Therefore, this test allows us to accurately identify prostate cancer patients likely to experience future recurrent disease immediately following removal of the primary tumor.Prostate cancer is the most common form of non-dermatological cancer among US men, with an increasing incidence due to the aging population. Patients diagnosed with clinically localized disease identified as intermediate or high-risk are often treated by radical prostatectomy. Approximately 33% of these patients will suffer recurrence after surgery. Identifying patients likely to experience recurrence after radical prostatectomy would lead to improved clinical outcomes, as these patients could receive adjuvant radiotherapy. Here, we report a new tool for prediction of prostate cancer recurrence based on the expression pattern of a small set of cooperation response genes (CRGs). CRGs are a group of genes downstream of cooperating oncogenic mutations previously identified in a colon cancer model that are critical to the cancer phenotype. We show that systemic dysregulation of CRGs is also found in prostate cancer, including a 4-gene signature  capable of differentiating recurrent from non-recurrent prostate cancer. To develop a suitable diagnostic tool to predict disease outcomes in individual patients, multiple algorithms and data handling strategies were evaluated on a training set using leave-one-out cross-validation (LOOCV). The best-performing algorithm, when used in combination with a predictive nomogram based on clinical staging, predicted recurrent and non-recurrent disease outcomes in a blinded validation set with 83% accuracy, outperforming previous methods. Disease-free survival times between the cohort of prostate cancers predicted to recur and predicted not to recur differed significantly (p = 1.38×10 Prostate cancer is the most common non-dermatological cancer among men in the United States, with an estimated 220,000 new diagnoses in 2015 . These pMost prediction models for prostate cancer recurrence are based on clinical features alone . Gene exCooperation Response Genes (CRGs) are a group of genes synergistically dysregulated in response to cooperating oncogenic mutations. They are critical to the cancer phenotype at about 50% frequency . Due to In this study, we analyzed CRG expression in a set of frozen prostate cancer samples. A gene signature differentially expressed in prostate cancer that later recurred was identified, and various predictive algorithms were evaluated using this signature. The best algorithm was then combined with a surgical nomogram that further increased predictive power. This was capable of predicting clinical outcomes of an independent blinded validation set with 83% accuracy, outperforming previous methods.Tissue from prostate cancer and benign prostate was collected from patients (n = 55) who underwent radical prostatectomy between 1990 and 2002. All patients had newly-diagnosed, clinically localized prostate cancer . Data coDifferences in CRG expression between prostate cancer and benign prostate were assessed post-normalization. 64% of CRGs were significantly dysregulated in prostate cancer compared to benign prostate  , and expThe samples were separated into training (n = 32) and validation (n = 23) sets, and disease outcomes were blinded in the validation set. The strategy was to test multiple methods of making predictions using the training set, identify the method that generated the most accurate predictions via cross-validation, and use this method to make predictions using the validation set.R.To make predictions on patient outcomes, we first normalized our gene expression data. In addition, a newly developed method for imputing missing gene expression values caused by PCR amplification failure was implemented . Two-taicentroid algorithm resulted in the most accurate predictions, with an accuracy of 75% averaged across all data handling conditions and p-value cutoffs  on the training set, while the nearest neighbor algorithm was inferior (accuracy = 69%)  using the training data set. Each combination consists of a data handling method, a p-value cutoff for inclusion in gene signature, and a prediction algorithm. Of the data-handling strategies we tested, predictions made using data normalized to BECN1 and then imputed to restore missing values caused by PCR amplification failure performed best, with accuracy of 86% averaged across the three algorithmic predictive methods and p-value cutoffs Figure . Predicts Figure . The dis) Figure . The hig) Figure . This cu% Figure . The lacOur chosen conditions accurately predicted 70% of the samples in our validation set, outperforming or equaling two sets of predictions made utilizing different cutoffs of Gleason score  and predictions made based on surgical stage (64%) Figure . For ourReceiver operating characteristic (ROC) curves were created to evaluate the sensitivity and specificity of predictions made using different discrimination thresholds for recurrence or non-recurrence. ROC curves generated using CRG-based predictions resulted in an area under the curve (AUC) of 0.67 Figure . When sa-6, log-rank test).Kaplan-Meier survival curves were created to visualize recurrences in our predicted high risk and low risk cohorts Figure . Of the To control for the possibility that the predictive power of the gene signature reported here may be affected by variation in stromal content of the prostate cancer specimens, we assessed the mRNA expression of two stromal markers, smooth muscle alpha-actin and vimentin, in our validation set. The two mRNAs are highly expressed in smooth muscle, fibroblasts, and myofibroblasts. Neither vimentin nor alpha-actin was significantly differentially expressed in recurrent vs non-recurrent cohorts using both non-normalized expression values   and exprAn estimated 50,000 radical prostatectomies are performed each year in the US, and about 15,000 of these patients will experience biochemical recurrence. Here we show that a four-gene signature based on HBEGF, HOXC13, IGFBP2, and SATB1 was able to identify patients whose prostate cancer recurred. The prediction algorithm that incorporated surgical information provided accurate prognostic information on patient outcomes with 83% accuracy. Most patients undergoing radical prostatectomy receive no additional treatment beyond regular monitoring of PSA. Adjuvant radiotherapy administered immediately after radical prostatectomy could improve outcomes for patients identified using the CRG signature to be at high risk for recurrence.in vitro, including the prostate cancer line PC-3ML [The four genes identified in our signature, HBEGF, HOXC13, IGFBP2, and SATB1, all play significant roles in the modification of cancer phenotypes. HOXC13, a homeobox-family transcription factor known to control cell proliferation and differentiation, was found to be upregulated in our recurrent prostate cancer samples. HOXC13 has been reported to be upregulated in metastatic melanoma compared to primary tumor tissue . Furthere PC-3ML . SATB1 ie PC-3ML . Publicle PC-3ML . Althouge PC-3ML . The stre PC-3ML . Finallye PC-3ML , 26, 27.e PC-3ML .The focus for this study was biochemical recurrence after radical prostatectomy. Biochemical recurrence after prostatectomy almost always requires the primary tumor to already have escaped the prostatic capsule, either invading local tissue or metastasizing to regional lymph nodes or distant organs. These behaviors are hallmarks of tumor aggressiveness, and suggest that the CRG signature may provide valuable information for assessing patient outcomes even in patients who have not undergone radical prostatectomy. Currently the best test to determine risk of aggressive disease is the Gleason score, a test with limited predictive power in many cases. Several groups have already identified gene signatures that are hallmarks of either indolent or aggressive disease , 28, 29.Irshad et al.  identifiThe success of this model to predict prostate cancer recurrence speaks to the importance of the CRGs in regulating human cancer behavior. As the CRGs were originally identified in a colon cancer background, it stands to reason that CRG expression may provide valuable prognostic information for colon cancer as well. Early-stage colon cancer is treated primarily with surgical resection, and assessing the likelihood of recurrence is an important clinical question. Future studies of the predictive potential of CRGs may provide greater insight into the likelihood of different disease outcomes, thus permitting better-informed decisions about treatment.17 All tissue samples were reviewed by a board certified anatomic pathologist to verify the diagnosis of prostatic adenocarcinoma and to estimate the percent neoplastic tumor nuclei. Data collected for each patient included standard prognostic variables, such as Gleason score, clinical (cTNM) and pathological (pTNM) stage, and PSA. All patients had at least 3 years of follow-up data. Biochemical progression was defined by the AUA guidelines of serum PSA of 0.2 ng/mL or greater (obtained 6 weeks - 3 months postoperatively), with a second confirmatory level of PSA greater than 0.2 ng/mL.All tissue samples were collected from radical prostatectomy specimens by the RPCI Pathology Resource Network with IRB approval. Patient demographic, clinical, pathology, and outcome data were collected through the Clinical Data Network, another shared resource at RPCI. All tissue specimens were immediately processed and snap frozen in liquid nitrogen within 30 minutes of prostatectomy by the Department of Urology.Total RNA was harvested from frozen tissue sections using a standard Trizol  application. Tissues were homogenized with Trizol reagent. RNA was precipitated from the aqueous phase using isopropanol and rehydrated using DEPC water. An RNA aliquot was run on an Agilent 2100 bioanalyzer to confirm RNA integrity by generating a RNA Integrity Number (RIN) value. RNA was converted to cDNA and quantified via Taq-Man Low Density Array (TLDA) RT-PCR.18 Statistical assessment of differential expression was performed using a t-test based on maximum likelihood estimates (MLEs) of the within group means and variances generated by the development version of the nondetects package (manuscript in preparation).Ct values were normalized to Becn1. Non-detects were imputed based on estimation of a non-random missing data mechanism using the nondetects R/Bioconductor package.Two-tailed t-tests were performed on normalized data to identify genes differentially regulated between biochemically recurrent and non-recurrent cohorts. Multiple p-value cutoffs were tested to assess the relative success of different sizes of gene signature. Three prediction algorithms based on clustering techniques were generated and software was written to implement each in R. Algorithms incorporated a gene signature of size N and evaluated samples as points in N-dimensional space. The “distance” algorithm generates recurrence and non-recurrence scores by comparing the Euclidian distance between the sample point and all points in the recurrent and non-recurrent groups respectively. The “centroid” algorithm generates recurrence and non-recurrence scores by comparing the distance between the sample point and the centroids of the recurrent and non-recurrent group. The “nearest neighbor” algorithm generates recurrence and non-recurrence scores by comparing the distance between the sample point and the closest member in both the recurrent and non-recurrent groups. Predictions are made by selecting the lower of either the recurrence or non-recurrence scores.recurrence scorenon-recurrence scoreI = # genes in signatureN = # samples in training setn(i) = expression of the ith gene in the signature in the nth recurrent sample in the training setRn(i) = expression of the ith gene in the signature in the nth non-recurrent sample in the training setNRi = expression of the ith gene in the signature in the current sample in the test setXrecurrence scorenon-recurrence scoreI = # genes in signatureN = # samples in training setn(i) = average expression of the ith gene in the signature in the nth recurrent sample in the training setRn(i) = average expression of the ith gene in the signature in the nth non-recurrent sample in the training setNRi = expression of the ith gene in the signature in the current sample in the test setXrecurrence scorenon-recurrence scoreI = # genes in signaturen(i) = expression of the ith gene in the signature in the closest recurrent sample in the training setRn(i) = expression of the ith gene in the signature in the closest non-recurrent sample in the training setNRi = expression of the ith gene in the signature in the current sample in the test setXPredictions were made on a 32-sample training set with 16 biochemically recurrent and 16 non-recurrent tumors. Each permutation of normalization method, p-value cutoff for gene signature, and prediction algorithm was evaluated using Leave-one-out cross-validation (LOOCV).The centroid algorithm was used to make predictions about a 23-sample validation set using the imputed and BECN1-normalized data with a CRG signature generated using a p-value cutoff of < 0.01.Surgical information was incorporated into the prediction decision procedure to improve specificity. Tumors with clinical stage of T2bN0M0 or below, with negative surgical margins were classified as non-recurrent. All other predictions were generated algorithmically as before.ROC curves were created using predictions made by varying the discrimination threshold between a prediction of recurrence vs non-recurrence. The model is altered by adding a modifier to the recurrence score before making predictions. When a modifier of -10 is added, all samples were predicted to be non-recurrent; when a modifier of +10 is added, all samples were predicted to be recurrent. Sensitivity and specificity of predictions were measured and plotted for each value of the modifier at which a prediction changes.Kaplan-Meier survival curves were taken by plotting time from prostatectomy to biochemical recurrence for patients predicted to recur and patients predicted not to recur respectively. Statistical analysis was done via log-rank test using the R package “survdiff”.


