bluuebunny/crossref_metadata_2025_split huggingface dataset:
Development of a Rapid and Sensitive Test for Identification of Major Pathogens in Bovine Mastitis by PCR
Authors: Riffon Renée , Sayasith Khampoune , Khalil Hayssam , Dubreuil Pascal , Drolet Marc , Lagacé Jacqueline 
Published: July 2002

ABSTRACT

Bovine mastitis is the most important source of loss for the dairy industry. A rapid and specific test for the detection of the main pathogens of bovine mastitis is not actually available. Molecular probes reacting in PCR with bacterial DNA from bovine milk, providing direct and rapid detection of
Escherichia coli
,
Staphylococcus aureus
,
Streptococcus agalactiae
,
Streptococcus dysgalactiae
,
Streptococcus parauberis
, and
Streptococcus uberis
, have been developed. Two sets of specific primers were designed for each of these microorganisms and appeared to discriminate close phylogenic bacterial species (e.g.,
S. agalactiae
and
S. dysgalactiae
). In addition, two sets of universal primers were designed to react as positive controls with all major pathogens of bovine mastitis. The sensitivities of the test using
S. aureus
DNA extracted from milk with and without a pre-PCR enzymatic lysis step of bacterial cells were compared. The detection limit of the assay was 3.125 × 10
2
CFU/ml of milk when
S. aureus
DNA was extracted with the pre-PCR enzymatic step compared to 5 × 10
3
CFU/ml of milk in the absence of the pre-PCR enzymatic step. This latter threshold of sensitivity is still compatible with its use as an efficient tool of diagnosis in bovine mastitis, allowing the elimination of expensive reagents. The two PCR tests avoid cumbersome and lengthy cultivation steps, can be performed within hours, and are sensitive, specific, and reliable for the direct detection in milk of the six most prevalent bacteria causing bovine mastitis.
Comparison of a Multiplex Reverse Transcription-PCR-Enzyme Hybridization Assay with Conventional Viral Culture and Immunofluorescence Techniques for the Detection of Seven Viral Respiratory Pathogens
Authors: Liolios Lisa , Jenney Adam , Spelman Denis , Kotsimbos Tom , Catton Michael , Wesselingh Steve 
Published: July 2002

ABSTRACTA multiplex reverse transcription-PCR-enzyme hybridization assay (RT-PCR-EHA; Hexaplex; Prodesse Inc., Waukesha, Wis.) was used for the simultaneous detection of human parainfluenza virus types 1, 2, and 3, influenza virus types A and B, and respiratory syncytial virus types A and B. One hundred forty-three respiratory specimens from 126 patients were analyzed by RT-PCR-EHA, and the results were compared to those obtained by conventional viral culture and immunofluorescence (IF) methods. RT-PCR-EHA proved to be positive for 17 of 143 (11.9%) specimens, whereas 8 of 143 (5.6%) samples were positive by viral culture and/or IF. Eight samples were positive by both RT-PCR-EHA and conventional methods, while nine samples were RT-PCR-EHA positive and viral culture and IF negative. Eight of the nine samples with discordant results were then independently tested by a different multiplex RT-PCR assay for influenza virus types A and B, and all eight proved to be positive. In comparison to viral culture and IF methods, RT-PCR-EHA gave a sensitivity and a specificity of 100 and 93%, respectively. Since RT-PCR-EHA was able to detect more positive samples, which would otherwise have been missed by routine methods, we suggest that this multiplex RT-PCR-EHA provides a highly sensitive and specific means of diagnostic detection of major respiratory viruses.
Automated Multiplex Assay System for Simultaneous Detection of Hepatitis B Virus DNA, Hepatitis C Virus RNA, and Human Immunodeficiency Virus Type 1 RNA
Authors: Meng Q. , Wong C. , Rangachari A. , Tamatsukuri S. , Sasaki M. , Fiss E. , Cheng L. , Ramankutty T. , Clarke D. , Yawata H. , Sakakura Y. , Hirose T. , Impraim C. 
Published: July 2002

