Predictors of concurrent heroin use among patients on opioid maintenance treatment in France: a multilevel study over 11 years.

BACKGROUND: Consistent reports from health professionals suggest that heroin is commonly used by patients undergoing opioid maintenance treatment (OMT) in France, potentially jeopardizing their recovery process. However, there has been no formal epidemiological assessment on the matter. METHODS: We use a yearly updated compendium retrieving information on patients admitted in treatment centres in France between 2010 and 2020. Given the hierarchical nature of the data collection, we conduct 2-level modified Poisson regressions to estimate the risks of past month heroin use among patients on OMT. RESULTS: Despite an overall decreasing trend over time, heroin use among patients on OMT is indeed common, with half of patients declaring concurrent use. Our study unveils differentiated risks of heroin use vary according to the type of OMT, with patients on methadone more likely to use heroin compared to those on buprenorphine. The use of multilevel-related measures also uncovers high heterogeneity among patients' profiles, reflecting different stages in the treatment process, as well as differentiated practices across treatment centres. CONCLUSION: Opioid maintenance treatment is associated with heroin use, in particular when methadone is involved. The heterogeneity among patients on OMT should be given particular attention, as it underscores the need for tailored interventions.

Correlates of Stimulant Use among People Who Use Heroin Undergoing Treatment in Out-Patient Facilities in France, 2010-2020.

Background: Polydrug use has been implicated in driving a "fourth wave" of the overdose crisis in North America, specifically through concurrent use of stimulants and opioids, especially fentanyl. In France, however, heroin has historically been and remains the easiest-to-access opioid, accounting for most drug treatment demand. Whether similar polydrug use is increasing in Western Europe remains understudied, despite severe health implications and potential inadequate public health responses.Methods: We take advantage of a nation-wide dataset containing information on all patients serviced in treatment centers in France from 2010 to 2020. We conduct Poisson regression to determine the main predictors of stimulant use among people who use heroin (PWUH) and opioids (PWUO) generally.Results: Heroin remains the primary opioid within drug treatment in France. A decreasing number of out-patients seeking treatment for heroin use has been accompanied by an increasing trend of stimulant use over time, most commonly with powder cocaine. Our results suggest a significant increase of crack cocaine use among the most vulnerable PWUH. Concurrent use of stimulants among PWUH was positively associated with use of alcohol, cannabis, unprescribed psychotropics and hallucinogens, and negatively with tobacco. Similar results were found for all in-treatment PWUO.Conclusions: Our results uncover heterogeneity in the profiles of PWUH that should be fully acknowledged to ensure better efficiency in substance use clinical practices and policy, while simultaneously drawing attention to trends in concurrent opioid-stimulant use outside North America. We advocate for an extension of the generalized risk framework and its implementation in prevention programs.

A detailed workflow to develop QIIME2-formatted reference databases for taxonomic analysis of DNA metabarcoding data.

BACKGROUND: The DNA metabarcoding approach has become one of the most used techniques to study the taxa composition of various sample types. To deal with the high amount of data generated by the high-throughput sequencing process, a bioinformatics workflow is required and the QIIME2 platform has emerged as one of the most reliable and commonly used. However, only some pre-formatted reference databases dedicated to a few barcode sequences are available to assign taxonomy. If users want to develop a new custom reference database, several bottlenecks still need to be addressed and a detailed procedure explaining how to develop and format such a database is currently missing. In consequence, this work is aimed at presenting a detailed workflow explaining from start to finish how to develop such a curated reference database for any barcode sequence. RESULTS: We developed DB4Q2, a detailed workflow that allowed development of plant reference databases dedicated to ITS2 and rbcL, two commonly used barcode sequences in plant metabarcoding studies. This workflow addresses several of the main bottlenecks connected with the development of a curated reference database. The detailed and commented structure of DB4Q2 offers the possibility of developing reference databases even without extensive bioinformatics skills, and avoids 'black box' systems that are sometimes encountered. Some filtering steps have been included to discard presumably fungal and misidentified sequences. The flexible character of DB4Q2 allows several key sequence processing steps to be included or not, and downloading issues can be avoided. Benchmarking the databases developed using DB4Q2 revealed that they performed well compared to previously published reference datasets. CONCLUSION: This study presents DB4Q2, a detailed procedure to develop custom reference databases in order to carry out taxonomic analyses with QIIME2, but also with other bioinformatics platforms if desired. This work also provides ready-to-use plant ITS2 and rbcL databases for which the prediction accuracy has been assessed and compared to that of other published databases.

