bluuebunny/crossref_metadata_2025 huggingface dataset:
Plasticization-Enhanced Hydrogen Purification Using Polymeric Membranes
Authors: Lin Haiqing , Van Wagner Elizabeth , Freeman Benny D. , Toy Lora G. , Gupta Raghubir P.
Published: February 2006

Polymer membranes are attractive for molecular-scale separations such as hydrogen purification because of inherently low energy requirements. However, membrane materials with outstanding hydrogen separation performance in feed streams containing high-pressure carbon dioxide and impurities such as hydrogen sulfide and water are not available. We report highly permeable, reverse-selective membrane materials for hydrogen purification, as exemplified by molecularly engineered, highly branched, cross-linked poly(ethylene oxide). In contrast to the performance of conventional materials, we demonstrate that plasticization can be harnessed to improve separation performance.
Evolution of a Polyphenism by Genetic Accommodation
Authors: Suzuki Yuichiro , Nijhout H. Frederik
Published: February 2006

Polyphenisms are adaptations in which a genome is associated with discrete alternative phenotypes in different environments. Little is known about the mechanism by which polyphenisms originate. We show that a mutation in the juvenile hormone-regulatory pathway in
Manduca sexta
enables heat stress to reveal a hidden reaction norm of larval coloration. Selection for increased color change in response to heat stress resulted in the evolution of a larval color polyphenism and a corresponding change in hormonal titers through genetic accommodation. Evidently, mechanisms that regulate developmental hormones can mask genetic variation and act as evolutionary capacitors, facilitating the origin of novel adaptive phenotypes.
Resolving the Motional Modes That Code for RNA Adaptation
Authors: Zhang Qi , Sun Xiaoyan , Watt Eric D. , Al-Hashimi Hashim M.
Published: February 2006

Using a domain elongation strategy, we decoupled internal motions in RNA from overall rotational diffusion. This allowed us to site-specifically resolve a manifold of motional modes in two regulatory RNAs from HIV-1 with the use of nuclear magnetic resonance spin relaxation methods. Base and sugar librations vary on a picosecond time scale and occur within helical domains that move collectively at diffusion-limited nanosecond time scales. Pivot points are short, functionally important, and highly mobile internal loops. These spontaneous changes in RNA conformation correlate quantitatively with those that follow adaptive recognition of diverse targets. Thus, ligands may stabilize existing RNA conformations rather than inducing new ones.
Natural and Experimental Evidence of Melt Lubrication of Faults During Earthquakes
Authors: Di Toro Giulio , Hirose Takehiro , Nielsen Stefan , Pennacchioni Giorgio , Shimamoto Toshihiko
Published: February 2006

Melt produced by friction during earthquakes may act either as a coseismic fault lubricant or as a viscous brake. Here we estimate the dynamic shear resistance (τ
f
) in the presence of friction-induced melts from both exhumed faults and high-velocity (1.28 meters per second) frictional experiments. Exhumed faults within granitoids (tonalites) indicate low τ
f
at 10 kilometers in depth. Friction experiments on tonalite samples show that τ
f
depends weakly on normal stress. Extrapolation of experimental data yields τ
f
values consistent with the field estimates and well below the Byerlee strength. We conclude that friction-induced melts can lubricate faults at intermediate crustal depths.
The Large-Scale Axisymmetric Magnetic Topology of a Very-Low-Mass Fully Convective Star
Authors: Donati Jean-François , Forveille Thierry , Cameron Andrew Collier , Barnes John R. , Delfosse Xavier , Jardine Moira M. , Valenti Jeff A.
Published: February 2006

Understanding how cool stars produce magnetic fields within their interiors is crucial for predicting the impact of such fields, such as the activity cycle of the Sun. In this respect, studying fully convective stars enables us to investigate the role of convective zones in magnetic field generation. We produced a magnetic map of a rapidly rotating, very-low-mass, fully convective dwarf through tomographic imaging from time series of spectropolarimetric data. Our results, which demonstrate that fully convective stars are able to trigger axisymmetric large-scale poloidal fields without differential rotation, challenge existing theoretical models of field generation in cool stars.
Structure of Human Urokinase Plasminogen Activator in Complex with Its Receptor
Authors: Huai Qing , Mazar Andrew P. , Kuo Alice , Parry Graham C. , Shaw David E. , Callahan Jennifer , Li Yongdong , Yuan Cai , Bian Chuanbing , Chen Liqing , Furie Bruce , Furie Barbara C. , Cines Douglas B. , Huang Mingdong
Published: February 2006

The urokinase plasminogen activator binds to its cellular receptor with high affinity and initiates signaling cascades that are implicated in pathological processes including tumor growth, metastasis, and inflammation. We report the crystal structure at 1.9 angstroms of the urokinase receptor complexed with the urokinase amino-terminal fragment and an antibody against the receptor. The three domains of urokinase receptor form a concave shape with a central cone-shaped cavity where the urokinase fragment inserts. The structure provides insight into the flexibility of the urokinase receptor that enables its interaction with a wide variety of ligands and a basis for the design of urokinase-urokinase receptor antagonists.
Effective Seed Dispersal Across a Fragmented Landscape
Authors: Bacles Cecile F. E. , Lowe Andrew J. , Ennos Richard A.
Published: February 2006

The role of seed dispersal in maintaining genetic connectivity among forest fragments has largely been ignored because gene flow by pollen is expected to predominate. By using genealogical reconstruction, we investigated gene flow after establishment of seeds in a wind-pollinated, wind-dispersed tree. Our data show that seed dispersal is the main vector of gene flow among remnants and that long-distance dispersal is common across a chronically fragmented landscape. The relative importance of seed-mediated gene flow may have been underemphasized in other fragmented systems, and diagnosing the response of forest trees to current anthropogenic disturbances requires the assessment of phenomena after establishment.
Plastic Deformation of MgGeO
3
Post-Perovskite at Lower Mantle Pressures
Authors: Merkel Sébastien , Kubo Atsushi , Miyagi Lowell , Speziale Sergio , Duffy Thomas S. , Mao Ho-kwang , Wenk Hans-Rudolf
Published: February 2006

