Little is known on the cost-effectiveness of novel regimens for hepatitis C virus (HCV) compared with standard-of-care with pegylated interferon (pegIFN) and ribavirin (RBV) therapy in developing countries. We evaluated cost-effectiveness of sofosbuvir/ledipasvir for 12 weeks compared with a 48-week pegIFN-RBV regimen in Chinese patients with genotype 1b HCV infection by economic regions.A decision analytic Markov model was developed to estimate quality-adjusted-life-years, lifetime cost of HCV infection and incremental cost-effectiveness ratios (ICERs). SVR rates and direct medical costs were obtained from real-world data. Parameter uncertainty was assessed by one-way and probabilistic sensitivity analyses. Threshold analysis was conducted to estimate the price which can make the regimen cost-effective and affordable.Sofosbuvir/ledipasvir was cost-effective in treatment-experienced patients with an ICER of US$21,612. It varied by economic regions. The probability of cost-effectiveness was 18% and 47% for treatment-naive and experienced patients, and it ranged from 15% in treatment-naïve patients in Central-China to 64% in treatment-experienced patients in Eastern-China. The price of 12-week sofosbuvir/ledipasvir treatment needs to be reduced by at least 81% to US$18,185 to make the regimen cost-effective in all patients at WTP of one time GDP per capita. The price has to be US$105 to make the regimen affordable in average patients in China.Sofosbuvir/ledipasvir regimen is not cost-effective in most Chinese patients with genotype 1b HCV infection. The results vary by economic regions. Drug price of sofosbuvir/ledipasvir needs to be substantially reduced when entering the market in China to ensure the widest accessibility. In the last two years, new direct-acting antivirals (DAAs) were approved by the US Food and Drug Administration (FDA) including the first-in-class nucleotide polymerase inhibitor sofosbuvir (SOF), the NS5A inhibitor daclatasvir (DCV) as well as fixed-dose combinations of ledipasvir (NS5A inhibitor)/sofosbuvir. Their efficacy and safety, with and without the addition of pegylated interferon (pegIFN) and/or ribavirin (RBV), have been proven to be very high across patient groups in clinical trials–3. HowevChina has largest number of HCV infection, with a minimum estimation of 10 million patients with chronic hepatitis C (CHC). GenotypDAAs are not current available in China. Clinical trials using DAAs have recently been initiated, and the market prices are not settled. China is a large and diverse country, with regions varying significantly in economic development (gross domestic product (GDP) per capita varies widely across the country) and socio-demographic profiles. The health financing system in China is quite decentralized, exacerbating rather than mitigating regional disparity. Though three public medical insurance programs , Urban Resident Basic Medical Insurance (URBMI) and New Rural Cooperative Medical Scheme (NRCMS)) have covered 95% of the population by 2011. All of Our study aimed to evaluate the cost-effectiveness of DAAs in Chinese patients with CHC GT1b, based on real-world practice in a Hong Kong-Beijing special hepatitis C clinic . We, taking fixed-dose combinations of sofosbuvir/ledipasvir  as an example, did the evaluation by four economic regions , assessed the budget impact and affordability of the regimen, and attempted to determine the price where the regimen is cost-effective and affordable from a government payer’s perspective.A decision analytic Markov model based on the natural history of HCV infection in the published literature was developed to simulate the progression of a 50-year-old GT1b cohort by treatment history, sex and fibrosis stages (F0-F4 defined by METAVIR score), using Since Chinese patients may respond differently compared with patients studied in global clinical trials of DAAs, effectiveness data was from a real-world practice in a Hong Kong-Beijing special hepatitis C clinic . The SVR rate was 100% for patients treated with Harvoni® regardless of treatment history and cirrhosis status. The 12-week drug price of Harvoni® was RMB600,000, reflected the actual price paid by patients. The 48-week drug price of pegIFN and RBV was RMB46,447. Both the prices were converted to 2014 U.S. dollars . All patThe retrospective study was approved by Hong Kong Clinical Research Ethics Committee (HKCREC), Hong Kong. The patients record and information was anonymized and de-identified prior to the analysis.Age-specific background mortality rates in China were used in each model, 25. We Baseline fibrosis before treatment and annual progression probabilities were obtained from a meta-analysis. To accoIn China, a large share of outpatient visits is to secondary and tertiary hospital outpatient departments which are government owned and managed. Bureau of Commodity Prices determines the prices for medical assessments and treatments in these hospitals in each province. For the same medical procedure, the prices within a city are relatively uniform across all hospitals. We obtained annual direct medical costs associated with disease states F0-F4, DC, and HCC from major hospitals in each economic region: Northeast (Shenyang), Central (Zhengzhou), East , and West . Annual medical costs included costs for liver-related and other laboratory tests , IFNL4 (rs368234815), genotyping, complete blood count, comprehensive metabolic panel, thyroid-stimulating hormone, PT-INR), procedures , medications, and hospitalizations. Costs that were not available from a particular city were replaced with the mean cost for that region. It was found that healthcare costs were significantly lower for patient with SVR than for those without SVR[79860, IFBecause utility values for Chinese patients were not available, we used the values for each disease state from a systematic review including 19 studies with direct HRQOL assessment using the Short Form-36 in HCV patients. UtilityThe incremental cost-effectiveness ratio (ICER) was calculated by dividing the difference in lifetime costs between sofosbuvir/ledipasvir and SOC by the difference in quality-adjusted life-years (QALYs), separate for treatment-naïve and -experienced patients with and without cirrhosis. We do not have a determined willingness-to-pay (WTP) threshold as that in the U.S.  or U.K. . According to WHO guidelines, three tWe calculated the life expectancy from different disease states (non-cirrhosis and cirrhosis) and the percentage of patients who progressed to cirrhosis, and compared them with published data to validate the model, 32. DetOne-way sensitivity analysis was conducted to assess the impact of changes of individual model parameter and assumptions on the model results. Whenever available, the 95% confidence interval (95%CI) reported were used . OtherwiWe estimated the budget required to treat all eligible patients in China which are a minimum of 10 million patients. By usinWe evaluated the affordability of sofosbuvir/ledipasvir using the average daily wage of urban employees. As suggested by WHO, treatments costing one day’s wage or less for a full course of treatment for an acute condition or a 30-day supply of medicine for chronic diseases are generally considered affordable.In order to assess the impact of price of sofosbuvir/ledipasvir on the cost-effectiveness, we performed a threshold analysis. Keeping all the other parameters constant, we estimated the maximum price where the regimen would be cost-effective compared to SOC. The price where the average patients can afford was also estimated.Depending on the treatment history, cirrhosis status, and economic regions, the ICERs ranged from US$8,950 to US$89,470 per QALY gain when comparing sofosbuvir/ledipasvir to SOC . By the Using one-way sensitivity analysis, we identified 10 parameters that had the largest impact on ICERs comparing sofosbuvir/ledipasvir to SOC for each region . The ICEProbabilistic sensitivity analysis showed that the probability that sofosbuvir/ledipasvir was cost-effective at a US$22,770 WTP was 18% and 47% for treatment-naïve and -experienced patients respectively in China . The proWe estimated that 5.3 million GT1b CHC patients would be eligible for treatment with a 95% health insurance coverage. Among 5The average daily wage for urban employees in China in 2014 was US$35. Averagely, an employee needs to work 2,776 days (96972/35) to receive a 12-week treatment of sofosbuvir/ledipasvir (or 923 days to purchase one bottle of sofosbuvir/ledipasvir). It is far above the affordable benchmark indicated by WHO.The price of 12-week sofosbuvir/ledipasvir treatment needs to be reduced by at least 69% to US$29,793 make the regimen cost-effective in all patients at WTP of three times GDP per capita . The priOur cost-effectiveness analysis of sofosbuvir/ledipasvir in Chinese CHC GT1b patients by regions shows that the regimen might not be cost-effective in all patient groups in different economic regions relative to their GDP per capita. Price is the major factor influencing the cost-effectiveness. At current price, neither government nor individual can afford the drug. The price for a 12-week course of treatment needs to be reduced by at least 81% to US$18,185 from current market price to make the regimen cost-effective.High prices is the main barrier to accessibility to affordable DAAs, especially in upper-middle-income countries like China. High-inAs our analysis showed that substantial reduction  from current market price of sofosbuvir/ledipasvir  is required to make the regimen cost-effective in all patients. A further reduction is needed to ensure the access to affordable treatment for each individual (down to US$105 as estimated). Price needs to be carefully settled when DAAs enter China market. Because of the restriction on accessibility to DAAs currently in China, patients seek for low-priced products by illegal means. They can either purchase the DAAs sold online or join the tourist groups to the neighboring countries like India, where generic products are available, to purchase the DAAs in person. It undermines the license agreement between generic manufactures and the pharmaceutical companies, and has potential risk of jeopardizing patients’ health if the drug is counterfeited as WHO recently alerted.In order to get access to affordable DAAs, the government needs to negotiate with pharmaceutical companies with much lower prices to afford the purchase and include them in the National Essential Medicines List as WHO did. In ordeThe cost-effectiveness of the new DAAs treatment relies on the society’s willingness to pay for improvements in health. As suggested by WHO, we adopted a threshold of three times GDP per capita as the decision rule in our analysis, althougOur study has several limitations. We lacked Chinese-specific data for many parameters and had to rely on estimates from the published literature. Small sample size for the primary efficacy measurement could have contributed to parameter uncertainty with respect to the expected SVR rate. But our analysis showed that even with a 100% SVR rate, sofosbuvir/ledipasvir had low probabilities to be cost-effective in most patients. We restricted our analysis to direct medical costs and did not include indirect costs (e.g. productivity loss) and non-medical costs . But this may lead to estimates that are more conservative. We did not model the future possibility of retreatment with the new regimens (either due to treatment failure or reinfection) nor the potential benefit of preventing incident HCV infectious through reductions in the number of infected patients from the population. In addition, the natural history after treatment with these new regimens is uncertain but we have comprehensively tested the natural history parameters in our sensitivity analyses.Our analysis demonstrated that sofosbuvir/ledipasvir might not be cost-effective in all Chinese GT1b patients infected with HCV at current price. At current price, it would pose a huge burden to government’s healthcare finance. Drug price of sofosbuvir/ledipasvir needs to be substantially reduced when entering the market in China. Hopefully current exercise will set a benchmark for upper-middle-income Asian countries to settle the price of DAAs and to ensure the widest access to DAAs.S1 FigTornado diagram of one-way sensitivity by previous treatment history in A: All China, B: Northeastern-China, C: Central-China, D: Eastern-China and E: Western-China.(DOCX)Click here for additional data file.S1 FileModel validation: life expectancies for chronic liver disease stage and general population (Table A). Validation of the natural history of our model (Table B).(DOCX)Click here for additional data file.


