bluuebunny/crossref_metadata_2025 huggingface dataset:
Prevalence of the ST239 Clone of Methicillin-Resistant
Staphylococcus aureus
and Differences in Antimicrobial Susceptibilities of ST239 and ST5 Clones Identified in a Korean Hospital
Authors: Cha Hwa Yun , Moon Dong Chan , Choi Chul Hee , Oh Jae Young , Jeong Young Sook , Lee Yoo Chul , Seol Sung Yong , Cho Dong Taek , Chang Hyun-Ha , Kim Shin-Woo , Lee Je Chul
Published: August 2005

A total of 188 nonduplicate methicillin-resistant
Staphylococcus aureus
(MRSA) isolates obtained between 2001 and 2004 in a university hospital in Daegu, Korea, were analyzed for their clonal types by molecular typing techniques, including multilocus sequence typing,
spaA
typing, staphylococcal chromosomal cassette
mec
(SCC
mec
) typing, and pulsed-field gel electrophoresis (PFGE). They were examined for their antimicrobial susceptibilities. The majority (87%) of MRSA isolates belonged to sequence type 239 (ST239;
n
= 100; 53%) and ST5 (
n
= 63, 34%) on the basis of sequence typing. MRSA isolates belonging to ST239 were genotypically homogeneous, while those belonging to ST5 showed variations in
spaA
type, SCC
mec
type, and PFGE patterns. The rates of resistance of the MRSA isolates belonging to ST239 to trimethoprim, sulfamethoxazole, tobramycin, gentamicin, erythromycin, and tetracycline were significantly higher than those of the isolates belonging to ST5 (
P
< 0.05). This study demonstrated that the ST239 clone, while rarely detected in Korea, was prevalent and that the antimicrobial susceptibility of the ST239 clone was significantly different from that of the ST5 clone.
First Nosocomial Outbreak of Vancomycin-Resistant
Enterococcus faecium
Expressing a VanD-Like Phenotype Associated with a
vanA
Genotype
Authors: Naas Thierry , Fortineau Nicolas , Snanoudj Renaud , Spicq Colette , Durrbach Antoine , Nordmann Patrice
Published: August 2005

Although enterococci expressing acquired vancomycin resistance phenotype have been reported increasingly worldwide, they have been rarely reported in France. From August to December 2004 we faced an outbreak of vancomycin-resistant
Enterococcus faecium
(VRE) isolates in the nephrology department at Bicêtre Hospital (K.-Bicêtre, France). The expression of the glycopeptide resistance varied among the 26 VRE isolates, with vancomycin MICs ranging from 12 to >256 μg/ml, whereas teicoplanin MICs ranged from 4 to 48 μg/ml. However, several strains appeared to be susceptible to glycopeptides according to disk diffusion testing and expressed resistance only after subculture with glycopeptides. In addition, a heterogeneous expression of glycopeptide resistance was also observed. This so-called VanD-like phenotype of resistance (low-level resistance to vancomycin and mostly susceptibility to teicoplanin) was surprisingly associated with a
vanA
gene. Plasmid extraction and mating-out experiments indicated that the
vanA
gene was located on a 200-kb self-transferable plasmid. Pulsed-field gel electrophoresis identified mostly dissemination of a single clone, whereas diffusion of the VanA-positive plasmid in different genomic backgrounds had also occurred. The
vanA
gene was part of a
vanA
-type operon for expression of resistance located on a Tn
1546
-like transposon. Sequencing of this transposon identified insertion of insertion sequence IS
16
in the
vanY
gene that encodes a
d
,
d
-carboxypeptidase that might explain in part the peculiar VanD-type phenotype of resistance. This report is the first description of a VRE outbreak in France and underlines the difficulty in detecting this organism due to variability on the expression of the glycopeptide resistance trait, if any.
Sensitivity of PCR Targeting the IS
2404
Insertion Sequence of
Mycobacterium ulcerans
in an Assay Using Punch Biopsy Specimens for Diagnosis of Buruli Ulcer
Authors: Phillips R. , Horsfield C. , Kuijper S. , Lartey A. , Tetteh I. , Etuaful S. , Nyamekye B. , Awuah P. , Nyarko K. M. , Osei-Sarpong F. , Lucas S. , Kolk A. H. J. , Wansbrough-Jones M.
Published: August 2005

Punch biopsy specimens from
Mycobacterium ulcerans
disease lesions were used to compare the sensitivities and specificities of direct smear, culture, PCR, and histopathology in making a diagnosis of
M. ulcerans
disease in a field setting. PCR for the insertion element IS
2404
was modified to include uracil-
N
-glycosylase and deoxyuridine triphosphate instead of deoxythymidine triphosphate to reduce the risk of cross contamination. The “gold standard” for confirmation of clinically diagnosed Buruli ulcer was a definite histological diagnosis, a positive culture for
M. ulcerans
, or a smear positive for acid-fast bacilli (AFB), together with a possible histological diagnosis. For 70 clinically diagnosed cases of
M. ulcerans
disease, the modified PCR was 98% sensitive and gave a rapid result. The sensitivities of microscopy, culture, and histology were 42%, 49%, and 82%, respectively. The use of a 4-mm punch biopsy specimen was preferred to a 6-mm punch biopsy specimen since the wound was less likely to bleed and to need stitching. Given adequate technical expertise and the use of controls, the PCR was viable in a teaching hospital setting in Ghana; and in routine practice, we would recommend the use of Ziehl-Neelsen staining of biopsy specimens to detect AFB, followed by PCR, in AFB-negative cases only, in order to minimize costs. Histology and culture remain important as quality control tests, particularly in studies of treatment efficacy.
Corynebacterium resistens
sp. nov., a New Multidrug-Resistant Coryneform Bacterium Isolated from Human Infections
Authors: Otsuka Yoshihito , Kawamura Yoshiaki , Koyama Takashi , Iihara Hirotoshi , Ohkusu Kiyofumi , Ezaki Takayuki
Published: August 2005