To investigate whether elevated urinary HAI-1, EpCAM and EGFR are independent prognostic biomarkers within non-muscle-invasive bladder cancer (NMIBC) patients, and have utility for risk stratification to facilitate treatment decisions.After accounting for EAU risk group in NMIBC patients, the risk of BC-specific death was 2.14 times higher (95% CI: 1.08 to 4.24) if HAI-1 was elevated and 2.04 times higher (95% CI: 1.02 to 4.07) if EpCAM was elevated. The majority of events occurred in the high-risk NMIBC group and this is where the biggest difference is seen in the survival curves when plotted for EAU risk groups separately. In MIBC patients, being elevated for any of the three biomarkers was significantly associated with BC-specific mortality after accounting for other risk factors, HR = 4.30 (95% CI: 1.85 to 10.03).Urinary levels of HAI-1, EpCAM and EGFR were measured by ELISA in 683 and 175 patients with newly-diagnosed NMIBC and MIBC, respectively, recruited to the Bladder Cancer Prognosis Programme. Associations between biomarkers and progression, BC-specific mortality and all-cause mortality were evaluated using univariable and multivariable Cox regression models, adjusted for European Association of Urology (EAU) NMIBC risk groups. The upper 25% of values for each biomarker within NMIBC patients were considered as elevated. Exploratory analyses in urine from MIBC patients were also undertaken.Urinary HAI-1 and EpCAM are prognostic biomarkers for NMIBC patients. These biomarkers have potential to guide treatment decisions for high-risk NMIBC patients. Further analyses are required to define the roles of HAI-1, EpCAM and EGFR in MIBC patients. High-risk non-muscle-invasive bladder cancer (HR-NMIBC ) represeShedding of the extracellular domains (ectodomains) of transmembrane proteins is recognised as an important cancer-related phenomenon –11. We hThe present study explores the associations between urinary levels of HAI-1, EpCAM and EGFR and disease progression and mortality in a cohort of 858 patients (683 NMIBC patients and 175 MIBC patients) from the West Midlands’ Bladder Cancer Prognosis Programme (BCPP ) in ordeAfter exclusions, there were 858 patients remaining in the dataset, of which 683 were diagnosed with NMIBC and 175 with MIBC , 359 (41.8%) and 387 (45.1%) had been used in previous analyses of urinary EGFR, EpCAM, HAI-1 in BC, respectively –14. The Using the upper quartile of biomarker values in NMIBC patients, the cut-offs for an elevated biomarker result were defined as 2 ng/mg creatinine, 29 pg/mg creatinine and 392 pg/mg creatinine for HAI-1, EpCAM and EGFR, respectively. The number and proportion of elevated biomarker values across EAU risk groups is shown in Table The association of elevated biomarker levels with the risk of progression from NMIBC to MIBC is weaker than for death, HAI-1 HR = 1.85 (95% CI: 1.07 to 3.20) unadjusted and HR = 1.69 (95% CI: 0.97 to 2.93) adjusted for EAU risk group; neither EpCAM nor EGFR reached significance  [We have demonstrated that elevated levels of urinary HAI-1 and EpCAM are associated with BC-specific mortality in high-risk NMIBC patients: each biomarker is associated with increased risk of death from disease within 5-years (adjusted for EAU risk group). Exploratory analyses demonstrate an association between elevated urinary HAI-1 and EGFR and increased risk of death in MIBC patients. For HAI-1, these associations have not been reported previously.The functions and biology of EpCAM and EGFR have been described elsewhere –14. HAI-Despite extensive efforts at the genomic, epigenomic and transcriptomic analysis of high-risk NMIBC, no validated tissue-based prognostic biomarkers have emerged. We suggest that urinary HAI-1 and EpCAM could be used to facilitate decision-making regarding the treatment of patients with high-risk NMIBC – a simple stratification tool, with an elevated test guiding patients and clinicians towards more radical therapy instead of bladder preservation. Furthermore, recruiting and randomising BC patients to trials of bladder preservation versus radical cystectomy is notoriously difficult . In thesFor MIBC, the clinical utility of our biomarkers for patient and treatment stratification is less obvious. We caution against over-interpretation since these were exploratory analyses; we would have liked to adjust for more appropriate risk factors had they been available, but BCPP is a NMIBC-focused study . HoweverWe appreciate that there are limitations to this study. We chose an arbitrary cutpoint to define an elevated test result (upper quartile); however, we felt that this approximated to the proportion of UK patients who undergo cystectomy as the initial primary treatment of high-risk NMIBC (c.25%). The number of progression events and BC-deaths were also low, resulting in low power to detect associations. However, although not always statistically significant, the direction of associations was consistent for the biomarkers with each of the outcomes, and within NMIBC and MIBC groups. Also due to the low number of events, we could not include many variables in the models individually and decided to simply adjust for EAU NMIBC risk group. Future work in larger cohorts with more events should additionally adjust for other potential confounders. In addition, treatment data for high-risk NMIBC patients were reported inconsistently and we could not analyse them reliably, nor incorporate the data into multivariable analyses; however, a simple observational analysis using the data that were recorded suggested similar proportions of normal and elevated biomarker levels within each treatment category and a low probability of influencing the biomarker relationships observed. Clearly, validation in an independent cohort is required as the next step of translation, alongside further work to determine a suitable biomarker cut-off value that assists treatment decisions.Furthermore, since patients’ biospecimens were only collected at the time of diagnosis, it remains unknown whether these biomarkers also have prognostic utility when applied at the time of NMIBC recurrence or progression. Finally, further evaluation of these biomarkers is required in MIBC patients as the clinical utility is less obvious than for NMIBC.Urine samples were obtained from the prospective BCPP biospecimen collection  . PatientUrine samples were obtained prior to transurethral resection of bladder tumour(s) (TURBT) specifically for biomarker analyses . SamplesTumour grade and stage records were amended according to results of re-resection or cystectomy (where performed). We used the 1973 grade classification as it was in universal use in the UK at the time of patient recruitment, and is also the basis for the EORTC and EAU NMIBC risk tables [ELISAs were performed as previously described –14. BrieOur previous work analysed urine samples for HAI-1, EpCAM and EGFR levels in subsets of patients –14. In tIndividuals were excluded from analysis if they were determined to not have bladder cancer or it was unknown. A complete case analysis was used; therefore, individuals were also excluded if they did not have biomarker measurements, were missing follow-up information, or were missing risk factor information for stage, grade, size, number of tumours, or presence or absence of CIS. Individuals with solitary CIS were excluded. Biomarkers were measured in all patients who provided a sufficient volume of urine for analysis; however, patient characteristics were compared between patients with and without biomarker measurements .Three outcomes were considered: progression to MIBC (for NMIBC patients), bladder cancer specific mortality and all-cause mortality. Survival times were calculated from the date of registration into the BCPP study until the date of progression, date of death , or date of death from bladder cancer . Individuals were censored at the date last known to be progression-free (for progression), alive, or additionally at the date of death from other causes for bladder cancer specific mortality.Patient characteristics were summarised using frequencies and percentages for categorical variables and medians with interquartile ranges (IQR) for skewed continuous variables. Follow-up was summarised using the median duration and 95% confidence interval (CI) for the median.For each biomarker, the cut-off for an elevated result was set at the 75th percentile of all values within NMIBC patients, therefore fixing 25% of NMIBC patients to be ‘elevated’ and approximating to the proportion of UK patients who undergo cystectomy as the initial primary treatment of high-risk NMIBC. Patients were categorised into EAU risk groups  . Kaplan-The biomarkers were also evaluated in MIBC patients using Kaplan-Meier plots and Cox models as for NMIBC patients. However, EAU risk groups are specific to NMIBC patients; therefore, in the multivariable models, adjustment was made for the individual factors that make up the EAU risk groups: stage, grade, size, number of tumours and presence or absence of CIS.Principal treatment was summarised for the high-risk NMIBC group using frequencies and percentages for normal and elevated values for each biomarker.Statistical analyses were performed using Stata MP 14.0 .Elevated levels of urinary HAI-1 and EpCAM are independent prognostic factors for BC-specific survival in NMIBC patients, and may be useful for informing both clinician and patient decisions regarding the initial management of HR-NMIBC by primary cystectomy or bladder-preservation.


