HIV/AIDS treatment in Nigeria may require that the cost of antiretroviral drugs be borne in part by infected people and their families.The current trend of withdrawal of donor support for ARVs) expressed by clients receiving treatment for HIV/AIDS in a tertiary hospital.This study was conducted to determine the economic value for free antiretroviral drugs  for ARVs for themselves and members of their households. Ordinary least squares (OLS) multiple regression analysis was used to test the construct validity of elicited WTP amounts.The contingent valuation method was used to elicit the values attached to free WTP for monthly supply of ARVs for self was US$15.32 and for household member was US$15.26 (1US$=₦160). OLS regression analysis showed that employment status and higher socio‐economic status were positively associated with higher WTP. OLS showed that age and transport cost per clinic visit were negatively related to WTP. Knowing the risks of not adhering to treatment protocol was positively related to WTP.About a third of the respondents were willing to pay for a monthly supply of ARVs for themselves and household members. The mean ARVs. This calls for greater financial support for the sustainable provision of the treatment service. However, holistic financing mechanisms should be explored to ensure sustained funding in the event of complete withdrawal of donor support.The respondents positively valued the free  In order to ensure programme sustainability, it is important to maintain a balance between the social goal of making services available to low income clients and the survival of the programme over time.It is estimated that the first‐line regimen of antiretroviral treatment for adults and children costs about US$368 per person per year (about 1US$ per day).Presently, clinical management of HIV/AIDS requires lifelong treatment, and infected people would most likely commit to this if they perceive the benefits of treatment to outweigh the cost. An estimation of how much money an individual is willing to pay for goods/services gives an idea of how much value they attach to that product, although this is often limited to one's wealth.Although international funding allows patients to access antiretroviral treatment (ART) for free, assessment of patients WTP for ARVs will play a vital role in preparing an appropriate ART policy once external financial support becomes unavailable or limited.This study provides evidence that could contribute to planning for sustainability of funding, to understand the values that the current beneficiaries attach to free ARVs, so as to determine its worthwhileness. Secondarily, it provides some information that is important for holistic funding of the treatment from all financing sources if donors withdraw or reduce their support—by estimating clients' WTP for ARVs in order to maintain lifelong therapy. WTP was elicited using the contingent valuation method (CVM) which has strong theoretical underpinning in welfare economics. The CVM can be used to monetize the values or monetize the benefits that people derive from or associate with services2This study was conducted in south‐east Nigeria at the Federal Teaching Hospital Abakaliki (FETHA), Ebonyi state. FETHA is a public tertiary hospital that offers a variety of specialist and non‐specialist health services including prevention, treatment and control of communicable diseases. In collaboration with the Centre for Clinical Care and Research Nigeria, FETHA provides treatment services for HIV/AIDS clients through its centre for communicable disease control and AIDS relief. HIV/AIDS treatment services are provided by medical doctors and trained nurses. As at the time of this study, a total of 700 clients had enrolled and were receiving treatment from this centre. The centre runs a 4‐day clinic from Monday to Thursday and the daily patient load averages about 25.2.1The study population comprised of adults who were enrolled in and receiving treatment for HIV/AIDS from the communicable disease control and AIDS relief centre of the hospital during a 3‐week period in May 2014. An adult was defined as someone who was 18 years old or more. Sample size was determined using 95% confidence level, 5% precision and 29% as the proportion of clients who are willing to pay for HIV/AIDS treatment.2.2A pre‐tested structured questionnaire was used to collect information from a sample of HIV/AIDS clients. The interviews were conducted by three undergraduate medical students who also participated in developing and pre‐testing the questionnaire. A 1‐day training was organized for them on quantitative interviewing using a WTP questionnaire. Role plays were performed in English and the local dialect. Clients were interviewed face‐to‐face as they exited the consulting room, using paper‐based questionnaires. Self‐ or interviewer‐administration of questionnaires was performed depending on client's literacy in English. Information was collected on respondent's demographic characteristics; awareness of ARV treatment procedures; ARV consumption patterns and expenditure; WTP for ARV drugs for HIV and AIDS; the amount they are willing to pay for themselves and for a member of their household. Information on household income, assets and food expenditure was collected.2.2.1The two non‐market valuation methods  often provide statistically convergent WTP values, but we chose contingent valuation because it has advantage of face‐to‐face contact which reduces misunderstanding and could make spontaneous questions possible unlike DCE that lacks direct valuation question.Willingness to pay for self alone was elicited first, followed by WTP for an infected household member or spouse alone. In both cases, the respondents were first told that the price of monthly supply of ARVs for one person is ₦5000 (starting bid), and then asked whether they would be willing to pay for their own ARVs assuming they were not given free. The bidding game iteration that was used in the study was as follows:The price of monthly supply of ARV is ₦5000, are you willing to pay?  1=yes (go to Q3), 0=no (go to Q2). 2=do not know (go to Q2).What is the maximum amount you are willing to pay?  .What if the cost of ARV is ₦10 000 will you be willing to pay? 1=yes , 0=no  What really is the maximum amount you are willing to pay for the ARV?  .The amount that you have quoted is too low, and cannot cover the cost of the drugs, and so you will have to increase the amount if you really want to be on treatment. So what is the final maximum amount you are willing to pay per month to continue your treatment? [₦ ]. .If due to inflation or other uncertainties, the cost of the ARV increases, what is the maximum amount you are very certain to pay bearing in mind your average monthly household income and money you spend on various items?[ ₦ ].2.3Frequencies and proportions were calculated for categorical variables while means were calculated for numeric variables. Households were grouped into four wealth quartiles namely poorest, very poor, poor and least poor, and this was calculated using principal component analysis based on household asset ownership  and weekly food expenditure per capita. Demographic characteristics and wealth quartiles were cross‐tabulated with participants' WTP to determine associations. Appropriate statistical tests of association were performed at 95% confidence. The mean WTP amounts, for both respondents WTP for self and for others, respectively, were computed from the final stated WTP amounts to the last question on the bidding game iteration. The theoretical validity of the elicited respondents' WTP for self and for others was assessed using ordinary least squares (OLS) multiple regression analysis. The dependent variables were mean WTP and some variables that were hypothesized to explain WTP were the independent variables Table . The ind2.4This research proposal and consent procedure were approved by a Health Research and Ethics Committee, and the registration number is FETHA/HREC/22/04/2014. Participants were informed of the purpose of the study, their roles, benefits and rights as participants, and possible risks from participation. Verbal informed consent was obtained from all participants, and their anonymity was maintained by non‐inclusion of self‐identifiers in the questionnaire. Participant consent was documented using an informed consent form with provision for ticking (√) in a “yes” or “no” box. The date of obtaining consent was also documented in the form. Verbal consent was preferred to written consent because some of the respondents did not want to append a signature or any form of identification on the form. Participants were interviewed singly in empty consulting rooms to ensure their privacy.3A total of 144 clients were approached for interview, 125 consented and were interviewed, but 123 questionnaires were appropriate for analysis.The participants had a mean age of 32.29 (±7.2) years. Other demographic characteristics are shown in Table A total of 120 (97.6%) of the respondents stated they knew the price of a monthly supply of their ARVs. As shown in Table Table P<.05). Participants in the higher wealth quartiles were more willing to pay ₦5000 (US$31.25) for their monthly supply of ARVs for HIV/AIDS than those in the lower quartiles (P<.001).Associations between participants WTP ₦5000 (US$31.25) and ₦10 000 (US$62.5) for ARVs for themselves and a household member or spouse are presented in Table 3.1Table 4This study examined whether people with HIV/AIDS who have been receiving treatment free of charge would be willing to pay for their ARVs and how much they are willing to pay should the need arise. Having told them how much it would cost to purchase their monthly supply of ARVs, one‐third of our study participants expressed WTP for their own drugs. When asked whether they would be willing to pay the same amount for an infected household member or spouse to get their monthly supply of drugs, about one‐third said they would. The regression analysis showed that the elicited WTP values were construct‐valid. In addition, there was no evidence of starting‐point bias because there was no clustering of WTP value around the starting bid.Although over 90% of our participants knew the cost of ARVs for HIV/AIDS, their maximum mean WTP for a monthly supply of these drugs was less than half of the actual cost of the drugs, implying that knowledge of the cost of ARVs does not necessarily translate into WTP, rather WTP is subject to socio‐economic status of participants. Thus, the knowledge of cost of ARV does not have anchor effect or starting‐point bias because if the participant's bid reflects his or her true value of the good or service, then it should not matter what initial amount was used to begin the bidding game.It was found that being willing to pay ₦5000 US$31.25) per month for ARVs had a positive statistical significant association with employment status of clients. Those who had more regular sources of income (paid employment) expressed more WTP than those whose income sources were unreliable (self‐employed) probably because in order to be able to give up a certain amount of money on a regular basis, an individual needs to be clear about how much they earn and how often their earnings come in. People who have steady sources of income are more able to make regular payments than people who are uncertain when the next money will come and how much that would be. Thus, confirming Nguyen et al. that individual capacity to pay for antiretroviral drug is strongly influenced by socio‐economic factors such as monthly income, household economic condition and marital status. Indeed, as socio‐economic status increases, level of WTP also increases.1.25 per Considering that only 51.8% of Nigerians are employed and that civil servants made up less than a quarter of people receiving treatment for HIV/AIDS in our study site, we can conclude that over three quarters of people who are infected with HIV/AIDS may not be able to regularly purchase their ARVs if they had to do so. As one of the major contributing factors to this inability to pay was the patients' unemployment situation, which leads to low, and often unstable, incomes.A limitation of the study was that hypothetical WTP for ARVs was elicited which may differ from revealed preferences through actual observations of how much is paid. It has been argued that stated preferences when compared to revealed preferences may produce under‐ or overestimates depending on the social and economic contexts at the time. Willingness to pay surveys allow programme managers to simulate price‐related changes in demand without actually changing prices, thereby helping them to make decisions based on empirical information.5Only a third of clients receiving treatment for HIV/AIDS are willing to pay for their ARVs and that of a family member or spouse. Their mean WTP for a monthly supply of ARVs for HIV/AIDS falls below the estimated cost of the drugs. Regular source of income and higher socio‐economic status are significantly associated with WTP of US$31.25 per month to purchase ARVs for HIV/AIDS. As less than a quarter of the clients surveyed have regular sources of income, charging user fees for HIV/AIDS treatment could lead to financial inaccessibility for most infected people. Governments need to consider and put in place more sustainable measures to ensure stable funding for prevention and control of HIV/AIDS. A solution that has been suggested is a co‐payment system for ART. However, in order for such a system to be effective, patients must be willing to pay for their share of services received. The policy implications of this study indicate that to get a greater number of individuals on ART government would have to find other mechanisms of lowering the price of the drugs or subsidizing treatment cost for HIV/AIDS patients.The authors declare that they have no conflict of interests.


