bluuebunny/crossref_metadata_2025_split huggingface dataset:
Core Promoter Structure in the Oomycete
Phytophthora infestans
Authors: McLeod Adele , Smart Christine D. , Fry William E. 
Published: February 2004

ABSTRACT

We have investigated the core promoter structure of the oomycete
Phytophthora infestans
. The transcriptional start sites (TSS) of three previously characterized
P. infestans
genes,
Piexo1
,
Piexo3
, and
Piendo1
, were determined by primer extension analyses. The TSS regions were homologous to a previously identified 16-nucleotide (nt) core sequence that overlaps the TSS in most oomycete genes. The core promoter regions of
Piexo1
and
Piendo1
were investigated by using a transient protoplast expression assay and the reporter gene β-glucuronidase. Mutational analyses of the promoters of
Piexo1
and
Piendo1
showed that there is a putative core promoter element encompassing the TSS (−2 to + 5) that has high sequence and functional homology to a known core promoter element present in other eukaryotes, the initiator element (Inr). Downstream and flanking the Inr is a highly conserved oomycete promoter region (+7 to + 15), hereafter referred to as FPR (flanking promoter region), which is also important for promoter function. The importance of the 19-nt core promoter region (Inr and FPR) in
Piexo1
and
Piendo1
was further investigated through electrophoretic mobility shift assays (EMSA). The EMSA studies showed that (i) both core promoters were able to specifically bind a protein or protein complex in a
P. infestans
whole-cell protein extract and (ii) the same mutations that reduced binding of the EMSA complex also reduced β-glucuronidase (GUS) levels in transient expression assays. The consistency of results obtained using two different assays (GUS transient assays [in vivo] and EMSA studies [in vitro]) supports a convergence of inference about the relative importance of specific nucleotides within the 19-nt core promoter region.
Analysis of Invasive
Haemophilus influenzae
Infections after Extensive Vaccination against
H. influenzae
Type b
Authors: Campos José , Hernando Margarita , Román Federico , Pérez-Vázquez María , Aracil Belén , Oteo Jesús , Lázaro Edurne , de Abajo Francisco ,   the Group of Invasive Haemophilus Infections of the Autonomous Community of Madrid, Spain
Published: February 2004

ABSTRACT

Little clinical and microbiological information is available about invasive
Haemophilus influenzae
infection after widespread vaccination against
H. influenzae
type b (Hib). We conducted an active community surveillance study on invasive
H. influenzae
during a 2-year period in a community of more than 5 million people after vaccination against Hib in children was introduced. The median incidence was 16.3 cases/100,000 persons per year in children less than 1-year-old and 4.41 cases/100,000 persons in children less than <5 years old. The highest incidence in adults was observed in patients greater than 70 years old. Clinical diagnoses included bacteremia, pneumonia, and meningitis. Of the
H. influenzae
-infected patients, 74.3% had underlying predisposing conditions, including impaired immunity and respiratory diseases. A total of 73.6% of the isolates were nontypeable and 16.5, 6.6, and 3.3% were types b, f, and e, respectively. Infections due to capsulated strains b, e, and f were evenly distributed between children and adults. Ampicillin and cotrimoxazole resistance occurred at frequencies of 24.2 and 48.4%, respectively. Antibiotic resistance was more prevalent in capsulated than in noncapsulated
H. influenzae
. Invasive isolates were highly resistant to antibiotics that were used infrequently in the community. Nontypeable
H. influenzae
were genetically much more heterogeneous than capsulated strains. Capsule-deficient mutants (b
−
) were not detected. Plasmid carriage was linked to antibiotic resistance and capsulated strains. Over the study period, the incidence of invasive
H. influenzae
infections, either encapsulated or not, did not increase. In the post-Hib vaccination era, most invasive infections were due to noncapsulated strains and occurred in the extreme ages of life in patients with predisposing conditions.
Use of Insertion Sequence Element IS
1126
in a Genotyping and Transmission Study of
Porphyromonas gingivalis
Authors: Park Ok-Jin , Min Kyung-Man , Choe Son-Jin , Choi Bong-Kyu , Kim Kack-Kyun 
Published: February 2004

