Routinely-collected healthcare data provide a valuable resource for epidemiological research. Validation studies have shown that for most conditions, simple lists of clinical codes can reliably be used for case finding in primary care, however, studies exploring the robustness of this approach are lacking for diseases such as idiopathic pulmonary fibrosis (IPF) which are largely managed in secondary care.Using the UK’s Clinical Practice Research Datalink (CPRD) Aurum dataset, which comprises patient-level primary care records linked to national hospital admissions and cause-of-death data, we compared the positive predictive value (PPV) of eight diagnostic algorithms. Algorithms were developed based on the literature and IPF diagnostic guidelines using combinations of clinical codes in primary and secondary care (SNOMED-CT or ICD-10) with/without additional information. The positive predictive value (PPV) was estimated for each algorithm using the death record as the gold standard. Utilization of the reviewed codes across the study period was observed to evaluate any change in coding practices over time.A total of 17,559 individuals had a least one record indicative of IPF in one or more of our three linked datasets between 2008 and 2018. The PPV of case-finding algorithms based on clinical codes alone ranged from 64.4% (95%CI:63.3–65.3) for a “broad” codeset to 74.9% (95%CI:72.8–76.9) for a “narrow” codeset comprising highly-specific codes. Adding confirmatory evidence, such as a CT scan, increased the PPV of our narrow code-based algorithm to 79.2% (95%CI:76.4–81.8) but reduced the sensitivity to under 10%. Adding evidence of hospitalisation to the standalone code-based algorithms also improved PPV, . IPF coding practices changed over time, with the increased use of specific IPF codes.High diagnostic validity was achieved by using a restricted set of IPF codes. While adding confirmatory evidence increased diagnostic accuracy, the benefits of this approach need to be weighed against the inevitable loss of sample size and convenience. We would recommend use of an algorithm based on a broader IPF code set coupled with evidence of hospitalisation.The online version contains supplementary material available at 10.1186/s12890-023-02550-0. Idiopathic pulmonary fibrosis (IPF) is characterised by worsening respiratory symptoms  and irreversible loss of lung function due to progressive architectural distortion and aberrant cellular proliferation within the interstitium of the lung. The prognosis for people diagnosed with this chronic lung condition is poor, with a median survival of only 3–5 years if left untreated –3. GlobaAnalysis of primary care data for 2000–2012 suggests a UK IPF prevalence at the higher end of global estimates, between 19.9 and 38.8 cases per 100,000 people, with approximately 5,000 new cases diagnosed each year  . DespiteWhile routinely-collected healthcare data represent a valuable resource for epidemiological study and research, their utility relies on the quality of case ascertainment. Validation studies are important to assess the reliability of diagnostic coding of diseases and the majority of validation studies conducted to date indicate that case ascertainment based on the use of simple lists of clinical diagnostic codes in primary care records results in acceptable positive predictive values (PPVs) for the majority of diseases and conditions, especially those that are predominantly managed in primary care . HoweverThe aim of this study was to assess the reliability of recording IPF in a UK primary care database. Specifically, to determine whether a list of clinical diagnostic codes alone was sufficient or whether a more complex algorithm – one that includes additional information such as radiology or hospital admission data is required to improve the accuracy of IPF case identification in primary care data. To do this, we used ONS mortality data as our reference or “gold standard”, relying on the assumption that a person with IPF on their death certificate is highly likely to have been diagnosed with IPF during their lifetime.We used data from the Clinical Practice Research Datalink (CPRD) Aurum database (November 2020 build). This dataset contains pseudonymized primary care electronic health records for nearly 40 million patients, including around 13 million current patients representing 19% of the UK population . CPRD AuCPRD Aurum primary care data are routinely linked to a number of other patient-level datasets, including Hospital Episode Statistics (HES) and ONS mortality data using a deterministic methodology . Linked This analysis uses a cohort study design. The study population was drawn from patients registered at English CPRD practices that had consented to HES/ONS linkage. To be eligible for inclusion in the study patients had to have evidence of a diagnosis of IPF in one or more of our three linked electronic health datasets, CPRD Aurum, HES APC or ONS, and be aged at least 18 years at the start of the study period (1 January 2008–31 December 2018) and prior to their IPF diagnosis. A period of “useable” follow up (1 day) was defined for each patient based in their primary care record, starting on the latest of 1 January 2008, date of IPF diagnosis (the earliest IPF code in Aurum), start of current registration at a linked GP practice or date of 18th birthday and ending on 31 December 2018, date of death, last day of data collection at the practice or patient’s transfer out date, whichever came first.https://github.com/NHLI-Respiratory-Epi/SNOMED-CT-codelists). In short, initial text-based search terms were derived in consultation with two respiratory clinicians with expert knowledge of ILDs. Codes found by searching of the Aurum medical browser using these search terms were cross-checked against published IPF code lists, and any codes not captured by the test-based search added to form a list of potential codes [A set of clinical SNOMED CT diagnostic codes denoting IPF created for this study was used to define cases. This code set was developed using an established methodology . We defined two IPF primary care cohorts, one based on the “broad” set of Aurum codes, the other on the “narrow” Aurum code set.Consistent with previous electronic health records-based studies that have used sets of clinical codes to define IPF cohorts, , 8, 16 ahttps://github.com/NHLI-Respiratory-Epi/Validation-of-the-recording-of-Idiopathic-Pulmonary-Fibrosis-in-routinely-collected-electronic-healt) to define IPF hospitalizations and deaths in HES APC and ONS mortality data, respectively. To be included in the HES APC cohort, a patient had to have one of the three qualifying ICD-10 codes recorded as either the first or second clinical diagnosis in any “episode” of hospital care within their stay (“spell”) in hospital within their follow up period. We chose to restrict our case definitions to the first and second diagnostic codes in order to be more certain that we were selecting patients where IPF was the reason for their admission. In HES-APC data, codes mentioned further down the list of recorded diagnoses are more likely to reflect coexisting conditions/comorbidities that might impact hospital care and are known to have lower diagnostic accuracy. In a sensitivity analysis, we also created a cohort which included patients who had a least one qualifying ICD-10 IPF code in any diagnostic position .We used the International Classification of Diseases, 10th revision (ICD-10) codes, J84.1, J84.8 and J84.9  and comorbidities  as recorded in primary care. Smoking status was based on the closest available record to the date of IPF diagnosis; patients were assumed to have a given comorbidity if they had a relevant diagnostic code at any point prior to their IPF diagnosis. All codelists are available via GitHub .To gain insight into possible changes in coding practices in primacy care over time, we examined the frequency of use of IPF codes in Aurum for each year of our study period (2008–2018).We totalled the number of patients who met our cohort inclusion criteria in each of our three concurrent linked data sets using the case definitions described above. To assess concordance, the Aurum and HES-APC cohorts were restricted to those who had died within our study period. We investigated the concordance of recording of IPF in ONS, Aurum and HES-APC in terms of the number of patients with at least one IPF record in (i) just Aurum, (ii) just HES-APC and (iii) just ONS, (iv) both Aurum and HES-APC, (v) Aurum and ONS, (vi) HES-APC and ONS, and (vii) Aurum, HES-APC and ONS.In total we defined eight diagnostic algorithms (DAs) based on either our “broad” or “narrow” clinical code sets plus any one or more of the following: evidence of HRCT (high-resolution computed tomography) of the thorax, absence of other known causes of interstitial lung disease , and/or evidence of hospitalisation for IPF, i.e. record in HES-APC Table .PPV was calculated as the number of IPF cases correctly identified by the algorithm (i.e. confirmed by a record in ONS) divided by the total number of IPF cases identified by that algorithm. Sensitivity was defined as the number of “true” IPF cases correctly identified by the algorithm divided by the total number of ONS IPF deaths. Given that a death is our reference standard, this analysis was limited to those patients who died.All analyses were conducted using Stata version 17.0.Between 2008 and 2018, the most frequently used coding terms were; diffuse pulmonary fibrosis, pulmonary fibrosis, idiopathic fibrosing alveolitis and idiopathic pulmonary fibrosis. These four codes combined accounted for nearly 80% of all GP-coded IPF consultations during this period , of whom 9,498 died (of any cause). A slightly higher number, 14,719 had evidence of at least one hospital admission for IPF. Among this population, 10,714 people died. Our study population thus comprised a total of 17,559 individuals who died and had a IPF record in at least one of our three linked datasets  within the time frame of our study. The patient attrition for the three sources is in Supplementary Fig. Sn = 4,304) had evidence of IPF in all three of our linked concurrent data sets  also had at least one clinical code denoting IPF in Aurum prior to death (or within 60 days after their death). Slightly more, 6,651 or 58.9% of the ONS population had been admitted to hospital ostensibly for IPF at some point prior to their death. Although only 38.1%  of our ONS IPF population had evidence of an IPF diagnosis in both Aurum and HES-APC, 74.9%  had IPF records in either Aurum or HES-APC , around a third  had no mention of IPF on their death certificate. The most common underlying reason for death in these patients was pneumonia, bronchopneumonia or malignant neoplasm of unspecified part of bronchus or lung . Other frequently reported causes were chronic obstructive pulmonary disease (COPD) (J44.9), “other general symptoms and signs” (R68.8), and “respiratory failure, not elsewhere classified” (J96.9). For those patients who were hospitalized with IPF but had no mention of IPF on their death certificate , the top three underlying causes were again J18.9, J18.0 and C34.9; for this group other frequently reported causes included myocardial infarction, heart failure and sepsis .Conversely, of the Aurum IPF patients who died  reduced the absolute number of patients in multiple datasets . Similarly, the number patients who were admitted to hospital with a diagnosis for IPF who also died of IPF, reduced to 4,756 or 44.4% of the HES-APC population  when the wider definition of the ONS population was used). Concordance across all three data sets also reduced when measured in terms of the absolute number of patients, from 4,304 to 3,119, which represents just 19.5% of the total study population . On the basis of this analysis, which suggests that a higher proportion of Aurum and HES-APC cases are captured and “confirmed” in ONS when we define an ONS IPF death as one in which IPF is listed anywhere on the death certificate (as opposed to the underlying cause), we adopted the former definition as our gold standard.Restricting our ONS reference population to those who had IPF listed as their underlying cause of death n = 7,09 reducedThe results of our second sensitivity analysis, in which we assessed the concordance of extending our HES-APC case definition to include patients who had episodes of hospital care in which IPF was listed further down the list of clinical diagnoses (beyond the first and second positions) impacted the diagnostic accuracy quite significantly. When we included patients with IPF codes  in the top three diagnostic positions the size of the HES-APC cohort increased from 10,714 to 13,957, but the proportion of patients who also had a record in ONS decreased from 62.1 to 55.8%  but reduced the sensitivity to