bluuebunny/crossref_metadata_2025_split huggingface dataset:
Repetitive-Sequence-PCR-Based DNA Fingerprinting Using the DiversiLab System for Identification of Commonly Encountered Dermatophytes
Authors: Pounder June I. , Williams Sheri , Hansen Dewey , Healy Mimi , Reece Kristy , Woods Gail L. 
Published: May 2005

ABSTRACT

The performance of repetitive-sequence-based PCR (rep-PCR) using the DiversiLab system for identification of dermatophytes commonly isolated in a clinical laboratory was assessed by comparing results to those of conventional tests (colony morphology, microscopic examination of slide cultures, and, for suspected
Trichophyton
species, use of additional media). Sixty-one cultures were tested in phase 1, the feasibility portion of the study; 64 additional cultures were tested in phase 2, the validation portion conducted to assess reproducibility and confirm accuracy. Discrepancies were resolved by repeating rep-PCR and conventional tests and, in phase 2, sequencing the internal transcribed spacers. After initial testing of the cultures in phase 1 (excluding one contaminated culture), agreement between conventional tests and rep-PCR was 90% (54 of 60). Agreement was 98.3% after resolution of discrepancies, and in all but one case the initial rep-PCR result was correct. After initial testing of cultures in phase 2 (excluding one discarded and one contaminated culture), agreement between rep-PCR and conventional testing was 88.7% (55 of 62). After discrepancies were resolved, agreement was 100%. Initial rep-PCR results were correct, except for one
Microsporum canis
culture containing two colony variants, which could not be initially identified by rep-PCR. The performance of the DiversiLab system for identification of the dermatophytes commonly encountered in a clinical mycology laboratory—
Trichophyton mentagrophytes
,
Trichophyton rubrum
,
Trichophyton tonsurans
, and
M. canis
—was excellent. Moreover, the DiversiLab system is technically simple and provides results in <24 h once a pure culture is available for testing, which is considerably more rapid than conventional identification tests.
Real-Time-PCR Assay for Diagnosis of
Entamoeba histolytica
Infection
Authors: Roy Shantanu , Kabir Mamun , Mondal Dinesh , Ali Ibne Karim M. , Petri William A. , Haque Rashidul 
Published: May 2005

ABSTRACT

We developed a real-time-PCR assay utilizing a molecular-beacon probe for the detection of
Entamoeba histolytica
and compared its sensitivity to stool antigen detection and traditional PCR. A total of 205 stool and liver abscess pus specimens from patients and controls were used for this purpose, 101 (49%) of which were positive by the TechLab
E. histolytica
-specific antigen detection test, while the other 104 (51%) stool and liver abscess pus specimens were negative by the antigen detection test. DNA was extracted from the stool and liver abscess pus specimens by the QIAGEN method and the small-subunit rRNA gene of
E. histolytica
and then amplified by traditional and real-time PCR. Out of these 205 stool and liver abscess pus specimens, 124 were positive by the real-time-PCR assay and 90 were positive by the traditional-PCR test. Compared to the real-time-PCR assay, the antigen detection test was 79% sensitive and 96% specific. When the traditional-PCR test results were compared to the real-time-PCR assay, the sensitivity of traditional PCR was 72% and the specificity was 99%. In conclusion, all three methods for the detection of
E. histolytica
were highly specific, with real-time PCR being the most sensitive.
Comparison of Non-Culture-Based Methods for Detection of Systemic Fungal Infections, with an Emphasis on Invasive
Candida
Infections
Authors: White P. Lewis , Archer Alice E. , Barnes Rosemary A. 
Published: May 2005

ABSTRACT

The accepted limitations associated with classic culture techniques for the diagnosis of invasive fungal infections have lead to the emergence of many non-culture-based methods. With superior sensitivities and quicker turnaround times, non-culture-based methods may aid the diagnosis of invasive fungal infections. In this review of the diagnostic service, we assessed the performances of two antigen detection techniques (enzyme-linked immunosorbent assay [ELISA] and latex agglutination) with a molecular method for the detection of invasive
Candida
infection and invasive aspergillosis. The specificities for all three assays were high (≥97%), although the
Candida
PCR method had enhanced sensitivity over both ELISA and latex agglutination with values of 95%, 75%, and 25%, respectively. However, calculating significant sensitivity values for the
Aspergillus
detection methods was not feasible due to a low number of proven/probable cases. Despite enhanced sensitivity, the PCR method failed to detect nucleic acid in a probable case of invasive
Candida
infection that was detected by ELISA. In conclusion, both PCR and ELISA techniques should be used in unison to aid the detection of invasive fungal infections.
Clinical Impact Associated with Corrected Results in Clinical Microbiology Testing
Authors: Yuan Shan , Astion Michael L. , Schapiro Jeff , Limaye Ajit P. 
Published: May 2005

