bluuebunny/crossref_metadata_2025_split huggingface dataset:
Real-Time PCR System for Detection of Orthopoxviruses and Simultaneous Identification of Smallpox Virus
Authors: Olson Victoria A. , Laue Thomas , Laker Miriam T. , Babkin Igor V. , Drosten Christian , Shchelkunov Sergei N. , Niedrig Matthias , Damon Inger K. , Meyer Hermann 
Published: May 2004

ABSTRACT

A screening assay for real-time LightCycler (Roche Applied Science, Mannheim, Germany) PCR identification of smallpox virus DNA was developed and compiled in a kit system under good manufacturing practice conditions with standardized reagents. In search of a sequence region unique to smallpox virus, the nucleotide sequence of the 14-kDa fusion protein gene of each of 14 variola virus isolates of the Russian World Health Organization smallpox virus repository was determined and compared to published sequences. PCR primers were designed to detect all Eurasian-African species of the genus
Orthopoxvirus
. A single nucleotide mismatch resulting in a unique amino acid substitution in smallpox virus was used to design a hybridization probe pair with a specific sensor probe that allows reliable differentiation of smallpox virus from other orthopoxviruses by melting-curve analysis. The applicability was demonstrated by successful amplification of 120 strains belonging to the orthopoxvirus species variola, vaccinia, camelpox, mousepox, cowpox, and monkeypox virus. The melting temperatures (
T

m

s) determined for 46 strains of variola virus (
T

m

s, 55.9 to 57.8°C) differed significantly (
P
= 0.005) from those obtained for 11 strains of vaccinia virus (
T

m

s, 61.7 to 62.7°C), 15 strains of monkeypox virus (
T

m

s, 61.9 to 62.2°C), 40 strains of cowpox virus (
T

m

s, 61.3 to 63.7°C), 8 strains of mousepox virus (
T

m

, 61.9°C), and 8 strains of camelpox virus (
T

m

s, 64.0 to 65.0°C). As most of the smallpox virus samples were derived from infected cell cultures and tissues, smallpox virus DNA could be detected in a background of human DNA. By applying probit regression analysis, the analytical sensitivity was determined to be 4 copies of smallpox virus target DNA per sample. The DNAs of several human herpesviruses as well as poxviruses other than orthopoxviruses were not detected by this method. The assay proved to be a reliable technique for the detection of orthopoxviruses, with the advantage that it can simultaneously identify variola virus.
mecA
Locus Diversity in Methicillin-Resistant
Staphylococcus aureus
Isolates in Brisbane, Australia, and the Development of a Novel Diagnostic Procedure for the Western Samoan Phage Pattern Clone
Authors: Huygens Flavia , Stephens Alex J. , Nimmo Graeme R. , Giffard Philip M. 
Published: May 2004

ABSTRACT

An emerging public health phenomenon is the increasing incidence of methicillin-resistant
Staphylococcus aureus
(MRSA) infections that are acquired outside of health care facilities. One lineage of community-acquired MRSA (CA-MRSA) is known as the Western Samoan phage pattern (WSPP) clone. The central aim of this study was to develop an efficient genotyping procedure for the identification of WSPP isolates. The approach taken was to make use of the highly variable region downstream of
mecA
in combination with a single nucleotide polymorphism (SNP) defined by the
S. aureus
multilocus sequence typing (MLST) database. The premise was that a combinatorial genotyping method that interrogated both a highly variable region and the genomic backbone would deliver a high degree of informative power relative to the number of genetic polymorphisms interrogated. Thirty-five MRSA isolates were used for this study, and their gene contents and order downstream of
mecA
were determined. The CA-MRSA isolates were found to contain a truncated
mecA
downstream region consisting of
mecA
-HVR-IS
431 mec
-dcs-Ins117, and a PCR-based method for identifying this structure was developed. The hospital-acquired isolates were found to contain eight different
mecA
downstream regions, three of which were novel. The Minimum SNPs computer software program was used to mine the
S. aureus
MLST database, and the
arcC
272G polymorph was identified as 82% discriminatory for ST-30. A real-time PCR assay was developed to interrogate this SNP. We found that the assay for the truncated
mecA
downstream region in combination with the interrogation of
arcC
position 272 provided an unambiguous identification of WSPP isolates.
Development and Evaluation of a Novel Loop-Mediated Isothermal Amplification Method for Rapid Detection of Severe Acute Respiratory Syndrome Coronavirus
Authors: Thai Hong Thi Cam , Le Mai Quynh , Vuong Cuong Duc , Parida Manmohan , Minekawa Harumi , Notomi Tsugunori , Hasebe Futoshi , Morita Kouichi 
Published: May 2004

