bluuebunny/crossref_metadata_2025_split huggingface dataset:
Use of Recombinant ESAT-6:CFP-10 Fusion Protein for Differentiation of Infections of Cattle by
Mycobacterium bovis
and by
M. avium
subsp.
avium
and
M. avium
subsp.
paratuberculosis
Authors: Waters W. R. , Nonnecke B. J. , Palmer M. V. , Robbe-Austermann S. , Bannantine J. P. , Stabel J. R. , Whipple D. L. , Payeur J. B. , Estes D. M. , Pitzer J. E. , Minion F. C. 
Published: July 2004

ABSTRACT

Immunological diagnosis of
Mycobacterium bovis
infection of cattle is often confounded by cross-reactive responses resulting from exposure to other mycobacterial species, especially
Mycobacterium avium
. Early secretory antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) are dominant gamma interferon (IFN-γ)-inducing antigens of tuberculous mycobacteria, and they are absent from many environmental nontuberculous mycobacteria. Because
M. avium
exposure is the primary confounding factor in the diagnosis of
M. bovis
-infected animals, in vitro responses to a recombinant ESAT-6:CFP-10 (rESAT-6:CFP-10) fusion protein by blood leukocytes from cattle naturally exposed to
M. avium
or experimentally challenged with
Mycobacterium avium
subsp.
avium
or
Mycobacterium avium
subsp.
paratuberculosis
were compared to responses by
M. bovis
-infected cattle. Responses to heterogeneous mycobacterial antigens (i.e., purified protein derivatives [PPDs] and whole-cell sonicates [WCSs]) were also evaluated. Tumor necrosis factor alpha (TNF-α), IFN-γ, and nitric oxide responses by
M. bovis
-infected cattle to rESAT-6:CFP-10 exceeded (
P
< 0.05) the corresponding responses by cattle naturally sensitized to
M. avium
. Experimental infection with
M. bovis
,
M. avium
, or
M. avium
subsp.
paratuberculosis
induced significant (
P
< 0.05) IFN-γ and nitric oxide production to WCS and PPD antigens, regardless of the mycobacterial species used for the preparation of the antigen. Responses to homologous crude antigens generally exceeded responses to heterologous antigens. Nitric oxide and IFN-γ responses to rESAT-6:CFP-10 by blood leukocytes from
M. bovis
-infected calves exceeded (
P
< 0.05) the corresponding responses of noninfected,
M. avium
-infected, and
M. avium
subsp.
paratuberculosis
-infected calves. Despite the reported potential for secretion of immunogenic ESAT-6 and CFP-10 proteins by
M. avium
and
M. avium
subsp.
paratuberculosis
, it appears that use of the rESAT-6:CFP-10 fusion protein will be useful for the detection of tuberculous cattle in herds with pre-existing sensitization to
M. avium
and/or
M. avium
subsp.
paratuberculosis
.
Bacterial Clearance and Cytokine Profiles in a Murine Model of Postsurgical Nosocomial Pneumonia
Authors: Manderscheid Patricia A. , Bodkin Ryan P. , Davidson Bruce A. , Jensen Erik , Russo Thomas A. , Knight Paul R. 
Published: July 2004

