bluuebunny/crossref_metadata_2025_split huggingface dataset:
Origin Binding Activity of the Mycobacterial Plasmid pAL5000 Replication Protein RepB Is Stimulated through Interactions with Host Factors and Coupled Expression of
repA
Authors: Basu Abhijit , Chawla-Sarkar Mamta , Chakrabarti Santanu , Das Gupta Sujoy K. 
Published: July 2002

ABSTRACT

The minimal replication region of the mycobacterial plasmid pAL5000 encompasses the replication origin (
ori
) and two tandemly organized replication genes,
repA
and
repB,
the functions of which are not clearly known. It was observed that when the
repA
and
repB
genes were expressed in
Escherichia coli,
a strong
ori
binding activity was generated in the host cells. Inactivation of
repB
led to a complete loss of activity, whereas inactivation of
repA
had a partial effect, indicating that while
repB
plays an important role in the process, its activity is stimulated through coexpression of
repA
. However, this stimulatory effect could be demonstrated only when expression of
repA
and that of
repB
were coupled. At a relatively high concentration (1,000 nM), the purified RepB protein was found to form an
ori
complex with low specificity, which was sensitive to high salt concentrations and challenge by a nonspecific competitor. In contrast, the complex formed by an extract of
repA-repB
-expressing cells was highly specific and was resistant to both types of challenges. At a 10-fold-lower concentration, RepB did not exhibit
ori
binding activity, but it could nevertheless form a salt-resistant
ori
complex in vitro, provided that host factors were present. Antibody supershift experiments indicated that RepB is a key component of the specific complex formed by extracts prepared from
E. coli
cells expressing the
repA
and
repB
genes and also from mycobacterial cells harboring pAL5000-derived vectors. The results indicate that in vivo RepB interacts with host factors and forms an
ori
complex, but this activity is maximal only when there is coupled expression of
repA
.
Simultaneous Detection and Typing of Influenza Viruses A and B by a Nested Reverse Transcription-PCR: Comparison to Virus Isolation and Antigen Detection by Immunofluorescence and Optical Immunoassay (FLU OIA)
Authors: Herrmann Björn , Larsson Christine , Zweygberg Benita Wirgart 
Published: July 2002

ABSTRACT
A nested reverse transcription (RT)-PCR was developed for simultaneous detection and typing of influenza viruses A and B. The detection limit for influenza virus A subtypes H1 and H3 and that for influenza virus B were between 1 and 4 target gene copies per reaction for each type. The clinical benefit of the RT-PCR method was evaluated by comparing the results with virus isolation and direct immunofluorescence (IF) assays on 215 nasopharyngeal aspirates from patients with suspected influenza virus infection. The RT-PCR detected 83 cases of influenza A, compared to 66 cases detected by virus isolation and 68 cases detected by IF assay. The corresponding figures for the detection of influenza B were 15, 12, and 11 cases, respectively. In total, 16 out of 98 RT-PCR-positive specimens were negative by virus isolation and IF. An optical immunoassay for rapid detection of influenza A and B (FLU OIA; Bio Star Inc., Boulder, Colo.) was compared to RT-PCR and IF on 105 nasopharyngeal aspirates and 79 swabs. The sensitivity for the OIA was 40.4% compared to PCR and 48.8% compared to IF assay, when nasopharyngeal aspirates were examined. The specificities were 94.3 and 93.9%, respectively. The sensitivity was higher for OIA on nasopharyngeal swabs, 77.5% and 86.6% compared to PCR and IF, respectively, while the specificity was lower, 82.0% and 75.5%, respectively. The RT-PCR provides a sensitive and specific method for detecting and typing influenza viruses A and B. The rapid OIA is useful as a complementary test, but it cannot replace established methods without further evaluation.
Contemporary Assessment of Antimicrobial Susceptibility Testing Methods for Polymyxin B and Colistin: Review of Available Interpretative Criteria and Quality Control Guidelines
Authors: Gales Ana C. , Reis Adriana O. , Jones Ronald N. 
Published: July 2002