We described the role and feasibility of the iACT service in guiding individualized antibiotic combination selection in patients with XDR-GNB infections.Combination therapy is increasingly utilized against extensively-drug resistant (XDR) Gram negative bacteria (GNB). However, choosing a combination can be problematic as effective combinations are often strain-specific. An iACT for clinical management were included. The feasibility and role of the prospective iACT service was evaluated. The following patient outcomes were described: (i) 30-day in-hospital all-cause and infection-related mortality, (ii) clinical response, and (iii) microbiological eradication in patients with bloodstream infections.A retrospective case review was performed in two Singapore hospitals from April 2009–June 2014. All patients with XDR-GNB and antibiotic regimen guided by iACT service was requested by Infectious Disease physicians for 39 cases . Bloodstream infection was the predominant infection (36%), followed by pneumonia (31%). All iACT recommendations were provided within 48h from request for the service. Prior to iACT-guided therapy, most cases were prescribed combination antibiotics empirically (90%). Changes in the empiric antibiotic regimens were recommended in 21 (54%) cases; in 14 (36%) cases, changes were recommended as the empiric regimens were found to be non-bactericidal in vitro. In 7 (18%) cases, the number of antibiotics used in combination empirically was reduced by the iACT service. Overall, low 30-day infection-related mortality (15%) and high clinical response (82%) were observed. Microbiological eradication was observed in 79% of all bloodstream infections.From 2009–2014, the iACT service can be feasibly employed to guide the timely selection of rationally optimized combination regimens, and played a role in reducing indiscreet antibiotic use.The  The emergence of extensively-drug resistant (XDR) Gram negative bacteria (GNB) is increasingly recognized as one of the greatest threat to healthcare . InfectiPseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae [in vitro and/or animal infection models, and should be individualized depending on organism, susceptibility profile, infection site and the clinical characteristics of each patient [In the recent years, there have been an increasing number of physicians employing combination antibiotic therapy against XDR-GNB, in particularly against eumoniae . The coneumoniae . On the eumoniae . This ha patient , 7.in vitro antibiotic combination testing (iACT) service, with a rapid turn-around time of 48 hours, was developed. The objective of the iACT service is to guide physicians in the selection of an individualized and rationally optimized combination therapy, taking into account the in vitro combination testing results of each strain and the patient’s clinical and pharmacokinetic (PK) parameters. In this study, we described the role and feasibility of the iACT service in guiding the selection of individualized and rationally optimized combinations in patients with XDR-GNB infections. In addition, the clinical and microbiological outcomes of patients receiving iACT-guided therapy were described.In view of the current antimicrobial landscape, a prospective st April 2009 to 30th June 2014 with microbiologic evidence of XDR-GNB or pan-drug resistant (PDR) GNB isolation causing clinical infections and treated with antibiotic regimen guided by iACT were identified from the service’s electronic database and retrospectively reviewed. The use of iACT to guide antibiotic combination selection in these patients was elected by their attending ID physicians as part of their clinical management strategy, out of desperate measures. Patients with infections due to an XDR-/PDR-GNB requiring iACT-guided therapy that were classified as cured, and then developed a subsequent infection after a 90-day period due to a different GNB requiring iACT-guided therapy were analyzed as two different cases. This study was approved by the ethics review board of Singapore General Hospital (2012/818/D) and Mount Elizabeth Hospital (PIEC/2012/038) prior to study initiation. The ethics review board of both institutions waived the need for informed consent as the study involved only a retrospective clinical notes review, and all data collected was analyzed anonymously.A retrospective case review was conducted in two Singapore hospitals: Singapore General Hospital, a 1700-bed public hospital and Mount Elizabeth Hospital, a 272-bed private hospital. All adults admitted from 1iACT service, the service was introduced to the Infectious Diseases (ID) physicians in the hospitals, to increase awareness of and provide education about the service. Prior to request of the iACT service for a patient with an infection caused by an XDR- or PDR-GNB isolate, the attending ID physician informed each patient (or immediate family member if the patient was incapacitated) of (i) the poor prognostic outcomes of such resistant infections which most or all antibiotics were not effective against, (ii) the nature of the in vitro testing method as a last-resort measure to guide treatment, (iii) alternative treatment strategies , iv) the clinical aims of the iACT guided treatment, and (v) the potential toxicities of the treatment. Verbal consent was sought and documented in clinical notes by the attending ID physician before iACT was requested.Before the implementation of the iACT service is detailed in In vitro combination testing was carried out immediately upon request of iACT for an individual patient. Simultaneously, a clinical ID pharmacist would review the patient’s clinical records and document all relevant clinical information required to develop rationally optimized combination dosage regimens, based on pharmacokinetics (PK) and pharmacodynamics (PD) principles. At 24h, combinations that were at least bacteriostatic (non-cloudy wells) in vitro were identified and fed back as preliminary results to the ID pharmacist. Preliminary recommendations to change antibiotic combinations were made by the ID pharmacist to the attending ID physician, if the initial empiric regimen was not at least bacteriostatic in vitro. At 48h, combinations with bactericidal activity in vitro were elucidated. Based on these in vitro results, the ID pharmacist would select and recommend the most optimal combination(s), with corresponding dosage regimens, to the attending ID physician. Selection was based on the patient’s clinical characteristics, site of infection, PK/PD parameter for efficacy of each antibiotic and the probabilities of PK/PD target attainment at infected sites. Clinical ID pharmacist followed-up and reviewed the patient’s case notes within 24h post-recommendation, to ensure that the patient was placed on the recommended antibiotic combination regimens.The process of the A. baumannii isolates were screened for blaOXA-23-like, blaOXA-24-like, blaOXA-51-like, and blaOXA-58-like genes using a multiplex PCR assay [P. aeruginosa and K. pneumoniae isolates, PCR was employed to detect genes encoding commonly acquired metallo-β-lactamases (MBLs)  [K. pneumoniae [K. pneumoniae using RT-PCR, and presence of efflux pump was determined using efflux pump inhibitor phenyl-arginine-β-naphthylamide (PABN) [Genus identity was determined using VITEK GNI+ cards . MICs to various antibiotics were determined using commercial microbroth dilution panels , and susceptibility was defined as per CLSI . All A. CR assay . For P.  blaSPM) , 11. Anaeumoniae , 11. Chae (PABN) .In vitro combination testing was carried out in clear 96-well microtiter plates, modified from a method by Aaron et al [5 CFU/mL (1 × 105–5 × 105 CFU/mL) of bacteria in each well. The wells were covered and incubated at 35°C for 24h. At 24h, bacteriostatic combinations were identified based on presence/absence of visible growth in each well. The contents of each well were then sampled and measured to ensure <10% loss in volume. The measured contents were centrifuged at 10,000 × g for 15 minutes and the pellet reconstituted to original volume to minimize drug carry-over. Bacterial count was quantified by depositing serial ten-fold dilutions of the sample onto Mueller-Hinton agar plates , incubated at 35°C for 24h, and enumerated visually to determine presence of bactericidal activity. The lower limit of detection was 3.9 × 102 CFU/ml.on et al . Brieflyon et al –25. Thisin vitro susceptibility data and concurrent infections) and antibiotic use (type and duration of antibiotics used) was also collected. The following roles of the iACT service were documented: (i) confirmation of bactericidal activity in the empirically selected regimen  (ii) change in empiric therapy, based on iACT results, or (iii) reduction of number of antibiotics empirically employed in combination, based on iACT results. If the iACT service recommended changes in an empiric therapy, the reasons for the recommendations were investigated. The time from culture isolation to request for iACT service, as well time from request for iACT service to time of provision of preliminary and final recommendations by the iACT service for each case was also documented.All data were extracted from patients’ inpatient charts and electronic medical records and documented in a structured form. Baseline characteristics collected included basic demographics, length of stay, presence of co-morbidities, APACHE II score at time of XDR-GNB isolation, history of intensive care unit stay or hospital stay within 3 months of XDR-/PDR-GNB isolation, and history of antibiotic use within 3 months of XDR-/PDR-GNB isolation. Details pertaining to the infection  30-day in-hospital all-cause and infection-related mortality, (ii) clinical response, and (iii) microbiological eradication (assessed only in patients with bloodstream infections as repeat cultures were not routinely taken for other sites). Adverse drugs events potentially attributable to iACT-guided therapy, selected by the attending ID physician based on individual preference, anecdotal experience or recommendations from published literature. Infections were defined in accordance to definitions provided by the Centers for Disease Control and Prevention (CDC) [3) or transplant