bluuebunny/crossref_metadata_2025_split huggingface dataset:
Purification and Characterization of Nipah Virus Nucleocapsid Protein Produced in Insect Cells
Authors: Eshaghi Majid , Tan Wen Siang , Ong Swee Tin , Yusoff Khatijah 
Published: July 2005

ABSTRACT

The nucleocapsid (N) protein of Nipah virus (NiV) is a major constituent of the viral proteins which play a role in encapsidation, regulating the transcription and replication of the viral genome. To investigate the use of a fusion system to aid the purification of the recombinant N protein for structural studies and potential use as a diagnostic reagent, the NiV N gene was cloned into the pFastBacHT vector and the His-tagged fusion protein was expressed in Sf9 insect cells by recombinant baculovirus. Western blot analysis of the recombinant fusion protein with anti-NiV antibodies produced a band of approximately 62 kDa. A time course study showed that the highest level of expression was achieved after 3 days of incubation. Electron microscopic analysis of the NiV recombinant N fusion protein purified on a nickel-nitrilotriacetic acid resin column revealed different types of structures, including spherical, ring-like, and herringbone-like particles. The light-scattering measurements of the recombinant N protein also confirmed the polydispersity of the sample with hyrdrodynamic radii of small and large types. The optical density spectra of the purified recombinant fusion protein revealed a high
A
260
/
A
280
ratio, indicating the presence of nucleic acids. Western blotting and enzyme-linked immunosorbent assay results showed that the recombinant N protein exhibited the antigenic sites and conformation necessary for specific antigen-antibody recognition.
Cysticercosis Immunodiagnosis Using 18- and 14-Kilodalton Proteins from
Taenia crassiceps
Cysticercus Antigens Obtained by Immunoaffinity Chromatography
Authors: Espíndola Noeli Maria , Iha Alberto Hiroshi , Fernandes Irene , Takayanagui Osvaldo Massaiti , Machado Luís dos Ramos , Livramento José Antônio , Mendes Maia Antônio Augusto , Peralta José Mauro , Vaz Adelaide José 
Published: July 2005

ABSTRACT

Monoclonal antibodies (MAb) against
Taenia crassiceps
and
Taenia solium
cysticerci were produced and showed cross-reactivity with a 14-kDa protein from
T. solium
and with 18- and 14-kDa proteins from
T. crassiceps
. These MAbs and antibodies from cerebrospinal fluid (CSF) as well as serum samples from patients with neurocysticercosis (NC) reacted with 18- and 14-kDa
T. crassiceps
proteins purified by immunoaffinity chromatography using a Sepharose column coupled with MAbs (anti-excretory/secretory or anti-vesicular fluid antigens). Immunoaffinity-purified 18- and 14-kDa proteins were used in the design of a diagnostic enzyme-linked immunosorbent assay (ELISA) to detect antibodies in 23 CSF and 20 serum samples from patients with NC, showing 100% sensitivity. The test specificity was determined using 42 noninflammatory CSF samples and 70 inflammatory CSF samples from patients with other neurological disorders (OND), showing 100% and 99.1% (confidence interval, 97.3% to 100%) specificity, respectively. A false-positive CSF sample result in the OND group was from a human immunodeficiency virus-positive patient with meningoencephalitis. By using serum samples from 194 healthy individuals, the specificity was 100%. Analysis of an additional 16 serum samples from individuals with other parasitic diseases (13 with intestinal parasitosis and 3 with schistosomiasis) showed negative results. Three (10%) serum samples from patients with hydatidosis were positive in our ELISA and in ELISA with
T. solium
cysticerci antigens. Two of them were also positive by immunoblotting. The use of 18- and 14-kDa
T. crassiceps
immunoaffinity-purified proteins for detection of anti-cysticercus antibodies in CSF and/or serum samples using an ELISA system showed a good performance and high specificity for serum samples, dispensing with the use of confirmatory tests, such as immunoblotting, for checking specificity.
Genomic Deletions Classify the Beijing/W Strains as a Distinct Genetic Lineage of
Mycobacterium tuberculosis
Authors: Tsolaki Anthony G. , Gagneux Sebastien , Pym Alexander S. , Goguet de la Salmoniere Yves-Olivier L. , Kreiswirth Barry N. , Van Soolingen Dick , Small Peter M. 
Published: July 2005