Polycrystalline MgGeO
3
post-perovskite was plastically deformed in the diamond anvil cell between 104 and 130 gigapascals confining pressure and ambient temperature. In contrast with phenomenological considerations suggesting (010) as a slip plane, lattice planes near (100) became aligned perpendicular to the compression direction, suggesting that slip on (100) or (110) dominated plastic deformation. With the assumption that silicate post-perovskite behaves similarly at lower mantle conditions, a numerical model of seismic anisotropy in the D″ region implies a maximum contribution of post-perovskite to shear wave splitting of 3.7% with an oblique polarization.
Comment on "Neutral Ecological Theory Reveals Isolation and Rapid Speciation in a Biodiversity Hot Spot"
Authors: Etienne Rampal S. , Latimer Andrew M. , Silander John A. , Cowling Richard M.
Published: February 2006

Latimer
et al
. (Reports, 9 September 2005, p. 1722) used an approximate likelihood function to estimate parameters of Hubbell's neutral model of biodiversity. Reanalysis with the exact likelihood not only yields different estimates but also shows that two similar likelihood maxima for very different parameter combinations can occur. This reveals a limitation of using species abundance data to gain insight into speciation and dispersal.
Rats Smell in Stereo
Authors: Rajan Raghav , Clement James P. , Bhalla Upinder S.
Published: February 2006

It has been hypothesized that rats and other mammals can use stereo cues to localize odor sources, but there is limited behavioral evidence to support this hypothesis. We found that rats trained on an odor-localization task can localize odors accurately in one or two sniffs. Bilateral sampling was essential for accurate odor localization, with internasal intensity and timing differences as directional cues. If the stimulus arrived at the correct point of the respiration cycle, internasal timing differences as short as 50 milliseconds sufficed. Neuronal recordings show that bulbar neurons responded differentially to stimuli from the left and stimuli from the right.
A Compassionate Universe?
Authors: Sternberg Esther
Published: February 2006

The Universe in a Single Atom
. The Convergence of Science and Spirituality.
By the Dalai Lama
. Morgan Road, New York, 2005. 224 pp. $24.95, C$32.95. ISBN: 0-7679-2066-X.

The author offers a Buddhist perspective on the relation between science and religion through discussions of such contentious topics as the origin of the universe, human consciousness, evolution, and genetic engineering.
Purified Excreted-Secreted Antigens from
Trypanosoma cruzi
Trypomastigotes as Tools for Diagnosis of Chagas' Disease
Authors: Berrizbeitia Mariolga , Ndao Momar , Bubis José , Gottschalk Marcelo , Aché Alberto , Lacouture Sonia , Medina Mehudy , Ward Brian J.
Published: February 2006

There is currently no “gold standard” test for the diagnosis of late-stage Chagas' disease. As a result, protection of the blood supply in areas where Chagas' disease is endemic remains problematic. A panel of 709 serum samples from subjects with confirmed Chagas' disease (
n
= 195), healthy controls (
n
= 400), and patients with other parasitic diseases (
n
= 114) was used to assess enzyme-linked immunosorbent assays (ELISAs) based on a concentrated extract of excretory-secretory antigens from either Brazil or Tulahuen strain
Trypanosoma cruzi
trypomastigotes (total trypomastigote excretory-secretory antigens [TESAs]). The total TESA-based assays had excellent overall sensitivity (100%) and specificity (>94%), except for cross-reactivity with
Leishmania
-infected sera. In an attempt to increase the specificity of the assay, immunoaffinity chromatography was used to purify the TESA proteins (TESA
IA
proteins). By Western blotting, a series of polypeptide bands with molecular masses ranging from 60 to 220 kDa were recognized by pooled sera positive for Chagas' disease. An ELISA based on TESA
IA
proteins had a slightly lower sensitivity (98.6%) but an improved specificity (100%) compared to the sensitivity and specificity of the total TESA protein-based ELISAs. A 60-kDa polypeptide was identified as a major contributor to the cross-reactivity with
Leishmania
. These data suggest the need for field validation studies of TESA- and TESA
IA
-based assays in regions where Chagas' disease is endemic.
Restricted Infectivity of a Human-Lineage H3N2 Influenza A Virus in Pigs Is Hemagglutinin and Neuraminidase Gene Dependent
Authors: Landolt Gabriele A. , Karasin Alexander I. , Schutten Melissa M. , Olsen Christopher W.
Published: February 2006

Influenza A viruses cause pandemics at sporadic intervals. Pandemic viruses can potentially be introduced into the human population through in toto transfer of an avian influenza virus or through reassortment between avian and human strains. Pigs are believed to play a central role in the creation of pandemic viruses through reassortment because of their susceptibility to infection with both avian and human influenza viruses. However, we recently found that a human-lineage H3N2 influenza virus was highly restricted in its ability to infect pigs after intranasal inoculation. We hypothesized that this restricted infectivity phenotype was controlled by the hemagglutinin (HA) and neuraminidase (NA). To test this, we infected pigs with reverse genetics-created HA plus NA reassortant viruses. Specifically, introduction of the HA and NA genes of a contemporary H3N2 swine virus into the genetic background of the wholly human virus resulted in a significant increase in virus shedding and pathogenicity. These data indicate that the HA/NA can play important roles in controlling human influenza virus infectivity in pigs. The results further support the premise that a barrier exists to human influenza virus infection in pigs, which may limit the role of pigs in pandemic virus creation through reassortment of human and avian influenza viruses.
Rapid Detection of
Mycobacterium tuberculosis
Beijing Genotype Strains by Real-Time PCR
Authors: Hillemann Doris , Warren Rob , Kubica Tanja , Rüsch-Gerdes Sabine , Niemann Stefan
Published: February 2006

