bluuebunny/crossref_metadata_2025 huggingface dataset:
Assessment of Geriatric Information on the Drug Label for Commonly Prescribed Drugs in Older People
Authors: Steinmetz Karen L. , Coley Kim C. , Pollock Bruce G.
Published: May 2005

Older people contribute to a significant portion of all prescription drug use and expenditures in the United States. Despite this, older people are often excluded from clinical trials examining the safety and efficacy of drugs. It is unclear to what extent drugs commonly used in older people contain information about prescribing in older people on their product labeling. The objective of this study was to determine the availability of pharmacokinetic, safety, and dosing information with reference to older people on product labels or package inserts (PIs) for commonly prescribed drugs in patients aged 65 and older. The top 50 oral drugs prescribed at the University of Pittsburgh Medical Center were identified through a computerized search of pharmacy records. PIs for these drugs were evaluated for information on elderly patients. Information on drug use in older people was found in 41 (82%) of the PIs. Drugs marketed after 1990 were more likely to contain information on geriatric use, with pharmacokinetic information being the most common type. Approximately 50% of the PIs contained precautionary statements for older people, but most did not provide any specific problems that might be encountered in this population. Only 28 (56%) PIs had dosing information available, and of these, just eight provided specific milligram recommendations for elderly patients. This analysis provides evidence that more should be done to improve the availability of prescribing information, particularly dosing and safety, on drug labels for older people. Additional regulatory action requiring this information to be included on drug product labels may be warranted.
Development and Evaluation of a Multitarget Real-Time Taqman Reverse Transcription-PCR Assay for Detection of the Severe Acute Respiratory Syndrome-Associated Coronavirus and Surveillance for an Apparently Related Coronavirus Found in Masked Palm Civets
Authors: Hu Wenqian , Bai Bingke , Hu Zhihong , Chen Ze , An Xuefang , Tang Lijun , Yang Jihong , Wang Hualin , Wang Hanzhong
Published: May 2005

Severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) is the etiological agent of SARS. It is believed that SARS-CoV originates from wild animals. We have developed a multitarget real-time Taqman reverse transcription-PCR (RT-PCR) assay for the quantitative detection of SARS-CoV. The sequences of the Taqman probes with a minor groove binder and the corresponding primers were based on the sequences of the N gene, open reading frame (ORF) 3, and ORF 8. The overall linear range of this assay was from at least 10
1
to 10
6
copies per reaction, and the detection limit could reach less than 10 copies per reaction. The quantification results for SARS-CoV from cell culture correlated well with those of the RT-PCR by using any two of the three sets of primer and probe used in this assay. However, the results of quantification of SARS-CoV obtained by using a few available throat swab specimens from SARS patients and the N gene as the target were almost 10 times higher than those obtained by using ORF 3 and ORF 8. Using this assay, we also detected an apparently SARS-CoV-related coronavirus in the throat swab specimens from masked palm civets in the west part of Hubei Province, People's Republic of China.
Detection and Typing of Herpes Simplex Virus (HSV) in Mucocutaneous Samples by TaqMan PCR Targeting a gB Segment Homologous for HSV Types 1 and 2
Authors: Namvar Lilly , Olofsson Sigvard , Bergström Tomas , Lindh Magnus
Published: May 2005

Herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) are major causes of mucocutaneous lesions and severe infections of the central nervous system. Here a new semiautomated method for detecting and typing of HSV was used to analyze 479 mucocutaneous swab samples. After DNA extraction using a Magnapure LC robot, a 118-bp segment of the gB region was amplified by real-time PCR utilizing type-specific TaqMan probes to identify HSV-1 or HSV-2. HSV detection in a single well using probes labeled with carboxyfluorescein (FAM) for HSV-1 and JOE (6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein) for HSV-2 had a sensitivity similar to that seen in separate reactions. All but one of 217 samples (99.5%) that had been positive by virus culture were positive by TaqMan PCR, with a correct identification of type in all cases. Out of 262 samples negative by virus culture, 48 (18.3%) were positive by TaqMan PCR, with higher

