bluuebunny/crossref_metadata_2025_split huggingface dataset:
Controlled Clinical Comparison of BACTEC Plus Anaerobic/F to Standard Anaerobic/F as the Anaerobic Companion Bottle to Plus Aerobic/F Medium for Culturing Blood from Adults
Authors: Wilson Michael L. , Mirrett Stanley , Meredith Frances T. , Weinstein Melvin P. , Scotto Vincenzo , Reller L. Barth 
Published: July 2002

ABSTRACT

To determine the optimal anaerobic companion bottle to pair with BACTEC Plus Aerobic/F medium for recovery of pathogenic microorganisms from adult patients with bacteremia and fungemia, we compared Plus Anaerobic/F bottles with Standard Anaerobic/F bottles, each of which was filled with 4 to 6 ml of blood. The two bottles were paired with a Plus Aerobic/F bottle filled with 8 to 12 ml of blood. A total of 14,011 blood culture sets were obtained. Of these, 11,583 sets were received with all three bottles filled adequately and 12,257 were received with both anaerobic bottles filled adequately. Of 818 clinically important isolates detected in one or both adequately filled anaerobic bottles, significantly more staphylococci (
P
< 0.001), streptococci (
P
< 0.005),
Escherichia coli
isolates (
P
< 0.02),
Klebsiella pneumoniae
isolates (
P
< 0.005), and all microorganisms combined (
P
< 0.001) were detected in Plus Anaerobic/F bottles. In contrast, significantly more anaerobic gram-negative bacilli were detected in Standard Anaerobic/F bottles (
P
< 0.05). Of 397 unimicrobial episodes of septicemia, 354 were detected with both pairs, 30 were detected with Plus Aerobic/F–Plus Anaerobic/F pairs only, and 13 were detected with Plus Aerobic/F–Standard Anaerobic/F pairs only (
P
< 0.05). Significantly more episodes of bacteremia caused by members of the family
Enterobacteriaceae
(
P
< 0.05) and aerobic and facultative gram-positive bacteria (
P
< 0.025) were detected with Plus Anaerobic/F bottles only. In a paired-bottle analysis, 810 of 950 isolates were recovered from both pairs, 90 were recovered from Plus Aerobic/F–Plus Anaerobic/F pairs only, and 50 were recovered from Plus Aerobic/F–Standard Anaerobic/F pairs only (
P
< 0.001). Paired Plus Aerobic/F–Plus Anaerobic/F bottles yielded significantly more staphylococci (
P
< 0.001), streptococci (
P
< 0.05), and members of the family
Enterobacteriaceae
(
P
<0.001). We conclude that Plus Anaerobic/F bottles detect more microorganisms and episodes of bacteremia and fungemia than Standard Anaerobic/F bottles as companion bottles to Plus Aerobic/F bottles in the BACTEC 9240 blood culture system.
Nested Restriction Site-Specific PCR To Detect and Type Hepatitis C Virus (HCV): a Rapid Method To Distinguish HCV Subtype 1b from Other Genotypes
Authors: Krekulova Laura , Rehak Vratislav , Wakil Adil E. , Harris Eva , Riley Lee W. 
Published: July 2002

ABSTRACT
Genotypic differentiation of hepatitis C virus (HCV) has become an integral part of clinical management and epidemiologic studies of hepatitis C infections. Thus, it is extremely important in areas such as the Czech Republic, where current instrumentation and kits for assessing HCV infection are too costly for widespread use. We describe a new and relatively inexpensive method called nested restriction site-specific PCR (RSS-PCR) that generates a “fingerprint” pattern to represent an HCV genotype without the use of restriction endonucleases and that specifically differentiates HCV genotype 1b from the other HCV genotypes. The RSS-PCR method was applied directly to serum samples from patients with hepatitis C from the Czech Republic and from patients with known HCV genotypes from the United States. The method was validated by comparison of the subtype determined by RSS-PCR to the subtype determined from analysis of the 5′ noncoding region (NC) or the nonstructural protein gene (NS5b) nucleotide sequence of HCV in these clinical samples. From 75 Czech samples containing HCV RNA, three distinct RSS-PCR patterns were observed; 54 were predicted to contain subtype 1b, 19 were predicted to contain subtype 1a, and 2 were predicted to contain subtype 3a. Among 54 samples predicted to contain HCV genotype 1b, all were confirmed by their 5′ NC or NS5b sequences to be subtype 1b. Thus, both the sensitivity and specificity of the RSS-PCR test for the differentiation of HCV subtype 1b from the others were 100%. While the assay described here was designed to specifically differentiate HCV subtype 1b from the other HCV genotypes, the RSS-PCR method can be modified to differentiate any HCV genotype or subtype of interest. Its simplicity and speed may provide new opportunities to study the epidemiology of HCV infections and the relationship between HCV genotypes and clinical outcome by more laboratories throughout the world.
Practical Strategies for Detecting and Confirming Vancomycin-Intermediate
Staphylococcus aureus
: a Tertiary-Care Hospital Laboratory's Experience
Authors: Marlowe Elizabeth M. , Cohen Martin D. , Hindler Janet F. , Ward Kevin W. , Bruckner David A. 
Published: July 2002