Community- and individual-level risk factors of past month e-cigarette use among adolescents in France.

PURPOSE: Studies of adolescent e-cigarette use infrequently consider how environmental effects impact use. Adolescent e-cigarette use in France is also understudied, yet an important contrast since e-cigarette use rarely precedes conventional tobacco use and daily tobacco use is common. We examine whether there is significant variation in e-cigarette use across the geographic unit of départements (n = 95), and whether community factors explain these differences and individual-level probabilities of e-cigarette use. METHODS: The ESCAPAD survey is a cross-sectional, nationally representative survey collected at a day of civic and military information mandatory for French 17-year-olds. We use the 2014 (n = 22,023) and 2017 (n = 39,115) surveys and geographic information from Eurostat and INSEE. Multilevel, multiple logistic regression models examine any and daily past month e-cigarette use. RESULTS: We find significant département-level variation in both outcomes, with a considerable proportion of this variation explained by département-level factors. Net of numerous significant individual-level covariates, département-level unemployment (OR = 1.049, p < .05), poverty (OR=0.975, p < .05), age structure (OR=0.720, p < .01), and population growth (OR=0.987 p < .01) were associated with any past month use. The département-level percentage of adolescents using conventional tobacco daily was associated with individual-level any (OR=1.029, p < .001) and daily (OR=1.033, p < .01) e-cigarette use. Predicted probabilities demonstrate that département-level and individual-level tobacco use together were associated with e-cigarette use. CONCLUSIONS: Researchers should incorporate community effects into studies of e-cigarette use. Particularly, the tobacco use environment contributes to risk of e-cigarette use. For policymakers, resources may be mobilized to address local socioeconomic, demographic, and tobacco use patterns to potentially affect adolescent e-cigarette use.

Detection of ornamental transgenic fish by real-time PCR and fluorescence microscopy.

Numbers of ornamental transgenic fish are increasing, and some of them are illegally imported into Europe. The fish are modified to display different fluorescent colors under UV light. In this article, we propose real-time PCR methods to detect fish presenting green, yellow and red fluorescent coloring. The methods were tested with success and were able to detect illegally imported fish in two European countries. The article also discusses some practical information that can be useful for routine analysis. In addition, a real-time PCR test able to highlight the presence of fish DNA in general is proposed to check the amplifiability of the DNA extracted from common bony fish species of the teleost subclass. Finally, as the testing by PCR can take several days and rapid decisions must be taken with living organisms, we explored the potential of fluorescence microscopy as a screening test to determine whether animals are suspect or can be released.

Increasing socioeconomic disparities in tobacco smoking decline among French adolescents (2000-2017).

BACKGROUND: This paper studies the evolution of transitions from first cigarette use to daily use by socioeconomic status (SES) among French adolescents over the course of 17 years, in a context of decreasing prevalence of tobacco use. METHODS: A total of 182 266 adolescents participated in the nationally representative ESCAPAD survey at nine different time points between 2000 and 2017. Discrete time-event analysis was used to model the transition to daily cigarette use as a function of SES, gender, age at onset and the use of other psychoactive substances. RESULTS: Although lifetime cigarette smoking and daily cigarette smoking decreased significantly over the studied time span, suggesting a positive impact of prevention policies, disadvantaged adolescents were consistently more prone to engage in daily cigarette smoking, more so in 2017 than 15 years earlier. In the same time span, transitions from initiation to daily cigarette smoking have shortened, with an accelerated pace among underprivileged adolescents. CONCLUSIONS: Accelerated transitions from initiation to daily cigarette use are a prevalent trend among disadvantaged adolescents in France. Efforts to mitigate the impact of marketing strategies and to promote health literacy should be pursued to reduce social inequalities in health.

Estimating the size of crack cocaine users in France: Methods for an elusive population with high heterogeneity.

