bluuebunny/crossref_metadata_2025_split huggingface dataset:
Evaluation of Molecular Tools for Detection and Drug Susceptibility Testing of
Mycobacterium tuberculosis
in Stool Specimens from Patients with Pulmonary Tuberculosis
Authors: Cordova Julianna , Shiloh Ron , Gilman Robert H. , Sheen Patricia , Martin Laura , Arenas Fanny , Caviedes Luz , Kawai Vivian , Soto Giselle , Williams Diana L. , Zimic Mirko , Escombe A. Roderick , Evans Carlton A. 
Published: March 2010

ABSTRACT

Pulmonary tuberculosis diagnosis is difficult when patients cannot produce sputum. Most sputum is swallowed, and tuberculosis DNA can survive intestinal transit. We therefore evaluated molecular testing of stool specimens for detecting tuberculosis originating from the lungs. Paired stool and sputum samples (
n
= 159) were collected from 89 patients with pulmonary tuberculosis. Control stool samples (
n
= 47) were collected from patients without tuberculosis symptoms. Two techniques for DNA extraction from stool samples were compared, and the diagnostic accuracy of the PCR in stool was compared with the accuracy of sputum testing by PCR, microscopy, and culture. A heminested IS
6110
-PCR was used for tuberculosis detection, and IS
6110
-PCR-positive stool samples then underwent rifampin sensitivity testing by universal heteroduplex generator PCR (heteroduplex-PCR) assay. For newly diagnosed pulmonary tuberculosis patients, stool IS
6110
-PCR had 86% sensitivity and 100% specificity compared with results obtained by sputum culture, and stool PCR had similar sensitivities for HIV-positive and HIV-negative patients (
P
= 0.3). DNA extraction with commercially available spin columns yielded greater stool PCR sensitivity than DNA extraction with the in-house Chelex technique (
P
= 0.007). Stool heteroduplex-PCR had 98% agreement with the sputum culture determinations of rifampin resistance and multidrug resistance. Tuberculosis detection and drug susceptibility testing by stool PCR took 1 to 2 days compared with an average of 9 weeks to obain those results by traditional culture-based testing. Stool PCR was more sensitive than sputum microscopy and remained positive for most patients for more than 1 week of treatment. In conclusion, stool PCR is a sensitive, specific, and rapid technique for the diagnosis and drug susceptibility testing of pulmonary tuberculosis and should be considered when sputum samples are unavailable.
Comparison of the Plating Efficiencies and Shelf Lives of Three Different Commercial Buffered Charcoal Yeast Extract Media Supplemented with α-Ketoglutaric Acid
Authors: Edelstein Paul H. , Edelstein Martha A. C. 
Published: March 2010

ABSTRACT

The plating efficiencies and shelf lives of locally made buffered charcoal yeast extract medium supplemented with α-ketoglutaric acid (BCYEα) were compared to those of media made by BD, Hardy, and Remel. Lung homogenates from guinea pigs infected with
Legionella pneumophila
were plated monthly onto different medium lots. All media performed equally well and had shelf lives of at least 12 months.
Comparison of Self-Collected and Physician-Collected Vaginal Swabs for Microbiome Analysis
Authors: Forney Larry J. , Gajer Pawel , Williams Christopher J. , Schneider G. Maria , Koenig Sara S. K. , McCulle Stacey L. , Karlebach Shara , Brotman Rebecca M. , Davis Catherine C. , Ault Kevin , Ravel Jacques 
Published: March 2010

ABSTRACT
To our knowledge, no data are available on whether the microbial species composition and abundance sampled with self-collected vaginal swabs are comparable to those of swabs collected by clinicians. Twenty healthy women were recruited to the study during a routine gynecological visit. Eligible women were between 18 and 40 years old with regular menstrual cycles. Participants self-collected a vaginal swab using a standardized protocol and then were examined by a physician, who collected an additional five swabs from the lateral wall of the mid-vagina. In this study, the self-collected and three physician-obtained swabs were analyzed and compared using terminal restriction fragment length polymorphism and sequence analyses of the 16S rRNA genes. Vaginal microbial community comparative statistical analyses of both T-RFLP and 16S rRNA gene sequence datasets revealed that self-collected vaginal swabs sampled the same microbial diversity as physician collected swabs of the mid-vagina. These findings enable large-scale, field-based studies of the vaginal microbiome.
Replacing Reverse Line Blot Hybridization Spoligotyping of the
Mycobacterium tuberculosis
Complex
Authors: Honisch Christiane , Mosko Michael , Arnold Catherine , Gharbia Saheer E. , Diel Roland , Niemann Stefan 
Published: March 2010

