bluuebunny/crossref_metadata_2025_split huggingface dataset:
Quantitation of Human Immunodeficiency Virus Type 1 in Breast Milk
Authors: Ghosh M. K. , Kuhn L. , West J. , Semrau K. , Decker D. , Thea D. M. , Aldrovandi G. M. 
Published: June 2003

ABSTRACT

The distribution and stability of human immunodeficiency virus type 1 (HIV-1) in breast milk (BM) components remain largely unknown. Inhibitory effects, if any, of BM on HIV RNA and DNA PCR amplification are poorly understood. We have addressed these issues by using virus-spiked BM samples from HIV-negative women. BM samples from HIV-negative women were spiked with HIV-1 virions or cells containing a single integrated copy of HIV DNA (8E5/LAV). After incubation under different experimental conditions, viral RNA was detected by the Roche Amplicor UltraSensitive assay in whole-milk, skim milk, and lipid fractions. We found excellent correlation between HIV-1 input copy and recovery in whole milk (
r
= 0.965,
P
< 0.0001), skim milk (
r
= 0.972,
P
< 0.0001), and the lipid fraction (
r
= 0.905,
P
< 0.001). PCR inhibition was observed in less than 10% of the spiked samples. Similar levels of inhibition were noted in BM samples collected from HIV-infected women. HIV proviral DNA was detected in BM samples using real-time PCR (linear correlation between the threshold cycle versus log DNA copy number, >0.982). The effects of incubation duration and temperature and repeated freeze-thaw cycles on HIV RNA recovery were analyzed. HIV RNA levels were remarkably stable in whole milk after three freeze-thaw cycles and for up to 30 h at room temperature. Our findings improve the understanding of the dynamics of HIV detection in BM and the conditions for BM sample collection, storage, and processing.
Identification by Subtractive Hybridization of a Novel Insertion Element Specific for Two Widespread
Burkholderia cepacia
Genomovar III Strains
Authors: Liu Lixia , Spilker Theodore , Coenye Tom , LiPuma John J. 
Published: June 2003

ABSTRACT

Species of the
Burkholderia cepacia
complex cause chronic and life-threatening infections in persons with cystic fibrosis. Epidemic strains infect multiple patients, reside primarily in genomovar III, and have an apparent enhanced capacity for human infection and/or interpatient transmission. By using subtractive hybridization, a novel insertion element, designated IS
1363
, was identified in epidemic strain PHDC, known to infect many cystic fibrosis patients in the mid-Atlantic region of the United States. IS
1363
was also found in most isolates of the ET12 lineage, responsible for infecting large numbers of patients in Ontario, Canada, and the United Kingdom. Southern blot analysis demonstrated that whereas multiple copies of IS
1363
were present in strain PHDC, only one copy was present in ET12 isolates. IS
1363
was used to probe a collection of 943
B. cepacia
complex isolates, representing all nine genomovars, recovered from 761 cystic fibrosis patients or the natural environment. IS
1363
was not found in other genomovar III strains and, with the exception of
B. ambifaria,
was absent from other
B. cepacia
complex species. Genotyping analyses of all IS
1363
-positive isolates demonstrated that strain PHDC was more widely distributed in the United States than previously appreciated; 212 cystic fibrosis patients in 24 states were identified as being infected with PHDC.
Dissemination of SHV-12 and Characterization of New AmpC-Type Beta-Lactamase Genes among Clinical Isolates of
Enterobacter
Species in Korea
Authors: Lee Sang Hee , Kim Jae Young , Shin Sang Heum , An Young Jun , Choi Young Wook , Jung Yeun Chang , Jung Ha Il , Sohn Eui Suk , Jeong Seok Hoon , Lee Kye Joon 
Published: June 2003

