bluuebunny/crossref_metadata_2025_split huggingface dataset:
High-Throughput Identification of Clinical Pathogenic Fungi by Hybridization to an Oligonucleotide Microarray
Authors: Huang Aihua , Li Jun-Wen , Shen Zhi-Qiang , Wang Xin-Wei , Jin Min 
Published: September 2006

ABSTRACT
Here we report the development of an oligonucleotide microarray method that can identify fungal pathogens in a single reaction. Specific oligonucleotide probes targeted to internal transcribed spacer 2 were designed and synthesized. Fungal DNA was amplified by universal primers, and the PCR product was hybridized with the oligonucleotide microarray. A series of specific hybridization profiles corresponding to species were obtained. The 122 strains of fungal pathogens, including standard and clinically isolated strains, used to test the specificity, stability, and sensitivity of the microarray system belonged to 20 species representing 8 genera. We found that the microarray system can successfully discriminate among the fungal pathogens to the species level, with high specificity and stability. The sensitivity was 15 pg/ml of DNA. This oligonucleotide microarray system represents a rapid, simple, and reliable alternative to conventional methods of identifying common clinical fungal isolates.
Evaluation of the New VITEK 2 Extended-Spectrum Beta-Lactamase (ESBL) Test for Rapid Detection of ESBL Production in
Enterobacteriaceae
Isolates
Authors: Spanu Teresa , Sanguinetti Maurizio , Tumbarello Mario , D'Inzeo Tiziana , Fiori Barbara , Posteraro Brunella , Santangelo Rosaria , Cauda Roberto , Fadda Giovanni 
Published: September 2006

ABSTRACT

Extended-spectrum beta-lactamases (ESBLs) are a large, rapidly evolving group of enzymes that confer resistance to oxyimino cephalosporins and monobactams and are inhibited by clavulanate. Rapid reliable detection of ESBL production is a prerequisite for successful infection management and for monitoring resistance trends and implementation of intervention strategies. We evaluated the performance of the new VITEK 2 ESBL test system (bioMérieux, Inc, Hazelwood, Mo.) in the identification of ESBL-producing
Enterobacteriaceae
isolates. We examined a total of 1,129 clinically relevant
Enterobacteriaceae
isolates (including 218 that had been previously characterized). The ESBL classification furnished by the VITEK 2 ESBL test system was concordant with that of the comparison method (molecular identification of beta-lactamase genes) for 1,121 (99.3%) of the 1,129 isolates evaluated. ESBL production was correctly detected in 306 of the 312 ESBL-producing organisms (sensitivity, 98.1%; positive predictive value, 99.3%). False-positive results emerged for 2 of the 817 ESBL-negative isolates (specificity, 99.7%; negative predictive value, 99.3%). VITEK 2 ESBL testing took 6 to 13 h (median, 7.5 h; mean ± SD, 8.2 ± 2.39 h). This automated short-incubation system appears to be a rapid and reliable tool for routine identification of ESBL-producing isolates of
Enterobacteriaceae
.
Fluorescence In Situ Hybridization for Rapid Identification of
Achromobacter xylosoxidans
and
Alcaligenes faecalis
Recovered from Cystic Fibrosis Patients
Authors: Wellinghausen Nele , Wirths Beate , Poppert Sven 
Published: September 2006

ABSTRACT

Achromobacter xylosoxidans
is frequently isolated from the respiratory secretions of cystic fibrosis (CF) patients, but identification with biochemical tests is unreliable. We describe fluorescence in situ hybridization assays for the rapid identification of
Achromobacter xylosoxidans
and
Alcaligenes faecalis
. Both assays showed high sensitivities and high specificities with a collection of 155 nonfermenters from CF patients.
Rapid and Sensitive Detection of Single
Cryptosporidium
Oocysts from Archived Glass Slides
Authors: Sunnotel O. , Snelling W. J. , Xiao L. , Moule K. , Moore J. E. , Millar B. Cherie , Dooley J. S. G. , Lowery C. J. 
Published: September 2006

