Meiotic recombination results in the exchange of genetic material between homologous chromosomes. Recombination rate varies between different parts of the genome, between individuals, and is influenced by genetics. In this paper, we assessed the genetic variation in recombination rate along the genome and between individuals in the pig using multilocus iterative peeling on 150,000 individuals across nine genotyped pedigrees. We used these data to estimate the heritability of recombination and perform a genome-wide association study of recombination in the pig.RNF212, SHOC1, SYCP2, MSH4 and HFM1.Our results confirmed known features of the recombination landscape of the pig genome, including differences in genetic length of chromosomes and marked sex differences. The recombination landscape was repeatable between lines, but at the same time, there were differences in average autosome-wide recombination rate between lines. The heritability of autosome-wide recombination rate was low but not zero . We found six genomic regions that are associated with recombination rate, among which five harbour known candidate genes involved in recombination: Our results on the variation in recombination rate in the pig genome agree with those reported for other vertebrates, with a low but nonzero heritability, and the identification of a major quantitative trait locus for recombination rate that is homologous to that detected in several other species. This work also highlights the utility of using large-scale livestock data to understand biological processes.The online version contains supplementary material available at 10.1186/s12711-021-00643-0. Meiotic recombination results in the exchange of genetic material between homologous chromosomes. After chromosomes have paired up and duplicated, they can break and exchange segments of chromosomes. Such recombination events are not evenly distributed along the chromosomes and result in a variable landscape of recombination rate across the genome, with peaks and troughs.The landscapes of recombination rate among vertebrate genomes share several features. Recombination rate tends to be lower in the middle of chromosomes and higher near their ends . RecombPRDM9 gene that displays rapid evolution of its DNA-binding zinc finger array, which suggests that the pig genome contains PRDM9-dependent recombination hotspots [RNF212, MSH4, REC8 and PRDM9 .Analysis of the genetic basis of recombination rate requires estimates of recombination rate from a large number of related individuals. Recombination rate can be estimated by phasing genotypes in pedigrees , 34–36, In this work, we assessed genetic variation in recombination rate along the genome and between individuals in the pig using multilocus iterative peeling on 150,000 individuals across nine genotyped pedigrees. We used these data to estimate the heritability of recombination and perform a genome-wide association study of recombination in the pig.We analysed the landscape of recombination rate in the genome of nine lines of pigs from a commercial breeding programme. We performed six analyses: (1) an analysis of the average number of recombination events on each chromosome (the genetic length of chromosomes) to estimate between-sex and between-line differences in genetic length and then compared these estimates to previously published estimates; (2) an analysis of the distribution of recombination events along the chromosomes (landscapes of recombination rates) to estimate between-line and between-sex differences; (3) estimation of the correlation between recombination rate and DNA sequence features that are known to correlate with recombination rate; (4) estimation of pedigree-based and genomic heritabilities of recombination rate; (5) a genome-wide association study to detect chromosomal regions associated with recombination rate; and (6) a simulation to test the accuracy of the inference method.The data consisted of single nucleotide polymorphism (SNP) chip genotype and pedigree data from nine commercial pig breeding populations of varying sizes with overlapping generations from the Pig Improvement Company breeding programme, covering 20–30 generations. These lines represent broadly-used populations, including animals of Large White, Landrace, Duroc, Hampshire and Pietrain heritage. Table Multilocus iterative peeling was used to estimate the number and location of recombination events in each individual –40. MultThe joint distribution of segregation values depends on chromosome length (in cM). To estimate chromosome length, we started with a length of 100 cM (on average 1 recombination per chromosome), and then refined this estimate in a series of steps. At each step, we calculated the expected number of recombination events for each individual at each locus, and set the chromosome length based on the average population recombination rate. This step was repeated four times. In preliminary simulations, we found that the estimates of chromosome length converged after four iterations and that the estimates of recombination rate for target individuals were insensitive to the assumed chromosome length.After estimation of recombination rates, we filtered the data by removing individuals without genotyped parents and grandparents in order to focus on individuals with high-quality estimates of recombination rate. An additional seven individuals with extremely high average recombination rate estimates (> 5 cM/Mbp) were also removed. These filtering steps reduced the number of pigs to 145,763. Table To compare the recombination landscapes of the nine lines, we calculated between-line pairwise correlations of the estimated recombination rates at each marker interval, within each sex. To compare the recombination landscapes between females and males, we calculated the correlation of recombination rates between each pair of SNPs between sexes within each line. We compared genetic map lengths between lines using a linear model by fitting the number of recombination events observed on a chromosome as response variable and fixed effects for each line and chromosome. Lines were compared separately for each sex. To compare the recombination landscapes obtained in our study to results in the literature, we plotted the genetic map length for each chromosome against published genetic map lengths .https://bioconductor.org/packages/Biostrings) and TFBSTools version 1.22.0 [PDRM9 consensus motif CCNCCNTNNCCNC [PRDM9 motif; and (4) the count of the CCCCACCCC motif, which was the most strongly associated motif with recombination rate in the pig reported by[To investigate the relationship of local recombination rates with genomic features, we divided the autosomal part of the Sscrofa11.1 genome  into 227n 1.22.0  in the RNTNNCCNC ; (3) theported by.2His2 Zinc Finger predictor of [In order to predict the porcine PRDM9 motif, we used the online Cysictor of  and the ictor of  as pig Phttp://www.repeatmasker.org) [We used repeat data from RepeatMasker (ker.org)  from theTo find putative pericentromeric regions, we used the inferred centromere positions from . For chrWe estimated the narrow-sense heritability of the autosome-wide recombination rate per Mb of parents that had genotyped offspring using the animal model in the MCMCglmm package  version We performed genome-wide association studies of autosome-wide recombination rates using a hierarchical linear mixed model in the RepeatABEL package  version ervative , 52) as ervative . To do tervative  and repo−8.We performed a meta-analysis of the genome-wide association studies by combining the lines but analysing sexes separately, using the meta R package 4.17-0 . It is bTo demonstrate that the method for estimating recombination rate by multilocus peeling works, we tested it first on a simulated dataset with features similar to the real data. We simulated genotype data using the AlphaSimR 0.10.0 software  for one PRDM9 consensus motif; (4) the heritability of recombination rate was on average 0.07 for females and 0.05 for males; and (5) six regions of the genome were associated with recombination rate, of which five contained known candidate genes, i.e. RNF212, SHOC1, SYCP2, MSH4 and HFM1.Our results show that: (1) the genetic length of chromosomes differs between sexes and lines; (2) the recombination landscape is similar between lines but differs between sexes; (3) as previously reported, the local recombination rate is correlated with GC content, repeat content, the CCCCACCCC sequence motif, but we do not confirm the previously described correlation with the In the simulation analysis, we found that multilocus iterative peeling could estimate the number of recombination events per individual with an accuracy of 0.7 for dams and 0.5 for sires, as well as the average recombination landscape along a chromosome, but with a tendency to overestimate the genetic length.The genetic length of chromosomes differed between lines and sexes. Figure Our estimates of the genetic lengths of chromosomes were comparable to previously reported estimates, but tended to be longer. We found that females had a higher recombination rate, except on chromosome 1, for which the male recombination rate was higher, and on chromosome 13, for which the recombination rate was similar for both sexes. This confirms previous results .The pattern of the recombination landscape was similar between lines but differed between the sexes. Figure PRDM9 consensus motif CCNCCNTNNCCNC , negatively correlated with counts of the predicted porcine PRDM9 motif , but moderately positively correlated with counts of the CCCCACCCC motif , which was previously reported to be enriched in high recombination regions in the pig genome [Figure Figure Genome-wide association studies, performed separately for each line, revealed three regions of the pig genome that contained SNPs that were associated with the autosome-wide recombination rate. Figure The meta-analysis of the genome-wide association studies detected two of the above-mentioned regions (on chromosomes 8 and 17) and three other regions that