bluuebunny/crossref_metadata_2025 huggingface dataset:
Meanings of a Meltdown, After the Facts
Authors: Rochlin Gene I.
Published: July 2004

Three Mile Island
A Nuclear Crisis in Historical Perspective. by J. Samuel Walker. University of California Press, Berkeley, 2004. 315 pp. $24.94, £16.95. ISBN 0-520-23940-7.

The author describes the 1979 accident at Three Mile Island, places it in the context of the debates over nuclear power at that time, and then explores the social, technical, and political issues it raised.
Watching the Growth of Bulk Grains During Recrystallization of Deformed Metals
Authors: Schmidt S. , Nielsen S. F. , Gundlach C. , Margulies L. , Huang X. , Jensen D. Juul
Published: July 2004

We observed the in situ growth of a grain during recrystallization in the bulk of a deformed sample. We used the three-dimensional x-ray diffraction microscope located at the European Synchrotron Radiation Facility in Grenoble, France. The results showed a very heterogeneous growth pattern, contradicting the classical assumption of smooth and spherical growth of new grains during recrystallization. This type of in situ bulk measurement opens up the possibility of obtaining experimental data on scientific topics that before could only be analyzed theoretically on the basis of the statistical characterization of microstructures. For recrystallization, the in situ method includes direct measurements of nucleation and boundary migration through a deformed matrix.
Immunotherapy: Bewitched, Bothered, and Bewildered No More
Authors: Steinman Ralph M. , Mellman Ira
Published: July 2004

The field of immunotherapy holds clear promise not only for the development of new approaches to cancer and other diseases, but also for providing fundamental insight into the human immune response. In order for this promise to be realized, however, the scientific community must overcome an array of challenges. These challenges reflect not only the difficulties inherent in conducting investigations in human patients, but also difficulties created by the culture and practice of our own institutions, reward structure, and funding mechanisms. We suggest steps to be taken to reinvigorate basic research in human subjects as part of the mainstream of science.
Longitudinal Profile of Immunoglobulin G (IgG), IgM, and IgA Antibodies against the Severe Acute Respiratory Syndrome (SARS) Coronavirus Nucleocapsid Protein in Patients with Pneumonia Due to the SARS Coronavirus
Authors: Woo Patrick C. Y. , Lau Susanna K. P. , Wong Beatrice H. L. , Chan Kwok-hung , Chu Chung-ming , Tsoi Hoi-wah , Huang Yi , Peiris J. S. Malik , Yuen Kwok-yung
Published: July 2004

By using a recombinant severe acute respiratory syndrome coronavirus (SARS-CoV) nucleocapsid protein-based enzyme-linked immunosorbent assay (ELISA) and serum specimens serially collected (from day 0 to day 240 after symptom onset) from patients with pneumonia due to SARS-CoV, we analyzed the longitudinal profiles of immunoglobulin G (IgG), IgM, and IgA antibodies against the SARS-CoV nucleocapsid protein in patients with pneumonia due to SARS-CoV. For IgG, the median optical density at 450 nm (OD
450
) turned positive at day 17 and a biphasic response was observed. At day 240, all patients were still positive for anti-nucleocapsid protein IgG antibody. For IgM, the median OD
450
turned positive at day 20.5, peaked at about day 80, and fell to below the baseline level at about day 180. At day 240, 36% of the patients were still positive for anti-nucleocapsid protein IgM antibody. For IgA, the median OD
450
turned positive at day 17, peaked at about day 50, and fell to below the baseline level at about day 180. At day 240, 36% of the patients were still positive for anti-nucleocapsid protein IgA antibody. The time of seroconversion detected by the recombinant SARS-CoV nucleocapsid protein-based ELISA and that detected by indirect immunofluorescence assay were similar. The median times of seroconversion for IgG, IgM, and IgA detected by the indirect immunofluorescence assay were 17 days (17 days by ELISA), 16.5 days (20.5 days by ELISA), and 17.5 days (17 days by ELISA), respectively, after disease onset. One, four, and one of the six patients who died did not produce any IgG, IgM, and IgA antibodies against the nucleocapsid protein of SARS-CoV, respectively, although these antibodies were detected in all six patients by the indirect immunofluorescence assay. Further studies should be performed to see whether SARS-CoV nucleocapsid protein antibody positivity has any prognostic significance.
Reliability of Immunoglobulin G Antitoxoplasma Avidity Test and Effects of Treatment on Avidity Indexes of Infants and Pregnant Women
Authors: Flori Pierre , Tardy Laetitia , Patural Hugues , Bellete Bahrie , Varlet Marie-Noëlle , Hafid Jamal , Raberin Hélène , Sung Roger Tran Manh
Published: July 2004

