Emerging evidence suggests that nonmedical use (NMU) of prescription attention deficit/hyperactivity disorder (ADHD) medications is rising, but many previous investigations have used clinical or regionally based samples or limited their investigations to stimulants rather than to medications specifically used to treat ADHD. Using an Internet-based epidemiological survey, this paper advances understanding of the prevalence and correlates of NMU of medications used to treat ADHD, sources of diverted medications, motivations for use, and consumption patterns.The study used a self-administered Internet survey of civilian, noninstitutionalized adults  aged 18 to 49 in the United States. National-level estimates were created using propensity scoring methods and weighting procedures using data from three nationally representative probability surveys: a random-digit dialed telephone survey, the current U.S. Census, and the National Survey on Drug Use and Health (NSDUH).Past-year prevalence of NMU of ADHD medications was approximately 2%, with 4.3% reported among those aged 18 to 25 and 1.3% among those aged 26 to 49. Most respondents reporting NMU used on multiple occasions. Receipt of medications for ADHD was a significant correlate of past-year NMU, though most nonmedical users never had a prescription. Among persons who had never been prescribed medication to treat ADHD, friends or family members were the most common source. Productivity was the most frequently endorsed reason for NMU. Alcohol was the substance most commonly used in combination with ADHD drugs.Because most prescription ADHD medications currently are highly regulated, policy options for supply-side reduction of nonmedical use may include identifying those medications with lower abuse liability for inclusion on insurance formularies. Patient and physician education programs also may be useful tools to heighten awareness of intentional and unintentional diversion of ADHD medications for nonmedical purposes. Clinician awareness of the presence and burden of attention deficit/hyperactivity disorder (ADHD) has increased in the United States and internationally -4. In paWhat is largely known about NMU of ADHD drugs is derived from data sources that may not focus specifically on ADHD medications or from samples with limited generalizability. Most of these investigations have grouped a variety of prescription stimulants  as a broad therapeutic class. A study that specifically identified the NMU of prescription ADHD stimulants was conducted by Kroutil and colleagues in 2006 using the 2002 National Survey on Drug Use and Health (NSDUH) . Except Other studies have observed higher rates of stimulant misuse among those aged 18 to 25, college students, and polydrug users . Past stCompared to studies examining the prevalence and correlates of nonmedical ADHD drug use, fewer studies have specifically examined dimensions of access and motivation for NMU of ADHD medications. McCabe et al.  reportedSome of these investigations used large, epidemiological surveillance systems  that offer considerable breadth but may lack sufficient depth for examining, in detail, more focused topics such as NMU of ADHD medications. In contrast, a small but insightful number of investigations focusing on patterns of diversion and motivation for NMU of ADHD medications have relied on small, regionally based or clinical samples ,21. The Internet surveys have emerged as an alternative data collection method when in-person or telephone surveys could be costly or time-consuming. Berrens et al.  list sevIn light of these issues, this diversion study reports findings using an existing opt-in Internet sample recruited from the entire United States for the following purposes: (1) provide estimates of NMU of ADHD medications by identifying specific medications used in the treatment of ADHD rather than using a general question about "prescription stimulants" commonly used, (2) identify the subpopulations  at greatest risk for NMU, (3) identify important sources of diverted medications, (4) examine motivations for NMU, and (5) estimate the patterns of alcohol and other drug use in combination with ADHD medications.Participants were drawn from Harris Interactive's Harris Poll Online (HPOL) panel, which consists of several million members internationally who consented to be contacted for public opinion surveys administered through the Internet. Members are added to the database after they have completed a two-stage registration process: first by signing on to the HPOL Web site and then by responding to a confirmation e-mail message providing a confidential password. Data are collected through an encrypted, password-protected Web portal. Eligible panelists were noninstitutionalized civilian adults aged 18 to 49 living in the United States (50 states and the District of Columbia). A minimum age of 18 was chosen because the time and costs of obtaining active parental consent for minors likely would have offset the benefits of online data collection. The maximum age was set at 49 to increase the efficiency of the sampling design, taking into account the low prevalence of NMU of prescription drugs among persons aged 50 or older  and the The survey was conducted during August 2005. Study enrollment and consent involved a two-part process. First, e-mail invitations describing the study were sent to panel members who initially met eligibility criteria according to data in the Harris Interactive (HI) database on panel members' age group and country of residence. These criteria were based on the NSDUH, which collects information from residents of households, noninstitutional group quarters , and civilians living on military bases. Persons excluded from the survey include homeless persons who do not use shelters, active-duty military personnel, and residents of institutional group quarters, such as prisons and long-term hospitals. Respondents completed a short screener that verified these characteristics. The screener also asked about respondents' military status, living situation, and substance use history, including NMU of ADHD medications; target groups were filled according to screening responses. For consistency with the NSDUH, active-duty military personnel and residents of institutional group quarters  were excluded from the study. Eligible respondents  were routed to a more detailed consent statement for participation in the full survey. Respondents who otherwise would have been eligible but whose target groups had already been filled did not complete the full survey. The survey took an average of 20 minutes to complete if respondents reported NMU.A total of 295,414 e-mail invitations were sent out, and 11,200 responses were received, regardless of outcome, or about 3.8% of the invitations. Of the 11,200 HPOL members who responded, 4,541 met the eligibility criteria and were needed for the study . Among this latter group, 4,297 (94.6%) completed the survey, and 244 (5.4%) declined to participate or did not complete the survey by the close of data collection.We employed a two-part methodology to adjust for Internet responses relative to the U.S. population of adults aged 18 to 49. First, propensity scoring methods were used to weight the data to approximate results for a probability-based telephone survey. Second, the data were further weighted to match the U.S. target population distribution by general demographic characteristics and to match the distribution of past-month cigarette use and past-month binge alcohol use estimated from 2003 NSDUH, which were the most current publicly available data during the data processing phase of this study, in 2005 . These pThe first step in the weighting procedure involved propensity scoring to reduce the bias attributable to collecting data from only HPOL members. The propensity score was included to adjust for self-selection into the online population and the panel, as well as for survey nonresponse that may not be explained by demographic differences. HI periodically conducts parallel telephone and Web surveys to measure attitudinal and behavioral variables that predict one's propensity to be online; the telephone survey is based on random-digit dialing (RDD) probability sampling. Using the combined telephone and Web surveys, HI created a propensity score model  for onliThe second step was that the propensity score-adjusted weights that RTI obtained from HI underwent further calibration using the generalized exponential model  using daWe also evaluated bias by comparing estimates in our survey with those for analogous variables in the 2005 NSDUH. Correspondence between the 2005 Diversion II estimates and the 2005 NSDUH suggests that the propensity model developed using 2003 NSDUH data, a follow-up telephone survey, and the U.S. census successfully reduced bias in the estimates, thus providing a large degree of confidence in the resulting estimates.Table Table Demographic and substance use items were based directly on public-domain NSDUH questions to establish comparability in the estimates between the two studies. NMU of prescription drugs was defined as use without a prescription or for the feeling or experience; examples of exact question wordings in the diversion study are shown in the appendix. Consistent with NSDUH, respondents were asked about the most recent NMU, including NMU in the 12-month period prior to taking the survey .NSDUH asks about lifetime NMU of a subset of ADHD medications but does not ask specifically about most recent NMU of ADHD medications. Therefore, we adapted NSDUH questions to ask about the most recent NMU of a wider range of ADHD medications. Similar to NSDUH, pictures of prescription pills were used to facilitate recall. For comparability with NSDUH, we asked respondents about NMU of brand-name drugs followed by their generic equivalents, or about NMU of specific brand-name drugs only. In addition, we asked respondents about NMU of a fictitious drug  to gauge the potential for indiscriminate reporting of NMU . After cand whether they ever received prescription medication for their condition. Among respondents who reported that they had received prescriptions for ADHD in the past year, we excluded four who reported that they obtained some medications by misrepresenting their symptoms or by going to doctors with lenient prescribing practices. We defined remaining respondents who received prescriptions for their ADHD in the past year as having "legitimate" prescriptions because they did not report NMU, or if they did, they did not obtain their medications fraudulently from physicians.NSDUH also does not capture information on ADHD symptomatology. Therefore, we created items to capture whether respondents reported ever receiving a diagnosis of ADHD from a medical professional Using existing items from prior studies ,34, respDescriptive statistics include prevalence estimates (percentages), the corresponding standard errors, and the estimated numbers of persons in the population. These