Togaviridae, Flaviviridae, Filoviridae, Arenaviridae, Hantaviridae, Peribunyaviridae, Phenuiviridae, Nairoviridae and Orthomyxoviridae families. Here, we summarize inactivation methods for these virus families that allow for subsequent molecular and serological analysis or vaccine development. The techniques identified here include: treatment with guanidium-based chaotropic salts, heat inactivation, photoactive compounds such as psoralens or 1.5-iodonaphtyl azide, detergents, fixing with aldehydes, UV-radiation, gamma irradiation, aromatic disulfides, beta-propiolacton and hydrogen peroxide. The combination of simple techniques such as heat or UV-radiation and detergents such as Tween-20, Triton X-100 or Sodium dodecyl sulfate are often sufficient for virus inactivation, but the efficiency may be affected by influencing factors including quantity of infectious particles, matrix constitution, pH, salt- and protein content. Residual infectivity of the inactivated virus could have disastrous consequences for both laboratory/healthcare personnel and patients. Therefore, the development of inactivation protocols requires careful considerations which we review here.The handling of highly pathogenic viruses, whether for diagnostic or research purposes, often requires an inactivation step. This article reviews available inactivation techniques published in peer-reviewed journals and their benefits and limitations in relation to the intended application. The bulk of highly pathogenic viruses are represented by enveloped RNA viruses belonging to the  Togaviridae, Flaviviridae, Filoviridae, Arenaviridae, Hantaviridae, Peribunyaviridae, Phenuiviridae, Nairoviridae and Orthomyxoviridae families. Techniques intended for molecular-, serological assessments and vaccine development research are discussed in separate sections  and the nature of the intended downstream application. While serological analysis and vaccine development research set high demands on the preservation of protein structure, molecular biology tests merely require virus genome integrity. The focus of this article is to present the available inactivation techniques for viruses belonging to sections .Togaviridae , Western equine encephalitis virus (WEEV) and Eastern equine encephalitis virus (EEEV)), Flaviviridae (West Nile virus (WNV), all Dengue virus serotypes (DENV)), Filoviridae (Ebola virus (EBOV), Marburgvirus (MARV)) and Phenuiviridae  [Arenaviridae such as Lassa virus (LASV) [Guanidium thiocyanate (GTC) constitutes the main component of nucleic acid extraction kits. GTC-containing buffers are extremely caustic and can be used to destabilize the viral envelope and eliminate cellular nucleases, while maintaining the structure of RNA/DNA for subsequent molecular biology analysis in lower-levels containment facilities ,33,34,69s (RVFV) . The dils (LASV) .The guanidium-based agents commercially available may include different ratios and concentrations of active ingredients that affect their performance. In addition, their efficiency may be influenced by the nature and concentration of the pathogen, the sample matrix and the concentration and contact time of the inactivating agent with the sample . Some stRosenstierne et al., 2016, developed another rapid and simple bedside method for EBOV inactivation . The comAll the above studies concur with PAHO/WHO guidelines 2015 for handling of Ebola and other BSL-4 pathogens . The aimSARS-CoV-2 is effectively inactivated after 5 min incubation of Trizol LS (1:4 ratio of virus:Trizol), or with a detergent, 0.5% sodium dodecyl sulfate (SDS) or with Triton X-100 incubated for 30 min at room temperature ,73. ThisArenaviridae) and other viruses such as Ebola and Marburg viruses (Filoviridae), Alkhurma hemorrhagic fever virus (Flaviviridae) as well as Chikungunya and Ross River virus (Togaviridae), has been demonstrated after exposure to 60 °C heat for 60 min [Thermal inactivation has been used as a method for inactivation of viruses and other infectious agents for 250 years. High temperatures alter viral structure (envelope and capsid) and proteins responsible for recognition and binding to the host cell, leading to inhibition of infection . The ther 60 min ,6,13,74.r 60 min ,14,15,26r 60 min . A subser 60 min . Genomicr 60 min . Conflicr 60 min ,77.Filoviridae family, for instance, requires a significantly higher dose than other virus families. In one study, EBOV and MARV were completely inactivated after exposure to 20 µg/mL of AMT and UV-A energy level of 1000 µW/cm2 for 120 and 150 min respectively, with total energy exposures of 120,000 and 150,000 µW/cm2 [Togaviridae) and WNV (Flaviviridae) were inactivated after a total energy exposure of 2000 µW/cm2. All the tested Flaviviridae  required a considerably lower dose (over 100 times) than Filoviridae for inactivation [Psoralens such as Amotosalen (previously known as S-59) and 4′-aminomethyl-trioxsalen (AMT) have been shown to inactivate a broad range of pathogens through inhibition of transcription and replication of nucleic acids ,38,39,400 µW/cm2 . In comptivation .Arenaviridae as well as RVFV (Phenuiviridae) and EBOV and MARV (Filoviridae), showing that RNA was sufficiently conserved for molecular analyses [Five RT-qPCR assays designed for DENV genomes were successful following AMT-UV-A inactivation, indicating that the photoadducts created in the viral genome did not inhibit reverse transcription . Moreoveanalyses   causes inactivation through damage of nucleic acids and proteins, either by direct or indirect pathways. The formation of crosslinks between pyrimidines and between nucleic acids and proteins inhibits viral transcription ,79,80,81r higher .2) or to a chemical agent . UV-irradiation fully inactivated HTNV after 3 min. Incubation in absolute methanol for 8 min, 1% paraformaldehyde for 20 min, or acetone/methanol (1:1) for 10 min also successfully inactivated the virus [A. A. Kraus et al., 2005 examined the ability of UV light to inactivate Hantaan orthohantavirus (HTNV) . HTNV-inhe virus . The authe virus  Table 22 or to aMethanol and acetone are both lipid solvents that cause destruction of the virion envelope through denaturation or coagulation of proteins . The abiFormaldehyde and paraformaldehyde  react with nucleic acids (both RNA and DNA) to form crosslinks between nucleic acids and proteins and between proteins ,21. ThisThermal inactivation can affect serological tests at temperatures exceeding 56 °C . For exaThe application of this protocol to flaviviruses is supported by several studies and is based on the structural similarity of these viruses ,82. The Flaviviridae, such as Togaviridae, however, is challenging [Togaviridae Ross River virus (RRV), Barmah forest virus (BFV), and O’nyong-nyong virus (ONNV) [Heat inactivation of viruses with a higher thermostability than llenging ,84. Chikllenging  which ans (ONNV) . While Bs (ONNV) .WHO guidelines for BSL 3–4 pathogens suggest the combination of inactivation methods, such as detergent and heat, for serologic testing ,15,26. FTween-20 and Tween-80 are mild, relatively non-toxic detergents that do not denature proteins ,11,16. UArenaviridae have been shown to require higher doses of radiation for complete inactivation, than for example Filoviridae [10 [Gamma irradiation can also be used to inactivate viruses, but because of multiple influencing factors, such as solute protein content, virus concentration, temperature and possibly the air volume , it requires confirmatory tests of inactivation ,85,86. Soviridae . Gamma-ioviridae ,60,61. Tidae [10 . Susceptidae [10 ,85. Gammidae [10 .Toga-, Flavi-, Fili-, Peribunya-, Arena-, Nairo-, Phenui-, and Orthomyxoviridae. Antigenicity and immunogenicity were specifically tested for DENV, LASV and Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV) representing Flaviviridae, Arenaviridae and Nairoviridae families respectively [Psoralens are photo active agents causing cross-linking of nucleic acid pyrimidine residues upon exposure to UV-A ,33,38. Aectively ,33,38.1.5-iodonaphtyl azide (INA) is another photoactive chemical known for its ability to inactivate enveloped viruses without affecting surface epitopes. It has primarily attracted attention for vaccine development (see section on vaccine development), but is equally useful where serological studies are required .Arenaviridae) can be inactivated in a similar manner [Aromatic disulfides (Ph-S-S-Ph) and diathianes  represent a different approach towards virus inactivation . They arr manner ,65,91. Tr manner ,90. Antir manner .The aromatic disulfide NSC20625 has been described as the most effective disulfide agent ,64. The Electron microscopy and SDS-PAGE analyses demonstrated that the morphology and function of AHFV surface proteins GP1 and GP2 is preserved  . The inaArenaviridae.Subsequently, immunoprecipitation tests were performed using hyperimmune anti-AHFV serum and demonstrated antibody binding with similar avidity to both inactivated and infectious virus. This suggests that antigenicity and immunogenicity are not altered. The method is thus a promising approach for serological analysis of 2O2) is a strong oxidizing agent that causes genomic damage thus impeding the ability of the virus to replicate. It is effective against both RNA and DNA viruses [2O2 has attracted attention for serologic analysis and in recent years additionally as an alternative for vaccine development (see section on vaccine development) [Hydrogen peroxide  ,49,50.w/v formaldehyde (13.3 M) equals 100% formalin [Vaccine development sets very specific demands on virus inactivation. The structure of the viral proteins must be sufficiently preserved to induce an effective host immune response. Specifically, the ability to elicit a neutralizing antibody response is often essential ,23,50. Sformalin   for instance, inactivation requires 50–60 days at 4 °C with formaldehyde concentration of 1/2000 [Formaldehyde and Beta-propiolactone  under group 2A and 2B respectively ,103. In f 1/2000 . The timf 1/2000 ,110. Thof 1/2000 . For vacf 1/2000 .2 for 10 min, using 310 nm UV light and was completely inactivated. [1.5-iodonaphtyl azide (INA) is a photoactive hydrophobic azide molecule that has recently attracted increased interest for vaccine application . Upon extivated. . Immunogtivated. . Antibodtivated. . The mortivated. . T-cell tivated. .2) or 35 µM AMT with exposure to UV  [A comparative study on the effect of formaldehyde  and the photochemical agents INA and AMT on antigenicity and immunogenicity of inactivated DENV  . DENV-2  mW/cm2) . Virus i mW/cm2) . Direct  mW/cm2) . Antigen mW/cm2) . Mice va mW/cm2) . High an mW/cm2) .This study supports previous studies showing AMT is an effective inactivating agent that can be used on highly pathogenic viruses of several virus groups (both DNA and RNA viruses) and provide a platform for viral vaccine development . The resBeta-propiolactone (BPL) is the second most commonly used inactivation method for vaccine development after formaldehyde ,26,42. IConsistent with these findings, Jonges et al., 2010, also reported a negative effect on both hemagglutinin and neuraminidase functions of the H3N2 virus tested after inactivation by 0.1% BPL ,110. WhiIn another study, nine avian influenza (AI) viruses were tested for susceptibility to BPL, ether and formaldehyde: four highly pathogenic avian influenza (HPAI) H5N1 viruses, two reverse genetically modified H5N1 and three low pathogenic avian influenza (LPAI) viruses  . Virus iThe results showed that all the tested viruses were inactivated after incubation in 0.1% BPL (for 16 h) at 4 °C and in 0.04% formalin (for 16 h) at 37 °C respectively. The concentration of formalin could be decreased to 0.02% if incubation time was increased to 48 h at 37 °C. Correspondingly, increasing the concentration reduced inactivation time, but adversely also altered the HA-titers. The tests using ether were variable, and non-effective against three of the HPAI viruses tested. When combined with Tween-20 (0.2 or 0.5%) all