The immunoglobulin G antitoxoplasma avidity test (Vidas; BioMérieux) is an immunoenzymatic test useful for excluding acute infection after the onset of pregnancy. The avidity index (AI) is the ratio of the signal in a test sample washed with urea, which disrupts low-avidity complexes, to that washed without urea. An AI of >0.3 is taken to mean that infection had occurred more than 4 months ago. The increase of the AI with time and the influence of the different treatments given to pregnant women and their newborns were evaluated. A total of 59 pregnant women (271 sera) and their 60 neonates (199 sera) were tested from 1998 to 2002. There were five groups of women based on the type and duration of treatment given. Thirteen pregnant women (group 1) did not receive any treatment, 15 (group 2), 11 (group 3), and 17 (group 4) women received treatment with spiramycin (9 MIU/day) for 0.5 to 2, 2.5 to 5, and 5.5 to 8 months, respectively, and the last 3 women (group 5) received tritherapy (pyrimethamine-sulfonamide and spiramycin alternatively) for 1.5 to 2.5 months. All of the maternal sera collected in the first 6 months had an AI of <0.30, with a mean of 0.07 (range, 0.01 to 0.21). The increase was slow (0.02/month), and there was no significant difference when comparisons were made between the treatment groups. Neonates with proven maternofetal transmission had an increasing AI, unlike those without transmission. However, long-term therapy with pyrimethamine-sulfonamide, as opposed to treatment with spiramycin alone, was found to slow down the progression of the AI. An AI of >0.2 is sufficient to exclude acute infection in pregnant women. In neonates, it is not of major use to diagnose congenital infection; however, it could be a good indicator of compliance and efficacy of treatment of infected infants.
Delineation of the Role of Platelet-Activating Factor in the Immunoglobulin G2 Antibody Response
Authors: Al-Darmaki Salma , Knightshead Kandi , Ishihara Yuichi , Best Al , Schenkein Harvey A. , Tew John G. , Barbour Suzanne E.
Published: July 2004

Localized aggressive periodontitis (LAgP) is a chronic inflammatory disease characterized by severe destruction of periodontal tissues surrounding the first molars and incisors. LAgP subjects produce large amounts of immunoglobulin G2 (IgG2) antibody against oral pathogens, and this response is inversely correlated with the severity of disease. We previously demonstrated that platelet-activating factor (PAF) is required for optimal IgG2 responses. The present investigation was designed to determine the mechanism of IgG2 induction by PAF. Exogenous PAF acetylhydrolase suppressed approximately 80% of pokeweed mitogen-stimulated IgG2 production, confirming that PAF is essential for optimal responses. PAF-activated leukocytes produced gamma interferon (IFN-γ), a Th1 cytokine that has been associated with IgG2 responses in previous studies. The monocyte-derived cytokines interleukin-12 (IL-12) and IL-18 are upstream of IFN-γ production, and IgG2 production was suppressed by neutralizing antibodies against these proteins. In addition, PAF induced monocyte-derived dendritic cells (DC) but not macrophages (MΦ) to secrete IL-12 and IL-18. This observation was interesting because monocyte differentiation in LAgP subjects is skewed to the DC phenotype. Although other investigators have implicated IFN-γ in IgG2 production, its precise role in this response is controversial. Our studies suggest that IFN-γ induces isotype switching to IgG2 but only in concert with the Th2 cytokine IL-4. Thus, it appears that the unique PAF metabolism of LAgP monocytes or DC promotes Th1 responses that are essential for optimal IgG2 antibody production. As IgG2 antibodies opsonize oral bacteria and promote their clearance and destruction, these alterations in PAF metabolism may be essential for limiting disease severity in LAgP patients.
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

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

The 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

The 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

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

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

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

Data 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

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

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

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

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

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

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.
Endemic, Epidemic Clone of
Salmonella enterica
Serovar Typhi Harboring a Single Multidrug-Resistant Plasmid in Vietnam between 1995 and 2002
Authors: Le Thi Anh Hong , Lejay-Collin Monique , Grimont Patrick A. D. , Hoang Thuy Long , Nguyen Thi Vinh , Grimont Francine , Scavizzi Maurice R.
Published: July 2004

