There is limited knowledge on durability of neutralization capacity and antibody affinity maturation generated following two versus three doses of SARS-CoV-2 mRNA vaccines in naïve versus convalescent individuals (hybrid immunity) against the highly transmissible Omicron BA.1, BA.2 and BA.3 subvariants. Virus neutralization titers against the vaccine-homologous strain (WA1) and Omicron sublineages are measured in a pseudovirus neutralization assay (PsVNA). In addition, antibody binding and antibody affinity against spike proteins from WA1, BA.1, and BA.2 is determined using surface plasmon resonance (SPR). The convalescent individuals who after SARS-CoV-2 infection got vaccinated develop hybrid immunity that shows broader neutralization activity and cross-reactive antibody affinity maturation against the Omicron BA.1 and BA.2 after either second or third vaccination compared with naïve individuals. Neutralization activity correlates with antibody affinity against Omicron subvariants BA.1 and BA.2 spikes. Importantly, at four months post-third vaccination the neutralization activity and antibody affinity against the Omicron subvariants is maintained and trended higher for the individuals with hybrid immunity compared with naïve adults. These findings about hybrid immunity resulting in superior immune kinetics, breadth, and durable high affinity antibodies support the need for booster vaccinations to provide effective protection from emerging SARS-CoV-2 variants like the rapidly spreading Omicron subvariants. Here the authors show that a third SARS-CoV-2 vaccination significantly boosts neutralizing antibodies against Omicron subvariants and that hybrid immunity (infection and vaccination) results in broader neutralization activity and cross-reactive antibody affinity maturation. Viral spike protein of the Omicron variant contains higher number (>30) of mutations compared to other variants, raising concerns that Omicron is resistant to neutralizing antibodies generated following SARS-CoV-2 vaccination or infection, and a third vaccine dose was recommended to boost immunity.The emergence of Omicron (B.1.1.529/BA.1) variant of SARS-CoV-2 in November 2021, and its rapid spread across the globe, resulted in designation of Omicron as a variant of concern (VOC)2. Thus far, the BA.2 demonstrates higher transmissibility compared with BA.1 and has reached dominance in several countries in Europe and Africa3. However, limited knowledge exists regarding vaccine-induced neutralizing and antibody affinity following two doses vs. three doses of mRNA vaccination in naïve vs. COVID-19 recovered individuals against SARS-CoV-2 Omicron BA.2 and BA.3 lineages and the durability of the vaccine-induced immunity. Therefore, it is critical to know the persistence of cross-reactive immunity generated following SARS-CoV-2 vaccination to neutralize the SARS-CoV-2 omicron subvariants BA.1, BA.2 and BA.3.Since then, several subvariants of Omicron emerged (BA.2 and BA.3), each with its own set of mutations  vaccination in naïve versus convalescent individuals (infection before vaccination) both at one-month after the second and third vaccinations and at 4 months post-3rd vaccination against vaccine-homologous WA1 and Omicron BA.1, BA.2 and BA.3 subvariants.N = 50) or SARS-CoV-2 convalescent  individuals both at peak (1 month) immune response after the second and third vaccination, as well as at 4 months post-3rd vaccination, against vaccine-homologous WA1 and Omicron BA.1, BA.2 and BA.3 subvariants. There were no significant differences for age, sex, race, ethnicity, body mass index (BMI) or vaccine type between the two cohorts  vaccination in a cohort of 81 adults: either naïve  against the SARS-CoV-2 vaccine homologous WA1, as well as Omicron BA.1, BA.2 and BA.3 subvariants that were previously shown to correlate well with neutralization titers measured with authentic SARS-CoV-2 in plaque reduction neutralization testsNinety-four percent (94%) of previously naïve individuals had positive WA1-neutralizing antibody titers (>1:60) at 1 month after the second vaccination with geometric mean titers (GMT) of 396. Following a third vaccine dose, 100% of naïve adults were seropositive with 5.9-fold average increase in neutralization titers (GMT = 2333) compared with the second vaccination. However, by four months after the third vaccine dose, the neutralization titers were 3.2-fold lower (GMT = 729) with 96% naïve individuals with PsVNA50 > 1:60 Fig. . In compNeutralization titers were significantly reduced against all three Omicron subvariants compared with WA1 Fig. . After trd vaccine dose  but not at 4 months after the third vaccine dose , and at 1 month (p = 0.0005) as well as 4 months (p = 0.0005) after the third vaccine dose  on the chip such as to measure monovalent interactions independent of the antibody isotype, as described before11.Surface Plasmon Resonance (SPR) was used to measure total antibody binding against recombinant spike proteins derived from vaccine homologous WA1 and currently predominant circulating Omicron BA.1 and BA.2 subvariants as determined by Resonance Units (RU) Fig. 7. The pup < 0.0001) and BA.2 (p = 0.0211) spikes, and against BA.1 spike at 4 months after the third vaccine dose (p = 0.0068)  Fig. .14. Previously, we showed that antibody kinetics measured under optimized SPR conditions primarily represent the monovalent interactions between the antibody-antigen complex, as antigen-antibody binding off-rates of the IgG and Fab interaction with protein antigens were similar14.To determine qualitative difference in antibody avidity maturation against SARS-CoV-2 spike proteins induced following mRNA vaccination, antibody dissociation kinetics (off-rate constants), which describe the stability of the antigen-antibody complex, i.e., the fraction of complexes that decay per second in the dissociation phase, that are independent of antibody concentration were used as a surrogate for overall average avidity of polyclonal antibody were determined directly from the human polyclonal sample interaction with recombinant purified SARS-CoV-2 spike proteins of vaccine homologous WA1 as well as Omicron BA.1 and BA.2 using SPR in the dissociation phase only for the sensorgrams with Max RU in the range of 10–150 RU, as described beforeAntibody binding dissociation rates against WA1 spike were slower indicating higher binding affinity after the third vaccination compared with the second vaccination against the vaccine spike protein Fig. . The antnd dose , one month after the 3rd dose  and at four months after the 3rd dose (p = 0.0024 for BA.2 spike)  was observed one month after the 3rd dose compared with post-2nd vaccine dose that was maintained at four months post-3rd vaccination  Fig.  and S7.The antibody evolution after mRNA vaccinations and their decay followed parallel trajectories with good correlation between PsVNA50 neutralizing antibody titers and spike antibody binding  are determined by multiple factors including immune escape due to specific amino acid mutations in the spike, the levels (specificity and affinity) of cross-reactive polyclonal antibodies elicited by prior infection and/or vaccination, and waning immunity18, as well as BA.3. But in our study, convalescent individuals with hybrid immunity showed better antibody response against the rapidly spreading Omicron BA.1, BA.2 and BA.3 compared with vaccinated and boosted individuals with no history of prior infection. Our data and other studies suggest that Omicron BA.3 may evade immunity acquired from vaccination slightly more efficiently than BA.1 and BA.219. Therefore, the need for an additional booster vaccination may vary based on the individual’s infection/vaccination histories and the circulating strains, in addition to various risk factors. In addition, our study demonstrated a strong correlation between SARS-CoV-2 neutralization titers and spike-binding antibody affinity against Omicron subvariants BA.1 and BA.2 spikes. Importantly, even at four months after the third dose the antibody binding affinities against the Omicron subvariants BA.1 and BA.2 were maintained and trended higher for the individuals with hybrid immunity. Recently, Wratil et al., reported similar findings but our data expands the antibody analyses, including delineating antibody affinity maturation and persistence of protective immunity to the Omicron subvariants up to 4 months post-third SARS-CoV-2 mRNA vaccination20. Although hybrid immunity due to prior SARS-CoV-2 infection followed by vaccination leads to broader antibody responses, it is important to understand that any infection still comes with a risk of complications. The gradual drop in antibody titers following hybrid immunity, irrespective if the infection took place before or after vaccination, suggest the immunity wanes at similar rates following vaccination and breakthrough infections with eventual loss of protection against circulating SARS-CoV-2 strains23. A possible role of immune imprinting in SARS-CoV-2 immune response due to prior SARS-CoV-2 infection/vaccination or the original antigenic sin (OAS) hypothesis, whereby adults with B-cell memory due to prior exposure to seasonal coronaviruses24 requires further investigation. Recently, we observed anti-S2 cross-reactivity in naive older children but not in the younger children (<4 years old), who share homology with HKU1, 229E and OC4325. OAS was also observed in mice immunization studies with seasonal CoV followed by SARS-CoV-2 spike26. Most of these cross-reactive antibodies do not neutralize SARS-CoV-2 and do not contribute to SARS-CoV-2 neutralization.Our study demonstrates that a third vaccination significantly boosts neutralizing antibodies against the Omicron subvariants, including BA.1 and BA.2, as recently reported32. In naïve individuals, antibody affinity maturation post vaccination lags the convalescent group but may continue to evolve in the weeks following additional vaccine boosts. The breadth of variant recognition is likely to be impacted both by the antigenic distance between the infecting and vaccination strains, the rate of antibody affinity maturation through repeated GC seedings of memory B cells, and the time postinfection and last vaccination34.Our data along with previous studies suggest the presence of circulating memory B cells with expanded affinity-matured repertoires in convalescent individuals that are recalled by vaccination and likely to re-enter secondary germinal centers to undergo further somatic hypermutation (SHM) and antibody affinity maturation35. In previous studies, we had demonstrated a strong correlation between