Taxonomic and diagnostic markers for identification of Coccidioides immitis and Coccidioides posadasii.

The ribosomal Internal Transcribed Spacer (ITS) regions of the two recognized species of Coccidioides were studied using a reference set of strains that had been previously identified with species defining microsatellite polymorphisms. Unambiguous identification of the two species proved to be possible by amplifying and sequencing the ITS region. PCR-reactions are sensitive to amplification conditions requiring their careful optimization. Stable amplification and sequencing was achieved with primers ITS3 and 4, enabling species diagnosis. Alternatively, Restriction Fragment Length Polymorphism (RFLP) of the entire ITS region using an annealing temperature of 52 degrees C with the restriction enzymes BsrI and XcmI can also distinguish the species. Three strains typifying the species, Glenospora meteuropaea, G. metamericana and Geotrichum louisianoideum, were analyzed and found to be conspecific with C. posadasii. Although these species have nomenclatural priority over C. posadasii, the latter will be proposed for conservation as it has been included in the US select agent list. In addition, Coccidioides immitis is neotypified in this report. Results of antifungal susceptibility testing did not reveal differences between the two species.     

Biocompatibility of six elastomers in vitro

The biocompatibility of two silicone rubbers, Silastic and Dow Corning Elastomer, and of a polyether and a polyester urethane, a polyether polyester copolymer, and polypropylene oxide was assessed in vitro. These elastomers were selected for assessment as a possible alloplastic tympanic membrane. For these studies use was made of rat middle ear mucosa explants and serially cultured epithelium. The quantitative results were based on epithelial growth curves, the morphological picture was based on the findings in epithelium, and the aging of a biomaterial was simulated. Epithelium morphology was investigated by scanning and transmission electron microscopy and x-ray microanalysis. Quantitative results showed that on Dow Corning Elastomer and polypropylene oxide, cell proliferation was significantly lower compared to normal growth curves. The morphological findings were negative for polypropylene oxide, and did not discriminate between the other biomaterials under study. The simulation results indicated better biocompatibility for the polyurethanes and the polyether polyester copolymer compared with that of polypropylene oxide and both silicone rubbers. Under the simulation conditions, cells exposed to Silastic showed silicon-containing inclusions. These in vitro results suggest that the biocompatibility of the polyurethanes and the polyether polyester copolymer is better than that of both silicone rubbers and polypropylene oxide.     

K88 fimbriae as carriers of heterologous antigenic determinants.

The K88 fimbriae of enterotoxigenic Escherichia coli are strongly immunogenic antigens that can be used to evoke protective immunity. To find out whether these fimbriae can be used as carriers for foreign epitopes, a highly variable region present in the primary structure of the different K88 variants was replaced with five different heterologous epitopes to investigate to what extent these insertions affected the expression, assembly (biogenesis), stability and immunogenic properties of the resulting hybrid fimbriae. Amino acid residues 163-173, were replaced using site-directed in vitro mutagenesis and the hybrid fimbriae were tested for these aspects using ELISA, immunoelectronmicroscopy and immunoblotting. Replacement of this highly variable region did not affect the biosynthesis of fimbriae, although all mutations tested resulted in a reduced expression depending on the epitope inserted. Testing of the different hybrid fimbriae with a panel of monoclonal antibodies raised against the various K88 serotypes K88ab, K88ac and K88ad indicated that replacement of amino acid sequence 163-173 did not affect conserved or K88ab specific epitopes but the K88ac and K88ad specific conformation was lost. Immunization with hybrid fimbriae raises antibodies specific for the inserted heterologous epitopes.     

The motivation of at-risk individuals and their partners in deciding for or against predictive testing for Huntington''s disease

Sixty-six percent of the at-risk persons and 74% of the partners in a large survey in Belgium have the intention of making use of predictive testing for Huntington's disease. One third of them, however, have expressed the intention of postponing the final decision for various reasons. The intention to be tested is not at all related to sociodemographic characteristics. A thorough exploration of the reasons for being in favour of or against taking the test reveals that the motivation inspiring this very personal decision is very complex. In the group of at-risk persons, less than half of the variation in the intention to be tested is explained by the role of a series of specific reasons as predictor variables in a regression analysis. The proportion of explained variation is slightly higher in the group of partners. 'To have certainty about my own future' and 'to make arrangements for the future' play a major part in the decision of the total group. 'Making decisions concerning children' and to a larger extent 'informing children about their risk status' are important factors in deciding in favour of the test.     

Determination of the parent of origin in nine cases of prenatally detected chromosome aberrations found after intracytoplasmic sperm injection

Prenatal cytogenetic analysis of 71 fetuses conceived by intracytoplasmic sperm injection (ICSI) resulted in the detection of nine (12.7%) chromosome aberrations including two cases of 47,XXY, four cases involving a 45,X cell line and three autosomal trisomies. Molecular analysis of the parental origin of the deleted or supernumerary chromosome was performed by using polymorphic microsatellite markers. Six cases involving a sex chromosome abnormality were found to be of paternal origin while the two trisomic cases that could be analysed were of maternal origin. Two cases involved the same infertile couple who had two consecutive ICSI pregnancies terminated because of a chromosome abnormality. The replaced embryos in both cases originated from a single batch of ICSI fertilized oocytes of which part was used to initiate the first pregnancy and part was cryopreserved and used to initiate the second pregnancy.      

