bluuebunny/crossref_metadata_2025_split huggingface dataset:
Detection, Isolation, and Molecular Subtyping of
Escherichia coli
O157:H7 and
Campylobacter jejuni
Associated with a Large Waterborne Outbreak
Authors: Bopp Dianna J. , Sauders Brian D. , Waring Alfred L. , Ackelsberg Joel , Dumas Nellie , Braun-Howland Ellen , Dziewulski David , Wallace Barbara J. , Kelly Molly , Halse Tanya , Musser Kimberlee Aruda , Smith Perry F. , Morse Dale L. , Limberger Ronald J. 
Published: January 2003

ABSTRACT

The largest reported outbreak of waterborne
Escherichia coli
O157:H7 in the United States occurred in upstate New York following a county fair in August 1999. Culture methods were used to isolate
E. coli
O157:H7 from specimens from 128 of 775 patients with suspected infections.
Campylobacter jejuni
was also isolated from stools of 44 persons who developed diarrheal illness after attending this fair. There was one case of a confirmed coinfection with
E. coli
O157:H7 and
C. jejuni
. Molecular detection of
stx
1
and
stx
2
Shiga toxin genes, immunomagnetic separation (IMS), and selective culture enrichment were utilized to detect and isolate
E. coli
O157:H7 from an unchlorinated well and its distribution points, a dry well, and a nearby septic tank. PCR for
stx
1
and
stx
2
was shown to provide a useful screen for toxin-producing
E. coli
O157:H7, and IMS subculture improved recovery. Pulsed-field gel electrophoresis (PFGE) was used to compare patient and environmental
E. coli
O157:H7 isolates. Among patient isolates, 117 of 128 (91.5%) were type 1 or 1a (three or fewer bands different). Among the water distribution system isolates, 13 of 19 (68%) were type 1 or 1a. Additionally, PFGE of
C. jejuni
isolates revealed that 29 of 35 (83%) had indistinguishable PFGE patterns. The PFGE results implicated the water distribution system as the main source of the
E. coli
O157:H7 outbreak. This investigation demonstrates the potential for outbreaks involving more than one pathogen and the importance of analyzing isolates from multiple patients and environmental samples to develop a better understanding of bacterial transmission during an outbreak.
Nested PCR Assays for Detection of
Blastomyces dermatitidis
DNA in Paraffin-Embedded Canine Tissue
Authors: Bialek Ralf , Cascante Cirera Anna , Herrmann Tanja , Aepinus Christian , Shearn-Bochsler Valerie I. , Legendre Alfred M. 
Published: January 2003

ABSTRACT

A
Blastomyces dermatitidis
nested PCR assay targeting the gene encoding the Wisconsin 1 (WI-1) adhesin was developed and compared with a nested PCR targeting the 18S rRNA gene (rDNA) of members of the family
Onygenaceae
. We examined 73 paraffin-embedded tissue samples obtained from nine dogs which died of blastomycosis and nine dogs which succumbed to lymphosarcoma according to autopsy findings; amplifiable canine DNA was extracted from 25 and 33 specimens from the two groups, respectively. The
B. dermatitidis
PCR amplified DNA from 8 of 13 tissue samples in which yeast cells were detected by microscopy. Sequencing revealed that all PCR products were homologous to the
B. dermatitidis
WI-1 adhesin gene. No PCR product was amplified from 12 microscopically negative biopsy specimens from dogs with blastomycosis or from 33 biopsy specimens from dogs with lymphosarcoma. The 18S rDNA PCR amplified DNA from 10 and 9 tissue samples taken from dogs which died of blastomycosis and lymphosarcoma, respectively. Only six products were identified as being identical to
B. dermatitidis
18S rDNA; they were exclusively obtained from specimens positive by the
B. dermatitidis
nested PCR. For specificity testing, 20 human biopsy specimens proven to have histoplasmosis were examined, and a specific
H. capsulatum
product was amplified by the 18S rDNA PCR from all specimens, whereas no product was obtained from any of the 20 samples by the
B. dermatitidis
PCR assay. In conclusion, the PCR targeting a gene encoding the unique WI-1 adhesin is as sensitive as but more specific than the PCR targeting the 18S rDNA for detection of
B. dermatitidis
in canine tissue.
Evaluation of an Enzyme Immunoassay Technique for Detection of Antibodies against
Treponema pallidum
Authors: Castro Rita , Prieto Emília S. , Santo Irene , Azevedo Jacinta , Exposto Filomena da L. 
Published: January 2003

