bluuebunny/crossref_metadata_2025 huggingface dataset:
Good practice in plasma collection and fractionation
Authors: Schärer C.
Published: June 2010

The control strategy to ensure safety of blood products includes a combination of measures focusing on ensuring the quality and safety of starting material by careful donor selection and testing strategies at different levels, together with validated manufacturing processes, including steps to inactivate or remove potential contaminating agents. Using an approach based on good manufacturing practice (GMP) provides a manufacturing model that allows for a documented system of incorporating quality throughout the entire manufacturing process and describes the activities and controls needed to consistently produce products that comply with specifications and are safe for use. There are no doubts that the aim of providing safe and high‐quality product to the patients should be the same for all products derived from human blood, independent of its use either as a blood component for direct transfusion or as industrially manufactured product. It would be difficult to justify whether for blood components the good practice standards and for plasma derivatives the GMP standards for manufacturing would not ensure equivalent levels of quality and safety. To ensure a high level of quality and safety of blood components and plasma derivatives, the implementation of double standards in blood establishments and fractionation industry would not be effective and should be avoided. Harmonized standards and good practices for collection and fractionation, based on the principles of GMP, should be envisaged in the whole chain of manufacturing blood components and plasma derivatives. Global initiatives to further promote the implementation of harmonized GMP for the collection in blood establishments and a stringent regulatory control are ongoing. This would further contribute to the global availability of plasma‐derived medicinal products.
When do I (not) release cellular products?
Authors: Humpe A. , Buwitt‐Beckmann U. , Gramatzki M.
Published: June 2010

Harvest, production, storage and release of cellular products are regulated by international guidelines like the EU guidelines 2004/23/EC or 2006/17/EC. In addition, worldwide effective guidelines exist with the FACT‐JACIE standards. As cellular products such as haematopoietic progenitor cells (HPC) are usually applied to critically ill patients requirements for release must be well defined and high.

The determination of criteria for release requires a prior definition of specifications of parameters for the product. Such specifications and the question of product release with parameters out of specification will be addressed here exemplarily for autologous and allogeneic HPC grafts.

The harvest of cellular products must be preceded by an examination of the healthy donor or in case of an autologous transplantation of the patient. Apart from the medical history and the clinical condition of the donor, results of the testing of infectious disease markers (IDM) (HCV, HBV, HIV1,2 and syphilis at the minimum) are decisive for the judgment on the donor’s eligibility for donation. In case of an autologous donor, even positive test results for the IDM do not necessarily exclude a donation. After harvest and processing of the cellular product, the processing records must be complete and parameters like the content of the active pharmaceutical ingredient, i.e. CD34+ cells for HPC grafts, the content of possible cellular contaminants such as CD3+ cells in the allogeneic setting or tumour cells in the autologous setting, are relevant and need to be specified. For CD34+ cells, a minimum amount of 2 × 106 cells/kg of body weight for autologous or 4 × 106 cells/kg of body weight for allogeneic grafts is required. This may sometimes only be achieved by more than one harvest. Regarding CD34+ cells, the quality of the cells defined by viability and optional by analysis of clonogenic growth has to be evaluated. Such parameters need to be measured especially after manipulations such as cryopreservation or selection or depletion procedures. In addition, microbial contaminations, i.e. potential contaminations by aerobe or anaerobe bacteria or fungi for HPC products, have to be detected. Cellular grafts undergoing incubation steps need to be examined for contaminations by mycoplasma or viruses in addition. Furthermore, incubation or activation steps of cellular products require examinations regarding functionality, genomic stability and potential malignant transformation of the cultivated cells.

The final decision to release a cellular product in case of parameters being out of the specifications should be guided by the consideration that benefits outweigh potential risks for the recipient when administering the product. Furthermore, the type of the product, HPC or other product like donor lymphocyte infusions, and its importance in a given medical situation of the patient, the documented urgent medical need, will influence the decision of release.
Side‐effects of blood products
Authors: Sachs U. J. H.
Published: June 2010

As infectious complications from blood transfusion have decreased because of sophisticated blood screening, non‐infectious side‐effects have emerged as the most common complications of transfusion in industrialized countries. Despite making every endeavour, quite a number of these side‐effects are very difficult to control. Some of these side‐effects present as acute transfusion reactions, the most important of which are transfusion‐related acute lung injury, circulatory overload, sepsis, and allergic and anaphylactic reactions. Acute adverse events require immediate action, but are often difficult to evaluate. On the other hand, transfusion recipients may experience delayed non‐infectious side effects, including, non‐ABO haemolytic transfusion reactions, graft‐versus‐host disease, and post‐transfusion purpura. In addition, a not well‐defined risk of increased mortality following blood transfusion has been recognized. Some aspects regarding the clinical presentation, the physiology behind, and possible preventive measures are summarized here.
Natural killer cells: alloreactive effects in hematopoietic stem cell transplantation
Authors: Hsu K. C.
Published: June 2010

Background and Objectives  Natural Killer (NK) cells are capable of recognizing target cells altered by pathologic states such as viral infection and malignant transformation. The NK cell’s capacity to recognize and kill abnormal cells while sparing normal cells relies on the integration of activating signals and tolerance to cells expressing self‐MHC molecules. Our understanding of how NK receptors mediate these signals and control NK cell function has advanced to the point where models of NK biology can be tested and revised in the clinical setting of hematopoietic stem cell transplantation.

