Detection of virulence genes of Clostridium difficile by multiplex PCR

Antikainen J, Pasanen T, Mero S, Tarkka E, Kirveskari J, Kotila S, Mentula S, Könönen E, Virolainen‐Julkunen A‐R, Vaara M, Tissari P. Detection of virulence genes of Clostridium difficile by multiplex PCR. APMIS 2009; 117: 607–13. Clostridium difficile strains belonging to the PCR ribotype 027, pulse‐field gel electrophoresis (PFGE) type NAP1, toxinotype III and restriction endonuclease analysis group BI harbouring mutations in the tcdC gene and possessing binary toxin components A and B have been described to cause epidemics with increased morbidity and mortality. In the present study we developed a conventional multiplex PCR designed to detect selected virulence associated markers of the hypervirulent C. difficile PCR ribotype 027. The multiplex PCR assay detected the major toxins A and B, binary toxin components A and B as well as a possible deletion in the tcdC gene: a characteristic pattern of amplification products for the PCR ribotype 027 strains was detected. This rather simple method was specific for the screening of this hypervirulent C. difficile strain. The correlation between the multiplex PCR and PCR ribotyping methods was excellent. The sensitivity and specificity were 100% in our epidemiological situation. In conclusion, this multiplex PCR was found useful in the preliminary screening for the hypervirulent C. difficile PCR ribotype 027.     

Real‐time multiplex PCR assay for detection of Yersinia pestis and Yersinia pseudotuberculosis

A multiplex real‐time polymerase chain reaction (PCR) assay was developed for the detection of Yersinia pestis and Yersinia pseudotuberculosis. The assay includes four primer pairs, two of which are specific for Y. pestis, one for Y. pestis and Y. pseudotuberculosis and one for bacteriophage λ; the latter was used as an internal amplification control. The Y. pestis‐specific target genes in the assay were ypo2088, a gene coding for a putative methyltransferase, and the pla gene coding for the plasminogen activator. In addition, the wzz gene was used as a target to specifically identify both Y. pestis and the closely related Y. pseudotuberculosis group. The primer and probe sets described for the different genes can be used either in single or in multiplex PCR assays because the individual probes were designed with different fluorochromes. The assays were found to be both sensitive and specific; the lower limit of the detection was 10–100 fg of extracted Y. pestis or Y. pseudotuberculosis total DNA. The sensitivity of the tetraplex assay was determined to be 1 cfu for the ypo2088 and pla probe labelled with FAM and JOE fluorescent dyes, respectively.     

Mental disorders and suicide prevention

Abstract In the research phase of the National Suicide Prevention Project, all suicides ( n = 1397) in Finland between March 1987 and April 1988 were examined retrospectively using the psychological autopsy method. Careful retrospective diagnostic evaluation of the victims according to DSM-III-R criteria was done by weighing and integrating all available information. A series of studies addressing the mental disorders among suicide victims, the treatment received before death and the life events is now reviewed.
Among a random sample of suicide victims from the nationwide suicide population, at least one psychiatric diagnosis was made for 93% of the victims. The most prevalent disorders were depressive syndromes (66%) and alcohol dependence/abuse (43%). The prevalence of major depression was higher among women than among men. Major depression as the principal diagnosis was more common among the elderly suicides. Among adolescent victims, depressive syndromes were also the most prevalent disorders. Adjustment disorders were common (25%) among male adolescent suicides. The majority of suicide victims of all ages suffered from comorbid mental disorders.
Among suicide victims who had had contact with a health carer before death, inadequacy of treatment for mental disorders seems to have been common. Of the major depressive victims only 3% were found to have received adequate psychopharmacological treatment, and only 7% received weekly psychotherapy by a trained therapist.
The analysis of the massive database collected in the research phase of the National Suicide Prevention Project in Finland is still ongoing, and the implications of the findings for suicide prevention will be refined during the research process. The necessity to improve recognition and treatment for comorbid depressive disorders in all age groups seems evident already.     