Enteroviruses (EVs) cause severe outbreaks of respiratory and neurological disease as illustrated by EV-D68 and EV-A71 outbreaks, respectively. We have mapped European laboratory capacity for identification and characterisation of non-polio EVs to improve preparedness to respond to (re)-emerging EVs linked to severe disease. An online questionnaire on non-polio EV surveillance and laboratory detection was submitted to all 30 European Union (EU)/European Economic Area (EEA) countries. Twenty-nine countries responded; 26 conducted laboratory-based non-polio EV surveillance, and 24 included neurological infections in their surveillance. Eleven countries have established specific surveillance for EV-D68 via sentinel influenza surveillance (n = 7), typing EV-positive respiratory samples (n = 10) and/or acute flaccid paralysis surveillance (n = 5). Of 26 countries performing non-polio EV characterisation/typing, 10 further characterised culture-positive EV isolates, whereas the remainder typed PCR-positive but culture-negative samples. Although 19 countries have introduced sequence-based EV typing, seven still rely entirely on virus isolation. Based on 2015 data, six countries typed over 300 specimens mostly by sequencing, whereas 11 countries characterised under 50 EV-positive samples. EV surveillance activity varied between EU/EEA countries, and did not always specifically target patients with neurological and/or respiratory infections. Introduction of sequence-based typing methods is needed throughout the EU/EEA to enhance laboratory capacity for the detection of EVs. Enterovirus genus (HRV-A to HRV-C) [A total of 116 enterovirus (EV) types have been identified from humans, and of these, 45 have been discovered in the past 10 years -4. Rhinoo HRV-C) ,3.EVs cause a wide spectrum of infections in humans, including non-specific febrile illness and viral exanthema, respiratory infections, hand, foot and mouth disease (HFMD), myocarditis, meningitis, encephalitis and, rarely, acute flaccid paralysis (AFP) . SpeciesThe classical method to diagnose EV infection has been virus isolation by cell culture from clinical specimens, followed by neutralisation assay to determine the serotype . AlthougEnterovirus genus and therefore enables detection of most 116 EV types within one assay with comparable sensitivity [Reverse transcriptase polymerase chain reactions (RT-PCR) used for diagnosing EV infections usually target the 5’ untranslated genomic region (5’UTR), which is highly conserved within the sitivity . Some ofsitivity ,29. Amplsitivity .Although EV surveillance is mainly recommended by the World Health Organization (WHO) as a component of the Global Poliovirus Elimination Action Plan, it also provides parallel data on non-polio EV detection. The main principal public health needs for non-polio EV surveillance include outbreak detection and response, and monitoring of EV types associated with severe disease . HoweverPolioviruses, like most other EVs, are transmitted mostly via the faecal–oral route . PolioviTo our knowledge, there are no previous Europe-wide data available on the methods currently used for EV surveillance, detection and typing. The main aim of this study was to record European capability for identification and characterisation of non-polio EVs in order to improve the laboratory response to (re)-emerging EVs linked to severe disease.An online questionnaire on non-polio EV surveillance and laboratory detection was submitted to all European Union (EU)/European Economic Area (EEA) countries via the National Coordinators of the European Centre for Disease Prevention and Control (ECDC) Coordinating Competent Bodies. Questionnaires were also sent to the Operational Contact Points for Influenza Surveillance as well as to the National Focal Points for Influenza, Microbiology and Vaccine Preventable Diseases. The National Coordinators were asked to assign a person or persons from their country to fill in the survey from the national perspective. Specific questions were asked about the laboratory methods used for detection and typing/characterisation of non-polio EVs and it was recommended that a person with detailed understanding of laboratory methods should respond to the questionnaire. Two reminders were sent to the National Coordinators and Operational Contact Points. Only one survey response per country was considered in the final analysis; data obtained from multiple responses were merged into a single country-specific response. The questionnaire was sent on 11 April 2016, and the responses were requested by 30 April 2016.The questionnaire was divided into four parts. The first part recorded the respondents’ background information and the second part focused on the current non-polio EV surveillance systems in place. Information was collected on whether the country’s EV surveillance included identification of poliovirus circulation via typing/characterisation of EV-positive stool samples, and identification of poliovirus and other EV via typing/characterisation of variety of clinical EV-positive specimens, and whether the country had reporting and/or surveillance systems for HFMD and EV-D68. Furthermore, we also specifically asked whether surveillance for EV-D68 was performed via: (i) sentinel surveillance of influenza-like illness (ILI) and/or other respiratory virus infections; (ii) AFP surveillance; (iii) EV surveillance including respiratory samples; (iv) laboratory reporting of EV-D68 positive samples or (v) in another way. The third part was on laboratory methods used for detection of non-polio EV, and the fourth on further typing/characterisation of EV-positive specimens. Countries were also asked to estimate how many EV-positive samples were referred for typing/characterisation annually, and to provide exact numbers for 2015 if available. We also calculated the proportion of EV-positive samples typed per 100,000 inhabitants for the countries that were able to provide these data. If the country was unable to provide an exact number, we used the upper limit of estimation for the calculations.Data on primers and probes used for non-polio EV detection were collected either directly from the survey answers, and/or from the references provided by responders. These were compared with the published sequences of all known EV types to identify potential mismatches. The alignment was performed in Simple Sequence Editor (SSE) version 1.2 .A total of 29/30 EU/EEA countries responded to the survey (97% response). The majority of respondents (n=27) were microbiologists, and several of these (n=7) were additionally trained in epidemiology. Most respondents worked at their national EV laboratory (n = 25), and some were also associated with the influenza and other respiratory viruses reference laboratory (n = 9).The properties of national surveillance systems and laboratory methods used for non-polio EVs in 29 EU/EEA countries are shown in Twenty-six countries reported that they conducted non-polio EV surveillance based on typing of EVs detected from variety of clinical specimens . Most peEleven countries had an additional reporting system for EV-D68, and 11 countries have specific surveillance for EV-D68 . This suAs an indicator for surveillance activity, respondents were asked to provide the number of typed EV samples in their country during 2015; over 5,000 EV-positive specimens were reported to have been typed in the EU/EEA countries in 2015 . The estimated number of typed EV specimens was < 50 in 11 countries , whereas six countries successfully typed over 300 EV specimens each in 2015, mostly through sequencing .A total of 28 countries provided EV testing at the national level, including primary EV testing of clinical specimens in 25 and EV typing in 26 countries ,6,7,8. TAll except one country performed virus isolation at the national level, and 10 countries also performed it at the local level. RT-PCR and/or commercial methods were used for EV detection by all 26 countries performing primary EV testing  and 6. RA total of 20 countries use RT-PCR for EV detection; four of them use the WHO-recommended screening primers targeting the VP4 region  and the We provide an overview of national surveillance and detection systems currently used for non-polio EVs within the EU/EEA. Although laboratory-based non-polio EV surveillance is currently in place in most of the countries (26/29), it does not always include neurological infections and the surveillance activity measured as the number of reported EV-positive samples typed in 2015 varied remarkably between countries. It is of concern that 11 countries had typed fewer than 50 EV-positive samples. This is likely to be an insufficient number, taking into account the large variety of circulating EV types .According to the current WHO guidance, an effective EV surveillance system should investigate a ‘significant proportion’ of reported clinical cases, and 80% is given as an example of a significant proportion in that document . A recenSystematic reporting and/or surveillance for EV-D68 has not been implemented in the majority of EU/EEA countries; only 11 countries have introduced a surveillance system for EV-D68 infections using existing sentinel surveillance systems, by typing EV-positive respiratory samples or via AFP surveillance . CurrentHFMD surveillance in the EU/EEA countries has rarely been implemented in an effective way, with only two countries possessing an established specific surveillance system for patients presenting with this disease. On the other hand, 24 countries with laboratory-based EV surveillance always included aseptic meningitis and other neurological infections in their surveillance system, and also subjected EV-positive CSF samples to further typing. This would mean that although data on uncomplicated HFMD infections are not systematically collected throughout the region, the current non-polio EV surveillance would potentially capture the severe neurological presentations associated with HFMD and/or EV-A71, or any other EV type. Over 80 cases of EV-A71 infections associated with neurological symptoms were recently identified in Spain via the non-polio EV surveillance . HoweverThe use of appropriate testing methodologies is of the greatest importance for any laboratory-based surveillance system. All national laboratories performing