bluuebunny/crossref_metadata_2025_split huggingface dataset:
Monitoring Cytomegalovirus Infection in Adult and Pediatric Bone Marrow Transplant Recipients by a Real-Time PCR Assay Performed with Blood Plasma
Authors: Leruez-Ville Marianne , Ouachée Marie , Delarue Richard , Sauget Anne-Sophie , Blanche Stéphane , Buzyn Agnès , Rouzioux Christine 
Published: May 2003

ABSTRACT

The objective of this study was to evaluate the advantages of cytomegalovirus (CMV) real-time PCR in blood plasma to monitor CMV infection in a population of adult and pediatric bone marrow recipients in comparison with the pp65 antigenemia method. Fifty allogeneic bone marrow transplant recipients from our center, including 23 adults and 27 children, were enrolled. A CMV real-time PCR designed to amplify a well-conserved region of the UL123 gene was evaluated for its results with whole blood and blood plasma. The CMV real-time PCR assay and the CMV antigenemia method were performed in parallel with 558 blood samples. The results obtained by the two techniques were significantly correlated (
r
= 0.732;
P
< 0.0001). Twenty patients developed at least one episode of CMV replication, with a total of 24 episodes detected by CMV PCR; antigenemia assays were positive in 17 of these 24 episodes. The first positive PCR test preceded the first positive antigenemia by a median of 8 days. The median time interval necessary to obtain a negative CMV PCR test after implementation of preemptive treatment was 28 days. CMV PCR of plasma was positive in two children with CMV disease (one with early CMV pneumonia and one with CMV gastroenteritis), while CMV antigenemia remained negative. The use of CMV PCR with plasma to guide both implementation and discontinuation of CMV preemptive therapy might reduce the risk of occurrence of CMV disease since patients would be treated earlier, and it might also help to reduce the duration of treatment, which could attenuate the side effects of antiviral drugs.
Epidemiology of
Cyclospora cayetanensis
and Other Intestinal Parasites in a Community in Haiti
Authors: Lopez Adriana S. , Bendik Jean M. , Alliance Jean Y. , Roberts Jacquelin M. , da Silva Alexandre J. , Moura Iaci N. S. , Arrowood Michael J. , Eberhard Mark L. , Herwaldt Barbara L. 
Published: May 2003

ABSTRACT

We conducted an exploratory investigation in a community in Haiti to determine the prevalence of
Cyclospora cayetanensis
infection and to identify potential risk factors for
C. cayetanensis
infection. In 2001, two cross-sectional stool surveys and a nested case-control study were conducted. In 2002, a follow-up cross-sectional stool survey was conducted among children ≤10 years of age. Stool specimens from study participants and water samples from their wells were examined for
Cyclospora
and other intestinal parasites. In stools, the prevalence of infection with
Cyclospora
in persons of all ages decreased from 12% (20 of 167 persons) in February 2001 to 1.1% (4 of 352 persons) in April 2001, a 90.8% decrease. For children ≤10 years of age, the prevalence rates were 22.5% (16 of 71 children) in February 2001, 3.0% (4 of 135 children) in April 2001, and 2.5% (2 of 81 children) in January 2002. Use of the water from the artesian well in the northern region of the community versus the one in the south was the only risk factor associated with
Cyclospora
infection in multivariate analyses (odds ratio, 18.5; 95% confidence interval, 2.4 to 143.1). The water sample from one of the nine wells or water sources tested (one sample per source) in January 2001, shortly before the investigation began, was positive for
Cyclospora
by UV fluorescence microscopy and PCR. None of the water samples from the 46 wells or water sources tested during the investigation (one sample per source per testing period, including the artesian wells) were positive for
Cyclospora
. Further studies are needed to assess the role of water as a possible risk factor for
Cyclospora
infection in Haiti and other developing countries.
Applications of Competitor RNA in Diagnostic Reverse Transcription-PCR
Authors: Kleiboeker Steven B. 
Published: May 2003