patients on immunosuppressive agents.XDR and PDR were defined as non-susceptibility to at least one agent in all but two or fewer antibiotic categories and non-susceptibility to all agents in all antimicrobial categories respectively . Bacterion (CDC) . PatientThirty-day in-hospital all-cause and infection-related mortality was defined as all-cause and infection-related death, as determined by the ID physician, that occurred 30 days from isolation of XDR-/PDR-GNB. Clinical response was defined as partial or complete resolution of infection symptoms as determined by the ID physician, irrespective of GNB eradication, while therapeutic failure was defined as worsening at any time or no improvement of clinical conditions by day 14 of therapy as stated by the ID physician. Microbiologic eradication was only assessed for bloodstream infections, and was defined as the clearance of XDR-/PDR-GNB in all follow-up blood cultures. Nephrotoxicity was defined as per RIFLE criteria .Categorical data were presented as numbers and percentages; continuous data were presented as mean ± standard deviation, or median (range), depending on the validity of normality assumption. All data were analyzed using IBM SPSS Statistics 21 .iACT-guided therapy. Of these, three patients were excluded as medical records were not available. Two patients developed two episodes of XDR-/PDR-GNB infection more than 90 days apart and were each analyzed as two different cases. Hence, a total of 39 cases were included.From April 2009 to June 2014, 40 patients received The clinical features of the patients were summarized in in vitro susceptibilities of the pathogens. Of the 39 isolates , 31 (79.5%) were XDR and 8 (20.5%) were PDR . Of the 31 XDR isolates, 19 (61.3%), were sensitive to polymyxin B only. All A. baumannii harbored blaOXA23 and blaOXA51 carbapenemase genes, while 60% (12/20) of the P. aeruginosa isolates harbored genes encoding MBLs . A large variety of mechanisms mediating carbapenem resistance was observed for K. pneumoniae isolates. Amongst the six K. pneumoniae isolates, four (66.7%) K. pneumoniae isolates harbored both genes encoding NDM as well as OXA-181 carbapenemases. Of these, one isolate also demonstrated reduction in porin gene expression, while another had presence of efflux pump as determined by phenotypic methods.iACT service  was observed. This delay was attributed to the fact that several ID physicians elected to use empirically selected combinations first upon culture isolation, and only requested for iACT service after the patients failed to respond to the empirically selected combinations. Upon request and isolate receipt, the iACT service provided all preliminary results (based on well turbidity) to the ID physicians within 24h. All final iACT recommendations were provided to the ID physicians within 48h.A delay between the time of culture isolation and request for iACT-guided therapy, most cases were empirically prescribed combination therapy   cases , or that a more appropriate combination regimen, based on the probabilities of PK/PD target attainment at infected sites, could be used . In 7 (17.9%) patients, the number of antibiotics used in combination empirically was reduced. The iACT service recommended dose changes in 8 (20.5%) patients; most recommendations involved increasing doses or prolonging infusion time to optimize probability of PK/PD target attainment.Prior to , 89.7%) . More th%) cases –the commiACT-guided therapy prescribed . Nebulized colistin was initiated in all patients with pneumonia who were prescribed intravenous polymyxin B . The most common combination recommended against A. baumannii infections were polymyxin B plus a carbapenem . Against the P. aeruginosa isolates, polymyxin B plus carbapenem plus aminoglycosides were most commonly recommended . Antibiotic combination regimens recommended for K. pneumoniae infections were highly strain-specific; no single regimen was universally effective against all K. pneumoniae strains.Polymyxin B-based two- or three-drug combinations were the commonest iACT-guided therapy (APACHE II score at time of iACT-guided therapy ranged from 17–30 in these patients). For the remaining patient, the initial episode of ventilator-associated pneumonia resolved upon iACT-guided therapy; however, the patient developed a new episode of sepsis within 30 days and died. Clinical response (cure or improvement) was observed in 32 (82.1%) patients. Of note, clinical response was observed for all seven patients in whom the number of antibiotics used in combination was reduced. Out of the 14 patients with bloodstream infections, 11 (78.6%) had documented microbiological eradication. Thirteen (33.3%) patients developed adverse drugs events: 10 (25.6%) developed nephrotoxicity likely secondary to polymyxin B (n = 8) or polymyxin B plus amikacin (n = 2), two (5.1%) developed non-Clostridium difficile-associated-diarrhea, and one (2.6%) developed confusion secondary to levofloxacin. No death related to the adverse events was observed. Adverse events resolved in all patients who survived .The outcomes according to infection type and responsible pathogen are detailed in in vitro combination testing service in our local setting. We found that the iACT service could provide individualized recommendations for the most optimal combination within a rapid turn-around time of 48h and provided an attractive alternative to selecting antibiotic combinations merely based on individual physician’s anecdotal experience. In addition, we observed promising rates of clinical response and infection-related mortality in patients prescribed iACT-guided combination therapy.This study explored the role and feasibility of a prospective The emergence of XDR-GNB worldwide and in Singapore has caused several paradigm shifts in antibiotic therapy . In addiiACT service was developed to guide physicians in managing patients with XDR-/PDR-GNB infections. The service employed a bench-to-bedside approach to design rationally optimized and individualized antibiotic combination regimens based on PK and PD principles. An in vitro combination testing method, designed to systematically test 80 two- and three-drug combinations within 48h, was first employed to prospectively determine the bactericidal activity of antibiotic combinations in vitro against individual strains [in vitro results, clinical ID pharmacists then selected the most appropriate antibiotic combination, taking into consideration all relevant PK parameters, such as the patient’s comorbidity status, patient’s renal and hepatic function and distribution of the antibiotics to the site of infection. Recommendations made by the ID pharmacists do not merely consist of specific antibiotic combinations; instead, individualized dosage regimens , optimized to achieve PK/PD targets at the site of infection, were recommended to best maximize efficacy and decrease risk of resistance emergence.The prospective  strains . LeveragiACT-guided therapy, selected based on previously published literature or based on individual physicians’ past experience [in vitro, highlighting the inadequacy of selecting combinations based on anecdotal experience in our local setting. Considerable delay was noted between isolation of XDR-GNB and request for iACT service. This is mainly attributable to the fact that the iACT service was not mandatory; as such, several physicians opted to request for iACT service only after patients failed to respond to the initial empiric combination. This can result in the potential benefits conferred by the iACT-guided combination therapy being negated by the delay in time to appropriate therapy, especially in the critically-ill patients. Moving forward, continued education will be required to reinforce the importance of early referral to the iACT service, to ensure the timely prescription of the most appropriate antibiotic combinations.In this study, we observed that most physicians empirically prescribed combination therapy prior to perience , 29. UnfiACT service employed an in vitro combination testing method to guide the selection of antibiotic combinations against each XDR-GNB strain. This can reveal any increased or decreased killing brought about by potential synergy (e.g. mechanistic or subpopulation synergy) or antagonism respectively when two or more antibiotics are combined [iACT service potentially provided other functionalities. In 20% of the patients, the iACT service reduced the number of antibiotics initially used in combination. This suggests that the iACT service plays a role in antimicrobial stewardship, reducing unnecessary or indiscreet antibiotic use.Our combined . In addiet al, all-cause in-hospital mortality was 47% in patients infected with carbapenem-resistant GNB and treated with polymyxin B-based combinations [et al [in vitro combination testing results as per our study. More than 80% of patients receiving iACT guided combination therapy had documented clinical response in our study. Of note, clinical response was observed for all patients, in whom the number of combination antibiotics used empirically was reduced, suggesting that reducing the number of antibiotics in combination based on iACT did not compromise patients’ outcomes.Overall, we observed an all-cause mortality rate of 21% in our study patients, which compared favorably to mortality rates reported in other studies. In a study by Crusio inations . Similars [et al . We acknet al, a break-point checkerboard plate technique was employed to guide combination selection against multi-drug resistant P. aeruginosa [in vitro testing, and did not take into account patient’s PK/PD parameters. Furthermore, the study only reported patients with clinical improvement. In contrast, our study assessed all patients whose antibiotic regimen was prospectively guided by iACT service; in addition to in vitro testing, PK/PD principles were applied as the basis for rational selection of antibiotic combinations and dosing regimens.To date, only one study explored the use of prospective combination testing to guide therapy in non-cystic fibrosis patients. In a case series by Nakamura ruginosa . The stuiACT service on patient outcomes. Moving forward, a randomized trial will be required to fully evaluate the utility of the service in improving patient outcomes. In addition, given that the iACT service was not mandatory, physicians may be inclined to request iACT service only for certain subset of patients with XDR-GNB infections , which can result in selection bias.Despite our promising findings, this study had several limitations. While we observed promising rates of clinical success and infection-related mortality in our study, we acknowledged that the study was not designed to examine outcomes, as no control group was employed for outcome comparison. Hence, no conclusion could be made with regards to the impact of the iACT service can be feasibly implemented to guide the timely selection of rationally optimized combination regimens against XDRGNB, and played an additional role in reducing indiscreet antibiotic use. While further randomized trials will be required to fully evaluate the utility of the service, it is hoped that the prospective iACT service can be continually employed to guide the rational selection of antibiotic combinations, and to reduce indiscreet use of antibiotic combinations.We found that the prospective


