bluuebunny/crossref_metadata_2025_split huggingface dataset:
Direct Detection of Rifampin- and Isoniazid-Resistant
Mycobacterium tuberculosis
in Auramine-Rhodamine-Positive Sputum Specimens by Real-Time PCR
Authors: Ruiz Maite , Torres Maria J. , Llanos Ana C. , Arroyo Aurelio , Palomares Jose C. , Aznar Javier 
Published: April 2004

ABSTRACT

Our objective was to evaluate the feasibility of a molecular assay based on a real-time PCR technique, carried out with a LightCycler instrument (Roche Biochemicals), to identify
Mycobacterium tuberculosis
bacilli and to detect rifampin and isoniazid resistance in DNA extracts from sputum samples. We studied three genes:
rpoB
, which is associated with rifampin resistance, and
katG
and
inhA
, which are associated with isoniazid resistance. A total of 205 sputum samples collected from 108 patients diagnosed with pulmonary tuberculosis with positive auramine-rhodamine-staining (AR) sputum samples, were tested. The sensitivities of the LightCycler PCR assay for the positive AR specimens was 97.5% (200 of 205) for
rpoB
and
inhA
genes and 96.5% (198 of 205) for the
katG
gene. For the total number of patients tested, the sensitivity was 100% (108 of 108 patients) for rifampin, whereas the sensitivity was 98.1% (106 of 108 patients) for isoniazid. Full agreement was found with the Bactec MGIT 960 method and the genotype inferred from the LightCycler data for rifampin. The phenotypic method for isoniazid reported 13 resistant strains (≥0.1 μg/ml). In seven (53.8%) strains there was a concordance between both methods, but we found that six (46.2%) strains reported as resistant by the phenotypic method were determined to be susceptible by real-time PCR. For the 75 strains reported as susceptible by the phenotypic method, the concordance with the LightCycler data was 100%. Our results demonstrate that rifampin-resistant
M. tuberculosis
could be detected in DNA extracted from auramine-rhodamine-positive sputum samples in a single-tube assay that took less than 3 h to perform for a collection of auramine-rhodamine-positive specimens obtained from patients with culture-documented pulmonary tuberculosis. Similarly, this occurs in half of the isoniazid-resistant
M. tuberculosis
DNA extracted from auramine-rhodamine-positive specimens.
Endocarditis with Ruptured Cerebral Aneurysm Caused by
Cardiobacterium valvarum
sp. nov
Authors: Han Xiang Y. , Meltzer Michelle C. , Woods Joan T. , Fainstein Victor 
Published: April 2004

ABSTRACT

A fastidious gram-negative bacterium was isolated from the blood of a 37-year-old man who had insidious endocarditis with a sudden rupture of a cerebral aneurysm. Characterization of the organism through phylogenetic and phenotypic analyses revealed a novel species of
Cardiobacterium
, for which the name
Cardiobacterium valvarum
sp. nov. is proposed.
C. valvarum
will supplement the current sole species
Cardiobacterium hominis
, a known cause of endocarditis. Surgeries and antibiotic treatment cured the patient's infection and associated complications. During cardiac surgery, a congenital bicuspid aortic valve was found to be the predisposing factor for his endocarditis.
Chromatography Paper Strip Method for Collection, Transportation, and Storage of Rotavirus RNA in Stool Samples
Authors: Rahman Mustafizur , Goegebuer Truus , De Leener Karolien , Maes Piet , Matthijnssens Jelle , Podder Goutam , Azim Tasnim , Van Ranst Marc 
Published: April 2004