were calculated by applying weights to generalize the answers of the respondents to the population of U.S. civilian, noninstitutionalized adults aged 18 to 49. Statistical tests, such as chi-squares, odds ratios, and corresponding 95% confidence intervals (CIs) were used to examine differences in the distribution of outcomes within demographic and substance use subgroups. Bivariate associations are presented because our primary interest was to identify vulnerable populations rather than build explanatory models using multivariate statistical modeling approaches. However, we also estimated a single multivariable  model using logistic regression. The independent variables included demographics, ADHD status, and drug use history. We present the significance effect of each characteristic after adjusting for potential confounders in the model. All analyses used SUDAAN (Release 9.01) to account for the sampling design by making appropriate use of the calculated sample weights . As thisAn estimated 7.1% of U.S. adults aged 18 to 49 used nonmedically any ADHD medication, which includes both stimulants and nonstimulants, at least once in their lifetime, and 2% did so in the past year Table  should bBivariate data in Table Higher rates of NMU of ADHD medications also were observed for those who self-reported that they had received a legitimate prescription for ADHD medications in the past year (OR = 22.1) or had received medications in their lifetime but not in the past year (OR = 6.3). Although ADHD apparently elevated the risk of NMU, 1.9 million of the 2.6 million persons aged 18 to 49 (73%) who participated in NMU had never been diagnosed and treated pharmacologically for ADHD. Further, fewer than 5% of past-year users who used ADHD medications nonmedically were prescribed ADHD medications in that period.NMU of ADHD medications was more prevalent among young adults aged 18 to 25 (4.3%) than among adults aged 26 to 49 (1.3%). No significant differences were observed in key demographic subgroups, including gender, race, student status, and insurance status.Similar to the bivariate models, in the multivariate logistic regression model, the likelihood of past-year NMU of ADHD drugs was higher for past-year marijuana users and binge drinkers than for their nonusing counterparts. Receipt of ADHD medications in the past year or lifetime also remained significantly associated with NMU of ADHD drugs in the multivariate model. However, the association for past-year methamphetamine use became nonsignificant in the multivariate model, suggesting that the strong bivariate association between methamphetamine use and NMU of ADHD drugs may be accounted for largely by other demographic, psychosocial, and substance use characteristics. This could be due either to confounding in which NMU and methamphetamine use are related to a third demographic or substance use factor, or a mediational pathway in which the exogenous factors influence methamphetamine use, which influences NMU of ADHD medications. The effect of age also became nonsignificant in the multivariate model, suggesting a possible mediational pathway in which age may influence ADHD symptoms and substance use behaviors such as binge alcohol use and marijuana or cocaine use. In turn, other drug use and ADHD symptoms may influence the likelihood of past-year NMU of ADHD medications.Figure Principal motivations among past-year NMUs were productivity (40%) and staying awake 23%)  differences in the rates of NMU of ADHD medications between young adult students (4.6%) and young adult nonstudents (4.2%), although we considered a broader range of medications and focused on ADHD drugs rather than on prescription stimulants in general. It also should be noted that our definition of student status included technical students and did not distinguish between full-time and part-time students. Thus, future studies should probe for differences in NMU by student enrollment status, including full-time, part-time, public, private, technical, and community college.There also is significant interest in the rates of NMU among students. Recent studies suggest that young adults, and particularly students, may account for much of the NMU of prescription stimulants. For example, Herman-Stahl et al.  found inAnother important contribution of this study involves examination of the patterns of polydrug use among NMUs of ADHD medications. Binge alcohol use 69%) and marijuana use (54%) were fairly common among past-year NMUs, which also has been found in a previous study  Please select the "NEXT" button at the bottom of your screen to continue with the survey.last used the drugs shown below when they were not prescribed for you or you took them only for the experience or feeling they caused?" Respondents were shown only drugs that they reported ever using nonmedically. Valid response choices were (1)within the past 30 days – that is, since MONFILL ; (2)more than 30 days ago but within the past 12 months; (3)more than 12 month ago but within the past 3 years; and (4)more than 3 years ago. Respondents who reported that they last used an ADHD medication nonmedically "in the past 30 days" or "more than 30 days ago but within the past 12 months" were defined as past-year NMUs.To identify past-year NMU, respondents were asked specifically, "How long has it been since you During the past 12 months, what is the main reason you used [this drug/these drugs] without a doctor's prescription or only for the experience or feeling [it/they] caused?1) For fun, kicks, or excitement2) To relax or relieve tension3) To feel good or get high4) To have a good time with friends5) To help yourself be more productive6) To stay awake7) To fit in with a group you like8) (IF PAST YEAR ALCOHOL USE REPORTED) To be able to drink more alcohol9) Some other reason(NOTE: Respondents not asked to specify the "other" reason.)98 Not sure99 Decline to answerQuestions on self-reported ADHD diagnosis and medications are shown below. Questions AD01 and AD02 appeared fairly early in the survey, but after questions DR01 through DR13 about lifetime nonmedical use of specific prescription medications. Question ADRXYR appeared toward the end of the survey, after all questions about nonmedical use of ADHD drugs, nonmedical use of other stimulants, and problems associated with nonmedical use of prescription drugs.These next questions are about attention-deficit/hyperactivity disorder, also known as ADD or ADHD.ever tell you that you had ADD or ADHD?AD01. Did a doctor or other medical professional 1 Yes2 No8 Not sure9 Decline to answerAD02. (If AD01 = 1) Did a doctor or other medical professional ever prescribe any medications for your ADD or ADHD?1 Yes2 No8 Not sure9 Decline to answer


To the Editor: During April–May 1999, 3 Bartonella isolates  were cultivated and established from the blood of 5 Macropus giganteus gray kangaroos from central coastal Queensland, Australia. We used multigene sequencing to evaluate whether these Bartonella isolates fulfill the minimum requirements for classification as a new species.Bartonella isolate was extracted by using the QIAamp tissue kit  according to the manufacturer’s instructions. Partial PCR amplification and sequencing of the genes encoding the 16S rDNA (rrs), citrate synthase (gltA), β-subunit of the RNA polymerase (rpoB), and cell division protein (ftsZ), as well as for the 16S–23S rDNA intergenic spacer (ITS) were attempted by using previously described primers and conditions  from 5 Macropus giganteus gray kangaroos, Australia, 1999*


Attention deficit/hyperactivity disorder (ADHD) and lead exposure are high-prevalence conditions among children.Our goal was to investigate the association between ADHD and blood lead levels (BLLs) in Chinese children, adjusting for known ADHD risk factors and potential confounding variables.Diagnostic and Statistical Manual of Mental Disorders, 4th ed., revised criteria (DSM-IV-R). We evaluated the association between BLLs and ADHD using the Pearson chi-square test for categorical variables and the Student t-test for continuous data. We then performed conditional multiple variables logistic regression analyses with backward stepwise selection to predict risk factors for ADHD.We conducted a pair-matching case–control study with 630 ADHD cases and 630 non-ADHD controls 4–12 years of age, matched on the same age, sex, and socioeconomic status. The case and control children were systematically evaluated via structured diagnostic interviews, including caregiver interviews, based on the p < 0.01; 5–10 μg/dL vs.≤ 5 μg/dL, OR = 4.9; 95% CI = 3.47–6.98, p < 0.01]. These results were not modified by age and sex variables.There was a significant difference in BLLs between ADHD cases and controls. ADHD cases were more likely to have been exposed to lead during childhood than the non-ADHD control subjects, with adjustment for other known risk factors [children with BLLs ≥ 10 μg/dL vs. ≤ 5 μg/dL; OR = 6.0; 95% confidence interval (CI) = 4.10–8.77, This was the largest sample size case–control study to date to study the association between BLLs and ADHD in Chinese children. ADHD may be an additional deleterious outcome of lead exposure during childhood, even when BLLs are < 10 μg/dL. Attention deficit hyperactivity disorder (ADHD) is one of the most common childhood psychiatric disorders, characterized by developmentally inappropriate levels of inattention, impulsivity, and hyperactivity . PrevaleAlthough the causes of ADHD remain unclear, both genetic and environmental factors are thought to influence the etiology of ADHD . Family,Lead is one of the well-established environmental poisons, and its general toxic effects, particularly in children, continue to be a major public health issue worldwide . It is wInternational Classification of Diseases, 10th Revision (ICD-10)   or in Di(ICD-10)  (Burns e(ICD-10) . Second,(ICD-10) . Third, (ICD-10)  and thusTherefore, in an effort to evaluate the association between BLLs and the risk for ADHD in Chinese children, we performed a case–control study involving large samples of children 4–12 years of age. We also considered known potential variables in the etiology of ADHD, such as delivery characteristics, perinatal factors, parental psychosocial factors, and prenatal exposure to tobacco and alcohol.This study was designed as a pair-matching case–control study. ADHD subjects were consecutively recruited from children coming for initial or follow-up assessment from October 2003 to August 2007 in two pediatric clinics at the Anhui Provincial Children’s Hospital and the Institute of Anhui Traditional Chinese Medicine in Anhui Province, People’s Republic of China. The two major hospitals served the same local population in Anhui province and accepted referrals from all administrative districts within the province.All ADHD  subjects were children of Chinese Han nationality 4–12 years of age at the time of investigation and had a lifetime medical history that fully met the DSM-IV-R criteria  for