High prevalence of mental disorders in primary care

OBJECTIVE: To determine the prevalence of common mental disorders in an adult primary care population. DESIGN: Cross-sectional survey in randomly selected subjects, using the PRIME-MD questionnaire. SETTING: Eighty-six general practices in Belgium. SUBJECTS: A total of 2316 randomly selected patients, aged 18 years or older and consulting their general practitioner for other than administrative reasons alone, with slightly more women (58.1%) than men (41.3%). MAIN OUTCOME RESULT: Prevalence rates of mental disorders most commonly seen in primary care practice (mood, anxiety, somatoform, eating and alcohol disorders). METHODS: To facilitate data collection and processing, the entire PRIME-MD questionnaire was programmed on a handheld computer. Patient answers and physician assessments were immediately electronically recorded during the interview. All investigators were trained on the use of the PRIME-MD. The recruitment period lasted 6 weeks: from 15 February to 25 March 1999, and patients were randomly selected for the interview based on a computerized procedure. RESULTS: Although only 5.4% of all patients consulted for a psychiatric reason, a threshold/subthreshold psychiatric disorder was detected in 42.5% of all patients. Most commonly detected disorders were mood disorders in 31.0% (major depressive disorder, 13.9% and dysthymia, 12.6%), anxiety disorders in 19.0% (generalized anxiety disorder, 10.3%), somatoform disorders in 18.0% and probable alcohol abuse/dependence in 10.1%. The results also showed the important rate of comorbidity between these disorders. CONCLUSION: The present study confirms the high prevalence of mental disorders in a general practice setting, and their frequent association. Prevalence rates of our study are even higher than those obtained in previously conducted trials. Our study also demonstrates the utility of the PRIME-MD as a screening tool for mental disorders in primary care. In addition the use of the handheld computer software version of the PRIME-MD allowed us to screen for mental disorders in patients who are unable to attend the GP office and are seen during 'home' visits.     

DAT1, DRD4, and DRD5 polymorphisms are not associated with ADHD in Dutch families.

Recent meta-analyses have indicated that the dopamine transporter gene (DAT1) and the dopamine receptor genes D4 (DRD4) and D5 (DRD5) are associated with attention-deficit hyperactivity disorder (ADHD), although single studies frequently failed to show significant association. In a family-based sample of 236 Dutch children with ADHD, we have investigated the previously described variable number of tandem repeat (VNTR) polymorphisms and two additional microsatellites at the DAT1 and DRD4 loci. DRD5 was investigated using the microsatellite that was previously found to be associated. Transmission disequilibrium tests (TDTs) did not show preferential transmission of alleles or two-marker haplotypes to affected offspring. These data suggest that DAT1, DRD4, and DRD5 do not contribute substantially to ADHD in the Dutch population.     

Subtelomeric deletions detected in patients with idiopathic mental retardation using multiplex ligation-dependent probe amplification (MLPA)

Subtelomeric rearrangements are responsible for 5% to 10% of cases of unexplained mental retardation. Despite their clinical relevance, methods to screen for these cytogenetically invisible abnormalities on a routine base are scarce. We screened patients with idiopathic mental retardation for subtelomeric aberrations using multiplex ligation-dependent probe amplification (MLPA). This recently developed technique is based on PCR amplification of ligated probes hybridized to chromosome ends. Currently, 41 telomeres can be screened in just two multiplex reactions. Four subtelomeric rearrangements (5.3%) were detected in a group of 75 patients with mild to severe mental retardation in combination with dysmorphic features and/or a familial history of mental retardation: two terminal 1p deletions, a terminal 1q deletion, and a terminal 3p deletion. Deletions could be verified by FISH and marker analysis. In one case the MLPA indicated a terminal 21q deletion due to a 3-bp deletion at the site of the probe, giving a false-positive rate of 1.3%. This study demonstrates that MLPA is a fast and reliable screening method, potentially suitable for use in routine diagnostics.     

Correlation between fluorescence in-situ hybridization analyses and in-vitro development to blastocyst stage of embryos from Robertsonian translocation (13;14) carriers

BACKGROUND: Little is known about the extent and timing of selection against the embryos that are carriers of unbalanced translocations. METHODS: Fluorescence in-situ hybridization (FISH) with probes for chromosomes 13, 14 and 18 was performed, mostly on day 3, on 69 human embryos which were then allowed to develop further in culture to day 5, from five carriers of Robertsonian translocation (RT) t(13;14). RESULTS: Twelve normal/balanced blastocysts were replaced in seven consecutive cycles (day 5). Three cycles resulted in clinical pregnancies. The proportion of blastocysts displaying a normal/balanced karyotype was 56%, while only the 20% of blocked embryos were normal/balanced (chi(2): P < 0.05). All the embryos analysed on day 5, except one, displayed mosaicism. The percentages of diploid cells for chromosomes 13 and 14 were significantly lower than for chromosome 18 (chromosome 13: 49.0 +/- 28.0; chromosome 14: 53.0 +/- 31.8; chromosome 18: 75.7 +/- 20.4; Mann-Whitney test: P < 0.01). The embryos displaying vertical line 62% of diploid cells for at least two of the three chromosomes analysed, more frequently reached the blastocyst stage (blocked embryos: blastocysts chromosome 13: 43.1 +/- 30.3, 64.9 +/- 29.0; chromosome 18: 64.9 +/- 29.0, 83.0 +/- 12.9; Mann-Whitney test: P < 0.01). CONCLUSIONS: Normal/balanced embryos developed better but the proportion of abnormal blastocysts was still high. Preimplantation genetic diagnosis is recommended to select normal/balanced embryos from RT t(13;14) carriers.