bluuebunny/crossref_metadata_2025 huggingface dataset:
New Microsatellite Multiplex PCR for
Candida albicans
Strain Typing Reveals Microevolutionary Changes
Authors: Sampaio Paula , Gusmão Leonor , Correia Alexandra , Alves Cíntia , Rodrigues Acácio G. , Pina-Vaz Cidália , Amorim António , Pais Célia
Published: August 2005

Five new microsatellite loci were described and characterized for use as molecular markers for the identification and genetic differentiation of
Candida albicans
strains. Following the typing of 72 unrelated clinical isolates, the analysis revealed that they were all polymorphic, presenting from 5 to 30 alleles and 8 to 46 different genotypes. The discriminatory power obtained by combining the information generated by three microsatellites used in a multiplex PCR amplification strategy was 0.99, the highest ever reported. The multiplex PCR was later used to test a total of 114
C. albicans
strains, including multiple isolates from the same patient collected from different body locations and along episodes of vulvovaginal infections. Three different scenarios for strain relatedness were identified: (i) different isolates that were revealed to be the same strain, (ii) isolates that were the same strain but that apparently underwent a process of microevolution, and (iii) isolates that corresponded to different strains. Analysis of the microevolutionary changes between isolates from recurrent infections indicated that the genotype alterations observed could be the result of events that lead to the loss of heterozygosity (LOH). In one case of recurrent infection, LOH was observed at the CAI locus, and this could have been related to exposure to fluconazole, since such strains were exposed to this antifungal during treatment. The analysis of microsatellites by a multiplex PCR strategy was found to be a highly efficient tool for the rapid and accurate differentiation of
C. albicans
strains and adequate for the identification of fine microevolutionary events that could be related to strain microevolution in response to environmental stress conditions.
Phylogenetic and Pathotypic Comparison of Concurrent Urine and RectalEscherichia coliIsolates from Men with Febrile Urinary Tract Infection
Authors: Johnson James R. , Scheutz Flemming , Ulleryd Peter , Kuskowski Michael A. , O'Bryan Timothy T. , Sandberg Torsten
Published: August 2005

Among men with febrile urinary tract infection (FUTI), whether the host's fecal flora is the source for the urine strain (“fecal-urethral” hypothesis), and whether pathogenesis is driven by prevalence versus special pathogenicity, are unknown. Accordingly, pretherapy urine isolates from 65 men with FUTI were compared with concurrent rectal isolates from the same hosts according to serotype, genomic profile, phylogenetic group, and virulence genotype. The host's multiple rectal colonies included only the urine clone in 25% of subjects, the urine clone plus additional clones in 22%, and only nonurine clones in 54%. Compared with the 67 unique rectal clones, the 65 urine isolates were significantly enriched for phylogenetic group B2, virulence-associated serotypes, and specific virulence genes and contained more virulence genes (median, 10 versus 6:P< 0.001). In multivariable models, phylogenetic group B2,hlyD(hemolysin),cnf1(cytotoxic necrotizing factor),iroN(siderophore receptor),ompT(outer membrane protease), andmalX(pathogenicity island marker) most strongly predicted urine source. These findings challenge the fecal-urethral and prevalence hypotheses for FUTI pathogenesis and instead strongly support the possibility of alternate infection routes in some men and the special pathogenicity hypothesis. They also identify specific bacterial traits as potential targets for anti-FUTI interventions.
Detection and Quantification of Human Immunodeficiency Virus Type 1 p24 Antigen in Dried Whole Blood and Plasma on Filter Paper Stored under Various Conditions
Authors: Li Chung-Chen , Seidel Kristy D. , Coombs Robert W. , Frenkel Lisa M.
Published: August 2005

The quantification of human immunodeficiency virus type 1 (HIV-1) by an assay measuring heat-dissociated (HD) p24 antigen (Ag) in specimens of whole blood and plasma stored on filter paper, and of plasma stored in tubes, was compared to HIV-1 RNA plasma levels determined by real-time reverse transcription (RT)-PCR. The stability of p24 Ag on filter paper under conditions simulating specimen transport was also evaluated. The HD p24 Ag in both plasma and whole-blood specimens stored on filter paper correlated with plasma HIV-1 RNA levels (Spearman rank ρ = 0.74 [
P
< 0.0001] and ρ = 0.56 [
P
= 0.0001], respectively). The sensitivity of the HD p24 Ag assay was similar when plasma and whole blood on filter paper were contrasted to the real-time RT-PCR assay (80% versus 82.5% and 78.6% versus 83.3%, respectively). However, while the specificity of the HD p24 Ag assay of plasma on filter paper was 100%, the specificity was diminished in whole-blood specimens. The storage of specimens on filter paper for 2 weeks at 37°C, 24°C, or 0°C did not alter the detection or quantification of HD p24 Ag. These results suggest that transport and storage of plasma on filter paper and quantification of HD p24 Ag may be a reliable method for HIV-1 load monitoring.
Identification of Candidate Periodontal Pathogens and Beneficial Species by Quantitative 16S Clonal Analysis
Authors: Kumar Purnima S. , Griffen Ann L. , Moeschberger Melvin L. , Leys Eugene J.
Published: August 2005