ADHDSchedule for Affective Disorders and Schizophrenia for School-Age Children (K-SADS-E) (The diagnoses of ADHD were derived from a structured diagnostic interview based on -SADS-E) , which w-SADS-E) , and tes-SADS-E) . The intThe non-ADHD controls were randomly selected from computerized lists of outpatients admitted for acute upper respiratory infection at the same two pediatric medical clinics during the daytime and during the same study period. By the pair-matched design, each ADHD case and control set had the same sex, the same age (difference between birthdays within 6 months), and almost the same level of socioeconomic status (SES). The controls were given the same full diagnostic assessment as the ADHD cases and screened only for the absence of ADHD without exclusion of any other diagnosis except for the same exclusion criteria applied to cases. SES is measured by poverty-to-income ratio (PIR), and PIR is the ratio of family income to the poverty threshold for the year of the interview. Low SES was defined as having PIR values < 1 in our analysis and high SES as having PIR > 3. The others were regarded as middle SES. The study was approved by our institutional review boards and complied with all applicable requirements of the United States. The parents of all the children in this study provided written informed consent at enrollment.Blood samples (2 mL/child) were collected in heparinized syringes. Lead concentrations were measured by anodic stripping voltametry through a blood lead analysis instrument  after the blood samples were digested with an organic tissue solubilizer. The limit of detection was 1.0 μg/dL. No detectable values were given values of 0.70 (1.0 divided by the square root of 2). Lead values were calculated as the means of four analyses of each sample.Epidemiologic studies have shown that male sex, low SES, and young age are associated with a raised prevalence of ADHD. Moreover, its prevalence falls with age . To addrIn addition, we considered multiple covariates and potential confounders for the association of lead exposure and ADHD in our study. They were based on established predictors of child behavioral problems and those widely used in studies of pediatric lead exposure . The folStudy factors were defined as binary variables or categorical variables. For example, maternal smoking and drinking habits during pregnancy were recorded as binary variables—that is, drinker or nondrinker, cigarette smoker or nonsmoker. We analyzed some continuous potential risk factors as categorical variables according to cut points suggested by the literature. For example, low birth weight is typically defined as < 2,500 g . Maternat-test for continuous data. We also performed a conditional logistic regression analysis with a binary outcome of ADHD in relation to BLLs, adjusting for other potential confounding factors . In addition, 49.8% of non-ADHD children had BLLs > 5 μg/dL, whereas of the ADHD cases, 74.7% had BLLS > 5 μg/dL .p < 0.01 for children with BLLs 5–10 μg/dL; OR = 7.15, p < 0.01 for with BLLs ≥ 10 μg/dL, using the sample with BLLs ≤ 5 μg/dL as referent) and family history of ADHD . The risk for ADHD decreased as the mother’s education level increased . The analysis also found an association between ADHD and maternal smoking during pregnancy, but the p-value is near the significant threshold . The remaining confounding variables in our analysis were not associated with ADHD status  Our sample size was the largest to date in case–control studies to investigate BLLs and ADHD. b) The ADHD diagnosis was made through an extensive clinical evaluation based on the DSM-IV-R diagnostic instrument and performed by child and adolescent psychiatrists. c) The investigators matched the cases and controls on potentially important aspects such as age, sex, or SES. This is important because lead levels are highest among younger children compared with adolescents, and ADHD is of higher prevalence among children of elementary and middle-school age.The strengths of this study are as follows: This study suggests that there is a link between ADHD and BLLs, and the results reinforced findings from previous studies . If on fMoreover, BLLs < 10 μg/dL also indicated a risk factor. This may suggest that the lower standard may effectively protect children from the harmful effects of lead, which is consistent with previous investigations .Some studies  found anin utero tobacco and alcohol exposure may be susceptible to recall bias. Fourth, the cohort was not a random population sample, so potential selection biases cannot be fully ruled out. In addition, because all our sample was Chinese Han, no children living in foster families were included, and none of the sample had health insurance, our results may not generalize to children with different socioeconomic or ethnic backgrounds. Finally, it is interesting that This study has several limitations. First, temporality between high BLLs and ADHD could not be ascertained definitely in this case–control design. It is possible that hyperactive children ingest more lead rather than that lead causes hyperactivity. Therefore, further studies with serial lead measurements, even from the antenatal to the postnatal period, and more continuous measures of ADHD symptoms or of specific underlying neurobehavioral domains may be required to document the temporal relationship between lead exposure and development of ADHD. Second, some might argue that concurrent blood lead tests are not an adequate biomarker of a child’s lifetime exposure. However, recent studies indicate that concurrent BLLs are a stronger predictor of lead-associated IQ decrements than blood lead measured during early childhood . Third, This hospital-based case–control study suggests a strong relationship exists between ADHD and lead exposure, even low-level lead exposure (< 10 μg/dL BLL), in Chinese children. If this finding is confirmed in future studies, the potential to prevent ADHD by reducing childhood lead exposure should be considered.


These workshops bring together clinicians, researchers, scholars, and advocates with diverse perspectives and from diverse fields. Our first commentary in CAPMH, which grew out of our first workshop, explained our method and explored the controversies in general. This commentary, which grows out of our second workshop, explains why informed people can disagree about ADHD diagnosis and treatment. Based on what workshop participants said and our understanding of the literature, we make 8 points. (1) The ADHD label is based on the interpretation of a heterogeneous set of symptoms that cause impairment. (2) Because symptoms and impairments are dimensional, there is an inevitable "zone of ambiguity," which reasonable people will interpret differently. (3) Many other variables, from different systems and tools of diagnosis to different parenting styles and expectations, also help explain why behaviors associated with ADHD can be interpreted differently. (4) Because people hold competing views about the proper goals of psychiatry and parenting, some people will be more, and others less, concerned about treating children in the zone of ambiguity. (5) To recognize that nature has written no bright line between impaired and unimpaired children, and that it is the responsibility of humans to choose who should receive a diagnosis, does not diminish the significance of ADHD. (6) Once ADHD is diagnosed, the facts surrounding the most effective treatment are complicated and incomplete; contrary to some popular wisdom, behavioral treatments, alone or in combination with low doses of medication, can be effective in the long-term reduction of core ADHD symptoms  The US Centers for Disease Control estimates that approximately 4.6 million (8.4%) American children aged 6–17 years have at some point in their lives received a diagnosis of Attention-Deficit/Hyperactivity Disorder (ADHD). Of these children, nearly 59% are reported to be taking a prescription medication -4. IndeeNot just school-age children are being treated with stimulants. Stimulant use among preschool children is also greater in the US than elsewhere: 0.44% of preschoolers in the US are prescribed stimulants, compared with 0.05% of preschoolers in the Netherlands, 0.02% of preschoolers in Germany, and 0% of preschoolers in the UK .The duration of treatment and complexity of the treatment regimen is also growing. Before 2000, most children treated for ADHD received short-acting drugs, during school, for 1 or 2 years. Today many receive long-acting drugs while in – and out – of school and the prevailing recommendation from ADHD experts is to start medication early and to continue as long as medication is needed. This suggests that, if they adhere to their regimens, many American children diagnosed with ADHD will receive far higher lifetime doses than similar children in the past . Even ouEven without any further increase in the rate of stimulant use  have been recognized as problematic for the last 100 years – and, arguably, for much longer. Generally, children are brought to their physicians because parents or teachers are concerned that the child's behavior is preventing him or her from functioning normally at home, in school, or in other settings. In the majority of cases, teachers are the first to suggest that a child might have ADHD . InitialAccording to the fourth edition of DSM "the essential feature of Attention-Deficit/Hyperactivity Disorder is a persistent pattern of inattention and/or hyperactivity-impulsivity that is more frequent and severe than is typically observed in individuals at a comparable level of development" . CurrentDSM IV lists 18 core symptoms of ADHD, which are divided into 2 major behavioral domains: (1) inattention and (2) impulsivity-hyperactivity. Among the 9 symptoms of inattention are: often makes careless mistakes, often has difficulty sustaining attention in play or other activities, and often does not seem to listen when spoken to directly. Among the 9 symptoms of hyperactivity-impulsivity are: often fidgets or squirms, often can not stay seated, blurts out, and is impatient.To bring conceptual order to this heterogeneous set of behaviors, clinicians in the US currently distinguish among 3 subtypes of ADHD: Predominantly Inattentive Type, Predominantly Hyperactive-Impulsive Type, and Combined Type. ADHD Not Otherwise Specified (ADHD NOS) is a fourth category, and includes children who exhibit fewer symptoms of inattention or hyperactivity-impulsivity than children who meet the criteria for one of the other 3 subtypes, but are nevertheless significantly impaired as a result of their symptoms. Findings from genetics and neurobiology have shed some light on the genetic and neurological correlates of the behaviors associated with ADHD ,19. For For one thing, geneticists today are grappling with the fact that, in general, single gene variants by themselves are less helpful in explaining the emergence of complex phenotypes than was once hoped ,23. MoreThe