bluuebunny/crossref_metadata_2025_split huggingface dataset:
Development and Evaluation of Serotype- and Group-Specific Fluorogenic Reverse Transcriptase PCR (TaqMan) Assays for Dengue Virus
Authors: Callahan Johnny D. , Wu Shuenn-Jue L. , Dion-Schultz Amanda , Mangold Beverly E. , Peruski Leonard F. , Watts Douglas M. , Porter Kevin R. , Murphy Gerald R. , Suharyono Wuryadi , King Chwan-Chuen , Hayes Curtis G. , Temenak Joseph J. 
Published: July 2002

ABSTRACT
Five fluorogenic probe hydrolysis (TaqMan) reverse transcriptase PCR (RT-PCR) assays were developed for serotypes 1 to 4 and group-specific detection of dengue virus. Serotype- and group-specific oligonucleotide primers and fluorogenic probes were designed against conserved regions of the dengue virus genome. The RT-PCR assay is a rapid single-tube method consisting of a 30-min RT step linked to a 45-cycle PCR at 95 and 60°C that generates a fluorogenic signal in positive samples. Assays were initially evaluated against cell culture-derived dengue stock viruses and then with 67 dengue viremic human sera received from Peru, Indonesia, and Taiwan. The TaqMan assays were compared to virus isolation using C6/36 cells followed by an immunofluorescence assay using serotype-specific monoclonal antibodies. Viral titers in sera were determined by plaque assay in Vero cells. The serotype-specific TaqMan RT-PCR assay detected 62 of 67 confirmed dengue virus-positive samples, for a sensitivity of 92.5%, while the group-specific assay detected 66 of 67 confirmed dengue virus-positive samples, for a sensitivity of 98.5%. The TaqMan RT-PCR assays have a specificity of 100% based on the serotype concordance of all assays compared to cell culture isolation and negative results obtained when 21 normal human sera and plasma samples were tested. Our results demonstrate that the dengue virus TaqMan RT-PCR assays may be utilized as rapid, sensitive, and specific screening and serotyping tools for epidemiological studies of dengue virus infections.
Evaluation of Typing of
Vibrio
parahaemolyticus
by Three PCR Methods Using Specific Primers
Authors: Wong Hin-Chung , Lin Chih-Hsueh 
Published: July 2002

ABSTRACT

Vibrio
parahaemolyticus
is a halophilic bacterium frequently involved in human outbreaks of seafood-associated gastroenteritis. For epidemiological purposes, different molecular typing methods, such as pulsed-field gel electrophoresis (PFGE) or ribotyping, have been developed for this pathogen; however, these methods are mostly labor-intensive and time-consuming. In this work, we designed and evaluated three rapid PCR typing methods for this pathogen using primers designed on the basis of the following specific sequences: conserved ribosomal gene spacer sequence (RS), repetitive extragenic palindromic sequence (REP), and enterobacterial repetitive intergenic consensus sequence (ERIC). Typing patterns and clustering analysis indicated that these methods apparently differentiated
V
.
parahaemolyticus
strains from reference strains of interspecific
Escherichia
coli
,
V
.
cholerae
, and
V
.
vulnificus
and were also valuable in subspecies typing of this pathogen. Forty domestic strains of
V
.
parahaemolyticus
, representing a wide range of PFGE patterns, were grouped into 15, 27, and 27 patterns, with discrimination indexes of 0.91, 0.97, and 0.98, by RS-, REP-, and ERIC-PCR, respectively. The discriminative abilities of these PCR methods closely approached or even exceeded those of PFGE and ribotyping. REP-PCR is preferable to ERIC-PCR because of the greater reproducibility of its fingerprints, while RS-PCR may be a practical method because it generates fewer amplification bands and patterns than the alternatives.
Antimicrobial Resistance and Bacterial Identification Utilizing a Microelectronic Chip Array
Authors: Westin Lorelei , Miller Carolyn , Vollmer Dana , Canter David , Radtkey Ray , Nerenberg Michael , O'Connell James P. 
Published: July 2002