Most studies of the bacterial etiology of periodontitis have used either culture-based or targeted DNA approaches, and so it is likely that pathogens remain undiscovered. The purpose of this study was to use culture-independent, quantitative analysis of biofilms associated with chronic periodontitis and periodontal health to identify pathogens and beneficial species. Samples from subjects with periodontitis and controls were analyzed using ribosomal 16S cloning and sequencing. Several genera, many of them uncultivated, were associated with periodontitis, the most numerous of which were gram positive, including
Peptostreptococcus
and
Filifactor
. The genera
Megasphaera
and
Desulfobulbus
were elevated in periodontitis, and the levels of several species or phylotypes of
Campylobacter
,
Selenomonas
,
Deferribacteres
,
Dialister
,
Catonella
,
Tannerella
,
Streptococcus
,
Atopobium
,
Eubacterium
, and
Treponema
were elevated in disease.
Streptococcus
and
Veillonella
spp. were found in high numbers in all samples and accounted for a significantly greater fraction of the microbial community in healthy subjects than in those with periodontitis. The microbial profile of periodontal health also included the less-abundant genera
Campylobacter
,
Abiotrophia
,
Gemella
,
Capnocytophaga
, and
Neisseria
. These newly identified candidates outnumbered
Porphyromonas gingivalis
and other species previously implicated as periodontopathogens, and it is not clear if newly identified and more numerous species may play a more important role in pathogenesis. Finally, more differences were found in the bacterial profile between subjects with periodontitis and healthy subjects than between deep and shallow sites within the same subject. This suggests that chronic periodontitis is the result of a global perturbation of the oral bacterial ecology rather than a disease-site specific microbial shift.
Model for Assessment of Proficiency of Human Immunodeficiency Virus Type 1 Sequencing-Based Genotypic Antiretroviral Assays
Authors: Huang Diana D. , Bremer James W. , Brambilla Donald J. , Palumbo Paul E. ,   for the Pediatric ACTG Sequencing Working Group
Published: August 2005

Use of sequencing-based genotyping as a diagnostic assay for human immunodeficiency virus (HIV) antiretroviral resistance is increasing. Periodic evaluation of the proficiency of laboratories performing this assay should be established. It is important to identify components of the assay that influence the generation of reliable sequencing data and that should and can be monitored. A model was developed to determine what parameters were reasonable and feasible for assessing the performance of genotyping assays. Ten laboratories using the genotyping platform, HIV-1 Genotyping System (HGS) v. 1 and software versions 1.1 or 2.0, participated in two rounds of testing. For each round, each group was sent a panel consisting of three clinical samples to sequence in real time. Six months later, seven laboratories using the TRUGENE HIV-1 Genotyping Kit participated in a separate round, working with both panels at the same time. Analysis of the data showed that one main indicator of genotyping proficiency was achievement of ≥98% sequence homology of a sample tested to a group consensus sequence for that sample. A second was concordant identification of codons at sites identified with resistance mutations in the sample, although scoring of these criteria is still undetermined from this study. These criteria are applicable to all sequence-based genotyping platforms and have been used as a baseline for assessing the performance of genotyping for the determination of antiretroviral resistance in our ongoing proficiency program.
Statistical Analyses of Complex Denaturing Gradient Gel Electrophoresis Profiles
Authors: Gafan Gavin P. , Lucas Victoria S. , Roberts Graham J. , Petrie Aviva , Wilson Michael , Spratt David A.
Published: August 2005

Studies using molecular techniques have demonstrated that a culture-based approach can severely underestimate the bacterial diversity in most environments. One of the molecular techniques that has been applied in microbial ecology is denaturing gradient gel electrophoresis (DGGE). The purpose of this study was to investigate differences in the microbiota of plaque, using a number of analysis techniques, from children without gingivitis (
n
= 30) and from those with gingivitis (
n
= 30). Extracted DNA from gingival margin plaque was subjected to PCR targeting the 16S rRNA gene using universal primers. DGGE profiles were analyzed in three ways. (i) Bacterial diversity was compared between cohorts by using the Shannon-Wiener index (also known as the Shannon-Weaver index). (ii) A hierarchical cluster analysis of the banding patterns was calculated and expressed as a dendrogram. (iii) Individual DGGE bands and their intensities for both cohorts were compared using a logistic regression analysis. The Shannon-Wiener indices demonstrated a greater bacterial diversity associated with no-gingivitis plaque (
P
= 0.009). Dendrograms demonstrated that seven clades associated with gingivitis and five clades associated with no gingivitis. The logistic regression demonstrated that one band was significantly associated with no gingivitis (
P
= 0.001), while two bands were significantly associated with gingivitis (
P
= 0.005 and
P
= 0.042). In conclusion, this study demonstrates that the development of gingivitis might be accompanied by a decrease in bacterial diversity. Furthermore, we have demonstrated that logistic regression is a good statistical method for analyzing and characterizing DGGE profiles.
Comparative Sequencing of the Serine-Aspartate Repeat-Encoding Region of the Clumping Factor B Gene (
clfB
) for Resolution within Clonal Groups of
Staphylococcus aureus
Authors: Koreen Larry , Ramaswamy Srinivas V. , Naidich Steven , Koreen Irina V. , Graff Gavin R. , Graviss Edward A. , Kreiswirth Barry N.
Published: August 2005