ABSTRACT

The development and evaluation of a one-step single-tube accelerated real-time quantitative reverse transcription (RT) loop-mediated isothermal amplification (LAMP) assay is reported for rapid detection of the severe acute respiratory syndrome coronavirus (SARS-CoV) replicase gene. A total of 49 samples (15 throat washes, 13 throat swabs, and 21 combined throat and nasal swabs) collected from patients admitted to the Hanoi-French and Ninhbinh hospitals in Vietnam during the SARS epidemic were evaluated and compared to conventional RT-PCR. The RT-LAMP assay demonstrated 100-fold-greater sensitivity, with a detection limit of 0.01 PFU. The sensitivity and specificity of RT-LAMP assay for detecting viral RNA in clinical specimens with regard to RT-PCR were 100 and 87%, respectively. The specificity of the RT-LAMP assay was further validated by restriction analysis as well as nucleotide sequencing of the amplified product. The concentration of virus in most of the clinical samples was 0.1 PFU (0.1 to 10
2
PFU), as determined from the standard curve of SARS RT-LAMP and based on the time of positivity. The assay procedure is quite simple, wherein the amplification is carried out in a single tube under isothermal conditions at 63°C, and the result can be obtained in less than 1 h (as early as 11 min). Thus, the RT-LAMP assay reported here has the advantages of rapid amplification, simple operation, and easy detection and will be useful for rapid and reliable clinical diagnosis of SARS-CoV in developing countries.
Variable Ratio of Hepatitis C Virus RNA to Viral Core Antigen in Patient Sera
Authors: Schüttler Christian G. , Thomas Christine , Discher Thomas , Friese Georg , Lohmeyer Jürgen , Schuster Ralph , Schaefer Stephan , Gerlich Wolfram H. 
Published: May 2004

ABSTRACT

Quantification of hepatitis C virus (HCV) core antigen and RNA in serum samples leads to a highly variable ratio of both. It is not clear whether this is due to the inaccuracy of RNA quantification or whether both are independent parameters in a certain range. We established a real-time reverse transcription (RT)-PCR for HCV RNA that combines very high sensitivity with a large dynamic range and minimal standard deviations. The assay was calibrated with the first international standard, 96/790, and the international genotype panel for HCV from the National Institute of Biological Standardisation and Control. A linear readout was obtained between 200 and 5 × 10
7
IU/ml. The detection limit was 80 IU/ml, the reproducibility was <0.05 log, and the standard error within one run was <0.01. Comparison of the method with the Roche Monitor competitive RT-PCR revealed its high accuracy. The core protein concentration was determined within a range from 1.5 to 400 pg/ml by using the preliminary trak-C assay from Ortho Clinical Diagnostics. Correlating the HCV RNA levels with core antigen concentrations in 197 serum samples from 23 interferon-treated patients, a average ratio of 7,900 IU of HCV RNA per pg of core antigen was estimated, but the variability of this ratio exceeded largely the variability of the two assays, ranging from 50 to 20,000 IU/pg. Theoretically, HCV should contain ca. 43,000 IU of RNA/pg core. In conclusion, the core antigen assay seems to detect, in addition to complete virions, RNA-free core protein structures, which enhances its sensitivity (98% in this group). The variable ratio of RNA and core protein is not mainly due to standard deviations of quantification but could be an additional parameter for treatment follow-up and state of viral replication.
Amplification of Coccidioidal DNA in Clinical Specimens by PCR
Authors: Johnson Suzanne M. , Simmons Keira A. , Pappagianis Demosthenes 
Published: May 2004

ABSTRACT

Coccidioides
DNA was amplified from serum by a PCR using coccidioid-specific primers. A 239-bp product was visualized when 10 fg of exogenous coccidioidal DNA was subjected to amplification. This product was demonstrated in some human and mouse sera prior to the detection of coccidioidal antibodies.
Use of Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem Repeat Typing To Examine Genetic Diversity ofMycobacterium tuberculosisin Singapore
Authors: Sun Yong-Jiang , Bellamy Richard , Lee Ann S. G. , Ng Sze Ta , Ravindran Sindhu , Wong Sin-Yew , Locht Camille , Supply Philip , Paton Nicholas I. 
Published: May 2004

