bluuebunny/crossref_metadata_2025_split huggingface dataset:
Diversity of Bacterial Populations on the Tongue Dorsa of Patients with Halitosis and Healthy Patients
Authors: Kazor C. E. , Mitchell P. M. , Lee A. M. , Stokes L. N. , Loesche W. J. , Dewhirst F. E. , Paster B. J. 
Published: February 2003

ABSTRACT

The primary purpose of the present study was to compare the microbial profiles of the tongue dorsa of healthy subjects and subjects with halitosis by using culture-independent molecular methods. Our overall goal was to determine the bacterial diversity on the surface of the tongue dorsum as part of our ongoing efforts to identify all cultivable and not-yet-cultivated species of the oral cavity. Tongue dorsum scrapings were analyzed from healthy subjects with no complaints of halitosis and subjects with halitosis, defined as an organoleptic score of 2 or more and volatile sulfur compound levels greater than 200 ppb. 16S rRNA genes from DNA isolated from tongue dorsum scrapings were amplified by PCR with universally conserved bacterial primers and cloned into
Escherichia coli
. Typically, 50 to 100 clones were analyzed from each subject. Fifty-one strains isolated from the tongue dorsa of healthy subjects were also analyzed. Partial sequences of approximately 500 bases of cloned inserts from the 16S rRNA genes of isolates were compared with sequences of known species or phylotypes to determine species identity or closest relatives. Nearly complete sequences of about 1,500 bases were obtained for potentially novel species or phylotypes. In an analysis of approximately 750 clones, 92 different bacterial species were identified. About half of the clones were identified as phylotypes, of which 29 were novel to the tongue microbiota. Fifty-one of the 92 species or phylotypes were detected in more than one subject. Those species most associated with healthy subjects were
Streptococcus salivarius
,
Rothia mucilaginosa
, and an uncharacterized species of
Eubacterium
(strain FTB41).
Streptococcus salivarius
was the predominant species in healthy subjects, as it represented 12 to 40% of the total clones analyzed from each healthy subject. Overall, the predominant microbiota on the tongue dorsa of healthy subjects was different from that on the tongue dorsa of subjects with halitosis. Those species most associated with halitosis were
Atopobium parvulum
, a phylotype (clone BS095) of
Dialister
,
Eubacterium sulci
, a phylotype (clone DR034) of the uncultivated phylum TM7,
Solobacterium moorei
, and a phylotype (clone BW009) of
Streptococcus
. On the basis of our ongoing efforts to obtain full 16S rRNA sequences for all cultivable and not-yet-cultivated species that colonize the oral cavity, there are now over 600 species.
Serotyping of
Listeria monocytogenes
by Enzyme-Linked Immunosorbent Assay and Identification of Mixed-Serotype Cultures by Colony Immunoblotting
Authors: Palumbo Jeffrey D. , Borucki Monica K. , Mandrell Robert E. , Gorski Lisa 
Published: February 2003

ABSTRACT

Routine analysis of
Listeria monocytogenes
by serotyping using traditional agglutination methods is limited in use because of the expense and limited availability of commercially prepared antisera and intra- and interlaboratory discrepancies arising from differences in antiserum preparation and visual determination of agglutination. We have adapted a commercially available set of
L. monocytogenes
antisera to an enzyme-linked immunosorbent assay (ELISA) format for high-throughput, low-cost serotype determination. Rather than subjective visualization of agglutination, positive antigen and antiserum reactions were scored by a quantitative, colorimetric reaction. ELISA serotyping of 89 of 101
L. monocytogenes
isolates agreed with slide agglutination serotyping data, and 100 previously uncharacterized isolates were serotyped unambiguously by the ELISA method. In addition, mixed-serotype cultures of
L. monocytogenes
were identified by a colony immunoblot procedure, in which serogroup 1/2 and serogroup 4 colonies were discriminated by differential staining.
Quantitative Assay of Hepatitis C Virus RNA Using an Automated Extraction System for Specific Capture with Probes and Paramagnetic Particle Separation
Authors: Miyachi Hayato , Masukawa Atsuko , Asai Satomi , Miura Toshiaki , Tamatsukuri Shigeru , Hirose Toru , Ando Yasuhiko 
Published: February 2003