Molecular
techniques such as
spa
typing and multilocus sequence typing
use DNA sequence data for differentiating
Staphylococcus
aureus
isolates. Although
spa
typing is capable of
detecting both genetic micro- and macrovariation, it has less
discriminatory power than the more labor-intensive pulsed-field gel
electrophoresis (PFGE) and costly genomic DNA microarray analyses. This
limitation hinders strain interrogation for newly emerging clones and
outbreak investigations in hospital or community settings where robust
clones are endemic. To overcome this constraint, we developed a typing
system using DNA sequence analysis of the serine-aspartate (SD)
repeat-encoding region within the gene encoding the
keratin- and fibrinogen-binding clumping factor B (
clfB
typing) and tested whether it is capable of discriminating within
clonal groups. We analyzed 116
S. aureus
strains, and the
repeat region was present in all isolates, varying in sequence and in
length from 420 to 804 bp. In a sample of 36 well-characterized
genetically diverse isolates,
clfB
typing subdivided identical
spa
and PFGE clusters which had been discriminated by
whole-genome DNA microarray mapping. The combination of
spa
typing and
clfB
typing resulted in a discriminatory power
(99.5%) substantially higher than that of
spa
typing alone and
closely approached that of the whole-genome microarray (100.0%).
clfB
typing also successfully resolved genetic differences
among isolates differentiated by PFGE that had been collected over
short periods of time from single hospitals and that belonged to the
most prevalent
S. aureus
clone in the United States.
clfB
typing demonstrated in vivo, in vitro, and interpatient
transmission stability yet revealed that this locus may be
recombinogenic in a primarily clonal population structure. Taken
together, these data show that the SD repeat-encoding region of
clfB
is a highly stable marker of microvariation, that in
conjunction with
spa
typing it may serve as a DNA
sequence-based alternative to PFGE for investigating genetically
similar strains, and that it is useful for analyzing collections of
isolates in both long-term population-based and local epidemiologic
studies.
Regulated Expression of the Beta2-Toxin Gene (
cpb2
) in
Clostridium perfringens
Type A Isolates from Horses with Gastrointestinal Diseases
Authors: Waters Michael , Raju Deepa , Garmory Helen S. , Popoff Michel R. , Sarker Mahfuzur R.
Published: August 2005

Recent epidemiological studies suggested that
cpb2
-positive
Clostridium perfringens
isolates are associated with gastrointestinal (GI) diseases in horses. These putative relationships, indicated by PCR genotyping, were tested in the present study by further genotyping and phenotyping of 23
cpb2
-positive
C. perfringens
isolates from horses with GI disease (referred to hereafter as horse GI disease isolates). Our beta2-toxin (CPB2) Western blot analyses demonstrated that all of the tested isolates were unable to produce detectable levels of CPB2. However, Southern blot and nucleotide sequencing analyses identified intact
cpb2
open reading frames in all of our surveyed horse GI disease isolates. Furthermore, reverse transcriptase PCR and Northern blot analyses showed that
cpb2
genes in all of our surveyed horse GI disease isolates were transcriptionally active, i.e., an ∼1.2-kb
cpb2
-specific mRNA was identified in total RNA from our surveyed isolates. The levels of
cpb2
mRNA in CWC245 (a high-CPB2-producing pig strain) and our surveyed horse GI disease isolates differed to such an extent (35-fold) that this difference could be considered as a major cause of the difference in levels of CPB2 production by CWC245 and horse GI disease isolates. This finding received further support from our observation that the complementing strain 106902(pMRS140), which produced significantly higher levels of mRNA than strain 106902, produced high levels of CPB2. Collectively, our results indicated that there is a positive correlation between
cpb2
transcription levels and the amount of CPB2 produced by a
C. perfringens
cell and that decreased transcription and/or message instability may be involved, at least in part, in the low CPB2 production noted for horse GI disease isolates in comparison to that noted for pig GI disease isolate CWC245.
Use of the Roche LightCycler Strep B Assay for Detection of Group B Streptococcus from Vaginal and Rectal Swabs
Authors: Uhl James R. , Vetter Emily A. , Boldt Kristi L. , Johnston Bruce W. , Ramin Kirk D. , Adams Marla J. , Ferrieri Patricia , Reischl Udo , Cockerill Franklin R.
Published: August 2005

The
results for a real-time PCR assay, using the LightCycler Strep B
analyte-specific reagents (Roche Diagnostics Corporation, Indianapolis,
Ind.), were compared to a direct plate method combined with a broth
enrichment culture method for detection of group B streptococcus
colonization in pregnant women. Two separate evaluations
were conducted using two different automated nucleic extraction
instruments, the MagNA Pure LC instrument (Roche Diagnostics
Corporation) and the lower-capacity MagNA Pure Compact instrument
(Roche Diagnostics Corporation). The sensitivities, specificities, and
positive and negative predictive values for the different evaluation
methods were as follow: for the LightCycler Strep B assay with MagNA
Pure LC, 100, 97, 90, and 100%, respectively; for the LightCycler Strep
B assay with MagNA Pure Compact, 92.5, 99, 97, and 97.5%, respectively.
The LightCycler Strep B assay combined with either MagNA Pure LC or
MagNA Pure Compact extraction is a suitable method for detecting group
B streptococcus colonization in pregnant women. An advantage of the
LightCycler assay over culture is the considerably reduced turnaround
time for
results.
Phenotypic and Molecular Characterization of
Candida nivariensis
sp. nov., a Possible New Opportunistic Fungus
Authors: Alcoba-Flórez Julia , Méndez-Álvarez Sebastián , Cano Josep , Guarro Josep , Pérez-Roth Eduardo , del Pilar Arévalo María
Published: August 2005

The new species
Candida nivariensis
, isolated from the clinical samples of three patients in Spain over a 3-year period, is presented here. This species can be easily differentiated from
Candida glabrata
, the closest genetic species, by different colony color on CHROMagar and by its ability to ferment trehalose. The analyses of the internal transcribed spacer region and the D1-D2 region of the 26S rRNA gene sequences support a new species designation.
Photorhabdus asymbiotica
, a Pathogen Emerging on Two Continents That Proves that There Is No Substitute for a Well-Trained Clinical Microbiologist
Authors: Weissfeld Alice S. , Halliday Rhonda J. , Simmons Dawn E. , Trevino Ernest A. , Vance Paula H. , O'Hara Caroline M. , Sowers Evangeline G. , Kern Rebecca , Koy R. Dudley , Hodde Kathy , Bing M. , Lo Carson , Gerrard John , Vohra Renu , Harper Jacqueline
Published: August 2005