ABSTRACT
We have developed an automated multiplex system for simultaneously screening hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type 1 (HIV-1) in blood donations. The assay, designated AMPLINAT MPX HBV/HCV/HIV-1 Test (AMPLINAT MPX), consists of virus extraction and target sequence-specific probe capture on specimen preparation workstation GT-X (Roche Diagnostics K.K., Tokyo, Japan) and amplification and detection by TaqMan PCR on the ABI PRISM 7700 Analyzer (Perkin-Elmer Applied Biosystems, Foster City, Calif.). An internal control (IC) is incorporated in the assay to monitor the extraction, target amplification, and detection processes. The assay yields qualitative results without discrimination of the three targets. Detection limits (95% confidence interval) are 22 to 60 copies/ml for HBV, 61 to 112 IU/ml for HCV, and 33 to 66 copies/ml for HIV-1, using a specimen input volume of 0.2 ml. The AMPLINAT MPX assay detects a broad range of genotypes or subtypes for all three viruses and has a specificity of 99.6% for all three viruses with seronegative specimens. In an evaluation of seroconversion panels, the AMPLINAT MPX assay detects HBV infection an average of 24 days before the detection of HBsAg by enzyme immunoassay. HCV RNA was detected an average of 31 days before HCV antibody. HIV-1 RNA was detected an average of 14 days before HIV-1 antibody and an average of 9 days before p24 antigen. The Japanese Red Cross has been evaluating the AMPLINAT MPX system since October 1999. The clinical performance indicates that the AMPLINAT MPX system is robust, sensitive, and reproducible, with a high percentage of valid assay runs (96.8%), a low false-positive rate (0.34%), and a low IC failure rate (0.24%).
Mutations in the
rpoB
Gene of Multidrug-Resistant
Mycobacterium tuberculosis
Clinical Isolates from India
Authors: Mani Cheruvu , Selvakumar N. , Narayanan Sujatha , Narayanan P. R. 
Published: July 2002

ABSTRACT

Mutations in the 81-bp rifampin resistance-determining region (RRDR) of the
rpoB
gene were analyzed by DNA sequencing of 50
Mycobacterium tuberculosis
clinical isolates (44 resistant and 6 sensitive) from various parts of India. Fifty-three mutations of 18 different kinds, 17 point mutations and one deletion, were observed in 43 of 44 resistant isolates. Three novel mutations and three new alleles within the RRDR, along with two novel mutations outside the RRDR, are reported in this study.
Identification of Novel
Helicobacter
Species in Pig Stomachs by PCR and Partial Sequencing
Authors: Choi Young K. , Han Jeong H. , Joo Han S. 
Published: July 2002

ABSTRACT

Evidence of infection with
Helicobacter
species in pig stomach was investigated by the use of a PCR with
Helicobacter
genus-specific primers. Forty pig stomachs, each of four different ulcer lesion grades, 0, 1, 2, and 3 in the
pars esophagea
area, were collected from a slaughterhouse in Minnesota. Of 160 stomach samples examined, 102 (63.8%) were positive by the PCR assay. The 40 samples each of lesion grades 0, 1, 2, and 3 showed 22.5, 52.5, 85.0, and 95.0% PCR-positive results, respectively. There was a significant trend (
P
≤ 0.01) in the proportions of PCR-positive cases relative to severity of the lesion. About 80% of the 16S rRNA gene was amplified, and PCR-restriction fragment length polymorphism (RFLP) patterns were analyzed. Of 102 PCR-positive samples, the PCR-RFLP patterns resulted in four different types, 32 samples being classified into type MN 1, 16 samples into type MN 2, 43 samples into type MN 3, and 11 samples into type MN 4. When the sequences of each RFLP type were compared to those reported in databases by using BLAST software, types MN 1, MN 2, MN 3, and MN 4 showed homologies of 97.3, 95.4, 96.7, and 99.5% with the 16S ribosomal DNA of
Helicobacter flexispira
taxon 3,
Helicobacter
sp. strains MIT 94-022 and MZ 640285, and
Helicobacter suis
, respectively. None of the 102 samples positive for the
Helicobacter
genus were positive with a primer set specific for
Helicobacter pylori
. Attempts to culture the organisms from selected stomachs in vitro were unsuccessful.
Molecular Analysis of
Neorickettsia risticii
in Adult Aquatic Insects in Pennsylvania, in Horses Infected by Ingestion of Insects, and Isolated in Cell Culture
Authors: Mott Jason , Muramatsu Yasukazu , Seaton Elizabeth , Martin Carol , Reed Stephen , Rikihisa Yasuko 
Published: July 2002

