bluuebunny/crossref_metadata_2025_split huggingface dataset:
Usefulness of a New Mycobacteriophage-Based Technique for Rapid Diagnosis of Pulmonary Tuberculosis
Authors: Alcaide Fernando , Galí Nuria , Domínguez José , Berlanga Pilar , Blanco Silvia , Orús Pilar , Martín Rogelio 
Published: July 2003

ABSTRACT

A new mycobacteriophage-based technique (PhageTek MB) was compared with standard culture and staining techniques for diagnosis of pulmonary tuberculosis. A total of 2,048 respiratory specimens from 1,466 patients collected from February 2000 to March 2001 were studied by both (i) conventional methods (direct microscopic examination [auramine-rhodamine fluorochrome], and culture in BacT/ALERT 3D and solid media) and (ii) the PhageTek MB assay. This phenotypic test utilizes specific mycobacteriophages to detect the presence of live
Mycobacterium tuberculosis
complex organisms within a decontaminated clinical sample. Overall, 205 (10%) specimens were positive for mycobacteria (134 patients): 144 (70.2%)
M. tuberculosis
isolates and 61 (29.8%) nontuberculous mycobacterium isolates (30
Mycobacterium kansasii
, 12
Mycobacterium xenopi
, 9
Mycobacterium gordonae
, 7
Mycobacterium avium
complex, 2
Mycobacterium chelonae
, and 1
Mycobacterium fortuitum
isolate). PhageTek MB was more likely to give a positive result with specimens in which high numbers of acid-fast bacilli were observed on the smear. The sensitivity, specificity, and positive and negative predictive values of this mycobacteriophage-based technique versus culture for
M. tuberculosis
were 58.3, 99.1, 83.2, and 96.9%, respectively. PhageTek MB is a rapid (48-h), specific, safe, and easy-to-perform test. According to the prevalence of the disease in the population studied, the test would require improved sensitivity in order to be used as a screening test for routine diagnosis of respiratory tuberculosis in our setting.
Different Cytomegalovirus Glycoprotein B Genotype Distribution in Serum and Cerebrospinal Fluid Specimens Determined by a Novel Multiplex Nested PCR
Authors: Tarragó David , Quereda Carmen , Tenorio Antonio 
Published: July 2003

ABSTRACT

A novel and highly sensitive multiplex nested PCR assay has been developed for the simultaneous glycoprotein B (gB) typing of four gB genotypes of human cytomegalovirus (CMV) directly from clinical specimens. Specifically, a pair of primers to conserved regions of all gB genotypes within the gB gene (
gB
and
gpUL55
) was used for primary amplification. A mixture of nested primers to specific and conserved regions of each gB genotype was used for a secondary PCR amplification, yielding amplicons of different sizes for each gB genotype that could easily be differentiated by agarose gel electrophoresis. A total of 40 of 44 serum specimens and 26 of 26 cerebrospinal fluid (CSF) samples, which had previously tested positive for CMV in 66 of 70 AIDS patients with different CMV disease conditions, were gB genotyped by this novel assay. Significant differences in glycoprotein B genotype distribution between serum and CSF specimens were found (
P
= 0.001). gB genotype 3 (gB3) and gB2 were the most prevalent genotypes in sera (42.5%) and CSF (38.5%), respectively. A different distribution was also observed when only patients with CMV retinitis were studied (
P
= 0.005), suggesting a gB2 neuron tropism. Neither CMV disease nor any particular clinical manifestation of CMV disease was associated with gB genotypes (
P
> 0.05). A high incidence of mixed infection with the gB1 and gB3 genotypes (27.5%) was detected in serum specimens, indicating that reinfection and reactivation are common traits in advanced AIDS patients.
Widespread Pyrazinamide-Resistant
Mycobacterium tuberculosis
Family in a Low-Incidence Setting
Authors: Nguyen Dao , Brassard Paul , Westley Jennifer , Thibert Louise , Proulx Melanie , Henry Kevin , Schwartzman Kevin , Menzies Dick , Behr Marcel A. 
Published: July 2003

