bluuebunny/crossref_metadata_2025 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

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

Swine 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

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

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

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

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

We 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

The 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

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

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

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

Sequencing-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

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.
Nucleotide Sequence-Based Multitarget Identification
Authors: Vinayagamoorthy T. , Mulatz Kirk , Hodkinson Roger
Published: July 2003

MULTIGEN technology (T. Vinayagamoorthy, U.S. patent 6,197,510, March 2001) is a modification of conventional sequencing technology that generates a single electropherogram consisting of short nucleotide sequences from a mixture of known DNA targets. The target sequences may be present on the same or different nucleic acid molecules. For example, when two DNA targets are sequenced, the first and second sequencing primers are annealed to their respective target sequences, and then a polymerase causes chain extension by the addition of new deoxyribose nucleotides. Since the electrophoretic separation depends on the relative molecular weights of the truncated molecules, the molecular weight of the second sequencing primer was specifically designed to be higher than the combined molecular weight of the first sequencing primer plus the molecular weight of the largest truncated molecule generated from the first target sequence. Thus, the series of truncated molecules produced by the second sequencing primer will have higher molecular weights than those produced by the first sequencing primer. Hence, the truncated molecules produced by these two sequencing primers can be effectively separated in a single lane by standard gel electrophoresis in a single electropherogram without any overlapping of the nucleotide sequences. By using sequencing primers with progressively higher molecular weights, multiple short DNA sequences from a variety of targets can be determined simultaneously. We describe here the basic concept of MULTIGEN technology and three applications: detection of sexually transmitted pathogens (Neisseria gonorrhoeae,Chlamydia trachomatis, andUreaplasma urealyticum), detection of contaminants in meat samples (coliforms, fecal coliforms, andEscherichia coliO157:H7), and detection of single-nucleotide polymorphisms in the humanN-acetyltransferase (NAT1) gene (S. Fronhoffs et al., Carcinogenesis 22:1405-1412, 2001).
Rapid Detection of
Candida albicans
in Clinical Blood Samples by Using a TaqMan-Based PCR Assay
Authors: Maaroufi Younes , Heymans Corine , De Bruyne Jean-Marc , Duchateau Valerie , Rodriguez-Villalobos Hector , Aoun Michel , Crokaert Françoise
Published: July 2003

We describe a rapid and reproducible PCR assay for quantitation of the
Candida albicans
ribosomal DNA (rDNA) in clinical blood samples based on the TaqMan principle (Applied Biosystems), in which a signal is generated by cleavage of a template-specific probe during amplification. We used two fluorogenic probes based on universal, fungus-specific primers, one for the detection of
C. albicans
species DNA and one for the detection of all
Candida
genus DNA.
C. albicans
blastoconidia mixed with whole blood in a titration experiment yielded a linear PCR signal over a range of 3 orders of magnitude. The TaqMan-based PCR assay for
C. albicans
exhibited a low limit of detection (5 CFU/ml of blood) and an excellent reproducibility (96 to 99%). While the
C. albicans
species-specific probe had 100% specificity for
C. albicans
, all
Candida
genus-specific probes cross-reacted with other organisms likely to coinfect patients with
C. albicans
infections. On the basis of these data, we determined the
C. albicans
loads with a species-specific probe from 122 blood samples from 61 hematology or oncology patients with clinically proven or suspected systemic
Candida
infections. Eleven positive samples exhibited a wide range of
C. albicans
loads, extending from 5 to 100,475 CFU/ml of blood. The sensitivity and specificity of the present assay were 100 and 97%, respectively, compared with the results of blood culture. These data indicate that the TaqMan-based PCR assay for quantitation of
C. albicans
with a species-specific probe provides an attractive alternative for the identification and quantitation of
C. albicans
rDNA in pure cultures and blood samples.
Rapid Detection of Human Pathogenic Orthobunyaviruses
Authors: Weidmann Manfred , Rudaz Veronique , Nunes Marcio R. T. , Vasconcelos Pedro F. C. , Hufert Frank T.
Published: July 2003