creators of the DSM system of diagnosis had several aims. They wanted to develop an algorithm that could quickly and reliably identify individuals who needed help. Such a neatly laid-out system would facilitate getting reimbursement to deliver services. And the careful description of symptoms was intended to help physicians and researchers in different places feel confident that they were indeed studying the same disease entity .While the DSM system achieves those aims, it also entails significant difficulties. First, a single diagnostic label – ADHD – is used to name children who have different collections and levels of symptoms and who suffer different levels of overall impairment. Moreover, as workshop participant and child psychiatrist Gabrielle Carlson suggested, like many conditions, ADHD is expressed differently in different children and it differs in severity from mild, to moderate, to severe .Because ADHD does not have a single, simply identifiable form, diagnosing it requires an observer's interpretation. While many physicians will agree that one particular child warrants an ADHD diagnosis and another child does not, many children will occupy what we will call a "zone of ambiguity." Physicians, teachers, and parents may well disagree about whether children in the zone of ambiguity exhibit the symptoms and suffer severe-enough impairment from those symptoms to warrant the ADHD diagnosis. Increases in the rates of ADHD diagnoses and the use of stimulant treatment have fueled the concern that too many children in the zone of ambiguity are today given an ADHD diagnosis rather than considered simply "different" or "spirited," and that drugs are too often the treatment of choice for these children.dimensional, not categorical. Children with and without ADHD do not occupy cleanly separate categories; rather, they occupy different places on one or more dimensions (or continua or spectra) of behavior. Bright lines do not separate children whose attention, impulsivity, or activity levels are normal from those whose are not . All children lie somewhere on these behavior spectra and many will fall in the zone of ambiguity. Despite the reminder in its Introduction that psychiatric disturbances are dimensional, DSM is a categorical system. Because DSM is so important for diagnosis and reimbursement, users often adopt its language and categories without recalling its limitations.The Introduction to DSM IV addresses some of this difficulty by acknowledging that many psychiatric diagnoses give labels to phenomena that are and children with fewer symptoms but greater impairment may go undiagnosed and untreated.According to several workshop members, yet another problem is that, even though DSM explicitly states that diagnoses should only be made if symptoms cause "clinically significant impairment in social, academic, or occupational functioning," clinicians sometimes base their diagnoses on the presence of symptoms alone. In one study, researchers reduced a 16.8% ADHD prevalence rate to 6.8% by adding an impairment criterion . While iOur description of these complexities and the blurriness of the lines is not to suggest that ADHD is not real. The symptoms of ADHD can cause significant suffering in children, families, and schools -28 and sTo invoke a term that no fewer than three psychiatrists – Michael First, Steven Hyman, and Benedetto Vitiello – used at our first workshop, we need to avoid the "reification" of the DSM categories. These categories are abstractions we have created, not entities we have discovered in nature. Diagnostic categories can be useful tools to help us talk about childhood behavioral and emotional disturbances, but we need to remember that they are tools created by us. We – doctors, parents, teachers, and others – set the threshold between behaviors and moods in need of pharmacological or behavioral treatment and differences that should be left alone or dealt with in other ways.One explanation for increased rates of diagnosis and stimulant use, therefore, is that we are setting ever lower diagnostic thresholds. This explanation concerned many workshop participants, including sociologist Peter Conrad and pediatrician William Carey. When they see data documenting an increase in diagnostic and treatment rates, they see a troubling decrease in societal tolerance of the behaviors and impairment associated with the ADHD diagnosis.We have already observed that stimulant medications are used at different rates in different countries. Specifically, they are used at higher rates in the US than in culturally similar places like Germany, the Netherlands, and the UK.  But even within the US there is significant variation in diagnostic and treatment rates. Beyond the phenomenological and etiological complexity and the zone of ambiguity we described earlier, why would rates of ADHD diagnosis be higher in some places than others?th edition of the World Health Organization's International Classification of Diseases (ICD 10).At least when it comes to understanding the difference between the rates at which ADHD is diagnosed in the US and Europe, it helps to notice that clinicians in those two geographical regions use closely related – but importantly different – systems of disease classification. Whereas clinicians in the US currently use the DSM IV, clinicians in Europe use the 10ICD 10 refers to Hyperkinetic Disorder (HD), whereas DSM IV speaks of ADHD. And while DSM IV and ICD 10 use very similar lists of symptoms for ADHD and HD respectively, their approaches to diagnosis are different in some important ways. DSM IV requires a child to exhibit only 6 symptoms in 1 of 2 broad domains (inattention or hyperactivity-impulsivity), while ICD 10 requires a child to exhibit 10 symptoms, including at least 1 in each of 3 domains . Whereas DSM IV requires that some impairment be present in more than 1 setting , ICD 10 requires that all criteria must be met in at least 2 settings. In short, the DSM system casts a wider net than does the ICD, so that "ADHD prevalence rates based on DSM-IV are expected to be higher than those based on ICD-10" . Indeed,and one or more coexisting conditions, such as an anxiety, mood, or developmental disorder. According to ICD, however, if one of those coexisting conditions is diagnosed, then HD cannot be diagnosed.The ICD and DSM approaches to coexisting conditions in a single child are also importantly different. Under DSM, a child can be diagnosed with ADHD However, even in the US, where all clinicians presumably use the DSM approach, there is variation. As with other disorders, community-based ADHD prevalence rates from treatment data vary according to demographic factors such as age, gender, and race/ethnicity . As workAs with other disorders, there is also considerable regional variation. Workshop participant and child psychiatrist Regina Bussing pointed to Centers for Disease Control data showing that the heaviest use of stimulants to treat ADHD occurs in southern US states, followed by states in the upper Midwest ,34. TherLaurence Greenhill pointed to still other variables to help explain international and regional variation in diagnosis and use of prescription medication. Clinicians in different places rely on different informants  and use different diagnostic guidelines  and diagWe also know that children's home, school, and community environments can differ greatly, including their sleep patterns, diets, physical exercise opportunities, and levels of exposure to television and other media. Anthropologist Sara Harkness cited studies she and child psychologist Charles Super have conducted comparing Dutch and American parenting styles: "The Dutch parents we studied were very closely attuned to their children's state of arousal and self-regulation, making sure that the child got plenty of sleep and that the environment was not overly stimulating. For Dutch parents, this was just a normal aspect of good parenting, whereas for American parents this approach might seem somewhat extreme, called for only when the child is really out of control."Harkness went on to note that Dutch schools are very concerned with each child's ability to pay attention: "Virtually every classroom I visited had two or three children's desks that were placed away from the others (touching the teacher's desk in a couple of cases), in order to help children who seemed more distractible than others." Harkness and Super also noted differences at a systems level; children who had difficulty learning due to behavioral problems were transferred to special schools, and the school day included a lunch break long enough for children to go home and spend time outside in unstructured play. While the environmental causes of the behaviors considered symptoms of ADHD are not well understood, many workshop participants agreed that the child's environment can influence the development of such behaviors.In addition, because different cultures have what Roy Martin called "different local normative expectations," different environments will be more or less tolerant of active, distractible children, and will be more or less prone to see impairment from those behaviors. To put the point in diagnostic terms: observing the same behaviors at the same rates in children around the world is one thing, but these children will not meet the diagnostic criteria unless they are also impaired by those behaviors in that culture. People in some cultures are also more likely than people in others to seek medical assistance and accept medical  treatments .Many workshop participants were concerned that cultural expectations in the US have grown intolerant of children exhibiting the behaviors currently associated with ADHD. Peter Conrad spoke of the "medicalization of underperformance," and psychiatrist John Sadler worried that changes in expectations about the conduct of classroom education and the pace of educational achievement make it more likely that the active, distractible child will be considered a problem. That said, many workshop participants agreed that children with the most severe forms of ADHD would be impaired in virtually any culture, community, or context.Bearing in mind the myriad factors that can affect how different people interpret the same behaviors, and remembering the phenomenological heterogeneity and etiological complexity associated with ADHD as well as the zone of ambiguity, it is hardly surprising that rates of diagnosis are different in different places. More specifically, setting aside the debates about the particulars of the DSM approach, we can see why there are concerns about both over- and under-diagnosis.The Great Smoky Mountain study examined the prevalence of serious emotional and behavioral disturbances, including ADHD, in children in the western region of North Carolina . In the not warrant an ADHD diagnosis nevertheless received stimulants. While 4.5% is a small percentage, it is 4.5% of all the children in the study who did not have ADHD, which is a large