Mutations in the human RecQ helicase, BLM, causes Bloom Syndrome, which is a rare autosomal recessive disorder and characterized by genomic instability and an increased risk of cancer. Fanconi Anemia (FA), resulting from mutations in any of the 19 known FA genes and those yet to be known, is also characterized by chromosomal instability and a high incidence of cancer. BLM helicase and FA proteins, therefore, may work in a common tumor-suppressor signaling pathway. To date, it remains largely unclear as to how BLM and FA proteins work concurrently in the maintenance of genome stability. Here we report that BLM is involved in the early activation of FA group D2 protein (FANCD2). We found that FANCD2 activation is substantially delayed and attenuated in crosslinking agent-treated cells harboring deficient Blm compared to similarly treated control cells with sufficient BLM. We also identified that the domain VI of BLM plays an essential role in promoting FANCD2 activation in cells treated with DNA crosslinking agents, especially ultraviolet B. The similar biological effects performed by ΔVI-BLM and inactivated FANCD2 further confirm the relationship between BLM and FANCD2. Mutations within the domain VI of BLM detected in human cancer samples demonstrate the functional importance of this domain, suggesting human tumorigenicity resulting from mtBLM may be at least partly attributed to mitigated FANCD2 activation. Collectively, our data show a previously unknown regulatory liaison in advancing our understanding of how the cancer susceptibility gene products act in concert to maintain genome stability. The effective repair of DNA damage, caused by exogenous agents or during DNA replication, confers protection from neoplastic transformation. Several genetic disorders that perturb the repair of DNA damage result in an increased predisposition to cancer –5. One sBloom Syndrome (BS) , similarA functional link between BLM and the FA pathway has now been suggested for a decade. However, there is a lack of evidence to demonstrate a direct functional connection between BLM and the FA proteins. Thus we utilized a pair of human fibroblast cell lines, PSNG13 derived from a BS patient and its derivative PSNF5, complemented with functional BLM  to examiBLM helicase is well acknowledged at many aspects of DNA metabolism including DNA replication, repair, recombination, and transcription. To this point, we wanted to see if this novel regulatory link was restricted to BS cells. We generated a set of stable cell pairs isogenic to the level of Blm expression using human osteosarcoma U2OS cells. This was achieved via lentivirus-mediated delivery of shRNA targeting Blm mRNA Figure , noting 2) to screen which motif of BLM influenced FANCD2 monoubiquitination. Cells carrying ΔVI-BLM were observed to have an effect on FANCD2 monoubiquitination/activation . As shown in Figure The core functional domain of the BLM protein is the RecQ helicase domain. This domain is composed of seven conserved motifs designated I, Ia, II-VI. The structural model for the BLM helicase demonstrates that Ia, III and V directly bind to the ssDNA of the helicase substrate, while I, II, III, IV and VI form the binding pocket for ATP binding and hydrolysis . To gainn Figure  bottom. To explore the functional significance of this novel signaling link, we searched various available databases for BLM mutations occurring in BS patients. We found there were deletion and/or point mutations in the Y974Q975 region within the motif VI of BLM protein that were documented in human cancers , 33. FurTo further confirm this previously unknown signaling link between BLM and FANCD2 activation, we anticipated that cells harboring the insufficient function of either BLM or FANCD2 would show similar biological effects. To this end, we performed a complete set of proliferation assays via U2OS derivative cells, isogenic to the status of FANCD2 activation or to the normal functional level of BLM. As shown in Figure p value of 1E-06  respectively Figure  . Antibodies against the FANCD2 and BLM were obtained from NOVUS. The anti-Flag and β-actin antibodies as well as the chemicals Mitomycin C (MMC), Cisplatin, crystal violet solution and Puromycin were purchased from Sigma.PSNF5 and PSNG13 are two BS derivative cell lines isogeneic to BLM expression. PSNF5 is a complemented BS cell line with wild-type BLM cDNA from PSNG13 BS line , 43. U2OFor lentiviral transduction essentially as described previously : a set oAs described previously , 46: we 3) were seeded onto sterilized black well slides and fixed with pre-chilled 3.7% paraformaldehyde/PBS for 15 min at 4°C and quenching with 125 mM of glycine for 5–10 min at room temperature (RT). Next, these cells were permeabilized with 0.2% pre-chilled Triton-X 100/PBS for 5 min at 4°C, blocked with 3% horse serum/PBS for 30 min at RT, and subsequently incubated with FANCD2 antibodies at 4°C overnight. After washing, cells were further incubated with anti-rabbit secondary antibodies at 1:1000 dilutions for 30 min at RT. After the incubation with secondary antibodies, cells were washed and mounted in Vectashield mounting media with DAPI . Cells were observed with a 40X objective lens under an Olympus BX-51 microscope equipped with a SenSys fluorescence CCD camera. Images were acquired and analyzed using MetaMorph version 4.5 Premier.The Immunofluorescence staining was performed as previously described . PSNF5, Equal numbers of U2OS and PA-1 stable cells were seeded into 35 mm dishes one day prior to MMC treatment. Cells were treated with MMC at concentrations of 50 ng/ml, 25 ng/ml, 12.5 ng/ml, or 6.25 ng/ml MMC for 5 days. Resulting colonies were then fixed and stained with crystal violet solution. The numbers of colonies were counted (Each drug dose was tested in triplicate) using a GelDoc with Quantity One software (BioRad).We designed a set of primers 50bp up and down the region encoding Y974Q975 of BLM and FAM-probe (18 mers) covering right on the region. Oligos  were ordered from IDT. 1 pg of wtBLM or ΔY974Q975-Blm cDNA was used as the template for the controls to set up the assay condition. The TaqMan assay was performed using 0.5°C increments for 5 seconds from 30 to 70°C until the final annealing stage. The intensity of FAM-florescence was plotted in referring to the increment of Tm. The peak of florescence was clearly separated using our modified program at 52.6°C for wtBLM cDNA and 46.8°C for Y974Q975-Blm cDNA.Frozen dissected ovarian tumor specimens, examined by two independent pathologists, contained 80–90% tumor cells, were pulverized manually in liquid nitrogen for extraction of total RNA following a standard phenol–chloroform protocol. The RNA samples were treated with DNase to remove genomic DNA contamination and were concentrated using the RNeasy MinElute Cleanup kit . The quality of ribonuclease-free deoxyribonuclease-treated total RNA was determined using 1% agarose gel electrophoresis for the integrity of 18s and 28s ribosomal RNAs. 1 μg of total RNA from each sample was processed for conversion to cDNA using the Cloned AMV First-Strand cDNA Synthesis kit . cDNA samples from 191 patients were used for the TaqMan assay. Primers  were designed in-house and chemically synthesized by Integrated DNA Technologies . The PCR conditions were modified as described above for our modified TaqMan assay. The calculation of BLM products from the real-time PCR was independently performed, and performers were blinded to the clinical data.p-value < 0.05 was considered statistical significance. All analyses were processed by SPSS 20 software.The correlation between the Tm's and the cancer stage was analyzed using the Chi-square test. A


Listeria monocytogenes and Salmonella enterica serovar Thompson was achieved by exposure to 12 and 3 ppm chlorine, respectively. The pathogens were subjected to chlorine washing following incubation on spinach leaves. Culture data revealed that total viable L. monocytogenes and Salmonella Thompson populations became VBNC by 50 and 100 ppm chlorine, respectively, while enumeration by direct viable counting found that chlorine caused a <1-log reduction in viability. The pathogenicity of chlorine-induced VBNC L. monocytogenes and Salmonella Thompson was assessed by using Caenorhabditis elegans. Ingestion of VBNC pathogens by C. elegans resulted in a significant life span reduction (P = 0.0064 and P < 0.0001), and no significant difference between the life span reductions caused by the VBNC and culturable L. monocytogenes treatments was observed. L. monocytogenes was visualized beyond the nematode intestinal lumen, indicating resuscitation and cell invasion. These data emphasize the risk that VBNC food-borne pathogens could pose to public health should they continue to go undetected.The microbiological safety of fresh produce is monitored almost exclusively by culture-based detection methods. However, bacterial food-borne pathogens are known to enter a viable-but-nonculturable (VBNC) state in response to environmental stresses such as chlorine, which is commonly used for fresh produce decontamination. Here, complete VBNC induction of green fluorescent protein-tagged  Listeria monocytogenes and Salmonella enterica. It was also found that chlorine is ineffective at killing total populations of the pathogens. A life span reduction was observed in Caenorhabditis elegans that ingested these VBNC pathogens, with VBNC L. monocytogenes as infectious as its culturable counterpart. These data show that VBNC food-borne pathogens can both be generated and avoid detection by industrial practices while potentially retaining the ability to cause disease.Many bacteria are known to enter a viable-but-nonculturable (VBNC) state in response to environmental stresses. VBNC cells cannot be detected by standard laboratory culture techniques, presenting a problem for the food industry, which uses these techniques to detect pathogen contaminants. This study found that chlorine, a sanitizer commonly used for fresh produce, induces a VBNC state in the food-borne pathogens  Entry into a viable-but-nonculturable (VBNC) state has been identified in a wide range of bacterial species and environmental stressors, including starvation, low temperature, antibiotic pressure, and oxidative stress –3. This –Listeria monocytogenes and Salmonella spp. were responsible for more than half of these deaths following gastrointestinal infection . The potential for infection by VBNC pathogens was then determined by using the animal model Caenorhabditis elegans.This study simulated the passage of spinach contaminated with L. monocytogenes and Salmonella Thompson were visualized by episcopic differential interference contrast (EDIC)-epifluorescence (EF) microscopy following 24 h of incubation on the spinach phylloplane. Green fluorescence indicated that the pathogens were localized primarily inside the spinach stomata and at cell junctions. Compared with uninoculated control spinach leaves, both inoculated spinach samples possess a rough, uneven surface indicative of biofilm growth  and a >4-log reduction by 6 ppm (P = 0.0075).by 6 ppm . BetweenSalmonella Thompson became fully VBNC after 2 min of exposure to 3 ppm chlorine (P < 0.0001). Each increase in the chlorine