bluuebunny/crossref_metadata_2025_split huggingface dataset:
Multilocus Sequence Typing Is a Reliable Alternative Method to DNA Fingerprinting for Discriminating among Strains of
Candida albicans
Authors: Robles Juan C. , Koreen Larry , Park Steven , Perlin David S. 
Published: June 2004

ABSTRACT

Multilocus sequence typing (MLST) has emerged as a powerful new DNA-typing tool for the evaluation of intraspecies genetic relatedness. This method relies on DNA sequence analysis of nucleotide polymorphisms in housekeeping genes and has shown a high degree of intraspecies discriminatory power for bacterial and fungal pathogens. However, the results of the MLST scheme for
Candida albicans
have heretofore never been formally compared to those of other established typing techniques. To assess the value of MLST relative to those of other DNA fingerprinting tools for discriminating among strains of
C. albicans
, we applied it to a previously well-characterized set of 29
C. albicans
isolates evaluated by the random amplified polymorphic DNA (RAPD), multilocus enzyme electrophoresis (MLEE), and Ca3 Southern hybridization probe techniques. MLST identified three clusters of genetically related isolates, with 82.3% direct concordance with MLEE, 82.7% with RAPD analysis, and 86.2% with the Ca3 Southern hybridization technique. When MLST was applied to a subset of 22 isolates of unrelated origins, it identified 21 independent diploid sequence types (DSTs), resulting in a discriminatory power of 99.6%. These DSTs were 96.9, 99.6, and 99.6% concordant with the genotypes identified by RAPD analysis, MLEE, and Ca3 Southern hybridization, respectively. These results demonstrate that MLST is a highly effective technique that performs at least comparably to other established DNA fingerprinting techniques.
Quantification of Intestinal Bacterial Populations by Real-Time PCR with a Universal Primer Set and Minor Groove Binder Probes: a Global Approach to the Enteric Flora
Authors: Ott Stephan J. , Musfeldt Meike , Ullmann Uwe , Hampe Jochen , Schreiber Stefan 
Published: June 2004

ABSTRACT

The composition of the human intestinal flora is important for the health status of the host. The global composition and the presence of specific pathogens are relevant to the effects of the flora. Therefore, accurate quantification of all major bacterial populations of the enteric flora is needed. A TaqMan real-time PCR-based method for the quantification of 20 dominant bacterial species and groups of the intestinal flora has been established on the basis of 16S ribosomal DNA taxonomy. A PCR with conserved primers was used for all reactions. In each real-time PCR, a universal probe for quantification of total bacteria and a specific probe for the species in question were included. PCR with conserved primers and the universal probe for total bacteria allowed relative and absolute quantification. Minor groove binder probes increased the sensitivity of the assays 10- to 100-fold. The method was evaluated by cross-reaction experiments and quantification of bacteria in complex clinical samples from healthy patients. A sensitivity of 10
1
to 10
3
bacterial cells per sample was achieved. No significant cross-reaction was observed. The real-time PCR assays presented may facilitate understanding of the intestinal bacterial flora through a normalized global estimation of the major contributing species.
Genomic Characterization of an EndemicMycobacterium tuberculosisStrain: Evolutionary and Epidemiologic Implications
Authors: Nguyen Dao , Brassard Paul , Menzies Dick , Thibert Louise , Warren Rob , Mostowy Serge , Behr Marcel 
Published: June 2004

ABSTRACTIn a study of 302Mycobacterium tuberculosisclinical isolates from the low-incidence Canadian-born population of Quebec, we characterized a large endemic strain family by using genomic deletions. The DS6Quebecdeleted region (11.4 kb) defined a strain family of 143 isolates encompassing two subgroups: one characterized by pyrazinamide (PZA) susceptibility and the other marked by a PZA-monoresistant phenotype. A second deletion (8 bp) in thepncAgene was shared by all 76 isolates with the PZA resistance phenotype, whereas a third DRv0961 deletion (970 bp) defined a further subset of 15 isolates. From their deletion profiles, we derived a most parsimonious evolutionary scenario and compared multiple standard genotyping modalities (using IS6110restriction fragment length polymorphism [RFLP], spoligotyping, and mycobacterial interspersed repetitive units [MIRU]) across the deletion-based subgroups. The use of a single genotyping modality yielded an unexpectedly high proportion of clustered isolates for a high IS6110copy strain (27% by IS6110RFLP, 61% by MIRU, and 77% by spoligotyping). By combining all three modalities, only 14% were genotypically clustered overall, a result more congruent with the epidemiologic profile of reactivation tuberculosis, as suggested by the older age (mean age, 60 years), rural setting, and low proportion of epidemiologic links. These results provide insight into the evolution of genotypes in endemic strains and the potential for false clustering in molecular epidemiologic studies.
Bovine Tuberculosis (
Mycobacterium bovis
) in Wildlife in Spain
Authors: Aranaz Alicia , de Juan Lucía , Montero Natalia , Sánchez Celia , Galka Margarita , Delso Consuelo , Álvarez Julio , Romero Beatriz , Bezos Javier , Vela Ana I. , Briones Victor , Mateos Ana , Domínguez Lucas 
Published: June 2004

