The prevalence of having coeliac disease in children with type 1 diabetes was not significantly higher during the Swedish coeliac epidemic.

AIM: From 1986 to 1996, there was a four-fold increase in coeliac disease among young Swedish children, known as the Swedish coeliac epidemic. Children with type 1 diabetes have an increased risk of developing coeliac disease. We studied whether the prevalence of coeliac disease differed in children with type 1 diabetes born during and after this epidemic. METHODS: We compared national birth cohorts of 240 844 children born in 1992-1993 during the coeliac disease epidemic and 179 530 children born in 1997-1998 after the epidemic. Children diagnosed with both type 1 diabetes and coeliac disease were identified by merging information from five national registers. RESULTS: There was no statistically significant difference in the prevalence of coeliac disease among children with type 1 diabetes between the two cohorts: 176/1642 (10.7%, 95% confidence interval 9.2%-12.2%) in the cohort born during the coeliac disease epidemic versus 161/1380 (11.7%, 95% confidence interval 10.0%-13.5%) in the post-epidemic cohort. CONCLUSION: The prevalence of having both coeliac disease and type 1 diabetes was not significantly higher in children born during, than after, the Swedish coeliac epidemic. This may support a stronger genetic disposition in children who develop both conditions.

Influenza A(H7N9) virus acquires resistance-related neuraminidase I222T substitution when infected mallards are exposed to low levels of oseltamivir in water.

Influenza A virus (IAV) has its natural reservoir in wild waterfowl, and new human IAVs often contain gene segments originating from avian IAVs. Treatment options for severe human influenza are principally restricted to neuraminidase inhibitors (NAIs), among which oseltamivir is stockpiled in preparedness for influenza pandemics. There is evolutionary pressure in the environment for resistance development to oseltamivir in avian IAVs, as the active metabolite oseltamivir carboxylate (OC) passes largely undegraded through sewage treatment to river water where waterfowl reside. In an in vivo mallard (Anas platyrhynchos) model, we tested if low-pathogenic avian influenza A(H7N9) virus might become resistant if the host was exposed to low levels of OC. Ducks were experimentally infected, and OC was added to their water, after which infection and transmission were maintained by successive introductions of uninfected birds. Daily fecal samples were tested for IAV excretion, genotype, and phenotype. Following mallard exposure to 2.5 µg/liter OC, the resistance-related neuraminidase (NA) I222T substitution, was detected within 2 days during the first passage and was found in all viruses sequenced from subsequently introduced ducks. The substitution generated 8-fold and 2.4-fold increases in the 50% inhibitory concentration (IC50) for OC (P < 0.001) and zanamivir (P = 0.016), respectively. We conclude that OC exposure of IAV hosts, in the same concentration magnitude as found in the environment, may result in amino acid substitutions, leading to changed antiviral sensitivity in an IAV subtype that can be highly pathogenic to humans. Prudent use of oseltamivir and resistance surveillance of IAVs in wild birds are warranted.

Oseltamivir-resistant influenza A (H1N1) virus strain with an H274Y mutation in neuraminidase persists without drug pressure in infected mallards.

Influenza A virus (IAV) has its natural reservoir in wild waterfowl, and emerging human IAVs often contain gene segments from avian viruses. The active drug metabolite of oseltamivir (oseltamivir carboxylate [OC]), stockpiled as Tamiflu for influenza pandemic preparedness, is not removed by conventional sewage treatment and has been detected in river water. There, it may exert evolutionary pressure on avian IAV in waterfowl, resulting in the development of resistant viral variants. A resistant avian IAV can circulate among wild birds only if resistance does not restrict viral fitness and if the resistant virus can persist without continuous drug pressure. In this in vivo mallard (Anas platyrhynchos) study, we tested whether an OC-resistant avian IAV (H1N1) strain with an H274Y mutation in the neuraminidase (NA-H274Y) could retain resistance while drug pressure was gradually removed. Successively infected mallards were exposed to decreasing levels of OC, and fecal samples were analyzed for the neuraminidase sequence and phenotypic resistance. No reversion to wild-type virus was observed during the experiment, which included 17 days of viral transmission among 10 ducks exposed to OC concentrations below resistance induction levels. We conclude that resistance in avian IAV that is induced by exposure of the natural host to OC can persist in the absence of the drug. Thus, there is a risk that human-pathogenic IAVs that evolve from IAVs circulating among wild birds may contain resistance mutations. An oseltamivir-resistant pandemic IAV would pose a substantial public health threat. Therefore, our observations underscore the need for prudent oseltamivir use, upgraded sewage treatment, and surveillance for resistant IAVs in wild birds.

