Degradability of creatinine under sewer conditions affects its potential to be used as biomarker in sewage epidemiology

Creatinine was proposed to be used as a population normalising factor in sewage epidemiology but its stability in the sewer system has not been assessed. This study thus aimed to evaluate the fate of creatinine under different sewer conditions using laboratory sewer reactors. The results showed that while creatinine was stable in wastewater only, it degraded quickly in reactors with the presence of sewer biofilms. The degradation followed first order kinetics with significantly higher rate in rising main condition than in gravity sewer condition. Additionally, daily loads of creatinine were determined in wastewater samples collected on Census day from 10 wastewater treatment plants around Australia. The measured loads of creatinine from those samples were much lower than expected and did not correlate with the populations across the sampled treatment plants. The results suggested that creatinine may not be a suitable biomarker for population normalisation purpose in sewage epidemiology, especially in sewer catchment with high percentage of rising mains.

The first application of wastewater-based drug epidemiology in five South Korean cities

Illicit drug consumption in five cities in South Korea was estimated by analyzing 17 drug residues in untreated wastewater samples collected during the Christmas and New Year period of 2012-13. Only methamphetamine, amphetamine, and codeine were detected at concentrations of tens of nanograms per liter or even lower concentrations in more than 90% of the samples. Other illicit drug residues (including cocaine, methadone, and benzoylecgonine) that have been detected frequently in wastewater from other countries were not found in this study. Methamphetamine was found to be the most widely used illicit drug in South Korea, and the estimated average consumption rate was 22 mg d−1 (1000 people)−1. This rate is, for example, 2-5 times lower than the estimated average consumption rates in Hong Kong and other parts of China and 4-80 times lower than the estimated average consumption rates in cities in Western countries. It should be noted that the wastewater samples analyzed in this study were collected during a holiday season, when daily consumption of illicit drugs is often higher than on an average day. The methamphetamine usage rates were calculated for different cities in South Korea, and the usage rates in smaller cities was higher (2-4 times) than the average.

Using quantitative wastewater analysis to measure daily usage of conventional and emerging illicit drugs at an annual music festival

Introduction and Aims Wastewater analysis provides a non-intrusive way of measuring drug use within a population. We used this approach to determine daily use of conventional illicit drugs [cannabis, cocaine, methamphetamine and 3,4-methylenedioxymethamphetamine (MDMA)] and emerging illicit psychostimulants (benzylpiperazine, mephedrone and methylone) in two consecutive years (2010 and 2011) at an annual music festival. Design and Methods Daily composite wastewater samples, representative of the festival, were collected from the on-site wastewater treatment plant and analysed for drug metabolites. Data over 2 years were compared using Wilcoxon matched-pair test. Data from 2010 festival were compared with data collected at the same time from a nearby urban community using equivalent methods. Results Conventional illicit drugs were detected in all samples whereas emerging illicit psychostimulants were found only on specific days. The estimated per capita consumption of MDMA, cocaine and cannabis was similar between the two festival years. Statistically significant (P < 0.05; Z = −2.0–2.2) decreases were observed in use of methamphetamine and one emerging illicit psychostimulant (benzyl piperazine). Only consumption of MDMA was elevated at the festival compared with the nearby urban community. Discussion and Conclusions Rates of substance use at this festival remained relatively consistent over two monitoring years. Compared with the urban community, drug use among festival goers was only elevated for MDMA, confirming its popularity in music settings. Our study demonstrated that wastewater analysis can objectively capture changes in substance use at a music setting without raising major ethical issues. It would potentially allow effective assessments of drug prevention strategies in such settings in the future.

Challenges and opportunities in using wastewater analysis to measure drug use in a small prison facility

Introduction and Aims Wastewater analysis (WWA) is intended to be a direct and objective method of measuring substance use in large urban populations. It has also been used to measure prison substance use in two previous studies. The application of WWA in this context has raised questions as to how best it might be used to measure illicit drug use in prisons, and whether it can also be used to measure prescription misuse. We applied WWA to a small regional prison to measure the use of 12 licit and illicit substances. We attempted to measure the non-medical use of methadone and buprenorphine and to compare our findings with the results of the prison's mandatory drug testing (MDT). Design and Methods Representative daily composite samples were collected for two periods of 12 consecutive days in May to July 2013 and analysed for 18 drug metabolites. Prescription data and MDT results were obtained from the prison and compared with the substance use estimates calculated from WWA data. Results Daily use of methamphetamine, methadone, buprenorphine and codeine was detected, while sporadic detection of ketamine and methylone was also observed. Overall buprenorphine misuse appeared to be greater than methadone misuse. Discussion and Conclusions Compared with MDT, WWA provides a more comprehensive picture of prison substance use. WWA also has the potential to measure the misuse of medically prescribed substances. However, a great deal of care must be exercised in quantifying the usage of any substance in small populations, such as in prisons.