Cross-national and longitudinal investigation of a short measure of workaholism

Abstract: The present study investigated the factor structure of the 10-item version of the Dutch Work Addiction Scale (DUWAS). The DUWAS-10 is intended to measure workaholism with two correlated factors: working excessively (WE) and working compulsively (WC). The factor structure of the DUWAS-10 was examined among multi-occupational samples from the Netherlands (n=9,010) and Finland (n=4,567) using confirmatory factor analysis (CFA). CFAs revealed that the expected correlated two-factor solution showed satisfactory fit to the data. However, a second-order factor solution, where WE comprised the first-order factors “working frantically” and “working long hours”, and WC the first-order factors “obsessive work drive” and “unease if not working”, showed significantly better fit to the data. The expectation of factorial group invariance of the second-order factor structure between the Dutch and Finnish samples was also supported. Moreover, factorial time invariance was observed across a two-year time lag in a sub-sample of Finnish managers (n=459). In conclusion, the DUWAS-10 was found to be a comprehensive measure of workaholism, meeting the criteria of factorial validity in multiple settings, and can thus be recommended for use in both research and practice.     

Low bone mass in patients with motor disability: prevalence and risk factors in 59 Finnish children

Aim Children with motor disabilities are at increased risk of compromised bone health. This study evaluated prevalence and risk factors of low bone mass and fractures in these children. Method This cross‐sectional cohort study evaluated bone health in 59 children (38 males, 21 females; median age 10y 11mo) with motor disability (Gross Motor Function Classification System levels II–V). Bone mineral density (BMD) in the lumbar spine was measured with dual‐energy X‐ray absorptiometry; BMD values were corrected for bone size (bone mineral apparent density [BMAD]) and skeletal maturity, and compared with normative data. Spinal radiographs were obtained to assess vertebral morphology. Blood biochemistry included vitamin D concentration and other parameters of calcium homeostasis. Results Ten children (17%) had sustained in total 14 peripheral fractures; lower‐limb fractures predominated. Compression fractures were present in 25%. The median spinal BMAD z‐score was ?1.0 (range ?5.0 to 2.0); it was ?0.6 in those without fractures and ?1.7 in those with fractures (p=0.004). Vitamin D insufficiency was present in 59% of participants (serum 25‐hydroxyvitamin D <50nmol/l) and hypercalciuria in 27%. Low BMAD z‐score and hypercalciuria were independent predictors for fractures. Interpretation Children with motor disability are at high risk of peripheral and vertebral fractures and low BMD. Evaluation of bone health and prevention of osteoporosis should be included in the follow‐up.     

Serum salbutamol concentrations during oral and intravenous treatment in pregnant women

Summary. The serum concentration of salbutamol was determined in 29 pregnant women during oral treatment (4 mg five times per day) and in seven during intravenous infusion (6–30 μg/min) because of premature labour. The concentration of salbutamol was determined by combined gas-liquid chromatography mass spectrometry. The mean concentration of serum salbutamol was twice as high during intravenous treatment (24; SD 9 ng/ml), than during oral treatment (12; SD 3 mg/ml). During oral treatment: the salbutamol levels were not correlated to maternal height, weight or the incidence or severity of side-effects. The serum concentrations of salbutamol in patients with twin pregnancies did not differ from those with singleton pregnancies. After stopping intravenous treatment, serum salbutamol levels remained high for several hours and oral treatment can be started 4–6h after stopping intravenous infusion.     

The ambulatory measurement of posture, thigh acceleration, and muscle tension and their relationship to heart rate

We compared the relative ability of continuous accelerometric, electromyographic (EMG), and hydrostatic posture measurements to diseriminate tasks involving variations in motor activity and posture and to predict heart rate (HR) variability. EMG was a more sensitive measure than accelerometry in differentiating the tasks. How-ever, accelerometry and EMG explained comparable amounts of HR variance. The hydrostatic posture was a stable measure that elearly differentiated postures and explained a significant amount of HR variance but less than accelerometry or EMG. Accelerometric and EMG measures of motor activity used either alone or in combination with the hydrostatic posture are valuable in discriminating activities and in controlling for the effects of motor activity and posture on HR during ambulatory measurement.     