number. In terms of absolute numbers, the study found that more children without ADHD received stimulants than did children with ADHD. The study concluded that in the community , a significant proportion of children with ADHD do not receive stimulants and a significant number of children without ADHD are prescribed stimulants.Two factors explain the Great Smoky Mountain study's findings. First, not all of the children who warranted an ADHD diagnosis had received stimulants; 72.2% of the children who warranted an ADHD diagnosis received stimulants and only 22.8% of children who warranted an ADHD-NOS diagnosis received stimulants. Second, 4.5% of children who did It is widely recognized that ADHD is over-diagnosed in some affluent communities, where "local expectations" are such that stimulants are just one more tool to promote performance in "the Academic Olympics" . Becausemild and very severe impairment due to the behaviors associated with ADHD, there always will be some children whose symptoms and impairment place them in the zone of ambiguity.While there frequently will be agreement among experts about whether to diagnose ADHD in children with very At least in part, views about where to set the threshold for diagnosing ADHD will be a function of peoples' differing conceptions of the proper goals of medicine in general or psychiatry in particular. Some observers are not alarmed by the tendency of medical institutions to treat ever more problems that seem to have more to do with someone's failure to meet social, cultural, or educational expectations than with a failure of physiological function. Others, like sociologist and workshop participant Peter Conrad, who are often alarmed by this tendency, label it medicalization and see both diagnosis and treatment as "social control for deviant behavior" .drugs to alter a child's temperament; he is urging caution about using any means to shape what he urges us to view as normal temperamental differences. He is equally concerned about the overuse of behavioral interventions, which he thinks should be reserved for responding to problematic behaviors (e.g. ignoring teacher requests) rather than problematic behavioral styles (e.g. a restless temperament)  they are cheaper than costly behavioral interventions."Given the prevalence of ADHD, philosopher and workshop participant Bonnie Steinbock also wondered whether a public health approach, which sought to understand the underlying causes and focused on prevention, might be warranted. Steinbock argued that an analogy might be made with obesity, rates of which have soared in the past few decades. If we understood better the causes of ADHD, we would not simply focus on diagnosis and treatment, but also on policy changes. That is, she brought us back to what Harkness and Super call the child's developmental niche , asking 1. The ADHD label refers to a heterogeneous set of symptoms that cause impairment.a. Until we have valid biological markers, diagnosis will depend upon observers' interpretations of children's behavior.2. The fact that symptoms are dimensional  helps explain disagreements about diagnosis.a. Most children will express some of the behaviors associated with ADHD, but not all children are impaired as a result.zone of ambiguity: i.e., a significant number of cases in which reasonable people will interpret the same child's behavior differently.b. While it will sometimes be clear to virtually all observers that one particular child is impaired and another child is not, the dimensional nature of behaviors means that there will be a large 3. ADHD is diagnosed at different rates in different places.a. Variation in rates of diagnosis is due to many variables, from different professional systems of diagnosis  to different styles of parenting and expectations of children.and over-diagnosed.b. Using DSM IV criteria, ADHD is both under- 4. People hold different – and reasonable – views about the proper goals of psychiatry and parenting, and thus worry less or more about treating children in the zone of ambiguity.5. There is nothing "mere" about social constructions.a. To recognize that there is no bright line written in nature between impaired and unimpaired children – to recognize that it is up to human beings to choose who should receive a diagnosis and who should not – is to acknowledge that ADHD is "a social construction." But acknowledging that does not make us diagnostic nihilists; rather, it means we understand that because nature does not show us where that line is, it is our weighty responsibility to decide where to draw it.6. The facts regarding different treatments are complicated and incomplete.and at improving many aspects of overall functioning.a. When a diagnosis is made, the facts surrounding the most effective treatment strategy are, unfortunately, less certain than caring physicians and distressed families would wish. For one thing, "efficacy" can be defined in different ways, from reduction in symptoms to improvement in academic achievement and peer and family relations. Recent research suggests that while stimulant medication is often effective in the short term at reducing the core symptoms of ADHD it is not always effective in the long term at improving a child's overall functioning. Moreover, it has been shown that behavioral treatments, alone or in combination with low doses of medication, can be effective in the long term at reducing the core symptoms of ADHD 7. People hold different – and reasonable – views about the proper means for psychiatry to employ in the treatment of ADHD.a. People who favor either medication or behavioral treatment can disagree about both the facts – which treatment most effectively reduces symptoms and improves overall functioning – and values – which treatment most effectively respects individual temperaments and improves children's lives.8. Truly informed decision-makinga. Parents  should be provided with the facts they need to make truly informed treatment decisions. Because many children will fall within the diagnostic zone of ambiguity, and because choosing a treatment plan requires grappling with uncertain facts about efficacy and long-term effects as well as with important values, that process will sometimes be difficult. To be truly informed, that process must be as insulated as possible from financial interests.b. The current US health care and education systems tend to favor pharmacological treatments, which are believed to be less expensive than behavioral treatments. Scaling up behavioral approaches would indeed require initially costly changes in classrooms, schools, and the education system, as well as in reimbursement policies. It would also entail opportunity costs to parents and children, who would need to work at learning new behavioral approaches. Alone or together with pharmacology, behavioral approaches may nonetheless more effectively improve overall functioning than pharmacological treatments alone.and behavioral therapy.c. Rather than debating whether medication is good or bad in itself, we should continue the dialogue about how to introduce changes in families, classrooms, schools, health care systems, and cultures so as to reduce the incidence of ADHD behaviors and reduce the likelihood that children who do have those behaviors will be impaired. When families consider it necessary to enlist medical assistance in treating impairing behaviors, they should be carefully informed of the benefits and limitations of medication Both authors contributed equally to all aspects of this article.


Objectives. This is a pilot study of the dietary intake and nutrient status of children with Attention Deficit Hyperactivity Disorder (ADHD). Method. Nutritional assessment of 43 children aged 6–12 with ADHD was performed using a 3-day food record, 24-hour recall, and serum assessors. Results. Macronutrient intake and consumption of Low-Nutrient Foods were comparable to population norms; however, 66% were found to be deficient in zinc and 23% in copper. Conclusions. This pilot study reports the food intake and nutrient status of children with ADHD and shows a predisposition for low zinc and copper status in ADHD. While diet and supplements in Attention Deficit Hyperactivity Disorder (ADHD) have and continue to remain a popular explanation of the disorder that has generated a wealth of research –4, thereThere are currently only four studies assessing dietary intake in ADHD; a study of ADHD preschoolers , two oveThere is now evidence to suggest that ADHD may be associated with low trace mineral status, specifically zinc and iron , 11. ThiSerum zinc levels have been found to be significantly lower in ADHD children compared to controls in several controlled studies around the world, including Poland , Turkey μg/L), significant improvement in ADHD symptoms was reported in ADHD children who received iron over a placebo [Several authors have also found an association between low serum ferritin and ADHD , 21, 22  placebo . The objective of this pilot study was to describe the dietary food intake and assess the nutrient status of DSM-IV diagnosed ADHD children aged 6–12 years. We hypothesized that children with ADHD have abnormal dietary intake and low micronutrient status.The University of British Columbia Clinical Research Ethics Board and the British Columbia Children's and Women's Hospital Research Committee approved the study protocol.Sequential patients at the Provincial ADHD Program in Vancouver, Canada were offered participation in the current study and 44/46 accepted. All parents gave informed consent, and children completed assent forms; the study was then carried out in accordance with the Declaration of Helsinki, revised. Eighteen (18) stimulant-treated, 9 atomoxetine-treated, and 17 treatment-naïve children aged 6–12 years participated. Inclusion criteria were a primary diagnosis of ADHD and proficiency in English. Those patients on ADHD medication had to have been stable on the current type and dose for at least 6 months and had to be taking medication 7 days a week. Exclusion criteria were the use of any additional medication known to alter food intake, other than stimulants or atomoxetine prescribed for ADHD, or any medical condition that could alter nutritional status. Female subjects had to be premenarchal.Diagnosis was based on a two-hour clinical interview and a semistructured diagnostic interview of childhood psychiatric disorders; the Kiddie Schedule for Affective Disorders and Schizophrenia (KSADS-E) , 28. ADHHeight and weight of ADHD children were assessed using calibrated stadiometers and electronic scales and compared with standard growth charts from the Centre for Disease Control (CDC) .6 analysis was carried out by Hospitals in Common (HIC). Comparative data received from the hospital laboratory provided the normal ranges by age and gender.Nonfasting, venous blood samples were collected by a registered phlebotomist at the Outpatient Laboratory of British Columbia Children's Hospital. Trace