ABSTRACT

Beijing/W strains of
Mycobacterium tuberculosis
are geographically widespread and hypervirulent. To enhance our understanding of their origin and evolution, we sought phylogenetically informative large sequence polymorphisms (LSPs) within the Beijing/W family. Comparative whole-genome hybridization of Beijing/W strains revealed 21 LSPs, 7 of which were previously unreported. We show that some of these LSPs are unique event polymorphisms that can be used to define and subdivide the Beijing/W family. One LSP (RD105) was seen in all Beijing/W strains and thus serves as a useful marker for the identification of this family of strains. Additional LSPs (RD142, RD150, and RD181) further divided this family into four monophyletic subgroups, demonstrating a deeper population structure than previously appreciated. All Beijing/W strains were also observed to have an intact
pks15/1
gene that is involved in the biosynthesis of a phenolic glycolipid, a putative virulence factor. A simple PCR assay using these Beijing/W strain-defining deletions will facilitate molecular epidemiological studies and may assist in the identification of the molecular basis of phenotypes associated with this important lineage of
M. tuberculosis
.
Genotyping of Methicillin-Resistant
Staphylococcus aureus
Strains from Two Hospitals in Bangalore, South India
Authors: Arakere Gayathri , Nadig Savitha , Swedberg Göte , Macaden Ragini , Amarnath Satish K. , Raghunath Dasarathy 
Published: July 2005

ABSTRACT

Methicillin-resistant
Staphylococcus aureus
(MRSA) is a major nosocomial pathogen in India, and up to 70% methicillin resistance has been reported from hospitals in various parts of India. Hospitals use phenotyping for the most part, and molecular genotyping is not done. Here we report on the genotyping of 82 single-patient isolates from two hospitals in Bangalore, South India, for the first time. Most of the strains possessed type III or IIIA staphylococcal cassette chromosome (
SCCmec
) cassettes, and we did not detect strains with type I, IA, or II cassettes. Most isolates also contained the type III cassette chromosome recombinase (
ccr
) AB region. Multilocus sequence typing (MLST) and staphylococcal protein A (
spa
) typing of a selected number of isolates have been carried out. Although most isolates that were chosen for MLST and
spa
typing had the same patterns, they were quite diverse in their pulsed-field gel electrophoresis (PFGE) patterns. PFGE, MLST, and
spa
typing of the Indian strains revealed that they are related to the previously described Hungarian and Brazilian clones.
Characterization of a Novel P[25],G11 Human Group A Rotavirus
Authors: Rahman Mustafizur , Matthijnssens Jelle , Nahar Sharifun , Podder Goutam , Sack David A. , Azim Tasnim , Van Ranst Marc 
Published: July 2005

ABSTRACT
A novel rotavirus strain (Dhaka6) isolated from a 21-year-old Bangladeshi male patient was characterized by sequence analysis of its VP7 and VP4 gene segments. Phylogenetic analysis of the VP7 gene of the Dhaka6 strain revealed a common evolutionary lineage with porcine G11 rotavirus strains. This isolate is the first reported G11 rotavirus strain infecting a human host. Comparison of the VP4 gene sequences with all currently recognized 24 different P genotypes revealed only low nucleotide (54 to 71%) and amino acid (52 to 76%) sequence identities. This lack of high sequence similarity in the VP4 gene indicates that the Dhaka6 isolate represents a new group A rotavirus P genotype, to which we propose assignment of the designation P[25].
Characterization of a Shiga Toxin-, Intimin-, and Enterotoxin Hemolysin-Producing
Escherichia coli
ONT:H25 Strain Commonly Isolated from Healthy Cattle
Authors: Sheng Haiqing , Davis Margaret A. , Knecht Hannah J. , Hancock Dale D. , Van Donkersgoed Joyce , Hovde Carolyn J. 
Published: July 2005