Five strains of an unknown, multidrug-resistant coryneform, gram-positive rod were isolated from blood, bronchial aspirate, and abscess specimens. Four of the five strains isolated were highly resistant to antimicrobial agents, including β-lactams, aminoglycosides, macrolides, quinolones, and tetracyclines, except for glycopeptides. In immunocompromised patients, bacteremia associated with this organism was rapidly fatal. This coryneform bacterium was nonmotile, lipophilic, and nonsaccharolytic. Lack of pyrazinamidase activity differentiated this organism from other lipophilic corynebacteria. Chemotaxonomic studies indicated that this multidrug-resistant coryneform bacterium belongs to the genus
Corynebacterium
. Comparative 16S rRNA gene sequencing and DNA-DNA hybridization analyses revealed that the five isolates were genetically identical and that they represent a new subline within the genus
Corynebacterium
, for which we propose the designation
Corynebacterium resistens
sp. nov. The type strain of
Corynebacterium resistens
is GTC 2026
T
(SICGH 158
T
, JCM 12819
T
, CCUG 50093
T
).
High Level of Sequence Diversity in the 16S rRNA Genes of
Haemophilus influenzae
Isolates Is Useful for Molecular Subtyping
Authors: Sacchi Claudio T. , Alber Dietmar , Dull Peter , Mothershed Elizabeth A. , Whitney Anne M. , Barnett Gwen A. , Popovic Tanja , Mayer Leonard W.
Published: August 2005

A molecular typing method based on the 16S rRNA sequence diversity was developed for
Haemophilus influenzae
isolates. A total of 330
H. influenzae
isolates were analyzed, representing a diverse collection of U.S. isolates. We found a high level of 16S rRNA sequence heterogeneity (up to 2.73%) and observed an exclusive correlation between 16S types and serotypes (a to f); no 16S type was found in more than one serotype. Similarly, no multilocus sequence typing (MLST) sequence type (ST) was found in more than one serotype. Our 16S typing and MLST results are in agreement with those of previous studies showing that serotypable
H. influenzae
isolates behave as highly clonal populations and emphasize the lack of clonality of nontypable (NT)
H. influenzae
isolates. There was not a 1:1 correlation between 16S types and STs, but all
H. influenzae
serotypable isolates clustered similarly. This correlation was not observed for NT
H. influenzae
; the two methods clustered NT
H. influenzae
isolates differently. 16S rRNA gene sequencing alone provides a level of discrimination similar to that obtained with the analysis of seven genes for MLST. We demonstrated that 16S typing is an additional and complementary approach to MLST, particularly for NT
H. influenzae
isolates, and is potentially useful for outbreak investigation.
Evaluation of the VACUTAINER PPT Plasma Preparation Tube for Use with the Bayer VERSANT Assay for Quantification of Human Immunodeficiency Virus Type 1 RNA
Authors: Elbeik Tarek , Nassos Patricia , Kipnis Patricia , Haller Barbara , Ng Valerie L.
Published: August 2005

Separation and storage of plasma within 2 h of phlebotomy is required for the VACUTAINER PPT Plasma Preparation Tube (PPT) versus 4 h for the predecessor VACUTAINER EDTA tube for human immunodeficiency virus type 1 (HIV-1) viral load (HIVL) testing by the VERSANT HIV-1 RNA 3.0 assay (branched DNA). The 2-h limit for PPT imposes time constraints for handling and transporting to the testing laboratory. This study compares HIVL reproducibility from matched blood in EDTA tubes and PPTs and between PPT pairs following processing within 4 h of phlebotomy, stability of plasma HIV-1 RNA at 24- and 72-h room temperature storage in the tube, and comparative labor and supply requirements. Blood from 159 patients was collected in paired tubes (EDTA/PPT or PPT/PPT): 86 paired EDTA tubes and PPTs were processed 4 h following phlebotomy and their HIVLs were compared, 42 paired PPT/PPT pairs were analyzed for intertube HIVL reproducibility, and 31 PPT/PPT pairs were analyzed for HIV-1 RNA stability by HIVL. Labor and supply requirements were compared between PPT and EDTA tubes. PPTs produce results equivalent to standard EDTA tube results when processed 4 h after phlebotomy. PPT intertube analyte results are reproducible. An average decrease of 13% and 37% in HIVL was observed in PPT plasma after 24 and 72 h of room temperature storage, respectively; thus, plasma can be stored at room temperature up to 24 h in the original tube. PPTs offer labor and supply savings over EDTA tubes.
Use of Fluorescent Probes To Determine MICs of Amphotericin B and Caspofungin against
Candida
spp. and
Aspergillus
spp
Authors: Peter Joanne , Armstrong Derek , Lyman Caron A. , Walsh Thomas J.
Published: August 2005