mineral analysis was conducted by the Outpatient Laboratory at Children's Hospital and pyridoxine/BThe 24-hour food recall was administered according to a multipass time interview format, the same method used in the NHANES comparative data . Any supThe three-day food record data served as an approximation of usual intake and was used to determine the proportion of the group below the Estimated Average Requirement (EAR) for specific micronutrients . DietaryA computer-generated randomization schedule of two weekdays and one weekend day was produced for up to 100 subjects and was utilized sequentially for all 43 subjects in this study in order to assign subjects to days in which to collect data for the three-day food record. Both parents of subjects and subjects were jointly instructed on portion estimation and provided with visual estimates to aid in three-day food record data collection . All dietary intake data was entered into a computer nutrient database  modified to include the Canadian Nutrient File.P < .05 level of significance. Chi-square tests were used to determine differences in categorical data between sample and populations. Independent sample t-tests were used to evaluate differences between groups of continuous, normally distributed variables. Comparisons of data obtained from the present study and previous population studies were conducted by first transforming the standard error given by population studies to a standard deviation statistic based on population size. Independent sample t-tests were then used to compare the results. As this study was conducted in Canada, local recent population dietary intake data were used as a basis for comparison [All statistical comparisons were performed using two-tailed tests set at mparison , 37. As mparison . Since tn = 23) and 9–12 years (n = 21). A compliance of 88.6 percent was obtained for the 3-day food record collection and 82% for the nutritional status analysis. The majority of children in the study were from two parent families (70.5%). The overall level of education of parents was high with 70% having obtained a trade or vocational certificate, a college certificate, or a university degree. The household gross income was greater than $60,000 per year for 61.4% of the families; with 27.3% of the families having an income greater than $100,000. Only two children were from families where the gross family income was less than $15,000.This study was composed of 37 males (84%) and 7 females (16%) with a mean age of participants being 8.5 years. Children were pooled into two groups; aged 6–8 years (n = 27) was similar to the mean height-for-age of drug-naïve subjects .Twenty percent of children were above the 97th percentile for height-for-age and twenty percent of children were above the 90th percentile for weight-for-age. The mean height-for-age of drug-treated subjects ; a vitamin-mineral supplement with iron only (n = 3); and a vitamin-only supplement (n = 7). Subjects that took the vitamin-mineral supplement had similar serum zinc and copper concentrations  compared to counterparts that did not consume a supplement. There was no significant difference in serum zinc and copper levels observed in our ADHD cohort and those obtained from laboratory norm values for children of similar age and gender without ADHD.Majority of the child cohort  took a dietary supplement that consisted of vitamin-mineral combination containing iron, zinc, and copper (Estimated Energy Requirements (EER) were calculated on subject age, physical activity, and height and weight. Estimated daily energy intake was determined from a three-day food record . Almost t (17) = 0.77, P = .46) that used NHANES II data [Approximately 60% of ADHD children aged 6–8 years followed in this study consumed less than 2 servings of Meat and Alternates per day. This result, when compared to a recent population data that indicated that Canadian children in this age group consume an average of 2.4 servings of Meat and Alternates per day , is rela II data . There w6 and all children in the study met the EAR for iron.In 6 in ADHD children were significantly (P < .001) below the population norms, while no differences in iron intake were found.Since there are no recent Canadian data available on mean dietary intakes of iron, zinc, and copper in children aged 6–12 years, data from this study was compared to the most recent US dietary intake data from NHANES III and is presented in t (34) = 2.48, P = .02), with 26% having serum zinc values below the 2.5th percentile of the NHANES II cutoffs for zinc deficiency for nonfasted individuals (≤10.1 μmol/L/66 μg/dL). The prevalence of zinc deficiency in ADHD children in this study was eight times greater than the normal prevalence of 3.3% reported for males and 3% for females [t (29) = 8.06, P < .001) and exhibited double the prevalence of low-serum zinc status (27.1%) [Serum zinc and copper concentrations of ADHD children examined in this study were found to be lower than the most recent US population data (NHANES II) for non-ADHD children aged 6–8 and 9–13 years . For exa females . Twenty 6 status of all children in this study were within lab normal range.Iron status and vitamin Br (36) = −0.13, P = .22), but there was a significant relationship between dietary intake of copper and serum copper status (r (36) = 0.420, P < .01) in ADHD children. Finally, no significant differences were found in dietary macro- and micronutrient intake between drug-naïve and drug-treated participants when assessed in terms of total caloric intake. This corresponded with the fact that mean height-for-age for drug-treated subjects was similar to that for drug-naïve subjects .Pearson correlations showed no relationship between dietary intake of zinc and serum zinc status (r (36) = −0.13, P = .22), but did show a relationship between dietary intake of copper and serum copper status (r (36) = 0.420, P < .01). Independent sample t-tests did not find a relationship between serum zinc status and meeting the recommendations of consumption from the Meat and Alternate food group (t (16) = 0.63, P = .54). Pearson correlations showed no relationship between dietary intake of zinc and serum zinc status . Among subjects that took vitamin-mineral supplements, one sample t-tests also showed no significant difference, between observed and lab normal values for serum zinc (t (23) = 1.54, P = .13), and copper (t (23) = 1.40, P = .17) values.  In subjects that took vitamin-mineral supplements, mean serum zinc and mean serum copper were 10.9 t (30) = 0.56, P = .58), carbohydrate (t (30) = 1.24, P = .23), and fat (t (30) = 0.36, P = .74) as assessed in calories per day or any difference in the mean dietary intake of calories (t (30) = 0.01, P = .94) between drug-naïve and drug-treated participants as determined by independent sample t-tests.There were no significant differences between drug-naïve and drug-treated participants in the dietary intake of protein  = 0.11, P = .91) between drug-treated subjects and drug-naïve subjects indicating that the medication status of the subjects examined in this study was not a factor. This may be because of the young age and low drug exposure of the children in the study. Independent sample This study found no significant difference between the ADHD children in this sample and population norms in mean energy intake, calories or proportion of calories derived from protein, fat, or carbohydrate as compared with data from the recent Canadian Community Health Study . ADHD chThis is the first study to demonstrate significantly lower dietary intakes of micronutrients in ADHD children compared to population norms . DespiteThis study is limited by the relatively small sample size and by lack of a specific normal control group. In addition, the study did not control for vitamin and mineral supplementation. That being said, all subjects had blood samples drawn in the morning in a nonfasted state and all analysis were performed at the same laboratory at BC Children's Hospital as the lab normal data with which the results were compared. This study adds to the growing literature demonstrating low serum zinc in ADHD children  and repoFurther research is needed to help identify the etiology, impact, and possible therapeutic implications of low micronutrient status in ADHD, given the essential nature of these micronutrients in the production of the neurotransmitters involved in ADHD.This study establishes that the dietary intake of these ADHD children from Canada is comparable to local population norms in the intake of calories and of macronutrients; however, ADHD children in this study consumed significantly lower quantities of zinc and copper, but not iron. Half of the children in the sample consumed less than the recommended servings of Meat and Alternates, a major source of zinc and copper and a significant source of iron.Since these ADHD children consumed the same percentage of Low-Nutrient-Density foods as children of the same age from the general population, it does not appear that displacement of trace minerals from excess “junk food” contributed to low dietary intakes of zinc and copper in these children.Serum micronutrient status in this study adds support to a previous reports of low zinc  in ADHD The importance of these findings is that zinc, iron, and copper are essential cofactors in the production of dopamine and norepinephrine , 45, 46;