primary EV diagnostic used either RT-PCR or commercial methods for EV detection. This should make it easier to introduce more uniform screening of EVs in clinical samples with well-defined sensitivities and specificities on a much greater scale. Laboratories may consider testing all CSF samples for EVs, as is already done in eight countries within the EU/EEA. However, molecular detection of EVs can be challenging due to the genetic diversity of EV types. As we identified a few potential mismatches with primers in terms of EV-D68 and EV-C104 detection, it is important to consider that not all previously published primers  can detect all EV types. Furthermore, molecular typing of EV-positive samples has also traditionally focused on species B EVs. It is important to note that use of additional primer pairs for typing species A and D EVs does enhance the success rate of molecular typing ,42-44. AAlthough direct molecular typing from clinical material has been well-established for EVs ,42-44, nAs the surveillance and detection systems applied may change from time to time, it is important to note that this study reflects the situation in individual countries as described in 2015 and 2016. This study was mainly based on the EV surveillance and detection activities performed within the national public health institutions; we have not collected information on laboratory capacities and performances at the sub-national level. Furthermore, we have not collected data on research activities in the field of EVs in each EU/EEA country. The potential regional variations in EV surveillance and/or detection activities within the individual EU/EEA countries have not been considered in this study either.The recent outbreaks of EV-D68 and EV-A71 infections in Europe emerging over the past 3 years have posed major concerns for local, national and international public health organisations. This is the first time information has been systematically gathered on the variety of existing surveillance systems and laboratory detection methods currently used for non-polio EVs within the EU/EEA countries. This survey highlighted substantial variability in non-polio EV surveillance and identified a clear need to strengthen non-polio EV detection capability and focus on neurological and respiratory infections. National guidance on testing should consider inclusion of respiratory and stool samples in addition to CSF samples for EV identification and characterisation in case of suspected neurological infections. Not only the introduction of molecular methods and sequencing for non-polio enterovirus detection and typing but also systematic data collection and monitoring would improve laboratory response to (re)-emerging EVs linked to severe disease.


In the in vivo studies, the topical application of cycloheterophyllin before UVA irradiation significantly decreased trans-epidermal water loss (TEWL), erythema, and blood flow rate. These results indicate that cycloheterophyllin is a photoprotective agent that inhibits UVA-induced oxidative stress and damage, and could be used in the research on and prevention of skin photoaging.Ultraviolet (UV) radiation, particularly ultraviolet A (UVA), is known to play a major role in photoaging of the human skin. Many studies have demonstrated that UV exposure causes the skin cells to generate reactive oxygen species and activates the mitogen-activated protein kinase (MAPK) pathway. Previous studies have also demonstrated that cycloheterophyllin has an antioxidant effect and can effectively scavenge free radicals. Extending the aforementioned investigations, in this study, human dermal fibroblasts were used to investigate the protective effect of cycloheterophyllin against UV-induced damage. We found that cycloheterophyllin not only significantly increased cell viability, but also attenuated the phosphorylation of MAPK after UVA exposure. Furthermore, cycloheterophyllin could reduce hydrogen peroxide (H Ultraviolet (UV) irradiation is recognized to play a major role in photoaging and skin cancer , 2. Many30H30O7), a prenylflavone  (C10018), as no interaction occurred with the subjects and no identifiable information was made available to the researchers. All animal tests were approved by the Fu Jen Catholic University’s Institutional Animal Care and Use Committee (IACUC) policy.Artocarpus heterophyllus Lam., as described previously . After sonication, the lysate was centrifuged  and FLO-N1  before UVA irradiation. The surface changes in the dorsal skin were recorded by photography. When the animals’ suffering clearly negated the need to continue with the experiment, they were dropped from the study and euthanized by CO2 in accordance with the Fu Jen Catholic University’s Institutional Animal Care and Use Committee (IACUC) policy. This study was reviewed and approved by the Fu Jen Catholic University’s IACUCEight-week-old male BALB/c mice were obtained from the National Laboratory Animal Center, Taipei, Taiwan. Animals were housed and handled according to institutional guidelines. Briefly, mice were housed one per cage with controlled temperature (21–25°C), humidity (60±5%), and light (12/12 h light/dark cycle) for one week. Alfalfa-free food  and water were administered Unless otherwise indicated, data are expressed as mean ± standard deviation (SD) by using the GraphPad Prism Program 6 software . Comparison of the mean survival rates of cells under UVA irradiation with and without cycloheterophyllin was made using one-way ANOVA followed by Dunnett’s t-test for multiple comparisons. We considered p < 0.05 to be statistically significant.2) exposure for 24 hours, we observed a dose-dependent decrease in cell viability (data not shown). We chose UVA 20 J/cm2 to perform further experiments. To investigate the photoprotective effects of cycloheterophyllin, fibroblasts were pretreated with different doses of cycloheterophyllin. Results showed that cycloheterophyllin increased cell viability after UVA irradiation (20 J/cm2) in a dose-dependent manner  exhibited low cytotoxicity after 24 hours of treatment . The efft manner .MAPK signaling is critically involved in UV-activated pathways and is known to regulate various downstream effects in the skin cells , 15–17. 2O2 induced a decrease in viability when compared to that of the control. Pretreating the cells with 1 μM cycloheterophyllin prior to the H2O2 treatment increased the cell survival rate, which suggests that cycloheterophyllin suppresses the H2O2-induced cytotoxicity , erythema, blood flow rate. To evaluate the low rate . These rA. heterophyllus. Previous studies have reported that cycloheterophyllin has anti-platelet, anti-oxidative, anti-inflammatory, anti-diabetic, and anti-tuberculosis activity [Cycloheterophyllin is one of the prenylflavones isolated from the root bark of activity , 23, 24.Previous studies noted that the main factor in the UVA-induced damage to the skin cells is the presence of ROS, including hydrogen peroxide; singlet oxygen; and hydroxyl radicals, which affect the activation of the downstream cellular signaling pathways and cause damage to the skin. In this study, cycloheterophyllin inhibited UVA- and ROS-induced phosphorylation of the MAPK pathway  in a dose-dependent manner. For these known signaling pathways, we also investigated the generation of ROS to explore the antioxidant and photoprotective effects of cycloheterophyllin treatment. We pretreated the fibroblasts with cycloheterophyllin to find that cycloheterophyllin effectively reduces the amount of ROS after UVA exposure and exhibits antioxidant capacity. However, we identified that cycloheterophyllin has a protective effect only on UVA-induced damage and not on UVB-induced damage, which causes inflammation of the skin , 26. TheMany studies on the protective effects of natural plant ingredients against UV and oxidative stress damage have been reported. For example, silymarin has hepatoprotective capabilities and catechin, which is abundantly present in green tea as (-)epigallocatechin gallate (EGCG) and (-)epicatechin gallate (ECG), possesses photoprotective effects against both UVA- and UVB-induced damage , 28, 29.In general, there are two types of melanin in the human skin: eumelanin and pheomelanin. The former is more abundant in humans, is a brownish-black pigment, and is present mainly in the skin and hair. The latter is a reddish yellow pigment , 34. NatNormal murine hair growth is cyclic, and the major stages can be classified into hair follicle growth (anagen), regression (catagen), and quiescence (telogen) –42. In a2O2-induced pathway would cause a downregulation of AQP-3 [in vivo experiments, applying cycloheterophyllin alone did not cause skin irritation, but significantly ameliorated UVA-induced skin peeling on the backs of the mice. In addition, we found that cycloheterophyllin could effectively reduce TEWL after UVA irradiation. TEWL measurements can be used as an indicator for the internal barrier properties of skin; a higher value indicates a poorer water-holding capacity of the stratum corneum. Because the skin structure deteriorates after UVA exposure, TEWL values increase significantly, and the cycloheterophyllin-pretreated group exhibited reduction in TEWL values. Cycloheterophyllin was also found to decrease UVA-induced skin redness and inflammation. These results may lay the foundation for clinical anti-photoaging treatments in the future.The skin is a metabolically active barrier; a previous study reports that a metabolic barrier by cytochrome P450 (CYPs) can regulate the homeostasis of a metabolic barrier through activation or inactivation of biologically relevant molecules , 49. Theof AQP-3 . Cyclohe