ABSTRACT

Porphyromonas gingivalis
is strongly associated with periodontal diseases and is regarded as one of the risk factors for periodontitis. Insertion sequence element IS
1126
-based PCR was used to investigate the genetic heterogeneity of
P. gingivalis
from periodontitis patients and to examine the frequency of the parent-child and spouse-spouse transmission. Two sets of IS
1126
-specific primers were used for the PCR. The inward primer set (PI1 and PI2), which amplifies the IS
1126
fragment of approximately 690 bp, was used to identify
P. gingivalis
. The outward primer set (PI1RC and PI2RC), which is reverse complementary to PI1 and PI2, respectively, and amplifies the gene fragments between the adjacent IS
1126
elements was used to characterize the genotypes of the
P. gingivalis
strains. PCR of
P. gingivalis
with PI1RC and PI2RC resulted in the production of two to seven amplicons, which showed a unique electrophoretic pattern in each strain (4 laboratory strains and 37 clinical isolates cultured from 12 patients with aggressive periodontitis). The usefulness of the method for transmission study was confirmed by detecting identical genotypes between the isolates and the plaque samples from which the isolates were cultured and between the plaque samples from different tooth sites in the same patient. Thirty probands with periodontal diseases and their thirty immediate family members were included in the transmission study. In 11 of 14 parent-child pairs (78.6%),
P. gingivalis
revealed an identical or similar band pattern, whereas 5 of 16 spouse pairs (31.25%) had this similarity. These results show that IS
1126
-based PCR for genotyping
P. gingivalis
has a highly discriminating potential with reproducible data and is a simple and reliable method for a transmission study.
Characterization of Cell-to-Cell Signaling-Deficient
Pseudomonas aeruginosa
Strains Colonizing Intubated Patients
Authors: Dénervaud Valérie , TuQuoc Patrick , Blanc Dominique , Favre-Bonté Sabine , Krishnapillai Viji , Reimmann Cornelia , Haas Dieter , van Delden Christian 
Published: February 2004

ABSTRACT

Cell-to-cell signaling involving
N
-acyl-homoserine lactone compounds termed autoinducers (AIs) is instrumental to virulence factor production and biofilm development by
Pseudomonas aeruginosa
. In order to determine the importance of cell-to-cell signaling during the colonization of mechanically ventilated patients, we collected 442
P. aeruginosa
pulmonary isolates from 13 patients. Phenotypic characterization showed that 81% of these isolates produced the AI-dependent virulence factors elastase, protease, and rhamnolipids. We identified nine genotypically distinct
P. aeruginosa
strains. Six of these strains produced AIs [
N
-butanoyl-homoserine lactone or
N
-(3-oxo-dodecanoyl)-homoserine lactone] and extracellular virulence factors (elastase, total exoprotease, rhamnolipid, hydrogen cyanide, or pyocyanin) in vitro. Three of the nine strains were defective in the production of both AIs and extracellular virulence factors. Two of these strains had mutational defects in both the
lasR
and
rhlR
genes, which encode the
N
-acyl-homoserine lactone-dependent transcriptional regulators LasR and RhlR, respectively. The third of these AI-deficient strains was only mutated in the
lasR
gene. Our observations suggest that most, but not all, strains colonizing intubated patients are able to produce virulence factors and that mutations affecting the cell-to-cell signaling circuit are preferentially located in the transcriptional regulator genes.
Experience with the MicroSeq D2 Large-Subunit Ribosomal DNA Sequencing Kit for Identification of Filamentous Fungi Encountered in the Clinical Laboratory
Authors: Hall Leslie , Wohlfiel Sherri , Roberts Glenn D. 
Published: February 2004

ABSTRACT
Described herein is our experience with the MicroSeq D2 large-subunit rDNA sequencing kit for the identification of filamentous fungi encountered in the mycology laboratory at the Mayo Clinic. A total of 234 filamentous fungi recovered from clinical specimens were used in the evaluation. All were identified by using phenotypic characteristics as observed macroscopically and microscopically on any medium or a combination of media, which included Sabouraud's dextrose, inhibitory mold, cornmeal, Czapek-Dox, potato dextrose, and V8 juice agars; all isolates were sequenced using the MicroSeq D2 large-subunit rDNA sequencing kit. Of the of 234 isolates, 158 were correctly identified to the appropriate genus or genus and species by using nucleic acid sequencing. Sequences for 70 (29.9%) of the isolates (27 genera) were not included in the MicroSeq library. Of the 80 dematiaceous and 154 hyaline fungi sequenced, 65 and 51.2%, respectively, gave results concordant with those determined by phenotypic identification. Nucleic acid sequencing using the MicroSeq D2 large-subunit rDNA sequencing kit offers promise of being an accurate identification system; however, the associated library needs to include more of the clinically important genera and species.
Evaluation of the RealArt Malaria LC Real-Time PCR Assay for Malaria Diagnosis
Authors: Farcas Gabriella A. , Zhong Kathleen J. Y. , Mazzulli Tony , Kain Kevin C. 
Published: February 2004

