bluuebunny/crossref_metadata_2025_split huggingface dataset:
Analysis of the Genetic Variability of Genes Encoding the RNA III-Activating Components Agr and TRAP in a Population of
Staphylococcus aureus
Strains Isolated from Cows with Mastitis
Authors: Gilot Philippe , Lina Gérard , Cochard Thierry , Poutrel Bernard 
Published: October 2002

ABSTRACT

The expression of
Staphylococcus aureus
virulence proteins is under the control of RNA III, a central pleiotropic regulator transcribed from the
agr
locus. RNA III is activated by at least two two-component systems, one encoded by the
agr
locus (AgrC-AgrA) and another encoded outside of this locus (TRAP-RAP). In this work, we developed new typing methods based on genes encoding these two systems, which we used to characterize a nonclonal population of
S. aureus
bovine mastitis isolates. Twelve
agr
restriction types were identified in this population, but the majority of strains (56.3%) were grouped in the R III-A1 type. No strain isolated from humans, whose
agr
sequence is available from GenBank, was found to belong to this major type. Restriction maps constructed for all of those
agr
variants allowed the linking of all types in an evolution scheme and their grouping in one of the four
agr
interference groups. This analysis indicates that groups 2, 3, and 4 probably evolved from the more frequently encountered type, which belongs to group 1.
agr
group 1 was also found to be the most prevalent (69.0% of the strains) and the most polymorphic interference group. By developing an
agr
group-specific multiplex PCR, we confirmed the above classification of strains in the
agr
interference groups. Four allelic variants of
trap
were also identified, indicating that this two-component system is also polymorphic. The majority of strains was grouped in the
trap
1 type (71.8%). Whereas no relationships between
agr
group and
trap
types were found, strains of similar
agr
restriction type were also of similar
trap
type (with the exception of strains belonging to the
agr
R IV-A5 and R VI-A8 types). Our analysis indicates that
S. aureus
isolated from cows has predominantly a clonal structure and that the highly prevalent
agr
R III-A1,
trap
1 type (56.3% of the strains) probably possesses a genetic background which endows it with superior ability to infect the bovine mammary gland.
Occult Hepatitis B Virus Infection and Clinical Outcomes of Patients with Chronic Hepatitis C
Authors: Kao Jia-Horng , Chen Pei-Jer , Lai Ming-Yang , Chen Ding-Shinn 
Published: October 2002

ABSTRACTAlthough occult hepatitis B virus (HBV) infections in individuals without detectable hepatitis B surface antigen (HBsAg) may occur and have been reported to be common in patients with chronic hepatitis C, the clinical relevance remains controversial. We searched for serum HBV DNA in 210 HBsAg-negative patients with hepatitis C virus (HCV)-related liver disease (110 patients with chronic hepatitis, 50 patients with cirrhosis, and 50 patients with hepatocellular carcinoma) by PCR. Most of the patients had detectable antibodies to HBsAg or HBV core antigen. All of the 110 chronic hepatitis C patients were treated with a combination therapy consisting of interferon plus ribavirin. In addition, 100 HBsAg-negative healthy adults served as controls. Thirty-one of the 210 patients (14.8%) had HBV DNA in their sera, as did 15 of the 100 healthy controls (15%). HBV DNA was not detected in the sera of those negative for serological markers of HBV infection. In patients with chronic HCV infection, the prevalence of occult HBV infection did not parallel the severity of liver disease (14.5% in patients with chronic hepatitis, 8% in patients with liver cirrhosis, and 22% in patients with hepatocellular carcinoma). In addition, the sustained response to combination therapy against hepatitis C was comparable between patients with and without occult HBV infection (38 versus 39%). In conclusion, these data suggest that occult HBV infection does not have clinical significance in chronic hepatitis C patients residing in areas where HBV infection is endemic.
Experimental versus In Silico Fluorescent Amplified Fragment Length Polymorphism Analysis of
Mycobacterium tuberculosis
: Improved Typing with an Extended Fragment Range
Authors: Sims Elizabeth J. , Goyal Madhu , Arnold Catherine 
Published: October 2002