Inclusion internal mammary lymph nodes as a part of regional nodal irradiation have a potential to reduce local recurrence, distant recurrence, and improve survival in breast cancer. However, the increased risk of cardiac toxicity and lungs injure associated with internal mammary lymph nodes irradiation has drew more and more attention. Estimating risk of metastasis in internal mammary lymph nodes based on axillary lymph nodes metastasis status is not always reliable: low-risk do not always mean negative in internal mammary lymph nodes and high-risk do not always indicate positive in internal mammary lymph nodes. Inaccurate prediction of in internal mammary lymph nodes metastasis might lead to over- or under-treatment of in internal mammary lymph node. Internal mammary sentinel lymph node biopsy is a minimally invasive technique which has a high potential to accurately evaluate the metastasis status in in internal mammary lymph nodes and improve accuracy of nodal staging. This technique might be a useful tool to guide individualized internal mammary lymph nodes irradiation. Internal mammary lymph nodes (IMN) metastasis has a similar prognostic importance as axillary lymph nodes (ALN) involvement in breast cancer patients. IMN metastases have been demonstrated to occur in 28–52% of ALN positive patients and 5–17% of ALN negative patients ) [It is worth noting that additional RNI including IMN was found to improve DFS, DDFS, and OS in stage I–III breast cancer in a meta-analysis of the MA.20, EORTC22922/10925, and French trials. The absolute benefits in OS were 1% in the MA.20 trial at 10 years, 1.6% in the EORTC22922/10925 trial at 10 years, and 3.3% in the French trial at 10 years . Subsequent to the results of the IMNI trials published, since 2016, NCCN Guidelines have updated recommendation of IMNI for patients with ≥ 4 positive ALNs as category 1, and strongly consider IMNI for patients with 1–3 positive ALNs (category 2A), both after mastectomy and lumpectomy.The ASCO-ASTRO-SSO panel also updated the ASCO guideline of postmastectomy radiotherapy which recommends treatment generally be administered to both the IMNs and the supraclavicular-axillary apical nodes in addition to the chest wall or reconstructed breast for patients with positive ALNs .Nevertheless, controversies persist regarding recommendation of IMNI in all patients with ALN metastasis. As a result, we need an accurate and effective procedure to detect subclinical metastases in IMN, in order to guide selection of high-risk population with IMN metastasis and best present the benefit of IMNIP < 0.05) if IMNI was performed without dose-volume constraints in organs-at-risk [P = 0.01) and lymphedema  [One of major concerns about IMNI is the increased dose-volume of cardiac and pulmonary irradiation –14. In t-at-risk , 15, 16.-at-risk , 17. The-at-risk . Similar-at-risk . The resExcept for increase dose-volume of cardiac and pulmonary irradiation, synergistic adverse effect of IMNI and systemic therapy on normal tissue has also led to specific concern. About 20–25% of breast cancer patients had human epidermal growth factor receptor 2 (HER2) over-expressive tumors, which could benefit from trastuzumab with increase in DFS and OS . SimilarIMNI was associated with significant increase in dose-volume irradiation of heart and lung when outdated techniques were used , 18, 26.Individual positioning is a feasible approach through which to reduce radiation dose to the OARs , 34. TheAlthough the 2016 NCCN Breast Cancer Clinical Practice Guidelines recommends IMNI for patients with ≥ 4 positive ALNs, and strongly considers IMN for patients with 1–3 positive ALNs, there is lack of evidence to establish the risk of IMN involvement . StudiesThe study by Veronesi et al. found that IMNI could improve the survival in patients with metastases in IMN identified by internal mammary lymph node biopsy. In this large series of 663 patients, 68  patients received IMNI for histologically proven IMN metastases. IMNI was effective yielded a 5-year OS of 95% to compare with IMN negative without IMNI .P < 0.001) [Currently, internal mammary sentinel lymph node biopsy (IM-SLNB) via intercostal space could be an accurate technique to guide personalized IMNI and minimize invasive staging in IMN , 49, 50.< 0.001) . The acc< 0.001) .Up to now, 313 patients with breast cancer received IM-SLNB guided by the modified radiotracer injection technique. The overall visualization rate of IM-SLN detected by preoperative lymphoscintigraphy and gamma probe was 70.9% (327/461). The success rate of IM-SLNB was 97.1% (304/313). In patients who performed IM-SLNB successfully, a total of 585 lymph nodes were removed, the median number of IM-SLNs was 2 (range 1–4 nodes). The IM-SLNs were located in the first , second , third  and forth  intercostal space. The positive IM-SLNs were located in the first , second , and the third  intercostal space. Our data showed that the IM-SLN involvement rate was 8.3% (18/218) in patient with ALN negative and 18.6% (16/86) in ALN positive patients. The significant meaning of IM-SLNB, is anticipated that, to detect the sub-clinical involvement of IMN, and thus to guide IMNI in patients who might not have been recommended for IMNI, as well as to guide not to give IMNI in patients who might been recommended for IMNI by current NCCN guideline. Our current data shows that, in patients with ≥4 positive ALNs, IMNI could be avoided in 57.1% cases (20/35) with negative IM-SLN. In patients with 1–3 positive ALNs, IMNI could be avoided in 90.6% cases (115/127) with negative IM-SLN. Our study is undergoing and needs a long-term follow-up to evaluate the benefit and the late side-effect of this method. The indication of IM-SLNB should be identified from person to person in the future . Currently, we are designing a clinical trial to explore whether IMNI following IM-SLNB should be performed to control the local recurrence in patients with IM-SLN metastasis . We hope the clinical trial will have a good result to guide the clinical practice of IMNI.As the part of the regional nodes irradiation for breast cancer radiotherapy, IMNI could partly contribute to a reduction in recurrence and improved survival in some population. The balance between the technical complexities and toxicities of IMNI and relative low frequency of clinically detected IMN involvement has posed therapeutic dilemma even if the NCCN guideline has upgraded its recommendation. An effective and accurate method is needed to individualize the patient selection of IMNI so that the benefit of IMNI can be performed in patients with IMN metastasis. IM-SLNB would make it possible which is a minimally invasive technique to evaluate the metastasis status in IMN, improve the nodal staging in IMN chain, and would be used to guide personalized IMNI.


Oryza sativa L.) breeding lines evaluated under rainfed drought-prone situations at three locations in eastern India from 2005 to 2007, a subset of 39 genotypes that were tested for two or more years was selected to develop a drought yield index (DYI) and mean yield index (MYI) based on yield under irrigated, moderate and severe reproductive-stage drought stress to help breeders select appropriate genotypes for different environments.Drought is the most severe abiotic stress reducing rice yield in rainfed drought prone ecosystems. Variation in intensity and severity of drought from season to season and place to place requires cultivation of rice varieties with different level of drought tolerance in different areas. Multi environment evaluation of breeding lines helps breeder to identify appropriate genotypes for areas prone to similar level of drought stress. From a set of 129 advanced rice (ARB 8 and IR55419-04 recorded the highest drought yield index (DYI) and are identified as the best drought-tolerant lines. The proposed DYI provides a more effective assessment as it is calculated after accounting for a significant genotype x stress-level interaction across environments. For rainfed areas with variable frequency of drought occurrence, Mean yield index (MYI) along with deviation in performance of genotypes from currently cultivated popular varieties in all situations helps to select genotypes with a superior performance across irrigated, moderate and severe reproductive-stage drought situations. IR74371-70-1-1 and DGI 75 are the two genotypes identified to have shown a superior performance over IR64 and MTU1010 under all situations.For highly drought-prone areas, a combination of DYI with deviation in performance of genotypes under irrigated situations can enable breeders to select genotypes with no reduction in yield under favorable environments compared with currently cultivated varieties. For rainfed areas with variable frequency of drought stress, use of MYI together with deviation in performance of genotypes under different situations as compared to presently cultivated varieties will help breeders to select genotypes with superior performance under all situations. The income of most farmers is low and they are challenged by erratic yields. In Asia, about 50% of all the rice land is rainfed and although rice yields in irrigated ecosystems have doubled and tripled over the past 30 years, only modest gains have occurred in rainfed rice systems S denote the yield of the ith genotype under stress, (Yi)NS the yield of the ith genotype under non-stress  conditions and yS and yNS the mean yields of all genotypes evaluated under stress and non-stress conditions, respectively.Yi)NS and (Yi)S. This index has an upward bias when the differences between non-stress and stress conditions are large and it favors genotypes with higher yield potential and lower stress tolerance. Higher values mean a higher rate of productivity. Mean relative performance is calculated as Let  to conditions with moderate to severe reproductive-stage drought in drought-prone eastern India. The lines were tested at three locations, Raipur, Hazaribag and Faizabad, in relatively similar shallow rainfed drought-prone ecosystems, from 2005 to 2007. At all sites, the experiments were planted under irrigated control and reproductive-stage drought stress. Experiments were planted in alpha lattice designs with three replicates. From the set of 134 genotypes, a subset of 39 genotypes , stress tolerance (TOL), stress tolerance index (STI) and geometric mean productivity (GMP), were calculated for both moderate and severe stress conditions. Correlations between these indices and with the drought yield index (DYI) were evaluated using the CORR procedure of SAS.Results of a conventional analysis of variance of the log-transformed data for grain yield indicated highly significant genotype x stress-level interaction (p<0.0001). The REML estimates of variance components and fixed effects are presented in Table -1 to 5.0 t ha-1 under non-stress, from 1.78 t ha-1 to 3.40 t ha-1 under moderate stress and from 0.45 t ha-1 to 2.19 t ha-1 under severe