bluuebunny/crossref_metadata_2025_split huggingface dataset:
Multiplex LightCycler PCR Assay for Detection and Differentiation of
Bordetella pertussis
and
Bordetella parapertussis
in Nasopharyngeal Specimens
Authors: Sloan Lynne M. , Hopkins Marlene K. , Mitchell P. Shawn , Vetter Emily A. , Rosenblatt Jon E. , Harmsen W. Scott , Cockerill Franklin R. , Patel Robin 
Published: July 2002

ABSTRACT

A rapid real-time multiplex PCR assay for detecting and differentiating
Bordetella pertussis
and
Bordetella parapertussis
in nasopharyngeal swabs was developed. This assay (LC-PCR-IS) targets the insertion sequences IS
481
and IS
1001
of
B. pertussis
and
B. parapertussis
, respectively, and is performed using the LightCycler (Roche Molecular Biochemicals, Indianapolis, Ind.). The analytical sensitivity is less than one organism per reaction. Results for
Bordetella
culture and/or direct fluorescent antibody testing and a second LightCycler PCR assay (target, pertussis toxin gene) were compared to results of the LC-PCR-IS assay for 111 nasopharyngeal swabs submitted for pertussis testing. Of the specimens, 12 were positive (9
B. pertussis
and 3
B. parapertussis
) and 68 specimens were negative by all methods. Three other specimens were positive for
B. pertussis
by at least two of the methods (including the LC-PCR-IS assay), and another 28 specimens were positive for
B. pertussis
by the LC-PCR-IS assay only. No specimens were negative by the LC-PCR-IS assay and positive by the other methods. A conventional PCR method (target, IS
481
) was also compared to the LC-PCR-IS assay for a different group of nasopharyngeal swab specimens (
n
= 96): 44 specimens were positive and 41 specimens were negative for
B. pertussis
with both PCR methods. Nine specimens were positive for
B. pertussis
by the LC-PCR-IS assay and negative by the conventional PCR assay, and two specimens were positive for
B. pertussis
by the conventional PCR assay and negative by the LC-PCR-IS assay. Positivity of the two assays was not significantly different (
P
= 0.0654). The insertion sequence IS
481
is also present in
Bordetella holmesii
; specimens containing
B
.
holmesii
may yield false-positive results. The LC-PCR-IS assay takes approximately 45 min to complete post-nucleic acid extraction, compared to 24 h for the conventional PCR assay previously used in our laboratory. The LC-PCR-IS assay is easier to perform than the conventional PCR assay, and the closed system decreases the chance of contamination. All of these characteristics represent a significant improvement in the detection of
B
.
pertussis
and
B
.
parapertussis
in nasopharyngeal specimens.
Multiserotype Enzyme-Linked Immunosorbent Assay as a Diagnostic Aid for Periodontitis in Large-Scale Studies
Authors: Pussinen P. J. , Vilkuna-Rautiainen T. , Alfthan G. , Mattila K. , Asikainen S. 
Published: July 2002