We investigated the utility of mechanism-based fluorescent probes for determination of MICs (FMICs) of amphotericin B and caspofungin against
Candida
spp. and
Aspergillus
spp. Amphotericin B was selected as a membrane-active antifungal agent, and caspofungin was selected as a cell wall-active agent. FMICs were also compared to the MIC determined by CLSI (formerly NCCLS) methods. Five isolates per species of
Candida albicans
,
Candida glabrata
,
Candida parapsilosis
,
Aspergillus fumigatus
, and
Aspergillus terreus
were studied with either amphotericin B or caspofungin. The fluorescent probes, carboxyfluorescein diacetate (CFDA) for cytoplasmic esterase activity and dihexyloxacarbocyanine iodide (DiOC
6
) for cell membrane potential, were each added to their respective plates. MICs and FMICs were determined in at least three separate experiments (in duplicate). Fluorescence was measured using a 96-well plate fluorometer. For amphotericin B and caspofungin, the FMIC end point was the lowest concentration of drug at which the percent growth inhibition from treated organisms versus control organisms displayed 80% inhibition for amphotericin B and 50% inhibition for caspofungin as measured by a fluorescent signal. The MIC for amphotericin B was defined as the lowest concentration of antifungal displaying no visible growth for both
Aspergillus
and
Candida
spp. The MIC for caspofungin was the lowest concentration of drug that displayed a minimum effective concentration for
Aspergillus
spp. For
Candida
spp., the MIC for caspofungin was defined as the concentration at which the antifungal agent significantly inhibits the organism. The FMICs of both antifungals, as measured by the DiOC
6
membrane probe, showed good agreement (83% to 100%), within one well dilution, with the MICs against amphotericin B and caspofungin for all species. Also, the FMICs measured by the CFDA cytoplasmic esterase probe reflecting damage due to cell wall or cell membrane showed strong agreement (79 to 100%) with the MICs of both amphotericin B and caspofungin for all species. There was no significant difference in comparisons of MIC and FMIC values (
P
≥ 0.05). The use of fluorescent probes provides a mechanism-based method of determination of MICs of amphotericin B and caspofungin against
Candida
spp. and
Aspergillus
spp. that correlates well with standard methods.
Human Immunodeficiency Virus (HIV) Reverse Transcriptase Activity Correlates with HIV RNA Load: Implications for Resource-Limited Settings
Authors: Sivapalasingam Sumathi , Essajee Shaffiq , Nyambi Phillipe N. , Itri Vincenza , Hanna Bruce , Holzman Robert , Valentine Fred
Published: August 2005

Measurement of human immunodeficiency virus type 1 (HIV-1) plasma RNA levels using Roche AMPLICOR version 1.5 (HIV RNA) is an integral part of monitoring HIV-infected patients in industrialized countries. These assays are currently unaffordable in resource-limited settings. We investigated a reverse transcriptase (RT) assay as a less expensive alternative for measuring viral burden that quantifies RT enzyme activity in clinical plasma samples. A comparison of RT and HIV RNA assays was performed on 29 paired plasma samples from patients living in the United States and 21 paired plasma samples from patients living in Cameroon. RT levels correlated significantly with plasma HIV RNA viral loads in plasma from U.S. patients (
r
= 0.898;
P
< 0.001) and Cameroonian patients, a majority of whom were infected with HIV-1 clade type CRF02_AG (
r
= 0.669;
P
< 0.01). Among 32 samples with HIV viral load of >2,000 copies/ml, 97% had detectable RT activity. One Cameroon sample had undetectable RNA viral load but detectable RT activity of 3 fg/ml. The RT assay is a simple and less expensive alternative to the HIV RNA assay. Field studies comparing these assays in resource-limited settings are warranted to assess the practicality and usefulness of this assay for monitoring HIV-infected patients on antiretroviral therapy.
In Vitro Susceptibilities of Clinical Isolates of
Candida
Species,
Cryptococcus neoformans
, and
Aspergillus
Species to Itraconazole: Global Survey of 9,359 Isolates Tested by Clinical and Laboratory Standards Institute Broth Microdilution Methods
Authors: Pfaller M. A. , Boyken L. , Hollis R. J. , Messer S. A. , Tendolkar S. , Diekema D. J.
Published: August 2005

The in vitro activity of itraconazole was determined against 7,299 isolates of
Candida
spp., 1,615 isolates of
Cryptococcus neoformans
, and 445 isolates of
Aspergillus
spp. obtained from over 200 medical centers worldwide. Itraconazole was active against all
Candida
spp. (96% of MICs were ≤1 μg/ml) with the exception of
C. glabrata
(77% of MICs were ≤1 μg/ml). Itraconazole inhibited 94% of
C. krusei
and 84% of other fluconazole-resistant
Candida
species, exclusive of
C. glabrata
, at a MIC of ≤1 μg/ml. Itraconazole was not active against fluconazole-resistant isolates of
C. glabrata
. Only modest activity was seen against
C. neoformans
(80% of MICs were ≤1 μg/ml); however, itraconazole showed excellent activity against
Aspergillus
spp. (94% of MICs were ≤1 μg/ml). These results provide an update on the antifungal activity of itraconazole against major opportunistic fungal pathogens. In light of the new intravenous formulation of itraconazole these data suggest that this agent remains a viable systemically active antifungal agent.
Impact of Human Immunodeficiency Virus Type 1 (HIV-1) Genetic Diversity on Performance of Four Commercial Viral Load Assays: LCx HIV RNA Quantitative, AMPLICOR HIV-1 MONITOR v1.5, VERSANT HIV-1 RNA 3.0, and NucliSens HIV-1 QT
Authors: Swanson Priscilla , de Mendoza Carmen , Joshi Yagnya , Golden Alan , Hodinka Richard L. , Soriano Vincent , Devare Sushil G. , Hackett John
Published: August 2005

Human immunodeficiency virus type 1 (HIV-1) evolution and changing strain distribution present a challenge to nucleic acid-based assays. Reliable patient monitoring of viral loads requires the detection and accurate quantification of genetically diverse HIV-1. A panel of 97 HIV-1-seropositive plasma samples collected from Cameroon, Brazil, and South Africa was used to compare the performance of four commercially available HIV RNA quantitative tests: Abbott LCx HIV RNA Quantitative assay (LCx), Bayer Versant HIV-1 RNA 3.0 (bDNA), Roche AMPLICOR HIV-1 MONITOR v1.5 (Monitor v1.5), and bioMérieux NucliSens HIV-1 QT (NucliSens). The panel included group M, group O, and recombinant viruses based on sequence analysis of
gag
p24,
pol
integrase, and
env
gp41. The LCx HIV assay quantified viral RNA in 97 (100%) of the samples. In comparison, bDNA, Monitor v1.5, and NucliSens quantified viral RNA in 96.9%, 94.8%, and 88.6% of the samples, respectively. The two group O specimens were quantified only by the LCx HIV assay. Analysis of nucleotide mismatches at the primer/probe binding sites for Monitor v1.5, NucliSens, and LCx assays revealed that performance characteristics reflected differences in the level of genetic conservation within the target regions.
Distinct Diversity ofvacA,cagA, andcagEGenes ofHelicobacter pyloriAssociated with Peptic Ulcer in Japan
Authors: Yamazaki Shiho , Yamakawa Akiyo , Okuda Tomoyuki , Ohtani Masahiro , Suto Hiroyuki , Ito Yoshiyuki , Yamazaki Yukinao , Keida Yoshihide , Higashi Hideaki , Hatakeyama Masanori , Azuma Takeshi
Published: August 2005

