bluuebunny/crossref_metadata_2025_split huggingface dataset:
Highly Sensitive Assay for Detection of Enterovirus in Clinical Specimens by Reverse Transcription-PCR with an Armored RNA Internal Control
Authors: Beld Marcel , Minnaar René , Weel Jan , Sol Cees , Damen Marjolein , van der Avoort Harry , Wertheim-van Dillen Pauline , van Breda Alex , Boom René 
Published: July 2004

ABSTRACT
The objective of the present study was the development of a diagnostic reverse transcription (RT)-PCR for the specific detection of enterovirus (EV) RNA in clinical specimens controlled by an internal control (IC) RNA. The IC RNA contains the same primer binding sites as EV RNA but has a different probe region. The IC RNA was packaged into an MS2 phage core particle (armored) and was added to the clinical sample to allow monitoring of both extraction efficiency and RT-PCR efficiency. Serial dilutions of the IC RNA were made, and the detection limit of the RT-PCR was tested in a background of EV RNA-negative cerebrospinal fluid. The sensitivity and specificity of the RT-PCR assay were tested by using all 64 known EV serotypes, several non-EV serotypes, and two Quality Control for Molecular Diagnostics (QCMD) Program EV proficiency panels from 2001 and 2002. In total, 322 clinical specimens were tested by RT-PCR, and to establish the clinical utility of the RT-PCR, a comparison of the results of viral culture and RT-PCR was done with 87 clinical specimens. The lower limit of sensitivity was reached at about 150 copies of IC RNA/ml. All 64 EV serotypes were positive, while all non-EV serotypes were negative. All culture-positive samples of the 2001 QCMD proficiency panel (according to the 50% tissue culture infective doses per milliliter) were positive by RT-PCR. Invalid results, i.e., negativity for both EV RNA and IC RNA, due to inhibition of RT-PCR were observed for 33.3% of the members of the 2002 QCMD proficiency panel and 3.1% of the clinical specimens. Inhibition of RT-PCR could be relieved by the addition of 400 ng of bovine α-casein per μl to both the RT reaction mixture and the PCR mixture. With this optimized protocol, the results for all samples of the 2002 QCMD proficiency panel and all clinical specimens except one fecal sample (0.3%) were valid. Evaluation of the clinical samples demonstrated that EV infection could be detected in 12 of 87 samples (13.8%) by RT-PCR, while viral culture was negative. Our data show that the RT-PCR with armored IC RNA offers a very reliable and rapid diagnostic tool for the detection of EV in clinical specimens and that the addition of bovine α-casein relieved inhibition of the RT-PCR for 99.7% of clinical specimens.
Comparative Evaluation of the New Version of the INNO-LiPA Mycobacteria and GenoType Mycobacterium Assays for Identification of
Mycobacterium
Species from MB/BacT Liquid Cultures Artificially Inoculated with Mycobacterial Strains
Authors: Padilla Eduardo , González Victoria , Manterola Jose María , Pérez Andrés , Quesada María Dolores , Gordillo Sergio , Vilaplana Cristina , Pallarés María Angeles , Molinos Sonia , Sánchez María Dolores , Ausina Vicente 
Published: July 2004

