bluuebunny/crossref_metadata_2025 huggingface dataset:
Observing Mother‐Child Relationships Across Generations: Boundary Patterns, Attachment, and the Transmission of Caregiving*
Authors: Kretchmar Molly D. , Jacobvitz Deborah B.
Published: May 2004

Grounded in both attachment and family systems theories, this study is one of the first to examine how relationship patterns observed in mothers' current relationships with their own mothers are recreated in their relationships with their infants. Mostly white, middle‐class families (N= 55), including maternal grandmothers, mothers, and infants, were observed when infants were 6, 9, and 18 months old. At 6 months, mothers and grandmothers completed self‐report assessments and worked together on discussion tasks. These interactions were coded using the Boundary Assessment Coding System, developed for the present study, which assessed three relational patterns: disengagement, balance, and entanglement. At 9 months, mothers were rated on sensitivity and intrusiveness while playing with and feeding their infants; and, at 18 months, infant‐mother attachment was assessed using the Strange Situation. Multiple regression analyses revealed, as predicted, that mothers who remembered being accepted by their mothers as children and who were in highly balanced relationships with their own mothers currently were more sensitive and less intrusive with their 9‐month‐old infants. Further, discriminant function analyses indicated that memories of acceptance, high levels of balance, and low levels of disengagement differentiated secure from insecure attachment, whereas memories of overprotection and high levels of entanglement distinguished resistant from secure and avoidant attachment. Discussion focuses on the theoretical hypothesis that mothers internalize relationship strategies experienced with their own caregivers and recreate these patterns with their infants.
Attachment, Mastery, and Interdependence: A Model of Parenting Processes*
Authors: Edwards Martha E.
Published: May 2004

A democratic nation needs an interdependent citizenry who are not only competent but who also can live together cooperatively with an eye toward what will benefit the whole as well as the self. In this article, the concept of interdependence is adopted as the central goal of parenting. The Parenting Processes Model is then presented, specifying how caregivers help children develop this interdependence. This work draws upon and integrates the work of a number of theoreticians, researchers, and clinicians, with the central focus on the work of John Bowlby, Alfred Adler, and Lev Vygotsky.
Balancing the Family and the Collective in Raising Children: Why Communal Sleeping in Kibbutzim Was Predestined to End*
Authors: Aviezer Ora , Sagi Abraham , Van Ijzendoorn Marinus
Published: May 2004

This article integrates research data about attachment in kibbutz‐raised children with a review of the socio‐historical processes that shaped the interrelations between the kibbutz family and the collective and influenced childrearing practices. It uses systems theory to evaluate the changing practices of kibbutz childrearing with particular focus on communal sleeping for infants and children away from their parents, and its impact on the formation of attachment relations to parents and caregivers, transmission of attachment across generations, and later school competence. It argues that artificial childrearing practices such as communal sleeping for infants and children created a unique and unprecedented “social experiment in nature,” which, from the perspective of attachment theory, was predestined to be discontinued because it betrayed the essential attachment needs of most parents and children.
Depression and Attachment in Families: A Child‐Focused Perspective
Authors: Herring Melissa , Kaslow Nadine J.
Published: May 2004

Given the social nature and developmental implications of attachment and depression, in this review we will examine the role of attachment and the effects of a depressed family member at each stage of a child's development. We discuss treatment strategies for families in which either a parent or a child is depressed. We propose a model that is based on the developmental phase of the children in the family, and that aims to strengthen attachment bonds among family members and alleviate depressive symptoms.
The Epigenesis of the Family System as a Context for Individual Development
Authors: Guttman Herta A.
Published: May 2004

In this article, the concept introduced by Lyman Wynne, that the individual develops epigenetically within the family system, is discussed and validated with data from a study of the characteristics and relationships of 27 women with borderline personality disorder and their parents. Each stage of the epigenetic process is impaired in one way or another, adversely affecting subsequent stages. Early impairment of attachment‐care‐giving processes is at least partly attributable to a lack of empathic parenting; effective communication is marred by family members' inability to experience or express feelings (alexithymia); this, in turn, makes it difficult to engage in joint family problem solving. Mutuality between family members does not occur in such a context, and there is an absence of intimacy between family members. These are often abusive family systems, with multiple abuse and intrafamilial sexual abuse more specifically directed at the daughter with BPD. The symptoms of the daughter can be understood systemically, as representing both predispositional characteristics and reactions to the family system. It is suggested that the epigenetic paradigm could be used to characterize the specific failure of developmental processes in many different disorders.
Cross‐Cultural Perspectives: Implications for Attachment Theory and Family Therapy
Authors: Minuchin Patricia
Published: May 2004

Cross‐cultural perspectives have always been useful for understanding behavior. They clarify the distinction between aspects that are essentially part of the human condition and those that are the most responsive to variation. The interesting article by Rothbaum and his colleagues is in that tradition, contrasting the cultural values and family patterns in Japanese society with those of Western cultures, including our own, and suggesting that these differences shape the nature and course of attachment. It stimulates questions about what we have taken for granted in our theories and in our evaluations of dysfunctional behavior.
Postmodern Society and Social Networks: Open and Anticipation Dialogues in Network Meetings
Authors: Seikkula Jaakko , Arnkil Tom Erik , Erikson Esa
Published: May 2004