ABSTRACT

Whole-genome fingerprinting fluorescent amplified fragment length polymorphism (FAFLP) data were compared with in silico data for the sequenced strains of
Mycobacterium tuberculosis
(H37Rv and CDC1551). For this G+C-rich genome, many predicted fragments were not detected experimentally. For H37Rv, only 108 (66%) of the 163 predicted
Eco
RI-
Mse
I fragments between 100 and 500 bp were visualized in vitro. FAFLP was also used to identify polymorphism in 10 clinical isolates of
M. tuberculosis
characterized previously by IS
6110
typing, examining fragments of up to 1,000 bp in size rather than up to 500 bp as was done previously. Five isolates had unique IS
6110
profiles and were not known to be epidemiologically related, two isolates were the same single-band IS
6110
type but were not known to be epidemiologically related, and the remaining three isolates were epidemiologically related with identical IS
6110
profiles. Analysis of fragments in the 500- to 1,000-bp range using nonselective primers differentiated better between strains than analysis of fragments in the 50- to 500-bp range using a set of four selective primers. Seventeen polymorphic fragments were identified between 500 and 1,000 bp in size compared with nine polymorphic fragments between 50 and 500 bp. Using the 500- to 1,000-bp analysis, a level of discrimination similar to that of IS
6110
typing was achieved which, unlike the IS
6110
typing, was able to differentiate the two
M. tuberculosis
strains, each of which had only a single copy of IS
6110
.
Method for Inactivating and Fixing Unstained Smear Preparations of
Mycobacterium tuberculosis
for Improved Laboratory Safety
Authors: Chedore Pamela , Th'ng Cecelia , Nolan Dennis H. , Churchwell George M. , Sieffert David E. , Hale Yvonne M. , Jamieson Frances 
Published: October 2002

ABSTRACT

The inactivation of smears that contain
Mycobacterium tuberculosis
for microscopy before removal of the material from a biosafety cabinet is an important safety factor in preventing the potential transmission of tuberculosis to laboratory workers. The fixing and inactivating properties of heat flaming, 70% ethanol, and 1, 3, and 5% phenol in ethanol for smears containing
M. tuberculosis
were investigated. Heat flaming failed to inactivate the smear material, whereas 5% phenol in ethanol successfully fixed and inactivated all smears containing
M. tuberculosis
both from concentrated sputum samples and from culture material.
Remarkable Genetic Polymorphism among
Entamoeba histolytica
Isolates from a Limited Geographic Area
Authors: Haghighi Ali , Kobayashi Seiki , Takeuchi Tsutomu , Masuda Gohta , Nozaki Tomoyoshi 
Published: October 2002

ABSTRACT

In order to understand genetic polymorphisms among
Entamoeba histolytica
strains in a limited geographic area and among restricted social populations, we studied nucleotide polymorphism in DNA regions that do not encode proteins (locus 1-2 and locus 5-6) and in genes coding for chitinase and for serine-rich
E. histolytica
protein. Thirty
E. histolytica
isolates from domestically infected Japanese amebiasis patients (male homosexuals and residents in institutions for the mentally handicapped) and four reference strains were examined. PCR revealed remarkable polymorphisms in both the number and size of the PCR fragments containing these loci. Polymorphisms in lengths, types, and numbers of internal repeat units were observed in locus 1-2 and the repeat-containing region of serine-rich
E. histolytica
protein among the Japanese isolates. In contrast, polymorphism at locus 5-6 was observed almost exclusively in the number of repeats of a 16-nucleotide unit. The repeat-containing region of chitinase appeared to be the least polymorphic among the four loci with a single dominant genotype representing 66% (20 out of 30) of all of the isolates. Isolates obtained from male homosexuals showed a more complex genetic polymorphism than those from residents in institutions. Considering all four polymorphic loci together, all 19 Japanese isolates from male homosexuals were distinct. In contrast, all isolates obtained from mass-infection cases at a single institution had an identical genotype, suggesting that these cases were caused by a single
E. histolytica
strain. No significant correlation was found between genotypes and zymodemes or between genotypes and clinical presentations, e.g., colitis or liver abscess. Certain genotypes were observed with higher frequencies in male homosexuals or residents of institutions. These data indicate that genotyping of the
E. histolytica
isolates by using these four polymorphic loci could serve as a tool to fingerprint individual isolates. We propose that genotyping of ameba isolates should help to determine geographic origins of isolates and routes of transmission.
Sensitive Detection of RNA Viruses Associated with Gastroenteritis by a Hanging-Drop Single-Tube Nested Reverse Transcription-PCR Method
Authors: Ratcliff Rodney Mark , Doherty James Christopher , Higgins Geoffrey David 
Published: October 2002

