bluuebunny/crossref_metadata_2025_split huggingface dataset:
Simplifying Collection of Corneal Specimens in Cases of Suspected Bacterial Keratitis
Authors: Kaye Stephen B. , Rao Prasad G. , Smith Godfrey , Scott John A. , Hoyles Sharon , Morton Clare E. , Willoughby Colin , Batterbury Mark , Harvey Graham 
Published: July 2003

ABSTRACT

Identification of the causative organisms in suspected bacterial keratitis traditionally involves collecting multiple corneal scrapes, which are plated directly onto different solid agar culture media. Difficulties have been reported with this practice, so the development of a simpler diagnostic method in suspected bacterial keratitis would be useful. It is unclear whether a single corneal scrape sent to the microbiology laboratory in a liquid transport culture medium (indirect method) is as reliable for the diagnosis of bacterial keratitis as inoculation of multiple scrapes directly onto agar plates (direct method). To investigate this, bacterial recovery was assessed following transfer and transport of different concentrations and types of bacteria from an artificially contaminated surgical blade into brain heart infusion (BHI). Bacterial recovery rates between the proposed (indirect) and standard (direct) method were then compared after the in vitro inoculation of pig corneas and following specimen collection in patients with presumed bacterial ulcerative keratitis. Recovery of bacteria from contaminated surgical blades was found to be the same from both solid and liquid culture media. There was no significant difference in the numbers of positive cultures from solid (direct) and liquid (indirect) culture media, both in the experimental pig cornea inoculation study (
P
= 0.34) and in experiments with patients with clinical infections (
P
= 0.4), with an 85.2% agreement between methods (kappa = 0.61,
P
< 0.0001). In conclusion, therefore, the collection of two corneal scrapes, one used for Gram staining and the other transported in BHI followed by plating and subculturing in an enrichment medium, provides a simple method for the investigation of presumed bacterial keratitis.
Pathogenic and Antigenic Properties of Phylogenetically Distinct Reassortant H3N2 Swine Influenza Viruses Cocirculating in the United States
Authors: Richt Jürgen A. , Lager Kelly M. , Janke Bruce H. , Woods Roger D. , Webster Robert G. , Webby Richard J. 
Published: July 2003

ABSTRACTSwine influenza is an acute respiratory disease caused by type A influenza viruses. Before 1998, swine influenza virus isolates in the United States were mainly of the classical H1N1 lineage. Since then, phylogenetically distinct reassortant H3N2 viruses have been identified as respiratory pathogens in pigs on U.S. farms. The H3N2 viruses presently circulating in the U.S. swine population are triple reassortants containing avian-like (PA and PB2), swine-like (M, NP, and NS), and human-like (HA, NA, and PB1) gene segments. Recent sequence data show that the triple reassortants have acquired at least three distinct H3 molecules from human influenza viruses and thus form three distinct phylogenetic clusters (I to III). In this study we analyzed the antigenic and pathogenic properties of viruses belonging to each of these clusters. Hemagglutination inhibition and neutralization assays that used hyperimmune sera obtained from caesarian-derived, colostrum-deprived pigs revealed that H3N2 cluster I and cluster III viruses share common epitopes, whereas a cluster II virus showed only limited cross-reactivity. H3N2 viruses from each of the three clusters were able to induce clinical signs of disease and associated lesions upon intratracheal inoculation into seronegative pigs. There were, however, differences in the severity of lesions between individual strains even within one antigenic cluster. A correlation between the severity of disease and pig age was observed. These data highlight the increased diversity of swine influenza viruses in the United States and would indicate that surveillance should be intensified to determine the most suitable vaccine components.
Use of 18S rRNA Gene-Based PCR Assay for Diagnosis of
Acanthamoeba
Keratitis in Non-Contact Lens Wearers in India
Authors: Pasricha Gunisha , Sharma Savitri , Garg Prashant , Aggarwal Ramesh K. 
Published: July 2003

