Determination of limit of detection (LOD) for loop-mediated isothermal amplification (LAMP) of human cytomegalovirus (hCMV) DNA.

The importance of cytomegalovirus in clinical practice remains and samples are monitored for CMV DNA titers to predict the development of disease. LAMP assays have gained increasing interest in the diagnosis of many pathogens since they do not require thermocycling, reduce the complexity of the required instrumentation as well as providing sensitivity and rapidity. So far, very few studies on CMV detection by LAMP have been reported in the literature and therefore the performance of LAMP CMV assays needs to be further characterized. In a set-up for biometrological evaluation of the suitability of the LAMP assay for CMV diagnosis, a LAMP assay was performed on a total of 192 samples with 24 replicates of 8 different hCMV DNA concentrations. The LOD was calculated as 39.09 copy/reaction (25.33 copy/reaction to 65.84 copy/reaction) with 95 % confidence, representing a range that is suitable for qualitative detection. Furthermore, the lower limit of quantification was estimated at approximately 100 copy/reaction. The LOD and LLOQ values obtained in this first study to assess the biometrological relevance of LAMP CMV tests are consistent when compared to studies published before. However further study under different conditions is needed for the use of LAMP tests in clinical applications.

Use of over-the-scope clips in the colon in clinical practice: results from a German administrative database.

BACKGROUND : The efficacy and safety of over-the-scope (OTS) clips in the colon is limited. This study aimed to evaluate OTS clip use in the colon in routine colonoscopy. METHODS: Using administrative data from a large health insurance company, patients with OTS clip placement during colonoscopy were identified and analyzed by specific administrative codes. Indication for OTS clipping was analyzed, and follow-up was evaluated for surgical and repeat endoscopic interventions. RESULTS: In 505 patients, indications for OTS clips were iatrogenic perforations (n = 80; Group A), polypectomy (n = 315; Group B), colonic bleeding (n = 51; Group C), and various underlying diseases (n = 59; Group D). In 11 Group A patients (13.8 %), surgical interventions occurred, mostly within 24 hours after clipping (n = 9), predominantly overstitching (n = 8). OTS clipping during polypectomy (Group B) was for complications (e. g. bleeding in 27 %) or was applied prophylactically. Only five patients required early surgery, three of whom had colorectal cancer. In four Group C patients (7.8 %), surgical resections were performed (persistent bleeding n = 1, colorectal cancer n = 2), while six patients underwent early repeat colonoscopy for recurrent bleeding. During further follow-up (days 11-30), 17 patients underwent resection for colonic neoplasms (n = 12) or persistent bleeding (n = 4), but only one case could be directly traced back to local OTS clip complication. CONCLUSION: Colonic OTS clipping appears safe and effective in selected indications and complications in clinical routine but must be anatomically and technically feasible, avoiding overuse.

Investigation of direct counting and sizing of DNA fragments in flow applying an improved data analysis and correction method.

Direct detection of single stained DNA fragments in flow is a very sensitive method for nucleic acid detection which does not need any amplification process. We have developed an instrument for direct counting and sizing of single DNA fragments (single or double stranded DNA) in flow with integrated sample volume measurement for concentration determination. As the method is a potential reference method for DNA quantification, processes affecting the measurement uncertainty are of major interest. Additionally, comparison of this method to the orthogonal method of digital PCR is useful with the restriction of low specificity of the direct detection method. In this study, we analysed raw detector signals and the sizing performance for target identification and the effect of coincidence detection concerning concentration measurements. We present data of purified artificial DNA samples measured with the home-built setup. Main emphasis was to develop an improved data analysis method to gain insight into and carefully correct for coincident detection of DNA fragments and for estimation of the amount of fragment dimers.

Robust versus consistent variance estimators in marginal structural Cox models.