ABSTRACT

In the present study, the performance of an enzyme-linked immunosorbent assay (ELISA) technique (Eti-syphilis-G and Eti-syphilis-M; DiaSorin) for detection of
Treponema pallidum
immunoglobulin M (IgM) and IgG antibodies for the laboratory diagnosis of syphilis was evaluated. Four hundred forty-one samples were studied. The sensitivity and specificity of the ELISA were 100 and 93%, respectively, compared with the results of a microhemagglutination assay for
Treponema pallidum
(MHA-TP) and 99.4 and 100%, respectively, compared with the results of the fluorescent treponemal antibody absorption (FTA-Abs) test. The results of the ELISA technique were concordant with those of MHA-TP for 98% of the samples tested, while the rate of concordance with the FTA-Abs test was 99.5%. The sensitivities of the rapid plasma reagin (RPR) test, MHA-TP, and the ELISA in the different phases of syphilis compared with the results of the FTA-Abs test were 92, 88, and 100%, respectively, for patients with primary syphilis; 100% for all tests evaluated for patients with secondary syphilis; 97.2, 99.4, and 100%, respectively, for patients with latent syphilis; and 57.9, 92.6, and 97.9%, respectively, for patients with past treated syphilis. The RPR test was reactive with 12 samples that were negative by all the specific tests. IgM antibodies were most frequently detected by the ELISA for IgM antibodies (32.8%) than by the FTA-Abs for IgM antibodies (28.4%). Detection of these antibodies by the FTA-Abs test and the ELISA for IgM antibodies decreased with the stage of disease (72 and 88%, respectively, for patients with primary syphilis to 17 and 19%, respectively, for patients with early latent syphilis). The high sensitivity and specificity of this ELISA technique during all stages of syphilis, together with the fact that it is a simple, objective, and easily automated method, lead us to believe that it could be used as a screening test for syphilis.
Rapid Detection of Methicillin-Resistant
Staphylococcus aureus
Directly from Sterile or Nonsterile Clinical Samples by a New Molecular Assay
Authors: Francois Patrice , Pittet Didier , Bento Manuela , Pepey Béatrice , Vaudaux Pierre , Lew Daniel , Schrenzel Jacques 
Published: January 2003

ABSTRACT

A rapid procedure was developed for detection and identification of methicillin-resistant
Staphylococcus aureus
(MRSA) directly from sterile sites or mixed flora samples (e.g., nose or inguinal swabs). After a rapid conditioning of samples, the method consists of two main steps: (i) immunomagnetic enrichment in
S. aureus
and (ii) amplification-detection profile on DNA extracts using multiplex quantitative PCR (5′-exonuclease qPCR, TaqMan). The triplex qPCR assay measures simultaneously the following targets: (i)
mecA
gene, conferring methicillin resistance, common to both
S. aureus
and
Staphylococcus epidermidis
; (ii)
femA
gene from
S. aureus
; and (iii)
femA
gene from
S. epidermidis.
This quantitative approach allows discrimination of the origin of the measured
mecA
signal. qPCR data were calibrated using two reference strains (MRSA and methicillin-resistant
S. epidermidis
) processed in parallel to clinical samples. This 96-well format assay allowed analysis of 30 swab samples per run and detection of the presence of MRSA with exquisite sensitivity compared to optimal culture-based techniques. The complete protocol may provide results in less than 6 h (while standard procedure needs 2 to 3 days), thus allowing prompt and cost-effective implementation of contact precautions.
Characterization of Heteroresistance to Fluconazole among Clinical Isolates of
Cryptococcus neoformans
Authors: Yamazumi T. , Pfaller M. A. , Messer S. A. , Houston A. K. , Boyken L. , Hollis R. J. , Furuta I. , Jones R. N. 
Published: January 2003

