Comparison of clonal relatedness and antimicrobial susceptibility of fecal Escherichia coli from healthy dogs and their owners.

OBJECTIVE: To determine prevalence of within-household sharing of fecal Escherichia coli between dogs and their owners on the basis of pulsed-field gel electrophoresis (PFGE), compare antimicrobial susceptibility between isolates from dogs and their owners, and evaluate epidemiologic features of cross-species sharing by use of a questionnaire. SAMPLE POPULATION: 61 healthy dog-owner pairs and 30 healthy control humans. PROCEDURES: 3 fecal E coli colonies were isolated from each participant; PFGE profiles were used to establish relatedness among bacterial isolates. Susceptibility to 17 antimicrobials was determined via disk diffusion. A questionnaire was used to evaluate signalment, previous antimicrobial therapy, hygiene, and relationship with dog. RESULTS: A wide array of PFGE profiles was observed in E coli isolates from all participants. Within-household sharing occurred with 9.8% prevalence, and across-household sharing occurred with 0.3% prevalence. No behaviors were associated with increased clonal sharing between dog and owner. No differences were found in susceptibility results between dog-owner pairs. Control isolates were more likely than canine isolates to be resistant to ampicillin and trimethoprim-sulfamethoxazole. Owners and control humans carried more multdrug-resistant E coli than did dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Within-household sharing of E coli was detected more commonly than across-household sharing, but both direct contact and environmental reservoirs may be routes of cross-species sharing of bacteria and genes for resistance. Cross-species bacterial sharing is a potential public health concern, and good hygiene is recommended.

Prevalence of urovirulence genes cnf, hlyD, sfa/foc, and papGIII in fecal Escherichia coli from healthy dogs and their owners.

OBJECTIVE: To determine the prevalence of 4 urovirulence genes in fecal Escherichia coli isolates from healthy dogs and their owners and to determine whether detection of E coli strains with these genes was associated with a history of urinary tract infection (UTI). SAMPLE POPULATION: 61 healthy dog-owner pairs and 30 healthy non-dog owners. PROCEDURES: A fecal specimen was obtained from each participant, and 3 colonies of E coli were isolated from each specimen. A multiplex PCR assay was used to detect 4 genes encoding virulence factors: cytotoxic necrotizing factor (cnf), hemolysin (hlyD), s-fimbrial and F1C fimbriae adhesin (sfa/foc), and pilus associated with pyelonephritis G allele III (papGIII). Human participants completed a questionnaire to provide general information and any history of UTI for themselves and, when applicable, their dog. RESULTS: 26% (16/61) of dogs, 18% (11/61) of owners, and 20% (6/30) of non-dog owners had positive test results for >or= 1 E coli virulence gene. One or more genes were identified in fecal E coli isolates of both dog and owner in 2% (1/61) of households. There was no difference in the detection of any virulence factor between dog-owner pairs. Female owner history of UTI was associated with detection of each virulence factor in E coli strains isolated from their dogs' feces. CONCLUSIONS AND CLINICAL RELEVANCE: Dogs and humans harbored fecal E coli strains possessing the genes cnf, hlyD, sfa/foc, and papGIII that encode urovirulence factors. It was rare for both dog and owner to have fecal E coli strains with these virulence genes.

Plasma serotonin in autism.

Serotonin is necessary for normal fetal brain development. Administration of serotonin inhibitors to pregnant rats results in offspring with abnormal behaviors, brain morphology, and serotonin receptor numbers. Low maternal plasma serotonin may contribute to abnormal brain development in autism. In this study, plasma serotonin levels in autism mothers and control mothers of typically developing children were compared, and plasma serotonin levels in children with autism (n = 17) and their family members were measured. Plasma serotonin levels in autism mothers were significantly lower than in mothers of normal children (P = 0.002). Plasma serotonin levels correlated between autism mothers and their children, but differed between autistic children and their fathers (P = 0.028) and siblings (P = 0.063). Low maternal plasma serotonin may be a risk factor for autism through effects on fetal brain development.

HLA-DR4 in families with autism.