were not significant in the separate analyses. Figure We tested the accuracy of the estimated recombination parameters by analysing a simulated dataset. Figure PRDM9 consensus motif; (2) we have shown that recombination rate in the pig is genetically variable and associated with alleles at the RNF212, SHOC1, SYCP2, MSH4, and HFM1 genes; and (3) we have demonstrated that multilocus iterative peeling is a compelling method for assessing recombination landscapes from large genotyped pedigrees, but that it tends to overestimate genetic map length.In this work, we have estimated the variation in recombination rate within the genome and between individuals in nine genotyped commercial pig breeding populations using multilocus iterative peeling. In this section, we discuss three main results: (1) we have confirmed the known features of the pig recombination landscape, but not the previously described correlation with the PRDM9 consensus binding motif and with density of some repeat classes differed from previously reported estimates.Our results recover some of the known features of recombination in the pig genome, including the relative chromosome genetic lengths and the marked sexual dimorphism. However, there are two notable exceptions: (1) our estimates of the overall genetic length of chromosomes are greater, and (2) correlations of recombination rates with density of the Regarding exception (1), we obtained total genetic map lengths that ranged from 18.5 to 21.7 M for males and 22.3–25.9 M for females, whereas Tortereau et al.  found sePRDM9 consensus binding motif was lower than previously reported and that the correlation between recombination rate and density of the porcine PRDM9 motif estimated from the pig PRDM9 amino acid sequence was negative. Because the PRDM9 protein targets recombination events to particular regions, thus determining the locations of a subset of recombination hotspots, the positive correlation between recombination rate and the consensus PRDM9 motif previously detected by Tortereau et al. [Regarding exception (2), we observed that the correlation between recombination rate and density of the u et al.  is biolou et al. . Furtheru et al. . Fundameu et al.  or by hiu et al. ). Thus, Associations of recombination rate with density of transposable elements varied with the type of transposable elements. We found an overall negative correlation of recombination rate with DNA repeats, in line with estimates reported for other species . The negRNF212 gene, for which a paralog has also been associated with recombination rate in deer [SHOC1 gene, which is essential for crossover formation and proper synapsis  [SHOC1 has not been associated with genetic variation in recombination rate before, it interacts with TEX11, which is associated with genome-wide recombination rates in mice [SYCP2 gene, which is required for assembly of the synaptonemal complex that connects homologous chromosomes [MSH4 gene, which is essential for proper chromosome pairing during meiosis [HFM1 gene, which is required for crossover formation [RNF212, SHOC1, SYCP2, and MSH4 are among those recombination-associated genes that have been found to evolve rapidly within mammals [Our results on variation in recombination rate in the pig genome agree with the general results in vertebrates, with a low but non-zero heritability and associated genomic regions that overlap with known meiosis-related candidate genes. In particular, these candidate genes are involved in the process that determines whether a double strand break resolves as a crossover or as a non-crossover. The significant region on chromosome 8 is homologous to regions that have been identified to be associated with recombination rate in humans –61, catt in deer , 29. The in deer . The locmeiosis) . While S in mice , 64. Theomosomes . One of  meiosis , 67 and  meiosis , 27. Finormation  and is aormation . The gen mammals .SHOC1 gene. For the significant region on chromosome 6, MSH4 is about 130 kb away from the most significant SNP in the female meta-analysis and is between the two most significant SNPs in the male meta-analysis. For the significant region on chromosome 4, HFM1 is about 260 kb away from the most significant SNP in the female meta-analysis. Finally, for the significant region on chromosome 17, the candidate gene SYCP2 is 13 kb away from the most significant SNP but it also contains the SPO11 gene, which is located about two Mb away from the most significant SNP. SPO11 encodes a key enzyme for creating the programmed double-strand breaks that initiate recombination [While these genes are suggestive candidates, one should keep in mind that the identified associated regions are large and overlap many genes. As shown in Fig. bination  and thatbination .Our results on recombination rate in the pig genome do not fully agree with those of a recent study by Lozada-Soto et al. , who alsWe observed differences in recombination rate between lines, which may be due to genetic differences. Given that livestock populations have relatively small effective population sizes, and assuming that variation in recombination rate has a rather simple genetic architecture, differences in recombination rate between lines might very well be due to genetic differences that have become fixed by chance. At the same time, all the lines studied here showed evidence of comparable genetic variation in recombination rate, and there was evidence that the major quantitative trait locus for recombination rate on chromosome 8 segregates in most lines.TEX11, a meiosis gene that evolves rapidly in vertebrates [One limitation of our study, and a possible avenue for future research, is that the study does not include the X chromosome; to do this a further development of our recombination inference method would be required. Both the association study and the estimation of recombination rates pertain only to the autosomes. The pig X chromosome is known to display regional variation in recombination rate, including one long “cold spot” of very low recombination rate , and diftebrates  and is atebrates , 64.A higher recombination rate could be beneficial for breeding, because it would reduce linkage disequilibrium between causative variants and release genetic variance. Simulations have suggested that a substantial increase in the genome-wide recombination rate could increase genetic gain . Based oUsing the distribution of estimated genome-wide recombination rates for the males in the largest line, the mean was 0.904 cM/Mb. If we selected the 10, 20 or 30% individuals with the highest recombination rate, the mean of the selected individuals Second, we estimated the increase in recombination rate if the favourable allele for the major quantitative trait locus on chromosome 8 that we detected in most of the lines was fixed. Again, using estimates from the largest line, the estimate of the additive effect of the locus was 0.0271 cM/Mb  and the frequency of the favourable allele was 0.332 . Thus, fixing this locus would increase the recombination rate by Compared to the simulation results of , which sIn this study, we have used multilocus iterative peeling to estimate recombination rates. Inferring recombination events and imputing genotypes simultaneously allows the use of large datasets without requiring high-density genotyping. However, the genotyping density does put a limitation on the resolution at which recombination events can be localised on the genome. In our simulation study, we found that multilocus iterative peeling could estimate the number of recombination events per individual with an accuracy of 0.7 for dams and 0.5 for sires, and the average recombination landscape along a chromosome. This is consistent with our analysis of the pig genome, for which we confirm previously known features of its average recombination landscape. However, the simulation results also show that our method overestimates the total genetic map length, which is also evident from comparisons with previously published estimates .Multilocus iterative peeling is a compelling technique for estimating recombination rate in large pedigree populations: it scales well to massive livestock pedigrees , does not require pre-phasing of the data, and handles individuals that may be genotyped on a range of platforms without requiring non-overlapping variants to be imputed beforehand. We evaluated the accuracy of imputation with multilocus iterative peeling with simulated data based on four of the pedigrees included in this study and found that it was high for individuals that were genotyped with at least 10,000 SNPs .One of the major downsides of multilocus iterative peeling is that it requires multiple generations of genotyped individuals to accurately phase and impute genotypes, and to estimate the recombination rate. Although this information may be available in pig or chicken breeding programmes , 78, andBy analysing 150,000 individuals from nine pig pedigrees, we were able to recover broad-scale patterns in genetic map lengths, landscapes of recombination rates, and sex differences in recombination rates. We also found that recombination rate had a low, but non-zero heritability and, by performing a genome-wide association study, we detected six regions that are associated with recombination rate. Our results highlight that large-scale pedigree and genomic data, as routinely collected in many closely-managed populations, can be used to infer and understand recombination and variation in recombination rate along the genome.Additional file 1: Figure S1. Predicted porcine PRDM9 binding site from the amino acid sequence, with reverse complement and the canonical PRDM9 motif for comparison. The sequence logos were generated with the online predictor of [ictor of .Additional file 2: Table S1. Male map of the landscape of pig recombination rate in 1-Mb windowsAdditional file 3: Table S2. Female map of the landscape of pig recombination rate in 1-Mb windows.Additional file 4: Table S3. Sex-averaged map of the landscape of pig recombination rate in 1-Mb windows.