ABSTRACTThe development of a nosocomial pneumonia is facilitated by alterations in host innate pulmonary antibacterial defenses following surgical trauma, which can result in decreased pulmonary bacterial clearance and increased morbidity and mortality. In a murine model of postoperative nosocomial infection, surgical stress (laparotomy) decreasedEscherichia coliclearance from the lungs of animals that underwent surgery. Consistent with previous studies, (i) pulmonary levels of tumor necrosis factor alpha at 6 h and of interleukin-1β (IL-1β), IL-6, and gamma interferon (IFN-γ) at 24 h post-bacterial infection (PBI) were decreased in animals that underwent laparotomy 24 h prior toE. coliinfection (LAP/E. coli) compared to animals that receivedE. colionly; (ii) KC and macrophage inhibitory protein 2 were elevated at 6 h PBI in LAP/E. colianimals compared toE. coli-only animals; however, at 24 h PBI, levels were higher in theE. coli-only group; (iii) at 24 h PBI, monocyte chemoattractant protein 1 was lower in the LAP/E. coligroup compared to theE. coli-only group; (iv) IL-10 levels were unaffected at all time points evaluated; and (v) the total number of neutrophils present in the lungs of LAP/E. colianimals at 6 h PBI was decreased in comparison to that inE. coli-only animals, resulting in decreased bacterial clearance and increased mortality in LAP/E. colianimals by 24 h PBI. Similar changes in cytokine profiles, pulmonary bacterial clearance, and mortality were consistent with reported findings in patients following surgical trauma. This model, therefore, provides a clinically relevant system in which the molecular and cellular mechanisms that lead to the development of nosocomial pneumonia can be further explored.
Normal Values for CD4 and CD8 Lymphocyte Subsets in Healthy Chinese Adults from Shanghai
Authors: Jiang Weiming , Kang Laiyi , Lu Hong-Zhou , Pan Xiaozhang , Lin Qingneng , Pan Qichao , Xue Yile , Weng Xinhua , Tang Yi-Wei 
Published: July 2004

ABSTRACTThe aim of this study was to establish reference ranges for lymphocyte subsets in Chinese adults. Venous blood specimens were obtained from 614 healthy, human immunodeficiency virus (HIV)-seronegative adults in Shanghai. Flow cytometry was used to determine percentages and absolute numbers of CD4 and CD8 T lymphocytes. Mean values for CD4 and CD8 lymphocytes were 727 and 540 cells/μl, respectively, yielding a CD4/CD8 ratio of 1.49. While CD8 lymphocyte values varied with age and gender, no significant differences in CD4 lymphocyte values were observed. Shanghai adults had approximately 100 fewer CD4 lymphocytes/μl on average than Caucasians, suggesting that lower CD4 lymphocyte cutoffs for classifying and monitoring HIV infection may be needed in China.
Reference System for Characterization of
Bordetella pertussis
Pulsed-Field Gel Electrophoresis Profiles
Authors: Advani Abdolreza , Donnelly Declan , Hallander Hans 
Published: July 2004

ABSTRACT

Pulsed-field gel electrophoresis (PFGE) has been used as an epidemiological tool for surveillance studies of
Bordetella pertussis
since the early 1990s. To date there is no standardized procedure for comparison of results, and therefore it has been difficult to directly compare PFGE results between laboratories. We propose a profile-based reference system for PFGE characterization of
B. pertussis
strain variation and to establish traceability of
B. pertussis
PFGE results. We initially suggest 35 Swedish reference strains as reference material for PFGE traceability. This reference material is deposited at the Culture Collection of the University of Gothenburg, Gothenburg, Sweden. Altogether, 1,810 Swedish clinical isolates from between 1970 and 2003 were studied, together with the Swedish Pw vaccine strain, six reference strains, and two U.S. isolates. Our system provides evidence that profiles obtained by using only one enzyme, i.e., XbaI, give enough data to analyze the epidemiological relationship between them. Characterization with one enzyme is far less labor intensive, yielding results in half the time than when a two-enzyme procedure is used. Also, we can see that there is a correlation between PFGE profile and pertactin type. One common PFGE profile, BpSR11 (
n
= 455), showed 100%
prn2
and 100% Fim3 when analyzed for pertactin type and serotype. On the other hand, strains with the same profile may express various serotypes when isolated over longer periods of time. Subculturing of the same isolate eight times or lyophilization caused no change in PFGE profile.
Molecular Typing of
Neisseria gonorrhoeae
Isolates by Pyrosequencing of Highly Polymorphic Segments of the
porB
Gene
Authors: Unemo Magnus , Olcén Per , Jonasson Jon , Fredlund Hans 
Published: July 2004