Does a gluten-free diet lead to better glycemic control in children with type 1 diabetes? Results from a feasibility study and recommendations for future trials.

BACKGROUND: Increasing evidence suggests a link between type 1 diabetes (T1D) and intake of gluten, but no controlled trials have examined whether a gluten-free diet (GFD) has positive effects on glycemic control in children with T1D. METHODS: We conducted a non-randomized feasibility study. Twenty-three children with newly diagnosed T1D were included and either followed a GFD (n = 14) or a normal diet (n = 9) for 12 months. Effects of diet on glycemic control were examined by measuring insulin production (c-peptide), hemoglobine A1c (HbA1c) and insulin dose adjusted A1c (IDAA1c). Degree of adherence to the GFD and effects on quality of life were also examined. RESULTS: Children on a GFD showed a statistically significantly lower HbA1c at six months (P = 0.042) compared with children on a normal diet and point estimate differences indicated better glycemic control in the GFD group at 6 and 12 months. Adherence to a GFD varied but was satisfactory for a majority of children. The GFD group reported poorer quality of life at inclusion and there was a non-significant difference for quality of life between groups throughout the study. CONCLUSIONS: A strict GFD can be maintained by children with newly diagnosed T1D and may have positive effects on glycemic control. Our findings should be interpreted carefully because of small samples and possible confounding. We provide recommendations for future trials and suggest using a randomized-controlled design with 30-40 participants in each arm.

Compliance to a Gluten-Free Diet in Swedish Children with Type 1 Diabetes and Celiac Disease.

Children with type 1 diabetes (T1D) are at increased risk of celiac disease (CD). The replacement of insulin in T1D, and the exclusion of gluten in CD, are lifelong, burdensome treatments. Compliance to a gluten-free diet (GFD) in children with CD is reported to be high, while compliance in children with both diseases has scarcely been studied. To examine compliance to a GFD in children with both T1D and CD, we analyzed tissue transglutaminase IgA-antibodies (tTGA). Moreover, associations between compliance and age, sex, glycemic control, ketoacidosis (DKA), body mass index (BMI), and time of CD diagnosis were investigated. Of the 743 children diagnosed with T1D in southern Sweden between 2005 and 2012, 9% were also diagnosed with CD. Of these, 68% showed good compliance to a GFD, 18% showed intermediate compliance, and 14% were classified as non-compliant. Higher age, poorer HbA1c, and more DKAs were significantly (p < 0.05) associated with poorer compliance. In conclusion, we found that compliance to a GFD in children with T1D and CD is likely be lower than in children with CD only. Our results indicate that children with both T1D and CD could need intensified dietary support and that older children and children with poor metabolic control are especially vulnerable subgroups.

Strategies for monitoring the emerging polar organic contaminants in water with emphasis on integrative passive sampling.

Although polar organic contaminants (POCs) such as pharmaceuticals are considered as some of today's most emerging contaminants few of them are regulated or included in on-going monitoring programs. However, the growing concern among the public and researchers together with the new legislature within the European Union, the registration, evaluation and authorisation of chemicals (REACH) system will increase the future need of simple, low cost strategies for monitoring and risk assessment of POCs in aquatic environments. In this article, we overview the advantages and shortcomings of traditional and novel sampling techniques available for monitoring the emerging POCs in water. The benefits and drawbacks of using active and biological sampling were discussed and the principles of organic passive samplers (PS) presented. A detailed overview of type of polar organic PS available, and their classes of target compounds and field of applications were given, and the considerations involved in using them such as environmental effects and quality control were discussed. The usefulness of biological sampling of POCs in water was found to be limited. Polar organic PS was considered to be the only available, but nevertheless, an efficient alternative to active water sampling due to its simplicity, low cost, no need of power supply or maintenance, and the ability of collecting time-integrative samples with one sample collection. However, the polar organic PS need to be further developed before they can be used as standard in water quality monitoring programs.

Contamination of surface, ground, and drinking water from pharmaceutical production.