C
t

values compared with culture positive samples (
P
< 0.0001). Overall, the

C
t

values for HSV-1 were lower than for HSV-2 (mean, 25.5 versus 27.9), but to some extent this could be due to weaker fluorescence by JOE. Lower

C
t

values for HSV-1 were seen also in the 202 genital samples (79 HSV-1, 122 HSV-2, 1 HSV-1 and HSV-2), indicating that HSV-1 replicates as well as HSV-2 in the genital area. HSV-1 constituted 40% of genital infections and was associated with lower mean age (29.2 versus 36.4 years), probably reflecting the fact that recurrent genital HSV-1 infections are rare.
Enhanced Enzyme Immunoassay with Negative-Gray-Zone Testing Compared to a Single Nucleic Acid Amplification Technique for Community-Based Chlamydial Screening of Men
Authors: Horner Paddy , Skidmore Sue , Herring Alan , Sell Jo , Paul Ian , Caul Owen , Egger Matthias , McCarthy Anne , Sanford Emma , Salisbury Chris , MacLeod John , Sterne Jonathan , Low Nicola
Published: May 2005

We evaluated a low-cost diagnostic strategy for detecting
Chlamydia trachomatis
in a low-prevalence population. We used an amplified enzyme immunoassay (EIA) with a reduced-cutoff “negative gray zone” to identify reactive specimens for confirmation by a nucleic acid amplification test. As part of the Chlamydia Screening Studies project, men provided a first-pass urine specimen, which they returned by post for testing. We tested 1,003 specimens by IDEIA PCE EIA (Dako) and Cobas PCR (Roche). There were 32 (3.2%) true positive specimens according to a combined standard using an algorithm requiring concordant results from at least two independent tests. All of these were positive by Cobas PCR and 24 were confirmed to be positive by PCE EIA, including 2 that gave results in the negative gray zone. There were 971 true negative specimens, 2 of which were positive by Cobas PCR and 19 of which were initially inhibitory for PCR. The relative sensitivity, specificity, positive predictive value, and negative predictive value of PCE EIA with PCR confirmation were 75.0% (95% confidence interval [CI], 56.6 to 88.5%), 100% (95% CI, 99.7 to 100%), 100% (95% CI, 88.3 to 100%), and 99.2% (95% CI, 98.4 to 99.6%), respectively. The corresponding values for Cobas PCR were 100% (95% CI, 89.1 to 100%), 99.8% (95% CI, 99.3 to 100%), 94.1% (95% CI, 76.9 to 98.2%), and 100% (95% CI, 99.6 to 100%), respectively, with 1.9% (19/1003) of the samples being initially indeterminate. When the prevalence of
C. trachomatis
is low, the use of an amplified EIA on urine specimens, with confirmation of results in the negative gray zone by use of a nucleic acid amplification technique, is not suitable for screening asymptomatic men. In addition, positive nucleic acid amplification test results should be confirmed and an inhibition control should be used.
Assessment of Ribosomal Large-Subunit D1-D2, Internal Transcribed Spacer 1, and Internal Transcribed Spacer 2 Regions as Targets for Molecular Identification of Medically Important
Aspergillus
Species
Authors: Hinrikson Hans P. , Hurst Steven F. , Lott Timothy J. , Warnock David W. , Morrison Christine J.
Published: May 2005