Modern detection and identification tools can help to provide answers to urgent questions about the incidence, prevalence, and epidemiology of currently emerging diseases. We developed highly sensitive one-step TaqMan reverse transcription-PCR assays with sensitivities ranging from 10
4
to 10
1
molecules for 11 human pathogens of the orthobunyaviruses. We compared the performances of these assays on three currently available cyclers (ABI-PRISM 7700, LightCycler, and SmartCycler). The assay for Oropouche virus (OROV) was tested using sera collected from days 1 to 5 after onset of OROV disease and was found to be greatly superior to an established nested PCR system. A mean copy number of 1.31 × 10
7
OROV RNA/ml of serum was detected. Diagnostic RNA detection can be used as early as day 1 after onset of OROV disease. The use of a mobile SmartCycler and a hands-on time of less than 3 h could help to intensify outbreak surveillance and control, especially in field studies.
Fluorescent Dye Terminator Sequencing Methods for Quantitative Determination of Replication Fitness of Human Immunodeficiency Virus Type 1 Containing the Codon 74 and 184 Mutations in Reverse Transcriptase
Authors: Nurpeisov Viktoria , Hurwitz Selwyn J. , Sharma Prem L.
Published: July 2003

The fluorescent dye-labeled dideoxynucleotide automated DNA sequencing system has been routinely used for monitoring the development of resistance mutations in human immunodeficiency virus type 1 reverse transcriptase (RT) and protease genes during therapy. This system has provided information regarding the presence of mixtures of nucleotides in the clinical samples but has not previously been validated for the quantitative determination between peak heights and relative DNA concentration. We evaluated this system by using various ratios of wild-type and mutated DNA fragments and by performing sequencing reactions at actual melting temperatures of specific primers. Several different ratios of purified DNA fragments containing mixtures of L74/V74 and M184/V184 were sequenced, and peak heights were measured. Regression analysis between ratios of peak heights and DNA concentration demonstrated a statistically significant linear correlation, suggesting that the quantification of two different species of DNA in a mixture could be achieved with the fluorescent dye-labeled dideoxynucleotide system. These strategies have broader implications for the quantification of replication fitness of viruses, particularly those containing RT mutations at codons 74 and 184.
Novel Selective Medium for Isolation of
Burkholderia pseudomallei
Authors: Howard K. , Inglis T. J. J.
Published: July 2003

Isolation of
Burkholderia pseudomallei
currently relies on the use of Ashdown's selective agar (ASA). We designed a new selective agar (
Burkholderia pseudomallei
selective agar [BPSA]) to improve recovery of the more easily inhibited strains of
B. pseudomallei. B. pseudomallei
,
Burkholderia cepacia
, and
Pseudomonas aeruginosa
were used to determine the selectivity and sensitivity of BPSA. BPSA was more inhibitory to
P. aeruginosa
and
B. cepacia
and should make recognition of
Burkholderia
species easier due to distinctive colony morphology. BPSA also inhibited
Enterococcus
,
Escherichia
,
Staphylococcus
, and
Streptococcus
. These results indicate that BPSA is a potential replacement for ASA.
First Isolation and Identification of
Rickettsia conorii
from Ticks Collected in the Region of Fokida in Central Greece
Authors: Psaroulaki Anna , Spyridaki Ioanna , Ioannidis Alexandros , Babalis Thomas , Gikas Achilleas , Tselentis Yannis
Published: July 2003

Three different spotted-fever group rickettsiae—
Rickettsia conorii
,
R. massiliae
, and
R. rhipicephali
—were detected and identified by PCR-restriction fragment length polymorphism analysis in
Rhipicephalus
ticks collected from domestic animals in the Fokida region of Greece, where a high seroprevalence of antibodies to
R. conorii
was previously demonstrated. The infection rate of ticks was 1.6%. Moreover,
R. conorii
was isolated from one
Rhipicephalus sanguineus
tick.
Legionella pneumophila
Serogroup 1 Strain Paris: Endemic Distribution throughout France
Authors: Aurell Helena , Etienne Jerome , Forey Françoise , Reyrolle Monique , Girardo Pascale , Farge Pierre , Decludt Bénédicte , Campese Christine , Vandenesch François , Jarraud Sophie
Published: July 2003

