Specific antisera produced by immunization with precipitin lines

Individual plasma proteins were precipitated, identified and isolated on the basis of line immunoelectrophoresis. A monospecific antibody response was induced by immunization of rabbits with less than 50 ng of precipitated antigen. Preservation of monospecificity was obtained by reimmunization with precipitates developed against the specific antisera.     

The ability of three global plasma assays to recognize thrombophilia

Three global assays, the Calibrated Automated Thrombogram (CAT), the ProC Global (PCG), and the Coagulation Inhibitor Potential (CIP) were performed in frozen plasma samples from 24 normal controls and 24 patients with inherited thrombophilia. Six patients had inherited antithrombin (AT) deficiency; 18 patients had abnormalities in the protein C/S anticoagulant system (protein C deficiency (n=3), protein S deficiency (n=10), homozygous FV Leiden mutation (n=5)). Nine of these twenty four patients carried additionally the heterozygous FV Leiden mutation. All three assays separated the thrombophilia group and the control group (P=0.083 for CAT, P<0.0001 for the other two assays) but there was considerable overlap, particularly in the CAT assay. The CAT assay separated all plasma samples with AT deficiency but was less sensitive to abnormalities in the protein C/S system. In contrast, ProC Global was more sensitive to abnormalities in the protein C system than to AT deficiency. The CIP assay was approximately equally sensitive to defects in both systems. Receiver operator characteristic (ROC) curves confirmed that the ProC Global and the CIP assays performed better than the CAT assay (P=0.0179 and P=0.0003, respectively). With the CIP assay ROC analysis showed that with a sensitivity of 100% the specificity was 87.5%. With the PCG assay, optimal threshold resulted in both a sensitivity and a specificity of 79.2%. Although our material is relatively small, the data suggest that at a cut-off value with a specificity of >80%, the CIP assay should be evaluated as a screening test for severe thrombophilia.     

Development of putative GABAergic neurons in the appendicularian urochordate Oikopleura dioica

Studying the developing brain of urochordates can increase our understanding of brain evolution in the chordate lineage. To begin addressing regional patterns of neuronal differentiation in appendicularian urochordates, we examined the development of putative GABAergic neurons in Oikopleura dioica using GABA immunohistochemistry and in situ hybridization for the GABA-synthesizing enzyme GAD. First, we assessed the developmental dynamics of neuron number and organization in the cerebral and caudal ganglia. We then identified and mapped the positions of putative GABAergic neurons using confocal microscopy. We found GAD mRNA-positive and GABA-immunopositive neurons in the first brain nerves and the cerebral and caudal ganglia, but not in the caudal nerve cord. In both ganglia GAD mRNA-positive and GABA-immunopositive neurons are found in the same characteristic intraganglionic locations. The differentiation of these GABAergic markers occurs first in the first brain nerves and the cerebral ganglion and then with a several-hour delay in the caudal ganglion. In all three structures GAD mRNA expression appears 2-3 hours prior to GABA expression. In general, GABA is expressed by the same number of neurons as express GAD. Several discrepancies suggest differential regulation of the GABAergic phenotype in different neurons, however. Our results show that the GABAergic phenotype has a stereotyped pattern of expression along the anteroposterior axis of the CNS. Given recent genome sequencing and developmental patterning gene studies in this species, the GABAergic neurons in O. dioica provide a good model for assessing, at the invertebrate-vertebrate transition, the molecular mechanisms that specify the GABAergic phenotype.     

Norwegian General Practitioners’ Perspectives on Implementation of a Guided Web-Based Cognitive Behavioral Therapy for Depression: A Qualitative Study

Background

Previous research suggests that Internet-based cognitive behavioral therapy (ICBT) has a positive effect on symptoms of depression. ICBT appears to be more effective with therapist support, but it is unclear what this support should comprise. General practitioners (GPs) have positive attitudes toward ICBT. However, ICBT is rarely used in regular care in general practice. More research is warranted to integrate the potential of ICBT as part of regular care.

Objective

The aim of this study was to explore aspects perceived by GPs to affect the implementation of guided ICBT in daily practice. Understanding their perspectives may contribute to improving the treatment of depression in the context of general practice.

Methods

A training package (3-day course) introducing a Norwegian translation of the ICBT program MoodGYM was developed and presented to GPs in Norway. Following training, GPs were asked to include guided ICBT in their regular care of patients with symptoms of depression by providing brief, face-to-face follow-up consultations between modules. We interviewed 11 GPs who had taken the course. Our interview guide comprised open questions that encouraged GPs to frame their responses using examples from their experiences when implementing ICBT. Thematic analysis was chosen to explore patterns across the data.

