bluuebunny/crossref_metadata_2025_split huggingface dataset:
Suspension Microarray with Dendrimer Signal Amplification Allows Direct and High-Throughput Subtyping of
Listeria monocytogenes
from Genomic DNA
Authors: Borucki Monica K. , Reynolds James , Call Douglas R. , Ward Todd J. , Page Brent , Kadushin James 
Published: July 2005

ABSTRACT

Listeria monocytogenes
is a significant cause of food-borne disease and mortality; therefore, epidemiological investigations of this pathogen require subtyping methods that are rapid, discriminatory, and reproducible. Although conventional microarray subtyping analysis has been shown to be both high resolution and genetically informative, it is still relatively low throughput and technically challenging. Suspension microarray technology eliminates the technical issues associated with planar microarrays and allows high-throughput subtyping of
L. monocytogenes
strains. In this study, a suspension array assay using dendrimer signal amplification allowed rapid and accurate serovar identification of
L. monocytogenes
strains using genomic DNA as a target. The ability to subtype genomic DNA without PCR amplification allows probes to be designed for many different regions within the bacterial genome and should allow high-resolution subtyping not possible with multiplex PCR.
Comparison between the Hybrid Capture II Test and a PCR-Based Human Papillomavirus Detection Method for Diagnosis and Posttreatment Follow-Up of Cervical Intraepithelial Neoplasia
Authors: Söderlund-Strand Anna , Rymark Per , Andersson Pia , Dillner Joakim , Dillner Lena 
Published: July 2005

ABSTRACT
Human papillomavirus (HPV) infection is the major cause of cervical cancer and its precursor, cervical intraepithelial neoplasia (CIN), and HPV testing has therefore been proposed for improved triaging and follow-up of women treated for CIN. We compared two common HPV DNA detection tests (Hybrid Capture II [HCII] and PCR-enzyme immunosorbent assay (EIA) using the primers GP5+/GP6+ followed by HPV typing with reverse dot blot hybridization) for sensitivity and specificity for detection of CIN and of CIN recurrence after treatment. Two hundred and thirty-nine women referred to the Department of Obstetrics and Gynaecology in Västerås, Sweden, were enrolled because of atypical Pap smears; 177 of these were later treated for dysplasia by conization or loop diathermy. Samples for HPV DNA testing were taken before and 4 to 6 months after treatment. There was substantial agreement between the HCII and PCR-EIA (kappa, 0.70 before treatment and 0.72 after treatment). The sensitivity for histopathologically confirmed CIN III was 100.0% for PCR-EIA and 95.6% for HCII. For patients with CIN II or worse (CIN II+), the sensitivities were 92.9% (PCR-EIA) and 91.8% (HCII). The specificities for CIN II+ in the pretreatment setting were 30.4% for PCR-EIA and 24.1% for HCII. After treatment, the sensitivities for CIN III in cytology were 100.0% by both methods, and for CIN II+, sensitivities were 80.0% by both methods. The specificities for CIN II+ in the posttreatment setting were 83.5% for PCR and 85.4% for HCII. In conclusion, the sensitivities of both PCR-EIA and HCII are high and almost equal, suggesting that both methods are suitable as tools for detection and posttreatment follow-up of CIN II-III.
Flow Cytometry-Based Assay for Titrating Dengue Virus
Authors: Lambeth C. R. , White L. J. , Johnston R. E. , de Silva A. M. 
Published: July 2005

ABSTRACTPlaque assays for titrating dengue virus (DENV) are time-consuming and not suitable for strains that do not plaque. Fluorescence-activated cell sorting (FACS) has been used to detect DENV-infected cells. Here we describe a FACS-based assay for titrating DENV. We determined that at 24 h postinfection, the number of infected cells detected by FACS represented the first round of infection and therefore could be used as a readout of the number of infectious particles in the inoculum. When the titers of different laboratory and clinical strains of DENV were compared using FACS, plaque, and endpoint dilution assays, for most strains the FACS titers were comparable to titers obtained by plaque or endpoint dilution assays. The FACS assay is an improvement over the plaque assay because the infection period is reduced from 5 to 7 days to 24 h and the assay can be used to titrate clinical isolates that frequently do not form clear plaques on cell monolayers. The novel FACS-based methods described here will facilitate laboratory studies of dengue.
Extended-Spectrum-β-Lactamase-Producing
Enterobacteriaceae
in Yaounde, Cameroon
Authors: Gangoué-Piéboji Joseph , Bedenic Branka , Koulla-Shiro Sinata , Randegger Corinne , Adiogo Dieudonné , Ngassam Pierre , Ndumbe Peter , Hächler Herbert 
Published: July 2005