Network therapy flourished in the U.S. during the 1970s, but has since dwindled there and begun to find new applications in Europe, especially in the Nordic countries. State social and healthcare systems, in developing deep vertical expertise, seems to build up a need for complementary horizontal expertise. The latest theories of sociology are used to analyze the need for networking, with the focus on language and dialogue as specific form. Two approaches developed in crisis service for psychotic patients (Open Dialogue) and in consultation for stuck cases in social care (Anticipation Dialogues), are dealt with. What becomes essential seems no longer to be the therapeutic method itself but the ability to see the polyphonic nature of clients' reality. In this respect, language–and dialogue as a specific form of being in language‐as the focus of treatment, makes the practical forms of different approaches secondary.
The Ecology of Attachment in the Family
Authors: Hill Jonathan , Fonagy Peter , Safier Ellen , Sargent John
Published: May 2004

In this article we outline a conceptualization of attachment processes within the family. We argue that the key elements of attachment processes are affect regulation, interpersonal understanding, information processing, and the provision of comfort within intimate relationships. Although these have been described and assessed primarily in terms of individual functioning and development, they are equally applicable in family systems, provided three farther steps are taken. First, the description of attachment processes at the individual level is applied to the family using the concept of shared frames or representations of emotions, cognitions, and behaviors. Second, there is an explicit formulation of the way in which individual and family processes are linked. Third, there is a conceptualization of the nature and quality of the dynamic between attachment and other processes in family life. In this “ecology” of family processes, those that entail heightened affect and a need to create certainty through action, particularly in response to threats to safety, attachment needs, and discipline challenges, are contrasted with exploratory processes characterized by low affect, tolerance of uncertainty, and opportunities to review existing assumptions and knowledge.
Bullying: The Consequences of Interparental Discord and Child's Self‐Concept
Authors: Christie‐Mizell C. André
Published: May 2004

The objective of this research is to explore how the relationship between interparental discord and child's self‐concept shapes participation in bullying behavior by elementary and middle‐school children. The main finding is that child's self‐concept mediates the effects of interparental discord on bullying behavior. Further, the results of the study support a symbolic interactionist view of child self‐development, in which children internalize the environment provided by parents. This internalization gives way to self‐concept, which guides behavior. This study adds to the growing body of literature that seeks to understand whether and how characteristics of children mediate the effects of parental attributes on behavioral outcomes. The proposed implications for the prevention of bullying include building children's self‐concept, intervening in parental conflict, and involving the entire family system in the intervention process.
“Living with” the Past: Coping and Patterns in Families of Holocaust Survivors
Authors: Chaitin Julia
Published: May 2004

This exploratory study looks at how families of Holocaust survivors work through the traumatic past by considering the coping patterns adapted by family members. Life‐story interviews (Rosenthal, 1993) with 57 individuals from 20 families, in which there were two to three generations, were used in order to learn about the significance they attach to the Holocaust past. The interviews were analyzed using Rosenthal's methods and Danieli's (1988) typology of post‐war adaptation (victim families, fighter families, those who made it, and numb families). Results showed that in order to differentiate between the coping styles exhibited by the families, two new categories had to be added to Danieli's typology. These were entitled “life goes on” and “split families.” It was concluded that survivor families exhibit heterogeneity in the ways in which they cope with the Holocaust past.
Habituation of an odorant‐induced startle response in Drosophila
Authors: Cho W. , Heberlein U. , Wolf F. W.
Published: May 2004

Habituation is a fundamental form of behavioral plasticity that permits organisms to ignore inconsequential stimuli. Here we describe the habituation of a locomotor response to ethanol and other odorants in Drosophila, measured by an automated high‐throughput locomotor tracking system. Flies exhibit an immediate and transient startle response upon exposure to a novel odor. Surgical removal of the antennae, the fly's major olfactory organs, abolishes this startle response. With repeated discrete exposures to ethanol vapor, the startle response habituates. Habituation is reversible by a mechanical stimulus and is not due to the accumulation of ethanol in the organism, nor to non‐specific mechanisms. Ablation or inactivation of the mushroom bodies, central brain structures involved in olfactory and courtship conditioning, results in decreased olfactory habituation. In addition, olfactory habituation to ethanol generalizes to odorants that activate separate olfactory receptors. Finally, habituation is impaired in rutabaga, an adenylyl cyclase mutant isolated based on a defect in olfactory associative learning. These data demonstrate that olfactory habituation operates, at least in part, through central mechanisms. This novel model of olfactory habituation in freely moving Drosophila provides a scalable method for studying the molecular and neural bases of this simple and ubiquitous form of learning.
Genetic and behavioral differences among five inbred mouse strains commonly used in the production of transgenic and knockout mice
Authors: Bothe G. W. M. , Bolivar V. J. , Vedder M. J. , Geistfeld J. G.
Published: May 2004