ABSTRACT

Strains of
Cryptococcus neoformans
expressing heteroresistance to fluconazole have been described previously. The present study was conducted to investigate the prevalence of heteroresistance among clinical isolates of
C. neoformans
and to characterize the heteroresistant phenotypes. A total of 107 clinical isolates of
C. neoformans
for which the MICs of fluconazole ranged from 0.25 to 32 μg/ml were selected. The isolates were chosen to represent a broad geographic distribution. Of the 107
C. neoformans
isolates tested, 4 grew on medium containing fluconazole at concentrations that were four to eight times higher than the MICs for each strain. A fifth isolate, for which the fluconazole MIC was 32 μg/ml, grew on agar with 64 μg of fluconazole per ml. These five isolates (4.7% of the total number) were confirmed to exhibit heteroresistant compositions by population analysis. The degree and frequency of resistance varied among the isolates. Stepwise selection by exposure to fluconazole resulted in subclones of all five strains for which the fluconazole MIC was >64 μg/ml. Subclones of three strains demonstrated a homogenous population of resistant cells on medium containing 64 μg of fluconazole/ml. The resistance was sensitive to incubation temperature, that is, heteroresistance was demonstrable only at 30°C by agar-based tests, and was reversible through serial transfers on fluconazole-free medium over a period of 8 days. These results suggest that the fluconazole-heteroresistant phenotype of
C. neoformans
exists in a significant proportion of clinical isolates and that fluconazole resistance can be developed by selection from heteroresistant clones and induction by exposure to fluconazole.
Nonencapsulated
Neisseria meningitidis
Strain Produces Amylopectin from Sucrose: Altering the Concept for Differentiation between
N. meningitidis
and
N. polysaccharea
Authors: Zhu Peixuan , Tsang Raymond S. W. , Tsai Chao-Ming 
Published: January 2003

ABSTRACT

Neisseria meningitidis
is the causative agent of meningococcal sepsis and meningitis.
Neisseria polysaccharea
is a nonpathogenic species.
N. polysaccharea
is able to use sucrose to produce amylopectin, a starch-like polysaccharide, which distinguishes it biochemically from the pathogenic species
N. meningitidis
. The data presented here indicate that this may be an insufficient criterion to distinguish between these two species. The nonencapsulated
Neisseria
strain 93246 expressed a phenotype of amylopectin production similar to that of
N. polysaccharea
. However, strain 93246 reacted with
N. meningitidis
serotype 4 and serosubtype P1.14 monoclonal antibodies and showed the
N. meningitidis
L1(8) lipo-oligosaccharide immunotype. Further analyses were performed on four genetic loci in strain 93246, and the results were compared with 7
N. meningitidis
strains, 13
N. polysaccharea
strains, and 2
N. gonorrhoeae
strains. Three genetic loci,
opcA
,
siaD
, and
lgt-1
in strain 93246, were the same as in
N. meningitidis
. Particularly, the
siaD
gene encoding polysialyltransferase responsible for biosynthesis of
N. meningitidis
group B capsule was detected in strain 93246. This
siaD
gene was inactivated by a frameshift mutation at the poly(C) tract, which makes strain 93246 identical to other nonencapsulated
N. meningitidis
strains. As expected, the
ams
gene encoding amylosucrase, responsible for production of amylopectin from sucrose, was detected in strain 93246 and all 13
N. polysaccharea
strains but not in
N. meningitidis
and
N. gonorrhoeae
strains. These data suggest that strain 93246 is nonencapsulated
N. meningitidis
but has the ability to produce extracellular amylopectin from sucrose. The gene for amylopectin production in strain 93246 was likely imported from
N. polysaccharea
by horizontal genetic exchange. Therefore, we conclude that genetic analysis is required to complement the traditional phenotypic classification for the nonencapsulated
Neisseria
strains.
Performance Evaluation of the VERSANT HCV RNA Qualitative Assay by Using Transcription-Mediated Amplification
Authors: Gorrin Gregg , Friesenhahn Michel , Lin Patsy , Sanders Marla , Pollner Reinhold , Eguchi Brandon , Pham Jimmykim , Roma Gianluca , Spidle Joseph , Nicol Susann , Wong Carol , Bhade Suvarna , Comanor Lorraine 
Published: January 2003

