bluuebunny/crossref_metadata_2025_split huggingface dataset:
Comparison of Six PCR Methods Using Peripheral Blood for Detection of Canine Visceral Leishmaniasis
Authors: Lachaud Laurence , Marchergui-Hammami Sarah , Chabbert Elisabeth , Dereure Jacques , Dedet Jean Pierre , Bastien Patrick 
Published: July 2002

ABSTRACTThe objectives of this study were to compare the sensitivities and reliabilities of different PCR methods for the diagnosis and epidemiological study of canine visceral leishmaniasis (CVL) using dog blood. We chose to work with peripheral blood, as this type of sampling is noninvasive, straightforward, and easy to repeat. Six PCR methods were compared: three primer pairs target genomic DNA, and the other three target kinetoplast (mitochondrial) DNA. Sensitivity, specificity, reproducibility, and ease of interpretation without hybridization were evaluated for each method. The assessment was first performed using artificial samples. All methods could detect less than one parasite per reaction tube. However, the sensitivities varied among the different methods by a factor of 500 on purified cultivated parasites and by a factor of 10,000 on seeded dog blood samples (i.e., from 10 to 10−3parasite per ml of blood for the latter). Only four methods were found sufficiently reliable for the diagnosis of CVL. They were tested on 37 dogs living in an area of endemicity and grouped according to clinical status and specific serology. Only the two methods targeting kinetoplast DNA (K13A-K13B and RV1-RV2) could detect the parasite in 100% of symptomatic infected dogs. Similarly, all seropositive dogs were found PCR positive by these methods versus 62% by the genomic-DNA-based methods. Finally, these kinetoplast-based methods proved clearly superior to the others in the detection ofLeishmaniain asymptomatic dogs. Our data allow the discussion of the advantages and drawbacks of highly sensitive versus moderately sensitive PCR methods in diagnosis and prevalence studies of CVL.
Discordance between Genotypic and Phenotypic Drug Resistance Profiles in Human Immunodeficiency Virus Type 1 Strains Isolated from Peripheral Blood Mononuclear Cells
Authors: Sarmati Loredana , Nicastri Emanuele , Parisi Saverio G. , d'Ettorre Gabriella , Mancino Giorgio , Narciso Pasquale , Vullo Vincenzo , Andreoni Massimo 
Published: July 2002

ABSTRACTThe aim of the study was to analyze the relationship between genotypic and phenotypic drug resistance profiles of human immunodeficiency virus type 1 (HIV-1) strains isolated from patients during double-analogue nucleoside therapy. A drug-resistant HIV strain was isolated from 20 out of 25 patients, with 16 (64%) subjects carrying a virus with multiple drug resistance mutations. The most frequent resistance mutations were M184V (18 isolates) and M41L (7 isolates). Discordance between the genotypic and phenotypic profile for at least one drug was detected in 16 out of 25 strains. Particularly, eight isolates had a discordant genotypic-phenotypic resistance pattern for two drugs and one isolate had such a pattern for three drugs. A genotypic resistance pattern with a phenotypic sensitivity profile was detected in six isolates (four resistant to zidovudine and two resistant to lamivudine). On the other hand for several strains a genotypic pattern of sensitivity pattern to abacavir (10 strains), didanosine (7 strains), stavudine (3 strains), zidovudine (2 strains), and lamivudine (1 strain) with a phenotypic resistance profile was detected. After a follow-up period of 8 months, an impairment of virological and immunological parameters was detected only in subjects with an HIV-1 isolate with a phenotypic resistance profile in despite of the genotypic results. Predicting resistance phenotype from genotypic data has important limitations. Despite the low number of patients and the short follow-up period, this study suggests that during failing therapy with analogue nucleosides, a phenotypic analysis could be performed in spite of an HIV genotypic sensitivity pattern.
Pseudoepidemic Due to a Unique Strain of
Mycobacterium szulgai
: Genotypic, Phenotypic, and Epidemiological Analysis
Authors: Zhang Qing , Kennon Randa , Koza Maureen A. , Hulten Kristina , Clarridge Jill E. 
Published: July 2002