ABSTRACT

Periodontitis is a common chronic oral infection caused by gram-negative bacteria, including
Actinobacillus actinomycetemcomitans
and
Porphyromonas gingivalis
. Periodontitis evokes inflammatory host response locally in the periodontium but also systemically. The systemic humoral antibody response against oral pathogens can conveniently be measured by an immunoassay. The aim of the study was to measure serum immunoglobulin G class antibodies against
A. actinomycetemcomitans
and
P. gingivalis
by an enzyme-linked immunosorbent assay (ELISA) in which mixtures of several serotypes of the pathogens were used as antigens to avoid biasing of the results in favor of a particular strain. For
A. actinomycetemcomitans
the antigen consisted of six strains representing serotypes a, b, c, d, and e and one nonserotypeable strain. In the
P. gingivalis
ELISA, antigens representing serotypes a, b, and c were used. Serum samples from 90 subjects, including 35 samples from patients with diagnosed periodontitis, 10 samples from periodontally healthy controls, and 45 samples from randomly selected apparently healthy volunteers (referred to as “healthy subjects”), were tested. For both pathogens the antibody levels (means ± standard deviations) of the patients—expressed as area under the dilution curve—were significantly higher than those for healthy controls or healthy subjects, with values for
A. actinomycetemcomitans
and
P. gingivalis
, respectively, as follows: patients, 22.60 ± 9.94 mm
2
and 26.72 ± 11.13 mm
2
; healthy controls, 9.99 ± 3.92 mm
2
and 6.90 ± 3.38 mm
2
; and healthy subjects, 16.85 ± 6.67 mm
2
and 8.51 ± 4.23 mm
2
. The serotype mixture ELISA is suitable for measuring antibodies against periodontal pathogens in large epidemiological studies in order to evaluate the role of periodontitis as a risk factor for other diseases.
Use of 16S rRNA Sequencing for Identification of
Actinobacillus ureae
Isolated from a Cerebrospinal Fluid Sample
Authors: Whitelaw A. C. , Shankland I. M. , Elisha B. G. 
Published: July 2002

ABSTRACT

Actinobacillus ureae
, previously
Pasteurella ureae
, has on rare occasions been described as a cause of human infection. Owing to its rarity, it may not be easily identified in clinical microbiology laboratories by standard tests. This report describes a patient with acute bacterial meningitis due to
A. ureae
. The identity of the isolate was determined by means of DNA sequence analysis of a portion of the 16S rRNA gene.
Enzyme-Linked Immunosorbent Assay Based on Soluble Promastigote Antigen Detects Immunoglobulin M (IgM) and IgG Antibodies in Sera from Cases of Visceral and Cutaneous Leishmaniasis
Authors: Ryan Jeffrey R. , Smithyman Anthony M. , Rajasekariah G-Halli , Hochberg Lisa , Stiteler John M. , Martin Samuel K. 
Published: July 2002

ABSTRACT
Leishmaniasis causes significant morbidity and mortality in areas where it is endemic. In areas where it is nonendemic, global travel and increased incidence of the disease in human immunodeficiency virus and intravenous-drug user populations are also causes for concern. The unavailability of rapid and reliable tests for diagnosis of the various leishmaniases makes patient management difficult. We have developed an enzyme-linked immunosorbent assay (ELISA) that can detect immunoglobulin M (IgM) and IgG antibodies in patients with visceral and cutaneous leishmaniasis. These practical assays are based on soluble antigens from promastigotes cultivated in a protein-free medium. In preliminary studies, 129 visceral (Brazil, Italy, North Africa, and Nepal) and 143 cutaneous (Brazil) leishmaniasis patients with controls were tested. Overall, the tests showed a sensitivity of 95.1%. In addition, the ELISA correctly identified 42 sera from Brazilian dogs with canine leishmaniasis and 10 healthy controls. Serological tests for the various clinical manifestations of leishmaniasis could be useful epidemiological and patient management tools in populations of areas of endemicity and nonendemicity.
Laccase and Melanization in Clinically Important
Cryptococcus
Species Other than
Cryptococcus
neoformans
Authors: Ikeda Reiko , Sugita Takashi , Jacobson Eric S. , Shinoda Takako 
Published: July 2002