ABSTRACT

Upon ingestion of adult aquatic insects, horses developed clinical signs of Potomac horse fever, and
Neorickettsia risticii
was isolated from the blood. 16S rRNA and 51-kDa antigen gene sequences from blood, isolates, and caddis flies fed to the horses were identical, proving oral transmission of
N. risticii
from caddis flies to horses.
Initial Concentration of
Staphylococcus epidermidis
in Simulated Pediatric Blood Cultures Correlates with Time to Positive Results with the Automated, Continuously Monitored BACTEC Blood Culture System
Authors: Haimi-Cohen Yishai , Vellozzi Ernestine M. , Rubin Lorry G. 
Published: July 2002

ABSTRACT

The relationship of initial concentration of
Staphylococcus epidermidis
in blood cultures and time to positivity (TTP) in an automated, continuously monitored blood culture system was assessed. Blood and 1 to 1,000 CFU of
S. epidermidis
per ml in stationary or exponential phase were inoculated in BACTEC Pediatric Plus F bottles and incubated. The TTP was inversely proportional to the initial inoculated concentration. Blood culture bottles with initial bacterial densities of <10 CFU/ml had a TTP of >20 h (upper limit of 95% prediction interval, 20.7 h) and bottles with initial bacterial densities of ≥50 CFU/ml had a TTP of ≤15 h (lower limit of 95% prediction interval, 13.4 h).
Enterococcus gilvus
sp. nov. and
Enterococcus pallens
sp. nov. Isolated from Human Clinical Specimens
Authors: Tyrrell Gregory J. , Turnbull LeeAnn , Teixeira Lúcia M. , Lefebvre Johanne , Carvalho Maria da Glória S. , Facklam Richard R. , Lovgren Marguerite 
Published: July 2002

ABSTRACT

Light yellow-pigmented (strain PQ1) and yellow-pigmented (strain PQ2), gram-positive, non-spore-forming, nonmotile bacteria consisting of pairs or chains of cocci were isolated from the bile of a patient with cholecystitis (PQ1) and the peritoneal dialysate of another patient with peritonitis (PQ2). Morphologically and biochemically, the organisms phenotypically belonged to the genus
Enterococcus
. Whole-cell protein (WCP) analysis and sequence analysis of a segment of the 16S rRNA gene suggested that they are new species within the genus
Enterococcus
. PQ1 and PQ2 displayed less than 70% identities to other enterococcal species by WCP analysis. Sequence analysis showed that PQ1 shared the highest level of sequence similarity with
Enterococcus raffinosus
and
E. malodoratus
(sequence similarities of 99.8% to these two species). Sequence analysis of PQ2 showed that it had the highest degrees of sequence identity with the group I enterococci
E. malodoratus
(98.7%),
E. raffinosus
(98.6%),
E. avium
(98.6%), and
E. pseudoavium
(98.6%). PQ1 and PQ2 can be differentiated from the other
Enterococcus
spp. in groups II, III, IV, and V by their phenotypic characteristics: PQ1 and PQ2 produce acid from mannitol and sorbose and do not hydrolyze arginine, placing them in group I. The yellow pigmentation differentiates these strains from the other group I enterococci. PQ1 and PQ2 can be differentiated from each other since PQ1 does not produce acid from arabinose, whereas PQ2 does. Also, PQ1 is
Enterococcus
Accuprobe assay positive and pyrrolidonyl-β-naphthylamide hydrolysis positive, whereas PQ2 is negative by these assays. The name
Enterococcus gilvus
sp. nov. is proposed for strain PQ1, and the name
Enterococcus pallens
sp. nov. is proposed for strain PQ2. Type strains have been deposited in culture collections as
E. gilvus
ATCC BAA-350 (CCUG 45553) and
E. pallens
ATCC BAA-351 (CCUG 45554).
Dynamics of Dissemination and Outer Surface Protein Expression of Different European
Borrelia burgdorferi
Sensu Lato Strains in Artificially Infected
Ixodes ricinus
Nymphs
Authors: Fingerle Volker , Rauser Sandra , Hammer Bettina , Kahl Olaf , Heimerl Christiane , Schulte-Spechtel Ulrike , Gern Lise , Wilske Bettina 
Published: July 2002