the tested viruses exposed to ether were effectively inactivated. There was no significant drop in HA-titers after treatment with BPL, ether or formalin, indicating preserved antigenicity of the inactivated virus. The HI-assay demonstrated antibody-titers at a significant level indicating that the virus had preserved immunogenicity .The alkylating agents formaldehyde and BPL were also investigated for antigenic and immunogenic properties upon inactivation of West Nile virus (WNV) . The virThese studies demonstrate that the preservation of antigenicity after inactivation by BPL is variable and relies on factors such as virus strain, BPL concentration, incubation time and pH.2O2 to replace formaldehyde and BPL as an inactivating agent for vaccine generation against Yellow Fever virus (YFV) and West Nile virus (WNV), was examined by Amanna et al. [2O2) is a strong oxidizing agent that causes genomic damage by attacking the carbon double bonds in the nucleosides or abstract hydrogen atoms [2O2 has the benefit of being environmentally safe as it is decomposed into oxygen and water and does not require complicated purification processes to be removed from vaccine preparations. Additionally, it has also been shown to preserve antigenic structures capable of inducing both humoral and cellular responses [2O2 was an extremely effective inactivating agent, causing 6-log10 reduction in titer in less than 2 h for both RNA and DNA viruses, after exposure to 3% aqueous solution of H2O2.The ability of Ha et al. . Hydrogeen atoms ,47. Thisen atoms . H2O2 haesponses ,48,49. T2O2 causes minimal damage to YFV viral epitopes. They compared inactivation of YFV by H2O2, BPL or formaldehyde. After coating to ELISA plates, inactivated virus was probed with YFV-immune serum from mice and the ability to bind to antibodies was assessed. 87–98% of the maximum antibody binding response was observed after 2 h of treatment with H2O2, while BPL- and formaldehyde-treated YFV had significantly reduced antigenicity [2O2 as an inactivating agent for vaccine development, mice were then vaccinated with vaccinia virus (VV) inactivated by either H2O2 or formaldehyde. A new injection was done on day 28 to boost the immune response. H2O2-inactivated virus induced significantly higher levels of virus-specific neutralizing antibody than the virus inactivated by formaldehyde. Mice immunized with H2O2-inactivated VV were then challenged with a lethal dose of VV and 100% of the vaccinated animals survived [2O2 inactivated WNV also induced antibody production in vaccinated mice exceeding that which was observed in human subjects 1 year after natural WNV infection. The vaccinated mice survived a lethal challenge with no sign of disease or weight loss. Full protective immunity was observed >280 days after vaccination, indicating the induction of long-term protective immunity [2O2- inactivated lymphocytic choriomeningitis virus (LCMV). 8 days post-vaccination, LCMV-specific CD8+ T cells reached a similar level to recombinant VV expressing LCMV nucleoprotein (NP)21 or glycoprotein (GP)22. The mice were challenged with 2 × 105 PFU of LCMV at 28 days after vaccination with H2O2-inactivated LCMV. The mice showed a >40-fold increase in LCMV -specific CD8+ T cells within 4 days. Induced T cells were cytolytic, polyfunctional and could provide protective immunity against chronic viral infection [The authors also found that Hgenicity . To assesurvived . H2O2 inimmunity . Furthernfection .2O2 on influenza virus with focus on antigenic and immunogenic properties [2O2 solution in PBS for 2 h in room temperature. The results showed that 2 h incubation in a concentration as low as 1% of H2O2 was sufficient to inhibit replication. Evidence of inhibition was assessed by infecting MDCK cells with inactivated virus and confirming there was no CPE. The virus replication was measured using PCR and showed no sign of replication [2O2. There was no difference in the production of INF-γ. The cytokine response was also tested using a microbead-based immunoassay. PBMCs were stimulated and the response measured by analyzing levels of IL-2, IL-10 and TNFα. The cytokine response for cells stimulated by untreated influenza virus was comparable with those stimulated by inactivated virus [A study performed by J. L. Dembinski et al., 2014 demonstrates the applicability of Hoperties . Three slication . Antigened virus . The humed virus .2O2 is an effective inactivating agent that can be used to provide a vaccine platform for several virus strains [Taken together these studies demonstrate that H strains ,48,49.2+-peptide antioxidants derived from bacteria Deinococcus radiodurans have been considered in the production of polio vaccines [Gamma irradiation has also been suggested as a possible inactivation method for vaccine production. Advantages include the capacity to inactivate large volumes with the virus contained in closed containers or even in a frozen state . Moreovevaccines . Though vaccines ,119. Inivaccines . Other svaccines ,118,119.The purpose of the article has been to identify available inactivating agents and demonstrate their applicability and limitations for specific viruses and settings, with a focus on highly pathogenic viruses. For strict nucleic acid analysis, guanidine based chaotropic salts remain suitable for most highly pathogenic viruses, but may require some caution upon selection ,26,32,70As complete inactivation for highly pathogenic viruses is so critical, several studies suggest the combination of inactivating techniques including at least two different modes of action, e.g., one physical and one chemical ,15,26. FPsoralen and 1.5-iodonaphtyl azide (INA) are alternatives for molecular biology analyses, but these have additionally, been shown to preserve virion structure sufficiently to allow for serologic testing and possibly even vaccine development ,35,96. HGamma irradiation has advantages as an inactivation technique including its high penetrative power and the lack of purification steps needed after inactivation with chemical counterparts. However, there are still some concerns regarding biosafety, reduced antigenicity and that the equipment is too bulky and expensive for some settings.Thermal inactivation is simple, follows first-order kinetics, but is affected by multiple factors such as serum protein content, pH, density of the solution, the presence of organic compounds and salts etc. and offers less reliable inactivation ,8. SimilArenaviridae, HIV and respiratory syncytial virus [Diathanes and disulfide-based compounds represent a totally different approach to virus inactivation. They are specifically targeted to cause alterations in zinc finger motifs within viral proteins, without affecting the integrity of the virus structure. The application, however, is limited to viruses containing such aprotein, such as al virus ,120. Oth2O2) stand out as particularly interesting agents for virus inactivation. They are fast acting, have broad application and an appealing safety profile. There are several photoactive compounds of which only psoralen and INA are mentioned here. Other alternatives not discussed here include methylene blue and riboflavin  is an extremely potent agent that dissolves into biologically safe compounds, while preserving antigenicity and immunogenicity on a number of different virus families [2O2 may be applied to form a vaccine platform against Orthomyxoviridae [Hydrogen peroxide (Hfamilies ,48,50. Joviridae .This article does not claim to be conclusive; the handling and inactivation of highly pathogenic viruses require careful considerations, as pointed out in this article. Our intention has been to create an overview over available inactivation techniques to aid in the selection and appropriate application.


Chemical modifications also have been revealed for virus derived RNAs several decades before, along with the potential of their regulatory roles in virus infection. Due to the dynamic changes of RNA modifications during virus infection, illustrating the mechanisms of RNA epigenetic regulations remains a challenge. Nevertheless, many studies have indicated that these RNA epigenetic marks may directly regulate virus infection through antiviral innate immune responses. The present review summarizes the impacts of important epigenetic marks on viral RNAs, including N6-methyladenosine (m This coas cap-0 . When a as cap-0 . Meanwhias cap-0 . mRNA caas cap-0 , includias cap-0 . Some vias cap-0 . Interesas cap-0 . Given tAfter the pseudouridine (ψ) was firstly identified in plant turnip yellow mosaic virus (TYMV) in 1998 , the folModified nucleotides may stabilize the functional RNA structures by reinforcing the hydrogen bond between Watson–Crick pairs, resulting in augmented thermal stability and reduced dynamics . Under oSensing the foreign molecules by the PRRs of the innate immune system serves as the initial step of the innate immune response . DiffereRetinoic acid-inducible gene I, melanoma differentiation-associated protein 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2) are the three major homologous helicases of RLRs . RIG-I a6A has been widely involved in the innate immune sensing process and thus regulates viral pathogenesis. In some cases, viral RNA-loaded m6A modification dampens the RIG-I mediated RNA sensing and activation of the downstream transcription factors such as IRF3 and IRF7, which depresses the type I interferon (IFN-I) gene expression  enhanced RIG-I sensing activity  infection. Due to the indispensable role of conformational change in RIG-I activation, m6A modifications in virus genome might block the binding of viral RNAs to RIG-I, which disabled the conformational change of RIG-I, as well as the subsequent MAVS-TBK1 pathways. In this case, m6A-deficient hMPV virion RNA induced much higher RIG-I expression  activity to cap their RNA, It has been showed that 2ʹ-O-MTases-deficient virus are highly sensitive to IFN-I  proteins. The phosphorylated STAT is then dimerized and binds to IRF9 to form an ISGF3 complex, a transcriptional factor after transfer into the nucleus. The ISGF3 regulates the expression of numerous kinds of IFN stimulating genes ISGs, which exert strong antiviral effects . HoweverWhenever ISGs are successfully expressed, they will perform diverse antiviral effects. More than an important effector in IFN-dependent antiviral immune response, some ISGs can also be upregulated directly and independent of IFNs after virus infection. Although ISGs have different effects, on the whole, they all can resist or control infectious pathogens . Previou6A is mainly regulated by a methyltransferase (writer), demethylase (eraser), and m6A binding protein . m6A modification of A1907 in HBV pgRNA is the key regulator of IFN α-mediated pgRNA decay. Further investigation showed that ISG20 selectively degraded the m6A HBV transcripts that are strictly regulated by m6A reader YTHDF2 , herpes simplex virus type 1 (HSV-1), and VSV infection. In these cases, YTHDF3 can inhibit the expression of ISGs by promoting the translation of transcriptional inhibitor FOXO3 . RAW264.nduction . Similared cells .6A modification on HBV pgRNA, it also has been indicated that m6A modification of tumor suppressor phosphatase and tensin homolog (PTEN) transcript is affected by HBV infection through invaliding PI3K/AKT pathway and inhibiting IRF-3 nuclear export  proteins to their RNA partners , Hundreds Talents Program of Hebei Province, China (E2020050011), and Advanced Talents Incubation Program of the Hebei University (521000981413) to JT, the National Natural Science Foundation of China (31971227), the Science and Technology Project of Hebei Education Department (ZD2021010), and the Natural Science Foundation of Hebei Province, China (C2020201039).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.https://www.frontiersin.org/articles/10.3389/fmicb.2022.845625/full#supplementary-materialThe Supplementary Material for this article can be found online at: Click here for additional data file.