ABSTRACTWe developed a novel method that uses sodium dodecyl sulfate-EDTA-treated chromatography paper strips to collect unconcentrated fresh stool samples. After the paper strips were stored for 4 months at room temperature, rotavirus RNA could be successfully amplified by using reverse transcriptase PCR. The use of filter paper strips as a specimen support allows (self-)collection of stool samples by untrained persons. Diarrheal stool samples from remote areas can be stored and transported to a central diagnostic laboratory without the need for freezers or special shipping conditions. This convenient and inexpensive rotavirus sample collection system can be of use in epidemiological surveillance studies and vaccine trials.
Diversity of Group A Human Rotavirus Types Circulating over a 4-Year Period in Madrid, Spain
Authors: Sánchez-Fauquier Alicia , Wilhelmi Isabel , Colomina Javier , Cubero Eusebio , Roman Enriqueta 
Published: April 2004

ABSTRACT
The incidence and distribution of human rotavirus G types among children under 5 years old with acute gastroenteritis were determined over a 4-year period (1998 to 2002) by using monoclonal antibodies and reverse transcription-PCR methods. Rotavirus was detected in 1,155 (31%) of 3,760 specimens tested. Rotavirus was studied in every month of the 48-month survey period. Rotavirus activity occurred mainly (51%) in the typically cooler months in Spain (November to February). The age distribution of rotavirus-positive cases showed that 90% of patients (1,038 of 1,155) were under 2 years old. Rotavirus types were determined for 576 of 1,155 patients (50%). G1 was the main genotype detected (53%), and the second most common was G4 (24%). The G2, G9, and G3 rotavirus types were detected in 14, 6, and 2% of the cases, respectively. Dual infections were detected in only 0.6%. The seasonal distribution of genotypes showed a significant genotypic shift: whereas G4 strains predominated (57%) during the 1998 to 2000 seasons, the G1 gradually increased to account for 75% in the 2000 to 2002 seasons. In addition, the present study reports the first detection of the G9 genotype in human fecal samples in Spain. Therefore, additional types may be required for vaccine development strategies that currently target only types G1 to G4.
Simple Algorithm for Identification of
Bordetella pertussis
Pertactin Gene Variants
Authors: Muyldermans Gaëtan , Piérard Denis , Hoebrekx Nathalie , Advani Reza , Van Amersfoorth Shirley , De Schutter Iris , Soetens Oriane , Eeckhout Leo , Malfroot Anne , Lauwers Sabine 
Published: April 2004

ABSTRACT

Studies performed in several countries have demonstrated the recent emergence and subsequent dominance of circulating
Bordetella pertussis
strains harboring pertactin and pertussis toxin variants not included in pertussis vaccines. Determination of the pertactin gene variants is commonly performed using a time-consuming and expensive sequence analysis. We developed a simple and reliable pertactin typing algorithm suitable for large-scale screening. The assay correctly identified all pertactin alleles in representative strains. The typing of 231 clinical strains of
B. pertussis
routinely isolated in Belgium showed that this algorithm was adequate to identify less-frequent
prn
types like
prn9
and
prn11
.
Lack of Longitudinal Intrapatient Correlation between p24 Antigenemia and Levels of Human Immunodeficiency Virus (HIV) Type 1 RNA in Patients with Chronic HIV Infection during Structured Treatment Interruptions
Authors: Prado Julia G. , Shintani Ayumi , Bofill Margarita , Clotet Bonaventura , Ruiz Lidia , Martinez-Picado Javier 
Published: April 2004

ABSTRACT

Structured treatment interruptions (STIs) have been proposed as a potential treatment strategy during human immunodeficiency virus type 1 (HIV-1) antiretroviral therapy. This still-experimental intervention requires a close monitoring of patients' plasma viremia and CD4
+
-T-cell counts during the treatment interruption phase. By using signal amplification of a heat-dissociated p24 antigen (p24Ag) assay, we compared p24Ag levels with levels of HIV RNA in plasma. One hundred seventy-four plasma samples were obtained from 51 chronically HIV-infected patients: 117 from patients who underwent STIs and 57 from patients who did not. Partial immune complex dissociation and clearance of those complexes by the erythrocytes were also investigated. A significant association between the two assays was observed (β = 0.23, 95% confidence interval = 0.18, 0.28;
P
< 0.0001), but the association was smaller in the subset of samples from patients undergoing STIs. Moreover, discordant results and lack of longitudinal intrapatient correlation between levels of p24Ag and HIV-1 RNA were higher in this group. Incomplete immune complex dissociation and binding of those complexes to erythrocytes could be contributing factors involved in the diminished detection of p24Ag. Therefore, signal amplification of a heat-dissociated p24Ag had a positive association with current HIV RNA assays in a population-based analysis. However, it might not be sensitive enough to monitor longitudinal intrapatient viremia during STIs in patients with high CD4
+
-T-cell counts potentially due to the production of high-affinity anti-p24 antibodies and clearance of immune complexes by erythrocytes.
Analysis of
Helicobacter pylori
Genotypes and Correlation with Clinical Outcome in Turkey
Authors: Saribasak Huseyin , Salih Barik A. , Yamaoka Yoshio , Sander Ersan 
Published: April 2004

