bluuebunny/crossref_metadata_2025 huggingface dataset:
Melatonin suppresses reactive oxygen species induced by UV irradiation in leukocytes
Authors: Fischer Tobias W. , Scholz Georg , Knöll Brunhilde , Hipler Uta‐Christina , Elsner Peter
Published: August 2004

Abstract  An investigation of the antioxidative UV protective effect of melatonin was performed in an in vitro irradiation model with leukocytes. Leukocytes were isolated from EDTA‐treated whole blood and taken up in phosphate‐buffered saline (PBS). Five of 10 aliquots were incubated with 2 mmol/L melatonin and 5 with PBS as a control. The samples were irradiated by UV light (280–360 nm, max: 310 nm) at doses between 75 and 300 mJ/cm2 or left unirradiated. Radical formation was measured using the chemiluminescence technique. Staining with trypan blue was performed to assess cell viability. Melatonin significantly suppressed radical formation in cell solutions irradiated from 75 to 300 mJ/cm2 (P ≤ 0.001). Controls showed an increase of reactive oxygen species (ROS) formation as a sign of oxidative stress when irradiated with increasing UV doses and a maximum ROS formation under 300 mJ/cm2 UV light. The cytotoxicity of UV light was reduced by melatonin up to a UV dose of 1.5 J/cm2. Leukocytes were suitable cells for the evaluation of the efficacy of melatonin as a radical scavenger under UV light. The results confirm that the clinically observed UV protective effects of melatonin may be at least partially based on its radical scavenging properties.
Influence of light–dark and lunar cycles on the ocular melatonin rhythms in the seagrass rabbitfish, a lunar‐synchronized spawner
Authors: Rahman Md. Saydur , Kim Byung‐Ho , Takemura Akihiro , Park Chang‐Bum , Lee Young‐Don
Published: August 2004

Abstract:  The aim of this study was to investigate the effects of light–dark (LD) cycles and lunar phases on ocular melatonin rhythms in the seagrass rabbitfish, Siganus canaliculatus, a lunar‐synchronized spawner. Under a natural 24‐hr LD (12.00:12.00) cycle, ocular melatonin levels were low during daylight hours. The levels significantly elevated to peak during the mid‐dark phase at 24.00 hr and then declined sharply in the early morning around 06.00 hr. These rhythms disappeared under either constant light (LL) or constant dark (DD) conditions. Melatonin levels remained low in LL compared with those in DD condition. These results suggest that ocular melatonin rhythms in the seagrass rabbitfish are suppressed in the presence of light. When fish were exposed to natural moon phases, ocular melatonin concentrations were higher around the new moon than both the first quarter and full moon phases. Exposure to experimental new moon conditions caused a significant increase in melatonin levels while those of the fish exposed to experimental full moon conditions were decreased. These results suggest that the seagrass rabbitfish perceives moonlight through the eye and that moonlight directly causes melatonin suppression.
Metamaterials and Negative Refractive Index
Authors: Smith D. R. , Pendry J. B. , Wiltshire M. C. K.
Published: August 2004

Recently, artificially constructed metamaterials have become of considerable interest, because these materials can exhibit electromagnetic characteristics unlike those of any conventional materials. Artificial magnetism and negative refractive index are two specific types of behavior that have been demonstrated over the past few years, illustrating the new physics and new applications possible when we expand our view as to what constitutes a material. In this review, we describe recent advances in metamaterials research and discuss the potential that these materials may hold for realizing new and seemingly exotic electromagnetic phenomena.
Beauty or Brains?
Authors: Ramachandran V. S.
Published: August 2004

The Psychology of Art and the Evolution of the Conscious Brain
. by Robert L. Solso. MIT Press, Cambridge, MA, 2004. 296 pp. $45, £29.95. ISBN 0-262-19484-8.

Drawing on cognitive science, anthropology, and art history, the author explores how human consciousness perceives and creates visual art.
Climatic Control of Riverine and Seawater Uranium-Isotope Ratios
Authors: Robinson Laura F. , Henderson Gideon M. , Hall Lisa , Matthews Iain
Published: August 2004

The large variation in the ratio of uranium-234 to uranium-238 (
234
U/
238
U) in rivers is not well understood, but may provide information about past weathering and rainfall and is important because it controls seawater (
234
U/
238
U). Here, we demonstrate the importance of physical weathering and rainfall for (
234
U/
238
U), using rivers from South Island, New Zealand. These data allow interpretation of an existing speleothem (
234
U/
238
U) record and suggest that New Zealand glacier advance 13,000 years ago was influenced by increased rainfall rather than by Younger Dryas–like cooling. A model of seawater (
234
U/
238
U) during glacial cycles indicates that rejection of corals based on modern (
234
U/
238
U) ± <0.01 is not merited and may reject the highest quality ages.
Structural Basis of Mitochondrial Tethering by Mitofusin Complexes
Authors: Koshiba Takumi , Detmer Scott A. , Kaiser Jens T. , Chen Hsiuchen , McCaffery J. Michael , Chan David C.
Published: August 2004

Vesicle fusion involves vesicle tethering, docking, and membrane merger. We show that mitofusin, an integral mitochondrial membrane protein, is required on adjacent mitochondria to mediate fusion, which indicates that mitofusin complexes act in trans (that is, between adjacent mitochondria). A heptad repeat region (HR2) mediates mitofusin oligomerization by assembling a dimeric, antiparallel coiled coil. The transmembrane segments are located at opposite ends of the 95 angstrom coiled coil and provide a mechanism for organelle tethering. Consistent with this proposal, truncated mitofusin, in an HR2-dependent manner, causes mitochondria to become apposed with a uniform gap. Our results suggest that HR2 functions as a mitochondrial tether before fusion.
A Plant Vacuolar Protease, VPE, Mediates Virus-Induced Hypersensitive Cell Death
Authors: Hatsugai Noriyuki , Kuroyanagi Miwa , Yamada Kenji , Meshi Tetsuo , Tsuda Shinya , Kondo Maki , Nishimura Mikio , Hara-Nishimura Ikuko
Published: August 2004