An analysis of 691 French clinical
Legionella
isolates showed that the endemic
L. pneumophila
serogroup 1 strain Paris was responsible for 12.2% of all cases of legionellosis and had a specific pulsed-field gel electrophoresis pattern. We also demonstrated the presence of this endemic clone throughout Europe.
Multilocus Sequence Typing of
Staphylococcus aureus
with DNA Array Technology
Authors: van Leeuwen Willem B. , Jay Corinne , Snijders Susan , Durin Nathalia , Lacroix Bruno , Verbrugh Henry A. , Enright Mark C. , Troesch Alain , van Belkum Alex
Published: July 2003

A newly developed oligonucleotide array suited for multilocus sequence typing (MLST) of
Staphylococcus aureus
strains was analyzed with two strain collections in a two-center study. MLST allele identification for the first strain collection fully agreed with conventional strain typing. Analysis of strains from the second collection revealed that chip-defined MLST was concordant with conventional MLST. Array-mediated MLST data were reproducible, exchangeable, and epidemiologically concordant.
Detection of
Legionella pneumophila
by Real-Time PCR for the
mip
Gene
Authors: Wilson Deborah A. , Yen-Lieberman Belinda , Reischl Udo , Gordon Steve M. , Procop Gary W.
Published: July 2003

A real-time PCR assay for the
mip
gene of
Legionella pneumophila
was tested with 27 isolates of
L. pneumophila
, 20 isolates of 14 other
Legionella
species, and 103 non-
Legionella
bacteria. Eight culture-positive and 40 culture-negative clinical specimens were tested. This assay was 100% sensitive and 100% specific for
L. pneumophila
.
Characterization of a Vancomycin-Resistant
Enterococcus faecalis
(VREF) Isolate from a Dog with Mastitis: Further Evidence of a Clonal Lineage of VREF in New Zealand
Authors: Manson Janet M. , Keis Stefanie , Smith John M. B. , Cook Gregory M.
Published: July 2003

We report here on the characterization of a vancomycin-resistant
Enterococcus faecalis
(VREF) isolated from a dog with mastitis. The isolate was positive for the
vanA
,
ermB
, and
tet
(M) genes, with
vanA
and
ermB
carried on the same transferable plasmid. Comparison of this isolate with VREF from poultry and human sources in New Zealand demonstrated identical
Sma
I macrorestriction patterns and Tn
1546
-like elements. This is further evidence of a clonal lineage of VREF in New Zealand.
Evaluation of [
13
C]Urea Breath Test and
Helicobacter pylori
Stool Antigen Test for Diagnosis of
H. pylori
Infection in Children from a Developing Country
Authors: Cardinali Luciana de Carvalho Costa , Rocha Gifone Aguiar , Rocha Andreia Maria Camargos , de Moura Sílvia Beleza , de Figueiredo Soares Taciana , Esteves Ana Maria Braz , Nogueira Ana Margarida Miguel Ferreira , Cabral Mônica Maria Demas Álvares , de Carvalho Anfrisina Sales Teles , Bitencourt Paulo , Ferreira Alexandre , Queiroz Dulciene Maria Magalhães
Published: July 2003

The [
13
C]urea breath test (
13
C-UBT) and
Helicobacter pylori
stool antigen test (HpSA) for the diagnosis of
H. pylori
infection in children were validated. The sensitivity, specificity, and positive and negative predictive values were 93.8, 99.1, 97.8, and 98.0%, respectively, for the
13
C-UBT and 96.9, 100, 100, and 98.0%, respectively, for HpSA. Both tests are appropriate for diagnosing
H. pylori
infection in children.
Comparison of Genotyping
Helicobacter pylori
Directly from Biopsy Specimens and Genotyping from Bacterial Cultures
Authors: Park Chang-Young , Kwak Minjung , Gutierrez Oscar , Graham David Y. , Yamaoka Yoshio
Published: July 2003