ABSTRACT

The laccase enzyme and melanin synthesis have been implicated as contributors to virulence in
Cryptococcus neoformans
. Since isolations of
Cryptococcus
species other than
C.
neoformans
from clinical specimens have been increasing, we examined the laccase activities of
C. albidus
,
C. laurentii
,
C. curvatus
, and
C. humicola
. Incubation of cells with epinephrine produced adrenochrome color in
C. albidus
,
C. laurentii
, and
C. curvatus
but not in
C. humicola
. Activity was always less than in
C. neoformans
. Laccase was detected in the soluble fractions of disrupted
C. albidus
,
C. laurentii
, and
C. curvatus
cells. Activity staining of partially purified enzyme after nondenaturing polyacrylamide gel electrophoresis revealed that laccases from
C. albidus
,
C. laurentii
, and
C. curvatus
migrated more slowly than that from
C. neoformans
. One strain of
C. curvatus
exhibited two melanin bands. Thus, several clinically emerging
Cryptococcus
species express laccase and can synthesize melanin.
Quantitative Detection of
Mycoplasma genitalium
from First-Pass Urine of Men with Urethritis and Asymptomatic Men by Real-Time PCR
Authors: Yoshida Takashi , Deguchi Takashi , Ito Masayasu , Maeda Shin-Ichi , Tamaki Masayoshi , Ishiko Hiroaki 
Published: July 2002

ABSTRACT

We developed a TaqMan-based real-time PCR assay for quantifying
Mycoplasma genitalium
. This assay is able to specifically quantify concentrations of the
M. genitalium
16S rRNA gene ranging from 10
7
to 10 copies/reaction. Using the TaqMan assay, we quantified the
M. genitalium
16S rRNA gene in first-pass urine of men with urethritis and asymptomatic men who were positive for
M. genitalium
by PCR- and phylogeny-based assay. Of 130 men with gonococcal urethritis (GU), five were positive for
M. genitalium
. The mycoplasma load for each specimen was <5 × 10 copies/ml. Of 84 men with chlamydial non-GU (CNGU), seven were positive for
M. genitalium
. One man had an
M. genitalium
load of <5 × 10 copies/ml, and six men had loads ranging from 1.1 × 10
7
to 2.7 × 10
2
copies/ml. Of 86 men with nonchlamydial NGU (NCNGU), 17 were positive for
M. genitalium
. The mycoplasma loads for these men ranged from 3.3 × 10
6
to 2.3 × 10
2
copies/ml. Of 76 asymptomatic men, only two were positive for
M. genitalium
. For these men, the loads were 2 × 10
2
and <5 × 10 copies/ml. The patients with NGU had significantly higher concentrations of
M. genitalium
in their first-pass urine than did men with GU (
P
< 0.01) or asymptomatic men (
P
< 0.05). In addition,
M. genitalium
loads were significantly higher in men with NCNGU than those in asymptomatic men (
P
< 0.05). The quantitative assessment of
M. genitalium
loads by the TaqMan assay will provide useful information for understanding the pathogenicity of this mycoplasma in the urogenital tract.
Comparison of Second- and Third-Generation Enzyme Immunoassays for Detecting Antibodies to Hepatitis C Virus
Authors: Abdel-Hamid Mohamed , El-Daly Mai , El-Kafrawy Sherif , Mikhail Nabiel , Strickland G. Thomas , Fix Alan D. 
Published: July 2002