Mycobacterium tuberculosis
strains of the Beijing genotype were first identified in China and neighboring countries and have attracted special attention due to their global emergence and association with drug resistance. To further analyze the spread and special characteristics of Beijing genotype strains, accurate, rapid and sensitive methods that overcome the drawbacks of the classical methods such as IS
6110
DNA fingerprinting or spoligotyping for the identification of strains of this genotype are needed. Based on the nucleotide sequences of
M. tuberculosis
SAWC0780 and H37Rv, primers and fluorogenic 5′ nuclease (TaqMan) probes for real-time PCR assays specific for Beijing and non-Beijing strains, respectively, were designed. The detection limits for the real-time PCR assays were about 5 and 10 copies of chromosomal DNA, respectively. In mixtures of Beijing and non-Beijing DNA, a multiplex assay was able to detect (i) one copy of Beijing DNA in approximately 1,000 copies of non-Beijing DNA and (ii) one copy of non-Beijing DNA in approximately 2,000 copies of Beijing DNA. In a blinded analysis of a collection of 103 multidrug-resistant strains isolated in Germany in 2001, all 62 Beijing and all 41 non-Beijing strains were correctly identified. In conclusion, the real-time assay allows for the rapid and specific detection of Beijing and non-Beijing strains. The major advantages of this test in comparison to other methods used for the identification of Beijing strains are its simplicity and sensitivity and the fact that amplification and detection occur within one reaction tube.
Isolation ofCorynebacterium tuscaniaesp. nov. from Blood Cultures of a Patient with Endocarditis
Authors: Riegel Philippe , Creti Roberta , Mattei Romano , Nieri Alfredo , von Hunolstein Christina
Published: February 2006

A strain of an unknown coryneform bacterium was repeatedly isolated in pure culture from the blood of a patient affected by endocarditis. Comparative 16S rRNA gene sequence analysis revealed that this isolate represented a new subline within the genusCorynebacterium. This new taxon can be identified by the presence of corynomycolic acids and its enzymatic activities and fermentation of sugars. Acid production from glucose and maltose, pyrazinamidase and alkaline phoshatase activities, and hippurate hydrolysis were the most characteristic phenotypic features of the bacterium. On the basis of both phenotypic and phylogenetic evidence, it is proposed that this isolate be classified as a novel species,Corynebacterium tuscaniaesp. nov. The type strain, ISS-5309, has been deposited in the American Type Culture Collection (ATCC BAA-1141) and in the Culture Collection of the University of Göteborg (CCUG 51321).
Shiga Toxins Present in the Gut and in the Polymorphonuclear Leukocytes Circulating in the Blood of Children with Hemolytic-Uremic Syndrome
Authors: Brigotti Maurizio , Caprioli Alfredo , Tozzi Alberto E. , Tazzari Pier Luigi , Ricci Francesca , Conte Roberto , Carnicelli Domenica , Procaccino Maria Antonietta , Minelli Fabio , Ferretti Alfonso V. S. , Paglialonga Fabio , Edefonti Alberto , Rizzoni Gianfranco
Published: February 2006

Hemolytic-uremic syndrome, the main cause of acute renal failure in early childhood, is caused primarily by intestinal infections from some
Escherichia coli
strains that produce Shiga toxins. The toxins released in the gut are targeted to renal endothelium after binding to polymorphonuclear leukocytes. The presence of Shiga toxins in the feces and the circulating neutrophils of 20 children with hemolytic uremic syndrome was evaluated by the Vero cell cytotoxicity assay and flow cytometric analysis, respectively. The latter showed the presence of Shiga toxins on the polymorphonuclear leukocytes of 13 patients, 5 of whom had no other microbiologic or serologic evidence of infection by Shiga toxin-producing
Escherichia coli
. A positive relationship was observed between the amounts of Shiga toxins released in the intestinal lumen and those released in the bloodstream. The toxins were detectable on the neutrophils for a median period of 5 days after they were no longer detectable in stools. This investigation confirms that the immunodetection of Shiga toxins on neutrophils is a valuable tool for laboratory diagnosis of Shiga toxin-producing
Escherichia coli
infection in hemolytic-uremic syndrome and provides clues for further studies on the role of neutrophils in the pathogenesis of this syndrome.
Evaluation of the Invader Assay for Genotyping Hepatitis C Virus
Authors: Germer Jeffrey J. , Majewski David W. , Yung Billy , Mitchell P. Shawn , Yao Joseph D. C.
Published: February 2006

