bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of a new candidate H5N1 avian influenza virus for pre‐pandemic vaccine production
Authors: Dong Jie , Matsuoka Yumiko , Maines Taronna R. , Swayne David E. , O’Neill Eduardo , Davis C. Todd , Van‐Hoven Neal , Balish Amanda , Yu Hong‐jie , Katz Jacqueline M. , Klimov Alexander , Cox Nancy , Li De‐xin , Wang Yu , Guo Yuan‐ji , Yang Wei‐zhong , Donis Ruben O. , Shu Yue‐long
Published: October 2009

Background  Highly pathogenic H5N1 avian influenza viruses currently circulating in birds have caused hundreds of human infections, and pose a significant pandemic threat. Vaccines are a major component of the public health preparedness for this likely event. The rapid evolution of H5N1 viruses has resulted in the emergence of multiple clades with distinct antigenic characteristics that require clade‐specific vaccines. A variant H5N1 virus termed clade 2.3.4 emerged in 2005 and has caused multiple fatal infections. Vaccine candidates that match the antigenic properties of variant viruses are necessary because inactivated influenza vaccines elicit strain‐specific protection.

Objective  To address the need for a suitable seed for manufacturing a clade 2.3.4 vaccine, we developed a new H5N1 pre‐pandemic candidate vaccine by reverse genetics and evaluated its safety and replication in vitro and in vivo.

Methods  A reassortant virus termed, Anhui/PR8, was produced by reverse genetics in compliance with WHO pandemic vaccine development guidelines and contains six genes from A/Puerto Rico/8/34 as well as the neuraminidase and hemagglutinin (HA) genomic segments from the A/Anhui/01/2005 virus. The multi‐basic cleavage site of HA was removed to reduce virulence.

Results  The reassortant Anhui/PR8 grows well in eggs and is avirulent to chicken and ferrets but retains the antigenicity of the parental A/Anhui/01/2005 virus.

Conclusion  These results indicate that the Anhui/PR8 reassortant lost a major virulent determinant and it is suitable for its use in vaccine manufacturing and as a reference vaccine virus against the H5N1 clade 2.3.4 viruses circulating in eastern China, Vietnam, Thailand, and Laos.
Tibial wedge ostectomy: complications of 300 surgical procedures
Authors: Kuan S , Smith B , Black A
Published: October 2009

Background Tibial wedge ostectomy (TWO) is a surgical procedure that aims to give functional stability during weight‐bearing in a hindlimb with cranial cruciate ligament deficiency, by reducing the slope of the tibial plateau angle.

Procedure Advantages of the TWO surgery are that it does not require dedicated equipment and can be performed in young dogs prior to closure of the physis. However, it is a technically demanding procedure and the potential for complications is high.

Results In this retrospective review of 249 dogs that had unilateral and 51 that had bilateral TWO surgery, the overall complication rate was 31.7% (95/300) and the rate of revision surgery was considerable (37 cases or 12.33%).

Conclusions The most important clinical complications were postoperative medial meniscal tears, tibial fractures and implant failures, necessitating repeat surgery.
Spinal dermoid sinus in a Burmese cat with paraparesis
Authors: Tong T , Simpson DJ
Published: October 2009

A 2‐year‐old, male, Burmese cat was evaluated for chronic progressive hindlimb weakness, ataxia and urinary incontinence. Radiographic examination, myelography and magnetic resonance imaging defined congenital vertebral anomalies and a space‐occupying intradural, extramedullary mass. A dermoid sinus was subsequently identified dorsal to the affected spine. Surgical excision of the tract necessitated a dorsal laminectomy and removal of a 1‐cm diameter intradural dermoid sinus ‘cyst’ that contained hair and sebaceous debris. The cat recovered hindlimb function after surgery and remains asymptomatic 50 months after surgery except for a persistent inability to urinate voluntarily.
Detection of Minchinia occulta in samples of pearl oysters Pinctada maxima infected by Haplosporidium hinei
Authors: Bearham D , Spiers ZB , Raidal SR , Jones JB , Nicholls PK
Published: October 2009

Objective  To determine if juvenile pearl oysters (Pinctada maxima) infected with Haplosporidium hinei are also infected with another haplosporidian parasite, Minchinia occulta.

Design  Archived samples of pearl oysters infected with H. hinei were examined using polymerase chain reaction (PCR) assays and in situ hybridisation (ISH) to analyse and identify haplosporidians. A 144‐bp and 220‐bp region of Minchinia DNA were targeted by PCR and amplified DNA from formalin‐fixed H. hinei‐infected pearl oyster samples was sequenced. A 25‐bp oligonucleotide probe targeting a variable section of the parasite's small subunit rRNA gene was used in ISH.

Results  The results of DNA‐based diagnostic assays supported each other. The sequences obtained by PCR were found to be almost identical to M. occulta from rock oysters and the ISH assay demonstrated infection with M. occulta in affected pearl oysters. ISH indicated a prevalence of infection of 26.7% in one of the previous outbreaks.

Conclusion  Pearl oyster spat are susceptible to infection by a Minchinia parasite, most likely M. occulta, which was recently identified in rock oysters within the pearl‐producing zones of Western Australia and is associated with mortalities of up to 80% in this species. The occurrence of haplosporidian co‐infections in pearl oysters suggests the immunocompetence of juvenile oysters may be an important factor in preventing infection and therefore preventing mortalities such as those occurring in the recent outbreaks of pearl oyster oedema disease.
Clinical field study to evaluate the efficacy and safety of the amino‐acetonitrile derivative, monepantel, compared with registered anthelmintics against gastrointestinal nematodes of sheep in Australia
Authors: Hosking BC , Griffiths TM , Woodgate RG , Besier RB , Le Feuvre AS , Nilon P , Trengove C , Vanhoff KJ , Kaye‐Smith BG , Seewald W
Published: October 2009

Objective  To determine the efficacy of monepantel, a developmental compound from the amino‐acetonitrile derivative class of anthelmintics, against field infections of gastrointestinal nematodes in sheep.