ABSTRACT

To determine the prevalence and genotype of an extended-spectrum beta-lactamase and new chromosomal AmpC beta-lactamases among clinical isolates of
Enterobacter
species, we performed antibiotic susceptibility testing, pI determination, induction tests, transconjugation, enterobacterial repetitive consensus (ERIC) PCR, sequencing, and phylogenetic analysis. Among the 51 clinical isolates collected from a university hospital in Korea, 6 isolates have been shown to produce SHV-12 and inducible AmpC beta-lactamases. These also included three isolates producing TEM-1b and one strain carrying TEM-1b and CMY-type beta-lactamases with a pI of 8.0. The results from ERIC PCR revealed that six isolates were genetically unrelated, suggesting that dissemination of SHV-12 was responsible for the spread of resistance to extended-spectrum beta-lactams in Korea. Six genes of inducible AmpC beta-lactamases that are responsible for the resistance to cephamycins (cefoxitin and cefotetan), amoxicillin, cephalothin, and amoxicillin-clavulanic acid were cloned and characterized. A 1,165-bp DNA fragment containing the
ampC
genes was sequenced and found to have an open reading frame coding for a 381-amino-acid beta-lactamase. The nucleotide sequence of four
ampC
genes (
bla
EcloK992004.1
,
bla
EcloK995120.1
,
bla
EcloK99230
, and
bla
EareK9911729
) shared considerable homology with that of AmpC-type class C beta-lactamase genes of gram-negative bacteria, especially that of the chromosomal
ampC
gene (
bla
EcloMHN1
) of
Enterobacter cloacae
MHN1 (99.9, 99.7, 99.6, and 99.6% identity, respectively). The sequences of two
ampC
genes (
bla
EcloK9973
and
bla
EcloK9914325
) showed close similarity to the chromosomal
ampC
gene (
bla
EcloQ908R
) of
E. cloacae
Q908R (99.7% identity). The results from phylogenetic analysis suggested that six
ampC
genes could originate from
bla
EcloMHN1
or
bla
EcloQ908R
.
Rapid Detection of
vanA
and
vanB
Genes Directly from Clinical Specimens and Enrichment Broths by Real-Time Multiplex PCR Assay
Authors: Palladino Silvano , Kay Ian D. , Flexman James P. , Boehm Ingrid , Costa Anna Maria G. , Lambert Erica J. , Christiansen Keryn J. 
Published: June 2003

ABSTRACT

A real-time PCR assay previously developed for use on the Roche LightCycler platform was investigated as an alternative to culture for the direct detection of vancomycin-resistant enterococci (VRE) in clinical specimens. PCR primers and fluorescence resonance energy transfer hybridization probes specific for the
vanA
and
vanB
genes were combined in a multiplex real-time PCR assay performed directly with fecal material obtained by rectal swabbing and with enrichment broth samples. DNA was prepared from the rectal swabs and enrichment broths with a commercially available DNA preparation column designed specifically for use with fecal specimens. One hundred eighty duplicate rectal swabs were obtained from 42 patients who were previously found to be positive for VRE and who were being monitored for carriage of VRE. Direct and enrichment broth cultures were performed with one swab, while PCR was performed with the other swab as well as any corresponding presumptive positive enrichment broth. In total, 100 specimens from 30 patients remained positive for VRE by at least one method. The multiplex real-time PCR was positive for 88 enrichment broths of rectal swabs from 27 patients but for only 45 rectal swabs from 15 patients. Direct culture was positive for VRE for only 43 specimens from 11 patients, while enrichment broth culture was positive for VRE for 75 specimens from 22 patients. Inhibition studies for the multiplex real-time PCR assay, performed by spiking the DNA extracts from 50 negative rectal swabs and the corresponding enrichment broths with between 1 and 10 CFU of a VanB
Enterococcus faecium
strain, detected inhibition rates of 55.1 and 10%, respectively. PCR performed directly with enrichment broths was found to be significantly more sensitive than enrichment broth culture (
P
< 0.025). Negative samples were identified significantly earlier by PCR than by culture alone.
High Percentage of Methicillin-ResistantStaphylococcus aureusIsolates with Reduced Susceptibility to Glycopeptides in The Netherlands
Authors: Van Griethuysen Arjanne , Van 't Veen Annemarie , Buiting Anton , Walsh Timothy , Kluytmans Jan 
Published: June 2003

ABSTRACTWhile testing the in vitro activities of 14 antimicrobial agents against 107 methicillin-susceptibleStaphylococcus aureus(MSSA) and 250 methicillin-resistantS. aureus(MRSA) isolates collected in The Netherlands, we found to our surprise that 19 (7.6%) MRSA isolates were suspected of having reduced susceptibilities to the glycopeptides when the Etest system (AB Biodisk, Solna, Sweden) was used with a large inoculum (no. 2 McFarland standard) and an extended incubation time (48 h) on brain heart infusion agar for MIC testing. Eventually, 15 of these isolates were classified as heterogeneously resistant to glycopeptides (heterogeneously glycopeptide-intermediateS. aureus[hGISA] isolates) according to the population analysis profile-area under the curve analysis. The MICs at which 50 and 90% of isolates are inhibited obtained with the Etest system with the large inoculum were as follows: for MSSA isolates, 3.0 and 4.0 μg/ml, respectively, for both teicoplanin and vancomycin; for MRSA isolates, 3.0 and 8.0 μg/ml, respectively, for teicoplanin, and 3.0 and 4.0 μg/ml, respectively, for vancomycin. This is the first report of hGISA isolates in The Netherlands.
Molecular Analysis of Human Herpesvirus 8 by Using Single Nucleotide Polymorphisms in Open Reading Frame 26
Authors: Endo Tokiomi , Miura Toshiyuki , Koibuchi Tomohiko , Nakamura Hitomi , Takahashi Takashi , Odawara Takashi , Goto Mieko , Ajisawa Atsushi , Iwamoto Aikichi , Nakamura Tetsuya 
Published: June 2003

