The development of high-throughput techniques has enabled profiling a large number of biomolecules across a number of molecular compartments. The challenge then becomes to integrate such multimodal Omics data to gain insights into biological processes and disease onset and progression mechanisms. Further, given the high dimensionality of such data, incorporating prior biological information on interactions between molecular compartments when developing statistical models for data integration is beneficial, especially in settings involving a small number of samples.cis-regulatory quantitative effects in the proposed model. The model, coined Cox-sMBPLS, exhibits superior prediction performance and improved feature selection based on both simulation studies and analysis of data from heart failure patients.We develop a supervised model for time to event data  that simultaneously accounts for redundant information within Omics profiles and leverages prior biological associations between them through a multi-block PLS framework. The interactions between data from different molecular compartments  were captured by using The proposed supervised Cox-sMBPLS model can effectively incorporate prior biological information in the survival prediction system, leading to improved prediction performance and feature selection. It also enables the identification of multi-Omics modules of biomolecules that impact the patients’ survival probability and also provides insights into potential relevant risk factors that merit further investigation. Therefore, the lasso method can poorly indicate this grouping information in the multi-Omics setting. Theoretical and practical explanations of this limitation are given in L1, L2) penalties on the regression coefficients including Cox model. A recent benchmark analysis  to integrate additional biological information to the training of the model. Note that the model and integrative strategy are general and can be easily adapted to other molecular compartments with appropriate modifications.In this paper, we propose a new integrative survival prediction model named supervised Cox sparse Multi-Block Partial Least Squares (Cox-sMBPLS) by simultaneously controlling the redundancy between Omics profiles from different molecular compartments, focusing on epigenomics, genomics and transcriptomics and incorporating To handle censoring in the survival outcome data, we employ a reweighting technique described in the section “Materials and Methods.” The high dimensionality of the Omics data under consideration is dealt with the use of regularization. The key objective of Cox-sMBPLS is to determine multi-Omics modules , that are most associated with disease progression and patient’s survival.cis-regulatory quantitative effects  to extract low dimensional Omics modules that are predictive of survival times is depicted in An overview of the multi-block data structure used as input, together with how associations between molecular compartments are captured through cis-regulatory quantitative effects  and updating Omics-blocks τ = [τ3.53.63.7b)( as jb)*( (updated based on Ωb) and y* in each block  using k number of latent components.Fit PLS regression to 3.9y* as Update 4. Final Cox-sMBPLS model: Fit a Cox-PH model with  and remaining latent components from the sMBPLS algorithm.iC indicate the independent true survival time and censoring time for the ith subject , respectively. The observed data consist of pairs {|i = 1, …, n} where i = 1 . To deal with the right censoring in the MBPLS algorithm, we use the reweighting method  = P(C > t), t ≥ 0. Then,Let g method  to constCS will be calculated using the intercept-only Cox-PH model (Kaplan-Meier) with the status indicator 1−δi (using survfit R function)1.This identity can be taken as the theoretical basis for estimating the mean observed survival time by the (weighted) sample average X (a n × p matrix of covariates) and y (a n × 1 matrix of response variable in a univariate case). In contrast to PCA, PLS uses both X and y to construct the latent components (T = XW) by maximizing a successive maximization problem. The objective function to find the weight vectors  is as follows:Partial least squares (PLS) regression, originally introduced by k is the number of the latent components (tuning parameter or fixed by user), and W is a p × k matrix of weights . The latent component T (a n × k matrix) is then calculated as T = XW.where Genome-wide quantitative trait loci (QTL) mapping enables the determination of genetic loci affecting other Omics, i.e., transcriptome, proteome, and metabolome. Some Omics markers do not directly contribute to the phenotype and affect the disease through other intermediates-Omics. Therefore, considering these QTL associations in each Omics layer can provide more functional information about disease-associated markers. In fact, a QTL-pair is a pair of different Omics  that are (highly) associated regarding their effects on the underlying disease (outcome). Hence involving both elements of a QTL-pair  in a regression model will not add much more information than involving only one of these elements (at the cost of degrees of freedom and multi-collinearity). One way around this is to consider one element of a QTL-pair in the regression model (such as SNP in an eQTL-pair), then regress out the effect of this element and replace the second element with the residuals. This way, we are considering unwanted/uncorrelated information besides the QTL information in the regression model, avoiding multicollinearity.As shown in b , a matrix of bp features/covariates measured for n samples. For ease of presentation, we consider the following B = 3 blocks . However, the proposed modeling framework can accommodate a larger number of blocks and thus other Omics types (such as proteomics and metabolomics) with minor modifications. Thus, let p1 genes, genotypes of p2 SNPs, and DNA methylation of p3 CpGs for n samples, respectively. Next, we split each Xb) is split by utilizing the eQTL information as follows:Let q1 is the number of the eQTL-genes . Whole blood eQTL data are extracted from the Genotype-Tissue Expression Project (GTEx)  and jth eQTL-gene (where t (GTEx)  that areWe then update The second block (genotypes) is split by utilizing the meQTL information as follows:q2 is the number of the meQTL-SNPs . Whole blood cis-meQTL data were extracted from BIOS QTL (where BIOS QTL  that areWe then update The third block (DNA methylations) is split by utilizing the eQTM information as follows:q3 is the number of the eQTM-CpGs . Whole blood cis-eQTM data were extracted from BIOS QTL . By regressing out the effect of these genes, the so-called “eQTM residuals” (jth" eQTM-CpG (where BIOS QTL  that areWe then update The illustration of the multi-Omics data structure and Omics-block updating procedure is presented in Xb)( = Xb)*( as explained in section “Integrating Cis-regulatory Quantitative Effects”) and y  be the covariate matrices (blocks) and response vector on the same n samples, respectively. In each block, the dimensionality of the block can be reduced by taking a linear combination of the covariates τb)( = Xb)(wb) that express the importance of each covariate on the latent component τb)( (a n×1 matrix). b)( (ωb)( > 0) is the weight for block b, which indicates the contribution of this block to the covariance structure of the input and response (y) data. We can then posit the following optimization problem for calculating the latent components across all blocks:Let L1 penalty on direction vector wb) to the sparse version could be obtained by adding an L1 constraint onto a surrogate of the direction vector. Therefore, a generalized version of the optimization problem using a combined L1&L2 regularization component becomes:In the case of PLS, 1 and λ2. cb) does not depend on and often needs a large λ2 value to be solved. Therefore, we use λ2 → ∞ which yields the special case of the elastic-net regularization, called univariate soft-thresholding . The algorithm is then followed by a PLS regression on the selected variables, and iterated with updating the response variable, y. The proof for the solution in equation (3) is provided in where X-space of selected variables (Xb)( ∈ Ωb)  is addressed by keeping the Krylov subsequence structure of the direction vectors in a restricted  ∈ Ω(b)) . Specifi ∈ Ω(b)) .Tn×1) in step 3.6. Blocks’ weight (ω) are calculated in Step 3.5. The so-called active variables set (Ωb) is then updated in step 3.7 followed by a PLS regression using active variables Xb)*( ∈ Ωb) is then updated in step 3.9. Note that Xb)*( and y* (step 3.9). The solution to the optimization function (2) also enables us to identify multi-Omics modules. These modules are linear combinations of multiple Omics profiles with large absolute values of wb) vectors are calculated in step 3.2 where λ is a non-negative (sparsity) tuning parameter which is tuned using a k-fold cross-validation . In step 3.3 the latent components for each block are calculated (k) and the sparsity (λ) hyper-parameters that lead to the best prediction performance. In principle, we can try different combinations of k (number of the latent components) and λ (sparsity). The chosen k and λ are the ones giving the highest model performance measures. For the real data analysis, we considered p is the total number of the covariates, ν is the fold number in the (k-fold) CV, and n is the sample size. This upper bound for the number of the latent components is suggested by Cross-validation (CV) is used to tune the number of components  at the University of Illinois at Chicago (UIC). All patients provided written, informed consent ; namely, with preserved ejection fraction (HFpEF) and reduced ejection fraction (HFrEF). Further, 47% of them experienced death or a hospitalization event. The original discovery cohort included 103 HF (HFpEF and HFrEF) patients with complete data of all Omics types , 12 of which were removed due to sex mismatch and  consent .R2 = 80% were excluded to keep only high-quality imputed profiles. We then performed linkage disequilibrium pruning (LDP). Thereafter, whole blood cis-eQTL data from the Genotype-Tissue Expression Project (GTEx) . We excluded SNPs with a missing rate ≥10%, monomorphic SNPs with MAF < 0.01% and SNPs on the negative strands. Additional genotypes were imputed based on a two-step approach. First, the samples were phased into a series of estimated haplotypes, and then, imputation was performed on them. After imputation, genotypes with t (GTEx)  were uset (GTEx) . In totaBIOS QTL  were thecis-regulatory quantitative effects), and the relevance of each Omics profile or a combination of them to explain the survival probabilities. We compared the proposed model to elastic-net Cox-PH (El-net Cox) , the association between the Omics profiles (via net Cox) , random net Cox) , Block Fnet Cox) , and mulnet Cox) . All modb = 1, 2, 3) of dimension bn = p, with fixed sample size n = 91. Matrices b = 1, 2, 3) are random samples (without replacement) from the real Omics data presented in the next section. For the construction of true latent components (τT), we assume that some of the features in each block have small or no effect on the response variable by specifying sparse (true) direction vector (wT). These weights are sampled from the distribution of the weights collected from a standard sparse PLS on a random sample of the Omics data. Therefore, true latent components are sparse across all simulations. The response variable  for sample i  is simulated using a flexible-hazard model . See k = 10). The results of the moderate and high levels of dimensionality are presented in We sample true predictor matrices rd model . We simuk = 2, 4% for k = 5 and k = 10; in El-net Cox decreased by 8%, 7%, and 11% for k = 2, 5, 10, respectively; in RSF decreased by 11%, 3%, and 10% for k = 2, 5, 10, respectively; in Block Forest decreased by 6%, 5%, and 5% for k = 2, 5, 10, respectively; in MCIA decreased by 3% and 0% for k = 2, 5, respectively, and increased by 2% for k = 10. Amongst these models, the proposed Cox-sMBPLS (2–4% decrease) and Block Forest (5–6% decrease) are more stable than El-net Cox (7-11% decrease), RSF (3–11% decrease), and