ABSTRACT

The clinical microbiology laboratory plays a critical role in the detection of
Staphylococcus aureus
with decreased susceptibility to vancomycin. Staff education and rapid laboratory response are of utmost importance. We report on our laboratory's experience and provide recommendations for the identification and confirmation of vancomycin-intermediate
S. aureus
.
Fluoroquinolone Resistance Is a Poor Surrogate Marker for Type II Topoisomerase Mutations in Clinical Isolates of
Streptococcus pneumoniae
Authors: Millichap John J. , Pestova Ekaterina , Siddiqui Farida , Noskin Gary A. , Peterson Lance R. 
Published: July 2002

ABSTRACT

The association between fluoroquinolone susceptibility and DNA mutations coding for amino acid substitutions in the quinolone resistance-determining region was assessed with 44 clinical isolates of
Streptococcus pneumoniae
. Twenty-three strains bore at least one amino acid substitution. Only seven strains with mutations were suggested by diminished susceptibility to ciprofloxacin (MIC, ≥2 μg/ml).
Detection and Identification of Fungal Pathogens by PCR and by ITS2 and 5.8S Ribosomal DNA Typing in Ocular Infections
Authors: Ferrer Consuelo , Colom Francisca , Frasés Susana , Mulet Emilia , Abad José L. , Alió Jorge L. 
Published: July 2002

ABSTRACT
The goal of this study was to determine whether sequence analysis of internal transcribed spacer/5.8S ribosomal DNA (rDNA) can be used to detect fungal pathogens in patients with ocular infections (endophthalmitis and keratitis). Internal transcribed spacer 1 (ITS1) and ITS2 and 5.8S rDNA were amplified by PCR and seminested PCR to detect fungal DNA. Fifty strains of 12 fungal species (yeasts and molds) were used to test the selected primers and conditions of the PCR. PCR and seminested PCR of this region were carried out to evaluate the sensitivity and specificity of the method. It proved possible to amplify the ITS2/5.8S region of all the fungal strains by this PCR method. All negative controls (human and bacterial DNA) were PCR negative. The sensitivity of the seminested PCR amplification reaction by DNA dilutions was 1 organism per PCR, and the sensitivity by cell dilutions was fewer than 10 organisms per PCR. Intraocular sampling or corneal scraping was undertaken for all patients with suspected infectious endophthalmitis or keratitis (nonherpetic), respectively, between November 1999 and February 2001. PCRs were subsequently performed with 11 ocular samples. The amplified DNA was sequenced, and aligned against sequences in GenBank at the National Institutes of Health. The results were PCR positive for fungal primers for three corneal scrapings, one aqueous sample, and one vitreous sample; one of them was negative by culture. Molecular fungal identification was successful in all cases. Bacterial detection by PCR was positive for three aqueous samples and one vitreous sample; one of these was negative by culture. Amplification of ITS2/5.8S rDNA and molecular typing shows potential as a rapid technique for identifying fungi in ocular samples.
Identification of the Hexon Region of an Adenovirus Involved in a New Outbreak of Keratoconjunctivitis
Authors: Imai Yasuo , Kameya Shuhei , Ohkoshi Masatoshi , Yamaki Kunihiko , Sakuragi Shozo 
Published: July 2002

ABSTRACT
We tested 15 adenovirus (Ad)-positive patients involved in a case of nosocomial spread of keratoconjunctivitis. A neutralization test, PCR-restriction fragment length polymorphism analysis, and sequencing of the hypervariable regions of the hexons were performed in order to identify the type of Ad involved. The serotype of the Ad was not identical to any published Ad sequence by either method.
Simplified Microneutralization Test for Serotyping Adenovirus Isolates
Authors: Malasig Marietta D. , Goswami Pulak R. , Crawford-Miksza Leta K. , Schnurr David P. , Gray Gregory C. 
Published: July 2002