ABSTRACTA commercially available automated specimen preparation instrument for specific probe capture and paramagnetic separation has been developed (AmpliCap/GT-12; Roche Molecular Systems). We evaluated assay performance of the AmpliCap/GT-12 in the quantitative assay for hepatitis C virus (HCV) RNA with the AMPLICOR HCV MONITOR Test (version 2.0). Assay linearity using serial dilutions from a serum panel was observed in the range of 500 to 850,000 IU/ml, with a slightly compromised slope in the higher viral titers. The overall within-run and between-run reproducibility of the entire detection process for 3 and 5 log10(IU/ml) of HCV RNA in samples had a standard deviation of <0.2, which was comparable to a manual method based on organic extraction and isopropanol precipitation (Roche Molecular Systems). Comparison of the test results with those obtained by the manual method showed a good correlation (R2= 0.972, n= 86). Using heparin (3, 6.5, and 13 U/ml), dextran sulfate (0.1, 1, and 5 mM), hemoglobin (1.13, 2.25, and 4.5 g/liter), conjugated or unconjugated bilirubin (7.5, 15, and 30 mg/dl), and ATP (1.25, 2.5, and 5.0 mM) as known inhibitors, inhibition was only detected at a dextran sulfate concentration of 1 mM with the manual method but not with the AmpliCap/GT-12 extraction. In summary, the AmpliCap/GT-12 system was shown to permit a stable extraction process and accurate results for the quantitative assay of HCV RNA, successfully eliminating the inhibitory effect of dextran sulfate. This automated extraction system provides reliable and reproducible test results and saves labor; thus, it is suitable for routine diagnostic PCR.
Diagnosing Herpesvirus Infections by Real-Time Amplification and Rapid Culture
Authors: van Doornum G. J. J. , Guldemeester J. , Osterhaus A. D. M. E. , Niesters H. G. M. 
Published: February 2003

ABSTRACT
Procedures using real-time technique were developed to demonstrate the presence of herpes simplex virus type 1 (HSV-1) and HSV-2, varicella zoster virus (VZV), and cytomegalovirus (CMV) in miscellaneous clinical specimens. The assays were compared to rapid culture using centrifugation followed by detection with monoclonal antibodies. A total of 711 consecutive samples were collected from different patient groups. Throat swabs were obtained from transplant patients; dermal or oral specimens were collected from patients suspected for VZV or HSV infection. Genital specimens were taken from patients who attended the Clinic for Sexually Transmitted Diseases at the Dijkzigt Hospital Rotterdam presenting with symptoms of a primary genital ulcer. Nucleic acid extraction was carried out using a MagnaPure LC instrument. The amplification steps were performed on the ABI Prism 7700 sequence detection system. To monitor the process of extraction and amplification, a universal control consisting of seal herpesvirus type 1 (PhHV-1) was added to the clinical specimens. By culture 127 of 668 (19%) samples were positive for HSV-1, 72 of 668 (10.8%) specimens were positive for HSV-2, and 17 of 366 (4.6%) were positive for VZV. Using real-time amplification the numbers of positive specimens were 143 of 668 (21.4%), 97 of 668 (14.5%), and 27 of 366 (7.4%), respectively. Eighty-six specimens were tested for CMV, 12 (14.0%) were positive by culture, and 17 (19.8%) were positive by real-time PCR. The clinical data of the patients with discrepant results were reviewed thoroughly. In all cases the patients with only real-time PCR-positive results could be considered as truly infected. We concluded that the real-time amplification technique is suitable for the detection of human herpesvirus infection. It offers a semiquantitative and reliable assay with a quick result that is more sensitive than rapid culture, especially for the diagnosis of HSV-2 and VZV infections.
Clonal Diversity among Streptogramin A-Resistant
Staphylococcus aureus
Isolates Collected in French Hospitals
Authors: Haroche Julien , Morvan Anne , Davi Marilyne , Allignet Jeanine , Bimet François , El Solh Névine 
Published: February 2003