ABSTRACT
We developed a strategy to determine the clinical impact associated with errors in clinical microbiology testing. Over a 9-month period, we used a sequential three-stage method to prospectively evaluate 480 consecutive corrected microbiology laboratory reports. The three stages were physician review of the corrected report, medical record review, and interview with the clinician(s) taking care of the patient. Of the 480 corrected reports, 301 (62.7%) were ruled out for significant clinical impact by physician review and an additional 25 cases (5.2%) were ruled out for clinical impact by medical record review. This left 154 cases (32.1%) that required clinician interview to determine clinical impact. The clinician interview revealed that 32 (6.7%) of the corrected reports were associated with adverse clinical impact. Of these 32 cases, 19 (59.4%) involved delayed therapy, 8 (25.0%) involved unnecessary therapy, 8 (25.0%) were associated with inappropriate therapy, and 4 (12.5%) were associated with an increased level of care. The laboratory was entirely responsible for the error in 28 (87.5%) of the 32 cases and partially responsible in the other 4 cases (12.5%). Twenty-six (81.3%) of the 32 cases involved potentially preventable analytic errors that were due to lack of knowledge (cognitive error). In summary, we used evaluation of corrected reports to identify laboratory errors with adverse clinical impact, and most of the errors were amenable to laboratory-based interventions. Our method has the potential to be implemented in other laboratory settings to identify and characterize errors that impact patient safety.
Comparison of Diagnostic Laboratory Methods for Identification of
Burkholderia pseudomallei
Authors: Inglis Timothy J. J. , Merritt Adam , Chidlow Glenys , Aravena-Roman Max , Harnett Gerry 
Published: May 2005

ABSTRACT

Limited experience and a lack of validated diagnostic reagents make
Burkholderia pseudomallei
, the cause of melioidosis, difficult to recognize in the diagnostic microbiology laboratory. We compared three methods of confirming the identity of presumptive
B. pseudomallei
strains using a collection of
Burkholderia
species drawn from diverse geographic, clinical, and environmental sources. The 95 isolates studied included 71
B. pseudomallei
and 3
B. thailandensis
isolates. The API 20NE method identified only 37% of the
B. pseudomallei
isolates. The agglutinating antibody test identified 82% at first the attempt and 90% including results of a repeat test with previously negative isolates. Gas-liquid chromatography analysis of bacterial fatty acid methyl esters (GLC-FAME) identified 98% of the
B. pseudomallei
isolates. The agglutination test produced four false positive results, one
B. cepacia
, one
B. multivorans
, and two
B. thailandensis
. API produced three false positive results, one positive
B. cepacia
and two positive
B. thailandensis
. GLC-FAME analysis was positive for one
B. cepacia
isolate. On the basis of these results, the most robust
B. pseudomallei
discovery pathway combines the previously recommended isolate screening tests (Gram stain, oxidase test, gentamicin and polymyxin susceptibility) with monoclonal antibody agglutination on primary culture, followed by a repeat after 24 h incubation on agglutination-negative isolates and GLC-FAME analysis. Incorporation of PCR-based identification within this schema may improve percentages of recognition further but requires more detailed evaluation.
Multilocus Sequence Typing Lacks the Discriminatory Ability of Pulsed-Field Gel Electrophoresis for Typing
Salmonella enterica
Serovar Typhimurium
Authors: Fakhr Mohamed K. , Nolan Lisa K. , Logue Catherine M. 
Published: May 2005