antibody affinity and protection from highly pathogenic avian influenza viruses37 and a correlation with clinical benefit in patients infected with Zika virus38, Ebola virus10, influenza virus39 and COVID-1914. Therefore, in addition to virus neutralization it is important to measure antibody affinity maturation against the SARS-CoV-2 spike not only for the vaccine strain, but also against spike proteins derived from the circulating variants of concern, that may influence the protective efficacy of vaccines against current and emerging SARS-CoV-2 variants of concern.The protective efficacy by vaccine-induced antibodies against emerging variants may be impacted by both specific amino acid mutations in the spike and the affinity of the polyclonal antibodies against the SARS-CoV-2 variants. An association was observed between high titers of low-affinity antibodies against RBD with the disease severity of COVID-19 patientsNo breakthrough infections with Omicron were reported in our cohorts. However, it will be important to demonstrate the impact of high-affinity anti-BA.1/BA.2 antibodies not only on severe outcome  but also on the clinical course and viral loads in Omicron breakthrough infections. Based on the waning immunity, it is expected that naïve adults will potentially require another vaccine booster earlier (4–6 months post-third vaccination) than individuals with hybrid immunity to generate protective strong neutralizing antibodies especially against the rapidly spreading Omicron subvariants BA.2.12.1, BA.4 and BA.5. In summary, our study demonstrates that hybrid immunity provides superior immune kinetics, breadth, and durable high-affinity antibodies vs. vaccine-only immunity against Omicron sublineages up to four months post-third vaccination with mRNA vaccines.Heat-inactivated de-identified samples were obtained from participants enrolled in the SPARTA  program in Athens, GA (USA) with written informed consent  or from Pfizer (BNT162b2) at 4-week or 3-week intervals between doses, respectively, between January and February 2021. All of them then received a third mostly homologous vaccine booster dose in September-October 2021. The primary series with the first two vaccine doses were homologous for each vaccinee. The boosters were either homologous or heterologous as shown in the Supplementary Table Post-SARS-CoV-2 mRNA vaccination serum samples were collected after the second vaccination and third vaccination from 31 convalescent individuals who were exposed to SARS-CoV-2 but not hospitalized (had confirmed SARS-CoV-2 infection between March and November 2020) prior to circulation of any of the Omicron lineages as well as naïve 50 healthy adults41.Sera were evaluated in a qualified SARS-CoV-2 pseudovirion neutralization assay (PsVNA) using SARS-CoV-2 WA-1 strain and the three Omicron variant lineages: BA.1 (B.1.1.529), BA.2 and BA.3 , pTrip-luc lentiviral vector and pcDNA 3.1 SARS-CoV-2-spike-deltaC19, using Lipofectamine 3000. The supernatants were harvested at 48 h post transfection and filtered through 0.45 µm membranes and titrated using 293T-ACE2-TMPRSS2 cells (HEK 293 T cells that express ACE2 and TMPRSS2 proteins)41.Pseudovirions were produced as previously described12. For the neutralization assay, 50 µL of SARS-CoV-2 S pseudovirions  were pre-incubated with an equal volume of medium containing serial dilutions  of heat-inactivated serum at room temperature for 1 h. Then 50 µL of virus-antibody mixtures were added to 293T-ACE2-TMPRSS2 cells (104 cells/50 μL)41 in a 96-well plate. The input virus with all SARS-CoV-2 strains used in the current study were the same (2 ×105 relative light units/50 µL/well). After a 3 h incubation, fresh medium was added to the wells. Cells were lysed 24 h later, and luciferase activity was measured using One-Glo luciferase assay system . Antibody data was collected in MS Excel version 16.57. The assay of each serum was performed in duplicate, and the 50% neutralization titer was calculated using Prism 9 (GraphPad Software). Controls included cells only, virus without any antibody and positive sera. The limit of detection for the neutralization assay is 1:20. Any sample that does not neutralize SARS-CoV-2 at 20-fold dilution was given a value of 10 for representation and data analysis purposes. Two independent biological replicate experiments were performed for each sample and variation in PsVNA50 titers was <9% between replicates.Neutralization assays were performed as previously describedSteady-state equilibrium binding of post-SARS-CoV-2 vaccinated human polyclonal sample was monitored at 25 °C using a ProteOn surface plasmon resonance (BioRad). The purified recombinant SARS-CoV-2 spike proteins were captured to a Ni-NTA sensor chip with 200 resonance units (RU) in the test flow channels. Serial dilutions  of freshly prepared samples in BSA-PBST buffer (PBS pH 7.4 buffer with Tween-20 and BSA) were injected at a flow rate of 50 µL/min (120 s contact duration) for the association, and disassociation was performed over a 600-s interval. Responses from the protein surface were corrected for the response from a mock surface and for responses from a buffer-only injection. Total antibody binding was calculated with BioRad ProteOn manager software (version 3.1). All SPR experiments were performed twice. In these optimized SPR conditions, the variation for each sample in duplicate SPR runs was <6%. The maximum resonance units (Max RU) shown in figures is for 10-fold diluted sample.14. Off-rate constants were determined from two independent SPR runs.Antibody off-rate constants, which describe the stability of the antigen-antibody complex (i.e. the fraction of complexes that decays per second in the dissociation phase) were determined directly from the interaction of human polyclonal samples with recombinant purified SARS-CoV-2 proteins using SPR in the dissociation phase only for the sensorgrams with Max RU in the range of 10–150 RU and calculated using the BioRad ProteOn manager software for the heterogeneous sample model as described beforeDescriptive statistics were performed to determine the geometric mean titer values and were calculated using GraphPad. All experimental data to compare differences among groups were analyzed using lme4 and emmeans packages in R (RStudio version 1.1.463).p value was less than 0.05.The demographic characteristics of these study participants are shown in Supplementary Table Samples were allocated randomly to each test group and tested blindly (researcher was blinded to sample identity) to minimize selection bias or detection bias. There were no exclusion criteria. All samples and data were used for analysis and presented in the study.Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Evaluating the serum cross-neutralization responses after breakthrough infection with various SARS-CoV-2 variants provides valuable insight for developing variant-proof COVID-19 booster vaccines. However, fairly comparing the impact of breakthrough infections with distinct epidemic timing on cross-neutralization responses, influenced by the exposure interval between vaccination and infection, is challenging. To compare the impact of pre-Omicron to Omicron breakthrough infection, we estimated the effects on cross-neutralizing responses by the exposure interval using Bayesian hierarchical modeling. The saturation time required to generate saturated cross-neutralization responses differed by variant, with variants more antigenically distant from the ancestral strain requiring longer intervals of 2–4 months. The breadths of saturated cross-neutralization responses to Omicron lineages were comparable in pre-Omicron and Omicron breakthrough infections. Our results highlight the importance of vaccine dosage intervals of 4 months or longer, regardless of the antigenicity of the exposed antigen, to maximize the breadth of serum cross-neutralization covering SARS-CoV-2 Omicron lineages.  •A modeling to estimate the effects on cross-neutralization by the exposure interval•The saturation time to generate a high neutralization titer differs for each variant•The optimal booster vaccination interval is estimated to exceed 4 months Immune system; Immunity; Immune response; Virology Compared to the SARS-CoV-2 ancestral strain, the BA.1 virus has more than 30 amino acid mutations in the spike protein, including insertions and deletions. The BA.2 spike protein differs from the BA.1 spike protein at 27 amino acid positions, whereas the BA.5 spike protein differs from the BA.2 spike protein by four amino acids, including a L452R mutation. BA.2.75 differs the from BA.2 spike protein at nine amino acid positions, including K147E, W152R, F157L, I210V, and G257S, which are located in the N-terminal domain, and G339H, G446S, N460K, and R493Q, which are located in the receptor-binding domain (RBD). BQ.1.1 differs from BA.5 spike protein at R346T, K444T, and N460K.At the end of 2021, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron (B.1.1.529) variant emerged and rapidly spread worldwide. Since then, the Omicron variant has evolved into multiple sub-lineages, with BA.1, BA.2, and BA.5, emerging sequentially as the globally dominant variant. In August 2022, BA.5 was the most common variant circulating worldwide. In India, the proportion of BA.5 infections increased in May 2022, but the proportion of BA.2.75 (a variant of the BA.2 sub-lineage) has increased since June 2022, suggesting that the transmissibility of BA.2.75 may be higher than that of BA.5.,,Because of the numerous mutations that have accumulated in the spike protein of Omicron variants, Omicron variants are antigenically distinct from the ancestral strain and have the capacity to evade immunity introduced by a primary series of the first-generation COVID-19 vaccine, including COVID-19 mRNA vaccine containing ancestral spike antigen alone. Among Omicron sub-lineages, BQ.1.1 shows the greatest immune evasion against serum neutralization.,,,,,,,,,,,,Recently, BA.1 and BA.2 breakthrough infections in individuals vaccinated with COVID-19 mRNA vaccines were found to increase broad serum neutralizing activity against Omicron BA.1, BA.2, and prior VOCs at levels comparable to those of the ancestral strain.,One approach to account for these differences is to use Bayesian hierarchical modeling. Bayesian hierarchical modeling constructs a hierarchical structure of sub-models, and by estimating probability distributions using Bayes' theorem, it enables the extension and estimation of parameters in multiple groups based on common parameters shared among these groups.In this study, cross-neutralizing activity against