bluuebunny/crossref_metadata_2025 huggingface dataset:
Perceptions of older people about falls injury prevention and physical activity
Authors: Snodgrass Suzanne J , Rivett Darren A , Mackenzie Lynette A
Published: June 2005

Objectives:  The purpose of this study was to explore the beliefs and perceptions of older people about falls injury prevention services, and to identify incentives and barriers to attending falls prevention services, including programs targeting physical activity.

Methods:  Seventy‐five people over age 60 years who were members of community groups completed a 23‐item survey that was returned by mail. Survey items included self‐reported falls and confidence when walking, awareness of falls prevention strategies, desirable programs for a falls prevention service, and incentives and barriers to participation in physical activity programs.

Results:  Twenty‐eight per cent of respondents had fallen within the previous 6 months, yet just over half (54%) were aware there were strategies to prevent falling, despite the majority of the sample being physically active (81%) and at least reasonably confident about walking (84%). The features that were considered most desirable for a falls prevention service were group exercise programs (61%), educational talks about health issues (57%), and vision/eye glasses assessment (52%). The most commonly reported incentives for attending a physical activity group included having a doctor advise them to attend (61%) or having a friend who attended the group (55%), and the most common barrier to attending a physical activity group was transportation (43%).

Conclusions:  Despite consistent numbers of older people experiencing falls, there is little awareness among older people that strategies exist to prevent falling. It appears that motivating older people to participate in physical activity with the aim of falls prevention will largely depend on the encouragement of their doctor or their peers.
Diverging regulation of pyruvate dehydrogenase kinase isoform gene expression in cultured human muscle cells
Authors: Abbot Emily L. , McCormack James G. , Reynet Christine , Hassall David G. , Buchan Kevin W. , Yeaman Stephen J.
Published: June 2005

The pyruvate dehydrogenase complex occupies a central and strategic position in muscle intermediary metabolism and is primarily regulated by phosphorylation/dephosphorylation. The identification of multiple isoforms of pyruvate dehydrogenase kinase (PDK1–4) and pyruvate dehydrogenase phosphatase (PDP1–2) has raised intriguing new possibilities for chronic pyruvate dehydrogenase complex control. Experiments to date suggest that PDK4 is the major isoenzyme responsible for changes in pyruvate dehydrogenase complex activity in response to various different metabolic conditions. Using a cultured human skeletal muscle cell model system, we found that expression of both PDK2 and PDK4 mRNA is upregulated in response to glucose deprivation and fatty acid supplementation, the effects of which are reversed by insulin treatment. In addition, insulin directly downregulates PDK2 and PDK4 mRNA transcript abundance via a phosphatidylinositol 3‐kinase‐dependent pathway, which may involve glycogen synthase kinase‐3 but does not utilize the mammalian target of rapamycin or mitogen‐activated protein kinase signalling pathways. In order to further elucidate the regulation of PDK, the role of the peroxisome proliferators‐activated receptors (PPAR) was investigated using highly potent subtype selective agonists. PPARα and PPARδ agonists were found to specifically upregulate PDK4 mRNA expression, whereas PPARγ activation selectively decreased PDK2 mRNA transcript abundance. PDP1 mRNA expression was unaffected by all conditions analysed. These results suggest that in human muscle, hormonal and nutritional conditions may control PDK2 and PDK4 mRNA expression via a common signalling mechanism. In addition, PPARs appear to independently regulate specific PDK isoform transcipt levels, which are likely to impart important metabolic mediation of fuel utilization by the muscle.
Evolutionary and experimental analyses of inorganic phosphate transporter PiT family reveals two related signature sequences harboring highly conserved aspartic acids critical for sodium‐dependent phosphate transport function of human PiT2
Authors: Bøttger Pernille , Pedersen Lene
Published: June 2005

The mammalian members of the inorganic phosphate (Pi) transporter (PiT) family, the type III sodium‐dependent phosphate (NaPi) transporters PiT1 and PiT2, have been assigned housekeeping Pi transport functions and are suggested to be involved in chondroblastic and osteoblastic mineralization and ectopic calcification. The PiT family members are conserved throughout all kingdoms and use either sodium (Na+) or proton (H+) gradients to transport Pi. Sequence logo analyses revealed that independent of their cation dependency these proteins harbor conserved signature sequences in their N‐ and C‐terminal ends with the common core consensus sequence GANDVANA. With the exception of 10 proteins from extremophiles all 109 proteins analyzed carry an aspartic acid in one or both of the signature sequences. We changed either of the highly conserved aspartates, Asp28 and Asp506, in the N‐ and C‐terminal signature sequences, respectively, of human PiT2 to asparagine and analyzed Pi uptake function in Xenopus laevis oocytes. Both mutant proteins were expressed at the cell surface of the oocytes but exhibited knocked out NaPi transport function. Human PiT2 is also a retroviral receptor and we have previously shown that this function can be exploited as a control for proper processing and folding of mutant proteins. Both mutant transporters displayed wild‐type receptor functions implying that their overall architecture is undisturbed. Thus the presence of an aspartic acid in either of the PiT family signature sequences is critical for the Na+‐dependent Pi transport function of human PiT2. The conservation of the aspartates among proteins using either Na+‐ or H+‐gradients for Pi transport suggests that they are involved in H+‐dependent Pi transport as well. Current results favor a membrane topology model in which the N‐ and C‐terminal PiT family signature sequences are positioned in intra‐ and extracellular loops, respectively, suggesting that they are involved in related functions on either side of the membrane. The present data are in agreement with a possible role of the signature sequences in translocation of cations.
Adenylyl cyclase Rv0386 from Mycobacterium tuberculosis H37Rv uses a novel mode for substrate selection
Authors: Castro Lucila I. , Hermsen Corinna , Schultz Joachim E. , Linder Jürgen U.
Published: June 2005

