Misdiagnosis and delayed diagnosis in progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) are common. Few studies have systematically evaluated the diagnostic process from symptom onset to death in representative cohorts.n = 28/2) and age-sex matched Parkinson’s disease (PD) cases (n = 30) were identified from a UK prospective incident Parkinsonism cohort. Medical and research records were reviewed to compare median times from first index symptom to key diagnostic milestones and the nature/timing of secondary care referral and review.All PSP/CBD cases (p < 0.001) and more impaired balance (p = 0.008) and falls (p = 0.004) in PSP/CBD. PD was diagnosed a median 0.96 years after index symptom. In PSP/CBD the median times from index symptom to identifying parkinsonism and then including PSP/CBD in the differential diagnosis and the final diagnosis were 1.88, 3.41 and 4.03 years, respectively . Survival from symptom onset in PSP/CBD and PD was not significantly different . More diagnoses (p < 0.001) were considered in PSP/CBD. Prior to diagnosis, PSP/CBD patients had more recurrent emergency attendances  and were referred to more specialities than PD (median 5 vs 2). Time to any outpatient referral  and to specialist movement disorder review  was longer in PSP/CBD.Index symptoms were similar apart from more tremor in PD (The duration and complexity of the diagnostic journey were greater in PSP/CBD than age-sex matched PD but can be improved. In this older cohort, there was little difference in survival from symptom onset in PSP/CBD and age-sex matched PD.The online version contains supplementary material available at 10.1007/s00415-023-11629-x. Progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) are rare, neurodegenerative tauopathies. Clinicopathological studies highlight the extent of misdiagnosis in PSP and CBD –4, but aThe PINE study is an incident cohort of PD and other forms of parkinsonism with prospective life-long annual follow-up in the North-East of Scotland . MultiplAll incident cases with a final clinical (or post-mortem) diagnosis of PSP or CBD in the PINE study were included in the study sample. Although the term CBD is increasingly restricted to pathologically confirmed cases due to the diversity of clinical presentations in those with CBD pathology, with the term corticobasal syndrome (CBS) preferred, the term CBD is used to maintain consistency with previous publications in the PINE cohort. PSP/CBD cases were age- and sex-matched to incident PD cases randomly selected from all recruited PD cases.At baseline and at subsequent reviews, the specific cause of the parkinsonian syndrome was made based on history, standardised examination , response to dopamine replacement treatment and available imaging findings guided by specific diagnostic criteria available at the time  case demographic and clinical characteristics were extracted. The full primary care, secondary care and research record for each case was systematically hand-searched retrospectively to identify relevant data. The onset of the diagnostic process was defined as the date of index symptom onset. Index symptom was defined as the first documented  symptom, in the primary or secondary care record, which persisted throughout the course of illness (to maximise specificity of symptoms relating to their parkinsonian diagnosis). A list of motor, non-motor and neuropsychiatric symptoms and signs systematically sought from the records are detailed in Appendix 1. At their baseline visit patients also recalled the onset of their first parkinsonian symptom, defined as patient recalled index symptom.A documented primary diagnosis was a clinician’s single best explanation for the patient’s clinical presentation, while differential diagnoses were documented alternative explanations for the patient’s presentation. Prior to PINE study referral clinicians were general practitioners (GPs) or hospital specialists (any), while after study entry patients had at least annual review by a movement disorder specialist in addition to other hospital specialists. Both syndromic and disease-specific diagnoses, mixed diagnoses, and exogenous factors such as alcohol or an infection (if a clinician’s single best explanation for symptoms) were considered discrete diagnoses. The date each primary diagnosis was first proposed or re-diagnosed if subsequent diagnostic revisions were made (to determine the frequency of changing diagnoses) was collated. If a specific diagnosis of PSP/CBD or PD was only made at post-mortem or following case note review after death, the date of death was used as the date of diagnosis.Secondary care referrals and attendances after index symptom onset were reviewed chronologically. Emergency attendances were defined as both unscheduled Accident and Emergency (A&E) assessments with subsequent discharge or those requiring hospital admission. Elective outpatient referrals were new referrals made by GPs or other secondary care physicians. Healthcare episodes which were the first documented occurrence of symptoms relevant to PSP/CBD were included. Follow-up secondary care appointments initiated prior to index symptom onset for unrelated co-morbidities were not counted as first secondary care contacts after index symptom onset. Outpatient referrals or elective inpatient episodes unequivocally due to unrelated specific symptoms/signs , were excluded.The proportion of patients with each individual symptom and sign at the onset of the diagnostic process was compared between PSP/CBD and PD using the Chi-square test. Using the dates associated with specific diagnoses, the average diagnostic time intervals  from index symptom to key diagnostic milestones including diagnosis of a parkinsonian syndrome, inclusion of a correct diagnosis of PSP/CBD or PD in differential diagnoses, PSP/CBD or PD as the final primary clinical diagnosis, and death, were calculated and compared between groups using the Mann–Whitney test. The average number  of primary diagnoses, differential diagnoses, and the number of chronological changes to a patient’s primary diagnosis from index symptom to death, and both before and after movement disorder specialist input (PINE study team review), were compared using the Mann–Whitney test.The mode of entry to secondary care, including the number of emergency attendances and elective referrals, subdivided by speciality, was compared between groups using the Chi square test. The timing, reason for attendance, outcome and duration of admission of the first unscheduled emergency contact in PSP/CBD and age-sex matched PD was compared using information from individually extracted episodes. For elective outpatient referrals, the documented specialty, referrer diagnosis and the primary diagnosis after each secondary care healthcare contact was also summarised. Time intervals  from index symptom onset to the first outpatient secondary care referral and first movement disorder review were compared using Mann–Whitney test.n = 200, 63.6%), DLB , vascular parkinsonism , PSP/CBD , MSA  and dementia with associated parkinsonism .Of 315 recruited to the PINE cohort with confirmed parkinsonism on follow-up, clinically diagnosed causes of parkinsonism at baseline included PD (n = 28), CBD (n = 2) and PD (n = 30), were deceased at the time of data extraction (February 2017). PSP and CBD were combined due to the small numbers of cases with CBD. All clinically diagnosed cases with PSP who came to post-mortem had PSP  while in five (17.9%) PSP cases, the correct diagnosis was only identified at post-mortem. The clinical diagnoses of nine (30.0%) sampled PD cases that underwent post-mortem were also all confirmed pathologically . At baseline assessment, there were no statistically significant differences between PSP/CBD and PD with respect to age, sex, ethnicity, marital status, education, socio-economic status or medical comorbidities  first identified in primary care. The average age at index symptom onset was 75.5 years (SD 9.4) in PSP/CBD and 74.6 years (SD 13.3) in PD. Only tremor in PD patients , and impaired balance  and falls  in PSP/CBD patients, were significantly different between groups at symptom onset, consistent with differences in index signs where, in addition to the above, rigidity was also more prevalent in PD  (Supplementary Table 1). The overall difference between patient recalled and documented index symptom onset (patient reported minus documented symptoms) was 0.00 (IQR − 0.72 to 2.49) years in PSP/CBD and − 0.55 (− 1.05 to − 0.01) years in PD .Both PSP/CBD and PD patients experienced a range of index symptoms Table , with moAll stages of the diagnostic process were significantly longer in PSP/CBD compared to PD Table . Overallp < 0.001) (Table Over this period (index symptom onset to death), individuals with PSP/CBD were given a median of 4.0 discrete primary diagnoses (both parkinsonian and non-parkinsonian diagnoses) compared with 2.0 primary diagnoses in the PD group , compared to 10.0% with PD  (p = 0.057) their first secondary care contact was an unscheduled healthcare episode. Most emergency presentations in both PSP and PD occurred at, or rapidly after, the date of index symptom onset. In the PSP/CBD subgroup the majority of first emergency presentations were due to falls (n = 8), half resulting in injury, and most (n = 8) requiring an inpatient stay (median duration 16.0 days). While all three PD emergency presentations also required admission, their admissions were significantly shorter . No unscheduled first secondary care contacts resulted in a diagnosis of parkinsonism, PSP/CBD or PD specifically. Unlike PD patients however, for whom their next secondary care contact was a scheduled outpatient review, individuals with PSP/CBD had a higher burden of recurrent emergency attendances  (p = 0.011). Of a total of 27 emergency attendances in the PSP/CBD group (Supplementary Table 4), prior to outpatient referral or inpatient diagnosis, over half were due to falls, many resulting in serious fall sequelae. Twenty-four (88.9%) of all PSP/CBD emergency attendances were admitted under Care of the Elderly (COTE) (7 [29.2%]), orthopaedics (5 [20.8%]), psychiatry (5 [20.8%]), general medicine (4 [16.7%]), or A&E (3 [12.5%]).From index symptom onset, in 33% of individuals with PSP/CBD , in approximately a third in both the PSP/CBD (n = 6) and PD group (n = 9), the date the outpatient referral was made was the date of their index symptom/sign. Overall, however, the median time interval from index symptom to primary care referral for elective outpatient review was significantly longer in PSP/CBD  compared to PD  (p = 0.025). In PD nearly all  referrals were sent to neurology or COTE, whereas in PSP/CBD over a third  of GP referrals were to specialists without movement disorder expertise, including orthopaedics (10.0% PSP/CBD vs 3.7% PD), ear nose and throat (5% PSP/CBD vs 3.7% PD), psychiatry (5% PSP/CBD vs 0% PD), or stroke medicine (10.0% PSP/CBD vs 0% PD) .Of those in whom their first contact with secondary care after symptom onset was in an outpatient setting  referrals, but 8 (29.6%) individuals were referred without a suspected diagnosis and 9 (33.3%) with non-parkinsonian diagnoses. By contrast, in the 30 referred PD cases, PD was proposed by 19 (63.3%) referrers, and parkinsonism in 2 (6.7%) cases. While 9 (30.0%) PD cases were referred without diagnosis, non-parkinsonian diagnoses were never proposed.n = 108), prior to movement disorder specialist review , at the initial outpatient review, parkinsonism was diagnosed in 11 (40.7%) in the PSP/CBD group and 20 (66.7%) in the PD group. A specific correct diagnosis was made in 3 (11.1%) initial outpatient reviews in the PSP/CBD group , and 18 (60.0%) PD initial outpatient reviews. Unsurprisingly, none of the referrals to specialities other than neurology or COTE resulted in a diagnosis of parkinsonism. Indeed, of all healthcare interactions in PSP/CBD in both primary care and secondary care . At their first movement disorder specialist review, 16 (53.3%) PSP/CBD patients received a specific diagnosis of PSP/CBD, compared to 22 (73.3%) PD patients (p = 0.18). Of these PSP/CBD patients diagnosed at their first movement disorder review, over a median follow-up of 3.2 (SD 2.6) years, the diagnosis remained unchanged in 14 (87.5%)  individuals. By their final movement disorder review, 23 (76.7%) PSP/CBD cases received their diagnosis compared to 100% of PD cases (p = 0.010). 