Molecular approaches are now being developed to provide a more rapid and objective identification of fungi compared to traditional phenotypic methods. Ribosomal targets, especially the large-subunit RNA gene (D1-D2 region) and internal transcribed spacers 1 and 2 (ITS1 and ITS2 regions), have shown particular promise for the molecular identification of some fungi. We therefore conducted an assessment of these regions for the identification of 13 medically important
Aspergillus
species:
Aspergillus candidus
,
Aspergillus
(
Eurotium
)
chevalieri
,
Aspergillus
(
Fennellia
)
flavipes
,
Aspergillus flavus
,
Aspergillus fumigatus
,
Aspergillus granulosus
,
Aspergillus
(
Emericella
)
nidulans
,
Aspergillus niger
,
Aspergillus restrictus
,
Aspergillus sydowii
,
Aspergillus terreus
,
Aspergillus ustus
, and
Aspergillus versicolor
. The length of ribosomal regions could not be reliably used to differentiate among all
Aspergillus
species examined. DNA alignment and pairwise nucleotide comparisons demonstrated 91.9 to 99.6% interspecies sequence identities in the D1-D2 region, 57.4 to 98.1% in the ITS1 region, and 75.6 to 98.3% in the ITS2 region. Comparative analysis using GenBank reference data showed that 10 of the 13 species examined exhibited a ≤1-nucleotide divergence in the D1-D2 region from closely related but different species. In contrast, only 5 of the species examined exhibited a ≤1-nucleotide divergence from sibling species in their ITS1 or ITS2 sequences. Although the GenBank database currently lacks ITS sequence entries for some species, and major improvement in the quality and accuracy of GenBank entries is needed, current identification of medically important
Aspergillus
species using GenBank reference data seems more reliable using ITS query sequences than D1-D2 sequences, especially for the identification of closely related species.
Molecular Epidemiology of Macrolide-Resistant Isolates of
Streptococcus pneumoniae
Collected from Blood and Respiratory Specimens in Norway
Authors: Littauer P. , Sangvik M. , Caugant D. A. , Høiby E. A. , Simonsen G. S. , Sundsfjord A. ,   the Norwegian Macrolide Study Group
Published: May 2005

Norway has a low prevalence of antimicrobial resistance, including macrolide-resistant
Streptococcus pneumoniae
(MRSP). In a nationwide surveillance program, a total of 2,200
S. pneumoniae
isolates were collected from blood cultures and respiratory tract specimens. Macrolide resistance was detected in 2.7%. M-type macrolide resistance was found in 60% of resistant isolates, and these were mainly
mef
(A)-positive, serotype-14 invasive isolates. The
erm
(B)-encoded macrolide-lincosamide-streptogramin B (MLS
B
) type dominated among the noninvasive isolates. One strain had an A2058G mutation in the 23S rRNA gene. Coresistance to other antibiotics was seen in 96% of the MLS
B
-type isolates, whereas 92% of the M-type isolates were susceptible to other commonly used antimicrobial agents. Serotypes 14, 6B, and 19F accounted for 84% of the macrolide-resistant isolates, with serotype 14 alone accounting for 67% of the invasive isolates. A total of 29 different sequence types (STs) were detected by multilocus sequence typing. Twelve STs were previously reported international resistant clones, and 75% of the macrolide-resistant isolates had STs identical or closely related to these clones. Eleven isolates displayed 10 novel STs, and 7/11 of these “Norwegian strains” coexpressed MLS
B
and tetracycline resistance, indicating the presence of Tn
1545
. The invasive serotype-14 isolates were all classified as ST9 or single-locus variants of this clone. ST9 is a
mef
-positive M-type clone, commonly known as England
14
-9, reported from several European countries. These observations suggest that the import of major international MRSP clones and the local spread of Tn
1545
are the major mechanisms involved in the evolution and dissemination of MRSP in Norway.
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

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

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

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

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

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

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

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

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

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

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

Since 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

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

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

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.
Staphylococcus aureus
Isolates Carrying Panton-Valentine Leucocidin Genes in England and Wales: Frequency, Characterization, and Association with Clinical Disease
Authors: Holmes A. , Ganner M. , McGuane S. , Pitt T. L. , Cookson B. D. , Kearns A. M.
Published: May 2005