It was based on the non-probabilistic sampling of suitability for cases selection. The T-student test, Chi-test and logistic regression were used in statistical analyses2, menopause , fœtal macrosomia , vaginal delivery  and perineal tears .five factors independently associated with genital prolapse were identifying: obesity with BMI≥30Kg/mthese factors independently associated with genital prolapse can be used for screening of high-risk women in gynecological and obstetrical consultations in order to improve the treatment of genital prolapse in our milieu. Consic level) ,24-28; ac level) ,24-27,29The lack of data about factors associated with genital prolapse in our milieu highlighted the need to conduct the present study with aims at identifying factors associated with genital prolapse in gynecology and obstetrics service (GOS) of Saint Joseph Hospital (SJH) of Kinshasa.Study design and setting: this was a case-control study in which cases were patients who suffered from genital prolapse and controls were patients who suffered from other disease in GOS of SJH of Kinshasa from January 1, 2008 to December 31, 2017. The GOS of SJH of Kinshasa was chosen because of the presence of formed medical staff, the frequent contact with patients who suffered from genital prolapse, and a free management through the fistula care program.Study population: we used medical files of patients who suffered from genital prolapse (cases) and from other disease (controls), treated in GOS of SJH of Kinshasa from January 1, 2008 to December 31, 2017 and paired according to the parity and the age. All medical files not found and those that contained less than 50% of studied variables were excluded. One hundred and forty-eight available cases and two hundred and ninety-six control have been included and thirteen cases were excluded because their medical files have not been found .Sampling: our sampling was non-probabilistic of suitability for cases selection. The sample size was calculated using the following formula:and T=nxc -26 where0: controls proportion; (P0=0.60), P1: cases proportion .Pα: Z-value for the risk of first species (1.645), α: risk of first species (0.05), Z1-β: Z-value for a power 1-β (1.282), 1-β: power wished (0.9) [T: total number of controls, Zed (0.9) -26. The Data collection: data were collected from registries of GOS and operating rooms, medical files of patients who suffered from genital prolapse (cases) and from other disease (control) at SJH and the data collection record. Variables for study were age of patients, weight, height, BMI, parity, menopausal state, vaginal delivery and their number, smoking, antecedent of foetal macrosomia, chronic pulmonary disease, perineal tears, personal and familial history for genital prolapse, pelvic surgery and spinal anomaly.Statistical analysis: all statistical analyses were performed using SPSS  software version 20. The T-student test and the Chi-square test were used to compare averages and proportions between groups respectively. The univariable logistic regression analysis was used to evaluate the strength of association between observed factors and genital prolapse´s appearance. The multivariable logistic regression analysis was used to identify factors associated with genital prolapse among variable with p-value of less than 0.2 in the univariable analysis. A p-value <0.05 was considered statistically significant.Ethical considerations: principles of medical ethics and documentary studies rules were respected: data were collected confidentially and treated anonymously.Frequency of genital prolapse: we registered 161 cases of genital prolapse out of 13957 patients in GOS of SJH, resulting in an overall frequency of 1.2%. We remind that 148 available cases have been included and 13 excluded because their medical files have not been found or were not available .Factors associated with genital prolapse: risk factors of which the proportion was significantly superior in the group of cases compared to this one of control are multiparity (parity≥4), obesity with body weight ≥90kg, obesity with BMI≥30kg/m2, menopause, foetal macrosomia, pelvic tear, vaginal delivery, vaginal delivery number≥4, genital prolapses surgery (2 (obesity 2), menopause, fœtal macrosomia, pelvic or perineal tears, vaginal delivery and vaginal delivery number ≥4 , the menopause , the fœtal macrosomia , the vaginal delivery  and perineal tears  as factors independently associated with genital prolapse  constituted a factor associated with genital prolapse and it multiply significantly the risk of genital prolapse´s appearance by 6. Our observation is in accordance not only with that of Erata who showed that the multiparity is a risk factor for genital prolapse , but als2was the factor associated with genital prolapse in our study. It augments the risk of genital prolapse by 4 for the body weigth ≥90Kg and by 2 for BMI significantly. Our observation corroborates not only those of Thubert´s [The obesity with body weigth ≥90Kg and BMI ≥30Kg/mhubert´s  and of Mhubert´s  who notehubert´s ,41-43. Thubert´s ,28,44,45hubert´s , and thehubert´s .The menopause was a factor associated with genital prolapse in our study. It multiply significantly the risk of genital prolapse´s occurrence by 2. Our results are similar to those of many authors in the literature ,32,38,39Following the example of other studies ,32,37-39The fœtal macrosomia was the factor associated with genital prolapse. Even if its OR was of 0.021, the fœtal macrosomia was significantly associated with genital prolapse in our series of cases. Our results are in line with those of many other authors in the literature where the fœtal macrosomia was significantly associated with genital prolapse too ,37-39,472, menopause, fœtal macrosomia, vaginal delivery and perineal tears. Our observation corroborates thoses of many authors in the literature [Factors independently associated with genital prolapse, in our study, were obesity with BMI≥30Kg/mterature -39,46-492, menopause, fœtal macrosomia, vaginal delivery and perineal tears. Our results warrant deepened studies upon genital prolapses in order to allow scientists to raise awareness upon genital prolapse’s studies and to improve its treatment in our milieu. These data could be used for screening of high-risk women in gynecological and obstetrical consultations in the GOS of SJH.Five factors associated with genital prolapse are identifying: obesity with BMI≥30Kg/mThe genital prolapse is the dynamic disease which can worsen or recede above all in the pregnant woman's in the postpartum period;It comprises a great recurrence's risk after surgical treatment and It caused several troubles  which hamper the quality of life of patients;The lack of data on the frequency and risk factors for genital prolapse in hospitals of Kinshasa, in the DR Congo.The frequency of genital prolapse is of 1.2%;2, menopause, fœtal macrosomia, vaginal delivery and perineal tears;Factors associated with genital prolapse include: obesity with BMI≥30Kg/mThese results could be used for screening of high-risk women in gynecological and obstetrical consultations.