ABSTRACT

We analyzed 62 clinical isolates of streptogramin A-resistant (SGA
r
)
Staphylococcus aureus
collected between 1981 and 2001 in 14 hospitals located in seven French cities. These isolates, including five with decreased susceptibility to glycopeptides, were distributed into 45 antibiotypes and 38
Sma
I genotypes. Each of these genotypes included between 1 and 11 isolates, the
Sma
I patterns of which differed by no more than three bands. Although numerous clones were identified, we observed the spread of monoclonal isolates either within the same hospital or within hospitals in distinct cities and at large time intervals. Hybridization with probes directed against 10 SGA
r
genes (
vatA
,
vatB
,
vatC
,
vatD
,
vatE
,
vgaA
,
vgaB
,
vgaAv
,
vgbA
, and
vgbB
) revealed six patterns:
vgaAv
(21 isolates),
vatA
-
vgbA
(24 isolates),
vgaAv
-
vatB
-
vgaB
(14 isolates),
vgaAv
-
vatA
-
vgbA
(1 isolate),
vgaAv
-
vatA
-
vgbA
-
vatB
-
vgaB
(1 isolate), and
vgaA
(1 isolate). We detected at least one SGA
r
determinant in all of the tested isolates.
vgaAv
, which is part of the recently characterized transposon Tn
5406
, was found in 59.7% of the tested isolates. Of the 16 streptogramin B-susceptible isolates, 14 carried
vgaAv
alone and were susceptible to the mixtures of streptogramins, whereas the 2 isolates carrying
vgaAv
-
vatB
-
vgaB
were resistant to these mixtures.
vatA
-
vgbA
was found on plasmids of the same apparent size in 26 (42%) of the tested clinical isolates from 18 unrelated
Sma
I genotypes. The possible dissemination of some of the multiple clones characterized in the present study with an expected increased selective pressure of streptogramins following the recent licensing of Synercid (quinupristin-dalfopristin) must be carefully monitored.
Comparison of a New Quantitative
ompA
-Based Real-Time PCR TaqMan Assay for Detection of
Chlamydia pneumoniae
DNA in Respiratory Specimens with Four Conventional PCR Assays
Authors: Apfalter Petra , Barousch Wolfgang , Nehr Marion , Makristathis Athanasios , Willinger Birgit , Rotter Manfred , Hirschl Alexander M. 
Published: February 2003