Materials and Methods  This review article highlights several of the seminal contributions to the field of NK biology and their relevance to allogeneic and autologous hematopoietic cell transplantation.

Results and Conclusions  Inhibitory NK receptors are critical for achieving tolerance to cells expressing self‐MHC molecules and recognizing target cells lacking self‐MHC class I molecules. Activating NK receptors can circumvent NK tolerance and directly signal activation. Both inhibitory and activating receptors play an important role in hematopoietic stem cell transplantation outcome for hematologic and solid tumors. Incorporation of NK receptor genotyping in donor selection algorithms may benefit recipients of both HLA‐matched and ‐mismatched allografts.
Pathogen inactivation/reduction of platelet concentrates: turning theory into practice
Authors: Gathof B. S. , Tauszig M. E. , Picker S. M.
Published: June 2010

Background  Pathogen reduction technology (PRT) has been proven to reduce the residual risk of transmission of infectious agents. Reduction of various contaminating bacteriae, viruses and parasites by few to several log steps and efficiency to prevent GVHD has been shown.

Aim  To evaluate and compare advantages and disadvantages of PRT available for practical application in platelets.

Materials and Methods  PRT for the treatment of platelets is currently offered by two formats: Amotosalen (INTERCEPT, Cerus, Concord, CA, USA) and vitamin B2 (Mirasol, Caridian, Denver, USA). Results from different studies and our own experiences with the two techniques are compared and discussed.

Results and Discussion  For both technologies, different groups of investigators have shown acceptable in‐vitro results with respect to functional and storage data for platelets stored for up to 5 days after production and before transfusion. Initial clinical studies showed no inferiority of the treated platelets in comparison to untreated controls in thrombocytopenic patients. However for both techniques a tendency towards lower CCI has been reported, which may be more pronounced in the platelets treated with the Intercept process. For introduction of PRT many countries require not only CE mark but licensing with the respective authorities since treatment for pathogen reduction is regarded as creating a ‘new’ blood product. With respect to a platelet loss during pathogen reduction it seems recommendable to increase the lower limit of platelet content of the product to 2.5 × 1011. Particularly for the Intercept system, where a considerable amount of platelets is lost in the purification of the product from Amotosalen, a change in the production process to increase the platelet yield may be necessary. Data from our group show a tendency for improved functional and storage parameters for platelets treated with the Mirasol process. Compared to conventional manufacturing of platelets by apheresis or pooling of buffy coats, pathogen reduction requires additional labour, space, and quality control. Shelf life of platelets is limited in most countries because of the risk of bacterial contamination (in Germany presently to 4 days). A prolongation to 5 or more days after pathogen reduction seems feasible but remains a topic for future studies.

Conclusion  Results of in vitro and clinical studies of pathogen reduced platelets are promising. Larger clinical trials will help to determine whether PRT proves to be beneficial (reduction of transmission of infections, less alloimmunisation) and overall cost effective (bearing in mind that additional costs may be compensated for by omission of gamma irradiation and potential longer shelf life).
Do I assist or supervise? Achievements of quality management implementation
Authors: Slopecki A.
Published: June 2010

Background and Objective To answer the question ‘Do I assist or supervise? Achievements of quality management implementation’ I illustrate how a Quality Management System used by a blood establishment can be derived based on simple common sense and is essentially there to control peoples actions to work consistently and safely.

Materials and Methods I briefly review the key principles in EU GMP to illustrate the importance of quality and people management and draw from events in my experience which have provided significant learning opportunities for my organisation.

Results The learning opportunities illustrate the importance of having real time data on which to base actions allowing you to work with suppliers to improve their products and your systems. That corrective and preventative action must be confirmed as effective. The systems you use need to be simple and effective and adhere to the principle of GMP. External inspectors are a valuable source of bench marking data which you can use to improve.

Conclusion If a quality management system is working effectively then as a Quality profession I would assist, however if the effectiveness of the system drifts or individuals/departments fail to play their part I reserve the right to supervise.
Relevance of parasite infections outside South and Central America: Chagas disease and malaria
Authors: Piron M.
Published: June 2010

The constant increase of population movements related to emigration, business or tourism, leads to the spread of infections until now restricted to endemic areas.

In the case of Central and South Americas, Chagas disease has become a matter of concern during the last decade in countries which receive Latino American immigrants. This disease is caused by a protozoan parasite, Trypanosoma cruzi, transmitted in endemic areas predominantly by a Triatomine vector. The probable routes of infection in non‐endemic countries, where the vector is non‐existent, are therefore blood transfusion, organ transplantation and vertical transmission. In Europe, Spain is the country that receives most part of immigrants proceeding from Central and South Americas. In Spanish blood banks, anti‐T. cruzi screening is mandatory since September 2005 in donors at‐risk for Chagas disease. From this date, this new selective screening allowed to identify 110 infected blood donors in the Catalonian Blood Bank. A large part of positive donors was from Bolivia, country with the highest seroprevalence of infection. Other positive donors were from Argentina, Paraguay, Ecuador etc., but also from Spain (mother born in endemic area, or donor born in endemic area or who had resided in endemic area). At the same time, look‐back procedures were initiated in those donors who had donated before and whose blood products had been transfused. Screening for T. cruzi infection is generally selective, only done in at‐risk donors, although the USA blood banks perform a universal screening since 2007. The selective screening strategy is finally discussed.