A 54-year-old ranch hand presented to the emergency room with an alleged spider bite and multiple abscesses. Both wound and blood cultures grew
Photorhabdus asymbiotica
, an enteric gram-negative rod that was initially misidentified by the hospital's rapid identification system. Clinical laboratories should be aware of the limitations of their rapid identification systems and always use them as an adjunct to analysis of morphological and phenotypic traits.
Septic Arthritis Caused by Vancomycin-Intermediate
Staphylococcus aureus
Authors: Lu Jang-Jih , Lee Shih-Yi , Hwa Su-Yang , Yang An-Hang
Published: August 2005

Four methicillin-resistant
Staphylococcus aureus
isolates were consecutively isolated from synovial fluid and a knee wound of a patient with septic arthritis. Determination of the MIC of vancomycin and population analysis confirmed these isolates as vancomycin-intermediate
S. aureus
(VISA). Results of this study also revealed that cell wall thickness and reduced susceptibility to Triton X-100 were characteristic features of VISA.
Inhibitor-Based Methods for Detection of Plasmid-Mediated AmpC β-Lactamases in
Klebsiella
spp.,
Escherichia coli
, and
Proteus mirabilis
Authors: Coudron Philip E.
Published: August 2005

Non-beta-lactam inhibitor-based methods were evaluated for detecting plasmid-mediated AmpC β-lactamases in
Klebsiella
spp.,
Escherichia coli
, and
Proteus mirabilis
. Using CLSI methodology and disks containing cefotetan alone and in combination with 400 μg of boronic acid, 9 of 10 positive control strains and 54 of 55 AmpC-PCR-positive clinical isolates were detected. Importantly 71% and 40% of these clinical isolates were susceptible by routine testing to ceftriaxone and ceftazidime, respectively. Boronic acid disks also enhanced detection of expanded-spectrum β-lactamases in AmpC producers.
Evaluation of β-Lactamase Inhibitors in Disk Tests for Detection of Plasmid-Mediated AmpC β-Lactamases in Well-Characterized Clinical Strains of
Klebsiella
spp
Authors: Black Jennifer A. , Thomson Kenneth S. , Buynak John D. , Pitout Johann D. D.
Published: August 2005

The diagnostic utility of the AmpC β-lactamase inhibitors LN-2-128, 48-1220, and Syn 2190 in combination with cefotetan (CTT) or cefoxitin in a disk test for the detection of clinical isolates of
Klebsiella
spp. producing plasmid-mediated AmpC β-lactamases (pAmpCs) was evaluated. The combination of Syn 2190 and CTT had a sensitivity of 91%, a specificity of 100%, and a reproducibility of 100% and showed the best potential of using an inhibitor for detection of
Klebsiella
spp. producing pAmpCs.
Reduced Rate of Diagnostic Positive Detection of JC Virus DNA in Cerebrospinal Fluid in Cases of Suspected Progressive Multifocal Leukoencephalopathy in the Era of Potent Antiretroviral Therapy
Authors: Marzocchetti Angela , Di Giambenedetto Simona , Cingolani Antonella , Ammassari Adriana , Cauda Roberto , De Luca Andrea
Published: August 2005

Fifty-nine human immunodeficiency virus (HIV)-infected patients with suspected progressive multifocal leukoencephalopathy and 224 controls were tested for JC virus (JCV) DNA in cerebrospinal fluid by PCR. The diagnostic positive detection rate dropped from 89.5% (95% confidence intervals of 75.5 to 103.5%) in the pre-highly active antiretroviral therapy (HAART) era to 57.5% (95% confidence intervals of 42.1 to 72.9%) in the HAART era; the specificity remained unchanged. Predictors of failure to detect JCV DNA were exposure to HAART at disease onset and higher CD4 counts.
Partial Excision of the Chromosomal Cassette Containing the Methicillin Resistance Determinant Results in Methicillin-SusceptibleStaphylococcus aureus
Authors: Donnio Pierre-Yves , Oliveira Duarte C. , Faria Nuno A. , Wilhelm Nathalie , Le Coustumier Alain , de Lencastre Herminia
Published: August 2005

We report a detailed characterization of methicillin-susceptibleStaphylococcus aureusisolates from five French hospitals negative for both themecAand theccrABloci but positive for the IS431::pUB110::IS431::dcsstructure, present in someStaphylococcuscassette chromosomemec(SCCmec) types. The presence of SCCmec-associated elements suggests that this unusual resistant phenotype is due to a partial excision of SCCmecfrom epidemic methicillin-resistantS. aureus. The hypothesis of a genetic relatedness is strengthened by common sequence andspatypes and similar susceptibility patterns.
Performance of a Rapid Immunochromatographic Screening Test for Detection of Antibodies to Human Immunodeficiency Virus Type 1 (HIV-1) and HIV-2: Experience at a Tertiary Care Hospital in South India
Authors: Vijayakumar Theophilus-Sunder , David Shoba , Selvaraj Kavitha , Viswanathan Thiruppavai , Kannangai Rajesh , Sridharan Gopalan
Published: August 2005

The performance characteristics of a rapid immunochromatographic-screening test, SD Bioline HIV-1/2 3.0 (Standard Diagnostics Inc., Kyonggi-do, South Korea) on 23,754 sera and 30 plasma samples are reported. The sensitivity and specificity for the assay on serum samples are 100% and 99.4%, respectively. The assay detected antibodies in individuals infected with human immunodeficiency virus type 1 (HIV-1) genotypes A and C and HIV-2. This straightforward assay is a reliable diagnostic tool for screening HIV in resource-poor settings.
Use of the MGB Eclipse System and SmartCycler PCR for Differentiation of
Mycobacterium chelonae
and
M. abscessus
Authors: Cloud Joann L. , Hoggan Karen , Belousov Evgeniy , Cohen Samuel , Brown-Elliott Barbara A. , Mann Linda , Wilson Rebecca , Aldous Wade , Wallace Richard J. , Woods Gail L.
Published: August 2005