7 (23.3%) PSP/CBD cases were only diagnosed after death, either by post-mortem (n = 5) or full case note review (n = 2). In four cases diagnosed with PSP at post-mortem only, their (ultimately incorrect) initial clinical diagnoses  remained unchanged over clinical follow-up.The median time from index symptom to movement disorder review was also significantly longer in PSP/CBD  compared to PD , it is notable that PSP/CBD appears not to be readily considered, even once the parkinsonian syndrome has been identified. The complexity of the diagnostic journey for individuals with PSP/CBD was also greater than for those with PD, with more differential diagnoses, primary diagnoses and changes to diagnosis overall. Individuals with PSP/CBD had more emergency admissions prior to diagnosis and a greater number of specialties involved in their diagnostic process with longer delays to a movement disorder specialist review, in particular, which was a key step in making a diagnosis.Some previous studies in PSP/CBD report similar time delays , 11, somr =  − 0.23, p < 0.001). The average age of our cohort was 78 years and so the time intervals reported may therefore be even worse in younger patients. In PD proposed determinants include sex (longer for men to present to primary care) [In general, there has been little research into the determinants of diagnostic delay in PSP/CBD. In one previous study, there was a significant correlation between diagnostic latency and age of onset, with older patients having shorter diagnostic latency overall (ry care) , motor pry care) , 35, andp = 0.012), for example, suggests that focal symptoms or signs in PSP/CBD are being identified but misattributed, perhaps due to a clinical reasoning process favouring common diseases. Careful assessment of the speed of symptom onset and symptom progression should broadly differentiate vascular and neurodegenerative pathologies. The greater number of neuropsychiatric diagnoses in PSP/CBD (p < 0.01), suggests training psychiatrists to perform targeted motor examinations in those with predominantly cognitive or behavioural presentations may reduce misdiagnosis in PSP/CBD or trigger movement disorder specialist referral, an approach previously suggested to differentiate DLB and AD [An initial appraisal of presenting symptoms/signs is a critical first step to systematically evaluate the greater diagnostic uncertainty and delay in PSP/CBD. The high prevalence of postural instability at symptom onset in PSP/CBD suggests it is underappreciated as a core feature of atypical parkinsonism. If realised earlier, this might narrow differential diagnoses and expedite movement disorder review. This is reflected in more recent changes to diagnostic criteria. Although postural instability, for example, is included in the UK PD Brain Bank criteria, it is not included in the 2015 Movement Disorder Society PD criteria as its presence in early disease should prompt consideration of alternative diagnoses, including PSP. Key identified categorical misdiagnoses, particularly vascular or primary psychiatric disorders, could be reduced by targeted history and examination. The higher frequency of TIA/stroke diagnoses in PSP/CBD (B and AD . CliniciB and AD . AtypicaB and AD .p = 0.028) and ophthalmology (p = 0.016) compared to age- and sex-matched PD [The transition from primary to secondary care is another key milestone in the diagnostic process. While there are no national referral recommendations for PSP/CBD in the UK, in PD national guidelines explicitly encourage urgent referral to movement disorder specialists to expedite diagnosis, determine prognosis, prevent distress arising from diagnostic delay, and allow early multidisciplinary involvement –41, a ratched PD .For nearly a third of PSP/CBD patients, predominantly women, their first secondary care contact after symptom onset is not due to a GP referral but an emergency presentation, three times higher than in their age-sex matched PD counterparts. Given the high fracture rate in those presenting with falls, the reported female preponderance in this group may reflect a higher fracture risk due to lower bone mineral density in women. Initial emergency presentations in PSP/CBD, unlike PD, also do not reliably trigger subsequent outpatient referrals to relevant specialities, resulting in a higher frequency of recurrent emergency attendances prior to outpatient referral and review. Such emergency attendances  result in a high rate of hospitalisation . The longer duration of hospitalisation in those with PSP/CBD compared to PD, suggests that the rehabilitation of PSP/CBD patients, prior to diagnosis, may already be slower than their age-sex matched PD counterparts. In addition, unscheduled admissions rarely resulted in inpatient diagnoses, a missed diagnostic opportunity. While patients with atypical parkinsonism have been previously identified as having a higher fracture risk in the 2 years preceding diagnosis , the curPSP/CBD is also difficult to diagnose even once individuals have reached secondary care unless there is movement disorder expertise. PSP/CBD was diagnosed in only 11.1% of initial outpatient reviews in the PSP/CBD group compared to 60.0% diagnosed with PD in the PD group, while parkinsonism was diagnosed in 40% of PSP/CBD cases and two-thirds of PD cases after an initial outpatient neurology or COTE review. Although PSP/CBD patients first reached secondary care by approximately 8 months on average, it took individuals with PSP/CBD nearly 2 years from symptom onset to receive specialist movement disorder input, approximately three times longer than PD patients. This is shorter than the time reported in a series of 16 pathologically confirmed PSP cases where the time to first movement disorder clinic visit was 2.9 years . In addiHaving reached a movement disorder specialist, approximately 50% of those with PSP/CBD were diagnosed at their first movement disorder assessment, reaching approximately 75% by their final review. Several studies have previously highlighted the improved diagnostic accuracy at final compared to initial review , 5, 37. Unexpectedly, a novel finding was that there was little difference in the overall survival from carefully documented symptom onset to death in those with PSP/CBD and their age- and sex- matched PD counterparts. In PD, older age and male sex have been identified as independent worse prognostic factors. Due to 1:1 sex-matching in PD to match the PSP/CBS sex ratio, survival in PD in our sample has likely been extended as relatively more women were included in the PD group. However, conversely, in that the median age in PSP (79.9 years) was also older than the entire incident PD population in PINE (73.8 years), age matching may have decreased survival in our matched PD population. At the time of the study, the NINDS-PSP criteria were used for diagnosis which did not permit the diagnosis of non-Richardson’s syndrome subtypes of PSP. Given the PINE study was an incident cohort of parkinsonism, cortical PSP subtypes (which may have greater diagnostic delay and a poorer prognosis) are likely under-represented potentially extending survival in our PSP cohort. However, the proportion of cognitive subtypes are relatively small , 48 and A particular strength of this study is the inclusion of the entire recruited PSP/CBD population of a community based, prospective incident study of parkinsonism in the UK, meaning PSP/CBD cases are likely to be representative of the motor predominant PSP/CBD population. Additional strengths include the detailed systematic review of initial symptoms and signs from primary and secondary care records, the availability of detailed longitudinal data from research and clinical records, and the comparative analysis in PSP/CBD and PD matching on variables identified as possible determinants of diagnostic delay.The study is not without limitations. PSP and CBD are distinct diseases with differentiating clinical features but were considered together for analysis due to small numbers (but akin to many studies in PSP/CBD due to their rarity). There is significant overlap between PSP and CBD clinically, but nonetheless with two CBD cases, our results mostly apply to those with PSP. The PINE study is an incidence study of parkinsonism so, while representative of the motor predominant PSP/CBD population, may under-represent those with a predominantly cognitive initial presentation. Primary and secondary care referrals of patients to a neurologist with a specialist interest in movement disorders were also actively sought as part of the PINE study, which may have reduced the time to movement disorder specialist and increased diagnostic accuracy compared to real-world practice in the UK. Unless explicitly documented, it is impossible to determine how frequently differential diagnoses or changes to primary diagnosis were communicated to patients, and the extent to which this impacted their perception and experience of their diagnostic journey. Pathological verification of all clinical diagnoses was not achieved . This is never possible outside brain bank studies which, as detailed previously, are highly selected and susceptible to bias. During life clinicians rely on clinical diagnosis to guide management, and so even without pathological confirmation of all cases, studies on best clinical diagnosis are important. In addition, final clinical diagnostic accuracy in our study is also likely to be high due to, at minimum, annual reviews from diagnosis to death and a full case note review after death. While, without post-mortem data in all cases, we may have missed some with atypical PSP, the effect of this potential bias would tend to make “true” delays in all cases of PSP/CBD even longer without better diagnostic tests. The PINE study also preceded the updated MDS diagnostic criteria for PSP . While tIn conclusion, individuals with PSP/CBD had a longer and more complex diagnostic pathway than individuals with age-sex matched PD. There are several ways this could be improved including greater recognition that balance issues may be parkinsonian, raised awareness of red flags for PSP including emergency admissions preceding diagnosis for unexplained falls/fractures and earlier access to movement disorder specialists. We found little difference in overall survival from symptom onset to death in PSP/CBD and age-sex matched PD. If replicated, this has important implications for our understanding of prognosis in PSP/CBD.Supplementary file1 (DOCX 26 KB)Below is the link to the electronic supplementary material.