ABSTRACT
Supplemental assays, such as recombinant immunoblot assays (RIBA), are used to confirm detection of antibodies to hepatitis C virus (HCV). However, due to their expense, they are not widely used in developing countries. The purpose of our study was to compare the results of second- and third-generation (G2 and G3, respectively) enzyme immunoassays (EIAs) and to resolve discordant results by using a supplemental assay to assess the reliability of G2 and G3 EIAs to confirm anti-HCV antibody-positive results. We performed both G2 and G3 EIAs for anti-HCV antibodies on 1,134 serum samples collected during the 2nd year of a longitudinal community-based study in Egypt; 35 samples with discordant results were tested by Abbott Laboratories Micro-Particle Immunoassay (M-EIA) and RIBA. Viremia was determined with an in-house nested reverse transcriptase PCR (RT-PCR) to detect HCV RNA. Concordance between the two assays (G2/G3) was 96.9%; 87 (7.7%) samples were positive and 1,012 (89.2%) were negative by both assays. For 17 samples, the discordant results were G2 assay negative and G3 assay positive, and for 18 samples, the discordant results were G2 assay positive and G3 assay negative. Among the 17 G2 assay-negative and G3 assay-positive samples, 15 were M-EIA positive and 7 were PCR positive. Among the 18 G2 assay-positive and G3 assay-negative samples, 2 were M-EIA positive and none were PCR positive. RIBA results from 24 discordant samples showed 87.5% agreement with the G3 EIA, 12.5% agreement with the G2 EIA, and 95.8% agreement with M-EIA. Eleven samples were indeterminate by RIBA and excluded from this analysis. Based on RIBA results, the sensitivity of the G3 EIA was 99%, compared to 89.8% for the G2 EIA, while the specificity of the G3 EIA was 99.8%, compared to 98.9% for the G2 EIA. These results show that the reliability of the G3 EIA in screening these sera is excellent, and the G3 assay can be used in the absence of supplemental tests where resources are limited. RIBA appears not to have advantages over the less expensive M-EIA screening assay. The main disadvantage of RIBA is the occurrence of indeterminate results, especially among problematic samples. Samples giving discordant results in multiple assays are often indeterminate with the RIBA.
PreservCyt Transport Medium Used for the ThinPrep Pap Test Is a Suitable Medium for Detection of
Chlamydia trachomatis
by the COBAS Amplicor CT/NG Test: Results of a Preliminary Study and Future Implications
Authors: Bianchi Anne , Moret François , Desrues Jean-Marc , Champenois Thierry , Dervaux Yves , Desvouas Orlane , Oursin André , Quinzat Dominique , Dachez Roger , Bathelier Christian , Ronsin Christophe 
Published: July 2002

ABSTRACT

The commercial COBAS Amplicor CT/NG test (Roche Diagnostic Systems, Meylan, France) is a sensitive and specific method for detection of
Chlamydia trachomatis
infections. This test currently consists of using a nucleic acid amplification method to detect
C. trachomatis
in first-void urine specimens and in endocervical swabs collected in 2-sucrose-phosphate (2SP) transport medium. We conducted a prospective study to determine whether the automated COBAS Amplicor CT/NG test can detect
C. trachomatis
in cervical specimens collected in PreservCyt transport medium (ThinPrep Pap Test; Cytyc Corporation, Boxborough, Mass.). PreservCyt medium is used to preserve cervical samples before the preparation of ThinPrep slides. We collected 1,000 cervical specimens from young women (age range, 15 to 25 years) during routine Pap smear tests. Only specimens with normal cytology and in which the gynecologist found no clinical evidence of urogenital infections were selected. The samples were stored in PreservCyt transport medium at 15 to 20°C.
C. trachomatis
was detected in 22 of the 1,000 cervical specimens that had been stored in PreservCyt. To confirm the positive samples, the test was repeated on new endocervical swab specimens collected in 2SP transport medium. Only 9 of the 22 positive patients agreed to undergo this control, but all 9 retested positive. To evaluate the influence of storage conditions on the sensitivity of the
C. trachomatis
PCR test, all of the positive samples were stored at 15 to 20°C in PreservCyt transport medium and were retested every 2 weeks for 6 weeks.
C. trachomatis
was successfully amplified from all 22 specimens for the whole 6-week period. The prevalence of
C. trachomatis
infection was 2.2% in our study population. These results demonstrate that PreservCyt transport medium is a suitable transport medium for detection of
C. trachomatis
by the COBAS Amplicor CT/NG test. The ThinPrep Pap Test may enable gynecologists to monitor for both cervical lesions and
C. trachomatis
infections with a single endocervical specimen.
Genotyping of Hepatitis C Virus Types 1, 2, 3, and 4 by a One-Step LightCycler Method Using Three Different Pairs of Hybridization Probes
Authors: Schröter Matthias , Zöllner Bernhard , Schäfer Peter , Landt Olfert , Lass Ulrich , Laufs Rainer , Feucht Heinz-Hubert 
Published: July 2002