In survival analyses, inverse-probability-of-treatment (IPT) and inverse-probability-of-censoring (IPC) weighted estimators of parameters in marginal structural Cox models are often used to estimate treatment effects in the presence of time-dependent confounding and censoring. In most applications, a robust variance estimator of the IPT and IPC weighted estimator is calculated leading to conservative confidence intervals. This estimator assumes that the weights are known rather than estimated from the data. Although a consistent estimator of the asymptotic variance of the IPT and IPC weighted estimator is generally available, applications and thus information on the performance of the consistent estimator are lacking. Reasons might be a cumbersome implementation in statistical software, which is further complicated by missing details on the variance formula. In this paper, we therefore provide a detailed derivation of the variance of the asymptotic distribution of the IPT and IPC weighted estimator and explicitly state the necessary terms to calculate a consistent estimator of this variance. We compare the performance of the robust and consistent variance estimators in an application based on routine health care data and in a simulation study. The simulation reveals no substantial differences between the 2 estimators in medium and large data sets with no unmeasured confounding, but the consistent variance estimator performs poorly in small samples or under unmeasured confounding, if the number of confounders is large. We thus conclude that the robust estimator is more appropriate for all practical purposes.

[Prescribing valproate to girls and women of childbearing age in Germany : Analysis of trends based on claims data]. / Valproatverordnungen bei Mädchen und Frauen im gebärfähigen Alter in Deutschland : Untersuchung zeitlicher Trends basierend auf Versichertendaten.

BACKGROUND: Measures to raise awareness of the teratogenic potential of valproate and restrict its use in girls/women of childbearing age have been intensified. For Germany, the impact of these measures on valproate prescription rates remains unknown. OBJECTIVES: Trends in prescribing valproate, the underlying treatment indication, and the specialty of the prescribing physician are analyzed. MATERIALS AND METHODS: With claims data from several statutory health insurance providers from 2004 to 2016 (approximately 3.5 million insured persons per year) considering treatment indication and medical specialties of prescribing physicians, we assessed the rate of girls/women (12 to 50 years) with at least one valproate dispensation per year. RESULTS: The age-standardized rate of girls/women with at least one valproate dispensation declined by 28% between 2004 and 2016 (2.91/1000 vs. 2.09/1000). For 2015, the indications were epilepsy (66.9%), bipolar disorder (13.6%), migraine/headache (5.6%), schizoaffective disorder (4.3%), and other mental disorders (8.9%). Among epilepsy patients, the proportion treated with valproate declined from 26.2 to 16.8%, but changed little in patients with bipolar disorder (9.3% vs. 8.0%). A total of 46.3% of valproate dispensations were issued by neurologists or psychiatrists and 29.6% by general practitioners, internal medicine specialists, or family doctors. CONCLUSIONS: Based on German claims data, a decline of valproate dispensations was shown for epilepsy patients of childbearing age, while the proportion in other indications has hardly changed since 2004.

Mapping the lack of public initiative against female genital mutilation in Denmark.

BACKGROUND: Female genital mutilation (FGM) is a harmful practice prevalent in 35 countries, mainly in Africa, as well as in some Middle Eastern countries and a few Asian countries. FGM comprises all procedures that involve partial or complete resection of, or other injury to, external female genitalia for non-medical reasons. The practice of FGM has spread to Western countries due to migration. The European Institute for Gender Equality recommend that FGM be combatted by nationally coordinated efforts through implementation of national action plans, guidelines for professionals as well as comprehensive research in the field. FGM was outlawed in Denmark 2003, but no national actions plan has been implemented. Instead, the task of combatting FGM is currently under the responsibility of local governments in the form of the 98 municipalities. The aim of this study is to investigate the Danish municipalities' efforts to prevent FGM on the local level, and whether these initiatives are in accordance with international recommendations and standards. METHODS: All 98 Danish municipalities were invited to respond to a questionnaire regarding FGM in their respective municipalities. The inclusion process and questionnaire was designed after a pilot study, which included 29 municipalities. The questionnaire consisted of four overall areas of focus: "action plan", "registration", "information material" and "preventive initiatives". Demographic data were gathered from the 2017 census by Statistics Denmark. Risk countries were defined as countries with a tradition for FGM, identified from the 2016 UNICEF definition. RESULTS: A total of 67 municipalities participated in the study. At the time of census, 1.8% of the Danish population was immigrants with origins in risk countries. A total of 10.4% of the responding municipalities indicated to have implemented a specific action plan against FGM. A total of 7,5% had implemented specific preventive initiatives against FGM. Registration of reported FGM cases were indicated to be performed in 73.1% of the responding municipalities; however, only 17.9% stated to perform registration of FGM specifically as such, and not as general child abuse. CONCLUSIONS: Our study shows that the current situation of FGM registration and prevention being under local administrative responsibility in the 98 Danish municipalities has led to a severe lack of coordinated public initiative against FGM.

