bluuebunny/crossref_metadata_2025_split huggingface dataset:
Comparison of Two Rapid Diagnostic Assays for Detection of Immunoglobulin M Antibodies to Dengue Virus
Authors: Wu Shuenn-Jue L. , Paxton Helene , Hanson Barbara , Kung Cheryl G. , Chen Timothy B. , Rossi Cindy , Vaughn David W. , Murphy Gerald S. , Hayes Curtis G. 
Published: April 2009

ABSTRACT
Two easy-to-use commercial diagnostic assays, a dipstick enzyme-linked immunosorbent assay (ELISA) (Integrated Diagnostics, Baltimore, Md.) and an immunochromatographic card assay (PanBio, Brisbane, Australia) were evaluated for detection of immunoglobulin M (IgM) antibody to dengue virus with an in-house IgM antibody capture microplate ELISA as a reference assay. The dipstick ELISA was based on the indirect-ELISA format using dengue 2 virus as the only antigen and enzyme-labeled goat anti-human IgM antibody as the detector. The total assay time was 75 min. The immunochromatographic card assay was based on the antibody capture format and separately measured both anti-dengue virus IgM and IgG in the same test. Colloidal-gold-labeled anti-dengue virus monoclonal antibody bound with dengue virus 1 to 4 antigen cocktail was the detector, and anti-human IgM and IgG were the capture antibodies. The total assay time was <10 min. Sera from 164 individuals classified as either anti-dengue virus IgM positive (94) or anti-dengue virus IgM negative (70) in the reference microplate ELISA with a dengue virus 1 to 4 antigen cocktail were tested in the two commercial assays. The dipstick ELISA missed 7 of 94 positive samples, for a sensitivity of 92.6%, while the immunochromatographic card assay missed two positive samples, for a sensitivity of 97.9%. Of the 70 negative samples, four were false positive by the dipstick ELISA and two were false positive in the immunochromatographic card assay, resulting in specificities of 94.3 and 97.1%, respectively. Both commercial assays provide sensitive and specific detection of anti-dengue virus IgM antibody and could prove useful in settings where the microplate ELISA is impractical.
Evaluation of the Quantiplex Human Immunodeficiency Virus Type 1 RNA 3.0 Assay in a Tertiary-Care Center
Authors: Gale Howard 
Published: April 2009

ABSTRACT

The Quantiplex human immunodeficiency virus type 1 RNA 3.0 Assay (bDNA) (Bayer Diagnostics, Walpole, Mass.) produced linear and reproducible (intra-assay and interassay) results over its quantification range of 50 to 500,000 copies/ml of plasma with 96% specificity. A threefold or 0.5-log
10
change or greater was clinically significant for serial patient samples.
Development of Positive Selectable Markers for the Fungal Pathogen
Cryptococcus neoformans
Authors: Hua Jun , Meyer Justin D. , Lodge Jennifer K. 
Published: April 2009

ABSTRACT

Cryptococcus neoformans
is an opportunistic fungal pathogen that causes meningitis in ∼10% of patients with AIDS. New selectable markers which confer resistance to G418 or phleomycin when transformed into
C. neoformans
were made. A hygromycin-selectable marker was modified to allow selection with a single copy of the marker.
Species-Specific Monoclonal Antibodies for Rapid Identification ofBartonella quintana
Authors: Liang Zhongxing , Raoult Didier 
Published: April 2009