ABSTRACTStrain typing using variable-number tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTR) is a powerful tool for studying the epidemiology and genetic relationships ofMycobacterium tuberculosisisolates. For this study, isolates from 291 patients in Singapore were genotyped by this method. One hundred sixty-six distinct MIRU-VNTR patterns were detected. One hundred sixty-two strains were grouped into 1 of 35 different MIRU-VNTR clusters and 131 isolates were unique. In this sample collection, 9 of the 12 MIRU-VNTR loci were moderately or highly discriminative according to their allelic diversities. The Hunter-Gaston discriminatory index was 0.975, indicating the high power of discrimination of MIRU-VNTR typing. By direct comparisons with previously typed MIRU-VNTR patterns and by genetic relationship analyses, we could identify and clearly define four epidemic groups ofM. tuberculosisin our sample, corresponding to the W/Beijing, East-Africa-Indian, Haarlem, and Delhi genotype families. Furthermore, MIRU-VNTR typing was able to clearly distinguish ancestral and modernM. tuberculosisstrains as defined by TbD1 genomic deletion analysis. These results indicate that MIRU-VNTR typing can be a useful first-line tool for studying the genetic diversity ofM. tuberculosisisolates in a large urban setting such as Singapore.
Reverse Transcriptase PCR Diagnostic Assay for the Coronavirus Associated with Severe Acute Respiratory Syndrome
Authors: Hui Raymond K. H. , Zeng Fanya , Chan Charis M. N. , Yuen K. Y. , Peiris Joseph S. M. , Leung Frederick C. C. 
Published: May 2004

ABSTRACT
Recent outbreaks of severe acute respiratory syndrome (SARS) have spurred intense research efforts around the world to deal with the serious threat to health posed by this novel coronavirus. A rapid, reliable diagnostic assay is needed for monitoring the spread of the disease. Here we report a method for eliminating false-negative results and increasing test sensitivity, based on the hypothesis that the message encoded by the nucleocapsid (N) gene is the most abundant during viral infection. Nasopharyngeal aspirates and stool samples were obtained from suspected SARS patients with major clinical symptoms and a significant history of close contact with infected patients. Total RNAs were extracted in a 96-well format, together with pig kidney epithelial (PK-15) cells as an internal control for extraction efficiency. PCR inhibitors were removed by ethanol precipitation, and a PCR for the pig β-actin gene was used as a positive control for all clinical samples. Samples were analyzed by a reverse transcriptase PCR assay. Northern blot analysis was performed to demonstrate differences in subgenomic transcripts of the virus, and a real-time quantitative PCR was employed to compare the sensitivities of two loci (1b and N). The detection rate of the assay reached 44.4% on day 9 after the onset of the disease. The diagnostic PCR amplifying the N gene gave an average of a 26.0% (6.3 to 60.0%) stronger intensity signal than that for the 1b gene. In conclusion, the nucleocapsid gene represents an additional sensitive molecular marker for the diagnosis of the SARS coronavirus and can be further adapted for use in a high-throughput platform assay.
Assessment of Virulence of Pigeon Isolates of
Salmonella enterica
subsp.
enterica
Serovar Typhimurium Variant Copenhagen for Humans
Authors: Pasmans Frank , Van Immerseel Filip , Hermans Katleen , Heyndrickx Marc , Collard Jean-Marc , Ducatelle Richard , Haesebrouck Freddy 
Published: May 2004

ABSTRACT

Salmonella enterica
serovar Typhimurium variant Copenhagen was isolated from 5 of 152 (3.3%) feral pigeons from the city of Ghent (Belgium) and from 26 pooled fecal samples from 114 pigeon lofts (22.8%). These isolates belonged to phage type (PT) 99. Seven of the pigeon isolates were further compared in vitro to five human variant Copenhagen isolates, 2 isolates of PT 208, 1 isolate each of PT 120 and U302, and a nontypeable isolate. No differences in invasiveness in human intestinal epithelial Caco-2 cells were found. The human strains, however, were able to multiply significantly more inside human THP-1 macrophages than the pigeon strains. After inoculation of mice with a pigeon PT 99 strain, high numbers of
Salmonella
bacteria were shed with the feces, the internal organs were heavily colonized, and the animals showed severe clinical symptoms resulting in death. In conclusion, the less-pronounced ability of the pigeon variant Copenhagen strains to multiply inside human macrophages than human strains as well as the lack of human PT 99 isolates during 2002, despite the relatively high frequency of this PT in the pigeon population, suggest these strains to be of low virulence to humans. However, the high virulence for mice of the tested strain implies that rodents may act as reservoirs.
Longitudinal Study of the Excretion Patterns of ThermophilicCampylobacterspp. in Young Pet Dogs in Denmark
Authors: Hald Birthe , Pedersen Karl , Wainø Michael , Jørgensen Jens Christian , Madsen Mogens 
Published: May 2004

