bluuebunny/crossref_metadata_2025_split huggingface dataset:
Evaluation of Laboratory Testing Methods for
Chlamydia trachomatis
Infection in the Era of Nucleic Acid Amplification
Authors: Battle Tamara J. , Golden Matthew R. , Suchland Kathleen L. , Counts Jon M. , Hughes James P. , Stamm Walter E. , Holmes King K. 
Published: July 2002

ABSTRACT

Diagnostic tests presently available for
Chlamydia trachomatis
have widely varying performance characteristics. To assess evolving laboratory testing practices since the introduction of nucleic acid amplification tests (NAAT), we surveyed laboratories in Washington State about their testing practices in 1998 and compared our findings to a similar survey conducted in 1995. Laboratory directors of 61 (87%) of 70 laboratories performing chlamydial tests in 1998 returned a survey. Between 1995 and 1998, 36 laboratories discontinued chlamydial testing, and the total number of laboratories performing tests in the state decreased from 92 to 70, a 24% decline. Of the 36 laboratories that discontinued testing, 25 (69%) had previously used rapid tests. While no laboratory routinely used NAAT in 1995, ligase chain reaction (LCR) was used in 23% of laboratories in 1998 and accounted for 113,624 (36%) of the 318,133 tests performed that year. Among the remaining 204,509 tests performed in 1998, other tests employed included DNA probe (29%), enzyme immunoassay (20%), culture (12%), direct fluorescent antibody assays (3%), and rapid tests (<1%). The majority (65%) of tests performed in 1998 using technologies other than LCR or culture were done in laboratories that did more than 10,000 tests. Cost and loss of revenue to laboratories were the most frequently cited reasons for not adopting NAAT. We conclude that in Washington State, NAAT have been rapidly adopted in larger laboratories, but most patients are still tested with much less sensitive technologies. Financial constraints represent the major barrier to more widespread use of DNA amplification tests.
Distribution of Clinically RelevantBorreliaGenospecies in Ticks Assessed by a Novel, Single-Run, Real-Time PCR
Authors: Rauter Carolin , Oehme Rainer , Diterich Isabel , Engele Matthias , Hartung Thomas 
Published: July 2002

ABSTRACTA LightCycler-based PCR protocol was developed which targets theospAgene for the identification and quantification of the differentBorrelia burgdorferisensu lato species in culture and in ticks, based on the use of a fluorescently labeled probe (HybProbe) and an internally labeled primer. The detection limit of the PCR was 1 to 10 spirochetes. A melting temperature determined from the melting curve of the amplified product immediately after thermal cycling allowed the differentiation of the three differentB. burgdorferisensu lato genospecies (B. burgdorferisensu stricto,Borrelia garinii, andBorrelia afzelii) that are clinically relevant in Europe in a single PCR run. This method represents a simplified approach to study the association of differentBorreliaspecies in ticks, the risk of Lyme borreliosis, and the putatively species-specific clinical sequelae. To determine the reliability of the real-time PCR protocol, we studied the prevalence ofB. burgdorferisensu lato infection inIxodes ricinusticks. A total of 1,055 ticks were collected by flagging vegetation in five different sites in the region of Konstanz (south Germany) and were examined for the distribution ofB. burgdorferispecies by real-time PCR. The mean infection rate was 35%. Of 548 adult ticks, 40% were positive, and of 507 nymphs, 30% were positive. The predominant genospecies (with 18% mixed infections) in the examined areas wasB. afzelii(53%), followed byB. garinii(18%) andB. burgdorferisensu stricto (11%); 0.8% of the infectingBorreliacould not be identified.
Identification of the
Brucella melitensis
Vaccine Strain Rev.1 in Animals and Humans in Israel by PCR Analysis of the
Pst
I Site Polymorphism of Its
omp2
Gene
Authors: Bardenstein Svetlana , Mandelboim Michal , Ficht Thomas A. , Baum Miriam , Banai Menachem 
Published: July 2002