SARS-CoV-2 variants, including the Omicron sub-lineages BA.1, BA.2, BA.5, BA.2.75, and BQ.1.1, was assessed using serum samples from individuals with breakthrough infections and booster vaccine recipients before or during the Omicron epidemic. Furthermore, Bayesian modeling was used to correct for the influence of different exposure intervals to enable estimation of the saturated serum cross-neutralizing responses against SARS-CoV-2 variants induced after breakthrough infection with the ideal exposure interval between vaccination and breakthrough infection.,We collected serum samples from individuals with breakthrough SARS-CoV-2 infections during the Omicron BA.1 wave and individuals who had received a booster dose of the first-generation mRNA COVID-19 vaccine containing the ancestral spike antigen alone . IndividNeutralization titers (NTs) were determined using live virus-based assays B. Serum To obtain an overall picture of the antigenicity of Omicron sub-lineages in serum samples from each exposure group, we performed antigenic cartography to locate the antigens and sera in a two-dimensional map D. Positi,,https://www.who.int/news/item/17-05-2022-interim-statement-on-the-use-of-additional-booster-doses-of-emergency-use-listed-mrna-vaccines-against-covid-19). Generally, an interval of 4–6 months after the second vaccination could be considered. In Japan, a vaccination interval of at least 3 months is recommended (https://www.mhlw.go.jp/stf/covid-19/booster.html). This vaccination strategy and the periods of pre-Omicron and Omicron waves resulted in distinct exposure intervals among the three exposure groups . Although a shorter vaccination interval during periods of transient surges in COVID-19 cases may benefit to the level of herd immunity, it may be insufficient to induce high levels of cross-neutralizing antibodies covering antigenically distinct variants. Previous studies have shown that in vaccine recipients, higher NTs were induced with a dose interval of 16 weeks  than with a dose interval of 4 weeks  between the first and second doses,,,,,An exposure interval of more than 128 days was required to induce broad cross-neutralizing activity against the BQ.1.1 variant, which was antigenically distant from the ancestral strain. As stated above, WHO recommends at least 4 months (approximately 120 days) after the second vaccination before booster vaccination, which is comparable to the exposure interval needed to induce broad cross-neutralizing responses. In contrast, the US Centers for Disease Control and Prevention recommends at least 2 months (approximately 60 days) between the second and third doses .Additional information and requests for resources and reagents should be directed to the lead contact, Tadaki Suzuki  under a material transfer agreement with the NIID, Tokyo, Japan.The characteristics of the participants are listed in MatchIt R functions .We used the SARS-CoV-2 ancestral strain WK-521 , Omicron BA.1 variant TY38-873 , Omicron BA.2 variant TY40-158 , Omicron BA.5 variant TY41-702 , Omicron BA.2.75 variant TY41-716 , and Omicron BQ.1.1 variant TY41-796  in this study. These variants were isolated using VeroE6/TMPRSS2 cells at NIID with ethics approval provided by the Medical Research Ethics Committee of NIID (#1178). More specifically, viruses belonging to the Omicron lineage were isolated at NIID using VeroE6/TMPRSS2 cells on respiratory specimens collected from individuals screened at airport quarantine stations in Japan and transferred to NIID for whole-genome sequencing.2.VeroE6/TMPRSS2 cells  were maintained in low-glucose Dulbecco’s modified Eagle’s medium (DMEM) containing 10% heat-inactivated fetal bovine serum (FBS), 1 mg/mL geneticin, and 100 U/mL penicillin/streptomycin at 37°C supplied with 5% COAntibody titers for the ancestral spike (S) RBD and nucleoprotein (N) were measured using Elecsys Anti-SARS-CoV-2 S and Elecsys Anti-SARS-CoV-2 kits according to the manufacturers’ instructions.,50) SARS-CoV-2 viruses, followed by incubation at 37°C for 1 hour. Before adding of the virus-serum mixtures, 1.0 x 104 VeroE6/TMPRSS2 cells were seeded in each well of the 96-well plates to ensure a consistent cell number across all wells. The virus-serum mixtures were then placed on the cells and cultured at 37°C with 5% CO2 for 5 days. The cells were then fixed with 20% formalin and stained with crystal violet solution. NTs were defined as the geometric mean of the reciprocal of the highest sample dilution that protected at least 50% of the cells from a cytopathic effect, using two to four multiplicate series. Because of the limited volume of serum samples from individuals with breakthrough infections, this assay was performed only once. All experiments using authentic viruses were performed in a biosafety level 3 laboratory at NIID.Live virus neutralization assays were performed as described previously.Racmacs R function with 2,000 optimizations, with the minimum column basis parameter set to “80”.,Antigenic maps based on NTs against SARS-CoV-2 viruses were created using the https://www.r-project.org/). Measurements below the detection limit were recorded as half the detection limit. One-way analysis of variance with Dunnett’s test or Tukey’s test were used to compare the antibody titers. Statistical significance was set at p < 0.05.Data analysis and visualization were performed using GraphPad Prism 9.3.1  and R 4.1.2 . We inferred population means . Four independent MCMC chains were run with 5,000 steps in the warm-up and sampling iterations, with subsampling every five iterations. We confirmed that all estimated parameters showed <1.01 R-hat convergence diagnostic values and >500 effective sampling size values, indicating that the MCMC runs were convergent. The fitted model closely replicated the observed NT increases in each exposure group (https://www.r-project.org/). Information on the estimated means of saturated NTs against SARS-CoV-2 variants is summarized in Parameter estimation was performed using a Markov chain Monte Carlo (MCMC) approach implemented in rstan 2.26.1 (re group A and 3A.


This study aimed to explore the influence of bone conduction hearing aid (BCHA) implantation on HRQoL and hearing disability in patients with hearing loss suffering from tinnitus. (2) Methods: Data were collected from an international hearing implant registry. Health Utilities Index Mark 3 (HUI-3), Spatial and Qualities of Hearing- 49 Questionnaire (SSQ) and self-reported tinnitus burden data for adult patients implanted with a BCHA (n = 42) who provided baseline as well as follow-up data 1-year post-implantation were extracted from the registry. Wilcoxon signed rank tests and paired samples t-tests were used to analyse outcomes data. (3) Results: Patients, with or without tinnitus, demonstrated clinically important mean improvements in HUI-3 multi-attribute utility scores, HUI-3 hearing attribute and SSQ scores. Hearing loss patients with tinnitus presented with a lower HRQoL than patients without tinnitus. (4) Conclusions: These findings demonstrate the importance of hearing rehabilitation in improving the quality of life and hearing disability of patients with or without tinnitus and in providing tinnitus relief in some patients with hearing loss and tinnitus. The term tinnitus describes a perceived auditory sensation in the absence of an associated external auditory stimulus . TinnituTinnitus is often directly related to age and hearing loss . HoweverCurrently, there is no cure for tinnitus; however, non-standardised interventions with varying efficacies exist for symptomatic relief, including auditory stimulation, education and reassurance, and cognitive behavioural therapy (CBT) . PsychotTinnitus is a severe condition comparable to other major chronic diseases . Its higAccording to the literature, some tinnitus sufferers have reported worsening symptoms in noisy areas; however, many have reported tinnitus aggravation from a lack of background noise, whereby its addition helped suppress their tinnitus ,25. TinnSubjective data on patient-reported outcomes using standardised measures on HRQoL such as the disease-specific Speech, Spatial and Qualities of Hearing Scale (SSQ) and the Health Utilities Index Mark 3 (HUI-3) are necessitated in addition to objective clinical outcome measures for optimal disease management . SSQ is Long-term follow-up based on acoustic hearing implantation influence on HRQoL in tinnitus patients are not readily available in the literature. Furthermore, these studies suffer from limited sample size and non-standardised reporting ,37. ThisThe study utilised retrospective data collected during the Cochlear Implant Recipient Observational Study (IROS). IROS was a prospective, longitudinal study that used repeated measures with intra-subject controls. The study used objective audiological measures and subjective evaluation tools such as the SSQ and HUI-3 to evaluate HRQoL and patient-related benefits of the use of implantable hearing devices, including BCHA devices. ClinicalTrials.gov with the identifier NCT02004353. The design, implementation and management of the registry have been published previously [The IROS is an international registry that also collected baseline and follow-up socio-economic information on adult recipients, including information on tinnitus burden. IROS is registered on eviously . The regThe presence of tinnitus, defined as noise or ringing in the head or ears, was assessed using a non-standardised form within the IROS patient questionnaire. Subjects were first asked to report pre-implantation if they experienced tinnitus. If yes, they were asked to complete follow-up questions based on how often it was experienced: always; sometimes; and do not know. The same questions were asked post-surgery, at the 1-year follow-up. Thereafter, a single question was asked based on how subjects would describe their tinnitus in comparison to pre-implantation: worse; same; better; and do not know. The registry contains baseline data, pre-implantation, and follow-up data for up to 3 years post-implantation, although there is significant attrition after 1-year follow-up. A total of 1164 subjects that received an implantable hearing device provided baseline data, but only 180 recipients provided data at the three-year follow-up visit . The study included all eligible adult patients receiving a BCHA device who have provided baseline as well as follow-up data after 1 year, including information on HUI-3, SSQ and self-reported tinnitus burden . EligiblThe IROS study was approved by the Ethical Review Boards of participating clinics in Colombia , Germany , Hungary , Poland , Spain , and South Africa  according to institutional and national research standards. All patients provided written informed consent prior to inclusion.Data were analysed using IBM SPSS version 29. Socio-demographic and other relevant clinical and nonclinical categorical data, including background characteristics at baseline, are presented descriptively. Categoric variables are represented as a number (n) and/or percentage. Continuous background data are presented as a mean value with standard deviation (SD).HUI-3 questionnaire data were analysed, and attribute and utility scores were calculated following standard methodology: Calculation Matrix HUI-3 Multi-attribute Utility HUI3 Multi-attribute Utility Function on Dead-healthy Scale Matrix .p-value of p < 0.05 was used to determine statistical significance.SSQ questionnaire scores from each domain were combined into mean domain-specific scores and a global mean score by taking the average score across each scale. HUI-3 and SSQ data were assessed graphically and by Kolmogorov–Smirnov and Shapiro–Wilk tests. These tests confirmed unequal distribution of the HUI-3 overall utility and hearing attribute scores pre- and post-implantation and normal distribution for the SSQ scores pre- and post-implantation. Therefore, the parametric Wilcoxon signed rank test for related samples was applied to determine mean utility and hearing attribute changes and paired t-tests were used for the SSQ score differences. A p-values are widely encouraged to enhance research quality [Effect size (d) values are presented as an additional measure to demonstrate the magnitude of the treatment effect, independent of sample size . Effect  quality . Point-biserial correlation tests were conducted to measure the strength of the association between tinnitus status and change in HUI-3 and SSQ scores pre- and post-implantation. Standard correlation value guidelines were applied whereby coefficient values between 0 and ±0.3 indicated a weak linear relationship, values between ±0.3 and ±0.7 indicate a moderate linear relationship, and values between ±0.7 and ±1 indicate a strong relationship. The positive or negative value indicates the direction of the strength of the relationship . HUI-3 mean overall HRQoL score differences of 0.03 or greater are considered clinically important, and differences of at least 0.01 may be meaningful and important in some contexts . DiffereThe study included 42 IROS participants who were implanted with a BCHA device. Forty patients received a transcutaneous device and the remaining 2 patients received percutaneous devices. The presence of tinnitus was reported by 23 participants at baseline, and 19 participants presented without tinnitus at baseline. IROS participants who had HUI-3 and SSQ data at baseline and one-year post-implantation were included. Participants who answered “do not know” for tinnitus-related questions at baseline or had missing tinnitus status data were excluded from the study.The study comprised about 60% males and 40% females. The mean age of the group was 40.21 (±14.58) years, and a majority of the participant group was based in Columbia (85.7%), with the remainder based in Poland (14.3%) . p = 0.218) and had a small effect size (d = 0.182). The HUI-3 mean utility score change for patients without tinnitus presented a clinically important mean improvement of 0.065 from pre-implantation (0.811) to post-implantation (0.876) (p = 0.277) and had a small effect size (d = 0.176).The HUI-3 mean scores for patients with tinnitus showed a clinically important mean improvement of 0.054 from pre-implantation (0.624) to post-implantation (0.678) . The Wil (0.876) ; howeverp = 0.139) and had a small effect size (d = 0.218). The HUI-3 hearing attribute mean score change for patients with tinnitus demonstrated a clinically relevant improvement of 0.051 from pre-implantation (0.865) to post-implantation (0.916) . Resultsp = 0.053) but displayed a medium effect size (d = 0.313).The HUI-3 hearing attribute mean score change for patients without tinnitus presented a clinically relevant improvement of 0.076 from pre-implantation (0.857) to post-implantation (0.933) . Resultsp < 0.001) and clinically relevant global SSQ mean improvement from pre (4.794) to post-implantation (6.566) for patients with tinnitus of 1.772 (p < 0.001) and clinically relevant global SSQ mean improvement from pre (5.036) to post-implantation (7.684) in patients without tinnitus of 2.648 (Results from the paired t-test in of 1.772  and dispof 2.648  and the p = 0.026) (p = 0.906). No other comparisons were statistically significant.Results show that there was a statistically significant difference in pre-operative HUI-3 score depending on tinnitus status in patients that were later implanted with a BCHA (= 0.026) , which wThe aim of this study was to explore the impact of BCHA on HRQoL and hearing disability in patients suffering from tinnitus by using standardised instruments in the form of HUI-3 and SSQ questionnaires. Subjective hearing assessments allow for a more comprehensive understanding of audiometrically assessed handicaps and provide information based on hearing ability pre- and post-intervention as well as additional hearing-related effects and its psychosocial impact ,46. The p = 0.218) and hearing attribute scores  for tinnitus patients and the overall utility change  and hearing attribute scores  for patients without tinnitus, however, yielded differences not reaching statistical significance, likely due to the small sample size. The mean score improvements between tinnitus (0.054) and non-tinnitus (0.065) patients are similar post-implantation and the correlation results show that there are no significant associations between tinnitus status and change in HUI-3 scores post-BCHA-implantation . This indicates that baseline tinnitus status is likely to have a negligible impact on change in HUI-3 multi-attribute scores between pre- and post-device implantation. Tinnitus status and pre-implant HUI-3 scores, however, are significantly negatively correlated . This indicates that patients with tinnitus are likely to present with lower HUI-3 scores before treatment than patients without tinnitus. This is also expected as individuals with untreated hearing loss without tinnitus have been shown to experience greater HRQoL than those with both untreated tinnitus and hearing loss [Important findings obtained from the HUI-3 measurements were that there were clinically relevant improvements in mean utility scores from pre- to post-implantation. The overall utility change  between tinnitus status and change in SSQ scores post-implantation. SSQ scores from a similar study were significantly lower in cochlear-implanted patients with tinnitus than without [The standardised SSQ system provides detailed information focused on patients’ auditory performance in everyday situations to further enhance the data analysis . There w without . Hearing without ,51.Results from the tinnitus perception assessment pre-and post-implant demonstrated that 29% of patients felt an improvement in tinnitus symptoms post-implant. Comparable findings based on hearing healthcare professionals’ survey results suggested that 60% experienced minor to major relief and 22% of patients experienced major tinnitus relief with hearing aids . Their sThe prevalence of tinnitus among BCHA patients (55%) is considerably higher than the global tinnitus prevalence rate. This can be expected for patients with hearing loss as they are more likely to experience distressing tinnitus symptoms than those able to experience a sound-filled environment ,22,28. TThe analysis of two standardised measures in the form of HUI-3 and SSQ questionnaires is a key strength of this study as they are valid instruments used to reliably measure both HRQoL as well as hearing disability ,32,57. HHearing loss patients with tinnitus present with a lower HRQoL than patients without tinnitus, however, the improvement in overall HRQoL post-implantation did not vary significantly between these patient groups. Bone conduction hearing implantation improves HRQoL and reduces hearing disability in patients with hearing loss, with or without tinnitus. Furthermore, tinnitus symptoms were improved in over a quarter of tinnitus patients 1 year post-device implantation. These findings demonstrate the importance of hearing rehabilitation in improving the quality of life and hearing disability of patients with or without tinnitus and in providing tinnitus relief in some patients with hearing loss and tinnitus.


Here, we synthesized and characterized a novel two-dimensional (2D) conjugated electron donor–acceptor (D-A) copolymer (PBDB-T-Ge), wherein the substituent of triethyl germanium was added to the electron donor unit of the polymer. The Turbo–Grignard reaction was used to implement the group IV element into the polymer, resulting in a yield of 86%. This corresponding polymer, PBDB-T-Ge, exhibited a down-shift in the highest occupied molecular orbital (HOMO) level to −5.45 eV while the lowest unoccupied molecular orbital (LUMO) level was −3.64 eV. The peaks in UV-Vis absorption and the PL emission of PBDB-T-Ge were observed at 484 nm and 615 nm, respectively. Polymer solar cells (PSCs), comprising bulk-heterojunction (BHJ) photoactive layers of electron donors (D) and electron acceptors (A), have attracted enormous attention due to their lightweight properties, solution-processability, low-temperature printing fabrication and mechanical flexibility ,5,6,7,8.sc), and the alignment of the energy levels between electron donors and acceptors was reported to control open-circuit voltage (Voc). The crystallinity and morphology of electron donor–acceptor pairs have also been reported to impact the fill factor (FF) dithiophene-4,8-dione, tin(ll) chloride dihydrate, anhydrous sodium sulfate (>99%), indium tin oxide (ITO) glass substrate, 1,3-Bis(5-bromo-2-thienyl)-5,7-bis(2-ethylhexyl)-4H,8H-benzo dithiophene-4,8-dione , tetrakis(triphenylphosphine) palladium (Pd(PPh3)4) and Y6 were purchased from Sigma-Aldrich . Triethyl germanium chloride and 2-iodothiophene were obtained from TCI . Ethanol was obtained from EMD . Isopropyl alcohol was purchased from VWR . All materials were used as received without further purification.Dichloromethane , isopropyl magnesium chloride lithium chloride complex , trimethyl tin chloride , acetone and deuterated chloroform (1) were added to the round-bottom flask under stirring condition. Then, iPrMgCl·LiCl  was added dropwise to the reaction mixture, which was left under stirring conditions for 3 h at room temperature. The mixture was quenched with cold DI water (30 mL), which was then extracted by DCM (25 mL) three times. The organic layer was collected and dried over anhydrous sodium sulfate (Na2SO4). The volatile solvent was removed under vacuum with mild temperature; then, the residue was purified by column chromatography using hexane: ethyl acetate (5:1 v/v) as an eluent with a retention factor (RF) of 0.0789 to yield the triethyl(thiophen-2-yl) germane as a colorless oil. Yield = 5.4 g (86%). 