ABSTRACT

In mid-1999, we noted multiple isolations at the Veterans Affairs Medical Center (VAMC) Houston Tex. of an unusual nonpigmented
Mycobacterium
species. Since, on the basis of 16S rRNA gene sequence analysis, the strains were identical to the
Mycobacterium szulgai
type strain and since
M. szulgai
has been reported only rarely as a commensal or environmental isolate, we were concerned about laboratory contamination, nosocomial spread, or even the possibility that this could be a novel organism associated with disease. Our investigation found that from 1999 to 2000, 37 strains of
M. szulgai
were isolated from patients at the VAMC (the base rate for the previous 10 years had been <1 isolation per year). We compared the phenotypic properties and genetic relatedness of these 37 strains (31 of which were nonpigmented) as well as eight stock strains and the
M. szulgai
type strain. All strains were similar in cellular fatty acid patterns, growth rates, and biochemical characteristics. However, we found three genogroups by gene sequence analysis. Genogroup I comprised the
M. szulgai
type strain, all the tested nonpigmented strains (27 of the 31 strains were tested), two pigmented strains isolated in 1999 and 2000, and five pigmented stock strains. Genogroup II comprised five pigmented strains: three were isolated from 1999 to 2000 and two were stock strains. The single strain (isolated in 1996) in genogroup III was pigmented and was the only strain associated with disease. Whereas the randomly amplified polymorphic DNA (RAPD) patterns of all nonpigmented strains were identical, indicating that they came from a common source (the pseudoepidemic strain), the RAPD patterns of the other strains were varied. In our investigation for a possible source, we found that there were no common reagents, specimen-processing or patient locations, or procedures linking the 31 pseudoepidemic strains. However, a nonpigmented
M. szulgai
strain with a gene sequence and RAPD pattern identical to those of the pseudoepidemic strain was recovered from a water storage tank serving the hospital. We concluded that the strains most likely originated from hospital water, which transiently inoculated our patients. Although no disease was associated with this cluster of isolates, the event was costly because identification was problematic and we could not easily discount the isolations, since most of the patients were immunocompromised and were candidates for opportunistic infection.
Population Structure and Genetic Diversity of
Actinobacillus actinomycetemcomitans
Strains Isolated from Localized Juvenile Periodontitis Patients
Authors: Kaplan Jeffrey B. , Schreiner Helen C. , Furgang David , Fine Daniel H. 
Published: July 2002

ABSTRACT

The phylogeny of 20
Actinobacillus actinomycetemcomitans
strains isolated from patients with localized juvenile periodontitis (LJP) was investigated by using partial sequence analysis of 16S rRNA genes, arbitrarily primed PCR (AP-PCR), and four additional PCR assays that amplified polymorphic regions in the leukotoxin (
lkt
), cytolethal distending toxin (
cdt
), major fimbrial subunit (
flp-1
), and serotype-specific O polysaccharide gene clusters. Our analysis also included four strains isolated from healthy subjects and nine reference strains. We found that
A. actinomycetemcomitans
strains comprised three major phylogenetic lineages. One lineage consisted of serotype b strains, a second lineage consisted of serotype c strains, and a third lineage consisted of serotype a, d, e, and f strains. 16S rRNA sequences within each lineage were highly conserved (<1% base substitutions), whereas sequences between lineages were exceptionally divergent (1.9 to 5.0% substitutions). Two strains exhibited 16S rRNA sequences that were even more distantly related to those of the three major lineages (2.7 to 6.7% substitutions), indicating that additional minor lineages or variants exist. The distribution of 16S rRNA sequences and
lkt
,
cdt
,
flp-1
, and AP-PCR genotypes was consistent with a clonal population structure, with little evidence of assortative recombination between strains of different serotypes. Strains from all three major lineages were recovered from LJP patients, suggesting that phylogenetically diverse strains of
A. actinomycetemcomitans
carry pathogenic potential.
New Yeast Species,
Malassezia dermatis
, Isolated from Patients with Atopic Dermatitis
Authors: Sugita Takashi , Takashima Masako , Shinoda Takako , Suto Hajime , Unno Tetsushi , Tsuboi Ryoji , Ogawa Hideoki , Nishikawa Akemi 
Published: July 2002

ABSTRACT

Malassezia
species are considered to be one of the exacerbating factors in atopic dermatitis (AD). During examination of the cutaneous colonization of
Malassezia
species in AD patients, we found a new species on the surface of the patients' skin. Analysis of ribosomal DNA sequences suggested that the isolates belonged to the genus
Malassezia
. They did not grow in Sabouraud dextrose agar but utilized specific concentrations of Tween 20, 40, 60, and 80 as a lipid source. Thus, we concluded that our isolates were new members of the genus
Malassezia
and propose the name
Malassezia dermatis
sp. nov. for these isolates.
Equine Abortion Associated with the
Borrelia parkeri
-
B. turicatae
Tick-Borne Relapsing Fever Spirochete Group
Authors: Walker R. L. , Read D. H. , Hayes D. C. , Nordhausen R. W. 
Published: July 2002