Verminephrobacter and less well-known Flexibacter-like bacteria. Verminephrobacter are present in almost all lumbricid earthworms, they are species-specific, vertically transmitted, and have presumably been associated with their hosts since the origin of lumbricids. Flexibacter-like symbionts have been reported from about half the investigated earthworms; they are also vertically transmitted. To investigate the evolution of this tri-partite symbiosis, phylogenies for 18 lumbricid earthworm species were constructed based on two mitochondrial genes, NADH dehydrogenase subunit 2 (ND2) and cytochrome c oxidase subunit I (COI), and compared to their symbiont phylogenies based on RNA polymerase subunit B (rpoB) and 16S rRNA genes. The two nephridial symbionts showed markedly different evolutionary histories with their hosts. For Verminephrobacter, clear signs of long-term host-symbiont co-evolution with rare host switching events confirmed its ancient association with lumbricid earthworms, likely dating back to their last common ancestor about 100 million years (MY) ago. In contrast, phylogenies for the Flexibacter-like symbionts suggested an ability to switch to new hosts, to which they adapted and subsequently became species-specific. Putative co-speciation events were only observed with closely related host species; on that basis, this secondary symbiosis was estimated to be minimum 45 MY old. Based on the monophyletic clustering of the Flexibacter-like symbionts, the low 16S rRNA gene sequence similarity to the nearest described species (<92%) and environmental sequences (<94.2%), and the specific habitat in the earthworm nephridia, we propose a new candidate genus for this group, Candidatus Nephrothrix.Nephridial (excretory organ) symbionts are widespread in lumbricid earthworms and the complexity of the nephridial symbiont communities varies greatly between earthworm species. The two most common symbionts are the well-described  Verminephrobacter, while most lumbricids have mixed nephridial communities , which was first discovered in the compost worm Eisenia fetida and shown to be vertically transmitted via the cocoon, together with Verminephrobacter and a member of the Microbacteriaceae  to investigate the putative host specificity of Flexibacter-like symbionts in more detail, and (ii) to elucidate the evolution of the tripartite symbiosis between earthworms, Verminephrobacter, and Flexibacter-like symbionts.Based on their presence in multiple earthworm species, putative host specificity , the gut-free body wall with attached nephridia was cut into smaller pieces (1–3 cm), fixed in 4% (w/v) paraformaldehyde for 2–3 h, washed twice in 1 × phosphate buffered saline (PBS), and stored in 70% ethanol/PBS at −20°C.A total of 18 earthworm species, 1–16 individuals of each , targeting all Flexibacter-like symbionts, and EUB338,-II,-III-FAM  for 10 min on ice, rinsed thoroughly with water, and immersed in a 3:1 mixture of Vectashield  and Citifluor . Single nephridia were dissected from the body wall and mounted on a glass slide prior to epifluorescence microscopy. Images were captured on an Axio Vert 200M epifluorescent light microscope, fitted with an Apotome for optical sectioning and an AxioCam MRm camera, all controlled by the AxioVision software .The localization of 2, 0.2 mM of each dNTP, 0.1 units/μL VWR Taq polymerase, inert red dye, and 0.2 μM of each primer. Primers and thermal cycling protocols are summarized in Supplementary Table DNA was extracted from the dissected nephridia using the DNeasy Blood & Tissue kit  following the protocol for animal tissue. DNA extracts were used for PCR amplification of both host and symbiont genes. All PCR reactions were run with the Taq master mix  containing 1.5 mM MgClTwo mitochondrially encoded genes, NADH dehydrogenase subunit II (ND2) and cytochrome c oxidase subunit I (COI), were targeted. These host genes are located adjacent to each other on the mitochondrial chromosome and were therefore amplified as one long fragment (ND2-COI) using the newly designed primers Lum-ND2-322F or Lum-ND2-370F, and Lum-COI-723R. Some COI sequences had been obtained in a previous study, in which cases only ND2 was amplified using the reverse primer Lum-COI-14R  and Flexibacter-like symbionts . rpoB was amplified, cloned using pGEM®-T easy vector system (Promega™) or TOPO® TA Cloning kit (Invitrogen™), and sequenced at GATC (Germany).Earthworms were screened for the presence of All obtained sequences have been submitted to GenBank under accession numbers KP420532-KP420720 and KM058238-KM058572   and substitution models were chosen according to jModelTest. Majority rule consensus trees were calculated, based on two simultaneous runs with four chains each, and a sample frequency of 500. After 2,000,000 generations the PSRF showed 1.000 indicating stationarity  and vegan  and the corresponding symbiont rpoB distance matrices (host vs. FrpoB and host vs. VrpoB). For constructing the distance matrices highly similar sequences were grouped following the terminal nodes in Figure Verminephrobacter and Flexibacter-like bacteria was confirmed in Al. chlorotica and E. andrei . In E. andrei the Verminephrobacter lines the lumen wall, as typically found in other earthworm species  and two Pontoscolecidae (Andiorrhinus sp. and Urobenus brasiliensis)  toward the basal nodes of the tree  between host and Verminephrobacter genetic distances based on ND2-COI and rpoB, respectively.All of the resolved groups in the earthworm phylogeny are reflected in the  Figures . As for  Figures , 4B. TherpoB-based tree of Flexibacter-like symbionts it is not possible to evaluate the degree of congruency between the two , the Flexibacter-like symbionts, Verminephrobacter, and the hosts, all have the same clustering of Al. chlorotica and Ap. icterica  between host and Flexibacter-like genetic distances based on ND2-COI and rpoB, respectively. Meanwhile, there are clear signs of host switching in the three worm species all having two distinct Flexibacter-like symbiont groups  to other known 16S rRNA gene sequences, combined with their unique habitat in earthworm nephridia and their conspicuous morphology, supports the establishment of a novel genus. In spite of numerous attempts, the symbionts could not be cultivated yet ; they can, however, be easily identified in situ by the newly developed FISH probe  hair], which reflects the specific habitat (earthworm nephridia) and the slender, hair-like morphology of these symbionts. As a first representative of the candidate genus, we suggest the specific symbiont of Eisenia fetida to be named “Ca. N. davidsonii” in honor of Seana Davidson, the scientist who first discovered the Flexibacter-like symbionts in earthworms.For the monophyletic Ca. Nephrothrix is an ancient co-evolving symbiont of earthworms due to the poor basal resolution of both phylogenies  Ca. Nephrothrix possibly co-diversified with its host  two distinct Ca. Nephrothrix rpoB variants are found  and tha Figures . PotentiVerminephrobacter (Allolobophora dacia) happened 95 MYA  , while Eisenia lucens diverged from E. fetida/E. andrei about 45 MYA . Thus, Ca. Nephrothrix has likely been associated with the Lumbricidae for a minimum of 45 MY. Furthermore, the intragroup genetic distance in the 16S rRNA gene in Verminephrobacter (7.3%) is higher than in Ca. Nephrothrix (4.8%), supporting that the genus Verminephrobacter is older than Ca. Nephrothrix.The age of the two symbiont groups can be estimated by comparison with the age of the earthworm hosts. In a recently published Lumbricidae phylogeny, the family was estimated to originate during the Cretaceous about 110 MYA  and the split to the most basal worm species with z et al. , O. tyrt, Verminephrobacter and Ca. Nephrothrix, the two bacterial partners have followed markedly different evolutionary trajectories. Symbionts from the novel candidate genus, Ca. Nephrothrix, are found in approximately half of all lumbricid earthworm species, they have apparently switched hosts, and subsequently adapted and become species-specific. In contrast, Verminephrobacter show signs of long-term cospeciation with their host, they are present in almost all lumbricid earthworms, and have rare host switching events. Verminephrobacter is thus likely to have originated in the last common ancestor of lumbricid earthworms 100 MYA (Lund et al., Ca. Nephrothrix is much younger with an estimated age of ≥45 MY. This scenario still allows for co-occurrence of the two symbionts in a range of earthworm species for at least 45 MY, and thus plenty of opportunity for interspecies genetic exchange as predicted by Kjeldsen et al. (Verminephrobacter genome evolution. In support of this possibility, natural competence and transformation of Verminephrobacter within earthworm cocoons has recently been demonstrated (Davidson et al., Ca. Nephrothrix has left its genetic trace in Verminephrobacter and vice versa remains to be shown.In the tripartite symbiosis between earthwormsn et al.  to explaThe 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.