1H NMR , δ (ppm): 7.62–7.59, 7.25–7.22, 7.20–7.19, 1–1.3. Triethyl(thiophen-2-yl) germane was synthesized via a method that was adopted from Sherborne G. et al. . Under t2) in THF (45 mL) was placed in a 100 mL, two-neck, round-bottom flask. The solution was cooled to 0 °C. n-BuLi  was added dropwise into the RBF and the mixture was kept at 0 °C for 40 min before the mixture was refluxed at 65 °C under stirring for 2 h. After we cooled the mixture down, 4,8-dehydrobenzo dithiophene-4,8-dione  was added in one portion, and the mixture was refluxed with stirring for another 2 h. Tin(ll) chloride dihydrate  in 10% HCl (15 mL) was added after the mixture was cooled down to room temperature and stirred overnight. The reaction was quenched using cold DI water (40 mL), and then the crude product was extracted using diethyl ether (25 mL) three times. The organic layer was collected and dried over anhydrous sodium sulfate (Na2SO4). The volatile solvent was removed under vacuum at a mild temperature and the crude product was purified using a column chromatography with hexane: ethyl acetate (5:1 v/v) as eluents to yield 4,8-bis-(5-(triethyl germanium)thiophene-2-yl)benzodithiophene as bright yellow gel. Yield = 0.8 g (36.2%). 1H NMR , δ (ppm): 7.67–7.65, 7.6–7.58, 7.47–7.45, 7.3–7.28, 1.00–1.4. Under the protection of argon, a solution of triethyl(thiophen-2-yl) germane  in THF (6 mL) was placed in a 25 mL round-bottom flask. The solution was cooled to −78 °C using dry ice with acetone, and then n-BuLi  was added dropwise. After the addition, the mixture was kept at −78 °C for 1 h; trimethyl tin chloride  was added dropwise. The resulting mixture was stirred overnight at room temperature. The mixture was then poured into cold DI water (6 mL) and extracted with diethyl ether (8 mL) three times; after drying over anhydrous sodium sulfate (Na2SO4), the volatile solvent was removed under vacuum at a mild temperature. Recrystallization was used for purification using ethanol as a solvent, and then dried for two days in a vacuum at a mild temperature to yield a yellowish white crystal of -2-trimethylstannylthieno[1]benzothiol-6-yl]-trimethylstannane. Yield = 0.092 g (15.6%). 1H NMR , δ (ppm): 7.49, 7.48–7.46, 7.2– 7.3, 1.2–0.9, 0.6–0.5. Under the protection of argon, a solution of 4,8-bis-(5-(triethyl germanium) thiophene-2-yl)benzodithiophene-4,8-dione  were dissolved in dry toluene (5 mL). The reaction container was purged with argon for 20 min, and then tetrakis(triphenylphosphine)palladium (Pd(PPh3)4)  was added. After another flushing with argon for 20 min, the reactants were heated up to reflux them at 120 °C for 24 h. The solution was cooled down to room temperature and poured into cold methanol (MeOH) (50 mL), and then washed three times using cold methanol. The product was dried under vacuum at a mild temperature for 24 h to yield a dark purple solid of PBDB-T-Ge. Yield 0.09 g. 3), respectively. The polydispersity (PDI) was observed to be 1.41. 1H NMR , δ (ppm): 7.6–7.75, 7.45–7.35, 7.2–7.3, 7.15–7.25, 6.95–7.05, 1.4–0.95, 0.9–0.6.In a 25 mL, double-neck, round-bottom flask, -2-trimethylstannylthieno[1]benzothiol-6-yl]-trimethylstannane  and 1,3-Bis(5-bromo-2-thienyl)-5,7-bis(2-ethylhexyl)-4d-chloroform as solvent and the internal chemical shift reference standard was selected based on the residual proton resonance of the solvent, which were acquired, processed, and analyzed using Bruker Topspin (4.1.4) software. The molecular weight of polymers was determined by gel permeation chromatography (GPC) relative to polystyrene standards with chloroform as the eluent at a concentration of 1.0 mg/ mL for at least 12 h. The optical properties were tested using a UV-Vis spectrometer (Shimadzu 3600 Plus). This characterization was performed using chloroform solution in a quartz cuvette using polymer film on a glass substrate. The polymer film samples were prepared using drop-casting on a glass substrate. The photoluminescence study was performed using Spectro-fluorophotometer (Shimadzu RF-5301PC model) using a chloroform solution. Cyclic voltammetry study was performed using a potentiostat  at room temperature to determine the redox reaction. The surface morphology characterizations on PBDB-T-Ge:Y6  composite film were performed using atomic force microscopy  using a taping mode for imaging. Further surface morphology was performed using scanning electron microscopy  with secondary electron for the imaging.The structure of PBDB-T-Ge was confirmed using an NMR spectrometer . The spectra were collected using n-butyl lithium to activate the ethylhexyl branch groups of PBDB-T-C and trialkyl silyl groups of PBDB-T-Si, as seen in Our work implemented germanium on the side branch of the PBDB-T-Ge. Previous reports utilized 1H NMR spectra in 2) is synthesized, where the three peaks between 7.1 and 7.7 ppm are assigned to the proton of the single-branched thiophene and the multiple peaks from 0.8 to 1.4 ppm are assigned to the alkyl groups. The 1H NMR spectra show that the implementation of triethyl germanium on the thiophene was successful, simplifying and enhancing product yields.onset) at 620 nm, and the maximum absorption (λmax) occurred at 477 nm. In comparison, the absorption peak of reference polymer  of 666 nm and maximum absorption (λmax) at 507 nm, while another reference polymer film  in acetonitrile as the supporting electrolyte and the ferrocene/ferrocenium as the internal reference. Cyclic voltammogram was obtained at a scan rate of 10 mV/s with a 1 mV sample interval.The HOMO and LUMO of PBDB-T-Ge were measured to evaluate the effect of the substitution of the triethyl germane sidegroup on the electronic energy levels using cyclic voltammetry. The electrochemical study was performed using conventional three electrodes configuration consisting of an Ag/AgCl electrode as a reference electrode, the glassy carbon electrode as the working electrode, and the platinum wire as the counter-electrode. We used tetraethylammonium tetrafluoroborate (Etoxonset and Eredonset), respectively, according to the Equations (1) and (2) [1/2.Fc/Fc+ is the half-potential of ferrocene/ferrocenium coupled to the electrochemical measurement system, and the energy level of Fc/Fc+ was taken as 4.8 eV below vacuum. Ferrocene/ferrocenium half-potential was measured to be 0.44 V ferrocene/ferrocenium, and Equations (3) and (4) are as follows:Solutions of PBDB-T-Ge polymer and Y6 were prepared by dissolving in chloroform and the films were deposited onto the glassy carbon button electrode via a drop-casting procedure. It was then air-dried. HOMO and LUMO energy levels were estimated from onset oxidation and reduction potentials (E and (2) .E (HOMOoxonset) and onset reduction potential (Eredonset) of PBDB-T-Ge were measured to be 1.09 and −0.72 V vs. Ag/AgCl, respectively. The HOMO energy level and LUMO energy level of PBDB-T-Ge were calculated to be −5.45 and −3.64 eV, respectively, as seen in sc, which is expected to enhance the photovoltaic performance. The data were collected from PBDB-T-C in 0.1 M tetrabutylammonium hexafluorophosphate (Bu4PF6) acetonitrile solution at a scan rate of 50 mV/s, while the data of PBDB-T-Si were collected using Ultraviolet Photoelectron Spectroscopy (UPS). The detailed electrochemical data for PBDB-T-Ge and its counterparts, Y6, are listed in oc, which is expected to enhance the PCE of PBDB-T-Ge-based PSC. It should be noted that the electrochemical bandgap (Egcv) of PBDB-T-Ge is 1.81 eV, which is similar to its optical bandgap of 1.86 eV.We noticed that the HOMO of PBDB-T-Ge was deepest among the three polymers and showed a smaller gap of 0.22 eV and 0.31 eV compared to PBDB-T-C and PBDB-T-Si, respectively. The small band gap is well-known to improve Jw/w) blend film was performed using atomic force microscopy (AFM) and electron-scanning microscopy (SEM), as depicted in Molecular-disorder-induced nanostructure variations in the BHJ are critical in optimizing photo-induced charge separation and transportation in OSCs. The surface morphology study and characterizations of PBDB-T-Ge:Y6  calculations were conducted to provide further insights into the fundamental aspects of the molecular architecture of germanium-based comonomers. All calculations were conducted with the Gaussian16 software package. Optimized geometries of all compounds were obtained at the B3LYP level of theory with the 6-31G(d) basis set for all atoms. Previous work ,40,41 shWe observed that the electron density distributions at the HOMO are highly localized on the electron-rich moiety of BDTT-Ge (M1) in the ground state while, in the excited state, it is distributed at the LUMO and highly localized in the electron-deficiency moiety BDD (M2). The transition of the electron density distributions at the HOMO and LUMO were observed from all cases of -C, -Si and -Ge.DFT calculations of Ge-based comonomer (triethyl-Ge) estimated the HOMO of −5.02 eV and LUMO of −2.36 eV with the band gap of 2.66 eV, while the band gaps for the counter-partner C-based comonomer (triethyl-C) and Si-based comonomer (triethyl-Si) were calculated to be 2.655 and 2.68 eV, respectively. The calculated electronic energy values of the HOMO, LUMO and band gap of the three co-monomers are summarized in A new 2D-conjugated donor–acceptor (D-A) copolymer (PBDB-T-Ge) was synthesized, where two reaction methods were compared to implement triethyl Ge: the conventional n-BuLi basis and Turbo–Grignard reactions. The Turbo–Grignard reaction was observed to be efficient in incorporating the group IV element to the side chain with a yield of 86%. The PBDB-T-Ge exhibited a LUMO of −3.64 eV, which was closer to that of Y6, an electron-acceptor. The gap between the LUMO values was calculated to be ca. 0.22 eV, which is sufficient to expect efficient photo-induced charge injection between the electron-donor and -acceptor in BHJ PSCs. PBDB-T-Ge and Y6 showed a complementary absorption, indicating a wide range of light-harvesting. DFT calculations on the comonomer verified that there were no significant changes in the band gap when the Ge sidegroup of the polymer replaces carbon- and silicon-base sidegroups.