Class III adenylyl cyclases usually possess six highly conserved catalytic residues. Deviations in these canonical amino acids are observed in several putative adenylyl cyclase genes as apparent in several bacterial genomes. This suggests that a variety of catalytic mechanisms may actually exist. The gene Rv0386 from Mycobacterium tuberculosis codes for an adenylyl cyclase catalytic domain fused to an AAA‐ATPase and a helix‐turn‐helix DNA‐binding domain. In Rv0386, the standard substrate, adenine‐defining lysine‐aspartate couple is replaced by glutamine‐asparagine. The recombinant adenylyl cyclase domain was active with a Vmax of 8 nmol cAMP·mg−1·min−1. Unusual for adenylyl cyclases, Rv0386 displayed 20% guanylyl cyclase side‐activity with GTP as a substrate. Mutation of the glutamine‐asparagine pair either to alanine residues or to the canonical lysine‐aspartate consensus abolished activity. This argues for a novel mechanism of substrate selection which depends on two noncanonical residues. Data from individual and coordinated point mutations suggest a model for purine definition based on an amide switch related to that previously identified in cyclic nucleotide phosphodiesterases.
Relationship between the structure of guanidines and N‐hydroxyguanidines, their binding to inducible nitric oxide synthase (iNOS) and their iNOS‐catalysed oxidation to NO
Authors: Lefèvre‐Groboillot David , Boucher Jean‐Luc , Stuehr Dennis J. , Mansuy Daniel
Published: June 2005

The binding of several alkyl‐ and aryl‐guanidines and N‐hydroxyguanidines to the oxygenase domain of inducible NO‐synthase (iNOSoxy) was studied by UV/Vis difference spectroscopy. In a very general manner, monosubstituted guanidines exhibited affinities for iNOSoxy that were very close to those of the corresponding N‐hydroxyguanidines. The highest affinities were observed for the natural substrates, l‐arginine and Nω‐hydroxy‐l‐arginine (Kd at the µm level). The deletion of either the CO2H or the NH2 function of their amino acid moiety led to dramatic decreases in the affinity. However, alkylguanidines with a relatively small alkyl chain exhibited interesting affinities, the best being observed for a butyl chain (Kd =20 µm). Arylguanidines also bound to iNOSoxy, however, with lower affinities (Kd > 250 µm). Many N‐alkyl‐ and N‐aryl‐N′‐hydroxyguanidines are oxidized by iNOS with formation of NO, whereas only few alkylguanidines led to significant production of NO under identical conditions, and all the arylguanidines tested to date were unable to lead to the production of NO. The kcat values of NO production from the oxidation by iNOS of the studied N‐hydroxyguanidines were found to vary independently of their affinity for the protein. The kcat values determined for the two‐step oxidation of alkylguanidines to NO were not clearly related to the Kd of these substrates toward iNOSoxy. However, there is a qualitative relationship between these kcat values and the apparent rate constants of dissociation of the complex between iNOSoxy and the corresponding N‐alkyl‐N′‐hydroxyguanidine (koffapp) that were determined by stopped‐flow UV/Vis spectroscopy. These data indicate that a key factor for efficient oxidation of a guanidine by iNOS to NO is the ability of the corresponding N‐hydroxyguanidine to bind to the active site without being too rapidly released before its further oxidation. This explains why 4,4,4‐trifluorobutylguanidine is so far the best non‐α‐amino acid guanidine substrate of iNOS with formation of NO, because the koffapp of the corresponding N‐hydroxyguanidine is particularly low. This suggests that the rational design of guanidines as new NO donors upon in situ oxidation by NOSs should take into account both thermodynamic and kinetic characteristics of the interaction of the protein not only with the guanidine but also with the corresponding N‐hydroxyguanidine.
Modular usage of the HLA‐DRA promoter in extra‐hematopoietic and hematopoietic cell types of transgenic mice
Authors: Giorda Ezio , Sibilio Leonardo , Martayan Aline , Feriotto Giordana , Bianchi Nicoletta , Mischiati Carlo , Di Rosa Francesca , Pozzi Laura , Gambari Roberto , Giacomini Patrizio
Published: June 2005

Class II MHC genes (for example, the human HLA‐DRA gene) are expressed at the cell surface in many professional and nonprofessional antigen‐presenting cells in a variety of anatomical locations. Here, we report about 13 mouse transgenic lines (11 of which have not been previously described) generated with four distinct sets of DRA transgenes carrying progressive, informative 5′ and 3′ deletions. DRA expression was assessed in B lymphocytes, dendritic cells, macrophages, and extra‐hematopoietic cells (particularly kidney epithelial cells). A compact transcriptional unit was identified that efficiently directs DRA expression [both constitutive and interferon (IFN)‐γ induced] in extra‐hematopoietic tissues and dendritic cells. It extends from position −266 upstream of the transcription initiation site to position +119 downstream of the last DRA exon. The same fragment, however, did not efficiently direct IFN‐γ‐induced DRA expression in macrophages, that required additional 5′ sequences. Thus, IFN‐γ uses distinct promoter segments and mechanisms to up‐regulate class II in different cell lineages. In contrast to previous results in transgenic mice expressing murine class II transgenes, we were unable to generate reproducible patterns of HLA‐DRA expression in B cells.
The Efficacy of Tadalafil in Clinical Populations
Authors: Lewis Ronald W. , Sadovsky Richard , Eardley Ian , O'Leary Michael , Seftel Allen , Wang Wei Christine , Shen Wei , Walker Daniel J. , Wong David G. , Ahuja Sanjeev
Published: June 2005

Objectives

To evaluate the efficacy of tadalafil in men with erectile dysfunction (ED) by demographic and ED characteristics, in patients having various comorbid medical conditions, and in patients receiving drug treatment for other medical conditions.