ABSTRACT
PCR-based methods have advantages over traditional microscopic methods for the diagnosis of malaria, especially in cases of low parasitemia and mixed infections. However, current PCR-based assays are often labor-intensive and not readily quantifiable and have the potential for contamination due to a requirement for postamplification sample handling. Real-time PCR can address these limitations. This study evaluated the performance characteristics of a commercial malaria real-time PCR assay (RealArt Malaria LC Assay; Artus GmbH, Hamburg, Germany) on the LightCycler platform for the detection of malaria parasites in 259 febrile returned travelers. Compared to nested PCR as the reference standard, the real-time assay had a sensitivity of 99.5%, specificity of 100%, positive predictive value of 100%, and negative predictive value of 99.6% for the detection of malaria. Our results indicate that the RealArt assay is a rapid (<45 min), sensitive, and specific method for the detection of malaria in returned travelers.
Association of Presence of
Aspergillus
Antibodies with Hemoptysis in Patients with Old Tuberculosis or Bronchiectasis but No Radiologically Visible Mycetoma
Authors: Chu Chung-ming , Woo Patrick C. Y. , Chong Ken T. K. , Leung Wah-shing , Chan Veronica L. , Yuen Kwok-yung 
Published: February 2004

ABSTRACT

Old tuberculosis and bronchiectasis are the two most important causes of chronic structural changes of lungs in our locality. In the absence of radiologically visible mycetoma, the cause of hemoptysis in these two groups of patients is largely unknown. A 17-month prospective study was carried out to compare the prevalence of
Aspergillus fumigatus
and
Aspergillus flavus
antibodies in hemoptysis patients with old tuberculosis or bronchiectasis but no radiologically visible mycetoma (cases,
n
= 38), hemoptysis patients with other diagnosis (control group 1,
n
= 29), and patients with old tuberculosis or bronchiectasis but no hemoptysis (control group 2,
n
= 47) by a recently developed sensitive and specific
A. fumigatus
and
A. flavus
antibody assay. There were a significantly larger number of patients with antibody against
A. fumigatus
or
A. flavus
among the cases than among the patients in control groups 1 and 2 (
P
< 0.05 in both comparisons). Molds were not recovered from any of the patients. Among the 10 cases with
Aspergillus
antibody, eight and two had antibody against
A. flavus
and
A. fumigatus
, respectively. We conclude that there was an association between the presence of
Aspergillus
antibodies and hemoptysis in patients with old tuberculosis or bronchiectasis, suggesting that these patients probably had occult infections caused by the corresponding fungi. Development of serological tests against other
Aspergillus
species as well as other causes of mycetoma will probably increase the detection of occult mold infections in patients with existing parenchymal lung diseases, and treatment of fungal microinvasion may help to alleviate hemoptysis in these patients with bronchiectasis or old tuberculosis who have
Aspergillus
antibodies.
Identification of Subgenus C Adenoviruses by Fiber-Based Multiplex PCR
Authors: Adhikary Arun Kumar , Inada Toshiki , Banik Urmila , Numaga Jiro , Okabe Nobuhiko 
Published: February 2004

ABSTRACT
Subgenus C human adenoviruses, which include serotypes 1, 2, 5, and 6, are often associated with respiratory illness, ocular infections, gastroenteritis, and systemic infection among immunocompromised patients. To address the problems associated with the conventional typing methods, we developed a fiber-based multiplex PCR assay for simple and specific identification of adenovirus type 1, 2, 5, and 6 field isolates. To design type-specific primers, adenovirus type 1 and 6 fiber genes were sequenced. The assay correctly identified prototype strains of adenovirus serotypes 1, 2, 5, 6, as well as 21 previously typed adenovirus field isolates. Mixing two different prototype DNAs produced two amplicons of different lengths, thus clearly distinguishing the prototypes. The results correlated 100% with serological tests and 95% with the previously described PCR-restriction fragment length polymorphism method. The detection of dual infection is an added benefit of the assay. No nonspecific amplification was detected with other adenovirus serotypes or with nonadenoviral DNA. Our fiber-based multiplex PCR assay will provide a convenient tool for type-specific identification of subgenus C adenovirus isolates in various clinical situations and in epidemiological investigations and is a better alternative than the hexon-based assay.
Use of TaqMan 5′ Nuclease Real-Time PCR for Quantitative Detection of
Mycoplasma genitalium
DNA in Males with and without Urethritis Who Were Attendees at a Sexually Transmitted Disease Clinic
Authors: Jensen Jørgen Skov , Björnelius Eva , Dohn Birthe , Lidbrink Peter 
Published: February 2004