Mycobacterium marinum is a prevalent nontuberculous mycobacterium (NTM)‐infecting teleosts. Conversely, little is known about mycobacteriosis in elasmobranchs, and M. marinum infection has never been reported from the subclass. This study investigated the histopathological characteristics and localization of this mycobacterium through molecular analysis of two captive sharks, a scalloped hammerhead Sphyrna lewini and a Japanese bullhead shark Heterodontus japonicus, exhibited in the same aquarium tank. We detected genital mycobacteriosis caused by M. marinum infection using molecular analyses, including polymerase chain reaction (PCR) and DNA sequencing targeting the 60 kDa heat‐shock protein gene (hsp65), and peptide nucleic acid–fluorescence in situ hybridization (PNA‐FISH) targeting the 16S rRNA gene. Both sharks showed granulomas in connective tissues of the gonads without central necrosis or surrounding fibrous capsules, which is unlike the typical mycobacterial granulomas seen in teleosts. This study reveals that elasmobranchs can be aquatic hosts of M. marinum. Because M. marinum is a representative waterborne NTM and a potential zoonotic agent, cautious and intensive research is needed to overcome a lack of data on the relationship between NTM and the aquatic environment in association with this subclass of Chondrichthyes. Peptide nucleic acid (PNA) probes have been applied to the detection of mycobacteria using clinical samples from humans  occurs circumglobally in warm‐temperate and tropical coastal seas .Here, we present the first reported cases of 22.1An approximately 10‐year‐old wild‐caught male scalloped hammerhead shark and a wild‐caught adult female Japanese bullhead shark of uncertain age were housed at the Shimane Aquarium in Shimane Prefecture, Japan. They were exhibited in a 1 million‐L tank exhibit along with other fish, including multiple teleost and elasmobranch species, and one sea turtle. Water pumped from the local sound (Sea of Japan), treated with pressure filtration, is used as the aquarium's water source. After 9 years and 9 months in captivity, the hammerhead shark presented anorexia for 1 month and was subsequently found dead in the tank. One month later, the bullhead shark was also found dead, but without any remarkable clinical signs. The captivity duration of this bullhead shark was unknown because Japanese bullhead sharks in the tank were not individually identified. Autopsies revealed marked hemocelom, multifocal congestions in the bilateral testes of the hammerhead shark and multifocal haemorrhages in the bilateral epigonal organ Figure . ApparenTo assess the microbial community distribution in the rearing environment, samples of water (1 L) and filtration sand, as well as swabs of biofilms from a wall and a water intake were provided. All samples were sent to the Laboratory of Aquatic Medicine at Nippon Veterinary and Life Science University in Tokyo.2.2Samples from the brain, alimentary canal, liver, kidney, spleen, pancreas and reproductive organs were collected from both sharks, and samples from the epigonal organ, eyes and skin were collected from the hammerhead shark. The reproductive organs from the bullhead shark were frozen on site and then fixed in 10% neutral‐buffered formalin at our laboratory; the other remaining samples were fixed in 10% formalin on site. All samples were routinely processed to create paraffin‐embedded tissue blocks, sectioned at 2‐μm thickness, mounted on frosted glass slides, stained with haematoxylin and eosin (HE), Giemsa, Gram and Ziehl–Neelsen (ZN) stains and subjected to histopathological examination.2.32.3.1Isolation of the etiological agent of mycobacteriosis was attempted from frozen organs, including the reproductive organs, kidney, spleen and liver, in both cases, and the epigonal organ from the hammerhead shark. Pretreatments were performed based on four methods, as follows: (1) non‐treatment; (2) decontamination by hydrochloric acid (HCl) solution, at a final concentration of 0.5 N , the beta subunit of RNA polymerase (rpoB) and superoxide dismutase (sodA), through the cycles described by Fukano et al. . The DNA was tested for the presence of mycobacterial DNA by PCR on the C1000TM Thermal Cycler (Bio‐Rad) using GoTaq® DNA Polymerase (Promega) and mycobacterial universal primers targeting the 16S ribosomal RNA (16S rRNA), 65 kDa heat‐shock protein , using the primer sets MU5–MU6 and PU4F–PU7Rbio for IS2404, and MU7–MU8 for IS2606 . The phylogenetic tree of the hsp65 sequences was reconstructed using the neighbour‐joining method with Kimura's 2‐parameter model, implemented in MEGA X software and evaluated further in a bootstrap analysis of 1000 replicates.Purified products were sequenced by the FASMAC Corporation using the specific primers described in Table 2.52.5.1M. marinum ATCC BAA‐535 and M. marinum JCM 17638T were used. To detect pathogens in the shark tissues, paraffin‐embedded tissue samples of the current cases, including the testes and epigonal organ of the hammerhead shark, and the ovary, uterus and shell gland of the bullhead shark, were used. As a negative tissue control, the ovary of another hammerhead shark, in which mycobacterial infection was not detected by ZN staining and by PCR assay, was also tested.To estimate the hybridizability and specificity of the designed PNA probes and to validate the PNA‐FISH procedure, mycobacterial isolates from the tissues of the current clinical cases and the reference strains 2.5.2M. marinum, Mycolicibacterium llatzerense (NJB1901), M. hodleri (NJB19061) and M. obuense (NJB19062). Each probe was designed not to cross‐hybridize with the non‐targeted genomic sequences of the other NTM candidates, and therefore, it acted as a negative control for the other three NTM species like a sense probe. The probes were chosen with regard to purine content, avoiding hairpin formations and inverted repeats. The N‐terminus of all probes were labelled with cyanine 3 (Cy3) fluorescent dye. All designed probes were custom‐synthesized by PANAGENE Inc.With the genome and partial sequence database using BLAST, and the results of sequence analyses of DNA extracted from the present samples, we designed four PNA probes Table , each of2.5.3The preparation of tissue sections and bacterial samples for FISH was performed as described in Lehtola et al.  and Lefm2.5.4Microscopy was performed with a fluorescence microscope (Keyence BZ‐X700). TRITC OP‐87764 and DAPI OP‐87762 filter sets (Keyence) were used to analyse the Cy3 and DAPI signals, respectively. Digital images were captured under the fluorescence microscope, and image analysis was performed with BZ‐H3 software (Keyence).33.1In both testes of the hammerhead shark, foamy macrophages infiltrated into the serosal connective tissues in the resorption zone mixing with large numbers of infiltrating lymphocytes Figure . NumerouAlthough it was somewhat difficult to distinguish inflammation cells since the sample was frozen after sampling, macrophage‐like cells with foamy cytoplasm infiltrated and accumulated in the ovarian connective tissue stroma of the bullhead shark Figure . ZN staiIn the hammerhead shark, in addition to mycobacteriosis, we diagnosed panophthalmitis, corneal trephination, enteritis, dermatitis, focal lymphocytic meningitis and suspected collagenofibrotic glomerulopathy. In the bullhead shark, we additionally diagnosed focal lymphocytic gastritis, focal lymphocytic interstitial nephritis, pancreatitis and necrotic splenitis. However, in both cases, no apparent pathological features of mycobacterial suspicious lesions were detected other than in the testes or ovaries.3.23.2.1In total, three AFB strains were isolated from the testis of the hammerhead shark (NJB1901) and the ovary of the bullhead shark (NJB19061 and NJB19062). NJB1901 was cultured using Middlebrook 7H11 agar without pretreatment; on day 21 after inoculation at 30°C, it formed a smooth, white colony. NJB19061 and NJB19062 were cultured using the same agar without pretreatments; on day 14 after inoculation at 25 and 30°C, respectively, each formed a smooth, yellow colony. All purified colonies of the three isolates showed rapid growth on day 3, with Middlebrook 7H11 agar, at 25°C (NJB19061) or 30°C .3.2.2Seven isolates of mycobacteria were cultured from the environmental samples. The colony characteristics of the isolates are presented in Table 3.33.3.1hsp65 gene, and from one part of the left testis (testis II) using primers to amplify the 16S rRNA, hsp65, rpoB and sodA genes. Sequence analyses indicated that M. marinum was the most probable candidate bacterial species detected in four portions of the testes  of the hammerhead shark, and in the ovary of the bullhead shark, based on genomic evidence, whereas M. llatzerense was the most probable candidate species in testis II of the hammerhead shark, based on the high degree of molecular homology  and from the ovary, using primers to amplify the hsp65, rpoB and sodA genes. The results of sequence analyses of the isolates showed that NJB1901, NJB19061 and NJB19062 were identical with M. llatzerense, M. hodleri and M. obuense, respectively , while apparent bands were observed with DNA extracted from the positive control (M. pseudoshottsii JCM15466)  did not show positive signals indicating the presence of the targeted NTM in the tissue sections of both samples. In the negative controls, no signal was detected in the tissue sections examined Figure . Also, n4hsp65 gene detected genomic evidence of nontuberculous mycobacteria (NTM) infection, with 100% nucleotide sequence identity to M. marinum. The nucleotide sequence of hsp65 gene of M. marinum shows a relatively high rate of similarity with mycolactone‐producing mycobacteria (MPM), such as M. pseudoshottsii. However, in most M. marinum strains reported previously, the insertion sequences IS2404 and IS2606 relating to mycolactone were not detected by PCR, unlike detection in MPM    (=Rhinobatos lentiginosus)  (Karsten & Rice, Mycobacteriosis cases in this subclass have rarely been reported (Anderson et al., Mycobacterium marinum is a representative NTM agent in aquatic animals; it is considered an important candidate infectious agent of aquatic zoonosis because M. marinum has been isolated from human lesions, including dermatitis (Tomas et al., M. marinum infection and the potential for zoonotic transmission from an elasmobranch species have not yet been shown. Nevertheless, the present results prove that an elasmobranch species can be a host of waterborne M. marinum and indicate that elasmobranchs, like teleosts, might act as a reservoir of M. marinum infection in humans, especially immunocompromised individuals (Streit et al., The progress of granuloma pathology is usually dependent on the duration and amount of infecting bacteria (Swaim et al., Mycobacterium spp. in association with granulomatous inflammation (Broussard & Ennis, M. marinum in the reproductive organs in the current cases clarified that mycobacteria could cause gonadal lesions in elasmobranchs as in teleosts.In teleost fishes, the ovary and testis are not major organs involved in mycobacterial infection (Novotny et al., In conclusion, our study indicates that mycobacteriosis can be suspected in elasmobranchs even without typical caseating granulomas and it should be added to potential zoonotic diseases in aquaria housing sharks.The authors have no conflicts of interest to declare.Appendix S1Click here for additional data file.