Methods

This is an analysis of 11 double-blind, placebo-controlled trials with 2,102 men with a broad spectrum of ED etiology and various comorbid medical conditions as participants. The variables analyzed in this report included race, age, body mass index (BMI), ED etiology, ED severity, ED duration, smoking, prior sildenafil use, presence of comorbid conditions (diabetes mellitus, hypertension, cardiovascular disease, hyperlipidemia, depression, benign prostatic hyperplasia), and treatment with antihypertensives or antidepressants. Patients were randomly assigned to receive tadalafil 10 mg (N = 321), tadalafil 20 mg (N = 1,143), or placebo (N = 638). The primary efficacy variables included mean changes from baseline in the erectile function (EF) domain score of the International Index of Erectile Function (IIEF) questionnaire, and the mean per-patient percentage of “yes” responses to the Sexual Encounter Profile (SEP) diary question 3 (SEP3––successful intercourse). The Global Assessment Question 1 (GAQ) was evaluated, as was the percentage of men attaining a normal IIEF EF domain score at end point.

Results

Patients taking tadalafil 10 mg or 20 mg demonstrated significant improvement (P < 0.005) from baseline to end point on the IIEF EF domain score in all subpopulations analyzed compared with patients receiving placebo. The mean-per-patient percentage of “yes” responses to SEP3 increased significantly in all subpopulations taking tadalafil compared with placebo (P < 0.05). Tadalafil-treated patients had a significantly greater positive response rate on the GAQ in all subpopulations analyzed compared with placebo-treated patients (P < 0.03) except for the tadalafil 10 mg cardiovascular subpopulation (placebo, 46.8%; tadalafil 10 mg, 71.0%; P = 0.127). The percentage of positive responses ranged from 72% to 91% for patients on tadalafil 20 mg and from 52% to 94% for tadalafil 10 mg compared with a range of 20% to 47% for placebo-treated patients.

Conclusions

Tadalafil was effective in improving erectile function across a wide spectrum of ED patients including patients with various comorbid conditions.
Testosterone Therapy Can Enhance Erectile Function Response to Sildenafil in Patients with PADAM: A Pilot Study
Authors: Shamloul Rany , Ghanem Hussein , Fahmy Ibrahim , El-Meleigy Amr , Ashoor Shedeed , Elnashaar Abdelrahman , Kamel Ihab
Published: June 2005

Introduction

Recent studies suggest a direct relationship between free testosterone and cavernous vasodilatation. Some men with erectile dysfunction (ED) associated with PADAM (partial androgen deficiency in aging men) might possibly benefit from testosterone undecanoate therapy (TRT).

Objectives

To determine the efficacy of testosterone undecanoate in facilitating the erectile response and patient satisfaction with sildenafil in men 40–70 years old with PADAM symptoms.

Design and Methods

Prospective study including 40 patients recruited after a sildenafil therapeutic trial. Total testosterone and sex hormone binding globulin (SHBG) were measured to calculate the free androgen index. Prostate specific antigen (PSA) was measured and repeated 2 months after treatment. A rating score was used for PADAM symptoms, and the 5-point abbreviated version of the International Index of Erectile Function (IIEF-5) to assess erectile function. Men failing to respond to sildenafil were randomized into two groups receiving sildenafil plus continuous TRT (group 1ST), and TRT (group 1T) alone. Men partially responding to sildenafil were randomized into two groups receiving sildenafil plus continuous TRT for 2 months (group 2ST), or sildenafil alone (group 2S). Treatment efficacy was assessed by analysis of between-group differences.

Results

Groups 1T, 2S, and 2ST showed significant improvement in PADAM scores (P < 0.05, Wilcoxon matched pairs test). Patients receiving both sildenafil plus continuous TRT (groups 1ST and 2ST) showed significant improvement in IIEF-5 scores (P < 0.5, paired t-test). No significant changes in serum levels of PSA were detected (paired t-test).

Conclusions

We conclude that TRT appears to be beneficial and safe in facilitating the erectile response and patient satisfaction with sildenafil in men with PADAM symptoms. Androgen supplementation should be carried out cautiously with careful monitoring to avoid possible adverse effects.
Association Between Erectile Dysfunction and Sleep Disorders Measured by Self-Assessment Questionnaires in Adult Men
Authors: Heruti Rafi , Shochat Tzipi , Tekes-Manova Dorit , Ashkenazi Itschak , Justo Dan
Published: June 2005

Background

Erectile dysfunction (ED) is often associated with sleep disorders and sleep apnea syndrome (SAS) in mostly middle-aged and elderly men. Sleep disorders and ED are also prevalent in younger men.

Purpose

To study the association between ED, sleep disorders, and SAS, particularly among adult men.

Methods

A health screening program is offered by the Israel Defense Force (IDF) for career servicemen older than 25 years, for the purpose of early detection of ED and sleep disorders, among other concealed morbidities. The Sexual Health Inventory for Man questionnaire (SHIM) was used to measure ED. The Sleep Quality (SQ) questionnaire, developed by the medical services of the IDF, was used to characterize SAS and other sleep disorders.

