Plasma biomarker discovery for early chronic kidney disease diagnosis based on chemometric approaches using LC-QTOF targeted metabolomics data.

Chronic kidney disease (CKD) is a progressive pathological condition in which renal function deteriorates in time. The first diagnosis of CKD is often carried out in general care attention by general practitioners by means of serum creatinine (CNN) levels. However, it lacks sensitivity and thus, there is a need for new robust biomarkers to allow the detection of kidney damage particularly in early stages. Multivariate data analysis of plasma concentrations obtained from LC-QTOF targeted metabolomics method may reveal metabolites suspicious of being either up-regulated or down-regulated from urea cycle, arginine methylation and arginine-creatine metabolic pathways in CKD pediatrics and controls. The results show that citrulline (CIT), symmetric dimethylarginine (SDMA) and S-adenosylmethionine (SAM) are interesting biomarkers to support diagnosis by CNN: early CKD samples and controls were classified with an increase in classification accuracy of 18% when using these 4 metabolites compared to CNN alone. These metabolites together allow classification of the samples into a definite stage of the disease with an accuracy of 74%, being the 90% of the misclassifications one level above or below the CKD stage set by the nephrologists. Finally, sex-related, age-related and treatment-related effects were studied, to evaluate whether changes in metabolite concentration could be attributable to these factors, and to correct them in case a new equation is developed with these potential biomarkers for the diagnosis and monitoring of pediatric CKD.

Detection of Staphylococcus aureus in cystic fibrosis patients using breath VOC profiles.

Staphylococcus aureus (S. aureus) is a common bacterium infecting children with cystic fibrosis (CF). Since current detection methods are difficult to perform in children, there is need for an alternative. This proof of concept study investigates whether breath profiles can discriminate between S. aureus infected and non-infected CF patients based on volatile organic compounds (VOCs). We collected exhaled breath of CF patients with and without S. aureus airways infections in which VOCs were identified using gas chromatography-mass spectrometry. We classified these VOC profiles with sparse partial least squares discriminant analysis. Multivariate breath VOC profiles discriminated infected from non-infected CF patients with high sensitivity (100%) and specificity (80%). We identified the nine compounds most important for this discrimination. We successfully detected S. aureus infection in CF patients, using breath VOC profiles. Nine highlighted compounds can be used as a focus point in further biomarker identification research. The results show considerable potential for non-invasive diagnosis of airway infections.

Identification of Pseudomonas aeruginosa and Aspergillus fumigatus mono- and co-cultures based on volatile biomarker combinations.

Volatile organic compound (VOC) analysis in exhaled breath is proposed as a non-invasive method to detect respiratory infections in cystic fibrosis patients. Since polymicrobial infections are common, we assessed whether we could distinguish Pseudomonas aeruginosa and Aspergillus fumigatus mono- and co-cultures using the VOC emissions. We took headspace samples of P. aeruginosa, A. fumigatus and co-cultures at 16, 24 and 48 h after inoculation, in which VOCs were identified by thermal desorption combined with gas chromatography - mass spectrometry. Using multivariate analysis by Partial Least Squares Discriminant Analysis we found distinct VOC biomarker combinations for mono- and co-cultures at each sampling time point, showing that there is an interaction between the two pathogens, with P. aeruginosa dominating the co-culture at 48 h. Furthermore, time-independent VOC biomarker combinations were also obtained to predict correct identification of P. aeruginosa and A. fumigatus in mono-culture and in co-culture. This study shows that the VOC combinations in P. aeruginosa and A. fumigatus co-microbial environment are different from those released by these pathogens in mono-culture. Using advanced data analysis techniques such as PLS-DA, time-independent pathogen specific biomarker combinations can be generated that may help to detect mixed respiratory infections in exhaled breath of cystic fibrosis patients.

Effect of constant strain rate, composed of varying amplitude and frequency, of early loading on peri-implant bone (re)modelling.