Although accurate in the identification of
Mycobacterium
species, partial 16S rRNA gene sequencing does not distinguish
Mycobacterium chelonae
from
M. abscessus
. Thus, we designed a SmartCycler PCR assay targeting the 16S-to-23S internal transcribed spacer (ITS) region with use of MGB Eclipse probes to distinguish each species. Comparison with PCR-restriction enzyme analysis of a 441-bp fragment of the
hsp65
gene resulted in 100% correlation with 25 isolates of
M. chelonae
and 25 isolates of
M. abscessus
. ITS PCR performed on 90 consecutive isolates identified by partial 16S rRNA gene sequencing (26 isolates of the
M. chelonae
-
M. abscessus
complex and 64 remaining isolates, including
Mycobacterium
species,
Nocardia
species, and other aerobic actinomycetes) showed 100% specificity and sensitivity. The ITS PCR assay is accurate and specific, easy to perform, and a good supplemental test when using partial 16S rRNA gene sequencing to identify
M. chelonae
and
M. abscessus
.
Variation of the Conserved Neutralizing Epitope in Influenza B Virus Victoria Group Isolates in Japan
Authors: Nakagawa Naoko , Kubota Ritsuko , Okuno Yoshinobu
Published: August 2005

For almost 20 years, the neutralizing-epitope site specific for influenza B virus Victoria group isolates was conserved at the “tip” of the hemagglutinin molecule; however, it was not detected in half of the isolates from the 2002-2003 epidemic in Japan. Amino acid substitutions (D164E or N165K) were observed at the “tip,” and the epitope was altered. The viral antigenicities were affected, and human antibodies did not substantially inhibit the hemagglutination in the hemagglutination inhibition tests. It is suspected that such variants will be important in future epidemics.
Decreased Prevalence of Virulence Factors among Ciprofloxacin-Resistant Uropathogenic
Escherichia coli
Isolates
Authors: Drews S. J. , Poutanen S. M. , Mazzulli T. , McGeer A. J. , Sarabia A. , Pong-Porter S. , Rzayev Y. , Willey B. , Green K. , Low D. E.
Published: August 2005

Ciprofloxacin resistance was identified in 18% and 6% of consecutively collected, clinically significant urinary tract isolates of
Escherichia coli
from inpatients and outpatients, respectively. In comparison to ciprofloxacin-susceptible isolates, there were fewer resistant isolates that expressed beta-hemolysis (outpatient, 9% versus 87%,
P
< 0.0001; inpatient, 4% versus 76%,
P
< 0.0001) and that had a
papEF
genotype, genes encoding P fimbriae (outpatient, 30% versus 70%,
P
= 0.0004; inpatient, 26% versus 70%,
P
< 0.0001).
Genetic Diversity of
Campylobacter
sp. Isolates from Retail Chicken Products and Humans with Gastroenteritis in Central Michigan
Authors: Fitch Brooke R. , Sachen Kacey L. , Wilder Stacey R. , Burg Matthew A. , Lacher David W. , Khalife Walid T. , Whittam Thomas S. , Young Vincent B.
Published: August 2005

Multilocus sequencing was used to compare
Campylobacter
sp. strains isolated from retail chicken products and humans with gastroenteritis in central Michigan. Sequence comparisons demonstrated overlapping diversity between chicken and human isolates.
Campylobacter jejuni
isolates from clinical sources had a greater diversity of flagellin alleles and a higher rate of quinolone resistance than isolates from retail chicken products.
Molecular Profiling and Phenotype Analysis of
Escherichia coli
O26:H11 and O26:NM: Secular and Geographic Consistency of Enterohemorrhagic and Enteropathogenic Isolates
Authors: Bielaszewska Martina , Zhang Wenlan , Tarr Phillip I. , Sonntag Anne-Katharina , Karch Helge
Published: August 2005

Fifty-eight enterohemorrhagic
Escherichia coli
O26:H11 or O26:NM (nonmotile) strains and 44 atypical enteropathogenic
E. coli
O26:H11 or O26:NM strains isolated from patients in 11 countries during 52 years share a common pool of non-
stx
virulence genes, fitness loci, and genotypic and phenotypic diagnostic markers. These findings indicate close relatedness between these pathotypes and provide a basis for their clinical laboratory diagnosis.
Monoclonal Antibody Routinely Used To Identify Avirulent Strains of Newcastle Disease Virus Binds to an Epitope at the Carboxy Terminus of the Hemagglutinin-Neuraminidase Protein and Recognizes Individual Mesogenic and Velogenic Strains
Authors: Alamares Judith G. , Li Jianrong , Iorio Ronald M.
Published: August 2005

Newcastle disease virus (NDV) strains are classified as having high (velogenic), intermediate (mesogenic), or low (lentogenic) pathogenesis and virulence in chickens. Recent studies have established that the hemagglutinin-neuraminidase (HN) protein plays an important role in viral tropism and virulence. A monoclonal antibody (AVS-I) has previously been shown to be specific for lentogenic strains of NDV (Srinivasappa et al., Avian Dis.30:562-567, 1986) and is routinely used to identify these strains. We have used competition antibody binding assays with a previously characterized panel of monoclonal antibodies, binding to chimeric HN proteins, and the characterization of an escape mutant to localize the binding site of AVS-I to the extreme carboxy terminus of the protein. In addition, we have shown that AVS-I does recognize at least one mesogenic strain and one velogenic strain of the virus, calling into question the potential of this antibody as a diagnostic reagent for avirulent NDV strains.
Evaluation of Two New Chromogenic Media, CHROMagar MRSA and S. aureus ID, for Identifying
Staphylococcus aureus
and Screening Methicillin-Resistant
S. aureus
Authors: Hedin Göran , Fang Hong
Published: August 2005

