bluuebunny/crossref_metadata_2025_split huggingface dataset:
Growth of a
Dehalococcoides
-Like Microorganism on Vinyl Chloride and
cis
-Dichloroethene as Electron Acceptors as Determined by Competitive PCR
Authors: Cupples Alison M. , Spormann Alfred M. , McCarty Perry L. 
Published: February 2003

ABSTRACT

A competitive PCR (cPCR) assay targeting 16S ribosomal DNA was developed to enumerate growth of a
Dehalococcoides
-like microorganism, bacterium VS, from a mixed culture catalyzing the reductive dehalogenation of
cis
-1,2-dichloroethene (cDCE) and vinyl chloride (VC), with hydrogen being used as an electron donor. The growth of bacterium VS was found to be coupled to the dehalogenation of VC and cDCE, suggesting unique metabolic capabilities. The average growth yield was (5.2 ± 1.5) × 10
8
copies of the 16S rRNA gene/μmol of Cl
−
(number of samples, 10), with VC being used as the electron acceptor and hydrogen as the electron donor. The maximum VC utilization rate (
q̂
) was determined to be 7.8 × 10
−10
μmol of Cl
−
(copy
−1
day
−1
), indicating a maximum growth rate of 0.4 day
−1
. These average growth yield and

q̂

values agree well with values found previously for dechlorinating cultures. Decay coefficients were determined with growth (0.05 day
−1
) and no-growth (0.09 day
−1
) conditions. An important limitation of this cPCR assay was its inability to discriminate between active and inactive cells. This is an essential consideration for kinetic studies.
Functional Expression of a Fungal Laccase in
Saccharomyces cerevisiae
by Directed Evolution
Authors: Bulter Thomas , Alcalde Miguel , Sieber Volker , Meinhold Peter , Schlachtbauer Christian , Arnold Frances H. 
Published: February 2003

ABSTRACT

Laccase from
Myceliophthora thermophila
(MtL) was expressed in functional form in
Saccharomyces cerevisiae
. Directed evolution improved expression eightfold to the highest yet reported for a laccase in yeast (18 mg/liter). Together with a 22-fold increase in
k
cat
, the total activity was enhanced 170-fold. Specific activities of MtL mutants toward 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) and syringaldazine indicate that substrate specificity was not changed by the introduced mutations. The most effective mutation (10-fold increase in total activity) introduced a Kex2 protease recognition site at the C-terminal processing site of the protein, adjusting the protein sequence to the different protease specificities of the heterologous host. The C terminus is shown to be important for laccase activity, since removing it by a truncation of the gene reduces activity sixfold. Mutations accumulated during nine generations of evolution for higher activity decreased enzyme stability. Screening for improved stability in one generation produced a mutant more stable than the heterologous wild type and retaining the improved activity. The molecular mass of MtL expressed in
S. cerevisiae
is 30% higher than that of the same enzyme expressed in
M. thermophila
(110 kDa versus 85 kDa). Hyperglycosylation, corresponding to a 120-monomer glycan on one N-glycosylation site, is responsible for this increase. This
S. cerevisiae
expression system makes MtL available for functional tailoring by directed evolution.
New PCR Assay Using Glucose-6-Phosphate Dehydrogenase for Identification of
Leishmania
Species
Authors: Castilho Tiago M. , Shaw Jeffrey Jon , Floeter-Winter Lucile M. 
Published: February 2003