The goal of this study was to investigate the incidence of departures from routine care at the postoperative week 1 (POW1) visit following uneventful resident-performed cataract surgery in asymptomatic patients who had a normal postoperative day 1 (POD1) examination., POD1 visit note, and POW1 visit note were evaluated and variables were recorded. Exclusion criteria consisted of any complications that would have necessitated close follow-up and a POW1 visit, whether discovered preoperatively, intraoperatively, at the POD1 visit, or leading up to the POW1 visit. The primary outcome measure was the incidence of unanticipated management changes at the POW1 visit following resident-performed cataract surgery.A retrospective chart review of phacoemulsification surgeries performed by the senior resident class at Montefiore Medical Center between June 20, 2018 and April 1, 2019 was performed. The most recent preoperative visit note, operative reportThe charts of 292 surgical cases of 234 patients that underwent phacoemulsification with intraocular lens implantation were reviewed. 226 cases (77%) had an uncomplicated pseudophakic fellow-eye history, with a routine surgery, and POD1 examination. 19 of these patients had symptomatic presentations at the POW1 timepoint, and an additional 30 had no POW1 visit at all. In total, 177 cases were included in the study, and only 4 of these cases (2.3%) had an unexpected management change at the POW1 visit.Asymptomatic patients who underwent uncomplicated cataract surgeries performed by resident surgeons followed by a routine POD1 visit had a low incidence of unexpected management changes at the POW1 visit. These results suggest that regularly scheduled POW1 visits could potentially be omitted for patients deemed to be at low risk for complications, and instead performed on an as-needed basis. In addition to providing care for large patient populations, academic teaching hospitals have the added obligation of training the future generation of physicians. Notwithstanding this additional responsibility, teaching hospitals, which can be defined by the presence of residents in training, have been shown to fare better in overall quality of care than nonteaching hospitals , 2. PrevCataract extraction with intraocular lens (IOL) implantation is the most frequently performed operation covered under the Medicare Part B insurance plan . Given tFollowing the performance of cataract surgery, physicians typically schedule their patients for three routinely timed follow-up visits: one at postoperative day 1 (POD1), another at postoperative week 1 (POW1), and a final visit 1 month after the surgery. While the first and third visits are deemed to be essential check-points for the monitoring of adequate progression following the procedure, the utility of the visit at POW1 has frequently been brought into question. Refraining from explicitly articulating the informally agreed upon 3-visit regimen, the Cataract in the Adult Eye Preferred Practice Pattern set forth the recommendation that patients have their first visit within 48 h in order to ensure appropriate recovery from surgery and an additional visit between 1 and 4 weeks after surgery for a more stabilized manifest refraction measurement [Although there is limited evidence for the ideal postoperative visit schedule, we are able to quantifiably analyze the value of the POW1 visit by examining the rate at which departures from routine postoperative care occur. A POW1 visit is indisputably required if there was a complicated cataract extraction or a clinically significant presentation that already required careful monitoring by POD1. However, whether it is cost-effective for an asymptomatic patient with an uncomplicated ocular history, uncomplicated surgery, and an uneventful POD1 visit to come back for a week 1 check-in, is important to understand on the resident level at academic teaching hospitals. In doing so, we can achieve a more data-driven post-operative schedule that has the ability to reduce costs for both the healthcare system and for patients who are at a low risk for any potential unanticipated management change.Departures from routine care, or unexpected management changes, can be defined as either a requirement for additional procedures, a need for referral to a specialty service, or a necessary change in postoperative drops. If a specific subset of patients has a minimal likelihood of requiring such an unexpected management change, then the removal of the week 1 visit as part of the routine protocol could have a substantial impact on healthcare costs. Instead of scheduled visits, these patients can be provided with adequate perioperative education along with an available line of communication to schedule an unplanned visit if any symptoms or complications do develop. Further, although the occurrence of complications is something that is inevitable, the timing of their pathologic manifestation is highly variable. It is therefore difficult to construct a schedule with visits whose purpose it is to detect these potential complications in a pre-symptomatic cohort of patients. A previous study has already shown that the POW1 visit in attending-performed cataract extractions has little function in a specific cohort of patients, shedding light on the potential for a more standardized and value-driven follow-up schedule . The purThis is a retrospective chart review of 234 patients who underwent cataract extraction by phacoemulsification with intraocular lens implantation at the resident cataract surgery service of Montefiore Medical Center between June 20, 2018 and April 1, 2019. The study was done in accordance with the Health Insurance Portability and Accountability Act. All experimental protocols were approved by the Institutional Review Board of Montefiore Medical Center and the Albert Einstein College of Medicine, and a waiver of informed consent was granted given the retrospective nature of our study.The electronic medical records of Montefiore Medical Center were searched to identify patients who underwent cataract extraction according to the Classification of Procedural Terminology codes 66,982  and 66,984 (extracapsular cataract extraction with insertion of intraocular lens prosthesis). A chart review evaluating the most recent preoperative visit note, the operative report, and visit notes from POD1 and POW1 was then conducted and variables were recorded. The timeframe of the POW1 visit was defined as any visit that occurred between post-operative days 5 and 14.Exclusion criteria consisted of findings that were present at each of these four different timepoints: preoperatively, intraoperatively, at the POD1 visit, or at the POW1 visit. Patients were excluded at the preoperative timepoint if they had a previous history of steroid intraocular pressure (IOP) response, persistent corneal edema, or rebound iritis following cataract surgery in the fellow eye. Intraoperative complications that led to exclusion included posterior capsular ruptures, anterior capsular rents (defined as any violation of the anterior capsule resulting in a discontinuous capsulorrhexis), performance of an anterior vitrectomy, zonular dehiscence, placement of a capsular tension ring, placement of the intraocular lens in the anterior chamber or sulcus, dropped nuclear fragments, or the performance of a concurrent vitreoretinal procedure. Patients were excluded at the POD1 timepoint if any unexpected clinical findings were noted that required a change in the standard post-operative care regimen of the primary surgeon. These included elevated IOP in the operative eye , intraocular lens out of position, severe corneal edema, corneal epithelial defects, a wound leak, a retained lens fragment, the performance of an anterior chamber paracentesis, or any change in the type or frequency of post-operative drops compared with the standard regimen. Patients were excluded at the POW1 timepoint if they presented with subjective ocular pathology-related symptoms either before or at the visit, including eye pain, redness, flashes, floaters, or decreased vision. Any patients that did not have a documented POD1 visit or a visit within the POW1 timeframe were also excluded.The primary outcome measure of this study was the incidence of departures from routine care, or unexpected management changes, at the POW1 visit. Specifically, unexpected management changes consisted of a requirement for additional procedures other than suture removal, a need for referral to a specialty service, or an unplanned change in the frequency or type of postoperative drops.Overall, the charts of 292 surgical cases of 234 patients that underwent phacoemulsification with intraocular lens implantation were reviewed. A total of 115 eyes were excluded; 4 eyes due to concerns from the contralateral eye at the preoperative timepoint, 18 due to intraoperative complications, 44 due to concerns at the POD1 timepoint, and 49 due to concerns at the POW1 timepoint  had a departure from routine care following an asymptomatic presentation at the POW1 visit  were scheduled for surgery at the POD1 visit and 30 eyes (29.1%) were scheduled at the POW1 visit Table .Table 5EThe overall incidence of unexpected management changes in asymptomatic patients with an uneventful perioperative history, routine cataract surgery, and unremarkable POD1 visit was 2.3%. This finding is consistent with those found in previous studies –11. HoweIn an older study that investigated cases of uncomplicated phacoemulsification, a clinical intervention rate of 2.8% was identified for all routine follow-up visits within a 120-day postoperative visit timeline . This leAlso focusing specifically on the POW1 visit, another study that included cases from an experienced group of 10 cataract surgeons determined the overall incidence of unexpected management changes in asymptomatic patients with uneventful cases to be 0.9% . AlthougOf note, a previous study done by a group in New Zealand whose data collection was performed more than 20 years ago determined the incidence of detected complications at the POW1 visit to be 4.1%, a value significantly higher than our reported findings . Like ouOur study excluded patients with POD1 findings that would have necessitated close observation in the follow-up period. Overall, 39 of 292 cases (13.4%) were excluded from our study for this reason. The most common cause for exclusion in this cohort was an elevation in IOP 61.5%). This corroborates the guidelines proposed by the American Academy of Ophthalmology that patients should have their first visit following cataract extraction within 48 h of the procedure. However, no mention is made of a POW1 timepoint, and previous studies have exhibited the potential lack of utility of this visit for an asymptomatic subset of patients. Our study further adds strength to these findings by substantiating their outcomes with strictly resident-performed cataract surgeries in an academic teaching hospital. It also adds to previous studies that have shown no significant difference in intraoperative and postoperative complication rates between resident-performed and attending-performed cataract surgeries %. This c.Our study has a few notable limitations. Many attending physicians at our institution already exclude POW1 visits from their standard postoperative follow-up schedule, and we therefore did not directly compare the incidence of unexpected management changes in resident-performed cases to attending-performed ones. As mentioned previously, however, our findings are consistent with rates reported in other studies in the literature. Additionally, although our study was sufficiently powered to analyze the incidence of unexpected POW1 management changes, the sample size was not large enough to adequately study the risk factors that could potentially necessitate a POW1 visit. Further studies could be done to identify such risk factors.In conclusion, we found very low rates of unexpected management changes at POW1 visits following resident-performed cataract surgery in a specific subset of patients. Our findings are in agreement with other recent studies in the literature which suggest that this visit may no longer be an essential part of routine cataract follow-up. Having found that nearly 30% of eligible fellow-eye cataract surgeries were booked at the POW1 appointment, it appears that the greatest utility of this visit may lie in its potential for coordinating future care. Future studies aimed at establishing alternative means of post-operative follow-up in lieu of an in-person encounter, such as telemedicine visits or video calls, could be valuable in expanding on these findings.