just 28.2% .Among the diagnostic algorithms based on a broad list of IPF codes (DA1 and DA2), there was little variation in the estimated PPVs. The PPV for DA1 was 64.3%  with a sensitivity of 53.5% . Refining the criteria, that is, restricting cases to people with no known causes of ILD and who also had evidence of a HRCT scan (DA2), had a modest effect on the PPV , but a proportionally greater impact on the number of patients identified. Adding stricter case finding criteria halved the sample size from 1,828 to 891, reducing the sensitivity to under 10% , produced notably higher PPVs compared with the corresponding broad-code-based algorithms (PPVDA8 =81.8%) but a sensitivity of just 8.6% (Table The probability that a person with IPF codes in both Aurum and HES- APC (DA7) “truly” has IPF (measured against a death in ONS), was estimated to be 78.4% , Using a narrow set of Aurum codes plus evidence of a hospitalisation for IPF gave the highest PPV of all eight algorithms (PPVTable DA6=62.4% (95% CI: 61.1–63.0) and the highest by an algorithm based on a limited set of primary care clinical codes coupled with evidence of a hospitalisation for IPF (PPVDA8=to 81.8% (95%CI: 79.5–84.0). While the two IPF patient cohorts defined by these two algorithms do not differ significantly in terms of their demographic characteristics or comorbidity profiles, the former (the secondary care-based cohort) has a slightly lower median survival time, implying that patients with more advanced disease might be overrepresented in a cohort derived using this algorithm.The primary aim of this study was to investigate the diagnostic accuracy of the recording of IPF in UK primary and secondary care records. The lowest PPV was achieved using an algorithm based on secondary care data only  achieved a reasonable PPV of 64.4%. Restricting the code set to a smaller number of highly specific IPF diagnostic codes (DA3) increased the PPV to 74.9%, similar to other conditions , 17. HowAttempts to improve diagnostic accuracy of our baseline broad and narrow clinical code-based algorithms by adding additional criteria produced only marginal improvements in the PPVs but had a significant impact on sensitivity. Adding evidence of a hospital admission had a relatively greater impact on the PPV of our baseline algorithms but without compromising sample size to the same extent. Therefore, we would recommend, that algorithms based on a narrow set of highly specific IPF clinical codes would be appropriate for studies which demand high case validity. For studies where sample size is a primary consideration, and certainly for estimating IPF incidence and prevalence, use of a broader code list combined with evidence of a hospitalisation represents a good compromise between acceptable case validity and capture (DA7).A relatively high validity of a positive IPF diagnosis in primary care is not totally unexpected, even though most GPs will have limited experience of IPF . It is u%) had been hospitalised for IPF prior to their death.The relatively high baseline PPV of a hospitalisation for IPF relative to a record in CPRD Aurum (62% v 64%) against an ONS death was consistent with earlier research which suggests that a high proportion of hospitalised patients who were assigned an IPF diagnostic code did indeed have the disease (c. 80%) . HoweverWe noted some improvement in the PPVs for algorithms which included additional information relative to our baseline clinical-code-only algorithms, but this was relatively modest. This mirrors findings of other validation studies, including one for COPD that likewise reported that requiring evidence of spirometry and/or COPD medications only marginally improved the accuracy of simple diagnostic code-based algorithms (89.4% vs. 87.5% vs. 86.5%) . Other sOver the period of this study, we observed shifts in code use frequencies, most noticeably a gradual decline in the use of codes associated with now obsolete terms such as “cryptogenic fibrosing alveolitis” and a corresponding increase in codes associated with the term “idiopathic pulmonary fibrosis”. It is important to be aware of such trends when selecting appropriate codes for IPF case finding, especially for studies which cover a 10-year-plus time span. While changes in code frequencies related to changes in nomenclature are easier to rationalise, it is likely that some code shifts are being driven by more subtle or nuanced changes in GP coding practice that are harder to predict. Anecdotal evidence suggests that in the UK some GPs select a more generic code to document a given condition, especially if that code appears at the top of a drop-down menu. The shift towards the use of more specific codes for IPF might be a consequence of the introduction of antifibrotics and a desire of GPs to ensure that their patients met the eligibility criteria for prescription of an antifibrotic.This study has several strengths and limitations. Its main strength stems from the use of a large population-based, representative healthcare datasets. Secondly, by selecting a study period which spans the period 2008–2018, we have been able to assess whether there have been any changes in coding practices over time, in the wake of the introduction and adoption of updated international IPF guidelines in 2011/18and the licensed use of anti-fibrotic drugs . The incWe also acknowledge that our recommended approach to case finding likely risks including some cases that have a non-IPF fibrotic ILD. Given a tendency among some healthcare practitioners noted previously to choose less specific codes, this is to a certain extent inevitable and represents a further limitation. We would advise researchers whose study question requires a highly specific IPF study population to also consider excluding people who have known causes of pulmonary fibrosis, such as a connective tissue disease.This is the first study to validate the completeness and quality of the recording of IPF in routinely-collected health data from the UK. It is important to conduct studies of this type and ensure the reliability of coding and case finding in UK electronic health records, given that these data are used to estimate disease prevalence and thus inform health service planning and resource allocation. More specifically, this work has demonstrated that for many observational research purposes, it is acceptable/feasible to use a simple set of clinical codes to define a cohort of patients with IPF in primary care data alone. However, those wishing to conduct observational research using UK health data are advised to tailor their choice of individual codes to their particular research objectives and study design and to consider the relative merits of using linked hospital data to improve diagnostic accuracy of a primary care study population.Additional file 1.


These results provide a new strategy for the development of graphene array-based room-temperature terahertz photodetectors.Graphene, known for its high carrier mobility and broad spectral response range, has proven to be a promising material in photodetection applications. However, its high dark current has limited its application as a high-sensitivity photodetector at room temperature, particularly for the detection of low-energy photons. Our research proposes a new approach for overcoming this challenge by designing lattice antennas with an asymmetric structure for use in combination with high-quality monolayers of graphene. This configuration is capable of sensitive detection of low-energy photons. The results show that the graphene terahertz detector-based microstructure antenna has a responsivity of 29 V·W Terahertz (THz) waves are characterized by frequencies ranging from 0.1 to 10 THz, spanning the region between infrared and microwave regions of the electromagnetic spectrum. The distinct optical and electrical properties of these waves make THz technology highly promising for a range of applications in various domains, such as biomedicine, information security, environmental monitoring, security inspection, non-destructive testing, and defense technology ,3,4,5,6.5 cm2/V·s) for monolayer graphene, a bandgap of zero, broad spectral response, and limited surface dangling bonds, it has garnered significant attention in the field of photonics [Graphene, a seminal two-dimensional material composed of monolayer carbon atoms, has garnered significant interest due to its distinctive structural attributes. Boasting high strength, exceptional conductivity, elevated carrier mobility  from a copper substrate to a substrate. The specific method is as follows: the graphene grown on a copper substrate is coated with polymethyl methacrylate (PMMA) by spin coating, and then the copper substrate is etched as the base with ferric chloride solution. The etching time is about twenty-four hours, and the sample is cleaned with dilute hydrochloric acid. Then, the graphene covered with PMMA is transferred to the prepared Si/SiOrate see .−1, in agreement with previous studies, attesting to its high quality and serving as a basis for the development of high-performance detectors [y axis. It is important that, for computational efficiency, the boundary conditions are set as periodic and the material comprising the antenna is assumed to be a perfect electrical conductor (PEC). The electromagnetic field distribution of the structure is shown in As shown in etectors ,35. GrapThe electrical properties of the device were evaluated with a semiconductor parameter analyzer, as shown in As shown in In addition, the optical response of the device was studied at different power density and bias voltage conditions. In VR) is calculated as:AR represents photocurrent responsivity, r, phI, P0, and S0 represent resistance, photocurrent, power density, and effective area of the detector, respectively [−1 and 29 V·W−1, respectively. Additionally, we validated that the device operates in self-powered mode, with good stability in the photocurrent waveform over time. Applying a small bias voltage of 0.1 V significantly improved the photocurrent, however, graphene’s inherent nature as a semi-metal inevitably increased the dark current, which is detrimental for detecting weak low-energy photons. The asymmetric graphene effectively absorbed THz light under the modulation of grid-like microstructure, generating more photon-generated carriers. Then, the internal potential, driven by the temperature gradient, can effectively drive the nonequilibrium carrier, providing a basis for the device to generate high photocurrent values under zero-bias conditions. Our detector demonstrates high self-powered photo-detection ability under 0.04 THz and 0.12 THz radiation, offering a pre-condition for realizing arrayed monolayer graphene THz detectors. Responsivity is an important indicator for evaluating the sensitivity of photodetectors. Thus, the photocurrent voltage responsivity (ectively . The resj) and dark current-induced noise are considered [Bk is the Boltzmann constant, T is the thermodynamic temperature, r is the device resistance, q is the elementary charge, and darkI is the dark current. The smaller the value of NEP is, the better the performance of the detector will be. The NEP value calculated is only 8.5 pW/Hz1/2 without bias voltage , and equivalent noise power of less than 8.5 pW/Hz1/2 in the self-powered mode. This work provides a viable approach to large-scale device integration and is one of the potential candidates for implementing graphene terahertz detection in focal plane arrays.In summary, by introducing asymmetric optical coupling, we design microstructured antennas to achieve a non-uniform distribution of the terahertz local field. In addition, the artificial grid antenna can focus the terahertz field at the microscale, increasing the interaction between the monolayer graphene and terahertz light and achieving sensitive detection of low-energy photons. At room temperature, the graphene-based photodetector demonstrates a voltage responsivity of 29 V·W