ABSTRACT

Glucose-6-phosphate dehydrogenase (G6PD) is one of the multilocus enzymes used to identify
Leishmania
by zymodeme analysis. The polymorphic pattern revealed by partial characterization of the gene encoding G6PD generated molecular markers useful in the identification of different
Leishmania
species by PCR. Initially degenerate oligonucleotides were designed on the basis of data on the conserved active center described for other organisms. Primers for reverse transcription-PCR experiments, designed from the nucleotide sequence of the PCR product, enabled us to characterize the 5′ and 3′ untranslated regions and the
G6PD
open reading frame of reference strains of
Leishmania
(
Viannia
)
braziliensis
,
Leishmania
(
Viannia
)
guyanensis
,
Leishmania
(
Leishmania
)
mexicana
, and
Leishmania
(
Leishmania
)
amazonensis
. Sets of paired primers were designed and used in PCR assays to discriminate between the parasites responsible for tegumentar leishmaniasis of the subgenera
Leishmania
(
Leishmania
) and
Leishmania
(
Viannia
) and to distinguish
L.
(
Viannia
)
braziliensis
from others organisms of the subgenus
Leishmania
(
Viannia
). No amplification products were detected for the DNA of
Crithidia fasciculata
,
Trypanosoma cruzi
, or
Leishmania
(
Sauroleishmania
)
tarentolae
or DNA from a healthy human control. The tests proved to be specific and were sensitive enough to detect parasites in human biopsy specimens. The successful discrimination of
L.
(
Viannia
)
braziliensis
from other parasites of the subgenus
Leishmania
(
Viannia
) opens the way to epidemiological studies in areas where more than one species of the subgenus
Leishmania
(
Viannia
) exist, such as Amazonia, as well as follow-up studies after chemotherapy and assessment of clinical prognoses.
Detection and Identification of Fungi from Fungus Balls of the Maxillary Sinus by Molecular Techniques
Authors: Willinger Birgit , Obradovic Alexandra , Selitsch Brigitte , Beck-Mannagetta Johann , Buzina Walter , Braun Hannes , Apfalter Petra , Hirschl Alexander M. , Makristathis Athanasios , Rotter Manfred 
Published: February 2003

ABSTRACT

The aim of this study was to find a reliable method for the detection and identification of fungi in fungus balls of the maxillary sinus and to evaluate the spectrum of fungi in these samples. One hundred twelve samples were obtained from patients with histologically proven fungal infections; 81 samples were paraffin-embedded tissue sections of the maxillary sinus. In 31 cases, sinus contents without paraffin embedding were sent for investigation. PCR amplification with universal fungal primers for 28S ribosomal DNA and amplicon identification by hybridization with species-specific probes for
Aspergillus fumigatus
,
Aspergillus flavus
,
Aspergillus niger
,
Aspergillus terreus
,
Aspergillus glaucus
,
Pseudallescheria boydii
,
Candida albicans
, and
Candida glabrata
were performed for all samples. Furthermore, PCR products were sequenced. Fresh samples were also cultivated. Fungal DNA was detected in all of the fresh samples but only in 71 paraffin-embedded tissue samples (87.7%). Sequence analysis was the most sensitive technique, as results could be obtained for 28 (90.3%) fresh samples by this method in comparison to 24 (77.4%) samples by hybridization and 16 (51.6%) samples by culture. However, sequence analysis delivered a result for only 36 (50.7%) of the paraffin-embedded specimens. Hybridization showed reliable results for
A. fumigatus
, which proved to be the most common agent in fungus balls of the maxillary sinus. Other
Aspergillus
species and other genera were rarely found.
Multiple Patterns of Resistance to Fluconazole in
Candida glabrata
Isolates from a Patient with Oropharyngeal Candidiasis Receiving Head and Neck Radiation
Authors: Redding Spencer W. , Kirkpatrick William R. , Saville Stephen , Coco Brent J. , White William , Fothergill Annette , Rinaldi Michael , Eng Tony , Patterson Thomas F. , Lopez-Ribot Jose 
Published: February 2003

ABSTRACT

Candida glabrata
has emerged in recent years as a significant cause of systemic fungal infection. We have previously reported on the first three patients receiving radiation for head and neck cancer to develop oropharyngeal candidiasis due to
C. glabrata
. The goal of this study was to track the development of increased fluconazole resistance in
C. glabrata
isolates and to evaluate previously described genetic mechanisms associated with this resistance from one of these three patients. The patient was a 52-year-old man with squamous cell carcinoma treated with radiation. At week 7 of his radiation, he developed oropharyngeal candidiasis, which was treated with 200 mg of fluconazole daily for 2 weeks. Serial cultures from this and three subsequent time points yielded
C. glabrata
. Isolates from these cultures were subjected to antifungal susceptibility testing, DNA karyotyping, and evaluation of the expression of genes previously associated with
C. glabrata
resistance to fluconazole,
CgCDR1
,
CgCDR2
, and
CgERG11
. Two strains (A and B) of
C. glabrata
were identified and found to display different patterns of resistance development and gene expression. Strain A developed resistance over a 2-week period and showed no overexpression of these genes. In contrast, strain B first showed resistance 6 weeks after fluconazole therapy was discontinued but showed overexpression of all three genes. In conclusion, development of resistance to fluconazole by
C. glabrata
is a highly varied process involving multiple molecular mechanisms.
Multicenter Evaluation of the VERSANT HCV RNA Qualitative Assay for Detection of Hepatitis C Virus RNA
Authors: Hendricks David A. , Friesenhahn Michel , Tanimoto Lorine , Goergen Bernd , Dodge Deborah , Comanor Lorraine 
Published: February 2003

