bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular Analysis of a Hospital Cafeteria-Associated Salmonellosis Outbreak Using Modified Repetitive Element PCR Fingerprinting
Authors: Johnson James R. , Clabots Connie , Azar Miguel , Boxrud David J. , Besser John M. , Thurn Joseph R. 
Published: July 2002

ABSTRACT

A hospital cafeteria-associated outbreak of gastroenteritis due to
Salmonella enterica
serotype Infantis was retrospectively evaluated using modified repetitive element PCR (rep-PCR) fingerprinting with the ERIC2 and BOXA1R primers and computer-assisted gel analysis and dendrogram construction. Rep-PCR yielded objective between-cycler, same-strain similarity values of from 92% (composite fingerprints) to 96% (ERIC2 fingerprints). The 70
Salmonella
isolates (which included 19 serotype Infantis isolates from the hospital outbreak, 10 other serotype Infantis isolates, and 41 isolates representing 14 other serotypes) were resolved well to the serotype level with each of the three fingerprint types (ERIC2, BOXA1R, and composite). Rep-PCR typing uncovered several historical serotyping errors and provided presumptive serotype assignments for other isolates with incomplete or undetermined serotypes. Analysis of replicate fingerprints for each isolate, as generated on two different thermal cyclers, indicated that most of the seeming subserotype discrimination noted in single-cycler dendrograms actually represented assay variability, since it was not reproducible in combined-cycler dendrograms. Rep-PCR typing, which would have been able to identify the presence of the hospital-associated serotype Infantis outbreak after the second outbreak isolate, could be used as a simple surrogate for serotyping by clinical microbiology laboratories that are equipped for diagnostic PCR.
Quantitative, Fluorogenic Probe PCR Assay for Detection of Human Herpesvirus 8 DNA in Clinical Specimens
Authors: Stamey Felicia R. , Patel Mitesh M. , Holloway Brian P. , Pellett Philip E. 
Published: July 2002

ABSTRACT
A quantitative, fluorescence-based PCR assay (TaqMan-based system) was developed for detection of human herpesvirus 8 (HHV-8) DNA in clinical specimens. Primers and probes chosen from each of five 10-kb segments from the unique region of the HHV-8 genome were evaluated for sensitivity with dilution series of DNA extracted from a cell line (BCBL-1) that harbors HHV-8 DNA. Although several of the primer-probe sets performed similarly with BCBL-1 DNA that had been diluted in water, their performance differed when target DNA was diluted in a constant background of uninfected cell DNA, an environment more relevant to their intended use. The two best primer-probe combinations were specific for HHV-8 relative to the other known human herpesviruses and herpesvirus saimiri, a closely related gammaherpesvirus of nonhuman primates. PCRs included an enzymatic digestion step to eliminate PCR carryover and an exogenous internal positive control that enabled discrimination of false-negative from true-negative reactions. The new assays were compared to conventional PCR assays for clinical specimens (saliva, rectal brushings, rectal swab specimens, peripheral blood lymphocytes, semen, and urine) from human immunodeficiency virus-positive patients with or without Kaposi's sarcoma. In all instances, the new assays agreed with each other and with the conventional PCR system. In addition, the quantitative results obtained with the new assays were in good agreement both for duplicate reactions in the same assay and between assays.
Ultrastructural Study ofChlamydia pneumoniaeIn a Continuous-Infection Model
Authors: Kutlin Andrei , Flegg Cameron , Stenzel Deb , Reznik Tamara , Roblin Patricia M. , Mathews Sarah , Timms Peter , Hammerschlag Margaret R. 
Published: July 2002

ABSTRACTWe have established an in vitro model of long-term continuousChlamydia pneumoniaeinfection in HEp-2 cells. Using transmission electron microscopy, we demonstrated the presence of spontaneous abnormal chlamydial inclusions similar in appearance to the persistent chlamydial forms induced in vitro by treatment with cytokines or antibiotics or by nutrient deprivation.
Multiplex PCR for Simultaneous Identification of
Staphylococcus aureus
and Detection of Methicillin and Mupirocin Resistance
Authors: Pérez-Roth E. , Claverie-Martı́n F. , Villar J. , Méndez-Álvarez S. 
Published: July 2002