ABSTRACT

Among bovine fecal and recto-anal mucosal swab samples cultured in our laboratory for
Escherichia coli
O157:H7, we frequently isolated
E. coli
organisms that were phenotypically similar to the O157:H7 serotype as non-sorbitol fermenting and negative for β-glucuronidase activity but serotyped O nontypeable:H25 (ONT:H25). This study determined the prevalence and virulence properties of the
E. coli
ONT:H25 isolates. Among dairy and feedlot cattle (
n
= 170) sampled in Washington, Idaho, and Alberta, Canada, the percentage of animals culture positive for
E. coli
ONT:H25 ranged from 7.5% to 22.5%, compared to the prevalence of
E. coli
O157:H7 that ranged from 0% to 15%. A longitudinal 8-month study of dairy heifers (
n
= 40) showed that 0 to 15% of the heifers were culture positive for
E. coli
O157:H7, while 15 to 22.5% of the animals were culture positive for
E. coli
ONT:H25. As determined by a multiplex PCR, the
E. coli
ONT:H25 isolates carried a combination of virulence genes characteristic of the enterohemorrhagic
E. coli
, including intimin, translocated intimin receptor, Stx2, and hemolysin (
eae-
β,
tir
,
stx
2vh-a
, and
hly
).
E. coli
ONT:H25 isolates from diverse geographic locations and over time were fingerprinted by separating XbaI-restricted chromosomal DNA by pulsed-field gel electrophoresis (PFGE) separation. Two strains of
E. coli
ONT:H25 were highly similar by PFGE pattern. Experimental inoculation of cattle showed that
E. coli
ONT:H25, like
E. coli
O157:H7, colonized the bovine recto-anal junction mucosa for more than 4 weeks following a single rectal application of bacteria.
Comparative Recovery of Microorganisms from BacT/ALERT Plastic and Glass FA and FN Blood Culture Bottles
Authors: Riley J. A. , Heiter B. J. , Bourbeau P. P. 
Published: July 2005

ABSTRACT

bioMerieux, Inc., has recently introduced plastic bottles to replace glass bottles for use in the BacT/ALERT blood culture system. We compared the performance of the plastic to the glass bottles in a large clinical evaluation. Two blood cultures were collected from each patient, one using glass FA (aerobic) and FN (anaerobic) bottles and one using plastic FA and FN bottles. Of the 4,040 sets of four bottles collected, 3,110 contained the recommended 8 to 12 ml of blood, yielding 524 microorganisms with 359 judged to be clinically significant. Of the 359 significant organisms, 255 were recovered in either one or two bottles from both pairs of bottles in a set while 56 organisms were recovered only from the glass bottles and 48 were recovered only from the plastic bottles (
P
, not significant [NS]). Of the 286 significant organisms recovered only in the FA bottles (glass and plastic), 180 were recovered in both bottles, 57 in the plastic bottles only, and 49 in the glass bottles only (
P
, NS). Of the 303 significant organisms recovered in the FN bottles only (glass and plastic), 212 were recovered in both bottles, 46 in the plastic bottles only, and 45 in the glass bottles only (
P
, NS). For individual organisms, the only significant difference in recovery was obtained for
Escherichia coli
, with more isolates recovered in the FN plastic than in the FN glass bottles (
P
= 0.02). These data suggest that recovery of microorganisms with plastic FA/FN bottles is at least equal to that with glass FA/FN bottles while offering greater safety for users.
Characterization of Vancomycin-Resistant
Enterococcus faecium
Isolates from Broiler Poultry and Pig Farms in England and Wales
Authors: Garcia-Migura L. , Pleydell E. , Barnes S. , Davies R. H. , Liebana E. 
Published: July 2005

ABSTRACT

This study aimed to investigate the occurrence and molecular epidemiology of vancomycin-resistant
Enterococcus faecium
(VREF) isolates on poultry and pig farms in England and Wales. A total of 217 VREF isolates were obtained from fresh feces and environmental swabs collected from conventional and organic farms. A predominant pulsed-field gel electrophoresis (PFGE) profile was found for each VREF-positive farm, together with less frequent types. All isolates presented the
vanA
genotype and were
esp
negative. Seventy-six percent of the VREF isolates were additionally resistant to nine or more antimicrobials, presenting a diverse range of resistance phenotypes. The multiresistance traits did not appear to be specific to individual farms or sample types (i.e., environmental or fecal), nor did they correlate with any specific PFGE type. Ninety-three percent of the isolates were resistant to penicillin, 89% were resistant to tetracycline, 87.5% were resistant to erythromycin, and 50% were resistant to quinupristin-dalfospristin (Synercid). The lack of clonality among these populations may suggest the horizontal transfer of resistance genes and/or a dynamic replacement of clonal lines rather than persistence.
Sensitive and Rapid Detection of Herpes Simplex Virus and Varicella-Zoster Virus DNA by Loop-Mediated Isothermal Amplification
Authors: Kaneko Hisatoshi , Iida Tomohiro , Aoki Koki , Ohno Shigeaki , Suzutani Tatsuo 
Published: July 2005