ABSTRACT

Mycobacterium bovis
infection in wildlife and feral species is a potential source of infection for livestock and a threat to protected and endangered species. The aim of this study was to identify Spanish wild animal species infected with
M. bovis
through bacteriological culture and spacer oligonucleotide typing (spoligotyping) of isolates for epidemiological purposes. This study included samples from red deer (
Cervus elaphus
), fallow deer (
Dama dama
), wild boar (
Sus scrofa
), Iberian lynx (
Lynx pardina
), hare (
Lepus europaeus
), and cattle (
Bos taurus
). They were collected in several geographical areas that were selected for their unique ecological value and/or known relationships between wildlife and livestock. In the areas included in this survey,
M. bovis
strains with the same spoligotyping pattern were found infecting several wild species and livestock, which indicates an epidemiological link. A locally predominant spoligotype was found in these areas. Better understanding of the transmission and distribution of disease in these populations will permit more precise targeting of control measures.
Multilocus Sequence Typing Analysis of Human and Animal
Clostridium difficile
Isolates of Various Toxigenic Types
Authors: Lemee Ludovic , Dhalluin Anne , Pestel-Caron Martine , Lemeland Jean-François , Pons Jean-Louis 
Published: June 2004

ABSTRACT

A multilocus sequence typing (MLST) scheme was developed to study the genetic relationships and population structure of 72
Clostridium difficile
isolates from various hosts, geographic sources, PCR ribotypes, and toxigenic types (determined by PCR targeting
tcdA
and
tcdB
genes). MLST was performed by DNA sequence analysis of seven housekeeping genes (
aroE
,
ddl
,
dutA
,
tpi
,
recA
,
gmk
, and
sodA
). The number of alleles ranged from five (
dutA
and
ddl
) to eleven (
recA
). Allelic profiles allowed the definition of 34 different sequence types (STs). These STs lacked correlation with geographic source but were well correlated to toxigenic type. The dendrogram generated from a matrix of pairwise genetic distances showed that animal isolates did not constitute a distinct lineage from human isolates and that there was no hypervirulent lineage within the population of toxigenic human isolates (isolates recovered from pseudomembranous colitis and antibiotic-associated diarrhea did not cluster in distinct lineages). However, A
−
B
+
variant isolates shared the same ST that appeared as a divergent lineage in the population studied, indicating a single evolutionary origin. The population structure was further examined by analysis of allelic polymorphism. The dendrogram generated from composite sequence-based analysis revealed a homogeneous population associated with three divergent lineages, one of which was restricted to A
−
B
+
variant isolates.
C. difficile
exhibited a clonal population structure, as revealed by the estimation of linkage disequilibrium (
Ia
) between loci. The analysis of alleles within clonal complexes estimated that point mutation generated new alleles at a frequency eightfold higher than recombinational exchange, and the congruence of the dendrograms generated from separate housekeeping loci confirmed the mutational evolution of this species.
Performance Characteristics of the Immunoglobulin G-Capture BED-Enzyme Immunoassay, an Assay To Detect Recent Human Immunodeficiency Virus Type 1 Seroconversion
Authors: Dobbs Trudy , Kennedy Susan , Pau Chou-Pong , McDougal J. Steven , Parekh Bharat S. 
Published: June 2004