ABSTRACT

Spoligotyping is a tool for the molecular characterization/typing of
Mycobacterium tuberculosis
complex (MTBC) strains based on target sequences (spacers) in the direct repeat (DR) region (14). The standard spoligotyping assay involves the hybridization of amplified sample DNA to nylon membrane-immobilized oligonucleotides whose sequences are representative of 43 spacer regions. Variations in the number of spacers as a result of deletions of adjacent blocks of repetitive units allow the differentiation of clinical isolates. In the present study, we developed a new multiplexed primer extension-based spoligotyping assay using automated matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) that improves the classical reverse line blot hybridization assay with respect to reproducibility, throughput, process flow, ease of use, and data analysis. Validation of the MALDI-TOF MS-based spoligotyping assay with two sample sets with a total of 326 samples resulted in 96.6% concordance (315/326) when the full spoligotype patterns were compared with the results of standard spoligotyping and 99.9% concordance when the results were compared with those of individual primer extension assays. Ten strains (including two
Mycobacterium canettii
strains) showed discordant results with one or two spacer differences from the membrane-based spoligotyping result. Most discordant samples were identified to be the result of ambiguities in the interpretation of weak hybridization signals in the reverse line blot assay and sequence variations in the spacer regions. We established a new automated primer extension assay and successfully validated it for use for the routine typing of MTBC strains in the research and public health laboratory environments. The present multiplex levels of up to 30 are extendable and allow the additional incorporation of controls and antibiotic resistance markers.
The
De Novo
Methyltransferases DNMT3a and DNMT3b Target the Murine Gammaherpesvirus Immediate-Early Gene 50 Promoter during Establishment of Latency
Authors: Gray Kathleen S. , Forrest J. Craig , Speck Samuel H. 
Published: March 2010

ABSTRACT

The role of epigenetic modifications in the regulation of gammaherpesvirus latency has been a subject of active study for more than 20 years. DNA methylation, associated with transcriptional silencing in mammalian genomes, has been shown to be an important mechanism in the transcriptional control of several key gammaherpesvirus genes. In particular, DNA methylation of the functionally conserved immediate-early replication and transcription activator (RTA) has been shown to regulate Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus Rta expression. Here we demonstrate that the murine gammaherpesvirus (MHV68) homolog, encoded by gene 50, is also subject to direct repression by DNA methylation, both
in vitro
and
in vivo
. We observed that the treatment of latently MHV68-infected B-cell lines with a methyltransferase inhibitor induced virus reactivation. In addition, we show that the methylation of the recently characterized distal gene 50 promoter represses activity in a murine macrophage cell line. To evaluate the role of
de novo
methyltransferases (DNMTs) in the establishment of these methylation marks, we infected mice in which conditional DNMT3a and DNMT3b alleles were selectively deleted in B lymphocytes. DNMT3a/DNMT3b-deficient B cells were phenotypically normal, displaying no obvious compromise in cell surface marker expression or antibody production either in naïve mice or in the context of nonviral and viral immunogens. However, mice lacking functional DNMT3a and DNMT3b in B cells exhibited hallmarks of deregulated MHV68 lytic replication, including increased splenomegaly and the presence of infectious virus in the spleen at day 18 following infection. In addition, total gene 50 transcript levels were elevated in the spleens of these mice at day 18, which correlated with the hypomethylation of the distal gene 50 promoter. However, by day 42 postinfection, aberrant virus replication was resolved, and we observed wild-type frequencies of viral genome-positive splenocytes in mice lacking functional DNMT3a and DNMT3b in B lymphocytes. The latter correlated with increased CpG methylation in the distal gene 50 promoter, which was restored to levels similar to those of littermate controls harboring functional DNMT3a and DNMT3b alleles in B lymphocytes, suggesting the existence of an alternative mechanism for the
de novo
methylation of the MHV68 genome. Importantly, this DNMT3a/DNMT3b-independent methylation appeared to be targeted specifically to the gene 50 promoter, as we observed that the promoters for MHV68 gene 72 (v-cyclin) and M11 (v-bcl2) remained hypomethylated at day 42 postinfection. Taken together, these data provide the first evidence of the importance of DNA methylation in regulating gammaherpesvirus RTA/gene 50 transcription during virus infection
in vivo
and provide insight into the hierarchy of host machinery required to establish this modification.
Human Cytomegalovirus IE2 86 and IE2 40 Proteins Differentially Regulate UL84 Protein Expression Posttranscriptionally in the Absence of Other Viral Gene Products
Authors: Sanders Rebecca L. , Spector Deborah H. 
Published: March 2010