concentration was met with a loss of Salmonella Thompson cells, with a 49% reduction between 0 and 15 ppm chlorine (P < 0.0001). There was also a 1.4-log difference between culturable cells and those enumerated by DVC (P < 0.0001) at 0 ppm chlorine  and by E. coli Op50 and VBNC L. monocytogenes (P = 0.0064), where the median life span of C. elegans feeding on E. coli Op50 was 12 days and only 9 days for both L. monocytogenes treatments. Similarly, ingestion of culturable (P < 0.0001) or VBNC (P < 0.0001) Salmonella Thompson significantly reduced the C. elegans life span compared with ingestion of the E. coli Op50 control. The median life spans of C. elegans worms that fed on culturable and VBNC Salmonella Thompson were 6 and 7 days, respectively, with a statistically significant difference observed between the two treatments (P = 0.0322) ( 0.0322) .C. elegans  fluorescence from each pathogen assessed was observed filling the intestinal lumen of  elegans  and, in  tissues . PathogeL. monocytogenes and Salmonella Thompson  the pathogen lost culturability more easily than L. monocytogenes . This indicates that while the pathogen is still virulent in the animal model, it does lose some infectivity in the VBNC state. Research on the cell invasion ability of VBNC Salmonella Typhimurium has indicated that VBNC cells have an impaired ability to invade epithelia . Conversely, Salmonella Thompson strain NCTC 2252 was shown to reduce the C. elegans life span  with Listeria selective supplement , and S. enterica was cultured on agar by using CHROMagar Salmonella Plus with its cognate supplement . E. coli Op50 was used as a nonpathogenic control in the nematode killing assay. It was cultured in Luria-Bertani broth  for 18 h at 37°C prior to use.The bacteria used in this study were nce gene . Both we7 CFU/ml of BHIB. Inoculated samples were shaken vigorously and incubated at 22°C for 24 h prior to being washed with chlorine.The leaf samples used were raw, unwashed spinach leaves supplied by Vitacress Salads Ltd., United Kingdom. Leaves were inoculated within 48 h of delivery. Twenty-five-gram leaf samples were placed in a Stomacher bag  and inoculated with 1 ml of bacteria at a concentration of 5 × 102O, which was further diluted in ddH2O to generate working solutions. Bacterial suspensions of 108 CFU in phosphate-buffered saline  were inoculated into 50 ml of ddH2O in a Stomacher bag to which 50 ml of the appropriate chlorine dilution was added. The sample was shaken vigorously for 2 min and then filtered through a 0.22-µm-pore-size mixed cellulose ester membrane  by vacuum filtration. Bacteria were removed from the membrane by placement in another Stomacher bag with 100 ml of PBS and shaken with a Pulsifier  for 30 s, producing a final concentration of 106 CFU/ml. Samples were then taken for culture and DVC.A stock solution of 2,500 ppm free chlorine was produced by dissolving one Haz-Tab  in 1 liter of ddH2O containing the appropriate volume of chlorine solution was added to inoculated spinach samples. Samples were vigorously shaken for 2 min, and the liquid was discarded, retaining the leaf samples; 225 ml of PBS was then added, and the bag was shaken in the Pulsifier for 30 s. Samples of the resulting bacterial suspension were then taken for culture and DVC.Following 24 h of incubation, 225 ml of ddH2O, 5 ml of R2 broth , and 10 µl of pipemidic acid at a concentration of 10 µg/ml. The suspension was incubated for 18 h at 22°C in darkness. The suspension was concentrated prior to DVC in the same manner as before.Samples taken for DVC were concentrated by centrifuging a 10-ml sample for 15 min at 4,000 rpm with a Heraeus Megafuge 1.0. The sample was then resuspended in 1 ml of PBS. To aid visualization, samples were subjected to cell elongation by a modification of the method of Juhna et al. . The 1-mAll samples were imaged by using EDIC-EF microscopy  and a QIC. elegans worms were maintained on 5-cm nematode growth medium (NGM) agar plates prepared in accordance with standard methods (E. coli Op50. To prepare an experimental plate, 50 µl of E. coli Op50, L. monocytogenes, or Salmonella Thompson culture was added to the center of the plate and it was incubated at 22°C for 24 h. To produce VBNC cells, cultures of L. monocytogenes and Salmonella Thompson were pelleted by centrifugation and resuspended in 10 ml of a 200 ppm chlorine solution for 30 min. Chlorinated water was removed by vacuum filtration as described above, and bacteria were removed from the membrane by vortexing in 1 ml of PBS for 2 min ( methods  with a lor 2 min , concentC. elegans worms were transferred to experimental plates at the L4 stage. Twenty worms were used per plate, and each condition was tested with at least four replicates. Nematodes were counted daily and transferred to fresh plates every other day. Nematodes that did not respond when prodded with a pick were considered dead.t tests. Nematode killing assay data were analyzed by using the survival curve comparison Mantel-Cox test. All statistical analyses were done with GraphPad Prism 7.Culture data and DVC were separately subjected to one-way analysis of variance with Tukey’s multiple-comparison test. Comparisons of culture and DVC data were done with multiple


Here we describe in detail marketing authorization and reimbursement procedures for medicinal products in Norway, with particular reference to nine novel antibiotics that received marketing authorization between 2005 and 2015. The description illustrates that, in places like Norway, with effective antibiotic stewardship policies and an associated low prevalence of antibiotic-resistant bacterial infection, there is little need for newer, more expensive antibiotics whose therapeutic superiority to existing compounds has not been demonstrated. Since resistance begins to emerge as soon as an antibiotic is used, Norway’s practice of leaving newer antibiotics on the shelf is consistent with the goal of prolonging the effectiveness of newer antibiotics. An unintended consequence is that the country has signalled to the private sector that there is little commercial value in novel antibiotics, which may nevertheless still be needed to treat rare or emerging infections. Every country aims to improve infection control and to promote responsible antibiotic use. However, as progress is made, antibiotic-resistant bacteria should become less common and, consequently, the need for, and the commercial value of, novel antibiotics will probably be reduced. Nevertheless, antibiotic innovation continues to be essential. This dilemma will have to be resolved through the introduction of alternative reward systems for antibiotic innovation. The DRIVE-AB (Driving re-investment in research and development and responsible antibiotic use) research consortium in Europe has been tasked with identifying ways of meeting this challenge. An antibiotic was regarded as novel if no antibiotic with the same generic chemical name had previously received Norwegian marketing authorization. Several new formulations of older antibiotics, such as tobramycin, received marketing authorization between 2005 and 2015, but they were not considered novel. Information on novel antibiotics was obtained by searching the Norwegian Medicines Agency’s medicines database,Norway is a member of the European Economic Area and participates in its common procedures for marketing authorization of medicinal products, which are coordinated by the European Medicines Agency (EMA). There are several ways of obtaining marketing authorization for pharmaceuticals in the European Economic Area. Typically, antibiotics are reviewed under the EMA’s so-called central procedure: an application for marketing authorization is sent to the EMA, which makes a binding decision for all countries in the European Economic Area based on the analyses of two national, regulatory agencies. Approval is valid for five years, after which the product must be reassessed. However, many older antibiotics received marketing authorization before the EMA’s central procedure had been established. As a result, there is some national variation across Europe in antibiotics that have been approved.Staphylococcus aureus (MRSA) infection, are regarded as so contagious and dangerous that the antibiotics used to treat them are always reimbursed.The Norwegian Medicines Agency is responsible for marketing authorization, pricing, reimbursement and other medicine-related services in Norway. Marketing authorization permits a medicinal product to be sold in community or hospital pharmacies but does not necessarily mean that its cost will be reimbursed by the Norwegian national health insurance scheme or that it will be used or purchased by health-care institutions. The national insurance scheme subsidizes the cost of prescription medicines prescribed through primary care to ensure that all critical medicines are accessible for Norwegian patients. Partial reimbursement of medication expenses in cases of severe or prolonged illness is included. Generally, the cost of antibiotics is not reimbursed because most infections are short-lived. In 2014, according to the Norwegian Prescription Database, only 13% of antibiotic prescriptions were reimbursed. However, some infections, such as methicillin-resistant After a medicine has received marketing authorization in Norway, the manufacturer must apply separately to the Norwegian Medicines Agency for preapproval for reimbursement . SubsequFailure to meet any one of these criteria results in rejection of the application for reimbursement. As a result, if the medicine is prescribed by a community-based physician, the patient must bear the full cost out of pocket. However, the physician has the opportunity to apply for a patient-specific exception and, if approved, the medicine is covered by the national insurance scheme.In parallel with the health technology assessment, the Norwegian Medicines Agency also determines a maximum price for the medicine. The agency uses an international reference pricing system, under which the manufacturer reports the price of the medicine for each indication in nine preselected countries: Austria, Belgium, Denmark, Finland, Germany, Ireland, the Netherlands, Sweden and the United Kingdom of Great Britain and Northern Ireland. The maximum price paid by the Norwegian national insurance scheme is the average of the three lowest prices applicable in these reference countries. Exchange rates are calculated using an average for the previous 6 months. If the application for reimbursement is approved, the manufacturer is permitted to sell its product in Norway at this maximum price or less.Norwegian hospitals have formed a pooled procurement mechanism, by which medicines are purchased through national tenders. Having a maximum price is useful for judging the savings offered by these tenders and the mechanism has secured lower total costs for both generic and patented medicines. For patented medicines, savings are typically made on products that can be easily substituted. Nursing homes, which are included in community services, may competitively outsource the provision of their medicines. In all instances, no medicine may be sold in Norway at greater than its maximum price.Securing national