Programmed cell death (PCD) in animals depends on caspase protease activity. Plants also exhibit PCD, for example as a response to pathogens, although a plant caspase remains elusive. Here we show that vacuolar processing enzyme (VPE) is a protease essential for a virus-induced hypersensitive response that involves PCD.
VPE
deficiency prevented virus-induced hypersensitive cell death in tobacco plants. VPE is structurally unrelated to caspases, although VPE has a caspase-1 activity. Thus, plants have evolved a regulated cellular suicide strategy that, unlike PCD of animals, is mediated by VPE and the cellular vacuole.
Chemistry of Rocks and Soils in Gusev Crater from the Alpha Particle X-ray Spectrometer
Authors: Gellert R. , Rieder R. , Anderson R. C. , Brückner J. , Clark B. C. , Dreibus G. , Economou T. , Klingelhöfer G. , Lugmair G. W. , Ming D. W. , Squyres S. W. , d'Uston C. , Wänke H. , Yen A. , Zipfel J.
Published: August 2004

The alpha particle x-ray spectrometer on the Spirit rover determined major and minor elements of soils and rocks in Gusev crater in order to unravel the crustal evolution of planet Mars. The composition of soils is similar to those at previous landing sites, as a result of global mixing and distribution by dust storms. Rocks (fresh surfaces exposed by the rock abrasion tool) resemble volcanic rocks of primitive basaltic composition with low intrinsic potassium contents. High abundance of bromine (up to 170 parts per million) in rocks may indicate the alteration of surfaces formed during a past period of aqueous activity in Gusev crater.
Mineralogy at Gusev Crater from the Mössbauer Spectrometer on the Spirit Rover
Authors: Morris R. V. , Klingelhöfer G. , Bernhardt B. , Schröder C. , Rodionov D. S. , de Souza P. A. , Yen A. , Gellert R. , Evlanov E. N. , Foh J. , Kankeleit E. , Gütlich P. , Ming D. W. , Renz F. , Wdowiak T. , Squyres S. W. , Arvidson R. E.
Published: August 2004

Mössbauer spectra measured on Mars by the Spirit rover during the primary mission are characterized by two ferrous iron doublets (olivine and probably pyroxene) and a ferric iron doublet (tentatively associated to nanophase ferric iron oxide). Two sextets resulting from nonstoichiometric magnetite are also present, except for a coating on the rock Mazatzal, where a hematite-like sextet is present. Greater proportions of ferric-bearing phases are associated with undisturbed soils and rock surfaces as compared to fresh rock surfaces exposed by grinding. The ubiquitous presence of olivine in soil suggests that physical rather than chemical weathering processes currently dominate at Gusev crater.
Wind-Related Processes Detected by the Spirit Rover at Gusev Crater, Mars
Authors: Greeley R. , Squyres S. W. , Arvidson R. E. , Bartlett P. , Bell J. F. , Blaney D. , Cabrol N. A. , Farmer J. , Farrand B. , Golombek M. P. , Gorevan S. P. , Grant J. A. , Haldemann A. F. C. , Herkenhoff K. E. , Johnson J. , Landis G. , Madsen M. B. , McLennan S. M. , Moersch J. , Rice J. W. , Richter L. , Ruff S. , Sullivan R. J. , Thompson S. D. , Wang A. , Weitz C. M. , Whelley P.
Published: August 2004

Wind-abraded rocks, ripples, drifts, and other deposits of windblown sediments are seen at the Columbia Memorial Station where the Spirit rover landed. Orientations of these features suggest formative winds from the north-northwest, consistent with predictions from atmospheric models of afternoon winds in Gusev Crater. Cuttings from the rover Rock Abrasion Tool are asymmetrically distributed toward the south-southeast, suggesting active winds from the north-northwest at the time (midday) of the abrasion operations. Characteristics of some rocks, such as a two-toned appearance, suggest that they were possibly buried and exhumed on the order of 5 to 60 centimeters by wind deflation, depending on location.
Molecular Serotyping ofKlebsiellaSpecies Isolates by Restriction of the Amplified Capsular Antigen Gene Cluster
Authors: Brisse Sylvain , Issenhuth-Jeanjean Sylvie , Grimont Patrick A. D.
Published: August 2004

The objective of the present work was to develop a molecular method that would enable determination of the capsular serotypes ofKlebsiellaisolates without the use of antiserum. PCR amplification of the capsular antigen gene cluster (cps) was followed by digestion with the restriction enzyme HincII (cpsPCR-restriction fragment length polymorphism [RFLP] analysis). The profiles (C patterns) obtained for 224 strains representing the 77 known K serotypes showed 3 to 13 fragments ranging in size from 0.2 to 4.4 kb. A total of 97 distinct C patterns were obtained; 100% of 61 pairs of samples tested twice showed reproducible C patterns. The C patterns were K-type specific; i.e., the C pattern(s) of any K serotype was distinct from the C patterns of all other K serotypes, with the only exceptions being serotypes K22 and K37, which are known to cross-react. For 12 of 17 K types for which at least two strains were included, C-pattern variations were found among strains with the same K serotype. Therefore,cpsPCR-RFLP analysis has a higher discriminatory power than classical K serotyping. C-pattern identity was observed among strains with a given K type that were collected many years apart and from distinct sources, indicating C-pattern stability. Only 4.5% of the strains were nontypeable, because of unsuccessful PCR amplification (whereas 8 to 23% are nontypeable by classical K serotyping). Three of four noncapsulated strains analyzed showed recognizable C patterns. The K serotypes of 18 (82%) of 22 recentKlebsiella pneumoniaeclinical isolates could be deduced from their C patterns. In conclusion,cpsPCR-RFLP analysis allows determination of the K serotype, while it is easier to perform and more discriminatory than classical serotyping.
Phenotypic and Genotypic Changes in
Salmonella enterica
subsp.
enterica
Serotype Typhimurium during Passage in Intestines of Broiler Chickens Fed on Diets That Included Ionophore Anticoccidial Supplements
Authors: Scalzo Salvatore , Corkill John E. , Shanks David J. , Rowan Timothy G. , Delaval José , Fleetwood Andrew , Murphy Martin , Hart C. Anthony
Published: August 2004