Since Immune response, nutritional status and Epstein–Barr Virus (EBV) DNA status have been confirmed to be relevant to the prognosis of patients with nasopharyngeal carcinoma (NPC), we believe that the combination of these factors is of great value for improving the predictive ability. LA , a novel indicator, had not been studied yet in NPC. We combined it with EBV DNA and used nomograms to increase the accuracy of prognosis.A total of 688 NPC patients were retrospectively reviewed and further divided into training and validation cohort randomly. Kaplan–Meier analyses were used to to distinguish the different survival outcomes. Multivariate Cox analyses were used to identify the independent prognostic factors for progression-free survival (PFS) and overall survival (OS). Calibration curves, concordance indexes (C-indexes) and decision curve analyses (DCA) were used to evaluate the nomograms’ predictive value.P < 0.005). In multivariate Cox analyses, LA and EBV DNA were both confirmed to be independent prognostic factors for PFS and OS . Prognostic nomograms incorporating LA and EBV DNA achieved ideal C-indexes of 0.69 (95% CI: 0.65–0.73) and 0.77 (95% CI: 0.71–0.82) in the prediction of PFS and OS. Otherwise, the calibration curves and DCA curves also revealed that our nomograms had pleasant predictive power.Patients with low LA and positive EBV DNA correlated with poorer 5-year PFS and OS (all LA is a novel and powerful biomarker for predicting clinical outcomes in NPC. Our nomograms based on LA and EBV DNA can predict individual prognosis more accurately and effectively.The online version contains supplementary material available at 10.1186/s12885-023-11691-8. Nasopharyngeal carcinoma (NPC), a distinctive head and neck cancer arising from nasopharyngeal epithelium, has a high reported incidence of 5.0/100,000 in Southeast Asia –4. ThereThe dynamic interaction between immune response and tumor microenvironment has become a popular focus of attention. Increasing evidence shows that the immune response of a patient contributes to cancer development and progression –7. LymphAs a specific pathogenic factor of NPC, the contribution of Epstein-Barr virus (EBV) DNA cannot be ignored . The detNomogram, a more precise tool for individual prediction of a clinical event than traditional TNM staging system, is commonly used for risk estimation in various types of cancers . In the Between January 2005 to December 2015, a retrospective study was performed including 688 patients with NPC, who were diagnosed at the Nanfang Hospital of Southern Medical University. The inclusion criteria were as follows: 1) histopathology confirmed NPC; 2) had complete medical records and baseline laboratory data including pretreatment albumin, lymphocytes and EBV DNA; 3) completed the entire treatment. The exclusion criteria were as follows: 1) developed distant metastasis or combined with other malignancies at diagnosis; 2) had a history of cancer treatment; 3) had serious complications; 4) had insufficient follow-up data. All patients were randomly divided into a training cohort (456 patients) and a validation cohort (232 patients). The 8th edition of the American Joint Committee on Cancer (AJCC) staging system was performed for the present study. The research was approved by the Ethics Committee of Nanfang Hospital of Southern Medical University .The peripheral blood test of including lymphocytes, albumin (ALB) were gathered within 1 week before treatment. The plasma EBV DNA was tested within 1 month before treatment. Both peripheral blood test and EBV DNA measurement were conducted in the Laboratory Medicine Center of Nanfang Hospital, Southern Medical University from the standard operating procedures  × serum albumin (g/L). The cutoff of LA was obtained via receiver operating characteristic (ROC) curve analyses.The BamH I-W region of EBV genome was amplified by real-time quantitative polymerase-chain reaction (RT-qPCR) technique to determine plasma EBV DNA levels. After PCR assay, the plasma EBV DNA level of ≥ 500 copies/ml was defined as positive, and the negative EBV DNA level was recorded as < 500 copies/ml. Referring to previous studies , 30, we Based on the National Comprehensive Cancer Network (NCCN) guidelines, intensity-modulated radiation therapy (IMRT) along was recommended for stage I patients, concurrent chemoradiotherapy (CCRT) was recommend for stage II patients, a combination of CCRT and induction chemotherapy (IC) with or without adjuvant chemotherapy (AC) was recommend for stage III/IV patients.  of nasopharynx and neck, a whole-body bone scan or the positron emission tomography and computed tomography (PET-CT). The recurrence of nasopharynx and neck tumor or metastasis of cervical lymph nodes was confirmed by the biopsy or needle biopsy in the suspected region. Our primary endpoint was progression-free survival (PFS), defined as the time from diagnosis to the date of disease progression, death from any cause or last follow-up. The secondary endpoint in current study was overall survival (OS), which was defined as the time between diagnosis to death from any cause or last follow-up.P value < 0.05 was considered statistically significant.Continuous variables were transformed into categorical variables, and chi-square test or Fisher’s exact test was performed to compare the clinical features of these two groups. The receiver operating characteristic (ROC) curve was used to determine the cut-off of LA. Survival curves were calculated using Kaplan–Meier method and compared by the log-rank test. Univariate and multivariate COX proportional hazards regression analyses were performed to determine independent prognostic factors of PFS and OS. Then, based on the significant prognostic factors from multivariate COX regression analysis in the training cohort, the OS and PFS nomogram were developed. To measure the discrimination performance of the nomogram, we calculated Concordance indexes (C-indexes). The calibration curve was used to evaluate the goodness of fit between the observed values and predicted values. Decision curve analysis (DCA) was performed to assess the clinical usefulness of the nomogram. All analyses were carried out using IBM SPSS 23.0, Graphpad Prism V6.0 and R software v4.2.1. All tests were two-tailed and a A total of 456 patients in the training cohort and 232 patients in the validation cohort were included in this study. The baseline characteristics of the training cohort and validation cohorts are shown in Table In the training cohort, there were 327 (71.7%) male and 129 (28.3) female patients with a median age of 47 years. The median follow-up duration was 61 months. During follow-up, 128 (28.1%) patients developed tumor progression and 58 (12.7%) patients died. In the validation cohort, there were 179 (77.2%) male and 53 (22.8%) female patients with a median age of 47 years. The median follow-up time was 61.5 months. During follow-up, 74 (31.9%) and 35 (15.1%) patients experienced tumor progression and mortality, respectively.P < 0.001; sensitivity: 0.688, specificity: 0.509)  and validation (P = 0.035) cohorts. However, in the validation cohort, a significant correlation between LA and age (P = 0.006) and EBV DNA (P = 0.036) was also observed.Table P < 0.001, Fig. P < 0.001, Fig. P = 0.003, Fig. P < 0.01, See Supplementary Figure 1, Additional File In the training cohort, the Kaplan–Meier survival analyses revealed that compared with the higher LA group, the low LA group had shorter PFS and OS  and OS (P < 0.010). The results were also observed in validation cohorts. . All variables with P values less than 0.05 in univariate analysis were included in multivariate Cox analysis.In univariate analyses, the variables of age, T stage, TNM stage, LA, and EBV DNA were significant predictors of PFS and OS. Gender was also a significant predictor of PFS (P < 0.050). In addition, age > 55 years was still found to be an independent risk factor for OS (P < 0.001). The complete results are presented in Tables Multivariate analyses demonstrated that LA, EBV DNA, and TNM stage were significant independent prognostic factors associated with OS and PFS , 121 (26.5%), 125 (27.4%), and 124 (27.2%) patients were assigned to each group. Our results revealed a significant survival difference in LA combined with EBV DNA. Patients in Group 4 had obviously worse PFS and OS than patients in the other groups  were integrated into the PFS nomogram Fig. a, while The calibration plots showed great consistency between actual observation and the nomogram prediction of 3-year and 5- year PFS and OS (See in Supplementary Figure 4, Additional File Owing to tumor heterogeneity, personalized treatment has become an important developing strategy for cancer treatment. Precise prognosis estimation helps clinicians tailor treatment options to improve survival outcomes. Hence, further refined risk stratification of patients with cancer is indispensable for individual treatment. Our study confirmed that LA was a strong independent factor for predicting clinical outcomes. The combination of LA and EBV DNA provided more detailed information for patient stratification. Moreover, we constructed comprehensive prognostic nomograms incorporating LA and EBV DNA for PFS and OS prediction. The C-index, calibration curve, and DCA curve demonstrated that this model achieved excellent predictive efficiency and clinical benefits.To the best of our knowledge, this is the first study to focus on the value of LA in NPC. We noticed that a low LA was obviously related to poorer 5-year PFS and OS in both the training and validation cohorts. This suggests that patients with low lymphocyte and low albumin levels had worse survival outcomes, which is consistent with previous studies , 31–33. The interactions between immune environment and cancer cells are dynamic and complex, and the immune biomarkers can reveal the prognosis of cancer patients . LymphocMoreover, proinflammatory cytokines can not only promote tumor invasion but also inhibit the synthesis of albumin and lead to its leakage by increasing microvascular permeability , 39, 40.Unlike LA, EBV DNA has been recognized as an important indicator of the prognosis of NPC. Studies indicated that EBV DNA was originated from NPC tumor cells, is a fragment of tumor cells necrosis and lysis, and its plasma load can reflect tumor load –44. SimiRecently, nomograms, with their simple, intuitive, and individual characteristics, have been widely used in cancer prognosis prediction. In studies conducted by Tang et al.  and ZhanHowever, there are some limitations to our study. First, this was a retrospective study. Although we conducted an internal validation to make the results more convincing, it is still far from practical clinical application. Second, in addition to LA, many other indicators such as LDH, CAR, SII and SIRI were reportedly associated with the prognosis of NPC patients. On account of the limited sample size, these relations have not been fully explored. Third, it was a single-center study with the absence of external validation. Hence, a large-scale and multi-center prospective study is required to further validate the value of LA.In conclusion, our study reported that LA was a simple and easily available biomarker, and when combined with EBV DNA, it was a stronger predictor of PFS and OS in NPC. The nomogram based on LA and EBV DNA is more effective and precise in predicting individual survival outcomes for NPC patients. It can serve as a good supplement to the TNM staging system to improve the identification of high risk of disease progression in NPC patients and guide more aggressive treatments for these patients to prolong their survival.Additional file 1: Supplementary file 1. The method of EBV DNA detection.Additional file 2: Supplementary file 2. The detailed protocols for radiotherapy and chemotherapy.Additional file 3: Supplementary Figure 1. Kaplan–Meier survival curves of PFS and OS in the validation cohort.Additional file 4: Supplementary Figure 2. Kaplan–Meier survival curves of PFS and OS in the subgroup analyses of TNM stage in the training cohort.Additional file 5: Supplementary Figure 3. Kaplan–Meier survival curves of PFS and OS in the subgroup analyses of TNM stage in the validation cohort.Additional file 6: Supplementary Figure 4. The calibration curves for predicting the 3-year PFS and OS.