Results

An overall belief that ICBT would benefit both the patients’ health and the GPs’ own work satisfaction prompted the GPs to take the ICBT course. ICBT motivated them to invest time and effort in improving treatment. The most important motivating aspects in MoodGYM were that a program based on cognitive behavioral therapy could add a structured agenda to their consultations and empower depressed patients. Organizational aspects, such as a lack of time and varied practice, inhibited the use of ICBT. Inadequate knowledge, recalling the program, and changing own habits were also challenging. The GPs were ambivalent about whether ICBT had a negative impact on the doctor–patient interaction in the module follow-ups. Generally, GPs made an effort to recommend MoodGYM, but the expected module follow-ups were often not provided to patients and instead the GPs returned to standard treatment.

Conclusions

GPs’ feedback in the present study contribute to our understanding of the challenges of changing treatment for depression. Our findings indicated that recommending ICBT could add to the GP’s toolkit. Offering training and highlighting the following aspects may increase recommendation of ICBT by GPs: (1) ICBT is theory-based and credible, (2) ICBT increases the GPs’ work satisfaction by having a tool to offer, and (3) ICBT facilitates empowerment of patients in their own health. In addition, the present study also indicated that complex aspects must be accommodated before module follow-ups can be incorporated into GPs’ treatment of depression.     

Unusual Distribution of Burkholderia cepacia Complex Species in Danish Cystic Fibrosis Clinics May Stem from Restricted Transmission between Patients

Forty-four of 48 Burkholderia cepacia complex strains cultured from Danish cystic fibrosis patients were Burkholderia multivorans, a distribution of species that has not been reported before. Although cases of cross infections were demonstrated, no major epidemic clone was found. The species distribution may represent the sporadic acquisition of bacteria from the environment.Burkholderia cepacia and related bacteria have emerged as significant pathogens in cystic fibrosis (CF) patients due to the risk of cepacia syndrome (a fatal necrotizing pneumonia with bacteremia), the organism''s innate multiresistance to antibiotics, and the transmissibility of bacterial strains between patients by social contact (10, 15). The genus Burkholderia encompasses more than 50 validly published species that can be divided into four groups (21). Strains colonizing the respiratory tract of CF patients are predominantly members of the B. cepacia complex (BCC), with 17 formally named species (23). Chronic infections typically involve a single strain, although strain displacements have been demonstrated (24).Most or all species of the BCC can colonize the lower airways of CF patients, although some of them are infrequently demonstrated. Studies from North America, Europe, and Australasia have shown that Burkholderia cenocepacia is the dominant species being recovered from 46 to 90% of colonized patients (4, 6, 13, 17, 19, 20). Different situations have been described in Lisbon, where contamination of saline solutions used in inhalant therapy by CF patients has resulted in a predominance of B. cepacia (5), and in France, where a small excess of B. multivorans (52%) over B. cenocepacia (45%) has been reported (3).Danish CF patients are treated in two centers, and respiratory cultures are routinely performed at the monthly visit to the outpatient clinic. Four hundred thirty-one patients were alive by 1 January 2007, and 24 (5.6%) were chronically infected with BCC species. A chronic infection was defined as the isolation of BCC bacteria from more than half of sputum cultures for more than 6 months (modified “Leeds criteria” for chronic Pseudomonas aeruginosa infection [14]), and/or the development of ≥2 precipitins measured by crossed immunoelectrophoresis (18). A total of 52 Danish CF patients are known to have been intermittently (n = 11) or chronically (n = 41) infected with BCC bacteria (Fig. ​(Fig.1).1). Intermittently colonized patients may be underrepresented in data from before the routine use of colistin-containing selective agar plates (9), and some of the recent BCC acquisitions may be reclassified as chronic infections with time. In retrospect, the first Danish patient was chronically infected with BCC in the late 1970s (18), but few cases were identified until 1990. The increased rate of BCC colonization after 1990 may be secondary to the widespread use of inhaled colistin for P. aeruginosa infection, which was introduced in the 1980s (11). Since 1993, the rate has stabilized at around three new cases per year (43 BCC acquisitions during 14 years) (Fig. ​(Fig.1).1). In the same time period, 174 Danish patients have been diagnosed with CF (on average, 12.4 ± 4.4 [mean ± standard deviation] new patients per year; range, 6 to 22).Open in a separate windowFIG. 1.Cumulative numbers of Danish CF patients experiencing a first-time isolation of BCC bacteria, separated by status (open bars, chronic infections; gray bars, intermittent colonizations).BCC isolates from 9 intermittently colonized and 39 chronically infected patients were available for characterization. One isolate per patient, cultured between 1994 and 2006, was included in the study. Allocation to species within the BCC was performed by partial atpD and recA sequencing (1); occasional isolates with no PCR product from either amplification were subjected to partial sequencing of fur (16). Two independent sequence-based identifications were thus obtained for all BCC isolates. Only three species were identified in Danish patients, and B. multivorans accounted for more than 90% of the isolates (Table ​(Table1).1). Pulsed-field gel electrophoresis (PFGE) genotypes were assessed after digestion with Xba and SpeI and interpreted as described previously (22). Thirty-eight BCC genotypes were disclosed by both enzymes, and five of the genotypes were identified in more than one patient (two to four patients). Some of these small clusters were epidemiologically related and probably reflect cases of cross infections. Two pairs of siblings each carried the same strain, and one additional patient harbored the same genotype as the two siblings treated in that CF center. Between 1994 and 2003, chronic infections with BCC of a single genotype were established in 4 patients treated in one center. A fourth cluster was composed of patients treated at both of the two Danish CF centers; a possible epidemiological relationship between these three patients was unknown. No patient-to-patient transmission could have occurred in the fifth cluster, where the same genotype was intermittently detected in two patients in 1994 and 1999, respectively, and established a chronic infection in a third patient in 2005. All BCC genotypes identified in more than one patient were B. multivorans.