Total acidity, syneresis, and redness value of the yogurts increased, parallel to the increase in the PJ concentration being added. Also, Streptococcus thermophilus count decreased significantly, whereas no significant effect was observed on the population count of Lactobacillus delbrueckii subsp. bulgaricus. Among PJ yogurt samples, the panelists selected the 13% PJ stirred yogurt as the best sample. PJ was observed to contain valuable bioactive compounds with functional and medicinal effects that culminate in health benefits.Pomegranate juice (PJ) (at concentrations of 13% and 17%) was added to yogurt and its physicochemical and microbial properties were investigated. PJ improved several features of yogurt, bringing an increase in total phenolic contents by 4.3–6.1 and 5.3–7.3 fold in response to 13% and 17% PJ, respectively. Also, there were increases in the total anthocyanin contents of yogurt by 2650–2870 and 3470–3820 fold in response to the said juice concentrations. These increases were observed in both set and stirred yogurts, whereas IC The addition of bioactive compounds, such as polyphenols, flavonoids, and anthocyanins, to the yogurt can make it healthier. Pomegranate juice is a valuable source of natural antioxidants, especially for its phenolic compounds, and can be added to the yogurt for improving its functional properties and to fulfill consumer expectations. The objective of this study was to optimize pomegranate juice concentrations (13% and 17%) by adding them to the yogurt, followed by an analysis of its physicochemical features and microbial properties. Iran, USA, Turkey, Egypt, Italy, India, Chile, and Spain are the leading countries in pomegranate production , with no visible external cuts or spoilage, were purchased from a local market in Shiraz, Iran. The pomegranate fruits were peeled manually and the arils were squashed in a blender . The resultant PJ was filtered through a cotton gauze to remove fruit debris and unwanted particles. Then, the fruits were stored at a frozen temperature (−18°C) until further analysis  were supplied by Sigma‐Aldrich. All other chemicals  and culture media were purchased from Merck. Milk powder was purchased from Pegah‐e‐Fars Dairy Company. YF‐L811 yogurt culture was obtained from Chr. Hansen Ltd. and directly used as starter culture.2.3Streptococcus thermophilus and Lactobacillus delbrueckii subsp. Bulgaricus; each count was ~1010 CFU/g). Then, PJ was added at 13% and 17% to the yogurts. Regarding the set yogurt, milk fermentation was carried out at 44 ± 1°C until a pH of 4.5–4.6 was achieved. The fermented milk batches were cooled and stored at 4°C for 24 h. For stirred yogurt, after fermentation, the fermented milk batches were cooled and stored at 4°C for 24 h. The obtained gels were removed from the refrigerator and gently stirred by a mechanical mixer  at 500 rpm for 1 min. Finally, 13% and 17% concentrations of PJ were added. Both stirred and set yogurts were stored at 4°C until further analysis. Physicochemical examinations were carried out after 1, 5, 15, and 20 days of refrigerated storage (4°C).According to our preliminary experiments (data not shown), the effects of different PJ concentrations were evaluated on primary texture, color, and taste of yogurts. Finally, 13% and 17% PJ concentrations were selected as optimum concentrations. Reconstituted milk was produced at 40 ± 1°C by a moderate mixing of milk powder with distilled water. The reconstituted milk contained 18.48% total solid and 3.03% fat. The milk dispersion was refrigerated at 4°C for 12 h to allow full hydration of the milk powder before usage. Reconstituted milk was pasteurized (85 ± 1°C for 30 min)  that was calibrated using pH 4.0 as reference along with 7.0 buffer solutions. The yogurt samples were blended before pH measurement. Also, total titratable acidity was determined by titration with 0.1 N NaOH until the pH value reached 8.1 and the amount of acidity was expressed as the percentage of lactic acid.2.4.2g for 10 min at 4°C. The clear supernatant was poured off, weighed, and finally expressed as the weight (%) relative to the original weight of the yogurt.Syneresis was analyzed during the storage period by a method proposed by Wang et al. . Forty g2.4.3L* (Lightness/darkness), a* (redness/greenness), and b* (yellowness/blueness).Color measurements were carried out using a HunterLab apparatus . The color values were expressed as 2.52.5.1Total phenolic content was measured according to the Folin‐Ciocalteu colorimetric method. For this purpose, 0.1 ml of each sample was mixed with 0.75 ml Folin‐Ciocalteu reagent which had been previously diluted 10 times in the presence of 0.75 ml sodium carbonate solution (2%). After being incubated at room temperature for 60 min, the absorbance was measured at 765 nm. Gallic acid was used at concentrations of 50, 100, 500, and 1000 mg/ml, as a measure of the standard curve for phenolic compounds .Total anthocyanin content was quantified by a pH differential method and calculated according to equations  and 2)  2) Fara. The abs2.6Ac is absorbance of the control reaction  and As is the absorbance of the sample after 60 min.Radical scavenging activity (RSA) was measured using DPPH°, as described by Farahmand et al. . A volum50) was determined from the equation and then obtained from the plotted inhibition curve.The sample that culminated in 50% inhibition  in a Stomacher bag. The suspension was homogenized for 1 min with a Stomacher Lab‐Blender . Serial dilutions were prepared using the same saline solution. 2.8Sensorial evaluation form was developed according to Arslaner et al.  with som2.9p < .05). Standard deviation values are presented in the tables and the standard deviation bars are provided in the figures.All experiments were performed in triplicate and the data were reported as mean values of the measurements. The data were analyzed by full factorial design using SAS software  for comparison among the mean values using Duncan's multiple range test , whereas the control samples of the stirred yogurts showed the highest L* value (92). This behavior can be related to the higher PJ concentration of yogurt, which increased the amount of red color in yogurt, so that its tendency decreased in being visualized as white. The type of yogurt (set or stirred) had no significant effect on the L* value. Nonetheless, at the end of the storage period, L* values of set and stirred yogurts decreased in the control , in 13% PJ‐yogurt , and in 17% PJ‐yogurt . In the course of the storage period, however, no significant difference (p < .05) was observed among the L* values.Color attributes of yogurt samples are shown in Figure n Figure . With 17a* increased parallel to the increase in PJ concentration from 13% to 17%, both in the set (5–7) and stirred (8–11) yogurts. The higher PJ concentration (17%) caused the highest a* values in the yogurts, which can be related to its higher TAC. At the end of the storage period, a* values of stirred yogurts reduced from 8 to 5 by 13% PJ and from 11 to 9 by 17% PJ. Also, a* values of set yogurts decreased from 5 to 4 by 13% PJ and from 7 to 5 by 17% PJ. Karaaslan et al.  had no significant effect on the b* value.By increasing the PJ concentration from 13% to 17%, 3.23.2.1Total phenolic contents of set and stirred yogurts are shown in Figure 3.2.2p < .05) by increasing the PJ concentration, confirming its direct effect on TAC. Also, TACs of the yogurts are reflected in the color properties of the samples. In agreement with our results, Jaster et al. , indicating the highest RSA, mainly due to higher TPC and TAC values. Phenolic compounds of PJ can act as antioxidants by their ability to donate hydrogen or electrons, by terminating chain reactions, or by chelating transition metal ions. During the storage period, the IC50 of all yogurt samples increased significantly, as a result of anthocyanin degradation  had no significant effect on RSA.The IC3.4L. delbrueckii in all samples revealed values higher than 6.00 log cfu/ml, although adding PJ had no significant effect (p < .05) on the counts of L. delbrueckii. Similarly, Cossu et al. (Cynara scolymus), strawberry (Arbutus unedo L.), and cherry (Prunus avium L.) can affect microbial properties of homemade yogurts. They reported that polyphenolic extracts had no significant effect on microbial properties of yogurt samples. Also, Jaster et al.  was observed in L. delbrueckii populations. This reduction can be attributed to the accumulation of ambient lactic acid that suppressed the microbial growth. Jaster et al. (p < .05) in L. delbrueckii count at the end of the storage period, as a result of an increase in acidity. According to Figure p < .05) on L. delbrueckii counts. Stirred yogurt hosted higher L. delbrueckii counts which could be seen as a result of adding PJ into the stirred yogurt . Contents of the PJ may facilitate the increase in the L. delbrueckii population.During the storage period, a significant decrease  compared to those of set PJ‐yogurts (8.32–9.02 cfu/ml × 108 cfu/ml), which can be related to the manufacturing process . In general, bacterial counts in the control samples (S. thermophilus and L. delbrueckii) were higher than those of PJ‐yogurt samples (both 13% and 17% PJ) during the storage period. Accordingly, adding PJ had a negative impact on the starter culture count, due to the acid injury. In accordance with our findings, Vinderola et al.  was observed in 17% PJ stirred yogurt, possibly because of its higher syneresis. On the other hand, the highest texture score (5) was observed in the control group of set yogurt, indicating that adding PJ to yogurts had negative effects on the texture score. Regarding the pleasantness, there were no significant differences between 13% PJ and control yogurt samples. However, 13% PJ stirred yogurt sample had a higher score (4.50) than the control sample (4.25). Our findings are in agreement with Rahman et al.  can be proposed as an excellent alternative for the purpose of producing flavored yogurt with high nutritional quality.The authors declare no conflict of interest.Mohammad‐Taghi Golmakani: Conceptualization ; Funding acquisition ; Project administration ; Resources ; Supervision ; Validation ; Writing‐review & editing . Mohammad Hadi Eskandari: Conceptualization ; Funding acquisition ; Project administration ; Resources ; Supervision ; Validation . Somayyeh Kooshesh: Formal analysis ; Methodology ; Software . Mahboobeh Pishan: Data curation ; Formal analysis ; Software ; Writing‐original draft .