ABSTRACT

Chlamydia pneumoniae
, an important respiratory pathogen, is difficult to culture, and detection rates by conventional PCRs vary considerably. A new quantitative
ompA
-based real-time PCR assay based on TaqMan technology for detection of
C. pneumoniae
in respiratory samples is described, and its performance in terms of sensitivity and reproducibility is compared with those of four published conventional PCRs (one single-step PCR targeting a cloned
Pst
I fragment; two nested PCRs, one targeting the 16S rRNA gene followed by hybridization and the other targeting the
ompA
gene; and a touchdown enzyme time-release [TETR] PCR also targeting the 16S rRNA gene). Both
ompA
-based PCRs showed the best analytical sensitivity. All five assays could detect even lower target levels from spiked sputum, with the 16S rRNA assays performing better than the
ompA
-based nested PCR (10
−6
inclusion-forming units [IFU] were detected in four of four and two of four replicates by the 16S rRNA TETR PCR and the 16S rRNA nested PCR, respectively). In general, the
ompA
-based real-time protocol produced the most consistent positive results for all replicates tested down to 10
−6
IFU. Eight of 45 patient sputum specimens (18%) were
C. pneumoniae
DNA positive in at least one of four replicates tested by at least one assay. Without taking into consideration the analytical sensitivity or the reproducibility of the test results, the numbers of
C. pneumoniae
DNA-positive sputum specimens (
n
= 8) were four, three, two, two, and one for the 16S rRNA TETR assay, the
Pst
I-based single-step PCR, the
ompA
-based real-time PCR, the
ompA
-based nested touchdown PCR, and the 16S rRNA-based nested PCR, respectively. However, the overall rate of concordance of positive results was low. Only one cell culture-positive sputum specimen was positive by four of five assays (14 of 16 replicates; mean cycle threshold value, 25; 10
8
particles/ml of sputum). Thirty-seven specimens were
C. pneumoniae
negative by all five assays for all replicates tested, as were all negative controls (
n
= 65 to 100 per testing panel). No PCR inhibitors were detected by real-time PCR or by the 16S rRNA-based nested assay. We confirm that the analytical sensitivity of an assay for the detection of
C. pneumoniae
does not necessarily predict its ability to detect its target in sputum. A quantitative, fast, and easy-to-handle diagnostic approach such as the
ompA
-based real-time TaqMan PCR described here might improve the detection of
C. pneumoniae
in respiratory samples.
Evaluation of Semiautomated Multiplex PCR Assay for Determination of
Streptococcus pneumoniae
Serotypes and Serogroups
Authors: Lawrence Elliot R. , Griffiths David B. , Martin Siobhán A. , George Robert C. , Hall Lucinda M. C. 
Published: February 2003

ABSTRACT

A semiautomated method for the determination of five serotypes and three serogroups in
Streptococcus pneumoniae
was developed. Primers specific for serotypes 1, 3, 14, 19F, and 23F and serogroups 6, 19, and 23 were combined in three multiplex PCRs. Products were separated by capillary electrophoresis with a 7-min run time, and a serotype or serogroup was assigned on the basis of fragment size. The method was used to test 93 clinical isolates, and all isolates of the serotypes concerned were correctly detected. The strategy would allow the detection of multiple serotypes in a single sample. Detection of additional serotypes could be included as capsule locus sequences become available.
Human Papillomavirus Type 16 Status in Cervical Carcinoma Cell DNA Assayed by Multiplex PCR
Authors: Lukaszuk Krzysztof , Liss Joanna , Wozniak Izabela , Emerich Janusz , Wójcikowski Czesław 
Published: February 2003

ABSTRACT

Integration of human papillomavirus (HPV) DNA into host genome occurs early in cancer development and is probably an important event in malignant transformation of cervical cancer. The HPV genome integration usually disrupts
E2
gene open reading frames. It results in the lack of
E2
gene suppressor of the synthesis of E6 and E7 products which, in turn, leads to the overexpression of
E6
and
E7
genes. The oncogenic HPV types (HPV16, -18, -45, and -58) can be present as episomes or may integrate into human chromosomes. Sixty-six cervical cancer patients positive for HPV16 were tested for the presence of
E6
,
E2
,
E1
, and
L1
genes. Multiplex PCR was carried out in all cases. Using cluster analysis, the calculated ratios of
E1/E6
,
E2/E6
,
L1/E6
,
E1/E2
, and
E2/
(
E1*E6
) gene amplification products were divided into two or three statistically different groups. These were used for statistical analysis of the prevalence of specific gene types in histological types of cancer, different levels of clinical staging, and histologically confirmed nodal metastases. The statistical analysis proved a significant correlation in the ratios of
E2/E6
and
E1/E2
only. The
E2/E6
and
E1/E2
were higher in carcinoma in situ than in advanced squamous cancers. The
E2/E6
ratios were lower in higher clinical stages. The multiplex PCR estimation of the
E2/E6
ratio could be a simple method for selecting patients with a high risk of a poor outcome in a standard stage-dependent treatment procedure.
Analysis of a Viridans Group Strain Reveals a Case of Bacteremia Due to Lancefield Group G Alpha-Hemolytic
Streptococcus dysgalactiae
subsp.
equisimilis
in a Patient with Pyomyositis and Reactive Arthritis
Authors: Woo Patrick C. Y. , Teng Jade L. L. , Lau Susanna K. P. , Lum Peggy N. L. , Leung Kit-Wah , Wong Kee-Lam , Li Kin-Wah , Lam Kui-Chun , Yuen Kwok-Yung 
Published: February 2003