ABSTRACT

Unfed
Ixodes ricinus
nymphs were infected with eight different strains and clones of
Borrelia afzelii
and
B. garinii
by capillary feeding. Except one
B. afzelii
clone, all expressed OspC in culture. Tick midguts and salivary glands were investigated at different time intervals for the presence of borreliae and for OspA and OspC phenotypes by immunofluorescence with simultaneous staining of OspA and OspC with monoclonal antibodies. Both species were transmittable to
I. ricinus
. All OspC-expressing strains and clones were able to disseminate into the salivary glands. In contrast, the OspC-negative
B. afzelii
clone was not detectable in the salivary glands, an indication that OspC plays an important role in dissemination. OspA-positive borreliae prevailed in the midgut. OspC positives were more frequent in the salivary glands than in the midgut. Notably, simultaneously OspA- and OspC-negative borreliae were detected in both organs. Kinetics of dissemination varied with the strains. The OspC-positive
B. afzelii
clone and all
B. garinii
OspA type 4 strains were detectable in the salivary glands right after feeding, while one
B. garinii
OspA type 6 strain invaded the salivary glands with a delay of 24 h. These findings support the hypothesis that OspA is abundantly expressed in unfed ticks while upregulation of OspC is also a prerequisite for dissemination in the vector for the Eurasian species
B. afzelii
and
B. garinii
. However, we found strain-specific dynamics of Osp expression and strain-specific kinetics of systemic infection in the vector tick and it appears that additional factors are involved in the initiation and regulation of the dissemination process.
Evaluation of the Abbott LCx Quantitative Assay for Measurement of Human Immunodeficiency Virus RNA in Plasma
Authors: de Mendoza Carmen , Alcamí José , Sainz María , Folgueira Dolores , Soriano Vincent 
Published: July 2002

ABSTRACT

Plasma human immunodeficiency virus RNA in 491 clinical specimens was measured by LCx. There was a strong correlation with the results provided by other methods (
r
2
values of 0.93 with Cobas-Monitor version 1.5 and of 0.95 with Quantiplex version 3.0). However, values were uniformly higher with LCx than with Quantiplex when non-B subtypes were tested.
Evaluation of the Directigen FluA+B Test for Rapid Diagnosis of Influenza Virus Type A and B Infections
Authors: Chan K. H. , Maldeis N. , Pope W. , Yup A. , Ozinskas A. , Gill J. , Seto W. H. , Shortridge K. F. , Peiris J. S. M. 
Published: July 2002

ABSTRACT
Directigen FluA+B (BD Diagnostic Systems, Sparks, Md.), a new rapid test for the detection of influenza virus types A and B, was evaluated with nasopharyngeal aspirate specimens collected from 250 patients in comparison with culture and direct fluorescent antigen (DFA) detection tests. The patients studied were predominantly children, 80% being ≤6 years old. Specimens negative by culture but positive by the Directigen FluA+B or DFA tests were analyzed by reverse transcription-PCR to resolve the discrepant results. The resolved sensitivity, specificity, and positive and negative predictive values of the Directigen FluA+B test for influenza virus type A were 96%, 99.6%, 96%, and 99.6%, respectively, and for influenza virus type B they were 87.5%, 96.8%, 80%, and 98%, respectively. Storage of nasopharyngeal aspirates in virus transport medium at 2 to 8°C for 48 h had little adverse effect on the detection of influenza virus type A, but diagnosis of influenza virus type B is best carried out with fresh specimens. The test detected a range of human and animal influenza virus A subtypes, including the H5N1 and H9N2 viruses that recently caused human disease in Hong Kong.
Epidemiological Dynamics of Hepatitis C Virus among 747 German Individuals: New Subtypes on the Advance
Authors: Schröter Matthias , Zöllner Bernhard , Schäfer Peter , Reimer Andreas , Müller Matthias , Laufs Rainer , Feucht Heinz-Hubert 
Published: July 2002