Five strains of mice commonly used in transgenic and knockout production were compared with regard to genetic background and behavior. These strains were: C57BL/6J, C57BL/6NTac, 129P3/J (formerly 129/J), 129S6/SvEvTac (formerly 129/SvEvTac) and FVB/NTac. Genotypes for 342 microsatellite markers and performance in three behavioral tests (rotorod, open field activity and habituation, and contextual and cued fear conditioning) were determined. C57BL/6J and C57BL/6NTac were found to be true substrains; there were only 12 microsatellite differences between them. Given the data on the genetic background, one might predict that the two C57BL/6 substrains should be very similar behaviorally. Indeed, there were no significant behavioral differences between C57BL/6J and C57BL/6NTac. Contrary to literature reports on other 129 strains, 129S6/SvEvTac often performed similarly to C57BL/6 strains, except that it was less active. FVB/NTac showed impaired rotorod learning and cued fear conditioning. Therefore, both 129S6/SvEvTac and C57BL/6 are recommended as background strains for targeted mutations when researchers want to evaluate their mice in any of these three behavior tests. However, any transgene on the FVB/NTac background should be transferred to B6. Habituation to the open field was analyzed using the parameters: total distance, center distance, velocity and vertical activity. Contrary to earlier studies, we found that all strains habituated to the open field in at least two of these parameters (center distance and velocity).
Genetic correlational analysis of glycogen synthase kinase‐3β and prepulse inhibition in inbred mice
Authors: Amar S. , Jones B. C. , Nadri C. , Kozlovsky N. , Belmaker R. H. , Agam G.
Published: May 2004

In humans, GSK‐3β activity is diminished in schizophrenic patients as is prepulse inhibition of the startle response (PPI). We performed a genetic correlational analysis between published PPI values and frontal cortex GSK‐3 activity analyzed in our laboratory in 10 inbred mouse strains. This methodology could indicate relevant parameters for study in an animal model. Indeed, we obtained significant correlations between the enzyme's activity and PPI measured by two different methods. This may indicate that investigation of the genetics of GSK‐3β regulation holds promise for understanding some of the biochemical underpinnings of schizophrenia.
The dark phase improves genetic discrimination for some high throughput mouse behavioral phenotyping
Authors: Hossain S. M. , Wong B. K. Y. , Simpson E. M.
Published: May 2004

Dark‐phase testing has previously been shown by others to improve the outcome of some ‘classical’ behavior test situations. However, the importance of such ethological correctness and the effect of the light/dark cycle on high throughput behavioral testing situations such as ‘mutant vs. wild type’ and ‘screening’, are less or unknown, respectively. These testing situations differ from the ‘classical’ in that they are designed primarily to discriminate between genetically different mice rather than provide a detailed assessment of ability or psychosocial state. Here we test the hypotheses that dark‐phase testing affects the outcome of high throughput behavioral tests and that dark‐phase testing improves discrimination between genetically distinct mice (C57BL/6J, 129S1/SvImJ and B6129F1) using high throughput behavioral tests. Our results demonstrate that, although all successful tests showed some effect of phase, only the SHIRPA primary screen, open‐field test and motor learning on the rotarod showed improved strain discrimination in the dark phase. Surprisingly, the social interaction test did not show a clear benefit to either phase, and interestingly, the tail‐flick test discriminated strains better in the light phase. However, since the preponderance of our data shows that dark‐phase testing improves, or does not affect, strain discrimination, we conclude that for these strains and tests, dark‐phase testing provided superior outcomes. If discrimination is not achieved in the dark phase, then light phase‐testing would be undertaken.
Reliable assessment of faecal loading in older adults by abdominal radiograph
Authors: Yates Mark , Day Keren , Mullany Jim , Harvey Jack
Published: May 2004

Objective: To produce a measure of faecal loading using plain abdominal radiograph that has both face validity and reliability. This formed part of the Ballarat Constipation Study, which aimed to establish a suite of objective assessment tools for the identification of constipation in residential and extended care facilities.

Methods: A 20‐point loading scale (five levels of loading × four segments of colon) was evaluated using 75 plain abdominal films of patients older than 65 years that were taken for various purposes. These were randomly ordered and five radiologists, following appropriate training, rated the films. Each was blinded to the others’ responses. To establish intra‐rater reliability, each radiologist rated 25 of the films for a second time.

Results: Reliability was assessed using Q‐type correlations for raw scores and Cohen's kappa for dichotomised scores. Inter‐rater correlations ranged from 0.57 confidence interval (CI) (0.38, 0.72) to 0.83 CI (0.74, 0.90). Inter‐rater kappas ranged from 0.28 CI (0.06, 0.50) to 0.72 CI (0.50, 0.94). Intra‐rater correlations ranged from 0.68 CI (0.38, 0.84) to 0.92 CI (0.82, 0.96) and intra‐rater kappas ranged from 0.26 CI (−0.08, 0.60) to 0.90 CI (0.70, 0.99).

Conclusion: This method of assessing and reporting faecal loading in older people has an acceptable level of reliability for four of the five radiologists. The scale was considered appropriate for use in the larger study, where its validity was tested.
Chronic medical conditions as predictors of functional disability in an older population in Taiwan
Authors: Chiu Herng‐Chia , Mau Lih‐Wen , Tasi Wen‐Ling , Hsieh Ying‐Hui , Liu Hong‐Wen
Published: May 2004

Objective: As there is little understanding of disability processes in Taiwanese elders, the present study aimed to identify medical predictors of the incidence of activities of daily living (ADL) disability.

Method: A total of 903 subjects were studied over a 4‐year period (1994–1998). The measurement instrument was the Chinese‐version Multidimensional Functional Assessment Questionnaire (CMFAQ). Only the subjects free of ADL disability at baseline were included in separate logistic regression models to predict disability in physical and instrumental ADL.