ABSTRACT

For prevention and control of gonorrhea, an objective, highly discriminating, and reproducible molecular epidemiological characterization of
Neisseria gonorrhoeae
is essential. In the present study, in pursuance of providing such qualities, pyrosequencing technology, a fast real-time DNA sequence analysis, was applied to six short, highly polymorphic
porB
gene segments, with subsequent genetic variant (genovar) determination of the bacterial isolates. The sequencing templates were obtained by real-time PCR amplification, which also included fluorescence melting curve analysis of the entire
porB
gene in order to determine the genogroup (
porB1a
or
porB1b
allele) prior to pyrosequencing analysis. The PSQ 96 MA system used allowed rapid (in approximately 1.5 h) determination of 96 sequences of 20 to 65 correct nucleotides each. The results were reproducible and mostly in concordance with the results of conventional Sanger dideoxy sequencing, with the exception of shorter read lengths and some uncertainty in determining the correct number of identical nucleotides in homopolymeric segments. The number of sequence variants identified in each of the six highly polymorphic segments of the
porB1a
and
porB1b
alleles (encoding surface-exposed amino acid loops of the mature PorB protein) ranged from 5 to 11 and from 8 to 39, respectively. Among
porB1a
isolates (
n
= 22) and
porB1b
isolates (
n
= 65), 22 and 64 unique genovars, respectively, were identified. All isolates were typeable. The present results provide evidence of a high discriminatory ability, practically the same as that for sequencing of the entire
porB
gene. In conclusion, the fast and high-throughput pyrosequencing technology can be used for molecular epidemiological characterization of
N. gonorrhoeae
.
Molecular Epidemiology of Tuberculosis among Immigrants in Hamburg, Germany
Authors: Diel Roland , Rüsch-Gerdes Sabine , Niemann Stefan 
Published: July 2004

ABSTRACT

To study the characteristics of tuberculosis (TB) in foreign-born individuals living in Hamburg, Germany, and to discover in what way foreign-born individuals contribute to the incidence of TB in Hamburg, an in-depth epidemiological study was performed by a combination of classical and molecular methods. In total, 796 patients with TB were analyzed between 1997 and 2002 (88.7% of all patients with culture-positive TB in the study period). Of this total, 334 were foreign-born patients from 43 different countries. Of these, only 31 cases were identified as a consequence of the screening of 12,176 asylum seekers at entry. Of the foreign-born patients, 41.9% had been living in Germany for more than 5 years. On the basis of the IS
6110
typing results for isolates from all patients, 246 patients (31%) were classified into 68 clusters, with each cluster containing from 2 to 38 patients. Among foreign-born individuals, 86 (26%) were represented in 40 clusters. In multivariate analyses, a previous history as a TB contact had the highest predictive risk for clustering among foreign-born patients, followed by drug addiction, alcohol dependence, being an asylum seeker, and unemployment. Epidemiological links verifying recent transmission could be confirmed for 39 of the 86 foreign-born members (45.3%) who formed a cluster, comprising 16 source patients and 23 directly infected patients. Of 2,227 previously known contacts of foreign-born patients subjected to traditional contact investigation, 14 foreign-born individuals (0.6%) subsequently contracted culture-confirmed TB, but only 9 transmissions could be confirmed by IS
6110
typing (39.1% of the 23 confirmed fresh infections retrospectively confirmed by IS
6110
typing). In conclusion, only a minority of TB cases among foreign-born individuals are detected by screening of asylum seekers or conventional contact tracing. Recent transmission does not play an important role in TB among immigrants in Hamburg.
Performance of Commercially Available Enzyme Immunoassays for Detection of Antibodies against Herpes Simplex Virus Type 2 in African Populations
Authors: Van Dyck Eddy , Buvé Anne , Weiss Helen A. , Glynn Judith R. , Brown David W. G. , De Deken Bénédicte , Parry John , Hayes Richard J. 
Published: July 2004