ABSTRACT

An unusually high prevalence of pyrazinamide (PZA) monoresistance in
Mycobacterium tuberculosis
has been observed in Quebec. In the absence of a recognized outbreak, we hypothesized that these isolates most likely represented reactivation of an old endemic strain in this low-incidence area. A case-control study of 77 PZA-resistant isolates with a specific Quebec mutation and 253 PZA-susceptible control
M. tuberculosis
isolates was undertaken. By molecular analysis, all 77 case isolates shared a unique mutation profile in the
pncA
gene which was not present in control isolates. While control isolates manifested diverse IS
6110
restriction fragment length polymorphism (RFLP) patterns, spoligotypes, and major genetic groups, case isolates had similar but nonidentical IS
6110
RFLP patterns, had common spoligotypes, and were confined to one major genetic group, suggesting a common clonal ancestor. By epidemiologic and geographic analyses, however, there were no significant differences between the cases and the controls. We conclude that a clonally related family of PZA-monoresistant
M. tuberculosis
isolates in Quebec represents historic rather than recent transmission.
Detection and Characterization of Verocytotoxin-Producing
Escherichia coli
by Automated 5′ Nuclease PCR Assay
Authors: Nielsen Eva Møller , Andersen Marianne Thorup 
Published: July 2003

ABSTRACT

In recent years increased attention has been focused on infections caused by isolates of verocytotoxin-producing
Escherichia
coli (VTEC) serotypes other than O157. These non-O157 VTEC isolates are commonly present in food and food production animals. Easy detection, isolation, and characterization of non-O157 VTEC isolates are essential for improving our knowledge of these organisms. In the present study, we detected VTEC isolates in bovine fecal samples by a duplex 5′ nuclease PCR assay (real-time PCR) that targets
vtx1
and
vtx2
. VTEC isolates were obtained by colony replication by use of hydrophobic-grid membrane filters and DNA probe hybridization. Furthermore, we have developed 5′ nuclease PCR assays for the detection of virulence factors typically present in VTEC isolates, including subtypes of three genes of the locus of enterocyte effacement (LEE) pathogenicity island. The 22 assays included assays for the detection of verocytotoxin genes (
vtx1
,
vtx2
), pO157-associated genes (
ehxA
,
katP
,
espP
, and
etpD
), a recently identified adhesin (
saa
), intimin (
eae
, all variants), seven subtypes of
eae
, four subtypes of
tir
, and three subtypes of
espD
. A number of reference strains (VTEC and enteropathogenic
E. coli
strains) and VTEC strains isolated from calves were tested to validate the PCR assays. The expected virulence profiles were detected for all reference strains. In addition, new information on the subtypes of LEE genes was obtained. For reference strains as well as bovine isolates, a consistent relationship between subtypes of the LEE genes was found, so that a total of seven different combinations of these were recognized (corresponding to the seven subtypes of
eae
). Isolates with 15 different serogroup-virulence profiles were isolated from 16 calves. Among these, 53% harbored LEE and 73% harbored factors carried by the large virulence plasmid. One LEE-negative isolate had the gene for the adhesin Saa. The most common virulence profile among the bovine isolates was
vtx1
,
eae
-ζ,
tir
-α,
ehxA
, and
espP
. This panel of assays offers an easy method for the extensive characterization of VTEC isolates.
Rapid Screening and Identification of Methicillin-Resistant
Staphylococcus aureus
from Clinical Samples by Selective-Broth and Real-Time PCR Assay
Authors: Fang Hong , Hedin Göran 
Published: July 2003