The Invader 1.0 assay (Invader HCV Genotyping Assay, version 1.0; Third Wave Technologies, Inc., Madison, WI) has been developed for the rapid differentiation of hepatitis C virus (HCV) genotypes 1 to 6 based on sequence variation within the HCV 5′ noncoding (NC) region. In the present study, we evaluated the compatibility of Invader 1.0 with the COBAS MONITOR (COBAS AMPLICOR HCV MONITOR Test, version 2.0; Roche Molecular Systems, Inc., Branchburg, NJ), COBAS AMPLICOR (COBAS AMPLICOR Hepatitis C Virus Test, version 2.0; Roche Molecular Systems, Inc.), and COBAS TaqMan (COBAS TaqMan HCV Test; Roche Molecular Systems, Inc.) assays. The minimum HCV RNA titers required for successful HCV genotyping (≥90% success rate) were 1,000 IU/ml for COBAS MONITOR, 100 IU/ml for COBAS AMPLICOR, and 10 IU/ml for COBAS TaqMan. Invader 1.0 results obtained from unpurified COBAS TaqMan amplification products of 111 retrospectively selected clinical serum specimens (genotypes 1 to 6, with virus titers ranging from 15.1 to 2.1 × 10
7
IU/ml) showed 98% concordance with results obtained from the TRUGENE
HCV 5
′
NC
Genotyping Kit (Bayer HealthCare LLC, Tarrytown, NY), used in conjunction with COBAS AMPLICOR. Although the assay is sensitive, accurate, and easy to perform, additional optimization of the Invader 1.0 interpretive software (Invader Data Analysis Worksheet) may be necessary to reduce potential misidentification of HCV genotypes in low-titer specimens. In summary, Invader 1.0 is compatible with a variety of commercially available PCR-based HCV 5′ NC region amplification assays and is suitable for routine HCV genotyping in clinical laboratories.
Emergence of a New Norovirus Genotype II.4 Variant Associated with Global Outbreaks of Gastroenteritis
Authors: Bull Rowena A. , Tu Elise T. V. , McIver Christopher J. , Rawlinson William D. , White Peter A.
Published: February 2006

Norovirus (NoV) is highly infectious and is the major cause of outbreak gastroenteritis in adults, with pandemic spread of the virus being reported in 1995 and 2002. The NoV genome is genetically diverse, which has hampered development of sensitive molecular biology-based methods. In this study we report on a nested reverse transcriptase PCR (nRT-PCR) that was designed to amplify the highly conserved 3′ end of the polymerase region and the 5′ end of the capsid gene of NoV genogroup II (GII). The nRT-PCR was validated with strains isolated from sporadic and outbreak cases between 1997 and 2004 in New South Wales, Australia. Phylogenetic analysis identified six genotypes circulating in New South Wales, GII.1, GII.3, GII.4, GII.6, GII.7, and GII.10, with GII.4 being the predominant genotype. In 2004, there was a marked increase in NoV GII activity in Australia, with a novel GII.4 variant being identified as the etiological agent in 18 outbreaks investigated. This novel GII.4 variant, termed Hunter virus, differed by more than 5% at the amino acid level across the capsid from any other NoV strain in the GenBank and EMBL databases. The Hunter virus was subsequently identified as the etiological agent in large epidemics of gastroenteritis in The Netherlands, Japan, and Taiwan in 2004 and 2005.
Molecular Identification of Zygomycetes from Culture and Experimentally Infected Tissues
Authors: Schwarz Patrick , Bretagne Stéphane , Gantier Jean-Charles , Garcia-Hermoso Dea , Lortholary Olivier , Dromer Françoise , Dannaoui Eric
Published: February 2006

Mucormycosis is an emerging infection associated with a high mortality rate. Identification of the causative agents remains difficult and time-consuming by standard mycological procedures. In this study, internal transcribed spacer (ITS) sequencing was validated as a reliable technique for identification of Zygomycetes to the species level. Furthermore, species identification directly from infected tissues was evaluated in experimentally infected mice. Fifty-four Zygomycetes strains belonging to 16 species, including the most common pathogenic species of
Rhizopus
spp.,
Absidia
spp.,
Mucor
spp., and
Rhizomucor
spp., were used to assess intra- and interspecies variability. Ribosomal DNA including the complete ITS1-5.8S-ITS2 region was amplified with fungal universal primers, sequenced, and compared. Overall, for a given species, sequence similarities between isolates were >98%. In contrast, ITS sequences were very different between species, allowing an accurate identification of Zygomycetes to the species level in most cases. Six species (
Rhizopus oryzae
,
Rhizopus microsporus
,
Rhizomucor pusillus
,
Mucor circinelloides
, and
Mucor indicus
) were also used to induce disseminated mucormycosis in mice and to demonstrate that DNA extraction, amplification of fungal DNA, sequencing, and molecular identification were possible directly from frozen tissues.
Correlation of Disease Severity with Fecal Toxin Levels in Patients with
Clostridium difficile
-Associated Diarrhea and Distribution of PCR Ribotypes and Toxin Yields In Vitro of Corresponding Isolates
Authors: Åkerlund Thomas , Svenungsson Bo , Lagergren Åsa , Burman Lars G.
Published: February 2006

We investigated in vivo and in vitro yields of toxins A and B from and PCR ribotypes of
Clostridium difficile
isolates from 164 patients with differing severities of
C. difficile
-associated diarrhea (CDAD) (patients were grouped as follows: <3 loose stools per day,
n
= 45; 3 to 10 per day,
n
= 97; >10 per day,
n
= 22). The median fecal toxin levels in each group were 0.5, 6.8, and 149 U/g feces (
P
< 0.001), respectively. Patients with severe diarrhea also had more-frequent occurrence of blood in stool and vomiting, but there was no association with fecal toxin levels per se. There was no correlation between fecal toxin level and toxin yield in vitro for the corresponding
C. difficile
isolate or between its PCR ribotype and disease severity. A broad range of toxin yields among isolates belonging to major PCR ribotypes indicated a presence of many subtypes. We hypothesize that bacterial and host factors that affect
C. difficile
toxin levels in feces are important determinants of symptoms in CDAD patients. An inverse correlation between toxin yield and spore count (
r
= 0.66) in stationary-phase cultures supported the notion that toxin production and sporulation represent opposite alternative survival strategies for
C. difficile
cells facing nutrient shortage.
Epidemiological and Genetic Correlates of Severe Acute Respiratory Syndrome Coronavirus Infection in the Hospital with the Highest Nosocomial Infection Rate in Taiwan in 2003
Authors: Chen Yi-Ming Arthur , Liang Shu-Yuan , Shih Yi-Ping , Chen Chia-Yen , Lee Yuan-Ming , Chang Ling , Jung Shiao-Ying , Ho Mei-Shang , Liang Kung-Yee , Chen Hour-Young , Chan Yu-Jiun , Chu Da-Chen
Published: February 2006