Colonization of the stomach mucosa byHelicobacter pyloriis a major cause of acute and chronic gastric pathologies in humans. SeveralH. pylorivirulence genes that may play a role in its pathogenicity have been identified. The most important determinants arevacAandcagAin thecagpathogenicity island (cagPAI) genes. In the present study, to consider the association of molecular genetics betweenvacAand thecagPAI regarding clinical outcome, we selectedH. pyloristrains with various genotypes ofvacAin Japan and sequenced full-lengthvacA,cagA, andcagEgenes. Sequencing ofvacAandcagAgenes revealed variable size, whereas thecagEgene was well conserved among strains. Each of the phylogenetic trees based on the deduced amino acid sequences of VacA, CagA, and CagE indicated that all three proteins were divided into two major groups, a Western group and an East Asian group, and the distributions of isolates exhibited similar patterns among the three proteins. The strains with s2 and s1a/m1avacAgenotypes and the Western-type 3′ regioncagAgenotype were classified into the Western group, and the strains with the s1c/m1bvacAgenotype and the East Asian-type 3′cagAgenotype were included in the East Asian group. In addition, the prevalence of infection with the Western group strain was significantly higher in patients with peptic ulcer (90.0%, 9/10) than in patients with chronic gastritis (22.7%, 5/22) (χ2 = 12.64,P= 0.00057). These data suggest that the molecular genetics ofvacAandcagPAI are associated and that the Western group withvacAandcagPAI genes is associated with peptic ulcer disease.
Design of Novel Conformational and Genotype-Specific Antigens for Improving Sensitivity of Immunoassays for Hepatitis C Virus-Specific Antibodies
Authors: Lin Sansan , Arcangel Phillip , Medina-Selby Angelica , Coit Doris , Ng Philip , Nguyen Steve , McCoin Colin , Gyenes Alex , Hu Celine , Tandeske Laura , Phelps Bruce , Chien David
Published: August 2005

The current commercially licensed enzyme-linked immunosorbent assays (ELISAs) for hepatitis C virus (HCV) mainly use recombinant proteins containing linear epitopes. There is evidence, however, that conformational epitopes of HCV are more immunoreactive. Thus, we have designed an HCV antibody assay that employs a conformational protein, NS3NS4a PI (with functional protease and helicase activities), and a linear fusion protein, multiple-epitope fusion antigen 7.1 (MEFA 7.1) or MEFA 7.2. We have shown that NS3NS4a PI detects early-seroconversion conformation-sensitive antibodies better than c33c antigen. The correct conformation of NS3NS4a PI also cross-reacts with different genotype samples better than the c33c antigen. MEFA 7.1 and MEFA 7.2 incorporate all the major immunodominant and genotype-specific epitopes of HCV core, E1, E2 hypervariable region 1 (HVR1), E2 HVR1-plus-HVR2 consensus, NS3, NS4, and NS5. Since MEFA 7.1 is degraded by the active NS3NS4a PI protease, we designed a second MEFA 7.2 construct in which the six protease cleavage sites found in MEFA 7.1 were eliminated by amino acid mutation. We demonstrate here that MEFA 7.2 remains intact in the presence of NS3NS4a PI and preserves the epitopes present in MEFA 7.1. Compared to currently licensed assays, an ELISA incorporating a combination of the two antigens NS3NS4a PI and MEFA 7.1 or 7.2 demonstrates better serotype sensitivity and detects seroconversion earlier in many commercially available panels. We believe that an assay using NS3NS4a PI and MEFA 7.1 or 7.2 may have the potential to replace current HCV immunoassays for better sensitivity.
Comparison of
Staphylococcus aureus
Genotypes Recovered from Cases of Bovine, Ovine, and Caprine Mastitis
Authors: Mørk T. , Tollersrud T. , Kvitle B. , Jørgensen H. J. , Waage S.
Published: August 2005

Staphylococcus aureus
is an important pathogen in domestic ruminants. The main objective of this study was to determine the similarity of epidemiologically unrelated
S. aureus
isolates from bovine, ovine, and caprine mastitis. By pulsed-field gel electrophoresis, 160 different pulsotypes (PTs) were identified among 905 isolates recovered from 588 herds in 12 counties in Norway. Based on estimates of similarity, using an 80% cluster cutoff, the isolates were assigned to 47 clusters. One cluster included 62% of all the isolates and more than 45% of the isolates from each host species. Twenty-three PTs included isolates from more than one host species; these 23 PTs represented 72% of all the isolates. The six most prevalent PTs included isolates from all host species and contained 45% of the bovine isolates, 54% of the ovine isolates, and 37% of the caprine isolates. Antimicrobial susceptibility testing of 373 of the isolates revealed resistance to penicillin in 2.9% and to streptomycin in 2.4%; only 1.9% were resistant to 1 of the other 11 antimicrobials tested. The results of this study suggest that a small number of closely related genotypes are responsible for a great proportion of
S. aureus
mastitis cases in cows, ewes, and goats in Norway and that these genotypes exhibit little or no host preference among these species. Selection due to antimicrobial resistance appears not to have contributed to the predominance of these genotypes.
Biochemical Differentiation and Comparison ofDesulfovibrioSpecies and Other Phenotypically Similar Genera
Authors: Warren Yumi A. , Citron Diane M. , Merriam C. Vreni , Goldstein Ellie J. C.
Published: August 2005

