bluuebunny/crossref_metadata_2025_split huggingface dataset:
Normalized Quantification of Human Cytomegalovirus DNA by Competitive Real-Time PCR on the LightCycler Instrument
Authors: Stöcher Markus , Berg Jörg 
Published: November 2002

ABSTRACTThe development of a novel normalized quantitative competitive real-time PCR on the LightCycler instrument (NQC-LC-PCR) and its application to the quantification of cytomegalovirus (CMV) DNA in clinical samples are described. A heterologous competitor DNA was spiked into test samples and served as an internal amplification control. The internal control (IC) DNA in the test samples was coamplified with the CMV DNA and was tested against a calibrator sample that contained equal amounts of IC DNA and CMV reference standard DNA. An algorithm was developed to normalize possible varying amplification efficiencies between the standard and the samples. After normalization, CMV DNA copy numbers were determined in absolute terms. In a routine clinical setting, normalized quantification by NQC-LC-PCR using a single IC concentration led to results ranging from 500 to 50,000 CMV DNA copies/ml. The results obtained with conventional real-time quantification on the LightCycler instrument were almost identical to those obtained with the NQC-LC-PCR-based quantification. This was the case only for samples in which the PCR was not inhibited. With partially inhibited samples, NQC-LC-PCR was still able to correctly quantify CMV DNA copy numbers even when the PCR was inhibited by about 70%. By analyzing 80 undefined clinical samples, we found that NQC-LC-PCR was suitable for the routine assessment of CMV DNA in clinical plasma samples. Since the ICs were already added to the samples during the DNA purification, almost the entire assay was controlled for sample adequacy. Thus, false negative results were precluded. The NQC-LC-PCR approach developed should be adaptable for additional microbiological applications.
Hepatitis E Virus (HEV) Capsid Antigens Derived from Viruses of Human and Swine Origin Are Equally Efficient for Detecting Anti-HEV by Enzyme Immunoassay
Authors: Engle R. E. , Yu C. , Emerson S. U. , Meng X.-J. , Purcell R. H. 
Published: November 2002

ABSTRACT
The recombinant truncated ORF2 (capsid) antigen derived from the Meng strain of swine hepatitis E virus (HEV) differs from that of the Sar-55 strain of human HEV by approximately 5% at the amino acid level. Serial serum samples from two chimpanzees and six rhesus monkeys experimentally infected with HEV were tested with one enzyme immunoassay (EIA) based on the Sar-55 antigen and with a second EIA based on the Meng antigen. We obtained 98% agreement (κ = 0.952) by direct comparison. The virtually identical results obtained with these antigens in detecting seroconversion following infection with HEV suggests that they were reacting with antibodies that detect the same or very similar epitopes of HEV. We then tested human and swine serum samples for anti-HEV in EIAs that utilized one or the other of the two ORF2 antigens and showed that these results were also virtually identical. The specimens tested included swine sera from the United States, Canada, China, Korea, and Thailand and sera from veterinarians, U.S. and non-U.S. volunteer blood donors, and U.S. and non-U.S. animal handlers. We tested 792 swine sera and obtained 93% agreement (κ = 0.839). We similarly tested 882 human sera and obtained 99% agreement (κ = 0.938). Moreover, we found virtually no difference in the levels of prevalence of anti-HEV as measured by the two tests, again suggesting that the antigens derived from human and swine HEV contain the same immunodominant epitopes.
Strain Typing of
Borrelia burgdorferi
,
Borrelia afzelii
, and
Borrelia garinii
by Using Multiple-Locus Variable-Number Tandem Repeat Analysis
Authors: Farlow Jason , Postic Danielle , Smith Kimothy L. , Jay Zack , Baranton Guy , Keim Paul 
Published: November 2002