ABSTRACT

Streptococcus dysgalactiae
is classified by a combination of phenotypic and genotypic characteristics into Lancefield group C alpha-hemolytic
Streptococcus dysgalactiae
subsp.
dysgalactiae
and Lancefield group C, group G, and group L beta-hemolytic
Streptococcus dysgalactiae
subsp.
equisimilis
. In this study, we report the isolation of a catalase-negative, alpha-hemolytic, optochin- and bacitracin-resistant viridans group strain, which does not grow in 10 or 40% bile, on MacConkey agar or bile esculin agar, or in 6% NaCl, from the blood culture of a 73-year-old woman with pyomyositis and poststreptococcal reactive arthritis. Lancefield grouping revealed that the strain was a group G streptococcus. The Vitek system (GPI) showed that it was unidentified, and the API system (20 STREP) showed that it was 95.7%
S. dysgalactiae
subsp.
dysgalactiae
. 16S rRNA gene sequencing showed that it was a strain of
S. dysgalactiae
. Based on phylogenetic affiliation with 16S rRNA gene or GroEL amino acid (another bacterial gene, in addition to 16S rRNA gene, that is highly conserved) sequences, the strain is most closely related to Lancefield group C beta-hemolytic
S. dysgalactiae
subsp.
equisimilis
. PCR amplification and sequencing of the streptolysin S structural gene (
sagA
) and M protein gene (
emm
) hypervariable region showed the presence of these suspected primary virulence factors. Further studies would delineate whether the isolate is just a hemolysin-deficient variant of group G beta-hemolytic
S. dysgalactiae
subsp.
equisimilis
or a novel type of
S. dysgalactiae
. The present case showed that group G alpha-hemolytic
S. dysgalactiae
subsp.
equisimilis
can be associated with serious invasive infection and poststreptococcal sequelae.
Evaluation of Three Commercial Assays for Detection of
Giardia
and
Cryptosporidium
Organisms in Fecal Specimens
Authors: Johnston Stephanie P. , Ballard Melissa M. , Beach Michael J. , Causer Louise , Wilkins Patricia P. 
Published: February 2003

ABSTRACT

There is an increasing demand for diagnostic testing for
Giardia intestinalis (G. lamblia)
and
Cryptosporidium parvum
, with a priority being placed on obtaining diagnostic results in an efficient and timely manner. Several commercial companies have developed rapid diagnostic tests that are simple to perform and can be completed in less time than traditional methods for detecting
Giardia
and
Cryptosporidium
. We compared one of these rapid tests, the ImmunoCard STAT! (Meridian Bioscience, Inc.) lateral-flow immunoassay, with the MERIFLUOR direct fluorescent-antibody (DFA) test, the ProSpecT EZ microplate assay for
Giardia
and the ProSpecT microplate assay for
Cryptosporidium
, and modified Kinyoun's acid-fast stained smears for the detection of
Cryptosporidium
using 246 specimens. The MERIFLUOR DFA (Meridian Bioscience, Inc.) test detected the largest number of cases (32
Giardia
and 37
Cryptosporidium
) infections and was used to calculate the sensitivity and specificity of the other tests. For
Giardia
, the sensitivities of the ImmunoCard STAT! and the ProSpecT
Giardia
EZ microplate assay (Alexon-Trend, Inc.) were 81 and 91%, respectively. For detection of
Cryptosporidium
, the sensitivities of the ImmunoCard STAT!, the ProSpecT
Cryptosporidium
microplate assay (Alexon-Trend, Inc.), and modified Kinyoun's acid-fast stained smears were 68, 70, and 78%, respectively. Test specificities were equal to or greater than 99%. Specimens with very small numbers of organisms were not detected by the ImmunoCard STAT!, the ProSpecT microplate assay or modified Kinyoun's acid-fast stained smears.
Characterization of Some
Actinomyces
-Like Isolates from Human Clinical Sources: Description of
Varibaculum cambriensis
gen. nov., sp. nov
Authors: Hall Val , Collins Matthew D. , Lawson Paul A. , Hutson Roger A. , Falsen Enevold , Inganas Elisabeth , Duerden Brian 
Published: February 2003