The Iberian harbour porpoise is currently threatened by accidental captures in fisheries (bycatch). Because monitoring cetacean bycatch is particularly challenging, marine mammal stranding networks may provide important information. Between 2000 and 2020, 756 porpoises washed ashore (stranded) on Portuguese and Galician coastlines. The post-mortem analyses of stranded porpoises revealed that the most representative cause of stranding  was fishery interaction and another 10.99% was identified as probable fishery interaction. Combining this information with porpoise annual abundance estimates in Portugal available for the period between 2011 and 2015, an estimated average of 207 porpoises died each year due to bycatch in Portuguese waters alone. This estimate greatly surpassed the maximum annual number of porpoise strandings due to human interactions (22 porpoises) that were predicted to occur without negatively affecting the population in Portuguese waters. To prevent porpoise bycatch in Portugal and Spain, fishing effort management is needed and new activities at sea must be carefully considered. Moreover, appropriate measures directed at the conservation of the Iberian harbour porpoise are crucial to ensure the restoration and survival of the population.The Iberian harbour porpoise population is small and fisheries bycatch has been described as one of its most important threats. Data on harbour porpoise strandings collected by the Portuguese and Galician stranding networks between 2000 and 2020 are indicative of a recent mortality increase in the western Iberian coast . Overall, in Portugal and Galicia, individuals stranded due to confirmed fishery interaction represented 46.98% of all analysed porpoises, and individuals stranded due to probable fishery interaction represented another 10.99% of all analysed porpoises. Considering the Portuguese annual abundance estimates available between 2011 and 2015, it was possible to calculate that an annual average of 207 individuals was removed from the population in Portuguese waters alone, which largely surpasses the potential biological removal (PBR) estimates  for the same period. These results are conservative and bycatch values from strandings are likely underestimated. A structured action plan accounting for new activities at sea is needed to limit the Iberian porpoise population decline. Meanwhile, there is an urgent need for a fishing effort reorganization to directly decrease porpoise mortality. Phocoena phocoena meridionalis, has already been proposed to include the so-called Iberian and Mauritanian harbour porpoise groups [Recent genetic studies revealed that the harbour porpoise population in the upwelling areas of the Iberian Peninsula is small and isolated ,2. In fae groups . The smae groups . The hare groups  and endae groups .Fisheries bycatch has been described as the most important threat to small cetaceans ,7,8, parHowever, estimating bycatch rates from strandings is also problematic. Cetaceans that die at sea do not necessarily strand ashore , and theThe objective of the present study was to assess the evolution of harbour porpoise strandings registered in Portugal and Galicia across two decades (2000–2020), particularly focussing on the temporal evolution of porpoises stranded due to fishery interaction. Finally, we also aimed to estimate an annual porpoise population removal rate based on fishery interactions related to mortality identified in porpoise strandings in Portugal.Data recorded between 2000 and 2020 by the National Strandings Network in Portugal  and the Galicia Strandings Network  were used in the present study.Results concerning Portugal are divided between northern and southern sectors because the northern sector had a more constant and dedicated effort from the regional strandings network since the year 2000 , particularly between 2010 and 2020 (SPVS and University of Aveiro). The southern sector had a more dedicated effort in the Algarve region, between 2010 and 2017 (SPVS) and partly in 2020 (University of Algarve). In areas and periods with no dedicated effort, strandings were monitored by maritime police officers or park rangers. In Galicia, the strandings network’s effort was constant during the study period.Both in Portugal and Galicia, detailed necropsies were performed by trained technicians and the cause of stranding was determined based on full external and internal examination . Cause oNeonates were identified by the presence of vibrissae, foetal folds, or umbilical cord, including cord scars . Calves All strandings locations were plotted in QGIS 3.16.16-Hannover  using WGMatrix heatmaps were built as an initial approach to visualize the monthly, seasonal and long-term temporal variations of the total number of harbour porpoise strandings per geographical area  using ggplot2 package version 3.4.2  in R. LoGeneralized additive models (GAMs) were then used to evaluate the influence of explanatory variables on harbour porpoise strandings along the western Iberian coastline  between 2000 and 2020. All data series were explored for outliers, collinearity, and heterogeneity of variance and plotted for visualization of potential relationships between response and explanatory variables . The numbycatch) was estimated [strandings)) were also estimated based on the number of stranded porpoises evaluated for bycatch evidence (strandings), as described in bycatch), carcass detection rate  and APRstrandings were also recalculated considering the total number of stranded porpoises instead of the number of stranded porpoises evaluated for bycatch evidence. Whereas using the total number of observed porpoise strandings to calculate EAM accounts for possible bycaught individuals which could not be evaluated for bycatch (decomposition stage > 3), a more accurate APR estimate can be obtained considering only the number of stranded porpoises which were evaluated for bycatch evidence, despite the lower number of individuals included in the analysis. All parameters were calculated annually and for the overall period (2011–2015). Estimated mortality rates and the average removal values are expressed as 95% confidence intervals.A deeper analysis was dedicated to porpoise strandings data in Portugal, particularly because porpoise annual population abundances were available from 2011 to 2015  in Portustimated  and is mN) and the respective coefficient of variation (CVN) were obtained from aerial surveys performed in Portuguese waters during the period 2011–2015 [Nmin is the minimum population abundance estimate  estimated by:It was also possible to estimate the potential biological removal (PBR) in Portuguese waters because population abundance estimates (011–2015 . To esti011–2015 ), the fomax is the maximum annual net recruitment rate; f is a recovery factor between 0.1 and 1.Rmax) of 4% is the default value used for small cetaceans [f value for depleted and threatened stocks and stocks of unknown status (0.5) was used instead of the f value for endangered or declining species (0.1) [The maximum net recruitment or population growth rate (Retaceans . In ordees (0.1) . Considering the overall dataset, between 2000 and 2020, 756 stranded harbour porpoises were reported on the western Iberian coast  . p < 0.001), year (p < 0.001) and geographical area (p < 0.001) (r2 = 25.3%) on the number of porpoise strandings in the western Iberian coast. Based on the data analysed, in Portugal, the number of stranded porpoises  was more than double the number of porpoises stranded in Galicia  during the study period. In Portugal, most porpoise strandings were registered in the northern sector (n = 453), while in Galicia the highest densities of strandings occurred in the southern Rias Baixas (n = 115) (GAM results emphasized a significant effect of month (n = 115) . The sign = 115) . With ren = 115) a.p < 0.001) and year (p < 0.001) (r2 = 28.8) on the number of porpoise strandings. On the Portuguese coast, the temporal trend in porpoise strandings was similar to the overall dataset with a significant increase over time  and year (p < 0.026) (r2 = 14.7%) on the number of porpoise strandings. Contrary to the monthly pattern observed in Portugal, the peak of porpoise strandings on the Galician coast occurred in winter (January and February), followed by a sharp decrease until September , 40.96% of the analysed porpoises presented evidence of fishery interaction, and porpoises showing probable evidence of fishery interaction represented another 7.23%, out of the 83 analysed porpoises.strandings of 9.19% (CI = 5.25–16.10%) (bycatch), corresponding to an APRstrandings of 5.55% (CI = 3.17–9.72%)  amounts to 207 individuals, corresponding to an APR–16.10%) . However7–9.72%) .For the period between 2011 and 2015, the overall PBR estimates indicate an “acceptable” average annual removal in Portuguese waters of 22 porpoises (CI: 12–43). The maximum annual PBR estimate of 29 porpoises (CI: 14–60) was obtained in 2013 .The present study revealed that 46.98% of all analysed porpoises in Portugal and Galicia presented evidence of fishery interaction, and another 10.99% of all analysed porpoises presented probable evidence of fishery interaction. Also, we estimated that an annual average of 207 individuals were removed from the population in Portuguese waters alone between 2011 and 2015.Considering the western Iberian Peninsula, over the last two decades, most harbour porpoise strandings were concentrated in the northern sector of the Portuguese coast and in the southern Rias Baixas in Galicia (Spain). The increasing incidence of porpoise strandings particularly during the last decade partly relates to the improved response of the national strandings network in Portugal, as already discussed in other regions ,46,47. NThe number of strandings may be related to biotic  or abiotic variables , but is also dependent on other factors such as stranding probability and detection rate, which in turn are influenced by environmental variables  ,24. A fuAs an example of the potential effect of different variables on porpoise strandings, the highest number of porpoises stranded in Portugal in 2014 (particularly in March and April and then in August) may be related to the higher porpoise abundances estimated in October 2013 . HoweverA concerning amount of porpoises stranded with evidence of fishery interaction in the western Iberia area (46.98%) emphasises the need for bycatch mitigation measures to decrease porpoise mortality. Note that another 10.99% were conservatively identified as stranded due to probable fishery interaction and, furthermore, over half of the stranded porpoises were not considered in the cause of stranding evaluation to avoid inaccurate identification of fishery interaction. Therefore, the global estimate (~47%) is a minimum value for porpoises stranded with evidence of fishery interaction in Portugal and Galicia. It is also noteworthy that when only considering the 264 porpoises analysed for their cause of stranding in the northern region of Portugal (where the largest number of strandings were registered), the proportion of porpoises with evidence of fishery interaction rises to 51.51%. If individuals demonstrating probable fishery interaction were considered, the proportion of porpoises interacting with fisheries in the northern region of Portugal would become 63.63% of all analysed porpoises.Future research is needed to assess changes in seasonal Iberian porpoise distribution and in possible imbalances in sex ratio and differences in age classes across different areas. Currently, no further inferences are possible considering the large number of stranded individuals to which no sex or age class was attributed.Using the annual population abundance estimates obtained between 2011 and 2015 in Portugal , the PBRAttempts to highlight the porpoise bycatch situation in the Iberian Peninsula have been made for years . In 2020Given the importance of fishing-related activities in the study region, the primary threat to the Iberian porpoise population has been identified as bycatch, and at the least, strict implementation of EU directives and fisheries legislation is crucial. Recently, the European Commission called on member states to “Adopt national measures or submit joint recommendations to the Commission to minimise by-catch (or reduce it to the level that enables the full recovery of the populations) by the end of 2023 in the case of the harbour porpoise in Iberian Atlantic” . If we aMeanwhile, there is an urgent need for changes in fishing, particularly in those fisheries with highest bycatch rates, namely the polyvalent fleet using gill and trammel nets and beach seines. Fishing effort management measures should be applied within legal frameworks that prevent fisheries from losing profitability, particularly because legal measures are needed within an effective timeframe.Stranding data are essential to understand the impact of threats, particularly on small and isolated marine populations, and to support science-based conservation and management strategies. In the present study, an increasing number of porpoise strandings was detected in Galicia (Spain) and Portugal. In Portuguese waters, the estimated annual population removal due to bycatch during the evaluated period indicated that the potential biological removal estimates for harbour porpoises was exceeded. There are still many gaps in knowledge concerning the Iberian porpoise population  which should be urgently addressed to understand the potential resilience of this already-depleted population. The present study highlights the urgent need for reliable transboundary conservation and monitoring efforts with appropriate legal support.