Both FLC and FTC were significantly lower in the Degenerated RV and Constricted RV groups compared with those in the Control and Overloaded RV groups. The T/L ratio was significantly lower in the Degenerated RV group (p = 0.008), while the Overloaded RV (p = 0.986) and Constricted RV (p = 0.582) groups had preserved T/L ratios, compared with the Control group. Transverse shortening contributes to RV function more significantly compared with longitudinal contraction. Impaired T/L ratios may reflect RV myocardial degeneration. RV fractional parameters may help precisely understand RV dysfunction.Studies of the usefulness of transverse right ventricular (RV) shortening are limited. We retrospectively analyzed the CMR images of 67 patients : n = 15, Constricted RV (pericarditis): n = 17, Degenerated RV (arrhythmogenic right ventricular cardiomyopathy): n = 15)  in a single center. We defined RV longitudinal  and transverse  contraction parameters. We assessed the FTC/FLC (T/L) ratio on four-chamber cine CMR views and compared the four groups regarding the fractional parameters. FTC had a stronger correlation (R CMR enables visualization of a complete four-chamber view without deficits, while echocardiography sometimes fails to visualize the part of the RV free wall that is located near the rib cage. However, RV volumetric assessment by CMR requires certain equipment and intensive labor for segmentation4. Therefore, clinically, RV volumetric assessment by CMR is difficult to perform routinely.Cardiovascular magnetic resonance (CMR) imaging is becoming an increasingly important tool in routine clinical practice, and this imaging is being applied for right ventricular (RV) volumetric assessment as the gold standard5. In contrast, in subjects with structural abnormalities in the RV, etiology-specific shortening patterns  have been reported rarely. Currently, the evaluation of strain assessment on CMR using feature tracking is increasing in clinical practice6. RV longitudinal strain has prognostic importance in various cardiac diseases7. Furthermore, while the clinical application of RV circumferential strain appears to be limited compared with that of longitudinal strain, the usefulness of evaluating RV circumferential strain has been reported in patients with pectus excavatum8 and in some SLE cases7. Through-plane motion may affects 2D measures of circumferential strain using a fixed imaging plane , overloaded RV , and degenerated RV (arrhythmogenic right ventricular cardiomyopathy: ARVC). The data for consecutive subjects were collected for each etiology of RV structural abnormality. All subjects were recruited in a single center (the Cardiovascular Imaging Clinic Iidabashi) and all were recruited consecutively in each group over a specified period for that group (as noted in the following sections). Considering RV disease rarity, the target sample size was set at approximately 20.The patients’ characteristics were obtained from the medical records, and all patients provided informed consent. All experiments were performed in accordance with the Declaration of Helsinki. The study was approved by the local ethical committee of the Cardiovascular Imaging Clinic Iidabashi Medical Corporation.11 (subjects with a “Not probable” classification were excluded), and they were strongly suspected of having ARVC in the CMR assessment. In all groups, subjects with images of poor quality or inadequate four-chamber view cine CMR results were excluded from the study. Each diagnosis was made by cardiologists certified by the Japan Cardiovascular Society who had each read more than 1,000 CMR images.Subjects referred by local medical institutes suspected as having any cardiac disease, but in whom cardiac disease was ruled out on CMR, with reported RV volumetric assessment, were recruited as the Control group. Subjects with constricted RV (pericarditis) were eligible if they had present or past symptoms of right heart failure, or were suspected of having pericarditis by computed tomography, and if marked thickening of the pericardium (maximum > 3 mm) was recognized on CMR. Subjects with overloaded RV (ASD) were eligible if left-to-right shunting was recognized in the interatrial septum, and the Qp/Qs ratio was > 1.5 by the phase-contrast method. Subjects with Ebstein anomaly were excluded. Subjects with degenerated RV (ARVC) were eligible if RV enlargement, asynergy, or fatty infiltration were recognized, they met at least one major or minor criteria of the 2010 task force criteria by CMRWe enrolled 20 consecutive subjects as the Control group, in accordance with the inclusion criteria. CMR was performed from June 2018 to September 2018 for this group. There were no excluded cases owing to poor image quality. Twenty-three subjects with ASD were identified in the clinical records from June 2016 to Aug 2020. After excluding 8 subjects , we enrolled 15 subjects in the Overloaded RV group.Twenty consecutive subjects with chronic pericarditis were identified in the clinical records from June 2017 to February 2020. After excluding 3 subjects , we enrolled 17 subjects in the Constricted RV group. Adhesions of the pericardium on cine or tagged images were identified in 14 subjects. Symptoms  were seen in 14 subjects. Two subjects were suspected as having coronary artery disease, and another 3 subjects had histories of cardiac surgery . Eighteen consecutive subjects suspected of having ARVC were identified in the clinical records from August 2016 to August 2020. After excluding 3 subjects , we enrolled 15 subjects in the Degenerated RV group. Nine subjects were classified as definite, 4 subjects were classified as borderline, and 2 subjects were classified as probable in accordance with the 2010 task force criteria for ARVC. A total of 67 subjects were finally enrolled in the present study.2 and each patient’s status. Cine four-chamber view slices were obtained by referencing the left ventricular (LV) short axis and two-chamber views. After the determination of the axis between the center of the mitral valve and the apex with the two-chamber view, a four-chamber view slice that passed across the acute margin of the RV free wall was identified. Short-axis cine images were used for the RV volumetric assessment.All examinations were performed using 1.5-T MRI scanners (Philips Achieva or Canon Vantage TITAN). The scan protocol was as follows: (Phillips/Canon): steady-state free precession cine sequence, 20/24 phase/beat, slice thickness: 8 mm, repetition time: 2.7/4.2 ms, echo time: 1.4/2.1 ms, flip angle: 60°/80°, field of view: 3 8 × 38/38 × 34 cm, and matrix: 208 × 193/192 × 240. The short axis slice was obtained with a 0 mm slice gap while the four-chamber view was generally obtained as a single slice, and other views  were performed in accordance with current guidelinesA workstation  was used for post-processing of the RV volumetric data. We calculated RV end-diastolic volume (RVEDV), end-systolic volume (RVESV), stroke volume (RVSV), and ejection fraction (RVEF). Papillary muscle volume was included as part of the blood pool. Another workstation, Zio2 Cardiac Function, was used for the LV volumetric assessment. We calculated LV end-diastolic volume (LVEDV), end-systolic volume (LVESV), ejection fraction (LVEF), stroke volume (LVSV), and LV myocardial mass. Volumetric data were indexed (I) by body surface area and labeled as RVEDVI, RVESVI, RVSVI, LVEDVI, LVESVI, LVSVI, and LV-myocardial mass index.The RV shortening parameters were calculated in the cine four-chamber view retrospectively, and the parameters were defined as follows. Figure The RV diameter was defined as the maximum distance between the septum and the RV wall parallel to the annulus of the trigeminal valve. We considered FTC to represent the parameter of transverse shortening.We calculated the FTC/FLC ) ratio to assess individual RV shortening patterns.p < 0.05 was defined as statistically significant.First, we compared the fractional parameters and RV volumetric parameters among the four groups  to examine whether these parameters have the same tendency as RVEF among disease groups using Tukey’s multiple comparison procedure. Second, we compared the T/L ratio between the four groups using Tukey’s multiple comparison procedure to clarify the differences in contraction patterns between the groups. Third, we examined which parameter, FLC or FTC, showed the highest correlation using linear regression analysis for all subjects. All data were analyzed using Stata 15 (Stata Corp LLC). Continuous variables were expressed as mean ± standard deviation, and differences between the four groups were analyzed using Tukey’s test for multiple comparisons. Assuming that RV contractility comprises longitudinal and transverse shortening, the relationships between RVEF and FLC or FTC were evaluated using single and multiple linear regression analyses. Intra- and interobserver reproducibility of the measurements of FLC and FTC were assessed in 10 patients by calculating the intraclass correlation coefficients (ICC). The patients’ characteristics are shown in Table Table Regarding LV function, the Degenerative RV group had higher LVEDVI values than those in the Constricted RV group as well as higher LVESVI values than those in the Overloaded RV group. There were no significant differences between the other groups for LVEDVI and LVESVI. LVEF was lower in the Degenerative RV and Constricted RV groups compared with that in the Overloaded RV group; however, there was no significant difference compared with the Control group. The Constricted RV group had the lowest LVSVI among the four groups.Regarding RV function, the Overloaded RV and Degenerative RV groups had higher RVEDVI values than those in the Control and Constricted RV groups. The Degenerative RV group had the largest RVESVI among the four groups. The Overloaded RV and Degenerated RV groups had larger RVESVI values compared with the Control and Constricted RV groups. The Control and Overloaded RV groups had higher RVEF values compared with the Constricted RV group, while the Degenerated RV groups had the lowest RVEF among the four groups. Among the four groups, the Overloaded RV group had the largest RVSVI value, while the Constricted RV group had the lowest RVSVI value.Regarding FLC and FTC values, the Control and Overloaded RV groups had significantly higher FLC values compared with the Constricted RV and Degenerative RV groups. The Constricted RV group had lower FTC values than those in the Control and Overloaded RV groups. The Degenerated RV group had significantly lower FTC values than those in the Constricted RV group, so that Degenerated RV had the lowest FTC value among the four groups. Finally, FTC showed the same tendency as that of RVEF Table .Regarding the T/L ratio, the Degenerated RV group had the lowest ratio among the four groups, with a T/L ratio of < 1 (FTC < FLC). Figure 2: 0.211; p < 0.001), while the correlation between FTC and RVEF was strong . As shown in Table p < 0.01) was a stronger explanatory variable compared with FLC .Figure Regarding the intraobserver reproducibility, the ICC was 0.903 (95% CI 0.677–0.974) for FLC and 0.961 (95% CI 0.861–0.990) for FTC. The time required to measure these parameters was 67.1 ± 15 s for operator A. Regarding interobserver reproducibility, the ICC was 0.819 (95% CI 0.453–0.951) for FLC and 0.894 (95% CI 0.653–0.972) for FTC. The time required to measure these parameters was 168 ± 28 s for operator B. Representative cases are shown in Fig. 13. MRI can visualize the entire morphology of the RV by cine stack; therefore, there may be controversy regarding the necessity of RV fractional parameters derived using only single-slice images. However, RV functional parameters derived by two-dimensional echocardiography were reported to be poorly correlated with MRI-derived RV volume, and these correlations improved only modestly when two-dimensional measurements were performed using MRI14. High intra- and interobserver reproducibility of LV and RV volume measurements on CMR was reported in several studies. However, these measurements require more than 20–30 min per case4. Therefore, some institutes are unable to analyze RV volumetry in all cases in clinical practice. In comparison, measurements of the fractional parameters described in the present study can be obtained within 2 min by a skilled operator, as shown in our results, without additional equipment. Left ventricular stroke volume should be used to assess the validity of RV volume analysis 2, which is difficult in the presence of valvular disease or intracardiac shunts. This new parameter may be able to predict right ventricular contractility within a certain range in every situation, and correct for errors in the analysis by measuring it beforehand.Our study demonstrated that the RV parameters, FLC and FTC, can evaluate RV shortening from a new perspective. Fractional shortening parameters for RV have been studied using echocardiography, and all studies showed moderate correlation with MRI-derived RV function15. We defined “CMR-derived TAPSE” as “RVLd-RVLs”, and ran the same analysis , replacing FLC with CMR-derived TAPSE  than FLC . The target population of the present study has a large variation in RV size. In such cases, standardizing the excursion distance by the length of the entire RV may lead to a more accurate assessment of contractility. And CMR-derived TAPSE failed to show a significant correlation in a multiple regression analysis with the FTC, as well as FLC.TAPSE (tricuspid annular plane systolic excursion) is a very widely used measure of RV longitudinal contraction as a standard. There have been some reports evaluating right ventricular function using CMR-derived TAPSEPSE Fig. . In univ16. On the epicardial surface, circumferentially-oriented fibers are present, which are shared with LV epicardial myofibers. In contrast, subendocardial myofibers form longitudinal lines, and these fibers are mainly responsible for longitudinal shortening, which generates the largest part of RV contraction in healthy subjects17. However, once RV pressure overload occurs, it may lead to ventricular hypertrophy and morphological changes to reduce the wall stress. Under these circumstances, the dominance of the circumferential fibers can be observed16. A comparative study of echocardiography and CMR involving subjects with precapillary pulmonary hypertension showed that correlations between tricuspid annular plane systolic excursion or