The effect of continuous in-feed administration of anticoccidial agents on antimicrobial sensitivity and the level of bacterial shedding in poultry experimentally infected with
Salmonella enterica
subsp.
enterica
serotype Typhimurium definitive type 104 (DT104) were investigated. On day 0, 1,200 1-day-old
Salmonella
-free broiler chicks were placed into 50 pens, and the pens were randomly allocated to one of five treatments: nonsupplemented (negative control; T1), monensin at 120 mg/kg of diet (T2), salinomycin at 60 mg/kg of diet (T3), semduramicin at 20 mg/kg of diet (T4), or semduramicin at 25 mg/kg of diet (T5). Each bird was inoculated with a well-characterized strain of serotype Typhimurium DT104 on day 10. On day 49, the birds were euthanatized humanely. Bulk fecal samples were collected on days 13, 43, and 48 and were examined for organisms which had acquired resistance. The genetic basis of acquired resistance was determined from representative samples of isolates. Of 784
Salmonella
-selective plates supplemented with antimicrobial agents, only 33 showed growth. These isolates came from all treatment regimens, including the nonsupplemented control. A number of phenotypic changes were observed; these included changes in motility, phage type, and agglutination properties. Supplementation of the diet with an anticoccidial drug does not appear to affect antimicrobial resistance or the level of excretion of salmonellae. Most of the changes observed do not seem to be related to the presence of a supplement in feed. Salmonellae appear to be capable of acquiring antimicrobial resistance and phenotypic changes independently of specific antimicrobial selection pressures.
Evaluation of Immunoglobulin M (IgM) and IgG Rapid Cassette Test Kits for Diagnosis of Melioidosis in an Area of Endemicity
Authors: Wuthiekanun Vanaporn , Amornchai Premjit , Chierakul Wirongrong , Cheng Allen C. , White Nicholas J. , Peacock Sharon J. , Day Nicholas P. J.
Published: August 2004

An enzyme-linked immunosorbent assay-based rapid cassette immunoglobulin G (IgG) and IgM immunochromogenic test kit was compared to the indirect hemagglutination test (IHA) for the diagnosis of acute melioidosis in northeastern Thailand. Admission sera from 70 culture-confirmed septicemic melioidosis patients and 30 patients with localized infections were tested. As a control group, 80 patients with other acute febrile illnesses (other bacterial infections, leptospirosis, or scrub typhus) and 119 healthy individuals were tested. The diagnostic sensitivity of the IgG and IgM tests and the IHA test were 79, 67, and 72%, respectively, with corresponding specificities of 90, 80, and 68%. This kit represents an improvement over IHA for the diagnosis of melioidosis an area of endemicity although, as with other serological tests, it has reduced diagnostic utility in a population with high background seropositivity.
Real-Time PCR Assay Using Molecular Beacon for Quantitation of Hepatitis B Virus DNA
Authors: Sum Simon Siu-Man , Wong Danny Ka-Ho , Yuen Man-Fung , Yuan He-Jun , Yu Jian , Lai Ching-Lung , Ho David , Zhang Linqi
Published: August 2004

Levels of hepatitis B virus (HBV) DNA in the blood serve as an important marker in monitoring the disease progression and treatment efficacy of chronic HBV infection. Several commercial assays are available for accurate measurement of HBV genomic DNA, but many of them are hampered by relatively low sensitivity and limited dynamic range. The aim of this study was to develop a sensitive and accurate assay for measuring HBV genomic DNA using real-time PCR with a molecular beacon (HBV beacon assay). The performance of this assay was validated by testing serial dilutions of the two EUROHEP HBV DNA standards (
ad
and
ay
subtypes) of known concentrations. The assay showed low intra-assay (<7%) and interassay (<5%) variations for both subtypes. Its dynamic range was found to be 10
1
to 10
7
copies per reaction (1.0 × 10
2
to 1.0 × 10
9
copies ml
−1
). The assay was further evaluated clinically using serum samples from 175 individuals with chronic hepatitis B. The HBV DNA level measured by this assay showed good correlation with that measured by the commercially available COBAS AMPLICOR HBV Monitor test (
r
= 0.901;
P
< 0.001). The higher sensitivity and broader dynamic range of this assay compared to the existing commercial assays will provide an ideal tool for monitoring disease progression and treatment efficacy in HBV-infected patients, in particular for those with low levels of HBV viremia.
High-Resolution Genotyping ofCampylobacter upsaliensisStrains Originating from Three Continents
Authors: Lentzsch P. , Rieksneuwöhner B. , Wieler L. H. , Hotzel H. , Moser I.
Published: August 2004