insurance reimbursement does not necessarily mean that a medicine will be prescribed; that will depend on its inclusion in national guidelines and on physicians’ adherence to those guidelines. Norway has two sets of national guidelines for antibiotics – one for hospitals and one for community practice. Both are based on: (i) the evidence available, which is evaluated using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach, where possible, to determine the quality of the evidence and the strength of the recommendations;Community antibiotic guidelines are updated regularly by the Norwegian Antibiotic Centre for Primary Care. Although physicians are not legally required to follow the guidelines, they are expected to document their reasons for any antibiotic prescribing that differs substantially from the guidelines. Some deviation is acceptable and there are regional differences in prescribing, which suggests that the guidelines are either not adhered to fully or interpreted differently in different places.Hospital antibiotic guidelines are updated regularly by the National Competence Center for Antibiotic Use in Specialist Health Services. Although price is not taken into account during guideline development, it may influence the physician’s decision, especially when an antibiotic is substantially more expensive but has not demonstrated therapeutic superiority. Most new antibiotics are approved on the basis of clinical trials designed to establish noninferiority, which means that the new antibiotic is not inferior to a comparable existing antibiotic. It is difficult to demonstrate the superiority of an antibiotic because, in general, existing alternatives remain highly effective while antibiotic resistance is still quite uncommon. At present, older antibiotics continue to perform relatively well. In the absence of evidence of superiority, other factors, such as cost, potential side-effects and ease of administration, have a dominant influence on clinical decisions. The strategy of using patented, better-targeted antibiotics  to slow the development of antibiotic resistance has, to date, not been a consideration in Norwegian hospital guidelines. The lack of certainty that resistance will emerge is too great to justify the high cost.Nine novel antibiotics received marketing authorization in Norway between 2005 and 2015 . All werClostridium difficile). In general, using a narrow-spectrum instead of a broad-spectrum antibiotic should reduce the likelihood of antibiotic-resistant bacteria developing, given that bacteria outside the spectrum of organisms covered would not be under selective pressure. Here, the use of these four antibiotics is examined in detail to give some insight into Norwegian physicians’ decision-making processes.Of the nine antibiotics, three are considered narrow-spectrum antibiotics  and one is considered pathogen-specific  is normally used only in hospitals and nursing homes. In the guidelines it is listed as an alternative to vancomycin (maximum price: US$ 146 per package) for patients with recurrent Retapamulin (maximum price: US$ 18 per package) is a narrow-spectrum antibiotic for the topical treatment of bacterial skin infections. Interestingly, it was the most frequently prescribed of the nine novel antibiotics, although its use was still small, 2107 packages in 2015. The relatively high use was probably due to its inclusion in community guidelines as a topical treatment for impetigo, which is most likely to occur among preschool children. The alternative treatment is an antiseptic ointment containing dibrompropamindin.Daptomycin (maximum price: US$ 178 per package) is listed in hospital guidelines as a treatment option for MRSA infections reported to the Norwegian Surveillance System for Infectious Diseases, typically as an alternative to vancomycin, as well as for vancomycin-resistant enterococci. In 2014, there were 833 cases of MRSA infection and 110 cases of infection with vancomycin-resistant enterococci. Health-care institutions dispensed 482 packages of daptomycin. Consequently, daptomycin probably has a substantial share of the small markets for these two indications.Tedizolid (maximum price: US$ 1778 per package) is a narrow-spectrum antibiotic against acute, bacterial, skin and soft tissue infections by Gram-positive bacteria. It received marketing authorization in 2015, primarily on the basis of clinical trials showing it was noninferior to linezolid (maximum price: US$ 707 per package).Human antibiotic use in Norway is among the lowest in the world:,Nevertheless, antibiotic resistance is gradually becoming more common in Norway for most pathogens and, as a result, the pattern of antibiotic consumption has altered.With current reimbursement models, it is difficult to provide an attractive return on investment in a country like Norway, where responsible use has diminished the size of the potential market for novel antibiotics to a negligible level. Today, however, probably only a handful of antibiotics markets resemble Norway’s. Yet, every country aspires to improve infection control and to promote responsible antibiotic use, thereby lowering the prevalence of antibiotic resistance. The need for novel, effective antibiotics will never disappear and there is the danger that, when a specific need arises in the future, it may be too late to develop novel drugs because medical innovation can be a lengthy process.The DRIVE-AB consortium has been tasked with identifying ways of meeting the challenge of antibiotic innovation while at the same time promoting responsible use and equitable access. The proposed solutions are intended to reward innovation rather than boost sales and to increase both public and private investment in antibacterial research and development. The consortium’s final recommendations will be announced by October 2017.The Norwegian marketing authorization and reimbursement systems for medicinal products have been designed to ensure that Norwegians receive the medicines they need at the lowest possible price. Norwegian antibiotic guidelines have been devised to promote evidence-based prescribing in the context of the local pattern of antibiotic resistance. The spread of resistance in Norway has been slowed by strong stewardship policies and adherence to guidelines. This has enabled the country to leave newer antibiotics on the shelf to be used in an emergency for rare or emerging infections. The unintended consequence is that Norway has signalled to the private sector that there is little commercial value in novel antibiotics. The example of Norway demonstrates the need for alternative reward systems for antibiotic innovation.


Surface wave velocity measurement of concrete using ultrasonic sensors requires testing on only one side of a member. Thus, it is applicable to concrete cast inside a form and is often used to detect flaws and evaluate the compressive strength of hardened concrete. Predicting the in situ concrete strength at a very early stage inside the form helps with determining the appropriate form removal time and reducing construction time and costs. In this paper, the feasibility of using surface wave velocities to predict the strength of in situ concrete inside the form at a very early stage was evaluated. Ultrasonic sensors were used to measure a series of surface waves for concrete inside a form in the first 24 h after placement. A continuous wavelet transform was used to compute the travel time of the propagating surface waves. The cylindrical compressive strength and penetration resistance tests were also performed during the test period. Four mixtures and five curing temperatures were used for the specimens. The surface wave velocity was confirmed to be applicable to estimating the concrete strength at a very early age in wall-like elements. An empirical formula is proposed for evaluating the early-age compressive strength of concrete considering the 95% prediction intervals. The setting and hardening of concrete are important processes during construction work and influence the form removal time. Recent advances in design and construction technology have expanded the construction market to super-high-rise buildings and long-span bridges, which require the placement of huge quantities of concrete within the shortest time possible. Accordingly, appropriate timing for form removal is important to reduce the construction period and costs.Concrete specifications in Korea prescribe removing the form when the concrete compressive strength reaches 5–8 MPa. For example, the Standard Concrete Specification , StandarTo monitor the early-age strength of concrete, various nondestructive evaluation methods such as active sensing methods using embedded piezoelectric transducers ,9,10 andStress waves are generated when pressure is induced by ultrasonic sensors attached to a concrete surface. Rayleigh waves have been adopted by many researchers for nondestructive testing purposes ,19,20,21To compare the surface wave velocity with the Rayleigh wave velocity, two types of FE models were developed depending on whether acrylic was installed. The surface wave velocity is determined by using the arrival time of surface waves between two ultrasonic sensors. There are several methods for determining the arrival time, such as using the first peak in the time domain , using ta is a scale parameter, and b is a shift parameter. Equation (2) is the Morlet wavelet function. It is widely used in the time-frequency analysis of elastic waves and shows good performance in time localization [Here, lization ,29. Figu3, respectively. Poisson’s ratio of concrete is assumed to be 0.3 to reflect the characteristics of early-age concrete. Rayleigh wave velocity along the free surface is estimated to be 857.29 m/s, whereas the surface wave velocity between the concrete and acrylic layer is 883.96 m/s. The difference is less than 3.2%. This indicates that the acrylic panel, used as a mounting device for the ultrasonic transducer, has a negligible effect on the velocity.3. The elastic modulus and Poisson’s ratio are assumed to be 6.23 GPa and 0.3, respectively. The velocity with a density of 2200 kg/m3 is 894.52 m/s. The higher the density is, the slower the waves propagate, as shown in 3, the change in velocity is less than 5%.Case 2 shows the surface wave velocities when considering differences in concrete density due to material uncertainty. The concrete density ranges from 2200 to 2400 kg/m3, respectively. The surface wave velocity with a Poisson’s ratio of 0.35 is 847.93 m/s. The lower the Poisson’s ratio is, the faster the waves propagate. For a Poisson’s ratio change of 0.16–0.35, the change in velocity is less than 5%.Case 3 shows the surface wave velocities considering differences in Poisson’s ratio due to material uncertainty. Poisson’s ratio ranges from 0.16 to 0.35. The elastic modulus and density of concrete are assumed to be 6.23 GPa and 2400 kg/mThe objective of the surface wave measurement is to evaluate the feasibility of using the surface wave velocity to predict the strength of concrete inside a form at a very early stage. A series of surface wave measurements on in situ concrete were conducted within the first 24 h after pouring by using the ultrasonic sensors. The