Predictors of non-drug psychiatric/psychotherapeutic treatment in children and adolescents with mental or behavioural disorders.

Children and adolescents with mental health problems need effective and safe therapies to support their emotional and social development and to avoid functional impairment and progress of social deficits. Though psychotropic drugs seem to be the preferential treatment, psychotherapy and psychosocial interventions are essential in mental health care. For Germany, current data on the utilization of psychotherapy and psychosocial interventions in children with mental health problems is lacking. To analyse why certain children and adolescents with mental or behavioural disorders do and others do not receive non-drug treatment, we assessed predictors associated with specific non-drug psychiatric/psychotherapeutic treatment including psychosocial interventions, psychotherapy and other non-drug treatments. The study is based on data of two large German health insurance funds, AOK and TK, comprising 30 % of the German child and adolescent population. Predictors of non-drug psychiatric/psychotherapeutic treatment were analysed for 23,795 cases and two controls for every case of the same age and sex in children aged 0-17 years following a new diagnosis of mental or behavioural disorder in 2010. Predictors were divided according to Andersen's behavioural model into predisposing, need and enabling factors. The most prominent and significant predictors positively associated with non-drug psychiatric/psychotherapeutic treatment were the residential region as predisposing factor; specific, both ex- and internalizing, mental and behavioural disorders, psychiatric co-morbidity and psychotropic drug use as need factors; and low area deprivation and high accessibility to outpatient physicians and inpatient institutions with non-drug psychiatric/psychotherapeutic department as enabling factors. In conclusion, the present study suggests that the residential region as proxy for supply of therapist and socioeconomic situation is an influencing factor for the use of psychotherapy. The analysis sheds further light on predisposing, need and enabling factors as predictors of non-drug psychotherapeutic/psychiatric treatment in children and adolescents with mental or behavioural health disorders in Germany. More research is needed to further understand the factors promoting the gap between the need and utilization of mental health care.

Psychopharmacological Prescriptions in Children and Adolescents in Germany.

BACKGROUND: In view of the well-known increase in prescriptions of stimulants for children and adolescents over the last 20 years, it is important to study trends in the prevalence and incidence of the use of other psychotropic drugs by this age group as well, to enable an early response to potential problems in the current care situation. METHODS: We used nationwide data from German statutory health insurance funds (Allgemeine Ortskrankenkasse [AOK], all insurees; Techniker Kranken - kasse [TK], a 50% randomized sample) concerning all insurees aged 0-17 years (5.0 million people in 2012) to study trends in the prevalence and incidence of psychotropic medication use as well as initially prescribing medical specialties over the period 2004-2012, both for the overall group of psychotropic drugs and for selected subgroups of drugs. RESULTS: From 2004 to 2012, the prevalence of psychotropic drug prescriptions (not including herbal and homeopathic substances) for children and adolescents rose from 19.6 to 27.1 per 1000 individuals. Marked rises were seen for stimulants (10.5 to 19.1 per 1000) and antipsychotic drugs (2.3 to 3.1 per 1000), while the prevalence of antidepressant prescriptions remained constant at about 2 per 1000. The rates of new prescriptions from 2006 to 2012 were generally constant or decreasing; for the overall group of (non-herbal, nonhomeopathic) psychotropic drugs, the rate of new prescriptions fell from 9.9 to 8.7 per 1000. There was a trend toward the issuance of new prescriptions by medical specialists, rather than by family physicians and pediatricians. CONCLUSION: The observed increased prevalence of psychotropic drug use among children and adolescents appears to be due not to an increased rate of initial prescriptions for these drugs, but rather to a rise in the number of patients who, once having received such drugs, were given further prescriptions for them in the years that followed.

Comparative risk for cardiovascular diseases of dipeptidyl peptidase-4 inhibitors vs. sulfonylureas in combination with metformin: Results of a two-phase study.