ABSTRACT

Human Lyme borreliosis (LB) is the most prevalent arthropod-borne infection in temperate climate zones around the world and is caused by
Borrelia
spirochetes. We have identified 10 variable-number tandem repeat (VNTR) loci present within the genome of
Borrelia burgdorferi
and subsequently developed a multiple-locus VNTR analysis (MLVA) typing system for this disease agent. We report here the successful application of MLVA for strain discrimination among a group of 41 globally diverse
Borrelia
isolates including
B. burgdorferi
,
B. afzelii
, and
B. garinii
. PCR assays displayed diversity at these loci, with total allele numbers ranging from two to nine and Nei's diversity (
D
) values ranging from 0.10 to 0.87. The average
D
value was 0.53 across all VNTR loci. A clear correlation exists between the repeat copy number and the
D
value (
r
= 0.62) or the number of alleles (
r
= 0.93) observed across diverse strains. Cluster analysis by the unweighted pair-group method with arithmetic means resolved the 30 observed unique
Borrelia
genotypes into five distinct groups.
B. burgdorferi
,
B. afzelii
, and
B. garinii
clustered into distinct affiliations, consistent with current 16S rRNA phylogeny studies. Genetic similarity and diversity suggest that
B. afzelii
and
B. garinii
are close relatives and were perhaps recently derived from
B. burgdorferi
. MLVA provides both phylogenetic relationships and additional resolution to discriminate among strains of
Borrelia
species. This new level of strain identification and discrimination will allow more detailed epidemiological and phylogenetic analysis in future studies.
Improvement of Differentiation and Interpretability of Spoligotyping for
Mycobacterium tuberculosis
Complex Isolates by Introduction of New Spacer Oligonucleotides
Authors: van der Zanden A. G. M. , Kremer K. , Schouls L. M. , Caimi K. , Cataldi A. , Hulleman A. , Nagelkerke N. J. D. , van Soolingen D. 
Published: November 2002

ABSTRACT

The direct repeat (DR) region in
Mycobacterium tuberculosis
complex strains is composed of multiple well-conserved 36-bp DRs interspersed with nonrepetitive DNA spacer sequences of similar size. Clinical isolates show extensive polymorphism in this DR region, and this has led to the development of a 43-spacer reversed line blot methodology: spoligotyping. Although this method has contributed significantly to the molecular epidemiology of tuberculosis in the last decade, the discriminatory power and the readability of this method were not found to be optimal. In order to improve the discriminatory power, the usefulness of 43 redesigned oligonucleotides and the usefulness of 51 new spacer oligonucleotides were evaluated. For 314
M. tuberculosis
complex strains isolated in the central part of The Netherlands over a 5-year period, 264 different IS
6110
RFLP types could be distinguished, and 160 different spoligotype patterns were identified by traditional spoligotyping. After the introduction of 51 new spacer oligonucleotides, 14 additional spoligotypes were recognized. This enabled us to split 11 clusters of isolates identified by the traditional spoligotyping. Furthermore, on the basis of the new spacer oligonucleotides a dichotomy was found among the Beijing genotype isolates. Among 76
Mycobacterium bovis
strains, 20 patterns were found by traditional spoligotyping and 30 patterns were found by novel probe spoligotyping, respectively. Nine
M. bovis
subsp.
caprae
isolates yielded six patterns by traditional spoligotyping and eight patterns by novel probe spoligotyping. A part of the redesigned oligonucleotides slightly improved the reading of spoligotype patterns. The reproducibility of spoligotyping, based on internal control probes, invariably yielded a high score; only 4 (1%) of the 314 patient isolates gave discrepant results. Analysis of a set of 31 duplicate
M. tuberculosis
complex strains demonstrated a 10% error rate for the identification of blinded duplicate samples. In a redundancy analysis, 40 essential spacer oligonucleotides of the 94-spacer sequences were selected, yielding the same number of spoligotype patterns. We propose to leave the traditional commercialized first-generation membrane for spoligotyping unchanged for current applications and to introduce a second-generation spoligotyping membrane whenever extended discrimination is required, e.g., for low-copy-number IS
6110
strains or for phylogenetic studies of Beijing genotype strains.
Simultaneous Identification of
Mycobacterium
Genus and
Mycobacterium tuberculosis
Complex in Clinical Samples by 5′-Exonuclease Fluorogenic PCR
Authors: García-Quintanilla Albert , González-Martín Julián , Tudó Griselda , Espasa Mateu , Jiménez de Anta María T. 
Published: November 2002