ABSTRACT

Mycoplasma genitalium
is a cause of nongonococcal urethritis, particularly in patients not infected with
Chlamydia trachomatis
. A quantitative 5′ nuclease assay (TaqMan PCR) was developed and validated. The assay detected a fragment of the MgPa adhesin gene by use of a TaqMan MGB (minor groove binder) probe and included an internal processing control to detect PCR inhibition. Urethral swab specimens and first-void urine samples from
M. genitalium
-positive men were examined, and the
M. genitalium
DNA load was correlated to symptoms and signs. The assay consistently detected <5 genome copies without cross-reactions with other mycoplasmas. Urine and urethral swab specimens from men with urethritis had higher
M. genitalium
DNA loads than specimens from men without urethritis. However, a very broad overlap of DNA loads between patients with and without urethritis was observed. Urethral swab specimens from patients with urethral discharge had a significantly higher DNA load than specimens from patients without discharge. This correlation was not found in first-void urine specimens.
High Genetic Diversity among
Stenotrophomonas maltophilia
Strains Despite Their Originating at a Single Hospital
Authors: Valdezate Sylvia , Vindel Ana , Martín-Dávila Pilar , Del Saz Begoña Sánchez , Baquero Fernando , Cantón Rafael 
Published: February 2004

ABSTRACT

The levels of genetic relatedness of 139
Stenotrophomonas maltophilia
strains recovered from 105 hospitalized non-cystic fibrosis patients (51% from medical wards, 35% from intensive care units, and 14% from surgical wards) and 7 environmental sources in the same hospital setting during a 4-year period were typed by the pulsed-field gel electrophoresis (PFGE) technique. A total of 99 well-defined distinct
Xba
I PFGE patterns were identified (Simpson's discrimination index, 0.996). The dendrogram showed a Dice similarity coefficient ranging from 28 to 80%. Two major clusters (I and II), three minor clusters (III, IV, and V), and two independent branches were observed when using a 36% Dice coefficient, indicating a high diversity of genetic relatedness. It is of note that 84% of strains were grouped within two major clonal lineages. No special cluster gathering was found among strains belonging to the same sample type specimen, patients' infection or colonization status, and ward of precedence. Despite this fact, three different clones (A, B, and C) recovered from respiratory samples from six, three, and two patients, respectively, and two clones, D and E, in two bacteremic patients each, were identified. Isolation of an
S. maltophilia
strain belonging to the clone A profile in a bronchoscope demonstrated a common source from this clone. This study revealed a high genetic diversity of
S. maltophilia
isolates despite their origin from a single hospital, which may be related to the wide environmental distribution of this pathogen. However, few clones could be transmitted among different patients, yielding outbreak situations.
Genotyping of
Mycobacterium leprae
on the Basis of the Polymorphism of TTC Repeats for Analysis of Leprosy Transmission
Authors: Matsuoka Masanori , Zhang Liangfen , Budiawan Teky , Saeki Keisuke , Izumi Shinzo 
Published: February 2004

ABSTRACT

The polymorphism of TTC repeats in
Mycobacterium leprae
was examined using the bacilli obtained from residents in villages at North Maluku where
M. leprae
infections are highly endemic (as well as from patients at North Sulawesi of Indonesia) to elucidate the possible mode of leprosy transmission. TTC genotypes are stable for several generations of passages in nude mice footpads and, hence, are feasible for the genotyping of isolates and epidemiological analysis of leprosy transmission. It was found that bacilli with different TTC genotypes were distributed among residents at the same dwelling in villages in which leprosy is endemic and that some household contacts harbored bacilli with a different genotype from that harbored by the patient. Investigations of a father-and-son pair of patients indicated that infections of bacilli with 10 and 18 copies, respectively, had occurred. Genotypes of TTC repeats were found to differ between a son under treatment and two brothers. These results reveal the possibility that in addition to exposure via the presence of a leprosy patient with a multibacillary infection who was living with family members, there might have been some infectious sources to which the residents had been commonly exposed outside the dwellings. A limited discriminative capacity of the TTC polymorphism in the epidemiological analysis implies the need of searching other useful polymorphic loci for detailed subdivision of clinical isolates.
Hepatitis C Virus (HCV) Genotypes in the Caribbean Island of Martinique: Evidence for a Large Radiation of HCV-2 and for a Recent Introduction from Europe of HCV-4
Authors: Martial Jenny , Morice Yoann , Abel Sylvie , Cabié André , Rat Christelle , Lombard Frédéric , Edouard André , Pierre-Louis Serge , Garsaud Philippe , Béra Odile , Chout Roger , Gordien Emmanuel , Deny Paul , Césaire Raymond 
Published: February 2004