ABSTRACTSeven species-specific monoclonal antibodies (MAbs) toBartonella quintanawere produced and characterized. The MAbs were of the immunoglobulin G class and reacted only with 13B. quintanastrains in indirect microimmunofluorescence and Western immunoblotting assays. They did not react with eight otherBartonellaspp., includingBartonella henselae, the most closely related species, and a selected MAb did also not react with nine other strains of gram-negative bacteria. The MAbs reacted mainly with a 34-kDa protein epitope ofB. quintanawhich was shown to be species specific by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Four of five body lice experimentally infected withB. quintanawere found to be positive for the organism in microimmunofluorescence assays with one MAb. These MAbs may provide a specific, simple, rapid, and low-cost tool for the identification ofB. quintanaand the diagnosis of infections due to the microorganism.
Characterization of a Monoclonal Antibody and Its Single-Chain Antibody Fragment Recognizing the Nucleoside Triphosphatase/Helicase Domain of the Hepatitis C Virus Nonstructural 3 Protein
Authors: Zhang Zhu-Xu , Lazdina Una , Chen Margaret , Peterson Darrell L. , Sällberg Matti 
Published: April 2009

ABSTRACTWe have produced a murine monoclonal antibody (MAb), ZX10, recognizing the NTPase/helicase domain of the hepatitis C virus (HCV) nonstructural 3 protein (NS3), from which we designed a single-chain variable fragment (ScFv). The ZX10 MAb recognized a discontinuous epitope of the NTPase/helicase domain, of which the linear sequence GEIPFYGKAIPL at residues 1371 to 1382 constitutes one part. cDNAs from variable regions coding for the heavy and light chains were cloned, sequenced, and assembled into the NS3-ScFv, which was inserted into procaryotic and eucaryotic expression vectors.Escherichia coli-expressed NS3-ScFv inhibited the binding of the ZX10 MAb to NS3, confirming a retained specificity. However, the ability to bind the peptide 1371–1382 had been lost. In vitro-translated NS3-ScFv and HCV NS3/NS4A were coprecipitated by antibodies to HCV NS4A, confirming the in vitro activity of the NS3 ScFv. Thus, we have designed a functional NS3 NTPase/helicase domain-specific ScFv which should be evaluated further with respect to disturbing enzymatic functions of the NS3 protein.
Measurement of Antinuclear Antibodies: Assessment of Different Test Systems
Authors: Kern P. , Kron M. , Hiesche K. 
Published: April 2009

ABSTRACT
The performance of rat liver and HEp-2 in the detection of antinuclear antibodies (ANA) was studied by two independent sites and compared against an ANA enzyme immunoassay (EIA) screen and EIA systems for the measurement of antibodies to double-stranded DNA (dsDNA) and ENA. Sixty-two sera from patients with connective tissue disease (CTD) and 398 from controls suffering from other disorders were included. The level of agreement was, for HEp-2 and rat liver (within one site), 82.0% (ANA positive/ANA negative) and 51.0% (ANA pattern); and for HEp2- and HEp-2 (between sites), 71.8 and 86.5%. On sera with the ANA homogeneous pattern, the measurement of anti-ENA EIA added little to the detection rate with anti-dsDNA EIA alone. On ANA speckled sera, the EIA reactivity depended on the reaction of the mitotic cells: while sera with positive mitoses reacted similarly to ANA homogeneous sera, in those with negative mitoses the measurement of anti-ENA added about 10% to the detection rate achieved with anti-dsDNA alone. The measurement of anti-Scl-70 and anti-Jo-1 did not markedly improve the positive rate with classical ENA (anti-SSA, -SSB, -Sm, and -RNP) alone, raising doubts about the cost efficiency of including these measurements in unselected sera. The ANA EIA identified patients with CTD at a rate similar to that for rat liver and HEp-2. However, up to 98% of the sera found to be negative by ANA EIA but positive by use of rat liver and HEp-2 were from controls. Thus, the ANA EIA may possible be used as an alternative screen, particularly in laboratories with a high frequency of sera from patients not suffering from CTD.
Detection of
Francisella tularensis
in Biological Specimens Using a Capture Enzyme-Linked Immunosorbent Assay, an Immunochromatographic Handheld Assay, and a PCR
Authors: Grunow Roland , Splettstoesser Wolf , McDonald Sahra , Otterbein Christian , O'Brien Tom , Morgan Cecilia , Aldrich Jennifer , Hofer Erwin , Finke Ernst-Jürgen , Meyer Hermann 
Published: April 2009