ABSTRACT

Adverse effects of strain persistence and secretion in milk have been encountered with the
Brucella melitensis
vaccine strain Rev.1. Field isolates obtained from vaccinated animals and from a human resembled the vaccine strain Rev.1 by conventional bacteriological tests. The lack of a specific molecular marker that could specifically characterize the commercial vaccine strain prevented confirmation of the homology of the Rev.1-like field isolates to the vaccine strain. The composition of the
omp2
locus from two gene copies with differences in their
Pst
I restriction endonuclease sites was used to establish an epidemiologic fingerprint for the
omp2
gene in the Rev.1 vaccine strain. Primers designed to amplify DNA sequences that overlap the
Pst
I site revealed a single 282-bp DNA band common to all
Brucella
spp. Agarose gel electrophoresis of the
Pst
I digests of the PCR products from strains 16M and the vaccine strain Rev.1 revealed a distinctive profile that included three bands: one band for the intact 282-bp fragment amplified from
omp2a
and two bands resulting from the digestion of the amplified
omp2b
gene fragment, 238- and 44-bp DNA fragments, respectively. Amplified fragments of 37 Rev.1-like isolates, including 2 human isolates, also exhibited this pattern. In contrast, DNA digests of all other Israeli field isolates, including atypical
B. melitensis
biotype 1 and representatives of the biotype 2 and 3 isolates, produced two bands of 238 and 44 bp, respectively, corresponding with the digestion of both
omp2a
and
omp2b
genes. This method facilitates identification of the Rev.1 vaccine strain in both animals and humans in Israel.
Roleof Picornaviruses in Flu-Like Illnesses of Adults Enrolled in anOseltamivir Treatment Study Who Had No Evidence of Influenza VirusInfection
Authors: Boivin Guy , Osterhaus Albert D. , Gaudreau Annie , Jackson Helen C. , Groen Jan , Ward Penelope 
Published: July 2002

ABSTRACT

The primary
objective of this study was to determine the role of picornavirus in
flu-like episodes (temperature of ≥38.0°C plus one respiratory and
one constitutional symptom) among otherwise healthy adults enrolled in a
placebo-controlled, double-blind, randomized oseltamivir treatment study.
Combined nasal and pharyngeal swabs were collected at baseline for
influenza cultures and picornavirus reverse transcription (RT)-PCR. In
addition, acute- and convalescent-serum samples were obtained for
serological studies of common respiratory pathogens. From a total of 719
subjects enrolled in the clinical trial within 36 h of the onset of
symptoms, 475 (66%) had evidence of recent influenza A or B virus
infections by means of culture and/or serological testing. Of the 244
remaining patients, 36 (15%) presented a seroconversion for at least one of
the common respiratory viruses or atypical pathogens. An RT-PCR assay for
the picornavirus 5" noncoding region (NCR) was positive in a subset of
15 (19%) of 78 patients with flu-like illnesses of undetermined etiology.
Sequence analysis of the picornavirus 5" NCR amplicons revealed that
14 (93%) of them had greater homology to rhinoviruses, whereas 1 (7%) was
related to enteroviruses. Interestingly, median total symptom scores and
oral temperatures of picornavirus-positive patients (
n
=
15) and placebo-treated influenza virus-positive patients (
n
=
161) were similar over a 3-week period. We conclude that, among
the influenza virus-negative preselected cases of this study, rhinoviruses
were relatively frequent pathogens associated with important respiratory
and systemic symptoms.
Rapid Detection of Pyrazinamide-Resistant
Mycobacterium tuberculosis
by a PCR-Based In Vitro System
Authors: Suzuki Yasuhiko , Suzuki Aya , Tamaru Aki , Katsukawa Chihiro , Oda Hajime 
Published: July 2002

ABSTRACT

Pyrazinamide (PZA), an analog of nicotinamide, is a prodrug for tuberculosis which requires conversion to the bactericidal compound pyrazinoic acid by bacterial pyrazinamidase activity. Mutations leading to a loss of pyrazinamidase activity cause PZA resistance in
Mycobacterium tuberculosis
. Thus, the detection of pyrazinamidase activity makes the discrimination of PZA-resistant tuberculosis possible. However, the detection of the pyrazinamidase activity of
M. tuberculosis
isolates needs a large amount of bacilli and is therefore time consuming. In this paper, we describe a new method for the detection of pyrazinamidase activity with a PCR-based system. The genes encoding pyrazinamidase (
pncA
genes) in 30 resistant clinical isolates were amplified by PCR by using forward primers containing bacteriophage T7 promoter sequences at their 5" ends. Then the PCR products were directly subjected to an in vitro transcription-translation coupled system. All of the PZA-resistant isolates tested showed reduced pyrazinamidase activity compared to susceptible
M. tuberculosis
type strain H37Rv. In contrast, all of the 15 susceptible clinical isolates exhibited pyrazinamidase activities similar to that of H37Rv. This fact suggested the possibility of the usefulness of this system for the rapid detection of PZA-resistant
M. tuberculosis
.
Macrolide Efflux Genes
mef
(A) and
mef
(E) Are Carried by Different Genetic Elements in
Streptococcus pneumoniae
Authors: Del Grosso M. , Iannelli F. , Messina C. , Santagati M. , Petrosillo N. , Stefani S. , Pozzi G. , Pantosti A. 
Published: July 2002