Procedures  Comparisons of efficacy (using standard faecal worm egg count reduction tests) and safety (on the basis of visual observations) were made in a large‐scale field study in Australia, between groups of sheep treated with either an oral solution of monepantel or a registered anthelmintic. The sheep were naturally infected with the major gastrointestinal nematode genera present in Australia.

Results  The post‐treatment efficacy results for monepantel were: at 7 days (±1 day) efficacy was >98%; at 14 days (±1 day) it was generally close to or >99%; and at 21 days (±1 day) efficacy was consistently >99%. A high proportion of the targeted nematode populations were confirmed as being resistant to one or more of the currently available anthelmintic classes.

Conclusions  Monepantel when used under field conditions at a minimum dose rate of 2.5 mg/kg was highly effective against mixed‐genus natural field infections of the major gastrointestinal nematode genera including Haemonchus, Teladorsagia (Ostertagia), Trichostrongylus, Nematodirus, Chabertia and Oesophagostomum. This result included efficacy against some populations resistant to the currently available broad‐spectrum anthelmintics. Few Cooperia spp. were present to allow confirmation of efficacy against this genus. On no occasion after treatment did any commercial anthelmintic‐treated groups have significantly lower faecal egg counts than the monepantel‐treated groups. Monepantel was safe for the target animals and human operators when used in a field situation.
Art Markets, Sociology and the Emotional Art Object
Authors: Herrero Marta
Published: October 2009

In its current state the sociology of art markets is characterized by an externalist approach to the analysis of art value in which the art object is the repository of beliefs, judgements given to it by art market actors. However, a review of the literature on art museums poses a challenge to this externalist approach by focusing on the mutually constitutive relationship between the art object, its exhibition and museum context, and viewers. The article reviews this literature exploring the advantages of this line of research for a meaningful sociology of art markets. It will argue for the need to overcome its current externalist focus with studies of the emotional dimension of art market objects as well as of the practices of art market actors.
The Janus‐Face of Whiteness: Toward a Cultural Sociology of White Nationalism and White Antiracism
Authors: Hughey Matthew W.
Published: October 2009

While the sociological study of white identity has traversed many stages, its most recent turn emphasizes the contextual heterogeneity of whiteness. Because of this increased attention to context and locality, the study of whiteness has never been more amenable to cultural analysis than it is today. Hence, an emphasis on different white racial formations that span a political spectrum from conservative to liberal and racist to antiracist is now dominant. In this vein, white nationalists and white antiracists represent the distinct polarities of contemporary inquisitions into white identity formation. Motivated by this academic milieu, this article first reviews the common perception that whiteness is in ‘crisis’ and polarizing into antagonistic political projects. Second, the article scans the literature on white nationalist and white antiracist groups, making explicit the relation to cultural theory. Third, the article questions why these two groups are consistently juxtaposed against one another and how such a conceptualization hinders, rather than advances, cultural analysis. Fourth and last, the article advanced a cultural sociological framework for understanding white racial identity formation that neither collapses white identities into a monolithic collective nor reifies white formations as a static typology. Such an approach considers the general processes and contexts which produce ‘whiteness’ and give it meaning, as well as illuminates the social relationships and practices in which white racial identity formations become embedded.
New methods to identify conserved microsatellite loci and develop primer sets of high cross‐species utility – as demonstrated for birds
Authors: DAWSON DEBORAH A. , HORSBURGH GAVIN J. , KÜPPER CLEMENS , STEWART IAN R. K. , BALL ALEXANDER D. , DURRANT KATE L. , HANSSON BENGT , BACON IDA , BIRD SUSANNAH , KLEIN ÁKOS , KRUPA ANDREW P. , LEE JIN‐WON , MARTÍN‐GÁLVEZ DAVID , SIMEONI MICHELLE , SMITH GEMMA , SPURGIN LEWIS G. , BURKE TERRY
Published: October 2009

We have developed a new approach to create microsatellite primer sets that have high utility across a wide range of species. The success of this method was demonstrated using birds. We selected 35 avian EST microsatellite loci that had a high degree of sequence homology between the zebra finchTaeniopygia guttataand the chickenGallus gallusand designed primer sets in which the primer bind sites were identical in both species. For 33 conserved primer sets, on average, 100% of loci amplified in each of 17 passerine species and 99% of loci in five non‐passerine species. The genotyping of four individuals per species revealed that 24–76% (mean 48%) of loci were polymorphic in the passerines and 18–26% (mean 21%) in the non‐passerines. When at least 17 individuals were genotyped per species for four Fringillidae finch species, 71–85% of loci were polymorphic, observed heterozygosity was above 0.50 for most loci and no locus deviated significantly from Hardy–Weinberg proportions.