ABSTRACTIt has previously been demonstrated that, during human cytomegalovirus infection, the viral IE2 86 and IE2 40 proteins are both important for the expression of an early-late viral protein, UL84. Here, we show that expression of the UL84 protein is enhanced upon cotransfection with either IE2 86 or IE2 40, although IE2 40 appears to play a more important role. The UL84 protein levels are tightly linked to the amount of IE2 40 present, but this does not appear to be true for IE2 86. RNA remains constant for all corresponding proteins, indicating posttranscriptional regulation of UL84. The first 105 amino acids of UL84 are necessary and sufficient for this phenotype, and this region is also required for an interaction with IE2 86 and IE2 40. Treatment with proteasome inhibitors shows that UL84 exhibits some proteasome-dependent degradation, and UL84 is not protected against this degradation when coexpressed with IE2 86 or IE2 40. UL84 also exhibits an inhibitory effect on IE2 86 and IE2 40 protein levels in these cotransfection assays. Further, we show that the amino acid sequence of UL84 is important for the enhancement governed by IE2 40. These results indicate that IE2 86, IE2 40, and UL84 serve to regulate protein expression in a posttranscriptional fashion and that this regulation is independent of other viral proteins.
Phenotypic and Functional Profile of HIV-Inhibitory CD8 T Cells Elicited by Natural Infection and Heterologous Prime/Boost Vaccination
Authors: Freel Stephanie A. , Lamoreaux Laurie , Chattopadhyay Pratip K. , Saunders Kevin , Zarkowsky David , Overman R. Glenn , Ochsenbauer Christina , Edmonds Tara G. , Kappes John C. , Cunningham Coleen K. , Denny Thomas N. , Weinhold Kent J. , Ferrari Guido , Haynes Barton F. , Koup Richard A. , Graham Barney S. , Roederer Mario , Tomaras Georgia D. 
Published: March 2010

ABSTRACT

Control of HIV-1 replication following nonsterilizing HIV-1 vaccination could be achieved by vaccine-elicited CD8
+
T-cell-mediated antiviral activity. To date, neither the functional nor the phenotypic profiles of CD8
+
T cells capable of this activity are clearly understood; consequently, little is known regarding the ability of vaccine strategies to elicit them. We used multiparameter flow cytometry and viable cell sorts from phenotypically defined CD8
+
T-cell subsets in combination with a highly standardized virus inhibition assay to evaluate CD8
+
T-cell-mediated inhibition of viral replication. Here we show that vaccination against HIV-1 Env and Gag-Pol by DNA priming followed by recombinant adenovirus type 5 (rAd5) boosting elicited CD8
+
T-cell-mediated antiviral activity against several viruses with either lab-adapted or transmitted virus envelopes. As it did for chronically infected virus controllers, this activity correlated with HIV-1-specific CD107a or macrophage inflammatory protein 1β (MIP-1β) expression from HIV-1-specific T cells. Moreover, for vaccinees or virus controllers, purified memory CD8
+
T cells from a wide range of differentiation stages were capable of significantly inhibiting virus replication. Our data define attributes of an antiviral CD8
+
T-cell response that may be optimized in the search for an efficacious HIV-1 vaccine.
Acceleration of Hepatitis C Virus Envelope Evolution in Humans Is Consistent with Progressive Humoral Immune Selection during the Transition from Acute to Chronic Infection
Authors: Liu Lin , Fisher Brian E. , Dowd Kimberly A. , Astemborski Jacquie , Cox Andrea L. , Ray Stuart C. 
Published: March 2010