ABSTRACT

Species-specific bacterial identification of clinical specimens is often limited to a few species due to the difficulty of performing multiplex reactions. In addition, discrimination of amplicons is time-consuming and laborious, consisting of gel electrophoresis, probe hybridization, or sequencing technology. In order to simplify the process of bacterial identification, we combined anchored in situ amplification on a microelectronic chip array with discrimination and detection on the same platform. Here, we describe the simultaneous amplification and discrimination of six gene sequences which are representative of different bacterial identification assays:
Escherichia coli gyrA
,
Salmonella gyrA
,
Campylobacter gyrA
,
E. coli parC
,
Staphylococcus mecA,
and
Chlamydia
cryptic plasmid. The assay can detect both plasmid and transposon genes and can also discriminate strains carrying antibiotic resistance single-nucleotide polymorphism mutations. Finally, the assay is similarly capable of discriminating between bacterial species through reporter-specific discrimination and allele-specific amplification. Anchored strand displacement amplification allows multiplex amplification and complex genotype discrimination on the same platform. This assay simplifies the bacterial identification process greatly, allowing molecular biology techniques to be performed with minimal processing of samples and practical experience.
Helicobacter pylori
Infection in an Urban African Population
Authors: Fernando Neluka , Holton John , Zulu Isaac , Vaira Dino , Mwaba Peter , Kelly Paul 
Published: July 2002

ABSTRACT

We have studied 221 adults drawn from an impoverished urban population with high human immunodeficiency virus (HIV) seroprevalence (35%) to determine the prevalence of gastroduodenal pathology and its relationship to serological markers of
Helicobacter pylori
virulence proteins and other potential environmental and immunological determinants of disease including HIV infection. Eighty-one percent were
H. pylori
seropositive, and 35% were HIV seropositive. Urban upbringing and low CD4 count were associated with a reduced likelihood of
H. pylori
seropositivity, as was current
Ascaris
infection, in keeping with recent evidence from an animal model. One hundred ninety-one adults underwent gastroduodenoscopy, and 14 had gastroduodenal pathology. Mucosal lesions were a major cause of abdominal pain in this population. While the majority of patients with gastroduodenal pathology (12 of 14) were seropositive for
H. pylori
, none were seropositive for HIV. Smoking was associated with increased risk of macroscopic pathology, and a history of
Mycobacterium bovis
BCG immunization was associated with reduced risk. Antibodies to
H. pylori
lipopolysaccharide were associated with pathology. HIV infection was associated with protection against mucosal lesions, suggesting that fully functional CD4 lymphocytes may be required for the genesis of gastroduodenal pathology.
Definition of a Divergent Epitope That Allows Differential Detection of Early Protein p41 from Human Herpesvirus 6 Variants A and B
Authors: Xu Yunhe , Linde Annika , Dahl Helena , Winberg Gosta 
Published: July 2002

ABSTRACT

The human herpesvirus 6 (HHV-6) early protein, p41, encoded by the U27 gene has been detected in oligodendrocytes of multiple sclerosis (MS) patients by using a monoclonal antibody (MAb to p41/38). We here report the antigenic epitope of HHV-6 p41 recognized by this MAb. First, we established that the MAb to p41/38 recognizes a nuclear antigen in HHV-6A strain GS-infected cells but not in HHV-6B strain Z29-infected cells. Secondly, we compared the reactivity of the MAb to p41/38 to that of another p41-specific MAb (MAb to p41) on immunoblots with purified p41-glutathione
S
-transferase fusion protein from strains GS and Z29 and GS- and Z29-infected-cell lysates. The two MAbs were tested in an enzyme-linked immunosorbent assay against a panel of synthetic peptides covering the amino acid substitutions between the GS- and Z29-derived p41 proteins, as determined by DNA sequencing of our cloned isolates of the U27 gene. The MAb to p41/38 reacted specifically with a peptide comprising p41 residues 321 to 340 from strain GS. The critical residue in this peptide was serine 328, as the substitution S328N in the Z29 strain rendered the corresponding peptide nonreactive. The p41 S328 marker was present in three of three HHV-6A strains, while four of four sequenced p41 genes from HHV-6B strains had N328. Our findings are of value for the interpretation of previous findings of p41 expression in brains of MS patients and may allow a more detailed analysis of the role of HHV-6 variants in other disorders.
Evaluation of Candida ID, a New Chromogenic Medium for Fungal Isolation and Preliminary Identification of Some Yeast Species
Authors: Fricker-Hidalgo H. , Orenga S. , Lebeau B. , Pelloux H. , Brenier-Pinchart M. P. , Ambroise-Thomas P. , Grillot R. 
Published: July 2002

ABSTRACT

Candida ID, a new chromogenic medium, allows identification of
Candida albicans
(blue colonies) and preliminary identification into a group of four species (pink colonies). In comparison with Albicans ID2 and Sabouraud gentamicin chloramphenicol on 446 fungal strains, Candida ID allowed the isolation of more species than Albicans ID 2 (95.5% versus 91.2%).
Development and Evaluation of Detection Systems for Staphylococcal Exfoliative Toxin A Responsible for Scalded-Skin Syndrome
Authors: Ladhani Shamez , Robbie Scott , Garratt Richard C. , Chapple Daniel S. , Joannou Christopher L. , Evans Robert W. 
Published: July 2002