ABSTRACT

Recently, we developed an immunoglobulin G (IgG)-capture BED-enzyme immunoassay (BED-CEIA) to identify recent human immunodeficiency virus (HIV) type 1 (HIV-1) seroconversion for use in incidence estimates. We have established an algorithm for its use; developed quality control reagents to monitor the assay; and evaluated its performance for interrun, intrarun, and operator variability. Analysis of 144 individual plates, which involved multiple plate lots and several operators over more than a year, indicated that the coefficients of variation (CVs) were between 10 and 15% for raw optical density (OD) values in the dynamic range between 0.5 and 2.0 OD units; the CVs decreased to 5 to 10% when the OD was normalized (OD-n; OD-n = specimen OD/calibrator OD). The intrarun CVs were generally in the range of 5 to 10% for specimens with ODs <0.5 and less than 5% for specimens with ODs >0.5. The level of concordance between multiple plate lots (
n
= 6) and multiple operators (
n
= 7) was quite high (
R
2
> 0.9). Comparison of the results of the initial and the confirmatory tests with specimens with OD-n values ≤1.5 demonstrated a high degree of correlation (
R
2
= 0.92); 566 (92%) of 615 of specimens tested in the two modes retained the same classification (recent or long-term infection). The values for those specimens with changed classifications (
n
= 49) were close to the cutoff (OD-
n
= 1.0), as expected. The twofold difference in the HIV IgG contents between the controls and the calibrator reagents was exploited to monitor individual plate runs by using a control plot, which was incorporated into the spreadsheet for data entry and run monitoring. This information provides baseline data for the successful transfer of BED-CEIA to other laboratories and the use of BED-CEIA for the detection of recent HIV seroconversion and the calculation of incidence estimates worldwide.
Sensitive and Specific Monoclonal Antibody-Based Capture Enzyme Immunoassay for Detection of Nucleocapsid Antigen in Sera from Patients with Severe Acute Respiratory Syndrome
Authors: Che Xiao-yan , Qiu Li-wen , Pan Yu-xian , Wen Kun , Hao Wei , Zhang Li-ya , Wang Ya-di , Liao Zhi-yong , Hua Xu , Cheng Vincent C. C. , Yuen Kwok-yung 
Published: June 2004

ABSTRACT
A rapid antigen test for the diagnosis of severe acute respiratory syndrome (SARS) is essential for control of this disease at the point of management. The nucleocapsid (N) protein of SARS-associated coronavirus (SARS-CoV) is abundantly expressed in infected-cell culture filtrate as demonstrable by Western blotting using convalescent-phase sera from patients with SARS. We used monoclonal antibodies specifically directed against N protein to establish a sensitive antigen capture sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of SARS-CoV. The assay employed a mixture of three monoclonal antibodies for capture and rabbit polyclonal antibodies for detection of serum antigen in 32 cases of clinically probable SARS as defined by the World Health Organization during the epidemic in Guangzhou, China. Recombinant N protein was used as a standard to establish a detection sensitivity of approximated 50 pg/ml. The linear range of detection in clinical specimens was from 100 pg/ml to 3.2 ng/ml. Using a panel of sera collected at different points in time, the amount of circulating N antigen was found to peak 6 to 10 days after the onset of symptoms. The sensitivity of the assay was 84.6% in 13 serologically confirmed SARS patients with blood taken during the first 10 days after the onset of symptoms (11 of 13). The specificity of the assay was 98.5% in 1,272 healthy individuals (1,253 of 1,272). There was no cross-reaction with other human and animal coronaviruses in this assay. In conclusion, a sensitive and quantitative antigen capture ELISA was established for the early diagnosis and disease monitoring of SARS-CoV infection.
Generation and Infectivity Titration of an Infectious Stock of Avian Hepatitis E Virus (HEV) in Chickens and Cross-Species Infection of Turkeys with Avian HEV
Authors: Sun Z. F. , Larsen C. T. , Huang F. F. , Billam P. , Pierson F. W. , Toth T. E. , Meng X. J. 
Published: June 2004

ABSTRACT

Avian hepatitis E virus (HEV), a novel virus identified from chickens with hepatitis-splenomegaly syndrome in the United States, is genetically and antigenically related to human HEV. In order to further characterize avian HEV, an infectious viral stock with a known infectious titer must be generated, as HEV cannot be propagated in vitro. Bile and feces collected from specific-pathogen-free (SPF) chickens experimentally infected with avian HEV were used to prepare an avian HEV infectious stock as a 10% suspension of positive fecal and bile samples in phosphate-buffered saline. The infectivity titer of this infectious stock was determined by inoculating 1-week-old SPF chickens intravenously with 200 μl of each of serial 10-fold dilutions (10
−2
to 10
−6
) of the avian HEV stock (two chickens were inoculated with each dilution). All chickens inoculated with the 10
−2
to 10
−4
dilutions of the infectious stock and one of the two chickens inoculated with the 10
−5
dilution, but neither of the chickens inoculated with the 10
−6
dilution, became seropositive for anti-avian HEV antibody at 4 weeks postinoculation (wpi). Two serologically negative contact control chickens housed together with chickens inoculated with the 10
−2
dilution also seroconverted at 8 wpi. Viremia and shedding of virus in feces were variable in chickens inoculated with the 10
−2
to 10
−5
dilutions but were not detectable in those inoculated with the 10
−6
dilution. The infectivity titer of the infectious avian HEV stock was determined to be 5 × 10
5
50% chicken infectious doses (CID
50
) per ml. Eight 1-week-old turkeys were intravenously inoculated with 10
5
CID
50
of avian HEV, and another group of nine turkeys were not inoculated and were used as controls. The inoculated turkeys seroconverted at 4 to 8 wpi. In the inoculated turkeys, viremia was detected at 2 to 6 wpi and shedding of virus in feces was detected at 4 to 7 wpi. A serologically negative contact control turkey housed together with the inoculated ones also became infected through direct contact. This is the first demonstration of cross-species infection by avian HEV.
Characterization of Novel Coding Sequences Specific toMycobacterium aviumsubsp.paratuberculosis: Implications for Diagnosis of Johne's Disease
Authors: Paustian Michael L. , Amonsin Alongkorn , Kapur Vivek , Bannantine John P. 
Published: June 2004