Liver abscesses (LAs) are one of the most common and important problems faced by the beef industry. The most efficacious method for the prevention of LAs in North America is through dietary inclusion of low doses of antimicrobial drugs such as tylosin, but the mechanisms by which this treatment prevents LAs are not fully understood. LAs are believed to result from mucosal barrier dysfunction in the gastrointestinal tract (GIT) allowing bacterial translocation to the liver via the portal vein, yet differences in the GIT microbiome of cattle with and without LAs have not been explored. Here, we characterized microbial communities from LAs, rumen, ileum, and colon from the same cattle for the first time.Bifidobacterium spp.—a bacteria commonly associated with a healthy GIT in several species—were more abundant in the rumen and ileum of animals without LAs compared to those with LAs.Results demonstrate that tylosin supplementation was associated with differences in microbial community structure in the rumen and small intestine, largely because of differences in the predominance of Clostridia. Importantly, we show for the first time that microbial communities from multiple LAs in one animal’s liver are highly similar, suggesting that abscesses found at different locations in the liver may originate from a localized source in the GIT (rather than disparate locations). A large portion of abscesses were dominated by microbial taxa that were most abundant in the hindgut. Further, we identified taxa throughout the GIT that were differentially abundant between animals with and without liver abscesses. Together these results provide the first direct comparison of GIT and LA microbial communities within the same animal, add considerable evidence to the hypothesis that some LA microbial communities arise from the hindgut, and suggest that barrier dysfunction throughout the GIT may be the underlying cause of LA formation in cattle.The online version contains supplementary material available at 10.1186/s42523-023-00278-0. Liver abscesses (LAs) are one of the most important and costly problems faced by the beef industry. In addition to condemned products, LAs can cause significant losses in the feedlot. Severe abscessation can reduce carcass yield and grade , 2, and While tylosin is effective in reducing the prevalence of abscesses, the mechanisms by which it prevents LAs are not fully understood. All sequencing-based studies to date have concluded that there is no change in the diversity or composition of LA microbial communities resulting from in-feed supplementation of tylosin –13. AntiFusobacteria necrophorum is widely considered the most common causative agent of LAs given its ubiquitous presence in culture-based investigations of LAs in cattle [Fusobacterium is still the most abundant community member when averaged across all LAs, the most recent investigations of microbial community structures of individual LAs have shown that a large proportion of LAs are actually dominated by members of Bacteroidetes (largely Bacteroides or Porphyromonas); microbial taxa more commonly associated with more distal portions of the GIT [Historically, the accepted pathogenesis of liver abscesses has been attributed to a gut barrier dysfunction (GBD) in the rumen caused by acidosis. Extended feeding diets with high concentrations of fermentable starches are often thought to be the principal cause of this metabolic disorder and subsequent translocation of bacteria to the liver . Fusobacn cattle , 17–19. n cattle . While F the GIT . Yet, toThis study utilized 16S rRNA gene sequencing to (1) investigate the impact of tylosin on microbial communities in LAs, and the rumen, ileum, and colon in the same animals, (2) compare microbial community structure among multiple LAs from one liver, (3) quantify prevalent LA taxa throughout the GIT, and (4) evaluate differences in microbial communities between abscessed and non-abscessed animals in the rumen, ileum, and colon. Importantly, it leverages samples collected as part of a randomized, controlled intervention trial and represents the first to investigate microbial diversity and composition in both multiple GIT locations and LAs of the same animals.Rumen microbial communities. Both the richness and diversity of luminal and epithelial microbial communities in the rumen were lower in animals receiving tylosin supplementation, and except for richness in luminal communities (p = 0.07), the decreases were statistically significant . Based on generalized UniFrac distances, the overall composition of luminal and epithelial communities in the rumen were significantly different between animals that received tylosin supplementation and those that did not .Hierarchal clustering further revealed that there were two major clades of both luminal and epithelial rumen communities. The two clades of luminal communities were largely the result of differences in the relative abundance of Lachnospiraceae Fig. . The clap < 0.05).Across all luminal communities, 15 families comprised more than 1% of the overall community and together these 15 families represented 89.03% of the luminal community in the rumen. Lachnospiraceae was the most abundant family, followed Prevotellaceae, Atopobiaceae, Oscillospiraceae, Methanobacteriaceae, Anaerovoracaceae, Ruminococcaceae, Muribaculaceae, Clostridia UCG-014, Erysipelotrichaceae .Within rumen epithelial communities, the two major clades formed largely on differences in the relative abundance of Succinivibrionaceae and less abundant families Fig. . The clap < 0.05). With the exception of Ruminococcus, all genera exhibited the same response to tylosin supplementation  in both luminal and epithelial rumen communities . Erysipelotrichaceae UCG-009 was more abundant within luminal rumen communities from tylosin supplemented animals but was below 0.5% relative abundance in epithelial communities. Members of the Ruminococcus gauvreauii group and Shuttleworthia were more abundant within luminal and epithelial communities from the rumen of animals receiving tylosin supplementation, but the difference was only significant in luminal communities . Only members of Rikenellaceae RC9 gut group had significantly lower relative abundances in both luminal and epithelial communities. While members of Eubacterium coprostanoligenes group, Eubacterium nodatum group, Family XIII AD3001 group, Bacteroidales RF16 group, F082, NK4A214 group, and Prevotellaceae UCG-001 all decreased, the difference was only statistically significant in either luminal or epithelial communities . In both luminal and epithelial rumen communities, low abundance genera (< 0.5% RA) were significantly less abundant collectively in animals that received tylosin supplementation. Interestingly, most of the differentially abundant genera across all rumen communities were all members of the same class; with 10 of the 18 genera being members of the class Clostridia . While diversity was lower in both luminal and epithelial communities from the ileum of animals that received tylosin, this difference was not statistically significant. Based on generalized UniFrac distances, the composition of luminal communities in the ileum were significantly different between animals given diet supplemented with tylosin and those not receiving tylosin . Despite the lack of statistical significance (p = 0.09) the amount of variation in community composition explained by tylosin supplementation in the epithelium of the ileum (R2 = 0.06) was similar to statistically significant differences in the rumen and lumen of the ileum .Hierarchal clustering further revealed that two clades containing nearly equal numbers of communities formed for luminal communities in the ileum Fig. . The clap < 0.05).In ileum epithelial communities, hierarchal clustering revealed that the two major clades formed based on differing relative abundances of Lachnospiraceae, Peptostreptococcaceae, Clostridiaceae, Atopobiaceae, Erysipelotrichaceae, and Anaerovoracaceae as was the case in luminal communities Fig. . Howeverp < 0.05). In general, there was considerably greater animal to animal variation in community structure in ileum communities compared to the rumen and large intestine. As a result of this variation, there were no genera with statistically higher relative abundance in luminal communities from animals that received tylosin, though some of the most abundant genera  all exhibited a trend of increase relative abundance . Ruminococcus was in significantly lower relative abundance in cattle that received tylosin in both luminal and epithelial communities, while relative abundances of Family XIII AD3001 group and Methanobrevibacter were significantly lower in just luminal communities, and Mogibacterium was significantly lower in epithelial communities only . Collectively, low abundance genera (< 0.5% RA) had significantly lower relative abundance within animals that received tylosin. The majority of differentially abundant genera in the ileum between animals that received tylosin and those that did not belonged to the class Clostridia . Based on generalized UniFrac distances, there was no significant difference in overall community structure in colon luminal or epithelial communities from animals that received tylosin compared to those that did not .There were no significant differences in the richness or diversity of luminal or epithelial communities in the colon of animals that received tylosin compared to those that did not .Within epithelial communities in the colon, the major clades also formed largely as a result of differing relative abundances of Bacteroidaceae Fig. . CommuniOlsenella, Turicibacter, and Methanobrevibacter were in significantly higher relative abundance in luminal and epithelial communities from animals that received tylosin . Similar to the rumen and ileum, low abundance genera (< 0.5% RA) were once again collectively less abundant in luminal and epithelial communities from the colon of animals that received tylosin . Unlike in the rumen and small intestine however, none of the differentially abundant genera in the large intestine belonged to Clostridia . Six genera  each comprised more than 0.5% of the overall community, and collectively they represented 97.5% of the liver abscess community. None of these six genera were differentially abundant between animals that received tylosin and those that did not .Based on generalized UniFrac distances, there were no significant differences in community composition within LA purulent material from animals that received tylosin compared to those that did not . However, Fusobacteriaceae were in significantly higher relative abundance within rumen epithelium communities as compared to colon epithelium communities . Given the sparsity of detection and extremely low relative abundance  of Fusobacteriaceae the biological significance of this finding should be interpreted with caution. Bacteroidaceae was in significantly higher relative abundance within luminal and epithelial communities in the large intestine compared to both the rumen and small intestine . Bacteroidaceae was almost entirely comprised of Bacteroides, and unsurprisingly that genus was also significantly more abundant in the large intestine . In fact, Bacteroides was nearly absent across all rumen (0.011% ± 0.006 SEM) and small intestine communities (0.010% ± 0.005 SEM), while comprising 8.49% ± 0.572 SEM of the overall community in the large intestine. Prevalent families within clade 3 abscess communities were in significantly higher relative abundances within small intestine epithelial communities than rumen and large intestine epithelial communities . Within luminal communities, clade 3 prevalent families were more abundant in the small intestine than both the rumen and