fractional longitudinal wall motion and RVEF were relatively mild when compared with correlations between functional area change and RVEF18. These findings indicate that both longitudinal contraction and transverse contraction must be considered in subjects with RV dysfunction or pulmonary hypertension.The present study also demonstrated that both longitudinal and transverse shortening play important roles in RV contraction, especially in subjects with RV-related disease. The RV myocardial structure mainly comprises two layers6, similar to the FLC value of the Control group in the present study of 23.4% ± 4.6%.As mentioned, our preliminary parameter, FTC, can determine maximum RV diameter visually in each cardiac phase on four-chamber cine CMR, and using the FTC value, we can minimize the influence of longitudinal shortening. Using this method, rating the fractional shortening parameters may pioneer disease- or individual-specific RV function analysis beyond conventional volumetry. Additionally, FLC may be approximated to RV longitudinal strain measured by feature tracking. A meta-analysis by Vo et al. showed that RV longitudinal strain varied between studies, and the mean value derived using a random effects model was − 21.8%5. We agree that the largest component of RV contraction in healthy subjects is longitudinal contraction. One of the ideas to resolve this discrepancy is that longitudinal contraction is not perpendicular to the AV valve, so the force is distributed vertically and parallel  to the annular surface. This creates transverse shortening even in normal subjects. In addition, Circumferential (or transverse) muscle tension will be essential as a supplementary factor even in healthy subjects with longitudinal-dominant contraction  is a condition in which the loss of pericardial elasticity causes restriction on diastolic filling of the heart21. Defects with shunts with Qp/Qs ratios > 1.5 are considered significant and are responsible for different degrees of right heart volume overload22. Our study demonstrated that the Overloaded RV group had preserved RVEF and the same range of contraction parameter (FLC/FTC) values as those of the Control group, and there were statistically significant differences in T/L ratios in the Overloaded RV group compared with those in the Degenerative RV group. These findings indicate that FLC, FTC, and T/L ratio can distinguish Overloaded RV and Degenerated RV, both of which present with RV enlargement.ASD is the most common congenital heart disease with left-to-right shunting in adults11. Te Riele et al. reported that “mutation-positive” ARVC describes a previously unrecognized characteristic pattern of disease involving the basal inferior and anterior RV, and the posterolateral LV, while the RV apex is involved in only advanced ARVC23. Our study, showing predominantly impairment of transverse contraction (FTC) compared with longitudinal contraction (FLC), may indicate some relationships with a specific distribution of lesions in subjects with ARVC.ARVC, which we classified as Degenerated RV, is a genetically determined heart muscle disorder that is characterized pathologically by fibro-fatty replacement of the right ventricular myocardium. The distribution of this structural abnormality is described as the “triangle of dysplasia” 24. AUC was 0.701 for FLC, 0.921 for FTC, 0.855 for T/L ratio, and 0.938 for RVEF, respectively. FTC was able to predict ARVC more accurately than FLC (p = 0.001), and T/L ratio showed comparable accuracy to RVEF (p = 0.170), considering that RVEF itself is included in the diagnostic criteria for ARVC, FTC and T/L ratio have high diagnostic power for “Degenerated RV”.After showing the characteristics of FLC, FTC, and T/L ratio in the subjects with ARVC, we additionally examined how accurately the FLC, FTC and T/L can predict "Degenerative RV" using ROC curves for the entire population of present study Fig. . EqualitOur study has some limitations. First, this was a single-center, cross-sectional study. Additionally, the rarity of each disease resulted in small sample sizes. Second, as mentioned, we could not assess echocardiography simultaneously because our institution is an outpatient imaging center. Third, RV contraction is affected by pressure overload; therefore, relationships between RV contraction patterns (T/L ratio) and RV pressure overload should be investigated further. Furthermore, we couldn’t obtain the data of strain analysis due to the lack of technical equipment. Further comparative study of strain analysis and present parameters is needed for the assessment of accuracy of the present preliminary parameters.In conclusion, transverse shortening plays an important role in RV contraction; more so than longitudinal shortening. Bi-directional RV fractional parameters are easily measured, reflect RV contraction to some extent, have the potential to differentiate the etiology of RV dysfunction and detect abnormal RV morphology.Supplementary Figure S1.Supplementary Legends.Supplementary Table S1.


To explore correlation between the dose of norepinephrine and the timing of starting enteral nutrition in septic shock (SS) patients.Totally 150 SS patients treated with enteral nutrition (EN) in Shiyan People’s Hospital from Dece20 to July 2022 were included in this retrospective analysis. Patients were divided into tolerance group (n = 97) and intolerance group (n = 53) according to whether EN was tolerated or not. The study indexes include baseline characteristics , clinical indexes , EN indexes , and gastrointestinal intolerance index . Student-t test and Mann-Whitney test were used for test of measurement data. Chi-square test and fisher exact test were used for comparison of categorical data.There were 51 (52.58%) male and 46 (47.42%) female patients with a median age of 66.4 ± 12.8 years old in tolerance group. There were 29 (54.72%) male and 24 (45.28%) female patients with a median age 67.3 ± 12.5 years old in intolerance group. The weight and BMI were significantly higher in intolerance group than those of tolerance group (both P < 0.001). There was no significant difference of comorbidity rate between two groups . Before the overlapping time of EN and norepinephrine, there were significantly more patients receiving gastrointestinal motility drugs in intolerance group compared with tolerance group . Patients in tolerance group had significantly less residual volume in gastric than that of intolerance group . The rate of residual volume in gastric > 250ml , vomiting  and aspiration were significantly lower in tolerance group than those of intolerance group. The BLA in tolerance group was significantly lower than that of intolerance group . There were significantly more patients with increased BLA  and > 2mmol BLA rising  in intolerance group than those of tolerance group. Patients in tolerance group had significantly lower time of starting EN , dose of NE, mortality in hospital  and mortality in ICU  compared with intolerance group. The EN target percent  and calorie of EN during overlapping period  in tolerance group were significantly higher than those of intolerance group.SS patients should be comprehensively evaluated according to their condition. Obese patients are more prone to EN intolerance, and those who can tolerate EN should be implemented as soon as possible. The use dose of NE is significantly related to EN tolerance. When the use dose is low, EN tolerance is greater. Septic shock (SS) is the most serious septicemia and the main cause of death in intensive care unit (ICU). It is emphasized that SS patients are not only with severe circulatory failure, but also combined with metabolism disorder and organ damage induced by infections, which results in higher death risks than spesis alone . Sepsis Enternal nutrition (EN) is a way supplying energy for patients, which not only protects gastrointestinal tract by maintaining mucous membrane completeness, decreasing permeability and increasing blood supply, but also improves prognosis, shortens in-hospital time and reduces complications –7. HowevThus, this study aimed to explore the effect of NE dose on EN tolerance and prognosis in SS patients.Totally 150 patients diagnosed as SS from December 2020 to July 2022 in ICU of Shiyan People’s Hospital were included in this retrospective analysis. Patients were divided into tolerance group (n = 97) and intolerance group (n = 53) according to whether EN was tolerated or not.Patients meeting the following criteria were included: (I) meet the diagnostic criteria of sepsis and SS ; (II) agPatients meeting the following criteria were excluded: (I) intestinal obstruction; (II) gastrointestinal bleeding ; (III) severe abdominal infection; (IV) acute pancreatitis; (V) diffuse peritonitis; (VI) gaslrointestinal perforation; (VII) contradiction of EN.The ethical approval for this study was waived by The ethical committee of Shiyan People’s Hospital (number: syrmyy2022-032) due to its retrospective nature. Informed consent was obtained from all the study subjects before enrollment. This study was performed in accordance with Declaration of Helsinki.Antibiotics, mechanical ventilation, fluid resuscitation, water electrolyte regulation and other treatments in accordance with the international guidelines for management of septic and sepsis shock (2021 Edition)  were rou(I) baseline characteristics: gender, age, weight, body mass index (BMI), scores of acute physiology and chronic health evaluation II system (APACHE II), comorbidity, time in-hospital, prognosis. (II) clinical indexes: mean arterial pressure (MAP), time of mechanical ventilation (MV), norepinephrine dose at the time of starting EN, using of sedative drug, gastrointestinal motility drugs and cardiotonic drugs. (III) EN indexes: timing of starting EN (from the time of patients with sufficient fluid infusion and use of NE), speed of EN infusion, calorie of EN per day, EN target percent. (IV) gastrointestinal intolerance indexes: residual gastric volume > 250 ml, vomiting, aspiration, gastrointestinal bleeding, blood lactic acid (BLA).All the data collected in this study were analyzed using SPSS 26.0 software. Normally distributed measurement data were expressed as mean ± standard deviation (SD), while non-normally distributed measurement data were expressed as median (interquartile range), and the comparisons were examined by Student-t test and Mann-Whitney test (non parametric distribution). The categorical data were expressed as n (%), and the differences between the two groups were examined by chi-square analysis or Fisher’s exact test. P < 0.05 was considered statistically significant.A total of 150 patients were included in the study, including 80 male patients (53.33%) and 70 female patients (46.67%), with an average age of 66.7 ± 12.7 years. The mean APACHE II score, MAP and BLA concentration were 21.35 ± 5.44, 74.41 ± 8.83 mmHg and 1.74 ± 0.67 mmol. There were 51 (52.58%) male and 46 (47.42%) female patients with a median age of 66.4 ± 12.8 years old in tolerance group. There were 29 (54.72%) male and 24 female (45.28%) patients with a median age 67.3 ± 12.5 years old in intolerance group. The weight and BMI were significantly higher in intolerance group than those of tolerance group (both P < 0.001). It revealed that obesity patients were more likely to be intolerant with EN. There were 18 (18.57%) patients with chronic kidney disease, 16 (16.49%) with heart failure, 20 (20.62%) with diabetes and 16 (16.49%) with hypertension in tolerance group, respectively. Corresponding number of patients in intolerance group were 17 (32.08%), 13 (24.53%), 12 (22.64%) and 12 (22.64%), respectively. There was no significant difference of comorbidity rate between two groups . Before the overlapping of EN and norepinephrine, the number of patients receiving gastrointestinal motility drugs in the intolerance group was significantly higher than that in the tolerance group . Besides, there was no significant difference of APACHE II score, MAP, BLA, using of mechanical ventilation, implementation of continuous renal replacement therapy, using of sedative drugs, using of cardiac agents and number of patient receiving more than 2 cardioactive drugs between these two groups  . The rate of residual volume in gastric > 250ml , vomiting  and aspiration were significantly lower in tolerance group than those of intolerance group. The BLA in tolerance group was significantly lower than that of intolerance group . There were significantly more patients with increased BLA  and > 2mmol BLA rising  in intolerance group than those of tolerance group. Besides, there was no significant difference of intestinal hemorrhage or perforation on image between two group (P > 0.05) , dose of norepinephrine , mortality in hospital  and mortality in ICU  compared with intolerance group. The EN target percent  and calorie of EN during overlapping period  in tolerance group were significantly higher than those of intolerance group. There was no significant difference of overlapping time, speed of norepinephrine infusion, EN energy density, time in-hospital and time in ICU between two groups  (Table The frequency distribution of different NE doses between the tolerance group and the intolerance group was shown in Fig. Septic shock is an intricate problem in the field of acute and critical medicine. There are about 189 of every 100,000 hospital sepsis patients dying of this disease. The hypermetabolic state caused by severe stress reaction and insufficient energy intake is easy to lead to malnutrition, and will affect the treatment and prognosis of these patients . AlthougAs one of the key process in treatment of SS, EN plays a vital role in improving intestinal mucous atrophy and villus damage, inhibiting enterogenous infection, and improving immunity , 21. HowThere were also several limitations in this study. Firstly, this were unavoidable biases in this analysis due to its retrospective nature. Secondly, this is a single center analysis with small sample size. Thus, all results in present study should be interpreted cautiously. Prospective study with large sample should be conducted in the future.In conclusion, SS patients should be comprehensively evaluated according to their condition. Obese patients are more prone to EN intolerance, and those who can tolerate EN should be implemented as soon as possible. The use dose of NE is significantly related to EN tolerance. When the use dose is low, EN tolerance is greater.