ABSTRACT

We report the results of a multicenter evaluation of a new assay for the detection of hepatitis C virus (HCV) RNA in human serum or plasma based on transcription-mediated amplification (HCV TMA). Analysis of combined data obtained from 15 independent sites, including 4 sites in the United States and 11 in Europe, by using preproduction kits showed a limit of detection of 9.8 IU/ml and an overall mean specificity of 97.9%. In addition, assay runs and samples were valid consistently (97.8% of assay runs and 98.0% of specimen results). Of the 15 sites that participated in the multicenter evaluation, 2 subsequently carried out additional performance studies with production kits in support of the assay's registration in France. Comparison of the findings from these two sites during the multicenter evaluation and during the registration studies showed an overall improvement in assay performance. A statistically significant (
P
< 0.001) improvement was achieved for both specificity and specimen validity in the registration studies, which were 99.4 and 98.1%, respectively. Combined data from the two sites showed a lower limit of detection of approximately 2.4 IU/ml and an improved assay validity of 100%, although the sample size was too small to show statistical significance at the 0.05 level. In summary, the performance characteristics of HCV TMA indicate that this assay is a reliable tool for the detection of HCV RNA in serum or plasma. Improvement in assay performance has been demonstrated with refinement of assay reagents, instrumentation, and operator experience.
Identification of Major Streptococcal Species by
rrn
-Amplified Ribosomal DNA Restriction Analysis
Authors: Schlegel Laurent , Grimont Francine , Grimont Patrick A. D. , Bouvet Anne 
Published: February 2003

ABSTRACT

Amplified ribosomal DNA restriction analysis (
rrn
-ARDRA) is based on PCR amplification and restriction of a fragment of rRNA genes including 16S and 23S genes and the intergenic spacer.
rrn
-ARDRA was evaluated for the identification of species within the genus
Streptococcus
. A total of 148 type and reference strains of pyogenic, oral, and group D streptococci were examined in order to construct a database for identification of streptococci. The amplified product was a single band approximately 4,500 bp long. This amplicon was digested separately with three (
Hha
I,
Mbo
II, and
Sau
3A) restriction endonucleases. Respectively, 27, 26, and 28 major patterns were observed after
Hha
I,
Mbo
II, and
Sau
3A restrictions. Streptococcal strains belonging to different species had different patterns or different combination of patterns. An identification system based upon a combination of the three restriction patterns in a single database was then proposed.
rrn
-ARDRA was successfully applied to 11 clinical isolates whose identification to the species level was difficult to obtain by phenotypic analysis. Using a database of well-characterized strains,
rrn
-ARDRA is a powerful method for the identification of streptococcal isolates.
Six Rapid Tests for Direct Detection of
Clostridium difficile
and Its Toxins in Fecal Samples Compared with the Fibroblast Cytotoxicity Assay
Authors: Turgeon David K. , Novicki Thomas J. , Quick John , Carlson LaDonna , Miller Pat , Ulness Bruce , Cent Anne , Ashley Rhoda , Larson Ann , Coyle Marie , Limaye Ajit P. , Cookson Brad T. , Fritsche Thomas R. 
Published: February 2003