ABSTRACTData are accumulating on the performance of enzyme immunoassays (EIAs) for the detection of herpes simplex virus type 2 (HSV-2) infection in North America and Europe, but little is known about their performance in other populations. Nine test kits were evaluated with 330 serum samples from sub-Saharan Africa. The tests were first compared to the monoclonal antibody (MAb) EIA (Central Public Health Laboratory, London, United Kingdom). Samples that gave discordant results in the MAb EIA and in the three tests that performed best compared to the MAb EIA were tested by Western blotting (University of Washington, Seattle). A random sample of concordant samples was also tested, and the sensitivities and specificities of the different tests were calculated, taking into account this sampling strategy. The sensitivities of the tests ranged from 86 to 100%; the specificities ranged from 47 to 99%. The tests that performed best were the Gull Premier EIA (sensitivity, 86.3%; specificity, 97.6%) and the Kalon Biological (sensitivity, 92.3%; specificity, 97.7%) and Biokit (sensitivity, 86.7%; specificity, 92.6%) tests. It cannot be assumed that enzyme immunoassays for the detection of HSV-2 infection that perform well in industrialized countries will perform equally well in other populations.
Use of Robotized DNA Isolation and Real-Time PCR To Quantify and Identify Close Correlation between Levels of
Neisseria meningitidis
DNA and Lipopolysaccharides in Plasma and Cerebrospinal Fluid from Patients with Systemic Meningococcal Disease
Authors: Øvstebø Reidun , Brandtzaeg Petter , Brusletto Berit , Haug Kari Bente Foss , Lande Knut , Høiby Ernst Arne , Kierulf Peter 
Published: July 2004

ABSTRACT

The present study, using robotized DNA isolation and quantitative PCR based on the
Neisseria meningitidis
-specific capsular transport A gene, demonstrates the ease, rapidity, specificity, and sensitivity of quantifying neisserial DNA in plasma (
n
= 65) and cerebrospinal fluid (CSF) (
n
= 12) from patients with systemic meningococcal disease. We found a close correlation between the levels of neisserial DNA and lipopolysaccharides in plasma (
r
= 0.905) and in CSF (
r
= 0.964). The median concentration of neisserial DNA in plasma in 23 patients with persistent shock was 2 × 10
7
copies/ml, versus <10
3
copies/ml in 42 nonshock patients. Furthermore, quantitative PCR made possible estimates of the total number of meningococci in plasma, as opposed to conventional blood cultures, suggesting about 1,000 dead meningococci for every viable bacterium. Finally, with logistic regression analyses, neisserial DNA may predict a patient's disease severity and outcome at hospital admission. The number of meningococci in plasma and CSF appears to be the main determinant of the lipopolysaccharide levels, clinical presentation, and outcome.
Assessment of Partial Sequencing of the 65-Kilodalton Heat Shock Protein Gene (
hsp65
) for Routine Identification of
Mycobacterium
Species Isolated from Clinical Sources
Authors: McNabb Alan , Eisler Diane , Adie Kathy , Amos Marie , Rodrigues Mabel , Stephens Gwen , Black William A. , Isaac-Renton Judith 
Published: July 2004