Salmonella enterica
serovar Typhi strains resistant to ampicillin, chloramphenicol, tetracyclines, streptomycin, and cotrimoxazole, isolated from sporadic cases and minor outbreaks in Vietnam between 1995 and 2002, were typed and compared. Plasmid fingerprinting, Vi bacteriophage typing, XbaI pulsed-field gel electrophoresis, and PstI ribotyping showed that endemic, epidemic multidrug-resistant typhoid fever was due, for at least 74.1% of the isolates, to one or two clones of serovar Typhi harboring a single resistance plasmid. PstI ribotyping was used as a basic technique to ensure that a serovar Typhi expansion was clonal.
Quantitative Analysis of Diverse
Lactobacillus
Species Present in Advanced Dental Caries
Authors: Byun Roy , Nadkarni Mangala A. , Chhour Kim-Ly , Martin F. Elizabeth , Jacques Nicholas A. , Hunter Neil
Published: July 2004

Our previous analysis of 65 advanced dental caries lesions by traditional culture techniques indicated that lactobacilli were numerous in the advancing front of the progressive lesion. Production of organic acids by lactobacilli is considered to be important in causing decalcification of the dentinal matrix. The present study was undertaken to define more precisely the diversity of lactobacilli found in this environment and to quantify the major species and phylotypes relative to total load of lactobacilli by real-time PCR. Pooled DNA was amplified by PCR with
Lactobacillus
genus-specific primers for subsequent cloning, sequencing, and phylogenetic analysis. Based on 16S ribosomal DNA sequence comparisons, 18 different phylotypes of lactobacilli were detected, including strong representation of both novel and gastrointestinal phylotypes. Specific PCR primers were designed for nine prominent species, including
Lactobacillus gasseri
,
L. ultunensis
,
L. salivarius
,
L. rhamnosus
,
L. casei
,
L. crispatus
,
L. delbrueckii
,
L. fermentum
, and
L. gallinarum
. More than three different species were identified as being present in most of the dentine samples, confirming the widespread distribution and numerical importance of various
Lactobacillus
spp. in carious dentine. Quantification by real-time PCR revealed various proportions of the nine species colonizing carious dentine, with higher mean loads of
L. gasseri
and
L. ultunensis
than of the other prevalent species. The findings provide a basis for further characterization of the pathogenicity of
Lactobacillus
spp. in the context of extension of the carious lesion.
Molecular and Ultrastructural Characterization of Porcine Hippurate-Negative
Brachyspira pilosicoli
Authors: Fossi Marja , Pohjanvirta Tarja , Sukura Antti , Heinikainen Sirpa , Lindecrona Rikke , Pelkonen Sinikka
Published: July 2004

Brachyspira pilosicoli
, the causative agent of porcine intestinal spirochetosis, usually has hippurate-cleaving capacity. We have regularly isolated hippurate-negative
B. pilosicoli
from cases of porcine diarrhea. In this study, we show that these biochemically atypical
B. pilosicoli
isolates can be classified as
B. pilosicoli
. 16S ribosomal DNA was partially sequenced from eight hippurate-negative and two hippurate-positive
B. pilosicoli-
like isolates from seven herds. The differences in nucleotide sequence with
B. pilosicoli
P43/6/78 type strain were not associated with hippurate cleavage. In 877 bp, the hippurate-negative isolates had a similarity of 98.63 to 100% to the type strain, with the corresponding figures for the two hippurate-positive isolates being 98.86 and 100%. The nucleotide sequences of hippurate-positive isolates were identical to the respective sequences of hippurate-negative isolates from one herd. The DNA macrorestriction patterns of a total of 20 hippurate-negative and -positive
B. pilosicoli
isolates were diverse, and no clustering in conjunction with the hippurate reaction was found. In two herds, hippurate-positive and -negative
B. pilosicoli
isolates had a common macrorestriction pattern. The ultrastructure of hippurate-negative isolates was similar to the type strain. In conclusion,
B. pilosicoli
can be either hippurate positive or negative and, thus, the scheme for biochemical differentiation of porcine
Brachyspira
should be revised to include identification of hippurate-negative
B. pilosicoli
.
Use of Swabs without Transport Media for the Gen-Probe Group A Strep Direct Test
Authors: Bourbeau Paul P. , Heiter Barbara J.
Published: July 2004