Following the spread of the Covid-19 pandemic in 2020, reports emerged on decreasing emergency department (ED) visits in many countries. Patients experiencing chest pain was no exception. The aim of the current study was to describe how the Covid-19 pandemic and the subsequential lockdown impacted the chest pain population in a Norwegian ED.th to August 30th, 2020, and compared to the corresponding period in 2019, assessing variations in the number of ED visits, severity, gender, and age.All patients presenting to the ED with chest pain during the study period were included. Data were collected retrospectively from the time period January 6Fewer patients with chest pain were seen in the ED following the national lockdown in Norway, compared to the corresponding 2019 period . By week 28, the rate normalized compared to 2019 levels. There was a relative increase in lower acuity patients among these patients, while fewer moderate acuity patients were seen. During the initial period following lockdown, the median age was lower compared to the corresponding 2019 period (58 years (IQR 25) vs 62 years (IQR 24), respectively). Admissions due to acute coronary syndromes (ACS) remained proportionally stable.Succeeding the Covid-19 outbreak and the subsequent national lockdown in Norway, fewer chest pain patients presented to the ED. Paradoxically, the patients seemed to be less severely ill and were on average younger compared to 2019 data. However, the proportion of patients admitted with ACS was stable during this period. This could imply that some patients may have failed to seek medical advice despite experiencing a myocardial infarction. The Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2) emerged in Wuhan, China in December 2019, and spread rapidly worldwide causing the Coronavirus Disease 2019 (Covid-19) pandemic [st, 2020. By March 12th, reported cases in Norway counted 621. In some cases, it was no longer possible to determine the source of infection. Comprehensive measures were implemented by the Norwegian Institute of Public Health (NIPH) the same day, March 12th, in an effort to contain the situation [th, the preventive measures in Norway were gradually lifted as the number of new cases decreased. The incidence of Covid-19 remained low over the summer, followed by a steady increase in the number of new cases throughout August.The first case of Covid-19 in Norway was reported on February 21ituation . From ApAs Covid-19 spread across the world, a growing number of reports were published on a substantial decline in the numbers of emergency department (ED) visits , 5. An iThe general decline in ED visits has also been found to include patients presenting with chest pain and possible acute coronary syndrome (ACS) in several international studies –9. This The trend of a general decline in ED visits as reported internationally was confirmed in an initial report from the ED at St. Olav’s University Hospital by Bjørnsen et al. . AlthougSt. Olav’s University Hospital is the local hospital in the city of Trondheim in central Norway, serving a population of approximately 300 000 inhabitants. Additionally, the hospital functions as a regional hospital in central Norway, covering more than 700 000 inhabitants. Annually, the university hospital manages more than 26 000 ED admissions in the main ED. As there is a separate ED for children, the patients managed in the main ED are primarily older than 16 years of age. The general rule is that patients must be referred to the ED by a physician, in most cases from their general practitioner (GP) or an urgent care center. The exception is situations when a patient’s condition requires emergency medical services (EMS). Therefore, the ED self-referral rate is low.th to August 30th) in 2020 were included in this retrospective observational study. The most comprehensive preventive measures implemented by the government commenced from March 12th (week 11) and were gradually lifted approaching the summer. As the number of ED visits in 2020 seemingly normalized compared to the 2019 data following week 27, weeks 11–27 were considered well suited to study the initial effects of Covid-19 and the preventive measures implemented by the government. The analysis is therefore focused on this period. Corresponding data from weeks 2–35 in 2019 were utilized for comparison, providing an opportunity to interpret data in light of seasonal variations and the increasing rates of patient visits to the ED over the recent years.All patients presenting to the ED at St. Olav’s University Hospital with chest pain as their chief complaint during weeks 2–35 . The Rapid Emergency Triage and Treatment System   is used All patients presenting with the chief complaint of chest pain according to the RETTS ESS #5 were included in the study Fig. . This apLogistic data on patients referred to the ED were retrieved from Central Norway Regional Health Authority's IT (Hemit) department’s data warehouse. This included data from the local ED database . Using the administrative tool NIMES Vis , supplementary data on the patients’ stay in hospital including discharge diagnoses and procedures were retrieved from the hospital’s patient and administration system . All data were linked by an automated algorithm, anonymized, and stored on a safe hospital server.Data on the total number of visits to the ED and the demographics of the population with regards to sex and age, was collected. The rate of patients transported to the ED by the EMS, the triage level, the in-hospital level of care, discharge diagnosis, in-hospital mortality, 30-day mortality, and the readmission rate within 30 days from the patients’ ED visit were included as surrogate markers of severity of the patents’ conditions. To account for the inconsistent reporting of how patients are transported to the ED (14.7% missing data), patients with a prehospital triage code were also included as being transported by the EMS. Regarding the remaining variables, 2.7% of patients had no recorded discharge diagnoses, 1.3% lacked data on possible readmissions and 30-day mortality, 1.0% of patients had no registered gender data and 0.3% lacked age information. These data are likely missing due to errors in the documentation process. However, the tools used for patient data collection in this study ensured a high level of accuracy to include the patients and match with their relevant clinical data. Therefore, there were generally very few missing data for the population. No data was missing for triage levels or level of care.When discharged from the ED or hospital, all patients receive one or more ICD-10 code(s) reflecting their diagnosis . Each paData were analyzed using STATA . Results are reported as numbers with percentages, mean with standard deviations in normally distributed data and median with interquartile range in skewed data. The numbers of ED visits can vary substantially from day to day . To reduth to August 30th) 1632 patients presented to the ED due to chest pain, compared to 1736 patients in the corresponding 2019 period  was observed. Only 36 patients presented to the ED due to chest pain in week 13, thus reaching a low-point representing a 38% decline compared to 58 patients in the corresponding week in 2019. This corresponds to a 30% decline from the weekly average 2019 control period (weeks 2–35) average of 51 (SD 8), and 35% fewer patients compared to the 2020 pre-lockdown (weeks 2–10) average of 55 (SD 8).Following the national lockdown in week 11, there were on average 11 (21%) fewer patients presenting to the ED due to chest pain per week during weeks 12–15 in 2020 compared to the same period in 2019. Compared to the pre-lockdown period in 2020 (weeks 2–10), there were on average 13 (24%) fewer weekly patients, and 9 (19%) fewer weekly patients compared to the 2019 control period (weeks 2–35). Over the next four weeks (16–19), the average number of patients per week was reduced by 8 (16%) compared to the corresponding 2019 period, and reduced by 12 patients (22%) per week compared to the early 2020 period (weeks 2–10). The marked decline in patients presenting to the ED due to chest pain during weeks 11–13 was followed by a gradual recovery until the number of patients normalized around week 28.The median age was 61 years (IQR 25) in 2020 compared to 63 years (IQR 23) in 2019. No apparent changes were found when comparing the Covid-19 period (weeks 11–27) with the corresponding weeks in 2019 overall, but there was an initial decline in the median age to 58 years (IQR 25) during weeks 11–15 as Covid-19 broke out in Norway  chest pain patients were triaged at the highest acuity level (RED) during the 2020 study period (weeks 2–35). In the 2019 control period, 133 out of 1736 (8%) were triaged at this level Fig. . FocusinThroughout the study period in 2020 (weeks 2–35), 862 out of 1632 (53%) patients were triaged at the second highest level (ORANGE). The corresponding numbers for 2019 were 1109 out of 1736 (64%). During weeks 11–27 in 2020, the proportion of patients at this triage level was 51% compared to 64% in the corresponding 2019 period.There were 641 (39%) patients triaged at the third highest level (YELLOW) in 2020 (weeks 2–35), corresponding to 484 (28%) patients in 2019. During the Covid-19 period , 43% of the patients were triaged as YELLOW, as opposed to the 28% in the corresponding 2019 period.There were five patients triaged as GREEN during the 2020 study period, and ten during the corresponding 2019 period.th to March 15th, 2020) compared to 19 out of 54 (35%) in the corresponding week in 2019.There was a majority of male patients both in 2019 (57%) and 2020 (56%). During the Covid-19 period , 260 out of 753 (35%) patients were transported to the ED by ambulance, compared to 342 out of 893 (38%) in the corresponding 2019 period. However, only 10 out of 47 (21%) patients presented to the ED during the week of the national lockdown (March 9The rate of outpatient visits increased from 549 out of 1736 (32%) in 2019 (weeks 2–35) to 593 out of 1632 (36%) in 2020 (weeks 2–35), the rate of patients admitted to hospital wards did not differ notably in the Covid-19 period  compared to the corresponding 2019 period  compared to 14 in the corresponding 2019 period. The 30-day mortality was 17 in the 2020 study period compared to 22 in the 2019 control period. There was no notable change in the readmission rate.Chest pain patients presenting to the ED at a regional University Hospital in Norway were analyzed in this study, examining how this patient population responded to the Covid-19 pandemic and subsequent national lockdown. The main findings were that 1) ED visits due to chest pain decreased substantially following the outbreak of Covid-19 and subsequent national lockdown. The abrupt decline persisted almost four months before normalizing. 2) Accompanying this decline was a relative decrease in elderly patients initially, but no apparent differences among sexes were found. 3) The marked drop from ORANGE to YELLOW triage levels imply that the patients on average presented with less severe symptoms Fig. .Fig. 5A Following the national lockdown in week 11, substantially fewer patients with chest pain presented to the ED at St. Olav’s University Hospital. The 35% decline in weekly visits following the national lockdown is compatible with the findings of a study by Myhre et al. . An initThe number of ED visits due to chest pain at St. Olav’s University Hospital gradually recovered from the initial decline, normalizing in week 28 compared to 2019 data. There were 16% fewer patients presenting to the ED due to chest pain throughout weeks 11–27 compared to 2019, corresponding well to the overall reduction in ACS admissions seen in England in the same period . The decRelatively fewer elderly patients over 60 years) presented to the ED at St. Olav’s University Hospital due to chest pain following the national lockdown in Norway weeks 11–15). While similar findings were reported in a general ED population in the US , the opp0 years p–15. WhilIt is possible that the same mechanisms were at play during the general staff holiday (weeks 28–30), where relatively fewer elderly patients visited the ED due to chest pain compared to 2019. While the median age remained stable at 60 throughout weeks 28–30 in 2020, it peaked at 72 in week 29 in the corresponding 2019 period. Further examinations of this unexpected finding by differentiating the study population into 10-year age groups, revealed a substantial rise in the number of patients over 70 years old. A similar rise did not take place in 2020, where only 48 patients over 70 years presented to the ED in this period compared to 64 in 2019. There were profound declines in the total ED visits due to chest pain throughout this period both in 2019 and 2020, thus it seems as though the surge of elderly patients in 2019 were compensated by an increase in younger patients in 2020. This increase could be caused by more younger people staying at home due to travelling restrictions, contributing to an impression of normalizing numbers of ED visits during the general staff holiday.Generally, chest pain patients sought out medical advice equally throughout the study period regardless of severity. The same trend was demonstrated in the general ED population at St. Olav’s University Hospital early on (6). Compatible findings are reported in the US , although Italian data suggest that the patients on average were more severely ill (4). This could be attributed to the immense pressure on the Italian healthcare system in an early phase of the pandemic 24). In the current study, the proportion of patients triaged as ORANGE dropped (64% to 51%) during the Covid-19 period  compared to 2019, while patients triaged as YELLOW increased (28% to 43%). This implies that chest pain patients presenting to the ED at St. Olav’s University Hospital during the Covid-19 period were less severely ill than the previous year. The opposite was noted in New Zealand regarding triage levels, where the proportion of low acuity presentations decreased significantly (21). Considering that other parameters indicating severity were more in line with the current study, this is difficult to interpret. One might speculate how well the triage levels reflect the severity of these cases. The Norwegian Directorate of Health reported an 18% increase in patient contacts with GPs, and a 34% increase in the use of urgent care centers nationally during March 2020 compared to March 2019 . In the . Other sChallenges regarding completeness of data and control over procedures for data collection are prominent when doing a retrospective register study. Additionally, such studies are not suited for concluding on causality. Thus, it cannot be concluded whether the decline in ED visits is due to a true decline in the incidence of chest pain or not based on this study. As the aim of the study was to provide insight into how the chest pain population in a Norwegian ED responded to the national lockdown and reopening in Norway following the spread of the Covid-19 pandemic, data on the subsequent outbreaks during the fall have not been gathered or analyzed. Including data from more prior years would provide an opportunity to account for seasonal variations and the increasing numbers of ED visits. The interpretation of more subtle changes in the study population could also become more conclusive. The inclusion process, where patients assessed after the RETTS  triage aThe number of chest pain patients presenting to the ED at St. Olav’s University Hospital declined considerably following the outbreak of Covid-19 and subsequent national lockdown. This decline was however not as pronounced as in many other European countries. The decline impacted the whole study population but seems to have affected the elderly more than the general population in the early phase. This suggests a need for increased preparedness targeting the elderly in similar situations in the future. Moreover, the shift toward lower triage levels suggests that the patients on average were less severely ill. Further studies will be needed to assess if the observed changes had any influence on morbidity and mortality.


