bluuebunny/crossref_metadata_2025_split huggingface dataset:
Citrobacterspp. Simultaneously HarboringblaCTX-M,blaTEM,blaSHV,blaampC, and Insertion Sequences IS26andorf513: an Evolutionary Phenomenon of Recent Concern for Antibiotic Resistance
Authors: Shahid M. 
Published: March 2010

ABSTRACTA collection of 40 confirmed isolates ofCitrobacterspp., obtained from the culture of 5,732 clinical samples during 2006 to 2008, was studied for the presence ofblaCTX-M,blaTEM,blaSHV, andblaampCby monoplex and multiplex PCRs. These isolates were also looked at for the presence of insertion elements IS26andorf513, which play an important role in the resistance gene pool.blaCTX-M,blaTEM,blaSHV, andblaampCwere noticed in 67.5%, 40%, 25%, and 40% of isolates, respectively. Sequencing for the specific CTX-M type revealed the presence of CTX-M-15-type extended-spectrum beta-lactamases (ESBL).sul1-type integrons were detected in 32.5% (13/40) of isolates, and a single plasmid of ca. ∼22 kb was also noticed in all the isolates harboringblagenes. A total of 48.2% (13/27) of isolates harboringblaCTX-Malleles were found to carry IS26elements and 53.4% (7/13) of isolates harboring thesul1-type integrons were found to carryorf513. Alarmingly, a few isolates simultaneously carried IS26andorf513, reflecting the occurrence of complex evolution. All theCitrobacterisolates were noticed to produce an amplicon of 400 bp in an IS26PCR, similar to that of the predominant UK CTX-M-15-producingEscherichia coliclone (clone A) and suggesting a probable genetic relatedness between the two, which could not be proved with British and IndianE. coliisolates in our earlier studies. Here, a first report is being presented describing the occurrence ofblaCTX-M-15in IndianCitrobacterspp. This is also the first report describing the simultaneous occurrence ofblaTEM,blaSHV, andblaampCalong withblaCTX-Min class 1 integrons. The recent findings indicate that the genetic environment ofblaCTX-M-15has changed through the insertion of IS26andorf513, although the consequences of these events remain uncertain. It perhaps also suggests a “turnover” of these mobile elements in the population over time.
Evaluation of an Immunochromatographic Test for Rapid and Reliable Serodiagnosis of Human Tularemia and Detection ofFrancisella tularensis-Specific Antibodies in Sera from Different Mammalian Species
Authors: Splettstoesser W. , Guglielmo-Viret V. , Seibold E. , Thullier P. 
Published: March 2010

ABSTRACTTularemia is a highly contagious infectious zoonosis caused by the bacterial agentFrancisella tularensis. Serology is still considered to be a cornerstone in tularemia diagnosis due to the low sensitivity of bacterial culture and the lack of standardization in PCR methodology for the direct identification of the pathogen. We developed a novel immunochromatographic test (ICT) to efficiently detectF. tularensis-specific antibodies in sera from humans and other mammalian species (nonhuman primate, pig, and rabbit). This new tool requires none or minimal laboratory equipment, and the results are obtained within 15 min. When compared to the method of microagglutination, which was shown to be more specific than the enzyme-linked immunosorbent assay, the ICT had a sensitivity of 98.3% (58 positive sera were tested) and a specificity of 96.5% (58 negative sera were tested) on human sera. On animal sera, the overall sensitivity was 100% (22 positive sera were tested) and specificity was also 100% (70 negative sera were tested). This rapid test preferentially detects IgG antibodies that may occur early in the course of human tularemia, but further evaluation with human sera is important to prove that the ICT can be a valuable field test to support a presumptive diagnosis of tularemia. The ICT can also be a useful tool to monitor successful vaccination with subunit vaccines or live vaccine strains containing lipopolysaccharide (e.g., LVS) and to detect seropositive individuals or animals in outbreak situations or in the context of epidemiologic surveillance programs in areas of endemicity as recently recommended by the World Health Organization.
Development and Preliminary Evaluation of a Rapid Oligochromatographic Assay for Specific Detection of New Human Influenza A H1N1 Virus
Authors: Pérez-Ruiz Mercedes , Navarro-Marí José-María , Bautista-Marín María-Fé , Pedrosa-Corral Irene , Sanbonmatsu-Gámez Sara , Camacho Ana G. , Rojas José , Ruiz-Ortiz Jorge , Rodríguez-Granger Javier , Carrillo José Antonio 
Published: March 2010