ABSTRACTThis study demonstrates the dynamics in the epidemiology of hepatitis C virus subtypes. Subtypes 3a and 4a have become increasingly prevalent in patients where an infection within recent years can be assumed. Evidence is presented that the subtypes observed among younger patients can spread rapidly and lead to significant changes in the subtype distribution.
Development of a Sensitive and Specific Enzyme-Linked Immunosorbent Assay for Detecting and Quantifying CMY-2 and SHV β-Lactamases
Authors: Hujer Andrea M. , Page Malcolm G. P. , Helfand Marion S. , Yeiser Bethany , Bonomo Robert A. 
Published: July 2002

ABSTRACT

Polyclonal rabbit antibodies against SHV-1 and CMY-2 β-lactamases were produced and characterized, and enzyme-linked immunosorbent assays (ELISAs) were developed. Immunoblots revealed that the anti-SHV-1 antibody recognized SHV-1 but did not recognize TEM-1, K-1, OXA-1, or any AmpC β-lactamase tested. The anti-CMY-2 antibody detected
Escherichia coli
CMY-2,
Enterobacter cloacae
P99,
Klebsiella pneumoniae
ACT-1, and the AmpC β-lactamases of
Enterobacter aerogenes
,
Morganella morganii
, and
Citrobacter freundii
. No cross-reactivity of the anti-CMY-2 antibody was seen against laboratory strains of
E. coli
possessing TEM-1, SHV-1, K-1, or OXA-1 β-lactamases. Operating conditions for performing ELISAs were optimized. Both anti-CMY-2 and anti-SHV-1 antibodies detected picogram quantities of purified protein in ELISAs. The reactivity of the anti-CMY-2 antibody was tested against a number of AmpC β-lactamases by assaying known quantities of purified enzymes in ELISAs (AmpC β-lactamases of
M. morganii
,
C. freundii
,
E. coli
, and
E. cloacae
). As the homology to CMY-2 β-lactamase decreased, the minimum level needed for detection increased (e.g., 94% homology recognized at 1 ng/ml and 71% homology recognized at 10 ng/ml). The ELISAs were used to assay unknown clinical isolates for AmpC and SHV β-lactamases, and the results were confirmed with PCR amplification of
bla
AmpC
and
bla
SHV
genes. Overall, we found that our ELISAs were at least 95% sensitive and specific for detecting SHV and AmpC β-lactamases. The ELISA format can facilitate the identification of AmpC and SHV β-lactamases and can be used to quantify relative amounts of β-lactamase enzymes in clinical and laboratory isolates.
Detection of Smallpox Virus DNA by LightCycler PCR
Authors: Espy Mark J. , Cockerill Franklin R. , Meyer Richard F. , Bowen Michael D. , Poland Gregory A. , Hadfield Ted L. , Smith Thomas F. 
Published: July 2002

ABSTRACT

A 300-bp plasmid fragment of the hemagglutinin gene was used as target DNA to develop a rapid real-time LightCycler (Roche Applied Science, Indianapolis, Ind.) PCR assay for laboratory detection of smallpox virus. PCR primers and probes were designed specifically for detection of smallpox virus DNA, but all viruses of the genus
Orthopoxvirus
tested could be detected by use of the hemagglutinin gene target sequence. Base pair mismatches in the 204-bp amplicon allowed discrimination of cowpox virus (melting temperature [