Aspergillus flavus, represent an important issue in food safety. Usually, non-toxigenic strains are exploited for biocontrol strategies to mitigate infections by toxigenic strains. To comprehend all the biological variables involved in the aflatoxin biosynthesis, and to possibly evaluate the interplay between A. flavus toxigenic and non-toxigenic strains during intraspecific biocompetition, the “virological” perspective should be considered. For these reasons, investigations on mycoviruses associated to A. flavus populations inhabiting specific agroecosystems are highly desirable. Here, we provide the first accurate characterization of the novel mycovirome identified within an A. flavus wild population colonizing the maize fields of northern Italy: a selection of A. flavus strains was biologically characterized and subjected to RNAseq analysis, revealing new mycoviruses and a peculiar geographic pattern distribution in addition to a 20% rate of infection. More interestingly, a negative correlation between viral infection and aflatoxin production was found. Results significantly expanded the limited existent data about mycoviruses in wild A. flavus, opening new and intriguing hypotheses about the ecological significance of mycoviruses.Microbial multi-level interactions are essential to control the success of spreading and survival of most microbes in natural environments. Phytopathogenic mycotoxigenic fungal species, such as  Cryphonectria hypovirus 1 (CHV1), it became clear that mycoviruses have relevant effects on the host behavior [Aspergillus is probably the most studied, because of its agricultural, clinical, and economic implications: the first mycoviruses reported in Aspergillus species dates back to 1970 [Nigri, Clavati, Circumdati, Fumigati, and Flavi sections have been described ([A. flavus were reported for the first time [A. flavus strain resulted in a stable suppression of aflatoxin biosynthesis. This was probably due to RNA interference (RNAi) mechanisms specifically directed against AF-related gene transcripts as a result of a structural similarity shared between viral dsRNA of non-toxigenic strains and AF-related genes [Partitiviridae family shed light on a possible role of some viruses in mycotoxin regulation, suggesting significant influences on the biocontrol potential exerted by some fungal hosts [A. flavus in the agroecosystem, particularly on cereal crops, and the associated repercussions for human and animal health in terms of harmful AF contamination of feed and food commodities, the biological importance of any interaction with mycoviruses deserves to be investigated in more detail. In Italy, there are more than 1 million hectares dedicated to maize cultivation, which are located mainly in northern Italy and which account for 16 million tons of final products . Since 2003, due to the ongoing climate change scenario, maize cultivation in northern Italy has started to suffer from high aflatoxin contamination, which is favored by both humid-hot summers and plant water stress [A. flavus spread and aflatoxin diffusion. However, despite the great potential of mycoviruses in the arsenal of biocontrol fungal agents, no accurate characterization of the virome of a wild A. flavus population has yet been carried out. The present work is aimed at investigating and describing, for the first time, the mycoviruses represented within an A. flavus wild population that colonize the maize fields of northern Italy.As important members of the microbial community, the interactions of fungi with other organisms variously influence different aspects of both agriculture and human health ,2,3. Thebehavior . Indeed,behavior ,11,12,13behavior ,15,16,17behavior ,19, whicbehavior . Despitebehavior ,22. Amon to 1970 , but to scribed , Emilia Romagna (Emi), Trentino Alto-Adige (Taa), Lombardia (Lom), Veneto (Ven), and Friuli Venezia-Giulia (Fri). Isolates were grown and maintained on YES agar medium  at 28 °C. All the strains are deposited in the laboratory collection (University of Parma) and available upon direct request to the authors. For gDNA analysis, mycelium cultured in YES liquid medium for 6 days at 28 °C, under stationary conditions, was used. For total fungal and viral RNA extraction, mycelium was obtained from conidial inocula cultured in YES liquid medium at 28 °C, in an orbital shaker at 110 rpm for 4 days. ex = 360 nm, λem = 465 nm, manual gain = 83, lag time = 0 µs, number of flashes = 3, and integration time = 200 µs. Each strain was analyzed in four biological replicates, and analysis for each replicate was repeated three times.The AF production ability of isolates was assessed in standard flat-bottom 96-well microplate  cultures, as previously described . Coconut5 conidia/mL suspension) of each A. flavus strain were centrally spotted on Czapek agar plates , according to [Point inoculations , according to the manufacturer’s instructions. Extracted samples were quantified with NanoDrop 2000 , and their quality was checked via electrophoresis on 1% agarose gel.® DNA polymerase Kit . The reaction was performed in 20 μL of PCR Flexi Buffer 5X , 25 mM of MgCl2, 25 mM of dNTPs, 10 μM of primer, and 0.5 U of Taq DNA-polymerase. Amplification parameters were: 4 min at 94 °C, for 35 cycles: 1 min at 94 °C, 20 s at 44 °C, and 2 min at 72 °C, and final extension for 6 min at 72 °C. Resulting RAPD-PCR patterns were visualized on 2% agarose gel.RAPD-PCR (Random Amplification of Polymorphic DNA) analysis was conducted using a degenerate oligonucleotide (5′-GAGAGAGAGAGAGAGAYG-3′), and 5 ng of gDNA was used as a template for the amplification reaction conducted with the GoTaqA. flavus mycelium following the TRIzol® protocol . RNA concentration was estimated using NanoDrop 2000 , while quality was checked on 1% agarose gel. To proceed with RNA-seq analysis, a mixed sample containing all the RNAs in equal amount was prepared, by mixing 500 ng of RNA from each isolate, as previously reported [Total RNA was extracted from reported . The poohttp://www.ncbi.nlm.nih.gov/gorf/orfig.cgi, accessed on 2 July 2021) and then blasted to NCBI nr databases.De novo assembly of sequenced RNA was achieved using high-quality and clean sequences selected using Trimmomatic . TrinityA. flavus strain, previously obtained RNA was used as a template for cDNA synthesis , following the manufacturer’s instructions. Amplification of A. flavus β-tubulin and viral RNAs was obtained with iTaq universal SYBR Green super-mix (Bio-Rad) and specific primers  were used for the purification of virus-like particles using a classic differential centrifugation protocol. The MN1 sample was homogenized with sterile glass and iron beads (4 mL of 0.5 mm in diameter glass beads and 1 mL of 1 mm iron beads) using a bead beater in 10 mL of extraction buffer . A first centrifugation at 1000× 0,000× g ,39. The For TEM observation of virus particles, samples were allowed to adsorb on carbon and formvar-coated grids for at least 5 min. After adsorption, grids were washed with water and then negatively stained with a 0.5% solution of uranyl acetate. Observations and image captures were made using a CM 10 electron microscope .p-value was derived from the hypergeometric function, where N represent the population size, M is the total number of AF-producing isolates, s is the subsample of virus-infected isolates, and k is the number of AF-producing isolates in the subsample.A hypergeometric-based test was used to assess the significance of correlations across samples between mycovirus infection and AF production. The rationale behind using the hypergeometric test was that if mycoviruses have functional association with the inability to produce AF, they would be found enriched in the subsample of virus-infected isolates. We tested under the null hypothesis that the viral infection in one sample is independent from the AF production within the same sample. The A. flavus strains were isolated from kernels belonging to northern Italian maize fields and were firstly subjected to random amplified polymorphic DNA (RAPD)-PCR analysis to define their molecular profile. A total of 250 isolates were screened, and strains with different RAPD amplification patterns were selected , https://talk.ictvonline.org/, accessed on 2 July 2021), and for this reason, we propose the name Aspergillus flavus deltaflexivirus 1 (AfDfV1)  and to Lentinula edodes deltaflexivirus 1 (QOX06047.1): it shows two conserved domains along the first ORF (from nucleotide 29 to nucleotide 6007), the first at the 5′ (about 1 Kb from the first nucleotide), which shows high homology to viral methyltransferases, and the second, which is located about 4 Kb from the first nucleotide, which encodes for a viral helicase. The genome then presents another two ORFs encoding for two proteins with no conserved domain and with no homology with other proteins in NCBI databases. The phylogenetic analysis of the RdRP sequence displayed a good relationship with Lentinula edodes deltaflexivirus 1 (QOX06047.1), confirming its relationship with the (AfDfV1) . Narnaviridae family [Aspergillus flavus narnavirus 1 (AfNV1) and Aspergillus flavus narnavirus 2 (AfNV2), respectively. Two other new viruses belonging to the Lenarviricota phylum were found: the first one is a 2863 nucleotides-long segment which displays a 2043 nucleotides ORF encoding a putative RdRP. The protein showed 88.14% identity to Spodoptera exempta virus TenAfr-2017 (QQZ00864.1), but no conserved domain was detected during the CDD/SPARCLE analysis. The second identified viral genome was a 3009 nt long sequence that exhibited a 2151 nucleotides ORF encoding for a putative RdRP, which shares 43.3% identity to Oidiodendron maius ourmia-like virus 1 (QNN89181.1) [Botourmiaviridae family [Aspergillus flavus magoulivirus 1 (AfMoV1) due to its phylogenetic relationship with the Magoulivirus genus, while the second one was named Aspergillus flavus scleroulivirus 1 (AfSoV1), due to its phylogenetic relationship with the Scleroulivirus genus (Two viruses showing homology to the e family  were idee family . The sece family . The phy89181.1) . The phyus genus .Partitiviridae family [Alphapartitivirus genus (Aspergillus flavus partitivirus 2 (AfPV2). Additionally, a virus with a bipartite genome was also identified: the first RNA (RNA1) is 1849 nt long and presents a 1701 nt ORF that encodes for a putative RdRP, as also suggested by the CDD/SPARCLE analysis that identified an RT-like domain. The closest proteins are two putative RdRP encoded by Hubei partiti-like virus 27 (APG78241.1)  and by Le family  and, morus genus ; for thiAspergillus flavus polymycovirus 1 (AfPMV1).We detected a second virus belonging to the dsRNA clade, which presents a multipartite genome made of 4 segments. The first segment (RNA1) is 2394 nt long (2331 nt length ORF) and codes for a protein with a conserved RT-like domain (cl02808). The second segment (RNA2), encoding a protein with a conserved methyltransferase domain, is 1931 nt long and presents a 1839 nt ORF. The third segment (RNA3—1222 nt in length) displays a 939 nt long ORF, and this ORF did not display any conserved domain, but it is related to several proline/alanine (PAS)-rich proteins of previously reported polymicoviruses. The last segment (RNA4—2257 nt) codes for a protein with no conserved domain and with no predicted function. Due to its similarity to other polymycoviruses and to its phylogenetic placement , we nameAspergillus fumigatus RNA virus 1 (txid2747487), a virus recently reported in NCBI [Plasmopara viticola. Similarly to what was reported for Plasmopara viticola lesion-associated vivivirus 1 [Aspergillus flavus vivivirus 1 (AfVV1). Finally, we identified a virus with three putative segments, the first one (RNA1) is 3592 nt long, displays a 3459 nt long ORF, and putatively codes for a protein with a conserved RdRP domain. The second is 3509 nt long with a 3273 nt long ORF and encodes a protein with two conserved domains: a methyltransferase at the 5′ and a viral helicase at the 3′. The third segment is 1942 nucleotides long, presents a 1671 nt long ORF, and encodes for a protein with no detected conserved domain. All three proteins displayed a relatively good degree of conservation with  1 txid27487, a viAspergillus flavus deltaflexivirus 1, and the second structure, revealed to be a small isometric and non-enveloped virus-like particle of about 14 nm in diameter, which was enriched after a further purification with a sucrose gradient, and which probably belongs to Aspergillus flavus vivivirus 1 , and the regulation of biosynthetic pathways for small bioactive molecules, critical for the interaction of Aspergilli with other organisms, that enable filamentous fungi to successfully exploit environmental resources by modifying the chemical diversity [A. flavus population is consistent with data previously reported [Due to its predominance amongst the fungal species