Bartonella vinsonii subsp. berkhoffii is an important, emerging, intravascular bacterial pathogen that has been recently isolated from immunocompetent patients with endocarditis, arthritis, neurological disease and vasoproliferative neoplasia. Vector transmission is suspected among dogs and wild canines, which are the primary reservoir hosts. This investigation was initiated to determine if pets and family members were infected with one or more Bartonella species.Bartonella alpha Proteobacteria growth medium (BAPGM) was used to determine infection status. Antibody titers to B. vinsonii subsp. berkhoffii genotypes I-III and B. henselae were determined using a previously described indirect fluorescent antibody test. Two patients were tested sequentially for over a year to assess the response to antibiotic treatment.PCR and enrichment blood culture in B. vinsonii subsp. berkhoffii genotype II and Bartonella henselae (Houston 1 strain) were confirmed in a veterinarian and his daughter by enrichment blood culture, followed by PCR and DNA sequencing. Symptoms included progressive weight loss, muscle weakness, lack of coordination (the father) and headaches, muscle pain and insomnia (the daughter). B. vinsonii subsp. berkhoffii genotype II was also sequenced from a cerebrospinal fluid BAPGM enrichment culture and from a periodontal swab sample. After repeated courses of antibiotics, post-treatment blood cultures were negative, there was a decremental decrease in antibody titers to non-detectable levels and symptoms resolved in both patients.Intravascular infection with B. vinsonii subsp. berkhoffii and B. henselae are zoonotic pathogens that can be isolated from the blood of immunocompetent family members with arthralgias, fatigue and neurological symptoms. Therapeutic elimination of Bartonella spp. infections can be challenging, and follow-up testing is recommended. An increasing number of arthropod vectors, including biting flies, fleas, keds, lice, sandflies and ticks have been confirmed or are suspected as the primary mode of transmission of Bartonella species among animal populations and may also pose a risk to human beings. Bartonella, rediscovered; numerous clinical, microbiological and pathological concepts related to disease causation and microbial pathogenesis are sequentially redefined. Subsequently, the medical relevance of the genus undergoes continued maturation; as knowledge of the organism, the host immune response, diagnostic test sensitivity and specificity, treatment efficacy and epidemiology expand. Since the early 1990s, this paradigm of discovery and ongoing biological and medical redefinition has clearly been applicable to the genus Bartonella. Prior to 1990, only two pathogenic Bartonella species, B. bacilliformis and B. quintana, were known to exist. Since 1990, greater than 22 Bartonella species have been described, of which at least half have been implicated or confirmed as human pathogens [When a genus of bacteria is discovered or, in the case of Bartonella vinsonii subsp. berkhoffii was initially isolated from a dog with endocarditis in 1993 [B. vinsonii subsp. berkhoffii were described, based upon analysis of blood samples from coyotes, dogs and foxes [B. vinsonii subsp. berkhoffii genotype II was documented in a healthy dog on 8 of 10 culture attempts spanning a 16-month period, thereby supporting the potential for persistent intravascular infection in pet dogs [B. vinsonii subsp. berkhoffii has been suggested, the mode(s) of transmission among canines has not been determined [B. vinsonii subsp. berkhoffii, domestic and wild felids represent the primary reservoir in nature for Bartonella henselae, an organism transmitted among cats by fleas ; a factor that contributes to a worldwide distribution for this Bartonella sp. [B. henselae for months to years [B. vinsonii subsp. berkhoffii in canines and B. henselae in felines, both of these two bacterial species can be pathogenic in both cats and dogs.nd foxes . To datend foxes -8. Previella sp. . Similarto years ,12. HoweB. vinsonii subsp. berkhoffii genotype II and B. henselae were foundin a veterinarian and his daughter. The father presented with progressive weight loss, muscle weakness and lack of coordination; his daughter had developed headaches, muscle pain and insomnia. Both individuals were being evaluated by a neurologist at the time of initial testing for evidence of Bartonella infection. Multiple courses of antibiotics were administered before the patients' clinical status improved and before microbiological, molecular and serological evidence of infection diminished or was negative.In this study, intravascular infection with Bartonella spp. DNA and enrichment culture of blood and serum samples in Bartonella alpha Proteobacteria growth medium (BAPGM) was used [Bartonella spp. following direct DNA extraction from patient blood and serum samples, PCR amplification following enrichment culture in BAPGM for 7 to 14 days, and PCR from isolates obtained following BAPGM subculture inoculation onto trypticase soy agar with 10% rabbit blood. Agar plates are incubated for 4 weeks and checked weekly for evidence of bacterial growth. To assess for potential laboratory contamination, an un-inoculated BAPGM culture flask was processed simultaneously and in an identical manner with each batch of patient blood and serum samples tested. Specifically, while establishing cultures using a batch of samples in the biosafety hood, the top was removed from the BAPGM un-inoculated control flask until all patient samples had been processed. Methods used for testing sample cultures, including DNA extraction, PCR amplification targeting the Bartonella 16S-23S intergenic spacer region (ITS), and sequencing procedures were performed using previously described methods [Bartonella DNA test control samples, consisting of bacteria-free blood DNA and DNA spiked with B. henselae genomic DNA at 0.5 genome copies per microliter, respectively, were used for each batch of DNA tested. For all results reported in this study, PCR products consistent in size with a Bartonella spp. (400-600 bp amplicon size) were sequenced to confirm the species and ITS strain. Sequences were aligned and compared with GenBank sequences using AlignX software .In October, 2007, the primary author was contacted by the father of a family residing in North Carolina, who requested Bartonella testing as a component of an IRB approved study . For all family members and pets  tested in this study, a previously described approach that combines PCR detection of Bartonella vinsonii subsp. berkhoffii and B. henselae antibodies were determined following traditional immunofluorescence antibody assay (IFA) practices with fluorescein conjugated goat anti-human IgG. Bartonella vinsonii subsp. berkhoffii genotypes I, II and III and B. henselae (Houston I strain) were passed from agar grown cultures of each organism into DH82 (a continuous canine histiocytic cell line) cultures to obtain antigens that would seemingly be expressed by an intracellular bacteria localized to erythrocytes or endothelial cells within the vasculature. Heavily infected cell cultures were spotted onto 30-well Teflon coated slides (Cel-Line/Thermo Scientific), air dried, acetone fixed and stored frozen. Serum samples were diluted in phosphate buffered saline (PBS) solution containing normal goat serum, Tween-20 and powdered nonfat dry milk to block non-specific antigen binding sites. Patient sera were screened at dilutions of 1:16 to 1:64. All sera that remained reactive at a titer of 1:64 were further tested with twofold dilutions out to a final dilution of 1:8192.Serology was performed using modifications of a previously described indirect fluorescent antibody test . BartoneThe father was a 50-year-old veterinarian whose symptoms began in 2006 with arthralgias and fatigue, which became progressively severe over ensuing 18 months. He described pain and stiffness of the joints, muscles, and neck that were most severe in the morning but improved throughout the day. He did not have fevers, but he suffered from profound fatigue. He had also experienced an 80-pound weight loss, though this was partially intentional. Beginning in September 2007, and of greatest concern to the patient, was progressive difficulty maintaining his balance while standing or ambulating. His history was notable for extensive occupational and domestic animal exposure. International travel was minimal. He was initially evaluated by a neurologist, and because of his exposure to zoonotic pathogens he was referred for infectious disease evaluation. He had not received any empiric courses of antibiotics.On physical examination, the patient had a blood pressure of 141/88 mm Hg, a pulse of 98 beats/min, and a temperature of 37°C. He was in no acute distress and had a normal sensorium. Notable abnormal findings included a positive Romberg sign and difficulty with heel-toe walking. Cranial nerves, muscle strength, sensation, and deep tendon reflexes were normal and symmetrical, and his funduscopic examination was normal. There was no lymphadenopathy, no organomegaly, and no rash. Lumbar puncture revealed normal cerebrospinal fluid indices and opening pressure. Magnetic resonance imaging of the brain was notable for an increase in signal intensity throughout the pons and upper medulla lateralizing to the left of the midline  failed to result in bacterial growth . Seroreactivity to B. vinsonii subsp. berkhoffii antigens remained essentially unchanged (within one dilution of previous results); however, there was a marked increase in the B. henselae antibody titer. The father reported a history of periodontal disease, which coincided with the onset of his illness. Sterile cotton swabs were used to obtain saliva and periodontal surface samples, after which ITS-PCR generated amplification products from both samples. Efforts to sequence the amplicon from saliva was not successful; however, B. vinsonii subsp. berkhoffii was amplified and sequenced from the periodontal swab. As B. vinsonii subsp. berkhoffii DNA was identified in three different sample sources  and at three different time points in the laboratory, he was treated for bartonellosis with doxycycline plus rifampin. During the first week of therapy he reported a worsening of symptoms, followed by gradual improvement. Following 3 weeks of antibiotics his B. henselae antibody had titer decreased fourfold, his B. vinsonii subsp. berkhoffii titers remained unchanged, and B. henselae (ITS Houston I strain) was amplified and sequenced from two 14-day BAPGM enrichment cultures (both EDTA and ACD-anti-coagulated blood samples were independently processed). Based upon this result, he received an additional 6 weeks of doxycycline plus rifampin. During the subsequent 11 months, PCR evidence of Bartonella infection was not detected in two additional blood cultures, the patient became non-seroreactive B. henselae and B. vinsonii subsp. berkhoffii. Post-treatment, the patient gradually regained body weight and no longer experienced arthralgias or neurological symptoms.th Table . By IFA The 7 1/2 year old daughter of the above patient first sought medical care for a similar constellation of symptoms in October, 2007. Her illness began suddenly one morning with severe neck pain. Over the next month the pain gradually improved but never fully remitted, and she additionally developed headaches, low-grade fevers, and general malaise. Her symptoms evolved to include intermittent weakness of her legs and paresthesias, which were so debilitating that she was no longer able to attend school. She was seen by a pediatric neurologist, and her vital signs and physical exam were noted to be normal. She did not have any objective neurologic deficits.B. vinsonii subsp. berkhoffii in the father's blood sample, a decision was made to test the daughter. The daughter was seroreactive to antigens of B. vinsonii subsp. berkhoffii genotypes II and III and to B. henselae . She began a 9-week course of doxycycline, and at week 6, Bartonella spp. DNA was no longer detectable from the extracted blood sample, from the BAPGM enrichment blood culture, or from the agar plate. Due to continued intermittent neck and back pain the BAPGM diagnostic platform was repeated approximately 6 weeks later and a bacterial subculture isolate was obtained. Because no product was amplified using the ITS primers, different primers were used. An Ochrobactrum sp. was amplified and sequenced from a subculture isolate, using primers targeting the RpoB gene. She remained symptomatic while off therapy for the next two