bluuebunny/crossref_metadata_2025_split huggingface dataset:
Production of a Recombinant Major Inner Capsid Protein for Serological Detection of Epizootic Hemorrhagic Disease Virus
Authors: Luo Lizhong , Sabara Marta I. 
Published: August 2005

ABSTRACT
Constructs of the major core protein, designated VP7, from epizootic hemorrhagic disease virus (EHDV) type 1 were made by amino- or carboxyl-terminal fusion of a six-histidine residue tag to the VP7-1 gene. The resulting fusion proteins were produced in a baculovirus expression system and purified by a rapid, one-step procedure using nickel-nitrilotriacetic acid technology. A high level of VP7-1 protein expression was detected with the N-terminal six-histidine tag fusion construct and was comparable to the level of expression observed with an untagged VP7-1 Bam construct. In contrast, the inclusion of a six-histidine tag at the C terminus adversely affected protein expression. The antigenicity of the N-terminal six-histidine tag EHDV VP7-1 product was identical to that observed with the native virus antigen and untagged EHDV VP7-1 recombinant protein, as determined by reactivity with EHDV specific antibodies in an enzyme-linked immunosorbent assay (ELISA) and Western blot. The high production and purity levels that can be attained for the N-terminal six-histidine tag VP7-1 protein and its reactivity with EHDV-specific sera in a competitive ELISA make it a suitable assay reagent.
Improvement of Lyme Borreliosis Serodiagnosis by a Newly Developed Recombinant Immunoglobulin G (IgG) and IgM Line Immunoblot Assay and Addition of VlsE and DbpA Homologues
Authors: Goettner Gereon , Schulte-Spechtel Ulrike , Hillermann Ruth , Liegl Gabi , Wilske Bettina , Fingerle Volker 
Published: August 2005

ABSTRACT

We developed and evaluated a recombinant
Borrelia
line immunoblot assay based on 18 homologues of seven different antigens, i.e., p100, p58, p41i, BmpA, VlsE, OspC, and DbpA. Each recombinant antigen can be detected separately and is distinct even from homologues with identical molecular weights. This blot was compared to the recently described recombinant
Borrelia
Western immunoblot assay (U. Schulte-Spechtel, G. Lehnert, G. Liegl, V. Fingerle, C. Heimerl, B. J. Johnson, and B. Wilske, J. Clin. Microbiol. 41:1299-1303, 2003). To verify sensitivity and specificity, both blots were evaluated for reactivity with
Borrelia
-specific immunoglobulin G (IgG) and IgM antibodies with 85 sera from patients with different manifestations of Lyme borreliosis and 110 controls. According to European interpretation criteria for
Borrelia
Western blots, which define a serum as positive when it recognizes at least two bands, sensitivity increased significantly from 70.6% (Western blot) to 84.7% (line blot) for IgG (
P
= 0.042) and from 40.0% (Western blot) to 73.8% (line blot) for IgM (
P
< 0.005). The increased sensitivity for IgG detection is due to the new line blot technique, whereas the improvement in detection of IgM is mainly achieved through incorporation of the additional antigens. Notably, the recombinant VlsE of
Borrelia garinii
strain PBi displayed the highest sensitivity of all antigens tested for IgG detection and is also one of the most useful antigens for IgM. Due to its excellent sensitivity and specificity combined with ease of evaluation, this line immunoblot assay offers a useful improvement in serodiagnosis of Lyme borreliosis.
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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT
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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACTColonization 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.