Low levels of pharmaceuticals are detected in surface, ground, and drinking water worldwide. Usage and incorrect disposal have been considered the major environmental sources of these microcontaminants. Recent publications, however, suggest that wastewater from drug production can potentially be a source of much higher concentrations in certain locations. The present study investigated the environmental fate of active pharmaceutical ingredients in a major production area for the global bulk drug market. Water samples were taken from a common effluent treatment plant near Hyderabad, India, which receives process water from approximately 90 bulk drug manufacturers. Surface water was analyzed from the recipient stream and from two lakes that are not contaminated by the treatment plant. Water samples were also taken from wells in six nearby villages. The samples were analyzed for the presence of 12 pharmaceuticals with liquid chromatography-mass spectrometry. All wells were determined to be contaminated with drugs. Ciprofloxacin, enoxacin, cetirizine, terbinafine, and citalopram were detected at more than 1 microg/L in several wells. Very high concentrations of ciprofloxacin (14 mg/L) and cetirizine (2.1 mg/L) were found in the effluent of the treatment plant, together with high concentrations of seven additional pharmaceuticals. Very high concentrations of ciprofloxacin (up to 6.5 mg/L), cetirizine (up to 1.2 mg/L), norfloxacin (up to 0.52 mg/L), and enoxacin (up to 0.16 mg/L) were also detected in the two lakes, which clearly shows that the investigated area has additional environmental sources of insufficiently treated industrial waste. Thus, insufficient wastewater management in one of the world's largest centers for bulk drug production leads to unprecedented drug contamination of surface, ground, and drinking water. This raises serious concerns regarding the development of antibiotic resistance, and it creates a major challenge for producers and regulatory agencies to improve the situation.

The enzyme toilet rim block 'pCure' does not efficiently remove drug residues in a hospital setting - exemplifying the importance of on-site implementation testing.

Introduction: Negative environmental effects of active pharmaceutical ingredients (APIs) are increasingly recognized, especially concerning antibiotics, and hospitals are important point sources. "pCure" is a toilet rim block containing API-degrading enzymes; the producing company claims positive in vitro results but no implementation studies have been performed. Materials and methods: In a university hospital setting, 16 weeks were randomized to installation or no installation of pCure in all 261 toilets connected to the same cesspit where sewage water was sampled daily. Ninety-six samples were analyzed for 102 APIs using liquid chromatography/tandem mass spectrometry. Results and Discussion: Fifty-one APIs were detected with a large variation in levels but no significant differences in the initial statistical analysis. More statistical testing of API level ratios (pCure installed/not installed) yielded some cases of significant decrease. Differences were small and not consistent when comparing means and medians. We cannot exclude a small pCure effect but clearly pCure has no effect of biological importance. Conclusion: pCure is not useful to reduce drug residue discharge in a hospital setting. In a bigger perspective, our study exemplifies that products claiming to reduce an environmental problem need to be tested in on-site implementation studies by independent researchers before reaching the market.

Vaginal Bleeding in Prepubertal Girls: Etiology and Clinical Management.

STUDY OBJECTIVE: We aimed to investigate the etiology and clinical management of vaginal bleeding in girls aged 0-9 years and to compare our results with previous publications. DESIGN, SETTING, PARTICIPANTS, INTERVENTIONS, AND MAIN OUTCOME MEASURES: The records of all girls younger than 10 years of age who were seen between 2001 and 2011 at Skåne University Hospital Lund for vaginal bleeding were retrospectively collected. RESULTS: We identified 86 girls with vaginal bleeding. Of those, 47 (54.7%) were diagnosed with a local lesion, the etiology was hormonal in 16 (18.6%), and in 23 (26.7%) the etiology was unclear. Trauma was the most frequent cause of local lesions and hormonal withdrawal of the newborn was the most common hormonal etiology. Two girls were diagnosed with a tumor, 1 with relapse of a vaginal rhabdomyosarcoma, and the other with recurrence of an ovarian granulosa cell tumor. There were large disparities in the clinical management of vaginal bleeding. A genital examination was conducted in 70 of 86 (81.4%), and colposcopy in only 8 of 86 of the patients (9.3%). CONCLUSION: This study confirmed vaginal bleeding as a rare finding in girls younger than 10 years of age. It is usually a benign symptom, but because there might be a serious underlying condition, proper investigation and follow-up are needed. Clinical management varied in our patient cohort. This might be because of insufficient knowledge and might indicate the need for general guidelines.

Fate of three anti-influenza drugs during ozonation of wastewater effluents - degradation and formation of transformation products.