ABSTRACTMycobacterium aviumsubsp.paratuberculosisis the causative agent of Johne's Disease, an economically important intestinal ailment of ruminants. Due to the considerable genetic and serologic cross-reactivity with closely related and ubiquitous members of theM. aviumcomplex, a species-specific method for the serological diagnosis of Johne's disease is unavailable. Computational and PCR-based analysis of the complete genome sequence ofM. aviumsubsp.paratuberculosisled to the identification of 13 open reading frames with no identifiable homologs. One of these sequences is a putative insertion element present in six copies on theM. aviumsubsp.paratuberculosisgenome. These novelM. aviumsubsp.paratuberculosisgenes were cloned intoEscherichia coliexpression vectors, and nine were successfully expressed as recombinant fusion proteins. Five of these proteins were purified in sufficient amounts to allow immunoblot analyses of their reactivity with sera from naturally infected cattle as well as mice and rabbits exposed toM. aviumsubsp.paratuberculosis. Fusion proteins representing MAP0862, MAP3732c, and MAP2963c were recognized by nearly all of the sera tested, including those from cattle in the clinical stages of disease. Notably, further analysis of the protein encoded by MAP0862 showed that it reacted with sera from additional infected cattle but not with sera from uninfected control animals. The fusion product of MAP0860c did not react with any of the sera tested, while the remaining four proteins were variably recognized by sera fromM. aviumsubsp.paratuberculosis-infected cattle. Collectively, the results of this study demonstrate the utility of genomic data to identify potential diagnostic sequences.
Prospective Comparison of the Diagnostic Potential of Real-Time PCR, Double-Sandwich Enzyme-Linked Immunosorbent Assay for Galactomannan, and a (1→3)-β-
d
-Glucan Test in Weekly Screening for Invasive Aspergillosis in Patients with Hematological Disorders
Authors: Kawazu Masahito , Kanda Yoshinobu , Nannya Yasuhito , Aoki Katsunori , Kurokawa Mineo , Chiba Shigeru , Motokura Toru , Hirai Hisamaru , Ogawa Seishi 
Published: June 2004

ABSTRACT

The establishment of an optimal noninvasive method for diagnosing invasive aspergillosis (IA) is needed to improve the management of this life-threatening infection in patients with hematological disorders, and a number of noninvasive tests for IA that target different fungal components, including galactomannan, (1→3)-β-
d
-glucan (BDG), and
Aspergillus
DNA, have been developed. In this study, we prospectively evaluated the diagnostic potential of three noninvasive tests for IA that were used in a weekly screening strategy: the double-sandwich enzyme-linked immunosorbent assay (ELISA) for galactomannan (Platelia Aspergillus), a real-time PCR assay for
Aspergillus
DNA (GeniQ-Asper), and an assay for BDG (β-glucan Wako). We analyzed 149 consecutive treatment episodes in 96 patients with hematological disorders who were at high risk for IA and diagnosed 9 proven IA cases, 2 probable IA cases, and 13 possible invasive fugal infections. In a receiver-operating characteristic (ROC) analysis, the area under the ROC curve was greatest for ELISA, using two consecutive positive results (0.97;
P
= 0.036 for ELISA versus PCR,
P
= 0.055 for ELISA versus BDG). Based on the ROC curve, the cutoff for the ELISA could be reduced to an optical density index (O.D.I.) of 0.6. With the use of this cutoff for ELISA and cutoffs for PCR and BDG that give a comparable level of specificity, the sensitivity/specificity/positive predictive value/negative predictive value of the ELISA and the PCR and BDG tests were 1.00/0.93/0.55/1.00, 0.55/0.93/0.40/0.96, and 0.55/0.93/0.40/0.96, respectively. In conclusion, among these weekly screening tests for IA, the double-sandwich ELISA test was the most sensitive at predicting the diagnosis of IA in high-risk patients with hematological disorders, using a reduced cutoff of 0.6 O.D.I.
Ileocolitis Associated with
Anaerobiospirillum
in Cats
Authors: De Cock H. E. V. , Marks S. L. , Stacy B. A. , Zabka T. S. , Burkitt J. , Lu G. , Steffen D. J. , Duhamel G. E. 
Published: June 2004