Staphylococcus aureus
isolates carrying the genes that encode for Panton-Valentine leucocidin (PVL), a highly potent toxin, have been responsible for recent outbreaks of severe invasive disease in previously healthy children and adults in the United States of America and Europe. To determine the frequency of PVL-positive isolates sent to the Staphylococcus Reference Unit (United Kingdom) for epidemiological purposes, we tested 515 isolates of
S. aureus
, and 8 (1.6%) were positive for the PVL locus. A further 470 isolates were selected to explore the association of PVL-positive
S. aureus
with clinical disease. Of these, 23 (4.9%) were PVL positive and most were associated with skin and soft tissue infections (especially abscesses). The PVL genes were also detected in isolates responsible for community-acquired pneumonia, burn infections, bacteremia, and scalded skin syndrome. Genotyping by pulsed-field gel electrophoresis and multilocus sequence typing revealed that the PVL-positive isolates were from diverse genetic backgrounds, although one prevalent clone of 12 geographically dispersed methicillin-resistant
S. aureus
(MRSA) isolates was identified (ST80). All 12 isolates were stapylococcal cassette chromosome
mec
type IVc, had an
agr
3 allele, and shared a common toxin gene profile (
sea-see
,
seg-sej
,
eta
,
etb
, and
tst
negative but
etd
positive). ST80 strains with similar genetic characteristics have been responsible for community-acquired infections in France and Switzerland. The remaining PVL-positive isolates were mostly methicillin-sensitive
S. aureus
and belonged to 12 different sequence types, including ST22 and ST30, which are closely related to the most prevalent MRSA clones in United Kingdom hospitals, EMRSA-15 and EMRSA-16, respectively.
Multilocus Sequence Typing of
Streptococcus uberis
Provides Sensitive and Epidemiologically Relevant Subtype Information and Reveals Positive Selection in the Virulence Gene
pauA
Authors: Zadoks Ruth N. , Schukken Ynte H. , Wiedmann Martin
Published: May 2005

Control of the bovine mastitis pathogen
Streptococcus uberis
requires sensitive and epidemiologically meaningful subtyping methods that can provide insight into this pathogen's epidemiology and evolution. Development of a multilocus sequence typing (MLST) scheme based on six housekeeping and virulence genes allowed differentiation of 40 sequence types among 50
S. uberis
isolates from the United States (
n
= 30) and The Netherlands (
n
= 20). MLST was more discriminatory than EcoRI or PvuII ribotyping and provided subtype data with better epidemiological relevance, e.g., by discriminating isolates with identical ribotypes obtained from different farms. Phylogenetic analyses of MLST data revealed indications of reticulate evolution between genes, preventing construction of a core phylogeny based on concatenated DNA sequences. However, all individual gene phylogenies clearly identified a distinct
pauA
-negative subtaxon of
S. uberis
for which housekeeping alleles closely resembled those of
Streptococcus parauberis
. While the average GC content for five genes characterized was between 0.38 and 0.40,
pauA
showed a considerably lower GC content (0.34), suggesting acquisition through horizontal transfer.
pauA
also showed a higher nonsynonymous/synonymous rate ratio (

d
N

/

d
S

) (1.2) compared to the other genes sequenced (

d
N

/

d
S

< 0.12), indicating positive selection in this virulence gene. In conclusion, our data show that (i) MLST provides for highly discriminatory and epidemiologically relevant subtyping of
S. uberis
; (ii)
S. uberis
has a recombinatorial population structure; (iii) phylogenetic analysis of MLST data reveals an
S. uberis
subtaxon resembling
S. parauberis
; and (iv) horizontal gene transfer and positive selection contribute to evolution of certain
S. uberis
genes, such as the virulence gene
pauA
.
Interrelationship between Polymorphisms ofincA, Fusogenic Properties ofChlamydia trachomatisStrains, and Clinical Manifestations in Patients in The Netherlands
Authors: Pannekoek Yvonne , Spaargaren Joke , Langerak Ankie A. J. , Merks Judith , Morré Servaas A. , van der Ende Arie
Published: May 2005

IncA variation among DutchChlamydia trachomatisisolates was investigated. Of 98 strains, two carried anincAwith a premature stop codon, lacked IncA, and were nonfusogenic, while 96 contained an intactincA, expressed IncA, and were fusogenic. Among these 96 strains, nine IncA sequence types were found, of which the three most frequently encountered (88% of the strains) were randomly distributed among symptomatic and asymptomatic patients.
Prevalence of Derepressed AmpC Mutants and Extended-Spectrum β-Lactamase Producers among Clinical Isolates of
Citrobacter freundii
,
Enterobacter
spp., and
Serratia marcescens
in Korea: Dissemination of CTX-M-3, TEM-52, and SHV-12
Authors: Kim Jungmin , Lim Yu-Mi
Published: May 2005