ABSTRACT

In this work, we describe a multiplex PCR assay for the detection of clinically relevant antibiotic resistance genes harbored by some
Staphylococcus aureus
isolates and for the simultaneous identification of such isolates at the species level. Conditions were optimized for the simultaneous detection of the 310-, 456-, and 651-bp regions of the
mecA
(encoding high-level methicillin resistance),
ileS
-
2
(encoding high-level mupirocin resistance), and
femB
(encoding a factor essential for methicillin resistance) genes, respectively, from a single colony in a single reaction tube. The
femB
PCR fragment allows the specific identification of
S
.
aureus
. Validation of the method was performed using 50 human isolates of methicillin-resistant
S
.
aureus
(MRSA) and the appropriate control strains. This assay offers a rapid, simple, feasible, specific, sensitive, and accurate identification of mupirocin-resistant MRSA clinical isolates and could be systematically applied as a diagnostic test in clinical microbiology laboratories, facilitating the design and use of antibiotic therapy.
Typing and Subtyping Influenza Virus Using DNA Microarrays and Multiplex Reverse Transcriptase PCR
Authors: Li Jiping , Chen Shu , Evans David H. 
Published: July 2002

ABSTRACTA model DNA microarray has been prepared and shown to facilitate typing and subtyping of human influenza A and B viruses. Reverse transcriptase PCR was used to prepare cDNAs encoding ∼500-bp influenza virus gene fragments, which were then cloned, sequenced, reamplified, and spotted to form a glass-bound microarray. These target DNAs included multiple fragments of the hemagglutinin, neuraminidase, and matrix protein genes. Cy3- or Cy5-labeled fluorescent probes were then hybridized to these target DNAs, and the arrays were scanned to determine the probe binding site(s). The hybridization pattern agreed perfectly with the known grid location of each target, and the signal-to-background ratio varied from 5 to 30. No cross-hybridization could be detected beyond that expected from the limited degree of sequence overlap between different probes and targets. At least 100 to 150 bp of homology was required for hybridization under the conditions used in this study. Combinations of Cy3- and Cy5-labeled DNAs can also be hybridized to the same chip, permitting further differentiation of amplified molecules in complex mixtures. In a more realistic test of the technology, several sets of multiplex PCR primers that collectively target influenza A and B virus strains were identified and were used to type and subtype several previously unsequenced influenza virus isolates. The results show that DNA microarray technology provides a useful supplement to PCR-based diagnostic methods.
Molecular Detection of
Mycoplasma pneumoniae
in Adults with Community-Acquired Pneumonia Requiring Hospitalization
Authors: Dorigo-Zetsma J. Wendelien , Verkooyen Roel P. , van Helden H. Pieter , van der Nat Hans , van den Bosch Jules M. 
Published: July 2002

ABSTRACT

Mycoplasma pneumoniae
infection was diagnosed in 18 (12.5%) of 144 adults hospitalized with community-acquired pneumonia. The infection was demonstrated by PCR in 15 patients and by serology, using two methods, in 10 patients. The mean age of the 8 patients with positive
M. pneumoniae
PCR and negative serology was significantly higher than that of the 10 patients with positive serology.
Evaluation of a Group-Specific 16S Ribosomal DNA-Based PCR for Detection of
Helicobacter bizzozeronii, Helicobacter felis
, and
Helicobacter salomonis
in Fresh and Paraffin-Embedded Gastric Biopsy Specimens
Authors: De Groote Dominic , Haesebrouck Freddy , van Doorn Leen-Jan , Vandamme Peter , Ducatelle Richard 
Published: July 2002

ABSTRACT

A new specific and sensitive 16S ribosomal DNA-based PCR assay was developed. The assay targets a 78-bp DNA fragment unique to
Helicobacter bizzozeronii, Helicobacter felis
, and
Helicobacter salomonis
and can be used with freshly frozen and formalin-fixed paraffin-embedded gastric biopsy specimens.
Molecular Evidence ofBartonellaspp. in Questing AdultIxodes pacificusTicks in California
Authors: Chang C. C. , Chomel B. B. , Kasten R. W. , Romano V. , Tietze N. 
Published: July 2002