months, and though her symptoms were milder than before, they persisted and she was retreated with a 6-week course of doxycycline. During the subsequent two months antibody titers remained unchanged and two BAPGM blood cultures failed to result in PCR detection of Bartonella spp. infection. However, despite becoming seronegative to all test antigens, B. henselae (Houston 1 strain) was amplified and sequenced from a BAPGM enrichment culture obtained 3 months later. She had a relapse of neck pain several months later, but she did not receive antibiotics that time. One year after testing was initiated, the girl was no longer symptomatic and remained seronegative and blood culture negative. She suffered a minor, unrelated head injury during this time. A CT scan and subsequent MRI of the brain incidentally revealed multiple curvilinear calcifications in the left posterior parietal lobe along the periphery, throughout the cerebrum in both the gray and white matter, and sparing the cerebellum  and for the two sons . In these three individuals, antibody titers ranged from 1:32 to 1:128 to antigens of B. henselae and B. vinsonii subsp. berkhoffii genotypes II and III. Bartonella spp. was not isolated nor PCR amplified from blood or serum samples obtained from these three other family members.Within the family, the mother and two sons were reportedly healthy. As described for the father and daughter, B. henselae and B. vinsonii subsp. berkhoffii antigens by IFA testing, whereas antibodies were not detectable in serum samples from the two newly acquired dogs (Table Bartonella henselae (ITS Houston 1 strain) was isolated from three of four cats. By DNA sequencing, the B. henselae ITS strains obtained from the father and daughter were identical to each of the B. henselae ITS sequences obtained from the three cats following direct extraction from blood, following BAPGM enrichment culture, and from agar plate derived isolates. The two English Springer Spaniel littermates were PCR negative for Bartonella spp. DNA following blood extraction, BAPGM enrichment cultures and no bacteria were isolated.The family had four domestic shorthair cats and two recently acquired one-year-old English Springer Spaniel littermates. Two other dogs, previously owned by the family, had been killed in an automobile accident, five months prior to the onset of illness in the father and daughter. All four cats had antibodies to gs Table . BartoneB. vinsonii subsp. berkhoffii infection in two family members who were experiencing neurological dysfunction. Bartonella vinsonii subsp. berkhoffii is an important emerging intravascular pathogen that has been isolated from patients with endocarditis, arthritis, neurological disease and vasoproliferative neoplasia [B. vinsonii subsp. berkhoffii genotype II strains and both were either co-infected or sequentially infected with B. henselae. Considering the sequential serological test results obtained for the father, it seems likely that he was co-infected, when the first blood culture was obtained. Initial B. henselae antibody titers for the father were 8 to 12 fold higher than the titers obtained using B. vinsonii subsp. berkhoffii genotype II antigens, whereas only a 6 fold difference was found in the initial two serum samples from the daughter. As there is a less convincing serological association supporting co-infection in the daughter and as three of four cats in the household were B. henselae bacteremic during the period in which the father and daughter were being treated for their B. vinsonii subsp. berkhoffii infections, it is possible that sequential infection with two different Bartonella spp. was documented in the daughter during the course of this study. As the isolation and molecular detection of these bacteria from patient samples remains microbiologically challenging, we were unable to clearly establish whether either patient was co-infected or sequentially infected at various time points.In this study, we report the simultaneous detection of eoplasia ,6,14,15.Bartonella sp. when two or more species are present in the extracted sample [Bartonella genes can enhance the possibility of detecting co-infection in patient samples [Bartonella spp. in co-infected individuals. It is also possible that BAPGM enrichment culture preferentially selects for the growth of one Bartonella spp. in a co-infected individual. Therefore both individuals may have been co-infected with B. vinsonii subsp. berkhoffii and B. henselae at the outset of this study.Previously, we described the preferential amplification of one d sample . The mec samples -19. PoteB. henselae strains obtained from the father and daughter's blood cultures were identical (B. henselae ITS strain Houston1) to the blood culture strains obtained from the three cats, the family cats were the presumed source of this infection.As the sequences of the B. henselae. The two newly acquired young dogs were not seroreactive to Bartonella antigens and both were blood culture negative; therefore it seems unlikely that these dogs played a role in transmission of B. henselae or B. vinsonii subsp. berkhoffii in the family. Although cats are the primary reservoir host, B. henselae has been isolated by blood culture from dogs and sequenced from dog saliva [B. vinsonii subsp. berkhoffii infection, as historically this organism has been isolated from domestic and wild canines and humans [B. vinsonii subsp. berkhoffii was recently described in a cat with recurrent osteomyelitis that was bacteremic over a 15-month time period [B. vinsonii subsp. berkhoffii bacteremia and potentially serve as a source of bacterial transmission to humans [B. vinsonii subsp. berkhoffii from the cats in this study using subspecies-specific primers were not successful. Over 100 years ago, B. quintana was transmitted to human volunteers, when saliva from a febrile patient was applied to escharified skin [Bartonella quintana, oral transmission of B. vinsonii subsp. berkhoffii from the father or a pet cat to the daughter through close family contact cannot be ruled out. Bartonella spp. DNA has now been reported in the saliva of cats, dogs and humans [Bartonella spp. transmission to humans and to their pets, but it seems prudent to recommend hygienic measures after contacting pet and perhaps human saliva.Although the mother and two sons were healthy and blood culture negative when tested on a single occasion, all three had serological evidence supporting prior exposure to g saliva ,9. It isd humans ,2. Infece period . Therefoo humans . Effortsied skin . As salid humans ,23. CleaBartonella spp. in cerebrospinal fluid. In a previous study, B. henselae was repeatedly detected by blood or cerebrospinal fluid culture in a 23 year-old girl who developed progressively severe seizures following a history of cat scratch disease [Bartonella spp. appear to target vascular endothelial cells, it is possible that B. vinsonii subsp. berkhoffii and B. henselae contributed to the nonspecific area of vascular injury reported on the father's MRI.The father in this report is the second patient in which enrichment culture enhanced the molecular detection of a  disease . In bothBartonella spp. following culture-enrichment of patient samples prior to performing PCR [B. vinsonii subsp. berkhoffii and B. henselae DNA were never directly amplified from extracted blood samples obtained from the daughter and were only detectable by PCR following enrichment culture. In our experience, at least a 7 to 14-day incubation period is required before the enriched sample is obtained from liquid BAPGM for DNA extraction. At no time during this study was Bartonella spp. DNA amplified from a DNA extraction control, a BAPGM un-inoculated enrichment culture control or a subculture agar plate control. Although not detailed in the table, it is not unusual for 7-day enrichment cultures and subcultures to be PCR negative for Bartonella spp. DNA, whereas the respective organism can be amplified and sequenced after a 14-day incubation period from the enriched liquid culture, the agar plate isolate, or both. Due to the high level of B. henselae bacteremia generally found in cats, the same strain could be detected in each cat following direct extraction of the blood sample, enrichment culture and the agar plate isolates  is used as the source of positive control DNA for the ITS PCR reaction; therefore, contamination with positive control DNA could not explain any PCR results obtained in these two patients. As over 500 species of bacteria have been estimated to inhabit the oral cavity, BAPGM enrichment culture was not attempted because rapidly growing organisms would negate efforts to increase Bartonella numbers via the enrichment process [B. vinsonii subsp. berkhoffii in close approximation of the periodontal surface reflects passive leakage of bacteria through inflamed and compromised vascular tissues or alternatively the establishment of an active foci of infection that contributed to the recent history of periodontitis. Detection of DNA in the oral cavity does not confirm the presence of viable bacteria; however, caution should be exercised by dentists and physicians when examining the oral cavity of an individual with chronic Bartonella spp. bacteremia.The father reported a recent history of severe periodontal disease. Two previous molecular microbiological studies identified ontitis. ,25. In aless man -28. Afteul Table . As iden process ,30. It iB. vinsonii subsp. berkhoffii and B. henselae are zoonotic pathogens that can be isolated from the blood of immunocompetent family members with arthralgias, fatigue and neurological symptoms. Therapeutic elimination of Bartonella spp. infections can be challenging, and follow-up testing is recommended. An increasing number of arthropod vectors, including biting flies, fleas, keds, lice, sandflies and ticks have been confirmed or are suspected as the primary mode of transmission of Bartonella species among animal populations and may also pose a risk to human beings.Bartonella species infection in animals and in human patient samples. Dr. Ricardo Maggi performed all molecular microbiological testing reported in this study and is the Scientific Technical Advisor and Laboratory Director for Galaxy Dx.In conjunction with Dr. Sushama Sontakke and North Carolina State University, Dr. Breitschwerdt holds U.S. Patent No. 7,115,385; Media and Methods for cultivation of microorganisms, which was issued October 3, 2006. He is the chief scientific officer for Galaxy Diagnostics, a newly formed company that provides diagnostic testing for the detection of EBB was involved in all aspects of this study, including generation of the initial draft of the manuscript; RGM performed all blood cultures, PCR, sequencing and molecular data analyses, PML and CWW were responsible for patient evaluation, medical record review and patient follow-up, BCH and JMB were responsible for serological testing. All authors contributed to the content and approved the final manuscript.