AIMS: The aim was to assess whether the use of additional data from the Disease Management Program (DMP) diabetes mellitus type 2 to minimize the potential for residual confounding will alter the estimated risk of either myocardial infarction, ischemic stroke or heart failure in patients with type 2 diabetes using sulfonylureas compared to dipeptidyl peptidase-4 (DPP-4) inhibitors in addition to metformin based on routine health care data. METHODS: We conducted a nested two-phase case-control study using claims data of one German health insurance from 2004 to 2013 (phase 1) and data of the DMP from 2010 to 2013 (phase 2). Adjusted odds ratios (ORs) for the combined cardiovascular event myocardial infarction, ischemic stroke or heart failure were calculated using a two-phase logistic regression. RESULTS: Phase 1 comprised 3179 patients (289 cases; 2890 controls) and phase 2 comprised 1968 patients (168 cases; 1800 controls). We observed an adjusted OR of 0.83 for the combined cardiovascular event (95% CI: 0.61-1.13). CONCLUSIONS: We observed a non-significantly reduced risk for cardiovascular diseases in patients using DPP-4 inhibitors compared to sulfonylureas in addition to metformin. This finding was not altered by the inclusion of additional information of the DMP in the analysis. However, due to the low power of this study, further studies are needed to reproduce our findings.

The costs and consequences of obesity in Germany: a new approach from a prevalence and life-cycle perspective.

With the steadily growing health burden of obesity in Germany, the measuring and quantification of its costs and relevant economic consequences have become increasingly important. The usual quantifications via previous cost-of-illness approaches mostly have several weaknesses, e.g., applying "indirect methods" by using "population-attributable fractions" to identify parts of costs that can be accrued to obesity, second using highly aggregated data and third often only displaying part of the costs. This article presents a new approach and a new estimation of the cost and consequences of obesity in Germany using claims data from a German health insurance company. A sample of 146,000 individuals was analyzed with both a prevalence and a life-cycle focus on the cost and consequences of obesity. With additional data sets, we calculate the deaths per year due to obesity, the excess costs per year and several intangible consequences usually referred to as "pain and suffering". Our results show that the cost estimations of obesity in Germany so far have been largely underestimated. The annual direct costs of obesity in Germany amount to approximately €29.39 billion and the indirect costs to an additional €33.65 billion. A total of 102,000 subjects die prematurely each year because of obesity, and there is a significant excess of unemployment, long-term nursing care, and pain and suffering due to obesity. From a lifetime perspective, every obese man is equal to an additional burden of €166,911 and each woman of €206,526 for the social security system in Germany. Obesity due to unhealthy eating is thus about to replace tobacco consumption in terms of costs and consequences as the main hazardous lifestyle factor and thus should be more intensively focussed by public health policy.

Healthcare costs and resource utilization of asthma in Germany: a claims data analysis.

INTRODUCTION: Asthma is associated with a substantial economic burden on the German Statutory Health Insurance. AIMS AND OBJECTIVES: To determine costs and resource utilization associated with asthma and to analyze the impact of disease severity on subgroups based on age and gender. METHODS: A claims database analysis from the statutory health insurance perspective was conducted. Patients with an ICD-10-GM code of asthma were extracted from a 10% sample of a large German sickness fund. Five controls for each asthma patient matched by age and gender were randomly selected from the same database. Costs and resource utilization were calculated for each individual in the asthma and control group. Incremental asthma-related costs were calculated as the mean cost difference. Based on prescribed asthma medication, patients were classified as intermittent or persistent. In addition, age groups of ≤ 5, 6-18, and >18 years were analyzed separately and gender differences were investigated. RESULTS: Overall, 49,668 individuals were included in the asthma group. On average, total annual costs per patient were €753 higher (p = 0.000) compared to the control group (€2,168 vs. €1,415). Asthma patients had significantly higher (p = 0.000) outpatient (€217), inpatient (€176), and pharmacy costs (€259). Incremental asthma-related total costs were higher for patients with persistent asthma compared to patients with intermittent asthma (€1,091 vs. €408). Women aged >18 years with persistent asthma had the highest difference in costs compared to their controls (€1,207; p < 0.0001). Corresponding healthcare resource utilization was significantly higher in the asthma group (p = 0.000). CONCLUSIONS: The treatment of asthma is associated with an increased level of healthcare resource utilization and significantly higher healthcare costs. Asthma imposes a substantial economic burden on sickness funds.

[Do nursing homes with higher quality ratings provide a better quality of care? : Empirical study based on administrative data]. / Bieten Pflegeheime mit besseren Pflegenoten auch eine bessere Qualität? : Empirische Analyse auf Basis von GKV-Routinedaten.