MCIA (0–3% decrease and/or 2% increase) against the censoring rate. The feature selection results showed less stability against censoring rate using other performance measures . I/D AUC, and Uno’s AUC tended to increase while increasing the censoring rate. This may be due to the relatively low number of events and the following low number of patient-pairs used to estimate the measures. k  and feature-selection performance (AUCs) of the proposed Cox-sMBPLS model remained higher than other models regardless of the different changing parameters. When increasing the censoring rate from 10 to 60%, the feature-selection performance (C/D AUC) of all models decreased (except for MCIA) : in Cox-s. k see –3. In mos. k see . In Tabls. k see  of lassoOverall, the proposed supervised Cox-sMBPLS method outperformed all competing methods  regarding the exact survival prediction and feature-selection power. Moreover, this method showed less sensitivity to the selection of the tuning parameters and censoring rate compared to competing methods.k = 15 multi-Omics modules . k is tuned using a 5-fold CV within the range of 1 = k = 73. The upper boundary (k=73) is calculated based on p is the total number of the covariates, ν is the fold number in the (k-fold) CV, and n is the sample size. Results are provided in p-value < 0.1) in detail. Module 10 contains 375 features  and module 13 contains 497 features . There are 122 Omics profiles , included in module 13 but not module 10. Details for these 122 features are also included in The supervised Cox-sMBPLS model (on HF cohort) retained We also compared the prediction performance of the Cox-sMBPLS model to El-net Cox , RSF Is, Block Fp = 0.097) and 13 (p = 0.059) are the two significant modules  also confirms the role of these genes in heart complications. Disease enrichment results similarly show that the genes in the multi-Omics modules are mainly enriched for heart disease, such as heart failure, cardiac hypertrophy, and myocardial failure. A brief discussion of the common genes follows.To interpret the biological relevance of the significant multi-Omics modules enrichment analysis of their Omics profiles using network-based resources and disease ontology is undertaken. Specifically, we performed a gene-set network analysis  using Geules see . There iPDPK1 (Phosphoinositide-dependent protein kinase-1) is the PDK1 protein coding gene and also a part of the AGC super family of protein kinases which have been well documented for playing a crucial role in heart complications (HRC) can affect Ca2+ cycling in the sarcoplasmic reticulum (SR) that could cause the mitochondrial death pathway and enhance cardiac function in failure heart (TAB2 (TAK1 binding protein-2) is known to play an important role in cardiac development and has recently received more attention in heart diseases. There has been recent research suggesting TAB2 and its signaling network (TAB2-TAK1) as novel therapeutic targets in heart complications  are required to identify novel biomarkers.These follow-up analyses suggest the biological relevance of the multi-Omics modules resulted from the proposed Cox-sMBPLS algorithm. However, further functional and validation studies  was developed. It also enables identification of multi-Omics modules -combinations of different Omics features- exhibiting a large effect on survival probabilities. The proposed modeling framework can easily accommodate a large number of blocks and thus other Omics types with minor modifications.A survival prediction model (Cox-sMBPLS) based on leveraging and integrating information across multi-Omics compartments via the cis-regulatory information and a censoring-reweighting technique in our proposed algorithm. The key output of the Cox-sMBPLS is to determine multi-Omics modules that are most associated with disease progression and patient survival.In the past decade, a large body of literature was developed to introduce methods relating Omics profiles and time to an event such as recurrence in cancer patients, death, etc. Cox-PH  is the mPDPK1 and TAB2 associated with HF which have been well documented for playing a crucial role in heart complications (Simulation studies showed that both the prediction and feature-selection performance of Cox-sMBPLS is significantly better than competing procedures (El-net Cox and RSF) across multiple settings , 2 and iications .A direction of future research is to enhance the incorporation of additional prior biological knowledge; e.g., include functional pathway information. On the validation front, analysis of data from animal studies can assist in identifying novel non-coding features prioritized by significant multi-Omics modules.juliod@cop.ufl.edu.The data analyzed in this study is subject to the following licenses/restrictions: Data are available from JD upon reasonable request. Requests to access these datasets should be directed to JD, The studies involving human participants were reviewed and approved by The University of Illinois at Chicago (UIC). The patients/participants provided their written informed consent to participate in this study.NV and GM designed the study and wrote the manuscript. NV, CM, AD, LC, and JD performed the multi-Omics data preparation and quality control. NV and JD implemented the analysis. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer HQ declared a shared affiliation with the authors NV, CM, LC, JD, and GM to the handling Editor at time of review.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Electronic cigarettes (e-cigarettes) are being increasingly used as a “safer” alternative to regular cigarettes as a method of de-addiction or a bridge to nicotine cessation. However, a multitude of pulmonary pathologies have been described associated with its use and have been clubbed under the category of e-cigarette or vaping use-associated lung injury (EVALI). This case describes a patient who started e-cigarette smoking in order to quit combustible cigarette smoking and developed adult-onset severe asthma. The clinical effect was initially reversible but later developed into persistent symptoms requiring inhaled and systemic therapy. Electronic cigarettes (e-cigarettes) have become a mainstay in today’s culture with many smokers utilizing them in their pursuit of smoking cessation or a “safer” alternative to traditional combustible cigarettes. E-cigarettes offer flexibility on the quantity of nicotine being used. The safety of e-cigarettes was called into question by the numerous cases of e-cigarette or vaping use-associated lung injury (EVALI), some of which were fatal. These cases highlighted the reality that e-cigarettes may not be the benign alternative to cigarettes that many were seeking . While EA 59-year-old female was referred to the pulmonary clinic for evaluation of wheezing, shortness of breath with exertion, and cough ongoing for the last five months. She was apparently asymptomatic before that. She denied chest pain, orthopnea, leg swelling, heartburn, sinus drainage, known environmental allergies, or any other complaints. The patient admitted to gaining 5 pounds (Lbs) of weight since the symptoms started (BMI 34.9). She started smoking cigarettes at the age of 16 and had smoked one1 pack per day since then till about six months ago (42 pack-years) when she started smoking e-cigarettes in a successful attempt to quit cigarette smoking. Incidentally, her partner continued to smoke cigarettes in the house. The patient’s indoor environment also consisted of three dogs and three cats, which she has had for at least five years. No indoor plants. She used to work as a beauty technician and had retired about a year back. No previous history of asthma or allergies. No family history of asthma or other lung-related ailments. No history of other significant organic or inorganic material exposures. The patient’s past medical history was significant for hypothyroidism, Asperger’s syndrome, Type II diabetes mellitus, peripheral neuropathy, dyslipidemia, endometriosis, and hypertension. The patient was diagnosed with asthma/chronic obstructive pulmonary disease (COPD) by her primary care physician and started on a tiotropium inhaler 18 mcg inhalation daily, beclomethasone 80 mcg inhaler, two puffs twice daily, and albuterol inhaler as needed for breakthrough symptoms. The patient was found eligible and enrolled in the lung cancer screening program. Her low dose computerized tomography scan of the chest (LDCT chest) showed mild emphysematous changes with no lung nodules. Pulmonary function testing (PFT) results are displayed in Table  Upon evaluation in the Pulmonary Clinic, her treatment regimen was optimized to include high dose inhaled corticosteroids (ICS), inhaled long-acting beta-agonists (LABA), inhaled long-acting muscarinic antagonists (LAMA), and short-acting inhaled beta-agonists (SABA). She was advised to discontinue e-cigarette use immediately and was started on nicotine patches as replacement therapy.At the one month follow-up clinic visit, the patient said that she had quit smoking e-cigarettes but had relapsed into conventional cigarette smoking. She denied the use of nicotine patches. Her symptoms were persistent despite new inhaler therapy after the last visit, which led her to quit e-cigarette use. After cessation, her symptoms had completely resolved. She was advised to continue regular use of the current asthma regimen till the next visit, where a step-down of therapy would be started if symptoms remained well controlled. She was counseled on smoking cessation.The patient missed her next follow-up visit and came back to the clinic five months later. She had restarted e-cigarette smoking. She complained of worsening respiratory symptoms despite being on the same bronchodilator regimen. ICS dose was increased, and the patient was given a taper of oral corticosteroid as a bridge. Counseling was provided to quit e-cigarette use using nicotine patches and behavioral therapy.Aspergillus fumigatus, and alder and birch trees. Total IgE was elevated at 615 kU/L. The absolute eosinophil count in peripheral blood was 210/ml. Due to persistent symptoms with multiple exacerbations requiring oral corticosteroid (OCS), the patient was started on scheduled OCS with a plan to switch to a biologic agent pending insurance approval. Unfortunately, she still continues to smoke cigarettes despite numerous attempts at smoking cessation with various modalities. Over the course of the next two years, montelukast and fluticasone nasal spray were added for asthma control in addition to inhaled therapies and several courses of oral corticosteroid. The patient had missed clinic appointments a number of times. For further workup, cardiac etiologies of dyspnea were ruled out. Sleep evaluation was done and the patient was started on bilevel positive airway pressure (BiPAP) therapy for obstructive sleep apnea and hypoventilation syndrome. Weight had fluctuated within 10 Lbs. The patient switched from e-cigarettes to cigarette smoking again and the symptoms did not improve this time. Follow-up PFT did not show any significant change. Serum allergen panel targeted towards common local allergens showed Immunoglobulin E (IgE) response to Despite the rising popularity of e-cigarettes as a commonly perceived safer alternative to tobacco cigarettes, the risks of harmful outcomes are not fully recognized by the majority of the population. Numerous studies have suggested that the use of e-liquid, with or without nicotine, decreases lung function, most likely due to the toxins and irritants found in the liquid itself. Furthermore, the metal coils used in the heating and cooling process of the e-liquid are a source of chromium, manganese, nickel, and lead, ultimately emitted as metallic nanoparticles and then inhaled . In our The use of e-cigarettes is not a safer alternative to combustible cigarette smoking and can lead to short and long-term implications, some of which can be refractory to conventional therapies. Our patient developed adult-onset asthma likely as a result of e-cigarette use, which initially was reversible, but later transformed into a refractory process requiring systemic corticosteroid use in addition to conventional inhaled therapies.