The resistance mechanism of extended-spectrum cephalosporins in clinical isolates of
Citrobacter freundii
,
Enterobacter
spp., and
Serratia marcescens
was studied. Of 152 isolates, 45 isolates (29.6%) were derepressed AmpC mutants and 39 isolates (25.7%) produced extended-spectrum β-lactamase (ESBLs). The most prevalent ESBLs were CTX-M enzymes, followed by TEM-52 and SHV-12.
Use of a Commercial Reagent Leads to Reduced Germ Tube Production by
Candida dubliniensis
Authors: Davis Leigh E. , Shields Christine E. , Merz William G.
Published: May 2005

The goal of this study was to determine the factor(s) explaining our inability to detect
Candida dubliniensis
. When germ tube-positive yeasts were tested for
C. dubliniensis
, no
C. dubliniensis
was detected; however, 58
C. dubliniensis
strains were detected when germ tube-negative
Candida albicans
strains were tested further. Since all 58
C. dubliniensis
strains detected were germ tube negative, these data implied that false-negative germ tube tests occurred with germ tube solution (GTS; Remel, Lenexa, KS). All 41 known
C. dubliniensis
strains tested were negative with GTS, whereas 40 were positive with rabbit serum (RS; Sigma-Aldrich, St. Louis, MO). Results for
C. albicans
were equivalent in GTS and RS. In conclusion, GTS cannot be used for the detection of
C. dubliniensis
, and switching from yeast to hyphae in
C. dubliniensis
is more restricted than in
C. albicans
.
Identification of
Shigella sonnei
Biotype
g
Isolates Carrying Class 2 Integrons in Italy (2001 to 2003)
Authors: Mammina Caterina , Pontello Mirella , Dal Vecchio Anna , Nastasi Antonino ,   the Shigella sonnei Working Group
Published: May 2005

Phenotyping and genotyping have been carried out on 64 epidemic and sporadic isolates of
Shigella sonnei
identified in Italy in the years 2001 to 2003. Class 2 integron carriage has been also investigated. Isolates from four of the five outbreaks and four of six sporadic cases were biotype
g
, pulsed-field gel electrophoresis type B, and class 2 integron positive, suggesting emergence and spread of an epidemic clone in Italy.
Comparison of Six Methods of Extracting
Mycobacterium tuberculosis
DNA from Processed Sputum for Testing by Quantitative Real-Time PCR
Authors: Aldous Wade K. , Pounder June I. , Cloud Joann L. , Woods Gail L.
Published: May 2005

Six methods of extracting
Mycobacterium tuberculosis
DNA from sputum for testing by quantitative PCR were compared: Tris-EDTA (TE) buffer, PrepMan Ultra, 2% sodium dodecyl sulfate (SDS)-10% Triton X with and without sonication, Infectio Diagnostics, Inc. (IDI) lysing tubes, and QIAGEN QIAamp DNA mini kit; all included a 15-min boiling step. Pooled digested and decontaminated sputum was spiked with
M. tuberculosis
ATCC 27294. Each extraction method was repeated eight times. Quantitative PCR was performed on the Smart Cycler and Rotor-Gene 3000 using primers targeting an 83-bp fragment of IS
6110
. An minor grove binding Eclipse probe with a fluorescent label was used for detection. An internal control was included to detect amplification inhibition. The limit of detection of
M. tuberculosis
DNA was 0.5 fg with both instruments. Calculated DNA concentrations (picograms) extracted using IDI, PrepMan, QIAGEN, and TE were 42.8, 30.4, 28.2, and 7.4, respectively, when run on the Smart Cycler, and 51.7, 20.1, 14.9, and 8.6, respectively, when run on Rotor-Gene. All extractions using SDS/Triton X with or without sonication were inhibited. Of the extraction methods evaluated, IDI lysis tubes provided the greatest yield of mycobacterial DNA, and the procedure can be completed in less than 1 h versus 2.5−3 h for the QIAGEN extraction.
Streptococcus pneumoniaeAntigen Test Using Positive Blood Culture Bottles as an Alternative Method To Diagnose Pneumococcal Bacteremia
Authors: Petti Cathy A. , Woods Christopher W. , Reller L. Barth
Published: May 2005