ABSTRACT

A screening method for methicillin-resistant
Staphylococcus aureus
(MRSA) by using selective broth and real-time PCR (broth-PCR) was developed and evaluated. The samples (
n
= 304) were cultured in the broth overnight, followed by
nuc
gene detection by real-time PCR.
nuc
-negative samples were further checked for the presence of
nuc
amplification inhibitors by a PCR internal inhibitor assay.
nuc
-positive samples and
nuc
-negative samples with PCR inhibitors were cultured onto plates and processed further. The diagnostic values for this MRSA screening method were 93.3% sensitivity, 89.6% specificity, 31.8% positive predictive value, and 99.6% negative predictive value. The application of the broth-PCR method will be able to report most of the negative samples (258 of 289 [89.3%]) on the next morning and can save as much as 84.9% (258 of 304) of the labor and cost spent on processing the
nuc
-negative specimens on plates. In the study, all the samples were processed in parallel by the broth enrichment method and the plating method for comparison. To identify MRSA, the isolated oxacillin-resistant
S. aureus
strains were tested by a duplex real-time PCR targeting the
mecA
gene and the
nuc
gene. A collection of MRSA, methicillin-susceptible
Staphylococcus aureus
, methicillin-resistant
Staphylococcus epidermidis
, and methicillin-susceptible
Staphylococcus epidermidis
strains and a panel of standard strains of 11 bacterial species other than
S. aureus
were also tested by this method, which was proved to be a valuable tool for MRSA identification in a routine microbiological laboratory.
Sources and Magnitude of Intralaboratory Variability in a Sequence-Based Genotypic Assay for Human Immunodeficiency Virus Type 1 Drug Resistance
Authors: Galli R. A. , Sattha B. , Wynhoven B. , O'Shaughnessy M. V. , Harrigan P. R. 
Published: July 2003

ABSTRACTWe assessed the intralaboratory reproducibility of a system for sequencing human immunodeficiency virus type 1 (HIV-1) protease (PR) and reverse transcriptase (RT) by using replicate subanalyses of 46 plasma samples collected from HIV-1-infected, antiretroviral-experienced patients in order to determine the relative contributions of the different procedural steps to final sequence variability. Complete sequence concordance between duplicates of each sample was 99.4%. Complete and partial mismatches occurred scattered throughout the PR-RT genome segment at >300 positions. Approximately 75% of the discordances involved mixtures, some of which appeared at key resistance sites. Most differences were the result of the first-round RT-PCR procedure. Inter-rater concordance for sequence analysis and assembly was >99.9%. There was no observed correlation between the number or frequency of mismatches and plasma viral loads. A separate longitudinal analysis of a single routine control sample sequenced 103 times over 9 months consistently gave highly reproducible sequences (median percentage of nucleotide discordances, 0.04%; range, 0 to 0.2%). Finally, sequence data from 168 sequential samples collected from 22 patients with long-term, predominantly wild type HIV showed that intrapatient nucleotide concordance with individual index sequences ranged from 96.5 to 100%. Together, these results confirm that sequence-based genotyping can be a precise and reliable tool for monitoring HIV drug resistance, and they suggest that efforts to reduce variability should focus on the first RT-PCR step. Consequently, the data suggest that the composition of external quality assessment panels should be based on clinical HIV isolates rather than DNA clones.
Identification of
Bacillus anthracis
by
rpoB
Sequence Analysis and Multiplex PCR
Authors: Ko Kwan Soo , Kim Jong-Man , Kim Jong-Wan , Jung Byeong Yeal , Kim Wonyong , Kim Ik Jung , Kook Yoon-Hoh 
Published: July 2003