While dysplastic liver nodules in cirrhosis are pre-malignant, little is known about the predictors of hepatocarcinogenesis of these lesions. This was a retrospective observational study of subjects with cirrhosis who had at least one hypervascular, non-malignant intrahepatic nodule on imaging while undergoing outpatient management by a tertiary hepatology referral centre between Jan 2009 and Jan 2019. Clinical and biochemical parameters were collected. The primary endpoint was transformation to hepatocellular carcinoma (HCC) as determined by Liver Imaging Reporting and Data System. During the study period, 163 non-malignant hypervascular nodules were identified in 77 patients; 147 had at least 6 months of follow up imaging and 16 received upfront radiofrequency ablation upon detection. During a median follow up of 38.5 months (IQR 16.5–74.5), 25 (17%) of the 147 hypervascular nodules being monitored transformed to HCC. On multivariate analysis, Child–Pugh grade was found to be the only independent predictor of nodule transformation into HCC (p = 0.02). Those with Child–Pugh B and C liver disease had a 10.1  and 32.6-fold  increased risk respectively for HCC transformation compared to Child–Pugh A subjects. This large, single centre study demonstrates that around 20% of dysplastic nodules in cirrhotic patients undergo hepatocarcinogenesis during follow up, and that Child Pugh grade is the only independent predictor of transformation to HCC. Additional prospective studies are warranted to better understand the risk profile of these nodules, and how best they should be managed. Around 90% of HCC cases arise in patients with liver cirrhosis5 with the annual incidence of HCC in cirrhosis being as high as 7%8. Although the independent predictors of HCC overall are well understood, these have not been well elucidated in the literature with specific reference to a cohort of cirrhotic patients. Nevertheless, retrospective studies have identified several clinical parameters that confer an increased risk of HCC in cirrhotic liver including the presence of dysplastic nodules10.Primary liver cancer is a leading cause of cancer-related death and the the seventh most common cancer worldwide14. They may be defined as focal and distinctly nodular lesions without clear malignant features based on imaging and/or histopathology17. However, there is no universally accepted definition of DN in the literature while the rate of malignant transformation remains poorly understood17. Radiologically, DN present as arterially enhancing (i.e. hypervascular) nodules on contrast imaging with computed tomography (CT) or magnetic resonance imaging (MRI) but without the salient feature of washout during the portal venous and/or delayed phases to connote HCC19. They are however often difficult to distinguish from early HCC’s predominantly due to both being vaguely nodular and having similar histologic features with the exact point of carcinogenesis not readily identifiable18.Dysplastic nodules (DN) are pre-neoplastic lesions that occur relatively commonly in cirrhosis with the reported prevalence being 14–37% and the annual incidence being around 1.5%20. Gadoxetic acid enhanced MRI was shown in a comparative 13 year systematic review and meta-analysis to have a high per-lesion sensitivity and positive predictive value of up to 85.6% and 94.2% respectively21. Similarly, contrast enhanced ultrasonography (CEUS) has been suggested as an option for further investigaton of indeterminate lesions22, having demonstrated overall per lesion sensitivity of 84.4% and positive predictive value of 89.3% for HCC21. Although CEUS is yet to be endorsed by current guidelines24 it can be considered as an advanced diagnostic tool in indeterminate lesions.In this context, the availability of MRI with newer contrast agents such as gadoxetic acid has been a major advancement in this field by enabling a better differentiation of dysplastic nodules from early HCC29. Small studies have suggested nodule characteristics such as size and morphology and change in size and/or vascular pattern throughout follow up are predictive, as is patient age32. In 2013 Iavarone et al.33 demonstrated a positive correlation toward hepatocarcinogenesis in histologically proven high grade nodules vs low grade nodules, but no significant clinical risk factors. Hence, there is insufficient published data overall to substantiate any clinical risk factors for hepatocarcinogenesis of these nodules.Currently, there is a paucity of information on the clinical and tumour risk factors for neoplastic change of premalignant lesions36. The most widely accepted course is close surveillance of these in order to monitor for malignant transformation over time. Similarly, there is no tangible data to assess whether these nodules should be actively managed when identified such as with local thermal ablation in order to prevent hepatocarcinogenesis. Thus, we undertook this observational study to evaluate a) the epidemiology and clinical course of de novo non-malignant hypervascular nodules in patients with cirrhosis, particularly in relation to malignant transformation, and b) the baseline factors predictive of the development of HCC in hypervascular nodules detected on contrast CT or MRI.In addition, there is limited data to determine how best to manage dysplastic nodules when newly identified in patients with cirrhosisst 2009 and January 31st 2019. Patients were identified from electronic medical records using coding for the terms “arterially enhancing”, “dysplastic”, or “hyper-vascular” and “hepatic nodule” or “liver nodule” on radiology reports related to multiphasic contrast scans with computed tomography (CT) or magnetic resonance imaging (MRI). The protocol and any amendments received Institutional Review Board approval prior to initiation of the study with submission being undertaken via a low risk ethics application.This was a retrospective, single-centre, observational study of subjects with cirrhosis who were found to have at least one hypervascular, non-malignant intrahepatic nodule while undergoing management of their liver disease by the hepatology service over a 10-year period between January 31To be eligible for study inclusion subjects needed to meet all the following eligibility criteria: (a) male and female patients at least 18 years of age; (b) documented history of cirrhosis; (c) presence of at least one non-malignant hypervascular intrahepatic nodule confirmed on abdominal imaging with multiphasic contrast CT and/or MRI, either at baseline or during follow up; and (d) be undergoing regular surveillance of the hypervascular nodule with imaging at 3–6 monthly intervals with at least 6 months of follow up on imaging. Subjects were excluded if they met any of the following exclusion criteria: (a) absence of cirrhosis; (b) undergone liver transplant within 6 months following detection of hyper-vascular nodule; (c) the presence of advanced HCC (macrovascular invasion and/or extrahepatic spread); and d) insufficient follow up of the nodule of < 6 months from initial detection unless treated prior. All cases with hypervascular nodules were discussed at a multi-disciplinary team meeting comprising hepatologists, diagnostic and interventional radiologists, and dedicated HCC nurse to confirm the diagnosis and determine initial management that in the vast majority of cases involved close observation and in select cases upfront ablation with RFA.37. Nodules that met the above radiologic criteria were only considered benign and included if they had shown stability in size and appearance for at least 6 months following detection. This reflects current international guidelines on the surveillance of a liver nodule thought to be benign36. Past HCC was defined as evidence of HCC being definitively treated without macrovascular invasion and/or extrahepatic spread prior to detection of a benign hypervascular nodule. Current HCC was defined as evidence of a hypervascular hepatic nodule(s) with evidence of washout on CT or MRI with primovist occurring either prior to or at the time of occurrence of the non-malignant nodule being included in the study. For this study definite HCC and probable HCC were defined in cirrhotics as a lesion that was LiRADS-5 and LiRADS-4 respectively.Cirrhosis was defined based on either (a) the combination of clinical, radiologic and laboratory criteria indicative of cirrhosis; (b) a liver stiffness measurement (LSM) > 12.5 kPa via vibration-controlled transient elastography (VCTE); or c) liver histology. Non-malignant hypervascular “dysplastic” nodules were defined on contrast-enhanced triphasic liver CT as hyperattenuating masses in the arterial phase with no washout on portal venous or delayed phases, and/or on MRI liver with primovist as the presence of a hyperattenuating mass post contrast and the absence of an increased T2 signal, restricted diffusion and washout on multiphasic post contrast imaging as described38.Data was extracted from electronic medical records and recorded on a standardised form. Data collection included: (1) Patient characteristics: age, gender, body mass index (BMI), diabetes history; (2) Liver disease: aetiology, Child–Pugh score, Model of End-stage Liver disease (MELD) score, ALBI score; (3) Laboratory parameters: serum alpha-fetoprotein (AFP), platelet count, serum albumin, INR, creatinine, bilirubin; (4) Nodule(s) characteristics: size (mm), location, number, imaging features including LiRADS status at baseline and during follow up; (5) Treatment received to nodules; (6) HCC status: past or current; and (7) Follow up details: patient survival, HCC status, nodule status . Transformation of nodules to HCC was determined by one of three experienced diagnostic radiologists  according to LiRADS criteriaThe primary end point of this study was the rate of malignant transformation to HCC in de novo hypervascular nodules in patients with cirrhosis. Further analysis was undertaken to determine the baseline factors predictive of the development of HCC in hypervascular nodules. The secondary endpoint was the role of radiofrequency ablation (RFA) at the time of identification of hypervascular