Results: The hypothesised associations between chronic conditions and future functional disability were cross‐nationally confirmed. Arthritis, diabetes and hypertension were significantly predictive of the onset of physical ADL (PADL) disability; diabetes was the only medical predictor of instrumental ADL (IADL) disability. Age and educational level were significant predictors of PADL and IADL.

Conclusions: Prevention programs for chronic disease need to pay more attention not only to the disabled population, but also to secondary prevention among those elders who have higher medical risk of developing disability.
Prevalence of coronary heart disease and cardiovascular risk factors in a sample of older Australians: Australian Longitudinal Study of Ageing
Authors: Harris Josephine , Giles Lynne , Finucane Paul , Andrews Gary
Published: May 2004

Objectives: To describe the prevalence of coronary heart disease (CHD) and characterise cardiovascular risk factors in elderly Australians.

Methods: At entry into the Australian Longitudinal Study of Ageing (ALSA), 1075 participants aged 70 years and over provided cardiovascular data by questionnaire, clinical assessment and laboratory measurements.

Results and Conclusion: A total of 201 participants (18.7%) were defined as having CHD, 138 participants (12.9%) had diabetes and 652 (61.3%) had systolic hypertension. Prevalent CHD was associated with male sex, older age, lower high‐density lipoprotein (HDL) cholesterol, low diastolic blood pressure and use of antihypertensive medication. While many ‘traditional’ cardiovascular risk factors were not significantly associated with CHD in this cross‐sectional analysis, follow‐up of this sample will allow a longitudinal assessment of their relationship with CHD in older Australians.
Trends in the prevalence of disability and chronic conditions among the older population: implications for survey design and measurement of disability
Authors: Wen Xingyan
Published: May 2004

A number of issues that are crucial for measuring disability and monitoring trends in disability prevalence include: (i) Why has a decline in the reported disability prevalence been accompanied by an increase in the reported prevalence of chronic diseases in some developed countries? (ii) Why have different trends (increases and decreases) in disability prevalence been reported among the Organisation for Economic Co‐Operation and Development (OECD) countries? (iii) While the cross‐nation comparison of level of disability prevalence is limited by the differences in survey design and methods, can trends in disability within each country be compared internationally on the basis of the existing survey data? The present paper discusses recent trends in reported prevalence of disability and chronic conditions among the older population and explores implications for population survey methods and measurement of disability. A comparison of examples from the USA and Australia indicates that the reported disability prevalence may be affected by whether the presence of any chronic conditions restricting everyday activity are included as part of the survey definition of disability. Focusing on long‐term and severe disability may increase the comparability of disability estimates from different countries, including estimates from time series data.
Periodic Signaling Controlled by an Oscillatory Circuit That Includes Protein Kinases ERK2 and PKA
Authors: Maeda Mineko , Lu Sijie , Shaulsky Gad , Miyazaki Yuji , Kuwayama Hidekazu , Tanaka Yoshimasa , Kuspa Adam , Loomis William F.
Published: May 2004

Self-regulating systems often use robust oscillatory circuits. One such system controls the chemotactic signaling mechanism of
Dictyostelium
, where pulses of adenosine 3′,5′-monophosphate (cAMP) are generated with a periodicity of 7 minutes. We have observed spontaneous oscillations in activation of the mitogen-activated protein (MAP) kinase ERK2 that occur in phase with peaks of cAMP, and we show that ERK2 modulates cAMP levels through the phosphodiesterase RegA. Computer modeling and simulations of the underlying circuit faithfully account for the ability of the cells to spontaneously generate periodic pulses during specific stages of development. Similar oscillatory processes may occur in cells of many different species.
GlyR α3: An Essential Target for Spinal PGE
2
-Mediated Inflammatory Pain Sensitization
Authors: Harvey Robert J. , Depner Ulrike B. , Wässle Heinz , Ahmadi Seifollah , Heindl Cornelia , Reinold Heiko , Smart Trevor G. , Harvey Kirsten , Schütz Burkhard , Abo-Salem Osama M. , Zimmer Andreas , Poisbeau Pierrick , Welzl Hans , Wolfer David P. , Betz Heinrich , Zeilhofer Hanns Ulrich , Müller Ulrike
Published: May 2004

Prostaglandin E
2
(PGE
2
) is a crucial mediator of inflammatory pain sensitization. Here, we demonstrate that inhibition of a specific glycine receptor subtype (GlyR α3) by PGE
2
-induced receptor phosphorylation underlies central inflammatory pain sensitization. We show that GlyR α3 is distinctly expressed in superficial layers of the spinal cord dorsal horn. Mice deficient in GlyR α3 not only lack the inhibition of glycinergic neurotransmission by PGE
2
seen in wild-type mice but also show a reduction in pain sensitization induced by spinal PGE
2
injection or peripheral inflammation. Thus, GlyR α3 may provide a previously unrecognized molecular target in pain therapy.
Cognitive Inflexibility After Prefrontal Serotonin Depletion
Authors: Clarke H. F. , Dalley J. W. , Crofts H. S. , Robbins T. W. , Roberts A. C.
Published: May 2004