ABSTRACT

Identification of
Acanthamoeba
cysts and trophozoites in ocular tissues requires considerable expertise and is often time-consuming. An 18S rRNA gene-based PCR test, highly specific for the genus
Acanthamoeba
, has recently been reported in the molecular diagnosis of
Acanthamoeba
keratitis. This PCR assay was compared with conventional microbiological tests for the diagnosis of
Acanthamoeba
keratitis. In a pilot study, the PCR conditions with modifications were first tested on corneal scrapings from patients with culture-proven non-contact lens-related
Acanthamoeba
, bacterial, and fungal keratitis. This was followed by testing of corneal scrapings from 53 consecutive cases of microbial keratitis to determine sensitivity, specificity, and predictive values of the assay. All corneal scrapings from patients with proven
Acanthamoeba
keratitis showed a 463-bp amplicon, while no amplicon was obtained from patients with bacterial or fungal keratitis. Some of these amplified products were sequenced and compared with EMBL database reference sequences to validate these to be of
Acanthamoeba
origin. Out of 53 consecutive cases of microbial keratitis included for evaluating the PCR, 10 (18.9%) cases were diagnosed as
Acanthamoeba
keratitis on the basis of combined results of culture, smear, and PCR of corneal scrapings. Based on culture results as the “gold standard,” the sensitivity of PCR was the same as that of the smear (87.5%); however, the specificity and the positive and negative predictive values of PCR were marginally higher than the smear examination (97.8 versus 95.6%, 87.5 versus 77.8%, and 97.8 versus 97.7%) although the difference was not significant. This study confirms the efficacy of the PCR assay and is the first study to evaluate a PCR-based assay against conventional methods of diagnosis in a clinical setting.
Comparative Evaluation of the Total Hepatitis C Virus Core Antigen, Branched-DNA, and Amplicor Monitor Assays in Determining Viremia for Patients with Chronic Hepatitis C during Interferon Plus Ribavirin Combination Therapy
Authors: Veillon Pascal , Payan Christopher , Picchio Gastón , Maniez-Montreuil Michèle , Guntz Philippe , Lunel Françoise 
Published: July 2003

ABSTRACT

An assay prototype designed to detect and quantify total hepatitis C virus [HCV] core antigen (HCV core Ag) protein in serum and plasma in the presence or absence of anti-HCV antibodies has been recently developed by Ortho-Clinical Diagnostics. The aim of the study was to evaluate the sensitivity, specificity, and reproducibility of the Total HCV core Ag assay in comparison with two quantitative assays for HCV RNA: Quantiplex HCV RNA 2.0 (bDNA v2.0) or Versant HCV RNA 3.0 (bDNA v3.0) assays and the Cobas Amplicor HCV Monitor version 2.0 (HCM v2.0) test. We have studied samples of a well-characterized panel and samples from patients with chronic hepatitis C treated with interferon alone or with ribavirin. We have also compared the kinetics of HCV core Ag and HCV RNA in the follow-up of treated patients. The HCV core Ag assay exhibited linear behavior across samples from different genotypes. The coefficients of variation for intra- and interassay performance were 5.11 and 9.95%, respectively. The specificity of the assay tested in blood donors was 99.5%. Samples from HCV-infected patients showed that the correlation between the HCV core Ag and the two HCV RNA quantitative assays (bDNA and HCM v2.0) was 0.8 and 0.7, respectively. This correlation was maintained across different genotypes of HCV (
r
2
= 0.64 to 0.94). Baseline HCV core Ag values were significantly lower in sustained responders to interferon (IFN) than in other groups of patients (5.31 log
10
[10
4
pg/ml] versus 5.99 log
10
[10
4
pg/ml];
P
< 0.001). In patients treated with IFN or combination therapy, we found an association between a decrease of more than 2 log IU/ml in viral load, undetectable HCV core Ag, and sustained response. Among sustained responders to IFN alone or combination therapy and among relapsers after IFN alone, 84 out of 101 (83.2%) had undetectable HCV core Ag, and 76 out of 96 (79.2%) had a viral load decrease of ≥2 log IU/ml, after 1 month of treatment. In conclusion, the Total HCV core Ag assay is a new useful test for the detection of HCV viremia and the monitoring of patients treated with IFN alone or in combination with ribavirin.
Real-Time PCR-Based System for Simultaneous Quantification of Human Papillomavirus Types Associated with High Risk of Cervical Cancer
Authors: Moberg Martin , Gustavsson Inger , Gyllensten Ulf 
Published: July 2003

ABSTRACT

We have previously shown that women with a high titer of human papillomavirus type 16 (HPV16) in cervical epithelial cells have an increased risk of developing cervical carcinoma in situ. In order to study the relationship between viral DNA amount and risk of cervical carcinoma for the HPV types most commonly found in cervical tumors, we developed a real-time PCR assay for the detection and quantification of HPV16, -18, -31, -33, -35, -39, -45, -52, -58, and -67. These HPV types are analyzed in two reaction tubes, allowing for independent quantification of three viral types, or groups of viral types, in each reaction. A separate reaction is used for estimating the number of a nuclear single-copy gene and is used to calculate the HPV copy number per genomic DNA equivalent in the sample. The system has a dynamic range from 10
2
to 10
7
HPV copies per assay and is applicable to both fresh clinical samples and DNA extracted from archival samples. Reconstitution experiments, made to mimic infections with several HPV types, shows that individual HPV types can be detected in a mixture as long as they represent 1 to 10% of the main type. The system was evaluated with respect to technical specificity and sensitivity, reproducibility, reagent stability, and sample preparation protocol and then used to analyze clinical samples. This homogeneous assay provides a fast and sensitive way for estimating the viral load of a series of the most frequent oncogenic HPV types in biopsies, as well as cervical smear samples.
Comparison of
Salmonella
Chromogenic Medium with DCLS Agar for Isolation of
Salmonella
Species from Stool Specimens
Authors: Cassar Robert , Cuschieri Paul 
Published: July 2003