ABSTRACT

Comparative sequence analysis was performed upon
Bacillus anthracis
and its closest relatives,
B. cereus
and
B. thuringiensis
. Portions of
rpoB
DNA from 10 strains of
B. anthracis
, 16 of
B. cereus
, 10 of
B. thuringiensis
, 1 of
B. mycoides
, and 1 of
B. megaterium
were amplified and sequenced. The determined
rpoB
sequences (318 bp) of the 10
B. anthracis
strains, including five Korean isolates, were identical to those of Ames, Florida, Kruger B, and Western NA strains. Strains of the “
B. cereus
group” were separated into two subgroups, in which the
B. anthracis
strains formed a separate clade in the phylogenetic tree. However,
B. cereus
and
B. thuringiensis
could not be differentiated. Sequence analysis confirmed the five Korean isolates as
B. anthracis
. Based on the
rpoB
sequences determined in the present study, multiplex PCR generating either
B. anthracis
-specific amplicons (359 and 208 bp) or
cap
DNA (291 bp) in a virulence plasmid could be used for the rapid differential detection and identification of virulent
B. anthracis
.
Detection and Verification of
Mycobacterium avium
subsp.
paratuberculosis
in Fresh Ileocolonic Mucosal Biopsy Specimens from Individuals with and without Crohn's Disease
Authors: Bull Tim J. , McMinn Elizabeth J. , Sidi-Boumedine Karim , Skull Angela , Durkin Damien , Neild Penny , Rhodes Glenn , Pickup Roger , Hermon-Taylor John 
Published: July 2003

ABSTRACT

Mycobacterium avium
subsp.
paratuberculosis
is a robust and phenotypically versatile pathogen which causes chronic inflammation of the intestine in many species, including primates.
M. avium
subsp.
paratuberculosis
infection is widespread in domestic livestock and is present in retail pasteurized cows' milk in the United Kingdom and, potentially, elsewhere. Water supplies are also at risk. The involvement of
M. avium
subsp.
paratuberculosis
in Crohn's disease (CD) in humans has been uncertain because of the substantial difficulties in detecting this pathogen. In its Ziehl-Neelsen staining-negative form,
M. avium
subsp.
paratuberculosis
is highly resistant to chemical and enzymatic lysis. The present study describes the development of optimized sample processing and DNA extraction procedures with fresh human intestinal mucosal biopsy specimens which ensure access to
M. avium
subsp.
paratuberculosis
DNA and maximize detection of these low-abundance pathogens. Also described are two nested PCR methodologies targeted at IS
900
, designated IS
900
[L/AV] and IS
900
[TJ1-4], which are uniquely specific for IS
900
. Detection of
M. avium
subsp.
paratuberculosis
in mucosal biopsy specimens was also evaluated by using mycobacterial growth indicator tube (MGIT) cultures (Becton Dickinson). IS
900
[L/AV] PCR detected
M. avium
subsp.
paratuberculosis
in 34 of 37 (92%) patients with CD and in 9 of 34 (26%) controls without CD (noninflammatory bowel disease [nIBD] controls) (
P
= 0.0002; odds ratio = 3.47).
M. avium
subsp.
paratuberculosis
was detected by IS
900
[L/AV] PCR in MGIT cultures after 14 to 88 weeks of incubation in 14 of 33 (42%) CD patients and 3 of 33 (9%) nIBD controls (
P
= 0.0019; odds ratio = 4.66). Nine of 15 (60%) MGIT cultures of specimens from CD patients incubated for more than 38 weeks were positive for
M. avium
subsp.
paratuberculosis
. In each case the identity of IS
900
from
M. avium
subsp.
paratuberculosis
was verified by amplicon sequencing. The rate of detection of
M. avium
subsp.
paratuberculosis
in individuals with CD is highly significant and implicates this chronic enteric pathogen in disease causation.
Genome-Wide DNA Microarray Analysis of
Francisella tularensis
Strains Demonstrates Extensive Genetic Conservation within the Species but Identifies Regions That Are Unique to the Highly Virulent
F. tularensis
subsp.
tularensis
Authors: Broekhuijsen Martien , Larsson Pär , Johansson Anders , Byström Mona , Eriksson Ulla , Larsson Eva , Prior Richard G. , Sjöstedt Anders , Titball Richard W. , Forsman Mats 
Published: July 2003