Seventeen human clinical isolates representing four species ofDesulfovibriowere characterized using 16S rRNA gene sequences and tests for catalase, indole, nitrate, bile, urease, formate-fumarate stimulation, desulfoviridin, motility, and hydrogen sulfide production, plus susceptibility to antimicrobial agents. Eighty additional strains representing 10 phenotypically similar genera (Bilophila,Selenomonas,Capnocytophaga,Campylobacter,Bacteroides,Sutterella,Anaerobiospirillum,Dialister,Veillonella, andMobiluncus) were included for comparison. AllDesulfovibriospecies produced H2S and were desulfoviridin positive, and allDesulfovibriospecies exceptD. pigerwere motile. The fourDesulfovibriospecies could be distinguished from each other using tests for catalase, indole, nitrate, urease, and growth on bile, with the following results (positive [+], negative [−], growth [G], and no growth [NG]): forD. piger, −,− , −, −, and G, respectively; forD.fairfieldensis, +, −, +, −, and G, respectively; forD. desulfuricans, −,− , +, +, and NG, respectively; and forD. vulgaris, −, +, −,− , and G, respectively. Resistance to the 10-μg colistin disk separated theDesulfovibriospecies from most of the other genera, which were usually susceptible. These simple tests were useful for characterizing theDesulfovibriospecies and differentiating them from other phenotypically similar genera.
Detection of
toxB
, a Plasmid Virulence Gene of
Escherichia coli
O157, in Enterohemorrhagic and Enteropathogenic
E. coli
Authors: Tozzoli Rosangela , Caprioli Alfredo , Morabito Stefano
Published: August 2005

The virulence plasmid of
Escherichia coli
O157 strain EDL933 carries a 10-kb putative virulence gene designated
toxB
. Little is known about the distribution of this gene among
E. coli
O157 strains or its presence in other enterohemorrhagic
E. coli
(EHEC) and enteropathogenic
E. coli
(EPEC) strains. We developed PCR and hybridization tools for the detection of the entire
toxB
sequence and investigated its presence in a collection of EHEC O157 strains and other EHEC and EPEC strains belonging to different serogroups and isolated from different sources. The EHEC O157 strains reacted with all of the PCR primers and probes used, thus indicating the presence of a complete
toxB
gene regardless of the human or bovine origin of the isolates. Similar positive reactions were observed for about 50% of the EHEC O26 strains tested and a few other EHEC and EPEC strains. However, the size of the DNA fragments hybridizing with the
toxB
probes differed from that of the positive fragments from EHEC O157, suggesting a polymorphism in the
toxB
genes present in the different
E. coli
serogroups. Moreover, several EHEC and EPEC strains belonging to different serogroups reacted with only some of the genetic tools used, suggesting either the existence of major variants of
toxB
or the presence of fragments of the gene. Southern blotting analysis showed that
toxB
sequences were located on large plasmids in EHEC and EPEC O26 as well.
Superiority of Molecular Techniques for Identification of Gram-Negative, Oxidase-Positive Rods, Including Morphologically Nontypical
Pseudomonas aeruginosa
, from Patients with Cystic Fibrosis
Authors: Wellinghausen Nele , Köthe Juliane , Wirths Beate , Sigge Anja , Poppert Sven
Published: August 2005

Phenotypic identification of gram-negative bacteria from Cystic Fibrosis (CF) patients carries a high risk of misidentification. Therefore, we compared the results of biochemical identification by API 20NE with 16S rRNA gene sequencing in 88 gram-negative, oxidase-positive rods, other than morphologically and biochemically typical
P. aeruginosa
, from respiratory secretions of CF patients. The API 20NE allowed correct identification of the bacterial species in 15 out of 88 (17%) isolates investigated. Agreement between the API and the 16S rRNA gene sequencing results was high only in isolates with an API result classified as “excellent identification.” Even API results classified as “very good identification” or “good identification” showed a high rate of misidentification (67% and 84%). Fifty-two isolates of morphological and biochemical nontypical
Pseudomonas aeruginosa
, representing 59% of all isolates investigated, were not identifiable or misidentified in the API 20NE. Therefore, rapid molecular diagnostic techniques like real-time PCR and fluorescence in situ hybridization (FISH) were evaluated in this particular group of bacteria for identification of the clinically most relevant pathogen,
P. aeruginosa
. The LightCycler PCR assay with a
P. aeruginosa
-specific probe showed a sensitivity and specificity of 98.1% and 100%, respectively. For FISH analysis, a newly designed
P. aeruginosa
-specific probe had a sensitivity and specificity of 100%. In conclusion, molecular methods are superior over biochemical tests for identification of gram-negative, oxidase-positive rods in CF patients. In addition, real-time PCR and FISH allowed identification of morphologically nontypical isolates of
P. aeruginosa
within a few hours.
Enhanced Expression of an α2,6-Linked Sialic Acid on MDCK Cells Improves Isolation of Human Influenza Viruses and Evaluation of Their Sensitivity to a Neuraminidase Inhibitor
Authors: Hatakeyama Shuji , Sakai-Tagawa Yuko , Kiso Maki , Goto Hideo , Kawakami Chiharu , Mitamura Keiko , Sugaya Norio , Suzuki Yasuo , Kawaoka Yoshihiro
Published: August 2005