ABSTRACT

Direct amplification and sequencing of the 16S rRNA gene and a variable region of the flagellin gene from fetal liver-associated spirochetes belonging to the
Borrelia parkeri
-
B. turicatae
tick-borne relapsing fever spirochete group with a late-term abortion in a mare are described.
Rapid PCR-Based Identification of Methicillin-Resistant
Staphylococcus aureus
from Screening Swabs
Authors: Jonas D. , Speck M. , Daschner F. D. , Grundmann H. 
Published: July 2002

ABSTRACT

A PCR identification of methicillin-resistant
Staphylococcus aureus
(MRSA), obviating the need for subculture on agar media, was investigated. The combination of MRSA detection by
mecA femB
PCR with prior enrichment in selective broth was tested for 439 swabs. PCR identified 36 MRSA-positive samples, in concordance with conventional methods.
Genotypic Characteristics of Two Serotypes ofBartonella henselae
Authors: La Scola Bernard , Liang Zhongxing , Zeaiter Zaher , Houpikian Pierre , Grimont Patrick A. D. , Raoult Didier 
Published: July 2002

ABSTRACTThe study of 16S rRNA gene sequences of all isolates ofBartonella henselaeobtained in our laboratory and others from human patients or cats has revealed two genotypes according to the sequence of the 16S rRNA gene. Two isolates of these genotypes have previously been related to two different serotypes, and lack of cross-protection of the two serotypes has been demonstrated in cats. We investigated the grouping of eight strains ofB. henselaeon the basis of 16S ribosomal DNA, 35-kDa protein, Pap 31 protein, and internal transcribed spacer (ITS) gene sequencing; sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles; and monoclonal antibody reactivity studies. Houston-1, 90-615, and SA2 strains showed the same patterns in SDS-PAGE, but they differed from the patterns ofB. henselaeisolates URBHLLY8, URBHLIE9, Cat6, Fizz, and CAL-1. Nine monoclonal antibodies derived from BALB/c mice immunized withB. henselaeHouston-1 strain reacted only with strains Houston-1, 90-615, and SA2, and not with any otherBartonellastrains. The two serogroups corresponded with two genotypes based on differences in the sequences of the genes encoding 16S rRNA, 35-kDa protein, and Pap 31 protein. Sequences of ITS genes were highly divergent among strains, as each had a unique sequence and the subdivision was not supported by DNA-DNA relatedness study. Study of 22 additional strains ofB. henselaeisolated from French bacteremic cats demonstrated that they all belong to one or the other of the proposed serotype or genotype.
Antibiotic Resistance, Virulence Gene, and Molecular Profiles of Shiga Toxin-Producing
Escherichia coli
Isolates from Diverse Sources in Calcutta, India
Authors: Khan Asis , Das S. C. , Ramamurthy T. , Sikdar A. , Khanam J. , Yamasaki S. , Takeda Y. , Nair G. Balakrish 
Published: July 2002

ABSTRACT

Antibiotic resistance, virulence gene, and molecular profiles of Shiga toxin-producing
Escherichia coli
(STEC) non-O157 strains isolated from human stool samples, cow stool samples, and beef samples over a period of 2 years in Calcutta, India, were determined. Resistance to one or more antibiotics was observed in 49.2% of the STEC strains, with some of the strains exhibiting multidrug resistance. The dominant combinations of virulence genes present in the strains studied were
stx
1
and
stx
2
(44.5% of strains) and
stx
1
,
stx
2
, and
hlyA
(enterohemorrhagic
E. coli
hemolysin gene) (19% of strains). Only 6.4% of the STEC strains harbored
eae
. The diversity of STEC strains from various sources was assessed by random amplification of polymorphic DNA (RAPD). STEC strains that gave identical or nearly similar DNA fingerprints in RAPD-PCR and had similar virulence genotypes were further characterized by pulsed-field gel electrophoresis (PFGE). Identical RAPD and PFGE profiles were observed in four sets of strains, with each set comprising two strains. There was no match in the RAPD and PFGE profiles between strains of STEC isolated from cows and those isolated from humans. It appears that the clones present in bovine sources are not transmitted to humans in the Calcutta setting although these strains showed evolutionary relatedness. Maybe for this reason, STEC has still not become a major problem in India.
Human Immunodeficiency Virus Type 1 Induces Apoptosis in CD4+but Not in CD8+T Cells in Ex Vivo-Infected Human Lymphoid Tissue
Authors: Grivel Jean-Charles , Malkevitch Nina , Margolis Leonid 
Published: July 2002