ABSTRACTMolecular epidemiological studies of hepatitis C virus (HCV) in the Caribbean may help to specify the origin and spread of HCV infection. Indeed, the Caribbean population is intermixed from European and African origins and geographically close to the American continent. We characterized HCV genotypes in the Caribbean island of Martinique. HCV genotypes were analyzed by sequencing or reverse hybridization in the 5′ noncoding region (5′NC) in 250 HCV-monoinfected and 85 HCV-human immunodeficiency virus (HIV)-coinfected patients. In addition, sequencing in the nonstructural 5B (NS5B) gene was required to determine the subtype or to perform phylogenetic analysis in selected samples. Genotypes 1 to 6 were found, respectively, in 84.4, 6.8, 5.2, 2.8, 0.4, and 0.4% of 250 HCV-monoinfected patients and in 71.7, 7.1, 15.3, 5.9, 0, and 0% of 85 HCV-HIV-coinfected patients. HCV-1b was found in 66.4% of the HCV-monoinfected patients and was associated with blood transfusion, whereas HCV-1a was detected in 41.2% of the HCV-HIV-coinfected patients and was associated with intravenous drug use (IVDU). The HCV-3 strains belonged to subtype 3a and were linked to IVDU. Phylogenetic analyses were focused on HCV-2 and HCV-4, which are common in Africa. Two opposite patterns were evidenced.NS5Bsequences from 19 HCV-2 isolates were affiliated with many different subtypes described either in Europe or in West Africa, suggesting an ancient radiation. In contrast, seven of the nine HCV-4NS5Bsequences ranged within HCV-4a and HCV-4d clusters spreading in continental France by the IVDU route. Epidemiological data demonstrate the recent introduction of HCV-4a and -4d subtypes into the Caribbean.
spa
Typing Method for Discriminating among
Staphylococcus aureus
Isolates: Implications for Use of a Single Marker To Detect Genetic Micro- and Macrovariation
Authors: Koreen Larry , Ramaswamy Srinivas V. , Graviss Edward A. , Naidich Steven , Musser James M. , Kreiswirth Barry N. 
Published: February 2004

ABSTRACT

Strain typing of microbial pathogens has two major aims: (i) to index genetic microvariation for use in outbreak investigations and (ii) to index genetic macrovariation for use in phylogenetic and population-based analyses. Until now, there has been no clear indication that one genetic marker can efficiently be used for both purposes. Previously, we had shown that DNA sequence analysis of the protein A gene variable repeat region (
spa
typing) provides a rapid and accurate method to discriminate
Staphylococcus aureus
outbreak isolates from those deemed epidemiologically unrelated. Here, using the hypothesis that the genetic macrovariation within a low-level recombinogenic species would accurately be characterized by a single-locus marker, we tested whether
spa
typing could congruently index the extensive genetic variation detected by a whole-genome DNA microarray in a collection of 36 isolates, which was recovered from 10 countries on four continents over a period of four decades, that is representative of the breadth of diversity within
S. aureus.
Using
spa
and
coa
typing, pulsed-field gel electrophoresis (PFGE), and microarray and multilocus enzyme electrophoresis (MLEE) data in molecular epidemiologic and evolutionary analyses, we determined that
S. aureus
likely has a primarily clonal population structure and that
spa
typing can singly index genetic variation with 88% direct concordance with the microarray and can correctly assign isolates to phylogenetic lineages.
spa
typing performed better than MLEE, PFGE, and
coa
typing in discriminatory power and in the degree of agreement with the microarray at various phylogenetic depths. This study showed that genetic analysis of the repeat region of protein A comprehensively characterizes both micro- and macrovariation in the primarily clonal population structure of
S. aureus
.
Comparison of LCx with Other Current Viral Load Assays for Detecting and Quantifying Human Immunodeficiency Virus Type 1 RNA in Patients Infected with the Circulating Recombinant Form A/G (CRF02)
Authors: Amendola Alessandra , Bordi Licia , Angeletti Claudio , Girardi Enrico , Ippolito Giuseppe , Capobianchi Maria R. 
Published: February 2004

ABSTRACT

LCx was compared to other assays in measuring human immunodeficiency virus type 1 (HIV-1) CRF02 viremia. LCx showed significant but low correlation with the other methods. Values of <2.60 log
10
cp/ml were observed in 29.6% of specimens with LCx and in only 14.8% with bDNA and PCR, suggesting suboptimal performance of LCx with CRF02.