AIM: Examine the effect of varying components of strain rate -- amplitude versus frequency -- while maintaining a constant strain rate of early controlled mechanical loading on implant stability, peri-implant bone mass and bone-to-implant contact. MATERIAL AND METHODS: Three groups of guinea-pigs received TiO2 -blasted implants in both tibiae. One week after installation test implants were loaded 5 days/week during 4 weeks. The contra-lateral implants were the unloaded controls. Strain rate was kept constant (1600 micro epsilon/s), while amplitude and frequency were varied per group. Implant stability was followed by resonance frequency analysis. Animals were sacrificed, and ground sections were prepared to rate bone-to-implant contact and bone mass. RESULTS: All implants (n=78) integrated uneventfully. A significant positive effect (p=0.03) of early loading on bone mass was observed in the distal medullar cavity. A significant difference in bone mass between test and control implants was evidenced between the groups (p=0.03 and 0.04). A significant increase in implant stability and bone-to-implant contact could not be shown. CONCLUSIONS: Early controlled stimulation of peri-implant bone is related to amplitude/frequency and not to strain rate as such, considering a constant stimulation time. An increase of bone mass around early-loaded implants was shown. This cortical bone model is most sensitive to low-frequency/high-amplitude stimulation.

Growth of self-perceived clinical competence in postgraduate training for general practice and its relation to potentially influencing factors.

OBJECTIVE: To examine the increase in self-perceived clinical competence during a three-year postgraduate training in general practice and to explore the relation between the growth of self-perceived competence and several background variables. DESIGN: Cohort, 1995-1998. SETTING: Three-year Postgraduate Training for General practice in the Netherlands PARTICIPANTS: All Dutch trainees who followed postgraduate training from September 1995 to September 1998 (N=191). INTERVENTION: We asked the trainees at the start and at the end of their postgraduate training to complete a questionnaire, which assessed their self-perceived knowledge, clinical skills and consultations skills. We collected information about potentially influencing background variables. Amongst these were variables such as: age, gender, prior medical experience, the effort someone has spent upon her/his education, insight in weak and strong areas of clinical competence and knowledge and skills levels. MAIN OUTCOME MEASURE: Self-perceived competence. RESULTS: A total of 127 trainees completed both questionnaires (190 at the first administration and 128 at the second one). We found statistically significant growth of self-perceived clinical competence. Self-perceived consultation skills increased more than self-perceived knowledge and clinical skills. The afore mentioned background variables did not relate in any way with the growth of self-perceived clinical competence. CONCLUSION: This study shows that a 3-year postgraduate training in general practice enhances self-perceived clinical competence. However, we still do not know how to explain this improvement. Further study into the theoretical concept of self-assessment in medical education and into the factors contributing to the feeling of being competent, is required.

Successes and challenges in a field-based, multi-method study of home telehealth.

We are conducting a three-year study of telehealth in 11 home care offices that serve rural clients in Alberta. Three hundred and twenty palliative home care clients are being recruited to participate in a randomized controlled trial (RCT) to answer three questions about the use of video-phones and their effect on symptom management, quality of life and cost, as well as readiness to use the technology. Both successes and challenges have been identified in three main areas: technology, people/organizational issues and study design. Maintaining study integrity has been the key factor in decision making, as adjustments from the original proposal are made. It is already clear that field-based RCTs are feasible, but require commitment and flexibility on the part of researchers and community partners to work through the study implementation.

Acquisition of communication skills in postgraduate training for general practice.

PURPOSE: The evidence suggests that a longitudinal training of communication skills embedded in a rich clinical context is most effective. In this study we evaluated the acquisition of communication skills under such conditions. METHODS: In a longitudinal design the communication skills of a randomly selected sample of 25 trainees of a three-year postgraduate training programme for general practice were assessed at the start and at the end of training. Eight videotaped real life consultations were rated per measurement and per trainee, using the MAAS-Global scoring list. The results were compared with each other and with those of a reference group of 94 experienced GPs. RESULTS: The mean score of the MAAS-Global was slightly increased at the end of training (2.4) compared with the start (2.2). No significant difference was found between the final results of the trainees and the reference group. According to the criteria of the rating scale the performance of both trainees and GPs was unsatisfactory. CONCLUSION: The results of this study indicate that communication skills do not improve in a three-year postgraduate training comprising both a rich clinical context and a longitudinal training of communication skills, and that an unsatisfactory level still exists at the end of training. Moreover, GPs do not acquire communication skills during independent practice as they perform comparably to the trainees. Further research into the measurement of communication skills, the teaching procedures, the role of the GP-trainer as a model and the influence of rotations through hospitals and the like, is required.

