treatment, diagnostics, and therapeutics . Among aAuNP biodistribution and toxicity are essentially associated to the way they are introduced into the human body—namely, orally, intravenously, or directly into the target cell. Nanomaterial compositions and sizes are important parameter that regulate the cellular uptake mechanisms, the intracellular localization of AuNP, and their chemical interaction with cells . A studyIntra-tumoral administration is a direct method of introducing AuNPs directly into the tumor site . A reseaThe intracellular responses, the biodistribution, and the cytotoxicity of the nanoparticles depend on several factors, such as the size and shape, surface conjugations, the target cell type, and administration methods . The cytAnother research study summarized the mechanism, the efficacy, and the toxicity of photothermal therapy by using AuNPs of different shapes and sizes . The resAuNPs have been used as an excellent system for the delivery of different types of drugs and biomolecules  to the target sites . The desThere is little research aimed at the direct use of AuNPs for cancer diagnostics and therapy  and evenThe enhanced permeability and retention (EPR) effect supports a clarification for the specific targeting of AuNPs in the tumor cells . As a co2+ cations building a framework. AuNPs have been also used for the detection of environmental pollutants, such as polycyclic aromatic hydrocarbons (PAHs), based on the powerful affinity adsorption among the AuNP unmodified surfaces and PAHs: their determination was conducted by means of laser-excited time-resolved Shpol’skii spectrometry [2+ cations [AuNPs can be employed as the Solid-Phase Extraction (SPE) adsorbent  as effictrometry . Furthertrometry . Some sttrometry . AuNPs htrometry . Surface cations , amino t cations , and mon cations . The amo cations . The res cations  and capi cations . Hybrid  cations . Polydop cations  have bee cations . The amp cations . The most used device, developed for the detection of biological molecules such as proteins, hormones, and pesticides, is based on a polymeric membrane sheet where the sample is analyzed through lateral diffusion. The AuNP surface is modified with specific antibodies for selective detection exploiting an immune reaction. Once the target molecules sample is poured on the polymeric membrane sheet, the antibodies move together with the mobile phase, binding their corresponding antigen. This bond inhibits antibodies to bind the antigen, linked on a test line membrane, covalently. If the sample solution does not contain antigen molecules, the antibody sites are free to bind with the test line membrane, and the consequent accumulation of AuNPs causes the test line color to change; an increase in the amount of antigens corresponds to a decrease in the amount of antibodies bonded to the test line, thus resulting in the color fading. This method is employed to detect and characterize compounds such as hormones, pesticides, or drugs . The sen3O4) and the optical properties of AuNP [Several imaging techniques exploit the surface plasmon resonance effect characteristic of AuNP. Larger nanoparticles (400 nm) can be detected using an optical microscope in the phase contrast mode, which involves only scattered light in dark-field microscopy. Small AuNPs can only absorb light, causing the local heating of the environment, which can be detected by photothermal imaging, by fluorescence microcopy, which allows for single particle level detection, by multiphoton SPR microscopy, etc. Immunostaining is a TEM imaging technique based on AuNP conjugated with antibodies that bind fixed and permeabilized cell antigens . The fie of AuNP . AuNP-as of AuNP . The int of AuNP .N-glycosylated peptides. In this procedure, the AuNPs were modified with glutathione, and afterwards, the carboxylic acid groups were derivatives with hydrazine to obtain AuNPs@hydrazide. The extraction and enrichment procedure of the N-glycosylated peptides was realized after the reaction of aldehyde groups of carbohydrates with hydrazine groups on AuNP surfaces. The analysis was performed by HPLC and QTOF mass spectrometer [cis-diol groups. These AuNPs showed significant selectivity for the glycopeptides [Biomarkers are exceptional and valuable indicators of a specific disorder. However, their analysis requires efficient sample preparation for a good selective extraction that needs to be suitable for the sensitive detection techniques . Oxidizetrometer . Likewispeptides . The AuNpeptides . For inspeptides . AuNPs hpeptides .Bio-barcodes are employed to quickly identify very low amounts of various proteins by using a series of reactions for the (i) specific detection, (ii) transcription, and (iii) amplification of the signal. The first