BACKGROUND: Despite several sources corroborating an expanding market and increased visibility and greater diversity in users' profiles, very little is known about the number of crack cocaine users in France. METHOD: The estimates rely on a single data source capture-recapture method. Annual data are extracted from treatment centres nationwide. To account for heterogeneity, we use an innovative zero-truncated geometric, regression-based estimator controlling for individual and centre characteristics. We use the well-known Zelterman estimator as a benchmark. RESULTS: The number of crack cocaine users received in treatment centres increased dramatically, from 3388 in 2010 to 5143 in 2017 (+52%). The estimated number of crack cocaine users is believed to have tripled over the course of the same period (from 9775, 95% CI [8288-11530] to 28983 [24876-33766], respectively), with prevalences below 1‰, similar to other European countries. The coverage rate (observed number/estimated number of users) decreased in a similar fashion, indicating lower utilization. In particular, females and younger users are underestimated by data from treatment centres. CONCLUSION: The prevalence of crack cocaine use is fairly low but steadily increasing. The diversity in users' profiles is a challenge to prevention and public health policies that should expand their scope to a more inclusive perspective of what defines crack cocaine users. Our method overcomes several methodological issues (data sources, data linkage, heterogeneity) and can be easily applied to a wide range of settings.

Doping among high school students: findings from the French ESPAD survey.

BACKGROUND: The increasing use of doping by youth is a growing public health concern. The present study aimed to calculate robust estimates of the prevalence of doping among French high school students and study factors related to the use of licit vs. banned agents. METHODS: The European School Project on Alcohol and other Drugs (ESPAD), a nationally representative school-based survey, was distributed across France from April to May 2015, generating a final sample size of 6642 students. Multilevel modelling was used to examine covariates of doping to enhance athletic performance in sport in this population. RESULTS: Overall, 2.3% of students used banned agents, and 6.1% used licit and banned substances to improve athletic performance. Significant gender differences existed for both, with males more prone to doping than females. Our findings provide support for the existing gateway hypothesis that nutritional supplementation leads to doping banned agents. Multilevel modelling revealed the presence of both school and class-level influences on individual use of licit substances, vs. solely class-level factors impacting the use of banned agents, suggesting a strong peer-effect. CONCLUSIONS: These findings support the need to engage in early doping prevention through high schools. Programmes should draw attention to the role of licit substances, including nutritional supplementation, in the progression to using banned agents and encompass the continuum of adolescent risk taking through a behaviour-based approach to doping prevention.

Age of Initiation and Patterns of Use among People Who Inject Drugs Welcomed in Harm Reduction Facilities in France from 2006 to 2015.

This exploratory analysis of time-series cross-sectional data provides insights on trends in age at first injection among people who inject drugs in France, and on associations with recent risky injecting behaviors. Data were collected from a national survey conducted in harm reduction facilities in five phases between 2006 and 2015. Standardized questionnaires collected information on demographics, substance use, and route of administration, as well as lifetime and past-month injection. Descriptive and multilevel models were applied to account for the hierarchical structure of the data. Prevalence of lifetime and past-month injection remained stable over time, while the prevalence of daily injection increased significantly. Mean age at first injection only appeared to increase for data collected after 2010. Gender differences in mean age at first injection decreased over time, suggesting the development of converging patterns of initiation independent of sex. After controlling for covariates, early initiation of injection was unrelated to daily injection or material sharing, and associated with the number of recently injected substances. Early initiation is likely a predictor of injected polysubstance use. Findings are relevant to the planning, implementation, and evaluation of prevention programs.

On the question of non-medical cognitive enhancers among in-school adolescents: prevalence, predictors and potential health-related harms.

Little is known about the use of non-medical cognitive enhancers (NCEs) in the general population, and even less among youth. The study utilises a nationally representative, cross-sectional survey of adolescents attending high schools to provide a comprehensive overview of NCEs and to assess risk factors such as socio-demographics, schooling, mental health and related substance use among French adolescents. A total of 6692 students attending high school (secondary schools) answered an anonymous questionnaire collecting information on demographics, health, psychoactive substance uses (neuroleptics, tobacco, alcohol, cannabis, illicit substances) and patterns of sociability. The use of cognitive enhancers appears to be an underestimated phenomenon among youth. Prevalence of use is heavily gender-influenced, with females twice as likely to use NCEs than males. More than daily school commitments, the use of cognitive enhancers is related to the proximity of the national secondary education examination. Moreover, mental health, use of prescribed anxiolytics and other psychoactive substances are significantly independently associated with the use of cognitive enhancers, particularly among females. The unregulated use of cognitive enhancers is a predictor of potential mental frailty and a substance-based response to stressful events, a behaviour likely to persist during adulthood. The study underpins the lack of contextual and comparable data. Systematic monitoring of younger students in neighbouring countries is required to develop reliable prevention programmes.