Results

From 2002 through 2003, 3,363 men (mean age, 36.1 ± 6.8 years) replied to the SHIM and SQ questionnaires. Of these men, 337 (10%) scored high (≥25) in the SQ questionnaire, suggesting moderate to severe sleep disorders, and 870 men (25.8%) scored low (≤21) in the SHIM questionnaire, suggesting ED. There was a negative correlation between the scores in the SHIM questionnaire and in the SQ questionnaire (r = −0.29, P < 0.0001)—even after age adjustment and after excluding men with risk factors for both ED and SAS (such as essential hypertension, diabetes mellitus, and obesity; r = −0.21, P < 0.0001). This correlation was consistent with all severity levels of ED and sleep disorders.

Conclusions

Sleep  disorders,  in  particular  SAS,  and  ED  are  prevalent  and  may  be  related  in adult  men.  Either  ED  or  sleep  disorders  should  be  considered  whenever  the  other  is  suspected in adult men. Sleep disorders and ED should also be investigated for the purpose of treating underlying systemic diseases and emotional disorders, and in order to prevent late complications of atherosclerosis.
The Use of the Sexual Function Questionnaire as a Screening Tool for Women with Sexual Dysfunction
Authors: Quirk Frances , Haughie Scott , Symonds Tara
Published: June 2005

Aim

To determine if the validated Sexual Function Questionnaire (SFQ), developed to assess efficacy in female sexual dysfunction (FSD) clinical trials, may also have utility in identifying target populations for such studies.

Methods

Data from five clinical trials and two general population surveys were used to analyze the utility of the SFQ as a tool to discriminate between the presence of specific components of FSD (i.e., hypoactive sexual desire disorder, female sexual arousal disorder, female orgasmic disorder, and dyspareunia).

Results

Sensitivity/specificity analysis and logistic regression analysis, using data from all five clinical studies and the general population surveys, confirmed that the SFQ domains have utility in detecting the presence of specific components of FSD and provide scores indicative of the presence of a specific sexual disorder.

Conclusions

The SFQ is a valuable new tool for detecting the presence of FSD and identifying the specific components of sexual functions affected (desire, arousal, orgasm, or dyspareunia).
Human color vision and the unsaturated blue color of the daytime sky
Authors: Smith Glenn S.
Published: June 2005

The usual answer to the question “Why is the sky blue?” is based only on Rayleigh scattering from the molecules in the atmosphere and makes little mention of the contribution of color vision. We supplement this answer with a quantitative discussion of the role color vision plays in determining the appearance of the daytime sky. The anatomy of the human eye is reviewed, and its response as a function of wavelength is described via the spectral sensitivities of the cones. Color matching is examined for a mixture of monochromatic lights and for the spectrum of the daytime sky. The spectral irradiance of skylight is shown to be a metameric match to unsaturated blue light. A simple experiment is described suitable for classroom use or a student project.
Refractive index measurement using total internal reflection
Authors: Van Keuren Edward Richard
Published: June 2005

An undergraduate level experiment is presented for demonstrating total internal reflection. The experiment yields a relatively precise value of the refractive index of a prism to be found from a measurement of the incident angle at which total internal reflection occurs. Analytic expressions are obtained for the refractive index as a function of the incident angle for both equilateral and right angle prisms. Students can observe not only the critical angle at which total internal reflection occurs, but measure and analyze the significant rise in reflected beam power. The addition of a small fluid reservoir attached to the side of the prism enables the determination of a liquid refractive index.
Increasing rate of water temperature due to an Ekman layer flow in a heated pot (fluid dynamics in the kitchen)
Authors: Ida K. , Yoshinuma M. , Nagaoka K.
Published: June 2005

A sharp increase in the local temperature of heated water at the center of an aluminum pot is demonstrated a few tens of second after rotating the heated water with a spoon. The stirring leads to a sharp gradient of the temperature in the heated water, which contradicts the general belief that stirring causes a uniform temperature distribution. This sharp increase of temperature is due to the flow of an Ekman layer near the heated bottom of the pot. When this flow is prevented by a barrier at the bottom of the pot, there is no sharp increase of the water temperature. The experiments using an aluminum pot in the kitchen aid in understanding the existence and importance of flow in an Ekman layer, which is a crucial concept in hydrodynamics.
Practical Methods Using Boronic Acid Compounds for Identification of Class C β-Lactamase-ProducingKlebsiella pneumoniaeandEscherichia coli
Authors: Yagi Tetsuya , Wachino Jun-ichi , Kurokawa Hiroshi , Suzuki Satowa , Yamane Kunikazu , Doi Yohei , Shibata Naohiro , Kato Haru , Shibayama Keigo , Arakawa Yoshichika
Published: June 2005