cylindrical compressive strength test and the penetration resistance test were also performed as the surface wave velocity was measured.The time-of-flight of the propagating surface waves between the concrete and mounting acrylic panel was used to derive the surface wave velocity for predicting the compressive strength of concrete inside the form. The mixing ratio of concrete can significantly affect the development of the concrete strength over time. Four mixture proportions were used and designated as MIX1–MIX4, given in Twenty concrete specimens were prepared to cover the various mixture proportions and curing temperatures for the surface wave velocity measurement. Freshly mixed concrete was placed in a steel mold to simulate formwork with dimensions of 400 mm × 300 mm × 300 mm as shown in Along with the surface wave velocity measurement, the penetration resistance and cylindrical compressive strength tests were conducted in accordance with KS F 2436  and KS FObservations showed that at a very early age there was considerable scatter and no well-defined trend in the computed velocities under 500 m/s. However, the plots in To further examine the relationship between the surface wave velocity and concrete strength at an early age, the measurement data for hardened concrete from previous studies ,19,20, aE was set as proportional to the square of the pulse velocity a and b are parameters determined by the least squares method to fit the measurement data in this study.To find the relationship between the early-age concrete strength and surface wave velocity, theoretical and empirical formulas were investigated. For the theoretical approach, the modulus of elasticity I 318-14  and KHBDI 318-14 , compresearchers ,34. Howeearchers ,36,37,38a and b of the exponential function presented in Equation (4) using a nonlinear regression method.In this study, the surface wave velocity measured with ultrasonic sensors was verified to be applicable to the monitoring of the solidification of concrete inside the form and an empirical formula was developed for evaluating early-age concrete strength according to the surface wave velocity. A pair of ultrasonic sensors was installed on the same side prior to placement. The sensors were used to measure the surface wave velocity of concrete. An acrylic panel was used to fix the position of the sensors and to increase the applicability on structures such as walls and towers. An FE simulation was conducted to compare the surface wave velocity with the Rayleigh wave velocity and the difference between them was less than 5%, even when material uncertainties were considered. The surface wave velocity for early-age concrete inside a form were comparable with the measured Rayleigh wave velocity for hardened concrete from previous studies. A series of experiments including surface wave velocity measurement, a cylindrical compressive strength test, and a penetration resistance test were conducted on in situ concrete during the first 24 h after placement. The penetration resistance test showed that a consistent surface wave velocity can be obtained prior to the initial setting and the measurement was sensitive to the developing strength up until the final setting time. Thus, the surface wave velocity is suitable for monitoring the hardening process of very young concrete. Based on the relationship between the measured surface wave velocity and the corresponding cylindrical compressive strength, an empirical exponential function was developed. The formula was obtained using all measurement data in this study. Then, a temperature coefficient, to account for the effect of construction in different seasons, was added to the formula considering the 95% prediction bound.The proposed formula can be used with the measured surface wave velocity for concrete inside a form to predict the compressive strength of concrete. The method can help with determining the appropriate form removal time for curing concrete, thereby contributing to the reduction of a construction period. The method has been developed based on the mix proportion of concrete used in this study and, therefore, further research is needed to investigate the applicability of the method for various mix proportions of concrete.


P = 0.007 and P = 0.011). In addition, dual immunofluorescence assay showed that immunoglobulin E and high affinity immunoglobulin E receptor staining exhibited a cytoplasmic granular pattern in IgG4-related lymphadenopathy, suggesting internalization of the antibodies and receptors. Our findings showed that mast cell activation might be involved in the pathogenesis of IgG4-related disease.Immunoglobulin G4 (IgG4)-related disease is characterized by elevated serum IgG4 levels and increased numbers of IgG4-positive cells. However, its pathogenesis is not fully understood. We previously suggested that mast cells may play an important role in IgG4-related disease. In this study, we confirmed the characteristics of mast cells in IgG4-related lymphadenopathy by using immunohistochemistry and dual immunofluorescence. We analyzed 23 cases of IgG4-related lymphadenopathy and compared them with 23 cases of non-specific lymphoid hyperplasia. The majority of patients with IgG4-related lymphadenopathy had cervical lesions with involvement of other organs. Immunohistologically, mast cells with strong cytoplasmic staining for immunoglobulin E and high affinity immunoglobulin E receptor were significantly increased in IgG4-related lymphadenopathy as compared to those in non-specific lymphoid hyperplasia (mean: 3.83 ± 3.99 cells per high power field and 7.14 ± 8.21 cells per high power field, respectively;  This is a fibro-inflammatory condition characterized by a tendency to form tumefactive lesions at multiple sites, with a distinctive histopathological appearance, and often, but not always, elevated serum IgG4 levels3. Although glucocorticoids are effective and the standard therapy for IgG4-RD, the disease is known to be likely to relapse during tapering or cessation of the medicine. Yamamoto et al.4 reported that more than half of patients with Mikulicz’s disease experience relapse during steroid therapy. Thus, IgG4-RD has been deemed incurable in Japan, and investigators have attempted to elucidate its pathogenesis within the last decade.Immunoglobulin G4-related disease (IgG4-RD) was initially described in 2001 as sclerosing pancreatitis with elevated serum IgG4 levels5, suggesting that the immune reaction mediated by these cytokines may be responsible for the lesions. These findings have also been confirmed in salivary glands, ocular adnexa, and kidneys8. However, to the best of our knowledge, only a few previous reports have investigated the mechanism underlying Th2 and Treg activation in IgG4-RD.There are many hypotheses regarding the pathogenesis of IgG4-RD that involve autoimmunity, molecular immunity, specific genotypes, intestinal bacterial flora, allergies, and other factors, which are yet to be proven. From a histopathological point of view, upregulation of T helper 2 (Th2)  and regulatory T (Treg) (IL-10 and transforming growth factor beta 1) cytokines was detected in affected tissues of patients with autoimmune pancreatocholangitis9. These findings in IgG4-related sialoadenitis were consistent with those of other previous reports11. In addition, we discerned that the infiltrating cells expressing Th2 and Treg cytokines were histomorphologically similar to mast cells and exhibited strong cytoplasmic staining for immunoglobulin E (IgE). Interestingly, although the number of c-kit-positive mast cells was not significantly increased in IgG4-related submandibular gland disease as compared to that in sialolithiasis and normal submandibular glands, the number of strongly cytoplasmic IgE-positive mast cells was significantly increased in IgG4-related submandibular gland disease. Based on these findings, we note mast cells exhibiting strong cytoplasmic staining for IgE as a key role in IgG4-RD. However, the underlying mechanisms and immunological significance of strong cytoplasmic IgE staining remain unclear.We previously reported a significant upregulation of IL-4, IL-10, IL-13, and transforming growth factor beta 1 in IgG4-related submandibular gland diseaseIn this study, we aimed to confirm whether mast cells exhibiting strong cytoplasmic staining for IgE were noted in IgG4-related lymphadenopathy cases and to identify the significance of the strong cytoplasmic staining.n = 23) of non-specific lymphoid hyperplasia were used as disease controls. All slides were jointly reviewed by K.N., M.T., and Y.S. Formalin-fixed paraffin-embedded specimens were used for immunohistochemistry and dual immunofluorescence. All tissue samples were obtained with the approval of the Institutional Review Board of Okayama University . Informed consent for the use of their samples in research was obtained from all patients.Tissue samples were obtained from 23 cases of IgG4-related lymphadenopathy. Tissue samples from an equal number of cases  with non-specific lymphoid hyperplasia. The specimens were fixed in 10.0% formaldehyde and embedded in paraffin. Serial 4-µm-thick sections were cut from the paraffin-embedded tissue blocks and stained with hematoxylin and eosin. Sections were immunohistochemically stained using an automated Bond Max stainer . The following primary antibodies were used: polyclonal IgG , IgG4 , KIT/CD117 , IgE , and high affinity IgE receptor (FcεRI) . Following immunohistochemical staining, the number of IgG4- and IgG-positive cells were estimated in areas with the highest density of IgG4-positive cells. In accordance with the consensus statement on the pathological features of IgG4-RD3, three different high power fields (HPFs)  were examined to calculate the average number of IgG4-positive cells per HPF and the IgG4-positive/IgG-positive cell ratio. IgE- and FcεRI-positive cells were counted in three different HPFs  that were determined to have the highest density of positive cells. The average number of positive cells per HPF was calculated.Excisional biopsies were obtained from lymph node lesions in 23 patients with IgG4-related lymphadenopathy and an equal number of patients  were used at a dilution of 1:400. The stained specimens were examined using a conventional immunofluorescence microscope .U test in the Statistical Package for the Social Sciences for Windows, software version 14.0 . A P < 0.05 was considered statistically significant.Data are presented as the means and standard deviations. All statistical analyses were performed using the Mann-Whitney The clinicopathological characteristics of the patients with IgG4-related lymphadenopathy are summarized in Table We confirmed that the tissue specimens from all 23 cases with IgG4-RD showed typical histological features of progressively transformed germinal center (PTGC)-type IgG4-related lymphadenopathy Fig. .Figure 1The specimens exhibited multiple lymphoid follicle formations with moderate lymphocytic infiltration of interfollicular areas. Compared to the histological features of IgG4-related lymphadenopathy, those of non-specific lymphoid hyperplasia showed no PTGC, and less infiltration of plasma cells and/or eosinophils. Dermatopathic and autoimmune disease-associated