ABSTRACT
A new oligochromatographic assay, Speed-Oligo Novel Influenza A H1N1, was designed and optimized for the specific detection of the 2009 influenza A H1N1 virus. The assay is based on a PCR method coupled to detection of PCR products by means of a dipstick device. The target sequence is a 103-bp fragment within the hemagglutinin gene. The analytical sensitivity of the new assay was measured with serial dilutions of a plasmid that contained the target sequence, and we determined that down to one copy per reaction of the plasmid was reliably detected. Diagnostic performance was assessed with 103 RNAs from suspected cases (40 positive and 63 negative results) previously analyzed with a reference real-time PCR technique. All positive cases were confirmed, and no false-positive results were detected with the new assay. No cross-reactions were observed when other viral strains or clinical samples with other respiratory viruses were tested. According to these results, this new assay has 100% sensitivity and specificity. The turnaround time for the whole procedure was 140 min. The assay may be especially useful for the specific detection of 2009 H1N1 virus in laboratories not equipped with real-time PCR instruments.
Identification of Atypical
Rhodococcus
-Like Clinical Isolates as
Dietzia
spp. by 16S rRNA Gene Sequencing
Authors: Pilares Lilian , Agüero Jesús , Vázquez-Boland José A. , Martínez-Martínez Luis , Navas Jesús 
Published: March 2010

ABSTRACT

Rhodococcus equi
and
Dietzia
spp. are closely related actinomycetes that show similar phenotypic properties. In humans,
R. equi
is an opportunistic pathogen associated with severe immunodeficiency.
Dietzia
spp. are environmental bacteria that have been isolated recently from clinical material and are presumptively associated with human infections. During the last 5 years, 15 bacterial isolates from human clinical samples collected at the Hospital Marqués de Valdecilla, Santander, Spain, were identified as
R. equi
by the API Coryne test. 16S rRNA gene sequencing confirmed seven isolates to be true
R. equi
strains, whereas the other eight were identified as members of the genus
Dietzia
, including
Dietzia maris
(four isolates),
Dietzia natronolimnaea
(two isolates), and
Dietzia timorensis
and
Dietzia
sp. (one isolate each). The eight
Dietzia
isolates were highly sensitive to 12 antimicrobial compounds.
Comparison of Inhibitory Mold Agar to Sabouraud Dextrose Agar as a Primary Medium for Isolation of Fungi
Authors: Scognamiglio Theresa , Zinchuk Riva , Gumpeni Pramod , Larone Davise H. 
Published: March 2010

ABSTRACT

Clinical specimens cultured on two selective fungal media, inhibitory mold agar (IMA) and Sabouraud dextrose agar (SDA), were compared with respect to recovery of fungi. Of the 840 fungal isolates recovered, 69.3% grew on both IMA and SDA; 24.9% grew only on IMA; and 5.8% grew only on SDA, showing that IMA is superior (
P
= 0.003).
Reliability of Antimalarial Sensitivity Tests Depends on Drug Mechanisms of Action
Authors: Wein Sharon , Maynadier Marjorie , Tran Van Ba Christophe , Cerdan Rachel , Peyrottes Suzanne , Fraisse Laurent , Vial Henri 
Published: March 2010

ABSTRACT

In vitro
antimalarial activity tests play a pivotal role in malaria drug research or for monitoring drug resistance in field isolates. We applied two isotopic tests, two enzyme-linked immunosorbent assays (ELISA) and the SYBR green I fluorescence-based assay, to test artesunate and chloroquine, the metabolic inhibitors atovaquone and pyrimethamine, our fast-acting choline analog T3/SAR97276, and doxycycline, which has a delayed death profile. Isotopic tests based on hypoxanthine and ethanolamine incorporation are the most reliable tests provided when they are applied after one full 48-h parasite cycle. The SYBR green assay, which measures the DNA content, usually requires 72 h of incubation to obtain reliable results. When delayed death is suspected, specific protocols are required with increasing incubation times up to 96 h. In contrast, both ELISA tests used (pLDH and HRP2) appear to be problematic, leading to disappointing and even erroneous results for molecules that do not share an artesunatelike profile. The reliability of these tests is linked to the mode of action of the drug, and the conditions required to get informative results are hard to predict. Our results suggest some minimal conditions to apply these tests that should give rise to a standard 50% inhibitory concentration, regardless of the mechanism of action of the compounds, and highlight that the most commonly used
in vitro
antimalarial activity tests do not have the same potential. Some of them might not detect the antimalarial potential of new classes of compounds with innovative modes of action, which subsequently could become promising new antimalarial drugs.
Novel Light-Upon-Extension Real-Time PCR Assay for Simultaneous Detection, Quantification, and Genogrouping of Group A Rotavirus
Authors: Nordgren Johan , Bucardo Filemón , Svensson Lennart , Lindgren Per-Eric 
Published: March 2010