This new set of microsatellite markers is of higher cross‐species utility than any set previously designed. The loci described are suitable for a range of applications that require polymorphic avian markers, including paternity and population studies. They will facilitate comparisons of bird genome organization, including genome mapping and studies of recombination, and allow comparisons of genetic variability between species whilst avoiding ascertainment bias. The costs and time to develop new loci can now be avoided for many applications in numerous species. Furthermore, our method can be readily used to develop microsatellite markers of high utility across other taxa.
Searching for DNA in museum specimens: a comparison of sources in a mammal species
Authors: CASAS‐MARCE M. , REVILLA E. , GODOY J. A.
Published: October 2009

The number of genetic studies that use preserved specimens as sources of DNA has been steadily increasing during the last few years. Therefore, selecting the sources that are more likely to provide a suitable amount of DNA of enough quality to be amplified and at the minimum cost to the original specimen is an important step for future research. We have compared different types of tissue (hides vs. bones) from museum specimens of Iberian lynx and multiple alternative sources within each type (skin, footpad, footpad powder, claw, diaphysis, maxilloturbinal bone, mastoid process and canine) for DNA yield and probability of amplification of both mitochondrial and nuclear targets. Our results show that bone samples yield more and better DNA than hides, particularly from sources from skull, such as mastoid process and canines. However, claws offer an amplification success as high as bone sources, which makes them the preferred DNA source when no skeletal pieces have been preserved. Most importantly, these recommended sources can be sampled incurring minimal damage to the specimens while amplifying at a high success rate for both mitochondrial and microsatellite markers.
When global environmentalism meets local livelihoods: policy and management lessons
Authors: Schelhas John , Pfeffer Max J.
Published: October 2009

Creation of national parks often imposes immediate livelihood costs on local people, and tensions between park managers and local people are common. Park managers have tried different approaches to managing relationships with local people, but nearly all include efforts to promote environmental values and behaviors. These efforts have had uneven results, and there is a need to improve our understanding of how environmental values develop, change, and influence behavior. We investigated this question in a study of communities in and adjacent to national parks in Costa Rica and Honduras, using a mental and cultural model approach. We found local people to be fluent in global environmental values, but some common values had limited impact on behavior due to conflicts with livelihood needs. However, new environmental values that integrated conservation and livelihood needs had developed that had a more direct relationship to behavior. We propose a general model of how local environmental values develop in the context of global environmentalism that can be used to guide outreach programs with park neighbors.
Visualizing Scientific Inference
Authors: Gooding David C.
Published: October 2009

The sciences use a wide range of visual devices, practices, and imaging technologies. This diversity points to an important repertoire of visual methods that scientists use to adapt representations to meet the varied demands that their work places on cognitive processes. This paper identifies key features of the use of visualization in a range of scientific domains and considers the implications of this repertoire for understanding scientists as cognitive agents.
Transient Starting Flow in a Cylinder With Counter-Rotating Endwall Disks
Authors: Hyun Jae Min
Published: October 2009

Spin-up from rest in a cylinder with top and bottom endwall disks rotating in opposite directions (ΩT and ΩB are the respective rotation rate, but S[≡ ΩT/ΩB] < 0) is investigated. The sidewall is fixed to the faster-rotating disk. A finite-difference numerical model is adopted to integrate the unsteady Navier-Stokes equations. We consider a cylinder of aspect ratio 0(1) and minute Ekman numbers. Numerical solutions are presented to show the transient azimuthal flow structures, axial vorticity profiles, and meridional flow patterns. An azimuthal velocity front, which separates the rotating from the nonrotating fluid, propagates radially inward from the sidewall. The appearance of the front is similar to the front propagation in spin-up in a rigid cylinder. As S decreases from zero, the direction of rotation in the bulk of the interior fluid becomes the same as that of the faster-rotating disk. The azimuthal velocities are still vertically uniform in the bulk of the interior. The scaled time to reach the steady state decreases. The angular velocities of the interior fluid near the central axis become very small. Under counter-rotation, the meridional circulation forms a two-cell structure. A stagnation point appears on the slower-rotating disk. During spin-up, the stagnation point moves from the sidewall to its steady-state position. As counter-rotation increases, the radial distance traveled by the stagnation point decreases.
Behavior of Air in the Rotor of a Model Mixed-Flow Pump Operating at Peak Efficiency
Authors: Fraser S. M. , Carey C. , Wilson G.
Published: October 2009

The behavior of air in a model of a mixed-flow bowl pump has been investigated using a laser-Doppler anemometer. Velocity measurements were obtained at a number of sections within, upstream and downstream of the rotor. All three components of velocity were determined, and related to the rotating frame of reference using a computerised data acquisition method. The results provide useful data relevant to some complex problems of incompressible flow in turbomachines, including annular casing boundary layer growth.
A New Diffuser Mapping Technique—Part 1: Studies in Component Performance
Authors: Japikse D.
Published: October 2009

A rigorous derivation of the K = Cpi − Cp diffuser recovery and loss equation is presented and the critical assumptions to yield this simplified relationship are critiqued. Qualified confirmation of the equation is achieved using precise laboratory data. A new diffuser loss-map is presented which facilitates engineering diffuser design work, even when a recovery map is unavailable. Numerous application examples for turbomachinery analysis are given including the application to a turbomachinery cascade with strong turning.
Internal Three-Dimensional Viscous Flow Solution Using the Streamlike Function
Authors: Hamed A. , Abdallah S.
Published: October 2009

This paper presents a new method for the three-dimensional elliptic solution of the Navier–Stokes equations. It is based on the streamlike-function vorticity formulation which was developed by the authors to study the development of secondary velocities and streamwise vorticity for inviscid flows in curved ducts. This formulation is generalized for viscous flows and used to predict the development of internal three dimensional flow fields. The computed results are presented and compared with experimental measurements for the three-dimensional viscous flow in a straight duct.
On the Development of Laminar Internal Flows With Mass Injection and Extraction
Authors: Busse C. A.
Published: October 2009

Logical criteria are presented for determining the effects of channel shape, viscous force, and mass injection/extraction on axisymmetric and planar velocity profiles.
Europe Rethinks Education
Authors: Léna Pierre
Published: October 2009