The Psychopathy Checklist and non-violent offender groups

Cleckley’s concept of psychopathy includes characteristics such as superficial charm, unreliability, and affective poverty. In this study, the Psychopathy Checklist (PCL) and personality questionnaires (MMPI, CPI, EPQ, and SSS) were used to assess 92 non-violent male offenders. The variable-based approach was applied in order to study the structure of the PCL and the relationships between the PCL, the PCL factors and the personality questionnaire scores. The results indicated that the personality scale scores failed to correlate positively with the PCL score, with the exception of the MMPI hypomania score. Two PCL factors emerged: factor 1 related to the core personality characteristics of the Cleckley psychopath, and factor 2 referred to a chronically unstable and antisocial lifestyle. In the person-oriented approach, three offender groups were distinguished based on the cluster analysis of the PCL items; cluster 1 subjects scored high on factor 1 items describing the personality characteristics of Cleckley’s psychopathy. They were psychopaths with a high level of fraud-like offences. Severely antisocial offenders, starting their criminal behaviour early and having an active criminal lifestyle, comprised cluster 2. Cluster 3 was a group of non-psychopathic but antisocial and experience-seeking offenders. The overall results advocated the importance of the PCL as a method of assessing the Cleckley psychopaths and the usefulness of personoriented analysis in differentiating them from others.     

OXIDATION OF GLUCOSE and D-B-OH-BUTYRATE BY THE EARLY HUMAN FETAL BRAIN

Abstract. Adam, P. A. J., Räihä, N., Rahiala, E.-L. and Kekomäki, M. (Departments of Pediatrics, Case Western Reserve University at Cleveland Metropolitan General Hospital, Cleveland, Ohio USA, and the University of Helsinki at the Children's Hospital, Helsinki, Finland). Oxidation of glucose and D-B-OH-butyrate by the early human fetal brain. Acta Paediatr Scand, 64:17, 1975.–The isolated brains of 12 previable human fetuses obtained at 12 to 21 weeks' gestation, were perfused through the interval carotid artery with glucose (3 mM) and/or DL-B-OH-butyrate (DL-BOHB), 4.5 mM, plus tracer quantities of either glucose-6-¹⁴C (G6¹⁴C) or β-OH-butyrate-3-¹⁴C (BOHB3¹⁴C). Oxidative metabolism was demonstrated by serial collection of gaseous ¹⁴CO₂ from the closed perfusion system, and from the recirculating medium. Glucose and BOHB were utilized at physiological rates as indicated (mean ±SEM): G6¹⁴C at 0.10±0.01 μmoles/min g brain (n=7) or 17.5±1.9 μmoles/ min kg fetus; and BOHB3¹⁴C at 0.16±0.05 μmoles/min g (n=5) or 27.3±7.4 μmoles/ min kg. Based on fetal weight, glucose metabolism by brain apparently accounted for about 1/3 of basal glucose utilization in the fetus. On a molar basis BOHB3¹⁴C was taken up at 1.47 times the rate of G6¹⁴C. Both BOHB3¹⁴C and G6 ¹⁴C were converted to ¹⁴C0₂. The rate of BOHB3¹⁴C conversion to ¹⁴CO₂ was equal to its rate of consumption, and exceeded the conversion of glucose to CO₂ because 45% of the G6¹⁴C was incorporated into lactate-¹⁴C. Accordingly, both substrates support oxidative metabolism by brain; and BOHB is a major potential alternate fuel which can replace glucose early in human development.     

A recurrent laminin 5 mutation in British patients with lethal (Herlitz) junctional epidermolysis bullosa: evidence for a mutational hotspot rather than propagation of an ancestral allele

The three genes (LAMA3, LAMB3 and LAMC2) that encode the anchoring filament protein, laminin 5, may all harbour pathogenetic mutations in the autosomal recessive blistering skin disorder, junctional epidermolysis bullosa (JEB). Recently, one particular mutation, R635X in the LAMB3 gene, has been found to account for approximately 40% of all JEB laminin 5 mutations (Kivirikko et al., Hum Mol Genet 1996; 5: 231-7). In this study, we assessed the frequency of this mutation in 12 British patients with lethal (Herlitz) JEB using PCR amplification of genomic DNA and restriction endonuclease digestion. The mutation R635X was found in seven of 24 (29%) mutant alleles, confirming its relative frequency within the British gene pool. In addition, haplotype analysis using intragenic polymorphisms showed that the mutation arose on at least four different haplotype backgrounds, suggesting it represents a mutational hotspot rather than propagation of a common British ancestral allele. These findings support the hypermutable nature of this CpG dinucleotide and have implications in screening for laminin 5 gene mutations in British and other patients with JEB.