For several years we used rayon or Dacron swabs with liquid transport media for collection and transport of throat swab specimens for testing with the Gen-Probe Group A Strep Direct Test (GASDT). A report of favorable results with a Dacron swab without any transport media for GASDT by another laboratory prompted us to compare detection of group A streptococci (GAS) with and without transport media (referred to as “wet” and “dry” swabs, respectively). Phase one of this study used swabs seeded with GAS. Initially, six recent clinical isolates of GAS were inoculated onto wet and dry swabs and stored at room temperature (RT). After 1, 2, and 3 days of storage, colony counting and GASDT were performed with the swabs. The results, expressed as the mean percentage of the results at zero time, were as follows: for GASDT with wet swabs at 1, 2, and 3 days, 62, 51, and 56%, respectively; for GASDT with dry swabs at 1, 2, and 3 days, 105, 80, and 85%, respectively; for colony counts with wet swabs at 1, 2, and 3 days, 52, 26, and 13%, respectively; for colony counts with dry swabs at 1, 2, and 3 days, 10, 0, and 0%, respectively. An additional six strains of GAS were tested in a similar manner, except that extracts of pharyngeal flora (PF) were added to the inocula. The results obtained with extracts of PF were comparable to those obtained with GAS alone. We also compared the performance of GASDT with wet and dry swabs stored at RT and 4°C. Ten strains of GAS were inoculated onto wet and dry swabs, and GASDT was performed each day for 9 days. The GASDT results for swabs on day 9, expressed as the mean percentage of the results obtained at zero time, were as follows: dry swab and 4°C, 59%; wet swab and 4°C, 31%; dry swab and RT, 33%; and wet swab and RT, 19%. In phase two of this study we conducted a clinical evaluation to determine whether the differences observed with seeded specimens would also be evident with patient specimens. We used a single dry Dacron swab paired with a single rayon Bacti-Swab with liquid Stuart transport medium for the clinical evaluation. Specimens were collected from 1,005 outpatients, plated onto a Strep Selective Agar plate, and then tested within 30 min by GASDT. If culture of GAS from the same swab is used to define a true-positive test result, the sensitivities and specificities were as follows: GASDT with wet swabs, 86.2 and 98.5%, respectively; GASDT with dry swabs, 90.7 and 98.1%, respectively. However, the use of culture as the “gold standard” may understate the actual performance characteristics of GASDT, particularly for the dry swabs. In conclusion, for GASDT the use of swabs without transport media may be preferable to the use of swabs with transport media.
Amplicon Sequencing and Improved Detection of Human Rhinovirus in Respiratory Samples
Authors: Deffernez Christelle , Wunderli Werner , Thomas Yves , Yerly Sabine , Perrin Luc , Kaiser Laurent
Published: July 2004

Improved knowledge of the genotypic characteristics of human rhinovirus (HRV) is required, as are nucleic detection assays with the capacity to overcome both the similarities between members of the family
Picornaviridae
and the wide diversity of different HRV serotypes. The goal of the present study was to investigate the variability and the genotypic diversity of clinical strains circulating in the community. Since most reverse transcription (RT)-PCR assays available cannot differentiate HRV from other members of the family
Picornaviridae
, we also validated an assay specific for HRV detection. The 5′ noncoding regions of 87 different HRV serotypes and clinical isolates were sequenced. On the basis of sequence analysis and phylogenetic determination, we first confirmed that all clinical isolates were HRV. We then validated a real-time RT-PCR assay that was able not only to detect all HRV serotypes and all clinical isolates tested but also to accurately discriminate between rhinovirus and other viruses from the family
Picornaviridae
. This assay was negative with isolates of coxsackievirus (types A and B), echovirus, enterovirus, parechovirus, and poliovirus, as well as nonpicornaviruses. Among a series of bronchoalveolar lavage specimens, 4% (7 of 161) were positive by culture, whereas 13% (21 of 161) were positive by RT-PCR. In the present study we showed that to specifically identify HRV in clinical specimens, diagnostic assays need to overcome both the diversities and the similarities of picornaviruses. By sequencing the 5′ noncoding regions of rhinoviruses recovered from clinical specimens, we designed probes that could specifically detect rhinovirus.
Molecular Genotyping of a Large, Multicentric Collection of Tubercle Bacilli Indicates Geographical Partitioning of Strain Variation and Has Implications for Global Epidemiology of
Mycobacterium tuberculosis
Authors: Ahmed Niyaz , Alam Mahfooz , Rao K. Rajender , Kauser Farhana , Kumar N. Ashok , Qazi Nazia N. , Sangal Vartul , Sharma V. D. , Das Ram , Katoch V. M. , Murthy K. J. R. , Suneetha Sujai , Sharma S. K. , Sechi Leonardo A. , Gilman Robert H. , Hasnain Seyed E.
Published: July 2004