lymphadenopathies, including rheumatoid arthritis, systemic lupus erythematosus, and Sjögren’s syndrome, were excluded.P = 0.369). The overall median age was 63 . There was no significant difference in sex between the two groups . Patients with IgG4-related lymphadenopathy had larger lymph nodes than patients with non-specific lymphoid hyperplasia . Lymphadenopathy occurred in the cervical region in 21 patients with IgG4-related lymphadenopathy and in 10 patients with non-specific lymphoid hyperplasia .The clinical differences between IgG4-related lymphadenopathy patients and those with non-specific lymphoid hyperplasia are shown in Table vs. 1.20 ± 1.79 cells per HPF, respectively; P = 0.007)   Fig. . Strongl11) Fig. .Figure 2In the IgG4-related lymphadenopathy specimens, IgE and FcεRI staining exhibited a cytoplasmic granular pattern Fig. . Intracy15. However, studies investigating the mechanism underlying cytokine activation in IgG4-RD are few. In this study, we focused on mast cells exhibiting strong cytoplasmic staining for IgE.Although many previous studies regarding IgG4-RD have been reported in the last decade, the pathogenesis of the disease remains unclear. Recent studies have reported that IgG4-RD is a Th2- and Treg-dominant disease, and the activation of these cytokines may be responsible for this disease17. Some patients with PTGC-type IgG4-related lymphadenopathy have normal serum IgG4 levels when measurements are obtained after biopsy, at which time there are no residual primary lesions18. In our current data, serum IgG4 levels in 16 cases were <135 mg/dl. Furthermore, the recurrence ratio of IgG4-RD has been reported at 21.3%17. Some patients with PTGC-type IgG4-related lymphadenopathy have also experienced either persistence or relapse of residual lymph nodes18. In this study, 10 of 23 patients progressed to the development of other lymph nodes or extranodal lesions with elevated levels of serum IgG4.Comprehensive diagnostic criteria consisted of elevated serum IgG4 concentrations (>135 mg/dl), but not in all cases9. Regarding the significance of strong cytoplasmic staining for IgE, we posit two hypotheses: overstaining and endocytosis.Here, we confirmed that mast cells exhibiting strong cytoplasmic staining for IgE, were increased in IgG4-related lymphadenopathy. This is consistent with our previous report of submandibular gland disease20. We previously reported that 52% of patients with IgG4-related lymphadenopathy also had allergic disease18. Our current study showed that 15 of 19 (78.9%) patients examined had allergic conditions. Furthermore, elevation of serum IgE levels, defined as IgE >250 IU/ml, was observed in more than half of patients with IgG4-RD17, suggesting that strong cytoplasmic staining for IgE might indicate only overstaining. However, this study confirmed that mast cells exhibiting strong cytoplasmic staining for FcεRI also increased, as did staining for IgE.IgG4-RD is frequently complicated by allergic conditions22. Accordingly, we added dual immunofluorescence assays, resulting in IgE and FcεRI staining exhibited as multiple cytoplasmic granules, some of which overlapped in IgG4-related lymphadenopathy. In contrast, staining in non-specific lymphoid hyperplasia was localized to the surface membrane. These results showed that the significance of strong cytoplasmic staining for IgE and FcεRI might reflect the internalization of IgE and FcεRI due to mast cell activation in IgG4-RD. Mast cells are activated by various biological substances , leading to degranulation and the production of mediators such as cytokines and eicosanoids23. Some previous studies have suggested that response to antigen presentation might be involved in the pathogenesis of IgG4-RD26. Our current study suggests that some antigens might activate mast cells. However, additional assays are needed to elucidate the functional significance of IgE and FcεRI internalization. Although previous reports clarified the internalized FcεRI trafficking in early endosomes23, no published reports have investigated the trafficking completely.Recent reports have shown that the activation of mast cells leads to internalization of IgE and FcεRI29. Mattoo et al.25 reported prominent clonal expansions of CD4+ SLAMF7+ cytotoxic T lymphocytes but not CD4+GATA3+ memory Th2 cells in patients with IgG4-RD. This strongly suggests that these cytotoxic T lymphocytes expanded in response to a specific causal antigen. Furukawa et al.29. suggested that M2 macrophages producing IL-33 are deeply involved in the activation of Th2 immune responses in IgG4-RD. Therefore, IgG4-RD might progress via the interconnected network of various Th subsets or immune cells.Other Th subsets (T follicular helper cells or CD4+ cytotoxic T lymphocytes) and other immune cells (M2 macrophages) have recently been reported to participate in the pathogenesis of IgG4-RDIn conclusion, we confirmed that mast cells exhibiting strong cytoplasmic staining for IgE and FcεRI were increased in IgG4-related lymphadenopathy, implying the activation of mast cells. Since mast cells “communicate” with various cells to produce a mediator, a better understanding of the role of mast cells could enable us to understand the pathogenesis of IgG4-RD.Supplementary Figure 1-2


Tor yingjiangensis Chen et Yang 2004, Tor qiaojiensis Wu et al. 1977, Garra qiaojiensis Wu et al. 1977, Garra bispinosa Zhang 2005, and Schizothorax oligolepis Huang 1985, originally described from the upper Irrawaddy (Ayeyarwaddy) River in China, are first reported herein as new records to Myanmar. Counts, measurements, descriptions, photographs, and distributions of the specimens of the five newly recorded species are provided.Freshwater fish from the Putao and Myitkyina areas were collected in three ichthyofaunal surveys of the Mali Hka River and tributaries in and around Khakaborazi National Park and Hponkanrazi Wildlife Sanctuary, Kachin State, from 2014-2016.  South and Southeast Asia are among the most speciose areas on the planet, containing 20% of all known freshwater vertebrate species and 25% of all known aquatic plants . There aOreinus cf. meridionalis , Garra aff. dulongensis (=Placocheilus dulongensis), and Pseudecheneis brachyurus.Putao (Hkamti Long) is the northern most district in Myanmar, and contains two protected areas, that is, Khakaborazi National Park and Hponkanrazi Wildlife Sanctuary . Twenty newly recorded species and at least three new species were reported by Oreoglanis hponkanensis was identified and published , Chinese Academy of Sciences (CAS), and Forest Research Institute (FRI) have organized continuous biological surveys in North Myanmar since 2014. After further study in the laboratory, identification of samples was confirmed by comparison with materials in the Kunming Natural History Museum of Zoology, Kunming Institute of Zoology (KIZ), CAS, Kunming, Yunnan, China. A new sisorid catfish species, ublished , and fivTor yingjiangensis  putitora: Material examined: SEABRI20140098-100, 3 ex., 70.5–81.5 mm SL, main stem of Mali Hka River, Irrawaddy Basin, Wurunga Village, Naung Mun Township, Putao District, Kachin State, Myanmar , collected by Xiao-Yong Chen and Tao Qin, 30 November to 7 December 2014; SEABRI20160086–089, 4 ex., 98.9–174.9 mm SL, from Myitkyina fish market, Myitkyina City, Kachin State, Myanmar, collected by Xiao-Yong Chen, Tao Qin and Shu-Sen Shu, 1 August 2016; SEABRI20160176, 1 ex., 75.5 mm SL, from Putao fish market, Putao District, Kachin State, Myanmar, collected by Xiao-Yong Chen, Tao Qin, and Shu-Sen Shu, 4 August 2016.Comparative material examined: Holotype: KIZ164401, 181 mm SL, paratypes: KIZ704404, KIZ764229, KIZ764235–236, 4 ex., 72.5–162 mm SL, Dayingjiang River (tributary of upper Irrawaddy River), Manyun Town, Yingjiang County, Yunnan, China; KIZ2006004189, 198.9 mm SL, Nanzhang River , Wangzishu Town, Longchuan County, Yunnan, China.Description: According to the original description of Tor yingjiangensis , V-LL 3 (vs. 3–3.5), circumpeduncular scales 10–12 (vs. 12), caudal-peduncle length 13.4%–15.8% SL (vs. 11.3–14.8), and caudal-peduncle depth 10.7%–12.7% SL (vs. 11.1–13.3).Tor (Tor) qiaojiensis Wu & Yao, in Material examined: Tor qiaojiensis, KIZ2014005930–931, 2 ex., 46.1–53.7 mm SL, stream near Rat Baw Village, Naung Mun Township, Putao District, Kachin State, Myanmar , collected by Xiao-Yong Chen and Tao Qin, 2 December 2014; KIZ2015006380–381, SEABRI20150277–281, SEABRI20150830–837, 15 ex., 62.8–189.7 mm SL, Zeyar Stream, Irrawaddy Basin, Zeyar Dan Village, Putao District, Kachin State, Myanmar , collected by Xiao-Yong Chen, Tao Qin and Shu-Sen Shu, 9 December 2015; SEABRI20150459–462, 4 ex., 58.2–62.8 mm SL, Nam Ru Stream, Irrawaddy Basin, War Sar Dan Village, Putao District, Kachin State, Myanmar , collected by Xiao-Yong Chen, Tao Qin, and Shu-Sen Shu, 17 December 2015; SEABRI20150464–466, 3 ex., 54.1–126.8 mm SL, Patheik Stream, Irrawaddy Basin, Putao District, Kachin State, Myanmar , collected by Xiao-Yong Chen, Tao Qin, and Shu-Sen Shu, 19 December 2015.Comparative material examined: KIZ2006012423–455, 39.9–104.8 mm SL, 33 ex., Guyong River , Guyong Town, Tengchong County, Yunnan, China; KIZ2006012418–422, 86.4–145.5 mm SL, 5 ex., Binglangjiang River , Guyong Town, Tengchong County, Yunnan, China.Description: Based on the literature , circumpeduncular scales 10 (vs. 10–12), head length 26.3%–31.4% SL (vs. 27.7–30.6), body depth 24.7%–28.1% SL (vs. 27.3–31.4), caudal-peduncle length 15.5%–17.4% SL (vs. 13.9–18.8), and caudal-peduncle depth 11.1%–12.5% SL (vs. 9.7–11).Garra qiaojiensis Wu & Yao, in Material examined: SEABRI20140185–193, 9 ex., 76.2–94.9 mm SL, main stem of Mali Hka River, Wurunga Village, Naung Mun Township, Putao District, Kachin State, Myanmar , collected by Xiao-Yong Chen and Tao Qin, 30 November to 7 December 2014; SEABRI20150231–234, SEABRI20150414, 5 ex., 118.8–165.9 mm SL, Zeyar Stream, Mali Hka Basin, Zeyar Dan Village, Putao District, Kachin State, Myanmar , collected by Xiao-Yong Chen, Tao Qin, and Shu-Sen Shu, 9 & 14 December 2015; KIZ2016007376-377, SEABRI20160188-190, 5 ex., 60.7–73.4 mm SL, from Putao fish market, Putao District, Kachin State, Myanmar, collected by Xiao-Yong Chen, Tao Qin, and Shu-Sen Shu, 6 August 2016.Comparative material examined: KIZ2005002680–694, 49.4–72.1 mm SL, 15 ex., Zhina River (tributary of Dayingjiang River), Zhina Town, Yingjiang County, Yunnan, China; KIZ2006011157, 92.9 mm SL, Binglangjiang River (upper Dayingjiang River), Guyong Town, Tengchong County, Yunnan, China; KIZ2006011169–174, 81.1–118.8 mm SL, 6 ex., Longchuanjiang River, Wuhe Town, Tengchong County, Yunnan, China; KIZ2006004475–476, 68.7–75.9 mm SL, 2 ex., Minglang River (tributary of Longchuanjiang River), Hehua Town, Tengchong County, Yunnan, China.Description: Specimens examined were identified as Garra qiaojiensis according to the following characters , collected by Xiao-Yong Chen and Tao Qin, 2 December 2014; SEABRI20150036–037, 2 ex., 79.6–96.5 mm SL, stream near Upper Chan Khaung Village, Putao District, Kachin State, Myanmar , collected by Xiao-Yong Chen, Tao Qin, and Shu-Sen Shu, 5 December 2015; SEABRI20150125–148, 23 ex., 69.6–95.3 mm SL  oligolepis: Mo, in Material examined: KIZ2015006382–383, SEABRI20150220–225, 