ABSTRACT

Human herpesvirus 8 (HHV-8) can be classified into distinct subtypes on the basis of sequence polymorphisms in several open reading frames (ORFs). We analyzed the subtypes of HHV-8 in 59 human immunodeficiency virus-infected Japanese patients by using polymorphisms in ORF26 and found that over two-thirds of the HHV-8 isolates fell into major subtype A. We also found that single nucleotide polymorphisms (SNPs) at nucleotide positions 1032 (C-to-A substitution) and 1055 (G-to-T substitution) in HHV-8 ORF26 were correlated with increased susceptibility to Kaposi's sarcoma, compared to the results obtained with HHV-8 with wild-type nucleotides at these positions (
P
= 0.0106). This observation suggests that molecular heterogeneity of the HHV-8 genome affects the biological properties of HHV-8, resulting in different clinical phenotypes of HHV-8 infection. Since sensitive PCR of ORF26 allowed us to analyze the SNPs by using peripheral blood from HHV-8-infected patients, the ORF26 SNPs will be a potent tool for investigating the pathogenesis of HHV-8 infection.
Analysis of Genetic Diversity of
Streptococcus suis
Clinical Isolates from Pigs in Spain by Pulsed-Field Gel Electrophoresis
Authors: Vela Ana I. , Goyache Joaquin , Tarradas Carmen , Luque Inmaculada , Mateos Ana , Moreno Miguel A. , Borge Carmen , Perea J. Anselmo , Domínguez Lucas , Fernández-Garayzábal José F. 
Published: June 2003

ABSTRACT

Pulsed-field gel electrophoresis (PFGE) was used to investigate the diversity of
Streptococcus suis
isolates of various serotypes recovered from swine clinical samples in Spain. Capsular types 9 (64.9%) and 2 (14.8%) were the most frequently isolated serotypes followed by serotype 7 (5.9%) and serotype 8 (4.3%). The PFGE results of this study with 60 different pulsotypes indicate a great genetic diversity among the
S. suis
isolates, which is consistent with the broad distribution of
S. suis
in the swine population. Forty-five percent of the pulsotypes corresponded to single isolates, no pulsotype was common to all farms, and at least 3 different pulsotypes were isolated in 56% of herds in which more than 3 clinical isolates were analyzed. These results reveal a great diversity both between and within herds throughout the strains of
S. suis
studied, demonstrating that different strains of
S. suis
are associated with infection in pigs. Some pulsotypes were more frequently isolated and exhibited a wider distribution over herds than others, and were the unique or predominant strains in several herds, suggesting the existence of a prevalent or a few prevalent clones responsible for a large proportion of clinical cases. Overall, the great genetic heterogeneity of the clinical strains of
S. suis
, the isolation of different strains within the same herd, and the predominance of particular strains in some herds are evidence that infection by
S. suis
is a dynamic process and reinforce the idea that the epidemiology of
S. suis
infection is very complex.
A Hospital-Associated Outbreak of Legionnaires' Disease Caused by
Legionella pneumophila
Serogroup 1 Is Characterized by Stable Genetic Fingerprinting but Variable Monoclonal Antibody Patterns
Authors: Bernander Sverker , Jacobson Kerstin , Helbig Jürgen Herbert , Lück Paul Christian , Lundholm Monica 
Published: June 2003