Detection of the resistance mediated by class C β-lactamases remains a challenging issue, considering that transferable plasmid-mediated class C β-lactamases are of worldwide concern. Methods for the identification of strains that produce extended-spectrum β-lactamases (ESBLs) or metallo-β-lactamases (MBLs) have been developed and applied for routine use in clinical microbiology laboratories, but no practical methods for identification of plasmid-mediated class C producers have been established to date. We therefore developed three simple methods for clinical microbiology laboratories that allow identification of plasmid-mediated class C β-lactamase-producing bacteria using a boronic acid derivative, 3-aminophenylboronic acid (APB), one of the specific inhibitors of class C β-lactamases. Detection by the disk potentiation test was based on the enlargement of the growth-inhibitory zone diameter (by greater than or equal to 5 mm) around a Kirby-Bauer disk containing a ceftazidime (CAZ) or a cefotaxime (CTX) disk in combination with APB. In a double-disk synergy test, the discernible expansion of the growth-inhibitory zone around the CAZ or the CTX disk toward a disk containing APB was indicative of class C β-lactamase production. A greater than or equal to eightfold decrease in the MIC of CAZ or CTX in the presence of APB was the criterion for detection in the microdilution test. By using these methods,Escherichia coliandKlebsiella pneumoniaeisolates producing plasmid-mediated class C β-lactamases, ACT-1, CMY-2, CMY-9, FOX-5, LAT-1, and MOX-1, were successfully distinguished from those producing other classes of β-lactamases, such as ESBLs and MBLs. These methods will provide useful information needed for targeted antimicrobial therapy and better infection control.
Flow Cytometry Susceptibility Testing for the Antifungal Caspofungin
Authors: Mitchell M. , Hudspeth M. , Wright A.
Published: June 2005

Rapid antifungal susceptibility testing for the antifungal agent caspofungin can be performed using flow cytometry (FC). An FC procedure using acridine orange provided minimum inhibitory concentration (MIC) results within 7 to 9 h which were compared with results obtained using the NCCLS M27-A2 protocol. To evaluate the consistency of this method, susceptibility testing using caspofungin was performed using 73 isolates of eight different species of
Candida
from various clinical samples in Central California. Macrotiter or microdilution tests were performed according to the NCCLS M27-A2 protocol, and the MICs were compared to those provided by our flow cytometry method. All isolates tested had results within the sensitive interpretive category, and 90% of the results compared within 1 dilution, showing excellent agreement between the methods. The MIC at which 50% of the isolates tested were inhibited (MIC
50
) and the MIC
90
of caspofungin for all eight
Candida
species were within 1 dilution. This flow cytometer 7-h protocol for testing the antifungal susceptibility of
Candida
species to caspofungin provided results equivalent to those obtained with the M27-A2 protocol.
Outbreak of Dysentery Associated with Ceftriaxone-Resistant
Shigella sonnei
: First Report of Plasmid-Mediated CMY-2-Type AmpC β-Lactamase Resistance in
S. sonnei
Authors: Huang I-Fei , Chiu Cheng-Hsun , Wang Mei-Hui , Wu Chan-Yao , Hsieh Kai-Sheng , Chiou Christine C.
Published: June 2005

We document the first report of plasmid-encoded CMY-2-type AmpC β-lactamase identified among
Shigella sonnei
isolates resistant to ceftriaxone and obtained after an outbreak of bacillary dysentery in Taiwan. One hundred eighty-two children in two elementary schools in Yu-Li, Taiwan, where an outbreak occurred after a typhoon hit this area in 2001, were enrolled in this study. Clinical and epidemiologic data on the infected children were collected. Pulsed-field gel electrophoresis (PFGE) was performed on the isolates to determine the genetic relatedness of outbreak strains. Plasmid analysis and PCR were performed to identify β-lactamase genes responsible for ceftriaxone resistance. Forty-seven children from the two elementary schools were culture positive for
S. sonnei
in this outbreak. Twenty-three children were asymptomatic. Of the total isolates 55.3% were resistant to ampicillin. One hundred percent of the isolates obtained from children in school A were initially susceptible to both ampicillin and ceftriaxone. Of isolates obtained from school B 96.2% were nonsusceptible to ceftriaxone. However, two isolates from school A developed resistance to ampicillin during the course of treatment. All 18 available isolates showed closely related PFGE patterns (4, 4a, 4b, and 4c). CMY-2-type AmpC β-lactamase was responsible for ceftriaxone resistance in ceftriaxone-nonsusceptible isolates; Southern blot hybridization confirmed that such a resistance gene was located on the plasmid. This is the first report of plasmid-mediated CMY-2-type AmpC β-lactamase in
S. sonnei
. Ampicillin-resistant isolates can develop during the course of antibiotic treatment.
High Frequency of Gastric Colonization with MultipleHelicobacter pyloriStrains in Venezuelan Subjects
Authors: Ghose C. , Perez-Perez G. I. , van Doorn L. J. , Domínguez-Bello M. G. , Blaser M. J.
Published: June 2005

MultipleHelicobacter pyloristrains may colonize an individual host. Using enzyme-linked immunosorbent assay and line probe assay (LiPA) techniques, we analyzed the prevalence of mixedH. pyloricolonization in 127 subjects from Venezuela, a country of highH. pyloriprevalence, from three regions representing different population groups: the Andes (Merida), where Caucasian mestizos predominate, a major city near the coast (Caracas), where Amerindian-Caucasian-African mestizos predominate, and an Amazonian community (Puerto Ayacucho), where Amerindians predominate and mestizos reflect Amerindian and Caucasian ancestry. Among 121H. pylori-positive persons, the prevalence ofcagA-positive strains varied from 50% (Merida) to 86% (Puerto Ayacucho) by LiPA. Rates of mixed colonization also varied, as assessed by LiPA of thevacA s(mean, 49%) andm(mean, 26%) regions. In total, 55% of the individuals had genotypic evidence of mixed colonization.vacA s1c, a marker of Amerindian (East Asian) origin, was present in all three populations, especially from Puerto Ayacucho (86%). These results demonstrate the high prevalence of mixed colonization and indicate that theH. pyloriEast AsianvacAgenotype has survived in all three populations tested.
Novel PCR-Restriction Fragment Length Polymorphism Method for Determining Serotypes or Serogroups of
Streptococcus pneumoniae
Isolates
Authors: Batt Sarah L. , Charalambous Bambos M. , McHugh Timothy D. , Martin Siobhan , Gillespie Stephen H.
Published: June 2005