ABSTRACT

The early detection of
Francisella tularensis
, the causative agent of tularemia, is important for adequate treatment by antibiotics and the outcome of the disease. Here we describe a new capture enzyme-linked immunosorbent assay (cELISA) based on monoclonal antibodies specific for lipopolysaccharide (LPS) of
Francisella tularensis
subsp.
holarctica
and
Francisella tularensis
subsp.
tularensis
. No cross-reactivity with
Francisella tularensis
subsp.
novicida
,
Francisella philomiragia
, and a panel of other possibly related bacteria, including
Brucella
spp.,
Yersinia
spp.,
Escherichia coli
, and
Burkholderia
spp., was observed. The detection limit of the assay was 10
3
to 10
4
bacteria/ml. This sensitivity was achieved by solubilization of the LPS prior to the cELISA. In addition, a novel immunochromatographic membrane-based handheld assay (HHA) and a PCR, targeting sequences of the 17-kDa protein (TUL4) gene of
F. tularensis
, were used in this study. Compared to the cELISA, the sensitivity of the HHA was about 100 times lower and that of the PCR was about 10 times higher. All three techniques were successfully applied to detect
F. tularensis
in tissue samples of European brown hares (
Lepus europaeus
). Whereas all infected samples were recognized by the cELISA, those with relatively low bacterial load were partially or not detected by PCR and HHA, probably due to inhibitors or lack of sensitivity. In conclusion, the HHA can be used as a very fast and simple approach to perform field diagnosis to obtain a first hint of an infection with
F. tularensis
, especially in emergent situations. In any suspect case, the diagnosis should be confirmed by more sensitive techniques, such as the cELISA and PCR.
Altered Ex Vivo Expression of Caspase 8, Caspase 9, and Bcl-2 Is Associated with T-Cell Hyporeactivity in Patients with Paracoccidioidomycosis
Authors: Cacere Camila R. , Mendes-Giannini Maria J. S. , do Valle Antonio Carlos F. , Duarte Alberto J. S. , Benard Gil 
Published: April 2009

ABSTRACT
To better understand the T-cell hyporesponsiveness of patients with paracoccidioidomycosis, we tested the hypothesis that the T cells were committed to apoptosis. We show here that T cells of patients with paracoccidioidomycosis overexpress caspase 9 and caspase 8 but express low Bcl-2 levels and that interleukin-2 was unable to revert the hyporesponsiveness. These data suggest that the T cells would in vivo be driven to a tolerant state and apoptosis.
Evaluation of a Commercial Blocking Enzyme-Linked Immunosorbent Assay To Detect Avian Influenza Virus Antibodies in Multiple Experimentally Infected Avian Species
Authors: Brown Justin D. , Stallknecht David E. , Berghaus Roy D. , Luttrell M. Page , Velek Katherine , Kistler Whitney , Costa Taiana , Yabsley Michael J. , Swayne David 
Published: April 2009