Revisiting Factors Associated With Screen Time Media Use: A Structural Study Among School-Aged Adolescents.

BACKGROUND: Screen-based media overuse has been related to harmful consequences especially among children and adolescents. Given their complex interrelationships, predictors of screen time (ST) should be analyzed simultaneously rather than individually to avoid incomplete conclusions. METHODS: Structural equation models were conducted to examine associations between media ST (television, video games, and computers) along with harmful consequences in adolescents' well-being, such as underweight and overweight, depression, and school failure. Predictors included individual (gender, age, and physical activity), family (structure and socioeconomic background), and substance use variables. We used the Health Behaviour in School-aged Children survey organized in 2014, including eighth- and ninth-grade students living in France (N = 3720). RESULTS: Students reported spending 3 hours per day in front of each media. Spending more than 2 hours behind each of those 3 media was associated with lower life satisfaction, less physical activity, active school bullying, and grade repetition. Socioeconomic status was the most important predictor of ST, whereas regular substance uses showed modest associations. CONCLUSION: The main implication of our findings is to sensitize parents and stakeholders about the limitation of ST, including their own use that adolescents are likely to mimic. Alternative measures such as off-line time should be encouraged.

Estimating the number of people who inject drugs: a proposal to provide figures nationwide and its application to France.

Background: This article provides an original method of estimating the prevalence of injecting drug users (IDU) in France. Method: In order to overcome legal restrictions hampering data linkage, the method relies on a single-source capture-recapture framework and makes use of individual data collected by a standardized survey conducted within treatment centres. Taking the hierarchical structure of data collection into consideration, a multilevel Zelterman regression model was used. Results: In 2014, last year IDU were thought to be 103 800 (85 300; 130 000), prevalence of 2.6‰ (2.1; 3.2), and last month IDU 86 000 (69 200; 110 400), prevalence of 2.2‰ (1.7; 2.7). Estimates have been broken down by gender, revealing a 3 to 1 ratio of male-to-female; and age, showing young adults less prone to regular use of injection. It is thought that one IDU out of three was seen in treatment centres in 2014. Conclusion: The number of last month IDU has remained stable when compared with the last available figures calculated in 2006. Nowadays IDU include heroin, buprenorphine, polydrug and stimulant users. This greater diversity ought to be taken into account by prevention policies, and underlines the need for perennial monitoring of IDU.

Detection by real-time PCR and pyrosequencing of the cry1Ab and cry1Ac genes introduced in genetically modified (GM) constructs.

The presence of genetically modified organisms (GMOs) in food and feed is mainly detected by the use of targets focusing on promoters and terminators. As some genes are frequently used in genetically modified (GM) construction, they also constitute excellent screening elements and their use is increasing. In this paper we propose a new target for the detection of cry1Ab and cry1Ac genes by real-time polymerase chain reaction (PCR) and pyrosequencing. The specificity, sensitivity and robustness of the real-time PCR method were tested following the recommendations of international guidelines and the method met the expected performance criteria. This paper also shows how the robustness testing was assessed. This new cry1Ab/Ac method can provide a positive signal with a larger number of GM events than do the other existing methods using double dye-probes. The method permits the analysis of results with less ambiguity than the SYBRGreen method recommended by the European Reference Laboratory (EURL) GM Food and Feed (GMFF). A pyrosequencing method was also developed to gain additional information thanks to the sequence of the amplicon. This method of sequencing-by-synthesis can determine the sequence between the primers used for PCR. Pyrosequencing showed that the sequences internal to the primers present differences following the GM events considered and three different sequences were observed. The sensitivity of the pyrosequencing was tested on reference flours with a low percentage GM content and different copy numbers. Improvements in the pyrosequencing protocol provided correct sequences with 50 copies of the target. Below this copy number, the quality of the sequence was more random.

Estimating the Number of Heroin Users in Metropolitan France Using Treatment Centers Data: An Exploratory Analysis.