ABSTRACT
Determination of hepatitis C virus (HCV) genotypes has become increasingly important during the last years for prediction of the clinical course and the outcome of antiviral therapy. Therefore, numerous different methods have been developed to enable HCV genotyping. However, many of them are very laborious and expensive, leading to limited usage in daily routine diagnostics. We have established a method which combines the speed of the new LightCycler technology with the use of amplification products generated for diagnostic quantitative HCV RNA determination. Differentiation of HCV genotypes is performed with these amplicons in a single step by using fluorophore-labeled hybridization probes. Although currently only two different acceptor fluorophores are available for the LightCycler, types 1, 2, 3, and 4, which are by far the prevailing HCV genotypes in Europe and the United States, can be distinguished. Genotypes of specimens from 190 chronically HCV-infected patients were determined by the LightCycler method and compared with the results of nucleotide sequencing. Concordant results were obtained for all samples. This new method offers a fast and convenient possibility to determine the quantitative HCV RNA load and the genotype in large-scale settings within about 4 h.
Initiation of DNA Replication within
oriP
Is Dispensable for Stable Replication of the Latent Epstein-Barr Virus Chromosome after Infection of Established Cell Lines
Authors: Norio Paolo , Schildkraut Carl L. , Yates John L. 
Published: July 2002

ABSTRACT

The 165-kb circularized chromosome of Epstein-Barr virus (EBV) is replicated in latently infected cells once per cell cycle by host proteins during S phase. Replication initiates at multiple sites on latent EBV chromosomes, including within a 1.8-kb region called
oriP
, which can provide both replication and stabilization for recombinant plasmids in the presence of the EBV-encoded protein, EBNA-1. Replication initiates at or near the dyad symmetry component (DS) of
oriP
, which depends on multiple EBNA-1 binding sites for activity. To test the importance of the replication function of
oriP
, the DS was deleted from the viral genome. EBV mutants lacking the DS and carrying a selectable gene could establish latent infections in BL30 cells, in which circular, mutant viral chromosomes were stably maintained. Analysis of replication fork movement using two-dimensional gel electrophoresis showed that the deletion of the DS reduced the initiation events to an undetectable level within the
oriP
region so that this segment was replicated exclusively by forks entering the region from either direction. A significant slowing or stalling of replication forks that occurs normally at the approximate position of the DS was also eliminated by deletion of the DS. The results confirm the DS as both a replication origin and a place where replication forks pause. Since the replication function of
oriP
is dispensable at least in certain cell lines, the essential role of EBNA-1 for infection of these cell lines is likely to be that of stabilizing the EBV chromosome by associating with the 30-bp repeats of
oriP
. The results also imply that in established cell lines, the EBV chromosome can be efficiently replicated entirely from origins that are activated by cellular factors. Presumably, initiation of replication at the DS, mediated by EBNA-1, is important for the natural life cycle of EBV, perhaps in establishing latent infections of normal B cells.
Characteristics of Bursal T Lymphocytes Induced by Infectious Bursal Disease Virus
Authors: Kim In-Jeong , You Seung Kwon , Kim Hyungee , Yeh Hung-Yeuh , Sharma Jagdev M. 
Published: July 2002