ABSTRACTTicks are the vectors of many zoonotic diseases in the United States, including Lyme disease, human monocytic and granulocytic ehrlichioses, and Rocky Mountain spotted fever. Most knownBartonellaspecies are arthropod borne. Therefore, it is important to determine if someBartonellaspecies, which are emerging pathogens, could be carried or transmitted by ticks. In this study, adultIxodes pacificusticks were collected by flagging vegetation in three sites in Santa Clara County, Calif. PCR-restriction fragment length polymorphism and partial sequencing of 273 bp of thegltAgene were applied forBartonellaidentification. Twenty-nine (19.2%) of 151 individually tested ticks were PCR positive forBartonella. Male ticks were more likely to be infected withBartonellathan female ticks (26 versus 12%,P= 0.05). None of the nine ticks collected at Baird Ranch was PCR positive forBartonella. However, 7 (50%) of 14 ticks from Red Fern Ranch and 22 (17%) of 128 ticks from the Windy Hill Open Space Reserve were infected withBartonella. In these infected ticks, molecular analysis showed a variety ofBartonellastrains, which were closely related to a cattleBartonellastrain and to several known human-pathogenicBartonellaspecies and subspecies:Bartonella henselae, B. quintana, B. washoensis,andB. vinsoniisubsp.berkhoffii. These findings indicate thatI. pacificusticks may play an important role inBartonellatransmission among animals and humans.
Comparison of Nucleic Acid Amplification, Serology, and Microbiologic Culture for Diagnosis ofRhodococcus equiPneumonia in Foals
Authors: Sellon Debra C. , Besser Thomas E. , Vivrette Sally L. , McConnico Rebecca S. 
Published: July 2002

ABSTRACTRecently, a technique was described for amplification ofRhodococcus equi-specific chromosomal andvapADNA from blood and tracheal wash fluids. It was hypothesized that this technique would be more sensitive than standard culture techniques or serology for diagnosis ofR. equipneumonia in foals. Tracheal wash fluid, nasal swabs, whole blood samples, and serum samples from 56 foals with pneumonia were analyzed. Final clinical diagnosis was determined by the attending clinician on the basis of final interpretation of all available information about each foal, including clinical presentation, diagnostic test results, response to therapy, and outcome. Clinical diagnosis was used as a final reference standard for calculation of sensitivity, specificity, and predictive values for PCR, serology using an agar gel immunodiffusion test, and tracheal wash fluid culture. PCR of tracheal wash fluid using primers that recognized thevapAvirulence plasmid ofR. equihad a diagnostic sensitivity of 100% and specificity of 90.6%. Sensitivity and specificity were 57.1 and 93.8%, respectively, for standard microbiologic culture of tracheal wash fluid and 62.5 and 75.9%, respectively, for serology. PCR of tracheal wash fluid is more sensitive and specific for diagnosis ofR. equipneumonia than are other available diagnostic tests.
Type-Specific Detection of Echovirus 30 Isolates Using Degenerate Reverse Transcriptase PCR Primers
Authors: Kilpatrick David R. , Quay Jacqueline , Pallansch Mark A. , Oberste M. Steven 
Published: July 2002

ABSTRACT
Following an approach used to specifically identify polioviruses and enterovirus 71, we have developed reverse transcriptase (RT) PCR primers containing mixed-base residues or deoxyinosine at positions of codon degeneracy. These primers permit specific RT-PCR amplification of echovirus 30 (E30) sequences by targeting sites that encode conserved amino acid motifs within the major capsid protein, VP1. All 221 E30 strains tested, isolated in 16 countries over a 44-year period, yielded the predicted 158-bp PCR product. No specific products were obtained by PCR assays containing templates from any of the other 63 EV serotypes. Inosine-containing degenerate primers may be widely applicable to the identification of echovirus serotypes by PCR.
Outer Membrane Proteins and DNA Profiles in Strains ofHaemophilus parasuisRecovered from Systemic and Respiratory Sites
Authors: Ruiz Alvaro , Oliveira Simone , Torremorell Montserrat , Pijoan Carlos 
Published: July 2002

ABSTRACTPolyserositis caused byHaemophilus parasuisis an important disease that affects mostly weaned pigs. Recent studies have shown that virulence can differ among strains recovered from distinct body sites and also that it may be related to the presence of certain outer membrane proteins (OMPs). The objective of this study was to compare the OMP and DNA profiles ofH. parasuisstrains isolated from systemic and respiratory sites from diseased and healthy pigs. Strains evaluated in this study were processed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and repetitive-PCR techniques. Two experiments were conducted in order to better define the relationship among genotype, phenotype, and site of isolation. Experiment 1 included 53H. parasuisisolates recovered from healthy and diseased pigs from unrelated herds. Experiment 2 included 31 isolates ofH. parasuisobtained from diseased pigs involved in an outbreak in a large, multifarm system. Results showed that strains recovered from systemic sites had more homogeneous OMP and DNA profiles than those isolated from respiratory sites. Evaluation of isolates involved in the multifarm outbreak showed that only twoH. parasuisstrains were causing disease. These strains had homogeneous OMP and DNA profiles. However, it was noted that these two parameters were unrelated, since strains classified in the same genotype group expressed different OMP profiles. The homogeneity of OMP and DNA profiles of strains isolated from systemic sites strongly suggests the existence of clonal relationships between virulent strains and also suggests that expression of certain OMP profiles may be related to virulence.
Use of Subgenic 18S Ribosomal DNA PCR and Sequencing for Genus and Genotype Identification of Acanthamoebae from Humans with Keratitis and from Sewage Sludge
Authors: Schroeder Jill M. , Booton Gregory C. , Hay John , Niszl Ingrid A. , Seal David V. , Markus Miles B. , Fuerst Paul A. , Byers Thomas J. 
Published: July 2002