stress  REI, MRP, STI, GMP and MPI and among (b) DYI, TOL, SSI and SSSI whereas the correlation between indices in groups (a) and (b) is highly negative. Table ARB 8 recorded the highest MYI value of 3.34, followed by IR74371-70-1-1, DGI 75, DGI307, IR74371-78-1-1, and IR55419-04 with MYI values of 3.30, 3.25, 3.21, 3.20 and 3.17, respectively.The deviations of genotype means from means of the cultivated high-yielding check varieties (IR64 and MTU1010) are presented in Table In our study, the significance of genotype x stress-level interaction was first investigated using an appropriate mixed model. The effect of stress level and genotypes was considered fixed because these were the only levels and genotypes of interest. The objective is to test the significance of genotype x stress interaction across all potential drought-prone sites and years. Interest is not specifically in those sites and years but the target population from which they were drawn. Therefore, years and sites were considered as random. When treatments are evaluated at different stress levels, they tend to have different levels of error variation. Therefore, heterogeneity of error variances at different stress levels was incorporated. The least squares means for the stress x genotype interaction were computed after appropriately accounting for all potential sources of variation. Then, pair wise stress-level comparison of means (non-stress vs moderate stress and non-stress vs severe stress) involving the same entry was carried out to look for those entries with statistically non-significant differences between means across the two stress levels.At the reproductive stage, yield reduction in rice is significant even with a moderate stress . HoweverA high positive correlation between the moderate and severe stress means indicates that the entries that performed well under moderate stress also performed well under severe stress. STI and GMP identified DGI75  and IR74371-70-1-1  among the top three stable performers. These two, along with ARB8  and IR55419-04 , were the top five. This implies that STI and GMP may be useful in identifying entries that yield well under non-stress and yield reasonably well under severe stress. The proposed DYI, TOL and SSI favor genotypes with good yield under stress. For these indices, DGI75, DGI307 or IR74371-70-1-1 do not stand out among the top performers. These indices are therefore more useful for identifying stress-tolerant genotypes that perform well in stress environments.ARB8 is again the top performer in the case of MRP and REI as well. This is because these indices are respectively the sum and product of two ratios, (i) genotype control mean/overall control mean and (ii) genotype severe-stress mean/overall severe-stress mean. The index values are inflated if either (i) or (ii) is higher. If (ii) is high, the entry enters the set of top performers though the performance under non-stress is not the topmost and vice versa. So, these indices are not very effective in distinctively discriminating entries that perform well under both non-stress and stress.For areas where severe stress is a recurrent phenomenon, selection of genotypes with high DYI can be useful. However, selection based on DYI may lead to the identification of genotypes with high yield in moderate or severe drought stress but not very high yield or yield equivalent to that of current cultivated varieties under normal irrigated situations. MYI that considers the performance of genotypes under different situations ranging from irrigated control to moderate drought stress and severe drought stress provides better opportunities for breeders to select genotypes with good performance under all situations. The careful study of the deviation in performance of 39 genotypes from two popular varieties (IR64 and MTU1010) revealed that the performance of only two genotypes (IR74371-70-1-1 and DGI 75) showed a positive deviation over the performance of both popular varieties under the three conditions. Even though genotypes such as ARB 8, DGI307, IR55419-04, and IR74371-78-1-1 showed a high MYI, they did not show a positive deviation in their performance under all situations due to their lower yield than IR64 under irrigated control situations. For areas with a variable rainfall pattern and with equal probability of occurrence of normal rainfall, moderate stress or severe stress situations, breeders should combine MYI with a positive deviation in the performance of genotypes under all situations in order to select a variety with better performance under all situations over current varieties. In fact, even for highly drought-prone areas, combinations of DYI and deviations in the performance of genotypes from that of current varieties will enable breeders to select genotypes with yield very near that of current cultivated varieties under irrigated normal situations in addition to their higher yield under moderate and severe drought situations.Many authors have reported that selection of stable genotypes should be based on a combination of indices. For regions with severe drought problems and higher frequency of drought occurrence, our study indicated that selection based on DYI will result in the identification of lines with significantly higher performance over current cultivated varieties under moderate to severe drought at the expense of slightly lower yield under normal irrigated situations. For regions where moderate to severe drought occurs once in three to four years and normal rainfall persists over other years, selection of genotypes based on a combination of MYI and a positive response over existing high-yielding varieties under all situations will enable breeders to select genotypes with higher yield over current varieties under all situations. From among 39 genotypes, the combination of MYI and deviation identified IR74371-70-1-1 and DGI 75 as two promising high-yielding drought-tolerant cultivars.


Introduction: Rapid scale-up of antiretroviral therapy (ART) in the context of financial and health system constraints has resulted in calls to maximize efficiency in ART service delivery. Adopting differentiated care models (DCMs) for ART could potentially be more cost-efficient and improve outcomes. However, no study comprehensively projects the cost savings across countries. We model the potential reduction in facility-level costs and number of health workers needed when implementing two types of DCMs while attempting to reach 90-90-90 targets in 38 sub-Saharan African countries from 2016 to 2020.Methods: We estimated the costs of three service delivery models: (1) undifferentiated care, (2) differentiated care by patient age and stability, and (3) differentiated care by patient age, stability, key vs. general population status, and urban vs. rural location. Frequency of facility visits, type and frequency of laboratory testing, and coverage of community ART support vary by patient subgroup. For each model, we estimated the total costs of antiretroviral drugs, laboratory commodities, and facility-level personnel and overhead. Certain groups under four-criteria differentiation require more intensive inputs. Community-based ART costs were included in the DCMs. We take into account underlying uncertainty in the projected numbers on ART and unit costs.Results: Total five-year facility-based ART costs for undifferentiated care are estimated to be US$23.33 billion . An estimated 17.5% (95% CI: 17.4%–17.7%) and 16.8% (95% CI: 16.7%–17.0%) could be saved from 2016 to 2020 from implementing the age and stability DCM and four-criteria DCM, respectively, with annual cost savings increasing over time. DCMs decrease the full-time equivalent (FTE) health workforce requirements for ART. An estimated 46.4% (95% CI: 46.1%–46.7%) fewer FTE health workers are needed in 2020 for the age and stability DCM compared with undifferentiated care.Conclusions: Adopting DCMs can result in significant efficiency gains in terms of reduced costs and health workforce needs, even with the costs of scaling up community-based ART support under DCMs. Efficiency gains remained flat with increased differentiation. More evidence is needed on how to translate analyzed efficiency gains into implemented cost reductions at the facility level. Global 90-90-90 targets and 2015 World Health Organization (WHO) guidelines call for universal access to and rapid scale-up in coverage of antiretroviral therapy (ART), at a time resources for HIV are constrained globally . While dA path to greater ART efficiency may be adopting service delivery models that reduce use of health system inputs while maintaining or increasing quality of care and hence patient outcomes. In this context, differentiated care models (DCMs) for ART, explored in this paper, have been suggested to potentially maximize quality of care efficiently ,12. ThesResearch on DCMs so far has examined the feasibility of bringing programs to scale and the costs and patient outcomes in pilot-level implementation. A 2014 study in Malawi, Mozambique, South Africa and the Democratic Republic of the Congo showed the benefits of four approaches to simplifying ART delivery for stable patients; from the patient perspective, travel and lost income was reduced, and from the health system perspective, clinic attendance was improved and retention in care remained high . There hWhile these studies suggest DCMs are cost-saving, no study projects the costs of increasing differentiation of ART service delivery across countries in an era of ART scale-up. This current study models the potential health system efficiency gains in terms of reduced facility-level costs and number of health workers needed for ART from implementing two types of DCMs compared to an undifferentiated care model in 38 sub-Saharan African countries from 2016 to 2020. We take into account underlying uncertainty in the projected numbers on ART and unit costs of interventions.Currently, most low- and middle-income African countries have limited patient differentiation in ART service delivery. Frequency of facility visits or laboratory testing is not necessarily based on response to treatment or other patient characteristics, and most ART services are offered in facility-based settings . As counThe 2016 WHO ART guidelines recommend differentiating four groups: PLHIV presenting for