ABSTRACT

Nontyphoidal salmonellae are among the leading causes of food-borne disease in the United States. Because of the importance of
Salmonella enterica
in food-borne disease, numerous typing methodologies have been developed. Among the several molecular typing methods, pulsed-field gel electrophoresis (PFGE) is currently considered the “gold standard” technique in typing
Salmonella
. The aim of this study was to compare the discriminatory power of PFGE to multilocus sequence typing (MLST) in typing
Salmonella enterica
serovar Typhimurium clinical isolates. A total of 85
Salmonella
Typhimurium clinical isolates from cattle were used in this study. PFGE using XbaI was performed on the 85 isolates by the Centers for Disease Control and Prevention method, and data were analyzed using the BioNumerics software package. Fifty PFGE profiles were observed among the isolates, and these grouped into three major clusters. For the MLST analysis, the
manB
,
pduF
,
glnA
, and
spaM
genes were amplified by PCR from the same 85 isolates. DNA sequencing of these four genes,
manB
,
pduF
,
glnA
, and
spaM
, showed no genetic diversity among the isolates tested, with a 100% identity in nucleotide sequence. Moreover, the DNA sequences of the aforementioned genes showed 100% identity to the sequence reported in GenBank for the
S. enterica
serovar Typhimurium LT2 strain. Therefore, MLST, using these genes, lacks the discriminatory power of PFGE for typing
Salmonella enterica
serovar Typhimurium.
Investigation of a Nosocomial Outbreak of Imipenem-Resistant
Acinetobacter baumannii
Producing the OXA-23 β-Lactamase in Korea
Authors: Jeon Byung-Chan , Jeong Seok Hoon , Bae Il Kwon , Kwon Su Bong , Lee Kyungwon , Young Dongeun , Lee Jung Hun , Song Jae Seok , Lee Sang Hee 
Published: May 2005

ABSTRACT

We investigated an outbreak of
Acinetobacter baumannii
in an intensive care unit and in the surgery, medicine, neurology, and urology wards of the Kosin University Gospel Hospital in Busan, Korea. The outbreak involved 36 cases of infection by
A. baumannii
producing the OXA-23 β-lactamase over an 8-month period and was caused by a single pulsed-field gel electrophoresis clone. The epidemic isolates were characterized by a modified cloverleaf synergy test. Isoelectric focusing of crude bacterial extracts detected one nitrocefin-positive band with a pI value of 6.65. PCR amplification and characterization of the amplicons by direct sequencing indicated that the epidemic isolates carried a
bla
OXA-23
determinant. The epidemic isolates were characterized by a multidrug resistance phenotype that remained unchanged over the outbreak, including penicillins, cephamycins, extended-spectrum cephalosporins, carbapenems, monobactams, and aminoglycosides. This study shows that the
bla
OXA-23
resistance determinant may become an emerging therapeutic problem.
Characterization of AmpC-Mediated Resistance in Clinical
Salmonella
Isolates Recovered from Humans during the Period 1992 to 2003 in England and Wales
Authors: Batchelor M. , Hopkins K. L. , Threlfall E. J. , Clifton-Hadley F. A. , Stallwood A. D. , Davies R. H. , Liebana E. 
Published: May 2005

ABSTRACT

The increase in AmpC-mediated resistance in salmonellae constitutes a serious public health concern, since these enzymes confer resistance to a wide range of β-lactams. One hundred six isolates were selected from 278,308
Salmonella
isolates based on resistance to ampicillin and cephalosporins and were subjected to further characterization. Nine isolates had a cefoxitin inhibition diameter ≤17 mm and were proven to be AmpC positive by multiplex PCR. Sequence analysis revealed the presence of
bla
DHA-1
,
bla
CMY-2
, and
bla
CMY-4
genes. All nine isolates presented different pulsed-field gel electrophoresis restriction profiles. The AmpC genetic determinants were present in transferable plasmids of around 11, 42, 70, 98, and 99 MDa. A combination of size and restriction fragment length polymorphism (RFLP) analysis showed that all the
bla
CMY
plasmids investigated in our study were different, which suggests that
bla
CMY
may be located in different plasmid environments. Some United Kingdom isolates linked to foreign travel showed RFLP plasmid patterns consistent with plasmids previously seen in the United States, which suggests that
bla
CMY-2
has also been disseminated through plasmid transfer. The fact that two of the domestically acquired United Kingdom isolates presented previously unseen RFLP plasmid patterns could indicate that these strains have followed routes different from those prevalent in North America or other parts of the world. This study represents the first report of
bla
CMY
genes in
Salmonella
isolates in the United Kingdom and the first report of CMY-4 in
Salmonella enterica
serotype Senftenberg worldwide.
Comparison of Genotypic and Phenotypic Methods for Species-Level Identification of Clinical Isolates of Coagulase-Negative Staphylococci
Authors: Heikens E. , Fleer A. , Paauw A. , Florijn A. , Fluit A. C. 
Published: May 2005