ABSTRACT

The performance of two DNA line probe assays, a new version of INNO-LiPA Mycobacteria (Innogenetics, Ghent, Belgium) and the GenoType Mycobacterium (Hain Diagnostika, Nehren, Germany), were evaluated for identification of mycobacterial species isolated from liquid cultures. Both tests are based on a PCR technique and designed for simultaneous identification of different mycobacterial species by reverse hybridization and line probe technology. The INNO-LiPA Mycobacteria v2 targeting the 16S-23S rRNA gene spacer region was developed for the simultaneous identification of 16 different mycobacterial species. The GenoType Mycobacterium, which targets the 23S rRNA gene, allows simultaneous identification of 13 mycobacterial species. Both tests were evaluated on 110 mycobacterial strains belonging to 22 different mycobacterial species (20 reference strains, 83 clinical strains, and 4
Mycobacterium kansasii
strains isolated from tap water) that were previously inoculated into MB/BacT bottles. The sensitivity of both methods, defined as the number of positive results obtained with the
Mycobacterium
genus probe together with an interpretable result on the number of samples tested was 110 of 110 (100%) for INNO-LiPA and 102 of 110 (92.7%) for GenoType. For samples with interpretable results, INNO-LiPA was able to correctly identify 109 of 110 samples (99.1%), whereas the GenoType correctly identified 100 of 102 samples (98.0%). Both tests were easy to perform, rapid, and reliable when applied to mycobacterial identification directly from MB/BacT bottles.
Molecular Epidemiology of
Serratia marcescens
in Two Hospitals in Danzig, Poland, over a 5-Year Period
Authors: Naumiuk Łukasz , Baraniak Anna , Gniadkowski Marek , Krawczyk Beata , Rybak Bartosz , Sadowy Ewa , Samet Alfred , Kur Józef 
Published: July 2004

ABSTRACT

The history of the
Serratia marcescens
population in two hospitals in Danzig, Poland, over a 5-year period was analyzed in a study that combined MIC evaluation, typing by randomly amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis, and analysis of extended-spectrum β-lactamases (ESBLs). We analyzed 354 isolates collected from 341 patients in two teaching hospitals in Danzig, Poland, from 1996 to 2000. The antimicrobial susceptibility profiles varied greatly, and for resistance to newer β-lactams, probable AmpC cephalosporinase derepression and ESBL production occurred in about 23 and 19% of the isolates, respectively. RAPD typing, by which 69 types were discerned altogether, revealed a high degree of clonal diversity among the populations. However, the four most prevalent types were highly predominant, grouping approximately 71% of the isolates studied. These clones were observed in the two hospitals and were strong contributors to both outbreaks and the background of endemicity of the
S. marcescens
infections. Some of the strains that were not so widely spread (12 RAPD types; ∼14% of the isolates) were responsible for several smaller outbreaks, and the remaining isolates represented unique RAPD types (53 types; ∼15% of the isolates) and were probably sporadic introductions from other environments. ESBLs were identified in several different clones, and some of these had most likely already been introduced into the hospitals as ESBL producers, whereas the others acquired the ESBL-encoding genes from other enterobacterial strains in these environments. The CTX-M-3 enzyme, which is widely observed in Poland, was the most common ESBL type among the
S. marcescens
isolates, followed by TEM-47 and SHV-5. The complex epidemiology of ESBLs, especially in 1999 and 2000, must have arisen from the introduction of ESBL producers from other centers, their clonal dissemination, and the constant penetration of the
S. marcescens
populations with plasmids with ESBL genes. Multiple
S. marcescens
isolates were obtained from 11 patients, who probably represented both patients with recolonizations and reinfections and patients with recurrences of infections with the evolution of the strain's susceptibility.
Geographic Variation in the Susceptibilities of Invasive Isolates of
Candida glabrata
to Seven Systemically Active Antifungal Agents: a Global Assessment from the ARTEMIS Antifungal Surveillance Program Conducted in 2001 and 2002
Authors: Pfaller M. A. , Messer S. A. , Boyken L. , Tendolkar S. , Hollis R. J. , Diekema D. J. 
Published: July 2004