Predictive validity of a written knowledge test of skills for an OSCE in postgraduate training for general practice.

PURPOSE: To examine the validity of a written knowledge test of skills for performance on an OSCE in postgraduate training for general practice. METHODS: A randomly-selected sample of 47 trainees in general practice took a knowledge test of skills, a general knowledge test and an OSCE. The OSCE included technical stations and stations including complete patient encounters. Each station was checklist rated and global rated. RESULTS: The knowledge test of skills was better correlated to the OSCE than the general knowledge test. Technical stations were better correlated to the knowledge test of skills than stations including complete patient encounters. For the technical stations the rating system had no influence on the correlation. For the stations including complete patient encounters the checklist rating correlated better to the knowledge test of skills than the global rating. CONCLUSION: The results of this study support the predictive validity of the knowledge test of skills. In postgraduate training for general practice a written knowledge test of skills can be used as an instrument to estimate the level of clinical skills, especially for group evaluation, such as in studies examining the efficacy of a training programme or as a screening instrument for deciding about courses to be offered. This estimation is more accurate when the content of the test matches the skills under study. However, written testing of skills cannot replace direct observation of performance of skills.

HLA-DQ polymorphism and degree of heteroplasmy of the A3243G mitochondrial DNA mutation in maternally inherited diabetes and deafness.

AIM: Maternally inherited diabetes and deafness (MIDD) associates with a mutation at position 3243 in mitochondrial DNA. Phenotypic expression of MIDD includes Type 1-like and Type 2-like diabetes. This study examined whether HLA-DQ phenotype and the degree of heteroplasmy in leucocyte and oral mucosa DNA influence clinical expression of the 3242 mutation. METHODS: In a group of 20 unrelated probands with MIDD, eight with Type 1- like diabetes, 12 with Type 2-like diabetes, HLA-DQ type and degree of heteroplasmy for the 3243 mutation were determined. HLA-DQA1/DQB1 phenotypes were categorized as predisposing, neutral or protective for autoimmune-mediated Type 1 diabetes. RESULTS: No differences were observed between Type 1 and Type 2-like MIDD groups with respect to the cumulative frequency of protective and predisposing HLA-DQ types. Predisposing HLA-DQ types are more prevalent in MIDD patients than in the control population (P < 0.05). Degrees of heteroplasmy for the 3243 mutation showed large variations in patients, ranging from 1 to 52% in leucocyte DNA. A strong correlation was seen between heteroplasmy in leucocyte DNA and DNA from oral mucosa cells (r = 0.89, P < 0.001). No correlation was observed between the degree of heteroplasmy and diabetic phenotype, even when group size was extended with diabetic relatives of patients with MIDD. The age of diagnosis of diabetes was not correlated with heteroplasmy, but the degree of heteroplasmy tended to decrease with age. CONCLUSIONS: The phenotype of diabetes in MIDD appears to be independent of HLA-DQ phenotype and degree of heteroplasmy in leucocyte and oral mucosa DNA indicating that other, as yet unknown, factors modulate clinical expression of the 3243 mutation.

Effect of a short skills training course on competence and performance in general practice.

OBJECTIVES: Evaluation of the efficacy of a short course of technical clinical skills to change performance in general practice. DESIGN: Subjects were self-selected general practitioners (n=59) who were unaware of the study design. They were assigned to the intervention group (n=31) or control group (n=28) according to their preference for course date. The course covered four different technical clinical skills (shoulder injection technique, PAP-smear, laboratory examination of vaginal discharge, ophthalmoscopic control in diabetes mellitus). Main outcome measures used were pre- and post-training scores on a knowledge test of skills (60 multiple choice items), and pre- and post-training performance of procedures in practice using a log-diary covering 20 days. SETTING: University of Maastricht, The Netherlands. SUBJECTS: Self-selected general practitioners. RESULTS: Competence, as measured by the knowledge test of skills, improved significantly as a result of the training and skills test scores were satisfactory after training. A significant effect on performance in practice was found for PAP-smear and shoulder injection technique, whereas no effect could be demonstrated for examination of vaginal discharge and ophthalmoscopic control in diabetes mellitus. CONCLUSIONS: A good degree of competence is a necessary but not always sufficient condition for a physician to change his performance in practice. While some skills training seems adequate to bring about desired changes, for other skills more complex interventions are probably needed.