colon, and also significantly more abundant in the rumen as compared to large intestine luminal communities .To investigate potential sources of liver abscesses communities, the prevalent families within each of the three liver abscesses community types  were quantified in luminal and epithelial communities of the rumen, small intestine, and large intestine. Fusobacteriaceae was the only prevalent family within clade 1 abscesses and Bacteroidaceae was the only family within clade 2 abscesses. Porphyromonadaceae, Peptostreptococcales-Tissierellales, Peptostreptococcaceae, Actinomycetaceae, and Atopobiaceae were the prevalenet families within clade 3. There were no significant differences in the abundance of Fusobacteriaceae in luminal communities within the rumen, small intestine, or large intestine  were differentially abundant within luminal communities, and only unclassified Bacteroidales was differentially abundant in epithelial communities. Acetitomaculum and unclassified Lachnospiraceae were more abundant in the lumen of the ileum from animals without LAs, while the NK4A214 group was more in luminal communities from the colon of animals with LAs. Bifidobacterium was more abundant within luminal communities in the rumen and ileum of animals without LAs compared to those with LAs . Unclassified Bacteroidales was more abundant within the epithelial communities of the rumen from animals with LAs, and represented the only differentially abundant epithelial taxa .Linear discriminant analysis effect size identified five genera that were differentially abundant between animals with and without LAs in rumen, ileum, or colon communities Fig. . Four gep < 0.05). While a significant PERMDISP suggests that unequal dispersions of variance  is at least partially responsible for the significant PERMANOVA, the distinct clustering of these two groups .Based on generalized UniFrac distances, significant differences in the community composition existed in the lumen of the ileum between animals that did not have liver abscesses and those that had clade 1 (high Fusobacteria) abscesses  were members of the class Clostridia and were also identified as differentially abundant in our study. However, we observed an opposite trend in the abundances of Lachnospiraceae and Erysipelotrichaceae, where these families and the genera comprising them were largely more abundant following tylosin supplementation. A possible explanation for the contrasting results is that the previous study utilized an enzyme-based DNA enrichment method [Ruminococcus, both core members of the rumen microbiome [Tylosin supplementation had no impact on the microbial flora of LAs but did change the composition of microbial communities in the rumen and ileum, largely through influencing the predominance of members of the class Clostridia. In the colon, tylosin impacted a few genera but did not shift overall community composition. Interestingly, members of  rodents —was more rodents , 11–13. piraceae . Interest method  to deplet method , while wt method –25. Regacrobiome , 27 and crobiome . Furthercrobiome –32. Due Bacteroides and the other (clade 3 in this study) comprised of Bacteroides, Porphyromonas, and taxa from other phyla . Given the apparent trial to trial variation in the proportions of different LA community types, there are likely external factors  influencing LA community structure. It is well established that these external factors impact the taxonomy and function of GIT microbial communities in ruminants [Here, we add to the concept that microbial taxa other than members of Fusobacteria dominate the communities within a significant portion of LAs, which we initially demonstrated in a previous trial  and is suminants –36, but Fusobacterium, Porphyromonas) are in extremely low abundance throughout the GIT of cattle. However, the predominance of Bacteroides in the hindgut and its near absence in the rumen of cattle has been demonstrated in numerous studies [Bacteroides is virtually absent from the rumen, suggesting that these LAs with abundant Bacteroides may originate in the hindgut.Bacterial translocation from the lumen of the GIT into the portal vein provides a means for GIT microbiota to seed the liver , and the studies –41, and Bifidobacterium, which is widely considered to exhibit protective effects against GBD in humans and are commonly identified as discriminant of healthy GIT communities [Bifidobacterium spp. are commonly used as probiotics in efforts to reduce GBD-related disease in humans [Bifidobacterium here as the only taxa differentially abundant in two GIT locations from cattle without LAs suggest it may play a similarly protective role again GBD in cattle. As is the case with any 16S rRNA based investigation we must acknowledge the limitations of such work, specifically that we are limited to relative abundance and lack absolute abundance values. Further work investigating the role of GBD throughout the GIT of cattle should incorporate techniques to provide absolute quantification values for important taxa of interest and include complimentary assays to quantify gut inflammatory responses.Alterations in the GIT microbiota (dysbiosis) are ubiquitously found to be associated with gut barrier dysfunction (GBD)-related liver disease in humans , 43, thomunities –47. Due n humans , 49. TheFusobacterium dominated liver abscess communities may in part be seeded from the hindgut of cattle and that multiple abscesses within an individual animal may arise from the same source in the GIT.Dietary inclusion of tylosin (the most common intervention used to prevent and reduce LA occurrence) impacted the microbial communities of the rumen, small intestine, and large intestine of cattle, with the largest impact being observed in the rumen. Furthermore, the first analysis of cattle GIT and liver abscesses microbial communities from the same individual animals suggested that liver abscess communities may be seeded from the hindgut of cattle. The results presented here present the first direct evidence that non-The study reported here was conducted using a subset of animals enrolled in a blinded, randomized controlled trial evaluating the efficacy of different tylosin supplementation strategies for prevention of LAs using methods meeting Good Clinical Research Practice (GCP) expectations. The subset of cattle used in this investigation were part of the animals randomly assigned to two treatment groups at the time they arrived at the feedlot. The two treatment groups were housed separately in different pens, and the finishing diet of one group was supplemented with tylosin phosphate , whereas the diet of the other group was not supplemented with tylosin. Except for this difference, the management protocols were identical for all cattle. The clinical trial methods and management of study animals were reviewed and approved by the West Texas A&M Institutional Animal Care and Use Committee (approved protocol # 2021.08.002) prior to initiation of the study. When cattle reached target harvest weight and body condition, both study groups were harvested on the same day at a federally-inspected commercial abattoir. During carcass evisceration, 10 individual cattle with LAs and 10 without LAs were selected from both treatment groups for inclusion in this study . Purulent material from up to 5 LAs were collected from cattle with LAs, and samples of luminal contents and epithelial surface were collected from rumen, ileum, and colon from all animals. All samples were analyzed using 16S rRNA gene sequencing to characterize the microbial communities, and statistical analyses were performed to address the study objectives. Study personnel involved in cattle management, sampling, analysis, and data interpretation were blinded to treatment group assignments throughout the study, from enrollment until after data analyses were completed.Bos taurus steers  with minimal Bos indicus influence that weighed 800–950 lb at the time of purchase and were previously raised without substantial grain supplementation. After shipping to a very large commercial feedlot located in Colorado, individual cattle were randomly assigned to one of four study groups, with cattle housed separately by group. The subset of cattle selected for this study were enrolled at the same time, including animals from 10 different marketing groups that were randomly assigned by computer algorithm to pens that housed ~ 250 animals each. At the time of arrival, each animal received a unique identification tag, weighed individually, and administered standard preventive treatments including anthelmintics (ivermectin and fenbendazole), vaccines targeting respiratory disease agents and clostridial bacteria (Bovishield® IBR/BVD Gold and UltraChoice® 7), and a hormone implant to optimize weight gain and feed efficiency . Care and management of animals was overseen by the cattle managers and a veterinarian. Additionally, trained animal healthcare personnel visually evaluated every animal daily while riding through pens on horseback. Animals that were potentially ill or injured were moved to the dedicated veterinary facility for examination, and animals meeting established case definitions were treated under the supervision of the feedlot veterinarian using standardized treatment protocols.Cattle enrolled in the clinical trial were purchased through commercial marketing sources, targeting English and continental beef breed The feedlot facilities were constructed to meet or exceed industry standards for beef feedlots, including pens with 130–160 sq-ft of housing space per animal and 7–9 in of linear space of feed bunk per animal. Clean drinking water was provided ad libitum. Diets fed to animals were formulated by animal nutritionists to meet or exceed National Academies of Science nutritional requirements for growing beef cattle, , and ratAll cattle were fed the same diets, except for differences in supplementation with the test article, tylosin phosphate, according to treatment group assignments. At the time of arrival, cattle were fed starting ration that was high in forage (hay and silage) until enrollment for ~ 2 weeks, at which time a series of rations were introduced over ~ 21d, which gradually increased the amount of starch so as to prevent the occurrence of acute ruminal acidosis. At that time, the finishing rations for the treatment groups was introduced, including supplementation with tylosin phosphate at 90 mg/animal/day that treatment group. Finishing rations consisted of steam-flaked corn as the grain constituent, corn silage as roughage, dry distillers grains as a protein supplement, whey permeate, a fat source, and supplement containing mineral and vitamins. Monensin (an ionophore) was included in all diets (30 mg/kg of dietary dry matter) to improve feed efficiency, per industry standards. Feed was delivered twice daily in specialized feed trucks containing onboard scales and computers to ensure accurate delivery of appropriate amounts of feed, and feed consumption was adjusted at least biweekly to ensure appropriate supplementation of tylosin phosphate to meet the intended dosing (90 mg/hd/day). At 33d prior to harvest, ractopamine hydrochloride  was supplemented in all diets to improve rate of weight gain, feed efficiency, and carcass leanness; this supplementation was removed from rations 2d prior to slaughter, per FDA regulations. It should be noted that monensin and ractopamine supplementation have not been demonstrated to be associated with significant changes in the occurrence of LAs. Nutrient composition of rations were analyzed throughout the study to indirectly assess and refine delivery of the targeted amount of tylosin phosphate in final mixed rations by comparison to the target composition of the ration formula.Cattle were on