A Model to Estimate the Population Contributing to the Wastewater Using Samples Collected on Census Day

An important uncertainty when estimating per capita consumption of, for example, illicit drugs by means of wastewater analysis (sometimes referred to as “sewage epidemiology”) relates to the size and variability of the de facto population in the catchment of interest. In the absence of a day-specific direct population count any indirect surrogate model to estimate population size lacks a standard to assess associated uncertainties. Therefore, the objective of this study was to collect wastewater samples at a unique opportunity, that is, on a census day, as a basis for a model to estimate the number of people contributing to a given wastewater sample. Mass loads for a wide range of pharmaceuticals and personal care products were quantified in influents of ten sewage treatment plants (STP) serving populations ranging from approximately 3500 to 500 000 people. Separate linear models for population size were estimated with the mass loads of the different chemical as the explanatory variable: 14 chemicals showed good, linear relationships, with highest correlations for acesulfame and gabapentin. De facto population was then estimated through Bayesian inference, by updating the population size provided by STP staff (prior knowledge) with measured chemical mass loads. Cross validation showed that large populations can be estimated fairly accurately with a few chemical mass loads quantified from 24-h composite samples. In contrast, the prior knowledge for small population sizes cannot be improved substantially despite the information of multiple chemical mass loads. In the future, observations other than chemical mass loads may improve this deficit, since Bayesian inference allows including any kind of information relating to population size.

Could wastewater analysis be a useful tool for China? — A review

Analysing wastewater samples is an innovative approach that overcomes many limitations of traditional surveys to identify and measure a range of chemicals that were consumed by or exposed to people living in a sewer catchment area. First conceptualised in 2001, much progress has been made to make wastewater analysis (WWA) a reliable and robust tool for measuring chemical consumption and/or exposure. At the moment, the most popular application of WWA, sometimes referred as sewage epidemiology, is to monitor the consumption of illicit drugs in communities around the globe, including China. The approach has been largely adopted by law enforcement agencies as a device to monitor the temporal and geographical patterns of drug consumption. In the future, the methodology can be extended to other chemicals including biomarkers of population health (e.g. environmental or oxidative stress biomarkers, lifestyle indicators or medications that are taken by different demographic groups) and pollutants that people are exposed to (e.g. polycyclic aromatic hydrocarbons, perfluorinated chemicals, and toxic pesticides). The extension of WWA to a huge range of chemicals may give rise to a field called sewage chemical-information mining (SCIM) with unexplored potentials. China has many densely populated cities with thousands of sewage treatment plants which are favourable for applying WWA/SCIM in order to help relevant authorities gather information about illicit drug consumption and population health status. However, there are some prerequisites and uncertainties of the methodology that should be addressed for SCIM to reach its full potential in China.

Monitoring substance use in prisons: Assessing the potential value of wastewater analysis

Prison substance use is a major concern for prison authorities and the wider community. Australia has responded to this problem by implementing the National Corrections Drug Strategy. Across Australia, the true extent of prison substance use cannot be determined. As a result, the effectiveness of the interventions employed as part of this strategy cannot be properly assessed. This has important implications for the allocation of corrective services resources and future policy development. This article explores the benefits and limitations, as well as the ethical and practical issues in using wastewater analysis (WWA) to measure levels of substance use in prisons. It reports results from the first application of WWA to an Australian prison, which supports the use of WWA in this context. Given the increasing concern for prescription misuse in prisons, we also highlight the novel use of WWA to measure the extent of prescription misuse by prisoners. The article concludes that as a result of its objectivity, sensitivity and cost-effectiveness, the use of WWA in prisons warrants further consideration in Australia.