ABSTRACT

An outbreak of 18 pneumonia cases caused by
Legionella pneumophila
serogroup 1 occurred at a Swedish university hospital 1996 to 1999. Eight clinical isolates obtained by culture from the respiratory tract were compared to 20 environmental isolates from the hospital and to 21 epidemiologically unrelated isolates in Sweden, mostly from patients, by using pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism analysis (AFLP), and monoclonal antibody (MAb) typing. All patients and most environmental isolates from the outbreak hospital belonged to the same genotypic cluster in both PFGE and AFLP. This genotype was distinctly different from other strains, including a cluster from a second hospital in a different part of the country. The MAb subtype of the outbreak clone was Knoxville except for three isolates that were Oxford. A variation in the MAb reactivity pattern was also found in a second genotypic cluster. These changes in the MAb reactivity pattern were due to the absence or presence of the
lag-
1 gene coding for an
O
-acetyltransferase that is responsible for expression of the lipopolysaccharide epitope recognized by MAb 3/1 of the Dresden Panel. In all MAb 3/1-positive strains, the
lag-
1 gene was present on a genetic element that was bordered by a direct repeat that showed a high degree of sequence homology. Due to this homology, the
lag-
1 gene region seemed to be an unstable element in the chromosome. MAb patterns are thus a valuable adjunct to genotyping methods in defining subgroups inside a genotypic cluster of
L. pneumophila
sg 1.
General Acquisition of Human Papillomavirus Infections of Skin Occurs in Early Infancy
Authors: Antonsson Annika , Karanfilovska Silvana , Lindqvist Pelle G. , Hansson Bengt Göran 
Published: June 2003

ABSTRACT
The human skin papillomaviruses (HPVs) represent a group of ubiquitous viruses detected at a high prevalence in the normal skin of healthy adults. In the present study, we analyzed skin swab samples from babies during their first days of life and from infants at various ages up to age 4 years. Specimens from their parents and, for the newborn babies, environmental samples were also investigated. HPV DNA was already detected on the day of birth in samples from 2 of the 16 babies, and 45% of the samples from the babies were positive for HPV in the days following birth. Seventy-seven percent of the skin samples collected from the mothers were HPV DNA positive. The prevalence of HPV DNA among children from the ages of 1 month to 4 years varied between 50 and 70%. The HPV DNA sequences detected revealed a great diversity of genotypes and putative genotypes. Among 115 samples from 38 infants and 31 parents and 7 environmental samples, a total of 73 HPV types or putative types were isolated. Of these, 26 putative HPV types have not been described before. Our data suggest that asymptomatic HPV infections of normal skin are acquired very early in infancy and are caused by a great multiplicity of HPV types.
Novel Reporter T-Cell Line Highly Susceptible to Both CCR5- and CXCR4-Using Human Immunodeficiency Virus Type 1 and Its Application to Drug Susceptibility Tests
Authors: Miyake Hiroshi , Iizawa Yuji , Baba Masanori 
Published: June 2003

ABSTRACT
CCR5-using (R5) human immunodeficiency virus type 1 (HIV-1) is a major viral population that is transmitted by sexual intercourse and that replicates in infected individuals during the asymptomatic stage of HIV-1 infection, suggesting that agents effective against R5 HIV-1 can be expected to prevent viral transmission and delay disease progression. However, R5 HIV-1 is unable to replicate in human T-cell lines, which is an apparent obstacle to efficient and reliable susceptibility tests of compounds for their activities against R5 HIV-1. To establish a simple and rapid assay system for the monitoring of R5 HIV-1 replication and drug susceptibility, we have established a novel reporter T-cell line, MOCHA (which represents MOLT-4 cells stably expressing CCR5 and carrying the HIV-1 long terminal repeat-driven secretory alkaline phosphatase). Cells of this cell line express CD4, CXCR4, and CCR5 on their surfaces and secrete human placental alkaline phosphatase into the culture supernatants during HIV-1 infection. MOCHA cells proved to be highly permissive for the replication of R5 HIV-1 as well as CXCR4-using (X4) HIV-1, and the alkaline phosphatase activity increased in parallel with increasing HIV-1 p24 antigen levels in the culture supernatants. When HIV-1 reverse transcriptase inhibitors, protease inhibitors, and entry inhibitors, including the CCR5 antagonist TAK-779 and the CXCR4 antagonist AMD3100, were examined for their inhibitory effects on R5 and X4 HIV-1 replication in MOCHA cells, the antiviral activities of these compounds were found to be almost identical to those previously reported in peripheral blood mononuclear cells. Thus, MOCHA cells are an extremely useful tool for detection of R5 and X4 HIV-1 replication and drug susceptibility tests.
Characterization ofTreponema phagedenis-Like Spirochetes Isolated from Papillomatous Digital Dermatitis Lesions in Dairy Cattle
Authors: Trott Darren J. , Moeller Michelle R. , Zuerner Richard L. , Goff Jesse P. , Waters W. Ray , Alt David P. , Walker Richard L. , Wannemuehler Michael J. 
Published: June 2003