Clostridioides difficile is a serious problem for the aging population. Aged mouse model of C. difficile infection (CDI) has emerged as a valuable tool to evaluate the mechanism of aging in CDI.We reviewed five published studies utilizing aged mice (7–28 months) for CDI model for findings that may advance our understanding of how aging influences outcome from CDI.Aged mouse models of CDI uniformly demonstrated more severe disease in the old compared to young mice. Diminished neutrophil recruitment to intestinal tissue in aged mice is the most consistent finding. Differences in innate and humoral immune responses were also observed. The effects of aging on the outcome of infection were reversed by pharmacologic or microbiota-targeted interventions.The aged mouse presents an important in vivo model to study CDI and elucidate the mechanisms underlying advanced age as an important risk factor for severe disease. Clostridioides difficile (formerly Clostridium difficile) is the most prevalent pathogen leading to healthcare-associated infections in the United States,1 accounts for an excess of a billion dollars to the healthcare system annually,2 and has been identified as one of the top five antibiotic resistance threats by the CDC.3 What is more striking is the toll on the aging population. C. difficile infection (CDI) is more likely to infect older patients,4 result in more recurrences,5 and more likely to end in death.6 Although people 65 and older make up half of the CDI patients, around 90% of deaths from CDI occur in this age group.6 Older patients also have poorer response to treatment for CDI.7 Even when controlling for confounding factors such as comorbidities and healthcare exposures, aging is an important factor to increase risk of worse outcomes.8 As the proportion of older people increases in the general population, it is even more compelling to pursue research evaluating the effect of age on CDI.in vivo research into pathogenesis of disease as well as for testing new treatment strategies. Animal models have been used to study the process of aging itself, utilizing a range of animals from Caenorhabditis elegans, Drosophila melanogaster, to mice and rats, up to primates.9 Rodent models have been used extensively due to the wealth of background knowledge, convenience of use, genetic manipulability, and expense.9 The rodent models of aging paved the way for aging research, such as the importance of caloric restriction on extending lifespan10 as well as for findings of certain genetic mutations being associated with longevity.11 Most studies have focused on conditions that accelerate aging or on measures that delay aging and increase longevity.9 In a limited capacity, aged mouse models have been used for infectious disease research as well. Especially in influenza infection, the aged mice have been used to test specific aspects of the pathogenesis,12 test new therapeutics,13 and test vaccines.15 However, the aged animal model was not utilized for research on CDI until recently. Over the past few years, studies utilizing aged animal models of CDI have been developed and published.16–20 The main challenge in development of an animal model of CDI is the microbiome. As seen in human disease, the normal intestinal microbiota must be disrupted before colonization and infection by C. difficile can occur.21 The development of varying models reflect the different methods utilized to cause susceptibility to C. difficile infection. One method would be to utilize gnotobiotic mice that do not possess a normal microbiome,16 while another method would be to utilize antibiotics to disrupt the normal intestinal microbiome.17–20 Different antibiotic regimens  were utilized in different models, while the age of the mice also differed from model to model. In this paper, we reviewed the features of each animal model and the key findings from the studies. The characteristics of each animal model and the discoveries on the changes in response with aging are summarized in Animal models have long been used for 16 At the time, there was no established, widely accepted mouse model of CDI, and this was one of the first mouse models developed to study CDI.16 The golden Syrian hamster model had been in use traditionally for CDI research, but with 100% mortality and lack of diarrhea, the concern was the difference in presentation from human disease.23 In this model, gnotobiotic C57BL/6 mice aged 7–14 months were infected with UVA13, a clinical NAP1/BI strain of C. difficile isolated from a patient in University of Virginia Health Center, by oral gavage.16 Compared to mice orally gavaged with a nontoxigenic strain, all of the mice given toxigenic C. difficile developed diarrhea then died or became moribund requiring euthanasia within 72 hours.16 Infected mice showed significant tissue damage in both cecum and colon.16 The levels of proinflammatory cytokines KC, MCP-1, IL-1β, and G-CSF were higher than controls, while IFN-γ, IL-12p40, IL-12p70, and IL-10 were lower compared to controls.16The first publication by Pawlowski et al. utilized an aged gnotobiotic mouse model of CDI.16 These findings hint at a more complex process than an excessive inflammatory response as the cause of the moribund state.Interestingly, the neutrophilic infiltration, while present, was not remarkably elevated in the infected group, nor were the levels of some cytokines, including TNFα, IL-17, and IL-6, while the levels of IFN-γ, IL-12p40, and IL-12p70 were lower than uninfected controls.16 and to more closely reproduce CDI seen in humans, models utilizing specific pathogen-free mice challenged with antibiotics have been developed. A mouse model of utilizing a cocktail of four antibiotics in the water before an additional injection with clindamycin to produce susceptibility to infection, described in Chen et al.24 came to be one of the most widely utilized models of CDI.26 The model had several advantages: a model which presents the spectrum of findings in human infection, with mice showing diarrhea, weight loss, and death, but without 100% mortality, making it possible to study a range of severity of the disease. van Opstal et al. published the first paper applying this model in aged mice17 and compared aged (18-month-old) mice to young (8-week-old) mice head-to-head.17 The mice were infected following exposure to a four antibiotic  cocktail in drinking water for 3 days followed by an intraperitoneal injection of clindamycin before orally infecting with 105 CFUs of VPI10463, a laboratory strain of CDI that is highly toxigenic.17 This model showed a significantly worse outcome in aged mice compared to young mice in terms of weight loss and mortality but without 100% mortality.17 The model was modified for a relapse model, in which the mice were treated with vancomycin by oral gavage for 5 days after infection.17 More severe disease during recurrent infection in aged mice was observed, which also correlated with lower levels of antibodies compared to young mice.17 Aged mice produced lower levels of anti-toxin A IgG antibodies, while the use of antibiotics seemed to decrease antibody levels in both aged and young mice, suggesting a role of antibiotic in perpetuating the vicious cycle of recurrent CDIs.27Due to the uniform mortality with CDI18 In Shin et al., the difference in weight loss was observed again, with worse outcomes in aged mice.18 In this study, the innate immune response was analyzed in detail, looking at cellular and cytokine responses, which revealed a lower proinflammatory response early in the infection (day 2 postinfection) compared to young mice.18 There was lower neutrophil recruitment to the intestinal tissue, along with lower levels of IL-1β, TNF-α, KC, MIP-2, IFN-γ, and IL-17.18 In addition to analyzing the outcome and immune responses, the microbiome was analyzed in this study.18 The aged mice microbiota had low alpha-diversity, but also had lower numbers of bacteria in the Bacteroidetes phylum. Further microbiome analysis down to genus level, revealed three distinct genera: Alistipes, Bacteroides, and rc4-4,18 differentiating aged from young mice. These deficiencies in microbial composition were restored by FMT from young mice to aged mice.18 What is more impressive is that FMT led to an improvement in outcome and reversed the distinction between aged and young mice immune responses.18 These findings strongly suggest an important role of microbiome-mediated host response in CDI pathogenesis in aging.18After establishment of the use of aged mice in a modified Chen mouse protocol, a follow-up study was performed which evaluated in more detail the innate immune response involved in CDI pathogenesis in the context of aging.19 which allows evaluation of the interaction of sex and aging, and provides potentially a more controlled comparison for evaluation of the effect of aging on the microbiome. The infection protocol followed the Chen protocol,24 utilizing the antibiotic cocktail and infection with VPI 10463.19 The main difference from the previous models utilizing antibiotic cocktails was that middle-aged mice (12–14 month old) were used with young mice (2–4 months old) as controls.19 Again, higher weight loss, signs of infection, and mortality were observed in the older mice.19 Consistent with earlier studies, infection in the aged mice showed lower neutrophil infiltration into the intestinal tissue, along with lower IL-6, IL-23, CXCL2, CXCR2, and MPO.19 An important finding from this study was the notable role of IL-22 in outcome of infection.19 IL-22 production was decreased in aged mice as measured by both RNA expression and protein levels, and the effect of IL-22 was confirmed by protection and improvement in outcome conferred by administering recombinant IL-22.19 Microbiome analysis revealed a significantly lower Bacteroidetes and higher Proteobacteria, Enterococcaceae, and Pseudomonadaceae in aged mice.19 Interestingly, the alpha-diversity, meaning diversity in the composition of the microbiome, was higher in middle-aged mice in this study,19 which is the opposite of what was seen in Shin et al., which showed a lower diversity in aged mice.18 These findings/inconsistency is interesting because of the complexity of the microbiome. Since CDI is only possible in a disrupted microbiome,21 and normal microbiome has a suppressive effect on C. difficile,28 a lower microbiome diversity is considered one of the factors predisposing to CDI, and has been associated with recurrences.29 However, it has been shown in human studies that the microbiome diversity generally tends to be higher in older individuals.30 As we will further