Serotyping
Streptococcus pneumoniae
is a technique generally confined to reference laboratories, as purchasing pneumococcal antisera is a huge investment. Many attempts have been made to modify serological agglutination techniques to make them more accessible, and more recently developments in serotyping have focused on molecular techniques. This paper describes a PCR assay which amplifies the entire capsulation locus between
dexB
and
aliA
. Amplicons are digested to produce serotype-specific patterns. We have shown, using 81 epidemiologically unrelated strains representing 46 different serotypes, that the patterns correlate with a 90 to 100% similarity range for the same serotype or serogroup. Prospective testing of 73 isolates of unknown serotype confirmed reliable serotype attribution, and serotype profiles are reproducible on repeated testing. Once our database contains all 90 serotypes, this technique should be fully portable, cost-effective, and useful in any laboratory with sufficient molecular experience.
Use of the Duplex TaqMan PCR System for Detection of Shiga-Like Toxin-Producing
Escherichia coli
O157
Authors: Hsu Ching-Fang , Tsai Tsung-Yu , Pan Tzu-Ming
Published: June 2005

Real-time PCR assays have been applied for the detection and quantification of pathogens in recent years. In this study two combinations of primers and fluorescent probes were designed according to the sequences of the
rfb

Escherichia coli
O157

and
stx
2
genes. Analysis of 217 bacterial strains demonstrated that the duplex real-time PCR assay successfully distinguished the
Escherichia coli
O157 serotype from non-
E. coli
O157 serotypes and that it provided an accurate means of profiling the genes encoding O antigen and Shiga-like toxin 2. On the other hand, bacterial strains that lacked these genes were not detected by this assay. The quantitative ranges of the real-time PCR assay for these two genes were linear for DNA concentrations ranging from 10
3
to 10
9
CFU/ml of
E. coli
O157:H7 in pure culture and milk samples. The real-time PCR allowed the construction of standard curves that facilitated the quantification of
E. coli
O157:H7 in feces and apple juice samples. The detection sensitivity of the real-time PCR assay ranged from 10
4
to 10
9
CFU/g (or 10
4
to 10
9
CFU/ml) for feces and apple juice and 10
5
to 10
9
CFU/g for the beef sample without enrichment. After enrichment of the food samples in a modified tryptic soy broth, the detection range was from 10
0
to 10
3
CFU/ml. The real-time PCR assays for

rfb
E. coli

O157
and
stx
2
proved to be rapid tests for the detection of
E. coli
O157 in food matrices and could also be used for the quantification of
E. coli
O157 in foods or fecal samples.
Molecular Epidemiology of EndemicClostridium difficileInfection and the Significance of Subtypes of the United Kingdom Epidemic Strain (PCR Ribotype 1)
Authors: Fawley Warren N. , Parnell Peter , Verity Paul , Freeman Jane , Wilcox Mark H.
Published: June 2005

We previously identified two subtypes of the epidemic strainClostridium difficilePCR ribotype 1, one clindamycin-sensitive strain (arbitrarily primed PCR [AP-PCR] type Ia) and a closely related clindamycin-resistant strain (AP-PCR type Ib) in our institution. We have now carried out prospective epidemiological surveillance for 4 years, immediately following the relocation of two acute medicine wards for elderly patients (wards A and B), to determine the clinical epidemiology of subtypes of the epidemicC. difficilePCR ribotype 1 group. To maximize the chance of strain discrimination, we used three DNA fingerprinting methods, AP-PCR, ribospacer PCR (RS-PCR), and pulsed-field gel electrophoresis (PFGE), to analyzeC. difficileisolates recovered from symptomatic patients and from repeated environmental samplings. On ward B the incidence ofC. difficileinfection correlated significantly with the prevalence of environmentalC. difficileboth in ward areas closely associated with patients and health care personnel (r= 0.53;P< 0.05) and in high-reach sites (r= 0.85;P< 0.05). No such relationships were found on ward A. Seventeen distinctC. difficilegenotypes were identified, 17 by AP-PCR, 12 by PFGE, and 11 by RS-PCR, but only 4 of 17 genotypes caused patient infection. Isolates recovered from the hospital ward environment were much more diverse (14 genotypes). AP-PCR type Ia represented >90% of theC. difficileisolates. In addition to this genotype, only two others were isolated from both patient feces and environmental surfaces. AP-PCR type Ib (clindamycin-resistant PCR ribotype 1 clone) was not associated with any cases ofC. difficileinfection and was isolated from the environment on only two occasions, after having been implicated in a cluster of sixC. difficileinfections 5 months before this study. The disappearance of this strain implies that differences in virulence and/or selective pressures may exist for this strain and the closely related, widespreadC. difficileAP-PCR type Ia strain. Our findings emphasize the need to understand the epidemiology and virulence of clinically significant strains to determine successful control measures forC. difficileinfections.
Novel Approach for Assessing Performance of PCR Cyclers Used for Diagnostic Testing
Authors: Schoder D. , Schmalwieser A. , Schauberger G. , Hoorfar J. , Kuhn M. , Wagner M.
Published: June 2005