ABSTRACTFour spirochete strains were isolated from papillomatous digital dermatitis (PDD) lesions in Iowa dairy cattle and compared with two previously described spirochete strains isolated from dairy cattle in California. These six strains shared an identical 16S ribosomal DNA sequence that was 98% similar toTreponema phagedenisand 99% similar to the uncultivated PDD spirochete sequence DDLK-4. The whole-cell protein profiles resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of these six strains were similar. However, these strains showed differences in the antigenic diversity of lipopolysaccharide (LPS). Genetic diversity was also detected by pulsed-field gel electrophoresis of genomic DNA digests, revealing differences among five of the six strains. Serum immunoglobulin G antibodies from dairy cattle with active PDD lesions reacted with the LPS of all but one PDD spirochete strain. Likewise, peripheral blood mononuclear cells from cattle with active PDD lesions produced blastogenic responses to one of the two California isolates. Both antibody and lymphocyte blastogenic responses were reduced in convalescent dairy cattle, suggesting the immune response to these spirochetes has short duration. These results demonstrate genetic and antigenic diversity amongT. phagedenis-like treponemes and provide further evidence for the involvement of these spirochetes in the pathogenesis of PDD.
Multilocus Sequence Typing System for Group B Streptococcus
Authors: Jones Nicola , Bohnsack John F. , Takahashi Shinji , Oliver Karen A. , Chan Man-Suen , Kunst Frank , Glaser Philippe , Rusniok Christophe , Crook Derrick W. M. , Harding Rosalind M. , Bisharat Naiel , Spratt Brian G. 
Published: June 2003

ABSTRACT

A multilocus sequence typing (MLST) system was developed for group B streptococcus (GBS). The system was used to characterize a collection (
n
= 152) of globally and ecologically diverse human strains of GBS that included representatives of capsular serotypes Ia, Ib, II, III, V, VI, and VIII. Fragments (459 to 519 bp) of seven housekeeping genes were amplified by PCR for each strain and sequenced. The combination of alleles at the seven loci provided an allelic profile or sequence type (ST) for each strain. A subset of the strains were characterized by restriction digest patterning, and these results were highly congruent with those obtained with MLST. There were 29 STs, but 66% of isolates were assigned to four major STs. ST-1 and ST-19 were significantly associated with asymptomatic carriage, whereas ST-23 included both carried and invasive strains. All 44 isolates of ST-17 were serotype III clones, and this ST appeared to define a homogeneous clone that was strongly associated with neonatal invasive infections. The finding that isolates with different capsular serotypes had the same ST suggests that recombination occurs at the capsular locus. A web site for GBS MLST was set up and can be accessed at http://sagalactiae.mlst.net. The GBS MLST system offers investigators a valuable typing tool that will promote further investigation of the population biology of this organism.
Species-Specific Identification of Campylobacters by Partial 16S rRNA Gene Sequencing
Authors: Gorkiewicz Gregor , Feierl Gebhard , Schober Caroline , Dieber Franz , Köfer Josef , Zechner Rudolf , Zechner Ellen L. 
Published: June 2003

ABSTRACT

Species-specific identification of campylobacters is problematic, primarily due to the absence of suitable biochemical assays and the existence of atypical strains. 16S rRNA gene (16S rDNA)-based identification of bacteria offers a possible alternative when phenotypic tests fail. Therefore, we evaluated the reliability of 16S rDNA sequencing for the species-specific identification of campylobacters. Sequence analyses were performed by using almost 94% of the complete 16S rRNA genes of 135 phenotypically characterized
Campylobacter
strains, including all known taxa of this genus. It was shown that 16S rDNA analysis enables specific identification of most
Campylobacter
species. The exception was a lack of discrimination among the taxa
Campylobacter jejuni
and
C. coli
and atypical
C. lari
strains, which shared identical or nearly identical 16S rDNA sequences. Subsequently, it was investigated whether partial 16S rDNA sequences are sufficient to determine species identity. Sequence alignments led to the identification of four 16S rDNA regions with high degrees of interspecies variation but with highly conserved sequence patterns within the respective species. A simple protocol based on the analysis of these sequence patterns was developed, which enabled the unambiguous identification of the majority of
Campylobacter
species. We recommend 16S rDNA sequence analysis as an effective, rapid procedure for the specific identification of campylobacters.