ABSTRACT

Salmonella
chromogenic medium (SCM; Oxoid, Basingstoke, United Kingdom), a new selective chromogenic medium, was compared to DCLS agar (Oxoid) for the detection and presumptive identification of
Salmonella
species from stool samples. This medium contains two chromogenic substrates, Magenta-cap (5-bromo-6-chloro-3-indolylcaprylate), which is hydrolyzed by
Salmonella
species to give magenta colonies, and X-Gal (5-bromo-4-chloro-3-indolyl-β
-d-
galactopyranoside), which is incorporated to visualize β
-d-
galactosidase-producing organisms as blue colonies. Thus, non-
Salmonella
organisms appear blue or are not stained by any of the chromogens of the medium. A total of 500 stool samples were investigated by plating them directly and after selenite enrichment on DCLS agar and SCM. A total of 44
Salmonella
-positive stool samples were detected. The sensitivities for direct plating and after enrichment were 22.7 and 81.8%, respectively, for DCLS agar, and for SCM these values were 34.1 and 100%, respectively. The specificities for direct plating and after enrichment were 82.5 and 72.8%, respectively, for DCLS agar and 98.5 and 95.8%, respectively, for SCM. According to these results, the sensitivities of SCM and DCLS agar were comparable on primary plating. However, the sensitivity of SCM was significantly higher after enrichment. In addition, the specificity of SCM was also significantly higher than that of DCLS agar both before and after enrichment. On the basis of these results, SCM can be recommended for the isolation of
Salmonella
species from stool samples in preference to DCLS agar.
Assessment by Meta-Analysis of PCR for Diagnosis of Smear-Negative Pulmonary Tuberculosis
Authors: Sarmiento Olga L. , Weigle Kristen A. , Alexander Janet , Weber David J. , Miller William C. 
Published: July 2003

ABSTRACTWe conducted a meta-analysis to assess the performance of PCR for the diagnosis of smear-negative pulmonary tuberculosis (SPT) and to identify factors that account for differences in the diagnostic accuracy of different studies. Studies published before February 2002 were included if sensitivity and specificity of PCR in smear-negative respiratory or gastric-aspirate specimens could be calculated. Analysis was conducted by using summary receiver operating characteristics models. Sensitivity and specificity ranged from 9 to 100% and from 25 to 100%, respectively. Fewer than 40% of the 50 studies reported results by number of patients, reported clinical characteristics of patients, or used as a reference standard combined culture and clinical criteria. Studies that included bronchial specimens showed higher accuracy than studies that evaluated only sputum specimens or included gastric aspirates. Studies that did not report that tests were applied blindly showed higher accuracy than those reporting blind testing. Increased sensitivity due to the use of DNA purification methods was associated with decreased specificity. Studies published after 1995, using Amplicor or dUTP-UNG, were associated with an increase in specificity at the expense of lower sensitivity. We concluded that PCR is not consistently accurate enough to be routinely recommended for the diagnosis of SPT. However, PCR of bronchial specimens could be useful in highly suspicious SPT cases. Studies not reporting blind testing are likely to overestimate accuracy of PCR. Future evaluation of PCR accuracy should be conducted by patient and type of respiratory specimen, blindly, by using a reference standard that combines culture and clinical criteria and addresses the issue of how patient characteristics affect PCR accuracy.
Rapid Identification ofRhodococcus equiby a PCR Assay Targeting thechoEGene
Authors: Ladrón Néstor , Fernández Marta , Agüero Jesús , Zörn Bruno González , Vázquez-Boland José A. , Navas Jesús 
Published: July 2003