ABSTRACTTheCampylobacterexcretion patterns of 26 domestic pet dogs were described in a longitudinal study. The dogs entered the study between 3 and 8 months of age and were monitored until 2 years of age. They were tested monthly forCampylobactercarriage in stool samples that were cultured on theCampylobacter-selective media CAT and modified CCDA agar at 37 and 42°C. This study comprised 366 fecal swab samples, of which 278 (76.2%) were found to beCampylobacterpositive, with the following distribution of species: 75.0%Campylobacter upsaliensis, 19.4%Campylobacter jejuni, 2.1%Campylobacter lari, 0.7%Campylobacter coli, and 2.8%Campylobacterspp. Isolates were typed by pulsed-field gel electrophoresis (PFGE) to elucidate the strain excretion pattern. All study dogs excretedCampylobacterspp. during the study period. At 3 months of age, 60% of the dogs carriedCampylobacter, increasing to nearly 100% carriers at 1 year of age, whereafter the carriage rate decreased to 67% at 24 months of age. The PFGE types showed that individual dogs were often colonized by unique strains ofC. upsaliensisfor several months, up to 21 months or longer. TheseC. upsaliensisstrains were either clonal (or underwent concurrent minor mutative changes) or independent strains. In contrast, the excretedC. jejuniisolates were much more diverse and, in most cases, only seen in one sample from each dog. A high degree of diversity among different dogs was seen. We conclude that young domestic pet dogs excretedCampylobacterspp. during the majority of their puppyhood and adolescent period. In generalC. upsaliensisstrains were excreted for months, with short-term interruptions by or cocolonization with other transitoryCampylobacterspp., predominantlyC. jejuni. C. jejuniwas more prevalent in dogs between 3 months and 1 year of age than in dogs between 1 and 2 years of age.
Detection and Differentiation of Human Parvovirus Variants by Commercial Quantitative Real-Time PCR Tests
Authors: Hokynar Kati , Norja Päivi , Laitinen Harri , Palomäki Pekka , Garbarg-Chenon Antoine , Ranki Annamari , Hedman Klaus , Söderlund-Venermo Maria 
Published: May 2004

ABSTRACT
Parvovirus B19 causes a variety of diseases in humans, with outcomes ranging from asymptomatic to severe, such as chronic anemia in immunocompromised patients or fetal hydrops and death after maternal infection during pregnancy. The virus may be transmitted via plasma-derived products. According to the results of solvent-detergent safety studies, an upper limit of B19 DNA in plasma pools was recently defined. To restrict the input of B19 virus into production pools, a quantitative nucleic acid test is a prerequisite. We examined the suitability of the two commercial quantitative B19 PCR tests, LightCycler-Parvovirus B19 quantification kit (Roche Diagnostics) and RealArt Parvo B19 LC PCR (Artus) for detection, quantification, and differentiation of the three known B19 genotypes, including the newly described erythrovirus variants (genotypes 2 and 3). The former kit was highly sensitive for genotype 1 but was not suitable for detection of genotype 2 or one of two genotype 3 strains. The latter kit detected and differentiated all three genotypes, albeit with lower sensitivity for one of the genotype-3 strains. We furthermore assessed the prevalence of the three B19 virus genotypes in blood donors, by screening pooled plasma samples derived from 140,160 Finnish blood-donor units. None of the pools contained detectable levels of B19 virus genotypes 2 or 3. The origin, mode of transmission, and clinical significance of these genotypes are unknown and deserve further study. The RealArt Parvo B19 LC PCR is suitable for detection, quantification, and differentiation of all three B19 virus genotypes in molecular and clinical research.
Expertise of French Laboratories in Detection, Genotyping, and Quantification of Hepatitis C Virus RNA in Serum
Authors: Lefrère Jean-Jacques , Roudot-Thoraval Françoise , Lunel Françoise , Alain Sophie , Chaix Marie-Laure , Dussaix Elisabeth , Gassin Michèle , Izopet Jacques , Pawlotsky Jean-Michel , Payan Christopher , Stoll-Keller Françoise , Thibault Vincent , Trabaud Mary-Anne , Bettinger Dominique , Bogard Marc , Branger Michel , Buffet-Janvresse Claudine , Charrois Anne , Defer Christine , Laffont Catherine , Lerable Joëlle , Levayer Thierry , Martinot-Peignoux Michèle , Mercier Bernard , Rosenberg Arielle R. 
Published: May 2004