ABSTRACTDetection of RNA viruses by reverse transcription (RT)-PCR has proven to be a useful approach for the diagnosis of infections caused by many viral pathogens. However, adequate controls are required for each step of the RT-PCR protocol to ensure the accuracies of diagnostic test results. Heterologous competitor RNA can be used as a control for a number of different aspects of diagnostic RT-PCR. Competitor RNA can be applied to assessments of the efficiency of RNA recovery during extraction procedures, detection of endogenous RT-PCR inhibitors that could lead to false-negative results, and quantification of viral template in samples used for diagnosis; competitor RNA can also be used as a positive control for the RT-PCR. In the present study, heterologous competitor RNA was synthesized by a method that uses two long oligonucleotide primers containing primer binding sites for RT-PCR amplification of porcine reproductive and respiratory syndrome virus or West Nile virus. Amplification of the competitor RNA by RT-PCR resulted in a product that was easily distinguished from the amplification product of viral RNA by agarose gel electrophoresis. Assessment of a variety of RNA samples prepared from routine submissions to a veterinary diagnostic laboratory found that either partial or complete inhibition of the RT-PCR could be demonstrated for approximately 20% of the samples. When inhibition was detected, either dilution of the sample or RNA extraction by an alternative protocol proved successful in eliminating the source of inhibition.
Development and Verification of an Automated Sample Processing Protocol for Quantitation of Human Immunodeficiency Virus Type 1 RNA in Plasma
Authors: Lee Brenda G. , Fiebelkorn Kristin R. , Caliendo Angela M. , Nolte Frederick S. 
Published: May 2003

ABSTRACT

We developed and verified an automated sample processing protocol for use with the AMPLICOR HIV-1 MONITOR test, version 1.5 (Roche Diagnostics, Indianapolis, Ind.). The automated method uses the MagNA Pure LC instrument and total nucleic acid reagents (Roche Applied Science, Indianapolis, Ind.) to extract human immunodeficiency virus type 1 (HIV-1) RNA from plasma specimens. We compared the HIV-1 load results for a dilution series (1 to 5 nominal log
10
copies/ml) and 175 clinical specimens processed by the automated method to those for the same samples processed by the manual methods specified by the manufacturer. The sensitivity, dynamic range, and precision of the viral load assay obtained by automated processing of specimens were similar to those obtained by an ultrasensitive manual processing method. The results were highly correlated (
R
2
, 0.95), and were in close agreement, with a mean difference of 0.09 log
10
(standard deviation, 0.292). The limits of agreement were ±0.58 log
10
for results for samples processed by both the manual and the automated methods. These performance characteristics were achieved with a smaller sample volume (200 versus 500 μl) and without a high-speed centrifugation step and required only 15 min of labor for a batch of 32 samples. In conclusion, the automated sample preparation protocol can replace both the standard and the ultrasensitive manual methods used with the AMPLICOR HIV-1 MONITOR test and can substantially reduce the labor associated with this test.
Multilocus Sequence Typing and Evolutionary Relationships among the Causative Agents of Melioidosis and Glanders,
Burkholderia pseudomallei
and
Burkholderia mallei
Authors: Godoy Daniel , Randle Gaynor , Simpson Andrew J. , Aanensen David M. , Pitt Tyrone L. , Kinoshita Reimi , Spratt Brian G. 
Published: May 2003

ABSTRACT

A collection of 147 isolates of
Burkholderia pseudomallei
,
B. mallei
, and
B. thailandensis
was characterized by multilocus sequence typing (MLST). The 128 isolates of
B. pseudomallei
, the causative agent of melioidosis, were obtained from diverse geographic locations, from humans and animals with disease, and from the environment and were resolved into 71 sequence types. The utility of the MLST scheme for epidemiological investigations was established by analyzing isolates from captive marine mammals and birds and from humans in Hong Kong with melioidosis. MLST gave a level of resolution similar to that given by pulsed-field gel electrophoresis and identified the same three clones causing disease in animals, each of which was also associated with disease in humans. The average divergence between the alleles of
B. thailandensis
and
B. pseudomallei
was 3.2%, and there was no sharing of alleles between these species. Trees constructed from differences in the allelic profiles of the isolates and from the concatenated sequences of the seven loci showed that the
B. pseudomallei
isolates formed a cluster of closely related lineages that were fully resolved from the cluster of
B. thailandensis
isolates, confirming their separate species status. However, isolates of
B. mallei
, the causative agent of glanders, recovered from three continents over a 30-year period had identical allelic profiles, and the
B. mallei
isolates clustered within the
B. pseudomallei
group of isolates. Alleles at six of the seven loci in
B. mallei
were also present within
B. pseudomallei
isolates, and
B. mallei
is a clone of
B. pseudomallei
that, on population genetics grounds, should not be given separate species status.
pgi
Genotyping Is a Surrogate for Serotyping of Encapsulated
Haemophilus influenzae
Authors: Anyanwu Juliana N. , Rodriguez Carina A. , Fleming Katherine E. , Adderson Elisabeth E. 
Published: May 2003