ABSTRACT

In this study we report on the development and application of a novel method for efficiently extracting and detecting single
Cryptosporidium
oocysts from archived glass slides. Laser capture microscopy was used to extract low numbers of oocysts from archived glass slides. Highly sensitive real-time PCR methods were then developed to enable the rapid detection and identification of
Cryptosporidium
oocysts from these samples. The method was applied to fecal smears stained with a variety of standard oocyst stains and water samples. This application, with samples derived from both public health and water service laboratories, highlighted the strong potential of this method to be used as a rapid high-throughput screening tool for the routine monitoring of
Cryptosporidium
and other medically important pathogens from clinical, veterinary, and environmental water samples. Importantly, the application of our protocol could be used to type
Cryptosporidium
and other pathogens from stored archived glass slides in public health and water service laboratories, providing vital epidemiological updates and helping to identify and trace pathogens and their routes of infection and ultimately improve their control.
Comparison of Transcription-Mediated Amplification and PCR Assay Results for Various Genital Specimen Types for Detection of
Mycoplasma genitalium
Authors: Wroblewski Jennifer K. H. , Manhart Lisa E. , Dickey Kathleen A. , Hudspeth Marie K. , Totten Patricia A. 
Published: September 2006

ABSTRACT

Mycoplasma genitalium
is now recognized as a possible cause of several idiopathic sexually transmitted disease (STD) syndromes. However, due to the difficulty of culture of this fastidious bacterium, nucleic acid amplification tests (NAATs) are necessary for its detection in patient specimens. In the current study we compared a newly developed research-only transcription-mediated amplification (TMA) assay (Gen-Probe Incorporated) to our in-house DNA-based PCR assay for detection of
M. genitalium
. The relative performance characteristics of these two NAATs were assessed with genital specimens from 284 women and 352 men reporting to an STD clinic in Seattle, WA. Among the women,
M. genitalium
was detected by the TMA and PCR assays in 36 (13%) and 39 (14%) vaginal swab specimens, respectively (κ = 0.923); 26 (9%) and 23 (8%) cervical swab specimens, respectively (κ = 0.843); and 25 (9%) and 28 (10%) urine specimens, respectively (κ = 0.687). Among the
M. genitalium
-positive women, the relative sensitivities of detection for the TMA and PCR assays were 84% and 91%, respectively, for vaginal swab specimens; 60% and 53%, respectively, for cervical swab specimens; and 58% and 65%, respectively, for urine specimens. By using an infected patient (a woman positive at any site by TMA assay and at any site by PCR) as a proxy for a “gold standard,” the specificities of detection were >99.5% for both the TMA and the PCR assays. Among the men,
M. genitalium
was detected in 24 urine specimens (6.8%) by the TMA assay, 26 (7.4%) urine specimens by PCR assay, and 32 urine specimens (9%) by either test (κ = 0.791). We conclude that the
M. genitalium
TMA and PCR assays are highly specific and that vaginal swab specimens are the most sensitive specimen type for the detection of
M. genitalium
in women.
Nucleotide Sequence-Based Analysis for Determining the Molecular Epidemiology of
Penicillium marneffei
Authors: Lasker Brent A. 
Published: September 2006

ABSTRACT

The dimorphic fungus,
Penicillium marneffei
, is an emerging opportunistic pathogen endemic in Southeast Asia, especially for those with impaired cellular immunity such as human immunodeficiency virus-infected persons. A discriminatory and reproducible method based on the analysis of nucleotide sequences would facilitate epidemiologic investigations of this fungus. Twenty-four clinical or environmental isolates of
P. marneffei
obtained from China, Thailand, and Vietnam were analyzed by nucleotide sequence analysis. A total of 3,803 bp, consisting of eight nuclear gene fragments (transcription factor [AbaA], catalase [CpeA]], homodomain transcription factor [StlA], isocitrate lyase [Icl1], polyaromatic amino acid biosynthesis [PAA], NADH-dependent glutamate synthase [NGS], lovastatin nonaketide synthase [LNS], a cell wall mannoprotein [MP1], and a gene fragment of the cytochrome oxidase subunit 1 gene [COX1] of the
P. marneffei
mitochondrial genome) were amplified by PCR and then sequenced. No polymorphic sites within the Cox1 gene fragment were observed. Likewise, no nucleotide sequence polymorphisms were observed for three gene fragments: StlA, AbaA, and NGS. Seven single-nucleotide polymorphisms were observed for three gene fragments, Icl1, CpeA, and PAA, providing only a low degree of discriminatory power (D = 0.747). In contrast, the gene fragment for an antigenic cell wall glycoprotein, MP1, a useful immunologic marker for infection, was observed to be highly polymorphic with 12 different MP1 types (D = 0.887). Single-nucleotide polymorphisms were observed at 21 different locations in the MP1 gene fragment. Indels of 3, 21, 24, and 42 bp were observed and were in frame for protein translation. The relatively high degree of MP1 polymorphisms suggests the sequence is rapidly evolving in order to evade host immune responses. After all polymorphic gene sequences were combined, a high degree of genetic variation was observed (D = 0.949) for a total of 16 different haploid sequence types with 11 genotypes represented by single isolates. Phylogenetic analysis detected clusters composed of isolates obtained only from China or Thailand, as well as clusters with a combination of isolates from these two countries, indicating some mixing or common descent. Identical sequences were observed for isolates passed in vitro for 8 weeks, suggesting good reproducibility. The low degree of nucleotide diversity in housekeeping and regulatory genes suggests the recent emergence and spread as a species or an evolutionary bottleneck. In summary, multilocus sequence typing demonstrated a high degree of discriminatory power and reproducibility and may provide a robust and reliable adjunct method for genotyping isolates of
P. marneffei
and facilitating interlaboratory comparisons.
Edema Disease Caused by a Clone of
Escherichia coli
O147
Authors: Helgerson Amy F. , Sharma Vijay , Dow Amie M. , Schroeder Regina , Post Karen , Cornick Nancy A. 
Published: September 2006