ABSTRACT

Organisms producing extended-spectrum β-lactamases (ESBLs) have been reported in many countries, but there is no information on the prevalence of ESBL-producing members of the family
Enterobacteriaceae
in Cameroon. A total of 259
Enterobacteriaceae
strains were isolated between 1995 and 1998 from patients at the Yaounde Central Hospital in Cameroon. Enterobacterial isolates resistant to extended-spectrum cephalosporin and monobactam were screened for ESBL production by the double-disk (DD) synergy test. Thirty-one (12%) of these
Enterobacteriaceae
strains were shown to be positive by the DD synergy test, suggesting the presence of ESBLs. Resistance to oxyimino-cephalosporins and monobactams of 12 (38.7%) of the 31 strains—i.e., 6
Klebsiella pneumoniae
, 4
Escherichia coli
, 1
Citrobacter freundii
, and 1
Enterobacter cloacae
strain—was transferred to
E. coli
HK-225 by conjugation. Resistance to gentamicin, gentamicin plus trimethoprim-sulfamethoxazole, or trimethoprim-sulfamethoxazole was cotransferred into 6, 2, and 1 of these transconjugants, respectively. All 12 transconjugants were resistant to amoxicillin, piperacillin, all of the cephalosporins, and aztreonam but remained susceptible to cefoxitin and imipenem. Crude extracts of β-lactamase-producing transconjugants were able to reduce the diameters of inhibition zones around disks containing penicillins, narrow- to expanded-spectrum cephalosporins or monobactams when tested against a fully susceptible
E. coli
strain but had no effect on such zones around cefoxitin, imipenem, and amoxicillin-clavulanate disks. The β-lactamases produced by the 12 tranconjugants turned out to be SHV-12 by DNA sequencing. Therefore, the ESBL SHV-12 is described for the first time in Cameroon.
Comparative Assessment of the Gelatin Particle Agglutination Test and an Enzyme-Linked Immunosorbent Assay for Diagnosis of Strongyloidiasis
Authors: Sithithaworn J. , Sithithaworn P. , Janrungsopa T. , Suvatanadecha K. , Ando K. , Haswell-Elkins M. R. 
Published: July 2005

ABSTRACT

The performances of the gelatin particle agglutination test (GPAT) and enzyme-linked immunosorbent assay (ELISA) for the diagnosis of strongyloidiasis with reference to the results of the agar plate culture technique (APCT) were evaluated with samples from 459 individuals from communities in northeast Thailand where strongyloidiasis is endemic. The prevalence of strongyloidiasis in five sample groups determined by GPAT varied between 29.3 and 61.5% (mean, 38.8%). ELISA and APCT, employed concurrently, gave lower prevalence rates of 27.5% (range, 21.6 to 42.1%) and 22.7% (range, 12.7 to 53.8%), respectively. By using APCT as the standard method, the sensitivity of GPAT was generally higher than that of ELISA (81 versus 73%). The specificity of GPAT was slightly lower than that of ELISA (74 versus 86%). The resulting GPAT titers exhibited positive linear relationships with the ELISA values (optical density at 490 nm) (
P
< 0.05), which suggests that the GPAT titer also reflects the levels of specific antibody comparable to those reflected by the ELISA values. Based on the relative ease and simplicity of use of the technique as well as the acceptable rates of sensitivity and specificity of the test, GPAT is more practical for screening for strongyloidiasis than the conventional ELISA.
Countrywide Spread of Community- and Hospital-Acquired Extended-Spectrum β-Lactamase (CTX-M-15)-Producing
Enterobacteriaceae
in Lebanon
Authors: Moubareck Carole , Daoud Ziad , Hakimé Noha I. , Hamzé Monzer , Mangeney Nicole , Matta Hiam , Mokhbat Jacques E. , Rohban Raymond , Sarkis Dolla Karam , Doucet-Populaire Florence 
Published: July 2005