ABSTRACT

Francisella tularensis
is a potent pathogen and a possible bioterrorism agent. Little is known, however, to explain the molecular basis for its virulence and the distinct differences in virulence found between the four recognized subspecies,
F. tularensis
subsp.
tularensis
,
F. tularensis
subsp.
mediasiatica
,
F. tularensis
subsp.
holarctica
, and
F. tularensis
subsp.
novicida
. We developed a DNA microarray based on 1,832 clones from a shotgun library used for sequencing of the highly virulent strain
F. tularensis
subsp.
tularensis
Schu S4. This allowed a genome-wide analysis of 27 strains representing all four subspecies. Overall, the microarray analysis confirmed a limited genetic variation within the species
F. tularensis
, and when the strains were compared, at most 3.7% of the probes showed differential hybridization. Cluster analysis of the hybridization data revealed that the causative agents of type A and type B tularemia, i.e.,
F. tularensis
subsp.
tularensis
and
F. tularensis
subsp.
holarctica
, respectively, formed distinct clusters. Despite marked differences in their virulence and geographical origin, a high degree of genomic similarity between strains of
F. tularensis
subsp.
tularensis
and
F. tularensis
subsp.
mediasiatica
was apparent. Strains from Japan clustered separately, as did strains of
F. tularensis
subsp.
novicida
. Eight regions of difference (RD) 0.6 to 11.5 kb in size, altogether comprising 21 open reading frames, were identified that distinguished strains of the moderately virulent subspecies
F. tularensis
subsp.
holarctica
and the highly virulent subspecies
F. tularensis
subsp.
tularensis
. One of these regions, RD1, allowed for the first time the development of an
F. tularensis
-specific PCR assay that discriminates each of the four subspecies.
Sequence-Based Typing of
Legionella pneumophila
Serogroup 1 Offers the Potential for True Portability in Legionellosis Outbreak Investigation
Authors: Gaia Valeria , Fry Norman K. , Harrison Timothy G. , Peduzzi Raffaele 
Published: July 2003

ABSTRACT

Seven gene loci of
Legionella pneumophila
serogroup 1 were analyzed as potential epidemiological typing markers to aid in the investigation of legionella outbreaks. The genes chosen included four likely to be selectively neutral (
acn
,
groES
,
groEL
, and
recA
) and three likely to be under selective pressure (
flaA
,
mompS
, and
proA
). Oligonucleotide primers were designed to amplify 279- to 763-bp fragments from each gene. Initial sequence analysis of the seven loci from 10 well-characterized isolates of
L. pneumophila
serogroup 1 gave excellent reproducibility (
R
) and epidemiological concordance (
E
) values (
R
= 1.00;
E
= 1.00). The three loci showing greatest discrimination and nucleotide variation,
flaA
,
mompS
, and
proA
, were chosen for further study. Indices of discrimination (
D
) were calculated using a panel of 79 unrelated isolates. Single loci gave
D
values ranging from 0.767 to 0.857, and a combination of all three loci resulted in a
D
value of 0.924. When all three loci were combined with monoclonal antibody subgrouping, the
D
value was 0.971. Sequence-based typing of
L. pneumophila
serogroup 1 using only three loci is epidemiologically concordant and highly discriminatory and has the potential to become the new “gold standard” for the epidemiological typing of
L. pneumophila
.
Molecular Epidemiology and Characterization of Plasmid-Encoded β-Lactamases Produced by Tunisian Clinical Isolates of
Salmonella enterica
Serotype Mbandaka Resistant to Broad-Spectrum Cephalosporins
Authors: Makanera Abdoulaye , Arlet Guillaume , Gautier Valérie , Manai Mohamed 
Published: July 2003