that produce aflatoxins, and the derived relevance of the socio-economic issues correlated to the diffusion of mycotoxins on food and feed commodities all around the world, genetics ,49,50. Miversity . Recentliversity , probablreported . HoweverA. flavus isolates. As already well-established for the human virome, which has been correlated to various disease and physiological states [Although attention has been paid to the environmental and cellular inputs driving the ecological features that characterize this species and its relationship with the environment, it is worth noting that very few studies looked at the role that mycoviruses can play in naturally occurring l states ,54,55,56l states ,57,58. A. flavus population isolated from a wide geographical region, and the virus-like particles associated with the recent newly reported group of viviviruses [A. flavus with the production of aflatoxin is quite limited. This is remarkable in consideration of the average Aspergillus species infection rate, which accounts for about 10% of the isolates [A. flavus strains were reported [A. flavus with a reduced conidiation and with an abnormal colony morphology [Here, we described for the first time the genome sequences of eight new mycoviral species infecting an iviruses . It is wisolates ,60. In cisolates , some myreported ,31,61,62rphology . In thisCryphonectria parasitica  and its virus Cryphonectria hypovirus 1. In more detail, it was demonstrated that the mycovirus infection produce a wide metabolic reprogramming of its host, modifying the whole primary and secondary metabolisms [A. flavus and the possible correlation with aflatoxins’ production could be found. In this respect, our work provides a first insight of viral infections into a naturally occurring population coming from a large-scale sampling survey. Interestingly, we observed that, in our population, the infected isolates were predominantly non-toxigenic . On the other hand, it is worth nothing that, on the contrary to aflatoxins, the sclerotia production seems not to be correlated with the viral infection: among the 13 virus-infected isolates, 6 are unable to produce sclerotia, whereas 7 are sclerotia-producing isolates. Further screening on a wider population will clarify if any correlation exists between the aflatoxigenic behavior and the virological state.Another important aspect of mycovirus infection is linked to the alteration of the primary and secondary metabolisms. An example of how mycoviruses can impact on fungal metabolisms is well-represented by the interaction between the model host abolisms ,64. The abolisms ,13,65. Fabolisms ,31. Initabolisms . UnfortuAspergillus flavus narnavirus 2, that was present both in Piemonte and Veneto regions, which do not share borders, all the other viruses resulted strictly associated to the indigenous population of specific areas. For example, Aspergillus flavus partitivirus 2 was found only in isolates from Friuli-Venezia Giulia, Aspergillus flavus deltaflexivirus was found associated to the Emilia-Romagna region, whereas Aspergillus flavus narnavirus 1 is associated to isolates from Lombardia. These observations are interesting under an ecological perspective, pointing to an apparently limited movement of the fungal population also between bordering territories. This evidence suggests that mycoviruses could thus be exploited as molecular markers to be used for the monitoring of the fungal populations’ movement among territories, possibly representing an alternative to the use, in the future, of the more laborious molecular analyses currently applied for this purpose [From a geographical perspective, it is interesting to observe that viruses seem to be associated with specific regions . With th purpose . A. flavus population, representative of a wide geographical area such as the Po Valley, this survey offered an opportunity to reinterpret some biological standpoints in light of the virus–host interaction principles in A. flavus. These include the role of mycoviruses in the environmental inter- and intra-specific competition, including fungal development, their potential as a tool for biocontrol strategies against phytopathogenic/mycotoxigenic fungi, and as therapeutics [A. flavus populations within a specific geographic region is another interesting aspect. Our data pose new questions about the interaction between mycoviruses and aflatoxin production, suggesting a negative correlation that was previously hypothesized in old studies. More detailed research concerning the biological characterization of the mycoviruses described here will enhance the possibility to understand this correlation. Additionally, the significance of these mycoviruses to fungal ecology and viral evolution, and the potential for mycoviruses-based biological control of A. flavus infection, could largely benefit from the findings reported in the present work. Finally, the observed geographical correlation of some viral species would suggest the possibility to develop population studies using mycoviruses as biomarkers, highlighting both the interaction between compatible isolates and the movement of one isolate from one region to another. By providing a likely accurate description of the mycovirome present in an apeutics . The pos


Although mechanisation has markedly reduced animal labour demand in agriculture, draught animals are still used in small production units located on terrains that do not favour agriculture mechanisation. Especially in Africa, Latin America, and Asia, they represent one of the main sources of sustenance for thousands of families who utilise animal labour in numerous agricultural tasks, such as ploughing and harvesting, as well as means of transport and hauling. Depending on the geographic area, the species involved are equids  and bovids . Draught animals proved to be sustainable in terms of global warming and the use of non-renewable energy as compared with agricultural machinery. However, critical points are the quality of human–animal interaction, due to the close contact between animals and humans while working, and the welfare of draught animals when transported and slaughtered, due to the high prevalence of injuries they suffer when subjected to these practices. Therefore, their use should be promoted in rural marginal areas where only low investments are usually feasible, and the energy of the animals can be obtained at a low cost by feeding them harvest residues and by-products.This study discusses scientific findings on the use of draught animals such as equids  and bovids  in rural labours. Relevant peer-reviewed literature published between 1980 and 2021 was retrieved from CAB Abstracts, PubMed, ISI Web of Knowledge, and Scopus databases. Although animals were used to produce draught power since their domestication and are still being used for this purpose, mechanisation has markedly reduced animal labour demand in agriculture. However, the process was uneven across continents according to economic constraints, and draught animals are currently concentrated in small production units located on terrains that do not favour agriculture mechanisation in Africa, Latin America, and Asia. Generally, equids can work at rates similar to those of bovids or faster but can sustain the work for shorter periods of time. In addition, buffaloes possess tough hooves and resistance to disease that make them suitable for working in wetlands and clay soils. Draught animals allow a marked reduction of both GHG emissions and non-renewable energy consumption as compared with agricultural machinery. In addition, they may allow obtaining profits from otherwise non-usable lands. Therefore, their use should be promoted in rural areas where low investments are usually the only ones feasible, and the energy of the animals can be obtained at a low cost by feeding them harvest residues and by-products. However, more attention should be paid to the quality of human–animal interactions—due to the close contact between animals and humans while working—and to the welfare of draught animals when transported and slaughtered—due to the high prevalence of injuries they suffer when subjected to these practices. Animals have been involved in the sociocultural and economic evolution of societies. Most significantly they participated in the process of utilization of natural resources to produce food and work in rural societies and, more broadly, they promoted the generation of wealth ,2.The most common species still used for transport and draught are equids, such as horses, mules, and donkeys, and bovids, such as cattle, including oxen, and buffaloes ,3,4,5. TAlthough no up-to-date data on the number of draught animals working in the world are available , draught animals persist as a primary source of livelihood for thousands of families in developing countries, and rural families, often including children, participate actively in the management and handling practices associated with draught animals, exerting a significant influence on their welfare .From a normative perspective, humans are obliged to ensure the quality of life of these animals ,9, not oOur capacity to perceive and understand the needs of other living beings has been a key feature of the process of providing protection for farm animals based on the knowledge of their physiological characteristics and behavioural needs, which are specific for draught animals. For these reasons, it is important to improve the attitude towards draught animals to provide an acceptable level of welfare to these animals in agreement with the principles of sustainable development . AccordiThe use of draught animals lightens human labour in cultivation, loading, and transport. However, the participation of draught animals to agricultural activities can also contribute to the farm economy . The addIt is from this perspective that this study discusses scientific findings on the use of draught animals such as equids  and bovids  in rural labours, the relations among draught animals, farmers, and rural families, the contribution of these animals to global warming, the quality of life of pack and traction animals, and their welfare conditions at the end of their working lives.Bubalus bubalis, oxen, equids, horse, donkey, mule, yoke, attaching animals to vehicles, land preparation, rural activities, greenhouse gas emission. Boolean strings were constructed based on meaningful combinations of search terms and operators. Search terms were repeatedly refined through a multistep process in several trials to make sure that the most effective search terms were used [Relevant peer-reviewed literature published between 1980 and 2021 was retrieved from CAB Abstracts, PubMed, ISI Web of Knowledge, and Scopus databases. The search was based on the following search terms: draught animals, pack animals, animal energy, draught power, working animals, animal welfare, human–animal relationship, feeding, economy, buffalo, ere used . The autSince prehistoric times, when hunters-gatherers changed their status to sedentary and settled, humans benefited from the muscular strength of large, domesticated animals to enhance their capacity for work in agricultural production and transport . ThroughThe specific characteristics of buffaloes are the strong and large hooves, flexible foot joints that enhance their work performance and allow higher efficiency in clayey agricultural soils subject to flooding, which demand greater traction effort, whereas camelids are preferred in Asian dry environments . The maiCurrently, the use of cattle as draught animals is quite common in Asia and Latin America, while in Africa these animals are used to pull carts or ploughs and their diffusion is only limited by dry environmental conditions and diseases . In geneMechanisation markedly reduced the labour demand in agriculture . HoweverNumerous studies compared the work produced by different animals  and genBoth animal and mechanical energy have positive impacts on production as compared with manual labour. However, mechanisation has proven to be more efficient even after taking into account the substantial investments required for inputs such as infrastructure and equipment (fixed costs), and the need to hire or train personnel to operate the equipment . HoweverCertainly, economic and productive efficiency improves with the incorporation of mechanised processes to replace animal energy. However, issues concerning sustainable production  should bThe main greenhouse gases (GHG) emitted from draught animals are carbon dioxide, methane, and nitrous oxide. The amount of GHG emitted by draught animals changes according to the species. Bovids show higher levels of emissions due to ruminal and enteric fermentations as compared to the monogastric equids . However2. Cerutti et al. [Mechanisation has markedly increased agriculture productivity and labour efficiency. However, only few studies