PCR for
vacA
and
cagA
genotypes of
Helicobacter pylori
using DNA isolated from infected gastric biopsy specimens was approximately equal to genotyping using bacterial DNA from cultures. Inconsistent results were associated with low
H. pylori
density in biopsies. A higher proportion of mixed infection was found when biopsies were used.
Comparison of Four Antimicrobial Susceptibility Testing Methods To Determine the In Vitro Activities of Piperacillin and Piperacillin-Tazobactam against Clinical Isolates of
Enterobacteriaceae
and
Pseudomonas aeruginosa
Authors: Karlowsky James A. , Weaver Mellany K. , Thornsberry Clyde , Dowzicky Michael J. , Jones Mark E. , Sahm Daniel F.
Published: July 2003

Susceptibility to piperacillin was similar to that to piperacillin-tazobactam (<1% difference) for 6,938 isolates of
Enterobacter aerogenes
and 13,954 isolates of
Enterobacter cloacae
tested using a Vitek system; for the same species, in contrast, susceptibility rates to piperacillin-tazobactam were 5.9 to 13.9% higher than to piperacillin using disk diffusion, MicroScan, and Vitek 2 testing. Unprecedented phenotypes (piperacillin susceptible and piperacillin-tazobactam intermediate; piperacillin intermediate and piperacillin-tazobactam resistant; piperacillin susceptible and piperacillin-tazobactam resistant) accounted for 6.1% of the results for
E. aerogenes
isolates and 6.0% of the results for
E. cloacae
isolates tested with the Vitek system.
Strain Variation among
Bordetella pertussis
Isolates from Québec and Alberta Provinces of Canada from 1985 to 1994
Authors: Peppler Mark S. , Kuny Sharee , Nevesinjac Anna , Rogers Christina , de Moissac Yvon R. , Knowles Kathleen , Lorange Manon , De Serres Gaston , Talbot James
Published: July 2003

Pulsed-field gel electrophoresis and gene typing were able to differentiate among 3,597
Bordetella pertussis
isolates circulating in Alberta and Québec Provinces, Canada, from 1985 to 1994 and distinguish them from the strains used in vaccine production. This study provides a baseline for continued surveillance of prevalent and emerging strains of
B. pertussis
in Canada.
Involvement of Adherence and Adhesion
Staphylococcus epidermidis
Genes in Pacemaker Lead-Associated Infections
Authors: Klug Didier , Wallet Frédéric , Kacet Salem , Courcol René J.
Published: July 2003

We explored three genes of attachment (
fbe
and
atlE
) and adhesion (
ica
) in 27 and 10
Staphylococcus epidermidis
strains involved in pacemaker-related infections (PMI) and intravascular-catheter-related infections (IVCI), respectively, and in 25 saprophytic strains. The detection rates of
fbe
and
atlE
were identical in PMI and IVCI strains, but
ica
detection rates were identical in PMI and saprophytic strains.
Evaluation of Use of Recombinant Em18 and Affinity-Purified Em18 for Serological Differentiation of Alveolar Echinococcosis from Cystic Echinococcosis and Other Parasitic Infections
Authors: Xiao Ning , Mamuti Wulamu , Yamasaki Hiroshi , Sako Yasuhito , Nakao Minoru , Nakaya Kazuhiro , Gottstein Bruno , Schantz Peter M. , Lightowlers Marshall W. , Craig Philip S. , Ito Akira
Published: July 2003

To further evaluate recombinant Em18 antigen (rEm18) for immunodiagnosis of human alveolar echinococcosis, 208 serum samples were examined by enzyme-linked immunosorbent assay (ELISA). To comparatively assess the results of rEm18-ELISA, ELISA and immunoblot analysis with two affinity-purified native antigens were also performed with 45 selected serum samples. The results indicate that rEm18 is highly useful for serodiagnosis.
Candida dubliniensis
Infections in a Pediatric Population: Retrospective Identification from Clinical Laboratory Isolates of
Candida albicans
Authors: Kim Jean O. , Garofalo Lucille , Blecker-Shelly Deborah , McGowan Karin L.
Published: July 2003