Taiwan experienced a series of outbreaks of nosocomial severe acute respiratory syndrome (SARS) infections in 2003. Two months after the final outbreak, we recruited 658 employees from the hospital that suffered the first and most severe SARS infections to help us investigate epidemiological and genetic factors associated with the SARS coronavirus (SARS-CoV). SARS-CoV infections were detected by using enzyme immunoassays and confirmed by a combination of Western blot assays, neutralizing antibody tests, and commercial SARS tests. Risk factors were analyzed via questionnaire responses and sequence-specific oligonucleotide probes of human leukocyte antigen (HLA) alleles. Our results indicate that 3% (20/658) of the study participants were seropositive, with one female nurse identified as a subclinical case. Identified SARS-CoV infection risk factors include working in the same building as the hospital's emergency room and infection ward, providing direct care to SARS patients, and carrying a Cw*0801 HLA allele. The odds ratio for contracting a SARS-CoV infection among persons with either a homozygous or a heterozygous Cw*0801 genotype was 4.4 (95% confidence interval, 1.5 to 12.9;
P
= 0.007).
Use of an Adaptation of a Commercially Available PCR Assay Aimed at Diagnosis of Chlamydia and Gonorrhea To Detect
Trichomonas vaginalis
in Urogenital Specimens
Authors: Van Der Pol Barbara , Kraft Colleen S. , Williams James A.
Published: February 2006

Trichomonas vaginalis
PCR using reagents from a commercially available assay for
Chlamydia trachomatis
and
Neisseria gonorrhoeae
was evaluated for detection of infection in women and men attending a sexually transmitted disease clinic. Evaluations included three primer sets, endocervical swabs, vaginal swabs and urine, and various storage conditions. The TVK3/TVK7 primer set was optimal in our hands with sensitivities ranging from 69.5 to 96.8%. In all comparisons,
T. vaginalis
PCR performed better than routine diagnostics using microscopy for women and culture for men (
P
> 0.05). The assay performed well for all sample types tested, and vaginal swabs were stable for up to 7 days at ambient temperature. Using samples prepared for, and reagents from, the
C. trachomatis-N. gonorrhoeae
PCR assay allowed incorporation of
T. vaginalis
PCR diagnosis into routine clinical testing.
Locus-Specific Mutational Events in a Multilocus Variable-Number Tandem Repeat Analysis of
Escherichia coli
O157:H7
Authors: Noller Anna C. , McEllistrem M. Catherine , Shutt Kathleen A. , Harrison Lee H.
Published: February 2006

Multilocus variable-number tandem repeat analysis (MLVA) is a validated molecular subtyping method for detecting and evaluating
Escherichia coli
O157:H7 outbreaks. In a previous study, five outbreaks with a total of 21 isolates were examined by MLVA. Nearly 20% of the epidemiologically linked strains were single-locus variants (SLV) of their respective predominant outbreak clone. This result prompted an investigation into the mutation rates of the seven MLVA loci (TR1 to TR7). With an outbreak strain that was an SLV at the TR1 locus of the predominant clone, parallel and serial batch culture experiments were performed. In a parallel experiment, none (0/384) of the strains analyzed had mutations at the seven MLVA loci. In contrast, in the two 5-day serial experiments, 4.3% (41/960) of the strains analyzed had a significant variation in at least one of these loci (
P
< 0.001). The TR2 locus accounted for 85.3% (35/41) of the mutations, with an average mutation rate of 3.5 × 10
−3
; the mutations rates for TR1 and TR5 were 10-fold lower. Single additions accounted for 77.1% (27/35) of the mutation events in TR2 and all (6/6) of the additions in TR1 and TR5. The remaining four loci had no slippage events detected. The mutation rates were locus specific and may impact the interpretation of MLVA data for epidemiologic investigations.
Validation of a Multiplex Pneumococcal Serotyping Assay with Clinical Samples
Authors: Lin Jisheng , Kaltoft Margit S. , Brandao Angela P. , Echaniz-Aviles Gabriela , Brandileone M. Cristina C. , Hollingshead Susan K. , Benjamin William H. , Nahm Moon H.
Published: February 2006

We have recently developed a rapid pneumococcal serotyping method called “multibead assay” (J. Yu et al., J. Clin. Microbiol.
43:
156-162, 2005) based on a multiplexed immunoassay for capsular polysaccharides in lysates of pneumococcal cultures. The multibead assay can identify 36 serotypes (1, 2, 3, 4, 5, 6A, 6B, 7A/7F, 8, 9L/9N, 9V, 10A/10B/39/33C, 11A/11D/11F, 12A/12B/12F, 14, 15B/5C, 17F, 18C, 19A, 19F, 20, 22A/22F, 23F, and 33A/33F). More than 90% of the U.S. isolates express one of these serotypes (J. B. Robbins et al., J. Infect. Dis.
148:
1136-1159, 1983). To validate the new assay, we examined 495 clinical isolates of pneumococci obtained in Brazil, Denmark, and Mexico. Pneumococci were serotyped by the Neufeld test in their countries of origin, and lysates of each strain were coded and mailed to the United States for the multibead assay at ambient temperature without any thermal protection. After breaking the code, 54 discrepancies (11% of samples) were noted, but 46 were due to nonreproducible technical problems or insufficient growth of the pneumococci. All of the isolates grew well for a second test, and therefore, the culture medium used for the multibead assay is adequate. The discrepancies persisted for eight isolates, involving the 6A, 11A, and 18C serotypes. Additional studies of the eight isolates showed that the discrepancies were due to differences in the reagents used in the multibead or Neufeld tests for these three serotypes. For instance, five isolates were typed as 6A with the Neufeld test but as nontypeable by the multibead assay. Selection of another new monoclonal antibody (Hyp6AG1) for the multibead assay resulted in all five discrepant isolates typing as 6A. This finding indicates the validity of the multibead assay and emphasizes the need to validate any new pneumococcal serotyping assay with a large number of clinical isolates from different locations. It also suggests the presence of serological subtypes among isolates expressing the 6A serotype.
Occurrence and Kinetics of False-PositiveAspergillusGalactomannan Test Results following Treatment with β-Lactam Antibiotics in Patients with Hematological Disorders
Authors: Aubry Alban , Porcher Raphael , Bottero Julie , Touratier Sophie , Leblanc Thierry , Brethon Benoît , Rousselot Philippe , Raffoux Emmanuel , Menotti Jean , Derouin Francis , Ribaud Patricia , Sulahian Annie
Published: February 2006