For societies to understand the consequences of vital issues such as climate change, education—especially science education—will play a critical role. Improving the quality of science education in primary and secondary schools is a challenge faced by nearly all countries. Europe has finally recognized the need for a trans-European effort to rejuvenate the scientific education of all students, and promising efforts are now under way.
Intraspecific Polymorphism to Interspecific Divergence: Genetics of Pigmentation in
Drosophila
Authors: Wittkopp Patricia J. , Stewart Emma E. , Arnold Lisa L. , Neidert Adam H. , Haerum Belinda K. , Thompson Elizabeth M. , Akhras Saleh , Smith-Winberry Gabriel , Shefner Laura
Published: October 2009

Drosophila
Body Color

Fly body color is controlled by a variety of genes and alleles. Now

Wittkopp
et al.

(p.
540
) describe how two genes at the ebony and tan genetic loci control body color among two closely related species,
Drosophila americana
and
D. novamexicana
. Variations at the tan locus and linked to the ebony locus also contribute to intraspecific pigmentation changes with geography in
D. americana
. The sequencing of multiple isolates suggests that some strains of
D. americana
carry alleles of tan and ebony that are more closely related to the
D. novamexicana
alleles than they are to other
D. americana
alleles. Thus, the genetic determinants of both inter- and intraspecies color variation is due to shared alleles.
A Cdc20-APC Ubiquitin Signaling Pathway Regulates Presynaptic Differentiation
Authors: Yang Yue , Kim Albert H. , Yamada Tomoko , Wu Bei , Bilimoria Parizad M. , Ikeuchi Yoshiho , de la Iglesia Núria , Shen Jie , Bonni Azad
Published: October 2009

Cdc20-APC in Synapse Formation

The E3 ubiquitin ligase Cdc20-anaphase promoting complex (Cdc20-APC) has important roles in the control of the cell division cycle.

Yang
et al.

(p.
575
) now show that Cdc20-APC also appears to be required for proper formation of synapses by developing neurons in the rat brain. When Cdc20-APC was depleted from cultured neurons or in the brains of developing rat pups, synapse formation was inhibited. The brain-enriched transcription factor NeuroD2 was shown to be a possible target of Cdc20-APC–stimulated degradation. NeuroD2 may act by promoting synthesis of Complexin II, a protein that regulates synaptic vesicle fusion.
Isolation of a C5-Deprotonated Imidazolium, a Crystalline “Abnormal” N-Heterocyclic Carbene
Authors: Aldeco-Perez Eugenia , Rosenthal Amos J. , Donnadieu Bruno , Parameswaran Pattiyil , Frenking Gernot , Bertrand Guy
Published: October 2009

Stretching Carbene Versatility

Stable N-heterocyclic carbene (NHC) molecules are versatile catalysts for organic reactions (see the Perspective by

Albrecht

).
Lavallo and Grubbs
(p.
559
) now show that these molecules can also catalyze organometallic transformations. Specifically, the carbenes induce coupling of monomeric iron olefin complexes to form clusters incorporating three or four bonded iron centers. Initial coordination of carbene to iron may facilitate formation of an iron-iron bond with a second complex. In a related development,

Aldeco-Perez
et al.

(p.
556
) prepared an NHC isomer in which the divalent carbon is shifted so that it no longer lies between the nitrogens. The compound forms stable complexes with both gold and CO
2
.
Fixing a Critical Climate Accounting Error
Authors: Searchinger Timothy D. , Hamburg Steven P. , Melillo Jerry , Chameides William , Havlik Petr , Kammen Daniel M. , Likens Gene E. , Lubowski Ruben N. , Obersteiner Michael , Oppenheimer Michael , Philip Robertson G. , Schlesinger William H. , David Tilman G.
Published: October 2009

Rules for applying the Kyoto Protocol and national cap-and-trade laws contain a major, but fixable, carbon accounting flaw in assessing bioenergy.
Smallest Algae Thrive As the Arctic Ocean Freshens
Authors: Li William K. W. , McLaughlin Fiona A. , Lovejoy Connie , Carmack Eddy C.
Published: October 2009

In the Arctic Ocean, phytoplankton cell sizes have decreased with warming temperatures and fresher surface waters.
Glimpsing the Weak Magnetic Field of Light
Authors: Giessen Harald , Vogelgesang Ralf
Published: October 2009

An instrument has been fabricated that can detect the weak magnetic field of infrared light.
Rejoice—RNAi for Yeast
Authors: Moazed Danesh
Published: October 2009

An RNA silencing mechanism with broad regulatory capability has finally been demonstrated in one of the most widely used model organisms.
Research Wolves of Yellowstone Killed in Hunt
Authors: Morell Virginia
Published: October 2009

On 3 October, a few weeks after Montana opened its first legal wolf-hunting season in decades, a hunter killed the alpha female of Yellowstone Park's Cottonwood Pack, whose behavior, travels, life history, and genealogy had been studied in detail by scientists for years. Her death, and that of five other pack members also shot outside Yellowstone, has irrevocably changed what had been a unique long-term study, the researchers say.
Hong Kong's Darwin Defenders Declare Victory in Teaching Fracas
Authors: Stone Richard
Published: October 2009

Scientists in Hong Kong are celebrating a partial victory in what is likely to be an ongoing war against proponents of teaching creationism and intelligent design in secondary schools.
Race for the Heavens
Authors: Bhattacharjee Yudhijit
Published: October 2009