ABSTRACT

The predominant
Helicobacter pylori
strains circulating among geographic locations differ in regard to genomic structure. The association of the
cagA
-positive,
vacA
s1 genotypes with peptic ulcer disease (PUD) and gastric cancer was reported in Western countries but not in East Asian countries. Strains from Western countries predominantly possessed
cagA
type 2a,
vacA
s1a or s1b/m1a, or
vacA
m2a genotypes, whereas strains from East Asia possessed
cagA
type 1a,
vacA
s1c/m1b, or
vacA
m2b genotypes. Whether the Turkish strains possessed such genotypes was investigated and correlated with the disease outcome. Seventy-three patients from Turkey were enrolled.
H. pylori
was detected in 65 (89%) patients (22 with gastritis, 33 with PUD, and 10 with gastric cancer) by any of the following tests:
Campylobacter
-like organism test, culture, or PCR. Among the
H. pylori-
positive patients, presence of the
cagA
gene (78%) was significantly associated with PUD (
P
< 0.00001), gastric cancer (
P
< 0.001), and
vacA
s1a genotypes (
P
< 0.0001). Multiple
vacA
genotypes were more prevalent in PUD and gastric cancer patients than in patients with gastritis. Restriction fragment length polymorphism analysis of the
cagA
gene revealed three different patterns with no significant association with clinical outcome. Turkish strains examined predominantly possessed
cagA
type 2a,
vacA
s1a/m1a, or
vacA
m2a genotypes, which were typical genotypes in strains from Western countries. This fact might be one of the reasons for the low prevalence of severe gastroduodenal diseases in Turkey compared to the East Asian countries.
Evaluation of Performance and Potential Clinical Impact of ProSpecT Shiga Toxin
Escherichia coli
Microplate Assay for Detection of Shiga Toxin-Producing
E. coli
in Stool Samples
Authors: Gavin Patrick J. , Peterson Lance R. , Pasquariello Anna C. , Blackburn Joanna , Hamming Mark G. , Kuo Kuo J. , Thomson Richard B. 
Published: April 2004