ABSTRACT
The detection of the human RNA viruses, calicivirus and astrovirus, requires high sensitivity and broad reactivity. A novel single-tube nested reverse transcription-PCR (RT-PCR) method is described here, in which all of the required reagents are included in the one tube; however, those required for the nested amplification are separated in a “hanging drop” in the cap to be introduced by centrifugation after the RT and first-round cDNA amplification steps. Broad reactivity was obtained by using primer cocktails covering the published sequence variation in the primer targets. The method was evaluated with clinical fecal samples from outbreak and sporadic cases. Norwalk-like virus types 1 and 2 and rotavirus were the causal agents in 10 of 12 outbreaks. A viral agent was detected in 44% of 197 samples from sporadic infections in patients presenting to community health centers and a children's hospital. Interestingly, whereas rotavirus was more common than astrovirus in patients presenting to the hospital (33 and 7.6%, respectively), the reverse was true for patients presenting to community health centers (4.2 and 34%, respectively).
Estimation of the Rate of Unrecognized Cross-Contamination with
Mycobacterium tuberculosis
in London Microbiology Laboratories
Authors: Ruddy M. , McHugh T. D. , Dale J. W. , Banerjee D. , Maguire H. , Wilson P. , Drobniewski F. , Butcher P. , Gillespie S. H. 
Published: October 2002

ABSTRACT
Isolates from patients with confirmed tuberculosis from London were collected over 2.5 years between 1995 and 1997. Restriction fragment length polymorphism (RFLP) analysis was performed by the international standard technique as part of a multicenter epidemiological study. A total of 2,779 samples representing 2,500 individual patients from 56 laboratories were examined. Analysis of these samples revealed a laboratory cross-contamination rate of between 0.54%, when only presumed cases of cross-contamination were considered, and 0.93%, when presumed and possible cases were counted. Previous studies suggest an extremely wide range of laboratory cross-contamination rates of between 0.1 and 65%. These data indicate that laboratory cross-contamination has not been a common problem in routine practice in the London area, but in several incidents patients did receive full courses of therapy that were probably unnecessary.
Human Cytomegalovirus DNA in Plasma and Serum Specimens of Renal Transplant Recipients Is Highly Fragmented
Authors: Boom René , Sol Cees J. A. , Schuurman Tim , van Breda Alex , Weel Jan F. L. , Beld Marcel , ten Berge Ineke J. M. , Wertheim-van Dillen Pauline M. E , de Jong Menno D. 
Published: October 2002

ABSTRACT
Quantitation of cytomegalovirus (CMV) DNA in plasma and serum by PCR is increasingly used to identify patients at risk for developing CMV disease and to monitor the efficacy of antiviral therapy. Although CMV DNA levels are generally interpreted as viral loads, the exact nature of the viral DNA in these specimens is unknown. We studied the state of CMV DNA in plasma and serum specimens obtained from three renal transplant recipients at peak viral DNA levels during primary CMV infection. For this purpose, DNA isolated from these specimens was fractionated by size, and CMV DNA levels in the resulting DNA fractions were measured by quantitative PCR targeted at large (578-bp) and small (134-bp) amplicons. These experiments showed that the molecular sizes of DNA fragments from which CMV DNA is amplified were small (<2,000 bp), indicating that CMV DNA in plasma and serum is highly fragmented. Furthermore, CMV DNA levels were consistently higher when targeted at the smaller amplicon, providing additional evidence for the fragmentation of viral DNA. In conclusion, the first results with three patients have shown that CMV DNA in plasma and serum is highly fragmented and does not necessarily reflect the amount of infectious virus. These observations have potential consequences for understanding CMV pathogenesis and interpreting CMV DNA levels in individual patient management.
Incidence of Human Astrovirus in Central Australia (1995 to 1998) and Comparison of Deduced Serotypes Detected from 1981 to 1998
Authors: Schnagl Roger D. , Belfrage Kate , Farrington Rachel , Hutchinson Kylie , Lewis Victoria , Erlich John , Morey Fran 
Published: October 2002