reaction involves recognition of the target protein by binding specific antibodies to a magnetic substrate, even though the free proteins are washed away a. DuringThis review described in detail the studies and experimental results concerning AuNPs. In particular, the paper described different types of synthesis and functionalization methods, as well as various characterization techniques and possible biomedical applications, concerning AuNPs. The prospects for studying AuNPs are very promising; their synthesis can be done by different methods with no toxic effects, obtaining singular optical, physicochemical, and biological features. AuNPs present great potential in the modern biomedical field, and this review collected different synthesis methodologies that are used in the reduction of gold ions into metallic gold and the consequent functionalizations. In this paper, the most-used characterization methods to determine the sizes of AuNPs and their eventual functionalization are also illustrated. AuNPs also require a further stabilization analysis in biological fluids for in vitro and in vivo testing. The applications of functionalized AuNPs in medicine and biotechnology have highly developed recently. The latest research works, conducted under different experimental conditions and protocols, show conflicting results. The relative toxicity of AuNPs is still the subject of scientific research. Accurate therapeutic dosages, the delivery mechanism, and the absence of a toxicity database need to be discussed before the usage of nanocarriers in clinical trials. Antitumor-targeted drug delivery and biological marker systems are among the most important application fields. Metallic nanoparticles—in particular, AuNPs—have achieved great attention because of their size-dependent features and biological behavior, improved biocompatibility, stability, and oxidation resistance. AuNPs are appropriate systems for targeted and controlled drug delivery or even to enhance the external treatment potential. AuNPs are suitable agents for drug delivery systems because of the tenability of nanoparticle surfaces with various molecules, such as amino acids and peptides, oligonucleotides, antibodies, etc., to facilitate the loading of a drug. Drug delivery systems in which AuNPs behave as carriers represent an interesting application that requires more investigation to overcome the limitations and to improve the effectiveness and efficiency of drug release at the desired site. Gold has different chemical and physical properties, such as a high electronegativity, its tendency to link with other gold atoms, and its variable colors of dispersed colloids. These features are related to its electronic structure, and they explain the resulting suitability for its many applications. This review focused on the principles of thermo-optical properties emerging from plasmon resonance, as well as on some possible applications in biology, drug delivery, and therapy.


Paragonimus heterotremus infection’s genotoxic potential was assessed in a rat model of simultaneous pulmonary and hepatic paragonimiasis by the alkaline version of single-cell gel electrophoresis (comet assay). Statistical analysis of comet parameters was based on the non-parametric Mann–Whitney U test. (2) Results: A clear and statistically significant increase in DNA damage was detected in the helminth-exposed group versus the control rats and the tissue areas adjacent to the parasite capsule versus remote ones; however, differences in DNA damage patterns between different tissues were not statistically significant. Infection resulted in up to 40% cells with DNA damage and an increased genetic damage index. (3) Conclusions: The data obtained contribute to understanding the pathogenesis mechanisms of paragonimiasis, suggesting oxidative stress as the most likely reason for DNA breaks; these findings allow us to consider P. heterotremus as a potentially cancerogenic species, and they are important for the monitoring and treatment of paragonimiasis.Parasites cause numerous health issues in humans, eventually leading to significant social and economic damage; however, the mechanisms of parasite-mediated pathogenesis are not well understood. Nevertheless, it is clearly evidenced that cancerogenic fluke-induced chronic inflammations and cancer are closely associated with oxidative stress. (1) Methods: The  Schistosoma haematobium is the causative agent of urinary bladder cancer; Clonorchis sinensis and Opisthorchis viverrini are unarguable risk factors for cholangiocarcinoma /(C0 + C1 + C2 + C3 + C4) .Statistical analysis was carried out using the STATISTICA software V. 7. First, data were tested for normality (Shapiro–Wilk test) and homogeneity of variance (Leven test), with visual examination