Two very different telescope projects are jostling to give the United States its biggest-ever eye on the sky. Can the country afford both?
Diverse Characteristics of the
cagA
Gene of
Helicobacter pylori
Strains Collected from Patients from Southern Vietnam with Gastric Cancer and Peptic Ulcer
Authors: Truong Bui Xuan , Mai Vo Thi Chi , Tanaka Hiroshi , Ly Le Thanh , Thong Tran Minh , Hai Hoang Hoa , Van Long Dao , Furumatsu Keisuke , Yoshida Masaru , Kutsumi Hiromu , Azuma Takeshi
Published: October 2009

The pathogenesis of gastroduodenal diseases is related to the diversity of
H
elicobacter
pylori
strains. CagA-positive strains are more likely to cause gastric cancer than CagA-negative strains. Based on EPIYA (Glu-Pro-Ile-Tyr-Ala) motifs at the carboxyl terminus corresponding to phosphorylation sites,
H. pylori
CagA is divided into East Asian CagA and Western CagA. The former type prevails in East Asia and is more closely associated with gastric cancer. The present study used full sequences of the
cagA
gene and CagA protein of 22
H. pylori
strains in gastric cancer and peptic ulcer patients from Southern Vietnam to make a comparison of genetic homology among Vietnamese strains and between them and other strains in East Asia. A phylogenetic tree was constructed based on full amino acid sequences of 22 Vietnamese strains in accordance with 54 references from around the world. The
cagA
gene was found in all Vietnamese
H. pylori
strains. Twenty-one of 22 (95.5%) strains belonged to the East Asian type and had similar characteristics of amino acid sequence at the carboxyl terminus to other strains from the East Asian region. From evidence of East Asian CagA and epidemiologic cancerous lesions in Vietnam,
H. pylori
-infected Vietnamese can be classified into a high-risk group for gastric cancer, but further studies on the interaction among environmental and virulence factors should be done. Finally, phylogenetic data support that there is a Japanese subtype in the Western CagA type.
Development and Evaluation of One-Step TaqMan Real-Time Reverse Transcription-PCR Assays Targeting Nucleoprotein, Matrix, and Hemagglutinin Genes of Equine Influenza Virus
Authors: Lu Zhengchun , Chambers Thomas M. , Boliar Saikat , Branscum Adam J. , Sturgill Tracy L. , Timoney Peter J. , Reedy Stephanie E. , Tudor Lynn R. , Dubovi Edward J. , Vickers Mary Lynne , Sells Stephen , Balasuriya Udeni B. R.
Published: October 2009

The objective of this study was to develop and evaluate new TaqMan real-time reverse transcription-PCR (rRT-PCR) assays by the use of the minor groove binding probe to detect a wide range of equine influenza virus (EIV) strains comprising both subtypes of the virus (H3N8 and H7N7). A total of eight rRT-PCR assays were developed, targeting the nucleoprotein (NP), matrix (M), and hemagglutinin (HA) genes of the two EIV subtypes. None of the eight assays cross-reacted with any of the other known equine respiratory viruses. Three rRT-PCR assays (EqFlu NP, M, and HA3) which can detect strains of the H3N8 subtype were evaluated using nasal swabs received for routine diagnosis and swabs collected from experimentally inoculated horses. All three rRT-PCR assays have greater specificity and sensitivity than virus isolation by egg inoculation (93%, 89%, and 87% sensitivity for EqFlu NP, EqFlu M, and EqFlu HA3 assays, respectively). These assays had analytical sensitivities of ≥10 EIV RNA molecules. Comparison of the sensitivities of rRT-PCR assays targeting the NP and M genes of both subtypes with egg inoculation and the Directigen Flu A test clearly shows that molecular assays provide the highest sensitivity. The EqFlu HA7 assay targeting the H7 HA gene is highly specific for the H7N7 subtype of EIV. It should enable highly reliable surveillance for the H7N7 subtype, which is thought to be extinct or possibly still circulating at a very low level in nature. The assays that we developed provide a fast and reliable means of EIV diagnosis and subtype identification of EIV subtypes.
Prevalence and Genotypes of
Helicobacter pylori
in Gastric Biopsy Specimens from Patients with Gastroduodenal Pathologies in the Cukurova Region of Turkey
Authors: Nagiyev Togrul , Yula Erkan , Abayli Bahri , Koksal Fatih
Published: October 2009

The effects of
Helicobacter pylori
genotypes on clinical prognosis in the Cukurova region of Turkey were investigated by PCR. The prevalence of type I strains carrying the
s1c
allele, unlike in neighboring regions and countries, was found to be significantly higher in patients with gastritis and/or gastric ulcers (
P
= 0.001), and that of type I strains carrying the
s1a
allele was found to be significantly higher in patients with duodenal ulcers (
P
< 0.001). The
cagA
gene was strongly associated with the more virulent
vacA
genotypes (
P
< 0.001).
CD64 Index Provides Simple and Predictive Testing for Detection and Monitoring of Sepsis and Bacterial Infection in Hospital Patients
Authors: Icardi M. , Erickson Y. , Kilborn S. , Stewart B. , Grief B. , Scharnweber G.
Published: October 2009