ABSTRACT
Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method in which reagents react rapidly and efficiently, with a high specificity, under isothermal conditions. We used a LAMP assay for the detection of herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), and varicella-zoster virus (VZV). The virus specificities of primers were confirmed by using 50 HSV-1, 50 HSV-2, and 8 VZV strains. The assay was performed for 45 min at 65°C. The LAMP assay had a 10-fold higher sensitivity than a PCR assay. An analysis of nucleotide sequence variations in the target and primer regions used for the LAMP assay indicated that 3 of 50 HSV-1 strains had single nucleotide polymorphisms. No HSV-2 or VZV strains had nucleotide polymorphisms. Regardless of the sequence variation, there were no differences in sensitivity with the HSV-1-specific LAMP assay. To evaluate the application of the LAMP assay for clinical diagnosis, we tested clinical samples from 40 genital herpes patients and 20 ocular herpes patients. With the LAMP assay, 41 samples with DNA extraction and 26 direct samples without DNA extraction were identified as positive for HSV-1 or HSV-2, although 37 samples with DNA extraction and just one without DNA extraction were positive by a PCR assay. Thus, the LAMP assay was less influenced than the PCR assay by the presence of inhibitory substances in clinical samples. These observations indicate that the LAMP assay is very useful for the diagnosis of HSV-1, HSV-2, and VZV infections.
Enterobacter hormaechei
subsp.
oharae
subsp. nov.,
E. hormaechei
subsp.
hormaechei
comb. nov., and
E. hormaechei
subsp.
steigerwaltii
subsp. nov., Three New Subspecies of Clinical Importance
Authors: Hoffmann Harald , Stindl Sibylle , Ludwig Wolfgang , Stumpf Anita , Mehlen Andre , Monget Daniel , Pierard Denis , Ziesing Stefan , Heesemann Jürgen , Roggenkamp Andreas , Schleifer Karl H. 
Published: July 2005

ABSTRACT

Six species and six additional genovars are combined within the so-called
Enterobacter cloacae
complex, with one of them being the species
Enterobacter hormaechei
. In a recent population genetic study, two genetic clusters were found in close phylogenetic proximity to the genetic cluster of
E. hormaechei
. In order to prove the hypothesis that these three genetic clusters belong to the same species, we performed cross-hybridization experiments in microplates with DNAs of representatives of each genetic cluster. The close phylogenetic relationship among the clusters was reflected by their relatively low Δ

T
m

values, ranging from 0.3 to 4.8, confirming the hypothesis that the clusters are parts of the same species. These clusters can be distinguished from the other species of the
E. cloacae
complex, which have Δ

T
m

values of 5.6 to 10.3. Forty-eight
E. hormaechei
strains from the different genetic clusters were phenotypically characterized with 129 biochemical tests. In this way,
E. hormaechei
could be differentiated from the other species of the
E. cloacae
complex because it tests negative in the 3-hydroxy-butyrate test. The three genetic clusters of
E. hormaechei
could also be differentiated from each other by using phenotypic tests. Hence, we propose three new subspecies of
E. hormaechei
corresponding to genetic clusters VI, VII, and VIII of the
E. cloacae
complex.
E. hormaechei
subsp.
hormaechei
comb. nov. corresponds to the original species description, as it gives negative results for the adonitol,
d
-arabitol,
d
-sorbitol, and
d
-melibiose tests and a positive result for the dulcitol test.
E. hormaechei
subsp.
oharae
subsp. nov. gives negative results for the dulcitol, adonitol, and
d
-arabitol tests and positive results for the
d
-sorbitol and
d
-melibiose tests.
E. hormaechei
subsp.
steigerwaltii
subsp. nov. gives a negative result for the dulcitol test and positive results for the adonitol,
d
-arabitol,
d
-sorbitol, and
d
-melibiose tests. Among the members of the
E. cloacae
complex,
E. hormaechei
seems to be the species most frequently recovered from clinical specimens.
Use of a Locked-Nucleic-Acid Oligomer in the Clamped-Probe Assay for Detection of a Minority
Pfcrt
K76T Mutant Population of
Plasmodium falciparum
Authors: Senescau Alice , Berry Antoine , Benoit-Vical Françoise , Landt Olfert , Fabre Richard , Lelièvre Joël , Cassaing Sophie , Magnaval Jean-François 
Published: July 2005

