bluuebunny/crossref_metadata_2025_split huggingface dataset:
Correlation of Penicillin Binding Protein 2a Detection with Oxacillin Resistance in
Staphylococcus aureus
and Discovery of a Novel Penicillin Binding Protein 2a Mutation
Authors: Bressler Adam M. , Williams Teresa , Culler Elizabeth E. , Zhu Wenming , Lonsway David , Patel Jean B. , Nolte Frederick S. 
Published: September 2005

ABSTRACT

We compared a rapid slide latex agglutination test (LAT; Oxoid, Basingstoke, United Kingdom) that detects penicillin binding protein 2a (PBP2a) with MicroScan conventional panels (Dade Behring, West Sacramento, CA) for detection of oxacillin resistance in
Staphylococcus aureus
. The PBP2a LAT demonstrated 99% agreement with MicroScan oxacillin MIC results for 388 isolates of
S. aureus
. All 249 oxacillin-resistant isolates gave strong positive reactions in the LAT (100% sensitivity). Three of the 139 oxacillin-susceptible isolates were also strongly positive and one was weakly positive in the LAT (97.1% specificity). The three oxacillin-susceptible isolates with strongly positive reactions were further characterized. The
mecA
gene was detected in all three by PCR; one isolate was determined to be resistant to oxacillin by reference broth microdilution testing (MIC, 8 μg/ml), one isolate was inducibly resistant to oxacillin (MIC of 16 μg/ml after overnight induction), and one isolate remained susceptible regardless of the method used for testing. Sequence analysis of a 2.1-kb gene fragment of the
mecA
gene from the susceptible isolate revealed a one-base substitution at nucleotide position 1449 which results in a Met-to-Ile change for amino acid residue 483. This amino acid substitution has not been previously reported and may be associated with a change in the function of PBP2a resulting in oxacillin susceptibility. An additional 487 isolates were tested in parallel with the both the LAT and MicroScan panels using criteria in which only strong (3 to 4+) or repeatedly weak (1 to 2+) LAT reactions were considered positive, and the results showed 99.4% agreement. The PBP2a LAT provided rapid and reliable detection of oxacillin resistance and proved a useful adjunct to the phenotypic method. Both methods provided reliable detection of oxacillin-resistant
S. aureus
and facilitated the discovery of a novel, functionally impaired form of PBP2a.
Salmonella enterica
Serovar Typhi O:1,9,12 Polysaccharide-Protein Conjugate as a Diagnostic Tool for Typhoid Fever
Authors: Zuñiga Jessica , Lillo Luis , Shin Junghee J. , Machavarapu Rajya L. , Quiroga Teresa , Goycoolea Manuela , Matsuhiro Betty , Aron-Hott L. , Godfrey Henry P. , Cabello Felipe C. 
Published: September 2005

ABSTRACT

Serologic tests play an important role in diagnosis of typhoid fever. In an effort to develop a more defined reagent for these tests, purified
Salmonella enterica
serovar Typhi (ST) O:1,9,12 polysaccharide was conjugated to human serum albumin (HSA), and the conjugate was purified chromatographically to yield a reagent with 2 moles ST O polysaccharide per mole HSA. In 40 patients with bacteriologically confirmed typhoid fever, significant dot immunobinding titers (≥20,000) were present in 28 (70%) tested with 100 ng of ST O antigen-HSA (ST O-HSA) conjugate, in 38 (95%) tested with 100 ng of ST lipopolysaccharide, and in 16 (40%) tested with purified unconjugated ST O chains. In sera from 22 patients with other nontyphoid fevers, 2 (9.1%) had such reactivities with 100 ng of ST O-HSA, 1 (4.5%) had such reactivity with 100 ng of ST lipopolysaccharide (4.5%), and none reacted with 100 ng of unconjugated ST O chains. None of the 17 healthy-control sera reacted significantly with any of the ST reagents. None of the patient or control sera reacted with unconjugated HSA. The sensitivity of dot immunobinding for typhoid fever was 70% with 100 ng of ST O-HSA, somewhat lower than that with 100 ng of ST lipopolysaccharide (95%) but similar to that of the Widal H agglutination test with a ≥1/160 cutoff (74%). Specificities of these tests were 91%, 95%, and 86%, respectively. These preliminary results suggest that ST O polysaccharide-protein conjugates could provide a nontoxic, easily quality-controlled synthetic reagent for analysis of human immune responses to ST as well as for the development of new diagnostics and vaccines for typhoid fever.
Use of Bacteriophage MS2 as an Internal Control in Viral Reverse Transcription-PCR Assays
Authors: Dreier Jens , Störmer Melanie , Kleesiek Knut 
Published: September 2005