ABSTRACT

Shiga toxin-producing
Escherichia coli
bacteria (STEC) are emerging pathogens capable of producing sporadic and epidemic diarrhea, hemorrhagic colitis, and potentially life-threatening hemolytic-uremic syndrome. Although the presence of
E. coli
O157 can be readily detected in stool by sorbitol-MacConkey agar culture (SMAC), STEC non-O157 serotypes cannot. In contrast to culture, testing for the presence of Shiga toxins 1 and 2 in stool detects both O157 and non-O157 STEC serotypes capable of causing disease. Over two consecutive summers, we evaluated the performance of the ProSpecT Shiga toxin
E. coli
Microplate assay (Alexon-Trend, Ramsey, Minn.), an enzyme immunoassay for the detection of Shiga toxins 1 and 2, on all stools submitted for culture of enteric pathogens, and the potential clinical impact of Shiga toxin detection. Twenty-nine stool specimens were STEC positive by ProSpecT assay. Twenty-seven of 29 STEC-positive isolates were confirmed by SMAC and serotyping or by a second enzyme immunoassay and PCR (positive predictive value, 93%). Thirteen of 27 confirmed Shiga toxin-producing strains were serotype O157. The remaining 14 strains represented 8 other serotypes. The ProSpecT assay was 100% sensitive and specific for detection of
E. coli
O157 in stool (7 of 7) compared to SMAC. In addition, the ProSpecT assay detected twice as many STEC as SMAC. Fifty-two percent of confirmed STEC-positive stools were nonbloody. Thus, in our population, screening strategies that test only visibly bloody stools for STEC would miss a majority of cases. Eleven (41%) STEC-positive patients were hospitalized, and eight (30%) developed severe disease (two developed hemolytic-uremic syndrome, and six developed hemorrhagic colitis). Prior to detection of STEC infection, seven (26%) and eight patients (30%) underwent unnecessary diagnostic procedures or received potentially deleterious empirical treatment, respectively. We propose that establishing a specific diagnosis of STEC may have prevented these potentially harmful interventions. We conclude that the ProSpecT assay is sensitive and specific for the detection of Shiga toxins 1 and 2 in stool and has potentially significant clinical impact for the individual patient and public health. Shiga toxin assays should be considered for routine use in settings where prevalence of STEC disease warrants testing.
Multiple Polymorphic Loci for Molecular Typing of Strains of
Mycobacterium leprae
Authors: Groathouse Nathan A. , Rivoire Becky , Kim Hansuk , Lee Hyeyoung , Cho Sang-Nae , Brennan Patrick J. , Vissa Varalakshmi D. 
Published: April 2004

ABSTRACT

The need for molecular tools for the differentiation of isolates of
Mycobacterium leprae
, the organism that causes leprosy, is urgent in view of the continuing high levels of new case detection, despite years of aggressive chemotherapy and the consequent reduction in the prevalence of leprosy. The slow onset of leprosy and the reliance on physical examination for detection of disease have restricted the epidemiological tracking necessary to understand and control transmission. Two genetic loci in several isolates of
M. leprae
have previously been demonstrated to contain variable-number tandem repeats (VNTRs). On the basis of these reports and the availability of the full genome sequence, multiple-locus VNTR analysis for strain typing has been undertaken. A panel of 11 short tandem repeat (STR) loci with repeat units of 1, 2, 3, 6, 12, 18, 21, and 27 bp from four clinical isolates of
M. leprae
propagated in armadillo hosts were screened by PCR. Fragment length polymorphisms were detected at 9 of the 11 loci by agarose gel electrophoresis. Sequencing of representative DNA products confirmed the presence of VNTRs between isolates. The application of nine new polymorphic STRs in conjunction with automated methods for electrophoresis and size determination allows greater discrimination between isolates of
M. leprae
and enhances the potential of this technique to track the transmission of leprosy.
New Approach for Serological Testing for Leptospirosis by Using Detection of
Leptospira
Agglutination by Flow Cytometry Light Scatter Analysis
Authors: Yitzhaki S. , Barnea A. , Keysary A. , Zahavy E. 
Published: April 2004

ABSTRACT

Leptospirosis is considered an important reemerging infectious disease worldwide. The standard and most widespread method for the diagnosis of leptospirosis is the microscopic agglutination test (MAT). This test is laborious and time-consuming, and the interpretation of the results is subjective. In the present work we describe an application of flow cytometry (FCM) as a tool for the serological diagnosis of leptospirosis. The analysis is based on the sensitivity of FCM to the size and shape of the bacteria analyzed by measurement of light scatter parameters: forward scatter (FSC) and side scatter (SSC). The addition of positive serum to an infecting leptospiral serovar results in a shift of the light scatter parameter to a different location with higher FSC and SSC values, indicating the formation of leptospiral aggregates. By using immunofluorescent staining, we have shown that the large particles formed are the agglutinated leptospires. Quantification of the agglutination process has been achieved by calculating an agglutination factor (

A
f

), based on changes in the light scatter parameters measured by FCM.