ABSTRACT

Wild birds of the orders
Anseriformes
and
Charadriiformes
are the natural reservoirs for avian influenza (AI) viruses. Traditionally, AI virus surveillance in wild birds has relied on virus identification strategies, including virus isolation and detection. To evaluate the accuracy of a commercial blocking enzyme-linked immunosorbent assay (bELISA) and the agar gel immunodiffusion (AGID) test for detection of antibodies in wild birds, which is indicative of AI virus infection, we tested 281 serum samples from various wild avian species that were experimentally infected with AI viruses. Included in these samples were 178 samples from birds with confirmed AI virus infections (122 infected with low-pathogenic AI [LPAI] viruses and 56 infected with highly pathogenic AI [HPAI] viruses) and 103 samples from birds that were uninfected, negative controls. The sensitivities of the bELISA and the AGID test were 0.820 (95% confidence interval [95% CI], 0.756 to 0.874) and 0.674 (95% CI, 0.600 to 0.742), respectively. Both tests had an estimated specificity of 1.00 (95% CI, 0.965 to 1.00). The bELISA was significantly more sensitive than the AGID test for both LPAI virus- and HPAI virus-infected birds. Both assays, however, had a higher sensitivity for birds infected with HPAI virus than for birds infected with LPAI virus. These results demonstrate the potential utility of the bELISA for detection of antibodies to both LPAI and HPAI viruses in multiple avian species, representing five avian orders and 17 genera. Additional studies are warranted to further evaluate the utility of the bELISA for use with naturally infected birds.
Serological Diagnosis of
Brucella
Infections in Odontocetes
Authors: Hernández-Mora Gabriela , Manire Charles A. , González-Barrientos Rocío , Barquero-Calvo Elías , Guzmán-Verri Caterina , Staggs Lydia , Thompson Rachel , Chaves-Olarte Esteban , Moreno Edgardo 
Published: April 2009

ABSTRACT

Brucella ceti
causes disease in Odontoceti. The absence of control serum collections and the diversity of cetaceans have hampered the standardization of serological tests for the diagnosis of cetacean brucellosis. Without a “gold” standard for sensitivity and specificity determination, an alternative approach was followed. We designed an indirect enzyme-linked immunosorbent assay (iELISA) that recognizes immunoglobulins G (IgGs) from 17 odontocete species as a single group. For the standardization, we used
Brucella melitensis
and
Brucella abortus
lipopolysaccharides, serum samples from seven resident odontocetes with no history of infectious disease displaying negative rose bengal test (RBT) reactions, and serum samples from seven dolphins infected with
B. ceti
. We compared the performance of the iELISA with those of the protein G ELISA (gELISA), the competitive ELISA (cELISA), and the immunofluorescence (IF) and dot blot (DB) tests, using 179 odontocete serum samples and RBT as the reference. The diagnostic potential based on sensitivity and specificity of the iELISA was superior to that of gELISA and cELISA. The correlation and agreement between the iELISA and the gELISA were relatively good (
R
i/g
2
= 0.65 and κ
i/g
= 0.66, respectively), while the correlation and agreement of these two ELISAs with cELISA were low (
R
i/c
2
= 0.46,
R
g/c
2
= 0.37 and κ
i/c
= 0.62, κ
g/c
= 0.42). In spite of using the same anti-odontocete IgG antibody, the iELISA was more specific than were the IF and DB tests. An association between high antibody titers and the presence of neurological symptoms in dolphins was observed. The prediction is that iELISA based on broadly cross-reacting anti-dolphin IgG antibody would be a reliable test for the diagnosis of brucellosis in odontocetes, including families not covered in this study.
Gene Expression of Nucleic Acid-Sensing Pattern Recognition Receptors in Children Hospitalized for Respiratory Syncytial Virus-Associated Acute Bronchiolitis
Authors: Scagnolari Carolina , Midulla Fabio , Pierangeli Alessandra , Moretti Corrado , Bonci Enea , Berardi Rosaria , De Angelis Daniela , Selvaggi Carla , Di Marco Paola , Girardi Enrico , Antonelli Guido 
Published: April 2009