Several reports have described a high rate of false-positiveAspergillusgalactomannan (GM) test results for patients treated with piperacillin-tazobactam. In this retrospective study, we first examined the relationships between intravenous administration of three β-lactam antibiotics and the occurrence of false-positive GM test results in hematology patients. We then estimated the kinetics of clearance of GM after the cessation of treatment. Sequential serum samples from 69 patients that had received β-lactams were analyzed by using a PlateliaAspergillustest. A significant association was found between GM positivity (≥0.5) and the administration of β-lactams (P< 0.0001). The direct role of β-lactams in patients' serum positivity was assessed by testing 39 batches of β-lactams, of which 27 were positive for GM. None of the latter were positive according to a fungus- andAspergillus-specific PCR. The kinetics of the decrease of GM was analyzed on sequential serum samples obtained after treatment. By use of a nonlinear regression model, the average time to negative antigen was assessed to be 5.5 days (95% confidence interval [CI], 4.1 to [7.0]), with a half-life of elimination of GM of 2.4 days (95% CI, 1.8 to 3.0). This study confirms that the administration of β-lactams containing GM is responsible for false-positive diagnostic results, even up to 5 days after the cessation of treatment.
Environmental Monitoring for Gastroenteric Viruses in a Pediatric Primary Immunodeficiency Unit
Authors: Gallimore Chris I. , Taylor Clive , Gennery Andrew R. , Cant Andrew J. , Galloway Angela , Iturriza-Gomara Miren , Gray Jim J.
Published: February 2006

The aim of this study was to determine if gastroenteric viruses were present on surfaces and equipment in a pediatric primary immunodeficiency unit (PPIU) by environmental sampling using swabs and subsequent nucleic acid extraction and reverse transcriptase PCR assays. A PPIU was chosen, and 11 swabs were taken at the same sites every 2 weeks for 6 months. Nested/heminested PCR assays were used to screen for astroviruses (AsV), noroviruses (NoV), and rotaviruses (RV). AsV, NoV, and RV were detected at multiple swab sites during the study period. NoV was the most frequently detected virus on environmental surfaces; however, RV was detected on 79% and NoV on 50% of swabbing dates during the study period. Toilet taps were the most contaminated sites. Fecal samples from selected patients in the unit were also screened during the study period, and patients excreted AsV, NoV, and RV at times during the study. New cleaning schedules and changes in some of the PPIU sanitary furniture have been suggested as a means of reducing environmental contamination.
High Analytical Sensitivity and Low Rates of Inhibition May Contribute to Detection of
Chlamydia trachomatis
in Significantly More Women by the APTIMA Combo 2 Assay
Authors: Chernesky Max , Jang Dan , Luinstra Kathy , Chong Sylvia , Smieja Marek , Cai Wenjie , Hayhoe Beth , Portillo Eder , MacRitchie Cindy , Main Cheryl , Ewert Ruth
Published: February 2006

The clinical sensitivity of nucleic acid amplification tests may be determined by analytical sensitivity and inhibitors in patient samples. We established endpoints for detection of propagated
Chlamydia trachomatis
L2 434, diluted according to swab and urine protocols for APTIMA Combo 2 (AC2), ProbeTec ET (PT), and Amplicor (AMP) assays. AC2 was 1,000-fold more sensitive than PT and 10-fold more sensitive than AMP on mock swab specimens. For urine, AC2 analytical sensitivity was 100-fold greater than those of the other assays. Spiking an aliquot of each clinical-trial sample from 298 women demonstrated inhibition rates in first-void urine (FVU), cervical swabs (CS), and vaginal swabs (VS) of 12.1%, 12.8%, and 10.4% for AMP; 27.2%, 2%, and 2%, for PT; and 0.3%, 1.7%, and 1.3% for AC2. Inhibition of our
C. trachomatis
spike and the PT or AMP amplification controls from the manufacturers showed less than 50% correlation. Using an infected-patient reference standard (a specimen positive in at least two tests or a single test positive in two of three samples) in AC2, the VS identified 68/69 (98.6%) infected women compared to CS (89.9%) or FVU (81.2%). Significantly fewer women were identified by PT (65.2%, 63.8%, and 66.7%) or AMP (65.2%, 59.4%, and 56.5%) with the three specimens. By individual specimen type, AC2 confirmed virtually all PT- and AMP-positive specimens, but rates of AC2 confirmation by AMP or PT ranged from 62.9 to 80.3%. The AC2 test identified significantly more women infected with
C. trachomatis
(
P
= 0.001). Vaginal swabs appear to be the specimen of choice for screening.
Application of Pulsed-Field Gel Electrophoresis To Identify Potential Outbreaks of Campylobacteriosis in New Zealand
Authors: Gilpin Brent , Cornelius Angela , Robson Beth , Boxall Naomi , Ferguson Alan , Nicol Carolyn , Henderson Tom
Published: February 2006