ABSTRACT

During the transition from acute to chronic infection in individuals persistently infected with hepatitis C virus (HCV), cellular responses initiate within the first 6 months of primary infection and collapse thereafter, whereas humoral responses activate later during the chronic phase. Whether and how this deviation of immune responses specifically influences HCV evolution are unknown. To determine the pattern of HCV evolution during this critical period, we conducted extensive sequence analysis on annual clonal hemigenomic sequences for up to 3 years in six well-characterized subjects, using statistical methods that accounted for repeated measures. Significantly different evolutionary rates were observed in envelope versus nonenvelope genes, with an increasing rate of nonsynonymous change (
dN
) in envelope genes and a stable
dN
in nonenvelope genes (
P
= 0.006). The ratio of the envelope to nonenvelope nonsynonymous rate increased from 2 in year 1 to 5 in years 2 and 3. Centripetal changes (reversions toward matching of the worldwide subtype 1a consensus sequence) were frequently observed during the 3-year transition from acute infection to chronicity, even in the presence of neutralizing antibody (NAb) pressure. Remarkably, sequences of hypervariable region 1 (HVR1) remained stable for up to 21 months in the absence of NAb pressure in one subject, followed by rapid changes that were temporally associated with the detection of NAb responses, which strongly suggests that HVR1 evolution is shaped by NAb pressure. These data provide the first systematic estimates of HCV evolutionary rates in multiple genes during early infection
in vivo
and provide additional evidence for deterministic, rather than random, evolution of HCV.
A Role for the C Terminus of Mopeia Virus Nucleoprotein in Its Incorporation into Z Protein-Induced Virus-Like Particles
Authors: Shtanko Olena , Imai Masaki , Goto Hideo , Lukashevich Igor S. , Neumann Gabriele , Watanabe Tokiko , Kawaoka Yoshihiro 
Published: March 2010

ABSTRACT
Arenaviruses are enveloped, negative-strand RNA viruses. For several arenaviruses, virus-like particle (VLP) formation requires the viral matrix Z protein. However, the mechanism by which viral ribonucleoprotein complexes are incorporated into virions is poorly understood. Here, we show that the expression of the Z protein and nucleoprotein (NP) of Mopeia virus, a close relative of the pathogenic Lassa virus, resulted in the highly selective incorporation of the NP protein into Z protein-induced VLPs. Moreover, the Z protein promoted the association of NP with cellular membranes, suggesting that the association of NP, Z, and the cellular membranes may facilitate the efficient incorporation of NP into VLPs. By employing a series of NP deletion constructs and testing their VLP incorporation, we further demonstrated an important role for the C-terminal half of NP in its incorporation into VLPs.
Influenza H1N1 A/Solomon Island/3/06 Virus Receptor Binding Specificity Correlates with Virus Pathogenicity, Antigenicity, and Immunogenicity in Ferrets
Authors: Xu Qi , Wang Weijia , Cheng Xing , Zengel James , Jin Hong 
Published: March 2010

ABSTRACT
Influenza viruses attach to cells via a sialic acid moiety (sialic acid receptor) that is α2-3 linked or α2-6 linked to galactose (α2-3SAL or α2-6SAL); sialic acid acts as a receptor for the virus. Using lectin staining, we demonstrated that the α2-6SAL configuration is predominant in the respiratory tract of ferrets, including trachea, bronchus, and lung alveolus tissues. Recombinant wild-type (rWT) influenza A/Solomon Island/3/06 (SI06) (H1N1) viruses were constructed to assess the impact of the hemagglutinin (HA) variations (amino acids 190 or 226) identified in natural variants on virus replication in the upper and lower respiratory tract of ferrets, as well as virus antigenicity and immunogenicity. A single amino acid change at residue 226 (from Gln to Arg) in the HA of SI06 resulted in the complete loss of binding to α2-6SAL and a concomitant loss of the virus's ability to replicate in the lower respiratory tract of ferrets. In contrast, the virus with Gln226 in the HA protein has a receptor binding preference for α2-6SAL and replicates efficiently in the lungs. There was a good correlation between viral replication in the lungs of ferrets and disease symptoms. In addition, we also showed that the 190 and 226 residues affected viral antigenicity and immunogenicity. Our data emphasize the necessity of thoroughly assessing wild-type influenza viruses for their suitability as reference strains and for carefully selecting the HA antigen for vaccine production during annual influenza vaccine evaluation processes.
Measles Virus-Induced Immunosuppression in SLAM Knock-In Mice
Authors: Koga Ritsuko , Ohno Shinji , Ikegame Satoshi , Yanagi Yusuke 
Published: March 2010