ABSTRACT

Study of the epidemiology of invasive infections caused by encapsulated
Haemophilus influenzae
has been complicated by the poor sensitivity and specificity of the serologic assays used to identify specific capsular polysaccharides. The population structure of these bacteria is highly clonal, however, and serotype is highly correlated with other genetic characteristics. We sought to determine if alleles of the highly conserved phosphoglucose isomerase (
pgi
) gene correspond to the serotypes of encapsulated
H. influenzae
strains.
pgi
alleles of 52 well-characterized encapsulated
H. influenzae
isolates were amplified by PCR, sequenced, and compared to one another and to additional previously reported
H. influenzae pgi
alleles. Overall, 83% of the strains possessed
pgi
alleles associated with the major serotype a, b, e, and f clonotypes that cause the most invasive disease in the United States. Six strains (four type a and two type f) had unusual
pgi
alleles, which suggested that these strains belonged to less common clonotypes of encapsulated bacteria or were actually nontypeable strains.
pgi
genotyping may provide a simple and stable surrogate for capsular serotyping. Further studies correlating
pgi
typing with the expression of capsule are likely to increase our understanding of the epidemiology and pathogenesis of these infections.
Evaluation of a New Serotyping Assay for Detection of Anti-Hepatitis C Virus Type-Specific Antibodies in Serum Samples
Authors: Gault Elyanne , Soussan Patrick , Morice Yoann , Sanders Lara , Berrada Assia , Rogers Brian , Dený Paul 
Published: May 2003

ABSTRACT
The performance of a new version (HC03) of the hepatitis C virus (HCV) serotyping 1-6 assay (Abbott Murex Laboratories), a specific test for serological determination of HCV types, was evaluated using a selected panel of 180 HCV RNA-positive sera. HC03 was more sensitive than the current HC02 version, typing 53 (37.6%) of 141 samples which were not typable with HC02. Furthermore, the HC03 specificity was 94.1% as evaluated with a panel of 22 genotyped samples. This new version of the test improves the quality of the serological approach to HCV type determination.
Genetic Characterization of a Novel, Naturally Occurring Recombinant Human G6P[6] Rotavirus
Authors: Rahman Mustafizur , De Leener Karolien , Goegebuer Truus , Wollants Elke , Van der Donck Ingrid , Hoovels Lieve Van , Van Ranst Marc 
Published: May 2003

ABSTRACT
A binary classification system has been established for group A rotaviruses, with the viral capsid protein VP7 defining G types and VP4 defining P types. At least 15 G types and 21 P types have been isolated globally with various G and P combinations. Most of the currently circulating human rotaviruses belong to G1P[8], G2P[4], G3P[8], and G4P[8]. We report a human rotavirus strain (B1711) with a novel genotypic VP7/VP4 combination of G6P[6]. This unique rotavirus was isolated from a 13-month-old human immunodeficiency virus (HIV)- negative child of an HIV-seropositive Malian mother that was hospitalized with severe diarrhea in Belgium after returning from a trip to Mali. The VP7 and VP4 genes of the rotavirus strain were sequenced, and phylogenetic trees were constructed. Nucleotide and amino acid sequence comparisons with 15 known G genotypes indicated that the VP7 sequence of strain B1711 was most closely related to an American (Se584) and an Italian (PA151) human G6 strain (95 to 96% nucleotide and 98% amino acid identity). Comparison of the VP4 sequence with 21 P types showed the closest similarity to P[6] genotypes, with greatest similarity to a G8P[6] Malawi strain (mw131) (97% nucleotide and 98% amino acid identity). The B1711 strain is the first reported rotavirus isolate with a G6P[6] genotypic combination. The discovery and surveillance of novel human and nonhuman rotavirus G or P types or of novel G/P combinations is essential for the design of future rotavirus vaccines and for our understanding of rotavirus diversity and evolution.
Evaluation of the Vitek 2 ID-GNB Assay for Identification of Members of the Family
Enterobacteriaceae
and Other Nonenteric Gram-Negative Bacilli and Comparison with the Vitek GNI+ Card
Authors: O'Hara Caroline M. , Miller J. Michael 
Published: May 2003