ABSTRACT

A prospective study was carried out to assess the extent of carriage of extended-spectrum β-lactamase (ESBL)-producing
Enterobacteriaceae
at both hospital and community levels in Lebanon. A total of 1,442 fecal samples were collected from hospital-based patients and 58 from health care workers of six Lebanese tertiary care general hospitals located in different areas of Lebanon between January and March 2003. A total of 382 fecal samples were also collected from healthy subjects between April and June 2003. The samples analysis led to the identification of 118 strains as ESBL producers based on the synergistic effects between clavulanate and selected β-lactams (ceftazidime and cefotaxime). These strains were isolated from 72 subjects: 61 patients, 2 health care workers, and 9 healthy subjects. One representative strain per subject was selected, and a total of 72 nonduplicate ESBL producers, including a high majority of
Escherichia coli
(
n
= 56),
Klebsiella pneumoniae
(
n
= 9),
Enterobacter cloacae
(
n
= 6), and
Citrobacter freundii
(
n
= 1), were characterized. The molecular analysis revealed that the majority of the strains (83%) express CTX-M-15 ESBL (pI 8.6). SHV-5a ESBL (pI 8.2) was produced by 18% of the strains. DNA macrorestriction analysis of ESBL-producing
E. coli
presented 38 different genotypes, revealing the absence of clonal link among these strains. In addition to the fact that the present study highlights the emergence and the countrywide dissemination of CTX-M-15-producing
E. coli
in Lebanon, it represents the first report of an SHV-5a-producing
C. freundii
.
Uncultivated Phylotypes and Newly Named Species Associated with Primary and Persistent Endodontic Infections
Authors: Siqueira J. F. , Rôças I. N. 
Published: July 2005

ABSTRACT

Endodontic infections have been traditionally studied by culture methods, but recent reports showing that over 50% of the oral microbiota is still uncultivable (B. J. Paster et al., J. Bacteriol. 183:3770-3783, 2001) raise the possibility that many endodontic pathogens remain unknown. This study intended to investigate the prevalence of several uncultivated oral phylotypes, as well as newly named species in primary or persistent endodontic infections associated with chronic periradicular diseases. Samples were taken from the root canals of 21 untreated teeth and 22 root-filled teeth, all of them with radiographic evidence of periradicular bone destruction. Genomic DNA was isolated directly from each sample, and 16S rRNA gene-based nested or heminested PCR assays were used to determine the presence of 13 species or phylotypes of bacteria. Species-specific primers had already been validated in the literature or were developed by aligning closely related 16S rRNA gene sequences. Species specificity for each primer pair was confirmed by running PCRs against a panel of several oral bacteria and by sequencing DNA from representative positive samples. All species or phylotypes were detected in at least one case of primary infections. The most prevalent species or phylotypes found in primary infections were
Dialister invisus
(81%),
Synergistes
oral clone BA121 (33%), and
Olsenella uli
(33%). Of the target bacteria, only these three species were detected in persistent infections. Detection of uncultivated phylotypes and newly named species in infected root canals suggests that there are previously unrecognized bacteria that may play a role in the pathogenesis of periradicular diseases.
Genetic Divergence of
Campylobacter fetus
Strains of Mammal and Reptile Origins
Authors: Tu Zheng-Chao , Eisner William , Kreiswirth Barry N. , Blaser Martin J. 
Published: July 2005