Besides Chagas disease, malaria is also a parasitic infection caused by Plasmodium and transmitted by the bite of Anopheles mosquito in endemic areas. Two of the five Plasmodium species, P. falciparum and P. vivax, are most often found in Central and South Americas. Cases of transfusion‐transmitted malaria are mostly associated to P. falciparum, although cases of transmission of P. malariae, vivax or ovale have also been described. The growing number of donors who travel to malaria endemic areas, or who were born in an endemic region increases the probability to fail to identify an at‐risk donor and to transfuse a contaminated blood component. In non‐endemic countries, the measures taken to prevent malaria transmission by transfusion generally consist in deferral of at‐risk donors, based on questionnaire reporting travels to or residence in malaria areas. Some blood bank use a screening test to detect anti‐Plasmodium antibodies, but have then to face the problem of confirmation of an initially reactive result and of donor’s counselling. To opt for a screening or for a deferral strategy could depend once again on the epidemiological profile of blood donors.
Error Sources in Atomic Force Microscopy for Dimensional Measurements: Taxonomy and Modeling
Authors: Marinello F. , Carmignato S. , Voltan A. , Savio E. , De Chiffre L.
Published: June 2010

This paper aimed at identifying the error sources that occur in dimensional measurements performed using atomic force microscopy. In particular, a set of characterization techniques for errors quantification is presented. The discussion on error sources is organized in four main categories: scanning system, tip-surface interaction, environment, and data processing. The discussed errors include scaling effects, squareness errors, hysteresis, creep, tip convolution, and thermal drift. A mathematical model of the measurement system is eventually described, as a reference basis for errors characterization, with an applicative example on a reference silicon grating.
Modeling of Recast Layer in Micro-Electrical Discharge Machining
Authors: Tan P. C. , Yeo S. H.
Published: June 2010

The thickness of recast layers produced during electrical discharge machining (EDM) is an important process performance measure as it may indicate an extent of crack propagation in a machined surface or thickness of a functional layer alloyed onto a machined surface. Thus, the availability of the recast layer thickness prediction models is needed to allow better control of machining outcomes, which becomes more vital for micro-EDM due to the microscale of machined features. The proposed numerical model, based on a multiple discharge approach for recast layer prediction, is developed to fill an existing gap in micro-EDM. The multiple discharge approach accounts for the overlapping nature by which craters are generated on the machined surface and considers the recast layer to be a combination of individual recast regions from individual craters. The numerical analysis, based on finite element methods, is used to determine the melting isotherms due to heat inputs on overlapping crater profiles. Then, a hemispherical-capped crater profile is estimated by applying a recast plasma flushing efficiency to the amount of molten material bounded by the melting isotherm. Finally, the recast region is defined to be bounded by the melting isotherm and crater profile. The model, developed for a peak discharge current of 1.45 A and pulse on time between 166 ns and 606 ns, predicted recast layer thicknesses of between 1.0 μm and 1.82 μm. It is then validated at pulse on time settings of 244 ns and 458 ns, which generated average recast layer thicknesses of 1.18 μm and 1.56 μm, respectively. Thus, the numerical model developed using the multiple discharge approach is suitable for estimation of recast layer thicknesses in micro-EDM.
Comparison of Nickel Nanoparticle-Assisted Diffusion Brazing of Stainless Steel to Conventional Diffusion Brazing and Bonding Processes
Authors: Tiwari S. K. , Paul B. K.
Published: June 2010

Transient liquid phase diffusion brazing is used in precision, hermetic joining applications as a replacement for diffusion bonding to reduce cycle times, reduce bonding pressure, and improve yields. In the present study, stainless steel 316L laminae are diffusion brazed with an interlayer of nickel nanoparticles and compared with samples joined by conventional diffusion bonding and electroplated nickel-phosphorous diffusion brazing. Comparison is made with regard to microstructural evolution, diffusional profile, and bond strength. All bonding was carried out in a uni-axial vacuum hot press at 1000°C with a heating rate of 10°C/min, a dwell time of 2 h and a bonding pressure of 10 MPa. Bond strength measurements show that the sample brazed with a nickel nanoparticle interlayer has the lowest void fraction at 4.8±0.9% and highest shear strength at 141.3±7.0 MPa. Wavelength dispersive spectroscopic analysis of sample cross-sections shows substantial diffusion of Ni and Fe across the nickel nanoparticle bond line. Scanning electron micrographs show no secondary phases along the nickel nanoparticle bond line.
Analysis of Molecular Beam Epitaxy Process for Growing Nanoscale Magnesium Oxide Films
Authors: Uddin Ghulam M. , Cai Zhuhua , Ziemer Katherine S. , Zeid Abe , Kamarthi Sagar
Published: June 2010