care at earlier stages of the disease, PLHIV presenting with advanced disease, patients stable on ART, and those unstable . The pacIn addition to the WHO guidelines, donors and global organizations, including PEPFAR; the Global Fund to Fight AIDS, Tuberculosis, and Malaria; UNAIDS; and Medécins Sans Frontières, have issued guidance on differentiated care ,14,23,24Based on this conceptual framework, we developed three stylized service delivery models for analysis . The firFor each model, we estimated the total costs of ARV drugs, laboratory commodities, facility-level personnel and overhead, and community-based ART support across 38 sub-Saharan Africa countries from 2016 to 2020 . We compWe modelled underlying uncertainty in the numbers of people on ART and the unit cost of treatment inputs per person. We conducted probabilistic sensitivity analysis and expected mean values were derived from sampling events in 5000 second-order Monte Carlo simulation trials performed using RiskAMP software (Structured Data LLC) integrated with Microsoft Excel . Each siThe study estimated the number of children (ages 0-9), adolescents ages 10–19), and adults (ages 20 and above) living with HIV from 2016 to 2020 in each country using projections from the uncertainty analysis tool within the Spectrum AIDS Impact Model (AIM) (see Supplementary File 1) . Annual 0–19, andFor the DCMs, the age-disaggregated number of people on ART were further divided into three categories – new, stable established, and unstable established patients – based on country- or regional-specific data on retention, mortality, and viral load suppression –33. We aWe accounted for uncertainty in unit costs for ARVs, laboratory tests, facility-based personnel and overhead using probabilistic sensitivity analysis based on ranges for current baseline costs and future reductions in costs (ARVs and laboratory only). Cost data were adjusted to constant 2015 US dollars . ARV regThe uncertainty analysis parameters for ARV costs were based on 2015 adult, adolescent and pediatric first- and second-line ARV costs, separated by income level and region, from the Global Price Reporting Mechanism (GPRM) database . The GPRWe modelled the unit cost of laboratory tests as uncertain based on recent literature on the reagent and consumables costs of viral load, CD4 cell count, haematology and clinical chemistry panel tests. Costs per test from 12 countries, categorized by country income level, were used in the analysis. Overall laboratory costs per patient per year  were basBased on a review of recent country-specific studies, we estimated ranges for the cost per clinical and ARV refill visit of facility-level overhead  and personnel costs . Data from 10 studies across 11 countries were included in the analysis –58. We aFew studies estimate community-based ART costs. Community-based ART cost ranges were derived from four studies ,55,59,60We estimate the number of children ages 0 to 9 on ART will decrease from 0.48 million  in 2016 to 0.42 million (95% CI: 0.40-0.44 million) in 2020 due to increased prevention of mother-to-child transmission, resulting in decreases in the future population of children living with HIV. The number of adolescents and adults on ART is estimated to increase from 12.9 million (95% CI: 12.6-13.2 million) in 2016 to 18.6 million (95% CI: 18.0-19.1 million) in 2020. The percentage of people on ART who are stable, established patients are estimated to increase over time . Key popUnder the undifferentiated care model, total five-year facility-based ART costs are estimated to be US$23,305 million . An estimated $5123 million (95% CI $5109-$5203 million) and $5,122 million (95% CI: $5108-$5201 million) in facility-based ART costs could be saved from 2016 to 2020 from implementing the age and stability DCM and four-criteria DCM, respectively. Differentiating care to certain groups requiring more intensive inputs under the four-criteria DCM prevents further efficiency gain compared to the age and stability DCM. When adding community-based ART support needed under DCMs, total cost savings from implementing DCMs decline slightly, but are still large with an estimated cost reduction of 17.5% (95% CI: 17.4-17.7%) and 16.8% (95% CI: 16.7-17.0%) under the age and stability DCM and four-criteria DCM, respectively. Annual cost savings from DCMs are estimated to increase over time .Figure Both DCMs result in similar levels of facility-level cost savings. Although the four-criteria model calls for increased clinical and refill visits for key versus general population adults, key populations represent a small proportion (2%) of all adults on ART and overall costs. Also, the cost savings from assumed lower refill visits for rural ART patients are nearly equal to the additional costs from assuming that urban ART patients will maintain more frequent refill visits. When factoring in community support costs, the difference in cost savings between the two DCMs becomes significant. This is a result of assuming a lower unit cost for community ART support among urban patients, who represent the majority of those on ART, compared with rural patients. We assumed urban ART patients who likely have better access to health facilities than those in rural areas would require fewer community ART support meetings and ARV distribution points.Cost savings in facility-based service delivery from DCMs are driven by a 44% reduction in overhead and personnel costs and an 11% reduction in laboratory costs. The laboratory cost savings assume CD4 count testing is discontinued under DCMs; however, even if annual CD4 testing was continued for 10-35% of those on ART with poor immune reconstitution, DCMs would save $176-$298 million in laboratory costs across all five years . ARV cosDue to the potentially wide range in community-based ART unit cost, we estimated the relationship between this unit cost and the savings from implementing DCMs, using the age and stability DCM as an example . CommuniDCMs also decrease the full-time equivalent (FTE) health workforce requirements for ART . An estiA previous analysis suggested that Eastern and Southern Africa alone would account for 61% of the global financing gap for facility-based ART over 2016-2020 [A practical implication of such efficiency gains is that additional ART patients could be managed with existing health resources and future needs would be reduced for investments in infrastructure or new health workers related to HIV treatment. Given competing priorities for government resources in the Sustainable Development Goals era, this is very desirable. Governments can then distribute increases in health spending across needs in multiple health domains. However, for our analysed gains in efficiency to translate into real reductions in additional health system resources needed as patient numbers increase, some conditions must be met. First, health worker time management at the facility level is critical. Initially, as the volume of patients’ clinic visits reduces once DCMs are implemented – that is, before more patients can be associated with each facility given ongoing scale-up towards 90-90-90 targets – “slack” in health worker capacity must be taken up. If certain health workers are pre-assigned and cannot be shifted to other interventions, for example, at standalone ART clinics, even those now not crowded, then efficiency gains are not readily realizable. Second, overall reduced use of facility overhead notionally allocated to ART can appear intangible to facility managers. Practically, only reduced crowding and waiting time at outpatient departments and reduced cost of consumables related to lower patient flow can be realized into tangible changes. For example, facility managers can increase or adjust clinic hours to allow more patients of other health areas to be seen.Gains in the efficiency of use for health system inputs such as health worker time and facility space and overhead accrue to the facility operators involved in ART services. There must be political will and policy flexibility to make meaningful adjustments in response to realizable efficiency gains.Implementing new service delivery models often require changes to guidelines, with stakeholder consultation and initial piloting. Health workers and service delivery managers require retraining in the revised processes and on identifying different patient groups appropriately for differentiation. Community-based interventions may require additional upfront and recurring investments to ensure that they yield the best outcomes for adherence and patient management. Identifying and keeping updated registers of stable vs. unstable patients, general vs. key population PLHIV, and other patient disaggregation require investments in information systems, including laboratory systems and epi-behavioural surveillance. Additional research is needed to understand DCM start-up costs as they are not estimated in this study; however, some of these costs could be absorbed by ongoing in-service training programs and periodic revision of service delivery guidelines. A limitation of our analysis is the focus on the health system funders’ perspective. We did not estimate costs from a full economic lens, which would include items such as transportation and opportunity costs faced by patients and caregivers under DCMs vs. undifferentiated care. The reductions in such costs may be significant. The overarching rationale for our analysis is that in addition to efficiency gains, DCMs may yield long-term benefits by improving patient management and hence health outcomes; however, this was not based on any modelling analysis.Our results suggest that there may be considerable basis for the widespread adoption of DCMs from a cost-efficiency and health workforce optimization standpoint. This argument is further strengthened if DCMs are beneficial to long-term health outcomes for patients by identifying those requiring specialized and/or more intensive care approaches. More evidence is needed on cost and cost-efficiency aspects of community-based ART support interventions that should be paired with DCMs. Our study is the first systematic and multi-country examination of the potential efficiency considerations of such DCMs – more analyses are needed using real-world data and to include other high HIV burden regions.