ABSTRACT

Edema disease is a systemic disease of weaned pigs caused by host-adapted strains of
Escherichia coli
, most commonly belonging to serogroup O138, O139, or O141. In the late 1990s,
E. coli
O147 strains containing the virulence genes
f18
,
sta
,
stb
, and
stx
2
were recovered from outbreaks of edema disease in the United States. Pulsed-field gel electrophoresis (PFGE) was used to determine that the majority of these strains (34/43) were closely related to one another. Subsequent analysis by multilocus restriction typing confirmed the PFGE results and indicated that the cluster of edema disease strains were only distantly related to other
E. coli
O147 strains. Serogrouping of edema disease isolates from the Iowa State University Veterinary Diagnostic laboratory recovered between 1996 and 2000 indicated that 42% belonged to serogroup O147. Our data suggest that these strains may be a common serotype of edema disease-causing
E. coli
in the United States.
Serodiagnosis Using Recombinant Nipah Virus Nucleocapsid Protein Expressed in
Escherichia coli
Authors: Yu Fuxun , Khairullah Nor Shahidah , Inoue Shingo , Balasubramaniam Vijayamalar , Berendam Stella Joan , Teh Leok Kin , Ibrahim Nik Shamsiah Wan , Abdul Rahman Sohayati , Hassan Sharifah Syed , Hasebe Futoshi , Sinniah Mangalam , Morita Kouichi 
Published: September 2006

ABSTRACT

Nipah virus nucleocapsid (NiV-N) protein was expressed in
Escherichia coli
and purified by histidine tag-based affinity chromatography. An indirect immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) for human and swine sera and an IgM capture ELISA for human sera were established using the recombinant NiV-N protein as an antigen. One hundred thirty-three suspected patient sera and 16 swine sera were used to evaluate the newly established ELISA systems in comparison with the CDC inactivated-virus-based ELISA systems. For the human sera, the NiV-N protein-based indirect IgG ELISA had a sensitivity of 98.6% and a specificity of 98.4%, and the NiV-N protein-based IgM capture ELISA had a sensitivity of 91.7% and a specificity of 91.8%, with reference to the CDC ELISA systems. The NiV-N-based IgM ELISA was found to be more sensitive than the inactivated-virus-based ELISA in that it captured eight additional cases. For the swine sera, the two test systems were in 100% concordance. Our data indicate that the Nipah virus nucleocapsid protein is a highly immunogenic protein in human and swine infections and a good target for serodiagnosis. Our NiV-N protein-based ELISA systems are useful, safe, and affordable tools for diagnosis of Nipah virus infection and are especially fit to be used in large-scale epidemiological investigations and to be applied in developing countries.
Serological Detection of Varicella-Zoster Virus-Specific Immunoglobulin G by an Enzyme-Linked Immunosorbent Assay Using Glycoprotein Antigen
Authors: Sauerbrei A. , Wutzler P. 
Published: September 2006