Like most nanomanufacturing processes, molecular beam epitaxy (MBE) processes are based on atomic-level control of growing films and thus are sensitive to subtle changes that make repeatability and reproducibility of desired performance indicators a nontrivial task. The gamut of challenges include insufficient understanding of atomic-level interactions, involvement of a large number of candidate process variables, lack of direct observation and measurement techniques for key performance indicators, and significant cost and time requirements for conducting experiments. A conventional design of experiment-based analysis becomes an unrealistic option due to its demand on extensive experimentation. In this paper, we present a hybrid approach that combines current process knowledge, artificial neural networks, and design of experiments (DOE) to make use of preliminary experimental data to analyze the process behavior, enhance process knowledge, and lay down foundations for cost effective systematic experimentation. Based on preliminary experimental data generated while exploring the MBE process for growing a MgO interface layer on 6H-SiC substrate, we developed a neural-network-based meta model that can interpolate and estimate the process responses to any combination of process variable settings within the input space. Using the neural-network model trained on preliminary experimental data, we estimate the process responses for a three-level full-factorial DOE runs. Based on these runs, the DOE based analysis is carried out. The results help explain the MgO film growth dynamics with respect to process variables such as substrate temperature, growth time, magnesium source temperature, and trace oxygen on the initial substrate surface. This approach can be expanded to statistically analyze the dynamics of other complex nanoprocesses when only the exploratory preliminary experimental data are available. This approach can also lay the foundation for efficient and systematic experimentation to further analyze and optimize the processes to address issues such as process repeatability and reliability.
Vibration Cancellation in a Plate Using Orthogonal Eigenstructure Control
Authors: Rastgaar M. A. , Ahmadian M. , Southward S. C.
Published: June 2010

Orthogonal eigenstructure control is a novel control method that can be used for vibration suppression in flexible structures. The method described in this study does not need defining the desired locations of the closed-loop poles or predetermining the closed-loop eigenvectors. The method, which is applicable to linear multi-input multi-output systems, determines an output feedback control gain matrix such that some of the closed-loop eigenvectors are orthogonal to the open-loop eigenvectors. Using this, the open-loop system’s eigenvectors as well as a group of orthogonal vectors are regenerated based on a matrix that spans the null space of the closed-loop eigenvectors. The gain matrix can be generated automatically; therefore, the method is neither a trial and error process nor an optimization of an index function. A finite element model of a plate is used to study the applicability of the method to systems with relatively large degrees of freedom. The example is also used to discuss the effect of operating eigenvalues on the process of orthogonal eigenstructure control. The importance of the operating eigenvalues and the criteria for selecting them for finding the closed-loop system are also investigated. It is shown that choosing the operating eigenvalues from the open-loop eigenvalues that are farthest from the origin results in convergence of the gain matrix for the admissible closed-loop systems. It is shown that the converged control gain matrix has diagonal elements that are two orders of magnitude larger than the off-diagonal elements, which implies a nearly decoupled control.
Angular Rate From the Nine Accelerometer Instrument
Authors: Vreeburg Jan P. B.
Published: June 2010

Instruments composed of arrangements of linear accelerometers are used to determine motion variables. With nine accelerometers, the angular rate of the instrument can be resolved algebraically rather than by the solution of a system of nonlinear differential equations. The algebraic equations consist of three quadratic forms in the angular rate and, in general, lead to multiple solutions. The number and distribution of the multiple roots have been analyzed. Examples illustrate the application of the analyses, for batch and real-time processing. Suitable algorithms have been developed and are detailed. Subjects for further study have been indicated.
Crucible aperture: An effective way to reduce source oxidation in oxide molecular beam epitaxy process
Authors: Kim Yong-Seung , Bansal Namrata , Oh Seongshik
Published: June 2010

Growing multielemental complex-oxide structures using a molecular beam epitaxy (MBE) technique requires precise control of each source flux. However, when the component elements have significantly different oxygen affinities, maintaining stable fluxes for easily oxidizing elements is challenging because of the source oxidation problem. Here, using Sr as a test source, the authors show that a crucible aperture insert scheme significantly reduces the source oxidation in an oxide-MBE environment. The crucible aperture insert was shaped like a disk with a hole at the center and was mounted inside the crucible; it blocks most of the oxygen species coming to the source, thus reducing the source oxidation. However, the depth of the aperture disk was critical for its performance; an ill-positioned aperture could make the flux stability even worse. With an optimally positioned aperture insert, the crucible exhibited more than four times improvement in Sr flux stability, compared to a conventional, nonapertured crucible.
Down-Regulation of a Host MicroRNA by a
Herpesvirus saimiri
Noncoding RNA
Authors: Cazalla Demián , Yario Therese , Steitz Joan A.
Published: June 2010

Herpes Virus MiRNA Modulation

Viruses use a number of strategies to manipulate the cells of their host to ensure a successful infection.
Herpesvirus saimiri
(HVS) generates highly conserved small noncoding RNAs
HSUR 1
and
HSUR 2
, which modulate expression of a number of proteins in infected primate T cells.

Cazalla
et al.