ABSTRACT

We assessed the ability of an in-house database, consisting of 111
hsp65
sequences from putative and valid
Mycobacterium
species or described groups, to identify 689 mycobacterial clinical isolates from 35 species or groups. A preliminary assessment indicated that
hsp65
sequencing confirmed the identification of 79.4% of the isolates from the 32 species examined, including all
Mycobacterium tuberculosis
complex isolates, all isolates from 13 other species, and 95.6% of all
M. avium
-
M. intracellulare
complex isolates. Identification discrepancies were most frequently encountered with isolates submitted as
M. chelonae
,
M. fortuitum
,
M. gordonae
,
M. scrofulaceum
, and
M. terrae
. Reexamination of isolates with discrepant identifications confirmed that
hsp65
identifications were correct in a further 40 isolates. This brought the overall agreement between
hsp65
sequencing and the other identification methods to 85.2%. The remaining 102 isolates had sequence matches below our acceptance criterion, had nondifferential sequence matches between two or more species, were identified by 16S rRNA sequencing as a putative taxonomic group not contained in our database, or were identified by
hsp65
and 16S rRNA gene sequencing as a species not in our biochemical test database or had conflicting identifications. Therefore, to incorporate the unconfirmed isolates it was necessary to create 29 additional entries in our
hsp65
identification database: 18 associated with valid species, 7 indicating unique sequences not associated with valid or putative species or groups, and 4 associated with unique, but currently described taxonomic groups. Confidence in the
hsp65
sequence identification of a clinical isolate is best when sequence matches of 100% occur, but our data indicate that correct identifications can be confidently made when unambiguous matches exceeding 97% occur, but are dependent on the completeness of the database. Our study indicates that for
hsp65
sequencing to be an effective means for identifying mycobacteria a comprehensive database must be constructed.
hsp65
sequencing has the advantage of being more rapid and less expensive than biochemical test panels, uses a single set of reagents to identify both rapid- and slow-growing mycobacteria, and can provide a more definitive identification.
Molecular Analysis of the Microflora Associated with Dental Caries
Authors: Munson M. A. , Banerjee A. , Watson T. F. , Wade W. G. 
Published: July 2004

ABSTRACT

Molecular techniques have revealed many novel, presumed unculturable, taxa in oral infections. The aim of this study was to characterize the bacterial community of the middle and advancing front of carious dental lesions by cultural and molecular analyses. Samples were collected with a hand excavator from five teeth with carious lesions involving dentine. Samples were cultured on blood agar and Rogosa agar incubated in air plus 5% CO
2
and on fastidious anaerobe agar anaerobically. DNA was also extracted directly from the samples and 16S rRNA genes were amplified by PCR with universal primers. PCR products were singularized by cloning, and the cloned inserts and cultured isolates were identified by 16S rRNA gene sequence analysis. We identified 95 taxa among the 496 isolates and 1,577 clones sequenced; 44 taxa were detected by the molecular method alone; 31 taxa were previously undescribed. Only three taxa,
Streptococcus mutans
,
Rothia dentocariosa
, and an unnamed
Propionibacterium
sp., were found in all five samples. The predominant taxa by anaerobic cultivation were the novel
Propionibacterium
sp. (18%),
Olsenella profusa
(14%), and
Lactobacillus rhamnosus
(8%). The predominant taxa in the molecular analysis were
Streptococcus mutans
(16%),
Lactobacillus gasseri
/
johnsonii
(13%), and
Lactobacillus rhamnosus
(8%). There was no significant difference between the compositions of the microflora in the middle and advancing front samples (
P
< 0.05, Wilcoxon matched pairs, signed ranks test). In conclusion, combined cultural and molecular analyses have shown that a diverse bacterial community is found in dentinal caries and that numerous novel taxa are present.
Improved Sensitivity of Nucleic Acid Amplification for Rapid Diagnosis of Tuberculous Meningitis
Authors: Johansen Isik Somuncu , Lundgren Bettina , Tabak Fehmi , Petrini Björn , Hosoglu Salih , Saltoglu Nese , Thomsen Vibeke Østergaard 
Published: July 2004