elucidate in the discussions section, the specific members of the microbiome may be more important. This is also demonstrated in Shin et al., where the FMT from young mice led to a higher proportion of Bacteroidetes and protection, but no change in the diversity of the total microbiome.18Another group of investigators  aged both male and female mice at their own institution,31 Abernathy-Close et al. utilized aged mice that were the oldest among the published studies (22–28 months old) and also bred in the same facility as young mice (2–3 months old).20 In addition to a different antibiotic treatment, two different strains, VPI10463 and 630, were used to explore the impact of aging on CDI outcomes by infecting with strains of different virulence.20 VPI10463, as described above, is a highly toxigenic strain that produces high levels of both toxin A and B, while 630, which is a clinical strain isolated from a patient, is considered low virulence due to the lower levels of toxin production.31 Both advanced age and strain VPI10463 were associated with more severe disease as demonstrated by clinical scores.20 Compared to infection with VPI10463, 630 resulted to mild symptoms and histopathologic scores were minimally changed and not different between young and aged mice.20 As was also seen in Shin et al.,18 the aged mice had lower histopathologic scores on day 2 postinfection when compared to young mice when infected with VPI10463.20 The neutrophil and eosinophil responses in the intestinal tissue in aged mice were significantly blunted compared to young mice, but there was actually a higher systemic eosinophil response with CDI in aged mice, which was not seen in young mice.20 In terms of the cytokine response, the aged mice had higher IL-17A but lower CXCL1 response compared to young mice.20 This study demonstrated consistent findings of the detrimental effect of aging on CDI in a different mouse model, with additional information on the eosinophil response, which has recently gained significant interest in binary-toxin producing strains.33Mouse model of CDI utilizing cefoperazone alone to induce dysbiosis has also been developed.34 High neutrophil count is associated with worse outcome to CDI,35 but neutrophil depletion has been shown to be associated with higher deaths from CDI in animal models.37 It is remarkable that across different aged mouse models utilizing different methodologies and different aged mice – Shin et al.:18 antibiotic cocktail model using NIA-aged mice; Peniche et al.:19 antibiotic cocktail model using investigator-aged mice; and Abernathy-Close et al.:20 cefoperazone model using investigator-aged mice – the neutrophil recruitment to infected tissue has been consistently seen to be depressed compared to young mice. Characterizing changes to the innate immune response with aging is complex. Studies to investigate the changes in neutrophil response with aging have not produced consistent results, with an increase in neutrophil recruitment to influenza infection in the lungs in a mouse model,38 no change of human neutrophils in migration in vitro,39 or a decrease of chemotaxis of human neutrophils in vitro,40 which makes the consistent trend with CDI even more striking. There are two questions that arise from these findings: (1) what caused the neutrophils to be lower in aged mice and (2) how do the lower neutrophils contribute to worse outcome in aged mice?Innate immune response is considered the main immune response to CDI.41 Shin et al. demonstrated lower levels of KC, MIP-2, IL-1β, and TNF-α on day 2 while Peniche et al. had lower levels of IL-6 and IL-23 on day 7.19 However, in Pawlowski et al., KC and IL-1β were higher on day 3.16 These contradictory findings suggest that recruiting chemokines or cytokines do not determine the age-related changes in neutrophil recruitment. Further research into the mechanism by which neutrophil recruitment is affected is needed.The difference in immune response to explain the lower neutrophil count in aged mice with CDI has not been elucidated yet. Chemokines such as KC (CXCL1) and MIP-2 (CXCL2) directly recruit neutrophils to infected tissue, while cytokines such as IL-1β, TNF-α, IL-17, IL-6, and IL-23 enhance inflammation.43 so the beneficial effect of neutrophils in this context is somewhat unexpected. There are data in animal models showing that depletion of neutrophils prior to infection leads to higher deaths45 as well as some human studies showing neutropenia as a risk factor for worse outcome.47 These conflicting data suggest that the benefit or harm from neutrophils may be context dependent. We could hypothesize that neutrophils, of the right quantity recruited into the right location at right timing, is required for protection, and that the dysregulation of this response lead to adverse outcomes. Therefore the more specific question would be “what is the protective effect of neutrophils in the early stage of the infection?” Clearance of C. difficile by neutrophils is a consideration, but bacterial burden has been shown to be not associated with outcome in human disease.48 Indeed in Shin et al., the infection burden was not seen to be different in mice even when there is a significant difference in outcome from CDI.18 Other functions of neutrophil response such as immune regulation can be considered, and should be investigated further. As noted in tumor immunology, special neutrophils or new populations of cells such as, low-density neutrophils (LDN)49 and myeloid-derived suppressor cells (MDSC),50 may have immune-regulatory role but these cells have not been investigated in CDI.The effect of the lower neutrophil recruitment is unknown at present. Human studies in CDI are more suggestive of elevated neutrophils as a risk factor for worse disease and higher deaths,C. difficile strains that produce binary toxin.51 Although the the protective effect of eosinophils was documented in these studies, the mechanism by which eosinophils protect the host from severe CDI has not been characterized. The protective effect of eosinophils has not been clearly demonstrated in the aged mouse as while there was lower number of eosinophils recruited to the intestinal tissue, the eosinophil count in the systemic circulation was actually higher in infected old mice in the studies by Abernathy-Close et al.20 The role of eosinophil recruitment to the intestinal tissue with CDI and effects of ILC2 and microbiota on this phenomenon in the context of aging is unclear.20Eosinophils have been shown to be important in protection against CDI, especially when infected with 53 In Hasegawa et al., a change in the complement system decreased circulation of pathogenic Enterobacteriaceae during CDI,52 while in Nagao-Kitamoto et al., IL-22 was shown to modify intestinal microbiota to prevent growth of C. difficile.54 One of the key findings of Peniche et al. was the importance of IL-22 in the aged mice CDI outcome, demonstrating a lower production of IL-22 in aged mice, and protection of the aged mice when given recombinant IL-22 during infection.19 Since in Peniche et al., recombinant IL-22 was given on day 1 of infection, suggesting a host response-mediated effect on outcome rather than a microbiome-mediated effect. In an animal model of inflammatory bowel disease (IBD) it has been shown that IL-22 produced by neutrophils leads to beneficial barrier function by increased epithelial cell proliferation and mucus production and leads to recovery.55 IL-22 is part of the immune response associated with type 3 innate lymphoid cells (ILCs) during CDI.56 With the other finding from aged animal models of CDI being the importance neutrophil response, the IL-22 may point to an important and possibly protective role in the immune response associated with ILC3.The IL-22 story is interesting in light of more recent studies pointing to a protective role of IL-22 in CDI pathogenesis.34 antibody response to C. difficile toxins play an important role in recurrent infections.5 Bezlotoxumab, a monoclonal antibody against C. difficile toxin B (TcdB) has been shown to be effective in decreasing recurrent CDI in patients with CDI.57 Older age is one of the risk factors for more frequent recurrent CDI. In van Opstal et al. recurrent CDI was tested in an aged mouse model by treating infected mice with vancomycin for 5 days.17 Consistent with human disease, recurrence was observed few days after discontinuation of vancomycin, with fatal infection occurring more in the aged mice.17 This poor outcome correlated with the humoral response, as aged mice had lower serum anti-toxin A IgM levels on day 6 post infection, as well as lower IgG and IgA levels on day 14 post-infection.17 An additional finding is that vancomycin led to lower levels of antibody production without a difference in C. difficile infection burden, pointing to a potential role of the resident intestinal microbiome on regulating the antibody response.58 Recurrence leads to significant quality-of-life issues, especially in the older population, and thus, vaccination and potentially, microbiota-targeted strategies to enhance humoral responses in the elderly warrant further studies.Although innate immunity is thought to be the main immune response to an initial infection,C. difficile because the normal microbiome has an inhibitory effect on C. difficile growth, referred to as “colonization resistance.”21 The fecal microbiome in aged individuals exhibited decreased colonization resistance compared to young subjects.28 Changes in microbiome with aging demonstrates a decreased relative abundance of Firmicutes phylum59 and a decrease in Bifidobacteria,30 but these studies showed a high individual variability. There are a number of beneficial organisms in in vitro and in vivo studies in protection against CDI such as Clostridium scindens which is suspected to play a role due to its unique bile acid metabolism,60 while bacteria in the Lachnospiraceae family have a suppressive effect on C. difficile as well.61 Changes in the composition of these organisms with aging have not been yet documented.The intestinal microbiota play an important role in CDI pathogenesis: disruption of the intestinal microbiome is a prerequisite for colonization by 17–19 In Shin et al., in addition to a lower alpha-diversity in aged mice compared to young mice, there was a notable lower proportion of Bacteroidetes phylum in the aged mice, which were supplemented with FMT from young mice.18 The other major phylum, Firmicutes, was not different between young and aged mice.18 Random forest analysis revealed important genera, two of which  belonged to