ABSTRACT

Early diagnosis of tuberculosis and screening of other mycobacteria is required for the appropriate management of patients. We have therefore developed a 5′-exonuclease fluorogenic PCR assay in a single-tube balanced heminested format that simultaneously detects
Mycobacterium tuberculosis
complex (MTC) and members of the
Mycobacterium
genus (MYC) using the 16S ribosomal DNA target directly on clinical samples. One hundred twenty-seven clinical samples (65 smear negative and 62 smear positive) with a positive culture result from 127 patients were tested, including 40 negative control specimens. The finding of both a positive MTC and probe value and a positive MYC probe value confirmed the presence of MTC or mycobacteria with a 100% positive predictive value. However, a negative value for MTC or MYC did not discount the presence of mycobacteria in the specimen. Interestingly, the addition of the MYC probe allowed the diagnosis of an additional 7% of patients with tuberculosis and rapid screening of nontuberculous mycobacteria (NTM). Thus, over 75% of the patients were diagnosed with mycobacterial disease by PCR. The sensitivity was much higher on smear-positive samples (90.3%) than smear-negative samples (49.2%) and was slightly higher for MTC than NTM samples. With regard to the origin of the sample, MTC pulmonary samples gave better results than others. In conclusion, we believe this test may be useful for the rapid detection of mycobacteria in clinical samples and may be a valuable tool when used together with conventional methods and the clinical data available.
Comparison of Two Commercial Assays with Expert Microscopy for Confirmation of Symptomatically Diagnosed Malaria
Authors: Iqbal Jamshaid , Khalid Nabila , Hira Parsotam R. 
Published: November 2002

ABSTRACT

Conventional light microscopy has been the established method for malaria diagnosis. However, recently several nonmicroscopic rapid diagnostic tests have been developed for situations in which reliable microscopy may not be available. This study was conducted to evaluate the diagnostic performance of a recently introduced ICT Malaria Pf/Pv test. This assay detects
Plasmodium falciparum
histidine-rich protein 2 antigen (PfHRP-2) for
P. falciparum
diagnosis and pan-malarial antigen for
P. vivax
diagnosis. In this study we compared the performance of ICT Malaria Pf/Pv with microscopy of Giemsa-stained blood films and with an OptiMAL test that detects
Plasmodium
lactate dehydrogenase (pLDH) antigen. A total of 750 clinically suspected malaria patients were examined at local health centers in Kuwait. Both the antigen tests had a high degree of specificity (>98%) for detection of malaria infection. However, they were less sensitive than microscopy. Compared with microscopy the ICT Malaria PF/pf test failed to detect malaria infection in 93 (34%) of 271 malaria patients (11% of patients with
P. falciparum
and 37% of patients with
P. vivax
) and the OptiMAL test failed to detect malaria infection in 41 (15%) of 271 malaria patients (7% of patients with
P. falciparum
and 13% of patients with
P. vivax
). The sensitivities of the ICT Malaria Pf/Pv and OptiMAL tests for detection of
P. falciparum
infection were 81 and 87%, and those for detecting
P. vivax
were 58 to 79%, respectively. The sensitivity of the ICT Malaria Pf/Pv and OptiMAL tests decreased significantly to 23 and 44%, respectively, at parasite densities of <500/μl. Both of the tests also produced a number of false-positive results. Overall, the performance of the OptiMAL test was better than that of the ICT Malaria Pf/Pv test. However, our results raise particular concern over the sensitivity of the ICT Malaria Pf/Pv test for detection of
P. vivax
infection. Further developments appear necessary to improve the performance of the ICT Malaria Pf/Pv test.
Identification ofListeriaSpecies by Microarray-Based Assay
Authors: Volokhov Dmitriy , Rasooly Avraham , Chumakov Konstantin , Chizhikov Vladimir 
Published: November 2002

ABSTRACTWe have developed a rapid microarray-based assay for the reliable detection and discrimination of six species of theListeriagenus:L. monocytogenes,L. ivanovii,L. innocua,L. welshimeri,L. seeligeri, andL. grayi. The approach used in this study involves one-tube multiplex PCR amplification of six target bacterial virulence factor genes (iap,hly,inlB,plcA,plcB, andclpE), synthesis of fluorescently labeled single-stranded DNA, and hybridization to the multiple individual oligonucleotide probes specific for eachListeriaspecies and immobilized on a glass surface. Results of the microarray analysis of 53 reference and clinical isolates ofListeriaspp. demonstrated that this method allowed unambiguous identification of all sixListeriaspecies based on sequence differences in theiapgene. Another virulence factor gene,hly, was used for detection and genotyping allL. monocytogenes, allL. ivanovii, and 8 of 11L. seeligeriisolates. Other members of the genusListeriaand threeL. seeligeriisolates did not contain thehlygene. There was complete agreement between the results of genotyping based on thehlyandiapgene sequences. AllL. monocytogenesisolates were found to be positive for theinlB,plcA,plcB, andclpEvirulence genes specific only to this species. Our data onListeriaspecies analysis demonstrated that this microarray technique is a simple, rapid, and robust genotyping method that is also a potentially valuable tool for identification and characterization of bacterial pathogens in general.
Apodemus
Species Mice Are Reservoir Hosts of
Borrelia garinii
OspA Serotype 4 in Switzerland
Authors: Huegli D. , Hu C. M. , Humair P.-F. , Wilske B. , Gern L. 
Published: November 2002