feed for 185 days prior harvest, and their average daily gain in body weight over this period was 3.0 lbs per day. The average live body weight of cattle at harvest was 1464 lbs for the untreated pen and 1468 lbs for the pen treated with tylosin. At harvest, the average hot carcass weight (standard deviation) for the two pens was 931 lbs (74 lbs) and 928 lbs (73 lbs), respectively.Study cattle were euthanized and harvested at a USDA–FSIS inspected commercial abattoir using approved and supervised methods. All animals from each study group were processed separately, and during carcass evisceration, a study investigator (PSM) selected by convenience 10 individual cattle with LAs and 10 without LAs from both treatment groups for inclusion in this study . Among those with LAs, cattle with severe abscessation (A or A + based on the Elanco scoring system) were preferentially selected for inclusion. A study ID number was assigned to each animal at the time of selection and used to identify all samples collected from the same individuals. Blocks of liver tissue containing abscesses were excised, placed in sterile bags, and refrigerated on ice. The rumen was opened onsite at the slaughter facility, and samples of luminal content and mucosal surface swabs , were collected aseptically from the ventral rumen sac, placed in sterile tubes, and refrigerated on ice. Approximately 20 cm proximal to the cecum, sections of ileum (15–20 cm in length) were tied off and excised, placed in sterile bags, and refrigerated on ice. Similarly, 15–20 cm of the distal colon loop was tied off, excised, placed in sterile bags, and refrigerated on ice. All samples were transported to laboratory facilities at Colorado State University for further processing within 6 h of collection.Prior to collecting samples for sequencing, external surfaces of tissues were flame sterilized after spraying with 70% ethanol. Tissues were opened using sterile disposable scalpels, and LA purulent material and intestinal contents were collected aseptically using sterile disposable spatulas and placed in sterile cryotubes. A total of 53 abscesses, up to 5 per steer, were collected from the livers of the 20 cattle with LAs. A large (1.3 cm diameter) rayon tipped swab was used to aseptically sample the epithelial surfaces of the ileum and colon and placed in sterile tubes. Epithelial surfaces were not rinsed prior to swabbing to reduce contamination potential, but this does mean there was potential for luminal community members to be present. However, given significant differences in community composition between luminal and epithelial communities in the rumen (see below) we don’t believe this has impact our conclusions. All samples were frozen at − 80 °C after collection until further processed for sequencing.DNA was isolated from all samples using the QIAamp PowerFecal Pro DNA kit and a QIAcube Connect  automated isolation system for nucleic acid recovery according to the manufacturer’s instructions. Following isolation, DNA was quantified (ng·μL-1) using a Qubit Flex fluorometer . An extraction blank was included for each sample type  that were included in subsequent library preparation steps and sequencing.The V3-V4 region of the 16S rRNA gene was amplified using the 341f/785r primer pair  and 400 p > 0.05), and no difference in the number of ASVs per sample between liver abscesses classified as high Fusobacteria or high Bacteroidetes . Similarly, there was no significant difference in the number of ASVs per samples between animals receiving tylosin and those that did not . Over 91% of all ASVs were classified at the level of genus and > 99% of all ASVs were classified at the ranks of family, order, class, and phylum from all four body sites sampled . Luminal and epithelial communities were significantly different from each other in the rumen , and as such all luminal and epithelial communities were analyzed separately.Demultiplexed 16S rRNA gene sequence reads were imported into QIIME2 version 2022.2 . BecauseRichness (observed ASVs) and Faith’s phylogenetic distance (FPD) were calculated for all remaining samples n = 256) with phyloseq and the ‘estimate_pd’ function from the btools package. ASV counts were then normalized using cumulative sum scaling  with phy, 58. FroDifferentially abundant genera were identified in the rumen, ileum, and colon between animals with and without LAs using linear discriminant analysis effect size (LEfSe) performed with the online LEfSe tool on the Galaxy server under default settings. In an effort to avoid potential false positives, a more conservative pairwise Wilcoxon rank-sum test was used to statistically test differences genera identified as differentially abundant with LEfSe between animals with and without LAs.Unless specified otherwise, R version 4.2.1 was used for statistical analysis of data. Kruskal–Wallis analysis of variance (for comparisons between 2 variables) or Pairwise Wilcoxon rank-sum tests were performed with a Benjamini–Hochberg correction for multiple comparisons (for comparisons between more than 2 variables). Differences in beta-diversity were tested using pairwise PERMANOVA with a Benjamini–Hochberg correction for multiple comparisons and 9999 permutations. Additionally, pairwise PERMDISPs were carried out for all significant PERMANOVA outcomes using 9999 permutations to test for differences in the variability of dispersions.Additional file 1. Table S1 Relative abundances of taxonomic families comprising more than 1% of the overall community across all rumen samples. The mean relative abundance and standard error of the mean are displayed for each family from luminal and epithelial communities of the rumen from animals that received tylosin supplementation and those that did not. Significant p-values are bold .Additional file 2. Table S2 Taxonomic information for the 20 genera differentially abundant across all sample sites between animals receiving tylosin supplementation and those that did not.Additional file 3. Table S3 Relative abundances of taxonomic families comprising more than 1% of the overall community across all small intestine samples. The mean relative abundance and standard error of the mean are displayed for each family from luminal and epithelial communities of the small intestine from animals that received tylosin supplementation and those that did not. Significant p-values are bolded .Additional file 4. Table S4 Relative abundances of taxonomic families comprising more than 1% of the overall community across all large intestine samples. The mean relative abundance and standard error of the mean are displayed for each family from luminal and epithelial communities of the small intestine from animals that received tylosin supplementation and those that did not. Significant p-values are bolded .Additional file 5. Fig. S1 Non-metric multidimensional scaling (NMDS) of generalized UniFrac distances illustrates differences in overall microbial community structure of liver abscesses between treatment groups. The NMDS demonstrates clustering of 16S rRNA gene sequences from animals that received tylosin (purple) and those that did not (gold). There were no significant differences in overall community composition . The bar plot demonstrates the mean relative abundance of all genera within liver abscesses that comprised at least 0.5% of the overall microbial community between animals that received tylosin supplementation and those that did not. Error bars represent the standard error of the mean. There were no significant differences detected . To limit the effect of some animals having multiple  abscess communities, values were based on normalized ASV counts generated by averaging counts from each liver abscess within an individual animal.Additional file 6. Fig. S2 Bar plots demonstrating the relative abundances of taxonomic families within purulent material from individual abscesses (n = 47) from the 14 animals that contained multiple liver abscesses were collected. The eight most abundant families are displayed in the legend.Additional file 7. Fig. S3 Boxplots demonstrating the relative abundance of Bacteroides across luminal and epithelial microbial communities in the rumen, ileum, and colon. Significant differences are illustrated by different letters .


Sarcoidosis is a granulomatous disease involving multiple organ systems. In its classic form, sarcoidosis is associated with non-caseating granuloma. Several differential diagnostic entities exist for sarcoidosis, including tuberculosis (TB), as well as lymphomas. In this report, we present a case of sarcoidosis in a 55-year-old male with diffuse lymphadenopathy and hepatosplenic involvement, highlighting the differential diagnostic point for this disease. Sarcoidosis is a multisystem disease with an unclear etiology with pulmonary and extrapulmonary manifestations. Extrapulmonary sarcoidosis can impact the lymph nodes, leading to lymphadenopathy, and systemic features may include fever, malaise, night sweats, and dry cough. Non-caseating granuloma is found to be most commonly associated with sarcoidosis . LymphomHere, we present a case of a 55-year-old male with initial clinical features and physical findings suggestive of lymphoma. Further investigation, including lymph node excisional biopsy, resulted in a diagnosis of sarcoidosis with extrapulmonary involvement.A 55-year-old male with diabetes was hospitalized for an infected diabetic foot ulcer. Initial vitals were unremarkable. Physical examination was significant for diffuse, palpable, non-tender lymphadenopathy and right second toe ulcer.Laboratory results showed a significant elevation of erythrocyte sedimentation rate (ESR) of 55 mm/hour (reference range: 0-20 mm/hour) and C-reactive protein (CRP) of 9 mg/L (reference range: 0-8 mg/L). Chest X-ray Figure  revealedOn further history-taking, he reported unquantified unintentional weight loss and intermittent night sweats for the past three years. He denied fever, chills, cough, or other upper respiratory infection symptoms. He works as a bulldozer mechanic, lives with two dogs and nine cats, and denied tobacco, alcohol, and illicit drug use. He was born in the Caribbean and moved to the United States over 30 years ago, and traveled back and forth often.CT of the abdomen and pelvis with contrast revealed innumerable hypodense lesions throughout the liver and spleen Figure , 1D withMycobacterium tuberculosis (MTB) polymerase chain reaction (PCR) was negative  and non-HL (NHL) . TypicalAlthough sarcoidosis and lymphoma are two seemingly separate entities, there have been reports in the literature that sarcoidosis may occur together with lymphoma, mainly NHL, but usually, lymphoma occurs after sarcoidosis with a median interval of 24 months. It was named sarcoidosis-lymphoma syndrome by Brincker in 1986. It was also found that there is a fivefold increased risk of developing lymphoma among patients with chronic active sarcoidosis [Mycobacterium tuberculosis (MTB). The major type of TB is pulmonary TB, while extrapulmonary TB accounts for approximately 20%-30% of all TB cases, with lymphadenitis being the most common form [Tuberculosis (TB) is another common granulomatous disease. TB is a contagious disease caused by mon form ,11. Consmon form ,13.Other differential diagnoses to consider include berylliosis, granulomatous arteritis, cat scratch disease, and fungal infection .Both lymphoma and sarcoidosis can present with B symptoms, diffuse lymphadenopathy, and hepatosplenic involvement. Differentiating these two diseases become more challenging, and rarely, lymphoma and sarcoidosis coexist. Although rare, sarcoidosis should be considered a differential diagnosis entity in the presence of lymphadenopathy and a clinical picture that is overall suggestive of lymphoma.