Government-led pollution prevention programs tend to focus on large businesses due to their potential to pollute larger quantities, therefore leaving a gap in programs targeting small and home-based businesses. In light of this gap, we set out to determine if a voluntary, peer education approach led by female, Hispanic community health workers (promotoras) can influence small and home-based businesses to implement pollution prevention strategies on-site. This paper describes a partnership between promotoras from a non-profit organization and researchers from a university working together to reach these businesses in a predominately Hispanic area of Tucson, Arizona. From 2008 to 2011, the promotora-led pollution prevention program reached a total of 640 small and home-based businesses. Program activities include technical trainings for promotoras and businesses, generation of culturally and language appropriate educational materials, and face-to-face peer education via multiple on-site visits. To determine the overall effectiveness of the program, surveys were used to measure best practices implemented on-site, perceptions towards pollution prevention, and overall satisfaction with the industry-specific trainings. This paper demonstrates that promotoras can promote the implementation of pollution prevention best practices by Hispanic small and home-based businesses considered “hard-to-reach” by government-led programs. The United States Environmental Protection Agency (US EPA) defines pollution prevention P2) as, “reducing or eliminating waste at the source, promoting the use of non-toxic or less-toxic substances, implementing conservation techniques, and re-using materials rather than putting them into the waste stream” . Formal  as, “redThe unique features of small and home-based businesses need to be considered when crafting P2 interventions to better target these businesses. Small businesses account for 99% of the total employer firms in the USA . Small bLow-wage workers typical of small and home-based businesses are disproportionately foreign-born, Hispanic, and women . Furtheretc.) [Given the challenges stated, P2 strategies need to be developed and customized for these types of businesses. We set out to determine whether a voluntary, peer education approach led by female, Hispanic community health workers (promotoras) can influence small and home-based businesses to change behaviors and apply P2 strategies on-site. In the literature, P2 outreach programs that target businesses are typically government agencies  or university groups that communicate P2 information unilaterally . Some P2etc.) ,15,16. Getc.)  suggest etc.)  determinetc.) , comprehBarriers in the implementation of best practices for long-term sustainability of P2 programs are important to study. These barriers highlight the potential pitfalls that can be considered when implementing a new P2 program. Identified barriers include declining public support, competing business priorities, and difficulty in documenting P2 progress . The lasi.e., politics, ethnicity, language, culture, and economics) partly determines how health disparities develop and how solutions can be applied [What is missing from traditional P2 approaches is a clear understanding of the social constructs in which small and home-based businesses reside. The eco-social context needs to be considered to implement effective P2 measures with long-term effects. A community’s ecology  grant, promotoras working for the Sonora Environmental Research Institute, Inc.  received complaints from community members about unusual chemical odors in their neighborhoods during promotora routine home visits. As part of this CARE grant, SERI developed a community-mapping project to identify high-risk areas based on environmental hazards by having promotoras walk neighborhoods and georeference tag areas of concern using global positioning system units. The maps developed showed the large number of small and home-based businesses in and around the neighborhoods and overlapped this information with the potential hazards based on the chemicals used at the businesses. Subsequent business interviews conducted by promotoras indicated a shortage of language and culturally appropriate information, lack of trust in governmental enforcement agencies, and perceived high cost of “becoming green” as barriers to implement P2 activities . Based oThis initial work pinpointed a community need, but for the program to become more formalized, promotoras needed technical trainings. To address this educational need, new collaborations were formed with academic and government organizations to move the program forward. The resulting multidisciplinary collaboration of academic, industry, and government environmental programs became an essential component of the promotora-led P2 program. Each collaborator’s contribution is reported in detail in the Experimental Section . This prIn Tucson there are an estimated 37,000 registered businesses of which 85%–90% are considered to be small businesses and many of which are Hispanic owned . The proThe promotora-led P2 program consisted of diverse activities that specifically included promotora-focused P2 technical trainings, P2 industry-specific workshops, P2 educational materials tailored to business and language, and face-to-face, peer education visits to small and home-based business. In addition, P2 outcomes and perceptions that were derived from the activities were captured via survey instruments . The University of Arizona (UA) Human Subjects Protection Program determined that this program is exempt due to the use of the data for evaluation and improvement purposes.The P2 technical trainings were developed to be accessible by promotoras and provided real-world applications (connecting basic science with action-oriented examples that could be easily translated to the targeted businesses). They consisted of a formal classroom-style lecture (one to two hours), hands-on activities, question and answer period (15 min), and business tour. Content experts carried out the trainings and all trainings were provided in Spanish (simultaneous translation into Spanish was provided if needed). SERI collaborated with UA’s Department of Atmospheric Sciences, Superfund Research Program (SRP), Dean Carter Binational Center for Environmental Health Sciences , and Arizona Water Institute to strengthen technical P2 trainings for promotoras. Government-based environmental and conservation collaborators housed in Pima County  and the City of Tucson (Tucson Water and Tucson Fire Department) provided additional technical trainings to promotoras as well as literature on existing small business assistance programs. This multidisciplinary component of the program worked to leverage P2 resources, expand the expert pool, and increase the knowledge base of promotoras. No evaluation tools were implemented to assess promotora knowledge gained or readiness.SERI promotoras worked closely with UA SRP and Binational Center to coordinate industry specific workshops for small and home-based businesses. Meetings were coordinated with industry-specific P2 experts to discuss the informational needs and accepted best practices of specific industries. Partnerships were developed with the Automotive Services Association, Printing Industry Association of Arizona, Arizona Lithographers, The Source Beauty Salon and Spa, The Green Edge Group, and Pure Aesthetics Natural Skincare School.At each industry-specific workshop, P2 technical advice, environmentally preferable product samples, and P2 informational packets were distributed. In general, an industry representative involved in either implementing P2 at his/her business or promoting P2 through a professional association delivered the majority of the technical advice. Bilingual (English and Spanish) P2 packets provided suggestions for reducing business waste, factsheets on industry-specific hazardous chemicals, and information on local P2 assistance services. Much of the outreach information was readily available from the US EPA Design for the Environment program, Arizona Department of Environmental Quality’s P2 for Automotive Maintenance and Repair Shops manuals, Occupational Safety and Health Administration safety information, and California Department of Toxic Substances Control P2 materials. This information was not always available in Spanish and lacked cultural nuances. The program translator not only worked with promotoras to develop culturally appropriate linguistic translations, but also had technical expertise that allowed for appropriate science translation. In addition, simultaneous translation into Spanish was provided at each industry-specific workshop.UA SRP and Binational Center partners developed a brief survey to apply at the industry-specific workshops. It was comprised of eight questions consisting of four-point Likert scale, three-point Likert scale, and open-ended. The purpose of this survey was to understand the workshop benefits, potential connections, practical application, and content satisfaction. Surveys that were not completed were not included in the analysis.SERI promotoras partnered with UA SRP and Binational Center to implement small and home-based businesses visits. Technically trained promotoras were the leads during these P2 peer education visits. Promotoras began each business visit by introducing themselves, providing the project goal, and discussing general concepts of P2. They used a survey to collect business information and observations . The survey also allowed promotoras to document their observations of the businesses as well as highlights from their conversations.The follow-up visit survey was structured to gauge the business’s progress towards implementing P2 practices, plans, and policies, obtaining measureable emission reductions, and utilizing the industry-specific packets and/or samples distributed during the initial visit. This follow-up survey was designed similar to the initial business visit survey, containing questions in the form of a script and checklist (yes or no). When an in-person follow-up visit was not feasible, the promotoras called the business and conducted the survey over the telephone.During follow-up business visits, behavior changes were self-reported and used to determine emission reductions. The emission reduction calculations for nail salons were based on average use of acetone per station. The calculations for solvent degreasers at auto repair shops were based on estimates for uncontrolled organic emissions from cold cleaner units.i.e., an established procedure for a given operation, can reduce emissions by 15%–45% [Control devices (methods and procedures used to reduce or prevent emission of pollutant) can reduce emissions 13%–38%, while operational procedures,  15%–45% . Reducti 15%–45% . A conseWe set out to determine whether the promotora-led P2 program, a voluntary peer education approach, would influence Hispanic small and home-based businesses to change behaviors and apply P2 strategies on-site. Survey instruments applied to both the small and home-based business visits and industry specific workshops allowed for analysis of outcomes. The results have been divided into the P2 activities mentioned A total of 17 SERI promotoras participated in basic P2 trainings for the auto repair, printing, and nail salon industries. Fifteen additional technical trainings were designed for these promotoras that included environmental exposure, contaminant transport of industrial solvents, air quality, water conservation, and business-specific P2 best practices .The promotora-led P2 program completed a total of 