T
m

], 56.40°C), monkeypox virus (

T
m

, 56.24°C), and vaccinia virus (

T
m

, 56.72°C), including the Dryvax vaccine strain, from smallpox virus (

T
m

, 62.45°C) by melting curve analysis. The analytical sensitivity was 5 to 10 copies of target DNA per sample. The assay was specific for members of the genus
Orthopoxvirus
; the DNAs of herpes simplex virus and varicella-zoster virus were not detected by the smallpox virus LightCycler PCR.
Effectiveness of Reverse Transcription-PCR, Virus Isolation, and Enzyme-Linked Immunosorbent Assay for Diagnosis of Influenza A Virus Infection in Different Age Groups
Authors: Steininger Christoph , Kundi Michael , Aberle Stephan W. , Aberle Judith H. , Popow-Kraupp Theresia 
Published: July 2002

ABSTRACT

The degrees of effectiveness of reverse transcription (RT)-PCR, virus isolation, and antigen enzyme-linked immunosorbent assay (ELISA) for the detection of influenza A virus were evaluated with nasopharyngeal swabs from 150 patients (1 week to 86 years old) with influenza A virus infection. RT-PCR had a sensitivity for influenza A virus in stock virus preparations 10
3
times higher than virus isolation and 10
6
to 10
7
times higher than ELISA. The detection rate achieved by RT-PCR in clinical samples was clearly higher (93%) than that by virus isolation (80%) and ELISA (62%). Despite low overall detection rates achieved by antigen ELISA, samples from patients younger than 5 years old yielded higher-than-average rates in this rapid assay (88%). The likelihood of negative results in the ELISA increased significantly with increasing age of the patient (
P
< 0.01). The degrees of effectiveness of RT-PCR and virus isolation were not influenced by the age of the patient. Neither influenza immunizations nor the interval between onset of symptoms and laboratory investigation (mean, 4.7 days; standard deviation, 3.3 days) affected results obtained by the three test systems. Our results demonstrate that the ELISA is reliable for rapid laboratory diagnosis of influenza in infants and young children, but for older patients application of RT-PCR or virus isolation is necessary to avoid false negative results.
Sindbis Virus Entry into Cells Triggers Apoptosis by Activating Sphingomyelinase, Leading to the Release of Ceramide
Authors: Jan Jia-Tsrong , Chatterjee Subroto , Griffin Diane E. 
Published: July 2002

ABSTRACTSindbis virus (SV) causes acute encephalomyelitis by infecting and inducing the death of neurons. Induction of apoptosis occurs during virus entry and involves acid-induced conformational changes in the viral surface glycoproteins and sphingomyelin (SM)-dependent fusion of the virus envelope with the endosomal membrane. We have studied neuroblastoma cells to determine how this entry process triggers cell death. Acidic sphingomyelinase was activated during entry followed by activation of neutral sphingomyelinase, SM degradation, and a sustained increase in ceramide. Ceramide-induced apoptosis and SV-induced apoptosis could be inhibited by treatment with Z-VAD-fmk, a caspase inhibitor, and by overexpression of Bcl-2, an antiapoptotic cellular protein. Acid ceramidase, expressed in a recombinant SV, decreased intracellular ceramide and protected cells from apoptosis. The data suggest that acid-induced SM-dependent virus fusion initiates the apoptotic cascade by inducing SM degradation and ceramide release.
Novel Tat-Encoding Bicistronic Human Immunodeficiency Virus Type 1-Based Gene Transfer Vectors for High-Level Transgene Expression
Authors: Srinivasakumar Narasimhachar , Schuening Friedrich 
Published: July 2002

ABSTRACTWe describe bicistronic single-exon Tat (72-amino-acid Tat [Tat72])- and full-length Tat (Tat86)-encoding gene transfer vectors based on human immunodeficiency virus type 1 (HIV-1). We created versions of these vectors that were rendered Rev independent by using the constitutive transport element (CTE) from Mason-Pfizer monkey virus (MPMV). Tat72-encoding vectors performed better than Tat86-expressing vectors in gene transfer experiments. CTE-containing vectors, produced in a Rev-independent packaging system, had gene transfer efficiencies nearly equivalent to those produced using a combination RNA transport (CTE and Rev-Rev response element)-based packaging system. The Tat72-encoding vectors could be efficiently transduced into a variety of cell types, showed higher levels of transgene expression than vectors with the simian cytomegalovirus immediate-early or the simian virus 40 early promoter, and provide an alternative to HIV-1 vectors with internal promoters.