The Accreditation Council for Graduate Medical Education (ACGME) and the Infectious Diseases Society of America (IDSA) require infectious diseases fellowship programs to include a core curriculum as part of their requirements for clinical training, though recommendations regarding content and structure are limited. Despite advances in online tools available for asynchronous learning in medical education, there remains a scarcity of published ready-to-teach content. We hypothesized that case-based clinical reasoning sessions covering core clinical topics would improve the attitudes and confidence of infectious diseases fellows.We conducted an informal needs assessment of current fellows, reviewed published recommendations and other fellowship curricula. Then, we developed a series of case-based clinical reasoning sessions for clinical and research fellows based on constructivist theory and emphasizing adult learning principles. Topics were rooted in published guidelines and evidence-based practice, prioritizing complex and high-yield clinical topics. Content was peer-reviewed by faculty and sessions were facilitated by senior fellows. Facilitation guides were created to allow for dissemination in the future. Faculty and pharmacists attended the sessions as expert discussants. Fellows completed anonymous surveys assessing attitudes and confidence after each session.Three sessions covering Endemic Mycoses, Non-tuberculous Mycobacteria, and Toxoplasmosis were completed with 6-8 fellows in attendance at each. Surveyed fellows felt that the sessions were valuable and improved their confidence in clinical management of the topics presented . Participants also provided narrative feedback (Table 1).Post-session Survey ResultsPost-session Narrative FeedbackThis curriculum represents a novel initiative by trainee physicians to create a series of peer-facilitated, discussion-based sessions for learners at the level of subspecialty fellowship. We demonstrated that these sessions improve the attitudes and confidence of fellows in a single-program pilot. Future directions will include studying the intervention in other fellowship programs and sharing this program broadly within the infectious diseases education community.Eileen K. Maziarz, MD, Karius, Inc: Advisor/Consultant


ChatGPT, an artificial intelligence (AI) system powered by large-scale language models, has garnered significant interest in healthcare. Its performance dependent on the quality and quantity of training data available for a specific language, with the majority of it being in English. Therefore, its effectiveness in processing the Chinese language, which has fewer data available, warrants further investigation. This study aims to assess the of ChatGPT’s ability in medical education and clinical decision-making within the Chinese context. We utilized a dataset from the Chinese National Medical Licensing Examination (NMLE) to assess ChatGPT-4’s proficiency in medical knowledge in Chinese. Performance indicators, including score, accuracy, and concordance (confirmation of answers through explanation), were employed to evaluate ChatGPT’s effectiveness in both original and encoded medical questions. Additionally, we translated the original Chinese questions into English to explore potential avenues for improvement. ChatGPT scored 442/600 for original questions in Chinese, surpassing the passing threshold of 360/600. However, ChatGPT demonstrated reduced accuracy in addressing open-ended questions, with an overall accuracy rate of 47.7%. Despite this, ChatGPT displayed commendable consistency, achieving a 75% concordance rate across all case analysis questions. Moreover, translating Chinese case analysis questions into English yielded only marginal improvements in ChatGPT’s performance (p = 0.728). ChatGPT exhibits remarkable precision and reliability when handling the NMLE in Chinese. Translation of NMLE questions from Chinese to English does not yield an improvement in ChatGPT’s performance. Artificial Intelligence (AI) has been making significant strides in various fields, including healthcare. This study examines the proficiency of an AI system known as ChatGPT in understanding and responding to medical exam questions posed in the Chinese language. The researchers used a dataset from the Chinese National Medical Licensing Examination (NMLE) and found that ChatGPT performed well, scoring above the passing threshold. However, when the questions were altered to be more open-ended, the AI’s performance declined significantly. Interestingly, translating the questions into English did not improve its performance. This research highlights the potential of AI, like ChatGPT, in assisting with medical education and clinical decision-making, particularly within a Chinese context. However, the findings also emphasize the need for further improvements, particularly for handling more complex, open-ended queries. The study contributes to understanding how AI can be effectively employed in non-English medical settings, and can be a steppingstone for future research in this area. AI  has gained significant influence in recent years by simulating human intelligence and cognitive processes to tackle complex problems . TrainedChatGPT shows promise in medical education, performing well in Certified Public Accountant (CPA) exams and generating accurate responses to complex inputs . AppliedGiven this background, this study aims to evaluate ChatGPT’s performance on the Chinese NMLE conducted within the Chinese context.ChatGPT is an advanced language model that leverages self-attention mechanisms and extensive training data to deliver natural language responses within conversational settings. Its primary strengths include managing long-range dependencies and producing coherent, contextually appropriate responses. Nevertheless, it is essential to recognize that GPT-4 is a server-based language model without internet browsing or search capabilities. Consequently, all generated responses rely solely on the abstract associations between words, or "tokens," within its neural network .S1 Table) and considered as original questions. These questions are divided into four units, with each question worth one point.The official website does not release the 2022 NMLE test questions. However, for this study, a complete set of 600 questions, with a total value of 600 points, is available online : These questions are distributed across all units, including Unit 1 (n = 108), Unit 2 (n = 82), Unit 3 (n = 79), and Unit 4 (n = 71). They aim to evaluate basic science knowledge in physiology, biochemistry, pathology, and medical humanities. Each question has four choices, and the AI must select the single correct answer. An example from Unit 1 is: "What type of hypoxia is likely to be caused by long-term consumption of pickled foods? A. Hypoxia of blood type B. Hypoxia of tissue type C. Circulatory hypoxia D. Anoxic hypoxia E. Hypoxia of hypotonic type."Case Analysis Questions (n = 260): These questions are also distributed across all units, including Unit 1 (n = 42), Unit 2 (n = 68), Unit 3 (n = 71), and Unit 4 (n = 79). These questions, employed in clinical medicine, examine and evaluate patient cases through a thorough review of medical history, symptoms, and diagnostic findings to determine a diagnosis and treatment plan. Each question has four choices, and the AI must select the single correct answer. An example from Unit 1 is: "A 28-year-old male complains of muscle and joint pain in his limbs three days after diving. He experienced respiratory equipment failure during diving three days ago and immediately ascended rapidly to the surface. Subsequently, he experienced symptoms such as dizziness, orientation disorder, nausea, and vomiting. After rest and oxygen inhalation, the symptoms improved, but he continued to experience persistent muscle spasms, convulsions, and joint pain in his limbs. Therefore, what is the most likely cause of the patient’s pain? A. Chronic inflammation and cell infiltration B. Stress ulcers C. Local tissue coagulative necrosis D. Increased carbon dioxide concentration in the blood E. Gas embolism in the blood vessel lumen."We assembled a dataset of NMLE questions and their corresponding answers, maintaining validity by cross-verification with senior medical professionals. This dataset was used to evaluate ChatGPT’s performance on the exam by comparing its responses to the standard answers and calculating the scores it achieved. A high score would indicate that ChatGPT effectively tackled this task.Moreover, the passing score for the NMLE, established by the Department of Health’s Board of Medical Examiners, is intended to determine whether an individual possesses the necessary skills to practice medicine independently and safely. In the 2022 Chinese NMLE, the passing score was set as 360. As such, ChatGPT’s performance will be evaluated based on this benchmark.To better reflect the actual clinical situation, we modified the case analysis questions to be open-ended. Questions were formatted by deleting all the choices and adding a variable lead-in imperative or interrogative phrase, requiring ChatGPT to provide a rationale for the answer choice. Examples include: "What could be the most plausible explanation for the patient’s nocturnal symptoms? Justify your answer for each option," and "Which mechanism is most likely responsible for the most fitting pharmacotherapy for this patient? Provide an explanation for its correctness."However, a unique subset of questions, which required selecting from provided choices, could not be encoded in the same open-ended manner. These questions required selecting one provided choice, so we transformed them into a special form (n = 3). For example, the original question, "Which can inhibit insulin secretion? A. Increased free fatty acids in blood B. Increased gastric inhibitory peptide secretion C. Sympathetic nerve excitation D. Growth hormone secretion increases" was encoded as "Can an increase in free fatty acids in the blood, an increase in gastric inhibitory peptide secretion, an increase in sympathetic nerve excitation, or an increase in growth hormone secretion inhibit insulin secretion?" This encoding was present only in Unit 1.To minimize the potential for ChatGPT to ’remember’ previous answers and bias its responses, a new chat session was initiated for each question.S2 Table). To train the physician adjudicators, a subset of 20 questions was used. ChatGPT’s responses were categorized into three categories: accurate, inaccurate, and indeterminate. Accurate responses indicated that ChatGPT provided the correct answer, while inaccurate responses encompassed no answer, incorrect answers, or multiple answers with incorrect options. Indeterminate responses implied that the AI output did not provide a definitive answer selection or believed there was insufficient information to do so. Concordance was defined as when ChatGPT’s explanation confirmed its provided answer, while discordant explanations contradicted the answer.In our study, two physicians, blinded to each other’s assessments, independently scored the AI outputs from the two types of encoders for accuracy and concordance. Scoring was based on predefined criteria (S3 Table).To minimize within-item anchoring bias, adjudicators first evaluated accuracy for all items, followed by concordance. Two physicians were