To evaluate the relationship between the fetal head‐circumference‐to‐maternal‐height (HC/MH) ratio measured shortly before delivery and the occurrence of Cesarean section (CS) for labor dystocia.This was a multicenter prospective cohort study involving four tertiary maternity hospitals. An unselected cohort of women with a singleton fetus in cephalic presentation, at a gestational age beyond 36 + 0 weeks and without any contraindication for vaginal delivery, was enrolled between September 2020 and November 2021. The MH and fetal HC were measured on admission of the patient to the labor ward. The primary outcome of the study was the performance of the HC/MH ratio in the prediction of CS for labor dystocia. Women who underwent CS for any indication other than failed labor progression, including fetal distress, were excluded from the final analysis.vs 164.5 ± 6.3 cm; P < 0.001), larger fetal HC  and a higher HC/MH ratio  compared with vaginal delivery. Multivariate logistic regression analysis showed that the HC/MH ratio was associated independently with CS for labor dystocia ; P < 0.001). The HC/MH ratio had an area under the receiver‐operating‐characteristics curve of 0.77 and an optimal cut‐off value for discriminating between vaginal delivery and CS for labor dystocia of 2.09, which was associated with a sensitivity of 0.62 , specificity of 0.79 , positive predictive value of 0.13  and negative predictive value of 0.98 .A total of 783 women were included in the study. Vaginal delivery occurred in 744 (95.0%) women and CS for labor dystocia in 39 (5.0%). CS for labor dystocia was associated with shorter MH (mean ± SD, 160.4 ± 6.6 Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.In a large cohort of unselected pregnancies, the HC/MH ratio performed better than did fetal HC and MH alone in identifying those cases that will undergo CS for labor dystocia, albeit with moderate predictive value. The HC/MH ratio could assist in the evaluation of women at risk for CS for labor dystocia. © 2022 The Authors.  abstract to view the translations.This article's abstract has been translated into Spanish and Chinese. Follow the links from the  What are the novel findings of this work?The newly described fetal head‐circumference‐to‐maternal‐height (HC/MH) ratio performs better than do fetal HC and MH alone in identifying cases that will undergo Cesarean section for labor dystocia, albeit with moderate predictive value.What are the clinical implications of this work?In an unselected population at low risk for labor dystocia enrolled within 72 h before delivery, the HC/MH ratio demonstrates a fair specificity and negative predictive value for vaginal delivery. Use of the HC/MH ratio could assist in the evaluation of women at risk for Cesarean section for labor dystocia.Labor dystocia is estimated to account for half of all primary Cesarean sections (CS)Over the years, several studies have investigated the role of maternal and fetal anthropometric characteristics, such as maternal height (MH)This was a multicenter prospective observational study conducted at four tertiary maternity hospitals, in Italy  and Germany , between September 2020 and November 2021. A non‐consecutive series of singleton pregnancies with a cephalic‐presenting fetus, gestational age (GA) ≥ 36 + 0 weeks and no contraindications for vaginal delivery were considered eligible for the study. Patients were approached on admission due to labor induction, premature rupture of the membranes or spontaneous labor. Informed consent for study participation was obtained upon enrolment. In all included cases, the fetal HC was measured on transabdominal ultrasound in the standard transventricular plane22. In detail, a protracted active phase of labor was defined as ≥ 6 cm of dilatation with ruptured membranes and failure to progress despite 4 h of adequate uterine activity or at least 6 h of oxytocin administration with inadequate uterine activity. Arrest of dilatation in the first stage requiring CS was diagnosed following two more hours of oxytocin administration with no cervical change. A diagnosis of arrest of labor in the second stage was made in the event that the duration of the active phase was at least 2 h in parous women and 3 h in nulliparous women on epidural analgesia, or 1 h and 2 h in parous and nulliparous women without epidural analgesia, respectively.A common protocol for labor management was shared across the participating units during the study period. Labor dystocia was defined based on the American College of Obstetricians and Gynecologists (ACOG)/Society for Maternal–Fetal Medicine (SMFM) recommendations for the safe prevention of primary CSAccording to the local protocol of the participating units, women diagnosed with labor dystocia underwent clinical examination by the senior obstetrician responsible for patient care. Obstetric intervention for labor dystocia was performed when the criteria for arrest of dilatation or arrest of labor in the second stage were fulfilledClinical data including maternal age, ethnicity, parity, height and body mass index (BMI), as well as GA at inclusion, mode of delivery and indications for CS, and neonatal outcomes, including birth weight, umbilical artery pH and 5‐min Apgar score, were extracted from patient case notes. The HC/MH ratio was computed as the ratio between the measurement of the HC, expressed in mm, and that of the MH, expressed in cm. All information was collected after birth using a dedicated form. Data were recorded and stored in a Microsoft Excel secured pseudonymized database , which was accessible only to members of the research team. The primary outcome of the study was the relationship between the HC/MH ratio within 72 h before birth and the occurrence of CS for labor dystocia.t‐test or the Mann–Whitney U‐test was used as appropriate for continuous variables. Results are presented as n (%), mean ± SD or median (range). Logistic regression analysis was used to control for potential confounding variables. The performance of MH, HC and the HC/MH ratio in predicting CS for labor dystocia was determined using receiver‐operating‐characteristics (ROC)‐curve analysis. The DeLong method was used for the comparison of areas under the ROC curve (AUCs). P < 0.05 was considered statistically significant. The study was approved by the local ethics committee of each participating unit and was reported in accordance with strengthening the reporting of observational studies in epidemiology (STROBE) guidelinesStatistical analysis was performed using Statistical Package for Social Sciences (SPSS) version 22 . The Kolmogorov–Smirnov test was used to assess the normality of the distribution of the data. The chi‐square test was used to compare categorical variables and Student's Overall, 836 women were eligible over the study period. Of these, 15 were excluded due to failed induction of labor and 38 due to CS for suspected intrapartum fetal distress, leaving 783 cases for data analysis , higher frequency of nulliparity  and epidural use , higher GA at inclusion , larger fetal HC , higher HC/MH ratio , higher birth weight  and shorter MH , compared with vaginal delivery.The demographic and perinatal characteristics of the study population overall are shown in Table P < 0.001), MH ; P < 0.001), fetal HC ; P < 0.001), parity , P = 0.04) and epidural use ; P = 0.03) were associated independently with CS for labor dystocia (Table P < 0.001) and 0.71 , respectively, while the HC/MH ratio had the highest AUC ; P < 0.001). This was higher than that of the MH and of the fetal HC alone  , 2.65 ; This study demonstrates that the HC/MH ratio is associated independently with CS for labor dystocia. The HC/MH ratio had the greatest accuracy in identifying cases that would undergo CS for labor dystocia, albeit with poor predictive value in our population of women at low risk for labor dystocia leading to CS.Several studies suggest an association between MH and mode of delivery, including CS for labor dystociaThe influence of fetal size on labor outcome has also been studied extensivelyThe present study suggests that predictive parameters or models aiming to identify those laboring women at risk for labor dystocia leading to CS should ideally include maternal and fetal anthropometric characteristics.Our results demonstrate a fair specificity and NPV of the HC/MH ratio, meaning that women with a HC/MH ratio below the index threshold have a good chance of delivering vaginally. Nonetheless, our data indicate that the HC/MH ratio has a poor sensitivity and PPV for CS for labor dystocia. On one hand, this can be explained by the fact that the size of the fetus and the pelvis do not represent the only determinants of obstructed labor, as malposition, cephalic malpresentation and asynclitism are among the factors which may cause a relative mismatch between size of the fetus and that of the birth canalth percentile for the given GAMoreover, the HC/MH ratio may also support obstetricians evaluating women at risk for fetal macrosomia close to term. A randomized controlled trial demonstrated that labor induction before 39 gestational weeks improved labor outcome in women at risk for fetal macrosomia based on an EFW above the 95The strengths of this study include its evaluation of a novel parameter and the prospective enrolment of a large cohort of cases. The HC/MH ratio demonstrated fair specificity and NPV for identifying cases with a good chance of vaginal delivery, which may have potential utility in the context of labor dystocia and the antenatal counseling of patients with a large‐for‐gestational‐age fetus. Another advantage of the parameter is that it can be quantified easily and quickly, and hence be readily available, even in emergency settings.Conversely, the non‐consecutive enrolment of patients could be seen as a limitation of the study findings. Furthermore, our results may not be generalizable to all centers, given that the study was conducted across four academic centers sharing a common protocol for labor management. The inclusion of the fetal scalp in the measurement of fetal HC could be also seen as a limitation, as it is not the standard recommendationIn a large cohort of women at low risk of labor dystocia enrolled within 72 h before delivery, the HC/MH ratio performed better than did fetal HC and MH alone in identifying those cases that will undergo CS for labor dystocia, albeit with poor predictive value. Further studies are needed to evaluate the performance of the HC/MH ratio in high‐risk settings, such as in the event of labor dystocia or antenatal suspicion of fetal macrosomia.