ABSTRACT

Early diagnosis of tuberculous meningitis (TBM) is essential for a positive outcome; but present microbiological diagnostic techniques are insensitive, slow, or laborious. We evaluated the standard BDProbeTec ET strand displacement amplification method (the standard ProbeTec method) for the detection of
Mycobacterium tuberculosis
complex organisms in parallel with the ProbeTec method with a modified pretreatment procedure with 101 prospectively collected cerebrospinal fluid specimens from 94 patients with suspected TBM. By the modified method, the sample-washing step was omitted. A definitive diagnosis was attained by culture. Thirteen specimens from 12 patients were culture positive for
M. tuberculosis
complex organisms; three specimens (23%) were microscopy positive for acid-fast bacilli. Among the culture-positive specimens, the standard ProbeTec method was positive for 8 (61.5%) and the modified assay was positive for 10 (76.9%). The overall specificity by both procedures was 98.8% compared to the results of culture. After discrepancy analysis, conducted by reviewing the patients' previous laboratory data, the specificity increased to 100%. If the cutoff value for respiratory specimens was adjusted from the recommended value of 3,400 to 1,000, the sensitivity of the modified procedure increased to 84.7%, with unchanged specificity. Results were obtained in 3 to 4 h. The new pretreatment procedure with the ProbeTec assay described here provides a rapid, simple, and sensitive tool for the diagnosis of TBM.
Widespread Dissemination in The Netherlands of the Epidemic Berlin Methicillin-Resistant
Staphylococcus aureus
Clone with Low-Level Resistance to Oxacillin
Authors: Wannet W. J. B. , Spalburg E. , Heck M. E. O. C. , Pluister G. N. , Willems R. J. L. , de Neeling A. J. 
Published: July 2004

ABSTRACT

Methicillin-resistant
Staphylococcus aureus
(MRSA) is an important human pathogen and represents a growing public health burden due to the emergence and spread of epidemic strains, particularly within the hospital environment. An epidemic MRSA clone, with characteristic low-level resistance to oxacillin, emerged in the year 2000 and became endemic in the Netherlands. Multilocus sequence typing characterized the strain as sequence type 45, which was previously designated the Berlin epidemic MRSA clone. In 2 years, this strain has become the predominant MRSA clone in the Netherlands.
Comparison of the APTIMA CT and GC Assays with the APTIMA Combo 2 Assay, the Abbott LCx Assay, and Direct Fluorescent-Antibody and Culture Assays for Detection of
Chlamydia trachomatis
and
Neisseria gonorrhoeae
Authors: Boyadzhyan B. , Yashina T. , Yatabe J. H. , Patnaik M. , Hill C. S. 
Published: July 2004

ABSTRACT

The Gen-Probe APTIMA Combo 2 (AC2) is a Food and Drug Administration-cleared nucleic acid amplification test (NAAT) for the detection of
Chlamydia trachomatis
and
Neisseria gonorrhoeae
from urine and urogenital swab specimens. The Centers for Disease Control and Prevention have recommended confirmation of positive NAAT results in low-prevalence populations. APTIMA CT (ACT) and APTIMA GC (AGC) are two discrete NAATs for
C. trachomatis
and
N. gonorrhoeae
detection that are suitable for confirming AC2-positive results because they target different nucleic acid sequences. Our objective was to determine if ACT and AGC could be used as confirmatory tests for AC2 and to correlate the APTIMA assays with culture, direct fluorescent-antibody (DFA), and LCx CT and GC assays. Urine and swab specimens (1,304) were initially tested with either culture, DFA, or LCx, followed by AC2. A subset (675) was then tested with ACT and AGC. There was absolute concordance between ACT-AGC and AC2. LCx did not detect 1 of 14 AC2-ACT- and 1 of 6 AC2-AGC-positive urine samples, and it yielded one
C. trachomatis
- and one
N. gonorrhoeae
-positive swab result that were not detected by AC2 and ACT-AGC. Culture failed to detect 5 of 20 AC2-ACT and 3 of 4 AC2-AGC positives, and DFA missed 4 of 4 AC2-ACT positives. Thus, ACT and AGC relative sensitivity compared to that of AC2 was 100%. All APTIMA assays detected more confirmed positive results than culture, DFA, and LCx. The performance of APTIMA assays was not altered by the use of various swab types and by long-term storage of specimens. All APTIMA assays are highly sensitive and rapid. ACT and AGC can be recommended for confirmation of positive results from other NAATs, such as AC2 and LCx.