nodules.Descriptive statistics of the cohort were performed with continuous variables assessed for normality and expressed as mean ± standard deviation (SD) or median (inter-quartile range) depending on the underlying data distribution. Categorical variables were summarized using frequencies or proportions. Variables were compared using Mann–Whitney test for continuous variables and Fishers exact test for categorical ones. Univariate and multivariate stepwise Cox regression were used to evaluate the baseline factors associated with development of HCC in the nodule. As an individual patient could have more than one dysplastic nodule, the Cox model was constructed with nodules nested within individual patients, with patients treated as a random variable . In addition, cumulative failure probability curves were used to examine malignant transformation of nodules among patients with cirrhosis according to baseline factors found to be significant on Cox proportional hazards analysis.This article does not contain any studies with animal subjects.All procedures followed were in accordance with the ethical standards of the responsible committee on human The protocol and any amendments received Institutional Review Board approval prior to initiation of the study with submission being undertaken via a low risk ethics application. This included a waiver of informed consent from all patients included in the study.The protocol and amendments received approval by The Alfred Hospital Human Research Ethics Committee prior to initiation of the study with submission being undertaken via a low risk ethics application. This include permission for publication of the data.This article does not contain any studies with plants.This was a retrospective cohort study that did not require clinical trials registration.2. A current or past history of HCC was present in 24 (14.7%) and 18 (11.0%) subjects respectively, while HCV infection was the most common aetiology for cirrhosis (46.3%) followed by HBV infection (25.2%) and alcohol (6.7%). Five (3.4%) nodules occurred in patients with dual HCV and HBV aetiology, and six cases (4.1%) with mixed alcohol and HCV liver disease. Median nodule size was 10.0 mm (IQR 7.0–13.0).We identified 163 eligible nodules in 77 patients. The baseline demographic, clinical and liver disease characteristics of the cohort at the time of the index liver nodule detection are shown in Table Sixteen 10%) of the 163 hypervascular nodules underwent definitive initial treatment with RFA. There were key differences between subjects with nodules that did and did not receive RFA at detection (see supplementary Table 0% of theDuring a median follow up of 38.5 months IQR 16.5–74.5), 25 (17%) of the 147 hypervascular nodules not receiving initial RFA transformed to HCC. The median time to transformation was 29.0 months (IQR 12.5–38.0 months). The 1-, 2-, and 3-year rate of transformation of dysplastic nodules to HCC was 4.3%, 9.5% and 15.7% respectively. The cumulative probability of progression to HCC of the overall cohort is shown in Fig. 6.5–74.5,The baseline clinical and liver disease characteristics of subjects according to whether transformation to HCC occurred over time or not are shown in Table On univariate analysis, dysplastic nodule transformation to HCC was associated with Child–Pugh score (p < 0.001) and grade (p < 0.001), MELD p = 0.002) and ALBI scores (p < 0.001). Notably, median AFP level at nodule diagnosis was not associated with malignant transformation . Other clinical parameters not significant on univariate analysis were age, sex, BMI, aetiology of liver disease, size of lesion, HCC status, and diabetes Table . On mult and ALBIOf the 122 nodules that did not transform, 47 (38.5%) spontaneously disappeared with a median time to disappearance of 31.0 months (IQR 12.0–36.5) while 75 (61.5%) remained stable, over a median follow up time of 59.0 months (IQR 33.0–95.0). Although the median MELD score was not dissimilar between the two groups (median 6 vs 8 respectively), nodules were more likely to disappear or remain stable throughout follow up with a lower MELD score (p = 0.02). Similarly, a lower ALBI Grade conferred a higher chance of spontaneous disappearance or stability of a nodule (p = < 0.001).In this real-world observational study, we evaluated the clinical course of de novo non-malignant hypervascular nodules in patients with cirrhosis particularly in relation to malignant transformation to HCC. Our clinical experience demonstrates that around one-fifth of these nodules transformed into HCC over time with the 1-, 2-, and 3-year rate of dysplastic nodule transformation to HCC being 4.3%, 9.5% and 15.7% respectively. In addition, we found that Child Pugh grade was the only independent predictor of nodule transformation into HCC (p = 0.02).25 who reported an overall HCC transformation rate of 18.8% over a median follow up time of 33.6 months. Similarly, Sato et al.27 found that 13.6% of dysplastic nodules transformed over a median follow up time of 31.9 months, while Seki et al.29 a rate of 12.5% over a median follow up period of 25 months. Our annual rate of HCC transformation of around 5% was lower than that of Iavarone et al.28 who reported a yearly rate of 9.2% over a similar follow up period of 39 months. Thus, it appears from published data that the annual incidence of HCC from these hypervascular at-risk nodules is somewhere between 5 and 9%.In our cohort, we found that 17% of dysplastic nodules that did not undergo RFA at detection transformed into HCC over a median follow up time of 38.5 months with the median time to hepatocarcinogenesis being 28 months. Given this represented 15.3% of the entire cohort, the results are similar to those of Kobayashi et al.33. In our study, we found that Child–Pugh grade was the only independent predictor of hepatocarcinogenesis on multivariate analysis although other markers of liver disease severity including MELD and ALBI were associated with HCC transformation on univariate analysis. Indeed, the risk of developing HCC in those with Child–Pugh C cirrhosis was around 30-fold higher compared to those with compensated Child–Pugh A cirrhosis. Moreover, Child–Pugh grade correlated with higher rates of hepatocarcinogenesis  of nodules disappear during follow up. While we did find an association between ALBI grade and nodule disappearance, currently there are no reliable predictor(s) of nodule disappearance, so consequently, all need follow-up imaging over time to monitor for malignant transformation.Another important finding of our study that is relatively under-appreciated in the literature is the disappearance on imaging of these seemingly “at-risk” nodules. We found that 38.5% of nodules disappeared on follow up ultrasound or CT with a median time to disappearance of 31 months. This is similar to that of Sato et al.vis a vis definitive management versus watchful observation40. While it appears intuitively obvious to ablate high-risk lesions when identified, Cho et al.34 found no distinct survival benefit of RFA of high grade dysplastic nodules that would support such an approach. In our cohort, sixteen hepatic nodules underwent definitive thermal ablation upon detection, many of whom occurred in patients with an active history of HCC at a separate site. During a median follow up of 13 months, none of nodules that were initially treated with RFA developed HCC at the ablation zone. However, eight (50%) of the nodules occurred in five (31%) individual patients with active HCC at a separate site and ten (62.5%) nodules occurred in patients with either past or current HCC. This highlights their high-risk status. In the absence of past HCC, upfront thermal ablation of an indeterminate or suspicious nodule could be curative for some patients and prevent the development of HCC. However, this invasive therapy must be carefully weighed up against its potential risks and there is currently no role for thermal ablation in patients with no past history of HCC. Clearly, more robust data is needed to guide clinical management in this space, and specifically, taking into account clinical risk factors of Child–Pugh status and past or current history of HCC. There is a randomised clinical trial underway to address the role of active therapy of dysplastic nodules43.To date, there is no consensus on the optimal management of hypervascular pre-malignant liver nodules when detected The strengths of this study include its sizeable cohort, well-established criteria for benign nodule inclusion and HCC transformation, independent radiological review using LiRADS to enhance diagnostic accuracy of HCC transformation, management of nodules via a multi-disciplinary team and appreciable follow up period that enabled a large number of nodules to be captured and assessed. In addition, it assessed the clinical and biochemical parameters influencing HCC transformation in a real-world context. However, there were important limitations to the study notwithstanding it’s retrospective design. This included the requirement for data retrieval on nodules, cirrhosis and hepatocellular carcinoma status from electronic medical records as opposed to predetermined criteria for the purposes of the study. In addition, there were no pre-defined protocolized criteria for management of these nodules in terms of ablation versus surveillance including monitoring frequency, although in all cases it was based on the opinion of the multi-disciplinary team managing these patients. Finally, being retrospective not all data was complete on clinical and biochemical parameters.In conclusion, this study has demonstrated for the first time a strong relationship between liver disease severity and hepatocarcinogenesis of dysplastic/ hypervascular liver nodules in patients with cirrhosis. Given the absence of clinical practice guidelines to inform clinicians of how dysplastic nodules should be managed, our findings highlight an urgent need for prospective studies of these nodules including randomised clinical trials particularly in those with advanced liver disease to determine the most appropriate course of action when they are detected.Supplementary Information.