The extensive use of neuraminidase (NA) inhibitors to treat influenza virus infections mandates close monitoring for resistant variants. Cultured cells do not provide a reliable means of evaluating the susceptibility of human influenza virus isolates to NA inhibitors. That is, the growth of such viruses in cell lines (e.g., Madin-Darby canine kidney [MDCK] cells) is not inhibited by these drugs, even though their sialidase activity is drug-sensitive. Matrosovich et al. (J. Virol.
77:
8418-8425, 2003) showed that an MDCK cell line overexpressing the human β-galactoside α2,6-sialyltransferase I (ST6Gal I) gene has the potential to assess the sensitivity of human influenza virus isolates to NA inhibitors, based on studies with a limited number of viruses. Here, we asked whether clinical isolates of influenza virus are universally sensitive to an NA inhibitor (oseltamivir) in an MDCK cell line expressing the ST6Gal I gene. The sensitivity of viruses to oseltamivir correlated with the sensitivity of viral sialidase to the compound, demonstrating the potential utility of this modified cell line for detecting NA inhibitor-resistant viruses. Moreover, in ST6Gal I-overexpressing cells, the growth of human influenza viruses was up to 2 logs higher than in MDCK cells. We conclude that the human ST6Gal I-expressing MDCK cell line is useful not only for evaluating their sensitivity to NA inhibitors, but also for isolation of influenza viruses from clinical samples.
Novel Application of the Masson-Fontana Stain for Demonstrating
Malassezia
Species Melanin-Like Pigment Production In Vitro and in Clinical Specimens
Authors: Gaitanis George , Chasapi Vassiliki , Velegraki Aristea
Published: August 2005

Melanin-like pigment produced in vitro and in vivo by
Malassezia
yeasts has not been described before. Masson-Fontana staining confirmed accumulation of black pigment on the cell walls of
l
-dihydroxyphenylalaline (
l
-DOPA)-cultured
Malassezia
species. Black pigment was also observed in cells and hyphae from hyperpigmented patient lesions with culture-confirmed pityriasis versicolor and seborrheic dermatitis.
Approaches to Bacterial RNA Isolation and Purification for Microarray Analysis of
Escherichia coli
K1 Interaction with Human Brain Microvascular Endothelial Cells
Authors: Di Cello Francescopaolo , Xie Yi , Paul-Satyaseela Maneesh , Kim Kwang Sik
Published: August 2005

We established a protocol for isolation of microarray-grade bacterial RNA from
Escherichia coli
K1 interacting with human brain microvascular endothelial cells. The extracted RNA was free of human RNA contamination. More importantly, microarray analysis demonstrated that no bias was introduced in the gene expression pattern during the RNA isolation procedure.
Affordable, Abbreviated Roche Monitor Assay for Quantification of Human Immunodeficiency Virus Type 1 RNA in Plasma
Authors: Fiscus Susan A. , Chen Shu , Hoover Donald , Kerkau Melissa G. , Alabanza Paul , Siharath Som , Schmitz John , Kumwenda Newton , Taha Taha E.
Published: August 2005

The cost for the Roche Monitor assay kit can be reduced 50% by using only the 1:1, 1:25, and 1:625 human immunodeficiency virus (HIV) and the 1:1 quantitation standard dilutions. This abbreviated test applied to 1,774 mostly African samples had results nearly identical to those obtained following the package insert instructions. To make this approach feasible, Roche would have to provide additional lysis buffer and master mix.
Dissemination ofSalmonella entericasubsp.entericaSerovar Typhimurium var. Copenhagen Clonal Types through a Contract Heifer-Raising Operation
Authors: Hegde Narasimha V. , Cook Michelle L. , Wolfgang David R. , Love Brenda C. , Maddox Carol C. , Jayarao Bhushan M.
Published: August 2005

Salmonella entericasubsp.entericaserovar Typhimurium var. Copenhagen isolates from a heifer-raising operation and from 11 dairy herds that had their calves contracted to the heifer-raising operation were examined for their phenotypic and genotypic characteristics. Results of the study showed that the heifer-raising operation could serve as a clearinghouse forSalmonellaserovar Typhimurium var. Copenhagen and perhaps otherSalmonellaserotypes.
Pathogen Evolution In Vivo: Genome Dynamics of Two Isolates Obtained 9 Years Apart from a Duodenal Ulcer Patient Infected with a Single
Helicobacter pylori
Strain
Authors: Prouzet-Mauléon Valérie , Hussain M. Abid , Lamouliatte Hervé , Kauser Farhana , Mégraud Francis , Ahmed Niyaz
Published: August 2005

The survival and microevolution of
Helicobacter pylori
strains in the niches of the stomach after eradication therapy have largely been unexplored. We analyzed genomic signatures for two successive isolates obtained 9 years apart from a duodenal ulcer patient who underwent eradication therapy for
H. pylori
. These isolates were genotyped based on 50 different parameters involving three different fingerprinting approaches and several evolutionarily significant and virulence-associated landmarks in the genome, including nine informative gene loci, the
cag
pathogenicity island and its right junction, members of the plasticity region cluster, and
vacA
and
iceA
alleles. Our observations reveal that the two isolates were derived from the same strain that colonized the patient for almost a decade and were almost identical. Microevolution, however, was observed in the
cagA
gene and its right junction, the
vacA
m1 allele, and a member of the plasticity region cluster (JHP926). These results suggest that
H. pylori
has a great ability to survive and reemerge as a microevolved strain posteradication, thereby hinting at the requirement for follow-up of patients after therapy.
Microbiology of Bartholin's Gland Abscess in Japan
Authors: Tanaka Kaori , Mikamo Hiroshige , Ninomiya Mochiyoshi , Tamaya Teruhiko , Izumi Koji , Ito Kunihiko , Yamaoka Kazukiyo , Watanabe Kunitomo
Published: August 2005

This study was conducted to determine the current epidemiology concerning the causative organisms for Bartholin's gland abscess in Japan. Microbiological examination of 224 cases showed positive results in 219 cases and negative results in 5 cases. Of all of the bacterial isolates, 307 and 118 were aerobes and anaerobes, respectively. The most frequently isolated bacterium was
Escherichia coli
. Of the anaerobes, the most frequently isolated organism was
Bacteroides
species, followed by
Prevotella
species. The organisms related to respiratory infectious diseases, such as
Streptococcus pneumoniae
and
Haemophilus influenzae
, including resistant bacteria, were sometimes involved between 2000 and 2004.
Molecular Detection of
Cellulosimicrobium cellulans
as the Etiological Agent of a Chronic Tongue Ulcer in a Human Immunodeficiency Virus-Positive Patient
Authors: Heym Beate , Gehanno Pierre , Friocourt Véronique , Bougnoux Marie-Elisabeth , Le Moal Michèle , Husson Corinne , Leibowitch Jacques , Nicolas-Chanoine Marie-Hélène
Published: August 2005