Since 2002, New Zealand's incidence of campylobacteriosis has exceeded 300 cases per 100,000 people per annum. To evaluate genetic variation in human isolates, 183
Campylobacter
isolates were collected from a single clinical laboratory in Christchurch: 77 during an 8-week period in spring, and the rest 3 months later over a second 8-week period in autumn. Isolates were identified to the species level and subtyped using Penner serotyping (
Campylobacter jejuni
only) and pulsed-field gel electrophoresis (PFGE) using both SmaI and KpnI. Approximately two-thirds of the isolates could be grouped into clusters of between 2 and 26 isolates with indistinguishable SmaI and KpnI patterns. Less than 10% of the isolates were of the same type between the two sampling periods. The epidemiological relevance of the PFGE clusters was supported by temporal clustering, some spatial clustering, and some statistically significant demographic similarities among cases in a cluster. Conversely, patient cases yielding isolates which did not cluster with isolates from other cases were more likely to report recent overseas travel and less likely to live within larger urban centers. To identify whether these clusters actually represent common-source outbreaks, however, would require the detailed, rapid, and reiterative epidemiological investigation of cases within a PFGE cluster. The combined and timely application of subtyping and epidemiological investigation would appear to be a promising strategy for understanding campylobacteriosis in New Zealand.
Partial Nucleotide Sequencing of themecAGenes ofStaphylococcus aureusIsolates from Cats and Dogs
Authors: Malik Seidu , Peng Haihong , Barton Mary D.
Published: February 2006

Methicillin-resistant staphylococci (MRS) pose a challenge to clinicians and health administrators in human medicine, but MRS infections in cats and dogs are not perceived as a problem in veterinary medicine. Ten methicillin-resistant staphylococcal isolates obtained from healthy and diseased cats and dogs were subjected to partial DNA sequencing of themecAgene. Sequence analysis shows that MRS isolates from both healthy and diseased cats and dogs can harbor themecAgene. ThemecAgenes of animal isolates were identical to that found in human MRS strains, and therefore the possibility of zoonotic transfer must be considered.
Comparison of Serum Hepatitis C Virus (HCV) RNA and Core Antigen Levels in Patients Coinfected with Human Immunodeficiency Virus and HCV and Treated with Interferon plus Ribavirin
Authors: Pivert A. , Payan C. , Morand P. , Fafi-Kremer S. , Deshayes J. , Carrat F. , Pol S. , Cacoub P. , Perronne C. , Lunel F.
Published: February 2006

Trak-C (Ortho-Clinical Diagnostics) is an enzyme-linked immunosorbent assay-based method capable of quantifying hepatitis C virus (HCV) core antigen (CA) in serum and could be an alternative to molecular detection and quantification of HCV RNA. We have evaluated the Trak-C assay in comparison with an HCV RNA quantitative assay (Versant HCV v3.0; Bayer Diagnostics) in the follow-up of 348 treated, human immunodeficiency virus (HIV)/HCV-coinfected patients included in the ANRS HC02 RIBAVIC trial. ANRS HC02 RIBAVIC is a therapeutic, multicenter, randomized protocol comparing the efficacy of alpha interferon 2b (IFN-α2b) (3 million units three times a week)-ribavirin (800 mg/day) to that of pegylated IFN-α2b (1.5 μg/kg of body weight/week)-ribavirin (800 mg/day) during 48 weeks of treatment of HIV/HCV-coinfected patients naïve to HCV treatment. Patients were assessed for virological analysis at day 0 and weeks 4, 12, 24, 48, and 72. Correlation of HCV RNA and HCV CA at the initiation of treatment was excellent (
r
= 0.92). HCV RNA and CA kinetics were similar during follow-up of HCV treatment from day 0 to week 72 whatever the group of response and genotype. The positive and negative predictive values of response to the treatment at week 4 were 59 and 94%, respectively, for HCV RNA load reduction of >2 log and 54 and 94%, respectively, for HCV CA below the threshold value (4.18 log
10
pg/ml · 10
4
). Trak-C, a new assay able to quantify CA in HIV/HCV-coinfected patients, correlates well with quantitative HCV RNA assays and is cheaper and easier to perform than molecular technology. HCV CA could be a valuable alternative test for therapeutic follow-up of coinfected patients treated with IFN plus ribavirin in developing countries.
Real-Time PCR Quantification of Genital Shedding of Herpes Simplex Virus (HSV) and Human Immunodeficiency Virus (HIV) in Women Coinfected with HSV and HIV
Authors: Legoff Jérôme , Bouhlal Hicham , Grésenguet Gérard , Weiss Helen , Khonde Nzambi , Hocini Hakim , Désiré Nathalie , Si-Mohamed Ali , de Dieu Longo Jean , Chemin Cécile , Frost Eric , Pépin Jacques , Malkin Jean-Elie , Mayaud Philippe , Bélec Laurent
Published: February 2006

The accuracy and usefulness of laboratory-developed real-time PCR procedures using a Light Cycler instrument (Roche Diagnostics) for detecting and quantifying human immunodeficiency virus type 1 (HIV-1) RNA and DNA as well as herpes simplex virus type 1 (HSV-1)/HSV-2 DNA in cervicovaginal secretions from women coinfected with HIV and HSV were evaluated. For HIV-1, the use of the NEC152 and NEC131 primer set and the NEC-LTR probe in the long terminal repeat gene allowed us to detect accurately the majority of HIV-1 subtypes of group M circulating in sub-Saharan Africa, including subtypes A, B, C, D, and G as well as circulating recombinant forms 02 and 11. The detection threshold of real-time PCR for HIV in cervicovaginal lavage samples was 5 copies per assay for both RNA and DNA; the intra- and interassay coefficients of variation of