The envelope (E) protein of the avian coronavirus infectious bronchitis virus (IBV) is a small-membrane protein present in two forms during infection: a monomer and a pentameric ion channel. Each form has an independent role during replication; the monomer disrupts the secretory pathway, and the pentamer facilitates virion production. The presence of a T16A or A26F mutation within E exclusively generates the pentameric or monomeric form, respectively. We generated two recombinant IBVs (rIBVs) based on the apathogenic molecular clone Beau-R, containing either a T16A or A26F mutation, denoted as BeauR-T16A and BeauR-A26F. The replication and genetic stability of the rIBVs were assessed in several different cell types, including primary and continuous cells, ex vivo tracheal organ cultures (TOCs) and in ovo. Different replication profiles were observed between cell cultures of different origins. BeauR-A26F replicated to a lower level than Beau-R in Vero cells and in ovo but not in DF1, primary chicken kidney (CK) cells or TOCs. Genetic stability and cytopathic effects were found to differ depending on the cell system. The effect of the T16A and A26F mutations appear to be cell-type dependent, which, therefore, highlights the importance of cell type in the investigation of the IBV E protein. Nidovirales, suborder Coronavirinae, family Coronaviridae, subfamily Orthocoronavirinae. Within the subfamily, there are four genera, Alphacoronavirus, Betacoronavirus, Gammacoronavirus and Deltacoronavirus. In humans, the Alphacoronavirus human coronavirus (HCoV) 229E and related coronaviruses are responsible for 18% of common cold cases -2-aminoethanesulfonic acid (BES) media [CK or Vero cells were seeded into 6-well plates to confluency. Cells were infected with 500 µL of Beau-R, BeauR-T16A or BeauR-A26F isolates at a titre of 1 × 10S) media . At 24 h4 or 1 × 105 PFU and incubated at 37 °C for 1 h with 5% CO2. The virus was removed, and the cells were washed twice with PBS followed by the addition of 3 mL BES media. The supernatant containing the infectious progeny was harvested at either 1, 24, 48, 72 and 96 hpi, to establish replication over multiple rounds of viral replication, or at 1, 2, 4, 6, 8, 10 and 11 hpi, to assess replication over the first round of viral replication. The quantity of infectious progeny was quantified in triplicate via plaque assay in CK cells.Confluent CK, DF1 or Vero cells, seeded in 6-well plates were infected with 500 µL of Beau-R, BeauR-T16A or BeauR-A26F isolates at a titre of either 1 × 104 PFU of Beau-R, BeauR-T16A or BeauR-A26F isolates. Mock infected TOCs were inoculated with 500 µL TOC medium. Tubes were incubated upright for 1 h at 37 °C, after which the inoculum was removed and the TOCs washed twice with PBS. 1ml of TOC infection media was added per tube, and the TOCs were incubated at 37 °C, 1 revolution per 7 min. The ciliary activity of each TOC was assessed at 24 h intervals using a light microscope, and the percentage of cilia beating was calculated as described [TOCs, singly plated in glass tubes, were washed twice with PBS. In replicates of ten, each TOC was inoculated with 500 µL TOC infection medium , containescribed ,39.4 PFU of Beau-R, BeauR-T16A-1, or BeauR-A26F-3. Tubes were incubated upright for 1 h at 37 °C after which the inoculum was removed and the TOCs washed twice with PBS. 1 mL of TOC infection media was added per tube, and the TOCs were incubated at 37 °C, 1 revolution per 7 min. The supernatant from each tube was harvested at 1, 24, 48, 72 and 96 hpi, and the quantity of infectious progeny was quantified in triplicate via plaque assay in CK cells.Six TOCs were plated per glass tube and were washed twice in PBS. Each tube was inoculated with 500 µL TOC infection medium containing 1 × 104 or 1 × 105 PFU of either Beau-R, BeauR-T16A or BeauR-A26F isolates. After 24 h, embryos were culled by refrigeration for a minimum of 4 h, and the allantoic fluid from each egg was harvested. The allantoic fluid was clarified using low-speed centrifugation. The quantity of infectious progeny was determined via plaque assay in triplicate in CK cells. The sequence of the E gene was determined as described in A protocol for propagation of IBV in eggs has been described . In replConfluent CK cells seeded into 6-well plates were washed once with PBS. In replicates of four, 500 μL of BES medium containing neat rIBV was added per well and incubated for 1 h at 37 °C. The inoculum was removed and replaced with 3 mL BES media and incubated for a further 23 h. The supernatant was harvested and diluted 1 in 10 for subsequent inoculation of the CK cells. The E gene of the resulting passaged isolates was Sanger sequenced at passage 5, 10 and 15 as described in RNA was extracted from either the cell supernatant or allantoic fluid using the Qiagen RNeasy kit following the manufacturer’s protocol for RNA clean up. RNA was reverse transcribed using random primer, 5′ GTTTCCCAGTCACGATCNNNNNNNNNNNNNNN 3′ and a SuperScript IV reverse transcription kit following the manufacturer’s protocol (Life Technologies). The E gene was amplified using recombinant Taq Polymerase (Life Technologies) using primers 5′-GCTGAAGATTGTTCAGGTGA-3′ and 5′-GCTGAACTGACTGTTCAAAG-3′. The PCR products were Sanger sequenced by Eurofins, and the resulting sequencing data was analysed using the Staden Package 2.0.0b11.5 PFU of Beau-R, BeauR-T16A or BeauR-A26F isolates or mock infected with BES media. At 24 hpi, the inoculated CK cells were imaged with an AMG EVOSTM XL Core microscope.CK cells seeded into 6-well plates were inoculated with 1 × 105 PFU. Mock infected cells were inoculated with the BES medium. At 24 h intervals, the viability of the cells was assessed using the luciferase assay CellTiter-Glo® kit (Promega), which measures the quantity of ATP present to quantify the number of viable cells following the manufacturer’s instructions, detailed in [Cells were seeded to confluency in 96-well plates and washed once with PBS prior to inoculation with a two-fold serial dilution, in BES medium, of Beau-R, BeauR-T16A or BeauR-A26F isolates starting at 1 × 10ailed in . The per5 PFU in BES medium. At 6 and 24 hpi, the cells were harvested, and RNA extracted using the Qiagen RNeasy kit including an on-column DNAse treatment, following the manufacturer’s (Qiagen) instructions. Reverse transcription was carried out using 1.25 µg of total RNA using Superscript IV reverse transcription kit according to the manufacturer’s protocol using random primer (5′ GTTTCCCAGTCACGATCNNNNNNNNNNNNNNN 3′). The resulting cDNA was diluted to ensure 100 µg was added per qRT-PCR reaction. TaqMan qPCR reagents were used to perform qPCR, using either TaqMan ® Fast Advance Master Mix or TaqMan ® Multiplex Mastermix (Life Technologies). Primers were used at 10 μM, and hydrolysis probes were diluted to 5 μM. A GeNorm was carried out and Beta-Actin was selected as an endogenous control; this control has been used in CK cells previously [CK cells were seeded to confluency in 6-well plates and were washed once with PBS. Cells were mock infected with BES media or inoculated with 500 µL of Beau-R, BeauR-T16A or BeauR-A26F isolates at a titre of 1 × 10eviously . SequencThe statistical analysis was assessed using GraphPad Prism 8.0. The standard deviation and normality were assessed prior to any statistical analysis.The E protein is divided into three domains; a short hydrophilic N terminal domain, a transmembrane domain, which is the focus of this study, and a long hydrophilic C terminal domain . A compaComplete genome sequences of the three isolates representing rIBVs BeauR-T16A and BeauR-A26F were assembled using NGS technologies. Consensus genomic sequences generated from stock viruses of all three isolates of both BeauR-T16A and BeauR-A26F were assembled and compared to the Beau-CK reference sequence (GenBank Accession number AJ311317). The stocks of BeauR-T16A and BeauR-A26F were generated at passages 4 and 3 in CK cells, respectively. In all isolates of both BeauR-T16A and BeauR-A26F, the consensus sequences generated confirmed the presence of the mutations T16A and A26F, respectively . In BeauThe replication of all isolates of BeauR-T16A and BeauR-A26F were investigated in primary CK cells A,B. All 9 PFU, which, although unusual, has previously been observed [The Beaudette strain of IBV has extended cell tropism ,45. The Beaudette rIBVs containing either the T16A or A26F mutations showed reduced plaque size in comparison to WT in Vero cells . ConversTo et al. indicated that, in Vero cells, the T16A and A26F mutations impede viral release . To estaIn vivo, IBV replication primarily occurs in ciliated tracheal epithelial cells . TOCs arTo investigate the genetic stability of both the T16A and A26F mutations, four replicates of Beau-R and the three independent isolates of BeauR-T16A and BeauR-A26F were serially passaged 15 times in CK cells. The E gene of progeny viruses was Sanger sequenced at passages 5, 10 and 15 .All isolates of BeauR-T16A maintained the T16A mutation at passage 5 and generated no other mutations within the E gene; however, mutations were observed at passage 10 A. A singThe BeauR-A26F isolates showed greater genetic stability than BeauR-T16A. Isolates BeauR-A26F-2 and BeauR-A26F-3 maintained the A26F mutation over all 15 passages and generated no compensatory mutations within the E gene B. BeauR-50) [4 or 105 PFU of either Beau-R, BeauR-T16A or BeauR-A26F were inoculated into allantoic cavities of 10-day-old SPF embryonated hen’s eggs. The quantity of infectious progeny present, within the allantoic fluid at 24 hpi was determined via titration in CK cells. Additionally, the sequence of the E gene of the isolated passaged viruses was determined via Sanger sequencing.The IBV strain Beaudette exhibits a non-pathogenic phenotype in birds . This is50) . To inveIn contradiction to previous research , all iso5 PFU of either Beau-R, BeauR-T16A or BeauR-A26F, and the CPE was observed. CK cells infected with the IBV show a prominent CPE, including syncytium formation (the fusion of virus infected cells with neighbouring cells resulting in a multinucleated cell) [To further examine the effect of the T16A and A26F mutations in a relevant cell type, CK cells were inoculated with 10ed cell) , cell roed cell) . Syncytied cell) . In CK c®, which measures ATP levels to quantify the number of metabolically active, and therefore viable, cells [5 PFU and serially diluted twofold. Cell viability was measured at 24 h intervals. At all the time points assessed, the cell viability was observed to be comparable between both rIBVs and the parental Beau-R, indicating infection results in equivalent cell cytotoxicity -1B and IL-6 [The E protein IC has been shown to be a pathogenicity factor in and IL-6 . In IBV,and IL-6 . The uprand IL-6 ,54,55,56The ability to successfully rescue rIBVs with either the mutation T16A or A26F within the E protein suggests that these residues are not required for viral replication in vitro, in line with earlier reports . It mustThe replication of BeauR-A26F was reduced in Vero cells in comparison to unmodified Beau-R C. This iThe potential for the different mechanistic actions of either the T16A or A26F mutations is further illustrated, as BeauR-A26F exhibited reduced replication in ovo in comparison to both BeauR-T16A and Beau-R A. SequenThe E protein is thought to play a key role in the assembly ,14,15 anA classical characteristic of viroporin activity is the modulation of the host response to infection. It has been shown that IC inactive mutants of both SARS-CoV  and IBV In conclusion, we have generated and characterised the T16A and A26F mutations within the hydrophobic domain of the E gene in the rIBV Beau-R. The rIBVs BeauR-T16A and BeauR-A26F are replication-competent, demonstrating that these residues are not essential for viral replication. The genetic stability of the T16A and A26F mutations, the replication of the rIBVs and the cytopathogenicity induced were assessed in a variety of systems, ranging from avian primary cell lines, continuous cell lines of both avian and mammalian origin, and in ovo and avian ex vivo TOCs. Differing results were observed through the assessment of the same parameter within different systems, highlighting the importance of cell-type or cell-system selection during IBV E protein research. Further studies in biologically relevant systems are therefore required to further elucidate the role of the IBV E protein during infection.