Effects of sewer conditions on the degradation of selected illicit drug residues in wastewater

The stability of five illicit drug markers in wastewater was tested under different sewer conditions using laboratory-scale sewer reactors. Wastewater was spiked with deuterium labelled isotopes of cocaine, benzoyl ecgonine, methamphetamine, MDMA and 6-acetyl morphine to avoid interference from the native isotopes already present in the wastewater matrix. The sewer reactors were operated at 20 °C and pH 7.5, and wastewater was sampled at 0, 0.25, 0.5, 1, 2, 3, 6, 9 and 12 h to measure the transformation/degradation of these marker compounds. The results showed that while methamphetamine, MDMA and benzoyl ecgonine were stable in the sewer reactors, cocaine and 6-acetyl morphine degraded quickly. Their degradation rates are significantly higher than the values reportedly measured in wastewater alone (without biofilms). All the degradation processes followed first order kinetics. Benzoyl ecgonine and morphine were also formed from the degradation of cocaine and 6-acetyl morphine, respectively, with stable formation rates throughout the test. These findings suggest that, in sewage epidemiology, it is essential to have relevant information of the sewer system (i.e. type of sewer, hydraulic retention time) in order to accurately back-estimate the consumption of illicit drugs. More research is required to look into detailed sewer conditions (e.g. temperature, pH and ratio of biofilm area to wastewater volume among others) to identify their effects on the fate of illicit drug markers in sewer systems.

Monitoring temporal changes in use of two cathinones in a large urban catchment in Queensland, Australia

Wastewater analysis was used to examine prevalence and temporal trends in the use of two cathinones, methylone and mephedrone, in an urban population (>200,000 people) in South East Queensland, Australia. Wastewater samples were collected from the inlet of the sewage treatment plant that serviced the catchment from 2011 to 2013. Liquid chromatography coupled with tandem mass spectrometry was used to measure mephedrone and methylone in wastewater sample using direct injection mode. Mephedrone was not detected in any samples while methylone was detected in 45% of the samples. Daily mass loads of methylone were normalized to the population and used to evaluate methylone use in the catchment. Methylone mass loads peaked in 2012 but there was no clear temporal trend over the monitoring period. The prevalence of methylone use in the catchment was associated with the use of MDMA, the more popular analogue of methylone, as indicated by other complementary sources. Methylone use was stable in the study catchment during the monitoring period whereas mephedrone use has been declining after its peak in 2010. More research is needed on the pharmacokinetics of emerging illicit drugs to improve the applicability of wastewater analysis in monitoring their use in the population.

Critical review on the stability of illicit drugs in sewers and wastewater samples

Wastewater-based epidemiology (WBE) applies advanced analytical methods to quantify drug residues in wastewater with the aim to estimate illicit drug use at the population level. Transformation processes during transport in sewers (chemical and biological reactors) and storage of wastewater samples before analysis are expected to change concentrations of different drugs to varying degrees. Ignoring transformation for drugs with low to medium stability will lead to an unknown degree of systematic under- or overestimation of drug use, which should be avoided. This review aims to summarize the current knowledge related to the stability of commonly investigated drugs and, furthermore, suggest a more effective approach to future experiments. From over 100 WBE studies, around 50 mentioned the importance of stability and 24 included tests in wastewater. Most focused on in-sample stability (i.e., sample preparation, preservation and storage) and some extrapolated to in-sewer stability (i.e., during transport in real sewers). While consistent results were reported for rather stable compounds (e.g., MDMA and methamphetamine), a varying range of stability under different or similar conditions was observed for other compounds (e.g., cocaine, amphetamine and morphine). Wastewater composition can vary considerably over time, and different conditions prevail in different sewer systems. In summary, this indicates that more systematic studies are needed to: i) cover the range of possible conditions in sewers and ii) compare results more objectively. To facilitate the latter, we propose a set of parameters that should be reported for in-sewer stability experiments. Finally, a best practice of sample collection, preservation, and preparation before analysis is suggested in order to minimize transformation during these steps.