Recovery ofStreptococcus pneumoniaefrom positive blood culture bottles may be difficult due to autolysis of pneumococci. Therefore, we evaluated the performance of the Binax NOWS. pneumoniaeantigen test with samples from positive blood culture bottles and defined the duration of detectable pneumococcal antigen in these bottles. Use of theS. pneumoniaeantigen test is an alternative method for identifyingS. pneumoniaefrom positive blood culture bottles and may enable a diagnosis of pneumococcal bacteremia despite negative subcultures.
Rahnella aquatilis
Bacteremia from a Suspected Urinary Source
Authors: Tash Kaley
Published: May 2005

A 76-year-old male with prostatic hyperplasia presented with acute pyelonephritis. Blood cultures yielded
Rahnella aquatilis
. Treatment with intravenous followed by oral levofloxacin resulted in cure. Important characteristics of this organism include its biochemical similarities to
Enterobacter agglomerans
, its apparent ability to cause bacteremia from a renal focus, and its response to quinolone therapy.
ON ALGEBRAS THAT HAVE ALMOST FINITE DIMENSIONAL REPRESENTATIONS
Authors: ELEK GÁBOR
Published: May 2005

We introduce the notion of almost finite dimensional representability of algebras and study its connection with the classical finiteness conditions.
A QUOTIENT CURVE OF THE FERMAT CURVE OF DEGREE TWELVE ATTAINING THE SERRE BOUND
Authors: KAWAKITA MOTOKO QIU
Published: May 2005

We find a new curve of genus four attaining the Serre bound over prime fields. It is defined by the equation y12=x4(1-x), which attains the bound over the finite field [Formula: see text] if and only if the prime number p satisfies p ≡ 1 mod 12, [Formula: see text] and [Formula: see text] with an integer n. Furthermore, we show that if a standard conjecture of prime numbers is true then infinitely many prime numbers satisfy these conditions.
THE BAD BEHAVIOR OF REPRESENTATION GROUPS
Authors: HIGGS R. J.
Published: May 2005

Let (H, A) be a primitive central extension of a finite group G. We show that A is not characteristic in H in general, and further demonstrate in a series of examples that results, which hold about inner automorphisms of H, do not extend to the full automorphism group of H. We also give some new results about isoclinism and representation groups of G in the case that H is capable. Finally we give an example of two non-isomorphic groups of the same order, which not only have a representation group in common, but also have identical projective character tables.
ADAPTIVE LEAK DETECTION AND LOCATION IN UNDERGROUND BURIED PIPELINES
Authors: WEN YUMEI , LI PING , YANG JIN , ZHOU ZHANGMIN
Published: May 2005

The vibrational acoustic signals collected on pipelines contain substantial information concerning the working status of pipelines. They can be used for pinpointing leaks in buried pipelines. However, because of the complexity of signal composition and the heavy corruption of signals by ambient noises, it is essential to set up an appropriate signal model and scheme of analysis and synthesis in order to extract leak signatures and specify leak locations. In addition, the features of vibrational acoustic signals vary with materials, sizes and buried conditions of tubular pipes. It is difficult to pre-determine the knowledge of signals, such as the spectral knowledge. Here, an adaptive detection and estimation strategy is proposed based on LMS adaptive filtering and modified Wavelet denoising. The feasibility leak detection and location estimation is first automatically analysed by the signal processing procedure without any prerequisite on signals and then the procedure adaptively finds better estimate. With the proposed schemes, even where collected signals are heavily blurred by bursting interferences and present non-stationary, the instrument may also carry on detection and achieve effective results.