Background: In this study, we use the case of medical doctors in the public health system in rural India to illustrate the nuances of how and why gaps in policy implementation occur at the frontline. Drawing on Lipsky’s Street Level Bureaucracy (SLB) theory, we consider doctors not as mechanical implementors of policies, but as having agency to implement modified policies that are better suited to their contexts.Methods: We collected data from primary care doctors who worked in the public health system in rural Maharashtra, India between April and September 2018 . We first sorted the data inductively into themes. Then we used the SLB theoretical framework to categorise and visualise relationships between the extracted themes and deepen the analysis.Results: Doctors reported facing several constraints in the implementation of primary care- including the lack of resources, the top-down imposition of programs that were not meaningful to them, limited support from the organization to improve processes as well as professional disinterest in their assigned roles. In response to these constraints, many doctors ‘routinized’ care, and became resigned and risk-averse. Most doctors felt a deep loss of professional identity, and accepted this loss as an inevitable part of a public sector job. Such attitudes and behaviours were not conducive to the delivery of good primary care.Conclusion: This study adds to empirical literature on doctors as Street Level Bureaucrats in lower and middle income countries. Doctors from these settings have often been blamed for not living up to their professional standards and implementing policies with rigour. This study highlights that doctors’ behaviours in these settings are ways through which they ‘cope’ with their loss of professional identity and organizational constraints; and highlights the need for appropriate interventions to counter their weak motivation. SLB theory posits that people at the frontline do not mechanistically comply to all imposed policy mandates. Nor do they implement these mandates in a vacuum. Instead, they implement a version of the policy mandate that appears to fit in with their everyday realities and contextual constraints. To cope with the wide range of contextual complexities they encounter, people at the frontline of policy delivery develop informal routines and practices. These routines and practices can be called ‘coping behaviours’ and lead to policies being implemented in ways that are different from the written policy rhetoric. Hence, people in the frontline of policy delivery have been called ‘Street Level Bureaucrats’ who create ‘policies as performed.’ It is this version of policies that the public ultimately experiences.Not all policy rhetoric translates into practice as originally intended. Even when top-down policy mandates are firmly imposed, gaps in implementation of policies can occur in the frontlines of service delivery. Why such gaps occur – and what can be done about these – have been questions of interest to policy analysts. Theories such as Lipsky’s Street Level Bureaucracy (SLB)enable policy analysts to deepen their understanding of such gaps better.In this paper, we have drawn on SLB theory to examine how medical doctors in public primary health centers  in one rural area in India delivered routine primary care. We have asked two specific questions:coping behaviours’ exhibited by doctors in primary health facilities in rural India?What were the ‘coping behaviours’?In what ways did the professional identities of doctors vis-à-vis the organisational constraints they faced shape these ‘ However, within LMIC literature, medical doctors working at the frontlines of care delivery have been less frequently been examined using SLB theory . In this paper, we have explicitly focussed on understanding the perspectives of medical doctors. This focus is particularly interesting since doctors often experience tensions between their twin identities as ‘professionals’ and as ‘bureaucrats,’ and struggle to balance these different identities in their actions.This paper is an attempt to add to theory-driven, actor-centric, empirical studies in the field of health policy implementation in low- and middle-income countries (LMICs). There has been a growing interest in understanding frontline health workers behaviours  in such settings. In this study, wehave treated the delivery of primary care by doctors as a “routine” policy, and have focussed on the decisions made by doctors in their day-to-day work.Another contribution of this study is its focus on the implementation of day-to-day primary care. Most studies from LMICs that have engaged with SLB theory focus on how staff in the frontline deal with newly instituted health policies. Very few studies have examined the implementation of policies long in existence and looked at routine practices within health systems (termed “coping” in this study) as important influencers of policy success on the ground. Located in peripheral areas, these centers are intended to deliver preventive as well as basic curative care in the public health system .The doctor at a primary health center  is considered as a social physician, responsible for the health of the entire service area under a primary health center. As the medical officer-in-charge, the doctor also serves as the administrative head of the health center, and manages the support staff . Other roles include the provision of primary-level clinical care, oversight of outreach care, implementation of all programs and schemes, and overall management of staff. and Indian Public Health Standards, 2012.Source: Bulletin of rural health statistics, 2017high production’ states for allopathic doctors in India, having 52 medical colleges of which 23 are public. A one-year rural service stint is compulsory for all undergraduate medical students from public colleges and this policy, to some extent, has enabled the filling of allopathic doctors’ posts at primary health centers in the state (unlike many other Indian states). Despite filled posts, primary health centers in Maharashtra often do not serve as first access care to communities. A 2014 national survey found that in Maharashtra, only 7.5% of people seeking ambulatory care utilized primary-level tiers in the public health system.This study was conducted in rural Maharashtra, a state in western India. Maharashtra is considered as one of the five ‘This study was undertaken in one small rural area. The name of the area as well as the district have been masked to protect the participants. This area had access to approximately 80 primary-level facilities, one district-level (secondary care) hospital and one non-for-profit tertiary care hospital. This area was chosen for the study since it had a mix of facilities with ‘good’ and ‘poor’ infrastructure  and posts for allopathic doctors was filled in most of the facilities. Process indicators from the health centers in this rural area for services like antenatal care and immunization were slightly better than that of state averages; and the area was considered to be overall, a ‘medium’ performing region (according to the state and district level authorities).). We also sought the opinion of 5 doctors who had earlier worked in primary care roles and had now moved on to other roles- to offer comparative perspectives on the different roles they performed.We visited 21 health facilities and interviewed 29 public sector doctors from the rural area we selected  appeared to resonate well with the theoretical lens of SLB, so we used concepts from this theory to deepen the analysis. We worked iteratively with the list of themes that emerged and constructed data displays (tables and charts) to organize and detect patterns of relationships in the data. We used NVivo version 12 to aid the coding process. The first author led the analysis, but the preliminary analytical summaries and data displays were shared with the other authors to allow for interpretation. During the last iteration of data collection, these summaries were shared with 2 additional doctors from the public system for further validation.Consent from all participants was taken; but ten doctors preferred to give verbal rather than written consent for the interviews (the doctors were willing to participate but did not want to sign a form). When given permission to record, interviews were audio-recorded. All recordings were deleted after being transcribed. All data in the transcripts has been anonymised. In the paper, we have not mentioned the name of the rural area or of the particular health centers so as to protect the identity of health system staff. Permissions for the study were granted by the state of Maharashtra as well as district-level authorities. These reactions were, thus, not “random acts of noncompliant behaviour,” but ways through which the doctors used their discretionary power and ‘coped.’ Lipksy originally categorised the coping behaviours of frontline workers as those pertaining to modifying client demands (thus maximising use of resources), modifying job objectives  and modifying perceptions of clients . Subsequent literature has tried to expand on these categories. For instance, Brehm and Gates categorised the behaviours of bureaucrats into three categories – ‘working’ , ‘sabotaging’  and ‘shirking’ . More recently, Tummers et al had put forth a typology of coping behaviours, synthesized from literature across several disciplines. Here, coping was defined as “the cognitive and behavioural efforts made by frontline workers to master, tolerate, or reduce external and internal demands and conflicts among them during policy implementation.” We found this typology to be comprehensive; and the categorization of coping behaviours used here seemed to fit well with the initial analytical themes that we had inductively extracted. We have adapted this typology for this study and have looked at coping in terms of:Lipsky’s original theory- set in contexts where frontline workers were blamed for being aggressive and irresponsive to client needs- contended that frontline workers exhibited these behaviours as responses to challenges in the public sector bureaucracy.specific instances of coping– specific examples of action observed or reported by doctors (like a doctor not doing an out-patient clinic that is mandated)coping strategies – mechanisms that surround coping instances (like a doctor rationing and routinising care) andadaptive processes – broader attitudes and behaviours that have become routine among doctors in the public primary care sector (like doctors becoming risk-averse or learning to game the system). We could not find instances of coping behaviours that moved against clients in our data, so we have used only the other two groups to report our findings.Tummers et al had also grouped coping behaviours as those that moved towards clients (made pragmatic adjustments to aid client needs), those that moved away from clients (actions that avoided meaningful interactions that can benefit clients) and those that moved against clients . Derived from these ideas, many efforts have been made to systematically understand factors that influenced street-level action. Previous literature from LMICs has categorised factors shaping coping behaviours of street level bureaucrats as: individual characteristics ; organizational characteristics ; community or patient characteristics ; and other broader socio-political systems. We have used similar categories to examine our findings. We have also looked specifically at the interactions between doctors’ professional identities and issues in the organizational context- for these interactions played out strongly in our data.After delineating the nature of coping behaviours by doctors, we also examined why such behaviours persisted in the frontlines of the public health system. Lipsky included constraints of resources, ambiguous policies, increased work burden and work stress as some of the issues encountered at the frontline.We have reported the findings of this paper in two sections. First, we have described various coping attitudes and behaviours exhibited by doctors in primary health centers (section 1). Then, we have examined why such coping behaviours persist in health system, thereby contributing to gaps in service delivery at primary health centers (section 2). Adapting from Tummers et al classification, we have discussed coping behaviours in terms of “moving away from patients” (doing actions/interactions that are less meaningful to the community) and “moving towards patients”  see .Our discussions with doctors revealed several coping behaviours at primary health centers that moved away from patients  in implementing these programs .