Anti-influenza drugs constitute a key component of pandemic preparedness plans against influenza. However, the occurrence of such drugs in water environments, the potential of resistance development in the natural hosts, and the risk for transmission of antiviral resistance to humans call for measures to increase removal in wastewater treatment plants (WWTPs). In this study, removal of three anti-influenza drugs; amantadine (AM), oseltamivir carboxylate (OC) and zanamivir (ZA), and formation/removal of their transformation products during ozonation of wastewater effluents from two Swedish WWTPs in Uppsala and Stockholm were studied. The removal profile of target antivirals and formation/removal of their transformation products were studied by liquid chromatography/high resolution mass spectrometry. 3.5 h of ozone exposure (total dose of ozone 5.95 g) led to complete removal of the three anti-influenza drugs with a degradation in the following order ZA > OC > AM. Two, five and one transformation products were identified and semi-quantified for AM, OC and ZA, respectively. Increasing and later decreasing transformation products concentration followed the decrease in concentration of target compounds. All transformation products detected, except one of AM in wastewater from Stockholm WWTP, were removed at the end of the experiment. The removal efficiency was higher for all studied compounds in wastewater from Uppsala WWTP, which had lower TOC and COD values, less phosphorus, and also higher pH in the water. Ozonation thus offers multiple benefits through its potential to degrade influenza antivirals, hence decrease the risk of environmental resistance development, in addition to degrading other pharmaceuticals and resistant microorganisms.

Oseltamivir Resistance in Influenza A(H6N2) Caused by an R292K Substitution in Neuraminidase Is Not Maintained in Mallards without Drug Pressure.

BACKGROUND: Wild waterfowl is the natural reservoir of influenza A virus (IAV); hosted viruses are very variable and provide a source for genetic segments which can reassort with poultry or mammalian adapted IAVs to generate novel species crossing viruses. Additionally, wild waterfowl act as a reservoir for highly pathogenic IAVs. Exposure of wild birds to the antiviral drug oseltamivir may occur in the environment as its active metabolite can be released from sewage treatment plants to river water. Resistance to oseltamivir, or to other neuraminidase inhibitors (NAIs), in IAVs of wild waterfowl has not been extensively studied. AIM AND METHODS: In a previous in vivo Mallard experiment, an influenza A(H6N2) virus developed oseltamivir resistance by the R292K substitution in the neuraminidase (NA), when the birds were exposed to oseltamivir. In this study we tested if the resistance could be maintained in Mallards without drug exposure. Three variants of resistant H6N2/R292K virus were each propagated during 17 days in five successive pairs of naïve Mallards, while oseltamivir exposure was decreased and removed. Daily fecal samples were analyzed for viral presence, genotype and phenotype. RESULTS AND CONCLUSION: Within three days without drug exposure no resistant viruses could be detected by NA sequencing, which was confirmed by functional NAI sensitivity testing. We conclude that this resistant N2 virus could not compete in fitness with wild type subpopulations without oseltamivir drug pressure, and thus has no potential to circulate among wild birds. The results of this study contrast to previous observations of drug induced resistance in an avian H1N1 virus, which was maintained also without drug exposure in Mallards. Experimental observations on persistence of NAI resistance in avian IAVs resemble NAI resistance seen in human IAVs, in which resistant N2 subtypes do not circulate, while N1 subtypes with permissive mutations can circulate without drug pressure. We speculate that the phylogenetic group N1 NAs may easier compensate for NAI resistance than group N2 NAs, though further studies are needed to confirm such conclusions.

Online solid phase extraction liquid chromatography using bonded zwitterionic stationary phases and tandem mass spectrometry for rapid environmental trace analysis of highly polar hydrophilic compounds - Application for the antiviral drug Zanamivir.

Zanamivir (Za) is a highly polar and hydrophilic antiviral drug used for the treatment of influenza A viruses. Za has been detected in rivers of Japan and it's environmental occurrence has the risk of inducing antiviral resistant avian influenza viruses. In this study, a rapid automated online solid phase extraction liquid chromatography method using bonded zwitterionic stationary phases and tandem mass spectrometry (SPE/LC-MS/MS) for trace analysis of Za was developed. Furthermore, an internal standard (IS) calibration method capable of quantifying Za in Milli-Q, surface water, sewage effluent and sewage influent was evaluated. Optimum pre-extraction sample composition was found to be 95/5 v/v acetonitrile/water sample and 1% formic acid. The developed method showed acceptable linearities (r(2)≥0.994), filtration recovery (≥91%), and intra-day precisions (RSD≤16%), and acceptable and environmentally relevant LOQs (≤20ngL(-1)). Storage tests showed no significant losses of Za during 20 days and +4/-20°C (≤12%) with the exception of influent samples, which should be kept at -20°C to avoid significant Za losses. The applicability of the method was demonstrated in a study on phototransformation of Za in unfiltered and filtered surface water during 28 days of artificial UV irradiation exposure. No significant (≤12%) phototransformation was found in surface water after 28 days suggesting a relatively high photostability of Za and that Za should be of environmental concern.