ABSTRACT

Among
Borrelia burgdorferi
sensu lato isolates, seven outer surface protein A (OspA) serotypes have been described: serotypes 1 and 2 correspond to
B. burgdorferi
sensu stricto and
Borrelia afzelii
, respectively, and serotypes 3 to 7 correspond to
Borrelia garinii
. In Europe, serotype 4 has never been isolated from
Ixodes ricinus
ticks until recently, although this serotype has been frequently isolated from cerebrospinal fluid from patients. In Europe,
B. afzelii
and
B. burgdorferi
sensu stricto were found associated with rodents and
B. garinii
was found associated with birds. In this study, the reservoir role of
Apodemus
mice for
B. garinii
OspA serotype 4 was demonstrated by xenodiagnosis.
Apodemus
mice are the first identified reservoir hosts for
B. garinii
OspA serotype 4.
New Serological Assay for Detection of Putative
Helicobacter pylori
Virulence Factors
Authors: Park Chang-Young , Cho Yong-Kyun , Kodama Tadashi , El-Zimaity Hala M. T. , Osato Michael S. , Graham David Y. , Yamaoka Yoshio 
Published: November 2002

ABSTRACT

We evaluated a new immunoblot assay (Helicoblot 2.1) for
Helicobacter pylori
infection and CagA and VacA status by using serum samples from 222 patients. The test accurately detected
H. pylori
infection and VacA status, but improvements in the interpretation criteria are required before it can be recommended for determination of CagA status.
Mycobacterium avium
subsp.
paratuberculosis
Strains from Cattle and Sheep Can Be Distinguished by a PCR Test Based on a Novel DNA Sequence Difference
Authors: Collins Desmond M. , De Zoete May , Cavaignac Sonia M. 
Published: November 2002

ABSTRACT

A DNA sequence differing between sheep and cattle types of
Mycobacterium avium
subsp.
paratuberculosis
was identified and used to develop a PCR test. The test unequivocally distinguished all sheep types from cattle types and was negative for a wide range of other strains from the
Mycobacterium avium
-
Mycobacterium intracellulare
complex. The test will be useful for epidemiological purposes, particularly in hosts such as deer that can be easily infected with either type.
Loss of a 29-Kilodalton Outer Membrane Protein in
Acinetobacter baumannii
Is Associated with Imipenem Resistance
Authors: Limansky Adriana S. , Mussi María Alejandra , Viale Alejandro M. 
Published: November 2002

ABSTRACT

We analyzed the possible causes of imipenem (IPM) resistance in multidrug-resistant isolates of
Acinetobacter baumannii
. Comparison of the outer membrane protein (OMP) profiles of two genomically related strains (Ab288 [IPM sensitive] and Ab242 [IPM resistant]) indicated the conspicuous loss of a 29-kDa polypeptide in the Ab242 strain. No carbapenemase activity was detected in any of these strains. The treatment of Ab288 with sodium salicylate resulted in IPM resistance and the loss of the 29-kDa OMP. In addition, IPM-resistant clones of Ab288 which were selected by repetitive culturing in increasing concentrations of this antibiotic also showed the absence of this 29-kDa OMP.
Roseomonas gilardii
Infection: Case Report and Review
Authors: Shokar Navkiran K. , Shokar Gurjeet S. , Islam Jamal , Cass Alvah R. 
Published: November 2002

ABSTRACT

Roseomonas gilardii
is a bacterium that has been indicated as a rare cause of human infections. The case of a patient presenting with cellulitis and bacteremia secondary to
R. gilardii
is described together with the clinical characteristics of infection with this organism obtained from a review of cases previously reported.
Rotavirus Gastroenteritis and Central Nervous System (CNS) Infection: Characterization of the VP7 and VP4 Genes of Rotavirus Strains Isolated from Paired Fecal and Cerebrospinal Fluid Samples from a Child with CNS Disease
Authors: Iturriza-Gómara M. , Auchterlonie I. A. , Zaw W. , Molyneaux P. , Desselberger U. , Gray J. 
Published: November 2002