ABSTRACT

Since the introduction of varicella vaccination in several countries, there has been an urgent need for commercially available test procedures that allow highly sensitive and specific quantitative determination of the varicella-zoster virus (VZV)-specific immune status, including immunity postimmunization. This study compared the performance of two enzyme-linked immunosorbent assays (ELISAs) for the sensitive and specific determination of VZV-specific immunoglobulin G (IgG) in seronegative and latently infected persons, as well as in vaccinees. One ELISA is based on the detection of antibody to VZV-specific envelope glycoproteins (gp), and the other comprises the whole antigen extract prepared from VZV-infected cells. A modified standard fluorescent-antibody-to-membrane-antigen (FAMA) assay was used as a reference. An excellent sensitivity (100%) in relation to FAMA was demonstrated for the gpELISA (Virion\Serion), while the non-gpELISA (Dade Behring) had a lower sensitivity (83%) when sera from latently infected persons were tested. After postvaccinal immunity was measured, a sensitivity of 87% was achieved with gpELISA, whereas the ELISA incorporating antigen extract of VZV-infected cells had a sensitivity of 78%. Excellent specificity (100%) was calculated for both the gpELISA and the non-gpELISA. In conclusion, SERION ELISA
classic
VZV IgG is useful for the sensitive and specific quantitative determination of VZV immune status after natural infection. The test can also be recommended for measuring antibody response after varicella vaccination, particularly after the cutoff value was optimized.
Molecular Epidemiology of Multidrug-ResistantMycobacterium bovisIsolates with the Same Spoligotyping Profile as Isolates from Animals
Authors: Romero Beatriz , Aranaz Alicia , Juan Lucía de , Álvarez Julio , Bezos Javier , Mateos Ana , Gómez-Mampaso Enrique , Domínguez Lucas 
Published: September 2006

ABSTRACTPCR-based characterization techniques have been adopted in most laboratories forMycobacterium bovistyping. We report a molecular characterization of human multidrug-resistantM. bovisisolates and three bovine isolates that share the spoligotyping profile. The analysis of the direct repeat region showed that both groups differed in the presence of spacers not included in the current membrane. They were also distinguished by two out of the nine mycobacterial interspersed repetitive unit variable-number tandem repeat loci tested, indicating that the human infection was not acquired from the cattle from which isolates were obtained. These results highlight that a combination of techniques is required for appropriate discrimination, even for those spoligotypes that have a low frequency.
Peptide Nucleic Acid Fluorescence In Situ Hybridization-Based Identification of
Candida albicans
and Its Impact on Mortality and Antifungal Therapy Costs
Authors: Forrest G. N. , Mankes K. , Jabra-Rizk M. A. , Weekes E. , Johnson J. K. , Lincalis D. P. , Venezia R. A. 
Published: September 2006

ABSTRACT

The impact of rapid identification of
Candida albicans
blood isolates by peptide nucleic acid fluorescence in situ hybridization (PNA FISH) on the selection and expenditure of antifungal therapy was evaluated. PNA FISH was 100% sensitive and specific in the rapid identification of 31 out of 72 candidemias as
C. albicans
and resulted in a significant reduction of caspofungin usage, with an overall cost savings of $1,729 per patient.
Prevalence of Newer β-Lactamases in Gram-Negative Clinical Isolates Collected in the United States from 2001 to 2002
Authors: Moland Ellen S. , Hanson Nancy D. , Black Jennifer A. , Hossain Ashfaque , Song Wonkeun , Thomson Kenneth S. 
Published: September 2006

ABSTRACT

Newer β-lactamases such as extended-spectrum β-lactamases (ESBLs), transferable AmpC β-lactamases, and carbapenemases are associated with laboratory testing problems of false susceptibility that can lead to inappropriate therapy for infected patients. Because there appears to be a lack of awareness of these enzymes, a study was conducted during 2001 to 2002 in which 6,421 consecutive, nonduplicate clinical isolates of aerobically growing gram-negative bacilli from patients at 42 intensive care unit (ICU) and 21 non-ICU sites across the United States were tested on-site for antibiotic susceptibility. From these isolates, 746 screen-positive isolates (11.6%) were referred to a research facility and investigated to determine the prevalence of ESBLs in all gram-negative isolates, transferable AmpC β-lactamases in
Klebsiella pneumoniae
, and carbapenemases in
Enterobacteriaceae
. The investigations involved phenotypic tests, isoelectric focusing, β-lactamase inhibitor studies, spectrophotometric assays, induction assays, and molecular analyses. ESBLs were detected only in
Enterobacteriaceae
(4.9% of all
Enterobacteriaceae
) and were found in species other than those currently recommended for ESBL testing by the CLSI (formerly NCCLS). These isolates occurred at 74% of the ICU sites and 43% of the non-ICU sites. Transferable AmpC β-lactamases were detected in 3.3% of
K. pneumoniae
isolates and at 16 of the 63 sites (25%) with no difference between ICU and non-ICU sites. Three sites submitted isolates that produced class A carbapenemases. No class B or D carbapenemases were detected. In conclusion, organisms producing ESBLs and transferable AmpC β-lactamases were widespread. Clinical laboratories must be able to detect important β-lactamases to ensure optimal patient care and infection control.
Molecular Differentiation ofTreponema pallidumSubspecies
Authors: Centurion-Lara Arturo , Molini Barbara J. , Godornes Charmie , Sun Eileen , Hevner Karin , Van Voorhis Wesley C. , Lukehart Sheila A. 
Published: September 2006