ABSTRACT

Campylobacter fetus
is a gram-negative bacterial pathogen of both humans and animals. Two subspecies have been identified,
Campylobacter fetus
subsp.
fetus
and
Campylobacter fetus
subsp.
venerealis
, and there are two serotypes, A and B. To further investigate the genetic diversity among
C. fetus
strains of different origins, subspecies, and serotypes, we performed multiple genetic analyses by utilizing random amplification of polymorphic DNA (RAPD), pulsed-field gel electrophoresis (PFGE), and DNA-DNA hybridization. All 10 primers used for the RAPD analyses can distinguish
C. fetus
strains of reptile and mammal origin, five can differentiate between
C. fetus
subsp.
fetus
and
C. fetus
subsp.
venerealis
strains, and four showed differences between type A and type B isolates from mammals. PFGE with SmaI and SalI digestion showed varied genome patterns among different
C. fetus
strains, but for mammalian
C. fetus
isolates, genome size was well conserved (mean, 1.52 ± 0.06 Mb for SmaI and 1.52 ± 0.05 Mb for SalI). DNA-DNA hybridization demonstrated substantial genomic-homology differences between strains of mammal and reptile origin. In total, these data suggest that
C. fetus
subsp
fetus
strains of reptile and mammal origin have genetic divergence more extensive than that between the two subspecies and that between the type A and type B strains. Combining these studies with sequence data, we conclude that there has been substantial genetic divergence between
Campylobacter fetus
of reptile and mammal origin. Diagnostic tools have been developed to differentiate among
C. fetus
isolates for taxonomic and epidemiologic uses.
Emergence of Virulent Methicillin-Resistant
Staphylococcus aureus
Strains Carrying Panton-Valentine Leucocidin Genes in The Netherlands
Authors: Wannet W. J. B. , Spalburg E. , Heck M. E. O. C. , Pluister G. N. , Tiemersma E. , Willems R. J. L. , Huijsdens X. W. , de Neeling A. J. , Etienne J. 
Published: July 2005

ABSTRACT

Methicillin-resistant
Staphylococcus aureus
(MRSA) strains carrying the Panton-Valentine leucocidin (PVL) genes have been reported worldwide and are a serious threat to public health. The PVL genes encode a highly potent toxin which is involved in severe skin infections and necrotizing pneumonia, even in previously healthy individuals. We assessed the prevalence of PVL-positive MRSA in The Netherlands for two periods of time: (i) 1987 through 1995 and (ii) 2000 and 2002, and determined their characteristics by using multilocus sequence typing and staphylococcal chromosome cassette (SCC
mec
) typing. It was found that up to 15% of all MRSA isolates detected in The Netherlands harbored the PVL genes. Most PVL-positive MRSA isolates were obtained from severe soft tissue infections in relatively young individuals. The first PVL-positive MRSA described in The Netherlands, isolated in 1988, was a single-locus variant of the “Berlin” epidemic MRSA clone. The 20 PVL-positive MRSA isolates studied in 2000 and 2002 consisted of five different sequence types (STs) that belonged to four clonal complexes. One of the STs, ST80, is considered to be a widespread European clone and was the most predominant ST (60%) in this study, while ST37 had never been found to be associated with PVL-positive MRSA. Most isolates harbored SCC
mec
type IV, a supposed marker for community-acquired MRSA. The number and type of virulence-associated genes varied among the different STs.
A Panton-Valentine Leucocidin (PVL)-Positive Community-Acquired Methicillin-Resistant
Staphylococcus aureus
(MRSA) Strain, Another Such Strain Carrying a Multiple-Drug Resistance Plasmid, and Other More-Typical PVL-Negative MRSA Strains Found in Japan
Authors: Takizawa Yoko , Taneike Ikue , Nakagawa Saori , Oishi Tomohiro , Nitahara Yoshiyuki , Iwakura Nobuhiro , Ozaki Kyoko , Takano Misao , Nakayama Teruko , Yamamoto Tatsuo 
Published: July 2005