ABSTRACT
Measles virus (MV) causes transient severe immunosuppression in patients, which may lead to secondary viral and bacterial infections, largely accounting for measles-related morbidity and mortality. MV is known to infect immune cells by using the human signaling lymphocyte activation molecule (SLAM; also called CD150) as a cellular receptor, but the mechanism by which MV causes immunosuppression is not well understood. We show that MV infection of SLAM knock-in mice, in which the V domain of mouse SLAM was replaced by the V domain of human SLAM, crossed with alpha/beta-interferon receptor knockout mice, reproduced many immunological alterations observed in human patients. These included lymphopenia, inhibition of T-cell proliferation and antibody production, increased production of the Th2 cytokine interleukin-4 (IL-4) and the immunosuppressive cytokine IL-10, and suppression of contact hypersensitivity. Gross redistribution of lymphocytes among lymphoid tissues was not apparent in infected mice, nor was an increase of regulatory T cells. The numbers of lymphocytes in lymph nodes remained almost unchanged after MV infection, despite enhanced apoptosis, suggesting that lymph nodes were replenished with lymphocytes from the peripheral blood, which may have contributed to the observed lymphopenia in the spleen. Blocking of IL-10 by use of an anti-IL-10 receptor antibody ameliorated suppression of contact hypersensitivity in infected mice. These results indicate that SLAM knock-in mice lacking the expression of the alpha/beta-interferon receptor serve as a useful small animal model with which to elucidate MV-induced immunosuppression.
Gene Expression Profiling Indicates the Roles of Host Oxidative Stress, Apoptosis, Lipid Metabolism, and Intracellular Transport Genes in the Replication of Hepatitis C Virus
Authors: Blackham Samantha , Baillie Andrew , Al-Hababi Fadel , Remlinger Katja , You Shihyun , Hamatake Robert , McGarvey Michael J. 
Published: March 2010

ABSTRACTHepatitis C virus (HCV) is a leading cause of chronic liver disease. The identification and characterization of key host cellular factors that play a role in the HCV replication cycle are important for the understanding of disease pathogenesis and the identification of novel antiviral therapeutic targets. Gene expression profiling of JFH-1-infected Huh7 cells by microarray analysis was performed to identify host cellular genes that are transcriptionally regulated by infection. The expression of host genes involved in cellular defense mechanisms (apoptosis, proliferation, and antioxidant responses), cellular metabolism (lipid and protein metabolism), and intracellular transport (vesicle trafficking and cytoskeleton regulation) was significantly altered by HCV infection. The gene expression patterns identified provide insight into the potential mechanisms that contribute to HCV-associated pathogenesis. These include an increase in proinflammatory and proapoptotic signaling and a decrease in the antioxidant response pathways of the infected cell. To investigate whether any of the host genes regulated by infection were required by HCV during replication, small interfering RNA (siRNA) silencing of host gene expression in HCV-infected cells was performed. Decreasing the expression of host genes involved in lipid metabolism (TXNIP and CYP1A1 genes) and intracellular transport (RAB33b and ABLIM3 genes) reduced the replication and secretion of HCV, indicating that they may be important factors for the virus replication cycle. These results show that major changes in the expression of many different genes in target cells may be crucial in determining the outcome of HCV infection.
Analysis of Apoptosis of Memory T Cells and Dendritic Cells during the Early Stages of Viral Infection or Exposure to Toll-Like Receptor Agonists
Authors: Bahl Kapil , Hüebner Anette , Davis Roger J. , Welsh Raymond M. 
Published: March 2010

ABSTRACTProfound type I interferon (IFN-I)-dependent attrition of memory CD8 and CD4 T cells occurs early during many infections. It is dramatic at 2 to 4 days following lymphocytic choriomeningitis virus (LCMV) infection of mice and can be elicited by the IFN-inducing Toll receptor agonist poly(I:C). We show that this attrition occurs in many organs, indicating that it is due to T cell loss rather than redistribution. This loss correlated with elevated intracellular staining of T cellsex vivofor activated caspases but with only low levels ofex vivostaining with annexin V, probably due to the rapid clearance of apoptotic cellsin vivo. Instead, a high frequency of annexin V-reactive CD8α+dendritic cells (DCs), which are known to be highly phagocytic, accumulated in the spleen as the memory T cell populations disappeared. After shortin vitroincubation, memory phenotype T cells isolated from LCMV-infected mice (day 3) or mice treated with poly(I:C) (12 h) displayed substantial DNA fragmentation, as detected by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, compared to T cells isolated from uninfected mice, indicating a role for apoptosis in the memory T cell attrition. This apoptosis of memory CD8 T cells early during LCMV infection was reduced in mice lacking the proapoptotic molecule Bim. Evidence is presented showing that high levels of T cell attrition, as found in young mice, correlate with reduced immunodomination by cross-reactive memory cells.