Tuberculosis continues to be a major killer disease, despite an all-out effort launched against it in the postgenomic era. We describe here the population structure of
Mycobacterium tuberculosis
strains, as revealed by a chromosome-wide scan of fluorescent amplified fragment length polymorphisms (FAFLPs), for more than 1,100 independent isolates from 11 different countries. The bacterial strains were genotyped based on a total of 136
±
1 different FAFLP markers at the genome sequence interface, with details on IS
6110
profiles, drug resistance status, clinicopathological observations, and host status integrated into the analysis process. The strains were found to cluster with possible geographic affinities, including the parameters of host species type, IS
6110
profile, and drug susceptibility status. Of the five most commonly amplified fragment sets (or amplitypes), type A predominated in strains of mixed origin, deposited in The Netherlands; type B was exclusively observed for Indian isolates; type C was found mainly in strains from Peru and Australia; and types D and E predominated in European strains from France and Italy. The amplitypes were independent of certain large sequence polymorphisms representing two important deletions, TbD1 and Rd9. It appears that
M. tuberculosis
has a high genomic diversity with a possible geographic evolution. This may have occurred due to specific genomic deletions and synonymous substitutions selected rigorously against host defenses and environmental stresses on an evolutionary timescale. The genotypic data reported here are additionally significant for genotype-phenotype correlations and for determining whether pathogen diversity is a reflection f the host population diversity.
High Genetic Diversity Revealed by Variable-Number Tandem Repeat Genotyping and Analysis ofhsp65Gene Polymorphism in a Large Collection of “Mycobacterium canettii” Strains Indicates that theM. tuberculosisComplex Is a Recently Emerged Clone of “M. canettii”
Authors: Fabre Michel , Koeck Jean-Louis , Le Flèche Philippe , Simon Fabrice , Hervé Vincent , Vergnaud Gilles , Pourcel Christine
Published: July 2004

We have analyzed, using complementary molecular methods, the diversity of 43 strains of “Mycobacterium canettii” originating from the Republic of Djibouti, on the Horn of Africa, from 1998 to 2003. Genotyping by multiple-locus variable-number tandem repeat analysis shows that all the strains belong to a single but very distant group when compared to strains of theMycobacterium tuberculosiscomplex (MTBC). Thirty-one strains cluster into one large group with little variability and five strains form another group, whereas the other seven are more diverged. In total, 14 genotypes are observed. The DR locus analysis reveals additional variability, some strains being devoid of a direct repeat locus and others having unique spacers. Thehsp65gene polymorphism was investigated by restriction enzyme analysis and sequencing of PCR amplicons. Four new single nucleotide polymorphisms were discovered. One strain was characterized by three nucleotide changes in 441 bp, creating new restriction enzyme polymorphisms. As no sequence variability was found forhsp65in the whole MTBC, and as a single point mutation separatesM. tuberculosisfrom the closest “M. canettii” strains, this diversity within “M. canettii” subspecies strongly suggests that it is the most probable source species of the MTBC rather than just another branch of the MTBC.
Purification of
Enterocytozoon bieneusi
Spores from Stool Specimens by Gradient and Cell Sorting Techniques
Authors: Kucerova Zuzana , Moura Hercules , Leitch Gordon J. , Sriram Rama , Bern Caryn , Kawai Vivian , Vargas Daniel , Gilman Robert H. , Ticona Eduardo , Vivar Aldo , Visvesvara Govinda S.
Published: July 2004

A three-step method for the purification of
Enterocytozoon bieneusi
spores from stool specimens was developed. The primary process of purification of the spores from bacterial contaminants involved Percoll gradient centrifugation followed by additional separation using cesium chloride density gradient centrifugation. The cesium chloride-isolated spores were further purified using a flow cytometer with cell sorting capabilities. Sorting was performed without the use of antibodies, fluorochromes, or dyes, leaving the sorted spores in their native state, which appears to be less destructive for spores. When quantified by flow cytometry using tubes with known numbers of highly fluorescent polystyrene beads, the sorted material showed a slight decrease in light scatter characteristics compared with the slightly larger
Encephalitozoon
species spores. Although the overall recovery of the
E. bieneusi
spores was low, calcofluor and Gram chromotrope staining, indirect immunofluorescence assay, and transmission electron microscopy revealed that the sorted material was highly purified and contained large numbers of
E. bieneusi
spores and relatively few bacteria and other debris. The sorted material appeared to be sufficiently pure and could be used for in vitro culture and for the development of a variety of diagnostic reagents as well as in studying the genome of
E. bieneusi
and host-parasite interactions.