ABSTRACT
A simplified microneutralization procedure is described that uses an empirically determined virus challenge dose, a single dilution of antiserum, and observation of cytopathic effect to determine the adenovirus serotype. The simplified test has faster turnaround time and was 96% concordant with a confirmatory test using serial dilutions of type-specific sera. This method will find utility in high-volume serotyping work.
Fatal Pulmonary
Nocardia farcinica
Infection
Authors: De La Iglesia Pedro , Viejo Guillermo , Gomez Beatriz , De Miguel Dolores , Del Valle Asuncion , Otero Luis 
Published: July 2002

ABSTRACT

Nocardia farcinica
infections are rare and potentially life threatening. Identification is based on growth at 45°C, opacification of Middlebrook 7H10 agar, and resistance to antibiotics. We describe a case of fatal pulmonary
N. farcinica
infection in a patient with pneumoconiosis that was diagnosed by culture of sputum onto selective media.
One-Point Population Analysis and Effect of Osmolarity on Detection of Hetero-Vancomycin-Resistant
Staphylococcus aureus
Authors: Jung Sook-In , Kiem Sungmin , Lee Nam Yong , Kim Yeon-Sook , Oh Won Sup , Cho Hwang Lae , Peck Kyong Ran , Song Jae-Hoon 
Published: July 2002

ABSTRACT

The original screening test and one-point population analysis (OPPA) were compared for the detection of hetero-vancomycin-resistant
Staphylococcus aureus
, and the influence of osmolarity on those tests was evaluated. The positivity rates and the reproducibilities were similar for both tests. The addition of NaCl increased the MIC as well as the positivity rates for both tests and the reproducibility of OPPA.
Genetic Analysis of a Poliovirus/Hepatitis C Virus (HCV) Chimera: Interaction between the Poliovirus Cloverleaf and a Sequence in the HCV 5′ Nontranslated Region Results in a Replication Phenotype
Authors: Zhao Wei Dong , Lahser Frederick C. , Wimmer Eckard 
Published: July 2002

ABSTRACT
Internal ribosomal entry sites (IRESs) can function in foreign viral genomes or in artificial dicistronic mRNAs. We describe an interaction between the wild-type hepatitis C virus (HCV)-specific sequence and the poliovirus (PV) 5′-terminal cloverleaf in a PV/HCV chimeric virus (containing the HCV IRES), resulting in a replication phenotype. Either a point mutation at nucleotide (nt) 29 or a deletion up to nt 40 in the HCV 5′ nontranslated region relieved the replication block, yielding PV/HCV variants replicating to high titers. Fortuitous yet crippling interactions between an IRES and surrounding heterologous RNA must be considered when IRES-based dicistronic expression vectors are being constructed.
Functional Characterization of the Dimer Linkage Structure RNA of Moloney Murine Sarcoma Virus
Authors: Ly Hinh , Nierlich Donald P. , Olsen John C. , Kaplan Andrew H. 
Published: July 2002

ABSTRACT
Several determinants that appear to promote the dimerization of murine retroviral genomic RNA have been identified. The interaction between these determinants has not been extensively examined. Previously, we proposed that dimerization of the Moloney murine sarcoma virus genomic RNAs relies upon the concentration-dependent interactions of a conserved palindrome that is initiated by separate G-rich stretches (H. Ly, D. P. Nierlich, J. C. Olsen, and A. H. Kaplan, J. Virol. 73:7255–7261, 1999). The cooperative action of these two elements was examined using a combination of genetic and antisense approaches. Dimerization of RNA molecules carrying both the palindrome and G-rich sequences was completely inhibited by an oligonucleotide complementary to the palindrome; molecules lacking the palindrome could not dimerize in the presence of oligomers that hybridize to two G-rich sequences. The results of spontaneous dimerization experiments also demonstrated that RNA molecules lacking either of the two stretches of guanines dimerized much more slowly than the full-length molecule which includes the dimer linkage structure (DLS). However, the addition of an oligonucleotide complementary to the remaining stretch of guanines restored the kinetics of dimerization to wild-type levels. The ability of this oligomer to rescue the kinetics of dimerization was dependent on the presence of the palindrome, suggesting that interactions within the G-rich regions produce changes in the palindrome that allow dimerization to proceed with maximum efficiency. Further, unsuccessful attempts to produce heterodimers between constructs lacking various combinations of these elements indicate that the G-rich regions and the palindrome do not interact directly. Finally, we demonstrate that both of these elements are important in maintaining efficient viral replication. Modified antisense oligonucleotides targeting the DLS were found to reduce the level of viral vector titer production. The reduction in viral titer is due to a decrease in the efficiency of viral genomic RNA encapsidation. Overall, our data support a dynamic model of retroviral RNA dimerization in which discrete dimerization elements act in a concerted fashion.
Inhibition of PKR Activation by the Proline-Rich RNA Binding Domain of the Herpes Simplex Virus Type 1 Us11 Protein
Authors: Poppers Jeremy , Mulvey Matthew , Khoo David , Mohr Ian 
Published: July 2002