ABSTRACT

Susceptibilities to macrolides were evaluated in 267
Streptococcus pneumoniae
isolates, of which 182 were from patients with invasive diseases and 85 were from healthy carriers. Of the 98 resistant isolates, 20 strains showed an M phenotype and carried
mef
. Strains that carried both
mef
(A) and
mef
(E) were found: 17 strains carried
mef
(A) and 3 carried
mef
(E). The characteristics of the strains carrying the
mef
genes and the properties of the
mef-
containing elements were studied. Strains carrying
mef
(A) belonged to serotype 14, were susceptible to all the antibiotics tested except erythromycin, and appeared to be clonally related by pulsed-field gel electrophoresis (PFGE). The three
mef
(E) strains belonged to different serotypes, showed different susceptibility profiles, and did not appear to be related by PFGE. The sequences of a fragment of the
mef-
containing element, which encompassed
mef
and the
msr
(A) homolog, were identical among the three
mef
(E)-positive strains and among the three
mef
(A)-positive strains, although there were differences between the sequences for the two variants at 168 positions. In all
mef
(A)-positive strains, the
mef
element was inserted in
celB
, which led to impairment of the competence of the strains. In line with insertion of the
mef
(E) element at a different site, the competence of the
mef
(E)-positive strains was maintained. Transfer of erythromycin resistance by conjugation was obtained from two of three
mef
(A) strains but from none of three
mef
(E) strains. Due to the important different characteristics of the strains carrying
mef
(A) or
mef
(E), we suggest that the distinction between the two genes be maintained.
Extended-Spectrum β-Lactamase-ProducingKlebsiellapneumoniaein a Neonatal Intensive Care Unit in the High-Prevalence Area of Athens, Greece
Authors: Lebessi E. , Dellagrammaticas H. , Tassios P. T. , Tzouvelekis L. S. , Ioannidou S. , Foustoukou M. , Legakis N. J. 
Published: July 2002

ABSTRACTExtended-spectrum β-lactamase-producingKlebsiellapneumoniae(EPKP) strains are frequently implicated in outbreaks in neonatal intensive care units (NICUs). During the period from 1997 to 1998, 21 infections and 23 colonizations with EPKP were recorded in the NICU of a children's hospital in Athens, Greece. Seventeen of the infected and 12 of the colonized neonates had been referred from other hospitals. The remaining infections and colonizations occurred during the current hospitalization. Pulsed-field gel electrophoresis typing showed that the latter cases were due to an outbreak strain that persisted in the unit, while the repeated introduction of EPKP carriers was mostly due to clonal outbreaks in two maternity hospitals.
Quantitative Detection ofNeospora caninumin Bovine Aborted Fetuses and Experimentally Infected Mice by Real-Time PCR
Authors: Collantes-Fernández Esther , Zaballos Ángel , Álvarez-García Gema , Ortega-Mora Luis M. 
Published: July 2002

ABSTRACTWe report the development of a real-time PCR assay for the quantitative detection ofNeospora caninumin infected host tissues. The assay uses the double-stranded DNA-binding dye SYBR Green I to continuously monitor product formation. Oligonucleotide primers were designed to amplify a 76-bp DNA fragment corresponding to the Nc5 sequence ofN. caninum. A similar method was developed to quantify the 28S rRNA host gene in order to compare the parasite load of different samples and to correct for the presence of potential PCR-inhibiting compounds in the DNA samples. A linear quantitative detection range of 6 logs with a calculated detection limit of 10−1tachyzoite per assay was observed with excellent linearity (R2= 0.998). Assay specificity was confirmed by using DNA from the closely related parasiteToxoplasma gondii. The applicability of the technique was successfully tested in a variety of host brain tissues: (i) aborted bovine fetuses classified into negative or positiveNeospora-infected animals according to the observation of compatible lesions by histopathological study and (ii) experimentally infected BALB/c mice, divided into three groups, inoculated animals with or without compatible lesions and negative controls. All samples were also tested by ITS1Neosporanested PCR and a high degree of agreement was shown between both PCR techniques (κ= 0.86). This technique represents a useful quantitative diagnostic tool to be used in the study of the pathogenicity, immunoprophylaxis, and treatment ofNeosporainfection.
Epidemiology of Extended-Spectrum β-Lactamase-Producing
Enterobacter
Isolates in a Spanish Hospital during a 12-Year Period
Authors: Cantón Rafael , Oliver Antonio , Coque Teresa M. , Varela María del Carmen , Pérez-Díaz José Claudio , Baquero Fernando 
Published: July 2002