ABSTRACT

We have developed a light-upon-extension (LUX) real-time PCR assay for detection, quantification, and genogrouping of group A rotavirus (RV), the most common cause of acute gastroenteritis in children. The LUX system uses a fluorophore attached to one primer and having a self-quenching hairpin structure, making it cost-effective and specific. We designed genogroup-specific primers having different fluorophores, making it possible to differentiate between the two main genogroups of human group A RVs. The assay was applied on clinical stool specimens from Sweden and Central America (
n
= 196) and compared to immunological and conventional PCR assays. The genogrouping ability was further validated against a subset of clinical specimens, which had been genogrouped using monoclonal antibodies. Our real-time PCR assay detected and quantified all positive specimens (
n
= 145) and exhibited higher sensitivity than immunological assays and conventional PCR. The assay exhibited a wide dynamic range, detecting from 5 to >10
7
genes per PCR, resulting in a theoretical lower detection limit of <10,000 viruses per gram of stool. No cross-reaction was observed with specimens containing norovirus, sapovirus, astrovirus, or adenovirus. In total, 22 (15%) of the positive clinical specimens were identified as genogroup I, 122 (84%) were identified as genogroup II, and 1 specimen was found to contain a mix of both genogroups. All genogroup I-positive specimens were associated with capsid glycoprotein 2 (G2). No significant difference in viral load was found between genogroups or geographic region. The detection and quantification, combined with the genogrouping ability, make this assay a valuable tool both for diagnostics and for molecular epidemiological investigations.
Comparison of the Vitek 2 Antifungal Susceptibility System with the Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) Broth Microdilution Reference Methods and with the Sensititre YeastOne and Etest Techniques forIn VitroDetection of Antifungal Resistance in Yeast Isolates
Authors: Cuenca-Estrella Manuel , Gomez-Lopez Alicia , Alastruey-Izquierdo Ana , Bernal-Martinez Leticia , Cuesta Isabel , Buitrago Maria J. , Rodriguez-Tudela Juan L. 
Published: March 2010

ABSTRACTThe commercial technique Vitek 2 system for antifungal susceptibility testing of yeast species was evaluated. A collection of 154 clinical yeast isolates, including amphotericin B- and azole-resistant organisms, was tested. Results were compared with those obtained by the reference procedures of both the CLSI and the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Two other commercial techniques approved for clinical use, the Etest and the Sensititre YeastOne, were included in the comparative exercise as well. The average essential agreement (EA) between the Vitek 2 system and the reference procedures was >95%, comparable with the average EAs observed between the reference procedures and the Sensititre YeastOne and Etest. The EA values were >97% forCandidaspp. and stood at 92% forCryptococcus neoformans. Intraclass correlation coefficients (ICC) between the commercial techniques and the reference procedures were statistically significant (P< 0.01). Percentages of very major errors were 2.6% between Vitek 2 and the EUCAST technique and 1.6% between Vitek 2 and the CLSI technique. The Vitek 2 MIC results were available after 14 to 18 h of incubation for allCandidaspp. (average time to reading, 15.5 h). The Vitek 2 system was shown to be a reliable technique to determine antifungal susceptibility testing of yeast species and a more rapid and easier alternative for clinical laboratories than the procedures developed by either the CLSI or EUCAST.
The Structure of Sindbis Virus Produced from Vertebrate and Invertebrate Hosts as Determined by Small-Angle Neutron Scattering
Authors: He Lilin , Piper Amanda , Meilleur Flora , Myles Dean A. A. , Hernandez Raquel , Brown Dennis T. , Heller William T. 
Published: March 2010