ABSTRACT

We evaluated the Vitek 2 ID-GNB identification card (bioMérieux, Inc., Durham, N.C.) for its ability to identify members of the family
Enterobacteriaceae
and other gram-negative bacilli that are isolated in clinical microbiology laboratories. Using 482 enteric stock cultures and 103 strains of oxidase-positive, gram-negative glucose-fermenting and nonfermenting bacilli that were maintained at −70°C and passaged three times before use, we inoculated cards according to the manufacturer's directions and processed them in a Vitek 2 instrument using version VT2-R02.03 software. All panel identifications were compared to reference identifications previously confirmed by conventional tube biochemical assays. At the end of the initial 3-h incubation period, the Vitek 2 instrument demonstrated an accuracy of 93.0% for the identification of enteric strains; 414 (85.9%) were correctly identified at probability levels ranging from excellent to good, and an additional 34 (7.1%) strains were correctly identified but at a low level of discrimination. Nineteen (3.9%) strains were unidentified, and 15 (3.1%) were misidentified. The 19 unidentified strains were scattered among 10 genera. Three of the 15 misidentified strains were lactose-positive
Salmonella
spp. and were identified as
Escherichia coli
; another was a lactose-positive, malonate-negative
Salmonella enterica
subsp.
arizonae
strain that was identified as
E. coli
. Of the 103 glucose-fermenting and nonfermenting nonenteric strains, 88 (85.4%) were correctly identified at probability levels ranging from excellent to good, and 10 (9.7%) were correctly identified, but at a low level of discrimination, for a total of 95.1% accuracy with this group. Two strains were unidentified and three were misidentified. The errors occurred for strains in three different genera. With the increased hands-off approach of the Vitek 2 instrument and accuracies of 93% for the identification of enteric organisms and 95.1% for the identification of nonenteric organisms with the ID-GNB card, use of this product presents an acceptable method for the identification of most gram-negative organisms commonly isolated in the clinical laboratory. A comparison of these results to those obtained by testing 454 of the same strains with the Vitek GNI+ card revealed no significant difference in the abilities of the two cards to identify these organisms accurately.
Recombinant VP7-Based Enzyme-Linked Immunosorbent Assay for Detection of Immunoglobulin G Antibodies to Colorado Tick Fever Virus
Authors: Mohd Jaafar Fauziah , Attoui Houssam , Gallian Pierre , Biagini Philippe , Cantaloube Jean-François , de Micco Philippe , de Lamballerie Xavier 
Published: May 2003

ABSTRACTVP6, VP7, VP9, VP10, VP11, and VP12 of Colorado tick fever virus (CTF virus), a virus member of the genusColtivirus, familyReoviridae, were expressed in bacteria with the pGEX-4T-2 vector. A partial sequence of VP7 (designated pVP7) was chosen to elaborate an enzyme-linked immunosorbent assay (ELISA) for detecting anti-CTF virus immunoglobulin G (IgG) antibodies in humans. This was based on two observations: (i) among all expressed proteins, pVP7 showed the highest immunoreactivity to an anti-CTF virus hyperimmune ascitic fluid; (ii) to provide the highest selectivity of antibody detection, the expressed sequence was chosen within a region which is highly divergent (49% amino acid identity) from the homologous sequence of another coltivirus, the Eyach virus. The pVP7 ELISA was evaluated with 368 serum samples from French blood donors and found to provide 98.1% specificity. Assays with the Calisher set of human serum samples, positive for anti-CTF virus antibodies (C. H. Calisher, J. D. Poland, S. B. Calisher, and L. A Warmoth, J. Clin. Microbiol.22:84-88, 1985), showed that the pVP7 ELISA provided 100% sensitivity for the tested population. After elaboration of recombinant-protein-based ELISAs for diagnosis of infections with members of the viral generaOrbivirus,Orthoreovirus, andRotavirus, it was shown that a recombinant protein could be used to detect antibodies to the human pathogen Colorado tick fever virus.
Identification and Characterization of a New Variant of Shiga Toxin 1 in
Escherichia coli
ONT:H19 of Bovine Origin
Authors: Bürk Christine , Dietrich Richard , Açar Gabriele , Moravek Maximilian , Bülte Michael , Märtlbauer Erwin 
Published: May 2003

ABSTRACT

A new variant of Shiga toxin 1 (Stx1), designated Stx1d, which deviates considerably more than any other known variant from Stx1 encoded by phage 933J, was identified in an
Escherichia coli
strain, ONT:H19, isolated from bovine feces. The complete
stx
1
gene of this strain was amplified and sequenced. Nucleotide sequence homology with
stx
1
from phage 933J was only 91%, resulting in the substitution of 20 amino acids in the A subunit and 7 amino acids in the B subunit of the protein. Cell culture supernatant of this strain, which was negative for
stx
2
by PCR testing, was cytotoxic to Vero cells and gave positive results in two commercial enzyme-linked immunosorbent assays for Stx. PCR primers were constructed for the specific detection of the new variant. The findings of this study suggest that Stx1 is not as conserved as thought before and that there might be more variants which cannot be detected by commonly used PCR methods.
Rapid Identification of
Escherichia coli
Pathotypes by Virulence Gene Detection with DNA Microarrays
Authors: Bekal Sadjia , Brousseau Roland , Masson Luke , Prefontaine Gabrielle , Fairbrother John , Harel Josée 
Published: May 2003