ABSTRACT

Staphylococcal scalded-skin syndrome is usually diagnosed clinically by its characteristic exfoliating rash. Isolation of
Staphylococcus aureus
from the patient further supports the diagnosis. Several detection systems have been developed to determine whether the isolated strain produces exfoliative toxin, but none are routinely available in hospital laboratories. In a novel approach, we used computer models to predict the structure of the exfoliative toxins based on other serine proteases and to identify surface epitopes for the production of antibodies that specifically bound the exfoliative toxin A (ETA) serotype. Several rapid immunologically based diagnostic tests for ETA were developed with these antibodies and compared with existing systems. Our results showed that Western blot analysis using these antibodies was in complete correlation with PCR, which has been validated against the “gold standard” mouse model. On the other hand, the double-antibody enzyme-linked immunosorbent assay (ELISA) and Ouchterlony immunodiffusion assay gave unacceptably high false-positive results due to interference by staphylococcal protein A. This problem was successfully overcome by the development of a F(ab′)
2
fragment ELISA, which was rapid and reproducible and was as sensitive and specific as PCR and Western blot analysis. The F(ab′)
2
fragment ELISA is superior to existing diagnostic systems because it is quantitative, which may be related to the severity of the condition, and can detect amounts of exfoliative toxin in the picogram range directly from serum. This is the first detection system with the potential to confirm the diagnosis of staphylococcal scalded-skin syndrome from a routine blood test within 3 h of presentation.
Basis of the Superiority of Cefoperazone Amphotericin Teicoplanin for Isolating
Campylobacter upsaliensis
from Stools
Authors: Byrne Catherine , Doherty Deirdre , Mooney Adele , Byrne Margaret , Woodward David , Johnson Wendy , Rodgers Frank , Bourke Billy 
Published: July 2002

ABSTRACT

The optimum method for isolating
Campylobacter upsaliensis
from stools has not been clearly defined. In a preliminary study, cefoperazone amphotericin teicoplanin (CAT) selective medium isolated six
C. upsaliensis
strains which were not detected using modified cefoperazone charcoal deoxycholate (mCCDA). In order to identify the factors that underlie the superiority of CAT over mCCDA for isolating
C. upsaliensis
, we examined the effect of incubation time and antibiotic content of culture media on the growth of
C. upsaliensis
isolates using semiquantitative methods. The recovery of a subgroup of
C. upsaliensis
isolates from seeded stool specimens was also evaluated. Differences in growth of
C. upsaliensis
on CAT and mCCDA were modest and were not explained by the antibiotic profiles of the two media. Recovery of
C. upsaliensis
from spiked human feces on CAT was superior to that on mCCDA at lower concentrations of organisms (10
3
CFU/ml). We conclude that although CAT is more suitable than mCCDA for the isolation of
C. upsaliensis
from stools, the superiority of CAT for detecting this organism is not accounted for by the antibiotic composition of the medium.
Pneumococcal Carriage in Children in The Netherlands: a Molecular Epidemiological Study
Authors: Bogaert Debby , Engelen Marlene N. , Timmers-Reker Anja J. M. , Elzenaar Kees P. , Peerbooms Paul G. H. , Coutinho Roel A. , de Groot Ronald , Hermans Peter W. M. 
Published: July 2002

ABSTRACT

In 1999, Engelen and coworkers investigated colonization in Amsterdam among 259 children attending 16 day-care centers (DCCs) and among 276 children who did not attend day-care centers (NDCCs). A 1.6- to 3.4-fold increased risk for nasopharyngeal colonization was observed in children attending DCCs compared with NDCC children, while no difference in antibiotic resistance was found between groups. The serotype and genotype distributions of 305 nasopharyngeal
Streptococcus pneumoniae
isolates of the latter study were investigated. The predominant serotypes in both the DCC and the NDCC groups included 19F (19 and 18%, respectively), 6B (14 and 16%, respectively), 6A (13 and 7%, respectively), 23F (9 and 7%, respectively), and 9V (7 and 7%, respectively). The theoretical vaccine coverage of the 7-valent conjugate vaccine was 59% for the DCC children and 56% for the NDCC group. Genetic analysis of the pneumococcal isolates revealed 75% clustering among pneumococci isolated from DCC attendees versus 50% among the NDCC children. The average pneumococcal cluster size in the DCC group was 3.8 and 4.6 isolates for two respective sample dates (range, 2 to 13 isolates per cluster), while the average cluster size for the NDCC group was 3.0 (range, 2 to 6 isolates per cluster). Similar to observations made in other countries, these results indicate a higher risk for horizontal spread of pneumococci in Dutch DCCs than in the general population. This study emphasizes the importance of molecular epidemiological monitoring before, during, and after implementation of pneumococcal conjugate vaccination in national vaccination programs for children.
Phylogeny-Based Rapid Identification of Mycoplasmas and Ureaplasmas from Urethritis Patients
Authors: Yoshida Takashi , Maeda Shin-Ichi , Deguchi Takashi , Ishiko Hiroaki 
Published: July 2002