ABSTRACT
A preclinical evaluation of a qualitative assay for the detection of hepatitis C virus (HCV) RNA by transcription-mediated amplification (TMA) was conducted according to the guidelines of the National Committee for Clinical Laboratory Standards and the U.S. Food and Drug Administration. Our results showed that this assay, HCV TMA, detected 95% of samples with HCV RNA concentrations of 5.3 IU/ml and 29 copies/ml. HCV TMA showed an overall specificity of 99.6% and was highly reproducible, detecting 99.3% of samples with HCV RNA concentrations of 50 copies/ml across seven different lots of reagents. Experiments with clinical samples showed that HCV TMA detected all HCV genotypes with similar efficiencies, detecting ≥95% of samples at 50 HCV RNA copies/ml from patients infected with HCV genotypes 1a, 2b, 3a, 4a, 5a, and 6a. In experiments with RNA transcripts, HCV TMA detected ≥96.6% of transcripts derived from HCV genotypes 1a, 1b, 2a, 2c, 3a, 4a, 5a, and 6a at 50 HCV RNA copies/ml. Detection of transcripts derived from HCV genotype 2b was slightly lower (88.4%) at 50 copies/ml but was 97.0% at 75 copies/ml. In addition, HCV TMA exhibited robust performance in detecting HCV RNA in samples subjected to various conditions commonly encountered in a clinical laboratory, including long-term storage, multiple freeze-thaw cycles, different collection tubes, and the presence of endogenous substances, commonly prescribed drugs, or other microorganisms and viruses. With its high sensitivity, specificity, reproducibility, and equivalent genotype reactivity, HCV TMA may provide an attractive alternative for routine qualitative HCV RNA testing in clinical laboratories.
Rapid Enterovirus RNA Detection in Clinical Specimens by Using Nucleic Acid Sequence-Based Amplification
Authors: Landry Marie L. , Garner Robin , Ferguson David 
Published: January 2003

ABSTRACT
Enterovirus (EV) detection by nucleic acid sequence-based amplification was compared with EV isolation in cell culture. The NucliSens Basic kit (bioMerieux) was utilized for RNA detection. For virus isolation, samples were inoculated into MRC-5, primary rhesus monkey kidney, A549, rhabdomyosarcoma, and/or Buffalo green monkey kidney cells.
Detection of North American Eastern and Western Equine Encephalitis Viruses by Nucleic Acid Amplification Assays
Authors: Lambert Amy J. , Martin Denise A. , Lanciotti Robert S. 
Published: January 2003