Serotonergic dysregulation within the prefrontal cortex (PFC) is implicated in many neuropsychiatric disorders, but the precise role of serotonin within the PFC is poorly understood. Using a serial discrimination reversal paradigm, we showed that upon reversal, selective serotonin depletion of the marmoset PFC produced perseverative responding to the previously rewarded stimulus without any significant effects on either retention of a discrimination learned preoperatively or acquisition of a novel discrimination postoperatively. These results highlight the importance of prefrontal serotonin in behavioral flexibility and are highly relevant to obsessive-compulsive disorder, schizophrenia, and the cognitive sequelae of drug abuse in which perseveration is prominent.
Structure of Nerve Growth Factor Complexed with the Shared Neurotrophin Receptor p75
Authors: He Xiao-lin , Garcia K. Christopher
Published: May 2004

Neurotrophins are secreted growth factors critical for the development and maintenance of the vertebrate nervous system. Neurotrophins activate two types of cell surface receptors, the Trk receptor tyrosine kinases and the shared p75 neurotrophin receptor. We have determined the 2.4 Å crystal structure of the prototypic neurotrophin, nerve growth factor (NGF), complexed with the extracellular domain of p75. Surprisingly, the complex is composed of an NGF homodimer asymmetrically bound to a single p75. p75 binds along the homodimeric interface of NGF, which disables NGF's symmetry-related second p75 binding site through an allosteric conformational change. Thus, neurotrophin signaling through p75 may occur by disassembly of p75 dimers and assembly of asymmetric 2:1 neurotrophin/p75 complexes, which could potentially engage a Trk receptor to form a trimolecular signaling complex.
Shapes of a Wedge
Authors: Olson Steve
Published: May 2004

Darwinism, Design, and Public Education
. John Angus Campbell and Stephen C. Meyer, Eds. Michigan State University Press, East Lansing, MI, 2004. 672 pp. $84.95. ISBN 0-87013-670-4. Paper, $28.95. ISBN 0-87013-675-5.

God, the Devil, and Darwin
A Critique of Intelligent Design Theory. by Niall Shanks. Oxford University Press, New York, 2004. 289 pp. $29.95. ISBN 0-19-516199-8.

Creationism's Trojan Horse
The Wedge of Intelligent Design. by Barbara Forrest and Paul R. Gross. Oxford University Press, New York, 2004. 411 pp. $40. ISBN 0-19-515742-7.

These three additions to a rapidly expanding literature discuss intelligent design creationism and some of the factors behind its recent political successes.
New Real-Time PCR Assay for Rapid Detection of Methicillin- ResistantStaphylococcus aureusDirectly from Specimens Containing a Mixture of Staphylococci
Authors: Huletsky A. , Giroux R. , Rossbach V. , Gagnon M. , Vaillancourt M. , Bernier M. , Gagnon F. , Truchon K. , Bastien M. , Picard F. J. , van Belkum A. , Ouellette M. , Roy P. H. , Bergeron M. G.
Published: May 2004

Molecular methods for the rapid identification of methicillin-resistantStaphylococcus aureus(MRSA) are generally based on the detection of anS. aureus-specific gene target and themecAgene. However, such methods cannot be applied for the direct detection of MRSA from nonsterile specimens such as nasal samples without the previous isolation, capture, or enrichment of MRSA because these samples often contain both coagulase-negative staphylococci (CoNS) andS. aureus, either of which can carrymecA.In this study, we describe a real-time multiplex PCR assay which allows the detection of MRSA directly from clinical specimens containing a mixture of staphylococci in <1 h. Five primers specific to the different staphylococcal cassette chromosomemec(SCCmec) right extremity sequences, including three new sequences, were used in combination with a primer and three molecular beacon probes specific to theS. aureuschromosomalorfXgene sequences located to the right of the SCCmecintegration site. Of the 1,657 MRSA isolates tested, 1,636 (98.7%) were detected with the PCR assay, whereas 26 of 569 (4.6%) methicillin-susceptibleS. aureus(MSSA) strains were misidentified as MRSA. None of the 62 nonstaphylococcal bacterial species or the 212 methicillin-resistant or 74 methicillin-susceptible CoNS strains (MRCoNS and MSCoNS, respectively) were detected by the assay. The amplification of MRSA was not inhibited in the presence of high copy numbers of MSSA, MRCoNS, or MSCoNS. The analytical sensitivity of the PCR assay, as evaluated with MRSA-negative nasal specimens containing a mixture of MSSA, MRCoNS, and MSCoNS spiked with MRSA, was ∼25 CFU per nasal sample. This real-time PCR assay represents a rapid and powerful method which can be used for the detection of MRSA directly from specimens containing a mixture of staphylococci.
Use of Genome Level-Informed PCR as a New Investigational Approach for Analysis of Outbreak-Associated
Mycobacterium tuberculosis
Isolates
Authors: Rajakumar Kumar , Shafi Jamila , Smith Rebecca J. , Stabler Richard A. , Andrew Peter W. , Modha Deborah , Bryant Gerry , Monk Philip , Hinds Jason , Butcher Philip D. , Barer Michael R.
Published: May 2004