ABSTRACT

Community-acquired methicillin-resistant
Staphylococcus aureus
(CA-MRSA) was collected from children with bullous impetigo in 2003 and 2004. One strain collected in 2003 was Panton-Valentine leucocidin (PVL) positive. In 2004, a multiple-drug-resistant PVL
+
CA-MRSA strain was isolated from an athlete with a cutaneous abscess. These strains were analyzed by multilocus sequence typing,
spa
typing,
agr
typing, coagulase typing, staphylococcal cassette chromosome
mec
(SCC
mec
) typing, PCR assay for 30 virulence genes, drug susceptibility testing, pulsed-field gel electrophoresis, and for plasmids. The two Japanese PVL
+
CA-MRSA strains belonged to the globally extant (“pandemic”) sequence type 30 (ST30) with SCC
mec
IV. A transmissible, multiple-drug resistance plasmid emerged in such ST30 strains. The PVL
−
CA-MRSA strains (“domestic” CA-MRSA) accumulated for bullous impetigo, exhibiting new genotypes. Hospital-acquired MRSA of ST91 (but not pandemic ST5) shared common features with the PVL
−
CA-MRSA strain.
Differential Distribution and Expression of Panton-Valentine Leucocidin among Community-Acquired Methicillin-Resistant
Staphylococcus aureus
Strains
Authors: Saïd-Salim Battouli , Mathema Barun , Braughton Kevin , Davis Stacy , Sinsimer Daniel , Eisner William , Likhoshvay Yekaterina , DeLeo Frank R. , Kreiswirth Barry N. 
Published: July 2005

ABSTRACT

Community-acquired methicillin-resistant
Staphylococcus aureus
(CA-MRSA) is an emerging threat worldwide. CA-MRSA strains differ from hospital-acquired MRSA strains in their antibiotic susceptibilities and genetic backgrounds. Using several genotyping methods, we clearly define CA-MRSA at the genetic level and demonstrate that the prototypic CA-MRSA strain, MW2, has spread as a homogeneous clonal strain family that is distinct from other CA-MRSA strains. The Panton-Valentine leucocidin (PVL)-encoding genes,
lukF
and
lukS
, are prevalent among CA-MRSA strains and have previously been associated with CA-MRSA infections. To better elucidate the role of PVL in the pathogenesis of CA-MRSA, we first analyzed the distribution and expression of PVL among different CA-MRSA strains. Our data demonstrate that PVL genes are differentially distributed among CA-MRSA strains and, when they are present, are always transcribed, albeit with strain-to-strain variability of transcript levels. To directly test whether PVL is critical for the pathogenesis of CA-MRSA, we evaluated the lysis of human polymorphonuclear leukocytes (PMNs) during phagocytic interaction with PVL-positive and PVL-negative CA-MRSA strains. Unexpectedly, there was no correlation between PVL expression and PMN lysis, suggesting that additional virulence factors underlie leukotoxicity and, thus, the pathogenesis of CA-MRSA.
Spatial Organization and Composition of the Mucosal Flora in Patients with Inflammatory Bowel Disease
Authors: Swidsinski Alexander , Weber Jutta , Loening-Baucke Vera , Hale Laura P. , Lochs Herbert 
Published: July 2005

ABSTRACT

The composition and spatial organization of the mucosal flora in biopsy specimens from patients with inflammatory bowel disease (IBD; either Crohn's disease or ulcerative colitis), self-limiting colitis, irritable-bowel syndrome (IBS), and healthy controls were investigated by using a broad range of fluorescent bacterial group-specific rRNA-targeted oligonucleotide probes. Each group included 20 subjects. Ten patients who had IBD and who were being treated with antibiotics were also studied. Use of nonaqueous Carnoy fixative to preserve the mucus layer was crucial for detection of bacteria adherent to the mucosal surface (mucosal bacteria). No biofilm was detectable in formalin-fixed biopsy specimens. Mucosal bacteria were found at concentrations greater than 10
9
/ml in 90 to 95% of IBD patients, 95% of patients with self-limiting colitis, 65% of IBS patients, and 35% of healthy controls. The mean density of the mucosal biofilm was 2 powers higher in IBD patients than in patients with IBS or controls, and bacteria were mostly adherent.
Bacteroides fragilis
was responsible for >60% of the biofilm mass in patients with IBD but for only 30% of the biofilm mass in patients with self-limiting colitis and <15% of the biofilm mass in patients with IBS. In contrast, bacteria which positively hybridized with the probe specific for
Eubacterium rectale
-
Clostridium coccoides
accounted for >40% of the biofilm in IBS patients but for <15% of the biofilm in IBD patients. In patients treated with (5-ASA) or antibiotics, the biofilm could be detected with 4,6-diamidino-2-phenylindole but did not hybridize with fluorescence in situ hybridization probes. A
Bacteroides fragilis
biofilm is the main feature of IBD. This was not previously recognized due to a lack of appropriate tissue fixation. Both 5-ASA and antibiotics suppress but do not eliminate the adherent biofilm.
Rapid Diagnosis of Bacterial Meningitis by Real-Time PCR and Fluorescence In Situ Hybridization
Authors: Poppert Sven , Essig Andreas , Stoehr Barbara , Steingruber Adelinde , Wirths Beate , Juretschko Stefan , Reischl Udo , Wellinghausen Nele 
Published: July 2005