ABSTRACT
Given the critical role of pattern recognition receptors (PRRs) in acid nucleic recognition in the initiation of innate immunity and the orchestration of adaptive immunity, the aim of this study was to determine whether any heterogeneity of PRR expression in the airway tracts of infants with respiratory syncytial virus (RSV) infection might explain the broad clinical spectrum of RSV-associated bronchiolitis in infants. For this purpose, the levels of melanoma differentiation-associated protein-5 (MDA-5), retinoic acid inducible gene-1 (RIG-1), and Toll-like receptor 3 (TLR-3), TLR-7, TLR-8, and TLR-9 mRNAs were evaluated, using TaqMan quantitative reverse transcription-PCR, in cells from nasopharyngeal washes collected from 157 infants suffering from acute bronchiolitis whether or not they were associated with respiratory viruses. High interindividual variability was observed in both virus-positive and -negative infants; however, the relative gene expression levels of MDA-5, RIG-1, TLR-7, and TLR-8 were significantly higher in the virus-infected group, whereas the expression levels of TLR-3 and TLR-9 were not significantly different. The differences in the gene expression of MDA-5, RIG-1, TLR-7, and TLR-8 were more evident in infants with RSV infection than in those with bocavirus or rhinovirus infection. In RSV-infected infants, PRR-mRNA levels also were analyzed in relation to interferon protein levels, viral load, clinical severity, days of hospitalization, age, and body weight. A significant positive correlation was observed only between RSV viral load and RIG-1 mRNA levels. These findings provide the first direct evidence that, in infants with respiratory virus-associated bronchiolitis, especially RSV, there are substantial changes in PRR gene expression; this likely is an important determinant of the clinical outcome of bronchiolitis.
HbaR, a 4-Hydroxybenzoate Sensor and FNR-CRP Superfamily Member, Regulates Anaerobic 4-Hydroxybenzoate Degradation by
Rhodopseudomonas palustris
Authors: Egland Paul G. , Harwood Caroline S. 
Published: April 2009

ABSTRACT

Under anaerobic conditions, structurally diverse aromatic compounds are catabolized by bacteria to form benzoyl-coenzyme A (benzoyl-CoA), the starting compound for a central reductive pathway for aromatic ring degradation. The structural genes required for the conversion of 4-hydroxybenzoate (4-HBA) to benzoyl-CoA by
Rhodopseudomonas palustris
have been identified. Here we describe a regulatory gene,
hbaR
, that is part of the 4-HBA degradation gene cluster. An
hbaR
mutant that was constructed was unable to grow anaerobically on 4-HBA. However, the mutant retained the ability to grow aerobically on 4-HBA by an oxygen-requiring pathway distinct from the anaerobic route of 4-HBA degradation. The effect of the HbaR protein on expression of
hbaA
encoding 4-HBA-CoA ligase, the first enzyme for 4-HBA degradation, was investigated by using
hbaA
::′
lacZ
transcriptional fusions. HbaR was required for a 20-fold induction of β-galactosidase activity that was observed with a chromosomal
hbaA
::′
lacZ
fusion when cells grown anaerobically on succinate were switched to anaerobic growth on succinate and 4-HBA. HbaR also activated expression from a plasmid-borne
hbaA
-′
lacZ
fusion when it was expressed in aerobically grown
Pseudomonas aeruginosa
cells, indicating that the activity of this regulator is not sensitive to oxygen. The deduced amino acid sequence of HbaR indicates that it is a member of the FNR-CRP superfamily of regulatory proteins. It is most closely related to transcriptional activators that are involved in regulating nitrate reduction. Previously, it has been shown that
R. palustris
has an FNR homologue, called AadR, that is also required for 4-HBA degradation. Our evidence indicates that AadR activates expression of
hbaR
in response to anaerobiosis and that HbaR, in turn, activates expression of 4-HBA degradation in response to 4-HBA as an effector molecule.
flhDC
, the Flagellar Master Operon of
Xenorhabdus nematophilus
: Requirement for Motility, Lipolysis, Extracellular Hemolysis, and Full Virulence in Insects
Authors: Givaudan Alain , Lanois Anne 
Published: April 2009