TABLE 1.

Specific identification of 48 BCC strains isolated from Danish CF patients

Evaluation of PCR-based preimplantation genetic diagnosis applied to monogenic diseases: a collaborative ESHRE PGD consortium study

Preimplantation genetic diagnosis (PGD) for monogenic disorders currently involves polymerase chain reaction (PCR)-based methods, which must be robust, sensitive and highly accurate, precluding misdiagnosis. Twelve adverse misdiagnoses reported to the ESHRE PGD-Consortium are likely an underestimate. This retrospective study, involving six PGD centres, assessed the validity of PCR-based PGD through reanalysis of untransferred embryos from monogenic-PGD cycles. Data were collected on the genotype concordance at PGD and follow-up from 940 untransferred embryos, including details on the parameters of PGD cycles: category of monogenic disease, embryo morphology, embryo biopsy and genotype assay strategy. To determine the validity of PCR-based PGD, the sensitivity (Se), specificity (Sp) and diagnostic accuracy were calculated. Stratified analyses were also conducted to assess the influence of the parameters above on the validity of PCR-based PGD. The analysis of overall data showed that 93.7% of embryos had been correctly classified at the time of PGD, with Se of 99.2% and Sp of 80.9%. The stratified analyses found that diagnostic accuracy is statistically significantly higher when PGD is performed on two cells versus one cell (P=0.001). Se was significantly higher when multiplex protocols versus singleplex protocols were applied (P=0.005), as well as for PGD applied on cells from good compared with poor morphology embryos (P=0.032). Morphology, however, did not affect diagnostic accuracy. Multiplex PCR-based methods on one cell, are as robust as those on two cells regarding false negative rate, which is the most important criteria for clinical PGD applications. Overall, this study demonstrates the validity, robustness and high diagnostic value of PCR-based PGD.     

Histological evidence of testicular dysgenesis in contralateral biopsies from 218 patients with testicular germ cell cancer

This study was prompted by a hypothesis that testicular germ cell cancer may be aetiologically linked to other male reproductive abnormalities as a part of the so-called 'testicular dysgenesis syndrome' (TDS). To corroborate the hypothesis of a common association of germ cell cancer with testicular dysgenesis, microscopic dysgenetic features were quantified in contralateral testicular biopsies in patients with a testicular germ cell tumour. Two hundred and eighty consecutive contralateral testicular biopsies from Danish patients with testicular cancer diagnosed in 1998-2001 were evaluated retrospectively. Two hundred and eighteen specimens were subsequently included in this study, after 63 patients who did not meet inclusion criteria had to be excluded. The presence of carcinoma in situ (which is believed to originate from transformed gonocytes) was detected in 8.7% of biopsies. The incidence of other dysgenetic features was immature tubules with undifferentiated Sertoli cells, 4.6%; microcalcifications (microliths), 6.0%; and the presence of a Sertoli-cell-only pattern in at least a few tubules, 13.8%. The cumulative incidence of one or more signs of testicular dysgenesis was 25.2%. In a few patients, areas with immature and morphologically distorted tubules were also noted. Spermatogenesis was qualitatively normal in 51.4%, whereas 11.5% had very poor or absent spermatogenesis. It is concluded that microscopic testicular dysgenesis is a frequent feature in contralateral biopsies from patients presenting with testicular germ cell neoplasms of the adolescent and young type. The findings therefore support the hypothesis that this cancer is part of a testicular dysgenesis syndrome. The presence of contralateral carcinoma in situ was higher in the present study than previously reported.     