ABSTRACT

Some strains of mycoplasmas and ureaplasmas (family
Mycoplasmataceae
) are associated with nongonococcal urethritis (NGU) or other genitourinary infections. We have developed a rapid and reliable method of identifying the presence and prevalence of mycoplasmas and ureaplasmas in men with NGU. This method is based on the amplification of a part of the 16S rRNA gene by PCR and phylogenetic analysis. A portion of the 16S rRNA gene from 15 prototype strains was amplified with a set of common primers, and their nucleotides were sequenced. The nucleotide sequence of the V4 and V5 regions was analyzed by the neighbor-joining method. The 15 prototype strains were grouped into three distinct clusters, allowing us to clearly segregate the strains into distinct lineages. To determine the prevalence of these pathogens among patients with NGU, this protocol was tested with 148 urine samples. Amplifications were observed for 42 samples, and their nucleotide sequences were analyzed along with those of the 15 prototype strains. The phylogenetic tree thus constructed indicated that 15 of the 42 formed a cluster with
Mycoplasma genitalium
. Among the remaining specimens, 2 formed a cluster with
Mycoplasma hominis
, 19 with
Ureaplasma urealyticum
, and 5 with
Ureaplasma parvum
; the remaining sample contained both
M. genitalium
and
U. urealyticum
. This phylogeny-based identification of mycoplasmas and ureaplasmas provides not only a powerful tool for rapid diagnosis but also the basis for etiological studies of these pathogens.
Molecular Identification of Enterovirus by Analyzing a Partial VP1 Genomic Region with Different Methods
Authors: Palacios G. , Casas I. , Tenorio A. , Freire C. 
Published: July 2002

ABSTRACT
VP1 is the most suitable region for use in the identification of enterovirus. Although VP1 sequencing methods may vary, it is necessary to agree on a common strategy of sequence analysis. Identification of a strain type may be achieved by three different approaches: pairwise sequence alignment, multiple-sequence alignment, and phylogenetic inference. Other methods are also available, but they are not simple enough to be performed at a virology laboratory. The performances of these methods were evaluated with nucleotide and protein sequences obtained from 32 original samples, 8 enterovirus isolates, and 64 GenBank sequences. Pairwise sequence alignment methods had very different results. The DNASTAR package identified only 28.8% of enterovirus strains, while the Genetics Computer Group package identified 50.0 or 72.1% of enterovirus strains when nucleotide or amino acid sequences were analyzed, respectively. Multiple-sequence alignment methods identified 94.2% (Clustal W program) or 92.3% (Pileup program) of the enterovirus strains, while the phylogenetic method increased this rate to 99.0%. Comparative evaluation of these analysis methods showed that the Clustal W program (version 1.81), a freely available multiple-sequence alignment program, presented one of the best performances when used with the correct criteria. Other commercial and expensive programs did not achieve the same performances, making them less suitable for molecular typing of enteroviruses. Finally, although phylogenetic inference is the most demanding method in terms of knowledge of the user, it remained the best option analyzed.
Balanced Hemagglutinin and Neuraminidase Activities Are Critical for Efficient Replication of Influenza A Virus
Authors: Mitnaul Lyndon J. , Matrosovich Mikhail N. , Castrucci Maria R. , Tuzikov Alexander B. , Bovin Nikolai V. , Kobasa Darwyn , Kawaoka Yoshihiro 
Published: July 2002