ABSTRACTThe actinomyceteRhodococcus equiis an important pathogen of horses and an emerging opportunistic pathogen of humans. Identification ofR. equiby classical bacteriological techniques is sometimes difficult, and misclassification of an isolate is not uncommon. We report here on a specific PCR assay for the rapid and reliable identification ofR. equi. It is based on the amplification of a fragment of thechoEgene encoding cholesterol oxidase. ThechoE-based PCR was assessed by using a panel of strains comprising 132 isolates from different sources and of different geographical origins, all initially identified biochemically asR. equi, and 30 isolates of representative non-R. equiactinomycete species, including cholesterol oxidase producers. The expected 959-bp amplicon was observed only withR. equiisolates, as confirmed by sequencing of a variable region of the 16S RNA gene from a random sample of 20 PCR-positive isolates. AllR. equiisolates gave a positivechoE-based PCR result, which correlated with a high degree of conservation of thechoEgene. Three of the 132 strains originally identified asR. equiwere negative for thechoEgene, and subsequent analysis of their 16S RNA gene sequences confirmed that they belonged to other bacterial species (Dietzia maris,Mycobacterium peregrinum, andStaphylococcus epidermidis). All non-R. equiisolates were negative by thechoE-based PCR. ATCC 21387, the only known isolate ofBrevibacterium sterolicum, gave a 959-bp amplicon whose DNA sequence was virtually identical to that ofR. equi choE. Comparison of the 16S RNA genes indicated that ATCC 21387 should be considered anR. equiisolate.
Real-Time PCR Assay for Detection of Fluoroquinolone Resistance Associated with
grlA
Mutations in
Staphylococcus aureus
Authors: Lapierre Pascal , Huletsky Ann , Fortin Véronique , Picard François J. , Roy Paul H. , Ouellette Marc , Bergeron Michel G. 
Published: July 2003

ABSTRACT

Resistance to fluoroquinolones among clinical isolates of
Staphylococcus aureus
has become a clinical problem. Therefore, a rapid method to identify
S. aureus
and its susceptibility to fluoroquinolones could provide clinicians with a useful tool for the appropriate use of these antimicrobial agents in the health care settings. In this study, we developed a rapid real-time PCR assay for the detection of
S. aureus
and mutations at codons Ser-80 and Glu-84 of the
grlA
gene encoding the DNA topoisomerase IV, which are associated with decreased susceptibility to fluoroquinolones. The detection limit of the assay was 10 genome copies per reaction. The PCR assay was negative with DNA from all 26 non-
S. aureus
bacterial species tested. A total of 85
S. aureus
isolates with various levels of fluoroquinolone resistance was tested with the PCR assay. The PCR assay correctly identified 100% of the
S. aureus
isolates tested compared to conventional culture methods. The correlation between the MICs of ciprofloxacin, levofloxacin, and gatifloxacin and the PCR results was 98.8%. The total time required for the identification of
S. aureus
and determination of its susceptibility to fluoroquinolones was about 45 min, including DNA extraction. This new rapid PCR assay represents a powerful method for the detection of
S. aureus
and its susceptibility to fluoroquinolones.
Polymorphic Nucleotide within the Promoter of Nitrate Reductase (NarGHJI) Is Specific for
Mycobacterium tuberculosis
Authors: Stermann Marion , Bohrssen Antje , Diephaus Catharina , Maass Silvia , Bange Franz-Christoph 
Published: July 2003

ABSTRACT

Mycobacterium tuberculosis
rapidly reduces nitrate, leading to the accumulation of nitrite. This characteristic served for the past 40 years to differentiate
M. tuberculosis
from other members of the
Mycobacterium tuberculosis
complex (MTBC), such as
Mycobacterium bovis
(non-BCG [referred to here as simply “
M. bovis
”]),
Mycobacterium bovis
BCG,
Mycobacterium africanum
, or
Mycobacterium microti.
Here, a
narG
deletion in
M. tuberculosis
showed that rapid nitrite accumulation of
M. tuberculosis
is mediated by
narGHJI
. Analysis of
narG
mutants of
M. bovis
and
M. bovis
BCG showed that, as in
M. tuberculosis
, nitrite accumulation was mediated by
narGHJI
, and no other nitrate reductase was involved. However, in contrast to
M. tuberculosis
, accumulation was delayed for several days. Comparison of the
narGHJI
promoter revealed that, at nucleotide −215 prior to the start codon of
narG
,
M. tuberculosis
carried a thymine residue, whereas the bovine mycobacteria carried a cytosine residue. Using LightCycler technology we examined 62 strains of
M. tuberculosis
,
M. bovis
,
M. bovis
BCG,
M. microti
, and
M. africanum
and demonstrated that this single nucleotide polymorphism was specific for
M. tuberculosis.
For further differentiation within the MTBC, we included, by using LightCycler technology, the previously described analysis of
oxyR
polymorphism, which is specific for the bovine mycobacteria, and the RD1 polymorphism, which is specific for
M. bovis
BCG. Based on these results, we suggest a LightCycler format for rapid and unambiguous diagnosis of
M. tuberculosis
,
M. bovis
, and
M. bovis
BCG.
Optimization of Media for Detection of Hydrogen Peroxide Production by
Lactobacillus
Species
Authors: Rabe L. K. , Hillier S. L. 
Published: July 2003