ABSTRACT

We examined the susceptibilities to amphotericin B, flucytosine, fluconazole, posaconazole, ravuconazole, voriconazole, and caspofungin of 601 invasive isolates of
Candida glabrata
and grouped the isolates by geographic location: North America (331 isolates), Latin America (58 isolates), Europe (135 isolates), and Asia-Pacific (77 isolates). Caspofungin (MIC at which 90% of isolates tested are susceptible [MIC
90
], 0.12 μg/ml; 100% of strains are susceptible [S] at a MIC of ≤1 μg/ml) and flucytosine (MIC
90
, 0.12 μg/ml; 99.2% S) were the most active agents in all geographic regions. Fluconazole susceptibility was highest in the Asia-Pacific region (80.5% S, 3.9% resistant [R]) and lowest in North America (64% S, 10.3% R) and Latin America (62.1% S, 3.4% R). The extended-spectrum triazoles were most active in the Asia-Pacific region (90 to 96.1% S) and least active in North America (82.5 to 90.3% S). All 46 isolates that were resistant to fluconazole were susceptible to caspofungin (MIC
90
, 0.06 μg/ml) and flucytosine (MIC
90
, 0.12 μg/ml) and exhibited variable cross-resistance to posaconazole, ravuconazole, and voriconazole.
Detection and Typing of Human Papillomavirus by E6 Nested Multiplex PCR
Authors: Sotlar K. , Diemer D. , Dethleffs A. , Hack Y. , Stubner A. , Vollmer N. , Menton S. , Menton M. , Dietz K. , Wallwiener D. , Kandolf R. , Bültmann B. 
Published: July 2004

ABSTRACT

A nested multiplex PCR (NMPCR) assay that combines degenerate E6/E7 consensus primers and type-specific primers was evaluated for the detection and typing of human papillomavirus (HPV) genotypes 6/11, 16, 18, 31, 33, 35, 39, 42, 43, 44, 45, 51, 52, 56, 58, 59, 66, and 68 using HPV DNA-containing plasmids and cervical scrapes (
n
= 1,525). The performance of the NMPCR assay relative to that of conventional PCR with MY09-MY11 and GP5+-GP6+ primers, and nested PCR with these two primer sets (MY/GP) was evaluated in 495 cervical scrapes with corresponding histologic and cytologic findings. HPV prevalence rates determined with the NMPCR assay were 34.7% (102 of 294) in the absence of cervical intraepithelial neoplasia (CIN 0), 94.2% (113 of 120) in the presence of mild or moderate dysplasia (CIN I/II), and 97.8% (44 of 45) in the presence of severe dysplasia (CIN III). The combination of all four HPV detection methods applied in the study was taken as “gold standard”: in all three morphological subgroups the NMPCR assay had significantly (
P
< 0.0001) higher sensitivities than the MY09-MY11 and GP5+-GP6+ assays and sensitivities comparable or equal to those of the MY/GP assay. All 18 HPV genotypes investigated were detected among the clinical samples. The ratio of high- to low-risk HPV genotypes increased from 4:1 (80 of 103) in CIN 0 to 19:1 (149 of 157) in CIN I to III. Multiple infections were detected in 47.9% (124 of 259) of the patients. In conclusion, the novel NMPCR method is a sensitive and useful tool for HPV DNA detection, especially when exact HPV genotyping and the identification of multiple HPV infections are required.
Discovery of Novel Human and Animal Cells Infected by the Severe Acute Respiratory Syndrome Coronavirus by Replication-Specific Multiplex Reverse Transcription-PCR
Authors: Gillim-Ross Laura , Taylor Jill , Scholl David R. , Ridenour Jared , Masters Paul S. , Wentworth David E. 
Published: July 2004