ABSTRACT

This study identified subgenic PCR amplimers from 18S rDNA that were (i) highly specific for the genus
Acanthamoeba
, (ii) obtainable from all known genotypes, and (iii) useful for identification of individual genotypes. A 423- to 551-bp
Acanthamoeba
-specific amplimer ASA.S1 obtained with primers JDP1 and JDP2 was the most reliable for purposes i and ii. A variable region within this amplimer also identified genotype clusters, but purpose iii was best achieved with sequencing of the genotype-specific amplimer GTSA.B1. Because this amplimer could be obtained from any eukaryote, axenic
Acanthamoeba
cultures were required for its study. GTSA.B1, produced with primers CRN5 and 1137, extended between reference bp 1 and 1475. Genotypic identification relied on three segments: bp 178 to 355, 705 to 926, and 1175 to 1379. ASA.S1 was obtained from single amoeba, from cultures of all known 18S rDNA genotypes, and from corneal scrapings of Scottish patients with suspected
Acanthamoeba
keratitis (AK). The AK PCR findings were consistent with culture results for 11 of 15 culture-positive specimens and detected
Acanthamoeba
in one of nine culture-negative specimens. ASA.S1 sequences were examined for 6 of the 11 culture-positive isolates and were most closely associated with genotypic cluster T3-T4-T11. A similar distance analysis using GTSA.B1 sequences identified nine South African AK-associated isolates as genotype T4 and three isolates from sewage sludge as genotype T5. Our results demonstrate the usefulness of 18S ribosomal DNA PCR amplimers ASA.S1 and GTSA.B1 for
Acanthamoeba
-specific detection and reliable genotyping, respectively, and provide further evidence that T4 is the predominant genotype in AK.
Evaluation of PCR Using TRC
4
and IS
6110
Primers in Detection of Tuberculous Meningitis
Authors: Narayanan Sujatha , Parandaman Vijayalakshmi , Narayanan P. R. , Venkatesan P. , Girish C. , Mahadevan S. , Rajajee Sarala 
Published: July 2002

ABSTRACT

We have evaluated a new set of primers (TRC
4
) in comparison with the IS
6110
primers commonly used in PCR to detect tuberculous meningitis among children. The levels of concordance between the results of IS
6110
PCR and TRC
4
PCR with cerebrospinal fluid specimens from patients with clinically confirmed tuberculous meningitis were 80 and 86%, respectively. Results with the two primer sets were concordant for 55 positive and 22 negative specimens (
n
= 98). We conclude that the sensitivity of PCR can be increased by using both IS
6110
and TRC
4
primers.
Rapid Receptor-Clustering Assay To Detect Uropathogenic and Diarrheal
Escherichia coli
Isolates Bearing Adhesins of the Dr Family
Authors: Goluszko P. , Selvarangan R. , Nowicki B. J. , Nowicki S. , Hart A. , Pawelczyk E. , Nguyen K. 
Published: July 2002

ABSTRACT

Infections caused by
Escherichia coli
isolates expressing adhesins of the Dr family are associated with diarrhea and urinary tract infections, and these
E. coli
strains recognize the complement regulatory protein decay-accelerating factor (DAF) as their receptor. Clustering of the DAF receptor at the sites of bacterial adherence to epithelial cells is proposed as an alternative to PCR assay for rapid detection of Dr-positive
E. coli
.
Standardization of Antifungal Susceptibility Variables for a Semiautomated Methodology
Authors: Rodrı́guez-Tudela Juan L. , Cuenca-Estrella Manuel , Dı́az-Guerra Teresa M. , Mellado Emilia 
Published: July 2002