Our results suggest that compensatory mutations are not a major contributor to the current epidemic of MDR-TB.Compensatory mutations have been suggested to promote multidrug-resistant tuberculosis (MDR-TB) transmission, but their role in facilitating the recent transmission of MDR-TB is unclear. To investigate the epidemiological significance of compensatory mutations, we analyzed a four-year population-based collection of MDR-TB strains from Shanghai (the most populous city in China) and 1346 published global MDR-TB strains. We report that MDR-TB strains with compensatory mutations in the  Mycobacterium tuberculosis (M. tb)1, and the resulting reductions in virulence and transmissibility would prevent multidrug-resistant tuberculosis (MDR-TB) strains from disseminating widely3. Early mathematical models predicted that MDR-TB should remain a local problem3, but the steady growth of the MDR-TB epidemic worldwide has contradicted these early expectations. A subsequent study suggested that even if the average fitness of MDR-TB strains is low, a small proportion of resistant strains that are relatively more fit will outcompete the less fit and drug-susceptible strains4. These more fit, resistant strains were thought to contain compensatory mutations that would restore fitness and thus constitute an important factor in the spread of MDR-TB strains6, and MDR-TB outbreaks in HIV-negative patients were regarded as successful examples of compensatory evolution7.It had been thought that drug-resistance mutations would introduce a fitness cost into resistant 9 and found that putative compensatory mutations in rpoC and rpoA were more common in VNTR-clustered strains. However, these studies did not determine whether the putative compensatory mutations accumulated before or after the MDR-TB strains were transmitted. If these compensatory mutations accumulated after transmission, they should not be considered as factors that promote or facilitate transmission. Moreover, the sampling methods used in these studies were not population based, and the IS6110/MIRU-VNTR defined clusters could have perhaps been further separated by whole-genome sequencing (WGS). To avoid these pitfalls, we examined the role of compensatory mutations in MDR-TB transmission by using WGS to determine transmission clusters in the MDR-TB strains collected in Shanghai over a 4-year period (2009–2012). Our findings contradict the previous inferences and do not support a significant role of compensatory mutations in promoting the ongoing MDR-TB epidemic.Recent studies have investigated the role of compensatory mutations in MDR-TB transmission by comparing the presence of compensatory mutations in clustered or nonclustered strains10. Here, to determine the presence of compensatory mutations in VNTR nonclustered MDR-TB strains, we randomly selected 105 isolates from the remaining 202 VNTR nonclustered MDR-TB isolates to sequence the full-length genes rpoA, rpoB, and rpoC. The combined results of the 227 Shanghai MDR-TB strains were termed the “Shanghai dataset”. Meanwhile, we obtained WGS records of 8331 M. tb isolates of global origin from the European Nucleotide Archive (ENA) (Supplementary Table 1), and 1346 of these strains were identified as MDR-TB  hospitals in Shanghai, China. All of these isolates were MIRU-VNTR genotyped, and 122 clustered MDR-TB strains had undergone WGS previouslyrpoA, rpoB, or rpoC genes that had arisen at least twice in parallel and were identified as putative compensatory mutations  difference were considered to constitute a transmission cluster. A total of 36 such clusters were found in the Shanghai dataset, and 117 clusters were identified in the Global dataset.Through phylogenetic reconstruction of the MDR-TB strains obtained above, we found 60 nonsynonymous mutations in the ons Fig. . Of thes11. In the Shanghai dataset, 12 transmission clusters were determined as C-type, 18 as N-type, and six as M-type  C    Fig. . An anally) Fig. . Notablyly) Fig. . Moreove9. However, these studies treated “compensatory mutations” as a static feature and ignored the possibility that compensatory mutations could occur in secondary cases that were initially infected with noncompensated MDR-TB strains. This is probably what has occurred in the M-type clusters . One explanation might be that new MDR-TB strains are continuously emerging during antibiotic treatment, and these new MDR-TB strains might not have had enough time to accumulate compensatory mutations. For the same reason, within the 4-year duration of the study, they might not have had enough time to cause secondary TB cases Fig. . Thus, tt-test, P < 0.0001, Fig. M. tb strain can accumulate multiple mutations that confer resistance to a single antibiotic (evolution of high-level resistance)14, we further compared the numbers of drug-resistance-conferring mutations in these two groups. Consistent with our prediction, the clustered MDR-TB strains had, on average, more drug-resistance mutations than the nonclustered MDR-TB strains did , while for clustered MDR-TB strains it was 5.13 (5.05 ~ 5.21)  for the isolates in the clustered versus nonclustered groups. We found that the isolates in the nonclustered group had a shorter average time since collection than the isolates in the clustered group did  were further separated by WGS. Second, we set strict criteria to identify putative compensatory mutations and excluded phylogenetic/lineage mutations or neutral polymorphisms. Third, our analysis discriminated between the accumulation of compensatory mutations before or after the transmission of MDR-TB strains  in the analysis of cluster size and the ratio of compensatory mutations.We believe in the accuracy of the results of our study because it incorporated several methodological improvements. First, we used WGS to identify transmission clusters, which is more precise than IS17, and different studies have reported heterogeneous results suggesting that MDR-TB strains can be ten times more or ten times less transmissible than drug-susceptible strains18. However, the reasons underlying these variations could simply reflect the differences in study settings and methodologies employed18; the drug-resistant strains reported to have reduced transmissibility were found in settings with effective TB control11, while strains with increased transmissibility were reported from regions with high TB burdens18. Thus, it seems more likely that the heterogeneous and discordant published results serve to demonstrate that TB transmission is a process that is primarily influenced by environmental factors such as TB control policy, time with the illness before diagnosis, treatment efficacy, and general quality of the health-care system19.The effect of drug resistance on transmission has been long debated21. Second, MDR-TB patients are, on average, infectious for longer periods due to the long course of treatment and high treatment failure rates23. Thus, there might be more chances for MDR-TB patients to generate secondary cases, which could conceivably compensate for a modest decrease in fitness. Third, the most common drug-resistance mutations carried by clinical isolates are associated with the lowest fitness cost in vitro, and such slight decreases in fitness may not be sufficient to affect transmission6. Moreover, positive epistatic effects between different drug-resistance mutations could further ameliorate the fitness cost imposed by individual drug-resistance mutations24. Fourth, it is still possible that the fitness cost measured in vitro might not reduce the ability of M. tb to transmit and establish in a new host in vivo26. This idea was supported by a previous study using tuberculin skin testing to trace infection that found an equal prevalence of infection among contacts exposed to patients harboring both drug-resistant and drug-susceptible strains27.We consider that there are at least four factors that can obscure the function of compensatory mutations in MDR-TB transmission. First, the efficacy of the TB control program. In countries or regions with high MDR-TB prevalence and poor management of MDR-TB patients, the transmission of MDR-TB strains is highly likely, even in the absence of compensatory mutations. In such circumstances, outbreaks and epidemics of MDR-TB are mainly driven by environmental factors28, and the 4-year observation window should have captured most of the transmissions that occurred within the first 2 years of the study. For transmissions that occurred during the last 2 years of the study, the secondary cases might have developed TB disease after our observation window. ow. Fig. . HoweverIn conclusion, we did not observe a promoting influence of compensatory mutations on the transmission of MDR-TB, and we suggest that the putative contribution of compensatory mutations may be overwhelmed by the complex and more powerful effects of environmental factors.10. MIRU-VNTR genotyping (12 loci) was performed for all MDR-TB strains, and the clustered strains were further subjected to WGS10. In the present study, we further sequenced the entire gene sequences of the rpoA, rpoB, and rpoC genes in 105 nonclustered MDR-TB strains to detect putative compensatory mutations. The PCR amplicons were sent for Sanger sequencing, and the results were analyzed to detect putative compensatory mutations using Geneious (http://www.geneious.com/).A total of 324 MDR-TB isolates were collected from 1 January 2009 to 31 December 2012, in Shanghai (the most populous city in China) during a population-based observational studyM. tb strains were downloaded from the ENA (http://www.ebi.ac.uk/ena). The quality criteria for data inclusion were set as follows: (1) the average sequencing depth should be above 10-fold; (2) the genome coverage rate should be above 95%. To avoid false transmission clustering, we included only the last isolate of longitudinal isolates collected from the same patient. Information about the geographic isolation of these isolates was obtained from the articles or the authors.A total of 8331 published whole-genome sequences of 29. In brief, the Sickle30 tool was used for trimming WGS data. Sequencing reads with Phred base quality above 20 and reads length longer than 30 were retained for analysis. The whole-genome sequence of the M. tb H37Rv strain (NC_000962.2) was used as the reference template for mapping reads. Sequencing reads were mapped to the reference genome using Bowtie2 (v2.2.9)31. SAMtools (v1.3.1)32 was used for SNP calling with mapping quality greater than 30. Fixed mutations (frequency ≥75%) were identified using VarScan (v2.3.9)33 with at least ten reads supporting and strand bias filter option on. We excluded all SNPs located in noise or repetitive regions of the genome .We used a validated pipeline for the mapping of sequencing reads to the reference genomeMEGA (v6.06)34, and the phylogenetic tree was further visualized by FigTree (http://tree.bio.ed.ac.uk/software/figtree/). Putative compensatory mutations in the rpoA, rpoB, or rpoC genes were identified through the following criteria: (1) nonsynonymous mutations in the rpoA, rpoB or rpoC genes that were present in only rifampicin-resistant strains; (2) each putative compensatory mutation must have arisen at least twice independently . We wrote a Python script to implement these criteria with the phylogenetic tree and a mutation matrix, and the Python script was uploaded to GitHub.The maximum likelihood method was used to reconstruct the phylogenetic trees with 500 bootstrap repeats using M. tb were obtained from the database TBDReaMDB and articles reporting rifampicin-resistance mutations  by inspection of their whole-genome sequences. Any isolates with genetic distances of less than 12 SNPs were classified into a cluster10. To compare the collection time distributions between the isolates in the clustered and nonclustered groups, we determined the collection time of each isolate as “days to the end of the study duration (31 December 2012)”. For example, if an isolate was collected on 1 January 2011, then its collection would be counted as “365 (days in 2010) + 366 (days in 2012) = 731 days”.Drug-resistance-associated mutations in A Python script was written for screening transmission clusters in both the Shanghai and Global datasets, and this Python script was uploaded to GitHub.t-test were used to compare discrete variables between groups. Chi-square analysis was performed to compare the ratios of compensatory mutations in different groups. All statistical analyses were performed and visualized in RStudio (https://www.rstudio.com/).The Wilcoxon nonparametric rank-sum test and Student’s The institutional review board of the Shanghai CDC approved the analysis with the anonymous dataset.https://github.com/StopTB/Compen_Muta_Transmit).The analysis scripts that were written and used in this study are available online at GitHub (Supplementary tables