As part of a large international project for validation and standardization of PCR, the influence of thermocyclers on PCR was tested. Six brand-new, Peltier technology-driven 96-well thermocyclers were subjected to a novel and stringent in-tube (not block) physical testing. The temperature was directly monitored in PCR tubes containing 50 μl of distilled water at 13 different block positions. The certified temperature accuracy of the measurement system was ±0.3°C. Finally, the results of the physical testing were compared to those of an amplification efficiency study running an in-house PCR assay. The cyclers did not perform within the manufacturer′s specification. Premature timing, under- and overshooting, and spatial variation of heat transfer were found to be the critical factors. The physical testing allowed us to distinguish accurate from less-accurate (2/6) cyclers. The lack of thermal homogeneities became most evident at the denaturation level during the first 15 s. At the time point zero, the accurate cyclers showed temperature deviations of 0.5 to 1.5°C, whereas less-accurate cyclers failed to reach the set temperature by 13 to 20°C. Consequently, the two less-accurate cyclers could not gain positive PCR results by running an in-house PCR assay. However, by modifying the original temperature protocol by increasing the denaturation temperature and time, the amplification efficiency of these two cyclers could be improved significantly. The results have implication for laboratories using diagnostic PCR testing.
Increase in Genetic Diversity of
Haemophilus influenzae
Serotype b (Hib) Strains after Introduction of Hib Vaccination in The Netherlands
Authors: Schouls Leo M. , van der Ende Arie , van de Pol Ingrid , Schot Corrie , Spanjaard Lodewijk , Vauterin Paul , Wilderbeek Dorus , Witteveen Sandra
Published: June 2005

Recently, there has been an increase in The Netherlands in the number of cases of invasive disease caused by
Haemophilus influenzae
serotype b (Hib). To study a possible change in the Hib population that could explain the rise in incidence, a multiple-locus variable number tandem repeats analysis (MLVA) was developed to genotype
H. influenzae
isolates. The MLVA enabled the differentiation of
H. influenzae
serotype b strains with higher discriminatory power than multilocus sequence typing (MLST). MLVA profiles of noncapsulated
H. influenzae
and
H. influenzae
serotype f strains were more heterogeneous than serotype b strains and were distinct from Hib, although some overlap occurred. The MLVA was used to genotype a collection of 520
H. influenzae
serotype b strains isolated from patients in The Netherlands with invasive disease. The strains were collected from 1983 from 2002, covering a time period of 10 years before and 9 years after the introduction of the Hib vaccine in the Dutch national vaccination program. MLVA revealed a sharp increase in genetic diversity of Hib strains isolated from neonates to 4-year-old patients after 1993, when the Hib vaccine was introduced. Hib strains isolated from patients older than 4 years in age were genetically diverse, and no significant change in diversity was seen after the introduction of the vaccine. These observations suggest that after the introduction of the Hib vaccine young children no longer constitute the reservoir for Hib and that they are infected by adults carrying genetically diverse Hib strains.
Comparative Analysis of the Diagnostic Performance of Six Major
Echinococcus granulosus
Antigens Assessed in a Double-Blind, Randomized Multicenter Study
Authors: Lorenzo Carmen , Ferreira Henrique B. , Monteiro Karina M. , Rosenzvit Mara , Kamenetzky Laura , García Hector H. , Vasquez Yessika , Naquira Cesar , Sánchez Elizabeth , Lorca Myriam , Contreras María , Last Jerry A. , González-Sapienza Gualberto G.
Published: June 2005

The serodiagnosis of hydatid disease is a valuable instrument for clinical diagnosis and epidemiological surveillance of high-risk populations. In the past decade a wealth of reports on the diagnostic performance of numerous antigens have been produced. However, their diagnostic value has been estimated under different conditions, using different serum collection, therefore precluding their direct comparison. Here we report an unbiased comparison of the same batch of six major
E. granulosus
antigens, namely, hydatid cyst fluid (HCF), native antigen B (AgB), two recombinant AgB subunits, an AgB-derived synthetic peptide, and recombinant cytosolic malate dehydrogenase from
E. granulosus
(EgMDH), against the same serum collection. The double-blind analysis was performed using a standardized protocol and receiver operating characteristic (ROC) data analysis by a network of six South American laboratories. High intercenter reproducibility was attained, and the intralaboratory analysis allowed the comparative ranking of the antigen panel. HCF, AgB, and its AgB8/1 subunit exhibited equivalent diagnostic efficiencies, 81.4% ± 0.5%, 81.3% ± 0.6%, and 81.9% ± 2.0%, respectively; with a more favorable balance toward specificity in the case of the last antigen. The diagnostic efficiencies for the other three antigens were 76.8% ± 6.8%, 69.1% ± 2.7%, and 66.8% ± 2.1%, for the peptide, the AgB8/2 subunit, and the EgMDH, respectively. The study also included an analysis of batch-to-batch variation in the diagnostic performance of different HCF regional preparations. Based on these results, a suggested recommendation on the use of these antigens was drawn.
Discrimination between Primary and Secondary Dengue Virus Infection by an Immunoglobulin G Avidity Test Using a Single Acute-Phase Serum Sample
Authors: Matheus Séverine , Deparis Xavier , Labeau Bhety , Lelarge Josiane , Morvan Jacques , Dussart Philippe
Published: June 2005