ABSTRACTInfectious bursal disease virus (IBDV) is an avian lymphotropic virus that causes immunosuppression. When specific-pathogen-free chickens were exposed to a pathogenic strain of IBDV (IM), the virus rapidly destroyed B cells in the bursa of Fabricius. Extensive viral replication was accompanied by an infiltration of T cells in the bursa. We studied the characteristics of intrabursal T lymphocytes in IBDV-infected chickens and examined whether T cells were involved in virus clearance. Flow cytometric analysis of single-cell suspensions of the bursal tissue revealed that T cells were first detectable at 4 days postinoculation (p.i.). At 7 days p.i., 65% of bursal cells were T cells and 7% were B cells. After virus infection, the numbers of bursal T cells expressing activation markers Ia and CD25 were significantly increased (P< 0.03). In addition, IBDV-induced bursal T cells produced elevated levels of interleukin-6-like factor and nitric oxide-inducing factor in vitro. Spleen and bursal cells of IBDV-infected chickens had upregulated gamma interferon gene expression in comparison with virus-free chickens. In IBDV-infected chickens, bursal T cells proliferated in vitro upon stimulation with purified IBDV in a dose-dependent manner (P< 0.02), whereas virus-specific T-cell expansion was not detected in the spleen. Cyclosporin A treatment, which reduced the number of circulating T cells and compromised T-cell mitogenesis, increased viral burden in the bursae of IBDV-infected chickens. The results suggest that intrabursal T cells and T-cell-mediated responses may be important in viral clearance and promoting recovery from infection.
Isolation of Transformation Suppressor Genes by cDNA Subtraction: Lumican Suppresses Transformation Induced by v-
src
and v-K-
ras
Authors: Yoshioka Naohisa , Inoue Hirokazu , Nakanishi Kazuyoshi , Oka Kiyomasa , Yutsudo Masuo , Yamashita Atsuko , Hakura Akira , Nojima Hiroshi 
Published: July 2002

ABSTRACT

We have reported that suppressive factors for transformation by viral oncogenes are expressed in primary rat embryo fibroblasts (REFs). To identify such transformation suppressor genes, we prepared a subtracted cDNA library by using REFs and a rat normal fibroblast cell line, F2408, and isolated 30 different cDNA clones whose mRNA expression was markedly reduced in F2408 cells relative to that in REFs. We referred to these as TRIF (transcript reduced in F2408) clones. Among these genes, we initially tested the suppressor activity for transformation on three TRIF genes, TRIF1 (neuronatin), TRIF2 (heparin-binding growth-associated molecule), and TRIF3 (lumican) by focus formation assay and found that lumican inhibited focus formation induced by activated H-
ras
in F2408 cells. Colony formation in soft agar induced by v-K-
ras
or v-
src
was also suppressed in F2408 clones stably expressing exogenous lumican without disturbing cell proliferation. Tumorigenicity in nude mice induced by these oncogenes was also suppressed in these lumican-expressing clones. These results indicate that lumican has the ability to suppress transformation by v-
src
and v-K-
ras
.
The r144 Major Histocompatibility Complex Class I-Like Gene of Rat Cytomegalovirus Is Dispensable for both Acute and Long-Term Infection in the Immunocompromised Host
Authors: Beisser Patrick S. , Kloover Jeroen S. , Grauls Gert E. L. M. , Blok Marinus J. , Bruggeman Cathrien A. , Vink Cornelis 
Published: July 2002

ABSTRACTThe rat cytomegalovirus (RCMV) r144 gene encodes a polypeptide homologous to major histocompatibility complex class I heavy chains. To study the role of r144 in virus replication, an RCMV r144 null mutant strain (RCMVΔr144) was generated. This strain replicated with efficiency similar to that of wild-type (WT) RCMV in vitro. Additionally, WT RCMV and RCMVΔr144 were found not to differ in their replication characteristics in vivo. First, the survival rate was similar among groups of immunosuppressed rats infected with either RCMVΔr144 or WT RCMV. Second, the dissemination of virus did not differ in either RCMVΔr144- or WT RCMV-infected, immunosuppressed rats, either in the acute phase of infection or approximately 1 year after infection. These data indicate that the RCMV r144 gene is essential neither for virus replication in the acute phase of infection nor for long-term infection in immunocompromised rats. Interestingly, in a local infection model in which footpads of immunosuppressed rats were inoculated with virus, a significantly higher number of infiltrating macrophage cells as well as of CD8+T cells was observed in WT RCMV-infected paws than in RCMVΔr144-infected paws. This suggests that r144 might function in the interaction with these leukocytes in vivo.