ABSTRACT
Diagnostic systems based on reverse transcription (RT)-PCR are widely used for the detection of viral genomes in different human specimens. The application of internal controls (IC) to monitor each step of nucleic acid amplification is necessary to prevent false-negative results due to inhibition or human error. In this study, we designed various real-time RT-PCRs utilizing the coliphage MS2 replicase gene, which differ in detection format, amplicon size, and efficiency of amplification. These noncompetitive IC assays, using TaqMan, hybridization probe, or duplex scorpion probe techniques, were tested on the LightCycler and Rotorgene systems. In our approach, clinical specimens were spiked with the control virus to monitor the efficiency of extraction, reverse transcription, and amplification steps. The MS2 RT-PCR assays were applied for internal control when using a second target hepatitis C virus RNA in duplex PCR in blood donor screening. The 95% detection limit was calculated by probit analysis to 44.9 copies per PCR (range, 38.4 to 73.4). As demonstrated routinely, application of MS2 IC assays exhibits low variability and can be applied in various RT-PCR assays. MS2 phage lysates were obtained under standard laboratory conditions. The quantification of phage and template RNA was performed by plating assays to determine PFU or via real-time RT-PCR. High stability of the MS2 phage preparations stored at −20°C, 4°C, and room temperature was demonstrated.
Human Coronavirus NL-63 Infections in Children: a 1-Year Study
Authors: Bastien Nathalie , Robinson Joan L. , Tse Alena , Lee Bonita E. , Hart Laura , Li Yan 
Published: September 2005

ABSTRACT
Human coronavirus NL63 (HCoV-NL63), a newly discovered coronavirus, has been associated with acute respiratory tract infections (ARI). Important questions pertaining to the contribution of HCoV-NL63 to ARI and its impact on public health remain. We reviewed HCoV-NL63 in specimens collected from 13 November 2002 to 31 December 2003 from the Stollery Children's Hospital on patients of <17 years of age to assess the role of this virus in ARI in children. Twenty-six of 1,240 specimens (2.1%) from seven outpatients and 19 inpatients aged 7 days to 9.5 years tested positive for HCoV-NL63 by reverse transcription-PCR. The majority of outpatients (86%) had upper respiratory tract infections, while the majority of inpatients (58%) had bronchiolitis. Peak HCoV-NL63 activity occurred in March. These results provide further evidence of the importance of HCoV-NL63 in ARI in children.
Depletion of Resident
Chlamydia pneumoniae
through Leukoreduction by Filtration of Blood for Transfusion
Authors: Ikejima Hideaki , Friedman Herman , Leparc German F. , Yamamoto Yoshimasa 
Published: September 2005