Intra- and inter-pandemic variations of antiviral, antibiotics and decongestants in wastewater treatment plants and receiving rivers.

The concentration of eleven antibiotics (trimethoprim, oxytetracycline, ciprofloxacin, azithromycin, cefotaxime, doxycycline, sulfamethoxazole, erythromycin, clarithromycin, ofloxacin, norfloxacin), three decongestants (naphazoline, oxymetazoline, xylometazoline) and the antiviral drug oseltamivir's active metabolite, oseltamivir carboxylate (OC), were measured weekly at 21 locations within the River Thames catchment in England during the month of November 2009, the autumnal peak of the influenza A[H1N1]pdm09 pandemic. The aim was to quantify the pharmaceutical response to the pandemic and compare this to drug use during the late pandemic (March 2010) and the inter-pandemic periods (May 2011). A large and small wastewater treatment plant (WWTP) were sampled in November 2009 to understand the differential fate of the analytes in the two WWTPs prior to their entry in the receiving river and to estimate drug users using a wastewater epidemiology approach. Mean hourly OC concentrations in the small and large WWTP's influent were 208 and 350 ng/L (max, 2070 and 550 ng/L, respectively). Erythromycin was the most concentrated antibiotic measured in Benson and Oxford WWTPs influent (max=6,870 and 2,930 ng/L, respectively). Napthazoline and oxymetazoline were the most frequently detected and concentrated decongestant in the Benson WWTP influent (1650 and 67 ng/L) and effluent (696 and 307 ng/L), respectively, but were below detection in the Oxford WWTP. OC was found in 73% of November 2009's weekly river samples (max=193 ng/L), but only in 5% and 0% of the late- and inter-pandemic river samples, respectively. The mean river concentration of each antibiotic during the pandemic largely fell between 17-74 ng/L, with clarithromycin (max=292 ng/L) and erythromycin (max=448 ng/L) yielding the highest single measure. In general, the concentration and frequency of detecting antibiotics in the river increased during the pandemic. OC was uniquely well-suited for the wastewater epidemiology approach owing to its nature as a prodrug, recalcitrance and temporally- and spatially-resolved prescription statistics.

Pathobiology and virus shedding of low-pathogenic avian influenza virus (A/H1N1) infection in mallards exposed to oseltamivir.

Low-pathogenic avian influenza (LPAI) viruses in wild birds are important as they can constitute the basis for the development of highly pathogenic avian influenza viruses or form part of human-adapted strains with pandemic potential. However, the pathogenesis of LPAI viruses is not well characterized in dabbling ducks, one of the natural reservoirs of LPAI viruses. Between 21 September 2009 and 21 December 2009, we used real-time reverse transcriptase polymerase chain reaction (q-PCR), histopathology, and immunohistochemistry (IHC) to study Mallards (Anas platyrhynchos) infected with an influenza A/H1N1 virus isolated from a wild Mallard in Sweden. The ducks were either inoculated intraesophageally ("artificial infection") or infected by virus shed by other ducks in the experiment ("contact infection"). The ducks were subjected to three low concentrations (80 ng/L, 1 µg/L, and 80 µg/L) of the active metabolite of oseltamivir (Tamiflu(®)), oseltamivir carboxylate (OC), which resulted in the development of the viral resistance mutation H274Y at 1 and 80 µg/L. The LPAI virus infection was localized to the intestinal tract and cloacal bursa except in one Mallard. The exception was a duck euthanized 1 day postinoculation, whose infection was located solely in the lung, possibly due to intratracheal deposition of virus. The intestinal infection was characterized by occasional degenerating cells in the lamina propria and presence of viral antigen as detected by IHC, as well as positive q-PCR performed on samples from feces and intestinal contents. Histopathologic changes, IHC positivity, and viral shedding all indicated that the infection peaked early, around 2 days postinfection. Furthermore, more viral antigen and viral RNA were detected with IHC and q-PCR in the proximal parts early in the infection. There was no obvious difference in the course of the infection in artificial versus contact infection, when the level of OC was increased from 80 ng/L to 1 µg/L (based on IHC and q-PCR), when the level of OC was increased to 80 µg/L, or when the resistance mutation H274Y developed (based on q-PCR).