Ninety-sixCampylobacter upsaliensisstrains that originated from Australia, Canada, and Europe (Germany) and that were isolated from humans, dogs, and cats were serotyped for their heat-stable surface antigens. All of them were genotyped by enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) profiling, and 83 strains were genotyped by macrorestriction analysis with the endonuclease XhoI. Eighty-four percent of the strains belonged to five different serotypes (serotypes OI, OII, OIII, OIV, and OVI), with the proportions of strains in each serotype being comparable among the groups of strains from all three continents. Two serotypes, OIII and OIV, were prevalent at rates of 35 to 40%. Serotypes OI, OII, and OVI were detected at rates of 1.5 to 15%. Between 10 and 17.7% of the strains did not react with the available antisera. Analysis of the ERIC-PCR profiles revealed two distinct genotypic clusters, which represented the German and the non-European strains, respectively. XhoI macrorestriction yielded two genotypic clusters; one of them contained 80.2% of the German strains and 34.6% of the non-European strains, and the second cluster consisted of 65.4% of the non-European strains and 19.8% of the German strains. Fourteen strains from all three continents were analyzed for their 16S rRNA gene sequences. Only two minor variations were detected in four of the strains. In conclusion,C. upsaliensishas undergone diverging processes of genome arrangement on different continents during evolution without segregating into different subspecies.
Molecular Typing and Distribution of
Staphylococcus aureus
Isolates in Eastern Canadian Dairy Herds
Authors: Sabour P. M. , Gill J. J. , Lepp D. , Pacan J. C. , Ahmed R. , Dingwell R. , Leslie K.
Published: August 2004

Macrorestriction analysis of SmaI-digested chromosomal DNA, using pulsed field gel electrophoresis (PFGE) was performed to type and estimate genetic relationships among 288
Staphylococcus aureus
isolates recovered from 58 Eastern Canadian dairy herds. In addition, a subset of the collection was phage typed and evaluated for sensitivity to 10 antimicrobial compounds. Of 288 isolates recovered, 29 distinct PFGE types were identified. Based on estimates of genetic relationships, the PFGE types were assigned to six lineage groups, designated A through F. Of all of the isolates, ca. 93% were assigned to lineage groups A, D, or F. In 58.6% of herds, only a single PFGE type was recovered, while the remainder had two to four types. Of the 212 isolates evaluated for antimicrobial resistance, 24.5% were resistant to one or more antimicrobials. Resistance to penicillin (9.9%) was most common, followed by resistance to sulfadimethoxine (7.5%). Isolates resistant to multiple antibiotics were rare. A total of 63% of isolates responded to phages from groups 1 and 3, and 32.8% could not be typed with any of the phage strains used. The other 4.1% belonged to a variety of phage types. Most of the PFGE lineage group A and F isolates corresponded to phage groups 3 and 1, respectively, and most group D isolates were not typeable. PFGE typing had better discriminatory power than phage typing in defining the relatedness of the
S. aureus
isolates. Distribution of PFGE types and phage types was independent across regions and within herds.
Interlaboratory Comparison of Results of Susceptibility Testing with Caspofungin against
Candida
and
Aspergillus
Species
Authors: Odds Frank C. , Motyl Mary , Andrade Roberto , Bille Jacques , Cantón Emilia , Cuenca-Estrella Manuel , Davidson Amanda , Durussel Christian , Ellis David , Foraker Elyse , Fothergill Annette W. , Ghannoum Mahmoud A. , Giacobbe Robert A. , Gobernado Miguel , Handke Rosemary , Laverdière Michel , Lee-Yang Wendy , Merz William G. , Ostrosky-Zeichner Luis , Pemán Javier , Perea Sophia , Perfect John R. , Pfaller Michael A. , Proia Laurie , Rex John H. , Rinaldi Michael G. , Rodriguez-Tudela Juan-Luis , Schell Wiley A. , Shields Christine , Sutton Deanna A. , Verweij Paul E. , Warnock David W.
Published: August 2004

Seventeen laboratories participated in a study of interlaboratory reproducibility with caspofungin microdilution susceptibility testing against panels comprising 30 isolates of
Candida
spp. and 20 isolates of
Aspergillus
spp. The laboratories used materials supplied from a single source to determine the influence of growth medium (RPMI 1640 with or without glucose additions and antibiotic medium 3 [AM3]), the same incubation times (24 h and 48 h), and the same end point definition (partial or complete inhibition of growth) for the MIC of caspofungin. All tests were run in duplicate, and end points were determined both spectrophotometrically and visually. The results from almost all of the laboratories for quality control and reference
Candida
and
Aspergillus
isolates tested with fluconazole and itraconazole matched the NCCLS published values. However, considerable interlaboratory variability was seen in the results of the caspofungin tests. For
Candida
spp. the most consistent MIC data were generated with visual “prominent growth reduction” (MIC
2
) end points measured at 24 h in RPMI 1640, where 73.3% of results for the 30 isolates tested fell within a mode ± one dilution range across all 17 laboratories. MIC
2
at 24 h in RPMI 1640 or AM3 also gave the best interlaboratory separation of
Candida
isolates of known high and low susceptibility to caspofungin. Reproducibility of MIC data was problematic for caspofungin tests with
Aspergillus
spp. under all conditions, but the minimal effective concentration end point, defined as the lowest caspofungin concentration yielding conspicuously aberrant hyphal growth, gave excellent reproducibility for data from 14 of the 17 participating laboratories.
Rapid Differentiation of
Aspergillus
Species from Other Medically Important Opportunistic Molds and Yeasts by PCR-Enzyme Immunoassay
Authors: de Aguirre Liliana , Hurst Steven F. , Choi Jong Soo , Shin Jong Hee , Hinrikson Hans Peter , Morrison Christine J.
Published: August 2004