ABSTRACT

The severe acute respiratory syndrome coronavirus (SARS-CoV) is the causative agent of the recent outbreak of severe acute respiratory syndrome. VeroE6 cells, fetal rhesus monkey kidney cells, and human peripheral blood mononuclear cells were the only cells known to be susceptible to SARS-CoV. We developed a multiplex reverse transcription-PCR assay to analyze the susceptibility of cells derived from a variety of tissues and species to SARS-CoV. Additionally, productive infection was determined by titration of cellular supernatants. Cells derived from three species of monkey were susceptible to SARS-CoV. However, the levels of SARS-CoV produced differed by 4 log
10
. Mink lung epithelial cells (Mv1Lu) and R-Mix, a mixed monolayer of human lung-derived cells (A549) and mink lung-derived cells (Mv1Lu), are used by diagnostic laboratories to detect respiratory viruses (e.g., influenza virus); they were also infected with SARS-CoV, indicating that the practices of diagnostic laboratories should be examined to ensure appropriate biosafety precautions. Mv1Lu cells produce little SARS-CoV compared to that produced by VeroE6 cells, which indicates that they are a safer alternative for SARS-CoV diagnostics. Evaluation of cells permissive to other coronaviruses indicated that these cell types are not infected by SARS-CoV, providing additional evidence that SARS-CoV binds an alternative receptor. Analysis of human cells derived from lung, kidney, liver, and intestine led to the discovery that human cell lines were productively infected by SARS-CoV. This study identifies new cell lines that may be used for SARS-CoV diagnostics and/or basic research. Our data and other in vivo studies indicate that SARS-CoV has a wide host range, suggesting that the cellular receptor(s) utilized by SARS-CoV is highly conserved and is expressed by a variety of tissues.
Performance of the MagNA Pure LC Robot for Extraction of
Chlamydia trachomatis
and
Neisseria gonorrhoeae
DNA from Urine and Swab Specimens
Authors: Dalesio Nicholas , Marsiglia Vince , Quinn Andrew , Quinn Thomas C. , Gaydos Charlotte A. 
Published: July 2004

ABSTRACT

DNA from uncentrifuged urines (
n
= 195 and
n
= 202) and cervical swabs (
n
= 221 and
n
= 601) was extracted by the MagNA Pure LC robot and the Amplicor method to validate the robot's extraction for
Chlamydia trachomatis
and
Neisseria gonorrhoeae
testing by Roche PCR. The robot provided a highly sensitive and specific method of DNA extraction.
Detection of
Pneumocystis jiroveci
in Respiratory Specimens by Four Staining Methods
Authors: Procop G. W. , Haddad S. , Quinn J. , Wilson M. L. , Henshaw N. G. , Reller L. B. , Artymyshyn R. L. , Katanik M. T. , Weinstein M. P. 
Published: July 2004

ABSTRACT

We examined four staining methods on replicate smears of 313 respiratory specimens submitted for
Pneumocystis jiroveci
examination. The sensitivity and specificity of Calcofluor white stain (CW) were 73.8 and 99.6%, respectively. The sensitivity and specificity of Grocott-Gomori methenamine silver stain (GMS) were 79.4 and 99.2%, respectively. The sensitivity and specificity of Diff-Quik stain were 49.2 and 99.6%, respectively. The sensitivity and specificity of Merifluor Pneumocystis stain were 90.8 and 81.9%, respectively. Only CW and GMS had positive and negative predictive values of >90%.
Clinical and Microbiological Analysis of Bloodstream Infections Caused by
Chryseobacterium meningosepticum
in Nonneonatal Patients
Authors: Lin Pen-Yi , Chu Chishih , Su Lin-Hui , Huang Chung-Tsui , Chang Wen-Ya , Chiu Cheng-Hsun 
Published: July 2004

ABSTRACT

Chryseobacterium meningosepticum
bloodstream infections in 11 nonneonatal patients were reported. More than half of the infections were community acquired. PCR assays indicated that the organisms produced extended-spectrum β-lactamases as well as metallo-β-lactamases. Genotyping showed diverse fingerprints among the isolates. Six patients survived without appropriate antibiotic treatment. Host factors are the major determinant of the outcomes of
C. meningosepticum
infections.
Prosthetic Mitral Valve Endocarditis Due to
Ochrobactrum anthropi
: Case Report
Authors: Romero Gómez M. P. , Peinado Esteban A. M. , Sobrino Daza J. A. , Sáez Nieto J. A. , Alvarez D. , Peña García P. 
Published: July 2004