Virulence characteristics and epidemiology of Yersinia enterocolitica and Yersiniae other than Y-pseudotuberculosis and Y-pestis isolated from water and sewage

Aims: To determine the species, bio-sero-phagetypes, antimicrobial drug resistance and also the pathogenic potential of 144 strains of Yersinia spp. isolated from water sources and sewage in Brazil.Methods and Results: the 144 Yersinia strains were characterized biochemically, serologically and had their antibiotic resistance and phenotypic virulence markers determined by microbiological and serological standard techniques. The Y. enterocolitica strains related to human diseases were also tested for the presence of virulence genes, by the PCR technique. The isolates were classified as Y. enterocolitica, Y. intermedia, Y. frederiksenii, Y. kristensenii and Yersinia biochemically atypical. The 144 isolates belonged to various bio-serogroups. Half of the strains showed resistance to three or more drugs. The Y. enterocolitica strains related to human diseases exhibited phenotypic virulence characteristics and virulence genes.Conclusions: Water from various sources and sewage are contaminated with Yersinia spp. in Brasil. Among these bacteria, virulent strains of Y. enterocolitica were found, with biotypes and serogroups related to human diseases.Significance and Impact of the Study: This is the first documented description of the occurrence of pathogenic Y. enterocolitica in water sources and sewage in Brazil. The occurrence of virulence strains of Y. enterocolitica shows that the environment is a potential source of human infection by this species in this country.

Epidemiology and comparative analysis of Yersinia in Ireland

Yersiniosis is an acute or chronic enteric zoonosis caused by enteropathogenic Yersinia species. Although yersiniosis is predominantly associated with gastroenteric forms of infection, extraintestinal forms are often reported from the elderly or patients with predisposing factors. Yersiniosis is often reported in countries with cold and mild climates (Northern and Central Europe, New Zealand and North of Russian Federation). The Irish Health Protection Surveillance Centre (HPSC) currently records only 3-7 notified cases of yersiniosis per year. At the same time pathogenic Yersinia enterocolitica is recovered from pigs (main source of pathogenic Y. enterocolitica) at the levels similar to that observed in Yersinia endemic countries. Introduction of Yersinia selective culture procedures may increase Yersinia isolation rates. To establish whether the small number of notifications of human disease was an underestimate due to lack of specific selective culture for Yersinia we carried out a prospective culture study of faecal samples from outpatients with diarrhoea, with additional culture of appendix and throat swabs. Higher levels of anti-Yersinia seroprevalence than yersiniosis notification rates in endemic countries suggests that most yersiniosis cases are unrecognised by culture. Subsequently, in addition to a prospective culture study of clinical specimens, we carried out serological screening of Irish blood donors and environmental screening of human sewage. Pathogenic Yersinia strains were not isolated from 1,189 faeces samples, nor from 297 throat swabs, or 23 appendix swabs. This suggested that current low notification rates in Ireland are not due to the lack of specific Yersinia culture procedures. Molecular screening detected a wider variety of Y. enterocolitica-specific targets in pig slurry than in human sewage. A serological survey for antibodies against Yersinia YOP (Yersinia Outer Proteins) proteins in Irish blood donors found antibodies in 25%, with an age-related trend to increased seropositivity, compatible with the hypothesis that yersiniosis may have been more prevalent in Ireland in the recent past. Y. enterocolitica is a heterogeneous group of microorganisms that comprises strains with different degree of pathogenicity. Although non-pathogenic Y. enterocolitica lack conventional virulence factors, these strains can be isolated from patients with diarrhoea. Insecticidal Toxin Complex (ITC) and Cytolethal Distending Toxins can potentially contribute to the virulence of non-pathogenic Y. enterocolitica in the absence of other virulence factors. We compared distribution of ITC and CDT loci among pathogenic and non-pathogenic Y. enterocolitica. Additionally, to demonstrate potential pathogenicity of non-pathogenic Y. enterocolitica we compared their virulence towards Galleria mellonella larvae (a non-mammalian model of human bacterial infections) with the virulence of highly and mildly pathogenic Y. enterocolitica strains. Surprisingly, virulence of pathogenic and non-pathogenic Y. enterocolitica in Galleria mellonella larvae observed at 37°C did not correlate with their pathogenic potential towards humans. Comparative phylogenomic analysis detects predicted coding sequences (CDSs) that define host-pathogen interactions and hence providing insights into molecular evolution of bacterial virulence. Comparative phylogenomic analysis of microarray data generated in Y. enterocolitica strains isolated in the Great Britain from humans with diarrhoea and domestic animals revealed high genetic heterogeneity of these species. Because of the extensive human, animal and food exchanges between the UK and Ireland the objective of this study was to gain further insight into genetic heterogeneity and relationships among clinical and non-clinical Y. enterocolitica strains of various pathogenic potential isolated in Ireland and Great Britain. No evidence of direct transfer of strains between the two countries was found.