ABSTRACT

Real-time PCR and fluorescence in situ hybridization (FISH) were evaluated as rapid methods for the diagnosis of bacterial meningitis and compared to standard diagnostic procedures. For PCR, a LightCycler approach was chosen, implementing eubacterial and specific PCR assays for the most relevant bacteria. For FISH, a similar probe set containing eubacterial and specific probes was composed of published and newly designed probes. Both methods were evaluated by use of cerebrospinal fluid (CSF) samples from patients with suspected bacterial meningitis. For all microscopy- and culture-positive samples (
n
= 28), the eubacterial PCR was positive. In addition, all identifiable pathogens were detected with specific PCR assays, according to an algorithm based on the Gram stain. The FISH method detected the pathogen in 13 of 18 positive samples. While the FISH method remained negative for all microscopy- and culture-negative samples (
n
= 113), the eubacterial PCR was positive for five of these samples. Sequencing of the amplicon revealed the presence of
Neisseria meningitidis
,
Streptococcus agalactiae
, and
Haemophilus influenzae
in three of these five samples. In addition, samples with discordant results by culture and microscopy were successfully investigated by PCR (10 samples) and FISH (5 samples). In conclusion, PCR is a highly sensitive tool for rapid diagnosis of bacterial meningitis. FISH is less sensitive but is useful for the identification of CSF samples showing bacteria in the Gram stain. Based on our results, an approach for laboratory diagnosis of meningitis including PCR and FISH is discussed.
Molecular Diversity of
Bacillus anthracis
in Italy
Authors: Fasanella Antonio , Van Ert Matthew , Altamura Settimia A. , Garofolo Giuliano , Buonavoglia Canio , Leori Guido , Huynh Lynn , Zanecki Shaylan , Keim Paul 
Published: July 2005

ABSTRACT

We used multiple-locus variable-number tandem repeat analysis (MLVA) to type 64
Bacillus anthracis
isolates from outbreaks that have occurred during the past 40 years in Italy. MLVA of the 64 isolates revealed 10 unique genotypes; 9 of these genotypes and the majority of isolates (63/64) belonged to the previously described genetic cluster A1.a. Within the A1.a isolates, two previously described genotypes (G1 and G3), which differ by a single mutation in the pX01 locus, account for the majority of isolates in the country (53/63). The low diversity of
B. anthracis
genotypes in Italy suggests a single, dominant historical introduction, followed by limited localized differentiation.
Potential Secondary Pathogenic Role for Bovine Herpesvirus 4
Authors: Donofrio Gaetano , Cavirani Sandro , van Santen Vicky , Flammini Cesidio Filippo 
Published: July 2005

ABSTRACT
Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus with no clear disease association. Previous studies have demonstrated that macrophages can harbor persistent BoHV-4. We found that the addition of prostaglandin E2 (PGE2) to bovine macrophage cells persistently infected with BoHV-4 increases viral replication. Because opportunistic infection can increase PGE2 production, we propose a link between opportunistic infection, PGE2 production, and BoHV-4 replication.