ABSTRACT

The emergence of infections caused by multidrug-resistant
Pseudomonas aeruginosa
and
Acinetobacter
spp. has necessitated the search for alternative parenteral agents such as the polymyxins. The National Committee for Clinical Laboratory Standards (NCCLS) documents do not currently provide interpretative criteria for the testing of the polymyxins, colistin and polymyxin B. Therefore, an evaluation of the antimicrobial activity of colistin and polymyxin B was initiated using 200 bloodstream infection pathogens collected through the SENTRY Antimicrobial Surveillance Program. All susceptibility tests were performed according to the NCCLS recommendations. Polymyxin B and colistin displayed a nearly identical spectrum of activity, exhibiting excellent potency against
P. aeruginosa
(MIC
90
, 2 μg/ml) and
Acinetobacter
sp. (MIC
90
, 2 μg/ml). In contrast, they showed limited activity against some other nonfermentative bacilli such as
Burkholderia cepacia
(MIC
90
, ≥128 μg/ml). Excellent correlation was achieved between broth microdilution and agar dilution tests (
r
= 0.96 to 0.98); 94.3% of the results were ±1 log
2
dilution between the methods used for both compounds. At a resistance breakpoint of ≥4 μg/ml for both agents, unacceptable false-susceptible or very major errors were noted for colistin (5%) and polymyxin B (6%). Modified zone criteria for colistin (≤11 and ≥14 mm) and polymyxin B (≤10 and ≥14 mm) were suggested, but some degree of error persisted (≥3.5%). It is recommended that all susceptible disk diffusion results be confirmed by MIC tests using the preferred reference NCCLS method. The quality control (QC) ranges listed in the product package insert require an adjusted range by approximately 3 mm for both NCCLS gram-negative quality control strains. This evaluation of in vitro susceptibility test methods for the polymyxin class drugs confirmed continued serious testing error with the disk diffusion method, the possible need for breakpoint adjustments, and the recalculation of disk diffusion QC ranges. Clinical laboratories should exclusively use MIC methods to assist the therapeutic application of colistin or polymyxin B until disk diffusion test modifications are sanctioned and published by the NCCLS.
Fecal Leukocyte Stain Has Diagnostic Value for Outpatients but Not Inpatients
Authors: Savola Kristen L. , Baron Ellen Jo , Tompkins Lucy S. , Passaro Douglas J. 
Published: July 2002

ABSTRACT

The methylene blue stain for fecal leukocytes (FL) is widely used as an adjunct to slower but more accurate tests of diarrheal etiology, such as stool culture (SCx) or toxin assays for
Clostridium difficile
. Prior studies investigating the utility of FL for predicting SCx and
C. difficile
toxin assay (CDTA) results did not evaluate the importance of inpatient versus outpatient status. We conducted a study of patients who submitted a stool specimen to the Stanford Hospital Microbiology Laboratory between May 1998 and April 1999. The results for stool specimens that were tested by FL and by a confirmatory test (either SCx or CDTA) were used to determine whether the FL method helped to predict the results of these tests. Of 797 stools that were tested by FL method and at least one confirmatory test, 502 stools were tested by CDTA, and 473 stools were cultured. The FL test was 14% sensitive and 90% specific for
C. difficile
with a diagnostic threshold of one white blood cell/high-power field (WBC/HPF). The overall likelihood ratio (LR) for a positive CDTA was 1.4 with a 95% confidence interval (CI) of 0.5 to 3.7 (
P
= 0.5) and was similar among inpatients and outpatients. In contrast, the presence of ≥1 WBC/HPF was 52% sensitive and 88% specific for the 27 positive SCx results and helped to predict a positive SCx result (LR, 4.2; 95% CI, 2.7 to 6.5;
P
< 0.001). The sensitivity of ≥1 WBC/HPF was 57%, and its predictive value for SCx was higher among outpatients (outpatient LR, 5.0; 95% CI, 2.9 to 8.6;
P
< 0.001; inpatient LR, 1.9; 95% CI, 0.3 to 10.8;
P
= 0.5). Among inpatients, only 4 (1.5%) of the 273 SCx results were positive, and the presence of ≥1 WBC/HPF was insensitive (25%) and did not predict a positive SCx (LR, 1.9; 95% CI, 0.3 to 10.8;
P
= 0.5). When the data were reanalyzed using a diagnostic threshold of five WBC/HPF for FL, the predictive power of the FL method was similar. Thus, FL was of no value in predicting CDTA positivity, nor was it helpful in predicting SCx results for inpatients. Neither SCx nor the FL method should routinely be performed on samples from inpatients. Among outpatients, presence of FLs should suggest a bacterial diarrhea in clinically compatible cases.
TaqMan-Based Detection of
Trichomonas vaginalis
DNA from Female Genital Specimens
Authors: Jordan Jeanne A. , Lowery Donna , Trucco Massimo 
Published: July 2002