ABSTRACTProgression of human immunodeficiency virus (HIV) disease is associated with massive death of CD4+T cells along with death and/or dysfunction of CD8+T cells. In vivo, both HIV infection per se and host factors may contribute to the death and/or dysfunction of CD4+and CD8+T cells. Progression of HIV disease is often characterized by a switch from R5 to X4 HIV type 1 (HIV-1) variants. In human lymphoid tissues ex vivo, it was shown that HIV infection is sufficient for CD4+T-cell depletion. Here we address the question of whether infection of human lymphoid tissue ex vivo with prototypic R5 or X4 HIV variants also depletes or impairs CD8+T cells. We report that whereas productive infection of lymphoid tissue ex vivo with R5 and X4 HIV-1 isolates induced apoptosis in CD4+T cells, neither viral isolate induced apoptosis in CD8+T cells. Moreover, in both infected and control tissues we found similar numbers of CD8+T cells and similar production of cytokines by these cells in response to phorbol myristate acetate or anti-CD3–anti-CD28 stimulation. Thus, whereas HIV-1 infection per se in human lymphoid tissue is sufficient to trigger apoptosis in CD4+T cells, the death of CD8+T cells apparently requires additional factors.
Immunogenicity of Mutations Induced by Nucleoside Reverse Transcriptase Inhibitors for Human Immunodeficiency Virus Type 1-Specific Cytotoxic T Cells
Authors: Samri Assia , Haas Gaby , Duntze Joerg , Bouley Jean-Marc , Calvez Vincent , Katlama Christine , Autran Brigitte 
Published: July 2002

ABSTRACTThe impact of drug resistance mutations induced by nucleoside reverse transcriptase (RT) inhibitors (NRTI) on cytotoxic T-lymphocyte (CTL) recognition of human immunodeficiency virus type 1 strain LAI (HIV-1LAI) RT was addressed in 35 treated or untreated patients. Two HIV-1LAIRT regions encompassing mutation M41L, L74V, M184V, and T215Y/F were recognized in 75 and 83% mutated and in 33 and 42% unmutated samples, respectively. A total of 41 new CTL epitopes overlapping these mutations were predicted. Mutations enhanced HLA-binding scores of 17 epitopes, decreased scores of 5, and had no effect in 19. Four predicted epitopes containing mutations 41, 74, and 184 were tested and recognized by CD8 cells from mutated or unmutated samples, with frequencies up to 270 gamma interferon spot-forming cells per 106peripheral blood mononuclear cells. Therefore, RT mutations induced by NRTI can increase the immunogenicity of RT for CTL and might allow a better immune control of resistant viruses in vivo, suggesting that specific immune therapy might help prevent these mutations.
Efficacy of Postexposure Prophylaxis after Intravaginal Exposure of Pig-Tailed Macaques to a Human-Derived Retrovirus (Human Immunodeficiency Virus Type 2)
Authors: Otten Ron A. , Smith Dawn K. , Adams Debra R. , Pullium Jennifer K. , Jackson Eddie , Kim Caryn N. , Jaffe Harold , Janssen Robert , Butera Sal , Folks Thomas M. 
Published: July 2002

ABSTRACT

Postexposure prophylaxis (PEP) after intravaginal exposure to human immunodeficiency virus (HIV) was investigated using the HIV type 2 (HIV-2)/pig-tailed macaque transmission model. PEP for 28 days with the reverse transcriptase inhibitor (
R
)-9-(2-phosphonylmethoxypropyl)adenine (PMPA; tenofovir) was initiated 12 to 72 h following HIV-2 exposure. Systemic infection was not evident in the 12- and 36-h groups, as defined by plasma viremia, cell-associated provirus, antibody responses, and lymph node virus. Breakthrough infection in the 72-h group was detected at week 16 post-virus exposure. These results demonstrate for the first time using a vaginal transmission model that early intervention after high-risk sexual exposures may prevent infection.
Evolution of Bovine Respiratory Syncytial Virus
Authors: Valarcher Jean-François , Schelcher François , Bourhy Hervé 
Published: July 2002