BACKGROUND: An updated prevalence estimate for heroin users in France is lacking. Accurate figures are needed to estimate the potential need for treatment. METHOD: This research relies on a capture-recapture framework and, for the first time, makes use of individual data collected by a standardized survey conducted within treatment centers. Taking the hierarchical structure of data collection into consideration, a multilevel Zelterman regression model was used, thus enabling estimates at national level. RESULTS: The number of heroin users was estimated to be 76,000, 95% CI = [68,000; 85,000] in 2009 (prevalence of 1.9‰ [1.7-2.1]), 101,000 in 2011, 95% CI = [66,000-137,000] (prevalence of 2.5‰ [1.6-3.4]), and 107,000 in 2013, 95% CI = [85,000-124,000] (prevalence of 2.6‰ [2.1-3.1]), with a 4:1 ratio of male:female. Less than one heroin users out of three was seen in treatment centers in 2013. CONCLUSIONS: The rise in the number of heroin users is related to an increasing availability of heroin, increasing use in more remote areas, and providers targeting new types of users. The figures, however, should be completed with alternative estimates and enhance the need for a perennial monitoring.

Determinants of heroin retail prices in metropolitan France: Discounts, purity and local markets.

INTRODUCTION AND AIMS: Field studies have indicated a recent increase in heroin availability and use in France, and yet very little is known about the mechanisms underlying heroin retail prices. This paper offers a first attempt at identifying the determinants of heroin pricing, to measure quantity discounts and assess the influence of purity on street prices, while controlling for a geographical effect. DESIGN AND METHODS: Data on heroin samples were collected during 2011 in seven urban areas of metropolitan France. Ordinary least squares regression was used to model the associations between price, quantity, purity and other independent variables. RESULTS: Quantity remains the most influential variable on heroin pricing. We estimate that a 10% increase in the size of a transaction leads to a 2.3% decrease in the unit price. Assessed purity proved to be significant, although in modest proportion. Sociodemographic characteristics, such as gender, users' experience and relationships with dealers, proved to be insignificant. Heroin retail prices vary according to a geographical gradient related to the routes of entry and distribution. DISCUSSION AND CONCLUSIONS: As a credence good, heroin retail prices in France are affected by more than simply the traditional supply and demand relationship. The results of this study also underline the limitations of a quantitative framework and should be complemented by further ethnographic research to obtain an in-depth understanding of local markets. Policies should be designed to better take local disparities into account.[Lahaie E, Janssen E, Cadet-Taïrou A. Determinants of heroin retail prices in metropolitan France:Discounts, purity and local markets. Drug Alcohol Rev 2016;35:597-604].

The GMOseek matrix: a decision support tool for optimizing the detection of genetically modified plants.

BACKGROUND: Since their first commercialization, the diversity of taxa and the genetic composition of transgene sequences in genetically modified plants (GMOs) are constantly increasing. To date, the detection of GMOs and derived products is commonly performed by PCR-based methods targeting specific DNA sequences introduced into the host genome. Information available regarding the GMOs' molecular characterization is dispersed and not appropriately organized. For this reason, GMO testing is very challenging and requires more complex screening strategies and decision making schemes, demanding in return the use of efficient bioinformatics tools relying on reliable information. DESCRIPTION: The GMOseek matrix was built as a comprehensive, online open-access tabulated database which provides a reliable, comprehensive and user-friendly overview of 328 GMO events and 247 different genetic elements (status: 18/07/2013). The GMOseek matrix is aiming to facilitate GMO detection from plant origin at different phases of the analysis. It assists in selecting the targets for a screening analysis, interpreting the screening results, checking the occurrence of a screening element in a group of selected GMOs, identifying gaps in the available pool of GMO detection methods, and designing a decision tree. The GMOseek matrix is an independent database with effective functionalities in a format facilitating transferability to other platforms. Data were collected from all available sources and experimentally tested where detection methods and certified reference materials (CRMs) were available. CONCLUSIONS: The GMOseek matrix is currently a unique and very valuable tool with reliable information on GMOs from plant origin and their present genetic elements that enables further development of appropriate strategies for GMO detection. It is flexible enough to be further updated with new information and integrated in different applications and platforms.

Opposite social gradient for alcohol use and misuse among French adolescents.