Candida dubliniensis
is a recently described species that shares many phenotypic and morphological features with
Candida albicans
. The clinical significance of isolating
C
.
dubliniensis
from the pediatric population is not clear, as most clinical isolates have been recovered from the oral cavities or bloodstreams of adults infected with human immunodeficiency virus. In order to understand further the epidemiology of
C
.
dubliniensis
in our pediatric population, we identified
C
.
dubliniensis
isolates from clinical isolates previously identified in the laboratory as
C
.
albicans
and conducted a retrospective chart review of cases of
C
.
dubliniensis
infections. A total of 205 isolates from 183 patients were tested, and only 14 (6.8%) were identified as
C
.
dubliniensis
. In 5 of the 14 positive cultures,
C
.
dubliniensis
was the sole organism isolated (two respiratory tract specimens, one tongue specimen, one vaginal specimen, and one skin specimen). A case review showed that there were no adverse outcomes for any of the patients, and only one of the patients with cultures positive for
C
.
dubliniensis
was immunocompromised. In our pediatric population, the distinction of
C
.
dubliniensis
from
C
.
albicans
did not prove to have significant clinical relevance. Data from further investigations may help to define better the role of
C
.
dubliniensis
as a potential pathogen in children.
Rapid Molecular Diagnosis of Posttraumatic Keratitis and Endophthalmitis Caused byAlternaria infectoria
Authors: Ferrer Consuelo , Montero Javier , Alió Jorge L. , Abad José L. , Ruiz-Moreno José M. , Colom Francisca
Published: July 2003

The first case ofAlternaria infectoriaocular infection is reported. Keratitis and endophthalmitis developed after eye-perforating trauma from a lemon tree branch. Two months after surgery and empirical steroid and antibiotic treatment, diagnosis by molecular methods was performed. PCR amplification was positive for a fungus after 4 h. Antifungal treatment with amphotericin B and fluconazole was initiated immediately. DNA sequence analysis showedAlternaria infectoriato be the causal agent. After topical and systemic administration of antifungal treatment, ocular inflammation disappeared and visual acuity improved. DNA typing was found to be a useful tool to achieve early identification of the causal agent.
Evaluation of the Clinical Sensitivities of Three Viral Load Assays with Plasma Samples from a Pediatric Population Predominantly Infected with Human Immunodeficiency Virus Type 1 Subtype G and BG Recombinant Forms
Authors: Antunes Rute , Figueiredo Sofia , Bártolo Inês , Pinheiro Manuel , Rosado Lino , Soares Isabel , Lourenço Helena , Taveira Nuno
Published: July 2003

The viral load assays AMPLICOR HIV-1 Monitor Test 1.5, Nuclisens HIV-1 QT, and Quantiplex HIV RNA 3.0 (bDNA) were evaluated for their abilities to quantify human immunodeficiency virus type 1 (HIV-1) RNA in 64 plasma samples from 21 children infected in Portugal. The children were infected with HIV-1 subtypes A1, B, F1, G, and BG recombinant virus. AMPLICOR v1.5 and Quantiplex v3.0 detected all samples, and there was a good correlation of results between the two kits. Thirty-eight specimens containing HIV-1 subtype B, G, or recombinant BG, could not be detected by Nuclisens HIV-1 QT. We also evaluated the new Retina HIV-1 assay on 21 samples that were HIV-1 positive; Retina HIV-1 failed to detect 5 of 11 subtype G specimens. AMPLICOR v1.5 and Quantiplex v3.0 assays may be used for HIV-1 RNA quantification in Portugal, whereas an improvement in sensitivity for subtype G and recombinant BG is required for Nuclisens HIV-1 QT and Retina HIV-1.
Tsukamurella
Conjunctivitis: a Novel Clinical Syndrome
Authors: Woo Patrick C. Y. , Ngan Antonio H. Y. , Lau Susanna K. P. , Yuen Kwok-Yung
Published: July 2003