Our aim was to explore the correlation between ultrasound attenuation parameter (UAP) and liver stiffness measurement (LSM) based on FibroTouch (China) and clinical features in patients with non-alcoholic fatty liver disease (NAFLD), so as to provide a certain basis for the clinical application of FibroTouch in NAFLD. Hepatic steatosis and fibrosis in patients with NAFLD were graded according to FibroTouch, and the relationship between steatosis and fibrosis levels and clinical characteristics was retrospectively analyzed. Hepatic steatosis was positively related with weight, BMI, waist, hyperlipidemia, hyperuricemia, FBG, UA, TG, ALT, AST, GGT, LSM and hepatic fibrosis grading, and was negatively related with gender , age and AST/ALT ratio. Hepatic fibrosis was positively related with age, BMI, waist, hypertension, FBG, ALT, AST, GGT, NFS, APRI, FIB-4, UAP and hepatic steatosis grading, and was negatively related with blood platelet (PLT) counts. Moreover, BMI, waist, TG, ALT and LSM were independent risk factors of hepatic steatosis, while decreased PLT counts, AST and UAP were independent risk factors of hepatic fibrosis. Body mass parameters, metabolic risk factors and liver function indicators increase the risk of hepatic steatosis and fibrosis in patients with NAFLD, and UAP and LSM can interact with each other. The global prevalence of NAFLD is 25.24%, with 40.76% progression to fibrosis and 0.09% mean annual rate of progression in NASH2. The highest rates were reported from South America and the Middle East, followed by Asia, the United States and Europe3. The annual medical cost attributed to NAFLD exceeds €35 billion in Europe  and $100 billion in the United States4.Non-alcoholic fatty liver disease (NAFLD) is defined as the accumulation of triglycerides within hepatocytes that exceeds 5% of liver weight in non-alcoholics5. NAFLD has emerged as the most frequent cause of cirrhosis and hepatocellular cancer (HCC)6, and has been the leading cause of chronic liver disease-related mortality3.NAFLD is the most common hepatic manifestation of metabolic syndromes and is strongly associated with obesity and insulin resistance. The change in nomenclature has been proposed from NAFLD to metabolic associated fatty liver disease (MAFLD)8. FibroTouch is an alternative of FibroScan developed by Wuxi Hisky Medical Technologies of China since 2010, and has been widely used in the evaluation of liver disease and liver health screening of high-risk populations in China. FibroTouch has good diagnostic performance for hepatic steatosis and fibrosis9.Among the available non-invasive tests, transient elastography by FibroScan®  is commonly used by hepatologists in Europe and Asia, and has been approved by the United States Food and Drug Administration for the diagnosis of liver disease in 2013. The controlled attenuation parameter (CAP) and liver stiffness measurement (LSM) determined by FibroScan are the evidence-based non-invasive indicators of hepatic steatosis and fibrosisHere, we enrolled 352 Chinese patients with NAFLD. Liver steatosis and stiffness were determined by FibroTouch and clinical characteristics were collected including demographics, body mass parameters, medical history, Laboratory parameters and noninvasive fibrosis markers. The aim was to evaluate the potential association between liver status as measured by FibroTouch and clinical features of patients with NAFLD.We conducted a retrospective analysis to explore the correlation between the ultrasound attenuation parameter (UAP) and LSM based on the vibration-controlled instantaneous elastography measured by FibroTouch  and clinical features in patients with NAFLD, so as to provide a certain basis for the clinical application of FibroTouch in NAFLD. All methods were carried out in accordance with the Declaration of Helsinki. All experimental protocols were approved by the Medical Ethics Committee of Wuhan Integrated TCM and Western Medicine Hospital, and the ethics approval number was 2022–51. The Medical Ethics Committee of Wuhan Integrated TCM and Western Medicine Hospital waived the need for written and verbal approved informed consent from the enrolled participants because this was a retrospective observational study using only existing information.We enrolled 352 consecutive patients with NAFLD at Wuhan No. 1 Hospital between January 1, 2021 and August 31, 2022. Inclusion criteria included (i) being diagnosed with fatty liver disease by any of three techniques: conventional ultrasonography, CT or MRI, (ii) aged 18 to 75 years and (iii) having well-documented clinical information. The exclusion criteria included (i) presence of alcohol consumption ≥ 30 g/day for men or ≥ 20 g/day for women, (ii) presence of concomitant viral hepatitis or other liver diseases including drug-induced liver disease, hepatolenticular degeneration and autoimmune liver disease, and (iii) presence of total parenteral nutrition, inflammatory bowel disease, celiac disease, hypothyroidism, Cushing's syndrome, and other specific diseases leading to steatosis of the liver. Demographic, clinical and biochemical assessments and liver steatosis and stiffness measurements by FibroTouch were conducted within two weeks after diagnosis of NAFLD.The demographic data were recorded including gender and age. Body mass parameters were collected including height, weight, body mass index (BMI), and waist. Participants who had been diagnosed with hypertension, diabetes, hyperlipidemia and hyperuricemia, or were taking relevant medication, were defined as having a history of these diseases. After an overnight fast, blood samples were collected and analyzed following standard laboratory procedures to determine the parameters including alanine transaminase (ALT), aspartate aminotransferase (AST), AST/ALT ratio, alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), serum total cholesterol (Chol), low-density lipoprotein cholesterol (LDL-C), Total serum triglycerides (TG), blood uric acid (UA), fasting blood glucose (FBG), albumin (ALB) and blood platelet counts (PLT).11.Four non-invasive fibrosis markers including the NAFLD fibrosis score (NFS), aminotransferase to platelet ratio index (APRI), fibrosis-4 index (FIB-4) and BARD were computed using the available parametersFibroTouch measurements were performed by experienced and certificated physicians who had performed more than 500 examinations without knowledge of the results of ultrasonography, CT or MRI. FibroTouch was to determine liver UAP and LSM values on the basis of two-dimensional ultrasound image-guided positioning system, so that the measurement points could effectively avoid blood vessels, cysts, nodules and other factors that might interfere with the accuracy of detection. This technology measured the speed of the shear wave propagation which reflected the stiffness. Meanwhile, the energy attenuation of ultrasound signal during the propagation process was tracked, and the quantitative result of UAP was obtained which indicated the degree of liver steatosis. Each patient was placed supine with right arm raised behind his/her head and remained still during the procedure. The intercostal space was fully exposed, and three appropriate measurement points located in different intercostal spaces were selected in the liver of the same subject. The measured values with at least 10 valid measurements, a success rate of at least 60%, and an interquartile rang / median (IQR/median) of ≤ 30% were considered reliable, as suggested according to manufacturer's instructions. The median values of the 10 acceptable UAP (dB/m) and LSM (kPa) were expressed as the representative measurement of FibroTouch. According to Chinese guidelines and manufacturer's recommendations, the grading criteria for steatosis were mild steatosis (UAP ≤ 265 dB/m), moderate steatosis (265 dB/m < UAP ≤ 295 dB/m), and severe steatosis (UAP > 295 dB/m), and the grading criteria for fibrosis were F0–F1  (LSM < 6.7 kPa), F2 (mild hepatic fibrosis) (6.7 kPa ≤ LSM < 9.8 kPa), and F3 (progressive hepatic fibrosis) (LSM ≥ 9.8 kPa).P value of < 0.05 was considered statistically significant.All statistical tests were performed using IBM SPSS Statistics 25 . Continuous variables were expressed as mean ± standard deviation (SD) for normal distribution, and one-way analysis of variance was used to compare differences following by Bonferroni test of pairwise comparison. Continuous variables were expressed as medians  for abnormal distribution, and Kruskar-wallis test was used to compare differences. Categorical variables were expressed as frequency (percentage). Binary variables were compared by Chi-square test and rank variables by Kruskal–wallis test following by Mann–Whitney test of pairwise comparison as appropriate. The correlation analysis was assessed using Spearman correlation analysis, and the regression analysis was assessed using ordered logistic regression. This is a retrospective observational study, which was reviewed and deemed exempt by medical Ethics Committee of Wuhan No.1 Hospital. This study fully protects the information and privacy of subjects. After full discussion by the Ethics Committee, informed consent can be exempted after weighing the risk benefit ratio.2DM), 273 (77.6%) had hyperlipidaemia, and 138 (39.2%) had hyperuricemia based on history of diseases. Body mass parameters, laboratory parameters, and non-invasive fibrosis markers of enrolled patients were shown in Table A total of 352 NAFLD patients were enrolled from January 2021 to August 2022. Baseline characteristics of the patients were shown in Table P < 0.001). There was a weak positive correlation between hepatic steatosis and fibrosis .As shown in Table P < 0.05). Patients in the severe steatosis group were younger than those in the mild and moderate groups (P < 0.05). The weight, BMI