Inguinal hernia repair is one of the most commonly performed operations in general surgery. A total of 130.000 inguinal hernia repairs are performed yearly in Italy, and approximately 20 million inguinal hernias are treated worldwide annually. This report represents the trend analysis in inguinal hernia repair in Italy from a nationwide dataset for the 6-year period from 2015 to 2020.Based on regional hospital discharge records, all the inguinal hernia repairs performed in public and private hospitals in Italy between 2015 and 2020 were reviewed based on diagnosis and procedure codes. For the aim of this study, data from the AgeNas  data source were analyzed.Elective inguinal hernia repairs outnumbered urgent operations over the 6-year study period, ranging from 122,737 operations in 2015 to 65,780 in 2020 as absolute numbers, and from 87.96 to 83.3% of total procedures in 2019 and 2020 respectively, with an annual change ranging from − 66.58%, between 2020 and 2019, to − 2.49%, between 2019 and 2018 .This large-scale review of groin hernia data from a nationwide Italian dataset provides a unique opportunity to obtain a snapshot of open groin hernia repair activity. More specifically, there is a trend to perform more elective than urgent procedures and there is a steady decrease in the amount of open hernia repairs in favor to laparoscopy.The online version contains supplementary material available at 10.1007/s10029-023-02902-z. Inguinal hernia repair is one of the most commonly performed operations in general surgery. A total of 130,000 inguinal hernia operations are performed annually in Italy, and approximately 20 million are treated worldwide .The choice of surgical technique for inguinal hernia repair remains controversial. Quality assessments of inguinal hernia repair have previously been conducted using two different methods: either through analyzing dedicated regional/national databases (DD) –7 or revAnalyzing the data from the AgeNas  data source, the present study aimed to offer the first analysis of open inguinal hernia repairs in Italy covering the 6-year period from 2015 to 2020.Using the Hospital Discharge records regional Databases (HDD), all inguinal hernia repairs carried out in public and private hospitals in Italy in patients over 18 years between 2015 and 2020 were retrieved based on diagnosis and procedure codes. The HDD collected clinical and administrative informations regarding all hospital admissions for patients discharged from any hospital in Italy. The AgeNaS led the management and analysis of data. All hospital admissions for inguinal hernia repair that occurred between 2015 and 2020 were analyzed. The data source included patient demographic data  and admission and discharge data with up to six discharge diagnoses , as well as living status at discharge . It did not provide information on size of the hernia defect, individual surgical expertise, number of surgeons or number of procedures performed per surgeon. It gave information on the use of a mesh for inguinal hernia repair and allowed registration of all readmissions regardless of the primary site of surgery and type of institution (public or private). It did not provide information on hernias treated as outpatient procedures.The validity of this data system with respect to the type of surgical procedure had been already established for several surgical procedures –8. This Informations on consecutive admissions for inpatient inguinal hernia repair in Italy from 2015 to 2020 for patients with a diagnosis of unilateral and bilateral inguinal or femoral hernia were retrieved from the database.Sex, age and preexisting comorbidities were analyzed. Comorbidities were further divided into general comorbidities and neurological comorbidities. The presence of bowel obstruction at the time of the operation was also registered. The search coding system is summarized Table The discrimination between laparoscopic or open groin hernia repair was identified as the presence or the absence of the ICD-9-CM code 54.21 (laparoscopy). The association of inguinal hernia repair with other surgical procedures was defined by the wording "AND cholecystectomy" or "AND adhesiolysis". The presence of an association with another type of surgical procedure other than cholecystectomy and adhesiolysis was grouped as "other". The analysis included total hospital stay, readmission rate within 30 days from the operation, early mortality rate (during the index admission or within 30 days from the operation), late mortality rate  and 30-day morbidity.Data concerning in-hospital and 30-day mortality were obtained linking the hospital discharge records and the regional registry of mortality through unique patient codes. Complication rates were defined using pre-defined codes either as primary diagnosis or among the first five secondary diagnoses Table .Data were processed using the MedCal statistical package (version 12.5). Qualitative variables were summarized by absolute numbers and percentages, while quantitative variables were described by the median and standard deviation (SD) or range min–max, for normally or non-normally distributed variables respectively.Statistical analysis was performed using Student’s t test and the Cochran Armitage test for trends, as appropriate. A two-tailed p value < 0.05 was considered statistically significant.st of December for the period from 2015 to 2020, as reported by the Italian National Institute of Statistics (ISTAT)  per 100,000 population was calculated, assessing the changes in the considered period. The sample size was the Italian population, reported by region, according to the average yearly population on the 31), with an annual change ranging from a minimum of − 2.26% between 2016 and 2015 to a maximum of – 60.63% between 2019 and 2020 . The mean annual change was—2.85%  from 2015 to 2019 . Mean age ranged from 59 ± 20 years in 2015 to 61 ± 19 years in 2020. Overall comorbidity rates were stable during the study period. More details are shown in Fig. 2Of this cohort, 0.02% patients (n = 171 over time) presented with bowel obstruction.A total of 757,558 inpatient inguinal hernia repairs were performed in Italy from 2015 to 2020. Of these, 723,633 (95.5%) were open repairs , but, excluding 2020, the numbers of open procedures decreased steadily with a mean annual change of − 2.75%   to a maximum of 7,146 . The mean annual change was 8.26% (95%CI 2.35%—14.17%) from 2015 to 2019, while a mean annual change of − 0.98% (95%CI: − 19.68%—17.72%) was observed over the whole time period  . The mean annual decrease was – 1.95%  from 2015 to 2019  inguinal hernia repairs were associated with additional surgical procedures between 2015 and 2020. Of these, 2,914 (0.43%) were associated with cholecystectomy, 1,477 (0.20%) with adhesiolysis, and 9,601 (1.42%) with other types of open surgical procedures. The mean annual change of this trend was − 1.23% (95%CI − 4.45%—0.55%) if considering the total number of the associated procedures.The trend of associated cholecystectomies varied with a mean annual decrease of – 11.32  over time, and of − 3.12%  from 2015 to 2019. The association of open inguinal repair and adhesiolysis demonstrated a mean annual change of 3.55% , decreasing to 2.85%  from 2015 to 2019.A total of 3461 complications were registered, ranging from 362 in 2020 to 706 in 2015 as absolute numbers and from 0.43% to 0.52% in relative frequencies of overall procedures in 2018 and 2015, respectively. Complication rates decreased over time, with an annual change ranging from − 6.4% between 2016 and 2015 to − 65% between 2020 and 2019 . The mean annual change was − 9.18%  from 2015 to 2019 . The mean annual change was − 1.96%  between 2015 and 2019  within 30 days of the operation and 1153 after 30 days (42.7%), with a mean annual change ranging from − 13.3% between 2016 and 2015, and 0.25% between 2020 and 2019 . The mean annual change was − 4.60%  from 2015 to 2019.Early mortality ranged from 194 in 2016 to 254 in 2015 as absolute frequencies and from 0.48% to 0.57% in relative frequencies of overall procedures in 2018 and 2020 respectively, with an annual change ranging from − 30.9% between 2016 and 2015 to 10.29% between 2018 and 2017 . The mean annual decrease was − 5.92%  from 2015 to 2019.Late mortality ranged from 168 in 2020 to 211 in 2016 as absolute frequencies and from 0.14% to 0.21% in relative frequencies of overall procedures in 2017 and 2020 respectively, with a mean annual change ranging from − 16.76% between 2018 and 2017 to 13.39% between 2018 and 2017 . The mean annual change was − 4.26%  if considering from 2015 to 2019. Figure 6Concerning the type of healthcare provided, the number of procedures performed in inpatient admission ranged from 37,093 in 2020 to 61,952 in 2015 and from 43.69% to 48.38% of overall procedures in 2019 and 2020 respectively, with a mean annual change ranging from a maximum of − 44.4% between 2020 and 2019 to a minimum of − 2.56% between 2018 and 2017  . The mean annual change was − 2.05% (95%CI − 4.43% – 0.33%) between 2015 and 2019. Figure 7.Mean hospital stay was 5 days between 2015 and 2019 and 4 in 2020Regional data are provided as supplemental material.This study provides a picture of the epidemiological trends over a six-year period for open inguinal hernia repair surgery in Italy. Although there have been previous studies involving ‘‘hernia audits’’ in regional populations , 9, 10, Currently, however, the treatment of inguinal hernias is not standardized across Europe and the choice of the surgical approach remains debatable and subject to bias, including individual surgeon experience and preference. The open approach remains prevalent in both elective and emergency settings worldwide –19, consThe results reported in both elective and emergency inguinal hernia repairs performed between 2015 and 2020 are consistent with the literature , 20, 21.Primarily, there was a gradual and significant decline in the number of open hernia repairs both in elective (p < 0.0001) and emergent setting (p < 0.0001). This decrease coincided, in part, with the increase in use of laparoscopy, and, additionally, with the gradual decline of inpatient hospital stay for groin repair (p < 0.001). Similarly, a reduction in the number of admissions for inpatient surgery was noted (p < 0.0001). Other international studies have noted a similar reduction in the use of inpatient procedures due to several factors, including broader use of local anesthesia for elective inguinal hernia repair , which hThe reduction in inpatient directed care, probably coincided with an increase in the ambulatory service. The choice of ambulatory service could be further investigated; however, other authors have reported the steady implementation of ambulatory services into their routine practice , 22.The otherwise well-established ambulatory set-up for elective groin hernia repairs , 26–28 hComplications and readmission rates did not significantly change in the considered period, when the decrease observed reflects the overall decrease of the overall procedures. A large study conducted in Denmark identified a hospital readmission rate for hernia of 1.8% . In our Our data also demonstrated that the number of days spent in hospital for elective admissions did not change over the decade considered. A likely explanation for this is that, despite gradual improvements in healthcare provision, the greater selection of patients referred for elective hospital admissions over the years has altered the case mix.In-hospital mortality remained relatively constant. A significant reduction in both early and late mortality rates was observed across the whole period but more significantly decreased in the period between 2015 and 2019. This trend reflected the drastic drop of hernia cases in 2020.The global necessity to adapt to the unprecedented challenges associated with the rapid spread of Sars-Cov-2 in early 2020 resulted in a significant disruption in all elective and benign surgery. Over a few short weeks, surgeons witnessed a dramatic change in their practice, with a rapid decrease in the number of surgical interventions reported worldwide .General surgeons were particularly affected due to the wide variety of procedures they perform, many of which are carried out routinely, many in outpatient settings .A nationwide survey  highlighThe limit of this paper is intrinsic in the search methodology and the data source. The system involves AD that collect data from all public and private hospitals of a given region or country, obtaining such information from hospital admissions and discharge charts. The main advantage of the AD system is the ability to retrospectively record almost 100% of the surgical procedures performed using the coding system based on the ICD-9-CM. Additionally, AD are readily accessible at low cost. However, they often result in inaccurate data registration as well as inexact follow-up and recurrence/complication information; additionally, they often lack case mix adjustment.Inguinal hernia repair could be used as an indicator of the surgical quality offered in different health institutions and countries, thereby establishing a scientific basis from which to make decisions, given the rate with which day surgery and outpatient surgery are developing. Such solutions could help improve elective surgery efficiency, better sustainability across healthcare services, and offer elective patients shorter waiting times. Finally, this dataset can be used as a tool to help present validated recommendations for the management of patients with groin hernias, with a potential to implement a registry of ambulatory hernia repairs, following the example of other European countries.This large-scale review of inguinal hernia data from a nationwide Italian dataset provides a unique opportunity to obtain a snapshot of inpatient open hernia repair activity. Generally, a steady substantial decrease of open procedures could be observed over the six-year period taken into consideration and a simultaneous increase of laparoscopic adoption, in the setting of a global reduction observed in 2020 due to the COVID pandemic. The trend of day care surgeries remained stable throughout the whole period.Supplementary file1 (DOCX 19 KB)Supplementary file2 (DOCX 25 KB)Supplementary file3 (DOCX 28 KB)Supplementary file4 (DOCX 944 KB)Supplementary file5 (DOCX 630 KB)Supplementary file6 (DOCX 942 KB)Supplementary file7 (DOCX 941 KB)Supplementary file8 (DOCX 19 KB)Below is the link to the electronic supplementary material.