ABSTRACT

To compare commonly used phenotypic methods with genotypic identification methods 47 clinical isolates of coagulase-negative staphylococci (CONS), 10 CONS ATCC strains, and a
Staphylococcus aureus
clinical isolate were identified using the API Staph ID test, BD Phoenix Automated Microbiology System, and 16S rRNA gene and
tuf
gene sequencing. When necessary part of the
sodA
gene was sequenced for definitive identification. The results show that
tuf
gene sequencing is the best method for identification of CONS, but the API Staph ID test is a reasonably reliable phenotypic alternative. The performance of the BD Phoenix Automated Microbiology System for identification of CONS is poor. The present study also showed that although genotypic methods are clearly superior to phenotypic identifications, a drawback of sequence-based genotypic methods may be a lack of quality of deposited sequences in data banks. In particular, 16S rRNA gene sequencing suffers from the lack of high quality among sequences deposited in GenBank. Furthermore, genotypic identification based on 16S rRNA sequences has limited discriminating power for closely related
Staphylococcus
species. We propose partial sequencing of the
tuf
gene as a reliable and reproducible method for identification of CONS species.
Extended Multilocus Sequence Typing System for
Campylobacter coli
,
C. lari
,
C. upsaliensis
, and
C. helveticus
Authors: Miller William G. , On Stephen L. W. , Wang Guilin , Fontanoz Samarpita , Lastovica Albert J. , Mandrell Robert E. 
Published: May 2005

ABSTRACT

A multilocus sequence typing (MLST) system has been reported previously for
Campylobacter jejuni
to both differentiate strains and identify clonal lineages. However, sequence variation at the MLST loci prevents its use for closely related
Campylobacter
species. We describe herein an expanded MLST method to include three clinically relevant
Campylobacter
species,
C. coli
,
C. lari
, and
C. upsaliensis
, and a fourth
Campylobacter
species,
C. helveticus
. The
C. coli
and
C. helveticus
methods use the same seven
C. jejuni
loci (
aspA
,
atpA
,
glnA
,
gltA
,
glyA
,
pgm
, and
tkt
); however,
adk
and
pgi
were substituted for
aspA
and
gltA
in
C. lari
and for
gltA
and
pgm
in
C. upsaliensis
. Multiple
C. coli
(
n
= 57),
C. lari
(
n
= 20),
C. upsaliensis
(
n
= 78), and
C. helveticus
(
n
= 9) isolates, representing both clinical and environmental sources, were typed. All four species were genetically diverse: the majority (>80%) of the isolates had unique sequence types (STs). Using this method, mixed
C. lari
,
C. upsaliensis
, and
C. helveticus
isolates were identified; upon separation, each isolate was shown to contain two strains of the same species with distinct STs. Additionally, the expanded MLST method was able to detect potential lateral transfer events between
C. jejuni
and either
C. coli
or
C. lari
and between
C. upsaliensis
and
C. helveticus
. Thus, the expanded MLST method will prove useful in differentiating strains of five
Campylobacter
species, identifying mixed
Campylobacter
cultures, and detecting genetic exchange within the genus.
Genomic Diversity inCampylobacter jejuni: Identification ofC. jejuni81-176-Specific Genes
Authors: Poly Frédéric , Threadgill Deborah , Stintzi Alain 
Published: May 2005

ABSTRACTSince the publication of the complete genomic sequence ofCampylobacter jejuniNCTC 11168 in February 2000, evidence has been compiling that suggestsC. jejunistrains exhibit high genomic diversity. In order to investigate this diversity, the unique genomic DNA sequences from a nonsequencedCampylobacterstrain,C. jejuni81-176, were identified by comparison withC. jejuniNCTC 11168 by using a shotgun DNA microarray approach. Up to 63 kb of new chromosomal DNA sequences unique to this pathogen were obtained. Eighty-six open reading frames were identified by the presence of uninterrupted coding regions encoding a minimum of 40 amino acids. In addition, this study shows that the whole-plasmid shotgun microarray approach is effective and provides a comprehensive coverage of DNA regions that differ between two closely related genomes. The two plasmids harbored by thisCampylobacterstrain, pTet and pVir, were also sequenced, with coverages of 2.5- and 2.9-fold, respectively, representing 72 and 92% of their complete nucleotide sequences. The unique chromosomal genes encode proteins involved in capsule and lipooligosaccharide biosynthesis, restriction and modification systems, and respiratory metabolism. Several of these unique genes are likely associated withC. jejuni81-176 fitness and virulence. Interestingly, the comparison ofC. jejuni81-176 unique genes with those ofC. jejuniATCC 43431 revealed a single gene which encodes a probable TraG-like protein. The product of this gene might be associated with the mechanism ofC. jejuniinvasion into epithelial cells. In conclusion, this study extends the repertoire ofC. jejunigenes and thus will permit the construction of a composite and more comprehensive microarray ofC. jejuni.
Microbiological Aspects of the Investigation That Traced the 1998 Outbreak of Listeriosis in the United States to Contaminated Hot Dogs and Establishment of Molecular Subtyping-Based Surveillance for
Listeria monocytogenes
in the PulseNet Network
Authors: Graves Lewis M. , Hunter Susan B. , Ong Anna Rae , Schoonmaker-Bopp Diana , Hise Kelley , Kornstein Laura , DeWitt Wallis E. , Hayes Peggy S. , Dunne Eileen , Mead Paul , Swaminathan Balasubramanian 
Published: May 2005