ABSTRACT

A double-labeled fluorescent probe was designed and evaluated for detecting
Trichomonas vaginalis
DNA in a 5′ nuclease (TaqMan) assay. The
T. vaginalis
-specific probe contains a 5′-fluorescein (5′-FAM) and a 3′-rhodamine (TAMRA) derivative. Female genital secretions were collected on Amplicor (Roche Molecular, Indianapolis, Ind.) swabs and by a transport system used for
Chlamydia trachomatis
and/or
Neisseria gonorrhoeae
DNA detection by PCR. Five hundred fifty-two female genital specimens, of which 248 (45%) were vaginal specimens and 304 (55%) were introital, were tested for both
T. vaginalis
DNA and viable microorganisms using the 5′ nuclease assay and broth culture, respectively. Of these, 304 of 552 (55%) were also evaluated by direct microscopic examination for the characteristic motile organism. After resolving discrepancies, the comparisons produced an analytical sensitivity and specificity for the TaqMan-based PCR assay of 97.8 and 97.4%, respectively. As a result, ΔRQ values (differences in fluorescence due to probe hybridization and resulting 5′-FAM cleavage from the specific PCR product) of ≥2.0 and ≤1.5 were established for
T. vaginalis
-positive and -negative cutoffs, respectively. ΔRQ values between 1.5 and 2.0 were considered indeterminate. Overall findings revealed a high level of agreement between PCR and culture for detecting
T. vaginalis
. Potential benefits of the 5′ nuclease assay include a greater sensitivity compared to direct microscopic examination and the ease of testing large numbers of clinical specimens in a significantly shorter turnaround time compared to culture.
Combined PCR-Heteroduplex Mobility Assay for Detection and Differentiation of Influenza A Viruses from Different Animal Species
Authors: Ellis Joanna S. , Zambon Maria C. 
Published: July 2002

ABSTRACT
Transfer of influenza A viruses from animal hosts to man may lead to the emergence of new human pandemic strains. The early detection and identification of such events are therefore paramount in the surveillance of influenza viruses. To detect and partially characterize influenza A viruses from different animal species, a combined reverse transcription (RT)-PCR heteroduplex mobility assay (HMA) was designed. This M gene RT-PCR was shown to be sensitive and specific for the detection of human, avian, and swine influenza A viruses. PCR amplicons from human, avian, and swine viruses of 15 different subtypes, with between 1.9 and 21.4% nucleotide divergence, were differentiated by HMA. Sequencing of the amplicons showed that the heteroduplex mobility patterns correlated with the sequence divergence between test and reference DNA. The application of the RT-PCR HMA method for rapid screening of samples was assessed with a reference panel of viruses of human, avian, and swine origin. The avian H9N2 virus A/HongKong/1073/99, which crossed the species barrier to humans, was screened against the reference panel. It was found to be most closely related to the avian A/Quail/HongKong/G1/97 H9N2 reference PCR product. Sequence analysis showed a nucleotide divergence of 1.1% between the A/Quail/HongKong/G1/97 and A/HongKong/1073/99 amplicons. From the results of our work, we consider the RT-PCR HMA method described to offer a rapid and sensitive means for screening for novel or unusual influenza viruses.
Detection and Differentiation of Human Polyomaviruses JC and BK by LightCycler PCR
Authors: Whiley David M. , Mackay Ian M. , Sloots Theo P. 
Published: July 2002