ABSTRACT
Before initiating new large-scale therapeutic trials for hepatitis C virus (HCV)-infected patients, the French Health Authorities for HCV research decided to organize an evaluation of the expertise of laboratories that could be engaged to undertake molecular biology assays in such trials; 21 experienced laboratories participated in this national evaluation of laboratory expertise, which was performed in two successive rounds. The first round evaluated the laboratories for their abilities to detect HCV RNA in serum, determine genotypes, and quantify HCV RNA loads. The results observed by qualitative assays for HCV RNA detection were 100% sensitivity and 100% specificity for all laboratories. The genotyping results were 100% concordant for 9 laboratories and greater than 90% for 10 laboratories. By contrast, large coefficients of variation were observed for quantitative determination of HCV RNA loads, leading to a second round with standardized quantitative assays only. The dispersion of the results was larger by the AMPLICOR HCV Monitor assay than by the branched-DNA assay (mean coefficients of variation, 57.4 and 16.9%, respectively). In the majority of cases, discrepancies between the results of the two tests were found for samples with high viral loads. These results indicate the usefulness of validating, by controlling for expertise, both the reliabilities of laboratories involved in multicenter work and the standardized assays chosen for use in the evaluation of the biological impacts of new therapies.
DNA Macroarray for Identification and Typing of
Staphylococcus aureus
Isolates
Authors: Trad Salim , Allignet Jeanine , Frangeul Lionel , Davi Marilyne , Vergassola Massimo , Couve Elisabeth , Morvan Anne , Kechrid Amel , Buchrieser Carmen , Glaser Philippe , El Solh Névine 
Published: May 2004

ABSTRACT

A DNA macroarray containing 465 intragenic amplicons was designed to identify
Staphylococcus aureus
at the species level and to type
S. aureus
isolates. The genes selected included those encoding (i)
S. aureus
-specific proteins, (ii) staphylococcal and enterococcal proteins mediating antibiotic resistance and factors involved in their expression, (iii) putative virulence proteins and factors controlling their expression, and (iv) proteins produced by mobile elements. The macroarray was hybridized with the cellular DNAs of 80
S. aureus
clinical isolates that were previously typed by analyses of their antibiograms and SmaI patterns. The set selected contained unrelated, endemic, and outbreak-related isolates belonging to 45 SmaI genotypes. In a gene content dendrogram, the 80 isolates were distributed into 52 clusters. The outbreak-related isolates were linked in the same or a closely related cluster(s). Clustering based on gene content provided a better discrimination than SmaI pattern analysis for the tested
mecA
+
isolates that were endemic to Europe. All of the antibiotic resistance genes detected could be correlated with their corresponding phenotypes, except for one isolate which carried a
mecA
gene without being resistant. The 16 isolates responsible for bone infections were distinguishable from the 12 isolates from uninfected nasal carriers by a significantly higher prevalence of the
sdrD
gene coding for a putative SD (serine-aspartate) adhesin (in 15 and 7 isolates, respectively). In conclusion, the macroarray designed for this study offers an attractive and rapid typing method which has the advantage of providing additional information concerning the gene content of the isolate of interest.
Comparison of Conventional and Molecular Methods for Identification of Aerobic Catalase-Negative Gram-Positive Cocci in the Clinical Laboratory
Authors: Bosshard P. P. , Abels S. , Altwegg M. , Böttger E. C. , Zbinden R. 
Published: May 2004

ABSTRACT

Over a period of 18 months we have evaluated the use of 16S ribosomal DNA (rDNA) sequence analysis as a means of identifying aerobic catalase-negative gram-positive cocci in the clinical laboratory. A total of 171 clinically relevant strains were studied. The results of molecular analyses were compared with those obtained with a commercially available phenotypic identification system (API 20 Strep system; bioMérieux sa, Marcy l'Etoile, France). Phenotypic characterization identified 67 (39%) isolates to the species level and 32 (19%) to the genus level. Seventy-two (42%) isolates could not be discriminated at any taxonomic level. In comparison, 16S rDNA sequencing identified 138 (81%) isolates to the species level and 33 (19%) to the genus level. For 42 of 67 isolates assigned to a species with the API 20 Strep system, molecular analyses yielded discrepant results. Upon further analysis it was concluded that among the 42 isolates with discrepant results, 16S rDNA sequencing was correct for 32 isolates, the phenotypic identification was correct for 2 isolates, and the results for 8 isolates remained unresolved. We conclude that 16S rDNA sequencing is an effective means for the identification of aerobic catalase-negative gram-positive cocci. With the exception of
Streptococcus pneumoniae
and beta-hemolytic streptococci, we propose the use of 16S rDNA sequence analysis if adequate species identification is of concern.