ABSTRACT

We describe a case of infective endocarditis in a prosthetic mitral valve due to
Ochrobactrum anthropi
. Although
O. anthropi
is an emerging pathogen in immunocompromised patients, infections with the bacterium have very rarely been documented in healthy hosts, and endocarditis is rare. To our knowledge, only two cases of
O. anthropi
endocarditis have been reported in the medical literature.
Characterization of acute lymphoblastic leukemia progenitor cells
Authors: Cox Charlotte V. , Evely Roger S. , Oakhill Anthony , Pamphilon Derwood H. , Goulden Nicholas J. , Blair Allison 
Published: July 2004

Abstract
Only some acute lymphoblastic leukemia (ALL) cells are thought to be capable of proliferating to maintain the leukemic clone, and these cells may be the most relevant to target with treatment regimens. We have developed a serum-free suspension culture (SC) system that supported growth of B-ALL cells from 33 patients for up to 6 weeks. ALL cells from 28 cases (85%) were expanded in this system, and growth was superior in SC than in long-term bone marrow culture. To characterize ALL progenitors, cells were sorted for expression of CD34 and CD10 or CD19 and the subfractions assayed in SC and in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Cells capable of long-term proliferation in vitro and NOD/SCID repopulation were derived only from the CD34+/CD10- and CD34+/CD19- subfractions, and these cells could engraft secondary recipients. The engrafted cells had the same immunophenotype and karyotype as was seen at diagnosis, suggesting they had differentiated in vivo. These results demonstrate that ALL cells capable of long-term proliferation in vitro and in vivo are CD34+/CD10-/CD19-. This suggests that cells with a more immature phenotype, rather than committed B-lymphoid cells, may be the targets for transformation in B-ALL.
The absence of Grb2-associated binder 2 (Gab2) does not disrupt NK cell development and functions
Authors: Zompi Simona , Gu Hahiua , Colucci Francesco 
Published: July 2004

AbstractScaffolding molecules bind simultaneously and link together various components of signal-transduction pathways. Grb2-associated binder 2 (Gab2) is a scaffolding protein required for FcγR-initiated allergic responses in mast cells and FcγR-mediated phagocytosis in macrophages, where it links IgE and IgG receptors to the phosphatidylinositol-3 kinase (PI-3K) pathway. The FcγR expressed by natural killer (NK) cells triggers antibody-dependent cellular cytotoxicity (ADCC). We show here that mouse NK cells express Gab2 and that although PI-3K was required for ADCC, this FcγR-mediated function was normal in Gab2−/− NK cells. Moreover, NK cell development, spontaneous cytotoxicity, and responses to and production of cytokines were not perturbed in Gab2−/− mice. Considering the striking differences between the signaling requirements of FcγR in macrophages and NK cells, our findings suggest that the organization of signal transduction downstream of the same FcR can be cell type-specific. Conversely, Gab family members Gab1, Gab2, and Gab3 may play specific roles in different leukocytes. As pharmacological targeting of Gab2 in mast cells is a potential strategy to treat allergy, our results suggest prudence, as NK cells may participate in IgE-mediated anaphylaxis in a Gab2-independent manner.
Development of intestinal inflammation in double IL-10- and leptin-deficient mice
Authors: Siegmund Britta , Sennello Joseph A , Lehr Hans A , Batra Arvind , Fedke Inka , Zeitz Martin , Fantuzzi Giamila 
Published: July 2004