ABSTRACT
The SD0 mutant of influenza virus A/WSN/33 (WSN), characterized by a 24-amino-acid deletion in the neuraminidase (NA) stalk, does not grow in embryonated chicken eggs because of defective NA function. Continuous passage of SD0 in eggs yielded 10 independent clones that replicated efficiently. Characterization of these egg-adapted viruses showed that five of the viruses contained insertions in the NA gene from the PB1, PB2, or NP gene, in the region linking the transmembrane and catalytic head domains, demonstrating that recombination of influenza viral RNA segments occurs relatively frequently. The other five viruses did not contain insertions in this region but displayed decreased binding affinity toward sialylglycoconjugates, compared with the binding properties of the parental virus. Sequence analysis of one of the latter viruses revealed mutations in the hemagglutinin (HA) gene, at sites in close proximity to the sialic acid receptor-binding pocket. These mutations appear to compensate for reduced NA function due to stalk deletions. Thus, balanced HA-NA functions are necessary for efficient influenza virus replication.
Recovery of Pathogenic Measles Virus from Cloned cDNA
Authors: Takeda Makoto , Takeuchi Kaoru , Miyajima Naoko , Kobune Fumio , Ami Yasushi , Nagata Noriyo , Suzaki Yuriko , Nagai Yoshiyuki , Tashiro Masato 
Published: July 2002

ABSTRACT
Reverse genetics technology so far established for measles virus (MeV) is based on the Edmonston strain, which was isolated several decades ago, has been passaged in nonlymphoid cell lines, and is no longer pathogenic in monkey models. On the other hand, MeVs isolated and passaged in the Epstein-Barr virus-transformed marmoset B-lymphoblastoid cell line B95a would retain their original pathogenicity (F. Kobune et al., J. Virol. 64:700–705, 1990). Here we have developed MeV reverse genetics systems based on the highly pathogenic IC-B strain isolated in B95a cells. Infectious viruses were successfully recovered from the cloned cDNA of IC-B strain by two different approaches. One was simple cotransfection of B95a cells, with three plasmids each encoding the nucleocapsid (N), phospho (P), or large (L) protein, respectively, and their expression was driven by the bacteriophage T7 RNA polymerase supplied by coinfecting recombinant vaccinia virus vTF7-3. The second approach was transfection with the L-encoding plasmid of a helper cell line constitutively expressing the MeV N and P proteins and the T7 polymerase (F. Radecke et al., EMBO J. 14:5773–5784, 1995) on which B95a cells were overlaid. Virus clones recovered by both methods possessed RNA genomes identical to that of the parental IC-B strain and were indistinguishable from the IC-B strain with respect to growth phenotypes in vitro and the clinical course and histopathology of experimentally infected cynomolgus monkeys. Thus, the systems developed here could be useful for studying viral gene functions in the context of the natural course of MeV pathogenesis.
Detection and Quantification of CD4+T Cells with Specificity for a New Major Histocompatibility Complex Class II-Restricted Influenza A Virus Matrix Protein Epitope in Peripheral Blood of Influenza Patients
Authors: Linnemann Thomas , Jung Günther , Walden Peter 
Published: July 2002

ABSTRACTFVFTLTVPS was identified as the core sequence of a new major histocompatibility complex class II-restricted T-cell epitope of influenza virus matrix protein. Epitope-specific CD4+T cells were detected in the peripheral blood of patients with frequencies of up to 0.94%, depending on the number of additional terminal amino acids.
Severe Murine Lung Immunopathology Elicited by the Pathogenic Equine Herpesvirus 1 Strain RacL11 Correlates with Early Production of Macrophage Inflammatory Proteins 1α, 1β, and 2 and Tumor Necrosis Factor Alpha
Authors: Smith Patrick M. , Zhang Yunfei , Grafton Warren D. , Jennings Stephen R. , O'Callaghan Dennis J. 
Published: July 2002

ABSTRACTThe CBA mouse model was used to investigate the immunopathology induced in the lung by the pathogenic equine herpesvirus 1 (EHV-1) strain RacL11 in comparison to infection with the attenuated vaccine candidate strain KyA. Intranasal infection with KyA resulted in almost no inflammatory infiltration in the lung. In contrast, infection with the pathogenic RacL11 strain induced a severe alveolar and interstitial inflammation, consisting primarily of lymphocytes, macrophages, and neutrophils. Infection with either EHV-1 strain resulted in the accumulation of similar numbers and ratios of CD4 and CD8 T lymphocytes in the lung and bronchoalveolar lavage (BAL) fluid. Further analysis of these T-cell populations revealed identical EHV-1-specific cytotoxic T-lymphocyte responses. RNase protection analysis of RNA isolated from the BAL fluid of RacL11-infected mice on day 3 postinfection revealed much higher levels of RNA specific for macrophage inflammatory protein 1α (MIP-1α), MIP-1β, and MIP-2 than were observed for KyA-infected mice. Furthermore, significantly higher levels of transcripts specific for tumor necrosis factor alpha were induced on day 3 postinfection with RacL11 compared with KyA. These findings suggest that the early production of proinflammatory beta chemokines plays a major role in the severe, most often lethal, respiratory inflammatory response induced by the pathogenic EHV-1 strain RacL11.