and waist circumference were the highest in the severe steatosis group, followed by the moderate and mild group (P < 0.05). The serum TG, ALT, AST and GGT levels and LSM values of the severe steatosis group were higher than those in mild and moderate groups (P < 0.05). The AST/ALT ratio in the severe steatosis group were lower than those in the mild and moderate groups (P < 0.05). The proportion of patients with a history of hyperuricemia and the FBG levels were higher in the severe steatosis group than those in the mild group (P < 0.05). The serum UA levels of moderate and severe steatosis groups were higher than those of mild group (P < 0.05). In the severe steatosis group, F2 stage was more common, while in the mild and moderate groups, F0–F1 stage was more common (P < 0.05). There were no significant differences among the three groups in the proportions of patients with the history of hypertension, diabetes or hyperlipidemia, PLT counts, the levels of Chol, LDL-C and ALP in the blood (P > 0.05).As shown in Table P < 0.05)  and age, a moderate negative correlation with AST/ALT ratio, a weak positive correlation with hyperlipidemia, hyperuricemia, FBG, UA, TG, AST, GGT, LSM and staging of hepatic fibrosis, and a moderate positive correlation with weight, BMI, waist and ALT (5) Table . There w5) Table .Table 3CP < 0.05) (Table P < 0.05) (Table P < 0.05)  Table . The hig Table . P < 0.05). There was no significant difference between F0–F1 and F2 groups or between F2 and F3 groups (P > 0.05) (Table P < 0.05), while there were no significant differences between F0–F1 and F2 groups (Table P < 0.05) (Table P < 0.05) (Table P < 0.05) (Table P < 0.05)  Table . The props Table . The ser5) Table . There w5) Table . The GGT5) Table . In F0–F5) Table . There w5) Table .Table 6CP < 0.05)  Table . There w5) Table .Table 7CP < 0.05)  Table . The inf12. The presence of advanced fibrosis is an important criterion for evaluating the severity of chronic liver disease. 948217 patients with NAFLD from 103 observational studies were analyzed, suggesting NAFLD significantly increased the risk of HCC , and increased HCC-related mortality risk 13. In addition, it is well known that the degree of hepatic steatosis is linked to the metabolic syndrome and the cardiovascular risk15. Therefore, an accurate estimation of the degree of hepatic steatosis and fibrosis is important to assess the hepatic condition of NAFLD. Histopathology is the gold standard for the diagnosis with the limitations including traumatic procedure, subjective nature and sampling variabilities16. The detection rate of mild hepatic steatosis (fat content > 5%) by commonly used ultrasound B-mode is low with reported sensitivity of 60.9–65%18, and a significant intra- and inter-observer variability has been reported20.NAFLD is the leading cause of diffuse liver disease. Hepatic steatosis plays an important role in fibrosis progression16. Meanwhile, it was suggested that patients with an LSM < 8 kPa was at low risk of progressive fibrosis, an LSM between 8 and 10 kPa was at intermediate risk, and an LSM ≥ 10 kPa was at high risk21. In patients with NASH-related cirrhosis, LSM not only is a useful tool to reflect the degree of liver fibrosis but also predicts portal hypertension, varices requiring treatment, cirrhotic complications and hepatocellular carcinoma and liver-related death22. As one of the transient elastography techniques, FibroTouch has a rich application experience in the diagnosis of hepatic steatosis and fibrosis in China, its effectiveness has been fully verified, and it was recommended by domestic experts consensus. With its two-dimensional image-guided positioning system, FibroTouch can avoid interference factors such as blood vessels, cysts, nodules and others, and can reduce errors caused by blind measurement, and improve detection speed, success rate and accuracy. The UAP measured by FibroTouch is calculated based on BMI variables and the spectral analysis of ultrasonic echo signals, and determines skin capsular distance which can affect the accuracy of FibroScan in patients with NAFLD. This algorithm can reduce the effect of subcutaneous fat on UAP, so FibroTouch may have certain advantages in quantifying liver fat content. The diagnosis accuracy and consistency of FibroTouch for staging fibrosis in chronic liver disease was comparable with FibroScan. There was a significant correlation  and similar cut-off values between the FibroTouch and FibroScan for liver stiffness23. Compared with the internationally recognized FibroScan technology, FibroTouch still has certain limitations, and its diagnostic efficacy and threshold for different types of liver disease still need more evidence to support.FibroScan has been widely used clinically to assess liver fat quantification by measuring CAP in the last decade, with liver stiffness assessment by LSM simultaneously. Validated against liver biopsy, CAP has been shown to have excellent diagnostic accuracy for detecting S1, S2, and S3 hepatic steatosis9. Validated by pathology, UAP was positively correlated with hepatic steatosis, and was significantly superior to the hepatic steatosis index, and LSM was positively correlated with degree of fibrosis and NAFLD activity score (NAS)9. Therefore, FibroTouch was selected to evaluate the liver condition of NAFLD patients in this study, which had a rich basis for clinical and scientific research.In patients with NAFLD, FibroTouch has a high diagnostic success rate, and can well distinguish the steatosis degree and fibrosis stage24. The proportion of patients with advanced fibrosis in Chinese adults with NAFLD from Hong Kong is low (3.7%)25. There is a lack of large epidemiological data on patients with different subtypes of NAFLD in China. Our study randomly selected patients with NAFLD diagnosed by imaging in a large general hospital in Wuhan, China. Patients with NAFL, NASH, and fibrosis were included, and patients with cirrhosis were excluded because the proportion of these patients was very small. Among these patients, 27.6% had mild steatosis, 47.4% had moderate steatosis, 24.7% had severe steatosis, 61.6% had no fibrosis, 30.4% had mild fibrosis and 28% had progressive fibrosis. The median UAP of these patients with mild, moderate and severe steatosis were 249 dB/m, 281 dB/m and 306 dB/m, respectively, and the median LSM with F0–F1, F2 and F3 were 5.2 kPa, 7.3 kPa and 11.3 kPa, respectively.The prevalence of NAFLD among adults in the general population in China is approximately 15% (6.3–27.0%)26. Some studies have found that waist circumference was a more accurate predictor of fatty liver than BMI26. Our study found that in non-cirrhotic patients with NAFLD, risk factors for the aggravation of liver steatosis included male, younger age, weight, BMI, waist circumference, history of hyperuricemia, FBG, UA, TG, ALT, AST, low AST/ALT ratio, GGT and LSM. The risk factors for the aggravation of hepatic steatosis included BMI, waist, TG, ALT and LSM. A prospective study of 5,323 FibroScan examinations found similar results, in which factors significantly associated with CAP were BMI, metabolic syndrome and LSM, and CAP increased with the number of these parameters28.Several risk factors for NAFLD have been identified from Chinese studies, including advancing age, male gender, recent weight gain, expanding waist circumference and metabolic disorders31. We found that risk factors for hepatic fibrosis in non-cirrhotic patients with NAFLD were decreased PLT count, increased AST and UAP values.Previous studies and systematic reviews showed that the variables associated with NASH and progressive fibrosis in patients with NAFLD were obesity , increasing BMI, hypertension, T2DM, AST/ALT ratio > 1 and decreased PLT count34. These non-invasive tests can reliably exclude advanced fibrosis and reduce the need for liver biopsy. In a cohort of NAFLD patients from Hong Kong and France, liver stiffness measurement by FibroScan was accurate for excluding advanced fibrosis (Kleiner stage 3–4) and performed better than non-invasive scores 35. A prospective study from China showed that the diagnosis accuracy and consistency of FibroTouch for staging fibrosis in chronic liver disease was better than APRI and FIB-423. Our study found that the staging of hepatic fibrosis according to FibroTouch was positively correlated with NFS, APRI and FIB-4, but the correlation was weak. The first three markers, but not BARD, differed among the different stages.Several non-invasive fibrosis markers have been used to assess hepatic fibrosis. In NAFLD patients, the negative predictive value (NPV) of these non-invasive indicators including FIB-4 index, APRI, NFS and BARD score for advanced fibrosis was about 90%, while the positive predictive value (PPV) was not high, among which FIB-4 index had the best predictive consistency41.Although transient elastography techniques such as FibroTouch and FibroScan provide good assessment of hepatic steatosis and fibrosis, their accuracy is affected by several confounding factors. The factors leading to the increase of LSM include hepatic inflammation, congestion and cholestasis, alcohol and food intakeIn conclusion, as an alternative of FibroScan, FibroTouch has good diagnostic performance for hepatic steatosis and fibrosis. Even so, there were some deficiencies in this study. Firstly, we did not expand the comparison between non-NAFLD and NAFLD. Secondly, the number of people with severe fatty liver and fibrosis included in this study was too small, which may affect the judgment of the results. Thirdly, because there were few people clinically diagnosed as liver cirrhosis caused by fatty liver in our hospital, this study does not include this kind of people, and future researchers can appropriately improve, so as to make the study more comprehensive.