ABSTRACT

Clostridium difficile
is one of the most frequent causes of nosocomial gastrointestinal disease. Risk factors include prior antibiotic therapy, bowel surgery, and the immunocompromised state. Direct fecal analysis for
C. difficile
toxin B by tissue culture cytotoxin B assay (CBA), while only 60 to 85% sensitive overall, is a common laboratory method. We have used 1,003 consecutive, nonduplicate fecal samples to compare six commercially available immunoassays (IA) for
C. difficile
detection with CBA: Prima System
Clostridium difficile
Tox A and VIDAS
Clostridium difficile
Tox A II, which detect
C. difficile
toxin A; Premier Cytoclone A/B and Techlab
Clostridium difficile
Tox A/B, which detect toxins A and B; and ImmunoCard
Clostridium difficile
and Triage Micro
C. difficile
panels, which detect toxin A and a species-specific antigen. For all tests, Triage antigen was most sensitive (89.1%; negative predictive value [NPV] = 98.7%) while ImmunoCard was most specific (99.7%; positive predictive value [PPV] = 95.0%). For toxin tests only, Prima System had the highest sensitivity (82.2%; NPV = 98.0%) while ImmunoCard had the highest specificity (99.7%; PPV = 95.0%). Hematopoietic stem cell transplant (HSCT) patients contributed 44.7% of all samples tested, and no significant differences in sensitivity or specificity were noted between HSCT and non-HSCT patients. IAs, while not as sensitive as direct fecal CBA, produce reasonable predictive values, especially when both antigen and toxin are detected. They also offer significant advantages over CBA in terms of turnaround time and ease of use.
Longitudinal Reliability of Focus Glycoprotein G-Based Type-Specific Enzyme Immunoassays for Detection of Herpes Simplex Virus Types 1 and 2 in Women
Authors: Cherpes Thomas L. , Ashley Rhoda L. , Meyn Leslie A , Hillier Sharon L. 
Published: February 2003

ABSTRACT

Serologic assays that utilize herpes simplex virus (HSV) type-specific glycoproteins G-1 (HSV-1) and G-2 (HSV-2) to discriminate between antibodies against HSV-1 and HSV-2 are sensitive and specific. However, the high rates of seroreversion, defined as the change in an individual's antibody status from positive to negative over time, previously reported in longitudinal evaluations of glycoprotein G type-specific tests suggests that their use in HSV acquisitional studies would be problematic. To further explore the reliability of the glycoprotein G-based serologic tests, we evaluated HSV-1 and HSV-2 enzyme immunoassays from Focus Technologies in a longitudinal cohort of 1,207 young women from Pittsburgh, Pa. On enrollment of the women in the study, HSV-1 and HSV-2 antibodies were detected in 46.6 and 24.9% of the women, respectively. Among the women with at least three visits, 3.4% (15 of 447) of those who were HSV-1 antibody positive had a subsequent negative result while fewer than 1% (2 of 227) of those who were HSV-2 antibody positive seroreverted. The median of mean positive index values for women who seroreverted to HSV-1 antibody was lower than that for women who remained seropositive (1.25 versus 7.06;
P
< 0.001). Similarly, the median of mean positive index values for women whose HSV-2 antibody status reverted from positive to negative was lower than that for those women who did not serorevert (1.83 versus 7.46;
P
= 0.02). Comparative Western blot analysis demonstrated that the lower positive index values, seen more often among the HSV seroreverters, often signified false-positive immunoassay results. Overall, the seroreversion rates were low; the use of glycoprotein G-based serologic tests for the measurement of HSV-1 and HSV-2 antibodies in incidence studies therefore appears warranted.
Multilocus Sequence Typing Reveals a Lack of Diversity among
Escherichia coli
O157:H7 Isolates That Are Distinct by Pulsed-Field Gel Electrophoresis
Authors: Noller Anna C. , McEllistrem M. Catherine , Stine O. Colin , Morris, J. Glenn , Boxrud David J. , Dixon Bruce , Harrison Lee H. 
Published: February 2003

ABSTRACT

Escherichia coli
O157:H7 is a major cause of foodborne illness in the United States. Pulsed-field gel electrophoresis (PFGE) is the molecular epidemiologic method mostly commonly used to identify food-borne outbreaks. Although PFGE is a powerful epidemiologic tool, it has disadvantages that make a DNA sequence-based approach potentially attractive. Multilocus sequence typing (MLST) analyzes the internal fragments of housekeeping genes to establish genetic relatedness between isolates. We sequenced selected portions of seven housekeeping genes and two membrane protein genes (
ompA
and
espA
) of 77 isolates that were diverse by PFGE to determine whether there was sufficient sequence variation to be useful as an epidemiologic tool. There was no DNA sequence diversity in the sequenced portions of the seven housekeeping genes and
espA
. For
ompA
, all but five isolates had sequence identical to that of the reference strains.
E. coli
O157:H7 has a striking lack of genetic diversity in the genes we explored, even among isolates that are clearly distinct by PFGE. Other approaches to identify improved molecular subtyping methods for
E. coli
0157:H7 are needed.
Detection ofFrancisella tularensiswithin Infected Mouse Tissues by Using a Hand-Held PCR Thermocycler
Authors: Emanuel Peter A. , Bell Ryan , Dang Jessica L. , McClanahan Rebecca , David John C. , Burgess Robert J. , Thompson Joseph , Collins Lisa , Hadfield Ted 
Published: February 2003