ABSTRACT
Human polyomaviruses JC and BK may cause several clinical manifestations in immunocompromised hosts, including progressive multifocal leukoencephalopathy and hemorrhagic cystitis. Molecular detection by PCR is recognized as a sensitive and specific method for detecting human polyomaviruses in clinical samples. In this study, a real-time PCR assay using the LightCycler platform was evaluated and compared to an “in-house” PCR assay using a conventional detection method. A total of 122 urine specimens were tested, and human polyomavirus was detected in 49 specimens (40%) by both conventional PCR and LightCycler PCR. The remaining 73 specimens (60%) were found negative by both assays. For 46 of the 49 positive specimens, LightCycler PCR and conventional PCR identified the same polyomavirus type. These samples included 30 samples with JC virus (JCV), 14 samples with BK virus (BKV), and 2 samples in which both viruses were detected. In the remaining three samples, both JCV and BKV were detected by the conventional assay, but only JCV was detected by the LightCycler assay. The results of this study show that the LightCycler PCR assay displays sensitivity and specificity similar to those of a conventional PCR assay. These data, combined with its rapid turnaround time for results and decreased hands-on time, make the LightCycler PCR assay highly suitable for the rapid detection and differentiation of JCV and BKV in the clinical laboratory.
Use of BACTEC 9240 Blood Culture System for Detection of
Brucella melitensis
in Synovial Fluid
Authors: Yagupsky Pablo , Peled Nechama , Press Joseph 
Published: July 2002

ABSTRACT

Synovial fluid specimens aspirated from patients with arthritis were inoculated into an aerobic Peds Plus blood culture bottle and monitored by the BACTEC 9240 instrument for 4 weeks. A total of 1,072 synovial fluid cultures were processed, and 15 (0.14%) were positive for
Brucella melitensis
. A single culture, harboring 1.3 CFU of viable organisms per ml, was missed by the instrument. All 14 positive BACTEC cultures were detected within 3 to 7 days.
Isolation and Characterization of a
Salmonella enterica
Serotype Typhi Variant and Its Clinical and Public Health Implications
Authors: Woo Patrick C. Y. , Fung Ami M. Y. , Wong Samson S. Y. , Tsoi Hoi-Wah , Yuen Kwok-Yung 
Published: July 2002

ABSTRACT

We report the isolation and characterization of a member of the family
Enterobacteriaceae
isolated from the gallbladder pus of a food handler. Conventional biochemical tests suggested
Salmonella enterica
serotype Typhi, but the isolate agglutinated with poly(O), 2O, 9O, and Vi
Salmonella
antisera but not with poly(H) or any individual H
Salmonella
antisera. 16S rRNA gene sequencing showed that there were two base differences between the isolate and
Salmonella enterica
serotype Montevideo, four base differences between the isolate and serotype Typhi, five base differences between the isolate and
Salmonella enterica
serotype Typhimurium, and six base differences between the isolate and
Salmonella enterica
serotype Dublin, indicating that the isolate was a strain of
S. enterica
. Electron microscopy confirmed that the isolate was aflagellated. The flagellin gene sequence of the isolate was 100% identical to that of the H1-d flagellin gene of serotype Typhi. Sequencing of the
rfbE
gene, which encoded the CDP-tyvelose epimerase of the isolate, showed that there was a point mutation at position +694 (G→T), leading to an amino acid substitution (Gly→Cys). This may have resulted in a protein of reduced catalytic activity and hence the presence of both 2O and 9O antigens. We therefore concluded that the isolate was a variant of serotype Typhi. Besides antibiotic therapy and cholecystectomy, removal of all stones in the biliary tree was performed for eradication of the carrier state.
Infection withBartonella weissiiand Detection ofNanobacteriumAntigens in a North Carolina Beef Herd
Authors: Breitschwerdt Edward B. , Sontakke Sushama , Cannedy Allen , Hancock Susan I. , Bradley Julie M. 
Published: July 2002