ABSTRACT

Current studies indicate that a significant percentage of healthy blood donors carry
Chlamydia pneumoniae
in their blood. Although the clinical significance of such findings is unknown, eradication of such bacteria from blood components may contribute to transfusion safety. Deletion of
C. pneumoniae
in Red Blood Cell (RBC) units was accomplished through leukoreduction by filtration. The presence of bacteria in RBC units before and after leukoreduction was assessed by real-time PCR using primers specific for
C. pneumoniae
16S rRNA. The eluates of filters used for leukoreduction were also assessed by PCR and immunostaining with fluorescein isothiocyanate-conjugated chlamydial monoclonal antibodies specific for
C. pneumoniae
determination. Nineteen of 30 RBC units tested showed the presence of
C. pneumoniae
DNA. Leukofiltration resulted in a marked reduction of leukocytes as well as
C. pneumoniae
in terms of bacterial number and positive rate for the bacteria. The eluates of filters showed trapped bacteria determined by both PCR and immunostaining assays. Thus, leukoreduction with a filter is an effective method to significantly reduce resident
C. pneumoniae
levels in RBC components but may not be completely sufficient for total eradication of this pathogen.
Phenotypic and Genotypic Characteristics of
Streptococcus porcinus
Isolated from Human Sources
Authors: Duarte Rafael S. , Barros Rosana R. , Facklam Richard R. , Teixeira Lúcia M. 
Published: September 2005

ABSTRACT

The phenotypic and genotypic characteristics of 25
Streptococcus porcinus
isolates recovered from human sources were investigated and compared to the characteristics of 17 reference strains obtained from nonhuman sources. All of the
S. porcinus
isolates were beta-hemolytic (wide zones), susceptible to vancomycin, gave positive results for the leucine aminopeptidase and
l
-pyrrolidonylarylamidase tests, and produced acids from mannitol and sorbitol. Most of them were positive for the CAMP test and resistant to bacitracin. The isolates were susceptible to most of the 14 antimicrobials tested, except for tetracycline, for which 80% of the human isolates and 35.2% of the nonhuman strains were resistant. The
tet
(M) and the
tet
(O) genes were detected in 23 (88.5%) and 8 (30.8%) of the 26 tetracycline-resistant isolates, respectively. Analysis of whole-cell protein profiles obtained after sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a high similarity among the profiles. Chromosomal DNA was analyzed by pulsed-field gel electrophoresis (PFGE) after digestion with SmaI and by random(ly) amplified polymorphic DNA (RAPD)-PCR using primer 1254. Analysis of SmaI-restricted genomic DNA revealed the substantial genetic diversity among
S. porcinus
isolates from nonhuman sources, which were also serologically more diverse. Most of the human isolates belonged to serogroup NG1 and shared highly related PFGE profiles that were distinct from profiles of isolates from nonhuman sources. These results were in agreement with those obtained by analysis of amplicons after RAPD-PCR, indicating the potential ability of these techniques for typing
S. porcinus
and suggesting the occurrence of a few clonal groups of
S. porcinus
strains adapted to the human host.
Comparison of Methods for Detection of Vaccinia Virus in Patient Specimens
Authors: Fedorko Daniel P. , Preuss Jeanne C. , Fahle Gary A. , Li Li , Fischer Steven H. , Hohman Patricia , Cohen Jeffrey I. 
Published: September 2005

ABSTRACTWe analyzed a shell vial culture assay (SVA), real-time PCR, and a direct fluorescent antibody assay (DFA) for rapid detection of vaccinia virus from vaccination sites of Dryvax vaccine recipients. Of 47 samples assayed, 100% were positive by PCR, 89% were positive by SVA, and 40% were positive by DFA. DFA was limited by the need for adequate numbers of cells, with 32% of samples inadequate for interpretation. DFA performed better with specimens from patients who had not previously received the vaccine. PCR was positive for longer times postvaccination than was SVA. Infectious virus could be recovered after 45 min of acetone fixation of shell vial coverslips. Commercially available polyclonal antibodies cross-reacted with other orthopoxviruses and herpes simplex 1, but commercially available monoclonal antibodies were specific for vaccinia virus. In summary, PCR was the most sensitive test for detecting vaccinia virus in clinical specimens, while the DFA was the most rapid but the least sensitive test.
Utility of Amsel Criteria, Nugent Score, and Quantitative PCR for
Gardnerella vaginalis
,
Mycoplasma hominis
, and
Lactobacillus
spp. for Diagnosis of Bacterial Vaginosis in Human Immunodeficiency Virus-Infected Women
Authors: Sha Beverly E. , Chen Hua Y. , Wang Qiong J. , Zariffard M. Reza , Cohen Mardge H. , Spear Gregory T. 
Published: September 2005