Thirty-nine methicillin-resistant
Staphylococcus aureus
(MRSA) isolates with diverse genetic backgrounds and two reference strains were correctly identified as
S. aureus
on CHROMagar MRSA and S. aureus ID media. Growth inhibition on CHROMagar MRSA was noted. A combination of cefoxitin disk and S. aureus ID was found suitable for rapid MRSA screening.
Stability of Human Immunodeficiency Virus Type 1 Proviral DNA in Whole-Blood Samples
Authors: Jennings Cheryl , Danilovic Aleksandra , Scianna Salvatore , Brambilla Donald J. , Bremer James W.
Published: August 2005

The Roche Amplicor HIV-1 Test requires that whole blood be processed within four days of collection. However, this requirement may be too limiting for use in international settings. Thus, we demonstrate that blood may be processed up to 10 days after collection if maintained under ambient conditions (2 to 25°C).
Dynamic Range and Reproducibility of Hepatitis B Virus (HBV) DNA Detection and Quantification by Cobas Taqman HBV, a Real-Time Semiautomated Assay
Authors: Lindh Magnus , Hannoun Charles
Published: August 2005

The Cobas Taqman assay for hepatitis B virus (HBV) DNA showed linear detection over 7 logs for genotypes A to D. The coefficient of variation was 1.2% at ≥1,000 IU/ml and 22.0% at 10 IU/ml. In 97 clinical samples, the log HBV DNA/ml differed by 0.11 between Cobas Amplicor and Cobas Taqman (
r
2
= 0.97).
Performance of Single-Step Gel-Based Reverse Transcription-PCR (RT-PCR) Assays Equivalent to That of Real-Time RT-PCR Assays for Detection of the Severe Acute Respiratory Syndrome-Associated Coronavirus
Authors: Inoue Masafumi , Barkham Timothy , Keong Lee Kok , Gee Lim Seng , Wanjin Hong
Published: August 2005

Simple gel-based one-step reverse transcription-PCR (RT-PCR) assays, used to investigate patients during the 2003 severe acute respiratory syndrome (SARS) outbreak in Singapore, were found to be as sensitive as commercial and in-house real-time RT-PCR assays. The detection limit was approximately 1 genome equivalent (GE) per 5 μl PCR mixture. One PFU of SARS coronavirus was estimated to be 258 ± 46 GE.
Phenotypic and Genotypic Mupirocin Resistance among Staphylococci Causing Prosthetic Joint Infection
Authors: Rotger Margalida , Trampuz Andrej , Piper Kerryl E. , Steckelberg James M. , Patel Robin
Published: August 2005

Mupirocin MICs and
mupA
presence were determined in 108 staphylococci causing prosthetic joint infection. Zero of 35 isolates (0%) of methicillin-susceptible
Staphylococcus aureus
, 4/15 (27%) methicillin-resistant
S. aureus
isolates, 3/16 (19%) methicillin-susceptible coagulase-negative staphylococci, and 11/42 (26%) methicillin-resistant coagulase-negative staphylococci were mupirocin resistant.
mupA
was detected in all five high-level mupirocin-resistant staphylococci and one mupirocin-susceptible staphylococcus.
Clindamycin-Resistant
Fusobacterium varium
Bacteremia and Decubitus Ulcer Infection
Authors: Legaria M. C. , Lumelsky G. , Rodriguez V. , Rosetti S.
Published: August 2005

Bacteremia due to
Fusobacterium
spp. is unusual (<10% of cases of anaerobic bacteremia), and the isolation of
Fusobacterium varium
is especially uncommon. The most probable sources of
Fusobacterium
bacteremia are the respiratory, the gastrointestinal, and the genitourinary tracts. A.-M. Bourgault et al. (Clin. Infect. Dis. 25[Suppl. 2]:181-183) described 40 patients with
Fusobacterium
bacteremia; only 3 had
Fusobacterium varium
, and no one had decubitus scars as the portal of entry. In another published series (S. Henry, A. De Maria, and W. R. McCabe, Am. J. Med. 75:225-231, 1983) of 26 cases, two patients had concomitant pulmonary lesions and decubitus ulcers but there was no identification to the species level mentioned. We report a case of
Fusobacterium varium
bacteremia and infected sacral decubitus ulcer in an elderly patient.
Unintentional injury depictions in popular children’s television programs
Authors: Glik D , Kinsler J , Todd W A , Clarke L , Fazio K , Miyashiro R , Perez M , Vielmetter E , Flores R C
Published: August 2005

Objectives:The frequency and magnitude of depictions of risky events and unintentional injuries in top ranked children’s animated and live action television programs was assessed.

Methods:Qualitative and quantitative media content analysis were used. Scenes from television episodes were examined for the depiction of risky events and unintentional injuries.

Results:A total of 201 actual and potential injury related events were tabulated in 99 episodes aired in 2003 and 2004. Only 26% of the risky events depicted led to an injury or physical consequence. More than 80% of the events involved behavioral or environmental risk factors. Safety factors were not present in over 90% of risky events depicted. The general tone and context of events was humor or indifference rather than concern or action, with little or no relevance to the plot.

Conclusion:Unintentional injuries are the leading cause of death and disability for children, yet animated and live action television programs marketed to children continue to portray injury risk inaccurately. Working with the children’s television production industry to improve injury depiction is a much needed advocacy activity.
People and guns involved in denied and completed handgun sales
Authors: Wright M A , Wintemute G J , Claire B E
Published: August 2005

Objective:Denial of handgun purchases by prohibited people and knowledge of the structure of gun commerce have helped to deter and prevent firearm violence. The authors hypothesize that handguns involved in a denied purchase would more closely resemble those used in crime compared with handguns sold.