ABSTRACT

A healthy vaginal ecosystem has been shown to be protective against the acquisition of human immunodeficiency virus and gonorrhea, and women who are colonized with H
2
O
2
-producing lactobacilli are more likely to maintain a normal vaginal flora than women with lactobacilli that do not produce H
2
O
2
. The purpose of this study was to formulate a testing medium that better supports the growth and detection of H
2
O
2
by a broader range of lactobacilli than a published, widely used agar formulation (TMB). The new medium (TMB-Plus) consists of brucella agar base, 3,3′,5,5′-tetramethylbenzidine, horseradish peroxidase, starch, vitamin K, hemin, magnesium sulfate, manganese sulfate, and horse serum. To validate the new formula, 256 vaginal isolates and ATCC strains were inoculated onto TMB-Plus and, for comparison, onto TMB. Growth was enhanced for 69% of the isolates on TMB-Plus, and 48% had enhanced color production. The percentage of H
2
O
2
-positive isolates increased from 71% on TMB to 79% on TMB-Plus. Formulations using Rogosa or MRS agar base in combination with peroxidase and a chromogen did not support the growth of all of the strains of
Lactobacillus
, and fewer H
2
O
2
-producing strains were detected on these formulations than on TMB-Plus. This new medium better supports the growth of a wider range of
Lactobacillus
strains isolated from the vagina and enhances the color production of H
2
O
2
-producing strains.
Evaluation of the Editing Process in Human Immunodeficiency Virus Type 1 Genotyping
Authors: Huang Diana D. , Eshleman Susan H. , Brambilla Donald J. , Palumbo Paul E. , Bremer James W. 
Published: July 2003

ABSTRACTSequencing-based human immunodeficiency virus type 1 (HIV-1) genotyping assays require subjective interpretation (editing) of sequence data from multiple primers to form consensus sequences and identify antiretroviral drug resistance mutations. We assessed interlaboratory variations in editing and their impact on the recognition of resistance mutations. Six samples were analyzed in a central laboratory by using a research-use-only HIV-1 genotyping system previously produced by Applied Biosystems. The electronic files of individual primer sequences from the samples were sent to 10 laboratories to compare sequence editing strategies. Each sequence data set included sequences from seven primers spanning protease codons 1 to 99 and reverse transcriptase codons 1 to 320. Each laboratory generated a consensus sequence for each sample and completed a questionnaire about editing strategy. The amount of editing performed, the concordance of consensus sequences among the laboratories, and the identification of resistance mutations were evaluated. Sequence agreement was high among the laboratories despite wide variations in editing strategies. All laboratories identified 66 (88%) of 75 resistance mutations in the samples. Nonconcordant identifications were made for 9 (12%) of the 75 mutations, all of which required editing for identification. These results indicate a need for standardized editing guidelines in genotyping assays. Proficiency in editing should be assessed in training and included in quality control programs for HIV-1 genotyping.
Detection and Identification of Ciprofloxacin-Resistant
Yersinia pestis
by Denaturing High-Performance Liquid Chromatography
Authors: Hurtle William , Lindler Luther , Fan Wei , Shoemaker David , Henchal Erik , Norwood David 
Published: July 2003

ABSTRACT

Denaturing high-performance liquid chromatography (DHPLC) has been used extensively to detect genetic variation. We used this method to detect and identify
Yersinia pestis
KIM5 ciprofloxacin-resistant isolates by analyzing the quinolone resistance-determining region (QRDR) of the gyrase A gene. Sequencing of the
Y. pestis
KIM5 strain
gyrA
QRDR from 55 ciprofloxacin-resistant isolates revealed five mutation types. We analyzed the
gyrA
QRDR by DHPLC to assess its ability to detect point mutations and to determine whether DHPLC peak profile analysis could be used as a molecular fingerprint. In addition to the five mutation types found in our ciprofloxacin-resistant isolates, several mutations in the QRDR were generated by site-directed mutagenesis and analyzed to further evaluate this method for the ability to detect QRDR mutations. Furthermore, a blind panel of 42 samples was analyzed by screening for two mutant types to evaluate the potential diagnostic value of this method. Our results showed that DHPLC is an efficient method for detecting mutations in genes that confer antibiotic resistance.