We developed a PCR-based assay to differentiate medically important species of
Aspergillus
from one another and from other opportunistic molds and yeasts by employing universal, fungus-specific primers and DNA probes in an enzyme immunoassay format (PCR-EIA). Oligonucleotide probes, directed to the internal transcribed spacer 2 region of ribosomal DNA from
Aspergillus flavus
,
Aspergillus fumigatus
,
Aspergillus nidulans
,
Aspergillus niger
,
Aspergillus terreus
,
Aspergillus ustus
, and
Aspergillus versicolor
, differentiated 41 isolates (3 to 9 each of the respective species;
P
< 0.001) in a PCR-EIA detection matrix and gave no false-positive reactions with 33 species of
Acremonium
,
Exophiala
,
Candida
,
Fusarium
,
Mucor
,
Paecilomyces
,
Penicillium
,
Rhizopus
,
Scedosporium
,
Sporothrix
, or other aspergilli tested. A single DNA probe to detect all seven of the most medically important
Aspergillus
species (
A. flavus
,
A. fumigatus
,
A. nidulans
,
A. niger
,
A. terreus
,
A. ustus
, and
A. versicolor
) was also designed. Identification of
Aspergillus
species was accomplished within a single day by the PCR-EIA, and as little as 0.5 pg of fungal DNA could be detected by this system. In addition, fungal DNA extracted from tissues of experimentally infected rabbits was successfully amplified and identified using the PCR-EIA system. This method is simple, rapid, and sensitive for the identification of medically important
Aspergillus
species and for their differentiation from other opportunistic fungi.
Capillary Electrophoretic Restriction Fragment Length Polymorphism Patterns for the Mycobacterial
hsp65
Gene
Authors: Ho Hsin-Tsung , Chang Po-Ling , Hung Chia-Chien , Chang Huan-Tsung
Published: August 2004

PCR-restriction fragment length polymorphism (RFLP) analysis is a nonprobe method for the rapid identification of
Mycobacterium
species. We demonstrate the separation of DNA or restriction fragments digested from the mycobacterial gene encoding the 65-kDa heat shock protein (
hsp65
) by capillary electrophoresis (CE). By using a pair of unlabeled primers, Tb11 and Tb12, and only one restriction enzyme, HaeIII, we investigated a total of 52 reference and clinical strains encompassing 12
Mycobacterium
species. The electrophoretic separation of high-resolution CE required <20 min and was capable of identifying fragments as small as 12 bp. A good agreement of measurement was observed between the sizes of restriction fragments resolved by CE, and the real sizes were deduced from the sequence analysis. Distinct differentiations were also well demonstrated between some species and subspecies by an extra HaeIII digestion site. With the advantage of the complete RFLP pattern available from CE, it appears to be more convenient to use an electropherogram rather than performing the cumbersome slab gel electrophoresis plus diagnostic algorithm to identify
Mycobacterium
species. Beyond the agarose and polyacrylamide gel electrophoresis, high-resolution CE provides an alternative for rapid identification of
Mycobacterium
species that is feasible for automation and routine use without the need for costly probes.
Use of 16S Ribosomal DNA PCR and Denaturing Gradient Gel Electrophoresis for Analysis of the Microfloras of Healing and Nonhealing Chronic Venous Leg Ulcers
Authors: Davies Charlotte E. , Hill Katja E. , Wilson Melanie J. , Stephens Phil , Hill C. Michael , Harding Keith G. , Thomas David W.
Published: August 2004

The bacterial microfloras of 8 healing and 10 nonhealing chronic venous leg ulcers were compared by using a combination of cultural analysis and denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene products. Cultural analysis of the microflora revealed that the majority of both wound types carried the aerobes
Staphylococcus
and
Pseudomonas
spp. (89 and 80%, respectively). Sequencing of 16S ribosomal DNAs selected on the basis of DGGE profiling allowed the identification of strains not detected by cultural means. Of considerable interest was the finding that more than 40% of the sequences represented organisms not cultured from the wound from which they were amplified. DGGE profiles also revealed that all of the wounds possessed one apparently common band, identified by sequencing as
Pseudomonas
sp. The intensity of this PCR signal suggested that the bacterial load of nonhealing wounds was much higher for pseudomonads compared to healing wounds and that it may have been significantly underestimated by cultural analysis. Hence, the present study shows that DGGE could give valuable additional information about chronic wound microflora that is not apparent from cultural analysis alone.
Use of a Genus- and Species-Specific Multiplex PCR for Identification of Enterococci
Authors: Jackson Charlene R. , Fedorka-Cray Paula J. , Barrett John B.
Published: August 2004

The 16S rRNA gene has previously been used to develop genus-specific PCR primers for identification of enterococci. In addition, the superoxide dismutase gene (
sodA
) has been identified as a potential target for species differentiation of enterococci. In this study,
Enterococcus
genus-specific primers developed by Deasy et al. (E1/E2) were incorporated with species-specific primers based upon the superoxide dismutase (
sodA
) gene for development of a multiplex PCR. This assay provides simultaneous genus and species identification of 23 species of enterococci using seven different reaction mixtures. Accuracy of identification of the multiplex PCR was determined by comparisons to standard biochemical testing, the BBL Crystal kit, VITEK, and API Rapid ID 32 Strep. Isolates from swine feces, poultry carcasses, environmental sources, and retail food were evaluated and, overall, results for 90% of the isolates tested by PCR agreed with results obtained using standard biochemical testing and VITEK. Eighty-five percent and 82% of PCR results agreed with results from the API Rapid ID 32 Strep and BBL Crystal tests, respectively. With the exception of concurrence between identification using standard biochemical testing and VITEK (85%) and between BBL Crystal and VITEK (83%), the percent agreement for PCR was higher than or equal to any other pairwise comparison. Multiplex PCR for genus and species determination of enterococci provides an improved, rapid method for identification of this group of bacteria.
Use of Fatty Acid RPMI 1640 Media for Testing Susceptibilities of Eight
Malassezia
Species to the New Triazole Posaconazole and to Six Established Antifungal Agents by a Modified NCCLS M27-A2 Microdilution Method and Etest
Authors: Velegraki Aristea , Alexopoulos Evangelos C. , Kritikou Stavroula , Gaitanis George
Published: August 2004