The rapid diagnosis and management of bacterial infection are heavily dependent upon clinical assessment. Blood culture may take up to 2 days for results and may be suspect. Surface neutrophil CD64 expression has been shown to be upregulated in cases of bacterial infection. Recently, a standardized kit for the CD64 index was used in neonatal intensive care units, showing high sensitivity and specificity for bacterial infections. Our study was designed to confirm and extend these results to adult hospital patients and to determine the impact of this testing on a clinical laboratory's finances and staffing. CD64 indices were performed with peripheral blood drawn in tandem with blood cultures from 109 patients over a 2-month period. We found that a CD64 index of ≤1.19 was predictive of “no growth” blood culture results. An index of >1.19 was predictive of an ultimate clinical and/or culture diagnosis of infection with a sensitivity and specificity of 94.6% and 88.7%, respectively. Positive and negative predictive values were 89.8% and 94%, respectively. The CD64 index was easily performed using our flow cytometer and staff, producing minimal alteration in clinical workflow. A 7-day-a-week testing schedule will result in some additional expense but will be more than offset by the expected cost savings. The CD64 index is a useful and inexpensive test for improving the diagnosis and management of hospital patients with bacterial infection. It can be readily performed by clinical laboratories and could result in considerable savings for the institution.
Improved Performance of a Rapid Office-Based Stool Test for Detection of
Helicobacter pylori
in Children before and after Therapy
Authors: Prell C. , Osterrieder S. , Lottspeich C. , Schwarzer A. , Rüssmann H. , Ossiander G. , Koletzko S.
Published: October 2009

A modified version of a rapid office based one-step monoclonal immunoassay for detection of
Helicobacter pylori
antigen in stool samples from children was evaluated against biopsy specimen-based methods and compared to a monoclonal enzyme immunoassay using the same antigen. Blinded stool samples from 185 children (0.3 to 18.2 years) were investigated at the time of upper endoscopy prior to anti-
H. pylori
therapy; 62 children were
H. pylori
infected and 123 noninfected according to predefined reference standards. Samples obtained 6 to 8 weeks after anti-
H. pylori
therapy were available from 58 children (3.8 to 17.7 years) and were compared to results of the [
13
C]urea breath test (14/58 were positive). The rapid stool tests were performed by two independent readers. Of 243 rapid tests performed, 1 (0.4%) was invalid for technical reasons. Equivocal results (very weak line) were reported 16 times by reader 1 and 27 times by reader 2. When equivocal results were considered positive, the two observers agreed on 76 positive and 160 negative results and disagreed on 7 samples (2.9%). The sensitivity was 90.8% for reader 1 and 85.5% for reader 2, and the specificity was 91.0% and 93.4%, respectively. The monoclonal enzyme immunoassay revealed a sensitivity and specificity of 94.7% and 97.6%, respectively. The modified chromatographic immunoassay is a good alternative in settings or situations when the monoclonal enzyme immunoassay or the [
13
C]urea breath test are not available or feasible. In order to improve sensitivity, very weak lines should be considered positive test results.
Pyrosequencing for Rapid Detection of
Mycobacterium tuberculosis
Resistance to Rifampin, Isoniazid, and Fluoroquinolones
Authors: Bravo Lulette Tricia C. , Tuohy Marion J. , Ang Concepcion , Destura Raul V. , Mendoza Myrna , Procop Gary W. , Gordon Steven M. , Hall Geraldine S. , Shrestha Nabin K.
Published: October 2009

After isoniazid and rifampin (rifampicin), the next pivotal drug class in
Mycobacterium tuberculosis
treatment is the fluoroquinolone class. Mutations in resistance-determining regions (RDR) of the
rpoB
,
katG
, and
gyrA
genes occur with frequencies of 97%, 50%, and 85% among
M. tuberculosis
isolates resistant to rifampin, isoniazid, and fluoroquinolones, respectively. Sequences are highly conserved, and certain mutations correlate well with phenotypic resistance. We developed a pyrosequencing assay to determine
M. tuberculosis
genotypic resistance to rifampin, isoniazid, and fluoroquinolones. We characterized 102
M. tuberculosis
clinical isolates from the Philippines for susceptibility to rifampin, isoniazid, and ofloxacin by using the conventional submerged-disk proportion method and validated our pyrosequencing assay using these isolates. DNA was extracted and amplified by using PCR primers directed toward the RDR of the
rpoB
,
katG
, and
gyrA
genes, and pyrosequencing was performed on the extracts. The
M. tuberculosis
H37Rv strain (ATCC 25618) was used as the reference strain. The sensitivities and specificities of pyrosequencing were 96.7% and 97.3%, 63.8% and 100%, and 70.0% and 100% for the detection of resistance to rifampin, isoniazid, and ofloxacin, respectively. Pyrosequencing is thus a rapid and accurate method for detecting
M. tuberculosis
resistance to these three drugs.
Multicenter Study of Prevalence of Nontuberculous Mycobacteria in Patients with Cystic Fibrosis in France
Authors: Roux Anne-Laure , Catherinot Emilie , Ripoll Fabienne , Soismier Nathalie , Macheras Edouard , Ravilly Sophie , Bellis Gil , Vibet Marie-Anne , Le Roux Evelyne , Lemonnier Lydie , Gutierrez Cristina , Vincent Véronique , Fauroux Brigitte , Rottman Martin , Guillemot Didier , Gaillard Jean-Louis
Published: October 2009

We performed a multicenter prevalence study of nontuberculous mycobacteria (NTM) involving 1,582 patients (mean age, 18.9 years; male/female ratio, 1.06) with cystic fibrosis in France. The overall NTM prevalence (percentage of patients with at least one positive culture) was 6.6% (104/1,582 patients), with prevalences ranging from 3.7% (in the east of France) to 9.6% (in the greater Paris area).
Mycobacterium abscessus
complex (MABSC; 50 patients) and
Mycobacterium avium
complex (MAC; 23 patients) species were the most common NTM, and the only ones associated with fulfillment of the American Thoracic Society bacteriological criteria for NTM lung disease. The “new” species,
Mycobacterium bolletii
and
Mycobacterium massiliense
, accounted for 40% of MABSC isolates. MABSC species were isolated at all ages, with a prevalence peak between 11 and 15 years of age (5.8%), while MAC species reached their highest prevalence value among patients over 25 years of age (2.2%).
Evaluation of Automated and Manual Commercial DNA Extraction Methods for Recovery of
Brucella
DNA from Suspensions and Spiked Swabs
Authors: Dauphin Leslie A. , Hutchins Rebecca J. , Bost Liberty A. , Bowen Michael D.
Published: October 2009