ABSTRACTThe diagnosis of human cases of tularemia often relies upon the demonstration of an antibody response toFrancisella tularensisor the direct culturing of the bacteria from the patient. Antibody response is not detectable until 2 weeks or more after infection, and culturing requires special media and suspicion of tularemia. In addition, handling liveFrancisellaposes a risk to laboratory personnel due to the highly infectious nature of this pathogen. In an effort to develop a rapid diagnostic assay for tularemia, we investigated the use of TaqMan 5′ hydrolysis fluorogenic PCR to detect the organism in tissues of infected mice. Mice were infected to produce respiratory tularemia. ThefopAandtul4genes ofF. tularensiswere amplified from infected spleen, lung, liver, and kidney tissues sampled over a 5-day period. The samples were analyzed using the laboratory-based Applied Biosystems International 7900 and the Smiths Detection-Edgewood BioSeeq, a hand-held portable fluorescence thermocycler designed for use in the field. A comparison of culturing and PCR for detection of bacteria in infected tissues shows that culturing was more sensitive than PCR. However, the results for culture take 72 h, whereas PCR results were available within 4 h. PCR was able to detect infection in all the tissues tested. Lung tissue showed the earliest response at 2 days when tested with the ABI 7900 and in 3 days when tested with the BioSeeq. The results were in agreement between the ABI 7900 and the BioSeeq when presented with the same sample. Template preparation may account for the loss of sensitivity compared to culturing techniques. The hand-held BioSeeq thermocycler shows promise as an expedient means of forward diagnosis of infection in the field.
Candida tropicalis
in a Neonatal Intensive Care Unit: Epidemiologic and Molecular Analysis of an Outbreak of Infection with an Uncommon Neonatal Pathogen
Authors: Roilides Emmanuel , Farmaki Evangelia , Evdoridou Joanna , Francesconi Andrea , Kasai Miki , Filioti Joanna , Tsivitanidou Maria , Sofianou Danai , Kremenopoulos George , Walsh Thomas J. 
Published: February 2003

ABSTRACT

From June to July 1998, two episodes of
Candida tropicalis
fungemia occurred in the Aristotle University neonatal intensive care unit (ICU). To investigate this uncommon event, a prospective study of fungal colonization and infection was conducted. From December 1998 to December 1999, surveillance cultures of the oral cavities and perinea of the 593 of the 781 neonates admitted to the neonatal ICU who were expected to stay for >7 days were performed. Potential environmental reservoirs and possible risk factors for acquisition of
C. tropicalis
were searched for. Molecular epidemiologic studies by two methods of restriction fragment length polymorphism analysis and two methods of random amplified polymorphic DNA analysis were performed. Seventy-two neonates were colonized by yeasts (12.1%), of which 30 were colonized by
Candida albicans
, 17 were colonized by
C. tropicalis
, and 5 were colonized by
Candida parapsilosis
. From December 1998 to December 1999, 10 cases of fungemia occurred; 6 were due to
C. parapsilosis
, 2 were due to
C. tropicalis
, 1 was due to
Candida glabrata
, and 1 was due to
Trichosporon asahii
(12.8/1,000 admissions). Fungemia occurred more frequently in colonized than in noncolonized neonates (
P
< 0.0001). Genetic analysis of 11 colonization isolates and the two late blood isolates of
C. tropicalis
demonstrated two genotypes. One blood isolate and nine colonization isolates belonged to a single type. The fungemia/colonization ratio of
C. parapsilosis
(3/5) was greater than that of
C. tropicalis
(2/17,
P
= 0.05), other non-
C. albicans Candida
spp. (1/11,
P
= 0.02), or
C. albican
s (0/27,
P
= 0.05). Extensive environmental cultures revealed no common source of
C. tropicalis
or
C. parapsilosis
. There was neither prophylactic use of azoles nor other risk factors found for acquisition of
C. tropicalis
except for total parenteral nutrition. A substantial risk of colonization by non-
C. albicans Candida
spp. in the neonatal ICU may lead to a preponderance of
C. tropicalis
as a significant cause of neonatal fungemia.