A novel formulation of RPMI 1640 medium for susceptibility testing of
Malassezia
yeasts by broth microdilution (BMD) and Etest is proposed. A modification of the NCCLS M27-A2 BMD method was used to test 53 isolates of
Malassezia furfur
(12 isolates),
M. sympodialis
(8 isolates),
M. slooffiae
(4 isolates),
M. globosa
(22 isolates),
M. obtusa
(2 isolates),
M. restricta
(2 isolates),
M. pachydermatis
(1 isolates), and
M. dermatis
(2 isolates) against amphotericin B, ketoconazole, itraconazole, fluconazole, voriconazole, terbinafine, and posaconazole by BMD and Etest. RPMI and antibiotic medium 3 (AM3) were supplemented with glucose, bile salts, a mixture of fatty acids, and
n
-octadecanoate fatty acids and Tween 20.
M. furfur
ATCC 14521 and
M. globosa
ATCC 96807 were used as quality control strains. Depending on the species, MICs were read after 48 or 72 h of incubation at 32°C. Low azole and terbinafine MICs were recorded for all
Malassezia
species, whereas amphotericin B displayed higher MICs (≥16 μg/ml) against
M. furfur
,
M. restricta
,
M. globosa
, and
M. slooffiae
strains, which were AM3 confirmed. Agreement of the two methods was 84 to 97%, and intraclass correlation coefficients were statistically significant (
P
< 0.001). Because of higher amphotericin B MICs provided by Etest for strains also displaying high BMD MICs (≥
1
μg/ml), agreement was poorer. The proposed media are used for the first time and can support optimum growth of eight
Malassezia
species for recording concordant BMD and Etest MICs.
Genomic Analysis Distinguishes
Mycobacterium africanum
Authors: Mostowy Serge , Onipede Anthony , Gagneux Sebastien , Niemann Stefan , Kremer Kristin , Desmond Edward P. , Kato-Maeda Midori , Behr Marcel
Published: August 2004

Mycobacterium africanum
is thought to comprise a unique species within the
Mycobacterium tuberculosis
complex.
M. africanum
has traditionally been identified by phenotypic criteria, occupying an intermediate position between
M. tuberculosis
and
M. bovis
according to biochemical characteristics. Although
M. africanum
isolates present near-identical sequence homology to other species of the
M. tuberculosis
complex, several studies have uncovered large genomic regions variably deleted from certain
M. africanum
isolates. To further investigate the genomic characteristics of organisms characterized as
M. africanum
, the DNA content of 12 isolates was interrogated by using Affymetrix GeneChip. Analysis revealed genomic regions of
M. tuberculosis
deleted from all isolates of putative diagnostic and biological consequence. The distribution of deleted sequences suggests that
M. africanum
subtype II isolates are situated among strains of “modern”
M. tuberculosis
. In contrast, other
M. africanum
isolates (subtype I) constitute two distinct evolutionary branches within the
M. tuberculosis
complex. To test for an association between deleted sequences and biochemical attributes used for speciation, a phenotypically diverse panel of “
M. africanum-
like” isolates from Guinea-Bissau was tested for these deletions. These isolates clustered together within one of the
M. africanum
subtype I branches, irrespective of phenotype. These results indicate that convergent biochemical profiles can be independently obtained for
M. tuberculosis
complex members, challenging the traditional approach to
M. tuberculosis
complex speciation. Furthermore, the genomic results suggest a rational framework for defining
M. africanum
and provide tools to accurately assess its prevalence in clinical specimens.
Amplified Fragment Length Polymorphism Reveals Genomic Variability among
Mycobacterium avium
subsp.
paratuberculosis
Isolates
Authors: O'Shea B. , Khare S. , Bliss K. , Klein P. , Ficht T. A. , Adams L. G. , Rice-Ficht A. C.
Published: August 2004

Ninety-six primer sets were used for amplified fragment length polymorphism (AFLP) to characterize the genomes of 20
Mycobacterium avium
subsp.
paratuberculosis
field isolates, 1 American Type Culture Collection (ATCC)
M. avium
subsp.
paratuberculosis
isolate (ATCC 19698), and 2
M. avium
subsp.
avium
isolates (ATCC 35716 and Mac 104). AFLP analysis revealed a high degree of genomic polymorphism among
M. avium
subsp.
paratuberculosis
isolates that may be used to establish diagnostic patterns useful for the epidemiological tracking of
M. avium
subsp.
paratuberculosis
isolates. Four
M. avium
subsp.
paratuberculosis
-polymorphic regions revealed by AFLP were cloned and sequenced. Primers were generated internal to these regions for use in PCR analysis and applied to the
M. avium
subsp.
paratuberculosis
field isolates. An appropriate PCR product was obtained in 79 of 80 reactions, while the
M. avium
subsp.
avium
isolates failed to act as templates for PCR amplification in seven of eight reactions. This work revealed the presence of extensive polymorphisms in the genomes of
M. avium
subsp.
paratuberculosis
and
M. avium
subsp.
avium
, many of which are based on deletions. Of the
M. avium
subsp.
paratuberculosis
-specific sequences studied, one revealed a 5,145-bp region with no homologue in the
M. avium
subsp.
avium
genome. Within this region are genes responsible for integrase-recombinase function. Three additional
M. avium
subsp.
paratuberculosis
-polymorphic regions were cloned, revealing a number of housekeeping genes; all were evaluated for their diagnostic and epidemiological value.
Comparison of Two Urinary Antigen Tests for Establishment of Pneumococcal Etiology of Adult Community-Acquired Pneumonia
Authors: Strålin Kristoffer , Kaltoft Margit Staum , Konradsen Helle Bossen , Olcén Per , Holmberg Hans
Published: August 2004