ABSTRACT
We have developed nucleic acid sequence-based amplification (NASBA), standard reverse transcription PCR (RT-PCR), and TaqMan nucleic acid amplification assays for the rapid detection of North American eastern equine encephalitis (EEE) and western equine encephalitis (WEE) viral RNAs from samples collected in the field and clinical samples. The sensitivities of these assays have been compared to that of virus isolation. While all three types of nucleic acid amplification assays provide rapid detection of viral RNAs comparable to the isolation of viruses in Vero cells, the TaqMan assays for North American EEE and WEE viral RNAs are the most sensitive. We have shown these assays to be specific for North American EEE and WEE viral RNAs by testing geographically and temporally distinct strains of EEE and WEE viruses along with a battery of related and unrelated arthropodborne viruses. In addition, all three types of nucleic acid amplification assays have been used to detect North American EEE and WEE viral RNAs from mosquito and vertebrate tissue samples. The sensitivity, specificity, and rapidity of nucleic acid amplification demonstrate the usefulness of NASBA, standard RT-PCR, and TaqMan assays, in both research and diagnostic settings, to detect North American EEE and WEE viral RNAs.
Stability of Serotypes during Nasopharyngeal Carriage of
Streptococcus pneumoniae
Authors: Meats Emma , Brueggemann Angela B. , Enright Mark C. , Sleeman Karen , Griffiths David T. , Crook Derrick W. , Spratt Brian G. 
Published: January 2003

ABSTRACT

Serotype changes among natural isolates of
Streptococcus pneumoniae
are well documented and occur by recombinational exchanges at the capsular biosynthetic locus. However, the frequency with which this phenomenon occurs within the nasopharynx of children is not clear and is likely to be highest in the nasopharynx of children, who have high rates of pneumococcal carriage. A birth cohort of 100 infants was studied, and pneumococci were recovered from nasopharyngeal samples taken at monthly intervals during the first 6 months of life and then at 2-monthly intervals until the age of 2 years. Among the 1,353 nasopharyngeal samples were 523 that contained presumptive pneumococci, and three colonies from each were serotyped. A total of 333 isolates, including all isolates of differing serotypes from the same child, were characterized by multilocus sequence typing. Sixty-eight children carried multiple serotypes during the first 2 years of life. Two children carried a typeable and a nonserotypeable pneumococcus of identical genotype, and five children carried genetically indistinguishable isolates of serotypes 15B and 15C. These isolates were considered, respectively, to be due to loss of capsule expression and the known ability of serotype 15B and 15C pneumococci to interconvert by loss or gain of an acetyl group on the capsular polysaccharide. In all other cases, isolates from the same children that differed in serotype also differed in genotype, indicating the acquisition of a different pneumococcal strain rather than a change in capsular type. There was therefore no evidence in this study for any change of serotype due to recombinational replacements at the capsular locus among the pneumococci carried within the nasopharynges of the children.
Real-Time PCR Assay for Rapid and Accurate Detection of Point Mutations Conferring Resistance to Clarithromycin in
Helicobacter pylori
Authors: Oleastro Monica , Ménard Armelle , Santos Adriana , Lamouliatte Hervé , Monteiro Lurdes , Barthélémy Philippe , Mégraud Francis 
Published: January 2003

ABSTRACT

The main cause of failure of
Helicobacter pylori
eradication therapy is resistance to clarithromycin. The resistance is due to three point mutations in two positions on the 23S rRNA (A2142C, A2142G, and A2143G). Our aim was to develop a rapid and accurate method to detect these mutations directly on biopsy specimens. We developed a real-time PCR that included a simultaneous detection of the amplicons by hybridization of two probes labeled with LC-Red and fluorescein by using the fluorescence resonance energy transfer (FRET) technology and melting curve analysis with the LightCycler thermocycler. The assay was first applied successfully on reference strains, reference plasmids, and
H. pylori
-negative biopsies. Biopsies from 200 patients having failed a first eradication attempt and for whom the
H. pylori
strain was available were then tested with the new assay. A result was obtained in 199 cases; a single genotype was detected in 157 cases, two genotypes were detected in 41 cases, and three genotypes were detected in one case. There were, in total, seven discrepancies between the real-time PCR and the phenotypic method of determination of clarithromycin susceptibility, and in an additional four cases the two phenotypic methods were in disagreement. PCR-restriction fragment length polymorphism was applied to a sampling of biopsies, including all of the cases with multiple genotypes and all the cases with discrepant results. Finally, in four cases with discrepant results, the real-time PCR detected the resistant population at a concentration so low that it could not be detected by the phenotypic method, while in three cases other mutations could be involved. This assay had an accuracy at least as satisfactory as that of the phenotypic tests and could be performed within 2 h, allowing it to be used before the administration of therapy in the case of a first
H. pylori
eradication.
Determination of Immunoglobulin A against
Gardnerella vaginalis
Hemolysin, Sialidase, and Prolidase Activities in Vaginal Fluid: Implications for Adverse Pregnancy Outcomes
Authors: Cauci Sabina , Thorsen Poul , Schendel Diana E. , Bremmelgaard Annie , Quadrifoglio Franco , Guaschino Secondo 
Published: January 2003