ABSTRACT

One approach to the accurate determination of the pathogenic potential (pathotype) of isolated
Escherichia coli
strains would be through a complete assessment of each strain for the presence of all known
E. coli
virulence factors. To accomplish this, an
E. coli
virulence factor DNA microarray composed of 105 DNA PCR amplicons printed on glass slides and arranged in eight subarrays corresponding to different
E. coli
pathotypes was developed. Fluorescently labeled genomic DNAs from
E. coli
strains representing known pathotypes were initially hybridized to the virulence gene microarrays for both chip optimization and validation. Hybridization pattern analysis with clinical isolates permitted a rapid assessment of their virulence attributes and determination of the pathogenic group to which they belonged. Virulence factors belonging to two different pathotypes were detected in one human
E. coli
isolate (strain H87-5406). The microarray was also tested for its ability to distinguish among phylogenetic groups of genes by using gene probes derived from the attaching-and-effacing locus (
espA
,
espB
,
tir
). After hybridization with these probes, we were able to distinguish
E. coli
strains harboring
espA
,
espB
, and
tir
sequences closely related to the gene sequences of an enterohemorrhagic strain (EDL933), a human enteropathogenic strain (E2348/69), or an animal enteropathogenic strain (RDEC-1). Our results show that the virulence factor microarray is a powerful tool for diagnosis-based studies and that the concept is useful for both gene quantitation and subtyping. Additionally, the multitude of virulence genes present on the microarray should greatly facilitate the detection of virulence genes acquired by horizontal transfer and the identification of emerging pathotypes.
Real-Time PCR Assay of Individual Human Immunodeficiency Virus Type 1 Variants in Coinfected Human Lymphoid Tissues
Authors: Ito Yoshinori , Grivel Jean-Charles , Margolis Leonid 
Published: May 2003

ABSTRACT
In the course of infection, human immunodeficiency virus type 1 (HIV-1) mutates, diverging into a “swarm” of viral quasispecies, and the predominance of CCR5- or CXCR4-utilizing quasispecies is strongly associated with the pattern of disease progression. Quantification of CCR5- and CXCR4-utilizing viruses in viral swarms is important in the investigation of the mechanisms of this phenomenon. Here, we report on a new real-time PCR-based methology for the evaluation of replication of individual CCR5- and CXCR4-utilizing variants. The assay is highly reproducible, with a coefficient of variation of <3%, and it accurately estimates the numbers of virus-specific RNA copies even when their difference in the mixture is 2 orders of magnitude. We demonstrate that replications of CCR5- and CXCR4-utilizing variants can be evaluated and distinguished in experimentally coinfected human lymphoid tissue. The assay we developed may facilitate study of the mechanisms of the R5-to-X4 switch in viral swarms in human tissues infected with HIV-1.
Comparison of Lateral-Flow Immunoassay and Enzyme Immunoassay with Viral Culture for Rapid Detection of Influenza Virus in Nasal Wash Specimens from Children
Authors: Cazacu Andreea C. , Greer Jewel , Taherivand Moe , Demmler Gail J. 
Published: May 2003

ABSTRACT
The performance of two commercially available rapid test kits for influenza virus detection was compared to that of viral culture by using 356 nasal wash specimens collected during the 2001 to 2002 influenza season. Overall, the two rapid tests were easy to perform and showed comparable sensitivities (70.4 and 72.2%) and specificities (97.7 and 98.3%); for both test kit groups, most of the specimens that yielded false-negative results were found to be growing influenza B virus.
Failure To Genotype Herpes Simplex Virus by Real-Time PCR Assay and Melting Curve Analysis Due to Sequence Variation within Probe Binding Sites
Authors: Anderson Trevor P. , Werno Anja M. , Beynon Kirsten A. , Murdoch David R. 
Published: May 2003

ABSTRACT
Real-time PCR with melting curve analysis of PCR products is a rapid procedure for detecting and genotyping herpes simplex virus (HSV). When testing mucocutaneous samples for HSV by a real-time PCR assay targeting the DNA polymerase gene, we found that some PCR products had atypical melting curves that did not conform to the expected melting temperatures for HSV type 1 or 2. Sequence analysis showed that these strains had base-pair mismatches over the probe binding sites. An alternative assay is required to type such atypical isolates.