11 industry-specific workshops for auto repair, hair and nail salons, and printing and lithography. A total of 313 business representatives participated in these workshops of which 74 requested on-site visits. Eight industry-specific packets were developed that targeted auto repair, hair salons, woodworking, business offices, nail salons, dry cleaning, print shops, and auto body and paint shops.Industry-specific P2 workshop surveys were provided at both the printing and lithography (28 participants) and hair and nail salon (17 participants) workshops. Survey responses highlighted that participants were satisfied with the workshops and that the P2 best practice information provided was relevant to their business. Of the 17 attendees of the hair and nail salon workshop, 11 stated that they were going to switch to ammonia-based hair dye and all printing and lithography workshop attendees reported that they would implement P2 suggestions. Information on whether these businesses actually implemented P2 strategy was not collected.Of the 17 technically trained promotoras, 10 of them conducted business visits and became actively involved in the P2 program. These participating promotoras engaged a total of 640 different small and home-based businesses in face-to-face peer education through initial visits. Of the 640 businesses, 105 were initially contacted under the CARE grant. After promotoras received technical training under this P2 program, they revisited these 105 businesses to share their newly acquired P2 technical knowledge.In addition to the initial business visits, 72 businesses received follow-up visits and 493 received follow-up telephone calls. As already mentioned, eight industry-specific packets were developed for the targeted businesses. A total of 662 of these packets were distributed at either initial or follow-up visits. All businesses received an industry-specific packet during the initial visit, and 22 businesses requested an additional packet during the follow-up visit. Of the total businesses, fewer than 20 were determined to be home-based businesses. Although the number is low, interacting with home-based businesses is noteworthy and was not originally anticipated as a business type. The businesses that participated in the survey identified themselves as managers (52%), owners (32%), or employee/receptionist/worker (14%). Two percent did not identify themselves at all. All businesses that received an initial visit completed the survey. The initial business visit survey revealed that 90% of the businesses were willing to take part in the voluntary promotora-led P2 program. Also, the initial business visit survey identified preferred incentives that would entice them to implement P2 best practices .Qualitative observations were also made during initial business visits. Promotoras detected that businesses did not want to participate in workshops because of the perceived lack of time and value. To encourage participation, promotoras personally visited businesses and invited them to events. Events were scheduled at different times throughout the day, and when possible, events coincided with new air quality requirements. Most small businesses believed that it took money to implement P2 measures and requested grants to purchase equipment. We emphasized low-cost methods that any business could implement and distributed information that demonstrated the cost savings over time; however, we found it difficult to convince many small businesses to purchase new equipment based solely on P2. Promotoras observed that many business owners and managers did not see the value in taking time to develop new plans or polices, as they were busy completing day-to-day operations and in general did not have business policies. As part of the program, examples of business plans were provided that could be easily modify for their facilities.During follow-up business visits, promotoras documented using the survey  any reported or observed P2 changes. When it came to actually actively implementing any P2 practices suggested, 288 (45%) of the total businesses accomplished this goal. These practices ranged from recycling to substitution of products and were variable from business to business. The follow-up business visit survey was used to determine reported emission reductions using recommended best practices provided to the business via the program . A totalFor automotive repair and paint and body shops, 203 businesses reported they began covering solvent degreaser containers when not in use. Covering a cold solvent degreaser and associated drainage facility reduces emissions between 13% and 38% . Using tThe promotora-led P2 program reached 640 small and home-based businesses in Tucson and 288 of these businesses reported implementing some kind of P2 practice that they had learned about through the program. Promotoras who are technically trained can influence small and home-based businesses’ environmental operation decisions. In this program, using the right culture and language, small and home-based businesses were encouraged to participate in the program and as a result became aware of the opportunities that P2 could offer. Through the promotora-led P2 program, businesses trusted both the messenger and the information, making them more amenable to suggested P2 practices. Since the information was “translated” with the community’s ecology in mind, the businesses could both better understand the information and engage in P2 practices more readily. P2 programs in other settings can benefit by applying similar community ecology principles. If promotora groups have not been established, community leaders also known as “sparkplugs” or “champions” can provide the structure needed to develop such a program. The benefits of incorporating a community’s ecology, demonstrated in the Tucson promotora-led P2 program and in the Boston Safe Shops model, are apparent—trusted community members could work successfully with small businesses to promote P2.Program findings reinforce and echo previous P2 findings, specifically the importance of cost avoidance and profitability ,39. It wi.e., follow-up in-depth interviews and content analysis) to obtain information not previously anticipated or code interviews to derive additional conclusions. In addition, more detailed information could have been gathered pertaining to how the promotoras reduced specific barriers and what were the motives for adopting practices after taking part in the P2 program (long-term perspective).Surveys were the primary method of gathering program information and evaluating outcomes. As mentioned previously, these surveys helped gather the perception of participants in the initial and follow-up business visits and the industry-specific workshops. The usefulness of these surveys was limited since only prescribed information was obtained. The program evaluation could be improved by applying a mixed methods approach  can result in improved P2 outreach. Specifically, technically trained female, community health workers can be effective at convincing small and home-based businesses in applying P2 practices on-site and demonstrate that emission reductions can be achieved by low-cost interventions. This type of voluntary approach is complimentary to government-based programs, since it extends the reach of existing resources and infrastructure.We recommend that similar programs should be implemented to fill the gap left by large-scale government programs. This process described here to achieve P2 can assist in decreasing community-wide pollution emissions that are greatly amplified in minority neighborhoods. It is important to keep in mind that this type of intervention is only one piece of the diverse partnerships needed to implement a sustainable P2 culture.


As one of the most abundant agricultural wastes, sugarcane bagasse is largely under-exploited, but it possesses a great potential for the biofuel, fermentation, and cellulosic biorefinery industries. It also provides a unique ecological niche, as the microbes in this lignocellulose-rich environment thrive in relatively high temperatures (50°C) with varying microenvironments of aerobic surface to anoxic interior. The microbial community in bagasse thus presents a good resource for the discovery and characterization of new biomass-degrading enzymes; however, it remains largely unexplored.We have constructed a fosmid library of sugarcane bagasse and obtained the largest bagasse metagenome to date. A taxonomic classification of the bagasse metagenome reviews the predominance of Proteobacteria, which are also found in high abundance in other aerobic environments. Based on the functional characterization of biomass-degrading enzymes, we have demonstrated that the bagasse microbial community benefits from a large repertoire of lignocellulolytic enzymes, which allows them to digest different components of lignocelluoses into single molecule sugars. Comparative genomic analyses with other lignocellulolytic and non-lignocellulolytic metagenomes show that microbial communities are taxonomically separable by their aerobic “open” or anoxic “closed” environments. Importantly, a functional analysis of lignocellulose-active genes (based on the CAZy classifications) reveals core enzymes highly conserved within the lignocellulolytic group, regardless of their taxonomic compositions. Cellulases, in particular, are markedly more pronounced compared to the non-lignocellulolytic group. In addition to the core enzymes, the bagasse fosmid library also contains some uniquely enriched glycoside hydrolases, as well as a large repertoire of the newly defined auxiliary activity proteins.Our study demonstrates a conservation and diversification of carbohydrate-active genes among diverse microbial species in different biomass-degrading niches, and signifies the importance of taking a global approach to functionally investigate a microbial community as a whole, as compared to focusing on individual organisms.The online version of this article (doi:10.1186/s13068-015-0200-8) contains supplementary material, which is available to authorized users. Lignocellulose is a basic constituent of plant biomass and represents one of the most abundant sources of renewable carbon in the biosphere. Its complex structure consists mainly of carbohydrate polymers: cellulose, hemicellulose, and lignin. In nature, the degradation of lignocellulose requires multiple enzymes produced by diverse microorganisms, which act corporately and attack the complex structure of lignocellulosic biomass ,2. The gAn industrial bagasse collection site at sugar mills represents a unique ecological niche for lignocellulose decomposition due to its high enrichment of lignocellulosic materials under high temperature and low nitrogen, with varying microenvironmental conditions, from the aerobic pile surface to the anoxic interior region. Recent studies have showed complexity in the bagasse microbial community and inherent metabolic potential in plant biomass decomposition, which provides novel genetic resources for biotechnological exploration ,8,9. HowComparative metagenomic studies have been used to investigate the microbial communities in different environments in terms of taxonomy, gene contents, and also biochemical and metabolic potentials -14. CultIn this study, a fosmid library of a microbial community extracted from industrial sugarcane bagasse was constructed and analyzed by shotgun pyrosequencing to characterize and catalog the biodiversity of the microbe community, as well as its lignocellulolytic enzyme potential in biomass decomposition. The metagenome sequenced from this bagasse fosmid