ABSTRACT
The incidence of astrovirus infection was determined among infants and young children admitted to Alice Springs Hospital (central Australia) with gastroenteritis from 1995 to 1998. Astrovirus was detected by reverse transcription-PCR in 33 of 495 stool samples, and this represented 4.3% of a total of 774 stool samples tested for astrovirus, rotavirus, and Norwalk-like viruses. Astrovirus incidence was substantially lower than that of rotavirus but higher than that of Norwalk-like viruses both overall and in each of the 4 years individually. Over the period from 1981 to 1998, including the period from 1981 to 1994 during which astrovirus was identified only by electron microscopy, astrovirus serotypes (deduced from genotypes) 1, 2, 3, and 4 were identified. Deduced serotypes 1, 3, and 4 all appeared regularly over this 18-year period. Also over this period, nucleotide variation (in some cases substantial) in a section of the capsid protein precursor region of the virus genome was evident among strains of all four of the deduced central Australian serotypes. Consequent amino acid changes were, however, only evident among deduced serotype 3 strains. Geographic variation at both the genome and the resultant amino acid levels was evident among strains of all four of the deduced central Australian serotypes and their respective prototypes isolated in the United Kingdom.
Detection of
gyrA
Mutations in Quinolone-Resistant
Salmonella enterica
by Denaturing High-Performance Liquid Chromatography
Authors: Eaves Deborah J. , Liebana Ernesto , Woodward Martin J. , Piddock Laura J. V. 
Published: October 2002

ABSTRACT

Denaturing high-performance liquid chromatography (DHPLC) was evaluated as a rapid screening and identification method for DNA sequence variation detection in the quinolone resistance-determining region of
gyrA
from
Salmonella
serovars. A total of 203 isolates of
Salmonella
were screened using this method. DHPLC analysis of 14 isolates representing each type of novel or multiple mutations and the wild type were compared with LightCycler-based PCR-
gyrA
hybridization mutation assay (GAMA) and single-strand conformational polymorphism (SSCP) analyses. The 14 isolates gave seven different SSCP patterns, and LightCycler detected four different mutations. DHPLC detected 11 DNA sequence variants at eight different codons, including those detected by LightCycler or SSCP. One of these mutations was silent. Five isolates contained multiple mutations, and four of these could be distinguished from the composite sequence variants by their DHPLC profile. Seven novel mutations were identified at five different loci not previously described in quinolone-resistant salmonella. DHPLC analysis proved advantageous for the detection of novel and multiple mutations. DHPLC also provides a rapid, high-throughput alternative to LightCycler and SSCP for screening frequently occurring mutations.
Single-Tube Nested PCR for Detection ofTritrichomonasfoetusin Feline Feces
Authors: Gookin Jody L. , Birkenheuer Adam J. , Breitschwerdt Edward B. , Levy Michael G. 
Published: October 2002

ABSTRACTTritrichomonasfoetus, a venereal pathogen of cattle, was recently identified as an inhabitant of the large intestine in young domestic cats with chronic diarrhea. Recognition of the infection in cats has been mired by unfamiliarity withT.foetusin cats as well as misdiagnosis of the organisms asPentatrichomonashominisorGiardiasp. when visualized by light microscopy. The diagnosis ofT.foetuspresently depends on the demonstration of live organisms by direct microscopic examination of fresh feces or by fecal culturing. AsT.foetusorganisms are fastidious and fragile, routine flotation techniques and delayed examination and refrigeration of feces are anticipated to preclude the diagnosis in numerous cases. The objective of this study was to develop a sensitive and specific PCR test for the diagnosis of felineT.foetusinfection. A single-tube nested PCR was designed and optimized for the detection ofT.foetusin feline feces by using a combination of novel (TFITS-F and TFITS-R) and previously described (TFR3 and TFR4) primers. The PCR is based on the amplification of a conserved portion of theT.foetusinternal transcribed spacer (ITS) region (ITS1 and ITS2) and the 5.8S rRNA gene. The absolute detection limit of the single-tube nested PCR was 1 organism, while the practical detection limit was 10 organisms per 200 mg of feces. Specificity was examined by usingP.hominis,Giardialamblia,and feline genomic DNA. Our results demonstrate that the single-tube nested PCR is ideally suited for (i) diagnostic testing of feline fecal samples that are found negative by direct microscopy and culturing and (ii) definitive identification of microscopically observable or cultivated organisms.
Detection of Antibodies Directed against Human Herpesvirus 6 U94/REP in Sera of Patients Affected by Multiple Sclerosis
Authors: Caselli Elisabetta , Boni Michela , Bracci Arianna , Rotola Antonella , Cermelli Claudio , Castellazzi Massimiliano , Di Luca Dario , Cassai Enzo 
Published: October 2002