ABSTRACT

Xenorhabdus
is a major insect pathogen symbiotically associated with nematodes of the family Steinernematidae. This motile bacterium displays swarming behavior on suitable media, but a spontaneous loss of motility is observed as part of a phenomenon designated phase variation which involves the loss of stationary-phase products active as antibiotics and potential virulence factors. To investigate the role of one of the transcriptional activators of flagellar genes, FlhDC, in motility and virulence, the
Xenorhabdus nematophilus flhDC
locus was identified by functional complementation of an
Escherichia coli flhD
null mutant and DNA sequencing. Construction of
X. nematophilus flhD
null mutants confirmed that the
flhDC
operon controls flagellin expression but also revealed that lipolytic and extracellular hemolysin activity is
flhDC
dependent. We also showed that the
flhD
null mutant displayed a slightly attenuated virulence phenotype in
Spodoptera littoralis
compared to that of the wild-type strain. Thus, these data indicated that motility, lipase, hemolysin, or unknown functions controlled by the
flhDC
operon are involved in the infectious process in insects. Our investigation expands the view of the flagellar regulon as a checkpoint coupled to a major network involving bacterial physiological aspects as well as motility.
Identification of a New Class of 5′-Adenylylsulfate (APS) Reductases from Sulfate-Assimilating Bacteria
Authors: Bick Julie Ann , Dennis Jonathan J. , Zylstra Gerben J. , Nowack Jason , Leustek Thomas 
Published: April 2009

ABSTRACT

A gene was cloned from
Burkholderia cepacia
DBO1 that is homologous with
Escherichia coli cysH
encoding 3′-phosphoadenylylsulfate (PAPS) reductase. The
B. cepacia
gene is the most recent addition to a growing list of
cysH
homologs from a diverse group of sulfate-assimilating bacteria whose products show greater homology to plant 5′-adenylylsulfate (APS) reductase than they do to
E. coli
CysH. The evidence reported here shows that the
cysH
from one of the species,
Pseudomonas aeruginosa
, encodes APS reductase. It is able to complement an
E. coli cysH
mutant and a
cysC
mutant, indicating that the enzyme is able to bypass PAPS, synthesized by the
cysC
product. Insertional knockout mutation of
P. aeruginosa cysH
produced cysteine auxotrophy, indicating its role in sulfate assimilation. Purified
P. aeruginosa
CysH expressed as a His-tagged recombinant protein is able to reduce APS, but not PAPS. The enzyme has a specific activity of 5.8 μmol · min
−1
· mg of protein
−1
at pH 8.5 and 30°C with thioredoxin supplied as an electron donor. APS reductase activity was detected in several bacterial species from which the novel type of
cysH
has been cloned, indicating that this enzyme may be widespread. Although an APS reductase from dissimilatory sulfate-reducing bacteria is known, it shows no structural or sequence homology with the assimilatory-type APS reductase reported here. The results suggest that the dissimilatory and assimilatory APS reductases evolved convergently.
Cysteine Biosynthesis Pathway in the Archaeon
Methanosarcina barkeri
Encoded by Acquired Bacterial Genes?
Authors: Kitabatake Makoto , So Man Wah , Tumbula Debra L. , Söll Dieter 
Published: April 2009

ABSTRACT

The pathway of cysteine biosynthesis in archaea is still unexplored. Complementation of a cysteine auxotrophic
Escherichia coli
strain NK3 led to the isolation of the
Methanosarcina barkeri cysK
gene [encoding
O
-acetylserine (thiol)-lyase-A], which displays great similarity to bacterial
cysK
genes. Adjacent to
cysK
is an open reading frame orthologous to bacterial
cysE
(serine transacetylase) genes. These two genes could account for cysteine biosynthesis in this archaeon. Analysis of recent genome data revealed the presence of bacteria-like
cysM
genes [encoding
O
-acetylserine (thiol)-lyase-B] in
Pyrococcus
spp.,
Sulfolobus solfataricus
, and
Thermoplasma acidophilum
. However, no orthologs for these genes can be found in
Methanococcus jannaschii
,
Methanobacterium thermoautotrophicum
, and
Archaeoglobus fulgidus
, implying the existence of unrecognizable genes for the same function or a different cysteine biosynthesis pathway.