283–291, 17 ex., 61.2–91.6 mm SL, Zeyar Stream, Irrawaddy Basin, Zeyar Dan Village, Putao District, Kachin State, Myanmar , collected by Xiao-Yong Chen, Tao Qin, and Shu-Sen Shu, 9 December 2015; SEABRI20150347, 350–351, 355, 362, 364, 375–377, 9 ex., 66.4–152.1 mm SL, upper tributary of Ponyin Stream, Zeyar Dan Village, Putao District, Kachin State, Myanmar , collected by Tao Qin and Shu-Sen Shu, 11 & 12 December 2015; SEABRI20150408–413, 6 ex., 72.6–84.4 mm SL, Ponyin Stream, Zeyar Dan Village, Putao District, Kachin State, Myanmar , collected by Xiao-Yong Chen, Tao Qin, and Shu-Sen Shu, 14 December 2015; SEABRI20150497–507, 11 ex., 57–100.5 mm SL, Zan Shaw Stream, War Sar Dan Village, Putao District, Kachin State, Myanmar , collected by Xiao-Yong Chen, Tao Qin, and Shu-Sen Shu, 18 December 2015.Comparative material examined: KIZ2016002803, 905–913, 920, 11 ex., 48.5–132.3 mm SL, Erganya River (tributary of Dayingjiang), Tongbiguan Town, Yingjiang County, Yunnan, China; KIZ2014005075-082, 62.3–91.6 mm SL, 8 ex., Dazhupeng River (tributary of Dayingjiang River), Zhina Town, Yingjiang County, Yunnan, China; KIZ2014004705–709, 48.8–71.6 mm SL, 5 ex., Zhongling River (tributary of Dayingjiang River), Zhina Town, Yingjiang County, Yunnan, China.Description: Combined with the diagnostic characters from the original description, the examined specimens can be distinguished as Schizothorax oligolepis based on the following characters: Body moderately elongate, head and caudal peduncle compressed, dorsal profile arched more than ventral , Yun-Hong Tan and Bin Yang from the Xishuangbanna Tropical Botanical Garden (XTBG), CAS, and Shwe Lone and Kyi Kyi Khaing from the Forest Research Institute, Forest Department, Ministry of Environmental Conservation and Forestry, Myanmar, for their contribution to the field survey. We especially thank Rui-Chang Quan and Ren Li from XTBG, CAS, for their heartfelt support in the field.


Current organizational guidelines regarding use of antibiotics during urinary tract catheterization are based on limited evidence and are not directly applicable to the pediatric urology population. We seek to improve understanding of this population by first evaluating current practices. This study aims to investigate practice patterns and attitudes of pediatric urologists regarding the use of antibiotics in the setting of urinary tract catheterization.An online survey was sent to members of the Society for Pediatric Urology. Questionnaire sections included demographics, general questions about antibiotic use with catheterization, and specific clinical scenarios. Descriptive statistics were used, and chi-square analysis was performed to examine associations between demographics and specific responses.p = 0.02, p = 0.03), but no other significant associations between demographic characteristics and antibiotic use were found.Of 448 pediatric urologists surveyed, 154 (34%) responded to the survey. A majority of surveyed urologists (78%) prescribe daily prophylactic antibiotics with a hypospadias stent in place, but extensive variation in use of antibiotics was reported with other catheters and tubes. Extensive variation in practice patterns was also reported for three case scenarios regarding antibiotic prophylaxis with catheterization. Urologists > 50 years of age and fellowship-trained urologists were more likely to prescribe antibiotics for hypospadias stents  contains supplementary material, which is available to authorized users. This manuscript was presented at the American Urological Association annual meeting in 2016 (MP55–15), and is published in abstract form [Catheter-associated urinary tract infections (CAUTIs) are the most common nosocomial infection in the United States and lead to increased cost of care as well as patient length-of-stay, morbidity, and mortality . Recent Alongside pressure to decrease infectious complications there has been an increasing focus on the problems caused by antibiotic resistance . Accordihttp://project-redcap.org) electronic data capture tools hosted at Northwestern University [A 20-item online questionnaire regarding the use of prophylactic antibiotics with urinary catheterization was sent to 315 active and affiliate members of the Society for Pediatric Urology (SPU). The original request was sent via email in August, 2015 and two follow-up reminders were sent in September, 2015. Questionnaire sections included: (1) Demographics, (2) General questions about antibiotic use with urinary catheterization, and (3) Specific clinical scenarios .p = 0.28). The majority of respondents (91%) were fellowship-trained in pediatric urology or were currently in fellowship. Sixty-six percent of respondents practiced in an academic setting, while the remainder practiced in private practice or as a hospital employee.Of 448 members of the SPU surveyed, 154 (34%) responded to the survey (Table The majority of respondents (78%) prescribe prophylactic antibiotics the entire time a hypospadias stent is in place Fig. . HoweverThe majority of respondents do not obtain urine cultures prior to removal of a hypospadias stent (90%), Foley catheter (75%), PCN (59%), SPT (69%), or internal double-J ureteral stent (67%) Fig. . If a urSixty-one percent of respondents reported prescribing no prophylactic antibiotics for patients undergoing a voiding cystourethrogram, while the remainder (39%) prescribe prophylactic antibiotics at least some of the time Fig. . SimilarSubstantial variation in practice patterns was reported for three case scenarios with indwelling catheters and no evidence of infection Fig. . Full caFor an 8-year old male undergoing a direct visual internal urethrotomy and a subsequent indwelling catheter for 3 days, 66% of respondents prescribe antibiotics the entire time the catheter is in place, while 18% would prescribe antibiotics only at catheter removal, and 9% would prescribe no antibiotics.For a 12-year old female being discharged from the hospital with a newly created continent catheterizable channel and an indwelling catheter through the channel for 2 weeks, 47% of respondents would prescribe antibiotics the entire time the catheter is in place, while 13% would prescribe antibiotics only at catheter removal, and 34% would prescribe no antibiotics.Finally, for a 2-year old female with ureteropelvic junction obstruction and a PCN for 2 weeks, 32% of respondents prescribe antibiotics the entire time the PCN is in place, while 34% would prescribe antibiotics only at PCN removal, and 27% would prescribe no antibiotics.Twenty-five percent of respondents reported having a patient with a serious complication (requiring an intensive care unit or an invasive procedure) or death related to a CAUTI. Thirty-one percent of these respondents reporting changing their practice based on this event, while 59% reported not changing their practice based on this event, and 10% reported being unsure if their practice changed based on this event.p = 0.02; 95% vs. 79%, p = 0.03). Respondents who reported changing practice patterns based on a serious complication or death related to CAUTI were more likely to prescribe antibiotics > 50% of the time with a ureteral double-J stent and for the entire time the catheter is in place for Case Scenario #2 . There was also a possible association noted for respondents who reported changing practice patterns based on an infectious complication to prescribe antibiotics > 50% of the time with a SPC, but this was not statistically significant . There was no difference in prescribing pattern based on gender, size of practice, practice setting, or AUA section.Urologists > 50 years of age and fellowship-trained urologists were more likely to prescribe antibiotics > 50% of the time for hypospadias stents . In addition, there was a possible association between respondents who changed their practice based on an infectious complication and more antibiotic use in patients with a SPC (p = 0.06). This suggests that the lack of evidence regarding antibiotic use in these populations may allow the availability bias of a prior adverse event to drive decision making.Interestingly, in our study, respondents who reported changing their practice patterns based on an infectious complication were significantly more likely to prescribe antibiotics for internal double-J stents and for a newly created catheterizable channel .In our study, urologists > 50 years of age and fellowship-trained urologists were more likely to prescribe antibiotics at least 50% of the time for hypospadias stents. Several studies have investigated the topic of antibiotic use with hypospadias stents, which may be why this was the only scenario with a clear consensus in our study, and especially for older urologists and fellowship-trained urologists, who may be more familiar with the literature. In 2004, Meir et al. reported a decreased risk of complicated UTI and a non-significant suggestion of a decreased rate of urethrocutanous fistula formation with antibiotic use while a hypospadias stent is in place . HoweverThe relative lack of evidence guiding antibiotic use with catheterization in the pediatric population may explain the practice variation reported in our study. While there is some evidence that antibiotic use at the time of catheter removal can decrease symptomatic UTIs in adults , 23, theVariation in antibiotic use in our study may also be explained by a lack of specific management guidelines in this area. Organizational guidelines from the AUA, EAU, and others have limited applicability to the pediatric urology population, although they include the option to give antibiotics in complex scenarios. The AUA Best Practice Policy Statement on Urologic Surgery Antimicrobial Prophylaxis recommends antibiotic prophylaxis for removal of external urinary catheters and for urodynamics if risk factors such as urinary anatomic abnormalities, immunodeficiency, externalized catheters, colonized exogenous or endogenous material, and prolonged hospitalization are present . Many peAlthough current guidelines include the option to give antibiotics in complex patients, there remains little guidance on which of these patients will actually benefit from antibiotic use. Our study shows that these patients are managed much differently by different practitioners – some with antibiotics, and some without. The risks of overuse of antibiotics are well-documented and include cost, unnecessary drug exposure, potential for allergic reactions, and antimicrobial resistance , 4. ClinSeveral limitations of this study should be acknowledged. First, only one-third of SPU members responded to our survey; however, this response rate is similar to other previous SPU surveys, and all sections of the AUA were represented in our sample , 31. AlsOur results indicate there is substantial variation in practice patterns among surveyed pediatric urologists regarding prophylactic antibiotic use with urinary catheterization and minor lower urinary tract procedures such as urodynamics, retrograde urethrogram, and voiding cystourethrogram. This lack of consensus in current management of these complex patients highlights the need for further research in this area and for the development of management guidelines for this unique population.