Bacteroidetes.18 Interestingly, cage switching was the method for FMT in this study, which would lead to an exchange of the microbiome between the young and aged mice, but this did not lead to a significant change in the microbiome composition in young mice while the aged mice underwent significant changes and became closer to young mice.18 This would suggest that there was a deficiency in the microbiome of the aged mice that were supplemented by that from the young mice, and that the young mouse microbiome was more robust and resistant to change. Another interesting finding was that the microbiome difference between young and aged mice did not lead to a difference in C. difficile colonization, suggesting a mechanism other than colonization resistance in mediating the difference in outcome.18 Peniche et al. did not find any major differences between aged and young mice in terms of microbiome composition, although the alpha-diversity of the microbiota was significantly increased in the older mice compared with young controls,19 which is the opposite finding from Shin et al.18 Even though the two studies showed different results regarding the microbiome – Shin et al. showed the microbiome changing with aging while Peniche et al. did not show any differences in microbiome with aging – the difference in the innate immune responses, especially the neutrophil response, was consistent across different models.19 With only two studies looking at the microbiome, it is difficult to draw a conclusion on whether aging has a specific effect on the intestinal microbiome. However, we can conclude that the immune responses changes are seen with aging regardless of whether there is a microbiome difference with aging.The microbiome was investigated in three of the studies, van Opstal et al., Peniche et al. and Shin et al.18 CDI itself is a disease that only occurs in the presence of dysbiosis, and therefore it is plausible that there is a microbiome factor associated with aging. Further studies to evaluate aging effects on microbiome is needed.Does this mean that the changes with aging is not related to the microbiome? The answer is not so simple. In Shin et al., FMT from young mice lead to protection of aged mice from death by CDI and a change in its immune responses.C. difficile by blast analysis against NCBI 16SrRNA gene database, was elevated in aged mice compared to young mice.19 Although this is not exactly analogous to the absolute C. difficile infection burden measured by qPCR of the tcdB gene as performed in Shin et al. and Abernathy-Close et al., it is interesting that the C. difficile infection burden measured in Shin et al. and Abernathy-Close et al. was not different between aged and young mouse groups,20 suggesting a different trend than Peniche et al.19 In van Opstal et al., the baseline microbiome in aged mice was not different from young mice in total numbers or numbers of Bacteroidetes and Enterobacteriaceae, while the Firmicutes were lower with aged mice.17 After infection with C. difficile, there is gradual decrease in overall bacterial numbers and the Bacteroidetes and Enterobacteriaceae, with Bacteroidetes significantly lower in aged mice especially lower with antibiotic treatment.17 There was an increase in Firmicutes numbers, likely reflecting the increase in C. difficile which is also a member of the Firmicutes phylum.17 The finding from this study that correlates with Shin et al. is the trend of the Bacteroidetes phylum18 Bacteroidetes numbers decreased with infection, but were even lower in aged mice, especially with vancomycin treatment.17 Bacteroidetes phylum is lower,18 or more susceptible to external stressors,17 which suggests the possibility of a protective function by members of the Bacteroidetes phylum against CDI, which is lost with age.Additional information from these studies regarding the microbiome is the dynamics of change of the microbiome with CDI in the context of aging. The main finding from Peniche et al. after infection was that OTU_11, the representative sequence of which demonstrated 99% identity to 62 Aged mice can be purchased from commercial laboratories, but is significantly higher in cost.63 Another option is to start an aged mouse colony, but this is also difficult due to the time and cost required. Aged mice utilized in the aged mouse models are around 12–24 months of age, necessitating maintaining the colonies for 1–2 years before any experiments can be performed, therefore considerable time and resources are committed before any results are seen. These difficulties raise the question of the utility of aged animal models of infection, whether the benefit seen from these studies outweigh the cost of the experiments.An aged animal model poses significant challenges. Aged mice from the National Institute on Aging aged mice colony are limited in number, therefore the access has been limited to researchers with aging biology grants.8 There are many confounding factors that need to be considered, and cannot always be controlled in clinical studies.8 Utilizing an aged animal model allows us to control for some conditions, such as environmental factors and diet, to evaluate the effect of aging on pathogenesis of CDI. As aging is associated with worse outcomes in multitudes of illnesses including infections such as influenza64 and COVID-19,65 findings from utilizing the aged mouse model could have far-reaching applications in the study of aging. Another advantage of an aged mouse model of CDI is its use in testing therapeutic options against CDI, as the elderly will be the main target patient population to use new therapeutics in.The value of the aged animal model comes from the phenomenon that is being studied: aging. Even though aging is one of the most important factors to consider in CDI, it is also quite difficult to study due to the multifactorial nature of aging.C. difficile bacteria.60 However, there have been studies linking the intestinal microbiome with immune response even in infections outside of the gastrointestinal tract.67 The finding that not only is the intestinal microbiome of the aged mouse distinct from that of the young mice, but also that transfer of the young mice microbiome into the aged mice reverses the effects of aging has major implications in our understanding of aging and immunity.18 This opens the doors to research on the interaction of the intestinal microbiome and immune response to a number of different conditions including various infections, malignancy, and autoimmune disorders. The possibility of microbiome-based therapeutics in improving health in the aging population would be relevant to commercial efforts for drug development.One finding from aged mouse model studies of CDI is the ability of the intestinal microbiome to lead to changes in disease outcome and immune responses in aged mice. Intestinal microbiome has always been considered important due to its effect on spore germination and colonization by the 69 the physiologic and immunologic basis by which aging leads to worse outcome with infections becomes more important than ever as a research goal. At the present, aged animal models of CDI have been quite promising, showing consistent results of aging on CDI outcome as well as showing promising leads to investigate for mechanisms of aging: neutrophil or other inflammatory cell recruitment or activity, innate immunity, and antibody response. The interaction of microbiome with immune response is also demonstrated in Shin et al., which could be relatable to other infections and settings other than older age.18 That said, it is fair to say the research in this field is in its infancy, with significant gaps remaining in our understanding regarding the effect of aging on CDI. The effect of aging on the microbiome, the mechanism of action by which the microbiome modulates the immune response, the mechanism by which the relative neutrophil deficiency in the aged host leads to worse outcome, the role of IL-22 in the aging effect on CDI, the interaction of the microbiome and the humoral response, are some of the burning questions raised by these studies. In addition, studies to bring the knowledge back to older patients for translational research are needed.Current research gaps in the research of aging and CDI are quite large. The mechanism of action by which older age leads to worse outcome has not been elucidated. In the light of the COVID-19 pandemic, which affects older patients disproportionately,


To create an aerosol containment mask (ACM) that contains aerosols during common otolaryngologic endoscopic procedures while protecting patients from environmental aerosols.Bench testing.Mannequin testing.The mask was designed in SolidWorks and 3-dimensional printed. Mannequins were fitted with a nebulizer to generate aerosols. Commercial particle counters were used to measure mask performance.The ACM has 2 ports on either side for instruments and endoscopes, a port for a filter, and a port that can evacuate aerosols contained within the mask via a standard suction pump. The mask contained aerosols on a mannequin with and without facial hair when the suction was set to 18.5 L/min. Other types of masks demonstrated substantial aerosol leakage under similar conditions. In a subsequent experiment, the ACM contained aerosols generated by a nebulizer up to the saturation of the particle detector without measurable leakage with or without suction.The ACM will accommodate rigid and flexible endoscopes plus instruments and prevent leakage of patient-generated aerosols, thus avoiding contamination of the room and protecting health care workers from airborne contagions.2. While one study found that laryngoscopy alone may not generate aerosols greater than that produced by breathing, laryngoscopy and nasal endoscopy are associated with increased risk of coughing and sneezing, which are aerosol-generating events.8 Currently in many practices, patients wear a surgical mask over their mouths during nasal endoscopy, although a regular surgical mask is insufficient to protect at close range against all particle transmission generated by simulated aerosol generation.Due to the spread of SARS-CoV-2, the virus responsible for COVID-19, clinicians and hospitals face difficult decisions regarding how to provide care for patients in clinics during procedures that may lead to the generation of aerosols. Airborne SARS-CoV-2 has been found in hospital rooms and ventilation systems where patients with COVID-19 have been treated.10 These cleaning and time requirements, compounded by limited testing capacity with variable time to results, can severely diminish