ABSTRACT
Upon activation by double-stranded RNA in virus-infected cells, the cellular PKR kinase phosphorylates the translation initiation factor eukaryotic initiation factor 2 (eIF2) and thereby inhibits protein synthesis. The γ34.5 and Us11 gene products encoded by herpes simplex virus type 1 (HSV-1) are dedicated to preventing the accumulation of phosphorylated eIF2. While the γ34.5 gene specifies a regulatory subunit for protein phosphatase 1α, the Us11 gene encodes an RNA binding protein that also prevents PKR activation. γ34.5 mutants fail to grow on a variety of human cells as phosphorylated eIF2 accumulates and protein synthesis ceases prior to the completion of the viral life cycle. We demonstrate that expression of a 68-amino-acid fragment of Us11 containing a novel proline-rich basic RNA binding domain allows for sustained protein synthesis and enhanced growth of γ34.5 mutants. Furthermore, this fragment is sufficient to inhibit activation of the cellular PKR kinase in a cell-free system, suggesting that the intrinsic activities of this small fragment, notably RNA binding and ribosome association, may be required to prevent PKR activation.
Two Patches of Amino Acids on the E2 DNA Binding Domain Define the Surface for Interaction with E1
Authors: Chen Grace , Stenlund Arne 
Published: July 2002

ABSTRACT
The E1 and E2 proteins from bovine papillomavirus bind cooperatively to the viral origin of DNA replication (ori), forming a complex which is essential for initiation of DNA replication. Cooperative binding has two components, in which (i) the DNA binding domains (DBDs) of the two proteins interact with each other and (ii) the E2 transactivation domain interacts with the helicase domain of E1. By generating specific point mutations in the DBD of E2, we have defined two patches of amino acids that are involved in the interaction with the E1 DBD. These same mutations, when introduced into the viral genome, result in severely reduced replication of the viral genome, as well as failure to transform mouse cells in tissue culture. Thus, the interaction between the E1 and E2 DBDs is important for the establishment of the viral genome as an episome and most likely contributes to the formation of a preinitiation complex on the viral ori.
DNA Methylation of Helper Virus Increases Genetic Instability of Retroviral Vector Producer Cells
Authors: Young Won-Bin , Lindberg Gary L. , Link Charles J. 
Published: July 2002

ABSTRACT

Retroviral vector producer cells (VPC) have been considered genetically stable. A clonal cell population exhibiting a uniform vector integration pattern is used for sustained vector production. Here, we observed that the vector copy number is increased and varied in a population of established LTKOSN.2 VPC. Among five subclones of LTKOSN.2 VPC, the vector copy number ranged from 1 to approximately 29 copies per cell. A vector superinfection experiment and Northern blot analysis demonstrated that suppression of helper virus gene expression decreased Env-receptor interference and allowed increased superinfection. The titer production was tightly associated with helper virus gene expression and varied between 0 and 2.2 × 10
5
CFU/ml in these subclones. In one analyzed subclone, the number of integrated vectors increased from one copy per cell to nine copies per cell during a 31-day period. Vector titer was reduced from 1.5 × 10
5
CFU to an undetectable level. To understand the mechanism involved, helper virus and vectors were examined for DNA methylation status by methylation-sensitive restriction enzyme digestion. We demonstrated that DNA methylation of helper virus 5′ long terminal repeat occurred in approximately 2% of the VPC population per day and correlated closely with inactivation of helper virus gene expression. In contrast, retroviral vectors did not exhibit significant methylation and maintained consistent transcription activity. Treatment with 5-azacytidine, a methylation inhibitor, partially reversed the helper virus DNA methylation and restored a portion of vector production. The preference for methylation of helper virus sequences over vector sequences may have important implications for host-virus interaction. Designing a helper virus to overcome cellular DNA methylation may therefore improve vector production. The maintenance of increased viral envelope-receptor interference might also prevent replication-competent retrovirus formation.
Locus Control Region of the Human
CD2
Gene in a Lentivirus Vector Confers Position-Independent Transgene Expression
Authors: Kowolik Claudia M. , Hu Jun , Yee Jiing-Kuan 
Published: July 2002