ABSTRACT

Given the emergence of drug resistance and the high rate of polyclonal microorganism infections, the availability of a fast and sensitive test to detect minority mutant populations would be an improvement in the diagnosis of infectious diseases. A clamped-probe real-time PCR assay to diagnose the
Plasmodium falciparum
K76T mutation in clone populations was developed, using a wild-type-specific locked-nucleic-acid-containing oligomer to suppress wild-type PCR amplification and to enhance melting analysis with a mutation-specific detection probe.
Identification of Two Distinct Hybridization Groups in the Genus
Hafnia
by 16S rRNA Gene Sequencing and Phenotypic Methods
Authors: Janda J. Michael , Abbott Sharon L. , Bystrom Sue , Probert Will S. 
Published: July 2005

ABSTRACT

A collection of 52 strains belonging to the
Hafnia alvei
complex were subjected to molecular (16S rRNA gene sequencing) and biochemical analysis. Based upon 16S rRNA gene sequencing results, two genetic groups were identified which correspond with previously recognized DNA hybridization group 1 (ATCC 13337
T
and ATCC 29926;
n
= 23) and DNA hybridization group 2 (ATCC 29927;
n
= 29). Of 46 biochemical tests used to characterize hafniae, 19 reactions (41%) yielded variable results. Of these 19 tests, 6 were determined to have discriminatory value in the separation of DNA groups 1 and 2, with malonate utilization found to be the most differential test. Test results of malonate utilization alone correctly assigned 90% of
Hafnia
isolates to their correct DNA group.
Multilocus DNA Sequence Comparisons Rapidly Identify Pathogenic Molds
Authors: Rakeman Jennifer L. , Bui Uyen , LaFe Karen , Chen Yi-Ching , Honeycutt Rhonda J. , Cookson Brad T. 
Published: July 2005

ABSTRACT
The increasing incidence of opportunistic fungal infections necessitates rapid and accurate identification of the associated fungi to facilitate optimal patient treatment. Traditional phenotype-based identification methods utilized in clinical laboratories rely on the production and recognition of reproductive structures, making identification difficult or impossible when these structures are not observed. We hypothesized that DNA sequence analysis of multiple loci is useful for rapidly identifying medically important molds. Our study included the analysis of the D1/D2 hypervariable region of the 28S ribosomal gene and the internal transcribed spacer (ITS) regions 1 and 2 of the rRNA operon. Two hundred one strains, including 143 clinical isolates and 58 reference and type strains, representing 43 recognized species and one possible new species, were examined. We generated a phenotypically validated database of 118 diagnostic alleles. DNA length polymorphisms detected among ITS1 and ITS2 PCR products can differentiate 20 of 33 species of molds tested, and ITS DNA sequence analysis permits identification of all species tested. For 42 of 44 species tested, conspecific strains displayed >99% sequence identity at ITS1 and ITS2; sequevars were detected in two species. For all 44 species, identifications by genotypic and traditional phenotypic methods were 100% concordant. Because dendrograms based on ITS sequence analysis are similar in topology to 28S-based trees, we conclude that ITS sequences provide phylogenetically valid information and can be utilized to identify clinically important molds. Additionally, this phenotypically
validated database of ITS sequences will be useful for identifying new species of pathogenic molds.
Use of an Automated Multiple-Locus, Variable-Number Tandem Repeat-Based Method for Rapid and High-Throughput Genotyping of
Staphylococcus aureus
Isolates
Authors: Francois Patrice , Huyghe Antoine , Charbonnier Yvan , Bento Manuela , Herzig Sébastien , Topolski Ivan , Fleury Bénédicte , Lew Daniel , Vaudaux Pierre , Harbarth Stephan , van Leeuwen Willem , van Belkum Alex , Blanc Dominique S. , Pittet Didier , Schrenzel Jacques 
Published: July 2005

ABSTRACT

Fast and reliable genotyping methods that allow real-time epidemiological surveillance would be instrumental to monitoring of the spread of methicillin-resistant
Staphylococcus aureus
. We describe an automated variable-number tandem repeat-based method for the rapid genotyping of
Staphylococcus aureus
. Multiplex PCR amplifications with eight primer pairs that target gene regions with variable numbers of tandem repeats were resolved by microcapillary electrophoresis and automatically assessed by cluster analysis. This genotyping technique was evaluated for its discriminatory power and reproducibility with clinical isolates of various origins, including a panel of control strains previously characterized by several typing methods and collections from either long-term carriers or defined nosocomial outbreaks. All steps of this new procedure were developed to ensure a rapid turnaround time and moderate cost. The results obtained suggest that this rapid approach is a valuable tool for the genotyping of
S. aureus
isolates in real time.