AbstractLeptin-deficient (ob/ob) mice are resistant in different models of autoimmunity and inflammation, suggesting that leptin regulates immunity and inflammation. To investigate whether leptin deficiency modulates the spontaneous intestinal inflammation observed in interleukin (IL)-10-deficient mice, double IL-10- and leptin-deficient [IL-10 knockout (KO) ob/ob] mice were generated and compared with single IL-10 KO mice for colitis severity. Body weight in IL-10 KO ob/ob mice was significantly reduced compared with that of ob/ob mice. However, when compared with wild-type or IL-10 KO mice, IL-10 KO ob/ob mice were still markedly obese. IL-10 KO and IL-10 KO ob/ob mice developed colitis with a comparable time-course and severity in terms of macroscopic and histologic scores. Likewise, production of inter feron-γ, IL-6, and IL-13 from colon cultures and splenocytes did not differ among these two groups. Conversely, rates of apoptosis were higher in lamina propria lymphocytes obtained from the colon of IL-10 KO ob/ob compared with IL-10 KO mice. In conclusion, although leptin deficiency has been associated with resistance in models of autoimmunity and inflammation induced by exogenous stimuli, leptin appears not to play a significant role in the spontaneous colitis of IL-10 KO mice, although it modulates survival of intestinal lymphocytes.
Dendritic cells derived from BCG-infected precursors induce Th2-like immune response
Authors: Martino Angelo , Sacchi Alessandra , Sanarico Nunzia , Spadaro Francesca , Ramoni Carlo , Ciaramella Antonio , Pucillo Leopoldo Paolo , Colizzi Vittorio , Vendetti Silvia 
Published: July 2004

AbstractHuman monocytes can differentiate into dendritic cells (DCs) according to the nature of environmental signals. We tested here whether the infection with the live tuberculosis vaccine bacillus Calmette-Guerin (BCG), which is known to be limited in preventing pulmonary tuberculosis, modulates monocyte and DC differentiation. We found that monocytes infected with BCG differentiate into CD1a– DCs (BCG-DCs) in the presence of granulocyte macrophage-colony stimulating factor and interleukin (IL)-4 and acquired a mature phenotype in the absence of maturation stimuli. In addition, BCG-DCs produced proinflammatory cytokines (tumor necrosis factor α, IL-1β, IL-6) and IL-10 but not IL-12. BCG-DCs were able to stimulate allogeneic T lymphocytes to a similar degree as DCs generated in the absence of infection. However, BCG-DCs induced IL-4 production when cocultured with human cord-blood mononuclear cells. The induction of IL-4 production by DCs generated by BCG-infected monocytes could explain the failure of the BCG vaccine to prevent pulmonary tuberculosis.
Chemokine expression during the development and resolution of a pulmonary leukocyte response to influenza A virus infection in mice
Authors: Wareing Mark D , Lyon Ashley B , Lu Bao , Gerard Craig , Sarawar Sally R 
Published: July 2004

AbstractInfluenza A virus replicates in the respiratory epithelium and induces an inflammatory infiltrate comprised of mononuclear cells and neutrophils. To understand the development of the cell-mediated immune response to influenza and how leukocyte trafficking to sites of inflammation is regulated, we examined the chemokine expression pattern in lung tissue from A/PR/8/34-infected C57BL/6 mice using an RNase protection assay. Monocyte chemoattractant protein 1, macrophage inflammatory protein 1α (MIP-1α), MIP-1β, MIP-3α, regulated on activation, normal T expressed and secreted (RANTES), MIP-2, and interferon-inducible protein 10 (IP-10) mRNA expression was up-regulated between days 5 and 15 after infection, consistent with a role for these chemokines in leukocyte recruitment to the lung. Low levels of expression were detected for the CC chemokine receptors (CCR)2 and CCR5, whereas CXC chemokine receptor (CXCR)3 was significantly up-regulated by day 10 after infection, coinciding with peak inflammatory cell infiltration in the airways. As RANTES, IP-10, and their receptors were up-regulated during influenza virus infection, we investigated leukocyte recruitment and viral clearance in mice deficient in RANTES or CXCR3, the receptor for IP-10. Leukocyte recruitment and viral replication in influenza-infected RANTES knockout(−/−) mice were similar to that in control mice, showing that RANTES is not essential for the immune response to influenza infection. Similarly, leukocyte recruitment and viral replication in CXCR3−/− mice were identical to control mice, except at day 8 postinfection, where fewer lymphocytes, neutrophils, and eosinophils were detected in the bronchoalveolar lavage of CXCR3−/− mice. These studies suggest that although the chemokines detected may play a role in regulating leukocyte trafficking to the lung during influenza infection, some may be functionally redundant.