ABSTRACT
Rotavirus RNA was detected in the cerebrospinal fluid (CSF) of a child with central nervous system disease symptoms associated with rotavirus gastroenteritis. The rotavirus isolates from the fecal and CSF samples were genotyped as G1P[8]. Sequence analysis of the VP7 and VP4 proteins derived from the fecal and CSF samples were remarkably similar to each other and to G1P[8] rotavirus strains commonly circulating in the community and associated with gastroenteritis.
Pulmonary Localization of
Enterocytozoon bieneusi
in an AIDS Patient: Case Report and Review
Authors: Botterel F. , Minozzi C. , Vittecoq D. , Bourée P. 
Published: November 2002

ABSTRACT

Enterocytozoon bieneusi
is an agent of intestinal microsporidiosis leading to malabsorption syndrome and diarrhea in AIDS patients. Respiratory tract microsporidiosis due to
Encephalitozoon
spp. has been reported. To date, however, only two cases of pulmonary involvement of
E. bieneusi
have been documented for patients with intestinal microsporidiosis. We report here another pulmonary localization of
E. bieneusi
in a human immunodeficiency virus-infected patient. Clinical features of these three cases are reviewed.
E. bieneusi
can colonize the respiratory tract but could be considered a simple carriage associated with an intestinal infection.
Patterns of illness and injury encountered in amateur ocean yacht racing: an analysis of the British Telecom Round the World Yacht Race 1996–1997
Authors: Price C J S , Spalding T J W , McKenzie C 
Published: November 2002

Objectives:To quantify the incidence and type of medical problem arising during an amateur circumnavigation yacht race, the BT Global Challenge.Methods:All cases from 14 participating yachts in a confidential medical log completed by an appointed medic were reported.Results:A total of 685 cases were reported, of which 299 (43.6%) were injuries and 386 (56.4%) illnesses. The subtype of injury, illness, and three evacuations at sea are described.Conclusion:Injury and other forms of medical problem are relatively common in an amateur long distance ocean yacht race. Most can be adequately managed at sea, provided that optimal communication, training, and equipment are provided and maintained.
Prolonged Survival After Complete Resection of Disseminated Melanoma and Active Immunotherapy With a Therapeutic Cancer Vaccine
Authors: Hsueh Eddy C. , Essner Richard , Foshag Leland J. , Ollila David W. , Gammon Guy , O’Day Steven J. , Boasberg Peter D. , Stern Stacey L. , Ye Xing , Morton Donald L. 
Published: November 2002

PURPOSE: The curative effect of surgery in certain patients with metastatic melanoma suggests the presence of endogenous antitumor responses. Because melanoma is immunogenic, we investigated whether a therapeutic cancer vaccine called Canvaxin (CancerVax Corporation, Carlsbad, CA) could enhance antitumor immune responses and thereby prolong survival. PATIENTS AND METHODS: Of 263 patients who underwent complete resection of American Joint Committee on Cancer stage IV melanoma, 150 received postoperative adjuvant vaccine therapy and 113 did not. The overall survival (OS) for the two groups was compared by Cox regression. Further survival analysis was performed by matched-pair analysis according to three prognostic variables: sex, metastatic site, and number of tumor-involved organ sites. RESULTS: Five-year OS rates were 39% for vaccine and 19% for nonvaccine patients. On multivariate analysis, vaccine therapy was the most significant prognostic variable in this cohort (P = .0001). Analysis of 107 matched pairs of vaccine and nonvaccine patients revealed a significant OS advantage for vaccine therapy (P = .0009): 5-year OS was 39% for vaccine patients versus 20% for nonvaccine patients. There was a significant delayed-type hypersensitivity (DTH) response to adjuvant vaccine therapy (P = .0001), and OS was significantly correlated with DTH to vaccine (P = .0001) but not with DTH to purified protein derivative (PPD), a control antigen. CONCLUSION: Prolonged survival was observed in patients who received postoperative active immunotherapy with Canvaxin therapeutic cancer vaccine. The correlation of survival with vaccine-DTH responses but not PPD-DTH indicates a treatment-specific effect. These findings suggest that adjuvant active specific immunotherapy should be considered after cytoreductive surgery for advanced melanoma.