ABSTRACT

A multistate outbreak of listeriosis occurred in the United States in 1998 with illness onset dates between August and December. The outbreak caused illness in 108 persons residing in 24 states and caused 14 deaths and four miscarriages or stillbirths. This outbreak was detected by public health officials in Tennessee and New York who observed significant increases over expected listeriosis cases in their states. Subsequently, the Centers for Disease Control and Prevention (CDC) began laboratory characterization of clinical isolates of
Listeria monocytogenes
by serotyping and restriction fragment length polymorphism analysis using pulsed-field gel electrophoresis (PFGE). For the purpose of this investigation, outbreak-related isolates were defined as those that had a specific AscI-PFGE pattern and indistinguishable or highly similar (no more than 2 band difference in 26 bands) ApaI-PFGE patterns when their DNA was restricted by AscI and ApaI restriction enzymes. Timely availability of molecular subtyping results enabled epidemiologists to separate outbreak cases from temporally associated sporadic cases in the same geographic areas and facilitated the identification of contaminated hot dogs manufactured at a single commercial facility as the source of the outbreak. During the investigation of this outbreak, a standardized protocol for subtyping
L
.
monocytogenes
by PFGE was developed and disseminated to public health laboratories participating with CDC's PulseNet network; these laboratories were requested to begin routine PFGE subtyping of
L
.
monocytogenes
.
Characterization of Oral Strains of
Cardiobacterium valvarum
and Emended Description of the Organism
Authors: Han Xiang Y. , Falsen Enevold 
Published: May 2005

ABSTRACT

The description of the new species
Cardiobacterium valvarum
prompted a search for additional strains of the organism. Here we report characterization of four oral
Cardiobacterium
strains from the Culture Collection of the University of Goteborg. The 16S rRNA gene sequences of the organisms exhibited 99.6% to 99.3% homology with
Cardiobacterium valvarum.
The cellular fatty acid profiles, electrophoretic patterns of whole-cell proteins, growth rate and nutritional requirement, colonial and cellular morphology, and biochemical reactions were also similar to those of
C. valvarum
. These results thus classify these organisms as oral strains of
C. valvarum
. All strains were susceptible to many antibiotics tested. The description of the species was emended.
C. valvarum
is a rare cause of endocarditis, and its relationship with periodontal diseases may need investigation.
Detection of
Cyclospora cayetanensis
Oocysts in Human Fecal Specimens by Flow Cytometry
Authors: Dixon Brent R. , Bussey Jeff M. , Parrington Lorna J. , Parenteau Monique 
Published: May 2005

ABSTRACT

A diagnosis of cyclosporiasis typically involves stool examinations for the presence of
Cyclospora
oocysts by means of microscopy. In recent years, flow cytometry has been gaining in popularity as a novel method of detecting pathogens in environmental and clinical samples. The present study is an evaluation of a flow cytometric method for the detection and enumeration of
Cyclospora
oocysts in human fecal specimens associated with food-borne outbreaks of cyclosporiasis in Ontario, Canada. Flow cytometry results were generally very comparable to the original microscopy results for these specimens, in terms of both presence or absence of oocysts and relative oocyst concentrations. Of the 34 fecal specimens confirmed positive for
Cyclospora
by microscopy, 32 were also found positive by flow cytometry, and 2 others were considered equivocal. Of the eight fecal specimens reported to be negative by microscopy, two were found positive by flow cytometry and five others were considered equivocal. These two flow cytometry-positive samples and one of the equivocal samples were confirmed by microscopic reexamination, suggesting that flow cytometry may be more sensitive than microscopy. While the sample preparation time for flow cytometry is similar to or slightly longer than that for microscopy, the actual analysis time is much shorter. Further, because flow cytometry is largely automated, an analyst's levels of fatigue and expertise will not influence results. Flow cytometry appears to be a useful alternative to microscopy for the screening of large numbers of stool specimens for
Cyclospora
oocysts, such as in an outbreak situation.