ABSTRACT

A nested case-control study of low birth weight and preterm delivery was performed with singleton women. Immunoglobulin A (IgA) against the
Gardnerella vaginalis
hemolysin (anti-Gvh IgA) and sialidase and prolidase activities were determined in vaginal fluid at 17 weeks of gestation. Sialidase positivity and bacterial vaginosis with high prolidase activity were associated with 2- and 11-fold increased risks for low birth weight, respectively. No woman with bacterial vaginosis plus a strong anti-Gvh IgA response had an adverse outcome.
Use of Real-Time PCR To Detect and QuantifyMycoplasma haemofelisand “CandidatusMycoplasma haemominutum” DNA
Authors: Tasker Séverine , Helps Chris R. , Day Michael J. , Gruffydd-Jones Tim J. , Harbour Dave A. 
Published: January 2003

ABSTRACTA real-time PCR assay using Taqman probes was developed to detect and quantifyMycoplasma haemofelisand “CandidatusMycoplasma haemominutum” in feline blood samples. The assay was rapid and sensitive and was successfully used to monitor the in vivo kinetics of cats experimentally infected with each species.
Sequence Polymorphism of the 16S rRNA Gene of
Vibrio vulnificus
Is a Possible Indicator of Strain Virulence
Authors: Nilsson William B. , Paranjype Rohinee N. , DePaola Angelo , Strom Mark S. 
Published: January 2003

ABSTRACT

Vibrio vulnificus
exhibits considerable strain-to-strain variation in virulence. Attempts to associate phenotypic or genotypic characteristics with strain virulence have been largely unsuccessful. Based on a 17-nucleotide difference throughout the sequence of the small subunit 16S rRNA gene, there are two major groups of
V. vulnificus
designated types A and B. In a survey of the 16S rRNA genotype in 67
V. vulnificus
human clinical and nonclinical strains, we determined that the majority of nonclinical isolates are type A (31 of 33) and that there is a statistically significant association between the type B genotype and human clinical strains (26 of 34).
Identification of
Balamuthia mandrillaris
by PCR Assay Using the Mitochondrial 16S rRNA Gene as a Target
Authors: Booton Gregory C. , Carmichael Jennifer R. , Visvesvara Govinda S. , Byers Thomas J. , Fuerst Paul A. 
Published: January 2003

ABSTRACT

Balamuthia mandrillaris
is an opportunistic pathogen that causes granulomatous amebic meningoencephalitis in animals, including humans. Based on sequence analysis of mitochondrial small-subunit-rRNA genes, we developed primers that amplify a
Balamuthia
-specific PCR product. These primers will be useful for retrospective analyses of fixed tissues and possible identification of
Balamuthia
in vivo.
Evaluation of New Medium for Identification of Dermatophytes and Primary Dimorphic Pathogens
Authors: Gromadzki Sally , Ramani Rama , Chaturvedi Vishnu 
Published: January 2003

ABSTRACT
Only 25 of 77 dermatophytic isolates caused dermatophyte identification medium (DIM) to turn purple after incubation at the recommended temperature (37°C); the accuracy of the results was improved at 30°C (71 of 77 isolates yielded positive results). Many dimorphic pathogenic fungi also tested positive at both incubation temperatures. Thus, DIM has limited usefulness for presumptive identification of dermatophytes.