ABSTRACT

Fifteen strains of an anaerobic, catalase-negative, gram-positive diphtheroid-shaped bacterium recovered from human sources were characterized by phenotypic and molecular chemical and molecular genetic methods. The unidentified bacterium showed some resemblance to
Actinomyces
species and related taxa, but biochemical testing, polyacrylamide gel electrophoresis analysis of whole-cell proteins, and amplified 16S ribosomal DNA restriction analysis indicated the strains were distinct from all currently named
Actinomyces
species and related taxa. Comparative 16S rRNA gene sequencing studies showed that the bacterium represents a hitherto-unknown phylogenetic line that is related to but distinct from
Actinomyces
,
Actinobaculum
,
Arcanobacterium
, and
Mobiluncus
. We propose, on the basis of phenotypic and phylogenetic evidence, that the unknown bacterium from human clinical specimens should be classified as a new genus and species,
Varibaculum cambriensis
gen. nov., sp. nov. The type strain of
Varibaculum cambriensis
sp. nov. is CCUG 44998
T
= CIP 107344
T
.
Use of a Phage-Based Assay for Phenotypic Detection of Mycobacteria Directly from Sputum
Authors: Park D. J. , Drobniewski F. A. , Meyer A. , Wilson S. M. 
Published: February 2003

ABSTRACT

The phage amplified biologically assay is a new method for rapid and low-cost phenotypic determination of the drug sensitivities of
Mycobacterium tuberculosis
isolates and the detection of viable organisms in patient specimens. Infection of slowly growing mycobacteria with phage (phage D29) was followed by chemical virucide destruction of extracellular phage. Infected mycobacteria were mixed in culture with rapidly growing sensor cells, which the phage can also infect; i.e., lytic amplification of phage occurs. The aims of the present study were to optimize the speed and sensitivity of the assay and reduce its cost for developing countries by using an
M. tuberculosis
-spiked sputum model with (i) identification of inhibitory components of sputum and optimization of decontamination methods; (ii) simplification of the washing and development steps; (iii) reduction of the use of high-cost components, e.g., oleate-albumin-dextrose-catalase (OADC) supplement; and (iv) optimization of virucide treatment. The following results were obtained. (i) An inhibitory factor in sputum which could be removed by treatment of the sample with sodium dodecyl sulfate or NaOH decontamination was identified. (ii) A microcentrifuge-based approach with thixotropic silica as a bedding and resuspension agent was developed as an alternative to conventional centrifugation medium exchange. The yield was increased 228-fold, with increased speed and reduced cost. (iii) At present, after extracellular inactivation of phage, the ferrous ammonium sulfate (FAS) virucide is sequestered by dilution with an expensive supplement, OADC. Sodium citrate with calcium chloride was found to be a cost-effective after treatment with the FAS protectant and offered greater protection than OADC. Kinetic-lysis experiments indicated that an infection time of 1 to 3 h prior to FAS addition was optimal. (iv) Amplification of the signal (which corresponded to the burst size) was shown by allowing lysis prior to plating in a spiked medium model (up to 20-fold) and a spiked sputum model (up to 10-fold). A liquid culture detection method capable of detecting approximately 60 viable
M. tuberculosis
organisms in 1 ml of sputum was developed. Taken together, these improvements support the routine application of the assay to sputum specimens.