“But then this is way the system is. Nothing works. Who will make things better? Why? Best to keep quiet and work with what the government gives” .“clinician” hats. As one doctor put it, the work in primary health centers had become aboutFurther, most doctors got used to taking risk-averse decisions in primary health centers, as well as making professional compromises in the day-to-day work .What were the implications of doctors’ coping behaviours on the work of primary health centers? The resigned attitudes of doctors, their distancing from patients, the rationing and routinising of care along with the adoption of a risk-averse approach contributed to the delivery of poor-quality outpatient care. Further, the same attitudes of resignation and inability to change things for the better contributed to the perfunctory and diluted implementation of programs at primary health centers.“safe mode.” For instance, in We found only a few doctors who reported behaviours that moved towards patients. These doctors bent rules for the sake of patients rather than for personal benefit and tried to retain their professional identities  even when the situation demanded it; and often took risk-averse decisions. They referred more cases than clinically necessary – for doctors shared that a clinical mishap “gave the government a bad name,” following which they would be blamed for their actions not only by the patients, but also by the higher authorities. Disillusioned by their lack of support for being ‘good’ professionals, doctors felt helpless and unable to help the community. Hence, they often stopped trying to live up to the professional standards they had once recognised for themselves. In the course of doing so, they sacrificed their clinician hats. For most doctors, this was a great burden to bear .“Some instruction will come from above…people from the top pick up the phone and say ‘send this report it, that report’ that all has to be done at (name of the center). Nothing that uses my doctor skills”.“Down in the system, people create trouble, politicians create trouble and you can’t work. I so much prefer the (district level) hospital”.“Sometimes I feel really bad…with so much difficulty I have obtained this qualification…this big degree…and after this, what kind of service I am giving people? No medicines, no investigations…I don’t even have a IV line…why will a patient come to me? What can I do for them?”.“What change? Better to let things be as they are…how things are going on, let them go on the same way. Nothing is in our hands, right? See, like today, we have given a demand for drugs, I know nothing will come…still I demand…and we adjust with what is there”.paper-work” person). But such doctors were few, and most doctors in the public system appeared to have accepted their diminished professional status.However, we also encountered a few doctors in the system who had managed to retain vestiges of their professional identity and tried to ‘cope’ with the situation by adopting positive behaviours .“There are so many lacunae in government…so what can we do, you tell me? Medicines are not there…equipment is not there…we can do nothing. We are like you only…we can do nothing. It’s a bigger pattern outside of our control…”.“The issue is that once you get into the government, there is a danger of you getting institutionalised. The more time you spend in certain settings, you become like that…one has to be very careful not to fall into the rut of doing nothing”.“It is all government policy to make which policy, which scheme…and how to implement it…it has nothing to go with me…I just follow…I do my work”.make do with what he had’ and not to improve the implementation of programs. In fact, we encountered the idea that it was not the doctor’s job to improve implementation processes, many times during our conversations. Doctors also shared that doing anything “extra” while managing programs (that was beyond their reporting mandates) posed a “risk;” and such actions, though not formally prohibited, were not encouraged or supported in the public system. A few doctors reported failed attempts to improve this state of affairs. One doctor had tried to send a memo to district authorities for the suspension of a support staff member who had refused to do outreach work; but there was no action taken on his memo despite many reminders. That experience had taught him that his job was to ‘happy’ as long as some version of the NCD policy directive was getting implemented on the ground. One doctor said that improving implementation processes was subtly discouraged in the public system and he had learnt through experience that it was “not his business” to improve implementation. Most doctors had worked out a way to implement the scheme for “name-sake” only- that is, superficially, by completing minimally required screening targets.During the time of our discussions, doctors were asked to participate in a massive screening campaign for non-communicable diseases (NCDs); but no treatment was available post-diagnosis for patients as per policy. Many doctors felt that screening without providing drugs was unethical; some shared that they were not trained to screen; others said that the diagnosis of NCDs was beyond the scope of primary-level care. However, these implementation concerns were rarely discussed with higher-level managers. It was generally acknowledged that top-level administrators were ‘‘taught’by the more senior doctors that making efforts to change the way things worked in the public system was essentially pointless. More experienced doctors shared they had become used to this work and hence felt little distress when they compromised in care provision; for they had accepted these issues as an integral part of the job.Younger doctors sometimes shared that they felt uncomfortable not doing anything about the routine compromises they had to made during their work at primary health centers. However, they had been Below, we summarise the study findings from section 1 and 2. Doctors reported several challenges in delivering primary healthcare-including the lack of resources, the top-down imposition of programs that were not meaningful to them, limited support from the organization to improve processes as well as professional disinterest. These challenges contributed to the adoption of a range of coping attitudes and behaviours. Most doctors routinized and rationed care, and often became resigned and risk-averse. Most of them felt a deep loss of their professional identity, and accepted this loss as an inevitable part of a public sector job. Further, most doctors felt that they could not do justice to the administrative roles that they had been assigned. Such coping behaviours, on balance are likely to impact negatively on the quality of care provided from primary health centers.; allowing their bureaucratic identity to be subsumed into their professional one. Our study shows, however, that there is merit in looking at doctors’ actions using SLB theory. Through the SLB lens, doctors’ actions are not treated solely as professional decisions. Instead, the actions are seen as ways through which these professionals ‘cope’ with a wide range of contextual factors; and ‘coping’ causes them to sustain particular attitudes and work-routines in the long-run. Thus, the use of the SLB lens expands the understanding of doctors’ actions as contextually situated routines.While the volume of actor-centric, empirical studies in the field of policy implementation has been growing recently in LMICs, few studies have looked at explicitly at doctors - as Street Level Bureaucrats. One reason for this could be that doctors have typically been considered a group of highly-skilled professionals with inherent discretionary power and some empirical LMIC literature does touch on how frontline health staff ‘cope’ by adopting certain attitudes and actions  during policy implementation. However, the knowledge base is still limited and more studies that systematically categorise coping behaviours and elicit nuanced accounts of these behaviours are needed from LMIC health contexts. In this study, the consideration of coping at various levels- as specific instances, as strategies that surround these instances, and also as aggregated adaptive processes (adapted from Tummers and colleagues’ framework) – has enabled a systematic and detailed understanding of doctors’ coping behaviours in primary healthcare settings. To elaborate, examining individual coping instances helped to dissect doctors’ actions and these instances have served as illustrative ideas around which to deepen discussion. Coping strategies that are more abstract than instances  have also helped to understand the mechanisms that define coping instances. Broader adaptive processes clarified how certain patterns of attitudes and behaviours eventually become part of work-routines at primary health centers. Overall, examining coping through these three levels has added depth as well as clarity to our understanding of doctors’ actions. Our analysis suggests the usefulness of further empirical work from LMICs that similarly considers coping behaviours in this layered way.