an outpatient clinic’s capacity to treat patients.In principle, virus aerosolized during clinic procedures could infect not only the surgeon performing the procedure but others who enter the room. The Centers for Disease Control and Prevention recommends that procedure rooms without negative pressure remain vacant following any aerosol-generating procedure before undergoing deep cleaning. This period is typically deemed to be 6 times the room air turnover time. modification of Ambu, nasotracheal intubation face masks with negative pressure, or a modified N95 mask to decrease aerosol dispersion during diagnostic nasal endoscopy and laryngoscopy. We present a 3-dimensional (3D) printed negative pressure respiratory aerosol containment mask (ACM) that provides N95-level protection to the patient. The negative pressure is generated through a standard suction commonly found in otolaryngology clinics. We measured aerosol generation in the ACM and compared it with previously described masks with and without instrumentation.Prior authors have suggested negative pressure microenvironments,The study was approved by the University of Southern California Institutional Review Board (HS-20-00482).We created multiple design iterations by using SolidWorks (Dassault Systemes) and printing on a 3D printer (Ultimaker) with tough PLA . We tested initial prototypes on endoscopic surgery model heads to gauge access to the nasal cavity and ability to contain aerosols. The design was modified as issues were identified. The main considerations during the design phase were to appropriately position the blind grommet, find a gel cushion to seal the mask to various face shapes, and create a way to attach an easily replaceable HEPA filter (high-efficiency particulate air). The revision described in this article is the fifth. and ). Each blind grommet contains 2 openings, and an endoscope or suction can be passed through any of the 4 openings. All materials can be cleaned in Cidex OPA (Advanced Sterilization Products). A N95-level commercially available respirator filter can be attached to any of the 3 front ports and replaced between patients. A suction is attached to the suction port of the mask from a commercially available suction pump.The final design included a 3D printed body with 4 ports, a gel cushion for seal and comfort of fit, and custom blind grommets placed in 2 front ports plus a head strap  at approximately 2 cm anterior to the mask (sensor 1) and at 2 cm lateral to the mask near the area where its edge was against the face of the mannequin (sensor 2).A test bench Figure 1 (an Ambu mask fitted with suction tubing leading to HEPA suction) and a commercially available N95 mask.16 The flow rate was varied for the ACM and Ambu mask designs, but the N95 mask does not have the ability to apply suction. The amount of aerosol was measured at baseline for all 3 mask designs and at various flow rates for the Ambu and ACM, between –18.5 and 16.5 L/min, by varying the valve in front of the suction pump. Particles were measured for 2 minutes with 15-second sampling intervals, and each measurement was performed 5 times for each mask type.The level of aerosol around the ACM was compared with that from an Ambu mask designThe mannequin testing was repeated for the ACM .Standard t test was used to test if the mean average particle count minus the baseline particle count was significantly different from zero . A positive mean indicates leakage of aerosols. A negative mean implies that the mask is functionally cleaning the air near the sensor. On the mannequin head with no facial hair, no leakage was found under any conditions for the ACM. At 18.5 L/min, the sensor directly in front of the N95 filter (sensor 2) had a negative mean. This implies that the air near this sensor is cleaner when the suction is on. It may be that the filter is removing particles from the air in the vicinity of the sensor or that the air flow into the mask is drawing cleaner air into the room. The Ambu mask shows leakage at both sensors when the suction is 16.5 L/min but only at sensor 2 when the suction is –18.5 L/min. The N95 shows leakage at both sensors. On the mannequin head with facial hair, the ACM shows leakage when the suction is 16.5 L/min but not when the suction is set to 18.5 L/min, while the Ambu and surgical N95 masks show leakage under all conditions.Supplemental Table S2 (available online), with box plots of the other particle sizes in Supplemental Figure S1. Sensor 1 (within mask) showed a significant difference in average particle count with the suction on and off for both experiments. Sensor 2 (outside mask) showed no significant difference with the suction on or off for the mock endoscope experiment. Hence, in this configuration, even with a very high particle count within the mask, there was no detectable leakage with the suction on or off. In the experiment with the grommet removed, there was no significant difference with the suction on. As expected, though, when the suction is turned off, there is a significant increase in particle count, as aerosols leak from the mask.In these experiments, the particle count from the nebulizer had to be reduced as compared with the aforementioned experiments to avoid saturating sensor 1, which was measuring the count inside the mask. Nevertheless, the particle counts exceeded any of those measured on the human volunteers. A baseline was acquired just prior to turning on the nebulizer. This was subtracted from particle counts measured with the nebulizer on, the mask suction on and off, and the grommet opened and closed. The box plots in The ACM significantly decreased the spread of aerosol particles in mannequin testing. It outperformed previously described masks, especially on mannequins with facial hair. However, an N95 respirator with an incision lined with a cut piece of surgical glove  contained aerosol spread. When trialed on the mannequin, the unmodified N95 underperformed when compared with the devices with suction, especially on patients with facial hair. Other barrier devices, such as a hood or box, have been detailed but may be difficult to place, are not conducive to rigid endoscopy, and do not contain suction.18Prior studies found that regular surgical masks are insufficient in protecting against aerosol escape generated by sneezing. Similarly, no aerosols were detected with drilling of a cadaveric specimen or 3D sinonasal model once a flexible suction was placed, likely because the aerosols were directed toward the suction tip rather than exiting the nares. Similarly, Workman et al did not identify aerosol contamination when utilizing the microdebrider, which is attached to suction.16 Our findings are similar in that once suction is placed on the mask at a level sufficient to overcome the generation of the aerosols and the difficulties of fit with facial hair, then no particles are detected leaking from the mask.Prior studies of endoscopic surgery found that the addition of suction prevents the spread of aerosols. Dharmarajan et al found that, even in a cadaver model with an endotracheal tube in the nasopharynx attached to a nebulizer with B2 solution, no aerosols were detected visually or with a cascade impactor once a flexible suction was placed in the nasal cavity or the nasopharynx.19 Finally, modifications of existing masks, such as the Ambu mask or nasotracheal intubation masks, have been outlined.12 While the Ambu mask outperformed an unmodified N95 mask, it was difficult to place and allowed for less access to the nasal cavity and oropharynx as compared with the ACM.Creating negative pressure microenvironments around the patient to contain particles has been described. Prior studies also detailed box-like containers that can be placed around the patient for outpatient procedures or intubation, although these may be time-consuming in an outpatient setting.21 One of these articles also detailed a complete 3D printed mask; however, it did not include suction, had only a single midline port for flexible endoscopes, and had not been tested on a human.Additionally, prior studies have reported on the creation of 3D printed devices to contain aerosols. Two studies described a 3D printed vent that could be placed through a regular surgical mask, but the efficacy of these devices may be limited, as Workman et al found that surgical masks contain aerosolized particles poorly. We have not yet tested removal procedures, although we believe that most aerosols would be evacuated by the suction pump.The current study design has several limitations. The mask material is not clear. This necessitates scope guidance via a camera or the eye piece to drive the scope from the entrance of the mask into the nares. Future versions of the mask could be made with clear material through injection molding or chemical polishing of transparent 3D printed parts. While the mask allows access to the nose and oral cavity for diagnostic purposes and single-instrument procedures, such as suctioning and hand instruments, it does not allow for insertion of larger objects  without removing 1 of the grommets. Nevertheless, in a trial on the mannequin with the grommet off, we found no significant increase in aerosols external to the mask with the suction on Figure 4Studies are ongoing regarding whether a single suction pump, such as that from an SMR cart , can be split and continue to provide adequate suction to the mask and a surgical suction. This article describes testing of the mask on only a mannequin, but we have tested it on healthy volunteers and are in the process of completing a larger-scale clinical trial on patients presenting to an otolaryngology clinic.A negative pressure mask may allow for the passage of rigid and flexible endoscopes without leakage of particles outside the mask. This may help prevent contamination of the room and protect health care workers during viral pandemics that involve airborne contagion. A larger clinical study is ongoing.Click here for additional data file.Supplemental material, sj-docx-2-oto-10.1177_01945998211024944 for COVID-19 in the Clinic: Aerosol Containment Mask for Endoscopic Otolaryngologic Clinic Procedures by Elisabeth H. Ference, Wihan Kim, John S. Oghalai, Jee-Hong Kim and Brian E. Applegate in Otolaryngology–Head and Neck SurgeryClick here for additional data file.Supplemental material, sj-pdf-1-oto-10.1177_01945998211024944 for COVID-19 in the Clinic: Aerosol Containment Mask for Endoscopic Otolaryngologic Clinic Procedures by Elisabeth H. Ference, Wihan Kim, John S. Oghalai, Jee-Hong Kim and Brian E. Applegate in Otolaryngology–Head and Neck Surgery