ABSTRACT
The complex natural cycle of vectored viruses that transition between host species, such as between insects and mammals, makes understanding the full life cycle of the virus an incredibly complex problem. Sindbis virus, an arbovirus and prototypic alphavirus having an inner protein shell and an outer glycoprotein coat separated by a lipid membrane, is one example of a vectored virus that transitions between vertebrate and insect hosts. While evidence of host-specific differences in Sindbis virus has been observed, no work has been performed to characterize the impact of the host species on the structure of the virus. Here, we report the first study of the structural differences between Sindbis viruses grown in mammalian and insect cells, which were determined by small-angle neutron scattering (SANS), a nondestructive technique that did not decrease the infectivity of the Sindbis virus particles studied. The scattering data and modeling showed that, while the radial position of the lipid bilayer did not change significantly, it was possible to conclude that it did have significantly more cholesterol when the virus was grown in mammalian cells. Additionally, the outer protein coat was found to be more extended in the mammalian Sindbis virus. The SANS data also demonstrated that the RNA and nucleocapsid protein share a closer interaction in the mammalian-cell-grown virus than in the virus from insect cells.
Human Papillomaviruses Modulate Expression of MicroRNA 203 upon Epithelial Differentiation to Control Levels of p63 Proteins
Authors: Melar-New Marta , Laimins Laimonis A. 
Published: March 2010

ABSTRACT
Human papillomaviruses (HPV) link their life cycles to epithelial differentiation and induce productive replication of viral DNA in suprabasal cells. Viral-DNA amplification requires cells to remain active in the cell cycle upon differentiation. This is in contrast to normal cells, which lose proliferative capability upon differentiation. One factor that negatively regulates proliferative capability upon differentiation is microRNA 203 (miR-203), which is expressed primarily in suprabasal epithelial cells. Although HPVs do not encode their own microRNAs (miRNAs), they modulate expression of cellular miRNAs to regulate the activities of cellular proteins. We show that the HPV E7 protein downregulates miR-203 expression upon differentiation, which may occur through the mitogen-activated protein (MAP) kinase/protein kinase C (PKC) pathway. One target of miR-203 is the p63 family of transcription factors, and we demonstrate that HPV-positive cells maintain significantly higher levels of these factors upon differentiation than do normal keratinocytes. Several downstream targets of p63, CARM-1, p21, and Bax, were also increased in E7-expressing cells, and their levels were inversely correlated with amounts of miR-203. Introduction of expression vectors for miR-203 into keratinocytes that stably maintain HPV episomes resulted in short-term elevation of HPV genome copy numbers, but these were rapidly lost upon subsequent passage. When HPV-positive cells expressing high levels of miR-203 were induced to differentiate in methylcellulose, impaired genome amplification was observed. We conclude that high levels of miR-203 are inhibitory to HPV amplification and that HPV proteins act to suppress expression of this microRNA to allow productive replication in differentiating cells.
Visualizing Viral Dissemination in the Mouse Nervous System, Using a Green Fluorescent Protein-Expressing Borna Disease Virus Vector
Authors: Ackermann Andreas , Guelzow Timo , Staeheli Peter , Schneider Urs , Heimrich Bernd 
Published: March 2010

ABSTRACT
Borna disease virus (BDV) frequently persists in the brain of infected animals. To analyze viral dissemination in the mouse nervous system, we generated a mouse-adapted virus that expresses green fluorescent protein (GFP). This viral vector supported GFP expression for up to 150 days and possessed an extraordinary staining capacity, visualizing complete dendritic arbors as well as individual axonal fibers of infected neurons. GFP-positive cells were first detected in cortical areas from where the virus disseminated through the entire central nervous system (CNS). Late in infection, GFP expression was found in the sciatic nerve, demonstrating viral spread from the central to the peripheral nervous system.
Comparative Studies of Lepidopteran Baculovirus-Specific Protein FP25K: Development of a Novel
Bombyx mori
Nucleopolyhedrovirus-Based Vector with a Modified
fp25K
Gene
Authors: Nakanishi Tadashi , Goto Chie , Kobayashi Michihiro , Kang WonKyung , Suzuki Takehiro , Dohmae Naoshi , Matsumoto Shogo , Shimada Toru , Katsuma Susumu 
Published: March 2010