(p.
1563
; see the Perspective by

Pasquinelli

) observed complementarity between HSUR sequences and the seed regions of three different miRNAs—miR-142-3p, miR-27, and miR-16—and found that these HSURs could bind to the miRNAs. Furthermore, the level of mature miR-27 was modulated by binding to
HSUR 1
, which targeted the miRNA for degradation.
Deconstructing the Conveyor Belt
Authors: Lozier M. Susan
Published: June 2010

For the past several decades, oceanographers have embraced the dominant paradigm that the ocean’s meridional overturning circulation operates like a conveyor belt, transporting cold waters equatorward at depth and warm waters poleward at the surface. Within this paradigm, the conveyor, driven by changes in deepwater production at high latitudes, moves deep waters and their attendant properties continuously along western boundary currents and returns surface waters unimpeded to deepwater formation sites. A number of studies conducted over the past few years have challenged this paradigm by revealing the vital role of the ocean’s eddy and wind fields in establishing the structure and variability of the ocean’s overturning. Here, we review those studies and discuss how they have collectively changed our view of the simple conveyor-belt model.
Tilting at Connections, from Pole to Equator
Authors: Philander S. George
Published: June 2010

Over the past 3 million years, how did variations in sunlight near the poles cause sea surface temperatures near the equator to fluctuate?
Dating Pharaonic Egypt
Authors: Bruins Hendrik J.
Published: June 2010

Radiocarbon dating and modeling of Egyptian dynasties are strengthening the links between historical chronology and archaeological associations.
Better Reception in Europe?
Agora Alejandro Amenábar, director
Mod Producciones, Himenóptero, and Telecinco Cinema, Spain, 2009. 126 minutes. http://agorathemovie.com/
Océans Jacques Perrin and Jacques Cluzaud, directors
Galatée Films, France, 2009. 100 minutes. www.oceans-lefilm.com/
Oceans Jacques Perrin and Jacques Cluzaud, directors
Disneynature, USA, 2010. 87 minutes. http://disney.go.com/disneynature/oceans/
Authors: Firestein Stuart
Published: June 2010

Reviewing the films Agora and Océans, Firestein wonders why they seem to have had hard passages from Europe to the United States.
The Changing Oceans
Authors: Church John A.
Published: June 2010

The evolving disaster of the Gulf of Mexico oil spill reminds us that our welfare depends on a healthy marine ecosystem and that the oceans are vulnerable to human activities. The oceans sustain a vast wealth of biological diversity, deliver critical ecosystem services, supply valuable natural resources, and are a central component of the climate system. It is therefore critical that the current ocean-observing system be extended to cover a wider range of ocean properties.
Water Shortages Loom as Northern China's Aquifers Are Sucked Dry
Authors: Jiao Li
Published: June 2010

Of all China's environmental woes, the biggest threat to livelihoods and food security may be looming water shortages. The outlook is especially dire on the North China Plain, an area encompassing six provinces and the Beijing and Tianjin metropolitan areas.
Endosulfan's Exit: U.S. EPA Pesticide Review Leads to a Ban
Authors: Lubick Naomi
Published: June 2010

After a lengthy scientific review, the United States last week decided to ban the use of endosulfan, an inexpensive organochlorine pesticide that builds up in the environment.
Keeping Tabs on the Sea
Authors: Malakoff David
Published: June 2010

Efforts to monitor the ocean are getting bigger and more sophisticated, but vast swaths of the marine world remain unknown.
A Push for Quieter Ships
Authors: Malakoff David
Published: June 2010

Although sonar and air guns have grabbed headlines, researchers say the cacophony from ships creates far more ocean noise.
Down on the Shrimp Farm
Authors: Stokstad Erik
Published: June 2010

Can shrimp become the new chicken of the sea without damaging the ocean?
Oral Vaccine Formulations Stimulate Mucosal and Systemic Antibody Responses against Staphylococcal Enterotoxin B in a Piglet Model
Authors: Inskeep Tiffany K. , Stahl Chad , Odle Jack , Oakes Judy , Hudson Laura , Bost Kenneth L. , Piller Kenneth J.
Published: June 2010

Despite the potential for its use as an agent of biowarfare or bioterrorism, no approved vaccine against staphylococcal enterotoxin B (SEB) exists. Nontoxic, mutant forms of SEB have been developed; however, it has been difficult to determine the efficacy of such subunit vaccine candidates due to the lack of superantigen activity of native SEB in rodents and due to the limitations of primate models. Since pigs respond to SEB in a manner similar to that of human subjects, we utilized this relevant animal model to investigate the safety and immunogenicity of a triple mutant of SEB carrying the amino acid changes L45R, Y89A, and Y94A. This recombinant mutant SEB (rmSEB) did not possess superantigen activity in pig lymphocyte cultures. Furthermore, rmSEB was unable to compete with native SEB for binding to pig leukocytes. These
in vitro
studies suggested that rmSEB could be a safe subunit vaccine. To test this possibility, piglets immunized orally with rmSEB formulations experienced no significant decrease in food consumption and no weight loss during the vaccination regimen. Oral vaccination with 1-mg doses of rmSEB on days 0, 7, 14, and 24 resulted in serum IgG and fecal IgA levels by day 36 that cross-reacted with native SEB. Surprisingly, the inclusion of cholera toxin adjuvant in vaccine formulations containing rmSEB did not result in increased antibody responses compared to formulations using the immunogen alone. Taken together, these studies provide additional evidence for the potential use of nontoxic forms of SEB as vaccines.
Evaluation of Colorimetric Methods Using Nicotinamide for Rapid Detection of Pyrazinamide Resistance in
Mycobacterium tuberculosis
Authors: Mirabal Niuris C. , Yzquierdo Sergio L. , Lemus Dihadenys , Madruga Mariela , Milián Yoslaine , Echemendía Miguel , Takiff Howard , Martin Anandi , Van der Stuyf Patrick , Palomino Juan Carlos , Montoro Ernesto
Published: June 2010