ABSTRACTVery recently,Bartonellaorganisms have been isolated from large ruminants (deer, elk, and dairy and beef cattle) located in the United States and in France. In this study, we report the serologic, microbiologic, and molecular findings related to the isolation of aBartonellaspecies in North Carolina beef cattle and the detection of nanobacterial antigen using a commercially available enzyme-linked immunosorbent assay. Between August 1998 and September 1999, blood was collected from 38 cattle ranging in age from 1 month to 6.5 years. After a 1-month incubation period, aBartonellasp. was isolated on a 5% rabbit blood agar plate from three of six EDTA blood samples. PCR amplification of the 16S rRNA gene from all three isolates resulted in a DNA sequence that was 100% identical to that ofB. weissii16S rRNA (GenBank no.AF199502). By IFA testing, 36 of 38 cattle had antibodies (≥1:64) toBartonella weissii(bovine origin) antigens. Nanobacterial antigen was detected in 22 of 22 serum samples. We conclude that infection with an organism similar or closely related toB. weissiican occur in North Carolina cattle and that although their actual existence is still controversialNanobacteriumantigens were detected with a commercially available test kit. The epidemiology, vector biology, and potential pathogenicity of these organisms in cattle deserve future consideration.
Adaptation ofEscherichia colito the Bovine Mammary Gland
Authors: Bradley A. J. , Green M. J. 
Published: July 2002

ABSTRACTClinical mastitis in six Somerset dairy herds was monitored over a 12-month period.Escherichia coliwas implicated in 34.7% of all clinical cases. Forty-one percent of all clinicalE. colimastitis cases occurred in just 2.2% of the population. A total of 23.9% of clinicalE. colicases occurred in quarters suffering recurrent cases ofE. colimastitis. The genotypes of strains involved in recurrent cases of clinicalE. colimastitis were compared by DNA fingerprinting with enterobacterial repetitive intergenic consensus primers. In 85.7% of cases of recurrent quarterE. colimastitis, the same genotype was implicated as the cause of disease, suggesting persistence of the organism within the mammary environment. The same genotype as that in the original case was also implicated in 8.5% of recurrent cases occurring in different quarters of the same cow, suggesting spread between quarters. These findings challenge our current understanding of the epidemiology ofE. colimastitis and suggest that pathogen adaptation and host susceptibility may be playing a part in the changing pattern of clinical mastitis experienced in the modern dairy herd.
Development of a Real-Time PCR Assay for Detection ofToxoplasma gondiiin Pig and Mouse Tissues
Authors: Jauregui L. H. , Higgins J. , Zarlenga D. , Dubey J. P. , Lunney J. K. 
Published: July 2002

ABSTRACTA highly sensitive and specific method has been developed to reproducibly detect and quantitateToxoplasma gondiiburden in animal tissue samples usingT. gondiiITS1-derived primers and a fluorogenic probe via real-time PCR. Assay specificity was confirmed against a panel of DNA samples fromT. gondiiand other common protozoa as well as host animal tissue. This Toxo TaqMan assay was able to detect as little as 0.1 pg ofT. gondiigenomic DNA, which is equivalent to 1T. gondiibradyzoite, and has a dynamic range of detection of from 100 ng to 100 fg ofT. gondiiDNA. Tissues from experimentally infected mice and pigs as well as bradyzoite-spiked pig muscle samples were used to test and standardize this technique. Positive signals were obtained withT. gondiiparasite concentrations ranging from 4 to 3.7 × 105parasites per g of spiked pig tissue, with excellent linearity (R2= 0.9776). AllT. gondii-infected animals were correctly identified by this technique. Results indicate that this assay is applicable to swine carcasses and commercial pig products, is compatible with automation technology for potential slaughterhouse use, and will enable scientists to diagnose and quantitateT. gondiiin animal tissues.
New Immunochromatographic Rapid Test for Diagnosis of Acute Puumala Virus Infection
Authors: Hujakka Helena , Koistinen Vesa , Eerikäinen Pekka , Kuronen Ilpo , Mononen Ilkka , Parviainen Markku , Lundkvist Åke , Vaheri Antti , Närvänen Ale , Vapalahti Olli 
Published: July 2002