have assessed the effect of the shift from animals to machinery in terms of GHG emissions. Aguillera et al.  showed ti et al.  using a i et al. . In conti et al. ,43.The main factors negatively affecting the welfare of draught animals are listed in The interaction between humans and animals is a keystone of the domestication and subsequent use of farm animals in agriculture, and a good human-animal relationship is fundamental to improving the welfare of both humans and animals . HoweverDraught animals may suffer fatigue, malnutrition, and diseases that prevent them from achieving optimum performances in their labours and maintain satisfactory welfare levels. Hyperthermia may be a major factor causing the onset of fatigue . The oxyCurrent evaluations of the welfare of draught horses are based on parameters of health and behaviour, including lameness, injuries, and general body conditions . Around Cylicocyclus asini was the most frequent, identified in 91.7% of cases, followed by Cyathostomum spp. in 83.3% of the animals. These results clearly show that it is essential to include deworming procedures in the care of these animals to improve their health in the short term, whereas preventive measures should be taken in the medium term.Recently, Attia et al.  noted thWith respect to bovines employed as draught animals, a study designed to evaluate the management of oxen based on several parameters-health, feeding, housing, workload, care—showed that 78% of farmers did not provide regular veterinary care . As to fIn the Birbhum district of West Bengal (India) the health of 810 buffaloes randomly selected from herds of draught animals was examined and it was found that several buffaloes reported wounds caused by ploughs while working (16.96%), yoke gall (9.82%), and leg traumas (15.17%). In addition, due to overwork, buffaloes showed hypoglycaemia and nutritional deficits  (15.17%), which were accentuated in summer 17.44%) . Another% . AnothMost of these deleterious effects can be mitigated by using the appropriate equipment and proper maintenance and fastening procedures. When using animals for hauling or transport, special attention must be paid to balancing the weight of the cart or wagon and to correctly harnessing the animals. Handlers must strive to ensure that draught power is transmitted by pushing a collar with the least amount of pressure and drag possible on the animal’s back, neck, and shoulders . The desWhen the working efficiency of draught animals decreases, most of them are sent for slaughter. Differently from productive categories, draught animals often reach this stage when they are fatigued or even exhausted . TherefoA common management practice imposed to draught animals is nose piercing, which is performed inserting a hot iron rod through the septum and then passing a rope through the hole. This practice can cause tearing or chafing injuries to the nostrils, particularly during transport, loading and unloading when the rope is more often used to lead the animal . AccordiThe working capacity of draught animals depends largely on the availability and use efficiency of feed nutrients . HoweverIn hilly and upland areas of the Philippines and Mexico, maize stover supplied half of the diets of draught ruminants and equids for 6 months of the year ,65. In sAs a long-term effect, feeding working animals with unbalanced diets can lead to poor health status and metabolic disorders, such as an increasing body calcium removal from bone by oxalic acid, particularly abundant in cereal crops by-products ,74. ThusGlucose and long-chain fatty acids (LCFA) have been highlighted as critical nutrients to sustain working muscle activity and to restores body fat during resting periods . In partIn mature male working animals, needs of energy sources can be partially offset by catabolizing amino acids, which may be redirect to gluconeogenesis or, as observed in buffaloes , directlEnergy expenditure of draught animals varies according to the work performed. Factors such as the travelled distance, weight of the load, surface characteristics over which the animals move, and the duration of the working day can affect the extent of extra daily energy requirements over maintenance ,99. Net According to Dijkman and Lawrence , Bunaji A large body of literature has accumulated over the years around the effects of working on feed intake. Light to medium exercise has been shown to increase voluntary intake in rats and horses . HoweverConversely, milking cows may increase feed intake in response to work even if the feed encumbrance is high as in the case of hay-based diets. In Ethiopia working cows showed higher intakes of natural pasture hay (72 MJ of metabolizable energy/d at 90 d postpartum) than non-working cows (60 MJ of metabolizable energy/d at 90 d postpartum) . HoweverConflicting results are available on the effect of working on digestive functions, particularly in cattle ,114,117,Over time, the use of animals for traction and transport has diminished in importance in rural environments, especially in flat areas where intensive agricultural systems have developed, systems that require other energy sources and huge inputs. These industrialised systems based on intensive livestock and forest exploitation have a marked impact on the environment in terms of GHG emission and land occupation. Under these circumstances, interest in draught animals is increasing in mountainous areas and small-scale production units for such labours as traction and transport, and as valuable sources of food. Therefore, due to the energy crisis, the lack of resources and, in some cases, the tendency to adopt agro-ecological production models aiming at reducing the impact of agricultural practices on the environment, the use of working animals in certain regions should be promoted. This would allow to obtain profits from otherwise non-usable lands in a sustainable manner, as the amount of non-renewable fuels used in agriculture would be reduced along with the emission of GHG.The labours that these animals perform for humans are not, however, in balance with their quality of life. Although the research available on this topic is scarce, high incidences of cutaneous lesions, diseases, and injuries to hooves and snouts are clearly documented, which results in poor welfare of the animals and reduced working efficiency. The welfare of working animals has a central role in promoting human welfare, given the multiple economic and social functions they play, particularly in less advantaged areas. Appropriate handling procedures, adequate, well-maintained equipment, and improved veterinary care will all play a fundamental role in reducing the incidence of injuries and increasing the welfare of draught animals and their performances. Long period of intense working can also negatively impact the welfare of the animals both directly, due to exhaustion, and indirectly, due to reduced frequency of feed ingestion with consequent loss of weight and reduced working and, in the case of lactating animals, productive performances. The welfare of draught animals is also at the stake at the end of their working life. Differently from other productive categories, draught animals often reach this stage when they are fatigued or even exhausted. Therefore, they are potentially more exposed to suffer falls, blows, and slipping during transport to livestock markets and at slaughter. This implies that these animals should be included in public policies and specific regulations. For example, OIE has considered equids, but neglected other species, such as cattle and buffaloes. The inclusion of these species will allow specific health campaigns, animal censuses, and genetic conservation programmes devoted to draught animals.


Although intraocular anti-vascular endothelial growth factors (anti-VEGFs) are effective as treatment of neovascular age-related macular degeneration (nAMD), the (economic) burden on the healthcare system is considerable. A treat-and-extend (T&E) regimen is associated with a lower number of injections without compromising the effectiveness and can therefore help optimise nAMD treatment. This study investigates the per-patient costs associated with nAMD treatment, when using aflibercept, bevacizumab, or ranibizumab with a T&E regimen.In this cost-minimisation model, the per-patient costs in the Netherlands were modelled using a healthcare payers’ perspective over a 3-year time horizon with the assumption that efficacy of treatments is similar. Additionally, the break-even price of the different anti-VEGFs was calculated relative to the cheapest option and injection frequency.The injection frequency varied from 14.2 for aflibercept to 27.4 for bevacizumab in 3 years. Nonetheless, bevacizumab remains the cheapest treatment option , followed by aflibercept  and ranibizumab . The medication covers the majority of the per-patient costs for aflibercept and ranibizumab, while administration covers the majority of the per-patient costs for bevacizumab. The break-even prices of aflibercept and ranibizumab are respectively €507 and €60.58 per injection. Brolucizumab was included in the scenario analysis and was more expensive than aflibercept . Brolucizumab should reduce to 13.8 injections over 3 years to be as costly as aflibercept.Bevacizumab is the cheapest anti-VEGF treatment. The list prices of all anti-VEGFs should reduce to be as costly as bevacizumab. Aflibercept is the second-choice treatment and so far brolucizumab is not. In the period 2011–2018, the yearly prevalence of AMD in the Netherlands has increased by approximately 70% to 51,400 men and 81,600 women. It is expected that the prevalence will keep increasing as the aging of the population continues [Age-related macular degeneration (AMD) is globally the main cause of severe vision loss . In the ontinues .AMD is characterised by the presence of drusen in the early stages but may progress into geographic atrophy or the neovascular form of AMD associated with severe vision loss . Although only 10–15% of all AMD patients develop neovascular AMD (nAMD), it causes 90% of all AMD-related severe vision loss . Due to Intravitreal injections of anti-vascular endothelial growth factors (anti-VEGFs) are used as first-line treatment to prevent visual loss related to nAMD . CurrentMultiple regimens for anti-VEGF treatment are currently being used for the treatment of nAMD. Earlier clinical trials focused on periodic regimens: for bevacizumab and ranibizumab a monthly regimen, and for aflibercept a bimonthly regimen. Brolucizumab was introduced with the periodic 12 weekly regimen, which was adjusted to an 8 weekly regimen in case of disease activity (Q12W/Q8W regimen) , 21, 22.Optimisation of the T&E regimen could reduce pressure on the healthcare system and might be necessary as the number of people with ophthalmic disorders is expected to increase by 52% by 2040 [To optimise the T&E regimen, it is important to consider all direct healthcare costs when choosing between the anti-VEGFs, and not solely medication costs, as done in the NOG guidelines. This cost-minimisation study aims to compare the per-patient costs within a T&E regimen with the different anti-VEGFs in treatment-naïve patients with nAMD in the Netherlands. Our study focusses on treatment-naïve patients to minimize the influences of former treatments and to include a broad spectrum of patients. Additionally, the break-even price for the different anti-VEGFs relative to the anti-VEGF with the lowest costs is determined.A cost-minimisation model was developed in Microsoft Excel 2016  to model the per-patient costs of the different anti-VEGFs for the treatment of nAMD. The costs were determined for a one-eye treatment of a treatment-naïve nAMD patient in the Netherlands when using a T&E regimen. Cost-minimisation analyses assume that the effectiveness and safety of the treatments under evaluation are comparable and therefore that treatments have no clinical benefits over each other . This isThe course of disease of nAMD was modelled based on published literature. The model included costs for medication, administration, diagnosis, clinic visits, and adverse events. The break-even prices of the anti-VEGFs with the highest total costs were calculated based on the anti-VEGF with the lowest total costs per patient as reference. The assumptions that were made in the base case scenario are shown in Table We used the Dutch healthcare payer’s perspective, which entails all the direct healthcare-related costs in the Netherlands induced by anti-VEGF treatment of treatment-naïve nAMD patients. Direct costs are relevant for daily practice, as the choice between treatments is made by hospitals and other treatment centres. The per-patient costs and break-even prices were calculated for 3 years according to the Dutch guideline for budget-impact models in healthcare .NOG issued a guideline for the use of