BACKGROUND: This study investigates the association of the family occupational category (F-OC) with adolescent alcohol use and its potential variation according to the frequency of use. METHODS: A national survey representative of adolescents aged 17 living in continental France conducted in 2005 (n = 29,393). Three outcomes were considered: overall use describes the drinking status (lifetime abstinence, use before the month prior the survey, use in the month prior the survey) without considering the frequency of use; last month use and binge drinking detail the frequency of use (1-5 uses, 6-9, 10-19 and 20+ uses) and of binge drinking (0, 1-2, 3-5, 6+ episodes of 5+ glasses in a single occasion) of the previous month users. F-OC was described in 7 categories based on the highest occupational category of the parents (from managers/professionals to unemployed). Analysis used generalised logistic regressions, controlling for gender, F-OC, parental separation, autonomy, other substance use, being out of school and sociability. RESULTS: There was a double gradient: adolescents from high F-OC families were more often experimenters and drinkers during the previous month whereas those of low F-OC families were more often binge drinkers. Adolescents from farmers' families were the most at risk for frequent use and binge drinking in the last month. Interactions tests show that the effect of F-OC was not significantly related to gender. CONCLUSIONS: Except for gender, adolescents' patterns of use reflect those observed in the adult population. Mechanisms that favour and hinder progression in alcohol use should be studied in various socioeconomic groups.

Social gradient in initiation and transition to daily use of tobacco and cannabis during adolescence: a retrospective cohort study.

AIMS: This study explores whether the family socio-economic status (F-SES) and school situation could have an impact on tobacco and cannabis initiation and transition to daily use during adolescence. DESIGN AND SETTING: A French cross-sectional nation-wide survey conducted in 2005 containing retrospective data. PARTICIPANTS: French teenagers aged 17 (n = 29 393). MEASUREMENT: The F-SES was defined by the highest occupational category of either parent, with seven categories ranging from unemployed/inactive to managers/professionals. Ages at repeat school years, at leaving school, at the first episode of drunkenness and at initiation of illicit drug use were used to model tobacco and cannabis initiation and transition to daily use with time-discrete logistic regressions. FINDINGS: The risk for tobacco initiation was almost equally distributed across F-SES groups, but the risk of a progression to daily use was higher in every F-SES category compared to managers/professionals [odds ratio (OR) from 1.17 to 1.90]. Compared to managers/professionals, risk of cannabis initiation was lower in all F-SES categories (OR from 0.63 to 0.87), but all categories except farmers were at increased risk of transition to daily use: the OR range between 1.29 (intermediate) and 1.98 (unemployed/inactive). Repeating school years and leaving school predicted daily use of tobacco (OR = 2.00 and 2.37) and cannabis (4.58 and 2.07). CONCLUSIONS: Adolescents from the highest family socio-economic status categories are at risk for tobacco and cannabis experimentation but are less prone to engage in daily use. Psychological and social mechanisms that inhibit transition to daily use should be investigated, including school attainment and performance.

Drug-related deaths in France in 2007: estimates and implications.

Compared with other European countries, France's low number of drug use-related deaths may appear enviable. Previous studies have suggested significant underreporting. The three official records listing drug-related deaths in France in 2007 were cross-checked. Different capture-recapture techniques were applied. The estimated numbers of drug-related deaths differ from the official figures. Different explanations and proposals to improve the recordings are consequently discussed. However, there are doubts as to the reliability of at least one of the records and the consequent accuracy of the proportion of overlapping cases. Estimates should be handled with caution and interpreted as upper bounds.

Design of multiplex calibrant plasmids, their use in GMO detection and the limit of their applicability for quantitative purposes owing to competition effects.

Five double-target multiplex plasmids to be used as calibrants for GMO quantification were constructed. They were composed of two modified targets associated in tandem in the same plasmid: (1) a part of the soybean lectin gene and (2) a part of the transgenic construction of the GTS40-3-2 event. Modifications were performed in such a way that each target could be amplified with the same primers as those for the original target from which they were derived but such that each was specifically detected with an appropriate probe. Sequence modifications were done to keep the parameters of the new target as similar as possible to those of its original sequence. The plasmids were designed to be used either in separate reactions or in multiplex reactions. Evidence is given that with each of the five different plasmids used in separate wells as a calibrant for a different copy number, a calibration curve can be built. When the targets were amplified together (in multiplex) and at different concentrations inside the same well, the calibration curves showed that there was a competition effect between the targets and this limits the range of copy numbers for calibration over a maximum of 2 orders of magnitude. Another possible application of multiplex plasmids is discussed.