Ulcerations appeared on the tongue of a 48-year-old human immunodeficiency virus-positive man. Histological findings of the biopsy specimen and the fact that the patient had resided in Louisiana led us to suspect “American histoplasmosis.” A new ulcer appeared while the patient was being treated with itraconazole, and the gene for 16S rRNA of
Cellulosimicrobium cellulans
was amplified. The lesions healed during treatment with oral penicillin and azithromycin.
First Case of Infant Botulism Caused by
Clostridium baratii
Type F in California
Authors: Barash Jason R. , Tang Tania W. H. , Arnon Stephen S.
Published: August 2005

In late 2003 a severely hypotonic neonate, just 38 h old at onset of illness, was found to have infant botulism caused by neurotoxigenic
Clostridium baratii
type F. Environmental investigations failed to identify a source of this strain. This is the youngest patient reported to have infant botulism and the fifth instance of infant botulism caused by
C. baratii
type F.
4-ENGEL GROUPS ARE LOCALLY NILPOTENT
Authors: HAVAS GEORGE , VAUGHAN-LEE M. R.
Published: August 2005

Questions about nilpotency of groups satisfying Engel conditions have been considered since 1936, when Zorn proved that finite Engel groups are nilpotent. We prove that 4-Engel groups are locally nilpotent. Our proof makes substantial use of both hand and machine calculations.
SPACE OF HIGHER ORDER DIFFERENTIAL OPERATORS ON S1 AND INTEGRABLE FLOWS
Authors: GUHA PARTHA
Published: August 2005

We study the action of vector field Vect(S1) on the space of higher order differential operators [Formula: see text], and its connection to projective structure on S1. In particular, we study the Euler–Poincaré flows on the space of third order differential operators and its relation to Drienfeld–Sokolov and coupled KdV type systems. We also discuss the Boussinesq equation associated with the third order operator. We study the factorization of higher order operators and its compatibility with the action of Vect(S1). We obtain the generalized Miura transformation and its connection to the modified Boussinesq equation. Finally, we also discuss flows on the special higher order differential operators for all ui = f(u,ux,uxx…) and its connection to KdV equation.
Hemochromatosis: Genetics and Pathophysiology
Authors: Beutler Ernest
Published: August 2005

A number of genetic disorders can result in the accumulation of excess iron in the body. These causes of hereditary hemochromatosis include defects in genes encoding HFE, transferrin receptor 2, ferroportin, hepcidin, and hemojuvelin. Hepcidin, with its cognate receptor, ferroportin, has emerged as a central regulator of iron homeostasis; all of the known causes of hemochromatosis appear to prevent this system from functioning normally. The most common form of primary hemochromatosis is that caused by C282Y mutation of the HFE gene. This mutation is most prevalent among Northern Europeans. Although the frequency of the homozygous genotype is approximately 5 per 1000, the disease itself is quite rare because the clinical penetrance of the genotype is very low.
Acid increases MAPK-mediated proliferation in Barrett's esophageal adenocarcinoma cells via intracellular acidification through a Cl−/HCO3−exchanger
Authors: Sarosi George A. , Jaiswal Kshama , Herndon Emily , Lopez-Guzman Christie , Spechler Stuart J. , Souza Rhonda F.
Published: August 2005

Abundant epidemiological evidence links acid reflux to adenocarcinoma in Barrett's esophagus, but few studies have examined the cellular mechanisms by which acid promotes this neoplastic progression. We hypothesized that extracellular acid exposure causes intracellular acidification that triggers MAPK signaling and proliferation in Barrett's epithelial cells. We tested that hypothesis in a Barrett's-derived esophageal adenocarcinoma cell line (SEG-1). SEG-1 cells were exposed to varying concentrations of acid, and intracellular pH (pHi) was measured by 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein microfluorimetry. After acid exposure, ERK and p38 MAPK activation were measured by Western blot analysis and an immune complex kinase assay. Proliferation was measured by Coulter counter cell counts and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide incorporation assay. Exposure of SEG-1 cells to solutions with a pH between 3 and 6.5 caused a rapid, reversible decrease in pHito a level approximately equal to extracellular pH. Acid exposure caused a rapid activation of both ERK and p38 MAPKs and also resulted in pH-dependent increases in cell number, with a maximum increase of 41% observed at pH 6.0. The MAPK activation and proliferation in SEG-1 cells induced by acid exposure could be blocked by pretreatment with disodium 4,4′-diisothiocyanatostilbine-2,2′-disulfonate (DIDS), which prevents intracellular acidification by inhibiting the Cl−/HCO3−exchanger. In conclusion, in SEG-1 cells, extracellular acid exposure causes intracellular acidification, which activates MAPK and causes proliferation. The magnitude of these effects is pH dependent, and the effects can be inhibited by preventing intracellular acidification with DIDS.
Neural Mechanisms of Stimulus Velocity Tuning in the Superior Colliculus
Authors: Razak Khaleel A. , Pallas Sarah L.
Published: August 2005