library, called the bagasse metagenome herein, was analyzed and compared with several reported metagenomic datasets from both lignocellulolytic and non-lignocellulolytic ecological niches. As well as elucidating the taxonomic compositions of microorganisms in different metagenomes, we have identified the biomass-degrading genes conserved among different microbial communities and the unique genes that could be related to specific metagenomes, thus providing a basis for understanding the roles and interplays of different microbes and their enzymes in biomass degradation in different environments.We first constructed a fosmid library from the microbial DNA sequences obtained from the soil-contacting region of sugarcane bagasse collected from an industrial collection site (see Methods for more details). The fosmid clones were pooled and pyrosequenced on one full lane of a 454 Genome Sequencer FLX . Approximately one million raw reads were obtained, with an average read length of 570 bp Table . Low quaTo explore the phylogenetic diversity and complexity of the microbial community in the bagasse metagenome, we first identified taxonomic classification of singletons and contigs before removing duplicates, using BLASTN against the NCBI non-redundant nucleotide database (NT)  .Focusing on bacteria, the majority of reads were mapped to the Proteobacteria phylum , which is metabolically versatile and spanned a wide range of bacterial taxa capable of aerobic as well as fermentative anaerobic metabolisms. The predominance of Proteobacteria in the sample collected from the exterior of a bagasse pile is in line with our previous observation in tagged 16S rRNA of the bagasse samples . In our The next largest phyla are Bacteroidetes (10.2%) and Actinobacteria (7.9%), followed by relatively smaller amounts of DNA from Acidobacteria, Chloroflexi, and Firmicutes. Bacteroidetes are mostly anaerobic and are widely distributed in soil, sediment, aquatic habitats, and animal guts ,24-27. AWe then explored the repertoire of lignocellulose-degrading enzymes in the bagasse microbial community by assigning the predicted open reading frames (ORFs) with three carbohydrate-active enzyme families from the CAZy database : glycosiThe microbial community found in bagasse is capable of producing various types of enzymes required to convert cellulose, hemicellulose, and lignin into different types of monosaccharides that are essential energy sources for aerobic  as well as anaerobic bacteria (through fermentation processes). Of all the ORFs mapped to the GH families, 679 ORFs about 42%) are related to 27 GH families that have lignocellulose-degrading enzymatic activities  for hydrolysis of xylan, the most abundant hemicellulose in bagasse; endo-1,4-beta-mannosidase (GH26) for mannan; and endo-1,4-beta-galactosidase (GH16) for galactan. The genes encoding these enzymes are all present in the bagasse metagenome. The higher percentage of reads mapped to GH10 (2.6%) might reflect the requirement to digest the greater abundance of xylan, as compared to mannan  and galactan (1.5%). Further downstream, xylan is degraded by xylo-oligosaccharide hydrolyzing enzymes and side chain cleaving hydrolases such as beta-glucosidases and beta-xylosidases from multiple families, including GH3, 39, 43, and 52. The downstream decomposition of manno-oligosaccharides and their dimeric sugars is catalyzed by alpha-mannosidase (GH38), whereas the dimeric sugars from galactan are degraded by beta-galactosidases from GH2 and 35. We also observed a wide range of debranching enzymes such as alpha-L-arabinofuranosidase (GH62), alpha-glucuronidase (GH67), and alpha-L-rhamnosidase , reflecting corporate enzymatic functions in the degradation of hemicellulose.In addition to a variety of glycoside hydrolases that degrade cellulose and hemicellulose, we also observed a moderate fraction of ORFs mapped to carbohydrate-binding modules (CBMs), a group of non-catalytic proteins that promote the association of the enzymes and substrates. The majority of the CBM enzymes found in the metagenome were identified as CBM50 (24.2%) and CBM48 (17.4%), peptidoglycan-binding and glycogen-binding proteins, respectively, although they are not known to be directly related to lignocellulosic hydrolysis. We also found a number of a recently defined CAZy class of lignin-degrading enzymes known as auxiliary activities (AAs), which contains eight families of ligninolytic enzymes and three families of lytic polysaccharide mono-oxygenases. The majority of lignin-breakdown enzymes found are multicopper oxidase  and choline dehydrogenase , followed by smaller amounts of AA4, 5, 6, 7, and 9.In terms of the microorganisms producing carbohydrate-degrading enzymes, our results show that heterogeneous hemicellulose and cellulose are degraded by specific endo-acting enzymes produced by all major bacterial phyla: Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria Figure , except Proteobacteria is the most abundant phylum in the sugarcane bagasse community; however, smaller phyla such as Actinobacteria, Bacteroidetes, and Firmicutes contribute as much to the production of lignocellulolytic enzymes for the microbial community. Bacteroidetes, for instance, is the second largest bacterial phylum in our bagasse metagenome based on the number of mapped reads 10.2%), but is still far behind the most abundant phylum, Proteobacteria (66.1%). Intriguingly, the phylum Bacteroidetes produces the largest repertoire of many carbohydrate-degrading enzymes, especially cellulases , oligosaccharide-degrading enzymes (GH2), and endo-hemicellulases   and Sequence Read Archive (SRA) projects , based oIn brief, bagasse pile, farm soil, and compost are classified as active lignocellulose-degrading environments, comprising both aerobic and anoxic regions ,32. DecoP-values < 2.2×10-16, Fisher’s exact test, unless indicated otherwise). This is still true even when compared with other lignocellulolytic datasets combined   to quantitatively assess the similarity between the presence/absence patterns of bacterial phyla in different environments Figure B left, aP < 10-40, Additional file To gain an overall picture of the functions of genes possessed by the 12 metagenomes, we first classified the assembled reads to different Clusters of Orthologous Groups (COGs)  , many metabolic COGs including the carbohydrate transport and metabolism [G] genes are most enriched in three lignocellulolytic environments: compost (577 unique genes), bagasse (541), and farm soil 319), suggesting a greater diversity of carbohydrate-active genes in these three metagenomes. However, one might consider that the numbers of total reads in these datasets are slightly larger than in other datasets  pathways  .As expected, lignocellulolytic metagenomes contain a greater number of unique GH genes with lignocellulose-degrading enzymatic activities Figure , red, asIn a similar manner to the majority of the GH families, CBMs  are evidently most enriched in the farm soil and compost metagenomes, with the exception of CBM48 and 50, which are highly present in all the environments analyzed  and metabolic genes (KEGG), the lignocellulolytic group possesses highly similar lignocellulose-degrading core genes, which are produced by different types and abundances of microbes within different lignocellulose-degrading communities. That is, even though the species compositions of lignocellulolytic metagenomes are no more similar than when they are compared across the two groups, their carbohydrate-active enzyme compositions are significantly more conserved than those in non-lignocellulolytic groups. This exemplifies an important interplay between diverse microorganisms in the communities that contribute to the enzyme repertoires required to degrade lignocelluloses under mixed microenvironmental conditions, in different ecological systems.The sugarcane bagasse sample was collected from soil-contacting regions of a bagasse pile (one meter from the edge of the pile) at Phu Khieo Bio-Energy Chaiyaphum province, Thailand . The bagasse piles are normally approximately 10 m in height covering an area of several acres. The sample was rapidly frozen in liquid nitrogen and kept at -80°C for subsequent experiments. Metagenomic DNA was directly extracted from a sample by the SDS-based DNA extraction procedure , with slEscherichia coli EPI300-T1R. The transformants were selected on LB agar plates supplemented with 12.5 μg/ml of chloramphenicol. The library was stored at -80°C in 15% glycerol in the form of individual clones as well as pool libraries.A metagenomic fosmid library from the bagasse sample was constructed using a CopyControl™ Fosmid Library Production Kit  according to the manufacturer’s instructions, with slight modifications. The purified DNA was end-repaired to generate blunt 5’-phospholyrated ends and then ligated to the pCC1FOS vector at 25°C for 3 h. The ligated DNA was packaged using the lambda packaging extract supplied and subsequently transformed into http://www.repeatmasker.org). The vector and host sequences were filtered by BLASTN, with an E-value cutoff of 1e-3. The filtered reads were assembled using the Newbler assembly software, developed by 454 Life Sciences . Non-overlapping fragment singletons were clustered using the CD-HIT software [A total of 3,300 randomly selected fosmid clones were sequenced on one full lane of the 454 GS-FLX Genome Sequencer System using the Titanium platform  following the manufacturer’s protocol. Repeats in raw sequenced reads obtained were removed using RepeatMasker  by MetaGeneMark . The ShaP-values from Fisher’s exact test and odds ratios were derived using the module SciPy in Python (http://www.scipy.org/).We compared our bagasse metagenome to publicly available metagenomic projects obtained from NCBI Whole Genome Shotgun (WGS) and Sequence Read Archive (SRA) projects . All add


Dendritic cell (DC)-based cancer vaccines may have a significant benefit to patients with advanced non-small cell lung cancers (NSCLCs). However, variations among clinical studies make it difficult to compare clinical outcomes. J-SICT DC vaccine study group is composed of the multi-medical centers in Japan, has provided DC vaccines as a compassionate use under the unified regimens for cell production and patient treatment, and published a number of clinical data of patients treated with DC vaccines. A single medical institution of this group has recently published a potentially beneficial effect of DC vaccines on overall survival of 62 patients with advanced NSCLCs. Here weOf 337 patients who met the inclusion criteria, 260 patients who received by weekly more than 5-times of peptide-pulsed DC vaccines were analyzed.st vaccine  in uni- and multivariate analyses. Importantly, the size of erythema reaction was significantly correlated with ECOG-PS, neutrophil/lymphocyte (N/L) ratio, hemoglobin, C-reactive protein, and fever (over 38 oC) at the time of leukapheresis, suggesting that the good physical condition as well as less inflammatory reaction might be important to induce stronger reaction against DC vaccines.No serious adverse event related to DC vaccination. The mean survival time from diagnosis was 33.0 months , and that from the first vaccination was 13.8 months . Similar to our previous findings obtained in the analysis for advanced pancreatic cancer[This is the first report of a multicenter clinical study suggesting the feasibility and possible clinical benefit of DC vaccines in patients with advanced NSCLCs. These findings suggest the potential responders against DC vaccines, and need to be addressed in well-controlled prospective randomized trials.