ABSTRACTTreponema pallidumincludes three subspecies of antigenically highly related treponemes. These organisms cause clinically distinct diseases and cannot be distinguished by any existing test. In this report, genetic signatures are identified in twotprgenes which, in combination with the previously published signature in the 5′ flanking region of the tpp15 gene, can differentiate theT. pallidumsubspecies, as well as a simian treponeme.
External Quality Assessment of Molecular Typing of
Staphylococcus aureus
Isolates by a Network of Laboratories
Authors: Deplano A. , De Mendonça R. , De Ryck R. , Struelens M. J. 
Published: September 2006

ABSTRACT

A network of laboratories designated Centres for Molecular Diagnosis was funded in 2000 by Belgian National Health Insurance to provide clinically relevant molecular diagnostic tests. These included typing of nosocomial pathogens as a service to local hospital infection control programs. Two external quality assessment (EQA) surveys were performed in 2001 and 2003 to evaluate the proficiencies of the laboratories at
Staphylococcus aureus
typing. EQA panels included
S. aureus
isolates with either indistinguishable, clonally related, or unrelated pulsed-field gel electrophoresis (PFGE) patterns. A hypothetical hospital outbreak problem was also submitted for analysis. Typeability, reproducibility, discrimination (
D
) index, and epidemiological concordance were evaluated. Ten centers participated in each survey. Seven centers performed PFGE analysis, while others used repetitive-element or randomly amplified polymorphic DNA PCR, amplified fragment length polymorphism, or
spa
typing. Full typeability (100%) was achieved by all centers, and all but one showed 100% reproducibility. Discrimination was appropriate (
D
index, ≥96%) for centers performing PFGE analysis but not for all those using other methods (
D
index range, 72% to 97%). Correct answers to the epidemiological questions were provided by 7/10 and 10/10 centers in 2001 and 2003, respectively. Individual feedback of results was provided to each center together with specific technical recommendations for improving performance. Our findings indicate that surveys of lab proficiency are useful for validation and optimization of molecular typing services to local hospital infection control programs.
Molecular Epidemiology of Rabies Virus Isolates in India
Authors: Nagarajan T. , Mohanasubramanian B. , Seshagiri E. V. , Nagendrakumar S. B. , Saseendranath M. R. , Satyanarayana M. L. , Thiagarajan D. , Rangarajan P. N. , Srinivasan V. A. 
Published: September 2006

ABSTRACT
In India, rabies is enzootic and is a serious public health and economic problem. India has a large population of stray dogs which, together with a lack of effective control strategies, might have led to the persistence of rabies virus (RV) in the canine population. Our objective was to study the molecular epidemiology of RV isolates in India based on nucleotide sequence analysis of 29 RV isolates originating from different species of animals in four states. Here we have analyzed two sets of sequence data based upon a 132-nucleotide region of the cytoplasmic domain (CD) of the G gene (G-CD) and a 549-nucleotide region (Psi-L) that combines the noncoding G-L intergenic region (Psi) and a fragment of the polymerase gene (L). Phylogenetic analysis revealed that the RV isolates belong to genotype 1 and that they were related geographically but were not related according to host species. Five different genetic clusters distributed among three geographical regions were identified. Comparison of the deduced amino acid sequences of G-CD between RV isolates revealed three amino acid changes (amino acid 462G [aa462G], aa465H, and aa468K) that distinguished the Indian RVs from RV isolates in other parts of the world. Analysis of the data indicated that the dog rabies virus variants are the major circulating viruses in India that transmit the disease to other domestic animals and humans as well.