The Binax NOW immunochromatographic test (ICT) detecting the pneumococcal C polysaccharide and a serotype-specific latex agglutination (LA) test detecting 23 pneumococcal capsular antigens were evaluated for establishing pneumococcal etiology in community-acquired pneumonia (CAP) by use of nonconcentrated urine. ICT was considered to be strongly positive for result lines at least as intense as the control line and weakly positive for less intense result lines. When 215 adult CAP patients were tested, strong ICT, weak ICT, and LA positivity were found in 28, 24, and 16 patients, respectively; of these patients, 13 (46%), 6 (25%), and 13 (81%), respectively, had pneumococcal bacteremia and 27 (96%), 17 (71%), and 15 (94%), respectively, had
Streptococcus pneumoniae
isolated from blood, sputum, and/or nasopharynx. Among 108 controls tested, 2 (1.9%) were weakly ICT positive. When weak positivity was considered negative, the sensitivity of ICT decreased from 79% (19 of 24) to 54% (13 of 24), while the specificity increased from 83% (158 of 191) to 92% (176 of 191); no controls were false positive. The sensitivity and specificity of LA were 54% (13 of 24) and 98% (188 of 191), respectively. Eight of nine LA serotypes corresponded to culture serotypes. In conclusion, using nonconcentrated urine and dividing ICT-positive results into strongly and weakly positive results is a suitable way of performing ICT. While weak ICT positivity should be interpreted with caution, strong ICT positivity and LA positivity should be considered supportive of pneumococcal etiology in adult CAP. As such, these assays might have implications for antibiotic use in CAP. LA has promising potential for pneumococcal serotyping, although further evaluation is required.
MICs of Selected Antibiotics forBacillus anthracis,Bacillus cereus,Bacillus thuringiensis, andBacillus mycoidesfrom a Range of Clinical and Environmental Sources as Determined by the Etest
Authors: Turnbull Peter C. B. , Sirianni Nicky M. , LeBron Carlos I. , Samaan Marian N. , Sutton Felicia N. , Reyes Anatalio E. , Peruski Leonard F.
Published: August 2004

This paper presents Etest determinations of MICs of selected antimicrobial agents for 76 isolates ofBacillus anthracischosen for their diverse histories and 67, 12, and 4 cultures, respectively, of its close relativesB. cereus,B. thuringiensis, andB. mycoidesderived from a range of clinical and environmental sources. NCCLS breakpoints are now available forB. anthracisand ciprofloxacin, penicillin, and tetracycline; based on these breakpoints, theB. anthracisisolates were all fully susceptible to ciprofloxacin and tetracycline, and all except four cultures, three of which had a known history of penicillin resistance and were thought to originate from the same original parent, were susceptible to penicillin. Based on NCCLS interpretive standards for gram-positive and/or aerobic bacteria, all cultures were susceptible to amoxicillin-clavulanic acid and gentamicin and 99% (one with intermediate sensitivity) of cultures were susceptible to vancomycin. No group trends were apparent among the different categories ofB. cereus(isolates from food poisoning incidents and nongastrointestinal infections and food and environmental specimens not associated with illness). Differences betweenB. anthracisand the other species were as expected for amoxicillin and penicillin, with allB. anthraciscultures, apart from the four referred to above, being susceptible versus high proportions of resistant isolates for the other three species. Four of theB. cereusand one of theB. thuringiensiscultures were resistant to tetracycline and a further sixB. cereusand oneB. thuringiensiscultures fell into the intermediate category. There was a slightly higher resistance to azithromycin among theB. anthracisstrains than for the other species. The proportion ofB. anthracisstrains fully susceptible to erythromycin was also substantially lower than for the other species, although just a singleB. cereusstrain was fully resistant. The Etest compared favorably with agar dilution in a subsidiary test set up to test the readings, and it compared with other published studies utilizing a variety of test methods.
Use of 16S rRNA Gene Sequencing for Rapid Confirmatory Identification of
Brucella
Isolates
Authors: Gee Jay E. , De Barun K. , Levett Paul N. , Whitney Anne M. , Novak Ryan T. , Popovic Tanja
Published: August 2004

Members of the genus
Brucella
are categorized as biothreat agents and pose a hazard for both humans and animals. Current identification methods rely on biochemical tests that may require up to 7 days for results. We sequenced the 16S rRNA genes of 65
Brucella
strains along with 17 related strains likely to present a differential diagnostic challenge. All
Brucella
16S rRNA gene sequences were determined to be identical and were clearly different from the 17 related strains, suggesting that 16S rRNA gene sequencing is a reliable tool for rapid genus-level identification of
Brucella
spp. and their differentiation from closely related organisms.
Population Genetic Analysis ofBartonella bacilliformisIsolates from Areas of Peru Where Carrion's Disease Is Endemic and Epidemic
Authors: Hambuch Tina M. , Handley Scott A. , Ellis Barbara , Chamberlin Judith , Romero Sofia , Regnery Russell
Published: August 2004