In the Netherlands, screening for tick-borne pathogens is performed using real time PCR on DNA extracted from ticks, followed by sequencing of positive samples, thereby disallowing identification in case of double-infections. Until recently, testing was performed with several reverse line blots (RLBs), each containing ~10 different probes. Advantages of RLBs are the capabilities to differentiate between bacterial species and to detect double infections, disadvantages are that RLBs are fastidious, elaborate, and time-consuming.The aim of this work was to 'test drive' bead-based suspension arrays for the rapid detection and identification of tick-borne Borrelia species. The platform used is the flow cytometry-based xMAP technology of Luminex, in which fluorescent nanoparticles (beads) are used to build multiplex assays simply by mixing different bead sets that are covered with specific probes. Each bead set contains a different ratio of red and infrared fluorophores, which enables identification of beads that pick up a third fluorophore by hybridization to a labelled target molecule.Using standard chemistry, aminated oligonucleotides were covalently linked to carboxylated Luminex beads, resulting in bead sets with general and specific probes for the 23S-5S intergenic spacer (IGS) of Borrelia species. Presence of probes on the beads was verified by hybridization of complementary biotinylated oligonucleotides in presence of a mixture of 1000 beads per bead set, using a 30 minute incubation at 37°C in a NaCl/Tris buffer, followed by labelling with streptavidin-phycoerythrin. Of each mixture, a minumum of 100 beads per bead set were investigated by flow cytometry on a dedicated Luminex instrument.~20% were found to carry one or more Borrelia species (resp. 15 and 5%). The most prevalent species was B. afzelii, followed by B. burgdorferi sensu stricto, B. garinii, B. sensu lato (unidentified) and B. valaisiana. These results show the viability of bead-based suspension arrays for (relatively) rapid detection and identification of multiple species, but at this stage need verification by a 'gold standard'. Currently, confirmation of these results is being performed by next generation sequencing.To evaluate the suspension array, DNA of nearly 1000 ticks was isolated and used to generate amplicons with a universal PCR targeting the 23S-5S IGS of Borrelia spp. All PCR reactions were then used in a direct hybridization assay in presence of a mixture of probe-containing beads. Out of 981 ticks, this work was partly funded by the European Union Seventh Framework Programme (FP7/2007-2013) under Grant Agreement Number 222633 (WildTech).


There are several factors, like environmental agents, neurotrophic factors,serotonin and some hormones such as estrogen, affecting neurogenesis and neural differentiation. Regarding to importance of proliferation and regeneration in central nervoussystem, and a progressive increase in neurodegenerative diseases, cell therapy is anattractive approach in neuroscience. The aim of the present study was to investigate theeffects of sex steroid hormones and basic fibroblast growth factor (bFGF) on neuronal differentiation of mouse bone marrow-derived mesenchymal stem cells (BM-MSCs). This experimental study was established in Kharazmi Univer-sity. BM was isolated from the bones of femur and tibia of 4-6-week old Naval MedicalResearch Institute (NMRI) mice, and the cells were cultured. The cells were divided intofollowing 4 groups based on the applied treatments: I. control (no treatment), II. steroidhormones , III. bFGF and IV. combination ofsteroid hormones and bFGF. Immunocytochemistry and flow cytometery analyses wereapplied for beta III-tubulin (β-III tubulin) and microtubule-associated proteins-2 (MAP-2) in4 days of treatment for all groups. The cells treated with combination of bFGF and steroid hormones representedmore expressions of neural markers as compared to control and to other two groupstreated with either bFGF or steroid hormones.This study showed that BM-MSCs can express specific neural markers afterreceiving bFGF pretreatment that was followed by sex steroid hormones treatment. Moreinvestigations are necessary to specify whether steroid hormones and bFGF can be considered for treatment of CNS diseases and disorders. Studies have shown that BM cells have potential to differentiate into myocytes, hepatocytes and glial cells. Researchers reported that BM-MSCs of humans and mice can be induced and differentiate into neural cells under culture conditions  are multipotent adult stem cells with mesodermal origin. MSCs can be isolated from bone marrow, being expanded and genetically modified in culture medium in order to be prepared as cellular carriers for local or systemic treatments. BM-MSCs are considered as a potent cell source for this purpose, since they have a high potency in self-renewal, multi-differentiation, and producing functional structures nditions ,2. MSCs produce both growth factors and important cytokines which can facilitate the regeneration of damaged tissues. Growth factors are mitogenic polypeptides which play important roles in proliferation and differentiation of neuronal and astroglial cells in culture medium . Steroid hormones actively take part in bone metabolism, and have multiple regulatory functions indifferent cells, such as BM-MSCs. Dexamethasone ( DEX ) and estradiol are neurosteroids which play regulatory roles in neural cell lines, specifically in astroglial types . For insEstrogens interfere in neuronal and astroglial differentiation and are key hormones in neurodegenerative events . Estrogein vivo , dimethyl sulfoxide ( DMSO ), 2-mercaptoethanol, 3-isobutyl-1-methylxanthine ( IBMX ) and 5-azacytidine have been used alone or in combination. In some of these studies, differentiation was initiated by a 1-day first step using basic fibroblast growth factor ( bFGF ) as a pre-inductive molecule that has been previously described to control neural stem cell ( NSC ) self-renewal and differentiation ,15. According to previous works, using bFGF as a pre-treatment agent seems to be proper for BMMSCs cultures. By adding this growth factor into culture medium, neurite-like extensions are recognizable after 4 days and fully developed after 7 days. However, from day 7, cells readopt a fibroblastoid shape, demonstrating the fact that these conditions are not able to make long-term differentiation, but the idea of using some growth factors as pre-treatments for researchers seems to be reasonable ,15. Ther2. After 48 hours, non-adherent cells were discarded and the medium was replaced every 3-4 days. This experimental study was established in research center of Kharazmi University and all the procedures were approved by the local Ethics Committee at Kharazmi University. Four-six-week old Naval Medical Research Institute ( NMRI ) mice ( purchased from Razi Institute ) were sacrificed by cervical dislocation. In sterile conditions, BM-MSCs were harvested from the bone marrow of the femurs and tibias, and the cells were plated into a T-25 flask  with dulbecco's modified Eagle's medium  containing 15% fetal bovine serum  and 100 U/ml penicillin-streptomycin  and incubated at 37˚C in a humidified atmosphere containing 5% COThe cells were characterized by flow-cytometric analysis of specific surface antigens of cluster of differentiation ( CD ) CD45, CD31, CD44, and CD29. When reaching 80% confluency, the cells were passaged, and after 2 passages, they were seeded in poly-lysine coated 24-well plates . The cells were incubated without any treatment and after 24 hours, they were divided into 4 groups and the treatments were administered for 4 days. The first group was considered as control group, with no treatment. The cells in second group received the combination of hormones, β-estradiol, progesterone and testosterone , in equal concentrations of 90 µM/ each and the cells of group 3 were treated with bFGF . The cells in group 4 received bFGF ( 25 ng/ml ) pretreatment for first two-day and hormones  treatment for second two-day. Each group was treated for 4 days. Immunocytochemical and flow cytometric analysis were performed for neural markers of microtubule-associated proteins-2 ( MAP-2 ) and beta III-tubulin ( β-III-tubulin ), for all 4 groups after 4 days of treatment. Our findings showed that MSCs derived from bone marrow were in a heterogenous suspension phase with other cells in culture dish and gradually adhered to the flask surface. After 48 hours, the medium was replaced to remove the other undesired suspending cells. MSCs tend to form colonies and after some days, they reached their normal spindle shape in colonies. Flow cytometry analysis of surface antigens revealed that cells were negative for CD45 and CD31, while positive for CD44 and CD29  and endodermal ( hepatocytes ) tissues in vitro ,17. in vitro, and specifically play key roles in tissue engineering and MSCs modification as different regulators  and remain for a long time, they might lose many features of stemness and gain some novel characteristics to promote differentiation. Some researchers in 2004 observed that MSCs, after many passages in vitro, without any specific treatment expressed neural lineage markers. Although such changes in molecular marker expression are usually transient and is not considered as stable differentiation, they might mislead the researchers. These cells after being isolated from their main niche, full of normal signals and specific conditions, should not be maintained in culture medium for a long time, and the treatment should be administered soon after reaching a homogenous cell source (In addition, the studies have shown that when mouse MSCs transport into culture medium ( l source ,31. Our data suggested that sex steroid hormones supplement enhances the neural differentiation effects of bFGF on cultured BM-MSCs. These findings could be used in the cultivation of BMMSCs for cell-based strategies for CNS disease treatment. This study showed that BM-MSCs can express specific neural markers after receiving bFGF as a pretreatment that is followed by receiving sex steroid hormones as treatment. It can be concluded that some steroid hormones may be considered as a drug for treatment of CNS diseases and disorders in future studies.