The direct detection of pyrazinamide resistance in
Mycobacterium tuberculosis
is sufficiently difficult that many laboratories do not attempt it. Most pyrazinamide resistance is caused by mutations that inactivate the pyrazinamidase enzyme needed to convert the prodrug pyrazinamide to its active form. We evaluated two newer and simpler methods to assess pyrazinamidase activity, the nitrate reductase and malachite green microtube assays, using nicotinamide in place of pyrazinamide. A total of 102 strains were tested by these methods and the results compared with those obtained by the classic Wayne assay. Mutations in the
pnc
A
gene were identified by sequencing the
pnc
A
genes from all isolates in which pyrazinamide resistance was detected by any of the three methods. Both the nitrate reductase and malachite green microtube assays showed sensitivities of 93.75% and specificities of 97.67%. Mutations in the
pnc
A
gene were found in 14 of 16 strains that were pyrazinamide resistant and in 1 of 4 strains that were sensitive by the Wayne assay. Both of these simple methods, used with nicotinamide, are promising and inexpensive alternatives for the rapid detection of pyrazinamide resistance in limited-resource countries.
Multiple-Locus Variable-Number Tandem-Repeat Analysis of the Swine Dysentery Pathogen,
Brachyspira hyodysenteriae
Authors: Hidalgo Álvaro , Carvajal Ana , La Tom , Naharro Germán , Rubio Pedro , Phillips Nyree D. , Hampson David J.
Published: June 2010

The spirochete
Brachyspira hyodysenteriae
is the causative agent of swine dysentery, a severe colonic infection of pigs that has a considerable economic impact in many swine-producing countries. In spite of its importance, knowledge about the global epidemiology and population structure of
B. hyodysenteriae
is limited. Progress in this area has been hampered by the lack of a low-cost, portable, and discriminatory method for strain typing. The aim of the current study was to develop and test a multiple-locus variable-number tandem-repeat analysis (MLVA) method that could be used in basic veterinary diagnostic microbiology laboratories equipped with PCR technology or in more advanced laboratories with access to capillary electrophoresis. Based on eight loci, and when performed on isolates from different farms in different countries, as well as type and reference strains, the MLVA technique developed was highly discriminatory (Hunter and Gaston discriminatory index, 0.938 [95% confidence interval, 0.9175 to 0.9584]) while retaining a high phylogenetic value. Using the technique, the species was shown to be diverse (44 MLVA types from 172 isolates and strains), although isolates were stable in herds over time. The population structure appeared to be clonal. The finding of
B. hyodysenteriae
MLVA type 3 in piggeries in three European countries, as well as other, related, strains in different countries, suggests that spreading of the pathogen via carrier pigs is likely. MLVA overcame drawbacks associated with previous typing techniques for
B. hyodysenteriae
and was a powerful method for epidemiologic and population structure studies on this important pathogenic spirochete.
Epidemiological and Phylogenetic Analysis of Spanish HumanBrucella melitensisStrains by Multiple-Locus Variable-Number Tandem-Repeat Typing, Hypervariable Octameric Oligonucleotide Fingerprinting, andrpoBTyping
Authors: Valdezate Sylvia , Navarro Ana , Villalón Pilar , Carrasco Gema , Saéz-Nieto Juan A.
Published: June 2010

The severe morbidity of human brucellosis is one of the main reasons for using molecular typing in the epidemiological surveillance of this worldwide zoonosis. Multiple-locus variable-number repeat analysis (MLVA-16), hypervariable octameric oligonucleotide fingerprinting (HOOF-print), and the differences in the single nucleotide polymorphisms (SNPs) (codons 1249 and 1309) of the DNA-dependent RNA polymerase β subunit (rpoB) were used to type a humanBrucella melitensispopulation (108 strains) collected from throughout Spain over 13 years. Eighty-six MLVA types (discriminatory index, 0.99) were detected, with a wide-ranging genetic similarity coefficient (37.2 to 93.7%). The population clustered into the following groups: American, with genotypes 47 (1 strain), 48 (13 strains), 53 (12 strains), 55 (2 strains), 80 (1 strain), and a new genotype (2 strains), Western Mediterranean, with genotype 51 (9 strains), and Eastern Mediterranean, with genotypes 42 (60 strains), 43 (4 strains), and 63 (4 strains). Two profession-related and two foodborne acquisitions were confirmed. Distributed throughout Spain, Eastern Mediterranean genotype 42 was the most common (55%). The low MLVA-16 allelic polymorphism (genetic similarity range, 75 to 94%) of the genotype 42 strains suggests that they recently evolved from a common ancestor.rpoBtyping grouped the strains asrpoBtype 1 (1249-ATG/1309-CTG; 28.7%),rpoBtype 2 (1249-ATG/1309-CTA; 62.9%), andrpoBtype 3 (1249-ATA/1309-CTG; 8.3%). According to the MLVA-16 results, the population clustered byrpoBtype. Given the correlation betweenB. melitensisMLVA groups andrpoBtypes (American andrpoBtype 1, Eastern Mediterranean andrpoBtype 2, and Western Mediterranean andrpoBtype 3), therpoBtype could be used as an initial marker for the epidemiological surveillance of brucellosis.
Epidemiology and Clinical Presentations of the Four Human Coronaviruses 229E, HKU1, NL63, and OC43 Detected over 3 Years Using a Novel Multiplex Real-Time PCR Method
Authors: Gaunt E. R. , Hardie A. , Claas E. C. J. , Simmonds P. , Templeton K. E.
Published: June 2010