This study evaluated automated and manual commercial DNA extraction methods for their ability to recover DNA from
Brucella
species in phosphate-buffered saline (PBS) suspension and from spiked swab specimens. Six extraction methods, representing several of the methodologies which are commercially available for DNA extraction, as well as representing various throughput capacities, were evaluated: the MagNA Pure Compact and the MagNA Pure LC instruments, the IT 1-2-3 DNA sample purification kit, the MasterPure Complete DNA and RNA purification kit, the QIAamp DNA blood mini kit, and the UltraClean microbial DNA isolation kit. These six extraction methods were performed upon three pathogenic
Brucella
species:
B. abortus
,
B. melitensis
, and
B. suis
. Viability testing of the DNA extracts indicated that all six extraction methods were efficient at inactivating virulent
Brucella
spp. Real-time PCR analysis using
Brucella
genus- and species-specific TaqMan assays revealed that use of the MasterPure kit resulted in superior levels of detection from bacterial suspensions, while the MasterPure kit and MagNA Pure Compact performed equally well for extraction of spiked swab samples. This study demonstrated that DNA extraction methodologies differ in their ability to recover
Brucella
DNA from PBS bacterial suspensions and from swab specimens and, thus, that the extraction method used for a given type of sample matrix can influence the sensitivity of real-time PCR assays for
Brucella
.
Clinical Application of Real-Time PCR to Screening Critically Ill and Emergency-Care Surgical Patients for Methicillin-Resistant
Staphylococcus aureus
: a Quantitative Analytical Study
Authors: Herdman M. Trent , Wyncoll Duncan , Halligan Eugene , Cliff Penelope R. , French Gary , Edgeworth Jonathan D.
Published: October 2009

The clinical utility of real-time PCR screening assays for methicillin (meticillin)-resistant
Staphylococcus aureus
(MRSA) colonization is constrained by the predictive values of their results: as MRSA prevalence falls, the assay's positive predictive value (PPV) drops, and a rising proportion of positive PCR assays will not be confirmed by culture. We provide a quantitative analysis of universal PCR screening of critical care and emergency surgical patients using the BD GeneOhm MRSA PCR system, involving 3,294 assays over six months. A total of 248 PCR assays (7.7%) were positive; however, 88 failed to be confirmed by culture, giving a PPV of 65%. Multivariate analysis was performed to compare PCR-positive culture-positive (P+C+) and PCR-positive culture-negative (P+C−) assays. P+C− results were positively associated with a history of methicillin-sensitive
Staphylococcus aureus
infection or colonization (odds ratio [OR], 3.15; 95% confidence interval [CI], 1.32 to 7.54) and high PCR thresholds of signal intensity, indicative of a low concentration of target DNA (OR, 1.19 per cycle; 95% CI, 1.11 to 1.26). P+C− results were negatively associated with a history of MRSA infection or colonization (OR, 0.19; 95% CI, 0.09 to 0.42) and male sex (OR, 0.40; 95% CI, 0.20 to 0.81). P+C+ patients were significantly more likely to have subsequent positive MRSA culture assays and microbiological evidence of clinical MRSA infection. The risk of subsequent MRSA infection in P+C− patients was not significantly different from that in case-matched PCR-negative controls. We conclude that, given the low PPV and poor correlation between a PCR-positive assay and the clinical outcome, it would be prudent to await culture confirmation before altering infection control measures on the basis of a positive PCR result.
Incubation Alone Is Adequate as a Culturing Technique for Cardiac Rhythm Management Devices
Authors: Viola George M. , Mansouri Mohammad D. , Nasir Nadim , Darouiche Rabih O.
Published: October 2009

There exist no standardized methods for culturing cardiac rhythm management devices. To identify the most optimal culturing method, we compared various techniques that comprise vortex, sonication, and incubation or combinations thereof. Incubation alone yielded bacterial colony counts similar to those of other culturing combinations and is the least labor-intensive.
Evaluation of a New Commercial TaqMan PCR Assay for Direct Detection of the
Clostridium difficile
Toxin B Gene in Clinical Stool Specimens
Authors: Stamper Paul D. , Babiker Wisal , Alcabasa Romina , Aird Deborah , Wehrlin Jennifer , Ikpeama Ijeoma , Gluck Linda , Carroll Karen C.
Published: October 2009