ABSTRACT

Fifteen
Enterobacter
clinical isolates (11
Enterobacter cloacae
isolates, 3
Enterobacter aerogenes
isolates, and 1
Enterobacter gergoviae
isolate), representing 0.4% of all
Enterobacter
isolates recovered in our hospital from 1989 to 2000, were suspected of harboring an extended-spectrum β-lactamase (ESBL). These isolates were recovered from 14 different patients. ESBLs were transferred by conjugation into an
Escherichia coli
recipient strain. Pulsed-field gel electrophoresis (PFGE) revealed a single clone of
E. aerogenes
and six different clones of
E. cloacae
. Four of these
E. cloacae
clonal types were represented by only one isolate each, but the other two were represented by three and four isolates, respectively. Isoelectric focusing, susceptibility phenotyping, PCR analysis, and sequencing demonstrated the presence of three different ESBLs. The most frequent was the recently characterized CTX-M-10 ESBL, which was found in the
E. gergoviae
isolate and in all but one of the
E. cloacae
isolates. The remaining
E. cloacae
isolate harbored a TEM-27 ESBL, and the three
E. aerogenes
isolates harbored a TEM-24 ESBL. PFGE revealed that our
E. aerogenes
strain was indistinguishable from the French TEM-24-producing
E. aerogenes
endemic clone. Although a low prevalence of ESBL-producing
Enterobacter
isolates was found in our institution over a 12-year period, a diversity of nonepidemic
E. cloacae
clones was detected, as was the persistence of the CTX-M-10 β-lactamase. The presence of the TEM-24-producing
E. aerogenes
French clone in our institution also demonstrates the intercountry dissemination of ESBL-producing isolates.
Emerging Extended-Spectrum β-Lactamases in
Proteus mirabilis
Authors: Pagani Laura , Migliavacca Roberta , Pallecchi Lucia , Matti Cecilia , Giacobone Ernesto , Amicosante Gianfranco , Romero Egidio , Rossolini Gian Maria 
Published: July 2002

ABSTRACT

β-Lactamase production was detected in 147 (52%) of 282 consecutive nonduplicate
Proteus mirabilis
isolates obtained over a 1-year period from the S. Matteo Hospital of Pavia (northern Italy). Seventy isolates (48% of the β-lactamase producers) were found to produce extended-spectrum β-lactamases (ESBLs), identified as PER-1 (first report in this species) and TEM-52 in 52 and 18 isolates, respectively. Analysis of clonal diversity of the ESBL producers suggested different spreading patterns for the two ESBL determinants.
Molecular Epidemiology of Subgroup C Avian Pneumoviruses Isolated in the United States and Comparison with Subgroup A and B Viruses
Authors: Shin Hyun-Jin , Cameron Kjerstin T. , Jacobs Janet A. , Turpin Elizabeth A. , Halvorson David A. , Goyal Sagar M. , Nagaraja Kakambi V. , Kumar Mahesh C. , Lauer Dale C. , Seal Bruce S. , Njenga M. Kariuki 
Published: July 2002

ABSTRACT
The avian pneumovirus (APV) outbreak in the United States is concentrated in the north-central region, particularly in Minnesota, where more outbreaks in commercial turkeys occur in the spring (April to May) and autumn (October to December). Comparison of the nucleotide and amino acid sequences of nucleoprotein (N), phosphoprotein (P), matrix (M), fusion (F), and second matrix (M2) genes of 15 U.S. APV strains isolated between 1996 and 1999 revealed between 89 and 94% nucleotide sequence identity and 81 to 95% amino acid sequence identity. In contrast, genes from U.S. viruses had 41 to 77% nucleotide sequence identity and 52 to 78% predicted amino acid sequence identity with European subgroup A or B viruses, confirming that U.S. viruses belonged to a separate subgroup. Of the five proteins analyzed in U.S. viruses, P was the most variable (81% amino acid sequence identity) and N was the most conserved (95% amino acid sequence identity). Phylogenetic comparison of subgroups A, B, and C viruses indicated that A and B viruses were more closely related to each other than either A or B viruses were to C viruses.
Characterization of Influenza Virus NS1 Protein by Using a Novel Helper-Virus-Free Reverse Genetic System
Authors: Enami Masayoshi , Enami Kazue 
Published: July 2002