ABSTRACT

Bacterial vaginosis (BV) is a clinical syndrome presenting with a malodorous vaginal discharge and increased vaginal pH. Diagnosis has been based on clinical Amsel criteria and direct Gram stain of vaginal secretions. Human immunodeficiency virus (HIV)-infected participants in the Women's Interagency HIV Study contributed cervicovaginal lavage (CVL) samples. Lactobacilli,
Gardnerella vaginalis
, and
Mycoplasma hominis
in cervicovaginal lavage samples were quantified by PCR. Gynecologic evaluation included Nugent score and Amsel criterion assessment. We compared the gold standard Nugent score to Amsel criteria and quantitative bacterial PCR for diagnosing BV in 203 CVL samples from women with Nugent scores of 7 to 10 (BV group) and 203 samples from women with BV Nugent scores of 0 to 3 (“No-BV” group). Only 75 of the 203 CVL samples from women with Nugent scores of 7 to 10 met positive Amsel criteria. Increasing levels of
G. vaginalis
and
M. hominis
and decreasing levels of lactobacilli were significantly associated with BV by Nugent score. Of the group with Nugent scores of 7 to 10, 83% and 81% had log
10
G. vaginalis
counts and log
10
M. hominis
counts greater than 6.81 and 4.82, respectively, while only 30% and 31% of the group with Nugent scores of 0 to 3 were above these thresholds, respectively. There was significant overlap in the log
10
lactobacillus counts between the two groups. Utilizing all three log
10
bacterial counts (
G. vaginalis
,
M. hominis
, and lactobacilli) in our model improved the sensitivity and specificity to 83% and 78%, respectively, in comparison with Nugent score. In this cohort, Amsel criteria were poorly predictive of BV. PCR quantification of
G. vaginalis
and
M. hominis
from CVL is significantly more sensitive than Amsel criteria for diagnosing BV.
Reduced PCR Sensitivity Due to Impaired DNA Recovery with the MagNA Pure LC Total Nucleic Acid Isolation Kit
Authors: Schuurman Tim , van Breda Alex , de Boer Richard , Kooistra-Smid Mirjam , Beld Marcel , Savelkoul Paul , Boom René 
Published: September 2005

ABSTRACT
The increasing demand for molecular diagnostics in clinical microbiology laboratories necessitates automated sample processing. In the present study, we evaluated the performance of the MagNA Pure LC total nucleic acid isolation kit (M extraction) in comparison with the manual method (Si extraction) according to Boom et al. (R. Boom, C. J. A. Sol, M. M. M. Salimans, C. L. Jansen, P. M. Wertheim-van Dillen, and J. van der Noordaa, J. Clin. Microbiol. 28:495-503, 1990) for the detection of viral DNA by competitive quantitative PCR. Reconstruction experiments with HindIII-digested phage lambda DNA and HaeIII-digested φX174 DNA showed that the recovery of DNA from phosphate-buffered saline, cerebrospinal fluid, EDTA-anticoagulated plasma, and EDTA-anticoagulated whole blood by M extraction is, on average, 6.6-fold lower compared to Si extraction. PCR signals of spiked PCR control DNAs for Epstein-Barr virus and varicella-zoster virus were also between 1.9- and 14.2-fold lower after M extraction compared to Si extraction, also suggesting impaired DNA recovery. M extraction of spiked cytomegalovirus strain AD 169 in whole blood showed a 5- to 10-fold reduction in PCR sensitivity compared to Si extraction. This reduction of PCR sensitivity was also observed when clinical whole blood samples were processed by M extraction. Before implementing M extraction, the clinical consequences of the reduced recovery should first be considered, especially when maximal sensitivity is required.
Definitive Differentiation between Single and Mixed Mycobacterial Infections in Red Deer (Cervus elaphus) by a Combination of Duplex Amplification of p34 and f57 Sequences and Hpy188I Enzymatic Restriction of Duplex Amplicons
Authors: Godfroid Jacques , Delcorps Cathy , Irenge Leonid M. , Walravens Karl , Marché Sylvie , Gala Jean-Luc 
Published: September 2005