Population Normalization with Ammonium in Wastewater-Based Epidemiology: Application to Illicit Drug Monitoring

Fluctuations in ammonium (NH4+), measured as NH4-N loads using an ion-selective electrode installed at the inlet of a sewage treatment plant, showed a distinctive pattern which was associated to weekly (i.e., commuters) and seasonal (i.e., holidays) fluctuations of the population. Moreover, population size estimates based on NH4-N loads were lower compared to census data. Diurnal profiles of benzoylecgonine (BE) and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH) were shown to be strongly correlated to NH4-N. Characteristic patterns, which reflect the prolonged nocturnal activity of people during the weekend, could be observed for BE, cocaine, and a major metabolite of MDMA (i.e., 4-hydroxy-3-methoxymethamphetamine). Additional 24 h composite samples were collected between February and September 2013. Per-capita loads (i.e., grams per day per 1000 inhabitants) were computed using census data and NH4-N measurements. Normalization with NH4-N did not modify the overall pattern, suggesting that the magnitude of fluctuations in the size of the population is negligible compared to those of illicit drug loads. Results show that fluctuations in the size of the population over longer periods of time or during major events can be monitored using NH4-N loads: either using raw NH4-N loads or population size estimates based on NH4-N loads, if information about site-specific NH4-N population equivalents is available.

Molecular Epidemiology of Escherichia coli isolates from Environmental, Food and Clinical Sources

In the present study diversity of E. coli in the water samples of Cochin estuary were studied for a period of 3 years ranging from January 2010- December 2012. The stations were selected based on the closeness to satellite townships and waste input. Two of the stations (Chitoor and Thevara) were fixed upstream, two in the central part of the estuary namely Bolgatty and Off Marine Science Jetty, and one at the Barmouth. Diversity was assessed in terms of serotypes, phylogenetic groups and genotypes. Two groups of seafood samples such as fish and shellfish collected from the Cochin estuary were used for isolation of E. coli. One hundred clinical E. coli isolates were collected from one public health centre, one hospital and five medical labs in and around Cochin City, Kerala. From our results it was clear that pathogen cycling is occurring through food, water and clinical sources. Pathogen cycling through food is very common and fish and shellfish that harbour these strains might pose potential health risk to consumer. Estuarine environment is a melting pot for various kinds of wastes, both organic and inorganic. Mixing up of waste water from various sources such as domestic, industries, hospitals and sewage released into these water bodies resulting in the co-existence of E. coli from various sources thus offering a conducive environment for horizontal gene transfer. Opportunistic pathogens might acquire genes for drug resistance and virulence turning them to potential pathogens. Prevalence of ExPEC in the Cochin estuary, pose threat to people who use this water for fishing and recreation. Food chain also plays an important role in the transit of virulence genes from the environments to the human. Antibiotic resistant E. coli are widespread in estuarine water, seafood and clinical samples, for reasons well known such as indiscriminate use of antibiotics in animal production systems, aquaculture and human medicine. Since the waste water from these sources entering the estuary provides selection pressure to drug resistant mutants in the environment. It is high time that the authorities concerned should put systems in place for monitoring and enforcement to curb such activities. Microbial contamination can limit people’s enjoyment of coastal waters for contact recreation or shellfish-gathering. E. coli can make people sick if they are present in high levels in water used for contact recreation or shellfish gathering. When feeding, shellfish can filter large volumes of seawater, so any microorganisms present in the water become accumulated and concentrated in the shellfish flesh. If E. coli contaminated shellfish are consumed the impact to human health includes gastroenteritis, urinary tract infections (UTIs), and bacteraemia. In conclusion, the high prevalence of various pathogenic serotypes and phylogenetic groups, multidrug-resistance, and virulence factor genes detected among E. coli isolates from stations close to Cochin city is a matter of concern, since there is a large reservoir of antibiotic resistance genes and virulence traits within the community, and that the resistance genes and plasmid-encoded genes for virulence were easily transferable to other strains. Given the severity of the clinical manifestations of the disease in humans and the inability and/or the potential risks of antibiotic administration for treatment, it appears that the most direct and effective measure towards prevention of STEC and ExPEC infections in humans and ensuring public health may be considered as a priority.