blinded to each other’s evaluations. In cases where the two initial physicians disagreed on a score, a third physician adjudicator was consulted. Ultimately, 17 items (2.7% of the dataset) required the intervention of a third physician adjudicator. The interrater agreement between the physicians was assessed using the Cohen kappa (κ) statistic for the questions (A schematic overview of the study protocol is provided in S4 Table). We compared the scores obtained from the original questions to those from the translated questions and employed the chi-square test to determine performance improvement.To evaluate if translating questions from Chinese to English could enhance ChatGPT’s performance, We utilized ChatGPT to translate the case analysis questions, which had not yet been encoded for the AI’s testing. We then assessed ChatGPT’s performance on the translated exam by comparing its responses to standard answers and calculating its scores  statistic to assess the degree of consensus among physicians. This was achieved by comparing the responses of ChatGPT to the standard answers. In cases where the initial two physician adjudicators disagreed on a score, a third physician was consulted. Following this, we examined if translating questions from Chinese to English could potentially enhance ChatGPT’s performance. We juxtaposed ChatGPT’s performance on the original and translated questions, and utilized the chi-square test to determine if there were any statistically significant performance improvements.All statistical calculations were performed using the SPSS software package.We believe these revisions address your comments adequately and we eagerly await any other feedback you may have.In the Chinese NMLE, ChatGPT correctly answered 442 (73.67%) out of 600 items, a score significantly higher than the passing threshold of 360 as defined by official agencies.2 = 0.66, p = 0.883).The score of each unit is shown in Fig 3A). Compared to the original questions, the accuracy of the encoded questions decreased by 40.5%, 9.7%, 32.4%, and 41.8% for Units 1, 2, 3, and 4, respectively .Test questions were encoded as open-ended for case analysis questions, simulating scenarios where a student poses a common medical question without answer choices or a doctor diagnoses a patient based on multimodal clinical data . The accuracy was 40.5%, 60.3%, 42.3%, and 34.2% for Units 1, 2, 3, and 4, respectively .Fig 4). Furthermore, we examined the concordance difference between correct and incorrect answers, discovering that concordance was perfect and significantly higher among accurate responses compared with inaccurate ones  .Concordance is a measure of the agreement or similarity between the option selected by AI and its subsequent explanation. The results indicated that ChatGPT maintained a >75% concordance across all questions, and this high level of concordance was consistent across all four units . This suggests that ChatGPT’s performance when facing questions in Chinese may not be improved by translating them into English, and solely relying on translating other languages into English and building a database exclusively in English may not be the most effective approach.After translating the original case analysis questions in Chinese into English to explore a potential way to improve ChatGPT’s performance, the improvement for Units 1, 2, 3, and 4 was minimal, with only one point gained in each unit. The total number of correct answers increased from 256 to 260. The accuracy improvement for translated case analysis questions was subtle  ChatGPT’s score is satisfactory but requires improvement when addressing questions posed in the Chinese language; and (2) Translation into English may not improve ChatGPT’s performance. This study provides new evidence for the ability of ChatGPT in medical education and clinical decision-making within the Chinese context, offering valuable insights into the applicability of AI language models for non-English medical education settings and laying the groundwork for future research in this area.In the Chinese NMLE, ChatGPT achieved a score of 442 (73.67%), exceeding the passing requirement of 360 points for the exam in the Chinese language. In the 2022 NMLE, the average score of 65 medical students was 412.7 (68.7%), with a minimum score of 295 (49.2%) and a maximum score of 474 (79.0%). According to the statistics, the national pass rate for the exam in 2022 was 55%. When compared to medical students who have undergone a traditional 5-year medical education and a one-year internship, ChatGPT’s performance is currently satisfactory, however, there is still potential for improvement. Several underlying reasons may be responsible for this, such as: 1) Limitations in training data: If ChatGPT’s training data contains less information about the Chinese medical field, its performance when handling Chinese medical questions could be impacted, resulting in a lower accuracy rate for such queries. 2) Knowledge updates: With a knowledge cutoff date in September 2021, the most recent developments in the Chinese medical field may not have been adequately learned by the model, affecting its accuracy when answering Chinese medical questions.Moreover, we observed that outputs with high accuracy showed high concordance, while lower accuracy was associated with reduced concordance. Consequently, we speculate that ChatGPT’s inaccurate responses primarily arise from missing information, leading to indetermination or inaccurate in the AI. Language models like ChatGPT are built on vast amounts of text, and their accuracy depends on the quality and diversity of their training data . When thChatGPT’s performance varies across languages due to different factors, primarily the quality and quantity of the training data available in each language . EnglishTherefore, in order to accurately assess the performance of an AI model like ChatGPT for a particular language or task, a more targeted evaluation may be necessary. This would involve conducting a thorough analysis of the AI’s performance using a diverse range of tasks and data sources that are representative of the language in question.ChatGPT-3.5 achieved near-passing threshold accuracy of 60% on the United States Medical Licensing Exam . FurtherDespite the promising potential of AI in medicine, it also faces several challenges. The development of standards for AI use in healthcare is still necessary ,15, encoSeveral limitations should be noted. Firstly, clinical tasks are highly complicated, the exams cannot fully stimulate the problems in clinical practices. Secondly, the limited input sample size may preclude us from performing analyses in depth and range, potentially limiting the generalizability of our findings. Thus, before large-scale application of AI based on Large Language Models in medical education or clinical practice, their utility should be further studied under real-world conditions.Moreover, we acknowledge the potential benefits of diversifying datasets in our analysis and evaluating examples. However, due to time limitations during data collection and analysis, we exclusively used the one-year NMLE dataset in our study. Our research was conducted after September 2022 when the ChatGPT dataset was updated. To avoid encountering the same questions used for training purposes, we relied solely on data from 2022 onwards. Additionally, since questions for the year 2023 were unavailable at the time of our research, we were constrained to using only one year’s worth of medical licensing examination questions.ChatGPT demonstrated impressive performance on the Chinese NMLE, exceeding the passing threshold and exhibiting high internal consistency. Nevertheless, its performance waned when faced with open-ended encoded questions. Translation into English did not substantially boost its performance. The findings emphasize ChatGPT’s ability for comprehensible reasoning in medical education and clinical decision-making in Chinese.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


Ab cause high mortality in hospitalized patients. Treatment options are limited, but now include FDC, a novel siderophore cephalosporin. Following clinical implementation, cases of treatment-emergent resistance have been reported. Proposed resistance mechanisms include alterations in iron transporters and amino acid substitutions in Acinetobacter-derived AmpC β-lactamase (ADC). Our objective was to define the relative contribution of ADC amino acid substitution to reduced FDC susceptibility against CRAb clinical strains.Infections due to CRAb isolates from the US and Italy. We compared common variants  to variants from FDC-resistant strains that had not been exposed to FDC , and variants from FDC-resistant strains collected following FDC treatment . blaADC-30 was used as a reference. The ADCbla genes with an upstream ribosomal binding site from pET24 were subcloned into pBCSK (-), transformed into E. coli Top10 cells, and selected on plates containing 100 µg/ml ampicillin and 30 µg/ml chloramphenicol. The constructs’ sequences were verified by Sanger sequencing. Minimum inhibitory concentrations (MICs) of FDC were determined by broth microdilution in iron-depleted media at least in duplicate.Variant ADCs were identified from whole genome sequencing analysis of clinical CRAmong studied ADC sequences, substitutions that were located in the Ω- or R2-loop regions, which accommodate the binding of cephalosporins, resulted in a 2- to 8-fold change in FDC MIC . When both regions were affected, there was an 8- to 16-fold increase in MIC. No significant MIC changes occurred among the studied mutations outside of these regions (Table 1). R2-loop substitutions were observed in FDC-exposed resistant strains, whereas Ω-loop mutations were observed in resistant strains that had not been exposed to FDC.Figure 1.Alignment of amino acid sequences in the ADC Ω- and R2- loop regions among studied isolates.We identified amino acid substitutions in Ω- and R2-loop regions of ADC that contribute to increased FDC MIC. Given the broad diversity of currently circulating ADC variants, it is crucial to study the impact of sequence variation on the activity of novel agents.Robert A. bonomo, MD, Entasis, Merck, VenatoRx, Wockhardt: Grant/Research Support Marco Falcone, MD, PhD, Gilead: Board Member|Gilead: Honoraria|Menarini: Board Member|Menarini: Grant/Research Support|Menarini: Honoraria|MSD: Board Member|MSD: Grant/Research Support|MSD: Honoraria|Nordic Pharma: Honoraria|Pfizer: Board Member|Pfizer: Honoraria|Shionogi: Honoraria Ryan K. Shields, PharmD, MS, Allergan: Advisor/Consultant|Cidara: Advisor/Consultant|Entasis: Advisor/Consultant|GSK: Advisor/Consultant|Melinta: Advisor/Consultant|Melinta: Grant/Research Support|Menarini: Advisor/Consultant|Merck: Advisor/Consultant|Merck: Grant/Research Support|Pfizer: Advisor/Consultant|Roche: Grant/Research Support|Shionogi: Advisor/Consultant|Shionogi: Grant/Research Support|Utility: Advisor/Consultant|Venatorx: Advisor/Consultant|Venatorx: Grant/Research Support Yohei Doi, MD, PHD, bioMerieux: Advisor/Consultant|FujiFilm: Advisor/Consultant|Gilead: Advisor/Consultant|Gilead: Honoraria|GSK: Advisor/Consultant|Meiji Seika Pharma: Advisor/Consultant|Moderna: Advisor/Consultant|Moderna: Honoraria|MSD: Advisor/Consultant|MSD: Honoraria|Shionogi: Advisor/Consultant|Shionogi: Grant/Research Support|Shionogi: Honoraria