Resistance mutation R292K is induced in influenza A(H6N2) virus by exposure of infected mallards to low levels of oseltamivir.

Resistance to neuraminidase inhibitors (NAIs) is problematic as these drugs constitute the major treatment option for severe influenza. Extensive use of the NAI oseltamivir (Tamiflu®) results in up to 865 ng/L of its active metabolite oseltamivir carboxylate (OC) in river water. There one of the natural reservoirs of influenza A, dabbling ducks, can be exposed. We previously demonstrated that an influenza A(H1N1) virus in mallards (Anas platyrhynchos) exposed to 1 µg/L of OC developed oseltamivir resistance through the mutation H274Y (N2-numbering). In this study, we assessed the resistance development in an A(H6N2) virus, which belongs to the phylogenetic N2 group of neuraminidases with distinct functional and resistance characteristics. Mallards were infected with A(H6N2) while exposed to 120 ng/L, 1.2 µg/L or 12 µg/L of OC in their sole water source. After 4 days with 12 µg/L of OC exposure, the resistance mutation R292K emerged and then persisted. Drug sensitivity was decreased ≈13,000-fold for OC and ≈7.8-fold for zanamivir. Viral shedding was similar when comparing R292K and wild-type virus indicating sustained replication and transmission. Reduced neuraminidase activity and decrease in recovered virus after propagation in embryonated hen eggs was observed in R292K viruses. The initial, but not the later R292K isolates reverted to wild-type during egg-propagation, suggesting a stabilization of the mutation, possibly through additional mutations in the neuraminidase (D113N or D141N) or hemagglutinin (E216K). Our results indicate a risk for OC resistance development also in a N2 group influenza virus and that exposure to one NAI can result in a decreased sensitivity to other NAIs as well. If established in influenza viruses circulating among wild birds, the resistance could spread to humans via re-assortment or direct transmission. This could potentially cause an oseltamivir-resistant pandemic; a serious health concern as preparedness plans rely heavily on oseltamivir before vaccines can be mass-produced.

Compliance to oseltamivir among two populations in Oxfordshire, United Kingdom affected by influenza A(H1N1)pdm09, November 2009--a waste water epidemiology study.

Antiviral provision remains the focus of many pandemic preparedness plans, however, there is considerable uncertainty regarding antiviral compliance rates. Here we employ a waste water epidemiology approach to estimate oseltamivir (Tamiflu®) compliance. Oseltamivir carboxylate (oseltamivir's active metabolite) was recovered from two waste water treatment plant (WWTP) catchments within the United Kingdom at the peak of the autumnal wave of the 2009 Influenza A (H1N1)pdm09 pandemic. Predictions of oseltamivir consumption from detected levels were compared with two sources of national government statistics to derive compliance rates. Scenario and sensitivity analysis indicated between 3-4 and 120-154 people were using oseltamivir during the study period in the two WWTP catchments and a compliance rate between 45-60%. With approximately half the collected antivirals going unused, there is a clear need to alter public health messages to improve compliance. We argue that a near real-time understanding of drug compliance at the scale of the waste water treatment plant (hundreds to millions of people) can potentially help public health messages become more timely, targeted, and demographically sensitive, while potentially leading to less mis- and un-used antiviral, less wastage and ultimately a more robust and efficacious pandemic preparedness plan.

Pyrosequencing of antibiotic-contaminated river sediments reveals high levels of resistance and gene transfer elements.