ABSTRACTSevere emaciation and mortalities suggestive of mycobacterial infections were recently reported for both adult and young wild red deer (Cervus elaphus) in the southeastern part of Belgium. In deer, tuberculous lesions are not pathognomonic ofMycobacterium bovisinfection due to gross and microscopic similarities with lesions caused byMycobacterium aviumsubsp.paratuberculosisorM. aviumsubsp.avium. The aim of this study was to improve molecular methods for the species-specific identification ofM. bovis,M. aviumsubsp.avium, andM. aviumsubsp.paratuberculosisin mycobacterial infections of deer. DNA banding patterns were assessed prior to and after Hpy188I restriction of f57-upstream (us)-p34 duplex amplicons. The duplex f57-us-p34 PCR differentiatedM. bovisfromM. aviumsubsp.paratuberculosisandM. aviumsubsp.aviuminfections, whereas the restriction step differentiated singleM. aviumsubsp.paratuberculosisorM. aviumsubsp.aviuminfections from mixedM. aviumsubsp.paratuberculosis/M. aviumsubsp.aviuminfections. The endonuclease Hpy188I cleaves DNA between nucleotides N and G in the unique TCNGA sequence. This restriction site was found at position 168 upstream of the us-p34 initiation codon in allM. aviumsubsp.aviumstrains tested, regardless of their origin and the results of IS901PCR. In contrast, the restriction site was abrogated in allM. aviumsubsp.paratuberculosisstrains tested, independent of their origin, Mycobactin J dependency, and IS900PCR results. Consequently, a two-step strategy, i.e., duplex us-p34-f57 PCR and Hpy188I restriction, allowed us to excludeM. bovisinfection and to identify single (M. aviumsubsp.paratuberculosisorM. aviumsubsp.avium) or mixed (M. aviumsubsp.paratuberculosis/M. aviumsubsp.avium) infections in wild red deer in Belgium. Accordingly, we propose to integrate, in a functional molecular definition ofM. aviumsubsp.paratuberculosis, the absence of the Hpy188I restriction site from the us-p34 amplicon.
Increased Genetic Diversity of
Neisseria meningitidis
Isolates after the Introduction of Meningococcal Serogroup C Polysaccharide Conjugate Vaccines
Authors: Diggle Mathew A. , Clarke Stuart C. 
Published: September 2005

ABSTRACT
During the 1990s, the incidence of meningococcal disease was high in the United Kingdom. This was due primarily to an increase in serogroup C disease, particularly that within the ET-37/ST-11 genetic lineage. Serogroup C meningococcal polysaccharide conjugate vaccines were introduced in the United Kingdom in 1999, but the sequence types of meningococci causing disease since that time have not yet been reported. We have used serogrouping and multilocus sequence typing to characterize meningococci from patients with invasive disease over a 4-year period and show that there is a significant increase in genetic diversity but no genetic evidence of capsule switching.
Search for Localized Dysbiosis in Crohn's Disease Ulcerations by Temporal Temperature Gradient Gel Electrophoresis of 16S rRNA
Authors: Seksik Philippe , Lepage Patricia , de la Cochetière Marie-France , Bourreille Arnaud , Sutren Malène , Galmiche Jean-Paul , Doré Joël , Marteau Philippe 
Published: September 2005