A
f

enables us to determine the specificity of the serological reaction of the patient serum with each leptospiral serovar. In this work, 27 serum samples from 18 leptospirosis patients were tested by both the MAT and the FCM techniques, in which each serum sample was tested against 13 serovars. Twenty-six human serum samples derived from patients with a variety of other defined illnesses were used as negative controls and enabled us to define the

A
f

threshold value as <9.3 for negative patients, while any value higher than that would be a positive result for leptospirosis. Compared to MAT, the FCM technique was found to be more specific and sensitive, especially in identifying the serogroup in the acute phase of the disease. The whole process was found to be rapid and took less than 1.5 h. Moreover, FCM analysis is objective and can be automated for the handling of large numbers of samples.
Molecular Analysis of Adenovirus Isolates from Vaccinated and Unvaccinated Young Adults
Authors: Blasiole Daniel A. , Metzgar David , Daum Luke T. , Ryan Margaret A. K. , Wu Jianguo , Wills Christopher , Le Charles T. , Freed Nikki E. , Hansen Christian J. , Gray Gregory C. , Russell Kevin L. 
Published: April 2004

ABSTRACT
Infections of adenovirus type 4 (Ad4) and Ad7 were discovered among previously vaccinated individuals through febrile respiratory illness surveillance at military recruit camps. Genetic analysis was performed on these isolates and a sample of adenovirus isolates from unvaccinated patients. Antigenic regions of the adenovirus hexon gene from 21 vaccinated and 31 unvaccinated patients were sequenced and compared to homologous regions of Ad4 and Ad7 vaccine strains and of other representative hexon sequences archived in GenBank. The phylogenetic distribution of sequences from vaccinated individuals closely resembled those from unvaccinated individuals. The most common Ad7 strain was the Ad7d2 hexon genotype, and the most common Ad4 strain was a genotype nearly identical to the recently discovered Z-G 95-873 Ad4 variant. Near exclusive isolation of Ad4 since 1999 indicates that the Ad4 variant is currently responsible for the vast majority of adenovirus morbidity in military recruit camps. Different ratios of nonsynonymous to synonymous nucleotide substitution rates in known antigenic regions compared to nonantigenic regions indicated positive selection for diversity in the antigenic regions and purifying selection in the nonantigenic regions.
Dual Toxin-Producing Strain of
Clostridium botulinum
Type Bf Isolated from a California Patient with Infant Botulism
Authors: Barash Jason R. , Arnon Stephen S. 
Published: April 2004

ABSTRACT

A retrospective study of
Clostridium botulinum
strains isolated from patients from California with infant botulism identified the fourth known
C. botulinum
strain that produces both type B and type F botulinum toxins. This unique strain represented 0.12% of the California infant botulism case isolates from 1976 to 2003. The relative concentrations of type B and F toxins produced were temperature dependent.
Quality Control Limits for Voriconazole Disk Susceptibility Tests on Mueller-Hinton Agar with Glucose and Methylene Blue
Authors: Pfaller M. A. , Barry A. , Bille J. , Brown S. , Ellis D. , Meis J. F. , Rennie R. , Rinaldi M. , Rogers T. , Traczewski M. 
Published: April 2004

ABSTRACT

An international collaborative study was performed in order to propose quality control limits for voriconazole disk diffusion tests on Mueller-Hinton agar with 2% glucose and 0.5 μg of methylene blue per ml. The supplement may be added to the agar before autoclaving, or Mueller-Hinton agar plates may be flooded with a glucose-methylene blue solution. Replicate tests on both types of agar plates with 1-μg voriconazole disks generated data to propose zone size limits for tests of
Candida parapsilosis
ATCC 22019 (28 to 37 mm),
Candida albicans
ATCC 90028 (31 to 42 mm), and
Candida krusei
ATCC 6258 (16 to 25 mm).
Candida tropicalis
ATCC 750 was not useful for this purpose.
Comparison of Two Widely Used PCR Primer Systems for Detection of
Toxoplasma
in Amniotic Fluid, Blood, and Tissues
Authors: Chabbert Elisabeth , Lachaud Laurence , Crobu Lucien , Bastien Patrick 
Published: April 2004