aflibercept, bevacizumab, and ranibizumab. Bevacizumab was chosen as the preferred treatment option because of the relatively low medication costs . AfliberAccording to the T&E regimen in the NOG guideline, all patients should start with a loading dose of three times a month injection, followed by flexible treatment intervals which will be extended based on the treatment response, which is determined on the hand of (an increase of) intra- or subretinal fluid. Treatment intervals could be either extended  or shortened (in case of presence of fluid) by 2 weeks after every monitoring visit. The minimal interval is 4 weeks and the maximal interval is 12 weeks .The injection frequency input data included in the model were based on phase III and/or IV, prospective, randomised trials. Trials should study one-eye anti-VEGF treatment with a T&E regimen in treatment-naïve nAMD patients. The primary endpoint of the trial should focus on the improvement in vision of patients. A weighted average of the injection frequency was used when multiple trials were found for the same anti-VEGF. A summary of the included regimens and the clinical trials is shown in Table During the loading phase, we assumed one monitoring visit. During the maintenance phase, one monitoring visit is assumed for every injection . In yearThe four most prevalent adverse events were included in the model by risk per injection. Since all the adverse events included in this model are solely injection-related, the risk per injection was equal for all three anti-VEGFs. Moreover, none of the clinical trials found a significant difference in injection-related adverse events , 21, 22.The per-patient costs were calculated by taking the sum of all the direct healthcare costs related to treatment of one nAMD patient. All costs have been adjusted for inflation to November 2020 . All theThe medication costs of aflibercept and ranibizumab were based on the list prices , 36. No Some hospitals order fully prepared bevacizumab intravitreal injections by production pharmacies, which are more expensive compared to the preparation in the hospital itself. The effect of the use of this more expensive preparation was tested in the scenario analysis.. The utilisation of the monitoring visits was based on the NOG guideline. The diagnosis consists of optical coherence tomography (OCT), fundus photography, and fluorescence angiography (FA). Subsequent monitoring visits include an OCT only [For every injection, administration costs were taken into account. Administration costs were based on a previously published economic evaluation of anti-VEGFs in the Netherlands and consisted of all the costs corresponding to the injection (except medication costs) . The utiOCT only . The OCTOCT only . The funOCT only . The cosOCT only .After the determination of the per-patient cost, the break-even price of every anti-VEGF relative to the anti-VEGF with the lowest total costs was calculated. This is the drug price of the relatively more expensive anti-VEGFs to reach equal per-patient total costs as for the cheapest anti-VEGF when using a T&E regimen. The break-even price is calculated by subtracting the sum of the administration, clinic visit costs, and adverse event-related from the total per-patient costs of the cheapest anti-VEGF. This is divided by the total injection frequency, resulting in the break-even price per injection. Additionally, the difference between the break-even price and the list price was presented as the percentage reduction (discount). To determine the effect of deviating injection frequencies, also the break-even price relative to the injection frequency was calculated.A univariate sensitivity analysis was performed to assess the influence of the different model input parameters on the total costs per patient per anti-VEGF. The parameters were variated with an interval of + / − 25% . AdditioSolely the per-patient costs of the anti-VEGFs are compared as clinical trials show that despite their different treatment intervals, they pursue an equal clinical effect , 30, 39.Treatment with bevacizumab results in the lowest total per-patient costs and the break-even price is therefore calculated relative to bevacizumab. The break-even price is the drug price per injection that leads to equivalent per-patient costs as bevacizumab. The results are shown in Table 7. Ranibizumab has the lowest break-even price (€60.58) and should decrease by 92% in medication price to reach equal costs. The break-even price of aflibercept is €507 and should decrease by 36% to reach equal costs.Figure The outcomes of the univariate sensitivity analysis are presented in tornado diagrams Appendix . For allFor bevacizumab, the intravitreal injection costs have the second biggest influence while for the other anti-VEGFs the medication costs have the biggest influence. Other important costs are the administration costs, the costs for clinical visits, and OCT costs.Instead of self-preparation, some hospitals order fully prepared bevacizumab injections. Therefore, the scenario of ordering fully prepared bevacizumab injection was performed. As is visible in Table 7, the per-patient costs of bevacizumab in scenario 1 are slightly higher compared to the base case scenario. However, the total costs per patient are still lower than those for all the other anti-VEGFs.Scenario 2 assessed the per-patient costs of treatment with brolucizumab . The per-patient costs and break-even price of brolucizumab are higher than those of aflibercept because of the slightly higher injection frequency in the Q12W/Q8W regimen (Table 7). With higher adverse event costs, brolucizumab will be even more expensive than aflibercept. In Fig. Our study shows that despite its high injection frequency, bevacizumab is the cheapest treatment option for treatment-naïve nAMD patients, with per-patient costs of €14,215 per patient over a 3-year time horizon. This was mainly caused by the relatively low medication costs compared to those of aflibercept and ranibizumab. Bevacizumab is the only anti-VEGF with higher administration costs than medication costs. Aflibercept and ranibizumab lead to total per-patient costs of €18,202 and €31,048, respectively. The break-even prices of ranibizumab and aflibercept were respectively €60.58 and €507, which are 92% and 36% below their current list price. Aflibercept is cheaper than ranibizumab due to its relatively low injection frequency.Based on the list prices, bevacizumab seems the most economically favourable option. Choosing either aflibercept or ranibizumab will therefore lead to higher healthcare-related costs. However, by calculating the break-even prices, we aimed to provide insight into the necessary discounts to reach cost equality for the anti-VEGFs. Besides the difference in costs, the non-economical effect of a high injection frequency is also an important factor to consider when choosing between the anti-VEGFs. Intravitreal injections lead to anxiety in patients and the clinical visits are a burden on the patient as well .To optimise nAMD treatment for the patient and the healthcare system, this study focuses on a T&E regimen. In a flexible T&E regimen, fewer injections are needed than in fixed regimens to guarantee similar effectiveness. The effectiveness of a T&E regimen is shown to be higher than that of other flexible regimens , 51. A rIn the scenario analysis, it was shown that brolucizumab was, with 15.4 injections over 3 years, more expensive than aflibercept but cheaper than ranibizumab. No trials with a T&E regimen were available for brolucizumab; and therefore, these outcomes are based on the Q12W/Q8W regimen. Our results showed that in a T&E regimen, brolucizumab would be cheaper than aflibercept with fewer than 13.8 injections in 3 years. However, brolucizumab is clinically the fourth choice of treatment because of the suspected higher chance of intraocular inflammation. These adverse events were not included in our analysis due to a lack of clinical data. At the time of this study, the TALON trial was still ongoing . This trUsing the slightly more costly pre-filled bevacizumab injections in the scenario analysis still led to lower per-patient costs for bevacizumab compared to the other anti-VEGFs. The preparation method of bevacizumab seems to have little impact on the per-patient cost. The fully prepared bevacizumab injections were only €502 more expensive than the self-prepared injections over 3 years.Several pharmacoeconomic studies compare the cost-effectiveness between (some of) the different anti-VEGFs. However, this study is the only known pharmacoeconomic study that focuses on a T&E regimen and cost-minimisation. In most studies, bevacizumab has shown to be more cost-effective than ranibizumab or aflibercept because of its lower medication price and equivalent effectiveness –60. ThisAs shown in the univariate scenario analysis, injection frequencies are the main driver for the total per-patient costs. The injection frequencies used in the model were based on previously published phase III and IV clinical studies. However, the use of different sources has some implications. For example, the included trials used different patient populations, which might reduce the comparability of their results. The ALTAIR trial is a Japanese trial that includes Asian patients, whereas all other trials were conducted in Europe or North America. nAMD characteristics for Asian patients differ from those for white patients. A higher proportion of the polypoidal choroidal vasculopathy subtype and a lower prevalence of retinal angiomatous proliferation are seen in Asian patients than in white patients. It is unclear whether ethnicity impacts treatment outcomes, and thus if the ALTAIR trial outcomes are comparable with the other studies. The VIEW study compared aflibercept and ranibizumab treatments for both white and Asian patients. In a sub-analysis, there were almost no significant differences found between the patient groups. This study used a fixed regimen in the first year and a flexible Pro Re Nata regimen in the second year. The number of injections was similar for both patient groups . In a JaAll included studies analysed the effects of a T&E regimen but there were some differences within the regimens. Some of the regimens followed in the clinical trials deviate from the NOG guidelines (e.g. the LUCAS and ALTAIR study) , 15, 23.Although the RIVAL trial focused on a T&E regimen for aflibercept and ranibizumab, this trial is not included in our study. The RIVAL used geographic atrophy as primary endpoint and visual improvement and number of injections as secondary endpoints. The trial found more injections for aflibercept in a T&E regimen than in a bimonthly regimen, while a bimonthly regimen is the fixed regimen used for aflibercept . MoreoveOur study does not include real-world studies because not for all anti-VEGFs 2-year T&E regimen real-word data was available. The injection frequency differs strongly between studies but the weighted average showed a similar injection frequency for aflibercept in the first year and a higher injection frequency for aflibercept in the second year than the ALTAIR trial  , 37, 38.Although clinical trials suggest some (serious) non-injection-related adverse events, only the injection-related adverse events are considered in the model. This assumption was made because of the relatively small influence of adverse events costs, as shown in the univariate sensitivity analysis. Moreover, the risks for non-injection-related adverse events are considerably small and more importantly equivalent for aflibercept, bevacizumab, and ranibizumab , 22, 42.The major strength of this study is that it considers all available anti-VEGFs. Moreover, the study gives a clear overview of the differences in the (distribution of) costs and break-even prices. The focus on a T&E regimen is also a strength since this is likely to be the leading regimen in the future of nAMD treatment in the Netherlands. This study gives a realistic insight on the difference between the actual per-patient costs when using the different treatments, and thus contributes to the needed knowledge for making a more considerate choice between the use of the different anti-VEGFs.In conclusion, bevacizumab is the cheapest treatment option based on list prices. When considering the difference in injection frequency with a T&E regimen, the costs of treatment with aflibercept and ranibizumab will be cost neutral compared to bevacizumab at prices per injection of €507 and €61, respectively. Brolucizumab should reduce from 15.4 injections in a Q12W/Q8W regimen to 13.8 injections over 3 years in a T&E regimen in order to be as costly as aflibercept. Therefore, aflibercept is the second-choice treatment and so far there is no reason to consider brolucizumab as the second choice.ESM 1(DOC 70.0 KB)ESM 2(DOC 45.0 KB)ESM 3(DOC 32.0 KB)ESM 4(DOCX 28.0 KB)