Mycobacterium tuberculosis
strain CH, the index isolate linked to a major tuberculosis outbreak associated with high levels of transmissibility and virulence, was characterized by microarray analysis by use of a PCR product array representative of the genome of
M. tuberculosis
strain H37Rv. Seven potential genomic deletions were identified in CH, five of which were confirmed by PCR analysis across the predicted deletion points. The panel of five PCRs required to individually interrogate these loci was collectively referred to as the genome level-informed PCR (GLIP) assay. GLIP analysis was performed with CH, 12 other epidemiologically linked isolates, and 43 recent, non-outbreak-associated isolates derived from patients within the local area. All 13 outbreak-linked isolates showed a profile corresponding to the presence of all five deletions. These 13 isolates were also found to share common variable-number tandem repeat and mycobacterial interspersed repetitive unit profiles. None of the 43 non-outbreak-associated isolates exhibited the five-deletion profile. Although three individual deletions were present in upwards of 44% of the non-outbreak-associated isolates, no single-deletion isolates were detected. Interestingly, none of these deletions had been previously recognized, and sequence analysis of the immediate flanking regions in CH failed to identify a likely mechanism of deletion for four of the five loci. The GLIP assay also proved valuable in ongoing surveillance of the outbreak, rapidly identifying a further two outbreak-associated cases months after the initial cluster and, importantly, dismissing a further 12 epidemiologically suspect cases, which allowed the optimum deployment of public health resources.
Sequencing and Comparative Analysis of Flagellin Genes
fliC
,
fljB
, and
flpA
from
Salmonella
Authors: McQuiston J. R. , Parrenas R. , Ortiz-Rivera M. , Gheesling L. , Brenner F. , Fields P. I.
Published: May 2004

Salmonella
isolates have traditionally been classified by serotyping, the serologic identification of two surface antigens, O-polysaccharide and flagellin protein. Serotyping has been of great value in understanding the epidemiology of
Salmonella
and investigating disease outbreaks; however, production and quality control of the hundreds of antisera required for serotyping is difficult and time-consuming. To circumvent the problems associated with antiserum production, we began the development of a system for determination of serotype in
Salmonella
based on DNA markers. To identify flagellar antigen-specific sequences, we sequenced 280 alleles of the three genes that are known to encode flagellin in
Salmonella
,
fliC
,
fljB
, and
flpA
, representing 67 flagellar antigen types. Analysis of the data indicated that the sequences from
fliC
,
fljB
, and
flpA
clustered by the antigen(s) they encode not by locus. The sequences grouped into four clusters based on their conserved regions. Three of the four clusters included multiple flagellar antigen types and were designated the G complex, the Z4 complex, and the α cluster. The fourth cluster contained a single antigen type, H:z
29
. The amino acid sequences of the conserved regions within each cluster have greater than 95% amino acid identity, whereas the conserved regions differ substantially between clusters (75 to 85% identity). Substantial sequence heterogeneity existed between alleles encoding different flagellar antigens while alleles encoding the same flagellar antigen were homologous, suggesting that flagellin genes may be useful targets for the molecular determination of flagellar antigen type.
Prevalence and Characterization of a Binary Toxin (Actin-Specific ADP-Ribosyltransferase) from
Clostridium difficile
Authors: Gonçalves Carina , Decré Dominique , Barbut Frédéric , Burghoffer Béatrice , Petit Jean-Claude
Published: May 2004

In addition to the two large clostridial cytotoxins (TcdA and TcdB), some strains of
Clostridium difficile
also produce an actin-specific ADP-ribosyltransferase, called binary toxin CDT. We used a PCR method and Southern blotting for the detection of genes encoding the enzymatic (CDTa) and binding (CDTb) components of the binary toxin in 369 strains isolated from patients with suspected
C. difficile
-associated diarrhea or colitis. Twenty-two strains (a prevalence of 6%) harbored both genes. When binary toxin production was assessed by Western blotting, 19 of the 22 strains reacted with antisera against the iota toxin of
C. perfringens
(anti-Ia and anti-Ib). Additionally, binary toxin activity, detected by the ADP-ribosyltransferase assay, was present in only 17 of the 22 strains. Subsequently, all 22 binary toxin-positive strains were tested for the production of toxins TcdA and TcdB, toxinotyped, and characterized by serogrouping, PCR ribotyping, arbitrarily primed PCR, and pulsed-field gel electrophoresis. All binary toxin-positive strains also produced TcdB and/or TcdA. However, they had significant changes in the
tcdA
and
tcdB
genes and belonged to variant toxinotypes III, IV, V, VII, IX, and XIII. We could differentiate 16 profiles by using typing methods, indicating that most of the binary toxin-positive strains were unrelated.
Real-Time PCR System for Detection of Orthopoxviruses and Simultaneous Identification of Smallpox Virus
Authors: Olson Victoria A. , Laue Thomas , Laker Miriam T. , Babkin Igor V. , Drosten Christian , Shchelkunov Sergei N. , Niedrig Matthias , Damon Inger K. , Meyer Hermann
Published: May 2004