Design:Cross sectional.

Setting:Denied and completed handgun sales in California, 1998–2000.

Main outcome measures:Handgun and purchaser characteristics of denied and completed sales were compared. In particular, handgun characteristics most closely associated with crime guns (type, caliber, barrel length, price) were examined.

Results:Compared with handguns sold, handguns in denied sales were somewhat more likely to be semiautomatic pistols (74.6%v69.4%), to have short barrels (25.9%v22.2%), and be of medium caliber (48.9%v37.3%). Ten percent of the handguns in denied sales and 3.4% of handguns sold were identified as inexpensive.

Conclusions:The characteristics of denied handguns are similar to those seen among crime guns. Both groups of guns may reflect the desirability for criminal purposes of pistols, which have larger ammunition capacities than other handguns, and short barrels, which increase their ability to be concealed.
Validation of parent self reported home safety practices
Authors: Robertson A S , Rivara F P , Ebel B E , Lymp J F , Christakis D A
Published: August 2005

Objectives:To evaluate the validity of parents’ self reported home safety practices concerning smoke detectors, bike helmets, car seats, and water heater temperature.

Setting:Parents of children 12 years old and under whose child had made at least one visit to a study clinic in the years 2000–2003.

Methods:As part of a randomized controlled trial to improve patient provider communication and preventive practices, parents’ responses to telephone interview were compared with observations of safety practices during a home visit. Home visits were completed within nine weeks of the telephone interview. Parents were not told that the visit was part of a validation study and home visit observers were unaware of the interview responses. The authors calculated sensitivities, specificities, positive and negative predictive values, and their corresponding confidence intervals.

Results:Sensitivity (0.78 to 0.98) and positive predictive values (0.75 to 1.00) were high for all items. Specificities and negative predictive values were more variable and the highest estimates (specificity 0.95 to 1.00, negative predictive value 0.95 to 0.97) were for car seat types.

Conclusions:The results suggest that parent self report practice of certain injury prevention behaviors (owning a car seat, hot water temperatures) is reliable, whereas self reports on other practices (working smoke detectors, properly fitting bike helmets) may be overstated.
Molecular Interactions Between Tomato and the Leaf Mold Pathogen Cladosporium fulvum
Authors: Rivas Susana , Thomas Colwyn M.
Published: August 2005

The interaction between tomato and the leaf mold pathogen Cladosporium fulvum is controlled in a gene-for-gene manner. This interaction has provided useful insights to the molecular basis of recognition specificity in plant disease resistance (R) proteins, disease resistance (R) gene evolution, R-protein mediated signaling, and cellular responses to pathogen attack. Tomato Cf genes encode type I membrane-associated receptor-like proteins (RLPs) comprised predominantly of extracellular leucine-rich repeats (eLRRs) and which are anchored in the plasma membrane. Cf proteins recognize fungal avirulence (Avr) peptides secreted into the leaf apoplast during infection. A direct interaction of Cf proteins with their cognate Avr proteins has not been demonstrated and the molecular mechanism of Avr protein perception is not known. Following ligand perception Cf proteins trigger a hypersensitive response (HR) and the arrest of pathogen development. Cf proteins lack an obvious signaling domain, suggesting that defense response activation is mediated through interactions with other partners. Avr protein perception results in the rapid accumulation of active oxygen species (AOS), changes in cellular ion fluxes, activation of protein kinase cascades, changes in gene expression and, possibly, targeted protein degradation. Here we review our current understanding of Cf-mediated responses in resistance to C. fulvum.
mTOR function in skeletal muscle hypertrophy: increased ribosomal RNA via cell cycle regulators
Authors: Nader Gustavo A. , McLoughlin Thomas J. , Esser Karyn A.
Published: August 2005

The purpose of this study was to identify the potential downstream functions associated with mammalian target of rapamycin (mTOR) signaling during myotube hypertrophy. Terminally differentiated myotubes were serum stimulated for 3, 6, 12, 24, and 48 h. This treatment resulted in significant myotube hypertrophy (protein/DNA) and increased RNA content (RNA/DNA) with no changes in DNA content or indices of cell proliferation. During myotube hypertrophy, the increase in RNA content was accompanied by an increase in tumor suppressor protein retinoblastoma (Rb) phosphorylation and a corresponding increase in the availability of the ribosomal DNA transcription factor upstream binding factor (UBF). Serum stimulation also induced an increase in cyclin D1 protein expression in the differentiated myotubes with a concomitant increase in cyclin D1-dependent cyclin-dependent kinase (CDK)-4 activity toward Rb. The increases in myotube hypertrophy and RNA content were blocked by rapamycin treatment, which also prevented the increase in cyclin D1 protein expression, CDK-4 activity, Rb phosphorylation, and the increase in UBF availability. Our findings demonstrate that activation of mTOR is necessary for myotube hypertrophy and suggest that the role of mTOR is in part to modulate cyclin D1-dependent CDK-4 activity in the regulation of Rb and ribosomal RNA synthesis. On the basis of these results, we propose that common molecular mechanisms contribute to the regulation of myotube hypertrophy and growth during the G1 phase of the cell cycle.
Potential role for mast cell tryptase in recruitment of inflammatory cells to endothelium
Authors: Meyer Maureen C. , Creer Michael H. , McHowat Jane
Published: August 2005