ABSTRACT

We studied 31 clinical isolates of
Salmonella enterica
serotype Mbandaka resistant to broad-spectrum cephalosporins and recovered in Tunisia over a 5-year period. The transferability of this resistance was demonstrated by conjugation experiments. Thirty of the 31 isolates were positive in the double-disk synergy test. By isoelectric focusing analysis, all of the isolates were found to produce a band of β-lactamase activity with a pI of 5.9. Three of these isolates produced an additional band with a pI of 7.6. PCR and DNA sequencing identified these β-lactamases as TEM-4 and SHV-2a, respectively. The remaining isolate, highly resistant to ceftazidime but susceptible to cefepime, produced a β-lactamase that focused at pI 7.8. No synergy was detected by the double-disk synergy test. Sequence analysis of the
bla
gene amplified by PCR showed that the plasmid-mediated AmpC-type enzyme was ACC-1a. Fingerprinting analysis by repetitive-element PCR and enterobacterial repeat intergenic consensus-PCR suggested that 29 of the 31
Salmonella
serotype Mbandaka isolates belonged to the same clonal population.
Genetic Relationship between Methicillin-Sensitive and Methicillin-Resistant
Staphylococcus aureus
Strains from France and from International Sources: Delineation of Genomic Groups
Authors: Branger Catherine , Gardye Carole , Galdbart Jacques-Olivier , Deschamps Catherine , Lambert Nicole 
Published: July 2003

ABSTRACT

Cluster analysis of the
Sma
I patterns, generated by pulsed-field gel electrophoresis, of 44 methicillin-resistant (MRSA) and 118 methicillin-sensitive (MSSA)
Staphylococcus aureus
strains isolated in various French hospitals and 61 MRSA and 48 MSSA strains from 20 other countries revealed 20 genomic groups distributed into four distantly related phylogenic branches. Eighty-three of the 105 MRSA strains (79%) were clustered in the six genomic groups of phylogenic branch I; and 154 of the 166 MSSA strains (92.8%) were clustered in the 14 genomic groups of phylogenic branches II, III, and IV. Agreement between genomic group and two other markers, esterase type and phage group, was obtained, emphasizing the clonal structure of the population. The genomic groups were delineated by esterase type. The distribution of the strains within the genomic groups was independent of their geographical origin; French strains were clustered with strains from other countries. The three types of the staphylococcal cassette chromosome
mec
(SCC
mec
) complex were distributed according to genomic groups. Most of the time, type I and type II SCC
mec
complexes were found in the MRSA strains belonging to the same genomic groups. In contrast, the type III SCC
mec
complex was specific to the MRSA strains belonging to the three genomic groups characterized by a common esterase type.
Differentiation of Rickettsiae by
groEL
Gene Analysis
Authors: Lee Jung-Hee , Park Hyo-Soon , Jang Won-Jong , Koh Seong-Eun , Kim Jong-Moon , Shim Soo-Kyoung , Park Mi-Yeoun , Kim Yoon-Won , Kim Bum-Joon , Kook Yoon-Hoh , Park Kyung-Hee , Lee Seung-Hyun 
Published: July 2003

ABSTRACT

The nucleotide sequences (534 to 546 bp) of the
groEL
gene, which encodes the 60-kDa heat shock protein GroEL, from 15 rickettsial strains were determined and compared. In the phylogenetic tree created by the unweighted pair group method with arithmetic averages and the neighbor-joining method, rickettsial strains could be distinguished from
Ehrlichia
strains. Five spotted fever group strains, four typhus group strains, and six scrub typhus group (STG) strains were differentiated as distinct entities. Unlike
gltA
and
ompA
gene analyses, differentiation between members of the genus
Rickettsia
and the STG rickettsiae by
groEL
gene analysis was possible. In comparison with 16S rRNA gene analysis, the
groEL
gene has a higher degree of divergence among the rickettsiae. We therefore successfully developed rapid differentiation methods, PCR-restriction fragment length polymorphism analysis and a species-specific PCR, based on the
groEL
gene sequences. Four Korean isolates were identified by these methods and
groEL
gene analysis. The results suggest that the
groEL
gene is useful for the identification and characterization of rickettsiae
.