ABSTRACTUntil now, the analysis of the genetic diversity of bovine respiratory syncytial virus (BRSV) has been based on small numbers of field isolates. In this report, we determined the nucleotide and deduced amino acid sequences of regions of the nucleoprotein (N protein), fusion protein (F protein), and glycoprotein (G protein) of 54 European and North American isolates and compared them with the sequences of 33 isolates of BRSV obtained from the databases, together with those of 2 human respiratory syncytial viruses and 1 ovine respiratory syncytial virus. A clustering of BRSV sequences according to geographical origin was observed. We also set out to show that a continuous evolution of the sequences of the N, G, and F proteins of BRSV has been occurring in isolates since 1967 in countries where vaccination was widely used. The exertion of a strong positive selective pressure on the mucin-like region of the G protein and on particular sites of the N and F proteins is also demonstrated. Furthermore, mutations which are located in the conserved central hydrophobic part of the ectodomain of the G protein and which result in the loss of four Cys residues and in the suppression of two disulfide bridges and an α helix critical to the three-dimensional structure of the G protein have been detected in some recent French BRSV isolates. This conserved central region, which is immunodominant in BRSV G protein, thus has been modified in recent isolates. This work demonstrates that the evolution of BRSV should be taken into account in the rational development of future vaccines.
The EICP22 Protein of Equine Herpesvirus 1 Physically Interacts with the Immediate-Early Protein and with Itself To Form Dimers and Higher-Order Complexes
Authors: Derbigny Wilbert A. , Kim Seong K. , Caughman Gretchen B. , O'Callaghan Dennis J. 
Published: July 2002

ABSTRACT

The EICP22 protein (EICP22P) of
Equine herpesvirus 1
(EHV-1) is an early protein that functions synergistically with other EHV-1 regulatory proteins to transactivate the expression of early and late viral genes. We have previously identified EICP22P as an accessory regulatory protein that has the ability to enhance the transactivating properties and the sequence-specific DNA-binding activity of the EHV-1 immediate-early protein (IEP). In the present study, we identify EICP22P as a self-associating protein able to form dimers and higher-order complexes during infection. Studies with the yeast two-hybrid system also indicate that physical interactions occur between EICP22P and IEP and that EICP22P self-aggregates. Results from in vitro and in vivo coimmunoprecipitation experiments and glutathione
S
-transferase (GST) pull-down studies confirmed a direct protein-protein interaction between EICP22P and IEP as well as self-interactions of EICP22P. Analyses of infected cells by laser-scanning confocal microscopy with antibodies specific for IEP and EICP22P revealed that these viral regulatory proteins colocalize in the nucleus at early times postinfection and form aggregates of dense nuclear structures within the nucleoplasm. Mutational analyses with a battery of EICP22P deletion mutants in both yeast two-hybrid and GST pull-down experiments implicated amino acids between positions 124 and 143 as the critical domain mediating the EICP22P self-interactions. Additional in vitro protein-binding assays with a library of GST-EICP22P deletion mutants identified amino acids mapping within region 2 (amino acids [aa] 65 to 196) and region 3 (aa 197 to 268) of EICP22P as residues that mediate its interaction with IEP.
The Ends on Herpesvirus DNA Replicative Concatemers Contain
pac2 cis
Cleavage/Packaging Elements and Their Formation Is Controlled by Terminal
cis
Sequences
Authors: McVoy Michael A. , Nixon Daniel E. , Hur Jay K. , Adler Stuart P. 
Published: July 2002

ABSTRACT

Herpesviruses have large double-stranded linear DNA genomes that are formed by site-specific cleavage from complex concatemeric intermediates. In this process, only one of the two genomic ends are formed on the concatemer. Although the mechanism underlying this asymmetry is not known, one explanation is that single genomes are cleaved off of concatemer ends in a preferred direction. This implies that
cis
elements control the direction of packaging. Two highly conserved
cis
elements named
pac1
and
pac2
lie near opposite ends of herpesvirus genomes and are important for cleavage and packaging. By comparison of published reports and by analysis of two additional herpesviruses, we found that
pac2
elements lie near the ends formed on replicative concatemers of four herpesviruses: herpes simplex virus type 1, equine herpesvirus 1, guinea pig cytomegalovirus, and murine cytomegalovirus. Formation of
pac2
ends on concatemers depended on terminal
cis
sequences, since ectopic cleavage sites engineered into the murine cytomegalovirus genome mediated formation of
pac2
ends on concatemers regardless of the orientation of their insertion. These findings are consistent with a model in which
pac2
elements at concatemer ends impart a directionality to concatemer packaging by binding proteins that initiate insertion of concatemer ends into empty capsids.