ABSTRACT

Ileocolitis associated with spiral bacteria identified as an
Anaerobiospirillum
sp. was found in six cats. Two cats had acute onset of gastrointestinal signs characterized by vomiting and diarrhea in one cat and vomiting in another cat, one cat had chronic diarrhea that was refractory to medical therapy; one cat had acute onset of anorexia and lethargy, and two cats had clinical signs that were not related to the gastrointestinal tract. The presence of an
Anaerobiospirillum
sp. was demonstrated on the basis of ultrastructural morphology of spiral bacteria associated with intestinal lesions and PCR amplification of a genus-specific 16S rRNA gene from affected tissues from each cat. The colons of three clinically healthy cats without lesions and one cat with mild colitis not associated with spiral bacteria were negative for
Anaerobiospirillum
spp. in the same assay. Comparative nucleotide sequence analysis of cloned PCR products from three affected cats further suggested that the spiral bacteria were closely related to
Anaerobiospirillum succiniciproducens
.
Rapid Immunochromatographic Test for Serodiagnosis of Canine Leishmaniasis
Authors: Otranto D. , Paradies P. , Sasanelli M. , Spinelli R. , Brandonisio O. 
Published: June 2004

ABSTRACT
An rK39 immunochromatographic test and immunofluorescent-antibody test (IFAT) for serodiagnosis of canine leishmaniasis were evaluated. The two tests showed correlation for all but one of the sera obtained from 68 dogs confirmed as leishmaniasis cases and 40 dogs (22 healthy dogs and 18 dogs with other diseases) from areas where the disease is not endemic. Specificity was 100% for both tests, while sensitivity was 97% for the rapid test and 99% for IFAT.
Comparison of Versions 1.0 AND 1.5 of the UltraSensitive AMPLICOR HIV-1 MONITOR Test for Subjects with Low Viral Load
Authors: Jackson J. Brooks , Piwowar-Manning Estelle , Johnson-Lewis LeTanya , Bassett Roland , Demeter Lisa M. , Brambilla Donald 
Published: June 2004

ABSTRACT

We compared the performance of two UltraSensitive AMPLICOR HIV-1 MONITOR kits (version 1.5 [v1.5] versus v1.0) by retesting 404 plasma samples with low viral loads (<3,000 copies/ml) with both kits. With 292 samples that initially had <50 copies/ml by the v1.0 kit, the v1.5 assay was more sensitive than the v1.0 assay for samples with human immunodeficiency virus type 1 RNA near the 50-copy/ml cutoff (
P
= 0.0146). Median numbers of copies per milliliter were similar for 112 samples with 50 to 3,000 copies/ml with no difference in sensitivity with a 200-copy/ml cutoff.
A Decline in Mupirocin Resistance in Methicillin-ResistantStaphylococcus aureusAccompanied Administrative Control of Prescriptions
Authors: Walker Elaine S. , Levy Foster , Shorman Mahmoud , David Gerard , Abdalla Jehad , Sarubbi Felix A. 
Published: June 2004

ABSTRACTSusceptibility to mupirocin was assessed in methicillin-resistantStaphylococcus aureusisolates selected from eras corresponding to differences in usage rate and prescription policies at a Veterans Affairs medical center. The eras studied encompassed from the time of introduction of the drug to its widespread use, through recommended judicious use, and finally to subsequent stringent administrative control. Prescriptions declined from 3.0 to 0.1 per 1,000 patient days. Precipitous declines first in the numbers of isolates with high-level resistance (from 31% to 4%) and then in those with low-level resistance (from 26% to 10%) accompanied prescription control.
Identification of
Campylobacter jejuni
Multilocus Sequence Type ST-21 Clonal Complex by Single-Nucleotide Polymorphism Analysis
Authors: Best E. L. , Fox A. J. , Frost J. A. , Bolton F. J. 
Published: June 2004

ABSTRACT

Conserved single-nucleotide polymorphisms (SNPs) which characterize the allelic profile of the major epidemiological lineage ST-21 were identified from the alleles within the current
Campylobacter jejuni
multilocus sequence typing (MLST) database. Allelic discrimination assays were designed for the detection of SNPs, enabling rapid strain profiling for clonal complex ST-21. This method is suitable for epidemiological investigations and is complementary to full MLST.