Superior colliculus (SC)–mediated control of visuomotor behavior depends on neuronal selectivity for stimulus velocity. However, the mechanism responsible for velocity tuning in SC neurons is unclear. It was shown in a previous study of anesthetized, decorticate hamsters that the number and distribution of feed-forward retinal inputs are not critical for velocity tuning. Here the alternate hypothesis that inhibition from the surround determines velocity tuning of SC neurons was tested. Surround inhibition was present in 65% (43/66) of SC neurons recorded in the superficial gray layer. Neurons within this group that were selective for slowly moving stimuli exhibited spatially asymmetric surround inhibition, and their velocity tuning arose by preferential suppression of responses to rapidly moving stimuli. In the other 35% (23/66) of SC neurons recorded, surround inhibition was weak or absent and did not play a role in velocity tuning. Most neurons with surround inhibition were nonselective for the duration of stationary flashed stimuli, whereas neurons without surround inhibition were selective for stimulus duration. The majority of neurons that preferred intermediate or rapidly moving stimuli exhibited spatially symmetric surround inhibition. In these neurons, occluding the surround reduced velocity selectivity by enhancing responses to slowly moving stimuli. Based on these data, a model is proposed suggesting spatiotemporal interactions between inhibition and excitation that could underlie velocity tuning.
Application of Dye-Enhanced Laser Ablation for Liver Resection
Authors: Noritomi T. , Yamashita Y. , Kodama T. , Mikami K. , Hashimoto T. , Konno T. , Maekawa T. , Shirakusa T.
Published: August 2005

Background: Dye-enhanced laser ablation (DLA) using a low-power diode laser for indocyanine green (ICG)-stained tissue has proven its effectiveness in dye-enhanced laser photocoagulation of retinal vessels or endoscopic surgical mucosectomy. We have applied DLA in hepatectomy and described its histological distinction in comparison with the cavitron ultrasonic surgical aspirator (CUSA). Methods: A diode laser (UDL-60 Laser unit, Olympus, Tokyo, Japan) with 810 ± 20 nm wavelength was employed for this study. The ICG dye (Diagnogreen®, Daiichi Pharmaceutical, Tokyo, Japan) with a peak absorption wavelength at 800–810 nm was injected topically into the resection plane of the liver. The liver tissue was divided by touching the tip of the diode laser. Three different concentrations of ICG solution such as 2.0, 1.0 and 0.5 mg/ml were tested in the preliminary animal experiment. The use of a low-power diode laser at 10 W with an ICG concentration of 0.5 mg/ml was the appropriate combination for liver resection. In the clinical series, 27 hepatectomies were performed by DLA, and 10 with CUSA. Results: DLA demonstrated smooth cutting and good hemostasis in liver resection. Among the hepatectomy cases given DLA, no postoperative hemorrhage or bile leakage was noted. The postoperative hospital stay was significantly shorter in the DLA than the CUSA group. The cut surface of the liver was sealed microscopically with a layer of protein coagulum. Conclusions: A layer of protein sealant on the cut surface of the liver contributes to the short postoperative hospital stay when using DLA.
Surgical Anatomy of the Inguinal Region: Implications during Inguinal Laparoscopic Herniorrhaphy
Authors: Totté E. , van Hee R. , Kox G. , Hendrickx L. , van Zwieten K.J.
Published: August 2005

Introduction:In laparoscopic inguinal hernia repair the inguinal region is approached and hernia repair performed from the interior side instead of the classical open external access. Exploration and placement of staplers in the internal inguinal region during laparoscopic hernia repair may sever different anatomical structures, or induce specific complications such as nerve entrapment, neuralgia, hematomas or osteitis. The incidence of these complications may be reduced by careful dissection of the preperitoneal tissues and by placing a prosthetic mesh without the use of stapling. As laparoscopic techniques evolved, different sizes of meshes have been used. An exact determination of mesh size was hitherto not investigated. Aim: Cadaver studies of the topography of blood vessels and nerves in the preperitoneal tissue in this region were carried out in order to assess a safe position and adequate size of the prosthetic mesh. Methods: Dissection in 6 preserved human female cadavers was performed to define the actual surface of the internal inguinal region. A physical model was developed to formulate the ideal size of the prosthesis. Specific measurements were used to define the maximal size of the meshes, so as to place them without stapling, and without inducing neurovascular complications. Results: The designed physical formula defines the size of the mesh as a function of the maximum intra-abdominal pressure, the size of the abdominal wall defect and the abdominal wall tension. Conclusion: On mathematical and physical grounds our study points out that the size of the currently used prosthetic mesh (10 × 15 cm) is large enough to be placed without stapling so that with proper placement no recurrences should occur.
Safety Considerations of Adjuvant Therapy in Early Breast Cancer in Postmenopausal Women
Authors: Gradishar William J.
Published: August 2005

Because of its proven efficacy profile based on long-term data, tamoxifen has been the standard adjuvant endocrine therapy for hormone-sensitive early breast cancer for the past 30 years. However, there is well-established evidence that long-term use of tamoxifen is associated with serious side effects. As adjuvant endocrine therapy is generally administered for long periods of time, the safety and tolerability of agents used in this setting are of particular importance. Due to their superior efficacy over tamoxifen, newer agents, such as the third-generation aromatase inhibitors (AIs), are already established therapies for the treatment of advanced breast cancer. In addition, recent trials indicate that the AI anastrozole (‘Arimidex’) has improved efficacy compared with tamoxifen in the adjuvant setting in postmenopausal women. The other third-generation AIs have reported disease-free survival benefits but not in the absence of prior treatment with tamoxifen; letrozole (‘Femara’) has been compared with placebo following 5 years of tamoxifen therapy and exemestane (‘Aromasin’) has been compared with tamoxifen following 2–3 years of prior treatment with tamoxifen. Long-term safety data show that anastrozole also has a more favorable overall safety profile compared with tamoxifen, particularly in terms of life-threatening events such as endometrial cancer and thromboembolism. Anastrozole alone, therefore, provides a new option for adjuvant therapy in postmenopausal women with hormone-receptor-positive early breast cancer. The AIs have differing pharmacological profiles, which may translate into dissimilar adverse event profiles in the adjuvant treatment setting, but patient follow-up in most trials is relatively short to make a valid comparison. It cannot, therefore, be assumed that all AIs will be equally well tolerated in the adjuvant setting. Further data on the long-term safety of AIs other than anastrozole are therefore required to allow overall risk:benefit assessments on these agents to be made.