ABSTRACT

A new immunochromatographic rapid test,
POC
PUUMALA (Erilab Ltd., Kuopio, Finland), for detection of acute-phase Puumala virus (PUUV) infection was developed based on a highly purified baculovirus-expressed PUUV nucleocapsid protein antigen and lateral immunodiffusion techniques. After addition of sample (5 μl of serum, plasma, or fingertip blood) and buffer, PUUV-specific immunoglobulin M (IgM) antibodies, if present, together with the gold-conjugated anti-human IgM, formed a specific colored line in 5 min. The sensitivity and specificity of the test were evaluated with 200 serum samples and 30 fingertip blood samples. The reference method for the serum samples was a μ-capture enzyme immunoassay (EIA) for IgM and an immunofluorescence assay (IFA) for IgG antibodies. The analytical sensitivity and specificity of the rapid test were 100 and 99%, respectively, for unfrozen serum samples (
n
= 103; 12 PUUV IgM-positive samples). When freeze-thawed serum samples were used, the sensitivity and specificity were each 97.1% (
n
= 70; 35 PUUV IgM-positive samples). The specificity of the test was 96.2% for 27 serum samples with nonspecific IgM antibodies or rheumatoid factor (RF). The fingertip blood samples (
n
= 30) were negative, but they gave clear positive results when spiked with IgM-positive sera (
n
= 20). The results were in good agreement with the standard diagnostic methods. The rapid performance, the lack of need for refined laboratory equipment, and the high specificity with fresh serum and fingertip blood samples indicate that the developed
POC
PUUMALA rapid test is a useful tool for fast diagnosis of acute PUUV infection.
Identification and Expression of a 50-Kilodalton Surface Antigen ofBabesia gibsoniand Evaluation of Its Diagnostic Potential in an Enzyme-Linked Immunosorbent Assay
Authors: Fukumoto Shinya , Xuan Xuenan , Nishikawa Yoshifumi , Inoue Noboru , Igarashi Ikuo , Nagasawa Hideyuki , Fujisaki Kozo , Mikami Takeshi 
Published: July 2002

ABSTRACTA cDNA expression library prepared fromBabesia gibsonimerozoite mRNA was screened withB. gibsoni-infected dog serum. cDNA encoding a 50-kDa protein was cloned and designated the P50 gene. The complete nucleotide sequence of the P50 gene was 1,922 bp. Computer analysis suggested that the sequence of the P50 gene contained an open reading frame of 1,401 bp with a coding capacity of approximately 50 kDa. The complete genomic nucleotide sequence of the P50 gene has been analyzed and shown to contain a single intron of 37 bp. Southern blotting analysis indicated that the P50 gene was present at a single copy in theB. gibsonigenome. The native P50 protein ofB. gibsoniwith a molecular mass of 50 kDa was identified by Western blotting with anti-recombinant P50 mouse serum. Confocal laser microscopic analysis showed that the P50 protein was located on the surface ofB. gibsonimerozoites. The recombinant P50 protein expressed by baculovirus in insect cells was used as the antigen in an enzyme-linked immunosorbent assay (ELISA). The ELISA was able to differentiate betweenB. gibsoni-infected dog serum andB. canis-infected dog serum or noninfected dog serum. Furthermore, the antibody response against the recombinant P50 protein was maintained until the chronic stage of infection in dogs experimentally infected withB. gibsoniwas developed. These results demonstrate that the recombinant P50 protein might be a useful diagnostic reagent for detection of antibodies toB. gibsoniin dogs.