In this report, we describe the first three cases of
Tsukamurella
conjunctivitis in the literature. All three patients presented with congestion of one eye with small amounts of serous discharge for 1 to 2 days. All three recovered after 10 days of treatment with polymyxin B-neomycin or chloramphenicol eyedrops. Sequencing of the 16S rRNA genes of the three isolates recovered from the serous discharge of the three patients showed that they were all
Tsukamurella
species. The phenotypic characteristics of the isolate obtained from one patient best fit the phenotypic profile of
Tsukamurella pulmonis
, whereas those of the other two best fit that of
Tsukamurella tyrosinosolvens
.
Development of a Duplex PCR Assay for Detection of
Brachyspira hyodysenteriae
and
Brachyspira pilosicoli
in Pig Feces
Authors: La Tom , Phillips Nyree D. , Hampson David J.
Published: July 2003

A duplex PCR (D-PCR) amplifying portions of the
Brachyspira hyodysenteriae
NADH oxidase gene and the
B. pilosicoli
16S rRNA gene was developed and then tested on DNA extracted from 178 porcine fecal samples. The feces also underwent anaerobic culture and species-specific PCRs. Fecal extraction-D-PCR detected seven additional samples containing
B. hyodysenteriae
and five more containing
B. pilosicoli
.
Differences in the Frequency of Resistance to Antiretroviral Drug Classes among Human Immunodeficiency Virus Type 1 Clinical Isolates
Authors: Campo Rafael E. , Lichtenberger Paola N. , Rosa Isabella , Suarez German , Rivera Fernando A. , Rodriguez Allan E. , Jayaweera Dushyantha T. , Wahlay Natalie A. , Kolber Michael A.
Published: July 2003

Genotypic resistance to all antiretroviral classes was widespread among human immunodeficiency virus type 1 isolates failing therapy. Resistance to nonnucleoside reverse transcriptase inhibitors was found most frequently and resistance to protease inhibitors was found least frequently, most likely due to differences in the number of enzymatic amino acid substitutions leading to resistance to each particular drug class.
Sequence of the
Escherichia coli
O121 O-Antigen Gene Cluster and Detection of Enterohemorrhagic
E. coli
O121 by PCR Amplification of the
wzx
and
wzy
Genes
Authors: Fratamico Pina M. , Briggs Connie E. , Needle Danielle , Chen Chin-Yi , DebRoy Chitrita
Published: July 2003

The DNA sequence of the 15,155-bp O-antigen gene cluster of
Escherichia coli
O121 was determined, and 14 open reading frames were identified (all had the same transcriptional direction). Analyses of results indicated that the
wzx
(O-antigen flippase) and
wzy
(O-antigen polymerase) genes were
E. coli
O121 specific, so regions in these two genes were chosen for development of PCR assays. The PCR assays using DNA from 99
E. coli
O121 strains, strains representative of non-O121
E. coli
serogroups, and strains of other bacterial genera and PCR assays using DNA from seven enrichments of swine fecal samples naturally contaminated with
E. coli
O121 showed specificity for
E. coli
O121. Thus, the PCR assay can be employed to reliably identify
E. coli
O121 and to potentially detect the organism in food, fecal, and environmental samples.
Low-Stringency Single-Specific-Primer PCR as a Tool for Detection of Mutations in the
rpoB
Gene of Rifampin-Resistant
Mycobacterium tuberculosis
Authors: Carvalho Wania S. , Spindola de Miranda Silvana , Costa Kátia M. , Araújo José G.V.C. , Augusto Claudio J. , Pesquero João B. , Pesquero Jorge L. , Gomes Maria A.
Published: July 2003

By the low-stringency single-specific-primer PCR technique, a highly sensitive and rapid method for diagnosis of rifampin resistance in
Mycobacterium tuberculosis
was established. Seven rifampin-resistant and five rifampin-susceptible specimens were analyzed. Rifampin resistance was determined by MIC measurement. A complex electrophoretic pattern consisting of many bands was obtained for both susceptible and rifampin-resistant isolates. The same pattern was obtained for all of the susceptible specimens, but differences between resistant and susceptible isolates were found. DNA sequencing showed that a particular mutation produces a specific electrophoretic pattern.