Inappropriate use of antibiotics increases resistance and reduces their effectiveness. Despite evidence-based guidelines, antibiotics are still commonly used to treat infections likely caused by respiratory viruses. In this study, we examined the temporal relationships between antibiotic usage and respiratory infections in the Republic of Korea.The number of monthly antibiotic prescriptions and the incidence of acute respiratory tract infections between 2010 and 2015 at all primary care clinics were obtained from the Korean Health Insurance Review and Assessment Service. The monthly detection rates of respiratory viruses, including adenovirus, respiratory syncytial virus, influenza virus, human coronavirus, and human rhinovirus, were collected from Korea Centers for Disease Control and Prevention. Cross-correlation analysis was conducted to quantify the temporal relationship between antibiotic use and respiratory virus activities as well as respiratory infections in primary clinics.p < 0.01), 0.46 (p < 0.01), 0.40 (p < 0.01), and 0.35 (< 0.01), respectively. Furthermore, a significant correlation was found between acute bronchitis and antibiotics, including penicillin , macrolides , and quinolones , at the 0-month lag.The monthly use of different classes of antibiotic, including penicillins, other beta-lactam antibacterials, macrolides and quinolones, was significantly correlated with influenza virus activity. These correlations peaked at the 0-month lag with cross-correlation coefficients of 0.45 (Our findings suggest that there is a significant temporal relationship between influenza virus activity and antibiotic use in primary clinics. This relationship indicates that interventions aimed at reducing influenza cases in addition to effort to discourage the prescription of antibiotics by physicians may help to decrease unnecessary antibiotic consumption. Overuse and inappropriate use of antibiotics drive the emergence and spread of antimicrobial resistance , 2. In tTo prevent overuse and inappropriate use of antibiotics, it is essential to identify and understand antibiotic prescribing patterns and determining factors, however, little is known about antibiotic prescribing patterns in the Republic of Korea. The purpose of this study was to describe antibiotic prescription patterns in primary care clinics over a 6-year period and to identify its temporal relationship with respiratory viruses and ARTIs.International Classification of Diseases, Clinical Modification, 10th Revision [ICD-10-CM]), and prescription drugs [The National Health Insurance covers 98% of the total Korean population, providing near- complete coverage of all antibiotic prescriptions in the Republic of Korea. Reimbursement data from over 80,000 healthcare service providers in Korea were collected from the Korean Health Insurance Review and Assessment Service (KHIRA). The data covers 46 million patients annually, approximately 90% of the population of the Republic of Korea, and includes patients’ diagnoses  from the Korea Centers for Disease Control and Prevention. KINRESS collects nasopharyngeal specimens from patients with acute respiratory symptoms, including cough, rhinorrhea, and sore throat, from sentinel primary care clinics. This weekly laboratory-based surveillance system has been in operation since 2009 to measure respiratory virus activity at the community level, including adenovirus (ADV), influenza virus , human coronavirus , human rhinovirus (hRV), and respiratory syncytial virus . Laboratory confirmation of respiratory pathogens was performed using multiplex polymerase chain reaction (PCR) or real-time reverse transcription PCR , 14.We obtained the monthly number of ARTI diagnoses between 2010 and 2015 from the KHIRA database using ICD-10-CM codes. All patients diagnosed with an ARTI, regardless of age or gender, were included. ARTIs were defined as acute bronchitis and acute upper respiratory tract infection. In addition, we included acute tonsillitis and pneumonia as comparators as they are more likely to require antibiotics than other ARTIs [We used regression analysis to describe the trends of antibiotic use, respiratory virus activity, and the incidence of ARTIs, including acute bronchitis, overall.To identify the temporal relationship between antibiotic prescriptions and respiratory virus activity and the incidence of ARTIs, we performed a cross-correlation function test. This cross-correlation test is widely used in identifying the time lags of onetime series (respiratory virus) with the possible predictors of another time series (antibiotic use) , 17.The Box-Jenkins method was applied to fit time-series data to seasonal autoregressive moving average models , 19. Stap-values were 2-sided and considered significant at p < 0.05.The statistical package R, version 3.2.4  was used for all statistical analysis. All p < 0.01) . For primary clinics, the prescribing rate was 25.2  DID in 2010 and 26.9  in 2015 with a tendency to increase (01) Fig. .Fig. 1a The most commonly used classes of antibiotic were penicillin , other beta-lactams antibacterials , macrolides , fluoroquinolones , tetracyclines , and aminoglycosides  Fig. .Mean annual detection rates of respiratory viruses fluctuated highly in 2011 and 2012, but was largely stable in the other years, though estimated ranges were relatively large. In 2010, 47%  of isolates had a virus, while only 39%  were detected in 2015 Fig. .Fig. 2a The most commonly detected respiratory viruses were hRV , IFV , ADV , RSV , and hCoV  Fig. .p < 0.01)  per 100,000 individuals in 2010 to 4612  per 100,000 individuals in 2015 (01) Fig. . The aveMonthly time series of antibiotic use, respiratory virus detection, and incidence of ARTIs are presented in Fig. p < 0.01). In bivariate analyses, antibiotic use rates for the 4 most commonly used antibiotics  were significantly cross-correlated with influenza virus detection at the 0-month lag with cross-correlation coefficients of 0.45 (p < 0.01), 0.46 (p < 0.01), 0.40 (p < 0.01), and 0.35 (< 0.01), respectively (Table p = 0.04).The total monthly rate of antibiotic prescriptions was highly cross-correlated with the monthly detection rate of influenza virus  for penicillins, 0.69 (p < 0.01) for other beta-lactam antibacterials, 0.74 (p < 0.01) for macrolides and 0.45 (p < 0.01) for fluoroquinolones (Table p < 0.01), other beta-lactam antibacterials , macrolides , and fluoroquinolones  without a lag. Again, no cross-correlation was found between classes of antibiotics with lower-use rates (< 2 DID) and ARTIs.For ARTIs, the correlation coefficiencts of antibiotic use and the incidence of acute bronchitis were 0.73 (es Table . Acute up < 0.01), macrolides , aminoglycosides , and other beta-lactam antibacterials  without a lag. Furthermore, acute tonsillitis was significantly correlated with penicillin (0.69. p < 0.01), other beta-lactam antibacterials , macrolides , and fluoroquinolones  without a lag.For comparators that were more likely to require antibiotics than ARTIs, pneumonia was significantly correlated with penicillins  was significantly associated with the change in the activity of influenza in the community. Seasonal variation of antibiotic prescriptions has been documented in the United States , 20, CanAside from the correlation between hRV and tetracycline, other viruses were not significantly correlated with antibiotic use. This may be due to the low numbers of antibiotic use against hRV.p = 0.05). This is likely because of the low incidence of pneumonia . This is not surprising as pneumonia is an uncommon diagnosis in the outpatient setting compared to acute bronchitis . Pneumonia is a quite severe infection and often requires hospitalization for confirmation. Thus, many acute bronchitis prescriptions might reflect uncertainty on the clinician part as to whether the patient may have pneumonia, and are prescribing out of an abundance of caution despite the potential downside consequences of unnecessary antibiotic use.Regarding the cross-correlation between influenza virus activity and the incidence of pneumonia, no significant temporal relationship was found within a 1-month lag  at the 0-month lag. This result is consistent with previous literature documenting the most common cause of tonsillitis is viral infection including influenza virus [Regarding the relationship between influenza virus activity and the incidence of acute tonsillitis, a significant cross-correlation was found  and influenza virus activity as well as the incidence of acute bronchitis and acute upper respiratory tract infections. Our results indicate that interventions aimed at reducing influenza infections and discouraging the use of antibiotics by physicians and the public may help to decrease antibiotic consumption. Additional studies, including precise evaluations of the Korean Influenza National Immunization Program, on antibiotic prescription patterns, may identify additional opportunities to reduce antibiotic prescriptions.