Carrion's disease is caused by infection with the α-proteobacteriumBartonella bacilliformis. Distribution of the disease is considered coincident with the distribution of its known vector, the sand flyLutzomyia verrucarum. Recent epidemics ofB. bacilliformisinfections associated with atypical symptomatology in nonendemic regions have raised questions regarding the historic and present distribution of this bacterium and the scope of disease that infection causes. Phylogenetic relationships and genomic diversity of 18B. bacilliformisisolates (10 isolates from a region where Carrion's disease is epidemic, Cuzco, Peru, and 8 isolates from a region where Carrion's disease is endemic, Caraz, Peru) were assessed using genomic data generated by infrequent restriction site PCR and gene sequence analysis of the flagellingltAandialBgenes. A population genetic analysis of the genomic diversity suggests that what was once considered an epidemic region of Peru did not result from the recent introduction ofB. bacilliformis.
Evaluation of the QuickLab RSV Test, a New Rapid Lateral-Flow Immunoassay for Detection of Respiratory Syncytial Virus Antigen
Authors: Slinger Robert , Milk Rose , Gaboury Isabelle , Diaz-Mitoma Francisco
Published: August 2004

Rapid respiratory syncytial virus (RSV) diagnosis is vital to the prevention of nosocomial RSV infections. We evaluated a new rapid lateral-flow RSV immunoassay, the QuickLab RSV test, that requires use of only one reagent. We compared QuickLab to the Directigen RSV (DIR) assay, which requires six reagents, and direct fluorescent antibody (DFA) testing. DFA results were considered the “gold standard.” For 133 nasopharyngeal aspirates tested, DFA results were 77 (57.8%) positive, 47 (35.3%) negative, and 9 (6.8%) indeterminate. The sensitivities, specificities, positive predictive values, and negative predictive values of QuickLab and DIR tests were 93.3% (70 of 75) and 80.8% (59 of 73), 95.6% (43 of 45) and 100.0% (46 of 46), 97.2% (70 of 72) and 100.0% (59 of 59), and 89.6% (43 of 48) and 76.7% (46 of 60), respectively. QuickLab was significantly (
P
= 0.02) more sensitive than DIR; the difference in specificities was not significant. DFA was more sensitive than DIR (
P
< 0.001) but not more sensitive than QuickLab (
P
= 0.45). The results of DIR testing were initially uninterpretable and required retesting with 15% of the specimens compared to 3% of QL results (
P
< 0.001). We conclude that the QuickLab RSV test has sensitivity similar to that of the DFA assay and better than that of the DIR assay. QuickLab testing is also simpler to perform and interpret than both DFA and DIR testing.
Use of Positive Blood Cultures for Direct Identification and Susceptibility Testing with the Vitek 2 System
Authors: de Cueto Marina , Ceballos Esther , Martinez-Martinez Luis , Perea Evelio J. , Pascual Alvaro
Published: August 2004

In order to further decrease the time lapse between initial inoculation of blood culture media and the reporting of results of identification and antimicrobial susceptibility tests for microorganisms causing bacteremia, we performed a prospective study in which specially processed fluid from positive blood culture bottles from Bactec 9240 (Becton Dickinson, Cockeysville, Md.) containing aerobic media were directly inoculated into Vitek 2 system cards (bio-Mérieux, France). Organism identification and susceptibility results were compared with those obtained from cards inoculated with a standardized bacterial suspension obtained following subculture to agar; 100 consecutive positive monomicrobic blood cultures, consisting of 50 gram-negative rods and 50 gram-positive cocci, were included in the study. For gram-negative organisms, 31 of the 50 (62%) showed complete agreement with the standard method for species identification, while none of the 50 gram-positive cocci were correctly identified by the direct method. For gram-negative rods, there were 50% categorical agreements between the direct and standard methods for all drugs tested. The very major error rate was 2.4%, and the major error rate was 0.6%. The overall error rate for gram-negatives was 6.6%. Complete agreement in clinical categories of all antimicrobial agents evaluated was obtained for 19 of 50 (38%) gram-positive cocci evaluated; the overall error rate was 8.4%, with 2.8% minor errors, 2.4% major errors, and 3.2% very major errors. These findings suggest that the Vitek 2 cards inoculated directly from positive Bactec 9240 bottles do not provide acceptable bacterial identification or susceptibility testing in comparison with corresponding cards tested by a standard method.
Comparison of Real-Time PCR Signal-Amplified In Situ Hybridization and Conventional PCR for Detection and Quantification of Human Papillomavirus in Archival Cervical Cancer Tissue
Authors: Biedermann Karin , Dandachi Nadia , Trattner Maria , Vogl Georgia , Doppelmayr Hildegard , Moré Elena , Staudach Alfons , Dietze Otto , Hauser-Kronberger Cornelia
Published: August 2004

Archival paraffin-embedded tumor specimens offer a wealth of information for both cancer research and for routine clinical applications. However, the use of formalin-fixed, paraffin-embedded specimens for quantitative real-time PCR is not yet a standard diagnostic method in many laboratories, in particular for the quantification of human papillomavirus (HPV). Particularly high-risk HPV types are involved in almost 100% of the carcinogenesis of cervical cancer. We compared the diagnostic applicability and sensitivity of real-time PCR to that of chromogenic tyramide-signal-amplified in situ hybridization and conventional PCR for the detection of HPV from archival tissue in 164 cases of carcinoma in situ and cervical cancer. Furthermore, we examined whether the viral load of HPV is of prognostic relevance. Our findings indicate that patients in tumor stage I with a lower viral load of HPV type 16 (HPV16; up to 1,000 copies/ng of DNA) had a significantly better survival than HPV 16-negative patients (
P
= 0.037). We observed a greater sensitivity of both real-time PCR and conventional PCR for the detection of HPV16 and -18 compared to signal amplified in situ hybridization. We found a considerable concordance between HPV16 (κ = 0.661) and HPV18 (κ = 0.781) status as measured by real-time PCR and conventional PCR, indicating similar sensitivities. We recognized an inhibitory effect of formalin fixation and paraffin embedding on the evaluation of real-time PCR quantification.