However, in 2022, an alarming number of MPXV cases were reported globally, with evidence of person-to-person transmission. Because of this, the World Health Organization (WHO) declared the MPXV outbreak a public health emergency of international concern. The supply of MPXV vaccines is limited, and only two antivirals, tecovirimat and brincidofovir, approved by the U.S. Food and Drug Administration (FDA) for the treatment of smallpox, are currently available for the treatment of MPXV infection. Here, we evaluated 19 compounds previously shown to inhibit different RNA viruses for their ability to inhibit orthopoxvirus infections. We first used recombinant vaccinia virus (rVACV) expressing fluorescence (mScarlet or green fluorescent protein [GFP]) and luciferase (Nluc) reporter genes to identify compounds with antiorthopoxvirus activity. Seven compounds from the ReFRAME library  and six compounds from the NPC library  showed inhibitory activity against rVACV. Notably, the anti-VACV activity of some of the compounds in the ReFRAME library  and all the compounds from the NPC library  were confirmed with MPXV, demonstrating their inhibitory activity IMPORTANCE Despite the eradication of smallpox, some orthopoxviruses remain important human pathogens, as exemplified by the recent 2022 monkeypox virus (MPXV) outbreak. Although smallpox vaccines are effective against MPXV, access to those vaccines is limited. In addition, current antiviral treatment against MPXV infections is limited to the use of the FDA-approved drugs tecovirimat and brincidofovir. Thus, there is an urgent need to identify novel antivirals for the treatment of MPXV infection and other potentially zoonotic orthopoxvirus infections. Here, we show that 13 compounds, derived from two different libraries, previously found to inhibit several RNA viruses, also inhibit VACV. Notably, 11 compounds also displayed inhibitory activity against MPXV. In 2022, the World Health Organization (WHO) declared the monkeypox virus (MPXV) outbreak a public health emergency of international concern –3. Histo–5–Orthopoxvirus of the poxvirus family, which includes variola virus (VARV), cowpox virus, and vaccinia virus (VACV) (–23–MPXV belongs to the genus s (VACV) , 10. Poxs (VACV) . Currents (VACV) . To dates (VACV) , 14. MPXs (VACV) , 16. Dess (VACV) –21. Zoon (VACV) –, 22, 23.ACV) –23–.Orthopoxvirus  –33 and A–34–38–poxvirus , 43. Howpoxvirus . Due to poxvirus , but itspoxvirus –53. TherIn this study, we used a previously described bireporter cell-based assay based on the use of recombinant vaccinia virus (rVACV) expressing fluorescent proteins (green fluorescent protein [GFP] or mScarlet [Scarlet]) and luciferase (Nluc)  to asses55–50s) for rVACV Nluc/Scarlet and Nluc/GFP were similar, 0.031 and 0.026 μM, respectively, regardless of which fluorescence reporter was used for quantifications   , right. ctively) . Tecovirctively) . The decctively)  and MPXVctively) , 61, 62.50 Scarlet = 0.07 μM; EC50 GFP = 0.06 μM) or Nluc  expression. However, the SI value of antimycin A differed based on the median cytotoxic concentration (CC50) results from the MTT  and XTT  toxicity assays  or Nluc  expression. In the case of AVN-944, the SI value was not altered by the CC50 results from the MTT and XTT toxicity assays , and Zika virus (ZIKV) –59 also –y assays . AVN-944y assays . Other cy assays . Likewisy assays , top andy assays , bottom y assays . As descy assays  and tecoy assays  treatmeny assays . In the y assays . These ry assays  and NPC y assays  librariey assays –57, 59 a50 values obtained from the MTT and XTT toxicity assays . We found that five of the compounds tested from the ReFRAME library  and all y assays  and 4. TThe MPXV outbreak in early 2022 was declared a public health emergency of international concern by the WHO, with over 30,286 confirmed cases and 38 deaths in the United States as of March 2023  13. Alarmi5–50s and SI values.In this study, we first demonstrated the feasibility of using rVACV expressing both fluorescent proteins (Scarlet and GFP) and luciferase (Nluc) to easily identify compounds with inhibitory activity against VACV. Next, we used rVACV Nluc/Scarlet and rVACV Nluc-GFP to test the anti-VACV activity of compounds from the ReFRAME and NPC libraries previously reported to have antiviral activity against RNA viruses –59. Usin–Among the compounds in the ReFRAME library, antimycin A and AVN-944 were found to have the strongest inhibitory activity against VACV. It is worth noting that antimycin A has been described to inhibit infection of multiple viruses, including porcine reproductive and respiratory syndrome virus (PRRSV) , dengue 67–The NPC library compounds valinomycin, rotenone, and mubritinib also exhibited potent anti-VACV activity. Other compounds with inhibitory activity against VACV from the NPC library included buparvaquone, narasin, and monensin. Valinomycin has been described to have properties against tumors , bacteri79–Importantly, six of the compounds from the ReFRAME library and all of the compounds from the NPC library found to have inhibitory activity against VACV were confirmed to also inhibit MPXV. Some of the differences observed in the compounds’ inhibitory activities against VACV and MPXV likely reflect different readouts to assess their inhibitory activity. The ability to use fluorescence or luciferase reporter genes and the lower level of biocontainment required to work with VACV support the use of the bireporter expressing rVACV Scarlet/Nluc and rVACV GFP/Nluc in high throughput screening (HTS) format to identify compounds with inhibitory activity against orthopoxviruses. Importantly, the compounds from the ReFRAME and NPC libraries we tested are FDA approved for use in humans, which should facilitate the process of bringing them into the clinic.in vivo and to test their therapeutic potential. It should be noted that the compounds we tested target host factors, which will pose a high genetic barrier to the emergence of viral escape mutants, a common event with antivirals directly targeting a viral protein activity. This is illustrated by tecovirimat resistance, which is the result of mutations in the viral gene encoding the target protein F13  containment laboratories at Texas Biomedical Research Institute (TX Biomed) and were approved by the Institutional Biosafety Committee (IBC) at TX Biomed. All the researchers involved in studies using MPXV were vaccinated with the modified vaccinia virus Ankara JYNNEOS vaccine.l-glutamine). Cells were grown at 37°C in a 5% CO2 atmosphere.African green monkey kidney fibroblasts  and human adenocarcinoma alveolar basal epithelial cells  were maintained in Dulbecco’s modified Eagle’s medium  containing 5% fetal bovine serum (FBS) and 1% PSG  and luciferase (Nluc), rVACV Nluc/GFP and rVACV Nluc/Scarlet, respectively, were previously described and characterized . We usedThe compounds previously identified to have antiviral activity against RNA viruses in the ReFRAME library were previously described –58 and i–4 cells/well, in quadruplicate) were infected with 200 PFU/well of rVACV Nluc/Scarlet or rVACV Nluc/GFP for 1 h at 37°C. After virus absorption, the virus inoculum was removed, and cells were incubated with infection medium containing 3-fold serial dilutions of the indicated compounds . Mock-infected cells and cells infected in the absence of drug were included as internal controls. At 24 h postinfection (hpi), fluorescence expression (GFP and Scarlet) was visualized using a fluorescence microscope and quantified using a microplate reader (BioTek Synergy). Simultaneously, cell culture supernatants from the same infections were collected and used to measure Nluc expression using the Nano-Glo luciferase substrate (Promega) and the microplate reader (BioTek Synergy). Percent viral infection was determined based on fluorescent (GFP or Scarlet) and Nluc signals, as previously described (n = 4). EC50s were determined using sigmoidal dose response curves on GraphPad Prism (version 9).Confluent monolayers of human A549 cells  were infected with 200 PFU/well of MPXV for 1 h at 37°C. After virus absorption, the virus inoculum was removed, and cells were incubated with infection medium containing 3-fold serial dilutions  of the indicated compounds and 1% Avicel (Sigma-Aldrich). Mock-infected cells and cells infected in the absence of drug were included as internal controls. At 24 hpi, cells were fixed in 10% neutral buffered formalin for 24 h and then permeabilized with 0.5% Triton X-100 in phosphate-buffered saline (PBS) for 10 min at room temperature (RT). Then, cells were blocked with 2.5% bovine serum albumin (BSA) in PBS for 1 h, followed by immunostaining with an anti-VACV A33R polyclonal antibody , Vectastain ABC kit, and DAB  peroxidase substrate kit (Vector Laboratories), based on the manufacturer’s recommendations. Viral infections were determined based on the number of plaques present in each of the 96-well plates using an ImmunoSpot plate reader, as previously described (n = 4). Nonlinear regression curves and EC50s were determined using sigmoidal dose response curves in GraphPad Prism (version 9).Confluent monolayers of human A549 cells  were infected with rVACV Nluc/Scarlet, rVACV Nluc/GFP, or MPXV at an MOI of 0.01 or 3 for 1 h at 37°C. After virus absorption, the virus inoculum was removed, and cells were incubated with infection medium containing 10-fold serial dilutions of tecovirimat (starting concentration of 100 μM). Mock-infected cells and cells infected in the absence of drug were included as internal controls. At 24, 48, and 72 hpi, cell culture supernatant was collected, and Nluc activity was determined by adding Nano-Glo luciferase substrate (Promega) and quantified in a microplate reader (rVACV Nluc/Scarlet or rVACV Nluc/GFP). Viral titers in the cell culture supernatants were determined by plaque assay in CV-1 cells. Briefly, cells were infected with 10-fold serially diluted supernatants for 1 h. After viral adsorption, cells were overlaid with medium containing 1% Avicel (Sigma-Aldrich). Cells were fixed in 10% neutral buffered formalin for 24 h and then permeabilized with 0.5% Triton X-100 in PBS for 10 min at room temperature. Next, cells were blocked with 2.5% BSA in PBS for 1 h and immunostained with an anti-VACV A33R polyclonal antibody  and developed with an anti-rabbit immunoglobulin Vectastain ABC kit and a DAB peroxidase substrate kit (Vector Laboratories), following the manufacturer’s recommendations. Experiments were conducted using technical triplicates. Microsoft Excel was used to calculate the mean and SD of viral inhibition from triplicate wells (n = 3).Confluent monolayers of human A549 cells  were incubated with 100 μL of infection medium containing 3-fold dilutions of compounds  or with 0.1% DMSO as a vehicle control. Then, plates were incubated at 37°C with 5% CO2 for 48 h, treated with 15 μL of dye solution for the MTT assay or 50 μL of XTT labeling reagent for the XTT assay, and incubated at 37°C for an additional 4 h. Stop solution was then added to the MTT assay to halt the reaction. Absorbance (570 nm) in each of the wells was measured using a microplate reader (BioTek Synergy). Cell viability was determined as the percentage of values for DMSO vehicle-treated cells. Nonlinear regression curves and the CC50 at 48 h were determined using GraphPad Prism software (version 9).MTT  and XTT  assays were used to determine A549 cell viability as previously described . Briefly50 and EC50s were calculated using sigmoidal dose-response curves, and the selective index (SI) of each compound was determined by dividing the CC50 by the EC50s. Significance was determined by standard unpaired Student’s t test.GraphPad Prism software (version 9) was used for data analysis. CC