Four human coronaviruses (HCoV-229E, HCoV-HKU1, HCoV-NL63, and HCoV-OC43) are associated with a range of respiratory outcomes, including bronchiolitis and pneumonia. Their epidemiologies and clinical characteristics are poorly described and are often reliant on case reports. To address these problems, we conducted a large-scale comprehensive screening for all four coronaviruses by analysis of 11,661 diagnostic respiratory samples collected in Edinburgh, United Kingdom, over 3 years between July 2006 and June 2009 using a novel four-way multiplex real-time reverse transcription-PCR (RT-PCR) assay. Coronaviruses were detected in 0.3 to 0.85% of samples in all age groups. Generally, coronaviruses displayed marked winter seasonality between the months of December and April and were not detected in summer months, which is comparable to the pattern seen with influenza viruses. HCoV-229E was the exception; detection was confined to the winter of 2008 and was sporadic in the following year. There were additional longer-term differences in detection frequencies between seasons, with HCoV-OC43 predominant in the first and third seasons and HCoV-HKU1 dominating in the second (see Results for definitions of seasons). A total of 11 to 41% of coronaviruses detected were in samples testing positive for other respiratory viruses, although clinical presentations of coronavirus monoinfections were comparable to those of viruses which have an established role in respiratory disease, such as respiratory syncytial virus, influenza virus, and parainfluenza viruses. The novel multiplex assay for real-time pan-coronavirus detection enhances respiratory virus diagnosis, overcomes potential diagnostic problems arising through seasonal variation in coronavirus frequency, and provides novel insights into the epidemiology and clinical implications of coronaviruses.
Detection and Characterization of Human
Ureaplasma
Species and Serovars by Real-Time PCR
Authors: Xiao Li , Glass John I. , Paralanov Vanya , Yooseph Shibu , Cassell Gail H. , Duffy Lynn B. , Waites Ken B.
Published: June 2010

We designed primers and probes for the detection and discrimination of
Ureaplasma parvum
and
U. urealyticum
and their 14 serovars by real-time PCR. The analytical sensitivity and specificity of the multiplex species-specific PCR were determined by testing corresponding American Type Culture Collection (ATCC) type strains, 47 other microbial species, and human genomic DNA. The limits of the multiplex PCR were 2.8 × 10
−2
CFU/μl PCR mixture for detecting
U. parvum
and 4.1 × 10
−2
CFU/μl PCR mixture for detecting
U. urealyticum
. Clinical specificity and sensitivity were proven by comparison with culture and traditional PCR. For the detection of any
Ureaplasma
species, the clinical sensitivity and specificity of real-time PCR were 96.9% and 79.0%, respectively, using culture as a reference. Multiplex real-time PCR was also more sensitive than traditional PCR in discriminating the two
Ureaplasma
species in culture-positive subcultures. Each of the 14 monoplex serovar-specific PCR assays was specific for the corresponding ATCC type strain serovar. This new species identification PCR is specific and sensitive in the detection of
Ureaplasma
species in clinical specimens, and the serovar-specific PCR assays are the first set of complete genotypic assays to differentiate all 14 known
Ureaplasma
serovars. These assays provide quick and reliable means for investigating the epidemiology and pathogenicity of ureaplasmas at the serovar level.
Comparison of the Novel Partec Rapid Malaria Test to the Conventional Giemsa Stain and the Gold Standard Real-Time PCR
Authors: Nkrumah Bernard , Agyekum Alex , Acquah Samuel E. K. , May Jürgen , Tannich Egbert , Brattig Norbert , Nguah Samuel Blay , von Thien Heidrun , Adu-Sarkodie Yaw , Huenger Frank
Published: June 2010

Malaria remains the single most frequent cause of death in Africa, killing one child every 30 s, but treatment decisions are often made only on clinical diagnosis, as laboratory techniques to confirm the clinical suspicion are labor intensive and costly. In this study, we evaluated the recently developed Partec rapid malaria test (PM) for the detection of
Plasmodium
spp. in human blood from patients in an area where malaria is endemic and compared the results with those of thick blood film Giemsa stain (GS) in terms of its performance and operational characteristics, using real-time (RT) PCR as the gold standard. The sensitivities of the PM and the GS were 62.2% (95% CI, 56.3 to 67.8) and 61.8% (95% CI, 55.9 to 67.4), respectively, while the specificities were 96.0% (95% CI, 92.3 to 98.3) and 98% (95% CI, 95.0 to 99.5), respectively. There was an excellent agreement between the results for the PM and those of the GS (
k
[level of agreement] = 0.96;
P
< 0.001). The results for the PM were obtained more quickly and at less cost than those for the GS. The performance characteristics of the PM were almost equal to those of the GS, but the operational characteristics were better, and the PM can therefore be considered as an alternative method for GS.
Tick-Borne Encephalitis Virus Delays Interferon Induction and Hides Its Double-Stranded RNA in Intracellular Membrane Vesicles
Authors: Överby Anna K. , Popov Vsevolod L. , Niedrig Matthias , Weber Friedemann
Published: June 2010