A screening assay for real-time LightCycler (Roche Applied Science, Mannheim, Germany) PCR identification of smallpox virus DNA was developed and compiled in a kit system under good manufacturing practice conditions with standardized reagents. In search of a sequence region unique to smallpox virus, the nucleotide sequence of the 14-kDa fusion protein gene of each of 14 variola virus isolates of the Russian World Health Organization smallpox virus repository was determined and compared to published sequences. PCR primers were designed to detect all Eurasian-African species of the genus
Orthopoxvirus
. A single nucleotide mismatch resulting in a unique amino acid substitution in smallpox virus was used to design a hybridization probe pair with a specific sensor probe that allows reliable differentiation of smallpox virus from other orthopoxviruses by melting-curve analysis. The applicability was demonstrated by successful amplification of 120 strains belonging to the orthopoxvirus species variola, vaccinia, camelpox, mousepox, cowpox, and monkeypox virus. The melting temperatures (
T

m

s) determined for 46 strains of variola virus (
T

m

s, 55.9 to 57.8°C) differed significantly (
P
= 0.005) from those obtained for 11 strains of vaccinia virus (
T

m

s, 61.7 to 62.7°C), 15 strains of monkeypox virus (
T

m

s, 61.9 to 62.2°C), 40 strains of cowpox virus (
T

m

s, 61.3 to 63.7°C), 8 strains of mousepox virus (
T

m

, 61.9°C), and 8 strains of camelpox virus (
T

m

s, 64.0 to 65.0°C). As most of the smallpox virus samples were derived from infected cell cultures and tissues, smallpox virus DNA could be detected in a background of human DNA. By applying probit regression analysis, the analytical sensitivity was determined to be 4 copies of smallpox virus target DNA per sample. The DNAs of several human herpesviruses as well as poxviruses other than orthopoxviruses were not detected by this method. The assay proved to be a reliable technique for the detection of orthopoxviruses, with the advantage that it can simultaneously identify variola virus.
mecA
Locus Diversity in Methicillin-Resistant
Staphylococcus aureus
Isolates in Brisbane, Australia, and the Development of a Novel Diagnostic Procedure for the Western Samoan Phage Pattern Clone
Authors: Huygens Flavia , Stephens Alex J. , Nimmo Graeme R. , Giffard Philip M.
Published: May 2004

An emerging public health phenomenon is the increasing incidence of methicillin-resistant
Staphylococcus aureus
(MRSA) infections that are acquired outside of health care facilities. One lineage of community-acquired MRSA (CA-MRSA) is known as the Western Samoan phage pattern (WSPP) clone. The central aim of this study was to develop an efficient genotyping procedure for the identification of WSPP isolates. The approach taken was to make use of the highly variable region downstream of
mecA
in combination with a single nucleotide polymorphism (SNP) defined by the
S. aureus
multilocus sequence typing (MLST) database. The premise was that a combinatorial genotyping method that interrogated both a highly variable region and the genomic backbone would deliver a high degree of informative power relative to the number of genetic polymorphisms interrogated. Thirty-five MRSA isolates were used for this study, and their gene contents and order downstream of
mecA
were determined. The CA-MRSA isolates were found to contain a truncated
mecA
downstream region consisting of
mecA
-HVR-IS
431 mec
-dcs-Ins117, and a PCR-based method for identifying this structure was developed. The hospital-acquired isolates were found to contain eight different
mecA
downstream regions, three of which were novel. The Minimum SNPs computer software program was used to mine the
S. aureus
MLST database, and the
arcC
272G polymorph was identified as 82% discriminatory for ST-30. A real-time PCR assay was developed to interrogate this SNP. We found that the assay for the truncated
mecA
downstream region in combination with the interrogation of
arcC
position 272 provided an unambiguous identification of WSPP isolates.
Development and Evaluation of a Novel Loop-Mediated Isothermal Amplification Method for Rapid Detection of Severe Acute Respiratory Syndrome Coronavirus
Authors: Thai Hong Thi Cam , Le Mai Quynh , Vuong Cuong Duc , Parida Manmohan , Minekawa Harumi , Notomi Tsugunori , Hasebe Futoshi , Morita Kouichi
Published: May 2004

The development and evaluation of a one-step single-tube accelerated real-time quantitative reverse transcription (RT) loop-mediated isothermal amplification (LAMP) assay is reported for rapid detection of the severe acute respiratory syndrome coronavirus (SARS-CoV) replicase gene. A total of 49 samples (15 throat washes, 13 throat swabs, and 21 combined throat and nasal swabs) collected from patients admitted to the Hanoi-French and Ninhbinh hospitals in Vietnam during the SARS epidemic were evaluated and compared to conventional RT-PCR. The RT-LAMP assay demonstrated 100-fold-greater sensitivity, with a detection limit of 0.01 PFU. The sensitivity and specificity of RT-LAMP assay for detecting viral RNA in clinical specimens with regard to RT-PCR were 100 and 87%, respectively. The specificity of the RT-LAMP assay was further validated by restriction analysis as well as nucleotide sequencing of the amplified product. The concentration of virus in most of the clinical samples was 0.1 PFU (0.1 to 10
2
PFU), as determined from the standard curve of SARS RT-LAMP and based on the time of positivity. The assay procedure is quite simple, wherein the amplification is carried out in a single tube under isothermal conditions at 63°C, and the result can be obtained in less than 1 h (as early as 11 min). Thus, the RT-LAMP assay reported here has the advantages of rapid amplification, simple operation, and easy detection and will be useful for rapid and reliable clinical diagnosis of SARS-CoV in developing countries.
Variable Ratio of Hepatitis C Virus RNA to Viral Core Antigen in Patient Sera
Authors: Schüttler Christian G. , Thomas Christine , Discher Thomas , Friese Georg , Lohmeyer Jürgen , Schuster Ralph , Schaefer Stephan , Gerlich Wolfram H.
Published: May 2004