ABSTRACTThe association between human herpesvirus 6 (HHV-6) and multiple sclerosis (MS) is controversial. In fact, it is difficult to establish a causative role of HHV-6, due to the high prevalence of latently infected individuals in the healthy population. Therefore, the presence of virus sequences in tissue biopsy does not support a viral role, and serological assays do not show significant differences between MS patients and control populations. The only viral gene expressed during latency is U94/rep. Therefore, we have developed a serological assay for the detection of antibodies specifically directed against U94/REP protein. Different populations were analyzed by enzyme-linked immunosorbent assay, including healthy controls, MS patients, and subjects with diseases unrelated to HHV-6 infection, including other neurological diseases. The results show statistically significant differences (P> 0.01) between MS patients and control groups, both in antibody prevalence (87 and 43.9%, respectively) and in geometric mean titer (1:515 and 1:190, respectively). The detection of antibodies specific for HHV-6 U94/REP shows that the immune system is exposed to this antigen during natural infection. The higher prevalence and higher titers of antibodies to U94/REP suggest that MS patients and control groups might experience different exposures to HHV-6.
Performance Assessment of Two Commercial Amplification Assays for Direct Detection ofMycobacterium tuberculosisComplex from Respiratory and Extrapulmonary Specimens
Authors: Piersimoni Claudio , Scarparo Claudio , Piccoli Paola , Rigon Alessandra , Ruggiero Giuliana , Nista Domenico , Bornigia Stefano 
Published: October 2002

ABSTRACTThe new BDProbeTec ETMycobacterium tuberculosisComplex Direct Detection Assay (DTB) was compared with the enhancedM. tuberculosisAmplified Direct Test (AMTDII). The system is an automated walkaway system characterized by simultaneous DNA amplification (strand displacement amplification) and real-time fluorometric detection. It also contains an internal amplification control (IAC) designed to identify inhibition from the processed samples. The AMTDII assay amplifies rRNA by transcription-mediated amplification; it uses hybridization with a chemoluminescent probe as a detection system and is entirely manual. A total of 515N-acetyl-l-cysteine-sodium hydroxide-decontaminated respiratory (n= 331) and extrapulmonary (n= 184) sediments (from 402 patients) were tested in parallel by both assays. The results were compared with those of acid-fast staining and culture (solid plus liquid media), setting the combination of culture and clinical diagnosis as the “gold standard.” Culture results from the tested specimens were as follows: 121Mycobacterium tuberculosiscomplex (MTB) (98 smear-positive), 46 nontuberculous mycobacteria (38 smear-positive), and 338 culture-negative results. After resolution of the discrepant results, the percent sensitivity, percent specificity, and positive and negative likelihood ratios for AMTDII were 88%, 99.2%, 110, and 0.11 for respiratory specimens and 74.3%, 100%, 740, and 0.26 for extrapulmonary specimens, respectively. The corresponding values for DTB were 94.5%, 99.6%, 235, and 0.05 for respiratory specimens and 92.3%, 100%, 920, and 0.07 for extrapulmonary specimens, respectively. The cumulative difference for all tuberculosis-positive extrapulmonary specimens was significant (P= 0.03). The overall inhibition rate for DTB was 5% (26 specimens). We conclude that both amplification assays proved to be rapid and specific for the detection of MTB in clinical samples and particularly feasible for a routine laboratory work flow. DTB combines a labor-intensive specimen preparation procedure with a completely automated amplification and detection. Finally, differences between AMTDII and DTB sensitivities were associated with the presence of inhibitory samples that the former assay, lacking IAC, could not detect.