Bilateral thoracoscopic splanchnicectomy in patients with chronic pancreatitis.

BACKGROUND: Intractable pain, the most prominent feature of chronic pancreatitis, causes the patient great disability, and its treatment poses a difficult problem for gastroenterologist and surgeon alike. The main goal of treatment is to provide sufficient and lasting pain relief without the use of opiates. Conservative management, including stopping alcohol consumption, dietary measures, pancreatic enzyme suppletion and analgesics, is discussed. When these measures fail, surgery is often unavoidable. Indications, effect on pain relief, morbidity and mortality of drainage and resection procedures are described. Bilateral thoracoscopic splanchnicectomy, a newly developed operation to alleviate pain irrespective of the type of anatomic abnormality, is outlined in more detail. Early encouraging results of pain relief in patients with chronic pancreatitis after thoracoscopic splanchnicectomy are presented.

[A new subtype of diabetes mellitus: maternally inherited diabetes and deafness (MIDD)]. / Een nieuw subtype van diabetes mellitus: via de moeder overgeërfde diabetes en doofheid (MIDD).

Diabetes mellitus comprises many subtypes, the pathogenesis of each of which involves a combination of inherited and environmental factors. Recently a new subtype of diabetes mellitus was recognized in a Dutch pedigree, designated as 'maternally inherited diabetes and deafness' (MIDD). Impaired hearing is an associated phenomenon of the disease. Approximately 1.3% of all diabetic cases in the Netherlands exhibit the MIDD subtype. MIDD shows a strictly maternal heredity. In MIDD there is a guanine-for-adenine substitution at position 3243 in mitochondrial DNA. Mitochondria carrying this mutation exhibit a decreased functionality. In carriers of the MIDD mutation the insulin secretion by the pancreas in response to stimulation by glucose is impaired.

Mutation in mitochondrial tRNA(Leu(UUR)) gene associated with progressive kidney disease.

Several studies show an association of a guanine for adenine substitution (A-->G) at position 3243 in mitochondrial DNA (mtDNA) with a recently recognized diabetic subtype designated maternally inherited diabetes and deafness (MIDD). This mutation shows heterogeneity in its phenotypic expression as is apparent from its association with several other syndromes. Screening for the 3243A-->G mutation in mtDNA was performed in those diabetic patients attending the Leiden University Hospital diabetics clinic who had a history of maternally inherited diabetes, sensorineural hearing loss, or both. Four individuals from three unrelated families were identified who suffered from progressive nondiabetic kidney disease in association with diabetes mellitus and hearing loss. The mode of inheritance suggested maternal transmission. The combination of renal failure and hearing loss had been misdiagnosed as Alport syndrome in three of the four individuals. Therefore, in addition to these three families, another 63 unrelated patients with possible Alport syndrome were selected at random. DNA from peripheral blood and other tissues from members of the three families and from the 63 additional Alport syndrome patients was examined for the presence of the 3243A-->G mutation in mtDNA. The mutation was detected in heteroplasmic form in the four patients and their maternal relatives. Also, one of the 63 suspected Alport syndrome patients showed heteroplasmy for the 3243 mutation. These data show the existence of a kidney disease that is characterized by the presence of the A-->G mutation at position 3243 in the mtDNA.

Evaluation of cardiopulmonary resuscitation skills of general practitioners using different scoring methods.

In this study we evaluated the practical performance of 70 general practitioners in cardiopulmonary resuscitation (CPR) before and after instruction and compared checklist-based scores to mechanical recording scores in order to investigate which scoring method is preferable. Both checklist and recording strip-based scores showed significant improvement after instruction, but only 37% were judged proficient according to the American Heart Association standards (checklist scoring), and 47% according to the recording print-based scoring system, while rates judged 97% as satisfactory by general impression. Interrater reliability was highest for the recording print (0.97) and lower for the checklist (0.79), especially for CPR performance (0.56). Comparison of checklist and recording print showed that the checklist was specific but not very sensitive in identifying poor performance for cardiac compression rate, since observers overestimated performance. The correlation for CPR performance between checklist score and recording strip score was low (0.45), indicating that candidates were ranked differently. The correlation between diagnosis and performance score was low for checklist as well as recording print (0.22), indicating that the score on diagnosis was a poor predictor for the score on performance of CPR. These results support the use of the recording manikin as compared with the use of a checklist for formative evaluation of basic life support skills. However, as proficiency in diagnosis and performance in CPR are poorly correlated, assessment of diagnosis using a checklist must be included. Therefore we strongly recommend the combination of assessment by observers using a checklist for diagnostic procedures and the recording strip of the manikin for performance of CPR, as employed in most evaluation schemes.