Quantification of hepatitis C virus (HCV) core antigen and RNA in serum samples leads to a highly variable ratio of both. It is not clear whether this is due to the inaccuracy of RNA quantification or whether both are independent parameters in a certain range. We established a real-time reverse transcription (RT)-PCR for HCV RNA that combines very high sensitivity with a large dynamic range and minimal standard deviations. The assay was calibrated with the first international standard, 96/790, and the international genotype panel for HCV from the National Institute of Biological Standardisation and Control. A linear readout was obtained between 200 and 5 × 10
7
IU/ml. The detection limit was 80 IU/ml, the reproducibility was <0.05 log, and the standard error within one run was <0.01. Comparison of the method with the Roche Monitor competitive RT-PCR revealed its high accuracy. The core protein concentration was determined within a range from 1.5 to 400 pg/ml by using the preliminary trak-C assay from Ortho Clinical Diagnostics. Correlating the HCV RNA levels with core antigen concentrations in 197 serum samples from 23 interferon-treated patients, a average ratio of 7,900 IU of HCV RNA per pg of core antigen was estimated, but the variability of this ratio exceeded largely the variability of the two assays, ranging from 50 to 20,000 IU/pg. Theoretically, HCV should contain ca. 43,000 IU of RNA/pg core. In conclusion, the core antigen assay seems to detect, in addition to complete virions, RNA-free core protein structures, which enhances its sensitivity (98% in this group). The variable ratio of RNA and core protein is not mainly due to standard deviations of quantification but could be an additional parameter for treatment follow-up and state of viral replication.
Amplification of Coccidioidal DNA in Clinical Specimens by PCR
Authors: Johnson Suzanne M. , Simmons Keira A. , Pappagianis Demosthenes
Published: May 2004

Coccidioides
DNA was amplified from serum by a PCR using coccidioid-specific primers. A 239-bp product was visualized when 10 fg of exogenous coccidioidal DNA was subjected to amplification. This product was demonstrated in some human and mouse sera prior to the detection of coccidioidal antibodies.
Use of Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem Repeat Typing To Examine Genetic Diversity ofMycobacterium tuberculosisin Singapore
Authors: Sun Yong-Jiang , Bellamy Richard , Lee Ann S. G. , Ng Sze Ta , Ravindran Sindhu , Wong Sin-Yew , Locht Camille , Supply Philip , Paton Nicholas I.
Published: May 2004

Strain typing using variable-number tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTR) is a powerful tool for studying the epidemiology and genetic relationships ofMycobacterium tuberculosisisolates. For this study, isolates from 291 patients in Singapore were genotyped by this method. One hundred sixty-six distinct MIRU-VNTR patterns were detected. One hundred sixty-two strains were grouped into 1 of 35 different MIRU-VNTR clusters and 131 isolates were unique. In this sample collection, 9 of the 12 MIRU-VNTR loci were moderately or highly discriminative according to their allelic diversities. The Hunter-Gaston discriminatory index was 0.975, indicating the high power of discrimination of MIRU-VNTR typing. By direct comparisons with previously typed MIRU-VNTR patterns and by genetic relationship analyses, we could identify and clearly define four epidemic groups ofM. tuberculosisin our sample, corresponding to the W/Beijing, East-Africa-Indian, Haarlem, and Delhi genotype families. Furthermore, MIRU-VNTR typing was able to clearly distinguish ancestral and modernM. tuberculosisstrains as defined by TbD1 genomic deletion analysis. These results indicate that MIRU-VNTR typing can be a useful first-line tool for studying the genetic diversity ofM. tuberculosisisolates in a large urban setting such as Singapore.
Reverse Transcriptase PCR Diagnostic Assay for the Coronavirus Associated with Severe Acute Respiratory Syndrome
Authors: Hui Raymond K. H. , Zeng Fanya , Chan Charis M. N. , Yuen K. Y. , Peiris Joseph S. M. , Leung Frederick C. C.
Published: May 2004

Recent outbreaks of severe acute respiratory syndrome (SARS) have spurred intense research efforts around the world to deal with the serious threat to health posed by this novel coronavirus. A rapid, reliable diagnostic assay is needed for monitoring the spread of the disease. Here we report a method for eliminating false-negative results and increasing test sensitivity, based on the hypothesis that the message encoded by the nucleocapsid (N) gene is the most abundant during viral infection. Nasopharyngeal aspirates and stool samples were obtained from suspected SARS patients with major clinical symptoms and a significant history of close contact with infected patients. Total RNAs were extracted in a 96-well format, together with pig kidney epithelial (PK-15) cells as an internal control for extraction efficiency. PCR inhibitors were removed by ethanol precipitation, and a PCR for the pig β-actin gene was used as a positive control for all clinical samples. Samples were analyzed by a reverse transcriptase PCR assay. Northern blot analysis was performed to demonstrate differences in subgenomic transcripts of the virus, and a real-time quantitative PCR was employed to compare the sensitivities of two loci (1b and N). The detection rate of the assay reached 44.4% on day 9 after the onset of the disease. The diagnostic PCR amplifying the N gene gave an average of a 26.0% (6.3 to 60.0%) stronger intensity signal than that for the 1b gene. In conclusion, the nucleocapsid gene represents an additional sensitive molecular marker for the diagnosis of the SARS coronavirus and can be further adapted for use in a high-throughput platform assay.