ABSTRACT

The mucosa-associated microbiota lining the gut epithelium might play a central role in the activation and/or perpetuation of mucosal inflammation in Crohn's disease (CD). We sought for localized dysbiosis by comparing the biodiversity and composition of the microbiotas in ulcerated and nonulcerated mucosal samples from patients with CD. Biopsy samples (
n
= 75) of ulcerated and adjacent nonulcerated mucosa were collected during colonoscopy from 15 patients, from the ileum, right colon, left colon, and rectum. Temporal temperature gradient gel electrophoresis (TTGE) of bacterial 16S rRNAs was used to evaluate the dominant bacterial species. TTGE profiles were compared using software that calculates similarity percentages. For a given patient, average similarity indexes between ulcerated and nonulcerated mucosal TTGE profiles ranged from 95.2% ± 4.2% to 97.9% ± 1.7% (means ± standard deviations) for the different segments. The mean values did not differ significantly. Average interindividual similarity indexes for a given segment among the different patients ranged from 33.6% ± 15.5% to 42.0% ± 25.6%. In CD, the dominant microbiotas do not differ qualitatively between ulcerated and nonulcerated mucosae. Biodiversity remains high in ulcerated mucosa. This argues against a pathogenic role of localized qualitative dysbiosis in CD-associated ulceration.
Molecular Epidemiology of Caliciviruses Detected in Sporadic and Outbreak Cases of Gastroenteritis in France from December 1998 to February 2004
Authors: Bon F. , Ambert-Balay K. , Giraudon H. , Kaplon J. , Le Guyader S. , Pommepuy M. , Gallay A. , Vaillant V. , de Valk H. , Chikhi-Brachet R. , Flahaut A. , Pothier P. , Kohli E. 
Published: September 2005

ABSTRACT
We compiled sequence and epidemiological data from 172 caliciviruses detected in France from December 1998 to February 2004 in sporadic and outbreak cases. The results showed a cocirculation of strains with a majority of genogroup II (GII) noroviruses. Three groups of noroviruses, not detected before in our laboratory, emerged and spread during the period: the recombinant GGIIb and Norwalk-related strains not amplified in the polymerase gene in 2000 and a new Lordsdale variant in 2002. We observed that (i) GII-4 noroviruses were predominant in nursing home and hospital outbreaks but rare in oyster- and water-related outbreaks despite continuous circulation in the population; (ii) at the opposite, genogroup I strains were detected in the majority of environmental outbreaks; (iii) several strains were frequently found in oyster- and water-linked outbreaks (up to seven), whereas one single strain was detected when transmission was from person to person; and (iv) whereas GII noroviruses were predominant in sporadic cases where patients were under 15 years of age, GI strains were more frequent in outbreaks occurring in this age group. Finally, from a methodology point of view, this compilation shows that detection and characterization in the polymerase gene are not adequate in a significant number of cases and should be completed by amplification and sequencing in the capsid gene.
Multilocus Sequence Typing Scheme That Provides Both Species and Strain Differentiation for the
Burkholderia cepacia
Complex
Authors: Baldwin Adam , Mahenthiralingam Eshwar , Thickett Kathleen M. , Honeybourne David , Maiden Martin C. J. , Govan John R. , Speert David P. , LiPuma John J. , Vandamme Peter , Dowson Chris G. 
Published: September 2005

ABSTRACT

A single multilocus sequence typing (MLST) scheme was developed for precise characterization of the opportunistic pathogens of
Burkholderia cepacia
complex (BCC), a group composed of at least nine closely related species. Seven conserved housekeeping genes were selected after a comparison of five
Burkholderia
species, and a collection of strains was subjected to nucleotide sequence analysis using a nested PCR amplification approach for each gene. MLST differentiated all nine current BCC species and identified 114 sequence types within a collection of 119 strains. No differentiation was found between strains recovered from environmental or clinical sources. The improved resolution in strain identification offered by MLST was able to identify previously characterized epidemic strain lineages and also demonstrated the presence of four novel potential species groups within the complex. There was also evidence for recombination having an important role in the recent evolution of individual BCC species. This highly transferable, validated, MLST scheme provides a new means to assist in species identification as well as unambiguous strain discrimination of the BCC by a single approach. It is also the first MLST scheme designed at the outset to incorporate multiple species and should facilitate global epidemiological investigations of the BCC.