The high and sometimes inappropriate use of antibiotics has accelerated the development of antibiotic resistance, creating a major challenge for the sustainable treatment of infections world-wide. Bacterial communities often respond to antibiotic selection pressure by acquiring resistance genes, i.e. mobile genetic elements that can be shared horizontally between species. Environmental microbial communities maintain diverse collections of resistance genes, which can be mobilized into pathogenic bacteria. Recently, exceptional environmental releases of antibiotics have been documented, but the effects on the promotion of resistance genes and the potential for horizontal gene transfer have yet received limited attention. In this study, we have used culture-independent shotgun metagenomics to investigate microbial communities in river sediments exposed to waste water from the production of antibiotics in India. Our analysis identified very high levels of several classes of resistance genes as well as elements for horizontal gene transfer, including integrons, transposons and plasmids. In addition, two abundant previously uncharacterized resistance plasmids were identified. The results suggest that antibiotic contamination plays a role in the promotion of resistance genes and their mobilization from environmental microbes to other species and eventually to human pathogens. The entire life-cycle of antibiotic substances, both before, under and after usage, should therefore be considered to fully evaluate their role in the promotion of resistance.

Environmental levels of the antiviral oseltamivir induce development of resistance mutation H274Y in influenza A/H1N1 virus in mallards.

Oseltamivir (Tamiflu®) is the most widely used drug against influenza infections and is extensively stockpiled worldwide as part of pandemic preparedness plans. However, resistance is a growing problem and in 2008-2009, seasonal human influenza A/H1N1 virus strains in most parts of the world carried the mutation H274Y in the neuraminidase gene which causes resistance to the drug. The active metabolite of oseltamivir, oseltamivir carboxylate (OC), is poorly degraded in sewage treatment plants and surface water and has been detected in aquatic environments where the natural influenza reservoir, dabbling ducks, can be exposed to the substance. To assess if resistance can develop under these circumstances, we infected mallards with influenza A/H1N1 virus and exposed the birds to 80 ng/L, 1 µg/L and 80 µg/L of OC through their sole water source. By sequencing the neuraminidase gene from fecal samples, we found that H274Y occurred at 1 µg/L of OC and rapidly dominated the viral population at 80 µg/L. IC50 for OC was increased from 2-4 nM in wild-type viruses to 400-700 nM in H274Y mutants as measured by a neuraminidase inhibition assay. This is consistent with the decrease in sensitivity to OC that has been noted among human clinical isolates carrying H274Y. Environmental OC levels have been measured to 58-293 ng/L during seasonal outbreaks and are expected to reach µg/L-levels during pandemics. Thus, resistance could be induced in influenza viruses circulating among wild ducks. As influenza viruses can cross species barriers, oseltamivir resistance could spread to human-adapted strains with pandemic potential disabling oseltamivir, a cornerstone in pandemic preparedness planning. We propose surveillance in wild birds as a measure to understand the resistance situation in nature and to monitor it over time. Strategies to lower environmental levels of OC include improved sewage treatment and, more importantly, a prudent use of antivirals.

Detection of the antiviral drug oseltamivir in aquatic environments.

Oseltamivir (Tamiflu) is the most important antiviral drug available and a cornerstone in the defence against a future influenza pandemic. Recent publications have shown that the active metabolite, oseltamivir carboxylate (OC), is not degraded in sewage treatment plants and is also persistent in aquatic environments. This implies that OC will be present in aquatic environments in areas where oseltamivir is prescribed to patients for therapeutic use. The country where oseltamivir is used most is Japan, where it is used to treat seasonal flu. We measured the levels of OC in water samples from the Yodo River system in the Kyoto and Osaka prefectures, Japan, taken before and during the flu-season 2007/8. No OC was detected before the flu-season but 2-58 ng L(-1) was detected in the samples taken during the flu season. This study shows, for the first time, that low levels of oseltamivir can be found in the aquatic environment. Therefore the natural reservoir of influenza virus, dabbling ducks, is exposed to oseltamivir, which could promote the evolution of viral resistance.

The use of semipermeable membrane devices as passive samplers to determine persistent organic compounds in indoor air.

In the study reported here semipermeable membrane devices (SPMDs) were used to sample 28 PAHs and 19 PCBs in the gas phase in 15 single-family houses located in an area where domestic wood burning is widespread. Eight of the households used wood burning appliances whereas the others used other systems for residential heating. Most of the studied compounds were found in the houses: the PAHs at levels that were similar to or slightly higher than published SPMD-sampled levels for background or urban sites in Sweden, and the PCBs at levels that were somewhat lower than those recently found in both indoor and outdoor urban locations. A principal component analysis revealed that wood-burning heating systems may contribute to PAHs in indoor air. The sources may be emissions indoors or penetration from outdoors. The convenience of SPMD technology facilitates its use for semi-quantitative screening and monitoring of various persistent organic compounds indoors in dwellings and working environments.