Tick-borne encephalitis virus (TBEV) (family
Flaviviridae
, genus
Flavivirus
) accounts for approximately 10,000 annual cases of severe encephalitis in Europe and Asia. Here, we investigated the induction of the antiviral type I interferons (IFNs) (alpha/beta IFN [IFN-α/β]) by TBEV. Using strains Neudörfl, Hypr, and Absettarov, we demonstrate that levels of IFN-β transcripts and viral RNA are strictly correlated. Moreover, IFN induction by TBEV was dependent on the transcription factor IFN regulatory factor 3 (IRF-3). However, even strain Hypr, which displayed the strongest IFN-inducing activity and the highest RNA levels, substantially delayed the activation of IRF-3. As a consequence, TBEV can keep the level of IFN transcripts below the threshold value that would permit the release of IFN by the cell. Only after 24 h of infection have cells accumulated sufficient IFN transcripts to produce detectable amounts of secreted IFNs. The delay in IFN induction appears not to be caused by a specific viral protein, since the individual expressions of TBEV C, E, NS2A, NS2B, NS3, NS4A, NS4B, NS5, and NS2B-NS3, as well as TBEV infection itself, had no apparent influence on specific IFN-β induction. We noted, however, that viral double-stranded RNA (dsRNA), an important trigger of the IFN response, is immunodetectable only inside intracellular membrane compartments. Nonetheless, the dependency of IFN induction on IFN promoter stimulator 1 (IPS-1) as well as the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2α) suggest the cytoplasmic exposure of some viral dsRNA late in infection. Using ultrathin-section electron microscopy, we demonstrate that, similar to other flaviviruses, TBEV rearranges intracellular membranes. Virus particles and membrane-connected vesicles (which most likely represent sites of virus RNA synthesis) were observed inside the endoplasmic reticulum. Thus, apparently, TBEV rearranges internal cell membranes to provide a compartment for its dsRNA, which is largely inaccessible for detection by cytoplasmic pathogen receptors. This delays the onset of IFN induction sufficiently to give progeny particle production a head start of approximately 24 h.
Genetic Diversity and Histo-Blood Group Antigen Interactions of Rhesus Enteric Caliciviruses
Authors: Farkas Tibor , Cross Robert W. , Hargitt Edwin , Lerche Nicholas W. , Morrow Ardythe L. , Sestak Karol
Published: June 2010

Recently, we reported the discovery and characterization of Tulane virus (TV), a novel rhesus calicivirus (CV) (T. Farkas, K. Sestak, C. Wei, and X. Jiang, J. Virol.82:5408-5416, 2008). TV grows well in tissue culture, and it represents a new genus withinCaliciviridae, with the proposed name ofRecovirus. We also reported a high prevalence of CV antibodies in macaques of the Tulane National Primate Research Center (TNPRC) colony, including anti-norovirus (NoV), anti-sapovirus (SaV), and anti-TV (T. Farkas, J. Dufour, X. Jiang, and K. Sestak, J. Gen. Virol. 91:734-738, 2010). To broaden our knowledge about CV infections in captive nonhuman primates (NHP), 500 rhesus macaque stool samples collected from breeding colony TNPRC macaques were tested for CVs. Fifty-seven (11%) samples contained recovirus isolates. In addition, one NoV was detected. Phylogenetic analysis classified the recovirus isolates into two genogroups and at least four genetic types. The rhesus NoV isolate was closely related to GII human NoVs. TV-neutralizing antibodies were detected in 88% of serum samples obtained from primate caretakers. Binding and plaque reduction assays revealed the involvement of type A and B histo-blood group antigens (HBGA) in TV infection. Taken together, these findings indicate the zoonotic potential of primate CVs. The discovery of a genetically diverse and prevalent group of primate CVs and remarkable similarities between rhesus enteric CVs and human NoVs opens new possibilities for research involvingin vitroandin vivomodels of human NoV gastroenteritis.
Simian Immunodeficiency Virus Infection Induces Expansion of α4β7+and Cytotoxic CD56+NK Cells
Authors: Reeves R. Keith , Evans Tristan I. , Gillis Jacqueline , Johnson R. Paul
Published: June 2010

Herein we demonstrate that chronic simian immunodeficiency virus (SIV) infection induces significant upregulation of the gut-homing marker α4β7 on macaque NK cells, coupled with downregulation of the lymph node-trafficking marker, CCR7. Interestingly, in naïve animals, α4β7 expression was associated with increased NK cell activation and, on CD16+NK cells, delineated a unique dual-function cytotoxic-CD107a+/gamma interferon (IFN-γ)-secreting population. However, while SIV infection increased CD107a expression on stimulated CD56+NK cells, α4β7+and α4β7−NK cells were affected similarly. These findings suggest that SIV infection redirects NK cells away from the lymph nodes to the gut mucosae but alters NK cell function independent of trafficking repertoires.