ABSTRACT

Vectors derived from murine leukemia virus (MLV) have been used in many human gene therapy clinical trials. However, insertion of the locus control regions (LCRs) derived from the β-globin gene locus or the
CD2
gene into MLV vectors frequently led to vector rearrangement. Since the human immunodeficiency virus (HIV) sequence diverges significantly from the MLV sequence, we tested whether the LCR sequence is more stable in the context of an HIV vector. Clones derived from human fibrosarcoma line HT1080 cells transduced with an HIV vector containing the T-cell-specific CD2 LCR exhibit the same wide range of transgene expression as clones lacking the LCR. In contrast, Jurkat and primary T-cell clones derived from the transduction of the LCR-containing vector show, on average, a three- to fourfold increase in transgene expression relative to that of the control vector. This is consistent with previous observations that the CD2 LCR contains a T-cell-specific enhancer. In addition, the clones derived from the LCR-containing vector have a much lower clonal variation in transgene expression than those derived from the control vector. We also demonstrate that the level of transgene expression is proportional to the vector copy number. These results suggest that the human CD2 LCR sequence is compatible with HIV vector sequences and confers enhanced integration site-independent and copy number-dependent expression of the transgene. Thus, HIV vectors may represent the ideal vehicle to deliver genes controlled by various
cis
-acting elements such as LCRs.
Fidelity of Leader and Trailer Sequence Usage by the Respiratory Syncytial Virus and Avian Pneumovirus Replication Complexes
Authors: Marriott Anthony C. , Smith Joanne M. , Easton Andrew J. 
Published: July 2002

ABSTRACT
The specificity of usage of promoters for replication and transcription by the pneumoviruses human respiratory syncytial virus (HRSV) and avian pneumovirus (APV) was studied using minigenomes containing a reporter gene. When infectious HRSV or APV was used as helper virus, replication could occur only if both the leader and trailer regions (containing the replicative and transcriptional promoters) were derived from the helper virus. In contrast, when the HRSV replication complex was supplied from cDNA plasmids, a minigenome containing either the APV leader or trailer was recognized and substantial levels of replication and transcription occurred. These data suggest that in pneumovirus-infected cells, helper virus functions can discriminate between genomes on the basis of the terminal sequences and that there is an association between the leader and trailer required for productive replication. This association is required only in virus-infected cells, not when replication and transcription are mediated by plasmid-directed expression of the component proteins required for replication and transcription. The possible implications of this are discussed.
Biologic Studies of Chimeras of Highly and Moderately Virulent Molecular Clones of Simian Immunodeficiency Virus SIVsmPBj Suggest a Critical Role for Envelope in Acute AIDS Virus Pathogenesis
Authors: Haddrick Malcolm , Brown Charles R. , Plishka Ronald , Buckler-White Alicia , Hirsch Vanessa M. , Ginsberg Harold 
Published: July 2002

ABSTRACT
Previous studies identified three molecular clones of the acutely pathogenic SIVsmPBj strain that varied in terms of relative in vivo pathogenicity. One clone, SIVsmPBj6.6, reproducibly induced a rapidly fatal disease in pigtailed macaques. In contrast, a highly related clone (SIVsmPBj6.9) was only minimally pathogenic in macaques. PBj6.6 and PBj6.9 shared a tyrosine substitution at position 17 in the Nef protein that is a major determinant of virulence but differed at one residue in Vpx (C89R), three residues within the envelope (D119G, R871G, G872R), and a single residue in Nef (F252L). SIVsmPBj6.9 was less efficient in inducing proliferation of resting macaque peripheral blood mononuclear cells in vitro than SIVsmPBj6.6 and exhibited a marked reduction in infectivity relative to SIVsmPBj6.6. Chimeric viruses for each of these variable residues were constructed, and their biologic properties were compared to those of the parental strains. Differences in Vpx and Nef did not alter the basic biologic phenotype of the chimeras. However, the D119G substitution in the envelope of SIVsmPBj6.9 was associated with a marked reduction in the infectivity of this virus relative to SIVsmPBj6.6. An associated processing defect in gp160 of SIVsmPBj6.9 and chimeras expressing the D119G substitution suggests that a reduction in virion envelope incorporation is the mechanistic basis for reduced virion infectivity. In vivo studies revealed that substitution of the PBj6.9 amino acid into PBj6.6 (D119) abrogated the pathogenicity of this previously pathogenic virus. Introduction of the PBj6.9 G119, however, did not confer full virulence to the parental PBj6.9 virus, implicating one or all of the other four substitutions in the virulence of SIVsmPBj6.6.