The ProGastro Cd assay (Prodesse, Inc., Waukesha, WI) is a new commercial TaqMan PCR assay that detects
tcdB
. The ProGastro Cd assay was compared to the Wampole
Clostridium difficile
toxin B test (TOX-B test; TechLab, Blacksburg, VA), a cell culture cytotoxicity neutralization assay (CCCNA), and to anaerobic toxigenic bacterial culture, as the “gold standard,” for 285 clinical stool specimens. Assays were independently performed according to manufacturers' directions. A 1.0-ml sample was removed from the stool specimen, of which 20 μl was used for extraction on the NucliSENS easyMAG platform (bioMérieux, Inc., Durham, NC) for the Prodesse ProGastro Cd assay and 200 μl of the stool filtrate was used for the TOX-B CCCNA. Anaerobic toxigenic culture was done by heating an additional 1.0 ml of the stool sample to 80°C for 10 min before inoculation onto modified cycloserine, cefoxitin, and fructose agar with horse blood (Remel, Lenexa, KS) and into a prereduced chopped meat glucose broth (BBL, BD Diagnostics, Sparks, MD). The prevalence of toxin-producing strains of
C. difficile
was 15.7% (
n
= 44) as determined by anaerobic toxigenic culture. The sensitivity, specificity, and positive and negative predictive values of the Prodesse ProGastro Cd assay compared to the TOX-B test were 83.3%, 95.6%, 69.4%, and 98%, respectively. Compared to toxigenic culture, the sensitivity, specificity, and positive and negative predictive values of the Prodesse ProGastro Cd assay were 77.3%, 99.2%, 94.4%, and 95.9%, respectively, and those of the TOX-B test were 63.6%, 99.2%, 93.3%, and 93.6%, respectively. Although no statistical difference (Fisher's exact test) was detected (
P
= 0.242) between the sensitivities of the Prodesse ProGastro Cd assay and a standard CCCNA compared to anaerobic culture for the detection of toxigenic
C. difficile
, the Prodesse ProGastro Cd assay did detect more toxigenic
C. difficile
isolates than the CCCNA.
Comparison of PCR and Culture for Screening of Vancomycin-Resistant Enterococci: Highly Disparate Results for
vanA
and
vanB
Authors: Mak Anton , Miller Mark A. , Chong George , Monczak Yury
Published: October 2009

We compared PCR to conventional culture for the detection of vancomycin-resistant enterococci (VRE) in 30,835 rectal samples over a 3-year period. The positive and negative predictive values of
vanB
PCR were 1.42% and 99.9%, respectively. A positive
vanB
result by PCR is poorly predictive and necessitates culture for differentiation of VRE-positive and -negative individuals.
Evaluation of Adult Chronic Chagas' Heart Disease Diagnosis by Molecular and Serological Methods
Authors: Ramírez Juan David , Guhl Felipe , Umezawa Eufrosina Setsu , Morillo Carlos A. , Rosas Fernando , Marin-Neto Jose A. , Restrepo Silvia
Published: October 2009

Chagas' disease caused by
Trypanosoma cruzi
is endemic in Latin America.
T. cruzi
presents heterogeneous populations and comprises two main genetic lineages, named
T. cruzi
I and
T. cruzi
II. Diagnosis in the chronic phase is based on conventional serological tests, including indirect immunofluorescence (IIF) and enzyme-linked immunosorbent assay (ELISA), and diagnosis in the acute phase based on parasitological methods, including hemoculture. The objective of this study was to evaluate the diagnostic procedures of Chagas' disease in adult patients in the chronic phase by using a PCR assay and conventional serological tests, including TESA-blot as the gold standard. Samples were obtained from 240 clinical chronic chagasic patients. The sensitivities, compared to that of TESA-blot, were 70% for PCR using the kinetoplast region, 75% for PCR using the nuclear repetitive region, 99% for IIF, and 95% for ELISA. According to the serological tests results, we recommend that researchers assess the reliability and sensitivity of the commercial kit Chagatest ELISA recombinant, version 3.0 (Chagatest Rec v3.0; Wiener Lab, Rosario, Argentina), due to the lack of sensitivity. Based on our analysis, we concluded that PCR cannot be validated as a conventional diagnostic technique for Chagas' disease. These data have been corroborated by low levels of concordance with serology test results. It is recommended that PCR be used only for alternative diagnostic support. Using the nuclear repetitive region of
T. cruzi
, PCR could also be applicable for monitoring patients receiving etiologic treatment.
Differential Specificity and Immunogenicity of Adenovirus Type 5 Neutralizing Antibodies Elicited by Natural Infection or Immunization
Authors: Cheng Cheng , Gall Jason G. D. , Nason Martha , King C. Richter , Koup Richard A. , Roederer Mario , McElrath M. Juliana , Morgan Cecilia A. , Churchyard Gavin , Baden Lindsey R. , Duerr Ann C. , Keefer Michael C. , Graham Barney S. , Nabel Gary J.
Published: October 2009

A recent clinical trial of a T-cell-based AIDS vaccine delivered with recombinant adenovirus type 5 (rAd5) vectors showed no efficacy in lowering viral load and was associated with increased risk of human immunodeficiency virus type 1 (HIV-1) infection. Preexisting immunity to Ad5 in humans could therefore affect both immunogenicity and vaccine efficacy. We hypothesized that vaccine-induced immunity is differentially affected, depending on whether subjects were exposed to Ad5 by natural infection or by vaccination. Serum samples from vaccine trial subjects receiving a DNA/rAd5 AIDS vaccine with or without prior immunity to Ad5 were examined for the specificity of their Ad5 neutralizing antibodies and their effect on HIV-1 immune responses. Here, we report that rAd5 neutralizing antibodies were directed to different components of the virion, depending on whether they were elicited by natural infection or vaccination in HIV vaccine trial subjects. Neutralizing antibodies elicited by natural infection were directed largely to the Ad5 fiber, while exposure to rAd5 through vaccination elicited antibodies primarily to capsid proteins other than fiber. Notably, preexisting immunity to Ad5 fiber from natural infection significantly reduced the CD4 and CD8 cell responses to HIV Gag after DNA/rAd5 vaccination. The specificity of Ad5 neutralizing antibodies therefore differs depending on the route of exposure, and natural Ad5 infection compromises Ad5 vaccine-induced immunity to weak immunogens, such as HIV-1 Gag. These results have implications for future AIDS vaccine trials and the design of next-generation gene-based vaccine vectors.