Autoimmune disorders are observed with increased frequency among parents of individuals with autism, particularly mothers. Because there is evidence supporting an association between autoimmune disorders and specific alleles of the human leukocyte antigen (HLA) system, we examined HLA types and subtypes in families with autism. Two groups were studied: 16 families selected from a geographically defined area in eastern Tennessee have males with a diagnosis of autism; and 33 families selected across all regions in the United States have multiple males having autism diagnosis. The HLA-DR4 frequencies of mothers, fathers, and children in these two groups were compared with a reference series of 475 normal, unrelated Caucasians. Results of HLA typing indicated that mothers and their sons in the geographically defined group had a significantly higher frequency of DR4 than normal control subjects (odds ratio = 5.54, 95% confidence interval = 1.74-18.67 and odds ratio = 4.20, 95% confidence interval = 1.37-13.27, respectively). No significant difference in the distribution of HLA alleles was evident between the United States-all region group and control subjects. Findings of this study are consistent with a hypothesis that prenatal maternal-fetal immune interaction can affect fetal brain development in a population residing in a geographically defined region. Such immune interactions may involve HLA and related genes in both genetic and epigenetic mechanisms during pregnancy.

New directions in cytogenetic and molecular testing of the neonate.

The development of new diagnostic, and hence therapeutic possibilities, has brought the realization that genetic disease is now an integral part of medical practice. Advances in cytogenetic and molecular testing have drastically improved the ability to diagnose with certainty many previously unrecognized conditions. However, this advance in technology does not come without new questions. New tests are not always the most cost effective ones, some have significant diagnostic limitations, and others raise valid ethical issues surrounding the testing of minors. A working understanding of new advances in genetic diagnosis as well as their inherent limitations is crucial for the contemporary practitioner.

Establishment of stably EBV-transformed cell lines from residual clinical blood samples for use in performance evaluation and quality assurance in molecular genetic testing.

Positive control materials for clinical molecular genetic testing applications are currently in critically short supply or non-existent for many genetically based diseases of public health importance. Here we demonstrate that anonymous, residual, clinical blood samples are potential sources of viable lymphocytes for establishing Epstein-Barr virus (EBV)-transformed blood lymphocyte cell lines. We attempted to transform 34 residual blood samples, and analyzed transformation success with respect to sample age, anticoagulant, storage temperature, volume, hemolysis, and patient age and sex. In univariate analysis, sample age was significantly associated with transformation success (P = 0.002). The success rate was 67% (6 of 9) for samples 1 to 7 days old, 38% (3 of 8) for samples 8 to 14 days old and 0% for samples 15 to 21 (0 of 11) days old. When we controlled for sample age in multivariate logistic regression, anticoagulant and storage temperature approached significance (P = 0.070 and 0.087, respectively; samples in acid citrate dextrose (ACD) and refrigerated samples were more likely to transform). Based on these findings, we suggest that samples collected in either ACD or ethylene diamine tetraacetic acid, and up to 14 days old (refrigerated) or 7 days old (stored ambient), are reasonable candidates for EBV transformation. The transformation rate for samples that met these criteria was 63% (10 of 16). Implementation of this process could help alleviate the shortage of positive control materials for clinical molecular genetic testing.

Effects of processing techniques on the forensic DNA analysis of human skeletal remains.

Human remains processed by forensic anthropologists may potentially be used for genetic analysis. Therefore, the condition of the deoxyribonucleic acid (DNA) in processed remains may become an issue for future analysis. Processing techniques employed by anthropologists are highly variable and scanning electron microscopy reveals significant alterations to the bone surface depending upon the technique used. Such damage to the bone indicates differences may exist in quality and quantity of DNA extracted. This study assessed how five processing procedures used by major forensic anthropology laboratories around the country affects the amounts of DNA extracted from human rib bones and the subsequent DNA analysis. The DNA was analyzed using the short tandem repeat (STR) locus CSF1PO and amelogenin. The findings indicate processing procedures used by forensic anthropologists do not adversely affect DNA analysis but prolonged exposure to heat during processing may decrease the yield of information from the DNA.

Maternal antibrain antibodies in autism.