For clinical and epidemiological purposes, it is necessary to be able to classify serological responses during dengue virus infection. Thus, it is important to develop a test that can distinguish between primary and secondary serological responses. The hemagglutination inhibition (HI) test, which is currently recommended by the World Health Organization, is complicated to perform. We developed an enzyme-linked immunosorbent assay based on changes in the avidity of immunoglobulin G during the infectious episode. This test can discriminate between primary and secondary infections by using a single serum sample collected during the acute phase of infection. We took 1,140 avidity measurements with 118 pairs of serum samples or sequential samples taken from patients classified as having primary or secondary infection according to World Health Organization laboratory criteria. The mean percent avidity was significantly lower during primary infection (25.9%) than during secondary infection (66.3%) (Student
t
test,
P
< 0.001). The test had a sensitivity of 82.7% (95% confidence interval [CI] = 79.0 to 86.6) and a specificity of 77.5% (95% CI = 73.3 to 81.7). Based on analysis of only blood samples collected between the third and seventh days of the illness, during which most clinical complications occur, the sensitivity and specificity of the test were 95.1% (95% CI = 92.6 to 97.7) and 80.0% (95% CI = 75.3 to 84.7), respectively. This rapid and simple test appears to be an excellent alternative to the HI test for discriminating between primary and secondary dengue virus infections during the acute phase of dengue.
Molecular Epidemiology of Community- and Health Care-Associated Methicillin-Resistant
Staphylococcus aureus
in Manitoba, Canada
Authors: Wylie John L. , Nowicki Deborah L.
Published: June 2005

Recently, acquisition of methicillin-resistant
Staphylococcus aureus
(MRSA) has been increasingly seen in community settings. Beginning in 1995, we have routinely conducted pulsed-field gel electrophoresis (PFGE) of MRSA isolates received at Cadham Provincial Laboratory (CPL) in Manitoba, Canada. Our diverse collection of isolates coupled with molecular subtype information allowed us to assess the extent to which MRSA isolates in general were associated with community acquisition and whether specific PFGE types were more likely to be found in community settings. Forty percent of the MRSA isolates in our analysis were designated community associated (CA), with two of the six most common PFGE types showing a greater likelihood to be CA-MRSA. Overall, CA-MRSA were more likely to show multiple sensitivity to antibiotics and to be associated with younger age groups. Mapping of specific CA-MRSA types over successive 5-year periods showed rapid temporal shifts in prevalence in different parts of the province.
Optimized Diagnosis of Acute Dengue Fever in Swedish Travelers by a Combination of Reverse Transcription-PCR and Immunoglobulin M Detection
Authors: Lindegren Gunnel , Vene Sirkka , Lundkvist Åke , Falk Kerstin I.
Published: June 2005

The dengue viruses (genus
Flavivirus
, family
Flaviviridae
) are mosquito borne and cause 100 million cases of dengue fever each year in most tropical and subtropical areas of the world. Increased global travel has been accompanied by an increased import not only of dengue but also of severe fevers of unknown origin to Sweden. Fifty-seven Swedish travelers to dengue epidemic areas, with clinical and serologically diagnosed dengue fever, were included in this study. To find fast and reliable methods to diagnose dengue in the early phase of the disease, patient acute-phase sera were investigated for the presence of dengue-specific immunoglobulin M (IgM) antibodies by enzyme-linked immunosorbent assay (ELISA) and also for dengue serotype (DEN-1 to DEN-4)-specific RNA by different PCR assays. The results showed that 15/20 (75%) of the samples collected 5 days or later post onset of disease, but only 5/37 (14%) of the samples collected on days 0 to 4, contained dengue-specific IgM. Of the samples collected on days 0 to 4 post onset, dengue RNAs of subtypes 1, 2, and 3 were detected by multiplex and/or by TaqMan PCR in 29/37 (78%); of these PCR-positive samples, 93% (27/29) were found IgM negative. By a combination of IgM ELISA and PCR assays, 84% (48/57) of the acute-phase samples were found to be positive. Our results demonstrated that detection of dengue viral RNA by reverse transcription-PCR and Taq-Man PCR is an excellent tool for the early diagnoses of dengue fever and that the IgM assay is a reliable complement for samples collected from day 5 post onset.
Shifts of
Bordetella pertussis
Variants in Sweden from 1970 to 2003, during Three Periods Marked by Different Vaccination Programs
Authors: Hallander Hans O. , Advani Abdolreza , Donnelly Declan , Gustafsson Lennart , Carlsson Rose-Marie
Published: June 2005

The Swedish population of
Bordetella pertussis
strains was characterized from 1,247 isolates covering a whole-cell vaccine program up to 1979, a 17-year period without vaccination (1979 to 1996), and a period after the introduction of general vaccination among newborns with acellular pertussis vaccines (1997 to 2003). Strains were characterized by serotyping and genotyping of pertactin and
ptxA
and by means of pulsed-field gel electrophoresis (PFGE). With emphasis on vaccine-related markers, the vast majority of circulating strains were of nonvaccine type. There were shifts of serotype connected with shifts of vaccination program. Serotype Fim3 was most frequent during the periods with general vaccination schedules, whereas serotype Fim2 was predominant during the 17-year vaccine-free period. Pertactin 1 was predominant during the pertussis whole-cell (Pw) vaccine period but was thereafter replaced by
prn2
and has not reappeared after the introduction of acellular pertussis (Pa) vaccines.
ptxA
(1) was predominant over all three decades. There was a significant difference in the distribution of serotypes between vaccinated and unvaccinated individuals, but not for pertactin. A few PFGE profiles were predominant over the years: BpSR25 (serotype Fim3
prn1/7
) and BpSR18 (serotype Fim3
prn2
) during the Pw period, BpSR1 (serotype Fim2
prn
2) during the 17 years without general vaccination, and BpSR11 (serotype Fim3
prn
2) after the reintroduction of general vaccination in 1996. Despite differences between the pertactin and toxin types of Pa vaccines and circulating strains, there is no evidence that there is a threat, i.e., the vaccination program so far has been effective against whooping cough, and there seems to be no impact on the effectiveness of the vaccination program from the bacterial polymorphism.