C
T

values were 1.30% and 0.69% (HIV-1 RNA) and 1.84% and 0.67% (HIV-1 DNA), respectively. Real-time PCR for HSV using primers and probe targeting the HSV DNA polymerase gene allowed both detection and quantification of HSV DNA and also differentiation between HSV-1 and HSV-2 genotypes. The detection threshold of real-time PCR for HSV was 5 copies per assay; the intra- and interassay coefficients of variation of

C
T

values were 0.96% and 1.49%, respectively. Both manual and automated silica-based procedures were appropriate for combined extraction of HIV and HSV genomes from female genital secretions. Taken together, these findings indicate that real-time PCR may be used as a unique nucleic acid amplification procedure to detect and quantify HIV and HSV genomes in cervicovaginal secretions and thus to assess at reduced costs the genital shedding of both viruses in women included in intervention studies.
Sequencing ofhsp65Distinguishes among Subsets of theMycobacterium aviumComplex
Authors: Turenne Christine Y. , Semret Makeda , Cousins Debby V. , Collins Desmond M. , Behr Marcel A.
Published: February 2006

TheMycobacterium aviumcomplex consists of epidemiologically distinct subsets. The classification of these subsets is complicated by a number of factors, including the ambiguous results obtained with phenotypic and genetic assays and the recent appreciation that human and avian strains appear to be distinct. In previous work, sequencing based on a 441-bp portion of thehsp65gene has proven to efficiently classify isolates within theMycobacteriumgenus but provides low resolution for distinguishing among members of theM. aviumcomplex. Therefore, in this study, we have targeted the more variable 3′ region of thehsp65gene to determine whether it can effectively discriminateM. aviumcomplex isolates at the levels of species and subspecies. Primers designed for this target consistently generated amplicons for all organisms classified asM. aviumcomplex. Sequences obtained indicate thatM. intracellulareis genetically divergent fromM. aviumorganisms, and distinct sequevars were obtained forM. aviumsubsets, includingM. aviumsubsp.avium(bird type),M. aviumsubsp.hominissuis, andM. aviumsubsp.paratuberculosis. In addition, sequence differences served to distinguish bovine from ovine strains ofM. aviumsubsp.paratuberculosis. A unique profile forM. aviumsubsp.silvaticumwas not obtained. These results indicate that sequencing the 3′ region of thehsp65gene can simply and unambiguously distinguish species and subspecies of theM. aviumcomplex.
Molecular Analysis of an Echovirus 3 Strain Isolated from an Individual Concurrently with Appearance of Islet Cell and IA-2 Autoantibodies
Authors: Williams Çiğdem H. , Oikarinen Sami , Tauriainen Sisko , Salminen Kimmo , Hyöty Heikki , Stanway Glyn
Published: February 2006

Growing evidence has implicated members of the genus
Enterovirus
of the family
Picornaviridae
in the etiology of some cases of type 1 diabetes (T1D). To contribute to an understanding of the molecular determinants underlying this association, we determined the complete nucleotide sequence of a strain of echovirus 3 (E3),
Human enterovirus B
(HEV-B) species, isolated from an individual who soon after virus isolation developed autoantibodies characteristic of T1D. The individual has remained positive for over 6 years for tyrosine phosphatase-related IA-2 protein autoantibodies and islet cell autoantibodies, indicating an ongoing autoimmune process, although he has not yet developed clinical T1D. The sequence obtained adds weight to the observation that recent enterovirus isolates differ significantly from prototype strains and provides further evidence of a role for recombination in enterovirus evolution. In common with most HEV-B species members, the isolate exhibits 2C and VP1 sequences suggested as triggers of autoimmunity through molecular mimicry. However, comparisons with the E3 prototype strain and previously reported diabetogenic and nondiabetogenic HEV-B strains do not reveal clear candidates for sequence features of PicoBank/DM1/E3 that could be associated with autoantibody appearance. This is the first time a virus strain isolated at the time of commencement of beta-cell damage has been analyzed and is an invaluable addition to enterovirus strains isolated previously at the onset of T1D in the search for specific molecular features which could be associated with diabetes induction.
Use of the
espZ
Gene Encoded in the Locus of Enterocyte Effacement for Molecular Typing of Shiga Toxin-Producing
Escherichia coli
Authors: Gilmour Matthew W. , Tracz Dobryan M. , Andrysiak Ashleigh K. , Clark Clifford G. , Tyson Shari , Severini Alberto , Ng Lai-King
Published: February 2006

Infections with Shiga toxin-producing
Escherichia coli
(STEC) result in frequent cases of sporadic and outbreak-associated enteric bacterial disease in humans. Classification of STEC is by
stx
genotype (encoding the Shiga toxins), O and H antigen serotype, and seropathotype (subgroupings based upon the clinical relevance and virulence-related genotypes of individual serotypes). The
espZ
gene is encoded in the locus of enterocyte effacement (LEE) pathogenicity island responsible for the attaching and effacing (A/E) lesions caused by various
E. coli
pathogens (but not limited to STEC), and this individual gene (∼300 bp) has previously been identified as hypervariable among these A/E pathogens. Sequence analysis of the
espZ
locus encoded by additional STEC serotypes and strains (including O26:H11, O121:H19, O111:NM, O145:NM, O165:H25, O121:NM, O157:NM, O157:H7, and O5:NM) indicated that distinct sequence variants exist which correlate to subgroups among these serotypes. Allelic discrimination at the
espZ
locus was achieved using Light Upon eXtension real-time PCR and by liquid microsphere suspension arrays. The allele subtype of
espZ
did not correlate with STEC seropathotype classification; however, a correlation with the allele type of the LEE-encoded intimin (
eae
) gene was supported, and these sequence variations were conserved among individual serotypes. The study focused on the characterization of three clinically significant seropathotypes of LEE-positive STEC, and we have used the observed genetic variation at a pathogen-specific locus for detection and subtyping of STEC.