ABSTRACT

Lepidopteran baculovirus-specific protein FP25K performs many roles during the infection cycle, including functions in the production of occlusion bodies (OBs) and budded viruses (BVs), oral infection, and postmortem host degradation. To explore the common and specific functions of FP25K proteins among lepidopteran baculoviruses, we performed comparative analyses of FP25K proteins from group I and group II nucleopolyhedroviruses (NPVs) and granulovirus (GV). Using recombinant
Bombyx mori
NPVs (BmNPVs), we showed that the FP25Ks from NPVs were able to eliminate all the phenotypic defects observed in an infection with a BmNPV mutant lacking functional
fp25K
but that FP25K from GV did not show abilities to recover oral infectivity and postmortem host degradation. We also observed that introduction of
Autographa californica
multiple NPV (AcMNPV)
fp25K
into the BmNPV genome enhanced OB and BV production. According to these results, we generated a novel BmNPV-based expression vector with AcMNPV
fp25K
and examined its potential in BmN cells and
B. mori
larvae. Our results showed that the introduction of AcMNPV
fp25K
significantly increases the expression of foreign gene products in cultured cells and shortens the time for obtaining the secreted recombinant proteins from larval hemolymph.
Influenza Virus M2 Ion Channel Protein Is Necessary for Filamentous Virion Formation
Authors: Rossman Jeremy S. , Jing Xianghong , Leser George P. , Balannik Victoria , Pinto Lawrence H. , Lamb Robert A. 
Published: March 2010

ABSTRACT
Influenza A virus buds from cells as spherical (∼100-nm diameter) and filamentous (∼100 nm × 2 to 20 μm) virions. Previous work has determined that the matrix protein (M1) confers the ability of the virus to form filaments; however, additional work has suggested that the influenza virus M2 integral membrane protein also plays a role in viral filament formation. In examining the role of the M2 protein in filament formation, we observed that the cytoplasmic tail of M2 contains several sites that are essential for filament formation. Additionally, whereas M2 is a nonraft protein, expression of other viral proteins in the context of influenza virus infection leads to the colocalization of M2 with sites of virus budding and lipid raft domains. We found that an amphipathic helix located within the M2 cytoplasmic tail is able to bind cholesterol, and we speculate that M2 cholesterol binding is essential for both filament formation and the stability of existing viral filaments.
Anti-gp41 Antibodies Cloned from HIV-Infected Patients with Broadly Neutralizing Serologic Activity
Authors: Pietzsch John , Scheid Johannes F. , Mouquet Hugo , Seaman Michael S. , Broder Christopher C. , Nussenzweig Michel C. 
Published: March 2010

ABSTRACT
Most HIV-infected individuals develop antibodies to the gp120 and gp41 components of the viral spike; however, only a fraction of these individuals mount a broadly neutralizing serum response against HIV. We have cloned anti-HIV antibodies from the memory B-cell compartment of six individuals with variable viral loads and high titers of broadly neutralizing antibodies. Here, we report on the features of the anti-gp41 response in these patients. Competition experiments with previously characterized antibodies targeting defined epitopes on the gp41 ectodomain showed antibodies directed against the “immunodominant region” (cluster I), the carboxy-terminal heptad repeat (cluster II), and the membrane-proximal external region (cluster IV). On the other hand, antibodies directed against the amino-terminal part of the molecule, including the fusion peptide, polar region, and the N-terminal heptad repeat, were not detected. When all patients' data were combined, unique B-cell clones targeting cluster I, II, and IV accounted for 32%, 49%, and 53% of all anti-gp41-reactive B cells, respectively; therefore, no single region was truly immunodominant. Finally, although we found no new neutralizing epitopes or HIV-1-neutralizing activity by any of the gp41 antibodies at concentrations of up to 50 μg/ml, high concentrations of 7 out of 15 anti-cluster I antibodies neutralized tier 2 viruses.
A Human Herpesvirus MicroRNA Inhibits p21 Expression and Attenuates p21-Mediated Cell Cycle Arrest
Authors: Gottwein Eva , Cullen Bryan R. 
Published: March 2010

ABSTRACT
The oncogenic human gammaherpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) expresses 12 viral microRNAs (miRNAs) in latently infected cells. Here, we report that cellular mRNAs encoding the cellular cyclin-dependent kinase inhibitor p21, a key inducer of cell cycle arrest, are direct targets for KSHV miR-K1. Ectopically expressed KSHV miR-K1 specifically inhibited the expression of endogenous p21 in KSHV-negative cells and strongly attenuated the cell cycle arrest that normally occurs upon p53 activation, yet miR-K1 did not prevent the induction of other p53-responsive genes. Stable knockdown of miR-K1 in latently KSHV-infected human primary effusion lymphoma (PEL) B cells revealed a derepression of p21 expression and enhanced cell cycle arrest following activation of p53. Our data demonstrate that miR-K1 represses the expression of p21, a protein with known tumor suppressor functions, and suggest that this KSHV miRNA is likely to contribute to the oncogenic potential of this opportunistic viral pathogen.