BACKGROUND: In 2009 a new system for the objective evaluation of nursing homes was introduced in Germany. The so-called nursing transparency agreement (Pflege-Transparenzvereinbarungen) was introduced to provide a reliable tool for an objective comparison of inpatient (PTVS) and outpatient (PTVA) care; however, the new regulations have been the subject of a broad discussion regarding reliability, efficiency and objectivity. AIMS: To overcome the lack of objective health outcomes, this study used administrative data from Germany's largest health insurance fund, the Techniker Krankenkasse, in order to analyze the association between the quality ratings and objective quality measures on an individual level. This is the first study that provides empirical evidence on this topic using administrative data. MATERIAL AND METHODS: The administrative dataset contained information on several individual characteristics as well as data on injuries, poisoning and other extrinsic effects on care-dependent individuals over the age of 64 years who were living in a nursing home in 2009. Based on these data an objective measure was constructed to test whether higher quality ratings of nursing homes led to a better quality of care of the respective patients using non-linear regression models. RESULTS: The results of the estimated models showed no significant evidence of such a relationship, neither considering the probability nor the number of injuries, poisoning and other extrinsic effects. Significant effects were only observed for gender and specific diseases. CONCLUSION: The results of this study support the argument that the current rating procedure for nursing homes has to be refined. Using quality indicators in combination with the administrative data could possibly contribute to such an enhancement.

Exploring Health System Responsiveness in Ambulatory Care and Disease Management and its Relation to Other Dimensions of Health System Performance (RAC) - Study Design and Methodology.

BACKGROUND: The responsiveness of a health system is considered to be an intrinsic goal of health systems and an essential aspect in performance assessment. Numerous studies have analysed health system responsiveness and related concepts, especially across different countries and health systems. However, fewer studies have applied the concept for the evaluation of specific healthcare delivery structures and thoroughly analysed its determinants within one country. The aims of this study are to assess the level of perceived health system responsiveness to patients with chronic diseases in ambulatory care in Germany and to analyse the determinants of health system responsiveness as well as its distribution across different population groups. METHODS AND ANALYSIS: The target population consists of chronically ill people in Germany, with a focus on patients suffering from type 2 diabetes and/or from coronary heart disease (CHD). Data comes from two different sources: (i) cross-sectional survey data from a postal survey and (ii) claims data from a German sickness fund. Data from both sources will be linked at an individual-level. The postal survey has the purpose of measuring perceived health system responsiveness, health related quality of life, experiences with disease management programmes (DMPs) and (subjective) socioeconomic background. The claims data consists of information on (co)morbidities, service utilization, enrolment within a DMP and sociodemographic characteristics, including the type of residential area. DISCUSSION: RAC is one of the first projects linking survey data on health system responsiveness at individual level with claims data. With this unique database, it will be possible to comprehensively analyse determinants of health system responsiveness and its relation to other aspects of health system performance assessment. The results of the project will allow German health system decision-makers to assess the performance of nonclinical aspects of healthcare delivery and their determinants in two important areas of health policy: in ambulatory and chronic disease care.

Estimating colorectal cancer treatment costs: a pragmatic approach exemplified by health insurance data from Germany.

BACKGROUND: The cost of colorectal cancer (CRC) treatment is a crucial parameter to inform cost-effectiveness analyses on CRC screening but it is not readily available and therefore often lacking. We aimed to elaborate and exemplify a pragmatic approach to estimate CRC treatment cost based on health insurance data from Germany. METHODS: We included two groups of persons who were continuously health-insured between 2005-2010: A) Cases: Persons with a hospital discharge diagnosis of CRC (ICD C18-C20) between 2007-2010 and no such a diagnosis between 2005-2006 (to focus on incident CRC cases); B) Controls: Persons without a diagnosis of CRC during the observation period, matched to CRC cases by age and sex (matching factor: 1∶5). We considered in-patient, out-patient and drug costs and calculated incremental costs as the difference in means between cases and controls. We divided costs into three phases of care (initial, intermediate and end-of-life phase). RESULTS: The initial, the intermediate and the end-of-life phase included 12,792, 5,280, and 3,779 CRC cases, respectively, and 63,960, 26,400, and 18,895 controls. The mean incremental costs--annualized for each phase--were €26,000, €2,300, and €51,700, respectively. The costs of the initial phase of care were higher for rectal than for colon cancer. Annualized stage-specific cost estimates ranged from €15,000 to €21,300 for early stages and from €29,800 to €35,000 for late stages. CONCLUSION: This pragmatic and feasible approach provided plausible estimates of CRC treatment costs in Germany; being transferable to other settings, it may thus facilitate to weigh up potential savings in treatment costs against the resources required for CRC control programs in various countries.