ABSTRACT

Recently, the methodology that will serve as a basis of the standard for antifungal susceptibility testing of fermentative yeasts of the European Committee on Antibiotic Susceptibility Testing has been described. This procedure employs a spectrophotometric method for both inoculum adjustment and endpoint determination. However, the utilization of a spectrophotometer requires studies for standardization. The present work analyzes the following parameters: (i) accuracy of inoculum preparation, (ii) correlation between optical density and CFU per milliliter, (iii) influence of the wavelength on the endpoint determination, and (iv) influence of the dimethyl sulfoxide concentration on the growth kinetics. The main results can be summarized as follows: (i) inoculum preparation following the methodology recommended by the National Committee for Clinical Laboratory Standards is an exact procedure; (ii) the relationship between optical density and CFU per milliliter is linear (coefficient of determination,
r
2
= 0.84); (iii) MICs obtained by means of spectrophotometric readings at different wavelengths are identical (for amphotericin B, an intraclass correlation coefficient of 0.98 was obtained; for fluconazole, the intraclass correlation coefficient was 1); and (iv) a 2% concentration of dimethyl sulfoxide produces a significantly slower and lower growth curve of
Candida
spp. than other concentrations.
Development of a PCR Assay for Identification of Staphylococci at Genus and Species Levels
Authors: Martineau Francis , Picard François J. , Ke Danbing , Paradis Sonia , Roy Paul H. , Ouellette Marc , Bergeron Michel G. 
Published: July 2002

ABSTRACT

We have developed a PCR-based assay which allows the detection of staphylococci at the genus level by targeting the
tuf
gene, which encodes the elongation factor Tu. Degenerate PCR primers derived from consensus regions of several
tuf
genes were used to amplify a target region of 884 bp from 11 representative staphylococcal species. Subsequently, the entire nucleotide sequence of these amplicons was determined. The analysis of a multiple alignment of these sequences revealed regions conserved among staphylococci but distinct from those of other gram-positive bacteria genetically related to staphylococci. PCR primers complementary to these regions could amplify specifically and efficiently a DNA fragment of 370 bp for all of 27 different staphylococcal species tested. There was no amplification with genomic DNA prepared from 53 nonstaphylococcal species tested to verify the specificity of the assay (20 gram positive and 33 gram negative). Furthermore, this assay amplified efficiently all 27 American Type Culture Collection (ATCC) staphylococcal reference strains as well as 307 clinical isolates of staphylococci from the Québec City region. Analysis of the multiple sequence alignment for the 884-bp fragment for the 11 staphylococcal species as well as comparison of the sequences for the 370-bp amplicon from five unrelated ATCC and clinical strains for each of the species
S. aureus, S. epidermidis, S. haemolyticus, S. hominis
, and
S. saprophyticus
demonstrated sufficient interspecies polymorphism to generate genus- and species-specific capture probes. This sequence information allowed the development of
Staphylococcus
-specific and species-specific (targeting
S. aureus, S. epidermidis, S. haemolyticus, S. hominis
, or
S. saprophyticus
) capture probes hybridizing to the 370-bp amplicon. In conclusion, this PCR assay is suitable for detection of staphylococci at both genus and species levels.
Evaluation of a Novel Heminested PCR Assay Based on the Phosphoglucosamine Mutase Gene for Detection of
Helicobacter pylori
in Saliva and Dental Plaque
Authors: Goosen C. , Theron J. , Ntsala M. , Maree F. F. , Olckers A. , Botha S. J. , Lastovica A. J. , van der Merwe S. W. 
Published: July 2002

ABSTRACT

A novel heminested PCR protocol was developed for the specific detection of
Helicobacter pylori
at low copy numbers. A set of primers specific for the phosphoglucosamine mutase gene (
glmM
) of
H. pylori
produced a 765-bp fragment that was used as template for the heminested primer pair delineating a 496-bp fragment. By using agarose gel electrophoresis for detection of the heminested PCR-amplified products, amplification of
H. pylori
genomic DNA was achieved at concentrations as low as 0.1 pg, equivalent to 5 × 10
2
bacteria. A study was subsequently undertaken to evaluate the heminested PCR for detection of
H. pylori
in dental plaque and saliva. Specimens collected from 58 individuals were cultured, and PCR was subsequently performed on the oral cultures. Identification of
H. pylori
in the same series of saliva and dental plaque specimens was carried out with PCR using a primer pair specific for the
H. pylori
urease B gene and by the heminested PCR assay. The identity of the amplified products was confirmed by DNA sequencing. Our results demonstrate that the heminested PCR assay was specific for detection of
H. pylori
, yielding no false-positive results, and that
H. pylori
had a low prevalence (approximately 3%) in specimens obtained from the oral cavity.