A Common Variant of Staphylococcal Cassette Chromosome mec Type IVa in Isolates from Copenhagen,Denmark, Is Not Detected by the BD GeneOhm Methicillin-Resistant Staphylococcus aureus Assay

Rapid tests for detection of methicillin-resistant Staphylococcus aureus (MRSA) carriage are important to limit the transmission of MRSA in the health care setting. We evaluated the performance of the BD GeneOhm MRSA real-time PCR assay using a diverse collection of MRSA isolates, mainly from Copenhagen, Denmark, but also including international isolates, e.g., USA100-1100. Pure cultures of 349 MRSA isolates representing variants of staphylococcal cassette chromosome mec (SCCmec) types I to V and 103 different staphylococcal protein A (spa) types were tested. In addition, 53 methicillin-susceptible Staphylococcus aureus isolates were included as negative controls. Forty-four MRSA isolates were undetectable; of these, 95% harbored SCCmec type IVa, and these included the most-common clone in Copenhagen, spa t024-sequence type 8-IVa. The false-negative MRSA isolates were tested with new primers (analyte-specific reagent [ASR] BD GeneOhm MRSA assay) supplied by Becton Dickinson (BD). The ASR BD GeneOhm MRSA assay detected 42 of the 44 isolates that were false negative in the BD GeneOhm MRSA assay. Combining the BD GeneOhm MRSA assay with the ASR BD GeneOhm MRSA assay greatly improved the results, with only two MRSA isolates being false negative. The BD GeneOhm MRSA assay alone is not adequate for MRSA detection in Copenhagen, Denmark, as more than one-third of our MRSA isolates would not be detected. We recommend that the BD GeneOhm MRSA assay be evaluated against the local MRSA diversity before being established as a standard assay, and due to the constant evolution of SCCmec cassettes, a continuous global surveillance is advisable in order to update the assay as necessary.Methicillin-resistant Staphylococcus aureus (MRSA) is a common nosocomial pathogen in countries all over the world. In recent years, community-associated MRSA (CA-MRSA) has become increasingly prevalent and has shown potential to cause health care-associated bloodstream infections (8, 26). Screening and isolation of MRSA-positive patients is essential to control the transmission of MRSA in hospitals (16, 24). However, conventional detection of MRSA by culture takes at least 48 h before a preliminary result is available, and as patients in many countries are only isolated when they are recognized as MRSA positive, the risk of having already transmitted MRSA is high. The real-time PCR BD GeneOhm MRSA assay (Becton Dickinson [BD] Diagnostics GeneOhm; San Diego, CA), formerly called IDI-MRSA, is one of a number of commercial kits for rapid MRSA detection directly from nasal swabs (7) and is based on primers developed by Huletsky et al. (18). The forward primers bind to the J3 region of the staphylococcal cassette chromosome mec (SCCmec), and the reverse primer binds in the orfX region that is specific for Staphylococcus aureus. At least seven SCCmec types are known (types I to VII) (3), and several subtypes, especially of type IV, have been described (21, 27).The BD GeneOhm MRSA assay has been tested in a number of studies (4, 5, 10, 11, 13-15, 22, 23, 25, 29-31). Most studies screened hospitalized patients, but only two studies described the SCCmec types of their MRSA isolates (15, 25). Therefore, it is possible that only a few predominant hospital clones with the same SCCmec types were tested. In Denmark, different CA-MRSA clones dominate and MRSA isolates mainly harbor SCCmec types IV (85%) and V (6%) (2). In-house testing with the Huletsky primers (18) revealed that they did not amplify a PCR fragment from our most-common MRSA clone, spa t024-sequence type 8 (ST8)-IVa. Based on this finding and with the knowledge of the high number of type IV subtypes known, we were interested in finding out whether the BD GeneOhm MRSA assay could detect MRSA isolates from a collection that included mainly CA-MRSA strains. We tested 349 MRSA isolates representing variants of SCCmec types I to V. Furthermore, we chose MRSA isolates of different staphylococcal protein A (spa) types to have a broad range of genetic backgrounds, testing the hypothesis that the same SCCmec type might have minor differences in different MRSA lineages and that these differences could be in the primer regions of the assay.