Recent research suggests that activation of protease-activated receptors (PARs) on the surface of endothelial and epithelial cells may play a role in general mechanisms of inflammation. We hypothesized that mast cell tryptase activation of endothelial cell PAR-2 is coupled to increased calcium-independent PLA2 (iPLA2) activity and increased platelet-activating factor (PAF) production that may play a role in inflammatory cell recruitment at sites of vascular injury. Stimulation of human coronary artery endothelial cells (HCAEC) with 20 ng/ml tryptase increased iPLA2 activity, arachidonic acid release, and PAF production. These tryptase-stimulated responses were inhibited by pretreatment with the iPLA2-selective inhibitor bromoenol lactone (BEL; 5 μM, 10 min). Similar patterns of increased iPLA2 activity and PAF production were also seen when HCAEC were treated with SLIGKV, which represents the tethered ligand sequence for the human PAR-2 once the receptor is cleaved by tryptase. Tryptase stimulation also increased cell surface expression of P-selectin, decreased electrical resistance, and increased neutrophil adherence to the endothelial cell monolayer. The tryptase-stimulated increases in both cell surface P-selectin expression and neutrophil adhesion were also inhibited with BEL pretreatment. We conclude that tryptase stimulation of HCAEC contributes importantly to early inflammatory events after vascular injury by activation of iPLA2, leading to arachidonic acid release, PAF production, cell surface P-selectin expression, and increased neutrophil adherence.
Evidence for Strong Synaptic Coupling Between Single Tactile Afferents From the Sole of the Foot and Motoneurons Supplying Leg Muscles
Authors: Fallon James B. , Bent Leah R. , McNulty Penelope A. , Macefield Vaughan G.
Published: August 2005

It has been known for some time that populations of cutaneous and muscle afferents can provide short-latency facilitation of motoneuron pools. Recently, it has been shown that the input from individual low-threshold mechanoreceptors in the glabrous skin of the hand can modulate ongoing activity in muscles acting on the fingers via spinally mediated pathways. We have extended this work to examine whether such strong synaptic coupling exists between tactile afferents in the sole of the foot and motoneurons supplying muscles that act about the ankle. We recorded from 53 low-threshold mechanoreceptors in the glabrous skin of the foot via microelectrodes inserted percutaneously into the tibial nerve of awake human subjects. Reflex modulation of ongoing whole muscle electromyography (EMG) was observed for each of the four classes of low-threshold cutaneous mechanoreceptors (17 of 21 rapidly adapting type I; 2 of 4 rapidly adapting type II; 7 of 18 slowly adapting type I; and 4 of 10 slowly adapting type II). Reflex modulation of the firing probability in single motor units (5 of 11) was also observed. These results indicate that strong synaptic coupling between tactile afferents and spinal motoneurons is not a specialization of the hand and emphasizes the potential importance of cutaneous inputs from the sole of the foot in the control of gait and posture.
Production of Recombinant Porcine Lactoferrin Exhibiting Antibacterial Activity in Methylotrophic Yeast, Pichia pastoris
Authors: Chen Hsiao-Ling , Lai Yi-Wen , Yen Chih-Ching , Lin Yuh-Yih , Lu Chien-Yu , Yang Shang-Hsun , Tsai Tung-Chou , Lin Yi-Jiun , Lin Chin-Wen , Chen Chuan-Mu
Published: August 2005

Lactoferrin is a metal-binding glycoprotein exhibiting multifunctional immunoregulation of antibacterial, antioxidant, anti-endotoxin and antiviral activities. Uptake of porcine lactoferrin (PLF) has been shown to enhance resistance to diarrhea and anemia in neonatal piglets. In this study, the methylotrophic yeast, Pichia pastoris, was used to express a recombinant PLF (rPLF) gene from swine mammary gland. A synthetic secretion cassette was constructed using the inducible promoter of the alcohol oxidase-1 gene (AOX1) and the yeast α-mating factor signal peptide. After electroporation and Zeocin selection, several clones expressed high levels of rPLF protein which constitutes more than 30% of the total protein. A time-course study showed that rPLF mRNA transcripts are stably expressed during 120 h of culture induction. rPLF was exported into the culture supernatant at approximately 87 mg/l and a large portion of rPLF was accumulated in the cell cytoplasm at approximately 760 mg/l after 72 h of methanol induction. Recombinant PLF protein was purified via a heparin column using a fast protein liquid chromatography system. The glycosylation of P. pastoris-derived rPLF was analyzed and similar patterns to milk PLF were observed. Pepsin hydrolysate of rPLF displayed high bactericidal activity against Escherichia coli ATCC 25922 under scanning electron microscopy observation and minimal inhibitory concentration and minimal bactericidal concentration tests. Our results suggested that the methylotrophic yeast-inducible system is suitable for large-scale production of active antibacterial rPLF glycoprotein.
Carbonic Anhydrases Fused to Anion Transporters of the SulP Family: Evidence for a Novel Type of Bicarbonate Transporter
Authors: Felce Jeremy , Saier Jr. Milton H.
Published: August 2005

The sulfate permease (SulP) family of secondary carriers (TC #2.A.53) includes functionally characterized members that are inorganic anion:H+ symporters and anion:anion antiporters. We here describe members of this family that are fused to non-transporter domains, a relatively rare occurrence in prokaryotes. One subfamily includes members that are either fused to or are encoded within operons that also encode homologues of carbonic anhydrases, suggesting that these carriers function to take up bicarbonate or carbonate. Within another subfamily, a SulP homologue is fused to rhodanese, a thiosulfate:cyanide sulfotransferase, suggesting that this carrier functions in sulfate uptake. Some homologues are encoded in operons that also encode putative Na+/H+ antiporters of the NhaD family (TC #2.A.62) or putative Na+:HCO3– symporters of the SBT family (TC #2.A.83). SulP homologues present in fungi and some bacteria are fused to cyclic AMP-binding domains and STAS domains that presumably function in regulation or targeting. Phylogenetic analyses reveal the relationships of these proteins and protein domains to each other and show that in some cases, but not in others, the hydrophilic domains/proteins have coevolved with the transporters.