‘Coping’ has always been a central concept in SLB theory,practical norms” – that develop over time and persist in public sector organizations. Our findings indicate that these adaptive processes are not specific to any particular scheme or policy; rather, they underlie all activities implemented from primary health centers and form the basis for de facto policy. Other studies have also touched upon such adaptive behaviours. Health providers in public health systems have expressed concerns about being used as “scapegoats” by the system, have reported being scared of being subjected to censure by the media and the community; and also have expressed feeling “pursued rather than protected in the exercise of medical care;” all of which contributes to the risk-averse coping routines that persist in public health systems.In this study, we have paid particular attention to the broader adaptive processes of coping- resignation, risk-averseness and the sacrifice of professional ideals. This is because such processes present a window for understanding informal routines – the “collective divergence” of doctors from intended policy goals. Over time, most doctors seem to have learnt that survival in the system implies working mainly with formally imposed and heavily monitored policy mandates  and ignoring other services; and, as a result, have, over time, “drifted towards compatibility with the way (things are) evaluated” in the public sector. However, it is important to note that these imposed policy mandates appear only to influence “what” must be done at primary health centers, rather than the more nuanced “how” of the tasks involved- which is often, inevitably, left to the discretion of doctors. Thus, in a risk-averse environment with resigned attitudes and low levels of professional enthusiasm, policy mandates are often carried out in perfunctory ways. There is little ownership of tasks beyond obligatory reporting requirements; and as Stone puts it, there always seems to exist a “felt need to go on to the next client, the next report, or the next item of business.”Even exceptional providers who try hard to take ownership of tasks are limited by resources, lack of organizational support, and the constant informal pressure to be risk-averse; and these limitations often leaves them frustrated. These experiences result in the perfunctory and diluted implementation of primary healthcare policies.In our study, adaptive coping processes often seemed to form the basis for the “ Many of the organizational and local socio-political factors elicited in this study – resource constraints, structural deficits, issues with organizational culture and relationships, and lack of connect with the community - have been noted in other empirical studies on frontline policy implementation that involve doctors, as well as other cadres of frontline staff.This study also highlights a range of factors as influencing doctors’ practices in Indian primary health centers. We have categorised these as the underlying beliefs of doctors , professional , organizational  and local socio-political  factors. Similar categorizations of influencing factors have been used by others as well. most SLB studies underplay the influence of professional factors over frontline worker actions. Lipsky in his original work does refer to professional identities as playing a role in shaping frontline worker response, but studies from the LMIC health sector that use SLB theory have usually placed less emphasis on this aspect. Our findings underscore the importance of examining both roles  when considering frontline cadres (like doctors) that have a strong sense of professional identity. In our study, we found that most doctors felt they could not do justice to either of these roles due to the many organizational constraints they faced. This has led to long-term demotivation and inculcated the attitudes of resignation among doctors that seem to have contributed to the delivery of poor quality care.In our findings, the set of ‘professional’ factors play out strongly, and these interact in various ways with ‘organizational’ factors to shape doctors’ actions. Although one other study has noted these interactions, as well as the challenges that doctors face in dealing with the duality of being a professional (a clinician) as well as a bureaucrat, that were not fully discussed by study participants, and so have not been reported in this paper. One important point that aided frank conversations was the fact that the first author who conducted the interviews was a doctoral student. A letter from the university seemed to assure doctors that they were not being tested for their competence or being evaluated by the organization secretly. Although we observed doctors while waiting at primary health centers, the evidence presented in this paper is mostly self-reported actions which could not be triangulated with observations. Finally, although we did purposefully select female doctor respondents, the majority of doctors available in these rural regions are men and so the opinions we report are male-dominated.Some limitations of this study are noted. First, it was not easy to collect data on street-level coping behaviours. While doctors were often willing to talk about general challenges faced during work in the public system, it was not always easy for doctors to talk about specific instances. Some doctors preferred to speak in third person (about difficult situations faced by a colleague) rather than about themselves. Conversations that were not tape-recorded in general yielded better information, especially on certain themes pertaining to the public sector organization. However, even during untaped conversations, there were certain themes- corruption, taking and giving of bribes by doctors and private practice - known to exist in similar settings,This study was done in one rural area, and we do not claim that the experiences we have reported here are representative of all doctors in rural primary healthcare settings. However, the rich, in-depth data from our study offers interesting insights into the nature of coping behaviours in such settings and factors that shape these behaviours. Based on these insights, we reflect below on what the study findings could mean for primary healthcare policies in India and other similar LMIC health contexts. Even among those who work at primary health centers, frustration and lack of motivation has been documented. However, there have been few interventions in the public sector in India to remedy the situation, besides technical training and structured incentives. Our evidence clearly points to the need for a different set of interventions; those that can deal with the fear of blame and frustration, help deal with challenging relationships and institute oversight mechanisms beyond measurement of specific performance indicators.Policies in India have historically been concerned about the challenge of recruiting doctors for work at primary health centers; as many doctors prefer to opt for private practice.advocacy, awareness-raising, and debate at the frontline,’ thereby making policies more meaningful to health providers. Interventions suggested in the literature from other LMICs include values’ clarification workshops, supportive supervision, engaging staff in reflection, consulting with staff regarding policy changes rather than imposing them top-down, using appreciative enquiry techniques and generally strengthening leadership at the frontline. The need to create spaces for deliberative and reflective practice has been pointed out. Recognising informal practices among frontline health workers and bringing about changes in the organizational culture that allow frontline health providers better access to and control over resources has also been shown as important. In summary, the need for ‘new and more creative strategies’for motivating healthcare workers has been emphasized.While the exact nature of these interventions needs to be context-specific, the general principles that surround a set of interventions to improve provider behaviour would likely remain the same across resource-constrained settings. Other LMIC studies using the SLB lens have pointed out the need for interventions that deal with ‘To conclude, this study attempts to adds to the policy implementation literature in LMICs that applies the SLB lens. It illustrates that doctors’ decisions at primary health centers are shaped by their professional values as well as by formal and informal organizational requirements. It highlights that a range of informal practices have become ‘routinised’ in the health system, and that these can contribute to the disjuncture between policy goals and frontline realities. Thus, this study confirms the usefulness of the SLB lens in studying routine health policy implementation in LMICs.This paper has been funded through the Health Policy Analysis Fellowship programme, supported by the Alliance for Health Policy and Systems Research, Switzerland. We acknowledge the mentor team of the fellowship program-Maylene Shung-king, Irene Agyepong, Jeremy Shiffman and Zubin Shroff; and all the fellows of the program for their reviews and comments on this work. We thank the Center for Social Medicine, Pravara Institute of Medical Sciences, in Ahmad Nagar, Maharashtra, for logistical support during data collection- in particular, the interns who worked with us and faculty of the school, Dr. Somasundaram and Dr. Thitame. We thank the health staff who shared their valuable thoughts with us during the study. We particularly thank Professor T. Sundararaman, Independent consultant and former dean, School of Health Systems Studies, Tata Institute of Social Sciences, Mumbai and Professor Surinder Jaswal, Deputy Director (Research), Tata Institute of Social Sciences, Mumbai, for their inputs on the results of this study.Ethical approval for the study was taken from the Institutional Review Board at the Tata Institute of Social Sciences, Mumbai, India- in April 2018.Authors declare that they have no competing interests.SR designed this part of the study, with mentorship from LG and SM. Data collection was done by SR, and LG, SM, and NG commented on the analysis. The first draft of the paper was written by SR, and reviewed by LG, SM, and NG. The final draft of this paper has been reviewed by all four authors. SR is the guarantor of the paper.1School of Health Systems Studies, Tata Institute of Social Sciences, Mumbai, India. 2Division of Health Policy and Systems, University of Cape Town, Cape Town, South Africa. 3Department of Global Health and Development, London School of Hygiene and Tropical Medicine, London, UK. 4Center for Health and Social Sciences, School of Health Systems Studies, Tata Institute of Social Sciences, Mumbai, India.This study contributes to empirical literature from low- and middle-income countries (LMICs) that explains how the attitudes and behaviours of frontline health providers shape policies. In such settings, doctors have often been blamed for not implementing policies well and for not living up to professional expectations. But an alternate way of looking at these actions is by considering these as ways in which doctors ‘cope’ with the challenging situations they face during day-to-day work. Doctors face several constraints in the implementation of primary care – lack of professional interest in primary care roles, top-down policy mandates imposed on them, lack of community trust and systemically-engendered risk aversion. These constraints lead to demotivation, resignation and a loss of professional identity among doctors. Ultimately, they deliver a compromised version of primary care, different from that of policy expectations. Further technical training of doctors has often been offered as a means of improving the delivery of primary care in public health settings in LMICs. This study suggests that other interventions - such as supportive supervision, the use of appreciative enquiry techniques, as well as consultations and deliberation with staff regarding policy implementation-would also be important to improve care. In this study, we look at how health providers’ attitudes and behaviours shape policies, through the example of doctors who work in the public, primary health sector in India. Doctors reported that they had limited professional interest in delivering primary care, and were constrained by other issues such as the lack of infrastructure, the imposition of too many targets and a lack of connection with the community. Doctors’ also acknowledged that such constraints led them to adopt behaviours that could potentially comprise the delivery of primary care. These findings suggest that the provision of technical training or the mere imposition of top-down targets on health providers are not adequate to make policies succeed. To ensure policy success, people in the frontline have to be supported and motivated in other ways.