of QQ-plots. Due to the invalidation of at least one of the tests, the non-parametric Mann–Whitney U test was employed for comparison of different groups. A significance level of α = 0.05 was set for all statistical analysis.P. heterotremus and had a narrow range of values in the control groups. Furthermore, the ratio of these indices differs between the control and experimental animals , suggesting alterations in the host’s physiological status under infection . The levels of DNA damage were also statistically significant in comparisons of close and remote cells both in the lung and liver . However, no statistically significant differences in DNA damage were observed between tissues; for comparisons of control, remote, and close cells, p = 0.155, 0.571, and 0.211, respectively. Median values indicate approximately 2–3 times increase of DNA percentage in the tail for remote cells and 3–5 times increase for close cells.The genotoxic effects due to parasite exposure are strongly statistically supported . RelativThe distribution of cells over the standard classes of DNA damage also demonstrated the heterogeneous response among cells of the same tissues, in different tissues, and at different distances from the infection source within the same tissue type . The conA more detailed analysis of cells’ distribution by the degree of their damage  revealedp < 0.05) increase in GDI (1.7–6.8 times) when compared to the control group of rats unexposed to the parasite  are typical under stress due to various changes in environmental conditions. Thus, C2, as the main class of DNA damage, and its recovery by reoxygenation, was found in gill cells of scallops exposed to anoxic stress . TherefoToxoplasma gondii, Helicobacter pylori [Taenia solium, as well as officially non-carcinogenic Hymenolepis nana, Toxocara canis, and Trichinella spiralis [The emergence of Class C3 usually indicates high DNA damage, suggesting a damage rate greater than the rate of recovery observed during oxidative stress. Class C4 has an extremely high DNA damage level when DNA is almost entirely in the comet tail, and almost no cell recovery is possible, with cell death being the most likely event. Along with the lighter injuries described above, such damages were recorded for different animals; for example, after exposure to X-rays, benzene, heavy metals, or other toxins ,25, and r pylori ,27, and spiralis ,29.Taenia solium in a hamster model of taeniasis [Toxoplasma gondii in experimental toxoplasmosis in mice [Opisthorchis felineus in a hamster model of opisthorchiasis [DNA damage is known to be time dependent, i.e., it can accumulate over time. For example, such an effect was reported for infections caused by aeniasis , by Toxo in mice , and by rchiasis . DNA damrchiasis . These fP. heterotremus ESPs, are there specific molecules that prevent tumorigenesis? Indeed, despite the infection genotoxicity (recorded in this paper) in rats with chronic paragonimiasis, numerous histopathological changes in the lung and liver tissues were observed (including necrosis), with no malignant transformations [Another issue that needs to be addressed is whether the accumulation of oxidative damage or cellular apoptosis and/or necrosis predominates in chronic paragonimiasis; moreover, among rmations . These frmations ,6.P. heterotremus infection is similar to that reported for fascioliasis. At the acute stage of the disease in rabbits, the average comet tail length was significantly greater  in liver cells of the animals infected by F. gigantica versus controls. This liver fluke was proposed to be considered a potentially cancerogenic species [Helicobacter pylori infections [In general, the obtained results on DNA damage are comparable to those of clinical observations in patients with diseases of high prevalence, including chronic obstructive pulmonary disease and breast cancer. They have, on average, 2–3 times higher levels of DNA strand breaks in leukocytes versus healthy controls . The gen species  and Helifections .P. heterotremus infection possesses genotoxic potential in a small mammalian model, and this potential is most likely due to oxidative stress and an inflammatory environment. Therefore, this fluke can be considered a potentially cancerogenic species that matches existing opinions regarding the underestimated and dual role of parasitic infections in the development of human cancer diseases. Further studies regarding pathology in paragonimiasis and its underlying molecular mechanisms are required to treat parasite-associated diseases more successfully. The exploration of the genotoxic effect of this fluke in the dependence on tissues and cell types, as well as the intensity and duration of infection, could be of particular interest.This study has shown that