Performance-based assessment in continuing medical education for general practitioners: construct validity.

The use of performance-based assessment has been extended to postgraduate education and practising doctors, despite criticism of validity. While differences in expertise at this level are easily reflected in scores on a written test, these differences are relatively small on performance-based tests. However, scores on written tests and performance-based tests of clinical competence generally show moderate correlations. A study was designed to evaluate construct validity of a performance-based test for technical clinical skills in continuing medical education for general practitioners, and to explore the correlation between performance and knowledge of specific skills. A 1-day skills training was given to 71 general practitioners, covering four different technical clinical skills. The effect of the training on performance was measured with a performance-based test using a randomized controlled trial design, while the effect on knowledge was measured with a written test administered 1 month before and directly after the training. A training effect could be shown by the performance-based test for all four clinical skills. The written test also demonstrated a training effect for all but one skill. However, correlations between scores on the written test and on the performance-based test were low for all skills. It is concluded that construct validity of a performance-based test for technical clinical skills of general practitioners was demonstrated, while the knowledge test score was shown to be a poor predictor of competence for specific technical skills.

The molecular basis and clinical characteristics of Maternally Inherited Diabetes and Deafness (MIDD), a recently recognized diabetic subtype.

Diabetes mellitus comprises a number of diseases with hyperglycemia as hallmark. Currently, multiple genetic factors are being recognized which contribute to the development of diabetes or which may modulate its clinical expression. This review presents an overview of our current knowledge on a diabetic subtype which associates with a single mutation in mitochondrial DNA. Based on the triad of Maternal Inheritance, Diabetes and Deafness we propose the name Maternally Inherited Diabetes and Deafness (MIDD) for this syndrome. In Northwestern Europe MIDD affects approximately 1.3% of all diabetic individuals.

Heteroplasmy levels of a mitochondrial gene mutation associated with diabetes mellitus decrease in leucocyte DNA upon aging.

We showed previously that a mutation in the mitochondrial tRNALeu(UUR) gene at position 3243 associates with maternally inherited diabetes and deafness (MIDD). This mutation shows heteroplasmy in DNA from peripheral blood and other tissues. To examine whether heteroplasmy levels in peripheral blood DNA change upon aging, heteroplasmy levels were determined in DNA samples from peripheral blood, collected recently and 1.5-6 years ago, from 18 individuals carrying the 3243 mutation. It was found that 17 out of 18 carriers showed a decrease upon aging (P = 0.001), the average change being -0.69 +/- 0.61% per year. These data indicate a continuous selection against haematopoietic (precursor) cells carrying high levels of the 3243 mutation. Moreover, they imply that heteroplasmy levels may decrease below the detection limit if DNA from peripheral blood is analyzed from elderly individuals. DNA from oral mucosa cells was found to be a good alternative as heteroplasmy levels for the 3243 mutation are on the average 1.7 fold higher than in DNA from peripheral blood.

Assessment of competence in technical clinical skills of general practitioners.

Technical clinical procedures constitute an important part of the work of general practitioners. Assessment of competence in the relevant skills is important from the perspective of quality assurance. In this study, the psychometric characteristics of three different methods for assessment of competence in technical clinical skills in general practice were evaluated. A performance-based test (8 stations), a written knowledge test of skills (125 items) and a self-assessment questionnaire (41 items) on technical clinical skills were administered to 49 GPs and 47 trainees in general practice. The mean scores on the performance-based test and the written knowledge test of skills showed no substantial differences between GPs and trainees, whereas the GPs scored higher on the self-assessment questionnaire. While the correlation of the score on the knowledge test of skills with the score on the performance-based test was moderately high, the score on the self-assessment questionnaire showed a rather low correlation with the performance-based test. Although performance-based testing is obviously the best method to assess proficiency in hands-on skills, a written test can serve as a reasonable alternative, particularly for screening and research purposes.