ABSTRACT

We have developed a novel helper-virus-free reverse genetic system to genetically manipulate influenza A viruses. The RNPs, which were purified from the influenza A/WSN/33 (WSN) virus, were treated with RNase H in the presence of NS (nonstructural) cDNA fragments. This specifically digested the NS RNP. The NS-digested RNPs thus obtained were transfected into cells together with the in vitro-reconstituted NS RNP. The NS-digested RNPs alone did not rescue viruses; however, cotransfection with the NS RNP did. This protocol was also used to rescue the NP transfectant. We obtained two NS1 mutants,
dl
12 and N110, using this protocol. The
dl
12 NS gene contains a deletion of 12 amino acids at positions 66 to 77 near the N terminus. This virus was temperature sensitive in Madin-Darby bovine kidney (MDBK) cells as well as in Vero cells. The translation of all viral proteins as well as cellular proteins was significantly disrupted during a later time of infection at the nonpermissive temperature of 39°C. The N110 mutant consists of 110 amino acids which are the N-terminal 48% of the WSN virus NS1 protein. Growth of this virus was significantly reduced at any temperature. In the virus-infected cells, translation of the M1 protein was reduced to 10 to 20% of that of the wild-type virus; however, the translation of neither the nucleoprotein nor NS1 was significantly interfered with, indicating the important role of NS1 in translational stimulation of the M1 protein.
The Core of the Respiratory Syncytial Virus Fusion Protein Is a Trimeric Coiled Coil
Authors: Matthews Jacqueline M. , Young Thomas F. , Tucker Simon P. , Mackay Joel P. 
Published: July 2002

ABSTRACT

Entry into the host cell by enveloped viruses is mediated by fusion (F) or transmembrane glycoproteins. Many of these proteins share a fold comprising a trimer of antiparallel coiled-coil heterodimers, where the heterodimers are formed by two discontinuous heptad repeat motifs within the proteolytically processed chain. The F protein of human respiratory syncytial virus (RSV; the major cause of lower respiratory tract infections in infants) contains two corresponding regions that are predicted to form coiled coils (HR1 and HR2), together with a third predicted heptad repeat (HR3) located in a nonhomologous position. In order to probe the structures of these three domains and ascertain the nature of the interactions between them, we have studied the isolated HR1, HR2, and HR3 domains of RSV F by using a range of biophysical techniques, including circular dichroism, nuclear magnetic resonance spectroscopy, and sedimentation equilibrium. HR1 forms a symmetrical, trimeric coiled coil in solution (
K
3
≈ 2.2 × 10
11
M
−2
) which interacts with HR2 to form a 3:3 hexamer. The HR1-HR2 interaction domains have been mapped using limited proteolysis, reversed-phase high-performance liquid chromatography, and electrospray-mass spectrometry. HR2 in isolation exists as a largely unstructured monomer, although it exhibits a tendency to form aggregates with β-sheet-like characteristics. Only a small increase in α-helical content was observed upon the formation of the hexamer. This suggests that the RSV F glycoprotein contains a domain that closely resembles the core structure of the simian parainfluenza virus 5 fusion protein (K. A. Baker, R. E. Dutch, R. A. Lamb, and T. S. Jardetzky, Mol. Cell 3:309–319, 1999). Finally, HR3 forms weak α-helical homodimers that do not appear to interact with HR1, HR2, or the HR1-HR2 complex. The results of these studies support the idea that viral fusion proteins have a common core architecture.
An Enzymatic Footprinting Analysis of the Interaction of 40S Ribosomal Subunits with the Internal Ribosomal Entry Site of Hepatitis C Virus
Authors: Kolupaeva Victoria G. , Pestova Tatyana V. , Hellen Christopher U. T. 
Published: July 2002

ABSTRACT

Hepatitis C virus translation is initiated on a ∼330-nucleotide (nt)-long internal ribosomal entry site (IRES) at the 5′ end of the genome. In this process, a 43S preinitiation complex (comprising a 40S ribosomal subunit, eukaryotic initiation factor 3 (eIF3), and a ternary [eIF2-GTP-initiator tRNA] complex) binds the IRES in a precise manner so that the initiation codon is placed at the ribosomal P site. This binding step involves specific interactions between the IRES and different components of the 43S complex. The 40S subunit and eIF3 can bind to the IRES independently; previous analyses revealed that eIF3 binds specifically to an apical half of IRES domain III. Nucleotides in the IRES that are involved in the interaction with the 40S subunit were identified by RNase footprinting and mapped to the basal half of domain III and in domain IV. Interaction sites were identified in locations that have been found to be essential for IRES function, including (i) the apical loop residues GGG
266-268
in subdomain IIId and (ii) the pseudoknot. Extensive protection from RNase cleavage also occurred downstream of the pseudoknot in domain IV, flanking both sides of the initiation codon and corresponding in length to that of the mRNA-binding cleft of the 40S subunit. These results indicate that the 40S subunit makes multiple interactions with the IRES and suggest that only nucleotides in domain IV are inserted into the mRNA-binding cleft of the 40S subunit.