Treatment costs of attention deficit hyperactivity disorder in Germany.

BACKGROUND: Attention deficit hyperactivity disorder (ADHD) is one of the most common behavioural disorders among children and adolescents. The number of patients as well as prescriptions to treat this disease has continuously increased over the past few years. The aim of the present study was to analyse the costs for treating ADHD patients from the perspective of a major German health insurance fund. METHODOLOGY: Anonymised administrative claims data were available for the study. All services reimbursed by the health fund for the selected ADHD patients were recorded. Apart from the resource use attributed directly to ADHD, co-morbidities as well as incremental costs were described based on a control group design. RESULTS: A total of 30,264 ADHD patients were diagnosed in 2008. The total costs for these patients were euro 3,888, and the incremental costs were euro 2,902. The largest proportions of incremental costs were due to therapeutic devices and remedies like occupational therapy amounting to euro 1,270. Proportionate costs of euro 263 have been settled for pharmacotherapy with Methylphenidate and Atomoxetine. However, 41% of the patients were not treated with ADHD-related pharmaceuticals. CONCLUSIONS: ADHD costs are relevant from health insurance perspective. The expenses for occupational therapy constitute the cost driver. Compared to the findings of studies from the United States and contrarily to the backdrop of public discussions about considerably increased prescriptions of ADHD-specific drugs, the significantly higher additional expenses for occupational therapy services are impressing. This kind of therapy is internationally rather unknown and is therefore not acknowledged as a therapeutic standard.

Fall risk increasing drugs and injuries of the frail elderly - evidence from administrative data.

BACKGROUND: Society benefits on a large scale from improved medical care and pharmaceuticals. The prescription of pharmaceuticals, however, also carries risks such as the possibility of an increased hazard of falls, which may lead to severe injuries and increased health expenditures associated with these injuries. This study investigates the relationship of several fall risk increasing drugs (FRIDs) and injuries of elderly persons using multivariate regression models. METHOD: Routine data from the Techniker Krankenkasse of frail elderly persons aged ≥ 65 years is analysed for the year 2009 by estimating count data models, in order to take the data generating process of the number of injuries into account. The results of this model are compared to those from logistic regressions, which is the default regression model in this field of research. RESULTS: The empirical results suggest that the incidence of injuries strongly increases with doses of antidepressants, anxiolytics, hypnotics and sedatives, antiarrhythmics and drugs from the Priscus-list. However, antihypertensives and antiparkinsonian agents show no significant association and neuroleptics indicate a significant negative association. CONCLUSION: As recurrent injuries are common, the analysis of the number of injuries rather than just the probability of having an injury provides a more informative analysis of FRIDs. According to the empirical results, antidepressants, anxiolytics, hypnotics and sedatives, antiarrhythmics as well as drugs from the Priscus-list should be prescribed carefully, because these drugs are positively correlated with the incidence of injuries.

Angiotensin converting enzyme gene polymorphism and myocardial infarction a large association and linkage study.

The DD genotype of the angiotensin converting enzyme (ACE) polymorphism has been associated with myocardial infarction (MI). However, sample sizes of many case-control studies showing positive association were small and data were inconsistent. Furthermore, no family-based study is available. In a case-control study frequencies of the ACE genotypes were compared in 1319 unrelated patients with previous MI before 60 years of age (616 from the MONICA Augsburg region and 703 from rehabilitation centers in south Germany) and in 2381 population controls from the MONICA Augsburg study region). Furthermore, linkage and association of the ACE I/D polymorphism with MI were tested in 246 informative families using the sib-transmission/disequilibrium test (S-TDT).Overall, no excess of the D allele was found in MI patients (frequency 0.53 versus 0.57 in the general population; P=0.2). The ACE DD genotype was even slightly less frequent in groups with MI compared to the general population controls (0.26 versus 0.33 in women and 0.28 versus 0.33 in men). Similar results were also obtained in 247 men with low cardiovascular risk. In the family-based study, the frequency of the D allele was not different in siblings with or without previous MI (0.53 versus 0.50, respectively; S-TDT P=0.15) indicating no linkage or association of the D allele with MI. In a case-control study of MI patients and controls from the general population as well as a family study neither association nor linkage of the ACE D allele with MI was detected despite sample sizes that were among the largest samples studied so far.