Autism is a neurodevelopmental disorder of prenatal onset that is behaviorally defined. There is increasing evidence for systemic and neuroimmune mechanisms in children with autism. Although genetic factors are important, atypical prenatal maternal immune responses may also be linked to the pathogenesis of autism. We tested serum reactivity in 11 mothers and their autistic children, maternal controls, and several groups of control children, to prenatal, postnatal, and adult rat brain proteins, by immunoblotting. Similar patterns of reactivity to prenatal (gestational day 18), but not postnatal (day 8) or adult rat brain proteins were identified in autistic children, their mothers, and children with other neurodevelopmental disorders, and differed from mothers of normal children, normal siblings of children with autism and normal child controls. Specific patterns of antibody reactivity were present in sera from the autism mothers, from 2 to 18 years after the birth of their affected children and were unrelated to birth order. Immunoblotting using specific antigens for myelin basic protein (MBP) and glial acidic fibrillary protein (GFAP) suggests that these proteins were not targets of the maternal antibodies. The identification of specific serum antibodies in mothers of children with autism that recognize prenatally expressed brain antigens suggests that these autoantibodies could cross the placenta and alter fetal brain development.

Genetically characterized positive control cell lines derived from residual clinical blood samples.

BACKGROUND: Positive control materials for clinical diagnostic molecular genetic testing are in critically short supply. High-quality DNA that closely resembles DNA isolated from patient specimens can be obtained from Epstein-Barr virus (EBV)-transformed peripheral blood lymphocyte cell lines. Here we report the development of a process to (a) recover residual blood samples with clinically important mutations detected during routine medical care, (b) select samples likely to provide viable lymphocytes for EBV transformation, (c) establish stable cell lines and confirm the reported mutation(s), and (d) validate the cell lines for use as positive controls in clinical molecular genetic testing applications. METHODS: A network of 32 genetic testing laboratories was established to obtain anonymous, residual clinical samples for transformation and to validate resulting cell lines for use as positive controls. Three panel meetings with experts in molecular genetic testing were held to evaluate results and formulate a process that could function in the context of current common practices in molecular diagnostic testing. RESULTS: Thirteen laboratories submitted a total of 113 residual clinical blood samples with mutations for 14 genetic disorders. Forty-one EBV-transformed cell lines were established. Thirty-five individual point and deletion mutations were shown to be stable after 20 population doublings in culture. Thirty-three cell lines were characterized for specific mutations and validated for use as positive controls in clinical diagnostic applications. CONCLUSIONS: A process for producing and validating positive control cell lines from residual clinical blood samples has been developed. Sustainable implementation of the process could help alleviate the current shortage of positive control materials.

Bioelectronic sensor technology for detection of cystic fibrosis and hereditary hemochromatosis mutations.

CONTEXT: Bioelectronic sensors, which combine microchip and biological components, are an emerging technology in clinical diagnostic testing. An electronic detection platform using DNA biochip technology (eSensor) is under development for molecular diagnostic applications. Owing to the novelty of these devices, demonstrations of their successful use in practical diagnostic applications are limited. OBJECTIVE: To assess the performance of the eSensor bioelectronic method in the validation of 6 Epstein-Barr virus-transformed blood lymphocyte cell lines with clinically important mutations for use as sources of genetic material for positive controls in clinical molecular genetic testing. Two cell lines carry mutations in the CFTR gene (cystic fibrosis), and 4 carry mutations in the HFE gene (hereditary hemochromatosis). DESIGN: Samples from each cell line were sent for genotype determination to 6 different molecular genetic testing facilities, including the laboratory developing the DNA biochips. In addition to the bioelectronic method, at least 3 different molecular diagnostic methods were used in the analysis of each cell line. Detailed data were collected from the DNA biochip output, and the genetic results were compared with those obtained using the more established methods. RESULTS: We report the successful use of 2 applications of the bioelectronic platform, one for detection of CFTR mutations and the other for detection of HFE mutations. In all cases, the results obtained with the DNA biochip were in concordance with those reported for the other methods. Electronic signal output from the DNA biochips clearly differentiated between mutated and wild-type alleles. This is the first report of the use of the cystic fibrosis detection platform. CONCLUSIONS: Bioelectronic sensors for the detection of disease-causing mutations performed well when used in a "real-life" situation, in this case, a validation study of positive control blood lymphocyte cell lines with mutations of public health importance. This study illustrates the practical potential of emerging bioelectronic DNA detection technologies for use in current molecular diagnostic applications.