ABSTRACT

The PCR diagnosis of toxoplasmosis suffers from lack of standardization. Interlaboratory comparative studies of PCR methods have been performed, but intralaboratory comparisons are scarce. Here, we optimized and compared the technical performances of two PCR primer systems widely used for
Toxoplasma
detection. The differences between the two methods were visible only at low parasite concentrations (≤1
Toxoplasma
genome per reaction tube). Nevertheless, when clinical samples were tested, both methods significantly differed in their technical sensitivities and specificities. Only one method appeared adequate for samples containing blood or tissue.
Genotyping of Enteric Adenoviruses by Using Single-Stranded Conformation Polymorphism Analysis and Heteroduplex Mobility Assay
Authors: Soares Caroline C. , Volotão Eduardo M. , Albuquerque Maria Carolina M. , Nozawa Carlos M. , Linhares Rosa Elisa C. , Volokhov Dmitriy , Chizhikov Vladimir , Lu Xiaoyan , Erdman Dean , Santos Norma 
Published: April 2004

ABSTRACT
Single-stranded conformation polymorphism (SSCP) analysis and heteroduplex mobility assays (HMAs) were used to identify and genotype enteric adenoviruses (EAd). The results were compared to those of restriction endonuclease assays, species-specific PCRs, and direct nucleotide sequence analyses. Of the 31 stool samples tested, 15 isolates were identified as EAd and 7 were identified as nonenteric Ad by all methods. An agreement of 100% was found between the SSCP and HMA results.
Development of a Multiplex PCR Technique for Detection and Epidemiological Typing of
Salmonella
in Human Clinical Samples
Authors: Alvarez Juan , Sota Mertxe , Vivanco Ana Belén , Perales Ildefonso , Cisterna Ramón , Rementeria Aitor , Garaizar Javier 
Published: April 2004

ABSTRACT

We have developed a multiplex PCR assay for
Salmonella
detection and epidemiological typing. Six sets of primers were designed to detect the major
Salmonella
serotypes and phage types in Spain. An internal amplification control was designed in order to detect PCR inhibition. The different amplification profiles obtained allowed us to detect
Salmonella
bacteria and to distinguish the clinically prevalent
Salmonella enterica
serotypes Enteritidis, Typhimurium and subspecies I serotype 4,5,12:i:−. Using this method, we could detect a specific band for DT104 and U302 phage types in
Salmonella
serotype Typhimurium.
Salmonella enterica
serotype Hadar and other C2 serogroup strains showed two specific band profiles. In the validation stage, the assay was reproducible for all serotypes studied, apart from some C2 serogroup strains. When the technique was applied to clinical stool specimens, the prevalent serotypes Enteritidis and Typhimurium were detected with a sensitivity of 93%, specificity of 100%, and efficiency of 98%. Also, a low PCR inhibition rate (8%) was obtained. The overall agreement of the multiplex PCR with conventional culture-based techniques was 95% for
Salmonella
typing using Cohen's kappa index.
DNA Polymorphism Assay Distinguishes Isolates ofLeishmania donovaniThat Cause Kala-Azar from Those That Cause Post-Kala-Azar Dermal Leishmaniasis in Humans
Authors: Sreenivas Gannavaram , Subba Raju B. V. , Singh Ruchi , Selvapandiyan Angamuthu , Duncan Robert , Sarkar Dwijen , Nakhasi Hira L. , Salotra Poonam 
Published: April 2004

ABSTRACTLeishmania donovaniin India causes visceral infection (kala-azar) and dermal infection (post-kala-azar dermal leishmaniasis). We report here the identification of polymorphism in a well-defined genetic locus among theLeishmaniaparasites causing the visceral and dermal manifestations, in a comparison of 15 post-kala-azar dermal leishmaniasis and 12 kala-azar patient isolates.